Springer Series on Biofilms

Volume 4

Series Editor: J. William Costerton

Los Angeles, USA
Hans-Curt Flemming • P. Sriyutha Murthy
R. Venkatesan • Keith Cooksey
Editors

Marine and Industrial

Biofouling
Editors
Prof. Dr. Hans-Curt Flemming Dr. R. Venkatesan
Biofilm Centre Organising Secretary RAMAT
University of Duisburg-Essen Group Head Ocean Science
Geibelstraße 41 & Technology for Islands
47057 Duisburg National Institute of Ocean Technology
Germany Pallikaranai, Chennai
India

Dr. P. Sriyutha Murthy Prof. Dr. Keith Cooksey

Ocean Science & Technology for Islands Department of Microbiology
National Institute of Ocean Technology Montana State University
Ministry of Ocean Development 109 Lewis Hall
Velachery Tamabaram Main Road PO Box 173520
Narayanapuram Bozeman, MT 59717
Chennai 601 302 USA
Tamil Nadu
India

Series Editor
J. William Costerton
Director, Center for Biofilms
School of Dentistry
University of Southern California
925 West 34th Street
Los Angeles, CA 90089
USA

ISBN 978-3-540-69794-7 e-ISBN 978-3-540-69796-1

DOI: 10.1007/978-3-540-69796-1

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Preface

This book describes the state of the art in antifouling measures using both conven-
tional biocides and some advanced approaches. Related to biocides, the concept of
the “Biocide Product Directive” of the European Union is presented as an example
of an administrative instrument for curbing excessive use of environmentally unde-
sirable products that may cause ecological damage.
Biofouling is defined as the unwanted accumulation of biological material on
man-made surfaces. This definition includes biofilm-forming microorganisms such
as bacteria, fungi and algae as well as fouling by macroorganisms like hydroids, bar-
nacles, tubeworms and bivalves on submerged surfaces. The problem is site-, season-
and substratum-specific and the control methods effective at a given geographical
location may not hold good elsewhere. The definition is clearly operational, as not
every biofilm or barnacle is equivalent to biofouling but only after the effect exceeds
an arbitrarily given threshold of interference with a technical process. It is impossible
to have an immaculately clean surface and the time has come for realization of the
fact that we have to “live with biofilms and biofouling”. It is for the plant managers
to determine the tolerable threshold of interference, critical to plant operations, and
select a biocidal dose and regime to keep biofilms/biofouling at bay.
The problems in technical processes that are posed by biofouling are substantial.
An example is the interference with heat exchangers, where both macro- and micro-
fouling contribute to losses in heat transfer and to increases in fluid friction resist-
ance. In India, for example, in the next decade 15 large new power plants will be
built, all using seawater as a coolant. Designers and operators will have to over-
come serious fouling problems. The common concepts of biofouling control are
still based on the use of biocides, which only partially and transiently mitigate the
problem. With respect to macrofouling control, focussed research on the biocidal
dosages required to prevent/inhibit settlement are lacking compared to research on
the dosages required for killing established fouling communities. This is important
as dosages required to prevent settlement are far lower than dosages required for
killing established fouling communities. Oxidizing biocides are a better option than
non-oxidizing biocides due to their known mode of action and toxicity, and knowl-
edge of their by-products and degradation pathways. Cost–benefit analysis and
meeting the environmental regulations for discharge are vital parameters governing

v
vi Preface

the selection of biocides in power and desalination plants. Biofouling is a “surface-

associated phenomenon” and control measures should concentrate on this aspect.
For example, treating the entire bulk water with a biocidal concentration seems to
be an economically unviable practice. If technological advancements could be
achieved to deliver biocides at the surface on a continuous basis by the use of
porous polymeric materials, this would ensure a cleaner surface, reduced biocidal
requirements and reduced impact on the environment.
In seawater desalination by membrane filtration, a process meeting the equally
important and increasing demand for freshwater, biofouling also represents the
“Achilles’ heel” of the technology. Again, the use of biocides is still the state of the art,
but their use threatens the material properties of membranes and other equipment, as
well as causing environmental problems when disposed of. The scenario for biofoul-
ing control measures in the case of the shipping industry is in a transient stage where
foul-release coatings alone seem to be an effective alternative. Several alternative
replacement techniques for tributyl tin self-polishing coatings are emerging, but cur-
rently none have demonstrated their performance at the field level and can be trans-
lated into a technology.
The sequence of events leading to biofouling of surfaces comprises the formation
of (i) biofilms containing the initial colonizing organisms, causing serious problems,
and (ii) layers of the most visibly obvious foulers that succeed them, i.e. macroalgae
(Enteromorpha sp., Sargassum sp., Gracillaria sp.) and the hard-shelled foulants
(barnacles, hydroids, tubeworms and bivalves). These organisms colonize submerged
surfaces that already have a microbial film present. Whether there is a positive or
negative effect of the microbial film on the colonization success of the macroorgan-
isms depends on the make-up of the biofilm and the species of invertebrates involved.
Various aspects of this topic are covered in several chapters of the book.
The common approach used against fouling biofilms can be compared to a
“medical paradigm”: the system is considered to be infected and the cure is seen to
be the use of biocides. However, killing the organisms is not the solution, as the
problem is usually not caused by their physiological activity but by their mere pres-
ence as a physical barrier. Reduction of the extent of fouling layers is clearly more
important, but not yet generally the focus of countermeasures.
Antifouling measures are taken all over the world with very unequal levels of
success. There is no such thing as a universal solution to the biofouling problem
(as with the case of biocidal type, dose and regime) but there are many insights
acquired from various fields stricken by biofouling that should be taken into con-
sideration. This book is an attempt to collect some of these approaches and to
provide the opportunity to learn from scientific research on biofouling, as well as
from interesting approaches in various technical fields.
October 2008 H.-C. Flemming
P. Sriyutha Murthy
R. Venkatesan
K. Cooksey
Contents

Part I Microbial Biofouling and Microbially

Influenced Corrosion

Why Microorganisms Live in Biofilms

and the Problem of Biofouling ...................................................................... 3
Hans-Curt Flemming

The Effect of Substratum Properties on the Survival

of Attached Microorganisms on Inert Surfaces .......................................... 13
K.A. Whitehead and J. Verran

Mechanisms of Microbially Influenced Corrosion ...................................... 35

Z. Lewandowski and H. Beyenal

Industrial Biofilms and their Control .......................................................... 65

P. Sriyutha Murthy and R. Venkatesan

Biofilm Control: Conventional and Alternative Approaches ..................... 103

H.-C. Flemming and H. Ridgway

An Example: Biofouling Protection for Marine Environmental

Sensors by Local Chlorination...................................................................... 119
L. Delauney and C. Compère

Surface Modification Approach to Control Biofouling............................... 135

T. Vladkova

A Strategy To Pursue in Selecting a Natural

Antifoulant: A Perspective ............................................................................ 165
K.E. Cooksey, B. Wigglesworth-Cooksey, and R.A. Long

vii
viii Contents

Novel Antifouling Coatings: A Multiconceptual Approach ........................ 179

D. Rittschof

Concept and Consequences of the EU Biocide Guideline .......................... 189

H.-C. Flemming and M. Greenhalgh

Part II Macrofouling

Hydroides elegans (Annelida: Polychaeta):

A Model for Biofouling Research ................................................................. 203
Brain T. Nedved and Michael G. Hadfield

Marine Epibiosis: Concepts, Ecological Consequences

and Host Defence............................................................................................ 219
T. Harder

Larval Settlement and Surfaces: Implications

in Development of Antifouling Strategies .................................................... 233
P. Sriyutha Murthy, V.P. Venugopalan, K.V.K. Nair,
and T. Subramoniam

Macrofouling Control in Power Plants ........................................................ 265

R. Venkatesan and P. Sriyutha Murthy

Inhibition and Induction of Marine Biofouling by Biofilms ...................... 293

S. Dobretsov

A Triangle Model: Environmental Changes Affect

Biofilms that Affect Larval Settlement ......................................................... 315
P.Y. Qian and H.-U. Dahms

Index ................................................................................................................ 329

Contributors

Haluk Beyenal
School of Chemical Engineering and BioEngineering, Washington State
University, Pullman, Washington 99164-2710, USA
beyenal@wsu.edu
C. Compère
Ifremer-In Situ Measurements and Electronics, B.P. 70, 29280, Plouzané, France
K. E. Cooksey
Department of Microbiology, Montana State University, Bozeman, MT 59717,
USA and Environmental Biotechnology Consultants, Manhattan, MT 59741, USA
umbkc@gemini.msu.montana.edu
H.-U. Dahms
Department of Biology and Coastal Marine Lab Hong Kong, University of
Science and Technology, Clearwater Bay, Kowloon, Hong Kong, China and
National Taiwan Ocean University, Keelung, Taiwan
L. Delauney
Ifremer-In Situ Measurements and Electronics, B.P. 70, 29280, Plouzané, France
laurent.delauney@ifremer.fr
Sergey Dobretsov
Marine Science and Fisheries Dep., Agriculture and Marine Sciences College,
Sultan Qaboos University, Al-Khod 49, PO Box 123, Sultanate of Oman and
Benthic Ecology, IFM-GEOMAR, Kiel University, Düsternbrooker Weg 20,
24105, Kiel, Germany
sergey_dobretsov@yahoo.com, sergey@squ.edu.om
Hans-Curt Flemming
Biofilm Centre, University of Duisburg-Essen, Geibelstrasse 41, D-47057,
Duisburg, Germany and IWW Centre for Water, Moritzstrasse 26, 45476,
Muelheim, Germany
hanscurtflemming@compuserve.com

ix
x Contributors

Malcolm Greenhalgh
Malcolm Greenhalgh Consultancy Ltd (MGCL), Dale Cottage, Lower Park Royd
Drive, Ripponden, West Yorkshire. HX6 3HR, UK
malcolm.greenhalgh@btopenworld.com
Michael G. Hadfield
Kewalo Marine Laboratory, University of Hawaii, 41 Ahui Street, Honolulu, HI
96813, USA
hadfield@hawaii.edu
Tilmann Harder
Centre for Marine Bio-Innovation, University of New South Wales, Sydney,
NSW 2052, Australia
t.harder@unsw.edu.au
Zbigniew Lewandowski
Department of Civil Engineering and Center for Biofilm Engineering,
Montana State University, EPS Building, Room 310, Bozeman, MT 59717, USA
ZL@erc.montana.edu
R. A. Long
Department of Biological Sciences, University of South Carolina, Columbia SC
29208, USA
K. V. K. Nair
National Institute of Ocean Technology, Ministry of Earth Sciences, Government
of India, Velachery-Tambaram Main Road, Narayanapuram, Chennai 600 100,
India
Brian T. Nedved
Kewalo Marine Laboratory, University of Hawaii, 41 Ahui Street, Honolulu,
HI 96813, USA
P. Y. Qian
1. Department of Biology and Coastal Marine Lab Hong Kong, University of
Science and Technology, Clearwater Bay, Kowloon, Hong Kong, China
boqianpy@ust.hk
Harry Ridgway
Stanford University & AquaMem Scientific Consultants, Department of Civil
and Environmental Engineering, PO Box 251, Rodeo, New Mexico 88056, USA
ridgway@vtc.net
Dan Rittschof
Duke University Marine Laboratory, Nicholas School of the Environment,
135 Duke Marine Lab Road, Beaufort, NC 28516, USA
ritt@duke.edu
Contributors xi

P. Sriyutha Murthy
Biofouling and Biofilm Processes Section, Water and Steam Chemistry Division,
BARC Facilities, Indira Gandhi Center for Atomic Research Campus, Kalpakkam
603 102, India
psm_murthy@yahoo.co.in, psmurthy@igcar.gov.in
T. Subramoniam
National Institute of Ocean Technology, Ministry of Earth Sciences, Government
of India, Velachery-Tambaram Main Road, Narayanapuram, Chennai 600 100,
India
R. Venkatesan
National Institute of Ocean Technology, Ministry of Earth Sciences, Government
of India, Velachery-Tambaram Main Road, Narayanapuram, Chennai 600 100,
India
V. P. Venugopalan
Biofouling and Biofilm Processes Section, Water and Steam Chemistry Division,
BARC Facilities, Indira Gandhi Center for Atomic Research Campus, Kalpakkam
603 102, India
vpv@igcar.gov.in
J. Verran
School of Biology, Chemistry and Health Science, Manchester Metropolitan
University, Chester St, Manchester, M1 5GD, UK
j.verran@mmu.ac.uk
T. Vladkova
Department of Polymer Engineering, University for Chemical Technology and
Metallurgy, 8 “Kliment Ohridsky” Blvd., 1756 Sofia, Bulgaria
tgv@uctm.edu
K. A. Whitehead
School of Biology, Chemistry and Health Science, Manchester Metropolitan
University, Chester Street, Manchester, M1 5GD, UK
k.a.whitehead@mmu.ac.uk
B. Wigglesworth-Cooksey
Department of Microbiology, Montana State University, Bozeman, MT 59717,
USA and Environmental Biotechnology Consultants, Manhattan, MT 59741, USA
 Part I
Microbial Biofouling and Microbially
Influenced Corrosion
Why Microorganisms Live in Biofilms
and the Problem of Biofouling

Hans-Curt Flemming

Abstract Microbial biofouling is a problem of microbial biofilms. Biofouling

occurs in very different industrial fields and is mostly addressed individually.
However, the underlying phenomenon is much more general and in order to
understand the processes causing biofouling, it is good to understand the basics
of biofilm formation and development. Almost every surface can be colonized
by bacteria, forming biofilms. After adhesion, the cells embed themselves in a
layer of extracellular polymeric substances (EPS), highly hydrated biopolymers
of microbial origin such as polysaccharides, proteins, nucleic acids and others. In
this matrix they organize their life, develop complex interactions and resistance to
biocides. The resulting biofilm structure is highly heterogeneous and dynamic. It
is kept together by weak physicochemical interactions of extracellular polymeric
substances, which have to be overcome when cleaning is attempted. The ecological
advantages for the biofilm mode of life are so strong that almost all microorganisms
on earth live in biofilm-like microbial aggregates rather than as single organisms.

1 Biofouling

Slime on surfaces is the usual manifestation of a phenomenon called “biofouling”. It

occurs in a wide range of industrial processes and in all of them it is a nuisance, some-
times a very expensive one. It is fought against in each industrial area individually and
there are many “re-inventions of the wheel” and many common mistakes – although
the underlying problem is always the same: microbial biofilms. Five common mistakes
in conventional anti-fouling measures can be identified in most cases are:
1. No early warning systems: Biofouling is detected by losses in process perform-
ance or product quality – no monitoring system.

H.-C. Flemming
Biofilm Centre, University of Duisburg-Essen, Geibelstrasse 41, 47057, Duisburg, Germany
e-mail: hanscurtflemming@compuserve.com

Springer Series on Biofilms, doi: 10.1007/7142_2008_13 3 3

© Springer-Verlag Berlin Heidelberg 2008
4 H.-C. Flemming

2. No information on biofilm site/extent: Sampling is performed of the water phase,

which gives no information about site and extent of fouling films; sampling is
not performed on surfaces.
3. Disinfection is performed as a countermeasure: This is not cleaning, while in
most cases, the problem is caused by biomass – dead or alive. Biocides leave
dead biomass on surface, providing good regrowth.
4. No nutrient limitation is considered: However, nutrients are potential biomass
and are not reduced by biocides.
5. No optimization of countermeasures: Efficacy control is performed only by
process or product quality – see point 1.
In very diverse industrial fields, biofouling problems all originate from the same
cause: microbial biofilms. Biofilms follow common natural laws, which are impor-
tant to be understood for more effective countermeasures. Basically, in biofouling
the same processes occur as in biological filtration: microorganisms colonize sur-
faces, sequester nutrients from the water phase and convert them into metabolites
and new biomass. Industrial systems frequently offer large surface areas, which
invite colonization and subsequent use of biodegradable substances, leading to an
extent of biofilm development that interferes with process parameters or product
quality. Biofouling can be considered as a “biofilm reactor in the wrong place and at
the wrong time”. Therefore, detailed knowledge about biofilms is crucial for under-
standing and preventing biofouling as well as for successful anti-fouling measures.
The purpose of this chapter is to highlight the reasons why microorganisms form
biofilms. They are the most successful form of life on earth and it is not surprising,
that they cannot be eliminated easily. In many cases, microbial biofilms precede
macroorganismic settlement (e.g. by larvae, barnacles and mussels), a phenomenon
called macrofouling.

2 Microbial Biofilms

It is only few decades since microorganisms, sitting at the walls of microbiological

liquid cultures, on rocks, sediments, in soil, on leaves, skin, teeth, implants or in
wounds turned from a nuisance that could not be investigated by classical microbio-
logical methods into a highly active field of research in which biofilms were acknowl-
edged as the dominant form of life for microorganisms on earth (Flemming 2008). It
became obvious that microorganisms on earth generally do not live as single cells and
in pure cultures but do so in aggregates of mixed species. Such aggregates can consist
of microcolonies as well as of patchy or confluent films on surfaces, but also as thick
mats, sludge or flocks in suspension. By convention, all these phenomena are sub-
sumed under the (somehow vague) term “biofilm” (Donlan 2002). It was just a shift
of point of view that made it evident that this form of life could be found everywhere.
In fact, biofilms are the first form of life recorded on earth, dating back 3.5 billion
years (Schopf et al. 1983), and the most successful one. Biofilms are found even in
extreme environments, such as the walls of pores in glaciers, in hot vents, under pressure
Why Microorganisms Live in Biofilms and the Problem of Biofouling 5

of 1,000 bar at the bottom of the ocean, in ultra-pure water as well as highly salty
solutions, and on electrodes active through the entire range of thermodynamic water
stability. Biofilms occur as endolithic populations in minerals, on the walls of disin-
fectant concentrate pipes or even in highly radioactive environments such as nuclear
power plants. The surface of almost all living organisms is colonized by biofilms,
which provide in many cases a protective and supportive flora (e.g. skin flora), while
in other cases they cause transient, acute, chronic and even fatal diseases. Biofilms
are substantially involved in the biogeochemical cycles of carbon, oxygen, hydrogen,
nitrogen, sulphur, phosphorus and many metals (Ehrlich 2002). Enhancing mineral
weathering processes by microbial leaching, they mobilized metal ions that were vital
for further evolution. In biofilms, photosynthetic organisms evolved from originally
anaerobic conditions on earth, providing oxygen as a “waste gas” from photosynthe-
sis to the atmosphere of this planet and restricting the space for living of anaerobic
organisms, which first dominated life on earth, to oxygen-depleted areas. Predation
among biofilm organisms is thought to have led to endosymbionts and, eventually, to
the evolution of eukaryotic organisms and the concept of infection.
One of the reasons for the late acknowledgement of biofilms is certainly the insuf-
ficient suitability of conventional microbiological methods. The introduction of fluo-
rescence microscopy and confocal laser scanning microscopy, micro-electrodes,
advanced chemical analysis with particular respect to protein analysis, and, most pow-
erfully, molecular biology has allowed biofilm biology to be revealed in much greater
detail. As a consequence, the literature in this field has virtually exploded with at least
100,000 publications on biofilms currently. The advance of knowledge is immense and
fast, and this brief chapter can only superficially cover it. From a life science point of
view, the most exciting aspect is that microorganisms today cannot be viewed as blind
little individuals that compete as much as they can, but as complex communities with
division of labour and many aspects of multicellular life (Flemming 2008). This is
certainly a new understanding of microbiology with big consequences for biotechnol-
ogy, medicine and handling of microbial problems in technical processes.
The biofilm mode of life provides a range of advantages to the single cell plank-
tonic mode of life. One of the biggest advantages is the fact that the cells can
develop stable interactions, resulting in synergistic microconsortia. An example is
the close association of ammonia oxidizing and nitrite oxidizing bacteria. The
ammonia oxidizers produce nitrite, an inhibitory end product that is comfortably
used as substrate by the nitrite oxidizers. This process occurs in the environment
and has been employed in nitrification steps in waste water treatment for a long
time and with great success. There are many other examples of orchestrated degra-
dation of substrates by cascades of organisms.

3 Extracellular Polymeric Substances

A characteristic feature of biofilm organisms is that they are kept together and attached
to surfaces by means of their extracellular polymeric substances (EPS, Flemming and
Leis 2002). An example is shown in Fig. 1, which is a scanning electron micrograph
6 H.-C. Flemming

Fig. 1 Scanning electron micrograph of a biofilm of Pseudomonas putida on a mineral surface.

EPS (dehydrated for SEM sample preparation) are surrounding the cells, keeping them together
and on the surface

of Pseudomonas putida on a mineral surface. The sheet-like material that surrounds

the cells is EPS, dehydrated by sample preparation for SEM observation.
The EPS determine the immediate conditions of life of biofilm cells living in this
microenvironment by affecting porosity, density, water content, charge, sorption
properties, hydrophobicity and mechanical stability – all belonging to the parame-
ters on which the conditions of life in a biofilm depend (Branda et al. 2005). This
section represents a recent synopsis of the actual state of understanding of the role
of EPS (Flemming et al. 2007).
EPS are biopolymers of microbial origin in which biofilm microorganisms are
embedded. In fact, the biopolymers are produced by archaea, bacteria and eukaryotic
microbes. Contrary to common belief, they are certainly more than only polysac-
charides. Additionally, they comprise a wide variety of proteins, glycoproteins,
glycolipids and in some cases surprising amounts of extracellular DNA (e-DNA). In
environmental biofilms, polysaccharides are frequently only a minor component. All
EPS biopolymers are highly hydrated and form a matrix, which keeps the biofilm
cells together and retains water. This matrix interacts with the environment, e.g. by
attaching biofilms to surfaces and by its sorption properties, which allows for
sequestering dissolved and particulate substances from the environment providing
nutrients for biofilm organisms. The EPS influence predator–prey interactions, as
demonstrated in a system of a predatory ciliate and yeast cells. Grazing led to an
increase in biofilm mass and viability with EPS as preferred food source.
Why Microorganisms Live in Biofilms and the Problem of Biofouling 7

Curli as proteinaceous fibrils have gained more interest beyond infection as

curli-like fibrils have also been found to play an important role in natural biofilms
produced by a variety of different microorganisms. An abundance of amyloid adhe-
sions in natural biofilms has been found, which may contribute considerably to
their mechanical properties. Strengthening of biofilm structure is crucial for the
stability of the “house” and the continuation of synergistic interactions based on
spatial proximity of various biofilm organisms.
Cellulose has been found to be a constituent EPS component in amoebae, algae
and bacteria. In agrobacteria, cellulose is involved in attachment and it seems as if
cellulose plays an underestimated role in environmental EPS. It is formed by a
variety of organisms and influences biofilm structure. Cellulose is also important in
infectious processes when co-expressed with curli fimbriae in Escherichia coli
(Wang et al. 2007).
Biofilms are also an ideal place for exchanging genetic material and maintaining
a large and well-accessible gene pool. Horizontal gene transfer is facilitated as the
cells are maintained in close proximity to each other, not fully immobilized, and
can exchange genetic information. Significantly higher rates of conjugation in bac-
terial biofilms compared to planktonic populations have been reported (Hausner
and Wuertz 1999).
The EPS matrix is not only composed of a variety of components but, in addi-
tion, these are able to interact. One example is the retention of extracellular pro-
teins such as lipase by alginate. Such mechanisms are crucial for preventing the
wash-out of enzymes, keeping them close to the cells that produced them and
allowing for effective degradation of polymeric and particulate material. This
leads to the concept of an “activated matrix”. Activation is made even more
dynamic and versatile by the excretion of membrane vesicles (MVs). These highly
ordered nanostructures act as “parcels” containing enzymes and nucleic acids, sent
into the depth of the EPS matrix. Such vesicles, along with phages and viruses
(which are of similar size), can serve as carriers for genetic material and thereby
enhance gene exchange. Through their chemistry, the MVs may bind extraneous
components; their enzymes may help degrade polymers, providing nutrients or
inimical agents and thereby inactivating them. Furthermore, they seem to be part
of the “biological warfare” within biofilms, occurring as predatory vesicles con-
taining lytic enzymes. This biological warfare is also long-range as, in common
with other matrix material, MVs are shed from the biofilm. In this respect, vesicles
are “missiles” delivering, among others, virulence factors and cell-to-cell signals
(Schooling and Beveridge 2006).
The composition, architecture and function of the EPSmatrix reveal a very com-
plex, dynamic and biologically exciting view. First of all, the matrix is a network
providing sufficient mechanical stability to maintain spatial arrangement for micro-
consortia over a longer period of time. This stability is provided by hydrophobic
interactions, cross-linking by multivalent cations and entanglements of the biopoly-
mers with e-DNA as a newly appreciated structural component. The forces that
keep the biofilm matrixtogether are provided, thus by weak physicochemical
interactions such as hydrogen bonds, van der Waals forces and eletrostatical inter-
actions. They are schematically depicted in Fig. 2 (after Mayer et al. 1999).
8 H.-C. Flemming

CH2
OH
(i)
CH2 COO-
OH CH2
COO- OH
(iv) Ca2+
CH2 (ii)
OH
-
OOC
CH2 COO- + + + + +
+
OH +

OH
(i) - - - -
(iii) - -
+ + + + + -
CH2

- -
- - -

Fig. 2 Forces that keep the EPS matrix together: (i) hydrogen bonding, (ii) cation bridging,
(iii) van der Waals forces, (iv) repulsive forces (after Mayer et al. 1999)

The repulsive forces are of big importance for the biofilm structure as they prevent
a polymer network from collapsing. Water is equally important as it dilutes the macro-
molecules and limits the number of interacting groups. During desiccation, more inter-
action takes place and turns biofilms into practically insoluble structures (Fig. 3).
When microbial biofilms are to be removed from surfaces, as in the case of
cleaning, these weak binding forces have to be overcome. Although the individual
forces are low, the gross overall binding force can exceed that of covalent bonds,
but it is not a directed bond. Therefore, in response to shear forces, biofilm first
show characteristics of viscoelastic bodies, while when a breaking point is
exceeded, they have properties of viscous liquids (Körstgens et al. 2001). Cleaning
has to attempt weakening of the binding forces in order to support the efficacy of
shear forces. From this point of view, it is very obvious that killing of the biofilm
organisms will not contribute to cleaning unless the matrix structure is affected.
In conclusion, it seems as if “slime” has been very much underestimated and it
turns out that the EPS matrix is considerably more than simply the glue for
biofilms. Rather, it is a highly sophisticated system that gives the biofilm mode of
life particular and successful features.

4 Structure of Biofilms

The biofilm matrix is highly hydrated and very heterogeneous. The morphology of
a biofilm appears very variable. Figure 4 shows an artists view of various aspects
of evolving and mature biofilms, as developed from many recent findings in biofilm
research.
Why Microorganisms Live in Biofilms and the Problem of Biofouling 9

Fig. 3 Desiccated biofilm. The cohesive forces and the surface adhesion forces increase. Curling
of biofilms occurs and sand grains from mortar are ripped out, contributing to microbially influ-
enced weathering

The figure reveals structural aspects that make life in biofilms even more attrac-
tive. The porous architecture allows for convectional flow through the depth of the
biofilm, while within the EPS matrix only diffusional transport is possible. Organisms
10 H.-C. Flemming

Fig. 4 Structure and processes in a biofilm (permission of Peggy Dirkx, Center for Biofilm
Engineering, Montana State University, Bozeman, MT)

at the bottom of the biofilm, thus, can get access to nutrients without competition
from those at the interface to the bulk water phase. Strong gradients can occur in
biofilms, e.g. by actively respiring aerobic heterotrophic organisms, which consume
oxygen faster than it can diffuse through the matrix. This generates anaerobichabitats
just below highly active aerobic colonies in distances of less than 50 µm. Other gra-
dients, such as pH-value, redox potential and ionic strength are known within bio-
films. The result is complex interactions and a functionally structured system. The
ecological relevance of this heterogeneity has inspired Watnick and Kolter (2000) to
describe the biofilm as a “City of Microbes”.
Another feature of biofilm cells is the increased tolerance to biocides, compared
to planktonic cells (Schulte et al. 2005). It must be taken into consideration that
biofilms have existed for billions of years and have survived all kinds of adverse
conditions. Therefore, many different mechanisms have evolved for resistance, and
they are far from being fully understood (Lewis 2001). The fact is that resistance
genes can be exchanged and that biofilms have been observed even in disinfection
concentrate pipes. The resistance of biofilms is particularly problematic in medi-
cine where contaminations of implants, catheters or bones result in long-term infec-
tions, which in many cases can only be overcome by radical measures such as
exchange of implants and removal of bone parts. In drinking water systems, bio-
films can harbour hygienically relevant organisms that may even proliferate if
nutrients are provided. Even enhanced application of disinfectants such as chlorine
will not eradicate such biofilms.
Why Microorganisms Live in Biofilms and the Problem of Biofouling 11

5 Ecological Advantages of the Biofilm Mode of Life

From the above highlighted context, it is obvious that microorganisms gain clear
advantages from the biofilm mode of life. This has been summarized very well by
Costerton (2007), a biofilm pioneer. The ecological advantages of the biofilm mode
of life are quite a few more and can be summarized as follows:
• Formation of stable microconsortia
• Biodiversity: gradients create different habitats
• Gene pool and facilitated genetic exchange
• Retention of extracellular enzymes in the matrix
• Access to particulate biodegradable matter by colonization
• Recycling of nutrients because lysed cells are retained in the biofilm
• Protection against biocides and other stress
• High population density: threshold concentration of signalling molecules is eas-
ily reached, facilitating intercellular communication
These are good reasons explaining the preference for the biofilm mode of life of
most microorganisms on earth.

References

Branda SS, Vik A, Friedman L, Kolter R (2005) Biofilms: the matrix revisited. Trends Microbiol
13:20–26
Costerton JW (2007) The biofilm primer. Springer, Berlin Heidelberg New York
Donlan RM (2002) Biofilms: microbial life on surfaces. Emerg Infect Dis 8:881–890
Ehrlich HL (2002) Geomicrobiology, 4th edn. Marcel Dekker, New York
Flemming H-C (2008) Biofilms. In: Encyclopedia of life sciences. Wiley, Chichester, http://www.
els.net/, doi: 10.1002/9780470015902.a0000342
Flemming H-C, Leis A (2002) Sorption properties of biofilms. In: Bitton G (ed.) Encyclopedia of
environmental microbiology, vol 5. Wiley-Interscience, New York, pp. 2958–2967
Flemming H-C, Neu TR, Wozniak D (2007) The EPS matrix: the “House of biofilm cells”.
J Bacteriol 189:7945–7947
Hausner M, Wuertz S (1999) High rates of conjugation in bacterial biofilms as determined by
quantitative in-situ analysis. Appl Environ Microbiol 65:3710–3713
Körstgens V, Wingender J, Flemming HC, Borchard W (2001) Influence of calcium ion concentra-
tion on the mechanical properties of a model biofilm of Pseudomonas aeruginosa. Water Sci
Technol 43 (6) 49–57
Lewis K (2001) Riddle of biofilm resistance. Antimicrob Agents Chemother 45:999–1007
Mayer C, Moritz R, Kirschner C, Borchard W, Maibaum R, Wingender J, Flemming HC (1999)
The role of intermolecular interactions: studies on model systems for bacterial biofilms. Int J
Biol Macromol 26:3–16
Schooling SR, Beveridge TR (2006) Membrane vesicles: an overlooked component of the matri-
ces of biofilms. J Bacteriol 188:5945–5947
Schopf JW, Hayes JM, Walter MR (1983) Evolution on earth’s earliest ecosystems: recent progress
and unsolved problems. In: Schopf JW (ed.) Earth’s earliest biosphere. Princeton University
Press, New Jersey, pp. 361–384
12 H.-C. Flemming

Schulte S, Wingender J, Flemming H-C (2005) Efficacy of biocides against biofilms. In: Paulus
W (ed.) Directory of microbiocides for the protection of materials and processes. Kluwer,
Doordrecht, pp. 90–120
Wang XM, Rochon A, Lamprokostopoulou A, Lünsdorf H, Nimtz M, Römling U (2007) Impact
of biofilm matrix components on interaction of commensal Escherichia coli with the gastroin-
testinal cell line HT-29. Cell Mol Life Sci 63:352–2363
Watnick P, Kolter R (2000) Biofilms, city of microbes. J Bacteriol 182:2675–2679
The Effect of Substratum Properties
on the Survival of Attached Microorganisms
on Inert Surfaces
K.A. Whitehead(*
ü ) and J. Verran

Abstract Biofilm formation is dependent on the surrounding environmental

conditions and substratum parameters. Once a biofilm forms many factors may influence
cell survival and resistance. Cell adhesion to a surface is a prerequisite for coloniza-
tion. However, attached microorganisms may not be able to multiply, and may merely
be surviving on the surface, for example at a solid–air interface, rather than forming a
biofilm. Retention of attached cells is a key focus in terms of surface hygiene and bio-
film control. Factors that affect this retention may differ from those affecting biofilm
formed at the solid–liquid interface: the nature of the substratum, presence of organic
material, vitality of the attached microorganism, and of course the surrounding envi-
ronment. The majority of publications focus on the solid–liquid interface; literature
addressing the solid–air interface is considerably less substantial.

1 Introduction

Microbial attachment, adhesion, retention and subsequent biofilm formation are

major concerns in many settings where biofilms play a key role in ensuring the
survival of microorganisms and their resistance to a range of external “attacks” for
example by protozoa, environmental conditions or chemical agents. Mechanisms of
resistance to these external forces are diverse.
The literature concerning biofilms and resistance is significant, and the fact that
biofilms demonstrate significantly enhanced resistance is well recognized. This
paper focuses on the survival of attached cells rather than on biofilm. Donlan and
Costerton (2002) define a biofilm as “a microbially derived sessile community
characterized by cells that are irreversibly attached to a substratum or interface or
to each other and that are embedded in a matrix of extracellular polymeric substances

K.A. Whitehead
School of Biology, Chemistry and Health Science, Manchester Metropolitan University,
Chester St, Manchester, M1 5GD UK
e-mail: k.a.whitehead@mmu.ac.uk

Springer Series on Biofilms, doi: 10.1007/7142_2008_23 13

© Springer-Verlag Berlin Heidelberg 2008
14 K.A. Whitehead and J. Verran

that they themselves have produced. The cells in the biofilm may also exhibit an
altered phenotype with respect to growth rate and gene transcription”. Attached
cells on the other hand may be surrounded by preformed extracellular polymeric
substances (EPS), but will not produce more unless appropriate environmental
conditions are present. Attached cells rather than biofilm are therefore found at the
solid–air as well as the solid–liquid interface. Intermittent exposure of the substra-
tum to moisture, for example during cleaning of hygienic surfaces or external surface
exposure to rain, or at a meniscus (Fig. 1) generates a solid-liquid–air interface, at
which fouling is apparent.
Adhesion is a prerequisite for colonization. Microorganisms can survive in very
thin water films but attached microorganisms may not be able to multiply, particu-
larly if there is little moisture available. Thus many of the factors affecting the
survival of cells in a biofilm may not be applicable to cells retained on a surface in
the absence of moisture, but in the presence of organic material. Thus external factors,
such as the nature of the substratum and of the surrounding environment, will
significantly affect survival and “biotransfer potential” (Verran and Boyd 2001).
In this chapter a range of examples showing the effect of surface features on cell
survival and resistance will be discussed, focusing on the marine environment wher-
ever possible/appropriate, and addressing any differences between the solid–liquid and
solid–air interface.

Fig. 1 Plastic buoy that has biofilm growing on its surface, resulting in fouling of the material
The Effect of Substratum Properties on the Survival of Attached 15

2 Microbial Attachment to Surfaces

Viruses, bacteria, fungi, algae and protozoa may all be found in the marine biofilm
community, (Fig. 2) along with “macroorganisms”, such as barnacles and sea-
weeds. Many studies have attributed microbial survival and resistance of attach-
ment microorganisms to their cellular physiology but it is now thought that there
are a number of contributory physical and chemical factors involved. Physicochemical
parameters will affect initial attachment. Once the cells attach, the surface chemistry
will influence cell adhesion, whilst topographic features allow maximum cell-sur-
face binding, enhancing strength of attachment and thus retention.

Fig. 2 The scale of the surface roughness is important since the organisms that may be involved in the
formation of an environmental biofilm may vary greatly in size and shape (a) Staphylococcus aureus
(bacteria) around 1 μ m; (b) Candida albicans (yeast) around 3–5 μ m; (c) Cladsporium sp. (fungal
species) 12–18 μ m; d) planktonic algae around 25 μ m; (e) Aureobasidum pullulans hyphae can grow
to various lengths; (f) Stentor coeruleus (cilitae) >200 μ m
16 K.A. Whitehead and J. Verran

In an aqueous environment bacterial attachment to a surface occurs rapidly, over

a few seconds to a few minutes. Moreover, the binding of microorganisms to a
surface can confer advantages to cell survival, for example the attachment of cells
to solid surfaces has been reported to immediately upregulate alginate synthesis in
a strain of Pseudomonas aeruginosa (Davies et al. 1993), therefore strengthening
cell–substratum binding. Bright and Fletcher (1983) also gave evidence that sup-
ported the existence of a direct substratum influence on the assimilation of amino
acids in a marine Pseudomonas sp. thus enhancing nutrient availability to the cell.
If cells are deposited on a surface in the absence of a solid–liquid interface, for
example by direct contact, then the behaviour of the passively attached cells may
differ from that described above. However, in either case, since the surface of the
substratum is the primary contact for cell attachment, the study of cell–surface
interactions is of utmost importance.

3 Primary Adhesion to Surfaces

Much of the literature discusses microbial resistance and cell eradication once cells
have attached to a surface. However, it would seem that a more proactive approach
is to target the organisms in order to prevent initial cell attachment and thus subse-
quent retention to a surface. Primary cell adhesion to surfaces is dictated by a
number of parameters. In an aqueous environment (liquid–solid), cells will first
approach a surface by natural forces such as diffusion, gravitation, and Brownian
motion. However, once in the vicinity of a surface, physicochemical parameters
will come into play and the influence of Lifshitz–van de Waals forces, electrostatic
forces and hydrogen bonding will influence the cells approach and subsequent
attachment to the surface. It would seem obvious that the physicochemical, chemi-
cal and the topography have an influence on the properties of the substratum.
However, the properties of the cell surface also need to be considered. The cell is a
complex arrangement of different chemical species and topographies (on the micro-
and nanoscale), and hence comprises islands of different physicochemical and
chemical properties. Further, these properties will alter with changes in a given
environment. The substratum, once in an aqueous environment will become coated
with organic material, known as a conditioning film, as will cells, in addition to the
presence of EPS. The EPS also plays a paramount role in primary adhesion.
At the solid–air interface in an open environment, the initial transfer of cells to a
surface may occur in one fouling event where surfaces are contaminated by direct
contact with the fouling material. Despite the complexities of these initial attach-
ment scenarios it would seem logical to attempt to reduce/delay/prevent this initial
cell–surface interaction in preference to managing the subsequent attached biofilm,
for example by surface modification. There are a number of approaches that are
directed towards this phenomenon, including the modification of surface topography
(macro-, micro- and nanoscale), chemistry (self-assembled monolayers) and physi-
cochemistry (superhydrophobic surfaces). However, it is inevitable that practically
all surfaces will be colonized sooner or later (Flemming, personal communication).
The Effect of Substratum Properties on the Survival of Attached 17

4 Substratum Physicochemical Properties

Many different physicochemical interactions between microorganisms and the

solid surface have been described in the literature (Boonaert and Rouxhet 2000;
Simoes et al. 2007). If the physicochemical properties of a surface can be defined
and controlled, then cell attachment, survival and biofilm formation can in turn be
more easily managed. There is conflicting literature concerning the complex effect
of surface and/or microbial physicochemical properties on microbial attachment to
surfaces (Bos et al. 1999; Chen and Strevett 2001). Adhesion of vegetative cells
(Sinde and Carballo 2000), bacterial spores (Husmark and Ronner 1993) and fresh-
water bacteria (Pringle and Fletcher 1983) has been shown to increase with increas-
ing surface hydrophobicity. Other organisms have also been shown to preferentially
bind to hydrophobic surfaces: for example Enteromorpha spores (Callow et al.
2002). It has been suggested that cell attachment to hydrophobic plastics occurs
very quickly (Carson and Allsopp 1980) whereas cell attachment to hydrophilic
surfaces such as metallic oxides, glass and metals increases with longer exposure
times (Dexter 1979).
The surface free energy of a substratum is believed to be important in initial cell
attachment, but the interactions involved are complex. Biofilm formation coincides
with increased inorganic positively charged elements at the surface (Carlen et al.
2001), but positive substratum surface charge has also been shown to impede bacte-
rial surface growth despite initially promoting adhesion (Gottenbos et al. 2001). A
maximum detachment rate for marine biofilms or bacteria has been demonstrated
for surface free energies of 20–27 mN m−1 (Becker 1998; Pereni et al. 2006).
The surface energy distribution on substrata will be dependent on the surface
structure and will be affected by surface imperfections such as cracks or pores, and
also on the conditioning layer of the substratum, which is in turn defined by the
surrounding environment. It has been suggested that the differences observed
between surfaces in in-vitro hydrophobicityassessments may be due to changes in
the substratum characteristics that occur during the first few minutes of exposure to
the surrounding fluid, where a primary film of organic molecules known as the
conditioning film is adsorbed to the substratum (Pringle and Fletcher 1983). The
presence of this film clearly affects microbial retention, and also contributes to cell
interactions with the surface. It is likely that conditioning films may mask some
substratum properties.
This interaction may not be relevant to the attachment of cells on open surfaces,
where contact between the cell and the substratum may be achieved by transient wetting
or transfer between surfaces involving direct contact, or airborne transmission.
However, the retention of the cells will be affected by the cohesive forces and by the
area of contact between the cell and the substratum, be it conditioned or otherwise.
In an aqueous environment the conditioning of a surface by smaller molecules
and ions will occur before bacterial attachment, thus the film provides the linking
layer between the cells and the surface. A clear understanding of all interactions is
needed if a logical attempt at controlling surface fouling is to transpire. Antifouling
surfaces are possible but, since each fouling environment is essentially unique, it
18 K.A. Whitehead and J. Verran

may be that situations have to be addressed on an individual basis. The life span
and cost of production for any antifouling product must also be considered along-
side the expected antifouling benefit. It is unlikely that fouling can be completely
prevented, but if soil is more easily removed or if fouling is delayed then clear
economical, ecological or health-associated benefits may be derived.

5 Chemical Properties of Materials

The chemical properties of materials are defined by the elements that ultimately make
up the molecules of a surface. The surface chemistry, i.e. the chemical properties of the
materials, has been shown to directly affect microbial attachment (Verran and
Whitehead 2005: Whitehead and Verran 2007) and survival. A range of inert substrata
find use in environments where microbial attachment and biofilm formation are common.
The chemistry of the surface inevitably affects these interactions. Thus the choice of
material must be made depending on the intended properties of the surface (e.g.
immersed/exposed, high cleanability/low fouling, low wear, non-toxic, low cost etc.).

5.1 Metals

Cell attachment and thus biofilm formation can occur on metals, including aluminium
(Nickels et al. 1981), stainless steel (Mittelman et al. 1990) and copper (Geesey and
Bremer 1990). However, some metals such as aluminium or copper are considered
toxic to bacteria (Avery et al. 1996). It has been suggested that microbial resistance
to some metals, for example lead acetate, can be attributed to the high lead content of
disinfectants and antiseptics, whilst resistance to copper sulfate may be due to its use
as an algicide (Hiramatsu et al. 1997). However, even with concerns of increased
resistance of microorganisms, and the frequent necessity of moisture to enable the
antimicrobial action to occur, the incorporation of a range of metals into “antibacte-
rial” surfaces has been reported (Kielemoes et al. 2000). The location of these
surfaces, whether immersed, intermittently wet or dry, will clearly affect any intended
antimicrobial effect. In particular, silver and copper have received significant atten-
tion. Antimicrobial silver and/or copper reagents have been occasionally applied to
the water distribution system for inactivation of pathogens (Liu et al. 1998). However,
bacterial resistance against silver and other metals may lead to limitations in the effi-
cacy of these bactericide-releasing materials (Cloete 2003).
Copper has been shown to increase the growth rate of some bacteria (Starr and
Jones 1957), whilst reduced growth in response to copper has been demonstrated
for microbial populations (Jonas 1989). When compared to plastics and stainless
steel surfaces, copper has been shown to have inhibitory effects on various micro-
organisms (De Veer et al. 1994; Keevil 2001). Copper-containing alloys have also
shown increased antibacterial activity when compared to stainless steel and brasses,
The Effect of Substratum Properties on the Survival of Attached 19

with increasing copper content reducing cell survival time (Wilks et al. 2005). It has
been suggested that for biocidal purposes the use of copper alloyed surfaces should
be restricted to regularly cleaned surfaces (Kielemoes and Verstraete 2001), since
accumulation of non-microbial material and potential reaction of the cleaning agent
with the copper and the fouling material may interfere with the antimicrobial effect,
on open as well as closed surfaces (Airey and Verran 2007). Indeed, the ability of
any antimicrobial agent in a surface to affect cells in the biofilm above will depend
on the ability of the agent to diffuse through the biofilm from the substratum.
Conversely, any antimicrobial agent whose effect relies on direct contact will only
be active against those cells at the base of the biofilm. One might speculate that the
effectiveness of an antifouling surface is only predictable for a given period of time,
since once conditioning of the surface begins, surface properties will change. This
will result in loss of direct contact of the surface with the foulant and consequently
the loss of the surface antifouling effect. This has been demonstrated in the copper
pipes containing disinfectant concentrate where biofilms have been found (Exner et al.
1983). As the copper surface becomes fouled, antimicrobial properties become
diminished unless regularly cleaned (Airey and Verran 2007).

5.2 Polymers

Synthetic polymers may contain many additive chemicals, such as antioxidants,

light stabilizers, lubricants, pigments and plasticizers, added to improve the desired
physical and chemical properties of the material (Brocca et al. 2002). However,
these additives may leach into the surrounding environment and provide nutrient
for microorganisms present: phosphorus has been shown to increase the formation
of biofilms on polyvinyl chloride in phosphorus-limited water (Lehtola et al. 2002).
Several studies have shown that plastic materials can support the growth of bio-
films, but it has been suggested that growth in plastic pipes is usually comparable
with that on iron, steel or cement (Niquette et al. 2000). However, Bachmann and
Edyvean (2006) used Aquabacterium commune cells under continuous cultivation
with stainless steel and medium density polyethylene (MDPE) surfaces and found
that biofilm cell density on MDPE slides was four times greater than on stainless
steel. When various pipe materials were tested with chlorine and monochloramine
disinfection, it was found that cement-based materials supported fewer fixed bacteria
than plastic-based materials (Momba and Makala 2004).
Again, most of these surfaces are exposed to liquid and, potentially microorgan-
isms, at a solid–liquid interface, often in a closed system. On open surfaces, many
different properties of polymers can be exploited, depending on the intended end
use. The relative softness of these surfaces makes them susceptible to surface dam-
age, which will affect surface topography, and hence fouling and cleanability
(Verran et al. 2000). However, as with all surfaces, long-term studies are required
to assess the effect of surface wear and the effect of fouling, e.g. by humic substances,
oil or mineral particles.
20 K.A. Whitehead and J. Verran

5.2.1 Incorporation and Release of Antimicrobial Agents in Polymers

In attempts to prevent/reduce cell attachment and survival on surfaces, antimicro-

bial agents have been incorporated in and onto polymers. Clearly, the release of the
biocide/metal ions will be determined by the matrix and properties of the bulk
material and surrounding environment. Biocides can be encapsulated to facilitate
“delayed release”, thereby extending the intended antimicrobial effect (Lukaszczyk
and Kluczka 1995; Coulthwaite et al. 2005). Coatings and molecules extracted
from natural sources have been suggested for use to deter microbial survival on a
surface. On open surfaces, incorporation of antimicrobial agents such as biocides
(for example Microban) or metals (for example BioCote or Agion) are used to
achieve “antibacterial” properties, but the mechanism of the biocide action (e.g. is
moisture required), duration, spectrum, speed and magnitude of effect are all
important determinants of eventual effectiveness at intended point of use.
There are a number of important factors that need to be considered with respect
to the development of biocide-incorporated materials, including physical and envi-
ronmental aspects. The effectiveness of a biocide-incorporated surface is dependent
upon the ability of the biocide to be released from the bulk material into which it is
incorporated. This is a delicate balance, since if the blending, dispersion and binding
properties are incorrect then the biocide release rate may be too fast (shortened life
span of material), too slow (not effective), or non-existent. There is always a limited
lifetime to these materials since an infinite amount of biocide is not available.
The release of biocide into the environment should also be considered. The
Biocidal Product Directive (European Parliament 1998) was designed to review
existing substances and aimed to provide high levels of protection for humans, ani-
mals and the environment. Many antifouling paints used to reduce the attachment of
living organisms to the submerged surfaces of ships, boats and aquatic structures
have biocide-release mechanisms. Two common biocides in use are the triazine
herbicide Irgarol 1051 (N-2-methylthio-4-tert-butylamino-6-cyclopropylamino-
s-triazine), and diuron (1-(3,4-dichlorophenyl)3,3-dimethylurea), which are designed
to inhibit algal photosynthesis. It has been shown that due to leaching, environmen-
tal concentrations of the compounds pose significant risks to the plant species Apium
nodiflorum and Chara vulgaris (Lambert et al. 2006). With biocide-incorporated
materials there are also problems encountered with the targeted organisms, e.g.
increased tolerance and resistance to the active material. Resistance to many chemical
compounds including benzalkonium chloride, benzisothiazolone, chloroallyltri-
azine-azoniadamantane, dibromodicyanobutane, methylchloro/methylisothiazolone,
tetrahydrothiadiazinthione and trifluoromethyl dichlorocarbanilide has been detected
(Chapman 1998).
By definition, biocides will not assist in the accumulation and removal of
organic material present on the surface and in the surrounding aqueous environ-
ment. The result may be that, although micro- and macroorganisms that attach to a
surface may be inhibited or killed, the transfer of organic matter to the surface will
not be affected. Thus an organic material layer will gradually build up on the sur-
face over time, potentially masking any biocide effect.
The Effect of Substratum Properties on the Survival of Attached 21

If the biocide is not uniformly dispersed in the bulk material, then there will be
areas of the surface that may allow attachment of tolerant or resistant microorganisms.
Once this attachment occurs, microbial colonization and thus biofilm formation can
occur, potentially enveloping the surrounding areas of material that are higher in
biocide concentration. Thus although biocide-releasing surfaces may be a practical
solution for surfaces that are to be used in the short term, in the long term they may
be of limited value, particularly at the solid–liquid interface.

5.3 Paints

Coatings and paints intended for use on ships and underwater components or super-
structures are a complex mixture of compounds that may include binders, pigments,
extenders, solvents, thinners and additives (e.g. biocides) (Watermann et al. 2005).
The purpose of antifouling paints is primarily to prevent development of macro-
fouling, particularly barnacles. Since microorganisms on a surface can increase the
attachment of other organisms, inhibition of microbial biofilm development might
decrease subsequent development of barnacles on the surface (Tang and Cooney
1998). Thus it is of importance to test new formulations for the survival and resistance
of macro- and microorganisms.
As with blended polymers, the complex nature of the paint and its components
will affect the activity of biocide/antimicrobial used and thus the final antimicrobial
activity of the paint. To provide effective antifouling properties, organic biocides
such as Irgarol, are often added in conjunction with copper to control copper-
resistant fouling organisms (Voulvoulis et al. 1999). It has been shown that the
release rate of copper depends not only on the copper compound and its dissolution
properties, but also on the character of the paint matrix (Sandberg et al. 2007). The
underlying substrata may also affect the antifouling properties of paint. Work by
Tang and Cooney (1998) showed that coating surfaces with a marine paint
decreased the numbers of Pseudomonas aeruginosa on stainless steel but had little
effect on numbers of cells on fibreglass or aluminium. However, when they added
copper or tributyltin (TBT) to the paint the initial development of biofilms was
inhibited for 72–96 h. Biofilms that formed on surfaces coated with copper or TBT-
containing paint did not synthesize greater amounts of EPS, thus the biofilms may
have contained copper- or TBT-resistant cells.
There have been some attempts to use naturally extracted products as antifouling
agents in paints. Four bacterial isolates from a marine environment were used to produce
extracts that were formulated into ten water-based paints: nine showed activity against
a test panel of fouling bacteria (Burgess et al. 2003). Five of the paints were shown to
inhibit the settlement of barnacle larvae, Balanus amphitrite, and algal spores of Ulva
lactuca, and for their ability to inhibit the growth of Ulva lactuca when grown on paint
containing an extract from Pseudomonas sp. strain (Burgess et al. 2003).
It is interesting to note that manufacturers do not need to specify ingredients of
the paint that are below 1% weight, thus antifouling paints may include significant
22 K.A. Whitehead and J. Verran

amounts of metallic and non-metallic elements (Sandberg et al. 2007). Unfortunately,

some materials used in paints (such as both organotins and copper) can be toxic to
non-target marine species, such as the dog-whelk (Gibbs and Bryan 1986), oysters
(Axiak et al. 1995) and juvenile carp (de Boeck et al. 1995; Tang and Cooney
1998). The use of biocidal antifouling paints has been prohibited in some European
countries, such as Sweden, Denmark, Germany and France (Watermann et al.
2005). It should be noted that although copper is widely used in Europe, Sweden
has prohibited its use in antifouling paints on pleasure crafts in fresh water and
along the Swedish coast of the Baltic Sea (Sandberg et al. 2007). However, recent
investigations have shown that newly developed, “toxin-free” antifouling paints
that do not contain, e.g., copper, Irgarol or TBT may still be toxic towards marine
organisms (Karlsson and Eklund 2004).
On surfaces that are not submerged but are externally exposed, a solid–liquid–air
interface will form as rain droplets pass over the surface. The physical washing
effect, coupled with release of any intended antimicrobial properties, will thus help
reduce fouling on the surface. On internal surfaces, required properties might
encompass easy cleanability rather than specifically antimicrobial properties –
although in hygienic environments some biocidal effect would be desirable. Thus,
photocatalytic paints are finding applications. UV radiation is an effective, but
temporary photochemical method for disinfection, which requires a special irradia-
tion source within the UV (185–254 nm) band. Photocatalysis is an alternative to
direct UV disinfection and antimicrobial efficacy is possible with higher wave-
lengths, which are naturally present in ambient solar and artificial light (Erkan et
al. 2006). Large band gap semiconductors, such as titanium dioxide (TiO2), tin
oxide and zinc oxide, are suitable photocatalytic materials with their higher wave-
length UV absorption (320–400 nm) (Erkan et al. 2006). Titanium dioxide doped
with metals has demonstrated photocatalytic activity, leading to an increased rate
of destruction of organic compounds (Vohra et al. 2005) and microorganisms
(Sunada et al. 2003). There has been some work carried out on the effectiveness of
nanoparticle anatase titania on the destruction of bacteria (Allen et al. 2005 ;
Verran et al. 2007). An example of photocatalytic paint currently on the market is
Aoinn®. However, in situ information on the effectiveness of these materials is
limited. The effectiveness of the activity of photocatalytic paint on microorganisms
is further complicated by the interactions of the paint components interfering with
the active chemicals (Caballero et al., 2008).

6 Substratum Roughness

There are a number of engineering terms used to define surface roughness, but
the Ra, (the average of the peak and valley distances measured along a centre line)
is the most universally used roughness parameter for general quality control
(Verran and Maryan 1997) and in microbiological publications (Verran and
Boyd 2001) . An important consideration when describing surface topography is
The Effect of Substratum Properties on the Survival of Attached 23

that there are several scales that can be used to characterize material surfaces in
terms of surface waviness, roughness and topography (Table 1). Thus the surface
feature dimension should be considered alongside the dimensions of the organism
of concern.
Simplistically, an increase in surface roughness will increase the retention of
microorganisms on a surface (Boulange-Petermann et al. 1997; Verran and
Whitehead 2005; Whitehead and Verran 2006). However, there is some debate over
the phenomenon (Duddridge and Prichard 1983; Taylor and Holah 1996), which
may be accounted for by a consideration of the scale of topography, the “pattern-
ing” of the features on the surface and of the testing methodology used.
Electropolishinghas shown to be advantageous in minimizing initial bacterial
adhesion (Arnold et al. 2001) but, in the long-term, surface roughness has been
shown not to affect the development of mature biofilms (Hunt and Parry 1998)

Table 1 Descriptions of the different scale of surface topographies

Size of surface features Description
Macro-topography Ra> 10 μm Will include surface finishes produced by
industrial processes, e.g. the use of cutting
tools (uniform spacing of surface features
with a well-defined direction) or grinding
processes (usually directional in charac-
ter with generally of irregular spacing).
Roughening of a surface will increase the
area available for microbial adhesion and
retention; however, if the surface rough-
ness is greatly increased, this may result
in wash out of microorganisms
Micro-topography Ra ~ 1 μm Surfaces with features of micron dimension
are of importance if hygiene is of concern,
e.g. in food processing
Nano-topography Ra< 1 μm Procedures such as polishing, whereby fine
abrasives are used to produce a smooth
shiny surface, nevertheless, all surfaces
have a nanotopography. Nanotopographies
are likely to have little effect on the Ra or
other roughness values as usually meas-
ured, but may affect retention of organic
material
Angstrom-scale Surface features 1–10 Angstrom-sized surface features involve the
topography nm configuration and mobility or functional
groups, which may be of importance for
both the cell and the substratum, espe-
cially where dynamic surfaces are being
investigated
Molecular Molecules The charge on surface molecules ultimately
topography make up the overall charge on the micro-
bial or substratum surface and will affect
the initial cell–surface binding
24 K.A. Whitehead and J. Verran

– a property already noted in the impact of biocides. For surfaces deemed “hygi-
enic”, usually encompassing microorganisms at the surface–air interface where
“surface features” are smaller than the microorganisms, topography does not affect
the retention of microorganisms on a surface (Verran et al. 2001a), although the
cells tend to be immobilized on the features. Work by Hilbert et al. (2003) on stain-
less steel that was smoothed to Ra of 0.9–0.01 μm also found that the adherence of
microorganisms was not affected by differences in the surface roughness, but they
did conclude that surface roughness was an important parameter for corrosion
resistance of the stainless steel.
At the “macro” level it has been suggested that surface roughness may not pose
a major problem in terms of bacterial adherence: because the surface features are
so much larger than the bacterial cells, they can have no role in retention. However,
some fungal spores, algae, protozoa and larger organisms, such as those found in
marine environments and implicated in fouling, may be of significance. Thus an
effect of surface roughness on attachment has been demonstrated for algal spores
(Fletcher and Callow 1992) and invertebrate larvae (Crisp 1974). However, such
macrofeatures may well encompass a micro- or nanotopography, which can retain
smaller cells (Fig. 3). In situ, this means that the surface may become colonized
with smaller cells such as bacteria prior to eukaryotic colonization. Not only might
this provide anchorage points for the larger organisms but also for possible nutri-
ents, thus increasing the chances of survival of the larger cells (Pickup et al. 2006).
This succession of surface conditioning, micro- and macrofouling is a well-
described phenomenon in immersed aquatic systems. Both viable and non-viable
cells will contribute to this succession.

Fig. 3 Surface grooves with a macrotopography (30 μm). However, these large scale features
exhibit a micro- or nanotopography in the peaks between grooves. Cells are washed from large
grooves but are piled up on the top of the groove peaks R a = 0.35 μm (image courtesy of A.
Packer, MMU)
The Effect of Substratum Properties on the Survival of Attached 25

6.1 Stainless Steel: Surface Topography and Microbial Retention

Stainless steel is the most commonly used material for a number of industrial appli-
cations. Grade 316 stainless steel contains molybdenum, which increases resistance
to surface pitting in aggressive environments, therefore it is widely used in the
environment (Little et al. 1991). For stainless steel, different finishes will produce
surfaces with differing topographies, whilst retaining low Ra values below 0.8 μm,
the value used for describing “hygienic” surfaces (Flint et al. 1997). As noted
above, features of appropriate dimension will retain and protect microorganisms
(Fig. 4), and reduce surface cleanability and hygienic status.
On open surfaces that are regularly cleaned, biofilm formation is unlikely
(Verran and Jones 2000), but in closed environments, increased retention of viable
microorganisms may accelerate development of biofilm, even if more mature biofilm
is unaffected by the underlying surface topography (Verran and Hissett 1999).
Larger surface defects will potentially entrap accumulations of microorganisms in
both open and closed systems. Work by Boyd et al. (2002) demonstrated that on
stainless steel surfaces, lateral changes of 0.1 μm were sufficient to increase the
strength of bacterial attachment. Such surfaces should ideally be free from defects
and chemical inhomogeneity in order to minimize microbial attachment. However,
Bachmann and Edyvean (2006) suggested that electropolishing of stainless steel
pipes for drinking water installations was not necessary, although at joints, welds,
dead ends and other features on pipelines, polishing may be necessary since microbial
accumulation is more likely at these sites.

6.2 Controlling Topography to Manage Fouling

Recently, it has been noted that the shape of surface features is of importance in
microbiological binding to a surface (Edwards and Rutenberg 2001; Whitehead
et al. 2005, 2006). Since surface topography affects the amount and strength of attach-
ment and retention, several groups have produced surfaces with defined topogra-
phies in order to truly assess the interactions. Callow et al. (2002) showed that when
using textured surfaces consisting of valleys or pillars, the swimming spores of the

Fig. 4 SEM of a brushed finish stainless steel surface demonstrating microbial retention within
linear features
26 K.A. Whitehead and J. Verran

green fouling alga Enteromorpha settled preferentially in valleys and against pillars,
and that the number of spores that settled increased as the width of the valley
decreased. Surface textural features of 50–100 μm have been shown to be signifi-
cantly less fouled by barnacles (Andersson et al. 1999); features smaller than the
diameter of the barnacle prevented attachment. The attachment of barnacle larvae
has similarly been shown to be enhanced or reduced according to the scale, shape
and periodicity of surface roughness (Hills and Thomason 1998; Berntsson et al.
2000). Surface roughness has also been shown to influence the settlement behaviour
of fouling larvae (Howell and Behrends 2006).
On a smaller scale, a range of engineered surfaces with controlled topographical
features, i.e. pits (Whitehead et al. 2004) and grooves (Packer et al. 2007), has been
developed to demonstrate the effect of surface topography on cell binding. Using
microbial retention assays, Whitehead et al. (2005) demonstrated that with a range of
differently sized unrelated microorganisms, the dimensions of the surface feature are
important with respect to the size of the cell and its subsequent retention, with maximal
retention occurring when features were of a diameter comparable with the microorgan-
isms. This observation was supported when assessing the force of adhesion of the cells
on the surfaces using atomic force microscopy (Whitehead et al. 2006). Edwards and
Rutenberg (2001) have further recognized that the cross-sectional shape of a groove
will have a large effect on binding potential, which is especially important where flow
is concerned. Likewise, the orientation of features with respect to the flow or direction
of cleaning will affect retention. It should also be noted that, in order to simplify cal-
culations, cells are treated as rigid bodies whereas actually a living cell has a flexible
wall and can deform to fit surface features (Beach et al. 2002). The study of the interac-
tions occurring between cells and substratum features of defined dimensions is thus
contributing to our understanding of surface fouling at the earliest stages of biofilm
formation at both the solid–liquid and solid–air interface. Wear of materials may occur
on the nanometer scale (Verran and Boyd 2001). Nanoscale surface features have been
shown to affect both bacterial retention (Bruinsma et al. 2002) and cell behaviour
(Dalby et al. 2002; Fan et al. 2002; Curtis et al. 2004). It may be speculated that surface
nanofeatures will also invariably affect organic soil retention.

7 Substratum Conditioning

The first event that occurs when a surface comes into contact with a fluid is the adsorp-
tion of molecules to the surface; the molecules attach to the surface more rapidly than
the cells, and the composition of the conditioning film is dependent on the composition
of the bulk fluid (Hood and Zottola 1995) and of the substratum. Retained soil in sur-
face features may facilitate the attachment of microorganisms to the surface, provide
a nutrient source for microorganisms, be indicative of poor hygiene/cleaning processes
(Verran et al. 2001b), affect the susceptibility of microorganisms to sanitising agents
(Holah 1995), physically protect cells retained in surface defects (Kramer 1992) or
provide attachment foci for re-colonization (Storgards et al. 1999).
The Effect of Substratum Properties on the Survival of Attached 27

Considering the effect of initial surface “conditioning” on attachment, retention

and survival, adsorbed proteins have been found to either increase or decrease
attachment (Carballo et al. 1991; Helke et al. 1993). The specificity of adhesion–
receptor interaction is more relevant at solid–liquid interfaces, where the microor-
ganisms can move towards a more advantageous location. At the solid–air interface,
the immobilized cells tend to require another surface to facilitate transfer.
The presence of organic material may result in complexation and reduction in
activity of some antifouling agents. Previous investigations have shown the major-
ity (>80%) of the total copper in natural water to be complexed to organic matter
(Bruland et al. 2000). Once natural sediments bind to a surface and reduce the effect
of the antifouling agent, the surface becomes freely available for cell attachment to
take place. In-situ field measurements on ships hulls on both pleasure crafts and
navy vessels have shown lower release rates compared to laboratory tests on panels,
most probably as a result of biofilm formation (Valkirs et al. 2003)

8 Microbial Resistance, Tolerance and Persistence

To help prevent the development of bacterial resistance, it is essential to understand

the ramifications of the use of antimicrobial surfaces and/or cleaning and disinfection
products, and to maintain excellent cleaning or management/maintenance protocols.
If a cell is able to survive on a surface, resisting cleaning treatment, it can then be a
source for biotransfer potential. A number of research reports have expressed concern
that use of biocides may contribute to development of antibiotic resistance (Levy et
al. 2000; McDonnell et al. 1999). Several workers have reported that the number of
mercury-resistant bacteria in soil and aquatic environments varied according to the
mercury content of the environment, where in these strains heavy metal-resistance
properties were associated with multiple drug resistance (Misra 1992).
There is a vast difference between the magnitude of resistance, tolerance and
persistence (RTP) of microorganisms dependent on whether the cells are found in as
single units or as colonies, or if the cells are in the protective matrix of a biofilm.
When Pseudomonas aeruginosa was tested in suspension or following deposition
onto metallic or polymeric surfaces to determine the effectiveness of disinfectants
(Cavicide, Cidexplus, Clorox, Exspor, Lysol, Renalin and Wavicide) and non-for-
mulated germicidal agents (glutaraldehyde, formaldehyde, peracetic acid, hydrogen
peroxide, sodium hypochlorite, phenol and cupric ascorbate) it was found that cells
were on average 300-fold more resistant when present on contaminated surfaces
than in suspension (Sagripanti and Bonifacino 2000). Further, it was also shown
that the surface to which bacteria were attached influenced the effectiveness of
disinfectants.
The development of tolerance and resistance to antimicrobial agents is not the
focus of this chapter. However, although different challenges face cells at a solid–
air interface in comparison with biofilms at solid–liquid interfaces, in either case
the potential exists for survival, development of resistance and dissemination.
28 K.A. Whitehead and J. Verran

9 Conclusions

The attachment of microorganisms on inert substrata is a key to the development of

biofilm at solid–liquid interfaces, and also to the potential for transfer on open
surfaces at solid–air interfaces. Although the means for deposition of cells at the
surface in these two systems will vary, properties of the substratum such as surface
chemistry, surface topography, and the presence of organic (or inorganic) material
conditioning the surfaces are essentially common to both systems.
The chemical and physicochemical properties of the substratum are important in
initial cell attachment and adhesion, but once biofilm has formed, the underlying
substratum has little effect on development – although surface roughness can have
a significant effect on cell retention, especially under conditions of flow.
Surface modification designed to produce antifouling surfaces as an independent
entity needs to focus on management of initial organic material and cell deposition
in order to prevent, control or delay subsequent cell retention and multiplication.
Forces used in the cleaning need to overcome those interactions that are active in
adhesion of primary organic material and pioneer cells.
A variety of surface modification strategies are being explored, coupled with more
fundamental investigations of factors affecting interactions occurring between cells
and inert substrata. A multidisciplinary approach between biologists, chemists, physi-
cists, engineers and modellers will facilitate the development of well-engineered and
designed surfaces and systems, which are economically viable and environmentally
acceptable, to enable optimum control of microbial fouling of surfaces. Promising
approaches include those based on superhydrophobic surfaces. At these surfaces, the
interplay of surface topography and chemistry results in contact angles approaching
180°. The development of chemically modified surfaces may be advantageous, but the
use of chemical species that are detrimental to the surrounding environmental should
be avoided. Mass-produced generic “solutions” may not be realistic; antifouling surface
design needs to be tailored to individual applications. Although initially time-consuming,
this would result in successful application and long-term cost savings.

References

Airey P, Verran J (2007) Potential use of copper as a hygienic surface; problems assocviated with
cumulative soiling and cleaning. J Hosp Inf 67(3):271–277
Allen NS, Edge M, Sandoval G, Verran J, Stratton J, Maltby J (2005) Photocatalytic coatings for
environmental applications. Photochem Photobiol 81:279–290
Andersson M, Berntsson K, Jonsson P, Gatenholm P (1999) Microtextured surfaces: towards
macrofouling resistant coatings. Biofouling 14:167–178
Arnold JW, Boothe DH, Bailey GW (2001) Parameters of treated stainless steel surfaces
important for resistance to bacterial contamination. Trans ASAE 44:347–356
Avery SV, Howlett NG, Radice S (1996) Copper toxicity towards Saccharomyces cerevisiae:
dependence on plasma membrane fatty acid composition. Appl Environ Microbiol
62:3960–3966
The Effect of Substratum Properties on the Survival of Attached 29

Axiak V, Sammut M, Chircop P, Vella A, Mintoff B (1995) Laboratory and field investigations on
the effects of organotin (tributyltin) on the oyster, Ostrea edulis. Sci Total Environ
171:117–120
Bachmann RT, Edyvean RGJ (2006) AFM study of the colonisation of stainless steel by
Aquabacterium commune. Int Biodeter Biodeg 58(3–4):112–118
Beach E, Tormoen G, Drelich J, Han R (2002) Pull of force measurements between rough surfaces
by atomic force microscopy. J Coll Interface Sci 247:84–99
Becker K (1998) Detachment studies on microfouling in natural biofilms on substrata with different
surface tensions. Int Biodeter Biodeg 41(1):93–100
Berntsson KM, Andreasson H, Jonsson PR, Larsson L, Ring K, Petronis S, Gatenholm P (2000)
Reduction of barnacle recruitment on microtextured surfaces: analysis of effective topographic
characteristics and evaluation of skin friction. Biofouling 16:245–261
Boonaert CJP, Rouxhet PG (2000) Surface of lactic acid bacteria: Relationships between chemical
composition and physicochemical properties. Appl Environ Microbiol 66:2548–2554
Bos R, van der Mei HC, Busscher HJ (1999) Physicochemistry of initial microbial adhesive inter-
actions – its mechanisms and methods for study. FEMS Microbiol Rev 23:179–230
Boulange-Petermann L, Rault J, Bellon-Fontaine M-N (1997) Adhesion of Streptococcus ther-
mophilus to stainless steel with different surface topography and roughness. Biofouling
11(3):201–216
Boyd RD, Verran J, Jones MV, Bhakoo M (2002) Use of the atomic force microscope to determine
the effect of substratum surface topography on bacterial adhesion. Langmuir 18(6):2343–2346
Bright J, Fletcher M (1983) Amino acid assimilation and respiration by attached and free-living
populations of a marine Pseudomonas sp. Microbial Ecol 9:215–226
Brocca D, Arvin E, Mosbæk H (2002) Identification of organic compounds migrating from
polyethylene pipelines into drinking water. Water Res 36(15):3675–3680
Bruinsma GM, Rustema-Abbing M, de Vries J, Stegenga B, van der Mei HC, van der Linden ML,
Hooymans JMM, Busscher HJ (2002) Influence of wear and overwear on surface properties
of etafilcon a contact lenses and adhesion of Pseudomonas aeruginosa. IOVS
43:3646–3653
Bruland KW, Rue EL, Donat JR, Skrabal SA, Moffett JW (2000) Intercomparison of voltammetric
techniques to determine the chemical speciation of dissolved copper in a coastal seawater
sample. Anal Chim Acta 405(1–2):99–113
Burgess JG, Boyd KG, Armstrong E, Jiang Z, Yan LM, Berggren M, May U, Pisacane T, Granmo A,
Adams DR (2003) The development of a marine natural product-based antifouling paint.
Biofouling 19(Suppl.):197–205
Callow ME, Jennings AR, Brennan AB, Seegert CE, Gibson A, Wilson L, Feinberg A, Baney R,
Callow JA (2002) Microtopographic cues for settlement of zoospores of the green fouling alga
Enteromorpha. Biofouling 18(3):237–245
Carballo J, Ferreiros CM, Criado MT (1991) Importance of experimental design in the evaluation
of proteins in bacterial adherence to polymers. Med Microbiol Immunol 180:149–155
Caballero L, Whitehead KA, Allen NS, Verran J (2008) Inactivation of Escherichia coli on immo-
bilized TiO2 using fluorescent light. J Photochem Photobiol A (submitted)
Carlen A, Nikdel K, Wennerberg A, Holmberg K, Olsson J (2001) Surface characteristics and in
vitro biofilm formation on glass ionomer and composite resin. Biomaterials 22:481–487
Carson J, Allsopp D (1980) The enumeration of marine periphytic bacteria from a temperol sam-
pling series. In: Becker OG, Allsopp D (eds.) Biodeterioration: proceedings of the fourth
international biodeterioration symposium. Pitman, London, pp. 193–198
Chapman JS (1998) Characterizing bacterial resistance to preservatives and disinfectants. Int
Biodeter Biodeg 41:241–245
Chen G, Strevett KA (2001) Impact of surface thermodynamics on bacterial transport. Environ
Microbiol 3(4):237–245
Cloete TE (2003) Biofouling control in industrial water systems: what we know and what we need
to know. Mater Corros – Werkstoffe Korrosion 54(7):520–526
30 K.A. Whitehead and J. Verran

Coulthwaite L, Bayley K, Liauw C, Craig G, Verran J (2005) The effect of free and encapsulated
OIT on the biodeterioration of plasticised PVC during burial in soil for 20 months. Int Biodeter
Biodeg 56:86–93
Crisp DJ (1974) Factors influencing the settlement of marine invertebrate larvae. In: Grant PT,
Mackie AM (eds.) Chemoreception in marine organisms. Academic, New York, pp. 165–177
Curtis ASG, Gadegaard N, Dalby MJ, Riehle MO, Wilkinson CDW, Aitchison G (2004) Cells
react to nanoscale order and symmetry in their surroundings. IEEE Trans Nanobiosci
3(1):61–65
Dalby MJ, Riehle MO, Johnstone H, Affrossman S, Curtis ASG (2002) In vitro reaction of
endothelial cells to polymer demixed nanotopography. Biomaterials 23:2945–2954
Davies D, Chakrabarty A, Geesey G (1993) Exopolysaccharide production in biofilms: substratum
activation of alginate gene expression by Pseudomonas aeruginosa. Appl Environ Microbiol
59:1181–1186
de Boeck G, Nilsson G, Elofsson U, Vlaeminck A, Blust R (1995) Brain monoamine levels and
energy status in common carp (Cyprinus carpio) after exposure to sublethal levels of copper.
Aquat Toxicol 33:265–277
De Veer I, Wilke K, Ruden H (1994) Bacteria reducing properties of copper containing and non
copper containing materials: II. Relationship between microbiocide effect on copper contain-
ing materials and copper ion concentration after contamination with moist and dry hands.
Zentralblatt Hygiene Umweltmedizin 195:516–528
Dexter S (1979) Influence of substratum critical surface tension on bacterial adhesion – in situ
studies. Coll Interface Sci 70:346–354
Donlan R, Costerton W (2002) Biofilms: survival mechanisms of clinically relevant microorganisms.
Clin Micro Rev 15:167–193
Duddridge JE, Prichard AM (1983) Factors affecting the adhesion of bacteria to surfaces. In:
Microbial corrosion: proceedings from the joint National Physical Laboratory and Metal
Society conference. The Metals Society, London, pp. 28–35
Edwards KJ, Rutenberg AD (2001) Microbial response to surface microtopography: the role of
metabolism in localised mineral dissolution. Chem Geol 180:19–32
Erkan A, Bakir U, Karakas G (2006) Photocatalytic microbial inactivation over Pd doped SnO2
and TiO2 thin films. J Photochem Photobiol Chem 184(3):313–321
European Parliament and Council of the European Union (1998) Directive 98/8/EC of the
European Parliament and of the Council of 16 February 1998 concerning the placing of biocidal
products on the market (Biocidal Products Directive). http://ec.europa.eu/environment/biocides/
index.htm. Last accessed 14 July 2008
Exner M, Tuschewitzki G-J, Thofern E (1983) Investigations on wall colonization of copper pipes
in a central disinfection dosing system. Zbl Bakt Hyg I Abt Orig B 177:170–181
Fan YW, Cui FZ, Hou SP, Xu QY, Chen LN, Lee I-S (2002) Culture of neural cells on silicon
wafers with nanoscale topography. J Neurosci Meth 120:17–23
Fletcher M, Callow JA (1992) The settlement, attachment and establishment of marine algal
spores. Br Phycol J 27:303–329
Flint SH, Brooks JD, Bremer PJ (1997) The influence of cell surface properties of thermophilic
Streptococci on attachment to stainless steel. J Appl Microbiol 83(4):508–517
Geesey GG, Bremer PJ (1990) Applications of Fourier-transform infrared spectrometry to studies
of copper corrosion under bacterial biofilms. Marine Tech Soc J 24:36–43
Gibbs P, Bryan G (1986) Reproductive failure in populations of the dog-whelk, Nucella lapillus,
caused by imposex induced by tributyltin from antifouling paints. J Marine Biol Assoc UK
66:767–777
Gottenbos B, Grijpma DW, van der Mei HC, Feijen J, Busscher HJ (2001) Antimicrobial effects
of positively charged surfaces on adhering Gram-positive and Gram-negative bacteria .
J Antimicrob Chemo 48(1):7–13
Helke DM, Somers EB, Wong ACL (1993) Attachment of Listeria monocytogenes and Salmonella
typhimurium to stainless steel and buta-N in the presence of milk and individual milk components.
J Food Prot 56(6):479–484
The Effect of Substratum Properties on the Survival of Attached 31

Hilbert LR, Bagge-Ravn D, Kold J, Gram L (2003) Influence of surface roughness of stainless
steel on microbial adhesion and corrosion resistance. Int Biodeter Biodeg 52(3):175–185
Hills JM, Thomason JC (1998) The effect of scales of surface roughness on the settlement of
barnacle (Semibalanus balanoides) cyprids. Biofouling 12:57–69
Hiramatsu K, Hanaki H, Ino T, Yabuta K, Oguri T, Tenover F (1997) Methicillin-resistant
Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J Antimicrob
Chemo 40:135–136
Holah JT (1995) Progress report on CEN TC 216 working group 3: Disinfectant test methods for
food hygiene, institutional, industrial and domestic applications. Int Biodeter Biodeg
36(3–4):355–365
Hood SK, Zottola EA (1995) Biofilms in food processing. Food Control 6(1):9–18
Howell D, Behrends B (2006) A methodology for evaluating biocide release rate, surface rough-
ness and leach layer formation in a TBT-free, self-polishing antifouling coating. Biofouling
22(5):303–315
Hunt AP, Parry JD (1998) The effect of substratum roughness and river flow rate on the development
of a freshwater biofilm community. Biofouling 12:287–303
Husmark U, Ronner U (1993) Adhesion of Bacillus cereus spores to different solid surfaces:
cleaned or conditioned with various food agents. Biofouling 7:57–65
Jonas RB (1989) Acute copper and cupric ion toxicity in an estuarine microbial community. Appl
Environ Microbiol 55(1):43–49
Karlsson J, Eklund B (2004) New biocide-free anti-fouling paints are toxic. Marine Poll Bull
9(5–6):456–464
Keevil CW (2001) Antibacterial properties of copper and brass demonstrate potential to combat
toxic E. coli outbreaks in the food processing industry. Paper presented at the symposium on
copper and health, CEPAL, Santiago, Chile
Kielemoes J, Verstraete W (2001) Influence of copper alloying of austentic stainless steel of multi
species biofilm development. Lett Appl Microbiol 33:148–152
Kielemoes J, Hammes F, Verstraete W (2000) Measurement of microbial colonisation of two types
of stainless steel. Environ Tech 21(7):831–843
Kramer DN (1992) Myths: cleaning, sanitation and disinfection. Dairy Food Environ Sanit
12:507–509
Lambert SJ, Thomas KV, Davy AJ (2006) Assessment of the risk posed by the antifouling booster
biocides Irgarol 1051 and diuron to freshwater macrophytes. Chemosphere 63:734–743
Lehtola MJ, Miettinen IT, Martikainen PJ (2002) Biofilm formation in drinking water affected by
low concentrations of phosphorus. Can J Microbiol 48:494–499
Levy S (2000) Antibiotic and antiseptic resistance: impact on public health. Pediatr Infect Dis J
19:120–122
Little B, Wagner P, Mansfield F (1991) Microbiologically induced corrosion of metals and alloys.
Int Mater Rev 36:253–272
Liu ZM, Stout JE, Boldin M, Rugh J, Diven WF, Yu VL (1998) Intermittent use of copper-silver
ionization for Legionella control in water distribution systems: A potential option in buildings
housing individuals at low risk of infection. Clin Infect Dis 26:138–140
Lukaszczyk J, Kluczka M (1995) Modification of the release of biocides bound to carboxylic
exchange resin 1. Encapsulation by the ISP method and by evaporation from aqueous suspension.
Polimery 40(10):587–590
McDonnell G, Russell AD (1999) Antiseptics and disinfectants: activity, action, and resistance.
Clin Microbiol Rev 12(1):14
Misra TK (1992) Bacterial resistances to inorganic mercury salts and organomercurials. Plasmid
27(1):4–16
Mittelman MW, Nivens DE, Low C, White DC (1990) Differential adhesion, activity, and carbo-
hydrate – protein ratios of Pseudomonas-atlantica monocultures attaching to stainless-steel in
a linear shear gradient. Microb Ecol 19(3):269–278
32 K.A. Whitehead and J. Verran

Momba MNB, Makala N (2004) Comparing the effect of various pipe materials on biofilm forma-
tion in chlorinated and combined chlorine-chloraminated water systems. Water SA
30(2):175–182
Nickels JS, Bobbie RJ, Lott DF, Martz RF, Benson PH, White DC (1981) Effect of manual brush
cleaning on biomass and community structure of micro-fouling film formed on aluminum and
titanium surfaces exposed to rapidly flowing seawater. Appl Environ Microbiol
41(6):1442–1453
Niquette P, Servais P, Savoir R (2000) Impacts of pipe materials on densities of fixed bacterial
biomass in a drinking water distribution system. Water Res 34(6):1952–1956
Packer A, Kelly P, Whitehead KA, Verran J (2007) Effects of defined linear features on surface
hygiene and cleanability. Paper presented at the 50th SVC annual technical conference,
Kentucky
Pereni CI, Zhao Q, Liu Y, Abel E (2006) Surface free energy effect on bacterial adhesion. Coll
Surf B: Biointerfaces 48:143–147
Pickup RW, Rhodes G, Bull TJ, Arnott S, Sidi-Boumedine K, Hurley M, Hermon-Taylor J (2006)
Mycobacterium avium subsp paratuberculosis in lake catchments, in river water abstracted for
domestic use, and in effluent from domestic sewage treatment works: Diverse opportunities for
environmental cycling and human exposure. Appl Environ Microbiol 72(6):4067–4077
Pringle JH, Fletcher M (1983) Influence of substratum wettability on attachment of freshwater
bacteria to solid surfaces. Appl Environ Microbiol 45:811–817
Sagripanti JL, Bonifacino A (2000) Resistance of Pseudomonas aeruginosa to liquid disinfectants
on contaminated surfaces before formation of biofilms. J AOAC Int 83:1415–1422
Sandberg J, Wallinder IO, Leygraf C, Virta M (2007) Release and chemical speciation of copper
from anti-fouling paints with different active copper compounds in artificial seawater. Mater
Corros – Werkstoffe Korrosion 58(3):165–172
Simoes LC, Simoes M, Oliveira R, Vieira MJ (2007) Potential of the adhesion of bacteria isolated
from drinking water to materials. J Basic Microbiol 47:174–183
Sinde E, Carballo J (2000) Attachment of Salmonella spp. and Listeria monocytogenes to stainless
steel, rubber and polytetrafluorethylene: the influence of free energy and the effect of com-
mercial sanitizers. Food Microbiol 17(4):439–447
Starr TJ, Jones ME (1957) The effect of copper on the growth of bacteria isolated from marine
environments. Limnol Oceanog 2(1):33–36
Storgards E, Simola H, Sjoberg AM, Wirtanen G (1999) Hygiene of gasket materials used in food
processing equipment part 2: aged materials. Food Bioprod Proc 77(C2):146–155
Sunada K, Watanabe T, Hashimoto K (2003) Bactericidal activity of copper-deposited TiO2 thin
film under weak UV light illumination. Environ Sci Tech 37(20):4785–4789
Tang RJ, Cooney JJ (1998) Effects of marine paints on microbial biofilm development on three
materials. J Ind Microbiol Biotech 20(5):275–280
Taylor JH, Holah JT (1996) A comparative evaluation with respect to the bacterial cleanability of
a range of wall and floor surface materials used in the food industry. J Appl Bacteriol
81(3):262–266
Valkirs AO, Seligman PF, Haslbeck E, Caso JS (2003) Measurement of copper release rates from
antifouling paint under laboratory and in situ conditions: implications for loading estimation
to marine water bodies. Mar Pollut Bull 46(6):763–779
Verran J, Boyd RD (2001) The relationship between substratum surface roughness and microbio-
logical and organic soiling: a review. Biofouling 17(1):59–71
Verran J, Hissett T (1999) The effect of substratum surface defects upon retention of biofilm forma-
tion by microorganisms in potable water. In: Keevil CW, Godfree A, Holt D, Dow C (eds.)
Biofilms in the aquatic environment. The Royal Society of Chemistry, Cambridge, pp. 25–33
Verran J, Jones M (2000) Problems of biofilms in the food and beverage industry. In: Walker J,
Surmann S, Jass J (eds.) Industrial biofouling detection, prevention and control. Wiley,
Chichester, pp. 145–173
Verran J, Maryan CJ (1997) Retention of Candida albicans on acrylic resin and silicone of different
surface topography. J Prosthet Dent 77(5):535–539
The Effect of Substratum Properties on the Survival of Attached 33

Verran J, Whitehead K (2005) Factors affecting microbial adhesion to stainless steel and other
materials used in medical devices. Int J Art Organs 28(11):1138–1145
Verran J, Rowe DL, Cole D, Boyd RD (2000) The use of the atomic force microscope to visualise
and measure wear of food contact surfaces. Int Biodeter Biodeg 46(2):99–105
Verran J, Rowe DL, Boyd RD (2001a) The effect of nanometer dimension topographical features
on the hygienic surface of stainless steel. J Food Prot 64(7):1183–1187
Verran J, Boyd R, Hall K, West RH (2001b) Microbiological and chemical analyses of stainless
steel and ceramics subjected to repeated soiling and cleaning treatments. J Food Prot
64(9):1377–1387
Verran J, Sandoval G, Allen NS, Edge M, Stratton J (2007) Variables affecting, the antibacterial
properties of nano and pigmentary titania particles in suspension. Dyes Pigments
73:298–304
Vohra A, Goswami DY, Deshpande DA, Block SS (2005) Enhanced photocatalytic inactivation of
bacterial spores on surfaces in air. J Ind Microbiol Biotech 32(8):364–370
Voulvoulis N, Scrimshaw MD, Lester JN (1999) Analytical methods for the determination of 9
antifouling paint booster biocides in estuarine water samples. Chemosphere
38(15):3503–3516
Watermann BT, Daehne B, Sievers S, Dannenberg R, Overbeke JC, Klijnstra JW, Heemken O
(2005) Bioassays and selected chemical analysis of biocide-free antifouling coatings.
Chemosphere 60(11):1530–1541
Whitehead KA, Verran J (2006) The effect of surface topography on the retention of microorganisms:
a review. Food Bioprod Process 84:253–259
Whitehead KA, Verran J (2007) The effect of surface properties and application method on the
retention of Pseudomonas aeruginosa on uncoated and titanium coated stainless steel. Int
Biodeter Biodegradation 60:74–80
Whitehead KA, Colligon JS, Verran J (2004) The production of surfaces of defined topography
and chemistry for microbial retention studies using ion beam sputtering technology. Int
Biodeter Biodegradation 54:143–151
Whitehead KA, Colligon J, Verran J (2005) Retention of microbial cells in substratum surface
features of micrometer and sub-micrometer dimensions. Coll Surf B – Biointerfaces
41:129–138
Whitehead KA, Rogers D, Colligon J, Wright C, Verran J (2006) Use of the atomic force microscope
to determine the effect of substratum surface topography on the ease of bacterial removal. Coll
Surf B – Biointerfaces 51:44–53
Wilks SA, Michels H, Keevil CW (2005) The survival of Escherichia coli on a range of metal
surfaces. Int J Food Microbiol 105:445–454
Mechanisms of Microbially Influenced Corrosion

Z. Lewandowski (*
ü ) and H. Beyenal

Abstract The chapter demonstrates that biofilms can influence the corrosion of
metals (1) by consuming oxygen, the cathodic reactant; (2) by increasing the mass
transport of the corrosion reactants and products, therefore changing the kinetics of
the corrosion process; (3) by generating corrosive substances; and (4) by generating
substances that serve as auxiliary cathodic reactants. These interactions do not
exhaust the possible mechanisms by which biofilm microorganisms may affect the
corrosion of metals; rather, they represent those few instances in which we
understand the microbial reactions and their effect on the electrochemical reactions
characteristic of corrosion. In addition, we can use electrochemical and chemical
measurements to detect one or more products of these reactions. An important
aspect of quantifying mechanisms of microbially influenced corrosion is to
demonstrate how the microbial reactions interfere with the corrosion processes and,
based on this, identify products of these reactions on the surfaces of corroding
metals using appropriate analytical techniques. The existence of these products,
associated with the increasing corrosion rate, is used as evidence that the specific
mechanism of microbially influenced corrosion is active. There is no universal
mechanism of MIC. Instead, many mechanisms exist and some of them have been
described and quantified better than other. Therefore, it does not seem reasonable
to search for universal mechanisms, but it does seem reasonable to search for
evidence of specific, well-defined microbial involvement in corrosion of metals.

1 Recent Views on Microbially Influenced Corrosion

When it is suspected that a material failure was caused by microbial corrosion,

it is reasonable to ask: “How do we know that the corrosion process was influ-
enced by microorganisms?” To address this question, many research groups

Z. Lewandowski
Department of Civil Engineering and Center for Biofilm Engineering, Montana State University,
Room 310, EPS Building, Bozeman, MT 59717, USA
e-mail: ZL@erc.montana.edu

Springer Series on Biofilms, doi: 10.1007/7142_2008_8 35

© Springer-Verlag Berlin Heidelberg 2008
36 Z. Lewandowski and H. Beyenal

have attempted to find a fingerprint of microbially influenced corrosion(MIC),

i.e., specific characteristics distinguishing microbially stimulated corrosion
from ordinary galvanic corrosion (Beech et al. 2005; Javaherdashti 1999; Lee
et al. 1995; Little et al. 2000, 2007; Mansfeld and Little 1991; Videla and
Herrera 2005; Wang et al. 2006). Despite significant research effort, no such
fingerprint characteristic of MIC has yet been found, and there are good reasons
to believe that a universal mechanism of microbially stimulated corrosion does
not exist (Beech et al. 2005; Flemming and Wingender 2001; Miyanaga et al.
2007; Starosvetsky et al. 2007). Instead of a universal mechanism, several
mechanisms by which microorganisms affect the rates of corrosion have been
described, and the diversity of these mechanisms is such that it is difficult to
expect that a single unified concept can be conceived to bring them all together.
From what we now understand, and what has been demonstrated by numerous
researchers, accelerated corrosion of metals in the presence of microorganisms
stems from microbial modifications to the chemical environment near metal
surfaces (Beech et al. 2005; Geiser et al. 2002; Lee and Newman 2003;
Lewandowski et al. 1997). Such modifications depend, of course, on the prop-
erties of the corroding metal and on the microbial community structure of the
biofilm deposited on the metal surface (Beech and Sunner 2004; Dickinson et
al. 1996b; Flemming 1995; Olesen et al. 2000b, 2001). The conclusion that
there are many mechanisms of MIC, rather than a single one, is generally
accepted in the literature and can be exemplified by the paper by Starosvetsky
et al. (2007), who concluded that to uncover MIC in technological equipment
failures requires an individual approach to each case, and that an assessment of
the destructive role of the microorganisms present in the surrounding medium
is possible only by analyzing and simulating the corrosion parameters found in
the field (Dickinson and Lewandowski 1998). Quite succinctly, Beech et al.
(2005) describe MIC as a consequence of coupled biological and abiotic elec-
tron-transfer reactions, i.e., redox reactions of metals enabled by microbial
ecology. Hamilton (2003) attempted to generate a unified concept of MIC and
has found common features in only some of the possible mechanisms. It is
unlikely that a unified concept of MIC can be generated at all.
MIC is caused by microbial communities attached to surfaces, known as
biofilms. A biofilm is composed of four compartments: (1) the surface to which the
microorganisms are attached, (2) the biofilm (the microorganisms and the matrix),
(3) the solution of nutrients, and (4) the gas phase (Lewandowski and Beyenal
2007). Each compartment consists of several components, and the number of
components may vary depending on the type of study. For example, in some MIC
studies it is convenient to distinguish four components of the surface: (1) the bulk
metal, (2) the passive layers, (3) the biomineralized deposits on the surface, and (4)
the corrosion products. Microorganisms can modify each of these components in a
way that enhances corrosion of the metal surface. In addition, components of the
other compartments of the biofilm can be modified in ways that affect the corrosion
reactions as well. Modifications in the solution compartment may include the
chemical composition, hydrodynamics and mass transfer rates near the metal
Mechanisms of Microbially Influenced Corrosion 37

surface; modifications in the biofilm compartment may include the microbial

community structure and the composition of the extracellular polymeric substances
(EPS). Each of these modifications may be complex in itself, and each may affect
the corrosion reactions in many ways. The complexity and the multitude of the pos-
sible interactions among microorganisms, their metabolic reactions, the corrosion
reactions and the metal, such as those shown by Coetser and Cloete (2005), are the
reasons why it is unlikely that a unifying concept of MIC can be developed (Coetser
and Cloete 2005).
When biofilms accumulate on metal surfaces, reactants and products of micro-
bial metabolic reactions occurring in the space occupied by the biofilm affect the
solution chemistry and the surface chemistry, and both types of modification may
interfere with the electrochemical processes naturally occurring at the interface
between the metal and its environment. The reactants and products of electrochemi-
cal reactions occurring at a metal surface interact with the reactants and products
of microbial metabolic processes occurring in biofilms in a complex way. Some of
these interactions accelerate corrosion, and some may inhibit corrosion. The inter-
actions that accelerate corrosion, and are characteristic enough, are called mecha-
nisms of MIC, and much of this text is devoted to quantifying the mechanisms that
we now understand. To approach the task of quantifying these interactions in an
organized manner, we will start by describing the reactions characterized as
galvanic corrosion and then assess the effects of various metabolic reactions on
these reactions. Corrosion science has developed a succinct system of quantifying
various forms of corrosion, and we will use this system to quantify the effects of
microbial metabolic reactions on corrosion by referring to the principles of the
chemistry and electrochemistry of metals immersed in water solutions. Traditionally,
the mechanisms of corrosion are quantified using thermodynamics and kinetics,
and we will follow this tradition here.
The term corrosion can be defined in various ways, and there are many forms
of corrosion and many materials that can corrode – both metallic and nonmetal-
lic. Among the well-known processes of nonmetallic corrosion is the corrosion
of stone and its effect on ancient artifacts. Here, we restrict the meaning of cor-
rosion and define it as the anodic dissolution of metals. Among the many
anodic reactions that may occur at the surface of a metal, the one in which the
metal itself is the reactant subjected to oxidation is singled out and termed cor-
rosion. Noble metals, such as platinum and gold, do not undergo an oxidation
reaction and serve only to facilitate charge transfer between external redox spe-
cies. In contrast, active metals such as iron are oxidized and this process con-
tributes to the net anodic reaction rate, which is typically the dominant anodic
process for freely corroding metals. On corroding metals, anodic reactions are
coupled with cathodic reactions (reduction). In aerated water solutions, the
dominant cathodic reaction is the reduction of dissolved oxygen, while in
anaerobic solutions, the reduction of protons is the dominant cathodic reaction;
this is typically represented as the reduction of water.
Equations (1)–(6) show the relevant half reactions, followed by the corresponding
net reactions, for the corrosion of ironin aqueous media (Lewandowski et al. 1997).
38 Z. Lewandowski and H. Beyenal

Anaerobic

Fe → Fe 2 + + 2e − anodic (1)

2H 2 O + 2e − → H 2 + 2OH − cathodic (2)

2H 2 O + Fe → Fe(OH)2 + H 2 net (3)

Aerobic
In aerobic solutions, the basic anodic reaction is of course the same as the one
described by (1) – dissolution of iron – but the products of the reaction, ferric ions,
are hydrolyzed and further oxidized by the available oxygen, and all these reactions
are summarized as follows:

4OH − + 4Fe(OH)2 → 4Fe(OH)3 + 4e − anodic (4)

O2 + 2H 2 O + 4e − → 4OH − cathodic (5)

4Fe(OH)2 + O2 + 2H 2 O → 4Fe(OH)3 net (6)

These corrosion reactions can be modified by the metabolic reactions in biofilms in

many ways, and we will discuss four possible modifications here:
1. Biofilms create oxygen heterogeneities near a metal surface.
2. Biofilm matrix increases mass transport resistance near a metal surface.
3. Metabolic reactions in biofilms generate corrosive substances, such as acids.
4. Metabolic reactions in biofilms generate substances that serve as cathodic
reactants.
These four possible interactions do not exhaust the possible effects of microor-
ganisms on corrosion reactions. The reason we have selected these four interac-
tions is that they have been extensively studied, and so we know more about them
than we know about other interactions. Other mechanisms, both accelerating and
inhibiting corrosion, are continually proposed and studied. For obvious reasons,
using biofilms to inhibit the corrosion of metals stimulates imaginations, and
several authors have described such scenarios. For example, Jayaraman et al.
(1999) demonstrated axenic aerobic biofilms inhibiting generalized corrosion of
copper and aluminum. Similarly, in the work by Zuo et al. (2005), Al 2024 was
passive in artificial seawater in the presence of a protective biofilm of Bacillus
subtilis WB600. When antibiotics were added to the artificial seawater to kill the
bacteria in the biofilm, pitting occurred within a few hours (Zuo et al. 2005).
However, as summarized by Little and Ray (2002), most of the experiments on
inhibiting corrosion with biofilms were done in laboratories, and when the bio-
films were exposed to natural waters they failed to protect the material. Clearly,
the laboratory biofilms were different from those deposited in nature. One
assumption made in attempting to use biofilms to inhibit corrosion is that biofilm
Mechanisms of Microbially Influenced Corrosion 39

formation is predictable and controllable (Little and Ray 2002). This is not true.
Even pure culture biofilms in laboratory are not uniform and their structure
changes all the time (Lewandowski et al. 2004).
Corroding metals fall into two categories: active metals – such as iron, and
passive metals – such as stainless steels. These two types of materials are affected
by different types of corrosion. To demonstrate the possible microbial modifica-
tions of the corrosion reactions, we need to specify the reactions characteristic of
each type of corrosion affecting these materials.

2 Corrosion of Active Metals

2.1 Thermodynamics of Iron Corrosion

Using the terminology accepted in electrochemical studies, a metal immersed

in water is called an electrode. The potential of an electrode in an aqueous solu-
tion depends on the rates of the anodic (oxidation) and cathodic (reduction)
reactions occurring at the metal surface. When these rates are at equilibrium,
thermodynamics can be used to quantify the electrode potential. When these
rates are not at equilibrium, thermodynamics cannot be used to find the elec-
trode potential and it must be found empirically. Corrosion reactions are not at
equilibrium, and the potentials of corroding metals cannot be predicted from
thermodynamics.
To illustrate the thermodynamic principles of galvanic corrosion, we will select
a set of conditions and compute the potentials of the reactions participating in the
corrosion of iron. For the anodic reaction,
Fe 2 + + 2e − → Fe E 0 = −0.44 VSHE (7)

The Nernst equation quantifies the half-cell potential for iron oxidation as

0.059 ⎡ 1 ⎤
E = E0 − log ⎢ 2 + ⎥ (8)
n ⎣ [Fe ] ⎦

Iron is a solid metal and its activity equals one. Consequently, the potential of the
anodic half reaction depends on the concentration of ferrous ions in the solution and
is computed as
E = −0.44 + (0.059 / 2) log[Fe 2 + ] (9)

Selecting the concentration of ferrous iron, [Fe2+] = 10−6 M, the potential equals
E = −0.62 VSHE.
40 Z. Lewandowski and H. Beyenal

The cathodic reaction– the reduction of oxygen:

O2 + 2H 2 O + 4e − → 4OH − E 0 = +0.401VSHE (10)

The Nernst equation quantifies the half-cell potential for oxygen reduction:

0.059 ⎡ [OH − ]4 ⎤
E = E0 − log ⎢ ⎥
(11)
n ⎣ pO2 ⎦

The potential of this half reaction depends on the partial pressure of oxygen and on
the pH.

E = 0.401 + 0.059 / 4[log( pO 2 ) + 4(14 − pH)] (12)

Assuming that p(O2) = 0.2 atm and pH 7, the potential of the cathodic reaction is
E = 0.804 VSHE.
If only one of these reactions were occurring on the metal surface, the metal
would assume the respective potential specified for the reaction. For example, if
only the cathodic reaction were taking place, the metal would have the potential
+0.804 VSHE, and if only the anodic reaction were taking place, the metal would
have the potential −0.62 VSHE. This can be demonstrated in electrochemical studies
where the anode and the cathode can be separated, placed in different half-cells,
and studied in isolation. However, in corrosion, both reactions occur on the same
piece of metal and at the same time, and the potential of the metal can have only
one value. As a result, the potential of the corroding metal is somewhere between
the potential of the anodic half reaction, −0.62 VSHE, and the potential of the
cathodic half reaction, +0.804 VSHE. The exact potential of the corroding metal
depends on the kinetics (reaction rates) of the anodic and cathodic reactions, and
can be measured empirically and interpreted from the theory of mixed potentials.
Here, for the purpose of this simplified argument, it is enough to assume that the
potential of the corroding iron is between the potentials of the anodic and cathodic
half reactions, say, in the middle: E = (−0.62 + 0.804)/2 = 0.092 VSHE. Setting the
potential of the metal between the potential of the anodic and cathodic half reac-
tions has consequences: it sets the position of the equilibrium for each of the par-
ticipating reactions. If the potential were equal to that computed for either of the
half reactions, anodic or cathodic, this half reaction would be at equilibrium. If the
potential of the corroding iron is between the potentials computed for the two half
reactions, none of these half reactions (1)–(6) are at equilibrium and each of them
proceeds in the direction that approaches the equilibrium. To quantify the conse-
quences of this departure from the equilibrium, we can inspect the Nernst equation
describing potentials of the anodic and cathodic half reactions when the potentials
are shifted from their respective equilibrium potentials. If the potential of the cor-
roding iron is 0.092 VSHE, it is higher than the equilibrium potential for the anodic
reaction and lower than the equilibrium potential for the cathodic reaction. As a
Mechanisms of Microbially Influenced Corrosion 41

consequence, each reaction will proceed spontaneously toward reaching the equi-
librium determined by the potential of the metal, by adjusting the concentrations of
the reactants and products to satisfy the equilibrium for the given potential.
The anodic reaction, Fe2+ + 2e− → Fe, must adjust its potential to +0.092 V

+0.092 = −0.44 + (0.059 / 2) log[Fe 2 + ] (13)

If separated from the cathodic reaction, this reaction has a potential of E = −0.62 VSHE.
When connected to the cathodic reaction, this reaction has a new equilibrium poten-
tial, E = +0.092 VSHE. To reach the new equilibrium potential, the concentration of
ferric ions must increase. Consequently, the reaction proceeds to the left, to increase
the concentration of ferric ions in the solution. Iron dissolves in this reaction.
The cathodic reaction, O2 + 2H 2 O + 4e − → 4OH − must adjust its potential to
,
+0.0092 V as well:

+0.092 = 0.401 + 0.059 / 4[log( pO 2 ) + 4(14 − pH)] (14)

If separated from the anodic reaction, this reaction has a potential of E = +0.804 VSHE.
When connected to the anodic reaction, this reaction has a new equilibrium poten-
tial, E = +0.092 VSHE. To reach this new equilibrium potential, the reaction proceeds
to the right, to decrease the partial pressure of oxygen. Oxygen is consumed in this
reaction.
As a result of setting the metal potential between the equilibrium potentials for
the anodic and cathodic half reactions, the anodic reaction spontaneously proceeds
toward dissolution of the iron and the cathodic reaction spontaneously proceeds
toward reduction of the oxygen. Both reactions proceed until one of the reactants is
exhausted or until they both adjust the concentrations of their respective reactants
to reach the new equilibrium at 0.092 VSHE. The thermodynamics of the corrosion
processes explains why these processes occur but of course cannot predict the
anodic or cathodic reaction rates. Kinetic computations are needed to refine what
was said in the section dedicated to thermodynamic considerations.

2.2 Kinetics of Iron Corrosion

As discussed in the previous section, the anodic and cathodic processes occurring on
metal surfaces correspond to different half reactions, and the electrode potential is
used to predict the directions in which these reactions will proceed. Typically, the
corrosion reactions occurring on the surfaces of corroding metals are the dominant
redox reactions. However, the metal can always serve as a source or sink for electrons
satisfying the dissolved redox couples in the solution, and more than one redox reac-
tion can occur on the surface. There is a possibility that more than one reaction is
occurring on the metal surface at a time and that each of the reactions uses the elec-
trode as a source or a sink for the electrons needed to reach its own equilibrium
42 Z. Lewandowski and H. Beyenal

potential. The term “mixed potential” is used to describe this condition, to distinguish
it from the reduction–oxidation potential in which the anodic and cathodic reactions
are simply the forward and reverse parts of a single reaction. The mixed potential in
which the anodic reaction is metal oxidation is termed the corrosion potential, Ecorr.
Let us use, as an example, the reaction described by (1) as the electrode reaction –
an iron electrode immersed in a solution of ferrous ions. At equilibrium, the exchange
of electrical charges between the electronic conductor – the electrode – and the ionic
conductors – ferrous ions in the solution – is composed of two streams of electrical
charges moving in opposite directions, to and from the electrode. In the forward reac-
tion, ferrous ions from the metal lattice are dissolved in water. In the reverse reaction,
ferrous ions are reduced and deposited on the surface of the electrode as iron atoms.
At equilibrium, the rates of the charge transfers across the interface are equal to each
other, and there is no net current flow across the interface; the potential at equilibrium
is named Eeq, and the current flowing in opposite directions, named the exchange
current, is usually quantified as the exchange current density, i0. Once the electrode
potential departs from equilibrium and an overpotential is applied, the electrode reac-
tion is no longer at equilibrium and a net current flows in one direction. The direction
in which this net current flows is determined by the sign of the applied overpotential:
a negative sign is equivalent to cathodic polarization and a positive sign is equivalent
to anodic polarization. This can be summarized as follows:
From the definition of overpotential, E = Eeq + h , we assign cathodic
polarization:
h = ( E − Eeq ) < 0 (15)

Anodic polarization: h = ( E − Eeq ) > 0 (16)

The magnitude of the net current is determined by the extent of the overpotential
(h) and by the intrinsic properties of the system, summarized by the exchange
current density, i0. At this condition, even though the currents in the two direc-
tions are equal, in various systems these currents may have different magnitudes,
depending on the material of the electrode and the type of the electrode reaction.
Polarizing the electrode, i.e., applying an overpotential, favors the flow of elec-
tric charges in one direction and inhibits the flow in the opposite direction: posi-
tive polarization amplifies the anodic current and negative polarization amplifies
the cathodic current. The following equation, somewhat simplified, is known as
the Butler–Volmer equation, and it quantifies the net current, equal to the differ-
ence in rate of charge transfer between the anodic and cathodic directions:

⎡ ⎛ −aFh ⎞ ⎛ aFh ⎞ ⎤
i = ic − ia = i0 ⎢exp ⎜ ⎟⎠ − exp ⎜⎝ ⎟ (17)
⎣ ⎝ RT RT ⎠ ⎥⎦

where a is the symmetry coefficient and is assumed to be equal to 0.5, and the
remaining symbols – F, R, and T – have their usual meanings. When applied potential
Mechanisms of Microbially Influenced Corrosion 43

h is negative (cathodic polarization), the first exponential expression in the Butler–

Volmer equation becomes positive and the second becomes negative. As a result,
the second exponential expression is, for practical reasons, negligibly small when
compared with the first exponential expression; i.e., the anodic current is negligibly
small when compared with the cathodic current. The opposite is true when the
overpotential has a positive sign (anodic polarization). Figure 1 shows the relation
between the applied potential and the current: the potentiodynamic polarization
curve.

2.3 Microbially Stimulated Modifications of the Corrosion

of Iron and Active Metals

In corrosion, the anodic and cathodic reactions are not at equilibrium but they are
related to each other by two requirements:
1. The two reactions progress on the same piece of metal, and so they must have
the same potential.
2. Electrons extracted in the anodic reactions are used in the cathodic reactions;
therefore, the anodic and the cathodic currents must be equal.
These two requirements combined are used to quantify the thermodynamics and
kinetics of the corrosion process – the corrosion potential and the corrosion current,
as shown in Fig. 2.
The reactants and products of microbial metabolism in biofilms may interact with
the corrosion reactions, and these interactions may affect the thermodynamics of the
process, e.g., by introducing an additional cathodic reactant and thus altering the position

60
i (mA / cm2)

40

20

0
0.20 0.15 0.10 0.05 0.00 -0.05 -0.10 -0.15 -0.20
η (V)
-20

-40

-60

Fig. 1 Potentiodynamic polarization curve. The relationship between the overpotential (h), which
varied between −0.2 and +0.2 V, and the current density (i) for i0 = 1 mA cm−2 and a = 0.5
44 Z. Lewandowski and H. Beyenal

Cathodic
current

icorr ; Ecorr
Potential, E

Anodic
current

Log Current, log i

Fig. 2 The intersect of the extrapolated anodic and cathodic potentiodynamic polarization curves
demonstrates the meaning of the corrosion potential, Ecorr, and the corrosion current, icorr. The cor-
rosion potential, Ecorr, can be measured with respect to a suitable reference electrode (see also Fig.
4, where such a measurement is run for a real sample). The corrosion current, icorr, cannot be
measured directly, unless the anode and the cathode are separated, but it can be estimated using
other electrochemical techniques based on disturbing the potential of the electrode

of the equilibrium for the relevant reactions. These interactions may also affect the
kinetics of the process, e.g., by changing the concentrations of the reactants and prod-
ucts of the corrosion reactions, and thus the rates of the relevant reactions. All these
interactions can be presented as in Fig. 2, or a similar plot can be created for specific
reactions and reactants. If such a plot is used, modifications affecting the thermody-
namics change the locations of the equilibrium points on the vertical axis. For exam-
ple, replacing protons with oxygen as the cathodic reactant would raise the position
of the equilibrium for the cathodic reactions. This would have an effect on the kinetics
of the reaction by affecting the position of the intercept between the anodic and
cathodic parts of the corrosion reaction, thus affecting Ecorr and icorr. The kinetics of
the participating reactions are illustrated as the slopes of the lines in Fig. 2. For exam-
ple, a sudden increase in the rate of the cathodic reaction (higher cathodic current for
the same potential) would be reflected by a decrease in the slope of the line represent-
ing the cathodic reaction, and thus by a change in the position of the intercept deter-
mining Ecorr and icorr. Figure 2 shows the principles of the corrosion of active metals,
and the mechanisms of MIC explain the processes that can cause such changes.
Despite their resistance to general corrosion, passive metals and alloys can also be
affected by MIC. To evaluate the mechanisms of such effects, we will first discuss the
mechanisms of the corrosion of stainless steels and other passive metals and then, as
Mechanisms of Microbially Influenced Corrosion 45

we did for active metals, discuss the possible microbial effects that can modify these
mechanisms and accelerate corrosion.

3 Corrosion of Passive Metals

Passive metals and alloys show a different mechanism of corrosion than active metals
do. The best known passive alloys are stainless steels, and much research has been
done on MIC of these materials. The corrosion reactions for stainless steels are the
same as those for iron. However, stainless steels are alloys and some of their compo-
nents, when oxidized, form dense layers of oxides, passive layers, which prevent fur-
ther corrosion. Passivated alloys can resist corrosion in the presence of strong oxidants
that would cause corrosion on unalloyed metal. However, this protection works to a
certain extent only. When a cathodic reactant polarizing the metal has a high enough
oxidation potential, localized corrosion, called pitting, occurs as a result of localized
damage to the passive layer. The mechanism of this process is shown in Fig. 3.
As shown in Fig. 3, when a passivating alloy is subjected to anodic polarization,
i.e., to an increasing electrode potential, the corrosion current initially increases, fol-
lowing the increase in the polarization potential. This increase continues until the
polarization potential reaches a critical value, called the passivation potential. At this
potential, the alloying constituents of the metal are oxidized and form dense layers on
the metal surface, which slow down the corrosion of the metal, as is demonstrated by
the decreasing corrosion current. As the polarization potential increases further, the
metal first reaches a passive zone, in which it is immune to the increase in the polari-
zation potential until the polarization potential reaches a critical value, called the pit-
ting potential. When the polarization reaches, and exceeds, the pitting potential, the

Pitting potential Transpassive

Ep zone

Passive
zone
ESHE, V

Epp
−
+ +e Passivation
M potential
M®
Eo M/M+
M+
+
e-
®
M

Log(current, Amp)

Fig. 3 Potentiodynamic polarization curve of a passive metal; thermodynamic principles of pas-

sivation and of pitting corrosion
46 Z. Lewandowski and H. Beyenal

corrosion current gradually increases, as a result of localized damage to the passive

layers. The damaged areas are small compared to the surface of the metal; the damage
to the surface has the form of small pits, and this type of corrosion is called pitting
corrosion. Because the damaged areas become anodic sites, their small combined
surface area makes the localized corrosion process particularly dangerous for the
integrity of the metal. The anodic current densities can reach high values and local-
ized damage of the material can progress much faster than it does in cases of general
corrosion, in which the anodic current densities are much smaller.

4 Microbially Stimulated Modifications of the Corrosion

of Passive Metals and Alloys

Passive metals and alloys, such as stainless steels, can be used within the passive
zone, where the oxidation potentials of the available oxidants do not exceed the
pitting potential. Microbial interference that may accelerate the corrosion of such
surfaces is then necessarily related to two possible mechanisms:
1. Microbially generated oxidants (cathodic reactants) can have higher oxidation
potentials than the pitting potential.
2. Microbially stimulated localized damage to the passive layers can decrease the
pitting potential.
The first mechanism is related to the deposition of biomineralized manganese
oxides, which can subsequently raise the potential of the passive metal above the
pitting potential. The second mechanism is related to the damage of the passive
metal surface by microorganisms in biofilms. We will discuss these mechanisms in
more detail later in this chapter.
Figure 4 shows the relation among the corrosion potential, pitting potential, and
probability of pits initiation, redrawn from Sedriks (1996). The potential (Ecorr) of a
Potential against a reference

Corrosion potential, Ecorr

Pitting Pitting
potential, EP occurs
electrode

No pitting

Corrosion potential, Ecorr

Time

Fig. 4 When the corrosion potential, Ecorr, reaches the pitting potential, Ep (dashed line), of the
metal in the given solution, pits are initiated (redrawn from Sedriks, 1996)
Mechanisms of Microbially Influenced Corrosion 47

metal such as stainless steel is measured against time. If the potential of the stain-
less steel is higher than the pitting potential (EP), the stainless steel develops pits to
initiate corrosion. If the potential of the stainless steel is less than the pitting poten-
tial, pits cannot develop. The pitting potential can be determined using standard
electrochemical techniques described elsewhere (ASM Handbook Series 1987).

5 Mechanisms by Which Metabolic Reactions in Biofilms

can Interact with Corrosion Reactions

5.1 Mechanism 1: Biofilms Create Oxygen Heterogeneities

The interaction between metabolic activity in biofilms and corrosion reactions

appears to be trivial: microorganisms use the cathodic reactant, oxygen, which makes
it unavailable for the corrosion reactions and, as a result, the corrosion rate decreases.
If true, this mechanism would actually inhibit corrosion, and there is experimental
evidence that this occurs in some situations. Hernandez et al. (1990) reported a
decrease in the corrosion rate of mild steel in the presence of a uniform layer of bio-
film. This decrease was attributed to the respiration of the biofilm microorganisms,
resulting in a decline in oxygen concentration at the metal surface and an associated
decrease in the rate of the cathodic reduction of oxygen. These authors reproduced
their observations using synthetic seawater with Pseudomonas sp. S9 as well as with
Serratia marcescens (Hernandez et al. 1994). We now know that to inhibit corrosion
by this mechanism the biofilm must cover the surface of the metal uniformly and, in
principle at least, must have uniformly distributed microbial activity. As biofilms are
not uniform and microbial activity in biofilms is not uniformly distributed, this
mechanism can be demonstrated in the laboratory but is unlikely to persist in a natural
environment. Oxygen consumption rates and oxygen concentrations in biofilms vary
from one location to another (Lewandowski et al. 1997; Lewandowski and Beyenal
2007); this leads to a more interesting interaction, mechanism 2, which increases the
rate of corrosion, and there is some experimental evidence for it as well. White et al.
(1985), for example, found no accumulation of iron or other metals in EPS from bio-
films growing on corroding 304 stainless steel. They attributed the observed acceler-
ated corrosion to an inhomogeneous distribution of biofilm at the metal surface,
resulting in areas of differing cathodic activity, consistent with a differential aeration
cell. Areas covered with biofilm exhibit lowered oxygen concentrations and become
anodic, while those with less biofilm exhibit higher oxygen concentrations and
become cathodic. As a result, anodic and cathodic areas are fixed at the metal surface,
and this mechanism is appropriately called corrosion as a result of differential aera-
tion cells (Ford and Mitchell 1990).
Metal corrosion through the formation of differential aeration cells results from
different concentrations of oxygen occurring at different locations on the metal
surface. This effect, different concentrations of oxygen at different locations on the
48 Z. Lewandowski and H. Beyenal

metal surface, can be caused by the active consumption of oxygen by microorgan-

isms in biofilms nonuniformly distributed on the metal surface, but it can also be
caused by a passive mechanism in which oxygen access to some areas is physically
obstructed. Placing an o-ring on a metal surface is an example of such a mecha-
nism, but other, more subtle, scenarios are possible as well. One such scenario is
based on partially covering the metal surface with a material that has nonuniformly
distributed diffusivity for oxygen. The access of oxygen to some locations on the
metal surface is more difficult than its access to other locations on the same surface,
and differential aeration cells are formed.
These speculations lead to the question of whether depositing microbial EPS on a
metal surface can cause the formation of differential aeration cells, and to a more
general question: what is the role of EPS in MIC? It is well known that polysaccha-
rides, the main constituent of EPS, can be cross-linked with metal ions. In principle,
then, if EPS covers a corroding site, the metal ions can cross-link the polysaccharides
and affect the position of the equilibrium between the corroding metal and its ions,
thus accelerating corrosion. This mechanism is analogous to the formation of differ-
ential aeration cells, and in corrosion science both mechanisms are called differential
concentration cells. The metal concentration cells do not seem to affect MIC to a large
extent, at least based on the report by White et al. (1985), who found no accumulation
of iron or other metals in EPS from biofilms growing on corroding 304 stainless steel.
Doubt remains about the passive effect of EPS, in which it changes the access of
oxygen to various locations on the metal surface. Can differential aeration cells be
formed by this mechanism?
To address this question, we will first discuss the thermodynamic principles of
corrosion by differential aeration cells and determine the factors that must be meas-
ured to resolve whether this mechanism is active in biofilms.
If the anodic reaction is the oxidation of iron: Fe → Fe 2 + + 2e − , and the
cathodic reaction is the reduction of oxygen: O2 + 2H 2 O + 4e − → 4OH − , then
the overall reaction describing the process is

2Fe + O2 + 2H 2 O → 2Fe 2 + + 4OH − (18)

The Nernst equation quantifying the potential for this reaction is

0.059 [Fe 2 + ]2 [OH − ]4

E = E0 − log (19)
4 p(O2 )

If the oxygen concentrations at two adjacent locations on an iron surface are differ-
ent, then the cell potentials at these locations are different as well. Specifically, the
location where the oxygen concentration is higher will have a higher potential
(more cathodic) than the location where the oxygen concentration is lower (more
anodic). The difference in potential will give rise to current flow from the anodic
locations to the cathodic locations and to the establishment of a corrosion cell. This
is the mechanism of differential aeration cells, and the prerequisite to this mechanism
Mechanisms of Microbially Influenced Corrosion 49

is that the concentration of oxygen vary among locations (Acuna et al. 2006;
Dickinson and Lewandowski 1996; Hossain and Das 2005). Indeed, many measure-
ments using oxygen microsensors have demonstrated that oxygen concentrations in
biofilms can vary from one location to another (Lewandowski and Beyenal 2007).
This mechanism by which differential aeration cells are formed, in which a thin
layer of biofilm at the surface of the substratum is discontinuous, is consistent with
the current model of biofilm structure, shown in Fig. 5.
One of the most dangerous forms of localized corrosion of mild steel is tubercu-
lation, which is the development or formation of small mounds of corrosion
products. According to Herro (1991), tubercle formation originates from a differ-
ential oxygen concentration cell.

5.2 Mechanism 2: Biofilm Matrix Increases the Mass Transport

Resistance near the Metal Surface, Thus Changing
the Kinetics of the Corrosion Processes

Once the mechanism of differential aeration cell formation in biofilms had been
demonstrated and explained, the immediately following question was whether
microbial activity in biofilms is a necessary prerequisite to the formation of differ-
ential aeration cells, or perhaps, the presence of extracellular polymeric substances
on the surface suffices. The idea that the presence of EPS on the surface might suf-
fice is related to the known mechanisms of corrosion initiation based on different
resistances to mass transport for oxygen at various locations on metal surfaces,
similar to the initial stages of crevice formation. The possibility that the active
removal of oxygen by the biofilm microorganisms might not be necessary to initiate
a differential aeration cell was discussed by MIC researchers, but it was usually
dismissed on the grounds that extracellular polymers are composed of 98% water
and their layers on metal surfaces are only a few hundred micrometers thick, so that

500 µm

Fig. 5 Conceptual structure of biofilms (left) and a light microscopy image of a biofilm (right)
50 Z. Lewandowski and H. Beyenal

the increase in diffusion resistance expected as a result of depositing extracellular

polymer could not possibly be significant. Nevertheless, the hypothesis was formulated
that the deposition of extracellular polymer on a metal surface might form differen-
tial aeration cells, and an appropriate experiment was designed and executed (Roe
et al. 1996). As a model of extracellular polymer, calcium alginate was used.
Alginate is an extracellular biopolymer excreted by biofilm microorganisms. If
alginate initiates the differential aeration cell, then the oxygen concentrations at the
locations covered with alginate should be higher than those at the locations not
covered with alginate. Also, pH at the locations covered with alginate should be
higher than that at locations not covered with alginate. These expectations are con-
sistent with the anodic and cathodic reactions, in which the anodic reaction
decreases pH because ferrous ions hydrolyze and precipitate as hydroxides, and the
cathodic reaction increases pH because it consumes protons. Two drops of sodium
alginate were deposited on the surface of a corrosion coupon made of mild steel and
exposed to a calcium solution which cross-linked the sodium alginate and formed
a calcium alginate gel on the surface. To test whether depositing calcium alginates
can initiate differential aeration cells, the variations in oxygen concentration and
pH above these spots were measured using scanning microelectrodes. In addition,
a scanning vibrating electrode (SVE) was used to determine the distribution of the
electrical field above the surface, and it was expected that this electrode would
detect the positions of the anodic and cathodic sites. The results, shown in Fig. 6

Fig. 6 Two spots of calcium alginate deposited on a surface of mild steel fix anodic sites (Roe
et al. 1996)
Mechanisms of Microbially Influenced Corrosion 51

demonstrated that the mere deposition of a thin layer of alginate on mild steel is
enough to fix the anodic sites and initiate corrosion. All the characteristics of dif-
ferential aeration cells were present in the system: pH was lower near the sites cov-
ered with alginate than near the sites not covered; oxygen concentration was lower
near the sites not covered; and, as demonstrated by the image of the electric field
distribution provided by the scanning vibrating electrode, there were anodic and
cathodic sites fixed at the surface of the metal. This result, somewhat unexpected at
that time, had further implications: it demonstrated that merely killing biofilm
microorganisms using biocide(s) or antimicrobial(s) does not necessarily stop MIC.
Once the biopolymer has been deposited on the surface, the active consumption of
oxygen in the respiration reaction enhances the formation of differential aeration
cells, but even without it, differential aeration cells can be formed just because EPS
has been deposited on the surface. This conclusion coincides with the general
notion that removing the biofilmis more important than killing the biofilm
microorganisms.
Once the differential aeration cell has been established, the corrosion proceeds
according to the mechanism described by (Eq. 18), which is also illustrated in Fig. 7.

5.3 Mechanism 3: Metabolic Reactions in Biofilms Generate

Corrosive Substances, Exemplified by the Sulfate-Reducing
Bacteria Corrosion of Mild Steels

The mechanism of MIC due to the formation of differential aeration cells can be
called a nonspecific one, because it does not depend on the physiology of the micro-
organisms that deposited the extracellular polymers. There are, however, other mech-
anisms that are closely related to the type of microorganisms active in the biofilm and
to their metabolic reactions (Beech and Gaylarde 1999; Romero et al. 2004;Videla
and Herrera 2005; Xu et al. 2007). An example of such a mechanism is sulfate-reduc-
ing bacteria (SRB) corrosion (Ilhan-Sungur et al. 2007; Lee et al. 1995).

Aerated water

BIOFILM
O
O2 Aerobic 2 O2
O2
FILM

BIOFIL

O2 O2 Anaerobic O2 O2 O2
O2
O2 O2 O2
BIO

O2 Anaerobic O
M

2 O2
O2 O2 OH- OH-
OH- OH- M+ M+
Cathode - e- M+ e- Cathode
e e-

Anode
Metal

Fig. 7 (a) Biofilm heterogeneity results in differential aeration cells. This schematic shows pit
initiation due to oxygen depletion under a biofilm (Borenstein 1994). (b) An anodic site and pit
under the biofilm and corrosion products deposited on mild steel
52 Z. Lewandowski and H. Beyenal

The corrosion of mild steel caused by SRB is probably the most celebrated case
of MIC because it provides a direct, and easy to understand, link between microbial
reactions and electrochemistry (Javaherdashti 1999). Despite the progress in
research, and in understanding of the process, little has been done to prevent or stop
this type of corrosion once initiated, and SRB corrosion is still considered the main
type of MIC. For example, Bolwell in 2006 demonstrated that engine failures in gas
turbines were caused by SRB growing in the seawater lubricating oil coolers and
contaminating it (Bolwell 2006). The overall progress in understanding of MIC,
however, allows us to implicate other microorganisms as partners of SRB and
consider more complex scenarios of MIC, in which two types of microorganisms
modify the potential of the electrode in the opposite directions. For example, Rao
et al. (2000) found that in the cooling water system of a nuclear test reactor iron-
and manganese-oxidizing bacteria (MOB) (Leptothrix sp.) and SRB (Desulfovibrio
sp.) were responsible for the corrosion of carbon steel. It is interesting to notice that
these two types of microorganisms drive the redox potential in the opposite
directions, thus increasing the gap between the potential of the anodic reaction and
the potential of the cathodic reaction.
SRB produce hydrogen sulfide by reducing sulfate ions (Videla and Herrera
2005). According to the mechanism that was proposed by Von Wohlzogen Kuhr in
1934, SRB oxidize cathodically generated hydrogen to reduce sulfate ions to H2S,
thereby removing the product of the cathodic reaction and stimulating the progress
of the reaction (Al Darbi et al. 2005). Over the years it became obvious that the
mechanism must be more complex than that initially suggested, and it is now cer-
tain that the possible pathways for cathodic reactions are more complex and can,
for example, include sulfides and bisulfides as cathodic reactants (Videla 2001;
Videla and Herrera 2005).
Hydrogen sulfideitself can be a cathodic reactant (Antony et al. 2007; Costello
1974):

2H 2 S + 2e − → H 2 + 2HS− (20)

Ferrous iron generated from anodic corrosion sites (21) precipitates with the meta-
bolic product of microbial metabolism, hydrogen sulfide, forming iron sulfides,
FeSx.

Fe 2 + + HS− = FeS + H + (21)

This reaction may provide protons for the cathodic reaction (Crolet 1992).
The precipitated iron sulfides form a galvanic couple with the base metal. For
corrosion to occur, the iron sulfides must have electrical contact with the bare steel
surface. Once contact is established, the mild steel behaves as an anode and elec-
trons are conducted from the metal through the iron sulfide to the interface between
the sulfide deposits and water, where they are used in a cathodic reaction. What
exactly the cathodic reactants are is still debatable.
Surprisingly, the most notorious cases of SRB corrosion often occur in the
presence of oxygen. Since the SRB are anaerobic microorganisms, this fact has
Mechanisms of Microbially Influenced Corrosion 53

been difficult to explain. Our group believes that this effect is based on mecha-
nism 3: iron sulfides (resulting from the reaction between iron ions and sulfide
and bisulfide ions) are oxidized by oxygen to elemental sulfur, a substance known
to be a strong corrosion agent (Lee et al.1995). Biofilm heterogeneity plays an
important role in this process, because the central parts of microcolonies are
anaerobic while the outside edges remain aerobic (Lewandowski and Beyenal
2007). This arrangement makes this mechanism possible because the oxidation of
iron sulfides produces highly corrosive elemental sulfur, as illustrated by the
following reaction:

2H 2 O + 4FeS + 3O2 → 4S0 + 4FeO(OH) (22)

Hydrogen sulfide can also react with the oxidized iron to form ferrous sulfide and
elemental sulfur (Schmitt 1991), thereby aggravating the situation by producing
even more elemental sulfur, and closing the loop through production of the reactant
in the first reaction, FeS.

3H 2 S + 2FeO(OH) → 2FeS + S0 + 4H 2 O (23)

The product of these reactions – elemental sulfur – accelerates the corrosion rate.
Schmitt (1991) has shown that the corrosion rate caused by elemental sulfur can
reach several hundred mils per year. We have demonstrated experimentally that
elemental sulphur is deposited in the biofilm during the SRB corrosion (Nielsen
et al. 1993). It is also well known that sulfur disproportionation reaction that pro-
duces sulfuric acid and hydrogen sulfide is carried out by sulfur disproportionating
microorganisms (Finster et al. 1998):

4S0 + 4H 2 O → 3H 2 S + H 2 SO 4 (24)

In summary, according to this mechanism, SRB corrosion of mild steel in the pres-
ence of oxygen is an acid corrosion:

anodic reaction : Fe → Fe 2 + + 2e − (25)

cathodic reaction : 2H + + 2e − → H 2 (26)

It is worth noticing that hydrogen, the product of the cathodic reaction, can be oxi-
dized by some species of SRB to reduce sulfate and generate hydrogen sulfide, H2S:

H 2 SO 4 + 4H 2 → H 2 S + 4H 2 O (27)

Hydrogen sulfide dissociates to bisulfides:

H 2 S = H + + HS− (28)

which are used in the reaction described by (20).

54 Z. Lewandowski and H. Beyenal

Thus, this mechanism involves several loops in which reactants are consumed
and regenerated, and the process continues at the expense of the energy released by
the oxidation of the metal.
These reactions are linked with each other in a network of relations. To illustrate
the main pathways, Fig. 8 shows the main reactions and the effect of oxygen on the
SRB corrosion of mild steel.

5.4 Mechanism 4: Metabolic Reactions in Biofilms Generate

Substances That Serve as Cathodic Reactants

One of the most puzzling aspects of MIC is the change in electrochemical properties
of stainless steel that occurs as the metal surface is colonized by microorganisms in
natural water. The dominant effects of colonization are a several-hundred-millivolt
increase in corrosion potential (Ecorr) to values near +350 mV versus the saturated
calomel electrode (SCE) and 2–3 orders of magnitude increase in cathodic current
density at potentials between approximately −300 and +300 mVSCE. These effects,
known as ennoblement, were first observed in the mid-1960s (Crolet 1991, 1992).
Since then, numerous researchers (Braughton et al. 2001; Dickinson et al. 1997;
Linhardt 2006; Washizu et al. 2004) have shown that stainless steels and other passive
metals in natural waters exhibit a several-hundred-millivolt increase in corrosion
potential, accompanied by an increase in cathodic current drawn, upon mild polariza-
tion. This phenomenon has been observed in a wide variety of natural and engineered
environments. Washizu et al. (2004), Mattila et al. (1997), and Dexter and Gao (1988)
described ennoblement in seawater (Amaya and Miyuki 1994), Dickinson et al.

Fig. 8 Sulfate-reducing bacteria corrosion of mild steel in the presence of oxygen is an acid cor-
rosion (Lewandowski et al. 1997)
Mechanisms of Microbially Influenced Corrosion 55

(1996a) reported its occurrence in a freshwater stream, and Linhardt (1996) reported
it in a hydroelectric power plant.
The ennoblement of stainless steels in natural waters may influence material
integrity: as the corrosion potential approaches the pitting potential, the material
integrity may be compromised by localized (pitting and crevice) corrosion. This
sequence of events, from an increase in corrosion potential to pit initiation, is well
known to material scientists, although the microbial component is new. Because the
pitting potential of 316L stainless steel in seawater is around 200 mVSCE, the danger
of pitting initiation in such an environment is serious. There are, however, reports
of microbial involvement in pitting corrosion of stainless steels immersed in fresh
waters of much lower chloride concentration than that found in seawater
(Hakkarainen 2003; Linhardt 2004, 2006; Olesen et al. 2001).
Temporal changes in the corrosion potential of 316L stainless steel coupons
immersed in different natural water sources are illustrated by our results in Fig. 9. In
all cases the potentials of 316L stainless steel coupons increased, demonstrating
ennoblement of the stainless steel. Several hypotheses have been postulated to explain
the mechanism of ennoblement, all suggesting that it is caused by microbial coloniza-
tion of the metal surface. Mollica and Trevis (1976) attributed ennoblement to micro-
bially produced extracellular polymeric substances. Dexter and Gao (1988) suggested
that acidification of the metal–biofilm interface caused by protons derived from the
metabolic reactions in the biofilm increased the potential. Chandrasekaran and Dexter
(1993) proposed a combination of acidification and hydrogen peroxide production.
Eashwar and Maruthamuthu (1995) believed that ennoblement was caused by micro-
bially produced passivating siderophores. Although many authors have demonstrated

400

300
Ecorr (mV vs. SCE)

200

100

−100

−200
0 2 4 6 8 10 12 14 16
Time (Weeks)

Roskie Creek Hebgan Lake Bracket Creek

Fig. 9 Potential of 316L stainless steel coupons exposed to fresh water at three locations in Montana
for 4 months. The rate and extent of ennoblement roughly correlate with the amount of biomineral-
ized manganese recovered from the surface after 4 months (Table 1) (Braughton et al. 2001)
56 Z. Lewandowski and H. Beyenal

the relationship between ennoblement and biofilm formation, the proposed hypothe-
ses have not been supported by convincing experimental evidence unequivocally
demonstrating the mechanism of ennoblement.
We have demonstrated, in the laboratory and in the field, that stainless steels
and other passive metals ennoble when colonized by MOB (Braughton et al.
2001; Dickinson et al. 1996a, 1997; Dickinson and Lewandowski 1996). While
the origin of the manganese-rich material deposited on stainless steel coupons
exposed to Bozeman stream water was not rigorously established, mineral-
encrusted bacterial sheaths characteristic of Leptothrix sp. and mineralized cap-
sules characteristic of Siderocapsa treubii were abundant on the surface of the
ennobled stainless steel coupons, and MOB were isolated from the manganese-
rich deposits (Dickinson and Lewandowski 1996). In parallel with these findings,
Linhardt (1996) also demonstrated that manganese-oxidizing biofilms were
responsible for pitting corrosion of stainless steel. Although biomineralization of
manganese can be carried out by certain genera of the so-called iron and manga-
nese group – Siderocapsa, Leptothrix, and Crenothrix – in fact the property is
widely distributed in a variety of organisms, including bacteria, yeast, and fungi
(Caspi et al. 1998; Francis and Tebo 2002; Tebo et al. 1997, 2004, 2005). These
organisms can oxidize dissolved manganese to form highly enriched mineral–
biopolymer encrustations. Deposits of manganese oxides form on submerged
materials, including metal, stone, glass, and plastic, and can occur in natural
waters and sediments with manganese levels as low as 10–20 ppb (Dickinson
et al. 1996a, 1997; Dickinson and Lewandowski 1996).
Because biomineralized manganese oxides are in direct electrical contact with
the metal, the metal exhibits the equilibrium dissolution potential of the oxides. The
standard potentials (E0) for (Eq. 29)–(Eq. 31) were calculated using the following
energies of formation: ΔGfo Mn2+ = −54.5 kcal mol−1, Δ Gfo γ-MnOOH = −133.3 kcal
mol−1, and ΔGfo γ-MnO2 = −109.1 kcal mol−1.

MnO 2(s) + H + + e − → MnOOH (s)

′
E 0 = +0.81VSCE EpH = 7.2 = +0.383 VSCE
(29)

MnOOH (s) + 3H + + e − → Mn 2 + + 2H 2 O
′
E 0 = +1.26 VSCE EpH = 7.2 = +0.336 VSCE (30)

This leads to the following overall reaction:

MnO2(s) + 4H + + 2e − → Mn 2 + + 2H 2 O
′
E 0 = +1.28 VSCE EpH = 7.2 = +0.360 VSCE (31)

The potentials (E′) were calculated at a pH of 7.2 and [Mn2+] = 10−6. Dickinson
et al. (1996a) demonstrated that just a 6% surface coverage by manganese oxides
can increase the resting open circuit potential (OCP) of stainless steels (−200 mVSCE)
by some 500 mV, which coincides closely with the reported equilibrium potential
Mechanisms of Microbially Influenced Corrosion 57

of the oxides, +362 mVSCE at a pH of 7.2 (Dickinson and Lewandowski 1996;

Linhardt 1998).
The thermodynamic calculations are in good agreement with the observations as
the potential of stainless steel coupons exposed to river water rises to about 360 mV,
as predicted. Our results directly correlate the extent and rate of ennoblement with
the amount and rate of manganese oxides deposition on metal surfaces (Braughton
et al. 2001). To determine which environmental factors influence the rate of enno-
blement, we exposed 316L stainless steel coupons at three locations, two creeks
and a lake, for 100 days. The open circuit potential was monitored periodically,
about once a week (Fig. 9). The coupons in both creeks reached a potential of
+350 mVSCE in 3 weeks. The coupons in the lake reached a final potential of less
than +100 mVSCE and the ennoblement rate was very slow. Manganese oxides were
deposited on all metal coupons, and their amounts roughly correlated with the rate
of ennoblement, as can be seen in Table 1.
Figure 10 shows the potentiodynamic polarization curves of nonennobled, fully
ennobled, and MnO2-plated stainless steel coupons. Both the microbial ennoble-
ment and electroplating of MnO2 on the metal surface shift corrosion potentials by
∼300 mV in the noble direction and cause a corresponding increase in cathodic
current density at modest overpotentials (around −100 mV).
In our laboratory, Dickinson and colleagues studied the effects of MOB on
stainless steels and demonstrated that 3–5 % surface coverage by biofouling
deposits was enough to ennoble 316L stainless steel (Dickinson et al. 1996a,
1997; Dickinson and Lewandowski 1998). Chemical examination of the deposits
showed the presence of Fe(III) and Mn(IV), while epifluorescence microscopy
revealed the presence of manganese- and iron-oxidizing bacteria (Dickinson and
Lewandowski 1998). On the basis of these observations and other studies con-
ducted in our laboratory, we have suggested that MOB are involved in the corro-
sion of stainless steels through the following mechanism (Braughton et al. 2001;
Dickinson et al. 1996a, 1997; Dickinson and Lewandowski 1996; Geiser et al.
2002; Olesen et al. 2000a; Shi et al. 2002a,b): the divalent manganese (Mn2+) ions
are microbially oxidized to manganese oxyhydroxide, MnOOH, which is depos-
ited on the metal surface; then the solid MnOOH is further oxidized to manganese
dioxide, MnO2. Both reactions contribute to the increase in the open circuit
potential because the deposited oxides, MnOOH and MnO2, are in electrical con-
tact with the surface and their dissolution potential is determined by the equilib-
rium of the deposited minerals with the dissolved divalent manganese. The oxides
deposited on the surface are reduced to divalent manganese by electrons gener-
ated at anodic sites. However, reducing the manganese oxides does not stop the
ennoblement process, because the reduced products of this reaction, soluble divalent

Table 1 Amounts of biomineralized manganese recovered from the surfaces after 4 months
Source Bracket creek Roskie creek Hebgen lake
Mn recovered (µg/cm2) 9.3 33.6 1.7
58 Z. Lewandowski and H. Beyenal

0.6

0.4

0.2
1
Potential (VSCE)

2
0

−0.2 3
Ennobled coupon (1)

−0.4
Coupon as received (3)

−0.6 Coupon covered with electroplated

MnO2 (2)

−0.8
1.00E−10 1.00E−08 1.00E−06 1.00E−04 1.00E−02
log(Current density)

Fig. 10 Potentiodynamic polarization curves (316L stainless steel, 0.01 M Na2SO4, pH 8.30; scan
rate: 0.167 mV s−1) show typical behavior of nonennobled, fully ennobled, and MnO2-plated stain-
less steel coupons. (1) Biomineralized manganese on graphite electrode, (2) electrochemically
deposited manganese oxides on graphite electrode, (3) clean graphite electrode used to reduce
oxygen only. Both microbial ennoblement and MnO2 electroplating of the metal surface shift cor-
rosion potentials by ∼300 mV in the noble direction and cause a corresponding increase in
cathodic current density at modest overpotentials (around −100 mV)

manganese ions, are reoxidized by the MOB attached to the metal surface. The
described sequence of events, oxidation–reduction–oxidation of manganese, is a
hypothetical mechanism that produces renewable cathodic reactants, MnOOH
and MnO2, and their presence on the metal surface endangers material integrity.
This mechanism is illustrated in Fig. 11.
The suggested mechanism relies on the activity of MOB in biofilms deposited
on metal surfaces. The biomineralization of manganese can be carried out by a
variety of organisms, including bacteria, yeast, and fungi, but it is particularly asso-
ciated with genera of the so-called iron and manganese group – Siderocapsa,
Gallionella, Leptothrix-Sphaerotilus, Crenothrix, and Clonothrix. These bacteria
accelerate the oxidation of dissolved iron and manganese to form highly enriched
mineral–biopolymer encrustations. Deposits form on submerged materials, includ-
ing metal, stone, glass, and plastic, in natural waters with manganese levels as low
as 10–20 ppb.
Biomineralized manganic oxides are efficient cathodes and increase cathodic
current density on stainless steel by 2–3 orders of magnitude at potentials between
roughly −200 and +400 mVSCE. The extent to which the elevated current density can
be maintained is controlled by the electrical capacity of the mineral, which reflects
both total accumulation and the conductivity of the mineral–biopolymer assem-
blage (only material in electrical contact with the metal will be cathodically active).
Mechanisms of Microbially Influenced Corrosion 59

2e−
Microbial
deposition

Cathode
MnO2 Mn 2+

2e− Electrochemical
reduction

Anode Feo Fe2+ Iron

dissolution

Fig. 11 Redox cycling on metal surfaces: hypothetical mechanism of microbial involvement in

the corrosion of stainless steels and other passive metals (Olesen et al. 2000a)

Oxide accumulation is controlled by the biomineralization rate and by the corrosion

current, in that high corrosion currents will discharge the oxide as rapidly as it is
formed. It appears that this mechanism may result in redox cycling of manganese
on metal surfaces, producing a renewable cathodic reactant, which agrees well with
the notion that whenever biofilms accumulate on cathodic members of galvanic
couples, a significant increase in the reduction current can be expected
(Chandrasekaran and Dexter 1993). In conclusion, the accumulation of manganese
oxides can cause pitting corrosion, as demonstrated in Fig. 12.

5.5 Further Implications

Our observations also suggest that MOB may be directly involved in pit initiation,
in addition to the indirect effects caused by the biomineralized manganese oxides
(Geiser et al. 2002). Scanning electron microscopy and atomic force microscopy
images (Fig. 13) show micropits formed on 316L stainless steel ennobled by L.
discophora SP-6. This indicates that the pits were initiated at the sites of bacterial
attachment and then propagated because of the presence of manganese oxides driv-
ing the potential in the noble direction.
Our data show that the manganese oxides deposited on the surface elevate the
potential, create an environment where the pits initiated by microbes can not repas-
sivate. Because the pits are initiated at the sites of attachment, in this light, it
appears that the bacteria initiate the pits and the microbially deposited manganese
oxides stabilize the growth of the pits by maintaining a high potential.
60 Z. Lewandowski and H. Beyenal

Fig. 12 Corrosion pit on a stainless steel surface covered with biomineralized manganese oxides
and immersed in a 3.5% solution of NaCl

Fig. 13 Scanning electron microscopy and atomic force microscopy images of damage to a sur-
face caused by colonization by manganese-oxidizing bacteria L. discophora SP-6 growing on
316L stainless steel surface. The size and shape of the indentations closely resemble the size and
shape of the microorganism colonies on the surface (Geiser et al. 2002)

6 Summary and Conclusions

In conclusion, we have demonstrated that biofilms can influence the corrosion of

metals (1) by metabolic reactions in the biofilms consuming oxygen, the cathodic
reactant; (2) by controlling the mass transport of the corrosion reactants and prod-
ucts, therefore changing the kinetics of the corrosion process; (3) by generating
corrosive substances; and (4) by generating substances that serve as auxiliary
cathodic reactants.
Mechanisms of Microbially Influenced Corrosion 61

These interactions do not exhaust the possible mechanisms by which biofilm

microorganisms may affect the corrosion of metals; rather, they represent those few
instances in which we understand the mechanism from the thermodynamic point of
view. In addition, we can use electrochemical and chemical measurements to detect
one or more of their products. Other mechanisms implicated in MIC involve bacte-
ria that produce corrosive metabolites. For example Thiobacillus thiooxidans pro-
duces sulfuric acid and Clostridium aceticum produces acetic acid. These two
metabolic products dissolve the passive layers of oxides deposited on the metal
surface, which accelerates the cathodic reaction rate (Borenstein 1994). Other
mechanisms may be initiated by hydrogen-generating microorganisms causing
hydrogen embrittlement of metals or by iron-oxidizing bacteria, such as Gallionella.
An important aspect of quantifying these mechanisms is to demonstrate exactly
how they interfere with the corrosion processes. There is no universal mechanism
of MIC. Instead, many mechanisms exist and some of them have been described
and quantified better than others.
It does not seem reasonable to search for universal mechanisms, but it does seem
reasonable to search for evidence of specific, well-defined microbial involvement in
corrosion processes. For example, demonstrating the presence of elemental sulfur in
the corrosion of mild steel can be considered evidence of SRB corrosion, and dem-
onstrating the presence of manganese oxides in the corrosion of stainless steel can be
considered evidence of MOB corrosion. However, even in these examples there is a
possibility that some aspects of microbial participation escape our attention. The
deposition of manganese oxides is easy to demonstrate on stainless steels or other
passive metals because they are stable on such surfaces. However, if MOB deposit
manganese oxides on mild steel where the oxides are reduced at the same rate as they
are deposited, the corrosion rate may increase without the evidence of microbial par-
ticipation in the process, the deposits of manganese oxides, being detectable.

Acknowledgments This work was partially supported by the United States Office of Naval
Research (contract nos. N00014-99-1-0701 and N00014-06-1-0217). Beyenal was supported by
Washington State University (fund no. 9904) and 3M.

References

Acuna N, Ortega-Morales BO, Valadez-Gonzalez A (2006)Biofilm colonization dynamics and its

influence on the corrosion resistance of austenitic UNSS31603 stainless steel exposed to Gulf
of Mexico seawater. Mar Biotechnol 8:62–70
Al Darbi MM, Agha K, Islam MR (2005) Comprehensive modelling of the pitting biocorrosion
of steel. Can J Chem Eng 83:872–881
Amaya H, Miyuki H (1994) Mechanism of microbially influenced corrosion on stainless-steels in
natural seawater. Journal of the Japan Institute of Metals 58:775–781
Antony PJ, Chongdar S, Kumar P, Raman R (2007) Corrosion of 2205 duplex stainless steel in
chloride medium containing sulfate-reducing bacteria. Electrochim Acta 52:3985–3994
ASM Handbook Series (1987) Corrosion: Materials. ASM International
Beech IB, Gaylarde CC (1999) Recent advances in the study of biocorrosion – An overview. Rev
Microbiol 30:177–190
62 Z. Lewandowski and H. Beyenal

Beech IB, Sunner JA, Hiraoka K (2005) Microbe–surface interactions in biofouling and biocorro-
sion processes. Int Microbiol 8:157–168
Beech WB, Sunner J (2004) Biocorrosion: Towards understanding interactions between biofilms
and metals. Curr Opin Biotechnol 15:181–186
Bolwell R (2006) Understanding royal navy gas turbine sea water lubricating oil cooler failures
when caused by microbial induced corrosion (“SRB”). J Eng Gas Turbines Power – Trans
ASME 128:153–162
Borenstein SB (1994) microbiologically influenced corrosion handbook. Industrial Press, New York
Braughton KR, Lafond RL, Lewandowski Z (2001) The influence of environmental factors on the
rate and extent of stainless steel ennoblement mediated by manganese-oxidizing biofilms.
Biofouling 17:241–251
Caspi R, Tebo BM, Haygood MG (1998) c-Type cytochromes and manganese oxidation in
Pseudomonas putida MnB1. Appl Environ Microbiol 64:3549–3555
Chandrasekaran P, Dexter SC (1993) Mechanism of potential ennoblement on passive metals by
seawater biofilms (paper no. 493). CORROSION/93, NACE International, Houston, TX
Coetser SE, Cloete TE (2005) Biofouling and biocorrosion in industrial water systems. Crit Rev
Microbiol 31:213–232
Costello JA (1974) Cathodic depolarization by sulfate-reducing bacteria. S Afr J Sci 70:202–204
Crolet JL (1991) From biology and corrosion to biocorrosion. In: Sequeira CAC, Tiller AK (eds.)
Proceedings of the 2nd EFC workshop on Microbial Corrosion. HMSO, London, pp 50–60
Crolet JL (1992) From biology and corrosion to biocorrosion. Oceanol Acta 15:87–94
Dexter SC, Gao GY (1988) Effect of seawater biofilms on corrosion potential and oxygen reduc-
tion on stainless steels. Corrosion 44:717
Dickinson WH, Lewandowski Z (1996) Manganese biofouling and the corrosion behavior of
stainless steel. Biofouling 10:79–93
Dickinson WH, Lewandowski Z (1998) Electrochemical concepts and techniques in the study of
stainless steel ennoblement. Biodegradation 9:11–21
Dickinson WH, Caccavo F, Lewandowski Z (1996a) The ennoblement of stainless steel by
manganic oxide biofouling. Corros Sci 38:1407–1422
Dickinson WH, Lewandowski Z, Geer RD (1996b) Evidence for surface changes during ennoble-
ment of type 316L stainless steel: Dissolved oxidant and capacitance measurements.
Corrosion 52:910–920
Dickinson WH, Caccavo F, Olesen B, Lewandowski Z (1997) Ennoblement of stainless steel by
the manganese-depositing bacterium Leptothrix discophora. Appl Environ Microbiol
63:2502–2506
Eashwar M, Maruthamuthu S (1995) Mechanism of biologically produced ennoblement –
Ecological perspectives and a hypothetical model. Biofouling 8:203–213
Finster K, Liesack W, Thamdrup B (1998) Elemental sulfur and thiosulfate disproportionation by
Desulfocapsa sulfoexigens sp nov, a new anaerobic bacterium isolated from marine surface
sediment. Appl Environ Microbiol 64:119–125
Flemming HC (1995) Biofouling and biocorrosion – Effects of undesired biofilms. Chem Ing Tech
67:1425–1430
Flemming HC, Wingender J (2001) Relevance of microbial extracellular polymeric substances
(EPSs) – Part II: Technical aspects. Water Sci Technol 43:9–16
Ford T, Mitchell R (1990) The ecology of microbial corrosion. Adv Microb Ecol 11:231–262
Francis CA, Tebo BM (2002) Enzymatic manganese(II) oxidation by metabolically dormant
spores of diverse Bacillus species. Appl Environ Microbiol 68:874–880
Geiser M, Avci R, Lewandowski Z (2002) Microbially initiated pitting on 316L stainless steel. Int
Biodeterior Biodegrad 49:235–243
Hakkarainen TJ (2003) Microbiologically influenced corrosion of stainless steels – What is
required for pitting? Mater Corros/Werkstoffe Korrosion 54:503–509
Hamilton WA (2003) Microbially influenced corrosion as a model system for the study of metal
microbe interactions: A unifying electron transfer hypothesis. Biofouling 19:65–76
Mechanisms of Microbially Influenced Corrosion 63

Hernandez G, Pedersen A, Thierry D, Hermansson M (1990) Bacterial effects of corrosion of steel

in seawater. In: Dowling NJ, Mittelman MW, Danko JC (eds) Proceedings – Microbially
influenced corrosion and biodeterioration, University of Tennessee, Knoxville
Hernandez G, Kucera V, Thierry D, Pedersen A, Hermansson M (1994) Corrosion inhibition of
steel by bacteria. Corrosion 50:603–608
Herro HM (1991) Tubercle formation and growth on ferrous alloys (paper no. 84). NACE,
Cincinnati, Ohio
Hossain MA, Das CR (2005) Kinetic and thermodynamic studies of microbial corrosion of mild
steel specimen in marine environment. J Indian Chem Soc 82:376–378
Ilhan-Sungur E, Cansever N, Cotuk A (2007) Microbial corrosion of galvanized steel by a fresh-
water strain of sulphate reducing bacteria (Desulfovibrio sp.). Corros Sci 49:1097–1109
Javaherdashti RA (1999) Review of some characteristics of MIC caused by sulfate-reducing
bacteria: Past, present and future. Anti-Corros Methods Mater 46:173–180
Jayaraman A, Ornek D, Duarte DA, Lee CC, Mansfeld FB, Wood TK (1999) Axenic aerobic
biofilms inhibit corrosion of copper and aluminum. Appl Microbiol Biotechnol 52:787–790
Lee AK, Newman DK (2003) Microbial iron respiration: Impacts on corrosion processes. Appl
Microbiol Biotechnol 62:134–139
Lee W, Lewandowski Z, Nielsen PH, Hamilton WA (1995) Role of sulfate-reducing bacteria in
corrosion of mild-steel – A review. Biofouling 8:165–194
Lewandowski Z, Beyenal H (2007) Fundamentals of biofilm research. CRC
Lewandowski Z, Dickinson W, Lee W (1997) Electrochemical interactions of biofilms with metal
surfaces. Water Sci Technol 36:295–302
Lewandowski Z, Beyenal H, Stookey D (2004) Reproducibility of biofilm processes and the
meaning of steady state in biofilm reactors. Water Sci Technol 49:359–364
Linhardt P (1996) Failure of chromium-nickel steel in a hydroelectric power plant by manganese-
oxidizing bacteria. In: Heitz E, Flemming HC, Sand W (eds.) Microbially influenced corro-
sion of materials. Springer Verlag, Berlin Heidelberg, pp 221–230
Linhardt P (1998) Electrochemical identification of higher oxides of manganese in corrosion rele-
vant deposits formed by microorganisms. Electrochemical Methods in Corrosion Research
VI, Pts 1 & 2. Book Series: Materials Science Forum, Vol. 289–2:1267–1274
Linhardt P (2004) Microbially influenced corrosion of stainless steel by manganese oxidizing
microorganisms. Mater Corros/Werkstoffe Korrosion 55:158–163
Linhardt P (2006) MIC of stainless steel in freshwater and the cathodic behaviour of biomineral-
ized Mn-oxides. Electrochim Acta 51:6081–6084
Little B, Ray R (2002) A perspective on corrosion inhibition by biofilms. Corrosion 58:424–428
Little B, Lee J, Ray R (2007) A review of ‘green’ strategies to prevent or mitigate microbiologi-
cally influenced corrosion. Biofouling 23:87–97
Little BJ, Ray RI, Pope RK (2000) Relationship between corrosion and the biological sulfur cycle:
A review. Corrosion 56:433–443
Mansfeld F, Little BA (1991) Technical review of electrochemical techniques applied to microbio-
logically influenced corrosion. Corros Sci 32:247–272
Mattila K, Carpen L, Hakkarainen T, Salkinoja-Salonen MS (1997) Biofilm development during
ennoblement of stainless steel in Baltic Sea water: A microscopic study. Int Biodeterior
Biodegrad 40:1–10
Miyanaga K, Terashi R, Kawai H, Unno H, Tanji Y (2007) Biocidal effect of cathodic pro-
tection on bacterial viability in biofilm attached to carbon steel. Biotechnol Bioeng 97:
850–857
Mollica A, Trevis A (1976) Correlation entre la formation de la pellicule primaire et la modifica-
tion de le cathodique sur des aciers inoxydables experimentes en eau de mer aux vitesses de
0.3 A, 5.2 m/s. Proceedings of 4th international congress on marine corrosion and fouling,
Juan Les-Pins, France, 14–18 June 1976
Nielsen P, Lee WC, Morrison M, Characklis WG (1993) Corrosion of mild steel in an alternating
oxic and anoxic biofilm system. Biofouling 7:267–284
64 Z. Lewandowski and H. Beyenal

Olesen BH, Avci R, Lewandowski Z (2000a) Manganese dioxide as a potential cathodic reactant
in corrosion of stainless steels. Corros Sci 42:211–227
Olesen BH, Nielsen PH, Lewandowski Z (2000b) Effect of biomineralized manganese on the
corrosion behavior of C1008 mild steel. Corrosion 56:80–89
Olesen BH, Yurt N, Lewandowski Z (2001) Effect of biomineralized manganese on pitting corro-
sion of type 304L stainless steel. Mater Corros/Werkstoffe Korrosion 52:827–832
Rao TS, Sairam TN, Viswanathan B, Nair KVK (2000) Carbon steel corrosion by iron oxidising
and sulphate reducing bacteria in a freshwater cooling system. Corros Sci 42:1417–1431
Roe FL, Lewandowski Z, Funk T (1996) Simulating microbiologically influenced corrosion by
depositing extracellular biopolymers on mild steel surfaces. Corrosion 52:744–752
Romero JM, Angeles-Chavez C, Amaya M (2004) Role of anaerobic and aerobic bacteria in
localised corrosion: Field and laboratory morphological study. Corros Eng Sci Technol
39:261–264
Schmitt G (1991) Effect of elemental sulfur on corrosion in sour gas systems. Corrosion
47:285–308
Sedriks AJ (1996) Corrosion of stainless steel. Wiley, New York
Shi X, Avci R, Lewandowski Z (2002a) Microbially deposited manganese and iron oxides on pas-
sive metals – Their chemistry and consequences for material performance. Corrosion
58:728–738
Shi XM, Avci R, Lewandowski Z (2002b) Electrochemistry of passive metals modified by man-
ganese oxides deposited by Leptothrix discophora: Two-step model verified by ToF-SIMS.
Corros Sci 44:1027–1045
Starosvetsky J, Starosvetsky D, Armon R (2007) Identification of microbiologically influenced
corrosion (MIC) in industrial equipment failures. Eng Fail Anal 14:1500–1511
Tebo BM, Ghiorse WC, van Waasbergen LG, Siering PL, Caspi R (1997) Bacterially mediated
mineral formation: Insights into manganese(II) oxidation from molecular genetic and
biochemical studies. Geomicrobiology: Interactions between Microbes and Minerals. Book
Series: Reviews in Mineralogy, 35:225–266
Tebo BM, Bargar JR, Clement BG, Dick GJ, Murray KJ, Parker D, Verity R, Webb SM (2004)
Biogenic manganese oxides: Properties and mechanisms of formation. Annu Rev Earth Planet
Sci 32:287–328
Tebo BM, Johnson HA, McCarthy JK, Templeton AS (2005) Geomicrobiology of manganese(II)
oxidation. Trend Microbiol 13:421–428
Videla HA (2001) Microbially induced corrosion: An updated overview (reprinted). Int Biodeterior
Biodegrad 48:176–201
Videla HA, Herrera LK (2005) Microbiologically influenced corrosion: Looking to the future. Int
Microbiol 8:169–180
Wang W, Wang J, Xu H, Li X (2006) Some multidisciplinary techniques used in MIC studies.
Mater Corros/Werkstoffe Korrosion 57:531–537
Washizu N, Katada Y, Kodama T (2004) Role of H2O2 in microbially influenced ennoblement of
open circuit potentials for type 316L stainless steel in seawater. Corros Sci 46:1291–1300
White DC, de Nivens PD, Nichols J, Mikell AT, Kerger BD, Henson JM, Geesey G, Clarke CK
(1985) Role of aerobic bacteria and their extracellular polymers in facilitation of corrosion:
Use of Fourier transforming infrared spectroscopy and “signature” phospholipid fatty acid
analysis. In: Dexter SC (ed.) Biologically induced corrosion. NACE, Houston, p 233
Xu CM, Zhang YH, Cheng GX, Zhu WS (2007) Localized corrosion behavior of 316L stainless
steel in the presence of sulfate-reducing and iron-oxidizing bacteria. Mater Sci Eng A Struct
Mater: Properties Microstruct Process 443:235–241
Zuo RJ, Kus E, Mansfeld F, Wood TK (2005) The importance of live biofilms in corrosion protec-
tion. Corros Sci 47:279–287
Industrial Biofilms and their Control

P. Sriyutha Murthy (*
ü ) and R. Venkatesan

Abstract Biofilms are considered to be ubiquitous in industrial and drinking water

distribution systems. Biofilms are a major source of contribution to biofouling in
industrial water systems. The problem has wide ranging effects, causing damage to
materials, production losses and affecting the quality of the product. The problem
of biofouling is operationally defined as biofilm development that exceeds a given
threshold of interference. It is for the plant operators to keep biofilm development
below the threshold of interference for effective production and to work out values for
threshold limits for each of the technical systems. Industrial biofilms are quite diverse
and knowledge gained with a certain type of biofilm may not be applicable to others.
In recognition of this, the old concept of a universal/effective biocide is a misnomer
as physical, chemical and biological parameters of source water vary from site to site
and so do the interactions of biocides with these parameters. Control methods have to
be tailor-made for a given technical system and cannot be extrapolated. Because of the
wide-ranging complexity in industrial technical systems, understanding the biofilm
processes, detection, monitoring, control and management is imperative for efficient
plant operation. A successful antifouling strategy involves prevention (disinfecting
regularly, not allowing a biofilm to develop beyond a given threshold), killing of
organisms and cleaning of surfaces. Killing of organisms does not essentially imply
cleaning as most industrial systems deploy only biocides for killing, and the cleaning
process is not achieved. Cleaning is essential as dead biomass on surfaces provide a
suitable surface and nutrient source for subsequent attachment of organisms. A first
step in a biofilm control programme is detection and assessment of various biofilm
components, like thickness of slime layer, algal and bacterial species involved, extent
of extracellular polymeric substances and inorganic components. Prior to adopting a
biocidal dose and regime in an industrial system, laboratory testing of biocides using
side-stream monitoring devices, under dynamic conditions, should be carried out to
check their effectiveness. Online monitoring strategies should be adopted and biocidal

P.S. Murthy
Biofouling and Biofilm Processes Section, Water and Steam Chemistry Division, BARC Facilities,
Indira Gandhi Center for Atomic Research Campus Kalpakkam, 603 102, India
e-mails: psm_ murthy@yahoo.co.in, psmurthy@igcar.gov.in

Springer Series on Biofilms, doi: 10.1007/7142_2008_18 65 65

© Springer-Verlag Berlin Heidelberg 2008
66 P.S. Murthy and R. Venkatesan

dosing fine-tuned to keep biofilms under control. Literature on biofilm control strate-
gies in technical systems is rich; however, the choice of the control method often
depends on cost, time constraints and the cleanliness (threshold levels) required for
a technical process. Currently, there is a trend to use strong oxidizing biocides like
chlorine dioxide in cooling systems and ozone in water distribution systems as low
levels of chlorine have been found to be ineffective against biofilms. A number of
non-oxidizing biocides are available, which are effective but the long-term effects on
the environment are still unclear. New techniques for biofilm control like ultrasound,
electrical fields, hydrolysis of extracellular polymeric substances and methods alter-
ing biofilm adhesion and cohesion are still in their infancy at the laboratory level and
are yet to be successfully demonstrated in large industrial systems.

1 Introduction

Water drawn from natural sources is the main industrial coolant for dissipating
waste heat from heat exchangers and process systems. Use of “pure” water would
not eliminate biofouling problems as pure water systems still contain traces of
organic carbon and, thus, also face problems due to biofouling. Apart from this,
desalination plants also face biofouling problems related to accumulation of biofilms
on pipe and membrane surfaces. The problems due to fouling by biofilms are
more pronounced in the open and closed freshwater recirculating systems of
power plants and to an extent on desalination membranes, hence problems in
these systems are discussed in this chapter. However, with regard to the control
of biofilms, experiences in pure water distribution systems are also discussed
here as the principles and approaches are similar and they share a common goal,
i.e. eliminating biofilms.
The events leading to deterioration of surfaces are:
1. Natural waters contain a large number of macromolecules released by break-
down of dead organisms. These substances adsorb onto submerged surfaces
constituting a primary film (Busscher et al. 1995).
2. Initially bacteria are attracted towards this surface and are held to the substratum
by weak electrostatic forces, hydrogen bonds and van der Waals interactions
(Busscher et al. 1995).
3. As the bacteria grow, extracellular polymeric substances (EPS) are produced and
accumulate so that the bacteria are eventually embedded in a highly hydrated
matrix (Christensen and Characklis 1990; Flemming 2002). The polymeric mate-
rial is largely composed of polysaccharides, proteins, nucleic acids and lipids
(Flemming and Wingender 2002). It is frequently believed to represent a diffusion
barrier; however, this is not the case for small molecules such as biocides as the
main component of the EPS matrix is water. Therefore, the diffusion coefficients
of such molecules are very close to those in free water (Christensen and Characklis
1990) unless these molecules do interact with matrix components. This effect is
called diffusion–reaction limitation(Gilbert et al. 2001). The production of EPS
Industrial Biofilms and their Control 67

provides adhesion to the substrate and matrix cohesion and, thus, increases the
mechanical stability of biofilms.
4. Subsequently, diatoms and microalgae colonize the substratum and the biofilm
grows in thickness, further entrapping nutrients from bulk water (Flemming and
Leis 2002).
Control of biofilms in industrial systems is an important component of a successful
water treatment programme (Ludensky 2003). Theoretical approaches consider that
the primary step in biofilm inhibition is to prevent the initial adhesion of microor-
ganisms (Busscher et al. 1999). However, in practice, this does not work because
sooner or later surfaces in technical systems will eventually be colonized. Biofilms
serve as a source for production and release of microbial cells, which influences
microbial levels in the water column. Codony et al. (2005) reported an interesting
observation to this effect: intermittent chlorination resulted in a tenfold increase in
the release of microbial cells to the water phase in the absence of biocide. Hence,
it becomes more important to control biofilms. Routine monitoring procedures
assess the presence of planktonic bacteria, whereas the vast majority of bacteria
indigenous to aquatic environments exist attached to solid particles or industrial
surfaces and go unnoticed. A particular biocide may inactivate more than one type
of microorganism. With our current levels of understanding of the mechanisms of
biocidal action and of microbial resistance it is pertinent to consider whether it is
possible to explain why some biocides are effective while others are not. The factors
that affect antimicrobial activity most are contact time, concentration, temperature,
pH, the presence of organic matter and the type of microorganism. Hence, com-
parative assessments of different biocides are somewhat difficult. For industrial
operations, system size, cleanliness, service schedules and monitoring programmes
are important factors governing smooth operations.

2 Factors Influencing Biofilm Development

in Industrial Systems

Industrial biofilms are quite diverse due to a wide range of contributing factors such
as microbial species, temperature, nutrient availability, velocity, substratum physical
and chemical characteristics, organic loading, suspended solids and general water
chemistry. Therefore it is difficult to generalize about the types of biofilm that form
in these systems, let alone about their control methods.

2.1 Temperature

Growth of biofilm and of species colonizing a biofilm is dependent on the operating

temperatures of industrial equipment. An increase in temperature is found to favour
biofilm growth. Even a small change in temperature (5°C) can cause an increase in
68 P.S. Murthy and R. Venkatesan

biofilm thickness (Bott and Pinheiro 1977). In heat exchangers, raising the temperature
above design value can increase the rate of corrosion, rate of chemical reactions and,
for inverse solubility salts, raising the temperature might initiate deposition (Bott
1995). Heat transfer surfaces (titanium, admiralty or aluminium brass, and cupro-
nickel 90:10) in an industrial heat exchanger generally experience temperatures in the
range 28–45°C for auxiliary cooling systems and 60–70°C for condenser cooling,
where bacterial biofilms have been shown to occur (Nebot et al. 2007).

2.2 Nutrient Availability

The basic mechanism of biofilm development involves the conversion of dissolved

nutrients into accumulated biomass. Griebe and Flemming (1998) considered bio-
fouling as a “biofilm reactor in the wrong place” because the same laws apply to
both cases. The major factor controlling biofilm growth is nutrient availability. In
industrial and drinking water systems, mass transfer of nutrients to the biofilm will
tend to increase with flow velocity (Characklis 1990). The rough surfaces of bio-
films also aid in increased mass transfer of nutrients by as much as threefold com-
pared to a smooth surface (Characklis and Marshall 1990; Bott and Gunatillaka
1983). Nutrient limitation may be one way to control biofilm development without
increasing disinfectant dosing in potable water distribution systems (Griebe and
Flemming 1998; Chandy and Angles 2001; Flemming 2002). Adsorption of mac-
romolecular substances increases their availability to bacteria. Industrial cooling
systems offer a continuous flow of fresh water bringing in nutrients. A 400%
increase in biofilm thickness was observed at a given velocity of 1.2 m s–1 for an
increase in nutrient level from 4 mg L–1 to 10 mg L–1 (Melo and Bott 1997).
Removal of organic carbon resulted in greater persistence of chlorine (Chandy and
Angles, 2001).
Treatment of water to reduce the organic load is a non-viable option for power
plants as once-through seawater cooling systems on an average have an intake
capacity of 30 m3 s−1 (for 500 MW(e) plants) and freshwater recirculating systems
have a circulation rate of 80–120 m3 h−1 with an intake capacity of 10 m3 h−1.
However, this factor is included in this section in order to have a measure of the
influence of nutrient concentration on biofilm thickness and density, which have
direct implications in biocidal efficacy by reacting with the biocide dosed and neu-
tralizing it. The method of reducing the organic load and, thus, limiting nutrients
has been suggested and during the last few years has become more and more accepted
in practice as a viable alternative for membrane desalination plants, as the feed
water is devoid of biocides to protect the reverse osmosis membranes(Griebe and
Flemming 1998; Flemming 2002). The option is viable as these plants require far
less quantity of water (intake) and it may prove economical considering the
consequences to membranes of biofilms and considering the treatment (organic
load removal) required to meet the quality standards of permeate water, involving
Industrial Biofilms and their Control 69

infrastructure facilities like coagulation chambers, activated carbon adsorption and

cartridge filtration for reducing organic load.

2.3 Flow Velocity

In flowing systems, bacterial populations exist as complex, structurally heteroge-

neous biofilms attached to surfaces. Residence within these complex matrices
provides organisms with a higher localized nutrient concentration than that found
in normal waters. In the case of heat exchangers, biofilm growth can be controlled
if relatively high velocities are imposed, as shear effects are likely to have an impact
on biofilm development. Operating at high velocities to achieve increased shear
forces also results in erosion of material surface and hence results in increased damage.
An optimum shear force and temperature for minimal adhesion is yet to be worked
out specifically for heat exchanger operation. Biofilms have been described as a
viscoelastic material with plastic flow properties (Korstgens et al. 2001), based on
their response to the modulus of elasticity and yield strength. The viscoelastic prop-
erty of biofilms makes them mechanically stable and also enables them to resist
detachment (Rupp et al. 2005). The EPS functions as a network of temporary junc-
tion points and yield points, which above a certain threshold results in failure of the
gel system resulting in a highly viscous fluid (Korstgens et al. 2001). Hence it
would be of practical importance to obtain data on the flow velocities required to
either detach or induce such effects. Flow velocities of water in pure and cooling
water systems govern the development of biofilms, their density and have important
implications with respect to penetration of biocides.
Studies by Pujo and Bott (1991) have shown that the Reynolds number seems to
have a profound effect on biofilm thickness. For a given Reynolds numberof 11,000
and fixed nutrient conditions, a velocity of 0.5 m s−1 generated biofilms ten times
thicker than at a velocity of 2 m s−1 over a period of 15 days. An increase in Reynolds
number increased biofilm removal (24%), but total biofilm removal was not found
for all conditions (Simoes et al. 2005a) suggesting that biofilms were more
mechanically stable to shear forces. Treatment of biofilms with chemicals and sur-
factants like cetyltrimethyl ammonium bromide (CTAB), ortho-phthalaldehyde
(OPA), sodium hydroxide and sodium hypochlorite promoted weakening of biofilm
mechanical stability (Simoes et al. 2005a). Similarly, velocity is also known to affect
biofilm density. Experiments with unispecies P. fluorescens biofilms showed that an
increase in velocity from 0.1 to 0.5 m s−1 resulted in an increase in density of biofilm
from 26 kg m−3 to 76 kg m−3 (dry mass/wet volume) (Pinheiro et al. 1988). Qualitative
analysis of flow effects on biofilms grown from tap water at different velocities
showed that under laminar conditions biofilms were patchy and consisted of cell
clusters separated by interstitial voids. In contrast, biofilms developed under turbu-
lent flow were found to be filamentous (Stoodley et al. 1999). In flowing systems,
bacteria can adapt rapidly to hydrodynamic and chemical stresses (Suci et al. 1998)
70 P.S. Murthy and R. Venkatesan

and sessile cells are known to undergo complex physiological changes during the
process of attachment (Sauer and Camper 2001), which reduce their susceptibility
to control measures (Cloete et al. 1997; Gilbert et al. 2002).
Another factor of importance in industrial systems is shear stress on the substra-
tum caused by flowing water. High shear forces at the substratum result in (1)
increased flux of nutrients at the surface, (2) increased transport of disinfectants to
the surface, (3) a greater shearing of biofilms (Percival et al. 2000) and (4) altered
biofilm diversity (Rickard et al. 2004). An increase in flow velocities resulted in
re-suspension of biofilms and sediments in water from pipe surfaces (laboratory
study), which increased particle and turbidity counts in bulk fluid (Lehtola et al.
2006). The consequences of release of biofilm clumps from surfaces are beneficial
in once-through systems where the biofilm load decreases, whereas in recirculatory
and drinking water systems they pose problems of bacterial regrowth and suspen-
sion of toxic metals from the surface to bulk water. However, recent studies by Tsai
(2006) showed that shear stress (0.29 N m−1) and chlorination had no interaction on
biofilm formation, reinstating findings of an earlier study by Peyton (1996), who
observed no significant effects of flow rate on biofilm thickness. A probable reason
for the observed effect in these studies is that the shear stress achieved in these
studies was inadequate to remove biofilms.
It is necessary to arrive at shear stress values for biofilm removal on a variety
of surfaces. Studies by Cloete et al. (2003) showed that high velocities of 3–4
m s−1 were required to detach biofilms from surfaces. Alternatively, fouling depo-
sition was found to occur at a slow rate when a nominal flow velocity of (1.85 m
s−1) was maintained in the heat exchanger tubes (Nebot et al. 2007). Increasing the
velocity regime may offer some relief from the problem of biofilms in water dis-
tribution pipelines but with respect to heat exchangers, increased velocity would
increase the overall heat transfer coefficient (Bott 1995). This would mean addi-
tional surface area and increased capital costs. Further increase in velocity
increases the pressure drop(i.e. pressure drop is the square of the velocity) (Bott
1995). Hence, the use of flow velocity to prevent biofilm formation is not a viable
option for heat exchangers and industrial circuits because of technical problems
and energy consumption. In addition, the role of velocity effects on biofilm forma-
tion is yet to be clearly understood and a clear distinction between the two con-
trasting schools of thought, viz: shearing effects/biofilm stability, needs to be
investigated to improve our understanding of using flow velocity as a biofilm
control method.

2.4 Substratum Physical and Chemical Characteristics

The type of substratum has a pronounced effect on biofilm accumulation. Smooth

surfaces accumulate less biofilm mass than rough surfaces. The mechanism behind
this is that individual cells are much smaller than crevices (Bott 1999) and an irregular
rough surface would offer protection for cells from shear effects. However, such
Industrial Biofilms and their Control 71

surface irregularities have a measurable effect only during the initial stages of biofilm
development and biofilms are unavoidable in distribution systems (Veeran and Hissett
1999). When biofilm thickness exceeds roughness dimensions, roughness will no
longer be of influence for biofilm accumulation; however, it will assist in better
anchoring them to surfaces. Vieira et al. (1992) have shown that biofilms of
P. fluorescens were more pronounced on aluminium plates than on brass and copper.
Similarly, more biofilms were observed on polyethylene pipes than on copper pipes
(Lehtola et al. 2006). This is commonly attributed to the toxic effects of copper and
brass on microorganisms. However, in industrial situations, heat transfer surfaces of
copper, brass and cupronickel alloys have all been shown to accumulate biofilms.
Titanium heat exchanger tubes were shown to accumulate more fouling than brass
tubes (Nebot et al. 2007). From the literature, it is understood that no single surface
escapes fouling and that it is impossible to create smooth industrial surfaces as the
surface roughness of materials used in industries is dependent on the manufacturing
process. Low surface energy coatings, which are characterized by low adhesion forces
of the biofilm to the surface (Busscher and van der Mei 1997), could offer some
protection for structural materials like pipelines, whereas in heat exchangers chemical
control methods are the only alternative.

2.5 Suspended Solids

Industrial cooling water drawn from natural sources (seawater or freshwater)

contains common particulate material like sand, silt, clay or quartz and to a certain
extent metal oxides resulting from the corrosion of equipment upstream. Although
in industrial systems the presence of suspended particles is common, studies on
their interaction with biofilms are limited. Deposition of these particles onto sur-
faces from suspension flows is found to occur in consecutive steps. The presence
of particles in suspension influences biofilm growth by: (1) increasing the availa-
bility of nutrients to microorganisms, directly influencing their metabolism,
(2) the erosion effects of particles, resulting in removal or suppression of biofilm
formation and (3) the presence of biofilm enhances the capture of particulate mat-
ter from flowing systems, increasing accumulation on surfaces (Bott and Melo
1992). These mechanisms can be observed and are dependent on the shear force
and size of the particles. Particulate material in flowing water influences biofilm
thickness and growth. If the particle sizes are large, this results in a sloughing
effect on the biofilm whereas smaller particles are known to embed within bio-
films (Lowe et al. 1988).
In general, to ensure maintaining biofilms within the required threshold limits in
industrial circuits, the following are necessary: operating industrial systems at
velocities higher than 2–3 m s–1, without additional pumping cost or erosion prob-
lems; operating at minimum (ambient) temperatures; avoiding large open sunlit
areas; use of appropriate materials and surface coatings with a smooth finish; a
proper biocidal and cleaning programme.
72 P.S. Murthy and R. Venkatesan

3 Problems Associated with Biofilms and Their Control

in Industrial Systems

3.1 Heat Exchangers and Cooling Water Systems

In cooling water circuits, the presence of biofilms can restrict flow in pipelines
(Bott 1999), decrease heat transfer in heat exchangers, increase pressure drop (Bott
1994; Characklis and Marshall 1990), enhance corrosion (Bott 1995) and alter
surface roughness, which in turn can increase fluid frictional resistance resulting in
decreased flow and act as a source of contamination (Camper 1993).
Two main problems encountered in heat exchanger systems due to fouling by
biofilms are reduction in heat transfer (loss of thermal efficiency) and pressure drop
across the heat exchangers due to flow reduction by deposits (Characklis 1990). The
restrictions to flow imposed by the presence of biofilm deposits in heat exchanger
surfaces increases fluid frictional resistance and, for a given throughput, the velocity
will have to increase, which means additional pumping costs. In addition, the pres-
ence of biofilms may accelerate corrosion of materials in contact. Other operating
costs may accrue from the presence of biofilm deposits, such as increased mainte-
nance requirement and unplanned shutdowns for cleaning. As a result of these fac-
tors, the engineering design of heat exchangers usually incorporates allowances for
fouling to accommodate a more satisfactory annual cleaning schedule.
Recirculating systems (Fig. 1) are usually located at sites where adequate water
is not available for cooling purposes. In open recirculating systems, cooling water
drawn from the source (usually a freshwater body) is circulated through a heat
exchanger and is conveyed to a cooling tower where evaporation of some of the
water results in a cooling effect and lowering of the cooling water temperature for

Fig. 1 General schematic of an industrial recirculatory cooling system

Industrial Biofilms and their Control 73

further recirculation. After passage through the cooling towers, the water is held in
a temporary open reservoir where algal and bacterial growth occurs. In recirculatory
systems, both open and closed makeup water is added to compensate for the evapora-
tive losses as well as to maintain the quality of recirculatory water. The conductivity
of recirculatory water increases due to concentration of salts on evaporation. This
is usually measured as cycles of concentration. Usually, plants operate at two to
three cycles of concentration, as an increase of cycles of concentration above four
usually results in enhanced scaling and corrosion of equipment.
In open recirculating systems, the problems to be encountered are many as these
systems are large (with a resident water of 60–80,000 m3 for a 1,000 MW(e) power
plant). Large open areas and available nutrients in the recirculating water provide
adequate conditions for enhanced growth of algal species, resulting in eutrophica-
tion. This further leads to organic loading in the system as detrital matter
accumulates. Further, the incoming makeup water brings in fresh nutrients that are
continuously recycled in the systems.
In closed recirculating systems, the principles are as the name implies, the cool-
ing water is conveyed through pipelines to the heat exchangers and after passage
through the cooling tower is recirculated. However, even in these systems it is
inevitable to have an open storage point as large volumes of water are involved.
Closed recirculatory systems are not preferred as large capital investments have to
be made on infrastructure. Recirculating water systems are often designed with an
average flow velocity through the condenser tubes in the range of 1.8–2.4 m s–1.
Small heat exchangers in the process systems have lower velocities in the range of
0.3–0.6 m s–1, which are prone to fouling. Water filtration devices of various types
are always installed in cooling water systems fed by natural waters. These generally
consist of a band screen with a coarse grid (about 1–10 cm spacing) where the flow
rate is lower than 10 m3 s–1 or drums for higher flow rates. Specific debris filters are
also used to protect heat exchangers from clogging. An overview concerning con-
denser cooling circuits is given in Table 1.
Cooling towers of both open and closed recirculating systems face severe problems
due to algal and bacterial growth. Cooling towers represent complex ecological
niches and even different towers of identical design on a single site will generally
behave quite different microbiologically (Prince et al. 2002). Conventionally, the
splash-type cooling tower has been used, in which the heated discharge from
the condensers is ejected through fine nozzles from the top of the cooling towers.
The discharge trickles down splash bars (either concrete or wood) and collects in
the cooling tower basin from where it is pumped for recirculation. The disadvan-
tages of these splash-type towers are their extremely large size and low thermal
efficiency. This led to the development of high-performance forced or induced draft
cooling towers where the water trickles down through high film fills (polyvinyl
chloride) to the cooling tower basin. The high film fills are comprised of corrugated
parallel plates with distances of 3–5 mm between the plates. The corrugations or
chevron angles result in water being broken up into fine droplets or films by the
extended surfaces of the film fills. The corrugation increases the surface area and
has resulted in reducing the size of cooling towers. However, these high film fills
74 P.S. Murthy and R. Venkatesan

Table 1 Biocidal regimes practiced in industrial circuits for condenser cooling

Concentration
(mg L–1) Effect Reference
Low level > 0.2 Effective if targeted dosing Jenner and
targeted Cl2 is done at inlet to heat Khalanski
exchangers at EDF power (1998)
station France
Low level Cl2 0.2 TRC Pilot plant device at a Nebot et al.
550 MW plant in Spain (2007)
Low level Cl2 0.1 TRC Effective against planktonic Nebot et al.
cells of lake water (2007)
Discontinuous Cl2 0.5–1.0 Ineffective Ewans et al.
(30 min every 12 h) (1992)
Discontinuous Cl2 3.0 Effective at EDF Martigues- Jenner and
(for 1 h every 8 h) Ponteau power station on Khalanski
Mediterranean coast (1998)
Intermittent Cl2 0.2–0.3 Effective against biofilms at Jenner and
(4 h on/4 h off) Maasvlakte power station, Khalanski
Rotterdam (1998)
Intermittent Cl2 1.2 Required for biofilm control Murthy et al.
(30 min on/1 h off) on plate heat exchangers (2005)
Targeted Cl2 1.0 Recommended by EPRI for
condenser slime control
Chlorination (30 min 0.5 Effective for fouling control in Jenner and
day−1) Netherlands – KEMA Khalanski
(1998)
Chlorine dioxide 0.05–0.1 With residual (1 h day−1) or Petrucci and
without residuals (10–12 Rosellini
h day−1), effective for sea- (2005)
water condenser cooling in
Mediterranean coast
Ozone 0.1–0.15 Killing and detaching sessile Jenner and
cells. Followed in Hochst Khalanski
unit, Germany, fed with (1998)
River Main water

have been prone to both inorganic and biological fouling compared to conventional
low fouling, splash bar fills where algal growth is the major problem to be
overcome.
In large natural cooling towers, algae tend to develop in the following regions:
– The inner surface of the shell. The wet parts that are exposed to some sunlight
become covered with a cyanobacterial and algal layer. Sloughing and detachment
of algae during shutdowns leads to a great input of organic matter into the system.
– In the honeycomb-like packing structures of cooling tower fills. Exposure to
sunlight and the slow flow of water (0.2 m s–1) are causal factors for growth of
filamentous green algae and cyanobacteria where light has access.
– In the cooling tower basins and on concrete walls and pillars of the cooling tower.
Industrial Biofilms and their Control 75

Table 2 Biocidal regimes practiced in industrial cooling towers

Concentration
Regime (mg L−1) Effect References
Discontinuous shock 2.5 Effective in killing algae; inland Blank (1984)
chlorination power station CEGB, UK
Discontinuous mass 8.0 Exposures of 6 h were effective Lutz and Merle
chlorination for killing algae (1983)
Chlorine dioxide 1.5 Elimination of filamentous algae
in cooling towers
0.3 Requires extended time for Merle and
achieving similar results Montanat (1980)
ACTIV-OX 0.2–0.8 Chlorine dioxide treatment Harris (1999)
effective against Legionella
sp. in cooling towers

Generally the walls of the cooling tower basins are not protected. The biocide
dosed in the water phase is not effective as the water does not trickle through the
wall in forced/induced draft towers with film technology. As a result, thick layers
of cyanobacteria develop on the wall and act as source for further contamination.
Some of the cooling tower water containing the biocide may come in contact with
the walls. This kills the outer layers of the encrusting algae, turning the filaments
white, but does not penetrate into the deep layers of horizontal filaments adhering
to the walls. When the dead filaments have been washed off, the horizontal filament
system is once again exposed to the flow of cooling water and growth begins again.
It is important that the walls of the cooling tower basins be treated with a suitable
antifouling coating or foul release coating and are subjected to periodical cleaning
by high-pressure water jet and disposal of the algal debris. This will ensure smooth
operation of the towers.
Chlorine has been the most common biocide used in cooling towers. Biocidal
regimes practised in cooling towers are listed in Table 2. Chlorine and copper salts
have been used as popular methods for controlling bacterial growth in cooling tow-
ers (Fliermans et al. 1982). Chlorine (2–4 mg L–1), silver ions (0.02–0.04 mg L−1)
and copper ions (0.2–0.4 mg L−1) have been used for treating cooling towers
(Chambers et al. 1962; Cassels et al. 1995; Pedahzur et al. 1997; States et al. 1998;
Kusnetsov et al. 2001; Kim et al. 2002a, b). However, the use of metal ions for
biofouling control should always take into consideration the development of resistant
microbial populations (Schulte et al. 2005).
Legionella sp. is an important component in natural and artificial water environ-
ments, cooling towers, plumbing systems and evaporators of large air conditioning
systems, and remains a health hazard. Legionella sp. is known to occur in biofilms
in cooling towers, showers, humidifiers (Fields et al. 2002) and hence knowledge
about its response to control measures is important. These Gram-negative aerobic
rods have been shown to survive at temperatures of 20–50°C and are inactivated at
temperatures above 70°C (Kim et al. 2002a) and in a pH range of 5.5–8.1.The
organism is known to occur in stagnant warm water bodies (Sanden et al. 1989).
76 P.S. Murthy and R. Venkatesan

This aspect is important as power plant exhaust plumes are known sources of
Legionella deposits. Legionella resident within biofilms are a severe problem in
cooling tower systems using freshwater.
Several disinfection methods have been tried out. In the technical context, the
term “disinfection”is usually not used in the proper sense of the definition (inactiva-
tion of infecting microorganisms) but rather as getting rid of microbial problems.
Chemical treatments using chlorine were the most common and widely used. Free
chlorine concentrations of 1 mg L−1 were required for killing planktonic cells
whereas a fourfold increase in concentration was required to kill sessile cells (Kim
et al. 2002a). An adaptive feature exhibited by Legionella pneumophila associated
with biofilm protozoa showed that cells were found to be less susceptible to chlo-
rine (residual of 0.5 mg L−1) (Donlan et al. 2005). Resistance by Legionella biofilms
was also observed for the organic compound chloramine T (N-chloro-p-toluene
sulfonamide), obtained by chlorinating benzene sulfonamide or para-toluene, on
planktonic and sessile cells (Ozlem et al. 2007). In cooling systems of power plants
an organic compound 1-bromo-3-chloro-5,5-dimethylhydantoin (BCDMH)con-
taining bromine as an active ingredient has been used to control Legionella (Kim
et al. 2002a). Effective bromine concentrations were in the range 1.0–1.5 mg L−1.
However, a shock dose of 3–5 mg L−1 of ClO2 for a period of 1 h was required to
eliminate Legionella from dental chair water systems (Walker et al. 1995).

3.2 Case Study: Microbial Fouling of Cooling Tower Fills

in a Power Station

The Talcher super thermal power station (TSTPS) located in the Eastern state of
Orissa, India has six units each of 500 MW(e) capacities. The plant operates on an
open recirculatory mode with a residence volume of 3,600,000 m3 h−1 of cooling
water and a makeup water of 10,000 m3 h–1. Cooling water comes from the peren-
nial rivers Bahmini, Trika and Singaraj, which converge to form the “Triveni
Sangam” from which water is drawn and transported through underground pipelines
for approximately 10 km before it reaches the recirculation system. Prior to entering
the recirculation system of the plant, the water is aerated and biocide (chlorine) is
added before the water is softened using alum. The pH drop after the addition of
softening agent (alum) is revived by addition of lime (calcium). This results in a pH
value of 8.2–8.3 in the cooling water system. The water is then clarified by remov-
ing suspended solids and reaches the pump house feeding the condenser. In the
post-condenser section, the heated water from the condensers is fed into the cooling
towers. The cooling towers are of forced draft type with a counter-flow direction.
The water is then ejected through a fine nozzle below the demisters and falls by
gravity down over the PVC fills. The water trickles down the PVC film fills through
the “chevron” angle (with a flute size of 17 mm and a peak distance of 34 mm) by
gravity flow. Empirical velocity across the fills is estimated to be around 0.2 m s−1.
The bottom of the cooling tower is of an open type for air ingress. The water is
Industrial Biofilms and their Control 77

collected in a basin from where it is directed through an open channel to reach the
pump house.
Severe clogging of high efficiency polyvinyl chloride film fills by deposits
(Fig. 2a, b) was observed in the cooling towers (3, 4, 5 and 6) of the 4,000 MW(e)
TSTPS, resulting in a loss in condenser vacuum of 40 mbar and operation of the
cooling towers reaching criticality (Fig. 2c, d). The problem was found to be specific
to high efficiency film fills, and was not observed in splash-type cooling towers
(1 and 2) receiving the same waters. Further, the cooling towers connected in par-
allel and receiving the same water had different bacterial genera. Cooling towers
3 and 4 had predominantly heterotrophs and cyanobacteria (Fig. 2e), whereas iron
bacteria (Fig. 2f) dominated in cooling towers 5 and 6. The problem occurred
within 3 years of operation with an intermittent chlorination regime of 1.0 ± 0.1
ppm residuals for 12 h in place. The severity of the problem is reflected in the
quality of the recirculating water. As a result of insufficient cooling, an increase in
temperature (Fig. 3a) in the post-condenser section was observed. Reduction in
flow and heat load in the condensers resulted in an increase in conductivity levels
of recirculating water (Fig. 3b), further increasing the propensity of scaling in the
system.
Experimental data and observations revealed the problem to be a microbially
associated phenomenon. The sequence of events leading to the clogging of fills is:
(i) establishment of bacterial biofilms on PVC fill surfaces due to long layoff chlo-
rination periods and (ii) the anionic nature of the biofilms aids the entrapment of
suspended, airborne particulate matter and of dissolved nutrients like the carbon,
phosphate, nitrate and silicate essential for microbial growth. Estimation of bacte-
rial loads in the cooling water during biocide dosing did not reveal significant
differences between the pre- and post-condenser sections (Fig. 3c).
Chemical analysis of the high film fill deposits by X-ray photon spectroscopic
(XPS) analyses showed 30–45% of silica content, which is known to precipitate,
coagulate or adsorb at high concentration levels (Table 3). It is well known that
naturally occurring silica can polymerize to form amorphous silica or colloidal
silica under supersaturation conditions. The anionic nature of the biofilms resulted
in entrapment of this compound into the matrix. The situation was noticed by plant
operators when operation of the cooling towers became a concern.
The problem seems to have manifested during the layoff of biocidal dosing
(during the night) when bacterial numbers multiplied. Mechanical cleaning was not
performed because it is too labour-intensive, time-consuming and physically dam-
aging to the system. Further, the towers could not be taken offline for cleaning.
Based on the findings, the chlorination regime was switched over to a low-dose
continuous mode (0.2 ppm residuals) and with a shock dose of 5 ppm for 15 min
once a shift (8 h), coupled with increased blow-down and intake of makeup water.
This resulted in slow break-down of biofilms on the fills and helped solve the problem
online. Within 3 months the cooling towers had limped back to normality. The cooling
towers now have improved heat transfer efficiency and are inching towards normality.
Effective testing, good housekeeping during operation, proper maintenance and
prompt antifouling treatment can control microbial activity in the system. The
78 P.S. Murthy and R. Venkatesan

Fig. 2 Biofouling of cooling tower fills of Talcher super thermal power station. a) a high film fill
b) dry deposits on fills c) sagging of fills due to fouling load d) closer view of clogging of fills e)
Cyanobacteria on cooling tower walls f) Iron bacteria on cooling tower walls

study clearly demonstrated the inefficiency of intermittent chlorination and has also
shown that low-level continuous chlorination along with periodical shock chlorination
is effective in breaking down biofilms.
 Temperature (⬚C)

c
a

b
Total viable counts (CFU/ml) Conductivity (milli siemens)

0
10
20
30
40
50

0
10
20
30
40
50
R R
iv iv
er er 28
6. .1
In 9 In
ta

0.0E+00
2.0E+03
4.0E+03
6.0E+03
8.0E+03
ta
R Pu
m ke Pu ke 27
iv
e ph 16 m .8
26 ph
Pu Inta r 0 ou ou
m ke 19 C s 8. se 27
ph 20 l2 Ae e 9 C .9
C ou D ra l2 Ae
ra
l2 s 13 o t D
Al to 27
Al D Ae e 00 um sin or 8.
5
Al os
in r .7
g
Industrial Biofilms and their Control

um os ra um
in to D g
D g r 96
0 D po
o p os poin os
in
g t 8.
4 in int
27
C sing oin
la 16 1. C g .8
0 24 la Po C
rif Po t
i rif in la Po
0. i 14 rif 27
C Mix er o int 0 92 M er o t .2 ie int .5
C oo
oo lin d utle e C i xe ut M ro
c 0 0.
C
oo d le
t 14 ix ut
C ling g T lari t 99
oo ling cla .4 C ed le
t 27
oo T ow fie r oo c l .9
lin ow e 0 0. lin
g
To ifi C lin ar
g r r 99 C er 14 oo g ifi
To er B Inle oo To wer .2 lin To er 27
w a t 52
0
1. lin w I g w .9
27
g er nl C T e
C Co er B sin To Ba et 41 oo ow rI
oo n a 3 72 0. .9 lin nl
li 0 56 w si
n g er
B et 42
Sampling points in cooling circuit

de s A er .7
C ng t ns in 5 28 41 To as
oo o e A 0 0.
7
Ba 3A w i n

Sampling point in cooling circuit

s .4
lin we r in er 3 32

Sampling points in cooling circuit

g r l 60 0. Pu in 5 Ba A .3
to 3A et 9 m A 46
w w C ph .3 Pu sin
er a 50 on ou m 5A 29
.9
5A ll 00 de se C ph
w 22 ns 41 on
de
ou
se
al 50 er
l in ns 33
.2

in the cooling water systems of the 500 × 6 MW Talcher super thermal power station
le 41 er
t

0
1
in

0.2
0.4
0.6
0.8
1.2
1.4
le 33
t .2
Chlorine residuals (mg /L)

Fig. 3 Distribution of a temperature, b conductivity, c total viable counts and chlorine residuals
79
80 P.S. Murthy and R. Venkatesan

Table 3 X-ray photon spectroscopy analysis of deposits in cooling tower fills and
demisters
Sample A Sample B
Element Deposits on demisters (%) Deposits on high film fills (%)
Aluminium Al2O3 6.90 9.77
Calcium 4.5 3.75
Chlorine 0.0008 0.0008
Iron – –
Magnesium 0.63 0.70
Potassium 0.74 1.04
Silicon 31.05 41.39
Sodium 8.46 14.17
Sulfur 0.06 0.06

4 Management of Biofilms in Industrial Systems

Cooling and pure water circuits are typical ecosystems that provide an ideal environment
for growth of microorganisms. The steps involved in effective management of
industrial systems are: (1) detection of biofilms, (2) biocide dosing, (3) cleaning of
surfaces, (4) monitoring of the effectiveness of the management strategy and (5)
fine-tuning of biocidal dosing.

4.1 Detection of Biofilms

In industrial situations biofilms are visible to the naked eye as copious slime layers
on surfaces. Biofilms in industrial systems are detected indirectly by symptoms
noticed in the operational parameters (Flemming 2002) or failure to meet the
required standards in desalination and potable water systems. The first step in
detection of biofilms is sampling on surfaces, which can be a real challenge.
However, water samples reveal neither the site nor extent of biofouling layers
(Flemming 2002), as also demonstrated by Goysich and McCoy (1989) for cooling
towers. The type of sampling method used is critical for the data to be obtained.
Various methods have been used for collecting biofilm samples like sterile nylon
brushes, utility knife, swabbing and stomaching for removing them from surfaces.
Among the various methods, use of the stomaching procedure was found to be
efficient to culture biofilm cells (Gagnon et al. 1999).
Laboratory analysis of samples involves culturing of microorganisms in biofilms
and estimating the number of colony forming units. However, most of the bacteria
occurring in industrial circuits cannot be cultured by standard plate methods. An
European task force, with scientists from 18 different participating laboratories
under the French Association des Hygienistes et Technicians Municipaux (AGHTM)
Industrial Biofilms and their Control 81

Table 4 Methods for estimating components of biofilms

Biofilm parameters Method References
Direct cell counting Epifluorescence microscopy Daley and Hobbie (1975)
Biofilm thickness Light microscopy Blakke and Olson (1986)
Colony forming units Standard methods APHA (1995)
Total living biomass Adenosine triphosphate Chalut et al. (1995)
Fluorescein diacetate estimation Rosa et al. (1998)
Total biomass Total organic carbon
Dry weight Biofilm total suspended solids APHA (1995)
Algal biomass Chlorophyll and phaeophytin APHA (1995)
estimation
Total proteins Protein determination Bradford (1976)
Total sugars Carbohydrate determination Dubois et al. (1956)
Lipids GC-MS Geesey and White (1990)
Uronic acids Uronic acid determination Mojica et al. (2007)
Respiratory activity CTC staining method Schaule et al. (1993)

during the period 1996–1997, validated methods for evaluation of aqueous biofilms
and recommended the use of glass beads or slides and plate counts of cells for
quantifying biofilms (Keevil et al. 1999). Advances in microscopy, microfiltration
membranes (nucleopore or polycarbonate) and molecular staining techniques like
the Live/Dead BacLight assays are now available, which minimizes the errors in
estimating viable and dead bacterial cells. A comparison of microscopic methods
for biofilm examination has been reviewed by Surman et al. (1996). The use of
redox dyes like CTC, which forms fluorescent and insoluble crystals after reduc-
tion, also provide a more accurate quantification of microbial numbers and activity
in biofilms (Schaule et al. 1993). Several other biofilm measurement techniques or
methods have been listed by Donlan (2000) and Flemming and Schaule (1996);
however, in practice, results from the methods listed in Table 4 were found to be
more realistic in gaining an insight into the nature and extent of the deposits.

4.2 Biocide Dosing

Biocide addition in industrial systems (Table 5) is the main method of controlling

problems associated with microbial biofilm formation (Chen and Stewart 2000).
The use of biocides is a common response to biofouling problems, resulting from
a “medical paradigm” that implies that biofouling can be considered as a “technical
disease” and “cured” by substances that kill the causing bacteria. However, it always
should be kept in mind that killing of bacteria is not equivalent to cleaning. The
complexity involved in combating biofilms in industrial systems is wide ranging,
elements of which have been discussed by Flemming (2002), who has formulated
82 P.S. Murthy and R. Venkatesan

Table 5 Biocides used in industrial circuits

Oxidizing Non-oxidizing
Bromine Clamtrol: alkyl dimethyl benzyl ammonium chloride
Chlorine (ADBAC); Bulab 6002: poly[oxyethylene
Chlorine dioxide (dimethyliminio) ethylene-(dimethyliminio)
Ozone ethylene dichloride]; biguanides; β-bromo-β-
Hydrogen peroxide nitrostyrene; 2-bromo-2-nitropropano-1,3-diol
Para-acetic acid (BNPD); chlorophenols; H-130; dodecyl dimethyl
Bromine chloride ammonium chloride (DDAC); 2,2-dibromo-3-nitrilo-
1-Bromo-3-chloro-5, propionamide (DBNPA); 2-dithiobisbenzamide;
5-dimethylhydantoin (BCDMH) glutraldehyde; isothiazolone; kathion;
methylenebisthiocyanate; organic sulfur and sulfones;
phosphonium biocides; 2-(thiocyano-methylithio)-
benzothiazole (TCMTB); thiocarbomate

a toolbox for an integrated antifouling strategy. Various devices (Robbins device,

annular reactors, continuous stirred batch reactors, flow cells, mixed consortia
reactors) and processes (cooling water systems, drinking water systems, model
cooling towers, synthetic mediums containing high and low nutrients) have been
used for assessing biocide efficacies, and have been listed extensively by Donlan
(2000). Each of these systems and processes is unique and hence comparisons or
extrapolation of data to other systems is very difficult. Furthermore, knowledge
from these studies on the response of microorganisms to different biocides and
processes is difficult to utilize in choosing a biocide type, dosing or regime. For a
given industrial system and process, preliminary studies have to be carried out to
arrive at the biocidal dose and concentration with respect to the environmental and
hydro-biological conditions on site.
As indicated above, elimination of biofilms is an important task, and mechanical
cleaning has been found to be the most satisfactory method for removing biofilms
(Walker and Percival 2000) because the problems caused by biofilms in heat
exchanger systems are due to their physical presence and properties. However, in
most industrial systems, design and construction of equipment does not facilitate
mechanical cleaning, except for tubular heat exchangers where online and offline
cleaning techniques have been used. Several complexities are involved in the action
of biocides in controlling biofilms, which are discussed in this section.
Good housekeeping practices (cleaning regularly) along with appropriate bio-
cidal and surfactant or biodispersant dosings are required to keep biofilms under the
threshold of interference. Biocides aid only in killing of cells, and dead biomass
often accelerates the attachment process by offering a rough surface. Further, bio-
cides increase the biodegradable organic matter(BDOC) in treated water. Instead of
cleaning the system they actually increase the amount of nutrients available for
growth. In general, the cleanliness and the effectiveness of the microbial control
agent used should be periodically monitored using a combination of visual inspec-
tion and monitoring of differential bacterial counts like total autotrophs, hetero-
trophs, iron oxidizers, iron reducers, sulfate reducers, slime formers and pathogens
Industrial Biofilms and their Control 83

such as Legionella pneumophila in both bulk water and on surfaces in order to

determine the efficacy of the biocidal programme in practice.

4.2.1 Role and Action of Biocides on Microorganisms

The ideal biocide for a particular system would meet each of the following require-
ments: (1) active at a low concentration against a wide range of microorganisms,
(2) a low order of toxicity to humans and non-target aquatic life, (3) biodegradable,
(4) active in hard and soft water, (5) non-corrosive and (6) not readily inactivated
in the presence of a wide range of soils.
The essential duty of the microbiocide is both to prevent primary biofilm forma-
tion and to prevent excessive growth of microorganisms, which can either induce
corrosion (e.g. sulfate-reducing bacteria) or cause degradation of chemical addi-
tives (e.g. nitrifying bacteria).

4.2.2 Factors Influencing Efficacy of Biocides in Industrial Cooling Systems

In practice, the effectiveness of a biocidal programme is assessed by recovery of

process parameters in industrial systems (Flemming and Schaule 1996). In turn,
efficacy of biocides is determined by the Chick and Watson law (Chick et al. 1908;
Watson et al. 1908):

In(N/N∞) = -kCnt

where N/No is the ratio of surviving organisms at time t, C is the disinfectant con-
centration, and k and n are empirical constants (n is referred to the coefficient of
dilution).
The Chick and Watson law, with its concentration C multiplied by the contact
time t (Ct) factor, has been the basis for all subsequent models (LeChevallier et al.
1988). Further, the efficacy of a disinfectant programme can be assessed by the
recovery of process parameters (Flemming and Griebe 2000). In an industrial cool-
ing system or water distribution system, dosing of biocides is done to prevent bacte-
rial growth and colonization. However, experience over the years has shown that
maintaining a biocide residual alone could not result in preventing microbial
growth and biofilms in industrial systems. From a better understanding of the prin-
ciples of microbial adhesion, the action of biocides and the quality of abstracted
water, it is now becoming obvious that living with biofilms is imperative (Flemming
and Griebe 2000). Biofilms are ubiquitous and cannot be totally eradicated even at
a very high cost factor and for environmental safety. All systems in contact with
water carry biofilms, but not all have biofouling problems. It is now being increas-
ingly recognized that to control biofouling means to maintain biofilm development
below the threshold limits so that operations are not affected (Flemming 2002).
84 P.S. Murthy and R. Venkatesan

The Ct values for all biocides and disinfectants are affected by a number of
parameters including temperature, pH value and biocide demand as commonly
caused by organic matter and protective cell aggregations (Walker and Percival 2000).
Temperature and pH effects on oxidizing biocides have been well documented
(refer to White 1999), whereas the most important parameter responsible for
determining biocidal availability for killing is the organic content of water, which
is a site- and season-specific dynamic parameter for which no specific value could
be assigned. In this context the influence of organic matter on the efficacy of bio-
cides is of utmost practical importance. The presence of even small quantities
of organic matter reduces the efficiency of oxidizing biocides to varying degrees.
The types of action that may occur are as follows:
– The biocide may react chemically with the organic material, giving rise to a
complex that is in many instances non-biocidal, or it may form an insoluble
compound with the organic matter, thus rendering it inactive
– Particulate and colloidal matter in suspension may absorb biocides so that it is
subsequently, if not totally, removed from solution
– Naturally occurring fats, phospholipids etc. may dissolve or absorb biocides
preferentially, rendering them inactive
– Organic and suspended particulate matter may form a coating on the surface that
may render the fluid in the immediate vicinity rather more viscous, and so tend
to prevent the ready access or penetration of biocides to the cell before any
biocidal activity can occur
Antifouling efficacy on mixed population biofilms in low nutrient environments
revealed a relationship between the nature of organic matter and disinfection effi-
ciency. Chlorine was effective in removing natural biofilms with low organic car-
bon content, whereas it was ineffective with biofilms grown using amino acids and
carbohydrates as the nutrient source (Butterfield et al. 2002). Organic load requires
additional dosing of biocides to compensate for the demand in the system and to
make available the biocide for reaction with biofilms. The price will be an increased
concentration of chlorination by-products. Compared to chlorine, monochloramine-
was found to be stable and is used in many recirculating and drinking water systems
and is effective against biofilms (Murthy et al. 2008). Biofilm bacteria challenged
with monochloramine retained significant respiratory activity even though they
could not be cultured (Huang et al. 1995).
Application of biocides to industrial cooling water systems is done either on a
continuous or on an intermittent basis. It is important when applying biocides to a
cooling water circuit that the concentration developed within the system exceeds
the minimal inhibitory concentration for the microbiological contaminants present
and that it also has a sufficient contact time to exert its activity. Unless the system
has a low retention time, there will be little difference between the inhibitory con-
centrations, whether dosing is continuous or intermittent. Conventionally, before
the advent of surfactants, intermittent dosing along with an increase in velocity was
practised in industrial cooling water systems where fouling caused by biofilms was
found to be a problem to be overcome. This is dependent upon the generation of a
Industrial Biofilms and their Control 85

relatively high concentration of microbiocide within the system at regular intervals

of time and the use of high velocities intermittently to slough off biofilm layers.
Due to a wide diversity and varying population of microorganisms that can be
present in any cooling system, it is impossible to establish definitive dosage figures
that will have universal application. In general, however, high dosages are necessary in
the case of severe microbial fouling. In effect, dosages are frequently applied in a two-
phased manner. The initial dosage is usually high and aims at disrupting and dispersing
any biomass present in the system, in addition to reducing the microorganisms to an
acceptable level. Once the load is within the threshold limit then a lower concentration
of biocides will inhibit further growth. In this context, cooling systems operate on a
continuous low dose biocidal treatment with an intermittent shock dosing.

4.2.3 Efficacy of Biocides in Drinking Water Systems

Experience from drinking water systems can be adopted at least partially to biofouling
control of heat exchanger circuits. However, drinking water disinfection has a different
goal (i.e. the control of hygienically relevant microorganisms) while antifouling meas-
ures in heat exchanger systems do not have to meet such high hygienic standards but
rather focus on limitation of microbial growth. Therefore, the term “disinfection” has
a strictly hygienic connotation in drinking water, while in heat exchanger systems it
refers in a more loose sense to partially inactivating the overall microbial biofilm popu-
lation, while cells in suspension usually do not represent the dominant problem.
In drinking water distribution systems, growth of biofilms generally exceeds the
growth of their planktonic counterparts (Camper 1996). Biofilms in drinking water
systems are thin and patchy (Characklis 1988; Wingender and Flemming 2004).
Control of biofilms in potable water systems is straightforward and usually achieved
by establishing stable water through control of biologically degradable organic
carbon (BDOC). This keeps the naturally occurring microbial population in drinking
water in an oligotrophic situation. Furthermore, the drinking water industry is continu-
ally seeking novel disinfection strategies to control biofouling in distribution systems
where nutrient limitation cannot be secured.
Conventionally, chlorine and chloramines are used as disinfectants in potable
water distribution systems (US Environmental Protection Agency (US EPA) 1992).
The efficacy of different biocides on test organisms is listed in Table 6. The prob-
lem in drinking water distribution systems is similar to cooling circuits with respect
to the development of multi-species biofilms. Studies by Williams et al. (2005)
have shown that biofilm communities in distribution systems are capable of chang-
ing in response to disinfection practices. Comparing two different treatments using
monochloramine and chlorine it was found that after 2 weeks, increased dosing was
required to maintain monochloramine levels in the system. In monochloramine-
treated systems Mycobacterium and Dechloromonas were dominant whereas in
chlorine-treated systems proteobacteria were dominant. Hence, it is advisable to
use a combination of biocides or to alternate between biocides in distribution systems
in order to prevent microorganisms from developing resistance.
86 P.S. Murthy and R. Venkatesan

Table 6 Biocides used for disinfecting planktonic and sessile cells in drinking water systems
Test system and Concentration (mg
Biocide organism L−1) Effect
Planktonic cells
Cl2 E. coli 0.2 Bacterial survival even after 2
Legionella 4 weeks of continuous exposure
pneumophila Monochloramine (Williams et al. 2003)
δ- and
β-Proteobacteria
Ozone P. fluorescence 0.1 and 0.3 Effective at 10–3 min (Viera et al.
Laboratory cultures 1999)
Biofilms
Cl2 and NHCl2 K. pneumoniae 2 Respiratory activity observed deep
P. aeruginosa in biofilm with CTC stain
Steel surfaces (Huang et al. 1995)
Natural biofilms
Pipe surfaces
Chloramine T L. pneumophila 0.1 – 0.3% Reduction in planktonic cells only
(Ozlem et al. 2007)
Oxsil 320 N P. aeruginosa 3 Wood et al. (1996)
Potassium mono P. aeruginosa 20 Eliminated total viable counts
persulfate (Wood et al. 1996)
Oxsil 320N A tenfold increase in concentra-
tion required to eliminate ses-
sile cells (Surdeau et al. 2006)
Chlorine dioxide Diverse microbes in 0.25 Percentage kill of 73.8%
a Chemostat 1.0 Percentage kill of 88.4% (Walker
and Morales 1997)
1.5 Percentage kill of 99.3%
Heterotrophic 0.25 low Disinfection (Gagnon et al. 2005)
Biofilms 0.5 high
Chlorite ion Heterotrophic 0.1 low Disinfection (Gagnon et al. 2005)
Biofilms 0.25 high
Ozone Laboratory biofilms 0.15 Diminish sessile cell population
by three orders of magnitude
(Viera et al. 1999)

Another important observation is that discontinuous or intermittent addition of

biocides increased the release of cells from the biofilm to bulk water. A tenfold
increase in microbial cells in the water phase was observed in the absence of chlorine
dosing (Codony et al. 2005). Intermittent dosing of biocides resulted in planktonic
cells developing resistance, corresponding to the number of times layoff periods
occurred. Results indicated that intermittent biocidal dosings may accelerate the
development of microbial communities with reduced susceptibility to disinfection
in drinking water systems (Codony et al. 2005).
Maintenance of a chlorine residual level does not inactivate all bacteria in a
water distribution system (Momba et al. 1998). Biofilm formation was observed at
residuals of 16.5 mg L−1 hydrogen peroxide, 1 mg L−1 monochloramine and 0.2 mg
Industrial Biofilms and their Control 87

L−1 free chlorine (Momba et al. 1998). Studies with chlorine have shown that 3–5
mg L−1 (Nagy et al. 1982) and 10 mg L−1 (Exner et al. 1987) of free chlorine elimi-
nates biofilms in pure water systems.
Chlorine dioxide is another option for disinfection in distribution systems. Chlorite
ion, a by-product generated in systems dosed with chlorine dioxide, was found to be
less effective at concentrations between 0.20 and 0.34 mg L−1 in eliminating hetero-
trophic bacteria (Gagnon et al. 2005). Field trials at the East Bay Municipal Utility
District (EBMUD) in California comparing the efficiency of UV/ClO2, ClO2, UV/Cl2
and Cl2 for biofilm control showed that UV/ClO2 was most effective against sus-
pended and sessile heterotrophic bacteria. ClO2 was more effective than Cl2 against
suspended and sessile bacteria, and that UV treatment alone was not as efficient as
ClO2 and Cl2 treatments (Rand et al. 2007). On the other hand, ozone has been a very
effective agent for disinfecting potable water systems. The formation of by-products
like iodate and bromate has been observed with ozonated waters. A low drinking
water standard of 10 mg L−1 has been set for drinking water, and hence disinfection
strategies should be designed to operate at these ranges (Gunten 2003). It is generally
believed that increasing the concentration of a disinfectant should control regrowth
but many instances exist where the opposite is seen (LeChevallier et al. 1987; Martin
et al. 1982; Reilly and Kippen 1984; Oliveri et al. 1985).

4.2.4 Efficacy of Biocides and Resistance of Biofilm Organisms

It is well known that biofilm organisms display a resistance to biocides. For their
inactivation, sometimes more than two orders of magnitude higher concentrations
are required than for planktonic cells (for review see Schulte et al. 2005). The reasons
for this phenomenon are under research and not fully elucidated. Among the
mechanisms discussed in terms of increased resistance are:
– Influence of abiotic factors such as limited access of biocides to biofilms in
crevices or in dead legs of water systems, and attachment to particles
– Diffusion–reaction limitation, due to the reaction of oxidizing biocides with EPS
components (main inactivation factor for chlorine)
– Slow growth rate, which protects dormant organisms from biocides interfering
with physiological processes
– Biofilm-specific phenotypes that express, e.g., copious amounts of EPS in
response to biocides or enzymes such as catalase that inactivate hydrogen
peroxide
– Persister cells, which is the term for the small number of organisms in a population
that survive even the most extreme concentrations by mechanisms still unknown
Ranking of halogen biocides against biofilms of Pseudomonas fluorescens (a con-
taminant in cooling water circuits), Pseudomonas aeruginosa (a contaminant in
potable water distribution systems) and Klebsiella pneumoniae (a contaminant in potable
water distribution and hygiene systems) showed stronger resistance of biofilms than
of planktonic cells (Tachikawa et al. 2005). Results of this study showed that efficacy
88 P.S. Murthy and R. Venkatesan

of different biocides varied with respect to the microorganism. In the case of

P. fluorescens biofilms exposed to various biocides, survival increased as follows:
NH4Br > NH2Cl > HOCl > STARBEX® > Br2Cl
with K. pneumoniae biofilms, percentage survival increased as follows:
Br2Cl > HOCl > NH2Cl > STARBEX® > NH4Br (Tachikawa et al. 2005).
STARBEX is a stable liquid bromine-based antimicrobial compound marketed
by NALCO (Naperville, IL). It is imperative from the results to ascertain the dominant
microorganisms present in an industrial system before a biocidal regime can be put
into place. Further, bacterial species having a high inherent susceptibility to water-
treatment biocides become dominant in systems in the presence of biocides. This
has been attributed to the formation of resistant cells. The effect was demonstrated
by Brözel et al. (1995) with P. aeruginosa, Pseudomonas stutzeri and Bacillus
cereus sub-cultured repeatedly in the presence of sub-inhibitory concentrations of
biocides, and thus adapted to grow in the presence of increasing concentrations.
Hence, in industrial water systems it is advisable to alternate between biocides to
maintain biofilms within the threshold levels.
Ozone was found to be effective at concentrations between 0.1 and 0.3 ppm at
eliminating planktonic cells of Pseudomonas fluorescens (a contaminant in industrial
systems) (107–108 cells mL−1) within a contact period of 10–30 min, whereas ozone
at a concentration of 0.15 ppm was only able to diminish cells by two to three
orders of magnitude (Viera et al. 1999). Biofilms have also been reported to develop
resistance to quaternamonium compounds like benzalkonium chloride as a result of
an increase in hydrophilicity of the bacterial cell surface by the production of
exopolysacchrides in P. aeruginosa CIP A22. However, this change in hydropho-
bicity was intermediate as the cells returned to normalcy after washing (Campanac
et al. 2002). This study shows that bacteria have similar mechanisms of resistance
for oxidizing and non-oxidizing compounds, i.e. development of EPS. Quaternary
ammonia compounds dosed along with a domestic detergent did not induce microbial
resistance in long-term exposures (McBain et al. 2004).
Due to the enhanced resistance exhibited by biofilms towards biocides, novel
approaches like dosing a combination of biocides are currently under investigation.
A laboratory study by Son et al. 2005 using a mixture of biocides showed that
combinations of Cl2/O3, Cl2/ClO2 and Cl2/ClO2 showed enhanced efficiency (52%)
compared to a single biocide (Cl2) in killing Bacillus subtilis spores. In comparison,
a combination of Cl2/H2O2 was not found to be as effective. This approach of a
combination of biocides could be tried out in heat exchangers (targeted biocide
addition) where improvement in threshold levels of biofilm would amount to
significant savings. Another study supporting the concept of application of dual
biocides was by Rand et al. (2007), who tested a combination of UV/ClO2, UV/Cl2,
ClO2 and Cl2 and showed that the combination of UV/ClO2 was the most effective
against suspended (3.93 log reduction) and attached (2.05 log reduction) hetero-
trophic bacteria. In contrast, UV light alone was not effective in disinfecting
Industrial Biofilms and their Control 89

suspended or sessile bacteria compared to both ClO2 and Cl2. Pretreatment with UV
aided in increased disinfection efficiencies with both the biocides ClO2 and Cl2.
The approach of using a combination of biocideshas also been tested in pure
water systems. Comparison of the disinfection efficiency of chlorine and chlorine
dioxide against microbial cells revealed chlorine dioxide to be effective over a
wide range of pH (Junli et al. 1997a). Further, disinfection efficiency of ClO2 on
algae (Ulothrix Cl2 94.2%, ClO2 100%; Chlamydomonas Cl2 92.9%, ClO2 75%;
Microphorimidum Cl2 81.3%, ClO2 100%) was found to be the same or slightly bet-
ter than liquid chlorine. Enhanced disinfection was observed with ClO2 against
virusesand zooplankton (Junli et al. 1997b). Chlorine dioxide inactivation of
Bacillus subtilis spores in natural waters and spiked ultrapure waters were far more
effective than chlorine (Barbeau et al. 2005). Intermittent application of chlorine
dioxidewas found to be ineffective in disinfecting bacteria in dental unit water lines
(Smith et al. 2001). Comparison of efficacies of non-oxidizing biocides, e.g.
Macrotrol MT200, Microtreat AQZ2010 and Microbiocide 2594, assayed against
23 groups of bacteria showed susceptibility of Gram-positive (MIC < 4 mg L−1) and
Gram-negative bacteria (MIC <16 mg L−1) that were in ranges far lower than those for
alkylated naphthoquinonederivate molecules (MIC 1–64 mg L–1) (Chelossi 2005).
Another example of a multiple biocide strategy is using oxidizing biocides like
hydrogen peroxide and potassium monopersulfate and a surface-active agent
(copper and cobalt phthalocyanine) incorporated in the surface matrix, which
reduced the quantity of the biocide (potassium monopersulfate) required (Wood
et al. 1996). This successful approach, demonstrated for surfaces of medical impor-
tance, could be tried in industrial systems where a multiple strategy of using biocide
and a low surface energy antifouling coating in tandem would reduce the amount
of biocide to be dosed.

4.3 Cleaning of Surfaces: Role of Surfactants

or Surface-Active Agents

Biocides have been used for killing both planktonic as well as sessile cells (Chen
and Stewart 2000) but killing alone is not enough, as explained earlier. In addition
to the killing action, oxidizing biocides like chlorine, ozone, hydrogen peroxide and
peracetic acid are known to weaken the biofilm matrix (Flemming 2002). The basic
concept is to apply shear forces to a weakened biofilm matrix for removal. This can
be achieved by the use of mechanical forcessuch as an increase in water flow velocity
or flushing with air or steam. Basically, biofilms are kept together by weak physico-
chemical interactions (see above). Understanding the requirement, industry has
adopted the use of surfactants or surface-active agents (the majority of which are
biodegradable and less toxic) addressing van der Waals interactions, as well as
complex-forming substances such as citric acid in order to overcome electrostatic
interactions (Flemming et al. 1999).
90 P.S. Murthy and R. Venkatesan

Quaternary ammonium compounds(QACs) are amphoteric surfactants that are

widely used for the control of bacterial growth in clinical and industrial environments
(Brannon 1997). These are known to have broad-spectrum antimicrobial and surfactant
properties, which have made QACs such as benzalkonium chloride the favoured
agents (Shimizu et al. 2002). QACs are known to act on the cell membrane and
rupture cells (Simoes et al. 2005a). Cetyltrimethylammonium bromide(CTAB), a
cationic QAC is known to act on the lipid component of the membrane causing cell
lysis as secondary effect (Gilbert et al. 2002). These are usually applied to open or
closed recirculating systems and are non-toxic for short-term applications, against
non-target organisms. QACs are dosed in small closed recirculating cooling systems
where the water is inaccessible for potable and domestic purposes, as the effects of
these compounds on the biota are yet to be worked out completely. These biocides
are required at milligram levels and are dosed periodically, once a day or once in 8
h and are effective for short-term periods like 12 or 48 h after dosing.
Ortho-phthalaldehyde(OPA), an aromatic compound with two aldehyde groups
(McDonnell and Russell 1999) having excellent microbiocidal and sporicidal
activity (McDonnell and Russell 1999; Rutala and Weber 2001) has received clear-
ance by the FDA (US Food and Drug Administration in 1999) and is currently
being tested with different biofilm models (Simoes et al. 2003, 2007, 2008). Some
commercial products based on quaternamonium compounds are also available
(NALCO, Naperville, IL; GE-Betz Dearborn, Decatur, IL) for treating cooling
water systems. Several detergents are available for disinfecting medical equipment
for hygienic purposes, whereas their use in potable water distribution systems is
non-existent. Investigations on the mode of action of these surface-active agents on
biofilm components, their interaction with water systems and their degradation and
by-product formation all need to be carried out before these can be recommended
in real-time systems. The cleaning efficacyof QACs, however, is very limited.
Surfactants to a certain extent are also known to inactivate microbial cells, apart
from removing them from the surface. The efficacy of CTAB (cetyltrimethyl
ammonium bromide), a cationic surfactant, on Pseudomonas fluorescens biofilms
(Simoes et al. 2005a; 2006a) grown under laminar and turbulent conditions revealed
biofilms generated under laminar conditions to be more susceptible to CTAB than
biofilms generated under turbulent conditions. Total inactivation of cells was not
achieved for either flow condition. In comparison, an anionic surfactant sodium
dodecyl sulfate(SDS) was effective in inactivation at higher concentrations, but
neither CTAB nor SDS promoted detachment of biofilms from surfaces (Simoes
et al. 2006b). These results indicate that surfactants alone are not sufficient to
remove biofilms. Furthermore, post-surfactant treatments resulted in biofilms
recovering respiritory activity to levels found in untreated controls. Subsequent
studies demonstrated resistance of P. fluorescens cells attached to glass surfaces on
treatment with CTAB and the aldehyde OPA (Simoes et al. 2008). The low cell
detachment observed with CTAB treatments has been attributed to a change in the
bacterial cell surface charge (it acquired a positive charge) and increased electro-
static interaction of the microbe to the surface (Azeredo et al. 2003). In comparison,
the combined exposure to CTAB application and increased shear stresses promoted
Industrial Biofilms and their Control 91

increased biofilm removal, demonstrating physical and chemical forces to be effec-

tive in removing biofilms (Simoes et al. 2005a). Alternatively, the response of
biofilms to combined exposure to oxidizing biocides and surface-active agents
needs to be evaluated to improve our understanding of their efficacies. Screening
for biofilm detachment using other surface-active agents needs to be carried out and
their mechanism of action with respect to their molecular and antimicrobial proper-
ties needs to be studied.

5 Monitoring the Effectiveness of Biocidal Dosings

Monitoring is of particular importance when water treatment is the primary approach

to prevention of biofouling in industrial systems (Bruijs et al. 2001; Flemming
2002). Microbial growth can be prevented by a good biocidal (Maukonen et al.
2003; Simoes et al. 2005b; Meyer 2006) with a biofilm monitoring programme in
place (Flemming 2003). Control or prevention of microbial attachment may form the
basis of a successful treatment programme (Meyer 2003). Various monitoring tech-
niques are available of which the following would be of practical use in industrial
systems: (1) in-situ analysis where fouling deposits are collected and analyzed, (2)
online monitoring devices and (3) side-stream monitoring devices.
In-situ analysis is a labour-intensive job requiring special laboratory skills for
estimation of various physical, chemical and biological parameters. On the other
hand, online monitoring techniques are found to offer an indication of surface dete-
rioration to plant operators to review their dosing strategy (e.g., Flemming 2003;
Jahnknecht and Melo 2003). Characklis proposed as early as 1990 an online bio-
fouling monitoring system from which the data collected is relayed to a central
processor system. This would allow for early warning, effective countermeasures
and efficacy determination.
The requirements for online monitoring are very demanding: it should give the
information online, in real time, non-destructively, automatically and possibly
remotely sensed. In general, only physical methods can meet these requirements.
The problem is that they usually detect a deposit but not its nature. Therefore, they
will respond to abiotic fouling as well as to biofouling. This requires experience
and advanced application research, which is not often performed.
Different types of online monitoring systems are available and it is up to the operator
to choose between them. They have been systematically considered by Flemming
(2003). These involve in-place monitors like test substrates (Yohe et al. 1986; Donlan
et al. 1994); retractable bioprobes (Jones et al. 1993); an optical fouling monitor
(Wetegrove et al. 1997; Tamachkiarow and Flemming 2003); the BioGeorge electro-
chemical biofilm activity monitoring system (Bruijs et al. 2001); and the Bridger
Scientific Fouling Monitor described by Bloch and DiFranco (1995). Flemming et al.
(1998) have described the design features and functioning of some of the online indus-
trial fouling monitoring devices (fibre optical sensor FOS; differential turbidity meas-
urement device DTM; Fourier transformation infrared spectroscopy flow cell).
92 P.S. Murthy and R. Venkatesan

An important aspect brought out by Donlan (2000) on the use of online fouling
monitors in operational industrial units is that these devices may throw light on the
extent of deterioration occurring in the system with respect to the current levels of
biocidal dosing but may not mimic exactly the system condition (pipe or electri-
cally conductive surface) where biofilms have been accumulating for years. Each
of these online monitoring methods has its own strengths and weaknesses and the
type of monitor should be carefully chosen for a particular application. As pointed
out by Donlan (2000), online sensors and detection devices are indicative of surface
deterioration rather than the nature of fouling (biological, inorganic fouling), which
is essential in determining the biocidal action. These devices measure total
fouling,which includes clay/silt, corrosion and scale deposits, and biofilms.
Addressing this aspect the electrochemical sensor BioGeorge has been developed,
which measures the change in electrochemical reactions produced by biofilms on
stainless steel electrodes (Bruijs et al. 2001).
In spite of the large number of online devices available, the concept of online
monitoring has not been widely adopted by the industry, partly because there is no
real consensus on accepted biofilm monitoring techniques and the paucity of infor-
mation regarding the concentration of biocides required to control biofilms in
industrial systems (as opposed to laboratory data) (Donlan 2000). To resolve the
concern an “expert system approach” has been proposed by Donlan (2000) that
involves studies comparing biofilm levels using different techniques. In other
words, it is the threshold levels of interference for a particular technical system that
is the scope of an industrial operator and not the online monitoring equipment.
Hence, the expert system approach should involve a study of threshold levels of
interference on a site- and season-specific basis and the results extrapolated to the
online monitoring device for its effective usage.
Compared to online monitoring devices, side-stream monitoring devices are
more practical and offer data of real-time value to operators. Several types of side-
stream monitoring devices are available: Robbins device (McCoy and Costerton
1982), annular reactors (Chexal et al. 1997) and parallel plate flow-through systems
(Pedersen et al. 1982). Measuring devices (pressure gauges) for Dp would also
offer an indication of the effectiveness of the control measure in practice. However,
this method is more appropriate for macrofouling organisms. The use of different
methods to evaluate biocide efficacy can lead to different conclusions about the
effects caused by the biocide (Simoes et al. 2005b). Simple flow-through systems
housing the material of interest and connected to the main system would be the best
method of understanding fouling development.
A regular monitoring programmeshould be a part of an antifouling programme.
Since fouling follows an asymptotic pattern in industrial systems, this curve should
be established for a given system to arrive at the sampling strategy. The next step is
the sampling strategy, where three types of coupons needs to be introduced. For the
time being, the most common method is to expose a short-term coupon (exposed
for a period of 15–20 days in a system, retrieved and quantified). Later, a long-term
coupon is exposed for a period of 30–40 days, retrieved and quantified. The time
intervals cited are arbitrary and need to be standardized for a given geographical
Industrial Biofilms and their Control 93

location based on the asymptotic fouling curve. Short-term exposure refers to the
log phase of the curve (15–20 days) and long-term exposure refers to the plateau
phase where deposition levels off (30–40 days). The third is a permanent coupon
(for visual observation, which is to be observed by the naked eye to note seasonal
changes). This is a less expensive and effective method compared to a simulated
side-stream monitoring device where these sampling procedures can be overcome.
In power stations, when a more precise control over the process parameters is
required, side-stream monitors incorporating both Dp and Dt measuring devices to
determine the thermal resistance of fouling deposits are a more precise and accurate
method of evaluating the effectiveness of the biocide. Data from such monitors could
be logged and available online through a computer for operators to fine-tune their
biocidal programmes. However, monitoring remains a highly neglected field in
improvement of antifouling measurements and early warning systems, as well in mini-
mizing the environmental burden of biocides. Still, preventive overdosing of biocides
is very common, causing considerable damage to the environment due to interference
with biological treatment of waste water and to excessive formation of by-products.

6 Concluding Remarks

Every industrial cooling water system is unique with respect to biological, chemical
and process parameters. As pointed out in this chapter, cooling water treatment
programmes have to meet a compromise between cost, cleanliness and environ-
mental requirements, wherein the threshold factor is of importance. Biofilms are
ubiquitous in industrial systems and have been demonstrated to be mechanically
stable. Elimination of biofilms in industrial systems is not necessary. However, it is
vital to learn how to live with biofilms and how to prevent their excessive development.
For this point, the threshold of interference due to biofilms becomes important and
has to be ascertained in order to evolve suitable control measures. This level is up
to the subjective tolerance of the operator and is only operationally, not scientifi-
cally, based.
Increasing the biocide dose to combat biofilms is neither a sufficient nor a com-
pletely acceptable option as biofilms in industrial cooling circuits have been shown
to develop resistance to biocides in the long run. Alternating between biocides would
help solve the problem to a certain extent; however, it is not viable in huge industrial
circuits where capital investments are involved. Although several mechanisms of
resistance have been put forward, biofilm resistance observed in industrial systems
is mainly due to failure of the most popular biocide, i.e. chlorine, to penetrate into
deep biofilm layers before being consumed by EPS components and interaction with
the process fluid. Quite often, cells deep in the biofilm are unaffected and multiply
to reach levels expected in untreated systems. To tackle this problem, more persistant
biocides are used, like monochloramine or bromine chloride, or a stronger oxidant
like chlorine dioxide for power plants and ozone for potable water distribution
systems, along with a surface-active agent to remove the biomass from surfaces.
94 P.S. Murthy and R. Venkatesan

Increased biocidal doses would initiate other problems like corrosion and by-product
accumulation. Instead, fine-tuning of the biocide dose and regime based on continuous
monitoring or surveillance should be adopted to keep biofilms under the threshold
level. In addition, it must be kept in mind that killing is not cleaning and that it is
imperative to use surfactants in a fouling control programme.
Biomass offers copious nutrients for increased colonization and regrowth of
bacteria. Hence, cleaning of surfaces is an important aspect of an antifouling pro-
gramme. As a consequence, heat exchanger systems should be designed to be
cleaning-friendly, with surfaces easily accessible (e.g. for pigging) and with low
adhesion forces of biofilms. Cleaning is more important than killing the organisms
and leaving them in place. Nutrients are potential biomass but are not addressed by
biocides – some biocides make nutrients even more bioavailable (e.g., by chlorina-
tion of humic substances). Treating of water for removal of nutrients is a non-viable
option for power plants, whereas this can be used as a limiting factor for biofilm
prevention in desalination membranes. Suitable devices for removal of organic load
need to be developed for industrial applications. The concept of living with biofilms
is a reality to be accepted, and it can be achieved by understanding the laws of
biofilm development.
Currently, more is known about the action of oxidizing biocides like chlorine,
chlorine dioxide and ozone than about the organic and synthetic biocides that are
now flooding the market. Even though these organic biocides are toxic at low con-
centrations, long-term environmental effects on the receiving water bodies need to
be assessed. This leaves us with chlorine dioxide and ozone as the potential biocides
to replace chlorine because of increasing legislations on the upper discharge limits
of chlorine. Comparatively little literature is available on the type, action and effi-
ciencies of surfactants, the main reason being insufficient success. Under these
conditions, chlorine dioxide promises an interesting alternative due to it high oxidizing
nature and low by-product formation.
From earlier studies it is clear that biocides alone are not sufficient to control fouling.
For efficient industrial operations, an integrated antifouling programme involving a
practical and reliable monitoring programme, biocide dosing, biodispersant dosing,
online cleaning and, eventually, off-line cleaning has to be put into practice. Online
mechanical cleaning methods assist biocides in combating fouling. Offline cleaning
methods should be included in the design of industrial systems. The frequency of
offline cleaning is again dependent on the required threshold levels of interference.
Side-stream monitoring devices simulating Dp and Dt with online data recording are
a convenient method of fine-tuning biocide dosing with respect to spikes in biofilm
formation. Comparatively, the use of coupons for periodic monitoring would offer a
better understanding of the diversity and density of organisms at surfaces.
Basically, the most elegant way to prevent biofouling is always nutrient limitation.
Considering biofouling as a “biofilm reactor in the wrong place”, it can be put in
the “right” place by using a biological filter ahead of the system to be protected.
The biofilm develops here, “in the right place”, where it does not disturb the process
and can be handled easily (Flemming 2002). Of course, this cannot be achieved in
Industrial Biofilms and their Control 95

all situations but certainly much more often than it is done now. It requires nothing
but a little shift of perspective.

References

APHA (1995) Standard methods for the examination of water and wastewater 19th edn. American
Public Health Association/American Water Works Association/Water Environment Federation,
APHA Washington DC, 9:34–9.41
Azeredo J, Pacheco AP, Lopes I, Oliveira R, Vieira MJ (2003) Monitoring cell detachment by
surfactants in a parallel plate flow chamber. In: Proceedings of the 2002 international special-
ized conference on biofilm monitoring. Water Sci Technol 47:77–82
Barbeau B, Desjardin R, Mysore C, Prevost M (2005) Impacts of water quality on chlorine and
chlorine dioxide efficacy in natural waters. Water Res 39:2024–2033
Bloch KP, DiFranco P (1995) Preventing MIC through experimental on-line fouling monitoring.
National Association of Corrosion Engineers annual conference; paper no 257
Blank LW (1984) Control of algal biofouling at High Marnham; 1981–83. CERL note no TPRD/
L/2649/N84, Central Electricity Research Laboratories, Leatherhead
Blakke R, Olsson PQ (1986) Biofilm thickness measurements by light-microscopy. J Microbiol
Methods 5:93–98
Bott TR, Pinheiro MMVPS (1977) Biological fouling – velocity and temperature effects. Can J
Chem Eng 55:473
Bott TR, Gunatillaka M (1983) Nutrient composition and biofilm thickness In: Bryers RW, Cole SS
(eds.) Fouling of heat exchanger surfaces. United Engineering Trustees New York, pp. 727–734
Bott TR, Melo LF (1992) Particle–bacteria interactions in biofilms In: Melo LF, Bott TR, Fletcher M,
Capdeville B (eds.) Biofilms – science and technology. NATO ASI Series Kluwer Academic
Netherlands pp. 199–206
Bott TR (1994) The control of biofilms in industrial cooling water systems In: Wimpenny W,
Nichols D, Sticker, Lappin-Scott H (eds.) Bacterial biofilms and their control in medicine and
industry Bioline, Cardiff
Bott TR (1995) Fouling of heat exchangers Elsevier, Amsterdam p. 524
Bott TR (1999) Biofilms in process and industrial waters: the biofilm ecology of microbial bio-
fouling, biocide resistance and corrosion. In: Keevil CW, Godfree A, Holt D, Dow C (eds.)
Biofilms in the aquatic environment. Royal Society of Chemistry, London pp. 80–92
Brannon DK (1997) Cosmetic microbiology: a practical handbook. CRC, Boca Raton
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities
of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254
Bruijs MCM, Venhuis LP, Jenner HA, Licina GJ, Daniels D (2001) Biocide optimization using an
on-line biofilm monitor. Power Plant Chem 3(7):400–405
Brözel VS, Pietersen B, Cloete TE (1995) Resistance of bacterial cultures to non-oxidizing water
treatment bactericides by adaptation. Water Sci Technol 31(5–6):169–175
Busscher HJ, Handley PS, Bos R, van der Mei HC (1999) Physico-chemistry of microbial adhe-
sion from an overall approach to the limits. In: Baszkin A, Norde W (eds.) Physical chemistry
of biological interfaces Marcel Dekker, New York pp. 431–458
Busscher HJ, Bos HC, van der Mei H (1995) Initial microbial adhesion is a determinant for the
strength of biofilm adhesion. FEMS Microbiol Let 128:229–234
Busscher HJ, van der Mei H (1997) Physico-chemical interactions in microbial adhesion and
relevance for biofilm formation. Adv Dent Res 11:24–32
Butterfield PW, Camper AK, Ellis BD, Jones WL (2002) Chlorination of model drinking water
biofilm: implications for growth and organic carbon removal. Water Res 36:4391–4405
Campanac C, Pineau L, Payard A, Baziar-Mouysset G, Roques C (2002) Interactions between
biocide cationic agents and bacterial biofilms. Antimicrob Agents Chemother 46:1469–1474
96 P.S. Murthy and R. Venkatesan

Camper AK (1993) Coliform regrowth and biofilm accumulation in drinking water systems: a
review In: Geesey GG, Lewandowski Z, Flemming HC (eds.) Biofouling/biocorrosion in
industrial systems Lewis Chelsea, MI
Camper AK (1996) Factors limiting microbial growth in distribution systems: laboratory and
pilot-scale experiments. AWWA Research Foundation, Denver
Cassels JM, Yahya MT, Gerba CP, Rose JB (1995) Efficacy of a combined system of copper and
silver and free chlorine for inactivation of Naegleria fowleri amoebas in water. Water Sci
Technol 31:119–122
Chambers CW, Protor CM, Kabler PW (1962) Bactericidal effect of low concentrations of silver.
J Am Water Works Assoc 54:208–216
Chalut J, D’Arise K, Bodkin PM, Stodolka C (1995) Identification of cooling water biofilm using
a novel ATP monitoring technique and their control with the use of biodispersants. In:
Corrosion/88. NACE International Houston, paper no 211
Chandy JP, Angles ML (2001) Determination of nutrients limiting biofilm formation and the
subsequent impact of disinfectant decay. Water Res 35:2677–2682
Characklis WG (1988) Bacterial regrowth in distribution systems. AWWA Research Foundation,
Denver, p. 332
Characklis WG (1990): Microbial fouling. In: W.G. Characklis, K.C. Marshall (eds.) Biofilms.
Wiley, New York, 523–584
Characklis WG, Marshall K (1990) Biofilms: a basis for an interdisciplinary approach. Wiley New
York, pp. 3–15
Chen X, Stewart PS (2000) Biofilm removal caused by chemical treatments. Water Res
34:4229–4233
Chelossi E, Faimali M (2005) Comparative assessment of antimicrobial efficacy of new potential
biocides for treatment of cooling and ballast waters Sci Total Environ
Chexal B, Horowitz J, Munson D, Spalaris C, Angell P, Gendron T, Ruscak M (1997) Biofilm
monitoring in power plant waters for use in prediction and control of MIC EPRI. Presented at
tenth service water reliability improvement seminar, Denver, CO
Chick H (1908) An investigation of the laws of disinfection. J Hygiene 8:92–158
Christensen BE, Characklis WG (1990) Physical and chemical properties of biofilms. In:
Characklis WG, Marshall KC (eds.) Biofilms. Wiley, New York, pp. 93–130
Cloete TE, Jacobs L, Brozel VS (1997) The chemical control of biofouling in industrial water
systems. Biodegradation 9:23–37
Cloete TE, Westaard D, van Vuuren SJ (2003) Dynamic response of biofilm to pipe surface and
fluid velocity. Water Sci Technol 47(5):57–59
Codony F, Morato J, Mas J (2005) Role of discontinuous chlorination on microbial production by
drinking water biofilms. Water Res 39:1896–1906
Daley RJ, Hobbie JE (1975) Direct counts of aquatic bacteria by a modified Epifluorescence
technique. Limnology and Oceanography 20:875–883
Donlan RM (2000) Biofilm control in industrial water systems: Approaching an old problem in
new ways In: Evans LV (ed.) Biofilms: recent advances in their study and control Harwood
Academic Netherlands, pp. 333–360
Donlan RM, Pipes WO, Yohe TL (1994) Biofilm formation on cast iron substrata in water distri-
bution systems. Water Res 28:1497–1503
Donlan RM, Forster T, Murga R, Brown E, Lucas C, Carpenter J, Fields B (2005) Legionella
pneumophila associated with the protozoan Hartmannella vermiformis in a model multi-spe-
cies biofilm has reduced susceptibility to disinfectants. Biofouling 21(1):1–7
Dubois M, Gilles KA, Hamilton JK, Rebers A, Smith F (1956) Colorimetric method for determi-
nation of sugars and related substances. Anal Chem 28:350–356
Ewans DW, Griffiths JS, Koopmans R (1992) Options for controlling zebra mussels. Ontario
Hydro Res Rev 7:1–25
Exner M, Tuschewitski GJ, Scharnagel J (1987) Influence of biofilms by chemical disinfection
and mechanical cleaning. Zentrablatt Bakteriologie Mikrobiologie Hygeine B. 183:549–563
Industrial Biofilms and their Control 97

Fields BS, Benson RF, Besser RE (2002) Legionella and Legionnaire disease: 25 years of inves-
tigation. Clin Microbiol Rev 15:506–526
Flemming H-C (2002) Biofouling in water systems-cases causes and countermeasures. Appl
Microbiol Biotechnol 59(6):629–640
Flemming H-C (2003) Role and levels of real time monitoring for successful anti-fouling strate-
gies. Water Sci Technol 47 (5):1–8
Flemming H-C, Griebe T (2000) Control of biofilms in industrial waters and processes In: Walker J,
Surman S, Jass J (eds.) Industrial biofouling. Wiley, New York, pp. 125–141
Flemming H-C, Leis A (2002) Sorption properties of biofilms. In: Bitton G (ed.) Encyclopedia of
environmental microbiology, vol 5. Wiley, New York, pp. 2958–2967
Flemming H-C, Schaule G (1996) Biofouling. In: Heitz E, Sand W, Flemming HC (eds.)
Microbially influenced corrosion of materials-scientific and technological aspects. Springer,
Heidelberg Berlin New York, pp. 39–54
Flemming H-C, Wingender J (2002): Extracellular polymeric substances: structure, ecological
functions, technical relevance. In: Bitton G (ed.) Encyclopedia of environmental microbiology,
vol 3. Wiley, New York, pp. 1223–1231
Flemming H-C, Griebe T, Schaule G (1996) Antifouling strategies in technical systems: a short
review. Water Sci Technol 34(5–6):517–524
Flemming H-C, Tamachkiarowa A, Klahre J, Schmitt J (1998) Monitoring of fouling and biofoul-
ing in technical systems. Water Sci Technol 38(8–9):291–298
Flemming H-C, Wingender J, Moritz R, Mayer C (1999): The forces that keep biofilms together.
In: Keevil W, Godfree AF, Holt DM, Dow CS (eds.) Biofilms in aquatic systems. Royal
Society of Chemistry, Cambridge, pp. 1–12
Fliermans CB, Bettinger GE, Fynsk AW (1982) Treatment of cooling systems containing high
levels of Legionella pneumophila. Water Res 16(6):903–909
Gagnon GA, Stawson RM (1999) An efficient biofilm removal method for bacterial cells exposed
to drinking water. J Microbiol Methods 34:203–214
Gagnon GA, Rand JL, O’Leary KC, Rygel AC, Chauret C, Andrews RC (2005) Disinfectant
efficacy of chlorite and chlorine dioxide in drinking water biofilms. Water Res 39:1809–1817
Geesey GG, White GC (1990) Determination of bacterial growth and activity at solid–liquid
interfaces. Ann Rev Microbiol 44:579–602
Gilbert P, Das JR, Jones MB, Allison D (2001) Assessment of resistance towards biocides follow-
ing the attachment of micro-organisms to, and growth on, surfaces. J Appl Microbiol
91(2):248–254
Gilbert P, Allison DG, McBain AJ (2002) Biofilms in vitro and in vivo: do singular mechanisms
influx cross-resistance? J Appl Microbiol 92:98S–110S
Goysich MJ, McCoy WF (1989) A quantitative method for determining the efficacy of algicides
in industrial cooling towers. J Ind Microbiol 4:429–434
Griebe T, Flemming HC (1998) Biocide-free antifouling strategy to protect RO membranes from
biofouling. Desalination 118:153–156
Gunten Urs von (2003) Ozonation of drinking water: Part II. Disinfection and by-product forma-
tion in presence of bromide, iodide or chlorine. Water Res 37:1469–1487
Harris A (1999) Problems associated with biofilms in cooling tower systems. In: Keevil CV,
Godfree A, Holt D, Dow C (eds.) Biofilms in the aquatic environment. Springer, Berlin
Heidelberg New York, pp. 139–144
Huang CT, Yu FP, McFeters GA, Stewart PS (1995) Non-uniform spatial patterns of respiratory
activity with biofilms during disinfection. Appl Environ Microbiol 61:2252–2256
Jahnknecht P, Melo L (2003) Online biofilm monitoring. Rev Environ Sci Biotechnol.
2:269–283
Jenner HA, Whitehouse JW, Taylor CJL, Khalanski M (1998) Cooling water management in
European power stations: biology and control of fouling. Hydroecologie Appliquee
10(1–2)225:
98 P.S. Murthy and R. Venkatesan

Jones DS, O’Rourke PC, Caine CS (1993) Detection and control of microbiologically influenced
corrosion in a Rocky Mountain oil production system-a case history National Association of
Corrosion Engineers annual conference, paper no 311
Junli H, Li W, Nanqi R, Fang MA, Juli (1997a) Disinfection effect of chlorine dioxide on bacteria
in water. Water Res 31(3):607–613
Junli H, Li W, Nenqi R, Li LX, Fun SR, Guanle Y (1997b) Disinfection effect of chlorine dioxide
on viruses, algae and animal planktons in water. Water Res 31(3):455–460
Keevil CW, Mackerness CW, Colbourne JS (1990) Biocide treatment of biofilms. Int Biodeter
Biodeg 26:169–179
Keevil CW, Walker JT, Maule A, James BW (1999) Persistence and physiology of Escherichia coli
O157:H7 in the environment. In: Duffy G, Garvey P, Coia J, Wasteson W, McDowell D(eds.)
Verocytotoxigenic E. coli in Europe: survival and growth. Teagasc, Dublin, pp. 42–52
Kim BR, Anderson JE, Mueller SA, Gaines WA, Kendall AM (2002a) Literature review-efficacy
of various disinfectants against Legionella in water systems. Water Res 36:4433–4444
Kim J, Cho M, Chung WK, Kang SJ, Yoon JY (2002b) Biogrowth control in cooling tower with
EEKO-BALL. Fall conference of Korean Society of Water and Wastewater, abstract A-47
Korstgens V, Flemming HC, Wingender J, Borchard W (2001) Uniaxial compression measure-
ment device for investigation of the mechanical stability of biofilms. J Microb Method
46:9–17
Kusnetsov J, Livanainem E, Elomaa N, Zacheus O, Martikainen PJ (2001) Copper and silver ions
more effective against Legionella than against mycobacteria in a hospital warm water system.
Water Res 35:4217–4225
LeChevallier MW, Babcock TM, Lee RG (1987) Examination and characterization of distribution
system biofilms. Appl Environ Microbiol 53:2714–2724
LeChevallier MW, Cawthon CD, Lee RG (1988) Inactivation of biofilm bacteria. Appl Environ
Microbiol 54:2492–2499
Lehtola JM, Laxander M, Miettinen TI, Hirvonen A, Vartiainen T, Martikainen PJ (2006) The
effects of changing water flow velocity on the formation of biofilms and water quality in pilot
distribution system consisting of copper or polyethylene pipes. Water Res 40:2151–2160
Lowe MJ (1988) The effect of inorganic particulate materials on the development of biological
films. PhD thesis, University of Brimingham
Ludensky M (2003) Control and monitoring of biofilms in industrial applications. Int Biodeter
Biodeg 51:255–263.
Lutz P, Merle G (1983) Discontinuous mass chlorination of natural draft cooling towers. Water
Sci Tech15:197–213
Martin RS, Gates WH, Tobin RS, Sumarah R, White P, Forestall P (1982) Factors affecting colif-
orm bacterial growth in distribution systems. J Am Water Works Assoc 74:34–37
Maukonen J, Matto J, Wirtanen G, Raaska L, Matila-Sandholm T, Saarela M (2003) Methodologies
for the characterization of microbes in industrial environments: a review. J Ind Microbiol
Biotech 30:327–356
McBain AJ, Ledder RG, Moore LE, Catrenich CE, Gilbert P (2004) Effect of quaternary ammo-
nium based formulations on bacterial community dynamics and antimicrobial susceptibility.
Appl Environ Microbiol 3449–3456
McCoy WF, Costerton JW (1982) Fouling biofilm development in tubular flow systems. Dev Ind
Microbiol 23:551–558
McDonnell G, Russell AD (1999) Antiseptics and disinfectants: activity action and resistance.
Clin Microbial Rev 12:147–179
Merle G, Montanat M (1980) Essai d’utilisation du dioxyde de chlore dans la boucle TERA instal-
lee a Montereau: resultats des tests d’efficacite EDF DER report HE/31–80.045
Melo LF, Bott TR (1997) Biofouling in water systems. Exp Therm Fluid Sci 14:375–381
Meyer B (2003) Approaches to the prevention, removal and killing of biofilms. Int Biodeter
Biodeg 51:249–253
Meyer B (2006) Does microbial resistance to biocides create a hazard to food hygiene? Int J Food
Microbiol 112:275–279
Industrial Biofilms and their Control 99

Mojica K, Elsey D, Cooney MJ (2007) Quantitative analysis of biofilm EPS uronic acid content.
J Microb Methods 71(1):61–65
Momba MNB, Cloete TE, Venter SN, Kfir R (1998) Evaluation of the impact of disinfection proc-
esses on the formation of biofilms in potable surface water distribution systems. Water Sci
Technol 38(8–9):283–289
Murphy HM, Payne SJ, Gagnon GA (2008) Sequential UV- and chlorine-based disinfection to
mitigate Escherichia coli in drinking water biofilms. Water Res 42(8–9):2083–2092
doi:10.1016/j.watres.2007.12.020
Murthy PS, Venkatesan R, Nair KVK Inbakandan D, Syed Jehan S, Magesh Peter D, Ravindran
M (2005) Evaluation of sodium hypochlorite for fouling control in plate heat exchanger for
seawater application. Int Biodeter Biodeg 55:161–170
Nagy LA, Kelly AJ, Thun MA, Olson BH (1982) Biofilm composition formation and control in
the Los Angeles aqueduct system. In: Proceedings of the American Water Works Association
water quality technology conference. American Water Works Association, Denver, pp.
141–160
Nebot E, Casanueva JF, Casanueva T, Sales D (2007) Model for fouling deposition on power plant
steam condensers cooled with seawater: Effect of water velocity and tube material. Int J Heat
Mass Transfer 50:3351–3358
Oliveri VP, Bakalian AE, Bossung KW, Lowther ED (1985) Recurrent coliforms in water distribu-
tion systems and the presence of free residual chlorine. In: Jolley RL, Bull RJ, Davis WP, Katz S,
Roberts MH, Jacobs VA (eds.) Water chlorination chemistry environmental impact and health
effects Lewis Boca Raton
Ozlem N, Sanli-Yurudu, Ayten Kimiran-Erdem, Aysin C (2007) Studies on the efficacy of chlo-
ramine T trihydrate (N-chloro-p-toluene sulfonamide) against planktonic and sessile popula-
tions of different Legionella pneumophila strains. Int J Hyg Environ-Health 210:147–153
Pedahzur R, Shoval HI, Ulitzur S (1997) Silver and hydrogen peroxide as potential drinking water
disinfectants their bactericidal effects and possible modes of action. Water Sci Technol
35:87–93
Pedersen K (1982) Factors regulating microbial biofilm development in a system with slowly
flowing seawater. Appl Environ Microbiol 44:1196–1204
Percival SL (2000) Detection of biofilms in industrial waters and processes. In: Walker J, Surman S,
Jass J (eds.) Industrial biofouling detection prevention and control. Wiley, Toronto, pp.
103–124
Petrucci G, Rosellini X (2005) Chlorine dioxide in seawater for fouling control and ost disinfec-
tion in potable waterworks. Desalination 182:283–291
Peyton BM (1996) Effects of shear stress and substrate loading rate on Pseudomonas aeruginosa
biofilm thickness and density. Water Res 30(1):29–36
Pinheiro MM, Melo LF, Bott TR, Pinheiro JD, Leitao L (1988) Surface phenomena and hydrody-
namic effects on deposition of Pseudomonas fluorescens. Can J Chem Eng 66:63–67
Prince EL, Muir AVG, Thomas WM, Stollard RJ, Sampson M, Lewis JA (2002) An evaluation of
the efficacy of Aqualox for microbiological control of industrial cooling tower systems. J Hosp
Infect 52:243–249
Pujo M, Bott TR (1991) Effects of fluid velocities and Reynolds numbers on biofilm development
in water systems. In: Keffer JF, Shah RK, Ganie EN (eds.) Experimental heat transfer, fluid
mechanics and thermodynamics. Elsevier, New York pp. 1358–1362
Rand JL, Hofmann R, Alam MZB, Chauret C, Cantwell R, Andrews RC, Gagnon GA (2007) A
field study evaluation for mitigating biofouling with chlorine dioxide or chlorine integrated
with UV disinfection. Water Res 41:1939–1948
Reilly KJ, Kippen JS (1984) Relationship of bacterial counts with turbidity and free chlorine in
two distribution systems. J Am Water Works Assoc 75:309–314
Rickard HA, McBain AJ, Stead AT, Gilbert P (2004) Shear rate moderates community diversity
in freshwater biofilms. Appl Environ Microbiol 70:7426–7435
Rosa DES, Sconza F, Volterra L (1998) Biofilm amount estimation by fluorescein diacetate. Water
Res 32(9):2621–2626
100 P.S. Murthy and R. Venkatesan

Rupp CJ, Fux CA, Stoodley P (2005) Viscoelasticity of Staphylococcus aureus biofilms in
response to fluid shear allows resistance to detachment and facilitates rolling migration. Appl
Environ Microbiol 71(5):2175–2178
RutalaWA, Weber DJ (2001) New disinfection and sterilization methods. Emerging Infect Dis
7(2):348–353
Sanden G, Fields BS, Barbaree JM, Feeley JC (1989) Viability of Legionella pneumophila in
chlorine free waters at elevated temperatures. Curr Microbiol 18:61–65
Sauer K, Camper AK (2001) Characterization of phenotypic changes in Pseudomonas putida in
response to surface associated growth. J Bacteriol 183:6579–6589
Schaule G, Flemming HC, Ridgway HF (1993) Use of 5-cyano-2,3-ditolyl tetrazolium chloride
(CTC) for quantifying planktonic and sessile respiring bacteria in drinking water. Appl
Environ Microbiol 59:3850–3857
Schulte S, Wingender J, Flemming HC (2005) Efficacy of biocides against biofilms. In: Paulus W
(ed.) Directory of microbicides for the protection of materials and processes. Kluwer
Academic, Doordrecht, pp. 90–120
Shimizu MK, Okuzumi A, Yoneyama T, Kunisada M, Araake H, Ogawa, Kimura S (2002) In vitro
antiseptic susceptibility of clinical isolates from nosocomial infections. Dermatology
204(Suppl 1):21–27
Simoes M, Carvalho H, Pereira MO, Vieira MJ (2003) Studies on the behaviour of Pseudomonas
fluorescens biofilms after ortho-phthalaldehyde treatment. Biofouling 19:151–157
Simoes M, Pereira MO, Vieira MJ (2005a) Action of a cationic surfactant on the activity and
removal of bacterial biofilms formed under different flow regimes. Water Res 39:478–486
Simoes M, Pereira MO, Vieira MJ (2005b) Validation of respirometry as a short term method to
assess the toxic effect of a biocide. Biofouling 47:217–223
Simoes M, Pereira MO, Machado I, Simoes LC, Vieira MJ (2006a) Comparitive antibacterial
potential of selected aldehyde based biocides and surfactants against planktonic Pseudomonas
fluorescens. J Ind Microbiol Biotech 33:741–749
Simoes M, Simoes LC, Machado I, Pereira MO, Vieira MJ (2006b) Control of flow generated
biofilms with surfactants - Evidence of resistance and recovery. Inst Chem Eng
84(c4):338–345
Simoes LC, Simoes M, Oliveira R, Vieira M (2007) Potential of the adhesion of bacteria isolated
from drinking water to materials. J Basic Microbiol 47:174–183
Simoes M, Simoes LC, Cleto S, Pereira MO, Vieira MJ (2008) The effects of a biocide and a
surfactant on the detachment of Pseudomonas fluorescens from glass surfaces. Int J Food
Microbiol 121:335–341
Smith AJ, Bagg J, Hood J (2001) Use of chlorine dioxide to disinfect dental unit waterlines.
J Hosp Infect 49:285–288
Son H, Cho M, Kim J, Oh B, Chung H, Yoon J (2005) Enhanced disinfection efficiency of
mechanically mixed oxidants with free chlorine. Water Res 39:721–727
States S, Kuchta J, Young W, Conley L, Ge J, Costello M, Dowling J, Wadowsky R (1998)
Controlling Legionella using copper-silver ionization. J Am Water Works Assoc 90:122–129
Stoodley P, Boyle J, Cunningham AB, Dodds I, Lappin-Scott HM, Lewandowski Z (1999)
Biofilm structure and influence on biofouling under laminar and turbulent flows In: Keevil
CW, Godfree A, Holt D, Dow C (eds.) Biofilms in the aquatic environment. Royal Society of
Chemistry Cambridge pp. 13–24
Surdeau N, Laurent-Maquin X, Bouthors S, Gelle MP (2006) Sensitivity of bacterial biofilms and
planktonic cells to a new antimicrobial agent, Oxsil 320 N. J Hosp Infect 62:487–493
Suci PA, Vrany JD, Mittelman MW (1998) Investigation of interactions between antimicrobial
agents and bacterial biofilms attenuated total reflection Fourier transform infrared spectros-
copy. Biomaterials 19:327–339
Surman SB, Walker JT, Goddard DT, Morton LHG, Keevil CW, Weaver W, Skinner A, Hanson
K, Caldwell D, Kurtze J (1996) Comparison of microscope techniques for the examination of
biofilms. J Microbiol Method 2(5):57–70
Industrial Biofilms and their Control 101

Tamachkiarow A, Flemming H-C (2003) On-line monitoring of biofilm formation in a brewery

water pipeline system with a fibre optical device (FOS). Water Sci Technol 47(5):19–24
Tachikawa M, Tezuka M, Morita M, Isogai K, Okada S (2005) Evaluation of some halogen
biocides using a microbial biofilm systems. Water Res 39:4126–4132
Tsai YP (2006) Interaction of chlorine concentration and shear stress on chlorine consumption,
biofilm growth rate and particle number. Bioresource Technol 97:1912–1919
US Environmental Protection Agency (US EPA) (1992) Control of biofilm growth in drinking
water distribution systems. EPA/625/R-92/001. USEPA, Washington, DC
Verran J, Hissett T (1999) The effect of substratum surface defects upon retention of and biofilm
formation by micro-organisms from potable water. In: Keevil CW, Godfree A, Holt D, Dow C
(eds.) Biofilms in the aquatic environment. Royal Society of Chemistry Cambridge, pp. 25–33
Vieira MJ, Oliveira R, Melo LF, Pinheiro MM, van der Mei HC (1992) Biocolloids and biosur-
faces. J Dispersion Sci Tech 13(4):437–445
Viera MR, Guiamet PS, de Mele MFL, Videla HA (1999) Use of dissolved ozone for controlling
planktonic and sessile bacteria in industrial cooling systems. Int Biodeter Biodeg
44:201–207
Walker JT, Mackerness CW, Rogers J, Keevil CW (1995) Biofilm – a haven for waterborne patho-
gens. In: Lappin-Scott HM, Costerton JW (eds.) Microbial biofilms. Cambridge University
Press, London, pp. 196–204
Walker JT, Morales M (1997) Evaluation of chlorine dioxide (ClO2) for the control of biofilms.
Water Sci Technol 35 (11–12):319–323
Walker JT, Percival SL (2000) Control of biofouling in drinking water systems. In: Walker J,
Surman S, Jass J (eds.) Industrial biofouling. Wiley, New York, pp. 55–76
Watson HE (1908) A note on the variation of the rate of disinfection with change in the concentra-
tion of the disinfectant. J Hygiene 8:536
Williams MM, Braun Howland EB (2003) Growth of Escherichia coli in model distribution system
biofilm exposed to hypochlorous acid or monochloramine. Appl Environ Microbiol 5463–5271
Williams MM, Jorge W, Domingo S, Meckes MC (2005) Population diversity in model potable
water biofilms receiving chlorine or chloramines residual. Biofouling 219 (5/6):279–288
Wetegrove RL, Banks RH, Hermiller MR (1997) Optical monitor for improved fouling control in
cooling systems. J Cooling Tower Inst 18:52–59
White GC (1999) Handbook of chlorination and alternative disinfectants, 4th edn. Wiley, New
York, p. 1568
Wood P, Jones M, Bhakoo M, Gilbert P (1996) A novel strategy for control of microbial biofilms
through generation of biocide at the biofilm surface interface. Appl Environ Microbiol
2598–2602
Wingender J, Flemming HC (2004): Contamination potential of drinking water distribution net-
work biofilms. Water Sci Technol 49: 277–285
Yohe TL, Donlan RM, Kyriss KK (1986) Sampling device for determining conditions on the
interior surface of a water main. US patent no 4,631,961
Biofilm Control: Conventional
and Alternative Approaches

H.-C. Flemming (*
ü ) and H. Ridgway

Abstract “Biofouling” is referred to as the unwanted deposition and growth of

biofilms. This phenomenon can occur in an extremely wide range of opportuni-
ties ranging from colonization of medical devices, during production of ultrapure
drinking and process water, and fouling of ship hulls, pipelines and reservoirs.
Although biofouling occurs in such different areas, it has a common cause, which
is the biofilm. Biofilms are the most successful form of life on earth and tolerate
high concentrations of biocidal substances. Conventional anti-fouling approaches
usually rely on the efficacy of biocides, aiming for inhibition of biofilm growth. It is
important to keep in mind that killing of biofilm organisms usually does not solve
biofouling problems as mostly the biomass is the problem and must be removed.
Therefore, cleaning is at least equally important. However, for a sustainable anti-
fouling strategy, an advanced approach is suggested, which includes the analysis
of the fouling situation, a selection of suitable components of the “anti-fouling
menu” and an effective and representative monitoring of biofilm development. One
important part of this menu is nutrient limitation, which could be implemented
on a much broader scale than is practiced today. Other items on the menu include
methods to monitor unwanted biofilm development and assessment of the efficacy
of anti-fouling measures. Also, natural anti-fouling strategies are worth exploring
and learning from – and nature never relies on only one defence line but on inte-
grated approaches.

1 What is Biofouling?

The term “biofouling” is referred to as the undesired development of microbial layers

on surfaces. This operationally defined term has been adapted from heat exchanger
technology where “fouling” is defined generally as the undesired deposition of material
on surfaces, including:

H.-C. Flemming
IWW Centre for Water, Moritzstrasse 26, 45476, Muelheim Germany
e-mail: hanscurtflemming@compuserve.com

Springer Series on Biofilms, doi: 10.1007/7142_2008_20 103 103

© Springer-Verlag Berlin Heidelberg 2008
104 H.-C. Flemming and H. Ridgway

– Scaling, mineral fouling: deposition of inorganic material precipitating on a surface

– Organic fouling: deposition of organic substances (e.g. oil, proteins, humic
substances)
– Particle fouling: deposition of, e.g., silica, clay, humic substances and other
particles
– Biofouling: adhesion of microorganisms to surfaces and biofilm development
In the first three kinds of fouling, the increase of a fouling layer arises from the trans-
port and abiotic accumulation on the surface of material from the water phase. What
is deposited on the surface originates quantitatively from the water. In these cases, foul-
ing can be controlled by eliminating the foulants from the liquid phase. However, this
is different in the case of biofouling: microorganisms are “pseudo-particles”, which
can multiply. Even if 99.99% of all bacteria are eliminated by pre-treatment (e.g.
microfiltration or biocide application), a few surviving cells will enter the system,
adhere to surfaces, and multiply at the expense of biodegradable substances dissolved
in the bulk aqueous phase. Thus, microorganisms convert dissolved organic material
into biomass locally, through metabolic transformations. These metabolic processes,
i.e. biodegradation and surface growth, form the basis of biofilm reactors (e.g. mem-
brane bioreactors) that have been introduced in the past decade. Biofouling can be
considered as a “biofilm reactor in the wrong place and time”. Substances suitable as
nutrients, which would not act as foulants per se, will support fouling indirectly.
Although most current anti-fouling measures target the microorganisms directly (e.g.
chlorine disinfection of a potable water system), the role of nutrients as a potential
source of biomass is frequently overlooked.
Moreover, biocides tend not to decrease the nutrient level that ultimately supports
the biofilm. On the contrary, nutrients released into solution by the oxidative break-
down of normally recalcitrant organics can support rapid post-biocide (LeChevallier
1991). As it is virtually impossible to keep a common industrial system completely
sterile, microorganisms on surfaces will always be present, “waiting” for traces of nutri-
ents. Thus, all biodegradable substances must be considered as potential biomass.
Usually, the different kinds of fouling mentioned above occur together. The propor-
tion of biofouling can be considerable. An example is the development of dental
plaque, i.e. mineral depositions on teeth which is favoured by biofilms. In algal bio-
films, precipitation of calcium carbonate is increased, mainly due to the rise in pH
resulting from photosynthesis (Callow et al. 1988). However, other mechanisms may
also play a role such as changing of the water activity by EPS molecules. Generally,
biofouling has to be considered as a biofilm problem. In order to understand the effects
and dynamics of biofouling and to design appropriate countermeasures, it is important
to understand the natural processes of biofilm formation and development.
From a microbiological point of view, there is no “typical” fouling organism. If
excessive biomass or non-specific contamination of the water phase is the problem,
it will be the most abundant organism in a given site that will be the main fouling
organism. If metabolic products cause the problem, such as low-chain fatty acids,
hydrogen sulphide or inorganic acids, the organisms producing these substances will
cause the fouling. Again, “fouling” is an operational expression, which is defined by
the specific physicochemical and biological characteristics of a system.
Biofilm Control: Conventional and Alternative Approaches 105

Nearly all microorganisms are capable of forming biofilms as this is a universal

way of microbial life. Practical observations revealed that particularly slimy strains
of environmental bacteria may prevail in water system biofilms (Wingender and
Flemming, personal observation). Usually, the composition of fouling biofilms is
dominated by the autochthonic flora, which can differ profoundly with different
fouling sites and conditions, including those systems whose microbial flora has
been perturbed by the application of biocides.
Biofouling in the sense of the given definition can occur in extremely diverse
situations ranging from space stations (Koenig et al. 1997) to profane explanations
for religious miracles like that of Bolsena, which is attributed to the growth of
Serratia marcescens on sacramental bread and polenta. Communion cups have
been identified as potential infection risks due to biofilms on the chalices (Fiedler
et al. 1998). In medicine, implant devices such as catheters are prone to biofouling.
Dental waterlines can be seriously contaminated by pathogens (Barbeau et al. 1998).
In general, it is acknowledged now that biofilms are a common cause for infections
(Costerton et al. 1999). Cases, causes and countermeasures have been reviewed
(e.g., Flemming 2002) and more are presented in this book.
In technical systems, a less considered problem is the fact that biofilms can
provide a habitat for pathogenic microorganisms. Biofouling, therefore, may be not
only a technical problem but can also imply the exposure of working personnel to
such pathogens released from biofilms. The contamination risk can occur from skin
contact and from inhalation of aerosols. Klebsiella, Mycobacterium, Legionella,
Escherichia coli and coliform organisms have been found in water system biofilms
(LeChevallier et al. 1990) from where they can detach and will be found in the
water phase. In the presence of corrosion products, pathogens seem to be particu-
larly protected. This is the conclusion of the study of Emde et al. (1992), which
found a much higher variety of species in corrosion product deposits, called “tuber-
cles”, compared to the free water phase, even after extended periods of chlorination.
The fate of viruses in biofilms is still in question. Reasoner (1988) reports very
occasional incidence of pathogens in drinking water biofilms. This is confirmed by
a large study on drinking water distribution system biofilms carried out currently in
Germany. First results indicate that some pathogens seem to be even eliminated by
the autochthonic biofilms (Wingender and Flemming, personal observation). In
distribution systems, due to the surface to volume ratio, more than 95% of the entire
biomass is located at the walls and less than 5% in the water phase (Flemming
1998). These biofilms contribute considerably to the overall purification process
because they degrade diluted organic matter. There is no correlation between the
cell concentration in the water and in the biofilm, although most of the cells found
in the water phase originate from biofilms. However, ongoing large field research
reveals that biofilms developing on certified materials seem not to represent a threat
to drinking water and in general do not harbour potentially pathogenic organisms
(Kilb et al. 2003). The situation is different if materials are involved that support
microbial growth. An example is shown in Fig. 1a: massive biofilm formation on
synthetic elastomers in drinking water pipelines. Figure 1b shows a scanning electron
micrograph of the same biofilm. The size of the cells indicates very good growth
conditions, in contrast to starving microcolonies, which are usually found on
106 H.-C. Flemming and H. Ridgway

Fig. 1 a Massive biofilm development on an elastomer coating of a valve in a drinking water

system (Kilb et al. 2003). b Scanning electron micrograph of a section of a. Right: magnification
of a section of the micrograph on the left (courtesy of G. Schaule)
Biofilm Control: Conventional and Alternative Approaches 107

materials that do not leach nutrients. This biofilm harboured coliform bacteria,
which were detected in downstream drinking water samples.

2 Countermeasures

In biofouling cases, it is reasonable to follow a three step protocol:

1. Identification of the cause and localization of the problem
2. Sanitation (cleaning is as important as killing the microorganisms)
3. Prevention
This has been described in detail earlier (Flemming 2). Usually, if a problem arises
in a process, the diagnosis “biofouling” will be attributed if other causes do not
explain the phenomena. In order to design the most effective countermeasures, it is
important, to verify this diagnosis. This has to be performed by sampling of the
surfaces, which requires a set of more sophisticated techniques (Schaule et al.
2000) than sampling of the water phase, although the latter is unfortunately per-
formed exclusively in most cases. The most common countermeasure against
unwanted microbial growth is the use of biocides (Flemming and Schaule 1996).
This line of thinking expands a medical paradigm to technical systems: the
colonization by bacteria is considered as a kind of “illness” that has to be cured by
some means of disinfectant, antibiotic or other biocide. However, it is well known
that biofilm organisms display a much higher tolerance to biocidal agents than their
freely suspended counterparts (LeChevallier et al. 1991). Various mechanisms are
discussed that may protect biofilm organisms (McBain 2001). The most plausible
explanation is based on a diffusion-limitation of the biocide by the EPS matrix.
However, recent measurements have revealed that this cannot be the case. Small
molecules experience practically no diffusion limitation in a biofilm matrix. Only
if they react with EPS components (as is the case with oxidizing biocides such as
chlorine or ozone) is consumption of the biocide and, thus, a concentration gradient
observed, caused by reaction with EPS components (Schulte et al. 2005). Tolerance
against hydrogen peroxide is frequently accompanied by an enhanced catalase activity.
In general, enhanced biocide tolerance must be taken into account in anti-fouling
applications (Morton et al. 1998).

2.1 An Integrated Anti-fouling Strategy

A more complex and hopefully more effective approach to combating biofilms may
be stimulated by an increasingly restrictive legislation towards biocides, particularly
in the EU, although the relevant literature cannot be exhaustively reviewed here (for
further details see Flemming and Greenhalgh 2008). It is important not to rely only on
one “wonder weapon” but to analyse all fouling factors and to develop an integrated
108 H.-C. Flemming and H. Ridgway

approach, based on detailed knowledge of biofilm development. The basic idea is

“to live with biofilms”, an approach that may well inspire creativity in new directions
(Flemming 2002).
Biofouling is an operational definition, referring to that amount of biofilm devel-
opment that interferes with technical, aesthetic or economical requirements. Research
on reverse osmosis (RO) membrane biofouling revealed that biofilms commence
development within the first moments of operation, thereby contributing to the
demise of the separation process without any knowledge or forewarning that such
processes are at work (Griebe and Flemming 1998). Only after observing a certain
reduced membrane permeability is the “level of interference” passed and biofouling
is said to have occurred. This motif can be transferred to other water systems; they
practically all carry biofilms, but not all of them suffer from biofouling. Figure 2
shows schematically the development of biofilms in a system.
What are the options for keeping biofilm development in a system below the
individual level of interference? Basically, the extent of biofilm growth is grossly
ruled by the availability of nutrients and the shear forces. Thus, nutrients must be
considered as potential biomass. This is an important issue as, usually, biocidal
approaches do not take this aspect into account and do not limit nutrients; to the
contrary, some biocides increase the nutrient content by oxidizing recalcitrant
organics and rendering them more bioavailable (LeChevallier 1991). Nutrient limi-
tation has been demonstrated successfully as a countermeasure to biofouling (Griebe
and Flemming 1998). By using biological sand filters prior to RO membranes it was
possible to suppress the extent of biofilm growth below the threshold of interference,
although the membrane was not completely free of a biofilm (Table 1).

Fig. 2 Schematic of biofilm development. Dotted line arbitrary threshold of interference (after
Flemming et al. 1994)
Biofilm Control: Conventional and Alternative Approaches 109

Table 1 Deposit data from membranes before and after biological filter
Parameter Unit Before filter After filter
Cell number #/cm2 1.0 × 108 5.5 × 106
CFU #/cm2 1.0 × 107 1.2 × 106
Protein mg cm-2 78 4
Carbohydrates mg cm-2 26 3
Uronic acids mg cm-2 11 2
Humic substances mg cm-2 41 12
Biofilm thickness mm 27 3
Flux decline % 35 <2

Obviously, this approach cannot be applied in all cases. However, there still
remain plenty of opportunities where it provides a suitable and realistic alternative
to adding biocides for prevention of biofouling. This approach would certainly
reduce the burden of wastewater with environmentally problematic substances and
certainly deserves more attention.

2.1.1 Surface Design and Primary Adhesion

Clearly, rough surfaces are more prone to microbial colonization than smooth
surfaces. This has been confirmed with stainless steel surfaces (Faille et al. 2000).
However, even on the smoothest surface, bacteria can attach. This is the result of
unsuccessful approaches to prevent biofouling in heat exchangers by electropolishing.
In order to understand what happens when a bacterial cell comes into contact with
a surface, it is helpful to take the entire situation in account. As shown in Fig. 3 for
the example of a Gram-negative organism, cells are surrounded by extracellular
material. Also, surfaces immersed in water become within seconds covered with a
so-called conditioning film consisting of macromolecules such as humic sub-
stances, polysaccharides and proteins, which are present in trace amounts in water. This
has long since been known (Loeb and Neihof 1975) but not taken into account. The
cells do not need to be viable for adhesion, the already present EPS are sufficient for
adhesion (Flemming and Schaule 1988)
Many approaches have been followed in order to prevent microbial adhesion.
Until now, only three of them have been successful:
1. Tributyl tin anti-fouling compounds. However, these are so toxic to marine organ-
isms that they have been widely banned from use.
2. Natural anti-fouling compounds. Such compounds have been isolated mainly
from marine plants that are not colonized by bacteria (Terlezzi 2000). Steinberg
et al. (1997) have isolated signalling molecules from an Australian seaweed
exhibiting anti-colonizing activity. More marine anti-fouling products have been
investigated by Armstrong et al. (2000) and Tirrschof (2000). The problem with
all these compounds is that most of them are only scarcely available, they are
difficult to apply on a constant basis on a surface, and they will select for organisms
Biofilm Control: Conventional and Alternative Approaches 111

RO membranes could be coated with a polyether–polyamide copolymer (PEBAX

1657), which penetrated deeply into the membrane surface resulting in a smoother
hydrophilic surface. Compared to uncoated controls, the coated RO membranes
displayed a significant reduction in fouling by an oil/surfactant/water emulsion in
trials lasting more than 100 days.
A more novel approach to designing low-fouling surfaces that is still in its early
stages of development involves the application of molecular simulations to observe
and measure in silico the dynamics of surface fouling by macromolecular substances.
An example of this approach is illustrated in Fig. 4 in which a hydrated oligomer
of bacterial alginate is shown undergoing rapid adsorption to the “surface” of an
aromatic cross-linked polyamide RO membrane. The system potential energy is
shown to decline substantially in this molecular dynamics simulation (inset), sug-
gesting this type of adsorption interaction is energetically favourable. The aim of
such modelling exercises is to introduce chemical modifications into the (polyamide)
surface that will inhibit or impede such rapid macromolecular fouling.
The alginate oligomer is positioned initially (t = 0) above the membrane surface
fragment (left vertical panel). The t = 0 positions are viewed from three spatial
perspectives: side view (top), oblique view (middle), and top-down view (lower).

Fig. 4 Molecular dynamics (MD) simulation of alginate adsorption to a polyamide (PA) reverse
osmosis membrane “surface” (H. Ridgway, AquaMem Scientific Consultants and Stanford
University, unpublished data). The MD simulation shows rapid adsorption of a hydrated oligomer
of bacterial alginate (red) to the PA surface (brown). Water molecules associated with the alginate
are green. Membrane-associated water is blue. For more details see text. Frame capture times are
given in picoseconds
112 H.-C. Flemming and H. Ridgway

Water and sodium counter ions have been hidden in the t = 0 images to better
observe the alginate and membrane atoms. The right-hand upper panel indicates
that alginate adsorption occurred relatively rapidly over a period of about 300 ps.
As indicated by a decline in the system potential energy (inset graph), alginate
adsorption was thermodynamically favourable. Alginate and membrane bonds are
represented as sticks; water atoms are given as blue or green depending on whether
they were initially associated with membrane or alginate, respectively.

2.1.2 Biofilm Management

In actual practice in a variety of systems, biofilm development can be successfully

controlled through the application of a combination of cost-effective strategies,
Such a multi-factorial approach can be described as “biofilm management” and
focuses on the limitation of factors that support biofilm growth above the “level of
interference” (see Fig. 2). A successful example is the use of sand filters in order
to remove biodegradable matter from cooling water in order to protect membrane
units from biofouling (Griebe and Flemming 1998). Nutrient limitation is mean-
while an accepted approach to minimize fouling. A thorough fouling factor analysis
is necessary, which must include in the first place the assessment of the nutrient
situation. It has been explained earlier that nutrients have to be considered as poten-
tial biomass. High shear forces will limit excessive biofilm development, although
they will not prevent it. Under high shear stress, there will be a selection for organ-
isms that produce mechanically stable biofilms. Limiting the access of microorgan-
isms will also be helpful; however, it must be taken into account that cells are
particles that can multiply.
Cleaning is an important issue in biofilm management. For cleaning, cohesion
of the biofilm and adhesion to surfaces have to be overcome, which are both aspects
of the mechanical stability of biofilms. Koerstgens et al. (2001) have developed a
film rheometer that allows for the quantification of biofilm stability with the apparent
elasticity module e as a relevant parameter. This research revealed that the EPS
matrix is kept together by weak physicochemical interactions, which result in a
fluctuating network of adhesion points. In compression experiments it was shown
that until a yield point s is reached, biofilms behave as gels with constant partner
groups responsible for the adhesion. After exceeding s, the gel breaks down, the
partners of the adhesion points change and the biofilm behaves as a highly viscous
fluid. This is why biofilms are slippery. In a model system with Pseudomonas aeru-
ginosa, it was shown that Ca2+ ions increase the stability of the network by bridging
alginate molecules, which are the main component of P. aeruginosa EPS. Mg2+ did
not show such an effect, but Fe2+, Fe3+ and Cu2+ did. Most commercial cleaners and
biodispersants, however, proved ineffective in this testing system. An effective
weakening of the EPS matrix can be achieved by enzymes (Johansen et al. 1997).
However, this is not a fast effect and, in practice, it has proven transient and ineffective
in many cases (e.g. Klahre et al. 1998). This is not surprising as EPS, like other struc-
Biofilm Control: Conventional and Alternative Approaches 113

tural biopolymers, are not readily biodegradable. Also, continuous use of enzymes
will select for organisms producing EPS that are not susceptible to these enzymes.
An important aspect in cleaning is the use of surfaces to which biofilms do not
attach strongly. Such materials have been developed and tested for anti-fouling on
ship hulls and fishing nets, with silcones as a promising class of compound (Terlezzi
et al. 2000; Estarlich et al. 2000; Holm et al. 2000). Anti-fouling polymer coatings
were mentioned above (Louie et al. 2006). Modelling surface–foulant interactions
(as described above) should help elucidate how anti-fouling coatings and anti-
fouling surface treatments prevent primary macromolecular adsorption.
Electric fields have been used both for prevention of microbial adhesion and for
inhibition of biofilm growth (Matsunaga et al. 1998, Kerr et al. 1999; Schaule et al.,
2008). Practical observation, however, has shown that all kinds of electrodes immersed
into water can be colonized and fouled by biofilms. Another approach to slow down
biofouling processes and to facilitate cleaning is the use of coatings that can change
their surface properties reversibly, induced by external stimuli such as light, tem-
perature or pH value (Flemming, current research). Very interesting is the observa-
tion that surfaces with pulsed polarization show significantly lower biofilm growth
over time (Schaule et al. 2008).

2.1.3 Biofilm Monitoring

It is of great importance to monitor biofilm development in order to optimize the

time-course and effectiveness of countermeasures. This is not possible by sam-
pling of the bulk water phase. Such samples give no information about the site, the
extent and the composition of a biofilm and they generally underestimate by orders
of magnitude the true microbial (surface) burden of a system. Although biofilms
contaminate the water phase, they do so not on a constant basis but very irregularly.
Biofilm cells may erode, but sloughing events may happen as well, leading to intermit-
tent high cell numbers in the bulk water phase. Thus, biofilm monitoring must be
performed using representative surfaces.
Conventional methods rely on sampling of defined surface areas or on exposure of
test surfaces (“coupons”) with subsequent analysis in the laboratory. A classical example
is the “Robbins device” (Ruseska et al. 1982), which consists of plugs smoothly inserted
into pipe walls, experiencing the same shear stress as the wall itself. After given periods
of time, they are removed and analysed in the laboratory for all biofilm-relevant
parameters. The disadvantage of such systems is the time-lag between analysis and
result. Jacobs et al. (1996) described a simple spectrophotometric monitoring method
using a nucleotide fluorescent stain (DAPI) and automatic measurement.
Other methods have been invented that report biofilm growth on-line, in real
time and non-destructively. They all are based on physical methods. One example
is the fibre optical device (FOS), which is based on a light fibre integrated in the
test surface, measuring the scattered light of material deposited on the tip. The
principle of the sensor is schematically depicted in Fig. 5a, a typical graph is shown in
Fig. 5b (Tamachkiarow and Flemming 2003). Detection of autofluorescence of
114 H.-C. Flemming and H. Ridgway

Cleaning of sensor
2,5 Cleaning of water pipe
2
Arb. Units

1,5
1
0,5

Nov. Dec. Jan. Febr. March Apr. May June

b Time

Fig. 5 a Schematic depiction of a fibre optical device (FOS). The tip of the fibre is integrated into
the water-exposed surface. Light is coupled in by the sending fibre. Material deposited on the tip will
scatter light, which is collected in the reading fibre. b Typical graph of intensity of backscattered
light as provided by the FOS (after Tamachkiarow and Flemming 2003)

biomolecules by spectroscopy allows differentiation of biological material in the

deposit from abiotic material.
Another method uses two turbidity measurement devices, one of which is con-
stantly cleaned. The difference of the signals is proportional to the biomass developing
on the non-cleaned window (Klahre et al. 2000). Nivens et al. (1995) have given an
excellent overview on continuous non-destructive biofilm monitoring techniques,
including FTIR spectroscopy, microscopic, electrochemical and piezoelectric
techniques, which have also been systematically described by Flemming (2003).
Biofilm Control: Conventional and Alternative Approaches 115

3 Conclusions

An “integrated anti-fouling strategy”will not aim to kill all organisms in a system

but keep them below a threshold of interference. The strategy has to be based on:
1. Multi-factorial analysis of the fouling situation
2. Installation of early warning systems
3. Limiting nutrient availability where ever possible (raw water, materials, addi-
tives etc.)
4. Prioritizing cleaning over killing
5. Effective and representative monitoring of cleaning measures
Any step towards a better understanding of biofilm growth and properties will add
to the “menu” and expand the possibilities for a flexible, effective and environmen-
tally suitable response to biofouling.

References

Armstrong E, Boyd KG, Pisacane A, Peppiatt CJ, Burgess JG (2000) Marine microbial natural
products in antifouling coatings. Biofouling 16:215–224
Barbeau J, Gauthier C, Payment P (1998) Biofilms, infectious agents, and dental unit waterlines:
a review. Can J Microbiol 44:1019–1028
Bers AV, Wahl M (2004) The influence of natural surface microtopographies on fouling.
Biofouling 20:43–51
Callow ME, Pitchers RA, Santos R (1988) Non-biocidal anti-fouling coatings. In: Houghton DR,
Smith RN, Eggins HOW (eds) Biodeterioration, 7, Elsevier Applied Sciences, New York, pp.
43–48
Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persistent
infections. Science 284:1318–1322
Emde KME, Smith DW, Facey R (1992) Initial investigation of microbially influenced corrosion
(MIC) in a low temperature water distribution system. Water Res 26:169–175
Estarlich FF, Lewey SA, Nevell TG, Thorpe AA, Tsibouklis J, Upton AC (2000) The surface
properties of some silicone and fluorosilicone coating materials immersed in seawater.
Biofouling 16:263–275
Faille C, Membre JM, Tissier JP, Bellon-Fontaine MN, Carpentier B, Laroche MA, Benezech T
(2000) Influence of physicochemical properties on the hygienic status of stainless steel with
various finishes. Biofouling 15:261–274
Fiedler K, Lindner M, Edel B, Wallbrecht F (1998) Infection from the communion cup: an under-
estimated risk? Zbl Hyg Umweltmed 201:167–188
Flemming HC, Greenhalgh M (2008) Concept and consequences of the EU biocide guideline.
Springer Ser Biofilms. doi: 10.1007/7142_2008_12
Flemming HC, Schaule G (1996) Measures against biofouling. In: Heitz E, Sand W, Flemming
H-C (eds.) Microbially influenced corrosion of materials – scientific and technological
aspects. Springer, Heidelberg Berlin New York, pp. 121–139
Flemming HC, Tamachkiarowa A, Klahre J, Schmitt J (1998) Monitoring of fouling and biofouling
in technical systems. Water Sci Technol 38:291–298
Flemming HC, Schaule G (1988) Biofouling on membranes – a microbiological approach.
Desalination 70:95–119
116 H.-C. Flemming and H. Ridgway

Flemming H-C, Schaule G, McDonogh R, Ridgway HF (1994) Mechanism and extent of membrane
biofouling. In:Geesey GG, Lewandowski Z, Flemming H-C (eds.)Biofouling and biocorrosion
in industrial water systems.Lewis, Chelsea, MI, pp. 63–89
Flemming HC (2002) Biofouling in water systems – cases, causes, countermeasures. Appl Environ
Biotechnol 59:629–640
Flemming H-C (2003) Role and levels of real time monitoring for successful anti-fouling strategies.
Water Sci Technol 47(5):1–8
Griebe T, Flemming HC (1998) Biocide-free antifouling strategy to protect RO membranes from
biofouling. Desalination 118:153–156
Holm ER, Nedved BT, Phillips N, Deangelis KL, Hadfield MG Smith CM (2000) Temporal and
spatial variation in the fouling of silicone coatings in Pearl Harbour, Hawaii. Biofouling
15:95–107
Jacobs L, De Bruyn EE, Cloete TE (1996) Spectrophotometric monitoring of biofouling. Water
Sci Technol 34(5–6):533–540
Johansen C, Falholr P, Gram L (1997) Enzymatic removal and disinfection of bacterial biofilms.
Appl Environ Microbiol 63:3724–3728
Kerr A, Hodgkiess T, Cowling MJ, Smith MJ, Beveridge CM (1999) Effect of galvanically
induced surface potentials on marine fouling. Lett Appl Microbiol 29:56–60
Kilb B, Lange B, Schaule G, Wingender J, Flemming HC (2003) Contamination of drinking water
by coliforms from biofilms grown on rubber-coated valves. Int J Hyg Environ Health
206(6):563–573
Klahre J, Flemming HC (2000) Monitoring of biofouling in papermill water systems. Water Res
34:3657–3665
Klahre J, Lustenberger M, Flemming HC (1998) Microbial problems in paper production. Part III:
monitoring. Das Papier 52:590–596
Koenig DW (1997) Microbiology of the space shuttle water system. Water Sci Technol
35(11–12):59–64
Koerstgens V, Wingender J, Flemming HC, Borchard W (2001) Influence of calcium-ion concen-
tration on the mechanical properties of a model biofilm of Pseudomonas aeruginosa. Water
Sci Technol 43(6):49–57
LeChevallier MW (1990) Coliform regrowth in drinking water: a review. J Am Water Works
Assoc 92 (11):74–86
LeChevallier MW (1991) Biocides and the current status of biofouling control in water systems.
In: Flemming HC, Geesey GG (eds) Biofouling and biocorrosion in industrial water systems.
Springer, Heidelberg Berlin New York, pp. 113–132
Loeb FI, Neihof RA (1975) Marine conditioning films. In: Baier RE (ed) Applied chemistry at
protein interfaces. Am Chem Soc, Washington, pp. 319–335
Louie JS, Pinnau I, Ciobanu I, Ishida KP, Ng A, Reinhard M (2006): Effects of polyether–polyamide
block copolymer coating on performance and fouling of reverse osmosis membranes. J Membr
Sci 280:762–770
Matsunaga T, Nakayama T, Wake H, Takahashi M, Okochi M, Nakamura N (1998) Prevention of
marine biofoling using a condictive paint electrode. Biotechnol Bioeng 59:374–378
McBain AJ (2001) Do biofilms present a nidus for the evolution of antibacterial resistance?. In:
Gilbert P, Allison D, Brading M, Verran J, Walker J (eds.) Biofilm community interactions:
chance or necessity? BioLine Press, Cardiff, pp. 341–351
Morton LHG, Greenway DLA, Gaylarde CC, Surman SB (1998) Consideration of some implications
of the resistance of biofilms to biocides. Int Biodeterior Biodegradation 41:247–259
Neinhuis C, Barthlott W (1997) Characterization and distribution of water-repellent, self-cleaning
plant surfaces. Ann Bot 79:667–677
Nivens DE, Palmer RJ, White DC (1995) Continuous nondestructive monitoring of microbial
biofilms: a review of analytical techniques. J Ind Microbiol 15:263–276
Reasoner DJ (1988) Drinking water microbiology research in the United States: an overview of
the past decade. Water Sci Technol 20:101–107
Biofilm Control: Conventional and Alternative Approaches 117

Ruseska I, Robbins J, Lashen ES, Costerton JW (1982) Biocide testing against corrosion-causing
oilfield bacteria helps control plugging. Oil Gas J 253–264
Schaule G, Griebe T, Flemming HC (2000) Steps of biofilm sampling and characterization in
biofouling cases. In: Flemming HC, Griebe T, Szewzyk U (eds) Biofilms, investigative methods
and applications. Technomics, Lancaster, PA, pp. 1–21
Schaule G, Rumpf A, Weidlich C, Mangold C, Flemming H-C (2008) Influence of polarization of
electric conductive polymers on bacterial adhesion. Poster at IWA conference on biofilms, Wat
Sci Techn, in press
Schulte S, Wingender J, Flemming H-C (2005) Efficacy of biocides against biofilms. In: Paulus
W (ed.) Directory of microbicides for the protection of materials and processes. Kluwer,
Doordrecht, pp. 90–120
Steinberg PD, de Nys R, Kjelleberg S (1997) Chemical defenses of seaweeds against microbial
colonization. Biodegradation 8:211–220
Tamachkiarow A, Flemming HC (2003) On-line monitoring of biofilm formation in a brewery
water pipeline system with a fibre optical device (FOS). Water Sci Technol 47(5):19–24
Terlezzi A, Conte E, Zupo V, Mazzella L (2000) Biological succession on silicone fouling-release
surfaces: long term exposure tests in the harbour of Ischia, Italy. Biofouling 15:327–342
Tirrschof D (2000) Natural product antifoulants: one perspective on the challenges related to
coatings development. Biofouling 15:119–127
An Example: Biofouling Protection for Marine
Environmental Sensors by Local Chlorination

L. Delauney (*
ü ) and C. Compère

Abstract These days, many marine autonomous environment monitoring networks

are set up in the world. Such systems take advantage of existing superstructures
such as offshore platforms, lightships, piers, breakwaters or are placed on specially
designed buoys or deep sea fix stations. The major goal of these equipments is to
provide in real time reliable measurements without costly frequent maintenance.
These autonomous monitoring systems are affected by a well-known phenomenon
in seawater condition, called biofouling. Consequently, such systems without
efficient biofouling protection are hopeless. This protection must be applied to
the sensors and to the underwater communication equipments based on acoustic
technologies. This paper presents the results obtained in laboratory and at sea, with
various instruments, protected by a localised chlorine generation system. Two other
major protection techniques, wipers and copper shutters, are presented as well.

1 Introduction

Monitoring networks commonly use various sensors such as dissolved oxygen,

turbidity, conductivity, pH or fluorescence units and, for specific matters, some
underwater video systems such as cameras, video equipments and lights. For
surface application the data gathered are generally transmitted in real time via
satellite and for deep sea application data logger or wired networks are involved.
In most cases the monitoring stations are autonomous, especially concerning
the energy needs.
In addition to the numerous environmental monitoring stations used along
continents, some specific measuring stations are deployed for other purposes in
some specific areas where biofouling is very much present.

L. Delauney
Ifremer–In Situ Measurements and Electronics, B.P. 70, 29280, Plouzané, France
e-mail: laurent.delauney@ifremer.fr

Springer Series on Biofilms, doi: 10.1007/7142_2008_9 119

© Springer-Verlag Berlin Heidelberg 2008
120 L. Delauney and C. Compère

For example, systems for the monitoring of polluting wrecks (Marvaldi et al.
2006) are based on autonomous and real-time stations deployed in order to measure
critical data nearby wrecks and to transmit them. These stations are equipped with
conventional seawater physicochemical sensors and with acoustic transducers for
underwater data communication (for an example, see Fig. 1). They are generally
deployed from 15 m depth down to whatever is needed, and for long-term monitor-
ing they are deployed for 1 month up to 6 months during which no maintenance is
possible.
For deep sea research, down to 3,000 m, specialized autonomous stations measure
physicochemical parameters and record pictures and movies. Some areas of interests
are, for example, fumes of hydrothermal sites (Sarrazin et al. 2007). For these appli-
cations the autonomy must be provided up to 1 year. The compactness of these
stations is crucial, since the equipment is deployed by a remotely operating vehicle.
These autonomous monitoring systems are affected by a well-known phenome-
non in seawater condition, called biofouling. The major goal of these equipments
is to provide in real time reliable measurements without costly frequent maintenance.
In deep sea conditions this maintenance is nearly impossible to provide. For coastal
applications it is quite well accepted now, that for economically viable in situ moni-
toring systems, the maintenance must not be performed more frequently than
2 month (Blain et al. 2004). Consequently, such systems without efficient biofouling
protection are hopeless. This protection must be applied to the sensors and to the
underwater communication equipments that are based on acoustic technologies.
Biofouling in seawater, during productive period, can occur very rapidly and
lead to poor data quality in less than 2 weeks. As shown in Figs. 2 and 3, the bio-
fouling species involved differ very much from one location to another (Le Haitre
et al. in press).
Very often, this biofouling gives rise to a continuous shift in the measurements.
Consequently, the measurements can be out of tolerance and the data become
useless. Video systems such as cameras, video equipments and lights are also

Messenger ADCP
rack Electronics
Container
Free use
volume

Energy Junction
Container Box

Deadweight
200 - 500 kg

Fig. 1 Autonomous monitoring station for polluting wreck surveillance (Ifremer – ROSE project)
An Example: Biofouling Protection for Marine Environmental 121

Fig. 2 Fluorometer after 30 days in Helgoland (Germany) during summer

Fig. 3 Transmissometer after 40 days in Throndheim harbour (Norway) during summer

122 L. Delauney and C. Compère

Drift of unprotected fluorometer due to biofouling

30 May June July
29
28
27 Drift starts
26
25
24
23 Unprotected
22 Instrument
Fluorescence intensity

21 Protected
20 Instrument
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65
days

Fig. 4 Drift of an unprotected fluorometer due to biofouling development on the optics

compromised by biofouling. Pictures become blurred or noisy, and lights lose

efficiency since their intensity decreases because of the scattering effect of biofilm
and macro-fouling.
As shown in Fig. 4, after 7 days, owing to biofouling on the sensitive part of the
sensor, a drift can be observed in the measurements produced by a fluorescence
sensor (Delauney and Cowie 2002). This kind of optical sensor is very sensitive to
biofouling since even a very thin biofilm on the optics interferes with the measure-
ment process and gives rise to over-evaluated measurements.

2 Biofouling Protection Methods for Oceanographic Sensors

Biofouling protection for oceanographic sensors is a difficult task requiring speci-

fications driven by three important characteristics:
1. It should not affect the measurement.
2. It should not consume too much energy in order to preserve the initial autonomy
of the autonomous monitoring system.
3. It should be reliable even in aggressive conditions for technological systems
(seawater corrosion, sediments, hydrostatic pressure, etc.).
Consequently, few techniques are actually used, and none of them are based on an anti-
fouling paint because the area to be protected cannot be coated with any opaque substance.
An Example: Biofouling Protection for Marine Environmental 123

Otherwise the measurements taken by the sensor can be completely compromised. We

must know that the goal of a biofouling protection is to limit as best as possible the growth
of biofouling on the sensitive part of the sensor. For every type of sensor, such as optical
sensors (fluorometer, turbidimeter, transmissometer, dissolved oxygen), membrane
sensors (pH, dissolved oxygen) or electrochemical sensors (conductivity), the interface
between the media to measure and the sensitive area of the sensor must remain clear.
Currently, three biofouling protection systems for oceanographic sensors are in
use for operational deployments:
4. Strictly mechanical devices – wipers
5. An “uncontrolled” biocide generation system based on a copper auto-corrosion
mechanism
6. A “controlled” biocide generation system based on a localized seawater electro-
chlorination system
These three techniques are commonly used on oceanographic sensors, each having
both advantages and disadvantages.

2.1 Mechanical In Situ Wiper Systems

Biofouling protection system using wipers are based on a mechanical process that
has to be adapted to the instrument from the early stages of design. Consequently,
such systems can be found on the instruments if the sensor manufacturers have
taken biofouling problem into account. Figure 5 shows such a biofouling protection
system based on wipers on a commercial multiparameter probe (YSI 6600 EDS).

Fig. 5 Multiparameter probe with a mechanical biofouling protection based on wipers (photo:
L. Delauney, Ifremer, France)
124 L. Delauney and C. Compère

Fig. 6 Wiper biofouling protection after 150 days of operation (photo: L. Delauney, Ifremer,
France)

The device consists of two distinct wipers that use three “scrapers”. Two of them
are made of sponge and directly wipe the sensors’ optical interfaces for fluores-
cence and turbidity measurements and a brush with long bristles has been imple-
mented to clean the non optical sensors such as pH and oxygen sensors that are
based on membrane techniques.
This biofouling protection technique is effective as long as the scrapers are in
good condition and as long as the geometry of the sensor head is suitable for this
cleaning process. The problems with this technique are mainly the mechanical
complexity of the system. The water tightness of the wipers’ axles as well as the
short-term robustness of the wiper motion device are major weaknesses. As said
earlier, sensors with a non-flat measurement interface, as shown on top right of Fig. 6,
cannot be protected with this technique. Currently, sensor manufacturers are
searching for new, alternative biofouling protection techniques to simplify their
instruments and consequently to improve their reliability.

2.2 Biofouling Protection by “Uncontrolled” Biocide

Generation: Copper Release

Copper is known for its biocide properties (Manov et al. 2004). As copper corrodes
in seawater, oxidized molecules are released into the water rather than remaining
on the metal surface. Copper interferes with enzymes on cell membranes and
prevents cell division.
An Example: Biofouling Protection for Marine Environmental 125

Copper is toxic at high concentrations, and to achieve this, the principle is to

catch in a “copper cell” a small volume of seawater on the sensor measurement
interface. In this way, the sensor interface will be in contact with a solution having
increasing concentration of Cu2+ ions as long as the cell is closed.
Many manufacturers use this protection technique. Some of them build the sen-
sor head totally in copper and add a wiper system to scrap the optics.
A specific equipment can be found that allows to equip any sensor with a copper
cell system more commonly named a “copper shutter.” A motor drives the mecha-
nism for shutters that open for measurements and close for biofouling protection
over the optical windows. It keeps the sensor very close to the copper system releas-
ing toxic copper, and the closed cell allows darkness, thereby reducing biofouling.
Such protection is not easy to implement on an existing sensor. The copper
screen with the stepper motor needs to be placed on the sensor in a way that the
copper screen includes a small volume of water over the sensor measurement inter-
face. An example of such a system on a Seapoint fluorometer is shown in Fig. 7.
To maximize the effectiveness of the protection, it was necessary to implement a
copper cell and to coat the entire sensor head with copper.
Results obtained with such a system (Delauney and Compère 2006), when the
implementation is made exactly as described earlier, can be satisfactory for long-
term deployment. The optics remained clean during the 3 months of deployment in
coastal area in Brest (FR) and during summer season.
Some results obtained with copper tubing and copper shutter on optical instru-
ments are presented by Manov et al. (2004). They conclude that “copper-based
antifouling systems have shown marked improvement in obtaining long-term dataset
for acquisition of optical measurement.”
However, this method can lead to the following problems:
● Copper corrosion produces copper oxide precipitates, which can interfere with
the measurements.
● Copper corrosion produces bubbles on the copper-coated surfaces, which are
trapped in the copper cell close to the measurement interface. This can interfere

Fig. 7 Biofouling protection with a HOBI Labs copper shutter HydroShutter-HS (HOBI Labs,
http://www.hobilabs.com) (photo and drawing: L. Delauney, Ifremer, France)
126 L. Delauney and C. Compère

with the measurements, especially if the sensor is based on an optical technology.

Bubbles are trapped easily since the system is based on a closed cell.
● When the copper screen is closed, it is of course impossible to take any measure-
ments. The screen must be closed for sufficient time in order to get an effective
protection, but then, it is impossible to take high-frequency measurements. And
actually, with the wide band data transmission systems getting more and more
common, and because of tide duration for which scientists need a good time
resolution, copper shutter can be a limitation.
● The mechanical system involved is quite fragile. It is based on a stepper motor
that cannot tolerate any mechanical obstacles; otherwise, the fragile gear box
system will break. Consequently, the copper screen must be adjusted very pre-
cisely in order to fit sufficiently watertight to the copper cell. Any misplacement
of the copper screen with the copper cell can lower the biofouling protection or
interfere with the mechanism.

2.3 Biofouling Protection by “Controlled” Biocide Generation:

Localized Seawater Electro-Chlorination System

This technique is the adaptation for biofouling protection of in situ oceanographic

sensors, of a largely used technique to protect seawater cooling system for industry
(Satpathy 2006). For our application, only the sensor transducing interface area will
be protected, which explains the term “localized.” Biocide generation is obtained
by seawater electrolysis. With this technique, we can achieve a powerful biocide
generation, hypochlorous acid, which can be concentrated as best as possible, on
the sensor transducing interface area.
This technique has many advantages:
● Biocide generation is controlled. Consequently, the biocide quantity can be
adjusted and on/off periods can be arranged as needed. On/Off periods are useful
in arranging biocide-free periods so as to obtain the measurements in good envi-
ronmental conditions. Moreover, the control of the biocide generation intensity
is very important in order to adapt the biocide generation in function of the bio-
fouling colonization.
● The energy needed for such systems is fully compatible with autonomous
coastal monitoring systems and deep sea autonomous monitoring stations.
● The system is very robust and reliable since no mechanical parts are in motion.
● The system is easily adaptable to existing sensors even for usage at high depth.
● The system can be integrated to the sensors by manufacturers.
As shown in Fig. 8, the system is made of an electrode placed around the sensor
transducing interface area, in this case the optic. This electrode is connected to an
electro-chlorination unit. This unit can be a separate electronic container as shown
in Fig. 8 or can be integrated inside the instrument.
An Example: Biofouling Protection for Marine Environmental 127

Fig. 8 Biofouling protection of a fluorometer by localized seawater electro-chlorination

(©Ifremer) – Protection system under Ifremer licence – NKE, Hennebont (56), France (photo:
Ifremer, France)

This biofouling protection technique has been successfully used for many in situ
coastal monitoring systems (Delauney et al. 2002) even immersed at low depth,
2 or 3 m, where biofouling development is intense (cf. Fig. 4), as well as for
medium-depth (15–100 m) stations (Marvaldi et al. 2006) or even for high-depth
stations down to 2,000 m where biofouling can appear close to hydrothermal vents
(Sarrazin et al. 2007).

3 Localized Electro-Chlorination Biofouling Protection Results

The local chlorination technique was applied on various instrumental technologies,

optic (turbidity, fluorescence, oxygen), electrodes (conductivity) and glass mem-
brane (pH). For every test, in the laboratory or at sea, two sensors were placed
simultaneously, one unprotected and one protected by the local chlorination device.
The measurements were internally recorded or when possible recorded by a laptop
for real time data analysis. When possible, some water was sampled and reference
analysis was done in order to follow the eventual drift of the sensors in real time.

3.1 Determination of Possible Interference

of Electro-Chlorination with the Measurement

Before implementing the system on the instruments, it is necessary to check possible

interfering effects on the measurements. Electrodes in the vicinity of a sensor may
perturb measurement. Consequently, a “Laboratory check” and a specific calibration
128 L. Delauney and C. Compère

is necessary. In the same way, biocide molecules can interfere with membranes or
induce local water property modifications, and this effect must be studied even if it
can be overcome by scheduled chlorination.
All instruments have been tested in the laboratory with standard solutions or
standard analytical methods in order to calibrate the signal of a protected instru-
ment vs. an unprotected one (Delauney and Compère 2006). Depending on the
parameter measured, the following standard methods were used:
● Oxygen: Winkler titration (Aminot and Kerouel 2004)
● Fluorescence: Ifremer Fluorescein protocol (Delauney and Le Guen 2003)
● Conductivity: natural seawater sampling and Reference Guildline salinometer
analysis (Aminot and Kerouel 2004; Fofonoff and Millard 1983)
The laboratory check for interference of chlorine with the measurement consists in
comparing the responses of two instruments, one of them equipped with the local
chlorination device. Two steps are involved. The first one determines the adverse
effect of the local chlorination hardware. This can possibly be overcome by a specific
calibration. The second determines the adverse effect of the chlorine generation,
which can possibly be overcome by a scheduling of the chlorination generation.

3.2 Biofouling Protection Field Test on Conductivity Sensor

Local chlorination protection device was tested on the conductivity instrument at

St. Anne du Portzic, Brest, in France. Two instruments were placed on site, one
protected and one unprotected. The local chlorination scheduler is adjusted to last
for 3 months with no maintenance.
Figure 9 shows the measurement obtained during the field test in St. Anne du
Portzic, Brest, in France. The dark top curve shows measurement from the pro-
tected instrument. The light top curve that then drops shows measurement from the
unprotected instrument. The bottom curve shows the difference between the two
signals. The drift started after 80 days; it remained linear up to the 110th day and
then became exponential until the end (133 days).
The reference measurements obtained from water withdrawn and subjected to
Guildline salinometer conductivity analysis (large dots in Fig. 9) show a slight shift
of the protected instrument (0.5 PSU). This drift is probably due to a stop in the chlo-
rination process after the 100th day due to a lack of energy (failure of battery).
Figure 10 shows the unprotected conductivity sensor (left) and the protected one
(right) after 133 days of deployment. Visually, we can perceive the effectiveness of
the biofouling protection. It is even surprising how the local chlorination system
placed inside the white probe housing has protected the outside.
The local chlorination biofouling protection for the conductivity sensor is
efficient as was clearly shown during St. Anne du Portzic Brest test for a continu-
ous period of 133 days. The drift of the unprotected instrument started after 80
days, in August.
 Hours
0 500 1000 1500 2000 2500 3000 3500
36 9.00

35 8.00
Protected
34 7.00
References
UnProtected
33 6.00

32 5.00

31 4.00

Salinity (PSU)
30 3.00
Difference (PSU)

Immersion in renewed seawater tan

29 2.00
An Example: Biofouling Protection for Marine Environmental

28 Unprotected - 1.00
Protected
27 0.00
Drift starts
26 −1.00
0 20 40 60 80 100 120 140
Days

Fig. 9 Conductivity sensor in situ results (133 days duration), 3 June–16 October 2003, Brest
129
130 L. Delauney and C. Compère

Fig. 10 Conductivity sensor: unprotected (left), protected (right) (photo: Ifremer, France)

3.3 Biofouling Protection Field Test on Optical Oxygen Sensor

The local chlorination protection device was tested on the oxygen instrument at
St. Anne du Portzic, Brest, France. Two instruments were placed on site, one
protected and one unprotected. As previously, the local chlorination scheduler was
adjusted to last for 3 months with no maintenance.
Figure 11 shows the measurement obtained from day 110 to day 140 during the
field test in St. Anne du Portzic, Brest.
The top dark curve shows measurement from the protected instrument. The top
light curve that then drops shows measurement from the unprotected instrument.
The bottom curve shows the difference between the two signals. The drift started
after 127 days. The protected optode signal is very good up to day 160. The
Winkler analysis, which was done until the 160th day, confirms this result.
The local chlorination biofouling protection for the oxygen optode sensor is
efficient as was clearly shown during St. Anne du Portzic Brest test for a continuous
period of 160 days. The drift started after 130 days, in April. The protected sensor
showed a temporary failure from day 160 to day 170.

3.4 Biofouling Protection Field Test on Fluorescence Sensor

A local chlorination protection device was tested on fluorescence measurement

instruments at Millport island, Scotland, for 100 days. Two instruments were placed
on site, one protected and one unprotected. The local chlorination scheduler was
adjusted to last for 3 months with no maintenance.
Figure 12 shows the measurement obtained during a field test at Millport Island.
For this experiment the two fluorometers were immersed at 1.5 m depth on Millport
island (Scotland) in 2004, with the collaboration of Dr P. Cowie (GMTC, UK) during
the European BRIMOM Project. The dark curve shows measurement from the
unprotected instrument. The light curve shows measurement from the protected
 14.0 16.0
13.5
13.0 14.5
12.5 UnProtected optode Protected optode
12.0 13.0
11.5
11.0 11.5
10.5
10.0 10.0
9.5
9.0 8.5
8.5
8.0 7.0
7.5
7.0 5.5
6.5
6.0 Drift starts 4.0
5.5
5.0 Difference btw 2.5
Difference (mg / L)

4.5 the 2 optodes

4.0 1.0
An Example: Biofouling Protection for Marine Environmental

3.5 0 mg/L
3.0 −0.5
2.5

Oxygen concentration (mg / L)

2.0 −2.0
1.5
1.0 −3.5
0.5
0.0 −5.0
110.00 115.00 120.00 125.00 130.00 135.00 140.00 145.00
Duration (days)
Fig. 11 Oxygen optode sensor in situ results (190 days duration), January–July 2004, Brest
131
 132

Drift of unprotected fluorometer due to biofouling

May June July August
30
29
28
27
26 Protection stopped
25
24 during 7 days
23 Unprotected
22 Instrument
21 Protected
20 Instrument
19
18
17
16
15
14
13
12
11
10

Fluorescence intensity
9
8
7
6
5
4
3
2
1
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
days

Fig. 12 Fluorescence sensor in situ results (100 days duration), May–August 2004, Millport Island
L. Delauney and C. Compère
An Example: Biofouling Protection for Marine Environmental 133

instrument. The unprotected fluorometer starts to drift after 7 days of immersion and
keeps drifting during the 80 days of experiment. The protected fluorometer does not
show any drift until day 70. It is very interesting to see that after day 70, the protected
instrument was not protected for 7 days. Consequently, biofouling started and a non-
negligible measurement drift was observed. This incident shows that if the protection
system is not mechanical, but rather chemical, any beginning biofouling will induce
a bias on the measurement which will be difficult to remove.
The local chlorination biofouling protection for the fluorometer sensor was
effective as was clearly shown during the Millport Island test for a continuous
period of 70 days. After 70 days, the small drift observed is mainly due to a chlo-
rinator batteries failure for 7 days.

4 Conclusion

For the last 10 years, oceanographic sensor biofouling protection has improved
quite a lot. Owing to the intense technological development of in situ autonomous
monitoring systems, the biofouling problem for such systems has been a techno-
logical one which needed to be solved. The prohibition of tributyl tin as a biocide,
which was used by some manufacturers to protect their sensors, has pushed
researchers to find alternative methods to protect sensors from biofouling.
Wipers, scrapers, and other mechanical systems are interesting solutions but
very often lead to mechanical failure, resulting in water leakage inside the instru-
ments and, thus, destroying the entire equipment.
Copper shutter scan work quite well but are still complicated to implement, and
the biocide generation is uncontrolled, which can lead to problems if the biocide
formation interferes with the sensor measurements.
Localized electro-chlorination biofouling protection is actually a promising and
an advanced solution for in situ oceanographic sensors, since many successful in
situ results have been obtained and sensor manufacturers can integrate in their
instruments a compact, simple, robust and low energy requiring solution.
This technique has been tested on many oceanographic instruments for coastal
and deep sea monitoring. Very encouraging results have been obtained for the
parameters commonly measured for marine monitoring. Every deployment has
been a success for a duration of up to 160 days. Various types of biofouling, such
as biofilm, algae, and barnacles, have been prevented on different types of instru-
ments and different types of measurement technologies. The system can be adapted
to many kinds of instruments quite easily. The energy requirement is compatible
with autonomous monitoring.
Special care should be taken for some sensitive parameters such as oxygen or
fluorescence. The chlorination period must be scheduled in order to leave free time
intervals to take the measurements. The system is now used by Ifremer for autono-
mous coastal monitoring and allows a reasonable maintenance frequency of 3 months,
with high-quality measurements obtained.
134 L. Delauney and C. Compère

Acknowledgements Ifremer thanks Phil Cowie from UMBS, Millport, Scotland, for taking very
good care of the Fluorometer experiment in Millport.

References

Aminot A, Kerouel R (2004) Hydrologie des écosystèmes marins, paramètres et analyses, mesure
de la concentration en oxygène dissous, Chap. VII. pp 92–118. Publisher: INRA Edition.
F-78026 Versailles France
Blain S, Guillou J, Tréguer P, Woerther P, Delauney L (2004) High frequency monitoring of the
coastal marine environment using the MAREL buoy. J Environ Monit 6:569–575
BRIMOM (biofouling resistant infrastructure for measuring, observing and monitoring), project
no. EVRI-CT-2002-40023
Delauney L, Compère C (2006) Biofouling protection for marine environmental optical sensors.
International conference on recent advances in marine antifouling technology (RAMAT),
Chennai (Madras), India. Proceedings of the International Conference on recent advances in
marine antifouling technology. Allied Publishers Pvt. Ltd
Delauney L, Cowie P (2002) BRIMOM (biofouling resistant infrastructure for measuring, observing
and monitoring) report (project no. EVR1-CT-2002-40023). http://cordis.europa.eu/data/
PROJ_FP5/ACTIONeqDndSESSIONeq112362005919ndDOCeq221ndTBLeqEN_PROJ.htm
Delauney L, LeGuen Y (2003) Calibration procedure for in-situ marine fluorometer. In:
International metrology conference 2003 proceedings, Toulon, France. CD Publisher: Collège
Français de métrologie. 75015 Paris
Delauney L, Lepage V, Festy D (2002) BRIMOM (biofouling resistant infrastructure for measur-
ing, observing and monitoring), project no. EVR1-CT-2002-40023. http://cordis.europa.eu/
data/PROJ_FP5/ACTIONeqDndSESSIONeq112362005919ndDOCeq221ndTBLeqEN_
PROJ.htm
Delauney L, Compère C, Lehaitre M (2005) Office for official publications of the european
communities - Luxembourg. Proceeding of the fourth international conference on EuroGOOS.
pp 397–403
Fofonoff NP, Millard RC (1983) Algorithms for computation of fundamental properties of seawater,
UNESCO Technical papers in marine science 44, UNESCO Division of Marine Science, Paris,
France, 53 pp
Lehaitre M, Delauney L, Compère C (in press) Oceanographic Methodology series, Real Time
Coastal Observing Systems for Marine Ecosystem Dynamics and Harmful Algal Blooms:
Theory, Instrumentation and Modelling. 2008, ISBN 978-92-3-104042-9. UNESCO Publishing
Manov DV, Chang GC, Dickey TD (2004) Methods for reducing biofouling on moored optical
sensors. J Atmos Ocean Technol 21(6):958–968
Marvaldi J, Coail Y, Legrand J (2006) ROSE, CMM’06 Caractérisation du Milieu Marin, 16–19.
SeatechWeek 2006 - Brest -France
Sarrazin J, Blandin J, Delauney L (2007) TEMPO: a new ecological module for studying deep-sea
community dynamics at hydrothermal vents (IEEE catalogue no. 07 EX). Oceans 2007 IEEE
conference proceedings, Aberdeen, Scotland, 18–21 June 2007
Satpathy KK (2006) Biofouling and its control in power plants cooling water system. International
conference on recent advances in marine antifouling technology (RAMAT), Chennai (Madras),
India. Proceedings of the international conference on recent advances in marine antifouling
technology. Allied Publishers Pvt. Ltd
YSI 6600 EDS (Extended Deployment System) – Clean SweepTM – http://www.ysi.com
Surface Modification Approach to Control
Biofouling

T. Vladkova

Abstract There are three principal approaches to control biofouling: (1) mechanical
detachment of biofoulers if possible; (2) killing or inactivation of biofouling organ-
isms using antibiotics, biocides, cleaning chemicals, etc. and (3) surface modification
turning the substrate material into a low-fouling or non-sticking (non-adhesive) one.
Such modification usually alters the surface chemical composition and morphology,
surface topography and roughness, the hydrophilic/hydrophobic balance, as well as
the surface energy and polarity.
In marine applications especially, current non-toxic biofouling control strategies
are based mainly on the third approach, i.e., on the idea of creating low-fouling or
non-adhesive material surfaces, an approach that includes development of strongly
hydrophilic “water-like” bioinert materials. Strongly hydrophobic low-energy surfaces
are preferable in industrial and marine biofouling control because of their relative
stability in aqueous media and reduced interactions with living cells.
This chapter presents a brief overview of some possibilities for biofouling control
by surface engineering. A number of related ideas will be discussed in this chapter,
including: (1) the use of protein adsorption as a mediator of bioadhesion and biofoul-
ing, (2) physicochemical parameters influencing these phenomena, (3) theoretical
aspects of cell/surface interactions, (4) some popular surface modification techniques,
and (5) examples of successful biofouling control approaches.

1 Introduction

Biofouling may be defined as any non-desirable accumulation and growth of living

matter on material surfaces (see Pasmore 2008). It is a worldwide problem affecting a
multitude of industrial water-based processes, including pulp and paper manufacturing,

T. Vladkova
Department of Polymer Engineering, University for Chemical Technology and Metallurgy,
8 “Kliment Ohridsky” Blvd. 1756, Sofia, Bulgaria
e-mail: tgv@uctm.edu

Springer Series on Biofilms, doi: 10.1007/7142_2008_22 135 135

© Springer-Verlag Berlin Heidelberg 2008
136 T. Vladkova

food processing and packaging, cooling towers, biomaterials production, membrane

technologies, underwater constructions and sensors, ship hulls, fishing farms, heat
exchangers, and water desalination systems. The cumulative cost of biofouling due
to lost production and only partially successful remedial efforts may run into bil-
lions of dollars per year worldwide, which explains the outstanding interest in the
development of effective and economical control measures.
There are three principal approaches to solving the biofouling problem: (1)
mechanical detachment of biofouling organisms and/or adsorbed biomolecules (i.e.,
biofoulers and biofoulants, respectively), (2) inactivating or killing the biofilm using
antibiotics, biocides, cleaning chemicals, etc., and (3) surface modification with the
aim of turning the substrate material into a low- or non-sticking (i.e., non-adhesive)
one. Such modification usually alters the surface chemical composition and morphol-
ogy, surface topography and roughness, the hydrophilic/hydrophobic balance, and the
surface energy and polarity. The most effective antifouling coatings available today
contain toxic biocides and will therefore be banned by the year 2008 (Brady 2003).
Current non-toxic biofouling control is based mainly on the third approach, i.e.,
on creation of low-fouling or non-adhesive material surfaces, an approach first
applied to the development of bioinert materials where strongly hydrophilic “water-like”
surfaces appear more promising. Strongly hydrophobic low-energy surfaces are
preferable in industrial and marine biofouling control settings because of their stability
in aqueous media and reduced interactions with living cells (Abarzua and Jacubowski
1995).
Biofouling is perceived as a multistage process starting with a “conditioning
film” (biofilm) formation in which the adhesion of microfoulers (i.e., bacteria,
diatoms, algae, etc.) to the surface is an important step. The industrial and marine
biofouling usually continues with settlement of soft and hard macrofoulers such as
algae, barnacles, mussels, tubeworms, etc. In principle, it should be possible to
prevent or at least reduce biofilm formation by creating material surfaces to which
bacteria cannot initially attach. In practice, synthetic materials that are capable of
preventing bacterial adsorption are still rather elusive, despite a significant volume
of research (Callow and Fletcher 1994).
Surface modification approaches leading to the creation of low-energy, low-
adhesive, non-sticking surfaces is accepted nowadays as the most promising
ecology-friendly alternative to the use of toxic biocides.

2 Biofouling and Bioadhesion

Upon submersion in a non-sterile aqueous liquid, most surfaces become rapidly colo-
nized by bacteria and other microorganisms. These attached cells and extracellular
polymeric substances (EPS), along with extracellular material (e.g., extracellular
biopolymers) constitute a biofilm (Costerton et al. 1987). Biofilm frequently forms
even on antifouling surfaces containing biocides. According to Brusscher et al.
(1995), initial (reversible) bacterial adhesion to a surface is the primary determinant
Surface Modification Approach to Control Biofouling 137

for biofilm formation. The reversible interactions between a bacterium and a substrate
depend on the physicochemical properties of the bacterial cells and substrate surface,
as well as the medium. On the other hand, bacteria adhering onto a surface usually
secrete a matrix (EPS), in order to cement themselves (irreversible adhesion) to the
surface (Van deVivere and Kirchman 1993; see Smeltzer 2008).
The nature of the bioadhesive interactions in the biofouling process is one of the
main questions of many current researchers because a fundamental understanding of
their molecular mechanisms can guide the creation of material surfaces preventing or
reducing biofouling. Several modes of adhesion bonding, such as chemical and elec-
trostatic interactions, mechanical interlocking, diffusion, etc. are currently known (see
Smeltzer 2008). The adhesion of microorganisms is very complicated because not just
one motif is followed, but rather a combination of modes has been demonstrated. In
addition, the types of adhesion mechanisms that are manifested by a biofilm are largely
dependent upon the species composition and physiology of the biofilm. The specific
surface structures of microorganisms such as pili, cell wall components, and EPS are
also known to influence biofilm formation. Finally, bioadhesion and biofouling can be
strongly dependent on surface hydrodynamic conditions (Casse and Swain 2006).
Ikada et al. (1984) theoretically predicted that the work of adhesion (i.e., the
interfacial surface energy) in aqueous media, W12,w, approaches zero when the water
contact angle, Q, approaches zero or 90°, i.e., there are two possibilities for a mate-

Fig. 1 Work of adsorption of bovine serum albumin (BSA) in water, W12,w against interfacial free
energy between water and polymer, g1w: 1 canine vein; 2 canine artery; 3 poly(vinyl alcohol); 4
cellulose; 5 polyurethane S; 6 polyurethane T; 7 polyurethane E; 8 poly(vinyl chloride); 9 nylon 6,6;
10 poly(methylmethacrylate); 11 poly(ethyleneterephtalate) A; 12 poly(ethy-leneterephtalate B; 13
PVC; 14 nylon 11; 15 poly(styrene); 16 poly-(styrene); 17 poly(trifluoroethylene); 18 paraffin; 19
PTFE; 20 PP; 21 LDPE; 22 MDPE; and 23 HDPE (Ikada et al. 1984)
138 T. Vladkova

rial surface to have W12,w approaching zero. In other words, the two possibilities to
be non-adhesive are (1) to create a super-hydrophilic, i.e., water-like surface (surface
energy, g1w~0) and (2) to create a super-hydrophobic surface (see Fig. 1)
This theoretical prediction, experimentally confirmed by BSA adsorption on
various polymer surfaces in phosphate buffer, represented a point of departure in
the development of strongly hydrophilic or strongly hydrophobic, low-adhesive,
protein-repellent, bioinert materials and non-sticking fouling release surfaces.
Many attempts to explain bioadhesion have been made in terms of the thermody-
namic theory of Derjaguin, Landau, Verwey and Overbeek (DLVO theory) that explains
the stability of lyophobic colloids at solid–liquid interfaces (Derjaguin 1955). In addi-
tion to DLVO forces (i.e., Van der Waals and electrostatic), other types of interactions
(such as hydrophobic interactions in polar media, ion bridging, and steric interactions)
as well as some external cellular appendages are thought to play an important role. The
net effect is a balance between all possible interactions (Oliviera 1997.)
Admittedly, such considerations treat the microorganisms as “living colloids,”
disregarding the specific roles of bacterial structures such as pili and cell wall com-
ponents that play an important role in bioadhesion at the latter stages of biofilm
formation (Morra and Cassinelli 1997). According to a number of authors, these
structural features are of less significance in the initial stages of the attachment
process than the intrinsic thermodynamic factorsinvolved (Van Loosdrecht et al.
1990; An and Friedman 1998; Morra and Cassinelli 1997). The study of Cunliffe
et al. (1999) shows that over the time period of protein adsorption and initial cell
attachment (1–24 h), this assumption is reasonable since the overall pattern of cell
adhesion to different substrates was similar among the various cell types, although
the absolute numbers varied considerably.
A review of bioadhesion-resisting surfaces(Kingshott and Griesser 1999) indicates
that, in spite of the very large amount of studies on polyethylene glycol (PEG) coat-
ings, there is still controversy about what exactly the properties and modes of action
of an “ideal” PEG coating should be. While some studies have reported no irreversible
protein adsorption, other very similar coatings appear less able to resist bioadhesion.
Vladkova et al. (1999) found that not only the amount but also the conformation of
proteins adsorbed on PEG layers is important for the cellular interactions.
The biofilm protects its habitants from predators, dehydration, biocides, and
other environmental extremes while regulating population growth and diversity
through primitive cell signals (see Smeltzer 2008). Dunne (2002) has demonstrated
that from a physiological standpoint, surface-bound bacteria behave quite differ-
ently from their planktonic counterparts.
Recently, some researchers have attempted to replace the relatively crude mac-
roscopic measurements used to describe bacterial adhesion to surfaces (e.g., cell
hydrophobicity via contact angle measurements or water–hexadecane partitioning)
with methods that directly measure cell-surface interaction forces (repulsive and
attractive) using, for example, atomic force microscopy (AFM) to observe homo-
geneity and topography of bacterial surfaces as well as to directly measure adhesion
forces (Velegol and Logan 2004; Li and Logan 2004; Xu and Logan 2005.)
Surface Modification Approach to Control Biofouling 139

Unfortunately, the mechanisms underlying the adhesion of bacteria to the material

surface are still not properly predictable from these theories. Electrostatic and Lifshitz–
van der Waals forces are usually considered responsible for the interactions at
the biomaterial interface. Studying the role of chemical interactions in bacterial adhe-
sion to polymer surfaces, Speranza et al. (2004) found an important role of the acid/
base (Lewis) interaction. Pedry (2005) employed contact angles to relate interfacial
free energies between the interacting surfaces. The thermodynamic approach treats the
bacteria/substrate interaction as an equilibrium process. This investigation indicates
that the more hydrophobic a substrate is, the less of an equilibrium process the interac-
tion becomes. The relationship obtained for the free energy change occurring when
bacteria attach to a surface considers only dispersion forces. But, other forces such as
electrostatic forces probably also contribute to the interaction and the simple model
should include other interactions in order to accurately model bacterial adhesion.
Lately, the concept of the “theta surface” (Baier 2006) has been developed as a
contribution to the need predicted by Larry Hench for a robust “general field”
theory that supports bioengineering solutions for biocompatibility and biofouling
control. For such a theory, there will be no need to know the names or identities of
specific biological substances that will be encountered, since all biological systems
share the same fundamental chemistry and pattern of events.
A “theta surface” defines the characteristic expression of outermost atomic fea-
tures least retentive of depositing proteins, and identified by the bioengineering criterion
of having measured critical surface tension(CST) between 20 and 30 mN m−1.
Material applications requiring strong bioadhesion must avoid this range, whereas
those requiring easy release of accumulating biomass should have “theta surface”
qualities. More than 30 years of empirical observations of the surface behavior of vari-
ous materials in biological settings, when correlated with contact angle-determined
CST for the same materials, support the definition of the “theta surface”. It derives
from the concept of “theta solvents” for macromolecules, that is, suspending liquid
phases that allow large, complicated molecules such as proteins to retain their ther-
modynamically most stable conformations, resisting “denaturation” in 3-dimensional
suspensions. The theta surface is that atomic force expression controlled from solid
surfaces, placed into aqueous biological media, that will least denature glycoproteina-
ceous macromolecules encountering those surfaces. It is the adsorbed configura-
tions, and strengths of binding/retention of biomass to contacting materials under
water, that determine resistance to shear-induced re-entrainment of that matter into
the biological stream. So, maintenance at the interface of near-solution-state confor-
mations of the first arriving macromolecules is the most effective approach to throm-
bus-resistant materials for long-term contact with flowing blood, and to fabrication of
“easy-release” coatingsfor exposure to any other biological system (from sea water to
dairy products, and from water purification units to sewage flow lines). Universal
features of all such systems are the presence of water and glycoproteinaceous macro-
molecules or their refractory remnants (e.g., surface-active humic substances in the
sea) as the dominant “conditioning film,” forming water-displacement agents entropi-
cally favored as the new interfacial occupants.
140 T. Vladkova

Bioadhesion manifests at every stage of biofouling, including the settlement of

macrofoulers such as barnacles, mussels, algae, etc. (see Flemming and Greenhalgh
2008; Nedved and Hadfield 2008; Harder 2008). Marine mussels are experts in
bonding to a variety of solid surfaces in wet, saline and turbulent environments.
The bonding is rapid, permanent, versatile, and protein based. In mussels, adhesive
bonding takes the form of a byssus (a bundle of extracorporal threads) each connected
to living tissues of the animal at one end and secured by an adhesive plaque at the
other. Trying to perform reverse engineering of bioadhesion in marine mussels, Waite
(1999) investigated the composition and formation of byssal plaques and threads with
the hope of discovering technologically relevant innovations in chemistry and materi-
als science. All proteins isolated from the byssus to date share the quality of contain-
ing the amino acid, 3,4-dihydroxyphenylalanine. This residue appears to have a dual
functionality, with significant consequences for adsorption and cohesion. On the one
hand, it forms a diverse array of weaker molecular interactions such as metal chelates,
H-bonds, and pi-cations: these appear to dominate in surface behavior (adsorption).
On the other hand, 3,4-dihydroxyphenylalanine and its redox couple, dopaquinone,
can mediate formation of covalent cross-links among byssal proteins (cohesion).
One of the challenges in making functional biomimetic versions of byssal adhesion
is to understand how these two reactivities are balanced.
Flammang and Jangoux (2004) have studied bioadhesion models of marine
invertebrates relating to biomechanical, morphological, biochemical, and molecular
processes involved in the adhesion. They have found that the adhesion of Cuvenian
tubules, which are specialized adhesive defense organs of some sea cucumber species,
is instantaneous. The results of this investigation suggest that the underwater adhe-
sive is in the form of a low molecular weight precursor protein in the secretory
granules of the adhesive cells. Upon release, these proteins instantly polymerize,
with no enzymatic curing required.
Holm et al. (2006) have found inter-specific variation in patterns of adhesion of
marine fouling, studying the adhesion of six hard fouling organisms (barnacles,
mollusks, and tubeworms) to 12 silicone fouling release surfaces. Removal stress
(adhesion strength) varied among the fouling species and among the surfaces. None
of the silicone materials generated a minimum in removal stress for all the organ-
isms tested. These results suggest that fouling release materials do not rank (in
terms of adhesion strength) identically for all fouling organisms, and thus develop-
ment of a globally effective hull coating will continue to require testing against a
diversity of encrusting species.

3 Physicochemical Parameters Influencing

Bioadhesion and Biofouling

Despite the fact that there is no general theory of bioadhesion, a lot of factors influ-
encing bioadhesion and biofouling are now known that could be used in biofouling
control. Even though the ability to resist protein, glycoprotein, and polysaccharide
adsorption remains imperative for a coating to prevent marine and industrial fouling,
Surface Modification Approach to Control Biofouling 141

it has been suggested that surface free energy, mechanical properties, and wettability
also play an important role in defining the extent to which a surface can resist bio-
fouling and facilitate fouling release (Finlay et al. 2002; Brady and Singler 2000;
Sigal et al. 1998).

3.1 Surface Energy and Related Parameters

Leading theories attempt to correlate the kind and intensity of biological responses
to surface and interfacial energetics (Brusscher et al. 1995). Surface thermody-
namic characteristics, such as hydrophobic/hydrophilic balanceand van der Waals
and donor/acceptor forces, are determined by contact anglemeasurements. The
surface free energy of the substratum is now accepted as one of the main factors
influencing microbial adhesion. Adhesion to surfaces with different surface free
energies has been studied by a large number of research groups. For a homogenous
solid, the critical surface tensiongc is the same as the surface free energy, i.e., sur-
face tension assuming that there are no other forced elastic strains on the solid and
no solvent adsorption (Good 1992).
Baier (1973) and Dexter (1979) were among the first researchers to correlate the
adhesion of fouling organisms with the surface free energy of the substratum. Hamza
et al. (1977) have showed that bacterial adhesion is less on hydrophobic surfaces with
a low surface energyand that they are easier to clean because of weaker binding at the
interface. McGuire and Swartzel (1987) found an optimum surface free energy, of
30–35 nM m−1, at which milk protein adsorption is minimal. There are also research-
ers that have drawn the opposite conclusion that hydrophilic membranes have smaller
biofouling tendency than hydrophobic ones (Pasmore et al. 2001).
Bacteria adhere to almost any surface, despite continuing arguments about the
importance of the physicochemical properties of substratum surfaces, such as
hydrophobicity and charge. Bos et al. (2000) demonstrated that bacteria do not have
a strong preference for adhesion to hydrophobic or hydrophilic surfaces but that the
substratum hydrophobicity is a major determinant of bacterial retention while it
hardly influences bacterial adhesion.
Studying the attachment of bacteria, like Salmonella, etc. to different surfaces
and the influence of their free energy, Sinde and Carballo (2000) found that the
bacterial adherence could not be correlated with surface free energies or contact
angles of bacteria.
In the case of soft fouling species (e.g., Ulva spores) using non-polar, self-
assembled monolayers, it has been shown that adhesion is strongly influenced by
critical surface tension (or “wettability”) (Finlay et al. 2002).
A generalized relationship between surface tension (i.e., the free energy of a
surface, which is commonly referred to as “surface energy”) and the relative
amount of bioadhesion has been established as shown in Fig. 2. This is commonly
known as the “Baier curve” (Anderson et al. 2003). The key feature of this curve is
that the minimum in the relative adhesion, at 22–24 nM m−1, (mJ m−2), does not
occur at the lowest surface energy.
142 T. Vladkova

Fig. 2 The “Baier curve” (Anderson et al. 2003)

Recently, Zhao et al. (2004) investigated the effect of surface free energy on
bacterial adhesion and reported the optimum surface free energy at which the bacte-
rial adhesion force is minimal to be about 20–30 nM m−1.
Schmidt et al. (2004) studyied adhesive and marine biofouling release properties
of coatings containing surface-oriented perfluoroalkyl groups andhave found that
the fouling release properties of the low-surface-energy surfaces cannot be evalu-
ated by using only static or advancing contact angle. Contact angle hysteresis
appears to be a direct indication of the liquid or adhesive penetration and to correlate
with marine biofouling resistance.
Dahlström et al. (2004) have shown that the initial surface wettabilityis of
importance in the settlement of macrofouling larvae, such as barnacles, bryozoans
and hydroids in both field and laboratory assays. Studying the settlement on surfaces
with different wettability, they concluded that the wettability might cause a biologi-
cal inhibition by interacting with chemo-receptors when the larva is making surface
contact, or that the inhibition might be of a physicochemical nature and, thus, surface
contact is impeded by repulsive chemical forces.
Holland et al. (2004) have found that fouling release poly(dimethylsiloxane)
(PDMS) coatings accumulate diatom slimes, which are not released even from vessels
operating at high speeds (>30 knots). Fouling diatoms adhere strongly to a hydro-
phobic PDMS surface and this feature maybe contributes to their successful
colonization of the fouling release coatings.
Studying the effect of substratum surface energy and chemistry on attachment
of marine bacteria and algal spores, Ista et al. (2004) and Walker et al. (2005) have
found that a number of macroorganisms, such as algae, exploit unicellular forms for
attachment and colonization of surfaces. Surface coverage by both macro- and
microorganisms depends initially on the ability of single cells to adsorb and adhere
to the attachment substratum.
Meyer et al. (2006) have confirmed that silicone coatings with critical surface
tension(CST) between 20 and 30 nM m−1 more easily release diverse types of
Surface Modification Approach to Control Biofouling 143

biofouling than materials of higher or lower CST. Oils added to these coatings
further selectively diminish the attachment strength of different marine fouling
organisms, without significant modification of the initial CST. They have also
demonstrated some contact angle anomalies indicating that surface-active eluates
from silicone coatings inhibit the adhesive mechanisms of fouling organisms.

3.2 Elastic Modulus

In order to control adhesion of biological organisms to a substrate, some funda-

mental fracture mechanics have to be considered. For prediction of the force
required to break an adhesive from a silicone elastomer substrate, basic fracture
mechanics should be examined. Griffith (1921) formulated the following equa-
tion for the critical stress (sc) required to propagating a crack in a plate for a
uniaxial direction:

s c = EGc / π a(1 − υ 2 ) (1)

where E, Gc, a, and u are the elastic modulus, Griffith’s critical fracture energy per
area, half the crack length, and Poisson’s ratio, respectively. Griffith then applied
this equation to the stress over a set crack area (A = pa2), known as the critical
pull-off force (Pc): (2)

Pc = π EGc a 3 / (1 − υ 2 ) (2)

A few years later, Kendall (1971, 1994), following Griffith’s fracture analysis
to model adhesion of elastomer substrates, derived the critical pull-off force
for thin elastomer film and radius of the disc being smaller than the size of the
elastomer film:
1
Pc = π a 2 (2Gc K / t ) 2 (3)

where: Gc, a, t and K are the critical fracture energy, radius of the contact area,
elastomer thin film thickness, and bulk modulus [K = E/3(1–2)]. These equations
show that there is a proportional relationship between the critical pull-off forceand
(EGc)1/2, which are material properties. In this case, fracture energy is directly
related to the work of adhesion, which is then equal to the critical surface tension
(gc) of the elastomer (Silberzan et al. 1994; Kendall 1994). As a result, the adhesion
correlates with (Egc)1/2. The elastic modulus and surface energy are parameters that
can be engineered with the material.
Thus, from a fracture mechanics study it has been shown that the elastic modulus
is a key factor in bioadhesion and the ability of organisms to release from a surface
(Brady and Singler 2000; Berglin et al. 2003).
Figure 3 demonstrates that the adhesion correlates better with (Egc)1/2 than
with either surface energy or elastic modulus on their own, despite some scatter
in the data.
144 T. Vladkova

Fig. 3 Relative adhesion as a function of the square root of the product of critical surface free
energy (gc) and the elastic modulus (E) (Brady and Singler 2000)

For this reason, siloxane elastomers are the major commercial candidates for
environmentally benign fouling release coatings, as they possess both low modu-
lus and low surface energy (Wynne et al. 2000). Commercial antifouling silicone
elastomers such as RTV11 or Intersleek have modules in the 3–1.4 MPa range
(Arce et al. 2003).

3.3 Thickness of Coating

Thickness is another characteristic of low surface energy coatings that plays an

important role in bioadhesion (Anderson et al. 2003; Sun et al. 2004). It has been
found that below ~100 mm dry film thickness, barnacles can “cut through” to the
underlying coats and thus establish firm adhesion. Above this thickness there is no
marked improvement in fouling release properties.
Determining the elastic modulus of silicon rubber coatings and films by depth-
sensing indentation, Zhili et al. (2004) have observed hard substrate effects when the
indentation displacement is less than 10% of the total coating thickness (of 1 mm).
Chaudhury et al. (2005) confirmed the effect of film thickness and modulus on
the release of adhered spores and sporelings of the green alga Ulva.

3.4 Surface Chemistry

Silicone polymer systems that have generally shown the lowest adhesion of biofou-
lants are characterized by a specific chemical structure that determines their special
behavior and, in particular, their durability and fouling release ability. Siloxane
polymers have a backbone of repeating (–Si–O–) units with saturated organic moie-
ties attached to the two non-backbone valences of the silicon. The Si–O bond is
Surface Modification Approach to Control Biofouling 145

stronger than a C–C bond (451 kJ mol−1 compared to 348 kJ mol−1), which helps to
explain the long-term durability of these compounds under field conditions. With a
length of 1.63 Å, Si–O is longer than the C–C bond in most organic polymers
(Baney and Voigt 1977).
This property presents large bond rotation and thus large chain mobility and
restructuring ability. As a result, the non-polar and polar groups can reorient on the
surface to their most favorable position depending on the environment (Hillborg and
Gedde 1999). PDMS) at its lowest energy state is in the all-trans conformation due
to this arrangement having the most favorable van der Waals interactions where the
methyl groups are separated by four bonds (Mark 1979). This ability for recovery to
its lowest energy state is beneficial when trying to predict the material properties
when exposed to diverse environments.
It has been found that incorporation of low molecular weight silicone polymers
(oils) enhances the fouling release properties of PDMS polymers (Milne 1977;
Stein, et al. 2003).
Krishnan et al. (2006) studied surfaces of novel block copolymers with
amphiphilic side chains for their ability to influence the adhesion of marine organ-
isms. The ability of the amphiphilic surface to undergo an environment-dependent
transformation in surface chemistry when in contact with the EPS is a possible
reason for its antifouling nature.

3.5 Slippage

The fracture mechanic equations assume that the applied force is in the normal direc-
tion to the elastomer surface. However, when the force is applied at an angle, as would
be the case when a ship is moving, there is the additional component of interfacial
slippage that has to be considered. According to Newby et al. (1995), the adhesion of
a viscoelastic adhesive on silicone elastomers is controlled heavily by interfacial
slippage rather than by thermodynamics. When peeling a viscoelastic adhesive, an
extension deformation occurs behind and contraction occurs in front of the moving
crack tip (Newby and Chaudhury 1997). If slippage is allowed to occur by the sub-
strate, then the work required to move the crack tip is lowered, which results in a lower
adhesion strength. The more mobile chains are on the substrate surface, the lower the
friction observed. When force is applied in the normal direction, the release mode is
peeling from nucleated voids within the contact area, but when force is directed at an
angle, failure occurs by a fingering process (like webbed fingers) as described by
Newby et al. (1995) that starts at the edges and moves inward (Kohl and Singler 1999).
These finger adhesion release deformations were also shown to increase in length (i.e.,
amplitude) slightly as the modulus or the thickness of the elastomer coating increased,
and significantly as the rigidity of the adhesive increased (Ghatak et al. 2000).
Friction/lubricity is a factor of biological adhesion. In order to engineer the elas-
tomer matrix with lubricity, various non-functional silicone oil additives have been
introduced into the bulk of the elastomer. Due to surface energetics, the lower surface
146 T. Vladkova

energy silicone oils migrate on the surface creating a lubricious layer (Homma et al.
1999). Oils by their nature are lubricants but this is not the main reason for their effi-
ciency. This is thought to be due to the surface tension and hydrophobicity changes
that the oils affect during the curing process and after immersion. It has been shown
that fouling release coatings do not rely on leaching of the oils for their fouling release
properties. Both laboratory studies (Truby et al. 2000) and ships’ trials have shown that
performance was maintained for up to 10 years in service (Anderson et al. 2003).

3.6 Surface Roughness and Topography

In addition to surface free energy, elastic modulus, and surface chemistry, other factors,
including surface roughness and topography, also significantly influence bacterial
adhesion. Therefore the mechanism is very complex. Surface roughness influences
the spreading of liquid cements secreted by organisms to increase adhesion on engi-
neered topography. With the fracture mechanisms discussed previously, the surfaces
were assumed to be completely smooth. However, even the smoothest substrate has
molecular roughness on the surface. Depending on the viscosity of the liquid, the
adhesive might not fill all the small crevices (Baier et al. 1968). When solidification
of the adhesive occurs, there are stress concentrations that occur at the focal points of
the roughness. The stress at these focal points is much higher than the applied force,
so less applied force is needed to fracture the adhesion. If the voids are relatively close
together then the fracture crack can propagate even more easily. The unfilled crevices
can be on the molecular level or micron scale depending on the size of the organism.
The trends observed from many studies are that as the roughness increased, the
advancing angle increased and the receding angle decreased. This means that when
static conditions are examined, as the roughness increases, the contact angle
increases and thus the critical surface tension calculated increases. However, this
statement does not consider the size, shape, and the exact location of the droplet
edge in reference to the rough features, but this roughness influences the spreading
of liquid adhesives. Many reports on the cellular responses to topographical cues
on both the nanometer and micrometer scales have appeared in the past few dec-
ades. However, it has been argued by a number of authors that these structural
features are of less significance in the initial stages of the attachment process than
the intrinsic thermodynamic factors involved (An and Friedman 1998; Morra and
Cassinelli 1997), and a number of detailed studies have been carried out to support
this assertion (Van Loosdrecht et al. 1990).
In the area of marine fouling, topography has been shown to alter settlement
of bacteria(Scheuerman et al. 1998), barnacles(Berntsson et al. 2000; Berntsson
2001; Berntsson and Jonsson 2003) and algae(Callow et al. 2002; Hoipkemeier-
Wilson et al. 2004) and to deter colonization of invertebrate shells(Scardino et al.
2003; Bers and Wahl 2004). The change in wettability of a surfacethat results
from surface roughness, i.e., topography, is likely to be contributing factor to
these responses.
Surface Modification Approach to Control Biofouling 147

Prior to adhesion, the swimming zoospore is able to select suitable surfaces on

the basis of surface characteristics, such as topography, or on the basis of physico-
chemical properties, such as contact angle (Callow et al. 1997, 2000, see Nedved
and Hadfield 2008).
Promising antifouling properties of microstructured surfaces have been reported
by Bohringer (2003) and Bers and Wahl (2004). Hoipkemeier-Wilson et al. (2004)
have studied the settlement and release of Ulva spores from microengineered
topographies.
Influence of nanoscale topography (Griesser et al. 2002) on hydrophobicity (the
contact angles and their hysteresis), including that of fluoro-based polymer thin
films (Gerbig et al. 2005), is reported in the special literature. Brennan et al. (2005)
have patented surface topography for non-toxic bioadhesion control.
Carman et al. (2006) have experimentally demonstrated the importance of wet-
tability models in predicting cellular contact guidance for engineered topographies,
but do not fully explain the process. Bioadhesion is a complex and specific process.
The material modulus and surface elasticity of cell membranes are other factors to
consider, in addition to the variety of adhesive proteins, glycoproteins, and polysac-
charides that organisms secrete. The wettability models are limited by the assumption
that the liquid droplet is much larger than the topographical features. This allows for
line tension effects to be neglected. Measurements with smaller drop sizes are
believed to enable the inclusion of line tension effects. Ultimately, the goal is to
improve the predictive quality of an energy-driven model for bioadhesion.

4 Physicochemical Parameters Influencing the Cell/

Material Surface Interaction

The ability of cells to adhere to each other or to the underlying substrate (called cell
adhesion) is their main property. Biological adhesion is not fully explained by physical
adhesion but is a much more general and complicated phenomenon determined by a
number of interacting biological processes such as cell attachment and mobility, cell
growth and differentiation, etc. (Bitton and Marshall 1980; Adams and Watts 1993).
Analyzing the special literature and studying experimentally the adhesion inter-
action of living cells with different model surfaces, Altankov (2003) has concluded
that the initial interaction of cells with biomaterials is governed by the efficiency of
the cell adhesion, the latter depending mainly on the surface properties of the substrate
and the adsorbed proteins. Hydrophilic surfaces support cell adhesion and prolif-
eration, cell growth, and the organization of the focal adhesion complex delivering
the signal via integrin receptors. An optimum interaction with cells usually appears
at moderate hydrophilicity (WCA ~ 60°). The chemical functional groups oppress
it in the following manner:
–NH2 > –OH > epoxy > –SO3 > –COOH > –CF3
A relationship between the efficiency of the cell interaction and the total negative
charge of the surface exists. This interaction is influenced not only by the chemically
148 T. Vladkova

grafted functional groups but also by the adsorbed ions. The synthesis and organization
of the fibronectin matrix by cells is better on surfaces that weakly bond fibronectin
compared to other matrix proteins. The conformation of the adsorbed adhesive
proteins also plays an important role in the adhesive interaction of strongly
hydrophilic non-charged PEG surfaces (Vladkova et al. 1999).
Properties of the substrate, such as hydrophobicity (Schackenraad et al. 1992),
hydrophilicity (Gölander 1986), steric hindrance (Kuhl et al. 1994), roughness
(Kiaie et al. 1995), and the existence of a “conditioning layer” at the surface
(Abarzua and Jacubowski 1995), are all thought to be important in the initial cell
attachment process.

5 Protein Adsorptionas Mediator of Bioadhesion

and Biofouling

Protein adsorption is the primary event in biofouling and in the interaction of foreign
surfaces with tissue, blood, and cells (Corpe 1970). The biological cascade of indus-
trial and marine biofouling as well as of all undesirable response reactions against
biomaterials begins with deposition of proteins. Therefore, low protein adsorption is
accepted now as the most important prerequisite for resistance against biofouling.

Fig. 4 The versatile nature of proteins (Hlady et al. 1985)

Surface Modification Approach to Control Biofouling 149

Because of their versatile nature (Fig. 4), different proteins can be adsorbed by
various mechanisms when presented with a complementary surface, which makes
the prevention of protein adsorption difficult.
Most investigations are devoted to the study of the adsorption of single well-defined
proteins, adsorption from multicomponent systems, or from blood plasma and are
aimed at identification of protein-repellent biomaterial surfaces (Gölander et al.
1986; Gölander 1986; Malmsten 1998; Pasche 2004; Atthoff 2006).
It is known that the protein adsorption and biocontact properties of polymers
depend on surface chemical composition and topography, surface hydrophilic/
hydrophobic balance and charge, the mobility of the surface functional groups, the
thickness and density of the modifying layer and its adhesion to the substrate, etc.
Hence, by changing some of these parameters we can control protein adsorption
(Gölander 1986).
According to Loeb and Neihof (1975), and Baier (1980) the adsorption of
organic molecules leads to formation of a “conditioning film” on a newly immersed
surface, altering the physicochemical properties of this surface and providing a
nutrient source for attachment of microbial flora. The primary mechanism in the
attachment of marine organisms to surfaces involves secretion of protein or glyco-
protein adhesives (Vreeland et al. 1998; Kamino et al. 2000; Stanley et al. 1999).
Therefore, it is no surprise that significant attention has been directed toward devel-
opment of efficient protein-resistant surfaces (Hester et al. 2002; Griesser et al.
2002; Ostuni et al. 2001, 2003; Bohringer 2003; Groll et al. 2004) for marine anti-
fouling (Youngblood et al. 2003) as well as for biomedical applications (Gölander
et al. 1984, 1986; Wagner et al. 2004; Vladkova 1995, 2001).
Identification of the type and amount of proteins adsorbing to the material surface
could provide important information for the rational development of new materials
that can resist biofouling. Adsorption of different organisms by adhesive proteins
undergoing subsequent underwater curing is thought to be a mediator of bioadhe-
sion and biofouling. Some recent investigations have focused on further study of
the curing mechanisms of bioadhesive proteins as well as on the mechanical properties
of bioadhesives such as spore adhesive glycoprotein of the green alga Ulva(Humphrey
et al. 2005; Walker et al. 2005).
Using biomolecules and green alga as probes, comparative evaluations have
been performed of the antifouling and fouling release properties of hyperbranched
fluoropolymer (HBFP)–poly(ethylene glycol) (PEG) composite coatings and
PDMS elastomers. The maximum resistance to protein, lipopolysaccharide, and
Ulva zoospore adhesion, as well as the best zoospore- and sporling-release properties
have been recorded for the HBFP–PEG coating containing 45%wt PEG. This mate-
rial also exhibited better performance than did a standard PDMS coating (Gudipati
et al. 2005).
It is expected that new analytical techniques and direct measurement of interfa-
cial forces between proteins and surfaces will improve understanding of protein/
surface interactions and open new possibilities for the guided design of surfaces
intended to resist bioadhesion.
150 T. Vladkova

6 Protein Repellent Surfaces

6.1 Strongly Hydrophilic Surfaces

Many strongly hydrophilic and hydrophobic surfaces have been developed to

decrease protein adsorption to biomaterials (Elbert and Hubbel 1996). A compara-
tive protein adsorption study of different strongly hydrophilic surfaces, including
positively charged (N-vinylpyrolidon), negatively charged (AA), and non-charged
(PEG) have clearly demonstrated the advantages of non-charged strongly hydrophilic
surfaces (Gölander et al.,1986).
PEGs, which currently represent the “gold standard” of biomaterials, are most
often used in the creation of bioinert material surfaces. The bioinertness of PEG mol-
ecules is utilized also in the prevention of marine biofouling using water-resistant
hybrid co-polymer networks containing PEG segments. Much research is devoted to
study of the protein adsorption resistance mechanisms of different PEG-coated surfaces,
for example surfaces with adsorbed PEG-graft copolymers (Pasche 2004).
The structural similarity of the –CH2CH2O– unit to water and the strong hydro-
gen bonding to the O-atom have been used to rationalize its miscibility with water.
The –CH2– groups are believed to be “caged” by a water network (Bailey and
Koleske 1976), see Fig. 5. Hence, when a foreign moiety approaches a PEG-coated
surface, that moiety behaves as if it was interacting with a hydrated surface and its
adsorption is minimized.

Fig. 5 “Molecular cilia” mechanism on PEG surface with hydrated poly(oxyethylene) chain
(Mori and Nagaoka 1982)
Surface Modification Approach to Control Biofouling 151

Fig. 6 Scheme showing the structural features of PEG layers obtained by different coating

A number of experimental techniques have been used to introduce PEG groups

on different polymer surfaces, such as PE, PVC, PMMA, NR, PDMS, PS, etc., by
wet chemistry or by plasma treatment (Vladkova 1995, 2001; Vladkova et al. 1999;
Harris 1992). Wet chemistry methods include deposition of photopolymer hydrogel
PEG coatings, including a two-step photopolymerization procedure to increase the
surface density of PEG chains (Gölander et al. 1984), grafting, or adsorption of PEG
chains on the substrate surface. Structural features of the PEG layers obtained in this
way are presented schematically in Fig. 6. Concentrating PEG chains through creation
of brush-type surface coatings using mono-functional PEG-acrylates and UV
polymerization has been used to prepare super-hydrophilic (water contact angle
<10°) surfaces with exclusively low (below 0.05 mg m–2) protein adsorption
(Gölander et al. 1984)
PEG-aldehyde (Gölander et al. 1987) and PEG-epoxide grafting or PEG-epoxide/
PEI copolymer (Fig. 7) quasi-irreversible adsorption (Vladkova et al. 1999) at optimal
reaction conditions also leads to the formation of surfaces with very low protein
adsorption of below 0.05 mg m−2 (by ellipsometry). Figure 8 shows a simple sketch
of PEG-aldehyde grafting by Schiff base reaction with surface NH2 groups.
The examples of PEG-coated surfaces described herein are only a small part of
those described in the special literature.
The bioinertness of PEG molecules is utilized in marine biofouling prevention
using water-resistant hybrid co-polymer networks containing PEG segments, oriented
toward the water in aqueous media (Gudipati et al. 2004, 2005). Surface-responsive
materialsfor non-stick coatings are prepared by linking perfluoropolyether
(hexafluoro-propylene oxide oligomer, PFPE), poly(dimethylsiloxane) (PDMS)
152 T. Vladkova

Fig. 7 Grafting of PEG by Schiff base reaction between PEG-CHO and surface-NH2

and PEG segments. Non-stick properties exhibited in air are due to the presence of
PDMS and PFPE at the air–solid interface. Upon exposure to water, this material
becomes non-stick due to migration of PEG segments to the water–solid interface
Russell (2002).

6.2 Protein-Repellent Plasma Films

Plasma treatment in vacuum or at normal pressure, in atmospheres of different gases,

as well as ion- or electron beam, etc., are referred to as “dry” chemistry methods, and
they represent another approach to surface modification aimed at creation of easily
cleaned or non-fouling material surfaces (Ratner et al. 1990; Chan 1993; Sheu et al.
1995; Chan et al. 1996; Vladkova 2001). Comparative studies of plasma-deposited
films indicated that both strongly hydrophobic silicon and strongly hydrophilic PEG
surfaces result in very low protein adsorption, unusually weak complement system
activation, and low cell and platelet adhesion (Kicheva et al. 1992; Vladkova 1995),
which is in agreement with the prediction of Ikada et al. (1984). Similar “dry” chemistry
also offers a possibility to turn the hydrophobic surfaces into hydrophilic surfaces and
the opposite, to combine the stability of the hydrophobic materials in water with the
advantages of the hydrophilic surfaces. For example, PDMS surface modification has
been performed to alter the hydrophilic–hydrophobic balance on the surface and
hence the interaction with living cells (Satriano et al. 2001, 2002; Vladkova et al.
Surface Modification Approach to Control Biofouling 153

Fig. 8 Chemical composition of plasma-deposited polymer films: diaminocyclohexane (DACH),

hydroxiethylmetacrylate (HEMA), hexamethyldisiloxane (HMDS), poly(ethylene oxide) (PEO)

2005). Radio frequency plasma discharge is considered as an important technique in

the creation of protein-repellent surfaces.

7 Low Surface Energy Coatings to Control Biofouling

Considerable attention in recent decades has been focused on the concept for the
creation of non-biocidal, non-toxic coating systems that prevent the attachment of
fouling organisms. The objective for these minimally adhesive “fouling release”
coatings was to create surfaces reducing the adhesion strength of attaching organ-
isms and hence causing their detachment under their own weight as they grow or
154 T. Vladkova

their dislodgement by water movement when a ship moves through the water
(Linder 1992). The initial interest in the development of such types of coatings was
focused on the fluoropolymers, but later it moved to the siloxane elastomers and
their copolymers because of the combination of lower elastic modulus with low
surface energy.
The most minimally adhesive polymer surfaces known currently are prepared
from siloxanes (Swain and Schultz 1996; Pike et al. 1996; Kohl and Singler 1999;
Uilk et al. 2002), fluoropolymers (Schmidt et al. 1994; Wang et al. 1997; Brady et al.
1999; Bunyard et al. 1999; Gan et al. 2003; Gudipati et al. 2004), and fluorosi-
loxanes (Johston et al. 1999; Mera et al. 1999; Uilk et al. 2002; Grunlan et al. 2004).
Their non-adhesive nature is attributed to low surface energy g, low storage modulus
G, and low glass-transition temperature Tg (Owen 1990; Newby et al. 1995; Brady
1999, 2000; Wynne et al. 2000). Low g values reduce polar and hydrogen-bonding
interactions with the marine organism’s adhesive, thereby decreasing the joint
strength. G is also significant because the rupture of an adhesive bond involves
viscoelastic flow at the coating surface (Kinloch and Young 1983).
Hybrid xerogel films have also been studied as novel coatings for antifouling and
fouling release (Tang et al. 2005). They were found to inhibit settlement of zoospores
of the marine fouling alga Ulva, hyperbranched fluoropolymer poly(ethylene oxide)
Hyperbranched fluoropolymer–poly(ethylene glycol) (HBFP–PEG) composite
coatings have been identified as material exhibiting better antifouling and fouling
release performance than standard PDMS coatings (Gudipati et al. 2005).
Minimally adhesive polymer surfaces (MAPSs) from star oligosiloxanes, star
oligofluorosiloxanes, and α,ω-bis(3-aminopropyl) PDMS have been prepared by
Grunlan et al. (2006). It was found that varying of the molecular weight of the star
oligosiloxanes and star oligofluorosiloxanes, as well as altering the ratio to α,ω-
bis(3-aminopropyl) PDMS, may enhance their fouling release behavior. Minimally
adhesive, fouling release applications of surface-enriched perfluoropolyether
(PFPE) graft terpolymer-based coatings are also in the research focus.
Silicone fouling release coatings, facilitating only weak adhesion of macrofouling
organisms such as barnacles, tubeworms, and macroalgae (Kavanagh et al. 2003;
Stein et al. 2003; Sun et al. 2004) currently represent the only viable commercial
alternative to biocide antifouling coatings.
Milne (1977a,b) was among the first researchers who pointed out the antifouling
properties of silicone polymers and also observed that the low molecular weight
silicone polymers (oils) greatly enhanced their fouling release properties. These
early observations constitute the basis of most silicone fouling release systems
commercially available today. Intersleek (International Coatings) was the first com-
mercial fouling release coating and its manufacturers recently celebrated the cover-
ing of their 100th ship. Hempasil (Hempel Company), EP 2000 and SN-1 HP (E
Paint Company), Eco-speed (Chugoku Marine Paints), Phase Coat URF, Si-Coat
560 and 561, etc. are other practically applicable low-surface-energy coatings.
However, none of them meet all of the desired performance characteristics.
The use of silicone fouling release coatingsis restricted to larger faster moving
vessels. Many lack the toughness to withstand the rigorous physical demands of the
Surface Modification Approach to Control Biofouling 155

marine environment, do not sufficiently self-clean or, due to polymer restructuring or

other degradation pathways, lose many of the desirable surface properties with time
and exposure to the marine environment. Therefore, many research groups are look-
ing for new possibilities for solving these problems. For example, the largest research
project, which includes 31 organizations across Europe (AMBIO 2006), aims to
develop new types of nanostructured fouling-prevention polymeric surface coatings
that mimic natural non-fouling surfaces (e.g., dolphin skin, lotus leaf effect).
More recently, Vladkova et al. (2006) succeeded in developing hard fouling-
preventing silicone coatings based on industrially produced room temperature
vulcanized (RTV) PDMS. Figure 9 demonstrates the biofouling of such sample
coatings after 1-year exposure in the Indian Ocean (at the Fishing Harbour,
Chennai) where the water salinity, temperature, and concentration of fouling organ-
isms are very high and thus the biofouling is very heavy. No macrofouling, only
slime formed mainly by brown diatoms, is observed on these samples exposed in
static conditions. Engineered coatings such as these that disallow development of
macrofouling under static conditions are suitable for macrofouling prevention
of the hulls of slowly moving ships and static underwater constructions.

Fig. 9 Fouling of silicone samples after 1-year exposure in the Indian Ocean, Chennai Fishing
Harbour
156 T. Vladkova

8 Conclusions

A general theory of bioadhesion remains illusive and the molecular details underly-
ing the adhesion mechanisms of fouling organisms remain unclear.
Many physicochemical factors influencing bioadhesion and biofouling are
unknown or remain nebulous, such as surface free energy and related parameters,
water contact angle and contact angle hysteresis, elastic modulus, surface chemis-
try, surface roughness and topography, and biological response, etc. Creation of a
“theta-surface” represents a new and fundamental antifouling concept, but it is still
at the initial step of development.
Surface patterningseems to be a very promising anti-biofouling strategy but
from a practical standpoint significant questions remain, such as ablation by water,
abrasion by sand particles, etc., under actual field applications. Recently developed
polymeric materials that resist such aggressive environmental impacts may eventually
make surface-patterning approaches pragmatic, but currently they are too expensive
to be commercially viable alternatives.
The creation of exceedingly smooth surfacesmay be a more realistic approach to
decreased biofouling. Manipulating the surface topography to enhance smoothness
and engineering local surface hydrodynamics should contribute to this approach.
The known fouling release coatings decrease the adhesion strength of the fouling
organisms but no one surface is known to prevent the formation of biofilm.
In depth study of the adsorption of adhesive proteins secreted by fouling
organisms may be the key to identification of a “universal” surface that would
release all biological fouling systems, since the biological cascade of biofouling
begins with deposition of such proteins. Surface modification to create material
surfaces with suitable composition and morphology would be necessary to
control biofouling.

References

Abarzua S, Jacubowski S (1995) Biotechnological investigation for the prevention of biofouling 1.

Biological and biochemical principles for the prevention of biofouling. Mar Ecol Prog Ser
123:301–312
Adams J, Watts F (1993) Regulation of development and differentiation by the extracellular
matrix. J Invest Dermatol 117:1183–1198
Altankov G (2003) Interaction of cells with biomaterial surfaces. DSc thesis, BAS, Institute of
Biophysics, Sofia
Altankov G, Groth T (1994) Reorganization of substratum bound fibronectin on hydrophilic and
hydrophobic materials is related to biocompatibility. J Mater Sci: Mater Med 5:732–737
AMBIO (2006) Advanced nanostructured surfaces for the control of biofouling. http://www.
ambio.bham.ac.uk/. Last accessed 14 July 2008
An YH, Friedman RJ (1998) Concise review of mechanisms of bacterial adhesion to biomaterial
surfaces. J Biomed Mater Res 43:338–348
Anderson C, Atlar M, Callow M, Candries M, Milne A, Townsin RL (2003) The development of
fouling-release coatings for seagoing vessels. J Marine Design B4:11–23
Surface Modification Approach to Control Biofouling 157

Arce FT, Avci R, Beech IB, Cooksey KE, Wigglesworth-Cooksey B (2003) A comparative study
of RTV11 and intersleek elastomers. J Chem Phys 119:1671–1682
Atthoff B (2006) Tailoring of biomaterials using ionic interactions. Synthesis, characterization and
application, PhD thesis, Uppsala University, Sweden
Baier RE (1973) Influence of the initial surface condition of materials on bioadhesion. In: Acker
RF, Brown BE, DePalma JR, Iverson WP (eds.) Proceedings third international congress on
marine corrosion and fouling. Northwestern University Press, Evanston, IL, pp. 633–639
Baier RE (1980) Substrata influences on the adhesion of microorganisms and their resultant new
surface properties. In: Bitton G, Marshal K (eds.) Adsorption of microorganisms to surfaces.
Wiley, New York, pp. 59–104
Baier RE (2006) Surface behavior of biomaterials: the theta surface for biocompatibility. J Mater
Sci: Mater Med 17:1057–1062
Baier RE, Shafrin EG, Zisman WA (1968) Adhesion: mechanisms that assist or impede it. Science
162:1360–1368
Bailey FE, Koleske JV (1976) Poly(ethylene oxide). Academic, New York
Baney RH, Voight CE, Mentele JW (1977) In: Harris FW, Seymour RB (eds.) Structure-solubility
relationships in polymers. Plenum, New York, pp. 225–232.
Berglin M, Lönn N, Gatenholm P (2003) Coating modulus and barnacle bioadhesion. Biofouling
195:63–69
Berntsson KM (2001) Larval behaviour of the barnacle Balanus improvisus with implications for
recruitment and biofouling control. PhD thesis, Dept. Marine Ecology, Göteborg University
Berntsson KM, Jonsson PR (2003) Temporal and spatial patterns in recruitment and succession of
a temperate marine fouling assemblage: a comparison of static panels and boat hulls during the
boating season. Biofouling 19:187–195
Berntsson KM, Jonsson PR, Lejhall M, Gatenholm P (2000) Analysis of behavioural reaction of
micro textured surfaces and implications for recruitment by the barnacle Balanus improvisus. J
Exp Mar Biol Ecol 251:59–83
Bers V, Wahl M (2004) The influence of natural surface micro topographies on fouling. Biofouling
20(1):43–51
Bitton G, Marshall KC (eds.) (1980) Adsorption of microorganisms to surfaces. Wiley, London
Bohringer KF (2003) Surface modification and modulation in microstructures: controlling protein
adsorption, monolayer desorption and micro-self-assembly. J Microtech Microeng
13:S1–S10
Bos R, van der Mei HC, Gold J, Busscher HJ (2000) Retention of bacteria on a substratum surface
with micro-patterned hydrophobicity. Microbiol Lett 189:311–315
Brady RF (1999) Properties which influence marine fouling resistance in polymers containing sili-
con and fluorine. Prog Org Coat 35:31–35
Brady RF (2000) Clean hulls without poisons: devising and testing nontoxic marine coatings. J
Coat Technol 72:44–56
Brady RF (2003) Antifouling coatings without organotin. J Protect Coat Linings 20(1):33–37
Brady RF, Singler IL (2000) Mechanical factors favoring release from fouling release coatings.
Biofouling 15(1–3):73–81
Brady RF, Bonafede SJ, Schmidt DL (1999) Self-assembled water-born fluoropolymer coatings for
marine fouling resistance. JOCCA-Surf Coat Int 82(12):582–585
Brennan AB, Baney RH, Carman ML, Estes TG, Feinberg AW, Wilson LH, Schumacher JF
(2005) Surface topography for non-toxic bioadhesion control. USA Patent 20060219143
Brusscher HJ, Bos R, van der Mei HC (1995) Initial microbial adhesion is determinant for the
strength of biofilm adhesion. FEMS Microbiol Lett 128:229–234
Bunyard WC, Romack TJ, DeSimone JM (1999) Perfluoropolyether synthesis in liquid carbon
dioxide by hexafluoropropylene photooxidation. Macromolecules 32:8224–8226
Callow ME, Fletcher RL (1994) The influence of low surface energy materials on bioadhesion: a
review. Int Biodeterior Biodegradation 34:333–343
Callow ME, Callow JA (2000) Substratum location and zoospore behavior in the fouling alga
Enteromorpha. Biofouling 15:49–56
158 T. Vladkova

Callow ME, Callow JA (2006) Biofilms. In: Fusetani N, Clare AS (eds.) Antifouling compounds.
Progress in molecular and submolecular biology, vol 32. Springer, Berlin Heidelberg New
York, pp. 141–169
Callow ME, Callow JA, Pickett-Heaps JD, Wetherbee R (1997) Primary adhesion of Enteromorpha
propagules: quantitative settlement studies in video microscopy. J Phycol 33:938–947
Callow ME, Callow JA, Ista LK, Coleman SE, Nolasco AC, Lopez GP (2000) The use of self-
assembled monolayers of different wettabilities to study surface selection and primary adhesion
process of green algal (Enteromorpha) zoospores. Appl Environ Microbiol 66:3249–3254
Callow ME, Jennings AR, Brennan AB, Seegert CE, Gibson A, Wilson L, Feinberg A, Baney R,
Callow JA (2002) Micro topographic cues for settlement of zoospores of the green fouling alga
Enteromorpha. Biofouling 18:229–236
Carman ML, Estes TG, Feinberg AW, Schumacher JF, Wilkerson W, Wilson LH, Callow ME,
Callow JA, Brenan AB (2006) Engineered antifouling microtopographies – correlating wet-
tability with cell attachment. Biofouling, 22:11–21
Casse F, Swain GW (2006) The development of microfouling on four commercial antifouling
coatings under static and dynamic immersion. Int Biodeterior Biodegradation 57:179–185
Chan CM (1993) Polymer surface modification and characterization, Chapters 5–7. HanserGardner,
Brookfield
Chan CM, Ko TM, Hiraoka H (1996) Polymer surface modification by plasmas and photons. Surf
Sci Rep 24(1–2):1–54
Chaudhury MK, Finlay JA, Chung JY, Callow ME, Callow JA (2005) The Influence of elastic
modulus and thickness of the release of the soft-fouling green alga Ulva linza from PDMS
model networks. Biofouling 21(1):41–48
Cooksey KE, Wigglesworth-Cooksey B (1995) Adhesion of bacteria and diatoms to surfaces in
the sea – a review. Aquat Microb Ecol 9:87–96
Corpe WA (1970) Attachment of marine bacteria to solid surfaces. In: Manly S (ed.) Adhesion in
biological systems.Academic, New York, pp. 73–87
Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, Marrie TJ (1987)
Bacterial biofilms in nature and disease. Ann Rev Microbiol 41:435–464
Cunliffe D, Smart CA, Alexander C, Vulfson EN (1999) Bacterial adhesion at synthetic surfaces.
Appl Environ Microbiol 65 (11):4995–5002
Dahlström M, Jonsson H, Jonsson PR, Elwing H (2004) Surface wettability as a determinant in the
settlement of the barnacle Balanus improvisus (DARWIN). J Exp Mar Biol Ecol 305:223–232
Derjaguin BV (1955) Theory of the heterocoagulation, interaction and adhesion of dissimilar
particles in solutions of electrolytes. Discuss Faraday Soc 18:85–86
Dexter SC (1979) Influence of substratum critical surface tension on bacterial adhesion in situ
studies. J Coll Interface Sci 70:346–354
Dunne WM (2002) Bacterial adhesion: seen any good biofilms lately? Clin Microbiol Rev
15:155–166
Elbert DL, Hubbel JA (1996) Surface treatments of polymers for biocompatibility. Annu Rev
Mater Sci 26:365–394
Finlay JA, Callow ME, Ista LK, Lopez GP, Callow JA (2002) The influence of surface wettability
on the adhesion strength of settled spores of the green alga Enteromorpha and the diatom
Amphora. Integ Comp Biol 42:1116—1125
Flammang P, Jangoux M (2004) Bioadhesion models from marine invertebrates: an integrated
study – biomechanical, morphological, biochemical, molecular – of the processes involved in
the adhesion of Cuvierian Tubules on sea cucumbers (Echiodermata, Holothuroidea). Mons-
Hainaut University, Belgium http://www.stormingmedia.us/86/8607/A860724.html. Last
accesses 14 July 2008
Flemming H-C, Greenhalgh M (2008) Concept and consequences of the EU biocide guideline.
Springer Ser Biofilms. doi: 10.1007/7142_2008_12
Gan D, Mueller A, Wooley KL (2003) Amphiphilic and hydrophobic surface patterns generated
from hyberbranched fluoropolymer/linear polymer networks: minimally adhesive coatings via
the crosslinking of s. J Polym Sci Part A: Polym Chem 41:3531–3540
Surface Modification Approach to Control Biofouling 159

Ghatak A, Chaudhury MK, Shenoy V, Sharma A (2000) Meniscus instability in a thin elastic
films. Phys Rev Lett 85:4329–4332
Gerbig YB, Phani AR, Haefke H (2005) Influence of nanoscale topography on the hydrophobicity
of fluoro-based polymer thin films. Appl Surf Sci 242:251–255
Good RG (1992) Contact angle, wetting and adhesion: a critical review. J Adh Sci Techn
6:1269–1302
Gölander C-G (1986) Preparation and properties of functionalised polymer surfaces. PhD thesis,
Royal Institute of Technology, Stockholm
60. Gölander C-G, Jönsson S-E, Vladkova T (1984) A surface coated article, process and means
for the preparation of thereof and use of thereof. Sweden patent no 8404866–9/28.09.1984;
Bulgarian Patent 67997/28.09.1984; European Patent 022966/28.09.84; PCT
SE85/00376/28.09.84
Gölander C-G, Jönsson S-E, Vladkova T, Stenius P, Eriksson J-C (1986) Preparation and protein-
adsorption properties of photo-polymerized hydrophilic films coating N-vinyl pyrollidone
(NVP), acrylic acid (AA) or ethylene oxide (EO) units as studied by ESCA. Coll Surf
21:149–165
Gölander C-G, Jönsson S-E, Vladkova T, Stenius P, Kisch E (1987) Protein adsorption on some
hydrophilic films. Presented at 31st IUPAC, 13–18 July 1987, Sofia
Griffith AA (1921) The phenomena of rupture and flow in solids. Phil Trans R Soc London A
221:163–198
Grinnell F, Milam M, Spree P (1972) Arch Biochem Biophys 153:193
Griesser HJ, Hartley PG, McArthur SL, McLean KM, Meagher L, Thissen H (2002) Interfacial
properties and protein resistance of nano-scale polysaccharide coatings. Smart Mater Struct
11:652–661
Groll J, Amirgoulova EV, Ameringer T, Heyes CD, Röcker C, Nienhaus GU, Möller M (2004)
Biofunctionalized ultrathin coatings of cross-linked star-shaped poly(ethylene oxide) allow
reversible folding of immobilized proteins). J Am Chem Soc 126:4234–4339
Grunlan MA, Lee NS, Gai G, Gedda T, Mabry JM, Mansfeld F, Kus E, Wendt DE, Kowalke GL,
Finley JA, Callow JA, Callow ME, Weber WP (2004) Synthesis of a,w-bis epoxy oligo
(1’H,1’H,2’H, 2’H-perfluoroalkyl siloxane)s and properties of their photo-acid cross-linked
films. Chem Mater 16:2433–2441
Grunlan MA, Lee NS, Mansfeld F (2006) Minimally adhesive polymer surfaces prepared from
star oligosiloxanes and star oligofluorosiloxanes. J Polym Sci Part A: Poly Chem
44:2551–2566
Gudipati CS, Greenlieaf CM, Johnson JA, Pornpimol P, Wooley KL (2004) Hyperbranched fluor-
opolymer and linear PEG based amphiphilic crosslinked networks as efficient anti-fouling
coatings: an insight into the surface compositions, topographies and morphologies. J Polym Sci
Part A: Poly Chem 42:6193–6208
Gudipati CS, Finlay JA, Callow JA, Callow ME, Wooley KL (2005) The antifouling and foulin-
release performance of hyperbranched fluoropolymer (HBFP)-poly(ethylene glycol) (PEG)
composite coatings evaluated by adsorption of biomacromolecules and the green fouling alga
Ulva. Langmuir 21:3044–3053
Hamza A, Pham VA, Matsuura T, Santerre JP (1977) Development of membranes with low sur-
face energy to reduce fouling in ultrafiltration applications. J Membr Sci 131:217–223
Harder T (2008) Marine epibiosis – concepts, ecological consequences and host defense. Springer
Ser Biofilms. doi: 10.1007/7142_2008_16
Harris JM (1992) Poly(ethylene glycol) chemistry. Biotechnical and biomedical applications.
Plenum , New York
Hester JF, Banerjee P, Won YY, Akthakul A, Acar MH, Mayers AM (2002) ATRP of amphiphilic
graft copolymers based on PVDF and their use as membrane additives. Macromolecules
35:7652–7661
Hillborg H, Gedde UW (1999) Hydrophobicity changes in silicone rubbers. IEEE Trans Dielect
Elect Insul 6:703–717
160 T. Vladkova

Hlady V, Van Vagenen RA, Andrade JD (1985) In: Andrade JD (ed.) Surface and interfacial
aspects of biomedical polymers, vol 2. Plenum , New York, p. 81
Holland R, Dugdale TM, Wetherbee R, Brennan AB, Finlay JA, Callow JA, Callow ME (2004)
Adhesion and motility of fouling diatoms on silicone elastomer. Biofouling 20:323–329
Holm ER, Kavanagh CJ, Meyer AE, Wiebe D, Nedved BT, Wendt D, Smith CM, Hadfield MG,
Swain G, Wood CD, Truby K, Stein J, Montemarano J (2006) Interspecific variation in pat-
terns of adhesion of marine fouling to silicone surfaces. Biofouling 22(3–4):233–243
Homma H, Kuroyagi I, Izumi K, Murley CL, Ronzello J, Boggs SA (1999) Diffusion of low
molecular weight siloxane from bulk to surface. IEEE Trans Dielect Elect Insul 6:370–375
Humphrey AJ, Finlay JA, Pettitt ME, Stanley MS, Callow JA (2005) Effect of Ellman’s reagent
and dithiothreitol on the curing of the spore adhesive glycoprotein of the green alga Ulva. J
Adhesion 81:791–803
Ikada Y, Suzuki M, Tamada Y (1984) Polymer surfaces possessing minimal interaction with blood
components. In: Shalaby SW, Hoffman AS, Ratner BD, Horbett TA (eds.) Polymers as bioma-
terials. Plenum, New York.
Ista LK, Callow M, Finlay S, Coleman E, Nolasco AC, Simons RH, Callow JA, Lopez GP (2004)
Effect of substratum surface chemistry and surface energy on attachment of marine bacteria
and algal spores. Appl Environ Microbiol 70(7):4151–4157
Johston E, Bullock S, Uilk J, Gatenhohnm P, Wynne KJ (1999) Networks from a,w-
dihydroxypoly(dimethylsiloxane) and (tridecafluoro-1,1,2,2-tetrahydrooctyl)triethoxysilane:
surface microstructures and surface characterization. Macromolecules 32:8173–8182
Kamino K, Inoue K, Maruyama T, Takamatsu N, Harayama S, Shizuri Y (2000) Barnacle cement
proteins: importance of disulfide bonds in their insolubility. J Biol Chem 275:27360–27365
Kavanagh CJ, Swain GW, Kovach BS (2003) The effect of silicone fluid additives and silicone
matrices on the barnacle adhesion strength. Biofouling 19 (6):381–390
Kendall K (1971) The adhesion and surface energy of elastic solids. J Phys D: Appl Phys
4:1186–1195
Kendall K (1994) Adhesion: molecules and mechanics. Science 263:1720–1725
Kiaie D, Hoffman AS, Horbett TA, Lew KR (1995) Platelet and monoclonal antibody binding to
fibrinogen adsorbed on glow discharge deposited polymers. J Biomed Mat Res 29:729–739
Kicheva J, Kostov V, Mateev M, Vladkova T (1992) Evaluation of the in vitro and in vivo biocom-
patibility of PVC materials with modified surfaces. In: Proceedings VI colloquium on bioma-
terials, Aachen, 24–25 Sept 1992, pp. 24–39
Kingshott P, Griesser HJ (1999) Surfaces that resist bioadhesion. Curr Opin Solid State Mater Sci
4 (4):403–412
Kinloch AJ, Young RJ (1983) Fracture behavior of polymers. Applied Science, London.
Klebe R (1974) Isolation of collagen-dependant cell attachment factor. Natura 250:248–252
Kohl JG, Singler IL (1999) Pull-off behaviour of epoxy bonded to silicone duplex coatings. Prog
Org Coat 36:15–20
Krishnan S, Callow JA, Fischer DA (2006) Anti-fouling properties of comb-like block copolymers
with amphiphilic side chains. Langmuir 22 (11):5075–5086
Kuhl TL, Leckband DE, Lasic DD, Israelachvili JN (1994) Modulation of interaction forces
between bi-layer exposing short-chained ethylene oxide head groups. Biophys J
66:1479–1488
Li X, Logan BE (2004) Analysis of bacterial adhesion using a gradient force analysis and colloid
probe atomic force microscopy. Langmuir 20(20):8817–8822
Linder E (1992) A low surface energy approach in the control of marine biofouling. Biofouling
6:193–205
Loeb GI, Neihof RA (1975) Marine conditioning films. Adv Chem 145:319–335
Malmsten M (1998) Biopolymers at interfaces. Marcel Dekker, New York.
Mark JE (1979) Interpretation of polymer properties in terms of chain conformations and spiral
configurations. Acc Chem Res 12:49–55
McGuire J, Swartzel KR (1987) Proceedings National Meeting American Institute Chemical
Engineers, Minneapolis, p. 31
Surface Modification Approach to Control Biofouling 161

Mera AE, Goodwin M, Pike JK, Wynne KJ (1999) Fluorinated silicone resin fouling release com-
posite. Polymer 40:419
Meyer A, Baier R, Wood CD, Stein J, Truby K, Holm E, Montemarano J, Kavanagh C, Nedved
B, Smith C, Swain G, Wiebe D (2006) Contact angle anomalies indicate that surface-active
eluates from silicone coatings inhibit the adhesive mechanisms of fouling organisms.
Biofouling 22(6):411–423
Milne A (1977a) Coated marine surfaces. UK Patent 1470465
Milne A (1977b) Antifouling marine compositions. US Patent 4025693
Mori Y, Nagaoka S (1982) A new antithrombogenic material with long poly(ethylene) oxide
chains. Trans Am Soc Artif Intern Organs 28:459
Morra M, Cassinelli C (1997) Bacterial adhesion to polymer surfaces: a critical review of surface
thermodynamic approaches. J Biomat Sci Polymer Ed 9:55–74
Nedved BT, Hadfield MG (2008) Hydroides elegans (Annelida: Polychaeta): a model for biofoul-
ing research. Springer Ser Biofilms. doi: 10.1007/7142_2008_15
Newby BZ, Chaudhury MK (1997) Effect of interfacial slippage on viscoelastic adhesion.
Langmuir 13:1805–1809
Newby BZ, Chaudhury MK, Brown HR (1995) Macroscopic evidence of effect of interfacial slip-
page on adhesion. Science 269:1407–1409
Oliviera R (1997) Understanding adhesion: a means for preventing fouling. Exp Thermal Fluid Sci
14:316–322
Ostuni E, Chen CS, Ingber DE (2001) Selective deposition of proteins and cells in arrays of micro-
wells. Langmuir 17:2828–2834
Ostuni E, Grzybowski BA, Mrksich M, Roberts CS, Whitesides GM (2003) Adsorption of proteins
to hydrophobic sites on mixed self-assembled monolayers. Langmuir 19(5):1861–1872
Owen MJ (1990) Silicon surface reactivity. In: Zeigler JM, Fearon FWG (eds.) Silicon-based poly-
mer science: a comprehensive resource. ACS Symposium Series 223. American Chemical
Society, Washington DC, pp. 709–717
Pasche S (2004) Mechanisms of protein resistance of adsorbed PEG-graft copolymers. DSc thesis,
Swiss Federal Institute of Technology, Zurich
Pasmore M (2008) Biofilms in hemodialysis. Springer Ser Biofilms. doi: 10.1007/7142_2008_5
Pasmore M, Todd P, Smith S, Baker D, Silverstein J, Coons D, Bowman CN (2001) Effect of
ultrafiltration membrane surface properties on Pseudomonas aeruginosa biofilm initiation for
the purpose of reducing biofouling. J Membr Sci 194:15–21
Pedry L (2005) Interaction of bacteria with hydrophobic and hydrophilic interfaces. PhD thesis,
Stanford University
Pike JK, Ho T, Wynne KJ (1996) Low surface energy fluorinated poly(amide urethane) block
copolymers and other low surface energy polymers. Chem Mater 8:856–860
Ratner BD, Chilkoti A, Lopez GP (1990) Plasma deposition and treatment for biomedical applica-
tions. In: d’Agustino R (ed.) Plasma deposition, treatment and etching of polymers. Academic,
San Diego, pp. 463–516
Russell TP (2002) Surface responsive materials. Science 279:964–967
Satriano C, Conte E, Marletta G (2001) Surface chemical structure and cell adhesion onto ion
beam modified polysiloxane. Langmuir 17:2243–2250
Satriano C, Carnazza S, Guglielmino S, Marletta G (2002) Differential cultured fibroblast behav-
ior on plasma and ion-beam-modified polysiloxane surfaces. Langmuir 18(24):9469–9475
Scardino A, de Nys R, Ison O, O’Connor W, Steinberg P (2003) Microtopography and antifouling
properties on the shell surface of the bivalve mollusks Mytilus galloprovincialis and Pictada
imbriticata. Biofouling 19:221–230
Schackenraad JM, Stokroos I, Bartels H, Busscher HJ (1992) Patency of small caliber, superhy-
drophobic E-PTFE vascular grafts: a pilot study in rabbit carotid artery. Cells Mater
2:193–199
Scheuerman TR, Camper AK, Hamilton MA (1998) Effects of substratum topography on bacterial
adhesion. J Coll Interface Sci 208:23–33
162 T. Vladkova

Schmidt DL, Coburn CE, DeKoven BM, Potter GE, Meyers GF, Fischer DA (1994) Water-based
non-stick hydrophobic coatings. Nature 368:39–41
Schmidt DL, Brady RF, Lam K, Schmidt DC, Chaudhury MK (2004) Contact angle hysteresis,
adhesion and marine biofouling. Langmuir 20(7):2830–2836
Sheu MS, Chen JY, Wang LP (1995) Biomaterials surface modification using plasma gas dis-
charge processes. In: Wise DL et-al. (eds.) Encyclopedic handbook of biomaterials and bioen-
gineering. Part A: Materials, vol 1. Marcer Dekker, New York, pp. 865–894
Sigal GB, Mrksich M, Whitesides GM (1998) Effect of surface wettability on the adsorption of
proteins and detergents. J Am Chem Soc 120:3464–3473
Silberzan P, Perutz S, Kramer EJ, Chaudhury MK (1994) Study of the self-adhesion hysteresis of
a siloxane elastomer using the JKR method. Langmuir 10:2466–2470
Sinde E, Carballo J (2000) Attachment of Salmonella and Listeria monocytogenes to stainless
steel, rubber and PTFE: the Influence of the free energy. Food Microbiol 17:439–447
Smeltzer MS (2008) Biofilms and aseptic loosening. Springer Ser Biofilms. doi:
10.1007/7142_2008_1
Speranza G, Gottardi G, Pederzolli C, Lunelli L, Carli E, Lui A, Brugnara M, Anderle M (2004)
Role of chemical interactions in bacterial adhesion to polymer surfaces. Biofouling
25(11):2029–2037
Stanley MS, Callow ME, Callow JA (1999) Monoclonal antibodies to adhesive cell coat glycopro-
teins secreted by zoospores of the green alga Enteromorpha. Planta 210:61–71
Stein J, Truby K, Wood CD (2003) Silicon foul release coatings: effect of interaction of oil and
coating functionalities on the magnitude of macro fouling attachment strengths. Biofouling
195:71–82
Sun Y, Akhremitchev B, Walker GC (2004) Using the adhesive interaction between atomic force
microscopy tips and polymer surfaces to measure the elastic modulus of compliant samples.
Langmuir 20:5837–5845
Swain GWJ, Schultz MP (1996) The testing and evaluation of non-toxic antifouling coatings.
Biofouling 10:187–197
Tang Y, Finlay JA, Kowalke GL (2005) Hybrid xerogel films as novel coatings for antifouling and
fouling release. Biofouling 21(1):59–71
Tidball JG, Albrecht DA (1998) Regulation of apoptosis by cellular interactions with the extracel-
lular matrix. In: Lockshin RA, Zakeri Z, Tilly JL (eds.) When cells die: a comprehensive evalu-
ation of apoptosis and programmed cell death. Wiley-Liss, New York, pp. 411–427
Truby K, Wood C, Stein J, Cella J, Carpenter J (2000) Evaluation of the performance enhancement
of silicone biofouling-release coatings by oil incorporation. Biofouling 15(1–3):141–150
Uilk J, Johnston EE, Bullock S, Wynne KJ (2002) Surface characterization, microstructure and
wetting of networks from a,w-dihydroxy(polydimethylsiloxane) and 1,1,2,2-tetrahydrotride-
cafluoro octyltriethoxysilane. J Macromol Chem Phys 203:1506–1511
Van deVivere P, Kirchman DL (1993) Attachment stimulates exopolysaccharide synthesis by a
bacterium. Appl Environ Microbiol 59:3280–3286
Van Loosdrecht MCM, Lyklemam J, Norde W, Zehnder AJB (1990) Hydrophobic and electro-
static parameters in bacterial adhesion. Aquat Sci 52:103–113
Velegol SB, Logan BE (2004) Correction to: “Contributions of bacterial surface polymers, elec-
trostatics and cell elasticity to shape of AFM force curves”. Langmuir 20:3820
Verwey EJW, Overbeek JTG (1948) Theory of stability of lyophobic colloids. Elsevier,
Amsterdam
Vladkova TG (1995) Modification of polymer surfaces for medical application. Presented at XIII
conference on modification of polymers, Kudowa Zdroj, Poland, 11–15 Sept 1995
Vladkova TG (2001) Some possibilities to polymer surface modification. UCTM, Sofia
Vladkova T, Krasteva N, Kostadinova A, Altankov GP (1999) Preparation of PEG-coated surfaces
and a study for their interaction with living cells. J Biomat Sci 10(6):609–615
Vladkova TG, Keranov Il, Dineff PD, Avramova IA, Altankov GP (2005) Plasma based Ar+ beam
assisted PDMS surface modification. Nucl Instrum Methods Phys Res B 236:552–562
Surface Modification Approach to Control Biofouling 163

Vladkova TG, Dineff PD, Zlatanov I, Katirolyi S, Venkatesan R, Murthy S (2006) Composition
coating for biofouling protection. Bulgarian Patent Appl no 109779; WO 2008/074102 A1
Vreeland V, Waite JH, Epstein L (1998) Polyphenols and oxidases in substratum adhesion by
marine algae and molluscs. J Phycol 34:1–8
Wagner VE, Koberstein JT, Bryers JD (2004) Protein and bacterial adhesion. Biomaterials
25:2247–2263
Waite JH (1999) Reverse engineering of bioadhesion in marine mussels. Ann N Y Acad Sci
18:301–309
Walker GC, Sun Y, Guo S, Finlay JA, Callow ME, Callow JA (2005) Surface mechanical proper-
ties of the spore adhesive of the green alga Ulva. J Adhesion 81:1101–1118
Wang J, Mao GP, Ober CK, Kramer EJ (1997) Liquid crystalline, semifluorinated side group
block copolymers with stable low energy surfaces: synthesis, liquid crystalline structure, and
critical surface tension. Macromolecules 30:1906–1914
Wynne KJ, Swain GW, Fox RB, Bullock S, Uilk J (2000) Two silicone nontoxic fouling release
coatings: hydrosilation cured PDMS and CaCO3 filled ethoxysiloxane cured RTV11.
Biofouling 16:277–288
Xu L-C, Logan BE (2005) Atomic force microscopy colloid probe analysis of interactions
between proteins and surfaces. Environ Sci Technol 39(10):3592–3600
Youngblood JP, Andruzzi L, Ober CK, Hexemer A, Kramer EJ, Callow JA (2003) Coatings based
on side-chain ether-linked poly(ethylene glycol) and fluorocarbon polymers for the control of
marine biofouling. Biofouling 19:91–97
Zhao Q, Wang S, Müller-Steinhagen H (2004) Tailored surface free energy of membrane diffusers
to minimize microbial adhesion. Appl Surf Sci 230:371–378
Zhili L, Brokken-Zijp JCM, de With G (2004) Determination of the elastic moduli of silicone
rubber coatings and films using depth-sensing indentation. Polymer 45:5403–5406
A Strategy To Pursue in Selecting a Natural
Antifoulant: A Perspective

K.E. Cooksey (*
ü ), B. Wigglesworth-Cooksey, and R.A. Long

Abstract With the ban of tributyl tin and its analogs and the fact that copper and
its derivatives are under legislative pressure, we must consider alternatives for the
control of biofouling. So-called fouling-release coatings are one potential solution,
but they do not control microfouling well. Compounds derived from macrobiota
that appear to be fouling-free appear to be suitable molecules for investigation as
components of antifouling coatings. If a molecule is to be active against a variety of
organisms, it is important that it inhibit some universal metabolic process. Ideally,
such a molecule would interfere with the surface sensing process itself and the
events that result from the reception of that signal. Cell signaling in all eukaryotes
is mediated by changes in the internal Ca2+ concentration. Therefore, a molecule
that interferes with Ca-mediated events would be an ideal candidate to inhibit cellu-
lar adhesion and thus fouling. Using an image analysis-directed assay with diatoms
and Ca-fluorophores to detect Ca fluxes, we report how 2-n-pentyl-4-quinolinol,
D-600,and trans, trans-2,4-decadienal influence diatom adhesion and motility.
All three molecules show activity as antifoulants.

1 Rationale

Here we report strategies to select anti-diatom agents since these organisms form a
major portion of the initial fouling biomass (Marszalek et al. 1979; Sieburth 1979;
Wetherbee et al. 1998). Trialkyl tins are no longer available for use in marine antifou-
lant coatings and the days of copper-based paints are numbered (Burgess et al. 2003)
Thus it behooves the marine antifouling coating community to search for alternate
strategies to control the deterioration of marine structures. One such strategy is to use
coatings that have a low surface energy, which allow organisms to settle, but not to
adhere strongly. The rationale for this approach is that poorly adhered organisms can

K.E. Cooksey
Department of Microbiology, Montana State University, Bozeman, MT 59717, USA and
Environmental Biotechnology Consultants, Manhattan, MT 59741, USA
e-mail: umbkc@gemini.msu.montana.edu

Springer Series on Biofilms, doi: 10.1007/7142_2008_11 165 165

© Springer-Verlag Berlin Heidelberg 2008
166 K.E. Cooksey et al.

then be removed by shear forces generated by a vessel when underway. Recent work
(Holm et al. 2006) showed that in a series of coatings, the most efficient in releasing
an invertebrate fouling burden depends on which organism is chosen as the test organ-
ism. Thus it is not likely that a coating with a particular surface energy will control
all types of macrofouling. In any event, it is well known that such coatings do not
control microfouling well. The problem is obviously exacerbated on slow-moving
vessels, stationary marine structures such as buoys, or optical surfaces. For instance,
it has been noted (Holland et al. 2004) that ships treated with a silicone elastomer
surface coating accumulated diatom fouling which was not released, even at 30 knots.
Terlizzi et al. (2000) and Jelic-Mrcelic et al. (2006) came to a similar conclusion, but
the observations from these latter authors were for classical antifouling paints.
Diatoms demonstrate the so-called Baier curve (after Robert E. Baier who first
published this information for Ehrlich ascites cells; Baier 1980). The curve in Fig. 1
indicates that the adhesion of diatoms on surfaces with surface energies of 15–70
dynes cm−1 shows a minimal adhesion value (~25 dynes cm−1), but that value is not
zero and represents about 30% of the maximal adhesion value (35 dynes cm−1)
(Characklis and Cooksey 1983). It is possible that the reason for this adhesive adapt-
ability in diatoms is because they secrete a glycoproteinaceous adhesive, which will
have hydrophilic and hydrophobic domains (Cooksey and Cooksey 1986). In more
recent work using atomic force microscopy (AFM), Arce et al. (2004) showed that the
works of removal of a species of Navicula from freshly-cleaved mica (very hydrophilic)
and Intersleek 425 paint (hydrophobic) were statistically equivalent. In this experiment
the AFM was operated in the bioprobe mode using a single diatom attached to the
cantilever surface of the AFM, so measurements of the adhesive power of the diatom
could be measured directly. Thus it is our opinion that no coating which depends only
on a particular surface energy will be successful in controlling microfouling.
The question then arises whether control of microfouling is really necessary
when classical thought is that invertebrates are the major culprits in producing
hydrodynamic drag on a vessel. We believe that it is not yet a settled question
whether microbes always prepare a surface for invertebrate settlement. In some
cases microbiota stimulate invertebrate larval adhesion, and in other cases, they
inhibit it (Dahms et al. 2004). We will not deal with that topic here since it has been
reviewed recently (Maki and Mitchell 2002; Dahms et al. 2004). However, it has
been shown by Bohlander (1991) that microfouling alone on a ship (US Navy
Frigate, USS Brewton) can cause a considerable fuel penalty. The fuel saving for
the ship at 26 knots was that required to produce the extra 4,500 HP needed for a
fouled ship. Thus there are at least two reasons to prevent microfouling: possibly to
break the settlement succession and to reduce biofilm-generated drag.

2 Approach

There are many marine organisms that appear not to support a surface layer of
microorganisms. Materials extracted from these organisms (both plant and animal)
can be tested to determine if they contain antifouling activity. It is possible to screen
A Strategy To Pursue in Selecting a Natural Antifoulant: A Perspective 167

Fig. 1 Adhesion of A. coffeaeformis on glass surfaces as measured by the chlorophyll content of

the attached cells. The surfaces were treated to modify their surface energy as follows: 1 radio-
frequency discharge cleaned and stored under sterile water; 2, as 1, but stored in air; 3 chloropro-
pyl trichlorosilane; 4 dichloromethyl silane; 5 perfluorinated silane (from Characklis and Cooksey
1983, with permission)

these extracts for the presence of compounds that interfere directly with cellular
adhesion, for processes related to adhesion such as motility, and with fundamental
physiological processes such as energy generation. Another physiological process
well worth consideration is the ability for one cell to communicate with another,
so-called quorum sensing. There is however a drawback to these approaches: often
the compounds responsible for the antifouling activity are available only from the
organism from which they were extracted originally and economic synthesis is
unlikely. The pioneering work of Rittschof and colleagues is a good example of this
168 K.E. Cooksey et al.

(Rittschof et al. 1986). Renillafoulin from Sea Pansies provided insights into the
adhesion process in barnacles, but as yet, there is no means to synthesize this mol-
ecule, nor is there a possibility to harvest this molecule on a grand scale from the
marine environment. Thus it is unlikely that this molecule will be used in coatings
for aircraft carriers and the like.

3 Development of an Assay Capable of Detecting and

Quantifying the Response of Diatoms to Compounds
with Potential Antifouling Properties

Since diatoms form a major (perhaps the major) component of the initial foulingfilm
(Marszalek et al. 1979), it is pertinent to use these organisms as model fouling
microorganisms. In fact they may well be the prime indicator to use as their physiology
is both plant- and animal-like (Webster et al. 1985; Wigglesworth-Cooksey and
Cooksey 1992; Armbrust et al. 2004; Vardi et al. 2006). Almost all of our work has
been with two species of diatoms, both of which can be found on fouled surfaces
in the sea (Callow 1986). At this point it is as well to consider just what we want
an antifoulant molecule to do. Do we wish to kill all cells arriving at a surface and
if so what time scale will we use? Should the active material allow initial adhesion-
but prevent cellular growth and thus colonization of the surface? Is it possible for
the potential antifoulant to prevent initial adhesion, or to allow initial adhesion but
promote cellular removal by engineering the properties of the surface? These ques-
tions must be taken into account in assessing potential activities of candidate mol-
ecules. A bioassay should provide this information but, in practical terms, it is just
not possible to screen compounds for all of these activities at one time in one type
of bioassay. In our assay, diatoms are allowed to attach to a clean microscope slide
cover glass, unattached cells are removed and the number of cells remaining is
determined by their chlorophyll fluorescence (Cooksey 1981). The assay also
allows an assessment of diatom motility (Cooksey and Cooksey 1980). An indica-
tion of the reproducibility of the adhesion assay is shown in Table 1. There are

Table 1 Adhesion of Amphora coffeaeformis to glass: a test of the reproducibility of

the assay
Unattached cell removal Cells remaining/mm2a % Remainingb
Not washed 841 ± 54 (6.4) 95
Washed ×1 824 ± 51 (6.2) 93
Washed ×2 806 ± 35 (4.3) 91
Washed ×3 841 ± 85 (10.1) 95
Modified from Wigglesworth-Cooksey et al. (1999)
a
n = 4 ± SD (coefficient of variation). Figures in brackets show % variation
b
Calculated from the cell concentration in the assay medium and their fluorescence
A Strategy To Pursue in Selecting a Natural Antifoulant: A Perspective 169

many variables in this assay which must be optimized. The level of the cell concentration
used is critical. If this is too large (>105 cells mL−1), the diatom biofilm will slough
from the glass substrate as a cohesive film at very low shear velocities. The experi-
mental surfaces must be reproducibly clean. Glow discharge cleaning is not recom-
mended as it produces a chemically unstable surface which quickly adsorbs
airborne contaminants (see Fig. 1). Various methods of removing poorly attached
cells have been investigated, but the shear produced by dipping the glass substrate
into medium was found to be the least invasive and thus the most reproducible. The
time frame in which the measurements are made is also critical and must be kept
constant as cells condition the surface, i.e., change its surface energy with time due
to the adsorption of materials from the aqueous milieu (Wigglesworth-Cooksey et
al. 1999). The way such a “conditioning film”influences the performance in the
marine environment of a coating with a low surface energy has been reviewed by
Maki and Mitchell (2002). A coating with a second strategy, such as the incorpora-
tion of a molecule which inhibits some essential metabolic process, may be less
susceptible to the influence of a conditioning film.
It can be seen that adhesion and motility (Table 2) are dependent on respiratory-
derived energy, not photosynthesis. This has been confirmed by Smith and
Underwood (1998). Both adhesion and motility are Ca-dependent (Cooksey and
Cooksey 1980; Cooksey 1981). Assays such as this are suitable for laboratory
screening studies and do not necessarily represent the conditions existing on the
hull of a ship that is underway. Adhesion assays using calibrated flumes are expen-
sive, time consuming, and do not facilitate testing of multiple samples simultane-
ously. Findlay et al. (2002) make the point that assays involving only the
measurement of diatom motility are not suitable for testing the efficacy of fouling
release coatings. We are proposing to test the activity of compounds that influence
diatom metabolic activity, so this observation does not apply here.
As we learned more about the adhesion of diatoms to surfaces, we realized that
adhered cells always were motile, at least initially, and loss of motility was usually
a precursor of cell detachment. Thus if we could measure motility quickly, we
could screen more efficiently. We had also noticed that cells that were compromised

Table 2 Influence of selected inhibitors on diatom adhesion and motility

Compound, concentration Motility Adhesion Site of action
DCMU, 2 µM 0a 0 Photosynthesis, PSII
Darkness 0 0 Photosynthesis
CCCP, 1.25 µM −b − All energy generation
Cycloheximide, 3.6 µM NT − Protein synthesis
Tunicamycin, 0.5 µg mL−1 − − Glycoprotein synthesis
Ca in medium reduced from 5 − − Secretion and all
to 0.25 mM signaling processes
DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea, CCCP carbonylcyanide m-chlorophenylhydra-
zone, NT not tested
a
0 indicates no effect
b
− indicates that the compound causes inhibition
170 K.E. Cooksey et al.

physiologically by the inclusion of some test substance in the medium appeared to

move more slowly. Initially, in our research, measurements were made manually
using a video camera, a video recorder, and a TV monitor (Cooksey and Cooksey
1988). Now all experiments are scored using an image analysis computer system
(Cell Trak, Motion Analysis Corp., Santa Rosa, CA). Even though the instrument
is capable of real-time assessments of diatom motility, it is convenient to preserve
all motility data using a video recorder and use the computer to assess the results
at a later time. The equipment allows measurement of speed in micrometers per
second, direction of travel as a compass bearing, turning velocity in degrees per
second, and changes in speed as micrometers per second per second of up to 80
cells in a single field simultaneously. These parameters make it possible to measure
cell behavior in response to a chemical challenge.
Using this equipment we determined that the speed of a diatom over a surface
was not constant, but varied from second to second. This is difficult to see with a
naked eye. Figure 2 shows the speed with time of Amphora coffeaeformis on clean
glass. Furthermore, we have observed cells that are in a medium with a low level
of toxicant “shunt,” i.e., they move backwards and forwards with no significant
change in their overall position. Tunicamycin causes this response at <0.5 µg mL−1
(Cooksey and Cooksey 1986). Others have proposed using diatom motility as an
indicator of toxicity of sediment elutriates (Cohn and McGuire 2000).

Fig. 2 Speed of a single cell of A. coffeaeformiswith time. Note the rhythmic variation in speed over
60 s. The horizontal line represents the average speed of the cell, i.e., 2.9 µm s−1 (±0.5 µm s−1)
A Strategy To Pursue in Selecting a Natural Antifoulant: A Perspective 171

4 Why a Calcium Antagonist Can be a Potential Antifoulant

The use of compounds that interfere with Ca homeostasis as active components

of antifouling coatings has not, to our knowledge been suggested previously
and therefore requires some justification. The impetus for this idea came from
our finding that the initial adhesion of diatoms to surfaces and their subsequent
motility requires a certain level of Ca in the external milieu. The Ca was shown
to act both externally to the cell as well as internally (Cooksey and Cooksey
1980; Cooksey 1981). Intracellular Ca concentration (Cai) regulates most
responses to external signals in all eukaryotes studied (Berridge et al. 1998).
Although initial studies in this field concerned excitable cells, it is now known
that the responses of non-excitable cells are also Ca controlled (e.g., Dolle and
Nultsch 1988 – Chlamydomonas; Andrejaustkast et al. 1985 – higher plants). In
fact Ca has been termed the “life or death signal” (Berridge et al. 1998) because
not only does its intracellular concentration modulate cellular processes such as
movement, it also controls cell death. To carry out these regulatory activities,
the Cai needs to be set within narrow limits. There are two sources of Cai: (a)
Ca that arises from outside the cell and crosses the plasma membrane to reach
the cytoplasm, and (b) Ca that is released from internal stores. These signals
have two functions. They can either activate localized cell processes or they can
involve channels throughout the cell, which in turn gives rise to waves of
increased Cai. Further examples relevant to diatom biology include vesicle
secretion (Webster et al. 1985) and, because of its ability to release bound intra-
cellular Ca, the fact that a chemotactic effector facilitates temporary cell motil-
ity in a Ca environment that is otherwise insufficient to support motility
(Cooksey and Cooksey 1988). It should be mentioned that the increases in Cai
referred to above are of the order of tenfold, i.e., 10–7–10–6 M Ca. Organisms
living in seawater, which is 10 mM Ca, must control Cai levels closely or
become inactive and/or die because elevated Cai is lethal. Major contributors to
this process in this environment are the membrane Ca-ATPases,which export
Ca from the cell. Cells avoid long periods of elevated Cai by delivering the Ca
signals as transient increases in Cai rather than one signal over a long period.
These are seen as Cai oscillations (see Fig. 2). In some cases, cells exhibiting Ca
waves in the proximity of other cells cause those cells also to exhibit Ca waves.
This ensures a tissue-like response. Examples from the diatom literature include
the directed cellular dispersal from a colony (Wigglesworth-Cooksey et al.
1999) and Ca wave propagation in Phaeodactylum tricornutum (Vardi et al.
2006). Thus we can conclude that any compound that interferes with the deli-
cately poised Ca homeostasis in a diatom is likely to reduce its ability to colo-
nize a surface. Such molecules would act as specific antifoulants for diatoms.
Since the role played by Cai in biological systems is universal, Ca homeostasis
antagonists may act generally on all fouling organisms.
172 K.E. Cooksey et al.

5 Some Results

It is possible to gauge changes in internal Ca concentration if diatom cells are

loaded with a fluorophore, the fluorescence of which depends on the intracellular
free Ca2+ concentration. We loaded cells of A. coffeaeformiswith the fluorophore
Ca-Green (Molecular Probes, Eugene, OR). The cells were washed and allowed to
attach to a glass surface, which was then placed in minimal medium containing
0.25 mM Ca2+ (Cooksey and Cooksey 1980) contained in a fluorometer cuvette.
Then, 0.1 M Ca2+ was added incrementally to a final concentration of 5 mM and
changes of cellular fluorescence were recorded with time (Fig. 3). It can be seen
from Figs. 2 and 3 that the changes in motility and Ca2+ fluorescence temporally
coincide, suggesting that these phenomena are linked and that Ca2+ waves, as
described for mammalian cells (Berridge et al. 1998), could be the signal system
controlling diatom motility. Incremental additions of Ca2+ synchronized the cellular
response; otherwise the signal would not have been so strong. From this and from
earlier work (Cooksey and Cooksey 1988) it is clear that signal response in diatoms
is controlled by internal Ca2+ levels, which have to arise from the concentration of
Ca2+ in the external milieu. We have investigated two examples of compounds that

Fig. 3 Fluorescence of synchronized population of A. coffeaeformis loaded with Calcium Green.

Note the oscillation of the fluorescence signal, which can be compared to that of the speed varia-
tion in a single cell (see Fig. 2)
A Strategy To Pursue in Selecting a Natural Antifoulant: A Perspective 173

are known to interfere with Ca homeostasis. The first of these D-600, or gallopamil,
is an anti-hypertensive drug related to the more commonly used verapamil. Its
action in mammalian cells is to prevent Ca2+ uptake via Ca2+ channels. Binding of
3
H-verapamil to plasmamembranes in Chlamydomonas reinhardtii(Dolle and
Nultsch 1988) and other plants (Andrejaustkast et al. 1985) has also been demon-
strated. D-600 also causes apoptosis in adenocarcenoma cells (Fleckenstein 1977).
At a concentration of 25 µM, D-600 prevented adhesion of Amphora and reduced
motility to 0–5% of the population in 2 h.
A second compound is from a natural source, although it is available in synthetic
form commercially (Sigma-Aldrich, St. Louis, MO). Trans, trans-2,4-decadienal
(DD) is produced by planktonic diatoms (Miralto et al. 1999). The compound has
been implicated as a chemical defense molecule as it impairs invertebrate grazing
of planktonic diatoms (Ianora et al. 2004; Romano et al. 2003; Ianora et al. 2006).
Vardi et al (2006) showed that treatment of the planktonic organisms Phaeoda-
ctylum tricornutum and Thalassiosira weissflogii, with DD caused intracellular Ca2+
transients similar to those we have seen with Amphora. Furthermore, DD generated
nitric oxide bursts, which produced a phenomenon akin to programmed cell death
(apoptosis). The same groups have proposed DD as an infochemical implicated in
phytoplankton bloom collapse. Caldwell et al. (2004) found that DD is toxic to the
developmental stages of a range of invertebrate species and microfilament and
microtubule related events are at the center of its activity. Since these same events
are also central to diatom adhesion and motility (Webster et al. 1985), it seemed
reasonable to us that DD could be the cellular dispersal agent (i.e., a negative
chemotactic effector) described previously (Wigglesworth-Cooksey et al.1999).
Although there has been considerable investigation of DD (see references in Vardi
et al. 2006), there are no studies on the use of DD as antifoulant, in spite of the fact
that it is commercially available. It appeared to us that a natural compound that
promotes programmed cell death and interferes with events involving the cytoskel-
etoncould be an ideal candidate for such a purpose. It may prevent the onset of cell
signaling and thus prevent the metabolic actions which take place subsequent to
arrival of a cell on a surface. In a preliminary study, we found that 66 µM DD
caused a loss in motilityin both Amphora and a species of Navicula in 2 h. In 20 h
all cells became permeable to Sytox Green (1 µM, 15 min) showing that their cell
membranes had become compromised (Figs 4a,b). Further work with more organ-
isms, including bacteria, is needed to establish that, most, if not all, cells are indeed
sensitive to this compound.
A natural compound that causes similar effects to DD in both diatoms and bac-
teria is 2-n-pentyl-4-quinolinol(PQ). This was isolated from an Aeromonas sp.
(Long et al. 2003) and has been synthesized (Long et al. 2003; Wratten et al. 1977).
Long et al. (2003) found that PQ was active in reducing growth and respiration in
planktonic bacteria and growth in planktonic diatoms. We have measured the
effects of PQ on three pennate biofilm-forming diatoms, i.e., A. coffeaeformis,
Navicula sp.,and an Auricula sp. (Wigglesworth-Cooksey et al. 2007). This group
of diatoms represent three types of adhesion mechanism: (a) secreted adhesive via
the raphe slit (Amphora, Webster et al. 1985), (b) adhesive secreted through pores in
174 K.E. Cooksey et al.

Fig. 4 Influence of decadienalon the proclivity of an Amphora biofilmto take up the vital stain Sytox
Green. a Control, untreated cells are red from chlorophyll fluorescence. The cell membrane is intact.
b Treated cells fluoresce green/yellow showing that their plasma membranes are compromised

the frustule (Navicula, Chamberlain 1976), and (c) adhesive secreted through a
raphe which is on a keel (Auricula, unpublished observation). In general, PQ inhib-
ited growth, adhesion, and motility in all three organisms, but the results were
species-specific. Whereas PQ prevented initial adhesion in Amphora (ED50 = 120
µM), it did not do so in Navicula. PQ caused cell membrane damage in most (94–
97%) cells (result similar to that depicted in Figs 4a,b for DD). Burgess et al. (2003)
investigated a similar molecule (a quinone, rather than a quinolinol). It showed
activity against barnacle larvae, macro-algal spores and some species of bacteria. It
was not tested against fouling diatoms.
A Strategy To Pursue in Selecting a Natural Antifoulant: A Perspective 175

Table 3 Effect of 100 µM PQ on diatom motility

Diatom Treatment Motile cellsa
Amphora Control 51 ± 3
+ PQ 0
Navicula sp.1 Control 60 ± 13
+ PQ 5±9
Auricula sp. Control 67 ± 1
+ PQ 0
a
Motile cells as a percentage of the population. Cells
moving at less than 0.25 µm s−1 were considered
non-motile

Motility in all three diatoms was reduced to near zero by 100 µM PQ (Table 3).
The difference between the adhesion results for Amphora and Navicula underscore
the need for care when generalizing from results with one organism. In this case
motility was a better indicator of inhibitory activity than the adhesion of the cell to
a surface. Thus it is important to use several organisms in the same type of assay
when screening potential antifoulants.

6 Conclusions and Thoughts for the Future

The motility of a diatom is a sensitive indicator of its metabolic health and can be
used to screen compounds for activity as antifoulants. Because of their animal-like
behavior, it is possible that results obtained with diatoms can be expanded to include
all fouling organisms. Although a fully automated diatom motility assessment is
convenient, purely manual measurements are possible. Compounds isolated from
nature are not likely to accumulate in the environment. Materials with potential for
commercial use should be able to be synthesized economically. Because of this,
compounds of fairly low molecular weight are likely to be of the most use. There are
many laboratories, both industrial and academic, and at least one national program
seeking to produce an environmentally benign antifouling coating. We suggest that
if the search to find new ways to protect structures in the sea is to accelerate and we
believe that it must, a forum for the exchange of ideas is needed.

References

Andrejaustkast E, Hertel R, Marme D (1985) Specific binding of the calcium antagonist[3H] vera-
pamil to membrane fractions from plants. J Biol Chem 260:5411–5414
Armbrust EV (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution and
metabolism. Science 306:79–86
176 K.E. Cooksey et al.

Arce FT, Avci R, Beech IB, Cooksey KE, Wigglesworth-Cooksey B (2004) A live bioprobe for
studying diatom-surface interactions. Biophys J 87:4284–4297
Baier RE (1980) Substrata influences on adhesion of microorganisms and their resultant new
surface properties. In: Bitton G, Marshall KC (eds.) Adsorption of microorganism to surfaces.
Wiley, New York, pp. 59–104
Berridge MJ, Bootman MD, Lipp P (1998) Calcium – a life or death signal. Nature 395:645–648
Bohlander GS (1991) Biofilm effects on drag: measurements on ships. Trans Inst Marine
Engineers (C) 103:135–138
Burgess JG, Boyd KG, Armstrong E, Jiang Z, Yan L, Berggren M, May U, Pisacane T, Adams DR
(2003) The development of a marine natural product-based antifouling paint. Biofouling
10:197–205
Caldwell GS, Bentley MG, Olive PJW (2004) First evidence of sperm motility inhibition by the
diatom aldehyde 2E,4E decadienal. Mar Ecol Progr Series 273:97–108.
Callow ME (1986) Fouling of “in service” ships. Botanica Marina 29:351–357
Chamberlain AHL (1976) Algal settlement and secretionof adhesive materials. In: Sharpley JM,
Kaplan AM (eds.) Proceedings of the third international biodegradation and biodeterioration
symposium, Applied Science, London, pp. 417–432
Characklis WG, Cooksey KE (1983) Biofilms and microbial fouling. Adv Appl Microbiol
29:93–138
Cohn SA, McGuire JR, (2000) Using diatom motility as an indicator of environmental stress:
effects of toxic sediment elutriates. Diatom Res 15:19–29
Cooksey KE (1981) Requirement for calciumin adhesion of a fouling diatomto glass. Appl
Environ Microbiol 41:1378–1382
Cooksey B, Cooksey KE (1980) Calcium is necessary for motility in the diatom Amphora coffe-
aeformis. Plant Physiol 65:129–131
Cooksey KE, Cooksey B (1986) Adhesion of fouling diatomsto surfaces: some biochemistry. In:
Evans LV, Hoagland KD (eds.) Algal biofouling. Elsevier, Amsterdam, pp. 41–53
Cooksey B, Cooksey KE (1988) Chemical signal-response in diatomsof the genus Amphora. J Cell
Biol 91:523–529
Dahms HU, Dobretsov S, Quian PY (2004) The effect of bacterial and diatom biofilms on the
settlement of the bryozoan Bugula neritina. J Exp Mar Biol Ecol 313:191–209
Dolle R, Nultsch W (1988) Specific binding of the calcium blocker[3H] verapamil to membrane
fractions of Chlamydomonas reinhardtii. Arch Microbiol 49:451–456
Findlay JA, Callow ME, Ista LK, Lopez GP, Callow JA (2002) The influence of surface wettabili-
tyon the adhesion strength of settled sporesof the green alga Enteromorpha and the diatom
Amphora. Integr Comp Biol 42:1116–1122
Fleckenstein A (1977) Specific pharmacology of calcium in myocardium, cardiac pacemakers and
vascular smooth muscle. Annu Rev Pharmacol 17:149–166
Holland R, Dugdale TM, Wetherbee R, Brennan AB, Finlay JA, Callow JA, Callow ME (2004)
Adhesion and motility of fouling diatoms on a silicone elastomer. Biofouling 20:323–329
Holm ER, Kavanagh CJ, Meyer AE, Wiebe D, Nedved BT, Wendt D, Smith CM, Hadfield GM,
Swain G, Wood CD, Truby K, Stein J, Montemarano J (2006) Interspecific variation in pat-
terns of adhesion of marine fouling to silicone surfaces. Biofouling 22:233–243
Ianora A, Miralto A, Poulet SA, Carotenuto Y, Buttino I (2004) Aldehyde suppression of copepod
recruitment in blooms of a ubiquitous planktonic diatom. Nature 429:403–407
Ianora A, Boersma M, Casotti R, Fontana A, Harder J, Hoffman F, Pavia H, Potin P, Poulet SA,
Toth G (2006) New trends in marine chemical ecology. Estuaries Coasts 29:531–551
Jelic-Mrcelic G, Sliskovic M, Antolic B (2006) Biofouling communities on test panels coated with
TBTand TBT-free copper-based antifouling paints. Biofouling 22:293–302
Long RA, Qureshi A, Faulkner DJ, Azam F (2003) 2-n-Pentyl-4-quinolinol produced by a marine
Alteromonas sp. and its potential ecological and biogeochemical roles. Appl Environ
Microbiol 69:568–576
Maki JS, Mitchell R (2002) Biofouling in the marine environment. In: Bitton G (ed.) Encyclopedia
of environmental microbiology. Wiley-Interscience, New York, pp. 610–619
A Strategy To Pursue in Selecting a Natural Antifoulant: A Perspective 177

Marszalek DS, Gerchacov SM, Udey LR (1979). Influence of substrate composition on marine
microfouling. Appl Environ Microbiol 38:987–995
Miralto A, Barone G, Romano G, Poulet SA, Ianora A (1999) The insidious effect of diatoms on
copepod reproduction. Nature 402:173–176
Rittschof D, Clare AS, Costlow JD (1986) Barnacle settlement inhibitors from Sea Pansies,
Renilla reniformis. Bull Mar Sci 39:376–382
Romano G, Russo GL, Buttino I, IanoraA, Mirealto A (2003) A marine diatom-derived aldehyde
induces apoptosis in copepod and sea urchin embryos. J Exp Biol 206:3484–3494
Sieburth JMc (1979) Sea microbes. Oxford University Press, New York, p. 489
Smith DJ, Underwood GJC (1998) Exopolymer production by intertidal epipelic diatoms. Limnol
Oceanogr 43:1578–1591
Terlizzi A, Conte E, Zupo V, Mazzella L (2000) Biological succession on silicone fouling-release-
surfaces: long-term exposure tests. Biofouling 15:327–342
Vardi A, Formiggini F, Casotti R, De Martino A, Ribalet F, Miralto A, Bowler C (2006) A stress
surveillance system based on calcium and nitric oxide in marine diatoms. PLOS Biol
4:411–419
Webster DR, Cooksey KE, Rubin RW (1985) An investigation of the involvement of cytoskeletal
structures and secretionin gliding motilityof the marine diatom Amphora coffeaeformis. Cell
Motility 5:103–122
Wetherbee R, Lind JL, Burke J, Quatrano RS (1998) The first kiss: establishment and control of
the initial adhesion of raphid diatoms. J Phycol 23:9–15
Wigglesworth-Cooksey B, Cooksey KE, (1992) Can diatoms sense surfaces? State of our knowl-
edge. Biofouling 5:227–238
Wigglesworth-Cooksey B, van der Mei H, Busscher HJ, Cooksey KE (1999) The influence of
surface chemistry on the control of cellular behavior: studies with a marine diatom and a wet-
tability gradient. Colloids Surf B Biointerfaces 15:71–79
Wigglesworth-Cooksey B, Long RA, Cooksey KE (2007) An antibiotic from the marine environ-
ment with antimicrobial fouling activity. Environ Toxicol 22:275–280
Wratten SJ, Wolfe MS, Anderson RJ, Faulkner DJ (1977) Antibiotic metabolites from a marine
pseudomonad. Antimicrob Agents Chemother 11:411–414
Novel Antifouling Coatings:
A Multiconceptual Approach

D. Rittschof

Abstract The development of novel antifouling and foul release coatings must be
considered in the context of business, government, and academic research. Existing
antifouling technology is based upon the use of broad-spectrum biocides. Foul
release technology is partially developed, has incompletely understood mecha-
nisms and unknown long term fates and effects. Business is structured to register,
generate, deliver, apply, and remove antifouling coatings based upon broad-spec-
trum biocides. Business is weak in biology and study of fates and effects beyond
those required for registration. Government is structured to regulate, respond to,
and support basic research. Government is not proactive or cooperative. Academics
are highly competitive, still relatively isolated from reality, strong in basic research,
and not well versed in business or government. Rapid progress in novel coatings
technology is unlikely in this environment. Business responses to regulations and
awareness of environmental responsibility will drive the process.

1 Introduction

The intent of this discourse is to provide a stationary target to promote conversations

on how best to make substantial progress toward environmentally benign antifouling
and foul release coatings. It is presented from the perspective of a researcher who
has dedicated 25 years to the development of environmentally benign antifouling
coatings. From that perspective, understanding four related topics is central to thinking
and planning new approaches. The four topics are:

D. Rittschof
Duke University Marine Laboratory, Nicholas School of the Environment, 135 Duke Marine
Lab Road, Beaufort, NC 28516, USA
e-mail: ritt@duke.edu

Springer Series on Biofilms, doi: 10.1007/7142_2008_21179 179

© Springer-Verlag Berlin Heidelberg 2008
180 D. Rittschof

1. Fouling, antifouling, and foul release concepts and technology (Costlow and
Tipper 1984; Kavanagh et al. 2003)
2. The role of business models and issues as they relate to antifouling and foul
release coatings(Champ 2000; Rittschof and Parker 2001)
3. The role of government and regulatory agencies and laws in novel coatings
development (Champ 2000; Rittschof 2000; International Maritime Organization
2003)
4. The role of academic research in the process (Clare 1997; Rittschof and Holm
1997; Rittschof and Parker 2001)
I argue that progress requires that all four topics be addressed simultaneously.
However, it is likely that other equally important yet to be conceived topics will be
added to this list.
I will make the argument that progress in developing novel coatings is unlikely
because the existing global, political, business, and research structures inhibit sharing
of expertise and the necessary cooperation between business, government, and
academia. Finally, although I am pessimistic about any hope of rapid progress due to
my perception and growing cynicism of human nature, I will suggest ways to facili-
tate progress in assessing developing environmentally benign solutions to fouling.

2 Fouling

In our context, fouling is the attachment and/or growth of undesirable molecules and
organisms to submerged surfaces. Fouling includes molecules, microbes, and mac-
roorganisms and spans the spectrum from true temporal succession (each subsequent
stage requiring a prior stage) to essentially random events determined by availability
of foulers. The actual process of fouling ranges from passive mechanisms comparable
to dust settling on a surface to highly specific mechanisms that require active behavior
(Fig. 1; Clare et al. 1992; Hadfield 1998). Biological fouling impacts the esthetics,
performance, and economics of stationary and mobile platforms.

3 Antifouling Coatings

Classically, antifouling is any of a large number of control measures that use broad
spectrum biocides to control fouling (Fisher et al. 1984; Preiser et al. 1984).
Antifouling coatings are coatings that control fouling by releasing broad-spectrum
biocides. Antifouling coating technology is closely tied to anticorrosion coating
technology. The two technologies are generally present on hulls in a multilayered
coating system. Development of these systems preceded global awareness of environ-
mental degradation due to build up and impact of biocides. Regulations have been
in general reactive rather than proactive (Brancato et al. 1999; Champ 2000;
International Maritime Organization 2003). Global business models, rules, regulations,
Novel Antifouling Coatings: A Multiconceptual Approach 181

Fig. 1 The spectrum of biological fouling (from Clare et al. 1992). Classically, biofouling was
thought to be an exclusively successional process (a). However, since the early 1990s evidence
has accumulated that supports the availability model (b), especially for the majority of the cosmo-
politan fouling organisms now found in the worlds ports. These foulers have been introduced
through shipping and dominate because they are analogous to terrestrial weeds

and performance expectations are tied to economics and performance of toxic

coatings (Champ 2000; Rittschof 2000).

4 Foul Release Coatings

The concept of developing foul release surfaces, surfaces that are easy to clean because
biological adhesives adhere to them poorly, has been popular for over three decades.
Originally, the concept was based upon fluoropolymers (Bultman et al. 1984) .
182 D. Rittschof

In the last several decades foul release coatings have been based upon silicone
polymers, which are much easier to clean than fluoropolymers (cf Brady 2000;
Stein et al. 2003). The physical/chemical mechanisms resulting in low adhesion of
silicones are incompletely understood. Poor physical properties of foul release
coatings are a major stumbling block. When physical properties are improved, the
ease of cleaning is reduced. Because foul release coatings are based upon coating
technology that is different from antifouling coatings, facilities that can apply either
kind of coating are unlikely at this time.

5 Antifouling–Foul Release Coatings

The notion of coatings that are hybrids of antifouling and foul release technology
is beginning to be a popular one. The basic concept is to deliver antifoulants
through foul release coatings. One popular idea is to improve the physical charac-
teristics of the foul release coating and then to add antifoulants that target perni-
cious foulers that are hard to clean. The overall concept may be based upon
observations that nontoxic silicone coatings containing organotin or other toxic
catalysts and additives such as silicone oils (Kavanagh et al. 2003) alter macrofouling
larval behavior (Afsar et al. 2003), inhibit fouling, and are routinely easy to clean.
Some of these coatings release sufficient toxic species that they may perform as
antifouling coatings for months (Rittschof and Holm 1997; Holm et al. 2005).

6 Business Models

Business approaches are harder to document. This section is based upon my

experience in working with a variety of businesses for over 20 years. The anti-
fouling coating industry has the mission of making money. Products are designed
for specific performance niches within the broader market. For example, the
small pleasure boat antifouling coating niche is for products that can be self-
applied and that are effective for 3–6 months, while the niche for coatings for
large ships is for products that are applied in specialized facilities and are expected
to maintain physical and antifouling properties up to for 10 years. The yacht and
intermediate-sized vessel market is for coatings with expectations that are
intermediate to the extremes.
The antifouling coatings business has three major components:
1. Antifouling additive suppliers, which develop and register biocides
2. Coatings manufacturers, which develop polymer systems, mix, deliver, and
develop protocols for product application
3. Coatings appliers, which apply and remove anticorrosive and antifouling coatings
The technical infrastructure is strong in protection of intellectual property, polymer
chemistry, anticorrosion chemistry, registration of toxic compounds, and customer
support. It is relatively weak in fouling biology and in environmental stewardship.
Novel Antifouling Coatings: A Multiconceptual Approach 183

The business of antifouling coatings is based upon coatings that control fouling
with broad-spectrum biocides. The biocides are delivered either by coatings (resin–
rosin systems) that act as slow release reservoirs from the bulk coating or by toxin
release that is controlled hydrolysis of the polymer (usually acrylates), which
expose and release new toxin over time. The highly effective but environmentally
damaging organotinpolymer films, which were voluntarily removed from global
markets in 2003, were of the ablative (hydrolytic) or self-polishing type. The base
polymers used in commercial antifouling coatings meet a spectrum of important
physical and anticorrosion characteristics (Preiser et al. 1984).
The coatings industry has a history of modifying its products when they are shown to
have unacceptable environmental impacts. More recently, environmental concerns have
resulted in regulatory pressure to reduce the release of copper from antifouling coatings.
The industry response to restriction of use of copper has been to reduce amounts of cop-
perand to supplement the coatings with broad-spectrum organic biocides(Gough 1994;
Liu et al. 1999; Readman 1996; Tolosa et al. 1996; Rittschof 2000).

7 Government

The role of government in regulating antifouling technology is centered on registration

of additives (national governments) and in making and enforcing laws and regulations
(cf International Maritime Organization 2003). Governments are, in general, reactive
and adversarial rather than proactive and cooperative. Even when government is well
informed by stakeholders such as business, scientists, and environmental groups,
political solutions are inadequate, expensive, and slow (cf Champ 2000). For example,
US registration of a new compound for use as an antifouling biocide can take over a
decade and cost over US$10,000,000 (Rittschof 2000).
However, governments also have a dramatic positive impact on fouling control
technology. Due to defense and economic and environmental considerations,
governments have a major role in supporting research in antifouling, foul release,
and environmental impacts(cf. Exploratory Research for Advance Technology,
ERATO, Biofouling Project Japan, 1990–1995; US Office of Naval Research
Antifouling program, Alberte et al. 1992; Nordic Council of Ministers, Dahllöf et
al. 2005; AMBIO 2006). Government involvement is central to funding of basic
research; training of government, industry and academic researchers; and development
of assessment techniques and concepts.

8 Academia

Academia and government researchers associated with antifouling are highly com-
petitive, strong in basic research and relatively weak in applied research. Academic
researchers are generally uninterested or uniformed about the workings of business
and government. In general, academics are isolated from business and from government
184 D. Rittschof

agencies charged with regulation. Historically, in the USA and many other developed
countries, academics from upper tier research universities were encouraged to
avoid interests in societally relevant research. More recently, there have been
gradual changes in attitudes in major research universities, associated with changes
in the scope of funding opportunities. Researchers have a role in initiating new
avenues of research. As ideas mature, often over the course of several decades,
potential productive avenues are identified and new research structures are generated
to move the concepts toward products. In the case of antifouling, these changes and
advances are on the horizon

9 Synopsis

Existing antifouling technology is based upon the use of broad-spectrum biocides.

Foul release technology is partially developed, has incompletely understood mech-
anisms and unknown long-term fates and effects. Business is structured to protect
intellectual property and to register, generate, deliver, apply, and remove antifouling
coatings based upon broad spectrum biocides. Business is weak in biology and in
the study of fates and effects beyond those required for registration. Government is
structured to regulate broad spectrum biocides and to enforce laws such as the US
Clean Water Act. Government is not proactive or cooperative. Academics are
highly competitive, still relatively isolated from reality, strong in basic research,
and not well versed in business or government. It is in this context that novel anti-
fouling coatings development should be considered.

10 Goals of Environmentally Benign Antifouling Coatings

Environmentally benign antifouling is a possibility that could be efficiently

approached if one could generate the necessary list of goals and their associated
assumptions and prioritize them. Given the context provided I suggest the following
list of goals and assumptions that all should be met by the first generation of envi-
ronmentally benign coatings:
1. Coating should be compatible with existing business models
2. Coating should use existing polymers, production, delivery, application methods,
and application facilities
3. Compatible with existing anticorrosion systems
4. Function comparably to coatings that are presently on the market
5. Deliver compounds with known minimal environmental impact
To make a long story short, in the context provided, the only solution that fits the
context established is a short-lived broad spectrum biocide. If one looks at the more
recent products on the market one can see that there are two business strategies that
Novel Antifouling Coatings: A Multiconceptual Approach 185

meet all but the assumption of known minimal environmental impact. Both strategies
use organic toxins to enable reduction in the level of copper released by antifouling
coatings:
1. Use of clearly long lived broad spectrum biocides such as Irgarol (Gough 1994; Liu
et al. 1999; Readman 1996; Tolosa and Readman 1996; Tolosa et al. 1996; New York
State Department of Environmental Conservation 1996) and Diuron (PAN 2008)
2. Use of shorter lived broad spectrum biocides, copper pyrithione (Dahllöf et al.
2005), and Sea 9–211 (Willingham and Jacobson 1996). These strategies are
clearly in line with the argument developed that business models can tolerate
only minor changes.
There are two related possibilities for the rationale of replacement of heavy
metal biocideswith long lived organic biocides: (1) Following the classical business
model to the letter by replacing one long lived biocide with another. Perhaps, it is
possible that these businesses could claim ignorance as an excuse for lack of concern
for environmental consequences. (2) Following the classical business model by
replacing one long lived biocide with another with knowledge of consequences, but
without legal, moral, or ethical responsibility for protecting the environment.
Independent of which possibility is correct, the result is the same high potential for
environmental degradation.
Similarly, using short lived broad spectrum biocides to replace long lived biocides,
could be unintentional or it could be a carefully considered decision that reflects a
company with a forward looking business model that includes responsibility for
environmental impacts. In these case there is documentation that the environment
was considered (cf. Callow and Willingham 1996; Galvin 1998; Willingham and
Jacobson 1996; Harrington et al. 2000; Dahllöf et al. 2005). Independent of which
option is correct, the end result is companies positioned to move more quickly away
from the old business model. It is these companies that should have a competitive
edge in a global community that will eventually recognize that business must take
responsibility for the environmental impacts of its products (Rittschof 2000).
In addressing development of novel antifoulant compounds from this perhaps
naive academic point of view, I came to understand one important concept. It is not
the half-life of the toxin that is important, it is whether or not the toxin will build-up
in the environment to deleterious levels. Although this kind of analysis is suffi-
ciently well developed to be standard fare in environmental chemistry textbooks (cf
Schwarzenbach et al. 2003), this kind of analysis is not to my knowledge part of
the regulatory structure. My sense is that business (most easily because they track
customer use and sales and know trade secrets) or academics could develop models
that could be used to predict conditions of use where there would be environmental
impacts. From an environmental perspective this would be a preferred alternative
to reporting dangerous build-ups (Gough 1994; Liu et al. 1999; Readman 1996;
Tolosa and Readman 1996; Tolosa et al. 1996) after the fact. Perhaps this will be
the next step in the process of developing environmental responsibility.
One inevitable conclusion from this intellectual exercise is that in the existing
global regulatory and business and research structure, novel antifouling technology
186 D. Rittschof

will evolve slowly from small changes in existing art. If one asks the question,
“Could the process be accelerated?” the answer is straightforward, but not easily
implemented. That answer is restructure business, government, and academia to
enable these sectors to work cooperatively by sharing expertise in working toward
a common goal (Rittschof and Parker 2001).
Such utopian restructuring is unlikely at the national level, but might be possible
at the level of an international organization such as the European Union or the
United Nations. The EU has a research and development structure, the Advanced
Nanostructured Surfaces for Control of Biofouling (AMBIO) project(AMBIO
2006), which meets many of these objectives. In reality, development of a multi-
functional cooperative structure would be a novel infrastructure that could be
charged with addressing a variety of global problems.

Acknowledgements Thanks to Murthy and Venkat and RAMAT for the stimulation and travel
funds to generate this document. The ideas represented were generated while working on projects
funded by the Office of Naval Research, agencies of the Government of Singapore, and several
chemical and coatings companies. LN polished the manuscript.

References

Afsar A, de Nys R, Steinberg P (2003) The effects of foul-release coatings on the settlement and
behaviour cyprid larvae of the barnacle Balanus amphitrite amphitrite Darwin. Biofouling
19(Suppl):105–110
Alberte RS, Snyder S, Zahuranec B (1992) Biofouling research needs for the United States Navy:
program history and goals. Biofouling 6:91–95
AMBIO (2006) Advanced nanostructured surfaces for the control of biofouling http://www.
ambio.bham.ac.uk/. Last accessed 14 July 2008
Brady RF, Singer IL (2000) Mechanical factors favouring release from fouling release coatings.
Biofouling 15(1–3):73–81
Brancato MS, Toll J, DeForest D, Tear L (1999) Aquatic ecological risks posed by tributyltin in
United States surface waters: pre-1989 to 1996 data. Environ Toxicol Chem 18:567–577
Bultman JD, Griffith JR, Field DE (1984) Fluopolymer coatings for the marine environment. In:
Costlow JD, Tipper RC (eds.) Marine biodeterioration: an interdisciplinary study. US Naval
Institute, Annapolis, MD, pp. 237–242
Callow ME, Willingham GL (1996) Degradation of antifouling biocides. Biofouling 10:239–249
Champ M (2000) A review of organotin regulatory strategies, pending actions, related costs and
benefits. Sci Total Environ 258:21–71
Clare AS (1997) Towards nontoxic antifouling (mini-review). J Mar Biotechnol 6:3–6
Clare AS, Rittschof D, Gerhart DJ, Maki JS (1992) Molecular approaches to nontoxic antifouling.
J Invert Reprod Dev 22:67–76
Costlow JDC, Tipper RC (1984) Marine biodeterioration: an interdisciplinary approach. US Naval
Institute, Annapolis, MD
Dahllöf I, Grunnet K, Haller R, Hjorth M, Maraldo K, Petersen DG (2005) Analysis, Fate and
toxicity of zinc and copper pyrithione in the marine environment. TemaNord 550–583
Fisher EC, Castelli VJ, Rodgers SD, Bleile HR (1984) Technology for control of marine biofouling
– a review. In: Costlow JD, Tipper RC (eds.) Marine biodeterioration: an interdisciplinary
study. US Naval Institute Press, Annapolis, MD, pp. 261–299
Galvin RM, Mellado JMR, Neihof RA (1998) A contribution to the study of the natural dynamics of
pyrithione (ii): deactivation by direct chemical and adsorptive oxidation. Eur Water Manag 4:61–64
Novel Antifouling Coatings: A Multiconceptual Approach 187

Gough MA, Fothergill J, Hendrie JD (1994) A survey of southern England coastal waters for the
s-triazine antifouling compound Irgarol 1051. Mar Pollut Bull 28:613–620
Hadfield MG (1998) Research on settlement and metamorphosis of marine invertebrate larvae:
past, present and future. Biofouling 12:9–29
Harrington JC, Jacobson A, Mazza LS, Willingham G (2000) Designing an environmentally safe
marine antifoulant. Presented at the10th international congress on marine corrosion and fouling,
DSTO, Melbourne
Holm ER, Orihuela B, Kavanagh CJ, Rittschof D (2005) Variation among families for characteristics
of the adhesive plaque in the barnacle Balanus amphitrite. Biofouling 21:121–126
International Maritime Organization (IMO) (2003) International Convention for the Prevention of
Pollution from Ships, 1973, as modified by the Protocol of 1978 relating thereto (cited 6 April
2003). http://www.imo.org/home.asp. Last accessed 14 July 2008
Kavanagh CJ, Swain GW, Kovach BS, Stein J, Darkangelo-Wood C, Truby K, Holm E,
Montemarano J, Meyer A, Wiebe D (2003) The effects of silicone fluid additives and silicone
elastomer matrices on barnacle adhesion strength. Biofouling 19:381–390
Liu D, Pacepavicius GJ, Maguire RJ, Lau YL, Okamura H, Aoyama I (1999) Survey for the occur-
rence of the new antifouling compound Irgarol 1051 in the aquatic environment. Water Res
33:2833–2843
New York State Department of Environmental Conservation (1996) NYSDEC Registration of
Irgarol algicide. http://pmep.cce.cornell.edu/profiles/herb-growthreg/fatty-alcohol-monuron/
irgarol/new-act-ing-irgarol.htmlLast accessed 14 July 2008
Pesticide Action Network (2008) Diuron identification, toxicity, use, water pollution potential, eco-
logical toxicity and regulatory information. PAN pesticides database. http://www.pesticideinfo.org/
Detail_Chemical.jsp?Rec_Id=PC33293 Last accessed 14 July 2008
Preiser HS, Ticker A, Bohlander GS (1984) Coating selection or optimum ship performance. In:
Costlow JD, Tipper RC (eds.) Marine biodeterioration: an interdisciplinary study. US Naval
Institute Press, Annapolis, MD, pp. 223–228
Readman JW (1996) Antifouling herbicides – a threat to the marine environment? Mar Pollut Bull
32:320–321
Rittschof D (2000) Natural product antifoulants: one perspective on the challenges related to coat-
ings development. Biofouling 15:199–207
Rittschof D, Holm ER (1997) Antifouling and foul-release: a primer. In: Fingerman M,
Nagabhushanam R, Thompson MF (eds.) Recent advances in marine biotechnology, vol I.
Endocrinology and reproduction. Oxford and IBH, New Delhi, pp. 497–512
Rittschof D, Parker KK (2001) Cooperative antifoulant testing: a novel multisector approach. In:
Fingerman M, Nagabhushanam R (eds.) Recent advances in marine biotechnology, vol VI.
Science, Einfield, pp. 239–253
Stein J, Truby K, Wood CD, Gardner M, Swain G, Kavanagh C, Kovach B, Schultz M, Wiebe D,
Holm E, Montemarano J, Wendt D, Smith C, Meyer A (2003) Silicone foul release coatings:
effect of the interaction of oil and coating functionalities on the magnitude of macrofouling
attachment strengths. Biofouling 19(Suppl):71–82
Schwarzenbach RP, Gschwend PM, Imboden DM (2003) Environmental organic chemistry, 2nd
edn. Wiley, Hoboken
Tolosa I, Readman JW (1996) Simultaneous analysis of the antifouling agents: tributyltin, tripheyltin
and Irgarol 1051 used in antifouling paints. Mar Pollut Bull 335:267–274
Tolosa IJ, Readman W, Blaevoet A, Ghilini S, Bartocci J, Horvat M (1996) Contamination of
Mediterranean (Cote d’Azur) coastal waters by organotins and Irgarol 1051 used in antifouling
paints. Mar Pollut Bull 32:335–341
Willingham GL, Jacobson AH (1996) Designing an environmentally safe marine antifoulant. ACS
Symp Ser 640224:–233
Concept and Consequences of the EU Biocide
Guideline

H.-C. Flemming (*
ü ) and M. Greenhalgh

Abstract The Biocide Product Directive (BPD) of the European Union is intended
to balance the efficacy of biocides in their intended use with their impact on human
and animal health and the environment. It legally organizes the process of putting
a biocide on the market and harmonizes the regulation of the EU Member States.
Biocides have to be subjected systematic tests for efficacy and risk before approval.
The BPD achieves its aims using a two-stage regime of rigorous evaluation of bio-
cidal active substances and products, to ensure they pose no unacceptable risks to
people, animals or the environment.
Ultimately only those biocidal products that contain an active substance that is
included in Annex I of the Directive will be authorized for use. Active substances
have to be evaluated to ascertain whether or not they will be included in Annex I.
This requires industry to submit data, which is evaluated by Member States with
decisions over Annex I inclusion being taken at the European level. Industry is charged
a fee for this process. Once an active substance has been included in Annex I,
national Competent Authorities can authorize products containing it in indi-
vidual Member States (providing that any necessary data have been supplied and
any conditions put on Annex I inclusion are met). Once a product has been author-
ized in the first Member State, it will be possible for it to be mutually recognized
and therefore authorized by other Member States (providing relevant conditions are
similar). However, there will have to be an application from other Member States,
and again there will be a fee for these processes.

1 Brief Historical Outline

By nature, biocidal products are directed against living organisms, frequently not
really restricted to “target organisms”. This implies that they inevitably also can harm
the health of non-target organisms such as humans or animals. An example is DDT,

H.-C. Flemming
Biofilm Centre, University of Duisburg-Essen, Geibelstrasse 41, D-47057, Duisburg, Germany
e-mail: hanscurtflemming@compuserve.com

Springer Series on Biofilms, doi: 10.1007/7142_2008_12 189 189

© Springer-Verlag Berlin Heidelberg 2008
190 H.-C. Flemming and M. Greenhalgh

which was very effective against mosquitoes spreading malaria but was bio-accumulated
and spread into the environment to an extent that made the further use of this substance
unacceptable. Furthermore, a wide variety of substances for biocidal use have been
developed and applied worldwide. They are authorized by regulations that are very
different in different countries, frequently incompatible, and based on (partially insuf-
ficient) systems of risk assessment. In order to overcome this situation, the European
Commission drafted legislation for harmonizing provisions for biocidal products, and
to ensure a more uniform and higher level of health and environmental protection
throughout the EU without compromising the internal market. This was (and still is) a
very challenging approach and the European Commission has gone further by co-operating
with other non-EU countries through the auspices of the Organisation for Economic
Co-operation and Development (OECD) to try to harmonize biocide regulations on a
global basis. The basic idea is that it is not enough just to develop a new and more
effective biocide but that the health and environmental issues involved in its applica-
tion are also considered. The concept of the authors of the guideline was to implement
a system that would force the chemical industry to behave in an ethically acceptable
way. In 1993, the first draft of a Directive concerning the placing of biocidal products
on the market was submitted by the European Commission. It was found that risk
assessment should be an integral part of the Directive. The Commission therefore
submitted a revised version in 1995. After long and controversial discussions, a final
text was adopted by the Council in 1998 as “Biocidal Products Directive” (BPD)
(European Parliament 1998). Each Member State appointed an agency to deal with the
new legislation: the so-called Competent Authority. The biocides industry transformed
dramatically with the implementation of the BPD. There has been much debate
amongst industry and the national Competent Authorities on how to operate the
scheme, within the idealistic legal framework of the Directive. Some Competent
Authorities themselves are considered by some to take an idealistic approach, whereas
others are more pragmatic and give higher priority to the needs of industry and biocide
users. Nevertheless, the BPD is significantly more stringent than any previous legisla-
tion, either within Europe or in the rest of the world. Knight and Cooke (2002) assume
that it may cost the industry well over 500 million €. Much of the information pro-
vided in this chapter is based on their excellent work.
From the point of view of health and environment, it clearly is a major step
forward to better protection, although the direction is very complicated in many
details and it is not free from discrepancies.

2 Scope of the Guideline

The BPD defines biocidal products as “preparations containing one or more active
substances that are intended to control harmful organisms by either chemical or
biological, but not physical, means”. This encompasses a wide range of products
including disinfectants, insect repellents, and anti-fouling paints. Despite the name
“biocide”, a biocidal product does not actually have to kill. If it is used to destroy,
Concept and Consequences of the EU Biocide Guideline 191

deter, make harmless, or control a harmful organism by chemical or biological

means, it maybe considered to be a biocide. For example a repellent used to “deter”
a mosquito could be considered to be a biocidal product.
The Directive will not apply to certain products already subject to European
legislation, including plant protection products, human medicines, veterinary medi-
cines, medical devices or cosmetics. Article 1 of the BPD lists those Directives that
are not covered within the scope of the BPD. The legislation also excludes the non-
biocidal uses of products and active substances
The official objective of the guideline is laid down in the Foreword, Chap. 1, of
the guideline:
“Whereas, in their resolution of 1 February 1993 on a Community programme of policy
and action in relation to the environment and sustainable development (4), the Council and
the representatives of the Governments of the Member States, meeting within the Council,
approved the general approach and strategy of the programme presented by the
Commission, in which the need for risk management of non-agricultural pesticides is
emphasised;”

In Chap. 3 of the Foreword the EU commits that biocides are necessary for the
control of organisms dangerous for the health of humans and animals:
“Whereas biocidal products are necessary for the control of organisms that are harmful to
human or animal health and for the control of organisms that cause damage to natural or
manufactured products; whereas biocidal products can pose risks to humans, animals and
the environment in a variety of ways due to the intrinsic properties and associated use
patterns;”

These are the official scientific reasons for implementing the BPD.
The biocidal products guideline is divided into three parts. The third and last
one, the Annexes, is potentially the most important because it lists the conditions
for placing biocidal products on the market. Annex V of the BPD classifies biocidal
products into four main groups: disinfectants and general biocides, preservatives,
pest controls, and other biocides as shown in Table 1 (Knight and Cooke 2002).
Ultimately only those biocidal products that contain an active substance that is
included in Annex I of the Directive will be authorized for use. An area of dispute
concerning scope is the regulation of in-situ generated biocides. These include
substances that are mixed together or otherwise generated by the consumer to cre-
ate the biocidal active ingredient. The EU Commission and Member States have
agreed that for example the in-situ generation of ozone is not covered.
When the Directive has been fully implemented in all Member States, existing
and new active substances will have to be evaluated to ascertain whether or not they
will be included in Annex I. Both processes will require industry to submit data,
with a system of data protection. The data will be evaluated by Member States with
decisions over Annex I inclusion being taken at the European level. Industry will
be charged a fee for this process.
Once an active substance has been included in Annex I, national Competent
Authorities can authorize products containing within individual Member States
(providing that any necessary data have been supplied and any conditions put on
Annex I inclusion are met).
192 H.-C. Flemming and M. Greenhalgh

Table 1 Products defined as biocides within the BPD

Main group 1: disinfectants and general biocides
1. Human hygiene products
2. Private and public health area disinfectants
3. Veterinary hygiene biocides
4. Food and feed area disinfectants
5. Drinking water disinfectants
Main group 2: preservatives
6. In-can preservatives
7. Film preservatives
8. Wood preservatives
9. Preservatives for fibre, leather and polymerized materials
10. Masonry preservatives
11. Preservatives for liquid cooling systems
12. Slimicides
13. Metal-working fluid preservatives
Main group 3: pest control
14. Rodenticides
15. Avicides
16. Molluscicides
17. Piscicides
18. Insecticides, acaricides and products to control other anthropods
19. Repellants and attractants
Main group 4: other biocides
20. Preservatives for food or feedstocks
21. Anti-fouling products
22. Embalming and taxidermist fluids
23. Control of vertebrates

Following product authorization in a first Member State, it is then possible for the
product to be mutually recognized and therefore authorized by other Member States
(providing relevant conditions are similar). However, there will have to be an applica-
tion to other Member States, and once again there will be a fee for these processes.
Each European Union Member State is responsible for implementing the BPD. In
Great Britain the Directive was implemented through the Biocidal Products Regulations
2001, which came into force on 6 April 2001. The Directive was implemented in
Northern Ireland through the Biocidal Products Regulations (Northern Ireland) 2001
on 16 January 2002. The Biocidal Products (Amendment) Regulations came into force
on 1 April 2003 and there has been a further amendment, the Biocidal Products
(Amendment) Regulations 2005, which came into force on 1 October 2005.

3 Approval Systems

To obtain authorization for the marketing of a biocide, the applicant must submit at
least two data packages: the first on the active substance, and the second on the
formulated product relating to the product type. For each additional product type
Concept and Consequences of the EU Biocide Guideline 193

for which authorization is sought, a further dossier is required at additional cost.

The BPD makes a pragmatic but arbitrary distinction between those biocidal active
substances on the market before 14 May 2000 (“existing” active substances) and
those placed on the market for the first time after this date (“new” active sub-
stances). The Member States and the Scientific Committee on Biocidal Products
review the scientific content of the dossier and make an appropriate recommenda-
tion to the Commission. If the recommendation is favourable, the Commission will
enter the active substance in an approved list (Annex I of the BPD). A review pro-
gramme is established in the BPD to assess systematically during a 10-year period
all the existing active substances (European Commission 2006).
Standard biocidal products containing active substances in Annex 1 of the BPD
require a full dossier of information, and applications for authorization are evalu-
ated by the national Competent Authority without undue delay. Biocidal products
containing a new active substance for which a decision for Annex 1 listing is pend-
ing may be provisionally authorized for up to 3 years. New products containing
existing active substances can be authorized under existing national schemes for up
to 10 years during the review programme. Under the BPD, applicants may use the
concept of “frame formulation” to facilitate authorization of re-branded biocidal
products. Frame formulations are groups of biocidal products with the same active
substance of the same technical specification and use, which differ only in minor
details of the formulation composition such as colour or perfume ingredients, and
hence have the same risk and efficacy. Once a Member State approves a biocidal
product, all other Member States must approve the product, according to the prin-
ciple of mutual recognition. If there are disputes between Member States by some
reason, the Standing Committeeon Biocidal Products will have to resolve them.
Member States may opt out of the mutual recognition procedure for avicide, pisci-
cide and vermin-control biocidal products.

4 Risk Assessment

Risk assessment is an important aspect of the regulatory process. It is performed for

both the intended use and a reasonable worst-case situation. The risk from a chemi-
cal substance is determined from its intrinsic hazardous properties and the likely
exposures of humans and the environment throughout its life-cycle. The intrinsic
chemical, health, and environmental hazardous properties can be quantified as a
hazard assessment. The hazard of the biocide is assessed predominantly through
toxicological testing in animal models (Annex IIA and IIB). Good quality human
data may also be available, perhaps from epidemiological studies. The hazard
assessment is combined with an exposure assessment to produce a risk assessment.
If the outcome is favourable, the substance will be recommended for Annex 1 list-
ing. If not, further information on toxicity or exposure in order to refine the risk
assessment may be demanded. If the risk remains unfavourable, a regulatory deci-
sion may be taken to implement risk management requirements, such as additional
labelling or restrictions to use, to permit product approval.
194 H.-C. Flemming and M. Greenhalgh

Exposure assessment is a more complex issue. There are two basic options:
measuring or modelling. Modelling can be carried out using generic data for chemi-
cal release. Estimates of environmental release are improved by gathering informa-
tion on the release of biocides from specific processes to develop emission
scenarios. Risk characterization is also conducted regarding animals kept and used
by humans. The humaneness of biocidal products targeted at vertebrates is also
considered, e.g. for biocides directed against rats.
The rule is that biocidal products can only be approved if, when used as pre-
scribed, they do not present unacceptable risks to man, animals or the environment,
are efficacious and use permitted active substances. Approval of biocidal products
requires that they are used properly at an effective but minimized application rate.
The regulatory authority also assesses the packaging, labelling and accompanying
safety data sheet.
Acute and repeat-dose toxicity, irritation and corrosivity, sensitization, muta-
genicity, carcinogenicity, toxicity for reproduction and the physicochemical prop-
erties of each active substance in the biocidal product are considered. If possible,
they are also quantified, preferably as a dose–response effect. This includes
the exposure of professionals, non-professionals, and those exposed indirectly via the
environment to each active substance in the biocidal product during its lifestyle.
Only as a last resort is the use of personal protective equipment taken into account
to enable a biocidal product to be used safely. Replacement of hazardous sub-
stances by non-hazardous ones is preferred. Biocidal products containing cate-
gory 1 or 2A or 2B carcinogens, mutagens or substances toxic to reproduction
cannot be approved for use by the general public. Carcinogens are defined after the
International Agency for Research on Cancer (1987):
Category 1 is for substances for which there is sufficient evidence for a causal
relationship with cancer in humans (confirmed human carcinogen)
Category 2A is for substances for which there is a lesser degree of evidence in
humans but sufficient evidence in animal studies, or degrees of evidence considered
appropriate to this category, e.g. unequivocal evidence of mutagenicity in mam-
malian cells (probable human carcinogen)
Category 2B is for substances for which there is sufficient evidence in animal
tests, or degrees of evidence considered appropriate to this category (possible
human carcinogen)

5 Costs for Authorization of a Biocidal Product

The Health and Safety Executive of the United Kingdom (2008) gives an interest-
ing look at the current costs for authorization of a biocidal product, which is avail-
able from its website (http://www.hse.gov.uk/biocides/index.htm).
For Annex I inclusion the HSE currently charges £10,000 (approximately US$
20,000) for a completeness check of a dossier prior to full evaluation, in addition to
an evaluation fee of £84,000 –89,000 for the full evaluation. The fees are based on
Concept and Consequences of the EU Biocide Guideline 195

actuals, meaning that if it was calculated that more time was spent on an evaluation,
a further fee to cover the work may be charged; consequently if less time was spent
on an evaluation the calculated difference could be refunded.
The UK figures quoted here, are estimates of the likely costs involved in evaluat-
ing a product dossier. At the time of writing there are three active agents authorized
in Annex 1, i.e. dichlofluanid, difethialone and sulphuryl fluoride, while carbon
dioxide has been authorized in Annex 1A. Actual costs are unknown, but costs for
product authorizations will vary depending on how relevant the data (that was sup-
plied for inclusion into Annex I of the BPD) is to a product. In the UK, the estimate
for the first product authorization after Annex inclusion could cost between £8,500
and £20,000. The fee will be based on the actual work done and will depend on how
much of the work was done at the Annex I inclusion stage. Once the initial product
has been authorized, fees will be lower. It is expected that products can be author-
ized that contain the same active ingredient and are the same formulation etc. as the
original product, providing the company seeking authorization holds the relevant
letters of access. Also it is expected that the cost for authorization of subsequent
products that have the same use, user type and contain the same active substance,
but with differences in composition from a previously authorized product that do
not affect the level of risk or efficacy associated with the product, is to be in the
region of £500.

6 Some Problems with the BPD

The given definition for biocides as given in the BPD is very broad. It includes
chemical compounds, formulations of compounds and also microoganisms and
viruses, which strictly are not chemical substances. The entire legal work is not without
inconsistencies and discrepancies. Thus, organisms such as Bacillus thuringiensis
are dealt within a guideline that was created to reduce the usage of hazardous sub-
stances such as the “Seveso toxin” dioxin (2,3,7,8-tetrachlordibenzo-p-dioxin and
2,3,7,8-tetrachlor-dibenzofuran).
Even substances that are commonly accepted as non-toxic are enclosed into this
definition: Ethanol for instance is not poisonous according to the guideline 67/EWG.
However, if a technical product (e.g. a cleaner) contains sufficient amounts of etha-
nol, it will be preserved against microbiological growth just by that ethanol. So it is
debatable whether ethanol is added to improve the cleaning results when applying
the product or whether ethanol is added to inhibit microbiological growth.
The problem leads to the transfer of judicial power to the public administration.
The administration may decide on the basis of the form sheet of the biocidal prod-
uct whether an ingredient is in fact a biocidal substance or not. The administration
uses the information provided when other users of biocidal products registered their
active ingredients before. When the notification process is completed, the public
administration will have had to deal with many more borderline cases like that
outlined above. So, the public administration will have to build up the capability to
196 H.-C. Flemming and M. Greenhalgh

evaluate biocidal compounds and biocidal products as well as decide which biocidal
compound or which biocidal product may be placed on the market and which may
not. Thus, the biocidal product guideline 98/8/EG not only imposes a heavy burden
on the chemical industry but also on the public administration simply because of
the immense administrative time and effort. The process of implementation is
designed in a way that the public authorities must learn and understand how bio-
cides act in a scientific way as well as the benefits they give to the user and the
damage they may potentially cause to human health and the environment. This all
takes a considerable time to accomplish.
The BPD guideline differentiates between biocidal substances and biocidal
products. Biocidal products are materials that are used to control harmful microor-
ganisms or wildlife, such as rodents. Biocidal substances are chemical substances
that have the ability to kill or inactivate target organisms and are used in biocidal
products for control of microorganisms or wildlife.
Biocidal substances (active agents) have to be listed in Annex 1 to be allowed to
be used in biocidal products. Biocidal substances that are not dangerous substances
according to the guideline 67/EWG may be listed in Annex IA. To have a substance
listed in Annex IA is more difficult than to have it listed in Annex 1, but the listing
of a biocidal product that contains the substance is easier. Biocidal substances that
are so common that their usage cannot be controlled by the administration will be
listed in Annex IB. The guideline gives some examples for such substances. Quoted
in the guideline itself are ethanol and carbon dioxide, used so widely that they are
listed in Annex IB.
To be placed in Annex 1, IA or IB, the items laid down in Annex 2 have to be
determined by tests and their results have to be disclosed to the public administra-
tion. To ensure that the results of the different tests from different laboratories are
comparable, test methods have to be developed and rules for the interpretation of
the test results have been published. A list of properties of the substances, which
have to be determined for substances to be listed in one of the chapters of Annex I
is given in Annex IIA. The Annex IA gives a list of data that have to be specified
by test results or by other data of equivalent significance and reliability. A major
weighting is placed upon data relating to the fate of the biocidal compound in the
environment. Data have to be submitted on the toxicity against aquatic organisms,
the fate of the compound in soil, in addition to data about the biological degrada-
tion. Biodegradation is especially important as biocidal compounds must not be
allowed to build up in the environment. The types of tests are legally determined,
for example OECD biodegradation study protocols. Some tests that have been
undertaken to allow chemical compounds to be placed on the US market will have
to be repeated as the US tests use different vertebrate species.
Annex IB lists the data necessary to place a biocidal product on the market,
which consists of biocidal compounds plus additional substances that might influ-
ence the activity of the biocidal substance.
The type of data necessary to be allowed to place a compound or a product on
the market depends on the intended use or the purpose of the biocidal compound or
biocidal product. Annex V lists 23 different purposes for biocidal compounds or
Concept and Consequences of the EU Biocide Guideline 197

products (product types). By legal definition this list of purposes is considered to

be complete so that all notified biocidal compounds or products have to be listed
within one of the defined 23 product classes (see Table 1). The decision whether a
biocidal substance may be placed on the market is made by one of the national
public administrations of the Member States of the EU. Currently most substances
that have been filed under the notification scheme have been on the market prior to
the introduction of the biocidal product Directive. All existing biocidal substances
(i.e. those placed on the market before 14 May 2000), which are to be notified and
supported, have been placed onto four lists with dossier call-in dates of 28 March
2004, 30 April 2006, 31 July 2007 and 31 October 2008. Once dossiers are received
the review process begins. The reviews have been distributed among the national
public administrations (Competent Authorities) according to their capabilities. The
amount of data that has to be controlled by the public administration to be able to
decide whether a substance may be placed on the market is so large that the admin-
istrations have yet to finish their work. One important date was 1 September 2006,
at which time all identified existing actives that were not notified as being sup-
ported through the authorization process must be withdrawn from the European
Union.
Once a decision is taken by one of these national public administrations it has to
be accepted by the other Member States, as long as there are no cogent scientific
reasons against it. It could be for instance, that one Member State rejects the deci-
sion of the administration of another one because the target organism, which should
be controlled by the compound, does not occur in the Member State, which then
rejects the decision. A compound can be notified for a period of not more than 10
years for non-toxic substances and not more then 5 years for toxic substances. After
that period the notification has to be renewed. If new data or new scientific knowl-
edge becomes known to the administration that are in contrast to the data presented
for the notification and make it desirable to withdraw the substance from the market
for safety or environmental reasons, the public administration has to withdraw the
notification and thus the substance from the market.

7 Consequences of the Guideline

In general, the BPD acts as a threshold for the development and diversification of
the biocide market. This is intended to control developments that might otherwise
lead to unwanted effects on health and the environment and takes responsibility for
preventing such effects. It represents a real threshold because generating the data
necessary to notify a compound or a product is very expensive and time-consuming.
Estimates range from about 2–10 million € (approximately US$ 15 million) for
testing and generation of the data in addition to the costs of the administration proc-
ess. Therefore, only highly profitable companies are able to afford the time-con-
suming process and the costs required to notify a substance. The lower the data
requirements, the lower the risk of losing money by the notification process due to
198 H.-C. Flemming and M. Greenhalgh

insufficient sales after putting the compound or the product on the market. This
doesn’t take into account the additional time and costs associated with the invention
and development of new active agents. Companies have tended to notify com-
pounds and products that are have already been on the market for a long time and
of which most of the data required by the administration have been generated and
paid for already. Consequently, the system of notification implemented by the bio-
cidal products guideline 98/8/EC is advantageous to old products. Given the market
size for the various biocidal product types and the barrier of the BPD (including the
substitution principle) most EU-based companies have withdrawn their new active
research programmes.
Initially more than 1,500 active substances were identified, of which around 800
were notified to the EU Commission as being supported through the authorization
process. Only approximately half of these dossiers have been submitted, leading to
the conclusion that only a total of approximately 400 will go through the review
process. As a consequence, the EU’s Competent Authorities have had to rearrange
their dossier review process.
Despite its complexity, the problems of internal coherence, and the political
problems within the EU the BPD still represents a serious and responsible compro-
mise to balance the intended effect of biocides (i.e. killing organisms) and the
protection of humans and the environment against the non-intended effects. On the
other hand, the BPD has certainly slowed down the development and implementa-
tion of new biocides.
Additional EU Directives such as the so-called VOC (volatile organic com-
pounds) Directive (European Parliament 1999), and REACH (registration, evalua-
tion, authorization and restriction of chemicals) (European Parliament 2006) have
complicated the issue of biocidal products. The aim of the VOC Directive is to
significantly reduce the use of VOCs within the EU. However, this has resulted in
more water-based products, which will require preservation. With REACH, aspects
of this Directive will impact on components of biocidal products. This Directive
seeks to regulate a further 30,000 chemicals found within the EU. It is estimated
that it will result in around 120,000 dossiers, something that will pose significant
strain on both industry and national authorities.
For the development of new biocides, the BPD represents a significant barrier
concerning the European market. This includes many chemical anti-fouling
approaches, which have to be thought over on this background. It is a wake-up call
against the unregulated dispersion of chemical agents, which tend not to be consid-
ered sufficiently. Therefore, alternative anti-fouling strategies such as nutrient limi-
tation, cleaning-friendly design and surfaces gain a specific advantage over
conventional chemical treatment.

References

Commission of the European Communities (2007) Commission Regulation (EC) no 1451/2007 of

4 December 2007 on the second phase of the 10-year work programme referred to in Article
Concept and Consequences of the EU Biocide Guideline 199

16(2) of Directive 98/8/EC of the European Parliament and of the Council concerning the
placing of biocidal products on the market. Off J Eur Union L325:11.12.2007
European Parliament and the Council of the European Union (1998) Directive 98/8/EC of the
European Parliament and of the Council of 16 February 1998 concerning the placing of bio-
cidal products on the market. Off J Eur Commun L123:24.04.1998 http://ecb.jrc.it/
legislation/1998L0008EC.pdf. Last accessed 13 July 2008
European Parliament and the Council of the European Union (1999) Council Direcive 1999/13/
EC of 11 March 1999 on the limitation of emissions of volatile organic compounds due to the
use of organic solvents in certain activities and installations. Off J Eur Commun
L85:29.3.1999
European Parliament and the Council of the European Union (2006) Regulation (EC) No
1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning
the registration, evaluation, authorisation and restriction of chemicals (REACH). Off J Eur
Union L396:30.12.2006
Health and Safety Executive of the United Kingdom (2008) Biocides and pesticides. http://www.
hse.gov.uk/biocides/index.htm. Last accessed 13 July 2008
International Agency for Research on Cancer, World Health Organisation (1994) IARC monographs
on the evaluation of carcinogenic risks to humans, vols 1–60, 1972–1994 and Suppl 7, 1987
Knight, DJ, Cooke, M (2002) Regulatory control of biocides in Europe. In: Knight, DJ, Cooke, M
(eds.) The biocide business. Wiley, Weinheim, pp. 45–74
 Part II
Macrofouling
Hydroides elegans (Annelida: Polychaeta):
A Model for Biofouling Research

Brian T. Nedved and Michael G. Hadfield(*

ü)

Abstract The small serpulid polychaete Hydroides elegans is a problem fouling

organism in warm water marine harbors around the world. Often the first significant
animal biofouler on newly submerged surfaces, its calcareous tubes can accumu-
late rapidly and create serious problems for ships. H. elegans is easily adapted for
laboratory biofouling research because of its rapid generation time (~3 wks) and
ease of propagation. The dioecious adult worms spawn readily in the laboratory,
and their metamorphically competent larvae develop in ~5 d at 25 oC. The larvae
of H. elegans settle in response to natural biofilms or films formed by many, but
not all, single marine bacterial species. Tubes of H. elegans adhere very tightly
to surfaces and are more resistant to dislodgement than many barnacles. Thus, H.
elegans is an excellent model organism for experimental studies, including tests of
newly formulated marine coatings.

1 Introduction

The fouling communities that occur on ships and other man-made structures
submerged in the sea are diverse assemblages of organisms (Carlton and Hodder
1995; Gollasch 2002; Godwin 2003). Due to that diversity, the variety of adhesives
that fouling organisms utilize to cement themselves to settlement substrata are
equally diverse (Naldrett and Kaplan 1997; Brady and Singer 2000; Wiegemann
2005; Smith and Callow 2006), posing a significant challenge for the development
of new coatings to combat biofouling processes (Holm et al. 2006). Minimizing
fouling on ship hulls is important because of the negative influence fouling has on
hull performance (Woods Hole Oceanographic Institution 1952), expenses associ-
ated with dry-docking, scraping and re-painting hulls, and the substantial costs
from propulsive fuel losses required to overcome the increased drag created by hull
fouling (Townsin 2003). Research to find new coatings to combat biofouling has

M.G. Hadfield
Kewalo Marine Laboratory, University of Hawaii, 41 Ahui Street, Honolulu, HI 96813, USA
e-mail: hadfield@hawaii.edu

Springer Series on Biofilms, doi: 10.1007/7142_2008_15 203 203

© Springer-Verlag Berlin Heidelberg 2008
204 B.T. Nedved and M.G. Hadfield

two major thrusts, one in chemically formulating experimental coatings and another
in testing these coatings in both field and laboratory settings. The serpulid polychaete
Hydroides elegans (Haswell 1883) has proven to be an excellent organism for
testing experimental coatings under both field and laboratory conditions.
Hydroides elegans is a common member of fouling communities throughout
tropical and subtropical seas (ten Hove 1974; Hadfield et al. 1994; Unabia and
Hadfield 1999; Bastida-Zavala and ten Hove 2002, 2003). H. elegans is a problem-
atic fouling organism because: (1) it quickly colonizes newly submerged surfaces
(Unabia and Hadfield 1999; Holm et al. 2000); (2) it grows as much as 1.5 mm
day−1 (Paul 1937); (3) it reaches sexual maturity in as short a time as 9 days in a
tropical harbor (Paul 1937); (4) it has a short larval period (Hadfield et al. 1994;
Carpizo-Ituarte and Hadfield 1998); and (5) aggregations of its calcified tubes can
accumulate to several centimeters thick on submerged surfaces in as short a time as
1–2 months in Pearl Harbor, Hawaii (Edmondson 1944) (Fig. 1).

Fig. 1 Dense accumulation of tubes of Hydroides elegans on Vexar after a 1-month submersion
in Pearl Harbor, HI
Hydroides elegans (Annelida: Polychaeta): A Model for Biofouling Research 205

Due to its importance as a fouler of ship hulls, there is a growing body of

research concerned with the natural inductive cues for recruitment of H. elegans
(Hadfield et al. 1994; Hadfield and Strathmann 1996; Walters et al. 1997; Unabia
and Hadfield 1999; Hadfield and Paul 2001; Lau and Qian 2001; Harder et al.
2002; Lau et al. 2002; Huang and Hadfield 2003; Lau et al. 2005; Shikuma and
Hadfield 2006), as well as the metamorphic cascades that are triggered by this
process (Carpizo-Ituarte and Hadfield 1998; Holm et al. 1998; Carpizo-Ituarte and
Hadfield 2003). Additionally, it has been employed in studies of neurogenesis
(Nedved and Hadfield 1998, and unpublished), muscular development (Nedved
and Hadfield 2001), segment development (Seaver and Kaneshige 2006; Seaver
et al. 2005), and the heritability of egg size (Miles et al. 2007). H. elegans will
undoubtedly find use as a research model in many other types of studies due to the
ease with which is can be maintained and reared in the laboratory.
Upon submersion in seawater, surfaces undergo a well-characterized progres-
sion from initial coating of adsorbed organic molecules (Zobell and Allen 1935), to
the formation of biofilms (Marshall 1981; Baier 1984) composed of a wide variety
of microorganisms that form highly organized communities (Costerton et al. 1999).
These complex biofilms provide settlement cues for larvae of many sessile marine
invertebrate species (reviewed by Hadfield and Paul 2001). Biofilm bacteria pro-
duce the inductive cue for settlement of competent larvae of H. elegans (Hadfield
et al. 1994; Unabia and Hadfield 1999; Lau and Qian 2001; Lau et al. 2002; Huang
and Hadfield 2003; Lau et al. 2005; Shikuma and Hadfield 2006). Laboratory evi-
dence that other biofilm organisms may produce inductive cues for larvae of
H. elegans (e.g. diatoms: Harder et al. 2002) remain provisionary, given the diffi-
culty of producing absolutely axenic cultures of such organisms for testing.
Larvae of H. elegans require a minimum bacterial density for the induction of
metamorphosis, and increased larval settlement positively correlates with the
density of bacteria in a biofilm (Hadfield et al. 1994; Huang and Hadfield 2003).
Settlement by H. elegans is greatly reduced or eliminated when multi-species
biofilms are treated with a variety of agents that act either as fixatives or antisep-
tics, demonstrating that the microorganisms within the biofilms must also be alive
for induction to occur (Unabia and Hadfield 1999). Recent studies by Lau et al.
(2005) and Shikuma and Hadfield (2006) using denaturing gradient gel electro-
phoresis (DGGE) have examined the effect that changes in the bacterial assem-
blages of biofilms have on the induction of metamorphosis of H. elegans, both
demonstrating a stronger positive correlation between settlement of H. elegans
and bacterial density than between settlement and differences in natural commu-
nity composition. However, the effectiveness of a bacterial biofilm as an inducer
of metamorphosis of H.elegans is not solely due to the sheer number of bacteria
residing in it. Huang and Hadfield (2003) demonstrated that single-strain, low-
density biofilms of Pseudoalteromonas luteoviolacea induced metamorphosis of
H. elegans (Fig. 2), while mono-specific biofilms of Flexibacter sp. and
Cytophaga sp. were non-inductive even though the cell densities of these biofilms
were 7–12 times greater (Fig. 3). These data indicate that induction of metamor-
phosis is due to specific chemical characteristics of P. luteoviolacea (Huang and
206 B.T. Nedved and M.G. Hadfield

Fig. 2 Induction of metamorphosis of Hydroides elegans by mono-specific biofilms on plastic

Petri dishes prepared from bacterial strains KMB1, KMB2, KMB3 and KMB4. Controls include:
(1) dishes similarly treated with filter-sterilized culture medium from each bacterial strain (S1, S2,
S3, S4); (2) natural biofilms (NB) allowed to accumulate on Vexar mesh in flowing seawater and
placed in a Petri dish of filtered seawater (FSW); (3) untreated Petri dishes filled with FSW; and
(4) dishes rinsed with fresh culture medium (M). KMB1 Pseudoalteromonas luteoviolacea, KMB2
Flexibacter sp, KMB3 Cytophaga sp, KMB4 Cytophaga lytica. Inoculation density of all strains
was approximately 10–8 cells mL−1. Bars represent mean percent of larvae that metamorphosed in
24 h + SD (n = 5 replicates per treatment) (reproduced from Huang and Hadfield 2003)

Hadfield 2003). Furthermore, production of this metamorphic cue is strain-spe-

cific; a different strain of P. luteoviolacea obtained from the American Type
Culture Collection (Manassas, VA) does not induce settlement of larvae of H.
elegans (unpublished personal observations).
Hydroides elegans is particularly well-suited for use in testing of experimental
coatings. The adhesive that secures the calcareous tubes of H. elegans appears to be
stronger than that of the balanoid barnacles Balanus eburneus and B. amphitrite,
two species often employed in testing of marine coatings. The mean removal force
for H. elegans that had settled on six different silicone coatings in Pearl Harbor was
nearly three times greater than the mean removal force required to remove B.
eburneus from replicate panels immersed in the Indian River Lagoon, FL (Fig. 4A, Stein
et al. 2003). Additionally, more spat of B. amphitrite than newly settled juveniles of
H. elegans are removed from the silicone coating RTV11 (General Electric,
New York) by a 4-min exposure to a wall-shear force equivalent to 100 Pa (unpub-
lished personal observations presented in Fig. 4B) in a turbulent flow apparatus
(described in Schultz et al. 2003).
Hydroides elegans (Annelida: Polychaeta): A Model for Biofouling Research 207

Fig. 3 Bacterial densities in single-species biofilms (see Fig. 2), counted under fluorescence
microscopy after formalin fixation and DAPI staining. KMB1, KMB2, KMB3 and KMB4 are the
bacterial strains studied; S1–S4 are dishes treated with the supernatants from each strain, respec-
tively; FSW and M are filtered seawater and culture-medium-only controls, respectively. KMB1
Pseudoalteromonas luteoviolacea, KMB2 Flexibacter sp, KMB3 Cytophaga sp, KMB4 Cytophaga
lytica. Inoculation density of all strains was approximately 10–8 cells mL−1. Bars represent bacte-
rial cell numbers × 103 mm−2 + SD (n = 25, consisting of five area counts per replicate and five
replicate dishes per treatment; replicate effects were not significant (reproduced from Huang and
Hadfield 2003)

Information on the occurrence and biology of H. elegans has been published under
several taxonomic names (e.g. Edmondson 1944; Wisely 1958). This confusion has
been resolved in taxonomic reviews by Zibrowius (1971), ten Hove (1974) and
Bastida-Zavala and ten Hove (2003), who concluded that H. norvegica is a species of
the northern Atlantic Ocean and the Mediterranean Sea and that the similar species in
warm seas around the world should be referred to as H. elegans. There are, of course,
other tropical species of Hydroides, and they may easily be confused with H. elegans
without careful observation of the operculum and setae, which are well illustrated in
ten Hove (1974), Bailey-Brock (1987) and Bastida-Zavala and ten Hove (2003).
According to ten Hove (1974), H. elegans is the only Hydroides species that forms
dense aggregations in warm water bays and estuaries worldwide.
The remainder of this chapter provides a concise summary of methods developed
in our laboratory for the culture of H. elegans for use in biofouling testing.
Our techniques have been used to successfully culture H. elegans elsewhere
(e.g. Bryan et al. 1997), including areas that do not have access to coastal waters.
208 B.T. Nedved and M.G. Hadfield

Fig. 4 Strength of adhesion of tubeworms and barnacles in the field (a) and a laboratory trial (b).
a Mean attachment strength of barnacles and tubeworms on test coatings. Data are for
Hydroides dianthus at Indian River and H. elegans at Pearl Harbor, and Balanus eburneus at
Indian River (reproduced with permission from Stein et al. 2003). b Percent of juveniles remain-
ing after exposure to a wall shear stress equivalent to 100 Pa for 4 min. Data are for H. elegans
and Balanus amphitrite (previously unpublished data from our laboratory)
Hydroides elegans (Annelida: Polychaeta): A Model for Biofouling Research 209

2 Collection and Care of Adults

In Pearl Harbor, larvae of Hydroides elegans settle on biofilmed surfaces throughout

the year and can reach high densities on both natural and man-made surfaces (Fig.
1). Several different materials have been used as artificial settlement substrata for the
field collection of H. elegans (Walters et al. 1997; Lau and Qian 2001; McEdward
and Qian 2001; Lau et al. 2002; Walters et al. 2003). We prefer to use small pieces
of extruded plastic mesh (Vexar) as settlement substrata for collecting H. elegans in
Pearl Harbor. These screens are hung from a pier approximately 1 m below the mean
low tide line, and within 3–4 weeks thousands of recruits have settled on them and
grown to reproductive maturity (Fig. 1). These dense populations of worms are then
transported back to the laboratory and kept in continuously flowing, unfiltered sea-
water for several weeks without a noticeable decrease in fecundity.
Vexar is preferred over solid substrata, because: (1) it greatly increases the surface
area of the material; (2) it provides crevices that may entrain larvae near the surface of
screen facilitating settlement on its surface; and (3) it allows water flow through the mesh
to bring food, oxygen, and remove wastes from settled worms (Walters et al. 1997).
Large populations of adult H. elegans can be kept in closed-system aquaria
containing either natural or artificial seawater. Additionally, individual worms can
be reared separately by allowing larvae to settle on small (1 × 1 cm) biofilmed chips
of polystyrene, removing all but one juvenile worm from each chip, and maintaining
the chips in individual wells of plastic ice cube trays (Eric Holm, personal com-
munication; Miles et al.2007). In both settings, adult worms survive and continue
producing gametes when fed Isochrysis galbana (6 × 104 cells mL−1).

3 Spawning

Spawning of H. elegans can be achieved using either destructive or non-destructive

methods. We typically use the destructive spawning method, because large numbers
of gravid worms are available to us throughout the year in Hawaii. When using this
method, we remove 30–40 worms from a piece of Vexar and place them in a small
glass dish containing 100 mL of 0.22 μm Milipore-filtered seawater (FSW). To
induce release of gametes, the calcareous tubes of the worms are broken in half
using forceps, and the abdominal segments of the worms are exposed. This process
causes release of thousands of small (~45 μm diameter) orange eggs or clouds of
sperm from the abdominal segments of the worm. We then repeat this process until
all the worms have been removed from their tubes. After 15–20 min, the fertilized
eggs are separated from the adult worms and debris by passing them through a 200
μm sieve (Nitex) into a 500-mL beaker. Filtered seawater is added to achieve a
volume of 200 mL. This addition of seawater dilutes the sperm concentration to
prevent polyspermy. Fertilization occurs within minutes of exposure of eggs to
sperm, and first cleavage occurs approximately 1 h after fertilization (23–26 C).
Using this method it is easy to obtain tens of thousands of embryos at a time.
210 B.T. Nedved and M.G. Hadfield

If it is not possible to sacrifice large numbers of adult worms to obtain gametes,

a non-destructive method may be used. In this method, worms whose tubes are still
attached to their substratum are placed in dishes containing FSW, and the aperture
of the worm’s tube is gently broken with fine forceps. This mechanical disturbance
causes release of eggs and sperm into the tube, and the worm then expels the gam-
etes from the tube by muscular peristaltic action. Generally, females induced to
spawn using this method release fewer eggs than can be obtained with the destruc-
tive method. After the worms have spawned, they can be placed back into their
individual containers where they will repair the apertures of their tubes, and can be
induced to spawn again in 2–3 days (personal observations). We have also noted
that when adult worms are kept individually in ice cube trays, they occasionally
release gametes spontaneously after the FSW is changed in the wells of the trays.

4 Feeding and Care of Larvae

4.1 Seawater

In our laboratory, natural coastal seawater (salinity 35 ‰) filtered through a 0.22

μm Millipore filter (FSW) is used in larval culture to minimize bacterial contamina-
tion. We have raised larvae of H. elegans in “MBL artificial seawater” (Cavanaugh
1975; Bidwell and Spotte 1985; Strathmann 1987) with no deleterious effects.
Antibiotics (60 μg mL−1 penicillin G and 50 μg mL−1 streptomycin sulfate) may be
used if larval mortality is high, but this mixture is generally not required to maintain
the larvae through metamorphic competence.

4.2 Temperature and Light

Larval cultures of H. elegans are maintained on the bench-top at room temperature

(23–26 C) and kept under the ambient lighting regime of the laboratory. However, main-
taining larval cultures at 25 C in an incubator provides a greater degree of synchrony of
early larval stages. At 24–25°C, larvae attain metamorphic competence after 5 days in
culture. Lower temperatures increase time to competence, at 20 C, larvae become com-
petent to metamorphose after 8–10 days in culture (Wisely 1958, as H. norvegica).

4.3 Culture Vessels

All glassware used for larval culture in our laboratory is scrubbed under running tap
water, rinsed several times with deionized water, and allowed to air dry prior to use.
Hydroides elegans (Annelida: Polychaeta): A Model for Biofouling Research 211

Additionally, all of our glassware is periodically soaked in a strong acid solution

(25% HCl and 25% H2SO4) to destroy any organic films that may have developed
on the interior surfaces of the containers. Adsorbed organic films hasten the devel-
opment of a biofilm in the culture vessels, which can provide a metamorphic cue
for competent larvae and may cause a substantial number of larvae to metamor-
phose on the walls of the glassware and the surface film. In our laboratory, larvae
are cultured in 1- or 2-L beakers at an initial density of 5–10 larvae mL−1. Cultures
of larval H. elegans are maintained without stirring or aeration with high levels of
larval survival through attainment of competence.
In order to prevent evaporation, beakers are covered with plastic wrap.

4.4 Changing Water in Cultures

After the second day, the larvae are transferred to clean beakers with fresh FSW.
To do this, each larval culture is poured into a small plastic beaker whose bottom
has been replaced by a piece of 41 μm Nitex sieve. The beaker is placed in a small
bowl of seawater in a sink and, as the larval culture is poured through the sieve, the
old culture water is allowed to run over the top of the bowl, and the larvae are con-
fined above the screen (see Strathmann 1987). The concentrated larvae are then
gently washed from the sieve into clean, acid-washed beakers containing fresh
FSW and phytoplankton. Care is taken to retain a small volume of water in the sieve
to prevent larvae from being crushed against it. This procedure is subsequently
repeated daily until larvae attain competence. Once attaining competence, larvae
can be maintained in this manner for several weeks (Unabia and Hadfield 1999).

4.5 Larval Food and its Culture

The unicellular alga Isochrysis galbana (Tahitian strain) is the most commonly
used food source for larvae of H. elegans (Hadfield et al. 1994; Carpizo-Ituarte
and Hadfield 1998; Holm et al. 1998; McEdward and Qian 2001; Carpizo-
Ituarte and Hadfield 2003; Huang and Hadfield 2003; Lau et al. 2005; Shikuma
and Hadfield 2006). However, other algal species have been utilized to raise larvae
of this species through metamorphosis (Wisely 1958 as H. norvegica; Hadfield
et al. 1994). We use I. galbana at a density of 6 × 104 cells mL−1. Larval cultures of
H. elegans are fed I. galbana daily by adding aliquots from our working alga
cultures; we do not attempt to separate the alga from its culture media.
In our laboratory, I. galbana is grown in a commercially produced, modified
Guillard’s f/2 media (Micro Algae Grow, Florida Aqua Farms, Dade City, FL). We
syringe filter (0.22 μm) Micro Algae Grow and use it at a working concentration of
1:1,000 in autoclaved seawater (salinity 25‰). A stock culture of I. galbana is
maintained in 50-mL screw-top Erlenmyer flasks and recultured bi-weekly. Algal
212 B.T. Nedved and M.G. Hadfield

cultures for larval cultures are started every 3 days and maintained in autoclaved
culture containers described in Switzer-Dunlap and Hadfield (1981). These cultures
are then used as a larval food source when the algal populations are in the later
portion of their growth phase. All cultures are bubbled and kept in continuous light
supplied by 20-W cool white fluorescent bulbs at room temperature.

4.6 Larval Development

Although the embryonic and larval development of H. elegans has been previously
described (Wisely 1958 as H. norvegica), the timing of larval development is highly
dependent on the culture conditions. Larvae cultured in our laboratory develop
more rapidly due to the higher ambient temperatures of Hawaii. Cell division
proceeds rapidly after first cleavage, and larvae hatch after about 4 h. Larvae of H.
elegans begin feeding as early trochophores approximately 9 h after fertilization
(25°C), and by 12 h they are well developed trochophores with an apical sensory
organ (ASO), a single eyespot on the right side of the larva (ES), a prototroch (Pro),
and a metatroch (Met) (Fig. 5a).
Larvae remain in the unsegmented prototroch stage for approximately 60 h
longer. Three days after fertilization, larvae have developed into metatrochophores
with a second eyespot on the left side of the larval episphere, rudiments of the

Fig. 5 Differential interference contrast microscopy images of larval development in Hydroides

elegans. a Lateral view of trochophore-stage larva (12 h post-fert at 25°C). b Ventral view of
metatroch-stage larva (72 h post-fert). Notice the appearance of a second eyespot in the episphere
of the larva, and the precocious development of the collar and the first three abdominal segments.
The segments are identifiable by the positioning of the paired setigers within each segment. c
Ventral view of a competent larva (~120 h post-fert). The larva has grown considerably since the
trochophore stage, and the hyposphere has become considerably longer. The horse-shoe shaped
cerebral ganglion have become quite developed, and the mid-gut of the larva has almost been
entirely displaced from the episphere of the larva. Larval mid-gut is easily visualized due to the
pigmented algal cells in its lumen. ASO apical sensory organ; CG cerebral ganglia; Col collar; ES
eyespot; Met metatroch; Pro prototrochal band; Set setae. Scale bars: 50 μm in all panels
Hydroides elegans (Annelida: Polychaeta): A Model for Biofouling Research 213

collar (Col), and elements of the first three abdominal segments including setae
(Set) (Fig. 5b).
Larvae of H. elegans become competent to metamorphose 5 days after fertiliza-
tion (25°C). The hyposphere of the larvae has lengthened considerably and the gut
has shifted posteriorly. The larval midgut (discernable by the algal cells within it,
visible in Figs. 5a–c) has been almost entirely displaced from the episphere by the
differentiating cerebral ganglia (CG, Fig. 5c). In competent larvae, the growth and
differentiation of the cerebral ganglia is accompanied with a change in a shape of
the larval episphere. The lateral margins of the episphere appear to constrict in the
region immediately anterior of the prototroch (compare Figs. 5b and 5c), so that the
previously hemispherical episphere becomes conical and provides a morphological
landmark for the development of competence.

4.7 Metamorphosis

In addition to exposure to biofilm bacteria, larvae of Hydroides elegans can be

artificially induced to metamorphose by the bath application of the cations K+ and
Cs+ and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX).
IBMX (0.1 mM) induces 80% of the larvae exposed to it to undergo metamor-
phosis (Holm et al. 1998). Potassium ions (50 mM excess in FSW) typically
induce metamorphosis in over 70% of larvae, but the response is much slower than
the rate of metamorphosis induced by biofilms (Carpizo-Ituarte and Hadfield
1998). Maximal induction by caesium ions occurs when 10 mM excess Cs+ is
applied in a 3.0–4.5 h pulse.
Carpizo-Ituarte and Hadfield (1998) described the morphogenic events associ-
ated with metamorphosis in larvae of H. elegans (Fig. 6). Competent larvae of H.
elegans may initiate metamorphosis almost immediately after contacting inductive
surfaces and begin the process by excreting a sticky thread from their posterior end
that serves to tether the larvae to the substratum. Almost immediately after, larvae
lie flat on the surface and begin secreting a primary tube from most or all of the
segments. They shape the newly secreted tube by rotating within it as they erect
their setae to push the primary tube away from their bodies (Carpizo-Ituarte and
Hadfield 1998). The secretion of the primary tube, which can be completed in as
little as 10 min after contact with a surface, is an irreversible process that perma-
nently attaches a larva to the substratum. As the primary tube is secreted, the collar
is everted, the area immediately surrounding the collar constricts, the larval body
elongates, and the metatroch is lost. Simultaneously, the pair of lobes that are the
precursors to the branchial radioles become apparent on the anterolateral margins
of the episphere of the juvenile. The primary tube is never calcified.
Secretion of the calcified secondary tube begins at the anterior margin of the
primary tube approximately 2 h after the commencement of metamorphosis, after
which new material is added to the secondary tube continuously. As the secondary
tube is secreted, the prototroch is resorbed, and the branchial radioles begin to
214 B.T. Nedved and M.G. Hadfield

differentiate from the anterior lobes. Metamorphosis is complete and juvenile

development has commenced by 11–12 h post-settlement (Carpizo-Ituarte and
Hadfield 1998). Because both the primary tube and the early portions of the secondary
tube are transparent, the events of metamorphosis and early juvenile development
are easily observed with relatively low power microscopy (Fig. 6).
In summary, the major tropical marine fouler H. elegans has proven to be a
near perfect laboratory-animal model for studies of biofouling processes.

Fig. 6 Time-course of metamorphosis in Hydroides elegans. Frames represent a competent larva

at the moment of induction to metamorphosis (0 h) and selected stages for the first 11.3 h after
induction: p prototroch; c collar; b branchial lobes; it initiation point of calcareous tube; br
branchial radioles; t calcareous tube covering the worm (Carpizo-Ituarte and Hadfield 1998)
Hydroides elegans (Annelida: Polychaeta): A Model for Biofouling Research 215

The information provided above should make it possible to employ this organism
for studies of biofouling or questions involving the development of polychaete
worms in most laboratories. If scientists attempting to use H. elegans in their
research find problems in its culture, we would be happy to communicate with
them to find solutions.

Acknowledgments We are grateful to many former graduate students and postdoctoral fellows
in the Hadfield laboratory who have contributed significantly to the development of the methods
described here, especially E. J. Carpizo-Ituarte, E. R. Holm, S. Huang, Y. Huang, N. Shikuma and
C. Unabia. The authors are grateful to M. J. Huggett and T. DuBuc for their assistance in creating
the figures in this manuscript. Research reported here has been supported by the US Office of
Naval research, currently grant no. N00014-05-1-0579 to MGH.

References

Baier RE (1984) Initial events in microbial film formation. In: Costlow JD, Tipper RC (eds.)
Marine biodeterioration: an interdisciplinary study. US Naval Institute, Annapolis, MD,
pp. 57–62
Bailey-Brock JH (1987) Phylum Annelida. In: DM Devaney and LG Eldredge (eds.) Reef and
shore fauna of Hawaii, Sections 2 and 3. Bishop Museum Press, Honolulu, pp. 213–454
Bastida-Zavala JR, ten Hove HA (2002) Revisions of Hydroides Gunnerus, 1768 (Polychaeta:
Serpulidae) from the Western Atlantic region. Beaufortia 52(9):103–178
Bastida-Zavala JR, ten Hove HA (2003) Revisions of Hydroides Gunnerus, 1768 (Polychaeta:
Serpulidae) from the Eastern Pacific region and Hawaii. Beaufortia 53(4):67–110
Bidwell JP, Spotte P (1985) Artificial seawaters: formulas and methods. Bartlett, Boston, p. 349
Brady Jr RF, Singer IL (2000) Mechanical factors favoring release from fouling release coatings.
Biofouling 15:73–81
Bryan PJ, Qian PY, Kreider JL, Chia FC (1997) Induction of larval settlement and metamorphosis
by pharmacological and conspecific associated compounds in the serpulid polychaete
Hydroides elegans. Mar Ecol Prog Ser 146:81–90
Carlton JT, Hodder J (1995) Biogeography and dispersal of coastal marine organisms: experimen-
tal studies on a replica of a 16th century sailing vessel. Mar Biol 121:721–730
Carpizo-Ituarte E, Hadfield MG (1998) Stimulation of metamorphosis in the polychaete Hydroides
elegans Haswell (Serpulidae). Biol Bull 194:14–24
Carpizo-Ituarte E, Hadfield MG (2003) Transcription and translation inhibitors permit metamor-
phosis up to radiole formation in the serpulid polychaete Hydroides elegans. Biol Bull
204:114–125
Cavanaugh GM (1975) Formulae and methods of the Marine Biological Laboratory Chemical
Room, 6th edn. Marine Biological Laboratory, Woods Hole, MA, 84 pp
Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persistent
infections. Science 284:1318–1322
Edmondson CH (1944) Incidence of fouling in Pearl Harbor. Occas Pap Bernice P Bishop Mus
18(1):1–34
Godwin LS (2003) Hull fouling of maritime vessels as a pathway for marine species invasions to
the Hawaiian Islands. Biofouling 19(Suppl.):123–131
Gollasch S (2002) The importance of ship hull fouling as a vector of species introductions into the
North Sea. Biofouling 18:105–121
Hadfield MG Paul VJ (2001) Natural chemical cues for settlement and metamorphosis of marine
invertebrate larvae. In: McClintock JB, Baker W (eds.) Marine chemical ecology. CRC Press,
Boca Raton, FL pp. 431–461
216 B.T. Nedved and M.G. Hadfield

Hadfield MG, Strathmann M (1996) Variability, flexibility and plasticity in life histories of marine
invertebrates. Oceanologica Acta 19(3–4):323–334
Hadfield MG, Unabia CC, Smith CM, Michael TM (1994) Settlement preferences of the ubiqui-
tious fouler Hydroides elegans. In: Thompson MF, Nagabhushanam R, Sarojini R, Fingerman M
(eds.) Recent developments in biofouling control. Oxford and IBH, New Delhi, pp. 65–74
Harder T, Lam C, Qian PY (2002) Induction of larval settlement in the polychaete Hydroides
elegans by marine biofilms: an investagation of monospecific diatom films as settlement cues.
Mar Ecol Prog Ser 229:105–112
Holm ER, Kavanagh CJ, Meyer AE, Wiebe D, Nedved BT, Wendt D, Smith CA, Hadfield MG,
Swain G, Darkangelo Wood C, Truby K, Stein J, Montemarano J (2006) Interspecific variation
in patterns of adhesion of marine fouling to silicone surfaces. Biofouling 22(4):233–243
Holm ER, Nedved BT, Carpizo-Ituarte E, Hadfield MG (1998) Metamorphic-signal transduction
in Hydroides elegans (Polychaeta: Serpulidae) is not mediated by a G-Protein. Biol Bull
195:21–29
Holm ER, Nedved BT, Phillips N, DeAngelis KL, Hadfield MG, Smith CM (2000) Temporal and
spatial variation in the fouling of silicone coatings in Pearl Harbor, Hawaii. Biofouling
15(1–3):95–107
Huang S, Hadfield MG (2003) Composition and density of bacterial biofilms determine larval
settlement of the polychaete Hydroides elegans. Mar Ecol Prog Ser 260:161–172
Lau SCK, Mak KKW, Chen F, Qian PY (2002) Bioactivity of bacterial strains isolated from
marine biofilms in Hong Kong waters for the induction of larval settlement in the marine
polychaete Hydroides elegans. Mar Ecol Prog Ser 226:301–310
Lau SCK, Qian PY (2001) Larval settlement in the serpulid polychaete Hydroides elegans in
response to bacterial films: an investigation of the nature of putative larval settlement cue. Mar
Biol 138(2):321–328
Lau SCK, Thiyagarajan V, Cheung CK, Qian PY (2005) Roles of bacterial community composi-
tion in biofilms as a mediator for larval settlement of three marine invertebrates. Aquat Microb
Ecol 38(1):41–51
Marshall KC (1981) Bacterial adhesion in natural environments. In: Berkeley RCW, Lynch JM,
Melling J, Rutter PR, Vincent B (eds.) Microbial adhesion to surfaces. Ellis-Horwood, New
York, pp. 187–196
McEdward LR, Qian PY (2001) Effects of the duration and timing of starvation during larval life
on the metamorphosis and initial juvenile size of the polychaete Hydroides elegans (Haswell).
J Exp Mar Bio Ecol 261(2):185–197
Miles CM, Hadfield MG, Wayne ML (2007) Estimates of heritability for egg size in the serpulid
polychaete Hydroides elegans. Mar Ecol Prog Ser 340:155–162
Naldrett MJ, Kaplan DL (1997) Characterization of barnacle (Balanus eburneus and B.crenatus)
adhesive proteins. Mar Biol 127:629–635
Nedved BT, Hadfield MG (1998) Neurogenesis in the larvae of the serpulid polychaete Hydroides
elegans. Am Zool 38:167A
Nedved BT, Hadfield MG (2001) Fate of larval muscles during metamorphosis of Hydroides
elegans. Am Zool 41:1536
Paul MD (1937) Sexual maturity of some organisms in the Mardras Harbor. Curr Sci Bangalore
5:478–479
Schultz MP, Finlay JA, Callow ME, Callow JA (2003) Three models to relate detachment of low
form fouling at laboratory and ship scale. Biofouling 19(Suppl.):17–26
Seaver EC, Kaneshige LM (2006) Expression of ‘segmentation” genes during larval and juvenile
development in the polychaetes Capitella sp. I and H. elegans. Dev Biol 289:179–194
Seaver EC, Thamm K, Hill SD (2005) Growth patterns during segmentation in the two polychaete
annelids, Capitella sp. I and Hydroides elegans: comparisons at distinct life history stages.
Evol Dev 7:312–326
Shikuma NJ, Hadfield MG (2006) Temporal variation of an initial marine biofilm community and
its effects on larval settlement and metamorphosis of the tubeworm Hydroides elegans.
Biofilms 2(4):231–238
Hydroides elegans (Annelida: Polychaeta): A Model for Biofouling Research 217

Smith AM, Callow JA (2006) Biological adhesives. Springer, Heidelberg Berlin New York, 284 pp
Stein J, Truby K, Wood CD, Stein J, Gardner M, Swain G, Kavanagh C, Kovach B, Schultz M,
Wiebe D, Holm E, Montemarano J, Wendt D, Smith C, Meyer A (2003) Silicone foul release
coatings: effect of the interaction of oil and coating functionalities on the magnitude of mac-
rofouling attachment strengths. Biofouling 19(Suppl.):71–82
Strathmann MF (1987) Reproduction and development of marine invertebrates of the Northern
Pacific Coast. University of Washington Press, Seattle, 670 pp
Switzer-Dunlap M, Hadfield MG (1981) Laboratory culture of Aplysia. In: Hinegardner RE, Fay
R (eds.) Marine invertebrates, laboratory animal management. National Academy of Sciences,
Washington, DC, pp. 199–216
ten Hove HA (1974) Notes on Hydroides elegans (Haaswell, 1883) and Mercierella enigmatic
Fauvel, 1923, alien serpulid polychaetes introduced to the Netherlands. Bull Zool Museum
(University of Van Amsterdam) 4(6):45–51
Townsin RL (2003) The ship hull fouling penalty. Biofouling 19(Suppl.):9–15
Unabia CRC, Hadfield MG (1999) Role of bacteria in larval settlement and metamorphosis of the
polychaete Hydroides elegans. Mar Biol 133:55–64
Walters LJ, Hadfield MG, del Carmen K (1997) The importance of larval choice and hydrody-
namics in creating aggregations of Hydroides elegans (Polychaeta: Serpulidae). Invert Biol
116(2):102–114
Walters LJ, Smith CM, Hadfield MG (2003) Recruitment of sessile marine invertebrates on
Hawaiian macrophytes: do pre-settlement or post-settlement processes keep plants free from
fouling? Bull Mar Sci 72(3):813–839
Wiegemann M (2005) Adhesion in blue mussels (Mytilus edulis) and barnacles (genus Balanus):
mechanisms and technical applications. Aquat Sci 67:166–176
Wisely B (1958) The development of a serpulid worm, Hydroides norvegica, Gunnerus
(Polychaeta). Aust J Mar Freshwater Res 9:351–361
Woods Hole Oceanographic Institution (1952) Marine fouling and its prevention. US Naval
Institute, Annapolis, MD, 243 pp
Zibrowius H (1971) Les espèces Méditerranéennes du genre Hydroides (Polychaeta Serpulidae):
remarques sur le prétendu polymorphisme de Hydroides uncinata. Tethys 2:691–746
Zobell CE, Allen EC (1935) The significance of marine bacteria in the fouling of submerged
surfaces. J Bacteriol 29:239–251
Marine Epibiosis: Concepts, Ecological
Consequences and Host Defence

T. Harder

Abstract The sessile mode of life is widespread in a variety of marine phyla.

Sessile life requires a stable substratum. On the benthos, motile life stages and ses-
sile adults compete for rigid surfaces making non-living, i.e. inanimate, hard sub-
stratum a limited resource. Epibiosis is a direct consequence of surface limitation
and results in spatially close associations between two or more living organisms
belonging to the same or different species. These associations can be specifically
guided by host chemistry resulting in species-specific symbiotic or pathogenic
assemblages. Most colonizers, however, are non-specific substratum generalists.
The ecological consequences for the overgrown host (basibiont) and the colonizer
(epibiont) can be positive and negative. The predominantly disadvantageous nature
of epibiosis by microorganisms for the basibiont has resulted in a variety of defence
mechanisms against microcolonizers, including physical and chemical modes of
action. Besides antimicrobial effects of secondary metabolites emanating from
the host, recent studies increasingly demonstrate that epibiotic bacteria associated
with the host deter growth and attachment of co-occurring bacterial species or new
epibiotic colonizers competing for the same niche.

1 Introduction

In the marine environment the sessile mode of life is dominant in the majority of
phyla. Owing to their low specific weight not only microorganisms (e.g. bacteria,
microalgae) but also the small motile life stages of macroorganisms (e.g. larvae and
spores) behave like passive propagules in this viscous, hydrodynamically dominated
environment. Clearly, for these species an attached filter-feeding mode of life is the
energetically advantageous and favourable state, although the lack of locomotion
necessitates a variety of new challenging survival strategies, such as reproduction
and various defence forms against consumers and overgrowth.

T. Harder
Centre for Marine Bio-Innovation, University of New South Wales, Sydney,
NSW 2052, Australia
e-mail: t.harder@unsw.edu.au

Springer Series on Biofilms, doi: 10.1007/7142_2008_16219 219

© Springer-Verlag Berlin Heidelberg 2008
220 T. Harder

Sessile life requires a stable substratum. On the benthos, motile life stages and
sessile adult forms compete for rigid surfaces making non-living (i.e. inanimate)
hard substratum a limited resource. Epibiosis (greek epi “on top” and bios “life”)
can be considered as a direct consequence of surface limitation and results in spatially
close associations between two or more living organisms belonging to the same or
different species. The substrate organism is considered the basibiont, while the
organism(s) growing attached to the animate surface is referred to as the epibiont.
Epibionts are further subdivided into epizoans (animals) and epiphytes (plants,
algae). Epibiotic assemblages are rarely species-specific; on the contrary, numerous
sessile organisms may live either as basibiont or as epibiont, or both simultaneously
(Wahl 1997). Attachment and growth on inanimate surfaces is usually considered
as fouling, although this term is frequently used synonymously in an epibiotic context
in the literature.
Epibiosis is a typical aquatic phenomenon although many examples of terrestrial
epibionts are known (e.g. algae, lichens). Depending on seasonality and location the
average millilitre of seawater contains 10–100 microscopic larvae and spores, 103
fungal cells, 106 bacteria and 107 viruses. Thus, the colonization pressure exerted
by meroplanktonic dispersal stages can be intense on submerged surfaces (Davis
et al. 1989) with severe ecological consequences for basibionts and epibionts.
The distinctive role of water as a food vector for sessile organisms is the main reason
why surface attachment, fouling and hence epibiotic associations predominate in
aquatic environments. A large variety of marine phyla have adopted the sessile mode
of life for at least one ontogenetic phase. The list includes many bacteria, protozoa,
diatoms, molluscs, tube-building polychaetes; most macroalgae, bryozoans, phoronids,
cnidarians; some echinoderms, crustaceans; all sponges and tunicates.

2 Ecological Consequences for Epibionts

Marine fouling is an omnipresent phenomenon and the list of foulers is long.

The different stages of the fouling process of dispersal stages on solid substrates have
been described in successional (Davis et al. 1989) and probalistic models (Clare et al.
1992; Maki and Mitchell 2002) and are presented in detail in this volume. Irrespective
of the fouling sequence, for most meroplanktonic larvae settlement is the ultimate
prerequisite for successful metamorphosis into sedentary juveniles (Hadfield and
Paul 2001). Also, microcolonizers such as bacteria, benthic diatoms and algal spores
often proliferate more rapidly or sometimes exclusively when fixed to a solid sub-
stratum (Grossart et al. 2003). Once attached, microcolonizers are challenged by
other members in the biofilm matrix (Fletcher and Callow 1992; Costerton et al.
1995). To successfully compete in biofilms, many representatives of the bacterial
genus Pseudoalteromonas release anti-bacterial products that aid the cells in the
colonization of host surfaces. Through the production of agarases, toxins, bacteriolytic
substances and other enzymes, bacterial cells are assisted in their competition for
nutrients and space as well as in their protection against predators grazing on surfaces
(Holmström and Kjelleberg 1999).
Marine Epibiosis: Concepts, Ecological Consequences and Host Defence 221

In densely populated marine environments where competition for space is high,

the advantage for colonizers in occupying empty animate surfaces is probably the
main reason for epibiosis (Wahl 1989; Todd and Keough 1994) although a variety
of other advantages for the colonizer may support specific host–epibiont associations.
For instance, settlement on raised or elevated hosts results in a hydrodynamically
favourable position of the epibiont (Keough 1986) as flow dynamics increase with
distance from the benthos (Butman 1987). Increased flow ensures better supply of
planktonic nutrients and more efficient removal of toxic excretory products such as
ammonia. An exposed habitat supports phototrophic epibionts, especially in deeper
or turbid waters where light penetration is weak (Brouns and Heijs 1986). While
filter-feeding epibionts profit from nutrient currents created by the host (Laihonen
and Furman 1986) deposit-feeding epibionts benefit from metabolites exuded by the
basibiont (Harlin 1973). Regarding the fate of colonizers, epibionts either benefit from
the host defence against consumers or other colonizers, a phenomenon termed “asso-
ciational defence” (Hay 1986), or the fates of epibiont and host are closely interlinked
and shared together, a phenomenon termed “shared doom” (Wahl and Hay 1995).
The predominantly advantageous associations of epibionts with host organisms
indicate that the mere presence of a surface is often not the only criterion for success-
ful colonization. Numerous colonizers are reported to be guided by specific “cues”
mediating the suitability of the settlement site (Rodriguez et al. 1993; Wieczorek
and Todd 1998; Steinberg et al. 2002). The recognition of appropriate cues activates
the genetically scheduled sequence of behavioural and physiological processes during
settlement (Morse 1990) and many larvae delay or even avoid settlement in the absence
of appropriate settlement cues (Coon et al. 1990; Qian and Pechenik 1998).

3 Settlement Cues

There is clear experimental evidence for physical settlement cues, such as surface
roughness (Berntsson et al. 2000) and wettability (Qian et al. 2000); environmental
conditions in direct proximity to the surface, such as irradiation (Maida et al. 1994)
and microhydrodynamics (Mullineaux and Butman 1991); and biogenic chemical
signals emanating from the basibiont or other epibionts (e.g. bacteria) already
present on the host surface (Johnson et al. 1991a,b; Krug and Manzi 1999). Several
authors have presented experimental evidence for selective settlement of both gen-
eralist and specialist epibionts in response to invertebrate or plant host cues. In most
of these studies, the host served as the obligate prey source for larvae or adults. This
raises the question of how planktonically dispersed larvae locate their patchily
distributed hosts. Given the large spatial scales that need to be screened by potential
colonizers, one would expect either strong or very distinct cues that govern such
host–epibiont associations. To address this question, a number of studies have
focused on the selective response of sea slugs to host corals. For example, water-
soluble cues from corals induce settlement and metamorphosis in larvae of the
opisthobranchs Phestilla sibogae (Hadfield and Scheuer 1985), Adalaria proxima
(Lambert and Todd 1997) and Alderia modesta (Krug and Manzi 1999). Other
222 T. Harder

well-investigated host plants are coralline algae that govern larval settlement of taxo-
nomically distinct invertebrates such as the sea urchin Holopneustes purpurascens
(Williamson et al. 2000), the starfish Acanthaster planci (Johnson et al. 1991b;
Johnson and Sutton 1994) and the mollusk Haliotis (Morse and Morse 1984; Hahn
1989). Other well-studied systems comprise obligate associations that seemingly
benefit from close proximity of conspecifics to enhance reproductive output, such as
in oysters (Tamburri et al. 1992; Turner et al. 1994) and barnacles (Clare and
Matsumura 2000). However, the settlement cue(s) involved in the establishment of
these systems were rarely identified at the molecular level.
In contrast to the numerous partially characterized inducers, only few settlement
cues isolated from natural sources were in fact chemically identified, e.g. delta-
tocopherols from Sargassum tortile that induce settlement of the hydroid Coryne
uchidai (Kato et al. 1975); jacarone isolated from the red alga Delesseria sanguinea
that induces settlement of the scallop Pecten maximus (Yvin et al. 1985); narains
and anthosamines A and B isolated from marine sponges and lumichrome isolated
from conspecifics that induce settlement of ascidian larvae (Tsukamoto et al. 1994,
1995, 1999); N-acylhomoserine lactone quorum sensing signal molecules that aid
zoospores of the green macroalgae Ulva to exploit a bacterial sensory system and
select permanent attachment sites by responding to bacteria already present on the
surface (Joint et al. 2002). In most cases, the ecological relevance of these compounds
in situ is not clear, either because the source of the settlement cue is not necessarily
related to the recruitment patterns of the organism (Yvin et al. 1985; Tsukamoto
et al. 1994, 1995), or because the availability of the cue to settling larvae has not
been demonstrated unequivocally (Tsukamoto et al. 1999).
Interestingly, there is a high similarity in host recognition by pathogens in
marine and terrestrial plants (Kolattukudy et al. 1995). For example, the pathogenic
filamentous green alga Acrochaete operculata recognized its host, the red alga
Chondrus crispus, by cell wall polysaccharides. C. crispus has an isomorphic life
history, in which the gametophytic and sporophytic generations differ only in minor
traits, such as sulfate-ester group distribution of their matrix polysaccharides, known
as κ- and λ-carrageenans. Remarkably, the sporophytic generation is highly susceptible
to infection whereas the gametophytic phase is naturally resistant. The virulence of
the green algal endophyte is modulated by the presence of λ-carrageenan, which
stimulates protein synthesis and elicits the production of specific polypeptides in
the pathogen (Bouarab et al. 2001).
Only recent years have witnessed some complete characterizations of marine
invertebrate larval settlement cues. In a series of investigations Matsumura et al. (1998)
identified the key molecule responsible for gregarious settlement in the fouling
barnacle Balanus amphitrite as a settlement-inducing protein complex (SIPC). This
protein complex has now been fully elucidated as a α2-macroglobulin-like glycoprotein
(Dreanno et al. 2006). Although the SIPC is regarded as an adult cue that is recog-
nized by the cyprid at settlement, it is also expressed in juveniles and in larvae,
where it may function in larva–larva settlement interactions. In another series of
investigations the structure and the different sources of coralline algae-derived
settlement cues for two larval species of sea urchins, Holopneustes purpurascens
Marine Epibiosis: Concepts, Ecological Consequences and Host Defence 223

and Heliocidaris erythrogramma, have been fully elucidated. The biogenic amine
histamine was isolated from the red alga Delisea pulchra by bioassay-guided frac-
tionation and identified as the inducer of settlement of H. purpurascens (Swanson
et al. 2004). The alga still evoked larval settlement after antibiotic treatments,
which effectively removed epiphytic bacteria on the algal surface, demonstrating
that histamine was indeed an alga-derived cue. In contrast, the coralline alga
Amphiroa anceps, which also stimulates larval settlement of H. purpurascens,
lacked detectable amounts of histamine. Interestingly, antibacterial treatment of
A. anceps removed the settlement cue, suggesting a bacterial origin of the cue from
this alga; indeed bacterial films of two isolates from the surface of A. anceps
induced settlement of H. purpurascens in laboratory assays (Swanson et al. 2006).
The role of algae-associated bacteria as producers of settlement cues has been
examined in more detail for the sea urchin H. erythrogramma (Huggett et al. 2006).
The hypothesis of a bacterially derived settlement signal was supported by the fact
that a variety of bacterial isolates from the surface of coralline algae triggered larval
settlement at levels comparable to those of the positive control of coralline algae.
One bacterial isolate from A. anceps, Thallasomonas viridans, is a known hista-
mine producer. Given that larvae of both urchin species settle in response to histamine,
these findings demonstrate a common settlement cue in coralline algae produced
by the host alga and/or by associated bacteria.

4 Ecological Consequences for Basibionts

Any potential basibiont, i.e. the majority of sessile, relatively long-lived organisms,
must either tolerate epibiosis or employ some sort of defence against this phenomenon.
While epibiosis entails both benefits and disadvantages for epi- and basibionts the
investment into defence depends on a finely tuned and often variable energy budget
of the basibiont (Wahl 1989). Epibiosis causes a variety of beneficial effects to the
basibiont, such as the induction of morphogenesis in macroalgae by symbiotic bacteria
(Tatewaki et al. 1983; Nakanishi 1999), the interaction between macroalgae and
nitrogen-fixing bacteria (Thevanathan et al. 2000), and the protection of seaweed
surfaces from bacterial colonizers by associated bacteria (Lemos et al. 1985).
A well-investigated example of a symbiotic association between host and epibiotic
bacteria is the embryo of the American lobster, Homarus americanus, which is
resistant to the fungus Lagenidium callinectes, a pathogen of many crustaceans.
The surfaces of healthy lobster embryos are covered almost exclusively by a single,
Gram-negative bacterium, which produces the antifungal substance 4-hydroxy-
phenethyl (Gil-Turness and Fenical 1992). Testing the effects of epibiosis on herbivory
and predation, research by Wahl and colleagues suggested that epibionts on the blue
mussel Mytilus edulis affected its susceptibility to predation by the shore crab
Carcinus maenas (Wahl et al. 1997). Similarly, epibiosis by a variety of plants and
animals altered the host susceptibility of the omnivorous sea urchin Arbacia
punctulata (Wahl and Hay 1995). Furthermore, Wahl and Mark (1999) investigated
224 T. Harder

the hypothesis that if the effects for epibiont and basibiont were predominantly
beneficial then co-evolution would be expected to lead to some sort of associational
specificity. However, by analyzing over 2000 patterns of epibiotic associations the
authors concluded that many colonizers are non-specific substratum generalists and
that epibiosis is predominantly facultative (Wahl and Mark 1999).
The adverse effects of epibiosis on the basibiont often outweigh the beneficial
ones (Table 1). For instance, soft-bodied marine invertebrates and algae are susceptible
to diseases and tissue necrosis induced by bacteria, fungi and microalgae (Mitchell
and Chet 1975; Bouarab et al. 2001; Cooney et al. 2002). The sometimes drastic
changes of pH and redox conditions created by microepibionts may attack chemically
sensitive surfaces of the basibiont (Terry and Edyvean 1981). Importantly, the adverse
effects of microbial epibiosis may reach beyond pathogenicity and virulence. Since
microbial films are important sources of chemical cues for larval settlement in many
benthic marine invertebrates (Lau et al. 2002; Harder et al. 2002), microbial epibiosis
may promote subsequent colonization by rigid crustose epibiotic macroorganisms,
which in turn significantly impair the basibiont’s ability to exchange gases and
nutrients (Jagels 1973), damage the tissue by increased weight, rigidity and drag
(Dixon et al. 1981), and decrease the growth rate of photosynthetic basibionts by
cutting surface irradiance levels (Sand-Jensen 1977; Silberstein et al. 1986). From
a nutritional perspective it is evident that if the host and the epibiont share the
same trophic requirements then planktonic nutrients reaching the basibiont may
already be partially depleted after their passage through the epibiotic barrier. As
epibionts may fall victim to predators of their hosts, so may basibionts suffer from

Table 1 Ecological consequences for epibiont and basibiont as a result of epibiotic associations
(summarized from Wahl 1989)
Advantages Disadvantages
Epibiont Colonization of new substrate Unstable, non-durable substrate
New surface due to growth Biologically variable substrate
of basibiont
Nutrient flow from basibiont Exposure to detrimental host defence
Favourable hydrodynamic conditions Shared doom
Favourable exposure to light
Associational resistance
Basibiont Camouflage Increased weight and drag
Insulation against desiccation Decreased elasticity
Nutrient flow from epibiont Increased surface roughness
Associational resistance Increased deposition of particulate
material
Insulation against exchange of gas and
waste products
Increased mechanical damage
Increased chemical damage
Decreased nutrient flow through
epibiotic filter
Marine Epibiosis: Concepts, Ecological Consequences and Host Defence 225

“shared doom”, i.e. damage due to grazers preying on epibionts (Dixon et al. 1981).
Table 1 summarizes the advantages and disadvantages of epibiosis for epi- and
basibionts.

5 Defence

Many marine invertebrates and plants have evolved a variety of physical and chemical
defence mechanisms to suppress epibiosis and/or remove epibionts. Epibiont removal
can be physically achieved by continuous or periodic surface renewal or by means
of mucus secretion (e.g. in cnidaria, algae, molluscs, echinoderms and tunicates)
and periodical shedding of the cuticula or epidermis (Sieburth and Tootle 1981;
Littler and Littler 1999; Nylund and Pavia 2005) (see Fig. 1).
To create unfavourable or toxic conditions at or immediately above the living
surface is a wide-spread adaptation of host organisms to cope with epibionts.

Fig. 1 Surface of the macroalga Laminaria digitata showing sloughing of the cuticle-containing
bacteria and diatoms to reveal an uncolonized algal surface. Scale bar: 30 μm
226 T. Harder

The brown alga Laminaria digitata and the red alga Gracilaria conferta react with an
oxidative burst to the presence of either alginate oligosaccharides or agar oligosac-
charides, both of which are degradation products of their own cell walls (Küpper et al.
2001), resulting in the efficient elimination of bacterial epiflora (Weinberger et al.
2000). Moreover, sessile marine organisms feature a variety of chemical defence
metabolites effective against different phyla of potential epibionts (reviewed by
Clare 1996; Faulkner 2000). There are numerous studies on the inhibition of micro-
and macroorganisms by extracts from diverse marine eukaryotes, such as corals,
sponges, tunicates, ascidians and macrophytes (e.g. Michalek and Bowden 1997;
Jensen et al. 1996; Wilsanand 1999; Slattery et al. 1995; Hellio et al. 2000; Dobretsov
et al. 2006). Mostly, these investigations were descriptive and did not result in the
purification and elucidation of inhibitory compounds. It remains unclear whether
these extracts deter epibiosis at or near surfaces in situ and, if so, at what concentra-
tions these effects are elicited. In this context, one of the better-studied models for
algal secondary metabolism is the Australian red alga Delisea pulchra, which produces
a range of structurally similar halogenated furanones (Steinberg et al. 2001). These
metabolites are encapsulated in vesicles in the gland cells of D. pulchra, which
provide a delivery mechanism to the surface of the alga at concentrations that deter
a wide range of prokaryote and eukaryote epibionts (Maximilien et al. 1998).
Being structurally related to acylated homoserine lactones (AHLs), halogenated
furanones inhibit bacterial colonization through direct antagonism of bacterial
cell-to-cell signalling. The AHL-mediated gene expression of bacteria is inhibited
when halogenated furanones occupy the AHL-binding site of LuxR-like proteins,
which represent the transcriptional activators in AHL regulatory systems (Manefield
et al. 1999).
Information on the localization, identity and surface concentration of
defence secondary metabolites is rapidly advancing (e.g. Salomon et al. 2001;
Kubanek et al. 2002; Nylund et al. 2005; Paul et al. 2006) and the relevance of
defence metabolites is increasingly discussed in a chemical ecological context.
Moreover, recent studies on the deterrence of microbial colonization highlight
that chemical antifouling defences cannot be generalized as broadly bacterio-
static or bactericidal, instead the effects are quite selective and targeted against
particular microbial species (Maximilien et al. 1998; Egan et al. 2000; Kubanek
et al. 2003).
Besides antimicrobial effects of secondary metabolites emanating from the
host, recent studies have increasingly demonstrated that epibiotic bacteria associated
with the host deter growth and attachment of co-occurring bacterial species or new
epibiotic colonizers competing for the same niche (Armstrong et al. 2001; Harder
et al. 2004a). A well-investigated bacterium in this context is Pseudoalteromonas
tunicata, which has been isolated from a tunicate and a green macroalga. P. tuni-
cata has been found to produce at least five extracellular compounds that inhibit
other organisms from establishing themselves in a epibiotic community by inhibit-
ing settlement of invertebrate larvae and algal spores, growth of bacteria and fungi,
and surface colonization by diatoms (Holmström and Kjelleberg 1999; Holmström
Marine Epibiosis: Concepts, Ecological Consequences and Host Defence 227

et al. 1996). Thus, in terms of the chemical ecology of host–epibiont associations,

it seems evident that there is a significant protective role of symbiotic microbial
epibionts, which in turn release antifouling compounds. However, after more than
20 years of research there is no experimental evidence demonstrating if and how
host organisms selectively attract such epibionts.
With the advancement of molecular biological tools to analyse the diversity and
abundance of bacteria in biofilms (Dahllöf 2002), several studies have demon-
strated that quantitative and qualitative bacterial occurrence on host organisms
differs significantly from inanimate reference surfaces (Harder et al. 2003, 2004b;
Lee and Qian 2004; Dobretsov et al. 2006; Rao et al. 2005). These findings firstly
suggest strong host defence mechanisms against non-culturable epibiotic bacteria but
also support the notion of potent effects of non-culturable epibiotic bacteria against
subsequent colonizers of host organisms. It will be interesting to see follow-up
studies that utilize advanced molecular biological tools, such as cloning techniques,
to directly test the metabolites of non-culturable symbiotic prokaryotes on epibiotic
eukaryotes.

References

Armstrong E, Yan L, Boyd KG, Wright PC, Burgess JG (2001) The symbiotic role of marine
microbes on living surfaces. Hydrobiologia 461:37–40
Berntsson KM, Jonsson PR, Lejhall M, Gatenholm P (2000) Analysis of behavioral rejection of
micro-textured surfaces and implications for recruitment by the barnacle Balanus improvisus.
J Exp Mar Biol Ecol 251:59–83
Bouarab K, Potin P, Weinberger F, Correa J, Kloareg B (2001) The Chondrus crispus Acrochaete
operculata host-pathogen association, a novel model in glycobiology and applied phycopathology.
J Appl Phycol 13:185–193
Brouns JJWM, Heijs FML (1986) Production and biomass of the seagrass Anhalus acovoides (Lf)
Royle and its epiphytes. Aquat Bot 25:21–45
Butman CA (1987) Larval settlement of soft-sediment invertebrates: the spatial scales of pattern
explained by active habitat selection and the emerging role of hydrodynamical processes.
Oceanogr Mar Biol A Rev 25:113–165
Clare AS (1996) Marine natural product antifoulants. Biofouling 9:211–229
Clare AS, Matsumura K (2000) Nature and perception of barnacle settlement pheromones.
Biofouling 15:57–71
Clare AS, Rittschof D, Gerhart DJ, Maki JS (1992) Molecular approaches to non-toxic antifouling.
Invert Reprod Dev 22:67–76
Coon SL, Fitt WK, Bonar DB (1990) Competence and delay of metamorphosis in the Pacific oyster
Crassostrea gigas. Mar Biol 106:379–387
Cooney RP, Pantos O, Le Tissier MDA, Barer MR, O’Donnell AG, Bythell JC (2002)
Characterization of the bacterial consortium associated with black band disease in coral using
molecular microbiological techniques. Environ Microbiol 4:401–413
Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM (1995) Microbial
biofilms. Annu Rev Microbiol 49:711–745
Dahllöf I (2002) Molecular community analysis of microbial diversity. Curr Opin Biotechnol
13:213–217
228 T. Harder

Davis AR, Targett NM, McConnell OJ, Young CM (1989) Epibiosis of marine algae and benthic
invertebrates: natural products chemistry and other mechanisms inhibiting settlement and
overgrowth. In: ScheuerPJ (ed.) Bioorganic marine chemistry, vol 3. Springer, Heidelberg
Berlin New York, pp. 85–114
Dixon J, Schroeter SC, Kastendick J (1981) Effects of encrusting bryozoan, Membranipora mem-
branacea on the loss of blades and fronds by the giant kelp, Macrocystis pyrifera (Laminariales).
J Phycol 17:341–345
Dobretsov S, Dahms HU, Harder T, Qian PY (2006) Allelochemical defense of the macroalga
Caulerpa racemosa against epibiosis: evidence of field and laboratory assays. Mar Ecol Prog
Ser 318:165–175
Dreanno C, Matsumura K, Dohmae N, Takio K, Hirota H, Kirby R, Clare AS (2006) An α-2-
macroglobulin-like protein is the cue to gregarious settlement of the barnacle Balanus
amphitrite. Proc Natl Acad Sci U S A 103:14396–14401
Egan S, Thomas T, Holmström C, Kjelleberg S (2000) Phylogenetic relationship and antifouling
activity of bacterial epiphytes from the marine alga Ulva lactuca. Environ Microbiol
2:343–347
Faulkner DJ (2000) Marine pharmacology. Antonie Van Leeuwenhoek 77:135–145
Fletcher RL, Callow ME (1992) The settlement, attachment and establishment of marine algal
spores. Br Phycol J 27:303–329
Gil-Turness MS, Fenical W (1992) Embryos of Homarus americanus are protected by epibiotic
bacteria. Biol Bull 182:105–108
Grossart HP, Kiørboe T, Tang K, Ploug H (2003) Bacterial colonization of particles: growth and
interactions. Appl Environ Microbiol 69:3500–3509
Hadfield MG, Scheuer D (1985) Evidence for a soluble metamorphic inducer in Phestilla sibogae:
ecological, chemical and biological data. Bull Mar Sci 37:556–566
Hadfield MG, Paul VJ (2001). Natural chemical cues for settlement and metamorphosis of
marine-invertebrate larvae: 431–461. In: McClintock JB, Baker BJ (eds.) Marine chemical
ecology. CRC, Boca Raton, pp. 1–610
Hahn KO (1989) Induction of settlement in competent abalone larvae. In HahnKO (ed.) Handbook
of culture of abalone and other marine gastropods. CRC, Boca Raton, pp. 101–112
Harder T, Lam C, Qian PY (2002) Induction of larval settlement of the polychaete Hydroides
elegans (Haswell) by marine biofilms: an investigation of monospecific diatom films as settlement
cues. Mar Ecol Prog Ser 229:105–112
Harder T, Lau SCK, Dobretsov S, Fang TK, Qian PY (2003) A distinctive epibiotic bacterial com-
munity on the soft coral Dendronephthya sp. and antibacterial activity of coral tissue extracts
suggest a chemical mechanism against bacterial epibiosis. FEMS Microbiol Ecol 43:337–347
Harder T, Dobretsov S, Qian PY (2004a) Waterborne, polar macromolecules act as algal antifoulants
in the seeweed Ulva reticulata. Mar Ecol Prog Ser 274:131–141
Harder T, Lau SCK, Tam WY, Qian PY (2004b) A bacterial culture-independent method to investigate
chemically mediated control of bacterial epibiosis in marine invertebrates by using TRFLP
analysis and natural bacterial populations. FEMS Microbiol Ecol 47:93–99
Harlin MM (1973) Transfer of products between epiphytic marine algae and host plants. J Phycol
9:243–248
Hay ME (1986) Associational plant defenses and the maintenance of species diversity: turning
competitors into accomplices. Am Nat 128:617–641
Hellio C, Bremer G, Pons AM, Le Gal Y (2000) Inhibition of the development of microorganisms
(bacteria and fungi) by extracts of marine algae from Brittany, France. Appl Microbiol
Biotechnol 54:543–549
Holmström C, Kjelleberg S (1999) Marine Pseudoalteromonas species are associated with higher
organisms and produce biologically active extracellular agents. FEMS Microbiol Ecol
30:285–293
Holmström C, James S, Egan S, Kjelleberg S (1996) Inhibition of common fouling organisms by
pigmented marine bacterial isolates. Biofouling 10:251–259
Marine Epibiosis: Concepts, Ecological Consequences and Host Defence 229

Huggett MJ, Williamson JE, De Nys R, Kjelleberg S, Steinberg PD (2006) Larval settlement of
the common Australian sea urchin Heliocidaris erythrogramma in response to bacteria from
the surface of coralline algae. Oecologia 149:604–619
Jagels R (1973) Studies of a marine grass, Thalassia testudinum I. Ultrastructure of the osmoregu-
latory leaf cells. Am J Bot 60:1003–1009
Jensen PR, Harvell CD, Wirtz K, Fenical W (1996) Antimicrobial activity of extracts of Caribbean
gorgonian corals. Mar Biol 125:411–419
Johnson CR, Sutton DC (1994) Bacteria on the surface of crustose coralline algae induce meta-
morphosis of the crown-of-thorns starfish Acanthaster planci. Mar Biol 120:305–310
Johnson CR, Muir DG, Reysenbach AL (1991a) Characteristic bacteria associated with surfaces
of coralline algae: a hypothesis for bacterial induction of marine invertebrate larvae. Mar Ecol
Prog Ser 74:281–294
Johnson CR, Sutton DC, Olson RR, Giddins R (1991b) Settlement of crown-of-thorns starfish:
role of bacteria on surfaces of coralline algae and a hypothesis for deepwater recruitment. Mar
Ecol Prog Ser 71:143–162
Joint I, Tait K, Callow ME, Callow JE, Milton D, Williams P (2002) Cell-to-cell communication
across the procaryote – eucaryote boundary. Science 298:1207
Kato T, Kumanireng AS, Ichinose I, Kitahara Y, Kakinuma Y, Nishihira M, Kato M (1975) Active
components of Sargassum tortile effecting the settlement of swimming larvae of Coryne uchidai.
Experientia 31:433–434
Keough MJ (1986) The distribution of a bryozoan on seagrass blades: settlement, growth and
mortality. Ecology 67:846–857
Kolattukudy PE, Rogers LM, Li D, Hwang CS, Flaishman MA (1995) Surface signaling in patho-
genesis. Proc Natl Acad Sci U S A 92:4080–4087
Krug PJ, Manzi AE (1999) Waterborne and surface-associated carbohydrates as settlement cues
for larvae of the specialist marine herbivore Alderia modesta. Biol Bull 197:94–103
Kubanek J, Whalen KE, Engel S, Kelly SR, Henkel TP, Fenical W, Pawlik JR (2002) Multiple
defensive roles for triterpene glycosides from two Caribbean sponges. Oecologia 131:125–136
Kubanek J, Jensen PR, Keifer PA, Sullards MC, Collins DO, Fenical W (2003) Seaweed resistance
to microbial attack: a targeted chemical defense against marine fungi. Proc Natl Acad Sci U S A
100:6916–6921
Küpper FC, Kloareg B, Guern J, Potin P (2001) Oligoguluronates elicit an oxidative burst in
brown algal kelp, Laminaria digitata. Plant Physiol 12:278–291
Laihonen P, Furman ER (1986) The site of settlement indicates commensalism between blue mussel
and its epibiont. Oecologia 71:38–40
Lambert WJ, Todd CD, Hardege JD (1997) Partial characterization and biological activity of a
metamorphic inducer of the dorid nudibranch Adalaria proxima (Gastropoda: Nudibranchia).
Invertebr Biol 116:71–81
Lau SCK, Mak KKW, Chen F, Qian PY (2002) Bioactivity of bacterial strains from marine biofilms
in Hong Kong waters for the induction of larval settlement in the marine polychaete Hydroides
elegans. Mar Ecol Prog Ser 226:301–310
Lee O, Qian PY (2004) Potential control of bacterial epibiosis on the surface of the sponge Mycale
adhaerens. Aquat Microb Ecol 34:11–21
Lemos ML, Toranzo AE, Barja JL (1985) Antibiotic activity of epiphytic bacteria isolated from
intertidal seaweeds. Microbiol Ecol 11:149–163
Littler MM, Littler DS (1999) Blade abandonment/proliferation: a novel mechanisms for rapid
epiphyte control in marine macrophytes. Ecology 80:1736–1746
Maida M, Coll JC, Samarco PW (1994) Shedding new light on scleractinian coral recruitment.
J Exp Mar Biol Ecol 180:189–202
Maki JS, Mitchell R (2002) Biofouling in the marine environment. In: Bitton G (ed.) Encyclopedia
of environmental microbiology. Wiley, New York, pp. 610–619
Manefield M, de Nys R, Kumar N, Read R, Givskov M, Steinberg PD, Kjelleberg S (1999)
Inhibition of LuxR-based AHL regulation by halogenated furanones from Delisea pulchra.
Microbiology 145:283–291
230 T. Harder

Matsumura K, Nagano M, Fusetani N (1998) Purification of a larval settlement-inducing protein

complex (SIPC) of the barnacle, Balanus amphitrite. J Exp Zool 281:12–20
Maximilien R, de Nys R, Holmström C, Gram L, Givskov M, Crass K, Kjelleberg S, Steinberg
PD (1998) Chemical mediation of bacterial surface colonization by secondary metabolites
from the red alga Delisea pulchra. Aquat Microb Ecol 15:233–246
Michalek K, Bowden BF (1997) A natural algacide from soft coral Sinularia flexibilis
(Coelenterata, Octocorallia, Alcyonacea). J Chem Ecol 23:259–273
Mitchell R, and Chet I (1975) Bacterial attack of corals in polluted seawater. Microb Ecol
2:227–233
Morse DE (1990) Recent progress in larval settlement and metamorphosis: closing the gaps
molecular biology and ecology. Bull Mar Sci 46:465–483
Morse ANC, Morse DE (1984) Recruitment and metamorphosis of Haliotis larvae induced by
molecules uniquely available at the surface of crustose red alga. J Exp Mar Biol Ecol
75:191–215
Mullineaux LS, Butman CA (1991) Initial contact, exploration and attachment of barnacle
(Balanus amphitrite) cyprids settling in flow. Mar Biol 110:93–103
Nakanishi K, Nishijima M, Nomoto AM, Yamazali A, Saga N (1999) Requisite morphologic
interaction for attachment between Ulva pertusa (Chlorophyta) and symbiotic bacteria. Mar
Biotech 1:107–111
Nylund G, Cervin G, Hermansson, Pavia H (2005) Chemical inhibition of bacterial colonization
by the red alga Bonnemaisonia hamifera. Mar Ecol Prog Ser 302:27–36
Nylund G, Pavia H (2005) Chemical versus mechanical inhibition of fouling in the red alga Dilsea
carnosa. Mar Ecol Prog Ser 299:111–121
Paul NA, de Nys R, Steinberg PD (2006) Chemical defence against bacteria in the red alga
Asparagopsis armata: linking structure with function. Mar Ecol Prog Ser 306:87–101
Qian PY, Pechenik JA (1998) Effects of larval starvation and delayed metamorphosis on juvenile
survival and growth of the tube-dwelling polychaete Hydroides elegans (Haswell). J Exp Mar
Biol Ecol 227:169–185
Qian PY, Rittschof D, Sreedhar B (2000) Macrofouling in unidirectional flow: miniature pipes
as experimental models for studying the interaction of flow and surface characteristics on
the attachment of barnacle, bryozoan and polychaete larvae. Mar Ecol Prog Ser
207:109–121
Rao D, Webb S, Kjelleberg S (2005) competitive interactions in mixed-species biofilms containing
the marine bacterium Pseudoalteromonas tunicata. Appl Environ Microbiol 71:1729–1736
Rodriguez SR, Ojeda FP, Inestrosa NC (1993) Settlement of benthic marine invertebrates. Mar
Ecol Prog Ser 97:193–207
Salomon CE, Deerinck T, Elissman MH, Faulkner DJ (2001) The cellular localization of dercita-
mide in the Palauan sponge Oceanapia sagittaria. Mar Biol 139:313–319
Sand-Jensen K (1977) Effect of epiphytes on eelgrass photosynthesis. Aquat Bot 3:55–63
Sieburth JM, Tootle JL (1981) Seasonality of microbial fouling on Ascophyllum nodosum (L.)
Lejol., Fucus vesiculosus L., Polysiphonia lanosa (L.) Tandy and Chondrus crispus Stackh.
J Phycol 17:57–64
Silberstein K, Chiffings AW, McComb AJ (1986) The loss of seagrass in Cockburn Sound,
Western Australia. 3. The effect of epiphytes on productivity of Posidonia australis. Hook F
Aquat Bot 24:355–371
Slattery M, McClintock JB, Heine JN (1995) Chemical defenses in Antarctic soft corals: evidence
for antifouling compounds. J Exp Mar Biol Ecol 190:61–77
Steinberg PD, De Nys R, Kjelleberg S (2001) Chemical mediation of surface colonization, In:
McClintock JB, Baker JB (eds.) Marine chemical ecology. CRC, Boca Raton, pp. 355–387
Steinberg PD, de Nys R, Kjelleberg S (2002) Chemical cues for surface colonization. J Chem Ecol
28:1935–1951
Swanson RL, Williamson JE, De Nys R, Kumar N, Bucknall MP, Steinberg PD (2004) Induction
of settlement of larvae of the sea urchin Holopneustes purpurascens by histamine from a host
alga. Biol Bull 206:161–172
Marine Epibiosis: Concepts, Ecological Consequences and Host Defence 231

Swanson RL, de Nys R, Huggett MJ, Green JK, Steinberg PD (2006) In situ quantification of a
natural settlement cue and recruitment of the Australian sea urchin Holopneustes purpurascens.
Mar Ecol Prog Ser 314:1–14
Tamburri MN, Zimmer-Faust RK, Tamplin ML (1992) Natural sources and properties of chemical
inducers mediating settlement of oyster larvae: a re-examination. Biol Bull 183:327–338
Tatewaki M, Provasoli L, Pintner IJ (1983) Morphogenesis of Monostroma oxyspermum
(Kuetz.) Doty (Chlorophyceae) in axenic culture, especially in bialgal culture. J Phycol
19:409–416
Terry LA, Edyvean RGJ (1981) Microalgae and corrosion. Bot Mar 24:177–183
Thevanathan R, Nirmala M, Manoharan A, Gangadharan A, Rajarajan R, Dhamotharan R,
Selvaraj S (2000) On the occurrence of nitrogen fixing bacteria as epibacterial flora of some
marine green algae. Seaweed Res Utiln 22:189–197
Todd CD, Keough MJ (1994) Larval settlement in hard substratum epifaunal assemblages: a
manipulative field study of the effects of substratum filming and the presence of incumbents.
J Exp Mar Biol Ecol 181:159–187
Tsukamoto S, Kato H, Hirota H, Fusetani N (1994) Narains: N,N-dimethylguanidinium styryl sul-
fates, metamorphosis inducers of ascidian larvae from a marine sponge Jaspis sp. Tetrahedron
Lett 35:5873–5874
Tsukamoto S, Kato H, Hirota H, Fusetani N (1995) Pipecolate derivatives, anthosamines A and B,
inducers of larval metamorphosis in ascidians, from a marine sponge Anthosigmella aff. raro-
microsclera. Tetrahedron 51:6687–6694
Tsukamoto S, Kato H, Hirota H, Fusetani N (1999) Lumichrome – a larval metamorphosis-
inducing substance in the ascidian Halocynthia roretzi. Eur J Biochem 264:785–789
Turner EJ, Zimmer-Faust RK, Palmer MA, Luckenback (1994) Settlement of oyster Crassostrea
virginica larvae: effects of water flow and a water-soluble chemical cue. Limnol Oceanogr
39:1579–1593
Wahl M (1989) Marine epibiosis. I. Fouling and antifouling: some basic aspects. Mar Ecol Prog
Ser 58:175–189
Wahl M (1997) Living attached: aufwuchs, fouling, epibiosis. In: Nagabushanam R, Thompson
MF (eds.) Fouling organisms of the Indian Ocean: biology and control technology. Oxford and
IBH, New Delhi, pp. 31–83
Wahl M, Hay ME (1995) Associational resistance and shared doom: effects of epibiosis on herbivory.
Oecologia 102:329–340
Wahl M, Mark O (1999) The predominantly facultative nature of epibiosis: experimental and
observational evidence. Mar Ecol Prog Ser 187:59–66
Wahl M, Hay ME, Enderlein P (1997) Effects of epibiosis on consumer-prey interactions.
Hydrobiologia 355:49–59
Weinberger F, Friedlander M (2000) Response of Gracilaria conferta (Rhodophyta) to oligoagars
results in defense against agar-degrading epiphytes. J Phycol 36:1079–1086
Wieczorek SK, Todd CD (1998) Inhibition and facilitation of the settlement of epifaunal marine
invertebrate larvae by microbial biofilm cues. Biofouling 12:81–93
Williamson JE, De Nys R, Kumar N, Carson DG, Steinberg PD (2000) Induction of metamorphosis
in the sea urchin Holopneustes purpurascens by a metabolite complex from the algal host
Delisea pulchra. Biol Bull 198332–345
Wilsanand V, Wagh AB, Bapuji M (1999) Effect of alcohol extracts of demospongiae on growth
of periphytic diatoms. Ind J Mar Sci 28:274–279
Yvin JC, Chevolet L, Chevolet-Maguer AM, Cochard JC (1985) First isolation of jacarone from an
alga, Delesseria sanguinea: a metamorphosis inducer of Pecten larvae. J Nat Prod 48:814–816
Larval Settlement and Surfaces: Implications
in Development of Antifouling Strategies

P. Sriyutha Murthy (*
ü ), V. P. Venugopalan, K. V. K. Nair,
and T. Subramoniam

Abstract Marine biofouling is a natural process that imposes technical operational

problems and economic losses on marine-related activities. Marine biofouling com-
munities are complex, diverse, highly dynamic ecosystems consisting of a range of
organisms. Larvae of these organisms spend a part of their lives in the planktonic
stage before settling on a surface. Passive transport and deposition of larvae were
considered responsible for the observed spatial variation in settlement pattern,
whereas breeding season and larval survival have been associated with temporal
fluctuations. Hydrodynamic conditions influence the transport and deposition of
larvae near the surface boundary layer, while dissolved environmental stimuli have
been associated with the induction of settlement and metamorphic behaviour. Over
the last two decades, chemical cues and physiological processing of the cue has
been the subject of study. Sufficient information has been obtained on the settle-
ment mechanisms, the nature of chemical substances, involvement of chemosen-
sory receptors and signal transduction pathways downstream. Knowledge on the
settlement mechanism is imperative for developing a suitable control strategy.
At present, more is known about chemosensory reception and downstream process-
ing of the sensory cue than the location of these receptors. The need to control
biofouling on underwater surfaces has given rise to many different technologies.
Conventional antifouling strategy employs the use of biocidal surface coatings.
The rationale behind these coatings is to kill everything. Historically, different solu-
tions for control of fouling have been employed. It was not until the development of
cold-plastic antifouling paints (copper oxide and tributyltin oxide or fluoride) in the
later part of the twentieth century that a truly long-lasting protection was achieved.
Unfortunately, the accumulation of slow-degrading organotin moieties in the water
column has resulted in sub-lethal effects on non-target organisms, which led to its
progressive abandonment. Insights into the larval sensory recognition of physical
cues and adhesion resulted in the development of foul release coatings based on low
surface energy phenomenon. Another alternative approach for control of biofouling

P.S. Murthy
Biofouling and Biofilm Processes Section, Water and Steam Chemistry Division, BARC
Facilities, Indira Gandhi Center for Atomic Research Campus, Kalpakkam 603 102, India
e-mail: psm_ murthy@yahoo.co.in, psmurthy@igcar.gov.in

Springer Series on Biofilms, doi: 10.1007/7142_2008_17 233 233

© Springer-Verlag Berlin Heidelberg 2008
234 P.S. Murthy et al.

and inhibition of larval settlement lies in inhibiting the neurophysiological

processes involved in larval settlement. This has been experimented upon using
natural bioactive molecules and synthetic analogues, which bind to specific
receptors inhibiting larval settlement. Several pharmacological compounds, natural
products and synthetic analogues that inhibit the metabolic processes underlying
settlement have been identified through laboratory bioassays. However, realization
of these compounds into commercial coatings is yet to happen. The reasons may
be attributed to reproducibility of laboratory results in ecologically realistic field
experiments. The need for suitable bioassays and knowledge of the broad-
spectrum activity of these compounds is obvious.

1 Introduction to Biofouling and Antifouling

The problem of biofouling presents a serious operational problem. In heat

exchangers it leads to reduced heat transfer efficiency, increased fluid frictional
resistance, additional maintenance and operational costs. On ship hulls the prob-
lem leads to increase in hydrodynamic drag and fuel consumption and decreases
the manoeuvrability of vessels. On submerged structures the problem leads to
hydrodynamic loading, material deterioration and failure of moored instruments
and the optical windows of sensors. The problem of biofouling is a surface-
associated phenomenon and control measures should be focussed on this aspect.
Conventionally, fouling mitigation in heat transfer equipment is carried out by the
addition of biocides (see Nymer et al. 2008). For protection of submerged struc-
tures and ship hulls most antifouling systems take the form of protective coatings.
Understanding the mechanisms of larval settlement and metamorphosis in benthic
invertebrates is important for developing suitable methods for interfering with the
settlement process and inhibiting settlement. In this chapter, molecular cues and
pathways involved in settlement of marine benthic organisms and the antagonistic
activity of synthetic and natural compounds in settlement prevention are eluci-
dated. In addition, an overview on the status of different antifouling strategies
being researched is also presented. Benthic marine communities are dominated by
diverse invertebrate fauna representing over 4,000 different species (Crisp 1974).
This chapter is limited to the dominant fouling organisms such as barnacles,
hydrozoans, tube-building polychaete worms and molluscs.

2 Physical Cues and Antifouling; Where do We Stand?

The possibility of larvae responding to environmental cues is debatable until

they are in close proximity with the near-surface boundary layer (Butman et al.
1988). Interaction of physical processes and hydrodynamics (Pawlik et al.
Larval Settlement and Surfaces 235

1991; Walters 1992; Millineaux and Garland 1993) assist in bringing the larvae
to the near-surface boundary layer, within the perception of physical or chemi-
cal cues. Sensory recognition of cues results in settlement, and the transforma-
tion of signals into the larval neuronal systems results in initiation of
differentiation and metamorphosis. Physical characteristics of the substratum
seem to be of secondary importance when compared to chemical and biologi-
cal characteristics (Mihm et al. 1981; LeTourneux and Bourget 1988) in set-
tlement induction. Physical factors such as light or shade, substratum type,
orientation depth, gravity, hydrostatic pressure and temperature are found to
influence larval behaviour and settlement in many invertebrate species (Thorson
1964; Crisp 1974; Ryland 1974; Sulkin 1984; Young and Chia 1987; Boudreau
et al. 1990; Kaye and Reiswig 1991; Anderson and Underwood 1994; Holiday
1996; Connell 1999; Galsby 1999, 2000; Forde and Raimondi 2004). In com-
parison to the above mentioned factors, physical characteristics of the substra-
tum like surface energy (wettability), roughness and microtopography have
been shown to have a measurable effect on larval settlementand can be probably
mimicked for producing antifouling surfaces. The mechanism of sensing physi-
cal cues like surface energy is thought to be initiated when the larvae abandon
swimming behaviour and commence surface exploration. Alternatively, it is
believed to be by the action of physiochemical forces between the surface and
the larval body or antennules. Laboratory and field studies conducted by
Rittschof and Costlow (1989a, b) and Roberts et al. (1991) (also, see Cooksey et al.
2008) showed similar trends in relation to surface energy by different larval
groups. Larvae of barnacles preferred high-energy surfaces, bryozoans and
ascidians preferred low-energy surfaces and larvae of hydroids settled
equally on surfaces of all energies. A species-specific difference was
observed with respect to Balanus amphitrite and Balanus improvisus cyprids.
B. amphitrite cyprids preferred unfouled high-energy surfaces (Rittschof et al.
1984) whereas B. improvisus cyprids preferred hydrophilic surfaces (Dahlstrom
et al. 2004). An intermediate change in surface wettability inhibited settlement
by 38% for this barnacle.
An interesting aspect of these studies is that no surface energy escapes settlement
by at least some group of macrofoulants. The surface energy principle has been
used successfully in the development of foul release silicone coatings where
additional lubricity has been provided by the addition of silicone oil. Decreasing
the surface elastic modulus to about 0.01–0.1 KPa significantly decreased larval
settlement in field trials as well as in laboratory experiments conducted with
bryozoan larvae (Natasha et al. 2002). This response to the elastic modulus may
be a result of mechanical deformation of membranes of the sensory cells used to
explore surfaces. Lower elastic modulussurfaces are also known to cause altera-
tion of ionic traffic across membranes and present a weaker signal for settlement
(Natasha et al. 2002). The use of surface energy and elastic modulus principles
for antifouling is still in its infancy. Species-specific data need to be generated
and correlated to obtain a value for these parameters to be incorporated into
antifouling coatings.
236 P.S. Murthy et al.

3 Induction Pathways and Physiological Events

on Perception of Cues

In each of the invertebrate phyla there are representations that have shown a unique
response to settlement and metamorphic cues and pathways. At the molecular and
cellular level, the receptors and signal transducers that control settlement and meta-
morphosis are proving to be structurally, functionally and evolutionarily closely
related to receptors and transducers of higher organisms. Most of the invertebrate
larvae are passive to the large-scale advective processes that bring the larvae near
settlement sites, whereas the irreversible phase of metamorphosis is dependent on the
sensory information processed by the larvae. Broadly, the exogenous and endogenous
events leading to settlement and metamorphosis (Fig. 1) can be classified into:
1. Induction by morphogenetic pathway
2. Induction by regulatory pathway
3. Induction by second messenger or diacylglycerol pathway
4. Induction by amino acid derivatives
5. Induction by catecholamine pathway
6. Induction by choline derivatives
7. Induction by ion-gated channels
8. Induction by nitric oxide synthase pathway
9. Induction by waterborne substances
10. Inducers associated with microbial biofilms
11. Inducers of biological or synthetic origin

3.1 Induction by Morphogenetic Pathway

The morphogenetic pathway proposed by Morse (1992) operates via larval sensory
recognition of exogenous inducing molecules by specialized chemosensory receptors,
resulting in the sequential activation of a larval membrane receptor (γ-aminobutyric
acid and dihydroxyphenylalanine receptors) and membrane-associated adenyl cyclase
cascade. This involves synthesis and activation of cyclic adenosine monophosphate
(cAMP), calcium-regulated protein kinase (PKA), protein phosphorylation and open-
ing of chloride or other anion channels in the chemosensory cell membrane and an
efflux of chloride or other anions, resulting in excitatory depolarization or firing of the
chemosensory cell (Fig. 1). This morphogenetic environmental stimulus is transduced
to an electrochemical signal that is propagated in the larval nervous system.

3.2 Induction by Regulatory or Amplifier Pathway and

the Second Messenger Diacylglycerol Pathway

Another signal transduction pathway involved in recognition of the chemical

signal is the amplifier or regulatory pathway (Morse 1992), which amplifies the
 SIGNAL SIGNAL
VOLTAGE VOLTAGE AMPLIFIER TRIGGER
GATED Ca2+ GATED k+ Natural Natural
CHLORIDE Sources Sources
INDUCER INDUCER DISS AMINO
ION Crustose Bacterial films
ACIDS
Excess Excess K+ INDUCER and
Ca2+ PEPTIDES Ivermectin INDUCER
A23187 (WATER BORNE) INDUCER L-DOPA
BLOCKER BACTERIAL & BLOCKERS GABA D-DOPA
BLOCKER S ALGAL PRODUCTS Picrotoxin SERATONIN
S TEA DISS EPINEPHRINE
METABOLITES NOREPINEPHRINE
BACTERI Diltiazem Na / K DOPAMINE
AL GABA
DIACTONOGLYCEROL DAPA / LYSINE RECEPTOR
2+ + RECEPTOR K+
Ca K
INDUCER DOPA
PLASMA
MEMBRANE Cholera RECEPTOR

anostilbene; TEA tetraethylammonium chloride

PK
PK CC
PIC
P PiP A
G
Cs+ INDUCER
Pro
DEPOLARIZATION Cl-
PK ATP IBMX
DA
Ca2+ FORSKOLIN
G THEOPHYLLIN
Pro cAMP E
EXCITATORY CALCIUM
DEPOLARIZATION
P
METAMORPHOSIS IONOPHORES

PKA FATTY ACIDS

Arachidonic
BEHAVIOUR Linoleic
GENE
EXPERESSION
ENDOGENOUS DIFFERENTIATI REGULATORY MORPHOGENETIC
ON PATHWAY PATHWAY
EVENTS
GROWTH

methylxanthine; K+ potassium ions; PiP2 phosphatidylinositol bisphosphate; PK protein kinase;

P protein phosphorylation; PL phospholipase; PDE phosphodiesterase; SITS sulfonyl isothiocy-
erol; DAPA diamino propionic acid; GABA γ-aminobutyric acid; G Pro G protein; IBMX isobutyl-
adenosine monophosphate; db-cAMP dibuteryl cyclic adenosine monophosphate; DG diacylglyc-
phore; ATP adenosine tri phosphate; Ca2+ calcium ions; CC chloride ion channel; cAMP cyclic
metamorphosis in various invertebrate larval models. AC adenyl cyclase; A23187 calcium iono-
Fig. 1 Schematic overview integrating different postulated pathways inducing settlement and
238 P.S. Murthy et al.

sensitivity of the larvae to the required morphogenetic signal by as much as 100-

fold. This pathway, rather than inducing irreversible changes, may act as ampli-
fier of the signal generated in the environment. The environmental signals
include lysine and related diamino acids dissolved in seawater, which are found
to induce settlement and metamorphosis in the abalone Haliotis rufescens. This
lysine receptor binding signal is transduced by a receptor-associated G protein,
which in turn leads to the activation of a diacylglycerol-stimulated calcium-
stimulated protein kinase C (PKC) that phosphorylates a specific target protein
(Fig. 1).

3.3 Induction by Amino Acid Derivatives

Gamma-aminobutyric acid (GABA), a product of glutamic acid decarboxylation, is

known to hyperpolarize postsynaptic membranes by increasing membrane perme-
ability to Cl− ions (Kuffler et al. 1984).

3.4 Induction by Catecholamine Pathway

Artificial inducers or neurotransmitters that mimic the action of natural compounds

have also been investigated in many marine invertebrate species. The ability of the
neurotransmitter to mimic chemical cues emanating from the substratum implies
the operation of neuronal receptors and the involvement of the nervous system in
the initial processes that trigger settlement (Baloun and Morse 1984a, b; Yool et al.
1986). Experimentation on neuroactive molecules commenced after natural inducer
substances eluded isolation and identification.

3.5 Induction by Choline Derivatives

Choline, a constituent of cell membranes and precursor of the neurotransmitter

acetylcholine has been found to induce settlement of larvae (1) by acting directly
on cholinergic receptors, (2) acting as a precursor of acetylcholine and (3) by stimu-
lating synthesis and release of catecholamines (Hirata and Hadfield 1986;
Pennington and Hadfield 1988).

3.6 Induction by Ion-Gated Channels

Another pathway by which larvae have been shown to settle and metamorphose
is through ion-gated channels, such as those involving K+, Ca2+, Mg2+ and Na+. ln
Larval Settlement and Surfaces 239

general, marine invertebrate larval settlement and metamorphosis have been

understood to be under possible nervous control (Hadfield 1978; Burke 1983;
Morse et al. 1984; Rittschof et al. 1986; Bonar et al. 1990). In invertebrate chem-
oreceptors, a stimulus-dependent increase in the cell membrane permeability can
transduce chemical stimuli into electrical impulses along the nervous system
(Morita 1972; Thurm and Wessel 1979; Kaissling and Thorson 1980). Perception
of signals by neuronal systems has been found to involve the depolarization of
specialized cells in response to an appropriate stimulus (Aidley 1978). Conduction
of electrical impulses in nervous tissues depends on the maintenance of an
electrical potential across the cell membrane, which is a function of differential
permeability of the membrane to Na+, K + and Cl− ions (Kuffler et al. 1984) .
The functioning of excitable cells involves the selective movement of ions across
specialized membranes. Perception of inductive cues by larvae is found to be
dependent on stimulus-mediated depolarization of cells in a sensory inductive
pathway (Baloun and Morse 1984a, b). Earlier studies on the nervous control of
larval metamorphosis have been carried out by exposing the larvae to altered
ionic composition and in the presence of neuropharmacological probes (Morse et
al. 1979; Baloun and Morse 1984a; Rittschof et al. 1986).

3.7 Induction by Nitric Oxide Pathway

Nitric oxide (NO) a neurotransmitter present in neural circuits of vertebrate and

arthropods has been demonstrated to play a role in larval settlement. NO (a signal-
ling molecule) production is effected by nitric oxide synthase (NOS), which
catalyses the conversion of l-arginine to l-citrulline. The pathway was first
proposed for the mollusc, where inhibition of NOS activity resulted in the induction
of metamorphosis in the marine snail llyanassa obsoleta (Froggett and Leise 1999).
This pathway has also been demonstrated in the sea urchin Lytechinus pictus
(Bishop and Brandhorst 2001).

4 Pharmacological Control of Larval Settlement

Several neurotransmitters and neuromodulators play inductive or inhibitory roles in

the pathways that govern larval settlement and metamorphosis. Environmental cues
influencing larval settlement have been studied but specific molecular details and
downstream neuroendocrine actions that control the events of larval metamorphosis
have often been bypassed. Pharmacological approaches are helpful in understand-
ing the internal biochemical pathways and this section deals with agonists and
antagonists that effectively block the neurotransmitter pathway, resulting in settle-
ment inhibition of fouling organisms.
240 P.S. Murthy et al.

4.1 Barnacles

B. amphitrite has been the most widely studied organism with respect to its
settlement induction and inhibition. Larval sensory recognition of environmental
cues in barnacles is by physical surface contact of antennules, wherein structures
proposed to be situated on the fourth antennular segment are employed for chemi-
cal recognition (Nott and Foster 1969; Hoeg et al. 1988; Clare and Nott 1994).
Multiple settlement induction pathways have been demonstrated for this species
of barnacle. Settlement-inducing protein complex(SIPC) is a glycoprotein of high
molecular mass consisting of three major subunits of 76, 88 and 98 kDa with a
lentil lectin (LCA)-binding sugar chain. It was found to be synthesized during
larval development and induced settlement in B. amphitrite cyprids (Matsumara
et al. 1998).
An increase in acetylcholine by partial inhibition of acetyl cholinesterase
activity increased settlement (Faimali et al. 2003) and induced muscular contrac-
tion and cement gland exocytosis in B. amphitrite cyprids. Inhibition of acetyl
cholinesterase (AchE), the lytic enzyme of acetylcholine, by inhibitors such as
eserine, methomyl and mercaptodimetur (carbamates) was found to promote set-
tlement. In contrast, settlement was inhibited by the cholinomimetic drugs atro-
pine, an alkaloid and natural antagonist of muscarinic receptors (mAChRs), and
nicotine, an agonist of nicotinic receptors (nAChRs) affecting cholinergic neuro-
transmission through binding of cholinergic receptors. Incidentally, a substance
that inhibits AcHE activity has been isolated from the sponge Reniera sarai
(Sepcic et al. 1998; Faimali et al. 2003) and can be used as a potential lead in
settlement inhibition of these barnacles. Apart from acetylcholine, cAMP was
also found to induce settlement in B. amphitrite through the morphogenetic
pathway (Clare et al. 1995).
Activation of adenylate cyclase with forskolin and the inhibition of phos-
phodiesterase with 3-isobutyl-1-methylxanthine, caffeine and theophylline sig-
nificantly increased the settlement of B. amphitrite cyprids. No significant
increase in settlement was observed with the cAMP analogues dibutyryl cAMP
(db-cAMP), dibutyryl cyclic GMP, 8-(4-chlorophenylthio) (CPT) cAMP or
papaverine(phosphodiesterase inhibitors), but db-cAMP induced metamorphosis
in B. amphitrite (Rittschof et al. 1986). Induction of metamorphosis by db-cAMP
by triggering the second messenger pathway was also observed in B. amphitrite
cyprids; however, the response was not significantly greater than with cAMP.
Treatment with forskolin significantly increased the cAMP titre of cyprids
(Clare et al. 1995). Miconazole nitrate, an adenylate cyclase inhibitor that inhib-
ited settlement by blocking cAMP production, could be used as an antifouling
agent. In addition, PKC has been shown to be involved in barnacle settlement
and metamorphosis (Yamamoto et al. 1995); specific inhibitors of these enzymes
may be effective antifouling agents.
Involvement of another group of receptors known as tyrosine kinase-linked
receptors and their transduction pathways in barnacle settlement induction was
Larval Settlement and Surfaces 241

demonstrated by Okazaki and Shizuri (2000). AG-879, a nerve growth factor

receptor-tyrosine kinase (NGFR-TK) inhibitor; tyrphostin 9, a platelet-derived
growth factor receptor-tyrosine kinase (PDGRF-TK) inhibitor; tyrphostin 25, an
epidermal growth factor receptor-tyrosine kinase (EGFR-TK) inhibitor; insulin
receptor-tyrosine kinase (InsR-TK) inhibitor; phospholipase C (PLC) inhibitor;
Wortmanin, a phoshatidylinositol-3 kinase (PI3K) inhibitor; and PD-98059, a
mitogen-activated protein kinase (MAPK) inhibitor all suppressed cyprid settle-
ment in a dose-dependent manner (Okazaki and Shizuri 2000). These compounds
and their synthetic analogues may be tried out in antifouling paint matrices to test
their efficacy in the field.
Metamorphosis is dependent on the settlement success of larvae in inverte-
brates. Competent barnacle cyprids engaged in active probing of substrata secrete
temporary cement, which enables the larvae to explore the substratum and also
acts as a settlement pheromone for interactions among larvae (Clare et al. 1994).
Both temporary and permanent adhesives are required for successful settlement of
larvae. Secretion of these cements from the cement glands to the antennular sacs
is under neuronal control (Okano et al. 1996). The catecholamines dopamine and
noradrenaline were found to initiate settlement in the barnacle Megabalanus rosa
(Okano et al. 1996). Secretion of dopamine and noradrenaline suggests the
involvement of adrenergic receptors in the settlement process. In comparison with
B. amphitrite, larvae showed that α-adrenergic receptor antagonist like phen-
tolamine inhibited settlement but induced metamorphosis without prior attach-
ment at concentrations of 0.1–10 mM (Yamamoto et al. 1998). These findings
resulted in the screening of various adregenergic antagonists like phentolamine,
prazosin, atipamezole, medetomidine, clonidine and idazoxanfor settlement inhi-
bition acti-vity. Medetomidine and clonidine repeatedly inhibited settlement of B.
amphitrite cyprids (Dahlstrom et al. 2000).
Medetomidine revealed a strong tendency to accumulate in solid/liquid phase
boundaries and seems to be an attractive candidate for incorporation into antifoul-
ing coatings. Surface affinity studies with medetomidine showed that the com-
pound adsorbed strongly to hydrophilic polystyrene compared to hydrophobic
polystyrene without change in chemical structure, which further provides an
attractive feature for incorporation into existing coatings as boosters (Dahlstrom
et al. 2004).
α-Adrenergic antagonists idozaxon and phentolamine inhibited settlement of
B. amphitrite larvae (Dahms and Qian 2004). An important feature of these verte-
brate α-adrenergic antagonists is their hydrophobic nature and their tendency to
accumulate at surfaces, which contributes to their successful inhibition of larval
settlement. Structural analogues of medetomidine and clonidine inhibited settle-
ment in barnacles. Among the seven analogues tested guanbenz [α(2)-agonist, I(2)
ligand], moxonidine [α(2)-agonist, I(1) ligand], BU 224 [I(2) ligand], metrazoline
[I(2) ligand], cirazoline [α(1)-agonist, I(2) ligand] and tetrahydrozoline [α-agonist
I(2) ligand] inhibited settlement of B. improvisus cyprids (Dahlstrom et al.
2005).
242 P.S. Murthy et al.

Another antifouling strategy is based on settlement inhibition of larvae by blocking

ion-gated channels. Excess potassium ion, magnesium ion and calcium ion inhibited
settlement in B. amphitrite (Rittschof et al. 1986). This effect may be due to changes
in the membrane potential caused by a potassium electrochemical gradient (Hodgkin
and Horowicz 1959). Potassium ion affected young cyprids while other cations had
more pronounced effects on older cyprids. Results indicate that firing of external
receptors becomes less important in aged cyprids and settlement inhibition may be
related to physiological stress on the increasingly fragile larvae. Delaying the settle-
ment of cyprids may result in natural inhibition and appears to be an easy method for
fouling control. Compounds that inhibit settlement and metamorphosis in invertebrate
larvae increase the probability of death, as the larvae have to spend more time in
the planktonic stage, which increases mortality. Settlement of B. amphitrite larvae was
inhibited by sulfonyl isothiocyanostilbene (SITS), a calcium channel blocker and TEA
(tetraethylammonium chloride), a potassium channel blocker, in the presence of a set-
tlement induction factor. Calmodulin, a major intracellular calcium-binding protein
present in adults and cyprids, was also found to be involved in the settlement process
(Yamamoto et al. 1998). Picrotoxin, an effector of chloride channels, was found to
induce settlement in barnacle cyprids (Rittschof et al. 1986). Antagonists of these
compounds could be used for inhibiting settlement of barnacle cyprids.

4.2 Hydrozoans

As for barnacles, multiple settlement induction pathways were observed in hydro-

zoans. Activation of Hydractinia echinata larvaetakes place in cells located at the
anterior end as a result of activation of a kinase C-like enzyme, which directly leads
to the closure of K+ channels. Closure of these channels causes depolarization and
thus release of an internal signal (Leitz and Klingmann 1990). Another induction
pathway is through the phosphotidylinositol/diacylglycerol/protein kinase C (PI/
DAG/PKC) system, where the metamorphic signal produced by bacterial cues is
transduced in membranes (Leitz and Muller 1987). Induction by bacterial films
causes an increase in endogenous diacylglycerol, the physiological activator of
PKC, suggesting that the bacterial inducer acts by activating receptor-regulated
phospholipid metabolism. Exogenous diacylglycerol leads to membrane transloca-
tion of PKC, indicative of activation (Schneider and Leitz 1994). Subsequent label-
ling studies by Leitz et al. (1994) showed that [14C]-arachidonic acid release was
also involved in the induction process.
Diacylglycerols such as 1,2,-sn-dioctanoylglycerol induced metamorphosis.
The inductive activity was suppressed by compounds like sphingosine and
K-252a, which inhibit mammalian PKC (Leitz and Klingmann 1990). An inves-
tigation with compounds known to block transmembrane ion transport has
enabled researchers to understand the role of these ions in the induction process.
Oubainblocks Na+/K+-ATPase, which maintains electrical potential across cell
membranes (Kuffler et al. 1984), and has been found to inhibit metamorphosis in
Larval Settlement and Surfaces 243

H. echinata larvae, which are induced by bacterial films (Muller and Buchal
1973). Other modulators of intracellular Ca2+ (inositol 1,4,5-triphosphate) or
compounds that regulate the calcium-dependent PKC have been implicated in the
cascade of events leading to metamorphosis of H. echinata (Leitz and Muller
1987; Leitz and Klingmann 1990).

4.3 Tubeworms

Among the tube-building polychaete worms, Hydroides elegansand Phragmatopoma

lapidosa californicaare the most extensively studied organisms (see Cooksey et al.
2008). Biofilms, adult extracts and dissolved free amino acids have all been shown to
induce settlement and metamorphosis in H. elegans (Beckmann et al. 1999; Harder and
Qian 1999). Structural and functional relationships have often been implied between the
neuroactive substances and the natural inducers. H. elegans failed to respond to
G-protein activator Gpp[NH]p or the inhibitor GDP-β-S, negating the involvement of
G protein-coupled receptors in the induction process (Holm et al. 1998). PKC and the
adenyl cylase pathway were not involved in the induction process.
The response of H. elegans larvae to inhibitors of mRNA and protein synthesis
revealed that larvae were induced to metamorphose by exposing them to bacterial
film or to a 3-h pulse of 10 mM CsCl in the presence of the gene transcription
inhibitor DRB (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole) or the transla-
tion inhibitor emetine. DRB and emetine inhibited the incorporation of radio-
labelled uridine into RNA and radiolabelled methionine into peptides, respectively,
indicating that they were effective in blocking the appropriate synthesis. Results of
this study demonstrated that induction of metamorphosis in H. elegans does not
require de novo transcription or translation (Carpizo-Ituarte and Hadfield 2003).
Abnormal metamorphosiswas observed with the neurotransmitters GABA,
choline chloride, 3,4-dihydroxyphenyl l-alanine(l-DOPA) and the ionic channel
blockers and potassium ions (Bryan et al. 1997). Neurotransmitter blockers ida-
zoxan and phentolamine inhibited settlement of H. elegans (Dahms and Qian 2004;
see Harder 2008). Several investigations revealed that density and specific species
of biofilm bacteria were preferred by these larvae. Incorporation of succinic acid, a
fungal metabolite isolated from sponge surface-associated fungus, was effective in
inhibiting settlement of these larvae (Yang et al. 2007). Abnormal metamorphosis
was induced by choline succinyl choline, serotonin and Oubain in P. lapidosa cali-
fornica larvae (Pawlik 1990). Larvae did not respond to GABA, dopamine, epine-
phrine and norepinephrine. Both l- and d-DOPA induced normal metamorphosis in
these larvae. Tetraethylammonium chloride (TEA), sulfonyl isothiocyanostilbene
(SITS) and picrotoxinhad no apparent effect on the larvae. The cAMP pathway was
also not active in this polychaete group, as evident from the failure to respond to
db-cAMP, cholera toxin and isobutylmethylxanthine (IBMX). The divalent cation
Ca2+ induced metamorphosis of the polycheate Phragmatopoma californica larvae.
Studies by Ilan et al. (1993) showed that the larvae were induced to settle by excess
244 P.S. Murthy et al.

calcium ions in the external medium (Ilan et al. 1993). This effect was found to be
specific for calcium ions and was not simply the result of osmotic changes, as an
excess of Mg2+ ions did not elicit this effect. The calcium ionophore A23187 and
the aromatic calcium channel blockers diltiazem, verapamil, D600 and nifidipine,
known to block Ca2+ channels in other systems (see Lewandowski and Beyenal
2008), also induced metamorphosis. These results indicate direct control of the
morphogenetic pathway by calcium ion and complexities of the calcium regulation
of this process (Ilan et al. 1993).

4.4 Molluscs

Chemosensory and molecular mechanisms governing the perception of cues and

internal transduction of the signal has been extensively studied in molluscs due to
their commercial importance. Among the bivalves, M. edulisand the oysters
Crassostrea virginica, C. gigasand C. madrasensisconstitute important aquaculture
and fouling species, and have been extensively studied with respect to their settlement
behaviour. The blue mussel is distributed in Europe and North America. l-DOPA was
found to induce settlement in the mussel, M. edulis (Cooper 1982; Dobretsov and
Qian 2003). Isobutylmethylxanthine also induced metamorphosis whereas the neuro-
transmitter GABA had no effect on this species (Dobretsov and Qian 2003).
Triacylglycerols (TAG) from the diatom Chaetoceros muelleriwere found to
improve the settlement rate of larvae of the blue mussel Mytilus sp. Free fatty acid
content was found to increase with TAG levels (Pernet et al. 2003). The ability of
the neuroactive compounds to influence metamorphosis in oysters has been exten-
sively studied. l-DOPA has been shown to induce larval metamorphosis in C. gigas
(Coon et al. 1985). l-DOPA and the catecholamines(namely dopamine, adrenaline
and noradrenaline) are derivatives of tyrosine and function as hormones, neuro-
transmitters and pigments. These tyrosine derivatives were found to induce settle-
ment of many mollusc larvae. l-DOPA was found to induce both settlement and
metamorphosis in the oyster C. gigas, whereas epinephrine and norepinephrine
were found to induce metamorphosis without settlement (Coon et al. 1985).
Dopamine was found to induce low levels of settlement of this species. Among the
neurotransmitters tested (l-DOPA, d-DOPA, dopamine, GABA, acetylcholine chlo-
ride, acetylcholine iodide and choline chloride) in the Indian oyster C. madrasensis,
l-DOPA alone induced settlement at a concentration of 10–5 M.
The cAMP pathway has not been found to be active in oyster species studied
viz: C. virginica (Bonar 1976), and C. madrasensis (Murthy 1999; Murthy et al.
1999). The other compounds tested were cAMP and db-cAMP, isobutylmethyl-
xanthine, forskolin, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and phorbol-
12–13-dibutyrate (PuDB), which did not induce settlement and metamorphosis of
C. madrasensis larvae (Murthy 1999; Murthy et al. 1999). Choline derivatives
tested against the larvae of the Pacific oyster C. gigas (Coon et al. 1985) showed
low and inconsistent levels of metamorphosis. Seratonin (5-hydroxytryptamine), a
Larval Settlement and Surfaces 245

derivative of tryptophan and a modulator of vertebrate and invertebrate nervous

systems (Kuffler et al. 1984), was found to induce metamorphosis of the mud snail
Ilyanassa obsoleta (Levantine and Bonar 1986; Couper and Leise 1996). In contrast,
the compound did not induce settlement in the larvae of the molluscs Haliotis rufes-
cens (Morse et al. 1979), Phestilla sibogae (Hadfield 1984) and C. gigas (Coon and
et al. 1985). The larvae of the Indian oyster C. madrasensis responded to changes in
external ionic concentration (Murthy 1999; Murthy et al. 1999). An increase in
settlement and metamorphosis was observed with an increase in calcium ion concen-
tration above that found in standard seawater (12.4 mM) up to 21 mM. Settlement and
metamorphosis decreased with further increase in concentration of the ion above
21 mM. Similarly, concentrations of the ion (1–6 mM) below those found in standard
seawater showed low levels of settlement compared to standard seawater. The larvae
failed to survive when placed in calcium-free seawater. Results suggest that the induc-
tion process may be due to an influx of Ca2+ ion across the larval cell membranes.
The carboxylic calcium ionophore A23187 induced settlement and metamor-
phtosis in the larvae in a concentration-dependent manner. Inhibition of settlement
and metamorphosis was observed with respect to calcium channel blockers(diltiazem,
verapamil and nifidipine). These results suggest the functional role of Ca2+ channels
and the presence of active sites on the larval surface, which mediate in the induction
process (Murthy 1999; Murthy et al. 1999).
Alteration in concentrations of potassium ion induced settlement in the Sydney
rock oyster Saccostrea commercialis (Neil and Holiday 1986). Baloun and Morse
(1984b) have demonstrated that induction by excess K+ ions could be attributed to
depolarization of certain “externally accessible excitable cells” by this ion. Larvae
of the prosobranch gastropod Crepidula fornicataare known to metamorphose in
response to dopamine. In contrast, blocking of dopamine receptors might prevent
excess potassium ions from stimulating metamorphosis as these receptors follow
the excitatory depolarization pathways, which could be used in an antifouling
strategy. Response of these larvae to putative dopamine antagonists varied with age
and time of exposure. Chlorpromazineblocked the inductive action by excess
potassium in the initial stages and D1 antagonist R(+)-Sch-23309 and D2 antagonist
spiperone(SPIP) had similar effects.
The latent induction by chlorpromazine (an inhibitor of nitric oxide synthase)
suggests that endogenous nitric oxide may play a natural role in inhibiting meta-
morphosis. In short, exposing larvae of C. fornicata to excess K+ leads to a shut-
down of NO synthesis via a dopaminergic pathway. Alternatively, the inductive
action by chlorpromazine may be due to the activation of the endogenous cAMP
concentrations, again acting downstream from the steps acted on directly by
excess K+ (Pechenik et al. 2002). Similarly, the mud snail L. obsoleta larvae meta-
morphose by the NO pathway. NO donors block pharmacologically induced
metamorphosis in the snail. In contrast, nitric oxide synthase (NOS) allows
competent larvae to become juveniles (Froggett and Leise 1999). Neuroanatomical
studies revealed endogenous NO levels to increase throughout the planktonic
stage and decrease with the onset of metamorphosis, suggesting the involvement
of the nitroergic signalling pathway in mollusc embryos (Leise et al. 2001).
246 P.S. Murthy et al.

Comparative analysis of induction pathways in various invertebrate larvae

indicates the involvement of biogenic amines (l-DOPA, norepinephrine, epine-
phrine, serotonin, lisuride, isoproterenol), which mediated neurotransmission to
play a major role in settlement of many of the invertebrate species. Although
these molecules many not have a commercial importance they do indicate that
chemicals or synthetic analogues that specifically alter biogenic amine-mediated
pathways may be promising antifouling compounds. A study on larval settle-
ment inhibition by pharmaceutical compounds and toxins with known modes of
action seems to be a viable solution. Laboratory bioassays using larval models,
and field assays using these molecules individually or in combination, would
yield substantial results.

5 Inhibition of Larval Settlement by Natural Products

and Synthetic Analogues

Many marine organisms are able to resist fouling by producing metabolites that
deter settlement of fouling organisms. This concept has been addressed repeat-
edly by academic researchers for incorporating active natural compounds into
antifouling coatings. Experimental approaches have demonstrated two types of
natural compounds: (1) compounds extracted from marine organisms and (2)
compounds released into the surrounding water bathing these organisms.
Research on natural product antifoulants aims to demonstrate the potency of an
organic extract from organisms to inhibit settlement of a target species. This is
followed by a bioassay-guided fractionation and identification of molecules.
From the perspective of potency, the US Navy program on natural antifouling
compounds has prescribed that extracts should be active at <25 μg mL−1 in static
bioassays (Rittschof 2001). The therapeutic ratio of the effective median concen-
tration to the 50% lethal concentration (EC50/LC50) should be larger than ten
(Rittschof 2001).
Further, secondary metabolites are complex molecules and the active component
involved in inhibition has often been difficult to identify. This poses a problem in
creating structural analogues for incorporation into antifouling coatings.
Experimental approaches to natural product-based antifoulants have rallied
around sponges and octocorals, which are a rich source of secondary metabolites.
Terpenoids, steroids, saponins, brominated tyrosine derivatives, sterols and fatty
acid-related compounds have all been shown to possess antifouling activity.
However, most of the studies have failed to elucidate the mode of action or mecha-
nism of inhibition by a particular natural compound.
It is worthwhile confronting the problem by conducting assays that involve
structure–activity relationships. Priorities should be to screen for natural com-
pounds possessing the ability to interfere with identified neuronal signalling path-
ways in larvae, the synthesis and screening of synthetic analogues to these molecules
Larval Settlement and Surfaces 247

and the enhancement of the activity of analogues through modification of their

chemical structure. Among hundreds of antifouling compounds isolated, some
examples of natural compounds belonging to different classes that show promising
activity and whose structure–activity relationships have been worked out are
outlined below.

5.1 Terpenoids

Examples of compounds whose structure–activity relationships have been stud-

ied are the isocyanoterpenoids, belonging to the terpenoid class identified by
Kitano et al. (2004); Kalihiene (Okino et al. 1995) ; 10β-formamidokalihinol;
10β-formamidokalihino-A (Hirota et al. 1998); Kalihinol A (Yang et al. 2006);
15-formamidokalihinene (Okino et al. 1996); 10-isocyano-4-cadinene (Okino et al.
1996) and 10-formamide-4-cadinene (Nogata et al. 2003). Isocyanoterpenoids, like
some of the natural terpenoids, are known to inhibit settlement of B. amphitrite
larvae at concentrations of 0.1 μg mL−1. A structure–activity analysis has shown
that an isocyano group and a hydrophobic site at a suitable position are important
in the expression of potent antifouling activity. Isocyanocylohexane synthesized
with an ester function showed potent antifouling activity against barnacles.
Synthetic derivatives of a sesquterpene hydroquinone avarol from the sponge
Dysidea avaraand the corresponding quinine avarone obtained by oxidation were
found to decrease settlement of B. amphitrite cyprids. Among these, avarol was
the most toxic and caused mortality of larvae (Tsoukatou et al. 2007).
Sesquiterpeneslike elatol, isolated from the red algae of the genus Laurencia,
showed potent activity against B. amphitrite cyprids (Steinberg et al. 1998).
Diterpenoids like dictyol, pachydictyol A and dictyodial isolated from brown
algae Dictyota spp. are antifouling against bryozoan Bugula neritinina lar-
vae (Schmitt et al. 1995). Higher concentrations (5 μg mL−1) were toxic to the
larvae. Pukalide, another diterepenoid isolated from the gorgonian Leptogorgia
virgulata, and renillafoulin A, isolated from the sea pen Renilla reniformis
(Rittschof et al. 1986), inhibited barnacle settlement.
11β,12β-Epoxypukalide, a cembranolide diterpene isolated from the sea fan
Phyllogorgia dilatata,was able to prevent mussel byssus attachment (Epifanio et al.
2006). Epoxypukalide isolated from a different species of gorgonian, namely
Leptogorgia virgulata, also inhibited the settlement of barnacle B. amphitrite
(Rittschof et al. 1985). Even though the source of these molecules differed (isolated
from different gorgonian species) the molecule (epoxypukalide) exhibited antifoul-
ing activity. However, both studies were carried out independently and on two dif-
ferent fouling species. Compounds that posses such multifunctional roles should
be taken up for subsequent field trials for commercialization. Labdane, a diterpene
isolated from the pulmonate Trimusculus reticulates, inhibited the settlement of
the tubeworm Phragmatopoma californica.
248 P.S. Murthy et al.

5.2 Alkaloids

Alkaloids have been shown to influence the central and peripheral nervous system;
nicotinic receptors and some pyridyl alkaloids also activate certain chemoreceptor
neurons. Anabaseine [2-(3-pyridyl)-3,4,5,6-tetrahydropyridyl] and 2,3′-bipyridyl
(2,3-BP) are two nemertine alkaloids that are known to influence crustaceans.
Structural isomers of bipyridyl (2,3′-BP) tested showed varying degrees of antifoul-
ing activity against B. amphitrite larvae (Kem et al. 2003).

5.2.1 3-Alkylpyridinium Compounds

Another group of molecules that have been successfully demonstrated to have

antifouling activity are the polymeric 3-alkylpyridinium salts (poly-APS) isolated
from the Mediterranean sponge Reniera sarai. The structure of the large poly-APS
isolated constituted of N-butyl(3-butylpyridinium) repeating subunits. This mole-
cule acts as an aetylcholinesterase inhibitor (Sepcic et al. 1998). The compounds
were toxic to the D-shaped veliger larvae of the mussel Mytilus galloprovincialis
(Faimali et al. 2003). The structure–activity relationship of poly-APS has revealed
a dual mechanism of action, viz: a detergent-like property exhibited by poly-APS
that means it could serve as a surfactant for cell membranes, and AChE inhibitory
activity that might be involved in the antifouling mechanism. Interestingly the
compound was not found to be toxic to cyprids, but inhibited settlement (Sepcic
and Turk 2006). The antifouling activity of natural poly-APS was far more effective
than the synthetic analogues of polymeric 3-alkylpyridinium salts developed
(Faimali et al. 2005). The low toxicity and high solubility and stability of poly-APS
make these compounds promising candidates for incorporation into antifouling
paints.

5.3 Peptide Analogues

Tegtmeyer and Rittschof (1989) demonstrated the involvement of synthetic ana-

logues of barnacle pheromone in settlement and metamorphosis of B. amphitrite
larvae as early as 1989. Compared to organic compounds, cyclopeptides isolated
from the sponge G. barrette, have also shown inhibition of settlement of the barna-
cle B. improvisus and the blue mussel Mytilus edulis in the field. Barettin, cyclo
[(6-bromo-8-en-tryptophan) arginine], and 8,9-dihydrobarettin constituted the class
of cyclopeptidestested. A significant aspect of this study by Sjogren et al. (2004) is
that the activities of these compounds are not lost on incorporation into self-
polishing marine paints. Among various synthetic analogues of barettin tested,
benzo(g)dipodazines inhibited settlement but were not toxic to cyprids (Sjogren
et al. 2006). Synthetic analogues of l-arginine, l-tryptophan, 5-bromo-d,l-tryptophan,
Larval Settlement and Surfaces 249

6-bromo-d,l-tryptophan and 6-fluoro-d,l-tryptophan were tested for structure–

activity relationships and did not shown any effect against B. improvisus cyprid
larvae (Sjogren and Bohlin 2004). As in the case of poly-APS, settlement inhi-
bition was reversible when incubated in normal seawater.

5.4 Sterols, Fatty Acids and Tyrosine Derivatives

Lactones, furansand furanones are some examples of compounds that have shown
multiple antifouling activities. Lactones prevented barnacle settlement through the
signal transduction pathway. Furanones exhibited maximum activity among the
polyketides isolated from the red algae Delisea pulchra (de Nys et al. 1995;
Steinberg et al. 2001). Among the steroids, epidioxysteroids isolated from the
marine sponge Acanthella cavernosa inhibited settlement and prolonged the swim-
ming time of cyprids (Tomono et al. 1998). 1-O-palmityl-sn-glycero-3-
phosphocholine (lyso-PAF) extracted from the marine sponge Crella incrustans
inhibited B. neritina larvae (Butler et al. 1996). Ceratinamides A and B and psam-
maplysin A derived from the sponge Psamplysia purpurea were effective against
barnacle cyprids (Tsukamoto et al. 1996). Moloka’iamine and its analogues also
exhibited settlement inhibitory activity in barnacles, with less toxicity to the cyprids
(Schoenfeld and Ganem 1998; Schoenfeld et al. 2002).
Oxime substituents, a form of bromotyrosine derivative (bastadin-3, hemibastadin-1,
aplysamine-2 and psammaplin A), inhibited settlement in the barnacle B. improvisus.
The synthetic analogue of hemibastadin-1, debromohemibastadin-1, inhibited cyprid
settlement more effectively than the natural compound hemibastadin-1. The mecha-
nism of action of the compound inhibiting settlement is being studied (Ortlepp et al.
2007). Heterocyclic compounds like pseudoceratidine from P. purpurea and mauri-
tiamine, an oroidin dimer from the sponge Agelas mauritiana, showed antifouling
activity (Tsukamoto et al. 1996). Among all these, 2,5,6-tribromo-1-methylgramine
isolated from the bryozoan Z. pellucidum showed a tenfold increase in antifouling
activity compared to tributiyl tin oxide (Kon-ya et al. 1994).
Over 500 analogues have been synthesized and tested for antifouling activity. A
structure–activity relationship study with 155 indole derivatives led to the discovery
of the non-toxic antifoulants 5–6-dichlorogramine, 5-chloro-2-methylgramineand
5,6-dichloro-1-methylgramine(DCMG). These were incorporated into silicone
paint matrices, which remained barnacle-free for a period of 1.5 years, which is
comparable to the commercial counterpart BIOX (Kawamata et al. 2006). An
advantage of this compound is that leaching of DCMG could be controlled by the
addition of an acrylic acid–styrene copolymer(ASP) to improve the performance of
the silicone coating(Kawamata et al. 2006).
Studies in search of non-toxic antifoulants over the last decade have yielded
more than 100 compounds and over 500 synthetic analogues being tested and quali-
fied in laboratory bioassays against target species. Another paradigm in this area is
that most of the compounds were identified using barnacle larval assays. However,
250 P.S. Murthy et al.

their activity on other invertebrate larval groups is still unknown. Only five of these
compounds have been tested in the field. These compounds need to be evaluated
under dynamic field conditions before ruling out their potential.
A problem encountered by researchers in this effort is incorporation of these
compounds into commercial coating formulations. In some cases simple matrices
like rosin, hydrogels and phytagels have been used to qualify extracts in the field.
But these are far away from commercial coatings, which come as a package con-
taining base, tie and topcoat systems, where the structural integrity of natural com-
pounds may be lost on incorporation into these coatings. These coatings further
vary in their physical and chemical properties. Compatibility of the compounds
with the coating matrix and interaction with additives during polymerization further
compound the problem and is difficult to predict the outcome of such procedures.
Academic–industry–research collaborations suggested by Rittschof (2001), (see
Lewandowski and Beyenal 2008) seem to be a practical way to solve the problem.
A few interesting leads have been obtained with compounds like isocyanoterpe-
noids and 3-alkylpyridinum salts (poly-APS), which inhibit acetyl cholinesterase
activity, that need to be pursued in field assays. Similarly, serotonin uptake blockers
and a-adrenergic antagonists, like medetomidine, also need to be qualified in field
assays. Partial success has been achieved with the compound furanone (halogen-
ated furanones from the Australian seaweed Delisea pulchra) incorporated into
polymer matrices and qualified in field trials (deNys et al. 1993; de Nys and
Steinberg 2002). However, commercialization of this product involves issues like
registration and environmental clearances. To date, more than 100 compounds pos-
sessing antifouling activity have been isolated and characterized, and their effective
inhibitory concentrations have been listed by Rittschof (2001) and Fusetani (2004).
However, these compounds have not yet been realized into antifouling coatings.
Several compounds have shown promising activity against one species of fou-
lant or another but the successful realization of these compounds into antifouling
coatings is yet to happen. This is because the identified compounds are specific in
action, inhibiting settlement of a particular larval form (narrow spectrum) whereas
in the field a multitude of organisms are found to settle (broad spectrum). A clas-
sical example of this is the study by Pereira et al. (2002), who adopted an alterna-
tive approach to screening and qualifying crude extracts through direct field
assays. Extracts of the gorgonian Phyllogorgia dilatata and sponges Aplysina flu-
vaand Mycale microsigmatosa were tested. A. fluva extracts failed whereas
M. microsigmatosa and P. dilatata extracts inhibited only barnacle settlement.
These results once again point to the narrow spectrum of activity exhibited by
natural products. A solution to this lies in conducting bioassays with larvae of the
major fouling organism to qualify and rank natural compounds. Alternatively,
compounds that act downstream from the initial sensory transduction, i.e., Ca2+
and K+ channel blockers, perhaps are broad spectrum and more likely to have less
organismic specificity.
Other problems concerned with natural products are the effective concentration
for incorporation into a coating matrix and the leaching rate of the compounds in
the environment. For realization of natural product antifoulants, research should
Larval Settlement and Surfaces 251

aim at determining the concentration of secondary metabolites experienced by set-

tlers in the field (Dobretsov et al. 2006; see Nedved and Hadfield 2008) to arrive at
the leaching rate of compounds from paint matrices. A more practical solution to
the problem would be to first identify potential candidate compounds (compounds
effective against different larval forms) and to incorporate them in a single coating
matrix in the field for testing natural products as antifoulants.

6 Current Status of Antifouling Methods and the Need

for Alternative Strategies

As a consequence of the 2001 International Maritime Organization restrictions on

the use of tributyltin (TBT), replacement coatings have to be environmentally
acceptable as well as maintain a certain lifetime. Tin-free self-polishing copolymer
(SPC) and foul release coatings are currently in use commercially. For a detailed
chronological description of developments in the coatings industry refer to the
review by Yebra et al. (2004). In the aftermath of restrictions on TBT, metallic spe-
cies like copper and zinc have again found increasing use and are delivered through
a self-polishing mechanism. The SPCs work on the principle of hydrolysis and ero-
sion. However, recently copper has also been under scrutiny. A copper concentra-
tion as low as 5 μg L−1 can be lethal to invertebrates and the USEPA has prescribed
a stipulated limit of 1,000 μg L−1 in drinking water (Chambers et al. 2006). With
copper being reviewed and with the increased tolerance exhibited by some macro-
phytes like Enteromorpha sp. to this metal, alternatives are being sought. Booster
biocides (pesticides and herbicides) have been incorporated into coatings to
increase the life and functionality of copper-based antifouling systems. Among the
various booster biocides used, Irgarol 1,051 and Diuron are facing regulation by
the UK Health and Safety Executive (Chesworth et al. 2004; Lambert et al. 2006).
The effectiveness of copper-based coatings depends on the ability of the coatings
to continuously leach booster biocides. Accumulation and persistence of these bio-
cides in sediments is being monitored and a worldwide environmental effect
(Konstantinou and Albanis 2004) of key booster biocides used in antifouling coat-
ings is also under scrutiny. Currently, booster biocides are offering an interim solu-
tion to the antifouling problems, but they face the threat of being phased out in the
near future due to their toxic effects on the environment.
On the other hand, foul release coatings have offered a promising alternative for
the industry. Two types of foul releasecoatings are in use, namely those based on
fluoropolymersand those based on silicones. These coatings are in use currently
and have been listed by Swain (1988) and Yebra et al. (2004). The coatings are
effective for fast-moving vessels and at high speeds (10–20 knots) but under static
conditions they do become fouled. The fouling layer is sloughed off once the vessel
is in motion. This is because of the poor adhesion strengthof organisms on these
coatings. These non-stick coatings aid removal of fouling through shear and tensile
stresses as well as their own weight by lowering the thermodynamic work of
252 P.S. Murthy et al.

adhesion. A combination of the critical surface energy (19–24 mN m−1) and low elas-
tic modulus allows the interfacial joint between the animal’s adhesive and the coat-
ing to fracture and fail (see Thomas et al. 2008). These coatings work on the
principle of cohesive failure of the bioadhesive to bind to the substratum.
Comparison of the adhesive strength of common foulants on RTV silicones showed
that oysters (Crassostrea virginica) and tubeworms (Hydroides dianthus) had higher
adhesion strength than barnacles (Balanus eburneus), (Kavanagh et al. 2001).
Continuous improvements in the surface properties of silicone coatings have
revealed critical surface tensions of 20–30 mN m−1 to easily release more biofoul-
ing than other materials at the same critical surface tension. Oils added to these
coatings selectively further diminish the attachment strength of organisms with-
out affecting the critical surface tension (Meyer et al. 2006). Cyprids showed an
inverted behaviour, preventing adhesion on coatings containing silicone oil at
concentrations ³ 5% (Afsar et al. 2003). A comparison of vessel speeds on foul
release properties showed that barnacles were removed at 7 knots and that
18 knots was required to remove weeds, whereas a speed of 30 knots was inef-
fective in removing slime films (Ryle 1999). Decreasing the surface energy of
materials renders them brittle, which is another problem to be overcome. These
disadvantages plus high cost and poor mechanical properties are yet to be solved.
Efforts are underway to improve these parameters but it remains to be seen
whether these coatings will move into the future or will be replaced by more
efficient competing technologies.

7 Status of Alternative Antifouling Methods

Challenges related to the development of alternative antifouling coatings depend on

the chemical nature of the compounds, developing polymer systems compatible
with the additives as well as with anticorrosive undercoats with appropriate
mechanical and application properties. Several alternatives are being actively
researched. These involve natural surface microtopographies, nanostructured sur-
faces, microbial metabolites, synthetic polymers and biomimetics. Increased atten-
tion is being directed to understanding the induction process and downstream
processing of the signal by larvae. Attention is also drawn towards natural antifoul-
ing strategies employed by living marine organisms and mimicking their effects.
Since bioadhesion and surface wettability are influenced by microscale topography,
investigators have mimicked the surfaces of marine organisms (see Thomas et al.
2008). One such proposition for study is the skin of pilot whales, which is free of
fouling. Studies have revealed a hydrated jelly nanorough surface characterized by
patterns of nanoridges with low pore sizes (Baum et al. 2003). Similarly, the skins
of porpoise and killer whales posses a surface rich in glycoproteinaceous material
with a low surface energy. The antifouling property of these surfaces may be due to
the high shear flow of water across the surface. Alternatively, the whales
Larval Settlement and Surfaces 253

may constantly secrete hydrolytic enzymes that may also help maintain the surfaces
clean. The effects of each of these individual components are being investigated.
Biomimics is another area of emerging interest in antifouling research. Novel
antifouling coatings were designed to mimic the placoid scales on shark skin sur-
face (Sharklet AF) and were effective in reducing fouling by the algae Ulva
(Carman et al. 2006). Engineered surface topographies of polydimethyl siloxanes
(feature height/feature width) showed that cyprid settlement was inhibited at high
aspect ratios. A barnacle-specific coating, Sharklet AF, which has a defined topog-
raphy (40 μm feature height with an aspect ratio of two), reduced cyprid settlement
by 97% (Schumacher et al. 2007). The presence of silicone oilsdid not reduce the
settlement of zoospores of the green fouling alga Ulva. However, the presence of
oils with altered topographic features reduced settlement of algae. The alga settled
more in channels than pillars (Hoipkemeier-Wilson et al. 2004).
The low surface energy of the polymer polyethylene glycol (PEG) has been the
target of recent investigations. These polymers are being manipulated for antifoul-
ing properties. Maintaining the mechanical properties and altering the surface
characteristics by using side group-modified polystyrene-based surface-active
block co-polymers (SABC) has been shown to inhibit zoospore settlement of
the green alga Enteromorpha (Youngblood et al. 2003). Modification of biomaterial
(l-DOPA) with poly(ethylene glycol) and subsequent coating on surfaces resulted
in surfaces that resisted cell attachment, exhibiting antifouling properties (Dalsin
and Phillip 2005). Conjugation of the methoxy-terminated poly(ethylene glycol)
polymer to the adhesive amino acid l-DOPA and coating on titanium surfaces gave
surfaces that performed well compared to glass, uncoated titanium and a poly-
dimethylsilicone elastomer. Reduced settlement of diatoms N. perminuta and
zoospores of the algae U. linza has shown that the biogenic amine is an effective
antifouling agent (Statz et al. 2006). Both of these studies have given potential leads
that need investigation in the field.
Surface properties (both physical and chemical) of mollusc shells and inverte-
brates are now being studied (Scardino et al. 2003; Scardino and de Nys 2004).
Surface microtopography is found to influence settlement. A homogenously ridged
surface (ridges with uniform distance of 1–2 μm with a mean depth of 1.5 μm) of
Mytilus galloprovincialis shell prevented settlement compared to the heterogeneous
surface of Pinctada imbricate with repeating structural patterns (Scardino et al.
2003). Scardino and de Nys (2004) demonstrated that the mussel M. galloprovin-
cialis was fouling-free, whereas high-resolution biomimics of shells and sanded
moulds fouled after 6–8 weeks. Results point to the fact that apart from surface
microtopography, other factors may be involved that contribute to fouling deterrence.
Species-specific fouling patterns with differential fouling between shell regions
were observed with the pearl oyster species Pinctada fucata, Pteria penguin and
Pteria chinensis. High resolution resin replicates of the surface of the blue mussel
Mytilus edulis and the crab Cancer paguruswith a surface microtopography of
<500 μm were effective in inhibiting settlement (Bers and Wahl 2004). Further,
larval settlement of the barnacle Semibalanus balanoides was reduced on isotropic
resin surfaces prepared from the molluscs M. edulis and Perna perna compared to
254 P.S. Murthy et al.

anisotropic surfaces (Bers et al. 2006). Periostracum of the mussels M. edulis and
Perna perna were found to possess a generic anti-settlement property against
cyprids of B. balanoides (Bers et al. 2006).
Involvement of surface-bound compounds in the antifouling property of shells
was demonstrated by Bers and Wahl (2004), where moderately polar and non-polar
fractions showed antifouling activity against B. amphitrite cyprids and the marine
bacteria Cobetia marina and Marinobacter hydrocarbonoclasticus. Moderate wet-
tability of surface structures of the sea stars Linckia laevigata, Fronia indica,
Cryptasterina pentagona and Archaster typicus are not fouled in nature (Guenther
and De Nys 2006). Mixed responses to fouling inhibition by shells were observed
in these studies. These studies have demonstrated that the micro-/nanotopography
of surfaces play a role in larval settlement. Micro- or nanopatterning of surfaces
through nanocoatings may provide interesting leads for antifouling.
A novel concept being researched is the use of enzymes as antifoulants. Two
approaches are being followed: (1) direct antifouling where enzymes themselves
are used as antifoulants and (2) indirect antifouling where certain enzymes are used
to release active components from paint matrixes. An example is the hydrolyzing
of adhesive polymers using commercial enzymes for biofouling prevention. This
showed that alcalase, a group of serine-proteases, exhibited antifouling activity
against the spores of the green alga Ulva linza and the barnacle B. amphitrite.
Results showed that effective hydrolysis could not be achieved once curing of the
juvenile barnacle cement commenced (Pettitt et al. 2004). However, problems that
plague natural product antifoulants seem to hold good for these compounds as no
efficient broad-spectrum antifouling coating based on a single or multiple enzymes
has yet been achieved. Research inputs have warranted the creation of new legisla-
tive issues, such as which part of the enzyme system should be considered as a
biocide for product registration purposes (Olsen et al. 2007).
Marine bacteria could provide a source of biologically active metabolites for the
antifouling industry ( Steinberg et al. 2001). The major advantage of bacterial
metabolites is the production of large water-borne (>100 kDa) polar compounds
(Dobretsov and Qian 2002; Dobretsov et al. 2004; Harder et al. 2004), compared to
secondary metabolites produced by macroorganisms with low solubility and activity
(Steinberg et al. 2002). Many studies have aimed at characterizing antifouling
compounds produced by bacteria isolated from epibionts like algae (Dobretsov and
Qian 2002; Dobretsov et al. 2004; Harder et al. 2004; Lee and Qian 2004), sponges
(Holmstrom et al. 1992; Kon-ya et al. 1995), ascidians (Olguin-Uribe et al. 1997;
Zapata et al. 2007) and seaweeds (Armstrong et al. 2000). Ubiquinoneis one example
of a compound produced by a species of Alteromonas, which colonizes the
surface of the sponge Halichondria okadali (Kon-ya et al. 1995). Bacterial extracts
were tested with acrylic base paint resin. These formulations were found to inhibit
marine fouling bacterial adhesion (Armstrong et al. 2000). As a further step,
Holmstrom et al. (2002) produced inhibitory “living paints”by incorporating live
cells of Pseudoalteromonas tunicata into hydrogels. Partial success was achieved,
as the paints were inhibitory for a period of 15 days. Investigations on the use of
microbial natural products are in their infancy compared to the large potential of
Larval Settlement and Surfaces 255

microbes to produce diverse compounds. The problems relate to compound supply;

simple molecules that allow easy structural modifications are advantageous with
microorganisms whose potential has not been fully utilized.
Another innovative approach for fouling control is creation of physical analogues
of natural processes (Cowling et al. 2000). The logic behind this approach is that
active substances that alter the characteristics, properties and behaviour of materials
may have potential in antifouling surfaces. Compounds like benzalkonium chloride
(BCI), propyl 4-hydroxygenzoate and 2,4-hexadienoic acid were incorporated into
the hydrophilic carrier 2-hydroxyethylmethacrylate gels. Mixed results were
obtained where polymers containing propyl 4-hydroxygenzoate and 2,4-hexadi-
enoic acid were found to accumulate microfouling within a period of 1 month,
whereas benzylalkonium chloride-incorporated polymers were fouling-free for up
to 5 months.

8 Concluding Remarks

From the studies reviewed here, two coating systems: self polishing copoly-mers
(SPC) containing a biocide (copper alone or with boosters) and foul release
coatings (polydimethyl siloxanes) working on the principle of low surface energy
are currently in use, even though they are not as effective as tributyltin paints.
Among these, the boosters containing SPC coatings face a threat of fresh regula-
tions by regional pesticide authorities, restricting their use due to the toxic nature
of their ingredients. Foul release coatings have been efficient on vessels operating
at high speeds. Versions of these coatings for small craft are currently being
released into the market. The disadvantage of these coatings is that they do not
resist fouling when the vessel is alongside and serve as a medium for transloca-
tion of alien species. The long-term fouling behaviour of these coatings under
different environmental conditions needs to be documented for assessing the suc-
cess of these coatings.
The biomimetic approach adopts the natural defence mechanisms of marine
organisms to solve the problem of biofouling. However, no single natural com-
pound or a particular microtextured or micropatterned surface has been shown to
exhibit a broad spectrum of activity as the settling preferences of marine inverte-
brates are diverse. Studies replicating natural antifouling mechanisms, wherein
both the physical and chemical defences exhibited by organisms are mimicked
together, would offer valuable inputs to antifouling research. Currently, polydime-
thyl siloxanes have been the focus of such investigations for incorporation of natu-
ral products, synthetic analogues, boosters, microtexturing, nanopatterning and
incorporation of nanoparticles to improve their efficiency. Fluoropolymers are
being researched as alternatives, and additives involving incorporation of natural
product analogues and physical modification of the surfaces using nanomaterials
are also being pursued. It is to be seen whether these coatings remain or are
replaced by more efficient technologies.
256 P.S. Murthy et al.

References

Afsar A, De Nys R, Steinberg P (2003) The effects of foul-release coatings on the settlement and
behaviour of cyprid larvae of the barnacle Balanus amphitrite, Darwin. Biofouling
19:105–110
Aidley DJ (1978) The physiology of excitable cells. Cambridge University Press, Cambridge, UK
Anderson MJ, Underwood AJ (1994) Effects of substratum on the recruitment and development
of an intertidal estuarine fouling assemblage. J Exp Mar Biol Ecol 184:217–236
Armstrong E, Boyd KG, Burgess JG (2000) Prevention of marine biofouling using natural com-
pounds from marine organisms. Biotechnol Annu Rev 6:221–241
Baloun AJ, Morse DE (1984a) Modulation by unsaturated fatty acids of norepinephrine and
adenosine induced formation of cyclic AMP in brain slices. J Neurochem 42:192–197
Baloun AJ, Morse DE (1984b) Ionic control of settlement and metamorphosis in larval Haliotis
rufescens (Gastropoda). Biol Bull 167:124–138
Baum C, Simon F, Meyer W, Fleischer I, Siebers D, Kacza J, Seeger J (2003) Surface properties
of the skin of the pilot Whale Globicephala melas. Biofouling 19:181–186
Beckmann M, Harder T, Qian PY (1999) Induction of larval attachment and metamorphosis in the
serpulid polychaete Hydroides elegans by dissolved free amino acids: mode of action in labo-
ratory bioassays. Mar Ecol Prog Ser 190:167–178
Bers A, Wahl M (2004) The influence of natural surface microtopographies on fouling. Biofouling
20(1):43–51
Bers AV, Prendergast GS, Zurn CM, Hansson L, Head RM, Thomason JC (2006) A comparative
study of the anti-settlement properties of Mytillid shells. Biol Lett 2:88–91
Bishop CD, Brandhorst BP (2001) NO/cGMP signaling and HSP90 activity represses metamor-
phosis in the sea urchin Lytechinus pictus. Biol Bull 201:394–404
Bonar DB (1976) Molluscan metamorphosis: a study in tissue transformation. Am Zool
16:573–591
Bonar DB, Coon SL, Walch M, Weiner RM, Fitt W (1990) Control of oyster settlement and meta-
morphosis by endogenous and exogenous chemical cues. Bull Mar Sci 46(2):484–498
Boudreau B, Bourget E, Simard Y (1990) Benthic invertebrate larval response to substrate char-
acteristics at settlement: shelter preferences of the American lobster Homarus americanus.
Mar Biol 106:191–198
Bryan PJ, Qian PY, Kreider JL, Chia FS (1997) Induction of larval settlement and metamorphosis
by pharmacological and conspecifics associated compounds in the serpulid polychaete
Hydroides elegans. Mar Ecol Prog Ser 146:81–90
Burke RD (1983) The induction of marine invertebrate larvae: stimulus and response. Can J Zool
61:1701–1719
Butler AJ, van Altena IA, Dunne SJ (1996) Antifouling activity of lyso-platelet-activating factor
extracted from Australian sponge Crella incrustans. J Chem Ecol 22:2041
Butman CA, Grassle JP, Webb CM (1988) Substrate choices made by marine larvae settling in still
water and in a flume flow. Nature 333:771–773
Carman ML, Estes TG, Feinberg AW, Schumacher JF, Wilkerson W, Wilson LH, Callow ME,
Callow JA, Brennan AB (2006) Engineered antifouling microtopographies-correlating wetta-
bility with cell attachment. Biofouling 22(1–2):11–21
Carpizo-Ituarte E, Hadfield MG (2003) Transcription and translation inhibitors permit metamor-
phosis up to radiole formation in the Serpulid polychaete Hydroides elegans. Haswell Biol Bull
204:114–125
Chambers LD, Stokes KR, Walsh FC, Wood RJK (2006) Modern approaches to marine antifoul-
ing coatings. Surf Coat Technol 201:3642–3652
Chesworth JC, Donkin ME, Brown T (2004) The interactive effects of the antifouling herbicides
Irgarol 1051 and Diuron on the seagrass Zostera marina (L). Aquat Toxicol 66:293–305
Clare AS, Nott JA (1994) Scanning electron microscopy of the fourth antennular segment of
Balanus amphitrite amphitrite. J Mar Biol Assoc UK 74:967–970
Larval Settlement and Surfaces 257

Clare AS, Freet RK, McClary Jr M (1994) On the antennular secretion of the cyprid of Balanus
amphitrite amphitrite and its role as a settlement pheromone. J Mar Biol Assoc UK
74:243–250
Clare AS, Thomas RF, Rittschof D (1995) Evidence for the involvement of cyclic AMP in the
pheromonal modulation of barnacle settlement. J Exp Biol 198:655–664
Connell SD (1999) Effects of surface orientation on the cover of Epibiota. Biofouling
14(3):219–226
Cooksey KE, Wigglesworth-Cooksey B, Long RA (2008) A strategy to pursue in selecting a natural
antifoulant: a perspective. Springer Ser Biofilms. doi: 10.1007/7142_2008_11
Coon SL, Bonar DB, Weiner RM (1985) Induction of settlement and metamorphosis of the pacific
oyster, Crassostrea gigas (Thunberg) by l-DOPA and catecholamines. J Exp Mar Biol Ecol
94:211–221
Cooper K (1982) A model to explain the induction of settlement and metamorphosis of eyed
pediveligers of the blue mussel Mytilus edulis L by chemical and tactile cues. J Shellfish Res
2:117
Couper JM, Leise EM (1996) Serotonin injections induce metamorphosis in larvae of the
Gastropod mollusk Ilyanassa obsolete. Biol Bull 191:178–186
Cowling MJ, Hodgkiess T, Parr ACS, Smith MJ, Marrs SJ (2000) An alternative approach to
antifouling based on analogues of natural processes. Sci Total Environ 258:129–137
Crisp DJ (1974) Factors influencing the settlement of marine invertebrate larvae. In: Chemoreception
in marine organisms. Academic, London, pp. 177–277
Dahlstrom M, Martensson LGE, Jonsson PR, Arnebrant T, Elwing H (2000) Surface active
adrenoceptor compounds prevent the settlement of cyprid larvae of Balanus improvisus.
Biofouling 16(2–4):191–203
Dahlstrom M, Jonsson H, Jonsson PR, Elwing H (2004) Surface wettability as a determinant in the
settlement of the barnacle Balanus improvisus (Darwin). J Exp Mar Biol Ecol 305:223–232
Dahlstrom M, Lindgren F, Berntsson K, Sjogren M, Martensson LG, Jonsson PR, Elwing H
(2005) Evidence for different pharmacological targets fro imidazoline compounds inhibiting
settlement of the barnacle Balanus improvisus. J Exp Zool 303(7):551–562
Dahms HU, Jin T, Qian PY (2004) Adrenoreceptor compounds prevent the settlement of marine
invertebrate larvae: Balanus amphitrite (Cirripedia), Bugula neritina (Bryozoa) and Hydroides
elegans (Polychaeta). Biofouling 20(6):313–323
Dalsin JL, Messersmith PB (2005) Bioinspired antifouling polymers. Mater Today 8:38–46
de Nys R, Steinberg PD (2002) Linking marine biology and biotechnology. Curr Opin Biotechnol
13(3):244–248
de Nys R, Wright AD, Koning GM, Sticher O (1993) New halogenated furanones from the marine
alga Delisea pulchara (fimbriata). Tetrahedron 49:11213–11220
de Nys R, Steinberg PD, Willemsen P, Dworjanyn SA, Gabelish CB, King RJ (1995) Broad spec-
trum effects of secondary metabolites from the red alga Delisea pulchra in antifouling assays.
Biofouling 8:259
Dobretsov S, Qian PY (2002) Effect of bacteria associated with the green alga Ulva reticulate on
marine micro- and macrofouling. Biofouling 18:217–228
Dobretsov S, Qian PY (2003) Pharmacological Induction of larval settlement and Metamorphosis
in the blue mussel Mytilus edulis L. Biofouling 19(1):57–63
Dobretsov S, Dahms H, Qian PY (2004) Antilarval and antimicrobial activity of waterborne
metabolites of the sponge Callyspongia (Euplacella pulvinata): evidence of allelopathy. Mar
Ecol Prog Series 271:133–146
Dobretsov S, Dahms HU, Qian PY (2006) Inhibition of biofouling by marine microorganisms and
their metabolites. Biofouling 22(1–2):43–54
Epifanio RA, da Gama BAP, Pereira RC (2006) 11b-12b-Epoxypukalide as the antifouling agent
from the Brazilian endemic sea fan Phyllogorgia dilatata Esper (Octocorallia, Gorgoniidae).
Biochem Syst Ecol 34:446–448
Faimali M, Falugi G, Gallus I, Piazza V, Tagliafierro G (2003a) Involvement of acetyl choline in
settlement of Balanus amphitrite. Biofouling 19:213–220
258 P.S. Murthy et al.

Faimali M, Sepcic K, Turk T, Geraci S (2003b) Non-toxic antifouling activity of polymeric

3-alkylpyridinium salts from the mediterranean sponge Reniera sarai (Pulitzer-Finali).
Biofouling 19 (1):47–56
Faimali M, Garaventa F, Mancini I, Sicurelli A, Guella G, Piazza V, Greco G (2005) Antisettlement
activity of synthetic analogues of polymeric 3-alkylpyridinium salts isolated from the sponge
Reniera sarai. Biofouling 21(1):49–57
Forde SE, Raimondi PT (2004) An experimental test of the effects of variation in recruitment
intensity on intertidal community composition. J Exp Mar Biol Ecol 301:1–14
Froggett SJ, Leise EM (1999) Metamorphosis in the marine snail Ilyanassa obsoleta, yes or no?
Biol Bull 196:57–62
Fusetani N (2004) Biofouling and antifouling. Nat Prod Rep 21:94–104
Galsby TM (1999) Effects of shading on subtidal epibiotic assemblages. J Exp Mar Biol Ecol
234:275–290
Galsby TM (2000) Surface composition and orientation interact to affect subtidal epibiota. J Exp
Mar Biol Ecol 248:177–190
Guenther J, DeNys R (2006) Differential community development of fouling species on the pearl
oysters Pinctada fucata, Pteria penguin and Pteria chinensis (Bivalvia, Pteriidae). Biofouling
22(3–4):163–171
Hadfield MG (1978) Metamorphosis in marine Molluscan larvae: an analysis of stimulus and
response. In: ChiaFS, Rice ME (eds.) Settlement and metamorphosis of marine invertebrate
larvae. Elsevier, New York, pp. 165–175
Hadfield MG (1984) Settlement requirements of Molluscan larvae new data on chemical and
genetic roles. Aquaculture 39:283–298
Harder T (2008) Marine epibiosis – concepts, ecological consequences and host defense. Springer
Ser Biofilms. doi: 10.1007/7142_2008_16
Harder T, Qian PY (1999) Induction of larval attachment and metamorphosis in the serpulid poly-
chaete Hydroides elegans by dissolved free amino acids: isolation and identification. Mar Ecol
Prog Ser 179:259–271
Harder T, Dobretsov S, Qian PY (2004) Waterborne polar macromolecules act as algal antifou-
lants in the seaweed Ulva reticulata. Mar Ecol Prog Ser 274:133–141
Hirata KY, Hadfield MG (1986) The role of choline in metamorphic induction of Phestilla
(Gastropoda: Nudibranchia). Comp Biochem Physiol 84C:15–21
Hirota H, Okino T, Yoshimura E, Fusetani N (1998) Five new antifouling sesquiterpenes from two
marine sponges of the genus Axinyssa and the Nudibranch Phyllidia pustulosa. Tetrahedron
54:13971–13980
Hodgkin AL, Horowicz P (1959) The influence of potassium and chloride ions on the membrane
potential of single muscle fibres. J Physiol 148:127–160
Hoeg J, Hosfeld B, Gram-Jensen P (1988) TEM studies on the lattice organs of cirripede cypris
larvae (Crustacea Thecostraca, Cirripedia). Zoomorphology 118:195–205
Hoipkemeier-Wilson L, Schumacher JF, Carman ML, Gibson AL, Feinberg AW, Callow ME,
Finlay JA, Callow JA, Brennan AB (2004) Antifouling potential of lubricious, micro-
engineered, PDMS elastomers against zoospores of the green fouling alga Ulva (Enteromorpha).
Biofouling 20(1):53–63
Holiday JE (1996) Effects of surface orientation and slurry coating on settlement of Sydney rock
Saccostrea commercialis oysters on PVC slats in hatchery. Aquaculture Eng 15(3):159–168
Holm ER, Nedved BT, Carpizo-Ituarte E, Hadfield MG (1998) Metamorphic signal transduction in
Hydroides elegans (Polychaeta: Serpulidae) is not mediated by a G protein. Biol Bull 195:21–29
Holmstrom C, Rittschof D, Kjelleberg S (1992) Inhibition of settlement by larvae of Balanus
amphitrite and Ciona intestinalis by a surface colonizing marine bacterium. Appl Env
Microbiol 2111–2115
Holmstrom C, Egan S, Franks A, McCloy S, Kjelleberg S (2002) Antifouling activities expressed
by marine surface associated Pseudoalteromonas species. FEMS Microbiol Ecol 41:47–58
Ilan M, Jensen RA, Morse DE (1993) Calcium control of metamorphosis in polycheate larvae.
J Exp Zool 267:423–430
Larval Settlement and Surfaces 259

Kaissling KE, Thorson J (1980) Insect olfactory sensilla: structural, chemical and electrical
aspects of the functional organizations. In: Satelle DB, Hall LM, Hildebrand JG (eds.)
Receptors for neurotransmitters, hormones and pheromones in insects. Elsevier/North Holland
Biomedical, New York, pp. 261–282
Kavanagh CJ, Schultz MP, Swain GW, Stein J, Truby K, Darkangelo-Wood C (2001) Variation in
adhesion strength of Balanus eburneus, Crassostrea virginica and Hydroides dianthus to
fouling-release coatings. Biofouling 17:155–167
Kawamata M, Kon-ya K, Miki W (2006) 5,6-Dichloro-1-methylgramine, a non-toxic antifoulant
derived from a marine natural product. Prog Mol Subcell Biol 42:125–139
Kaye HR, Reiswig HM (1991) Sexual reproduction in four Caribbean commercial sponges. III.
Larval behavior, settlement and metamorphosis. J Invert Reprod 19:25–35
Kem WR, Soti F, Rittschof D (2003) Inhibition of barnacle larval settlement and crustacean toxic-
ity of some hoplonemertine pyridyl alkaloids. Biomol Eng 20:355–361
Kitano Y, Nogata Y, Shinshima K, Yoshimura E, Chiba K, Tada M, Sakaguchi I (2004) Synthesis
and anti-barnacle activities of novel isocyanocylohexane compounds containing an ester or
ether functional group. Biofouling 20(2):93–100
Konstantinou IK, Albanis TA (2004) Worldwide occurrence and effects of antifouling paint
booster biocides in the aquatic environment review. Environ Int 30:235–248
Kon-ya K, Shimidzu N, Adachi K, Miki W (1994) 2,5,6-Tribromo-1-methylgramine, an antifoul-
ing substance from the marine bryozoan Zoobotryon pellucidum. Fish Sci 60(6):773–775
Kon-ya K, Wataru M, Endo M (1995) l-Tryptophan and related compounds induce settlement of
the barnacle Balanus amphitrite Darwin. Fish Sci 61(5):800–803
Kuffler SW, Nicholls JG, Martin AR (1984) From neuron to brain. Sinauer Associatea, Sunderland,
MA, pp. 651
Lambert SJ, Thomas KV, Davy AJ (2006) Assessment of the risk posed by the antifouling booster
biocides Irgarol 1051 and Diuron to freshwater. Chemosphere 63:734–743
Lee OO, Qian PY (2004) Potential control of bacterial epibiosis on the surface of the sponge
Mycale adherens. Aquat Microb Ecol 34:11–21
Leise EM, Thavradhara K, Durhan NR, Turner BE (2001) Serotonin and nitric oxide regulate
metamorphosis in the marine snail Ilyanassa obsoleta. Am Zool 41:258–267
Leitz T, Klingmann G (1990) Metamorphosis in Hydractinia: studies with activators and inhibi-
tors aiming at protein kinase C and potassium channels. Rouxs Arch Dev Biol 199:107–113
Leitz T, Muller WA (1987) Evidence for the involvement of PI-signalling and diacylglycerol
second messengers in the initiation of metamorphosis in the hydroid Hydractinia echinata.
Fleming Dev Biol 121:82–89
Leitz T, Beck H, Stephan M, Lehmann WD, Petrocellis LDE, Marzo VDI (1994) Possible involve-
ment of arachidonic acid and eicosanoids in metamorphic events in Hydractina echinata
(Coelenterata; Hydrozoa). J Exp Zool 269:422–431
LeTourneux F, Bourget E (1988) Importance of physical and biological settlement cues used at
different spatial scales by the larvae of Semibalanus balanoides. Mar Biol 97:57–66
Levantine PL, Bonar DB (1986) Metamorphosis of Ilyanassa obsoleta natural and artificial induc-
ers. Am Zool 26:14A
Lewandowski Z, Beyenal H (2008) Mechanisms of microbially influenced corrosion. Springer Ser
Biofilms. doi: 10.1007/7142_2008_8
Matsumara K, Mori S, Nagano M, Fusetani N (1998) Lentil lectin inhibits adult extract-induced
settlement of the barnacle Balanus amphitrite. J Exp Zool 280:213
Meyer A, Baier R, Wood CD, Stein J, Truby K, Holm E, Montemarano J, Kavanagh C, Nedved B,
Smith C, Swain G, Wiebe D (2006) Contact angle anomalies indicate that surface-active elu-
ates from silicone coatings inhibit the adhesive mechanisms of fouling organisms. Biofouling
22(5–6):411–423
Mihm JW, Banta WC, Loeb GI (1981) Effects of adsorbed organic and primary fouling films on
bryozoan settlement. J Exp Mar Biol Ecol 54:167–179
Millineaux LS, Garland ED (1993) Larval recruitment in response to manipulated field flows. Mar
Biol 116:667–683
260 P.S. Murthy et al.

Morita H (1972) Primary process of insect chemoreception. Adv Biophys 3:161–198

Morse DE, Hooker N, Duncan H, Jensen L (1979) Gamma-amino butyric acid – a neurotransmitter
induces planktonic abalone larvae to settle and begin metamorphose. Science 204:407–410
Morse ANC, Froyd CA, Morse DE (1984) Molecules from cyanobacteria and red algae that induce
larval settlement and metamorphosis in the mollusc Haliotis rufescens. Mar Biol 81:293–298
Morse DE (1992) Molecular mechanisms controlling metamorphosis and recruitment in abalone
larvae. In: Shepherd SA, Tegner MJ, Guzman del Proo SA (eds.) Abalone of the world.
Blackwell, Oxford, pp. 107–119
Muller WA, Buchal G (1973) Metamorphose-Induktion bei Planulalarven. II Induktion durch
monovalente Kationen: Die Bedeutung des Gibbs-Donnan-Verhältnisses und der Na+/K+-
ARPase. Wilhelm Roux Arch 173:122–135
Murthy PS (1999) Studies on the factors influencing larval settlement and metamorphosis in
Crassostrea madrasensis (Preston) and its control using bioactive compounds. PhD thesis,
University of Madras, pp. 105
Murthy PS, Venugopalan VP, Nair KVK, Subramoniam T (1999) Chemical cues inducing settlement
and metamorphosis in the fouling oyster Crassostrea madrasensis. J Ind Inst Sci 79:513–526
Natasha IG, William B, Loeb GI (2002) Aquatic biofouling larvae respond to differences in the
mechanical properties of the surface on which they settle. Biofouling 18 (4):269–273
Nedved BT, Hadfield MG (2008) Hydroides elegans (Annelida: Polychaeta): a model for biofoul-
ing research. Springer Ser Biofilms. doi: 10.1007/7142_2008_15
Neil JA, Holliday JE (1986) Effects of potassium and copper on the settling rate of Sydney Rock
Oyster (Saccostrea commercialis) larvae. Aquaculture 58:263–267
Nogata V, Yoshimura V, Shinshima K, Kitano Y, Sakaguchi I (2003) Antifouling substances
against larvae of the barnacle Balanus amphitrite from the marine sponge, Acanthella caver-
nosa. Biofouling 19:193–196
Nott JA, Foster BA (1969) On the structure of the antennular attachment organ of the cypris larva
of Balanus balanoides. Phil Trans R Soc 256:115–133
Nymer M, Cope E, Brady R, Shirtliff ME, Leid JG (2008) Immune responses to indwelling medi-
cal devices. Springer Ser Biofilms. doi: 10.1007/7142_2008_4
Okano K, Shimizu K, Satuito CG, Fusetani N (1996) Visualization of cement exocytosis in the
cypris cement gland of the barnacle Megabalanus rosa. J Exp Biol 199:2131–2137
Okazaki Y, Shizuri Y (2000) Effect of inducers and inhibitors on the expression of bcs genes
involved in cypris larval attachment and metamorphosis of the barnacles Balanus amphitrite.
Int J Dev Biol 44:451–456
Okino T, Yoshimura E, Hirota H, Fusetani N (1995) Antifouling kalihinenes from the marine
sponge Acanthella cavernosa. Tetrahedron Lett 36(47):8637–8640
Okino T, Yoshimura E, Hirota H, Fusetani N (1996) New antifouling sesquiterpenes from four
nudibranchs of the family Phyllidiidae. Tetrahedron 52(28):9447–9454
Olguin-Uribe G, Abou-Mansour E, Boulander A, Debard H, Francisco C, Combaur G (1997)
6-Bromoindole-3-carbaldehyde, form an Acinetobacter sp. bacterium associated with the
ascidian Stomoza murrayi. J Chem Ecol 23:2507–2521
Olsen SM, Pedersen LT, Laursen MH, Kill S, Dam-Hohansen K (2007) Enzyme-based antifouling
coatings: a review. Biofouling 23(5):369–383
Ortlepp S, Sjogren M, Dahlstrom M, Weber H, Ebel R, Edrada R, Thomas C, Schup P, Bohlin L,
Proksch P (2007) Antifouling activity of bromotyrosine-derived sponge metabolites and syn-
thetic analogues. Mar Biotechnol 23
Pawlik JR (1990) Natural and artificial induction of metamorphosis of Phragmatopoma lapidosa
californica (Polychaeta: Sabellariidae) with a critical look at the effects of bioactive com-
pounds on marine invertebrate larvae. Bull Mar Sci 46(2):512–535
Pawlik JR, Butman CA, Starczak VR (1991) Hydrodynamic facilitation of gregarious settlement
of a reef-building tube worm. Science 251:421–424
Pechenik JA, Li W, Cochrane DE (2002) Timing is everything: the effects of putative dopamine
antagonists on metamorphosis vary with larval age and experimental duration in the proso-
branch gastropod Crepidula fornicate. Biol Bull 202:137–147
Larval Settlement and Surfaces 261

Pennington KT, Hadfield MG (1988) Larvae of a Nudibranch mollusc (Phestilla sibogae) meta-
morphose when exposed to common organic solvents. Biol Bull 177:350–355
Pernet F, Tremblay R, Bourget E (2003) Settlement success, spatial pattern and behavior of mussel
larvae Mytilus sp. in experimental down-welling systems of varying velocity and turbulence.
Mar Ecol Prog Ser 260:125–140
Pereira RC, Carvalho AGV, Gama BAP, Countinho R (2002) Field experimental evaluation of
secondary metabolites from marine invertebrates as antifoulants. Braz J Biol 62(2):311–320
Pettitt ME, Henry SI, Callow ME, Callow JA, Clare AS (2004) Activity of commercial enzymes
on settlement and adhesion of cypris larvae of the barnacle Balanus amphitrite, spores of the
green alga Ulva linza, and the diatom Navicula perminuta Biofouling 20(6):299–311
Rittschof D (2001) Natural product antifoulants and coating development. In: McClintock JB,
Baker BJ (eds.) Marine chemical ecology. CRC, Boca Raton, pp. 543–566
Rittschof D, Costlow JD (1989a) Surface determination of macro invertebrate larvae settlement. In:
Klekowsky RZ, Styczynska-Jureqwicz E, Falkowsky J (eds.) Proceedings of the 21st European
marine biology symposium, Gdansk, 14–19 Sept 1996. Ossolineum, Gdansk, pp. 155–163
Rittschof D, Costlow JD (1989b) Bryozoan and barnacle settlement in relation to initial surface
wettability: a comparison of laboratory and filed studies. In: Ros JD (ed.) Topics in marine
biology: proceedings of 22nd European marine biology symposium, Barcelona, 17–22 August
1987: Scientia Marina, Barcelona, pp. 411–416
Rittschof D, Branscomb ES, Costlow JD (1984) Settlement and behavior in relation to flow and
surface in larval barnacle, Balanus amphitrite Darwin. J Exp Mar Biol Ecol 82:131–146
Rittschof D, Hooper IR, Branscomb ES, Costlow JD (1985) Inhibition of barnacle settlement and
behavior by natural products from whip corals, Leptogorgia virgulata (Lamarck, 1815).
J Chem Ecol 11:551–563
Rittschof D, Maki J, Mitchel R, Costlow JD (1986) Ion and neuropharmacological studies of
barnacle settlement. Neth J Sea Res 20:269–275
Roberts D, Rittschof D, Holm E, Schmidt AR (1991) Factors influencing initial larval settlement:
temporal, spatial and surface molecular components. J Exp Mar Biol Ecol 150:203–211
Ryland RS (1974) Behaviour, settlement and metamorphosis of bryozoan larvae: a review.
Thalassia Jugoslavica 10:239–262
Ryle M (1999) Are TBT alternatives as good? The Motor Ship. pp. 34–39
Scardino A, Nys RD, Ison O, O’Connor W, Steinberg P (2003) Microtopography and antifouling
properties of the shell surface of the bivalve molluscs Mytilus galloprovincialis and Pinctada
imbricate. Biofouling 19:221–230
Scardino AJ, de Nys R (2004) Fouling deterrence on the bivalve shell Mytilus galloprovincialis:
a physical phenomenon. Biofouling 20(4–5):249–257
Schmitt TM, Hay ME, Lindquist N (1995) Constraints on chemically mediated coevolution: mul-
tiple functions for seaweed secondary metabolites. Ecology 76:107–123
Schneider T, Leitz T (1994) Protein kinase C in hydrozoans: involvement in metamorphosis of
Hydractinia and in pattern formation of Hydra. Roux’s Arch Dev Biol 203:422–428
Schoenfeld RC, Ganem B (1998) Synthesis of ceratinamine and moloka’iamine: antifouling
agents from the marine sponge Pseudoceratina purpurea. Tetrahedron Lett 39:4147–4150
Schoenfeld RC, Conova S, Rittschof D, Ganem B (2002) Cytotoxic, antifouling bromotyramines:
a synthetic study on simple marine natural products and their analogues. Bioorg Med Chem
Lett 12:823–825
Schumacher JF, Aldred N, Callow ME, Finlay JA, Callow JA, Clare AS, Brennan AB (2007)
Species-specific engineered antifouling topographies: correlations between the settlement of
algal zoospores and barnacle cyprids. Biofouling 23(5):307–317
Sepcic K, Turk T (2006) 3-Alkylpyridinium compounds as potential non-toxic antifouling agents.
In: Fusetani N, Clare AS (eds.) Antifouling compounds. Progress in molecular and subcellular
biology, vol 42. Springer Berlin Heidelberg New York, pp. 105–124
Sepcic K, Marcel V, Klaebe A, Turk T, Síuput D, Fournier D (1998) Inhibition of acetylchloineste-
rase by an alkylpyridinium polymer from the marine sponge, Reniera sarai. Biochim Biophys
Acta 1387:217–225
262 P.S. Murthy et al.

Sjogren M, Dahlstrom M, Goransson U, Jonsson P, Bohlin L (2004) Recruitment in the field of

Balanus improvisus and Mytilus edulis in response to the antifouling cyclopeptides barettin
and 8,9-dihydrobarettin from the marine sponge Geodia barrette. Biofouling 20(6):291–297
Sjogren M, Johnson AL, Hedner E, Dahlstrom M, Goransson U, Shirani H, Bergman J, Jonsson
PR, Bohlin L (2006) Antifouling activity of synthesized peptide analogs of the sponge metabo-
lite. Barettin Peptides 27:2058–2064
Statz A, Finaly J, Dalsin J, Callow M, Callow JA, Messersmith PB (2006) Algal antifouling and
fouling-release properties of metal surfaces coated with a polymer inspired by marine mussels.
Biofouling 22(5–6):391–399
Steinberg PD, De Nys TD, Kjelleberg S (1998) Chemical inhibition of epibiota by Australian
seaweeds. Biofouling 12(1–3):227–244
Steinberg PD, De Nys R, Kjelleberg S (2001) Chemical mediation of surface colonization. In:
McClintock J, Baker B(eds.) Marine chemical ecology. CRC, Boca Raton, pp. 325–353
Steinberg PD, De Nys R, Kjelleberg S (2002) Chemical cues for surface colonization. J Chem
Ecol 28:1935–1951
Sulkin SD (1984) Behavioral basis of depth regulation in the larvae of brachyuran crabs. Mar Ecol
Prog Ser 15:181–205
Swain G (1988) Proceedings of the international symposium on sea water drag reduction. Naval
Undersea Warfare Center, Newport, pp. 155–161
Tegtmeyer K, Rittschof D (1989) Synthetic peptide analogs to barnacle settlement pheromone.
Peptides 9:1403–1406
Thomas JG, Corum L, Miller K (2008) Biofilms and ventilation. Springer Ser Biofilms. doi:
10.1007/7142_2008_7
Thorson G (1964) Light as an ecological factor in the dispersal and settlement of larvae of marine
bottom invertebrates. Ophelia 1:187–208
Thurm U, Wessel G (1979) Metabolism-dependant transepithelial potential differences at epider-
mal receptors of arthropods. J Comp Physiol 134A:119–130
Tomono Y, Hirota H, Fusetani N (1998) Antifouling compounds against barnacle (Balanus
amphitrite) larvae from the marine sponge Acanthella cavernosa. In: Watanabe Y, Fusetani N
(eds.) Sponge sciences – multidisciplinary perspectives. Springer Berlin Heidelberg New York,
pp. 413–424
Tsoukatou M, Marechal JP, Hellio C, Novakovic I, Tufegdzic S, Sladic D, Gasic MJ, Clare AS,
Vagias C, Roussis V (2007) Evaluation of the activity of the sponge metabolites Avarol and
Avarone and their synthetic derivatives against fouling micro- and macroorganisms. Molecules
12:1022–1034
Tsukamoto S, Kato S, Hirota H, Fusetani N (1996) Pseudoceratidine: a new antifouling spermi-
dine derivative from the marine sponge Pseudoceratina purpurea. Tetrahedron Lett
37(9):1439–1440
Walters LJ (1992) Field settlement locations on subtidal marine hard substrata: is active larval
exploration involved? Limnol Oceanogr 37:1101–1107
Yamamoto H, Satuito CG, Yamazaki M, Natoyama K, Tachibana A, Fusetani N (1998)
Neurotransmitter blockers as antifoulants against planktonic larvae of the barnacle Balanus
amphitrite and the mussel Mytilus galloprovincialis. Biofouling 13:69–82
Yamamoto H, Tachilbana A, Matsumura K, Fusetani N (1995) Protein kinase C (PKC) singal
transduction system involved in larval metamorphosis of the barnacle, Balanus amphitrite.
Zool Sci 12:391–396
Yang LH, Lee O, Jin T, Li XC, Qian PY (2006) Antifouling properties of 10b-formamidokalihi-
nol-A and kalihinol A isolated from the marine sponge Acanthella cavernosa. Biofouling
22(1–2):23–32
Yang LH, Lau SCK, Lee OO, Tsoi MMY, Qian PY (2007) Potential roles of succinic acid against
colonization by a tubeworm. J Exp Mar Biol Ecol 349:1–11
Yebra DM, Kiil S, Dam–Johansen K (2004) Antifouling technology – past, present and future
steps towards efficient and environmentally friendly antifouling coatings. Prog Org Coat
50:75–104
Larval Settlement and Surfaces 263

Yool AJ, Grau SM, Hadfield MG, Jensen RA, Markell DA, Morse DE (1986) Excess potassium
induces larval metamorphosis in four marine invertebrate species. Biol Bull 170:255–266
Young CM, Chia FS (1987) Abundance and distribution of pelagic larvae as influenced by preda-
tion behavior and hydrographic factors. In: Giese AC, Pearse JS (eds.) Reproduction of marine
invertebrates, vol 9. Blackwell, Palo Alto, pp. 385–453
Youngblood JP, Andruzzi L, Ober CK, Hexemer A, Kramer EJ, Callow JA, Finaly JA, Callow ME
(2003) Coatings based on side-chain ether-linked poly(ethylene glycol) and fluorocarbon poly-
mers for the control of marine biofouling. Biofouling 19:91–98
Zapata M, Silva F, Luza Y, Wilkens M, Riquelme C (2007) The inhibitory effect of biofilms pro-
duced by wild bacterial isolates to the larval settlement of the fouling ascidia Ciona intestinalis
and Pyura praeputialis. Electron J Biotechnol 10(1):149–159
Macrofouling Control in Power Plants
R. Venkatesan and P. Sriyutha Murthy (*
ü)

Abstract Macrofouling organisms readily colonize artificial man-made structures,

cooling water intake tunnels, culverts, pump chambers and heat exchangers.
Cooling water systems if not properly treated invariably become susceptible to
biofouling. The problem is particularly severe in the tropics and is site-, season-,
and substratum-specific. Further, cooling systems serve as a source of macrofoul-
ing organisms and breeding grounds wherein invertebrate larvae are produced and
colonize equipment downstream like pipelines, valves and heat exchangers. Once-
through seawater or freshwater systems encounter severe macrofouling-associated
problems like flow reduction, increased pressure drop across heat exchangers and
equipment breakdown. Biocidal dose and regime for cooling water systems and
heat-exchangers have to be tailor-made for a power plant and should be effective
in controlling microbial biofouling as well as hard foulants (barnacles, mussels,
tubeworms and oysters). With regard to macrofouling control in condenser-cooling
systems of power plants, chlorination has been the method of choice for fouling
control over the years due to its low cost, easy availability and handling, and known
degradation pathways. Increasing awareness on the toxic effects of chlorination
by-products and better understanding of the biocidal action, environmental issues
and higher dosages required for sanitization of surfaces has resulted in replacement
of chlorine by stronger oxidizing biocides like chlorine dioxide. Experimental studies
using coastal seawater in plate heat exchangers, has revealed a chlorine residual
of 1.0 ppm to prevent settlement of invertebrate larvae. However, an intermittent
chlorination dose of 1.2 ppm residuals at a frequency of 0.5–2 h was sufficient in
controlling slime formation. Side-stream monitoring of these heat exchangers in a
nuclear power plant cooling circuit revealed barnacle fouling in spite of continuous
chlorination of 0.2–0.3 ppm residuals and shock doses of 0.4–0.6 ppm twice a week
for 8 h. In an operational plant, continuous monitoring of the fouling situation using
side-stream monitoring devices is to be practised and the biocidal dose and regime

P.S. Murthy
Biofouling and Biofilm Processes Section, Water and Steam Chemistry Division, BARC Facilities,
Indira Gandhi Center for Atomic Research Campus, Kalpakkam, 603 102, India
e-mails: psm_murthy@yahoo.co.in, psmurthy@igcar.gov.in

Springer Series on Biofilms, doi: 10.1007/7142_2008_14 265 265

© Springer-Verlag Berlin Heidelberg 2008
266 R. Venkatesan and P.S. Murthy

altered to overcome any spikes in settlement. This is essentially because biocidal

doses required to kill established fouling communities are far higher than those for
inhibiting settlement. Even if killing is achieved, accumulation of dead shell biomass
(barnacles and tubeworms) often results in loading on equipment surfaces and
increases surface roughness, facilitating settlement of other fouling organisms.

1 Introduction

Industrial fouling involves inorganic, organic, particulate and biological fouling

(see Smeltzer 2008). Biofouling in industrial water systems is a recalcitrant prob-
lem not easily controlled and even then at a significant cost. Operationally, the
problem due to biofouling manifests when “biofilm development exceeds a given
threshold of interference” (Flemming and Griebe 2000). Microbial biofouling can
alternatively be considered as a biofilm reactor in the wrong place, as cooling
systems offer large surface areas for colonization and nutrients for growth
(Flemming 2002). Biofouling in recirculating freshwater systems is less pronounced
than in once-through seawater systems and generally manifests in the form of condenser
slime (biofilms) in power-plant cooling circuits.
About 150 species of macrofoulants have been listed for seawater-cooled
systems whereas Asiatic clams and Zebra mussels are the main macrofoulants in
freshwater-cooled systems (Claudi and Mackie 1994). Macrofouling in the cooling
water systems of power plants results in reduction of flow to the condenser tubes,
blockage of intake pipes, condenser tube blockage, mechanical damage to pumps
and condenser tubes, promotion of microfouling and enhanced corrosion of con-
denser tubes. Further settlement of hard-shelled fouling organisms causes damage
to material integrity and often results in failure of equipments. Compared to shell
and tube heat exchangers, plate heat exchangers (PHE) are finding increased appli-
cations recently in nuclear, thermal and desalination plants. Long-term data on the
use of these heat exchangers in power plant cooling circuits revealed accumulation
of corrosion products and particulate fouling on the plates. Initiation time for bio-
logical fouling in industrial systems ranges from a few hours to about 400 h (Bott
1993). For a successful antifouling strategy an integrated approach of monitoring
the surfaces, including analysis of fouling situations, is important (Flemming
2002). In seawater-cooled systems macrofouling is a predominant problem and
control measures should aim at a biocidal dose and regime to prevent settlement of
organisms and development of biofilms, i.e. restricting biofilm development at the
given threshold of interference. Environmental parameters such as fluid velocity,
temperature, pH value, nutrient levels, cell concentration and surface roughness
have all been demonstrated to have a measurable effect on the development of both
biofilms and settlement of macrofouling organisms. A biocide dose should be
effective from zero hours to keep the surface clean. The old approach of increasing
biocide dosage to remediate a biofouling problem frequently fails in practice. Since
biofouling is a surface-associated phenomenon it should be treated as such, by
Macrofouling Control in Power Plants 267

targeting treatments for controlling surface-associated or sessile organisms (Donlan

2000; Flemming 2002).
One of the major factors affecting power plant operation is the performance of the
cooling water system (Brankevich et al. 1990; Bell 1977). This has led to the develop-
ment of specific antifouling measures for power plants. The most common counter-
measure as practised in a majority of power plants comprises filtration of debris and
fouling organisms through intake and travelling water screens, biocide dosing (chlo-
rine) and thermal shock treatment. Compared to chlorine dioxide, the use of a much
stronger oxidizing agent such as ozone is not popular. Due to the high production cost
and considering the volume of water to be treated in once-through systems, this does
not seem to be a viable alternative. The success of these methods is dependent on the
nature of the fouling organisms in a given geographical location (Strauss 1989;
Sasikumar et al. 1992), quality of cooling water (Corpe 1977), thermal and biocidal
tolerance ranges of different size classes of macrofouling organisms existing in a
cooling circuit (Jenner 1980; Sasikumar et al. 1992; Rajagopal et al. 1995) and silta-
tion (Jenner et al. 1998). The development of a macrofouling community in the cool-
ing water system occurs as a result of passage of planktonic larvae of invertebrates
followed by a settlement phase (see Murthy et al. 2008), during which the organisms
metamorphose into adults by producing an outer shell. The high flow velocity ensures
a continuous supply of oxygen, food for the growth of macrofoulants, avoids the
accumulation of waste and results in increased colonization of macrofoulants (Jenner
1980). Over a period of time if the situation is left untreated a uniform fouling layer
accumulates on the surfaces with a thickness reaching 30 cm from the wall (Kovalak
et al. 1993). These cause problems in the condenser section as the fouling layer grows
in thickness they are sloughed off due to high velocities, and such clusters are depos-
ited in the heat exchangers blocking the flow (Kovalak et al. 1993).
The problem of macrofouling in heat exchangers leads to flow blockage in shell
and tube heat exchangers (Fig. 1a) and deposition in plate heat exchangers
(Fig. 1b). Fouling causes irreversible mechanical damage to the equipment surface
due to the hard calcareous shells of fouling organisms. A 5 mm Hg reduction in
condenser backpressure is equal to 0.5% improvement in turbine heat rate, which
is approximately equal to an additional 3 MW(e) of generating capacity (Drake
1977). On the other hand, fouling of heat exchanger surfaces results in reduced heat
transfer efficiency and increased fluid frictional resistance,resulting in additional
maintenance and operating costs (Bott and Tianqing 2004). Apart from macrofouling,
even a 250 micron-thick layer of slime may result in up to a 50% reduction in heat
transfer in heat exchangers (Goodman 1987). In the case of heat exchangers, a
decrease of the overall heat transfer coefficient due to fouling deposits leads to
overdesign, and increased energy and cleaning costs, which are substantial (Bott 1995).
As far as overdesign is concerned, fouling in plate exchangers leads to higher
operational costs compared to shell and tube exchangers because of their higher
efficiency (Muller-Steinhagen 1993; Hesselgreaves 2002; Kukulka and Devgun
2007). In seawater flow systems the fouling layer on a heat exchanger surface com-
prises chiefly of inorganic and biological fouling. If biofilm formation precedes that
of inorganic film, the inorganic film develops in the channels existing in the biofilm
268 R. Venkatesan and P.S. Murthy

Fig. 1 a Flow blockage of tubular heat exchangers (PSWHX) of Madras Atomic Power station
after one year of operation. b Fouling by particulate material and corrosion product on titanium
plate heat exchanger surface after 69 days of operation at 0.5 m s−1 velocity in a side-stream study
at the Madras Atomic Power Station, Kalpakkam

matrix. If inorganic fouling precedes organic fouling then it would attract adhesion
of bacteria (Sheikholeslami 2000). However, these mechanisms are yet to be sub-
stantiated through experimental data. Some of the factors influencing fouling of
heat exchangers are the material of construction of a heat exchanger, and its surface
roughness will influence the development of biofilms (Mott and Bott 1991; Mott
et al. 1994). Vieira et al. (1992) demonstrated that initial attachment of Pseudomonas
fluorescens was more pronounced on aluminium plates than on brass or copper,
which may be attributed to the release of toxic ions by these surfaces. Rabas et al.
(1993) demonstrated that fouling was higher on spirally indented tubes than plain
tubes. On the other hand, rough surfaces were more hospitable to microorganisms
than smooth surfaces (Reid et al. 1992). On a rough surface, valleys provide shelter
against removal by shear stress and hills act as nucleation sites; therefore the extent
of fouling is high on rough surfaces. Surface properties like adsorption, surface
charge and corrosiveness were also found to affect fouling (Epstein 1983).

2 Types and Features of Industrial Cooling Water Systems

Choice on the type of cooling water system is influenced by plant location and
availability of water suitable for cooling purposes. Once-through cooling systems
are used in plants sited beside large water bodies (sea, large flowing rivers and
estuaries) that have the ability to dissipate waste heat from the steam cycle. For
detailed system analysis and design of structures refer to Jenner et al. (1998),
Neitzel and Dalling (1984). Design characteristics of once-through systems may
allow or even increase the rate of fouling by promoting conditions that are conducive
to sedimentation, macrofoulants and corrosion. Intake structures of once-through
systems vary from plant to plant depending on environmental considerations and
flow requirements. Most of the flow-through systems comprise of an offshore
intake system (bored tunnel or a buried culvert), which conveys the water to a pump
Macrofouling Control in Power Plants 269

CONDENSER

PSWHX

PUMP HOUSE INTAKE

TRASH RACK
TWS MSL
INTAKE
SCREEN
OUTFALL SEA BED

INTAKE SHAFT
- 50 m - 50 m

468 m TUNNEL

Fig. 2 Schematic view of a cooling water system of a power station (Madras Atomic Power
Station, Kalpakkam, located on the east coast of India). TWS travelling water screens, PSWHX
process seawater heat exchangers, MSL mean sea level

house located onshore from where the water is pumped through the condensers, and
returns via a shore-based outfall (Fig. 2). Near shore intakes are not preferred as
they may result in a severe siltation problem. Some of the design features incorpo-
rate physical water treatment methods like water velocities in the cooling water
tunnel designed around 1.5–3.0 m s−1 to prevent sedimentation, and provision of
trash screens at the offshore intake point and travelling water screens before the
pumps. In addition, once-through systems typically have high flow velocities and
mass flow rates for minimizing temperature effects on receiving waters. A typical
500 MW(e) unit would have a flow of 30 m3 s−1 at an average velocity of 3 m s−1 in
the cooling water circuits. Design factors influencing macrofouling of once-through
systems are (1) flow velocity, (2) flow pattern, (3) frequency of use, (4) valve leaks,
(5) unreliable and ineffective biocidal systems, (6) compartment size, (7) system
configuration and (8) water temperature (Neitzel et al. 1984).

3 Economic Losses Due to Biofouling in Power Plants

An estimate of economic losses due to biofouling problems is large and emphasizes

the importance of biofouling control measures in power plants. Most of the literature
on losses due to biofouling dates to the late 1990s. Costs for one day of unplanned
outage of a 235 MW(e) power plant can be around 0.3% of the earning. Hence control
measures adopted to maintain cooling water cleanliness reflects on indirect earnings to
the industry. Losses as a result of shutdown of a 235 MW(e) power station due to
biofouling were estimated to be about Rs. 40 lakhs a day (about US$100,000)
(Venugopalan and Nair 1990). The cost of removing macrofouling organisms from
screening houses alone for two European power plants cost around US$ 25–30,000
270 R. Venkatesan and P.S. Murthy

every 2 years (Kovalak et al. 1993). In the USA, approximately 4% of failures in power
plants >600 MW(e) is due to biofouling of condensers (Meesters et al. 2003). Fouling
by Asiatic clams Corbicula asiatica in condenser tubes of power plants alone costs the
USA over US$1 billion annually (Chow 1987; Strauss 1989). The problem is more
pronounced in heat exchangers, where an increase in condenser backpressure due to
fouling in a 250 MW(e) plant costs about US$250,000 annually (Chow 1987).
Cleaning of biofouling organisms from cooling water-circuits of power plants is a
very expensive option for plant operations. Studies by Coughlan and Whitehouse
(1977) showed that between 1957 and 1964, some 4,000 condenser tubes failed due to
mussel fouling, leading to leakage of cooling water into the boiler. Apart from the loss
of power generation, these leaks contaminated the feed-water system and accelerated
the boiler waterside corrosion, resulting in boiler tube failures. This has necessitated
the inlet culverts to be drained for manual cleaning once a year. The average quantity
of mussels removed was estimated at 40 tons per year and the maximum was 130 tons
per year. Similarly, 300 tons of mussels were removed from the Pools power station
(Dorset). About 300 tons of mussels were removed following shock chlorination treat-
ment from the atomic power station intake tunnel in a single occasion (Rajagopal et al.
1996). Similarly, 4,000 man-hours were used to clean the circuits and remove mussels
(360 m3) at the power station at Dunkerque (Whitehouse 1985). Another example of
intense fouling is the Carmarthen Bay power station, where within a year of commis-
sioning the problem became so severe that the plant was shut down periodically (James
1985). The underwater cooling conduits of the Tanagwa power station in Japan
showed a fouling thickness of 70 cm (Kawabe and Treplin 1986).

4 Biofouling Control Methods in Once-Through

Seawater-Cooled Systems

An important consideration in the operation of equipment subject to biofouling is

its mitigation. Mitigation techniques broadly fall into (1) physical (Bott and
Tianqing 2004; Melo and Bott 1997) and (2) chemical methods (Sohn et al. 2004;
Meyer 2003; Ludensky 2003; Walsh et al. 2003; Rajagopal et al. 2003; Butterfield
et al. 2002a, b; Prince et al. 2002; Ormerod and Lund 1995).

4.1 Physical Control Methods

4.1.1 Flow

Water flow is a major factor influencing settlement of marine invertebrate larvae

(Table 1). Velocity is a design factor for cooling water systems (Strauss and
Puckorius 1984; Tuthill 1985; Johnson et al. 1986). Flow velocities in cooling
water conduits must be measured very close to the wall (1 mm) surface rather than
Macrofouling Control in Power Plants 271

Table 1 Velocity as an antifouling measure

Velocity (m s−1) Effects on organisms References
Less than 0.9 Allows sedimentation and microfouling
to occur
>0 but <0.3 Allows Asiatic clam larvae to settle Jenner et al. (1998)
>0.1 but <1.2 Allows blue mussel and oyster larval
settlement
>0.1 up to 1.5 Allows mussel settlement in intake tunnel Rajagopal et al. (2006)
Branchidontes variablis, B. striatulus
and Modiolus philippinarum
>3.0 Does not detach mussels Syrett and Coit (1983)
>0.01 up to 1.0 Allows Zebra mussel larvae to settle Tuthill (1985)
0.15 Allows Zebra mussel larvae to settle. Data Kovalak et al. (1993)
from ten European power plants
Above 2.0 Inhibits Zebra mussel larval settlement Leglize and Ollivier (1981)
<0.2; >0.6; >0.9 Settlement; growth inhibition; detachment Aprosi (1988)
of colonies of bryozoans
1.8–2.2 Allows settlement of mussels, barnacles, Jenner and Khalanski (1998)
hydroids in circular conduits
Up to 1.4 Allows mussel, barnacle, hydroid settle- Jenner et al. (1998)
ment in large rectangular conduits of
(5–11 m2)
>4 Required for preventing erosion corrosion Jenner et al. (1998)
of metal structures

the mean water velocity, as settling larvae are subjected to the velocity at the near
surface boundary rather than in the bulk water. At high flow rates, the shear stress
of the water often exceeds the shear strength of many organisms, hence they do not
settle (Collins 1964). However this is not the situation in operational power plants,
where barnacles were found to colonize even at velocities of 3.0 m s−1, making the
surface rough and creating sites for further settlement of mussels (Syrett and Coit
1983). Hence it is imperative to adopt a chemical control strategy to control the
settlement and growth of macrofoulants. Conventionally a 1,000 MW(e) capacity
power plant requires cooling water at the rate of 30 m3 s−1 (Whitehouse 1985),
which is drawn at a velocity of 2.0–3.0 m s−1 through inlet pipelines. In general,
flow across the heat exchanger tubes is maintained around 1.4–2.0 m s−1. Analysis
of operational and experimental data from different power plants shows that veloci-
ties in the range of 3.5–4.0 m s−1 are required to prevent settlement of marine organ-
isms. However, most power stations operate at velocities of 1.4–1.8 m s−1 across the
heat exchangers and at around 2.0–3.0 m s−1 in pipe sections and cooling water
circuits, which does not prevent the settlement of macrofoulants. The successional
pattern of macrofouling organisms in cooling water circuits are also known to be
driven by flow velocity. Larval forms capable of settling at high velocities are the
primary colonizers and these established organisms were found to baffle water cur-
rents, which enabled attachment of larvae that preferred low velocities to settle
(Corfield et al. 2004). An example of this effect was reported by Jenner (1980),
272 R. Venkatesan and P.S. Murthy

whose study showed that barnacles were found to settle before mussels as they can
attach at much higher velocities and their shells provide the roughness required for
mussels to settle. In practice, there are low flow regions associated with the geom-
etry of the cooling water circuit that may favour increased settlement, and it appears
impossible to maintain a constant velocity throughout the cooling water circuit.
Increasing the velocity requires additional pumping costs and does not seem to be
a viable method. A possible antifouling method postulated by Jenner (1983) for
controlling biofouling using velocity is to decrease the flow rate instead of increas-
ing the flow rate, taking into consideration the sinking rate of different organisms
at low flow velocities. However, this proposition is yet to be substantiated through
experimental studies to be adopted in power stations. Alternatively the use of high
velocities (1.5–2.5 m s−1) with a smooth finish to the surface of the culverts could
theoretically prevent settlement of mussel spat (Jenner 1982). With the advent of
foul release coatings this seems to be a viable option, along with the flow and bio-
cidal regimes in force. In general, the use of flow as an antifouling method is
unlikely to be effective in power stations.

4.1.2 Travelling Screens

Another problem encountered in once-through offshore/near shore intake systems

of power plants is impingement by large fish, driftwood, seaweed, jellyfish etc. The
problem is overcome by the provision of single or double trash racks for the offshore
intake systems, which serve as the first line of defense (Brankevich et al. 1990).
Usually, travelling water screens are provided ahead of the heat exchangers to filter
out the floating debris and adult macroorganisms. Screens of different mesh sizes
(10 mm UK and Japan; 4 mm France and Italy; 4–10 mm Netherlands; 10–25 mm
India) are in general use. Downstream of service water pumps, the water passes
through basket strainers for removing particles and the water is taken to the con-
densers for cooling. Seawater-cooled plants are designed to minimize the number
of components that interface directly with seawater because of the corrosive nature
of seawater. Intermediate closed-cycle loops filled with demineralized water are
used for cooling the auxiliary systems (process water heat exchangers). The use of
travelling water screens is indispensable for plant operation.

4.1.3 Mechanical Cleaning Techniques

In spite of the presence of physical control methods like travelling screens and
biocidal programmes, heat exchangers are often subjected to sedimentation and
biofilm accumulation. Mechanical online and offline methods are available for
cleaning of shell and tube heat exchangers. In the case of plate heat exchangers,
online mechanical cleaning has been found to be economically non-viable and
technically unfeasible warranting the use of chemical treatments. Ceramic, glass or
sponge rubber balls have been used for online cleaning. Brush type online cleaning
Macrofouling Control in Power Plants 273

devices are also available. Two types of automatic cleaning systems are employed
in power stations during normal plant operations: the Amertap system and the
American M.A.N. brushes system. The Amertrap system can be operated on an
intermittent or continuous basis depending on the severity of problem in the shell
and tube heat exchangers. The Amertrap system comprises sponge rubber balls,
slightly bigger in size than the tubes, that circulate along the length of the tubes
(Bell 1977; Brankevich et al. 1990; Fritsch et al. 1977). The constant rubbing action
keeps the surfaces clean and removes biofilms. The balls are collected in an outlet
chamber and are again pumped into the heat exchanger. The American M.A.N
system uses flow-driven brushes that are passed through the condenser tubes inter-
mittently by reversing the flow. The brushes abrasively remove fouling and corro-
sion products. Automatic online mechanical cleaning methods are the most
economical and are practised invariably in most of the power stations around the
world. Even though these are a crude methods, no alternative technology is avail-
able at hand and the sponge rubber ball cleaning is again an indispensable method
for microbial biofouling control in heat exchangers.
Offline cleaning is done by the hydrolazing method(specialized high pressure
water jet cleaning) with a pressure of 10,000–20,000 psi for cleaning heat exchanger
tubes. Tubes with scales showed that at a low pressure of 10,000 psi cleaning was
relatively poor compared to 20,000 psi. Cleaning of dry tubes was more efficient
with brass and spin grit brushes compared to wet tubes, which may be attributed to
the lubricating effect of water between brush tips and tube surface. Hydrolazing
was effective in cleaning wet tubes (Young et al. 2000). Other types of mechanical
cleaning techniques involve moulded plastic cleaners (pigs) that are useful for
cleaning light silt deposits. Spirally formed, indented or finned brushes are used for
cleaning tubes. Hard calcite depositsare difficult to clean even by acids; rotary cut-
ters similar to the ones used for cutting glass with a Teflon body are used for clean-
ing. Compressed airdriven devices are also available for cleaning of heat exchanger
tubes. Offline cleaning methods are practised in power stations when the thermal
resistance values in heat exchangers drop beyond an acceptable level. This results
in shutdown of the equipment and affects production costs.

4.1.4 Thermal Treatment

Cooling water requirement of power plants are sized according to the upper thermal
limits of discharge, i.e. 7–10°C above the ambient prevailing at a given location.
Thermal treatment of cooling water circuits and inlet conduits is an effective envi-
ronmentally friendly method for control of biofouling in power plants, wherein the
cooling water temperature is raised above the thermal tolerance level (Table 2) of
fouling organisms (Brankevich et al. 1990). The exact temperature and time
required for mortality of fouling organisms is dependent on many factors; the main
factor being the acclimation temperature, i.e. the difference between the ambient
and treated temperature. A second factor is the rate of acclimation: if the tempera-
ture increase is slow, the mussels are found to acclimatize to the rate. Another factor
274 R. Venkatesan and P.S. Murthy

Table 2 Thermal tolerancelevels of common fouling organisms

Temperatures (°C) Effect on organisms References
35–37 Kills most macrofouling organisms Jenner (1980); Gunasingh
et al. (2002)
37 for 30 min; 38 for Causes 100% mortality in the Jenner (1980)
15 min; 39 for 5 min mussel Mytilus edulis
43 for 30 min Causes 100% mortality in the green Rajagopal et al. (1995)
mussel Perna viridis
39 for 30 h; 43 for Tolerates; 100% mortality within Rajagopal et al. (1995)
30 min; >45 2.15 h; 100% mortality immedi-
ately for Branchidontes
striatulus
35–47 Causes 100% mortality for barnacle Sasikumar et al. (1992)
Megabalanus tintinabulum

is genetic variations in local populations (Claudi and Mackie 1994). Before imple-
menting a thermal treatment programme, the thermal tolerance of the major foulant
species at a particular facility needs to be determined. This can be derived through
simple experimentation and following the multiple regression formulae developed
by McMahon et al. (1993) for 50% LT50 and 100% LT100 mortality of fouling
organisms:

LT50=34.57–0.035(min/1°c) + 0.149(°C acclimation temperature)

LT100=36.10–0.040(min/1°c) + 0.147(°C acclimation temperature)

Thermal treatment procedures are very effective against macrofoulants compared to

microfoulants as they are known to exist in condenser tubes (condenser slime)
experiencing elevated temperatures (50–70°C). Thermal treatment methods have
been successfully implemented at the Commonwealth Edison Heat plant, where
100% mortality of mussels was achieved by raising the water temperature from
31.6 to 37.2°C and maintaining this temperature for a 6 h period (Claudi and
Mackie 1994). The method is also used in some power stations that have the option
of dual intake pipelines and facilities for recirculation of water from the heat
exchangers. One of the pipelines is used as an intake and the heated effluent from
the heat exchanger is circulated through the other. After a certain period the direc-
tion of flow is reversed in these pipelines (Jenner 1982). The thermal method has
been effective at Marsden B power station where the cooling circuits were treated
with elevated temperatures (51.7°C). However, the periodicity of such operations
depends on the intensity of fouling at a location. The effectiveness of thermal treat-
ment is also dependent on the appropriate choice of water temperature, duration of
exposure and frequency of exposure. For thermal treatment to be efficient, exposure
periods no longer than 3 h should be adopted if it is to be economically and environ-
mentally sustainable (Jenner 1982) for operational power plants. However, cooling
water systems have a variety of macrofouling organisms, with different tolerance
Macrofouling Control in Power Plants 275

levels. Knowledge of the thermal tolerance of fouling species and information

about the biological community that exists in a cooling water system is important
for designing treatment strategies. Major disadvantages of thermal shock treatments
are in regard to meeting the environmental regulations governing discharge of
heated water and the availability of the option for thermal backwashing and losses
involved in shutdown of the plant during the backwashing period.

4.2 Chemical Methods

4.2.1 Advantages and Disadvantages of Oxidizing Biocides

Oxidizing biocides are in use for treating cooling waters. In the oxidizing biocide
category, chlorine has been the most extensively used and cost-effective biocide.
The order of volatility is ozone > chlorine > chlorine dioxide > chloramines >
hypochlorous acid > hypobromous acid.
Efficient chlorination treatments suitable both for biofilm and macrofouling control
in condensers must be worked out for a given location. The addition of chlorine to water
can be viewed as an instantaneous reaction resulting in an equilibrium mixture of
hypochlorous acid (HOCl) and hypochlorite ions. Hypochlorous acid is the active
biocide and its stability is dependent on the pH of the solution. At a low pH value of
6.0–7.0 relatively more concentration of hypochlorous acid is present than at a seawater
pH value of 8.2. In addition, hypochlorous acid (HOCl) reacts with organic matter/
nitrogenous compounds and is consumed readily (chlorine demand). This necessitates
increased dosing to overcome the demand. In chlorination of natural waters, the chlorine
demand has to be ascertained before administering the biocide. Chlorine demand is also
found to vary seasonally and the demand of tropical coastal seawater usually varies
between 0.7 and 1.0 mg L−1 (Murthy et al. 2005). To reduce biofouling, chlorination of
seawater is usually practised, with typical applied doses of 0.5–1.0 mg L−1 (expressed as
Cl2) and a resultant residual oxidant level of 0.1 ± 0.3 mg L−1 in the cooling water.
Chlorination can be an effective control technique for both bivalves and microbial slime.
Different chlorine doses and regimes have been tested for fouling control (Jenner et al.
1998; Rajagopal et al. 1994, 2003; Rajagopal 1997; Gunasingh et al. 2002).
Some of the common chlorination practices adopted in power stations are:
– Low level continuous chlorination: Continuous application of chlorine at residuals
of 0.1–0.2 mg L−1 is used to deter larval forms from settling. Mussel larvae close
their shells in the presence of chlorine and the velocity in the system will flush
them out without allowing the larvae to colonize the substratum (Claudi and
Mackie 1994).
– Intermittent treatment: This method came into practice to reduce the cost of the
treatment programme and also to meet the biocide discharge criteria. In addition,
mussels are constrained to close their shell valves in response to continuous chlo-
rination. However, studies by Rajagopal et al. (2003) have shown the method to
276 R. Venkatesan and P.S. Murthy

be ineffective as mussels were able to tide over periods of chlorine dosages by

closing their shell valves. Alternatively, the more intelligent version of intermit-
tent chlorination, namely pulse chlorination, developed at KEMA (Poleman and
Jenner 2002) has been effective in achieving killing of mussels as well as reduc-
ing the biocide inventory and environmental burden.
– End of the season chlorination: This has been practised in some of the European
power stations (Jenner and Janssen-Mommen 1993) where chlorine levels of 0.5
mg L−1 were maintained for 2 weeks at the end of the breeding season to cause
95% mortality of newly settled mussels.
Growing concerns over the harmful effects of chlorination by-products, i.e.
trihalomethanes (THMs; volatile), haloacetonitriles (HANs; semi-volatile), halophe-
nols (HPhs) and haloacetic acids (HAAs), resulted in chlorination being disallowed
in several of the US, UK, Canadian and European power stations. Use of chlorine
is subjected to increasing environmental regulations (such as the new Biocidal
Product Directive, 98/8/CE, in European countries). The USEPA chronic and acute
marine water quality guidelines for chlorine are 0.0075 and 0.013 g m−3, respec-
tively (USEPA 2002). CORMIX modelling done by the National Institute of Water
and Atmospheric Research (NIWA) show that an eightfold dilution of the cooling
water plume occurs in the mixing zone (Oldman et al. 2004). If residual concentra-
tions in the cooing water outfall are in the range of 0.1 g m−3 after reasonable mixing,
the maximum chlorine concentration would be 0.013 g m−3, equivalent to the
USEPA guideline (Corfield et al. 2004). The Safe Drinking Water Act (1979)
enacted by the USA prescribes the maximum contaminated levels of total THM
(TTHM) to 0.10 mg L−1 (100 ppb) and the disinfectants and disinfectant by-product
rule has fixed the limits at 0.80 mg L−1 TTHM (USEPA 1994). Alternatives to
conventional chlorination in power stations depends on the cost of the products
proposed from the market, roughly these products are one to three orders of magnitude
costlier than sodium hypochlorite.
An alternative biocide for controlling biofouling in power plant cooling systems
is bromine. Bromine is a chemical halogen similar to chlorine and was introduced
commercially in 1980. Since then, plant chemists have had the option of choosing
either one or both biocides for their cooling systems. Bromination has been used
for some time along with chlorine and can significantly reduce the total disinfectant
and halogen application rates because bromine oxidants generated in water are
more effective for controlling biofouling than their chlorine counterparts at high pH
values, above the 8.0 found in seawater. Several forms of bromine are available,
which include activated bromine, sodium bromide, bromine chloride and proprie-
tary mixtures of bromine and chlorine. Commercial formulations like the Active
Bromide (NALCO Chemicals), BromiCide (Great Lakes Chemical Corporation)
and Starbex, a sodium hypobromite compound for microfouling control, have been
adopted by some power stations along with chlorine dosing. Sodium bromide can
be used to convert hypochlorous acid (HOCl) into hypobromous acid (HOBr).
Literature on the toxicity of this biocide to marine organisms is limited. However,
when used in combination with chlorination it is effective in reducing the total halogen
Macrofouling Control in Power Plants 277

load, and the bromine oxidants that are generated are more effective for controlling
biofouling at pH values above 8.0 (Fisher et al. 1999).
Currently, chlorine dioxide is being adopted in several European power stations
because of its effectiveness in killing macrofoulants as well as against microbial
biofouling and because of the lesser formation of organo-halogenated by-products.
Typical doses of ClO2 for seawater cooling systems range from 0.05 to 0.1 mg L−1
(Petrucci and Rosellini 2005). Another oxidizing biocide being used for fouling con-
trol is ozone. The use of ozone as a biocide is still a very expensive method, estimated
around 3.8 times that of the cost of sodium hypochlorite (Duvivier et al. 1996).
Oxidizing biocides have a similarity in their mode of action on biological organ-
isms. The toxicity of chlorine has been reported to be due to the destruction of the
respiratory membrane by oxidation (Bass and Heath 1977), oxidation of enzymes
containing a sulfhydryl moiety (Ingols et al. 1953) and ion imbalances (Vreenegoor
et al. 1977). An EPRI report (Electric Power Research Institute 1980) attributed the
toxic effect of chlorine on mussels to a weakening of the strength of the byssal threads.
The principle effect of chlorine was to depress the activity of the foot of mussels, lead-
ing to a reduction in the number of threads formed. Chlorinated mussels, with their
weaker attachment systems, were swept from the walls of the cooling system (Claudi
and Mackie 1994). In comparison, the biocidal action of ozone is on the bacterial mem-
brane glycoproteins, glycolipids and certain amino acids such as tryptophan. Ozone
also acts on the sulfhydryl groups of certain enzymes, resulting in disruption of normal
cellular enzymatic activity. Bacterial death is rapid and is often attributed to changes in
cellular permeability followed by cell lysis. Ozone also acts on the nuclear material,
modifying the purine and pyramidine bases of nucleic acids (Roy et al. 1981).
The choice of the biocide for cooling water systems is primarily governed by the
cost. The dose and regime depends on the nature and intensity of fouling organisms
at a given geographical location, and on environmental conditions. There is no such
concept as a best dose or a best biocide. Biocide doses and regimes must be tailor-
made for each of the cooling water systems. Chlorine is effective but may require very
high concentrations, which are not environmentally acceptable. Hence alternative
biocides like chlorine dioxide or ozone may be considered, but here cost becomes a
limiting factor. Hence, power plant operators have to strike a balance between cost
and the cleanliness required. A comparative account of the properties and effective-
ness of different oxidizing biocides are given in Table 3. The table has been synthe-
sized based on experience and on data published by Jenner et al. 1998; Claudi and
Mackie 1994; Corfield et al. 2004; Rajagopal et al. 1997; Cristiani 2005.

4.2.2 Biocidal Requirements for Prevention of Larval

Settlement in Cooling Water Systems

Usually the problem of biofouling gains attention when it interferes with the per-
formance of the station, even in the presence of a biocidal programme in place. This
is due to the inadequacy of the biocidal programme in overcoming sudden increases
in macrofouling settlement. Hence continuous surveillance, detection of fouling
 278

Table 3 Comparison of properties of different oxidizing biocides

Parameters Chlorine (Cl2) Bromine (Br) Chlorine dioxide (ClO2) Ozone (O3) Peraacetic acid
−1 −1 −1 −1
Conc. used in CWS 0.2–1.0 mg L 0.1–0.5 mg L 0.1–0.5 mg L 0.01–0.3 mg L 1.5–3.0 g m−3
(doses)
Activity Narrow spectrum at Moderately effective Broad spectrum at low Broad spectrum at low Moderately effective
low concentrations concentrations concentrations
Contact time Seconds to minutes Seconds to minutes Seconds to minutes Seconds Minutes
pH Not very effective at Effective up to pH 9.0 Very effective up to Not effective above Effective up to pH 9.0
pH higher than 7 pH 11 pH 8.5
Temperature effects Cannot be used at Cannot be used at Cannot be used at Cannot be used at Cannot be used at
higher temperatures higher temperatures higher temperatures higher temperatures higher temperatures
Corrosiveness Corrosive to handle Not very corrosive Not very corrosive Moderately corrosive Corrosive to iron sub-
strates
By-products Produces toxic tri- Used along with Does not produce toxic Bromate and assimila- Readily biodegradable
halomethanes; chlorine by-products;chlorite ble organic carbon
regulations on upper ions are generated
toxic levels
Reaction with organics Reacts with organics Does not react with Does not react with Reacts with NH3. Reacts with sulfites and
and is consumed. organics organics and NH3. Removes organic sulfides
Reacts with NH3 Reacts with second- matter, odour
ary amines
Storage Conc. decreases slowly Can be prepared fresh Conc. decreases rapidly Cannot be stored Can be stored
with time and dosed with time
Cost (arbitrary units) Cheap 2.0 times cost of 2.5 times cost of 3.8 times cost of 10–20% more than
chlorine chlorine chlorine chlorine
R. Venkatesan and P.S. Murthy
Macrofouling Control in Power Plants 279

organisms, monitoring of the efficiency of the biocide, and fine tuning the dosages
would help in reducing the biocidal requirement of power plants. Flow-through
power stations use different biocidal doses and regimes for control of macrofouling
organisms. In practice, a low level of continuous chlorination (0.1–0.2 mg L−1
residuals) coupled with shock dosing (0.5–1.0 mg L−1 residuals for 30 min once a
week) is employed in power stations. Studies on the dosages required to prevent
settlement of organisms are limited except for the available literature based on
operational experiences at power stations. The gap in knowledge is due to the com-
plexity of the cooling systems (geometry, flow, surface characteristics, diversity of
organisms, cost involved in a biocidal programme and knowledge about larval set-
tlement behaviour) encountered and to the interfacing of engineering aspects with
biology and toxicology. Further complexity arises in the scaling of laboratory results
to real-time cooling circuits. In general dosages required to inhibit or prevent settle-
ment would be far less compared to those required for killing established fouling
communities (Claudi and Mackie 1994).
Field observation on the effectiveness of continuous chlorination revealed a
residual of 0.25 mg L−1, sufficient for preventing attachment and growth of Mytilus
species at water velocities as low as 0.4 m s−1 (Elecric Power Research Institute
1980). Laboratory studies showed that a total residual oxidant (TRO) level of 0.1
mg L−1 prevented attachment of mussels to concrete panels at water velocities as
low as 0.76 m s−1 (Elecric Power Research Institute 1980). Alternatively, continuous
chlorination at 0.2 mg L−1 had no effect on settlement of the blue mussel Mytilus
edulis(L) at Maasvlakte power station, Rotterdam (Jenner 1983). Comparison of
results from these two studies reveal the inadequacy of chlorine, i.e. through inter-
action with organics and being unavailable for killing, and the site-specific require-
ments of biocidal doses. Levels of 0.2–0.5 mg L−1 delayed settlement of 30% of
mussel larvae (Khalanski and Bordet 1980). Compared to mussels, barnacles were
more resistant to continuous low-level chlorination and required higher dosages.
From the studies carried out at Astoria power station (NY, USA), a chlorine residual
of 0.1 mg L−1 for 1 week during the spat settlement season reduced the density of
settlement 15-fold. However, these concentrations were not effective in preventing
the settlement of the barnacle Balanus eburneus (Sarunac et al. 1994). Continuous
chlorination at concentrations above 0.8 mg L−1 were required to prevent settlement
of coelenterate Hydroids and tubeworms on steel surfaces in flow chambers
(Fig. 2a), whereas an intermittent chlorination of 1.2 mg L−1 with a 2 h-on/2 h-off
regime was effective in bringing down the settlement of these foulants on plate heat
exchanger surfaces (Murthy et al. 2005). In comparison, continuous application
of chlorine dioxide at residuals of 0.1–0.2 mg L−1 resulted in clean surfaces
(Ambrogi 1997) and elimination of the Mediterranean hydroid Laomedea flexuosaat
residuals of 0.1–0.2 mg L−1 (Geraci et al. 1993). Chlorine dioxide treatments (0.22
mg L−1) adopted at the Brindisi Nord power station on the Adriatic showed that test
panels placed inside the condenser boxes were clean of both macrofouling and
slime in comparison to periods before switching to chlorine dioxide, when 20 × 30
cm panels would accumulate a wet weight of 160 g over 3 months (Ambrogi 1997).
Similarly, at the Taranto steel plant located in the south of Italy, fouling biomass on
280 R. Venkatesan and P.S. Murthy

experimental panels were 60 kg m−2 year−1. Continuous treatment with chlorine

dioxide at a dose of 0.5 mg L−1 resulted in clean surfaces, as observed from foul-
ing collectors (Belluati et al. 1997). The higher biocidal action against macrofouling
organisms at low concentrations has resulted in many stations turning to chlorine
dioxide. At present, the studies at the Brindisi Nord power station and the Taranto
steel plant are the only literature available on dosages required to prevent settlement
of larvae by chlorine dioxide. Application of ozone to cooling water systems was
also found to be effective in preventing the settlement of mussel larvae by inhibition
of production of byssal thread at concentrations in the range 20 –30 mg L−1. Studies
by Lewis et al. (1993) have indicated that a minimum contact time of 5 h was
required for 100% mortality of veligers and post-veligers at concentrations of 0.5
mg L−1 at 15–20 C water temperatures. These biocides have to be evaluated under
dynamic conditions at varying velocities to assess their efficacy and to arrive at
some minimum dosages for cooling water systems.

4.2.3 Biocidal Requirements for Killing Established Fouling Communities

in Cooling Water Systems

Often in an operating plant the problem of biofouling gains attention and importance
when it leads to breakdown of equipment. The usual situation one encounters in an
operational plant is an established fouling community as a result of lack of surveil-
lance and monitoring and fine tuning of the biocidal dose and regime according to the
requirements to keep biofouling at bay. As a result, the biofouling load exceeds the
threshold limits and one is faced with the challenge of killing and removing the estab-
lished fouling communities. It is all the more important to keep the cooling water
systems clean from macrofouling as killing is not cleaning. An established fouling
community offers surface roughness for larvae to colonize the substratum. In the case
of macrofouling by hard-shelled organisms like barnacles, oysters and tubeworms
irreversible damage to the surface occurs and can be cleaned only by mechanical
methods like chiselling. Not all places in a cooling water systems are accessible to
cleaning and may result in replacement of the equipment. In many power stations,
bivalves are the most dominant of the fouling organisms. Dosages and regimes
required for preventing bivalve settlement are different to those required for removal
or killing of already settled mussels. Further, byssal threads of mussels dead or
detached tend to remain in the system leading to under-deposit corrosion and can
enhance attachment opportunities for incoming fouling larvae (Claudi and Mackie
1994). Discontinuous chlorination was not effective in killing mussels even at con-
centrations of 0.5–1.5 mg L−1 residuals. The biocidal action of chlorine in killing
mussels of the species Mytilus edulis and Mytilus galloprovincialis was found to be
dependent on temperature. Residuals of 0.2–1.0 mg L−1 required 15–135 days for
mortality (Lewis 1983). Toxicity modelling showed a tenfold decrease in the required
killing time for mussels, when comparing mortality rates at 10 C and 25 C. Low-level
continuous chlorination was more effective against mussel spat than on adults
(Travade and Khalanski 1986) Adult mussels were able to survive the continuous
Macrofouling Control in Power Plants 281

chlorination (residuals of 0.29 and 0.49 mg L−1) practised at the Gravelines plant.
Adult mussels survived up to 5 months whereas the recently settled mussels were far
more sensitive to chlorine. Settlement of spat was inhibited and a large portion of
existing spat were found to detach and die at continuous residuals of 0.49 mg L−1
within 20–30 days. At residuals of 0.29 mg L−1 growth inhibition and detachment of
spat was observed (Travade and Khalanski 1986).
Intermittent chlorinationwas ineffective in removing mussel community lodged
in the intake tunnel of the Madras atomic power station (MAPS), India.
Alternatively, a continuous high-level chlorination at residuals of 1.4 mg L−1 fol-
lowed by continuous low-level chlorination at 0.2 mg L−1 dislodged the mussel
community and about 187 tons of fouling biomass was collected in the travelling
water screens (Rajagopal et al. 1996). Low-level continuous chlorination of 0.2
mg L−1 led to reduction in the growth of the shell of the mussel Mytilus edulis as
observed from the growth rates of mussels in the cooling culverts (Thompson et al.
2000). The findings were consistent with introduced mussels also exhibiting the
same trend. High-level continuous chlorination has also proven to be effective in
eliminating mussels due to two processes: a decrease in water filtration rate, which
deprives the mussel of its food, or a progressive intoxication by oxidant com-
pounds absorbed within small amounts of seawater in the mantle cavity (Khalanski
and Bordet 1980). In comparison, low continuous chlorine residuals of 1.0 mg L−1
took 468 h (7 mm) and 570 h (25 mm) for 100% mortality in the brackish water
mussel Brachidontes striatulus, whereas high chlorine residuals of 5.0 mg L−1 took
102 h (7 mm) and 156 h (25 mm) for 100% mortality (Rajagopal et al. 1997).
In a cooling water system different species of mussels co-exist, hence species-
specific variability in tolerance of mussels to chlorination is an important aspect in
framing a treatment regime. Small sized mussels are more susceptible to chlorina-
tion than larger ones (Rajagopal et al. 2003). Similarly, response of different spe-
cies of tropical marine mussels, Perna viridis, Perna perna, Brachidontes striatulus,
Brachidontes variabilisand Modiolus philippinarum, to chlorination showed that
reduction in physiological activities is the lowest in P. viridis and the highest in
B. variabilis (Rajagopal et al. 2003). Mussels were able to tide-over continuous
low-level or intermittent chlorination by closing their shell valves to overcome the
period. Consequently, the technique of pulse chlorination(Poleman and Jenner
2002; European IPPC Bureau 2000) developed by KEMA has been found to be
effective in controlling bivalve fouling in European power stations as off-treatment
intervals occur when the mussels have shut their valves tight in response to the
biocide. Chlorine must be applied continuously at least during spawning seasons to
control bivalve settlement. On the other hand, semi-continuous treatments coupled
with high frequency treatments (i.e. 15 min-on/15 min-off and 15 min-on/30 min-
off) has shown good results for controlling mussels at residuals of 0.5 mg L−1
(Wiancko and Claudi 1994). Compared to mussels, oysters (Crassostrea madrasen-
sis) attach to surfaces by cementing one of the valves to the substratum, posing
severe problems. A continuous residual of 1.0 mg L−1 took 21 days for 100% mor-
tality in the size group 13 mm and 31 days for the size group 64 mm (Rajagopal et al.
2003). Compared to mussels, barnacles tolerated high chlorine residuals of 1.0 mg
282 R. Venkatesan and P.S. Murthy

L−1 for up to 15 days, where 75% of them survived up to 5 days only (Turner et al. 1948).
Another species of barnacles, Balanus improvisus, required 2.5 mg L−1 for 100%
mortality at short exposure times (5 min) (McLean 1973).
Further chlorine dioxide residuals of 0.2 mg L−1 were found to kill bivalve mussels
more rapidly than chlorine at concentrations of 1.1 mg L−1 (Jenner et al. 1998).
Comparison of the efficacy of chlorine and chlorine dioxide on killing mussels has
shown chlorine dioxide to be more effective at a concentration of 1.1 mg L−1. Long-
term semi-continuous addition of chlorine dioxide at residuals of 0.2 mg L−1 with
time intervals of 1 h-on and 2 h-off is as efficient as continuous treatment (Belluati
et al. 1997). Chlorine dioxide has also been reported to be effective against serpulid
worms at a concentration of 0.2 mg L−1. Experimental runs with chlorine and chlorine
dioxide conducted at the Vandellos II nuclear power station on the Mediterranean coast
of Catalonia in Spain showed that macrofouling was eliminated at chlorine dioxide
concentrations of 0.16–0.20 mg L−1 (residuals of 0.04 mg L−1) and chlorine at 1.1–1.2
mg L−1 (residuals of 0.3–0.4 mg L−1). However, the cost difference between chlorine
dioxide and electro-chlorination was found to be 30% (Jenner et al. 1998). In contrast
to chlorine dioxide, a far lower concentration of ozone (0.1 mg L−1) was required to
eliminate bryozoans (Plumatella emarginata) (Duvivier et al. 1996). Chlorine diox-
ide has shown to be effective against established fouling communities compared to
chlorine and seems a promising candidate for cooling water systems in the future. The
cost economics of application of chlorine dioxide needs to be worked out for the
treatment to be widely adopted. In comparison, studies using ozone for treating cool-
ing water systems in power plants are also limited. Concentrations of 0.25–0.5 mg L−1
were effective in eliminating the blue mussel (Mytilus) from European power stations
(Claudi and Mackie 1994). In another study, concentrations of 0.5 mg L−1 ozone were
required for a period of 7–12 days for 100% mortality of mussels (Lewis et al. 1993).
The features of ozone that make it attractive for treating once-through cooling water
systems are also its major drawbacks. One of the major disadvantages of ozone is that
it dissipates more rapidly in water, which in a way minimizes the downstream envi-
ronmental impact. However, the short life of ozone in water requires multiple injec-
tion points in the cooling water system to protect downstream equipment, which
would be probably very expensive and is the main reasons why this biocide is not so
popular for large once-through cooling systems.

4.2.4 Fouling Control in Once-Through Freshwater Cooling Systems

In freshwater systems, fouling by Asiatic clams, Zebra mussels and weeds poses a
severe problem. In once through systems using freshwater clogging of heat exchangers
by Asiatic clams has been related to changes in flow configuration in the service
water systems. Continuous chlorination of 0.6–0.8 mg L−1 was required for controlling
Asiatic clam settlement. Fouling by colonial hydroid Cordylophora caspiais a prob-
lem in several European and American power stations. Chlorine residuals of 0.2–5.0
mg L−1 with exposure time of 105 min and short intermittent exposure of 20 min did
not kills the animals but reduced their growth (Folino-Rorem and Indelicato 2005).
Macrofouling Control in Power Plants 283

Compared to marine mussels, the freshwater mussel Dreissena polymorphawas less

tolerant to chlorine. Continuous chlorination employed at the Ontario Hydro experi-
mental station on Lake Erie at Nanticoke showed that at residuals of 0.3 mg L−1,
attachment of the mussels was inhibited. Discontinuous treatments (half hour on, once
in 12 h) were ineffective even at higher dosages of 0.5–1.5 mg L−1. Similarly, another
power station (Cleveland Electric illuminating company installation on Lake Erie)
operating on a discontinuous mode failed to kill the mussels at 0.3 mg L−1 (Barton
1990). In contrast, semi-continuous chlorination has shown promising results at
residuals of 0.5 mg L−1 with high frequency treatments like 15 minon/15 min-off and
15 min-on/30 min-off (Wiancko and Claudi 1994). In comparison, response of the
Zebra mussels to shock chlorination showed that two successive shocks of 200 mg
L−1 once every 24 h resulted in 100% mortality of mussels in 9 days (Khalanski
1993). The action of chlorine in killing Zebra mussels was found to be dependent on
water temperature. For 95% mortality at 10°C, a time period of 42 days was required
as against 7 days at a water temperature of 25°C (Van Benschoten et al. 1993). The
study also demonstrated that compared to chlorine, chloramine concentrations above
1.5 mg L−1 were effective in controlling veligers of Zebra mussels in both static and
flow-through tests. Exposure times of 1,080 h at 0.25 mg L−1 and 252 h at 3.0 mg L−1
are required for 100% mortality of these mussels (Rajagopal et al. 2003). Continuous
chlorination at residuals of 0.5 mg L−1 was effective in killing the Asiatic clamsC.
fluminea with periods ranging from 2–3 weeks (Dohorty et al. 1986; Ramsey et al.
1988). In addition, monochloramine (NH2Cl) was found to be effective against the
Asiatic clams (Belanger et al. 1991). With respect to the freshwater Zebra mussel-
Dreissena polymorphabrief exposure to chlorine dioxide at a concentration of 10 mg
L−1 for 13 min or 50 mg L−1 for 3.2 min kills 50% of adult mussels, whereas at con-
centrations of 2 mg L−1 no mortality is observed (Montanat et al. 1980). Concentrations
of 5 mg L−1 in closed recirculating systems of the Seraing power station on the river
Meuse were found to be effective, giving 100% mortality of the bivalves (Corbicula sp.
and Dreissena sp.) over a period of 18 days (Jenner et al. 1998). Synthesis of the
above information reveals that biocidal requirements for fouling control in freshwater
once-through systems are far less than for seawater-cooled once-through systems.
In freshwater cooling systems (where Zebra mussels and Asiatic clams are the dominant
foulers) chlorine has been found to be the most effective and commonly used method
of mussel control in Europe, Asia and North America (Jenner et al. 1998; Claudi and
Mackie 1994; Rajagopal 1997).

5 New Approaches for Fouling Control in Heat Exchangers

5.1 Electrolytically Generated Biocides

Currently, electrochemical methods are being tested for treating industrial waters
with the goal of combating fouling without adversely affecting the environment.
Metal ions particularly silver, copper (Cu anodes) hydrogen peroxideand potassium
284 R. Venkatesan and P.S. Murthy

permanganate can be electrolytically generated (Martinez et al. 2004). In heat

exchangers made of titanium, anodic polarization by a current of some tens of
milliamps per square metre applied to titanium causes a low production of oxidant
species (chlorine or bromine) at the metal–seawater interface. However, with heavy
metal ions there is always the problem of occurrence of resistance in the organisms.
The current is low but is enough to inhibit the growth of titanium on plates and
tubes. Experiments to this effect at the Venetian Lagoon demonstrated the control
of settlement of macroorganisms and algae with a polarization of about 100–200
mA m−2. This technique is very interesting for heat exchangers considering the
effect of the pH decrease in the water close to the anodic polarized surface of tita-
nium (Cristiani 2005). Electrolytically generated biocides are particularly useful in
cooling systems to combat biofouling of sensors for temperature, conductivity and
pH. This technique is still in infancy and in-situ studies demonstrating this effect
are lacking. Further application of this technique to large cooling systems seems to
be an unviable proposition.

5.2 Surface Modification Approach to Control Biofouling

Surfactants or surface active agents alter the surface tension within the biofilm and at
the biofilm–substratum interface, allowing enhanced penetration by biocide molecules
and also more effective removal of the biofilm deposits from the surfaces. However,
they only address one of the forces that provide cohesion and adhesion of fouling lay-
ers. Several studies on the positive effects of the use of surfactants have been reported
from cooling water systems. Some of the most effective surfactants reported are ethylene
oxide/propylene oxide block copolymer (Donlan et al 1997), dimethlyamide (DMATO)
(Lutey et al. 1989), dinonylsulfosuccinate (Wright and Michalopoulos 1996), a com-
bination of peracetic acid with ethylene oxide/propylene oxide (Meade et al. 1997),
sodium dodecyl sulfate (SDS) in combination with urea (Whittaker et al. 1984), and
Tween 20 (Fletcher et al. 1991). Recently, low-energy surfaces have been prepared by
ion implantation (Yang et al. 1994). Low-energy surfaces can increase the induction
period of fouling and facilitate detachment of foulants (Yang et al. 1994; Forster et al.
1999) during which stable nucleation takes place at localized sites and the lateral
growth of individual nucleation sites results in a complete coverage of the surface.
Another study to minimize particle adhesion on stainless steel plate heat exchangers
used TiN sputter coatings, which decreased the surface energy and resulted in less
deposition of particles (Rosmaninho et al. 2005). Ion-sputtered diamond-like carbon
(Forster et al. 1999), self-assembled monolayers (SAMs) and electroless plated
surfaces (Yang et al. 2000b) have been used to mitigate fouling due to the weak adhe-
sion strength between the fouling layer and the heat transfer surface. SAMs of low
surface energy can prolong the induction period of fouling. Also, thermally resistant
(Yang et al. 2000a). SAM surfaces based on Si wafers exhibit no significant change
after heat treatment up to 200°C (Shin et al. 1999) and SAM films of hexadecyl
disulfide can withstand temperatures of up to 225°C (Nuzzo et al. 1987). SAMs can
Macrofouling Control in Power Plants 285

also protect metals against corrosion as they act as effective barriers against diffusion
of oxygen and water. tThe cross-linking SAMs can result in more robust films with
improved levels of protection (Itoh et al. 1995).Thus, SAM surfaces have great potential
for use as heat transfer surfaces to reduce fouling. The technique has not been used
widely in heat exchangers except for some small stations that require an improvement
in the rate of heat transfer. The technique as such seems to be interesting for plate-type
heat exchangers and needs to be evaluated under field conditions, provided the film
lasts over an extended period of time. However, a breakthrough against fouling has not
been achieved yet by the use of surfactants. Surfactants comprise only one of many
more components of integrated antifouling strategies.

6 Concluding Remarks

The incidence of macrofouling in cooling water circuits of power plants varies

considerably depending on the location and design of systems. The use of trash
racks at the offshore intake point and travelling water screens before the pumps is
a mandatory technique for removing debris and detached fouling biomass from
clogging the heat exchangers. Thermal treatment is an effective option but many
stations do not have the facility of recirculating effluent water in the cooling circuits.
Chlorine or sodium hypochlorite is in common use internationally and requires
high doses (0.5–1.0 mg L−1) to overcome the demand in water and for killing mac-
rofouling organisms. However, for effective plant operation the issue of killing
established macrofoulants is secondary to preventing their settlement and coloni-
zation, right from the initial stages of commissioning the cooling water circuit.
A fouled circuit provides a source of larvae, which colonize systems downstream,
and dosages required for inhibiting settlement are far less than those for killing
established communities. In general, power stations adopt low-level continuous
chlorination (with residuals of 0.1–0.2 mg L−1) at the outfall coupled with periodic
shock or booster doses of the biocide depending on the intensity of fouling. The use
of various techniques of chlorination, like shock chlorination and targeted chlorina-
tion of heat exchangers, has offered temporary relief to certain sections or equip-
ment in the circuit. With the advent of the technique of pulse chlorination, up to
50% reduction in chlorine consumption can be achieved and bring down the
environmental burden of toxic by-products (Poleman and Jenner 2002; European
IPPC Bureau 2000). Commercial variants of bromine are in use in some of the
European and Indian power stations. However, growing awareness of keeping bio-
fouling levels within the threshold to minimize plant shutdown and the increasing
regulations on effluent discharge has resulted in plant operators adopting the
stronger oxidant, i.e. chlorine dioxide. The low concentrations required for killing
and its environmentally safe nature have resulted in its use in power plants in spite
of the higher cost of this biocide. Prior to the commissioning of a power station,
effective biocidal dose and concentration need to be worked out on a site-specific
basis. Dosages worked out elsewhere will not be effective for a given location.
286 R. Venkatesan and P.S. Murthy

Side-stream monitors(Biobox) (Jenner et al. 1998) or electrochemical probes need

to be installed and monitored regularly. Spikes in settlement observed in side-stream
monitors can be taken as a signal to alter the biocide dose or regime to achieve
killing of new settlers. Continuous surveillance and monitoring of cooling water
and fine tuning of the biocidal programme will ensure that biofouling levels are
maintained well within the threshold limits. As biofouling is a surface-associated
phenomenon, a combined approach of treating the cooling water and surface pro-
tection by the use of foul release coatings would offer long term solutions to mac-
rofouling problems in cooling systems. Fouling release coatings have demonstrated
their ability to resist macrofouling at high water velocities and are a potential
option for cooling circuits (Leitch 1993; Kilgour and Mackie 1993; Claudi and
Mackie 1994). With regard to heat exchanger fouling, mechanical methods like
sponge rubber ball cleaning together with biocidal treatment is the only available
method of control. In shell and tube heat exchangers flow blockage due to clogging
of tubes by macrofoulants and biofilm is the primary problem. Contrary to the
concept that high shear forces created by chevron angles in plate heat exchangers
retard fouling, barnacle fouling on these heat exchangers has been observed.
Sedimentation and accumulation of corrosion products on these heat exchangers is
a problem to be overcome. Since online cleaning methods are not available for these
heat exchangers a control strategy should take into account a biocide, cleaners and
a corrosion inhibitor for optimum performance of these heat exchangers.

References

Ambrogi R (1997) Environmental impact of biocidal antifouling alternative treatments on seawa-

ter once-through cooling systems. In: Chlorine dioxide and disinfection. Proceedings of first
European symposium on chlorine dioxide and disinfection, Rome, 7–8 Nov 1996. Collana
Ambiente 17 pp. 119–132
Aprosi G (1988) Bryozoans in cooling water circuits of a power plant. Verh Int Verein Limnol
23:1542–1547
Barton LK (1990) Control of zebra mussels at CEI facilities. Annual meeting of the American
power conference, 23–25 April 1990, Chicago, pp. 1001–1005
Bass ML, Heath AG (1977) Cardiovascular and respiratory changes in rainbow trout, Salmo
gairdneri exposed intermittently to chlorine. Water Res 11:497–502
Belanger SE, Cherry DS, Farris JL, Sappington KG, Cairns J (1991) Sensitivity of the Asiatic
clam to various biocidal control agents. J Am Water Works Assoc 83:79–87
Bell KJ (1977) The effect of fouling on heat exchanger design, construction and operation. In:
Proceedings of the Ocean Thermal Energy Conversion (OTEC) biofouling and corrosion sym-
posium, pp. 19–29
Belluati M, Bartole L, Bressan G (1997) Chlorine dioxide and disinfection. In: Proceedings of first
European symposium on chlorine dioxide and disinfection, Rome, 7–8 Nov 1996. Collana
Ambiente 17
Bott TR (1993) Aspects of biofilm formation and destruction. Corrosion Rev 11(1–2):1–24
Bott TR (1995) Fouling of heat exchangers. Elsevier, Amsterdam, p. 576
Bott TR, Tianqing L (2004) Ultrasound enhancement of biocide efficiency. Ultrason Sonochem
11:323–326
Macrofouling Control in Power Plants 287

Brankevich GJ, De Mele MLF, Videla HA (1990) Biofouling and corrosion in coastal power plant
cooling water systems. MTS J 24:18–28
Butterfield PW, Camper AK, Ellis BD, Jones WL (2002a) Chlorination of model drinking water
biofilm: implications for growth and organic carbon removal. Water Res 36:4391–4405
Butterfield PW, Camper AK, Biederman A, Bargmeyer AM (2002b) Minimizing biofilm in the
present of iron oxides and humic substances. Water Res 36:3898–3910
Chow W (1987) Targeted chlorination schedules and corrosion evaluations. American Power
Conference Chicago
Claudi R, Mackie GL (1994) Practical manual for Zebra Mussel monitoring and control. Lewis,
Boca Raton, p. 227
Collins TM (1964) A method for designing seawater culverts using fluid shear for the prevention
of marine fouling. CERL report no RD/1/N93/64. Central Electricity Research Laboratories,
Leatherhead, pp. 1–5
Corfield J, Spooner D, Ray D, Clearwater S, Macaskill B, Roper D (2004) Biofouling issues for the
Marsden B power station cooling water system. NIWA client report HAM 2005–106 NIWA
Project MRP05251. National Institute of Water and Atmospheric Research, Wellington, NZ
Corpe WA (1977) Marine microfouling and OTEC heat exchangers. Proceedings of the Ocean
Thermal Energy Conversion (OTEC) biofouling and corrosion symposium, pp. 31–44
Coughlan J, Whitehouse J (1977) Aspects of chlorine utilization in the UK. Chesapeake Sci
18(1):102–111
Cristiani P (2005) Solutions to fouling in power station condensers. Appl Therm Eng
25:2630–2640
Dohorty FG, Farris JL, Cherry DS, Cairns Jr. J (1986) Control of freshwater fouling bivalve
Corbicula fluminea by halogenation. Arch Environ Contam Toxicol 15:535–542
Donlan RM (2000) Biofilm control in industrial water systems: approaching an old problem in
new ways. In: Evans LV (ed.) Biofilms: recent advances in their study and control. Harwood
Academic, pp. 333–361
Donlan RM, Elliott DL, Gibbon DL (1997) Use of surfactants to control silt and biofilm deposition
onto PVC fill in cooling water systems. International water conference, paper no IWC-97–73
Drake RC (1977) Increasing heat exchanger efficiency through continuous mechanical tube main-
tainance. In: Jensen LD (ed.) Biofouling control procedures technology and ecological effects.
Marcel Dekker, New York pp. 43–53
Duvivier L, Leynen M, Ollivier F, Van Damme A (1996) Fighting zebra mussel fouling with
ozone. In: Proceedings of the EPRI PSE Service water systems reliability improvement semi-
nar 25–27 June 1996, Daytona Beach, FL, p. 14
Electric Power Research Institute (EPRI) (1980) Review of open literature on effects of chlorine on
aquatic organisms. Report to the EPRI by Oak Ridge National Laboratory. EPRI, Palo Alto, CA
Engineering Sciences Data Unit (ESDU) (2000) Heat exchanger fouling in the pre-heat train of a
crude oil distillation unit. ESDU item 00016, ESDU, London
Epstein N (1983) Thinking about heat transfer fouling: a 5 × 5 matrix. Heat Transfer Eng.
14(1):25–36
European IPPC Bureau (2000) Document on the application of best available techniques to indus-
trial cooling systems, November 2000. BREF (11.00) Cooling systems. European IPPC
Bureau, Sevilla. http://eippcb.jrc.es. Last accessed 14 July 2008
Fisher DJ, Burton DT, Yonkos LT, Turley SD, Ziegler GP (1999) The relative acute toxicity of
continuous and intermittent exposures of chlorine and bromine to aquatic organisms in the
presence and absence of ammonia. Water Res 33(3):760–768
Flemming HC (2002) Biofouling in water systems-cases, causes and countermeasures. Appl
Microbiol Biotechnol 59:629–640
Flemming HC, Griebe T (2000) Control of biofilms in industrial waters and processes. In: Walker
J, Surman S, Jass (eds.) Industrial biofouling. Wiley, London, pp. 125–137
Fletcher M, Lessmann JM, Loeb GI (1991) Bacterial surface adhesives and biofilm matrix poly-
mers of marine and freshwater bacteria. Biofouling 4:129–140
288 R. Venkatesan and P.S. Murthy

Folino-Rorem NC, Indelicato J (2005) Controlling biofouling caused by the colonial hydroid
Cordylophora caspia. Water Res 39:2731–2737
Forster M, Aufustin W, Bohnet M (1999) Influence of the adhesion force crystal/heat exchanger
surface on fouling mitigation. Chem Eng Process 39:449–461
Fritsch A, Adamson W, Castelli V (1977) An evaluation of mechanical cleaning methods for
removal of soft fouling from heat exchanger tubes in OTEC power plants. Proceedings of the
Ocean Thermal Energy Conversion (OTEC) biofouling and corrosion symposium. pp.
159–166
Geraci S, Ambrogi R, Festa V, Piraino S (1993) Field and laboratory efficacy of chlorine dioxide
as antifouling in cooling systems of power plants. Oebalia 19:383–393
Goodman PD (1987) Effect of chlorination on materials for seawater cooling system: a review of
chemical reactions. Br Corros J 22(1):56–62
Gunasingh M, Jesudoss KS, Nandakumar K, Satpathy KK, Azariah J, Nair KVK (2002) Lethal
and sub-lethal effects of chlorination on green mussel Perna viridis in the context of biofouling
control in a power plant cooling water system. Mar Env Res 53:65–76
Hesselgreaves JE (2002) An approach to fouling allowances in the design of compact heat
exchangers. App Therm Eng 22:755–762
Ingols RS, Wyckoff HA, Kethley TW, Hodgden HW, Fincher EL, Hildebrand JC, Mandel JE
(1953) Bactericidal studies of chlorine. Ind Eng Chem 45:996–1000
Itoh M, Nishihara H, Aramaki K (1995) Preparation and evaluation of two dimensional polymer
films by chemical modification of an alkanethiol self assembled monolayer for protection of
copper against corrosion. J Electrochem Soc 142:3696–3704
James WG (1985) Mussel fouling and use of exomotive chlorination. Chem Ind 994–996.
Jenner HA (1980) The biology of the mussel Mytilus edulis in relation to fouling problems in
industrial cooling water systems. Janvier 434:13–19
Jenner HA (1982) Physical methods in the control of mussel fouling in seawater cooling systems.
Trib Cebedeau 35:287–291
Jenner HA (1983) Control of mussel fouling in The Netherlands: experimental and existing meth-
ods. In: Tous IAD, Miller MJ, Mussalli YG (eds.) Condenser macrofouling control technolo-
gies: the state of the art. Electric Power Research Institute, Hyannis. MA pp. 12–24
Jenner HA, Davis MH (1998) A new design in monitoring fouling: KEMA Biofouling Monitor
Fawley Biobox. In: Abstracts from the eighth international zebra mussel and other nuisance
species conference, Sacramento CA, 16–19 March 1998
Jenner HA, Janssen-Mommen JPM (1993) Monitoring and control of Dreissena polymorpha and
other macrofouling bivalves in the Netherlands. In: Nalepa TF, Schloesser DW (eds.) Zebra
mussels biology, impacts and control. Lewis, Boca Raton pp. 537–554
Jenner HA, Whitehouse JW, Taylor CJL, Khalanski M (1998) Cooling water management
in European power stations: biology and control of fouling. Hydroecologie Appliquee
10(1–2):225
Johnson KI, Henager CH, Page TL, Hayes PF (1986) Engineering factors influencing Corbicula
fouling in Nuclear Service Water Systems. In: Proceedings of the EPRI symposium on con-
denser macrofouling control technologies: the state of the art. Electric Power Research
Institute, Palo Alto, CA
Kawabe A, Treplin FW (1986) Control of macrofouling in Japan, existing and experimental meth-
ods. Res Rep 23:1–42
Khalanski M (1993) Testing of five methods for the control of zebra mussels in cooling circuits
of power plants located on the Moselle River. Proceedings: third international Zebra mussel
conference, Toronto, Canada
Khalanski M, Bordet F (1980) Effects of chlorination on marine mussels. In: Jolly RL, Brungs
WA, Cumming RB (eds.) Water chlorination chemistry environmental impacts and health
effects, vol 3. Ann Arbor Science, Ann Arbor, Michigan, pp. 557–567
Kilgour BW, Mackie GL (1993) Colonization of different construction materials by the Zebra
Mussel Dreissena polymorpha (Bivalvia: Dreissenidae), In: Nalepa TF, Schloesser DW (eds)
Zebra mussels biology, impacts and control. Lewis, Boca Raton, pp. 167–173
Macrofouling Control in Power Plants 289

Kovalak WP, Longton GD, Smithee RD (1993) Infestation of power plant water systems by the
zebra mussel (Dreissena polymorpha Pallas). In: Nalepa TF, Schloesser DW (eds) Zebra mus-
sels: biology, impacts and control. Lewis, Boca Raton, pp. 359–380
Kukulka DJ, Devgun M (2007) Fluid temperature and velocity effect on fouling. Appl Thermal
Eng 27:2732–2744
Leitch EG (1993) Evaluation of coatings for “Zebra Mussels”. Agenda and abstracts of third
international Zebra mussel conference, Toronto, Canada, 1993
Lewis BG (1983) Development of a preliminary model to predict time in days for 100% kill of
mussels subject to continuous chlorination. CERL note no TPRD/L/2469/N83, Central
Electricity Research Laboratories, Leatherhead
Lewis D, Van Benschoten JE, Jensen (1993) A study to determine effective ozone dose at various
temperatures for inactivation of adult Zebra mussels. Unpublished report for Ontario Hydro
Leglize L, Ollivier F (1981) Mise au point bi-bliographique sur la biologie et l’ecologie de
Dreissena polymorpha PALAS (1771) repartition geographique en France et dans les pays
limitrophes. EDF/DER Report HE/31–81.37
Ludensky M (2003) Control and monitoring of biofilms in industrial applications. Int Biodeter
Biodegr 51:255–263
Lutey RW, King VM, Gleghorn M (1989) Mechanisms of action of dimethylamides as a penetrant/
dispersant in cooling water systems. International water conference, paper no IWC-89–33
Martinez SS, Gallegos AA, Martinez E (2004) Electrolytically generated silver and copper ions to
treat cooling water: an environmentally friendly novel alternative. Int J Hydrogen Energy
29:921–932
McLean RI (1973) Chlorine and temperature stress on selected estuarine invertebrates. Am Zool
J Water Pollut Cont Fed 45:837–841
McMahon RF, Ussery TA, Miller AC (1993) Thermal tolerance in Zebra mussels (Dreissena poly-
morpha) relative to rate of temperature increase and acclimation temperature. In: Agenda and
abstracts of the third international Zebra mussel conference, Toronto, Canada, 1993.
pp 97–118
Meade RJ, Robertson LR, Taylor NR, LaZomby JG (1997) Method for preventing microbial
deposits in the papermaking process with ethylene oxide/propylene oxide copolymers. US
Patent No 5: 624, 575
Meesters KPH, Groenestijn JWV, Gerritse J (2003) Biofouling reduction in recirculating cooling
systems through biofiltration of process water. Water Res 37:525–532
Melo LF, Bott TR (1997) Biofouling in water systems. Exp Therm Fluid Sci 14:375–381
Meyer B (2003) Approaches to prevention, removal and killing of biofilms. Int Biodeter Biodegr
51:249–253
Montanat M, Merle G, Migeon B (1980) Essai d’utilisation du dioxide de chlore dans la boucle
TERA installee a Montereau. Resultats des tests de toxicite. EDF DER Report HE/3180.044
Mott IEC, Bott TR (1991) The adhesion of biofilms to selected materials of construction for heat
exchangers. Proceedings 9th international heat transfer conference, Jerusalem. 5:21–26
Mott IEC, Santos R, Bott TR (1994) The effect of surface on the development of biofilms.
Proceedings 3rd meeting European adhesion of micro-organisms to surfaces, Chatenay-
Malabry. C21:1–2
Muller-Steinhagen H (1993) Fouling: the ultimate challenge for heat exchanger design. In: Lee JS,
Chung SH, Kim KY (eds.) Transport phenomenon in thermal engineering, vol 2. Begell
House, pp 811–823
Murthy PS, Venkatesan R, Nair KVK, Inbakandan D, Syed Jehan S, Magesh Peter D, Ravindran
M (2005) Evaluation of sodium hypochlorite for fouling control in plate heat exchanger for
seawater application. Int Biodeter Biodegr 55:161–170
Murthy PS, Venkatesan R, Nair KVK, Subramoniam T (2008) Larval settlement and surfaces:
implications in development of antifouling strategies. Springer Ser Biofilms. doi:
10.1007/7142_2008_17
Neitzel DA, Johnson KI, Page TL, Young JS, Dalling PM (1984) Correlation of bivalve biological
characterestics and service water system design. Bivalve fouling of nuclear power plant service
290 R. Venkatesan and P.S. Murthy

water systems, vol 1. NUREG/CR-4070, PNL-5300, vol 1.US Nuclear Regulatory Commission,
Washington DC
Nuzzo RG, Fusco FA, Allara DL (1987) Spontaneously organized molecular assemblies 3.
Preparation and properties of solution adsorbed monolayers of organic disulfides on gold
surfaces. J Am Chem Soc 109:2358–2368.
Oldman J, Clearwater S, Hickey C, Macaskill B, Grange K, Handley S, Ray D (2004) Marsden B
coal fired power station: effects of cooling water abstraction and discharge. NIWA Client
Report HAM2004–108
Ormerod K, Lund Y (1995) The influence of disinfection processes on biofilm formation in water
distribution systems. Water Res 29:1013–1021
Petrucci G, Rosellini M (2005) Chlorine dioxide in seawater for fouling control and cost of disin-
fection in potable waterworks. Desalination 182:283–291
Poleman HJG, Jenner HJ (2002) Pulse chlorination the best available technique in macrofouling
mitigation using chlorine. Power Plant Chem 4:93–97
Prince EL, Muir AVG, Thomas WM, Stollard RJ, Sampson M, Lewis JA (2002) An evaluation of
the efficacy of aqualox for microbiological control of industrial cooling tower systems. J Hosp
Infect 52:243–249
Rabas TJ, Panchal CB, Sasscer DS, Schaefer R (1993) Comparison of river water fouling rates for
spirally indented and plain tubes. Heat Trans Eng 14(4):58–73
Rajagopal S (1997) The ecology of tropical marine mussels and their control in industrial cooling
water systems. Ph.D. thesis, University of Nijmegen, Nijmegen, The Netherlands
Rajagopal S, Van Der Velde G, Jenner HA (1994) Biology and control of brackish water mussel,
Mytilopsis leucophaeta in the Velsen and Hemweg power stations, The Netherlands. Part II: con-
trol measures. Report no 163871-KES/WBR 94–3128, KEMA Environmental Research, pp. 45
Rajagopal S, Venugopalan VP, Azariah J, Nair KVK (1995) Response of the green mussel Perna
viridis (L) to heat treatment in relation to power plant biofouling control. Biofouling
8:313–330
Rajagopal S, Nair KVK, Azariah J, Van der Velde G, Jenner HA (1996) Chlorination and mussel
control in the cooling conduits of a tropical power station. Mar Environ Res 41(2):201–221
Rajagopal S, Nair KVK, Van der Velde G, Jenner HA (1997) Response of mussel Brachidontes
striatulus to chlorination: an experimental study. Aquat Toxicol 39:135–149
Rajagopal S, Van der Velde G, Van der Gaag M, Jenner HA (2003) How effective is intermittent
chlorination to control adult mussel fouling in cooling water systems. Water Res 37:329–338
Rajagopal S, Venugopalan VP, van der Velde G, Jenner HA (2006) Mussel colonization of a high
flow artificial benthic habitat: byssogenesis holds the key. Mar Environ Res 62:98–115
Ramsey TGG, Tackett JH, Morris DW (1988) Effect of low-level continuous chlorination on
Corbicula fluminea.Environ Toxicol Chem 7:855–856
Reid DC, Bott TR, Miller R (1992) Biofouling in stirred tank reactors effect of surface finish. In:
Melo LF, Bott TR, Fletcher M, Capdeville B (eds.) Biofilms science and technology. Kluwer,
Dordrecht, pp. 521–526
Rosmaninho R, Rizzo G, Muller-Steinhagen H, Melo LF (2005) Antifouling stainless steel based
surfaces for milk heating processes. In: Muller-Steinhagen H, Malayeri MR, Watkinson AP
(eds.) Proceedings of 6th international conference on heat exchanger fouling and cleaning
challenges and opportunities, Kloster Irsee, Germany5–10 June 2005. ECI Symp Ser
RP2:97–102
Roy D, Wong PK, Engelbrecht RS, Chian ES (1981) Mechanism of enteroviral inactivation by
ozone. Appl Environ Microbiol 41:718–723
Sarunac N, Guida V, Weisman R, Zhang Z (1994) Integration of macrobiofouling control methods
into an effective macrobiofouling control strategy. Proceedings condenser technology confer-
ence EPRI Report TR-1034753.39–3.68
Sasikumar N, Nair KVK, Azariah J (1992) Response of barnacles to chlorine and heat treatment:
an experimental study for power plant biofouling control. Biofouling 6:69–79
Shin HJ, Wang YH, Sukenik CN (1999) Pyrolysis of self-assembled organic monolayers on oxide
substrates. J Mater Res 14:2116–2123
Macrofouling Control in Power Plants 291

Sheikholeslami R (2000) Composite fouling of heat transfer equipment in aqueous media a

review. Heat Transfer Eng 21:34–42
Smeltzer MS (2008) Biofilms and aseptic loosening. Springer Ser Biofilms. doi:
10.1007/7142_2008_1
Sohn J, Amy G, Cho J, Lee Y, Yoon Y (2004) Disinfectant decay and disinfection by-products
formation model development: chlorination and ozonation by-products. Water Res
38:2461–2478
Strauss SD (1989) New methods, chemical improve control of biological fouling. Power 51–52
Strauss SD, Puckorius PR (1984) Cooling water treatment for the control of scaling, fouling and
corrosion. Power S1–S24
Syrett BC, Coit RL (1983) Causes and prevention of power plant condenser tube failures. Mater
Perform 4:44–50
Thompson IS, Richardson CA, Seed R, Walker G (2000) Quantification of Mussel (Mytilus edulis)
growth from power station cooling waters in response to chlorination procedures. Biofouling
16(1):1–15
Travade F, Khalanski M (1986) Controle de la salissure biologique des circuits de centrals ther-
miques cotieres parles moules (Mytilus edulis). Etude d’optimisation de la chlorination a
Gravelines Haliotis 15:265–273
Turner HJ, Reynolds DM, Redfiled AC (1948) Chlorine and sodium pentachlorphenate as fouling
preventatives in sea water conduits. Ind Eng Chem 40:450–453
Tuthill AH (1985) Sedimentation in condensers and heat exchangers. Causes Effects Power Eng
89:46–49
US Environmental Protection Agency (USEPA) (1994) National primary drinking water regula-
tions: disinfectants and disinfection. USEPA, Washington, DC
US Environmental Protection Agency (USEPA) (2002) National recommended water quality
criteria 2002: EPA-822-R-02–047. USEPA, Office of Water, Washington DC
Van Benschoten UJE, Jensen JN, Lewis D, Brady TJ (1993) Chemical oxidants for controlling Zebra
mussels (Dreissena polymorpha): a synthesis of recent laboratory and filed studies. In: Nalepa TF,
Schloesser DW (eds.) Zebra mussels biology, impacts and control. Lewis, London, pp. 599–619
Venugopalan VP, Nair KVK (1990) Effects of a biofouling community on cooling water charac-
terestics of a coastal power plant. Can J Mar Sci 8:233–245
Vieira MJ, Oliveira R, Melo LF, Pinheiro MM, Van der Mei HC (1992) Biocolloids and biosur-
faces. J Dispersion Sci Tech 13(4):437–445
Vreenegoor SM, Block RM, Rhoderick JC, Gullans SR (1977) The effects of chlorination on the
osmoregulatory ability of the blue crab Callinectes sapidus Assoc. Southeastern Biol Bull 24–93
Walsh SE, Maillard JY, Russell AD, Catrenich CE, Charbonneau DL, Bartolo RG (2003)
Development of bacterial resistance to several biocides and effects on antibiotic susceptibility.
J Hosp Infect 55:98–107
Whitehouse JW (1985) Marine fouling and power stations. A collaborative research report by
CEGB, EDF, ENEL and KEMA, pp. 1–48
Whittaker C, Ridgeway HR, Olson BH (1984) Evaluation of cleaning strategies for removal of
biofilms from reverse osmosis membranes. Appl Environ Microbiol 48:395–403
Wiancko PM, Claudi R (1994) The Ontario Hydro final strategy for Zebra mussel control. Fourth
international Zebra mussel conference, Madison, Wisonsin, USA, pp. 225
Wright JB, Michalopoulos DL (1996) Method and composition for enhancing biocidal activity.
European Patent No EP0741109A2
Yang CF, Xu DQ, Shen ZQ (1994) A theoretical analysis and experimental study of the induction
period of calcium carbonate scaling. J Chem Ind Eng (China) 45:199–205
Yang QF, Ding J, Shen ZQ (2000a) Investigation on fouling behaviors of low energy surface and
fouling fractal characterestics. Chem Eng Sci 55:797–805
Yang QF, Ding J, Shen ZQ (2000b) Investigation of calcium carbonate scaling on ELP surface. J
Chem Eng Jpn 33:591–596
Young IC, Rong L, William JM, Lewis F (2000) Study of scale removal methods in a double pipe
heat exchanger. Heat Transfer Eng 21:50–57
Inhibition and Induction of Marine
Biofouling by Biofilms

S. Dobretsov

Abstract Microbial biofilms, predominantly composed of bacteria and diatoms,

affect the settlement of invertebrate larvae and algal spores by production of bioac-
tive compounds that can inhibit or induce settlement of biofoulers. In this review I
summarize the studies on the inductive and inhibitive properties of biofilms.
Particular attention has been given to antifouling and inductive compounds from
marine microorganisms and quorum sensing signaling in prokaryotes and eukaryotes.
Additionally, future research directions in the field of marine microbial biofouling
are highlighted.

1 Introduction: Biofilms and Biofouling

Any natural and man-made substrates in the marine environment are quickly
subject to biofouling, which is due to different species of micro- and macroorgan-
isms (Railkin 2004). The process of biofouling has three main stages: adsorption of
dissolved organic molecules, colonization by prokaryotes and eukaryotes, and
subsequent recruitment of invertebrate larvae and algal spores (Maki and Mitchell
2002). These stages can overlap, be successional, or occur in parallel.
Aggregates of microorganisms adhered to each other and/or to surfaces with a
distinctive architecture can be referred as biofilms (Maki and Mitchell 2002). In
marine environments biofilms mainly consist of numerous species of bacteria and
diatoms (Railkin 2004) incorporated into a matrix of extracellular polymers (EPS)
composed of high molecular weight polysaccharides (Donlan 2002). Other unicel-
lular organisms, like flagellates, yeasts, sarcodines and ciliates, contribute less than
1% to the total number of cells in biofilms (Railkin 2004).

S. Dobretsov
Marine Science and Fisheries Department, Agriculture and Marine Sciences College,
Sultan Qaboos University, Al-Khod 49, PO Box 123, Sultanate of Oman
Benthic Ecology, IFM-GEOMAR, Kiel University, Düsternbrooker Weg 20,
24105, Kiel, Germany
e-mail: sergey_dobretsov@yahoo.com, sergey@squ.edu.om

Springer Series on Biofilms, doi: 10.1007/7142_2008_10 293 293

© Springer-Verlag Berlin Heidelberg 2008
294 S. Dobretsov

Biofilm development is a multistep process dependent on the properties of the

substratum and environment and on the composition of the biofilm. At the same
time, all biofilms in their development pass through several stages, such as attach-
ment of cells and slime production, biofilm growth and, finally, biofilm sloughing
and cell detachment (Lewandowski 2000).
Every biofilm is unique and heterogeneous in space and time, ranging from
single layer of bacterial cells to multilayer biofilms containing numerous species of
bacteria, diatoms, Archaea, and Eucarya (Donlan 2002). Changes in environmental
conditions, such as water turbulence, temperature, salinity, light regime, and
amount of nutrients, immediately change the composition of biofilms (Wieczorek
and Todd 1998; Lau et al. 2005) and the production of chemical compounds (Miao
et al. 2006).
Bacteria in biofilms control their growth and densities by a regulatory mecha-
nism named quorum sensing (QS). It consists of production and release of low
molecular weight signal molecules that activate or de-activate target bacterial genes
responsible for cell division and adhesion. Quorum sensing signals can play a role
in interactions between bacteria and higher organisms, such as the squid E. scolopes
(Ruby and Lee 1998) and the alga Ulva (Enteromorpha) sp. (Tait et al. 2005). Some
marine organisms have the capacity to interfere with bacterial QS signals in order
to control biofilm formation on their surface (Zhang and Dong 2004).
Biofilms can have a substantial impact on biofouling communities by mediation
of protist’s colonization, the settlement of invertebrate larvae and macroalgal spores
(Qian 1999; Egan et al. 2002; Huang and Hadfield 2003). On the whole, the physical

14
Percentage of biofouling publications

12

10

0
1980 1985 1990 1995 2000 2005
a Years

Fig. 1 Marine biofouling-related publication trends in the scientific literature. To access the
frequency of marine biofouling-related papers, we ran a search on the Web of Science (Science
Citation Index) for the period 1980–2006. a Rate of marine biofouling publications. b Main topics
in marine biofouling studies. c Publications about the main marine biofouling organisms. Our
search terms were “marine” plus specific terms presented in this figure. Because some articles
considered multiple topics, the bars in b and c add up to more than 100%
Inhibition and Induction of Marine Biofouling by Biofilms 295

Percentage of biofouling studies

80

60

40

20

g
s
ae

es

es
ds
ilm

li n
iti

or
un
rv

ou
of

un
la

sp
po
bi

tif
m

m
an
m

b
co
co

30
Percentage of biofouling studies

25

20

Microfouling
15 Algae
Invertebrates

10

0
di ria

s
pr ngi
oa
ba gae

m les

sp els
hy ges

ec lyc a

de ta
tu ata

br ta

a
om

po rozo

zo
ca
no ae
oz
e

s
ac
fu

rm
on

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al
ct

us
at

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ot

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Fig. 1 (continued)

and chemical properties of biofilms, their composition, and accumulated chemical

compounds inside the EPS matrix affect formation of biofouling communities
(Maki and Mitchell 2002; Railkin 2004; Thiyagarajan et al. 2006).
Since 1972, the literature on biofouling has grown dramatically (Fig. 1a).
This can be explained by several reasons. Biofouling is a serious problem for
marine industries, aquaculture, and navies around the world (Railkin 2004;
Yebra et al. 2004). The most effective methods of biofouling control are based
296 S. Dobretsov

on the application of highly toxic substances, like tributyl tin (TBT) and copper
(Yebra et al. 2004). The recent ban of TBT and other tin-containing substances
in antifouling paints stimulated biofouling studies and increased the necessity to find
“environmentally friendly” non-toxic defensive compounds against biofouling.
According to the Web of Science, most biofouling publications deal with single
species of invertebrate larvae, while investigations of biofilms and biofouling com-
munities received less attention from researchers (Fig. 1b). Among biofilm studies,
bacteria-related publications dominate (Fig. 1c).
In this review, I shall mainly cover different aspects of microbe–larval interac-
tions. A number of reviews over the past years have presented information in part
and in full about the antifouling compounds from cyanobacteria (Dahms et al.
2006), bacteria (Maki and Mitchell 2002; Dobretsov et al. 2006), and marine organ-
isms (Railkin 2004, Fusetani et al. 2006), therefore these aspects will not be covered
here and the reader is directed to these publications. I here review the interactions
between microbes and macrofoulers with special emphasis on:
1. Induction and inhibition of larval settlement by biofilms
2. Chemical quorum sensing signaling between prokaryotes and eukaryotes
3. Directions for the future investigations

2 Effect of Biofilms on Larval Settlement

Biofilms are known to be important for the settlement of marine invertebrate larvae
and spores of macroalgae (see reviews Wieczorek and Todd 1998; Maki and
Mitchell 2002; Railkin 2004; Dobretsov et al. 2006). Marine biofilms can enhance
(Kirchman et al. 1982; Patel et al. 2003; Qian et al. 2003; Huang and Hadfield
2003), inhibit (Maki and Mitchell 2002; Holmström et al. 2002; Egan et al. 2001;
Dobretsov and Qian 2002, 2004) or have no effect on settlement of marine inverte-
brate larvae and macroalgal spores (Wieczorek and Todd 1998).
Different species of algae and invertebrates respond to biofilms differently.
Larvae of specialist species may settle only on biofilms with a specific microbial
composition, while generalists can settle on any kind of biofilm. The settlement
response of the generalist larvae of Hydroides elegans depends on the density of
bacteria in biofilms but has no relationship with bacterial community composition
(Qian 1999; Unabia and Hadfield 1999; Lau et al. 2005; Nedved and Hadfield
2008). In contrast, larvae of Balanus amphitrite and B. improvisus differentiated
the composition of intertidal and subtidal biofilms in experiments where there was
a choice of sensing different settling cues (Qian et al. 2003; Thiyagarajan et al.
2005). Analogously, larvae of the bryozoan Bugula neritina in all cases attached on
the subtidal biofilms when offered a choice of biofilms from different tidal regions
(Dobretsov and Qian 2006). Since microbial community composition in biofilms
varies substantially among tidal zones and substrata, the differential response of
specialist larvae to biofilm composition may allow the larvae to evaluate substrata
and then selected the suitable ones (Qian et al. 2003).
Inhibition and Induction of Marine Biofouling by Biofilms 297

2.1 Induction of Larval Settlement by Biofilms

Numerous studies demonstrated an induction of larval settlement by single species

of bacteria or diatoms (Table 1, see reviews: Wieczorek and Todd 1998; Maki and
Mitchell 2002; Railkin 2004). Additionally, multispecies biofilms promote settle-
ment and metamorphosis of polychaetes, hydroids, bryozoans, mollusks, tuni-
cates, and barnacles (Table 1). The data presented in this table, clearly show that
most of publications were focused on species with high economic significance,
either because they can cause a significant biofouling problem (e.g., barnacles and
polychaetes) or because they are commercially exploited (e.g., mollusks). The
suitability of biofouling species for bioassays is another criterion for the selection
of study objects. Most species that have been used can be reared and grown in the
laboratory (e.g., barnacles, mollusks, and polychaetes) or can release lecitotrophic
larvae (e.g., some bryozoa), which do not require intensive culturing and larval
feeding (Wieczorek and Todd 1998).
Up to now only a limited number of biofouling field studies have been per-
formed (Table 1). In most cases, investigators have studied the effect of monospe-
cies microbial films on larval settlement and metamorphosis in laboratory
experiments. These studies give only limited information about the role of biofilms
in the field and cannot adequately predict the settlement of larvae and spores in
response to mixed-species biofilms (Lau et al. 2002), in which most bacterial
strains cannot be cultivated (Dobretsov et al. 2006).
The effect of diatoms on settlement and metamorphosis of invertebrate larvae
has not been well documented (Table 1). Most of the diatoms isolated from
Hong Kong biofilms induced larval settlement of the tube worm Hydroides
elegans in laboratory experiments (Harder et al. 2002a). Further investigation
demonstrated that carbohydrates but not proteins from EPS of the diatoms
Achnanthes sp. and Nitzschia constricta induced larval settlement of H. elegans
(Lam et al. 2005). Larval attachment of the bryozoan Bugula neritina correlated
with the density of diatoms in biofilms (Kitamura and Hirayama 1987). No
evidence that zoo- or phytoflagellates can induce or inhibit larval settlement
was detected, despite the facts that flagellates are the third largest group in
marine biofilms and that the densities of flagellates correlate with those of mollusks
(Railkin 2004).
Water-soluble metabolites (Fitt et al. 1989; Rodriguez 1993; Dobretsov and
Qian 2002), surface-associated signals (Kirchman et al. 1982; Szewzyk et al. 1991;
Lam et al. 2005), and volatile molecules (Harder et al. 2002b) produced by bacteria
and diatoms induce larval settlement. Compared to the large number of chemical
compounds that have been isolated and identified from marine organisms, only a
few inductive compounds from microorganisms have been identified. These com-
pounds include lipids (Schmahl 1985), oligopeptides (Neumann 1979), glycocon-
jugates (Kirchman et al. 1982; Szewzyk et al. 1991), amino acids (Fitt et al. 1989),
alkanes, alkenes, and hydroxyketones (Harder et al. 2002b). Therefore, isolation
and identification of inductive compounds from microorganisms should be an
important target of future investigations.
298 S. Dobretsov

Table 1 Induction of larval settlement by microbial biofilms (bf)

Species of larvae Place of experi-
(Phylum/Class) ment Inductive biofilms References
Cnidaria
Aurelia aurita Laboratory Monospecies bf of Schmahl (1985)
Micrococcaceae
Cassiopea Laboratory bf of the bacterium Hofmann and Brand
andromeda Vibrio sp. (1978); Neumann
(1979)
Cyanea capitata Laboratory Multispecies bf Brewer (1976)
Hydractinia Laboratory bf of the Leitz and Wagner
echinata bacterium (1993)
Alteromonas
epejiana
Obelia loveni Laboratory Multispecies bf Dobretsov (1999)
Clava multicornis Laboratory Multispecies bf Orlov (1996)
Acropora millepora Laboratory bf of the bacterium Negri et al. (2001)
Pseudoalteromonas
Heteroxenia Laboratory bf of the bacteria Henning et al. (1991)
fuscenscens
Acropora Laboratory Multispecies bf Webster et al. (2004)
microphthalma
Mollusca
Mytilus Laboratory Multispecies bf Bao et al. (2007)
galloprovincialis
Mytilus edulis Laboratory, field Multispecies bf Bayne (1964)
Pinctada maxima Laboratory Multispecies bf; Zhao et al. (2003)
monospecies
bf of bacteria
Chlamys islandica Laboratory Multispecies bf Harvey et al. (1995)
Crassostrea gigas, Laboratory bf of the bacterium Fitt et al. (1989)
C. virginica Alteromonas
colwelliana
Saccostrea Laboratory Monospecies bf Anderson (1996)
commercialis of bacteria
Haliotis discus, Laboratory Multispecies bf; Morse et al. (1984);
H. rufescens, monospecies Roberts (2001)
H. laevigata bf of bacteria
and diatoms
Ostrea edulis Laboratory Multispecies bf Knight-Jones (1951)
Placopecten Laboratory Multispecies bf Parsons et al. (1993)
magellanicus
Concholepas Laboratory Monospecies bf Rodriguez et al. (1993)
concholepas of bacteria
Bryozoa
Bugula neritina Laboratory, field Multispecies bf; Mihm et al. (1981);
monospecies Kitamura and
bf of bacteria Hirayama (1987);
and diatoms Maki et al. (1989);
Dahms et al.
(2004); Dobretsov
and Qian (2006)
(continued)
Inhibition and Induction of Marine Biofouling by Biofilms 299

Table 1 (continued)
Species of larvae Place of experi-
(Phylum/Class) ment Inductive biofilms References
B. simplex, Laboratory Multispecies bf Brancato and
B. stolonifera, Woollacott (1982)
B. turrita
Annelida
Hydroides elegans Laboratory, field Multispecies bf; Hadfield et al. (1994);
monospecies bf of Lau et al. (2002);
bacteria and diatoms Harder et al.
(2002a); Lam et al.
(2005)
Pomatoceros lamarkii Laboratory Multispecies bf Hamer et al. (2001)
Janua brasiliensis Laboratory bf of the bacterium Kirchman et al. (1982)
Halomonas marina
Spirorbis borealis Laboratory Multispecies bf Williams (1964)
S. corrallinae, Laboratory Multispecies bf De Silva (1962)
S. tridentatus
S. spirorbis Laboratory, field Multispecies bf Wieczorek and Todd
(1998)
Arthropoda/Cirripedia
Balanus amphitrite Laboratory, field Multispecies bf; Maki et al. (1988);
monospecies bf of Khandeparker et al.
bacteria and diatoms (2002); Qian et al.
(2003); Patil and
Anil (2005)
Balanus trigonus Laboratory Multispecies bf Lau et al. (2005);
Thiyagarajan et al.
(2006)
Balanus cariosus, Field Multispecies bf Strathmann et al.
B. glandula (1981)
Notomegabalanus Field Multispecies bf Hentschel and Cook
algicola (1990)
Semibalanus Laboratory, field Multispecies bf Le Tourneux and
balanoides Bourget (1988);
Thompson et al.
(1998)
Echinodermata/Echinoidea
Heliocidaris Laboratory, Multispecies bf and Huggett et al. (2006)
erythrogramma field the bacterium
Pseudoalteromonas
luteoviolacea
Acanthaster planci Laboratory Bacterial monospecies bf, Johnson and Sutton
multispecies bf (1994)
Arbacia punctulata, Laboratory Multispecies bf Cameron and
Lytechinus pictus Hinegardner (1974)
Strongylocentrotus Laboratory Multispecies bf Pearce and Scheibling
droebachiensis (1991)
S. purpuratus Laboratory Multispecies bf Amador-Cano et al.
(2006)
(continued)
300 S. Dobretsov

Table 1 (continued)
Species of larvae Place of experi-
(Phylum/Class) ment Inductive biofilms References
Chordata/Ascidiacea
Ciona intestinalis Laboratory bf of the bacterium Szewzyk et al. (1991)
Pseudoalteromonas
sp.
Multispecies bf Wieczorek and Todd
(1998)

It remains unknown how biofilm-derived cues trigger settlement and metamor-

phosis of invertebrate larvae and algal spores. The neuronal and genetic bases of the
signal transduction pathways involved in this process have been studied only rarely.
Previous studies suggested that specific larval receptors may be involved in the set-
tlement process and that larval genes are differentially expressed during larval met-
amorphosis (Qian 1999). Lectins on the surface of the larvae of the polychaete
Janua brasiliensis (Kirchman et al. 1982), B. amphitrite (Khandeparker et al.
2002), Bugula spp. (Maki et al. 1989), the hydrozoan Obelia loveni (Railkin 2004),
and the green alga Dunaliella sp. (Mitchell and Kirchman 1984) play an important
role in the recognition of marine biofilms. For example, incubation of J. brasiliensis
larvae in solutions of d-glucose inhibited their settlement and metamorphosis
(Kirchman et al. 1982). The attachment of the larvae was also suppressed if bacte-
rial films in the experiment were pretreated with a solution of the lectin concanava-
lin A. This allowed the authors to formulate the hypothesis that lectins similar to
concanavalin A are located on the surface of the larvae and interact by the “lock-
and-key” scheme with bacterial polysaccharides and glycoproteins, which leads to
the settlement of larvae and their metamorphosis (Kirchman et al. 1982). Beside the
lectin receptors, G-protein-coupled receptors and two signal transduction systems
(adenylate cyclase/cylic AMP and phosphatidyl-inositol/diacylglycerol/protein
kinase C) play an important role in regulating metamorphosis of barnacles, mol-
lusks and, possibly, polychaetes (Baxter and Morse 1992; Clare et al. 1995).
Finding of larval receptors and identification of microbial cues involved in larval
and spore settlement will help us understand the signal transduction pathways and
facilitate the search for and development of new antifouling technologies.

2.2 Inhibition of Larval Settlement by Biofilms

Generally, the amount of inhibitive and inductive isolates in marine biofilms is

approximately equal (Lau et al. 2002; Dobretsov and Qian 2004). Initially, it had
been proposed that bacteria belonging mostly to the genus Pseudoalteromonas
inhibit larval and spore settlement (Egan et al. 2001; Holmström et al. 2002). Later,
it was shown that a wide range of bacterial taxa can inhibit larval settlement
(Burgess et al. 2003; Dobretsov et al. 2006; Table 2). For instance, the marine bacteria
Table 2 Antialgal and antilarval compounds isolated from marine biofilms
Effective
Antifouling concentrations Mode
Microbial strain compound (µg mL−1) of action Effective against Reference
Bacterium Alteromonas sp. Ubiquinone 12.5–25.0 NT Barnacle Balanus amphitrite Kon-ya et al. (1995)
(LE)
Bacterium Acinetobacter sp. 6-Bromoindole-3- 10 NT Barnacle B. amphitrite Olguin-Uribe et al.
carbaldehyde (1997)
Bacteria Halomonas Polysaccharides ? NT Barnacle B. amphitrite (LE) Maki et al. (1988)
(Deleya) marina, Vibrio
campbelli
Bacterium Vibrio Heat stable, polar 1,333–0.013 NT Barnacle B. amphitrite, Dobretsov and Qian
alginolyticus polysaccharide(s) polychaete Hydroides (2002); Harder
>200 kDa elegans, bryozoan Bugula et al. (2004)
neritina (LE)
Bacteria Vibrio sp. and Heat stable, polar ? NT Polychaete H. elegans, Dobretsov and Qian
an unidentified polysaccharides bryozoan B. neritina (LE) (2004)
α-Proteobacterium >100 kDa
Bacterium Streptomyces Diketopiperazines 25–100 NT Barnacle B. amphitrite (LE) Li et al. (2006)
Inhibition and Induction of Marine Biofouling by Biofilms

fungicidicus
Bacterium Pseudoalteromonas Proteolytic enzymes 0.001 NT Bryozoan B. neritina Dobretsov et al.
issachenkonii (LE), barnacle B. amphitrite, (2007b)
byozoans B. neritina,
Schizoporella sp. (FE)
Bacterium Shewanella 2-Hydroxymyri 10 R Alga Ulva pertusa spores Bhattarai et al. (2007)
oneidensis stic acid (LE), Ulva sp. (FE)
(continued)
301
Table 2 (continued)
302

Effective
Antifouling concentrations Mode
Microbial strain compound (µg mL−1) of action Effective against Reference
Bacterium Shewanella cis-9-Oleic acid 100 R Alga Ulva pertusa (LE), Bhattarai et al. (2007)
oneidensis Delisia fimbriata,
Sargassum sp., Ulva
pertusa, B. amphitrite,
Mytilus sp., Spirorbis
borealis (FE)
Cyanobacterium Phormidium Galactosyl ? T Antialgal (LE) Murakami et al. (1991)
tenue diacylglycerol
Cyanobacterium Nodularia Norharmalane 0.5–18.0 T Alga Nostoc insulare (LE) Volk (2006)
harveyana
Bacterium Pseudoalteromonas Heat-sensitive, ? ? Algae Polysiphonia sp., Ulva Egan et al. (2001)
tunicata polar compound lactuea (LE)
3–10 kDa
Cyanobacterium Aponin ? T Alga Gymnodinium breve (LE) McCoy et al. (1979)
Gomphosphaeria
aponina
Fungus 3-Chloro-2,5- 0.67–3.81 NT Tubeworm Hydroides elegans, Kwong et al. (2006)
Ampelomyces sp. dihydroxybenzyl barnacle B. amphitrite (LE)
alcohol
T toxic; NT non-toxic; R repellent; ? no information; LE laboratory experiment; FE field experiment
S. Dobretsov
Inhibition and Induction of Marine Biofouling by Biofilms 303

Halomonas (Deleya) marina (Maki et al. 1988) and bacteria belonging to the gen-
era Vibrio, Alteromonas, Flavobacterium, Micrococcus, Rhodovulum, and
Pseudomonas (Mary et al. 1993; Lau et al. 2003) inhibited larval attachment of the
barnacle B. amphitrite. There is no any predictive relationship between the phylo-
genetic affiliation of bacteria and their antifouling activity (Lau et al. 2002; Patel
et al. 2003; Dobretsov and Qian 2004).
Epibiotic bacteria associated with marine organisms have been proposed as an
important source of antifouling compounds since they may help to protect their hosts
from biofouling (Dobretsov and Qian 2002; Holmström et al. 2002). The bacterium
P. tunicata is one of the first isolates that produced a range of antilarval, antialgal,
antifungal, and antibacterial compounds (Holmström et al. 2002). Forty two bacte-
rial isolates from different marine organisms produced antibacterial compounds, and
one strain (Pseudomonas sp.) inhibited settlement of B. amphitrite larvae and Ulva
lactuca spores (Burgess et al. 2003). Recent studies confirmed the antifouling
activity of epibiotic bacterial strains isolated from marine sponges, corals, and algae
(Holmström et al. 2002; Dobretsov and Qian 2002; Dobretsov and Qian 2004).
These examples demonstrate that epibiotic bacteria associated with marine organ-
isms can be an important source of antifouling compounds.
Even though biofilms and their compounds are key factors in the establishment
of biofouling communities (Qian et al. 2003), only a few antifouling compounds
have been isolated from marine bacteria so far (Dobretsov et al. 2006; Fusetani
et al. 2006; Table 2). The first identified antilarval compound from marine bacteria
– ubiquinone – was isolated from Alteromonas sp. (Kon-ya et al. 1995). The mode
of action of this compound has not been discovered but the authors showed that
ubiquinone inhibited larval settlement of the barnacle B. amphitrite in a non-toxic
way (Table 2). In another study, 6-bromoindole-3-carbaldehyde isolated from the
γ-Proteobacteria Acinetobacter sp. showed antifouling activity against larvae of the
barnacle B. amphitrite (Olguin-Uribe et al. 1997). Several strains of cyanobacteria
produce cytotoxic compounds that affect algal growth and survival (Volk 2006).
Marine fungi have been shown to produce antifouling compounds as well (Kwong
et al. 2006; Table 2). Antilarval and antialgal compounds isolated from marine
microbes include lipids, polysaccharides, fatty acids, piperazines, and proteins
(Table 2). Recently, a proteolytic enzyme from the deep-sea bacterium
Pseudoalteromonas issachenkonii has been isolated (Dobretsov et al. 2007a). This
enzyme inhibited larval settlement of the bryozoan Bugula neritina at a concentra-
tion of 1 ng mL−1 and was non-toxic. This concentration is much lower than that of
other known antifouling compounds (Table 2, Dobretsov et al. 2006; Fusetani et al.
2006). This suggests that microbial enzymes may be a good alternative for toxic
antifouling compounds.
So far, it has been postulated that antifouling compounds produced by marine
organisms are mostly non-polar, poorly water-soluble secondary metabolites, which
are effective at low concentrations (Steinberg et al. 2001). However, the data presented
in Table 2 clearly show that microorganisms may produce both antifouling water-soluble
and non-water-soluble compounds. Therefore, these compounds need to be investi-
gated in the future.
304 S. Dobretsov

Most of the antifouling compounds from marine microorganisms have been

tested only under laboratory conditions (Table 2). Stability and performance of
antifouling compounds in the field may be different from laboratory conditions
(Dobretsov et al. 2006; Dahms et al. 2006). Until now, only a few antifouling com-
pounds from microorganisms have been incorporated into non-toxic paint matrixes
and tested in field experiments (e.g., Burgess et al. 2003; Dobretsov et al. 2007a;
Bhattarai et al. 2007). In future experiments it is necessary to test all potent anti-
fouling compounds from microbes both in the laboratory and in the field.

3 Quorum Sensing in Biofouling Communities

There are a number of different quorum sensing (QS) signaling systems employed
by bacteria, but overall the mechanism of QS remains consistent through all
prokaryotes (Whitehead et al. 2001). Generally, a small chemical compound
(“autoinducer” or “signal”) is produced by bacteria and then transported or diffused
outside the cell. So far, N-acetyl-l-homoserine lactones, furanosylborate, cyclic thi-
olactone, hydroxy-palmitic acid, methyl dodecenoic acid, and farnesoic acid have
been identified as QS signals (Parsek and Greenberg 2000). When the bacterial
density in biofilms is high enough, these molecules reach a threshold concentration
and begin to bind to a receptor protein. This process promotes the transcription of
a number of genes, which regulates cell division and controls biofilm formation and
composition (Parsek and Greenberg 2000).
Based on the properties of bacterial signal receptors, QS signaling can be grouped
into two categories (Fig. 2). Most Gram-negative bacteria represent one category
where N-acetyl-l-homoserine lactone (AHL) signal molecules, LuxR-type signal
receptor, and LuxR-type I synthase are the major components (Dong et al. 2002).
In contrast, cell-to-cell signaling in most Gram-positive bacteria occurs via a
phosphorylation–dephosphorylation mechanism that is mediated by a two-compo-
nent QS system. Here, oligopeptides signals are transported outside the cell and
detected by a membrane-bound sensor (Novick 2003), which affects a response
regulator by a phosphorelay (Zhang and Dong 2004).
Information concerning the presence of AHLs and other QS molecules in the marine
environment is scarce (Dobretsov et al. 2007b). In the light organ of the sepiolid
squid Euprymna scolopes, light emissions are regulated by AHLs of the marine
symbiotic bacterium Vibrio fisheri (Ruby and Lee 1998). Recently, the presence of
QS signals was demonstrated in marine snow (Gram et al. 2002). The production
of AHLs by bacteria associated with marine sponges was reported by Taylor et al.
(2004). The production of QS signals by tropical marine 2-, 4-, and 6-day-old subtidal
biofilms has recently been investigated (Huang et al. 2007). A QS inducer,
N-dodecanoyl-l-homoserine lactone, was detected in 6-day-old biofilms at a con-
centration of 3.36 mM L−1 by GC-MS. These findings suggest that QS signals
might be produced in situ, but more investigations are needed.
Inhibition and Induction of Marine Biofouling by Biofilms 305

Fig. 2 General scheme of quorum sensing (QS) in a Gram-negative and b Gram-positive bacterial cells
and its inhibition by chemical compounds. AHL N-Acetyl-l-homoserine lactone, AIP autoinducing
peptide, R R-protein, P phosphate

Any reagent that prevents accumulation or recognition between QS signals and

receptor proteins might block QS-dependent gene expression (Fig. 2; Zhang and
Dong 2004). This inhibits bacterial attachment and disrupts biofilm formation. For
306 S. Dobretsov

example, bacterial AHL signal degradation enzymes (AHL-lactonase and AHL-

acylase; Fig. 2) inhibit QS (Zhang and Dong 2004). Triclosan – a potent inhibitor
of the enoyl-ACP reductase that is involved in AHL biosynthesis – reduces AHL
production and blocks bacterial QS (Dobretsov et al. 2007b). Halogenated
furanones, produced by the red alga Delisea pulchra (Givskov et al. 1996), by
Streptomyces spp. (Cho et al. 2001) and by the magnolia Hortonia sp. (Bauer and
Robinson 2002) result in acceleration of LuxR synthase degradation and lead to QS
inhibition (Fig. 2a). There are several known mechanisms that interfere with the
two-component systems of Gram-positive bacteria (Fig. 2b). For example, several
phenolic inhibitors, such as closantel, cause structural alterations to the receptor
kinase and inhibit QS (Stephenson et al. 2000). Additionally, a truncated autoinduc-
ing peptide (AIP) lacking the N-terminal tail shows a wide QS inhibitory activity
towards all the four AIP-specific groups of Streptococcus aureus (Zhang and Dong
2004). These examples show that QS of Gram-positive and Gram-negative bacteria
can be effectively blocked by QS inhibitors.
Because biofilms enhance settlement of invertebrate larvae and algal spores (see
Table 1; Wieczorek and Todd 1998; Maki and Mitchell 2002; Railkin 2004), QS
blockers can control larval settlement indirectly by regulating the microbial com-
munity structure of biofilms and the density of bacteria, which in turn affects larval
behavior (Dobretsov et al. 2007b; Fig. 2). This result demonstrates the possibility
of using QS inhibitors for control of both micro- and macrofouling communities.

4 Conclusions and Future Perspectives

The data presented in this review clearly demonstrate that marine biofilms are a key
factor for the settlement of macrofoulers. Up to now, inhibitive and inductive
metabolites have been isolated predominantly from bacteria and diatoms (Tables 1
and 2). Only one inhibitive compound from a marine fungus has been isolated and
identified (Kwong et al. 2006). At the same time, biofilms consist of numerous
species of bacteria, diatoms, flagellates, fungi, sarcodines, and ciliates (Railkin
2004), which may produce both inhibitive and inductive compounds. Therefore,
less investigated groups of microorganisms, like marine microalgae, flagellates,
fungi, sarcodines, and ciliates, may have a high antifouling potential.
Natural biofilms have been shown to be complex and dynamic communities;
interactions within their components play an important role in the production of
chemical compounds. For example, the proportion of inductive, non-inductive, and
inhibitive strains of microorganisms in biofilms determines larval settlement (Lau
et al. 2002; Dahms et al. 2004). Nevertheless, most investigators have been dealing
with single species of microorganisms belonging to particular taxa (Tables 1, 2),
while the combined effect of different microbial taxa on larval settlement might be
different and should be explored in future studies.
Because only a limited amount of marine organisms can be cultivated in the
laboratory, it is necessary to investigate the performance of inductive or antifouling
Inhibition and Induction of Marine Biofouling by Biofilms 307

compounds under natural conditions on a variety of species. Up to now, only a lim-

ited number of field investigations of larval responses towards biofilms have been
carried out (Table 1), and only in a few cases have microbial antifouling compounds
been tested under natural conditions (i.e., Burgess et al. 2003; Bhattarai et al. 2007;
Dobretsov et al. 2007a). Thus, little is known about the antifouling performance of
biocides in nature, and in future experiments antifouling compounds should be
tested under field conditions.
The interaction between marine microbes and larvae is another area where more
information is needed. In several invertebrate species the larval receptors (Hadfield
et al. 2000; Jeffery 2002), the signal transduction pathways (Morse 1990; Carpizo-
Ituarte and Hadfield 2003; Amador-Cano et al. 2006), potential genes (Seaver et al.
2005; Frobius and Seaver 2006), and expressed proteins (Sanders et al. 2005;
Gallus et al. 2005) involved in larval settlement and metamorphosis have been
characterized. In future studies, it will be necessary not only to identify inductive
and inhibitive compounds from microorganisms but also to identify the genes
responsible for production of these compounds, as well as the larval receptors
involved in their detection. A better understanding of the signal transduction path-
ways of settlement processes will allow us to improve bioassay systems and find
new antifouling and inductive compounds.
Under different culture conditions marine microorganisms can produce different
chemical compounds. It has been shown that bacteria were inhibitive to the larvae
of B. amphitrite at salinities of 35 and 45 ‰ but were inductive at 15 and 25‰
(Khandeparker et al. 2002). Additionally, inductiveness of biofilms and production
of compounds varied at different temperatures (Miao et al. 2006). These results
indicate that the performance of antifouling compounds and their production by
microorganisms should be investigated under field conditions.
In the coming decades, the marine environment will be subject to profound abi-
otic changes, such as elevated water temperature, changes in salinity, decrease of
pH, and elevated ultraviolet radiation. These changes will affect not only the sur-
vival of propagules but also their recruitment, which is controlled by the quality and
quantity of settlement cues produced by marine microorganisms. Additionally, cli-
mate changes can modify the composition of microbial biofilms and their metabo-
lites, which in turn, can change propagule settlement. Therefore, it would be
interesting to investigate and predict possible effects of climate changes on micro-
bial biofilms, macrofouling communities, and their interactions.
Many marine organisms can control epibiosis on their surface by production of
chemical compounds (Harder 2008; Dobretsov and Qian 2002). Can we “learn
from nature” and manipulate biofilm properties in order to increase their antifoul-
ing or inductive properties? Several recent attempts have been made, which include
the immobilization of live bacterial cells (Holmström et al. 2000), deterrence of
microbes (Mitchell and Kirchman 1984, Railkin 2004), and inhibition of bacterial
QS signals (Dobretsov 2007b). In “living paints” the bacteria that release antifoul-
ing compounds are immobilized in polymers and maintained alive. Such coatings
would have an indefinite lifespan in comparison to traditional antifouling coatings,
which fail when they exhaust their reservoir of biocides. Theoretically, such
308 S. Dobretsov

technology is well within the realms of possibility, since the immobilization of

bacteria in artificial matrices is established (Holmström et al. 2000).
Overall, our data suggest that microorganisms are an important source of bio-
logically active metabolites for the antifouling industry and aquaculture. Additional
screening of different microbial taxa will result in the isolation of novel and potent
biofouling compounds. Future studies should include genetic, molecular, biochemi-
cal, and microbiological multidisciplinary approaches for the investigation of
microbe–larva interactions. Additionally, the future development of antifouling
coatings with microbial compounds requires a successful collaboration between
academic and industrial researchers (Rittschof).

Acknowledgemnts We thank Dr. F. Weinberger for his constructive comments on the manuscript.
The author’s studies were supported by an Alexander von Humboldt Fellowship.

References

Amador-Cano G, Carpizo-Ituarte E, Cristino Jorge D (2006) Role of protein kinase C, G-protein

coupled receptors, and calcium flux during metamorphosis of the sea urchin Strongylocentrotus
purpuratus. Biol Bull 210:121–131
Anderson MJ (1996) A chemical cue induces settlement of Sydney rock oysters, Saccostrea com-
mercialis, in the laboratory and in the field. Biol Bull 190:350–358
Bao W-Y, Satuito CG, Yang J-L, Kitamura H (2007) Larval settlement and metamorphosis of the
mussel Mytilus galloprovincialis in response to biofilms. Mar Biol 150:565–574
Bauer WD, Robinson JB (2002) Disruption of bacterial quorum sensing by other organisms. Curr
Opin Biotechnol 13:234–237
Baxter GT, Morse DE (1992) Cilia from abalone larvae contain a receptor-dependent G-protein
transduction system similar to that in mammals. Biol Bull 183:147–154
Bayne BL (1964) Primary and secondary settlement in Mytilus edulis L. (Mollusca). J Anim Ecol
33:513–523
Bhattarai HD, Ganti VS, Paudel B, Lee YK, Lee HK, Hong Y-K, Shin HW (2007) Isolation of
antifouling compounds from the marine bacterium, Shewanella oneidensis SCH0402. World
J Microbiol Biotechnol 23:243–249
Brancato MS, Woollacott RM (1982) Effect of microbial films on settlement of bryozoan larvae
(Bugula simplex, B. stolonifera, B. turrita). Mar Biol 71:51–56
Brewer RH (1976) Larval settling behaviour in Cyanea capillata (Cnidaria: Scyphozoa). Biol Bull
150:183–199
Burgess JG, Boyd KG, Armstrong E, Jiang Z, Yan L, Berggren M, May U, Pisacane T, Granmo
A, Adams DR (2003) The development of a marine natural product-based antifouling paint.
Biofouling 19:197–205
Cameron RA, Hinegardner RT (1974) Initiation of metamorphosis in laboratory cultured sea
urchins. Biol Bull 146:335–342
Carpizo-Ituarte EJ, Hadfield MG (2003) Transcription and translation inhibitors permit metamor-
phosis up to radiole formation in the serpulid polychaete Hydroides elegans Haswell. Biol
Bull 204:114–125
Cho KW, Lee HS, Rho JR, Kim TS, Mo SJ, Shin J (2001) New lactone-containing metabolites
from a marine-derived bacterium of the genus Streptomyces. J Nat Prod 64:664–667
Clare AS, Thomas RF, Rittschof D (1995) Evidence for the involvement of cyclic-AMP in the
modulation of barnacle settlement. J Exp Biol 198:655–664
Inhibition and Induction of Marine Biofouling by Biofilms 309

Dahms H-U, Dobretsov S, Qian P-Y (2004) The effect of bacterial and diatom biofilms on the
settlement of the bryozoan Bugula neritina. J Exp Mar Biol Ecol 313:191–209
Dahms H-U, Xu Y, Pfeiffer C (2006) Antifouling potential of cyanobacteria: a mini-review.
Biofouling 22:317–327
De Silva PHDP (1962) Experiments on the choice of substrate by Spirorbis larvae (Serpulidae). J
Exp Biol 39:483–490
Dobretsov SV (1999) Macroalgae and microbial film determine substrate selection in planulae of
Gonothyraea loveni (Allman 1859) (Cnidaria: Hydrozoa). Zoosystematica Rossica 1:109–117
Dobretsov S, Qian P-Y (2002) Effect of bacteria associated with the green alga Ulva reticulata on
marine micro- and macrofouling. Biofouling 18:217–228
Dobretsov S, Qian P-Y (2004) The role of epibotic bacteria from the surface of the soft coral
Dendronephthya sp. in the inhibition of larval settlement. J Exp Mar Biol Ecol 299:35–50
Dobretsov S, Qian P-Y (2006) Facilitation and inhibition of larval attachment of the bryozoan
Bugula neritina in association with mono-species and multi-species biofilms. J Exp Mar Biol
Ecol 333:263–264
Dobretsov S, Dahms H-U, Qian P-Y (2006) Inhibition of biofouling by marine microorganisms
and their metabolites. Biofouling 22:43–54
Dobretsov S, Xiong H, Xu Y, Levin LA, Qian PY (2007a) Novel antifoulants: inhibition of larval
attachment by proteases. Mar Biotech 9:388–397
Dobretsov S, Dahms H-U, Huang YL, Wahl M, Qian PY (2007b) The effect of quorum sensing
blockers on the formation of marine microbial communities and larval attachment. FEMS
Microbiol Ecol 60:177–188
Dong YH, Gusti AR, Zhang Q, Xu JL, Zhang LH (2002) Identification of quorum-quenching
N-acyl homoserine lactonases from Bacillus species. Appl Environ Microbiol 68:1754–1759
Donlan RM (2002) Biofilms: microbial life on surfaces. Emerg Infect Dis 8:881–890
Egan S, James S, Holmström C, Kjelleberg S (2001) Inhibition of algal spore germination by the
marine bacterium Pseudoalteromonas tunicata. FEMS Microbiol Ecol 35:67–73
Egan S, James S, Kjelleberg S (2002) Identification and characterization of a putative transcrip-
tional regulator controlling the expression of fouling inhibitors in Pseudoalteromonas tunica-
tae. Appl Environ Microbiol 68:372–378
Fitt WK, Labare MP, Fuqua WC, Walch M, Coon SL, Bonar DB, Colwell RR, Weiner RM (1989)
Factors influencing bacterial production of inducers of settlement behaviour of larvae of the
oyster Crassostrea gigas. Microb Ecol 17:287–298
Frobius AC, Seaver EC (2006) Capitella sp I homeobrain-like, the first lophotrochozoan member
of a novel paired-like homeobox gene family. Gene Expr Patterns 6:985–991
Fusetani N, Clare AS, Mueller WEG (eds.) (2006) Antifouling compounds. Progress in molecular
and subcellular biology, vol 42. Springer, Berlin Heidelberg New York
Gallus L, Ramoino P, Faimali M, Piazza V, Maura G, Marcoli M, Ferrando S, Girosi L, Tagliafierro
G (2005) Presence and distribution of serotonin immunoreactivity in the cyprids of the barna-
cle Balanus amphitrite. Eur J Histochem 49:341–347
Givskov M, deNys R, Manefield M, Gram L, Mayimilien R, Eberl L, Molin S, Steinberg PD,
Kjelleberg S (1996) Eukaryotic interference with homoserine lactone-mediated prokaryotic
signalling. J Bacteriol 178:6618–6622
Gram L, Grossart HP, Schlingloff A, Kiőrboe T (2002) Possible quorum sensing in marine snow
bacteria: production of acylated homoserine lactones by Roseobacter strains isolated from
marine snow. Appl Environ Microbiol 68:4111–4116
Hadfield MG, Unabia CC, Smith CM, Michael TM (1994) Settlement preferences of ubiquitous
fouler Hydroides elegans. In: Thompson M-F, Sarojini R, Nagabhushanam R (eds.) Recent
developments in biofouling control. Balkema, Rotterdam, pp. 65–74
Hadfield MG, Meleshkevitch EA, Boudko DY (2000) The apical sensory organ of a gastropod
veliger is a receptor for settlement cues. Biol Bull 198:67–76
Hamer JP, Walker G, Latchford JW (2001) Settlement of Pomatoceros lamarkii (Serpulidae) lar-
vae on biofilmed surfaces and the effect of aerial drying. J Exp Mar Biol Ecol 260:113–132
310 S. Dobretsov

Harder T, Lam C, Qian P-Y (2002a) Induction of larval settlement in the polychaete Hydroides
elegans by marine biofilms: an investigation of monospecific diatom films as settlement cues.
Mar Ecol Prog Ser 229:105–112
Harder T, Lau SCK, Dahms H-U, Qian PY (2002b) Isolation of bacterial metabolites as natural
inducers for larval settlement in the marine polychaete Hydroides elegans (Haswell). J Chem
Ecol 28:2029–2043
Harder T, Dobretsov S, Qian PY (2004) Waterborne polar macromolecules act as algal antifou-
lants in the seaweed Ulva reticulata. Mar Ecol Prog Ser 274:133–141
Harder T (2008) Marine epibiosis: concepts, ecological consequences and host defence. Springer
Series on Biofilms, doi:7142_2008_16
Harvey M, Miron G, Bourget E (1995) Resettlement of Iceland scallop (Chlamys islandica) spat
on dead hydroids: response to chemical cues from the protein-chitinous perisarc and associ-
ated microbial film. J Shellfish Res 14:383–388
Henning G, Benayahu Y, Hofmann DK (1991) Natural substrates, marine bacteria and a phorbol
ester induce metamorphosis of the soft coral Heteroxenia fuscenscens (Anthozoa:
Octocorallina). Verh Dtsch Zool Ges 84:486–487
Hentschel JR, Cook PA (1990) The development of marine fouling community in relation to the
primary film of microorganisms. Biofouling 2:1–11
Hofmann DK, Brand U (1978) Induction of metamorphosis in the symbiotic scyphozoan
Cassiopea andromeda: role of marine bacteria and of biochemicals. Symbiosis 4:99–116
Holmström C, Steinberg P, Christov V, Christie G, Kjelleberg S (2000) Bacteria immobilized in
gels: improved methodologies for antifouling and biocontrol applications. Biofouling
15:109–117
Holmström C, Egan S, Franks A, McCloy S, Kjelleberg S (2002) Antifouling activities
expressed by marine surface associated Pseudoalteromonas species. FEMS Microbiol Ecol
41:47–58
Huang S, Hadfield MG (2003) Composition and density of bacterial biofilms affect metamorpho-
sis of the polychaete Hydroides elegans. Mar Ecol Prog Ser 260:161–172
Huang Y-L, Dobretsov S, Ki J-S, Yang L-H, Qian P-Y (2007) Presence of acyl-homoserine lac-
tones in subtidal biofilms and the implication in inducing larval settlement of the polychaete
Hydroides elegans. Microb Ecol 54:384–392
Huggett MJ, Williamson JE, de Nys R, Kjelleberg S, Steinberg PD (2006) Larval settlement of the
common Australian sea urchin Heliocidaris erythrogramma in response to bacteria from the
surface of coralline algae. Oecologia 149:604–619
Jeffery CJ (2002) New settlers and recruits do not enhance settlement of a gregarious intertidal
barnacles in New South Wales. J Exp Mar Biol Ecol 275:131–146
Johnson CR, Sutton DC (1994) Bacteria on the surface of crustose coralline algae induce meta-
morphosis of the crown of thorns starfish Acanthaster planci. Mar Biol 120:305–310
Khandeparker L, Anil AC, Raghukumar S (2002) Factors regulating the production of different
inducers in Pseudomonas aeruginosa with reference to larval metamorphosis in Balanus
amphitrite. Aquat Microb Ecol 28:37–54
Kirchman D, Graham D, Reish D, Mitchell R (1982) Lectins may mediate in the settlement and
metamorphosis of Janua (Dexiospira) brasiliensis Grube (Polychaeta: Spirorbidae). Mar Biol
Lett 3:201–222
Kitamura H, Hirayama K (1987) Effect of primary films on the settlement of larvae of a bryozoan
Bugula neritina. Bull Jpn Soc Sci Fish 53:1377–1381
Knight-Jones EW (1951) Gregariousness and some other aspects of the settling behaviour in
Spirorbis. J Mar Biol Ass UK 30:201–222
Kon-ya K, Shimidzu N, Otaki N, Yokoyama A, Adachi K, Miki W (1995) Inhibitory effect of
bacterial ubiquinones on the settling of barnacle, Balanus amphitrite. Experientia
51:153–155
Kwong TFN, Li M, Li X, Qian PY (2006) Novel antifouling and antimicrobial compound from a
marine-derived fungus Ampelomyces sp. Mar Biotech 8:634–640
Lam C, Harder T, Qian P-Y (2005) Induction of larval settlement in the polychaete Hydroides
elegans by extracellular polymers of benthic diatoms. Mar Ecol Prog Ser 286:145–154.
Inhibition and Induction of Marine Biofouling by Biofilms 311

Lau SCK, Mak KK, Chen F, Qian P-Y (2002) Bioactivity of bacterial strains from marine biofilms
in Hong Kong waters for the induction of larval settlement in the marine polychaete Hydroides
elegans. Mar Ecol Prog Ser 226:301–310
Lau SCK, Thiyagarajan V, Qian P-Y (2003) The bioactivity of bacterial isolates in Hong Kong
waters for the inhibition of barnacle (Balanus amphitrite Darwin) settlement. J Exp Mar Biol
Ecol 282:43–60
Lau SCK, Thiyagarajan V, Cheung SCK, Qian P-Y (2005) Roles of bacterial community composi-
tion in biofilms as a mediator for larval settlement of three marine invertebrates. Aquat Microb
Ecol 38:41–51
Le Tourneux F, Bourget E (1988) Importance of physical and biological settlement cues used at
different scales by the larvae of Semibalanus balanoides. Mar Biol 97:57–66
Leitz T, Wagner T (1993) The marine bacterium Alteromonas espejiana induces metamorphosis
of the hydroid Hydractinia echinata. Mar Biol 115:173–178
Lewandowski Z (2000) Structure and function of biofilms. In: Evans LV (ed.). Biofilms: recent
advances in their study and control. Harwood Academic, Amsterdam, pp. 1–17
Li X, Dobretsov S, Xu Y, Xiao X, Hung OS, Qian PY (2006) Antifouling diketopiperazines pro-
duced by a deep-sea bacterium, Streptomyces fungicidicus. Biofouling 22:201–208.
Maki JS, Mitchell R (2002) Biofouling in the marine environment. In: Bitton G (ed.) Encyclopedia
of environmental microbiology. Wiley, New York, pp. 610–619
Maki JS, Rittschof D, Costlow JD, Mitchell R (1988) Inhibition of attachment of larval barnacles,
Balanus amphitrite, by bacterial surface films. Mar Biol 97:199–206
Maki JS, Rittschof D, Schidt AR, Snyder AG, Mitchell R (1989) Factors controlling settlement of
bryozoan larvae: a comparison of bacterial films and unfilmed surfaces. Biol Bull 177:295–302
Mary A, Mary V, Rittschof D, Nagabhushanam R (1993) Bacterial-barnacle interaction: potential
of using juncellins and antibiotics to alter structure of bacterial communities. J Chem Ecol
19:2155–2167
McCoy FL, David Jr I, Eng-Wilmont L, Martin DF (1979) Isolation and partial purification of a
red tide (Gymnodinium breve) Cytolytic factor(s) from cultures of Gomphosphaeria aponina.
J Agric Food Chem 27:69–79
Miao L, Kwong TFN, Qian PY (2006) Effect of culture conditions on mycelial growth, antibacte-
rial activity, and metabolite profiles of the marine-derived fungus Arthrinium c.f. sacchari-
cola. Appl Microb Biotechnol 72:1063–1073
Mihm JW, Banta WC, Loeb GI (1981) Effects of absorbed organic and primary biofilms on bryo-
zoan settlement. J Exp Mar Biol Ecol 54:167–179
Mitchell R, Kirchman D (1984) The microbial ecology of marine surfaces. In: Costlow JD, Tipper
RC (eds.) Marine biodeterioration: an interdisciplinary study. Naval Institute Press, Anapolis,
MA, pp. 49–56
Morse DE (1990) Recent progress in larval settlement and metamorphosis: closing gaps between
molecular biology and ecology. Bull Mar Sci 46:465–483
Morse ANC, Froyd CA, Morse DE (1984) Molecules from cyanobacteria and red algae that induce
larval settlement and metamorphosis in the mollusk Haliotis rufescens. Mar Biol 81:293–298
Murakami N, Morimoto T, Imamura H, Ueda T, Nagai S, Sakakibara J, Yamada N (1991) Studies
on glycolipids: III. glycoglycolipids from an axenically cultures cyanobacterium, Phormidium
tenue. Chem Pharm Bull 39:2277–2281
Nedved BT, Hadfield MG (2008) Hydroides elegans (Annelida: Polychaeta): a model for bio-
fouling research. Springer Series on Biofilms, doi:7142_2008_15
Negri AP, Webster NS, Hill RT, Heyward AJ (2001) Metamorphosis of broadcast spawning corals
in response to bacteria isolated from crustose algae. Mar Ecol Prog Ser 223:121–131
Neumann R (1979) Bacterial induction of settlement and metamorphosis in the planula larvae of
Cassiopea andromeda (Cnidaria: Schiphozoa, Rhizostomeae). Mar Ecol Prog Ser 1:21–28
Novick RP (2003) Autoinduction and signal transduction in the regulation of staphylococcal viru-
lence. Mol Microbiol 48:1429–1449
Olguin-Uribe G, Abou-Mansour E, Boulander A, Debard H, Francisco C, Combaut G (1997)
6-Bromoindole-3-carbaldehyde from an Acinetobacter sp. bacterium associated with the
ascidian Stomoza murrayi. J Chem Ecol 23:2507–2521
312 S. Dobretsov

Orlov D (1996) Observations on the settling behaviour of planulae of Clava multicornis Forskal
(Hydroidea, Athecata). Scientia Marina 60:121–128
Parsek MR, Greenberg EP (2000) Acyl-homoserine lactone quorum sensing in gram-negative
bacteria: a signaling mechanism involved in associations with higher organisms. Proc Natl
Acad Sci U S A 97:8789–8793
Parsons GJ, Dadswell MJ, Roff JC (1993) Influence of biofilm on settlement of sea scallop
Placopecten magellanicus (Gmelin, 1791), in Passamaquoddy Bay, New Brunswick, Canada.
J Shellfish Res 12:279–283
Patel P, Callow ME, Joint I, Callow JA (2003) Specificity in larval settlement modifying response
of bacterial biofilms towards zoospores of the marine alga Enteromorpha. Environ Microbiol
5:338–349
Patil JS, Anil AC (2005) Influence of diatom exopolymers and biofilms on metamorphosis in the
barnacle Balanus amphitrite. Mar Ecol Prog Ser 301:231–245
Pearce CM, Scheibling RE (1991) Effect of macroalgae, microbial films, and conspecifics on the
induction of metamorphosis of the green sea urchin Strongylocentrotus droebachiensis. J Exp
Mar Biol Ecol 147:147–162
Qian P-Y (1999) Larval settlement of polychaetes. Hydrobiologia 402:239–253
Qian P-Y, Thiyagarajan V, Lau SCK, Cheung SCK (2003) Bacterial community profile in biofilm
and attachment of the acorn barnacle Balanus amphitrite. Aquat Microb Ecol 33:225–237
Railkin AI (2004) Marine biofouling: colonization processes and defenses. CRC, Boca Raton,
FL
Roberts R (2001) A review of settlement cues for larval abalone (Haliotis spp.). J Shellfish Res
20:571–586
Rodriguez SR, Ojeda FP, Inestrosa NC (1993) Settlement of benthic marine invertebrates. Mar
Ecol Prog Ser 97:193–207
Ruby EG, Lee KH (1998) The Vibrio fischeri Euprymna scolopes light organ association: current
ecological paradigms. Appl Environ Microbiol 64:805–812
Sanders MB, Billinghurst Z, Depledge MH, Clare AS (2005) Larval development and vitellin-like
protein expression in Palaemon elegans larvae following xeno-oestrogen exposure. Integrat
Compar Biol 45:51–60
Seaver EC, Thamm K, Hill SD (2005) Growth patterns during segmentation in the two polychaete
annelids, Capitella sp I and Hydroides elegans: comparisons at distinct life history stages.
Evol Develop 7:312–326
Schmahl G (1985) Bacterial induced stolon settlement in the scyphopolyp of Aurelia aurita
(Cnidaria, Scyphozoa). Helgolander Meeresuntersuchungen 39:33–42
Steinberg PD, De Nys R, Kjelleberg S (2001) Chemical mediation of surface colonization. In:
McCkintock JB, Baker JB (eds.), Marine chemical ecology. CRC, Boca Raton, FL, pp.
355–387
Stephenson K, Yamaguchi Y, Hoch JA (2000) The mechanism of action of inhibitors of bacterial
two-component signal transduction systems. J Biol Chem 275:38900–38904
Strathmann RR, Branscomb ES, Vedder K (1981) Fatal errors in set as a cost of dispersal and the
influence of internal flora on set of barnacles. Oecologia 48:13–18
Szewzyk U, Holmstrom C, Wrangstadh M, Samuelsson MO, Maki JS, Kjelleberg S (1991)
Relevance of the exopolysaccharide of marine Pseudomonas sp. strain S9 for the attachment
of Ciona intestinalis larvae. Mar Ecol Prog Ser 75:259–265
Tait K, Joint I, Daykin M, Milton DL, Williams P, Camara M (2005) Disruption of quorum sens-
ing in seawater abolishes attraction of zoospores of the green alga Ulva to bacterial biofilms.
Environ Microbiol 7:229–240
Taylor MW, Schupp PJ, Baillie HJ, Charlton TS, de Nys R, Kjelleberg S, Steinberg PD (2004)
Evidence for acyl homoserine lactone signal production in bacteria associated with marine
sponges. Appl Environ Microbiol 70:4387–4389
Thiyagarajan V, Hung OS, Chiu JMY, Wu RSS, Qian P-Y (2005) Growth and survival of juvenile
barnacle Balanus amphitrite: interactive effects of cyprid energy reserve and habitat. Mar Ecol
Prog Ser 299:229–237
Inhibition and Induction of Marine Biofouling by Biofilms 313

Thiyagarajan V, Lau SCK, Cheung SCK, Qian PY (2006) Cypris habitat selection facilitated by
microbial films influences the vertical distribution of subtidal barnacle Balanus trigonus.
Microb Ecol 51:431–440
Thompson RC, Norton TA, Hawkins SJ (1998) The influence of epilithic microbial films on the
settlement of Semibalanus balanoldes cyprids – a comparison between laboratory and field
experiments. Hydrobiologia 376:203–216
Unabia CRC, Hadfield MG (1999) Role of bacteria in larval settlement and metamorphosis of the
polychaete Hydroides elegans. Mar Biol 133:55–64
Volk RB (2006) Antialgal activity of several cyanobacterial exometabolites. J Appl Phycol
18:145–151
Webster NS, Smith LD, Heyward AJ, Watts JEM, Webb RI, Blackall LL, Negri AP (2004)
Metamorphosis of a scleratinian coral in response to microbial biofilms. Appl Envir Microb
70:1213–1221
Wieczorek SK, Todd CD (1998) Inhibition and facilitation of the settlement of epifaunal marine
invertebrate larvae by microbial biofilm cues. Biofouling 12:81–93
Williams GB (1964) The effect of extracts of Fucus serratus in promoting the settlement of larvae
of Spirorbis borealis (Polychaetea). J Mar Biol Ass UK 44:397–414
Whitehead NA, Barnard AM, Slater H, Simpson NJ, Salmond GP (2001) Quorum sensing in
Gram-negative bacteria. FEMS Microbiol Rev 25:365–404
Yebra DM, Kiil S, Dam-Johansen K (2004) Antifouling technology – past, present and future
steps towards efficient and environmentally friendly antifouling coatings. Prog Org Coatings
50:75–104
Zhang LH, Dong YH (2004) Quorum sensing and signal interference: diverse implications. Mol
Microbiol 53:1563–1571
Zhao B, Zhang S, Qian P-Y (2003) Larval settlement of the silver- or goldlip pearl oyster Pinctada
maxima (Jameson) in response to natural biofilms and chemical cues. Aquaculture
220:883–901
A Triangle Model: Environmental Changes
Affect Biofilms that Affect Larval Settlement

P.Y. Qian (*
ü ) and H.-U. Dahms

Abstract Biofilms are ubiquitous – covering every exposed surface in marine

environment and thus playing a key role in mediating biotic interactions and
biogeochemical activities occurring on the surfaces. For the propogates of
marine organisms, biofilm attributes serve as inhibitive or inductive cues for the
attachment of settling larvae and algal spores of potential colonizers. Microbes
in biofilms are not only the sources of chemical cues but also consumers of
chemical cues. As microbes in biofilm are very sensitive to changes in ambient
environment, the production of chemical cues by the microbes will change in
response to spatio-temporal variation of microbial density, community structure,
topography, dynamics, and the microbial physiological conditions in biofilms.
These lead to changes in physical and chemical biofilm properties and in the bio-
activity of biofilm for attachment of marine propogates. While there have been a
number of reviews on the effect of biofilms on settlement of marine invertebrate
larvae and algal spores, the effects of environmental changes on microbial com-
munity structure dynamics and bioactivities of biofilms remain much unexplored.
Recent advances in molecular fingerprinting techniques have made it possible to
precisely study the linkage between environmentally driven changes in biofilms
and larval settlement. We are now gaining a better picture of the triangle relation-
ship between environmental variables, biofilm dynamics and bioactivity, and the
behavior of settling larvae or spores of marine organisms. Here, we would like
to formally introduce a triangle model to provide a conceptual framework for
interactions between environmentally induced biofilm changes that in turn affect
the settlement of dispersal propogates.

P.Y. Qian
Department of Biology and Coastal Marine Lab Hong Kong, University of Science
and Technology, Clearwater Bay, Kowloon, Hong Kong
e-mail: boqianpy@ust.hk

Springer Series on Biofilms, doi: 10.1007/7142_2008_19315 315

© Springer-Verlag Berlin Heidelberg 2008
316 P.Y. Qian and H.-U. Dahms

1 Introduction

Biofilms are surface-attached microbial communities consisting of multiple layers

of cells embedded in hydrated matrices (Kierek-Pearson and Karatan 2005). They
are ubiquitous on aquatic substrata. The physical, chemical, and biological events
leading to the establishment of attached communities of microorganisms as bio-
films have been thoroughly reviewed (Characklis and Marshall 1990; Caldwell
et al. 1993; Palmer and White 1997). Biofilms have a wide array of interactions
with other organisms: with interspecific interactions to even transphyletic microbiota
(archaea, eubacteria, fungi, unicellular eucaryotes; see Paerl and Pinckney 1996)
as well as with macroalgae and invertebrates (Hadfield and Paul 2001).
Several studies show that biofilms can either foster (Kirchman et al. 1982;
Callow and Callow 2000; Qian et al. 2003; Hung et al. 2005a, b; Lau et al. 2005)
or reduce settlement of marine invertebrate larvae (Holmstrøm et al. 1992;
Dobretsov and Qian 2004; Dahms and Qian 2005), or have no effect (Todd and
Keough 1994; Lau et al. 2003a). Microbes forming biofilms (e.g., bacteria, dia-
toms, fungi) attract other organisms by providing resources (e.g., food, habitat,
protection), by benefitting invertebrate larvae in neutralizing and marking adverse
substratum properties (Costerton et al. 1995), or by signaling habitats with different
fitness expectations (Gordon 1999). Biofilms may also provide important inte-
grated historical environmental information (Harder et al. 2002a). The physical,
chemical, and biotic properties of biofilms (Qian et al. 2003), their composition,
and the dynamics of resulting bioactivities, therefore, have a share in structuring
macrobenthic communities (Underwood and Fairweather 1989).
Larvae were shown to choose when and where to settle to some extent (Pawlik
1992; Qian et al. 2003). They can deferentiate between biofilms of different
density (e.g., Maki et al. 1988; Neal and Yule 1994), community composition (e.g.,
Holmstrøm et al. 1992; Lau et al. 2002; Patel et al. 2003; Qian et al. 2003; Lau et al.
2005), age (e.g., Szewzyk et al. 1991; Keough and Raimondi 1996), metabolic
activity (e.g., Wieczorek and Todd 1998; Holmstrøm and Kjelleberg 1999; Hung et al.
2005a, b; Lau et al. 2005), a wide variety of microbial products ranging from small-
molecule metabolites to high molecular weight extracellular polymers (Harder et al.
2002a; Lau et al. 2003a; Lam et al. 2005a, b), or different habitats (Keough and
Raimondi 1996; Thiyagarajan et al. 2005; Dobretsov et al. 2006). While some
invertebrate species only settle on biofilms with viable bacterial cells (Lau et al.
2003b), others can settle on biofilms of non-viable cells (Hung et al. 2005a). Saying
this, it has to be kept in mind that larvae respond differentially to biofilms also as a
function of their own (larval) metabolic activity, density, and composition
(Holmstrøm and Kjelleberg 1999). In addition, macroorganisms show different
taxonic, ontogenetic, and physiological patterns for settlement (Wahl 1989).
Whereas colonization cues, particularly with respect to invertebrate larval
settlement and metamorphosis, have been studied intensively (see reviews by Chia
1989; Pawlik 1992; Qian 1999; Rodriguez et al. 1993), the role of biofilms in larval
settlement have just begun to be explored (Wieczorek and Todd 1998; Holmstrøm
Environmental Changes Affect Biofilms that Affect Larval Settlement 317

and Kjelleberg 2000; Qian et al. 2003). This holds particularly for aspects of bio-
film dynamics, both in the process of biofilm development and at a mature stage.
Biofilms change phenotypically and genetically according to internal (e.g., age,
bioactive compounds, competition) and external environmental conditions (Todd
and Keough 1994; Cooksey and Wigglesworth-Cooksey 1995; Wimpenny 2000).
In this review, we pursue the hypothesis that environmentally caused variations
of biofilms are critical for settlement processes and the structure of resulting com-
munities. We put forward a conceptual triangle model to describe how the environ-
ment, microbes in biofilm, and the settling propogates of marine organisms can
interact. The model also describes how these interactions change structure, compo-
sition, and bioactivities of biofilms and, in turn, affect possible distribution patterns
of marine macroorganisms. Environmental factors could be biotic (e.g., grazing,
competition, physical disturbance) or abiotic, but we will focus on the latter. We
particularly attempt in this review to discuss, with respect to larval settlement, the
unavoidable phenomena of biofilm development in assay wells and the effects of
biofilms on compound uptake and compound production.

2 Triangle Model

A triangle model is introduced to provide a conceptual framework for interactions

between environmentally caused changes in biofilm bioactivity that in turn affect
the settlement of dispersal stage (Fig. 1). Biofilm cues that affect potential colonizers

(s
et Al
tle te
m ra
Environment en tio
tp n
ro
pe
rti
es
)

Macrofauna
Larval Macrofauna
Biofilm
Mediation -adults
- larvae bioactivity
settlement
- juveniles of settlement

Fig. 1 Elements of a triangle model that demarcate environmental effects on biofilm that affect
larval settlement of marine substrata
318 P.Y. Qian and H.-U. Dahms

differentially by providing either inhibiting or attracting colonization cues can be

caused by environmentally caused dynamic changes in biofilm properties, such as
in the abundance and composition of microorganisms in biofilms, and in the physical
structure, composition, and physiology of biofilms.

3 Biofilm Properties

Biofilm properties that are relevant for the mediation of larval settlementand are
prone to environmental changes include age, taxon diversity, density, quorum
sensing, extracellular polymers (EPS), and other metabolites (see reviews by
Dobretsov et al. 2006, Qian et al. 2007). Any environmental factor that affects such
biofilm properties in turn would also affect larval settlement.

3.1 Biofilm Taxon Diversity

Several studies indicate that settlement responses are determined by bacterial com-
munity composition rather than by cell density or biomass (Dahms et al. 2004; Lau
et al. 2005; Dobretsov and Qian 2006). So far, no correlation could be shown
between the phylogenetic affiliation of bacteria and their effects on larval or spore
settlement (Lau et al. 2002; Patel et al. 2003; Dobretsov and Qian 2004) although
more species in the genera Peudoaltermonos, Altermonos, Vibrio, Streptomyces and
Actenomyces appear to be bioactive (see review by Dobretsov et al. 2006).

3.2 Biofilm Density

Larval settlement of Hydroides elegans differ only slightly among biofilms developed
at different salinities, but not among those developed at different temperatures (Lau
et al. 2005). This settlement response was moderately correlated with bacterial den-
sity but had no relationship with bacterial community composition of the biofilm.

3.3 Biofilm Age

Maki et al. (1988) showed that the effect of bacteria from natural biofilms that inhibit
barnacle attachment depend on biofilm age. Laboratory experiments of Lau et al. (2003a)
demonstrated that inductive bacterial strains were more active in their stationary phase
than in their log phase. Since biofilm age is generally correlated with biofilm thickness
or bacterial density, the latter will provide a good indicator of substratum stability.
Environmental Changes Affect Biofilms that Affect Larval Settlement 319

3.3.1 Quorum Sensing

The capacity for intercellular communication has major effects on the formation
and community structure of biofilms (Dobretsov et al. 2007, Huang et al. 2007b).
Gram-negative bacteria may communicate between each other through the use of
quorum sensing signals, such as N-acetyl-l-homoserine lactones (AHL; see Parsek
et al. 1999; Huang et al. 2007a). These small molecules act as extracellular signals
that activate transcription and modulate physiological processes when accumulated
in the presence of increased cell densities (Parsek and Greenberg 2005).

3.4 Extracellular Polymers (EPS)

Microbial cells in biofilms are enmeshed in an extensive matrix of extracellular

polysaccharides (Decho 1990). Its extensive mucoid network facilitates the attach-
ment of bacteria (Stevenson and Peterson 1989) and other microbes as well as the
settlement of invertebrate larvae and algal spores (Holmstrøm and Kjelleberg 1999,
Lau et al. 2003a). The EPS production of diatoms is largely affected by environ-
mental parameters (Wolfstein and Stal 2002) such as the provision of nutrients and
light. Differences (i.e., molecular weight and monomer composition) in EPS
obtained from diatoms grown under different environmental conditions (tempera-
ture and salinity) are reflected in the larval settlement response (Lam et al. 2005b),
confirming the ability of larvae to distinguish between biofilms of varying compo-
sition, physiological condition, and growth phase (Wieczorek and Todd 1998).

3.5 Biofilm Chemical Compound Diversity

Marine microbes are a potent source of bioactive compounds. But, so far, only a
limited number of marine microbes have been screened and only a few antisettle-
ment compounds have been isolated and identified (Dobretsov et al. 2006). This
holds particularly for microbes enmeshed in biofilms. Recently, five antifouling
diketopiperazines were isolated from the deep sea bacterium Streptomyces fungi-
cidicus (Li et al. 2006) and more bioactive compounds were isolated from other
microbes (Yang et al. 2006; Xu et al. 2007).
The synthesis of bioactive metabolites by microbes changes with environmental
conditions (Kjelleberg et al. 1993, Miao et al. 2006; Yang et al. 2007). For example,
bacterial strains of the same species can produce different compounds under differ-
ent cultural or environmental conditions, and provide a variable mount of bioactive
compounds (Armstrong et al. 2001). In entire multispecies biofilms, changes in the
type and amount of compound production are likely to occur as well (Cooksey and
Wigglesworth-Cooksey 1995; Qian et al. 2007).
320 P.Y. Qian and H.-U. Dahms

4 Environmental Effects on Biofilm Development

and Variability

Biofilm community alterations can be caused by abiotic factors (e.g., depth,

illumination, exposure time, tidal height, flow regime, physical disturbance, latitude,
season, water chemistry, nutrient supply; see Characklis and Marshall 1990) or by
biotic factors (e.g., availability and physiological condition of colonizing species,
competition and cooperation among species, biological disturbance; see Clare et al.
1992; Fenchel 1998; Dahms and Qian 2005). Spatial variability of biofilms pro-
vides neocolonization possibilities that also affect the vertical distribution of
microbes (Caldwell et al. 1993; Costerton et al. 1995). Heterogeneity with vertical
depth is of particular relevance for the interpretation of successional events following
disturbances that create open spaces within natural aged communities, where space
is commonly limited (e.g., Butler and Chesson 1990). The conceptual frame of
“patch-dynamics’’ in metapopulations (Wright et al. 2004) suggests that distur-
bances provide neocolonization opportunities at any developmental stage of a com-
munity in a mosaic fashion (Butler and Chesson 1990). Such a stochastic concept is
much more applicable to the process of biofilm formation and subsequent overgrowth
by macroorganisms than any scenario of gradual succession (see Henschel and
Cook 1990). Colonization in situ is a dynamic process, where chance effects rather
than deterministic processes become prevailant. Some phases in the colonization
process may be accelerated or slowed down, occur reversely or simultaneously
(Palmer and White 1997).
Biofilm community succession can be affected by a number of ecological, bio-
logical, and physiological events initiated by primary colonizers, as well as by
surface modifications, which determine the types/species of microbes to be
recruited as secondary colonizers (Costerton et al. 1995). Synergistic and/or com-
petitive interactions among colonizers, together with the arrival of new recruits and/
or loss of previous colonists, continuously shape the biofilm community (Wimpenny
2000). As the thickness of the biofilm increases, sharp vertical gradients as well as
horizontal patches of pH, dissolved oxygen, and metabolic byproducts usually
develop within biofilms.
Colonization events are thus suggested to be ruled predominantly by the follow-
ing factors:
1. Qualitative and quantitative aspects of colonizers (i.e., which and how many can
approach and attach to a surface) that themselves provide particular biotic
functions
2. Physical and chemical conditions of the seawater/substratum interface
Biotic parameters also include microbes with good adhesion properties, which
would have a selective advantage even under turbulent conditions (Beech et al.
2000). This ability would be enhanced by the secretion of EPS that is resistant to
high fluid shear and chemical agents (Stewart 2002). Microbes may retain the ability
to detach from biofilms when conditions become unfavorable (Maki 1999).
Environmental Changes Affect Biofilms that Affect Larval Settlement 321

5 Abiotic Environmental Factors that Structure

Biofilms and their Bioactivity

The relationship between habitat and biofilm community is tight so that the structure,
composition, and/or physiology of a biofilm community will effectively reflect key
environmental factors at substratum interfaces. Environmental gradients and
changes in abiotic factors that affect the bioactivity of biofilms include: depth,
illumination, exposure time, latitude, season, water chemistry, nutrient supply, and
substratum characteristics.

5.1 Light

Photosynthesis in diatoms and cyanobacteria can result in extra amounts of EPS

exudates (Wolfstein and Stal 2002), with effects that are mentioned above. Hung
et al. (2005a, b) studied whether either UV-A or UV-B radiation can indirectly
affect larval attachment of barnacles by altering the settlement bioactivity of biofilms.
Both UV-A and UV-B caused a decrease in the percentage of respiring bacterial
cells in microbial films and this effect increased with an increase of UV energy.
At the same energy level, UV-B caused a greater decrease in respiring bacterial
cell densities than UV-A (Hung et al. 2005a, b). However, despite strong UV
radiation, the bioactivity of biofilm that mediates cyprid settlement remained
unchanged, indicating that increased UV radiation may not significantly affect the
barnacle recruitment by means of affecting the inductive larval attachment cues of
microbial films (Hung et al. 2005a). In contrast, larval settlementof Hydroides
elegans decreased with increased UV radiation, indicating that enhanced UV
radiation may have a significant effect on the larval settlement of H. elegans by
affecting a biofilm’s inductive cues (Hung et al. 2005b). These findings suggest
that the effects of light on biofilm bioactivity will depend on the larval/spore’s
response to biofilm properties.

5.2 Flow

Under turbulent conditions, bacteria that are capable of rapid adhesion have an
advantage for settling and growing. Adhesion is enhanced by the secretion of
EPS that is resistant to high fluid shear (Ophir and Gutnick 1994). This way,
flow can structure microbial communities that are shown to affect settlement
differentially. However, there is no study hitherto of hydrodynamic effects on
larval settlement (Qian et al. 1999, 2000) that has considered the effects of flow
on biofilms.
322 P.Y. Qian and H.-U. Dahms

5.3 Temperature and Salinity

Lau et al. (2005) studied temperature and salinity effects on the density and
total biomass of bacterial communities that in turn affected the settlement of
barnacles and a tubeworm. Larval settlement of Balanus amphitrite and B. trigonus
was induced by biofilms developed at high temperatures (23°C and 30°C), but
was unaffected (B. amphitrite) or inhibited (B. trigonus) by those developed at
low temperature (16°C). The settlement response of these barnacles did not
correlate with the biomass or the bacterial density of the biofilms, but did
coincide with marked differences in bacterial community compositions at dif-
ferent temperatures. Chiu et al. (2006) found variations in microbial commu-
nity structure of microbial films as determinants in the control of larval
metamorphosis. Microbial films that developed at higher temperatures (23°C
and 30°C) induced higher rates of larval metamorphosis than biofilms devel-
oped at lower temperatures (16°C). However, no significant conclusion on the
interactive effect between temperature and salinity and larval settlement could
be drawn.

5.4 Nutrients

The chemical composition (e.g., nutrient load) of ambient waters strongly deter-
mines the number, diversity, and metabolic states of planktonic bacteria, as well
as their tendency to adhere to surfaces (Schneider and Marshall 1994). Until now
there has been no study available that links nutrients, biofilms, and colonization
in situ. In the laboratory, Huang et al. (2007b) demonstrated that nutrient
availability and de novo protein synthesis mediated biofilm formation of
Pseudoalteromonas spongiae under static and starving conditions, which in turn
affected the inductiveness of biofilms for the larval settlementof H. elegans. The
effects of organic substances in the form of amino acids on biofilm bioactivity
were studied by Jin and Qian (2004, 2005). They found that aspartic acid and
glutamic acid significantly increased bacterial abundance, modified the bacterial
community structures of biofilms, and elevated the inductive effect of biofilms.
Alanine and asparagine increased, while isoleucine decreased, the bioactivity of
biofilms by changing their bacterial species composition, but not the bacterial
density. Leucine, threonine, and valine did not alter bacterial community struc-
tures or bioactivities of the biofilm in that study. In a recent study, Hung et al.
(2007) found that biofilm developed at the same intertidal height of different
habitats with contrasting environmental conditions showed remarkable differ-
ences in bioactivity for barnacle larval settlement, suggesting that the nutrient
condition at different habitats is the key factor governing the different bioactivi-
ties of those biofilms.
Environmental Changes Affect Biofilms that Affect Larval Settlement 323

5.5 Intertidal Versus Subtidal Biofilms

In experiments with Bugula neritina, larvae preferentially attached to subtidal

biofilms (Dobretsov and Qian 2006). In the latter study, subtidal biofilms, diatom
density, EPS thickness, and biofilm age, but not bacterial density, correlated
positively with enhanced larval attachment. Minchinton and Scheibling (1993)
showed that the diatom Achnantes parvula guided the attachment of the barnacle
Semibalanus balanoides on high intertidal regions. In a study by Qian et al.
(2003), cyprids of B. amphitrite preferred intertidal biofilms (i.e., 6-day old)
over unfilmed surfaces for attachment. Cyprids also preferred biofilms of mid-
intertidal height over high-intertidal or subtidal heights. There was no correla-
tion between attachment and any of the three biofilm attributes (i.e., biomass,
abundance of bacteria and diatoms). Qian et al. (2003) therefore concluded that
changes in bacterial community profiles in the biofilm affected the attractiveness
of the biofilm to barnacle larvae. In our previous study, we found that mid-
intertidal biofilms induced cyprid settlement of B. amphitrite, while subtidal
biofilms from the same site did not induce cyprid settlement of B. trigonus
(Thiyagarajan et al. 2005).

5.6 Seasonality

One possible cause of temporal variation of immigration responses to microbial

communities could be temporal differences in the “sensitivity” of biota to microbial
cues (i.e., a form of intrinsic variability). There is substantial evidence for behavioral
differences on the population level, as well as of cohorts or generations. Seasonally
distinct behavioral responses at immigration would appear to be adaptive where
these are related to variations in selection pressure, such as the seasonality of com-
petitors for resources or predators (Raimondi and Keough 1990). The composition,
quantity, and metabolic characteristics of biofilm communities change seasonally
in the field (e.g., Anderson 1995) and it seems reasonable that immigrating organisms
respond to these temporal alterations.
Wieczorek et al. (1996) showed marked seasonal variations in the effects of
biofilm cues on the larval settlement of certain marine invertebrate groups and taxa
to hard substrata under natural conditions, which are not to be explained by larval
availability alone. Also, a reversal of the biofilm effect on larval settlement response
with season, from inhibitory to facilitatory, was noted for certain species.
This may also hold for biochemical compounds (Cooksey and Wigglesworth-
Cooksey 1995; Qian et al. 2007), but has not been characterized as yet – neither
under laboratory nor under field conditions. It is necessary to monitor biofilm cue
production and release rate under natural conditions where the cells occur in
heterogeneous consortia within biofilms (Paerl and Pinckney 1996).
324 P.Y. Qian and H.-U. Dahms

6 Conclusions

It has to be emphasized that relevant settlement signals have not reasonably been
characterized as yet, neither for facilitative nor for inhibitory biofilms. Also,
responses of invading biota to monospecific biofilms in the laboratory may not
reflect responses to complex microbial communities in the field. This emphasizes
the need for long-term assessments of biofilm effects on settlement, under field
conditions, if appropriate conclusions are to be drawn about species-specific larval
responses to biofilms with the consequence of community alterations.
Besides knowing settlement-mediating chemical signals that are produced by
biofilms, the producers of inhibitive or inductive chemical cues need to be identified.
In addition, any synergistic effects in heterogeneous consortia within multispecies
biofilms need to be studied. As emphasized in this review, we particularly need to
investigate the environmental conditions that modify entire multispecies biofilm
bioactivity, since the settlement-mediating bioactivity of biofilms varies with envi-
ronmental conditions in the laboratory as under natural conditions. The investigation
of dynamic biofilm bioactivity is complicated by several biofilm properties, such as
cell density, biofilm thickness, structural alterations, microbial taxon diversity, bioactive
compound diversity, and the differential production of compounds. New methods
need to be developed that allow one to genetically identify and measure microbial
abundances and diversity, and to analyze such functions as the production and stor-
age of toxic, deterring, attracting or biocommunicative compounds that mediate the
colonization of invertebrate larval settlers in the marine environment.

Acknowledgements This contribution is supported by a RGC grant (HKUST6402/05M) and a

COMAR grant (COMRRDA06/07.SC01 and the CAS/SAFEA International Partnership Program for
Creative Research Team) to P-Y Qian.

References

Anderson MJ (1995) Variations in biofilms colonizing artificial surfaces: seasonal effects and
effects of grazers. J Mar Biol Assoc UK 75:705–714
Armstrong E, Yan L, Boy KG, Wright PC, Burgess JG (2001) The symbiotic role of marine
microbes on living surfaces. Hydrobiologia 461:37–40
Beech IB, Gubner R, Zinkevich V, Hanjangsit L, Avci R (2000) Characterisation of conditioning
layers formed by exopolymeric substances of Pseudomonas NCIMB 2021 on surfaces of AISI
316 stainless steel. Biofouling 16(2–4):93–104
Butler AJ, Chesson PL (1990) Ecology of sessile animals on subtidal hard substrata: the need to
measure variation. Aust J Ecol 15:521–531
Caldwell DE, Korber DR, Lawrence JR (1993) Analysis of biofilm formation using 2D vs 3D
imaging. J Appl Bacteriol Symp Suppl 74:52–66
Callow ME, Callow JA (2000) Substratum location and zoospore behavior in the fouling alga
Enteromorpha. Biofouling 15:49–56
Characklis WG, Marshall KC (1990) Biofilms: a basis for an interdisciplinary approach. In:
Characklis WG, Marshall KC (eds.) Biofilms. Wiley, New York, pp. 3–15
Environmental Changes Affect Biofilms that Affect Larval Settlement 325

Chia FS (1989) Differential larval settlement of benthic marine invertebrates. In: Ryland JS, Tyler
PA (eds.) Reproduction, genetics and distributions of marine organisms. Olsen and Olsen,
Fredensborg, Denmark, pp. 3–12
Chiu JMY, Thiyagarajan V, Tsoi MMY, Qian P-Y (2006) Qualitative and quantitative changes in
marine biofilms as a function of temperature and salinity in summer and winter. Biofilms 1–13
Clare AS, Rittschof D, Gerhart DJ, Maki JS (1992) Molecular approaches to nontoxic antifouling.
Invert Reprod Develop 22(1–3):67–76
Cooksey KE, Wigglesworth-Cooksey B (1995) Adhesion of bacteria and diatoms to surfaces in
the sea: a review. Aquat Microbial Ecol 9:87–96
Costerton JW, Lewandowski Z, de Berr D, Caldwell D, Kroner D, James G (1995) Microbial
Biofilms. Annu Rev Microbiol 49:711–745
Dahms HU, Qian PY (2005) Exposure of biofilms to meiofaunal copepods affects the larval set-
tlement of Hydroides elegans (Polychaeta). Mar Ecol Prog Ser 297:203–214
Dahms HU, Dobretsov S, Qian PY (2004) The effect of bacterial and diatom biofilms on the set-
tlement of the bryozoan Bugula neritina. J Exp Mar Biol Ecol 313:191–209
Decho AW (1990) Microbial exopolymer secretions in ocean environments: their role(s) in food
webs and marine processes. Oceanogr Mar Biol Ann Rev 28:73–153
Dobretsov S, Qian PY (2004) The role of epibotic bacteria from the surface of the soft coral
Dendronephthya sp. in the inhibition of larval settlement. J Exp Mar Biol Ecol 299:35–50
Dobretsov S, Qian PY (2006) Facilitation and inhibition of larval attachment of the bryozoan
Bugula neritina in association with mono-species and multi-species biofilms. J Exp Mar Biol
Ecol 333:263–264
Dobretsov S, Dahms HU, Qian PY (2006) Inhibition of biofouling by marine microorganisms and
their metabolites. Biofouling 22:43–45
Dobretsov S, Dahms HU, Huang YL, Wahl M, Qian PY (2007) The effect of quorum sensing
blockers on the formation of marine microbial communities and larval attachment. FEMS
Microbiol Ecol 60:177–188
Fenchel T (1998) Formation of laminated cyanobacterial mats in the absence of benthic fauna.
Aquat Microbiol Ecol 14:235–240
Gordon JW (1999) Genetic enhancement in humans. Science 283 (5410):2023
Hadfield MG, Paul VJ (2001) Natural chemical cues for settlement and metamorphosis of marine-
invertebrate larvae: 431–461. In: McClintock JB, Baker BJ (eds.) Marine chemical ecology.
CRC, Boca Raton, pp. 1–610
Harder T, Lam C, Qian PY (2002a) Induction of larval settlement in the polychaete Hydroides
elegans by marine biofilms: an investigation of monospecific diatom films as settlement cues.
Mar Ecol Prog Ser 229:105–112
Henschel JR, Cook PA (1990) The development of a marine fouling community in relation to the
primary film of microorganisms. Biofouling 2:1–11
Holmstrøm C, Kjelleberg S (1999) Marine Pseudoalteromonas species are associated with higher
organisms and produce active extracellular compounds. FEMS Microbiol Ecol 30:285–293
Holmstrøm C, Kjelleberg S (2000) Bacterial interactions with marine fouling organisms. In:
Evans LV (ed.) Biofilms: recent advances in their study. Harwood Academic, Amsterdam,
pp. 101–117
Holmstrøm C, Rittschof D, Kjelleberg S (1992) Inhibition of settlement by larvae of Balanus
amphitrite and Cliona intestinalis by a surface-colonizing marine bacterium. Appl Environ
Microb 58:2111–2115
Huang YL, Dobretsov S, Ki JS, Yang LH, Qian PY (2007a) Presence of acyl-homoserine lactone
in subtidal biofilm and the implication in larval behavioral response in the polychaete
Hydroides elegans. Microbial Ecol 54:384–392
Huang YL, Dobretsov SV, Xiong HR, Qian PY (2007b) Effect of biofilm formation by
Pseudoalteromonas spongiae on induction of larval settlement of the polychaete Hydroides
elegans. Appl Environ Microbiol 73:6284–6288
326 P.Y. Qian and H.-U. Dahms

Hung OS, Gosselin LA, Thiyagarajan V, Wu RSS, Qian PY (2005a) Do effects of ultraviolet radiation
on microbial films have indirect effects on larval attachment of the barnacle Balanus
amphitrite? J Exp Mar Biol Ecol 323:16–26
Hung OS, Thiyagarajan V, Wu RSS, Qian PY (2005b) Effect of ultraviolet radiation on biofilms
and subsequent larval settlement of Hydroides elegans. Mar Ecol Prog Ser 304:155–166
Hung OS, Thiyagarajan V, Zhang R, Wu RSS, Qian PY (2007) Attachment of Balanus amphitrite
larvae to biofilms originated from contrasting environments. Mar Ecol Prog Ser 333:229–242
Jin T, Qian PY (2004) Effect of mono-amino acids on larval metamorphosis of the polychaete
Hydroides elegans. Mar Ecol Prog Ser 267:223–232
Jin T, Qian PY (2005) Amino acid exposure modulates the bioactivity of biofilms for larval set-
tlement of Hydroides elegans by altering bacterial community components. Mar Ecol Prog Ser
297:169–179
Keough MJ, Raimondi PT (1996) Responses of settling invertebrate larvae to bioorganic films:
effects of large-scale variation in films. J Exp Mar Biol Ecol 207:59–78
Kierek-Pearson K, Karatan E (2005) Biofilm development in bacteria. Adv Appl Microbiol
57:79–111
Kirchman D, Graham D, Reish D, Mitchell R (1982) Lectins may mediate in the settlement and
metamorphosis of Janua (Dexiospira) brasiliensis Grube (Polychaeta: Spirorbidae). Mar Biol
Lett 3:201–222
Kjelleberg S, Albertson N, Flardh K, Holmquist L, Jouper-Jaan A, Marouga R, Ostling J,
Svenblad B, Weichart D (1993) How do non-differentiating bacteria adapt to starvation?
Antonie Leeuwenhoek 63:333–341
Lam C, Harder T, Qian PY (2005a) Induction of larval settlement in the polychaete Hydroides
elegans by extracellular polymers of benthic diatoms. Mar Ecol Prog Ser 286:145–154
Lam C, Harder T, Qian PY (2005b) Growth conditions of benthic diatoms affect quality and
quantity of extracellular polymeric larval settlement cues. Mar Ecol Prog Ser 294:109–116
Lau SCK, Mak KKW, Chen F, Qian PY (2002) Bioactivity of bacterial strains from marine bio-
films in Hong Kong waters for the induction of larval settlement in the marine polychaete
Hydroides elegans. Mar Ecol Prog Ser 226:301–310
Lau SCK, Harder T, Qian PY (2003a) Induction of larval settlement in the serpulid polychaete
Hydroides elegans (Haswell): role of bacterial extracellular polymers. Biofouling 19:197–204
Lau SCK, Thiyagarajan V, Qian PY (2003b) The bioactivity of bacterial isolates in Hong Kong
waters for the inhibition of barnacle (Balanus amphitrite Darwin) settlement. J Exp Mar Biol
Ecol 282:43–60
Lau SCK, Thiyagarajan V, Cheung SCK, Qian PY (2005) Roles of bacterial community composi-
tion in biofilms as a mediator for larval settlement of three marine invertebrates. Aquat Microb
Ecol 38:41–51
Li X, Dobretsov S, Xu Y, Xiao X, Qian PY (2006) Antifouling diketopiperazines produced by a
deep-sea bacterium Streptomyces fungicidicus. Biofouling 22:201–208
Maki JS, Rittschof D, Costlow JD, Mitchell R (1988) Inhibition of attachment of larval barnacles,
Balanus amphitrite, by bacterial surface films. Mar Biol 97:199–206
Maki JS (1999) The influence of marine microbes on biofouling. In: Fingerman M, Nagabhushanam R,
Thompson F (eds.) Recent advances in marine biotechnology, vol 3. Biofilms, bioadhesion,
corrosion, and biofouling. Science, Enfield, NH, pp. 147–171
Miao L, Kwong TFN, Qian PY (2006) Effect of culture conditions on the mycelial growth, anti-
bacterial activity and metabolite profiles of the marine-derived fungus Arthrinium cf saccharicola.
Appl Microbiol Biotechnol 72:1063–1073
Minchinton TE, Scheibling RE (1993) Variations in sampling procedure and frequency affect
estimates of recruitment of barnacle. Mar Ecol Prog Ser 99:83–88
Neal AL, Yule AB (1994) The tenacity of Elminius modestus and Balanus perforatus cyprids to
bacterial films grown under different shear regimes. J Mar Biol Assoc UK 74:251–257
Ophir T, Gutnick DL (1994) A role for exopolysaccharides in the protection of microorganisms
from dessication. Appl Environ Microbiol 60:740–745
Environmental Changes Affect Biofilms that Affect Larval Settlement 327

Paerl HW, Pinckney JL (1996) A mini-review of microbial consortia: their roles in aquatic production
and biogeochemical cycling. Microb Ecol 31:225–247
Palmer RJ, White DC (1997) Developmental biology of biofilms: implications for treatment and
control. Trends Microbiol 5:435–439
Parsek MR, Greenberg EP (2005) Sociomicrobiology: the connections between quorum sensing
and biofilms. Trends Microbiol 13:27–33
Parsek MR, Val DL, Hanzelka BL, Cronan JEJr, Greenberg EP (1999) Acyl homoserine-lactone
quorum-sensing signal generation. Proc Natl Acad Sci U S A 96(8):4360–4365
Patel P, Callow ME, Joint I, Callow JA (2003) Specificity in larval settlement modifying response
of bacterial biofilms towards zoospores of the marine alga Enteromorpha. Environ Microb
5:338–349
Pawlik JR (1992) Chemical ecology of the settlement of benthic marine invertebrates. Oceanogr
Mar Biol Ann Rev 30:273–335
Qian PY (1999) Larval settlement of polychaetes. Hydrobiologia 402:239–253
Qian PY, Rittschoff D, Sreedhar B, Chia FS (1999) Macrofouling in unidirectional flow: miniature
pipes as experimental models for studying the effects of hydrodynamics on invertebrate larval
settlement. Mar Ecol Prog Ser 191:141–151
Qian PY, Rittschof D, Sreedhar B (2000) Macrofouling in unidirectional flow: miniature pipes as
experimental models for studying the interaction of flow and surface characteristics on the
attachment of barnacle, bryozoan and polychaete larvae. Mar Ecol Prog Ser 207:109–121
Qian PY, Thiyagarajan V, Lau SCK, Cheung S (2003) Relationship between community and the
attachment of acorn barnacle Balanus amphitrite Darwin. Aquat Microbiol Ecol 33:225–237
Qian PY, Lau SCK, Dahms HU, Dobretsov S, Harder T (2007) Marine biofilms as mediators of
colonization by marine macroorganisms implications for antifouling and aquaculture. Mar
Biotech 9:399–410
Raimondi PT, Keough MJ (1990) Behavioural variability in marine larvae. Aust J Ecol 15:427–437
Rodriguez SR, Ojeda FP, Inestrosa NC (1993) Settlement of benthic marine invertebrates. Mar
Ecol Prog Ser 97:193–207
Schneider RP, Marshall KC (1994) Retention of the Gram negative marine bacterium SW8 on
surfaces – effects of microbial physiology, substratum nature and conditioning films. Colloids
Surf B: Biointerfaces 2:387–396
Stevenson RJ, Peterson CG (1989) Variation in benthic diatom (Bacillariophyceae) immigration
with habitat characteristics and cell morphology. J Phycol 25:120–129
Stewart PS (2002) Mechanisms of antibiotic resistance in bacterial biofilms. Int J Med Microbiol
292:107–113
Szewzyk U, Holmstroem C, Wrangstadh M, Samuelsson MO, Maki JS, Kjelleberg S (1991)
Relevance of the exopolysaccharide of marine Pseudomonas sp. strain S9 for the attachment
of Ciona intestinalis larvae. Mar Ecol Prog Ser 75:259–265
Thiyagarajan V, Hung OS, Chiu JMY, Wu RSS, Qian PY (2005) Growth and survival of juvenile
barnacle Balanus amphitrite: interactive effects of cyprid energy reserve and habitat. Mar Ecol
Prog Ser 299:229–237
Todd CD, Keough MJ (1994) Larval settlement in hard substratum epifaunal assemblages:
a manipulative field study of the effects of substratum filming and the presence of incumbents.
J Exp Mar Biol Ecol 181:159–187
Underwood AJ, Fairweather PG (1989) Supply side ecology and benthic marine ecology. Trends
Ecol Evol 4:16–20
Wahl M (1989) Marine epibiosis. I. Fouling and antifouling: some basic aspects. Mar Ecol Prog
Ser 58:175–189
Wieczorek SK, Todd CD (1998) Inhibition and facilitation of settlement of epifaunal marine
invertebrate larvae by microbial biofilm cues. Biofouling 12:81–118
Wieczorek SK, Murray AWA, Todd CD (1996) Seasonal variation in the effects of hard substratum
biofilming on settlement of marine invertebrate larvae. Biofouling 10:309–330
328 P.Y. Qian and H.-U. Dahms

Wimpenny J (2000) An overview of biofilms as functional communities. In: Allison D, Gibert P,

Lappin-Scott H, Wilson M (eds.) Community structure and co-operation in biofilms.
Cambridge University Press, Cambridge, UK, pp. 1–24
Wolfstein K, Stal LJ (2002) Production of extracellular polymeric substances (EPS) by benthic
diatoms: effect of irradiance and temperature. Mar Ecol Prog Ser 236:13–22
Wright JP, Gurney WSC, Jones CG (2004) Patch dynamics in a landscape modified by ecosystem
engineers. Oikos 105:336–348
Xu Y, Miao L, Li XC, Xiao X, Qian PY (2007) Antibacterial and antilarval activity of deep-sea
bacteria from sediments of the West Pacific Ocean. Biofouling 23:131–137
Yang LH, Li XC, Yan SC, Sun HZ, Qian PY (2006) Antifouling properties of 10b-formamidodalihinol-
A and kalihinol A isolated from the marine sponge Acanthella cavernosa. Biofouling
22:23–32
Yang LH, Xiong HR, Lee OO, Qi SH, Qian PY (2007) Effect of agitation on violacein production
in Pseudoalteromonas luteoviolacea isolated from a marine sponge. Lett Appl Microbiol
44:625–630
Index

A Balanus eburneus, 206, 208

Academia, 180, 183–184, 186 Barnacle larvae, 174
Actecholamine pathway Barnacles, 168, 265–266, 271–272, 279–282,
Active metal corrosion, 37, 39–45 295, 297, 300–303
Adhesion, 165–169, 171, 173–175, 294 Basibiont, 219–225
Adhesive, 203, 206 Bioadhesion, 135–141, 143, 147–149, 156
Advantages, ecological, 3, 11 Biocidal products, 189–198
AFM. See Atomic force microscopy Biocidal Products Directive, 190
American M.A.N. brushes, 273 Biocide generation, 123–127, 133
Amino acid derivatives, 236, 238 Biocides, 189–192, 194–198
Amphora coffeaeformis, 167, 168, 170, 172, 173 broad spectrum, 179, 180, 183, 184–185
Anaerobic habitat, 10 long-lived, 185
Animals, risk to, 189, 191, 194 metals, 185
Anodic current, density, 42, 43 organic, 185
Anodic reaction, 37–43, 48, 50, 52–53 registration, 183
Anti-diatom, 165 short-lived, 184–185
Antifoulant, 165–175 tolerance, 10
Antifouling, 179–186 Biodegradable organic matter (BDOC),
Antifouling coatings 82, 85
ablative, 183 Biodegradation, 196
business models, 180, 182–185 Biofilms, 3–11, 13–19, 21, 23, 25–28, 203,
development, 179, 180, 183–186 205–207, 209, 211, 213, 293–308
environmentally benign, 179–180, 184–186 management, 112–113
industry, 182–183 monitoring techniques, 114
resin, 183 protective, 38
technology, 179, 180, 182–185 removal, 49, 51
Antifouling compounds, 246, 247, 254 structure, 3, 7–10
Anti-fouling strategy, 107–115 thickness, 68–71, 81
Atomic force microscopy, 166 Biofilm attributes
Auricula sp., 173, 175 biofilm bioactivity, 315, 317, 321, 322, 324
Autonomous monitoring, 119, 120, 122, 126, 133 biofilm changes, 315, 317–319
Availability model, 181 biofilm dynamics, 315–317
biofilm properties, 315–318, 321, 324
community structure, 315, 319, 322
B dynamics, 315–317, 320
Balanus amphitrite, 206, 208 microbial communities, 315, 316, 321,
Bacillus thuringiensis, 195 323, 324
Bacteria, 219–227, 293–301, 303–308 microbial density, 315
Baier curve, 166 microbial physiological conditions, 315

329
330 Index

Biofilm attributes (cont.) Competent larvae, 203, 205, 210, 211, 213
microbial products, 316 Condenser backpressure, 267, 270
physical, chemical, and biotic properties Conditioning film, 109, 110, 169
of biofilms, 316 Conductivity sensor, 123, 128–130
Biofilmed surfaces, 209 Control
Biofouling, 3–11 Antimicrobial agents, 19, 20, 27
antifouling, 136 biocides, 20, 21, 24, 27
control, 135–156 Disinfection, 19, 22, 27
non toxic control, 135, 136, 153 TBT, 21, 22
operational definition, 108 Titanium dioxide, 22
release, 142, 153, 154 Cooling towers, 72–80, 82
Biofouling in seawater, 119, 120, Copper, 296
129–127 corrosion, 125
Biomimics, 253 release, 124–126
Bioprobe, 166 shutters, 119, 125, 126, 133
BPD, problems with, 195–197 Corrosion
Branchial radioles, 213, 214 current, 43–46, 59
Business, 179, 180, 182–186 definition of, 37
of iron, 37–38
metals, 38
C biofilms influence, 35, 36, 60
Ca-ATPases, 171 microbially stimulated, 36, 43–47
Ca-fluorophores, 165 potential, 42–44, 46, 54–55, 57, 58
Ca homeostasis, 171, 173 reactions, 36–39, 41, 43–45, 47–60
Ca-mediated, 165 Corrosivity, 194
cAMP-cyclic adenosine monophosphate, Cyanobacteria, 74, 75, 77, 78, 296, 303
236, 237
Carcinogenicity, 194
Cathodic polarization, 42–43 D
Cathodic reaction, 37, 40–44, 48, 50, 52–53, 61 D-600, 165, 173
Cell, 135–138, 140, 142, 146–148, 152 Defence, 219–227
Cell signaling, 165, 173 Dental plaque, 104
Chemical properties of material Diatoms, 220, 225, 226, 293–295,
copper, 18–22, 27 297–299, 306
metals, 17–20, 22, 27 Differential aeration cells, 47–51
paints, 20–22 Dihydroxyphenyl l-alanine (l-DOPA), 243
polymers, 19–21 Disinfectants, 190–192
Chemotactic, 171, 173 Dispersal, 171, 173
Chick and Watson law, 83 Drag, 166
Chlamydomonas, 171, 173
Chlorophyll, 167, 168, 174
Cleaning, 3, 4, 8, 103, 107, 112–115 E
Clean Water Act, 184 Effectiveness, 125, 128
Climate, 307 Electric fields, 113
Clostridium aceticum, 61 Electro-chlorination system, 126–127
Coating Electro-chlorination unit, 126, 127
antifouling, 136, 154 End of the season chlorination, 276
elastic modulus, 143–144, 147, 154 Ennoblement
fouling release, 142, 144, 146, 153, 154, 156 environmental factors, 54, 55, 57
silicone, 142, 143, 154, 155 rate and extent, 55
thickness, 144 of stainless steel, 54–59
Coatings, 307, 308 Environment, risk to, 189, 191, 194
Cohesion, 112 Environmental effects
Coliform organisms, 105 depth, 320, 321
Competent Authorities, 189–191, 193, 197, 198 exposure time, 30, 321
Index 331

flow, 320, 321 G

flow regime, 320 Gallionella, 58, 61
illumination, 320, 321 Glycoprotein, 166, 169
light, 319, 321 Government, 179, 180, 183–184, 186
nutrients, 319, 322 Green mussels, 274
nutrient supply, 320, 321 Guideline, consequences of, 189–198
physical disturbance, 317, 320
salinity, 319, 322
seasonality, 320, 321, 323 H
temperature, 319, 322 Hydrogen, 52, 53, 55, 61
tidal height, 320, 322, 323 Hydrogen peroxide, 82, 86, 87, 89
vertical depth, 320 Hydrogen sulfide, 52–53
water chemistry, 320, 321 Hydroids, 271, 279
Enzyme(s), 112, 113, 301, 303, 306 Hydrolazing, 273
Epibiont, 219–221, 223–227 Hydrophilic, 166
EPS, 1, 5–9 Hydrophobic, 166
function, 7
matrix, 293, 295
Escherichia coli, 105 I
Ethanol, 195, 196 Image analysis, 165, 170
European Union, 186 Inorganic fouling, 266–268
“Existing” active substances, 193 In-situ wiper, 123–124
Exposure, to biocides, 193, 194 Interference of electro-chlorination, 127–128
Extracellular polymeric substances, Intermittent chlorination, 67, 77, 78
1, 5–8 Invertebrate, 166, 169, 173, 174
Extracellular polymers, 293 Ion-gated channels, 236, 238–239, 242
Extrapolymeric substances (EPS), 66, 69, 87, Iron corrosion
88, 93 kinetics, 41–43
thermodynamics, 39–41
Iron sulfides, precipitated, 52
F Irritation, 194
Feeding and care of larvae, 210–215
Ferrous iron, 39, 41, 52
Fertilization, 209, 212, 213 J
Fibre optical device, 113, 114 Juvenile development, 214
Flagellates, 293, 297, 306
Fluid frictional resistance, 72
Fluorescence sensor, 122, 130–133 K
Fouling, 165, 166, 168, 169, 171, Klebsiella, 105
174, 175
communities, 203, 204
EPS, 14, 16, 21 L
organic material, 14, 16, 20, 27, 28 Lactones, 249
research, 179, 183 Larvae, 219–223, 226
Fouling management larval settlement, 315–324
environmentally benign, larval settlement and metamorphosis,
179, 180 316, 322
environmentally damaging, 183 settlement processes, 315, 316, 319
Foul-release coatings, 179–182 Larval development, 212–213
antifouling, 179, 180, 182, 184 Larval food, 211–212
development, 179, 181, 184 Larval settlement, 205, 206, 223–255
fluoropolymer, 181–182 Lectins, 300
silicone, 182 Legionella, 105
Furanones, 249, 250 Legionella sp, 75
Furans, 249 Life, multicellular, 5
332 Index

Life-cycle, of chemical, 193 O

Localized electro-chlorination, 127–133 Oceanographic sensor, 122–127
Lotus effect, 110 Oceanographic sensor biofouling protection,
Low level continuous chlorination, 275, 280, 122–127
281, 285 Once through systems, 70
Low surface energy coatings, 233 Organic fouling, 104, 268
Oxygen access, 48
Oxygen heterogeneities, 38, 47–49
M
Macrofoulants, 266–268, 271, 274, 277,
285–286 P
Macrofouling, 306, 307 Pseudomonas aeruginosa, 112
Manganese, biomineralization, 56, 58 Particle fouling, 104
Manganese oxides, 46, 56–61 Passive metal, corrosion, 39, 44–47, 54, 56,
Manganese-oxidizing bacteria, 52, 56–61 59, 61
Mass transport resistance, 38, 49–51 Peracetic acid, 284
Matrix, 3, 6–11 Pest control, 191, 192
activated, 7 Phaeodactylum tricornutum, 171, 173
cohesion, 9 Pitting, 38, 45–47, 55–56, 59
hydrated, 6, 8 Pitting potential, 45–47, 55
Mechanism, MIC, 35–61 Plate heat exchangers, 265–268, 272, 279,
Metamorphosis, 205, 206, 211, 213–215 284, 286
Metatrochophores, 212 Polarization curves, potentiodynamic, 43–45,
MIC, mechanisms of, 35–61 57–58
Microbial Polysaccharides, 293, 300, 301, 303
Adhesion, 13, 14, 16, 23, 26–28 Potable water systems, 80, 85, 87
attachment, 13, 15–18, 20, 21, 26–28 Preservatives, 191, 192
persistence, 27 Primary tube, 213, 214
resistance, 13, 15, 16, 18, 27 Protection, 119–134
retention, 13, 15, 17, 23, 25, 26–28 Protein
survival, 13–28 adsorption, 135, 138, 140, 141, 148–152, 156
tolerance, 27 plasma films, 152–153
Microbially influenced corrosion, 35–61 repellent, 138, 149–153
Microbial resistance, 67, 88 Protein kinase C, 238, 240, 242, 243
Microconsortia, 5, 7, 11 Prototroch stage, 212
Microtopography, 235, 252, 253 Pseudoalteromonas luteoviolacea, 205–207
Mineral fouling, 104 Pulse chlorination®, 276, 281, 285
Molecular simulations, 111
Monitoring, of biofilms, 113–114
Monochloramine, 84–86, 93 Q
Morphogenetic pathway, 236, 237, 240, 244 Quaternary ammonium compounds (QAC), 90
Motility, 165, 167–175 Quorum sensing, 167, 293, 294, 296,
Mutagenicity, 194 304–306
Mycobacterium, 105

R
N Recirculating systems, 66, 68, 72, 73, 90
N-acetyl-L-homoserine lactone, 304, 305 Regulatory pathway, 236, 237
Natural anti-fouling compounds, 109–110 Renillafoulin, 168
Navicula, 166, 173–175 Resistance, 3, 10
Neurotransmitters, 238, 239, 243, 244 Risk assessment, 190, 191, 193–194
“new” active substances, 191, 193 Risk characterization, 194
2-n-pentyl-4-quinolinol, 165, 173 Risks to humans, 191, 194
Nutrient limitation, 103, 108, 112 Robbins device, 113
Index 333

S Synthetic analogues, 234, 241, 246–251, 255

Salinometer, 128 Synthetic elastomers, 105
Scanning vibrating electrode, 50, 51 Sytox Green, 173, 174
Sea Pansies, 168
Secondary metabolites, 219, 226
Secondary tube, 213, 214 T
Second messenger diacylglycerol pathway, Thalassiosira weissflogii, 173
236–238, 240 Thiobacillus thiooxidans, 61
Settlement, 220–224, 226 Toxicity, for reproduction, 194
Shear forces, 69–71, 89, 166 Trans, 165, 173
Shell and tube heat-exchangers, 266–267, Trans-2,4-decadienalinfluence diatom, 165
272–273, 286 Travelling water screens, 267, 269, 272,
Silicones, 235, 249, 251–253 281, 285
Spawning, 209–210 Trialkyl tins, 165
Speed, 170, 172 Triangle Model
Sponge rubber balls, 272–273 biofilm bioactivity, 317
SRB corrosion, 51–54, 61 biofilms, 318
Standing committee, on biocidal products, 193 environmental changes, 317–318
Strength of adhesion, 208 larval settlement, 317
Substances, 189–191, 193–198 settlement of dispersal stage, 317
corrosive, 35, 38, 51–54, 60–61 Tributyl tin (TBT), 109, 133, 296, 233, 251,
Succession model, 181 255, 296
Sulfate-reducing bacteria, 51–54 Trochophores, 212
Sulfur disproportionation, 53 Tuberculation, 49
Surface Tubeworms, 265–266, 279–280
chemistry, 144–146, 152, 156 Turbulent flow apparatus, 206
design, 109–112
energy, 165–167, 169, 233, 235, 252,
253, 255 U
hydrodynamics, 137, 156 United Nations, 186
hydrophilic, 135, 136, 141, 147–152 U.S. Environmental Protection Agency
hydrophobic, 135, 136, 138, 141, 142, 146, (US-EPA), 85
149, 150, 152
interaction, 135, 137, 138, 147–149, 152
low energy, 135, 136 V
repellent, 138, 149–153 Verapamil, 173, 175
roughness, 135, 136, 146–148, 156, 235 Vesicle secretion, 171
tension, 139, 141–143, 146 Volatile organic compounds, 198
theta, 139, 156
topography, 135, 136, 138, 146–147, 149, 156
wettability, 141, 142, 146 W
physicochemistry Wipers, 119, 123–125, 133
hydrophobicity, 17
surface free energy, 17
surface topography, 16, 17 Z
Surfactants, 69, 82, 84, 89–91, 94 Zebra mussels, 266, 271, 282–283