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Biosurfactants: Classification, Properties and Environmental Applications

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 1104

Biosurfactants: Classification, Properties

and Environmental Applications
HUMBERTO B.S. SOBRINHO1,2, JULIANA M. LUNA1, RAQUEL D. RUFINO1,
ANA LÚCIA F. PORTO3 AND LEONIE A. SARUBBO1

ABSTRACT

Biosurfactants are natural products derived from bacteria, yeasts or

fungi. The complex chemical structures and physical properties of
biosurfactants generally result in properties equal to or exceeding many
synthetic surfactants. Biosurfactants demonstrate low toxicity to
freshwater, marine and terrestrial ecosystems and are potential
candidates for a variety of environmental applications. Research has
largely been focused on the enhancement of oil biodegradation and
microbial-enhanced oil recovery. The solubilisation and emulsification
of toxic heavy metals by biosurfactants have also been reported, assisting
in the recovery of such hazardous materials from contaminated sites.
The future success of biosurfactant technology in bioremediation
initiatives is promising, but will require the precise targeting of the
biosurfactant systems to reduce production costs and increase product
yield. The suitability to the physical conditions and chemical nature of
the pollution-affected site is also important the commercialisation of
these biomolecules.

1
Centro de Ciências e Tecnologia, Universidade Católica de Pernambuco, Rua do
Príncipe, n. 526, Boa Vista, CEP: 50050-900, Recife, PE, Brazil
2
Programa de Doutorado em Ciências Biológicas, Universidade Federal de Pernambuco,
Av. Prof. Moraes Rêgo, s/n, Cidade Universitária, CEP: 50.900-000, Recife, PE,
Brazil
3
Departamento de Fisiologia e Morfologia Animal, Universidade Federal Rural de
Pernambuco, Rua Dom Manoel de Medeiros, s/n, Dois Irmãos, CEP: 52171-900,
Recife, PE, Brazil
*Corresponding author: E-mail: leonie@unicap.br
2 Biotechnology Vol. 11: Bioremediation

Key words: Biosurfactants, Bioemulsifiers, Environment, Petroleum,

Industrial residues, Bioprocess

1. INTRODUCTION

Surfactants are a class of chemical compounds formed by molecular

structures with both hydrophilic and hydrophobic moieties that tend to
be distributed at the interface between liquid phases with different
degrees of polarity (oil/water) (Muthusami et al., 2008). Surfactants
reduce both surface and interfacial tension, leading to the capacity for
detergency, emulsification, lubrication, solubilisation and phase
dispersion (Deleu and Paquot, 2004; Gautam and Tyagi, 2006; Nitschke
et al., 2007). The commercial importance of surfactants is evident by
the market tendency to increase the production of these compounds
due to the diversity of industrial applications (Calvo et al., 2009), which
are classified based on use: 54% as detergents, 13% in the textile, leather
and paper industries, 10% in chemical processes, 10% in the
pharmaceutical and cosmetic industries, 3% in the food industry, 2% in
agriculture and 2% in other applications (Muthusamy et al., 2008).

Most commercially available surfactants are synthesised from

petroleum derivatives. However, concerns among consumers and current
environmental legislation have led to the search for natural surfactants
as an alternative to existing products (Nitschke and Pastore, 2002). A
number of natural compounds with tensioactive properties are
synthesised by living organisms, such as plants (saponins),
microorganisms (glycolipids) and even the human organism (bile salts)
(Maneerat, 2005). Compounds of a microbial origin (metabolic by-
products of bacteria, yeasts and filamentous fungi) that exhibit
surfactant properties (emulsification capacity and a reduction in surface
tension) are denominated biosurfactants (Singh et al., 2007).

The present review of data published in the literature offers a

summary of the main factors that exert an influence on the synthesis of
biosurfactants, their physicochemical and bioactive properties and
potential use in environmental applications.

2. ESTIMATION OF SURFACTANT ACTIVITY

Before addressing biosurfactants in detail, it is important to discuss

basic surface tension phenomena by which surfactants are evaluated.
Surface activity can be determined by measuring changes in surface
tension, interfacial tension, emulsion stabilisation/destabilisation and
the hydrophilic/lipophilic balance (HLB).
Biodegradation of Vegetable Waste for Cellulase Production 3

Surface tension is the most important property of tensioactive agents

and is the attraction force between the molecules in liquids (Pacwa-
P³ociniczak et al., 2011). Surface is defined as the limit between a liquid
and the surrounding air and interface is defined as the limit between
two liquids. The tension between the air/water and oil/water phases is
respectively known as the surface tension and interfacial tension (Banat
et al., 2010). Surface tension is measured quantitatively using a
tensiometer. This measurement is the bases of most initial evaluations
for the identification of the presence of a surfactant in the medium. The
air/water surface tension for distilled water is approximately 72 mN/m
(or dynes/cm). The interfacial tension for distilled water and n-
hexadecane is approximately 40 mN/m. Surfactants typically reduce
these values to 30-40 mN/m and 1 mN/m, respectively (Parkinson, 1985).

Surface tension diminishes with the increase in the concentration of

the surfactant in the aqueous medium and the formation of micelles.
Micelles are aggregated amphipathic molecules with hydrophilic
moieties positioned toward the outer part of the molecule and
hydrophobic moieties positioned toward the inner part. The critical
micelle concentration (CMC) corresponds to the minimum concentration
of surfactant necessary to achieve a maximum reduction in surface
tension. At concentrations above the CMC, biosurfactant molecules
associate to form micelles (Cortis and Ghezzehei, 2007; Van-Hamme et
al., 2006). Efficiency and effectiveness are essential characteristics of a
good surfactant. Efficiency is measured by the CMC, whereas
effectiveness is related to surface and interfacial tension (Barros et al.,
2007).

An emulsion is formed when a liquid phase is dispersed as microscopic

droplets in another continuous liquid phase. Two types of emulsions
can be formed: water in oil (surfactant that is more soluble oil) and oil
in water (surfactant that is more soluble in water). The stability of an
emulsion depends on a number of factors, such as the size of the
dispersed droplets, which is favoured by the reduction in surface tension.
The presence of an emulsifier or demulsifier respectively stabilises or
destabilises an emulsion. Emulsifying capacity is analysed by the ability
of a surfactant to cause turbidity due to the suspension of hydrocarbons,
such as n-hexadecane, in an aqueous system. Demulsifying capacity is
generally assessed by the effect of the demulsifying agent on normal
emulsions prepared with synthetic tensioative agents (Parkinson, 1985).

The HLB value indicates whether a surfactant will promote water-

in-oil or oil-in-water emulsions based on comparisons with the HLB
value of known surfactants. The HLB scale is based on a value of 1 for
oleic acid and 20 for sodium oleate, with intermediate values determined
4 Biotechnology Vol. 11: Bioremediation

proportionately by a mixture of the two. Emulsifiers with HLB less than

6 favour the stabilisation of water-in-oil emulsions and those with HLB
values between 10 and 18 favour the stabilisation of oil-in-water
emulsions (Parkinson, 1985).

3. BIOSURFACTANTS

Most known biosurfactants are produced on substrates that are insoluble

in water, such as solid and liquid hydrocarbons, oils and fats, although
many are obtained from soluble substrates or a combination of the two
(Van-Hamme et al., 2006). The production of bio0surfactants with the
use of renewable substrates and different microbial species as well as
the variation in culture parameters (incubation time, stirring speed,
pH of the medium and added nutrients) allow the acquisition of
compounds with distinct structural characteristics and physical
properties. This makes biosurfactants comparable to or even better than
synthetic surfactants in terms of efficiency, although production costs
do not yet allow greater competitiveness with their petrochemical
counterparts (Canet et al., 2002).

The CMC of biosurfactants (measure of efficiency) ranges from 1 to

2000 mg/L, whereas interfacial tension (oil/water) and surface tension
are around 1 and 30 mN/m, respectively. Data on the CMC of
biosurfactants are scarce and difficult to interpret or correlate. Table 1
offers a comparison of the CMC of biosurfactants and their chemical
counterparts, demonstrating much lower CMCs for the former group.
In principle, a lower CMC denotes greater efficacy of the surfactant
and, from the economic standpoint, favours its use in industrial processes
(Bognolo, 1999).

Table 1: Examples of critical micelle concentration of biosurfactants and chemical

surfactants
Surfactant CMC (mg/L)
Phosphatidylethanolamine 30
Phosphatic acid 70
Rhamnolipid 20
Surfactin 11
Alkylbenzene sulfonate 590
Sodium lauryl sulfate 2000 to 2900
Source: Bognolo, 1999

3.1 Classification

Synthetic surfactants are classified based on the ionic charge in the

polar part of the molecule. The presence/absence of an electrical charge
Biodegradation of Vegetable Waste for Cellulase Production 5

classifies surfactants as anionic, cationic, non-ionic or amphoteric

(Maneerat, 2005; Ron and Rosenberg, 2001). Most biosurfactants are
anionic or neutral, whereas only some are cationic, such as those that
contain amine groups. The hydrophobic moiety is characterised by long-
chain fatty acids and the hydrophilic moiety may be a carbohydrate,
amino acid, cyclic peptide, phosphate, carboxyl acid or alcohol (Bognolo,
1999).

Biosurfactants are commonly classified based on their biochemical

nature or the microbial producer species. With regard to structure,
these compounds are classified into five main groups (Rahman and
Gakpe, 2008).

• Glycolipids – the degree of polarity depends on the hydrocarbons

used as substrate; examples: rhamnolipids produced by
Pseudomonas aeruginosa and sophorolipids produced by species
of Candida.
• Lipopolysaccharides – which normally have a high molecular mass
and are soluble in water; example: emulsan, an extracellular
emulsifier produced fr om hydroc arbons by the bac teria
Acinotobacter calcoaceticus;
• Lipopeptides – example: surfactin produced by Bacillus subtilis
(one of the most potent biosurfactants reported in the literature);
• Phospholipids – structures common to many microorganisms;
example: biosurfactant from Corynebacterium lepus;
• Fatty acids, neutral lipids (some classified as glycolipids) and
hydrophobic proteins.

3.2 Producer Microorganisms

A large variety of microorganisms are able to produce biosurfactants

with different molecular structures (Deleu and Paquot, 2004). Bacteria
from the genera Pseudomonas and Bacillus are described in the literature
as large producers of biosurfactants. Rhamnolipids produced by
Pseudomonas aeruginosa have been studied extensively (Abdel-Mawgoud
et al., 2010; Chrzanowski et al., 2012; Henkel et al., 2012; Nitschke et al.,
2011). Composition and yield depend on the type of fermenter, pH,
nutrients, substrates and temperatures employed (Mulligan, 2005).
Bacillus subtilis produces lipopeptides, such as surfactin, which contains
seven amino acids linked to carboxyl and hydroxyl groups of C14 acid
(Barros et al., 2007; Lu et al., 2007). Concentrations of surfactin lower
than 0.005% reduce surface tension to 27 mN/m, making this compound
one of the most potent biosurfactants. On the other hand, the solubility
and surfactant capacity of surfactin depend on the type of substrate (HUE
6 Biotechnology Vol. 11: Bioremediation

Fig. 1: Illustrates typical structures of biosurfactants – emulsan (lipopolysaccharide),

rhamnolipid and sophorolipid (glycolipids).

et al., 2001). Among yeasts, species of the genus Candida have been
successfully employed in the fermentation of hydrocarbons and
consequent production of biosurfactants (Gusmão et al., 2010; Luna et
al., 2011a,b; 2013; Rufino et al., 2007; 2008; Sarubbo et al., 1999; 2001;
2006; 2007; Sarubbo and Campos-Takaki, 2011). Table 2 offers a summary
of the main classes of biosurfactants and respective producer
microorganisms described in the literature.

3.3 Properties

The physicochemical properties of biosurfactants, such as a reduction

in surface tension, foaming capacity, emulsifying capacity, stabilising
Biodegradation of Vegetable Waste for Cellulase Production 7

Table 2: Main classes of biosurfactants and respective producer microorganisms

Class/Type of Biosurfactant Microorganism
Glycolipids
Rhamnolipids Pseudomonas aeruginosa
Sophorolipids Torulopsis bombicola, T. apicola
Trehalolipids Rhodococcus erythropolis, Mycobacterium sp.
Lipopeptides and lipoproteins
Peptide–lipid Bacillus licheniformis
Viscosin Pseudomonas fluorescens
Serrawettin Serratia marcenscens
Surfactin Bacillus subtilis
Subtilisin Bacillus subtilis
Gramicidin Bacillus brevis
Polymyxin Bacillus polymyxia
Fatty acids, neutral lipids and phospholipids
Fatty acid Corynebacterium lepus
Neutral lipids Nocardia erythropolis
Phospholipids Thiobacillus thiooxidans
Polymeric surfactants
Emulsan Acinetobacter calcoaceticus
Biodispersan Acinetobacter calcoaceticus
Liposan Candida lipolytica
Carbohydrate–lipid–protein Pseudomonas fluorescens
Mannan–lipid–protein Candida tropicalis
Particulate surfactant
Vesicles Acinetobacter calcoaceticus
Cells Various bacteria
Source: Muthusamy et al., 2008; Pacwa–P³ociniczak et al., 2011

capacity, low CMC, solubility and detergency, are very important in

the evaluation of the performance and selection of microorganisms with
the potential to produce these agents (Deleu and Paquot, 2004). Despite
the diversity in the chemical composition and properties, a number of
characteristics are common to the majority of biosurfactants, many of
which offer advantages over conventional surfactants (Nitschke et al.,
2007):

• Surface and interfacial activity: Biosurfactants produce a lower

surface tension at a lower concentration, demonstrating greater
effectiveness and efficiency in comparison to conventional
surfactants. The CMC of biosurfactants (measure of efficiency)
ranges from 1 to 2000 mg/L, wheareas interfacial tension (oil/water)
and surface tension are around 1 and 30 mN/m, respectively.
• Tolerance to temperature, pH and ionic strength: Many
biosurfactants can be used under extreme conditions. For instance,
the lipopeptide from Bacillus licheniformis JF-2 is stable at
8 Biotechnology Vol. 11: Bioremediation

temperatures around 75°C for up to 140 hours and within a pH

range of 5 to 12. Biosurfactants also tolerate salt concentrations
up to 10%, whereas 2% NaCl is sufficient to inactivate conventional
surfactants.
• Biodegradability: Biosurfactants are easily degraded by bacteria
and other microorganisms in water or soil, which makes them
adequate for bioremediation applications and waste treatment.
• Low toxicity: Biosurfactants have received greater attention due
to the increasing concern on the part of the population regarding
the allergic effects of artificial product. Moreover, the low degree
of toxicity of these compounds allows their use in food, cosmetic
and pharmaceutical products.
• Availability: Biosurfactants can be produced from widely available
raw materials and can even be produced from industrial waste.
• Specificity: As complex organic molecules with specific functional
groups, biosurfactants are specific in their actions, which is of
considerable interest regarding the detoxification of specific
pollutants as well as in particular applications in the food, cosmetic
and pharmaceutical industries.
• Biocompatibility and digestibility: These properties allow the
application of biosurfactants in the food, cosmetic and
pharmaceutical industries.
Despite the advantages, the unfavourable points should also be
mentioned (Rahman and Gakpe, 2008):

• The large-scale production of biosurfactants is expensive. However,

this problem may be overcome with the combination of low-cost
substrates.
• The acquisition of products with a high degree of purity is difficult
due to the need for consecutive purification steps for the metabolic
broth.
• Super-producing species are rare. Known species are not capable
of producing high surfactant yields and require a complex culture
medium.
• The regulation of biosurfactant synthesis is not fully understood,
as these biomolecules may be produced as secondary metabolites
or in association with microbial growth.
• The increase in productivity is often impaired by the formation of
foam, which requires the use of a diluted medium.
Biodegradation of Vegetable Waste for Cellulase Production 9

3.4. Physiology

Biosurfactants are produced by a large variety of microorganisms either

through excretion or adhesion to cells, especially when cultivated on
substrates that are insoluble in water (Tan et al., 2000). 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. Direct contact between the cell and hydrocarbon
droplets has been described, along with interactions with the emulsified
droplets.

The main physiological role of biosurfactants resides in the fact that

these compounds allow microorganisms to grow on substrates that are
insoluble in water through the reduction in surface tension between
phases, making the substrate more available for ingestion and
metabolism. The molecular mechanisms of the ingestion of these
substrates (for example, alkanes) have not been fully clarified. The direct
ingestion 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 (Tan et al., 2000).
Besides the emulsification of the carbon source, biosurfactants are also
involved in the adhesion of microbial cells to the hydrocarbon.
Microorganism cell adsorption to insoluble substrates and the excretion
of surfactant compounds allow growth on these carbon sources.

3.5 Biosynthesis and Regulation

The primary metabolic pathways, namely hydr ocarbons and

carbohydrates, are respectively involved in the synthesis of the
hydrophobic and hydrophilic moieties (Desai and Banat, 1997). The
metabolic pathways of the synthesis of these two groups of precursors
are diverse and use specific enzymes. In many cases, the first enzymes
are regulatory. Consequently, despite the diversity, there are a number
of common principles regarding the synthesis and regulation of these
precursors. The metabolic pathways have been extensively studied and
documented in the literature. Syldatik and Wagner (1987) list the
following possibilities for the synthesis and regulation of the two moieties
of a biosurfactant: a) the hydrophilic and hydrophobic moieties are
synthesised by de novo synthesis through two different pathways; b)
the hydrophilic moiety is synthesised by de novo synthesis and the
hydrophobic moiety is induced by the substrate; c) the hydrophobic
moiety is synthesised by de novo synthesis and the hydrophilic moiety
is dependent on the substrate; and d) the synthesis of both moieties is
dependent on the substrate.
10 Biotechnology Vol. 11: Bioremediation

Three mechanisms (induction, repression and nitrogen/multivalent

ions) regulate the production of a biosurfactant. The literature reports
the induction of lipid synthesis by Torulopsis magnoliae through the
addition of long-chain fatty acids, hydrocarbons or glycerides to the
growth medium, the synthesis of trehalolipids by Rhodococcus
erythropolis through the addition of hydrocarbons and the production
of a glycolipid by Pseudomonas aeruginosa through the addition of
alkanes.

Regulation by nitrogen or metal ions also plays an important role in

the synthesis of surfactants. The synthesis of rhamnolipids by
Pseudomonas aeruginosa following the exhaustion of nitrogen in the
stationary growth phase has been reported by a number of researchers.
In contrast, the addition of a nitrogen source is reported to cause the
inhibition of the synthesis of rhamnolipids by P. aeruginosa cells in the
stationary phase. The limitation of multivalent cations also causes the
production of biosurfactants. According to Guerra-Santos et al. (1984),
restricted concentrations of magnesium, calcium, potassium, sodium
and trace elements lead to a high yield of rhamnolipids by Pseudomonas
aeruginosa. Iron restriction stimulates the production of biosurfactants
by P. fluorescens and P. aeruginosa, whereas the addition of iron and
manganese simulates the production of biosurfactants by B. subtilis
and Rhodococcus sp.

3.6 Economics in the Production of Biosurfactants

Economics is an important factor in biotechnological processes. The

successful production of biosurfactants depends on the selection of low-
cost substrates, which currently account for 10 to 30% of the overall
production cost. Biosurfactants must compete with petrochemical
surfactants in terms of function, production capacity and cost (Banat et
al., 2010; Cameotra and Makkar, 1998; Coimbra et al., 2009;
Muthusamy, 2008; Rufino et al., 2008). The high cost of biosurfactant
production can be absorbed when these compounds are used in small
amounts, such as in cosmetics, medications and food. However, the high
production cost hinders the use of biosurfactants in applications such
as oil recovery, which requires large amounts of surfactant (Cameotra
and Makkar, 1998).

Pattanathu et al. (2008) suggest four factors for reducing the cost of
biosurfactants: 1) microorganisms selected, adapted and cultivated for
large-scale production; 2) adjustments to the production process to
ensure low operational costs; 3) low-cost culture media; and 4) the
Biodegradation of Vegetable Waste for Cellulase Production 11

processing of recycled products. Biosurfactant production may be either

spontaneous or induced by the presence of lipophilic compounds,
variations in pH, temperature, aeration and agitation rate or when cell
growth is maintained under conditions of stress, such as low
concentrations of the nitrogen source (Desai and Banat, 1997).

Low-cost biosurfactant production is hampered when there is a need

for extensive refining. Thus, biosurfactants should be capable of being
recovered through the use of simple, inexpensive methods. Extraction
with solvents (chloroform-methanol, dichloromethane-methanol,
butanol, ethyl acetate, pentane, hexane, acetic acid, etc.) constitutes
the most common biosurfactant recovery process. However, ammonium
sulphate precipitation, crystallisation, centrifugation, adsorption and
fractional fermentation methods have also been described (Makkar and
Cameotra, 2002).

3.7 Use of Industrial Waste for Biosurfactant Production

Industrial waste had piqued the interest of researchers as a low-cost

substrate for biosurfactant production (Makkar and Cameotra, 2002).
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 (Makkar and Cameotra, 1999; Mercade
et al., 1994). According to Barros et al. (2007), the use of agroindustrial
waste is one of the steps toward the implantation of feasible biosurfactant
production on an industrial scale, for which studies on the different
variables involved are required to optimise the process. The literature
describes a number of waste products employed in biosurfactant
production, such as vegetable oils, oily effluents (Mercadé et al., 1993;
Sarubbo et al., 2007; Batista et al., 2010), starchy effluents (Fox and
Bala, 2000; Thompson et al., 2000), animal fat (Deshpande and Daniels,
1995; Maneerat, 2005), vegetable fat (Gusmão et al., 2010), vegetable
cooking oil waste (Alcantara et al., 2000; Cvengros and Cvengrosova,
2004; Haba et al., 2000; Maneerat, 2005), soapstock (Shabtai, 1990;
Benincasa et al., 2002; Maneerat, 2005), molasses (Ghurye and
Vipulanandan, 1994; Kalogiannis et al., 2003; LAZARIDOU et al., 2002;
Makkar and Cameotra, 1997), dairy industry waste (whey) (Sudhakar-
Babu et. al., 1996), corn steep liquor (Luna et al., 2011; 2012; Rufino et
al., 2007; Sobrinho et al., 2008), cassava flour wastewater (Nitshke et
al., 2004), oil distillery waste (Luna et al., 2011; 2012; Rufino et al.,
2007) and glycerine (Silva et al., 2010).
12 Biotechnology Vol. 11: Bioremediation

3.8 Environmental Applications

Due to their structures and properties, biosurfactants have applications

in different industrial processes as well as possible novel uses in the
future. Indeed, biosurfactants are expected to become known as the
“multifunctional materials” of the 21st century (Marchant and Banat,
2012; Muthusamy et al., 2008). Currently, the major market for
biosurfactants is the petroleum industry, in which these compounds
can be used in the cleanup of oils spills, the removal of oil residue from
storage tanks, microbial-enhanced oil recovery and the bioremediation
of soil and water (Gautam and Tyagi, 2006; Singh et al., 2007).

Environmental contamination caused by industrial activities is often

due to the accidental or deliberate release of organic and/or inorganic
compounds into the environment, which represents a problem for
remediation, as such compounds bond easily to soil particles and spread
in an aqueous medium. The aim of the application of biosurfactants in
the decontamination of organic compounds, such as hydrocarbons, is to
enhance the bioavailability of these compounds or mobilise and remove
the contaminants through pseudo-solubilisation and emulsification
during clean up. On the other hand, the application of biosurfactants in
the recovery of inorganic compounds, such as heavy metals, involves
chelation and the removal of these ions during the washing phase, which
is facilitated by chemical interactions between the amphipathic
compounds and metal ions (Banat et al., 2010; Olkowska et al., 2012).

3.8.1. Application of biosurfactants in bioremediation

Biorremediation regards the ability of living organisms to transform or

mineralise organic contaminants for the generation of less harmful
substances that can be integrated into the natural biogeochemical cycle.
However, the biodegradability of these contaminants is influenced by
factors such as oxygen, pH, the presence of macronutrients and
micronutrients, the physicochemical characteristics of the pollution
history of the contaminant and particles to which the organisms and
contaminants may be adsorbed (Margesin and Schinner, 2001).

Amphipathic compounds are capable of altering the physicochemical

conditions of the interfaces that affect the distribution of chemical
substances among the phases (Tiehm, 1994). For instance, a soil
contaminated by a hydrocarbon has at least six phases: bacteria, soil
particles, water, air, immiscible liquid and solid hydrocarbon.
Hydrocarbons can be partitioned among different states: solubilised in
the water phase, adsorbed/absorbed to soil particles, adsorbed to cell
Biodegradation of Vegetable Waste for Cellulase Production 13

surfaces and free/insoluble. Biosurfactants can interact with both abiotic

particles and bacterial cells, which affects the interaction mechanisms
with the environment through the emulsification of the organic
contaminant. Interactions between surfactant micelles and cells are
among the main alterations the bacterial component undergoes
(Volkering et al., 1998). These phenomena can either enhance the
bioavailability of soluble contaminants, thereby increasing the
biodegradation rate, or result in the inhibition of biodegradation. Despite
the numerous successful applications of biosurfactants, the literature
describes conflicting results with regard to bioremediation. For example,
rhamnolipids from P. aeruginosa stimulate the degradation of n-
hexadecane, but those produced by the genus Rhodococcus do not, which
demonstrates the specificity of mic roorganisms. However, a
biosurfactant from R. erythropolis 3C-9 was found to lead to a significant
increase in the degradation rate of n-hexadecane. Therefore, the effect
of a biosurfactant on bioremediation processes is unpredictable and the
efficacy of a given surfactant needs to be evaluated experimentally
(Franzetti et al., 2008).

Interactions among bacteria, contaminants and biosurfactants can

be interpreted from a functional standpoint, as the main role attributed
to biosurfactants is their involvement in trapping of hydrocarbons
(Perfumo et al., 2010). Microbial surfactants promote microbial growth
on hydrocarbons by increasing the surface area between the oil and
water and enhancing the pseudo-solubility of hydrocarbons through
partitioning among micelles (emulsification) (Miller and Zhang, 1997;
Volkering et al., 1998). Biosurfactants with a high molar mass
(bioemulsifiers) have considerable potential in stabilising emulsions
involving liquid hydrocarbons and water by increasing the surface area
available for bacterial biodegradation. However, such biosurfactants
have rarely been tested as stimulators of hydrocarbon degradation in
bioremediation systems. Moreover, conflicting findings are described
in the literature (Barkay et al., 1999; Franzetti et al., 2009). Above the
CMC, biosurfactants with a low molar mass partition a significant
portion of hydrophobic contaminants in the core of micelles. In some
cases, this results in the enhanced bioavailability of the contaminant to
pollutant-degrading microorganisms. Successful applications of
rhamnolipids and surfactin have been described in bioremediation
processes (Mulligan, 2009).

Microbial consortia and rhamnolipids have demonstrated potential

regarding the bioremediation of petroleum hydrocarbons (Rahman et
al., 2006). A rhamnolipid produced by Pseudomonas aeruginosa DS10-
129 enhanced the bioremediation of gasoline adsorbed to soil particles
14 Biotechnology Vol. 11: Bioremediation

(Rahamn et al., 2002). A number of studies report that biosurfactants

enhance the bioavailability of aromatic compounds with a low degree of
solubility, such as polycyclic aromatic hydrocarbons (Mulligan, 2005;
Singh et al., 2007). The use of biosurfactants in the biodegradation of
pesticides has also been the object of investigation. The degradation of
hexachlorocyclohexane and organochlorinated pesticides, such as DDT
and cyclodienes, by surfactants produced from species of the genus
Pseudomonas has been reported (Karanth et al., 1999).

As stated above, the specific modes of hydrocarbon adsorption are

not fully understood. Cameotra and Singh (2009) recently described
the n-hexadecane adsorption mechanism mediated by rhamnolipids from
P. aeruginosa, in which the rhamnolipids produced an emulsion with
hexadecane, facilitating contact between the hydrocarbon and bacteria.
The authors also found the absorption of the hydrocarbon droplets
covered with the biosurfactant, which suggests the occurrence of a
pinocytosis mechanism rather than bacterial capturing of the
hydrocarbon. In contrast, the presence of a surfactant can have a
negative effect on biodegradation. Micelles can retain organic
contaminants, forming a barrier between the microorganism and organic
molecules and resulting in the reduction of substrate availability. For
instance, a non-ionic surfactant from alcohol ethoxylate, denominated
witconol SN70, led to a reduction in the biodegradation rate of
hexadecane (Colores et al., 2000).

Another role proposed for biosurfactants in capturing hydrocarbons

regards the regulation of the bonding of cells to hydrophobic and
hydrophilic surfaces, thereby altering the hydrophobicity of the cell
surface (Franzetti et al., 2008; Rosenberg et al., 1987). This natural role
can be exploited through the use of biosurfactants to enhance the
hydrophobicity of microorganisms and facilitate the access of cells to
hydrophobic substrates (Shreve et al., 1995). Coimbra et al. (2008) report
studies involving hydrophobicity with biosurfactants from species of
the genus Candida. Chang et al. (2009) demonstrated an increase in
cell hydrophobicity due to the build-up of different fatty acids on the
cell surface during growth of R. erythropolis NTU-1 on hydrocarbons.
Kaczorek et al. (2008) report a correlation between the change in the
cell surface by saponins and the degree of hydrocarbon degradation.

3.8.2. Application of Biosurfactants in Microbial-Enhanced Oil

Recovery

Biosurfactants may also be used in microbial-enhanced oil recovery

(MEOR). MEOR methods are used to recover oil remaining in reservoirs
Biodegradation of Vegetable Waste for Cellulase Production 15

following primary (mechanical) and secondary (physical) recovery (Singh

et al., 2007; Packwa-P³ociniczak et al., 2011). Enhanced oil recovery is
an important tertiary process in which microorganisms or their
metabolites, such as biosurfactants, biopolymers, biomass, acids,
solvents, gases and enzymes, are used to further oil recovery from
drained deposits. The application of biosurfactants in MEOR is one of
the most promising methods for the recovery of a substantial portion of
residual oil. The remaining oil is often located in regions of the reservoir
that are difficult to access, where the oil is trapped in pores by capillary
pressure. Biosurfactants reduce the oil/water and oil/rock interfacial
tensions, which reduces the capillary forces that impede the oil from
moving through the pores of the rock. Biosurfactants can also strongly
bond with the oil/water interface to form an emulsion, which stabilises
the oil desorbed in the water and allows its removal through the injection
of water (Marchant and Banat, 2012).

Bordoloi and Konwar (2008) investigated the recovery of crude oil

from a saturation column under laboratory conditions. Laboratory
studies on MEOR typically use columns that contain the desired
substrate (generally sand), which is used to demonstrate the usefulness
of biosurfactants in the recovery of oil from reservoirs. For this effect, a
glass column packed with dry sand is saturated with crude oil and an
aqueous biosurfactant solution is poured into the column. The potential
of biosurfactants in MEOR is estimated by measuring the amount of oil
released from the column following the passage of the biosurfactant
solution through the column. The experiment should be carried out at
room temperature as well as at a high temperature, such as 70 to 90°C,
to assess the effect of temperature on biosurfactant-induced oil recovery.
The biosurfactants used in the experiment by Bordoloi and Konwar
(2008) were produced from strains of the bacterium P. aeruginosa
(MTCC7815, MTCC7814, MTCC7812 and MTCC8165). The
biosurfactants from the MTCC7815, MTCC7812 and MTCC8165 strains
recovered 49 to 54% of the crude oil from the packed columns at
temperatures of 52 to 70°C and 58 to 62% at 90ºC.

Bai et al. (1997) investigated the potential of an anionic rhamnolipid

isolated from P. aeruginosa in the removal of hexadecane adsorbed to
sand in packed columns, demonstrating an 84% removal rate from 20-
30 mesh sand (0.6 to 0.85 mm) and a 22% removal rate from 40-50 mesh
sand (0.3 to 0.42 mm). The removal capacity of the rhamnolipid was
compared to that obtained with synthetic surfactants [the anionic SDS
(CMC: 2360 mg/l) and non-ionic Tween 80 (polyoxyethylene sorbitan
mono-oleate 20; CMC: 13 mg/l)] used at a concentration of 500 mg/l for
40/50 mesh sand. SDS (472 mg/l) and Tween 80 (51 mg/l) removed 0 and
16 Biotechnology Vol. 11: Bioremediation

6% of the hexadecane, respectively. In contrast, SDS proved capable of

removing 95% of diesel fuel adsorbed to soil particles in a column, in
contrast to the 24.7% removal rate achieved with water (used as the
control); the influence of surfactant concentration and contact time on
the kinetics of the removal of the pollutant was also determined in the
study (Khalladi et al., 2009). High concentrations (2.5 and 5.0 g/l) of a
biosurfactant isolated from P. aeruginosa 57SJ (CMC: 400 mg/l) proved
necessary to remove 70% of pyrene adsorbed to soil particles measuring
2 mm (Bordas et al., 2007). Moreover, the cell-free broth containing P.
aeruginosa strains MTCC7815, MTCC7812 and MTCC8165 cultivated
in 2% glycerol removed 49 to 54% of crude oil from packed columns
(Bordoloi and Konwar, 2008).

Biosurfactants can also be used for other applications in the

petroleum industry. The demulsifying properties of some biosurfactants
can be used to break down emulsions that form in different steps of oil
extraction and processing, allowing greater recovery of the product. The
reduction in surface tension cause by microbial surfactants can also be
used to separate oil from the bottom of tanks (Perfumo et al., 2010;
Singh et al., 2007).

Although a number of field tests involving in situ applications of

MEOR are reported in the literature (SEN, 2008), it has not been fully
clarified whether the microorganisms involved are actually efficacious
in the oil recovery process or whether they compete with autochthonous
bacteria. The inability to compare tests performed with control wells
submitted to similar treatment procedures without the introduction of
living microorganisms or products hinders the establishment of valid
conclusions. Using molecular markers, Wang et al. (2008) found changes
in the microbial community in an oil reservoir during MEOR and
concluded that both exogenous and autochthonous bacteria seem to
stimulate enhanced oil recovery.

3.8.3. Washing of Soils with Biosurfactants

The application of biosurfactants for the removal of contaminants from

soils is less known than the advance application of these compounds in
bioremediation, as the removal efficiency is driven mainly by the
physicochemical properties of the biosurfactant and not by its effects
on metabolic activity or changes in the properties of the cell surface.
However, the mechanisms that affect the mobilisation and solubilisation
of hydrocarbons in soils are similar to those involved in the enhancement
of bioavailability for bioremediation (Banat et al., 2010; Franzetti et al.,
2009; Mulligan, 2009).
Biodegradation of Vegetable Waste for Cellulase Production 17

Biosurfactants enhance the bioremediation of hydrocarbons through

two mechanisms. One involves the increased bioavailability of the
substrate to microorganisms and the other involves interactions with
the cell surface, which enhances the hydrophobicity of the surface of
hydrophobic substrates, facilitating the association with bacterial cells
(Prieto et al., 2008). By reducing surface and interfacial tensions,
biosurfactants increase the surface area of insoluble compounds, which
leads to greater hydrocarbon mobility and bioavailability.

Biosurfactants enhance the biodegradation and removal of

hydrocarbons through mobilisation, solubilisation or emulsification
(Pacwa-P³ociniczak et al., 2011). Solubilisation capacity depends on the
ability of the surfactant to increase the solubility of hydrophobic
components in the aqueous phase. A considerable increase in this
capacity occurs above the CMC, which is attributed to the partitioning
of the hydrocarbon in the hydrophobic site of the micelles. In this process,
greater concentrations of surfactants are normally required, since the
solubility of the hydrocarbon components in the solution depends wholly
on this concentration of the surfactant (Bai et al., 1997).

Mobilisation occurs at concentrations below the CMC and is divided

into displacement and dispersion. Displacement consists of the release
of hydrocarbon droplets from the porous medium due to the reduction
in interfacial tension. Using a theoretical explanation, hydrocarbons
are removed if the interfacial tension between the aqueous and oil phases
is sufficiently reduced to overcome the forces of capillarity that cause
the formation of residual saturation. Dispersion is the process by which
a hydrocarbon is dispersed in the aqueous phase as tiny emulsions.
Emulsions are not generally thermodynamically stable, but may remain
stable for significant periods of time due to kinetic restrictions.
Dispersion is related to interfacial tension and surfactant concentration
and differs from displacement, as the displacement process is related
only to interfacial tension between the aqueous and hydrophobic phases,
with no formation of emulsions (Bai et al., 1997).

The efficiency of a surfactant in the removal of hydrophobic

compounds also depends on the pH and 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. Rhamnolipids are considered good
examples of anionic surfactants, as these compounds are retained less
by the soil than non-ionic or neutral surfactants due to the negative
surface charge of soil (Bordas et al., 2007).
18 Biotechnology Vol. 11: Bioremediation

A number of studies have described the use of biosurfactants for the

washing of soils. Sobrinho et al. (2008) demonstrated that a biosurfactant
isolated from C. sphaerica was capable of removing 65% of motor oil
adsorbed to sand. A biosurfactant from C. antarctica demonstrated the
ability to remove about 50% of oil adsorbed to sand (Adamczac and
Bednarski, 2000) and a biosurfactant isolated from C. glabrata achieved
an 84% removal rate of adsorbed motor oil (Luna et al., 2007). A
biosurfactant from C. lipolytica grown on agroindustrial waste was
successfully used in the removal of a petroleum derivative from soil of
the barrier formation (Rufino et al., 2011). Abu-Ruwaida et al. (1991)
report an 86% removal rate of residual crude oil adsorbed to sand using
a cell-free broth containing a biosurfactant produced by Rhodococcus.
A biosurfactant from P. aeruginosa UCP0992 grown on glycerine
removed high percentages of diesel adsorbed to sand (Silva et al., 2010).
Mulligan (2009) analysed the application of biosurfactants in the
washing of soils contaminated by hydrocarbons and metals. Franzetti
et al. (2009) report the efficient removal of crude oil from soil using and
extracellular bioemulsifier produced by Gordonia sp. BS29.

3.8.4. Application of biosurfactants in cleaning of oil reservoirs

The removal of heavy oil residue requires washing with solvents or even
manual washing, both of which are dangerous, slow and expensive
processes, since heavy oils that settle at the bottom of storage tanks are
highly viscous and cannot be removed with conventional pumping. The
use of biosurfactants in this cleaning process reduces the viscosity of
the product and promotes the formation of oil/water emulsions to
facilitate the pumping of the residue and recovery of the crude oil
following the breakdown of the emulsion (Singh et al., 2007; Mulligan
and Wang, 2004). The use of biosurfactants rather than conventional
surfactants in the cleaning of tanks is reported to allow the cleaning
and recovery of 90% of the hydrocarbons in the residue (Mulligan, 2004).

3.8.5. Application of Biosurfactants in the Removal of Heavy Metals

Heavy metals occur naturally in rock, soil, plants and animals and are
the inorganic pollutants that pose the greatest potential risk to human
health. Metals occur in different forms – as ions dissolved in water,
vapour or minerals in rock, sand and soil. Metals can also be bonded to
organic or inorganic molecules or trapped by air particles. Both natural
and anthropogenic processes release metals into the air and water
(Aguiar et al., 2002; Juwarkar et al., 2007).
Biodegradation of Vegetable Waste for Cellulase Production 19

Heavy metal contamination is the result of a number of industrial

activities, such as mining, metal forging, the manufacturing of
automobile batteries, emissions from vehicles and industrial waste
dumps and the dispersion of ash from incineration processes (Hong et
al., 2002; Hashim et al., 2011). The presence of heavy metals in soil
causes serious problems, since these substances are not biodegraded,
which leads to the contamination of biological systems and the subsoil
through the process of lixiviation (Morrrison, 1983). For instance, lead
is found in 15% of contaminated lands in the USA, followed by chrome,
cadmium and copper, which are found in 7 to 11% of soils.

A number of different methods have been developed and implemented

to reduce costs associated with the treatment of soil contaminated by
heavy metals (Peng et al., 2008). Two main technologies are employed
to treat contaminated soil. The first consists of immobilising heavy
metals in a solid matrix strongly bonded to the soil to minimise
migration. However, this technology is not a definitive solution for the
problem, as there is no possibility of reusing the soil and long-term
monitoring is required. The second technology promotes the mobility of
the metal and its migration to a liquid phase through desorption and
solubilisation. This technology is considered a permanent solution, as
it allows the recycling of the remediated soil and consequent reuse of
the land (Hashim et al., 2011). The flushing of soil with acids and
chelating agents, such as ethylenediaminetetraacetic acid (EDTA), are
the most widely applied methods. However, flushing with acids reduces
the fertility of the soil and leads to changes in the physicochemical
composition due to the dissolution of minerals (Reed et al., 1996).
Moreover, the use of EDTA is worrisome from the health and safety
standpoint due to its low degradation rate. The difficulty in recovering
the heavy metal from the metal-EDTA complex also restricts the use of
this method.

The use of surfactants is a potential solution for the remediation of

soils contaminated by metals and oils. Surfactants in solutions facilitate
the solubilisation, dispersion and desorption of the contaminants and
also allow the reuse of the soil (Hashim et al., 2011). A number of
synthetic surfactants have been evaluated in decontamination tests (Asçi
et al., 2008; Ellis, et al., 1985; Nash et al., 1987). However, the need to
replace synthetic compounds with natural surfactants has led to
research into the use of biosurfactants (Mulligan, 2009). In this respect,
studies have demonstrated the potential of surfactin, rhamnolipids (both
of a bacterial origin) and sophorolipids (of a yeast origin) (Dahrazma
and Mulligan, 2007; Herman et al., 1995; Mulligan et al., 1999; Neilson
et al., 2003; Ochoa-Loza et al., 2007; Tan et al., 1994). The ionic nature,
20 Biotechnology Vol. 11: Bioremediation

biodegradability, low degree of toxicity and excellent surface properties

make biosurfactants candidates for the removal of heavy metals from
soil and sediment. According to Mulligan (2009), removal is possible
with different concentrations of biosurfactants. Das et al. (2009) report
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 nearly complete removal of 100 ppm of
metallic ions. Wen et al. (2009) 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 metal.

The removal of metals by ionic biosurfactants is thought to occur in

the following sequence: 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 the micelles of the
biosurfactant. Heavy metals are trapped within the micelles through
electrostatic interactions and can be easily recovered through
precipitation or membrane separation methods (Kitamoto et al., 2002).

Anionic biosurfactants create non-ionic complexes with metal through

ionic bonds. Such bonds are stronger than those between the metal and
soil and the metal-biosurfactant complex is desorbed from the soil matrix
due to the reduction in interfacial tension. Cationic biosurfactants can
replace similarly charged metal ions through competition for some, but
not all, the negatively charged surfaces (ion exchange). Metal ions can
also be removed from the soil surface by surfactant micelles.

Biosurfactants offer indisputable advantages, since microorganisms

capable of producing surfactant compounds do not need to have the
ability to survive in the soil contaminated by a heavy metal, although
the continual addition of biosurfactant is required in the process
(Packwa-Plocinikzac et al., 2011). Biosurfactants have also been applied
in mining. 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 CaWO4 and 30% for CaCO3,
whereas conventional chemical reagents were not capable of separating
these two minerals (Nitschke and Pastore, 2002). Biosurfactants
produced by species of Candida have been successfully employed in the
floatation of heavy metals, demonstrating the ability to remove more
than 90% of cations from columns and in dissolved air floatation
processes (Menezes et al., 2011; Albuquerque et al., 2012). An anionic
polysaccharide produced by Acinetobacter calcoaceticus A2, denominated
biodispersan, has been used for the prevention of flocculation and
Biodegradation of Vegetable Waste for Cellulase Production 21

dispersion in mixture of calcareous rock and water (Ron and Rosenberg,

2002).

3.9 Prospects for Use

Many of the potential applications for biosurfactants and the expansion

of uses in the market depend on the economics of the production process.
A great deal of work is still necessary for the optimisation of biological
processes and engineering. The typical cost of biosurfactant production
ranges from US$ 10/mg for pure surfactin (98% purity), which is used
in medical research, to US$ 24/kg for formulas employing emulsan,
which was proposed in the early 1980s for the cleaning of tanks and
enhanced oil recovery. Estimates from the last ten years situate the
cost of biosurfactants between US$ 3 to 20/kg, whereas the production
cost of synthetic surfactants, suc h as ethoxylates and alkyl
polyglycosides, ranges from US$ 1 to 3/Kg (Bognolo, 1999).

While improvements in the technology of biosurfactant production

have allowed a 10-to-20-fold increase in productivity, it is likely that
further progress (albeit at a lesser order of magnitude) is necessary to
make this technology economically viable (Gautam and Tyagi, 2006).
The following parameters can be varied to optimise biosurfactant
production:

a) Selection of low-cost raw materials – enabling an adequate balance

of carbon, nitrogen, phosphorus and other oligoelements to
maximise the development and yield of microorganism strains
capable of metabolising waste by-products;
b) Bioprocessing – which can be optimised by the operational
conditions of the reactor and recycling of the medium;
c) Product isolation/recovery – most initial technologies involved
elaborate forms of purification and isolation. The possibility of in
situ development or the use of metabolic broths of crude
biosurfactant will lead to a substantial reduction in costs (Bognolo,
1999).

4. CONCLUSIONS

The success of the commercialisation of a biotechnological product largely

depends on the economics of its processing. The prices of microbial
surfactants are not currently competitive with those of synthetic
tensioactive agents due to the high production cost and low yield. It is
therefore important to optimise the biological production and
engineering of the process to obtain economically viable products.
22 Biotechnology Vol. 11: Bioremediation

Advances from studies on biosurfactant production have allowed a 10-

to-20-fold increase in productivity, but further studies are needed. The
use of low-cost substrates, the establishment of microbial growth under
ideal production conditions, the development of novel purification
methods and the use of hyperproducing microbial strains can make
biosurfactant production economically feasible. While a large number
of biosurfactant-producing microorganisms are reported in the
literature, studies on the increase in production have generally been
concentrated on few genera, such as Bacillus, Pseudomonas and
Candida. Biosurfactants are viable candidates for the replacement of
synthetic surfactants, especially in the oil industry. Investments in
strategies for improving the processing of these natural compounds will
pave the path to large-scale biosurfactant production.

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