INTERNATIONAL JOURNAL OF

MOLECULAR SCIENCES

Review
Biosurfactants: Multifunctional Biomolecules
of the 21st Century
1,2 2,3 2,3
Danyelle Khadydja F. Santos , Raquel D. Rufino , Juliana M. Luna , Valdemir A.
2,3 1,2,3,
Santos and Leonie A. Sarubbo *
1 Northeast Botechnology Network (RENORBIO), Federal Rural University of Pernambuco,
Rua Dom Manoel de Medeiros, s/n, Dois Irmãos, 52171-900 Recife-PE, Brazil; danykhadydja@hotmail.com
2 Center of Sciences and Technology, Catholic University of Pernambuco (UNICAP), Rua do
Príncipe, 526, Boa Vista, 50050-900 Recife-PE, Brazil; raqueldrufino@yahoo.com.br (R.D.R.);
julianamouraluna@gmail.com (J.M.L.); valdemir.alexandre@hotmail.com (V.A.S.)
3 Advanced Institute of Technology and Innovation (IATI), Rua Carlos Porto Carreiro, 70,
Derby, 50070-090 Recife-PE, Brazil
* Correspondence: leonie@unicap.br; Tel.: +55-81-2119-4084

Academic Editor: Andreas Taubert

Received: 21 January 2016; Accepted: 11 March 2016; Published: 18 March 2016

Abstract: In the era of global industrialisation, the exploration of natural resources has served as a
source of experimentation for science and advanced technologies, giving rise to the manufacturing
of products with high aggregate value in the world market, such as biosurfactants. Biosurfactants
are amphiphilic microbial molecules with hydrophilic and hydrophobic moieties that partition at
liquid/liquid, liquid/gas or liquid/solid interfaces. Such characteristics allow these biomolecules to
play a key role in emulsification, foam formation, detergency and dispersal, which are desirable
qualities in different industries. Biosurfactant production is considered one of the key technologies
for development in the 21st century. Besides exerting a strong positive impact on the main global
problems, biosurfactant production has considerable importance to the implantation of sustainable
industrial processes, such as the use of renewable resources and “green” products.
Biodegradability and low toxicity have led to the intensification of scientific studies on a wide range
of industrial applications for biosurfactants in the field of bioremediation as well as the petroleum,
food processing, health, chemical, agricultural and cosmetic industries. In this paper, we offer an
extensive review regarding knowledge accumulated over the years and advances achieved in the
incorporation of biomolecules in different industries.

Keywords: biosurfactant; surface tension; critical micelle concentration; biodegradability;

functional properties; physiology; kinetics; recovery; industrial applications

1. Introduction
Surfactants are amphipathic compounds with both hydrophilic and hydrophobic moieties that
preferentially partition between liquid interfaces with different degrees of polarity and hydrogen
bridges, such as oil/water or air/water interfaces. The apolar moiety is often a hydrocarbon chain,
whereas the polar moiety may be ionic (cationic or anionic), non-ionic or amphoteric [ 1,2], as
illustrated in Figure 1.

Int. J. Mol. Sci. 2016, 17, 401; doi:10.3390/ijms17030401 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2016, 17, 401 2 of 31
Int. J. Mol. Sci. 2016, 17, 401 2 of 30

Figure 1. Surfactant molecule with apolar (hydrophobic) and polar (hydrophilic) moieties.
Figure 1. Surfactant molecule with apolar (hydrophobic) and polar (hydrophilic) moieties.
Surfactants increase the solubility of hydrophilic molecules, thereby reducing both surface and Surfactants increase the
solubility of hydrophilic molecules, thereby reducing both surface and
interfacial tensions at the oil/water interface [3]. The critical micelle concentration (CMC) is the interfacial tensions at the oil/water interface [3]. The
critical micelle concentration (CMC) is the
concentration of surfactant at which organised molecular assemblies, known as micelles, are formed concentration of surfactant at which organised
molecular assemblies, known as micelles, are formed
(Figure 2) and corresponds to the point at which the tensioactive agent achieves the lowest stable (Figure 2) and corresponds to the point at which the
tensioactive agent achieves the lowest stable
surface tension (Figure 3) [4].
surface tension (Figure 3) [4].
Figure 1. Surfactant molecule with apolar (hydrophobic) and polar (hydrophilic) moieties.
Most currently produced surfactants are chemically derived from petroleum. However, such Most
Figurecurrently1.Surfactantproducedmoleculesurfactantswithapolararechemically(hydrophobic)derivedandpolarfrom(hydrophilic)petroleum.moietiesHowever,. such
synthetic tensioactive agents are generally toxic and difficult to break down through the action of synthetic tensioactive agents are generally toxic and
difficult to break down through the action of
Surfactantsmicroorganismsncrease.Inrecthentsolubilityyears,suchof hydrophilicproblemshavemolecules,les,motivatedtherebyscientificreducingommunitybothsurfacetoseekand
microorganismsSurfactants. iIn recentasetheyears,solubilitysuch problemsofhydrophilichave motivated therebyscientificreducingcommunitybothsurfacetoseekand
surfactants that are more environmentally friendly, such as thoseachieved through microbial interfacial tensions at the
interfacial tensions at the oil/water interfaceerface[ 3[3].The critical micelle concentration is the surfactants thatare more
. (CM(CMC) isthe
environmentally friendly, such as those achieved through microbial
production, known as biosurfactants [5]. Moreover, concerns regarding the environment on the part
concentrationproduction,concentrationofknownsurofactantsurfactasbiosurfactantsawhichatichorganised[5].Moreover,molecularoncernsassemregardingblies,knowntheenvironmentasasmicellmicelles,onaretheareformedpartformed
(Figure 2) and corresponds tothe point at which e the lowest stable
of consumers and new environmental control legislation have led to the development of natural (Figureof consumers2)and corresponds and newenvironmentaltothe point atcontrol which legislatiothe tetensisioactivehaveled agenttotheachievesdevelopment the oflowestnatural stable

surface tension (Figure 3)[4] .

surfactants surfactants asasanan alternativealternative to toexistingexistingproducts.products.
surface tension (Figure 3) [4].
Most currently produced surfactants are chemically derived from petroleum. However, such
synthetic tensioactive agents are generally toxic and difficult to break down through the action of
microorganisms. In recent years, such problems have motivated the scientific community to seek
surfactants that are more environmentally friendly, such as those achieved through microbial
production, known as biosurfactants [5]. Moreover, concerns regarding the environment on the part
of consumers and new environmental control legislation have led to the development of natural
surfactants as an alternative to existing products.

Figure 2. Schematic illustration of tensioactive agent and micelle formation.

Figure 2.Schematic of and formation .
Figure 2.Schematic illustrationof tensioactiveagent andmicelle formation .

Figure 2. Schematic illustration of tensioactive agent and micelle formation.

FigureFigure3.Illustration3.Illustrationofregionsofregionsinwhinchwhmichellemicelleformationformationoccursoccurs(critical(criticalmicellemicelleconcentrationCMC). Figure 3. Illustration of

regions in which micelle formation occurs (critical micelle concentration
CMC).
CMC).
Most currently produced surfactants are chemically derived from petroleum. However, such
synthetic tensioactive agents are generally toxic and difficult to break down through the action of
microorganisms. In recent years, such problems have motivated the scientific community to seek
surfactants that are more environmentally friendly, such as those achieved through microbial
production, known as biosurfactants [5]. Moreover, concerns regarding the environment on the part Figure 3.
Illustration of regions in which micelle formation occurs (critical micelle concentration
of consumers and new environmental control legislation have led to the development of natural CMC).
surfactants as an alternative to existing products.
Studies involving biosurfactants began in the 1960s and the use of these compounds has expanded
in recent decades [2,6]. Biosurfactants have drawn the interest of different industries due to advantages
such as structural diversity, low toxicity, greater biodegradability, ability to function in wide ranges of pH,
temperature and salinity as well as greater selectivity, lower CMC and production involving renewable
sources/industrial waste and industrial by-products [7–9]. The present review demonstrates
Int. J. Mol. Sci. 2016, 17, 401 3 of 31

the reasons for which biosurfactants are considered the multifunctional materials of the 21st century,
with a description of concepts, properties, classification, modes of production, physiology and uses
in the most diverse industries.

2. Producing Microorganisms
Microorganisms use a set of carbon sources and energy for growth. The combination of carbon
sources with insoluble substrates facilitates the intracellular diffusion and production of different
substances [10–12]. Microorganisms (yeasts, bacteria and some filamentous fungi) are capable of
producing biosurfactants with different molecular structures and surface activities [4]. In recent
decades, there has been an increase in scientific interest regarding the isolation of microorganisms
that produce tensioactive molecules with good surfactant characteristics, such as a low CMC, low
toxicity and high emulsifying activity [2].
The literature describes bacteria of the genera Pseudomonas and Bacillus as great biosurfactant
producers [2]. However, most biosurfactants of a bacterial origin are inadequate for use in the food
industry due to their possible pathogenic nature [13]. Candida bombicola and Candida lipolytica are
among the most commonly studied yeasts for the production of biosurfactants. A key advantage of using
yeasts, such as Yarrowia lipolytica, Saccharomyces cerevisiae and Kluyveromyces lactis, resides in their
“generally regarded as safe” (GRAS) status. Organisms with GRAS status do not offer the risks of toxicity
or pathogenicity, which allows their use in the food and pharmaceutical industries [4]. Table 1 displays a
list of microorganisms that produce biosurfactants.

3. Classification
Most biosurfactants are either anionic or neutral, whereas those that contain amine groups are
cationic. The hydrophobic moiety has long-chain fatty acids and the hydrophilic moiety can be a
carbohydrate, cyclic peptide, amino acid, phosphate carboxyl acid or alcohol. The molar mass of
biosurfactants generally ranges from 500 to 1500 Da [14]. Biosurfactants are generally categorised
by their microbial origin and chemical composition, as follows [3,5,15].

3.1. Glycolipids
Rhamnolipids, sophorolipids and trehalolipids are the best known glycolipids [16]. Rhamnolipids
were found as exoproducts of the pathogen P. aeruginosa and are a combination of - L-rhamnopyranosyl-
-L-rhamnopyranosyl- -hydroxydecanoyl- -hydroxydecanoate (Rha-Rha-C10-C10) and - L-
rhamnopyranosyl- -L-rhamnopyranosyl- -hydroxydecanoate (Rha-Rha-C10) as well as their mono-
rhamnolipid congeners (Rha-C10-C10 and Rha-C10) [17]. Sensitive analytical techniques have led to the
discovery of rhamnolipid congeners and homologues (approximately 60) produced at different
concentrations by species of Pseudomonas and bacteria belonging to other families, classes or even
phyla [16]. For instance, various species of Burkholderia have been shown to produce rhamnolipids that
have longer alkyl chains than those produced by P. aeruginosa [17–19]. Surface tensions values of 29
mN/m constitute a characteristic of such components, which can be produced using different substrates,
such as alkanes, pyruvate, citrates, fructose, glycerol, olive oil and glucose [20]. Most studies involving
rhamnolipids focus mainly on assessing the biodegradation efficiency of petroleum hydrocarbons [21,22].
Although researchers have found increased dissipation of target contaminant upon the addition of
rhamnolipids, a decrease in biodegradation efficiency or no effect following rhamnolipid supplementation
have also been reported [16,20]. The presence of surfactant molecules may induce changes in the
microbial community, which, in turn, correspond to different degradation patterns. Interestingly, although
rhamnolipids are considered biodegradable, few reports have demonstrated that these substances can
be co-degraded or solely utilised as a carbon and energy source by various monocultures [18].
Rhamnolipids are described as potentially toxic to natural vegetation [23], but have also been found to
reduce the toxicity of specific compounds by increasing hydrocarbon solubilisation, thereby facilitating
biodegradation [24,25].
Int. J. Mol. Sci. 2016, 17, 401 4 of 31

Table 1. Main classes of biosurfactants and respective producing microorganisms.

Biosurfactant Class
Glycolipids Polymeric Surfactants Lipopeptides Fatty Acids Particulate Surfactant Phospholipids
Producer microorganisms
Acinetobacter calcoaceticus
Alcanivorax borkumensis
Arthrobacter paraffineus
Arthrobacter sp.
Candida antartica
Candida apicola Acinetobacter calcoaceticus
Acinetobacter calcoaceticus Acinetobacter sp.
Candida batistae
Acinetobacter calcoaceticus Bacillus licheniformis
Candida bogoriensis
Acinetobacter calcoaceticus Bacillus pumilus
Candida bombicola
Bacillus stearothermophilus Bacillus subtilis
Candida ishiwadae
Candida lipolytica Candida Candida lipolytica
Candida lipolytica
utilis Halomonas eurihalina Gluconobacter cerinus
Lactobacillus fermentum Pseudomonas fluorescens
Nocardia sp. Mycobacterium
Serratia marcescens
Pseudomonas aeruginosa thermoautotrophium
Streptomyces sioyaensis
Pseudomonas sp. Sphingomonas paucimobilis
Thiobacillus thiooxidans
Rhodococcus erythropolis
Rhodotorula glutinus
Rhodotorula graminus
Serratia marcescens
Tsukamurella sp.
Ustilago maydis
Arthrobacter paraffineus
Capnocytophaga sp.
Corynebacterium
Acinetobacter calcoaceticus Acinetobacter sp.
insidibasseosum
Cyanobacteria Aspergillus
Corynebacterium lepus
Pseudomonas marginalis Corynebacterium lepus
Nocardia erythropolis
Penicillium spiculisporum
Talaramyces trachyspermus
Int. J. Mol. Sci. 2016, 17, 401 5 of 31

Sophorolipids are produced by yeasts that belong to the genus Candida [ 26,27]. These glycolipids
have a dimeric carbohydrate sophorose linked to a long-chain hydroxyl fatty acid through a glycosidic
bond. Sophorolipids and lactone form a sophorolipid that is preferable in many applications [28,29]. C.
bombicola stands out among the different types of yeasts used in the production of these biosurfactants.
Surface tension values of approximately 33 mN/m and a reduction in the surface tension of n-
hexadecane and water from 40 to 5 mN/m has been recorded for these agents [30]. Mannosylerythritol
lipids (MEL), which are yeast glycolipids, are one of the most promising biosurfactants known and are
abundantly produced from vegetable oils by Pseudozyma (previously Candida) antarctica [ 31].
Trehalolipids are produced by species of Mycobacterium, Nocardia and Corynebacterium. Trehalolipids
from Arthrobacter spp. and Rhodococcus erythropolis are able to lower surface and interfacial tensions in
culture broth to 25–40 and 1–5 mN/m, respectively [5].

3.2. Fatty Acids, Phospholipids and Neutral Lipids

Different bacteria and yeasts produce large amounts of fatty acids and phospholipid surfactants
during growth on n-alkanes. Phosphatidyl ethanolamine-rich vesicles are produced from
Acinetobacter spp. and form optically clear microemulsions of alkanes in water. These
biosurfactants are essential to medical applications. According to Gautam and Tyagi [28],
phospholipid protein complex deficiency is the major cause of respiratory failure in the children born
prematurely. The authors also suggest that the isolation and cloning of genes involved in the
production of surfactants can be used in fermentative processes [28].

3.3. Polymeric Biosurfactants

Emulsan, lipomanan, alasan, liposan and other polysaccharide protein complexes are the best-
studied polymeric biosurfactants. Emulsan is an emulsifier for hydrocarbons in water at concentrations as
low as 0.001% to 0.01% [31,32]. Liposan is an extracellular water soluble emulsifier synthesised by C.
lipolytica and is made up of 83% carbohydrates and 17% proteins. Chakrabarti [ 33] discuss the
application of liposan as an emulsifier in the food and cosmetic industries.

3.4. Particulate Biosurfactants

Particulate biosurfactants partition extracellular membrane vesicles to form a microemulsion
that exerts an influence on alkane uptake in microbial cells. The Acinetobacter spp. has vesicles
with a diameter of 20 to 50 nm and a buoyant density of 1.158 cubic gcm composed of proteins,
phospholipids and lipo-polysaccharides [5,33].
Figure 4 illustrates the chemical structure of the most studied microbial surfactants. Table 1 also
Int. J. Mol. Sci. 2016, 17, 401 6 of 30
displays biosurfactant classes and their producers.

Figure 4. Chemical structure of most studied microbial surface-active compounds. (a) Rhamnolipid;
Figure 4. Chemical structure of most studied microbial surface-active compounds. (a) Rhamnolipid; (b) Sophorolipid; (c)
Surfactin and (d) Emulsan.
(b) Sophorolipid; (c) Surfactin and (d) Emulsan.

4. Properties

It is necessary to submit a biosurfactant to conservation methods to evaluate its properties

(surface tension and dispersion) over a period of 120 days to estimate the commercial validity of the
product. Thus, heating methods are used separately or in combination with potassium sorbate, which
is a conservative that inhibits the growth of mould that is widely used in the production and
Int. J. Mol. Sci. 2016, 17, 401 6 of 31

4. Properties
It is necessary to submit a biosurfactant to conservation methods to evaluate its properties
(surface tension and dispersion) over a period of 120 days to estimate the commercial validity of the
product. Thus, heating methods are used separately or in combination with potassium sorbate,
which is a conservative that inhibits the growth of mould that is widely used in the production and
conservation of foods. Some characteristics are common to the majority of biosurfactants and have
advantages over conventional surfactants, as described below [5].

4.1. Surface and Interfacial Activity

Efficiency and effectiveness are essential characteristics of a good surfactant. Efficiency is
measured by the CMC, whereas effectiveness is related to surface and interfacial tensions [34]. The
CMC of biosurfactants ranges from 1 to 2000 mg/L, whereas interfacial (oil/water) and surface
tensions are respectively approximately 1 and 30 mN/m. Good surfactants are able to reduce water
surface tension from 72 to 35 mN/m and the interfacial tension of n-hexadecane from 40 to 1 mN/m.

4.2. Tolerance to Temperature, pH and Ionic Strength

Many biosurfactants can be used at high temperatures and pH values ranging from 2 to 12.
Biosurfactants also tolerate a salt concentration up to 10%, whereas 2% NaCl is enough to
inactivate synthetic surfactants.

4.3. Biodegradability
Biosurfactants are easily degraded by microorganisms in water and soil, making these
compounds adequate for bioremediation and waste treatment.

4.4. Low Toxicity

Low degree of toxicity allows the use of biosurfactants in foods, cosmetics and pharmaceuticals.
Low toxicity is also of fundamental importance to environmental applications.
Biosurfactants can be produced from largely available raw materials as well as industrial waste.

4.5. Specificity
Biosurfactants are complex molecules with specific functional groups and therefore often have
specific action. This is of particular interest in the detoxification of different pollutants and the de-
emulsification of industrial emulsions as well as specific food, pharmaceutical and cosmetic
applications.

4.6. Biocompatibility and Digestibility

These properties allow the use of biomolecules in different industries, especially the food,
pharmaceutical and cosmetic industries.

4.7. Emulsion Forming/Breaking

Biosurfactants can be either emulsifiers or de-emulsifiers. An emulsion is a heterogeneous system
consisting of an immiscible liquid dispersed in another liquid in the form of droplets, the diameter of which
generally exceeds 0.1 mm. There are two basic types of emulsion: oil-in-water (o/w) and water-in-oil
(w/o). Emulsions have minimal stability, but the addition of biosurfactants can lead to an emulsion that
remains stable for months or even years [35]. Liposan, which is a water-soluble emulsifier synthesised by
C. Lipolytica, has been used with edible oils to form stable emulsions. Liposan is commonly used in the
cosmetic and food industries for producing stable oil/water emulsions [4,36].
Int. J. Mol. Sci. 2016, 17, 401 7 of 31

5. Factors Affecting Biosurfactant Production

The production of biosurfactants can be either spontaneous or induced by the presence of
lipophilic compounds, variations in pH, temperature, aeration and agitation speed or when cell
growth is maintained under conditions of stress, such as a low concentration of nitrogen [37]. The
various physicochemical factors are discussed below [38].

5.1. Carbon Source

The carbon source plays an important role in the growth and production of biosurfactants by
microorganisms and varies from species to species. A very low yield was found when only either
glucose or vegetable oil was used for the production of a biosurfactant by T. bombicola, but the yield
increased to 70 g/L when both carbon sources were provided together [39]. At a concentration of 80
and 40 g/L of glucose and soybean oil, respectively, the maximum yield of sophorose lipids was
obtained by T. bombicola [40]. Even higher yields of sophorolipids (120 g/L) were produced with C.
bombicola in eight days when sugar and oil were used as carbon sources [41]. When canola oil and
glucose were used as carbon sources at concentrations of 10% each, maximum yield of
sophorolipids (8 g/L) was obtained from C. lipolytica [42]. Moreover, when industrial waste was used
for the production of a biosurfactant by C. lipolytica, the yield of the protein-lipid-carbohydrate
complex was 4.5 g/L, with a reduction in the surface tension of distilled water from 71 to 32 mN/m
[43]. A high production of bioemulsifier was obtained with C. lipolytica when supplemented with
1.5% glucose (w/v) [44]. C. antarctica and C. apicola yielded 13.4 and 7.3 g/L of sophorolipids,
respectively, when soapstock was used at a concentration of 5% (v/v) [45]. The resting cells of
Pseudozyma (C. antarctica) were found to covert C 12 to C18 n-alkanes into mannosylerythritol lipids
(MEL); the yield was 140 g/L after four weeks and the biosurfactant was able to emulsify soybean oil
[46]. A change in the fatty acid constitution of the final biosurfactant occurred when the fatty acid
composition was changed in the fermentation medium containing C. ingens [47].

5.2. Nitrogen Sources

This is the second most important supplement for the production of biosurfactants by
microorganisms. In fermentative processes, the C/N ratio affects the buildup of metabolites. High C/N
ratios (i.e., low nitrogen levels) limit bacterial growth, favouring cell metabolism towards the production of
metabolites. In constrast, excessive nitrogen leads to the synthesis of cellular material and limits the
buildup of products [48]. Different organic and inorganic nitrogen sources have been used in the
production of biosurfactants. Santa Anna et al. [49] describe the importance of nitrogen for the production
of a biosurfactant by P. aeruginosa cultivated in a mineral medium containing 3% glycerol. As NaNO 3

proved more effective than (NH 4)2SO4, nutritional limitations clearly guide the cell metabolism to the
formation of the product. Mulligan and Gibbs [50] report that P. aeruginosa uses nitrates, ammonium and
amino acids as nitrogen sources. Nitrates are first reduced to nitrite and then ammonium. Ammonium is
assimilated either by glutamate dehydrogenase (EC 1.4.1.4) to form glutamate or glutamine synthetase
(EC 6.3.1.2) to form glutamine. Glutamine and -ketoglutarate are then converted to glutamine by L-
glutamine 2-oxoglutarate aminotransferase (EC 1.4.1.13). However, lipid formation rather than sugar is
the rate-determining factor in the biosynthesis of rhamnolipids and nitrogen limitation can lead to the
accumulation of lipids. In comparison to ammonium, the assimilation of nitrate is slower and simulates
nitrogen limitation, which is favourable to the production of rhamnolipids. High yields of sophorose lipids,
which are biosurfactants produced by the fungi T. bombicola and C. Bombicola, have been achieved
using yeast extract and urea as the nitrogen source [51]. Moreover, high yields of mannosylerythritol lipid
by Candida sp. SY16, C. lipolytica and C. glabrata have been achieved with ammonium nitrate and yeast
extract [42,43,46,52–54].
Int. J. Mol. Sci. 2016, 17, 401 8 of 31

5.3. Growth Conditions

Growth conditions (temperature, pH, agitation speed and oxygen) also influence biosurfactant
production [37]. Species of the genus Candida produce maximum biosurfactant yields in a wide pH
range, such as pH 5.7 for C. glabrata UCP 1002, pH 7.8 for Candida sp. SY16, pH 5.0 for C. Lipolytica
and pH 6.0 for C. batistae [52,54–56]. Moreover, Pichia anamola and Aspergillus ustus produce
maximum biosurfactant yield at pH 5.5 and 7.0, respectively [57,58]. Different microbial processes are
affected by even a small change in temperature. The most favourable temperature for the production of
biosurfactants by different fungi is 30 C, as observed for different species of Candida, viz. Candida sp.
SY16, C. bombicola, C. batistae and T. bombicola [39,51,52,56]. In case of C. lipolytica, 27 C has been
found to be the best temperature. Incubation time also exerts a significant effect on biosurfactant
production. Microorganisms produce biosurfactants in different time intervals. Maximum biosurfactant
production by Aspergillus ustus was found after five days of incubation, whereas the incubation periods
for C. bombicola were seven, eight and 11 days [59,60]. Maximum biosurfactant production by C.
bombicola grown in animal fat was found after 68 h of incubation [49]. Moreover, an increase in agitation
speed favoured the accumulation of a biosurfactant by P. aeruginosa UCP 0992 grown in glycerol [ 61].
Oliveira et al. [62] studied the effect of a change in agitation speed of cultures from 50 to 200 rpm on P.
alcaligenes cultivated in palm oil. The authors found that the increase in rotation velocity favoured a
reduction in the surface tension of the cell-free broth to 27.6 mN/m. In contrast, Cunha et al. [ 63] found
that agitation had a negative effect regarding a reduction in surface tension using a biosurfactant from
Serratia sp. SVGG16 grown in a hydrocarbon culture.

6. Metabolic Pathways of Biosurfactant Production

Hydrophilic substrates are primarily used by microorganisms for cell metabolism and the synthesis
of the polar moiety of a biosurfactant, whereas hydrophobic substrates are used exclusively for the
production of the hydrocarbon portion of the biosurfactant [37,64]. Diverse metabolic pathways are
involved in the synthesis of precursors for biosurfactant production and depend on the nature of the main
carbon sources employed in the culture medium. For instance, when carbohydrates are the only carbon
source for the production of a glycolipid, the carbon flow is regulated in such as way that both lipogenic
pathways (lipid formation) and the formation of the hydrophilic moiety through the glycolytic pathway are
suppressed by the microbial metabolism, as illustrated in Figure 5 [65].
A hydrophilic substrate, such as glucose or glycerol, is degraded until forming intermediates of the
glycolytic pathway, such as glucose 6-phosphate, which is one of the main precursors of carbohydrates
found in the hydrophilic moiety of a biosurfactant. For the production of lipids, glucose is oxidised to
pyruvate through glycolysis and pyruvate is then converted into acetyl-CoA, which produces malonyl-CoA
when united with oxaloacetate, followed by conversion into a fatty acid, which is one of the precursors for
the synthesis of lipids [66]. When a hydrocarbon is used as the carbon source, however, the microbial
mechanism is mainly directed to the lipolytic pathway and gluconeogenesis (the formation of glucose
through different hexose precursors), thereby allowing its use for the production of fatty acids or sugars.
The gluconeogenesis pathway is activated for the production of sugars. This pathway consists of the
oxidation of fatty acids through -oxidation to acetyl-CoA (or propionyl-CoA in the case of odd chain fatty
acids). Beginning with the formation of acetyl-CoA, the reactions involved in the synthesis of
polysaccharide precursors, such as glucose 6-phosphate, are essentially the inverse of those involved in
glycolysis. However, reactions catalysed by pyruvate kinase and phosphofructokinase-1 are irreversible.
Thus, other enzymes exclusive to the process of gluconeogensis are required to avoid such reactions.
Figure 6 illustrates the main reactions through to the formation of glucose 6-phosphate, which is the main
precursor of polysaccharides and disaccharides formed for the production of the hydrophilic moiety of
glycolipids [67].
 Int. J. Mol. Sci. 2016, 17, 401 9 of 31
Int. J. Mol. Sci. 2016, 17, 401 9 of 30

Figure 5.Intermediate metabolism related to of precursors with use of

Figure 5.Intermediate metabolism related to synthesis of biosurfactant precursors with use of
carbohydratescarbohydratesassubstrate.assubstrate.EnzymeEnzymekeyskeysfor forcontrolcontrolofofcarboncarbonflow:flow: (A(A))phosphofructokinase;phosphofructokinase; (B)
(B) pyruvate kinase; (C) isocitrate dehydrogenase.
pyruvate kinase; (C) isocitrate dehydrogenase.
According to Sydatk and Wagner [68], the biosynthesis of a surfactant occurs through four
A hydrophilic substrate, such as glucose or glycerol, is degraded until forming intermediates of
different routes: (a) carbohydrate and lipid synthesis; (b) synthesis of the carbohydrate half while the
the glycolytic pathway, such as glucose 6-phosphate, which is one of the main precursors of
synthesis of the lipid half depends on the length of the chain of the carbon substrate in the medium; carbohydrates found in the hydrophilic moiety of a biosurfactant. For
the production of lipids,
(c) synthesis of the lipid half while the synthesis of the carbon half depends on the substrate employed;
glucose is oxidised to pyruvate through glycolysis and pyruvate is then converted into acetyl-CoA, and (d) synthesis of the carbon and
lipid halves, which are both dependent on the substrate. Therefore,
which produces malonyl-CoA when united with oxaloacetate, followed by conversion into a fatty the length of the n-alkane chain used as the carbon source
alters the biosynthesis of a surfactant.
acid, whichKitamotois etoneal. [of46]thestudiedprecursorstheproductionfortheofsynthesis manosilerythritoloflipidslipid[66](MEL).Whenby theahydrocarbonyeastC.antarcticaisusedin as the

carbonthepresencesource,of however,differentn-thealkanesmicrobialandfoundmechanismthatthisspeciesismainly does directednotgrow orto producethelipolyticabiosurfactantpathway and

inmediacontaining C toC . However, production occurred when thespecies wasgrown

gluconeogenesis (theformation 10 of18 glucose through different hexose precursors), thereby allowing its

use formedium theproductioncontaining ofC12fattytoC18acidsand oroctadekanesugars. Theassubstrategluconeogenesisledtothe greatestpathwayyield.isactivatedIncontrast,for the

production was minimal in media containing n-alkanes with more than 19 carbons.
production of sugars. This pathway consists of the oxidation of fatty acids through β-oxidation to
acetyl-CoA (or propionyl-CoA in the case of odd chain fatty acids). Beginning with the formation of
acetyl-CoA, the reactions involved in the synthesis of polysaccharide precursors, such as glucose 6-
phosphate, are essentially the inverse of those involved in glycolysis. However, reactions catalysed
by pyruvate kinase and phosphofructokinase-1 are irreversible. Thus, other enzymes exclusive to the
process of gluconeogensis are required to avoid such reactions. Figure 6 illustrates the main reactions
through to the formation of glucose 6-phosphate, which is the main precursor of polysaccharides and
disaccharides formed for the production of the hydrophilic moiety of glycolipids [67].
 Int. J. Mol. Sci. 2016, 17, 401 10 of 31
Int. J. Mol. Sci. 2016, 17, 401 10 of 30

Figure 6. Intermediate metabolism related to synthesis of precursors of biosurfactant Figure 6. Intermediate metabolism related to synthesis
of precursors of biosurfactant using

hydrocarbonsusinghydrocarbonsassubstrate.assubstrate.Key enzymes:Keyenzymes:(A) isocitrate(A)isocitratelyase;lyase;(B) (B) malate synthase; malate

(C) phosphoenolpyruvate; (D) fructose-1. synthase; (C)
phosphoenolpyruvate; (D) fructose-1.
7. Physiology
According to Sydatk and Wagner [68], the biosynthesis of a surfactant occurs through four different Biosrourfactantstes:(a)carbohydrateareproducedandby
lipidmicroorganismssynthesis;(b)eithersynthesisthroughof theexcretioncarbohydrateoradhesionhalf whiletocells,the
synthesis especiallyof thewhenlipidcultivatedhalfdependsonsubstratesonthe thatlengthareofinsolublethechain ofwaterthe. carbonWhilethesubstratefunctionininthemicrobmedium;al

(c) cellssynthesisnot ofyetthefullylipidunderstood,halfwhileit hasthe beensynthesispeculatedofthethcatrbonbiosurfactantshalfdependsare involvonthedsubstrateinthe

employed;emulsificationand of(d)insolublesynthesissubstratofthes carbon[37,69]. and lipid halves, which are both dependent on the

substrateThe.Therefore,mainphysiologicalthelengthroleof theofbiosurfactants-alkanechain is usedtoallowasthemicroorganismsarbonsourcetoaltersgrowtheon biosynthesissubstrates

areinsoluble inwater through areduction insurface tension betweenphases, making the
of thatsurfactant .Kitamoto etal .[46]studied theproduction of manosilerythritol lipid (MEL) by the

substrate more available for uptake and metabolism. The uptake mechanisms of these substrates yeast C. antarctica in the presence of different n-alkanes and found that this
species does not grow or
(such as, alkanes) are not yet fully clarified. The direct uptake of dissolved hydrocarbons in the produce a biosurfactant in media containing C10 to C18.
However, production occurred when the
aqueous phase, direct contact between cells and large hydrocarbon droplets, and the interaction with species was grown in a medium containing C12 to C18 and
octadekane as substrate led to the greatest
emulsified droplets (emulsion) have been described. Besides the emulsification of the carbon source, yield. In contrast, production was minimal in media containing
n-alkanes with more than 19 carbons.
biosurfactants are also involved in the adhesion of microbial cells to hydrocarbons, as discussed in
the following sections. Microorganism cell adsorption to insoluble substrates and the excretion of
7. Physiology
surfactant compounds allow growth on carbon sources [37].
Biosurfactants are produced by microorganisms either through excretion or adhesion to cells,
especially when cultivated on substrates that are insoluble in water. While the function in microbial
cells is not yet fully understood, it has been speculated that biosurfactants are involved in the
emulsification of insoluble substrates [37,69].
The main physiological role of biosurfactants is to allow microorganisms to grow on substrates that
are insoluble in water through a reduction in surface tension between phases, making the substrate more
available for uptake and metabolism. The uptake mechanisms of these substrates (such as, alkanes) are
not yet fully clarified. The direct uptake of dissolved hydrocarbons in the aqueous phase, direct contact
between cells and large hydrocarbon droplets, and the interaction with emulsified droplets (emulsion)
have been described. Besides the emulsification of the carbon source, biosurfactants are also involved in
the adhesion of microbial cells to hydrocarbons, as discussed in
Int. J. Mol. Sci. 2016, 17, 401 11 of 31

8. Fermentation Kinetics
Biosurfactant production kinetics has considerable variation among different systems. For
convenience, kinetic parameters are grouped as follows: (a) growth-associated production;
(b) production under growth-limiting conditions; (c) production by resting or immobilised cells; and
(d) production with precursor supplementation [37]. In growth-associated production, parallel
relationships are found between growth, the use of the substrate and biosurfactant production.
Production under growth-limiting conditions is characterised by an accentuated increase in
biosurfactant concentration as a result of the limitation of one or more medium components.
Production by resting or immobilised cells is a type of biosurfactant production in which there is no
cell multiplication; the cells nonetheless continue to use the carbon source for biosurfactant
synthesis. Investigators report that the addition of biosurfactant precursors to the medium leads to
qualitative and quantitative changes in the final product.

9. Raw Materials for Biosurfactant Production

Current society is characterised by an increase in expenditures, the need to reuse materials
and environmental concerns. Consequently, greater emphasis has been given to recovery, recycling
and reuse. Indeed, the need for environmental preservation has led to the reuse of different
industrial wastes. This is particularly valid for the food production industry, the waste products,
effluents and by-products of which can be reused [70]. Industrial waste had piqued the interest of
researchers as a low-cost substrate for biosurfactant production [71]. The selection of waste
products should ensure the proper balance of nutrients to allow microbial growth and consequent
biosurfactant production. Industrial waste with a high content of carbohydrates or lipids is ideal for
use as substrate [71]. According to Barros et al. [34], the use of agro-industrial waste is one of the
steps towards the implantation of feasible biosurfactant production on an industrial scale, for which
the optimisation of the different variables involved is required.
The literature describes a number of waste products employed in biosurfactant production,
such as vegetable oils, oily effluents [42,72,73], starchy effluents [74,75], animal fat [51,76–78],
vegetable fat [79], vegetable cooking oil waste [72,80–82], soapstock [76,83,84] molasses [85–88],
dairy industry waste (whey) [89], corn steep liquor [43,71,90–92], cassava flour wastewater [93], oil
distillery waste [43,90,94,95] and glycerol [61]. Some of the most commonly employed industrial
waste products for biosurfactant production are detailed below.

9.1. Olive Oil Mill Effluent (OOME)

Olive Oil Mill Effluent (OOME) is a concentrated black liquor with a water-soluble portion of ripe
olives and water that is used for the extraction of olive oil. OOME has polyphenols that represent a
challenge in terms of the environment disposal. However, it also contains nitrogen compounds (12
to 24 g/L), sugars (20 to 80 g/L), residual oil (0.3 to 5 g/L) and organic acids (5 to 15 g/L). Mercade
et al. [73] successfully employed OOME for the strain Pseudomonas sp. to produce rhamnolipids.

9.2. Animal Fat

Animal fat and lard can be obtained in large quantities from the meat processing industry and
have been used as a medium for cooking foods. Recently, however, such fats have lost a large part
of the market to vegetable oils due to the lower degree of harm to health caused by the latter [ 70].
Animal fat stimulates the production of sophorolipids by the yeast C. bombicola [51]. Using animal
fat and corn steep liquor, Santos et al. [77,78] achieved maximum glycolipid production by the yeast
C. lipolytica UCP 0988. The authors also report that the product has uses in bioremediation as well
as oil mobilisation and recovery.
Int. J. Mol. Sci. 2016, 17, 401 12 of 31

9.3. Frying Oils

Fry oil and edible fats are considered great carbon sources for biosurfactant production. Vegetable
oils constitute a lipid carbon source and are mainly comprised of saturated or unsaturated fatty acids with
chains of 16 to 18 carbon atoms [9]. Different oils are adequate substrates for biosurfactants. Babassu oil
(5% v/v) with a carbon source (1% glucose w/v) is a good medium for biosurfactant production. Sarubbo
et al. [96] found that two strains of C. lipolytica (1055 and 1120) produce biosurfactants toward the final of
the exponential growth phase and onset of the stationary phase. Sunflower and olive oils have proven to
be adequate energy and carbon sources for the production of biosurfactants. P. aeruginosa strains
produce a biosurfactant on residue from corn, soybean and canola oil plants [97,98]. Canola oil residue
and sodium nitrate has been reported adequate for microbial growth and the production of up to 8.50 g/L
of rhamnoipids. The combination of glucose and canola oil has been used for the successful production
of a biosurfactant by C. lipolytica [42].

9.4. Soapstocks
Oil cakes or soapstocks are produced from oilseed processing involving the refining of seed-
based edible oils with the use of chemicals [76]. Soapstock has been used together with sunflower
oil, olive oil or soybean oil as substates to produce rhamnolipids. Yields as high as 15.9 g/L have
been reported using P. aeruginosa in a soapstock medium [83]. Soapstock and oil refinery wastes
have been used with C. antarctica or C. apicola for biosurfactant production, achieving a greater
yield than that in the medium without oil refinery residue [45]. Shabtai [84] also report the production
of two extracellular heteropolysaccharides (emulsan and biodispersan) by A. calcoaceticus and A.
calcoaceticus, respectively, using soapstock as a carbon source.

9.5. Molasses
Molasses is a by-product of sugarcane and beet processing. This inexpensive substrate has
dry matter (75%), non-sugar organic matter (9%–12%), protein (2.5%), and potassium (1.5%–5.0%),
as well as magnesium, phosphorus and calcium ( 1%). The inositol, biotin, thiamine and pantothenic
acid contents (1%–3%) give molasses its thick consistency and brown colour. The high sugar
content (48%–56%) makes molasses adequate for biosurfactant production by different
microorganisms. Laboratories have used molasses for the production of different microbial
metabolites. According to Makkar and Cameotra [88], Bacillus subtilis in a minimal medium
supplemented with molasses as the carbon source produces a biosurfactant. Joshi et al. [99] used
molasses as well as other carbon sources to produce biosurfactants from strains of Bacillus.

9.6. Whey
The dairy industry produces large quantities of whey, such as whey waste, cheese whey, curd
whey and lactic whey, all of which can be used as substrates for the microbial production of
metabolites [70,100–102]. A high amount of lactose (approximately 75%) is found in lactic whey.
Other components, such as proteins, vitamins and organic acids, are good sources for microbial
growth and biosurfactant production [75]. Moreover, whey disposal represents a major pollution
problem, especially in countries that depend on a dairy economy [103]. Thus, the disposal of this by-
product represents a waste of a widely available substrate and an environmental hazard.

9.7. Corn Steep Liquor

The agro-industry of corn-based products through wet processing results in both solid and liquid by-
products, which, when disposed improperly, become a source of contamination and harm to the
environment. Corn steep liquor is a by-product of the washing water and soaking of kernels as well as
fractioning into starch and germen (oil) that contains 40% solid matter. This by-product consists of 21% to
2+ 2+
45% proteins, 20% to 26% lactic acid, approximately 8% ash (containing Ca , Mg ,
Int. J. Mol. Sci. 2016, 17, 401 13 of 31

+
K , etc.), approximately 3% carbohydrates and a low fat content (0.9% to 1.2%) [103–105]. Nut oil
refinery residue and corn steep liquor are low-cost nutrients for the production of glycolipids by C.
sphaerica (UCP 0995). The biosurfactant of this strain mobilises and removes up to 95% of motor
oil on sand, making it useful for bioremediation [89,94,106]. Silva et al. [107] also report the
production of a biosurfactant from P. cepacia grown in mineral medium supplemented with 2.0%
corn steep liquor and 2.0% soybean waste frying oil.

9.8. Starchy Substrates

Abundant starch-based substrates also constitute renewable carbon sources. The potato processing
industry produces significant amounts of starch-rich waste that are adequate for biosurfactant production.
Besides the approximately 80% water content, potato waste has carbohydrates (17%), proteins (2%) and
fats (0.1%) as well as inorganic minerals, trace elements and vitamins [103]. As an example, Fox and
Bala investigated a commercially prepared potato starch in a mineral salt medium for the production of a
biosurfactant by B. subtilis [74]. Cassava wastewater, which is another carbohydrate-rich waste product
generated in large amounts, has been used for the production of surfactin by B. Subtilis 35. Other starchy
wastes, such as rice water and cereal processing wastewater, have the potential to permit microbial
growth and biosurfactant production [108].

10. Recovery of Biosurfactants

The production of low-coast biosurfactants is unlikely due to the complicated recovery process.
Process development is conduted in order to obtain biosurfactants that can be recovered easily and
inexpensively. In many biotechnological processes, downstream processing accounts for 70%–80%
of production costs. For economic reasons, most biosurfactant production processes need to involve
spent whole-cell culture broths or other crude preparations [11,103,109]. Extraction with chloroform-
methanol, dichloromethane-methanol, butanol, ethyl acetate, pentane, hexane, acetic acid, ether,
etc. constitutes the most commonly used method in biosurfactant downstream processing. The most
widely employed products are different ratios of chloroform and methanol, which facilitate the
adjustment of the polarity of the extraction agent to the extractable target material. The
disadvantages of using organic solvents for biosurfactant recovery include the large amount of
solvent required and the increase in production costs due to the price of expensive solvents.
Chloroform is a toxic chloro-organic compound that is harmful to human health and the environment.
Thus, there is a need for inexpensive solvents with low toxicity for biosurfactant extraction processes
that are suitable for industrial applications. Other product precipitation techniques have also been
reported, such as precipitation with ammonium sulphate, centrifugation and adsorption.
Biosurfactant recovery depends mainly on the ionic charge, water solubility and location
(intracellular, extracellular or cell bond) [103,109]. Foam fractionation is a solvent-free method that
separates biosurfactant molecules adsorbed to air bubbles in the culture medium. Biosurfactant
production involves continuous foam formation due to the high surface activity. Foam in the broth
interferes with mass and heat transfer processes, thereby affecting productivity. However, foam is
beneficial to biosurfactant production, as it assists in the continuous removal of product, and
therefore production and recovery processes can be accomplished in a single stage [ 110].
Continuous foam fractionation in the fermentation process helps prevent the accumulation of
product that could otherwise inhibit biomass growth and product formation and also facilitates
extended biosurfactant production in fed-batch or continuous mode operations. Moreover,
biosurfactants do not readily undergo denaturation due to their small size and simple structure [111].
More research and development are required to optimise existing recovery processes to make
such processes both commercially viable and more competitive [39,103]. Table 2 lists the most
common biosurfactant recovery techniques and their advantages.
Int. J. Mol. Sci. 2016, 17, 401 14 of 31

Table 2. Downstream processes for recovery of important biosurfactants and respective advantages.

Biosurfactant Property Responsible for

Process Biosurfactant Type Advantages
Separation
Biosurfactants become insoluble at low Low cost, efficient in crude
Acid precipitation Surfactin
pH values biosurfactant recovery
Batch mode
Trehalolipids; Efficient in crude biosurfactant
Biosurfactants are soluble in organic
Organic solvent extraction Sophorolipids; recovery and partial purification,
solvents due to the hydrophobic end
Liposan reusable nature
Emulsan;
Salting-out of polymeric or Effective in isolation of certain type
Ammonium sulphate precipitation Biodispersan;
protein-rich biosurfactants of polymeric biosurfactants
Lipopeptides
Rhamnolipids;
Biosurfactants are adsorbed to activated Highly pure biosurfactants, cheaper,
Lipopeptides;
Adsorption to wood-activated carbon carbon and can be desorbed using reusability, recovery from
Glycolipids;
organic solvents continuous culture
Mannosylerythritol Lipids (MEL)
Continuous mode Rhamnolipids;
Biosurfactants are adsorbed to polystyrene Highly pure biosurfactants, cheaper,
Lipopeptides;
Adsorption to polystyrene resines resins and subsequently desorbed using reusability, recovery from
Glycolipids;
organic solvents continuous culture
MEL
Insoluble biosurfactants are precipitated due Reusable, effective in crude
Centrifugation Glycolipids
to centrifugal force biosurfactant recovery
Charged biosurfactants are attached to
High purity, reusability,
Ion-exchange chromatography Glycolipids ion-exchange resins and can be eluted
fast recovery
with buffer
Useful in continuous recovery
Foam fractionation Surfactin Biosurfactant form and partition into foam
processes, high purity of product
Biosurfactants form micelles above their
Fast, one-step recovery, high level of
Ultrafiltration Glycolipids critical micelle concentration (CMC), which
purity, reusability
are trapped by polymeric membranes
 Int. J. Mol. Sci. 2016, 17, 401 15 of 31

Int. J. Mol. Sci. 2016, 17, 401 15 of 30

11. Industrial Applications of Biosurfactants

11. Industrial Applications of Biosurfactants
Biosurfactants have a wide range of biotechnological applications in petroleum, foods, beverages,
cosmetics,Biosurfactantsdetergents,have atextilwides,rangepaints,ofmining,biotechnocellulose,ogica applicationspharmaceuticsin andpetroleum,nanotechnologyfoods,beverages,[112].

cosmetics,Currently,detergents,themain textiles,market ispaints,thepetroleummining, industrycellulose, . Biosurfactanpharmaceuticscanandbe usednanotechnologyforoilresidue[112].

Currently,recoverythefrommainstoragemarkt tanks,isthe peotroleumheroil industryrecovery. Biosurfactantsprocesses,hecancleanbeusedpofforoiloilspillsresidueandrecoverythe

frombioremediationstoragetanks,ofotherbothoilsoilrecoveryandwaterprocesses,[2113]. theTablecleanup3offersofaoilsummaryspillsandof the bioremediationusesofbiosurfactantsofbothinsoil

anddifferentwater[2,113]industries.Table.The3offersmainabiotechnologicalsummaryoftheapplicationsusesofbiosuarfactantsedetailedinindifferentthefollowingindustriessections.The. main

biotechnological applications are detailed in the following sections.

11.1. Petroleum Recovery

11.1. Petroleum Recovery
Petroleum is an essential energy source and driving force of economic development. The US
Department of Energy reports that fossil fuels constitute 83% of all primary energy sources in Petroleum is an essential energy source
and driving force of economic development. The US
the country and petroleum accounts for 57% of such products. Indeed, 19.2 million cubic metres Department of Energy reports that fossil fuels constitute 83% of all primary energy
sources in the country
3
of petroleum were consumed per day in 2010 [114]. The USA produces 870,000 m of crude and petroleum accounts for 57% of such products. Indeed, 19.2 million cubic
metres of petroleum were
3
oil from 530 thousand production wells, 35% of which produce 0.16 m /day and 79% produce consumed per day in 2010 [114]. The USA produces 870,000 m3 of crude oil
from 530 thousand production
< 3
1.59 m /day [115]. Through oil recovery processes, these oil wells produce only one third to wells, 35% of which produce 0.16 m3/day and 79% produce <
1.59 m3/day [115]. Through oil recovery
half of the petroleum originally present at the sites. Oil residue in small pores within petroleum processes, these oil wells produce only one third to half of the
petroleum originally present at the sites. Oil
reservoirs accounts for 50% to 65% of oil and is trapped by high forces of capillarity as well as residue in small pores within petroleum reservoirs accounts for
50% to 65% of oil and is trapped by high
interfacial tension between the hydrocarbon and aqueous phases. Different reductions in interfacial forces of capillarity as well as interfacial tension between the
hydrocarbon and aqueous phases. Different
tension are needed for the mobilisation of this hydrocarbon [ 116,117], which is only achieved with reductions in interfacial tension are needed for the
mobilisation of this hydrocarbon [116,117], which is only
the use of surfactant concentrations significantly higher than that required for the formation of achieved with the use of surfactant concentrations significantly
higher than that required for the formation
micelles [118,119]. In enhanced oil recovery, the use of heat, tensioactive agents, microbial processes of micelles [118,119]. In enhanced oil recovery, the use
of heat, tensioactive agents, microbial processes and
and gas injection leads to the recovery of a significant portion of the retained oil. However, the high gas injection leads to the recovery of a significant portion
of the retained oil. However, the high cost of
cost of chemical tensioactive agents hinders widespread use of surfactants in oil recovery processes. chemical tensioactive agents hinders widespread use of
surfactants in oil recovery processes. Thus,
Thus, biosurfactants have been employed to reduce the interfacial tension between oil/water and biosurfactants have been employed to reduce the interfacial
tension between oil/water and oil/rock, which
oil/rock, which leads to a reduction in the capillary forces that impede oil from moving through rock leads to a reduction in the capillary forces that impede oil
from moving through rock pores (Figure 7).
pores (Figure 7). Biosurfactants also form an emulsion at the oil-water interface, which stabilises the Biosurfactants also form an emulsion at the oil- water
interface, which stabilises the desorbed oil in water
anddesorbedallowsoiloilremovalinwateralongand allowswiththeoilinjectionremovalwateralong[1,8].with the injection water [1,8].

Figure 7. Enhanced oil recovery mechanism by biosurfactants.

Figure 7. Enhanced oil recovery mechanism by biosurfactants.
Int. J. Mol. Sci. 2016, 17, 401 16 of 31

Table 3. Applications of biosurfactants for industrial uses.

Industry Application Role of Biosurfactants References

Emulsification of oils, lowering of interfacial tension,
Bioremediation;
dispersion of oils, solubilisation of oils, wetting, spreading,
Environment Oil spill cleanup operations; [2,8]
detergency, foaming, corrosion inhibition in fuel oils and
Soil remediation and flushing
equipment, soil flushing.
Emulsification of oils, lowering of interfacial tension,
de-emulsification of oil emulsions, solubilisation of oils,
Enhanced oil recovery;
Petroleum viscosity reduction, dispersion of oils, wetting of solid surfaces, [8,120]
De-emulsification
spreading, detergency, foaming, corrosion inhibition in fuel oils
and equipment.
Heavy metal cleanup operations; Wetting and foaming, collectors and frothers, removal of metal
Mining Soil remediation; ions from aqueous solutions, soil and sediments, heavy metals [121]
Flotation sequestrants, spreading, corrosion inhibition in oils.
Solubilisation of flavoured oils, control of consistency,
Emulsification and de-emulsification;
Food emulsification, wetting agent, spreading, detergency, [4]
Functional ingredient
foaming, thickener.
Anti-adhesive agents, antifungal agents, antibacterial agents,
Microbiological;
Medicine antiviral agents, vaccines, gene therapy, [20,122,123]
Pharmaceuticals and therapeutics
immunomodulatory molecules.
Wetting, dispersion, suspension of powdered pesticides and
fertilisers, emulsification of pesticide solutions, facilitation of
Biocontrol;
Agriculture biocontrol mechanisms of microbes, plant pathogen [124]
Fertilisers
elimination and increased bioavailability of nutrients for
beneficial plant-associated microbes.
Emulsification, foaming agents, solubilisation, wetting agents,
Cosmetics Health and beauty products [5]
cleansers, antimicrobial agents, mediators of enzyme action.
Detergents and sanitisers for laundry, wetting, spreading,
Cleaning Washing detergents [3,5]
corrosion inhibition.
Preparation of fibres; Wetting, penetration, solubilisation, emulsification, detergency
Textiles Dyeing and printing; and dispersion, wetting and emulsification in finishing [3,103]
Finishing of textiles formulations, softening.
Nanotechnology Synthesis of nanoparticles Emulsification, stabilisation. [5,125]
Int. J. Mol. Sci. 2016, 17, 401 17 of 31

11.2. Bioremediation
Oil spills occur during cargo transportation or in the form of industrial oil and by-product spills.
Petroleum exerts a negative effect on cell membranes in living organisms, offering considerable risk
of contamination to both marine and terrestrial ecosystems [2,114].
The US Environmental Protection Agency proposes different physical, chemical and biological
technologies for the treatment of contaminated soil [126], one of the most studied of which is
bioremediation. This process involves the natural degradation capacity of plants and
microorganisms for either the partial conversion of contaminants into less toxic compounds or the
complete conversion of such substances into carbon dioxide and water.
Larger degrading microorganism populations lead to a quicker, more efficient bioremediation
processes. Therefore, this technique can be conducted through biostimulation, which consists of
stimulating the growth of microorganisms present at the contaminated site. The process involves the
introduction of specific electron receptors, oxygen and nutrients for the degradation of the
contaminant as well as substances to correct the pH. Bioremediation can also be performed through
bioaugmentation, in which indigenous (allochthonous) microorganisms are added to the
contaminated environment to accelerate and complete the degradation of the pollutant [114].
Bioremediation played an important role in the cleanup of the 41 million litre oil spill caused by the
oil tanker Exxon Valdez in the Gulf of Alaska in 1989, giving rise to the development of this technology
and demonstrating that there are good reasons to believe in the effective application of this treatment
method in future oil spills under the appropriate circumstances [114]. In the accident with the Exxon
Valdez, the first measure taken was physical washing with high-pressure water. Chemical surfactants
were then applied in polluted areas to accelerate the growth and activity of petroleum-degrading
microorganisms. Two or three weeks later, the regions treated with surfactants were significantly cleaner
than control areas. However, it was difficult to evaluate the exact effects of the treatment due to the
heterogeneity of the contamination. Nonetheless, subsequent studies have demonstrated the importance
of the use of surfactants to enhance the biodegradation of oil [114,127].
While bioremediation is an effective, environmentally friendly method, the time and costs
involved make this process unviable for the treatment of large amounts of waste [ 128]. Thus, the
use of biosurfactants emerges as a safe alternative for improving the solubility of hydrophobic
compounds by allowing the desorption and solubilisation of hydrocarbons and facilitating the
assimilation of these compounds by microbial cells [129].
The biodegradation of oil-derived hydrocarbons by biosurfactants occurs through two mechanisms.
The first involves an increase in the bioavailability of the hydrophobic substrate to microorganisms, with a
consequent reduction in surface tension of the medium around the bacterium as well as a reduction in
interfacial tension between the cell wall and hydrocarbon molecules. The other mechanism involves the
interaction between the biosurfactant and cell surface, leading to changes in the membrane, facilitating
hydrocarbon adherence (increase in hydrophobicity) and reducing the lipopolysaccharide index of the cell
wall without damaging the membrane. Thus, biosurfactants block the formation of hydrogen bridges and
allow hydrophobic-hydrophilic interactions, which cause molecular rearrangements and reduce the
surface tension of the liquid by increasing its surface area as well as promoting bioavailability and
consequent biodegradability [130,131]. Figure 8 illustrates the action of biosurfactants in increasing the
surface area of oil droplets as well as facilitating access to a greater number of bacteria and
consequently producing a greater biomass.

11.3. Removal of Hydrophobic Organic Pollutants

The application of biosurfactants for the removal of contaminants from soil is less well known than
the advanced application of these compounds in bioremediation processes, since removal efficiency is
driven mainly by the physicochemical properties of the biosurfactant rather than the effects on metabolic
activity or changes in the properties of the cell surface. However, the mechanisms that
 Int. J. Mol. Sci. 2016, 17, 401 18 of 31

affect the mobilisation and solubilisation of hydrocarbons in soils are similar to those involved in the
enhancement of bioavailability for bioremediation [131,132].
Int. J. Mol. Sci. 2016, 17, 401 18 of 30

Figure 8. Illustration of biosurfactant action on petroleum.

Figure 8. Illustration of biosurfactant action on petroleum.

11.3. RemovalBiosurfactantsofHydrophobicenhanceOrganicthe Pollutantsremoval of hydrocarbons through biodegradation, solubilisation,

mobilisation or emulsification [8]. Solubilisation capacity depends on the ability of the surfactant to The application of
biosurfactants for the removal of contaminants from soil is less well known than the increase the solubility of hydrophobic
components in the aqueous phase. A considerable increase in this
advanced application of these compounds in bioremediation processes, since removal efficiency is driven capacity occurs above the CMC due
to the partitioning of the hydrocarbon in the hydrophobic portion mainly by the physicochemical properties of the biosurfactant rather than the
effects on metabolic activity of the micelles. In this process, greater concentrations of surfactants are normally required, since the
or changes in the properties of the cell surface. However, the mechanisms that affect the mobilisation and solubility of the hydrocarbon
components in the solution depends wholly on the concentration of the solubilisation of hydrocarbons in soils are similar to those involved in the
enhancement of bioavailability surfactant [8]. Mobilisation occurs at concentrations below the CMC and is divided into displacement
for bioremediation [131,132].
and dispersion. Displacement consists of the release of hydrocarbon droplets from the porous medium
Biosurfactants enhance the removal of hydrocarbons through biodegradation, solubilisation, due to the reduction in interfacial tension. Using a
theoretical explanation, hydrocarbon removal
mobilisation or emulsification [8]. Solubilisation capacity depends on the ability of the surfactant to increase is possible when the interfacial tension
between the aqueous and oil phases is sufficiently reduced
the solubility of hydrophobic components in the aqueous phase. A considerable increase in this capacity to overcome the forces of capillarity
that cause the formation of residual saturation. Dispersion is a occurs above the CMC due to the partitioning of the hydrocarbon in the
hydrophobic portion of the micelles. process by which a hydrocarbon is dispersed in the aqueous phase as tiny emulsions. Emulsions are
In this process, greater concentrations of surfactants are normally required, since the solubility of the not generally thermodynamically stable, but may
remain stable for significant periods of time due to
hydrocarbon components in the solution depends wholly on the concentration of the surfactant [8]. kinetic restrictions. Dispersion is related to interfacial
tension and surfactant concentration and differs
Mobilisation occurs at concentrations below the CMC and is divided into displacement and dispersion. from displacement, which is related only to interfacial tension
between the aqueous and hydrophobic Displacement consists of the release of hydrocarbon droplets from the porous medium due to the reduction
phases, with no formation of emulsion [132].
in interfacial tension. Using a theoretical explanation, hydrocarbon removal is possible when the interfacial The efficiency of a surfactant in the removal of
hydrophobic compounds also depends on the pH
tension between the aqueous and oil phases is sufficiently reduced to overcome the forces of capillarity that and ionic strength of the solution,
which can alter the arrangement of the aggregated micelles and cause the formation of residual saturation. Dispersion is a process by which a
hydrocarbon is dispersed in sorption of the surfactant to the soil, which, in turn, limits the transport of the hydrocarbon by the
the aqueous phase as tiny emulsions. Emulsions are not generally thermodynamically stable, but may surfactant. Different biosurfactants have been tested
for the removal of petroleum-derived products
remain stable for significant periods of time due to kinetic restrictions. Dispersion is related to interfacial from contaminated soil and water.
Rhaminolipids have been successfully used in biotechnological tension and surfactant concentration and differs from displacement, which is
related only to interfacial decontamination processes [2,7,17]. Other surfactants produced by species of Pseudomonas [110,133],
tension between the aqueous and hydrophobic phases, with no formation of emulsion [132].
Bacillus [5,134], and Candida [109,113,135–137], have also been successfully used in the remediation
The efficiency of a surfactant in the removal of hydrophobic compounds also depends on the pH and of soil.
ionic strength of the solution, which can alter the arrangement of the aggregated micelles and sorption of
the surfactant to the soil, which, in turn, limits the transport of the hydrocarbon by the surfactant. Different 11.4. Removal of
Heavy Metals
biosurfactants have been tested for the removal of petroleum-derived products from contaminated soil and
Heavy metals and radionuclides are persistent soil contaminants. Increases in levels of water. Rhaminolipids have been successfully used in
biotechnological decontamination processes [2,7,17].
heavy metals in soil have been reported in many industrialised countries. Metals and metalloids, Other surfactants produced by species of Pseudomonas
[110,133], Bacillus [5,134], and Candida [109,113,135–
such as chromium, cadmium, mercury and lead, can threaten ecosystems and human health 137], have also been successfully used in the remediation of
soil.
through either the food chain or direct exposure to contaminated soil and water [1,12]. As
different technologies can be used in combination for the treatment of organic pollutants and 11.4. Removal of Heavy Metals
heavy metals, biosurfactants can be used in the removal of hydrophobic organic compounds and
Heavy metals and radionuclides are persistent soil contaminants. Increases in levels of heavy metals heavy metals [138–140]. Heavy metals mainly
adsorb to the surface of soil in the form of ions or
in soil have been reported in many industrialised countries. Metals and metalloids, such as chromium, the precipitation of metal compounds. Unlike organic
contaminants, heavy metals are removed
cadmium, mercury and lead, can threaten ecosystems and human health through either the food chain or direct
exposure to contaminated soil and water [1,12]. As different technologies can be used in combination for the
treatment of organic pollutants and heavy metals, biosurfactants can be used in the removal of
 Int. J. Mol. Sci. 2016, 17, 401 19 of 31

from soil through surfactant-associated complexation [141] and ion exchange [142]. Therefore,
surfactant-enhanced washing and surfactant-enhanced bio-extraction can be applied to the
remediation of soils contaminated with heavy metals.
Surfactants in solutions facilitate the solubilisation, dispersion and desorption of contaminants and
allow the reuse of the soil [143]. Decontamination tests have been performed with different synthetic
surfactants [144,145], but the desire to replace such compounds with natural surfactants has led to
research into the use of biosurfactants [119]. Studies have demonstrated the potential of surfactin,
rhamnolipids and sophorolipids [146–148]. The ionic nature, biodegradability, low toxicity and excellent
surface properties make biosurfactants adequate for the removal of heavy metals from sediment and
soil. According to Mulligan [119], removal is possible with different concentrations of biosurfactants. Das
et al. [149] found that the removal of cadmium using an aqueous solution also occurred at concentrations
below the CMC, while a concentration fivefold greater than the CMC resulted in the virtually complete
removal of 100 ppm of metal ions. Wen et al. [150] studied the degradation of a rhamnolipid in soils
contaminated by cadmium and zinc and found that this compound could remain in the soil long enough
to enhance the phytoextraction of the metals.
The removal of metals by ionic biosurfactants is thought to occur in the following order (Figure 9):
(1) sorption of the biosurfactant to the soil surface and complexation with the metal; (2) detachment of the
metal from the soil to the solution; and (3) association with micelles. Heavy metals are trapped
within the micelles through electrostatic interactions and can be easily recovered through precipitation
Int. J. Mol. Sci. 2016, 17, 401 20 of 30 or membrane separation methods [151].

Figure 9. Mechanism of heavy metal removal by biosurfactants.

Figure 9. Mechanism of heavy metal removal by biosurfactants.

Anionic biosurfactants create non-ionic complexes with metals through ionic bonds. As such 11.5. Food Industry
bonds are stronger than those between the metal and soil, the metal-biosurfactant complex is detached
Emulsification is important for the formation of consistency and texture in foods as well as phase from the soil due to the reduction in interfacial
tension. Cationic biosurfactants can replace similarly
dispersion and the solubilisation of aromas [4,159]. The general function of emulsifiers in food products is to
stabilise the emulsion by controlling the agglomeration of fat globules and stabilising aerated systems [4,152].
An emulsion has at least one immiscible liquid (discontinuous internal phase) dispersed in another (continuous
outer phase) in the form of droplets. The stability of this system is minimal, but can be enhanced by the
addition of a surfactant, which reduces surface energy between the two phases by a reduction in interfacial
tension, thereby preventing particle coalescence through the formation of steric and electrostatic
Int. J. Mol. Sci. 2016, 17, 401 20 of 31

charged metal ions through ion exchange (competition for negatively charged surfaces). Surfactant
micelles can also be used to remove metal ions from the soil surface [115].
Biosurfactants offer indisputable advantages, since surfactant-producing microorganisms do
not need to survive in contaminated soil, although the continual addition of biosurfactant is required
in the process [8]. Biosurfactants have also been applied in mining processes. Tensioactive
compounds produced by Pseudomonas sp. and Alcaligenes sp. have been used for the floatation
and separation of calcite and scheelite, with recovery rates of 95% for CaWO 4 and 30% for CaCO3,
whereas conventional chemical reagents are not capable of separating these two minerals [152].
Slizovskiy et al. [153] studied the enhanced remediation of soils contaminated with heavy metals
using the cationic surfactant 1-dodecylpyridinium chloride (DPC), the non-ionic surfactant oleyl
dimethyl benzyl ammonium chloride (trade name Ammonyx KP) and the ionic rhamnolipid
biosurfactant (trade name JBR-425); the latter substance exhibited the best elution with regard to Zn
(39%), Cu (56%), Pb (68%) and Cd (43%). Almeida et al. [154] studied the impact of surfactants on
the removal of Cu by the salt marsh plant Halimione portulacoides. TX-100 and SDS (sodium
dodecyl sulfate) were favourable to Cu harvesting and transportation in plant roots, but did not affect
the transportation of Cu in the stem and leaves. The results of this study suggest that surfactants
promote phytoremediation through a change in the membrane permeability of root cells. Therefore,
surfactants can promote the desorption of metals and uptake by plants [1].
Rhamnolipids and soapberry-derived saponin have recently been found to assist in the removal of
chromium and arsenic oxyanions from soils or the ore waste of mines [155,156]. Biosurfactants produced
by yeast of the genus Candida have been successfully used in heavy metal flotation, demonstrating the
ability to remove more than 90% of cations in columns and air-dissolved flotation processes [157,158]. C.
lipolytica produces a biosurfactant that has also been used for the removal of heavy metals and
petroleum byproducts using a soil barrier [90]. The biosurfactant significantly reduced soil permeability,
thereby demonstrating its usefulness in reactive barriers, with the removal of approximately 96% of Zn
and Cu as well as a reduction in Pb and Cd concentrations in groundwater.

11.5. Food Industry

Emulsification is important for the formation of consistency and texture in foods as well as
phase dispersion and the solubilisation of aromas [4,159]. The general function of emulsifiers in food
products is to stabilise the emulsion by controlling the agglomeration of fat globules and stabilising
aerated systems [4,152]. An emulsion has at least one immiscible liquid (discontinuous internal
phase) dispersed in another (continuous outer phase) in the form of droplets. The stability of this
system is minimal, but can be enhanced by the addition of a surfactant, which reduces surface
energy between the two phases by a reduction in interfacial tension, thereby preventing particle
coalescence through the formation of steric and electrostatic barriers. Examples of processed foods
that are emulsions include heavy cream, butter, mayonnaise, salad dressings, fillings, etc. [ 35].
Other uses for emulsifiers have been described, such as improving the texture and shelf life of
products containing starch, the formation of complexes, altering the rheological properties of wheat
flour and interactions with gluten as well as improving the consistency and texture of fat-based
products through the control of polymorphism and the crystalline structure of fats [4].
Biosurfactants can also be used as emulsifiers in the processing of raw materials, the control of fat
globule agglomeration, the stabilisation of aerated systems and an improvement in the consistency of fat-
based products. The use of rhaminolipids to improve the emulsifying properties of frozen desserts, butter
and croissants has also been reported [4,111]. For instance, Candida utilis produces a bioemulsifier used
in processed salad dressings [160]. A manoprotein produced by Saccharomyces cerevisiae stabilises
water/oil emulsions in cookies, mayonnaise and ice cream, etc. [13,161]. However, the food industry has
not yet made widescale use of biosurfactants. Many of the properties of biosurfactants and their
regulation as new ingredients for foods are pending approval.
Int. J. Mol. Sci. 2016, 17, 401 21 of 31

11.6. Medicine
Biosurfactants have also been used in different biological (therapeutic) applications due to their
fungicidal, bactericidal, insecticidal and anti-viral properties as well as use as anti-adhesive agents
and enzyme inhibitors [111,116,117]. A number of rhaminolipids exhibit antibacterial activity. For
instance, Abalos et al. [162] identified six rhaminolipids in cultures of P. aeruginosa AT10 grown on
soybean oil refinery residue and evaluated the antimicrobial properties of the solution. These
rhaminolipids exhibited excellent antifungal properties against different fungi at concentrations
ranging from 16 to 32 g/mL. C. bombicola-derived sophorolipids inhibited the growth of both Gram-
negative and Gram-positive bacteria with a minimum inhibitory concentration of approximately 30
and 1 mg/mL in a contact time of 2 and 4 h, respectively, for E. coli (ATCC 8739) and P. aeruginosa
(ATCC 9027) as well as 6 and 1 mg/mL in a contact time of 4 h for S. aureus (ATCC 6358) and B.
subtilis (ATCC6633), respectively [163].
Despite the number of publications describing the antimicrobial activity of biosurfactants and
patents related to their usage, real applications in the pharmaceutical, biomedical and health
industries remains quite limited [164]. Some lipopeptides, such as daptomycin, have reached a
commercial antibiotic status [165]. Daptomycin, is a branched cyclic lipopeptide isolated from
Streptomyces roseosporus cultures and is produced by Cubist Pharmaceuticals under the name
®
Cubicin [166]. This drug was approved in 2003 for the treatment of skin infections caused by
methicillin-resistant Staphylococcus aureus and other Gram-positive pathogens and approved in
2006 for the treatment of endocarditis and bacteraemia caused by S. aureus. Daptomycin had also
been reported to display strong antibacterial activity against other important pathogens, such as
penicillin-resistant Streptococcus pneumoniae, coagulase-negative Staphylococci, glycopeptide-
intermediate-susceptible S. aureus and vancomycin-resistant Enterococci [166].
Biofilms are groups of bacteria and other organic matter that has colonised/accumulated on a given
surface [117]. Bacterial adherence to the surface is the first step in the establishment of biofilm and is
affected by various factors, such as the type of microorganism, hydrophobicity, electrical charges of the
surface, environmental conditions and the ability of microorganisms to produce extracellular polymers
that assist the cells in anchoring to the surface [167]. Anti-adherent activity, which is the ability to inhibit
the adherence of pathogenic microorganisms to solid surfaces or infectious sites, has also been reported
for biosurfactants, leading to a reduction in hospital infections with no need for drugs or synthetic
chemical agents [112]. Meylheuc et al. [168] studied a biosurfactant obtained from P. fluorescens with
inhibitory properties regarding the adherence of Listeria monocytogenes to stainless steel and
polytetrafluoroethylene surfaces. C. sphaerica-derived lunasan inhibited the adherence of P.aeruginosa,
Streptococcus agalactiae and S. sanguis between 80% and 92% at a concentration of 10 mg/mL [95],
while the biosurfactant rufisan produced by C. lipolytica UCP 0988 demonstrated antimicrobial activities
against S. agalactiae, S. mutans, S. mutans NS, S. mutans HG, S. sanguis 12, S. oralis J22 at a
concentration above the CMC (0.3%). Moreover, the biosurfactant showed anti-adherent activity against
most of the microorganisms tested [137].
Deficiency of lung surfactant, which is a protein-phopholipid complex, is responsible for
respiratory failure in premature infants. Gene isolation for protein molecules in this surfactant and
cloning in bacteria allow fermentative production for medical applications [108]. Sophorolipids from
C. bombicola have been studied due to their spermicidal and cytotoxic activities as well as anti-HIV
action that can reduce the proliferation of acquired immunodeficiency syndrome (AIDS).
Sophorolipids have also been studied as anti-inflammatory agents for patients with immune
diseases [108,169]. Iturin is a lipopeptide produced by B. subtilis that has demonstrated antifungal
activity by affecting the morphology and structure of the cell membrane of yeasts. In vitro
experiments have demonstrated that surfactin can effectively inactivate the virus that causes herpes
as well as the retrovirus and other compact RNA and DNA viruses. The antiviral activity of surfactin
has been determined for a broad spectrum of viruses. Moreover, surfactin has been found to exert
an effect on insulin absorption in the lungs of laboratory rats [108].
Int. J. Mol. Sci. 2016, 17, 401 22 of 31

11.7. Nanaotechnology
Biosurfactants have been used in nanotechnology and nanoparticle synthesis is emerging as part of
green chemistry [118,170]. Nickel oxide (NiO) nano-rods can be produced by water-in-oil microemulsions
[171]. In one experiment, two microemulsions were formed with the addition of a nickel chloride solution
to a biosurfactant and heptane solution, with the addition of ammonium hydroxide to the same
hydrocarbon mixture. The centrifuged microemulsions and ethanol was then used to wash the
precipitates and remove the biosurfactant and heptane. The use of biosurfactants is a more ecofriendly
approach [119]. Reddy et al. [172] found that silver nanoparticle synthesis could be stabilised for two
months using surfactin, which is a biodegradable, renewable stabilising agent with low toxicity [ 3,119]. A
biosurfactant produced by P. Aeruginosa grown in a low-cost medium has been employed to stabilise
silver nanoparticles in the liquid phase [173]. The effect of a rhamnolipid on the electrokinetic and
rheological behaviour of nano-zirconia particles has also been analysed, although this is not strictly an
environmental application [174]. The biosurfactant increasingly adsorbs to zirconia with the increase in
concentration and can serve as an ecofriendly product for the flocculation and dispersion of high solid
contents of microparticles. New applications for biosurfactants are being developed in the field of
nanotechnology. Future research should focus on the stabilisation of the nanoparticles by biosurfactants
before addition during remediation processes [119].

12. Future Directions and Concluding Remarks

The biosurfactant industry has demonstrated remarkable growth in recent decades, although the
large-scale production of these biomolecules remains a challenge from the economic standpoint. This is
mainly due to the enormous difference between the financial investment required and viable industrial
production. Thus, the following are the main criteria to be considered for biosurfactant production to
become truly viable: (a) type of raw materials; (b) continuous provision of the same composition of
ingredients; (c) types of microorganisms; (d) the adequate design of industrial fermentors; (e) financial
investments; (f) the target market; (g) purification processes; (h) biosurfactant properties; (i) production
conditions, especially the time required for fermentation; (j) adequate production yields; and (k) the
processing of recycled products (minimal or able to sell for more than the drop in value).
The target market is of fundamental importance to the implantation of an industrial biosurfactant
production project. For cosmetic, medicinal and food products, production is only viable on a small-scale,
as the column chromatography methods required to separate molecules are not economical on a large
scale. Thus, the use of crude fermentation broths could be a viable solution, especially if the application
is in an environmental context, as biosurfactants in such cases do not need to be pure and can be
synthesized using a blend of inexpensive carbon sources, which would allow the creation of an
economically and environmentally viable technology for bioremediation processes.
According to Hazra et al. [140], sophorolipids are offered as sophoron TM from Saraya (Osaka,
Japan) and Soliance (Pomacle, France), whereas rhamnolipids are available from Ecover
(Boulogne-sur-Mer, France), Jeneil Biosurfactant Inc. (Saukville, Wisconsin, USA) and Rhamnolipid
Holdings Inc. (New york, USA). Sophorolipid production costs run from 2 to 5 /kg. Rhamnolipid
3
production costs US$ 20/kg at a volume of 20 m , but only US$ 5/kg when produced on a scale of
3
100 m , placing it closer to ethoxylate or alkyl polyglycoside (US$ 1 to 3/kg). The Exxon Company
spent more than US$ 10 million in bioremediation studies between 1993 and 1997 after the spillage
of petroleum (41 million litres) by the oil tanker Exxon Valdez in Alaska in 1989, leading to the
generation of seven patents and making bioremediation second only to enhanced oil recovery within
the initial years of use. Distribution in specific biosurfactant areas of the oil industry includes 17
patents for soil and water bioremediation as well as 20 for enhanced oil recovery [140,175].
Although improvements in biosurfactant technology have enabled a 10-to-20-fold increase in
the production of these biomolecules, it is likely that further, significant advances (even if of a
smaller magnitude) are needed to make this technology commercially viable.
Int. J. Mol. Sci. 2016, 17, 401 23 of 31

Acknowledgments: This study received funding from the Brazilian fostering agencies the State of Pernambuco
Foundation for the Assistance to Science and Technology (FACEPE); the Research and Development Program of
the Brazilian National Electrical Energy Agency (ANEEL); the National Council for Scientific and Technological
Development (CNPq), and the Federal Agency for the Support and Evaluation of Graduate Education (CAPES).
Author Contributions: Leonie A. Sarubbo proposed the theoretical frame; Danyelle Khadydja F. Santos,
Raquel D. Rufino, Juliana M. Luna, Valdemir A. Santos and Leonie A. Sarubbo wrote the paper; Leonie A.
Sarubbo performed manuscript editing and final improvement.
Conflicts of Interest: The authors declare no conflict of interest.

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