ENZYMA TIC PITCH CONTROL IN THE KRAFT

PULPING AND BLEACHING OF EUCALYPTUS SPP.

by

Gerhardus C. Scheepers

Thesis presented in partial fulfilment of the requirements for the degree of Master
of Wood Science at the University of Stellenbosch

March 2000

STUDY LEADER: Dr. B.J.H. Janse

CO-STUDY LEADER: Dr. T. Rypstra

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DECLARATION

I, the undersigned, hereby declare that the work contained in this thesis is my
original work and has not previously in its entirety or in part been submitted at any
university for a degree.

G.C. Scheepers
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SUMMARY

The extractive materials in wood often cause pitch problems in pulp mills. During pulping and
bleaching extractives are released from the wood and pulp and later stick to ceramic and metal
parts, forming pitch deposits. Pitch deposits impair both product quality and production rates. It
decreases the efficiency of pulp washing, screening, centrifugal cleaning, and refining, and can
disrupt many paper machine operations. The deposits also break loose from equipment and
cause spots in the final product. There are a few triggering mechanisms that induce pitch
deposition. Hydrodynamic or mechanical shear can destabilise the colloidal pitch emulsion,
causing pitch to agglomerate and deposits to form. Similarly, sudden temperature drops and/or
pH shocks and/or the introduction of water hardness ions from fresh water inlets or showers can
also cause pitch deposits by destabilising the colloidal pitch emulsion. Inorganic salts, such as
calcium carbonate, can catalyse pitch deposition by acting as the building blocks for the sticky
pitch. Calcium ions in the white water can react with fatty acids, forming insoluble, sticky
calcium soaps. Triglycerides have also been shown to be a major contributor to pitch deposition
in kraft pulping and bleaching mills. It forms a sticky deposit to which less sticky particles
attach.

To attain an improved understanding of pitch problems associated with the kraft pulping and
bleaching of Eucalyptus spp., various analyses were done on wood- and pulp extractives and
pitch from a South African kraft pulp mill. High molecular weight compounds (involatile)
constituted a large portion of the extracts and pitch. Approximately 40% of volatile Eucalyptus
grandis extract was f3-sitosterol, with fatty acids (22.8%) and triglycerides (15.5%) also making
a substantial contribution. Fatty acid amides were a prominent fraction of pulp extracts from the
latter stages of bleaching. The amides constituted 38.3% and triglycerides 10.1% to total volatile
pitch deposits.

Lipases hydrolyse triglycerides and could therefore help to reduce pitch problems. Consequently
381 filamentous fungi isolated from indigenous and commercial forests in South Africa were
screened for lipase activity on tributyrin and Tween 80. Eight strains were selected and the
tributyrin and Tween 80 assays were repeated by monitoring lipase activity over a seven-day

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period. The selected strains were also assayed for their activity toward p-nitrophenyl palmitate.
Ophiostoma piliferum Cartapip 58™ and Phanerochaete chrysosporium BKM-F-1767, two
strains known for respectively their biodepitching and biopulping ability, were' used as controls.
A few of the strains compared well and even outperformed the control strains, indicating their
potential for use in pitch control.

The effect of pretreatment with the eight selected fungal strains on E. grandis wood- and pulp
extractives was determined. Cartapip 58™ and P. chrysosporium BKM-F-1767 were used as
control strains. Several of the strains compared well to the control strains in their ability to
reduce the triglyceride content of wood extract. The South African isolate, white-rot fungus
Phanerochaete psuedomagnoliae nom. prov., reduced triglyceride content significantly.
Consequently it can act as an agent for both biopulping and biodepitching. The treated wood
samples had a lower triglyceride content than the sterile controls. Consequently more
triglycerides would be released into process waters by the sterile controls than the treated
samples. The effect of commerciallipases on deposited brown stock pulp extract was also
evaluated. The lipases did not reduce the triglyceride content of the deposited extract. The
addition of lipases in pulping and bleaching processes would therefore not affect already
deposited pitch.

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OPSOMMING

Die ekstrakstowwe van hout veroorsaak dikwels 'n neerslag tydens verpulping. Gedurende
verpulping en bleiking kom ekstrakstowwe van die hout enpulp vry en kleef aan keramiek- en
metaalonderdele. Gevolglik benadeel dié neerslag produkkwaliteit en produksietempo. Dit
verlaag die effektiwiteit van pulpwas, sifting, sentrifugale skoonmaakprosesse en suiwering, en
kan die werkverrigting van papiermasjiene ontwrig. Die neerslag kan ook later los breek en
kolletjies op die finale produk veroorsaak. Verskeie meganismes kan die neerslag veroorsaak.
Hidrodinamiese of meganiese wrywing kan die kolloïdale ekstrakstofemulsie destabiliseer en
sodoende die ekstrakstof laat konglomereer en neerslaan. Op soortgelyke wyse veroorsaak
skielike temperatuurverlaging en/of pH-skokke en/of die toevoeging van ione in varswater om
waterhardheid te beheer ook die neerslag deur die kolloïdale ekstrakstofemulsie te destabiliseer.
Anorganiese sout soos kalsiumkarbonaat kan neerslagvorming kataliseer omdat dit optree as
bousteen vir die klewerige, sementagtige ekstrakstowwe. Kalsiumione in die proseswater kan
ook reageer met vetsure om onoplosbare, klewerige kalsiumsepe te vorm. Dit is bewys dat
trigliseriede een van die hoofoorsake is in die vorming van die neerslag tydens kraft verpulping-
en bleikingprosesse.

Om die neerslagreaksie wat met die kraft verpulping en bleiking van Eucalyptus spp. geassosieer
word, beter te verstaan, is verskeie analises op hout- en pulpekstrakte asook die neerslag van 'n
Suid-Afrikaanse kraft verpulpingsaanleg uitgevoer. Hoë molekulêre massa (nie-vlugtige)
stowwe het 'n groot gedeelte van die ekstrakte en neerslag uitgemaak. Ongeveer 40% van die
vlugtige Eucalyptus grand is ekstrak bestaan uit ~-sitosterol met vet sure (22.8%) en trigliseriede
(15.5%) wat ook aansienlike bydraes lewer. Vetsuuramiede verteenwoordig 'n beduidende
komponent van pulpekstrak by die laaste stadiums van bleiking. Die amiede het 38.3% en
trigliseriede 10.1% tot die vlugtige fraksie van die neerslag bygedra.

Lipases hidroliseer trigliseriede en kan dus help om neerslagprobleme te voorkom. Gevolglik is

381 filamentagtige fungi geïsoleer uit inheemse en kommersiële woude van Suid-Afrika en hul
lipase-aktiwiteit op tributyrin en Tween 80 geëvalueer. Agt rasse is geselekteer en die tributyrin
en Tween 80 toetse is herhaal deur lipase-aktiwiteit oor 'n sewe-dag periode te monitor. Die
geselekteerde rasse is ook getoets vir lipase-aktiwiteit met p-nitrofenielpalmitaat. Ophiostoma

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piliferum Cartapip 58™ en Phanerochaete chrysosporium BKM-F-1767, twee rasse wat

daarvoor bekend staan vir onderskeidelik hul vermoë om houtekstrakstowwe te verminder en te
bioverpulp, is as kontroles gebruik. 'n Paar van die geselekteerde rasse het goed vergelyk en
selfs beter presteer as die kontrolerasse; 'n aanduiding van hul potensiaal om neerslagreaksies te
beheer.

Die effek van voorafbehandeling met die agt geselekteerde fungi rasse op E. grandis hout- en
pulpekstrak is vasgestel. Cartapip 58™ en P. chrysosporium BKM-F-1767 is gebruik as
kontrolerasse. Verskeie rasse het goed vergelyk met die kontrolerasse in hul vermoë om die
trigliseriedinhoud van die houtekstrak te verlaag. Die Suid-Afrikaanse isolaat,
witverrottingswam Phanerochaete pseudomagnoliae nom. prov., het ook die trigliseried inhoud
beduidend verminder. Gevolglik sou dit as 'n middel kon dien vir beide neerslagvoorkoming en
bioverpulping. Die trigliseriedinhoud van die behandelde monsters was laer as dié van steriele
kontroles. Gevolglik sal meer trigliseriede in proseswater vrygestel word deur die steriele
kontroles as die behandelde monsters. Die effek van kommersiële lipases op ongebleikte kraft
pulpekstrakneerslag is ook geëvalueer. Omdat lipases nie die trigliseriedinhoud van die neerslag
kon verlaag nie sal die gebruik van lipases dus nie die ekstrakstofneerslag in verpulpings- en
bleikingsprosesse beïnvloed nie.

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This dissertation is dedicated to the Lord Almighty, for I can do anything

through Him who gives me strength.

Hierdie proefskrif is opgedra aan God die Almagtige, want ek is tot alles in
staat deur Hom wat my krag gee.

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ACKNOWLEDGEMENTS

I wish to express my sincere gratitude and appreciation to the following persons and institutions
for their contribution to the successful completion of this study:

Dr. B.J.H. Janse and Dr. T. Rypstra, study leader and co-study leader, for the opportunity to
work with them, and for their encouragement and guidance in all circumstances;

Prof. G.F.R. Gerischer and Mr J.W. Hunt, internal and external examiners, for their effort and
time;

Mr P. Leah, Mondi Kraft Ltd., for his advice and support during this project;

The Foundation for Research Development, for supporting this project financially and
supplying bursaries;

The Technology and Human Resources for Industry Programme and

Mondi Kraft Ltd., for also supporting this project financially;

my family, friends and colleagues, for every contribution they made through the years, directly
or indirectly, to this achievement.

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PREFACE

This thesis is presented as a compilation of manuscripts. Chapters 1, 2 and 6 were formatted

according to the style of the scientific journal Biotechnology Letters. Each of Chapters 3 to 5 was
formatted according to the style of the journal to which it will be submitted.

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TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION " 1

1.1 Introduction 2

1.2 References 3

CHAPTER 2: LITERATURE REVIEW 4

2.1 Wood structure and characteristics with special reference to Eucalyptus

grandis 5
2.1.1 Wood structure 5
2.1.2 General information about Eucalyptus grandis 8
2.1.2.1 Macroscopic structure 8
2.1.2.2 Extractive content of various Eucalyptus species 8
2.1.2.3 Sapwood and Heartwood distribution 9

2.2 The extractives of wood 11

2.2.1 Terpenes and terpenoides 12
2.2.2 Fats, waxes and their components 12
2.2.3 Phenolic substances 14
2.2.4 Tannins 15
2.2.5 Other substances present in extractives 16

2.3 The kraft pulping process 17

2.3.1 Terminology 18
2.3.1.1 White liquor 18
2.3.1.2 Black liquor 18
2.3.1.3 Kappa number 19
2.3.2 The digestion process 19
2.3.3 The blow tank 21
2.3.4 Brown stock washing 21

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2.3.5 Bleaching 21
2.3.6 Knotters, rimers and screens 22
2.3.7 Refining : : 22

2.4 Pitch 23
2.4.1 Defining pitch 23
2.4.2 The composition ofpitch 23
2.4.3 Mechanisms of deposition 25
2.4.3.1 Deposition due to polymerisation 25
2.4.3.2 Metal soap deposition 26
2.4.3.3 Resin transfer from fibres 28
2.4.3.4 Fines and fibre deposition 28
2.4.3.5 Transfer of resin to the felts in press nips 29
2.4.3.6 Deposition due to evaporation 29
2.4.3.7 Deposition of resin due to film formation 29
2.4.3.8 Resin deposition in refiners and beaters 30
2.4.3.9 Temperature and pH fluctuations 30
2.4.3.10 Co-deposition of extraneous substances 30

2.5 Triglyceride breakdown with lipase 31

2.5.1 Lipase structure 32
2.5.2 Hydrolysis reaction mechanism 32
2.5.3 Homology oflipases from different species 36
2.5.4 Conditions for optimal lipase activity 39

2.6 Wood-decay fungi: classification and characteristics 41

2.6.1 Molds 43
2.6.2 Sap-stain fungi 43
2.6.3 White-rot fungi 44
2.6.4 Brown-rot fungi 46
2.6.5 Soft-rot fungi 47
2.6.6 Hyphal growth and cell wall composition 47

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2.7 Fungal growth in wood ~ 50

2.7.1 Nutrient sources in wood for fungi 50
2.7.2 Factors influencing colonisation of wood : 51
2.7.2.1 Physical and chemical properties of wood 51
2.7.2.2 Microclimate 54
2.7.3 Conditions in wood chip piles 56

2.8 Conclusions 58

2.9 References 60

CHAPTER 3: MANUSCRIPT SUBMITTED FOR PUBLICATION IN

HOLZFORSCHUNG 67

3.1 An Investigation into Pitch Deposition in a Eucalyptus spp. Kraft Pulping

and Bleaching Mill 68
3.1.1 Summary 68
3.1.2 Introduction 68
3.1.3 Experimental 69
3.1.3.1 Wood, pulp and pitch sampling 69
3.1.3.2 Dichloromethane extractions 70
3.1.3.3 Gel permeation chromatography (GPC) 70
3.l.3.4 Derivatization of extracts 70
3.l.3.5 Gas chromatography (GC) of diazomethane methylated extracts 70
3.l.3.6 Gas chromatography ofTMSH methylated extracts 71
3.l.3.7 Gas chromatography - Mass spectrometry analyses (GC-MS) 71
3.1.4 Results and discussion 71
3.l.4.1 The composition ofE. grandis wood extract.. 71
3.1.4.2 The composition of Eucalyptus spp. pulp extract 75
3.1.4.3 Pitch composition 79
3.l.5 Conclusions 80
3.l.6 Acknowledgements 81
3.l.7 References 81

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CHAPTER 4: MANUSCRIPT SUBMITTED FOR PUBLICATION IN

BIOTECHNOLOGY LETTERS 84

4.1 Lipase production and activity of fungi isolated from South African
forests ........................................................•.................................................... 85
4.1.1 Introduction 85
4.1.2 Materials and methods 86
4.1.2.1 Materials 86
4.1.2.2 Fungal strains 87
4.1.2.3 Tween 80 agar plate method 87
4.1.2.4 Tributyrin deep agar diffusion method : 87
4.1.2.5 Assay of enzyme activity with pNPP as substrate 88
4.1.3 Results and discussion 88
4.1.4 Conclusions 91
4.1.5 Acknowledgments 91
4.1.6 References 92

CHAPTER 5: MANUSCRIPT SUBMITTED FOR PUBLICATION IN

BIOTECHNOLOGY LETTERS 94

5.1 Enzymatic Pitch Control in the Kraft Pulping of Eucalyptus grandis 95

5.1.1 Introduction 95
5.1.2 Experimental 96
5.1.2.1 Wood and fungal strains 96
5.1.2.2 Dichloromethane extractions 96
5.1.2.3 Wood chip treatment trial 97
5.1.2.4 Pulping trial. 97
5.1.2.5 Lipase treatment of deposited brown stock pulp extractives 98
5.1.2.6 Gas chromatography of wood and pulp extracts 98
5.1.3 Results and discussion 99
5.1.3.1 Wood chip treatment trial 99
5.1.3.2 Pulping trial. 101

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5.1.3.3 Lipase treatment of brown stock pulp extract 101

5.1.4 Conclusions 103
5.1.5 Acknowledgements : : 104
5.1.6 References 104

CHAPTER 6: GENERAL DISCUSSION AND CONCLUSIONS 106

6.1 . General discussion and conclusions 107

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Chapter 1: Introduction
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1.1 Introduction

The vast majority of paper products are made from cellulose fibres, the· aggregate of
fibres being known as pulps. There are three main pulp categories: mechanical, semi-
chemical and chemical pulp. World pulp production statistics are summarised in Table 1.
The major products are mechanical and chemical pulps, with more than 80% of the latter
being manufactured by the kraft process (Sjostrom, 1993).

Table 1 World pulp production by grade in 1998 (James et al., 1999)

Type of pulp 1000tons

Chemical" 123680
Mechanical 34836
Other pulp" 17020

TOTAL: all pulps 175532

a Includes semi-chemical pulp

b For countries where breakdown by grade is not possible (i.e. not specified or
not estimated) total au put has been listed under the "Other pulp" category

The extractive materials in wood often deposit in pulp and bleach mills to form pitch.
Not all of the extractives are troublesome, most problems occurring in pulping and
papermaking when there are shifts in pH and/or temperature. Triglycerides are
considered to be the cause of pitch deposition (Farrell et al., 1997). During the pulping
process extractives are released from wood and later stick to ceramic and metal parts as
well as the wires of papermaking machines, forming pitch. The pitch also stains the felts
and canvas, and eventually reaches the dryer section. Pitch accumulation can cause paper
spotting and web breaks on the papermachine, causing downtime and reduced product
quality. In effect pitch deposition gives rise to a production cost increase while product
value decreases. The severity of pitch problems vary with wood species. Pitch from
several softwoods is known to cause severe problems. Hardwood pitch, particularly from
tropical hardwood species and Eucalyptus, can also be detrimental (Farrell et al., 1997).

Traditional methods to control pitch problems include seasoning of wood before pulping
and/or adsorption and dispersion of pitch particles in a pulp suspension with chemicals
like talc, dispersants and cationic polymers. Seasoning requires raw wood logs to be left
outdoors for several months or chips to be piled and left for weeks. Seasoning can

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potentially cause losses such as decreased pulp brightness and pulp yield due to
biological deterioration. It also increases working capital costs due to high wood
inventory and land use. Thus, this method is often unacceptable, especially in areas
where space is limited.

Recently two new and different methods of combating pitch, both of which are
biotechnological in basis, have been developed independently and are now used
industrially. One is the development of a pitch control method using the enzyme lipase,
which catalyses the hydrolysis of triglycerides (Fujita et al., 1992). Another is a pitch
control method using a fungus developed in a laboratory from the ascomycete sap-
staining organisms that cause natural ageing. This fungus· is colourless and avoids the
staining and decrease in brightness normally associated with aged wood (Farrell et al.,
1994).

The objective of this investigation was to relieve the pitch problems of Mondi Kraft Ltd.
using enzymatic means. The bleached kraft pulp mill utilises Eucalyptus species and is
situated in Richards Bay, South Africa.

1.2 References

Farrell, RL, Blanchette, RA, Brush, TS, Iverson, S and Fritz, AR (1994), In: Biological
Sciences Symposium, pp. 85-87: Tappi
Farrell, RL, Rata, K and Wall, MB (1997), Advances in Biochemical
Engineering/Biotechnology 57: 198-212
Fujita, Y, Awaji, R, Taneda, R, Matsukura, M, Rata, K, Shimoto, R, Sharyo, M,
Sakaguchi, H. and Gibson, K (1992), Tappi Journal 75: 117-122
James, R, Matussek, R, Janssens, I and Kenny, J (1999), Pulp and Paper International
47: 10-11
Sjostrom, E (1993), Wood Chemistry: Fundamentals and Applications, 2nd edn.,
Academic Press, San Diego

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Chapter 2: Literature review

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2.1 Wood stucture and characteristics with special reference to

Eucalyptus grandis

2.1.1 Wood structure

Softwoods are members of the Gymnospermae and hardwoods are members of the
Angiospermae. Although the terms softwood and hardwood were originally intended to
indicate the relative hardness of the timbers, it is not an appropriate distinction. A wood
anatomical difference is more descriptive. Softwoods and hardwoods have different
cellular structures when viewed with a hand lens or microscope. To provide support and
conducting pathways the softwoods have radial and longitudinal tracheids and the
hardwoods have respectively longitudinal fibres and longitudinal vessels (Siau, 1995). In
both softwoods and hardwoods parenchyma serve a storage function and can occur both
longitudinally or radially. Groups of radial parenchyma cells form rays.

(A) (B)

Figure 1 (A), Transverse and tangential longitudinal faces of a hardwood species. The wood comprises
axial vessels surrounded by longitudinal parenchyma and fibres and radial parenchyma in broad rays.
(B), Schematic structure of a woody cell wall, showing the middle lamella (ML), the primary wall (P), the
outer (51), middle (S2), and inner (S3) layers of the secondary wall, and the warty layer (W) (Cóté, 1967).

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Figure la shows an electron microscope image of a hardwood where all the cells can be
seen. When vessels are grouped predominantly in the earlywood, the pattern is described
as nng porous. When they are distributed throughout the growth ring, it is described as
diffuse porous.

Cellulose fibrils form a skeleton that is surrounded by other substances functioning as

matrix (hemicelluloses) and encrusting (lignin) materials. The length of a native
cellulose molecule is at least 5000 nm corresponding to a chain with about 10000 glucose
units. The smallest building element of the cellulose skeleton is considered to be an
elementary fibril. This is a bundle of 36 parallel cellulose molecules which are held
together by hydrogen bonds. The bonding between· cellulose molecules varies
intermittently between crystalline structures and amorphous regions along the length of
the fibril (Tsournis, 1991). The length of the crystallites can be 100-250 nm and the cross
section, probably rectangular, is on an average 3 nm x 10 nm. Elementary fibrils are
arranged into microfibrils that combine to larger fibrils and lamellae. Disordered
cellulose molecules as well as hemicelluloses and lignin are located in the spaces
between the microfibrils. The hernicelluloses are considered to be amorphous although
they apparently are oriented in the same direction as the cellulose microfibrils. Lignin is
both amorphous and isotropic (Sjostrom, 1993).

Figure lb shows the cell wall structure. The cell wall is built up by several layers,
namely middle lamella (M), primary wall (P), outer layer of the secondary wall (SI),
middle layer of the secondary wall (S2), inner layer of the secondary wall (S3) and warty
layer (W). These layers differ from one another with respect to their structure as well as
their chemical composition. The middle lamella is located between the cells and serves
the function of binding the cells together (Tsoumis, 1991).

The primary wall is a thin layer, 0.1-0.2 urn thick, consisting of cellulose, hemicelluloses,
pectin and protein, embedded in lignin. The microfibrils form an irregular network in the
outer portion of the primary wall; in the interior they are oriented nearly perpendicularly
to the cell axis. The middle lamella together with the primary walls on both sides, is
often referred to as the compound middle lamella. lts relative lignin concentration is

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-Ml

'iP
II,S
n.

Aperture ------.

Torus
Pit membrane
{ Margo

A B c
RAY CELLS

Figure 2 Types of pit pairs. A, bordered; B, Figure 3 Development of tyloses from ray
half bordered; C, simple. ML, middle lamella; cells in oak (Sjostrom, 1993).
P, primary wall; S, secondary wall (Sjostrom,
1993).

high, but because the layer is thin, only 20-25% of the total lignin in wood is located in
this layer (Sjostrom, 1993).

The secondary wall consists of three layers, thin outer (SI) and inner (S3) layers and a
thick middle (S2) layer. These layers are built up by lamellae formed by almost parallel
microfibrils between which lignin and hemicelluloses are located (Butterfield and
Meylan, 1980). The outer layer is 0.2-0.3 J..I.m
thick and contains 3-4 lamellae where the
microfibrils form a helix. The angle of orientation of the microfibril network varies
between 50 and 70° with respect to the fibre axis. The middle layer forms the main
portion of the cell wall. The inner layer is a thin layer of about 0.1 J..I.mconsisting of
several lamellae, which contain microfibrils in helices with a 50-90° angle (Sjostrom,
1993). The warty layer (W) is a thin amorphous membrane located in the inner surface
of the cell wall in all conifers and in some hardwoods, containing warty deposits of an
unknown composition (Butterfield and Meylan, 1980).

Water conduction in a tree is made possible by pits, which are recesses in the secondary
wall between adjacent cells. Two complementary pits normally occur in neighbouring
cells thus forming a pit pair (Wilson and White, 1986). Figure 2 shows the different
types of pit pairs. Radially oriented microfibril bundles form a netlike membrane
permeable to liquids (margo) in the pit. The central thickened portion of the pit
membrane (torus) is rich in pectic material and in pine and spruce also contains cellulose.
Bordered pit pairs are typical of softwood tracheids and hardwood fibres and vessels. In
softwood heartwood the pits can become aspirated when the torus becomes pressed

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against either side of the border (Siau, 1995). In some hardwoods the vessels in the
heartwood can become closed by tyloses (Figure 3) when a special tylose forming
membrane from adjacent parenchyma ray cells enters the vessel through the pits and
forms bubbles in its inside, covering both the vessel wall and the other pits (Walker,
1993).

2.1.2 General information about Eucalyptus grandis

The Eucalyptus species from the family Myrtaceae are hardwood species indigenous to
the eastern part of Australia. Because of widespread planting in South Africa, Eucalyptus
species have become of economic importance to both the South African sawmilling and
pulp and paper industries. They are planted on a fairly large scale in humid regions like
Mpumalanga and Kwazulu/Natal for sawmilling, pulping and secondary wood
processing. Commonly it is known as Rose gum or Saligna gum. It is a valuable wood
on account of its multiple uses. It is very popular for use in constructing mine props and
the treated wood is suitable for electricity poles, telephone poles and fencing. It is also
suitable for frames, panels, furniture, joinery and many other purposes. The colour of the
wood varies from almost white in young trees to dark red in the heartwood of old trees
(Dyer, 1989). The approximate density of 12-15 year old trees is 570 kg/nr' (Dyer,
1989). The density of 5-year-old trees is about 420 kg/rrr' (Bamber, 1985).

2.1.2.1 Macroscopic structure

E. grandis is a diffuse-porous wood. The vessels are solitary, numerous, moderately

large and visible to the naked eye. A distinct diagonal arrangement forms vessel lines.
Growth rings can be distinguished clearly because bands of fibre tissue containing vessels
are alternated by bands without vessels. Numerous rays are visible with a hand lens, as
light lines on a dark background. Parenchyma is visible with a hand lens as sheaths
around the pores (Dyer, 1989).

2.1.2.2 Extractive content of various Eucalyptus species

The extractives of the Eucalyptus genera have a detrimental effect in both pulping and
sawmilling (Chafe, 1987; Hillis and Carle, 1959; Nelson et al., 1970; Yazaki et al.,

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1993). Extractives of Eucalyptus increase chemical consumption and impair the colour
and brightness of unbleached pulp (Nelson et al., 197Ó). Table 1 gives the
alcohol/benzene/hot water extractive content of twelve species of the Eucalyptus genus.
The extractive content of these species is high with the exception of E. diversicolor,
E. delegatensis and E. marginata. E. globulus has a low dichloromethane extractives
content ofO.3 % (Wallis and Wearne, 1999).

It is generally assumed that lignin is responsible for the brown colour of raw pulp. In
Eucalyptus the polyphenols produce much more colour than does lignin during mild
alkaline pulping conditions (Hillis, 1971).

Table 1 Alcohol/benzene/hot water extractive content for

twelve species of the Eucalyptus genus (Hillis, 1962)

Species Extractive content (%)

E. diversicolor 7.6
E. delegatensis 8.3
E. longifolia 22.8
E. marginata 6.4
E. microcorys 17.5
E. obliqua 29.4
E. paniculata 19.4
E. polyanthemos 25.2
E. regnans 16.9
E. robusta 18.0
E. sideroxylon 34.1
E. sieberiana 19.3

2.1.2.3 Sapwood and Heartwood distribution

Extractives are usually present in greater amounts in the heartwood than in the sapwood
and the change in extractive content can be very abrupt at the heartwood periphery
(Breuil et al., 1994; Gao et al., 1995; Hillis, 1971; Holl and Poschenrieder, 1975; Sitholé
et al., 1992; Yanchuk et al., 1988). The distribution of extractives in the stem is
consequently mostly dependent on the ratio between sapwood and heartwood.

Studies done in Australia on 9Y2year old Eucalyptus grandis show that the heartwood
constitute 45% of the volume of the lower half of the stem. This percentage decreases
from bottom to top (Wilkins, 1991). It has also been shown that the stems of 5 and 50-
year-old Eucalyptus grand is have respectively 25% and 80% heartwood (Bamber, 1985).
There is an indication that the number of sapwood rings in a species is a heritable feature.
 Stellenbosch University http://scholar.sun.ac.za

For example, in the Eucalyptus grown in Australia there are about 5-6 sapwood rings for
the greatest portion of the genus (Hillis, 1971). Bamber (1985) also reported that
heartwood formation begins fairly early in Eucalyptus, probably in the fourth or fifth year
as measured from the cambium. While in softwoods there is a striking decrease in the
moisture content of the wood during heartwood transition, there is little change in the
Eucalyptus genera (Bamber, 1985). Eucalyptus heartwood tends to be more acidic
(± pH 4) than the sapwood (± pH 5) (Bamber, 1985).

The extractive content of heartwood from young fast grown Eucalyptus is, however, low
when compared to the heartwood of older slow grown trees (Hillis, 1971). Fast grown
trembling aspen (Populus tremuloides) also has a lower extractive content (Yanchuk et
al., 1988). Consequently the extractive content difference between the heartwood and
sapwood of young fast-grown Eucalyptus may be less pronounced. The lipid
composition of extractives may however differ from sapwood to heartwood. In
Lodgepole pine (Pinus contorta Dougl.), triglyceride content decrease from sapwood to
heartwood while fatty acid content increases (Gao et al., 1995). Similar results were
reported for aspen heartwood and sapwood (Breuil et al., 1994). It has been suggested
that the reason for the differences is the hydrolysis of triglycerides during the formation
of heartwood. This reaction effects an increase in fatty acids and mono-, and diglycerides
(Saranpaa and Nyberg, 1987).

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2.2 The extractives of wood

A large number of wood components, although usually representing a mmor mass

fraction, are soluble in neutral organic solvents or water. They are called extractives.
The composition of wood is illustrated in Figure 4. Extractives can be regarded as non-
structural wood constituents, almost exclusively composed of low molecular mass,
organic compounds (Jane, 1970). Extractives comprise an extraordinarily large number
of individual compounds of both lipophilic and hydrophilic types. Extractives occupy
certain morphological sites in the wood structure. Lipids are concentrated in ray
parenchyma cells while phenolic extractives are present mainly in heartwood and in bark.

Figure 4 The composition of wood (Fen gel and Wegener, 1989).

Different types of extractives are necessary to maintain the diversified biological

functions of the tree. Fats constitute the energy source of the wood cells, whereas lower
terpenoids, resin acids, and phenolic substances protect the wood against microbiological
damage or insect attacks. The term "resin" is used as a collective name for the lipophilic
extractives (with the exception of phenolic substances) soluble in non-polar organic
solvents but insoluble in water (Sjostrom, 1993). Dichloromethane is more selective for
extracting lipids than methanol or acetone (Wallis and Wearne, 1999).

Table 2 gives the typical composition of softwoods and hardwoods. Extractives make
out, on average, only 2-8% of the oven dry mass of hardwoods and 1-5% of softwoods.
Extractives constitute a valuable raw material for making organic chemicals and they
play an important role in the pulping and paper making processes (Sjostrom, 1993).

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Table 2 Typical composition of softwoods and hardwoods (Walker,

1993 .

Constituent Softwood (%) Hardwood (%)

Cellulose 42±2 45 ±2
Hemicelluloses 27 ±2 30 ±5
Lignin 28 ±3 20 ±4
Extractives 3±2 5±3

2.2.1 Terpenes and terpenoides

The terpene and terpenoide fraction of extractives is built up by a building block called
an isoprene unit. Their number of isoprene units present is used to classify the terpenes.
Figure 5 shows the basic structures of some terpenes. To date no mention has been made
of the presence of resin acids, which are diterpenes, in hardwoods. It is, however, an
abundant substance in softwoods (Biermann, 1993).

Number of
Name 5C-units Structure

Isoprene
(basic unit)
1 x SC ~(

Monoterpenes 2 xSC
2
Sesquiterpenes 3 x SC en
Diterpenes 4 x SC
~

Figure 5 Basic structures of several terpenes

(pengel and Wegener, 1989).

2.2.2 Fats, waxes and their components.

A fat is an ester of glycerol and one or more fatty acids. A wax is an ester of a long chain
fatty alcohol and fatty acids, thus its molecular mass is much higher than that of a fat.
Examples of fats, waxes and their components are given in Figure 6. The fats are glycerol
esters of fatty acids and occur in wood predominantly as triglycerides. More than thirty
different fatty acids, both saturated and unsaturated, have been identified in softwoods

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and hardwoods (Fengel and Wegener, 1989). Examples of the most common fatty acids
are shown in Table 3.

Fats H H
H-t--O-CO-R I
Hj--O-CO-RI
H-{--O-CO-R2 H -OH
H-C--O-CO-R3 H -OH
I I
H H
Triglyceride Monoglyceride
Waxes
Clli -(Clli ).rO-CO-(Clli )m-Clli

Fatty acids
~COOH

Hexadecanoic (palmitic) acid

~COOH

14-Methyl hexadecanoic acid

~COOH

Ocladecanoic (stearic) acid

~COOH

Ocladecaenoic (oleic) acid

~COOH

Ocladecadienoic (linoleic) acid

~COOH

Ocladecatrienoic (linolenic) acid

Alcohols C"H"OH Eicosanol (Arachidic alcohol)

C"H"OH Docosanol (Behenic alcohol)

C"H ..OH Tetracosanol (Lignoceric alcohol)

Figure 6 Fats, waxes and their components

(Fen gel and Wegener, 1989).

Table 3 Abundant fatty acid components of fats and waxes (Sjostrom, 1993)

Trivial name Systematic name Chain length

Saturated
Palmitic Hexadecanoic C16
Stearic Octadecanoic C18
Arachidic Eicosanoic C20
Behenic Docosanoic C22
Lignoceric Tetracosanoic C24

Unsaturated
Oleic cis-9-0ctadecanoic C18
Linoleic cis,cis-9,12-0ctadecadienoic C18
Linolenic cis, cis, cis-9, 12, 15-0ctadesatrienoic C18
Pinolenic cis, cis, cis-5, 9, 12-0ctadesatrienoic C18
Eicosatrienoic cis, cis, cis-5, 11, 14-Eicosatrienoic C20

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Fats and waxes (esters) are hydrolysed during kraft pulping. The fatty acids that are
liberated can be recovered together with resin acids as soap skimmings from the black
liquor (Biermann, 1993).

2.2.3 Phenolic substances

Extractives contain a large number of phenolic substances of simple and more complex
structure. Those of more complex structure are the lignans and quinones. Some examples
of the simple structure and complex phenols are displayed in Figure 7, Figure 8 and
Figure 9. Phenolics give wood its resistance against microbial decay (Walker, 1993).

~,&oc~ ~
o
2,6-OimethoX)'
benzoquinone
o
Lepechot ~
~ 1'1
SWlapaldahyd. Feruk aeid Propbgu.ï.eone Eu~WloI

o
p.-Oehydrolapachone

~co~o~
Tectcquinone o ~

Syringald.hyde Gualaco p-Cresol o

4-Melhoxy dalbergtone

Figure 7 Some simple phenols Figure 8 Kinones of hardwoods

(Fengel and Wegener, 1989). (Fengel and Wegener, 1989).

,OH

OH
Thornaslcadd
(Thomasldioic acid)

Figure 9 Well-known lignans of

hardwoods (Fengel and Wegener,
1989).

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2.2.4 Tannins

Two types, hydrolisable and condensed tannins, exist. Among the condensed tannins are
flavonoids, a substance of widespread occurrence in the plant kingdom. Hydrolisable
tannins can be viewed as polyesters of gallic acid and its dimers (Figure 10). Examples of
the hydrolisable tannins and flavonoids are displayed in Figure 11 and Figure 12 (Fengel
and Wegener, 1989).

Basic structure OH-(OCH3)·position

3,7,3',4'

0
OH OH

"O*OH HO*OH OH
7

•
Qi¢'
.
0
, "
3,5,7,4'
3,7,3',4',5'
3,5,7,3',4'
3,5,7, 'Z, 4'

I J=O Plavenes

o=c
O=~OH COOH

ód¢'
3,7,3',4'
Gallic acid 3, 4, 7, 3', 4'
7 , "
OH HOOC OH 3,5,7,3',4'
• , 3, 4, 5, 7, 3', 4'
Ellagic acid Digallic acid
Flavanes

Figure 10 Galluacid and its dimers (Fengel and Figure 11 Some flavonoids
Wegener, 1989), (Fengel and Wegener, 1989),

OH OH

V.sel*'-

Clst.l.gIn

O~&"
HO

Figure 12 Some hydrolisabie tannins (Fen gel and

Wegener, 1989).

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2.2.5 Other substances present in extractives

Saturated hydrocarbons (Fengel and Wegener, 1989) and free or bonded amino acids
(Hagglund, 1951) are also found in wood. The saturated hydrocarbons occur in a
homologous range from Cll to C33 and in addition to the amino acids, there are also
other nitrogen containing substances.

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2.3 The kraft pulping process

Pulp is the basic product of wood, predominantly used for papermaking, .but it is also
processed to various cellulose derivatives, such as rayon silk and cellophane. The main
purpose of wood pulping is to liberate the fibres, which can be accomplished either
chemically or mechanically or by combining these two types of treatments. The common
commercial pulps can be grouped into chemical, semi chemical, chemimechanical, and
mechanical types (Table 4). Chemical pulping is a process in which lignin is removed so
completely that the wood fibres are easily liberated on discharge from the digester or at
most after a mild mechanical treatment. Practically all of the production of chemical
pulps in the world today is still based on the sulphite and sulphate (kraft) processes, of
which the latter dominates. Pressurised alkaline cooking systems at high temperatures
were introduced in the 1850's. In 1870 AK. Eaton patented the use of sodium sulphate
instead of sodium carbonate.

Table 4 Commercial pulp types (Sjëstrëm, 1993)

Pulp type Yield (% of wood)

A. Chemical 35-65
Acid sulphite
Bisulphite
Multistage sulphite
Kraft
Polysulphide-kraft
Prehyd rolysi s-kraft
Soda

B. Semichemical 70-85
NSSC
Green liquor
Soda
C. Chemimechanical 85-95
Chemithermomechanical (CTMP)
Chemigroundwood (CGW)

D. Mechanical 93-97
Stone groundwood (SGW)
Pressure groundwood (PGW)
Refiner mechanical (RMP)
Thermomechanical (TMP)

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Calender stack

Head A +-- Wood chips

box
U Digester

Finished I
paper t
Beater Ó
c-9, ------ ~g/{g~~ser I~I+--
..... V Blowtank

D• Brownstock
tank

!
~

c:FE~t~~k+--~ ~~ ~~ ~~ ~~ ~
Knotters, rifflers L.....J ~ ~ ~ ~
and screens
o E o E CD

Figure 13 A schematic illustration of the kraft pulping process. Adapted from

Macdonald (1969). A CDEDED bleaching process is illustrated.

The kraft process has almost completely replaced the old soda process because of its
superior delignification selectivity resulting also in a higher pulp quality. More than 80%
of the chemical pulp produced in the world is kraft pulp (Sjostrom, 1993). A schematic
illustration of the kraft pulping process is given in Figure 13.

2.3.1 Terminology

2.3.1.1 White liquor

White liquor is the solution added to the chips in the digester and consists of sodium
hydroxide, sodium sulphide, water and sodium hydrosulphide (Macdonald, 1969).

2.3.1.2 Black liquor

The volatile wood extractives, consisting mainly of lower terpenes, are recovered during
kraft pulping. The resin acids and fatty acids are recovered as tall soap. After
acidification with sulphuric acid the resulting tall oil is finally purified and fractionated
by distillation. The remaining kraft spent liquor (black liquor) contains organic
constituents in the form of lignin and carbohydrate degradation products (Sjostrom,

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1981). Weak black liquor from the brown stock washers normally contains 12-18%
solids. It contains some residual alkali not consumed during the cooking and has a pH of
at least 12. Much of the sodium in the black liquor is in the form of sodium salts of
organic acids. These acids are formed when lignin arid carbohydrates dissolve during
digesting. Inorganic components in the liquor are sodium carbonate, sodium sulphate,
sodium thiosulphate, and sodium hydrosulphide. The sodium salts of resin and fatty
acids are an important part of the black liquor. They are soaps that can be recovered and
sold as the by-product tall oil (Mimms, 1989). Black liquor is used as a diluent of white
liquor in amounts varying from 10 to 60 per cent of the total volume of the liquor charge.
White liquor is added to the chips in the digester at the start of the process (Stephenson,
1950). The composition of softwood black liquor is given in Table 5.

Table 5 The composition of softwood black liquor (Sjostrom, 1981)

Component Content Composition

(% of dry solids) (% of hydroxy acids)

Lignin 47
Hydroxy acids: 28
Lactic 15
2-Hydroxybutanoic 5
2,5-Dihydroxypentanoic 4
Xyloisosaccharinic 5
a-Glucoisosaccharinic 15
p-Glucoisosaccharinic 36
Others 20

Formic acid 7
Acetic acid 4
Extractives 5
Other compounds 9

2.3.1.3 Kappa number

The kappa number gives an indication of the lignin content of the pulp and is used for
comparing different samples of pulp coming from the same wood species or wood
species mix (Biermann, 1993).

2.3.2 The digestion process

There are two basic digester designs: batch digesters and continuous digesters. The
principal operations in batch digesting include chip packing and steaming, liquor filling,

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slow temperature rise to assure complete penetration of the chips by liquor, relief of
gases, cooking at maximum temperatures, relief of pressure, and blowing the digester
(Mimms, 1989).

The digestion process is essentially the treatment of wood, in the form of chips, in a
pressure vessel called a digester under controlled conditions of temperature, pressure, and
time, with a liquor composed mainly of an aqueous solution of sodium hydroxide or of a
mixture of sodium hydroxide and sodium sulphide. The sodium hydroxide is responsible
for delignification (Stephenson, 1950). The sodium hydroxide and sodium sulphide are
considered to be the- only active chemicals. The cooking is done at a temperature of 170-
180°C for one to two hours in both batch and continuous digestors (Mimms, 1989). The
sulphide reacts with water to give sodium hydroxide (NaOH) and sodium hydrosulphide
(NaSH) (Equation 1).

Equation 1 The reaction of sodium sulphide with water (Mimms, 1989)

Na2S + H20 NaOH + NaSH

The reaction is reversible; thus the concentrations of the different substances will reach a
certain equilibrium state. As the cook proceeds, the sodium hydroxide would be
consumed and the concentrations would shift, thus tending to increase hydrolysis of
sodium sulphide to regain equilibrium (Stephenson, 1950). The sulphidity of the liquor
in sulphate mills is usually in the range of 20 to 30 per cent (Stephenson, 1950).
Constituents present in the cooking liquor are given in Table 6. Small amounts of sodium
chloride, potassium salts, silicate, and calcium are also present.

All constituents of the wood are simultaneously attacked in differing degrees in the
digestion process. Most of the extractives, fats, resins, etc., are saponified and dissolved
in the cooking liquor. Saponification is the chemical process by which esters (e.g. mono-
di- and triglycerides) are broken down into alcohols and fatty acids. Triglycerides are
saponified by sodium hydroxide either partially or totally (Equation 2).

Some substances belonging to the extractives group are very difficult to dissolve and
remain in the pulp. They are referred to as non-saponifiables (Stephenson, 1950). An
example of a non-saponifiable is wax.

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Equation 2 The reaction of triglyceride with sodium hydroxide (Stephenson, 1950)

glycerol + 3 x fatty acid

Table 6 The constituents of cooking liquor (Stephenson, 1950)

Name Formula Concentration range

(gil as Na20)

Sodium hydroxide NaOH 81-120

Sodium sulphide Na2S 30-40
Sodium carbonate Na2C03 11-44
Sodium sulphate Na2S04 4.4-18
Sodium sulphite Na2S03 unknown (small amount)
Sodium thiosulphate Na2S203 4.0-8.9

2.3.3 The blow tank

The digester contents are blown tangentially into the top of the blow tank, the stock
dropping into the tank and the steam and gases escaping from the top vent to a steam
condenser (Stephenson, 1950). It is not until the blowing process that the chips are fully
separated into fibres, if the kappa number has been lowered sufficiently. During blowing
the temperature and pressure are lowered quickly. The resuIt is that the liquor inside the
chips starts to boil and the resulting pressure is enough to separate the fibres in the chips
(Mimms, 1989).

2.3.4 Brown stock washing

The brown stock pulp coming from the digester has to be washed to recover process
chemicals. If chemical pulps are not properly washed, foaming will be a problem,
additional make-up chemicals will be needed, more bleaching chemicals will be required,
and additional pollutants will result (Biermann, 1993).

2.3.5 Bleaching

The goal of bleaching is to produce whiter pulp. Some of the bleaching agents used are
listed in Table 7. Between bleaching steps there are alkali extractions to wash out lignin
fragments. The following sequence is an example of a bleaching process: bleach with
chlorine, extraction with sodium hydroxide, bleach with chlorine dioxide, extraction with
sodium hydroxide and bleach chlorine dioxide (CEDED) (Haylock, 1974).

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Table 7 Nomenclature of bleaching (Walker, 1993)

Stage Description

o Delignification with oxygen

Z Delignification with ozone
C Delignification with chlorine, Cl2
H Delignification with hypochlorite, ocr
o Delignification with chlorine dioxide, CI02
E Extraction of lignin fragments with NaOH

2.3.6 Knotters, riffiers and screens

Knots are the uncooked wood particles, which are still not separated. The screens, rimers
and knotters serve to isolate and break down the knots into smaller particles (Mimms,
1989).

2.3.7 Refining

Refiners mechanically enlarge the specific area of the fibres by causing fibrillation and
cell wall collapse. The purpose of this is to better cohesion between fibres and thus
produce stronger paper. Different types of paper can be produced by controlling the
degree of refining (Haylock, 1974).

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2.4 Pitch

2.4.1 Defining pitch

Pitch problems arise at screens, filters, refining equipment, pulp washers and the paper
machine (Macdonald, 1969). It causes clogging of equipment and results in higher
production costs of paper. Allen (1988a) uses the definition of pitch as "the material in
wood or wood pulps which is insoluble in water, but soluble in neutral organic solvents".
This definition is also used in the book edited by Hillis (1962).

2.4.2 The composition of pitch

There are many variables (species used, specie mix, period of wood storage, stage of
process, etc.) determining the composition of pitch. Thus the composition of one pitch
sample would never match the other. However, an analysis of pitch does give an idea of
what is to be expected. Some substances could be recognisable in all samples in larger or
smaller quantities. Ohtani and Shigemoto (1991) analysed five pitch samples from four
different kraft pulp mills in Japan. Suckling and Ede (1990) performed a 13C nuclear
magnetic resonance analysis on pitch from a pulp and paper mill that produces
mechanical and kraft pulps.

Ohtani and Shigemoto (1991) analysed high molar mass components of machine and
kraft pitch. They defined kraft pitch as pitch that deposits after pulping and before paper
machine operations while machine pitch refers to pitch that deposits in paper machine
operations. The gel permeation chromatogram (GPC) of one of the kraft pitches newly
collected by these authors is displayed in Figure 14.

The machine pitch deposited in the Uhle box of a kraft paper machine was also examined
by GPc. The fraction with molar mass over 1000 accounted for 85% of the total CHCh-
solubles, similar to the situation in kraft pitches. The pitch was divided into four
fractions according to molar mass with the same methods as used in the case of kraft
pitches (Figure 14). Fraction IV (Figure 15) amounted to 15.5% of the CHCh-solubles.
Fraction III (Figure 16) accounted for 22.3% of the CHCh-solubles. Fraction II

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accounted for 14.5% and fraction I 47.5% of CHCh-solubles. Fractions I and II were
identified as polymerised fatty and resin acids.

High amounts of aluminium were found in machine pitch due to the use of alum. Based
on chemical composition, the mechanism of pitch deposition seemed to differ for these
two pitches. The high molar mass fraction of machine pitches was found to be a complex
consisting of aluminium and acidic wood extractives. Irrespective of difference in mill
locations and pulping stages, the high molar mass fractions of most of the laaft pitch
deposits gave similar carbon-IJ NMR spectra and were characterised as polymerised
aliphatic hydrocarbons (Ohtani and Shigemoto, 1991).

In their investigation Suckling and Ede (1990) dissolved the pitch deposit in chloroform.
Seventy nine percent of the dry mass of the deposit was soluble in chloroform, the
remainder consisted largely of paper fines. A NMR analysis showed that the pitch was
largely composed of organic compounds present in wood extractives. The pitch sample

#'~## Abetadien. Abietatrian.

~of2 f2 W~BQ5'
a.-C.dinol r-e.dino! ó-Cadinol Manoal Biformene

6000 2000 300

Molar mass
100
~~~~q
Figure 14 GPC of a kraft pitch sample Figure 15 Compounds from fraction
(Ohtani and Shigemoto, 1991). IV (Ohtani and Shigemoto, 1991).

Oihydrocydoeucalenol Sitostanol

Figure 16 Compounds from fraction III

(Ohtani and Shigemoto, 1991).

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contained 34% triglycerides by mass, 3% fatty acids and 29% resin acids. No significant
amounts of fatty acid esters other than triglycerides were present. The acids existed in salt
form in the pitch. The balance of the chloroform soluble material presumably includes
unsaponifiable extractives components, polymerised eind oxidised resin, other added
organic components such as defoamers or release aids and inorganic salts.

2.4.3 Mechanisms of deposition

2.4.3.1 Deposition due to polymerisation

Ohtani et al. (1986) investigated the chemical aspects of pitch deposits in five Japanese
kraft pulp mills and found that most of the pitch deposits contained high molecular mass
components in rather high proportions. Polymerised alkyl hydrocarbons found in pitch
deposits and wood chips were assumed to be derived from fatty acids and alcohols, which
exist in the wood. In a simulation to understand the chemical reactions of fatty acids and
alcohols in the interior of wood some experiments were conducted where these
compounds were heated in an oven at 105°C. The results are shown in Figure 17.
Stearyl alcohol and stearic acid underwent very little change. Oleyl alcohol, oleic acid,
and linoleic acid, all of which have one or two double bonds, gradually changed into
higher molecular mass substances, and they easily underwent condensation reactions.
The more double bonds they had, the faster the condensation reaction proceeded. Fatty
acids are polymerised faster than fatty alcohols. It was found that polymeric materials in
the pitch from four mills were condensed substances of unsaturated fatty acids or
alcohols. The condensation reaction was assumed to proceed during outside chip storage.
The Eucalyptus woods (mainly from Tasmania) pulped in Japanese mills contained large
amounts of polyphenols with lesser amounts of fatty acids or fatty alcohols. According
to Ohtani et al. (1986) most of the extractives from Eucalyptus spp. are dissolved in the
black liquor and are easily removed from pulp by washing. The polymerised alkyl
hydrocarbons derived from fatty acids and fatty alcohols are assumed not to be
eliminated to any extent by cooking or bleaching and therefore induce depositions of
pitch. Long-term storage of woods that contain large amounts of unsaturated fatty acids
and fatty alcohols may be unfavourable for pulping because of the formation of
polymeric substances, which can cause pitch deposits (Ohtani et al., 1986).

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100 --__ -_ __ Oleyl alcohol

.... .... ,
,,
'E \
~ 50
ot:

..«:
5 10 50 100

5 10 50 100

100 - -c-,

' ........
, , Linoleic acid
, \
\
\. -c
',/
_ ..-., '" "".' <;

5 10 50 100
Aging Time (h)
>6000 MM
-- 6000-2000 MM
--_. <2000 MM

Figure 17 Changes in unsaturated fatty

acids and alcohols during ageing (Ohtani et
a/., 1986). Key: MM molecular mass. =

2.4.3.2 Metal soap deposition

The saponifiable triglycerides and other lipids break down into fatty and resin acids and
alcohols in the extreme conditions of the digester. Under alkali conditions a resin or fatty
acid tends to lose a hydrogen atom and becomes a soap anion. When binding with a
metal ion it will form a soap (Equation 3).

Equation 3 The reaction of a metal ion with a soap anion (Allen, 1980)

Mn+ + nSoap- ~ M Soapril-

(Metal Ion) (Soap Anion) (Metal Soap)

The resulting metal soap, which is sticky and insoluble, is sometimes found in relatively
large concentrations in pitch deposits. The metal-to-soap bond appears to be strong, so
that the reaction depicted in Equation 3 is effectively irreversible, even when moderately
acidic conditions (e.g., pH 4) are created. Concentrations of metal ions and of fatty and

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resin acids in mill white waters are usually high compared to other components. The
metal ion that is most frequently involved in soap deposition in the kraft pulp mill, is
calcium (Allen, 1988 b). A cationic polymer spray is available on the market to combat
pitch deposition. It forms a complex with the anions and thus it hinders pitch deposition
where it is applied (Aston et al., 1992). The pitch particles are then fixated onto the
fibres, thus reducing the concentration of pitch particles in the process water (Hassler,
1988).

The extent of the reaction described in Equation 3 is controlled by the presence or

absence of soap anions in the white water and this, in tum, is controlled by pH. A pH of
6.5 and higher would result in a high concentration of anions·. Allen (1988b) stated that,
in all likelihood, the high temperature in the digester is responsible for a somewhat lower
pH at which dissolution begins than would be found at lower temperatures. Metal soap
deposition can constitute 30% of the deposited material in a part of a kraft mill where the
pH is over 10 (Allen, 1988b). A mechanism for the formation of calcium soaps has been
advanced where the soap anions are adsorbed onto calcium carbonate, which is also
present in unbleached kraft pulps, and are then converted into calcium soaps. This
mechanism is assumed to be more acceptable because the small amounts of dissolved
calcium ion in unbleached kraft pulp appear to be unavailable for reaction because they
are tied up with lignin (Douek and Allen, 1983).

A B c
Figure 18 Schematic diagrams of: (a) a soap
anion, (b) a micelle, (c) a micelle solubilising
unsaponifiable material in its interior (Allen, 1988a).

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If wood resm does not contain much unsaponifiable material the extent of pitch
deposition is less. The saponifiable material becomes soaps which aid in removing the
remainder of the resin, the unsaponifiable or non-soap portion, from the pulp. Figure 18a
is a schematic representation of a soap molecule, in solution, with its hydrocarbon tail
and carboxylate anion head group. Above a certain concentration, called the critical
micelle concentration, these soap anions cluster together with their hydrocarbon tails on
the. inside and the head groups on the outside to form micelles (Figure 18b). The
importance of this phenomenon, which always occurs in kraft digesters, is that these
micelles are capable of solubilising some of the unsaponifiable material. The
unsaponifiable material dissolves in the centres of the micelles (Figure 18c), where the
hydrocarbon tails are concentrated. This phenomenon is the basis of detergency and is an
important aspect of resin removal during kraft cooking. It is therefore appropriate to
consider the ratio of saponifiables to unsaponifiables in the extractives of the wood. This
is a good way to compare the deresination qualities of various different kinds of wood. It
is estimated that a ratio of less than three will lead to poor deresination (Allen, 1988a).
The waxes in hardwood resin are very difficult to saponify during alkaline pulping, but it
can be enzymatically hydrolysed during storage of the wood. Thus, some of the
unsaponifiables which are the most difficult to remove are hydrolysed during storage
(Allen, 1988a).

2.4.3.3 Resin transfer from fibres

Resin transfer from fibres to equipment occurs when fibres in a flowing pulp suspension
scrape or brush against the surfaces of equipment. In areas of intense hydrodynamic
shear, such as wires and comers, resin is transferred from fibre surfaces. The deposited
resin then flows along the surface to areas of less shear (Allen, 1980).

2.4.3.4 Fines and fibre deposition

When pressure is applied to a partially dried pulp such as at the press roll, it is possible
for fines and fibres to adhere to the surfaces of the rollers because of the sticky resin on
the surfaces involved (Allen, 1980).

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2.4.3.5 Transfer of resin to the felts in press nips

In the press section, the white water is squeezed from the paper into the felt (Figure 19)
and carries with it minor amounts of fibres, parenchyma cells, and dispersed resin.
Deposition can occur during the felt dewatering process on the Uhle box lips (Allen,
1980).

Figure 19 Transfer of resin from

paper to felt in a press nip (Allen,
1980).

2.4.3.6 Deposition due to evaporation

Resin would be left behind when white water containing resin and water is evaporated.
The resin will then form a deposit on the surface on which it was left behind. An
example of this is the evaporation of water off the dryer rolls (Allen, 1980).

2.4.3.7 Deposition of resin due to film formation

The dispersed resin in a storage tank where the process liquid is not moving would
emulsify and then rise to the surface of the tank. There it forms a ring on the tanks,
which frequently breaks off and disintegrates to form deposits downstream in the
equipment or on the finished product. Resin particles also attach themselves to foam or
air bubbles rising from the bottom of the tank. Thus the resin is taken to the surface, also
forming a ring, and the result is the same as previously mentioned (Allen, 1980).

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2.4.3.8 Resin deposition in refiners and beaters

During the refining process (beater, refiner, etc.) the stock is subjected to a treatment
severe enough to crush many parenchyma cells and emulsify the resin liberated both from
the cells and from the resin pockets and canals. The resin then deposits inside this
equipment and may break off later to cause problems downstream (Allen, 1980).

2.4.3.9 Temperature and pH fluctuations

Sudden temperature drops can decrease the solubility of pitch and cause it to precipitate.
Changes in pH can also cause pitch to become less soluble and cause it to deposit in the
machine system (Laubach and Greer, 1991).

2.4.3.10 Co-deposition of extraneous substances

Any material suspended in the pulp will adhere to a pitch deposit if it comes in contact
with it. For example, ray cells, fibres, sand, bark, and ink particles from recycled
newspaper all have a tendency to collect in pitch deposits, and these materials can on
occasion affect the volume, stickiness, viscosity, and appearance of the deposit. Certain
defoamers can cause deposition even in the absence of resin (Allen, 1980).

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2.5 Triglyceride breakdown with lipase

Triacylglycerol acylhydrolase (EC 3.1.1.3) is also known as triacylglycerol lipase or only

as lipase. It is an enzyme produced by humans and animals in the digestion canals as well
as by microorganisms for the breakdown of mainly triglycerides, but also di- and
monoglycerides. It can also cause ester synthesis in the presence of microamounts of
water. Figure 20 illustrates all the reactions lipase can catalyse. Lipase enzymes are of
common occurrence in fungi and are of two types: those for specific fatty acids chain
length and those exhibiting positional specificity. The latter enzymes are further of two
kinds. Type "a" hydrolyses a specific position in the acylglycerol molecule, whereas type
"b" is non-specific and hydrolises all fatty acyl ester bonds (Chopra and Khuller, 1984).
Lipase production is inducible in fungi by growing the organisms in a triglyceride-
containing medium (Akhtar et al., 1977, 1980). The enzyme may be membrane bound,
as in Saccharomyces cerevisiae (Schousboe, 1976), or extracellular, as in Penicillium
roqueforti (Lobyreva and Marchenkova, 1980) and Rhizopus japonicus (Aisaka and
Terada, 1981).

Triglyceride

1~
Diglyceride + Fatty acid

1~
Monoglyceride + Fatty acid

Glycerol + Fatty acid

Figure 20 Reactions catalysed by lipase

(Ghosh et al., 1996).

Lipase is an important enzyme in industry servmg as a biocatalyst for fat and oil
processing in medicine, food additives, diagnostic reagents and detergents. The lipases
used are usually of fungal or bacterial origin. Triglycerides have been shown to be major

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contributors to pitch deposition (Allen, 19'77 Fujita et al., 1992; Suckling and Ede, 1990).
Some success has been achieved in reducing the triglyceride content of wood, and
therefore a reduction in pitch formation, by inoculating wood chips with white-rot fungi
or by adding lipase to pulp (Blanchette et al., 1992; Brush et al., 1994; Fischer and
Messner, 1992).

2.5.1 Lipase structure

Lipase, like any other enzyme, is a protein which consists of a single or a few polypeptide
or amino acid chains, forming a complex folded structure. Twenty different amino acids
exist and these form the polypeptide chains. The folds of the chain or chains are stabilised
by disulphide bonds. The part of the enzyme that is responsible for its biocatalytic
potential is called the catalytic site. Only a substrate molecule, like triglyceride in the
case of lipase, which could fit into the enzyme conformation to get close enough to the
catalytic site, can be attacked. The chemical mechanism of an enzyme is induced by only
a few (e.g. three in the case of lipase) amino acids.

2.5.2 Hydrolysis reaction mechanism

The catalytic site of lipases usually consists of a Ser-His-Asp (serine, histidine, aspartic
acid) amino acid triad (Brady et al., 1990; Derewenda, 1994; Isobe and Nokihara, 1993;
Jaeger et al., 1993; Yamaguchi et al., 1991). However, a Ser-His-Glu triad has also been
identified for the lipase of the fungus Geotrichum candidum (Derewenda, 1994; Schrag
and Cygler, 1993). The order in which Ser-His-Glu is written does not indicate any
structural or chemical sequence.

Figure 21 shows the molecular structure of serine, histidine and aspartic acid. The
chemically functional groups can catalyse the hydrolysis of ester bonds when the amino
acids are positioned correctly, as in the case of lipase enzymes.

The following description of the mechanism of the catalytic triad is simplified, as it has to
serve as a model for all lipases. The details about the angles and orientations of the
different amino acids, substrate molecules, etc. differ for each type of lipase molecule.

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The catalytic triad of serine proteases is' chemically analogous to that of lipase and
hydrolises its substrate by a mechanism very similar to that used by lipases (Brady et al.,
1990; Winkler et al., 1990). Chymotrypsin is a serine protease. Figure 22 shows the
mechanism of the chymotrypsin catalytic triad and the following discussion describes the
mechanism with the help of this figure.

Some interactions between the catalytic triad are necessary to create the potential to
hydrolise ester bonds. One of these is the hydrogen bond that is formed between the
nucleophilic serine and histidine (Derewenda, 1994; Dressler and Potter, 1991) (Figure
22a). Another one of the vital interactions in any triad is that of the histidine imidazole
(the ring structure on the side chain of histidine) and a carboxylic acid. Generally a
hydrogen bonded Asp-His pair is found in the catalytic centers of a variety of enzymes
(Figure 22a). The two bonded side chains assist in a nucleophilic attack during
hydrolysis either directly by activating a serine hydroxyl (as in Figure 22a), a cysteine
thiol, or a water molecule, or indirectly through a metal ion that polarizes the carbonyl
attacked by a nucleophile (Derewenda, 1994). These two interactions appear to be
necessities for the creation of a nucleophilic active site.

The net effect of these two interactions is that hydrogen is transferred to the histidine, so
creating a nucleophilic serine. The nucleophilic serine then attacks the ester bond and
forms a tetrahedral intermediate (Figure 22b). The tetrahedral intermediate then
collapses (Figure 22c), the ester bond breaks up and an acyl-enzyme intermediate is
formed (Figure 22d). Histidine then donates a hydrogen atom to the released part of the

OH
I
HN"""'\\
~NH+
o
,/I
0-

CH2 CH2 CH2

I I I
+H3N- C -COO- +H3N- C -COO- +H3N- C -coo-
I I I
H H H

Ser His Asp

Figure 21 Serine, histidine and aspartic acid (Mathews and

Holde, 1990).

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substrate and returns to its original base state. Now the acyl-enzyme intermediate is
broken down through the Asp-His couple which draws a hydrogen atom away from a
nearby water molecule. A hydroxyl group is left over and it attacks the C=Q group, a
carboxyl group is formed and the remaining portion of the original substrate is released
from the serine (Dressler and Potter, 1991; Scott and Sigler, 1994). It is not a necessity
that an acyl-enzyme intermediate has to be formed, for in phospholipase A2 a conserved
water molecule hydrogen bonded to the catalytic histidine serves as the source of the
nucleophile (Scott and Sigler, 1994).

A unique property of lipases is their action at oil-water interfaces, the presence of which
greatly enhances their enzyme activity. This is called interfacial activation (Derewenda,
1994; Sarda and DesnuelleI958). However, a lipase from Pseudomonas aeruginosa has
been characterized which does not need interfacial activation (Jaeger et al., 1993). There
is a definite structural difference between this lipase and the other lipases that do possess
interfacial activation. Those that do have interfacial activation have a lid-like helical loop
structure that covers the active site (Brady et al, 1990; Schrag and Cygler, 1993; Winkler
et al, 1990). This loop is not present in Pseudomonas aeruginosa lipase (Jaeger et al.,
1993). In the right conditions of an oil-water interface, the loop would open up and thus
expose the active site (Schrag and Cygler, 1993).

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(3)f ®
+ ~l ~ ~2\~ ~3 ~ ~4 ~ + ~l ~ ~2 ~3 ~ ~ ~
H~-C-C-N--c -C-N -C--c-N-C-C-O- H~-C-C-N--c -C-C-N-C-C-O-
I I I I I I I I I I I I I I
H H H H H H H H H H H H H H

Formation of the acyl-enzyme intermediate

Nucleophilic attack by Collapse of and transfer of W from Histidine-57 to the
Serine-195 on the C=O the tetrahedral departing amino group. Restoration of the
group of the peptide bond intermediate histidine side chain to its original base (N:)
and transfer of hydrogen to
state
the side chain of Histidine-
57. Formation of the R 0 R
tetrahedralintermediate. + I 1 II I2 ~3 ~ ~ ~ + ~l ~ ~2 ~ <ID ~3 ~ ~ ~
H~-C-C-N--C -C--c-N-C-C-O- H~-C-C-N--c -C--- R-C-C-N-C-C-O-
I I I I I I I I I I I I I I
H H H H H H H H H H H H H H
Acyl enzyme intermediate substrate

Figure 22 The role of the catalytic triad in the hydrolysis of a peptide bond by chymotrypsin. The diagram shows the involvement of acid-base catalysis in the first stage of the
cleavage reaction, in which the acyl-enzyme intermediate is formed. The sequential numbering of the arrows is intended to make it easier to trace the logic of the reaction,
although, in reality, the electron rearrangements may occur in a concerted way so that the various steps in the reaction occur simultaneously (Dressler and Potter 1991).

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2.5.3 Homology of lipases from different species

The realisation of homology (similarity) in the gene and ammo acid sequences of
different species is vital for recombinant DNA technology. Tables 10 to 12 show the
homology in the amino acid sequences around the active Ser residue, the active Asp
residue, and the oxyanion hole. The oxyanion hole is a structurally and functionally
important region around the catalytic Serine residue, which is necessary for enzymatic
activity. It increases the affinity of the enzyme for lipid substrates and helps stabilise the
transition state intermediate during catalysis (Winkler et al., 1990). It is a strictly
conserved structural element in lipases, which is formed by a region that contains a
central Glycine residue (Jaeger et al., 1993). Table 8 gives the names and one- and three-
letter symbols of all the amino acids in order to simplify Table 9, Table 10 and Table Il.
The use of shading indicates the degree of homology.

Table 8 The amino acids and their symbols (Sambrook et aI.,

1989)

Amino acid One-letter Three-letter

symbol symbol

Alanine A Ala
Arginine R Arg
Asparagine N Asn
Aspartic acid D Asp
Cysteine C Cys
Glutamine Q Gin
Glutamic acid E Glu
Glycine G Gly
Histidine H His
Isoleucine I lie
Leucine L Leu
Lysine K Lys
Methionine M Met
Phenylalanine F Phe
Proline P Pro
Serine S Ser
Threonine T Thr
Tryptophan W Thr
Tyrosine Y Tyr
Valine V Val

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Table 9 A comparison of amino acid sequences of lipases from different origin. Homology is shown for regions around the
catalytic Ser residue (Jaeger et ai., 1993)

SOURCE

Table 10 A comparison of amino acid sequences of lipases from different origin. Homology is shown for
regions around the oxyanion hole (Jaeger et ai., 1993)

SOURCE Sequence homologies of lipases around the oxyanion hole

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Table 11 A comparison of amino acid sequences of lipases from different origin. Homology is shown
for regions around the catalytic Asp residue (Jaeger et a/., 1993)

SOURCE Sequence hom%gies of lipases around the active site Asp

These sequences show some homology around the catalytic site. Especially around
the active nucleophile, the Serine residue, homology is clear with the G-X-S-X-G
sequence present in all the sequences. The oxyanion hole also has a Glycine residue,
which is present in all the sequences listed. The sequences around the Aspartic acid
residue also show homology, especially with the Aspartic acid itself, which is replaced by
Glutamic acid in Geotrichum candidum and Candida rugosa. The active group of
Glutamic acid is precisely the same as that of Aspartic acid; the only difference is in the
chain length. Thus one can expect the Glutamic acid to fulfil the same role.

The full amino acid sequences of the lipases of 14 organisms were compared by multiple
alignment to produce a dendrogram (Figure 23). The dendrogram indicates how closely
related the lipases from the different organisms are. It is evident from this dendrogram
that the lipases are not closely related in respect to their whole amino acid sequences.
The number of amino acid residues of the lipases from these organisms varies from 173
to 679. Thermomyces lanuginoses is a thermophilic fungus that has also been isolated in
South Africa and is mentioned in chapters 4 and 5 of this thesis. It is a thermophilic
fungus and from the dendrogram it is clear that its lipase is not closely related to the
lipase of any of the other organisms.

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Aspergillus oryzae

Penicillium camembertii

Thermomyces lanuginoses

Rhizomucor miehei

100

Rhizopus niveus
100

Bacillus thermocatenulatus

100

Staphylococcus aureus

94 88

Pseudomonas aeruginosa

100

Pseudomonas tragi

Pseudomon as f1uorescens

100
96
Candida rugosa

100

Geotrichum candidum

Bacillus pu mi/us

100

Bacillus sub tilis

Figure 23 A dendrogram constructed from the multiple alignment of sequences of lipases from 14 different
organisms. The figure at each node indicates the distance to the previous node (root node of that node).
The sequences were obtained from the internet websites of the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/) and the National Biomedical Research Foundation
(http://www-nbrf.georgetown.edu/).

2.5.4 Conditions for optimal lipase activity

Most lipases possess interfacial activation and have an affinity for hydrophobic surfaces
since their substrates are encountered mostly in partially or fully emulsified state. The
best stimulant of lipase activity is the main substrate, triglycerides. Table 12 presents a

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companson of optimal conditions for lipase activity and stability of different fungal
species.

Table 12 A comparision of optimal conditions for lipase activity and stability of different fungal species

Species pH Temp. re» . Upid substrate Reference

Candida antarctica 70 Coulon et al., 1997

Trichosporon asteroides 5 60 3-10 <70 Dharmsthiti et al, 1997
Candida rugosa 6.7 40-50 TG with butyric Marangoni, 1994
acid
Emericella rugulosa 8 45 Venkateshwarlu and Reddy,
1993
Humicola sp. 8 45 Venkateshwarlu and Reddy,
1993
Thermomyces lanuginoses 8 45 Venkateshwarlu and Reddy,
1993
Penicillium purpurogenum 8 45 Venkateshwarlu and Reddy,
1993
Chrysosporium sulfureum 8 45 Venkateshwarlu and Reddy,
1993
Penicillium citrinum 8 34-38 1% Olive oil Pimentel et al., 1994
Ophiostoma piceae 5.5 37 Olive oil Gao and Breuil, 1995
Penicillium caseicolum 9 35 TG with short Alhir et al., 1990
chain FA
Calvatia gigantea 7 30 Christakopoulos et al., 1992
Byssochlamys fu/va 8.5 25 Maize oil Ku and Hang, 1994
Alternaria brassicicola 9 25 TGwith long Berto et al., 1997
chain FA
Microdochium nivale 20 Hoshino et al., 1996
Aspergillus niger 7.5 Pabai et al., 1995
Rhizopus oryzae 7.5 Pabai et aI., 1995

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2.6 Wood-decay fungi: classification and characteristics

The development of the electron microscope in the 1950s expanded the dimensions of the
microscopic realm a thousandfold and led to the discovery of two very different cell
types, prokaryotic and eukaryotic cells. Prokaryotic cells exhibit no mitosis or
cytoplasmic streaming and have no membrane-bound organelles or organised nuclei.
Eucaryotic cells are larger and are present in the higher life forms. These cells divide by
mitosis, exhibit cytoplasmic streaming, and have organised nuclei with double
membranes, mitochondria, and plastids (Zabel and Morrell, 1992). Figure 24a shows the
historical development of the living kingdoms.

(A)

4000 million years 2500 570 Present

(B)

/EUmYCot.~

Mastigomycotina Deuteromycotina

Zygomycotina Basidiomycotina

Ascomycotina
Figure 24 (a), The history of the living kingdoms (Kendrick, 1992) and (b), the classification of
Eumycota (Zabel and Morrell, 1992).

Additional evidence clearly established the distinctiveness of fungi from plants based on
differences in cell-wall composition, heterotrophy and the external mode of digestion

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(Lindenmayer, 1965). Eumycota include primarily the filamentous fungi which

incorporates wood-decay fungi. Fungal classification is based primarily on differences in
the types of the reproductive structures. Currently there are five subdivisions of
Eumycota, which are given in Figure 24b (Zabel and Morrell, 1992). The classification
of wood-decay groups is done firstly on the sequence of cell-wall constituents utilised or
altered. Secondary emphasis is placed on the mode of hyphal penetration of fibres and
tracheids and the types of tissue damaged (Zabel and Morrell, 1992). Table 13 lists the
major types of wood inhabiting micro-organisms as well as their anatomical and chemical
features.

Table 13 A summary of the anatomical and chemical features of the major types of wood inhabiting micro-
organisms (label and Morrell, 1992) .

Wood-inhabiting Cell-wall constituents used Anatomical features Causal agents

micro-organisms

Decayers

Simultaneous white- All Cell walls attacked Basidiomycotina

rots progressively from lumen Some Ascomycotina
surface

Sequential white-rots All, but hemicelluloses and Cell walls attacked Basidiomycotina
lignin used selectively progressively from lumen Some Ascomycotina
initially surface

Brown-rots Carbohydrates, but lignin Entire wall zone attacked Basidiomycotina

modified rapidly

Type 1 soft-rots Carbohydrates Longitudinal bore holes Ascomycotina

develop in secondary wall Deuteromycotina
Some bacteria

Type 2 soft-rots Carbohydrates Secondary wall erosion Ascomycotina

from lumen surface Deuteromycotina
Some bacteria

Nondecayers

Sapstainers Wood extractives Primarily invade Ascomycotina

parenchyma cells in Deuteromycotina
sapwood

Molds Wood extractives Surface growth on wet Ascomycotina

wood Deuteromycotina
lycomycotina

Scavengers Wood extractives and Penetrate wood cells Bacteria

decay residues primarily through pits Ascomycotina
Deuteromycotina
lycomycotina
Basidiomycotina

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The dominant available carbon sources in wood are cellulose, lignin and hemicelluloses,
which are also the major structural components of wood. The fungi that decay the cell
walls are divided into three classes; white-rot, brown-rot and soft-rot fungi. (Cooke and
Rayner, 1984).

2.6.1 Molds

Molds are capable of degrading wood extractives and grow best on wood that is very wet
or that has been exposed to very high humidity for a long time. On softwoods molds
mainly grow on wood surfaces. On hardwoods molds can enter the wood at exposed
parenchyma, vessels, and ruptured cells and can move throughout the wood by rupturing
pit membranes (Messner et al., 1992). Table 14 shows the effect of various molds on the
dichloromethane extractive content of non-sterile southern yellow pine when incubated at
room temperature for two weeks (Farrell et al., 1997). Phlebia roqueforti reduced the
extractive content substantially more than the other fungal species.

Table 14 Dichloromethane extractive content of non-sterile southern yellow pine treated with various
molds (Farrell et aI., 1997)

Fungal species Extractives (%) Extractives (%) Reduction (%)

Control Treatment

Phlebia roqueforti 3.34 2.16 35

Leptographium terrebrantis 2.27 1.92 15
Verticicladella truncata 2.27 1.96 14
Diplodia pinea 2.27 2.02 11
Codinaea sp. 2.27 2.07 9
Aureobasidium pullulans 3.34 3.26 2

2.6.2 Sap-stain fungi

This group is characterised by pigmentaion of the hyphal walls, which causes

discolouration of infected sapwood (Levy and Dickinson, 1981). The discolouration may
cause considerable reduction of the market value of wood (Tsoumis, 1991). Sap-stain
fungi rapidly colonise the sapwood of logs and wood chips. These fungi grow mainly in
ray parenchyma cells and are capable of deeply penetrating logs and wood chips. They
can penetrate simple and bordered pits. Sap-stain fungi are not capable of degrading the
major components of the wood cell wall, cellulose and lignin. Hemicelluloses are
degraded to a very slight degree. Extractives and simple sugars found in the parenchyma

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cells are the major nutrient source for these fungi. Sap-stain fungi cause a characteristic
staining of sapwood, resulting in a blue, black, grey, or brown discoloration of the wood
(Farrell el al., 1997).

A variety of sap-stain fungi were screened for their ability to degrade wood extractives
(Farrell el al., 1997). Sterile southern yellow pine was inoculated with the sap-stain fungi
listed in Table 15 and incubated for two weeks. Ceratocystis adiposa and Ophiostoma
piliferum reduced the dichloromethane extractives significantly.

Table 15 Dichloromethane extractive degradation by sap-stain fungi on sterile

southernyellow pine (Farrell et al., 1997)

Fungal species Extractives (%) Extractives (%) Reduction (%)

Control Treatment

Ceratocystis adiposa 2.13 1.26 41

Ophiostoma piliferum 3.34 2.27 32
Ceratocystis adjuncti 1.98 1.44 27
Ceratocystis minor 2.13 1.57 26
Ophiostoma piceae 2.13 1.57 26
Ophiostoma populina 2.13 1.62 24
Ophiostoma abiocarpa 2.13 1.61 24
Ceratocystis tremuloaura 2.13 1.65 20
Ophiostoma fraxinopennsylvanica 2.13 1.71 20
Ophiostoma plurianulatum 2.13 1.73 19
Europhium aereum 1.98 1.61 19
Ceratocystis ponderosa 2.19 1.86 18
Ceratocystis penicilIata 1.98 1.58 15
Ophiostoma olivaceum 2.27 1.93 15
Europhium clavigerum 2.13 1.84 14
Ceratocystis hunti 2.13 1.89 11
Ceratocystis ambrosia 2.13 1.92 10
Ophiostoma distorlum 2.27 2.07 9
Europhium robustum 2.27 2.06 9
Cladobotryum vireseens 2.27 2.11 7
Ophiostoma galeiformis 2.13 1.98 7
Ceratocystis coerulescens 2.13 2.13 o
Ophiostoma dryocetidis 2.24 2.27 o
Ophiostoma stenoceras 2.13 2.21 o
Xylaria. conudamae 2.44 3.16 o
Xylaria hypoxylon 2.44 2.88 o

2.6.3 White-rot fungi

White-rot fungi are able to attack and metabolise all major wood constituents. They are
unique among most microorganisms in their capacity to depolymerise and metabolise
lignin. The major components are used in varying orders and rates by different white-rot
fungi, suggesting that these fungi probably represent a heterogeneous group with widely
varying enzymatic capabilities (Zabel and Morrell, 1992). These differences were used to

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group white-rot fungi into the simultaneous rotters, which utilised the components
uniformly, and white-rotters, which utilised lignin initially more rapidly than cellulose
(Liese, 1970). Table 16 shows that white-rot fungi like Phanerochaete chrysosporium
not only attacks lignin, but also reduces the dichloromethane extractives content of
southern yellow pine.

All cell-wall components are presumably ultimately consumed, with the exception of the
minor minerals. There is considerable variation in the sequence and rate of component
utilisation by both species and fungal strains within a species. Weight losses may
approach 95-97% ofthe original wood material when prolonged exposures under optimal
decay conditions prevail. At all stages of decay, the residual wood has a low solubility in
1% sodium hydroxide, suggesting that the breakdown products of decay are utilised by
the fungi as rapidly as they are released. The cellulose, hemicelluloses and lignin
remaining in the undecayed portions of the wood appear to be essentially unaltered,
suggesting that white-rot fungi concentrate their attack on exposed cell-wall surfaces.
Thus the enzymes slowly erode their way into the cell walls from lumina surfaces (Zabel
and Morrell, 1992).

Table 16 Dichloromethane extractive content of sterile southern yellow pine treated with various
Basidiomycetes (Farrell et al., 1997)

Fungal species Control extractives (%) Treatment extractives (%) Reduction (%)

Phanerochaete chrysosporium 2.19 1.30 41

Phanerochaete subacida 3.34 2.01 40
Phanerochaete gigantea 3.34 2.03 39
Phanerochaete tremellosa 1.98 1.21 39
Hyphodontia setulosa 1.98 1.20 39
Coriolus versicolor 1.98 1.28 36
Inonotus rheades 3.34 2.18 34
Trichaptum abietinium 4.70 3.13 33
Ceriporiopsis subvermispora 3.34 2.18 29
Trichaptum biforme 4.70 3.13 24
Schizophylum commune 2.50 2.03 17
Sistotrerma brinkmanii 2.44 2.17 11
Pleurotus ostreatus 2.44 2.30 6
Alurodiscus sp. 2.44 3.70 0
Ganoderma collosum 2.29 1.75 0
Phellinus igniarius 2.44 2.48 0

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2.6.4 Brown-rot fungi

Brown rot fungi primarily decompose the cell-wall carbohydrates, leaving behind a
modified, demethoxylated lignin residu. The hemicelluloses are removed more rapidly
than cellulose by brown-rot fungi in the early decay states (Zabel and Morrell, 1992).
Brown rots differ from white rots in the extensive depolymerisation of the carbohydrates
in the secondary wall early in the decay process (Kirk and Highley, 1973). Whereas lysis
zones are closely associated with white-rot hyphae, substantial cell-wall damage at
distances up to several cell widths has been noted for some brown rots (Eriksson et al.,
1990). Figure 25 shows the mode of cell-wall attack for brown-rots, as well as white-rots
and two types of soft-rot.

All carbohydrates are ultimately consumed, leaving a residuum of modified lignin in the
cell wall. Large increases in water solubility and solubility in 1% NaOH occur at early
decay stages, owing to the rapid carbohydrate depolymerisation. Brown-rot fungi appear

(Progressive erosion of (Rapid chemical attack of all

all cell wall components cell wall carbohydrates)
from lumen surface)

(Progressive erosion of all (Selective localised attack

cell wall components of secondary wall and
innially in the 52) formation of lonqitudinal
bore holes)
• Sound cell wall
• Decayed cell wall
PW Primary wall
SW Secondary wall
H Hypha
L Lumen

Figure 25 Diagrams showing the various modes of cell-wall

destruction for white-rots, brown-rots, and the two types of soft-
rots (Zabel and Morrell, 1992).

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to depolymerise wood in the early decay stages much more rapidly than the decay
products can be metabolised. The decay process rapidly involves the SI and S2 layers of
the cell walls, but develops irregularly. There appears to be much less variation in the
sequential attack of cell-wall constituents by brown-rot fungi as compared to the white-
rotters (Zabel and Morrell, 1992).

2.6.5 Soft-rot fungi

Soft-rot fungi display considerable variation in their effects on cell-wall constituents

during decay development. For many species, the principle targets are the carbohydrates,
whereas lignin attack is limited to minor demethoxylation (Zabel and Morrell, 1992).
Some soft-rotters, however, selectively remove more lignin than carbohydrates from
coniferous wood, in a manner similar to that of some white-ratters (Eslyn et al, 1975).

Wood decayed by soft-rot fungi resemble that of white- rot-degraded wood in having low
alkali solubility, indicating that the degradation products are used at the same rate as they
are released (Zabel and Morrell, 1992). Initially, soft rots were defined by the formation
of unique longitudinal bore holes in the secondary cell wall, however, it was determined
that some non-Basidiomycotina eroded the secondary wall in a manner similar to that of
some white-ratters. Type 1 soft-rot fungi are able to degrade crystalline cellulose as
reflected by the formation of characteristic cavities in the S2 zone of the secondary wall.
The cell-wall eroders (hyphae lying on the S3 layer) are the Type 2 soft-rot fungi
(Corbett, 1965). In conifers, the S3 zone of the secondary wall is resistant to soft-rot
attack, but delignification substantially increases decay susceptibility and may shift the
fungus from the cavity formation (Type 1) to the wall erosion (Type 2) mode (Morrell
and Zabel, 1987).

2.6.6 Hyphal growth and cell wall composition

Fungi, like plants, possess firm cell walls that provide the rigidity needed for the
penetration of plant cell walls. Structurally, fungal cell walls consist of an inner network
of microfibrils embedded in an amorphous matrix that also forms the outer layers or often
lamellae of the wall (Burnett, 1979). Chemically the walls consist of 80 to 90%
polysaccharides, with the remainder composed of proteins and lipids (Zabel and Morrell,

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1992). Chitin is the principal skeletal framework material (Burnett, 1979). Chitin,
cellulose, and in a few cases chitosan form the microfibrils that serve as the skeletal
framework of the walls (Zabel and Morrell, 1992). Table 17 shows the lipid·composition
of various fungi, indicating the total lipid content as well as the contribution of different
lipid classes. According to this data the lipid composition of the biomass could be as
little as 3% and as much as 75% of the total dry weight.

Table 17 Lipid composition of selected fungi (Lësel, 1988)

Tota/lipid Percentag_e of tota/lie/ds

Fungi (%ofWd) NL PL PH GL FFA TG DG ST STE

Ascomycotina
Penicillium chrysogenum
carbon source: D-glucose 12.7
D-fructose 18.7
sucrose 19.0
Aspergil/us niger 4.0 31.3 68.7 40.9 27.2 4.7 1.9 19.1 3.7
Humico/a /anuginosa 37°C 75.1 84.4 15.6 41.0 45.6 5.5 3.0 4.9
52°C 8.5 84.9 15.1 5.8 45.0 31.3 7.7 10.2
45°C 17.0
Sclerotium roltsl! mycelium 10.0 40.0 40.0 20.0

Basidiomycotina
Agaricus bisporus mycelium 5.1 68.1 20.7 2.0 10.4 13.3 3.3 1.7
Armil/ariel/a mel/ea 11.7 46.5
Bo/etus edulis 13.4 26.3
Cantherel/us cibarius 48.6 51.4 49.6 1.7
Lactarus trivialis 12.2 23.3
Lepista nuda mycelium
3 days 15.8 80.0 20.0 18.0
4 days 20.0
6 days 30.6
12 days 47.7
Po/yporus ovinus 10.3 34.0
Rhizoctonia so/ani mycelium 2.9 16.3 32.7
Pisolithus tinctorius mycelium
Til/etia controversa mycelium 5.8 48.0
Ustilago zeae mycelium
14 days, 5% sucrose 27.8
22% sucrose 7.0

NL, neutral lipids; PL, polar lipids; PH, phospholipids; GL, glycolipids; FFA, free fatty acids; TG, triacylglycerols; DG,
diacylglycerols; ST, sterols; STE, sterol esters.

Hyphal growth occurs primarily by apical extension at the tips (Burnett, 1979). Vacuoles
usually develop a few cells behind the hyphal tip, and the related turgor pressure within
the cell protoplasts is the presumed driving force extending the plastic tip. Hence, some
stain fungi are able to penetrate thin silver or aluminum foils. Hyphal branching is
common and usually begins early behind the developing hyphal tips. The cytoplasmic

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streaming appears to move material steadily toward the tips, leaving empty or vacuolated
hyphae behind (Zabel and Morrell, 1992). Hyphae grow longitudinally along the lumina
surface and pass from cell to cell through the pits (Levy and Dickinson, 1981). Growth
rates longitudinally through the wood, under ideal conditions such as wood blocks in
decay chambers, approach growth rates on malt-agar medium. No correlation is evident
between the abundance of hyphae in cells and decay severity. The penetration modes of
wood-inhabiting fungi are probably primarily enzymatic for the decay fungi that often
form bore holes in the size range of hyphal diameters and primarily mechanical for the
Type 2 soft-rotters (Zabel and Morrell, 1992).

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2.7 Fungal growth in wood

There are several accounts on the experimental biotechnological trials of fungi on wood
(Brush et al., 1994, Chen et al., 1994, Farrell et al., 1994, Fischer et al., 1995, Wang et
al., 1995). The degree of and method of colonisation of wood, especially wood in the
form of chips or chip piles, is relevant when considering biotechnological applications.
The factors influencing the colonisation of wood by fungi are the physical and chemical
properties of the wood as well as the microclimate (Rayner and Boddy, 1988).

2.7.1 Nutrient sources in wood for fungi

Soluble sugars, lipids and peptides, together with the major storage compound starch,
provide the main supply of readily assimilable carbon sources for fungi growing in wood.
They enable development of certain micro fungi such as those causing blue stain in
conifers, which can not degrade cell-wall components, but are rapidly depleted during
fungal colonisation. The dominant available carbon sources for fungi growing in wood
are structural cell-wall components (Cooke and Rayner, 1984). The major types are
cellulose, hemicelluloses and lignin. The different enzymatic affinities of fungi for these
three major structural components of woody cell walls at least partially explain the three
characteristically different decay types known as white-rot, brown-rot and soft-rot.
Nitrogen, phosphorous and potassium are important minerals for fungal growth (Rayner
and Boddy, 1988).

The presence of extractives can affect the performance of fungi in three ways: they can
serve as carbon sources, they can be inhibitory or they may serve as growth stimulants.
Variations between fungi in relation to these effects may be a highly significant
determinant of selectivity towards different timber types (Rayner and Boddy, 1988). As
the heartwood of some trees (e.g. Eucalyptus) ages, there is a progressive breakdown of
toxic extractives to a less toxic form, with concentrations of inhibitory compounds being
least in inner heartwood (Rudman, 1964, 1965). Extractives are found largel y in the
lumen of parenchyma cells but can also be found in the lumen of vessels, fibres and
sometimes in specialised cells. They can also form coatings on the cell wall, in pits and in
capillaries in the cell wall (Rayner and Boddy, 1988).

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2.7.2 Factors influencing colonisation of wood

2.7.2.1 Physical and chemical properties of wood

To gain access to the nutrient sources that are held in the woody cell walls in a tree or
branch the fungus first has to gain entry to the wood itself and secondly use natural
passages in the wood. When colonising chips the fungus does not have to find ways to
gain entry to the wood itself for the wood is already unobstructed. The fungus can
achieve rapid axial access. The nature, distribution and size of the wood passages, and
the interconnections (pits) that occur between them are crucial in determining patterns of
colonisation. Fungal hyphae are capable of directly penetrating woody cells by their
capacity for exertion of forward mechanical pressure and extracellular enzyme action,
however the natural tendency is to move along avenues of least resistance. The natural
pathways are provided by the axial and radial cells that are connected by pits (Rayner and
Boddy, 1988).

In hardwoods the larger vessels provide the most effective channels, so that their size,
number and distribution are important in determining invasion patterns (Cooke and
Rayner, 1984). In diffuse-porous woods, like Eucalyptus spp., penetration is limited
during the early stages of colonisation when compared to ring-porous woods (Rayner and
Boddy, 1988). Since the wood is already easily accessible in the chip form this feature of
a specific wood specie would once again not play a significant role. The size, structure
and distribution of pits are significant factors affecting the accessibility of woody tissues
to fungal hyphae. When the pits are aspirated it reduces permeability (Siau, 1995), so
that the breakdown of pit-closing membranes represent an early priority during fungal
colonisation (Cooke and Rayner, 1984). Axial and ray wood parenchyma, with their
relatively unthickened walls and large lumina, together with the presence of assimilable
nutrients derived from their protoplasts, can be regarded as facilitating colonisation.
Fibres with their thick, less pitted walls and narrow lumina are ineffectual as access
routes, and when occurring together, form tissues which can be regarded as barriers to
both axial and transverse colonisation. Little precise information is available concerning
the role of parenchyma and fibres with regard to fungal colonisation (Rayner and Boddy,
1988).

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It has been reported that fatty acids can inhibit fungal growth (Garg and Muller, 1993).
In some wood species like aspen (Populus tremuloides) and lodgepole pine (Pinus
contorta) fungal growth is inhibited in the heartwood of the tree. The heartwood of aspen
has a much higher fatty acid concentration than the sapwood (Table 18). According to
literature it is, however, doubtful that the fatty acid content would be high enough to
inhibit fungal growth. Tests in liquid medium showed that fungi can easily grow in fatty
acid concentrations of 1% and the fatty acid concentrations of the aspen heartwood which
was inoculated was 0,83%. In this case fungal growth may have been inhibited because
the moisture content of the heartwood (33%) was lower than that of the sapwood (43%)
(Breuil et al., 1994). The natural durability of wood appears to be dependent on the
presence of certain phenolic extractives (Rudman, 1963), rather than on fatty acid
content. The decay resistance of aspen is increased after it has been submersed in the
methanol extracts of the heartwood of black locust (Robinia pseudoacacia), osage orange
(Mac/ura pomifera), redwood (Sequoia sempervirens) and Intsia bijuga (Kamdem,
1994).

Table 18 Contents of different lipid classes (% of oven dry weight) in the sap- and
heartwood of untreated aspen (Breuil et al.1994)

Upid class Aspen sapwood Aspen hearlwood

Triglycerides 1.45 ± 0.03 0.36 ± .01

Fatty acids 0.03 ± 0.01 0.83 ± 0.03
Waxes and steryl esters 0.62 ±0.01 0.52 ±0.02
Others 0.99 ±0.08 0.73 ± 0.08

The pH optima for growth of most wood decay fungi is 4 to 6, which corresponds to
the pH found in most woods. White-rot fungi show greater tolerance of high pH than
brown-rot fungi (Cooke and Rayner, 1984). Table 19 shows the pH values of a range of
hardwood tree species. The Myrtaceae family, which incorporates the Eucalyptus species,
is also included. There can be considerable differences between sapwood and heartwood
within a tree (Hartley et al., 1961). Generally, the sapwood would have a lower pH than
heartwood. However, the actual micro environmental conditions experienced at the
hyphal level and metabolic activity itself is likely to have both marked and highly
localised effects on the pH. Nonetheless, extremes of pH have sometimes been implicated
in the selectivity of fungi for particular timber types, examples being elm (Ulmus spp.)

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with a pH above 7 (Gray, 1958) and in oak (Quercus spp.), which may have a pH as low
as 3 and acid enough to corrode metals (Packman, 1960).

Table 19 The pH values for a range of hardwood tree species (Rayner and Boddy, 1988)

Fami/y Species pH range

Aceraceae Acer pseudoplatanus 4.05-6.0

Apocynaceae Dyera costulata 4.65
Aquifolaceae /lex aquifolium 5.5
Araliaceae Acanthopanax ricinofolius 6.7
Betulaceae Alnus glutinosa, Betula spp., Carpinus betulus 4.5-5.95
Bombaceae achroma lagopus 6.6
Burseraceae Aucoumea klaineana 4.2-4.45
Buxaceae Buxus sempervirens 5.05-6.0
Combretaceae Terminalia spp. 3.05-5.65
Compositae Brachylaena hutchins;; . 4.35-4.8
Dipterocarpaceae Anisoptera sp., Dipterocarpus spp., Shorea spp., Parashorea sp. 3.2-5.55
Ebenaceae Diospyros mespiliformis 5.7
Fagaceae Castanea sativa, Fagus sylvatica, Quercus spp., Nothofagus spp. 3.35-6.05
Flacourtiaceae Scoffelia coriacea 5.35-6.8
Gonystylaceae Gonystylus sp. 5.25-5.35
Guttiferae Calophyllum brasilienee 4.9
Juglandaceae Carya sp., Juglans regia 4.4-5.8
Lauraceae Eusideroxylon sp., Ocotea spp., Persea sp. 3.65-4.9
Lecythidaceae Cariniana sp. 3.3
Leguminosae 18 spp. tested 3.65-6.15
Melastomaceae Dactylocladus stenostalchys 4.55
Meliaceae 12 spp. tested 2.75-5.85
Moraceae Chlorophora exe/sa, Piratinera guianensis 5.4-7.25
Myrtaceae Eucalyptus spp., Tristania conferta 3.2-4.95
Ochnaceae Lophira alata 4.2-4.45
Olaceae Fraxinus spp., Olea sp. 3.55-6.25
Platanaceae Platanus acerifolia 4.6-6.0
Proteaceae Grr!JVillearobusta 4.95
Rhamnaceae Maesopsis emin;; 4.35
Rosaceae Prunus acium, Sorbus aucuparia 4.5-4.8
Rubiaceae Calycophyllum sp., Mitragyna sp. 3.85-5.75
Salicaceae Populus spp., Salix fragi/is 4.2-4.8
Sapotaceae Minusops heckelii 4.75-5.1
Steruliaceae Sterculia oblonga, Tarrieta spp. 4.9-5.5
Tiliaceae Cistanthera papaverifera, Tilia vulgaris 3.75-5.55
Triplotichonaceae Mansonia altissima, Triplochiton sc/eroxylon 4.05-6.75
Trochodendraceae Ceridophullum japonicum 5.05-5.55
Ulmaceae Ulmus spp. 6.45-7.15
Verbaceae Tectona grandis 4.75-4.9
Vochysiaceae Vochysia sp. 3.65
Zygophyllaceae Guaacum spp. 3.6-5.45

Different patterns of attack of the S 1, S2 and S3 layers occur. In white rots hyphae
typically penetrate into the cell lumen, where they lie on the inner surface of the wood
cell wall (Nasroun, 1971; Bravery, 1971, 1972, 1975, 1976; Levy and Dickinson, 1981).
Erosion of the cell wall may be generalised or localised to the immediate vicinity of the
hyphae, forming a groove or trough with a central ridge on which the hypha lies. This

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implies that the extracellular enzymes are bound to the hyphae by an external layer of
mucilage (Cooke and Rayner, 1984). Branching of the hyphae eventually results in
progressive cell-wall erosion from the lumen through the S3 and S2 layers {Rayner and
Boddy, 1988).

Soft rot fungi produce two distinct types of attack termed Type 1 and Type 2 (Corbett,
1963, 1965; Crossley, 1980; Nilsson, 1974, 1975, 1976, 1977; Zainal, 1976). Type 1
attack is characterised by the formation of chains of cavities with pointed ends in the S2
layer, which follow the orientation of the microfibrils (Savory, 1954). Type 2 attack is
similar to localised white-rot attack, in that it works outward from the lumen and results
in an eroded groove with the hypha lying on a central ridge .: With brown rot the hyphae
lie on the surface of the S3 layer in the lumen, but not within it. The hyphae, S3 layer and
compound middle lamella alter very little but the S2 and SI layers become extensively
hollowed out owing to removal of cellulose and hemicelluloses (Bravery, 1971; Crossley,
1980; Nasroun, 1971; Wilcox, 1968).

2.7.2.2 Microclimate

Wood, which is not in contact with the ground, absorbs water by precipitation through
rain, mist, hail or snow and also by absorption of water vapour from the air. The relative
humidity of the surrounding air is an important factor. Air has a certain maximum
potential saturation with water vapour and when the real water vapour content is
expressed as a percentage of the maximum potential water vapour content it is called
relative humidity. Wood will absorb water from the air or transfer water to the air until it
reaches an equilibrium moisture content that is governed by the relative humidity of the
air. The approximate equilibrium moisture contents of wood at varying relative humidity
values are given in Figure 26. At about 30% moisture content wood reaches the fibre
saturation point where the cell walls are saturated with water and the voids are empty.
Above 30% moisture content, free water exists in the lumen of woody cells. Below 30%
all moisture is bound to cell walls (Siau, 1995). Excessive water content can prevent
fungal colonisation (Cooke and Rayner, 1984).

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100

80

100

20

10 20
o

EIVIC ("lo) M:listure oontent (%)

Figure 26 The equilibrium moisture Figure 27 An approximation' of the cardinal moisture contents
content (EMC) of wood with varying for the rates of decay development of several common
relative humidity (RH) (Skaar, 1972). mesophilic basidiomycete decay fungi as judged from reports
in the literature and experience (Zabel Morrell, 1992).
Maximal moisture contents are calculated based on data from
Higgins (1957) and Skaar (1972).

Moisture content crucially affects the growth of wood-decaying fungi in either of two
ways. At low moisture contents limitations are imposed by restricted accessibility of
water supplies to growing hyphae. At high moisture contents the presence of water-filled
void space may impose a physical barrier to growth by influencing the exchange and
availability of air. Under normal temperature conditions the solubility of carbon dioxide
is 30-50 times greater than that of oxygen (Brock, 1966). Reduced supply of oxygen and
an elevated concentration of carbon dioxide have consistently been shown to reduce
growth (Jensen, 1967; Scánel, 1976). Water fill of void space would hinder oxygen
supply due to its relative insolubility in water. Above 30% equilibrium moisture content
the woody cells become filled with water. The lack of oxygen supply would become
limiting for fungal growth with an increase in moisture content above fiber saturation
point. Several species grow at lower extremes of moisture levels including Schizopora
paradoxa which survives at a relative humidity of 35% (Theden, 1961), which would
correspond to a wood moisture content of ± 8% (Figure 26). Table 20 shows the minimal
moisture levels for decay development. Optimal wood-moisture levels for most decay
fungi lie between 40 and 80% (Scheffer, 1973). Since the void volume of wood varies

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inversely with specific gravity, the upper moisture levels limiting fungal growth will be
much lower in the high-density woods (Figure 27).

Table 20 Minimal moisture levels for decay development (Zabel and Morrell, 1992)

Fungi Moisture level Reference

Wood-decay fungi 25-32% Snell et al., 1925

Xylobolus frustulatus 16-17% Bavendamm and Reichelt, 1938
Schizophyllum commune 16-17% Bavendamm and Reichelt, 1938
Antrodia sinuosa 26% Freyfeld, 1939
Common wood-decay fungi 22-24% Cartwright and Findlay, 1958
Ophiostoma piliferum 23% Lindgren, 1942
Wood-inhabiting fungi Slightly above fibre saturation point Etheridge, 1957

Generally, wood-decay fungi have been found to be mesophilic, with a growth range
within a-45°C and an optimum between 20 and 30°C. Phanerochaete chrysosporium,
however, has an optimum of about 40°C and a maximum of SO°C (Bergman and Nilsson,
1971).

2.7.3 Conditions in wood chip piles

Chip storage of pulpwood, because of its handling advantages and relatively low losses,
has now become the predominant method of wood storage. It decreases handling cost,
and requires smaller storage areas (Zabel and Morrell, 1992). In relation to the wood in a
tree a chip pile has less free surface for evaporation and dissipation of metabolic heat and
commonly gives rise to a self-heating system, in which the centre may be considerably
hotter and moister than the outside. Temperatures as high as 48,9°C may be reached after
seven days (Springer and Hajny, 1970). Further temperature rises depend, in part, on pile
features. Tall piles with excessive compaction, or accumulated layers of fines reduce air
circulation, and temperatures may rise to 60-71 "C. At these temperatures, slow heat
decomposition of wood begins, and acetic acid is released (Kubler, 1982). The
exothermic reaction further elevates the temperature, and the acetic acid increases wood
acidity, which may reach a pH of 3. Chips exposed at these acidity and temperature
levels turn brown and become soft. Losses from chemically degraded chips and
occasional fires from spontaneous combustion can occur in piles under these high
temperature conditions. The principle heat sources in chip piles and the factors affecting
various temperature levels are illustrated in Figure 28 (Zabel and Morrell, 1992). After

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the initial rise, temperatures decline in properly constructed and managed piles, but
remain higher than external ambient temperatures (Feist et al., 1973). Management
practices generally strive to maintain pile temperatures below 60°C.

Fungal colonisation by both stain-causing fungi and true decay species occurs mostly in
the outermost layers (Lindgren and Eslyn, 1961). While the growth of most
basidiomycetes is restricted, Phanerochaete chrysosporium has been repeatedly isolated
(Bergmann and Nilsson, 1971; Tansey, 1971; Ofosu-Asiedu and Smith, 1973). A range
of thermophilic fungi grows on chip piles (Tansey, 1971). Thermophilic or
thermotolerant fungi predominate in the interior zone of a chip pile (Zabel and Morrell,
1992).

82

60
Chemical 8utooxldetlon

Mir>mum compaction
Low pile height: 15m
~
~
::J 38
f!
~
E
Q>
f-

15 ,g '"
~ !!
61- "0
!'! ii
lJi;' i~
1il1il Bacteria and fungi

0:: "''''

3 5 6
Storage time (months)

Figure 28 The principle heat sources

in large piles of pulpwood chips and
the various factors affecting the
temperature levels and chip damage
(Zabel and Morrell, 1992).

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2.8 Conclusions

The temperature, pH fluctuations and chemicals used in the kraft pulping process coupled
with a bleaching regime induce the formation of pitch deposits from wood extractives.
Pitch deposits lead to the formation of a layer on tile and metal parts. This layer would
break up into flakes and form spots on the pulp sheets and paper produced and reduce its
value. Current chemical technologies of pitch reduction (e.g. surfactants and dispersants)
are costly and the use of biotechnology may decrease the use of chemicals while
improving product quality.

Triglyceride is the key to pitch formation in the pulping. process. The products of
triglyceride breakdown, fatty acids and glycerol, are more easily removable from the
pulping process. This prompts the use of lipase in combating pitch. In a pulping context
the use of a lipase secreting white-rot fungus is the ideal because of the capability of
white-rots to remove lignin. With genetic engineering in mind, the identification of any
lipase secreting fungi is of significance. Ophiostoma piliferum (Cartapip") is a sap stainer
and Ascomycete used industrially specifically for its ability to hydrolise triglycerides.
Although it has been reported that Ophiostoma piliferum improves chemical pulping
efficiency (due to the increased permeability of the wood) and mechanical pulp properties
(Kohler et al., 1995; Wall et al., 1994; Wall et al., 1995), a white-rot fungus which
hydrolyses triglycerides and lignin and increases wood permeability would still be the
ideal. The application of lipase biotechnology in the pulping process is already practised
in industry with success (Fujita et al., 1992).

Although Eucalyptus grand is is not a ring-porous but a diffuse porous hardwood, it is not
a factor in the chip piles of a pulp mill when considering the inclination of the wood
specie to be colonised by fungi. The presence of numerous rays in Eucalyptus grandis
makes the wood more inclined to fungal colonisation. The wood is already well exposed
in the chip form and the colonisation by fungi will be unconstrained. In addition to pitch
reduction it is probable that the permeability of the wood will be enhanced by reduction
of tyloses and opening up of pits (Siau, 1995), consequently ensuring that the wood is

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more evenly pulped. The necessity of holding large amounts of wood inventory in the
form of chip piles may be a limiting factor.

The optimal moisture content of wood for fungal growth lies between 40 and 80%.
Addition of water would have to be considered to maintain this moisture content
throughout a chip pile. The temperature optima for the fungus used, climate and self-
heating characteristic of a chip pile are factors to be considered when contemplating
temperature control of a chip pile. Aeration of chip piles may be necessary for
temperature control, depending on fungal strain used, as well as oxygen supply.

The development of a fungal or enzymatic biotechnological approach to combat pitch

problems in South African pulp mills that utilise Eucalyptus grand is could decrease
chemical consumption and improve product quality. The use of biotechnology can also
improve the marketability of pulp and paper products.

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2.9 References

Aisaka, K and Terada, 0 (1981), BioI. Chemo 89: 817

Akhtar, MW, Mirza, AQ and Chughtai, MID (1977), Pak. J. Biochem. 10: 82
Akhtar, MW, Mirza, AQ and Chughtai, MID (1980), Applied Environmental
Microbiology 40, 257
Alhir, S, Markakis, P and Chandan, RC (1990), Journal of Agricultural and Food
Chemistry 38: 598-601
Allen, LH (1977), Trend (pointe Claire, Canada) 26: 4-10
Allen, LH (1980), Tappi Journal63: 81-87
Allen, LH (1988a), Pulp and Paper Canada 89: 87-91
Allen, LH (1988b), Tappi Journal71: 61-64
Aston, DA, Fryer, MT and Lambert, CG (1992), Pulp and Paper Canada 93: 75-77
Bamber, RK (1985), Appita 38: 210-216
Bavendam, Wand Reichelt, H (1938), Arch. Mikrobiol. 9: 486-544
Bergman, 0 and Nilsson, T (1971), Studies on Outside Storage of Saw Mill Chips,
Research notes of the Royal College of Forestry, Stockholm
Berto, P, Belingheri, Land Dehorter, B (1997), Biotechnology Letters 19: 533-536
Biermann, CJ (1993), Essentials of Pulping and Papermaking, Academic Press, New
York
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Chapter 3: Manuscript submitted for

publication in Holzforschung

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3.1 An Investigation into Pitch Deposition in a Eucalyptus spp. Kraft

Pulping and Bleaching Mill

By G.C. Scheepers', T. Rypstra', T. H. de Koker2 and B.J.H. Janse'

'Department of Wood Science, University of Stellenbosch, Stellenbosch, 7602, South

Africa.
2Department of Microbiology, University of Stellenbosch, Stellenbosch, 7602, South
Africa.
3Mondi Forests, P. O. Box 39, Pietermaritzburg, 3200, South Africa.

3.1.1 Summary

Wood and pulp extracts as well as pitch deposits were collected and their composition
was analysed. Large portions of the samples were involatile and had a molecular weight
above 900 Da. Approximately 40% of volatile E. grandis extract was identified as
f3-sitosterol, with fatty acids (22.8%) and triglycerides (15.5%) also making a substantial
contribution. Amide derivatives of fatty acids 16:0, 18:2, 18:1 and 18:0 were a
prominent fraction of the pulp extracts from the later stages of bleaching. The amide
derivatives constituted 38.3% and triglycerides 10.1% oftotal volatile pitch deposits.

Keywords: Eucalyptus grandis, wood extractives, kraft pulp extractives, pitch, gas
chromatography, mass spectrometry, gel permeation chromatography

3.1.2 Introduction

The extractive materials in wood often cause pitch problems in pulp mills. During
pulping and bleaching, extractives are released from the wood and pulp and later stick to
ceramic and metal parts. These pitch deposits impair both product quality and production
rates. They decrease the efficiency of pulp washing, screening, centrifugal cleaning, and
refining, and can disrupt many paper machine operations (Dreisbach and Michalopoulos
1989). There are a few triggering mechanisms that induce pitch deposition.
Hydrodynamic or mechanical shear can destabilise the colloidal pitch emulsion causing
pitch to agglomerate and deposits to form. Similarly, sudden temperature drops, pH
shocks, or the introduction of water hardness ions from fresh water inlets or showers can

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also cause pitch deposition by destabilising the colloidal pitch emulsion. Inorganic salts,
such as calcium carbonate, can catalyse pitch deposition by acting as the building blocks
for the sticky pitch. Furthermore, calcium ions in the white water can react with fatty
acids, forming insoluble, sticky calcium soaps (Laubach and Greer 1991). Triglycerides
have been identified as the major contributors to pitch deposition in kraft pulping and
bleaching mills (Allen 1977; Fischer and Messner 1992; Fujita et al. 1992; Suckling and
Ede 1990).

The extractives of the Eucalyptus genera have also been reported to have a detrimental
effect in both pulping and sawmilling operations (Chafe 1987; Hillis and Carle 1959;
Nelson et al. 1970; Yazaki et al. 1993). To attain an improved understanding of pitch
problems associated with the kraft pulping and bleaching of Eucalyptus spp., various
analyses were done on pulp extractives and pitch from a South African kraft pulp mill in
this investigation. The aim of the study was to determine (a) the composition of the
wood and pulp extracts, (b) the change in pulp extract composition through the pulping
and bleaching process and (c) the composition of the small amounts of pitch still
depositing despite the use of pitch reducing additives.

3.1.3 Experimental

3.1.3.1 Wood, pulp and pitch sampling

Six year old Eucalyptus grandis logs were obtained from the logyard of a kraft pulp mill
in the KwaZululNatal region of South Africa. The logs were left in the plantations for six
weeks after harvesting prior to delivery to the pulp mill logyard. The logs were hand
debarked and chipped. The pulp mill feedstock comprises several Eucalyptus species and
hybrids with E. grand is comprising more than 50% of the total feedstock. The mill has a
C/DEoDED bleaching regime. Pulp samples were taken from the brown stock decker
mat, CD mat, Dl inlet, E2 mat and bleach stock decker mat. Pitch was sampled from the
bleach stock decker outlet. All samples were stored at -20DC until analysed.

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3.1.3.2 Dichloromethane extractions

Dichloromethane was selected as extraction solvent due to its ability to extract lipids
(Wallis and Wearne, 1999). The moisture and extractive contents of wood and pulp
samples were determined according to Tappi method T 204 om-88. The wood chips
were ground in a Wiley mill with a 0.40 mm (40 mesh) sieve. Two thimbles per wood
sample, each containing approximately two grams of air dry ground wood (2 g heartwood
or 2 g sapwood or 1 g heartwood and 1 g sapwood), were extracted with dichloromethane
for 5 hours. Two thimbles per pulp sample, each containing approximately 109 of air
dry pulp each, were extracted with dichloromethane for 5 hours. The extracts were air
dried in a fume hood, weighed to determine the total amount of extractives present and
analysed by gel permeation chromatography, gas chromatography and
gas chromatography - mass spectrometry.

3.1.3.3 Gel permeation chromatography (GPC)

Samples were dissolved in tetrahydrofuran. Analyses were done using a Spectra Physics
8875 autosampler, HPI100 isoeratic pump and Spectra Physics GPC software. The
molecular mass distribution of wood and pulp extracts were determined by passing
extracts through four in series 300 x 7.8 micron Phenogel 10columns from Phenomenex
with packing pore sizes of 100 A, 500 A, 1000 A and 10 000 A at a flow rate of
1.25 ml.min". Molecular masses were determined using polystyrene standards and an
Erma 7510 refractive index (RI) detector.

3.1.3.4 Dertvatization of extracts

Diazomethane was used to methylate all carboxylic acids present in the wood or pulp
extracts (Christie 1992). Extracts were also dissolved in chloroform and methylated by
the addition of trimethylsulphonium hydroxide (TMSH) as described by Butte (1983).
TMSH caused transesterification and methylation of carboxylic acids.

3.1.3.5 Gas chromatography (GC) of diazomethane methylated extracts

Gas chromatography analyses of diazomethane methylated extracts was carried out on a

Hewlett Packard 6890 gas chromatograph equipped with a flame ionisation detector.

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Column: Chrompack SimDist Viti Metal, 10 m x 0.53 mm i.d., 0.17 11m coating.
Carriergas: Nitrogen. Injection method: Samples were dissolved in 200 III
chloroform/methanol (2: 1) and 2 III was injected with a 20: 1 split ratio. .Temperature
program: Injector 250°C, initial column temperature 130°C, ramp rate 5°C.min-I, final
column temperature 390°C.

3.1.3.6 Gas chromatography ofTMSH methylated extracts

Gas chromatography analyses of TMSH treated extracts were carried out on a Hewlett
Packard 5890 Series II gas chromatograph equipped with a flame ionisation detector.
Column: Supelcowax-lO, 30 m x 0.53 mm i.d., 1 11m coating. Carriergas: Nitrogen.
Injection method: Samples (still dissolved in the methylation 'solvents) were injected with
a HP 7673 autosampler at a 40: 1 split ratio. Temperature program: Injector 220°C, initial
column temperature 145°C held for 3 min, ramp rate 3°C.min-I, final column temperature

245°C.

3.1.3. 7 Gas chromatography - Mass spectrometry analyses (GC-MS)

Gas chromatograph: GC-MS analysis was carried out on a Hewlett Packard 5890 Series
II gas "chromatograph coupled to a HP 5972 quadropole mass spectrometer. Column: HP-
5 60 m fused silica capillary, 0.28 mm i.d. 0.25 11m coating. Carriergas: He. Injection
method: Samples were dissolved in 200111 chloroform/methanol (2: 1) and 2 III was
injected with a 50: 1 split. Temperature program: Injector 250°C, initial column
temperature 130°C, ramp rate 6°C.min-I, final column temperature 320°C. Mass
spectrometer parameters: EMV 1447, scan 20 to 690 atomic mass units, GC-MS
interphase temperature 280°C.

3.1.4 Results and discussion

3.1.4.1 The composition of E. grandis wood extract

Ohtani et al. (1986) and Ohtani and Shigemoto (1991) reported that the high molecular
mass components of several hardwoods used in Japanese kraft pulping mills as well as
pitch from these mills were mostly polymerised aliphatic hydrocarbons. The polymerised

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aliphatic hydrocarbons were primarily unsaturated fatty acids and alcohols that
underwent condensation reactions. They found that the more double bonds the
hydrocarbons had, the faster the condensation reactions took place. Fatty acids were
polymerised faster than fatty alcohols. Extensively polymerised aliphatic hydrocarbons
are not soluble in organic solvents (Ohtani et al. 1986). Consequently, it can be expected
that some of the higher molecular weight polymerised compounds would not be removed
from wood or pulp by dichloromethane extractions.

Triglycerides, which have molecular weights in the range of 800-900 Da, were the
compounds with the' highest molecular weight that could be detected by GC analysis.
The <900 Da range comprised 71.4% of the total wood extract (Fig. I). The >900 Da
range accounted for 28.6% of total wood extract. The GPC peaks in the <200 Da range
represent terpenes and phenols and the one in the 200-900 Da range represents fatty
acids, sterols, mono-, di- and triglycerides. Gas chromatography showed that the major
contributors to the volatile component of the wood extract were fatty acids, sterols and
triglycerides (Table 1). One specific sterol, peak 12 in Fig. 2, comprised about 40% of
the total volatile wood extract. The mass spectra of sterols are characterised by among
other a combination of rnJz 43, rnJz 55 and rnJz 81 fragments. The mass spectrum of
~-sitosterol in particular is also characterised by the presence of fragments rnJz 303, rnJz
329 and the molecular ion at rnJz 414. The mass spectrum of peak 12 is illustrated in
Fig. 3 and exhibits the characteristic sterol and ~-sitosterol fragments, confirming that the

compound in question was ~-sitosterol. ~-sitosterol is reported to be a major fraction of

the sterol content of many hardwoods (Fengel and Wegener 1989).

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BID l!ID am BID 4ID 3ID am 'KID 0 am l!ID am BID 4ID 3ID am 1CIIJ 0
~ M:l8lJ<r.,..;gt

CDmipJipE!>d!a::t Dl ns ~edrat

am lID) am em
~
4ID 3ID am 'KID 0 am ita)
~~~-~
am BID 4:lD 3ID am 1<XD 0
M:HnJ..- \\ligt
Mieo.J ... wigt

E2 mi pJpe4Ja::t BEBtlsl!rJ<cI:de" mi pJpe4ra::t

~. ~~
BID lID) em em 4ID 3ID am 101) 0 BID lID) em em 4ID 3ID am 101) 0
M:ieaJa-...,;gt M::lectJa-....,;gt

Fig. 1. Gel permeation chromatograms showing the change in extractive composition dwing the pulping
and bleaching process.

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Table 1. A comparison of the composition of E. grandis

heartwood and sapwood extract
Heartwood Sapwood

Total extractives" 0.39 0.37

Terpenes and phenols 3.6 2.1

Free fatty acids 22.9 22.7
16:0 7.7 13.2
18:2 5.9 2.7
18:1 2.4 1.6
18:0 0.7 1.0
20:0 0.4 0.6
22:0 0.9 1.0
23:0 0.0 0.5
24:Q 1.6 1.7
26:0 2.0 0.4
28:0 1.2 0.0

Sterols 47.8 47.3

~-sitosterol 39.0 45.9

Monogl ycerides 3.0 2.9

Diglycerides 0.0 0.0
Triglycerides 15.9 15.1

"Total extractives are given as a percentage of oven dry

weight. The remainder is given as a percentage of total
volatile extract.

The terpene and phenol content of the heartwood extract was higher than that of the
sapwood extract. Total free fatty acid content was much the same, however, there was a
marked difference in the fatty acid composition of the heartwood and sapwood. Fatty
acids 16:0,18:2 and 18:1 were abundant in both, with 16:0 being much more prevalent in
the sapwood than the heartwood. Upon derivatisation with TMSH the fatty acid ratio
(16:0):(18:2):(18: 1):(18:0) would typically change from 9:8:2: 1 to 7: 10:3: 1, indicating an
increase in fatty acids 18:2 and 18:1 relative to 16:0 and 18:0. Consequently fatty acids
18:2 and 18:1 were the predominant esterified fatty acids in the wood extract.
Monoglycerides generally constituted about 3% of the total extract. Diglyceride content
of the wood extract was rarely higher than 3% and frequently none could be detected.
There was little difference in the triglyceride content of the sapwood and heartwood.
Triglyceride content was usually within the range of 15-30% of volatile wood extract.

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2 12
pA ..~
3

'"4
400

300

200

100

0
10 min

Fig. 2. Gas chromatogram of diazomethane treated E. grandis extract. Retention time ranges
determined with standards: 1-5.6, terpenes and phenols; 19.1-20.3, monoglycerides; 22.9-30, sterols; 32-
40, diglycerides; 40-46.5, triglycerides. Peaks identified by GC-MS and/or the use of standards: 1,
phenol; 2, fatty acid (16:0); 3, fatty acid (18:2); 4, fatty acid (18:1); 5, fatty acid (18:0); 6, fatty acid
(20:0); 7, fatty acid (22:0); 8, fatty acid (23:0); 9, fatty acid (24:0); l O, fatty acid (26:0); II, fatty a:cid
(28:0); 12, J3-sitosterol.

.Ab.lrrln:e
43
4&10
4000
EO
:mo ss
2IlO HO Molecular weight = 414.72

2lJO
Bl 1C5
15JO 145 M
119
414
1000
5lJ

rw'z->O 0

Fig. 3. The mass spectra of peak 12, exhibiting the fragmentation pattern
of J3-sitosterol. Key: M = molecular ion.

3.1.4.2 The composition of Eucalyptus spp. pulp extract

GC analysis of pulp and pitch revealed the formation of four compounds that did not
occur in the wood extract initially (Fig. 4). The first four unknown peaks occurred at a
4.6 min retention time offset from respectively the 16:0, 18:2, 18: 1 and 18:0 fatty acid
peaks, indicating that the compounds are derivatives of these fatty acids. The mass
spectrum of fatty acid 16:0 (palmitic acid) is compared with its corresponding peak at a
4.6 min offset in Fig. 5. The base peak of palmitic acid was formed due to the
McLafferty rearrangement where a hydrogen atom is donated, resulting in the formation
of fragment m/z 74 (Budzikiewicz et al. 1967). The mass spectrum of the peak

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corresponding to palmitic acid is an identical match to the spectrum of

N, N,-dimethylpalmitamide. The mass spectra of the other three peaks showed that they
were also amide derivatives of respectively fatty acids 18:2, 18:1 and 18:0. Amide
derivatives of fatty acids have also been detected in mechanical pulp effluents (Eriksson
1999). The origin of these compounds may be the additives utilised by the mill.

0(18:2)
0(16:0)
pA .... \ ~ 0(18:1)
16:0 . ._.;
120

100
<:
80

00

40

20

10 20 30 40 min

Fig. 4. Gas chromatogram ofE2 mat pulp extract showing the four fatty acid derivative peaks which occurred
at a 4.6 min offset from the corresponding fatty acid peaks. Key: D = derivative.

The presence of amide derivatives of fatty acids can be ascribed to the use of non-ionic
organic additives provided by Buckman Laboratories (Pty) Limited. BUBREAK® 4051
is a brown stock drainage aid that also possesses the properties of a washing aid. It is
added at the brown stock washing stage (1. 1 kg/ton dry pulp) to improve drainage of the
cooking liqour. It is added again after screening of the washed brown stock.
BUBREAK® 4051 is specifically engineered to stick to the pulp. BUSPERSE® 47 is a
pitch dispersant added before the screening decker (150 g/ton dry pulp), after the
screening decker (250 g/ton dry pulp), before the El bleaching stage (400 g/ton dry pulp)
and before the E2 bleaching stage (400 g/ton dry pulp). Tale is added after the E2 filter
(4.2 kg/ton dry pulp). BUSPERSE® 2219, a dispersant, is added after the D2 filter
(1.9 kg/ton dry pulp). The fact that the additives enter process waters at an early stage
was reflected in the analysis results. The washed brown stock pulp already contained
fatty acid amides (Table 2).

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Table 2. A comparison of the composition of EucalYPtus spp. pulp extracts from different stages
of the pulping and bleaching process.

Brown CD mat Dl inlet E2mat Bleach stock

stock decker mat

Total extractive content" 0.15 0.26 0.19 0.16 0.22

Terpenes and phenols 1.4 5.8 1.6 1.2 1.3

Fatty acids 23.7 25.0 24.0 13.9 24.0

16:0 11.6 12.5 13.1 6.7 13.7
18:2 0.5 0.4 0.8 1.0 0.0
18:1 6.4 0.5 1.0 l.l 1.1
18:0 1.6 2.1 2.7 1.3 2.7
20:0 0.6 1.2 1.4 0.7 1.3
22:0 0.8 2.6 3.4 1.0 1.3
23:0 0.6 0.5 0.7 0.0 1.2
24:0 1.1 3.6 4.6 1.6 1.9
26:0 0.6 1.6 2.4 0.6 0.8
28:0 0.0 0.0 0.0 0.0 0.0

Sterols 51.9 28.8 30.1 21.6 20.9

P-sitostero1 30.5 5.8 6.2 4.2 3.2

Monoglycerides 1.5 3.2 8.4 3.6 8.4

Dig1ycerides 2.3 1.0 0.0 0.0 0.0
Triglycerides 0.0 0.0 0.0 0.0 0.0

Amide derivatives 7.9 6.1 8.7 47.9 29.0

16:0 2.2 3.1 5.1 10.5 15.0
18:2 1.3 1.3 0.0 19.2 2.7
18:1 3.3 0.3 0.6 13.5 4.6
18:0 1.0 1.5 3.0 4.7 6.6

-Total extractives are gIven as a percentage of oven dry weight. The remainder IS gIVen as a
percentage of total volatile extract.

Non-ionic agents are extremely stable in acid and alkaline solutions. There are three
principle types: alkyl phenol-ethylene oxide, aliphatic poly hydric alcohol esters, and fatty
acid amides (Shreve 1956). The type used in this case is probably fatty acid amides,
which accounts for the fatty acid amides found in the pulp extracts and pitch. Fatty acid
amides used as detergents could be of poly hydroxy lie constitution (Webb 1964), in which
case there had to be some molecular modification of the additives, for the fatty acid
amides found had no hydroxyl groups. Molecular modification is, however, improbable
due to the stability of non-ionic additives. The only source for amide groups up to the
point of high density storage of washed brown stock was the amide derivatives added
after brown stock washing. There were not any extreme pH fluctuations after the
addition of the additives before the brown stock decker and the sampling of brown stock

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decker mat pulp. Taking this fact into account as well as the stability of the additives, it
follows that the fatty acid amides found were constituents of the additives.

o
74

llllXl
\,l ?
173-->74 ca,
2)1lXl
Molecular weight = 270.46

151lXl

101lXl
M
1n 185199 213227239 allo
nVz_>ol.--"..u...-'1'-~~~~~~~~~~~~~.

Molecular weight = 283.50

10000 M
~ ~~$rn~~ ~~a~~
<I) -10 £Il 00 100 1<lJ 1-10 1£1l 100 an zn 2-10 zn zn

Fig. 5. The mass spectrum of methyl palmitate, methyl ester of

fatty acid 16:0, and its amide derivative,
N, N-dimethylpalmitamide. The McLafferty rearrangement
(Budzikiewicz et al. 1967) caused the formation of fragment
mlz 74 in the case of methyl palmitate and fragment mlz 87 in
the case of N, N-dimethylpalmitamide. Key: M = molecular
ion.

Affleck and Ryan (1969) reported that the ethanol/benzene (1 :2) extractive content of the
pulp from a kraft mill with a CEDED bleach plant decreased significantly due to
washing, bleaching and caustic extraction. The total DCM extractive content of the
bleached pulp is higher than that of the brown stock pulp. The first bleaching stage, the
CD stage, had a significant effect on total extractive content and pulp extract composition
(Fig. 1). A large portion of the sterols was washed out (Table 2) and the reduction is
reflected in Fig. 1. The fatty acid amides constituted a large proportion of the pulp
extracts from the latter stages of bleaching. The fatty acid ratio
(16:0):(18:2):(18:1):(18:0) for the E2 mat pulp extract differs sharply with the ratios of
the other stages of bleaching. Fatty acid 16:0 content is lower relative to the other fatty
acids. Talc was added to the bleaching process after the E2 filter as it is known that talc

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reduces the stickiness of pitch particles by adsorbing onto the particles. The mechanism
does not involve attachment of particles to fibres (Hassler 1988). There was a significant
reduction in the proportion of amide derivatives and increase in the proportion of fatty
acids from E2 mat pulp extract to bleach stock decker mat pulp extract (Table 2).

Figure 1 shows the gel permeation chromatograms of E. grand is and pulp extracts from
different stages in the pulping and bleaching process. The same compounds as discussed
under E. grandis wood extract were present in the pulp extracts. However, in the latter
stages of bleaching a significant proportion of the 250-350 range was represented by
amide derivatives offatty acids 16:0, 18:2, 18:1 and 18:0 (Table 2). The first graph in
Fig. 1 shows the extractive composition of E. grand is and the second the composition of
brown stock decker inlet pulp extract. Due to the digestion process the contribution of
the >900 Da range changed from 28.6% to 30.1%. The >900 range comprised 51.6% of
bleach stock decker mat pulp extract, indicating that the high molecular weight
compounds were not easily removed.

3.1.4.3 Pitch composition

A lot of pulp fibres were attached to the sticky pitch deposits. Free fatty acids and fatty
acid amides constituted a large portion of the volatile fraction of pitch (Table 3). Most of
the amide derivatives were N, N-dimethylpalmitamide. It should be noted that the
additives did not intensify pitch deposition. Its presence in the small amounts of pitch
still depositing is only an artefact of its presence in process waters. The pulp mill
experienced minimal pitch deposition since the use of additives was instigated.

Triglycerides constituted 10.1% of the volatile pitch deposits. Pulp extracts contained no
triglycerides. This indicates either that triglycerides were in suspension in the white
water or that very small amounts carried by the pulp deposited over an extended period of
time. It is also possible that the triglycerides were deposited in periods when feedstock
with a high triglyceride content was pulped.

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Table 3. The composition of pitch. All

values are given as a percentage of total
volatile extract.
Component Content

Terpenes and 0.8

phenols
Free fatty acids 21.7
16:0 6.7
18:2 0.0
l8:l 0.0
18:0 1.7
20:0 0.6
22:0 2.0
23:0 2.1
24:0 4.6
26:0 3.6
28:0 0.4

Sterols 11.5
j3-sitosterol 8.5

Monogl ycerides 3.6

Diglycerides 3.8
Triglycerides 1O.l

Amide derivatives 38.3

16:0 20.0
18:2 1.2
18:1 9.8
18:0 7.2

3.1.5 Conclusions

The dichloromethane extractable levels remained much the same despite washing and
bleaching stages. High molecular weight compounds constituted a large portion of wood
and pulp extracts. Approximately 40% of volatile E. grand is extract was identified as
B-sitosterol. Triglycerides constituted 10% and amide derivatives of fatty acids 16:0,
18:2, 18:1 and 18:0 from additives made up 38.3% of the volatile fraction of pitch
deposits. Wallis and We arne (1999) found a substantial amount of steryl esters in the
dichloromethane extracts of E. globulus. In our investigation on E. grandis extracts, no
significant amounts of steryl esters were found, indicating that there could be significant
differences in the extract composition of different Eucalyptus species.

The composition of the E2 mat pulp extract differed considerably from pulp extracts from
other stages. The increase in fatty acid amides and decrease in fatty acids may be
ascribed to extreme conditions in the second caustic extraction stage. However, the first

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extraction stage does not have such a marked effect on the pulp extract. The decrease in
fatty acid ami des in the bleach stock decker mat pulp extract as well as the normalisation
of fatty acid levels may be attributed tb the addition of tale and the change in process
conditions.

Kraft cooking eliminated triglycerides from the pulp extract. Wallis and Wearne (1999)
also found that kraft cooking eliminated triglycerides from E. globulus pulp extract. The
fact that triglycerides were found in the pitch deposits indicate either that triglycerides
were in suspension in the white water or that minute amounts carried by the pulp
deposited over an extended period of time. It is also possible that the triglycerides were
deposited in periods when feedstock with a high triglyceride content was pulped. As
reported by Farrell et al. (1997), triglycerides may also be the key to pitch deposition in
this case. Due to their sticky nature only small amounts of triglycerides are needed to
induce pitch deposition.

3.1.6 Acknowledgements

The authors are grateful to Mondi Kraft Ltd., The Foundation for Research and
Development as well as The Technology and Human Resources for Industry Programme
for supporting this project.

3.1.7 References

Affleck, R.R. and R.G. Ryan. 1969. Pitch control in a kraft pulp mill. Pulp and Paper
Magazine of Canada 70: 107-111.
Allen, L.H. 1977. Pitch in wood pulps. Trend (pointe Claire, Canada) 26: 4-10.
Budzikiewicz, H., C. Djerassi and D.H. Williams. 1967. Mass Spectrometry of Organic
Compounds. Holden-Day, Inc. San Francisco.
Butte, W. 1983. Rapid method for the determination of fatty acid profiles from fats and
oils using trimethylsulphonium hydroxide for transesterification. Journal of
Chromatography 261: 142-145.
Chafe, S.C. 1987. Collapse, volumetric shrinkage, specific gravity and extractives in
Eucalyptus and other species. Wood Science and Technology 21: 27-41.

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 Stellenbosch University http://scholar.sun.ac.za

Christie, W.W. 1992. Gas Chromatography and Lipids: A Practical Guide. W. W.

Christie, ed. The Oily Press, Ayr, Scotland.
Dreisbach, D.D. and D.L. Michalopoulos. 1989. Understanding the behaviour of pitch in
pulp and paper mills. Tappi Journal 72 (6): 129-134.
Eriksson, KE. 1999. Personal Communication.
Farrell, RL., K Hata and M.B. Wall. 1997. Solving pitch problems in pulp and paper
processes by the use of enzymes or fungi. Advances in Biochemical
EngineeringlBiotechnology 57: 198-212. Springer-Verlag, Berlin.
Fengel, D. and G. Wegener. 1989. Wood: Chemistry, Ultrastructure, Reactions. Walter
de Gruyter, Berlin.
Fischer, K and K Messner. 1992. Reducing troublesome pitch in pulp mills by lipolytic
enzymes. Tappi Journal 75: 130-134.
Fujita, Y, H. Awaji, H. Taneda, M. Matsukura, K. Hata, H. Shimoto, M. Sharyo, H.'
Sakaguchi and K Gibson. 1992. Recent advances in enzymatic pitch control. Tappi
Journal 75: 117-122.
Hassler, T. 1988. Pitch deposition in papermaking and the function of pitch-control
agents. Tappi Journal 71(6): 195-20l.
Hillis, W.E. and A. Carle. 1959. The influence of extractives on Eucalypt pulping and
papermaking. Appita 13, 74-83.
Laubach, G.D. and C.S. Greer. 1991. Pitch deposit awareness and control. Tappi Journal
74 (6): 249-252.
Nelson, P.F., lG. Smith and W.D. Young. 1970. The influence of extractives on some
properties of Eucalypt Kraft pulp. Appita 24: 101-107.
Ohtani, Y and T. Shigemoto. 1991. Chemical aspects of pitch from Japanese pulp and
paper mills. Appita 44 (1): 29-32.
Ohtani, Y, T. Shigemoto and A. Okagawa. 1986. Chemical aspects of pitch deposits in
kraft pulping of hardwoods in Japanese mills. Appita 39 (4): 301-306.
Shreve, RN. 1956. The Chemical Process Industries. McGraw-Hill Book Company, New
York.
Suckling, I.D. and RM. Ede. 1990. A quantitative 13C nuclear magnetic resonance
method for the analysis of wood extractives and pitch samples. Appita 43: 77-80.

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 Stellenbosch University http://scholar.sun.ac.za

Wallis, AF.A and R.H. Wearne. 1999. Analysis of resin in eucalypt woods and pulps.
Appita 52: 295-299
Webb, F.C. 1964. Biochemical Engineering. D. Van Nostrand Company Ltd.London.
Yazaki, Y., P.l Collins and T. Iwashina. 1993. Extractives from Blackbutt (Eucalyptus
pilularis) wood which affect gluebond quality of phenolic resins. Holzforschung 47:
412-418.

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Chapter 4: Manuscript submitted for

publication in Biotechnology Letters .

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4.1 Lipase production and activity of fungi isolated from South African
forests

Gerhardus C. Scheepers'>, Tim Rypstra', Theodorus H. de Koker2 and Bernard J.H.

Janse'

IDepartment of Wood Science, University of Stellenbosch, Stellenbosch, 7600, South

Africa, email: 9303731@land.sun.ac.za, fax: +2721 8083611
2Department of Microbiology, University of Stellenbosch, Stellenbosch, 7600, South
Africa
3Mondi Forests, P. O. Box 39, Pietermaritzburg, 3200, South Africa.

Filamentous fungi isolated from indigenous and commercial forests in South Africa, were
screened for lipase activity on tributyrin and Tween 80. Eight strains were selected and
the tributyrin and Tween 80 assays were repeated by monitoring lipase activity over a
seven-day period. The selected strains were also assayed for their activity toward p-
nitro phenyl palmitate. Ophiostoma piliferum Cartapip 58™ and Phanerochaete
chrysosporium BKM-F-1767, two strains used commercially in the pulp and paper
industry, were used as controls. A few of the strains compared well and even
outperformed the control strains, indicating that there is potential for the use of these
strains in the pulp and paper industry for pitch control.

Keywords: fungi, lipase activity, triglycerides, Tween 80, tributyrin, p-nitrophenyl

palmitate, pitch control

4.1.1 Introduction

Lipases (acylglycerol acylhydrolases, EC 3.1.1.3) hydrolyse the ester bonds in tri-, di-,
and monoacylglycerols. Some will also degrade a fairly broad range of other compounds
containing an ester linkage. Lipases are important enzymes in industry, serving as
biocatalysts for fat and oil processing in medicine, food additives, diagnostic reagents and
detergents (Alhir et al., 1990; Derewenda, 1994; Jaeger et al., 1994; Soberón-Chávez and
Palmeros, 1994). Animals, plants and micro-organisms produce lipases. To date, the

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rnajonty of lipases used for biotechnological purposes have been isolated from
filamentous fungi since they produce extracellular enzymes (Comménil et al., 1995).

The extractive materials in wood often cause pitch problems in pulp mills. During the
pulping and bleaching process the extractives are released from the wood and pulp and
the organic deposits formed, called pitch, later stick to ceramic and metal parts as well as
the wires of the papermaking machines. Not all of the extractives are troublesome.
Wood triglycerides have been shown to be a major contributor to pitch deposition (Allen,
1977; Fujita et al., 1992; Suckling and Ede; 1990). Recently two new methods of
decreasing triglycerides in pulping and bleaching processes, and therefore combating
pitch, have been developed independently and are now used industrially. One is a
method using a fungus obtained through selective breeding from the sap-staining
organisms that are associated with weathering of wood. This Ascomycetous fungus is
non-pigmented arid avoids the staining and decrease in brightness normally associated
with the ageing of wood (Farrell, 1994). It is grown on wood chip piles prior to pulping.
The other method is the addition of lipase to the stock after the pulping process (Fujita,
1992). There are several reports describing the effectiveness of fungal biotechnology and
lipase use in decreasing the triglyceride content of wood extractives (Blanchette et al.,
1992; Brush et al., 1994; Farrell et al., 1997; Fischer and Messner, 1992; Fischer et al.,
1994; Fischer et al., 1995; Rocheleau et al., 1998). Consequently the investigation and
identification of fungi, especially white-rot fungi that produce lipase, can be of benefit to
the pulping industry. In this study, 381 fungal strains were screened for lipase production
and activity.

4.1.2 Materials and methods

4.1.2.1 Materials

Peptone, yeast extract, malt extract and potato dextrose agar were obtained from Merck.
Lipase from Rhizopus arrhizus, tributyrin, Tween 80 and p-nitrophenyl palmitate (pNPP)
were purchased from Sigma Chemical Co. All other reagents were analytical grade from
commercial sources.

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4.1.2.2 Fungal strains

Three hundred and eighty one fungal strains isolated from indigenous forests as well as
commercial Eucalyptus spp. and Pinus spp. plantations in South Africa (De Koker et al.,
1998) were screened for their lipase production and activity using the described Tween
80 and tributyrin methods. The eight best strains were re-evaluated over a seven-day
period with the same methods and were also assayed for their lipase production and
activity with pNPP as substrate. Cartapip 58™ (Ophiostoma piliferum), a commercial
fungal strain known for its depitching potential, was used as a control in the Tween 80,
tributyrin and pNPP assays. Phanerochaete chrysosporium BKM-F-1767, a fungal strain
known for its biopulping ability, was also used as control in the pNPP assay.

4.1.2.3 Tween 80 agar plate method

A basal medium consisting of 10 g peptone, 15 g potato dextrose agar,S g NaCl, 25 mg

bromocresol purple and 0.1 g CaCh.2H20 made up to 1 litre with distilled water was
prepared. The pH was adjusted to 5.4 with hydrochloric acid. A 10% (v/v) aqueous
Tween 80 solution was prepared by slowly adding 10 ml Tween 80 to 90 ml warmed (60-
70°C) distilled water. The agar and Tween 80 solutions were sterilised separately by
autoclaving for 15 min at 121°C. After the media cooled down to 65-70°C, one part
Tween 80 solution was added to nine parts basal medium. The medium was inoculated
with an agar plug and incubated for 7 days at 23°C except for the thermophilic fungi,
which were incubated at 50°C. A positive reaction was recorded as the development of a
purple zone. Every 24 hours the diameters of the purple zones were measured. Due to
the size of the plates, the growth and lipase active zones could only reach a maximum
diameter of 35 mm.

4.1.2.4 Tributyrin deep agar diffusion method

The medium, consisting of 0.5% peptone, 0.3% yeast extract, 1.5% potato dextrose agar,
and 0.1% tributyrin in distilled water, was sterilised by autoclaving for 15 min at 121°e.
The sterilised medium was dispensed into sterile test tubes (7 ml per test tube) and
allowed to cool down. The medium was inoculated with agar plugs of growing cultures
and incubated for 7 days at 23°C except for the thermophilic fungi, which were incubated

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at 50°C. A positive reaction was recorded as a clearing of the opaque medium. Every 24
hours the depth of the clearing zones was measured.

4.1.2.5 Assay of enzyme activity with pNPP as substrate

A pH 8 phosphate buffer containing 1 part 0.05 M KH2P04 and 19 parts 0.05 M

Na2HP04 was prepared. Thirty milligrams of pNPP was dissolved in 10 ml propan-2-ol.
A solution containing 0.207 g sodiumdeoxycholate (Sigma Chemical Co.) and 0.1 g gum
arabic (Sigma Chemical Co.) was dissolved in 90 ml of the prepared phosphate buffer.
The pNPP solution was added in small quantities to the sodiumdeoxycholate/gum arabic
solution while stirring continuously until all was dissolved. This solution, which served
as substrate, was dispensed into test tubes in 2.5 ml quantities. The pNPP substrate was
prewarmed at 37°C. Lipase containing culture supernatant (100 IJ.I) from the liquid
medium was added to the pNPP substrate and incubated at 37°C for 15 minutes. The
reaction was stopped by boiling the substrate for 3 minutes and then placed on ice.
Absorbance was measured at 410 nm against a blank consisting of water. The
commercial lipase from Rhizopus arrhizus was used to create calibration curves for lipase
activity. Fresh solutions were made up daily for the assay and to generate calibration
curves. According to Sigma Chemical Co. one unit of lipase will hydrolyse 1.0
microequivalent of fatty acid from a triglyceride in 1 hr at pH 7.7 at 37°C.

The selected strains were grown in 100 ml liquid medium containing 1% olive oil and
1.5% malt extract medium prior to inoculation in triplicate into the same medium for the
assay. The inoculum (10 ml) consisted of 10 day old fungal biomass which was
fragmented in a Waring blender. The medium was incubated for 7 days at 23°C except
for the thermophilic fungi, which were incubated at 50°C. Lipase activity in the culture
supernatant was measured throughout the incubation period.

4.1.2.6 Results and discussion

The preliminary Tween 80 agar plate trial results of the 381 screened fungal strains are
shown in Table 1. Most of the strains did not cause discolouration while 42 strains gave
a high degree of discoloration. In the following Tween 80 trial, the eight selected fungal
strains, except GTB 69, all gave a larger lipase active zone than the control Cartapip 58™

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(0. piliferum CAR 58) and all of them had a faster growth rate (Figs. 1 and 2). All the
strains, except GTB 69, MTZ 95 and P. pseudomagnoliae nom. provo did not clear the
medium as well as 0. piliferum CAR 58 (Table 1). From these results it is clear that most
of the strains tested, are better than the control strain 0. piliferum CAR 58.

Table 1 Results of the Tween 80 agar

plate screening of the 381 fungal strains.

Discoloration Number of strains

203
+ 91
++ 45
+++ 42

- no effect, + weak effect, ++ moderate

effect, +++ striking effect

The results of the eight selected strains and the control, 0. piliferum CAR 58, when
grown on tributyrin are shown in Figure 3 and listed in Table 2. The two thermophilic
Thermomyces lanuginoses strains, MBD 2D and MBD 4B 1, performed better than
Cartapip 58™ (0. piliferum CAR 58) in terms of lipase production (size of lipase active
zone) and was equal in lipase activity (clarity oflipase active zone). Strain MTZ 95 was
comparable in terms of the size of the lipase active zone, but did not match the lipase
activity of the control and the two thermophilic strains.

Table 2 Lipase activity of the eight selected strains and the control, O. piliferum
CAR 58, on tributyrin and Tween 80

Fungal strain Degree of clarity Degree of discoloration

(TributY[in) (Tween 80)

BLK 10A +++ +++

O. piliferum CAR 58 +++ ++
GTB69 ++ +
KWA 16 ++ +++
T. lanuginoses MEO 20 +++ +++
T. lanuginoses MEO 4B1 +++ +++
MTZ95 ++ +
MTZ97 +++ +++
p. e.seudoma9_noliae nom. [!rov. ++ +

+ weak effect; ++ moderate effect; +++ striking effect

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 Stellenbosch University http://scholar.sun.ac.za

40
35 __ BLKI0A __ BLK 10A
35
30 __ O. pilirerum CAR 58 ____0. pilifetUm CAR 58
__ GTB69 30
"'_GTB69
'E25 __ ¥MIA 16 E 25 __ I<JIVA 16
E
.s
i20 ~ T. lanuginoses MD 20
~ 20 ___ T lanuginoses MED 20

o
0
....... T. lanuginoses ~ 481
~ 15
15 -+- T lanuginoses MEO 481
lO --+-MlZ 95
~10 10
-+-MlZ95
~MlZ97
~MlZ97
- P. pseuclanegno/i ... nom 5
_Po pseudcmagJOliae
prov.
0 0 ~~~~ __~ ~ __--,n=~
0 4 6 0 2 4 6 8
Day
Day

Figure 1 The size of the zones of the Figure 2 Growth rate of the eight
eight selected strains and the control, selected strains and the control,
CAR 58, on Tween 80. CAR 58, on Tween 80.

0.00
30 0.00
__ BLKIOA 0.70
III
25 ___ O. pilifenJm CAR 58 'Ë O.ED
__ GTll69
; 0.!:iJ
E20
.s
Cl>
__ K'I\A 16
~ 0.40
__ T. lanuginosas M3:l 2D
515
N
an 0.3:1
____ T. lanuginoses M3:l4B'
Cl>
<Il
0.20
[10 --+-MlZ95
:::; 0.10
____ MlZ97
5
...,.........,_.........,'' ' ' ' L.,--J_
0.00 +""''''-r''--.-''''''''...,.....'''-r'' ..................
- P pseucbmagnoliae
nom prcw.
0
0 2 4 6 8
Day

Figure 3 The size of the zones of the Figure 4 Cell-associated (Day 1) and free
eight selected strains and the control, (Day 4) lipase activity of eight selected
CAR 58, on tributyrin. strains and the controls, CAR 58 and
BKM-F-1767, on pNPP.

Rapp and Backhaus (1992) reported that the rate of hydrolysis of pNPP by microbial
lipases was in the range of 0.1-6% of the activity toward triolein, indicating that pNPP is
a poor substrate for many microbial lipases. In our study, low absorbance values were
also detected. White-rot erosion of the wood cell wall may be widespread or localised to
the immediate vicinity of the hyphae, forming a groove or trough with a central ridge on
which the hypha lies. If erosion is localised, the extracellular enzymes are associated
with the hyphae by an external layer of mucilage (Rayner and Boddy, 1988). Figure 4
shows the lipase activity in culture supernatant inoculated with fragmented biomass on
day 1 as well as the activity in the culture supernatant on day 4. Due to the fragmentation

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of the biomass prior to inoculation, cell-associated lipases (intracellular and/or mucilage-

bound lipases) were released into the culture supernatant giving cell-associated lipase
activity on day 1. The lipase activity found in the culture supernatant 'on day 4 is
attributed solely to lipases released into the supernatant without any agitation of the
biomass. These lipases are defined as free lipases. Three of the strains; Thermomyces
lanuginoses MED 2D, T lanuginoses MED 4B 1 and P. pseudomagnoliae nom. prov.,
showed a higher lipase activity on day 4 than day 1, indicating higher free lipase activity.
The control strain, O. piliferum CAR 58, had both high cell-associated and free lipase
activities. The other control strain, Phanerochaete chrysosporium BKM-F-1767, which
is also known to reduce the amount of total dichloromethane wood extractives (Farrell et
al., 1997), has a high cell-associated lipase activity and a low free lipase activity. Since
both control strains reduce total dichloromethane wood extractives, the inability to
produce free lipases does not seem to have a negative effect on the ability of a fungus to
hydrolyse wood extractives while growing on wood.

4.1.3 Conclusions

The strains used in this study showed different levels of lipase activity toward the
different substrates, indicating that the lipases are substrate specific. A few of the strains
compared favourably and even outperformed the control strains, indicating the potential
for the use of these strains to break down the triglycerides in wood extractives.
Consequently these strains have the potential for application in the pulp and paper
industry for pitch control. The lipases from the Thermomyces lanuginoses strains,
MED 2D and MED 4B 1, have even more potential due to their ability to hydrolyse
triglycerides at the higher temperatures commonly found in industrial processes. The
eight strains isolated in South Africa will also be evaluated in our laboratory for their
biopulping and depitching potential.

4.1.4 Acknowledgments

The authors are grateful to Saralene Thomas for her technical assistance in screening the
381 fungal strains as well as Mondi Kraft Ltd., The Foundation for Research and

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Development, and The Technology and Human Resources for Industry Programme for
supporting this project.

4.1.5 References

Alhir, S, Markakis, P and Chandan, RC (1990), Journal of Agricultural and Food

Chemistry 38: 598-601
Allen, LH (1977), Trend (pointe Claire, Canada) 26: 4-10
Blanchette, RA, Farrell, RL, Burnes, TA, Wendler, PA, Zimmerman, W, Brush, TS and
Snyder, RA (1992), Tappi Journal75: 102-106
Brush, TS, Farrell, RL and Ho, C (1994), Tappi Journal77: 155-159
Comménil, P, Belingheri, L, Sancholle, Mand Dehorter, B (1995); Lipids 30: 351-356
De Koker, TH, Zhao, J, Allsop, SF and Janse, BJH (1998), In: Seventh International
Conference on Biotechnology in the Pulp and Paper Industry, Poster Presentations
Vol. B, pp. B89-B91
Derewenda, ZS (1994), Advances in Protein Chemistry 45: 1-52
Farrell, RL, Blanchette, RA, Brush, IS, Iverson, S and Fritz AR (1994), In: Biological
Sciences Symposium, Tappi, pp. 85-87
Farrell, RL, Hata, K and Wall, MB (1997), Advances in Biochemical
Engineering/Biotechnology 57: 197-212
Fischer, K, Akhtar, M, Blanchette, RA, Burnes, TA, Messner, K and Kirk, TK (1994),
Holzforschung 48: 285-290
Fischer, K, Akhtar, M, Messner, K, Blanchette, RA and Kirk, TK (1995), In: Proceedings
of the 6th International Conference on Biotechnology in the Pulp and Paper Industry:
Advances in Applied and Fundamental Research, E Srebotnik and K Messner, eds pp.
193-196, Facultas-Universitaetsverlag, Vienna, Austria
Fischer, K and Messner, K (1992), Tappi Journal75: 130-134
Fujita, Y, Awaji, H, Taneda, H, Matsukura, M, Hata, K, Shimoto, H, Sharyo, M,
Sakaguchi, H and Gibson, K (1992), Tappi Journal75: 117-122
Jaeger K, Ransac, S, Dijkstra, BW, Colson, C, Van Heuvel, M and Misset, 0 (1994),
FEMS Microbiology Reviews 15: 29-63
Rapp, P and Backhaus, S (1992), Enzyme and Microbial Technology 14: 938-943

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Rayner, ADM and Boddy, L (1988), Fungal Decomposition of Wood: Its Biology and
Ecology, Chichester, West Sussex: John Wiley and Sons Ltd.
Rocheleau, MJ, Sitholé, BB, Allen, Lil, Iverson, S, Farrell, Rand Noël, Y (1998),
Journal of Pulp and Paper Science 24: 37-42
Soberón-Chávez, G and Palmeros, B (1994), Critical Reviews in Microbiology 20:

95-105
Suckling, ID and Ede, RM (1990), Appita 43: 77-80

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Chapter 5: Manuscript submitted for

publication in Biotechnology Letters

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5.1 Enzymatic Pitch Control in the Kraft Pulping of Eucalyptus grandis

Gerhardus C. Scheepers', Tim Rypstra', Theodorus H. de Koker2 and Bernard J.H. Janse'

1 Department of Wood Science, University of Stellenbosch, Stellenbosch, 7602, South

Africa, email: 9303731@land.sun.ac.za, fax: +27218083611
2 Department of Microbiology, University of Stellenbosch, Stellenbosch, 7602, South
Africa
3 Mondi Forests, P. O. Box 39, Pietermaritzburg, 3200, South Africa

The effect of pretreatment with eight fungal strains isolated in South Africa on
Eucalyptus grandis wood- and pulp extractives was determined. Ophiostoma piliferum
Cartapip 58™ and Phanerochaete chrysosporium BKM-F-1767 were used as control
strains. Several of the strains compared well to the control strains in their ability to
reduce the triglyceride content of wood extract. The white-rot Phanerochaete
psuedomagnoliae nom. provo gave better results than both the control strains.
Consequently it can act as an agent for both biopulping and biodepitching. Treated
samples did not show a significant difference in pulp triglyceride content or pulp
characteristics compared to the controls. The effect of commercial lipases on deposited
brown stock pulp extract was also evaluated. The lipases did not reduce the triglyceride
content of the deposited extract.

Keywords: Fungi, biotechnology, pulp, pitch, extractives, triglycerides, lipase

5.1.1 Introduction

Because of extensive planting in the humid regions of South Africa, Eucalyptus species
have become of economic importance to the South African sawmilling and pulp and
paper industries. The extractives of the Eucalyptus genera have a detrimental effect
during both pulping and sawmilling operations (Chafe, 1987; Hillis and Carle, 1959;
Nelson et al., 1970; Yazaki et al., 1993). The extractives increase consumption of
pulping chemicals and impair the colour and brightness of unbleached pulp (Nelson et
al., 1970). Pitch is a sticky organic deposit found on pulping equipment that is caused by
wood extractives. During the pulping and bleaching process the extractives are released
from the wood and pulp and later stick to ceramic and metal parts as well as the wires of

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the papermaking machines. Wood triglycerides are major contributors to pitch deposition
(Allen, 1977; Fischer and Messner, 1992; Fujita et al., 1992; Suckling and Ede, 1990).
Recently two new methods of decreasing triglycerides in pulping and bleaching processes
have been developed independently and are now used industrially. Both methods are
aimed at hydrolysing the triglycerides. One is the use of fungal biotechnology to
decrease triglyceride content of wood (Blanchette et al., 1992; Brush et al., 1994; Farrell
et al., 1997; Fischer et al., 1994; Fischer et al., 1995; Rocheleau et al., 1998). The other
method is the addition of lipase to pulp in a watery suspension (Fujita et al., 1992;
Fischer and Messner, 1992).

The screening of fungi that hydrolyse wood triglycerides, especially white-rot fungi, is of
importance to the pulping industry. It would be ideal to use one fungal strain for both
biopulping and biodepitching. In this study the ability of fungal strains isolated in South
Africa to decrease wood and pulp triglyceride content was evaluated. Commercial
lipases decrease triglyceride content of pulp in suspension (Fujita et al., 1992; Fischer
and Messner, 1992). In this study the effect of lipase addition on deposited brown stock
pulp extract was determined as well.

5.1.2 Experimental

5.1.2.1 Wood and fungal strains

Freshly delivered 6 year old Eucalyptus grandis logs were obtained from the
KwaZulu/Natal region in South Africa. The logs are left in the plantations for a few
weeks before delivery. The logs were hand debarked and chipped. Eight fungal strains
isolated in South Africa were selected on the basis of their lipase activity (Scheepers et
al.,2000). Ophiostomapiliferum Cartapip 58™, a commercial fungal strain known for its
depitching potential, was used as a control strain in the evaluation of the effect of the
fungi on wood extractives. Phanerochaete chrysosporium BKM-F-1767, a fungal strain
known for its biopulping ability, was used as control strain in the pulping trial.

5.1.2.2 Dichloromethane extractions

The moisture and extractive contents of wood and pulp samples were determined
according to Tappi method T 204 om-88. The wood to be extracted was ground in a

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Wiley mill with a 0.40 mm (40 mesh) SIeve. Two thimbles per wood sample, each
containing approximately two grams of air dry ground wood (1 g heartwood and 1 g
sapwood), were extracted with dichloromethane for 5 hours. Two thimbles per pulp
sample, each containing approximately 109 of air dry pulp, were extracted with
dichloromethane for 5 hours. The extracts were dried in a fume hood, weighed to
determine the total amount of extractives present and analysed by gas chromatography.

5.1.2.3 Wood chip treatment trial

The strains used in this trial are listed in Table 1. The strains were incubated in triplicate
in liquid medium containing 1.5% malt extract, 0.5% yeast extract and 1% olive oil until
a mycelial mat has formed. The mycelia were fragmented in a Waring blender prior to
inoculation in triplicate on 100 g (oven dry equivalent) ofautoclaved (121°C for 15 min)
Eucalyptus grand is wood chips at 70% moisture content based on dry weight. The
samples were incubated for four weeks in a cotton wool stoppered container at 30°C
except for Thermomyces lanuginoses MED 2D and Thermomyces lanuginoses MED 4B 1,
which were incubated at 50°C. After incubation the total dichloromethane extractives of
each sample were determined. The dried extract was stored at -20°C for GC analysis.

Table 1 The strains used for the two fungal biotechnology trials

Strains used for wood chip treatment Strains used for pulping trial
trial

BLK 10A P. chrysosporium BKM-F-1767

O. pi/iferum CAR 58 BLK 10A
GTB69 Thermomyces lanuginoses MEO 4B1
KWA16 MTZ95
T. lanuginoses MEO 20 P. pseudomagnoliae nom. provo
T. lanuginoses MEO 4B1
MTZ95
MTZ97
P. psuedomagnoliae nom. prov

5.1.2.4 Pulping trial

The strains used in this trial are listed in Table 1. Inoculum was prepared in the same
way as with the wood chip treatment trial. 500 g (oven dry equivalent) of autoclaved
(121°C for 15 min) Eucalyptus grandis wood chips at 70% moisture content based on dry
weight was inoculated. The samples were incubated for four weeks in cotton wool
stoppered containers at 30°C except for Thermomyces lanuginoses MED 4B1, which was

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incubated at 50°C. Two hundred grams of the wood chips were removed after incubation
for dichloromethane extractions while the rest was kraft pulped. Triplicates were pooled
and pulped together to produce only one pulp sample per fungal strain. Pulp
characteristics were evaluated. The pulp was also extracted with dichloromethane.
Samples were stored at -20°C for GC analysis.

5.1.2.5 Lipase treatment of deposited brown stock pulp extractives

Three brown stock pulp extract samples were incubated with purified lipase from Mucor
javanicus and another three with purified lipase from Rhizopus arrhizus (Sigma Chemical
Co.). One negative control with only buffer and two positive controls with autoclaved
enzyme solutions from each of the lipases were used. Thirty milligrams of brown stock
pulp dichloromethane extractives was dried down in glass vials, forming a deposit on the
glass. The deposited extract was incubated with 0.5 mg of the lipases in 10 ml pH 7.7
phosphate buffer at 37°C for 4 hours. 0.5 mg yields 200 U of lipase from Mucor
javanicus and 195 U Rhizopus arrhizus lipase. One unit of lipase will hydrolyse 1.0
microequivalent of fatty acid from a triglyceride in 1 hr at pH 7.7 at 37°C. At these
conditions only 68 U would be needed to hydrolyse 30 mg of triglycerides in 1 hour.
Because of this and since triglycerides constitute a small fraction of the total brown stock
pulp extract, the amount of lipase used was more than adequate to hydrolyse all the
triglycerides present in 4 hours. After incubation all the samples were freeze dried, the
triplicate samples were combined and stored at -20°C.

5.1.2.6 Gas chromatography of wood and pulp extracts

All carboxylic acids present in the wood or pulp extracts were methylated by using
diazomethane. The methylation with and preparation of diazomethane was done
according to the methods of Christie (1992). GC analyses of diazomethane methylated
extracts were carried out on a Hewlett Packard 6890 gas chromatograph equipped with a
flame ionisation detector. Column: Chrompack SimDist Ulti Metal, 10 m x 0.53 mm i.d.,
0.17 11mcoating. Carriergas: Nitrogen. Injection method: 10 mg of each sample was
dissolved in 200 III chloroform/methanol (2: 1) and 2 III was injected with a 20: 1 split

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ratio. Temperature program: Injector 250°C, initial column temperature 130°C, ramp
rate 5°C min-I, final column temperature 390°C.

5.1.3 Results and discussion

5.1.3.1 Wood chip treatment trial

The extractive composition of the sterile controls varied considerably and made it
difficult to compare strains in their ability to reduce triglyceride content. The major
components of the extract of the E. grand is used in this study have been identified
(Scheepers et al., 2000). The wood extract consists of approximately 30% high
molecular weight compounds and 70% low molecular weight (volatile) compounds. GC
analyses only describe the composition of the low molecular weight compounds. Table 2
presents the effect of the different fungal strains on the composition of the volatile wood
extract. All eight strains were evaluated with Ophiostoma piliferum CAR 58 as control
strain. BLK lOA, GTB 69, MTZ 95 and P. pseudomagnoliae nom. provo all whitened the
wood during growth. None of the nine fungal strains decreased the total extractive
content although all, except T. lanuginoses MED 4Bl, decreased the triglyceride content.
The increase in extractive content could be attributed to the presence of fungal mycelia.

KW A 16 was the only fungal strain that decreased free fatty acid content, while
BLK lOA was the only fungus that decreased diglyceride content compared to the sterile
controls. Fatty acid 18: 1 increased substantially in most cases. Analyses done on
E. grand is wood extracts have shown that most of the esterified fatty acids are 18:1 and
18:2 fatty acids (Scheepers et al., 2000). Hydrolysis of triglycerides would therefore
result in an increase in fattty acid 18:1 and 18:2 content. 0. piliferum CAR 58 gave
results comparable to the white-rots. All the white-rots except GTB 69 decreased
triglyceride content to a lower final level than CAR 58. The final triglyceride content of
MTZ 97 treated samples was substantially lower than that of the other strains. The initial
triglyceride content of the treated samples may have been very low, giving a false
impression of the ability of the fungus to reduce the triglyceride content. T. lanuginoses

strains, MED 2D and MED 4B 1, had little effect on total extractive content. However,
the terpene and phenol as well as sterol contents were considerably reduced by the T.
lanuginoses strains. Both increased the triglyceride, diglyceride and fatty acid contents.

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with fatty acid 18:1 content changing most. It is important to note that the most abundant
fatty acids in fungi are 16:0,16:1,18:0,18:1,18:2 and 18:3 (Kock and Botha, 1998). A
dramatic increase in fatty acid 18:1, under conditions where the wood esters were not
hydrolysed, could therefore be linked to the fatty acid contribution of the fungus.
KWA16 decreased terpene and phenol, fatty acid and triglyceride content while
. .
mcreasmg sterol content. MTZ 97 increased total extractive content fourfold due to a
dramatic Increase in fatty acid content as well as diglyceride content. Terpene and
phenol, sterol and triglyceride content did not decrease much when the increase in total
extract is considered ..

Table2 Average extractive content and composition of sterile E grandis treated with nine different
fungal strains
BLK IDA 0.pil/forum GTB69 KWA 16 T. lanuginoses
CAR 58 ME02D
Sterile Treated Sterile Treated Sterile Treated Sterile Treated Sterile Treated

Total extractives
. controls

0.33
sameles

0.44
controls

0.31
sameles

0.32
controls

0.32
sameles

0.34
controls

0.31
sameles

0.31
controls

0.34
sameles

0.36

Terpenes and phenols 5.0 6.9 5.7 4.6 5.7 4.0 7.1 3.6 9.2 3.1

Free tatty acids total 23.4 30.6 23.9 25.4 25.0 34.4 28.7 19.1 25.9 33.9
16:0 8.8 10.9 6.3 8.4 8.4 7.8 10.9 3.3 8.1 6.4
18:2 7.4 7.6 8.9 6.6 8.1 7.0 9.0 5.5 7.6 5.3
18:1 1.6 5.8 2.0 4.0 1.9 12.8 2.2 4.4 2.3 14.1
18:0 0.5 0.8 0.6 0.5 0.6 12 0.8 0.5 0.9 1.4
20:0 0.3 0.4 0.4 0.3 0.4 0.5 0.4 0.4 0.5 0.6
22:0 0.6 0.6 0.7 0.7 0.7 0.7 0.7 0.7 0.8 0.7
23:0 0.6 0.5 0.4 0.4 0.4 0.4 0.6 0.5 0.6 0.8
24:0 1.4 1.3 1.5 1.5 1.6 1.4 1.4 1.2 1.8 1.6
26:0 1.3 1.2 1.7 1.5 1.7 1.4 1.2 1.3 2.3 1.9
28:0 1.0 1.5 1.4 1.4 1.2 1.2 1.6 1.3 0.8 1.0

Sterols total 33.5 44.6 42.1 43.6 39.3 32.0 40.2 55.9 43.4 34.4
~-sitosterol 29.2 32.2 33.7 34.7 32.7 23.6 32.7 30.7 33.0 26.0

Monoglycerides 2.4 1.9 2.6 1.9 2.7 1.8 3.0 1.8 3.0 2.2
Diglycerides 7.2 2.7 1.5 3.7 1.2 3.4 0.5 3.2 0.9 4.7
Triglïcerides 26.6 11.0 20.9 17.2 22.9 20.6 15.2 12.0 12.3 18.0

Table 2 continued Thermomyces MTZ 95 MTZ97 P. pseudomagnoliae

/anuginoses nom. provo
ME04Bl
Sterile Treated Sterile Treated Sterile Treated Sterile Treated
controls sameles controls same/es controls sameles controls sameles

Total extractives 0.33 0.33 0.31 0.39 0.32 1.28 0.33 0.36

Terpenes and phenols 8.9 3.4 6.3 9.9 6.9 2.6 4.2 4.5

Free tatty acids total 27.4 37.5 28.2 31.8 26.1 73.9 21.7 33.5
16:0 8.2 8.7 10.4 6.0 7.2 10.5 6.9 7.8
18:2 8.6 5.8 9.0 6.3 9.3 55.7 7.2 7.5
18:1 2.5 15.6 2.0 10.8 2.4 3.2 1.6 10.9
18:0 0.9 1.4 0.6 1.4 0.7 0.2 0.5 11
20:0 0.6 0.6 0.4 0.8 0.5 0.6 0.3 0.5
22:0 0.9 0.8 0.7 1.0 0.8 0.3 0.6 0.7
23:0 0.5 0.4 0.4 0.5 0.3 2.1 0.5 0.7
24:0 2.0 1.6 1.6 1.7 1.8 0.4 1.3 1.4
26:0 2.4 1.9 1.7 1.5 2.1 0.5 1.3 1.4
28:0 0.9 1.2 1.3 1.8 1.1 0.5 1.4 1.5

Sterols total 44.7 35.5 41.1 35.2 43.2 9.9 33.9 29.2
~-sitosterol 34.3 26.3 34.0 19.9 33.8 8.1 27.3 20.5

Monoglycerides 3.1 1.6 2.5 2.3 2.7 1.2 2.3 1.7

Diglycerides 0.6 3.3 0.6 2.7 1.1 7.1 6.3 10.2
Triglïcerides 10.0 15.0 17.6 12.4 15.5 2.9 29.4 16.5

• Total extractive content is given as a percentage of oven dry wood weight. All other values are given as a percentage of total volatile extract.

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5.1.3.2 Pulping trial

Four fungal strains were selected for the pulping trial with P. chrysosporium
BKM-F-1767 as control strain. Table 3 displays the effect of the strains on the wood
extract content and composition. The extractive content of the E. grand is used in this
trial was higher. Nevertheless, the effect of the selected fungal strains on the wood
extract was the same except for Thermomyces lanuginoses MED 4Bl, which decreased
the triglyceride content. Pulping halved the total extractive content with sterols being
most difficult to remove. Only P. chrysosporium BKM-F-1767 treated samples showed a
significant decrease' in pulp triglyceride content compared to the sterile controls
(Table 4). Consequently, more triglycerides would be released into process waters by the
sterile controls than the treated samples. There were no significant differences in pulp
properties of treated and sterile control samples except for the high percentage of rejects
and low percentage of screened yield in the P. chrysosporium BKM-F-1767 treated pulp
sample (Table 5). The abnormal amount of rejects and low screened yield could be
ascribed to a process problem rather than an effect from pretreatment.

Table3 Extractive content and composition of sterile E. grandis treated with five different fungal
strains
P. chrysosporium BLK 10A Thermamyces MTZ 95 P. pseudomagnoliae
BKM-F-1767 Januginoses nom. provo
MED4B1
Sterile Treated Sterile Treeted Sterile Treated Sterile Treated Sterile Treated
controls sameles controls sameles controls sametes controls sameles controls sameles

Total extractives • 0.41 0.48 0.42 0.40 0.44 0.36 0.42 0.46 0.45 0.45

Terpenes and phenols 8.7 9.7 7.9 6.8 7.5 6.5 7.6 9.2 6.6 9.0

Free fatty acids total 21.7 27.0 19.9 19.8 22.6 20.1 19.2 20.1 22.0 22.3
16:0 7.4 6.2 7.2 6.6 7.1 6.1 6.6 4.8 7.3 6.2
18:2 5.4 4.3 5.2 4.4 5.7 3.6 4.9 3.8 5.9 5.1
18:1 2.0 8.8 1.8 2.3 2.8 2.9 1.8 4.2 1.9 3.4
18:0 1.0 1.6 1.0 0.9 1.2 1.0 1.0 1.1 1.3 1.2
20:0 0.7 0.9 0.5 0.5 0.8 0.6 0.5 0.6 0.4 0.6
22:0 1.0 1.1 0.6 0.9 1.0 1.1 0.8 1.3 0.8 1.2
23:0 0.5 0.7 0.4 0.5 0.3 0.4 0.4 0.5 0.5 0.5
24:0 1.8 1.7 1.6 1.6 1.7 2.0 1.6 1.9 1.8 2.1
26:0 1.3 1.1 11 1.1 1.5 1.6 1.2 1.4 1.3 1.4
28:0 0.6 0.6 0.5 0.9 0.6 0.9 0.6 0.6 0.8 0.7

Sterols total 43.0 37.1 39.0 44.1 43.2 47.6 39.1 44.2 45.1 42.2
~-sitosterol 36.5 27.7 36.0 35.2 38.0 37.6 34.4 30.1 40.5 31.0

Monoglycerides 3.5 2.5 2.6 2.3 2.7 2.9 2.9 2.8 3.3 3.2
Diglycerides 0.0 2.7 2.4 1.2 0.0 1.8 2.4 2.8 0.0 1.5
Trigll'cerides 14.8 11.7 21.4 16.6 15.8 12.1 22.0 12.0 16.7 11.0

a Total extractive content is given as a percentage of oven dry wood weight All other values given as a percentage of total volatile extract.

5.1.3.3 Lipase treatment of brown stock pulp extract

Mucor javanicus and Rhizopus arrhizus lipase had no effect on the triglyceride content of
the deposited brown stock pulp extract (Table 6). The high molecular weight compounds

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of the extract as well as the other volatile compounds made the triglycerides inaccessible
for hydrolysis. Pitch deposits in a pulp mill would therefore be unaffected by the
addition of lipase.

Table 4 Extractive content and composition of the kraft pulp from the treated wood
P. chrysosporium BLK lOA Tbermomyces MTZ 95 P. pseudomagno/ia
BKM-F-1767 lanuginoses e nom. provo
MED4Bl
Sterile Treated Sterile Treated Sterile Treated Sterile Treated Sterile Treated
controls same/es controls samples controls samples controls same'es controls same'es

Total extractives • 0.26 0.26 0.21 0.24 0.23 0.21 0.21 0.21 0.26 0.27

Terpenes and phenols 2.5 3.8 0.9 1.8 1.4 1.3 0.9 1.0 2.5 2.2

Free fatty acids total 9.6 12.8 8.4 5.5 9.4 5.1 8.4 6.7 9.6 10.3
16:0 4.8 4.7 3.9 1.9 4.7 2.0 3.9 2.1 4.8 2.9
18:2 0.4 3.6 0.6 0.3 0.4 0.4 0.6 0.3 0.4 1.3
18:1 1.1 3.6 1.2 0.9 1.2 0.7 1.2 1.9 1.1 1.3
18:0 1.5 1.6 0.8 0.8 1.1 0.7 0.8 0.8 1.5 0.9
20:0 0.3 0.4 0.3 0.3 0.3 0.3 0.3 0.4 0.3 0.4
22:0 0.5 0.5 0.4 0.4 0.5 0.3 0.4 0.3 0.5 0.5
23:0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
24:0 0.8 1.1 0.9 0.6 0.8 0.5 0.9 0.6 0.8 12
26:0 0.3 0.5 0.2 0.3 0.4 0.2 0.2 0.2 0.3 0.5
28:0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.1

Sterols total 71.9 70.4 79.5 77.6 79.8 81.0 79.5 80.9 71.9 72.6
p-sitosterol 61.0 80.3 63.5 68.1 67.6 72.4 63.5 68.6 81.0 63.8

Monoglycerides 1.4 2.1 1.1 1.4 1.6 1.3 1.1 0.9 1.4 1.8
Diglycerides 0.9 0.0 1.3 0.2 0.7 0.8 1.3 1.3 0.9 0.4
Tri91~rides 8.6 5.6 4.9 9.7 2.7 7.2 4.9 4.8 8.8 9.1

• Total extractive content is given as a percentage ot oven dry wood weight. All other values given as a percentage ot total volatile extract.

Table 5 Properties of pulp from treated and control samples

Sample Residual Screened Rejects 1",(,) Kappa No. Weighted Fibre Fibres Per
Black Liqour Yield (%) Fibre Length Coarseness Gram
(gA) (mm) (mg/l00m) (millions)

P. chrysosporium BKM-F-1767 5.68 41.3 13.94 36.3 0.82 8.40 18.9

BLK10A 6.33 48.2 1.45 33.0 0.79 8.00 20.2
Thermomyces Januginoses MED 4B1 8.18 49.3 0.68 29.0 0.79 7.90 20.4
MTZ95 7.50 48.1 0.65 27.2 0.79 8.60 24.0
P.pseudomagnoliae nom. provo 8.89 55.1 0.50 28.2 0.78 6.50 24.8
3QOCsterile controls 8.99 50.5 0.81 30.6 0.80 7.10 22.4
3QOCsterile controls 5.91 43.9 3.37 37.0 0.86 8.80 17.2
500e sterile controls 8.90 55.4 0.42 28.2 0.79 6.50 24.0

Table6 The effect of commerciallipases on deposited brown stock pulp

extract
Rhizopus arrhizus lipase Mucor javanicus lipase
Negative Positive Trealed Posi&ve Trealed
control control same.'e control same.1e

Terpenes and phenols 0.8 0.3 0.5 0.4 0.4

Free tatty acids 20.1 19.6 18.4 17.7 18.8

18:0 6.8 7.2 6.4 5.8 6.2
18:2 4.6 4.5 3.9 4.2 4.3
18:1 4.6 4.5 39 4.2 4.3
18:0 1.1 1.1 1.2 1.0 1.2
20:0 0.5 0.5 0.6 0.5 0.6
22:0 0.8 0.7 0.7 0.7 0.6
23:0 0.0 0.0 0.1 0.0 0.1
24:0 1.1 0.8 0.9 0.9 0.9
28:0 0.8 0.4 0.7 0.5 0.6
28:0 0.0 0.0 0.0 0.0 0.0

Average sterols 61.4 59.9 59.1 61.2 57.2

p-sitosterol 44.0 47.1 46.0 49.1 45.3

Monoglycerides 1.4 1.8 2.2 2.0 2.6

Diglycerides 0.6 0.0 0.9 0.0 0.3
Trial~rides 5.2 7.8 7.8 7.5 8.8

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5.1.4 Conclusions

Although none of the strains reduced the total extractive content of the wood chips, the
triglyceride content was decreased by seven of the nine fungal strains. Wallis and
Weame (1999) obtained similar results when they seasoned Eucalyptus globulus wood
chips. The total extractive content they determined for E. globulus is similar to that of
the E. grand is of this study. This result indicates that the reduction of the total extractive
content can not be used as a measure of the ability of a fungal strain to combat pitch. The
effect on the triglyceride content should rather be employed as measure. More
triglycerides would be released into process waters by the sterile controls than the treated
samples due to the higher triglyceride content of sterile woodcontrols.

The apparent natural variation in extractive composition of the sterile controls made it
difficult to compare the ability of different fungal strains to reduce extractive and
triglyceride content. All the white-rot fungi compared well to 0. piliferum CAR 58 and
P. chrysosporium BKM-F-1767 in reducing the triglyceride content of E. grandis. The
South African isolate white-rot P. pseudomagnoliae nom. provo gave good results and
can therefore act as an agent for both biopulping (Cerff, 1999) and biodepitching.

Pulping results were variable. Treated pulp samples did not show a significant difference
in triglyceride content or pulp characteristics compared to the sterile controls.
Triglycerides were not eliminated from the pulp extract as reported in a previous study on
the effect of industrial pulping on pulp extracts (Scheepers et al., 2000). Laboratory scale
pulping gave variable results and did not correlate well with industrial scale pulping
results. Consequently, no outright conclusions could be made on the basis of laboratory
scale pulping results before industrial scale trials are done.

The addition of commerciallipases did not affect the triglyceride content of deposited
pulp extract. The addition of lipases in pulping and bleaching processes would therefore
not affect already deposited pitch.

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5.1.5 Acknowledgements

The authors are grateful to Mondi Kraft Ltd., The Foundation for Research and
Development as well as The Technology and Human Resources for Industry Programme
for supporting this project.

5.1.6 References

Allen, LH (1977), Trend26: 4-10

Blanchette, RA, Farrell, RL, Burnes, TA, Wendler, PA, Zimmerman, W, Brush TS, and
Snyder, RA (1992), Tappi Journal75: 102-106
Brush, TS, Farrell, RL and Ho, C (1994), Tappi Journal77, 1~5-159
Cerff C (1999), Kraft Biopulping of Eucalyptus grand is, M.Sc. thesis, University of
Stellenbosch, Stellenbosch, South Africa
Chafe, SC (1987), Wood Science and Technology 21: 27-41
Christie, WW (1992), Gas Chromatography and Lipids: A Practical Guide, WW
Christie, ed. The Oily Press, Ayr, Scotland
Farrell, RL, Blanchette, RA, Brush, TS, Iverson, S and Fritz AR (1994), In: Biological
Sciences Symposium, pp. 85-87, Tappi
Farrell, RL, Hata, K and Wall, MB (1997), Advances in Biochemical
Engineering/Biotechnology 57: 197-212
Fischer, K, Akhtar, M, Blanchette, RA, Burnes, TA, Messner, K and Kirk, TK (1994),
Holzforschung 48: 285-290
Fischer, K, Akhtar, M, Messner, K, Blanchette, RA and Kirk, TK (1995), In: Proceedings
of the 6th International Conference on Biotechnology in the Pulp and Paper Industry:
Advances in Applied and Fundamental Research, E Srebotnik and K Messner, eds
pp. 193-196, Facultas-Universitaetsverlag, Vienna, Austria
Fischer, K and Messner, K (1992), Tappi Journal75: 130-134
Fujita, Y, Awaji, H, Taneda, H, Matsukura, M, Hata, K, Shimoto, H, Sharyo, M,
Sakaguchi, H and Gibson, K (1992), Tappi Journal75: 117-122
Hillis, WE and Carle, A (1959), Appita 13: 74-83
Kock, JLF and Botha, A (1998), In: Chemical Fungal Taxonomy, JC Frisvad, PD Bridge
and DK Arora, eds pp. 219-246, Marcel Dekker, Inc., New York

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Nelson, PF; Smith, JG and Young, WD (1970), Appita 24: 101-107

Rocheleau, MJ, Sitholé, BB, Allen, LH, Iverson, S, Farrell, R and Noë!, Y (1998),
Journal of Pulp and Paper Science 24: 37-42
Scheepers, GC, Rypstra, T, de Koker, TH and Janse, BJH (2000), Holzforschung,
Submitted
Suckling, ID and Ede, RM (1990), Appita 43: 77-80
Wallis, AFA and Wearne, RH (1999), Appita 52: 295-299
Yazaki, Y, Collins, PJ and Iwashina T (1993), Holzforschung47: 412-418

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Chapter 6: General discussion and

conclusions

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6.1 General discussion and conclusions

In recent times the pulp and paper 'industry has looked to fungal and enzymatic
biotechnology to reduce chemical consumption and cut production costs. Some pulp
mills have already employed these technologies successfully. Pulping is no longer
confined to the cooking process in digesters. Since the advent of fungal biopulping,
pulping can commence on the chip piles, so reducing chemical requirements in the
caustic digestion process. Fungal growth on wood chip piles and lipase addition to pulp
in suspension can also decrease the use of chemical pitch control agents. The
identification of a fungal strain that performs the dual task of biopulping and depitching
would thus be beneficial to the pulping industry.

Chapter 2 introduces all the relevant factors to combating pitch with either fungal or
enzymatic means. Triglycerides are thought to be the key to pitch troubles. They tend to
adhere to hydrophobic surfaces and build up to become pitch deposits. Commercial
lipases are already employed cost effectively in some pulp mills to hydrolyse
triglycerides in a pulp suspension. The result is improved product quality and less
downtime to clean forming wires and other equipment. Wood structure is such that wood
is easily penetrable by fungal hyphae. Consequently, resin deposits incorporating
triglycerides are accessible to fungi. Some fungal strains can decrease the triglyceride
content of wood while growing on wood chip piles. In this way less triglycerides enter
the papermaking process and thus the propensity for pitch deposition is reduced. Pulp
mills in South Africa pulping Eucalyptus spp. could benefit from fungal pitch control,
especially those in the warm, humid climate areas of South Africa where conditions
favour accelerated fungal growth.

The results of analyses of wood and pulp extracts as well as pitch presented in Chapter 3
indicate either that triglycerides were in suspension in the white water or that very small
amounts carried by the pulp deposited over an extended period of time. As reported by
Farrell et al. (1997), triglycerides may also be the key to pitch deposition in this mill
situation. The presence of amide derivatives of fatty acids can be ascribed to the use of
non-ionic organic additives provided by Buckman Laboratories (Pty) Limited. The

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composition of the E2 mat pulp extract differed considerably from pulp extracts from
other stages. The increase in fatty acid ami des and decrease in fatty acids may be
ascribed to extreme conditions in the second caustic extraction stage. However, the first
extraction stage did not have such a marked effect on the pulp extract. The decrease in
fatty acid ami des in the bleach stock decker mat pulp extract as well as the normalisation
of fatty acid levels could be attributed to the addition of talc and the change in process
conditions.

The lipase production capability of several strains isolated in South Africa was compared
to that of commercial control strains (Phanerochaete chrysosporium BKM-F-1767 and
Ophiostoma piliferum CAR 58) in Chapter 4. P. chrysosporium BKM-F-1767 is a
biopulping and 0. piliferum CAR 58 a biodepitching strain. Results indicated that a few
of the strains compared favourably and even outperformed the control strains, indicating
the potential for the use of these strains to break down wood triglycerides. Some of the
isolated strains were white-rot fungi, which could also serve as biopulping agents.

Chapter 5 illustrates the effect of the isolated strains compared to the control strains on
wood extract composition. All the white-rot fungi compared well to 0. piliferum
CAR 58 and P. chrysosporium BKM-F-1767 in reducing the triglyceride content of
Eucalyptus grand is. The South African white-rot isolate Phanerochaete
pseudomagnoliae nom. prov., known for its biopulping potential, reduced the triglyceride
content substantially. Results indicate that the reduction of total extractive content can
not be used as a measure of the ability of a fungal strain to combat pitch. The effect on
the triglyceride content should rather be employed as measure. The addition of
commerciallipases did not affect the triglyceride content of deposited pulp extract. The
addition of lipases in pulping and bleaching processes would therefore not affect already
deposited pitch.

The South African isolate P. pseudomagnoliae nom. provo proved to be the best candidate
for combined biopulping and pitch control in one step. Its use could therefore reduce the
need for pulping and pitch control chemicals.

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