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Review of Literature

REVIEW OF LITERATURE

2.1 Surfactants and biosurfactants

Surfactants (SURFace ACTive AgeNTS) are amongst the most versatile process

chemicals used in agriculture, cosmetics, pharmaceuticals, detergents, food processing,

etc., owing to their wide range of properties including the lowering of surface and

interfacial tension of liquids. Surface tension is defined as the free surface enthalpy per

unit area and is the force acting on the surface of liquid leading to minimization of the

area of that surface. The surfactants are known to reduce the surface tension of water

from 74 to 27 mN m-1. Surfactants have both hydrophilic and hydrophobic/lipophilic

(non-polar) moieties in the molecule. They are amphipathic molecules that partition

preferentially at the interface between fluid phase with different degrees of polarities and

hydrogen bonding such as oil/water or air/water interfaces (Desai and Banat 1997). These

properties render surfactants capable of reducing surface/interfacial tension and forming

emulsions. Such characteristics confer excellent detergency, emulsifying, foaming and

dispersion traits, thereby making the market of surfactants extremely competitive.

Biosurfactants are biological compounds that exhibit surface-active properties.

There are many advantages of biosurfactants compared to their chemically synthesized

counterparts, a few are mentioned below:

• biodegradability and low toxicity

• have lower critical micelle concentration (CMC)

• better environmental compatibility

• can be produced from renewable substrates

• effectiveness at extreme temperatures, pH and salinity

The numerous advantages of biosurfactants have prompted applications not only

in food processing, cosmetics, agriculture, pharmaceuticals, detergents, textile

manufacturing, metal treatment, paper and pulp processing and paint industry, but in the

environmental protection as well (Singh et al. 2007). The use of surfactants to overcome

bioavailability-associated limitations during soil remediation applications has attracted

considerable attention (Volkering et al. 1998). The high emulsifying, dispersing or

solubilizing activities of biosurfactants may improve release of hydrophobic

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contaminants, which are tightly adsorbed to soil, and will facilitate their availability and

biodegradation (Kitamoto et al. 2002).

The data accumulated over the past two decades have reported a diverse range of

prokaryotic and eukaryotic microorganisms capable of producing surfactants of low- and

high-molecular weight (Table 2.1) (Bodour et al. 2004; Fukuoka et al. 2007, 2008; Lang

2002). The low-molecular weight types are generally glycolipids or peptidyl lipids

(lipopeptides) and are involved in the lowering of surface and interfacial tension of

liquids. The emulsions formed by these surfactants are not usually stable. One the other

hand, high-molecular weight biosurfactants i.e. (lipo)polysaccharides, (lipo)proteins or

combinations of these, are generally associated with the production of stable emulsions

but do not lower the surface tension (Christofi and Ivshina 2002).

The effective commercial use of microbially-produced surfactants will have to

compete with synthetic surfactants in three respects: cost, functionality and efficiency, so

that biosurfactants can meet the need of the intended varied applications. The different

ways to enhance the yield include: medium development, process optimization, strain

improvement and the use of alternative, inexpensive substrates (Mukherjee et al. 2006;

Patel and Desai 1997). Interestingly, biosurfactants have about a 10- to 40-fold lower

CMC (critical micelle concentration) than do chemical surfactants (having CMC value

ranging between 590-2120 mg l-1), thereby resulting in lower cost of application

(Christofi and Ivshina 2002). The potential of biosurfactants in different applications can

be further exploited by isolation and characterization of novel surface-active compounds,

by developing the economical strategies for their production and by using efficient

downstream processing (Bodour et al. 2004; Fukuoka et al. 2007).

2.2 Screening of potential biosurfactant-producing microorganisms

Recent advances in the field of microbial surfactants are largely attributed to the

development of quick, reliable and easy methods for screening biosurfactant-producing

microbes and assessing their surface-active properties. A brief outline of methods to

detect biosurfactant production by diverse microorganisms is discussed below.

2.2.1 Blood agar method:

The hemolytic activity of biosurfactants was first discovered when Bernheimer

and Avigad (1970) reported that the biosurfactant named surfactin produced by Bacillus

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subtilis lysed red blood cells. Blood agar lysis has been used to quantify surfactin (Moran

et al. 2002) and rhamnolipids (Johnson and Boese-Morrazzo 1980) and has been used to

screen new biosurfactant-producing isolates (Yonebayashi et al. 2000; Youseff et al.

2004).

Carrillo et al. (1996) found an association between hemolytic activity and

surfactant production, and they recommended the use of blood agar lysis as a primary

method to screen for biosurfactant activity. However, in one study only 13.5% of the

hemolytic strains lowered the surface tension below 40 mN m-1 (Youseff et al. 2004). In

addition, other microbial products such as virulence factors may lyse blood cells and

biosurfactants that are poorly diffusible may not lyse blood cells. Moreover, the extent of

hemolytic zone formation may be affected by divalent ions and other hemolysins

produced by the microbes under investigation (Seigmund and Wagner 1991; Thimon et

al. 1992). Thus, it is not clear whether blood agar lysis should be used to screen

microorganisms for biosurfactant production.

2.2.2 Surface activity:

There are a number of approaches that measure directly the surface activity of

biosurfactants. These include surface and/or interfacial tension measurement (Haba et al.

2000; McInerney et al. 1990), axisymmetric drop shape analysis profile (ADSA-P)

(Noordmans and Busscher 1991; vander Vegt et al. 1991), drop collapse method (Bodour

and Maier 1998; Jain et al. 1991), and the oil spreading technique (Morikawa et al.

2000).

2.2.2.1 Surface tension measurement:

The surface tension measurement has traditionally been used to detect

biosurfactant production as the standard method (Bosch et al. 1988; Neu and Poralla

1990; Persson and Molin 1987). However, the measurement of surface tension is time

consuming, which makes it inconvenient to use for screening of a large number of

isolates.

2.2.2.2 Drop collapse technique:

The drop collapse technique depends on the principal that a drop of a liquid

containing a biosurfactant will collapse and spread completely over the surface of oil

(Bodour andMaier 1998; Jain et al. 1991). The method is easy and can be used to screen

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large number of samples (Batista et al. 2006), but it has not been correlated to surface

tension reduction to confirm its reliability.

2.2.2.3 Oil spreading technique:

The oil spreading technique measures the diameter of clear zones caused when a

drop of a biosurfactant-containing solution is placed on an oil-water surface. Morikawa et

al. (2000) used this method to compare the activity of both cyclic and linear forms of

surfactin and arthrofactin. However, its ability to detect biosurfactant production in

diverse microorganisms has not been tested.

2.2.3 Emulsification activity:

Biosurfactant production has also been detected by measuring emulsification

(Makkar and Cameotra 1998; Van Dyke et al. 1993). The use of methods that measure

properties other than the surface activity can be problematic. Although, a direct

correlation was found between surface activity and emulsification activity (Cooper and

Goldenberg 1987; Denger and Schink 1995), the ability of a molecule to form a stable

emulsion is not always associated with surface tension lowering activity (Trebbau de

Acevedo and McInerney 1996; Willumsen and Karlson 1997).

2.2.4 Cell surface hydrophobicity:

Cell surface hydrophobicity is an important aspect in bacterial cell adhesion to

surfaces, since hydrophobic surfaces are usually associated with molecules of low surface

energy (Mozes and Rouxhet 1987; vander Mei et al. 1987). However, it is not clear that

whether the method for measuring cell surface hydrophobicity is appropriate for general

screening (Dillon et al. 1986). Neu and Poralla (1990) used this property to screen

microbes for biosurfactant production. Pruthi and Cameotra (1997) found a direct

correlation between hydrophobicity and biosurfactant production.

2.2.5 In situ thin-layer chromatography (TLC):

Some in situ techniques utilizing the physiological and chemical properties of

biosurfactants have also been developed for the detection of biosurfactant production. A

modified version of TLC has been reported for direct screening of bacterial colonies for

biosurfactant-producing variants (Matsuyama et al. 1991). Instead of spending days for

TLC sample preparation, this technique involves the direct application of bacterial mass

on a TLC plate. The plate containing the bacterial isolates was subsequently developed

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following the removal of adhering bacterial mass and drying. The assay was employed

successfully to identify and isolate the bacterial variants defective in biosurfactant

production. However, this direct-colony TLC has low sensitivity thus, may not be applied

to microbes producing low levels of biosurfactant.

2.2.6 Cetyltrimethylammonium bromide (CTAB) and methylene blue plate assay

method:

A semi-quantitative agar plate biosurfactant assay specific for anionic

biosurfactants using cetyltrimethylammonium bromide (CTAB) and methylene blue has

been reported (Seigmund and Wagner 1991). The assay is based on the property that the

concentration of anionic surfactants in culture broths can be determined by the formation

of insoluble ion pairs with various cationic substances. Under optimal conditions, dark

blue halos were observed around rhamnolipid-producing colonies and the diameter of

halos could be directly related to the concentration of rhamnolipid produced. The assay

was shown to be applicable to other anionic glycolipids such as sophorolipids and

cellobioselipids as well. The disadvantages of blood agar assay for screening of

biosurfactant-producers were not observed in this agar plate assay. However, further

modification of this assay with other cationic substitutes, such as N-cetylpyridinium

chloride or benzethonium chloride, may be necessary for other biosurfactant-producing

microorganisms, because CTAB inhibits the growth of most bacteria (Seigmund and

Wagner 1991). Since this approach is specific for anionic surfactants, it cannot be used as

a general method of screening for biosurfactant producers. Shulga et al. (1993) have also

described a colorimetric estimation of biosurfactants based on the ability of the anionic

surfactants to react with the cationic indicator to form a colored complex.

2.2.7 Other methods:

In 2007a, Chen et al. reported a high throughput analysis method using a 96-well

plate for the screening of potential biosurfactants. The method is based on the effect of

meniscus shape on the image of a grid viewed through the wells of a 96-well plate. The

assay is rapid, sensitive, easy to perform and also does not require specialized equipment.

Recently, Mukherjee et al. (2009) reported a simple turbidometric method for the

quantification of crude biosurfactants produced by diverse bacteria based on their

property to become insoluble at low pH values.

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2.3 Physico-chemical properties of biosurfactants

The major properties of microbial surfactants that are of a great interest are

discussed below.

2.3.1 Surface and interfacial activity:

The effectiveness of a surfactant is determined by its ability to lower the surface

tension, which is a measure of the surface free energy per unit area required to bring a

molecule from the bulk phase to the surface (Rosen 1978). Due to the presence of a

surfactant, less work is required to bring a molecule to the surface as the surface tension

is reduced. For example, a good surfactant can lower surface tension (ST) of water from

72 to 35 mN m-1 and the interfacial tension (IT) of water/hexadecane from 40 to 1 mN m-

1 (Mulligan 2005). Surfactin from B. subtilis can reduce ST of water to 25 mN m-1 and IT

of water/hexadecane to < 1 mN m-1 (Cooper et al. 1981b). The rhamnolipids from

Pseudomonas aeruginosa decreased ST of water to 26 mN m-1 and IT of

water/hexadecane to value < 1 mN m-1 (Syldatk et al. 1985), however, certain

rhamnolipid homologues have lower values (Nitschke et al. 2005b). The sophorolipids

from Candida bombicola were reported to reduce ST to 33 mN m-1 and IT to 5 mN m-1

(Cooper and Paddock 1984).

2.3.2 Critical micelle concentration (CMC):

The CMC is the minimum surfactant concentration required for reaching the

lowest surface or interfacial tension. Efficient surfactants have a low critical micelle

concentration i.e. less surfactant is required to decrease the surface tension. Microbial

culture broth or biosurfactants are diluted several-fold, surface tension is measured for

each dilution and the CMC is calculated from this value. The values of the surface

tension, interfacial tension and CMC of some known biosurfactants are listed in Table

2.2. It is evident from the data presented in Table 2.2 that biosurfactants have lower CMC

values than the commonly used chemical surfactants thereby, making them attractive

options for diverse applications.

At concentrations above the CMC, surface-active molecules associate readily to

form supramolecular structures such as micelles, bilayers and vesicles. The forces that

hold these structures together include hydrophobic, vander Waals, electrostatic and

hydrogen bonding interactions. Since no chemical bonds are involved, these structures

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are fluid-like and are easily transformed from one state to another as conditions such as

electrolyte concentration and temperature are changed. Lipids can form micelles

(spherical or cylindrical) or bilayers mainly based on the area of the hydrophilic head

group and the chain length of the hydrophobic tail. Molecules with small chain lengths

and large head groups generally form spherical micelles. Those with smaller head groups

tend to associate into cylindrical micelles, while those with long hydrophobic chains form

bilayers which, in turn, under certain conditions form vesicles (Israelachvili 1985). The

formation of micelles can result in the solubilization of oil or water in the other phase,

giving rise to a micro-emulsion. Micelle formation allows the partitioning of hydrophobic

structures into the central hydrophobic pseudo-phase core enabling ‘solubility’. This can

lead to increased dispersion of a compound in solution above its water solubility limit

(Rouse et al. 1994). This solubilization can also lead to mobilization of sorbed and

adsorbed hydrophobic soil contaminants by lowering of capillary forces (Tsomides et al.

1995).

2.3.3 Emulsification activity:

An emulsion is formed when the liquid phase is dispersed as microscopic droplets

in another liquid continuous phase. Biosurfactants may stabilize (emulsifiers) or

destabililize (deemulsifiers) the emulsion. The emulsification activity is assayed by the

ability of the surfactant to generate turbidity due to suspended hydrocarbons such as a

hexadecane: 2-methylnaphthalene mixture (Desai et al. 1988; Rosenberg et al. 1979) or

kerosene (Cooper and Goldenberg 1987), etc. in an aqueous system.

2.3.4 HLB values:

Another parameter frequently used for predicting surfactant behavior is the

hydrophilic and lipophilic balance (HLB). Surfactants can be classified according to their

HLB values that affect their physico-chemical properties (Tiehm 1994). In general, a

surfactant with a low HLB (< 9) is lipophilic, whereas a high HLB (> 11) confers better

water solubility (Sabatini et al. 1995). Most ionic surfactants have HLB values greater

than 20. In general, water-in-oil (W/O) emulsifiers exhibit HLB values in the range 3-8,

while oil-in-water (O/W) emulsifiers have HLB values of about 8-18. The HLB is an

indicator of the emulsifying characteristics of an emulsifier but not its efficiency

(Schramm et al. 2003).

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2.3.5 Tolerance to temperature, pH and ions:

The biosurfactants and their surface activities are not generally affected by

environmental conditions such as temperature and pH. Abu-Ruwaida et al. 1991(b)

reported that heat treatment (autoclaving at 120°C for 15 min) of some biosurfactants

caused no appreciable change in biosurfactant properties such as the lowering of surface

tension or interfacial tension and the emulsification efficiency. McInerney et al. (1990)

reported that lichenysin from B. licheniformis JF-2 was stable at temperature up to 50°C,

at pH range 4.5-9.0 and in the presence of NaCl and calcium concentrations up to 50 and

25 g l-1, respectively. The Antarctic psychrophilic strain Arthrobacter protophormiae

produced a biosurfactant that was thermostable (30-100°C) and was stable under wide pH

range of 2-12 (Pruthi and Cameotra 1997). A lipopeptide from B. subtilis LB5a was

stable even after autoclaving (121°C for 20 min) and after 6 months storage at –18°C.

The surface activity has been found to be stable in the range of pH 5.0 to pH 11.0 and

NaCl concentrations up to 20% (Nitschke and Pastore 2006). These properties make them

suitable candidates for applications in industrial processes frequently involving exposure

to extremes of temperature, pressure, pH and ionic strength; hence, there is a continuous

need to isolate new microbial-derived products able to function under these conditions.

2.3.6 Biodegradability:

Unlike synthetic surfactants, microbially-produced compounds are easily

degraded, thus are particularly suited for environmental applications such as

bioremediation (Mohan et al. 2006; Mulligan 2005).

2.3.7 Low toxicity:

Biosurfactants are generally considered low or non-toxic products and therefore,

appropriate for pharmaceutical, cosmetic and food uses (Edwards et al. 2003; Nitschke

and Costa 2007).

Rhamnolipid surfactants are presently produced at a commercial scale by Jeneil

Biosurfactant Corp. (www.biosurfactant.com) which offers diverse formulations for

different purposes. Additionally, the greater consumer awareness of adverse allergic

effects caused by artificial products stimulated the development of alternative

ingredients, thus opening an excellent opportunity to expand the use of natural surfactants

of microbial origin (Cameotra and Makkar 1998).

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2.4 Biosurfactant classification

Biosurfactants are categorized mainly by their chemical composition and their

microbial origin. In general, their structure includes a hydrophilic moiety consisting of

mono-, di-, or polysaccharides; amino acids or peptides; and a hydrophobic moiety

consisting of unsaturated or saturated fatty acids. Accordingly, the major classes of

biosurfactants include glycolipids, lipopeptides and lipoproteins, phospholipids and fatty

acids, polymeric surfactants, and particulate surfactants. The biosurfactant-producing

microbes are distributed among a wide variety of genera (Table 2.1).

2.4.1 Glycolipids:

Most known biosurfactants are glycolipids. They are carbohydrates in

combination with long-chain aliphatic acids or hydroxyaliphatic acids. Among the

glycolipids, the best known are rhamnolipids, trehalolipids, sophorolipids and

mannosylerythritol lipids (MEL).

2.4.1.1 Rhamnolipids:

Rhamnolipids, in which one or two molecules of rhamnose are linked to one or

two molecules of β-hydroxydecanoic acid, are the best-studied glycolipids. Glycolipids

containing rhamnose and β-hydroxydecanoic acid were first reported by Bergström et al.

(1946) in Pseudomonas pyocyanea grown on glucose. The authors were unable to

determine the molar ratio of the two components. This was achieved by Jarvis and

Johnson (1949), who demonstrated a glycosidic linkage of β-hydroxydecanoyl-β-

hydroxydecanoate with two rhamnose molecules after cultivation of P. aeruginosa on 3%

(v/v) glycerol. The structure was elucidated by Edwards and Hayeshi in 1965. Figure 2.1

shows the first rhamnolipid identified, rhamnolipid 2 (R2, RhaC10C10), as well as others.

Rhamnolipid 1 (R1, Rha2C10C10) was isolated from a culture of P. aeruginosa KY4025

grown in presence of 10% n-alkanes (Itoh et al. 1971). The methyl esters of the rhamnose

lipids R1 and R2 have been reported by Hirayama and Kato (1982). Two rhamnose lipids

that are similar to R1 and R2 but contain only one β-hydroxydecanoyl unit, rhamnolipid

3 (R3, Rha2C10) and 4 (R4, RhaC10) (Figure 2.1), were detected after experiments with

resting cells of Pseudomonas sp. DSM 2874 (Syldatk et al. 1985). Zhang and Miller

(1994) have reported the presence of mono-rhamnolipids with long fatty acid chains of

C18, C22 and C24. In fact, as many as 28 different homologues have been reported, with

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saturated and unsaturated acyl chains varying from C8-C14 and with branched sugar

moieties (Monteiro et al. 2007; Soberon-Chavez et al. 2005). The composition of the

congeners is related to many parameters, the most important being the strain, the culture

media composition, the culture conditions and the age of the culture (Mata-Sandoval et

al. 1999). The resulting mixture of congeners determines the properties of the

biosurfactant and even slight differences in the composition of the mixture can have great

consequences on its physico-chemical properties. For instance, mono-rhamnolipids are

less soluble, adsorb onto surface more strongly and bind cationic metals more strongly

than do the homologue di-rhamnolipids (Perfumo et al. 2006).

Rhamnolipid molecules show free carboxylic groups and behave as anions when

the pH is above 4.0. These compounds are soluble in methanol, chloroform, and ethyl

ether and also show good solubility in alkaline aqueous solutions (Zhang and Miller

1994). The surface-active properties of crude, purified, and specific homologue

compounds of Pseudomonas rhamnolipids have been summarized in the extensive

reviews by Monteiro et al. (2007) and Nitschke et al. (2005b).

2.4.1.2 Trehalolipids:

Several types of microbial trehalolipid biosurfactants have been reported (Lang

and Wagner 1987; Li et al. 1984). Disaccharide trehalose linked at C-6 and C-6' to

mycolic acids is associated with species of genera Mycobacterium, Nocardia and

Corynebacterium. Mycolic acids are long-chain, α-branched-β-hydroxyfatty acids.

Trehalolipids from different organisms differ in size and structure of mycolic acids, the

number of carbon atoms, and the degree of unsaturation (Assilineau and Assilineau 1978;

Lang and Wagner 1987; Syldatk and Wagner 1987). Trehalose lipids from R.

erythropolis have been reported to lower the surface tension of culture broth to 25-40 mN

m-1 (Kretschmer et al. 1982).

2.4.1.3 Sophorolipids:

Several yeasts are known to produce sophorolipids in large amounts from various

substrates such as carbohydrates (glucose, fructose, sucrose, and lactose), vegetable oils,

animal fats and n-alkanes. Sophorolipids, produced by yeasts such as Torulopsis

petrophilum (Cooper and Paddock 1983), T. bombicola (Cooper and Paddock 1984), and

T. apicola (Tulloch et al. 1967), consist of a dimeric carbohydrate sophorose linked to a

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long-chain hydroxy-fatty acid. These biosurfactants are a mixture of at least six to nine

different hydrophobic sophorosides. T. petrophilum produced sophorolipids on water-

insoluble substrates such as alkanes and vegetable oils (Cooper and Paddock 1983).

These sophorolipids, which were chemically identical to those produced by T. bombicola,

did not emulsify alkanes or vegetable oils. It was also observed that when T. petrophilum

was grown on a glucose-yeast extract medium, however, sophorolipids were not

produced, but an effective protein-containing alkane emulsifying agent was formed.

These showed remarkable stability towards pH and temperature changes (Cooper and

Paddock 1983). Although, sophorolipids can lower surface and interfacial tension, they

are not effective emulsifying agents (Cooper and Paddock 1984). C. bombicola KSM-36

produces sophorolipids at a yield of 100-150 g l-1 from palm oil and glucose. The lipid

derivatives with propylene glycols have excellent skin compatibility, and are

commercially used by Kao Corporation (Tokyo) as a skin moisturizer for cosmetics

(Banat et al. 2000). Jing et al. (2006) reported the production of sophorolipids from

Wickerhamiella domercqiae and the authors suggested that the purified sophorolipid had

cytotoxic effect on the cancer cells.

2.4.1.4 Mannosylerythritol lipids:

Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants

known (Kitamoto 2002). MELs are abundantly produced by different strains of yeast

from vegetable oils, for example Pseudozyma sp. produces 100 g l-1 of the biosurfactant

(Fukuoka et al. 2008; Morita et al. 2008; Rau et al. 2005). Evaluating the properties of

MELs from P. antarctica ATCC 20509, it was observed that the surface tension of

culture broth decreased to 35 mN m-1 (Adamczak and Bednarski 2000). Kitamoto et al.

(2001) demonstrated that MEL acts as a potential anti-agglomerating agent in an ice-

water slurry system to be used for cold thermal storage.

In the MEL producers such as P. antarctica (Kitamoto et al. 2001), P. rugulosa

(Morita et al. 2006) and P. aphidis (Rau et al. 2005), MEL-A is mostly produced and

comprises more than 70% of the total MELs. However, due to very low water-solubility

MEL-A has limited practical application (Kitamoto et al. 2002). However, amongst the

other known MELs, MEL-B, MEL-C and MEL-D, the deacetylated derivatives of MEL-

A have a higher hydrophilicity and lower critical aggregation concentrations (Imura et al.

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2006). They seem highly advantageous for the use of emulsifiers, dispersants, and/or

washing detergents. These not only show the excellent surface-active properties but also

possess versatile biochemical actions, thereby expanding the development of yeast

biosurfactants. Fukuoka et al. (2008) reported the production of MEL by Pseudozyma

tsukubaensis. The authors found that the biosurfactant had a different carbohydrate

structure in comparison to that of conventional MELs.

2.4.1.5 Other glycolipid biosurfactants:

Ustilago maydis ATCC 14826, a corn smut fungus, produces cellobiose lipids at

the yield of 15 g l-1 from coconut oil under the resting cell conditions (Frautz et al. 1986).

Tsukamurella sp. DSM 44370, which was isolated from an oil-containing soil sample,

produces a mixture of oligosaccharide lipids at the yield of 30 g l-1 from sunflower oil

(Vollbrecht et al. 1999).

Bodour et al. (2004) have described a new class of biosurfactants named

flavolipids produced by a soil isolate Flavobacterium sp. The new surfactant showed

strong surface activity and emulsifying ability, and exhibits a polar moiety that resembles

citric acid.

2.4.2 Lipopeptides and lipoproteins:

Lipopeptides represent a unique class of bioactive microbial secondary

metabolites, and many of them show attractive therapeutic and biotechnological

properties (Maget-Dana and Peypoux 1994; Rodrigues et al. 2006a).

Surfactin is an eight-member cyclic compound consisting of seven amino acids

and a β-hydroxydecanoic acid moiety (Figure 2.2). Surfactin produced by B. subtilis

ATCC 21332, is one of the most powerful biosurfactants. It lowers the surface tension

from 72 to 27.9 mN m-1 at concentrations as low as 0.005% (w/v) (Arima et al. 1968).

Although, surfactin was discovered about 35 years ago, there has been a revival of

interest in the compound over the past decade, triggered by an increasing demand for

effective biosurfactant (Von Dorren et al. 1997) and molecules with desirable biological

properties. The surfactin molecule has a range of biological activities viz. antimicrobial

(Vater 1986), antiviral (Naruse et al. 1990; Vollenbroich et al. 1997a), antitumoral

(Kameda et al. 1974), antimycoplasmic (Vollenbroich et al. 1997b) and hemolytic

activities (Bernheimer and Avigad 1970). Until now, despite many advantages of

15


surfactin over chemical agents, it is not much in use, mainly because of poor yields and

cost of substrates required to get the desired biomolecules (Desai and Banat 1997).

A large number of cyclic lipopeptides including decapeptide antibiotics

(gramicidins) and lipopeptide antibiotics (polymyxins), produced by B. brevis (Marahiel

et al. 1977) and B. polymyxa (Suzuki et al. 1965), respectively, possess remarkable

surface-active properties. Ornithine-containing lipids from P. rubescens (Yamane 1987)

and Thiobacillus thioxidans (Knoche and Shiveley 1972), cerilipin from Gluconobacter

cerinus IFO 3267 (Tahara et al. 1976a), and lysine-containing lipids from Agrobacterium

tumefaciens IFO 3058 (Tahara et al. 1976b) also exhibit excellent biosurfactant activity.

An aminolipid biosurfactant called serratamolide has been isolated from Serratia

marcescens NS.38 (Matsuyama et al. 1985).

Yakimov et al. (1995) have reported the production of a new lipopeptide

surfactant, lichenysin A by B. licheniformis BAS-50 containing long β-hydroxyfatty

acids. Lichenysin A reduces the surface tension of water from 72 to 28 mN m-1. The

detailed characterization of lichenysin A showed that isoleucine was the C-terminal

amino acid instead of leucine and an asparagine residue was present instead of aspartic

acid as in the surfactin peptide.

Viscosin is composed of a hydroxydecanoic moiety attached to a peptide of nine

amino acids, seven of which form a lactone ring has been reported. At a CMC of 4 mg l-1,

the surface tension of water is reduced to 27 mN m-1. The genetic control of viscosin

production in P. fluorescens PfA7B has been reported (Braun et al. 2001).

2.4.3 Fatty acids, phospholipids, and neutral lipids:

Several bacteria and yeasts produce large quantities of fatty acid and phospholipid

surfactant during growth on n-alkanes (Asselineau and Asselineau 1978; Cirigliano and

Carman 1985; Cooper et al. 1978; Robert et al. 1989). The production of phospholipids

has also been detected in Thiobacillus thioxidans (Beeba and Umbreit 1971). In

Acinetobacter sp. strain HO1-N phosphatidylethanolamine-rich vesicles are produced

(Kappeli and Finnerty 1979), which form optically clear micro-emulsions of alkanes in

water. Arthrobacter strain AK-19 (Wayman et al. 1984), and P. aeruginosa 44T1 (Robert

et al. 1989) accumulate up to 40 to 80% (w/w) of such lipids when cultivated on

hexadecane and olive oil, respectively.

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2.4.4 Polymeric biosurfactants:

The best studied polymeric biosurfactants are emulsan, biodispersan, liposan,

mannoprotein, and other polysaccharide-protein complexes. There are reports that

polymeric biosurfactants are good emulsifiers; however, they may not have a potential to

lower the surface tension significantly (Plaza et al. 2006; Willumsen and Karlson 1997).

Emulsan is a very effective emulsifying agent for hydrocarbons in water even at a

concentration as low as 0.001 to 0.01% (w/v). It is one of the most powerful emulsion

stabilizers known today. Kim et al. (2000) showed that the biological modification of the

fatty acid group (C8-C20) in emulsan influenced the emulsification activity.

Biodispersan is an extracellular dispersing agent produced by A. calcoaceticus A2

(Rosenberg et al. 1988). It is an anionic heteropolysaccharide containing four reducing

sugars, namely glucosamine, 6-methylaminohexose, galactosamine uronic acid, and an

unidentified amino sugar. Navonvenezia et al. (1995) described the isolation of alasan, an

anionic alanine-containing heteropolysaccharide-protein biosurfactant from

Acinetobacter radioresistens KA-53, which was found to be 2.5 to 3 times more active

after being heated at 100°C under neutral or alkaline conditions. Liposan is an

extracellular water-soluble emulsifier synthesized by Candida lipolytica and is composed

of 83% carbohydrate and 17% protein (Cirigliano and Carman 1984). The carbohydrate

portion is a heteropolysaccharide consisting of glucose, galactose, galactosamine, and

galactouronic acid.

Kappeli et al. (1984) have isolated a mannan-fatty acid complex from alkane-

grown Candida tropicalis; this complex stabilized hexadecane-in-water emulsions.

Cameron et al. (1988) reported the production of large amounts of mannoprotein by

Saccharomyces cerevisiae; this protein showed excellent emulsifying activity towards

several oils, alkanes, and organic solvents. The purified emulsifier contains 44% mannose

and 17% protein.

2.4.5 Particulate Biosurfactants:

Extracellular membrane vesicles partition hydrocarbon to form a microemulsion,

which plays an important role in alkane uptake by microbial cells. Vesicles of

Acinetobacter sp. strain HO1-N with a diameter of 20-50 nm are composed of protein,

phospholipids, and lipopolysaccharide (Kappeli and Finnerty 1979). Surfactant activity in

17


most hydrocarbon-degrading and pathogenic bacteria is attributed to several cell surface

components, which include structures such as M protein and lipoteichoic acid in the case

of group A streptococci, protein A in Staphylococcus aureus¸ layer A in Aeromonas

salmonicida, prodigiosin in Serratia spp., gramicidins in B. brevis spores, and thin

fimbriae in A. calcoaceticus RAG-1 (Desai 1987; Fattom and Shilo 1985; Lang and

Wagner 1987; Rosenberg 1986; Wilkinson and Galbraith 1975).

2.5 Physiological role of biosurfactants

Microbial surfactants have diverse structures, are produced by a wide variety of

microorganisms and possess different surface properties. Thus, it is expected that

biosurfactants have various roles, some of which are unique to the physiology and

ecology of the producing microorganisms. However, it is impossible to draw any

generalizations or to identify one or more functions that are clearly common to all

microbial surfactants.

2.5.1 Increasing the surface area of hydrophobic water-insoluble substrates:

Direct contact of cells with hydrocarbon droplets and their interaction with

emulsified droplets have been described by several authors (Francy et al. 1991;

Rosenberg 1986; Singh and Desai 1986). Emulsification is a cell-density dependent

phenomenon; i.e. the greater the number of cells, the higher the concentration of

extracellular product (Ron and Rosenberg 2001).

2.5.2 Increasing the bioavailability of hydrophobic water-insoluble substrates:

One of the most important reasons for the prolonged persistence of hydrophobic

compounds is their low water solubility, thereby increasing their sorption onto surfaces

and resulting in their limited availability to biodegrading microorganisms. Biosurfactants

can enhance growth on bound substrates by desorbing them from surfaces or by

increasing their apparent water solubility (Deziel et al. 1996). Surfactants that lower

surface tension/interfacial tension are particularly effective in mobilizing bound

hydrophobic molecules and making them available for biodegradation. Low-molecular-

weight surfactants that have low critical micelle concentrations (CMCs) increase the

apparent solubility of hydrophobic substrates by incorporating them into the hydrophobic

cavities of micelles (Miller and Zhang 1997).

18


2.5.3 Regulating the attachment-detachment of microorganisms to and from

surfaces:

Biosurfactant can form a conditioning film on an interface, thereby stimulating

certain microorganisms to attach to the interface, while inhibiting the attachment of

others (Neu 1996). For example, the cell surface hydrophobicity of P. aeruginosa was

greatly increased by the presence of cell-bound rhamnolipid (Zhang and Miller 1994),

whereas the cell surface hydrophobicity of Acinetobacter strains was reduced by the

presence of its cell-bound emulsifier (Rosenberg and Rosenberg 1983). The data

suggested that microorganisms can use their biosurfactants to regulate their cell surface

properties in order to attach or detach from surfaces according to the need. This has also

been demonstrated for A. calcoaceticus RAG-1 growing on crude oil (Rosenberg 1993).

2.5.4 Antimicrobial activity:

Several biosurfactants have shown antimicrobial action against bacteria, fungi,

algae and viruses. The lipopeptide iturin from B. subtilis showed potent antifungal

activity (Besson et al. 1976). Inactivation of enveloped virus such as Herpes and

Retrovirus was observed with surfactin (Vollenbroich 1997a). The mannosylerythritol

lipid (MEL), a glycolipid surfactant from Candida antarctica has demonstrated

antimicrobial activity particularly against Gram-positive bacteria (Kitamoto et al. 1993).

Rhamnolipids inhibit the growth of harmful bloom algae species at concentration ranging

from 0.4 to 10.0 mg l-1 (Wang et al. 2005). A rhamnolipid mixture obtained from

Pseudomonas spp. showed inhibitory activity against the bacteria and had excellent

antifungal properties (Abalos et al. 2001; Benincasa et al. 2004). There are reports that

sophorolipids and rhamnolipids were found to be effective antifungal agents against plant

and seed pathogenic fungi (Yoo et al. 2005). Mycelial growth of Phytophthora sp. and

Phythium sp. was 80% inhibited by 200 mg l-1 of rhamnolipids and 500 mg l-1 of

sophorolipids.

Besides their antimicrobial activity, new biological applications of biosurfactants

have been found and some reviews concerning the potential uses of microbial surfactants

in biomedical sciences have been published (Rodrigues et al. 2006a; Singh and Cameotra

2004).

19


2.5.5 Role in biofilm development:

Biosurfactants produced by P. aeruginosa play a role both in maintaining

channels between multicellular structures in biofilms and in dispersal of cells from

biofilms (Boles et al. 2005; Irie et al. 2005; Schooling et al. 2004). The studies suggested

that P. aeruginosa biosurfactants have multiple roles in biofilm development: (i) they are

necessary for initial microcolony formation, (ii) they facilitate surface-associated

bacterial migration and thereby, the formation of mushroom-shaped structures, (iii) they

prevent colonization of the channels between the mushroom-shaped structures (Davey et

al. 2003), and (iv) they play a role in biofilm dispersion.

Pamp and Tolker-Nielsen (2007) presented genetic evidence that during biofilm

development by P. aeruginosa biosurfactants promote microcolony formation in the

initial phase and facilitate migration-dependant structural development in the later stage.

2.6 Biosynthesis of surfactants

2.6.1 Rhamnolipid biosynthesis:

P. aeruginosa is an environmental bacterium that can be isolated from many

different habitats, including water, soil and plants, but it is also an opportunistic human

pathogen causing serious nosocomial infections (Costerton 1980; Lyczak et al. 2000).

This bacterium was reported to produce rhamnolipids by Jarvis and Johnson (1949),

which are amphiphilic molecules, composed of a hydrophobic fatty acid moiety and a

hydrophilic portion composed of one or two rhamnose. The synthesis of these surfactants

by cell-free extracts and the fact that they were secreted by bacteria in the stationary

phase of growth was described by Hauser and Karnovsky (1958). Rhamnolipid anabolic

precursors without the sugar moiety, 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs),

are also released by the bacteria and display tension-active properties (Deziel et al. 2003).

While the production of rhamnolipids is a characteristic of P. aeruginosa, some

isolates of the non-pathogenic pseudomonads P. putida and P chlororaphis as well as the

pathogen Burkholderia pseudomallei were also shown to produce a variety of

rhamnolipids (Gunther et al. 2005; Haussler et al. 1998, 2003; Tuleva et al. 2002).

Biosynthesis of rhamnolipids is dependant on central metabolic pathway (Figure 2.3),

such as fatty acid and deoxythymidine diphosphate (dTDP)-activated sugars synthesis

20


(Soberon-Chavez et al. 2005). The rhamnolipid biosynthetic pathway has also steps in

common with lipopolysaccharides (LPS) (Rahim et al. 2000), alginate (Olvera et al.

1999), and 4-hydroxy-2-alkylquinolines (HAQs) (Bredenbruch et al. 2005).

In liquid culture, P. aeruginosa produces primarily two forms of rhamnolipids:

rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (mono-rhamnolipid) and rhamnosyl-

rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (di-rhamnolipid). The biosynthesis

of these surface-active molecules proceeds by two sequential rhamnosyl-transfer

reactions, each catalyzed by a specific rhamnosyl-transferase (Rt1 and Rt2, respectively

with dTDP-L-rhamnose acting as the rhamnosyl donor in both reactions and β-

hydroxydecanoyl-β-hydroxydecanoate or mono-rhamnolipid acting as the respective

recipients (Burger et al. 1963, 1966).

The Rt1 enzyme is composed of two polypeptides that are encoded by the rhlA

and rhlB genes. RhlA is an inner membrane protein and the catalytic subunit, RhlB is

periplasmic (Ochsner et al. 1994). The gene (rhlC) encoding the Rt2 enzyme also seems

to be loosely bound to the inner membrane (Rahim et al. 2001).

Synthesis of the fatty acid moiety of rhamnolipids diverges from the P.

aeruginosa general fatty acid biosynthetic pathway at the level of ketoacyl reduction

(Campos-Garcia et al. 1998). RhlG is specifically involved in rhamnolipids production

and also affects polyhydroxyalkanoate (PHA) synthesis (Figure 2.3). It was recently

reported that RhlG is involved in providing acyl carrier protein (ACP) fatty acid

precursors for the synthesis of HAQs (Bredenbruch et al. 2005).

The donor of the rhamnosyl moiety for the synthesis of both mono- and di-

rhamnolipid is TDP-L-rhamnose. Rahim et al. (2000) reported that in P. aeruginosa, the

enzymes catalyzing the formation of TDP-L-rhamnose are encoded by rmlA, rmlB, rmlC

and rmlD, respectively and form the rmlBCAD operon. Olvera et al. (1999) have

described that AlgC through its phospho-gluco-mutase activity is also directly involved

in rhamnolipids biosynthesis (Figure 2.3).

2.6.2 Surfactin biosynthesis:

Studies on the biosynthesis of surfactin began with the work of Kluge et al.

(1988), who proposed a non-ribosomal mechanism catalyzed by multienzymatic thio-

21


templates constituting the surfactin synthetase. The scheme of biosynthesis is based on

the multiple-carrier concept, implying multiple 4'-phosphopantetheinyl co-factors (Vater

et al. 1997)

Surfactin synthetase consists of four subunits, which are found to be entirely

cytoplasmic (Menkhaus et al. 1993; Ullrich et al. 1991). These are constituted by

enzymes, E1A, E1B, E2 and E3 of 460 kDa, 435 kDa, 160 and 40 kDa, respectively. In vitro

experiments have indicated that β-hydroxyacyl L-glutamate is the initiation intermediate

of the biosynthesis and that the enzyme responsible for the first step is an acyltransferase

with E3 being involved. The subunits E1A and E1B catalyze the elongation of the initiation

product into lipo-tripeptide, then into lipo-hexapeptide through a series of thioester bond

cleavages and simultaneous transpeptidation reactions. Finally, the fraction E2 catalyses

the condensation of the leucine residue 7 and the release of the resulting lipo-

heptapeptidyl intermediate from the protein. The seven constituent amino acids are

activated by ATP-dependent adenylation in seven amino-acid-activating domains before

being covalently attached to the enzyme-associated sulfydryls by carboxythioester bonds.

2.7 Genetic regulation of biosurfactant production

2.7.1 Rhamnolipid synthesis:

The rhlA and rhlB genes are arranged as an operon and are clustered with rhlR

and rhlI, which encode proteins involved in their transcriptional regulation through the

quorum-sensing (QS) responses (Lazdunski et al. 2004). The rhlC gene is not linked in

the chromosome to other rhl genes and forms an operon with a gene whose function is

not known. This operon is regulated at the transcriptional level in a similar manner as

rhlAB (Rahim et al. 2001).

QS response regulates at the transcriptional level the production of several

virulence-associated traits, including rhamnolipids (Van Delden and Iglewski 1998). The

QS response depends on the production of two autoinducers, butanoyl-homoserine

lactone (C4-HSL) and 3-oxo-dodecanoyl-homoserine lactone (3-oxo-C12-HSL), they bind

to RhlR and LasI, respectively (Lazdunski et al. 2004; Soberon-Chavez et al. 2005). The

transcriptional activator, LasR, once bound to 3-oxo-C12-HSL, promotes the expression

of several genes, including the one coding for the transcription regulator RhlR (Medina et

22


al. 2003; Pesci et al. 1997). The second QS genetic circuit responds to RhlR that once

bound to C4-HSL (Ochsner and Reiser 1995), promotes the expression among others, of

rhlAB and rhlC (Ochsner et al. 1994; Rahim et al. 2001). The RhlR-dependent QS

response, including rhamnolipid production, is primarily expressed under conditions of

nutrient limitation (Pearson et al. 1997). It also regulates the stationary phase sigma

factor encoded by rpoS, involved in the regulation of numerous genes important for

survival under adverse conditions (Latifi et al. 1996). In addition, the transcriptional

regulator MvfR, which directs the synthesis of HAQs, influences the expression of

multiple RhlR-dependant genes, including rhlAB (Deziel et al. 2005).

2.7.2 Surfactin synthesis:

The spore-forming bacterium, B. subtilis is an important organism for the

molecular genetic study of peptide synthesis. A number of genetic studies were

undertaken to identify the genes required for the production of surfactin (Kakinuma et al.

1969; Kunst et al. 1997; Nakano et al. 1992). The studies of the genetics of surfactin

production suggest the involvement of three chromosomal genes: sfp, srfA and comA

(Peypoux et al. 1999). The sfp gene was found to be essential for surfactin production, as

its transfer to a surfactin-nonproducing strain makes the cells surfactin-positive (Nakano

et al. 1992). Moreover, mutation in comA (earlier designated srfB) blocks competence

development and renders sfp-bearing cells surfactin-negative (Nakano and Zuber 1989).

Weinrauch et al. (1989) showed that the comA product is a response-regulator type

protein which can be activated through phosphorylation by the comP histidine kinase

membrane sensor protein. Roggiani and Dubnau (1993) purified comA protein, and after

its phosphorylation, it acquired strong binding affinity to the srfA promoter. It has been

further suggested that two regions upstream of the srfA promoter are required for the

expression of srfA. According to Nakano and Zuber (1993), a cooperative interaction of

comA dimers and binding of such dimers upstream of the srfA promotor result in a

transcriptional activation of srfA genes.

Nakano et al. (1992) demonstrated that the srfA locus is a large operon of more

than 25kb of DNA that encodes multifunctional surfactin synthetase. Moreover, the srfA

operon was also shown to be involved in the production of pheromone-like peptide

23


factors which are responsible for the initiation of sporulation in Bacillus sp. The synthesis

of these regulatory peptides and surfactin appears to involve the same intermediate

(Nakano et al. 1991). A closely linked sfp locus encodes a 224-amino acid polypeptide

which is responsible for converting intermediates to surfactin (Nakano et al. 1992). This

polypeptide also decreases the transcription of the srfA operon, indicating that it has a

regulatory function in addition to its direct role in surfactin biosynthesis.

2.8 Factors affecting biosurfactant production

2.8.1 Carbon source:

The type of carbon source used in production medium can be divided into three

categories: carbohydrates, hydrocarbons and vegetable oils. Some microorganisms

produce biosurfactants by using hydrophobic carbon source such as hydrocarbon or

vegetable oils, others only use carbohydrates and still others support biosurfactant

production in presence of different carbon sources in combination or individually (Kim et

al. 1997).

Water-soluble carbon sources such as glycerol, glucose, fructose, mannitol and

ethanol can be used for rhamnolipid production by Pseudomonas spp. (Chayabutra et al.

2001; Matsufuji et al. 1997; Robert et al. 1989). Syldatk et al. (1985) demonstrated that

although different carbon sources in the medium affected the composition of

biosurfactant production in Pseudomonas spp. however, substrates with different chain

lengths exhibited no effect on the chain lengths of fatty acid moieties in biosurfactants.

On the other hand, qualitative variation was observed by Finnerty & Singer (1985) and

Neidleman & Geigert (1984) in the fatty acid moieties during biosurfactant production in

Acinetobacter sp. strains H13A and HO1-N, respectively.

A significant increase in the biosurfactant yield was observed by Arthrobacter

paraffineus ATCC 19558 cells grown on D-glucose and fed with hexadecane during the

stationary growth phase (Duvnjak et al. 1982). In 1985, Duvnjak and Kosaric showed the

presence of large amounts of biosurfactant bound to Corynebacterium lepus cells when

grown on glucose, and addition of hexadecane facilitated the release of surfactant from

cells. Glycolipid production by Torulopsis bombicola was stimulated by the addition of

vegetable oils during growth on D-glucose (10%) medium, giving a yield of 80 g l-1

24


(Asmer et al. 1988; Cooper et al. 1988). Davila et al. (1992) demonstrated a high yield of

sophorolipids by overcoming product inhibition in Candida bombicola CBS 6009 though

the addition of ethyl esters of rapeseed oil fatty acids in D-glucose medium. Using T.

apicola IMET 43747, Stuwer et al. (1987) achieved glycolipid yields as high as 90 g l-1

with a medium containing D-glucose and sunflower oil. In an interesting study, Lee and

Kim (1993) reported that in batch culture 37% of the carbon input was channeled to

produce 80 g of sophorolipid per liter by T. bombicola. However, in fed-batch cultures,

about 60% of the carbon input was incorporated into biosurfactant, increasing the yield to

120 g l-1.

Thus, it is evident that the nature of the carbon source available and time of its

addition to the medium has a significant effect on the type and the yield of biosurfactant

produced (Abouseoud et al. 2008; Das et al. 2009).

2.8.2 Nitrogen Source:

Medium constituents other than carbon source also affect the production of

biosurfactants. Among the inorganic salts tested, ammonium salts and urea were

preferred nitrogen sources for biosurfactant production by Arthrobacter paraffineus

(Duvnjak et al. 1983), whereas nitrate supported maximum surfactant production in P.

aeruginosa (Guerra-Santos et al. 1984; Robert et al. 1989) and Rhodococcus spp. (Abu-

Ruwaida et al. 1991a). Biosurfactant production by A. paraffineus increased by the

addition of L-amino acids such as aspartic acid, glutamic acid, asparagine, and glycine to

the medium (Duvnjak et al. 1983). The structure of surfactin was influenced by the L-

amino acid concentration in the medium to produce either Val-7 or Leu-7 surfactin

(Peypoux and Michel 1992). Similarly, lichenysin A production was enhanced two- and

four-fold in B. licheniformis BAS-50 (Yakimov et al. 1996) by addition of L-glutamic

acid and L-asparagine, respectively to the medium.

In P. aeruginosa, a simultaneous increase in rhamnolipid production and

glutamine synthetase activity was observed when growth slowed as the medium became

nitrogen limiting (Mulligan and Gibbs 1989). Similarly, nitrogen limitation resulted in

increased biosurfactant production in P. aeruginosa (Ramana and Karanth 1989; Suzuki

et al. 1974), Candida tropicalis IIP-4 (Singh et al. 1990), and Nocardia strain SFC-D

25


(Kosaric et al. 1990). It has been reported that nitrogen limiting conditions do not favor

rhamnolipids production per se, but production starts with the exhaustion of nitrogen

(Manresa et al. 1991; Ramana and Karanth 1989; Robert et al. 1989). Production of

rhamnolipids was inhibited by the presence of NH4+, glutamine, asparagine, and arginine

as nitrogen source and promoted by nitrate, glutamate and aspartate in the production

medium (Kohler et al. 2000; Mulligan and Gibbs 1989). Guerra Santos et al. (1984,

1986) reported maximum rhamnolipid production by P. aeruginosa after nitrogen

limitation at a C: N ratio of 16:1 to 18:1 and there was no surfactant production below a

C: N ratio of 11:1.

2.8.3 Environmental factors:

Environmental factors and growth conditions such as pH, temperature, agitation

and oxygen availability also affect biosurfactant production through their effects on

cellular growth or activity (Gerson and Zajic 1978; Kim et al. 1990).

Rhamnolipid production in Pseudomonas spp. was at its maximum in a range of

pH 6.0 to pH 6.5 and decreased sharply above pH 7.0 (Guerra-Santos et al. 1984). In

contrast, Powalla et al. (1989) showed that penta- and disaccharide lipid production in

Nocardia corynbacteroides is not affected in the pH range of 6.5 to 8. In addition, surface

tension and critical micelle concentration (CMC) of a biosurfactant product remained

stable over a wide range of pH values, whereas emulsification ability was observed over

a narrower pH range (Abu-Ruwaida et al. 1991b).

Wei et al. (2005) studied the effect of temperature (25-47°C) on rhamnolipid

production by Pseudomonas aeruginosa J4. The results suggested the optimal

temperature for the biosurfactant production by J4 strain was in the range of 30-37°C.

The authors have also reported that pH plays vital role in affecting the efficiency of

rhamnolipid production by the respective strain. Chen et al. (2007b) reported production

of rhamnolipids by P. aeruginosa S2 in a fermenter, in order to explore the effect of

environmental parameters such as temperature (30, 37 and 42°C) and pH (6.0, 6.5, 6.8,

7.0, 7.2, 7.5, and 8.0). From the results, it was observed that pH 6.8 and 37°C appeared to

be the most favorable for biosurfactant production by the strain.

26


There are reports in literature regarding the effect of agitation speed on

biosurfactant yields. An increase in agitation speed results in the reduction of

biosurfactant yield due to shearing effect in Nocardia erythropolis (Margaritis et al.

1979, 1980). On the other hand, in yeast, biosurfactant production increases when the

agitation and aeration rates are increased (Spencer et al. 1979). Wang and Wang (1990)

reported a decreased cell-bound polymer to dry-cell ratio in A. calcoaceticus RAG-1 as

the shear stress increased due to increased rate of agitation. Sheppard and Cooper (1990)

during their studies on the process optimization and scale-up of surfactin production in B.

subtilis concluded that oxygen transfer is one of the key parameters. Oliveira et al. (2006)

reported that the agitation rate of 100-150 rpm was optimal for the biosurfactant

production by P. aeruginosa FR strain.

2.8.4 Influence of metal ions and other additives on biosurfactant production:

The production of biosurfactants by microorganisms may be affected by other

medium components such as phosphates, metal ions and additives. Mulligan et al. (1989)

have reported that the production of biosurfactant might be affected by phosphate

metabolism. Palejwala and Desai (1989) reported that low phosphate concentration

stimulated bioemulsifier production in a Gram-negative bacterium during cultivation on

ethanol. Lin et al. (1993) reported increase in production of lipopeptide biosurfactant by

B. licheniformis JF-2 from 35 mg l-1 to 110 mg l-1 by reducing the phosphate

concentration. The phosphate, iron, magnesium and sodium supplement to the medium

significantly affected biosurfactant production by Rhodococcus sp. as compared to the

effect of either potassium or calcium (Abu-Ruwaida et al. 1991a). The addition of iron or

manganese salts has also been shown to increase the yield of surfactin production by B.

subtilis (Copper et al. 1981).

The effects of multivalent ions on biosurfactant production might be correlated to

their role in nitrogen metabolism (Sheppard et al. 1991). On the other hand, there are

reports that the limitation of multivalent cations causes overproduction of biosurfactants

by Pseudomonas spp. (Guerra-Santos et al. 1984; Syldatk et al. 1985). Chayabutra et al.

(2001) investigated the effects of different limiting nutrients (N, P, S, Mg, Ca, and Fe) on

rhamnolipid production potential of P. aeruginosa. They observed P limitation was the

27


most effective, giving four- to five-fold higher specific productivity than the conventional

N limitation. The limitation of sulphur was comparable to N limitation, whereas Mg

limitation was much less effective.

The elevated C/N (Guerra-Santos et al. 1984; Ramana and Karanth 1989) and C/P

(Mulligan et al. 1989) ratios promoted rhamnolipid production by Pseudomonas spp.,

while high concentrations of divalent cations, especially iron, were inhibitory (Guerra-

Santos et al. 1986). Iron concentration has a dramatic effect on rhamnolipid production

by P. aeruginosa, resulting in a three-fold increase in production when cells were shifted

from medium containing 36 µM iron to medium containing 18 µM iron (Guerra-Santos et

al. 1984, 1986). Sabra et al. (2002) proposed that P. aeruginosa produced rhamnolipids

to reduce oxygen transfer rate as a means to protect itself from oxidative stress, and

indicated that this mechanism was related to iron deficiency (Kim et al. 2003).

The yields of biosurfactant can be either enhanced or inhibited by the addition of

antibiotics such as penicillin or chloramphenicol (Rubinowitz et al. 1982). In some cases,

the addition of biosurfactant precursors can modify both the structure and yield of

biosurfactants (Peypoux and Michel 1992). Salt concentration also affected biosurfactant

production depending on its effects on cellular activity. Biosurfactant production,

however, was not affected by salt concentrations up to 10% (w/v) although, slight

reductions in the CMCs were detected (Abu-Ruwaida et al. 1991b).

2.9 Biosurfactant production using cheap and unconventional substrates

The major constraint in the widespread use of biosurfactants is the economics of

their production. An important point that should be considered for the development of

cheaper processes is the selection of cheap medium components, as these accounts for

10-30% of the overall costs (Cameotra and Makkar 1998). The agro-industrial by-

products or wastes generally containing high levels of carbohydrates or lipids can be a

good choice to support growth and surfactant synthesis by potential strains. Furthermore,

the treatment and disposal costs for these residues are significant to industries generating

them and they are invariably searching for alternatives to reduce, reuse, recycle, and

valorize their wastes. A great variety of alternative raw materials viz. vegetable oils

(Fukuoka et al. 2007), distillery and whey wastes (Dubey and Juwarkar 2001), starch-rich

28


wastes from potato processing industries (Fox and Bala 2000), etc. have been reported to

support biosurfactant production (Table 2.3).

2.9.1 Vegetable oils and oil wastes:

Several studies with plant-derived oils have shown that they can act as effective

and cheap raw materials for biosurfactant production, for example, rapeseed oil

(Trummler et al. 2003), babassu oil and corn oil (Pekin et al. 2005; Vance-Harrop et al.

2003). Oils such as sunflower and soybean oils have been reported to be used for the

production of rhamnolipid, sophorolipid, and mannosylerythritol lipid biosurfactants by

various microorganisms (Fukuoka et al. 2007; Kim et al. 2006; Rahman et al. 2002).

Apart from various vegetable oils, oil wastes from vegetable oil refineries and the food

industry have also been reported as good substrates for biosurfactant production

(Benincasa and Accorsini 2008). In addition, industrial oil wastes such as lard, free fatty

acids, marine oils and soapstock can potentially induce microbial growth and metabolite

production owing to their typical fatty acid composition. Furthermore, different waste

oils used for frying, in vegetable oil refineries or the soap industries were found to be

suitable for microbial growth and biosurfactant production (Abalos et al. 2001; Bednarski

et al. 2004; Benincasa et al. 2002, 2004; Nitschke et al. 2005a). These oils and oil wastes

are readily available in good amounts throughout the world. However, the oils used to

date for biosurfactant production are mostly edible oils and are not that cheap. Several

plant-derived oils, for example, castor oil, jojoba oil, etc. are not suitable for human

consumption due to their unfavorable odor, color and composition and are, therefore,

available at much cheaper rates. Incorporation of these cheaper oils and oil wastes in the

industrial production media might potentially reduce the overall costs of biosurfactant

production (Mukherjee et al. 2006).

Candida antarctica and C. apicola produced biosurfactants (glycolipids) in a

cultivation medium supplemented with oil refinery waste i.e. with soapstock (5-12% v/v)

or post-refinery fatty acids (2-5% v/v). It was observed that the production of

biosurfactant ranged from 7.3 to 13.4 g l-1 in the medium supplemented with soapstock,

while 6.6 to 10.5 g l-1 in post-refinery fatty acids-supplemented medium (Bednarski et al.

2004). Recently, Sobrinho et al. (2008) reported the use of groundnut oil refinery residue

29


(5.0% w/v) and cornsteep liquor (2.5% v/v) for the biosurfactant production by C.

sphaerica UCP0995. The yeast produced 4.5 g l-1 of the biosurfactant, which was later

characterized as an anionic glycolipid.

Frying oils is produced in large quantities for use, both in the food industry and at

the domestic scale. Haba et al. (2000) studied a screening process for the selection of

microorganisms capable of growing on frying oils (sunflower and olive) and

accumulating surface-active compounds in the culture medium. P. aeruginosa 47T2 was

selected, capable of producing rhamnolipid at a rate of 2.7 g l-1. In an another studies by

Benincasa et al. (2002), sunflower oil soapstock was assayed as the carbon source for

rhamnolipid production by P. aeruginosa LBI strain, giving a final surfactant

concentration of 12 g l-1 in shaker and 16 g l-1 in bioreactor experiments. Similarly,

Nitschke et al. (2005a) evaluated edible oil soapstocks as alternative low-cost substrates

for the production of rhamnolipid by P. aeruginosa LBI strain. The wastes obtained from

soybean, cotton seed, babassu, palm and corn oil refinery were tested. The soybean

soapstock waste was the best substrate, generating 11.7 g l-1 of rhamnolipids and a

production yield of 75%. Benincasa and Accorsini (2008) reported that P. aeruginosa

LBI produced rhamnolipids when cultivated on wastes from sunflower-oil refinery as a

substrate. The strain was able to produce 7.3 g l-1 of the biosurfactant at a C/N ratio of

8/1. The rhamnolipid produced was a mixture of six rhamnolipid homologues which

showed good surface and interfacial properties. Also, the emulsification potential of the

biosurfactant for different hydrophobic substrates indicated the possibility of using the

biosurfactant in bioremediation applications.

Bento and Gaylarde (1996) observed production of surfactants by P. aeruginosa

in the presence of mineral salts and glucose medium and found an increase in emulsifying

activities of the surfactant by the addition of sterile diesel oil to medium. In a similar

study, Muriel et al. (1996) observed production of extracellular biosurfactants by

Cladosporium resinae when growing on jet fuel JP8. The production of biosurfactants

was observed by the reduction of surface tension of the aqueous phase of the growth

medium, and by increase in emulsion and foaming properties. A partial purification gave

better physical properties.

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Thus, producing biosurfactants from vegetable oils, used vegetable oils and used

motor oil is a sound strategy of waste management for the food and auto industries to

reduce the generation of waste.

2.9.2 Lactic whey and distillery wastes:

The lactic whey from dairy industries has also been reported as a cheap and viable

substrate for biosurfactant production. The effluent from the dairy industry, known as

dairy wastewater, supports good microbial growth and is used as a cheap raw material for

biosurfactant production (Dubey and Juwarkar 2001, 2004). The studies showed that

lactic whey wastes might be comparatively better substrates for biosurfactant production

at the commercial scale. Furthermore, the potential use of dairy wastewater provides a

good alternative for the economical production of biosurfactants and efficient dairy

wastewater management. Thanomsub et al. (2006) described the chemical structure and

biological activities of rhamnolipids produced by P. aeruginosa B189 isolated from milk

factory waste. The culture produced two types of biosurfactants, Rha-RhaC10C10 and

Rha-RhaC10C12. From the results, it was observed that the rhamnolipids produced by the

strain inhibited insect and cancer cell lines. Thereby, indicating that the biosurfactant

produced by P. aeruginosa B189 could be used as anticancer drug.

The disposal of cheese whey is a continuing and growing problem to the diary

industry. A two-step batch cultivation process was developed to produce sophorolipids

from whey by C. bombicola and Cryptococus curvatus. In the first step, C. curvatus was

grown on deproteinized whey concentrate (DWC); the cultivation broth was disrupted

with a glass bead mill and it served as a medium for growth and sophorolipid production

by C. bombicola (Daniel et al. 1999).

Sudhakar Babu et al. (1996) performed batch kinetic studies on rhamnolipid

production from synthetic medium using distillery and whey wastes, as substrates. The

results indicated that the specific growth rate (µmax) and specific product formation rates

(Vmax) from both types of waste were comparatively better than the synthetic medium,

revealing that both of these industrial wastes (distillery and whey) can be successfully

utilized as substrates for biosurfactant production. Rodrigues et al. (2006c) screened

Lactobacillus strains for their ability to produce surfactants using whey. The lactic acid

31


bacteria, Lactobacillus casei, L. rhamnosus, L. pentosus and L. coryneformis torquens

were selected as surfactant producing organisms, with L. pentosus as the most promising

strain and whey as a potential alternative substrate.

2.9.3 Carbohydrate-rich residues:

Starchy waste substrates are also potential alternative raw materials for the

production of biosurfactants. The potatoes are composed of 80% water, 17%

carbohydrates, 2% protein, 0.1% fat and 0.9% vitamins, inorganic minerals and trace

elements. They are a rich source of carbon (in the form of starch and sugars), nitrogen

and sulphur (from protein), inorganic minerals, trace elements and vitamins. Fox and

Bala (2000) demonstrated that potato processing effluent was a suitable alternative

carbon source to generate surfactant from B. subtilis ATCC 21332. Das and Mukherjee

(2007) reported the production of lipopeptide biosurfactants by B. subtilis strains in

submerged (SmF) and solid state fermentation (SSF) systems using potato peels as cheap

carbon source. The biosurfactants produced exhibited appreciable thermostability and

strong emulsifying properties.

Cassava wastewater is another carbohydrate-rich residue, which is generated in

large amounts during the preparation of cassava flour. This residue proved to be an

appropriate substrate for biosurfactant synthesis, providing not only bacterial growth and

product accumulation, but also a surfactant that has interesting and useful properties with

potential for many industrial applications (Nitschke and Pastore 2003, 2004, 2006). These

wastes are obtained at low cost from the respective processing industries and can be used

as low-cost substrates for industrial level biosurfactant production. Several other starchy

waste substrates, such as cornsteep liquor, wastewater from the processing of cereals,

pulses and molasses, have tremendous potential to support microbial growth and

surfactant production.

Molasses is a co-product of sugar production, both from sugarcane as well as

from sugarbeet. It is defined as the run-off syrup from the final stage of crystallization, in

which further crystallization of sugar is uneconomical. Molasses is rich in various

nutrients besides sucrose (Makkar and Cameotra 2002). Average values for the

constituents of cane molasses (75% dry matter) are: total sugars, 48-56%; non-sugar

32


organic matter, 9-12%; protein, 2.5%; potassium, 1.5-5.0%; calcium, 0.4-0.8%;

magnesium, 0.06%; phosphorus, 0.06-2.0%; biotin, 1.0-3.0 mg/kg; pantothenic acid, 15-

55 mg/kg; inositol, 2500-6000 mg/kg; and thiamine 1.8 mg/kg. Molasses has been used

as the major raw material for production of pullulan (Lazaridou et al. 2002), xanthan gum

(Kalogiannis et al. 2003), citric acid (Ikram-Ul et al. 2004) as well as fructo-

oligosaccharides (Shin et al. 2004).

Two B. subtilis strains were able to produce lipopeptide surfactants using minimal

medium supplemented with molasses as a carbon source (Makkar and Cameotra 1997).

Molasses and cornsteep liquor were used as the primary carbon and nitrogen sources for

production of rhamnolipid by P. aeruginosa GS3 with an overall yield of 0.24 g

rhamnolipid l-1 (Patel and Desai 1997). Raza et al. (2007) have also reported 1.45 g l-1 of

rhamnolipids at 96 h on 2% total sugars-based molasses by a P. aeruginosa mutant,

which was 2-3 times higher than that achieved by wild-type strain.

2.10 Process optimization- the best combination of essential factors

Maximizing productivity or minimizing the production costs demands the use of

process-optimization strategies that involve optimization of multiple factors affecting the

productivity of strains. The classical method of medium optimization involves changing

one variable at a time, while keeping the others at fixed levels is a laborious, time

consuming process and does not guarantee the determination of the optimal conditions

for metabolite production. The optimization process can be made more predictable by the

statistical methods based on response surface methodology (RSM). There are reports in

the literature wherein various investigators have employed this method to determine the

optimum media, inoculum and environmental conditions for the enhanced production of

biosurfactants for B. subtilis (Sen 1997; Sen and Swaminathan 1997, 2004), P.

aeruginosa EM1 (Wu et al. 2008), P. aeruginosa AT10 (Abalos et al. 2002), by the

probiotic bacterial strains Lactococcus lactis and Streptococcus thermophilus (Rodrigues

et al. 2006b), and by B. licheniformis capable of concomitant production of biosurfactant

and protease RG1 using agro-products such as cornstarch and soy flour as carbon and

nitrogen sources (Ramnani et al. 2005). These optimization methods would help the

33


industry to design the best production media based on cheaper substrates and to use the

most favorable environmental conditions for improved biosurfactant production.

2.11 Recovery of biosurfactants

The optimization of medium components and physico-chemical conditions is

important, but the production process is still incomplete without an efficient and

economical means for the recovery of products. Thus, one important factor determining

the economic feasibility of a production process on a commercial scale is the availability

of suitable and economic recovery and downstream procedures. For many

biotechnological products, the downstream processing costs account for ~60% of the total

production costs (Mukherjee et al. 2006).

Several conventional methods for the recovery of biosurfactants, such as acid

precipitation, solvent extraction, crystallization, ammonium sulphate precipitation and

centrifugation, have been widely reported in the literature (Desai and Banat 1997). A few

unconventional and interesting recovery methods have also been reported. These

procedures take advantage of some of the other properties of biosurfactants – such as

their surface activity or their ability to form micelles and/or vesicles and are particularly

applicable for large-scale continuous recovery of extracellular biosurfactants from the

culture broth. A few examples of such biosurfactant recovery strategies include foam

fractionation (Davis et al. 2001; Noah et al. 2002; Sarachat et al. 2010), ultrafiltration

(Sen and Swaminathan 2005), adsorption-desorption on polystyrene resins and ion-

exchange chromatography (Reiling et al. 1986). One of the main advantages of these

methods is their ability to operate in a continuous mode for recovering biosurfactants

with high level of purity. Table 2.4 describes the biosurfactant recovery procedures in

addition to their working principles and advantages.

The solvents that are generally used for biosurfactant recovery, for example

acetone, methanol and chloroform, are toxic in nature and harmful to the environment.

Cheap and less toxic solvents such as methyl tertiary-butyl ether (MTBE) have been

successfully used in recent years to recover biosurfactants produced by Rhodococcus sp.

(Kuyukina et al. 2001). These types of low cost, less toxic, and easily available solvents

can be used to cut the recovery expenses and minimize the environmental hazards.

34


Often, a single downstream processing technique is not enough for the product

recovery and purification. In such a case, a multi-step recovery strategy, using a

combination of concentration and purification steps, is much more effective (Reiling et

al. 1986). In such a multi-step recovery for biosurfactants, it will be possible to obtain the

product of required degree of purity. Crude or impure biosurfactants obtained at the

initial stages of recovery process can be used for environmental applications, in oil

recovery, and the paint and textile industries to reduce the cost of application. On the

other hand, the highly pure biosurfactants required for the pharmaceutical, food and

cosmetic industries can be obtained by performing further purification steps.

Glazyrina et al. (2008) reported an automated harvesting and collection system

named ‘flounder’ for extraction of fengycin and bacillaene from liquid surface layer.

However, this technique is not suitable for extraction of surfactants from large volumes.

2.12 Potential commercial applications

Biosurfactants reported over the past few years have shown a diverse range of

applications (Table 2.5). The surface-active properties make surfactants one of the most

important and versatile class of chemical products, used in a variety of applications in

household, industry and agriculture (Deleu and Paquot 2004).

2.12.1 Biosurfactants as speciality product:

Rhamnolipids have been a source of stereospecific L-rhamnose, which is used in

the production of high quality flavor compounds and as starting material for the synthesis

of some organic compounds (Linhardt et al. 1989).

Ishigami et al. (1996) reported the synthesis of a pyrenacyl ester of rhamnolipids

(R-PE) for use in monitoring the polarity and fluidity of solid surfaces and the attendant

impact of coatings on the surface properties. The R-PE was synthesized to facilitate the

use of pyrene, which is one of the most effective fluorescent probes in monitoring the

micropolarity and microfluidity of surfaces. However, pyrene alone is difficult to use in

aqueous systems because of its extremely low aqueous solubility and its tendency to bind

to the hydrophobic surfaces.

35


2.12.2 Biosurfactants used in the food industry:

Emulsion forming and stabilization, antiadhesive and antimicrobial activities are

some properties of biosurfactants, which could be explored in food processing and

formulation.

An improvement of dough stability, texture volume and conservation of bakery

products was obtained by the addition of rhamnolipid surfactants (Van Haesendonck and

Vanzeveren 2004). The authors also suggested the use of rhamnolipids to improve the

properties of butter cream, croissants and frozen confectionary products. A lipopeptide

obtained from B. subtilis was able to form stable emulsions with soybean oil and coconut

fat, suggesting its potential as emulsifying agent in foods (Nitschke and Pastore 2006).

Iyer et al. (2006) reported a bioemulsifier isolated from a marine strain of Enterobacter

cloacae, which was described as a potential viscosity enhancing agents of interest in food

industry. L-Rhamnose has a considerable potential as a precursor for flavorings. It is

being used industrially as a precursor of high-quality flavor components like Furaneol

(Trademark of Firmevich SA, Geneva).

2.12.3 Biosurfactants as therapeutic agents:

The use and potential commercial application of biosurfactants in the medical

field has increased during the past decade. Their antimicrobial, antifungal and antiviral

activities make them relevant molecules for applications in combating many diseases and

as therapeutics (Table 2.6).

Rhamnolipids produced by P. aeruginosa (Abalos et al. 2002; Benincasa et al.

2004), lipopeptides produced by B. subtilis (Leenhouts et al. 1995; Sandrin et al. 1990;

Vollenbroich et al. 1997b) and B. licheniformis (Fiechter 1992; Jenny et al. 1991;

Yakimov et al. 1995) and mannosylerythritol lipids from Candida antarctica (Kitamoto

et al. 1993) have all been reported to have antimicrobial activities. Surfactin, one of the

earliest known biosurfactants, has various pharmacological applications such as

inhibiting fibrin clot formation and haemolysis (Bernheimer and Avigad 1970) and

formation of ion-channels in lipid membranes (Sheppard et al. 1991). It has also been

reported to have an antitumor activity against carcinoma cells (Kameda et al. 1974) and

having antifungal properties (Vater 1986b). Vollenbroich et al. (1997a) have reported a

36


potential use for surfactin in the virus safety enhancement of biotechnological and

pharmaceutical products. They also suggested that the antiviral action of surfactin is due

to physico-chemical interaction between the membrane-active surfactant and the virus

lipid membrane. Viscosin produced by Pseudomonas libanensis M9-3 and sophorolipid

produced by Wickerhamiella domercqiae have been reported to show anticancer

properties (Jing et al. 2006; Saini et al. 2008). There are several reports that

biosurfactants are a suitable alternative to synthetic medicines and antimicrobial agents

and may be used as safe and effective therapeutic agents against pathogens (Rodrigues et

al. 2006a; Singh and Cameotra 2004).

Possible applications of biosurfactants as emulsifying agents for drug transport to

the infection site, as agents supplementing the pulmonary surfactant and as adjuvant for

vaccines were suggested by Kosaric (1996). Another approach for the use of

biosurfactants in biomedical applications is the development of suitable anti-adhesion

biological coatings for implant materials (Busscher et al. 1997, 1999; Rodrigues et al.

2004).

2.12.4 Biosurfactants for agricultural use:

Concerns about pesticide pollution have prompted global efforts to find

alternative biological control technologies. Haferburg et al. (1987) successfully used a

1% (w/v) emulsion of rhamnolipids for the treatment of leaves of Nicotiana glutinosa

infected with tobacco mosaic virus. In one of the reports, Stanghellini and co-workers

(Stanghellini et al. 1996) showed the efficiency of some synthetic surfactants in control

of the root rot of cucumbers and peppers caused by Pythium aphanidermatum and

Phytophthora capsici. Further, in one of their experiments they observed rhamnolipids of

P. aeruginosa in the nutrient solution caused the lysis of zoospores (Stanghellini and

Miller 1997). The biosurfactant has zoosporicidal activity against species of Phythium,

Phytophthora and Plasmopara at concentrations ranging from 5 to 30 µg ml-1.

Biosurfactants have been used in formulating poorly-soluble pesticides. Two

Bacillus strains produced an emulsifier, possibly a glycolipopeptide, which was able to

form a stable emulsion in the presence of a pesticide Fenthion (Patel and Gopinathan

1986).

37


2.12.5 Biosurfactants in cosmetic and health care industries:

The cosmetic and health care industries use large amounts of surfactants for a

wide variety of products (Maier and Soberon-Chavez 2000). Biosurfactants in general are

considered to have some advantages over synthetic surfactant, such as low irritancy or

anti-irritating effects and compatibility with skin (Kleckner and Kosaric 1993).

Sophorolipids are commercially used by Kao Co. Ltd. as humectants in cosmetic

make-up brands such as Sofina (Yamane 1987). A product containing one mole

sophorolipid and 12 moles propylene glycol has specific compatibility to the skin and has

found commercial activity as a skin moisturizer. Rhamnolipids are also being used as

cosmetic additives in Japan (Iwata Co., Japan). There are currently patents for use of

rhamnolipids to make liposomes and emulsions, both important in the cosmetic industry

(Ishigami et al. 1988a, b).

2.12.6 Biosurfactants in mining:

Biosurfactants may be used for the dispersion of inorganic minerals in mining and

manufacturing processes. Rosenberg et al. (1988) described the production of an anionic

polysaccharide called biodispersan by Acinetobacter calcoaceticus A2, which prevented

flocculation and dispersed a 10% limestone in water mixture. Biodispersan served two

functions: dispersant and surfactant, and catalyzed the fracturing of limestone into small

particles. Rosenberg and Ron (1998) elucidated mechanism of action of biodispersan and

suggested that the pH should be alkaline (9-9.5) during the grinding process so that

surfactant is an anionic polymer at that pH. The polymer enters microdefects in the

limestone and lowers the energy required for cleaving the microfractures. Kao Chemical

Corporation (Japan) used Pseudomonas, Corynebacterium, Nocardia, Arthrobacter,

Bacillus and Alcaligenes to produce biosurfactants for the stabilization of coal slurries to

aid the transportation of coal (Kao 1984, Australian Patent 8317-8555).

2.12.7 Biosurfactants as corrosion inhibitors:

Dagbert et al. (2006) suggested the existence of protective properties of biological

surfactants against corrosion. They studied the corrosion behavior of AISI 304 stainless

steel in the presence of a biosurfactant produced by P. fluorescens (Pf 495). The surface

morphology of the corroded specimens was investigated using scanning electron

38


microscopy (SEM). The authors suggested the role of biosurfactants as environment

friendly corrosion inhibitors. Stadler et al. (2008) have also reported the applicability of

microbial extracellular polymeric substances (EPS) for corrosion protection of metal

substrates. For which, the authors cultivated various strains of bacteria including

sulphate-reducing bacteria (SRB) and Pseudomonas in order to harvest their EPS. From

the results, it was observed that Desulfovibrio alaskensis-EPS are promising substances

for corrosion inhibition by the biopolymers.

2.12.8 Synthesis of nanoparticles:

Xie et al. (2006) studied the possibility of synthesizing silver nanoparticles in

water-in-oil microemulsion stabilized by rhamnolipid. Thereby, providing a new example

for synthesizing inorganic nanoparticles by the use of biosurfactants as “green” template.

2.12.9 Other applications:

Some other potential commercial applications of biosurfactants are in the pulp

and paper industry (Rosenberg et al. 1989), textile, ceramics (Horowitz and Currie 1990)

and uranium ore processing (McInerney et al. 1990). Biodispersan from A. calcoaceticus

A2 has a potential use in paint industries (Rosenberg and Ron 1998).

2.13 Biosurfactants and bioremediation:

Bioremediation of soil contaminated with organic chemicals is a viable method

for clean-up and remediation of sites polluted with hazardous wastes. The final objective

in this approach is to convert the toxic pollutant to a readily biodegradable product,

which is harmless to human health and/or the environment (Mulligan 2005, 2009). The

dispersion or solubilization of water-insoluble pollutants is an important step in

bioremediation. Surfactants are required to remove organic compounds from soil and to

increase their bioavailability. The chemical surfactants mostly have higher CMC (critical

micelle concentration) values usually more than 600 mg l-1 (Chritsofi and Ivshina 2002).

Thus, they are required at higher concentrations to get the desired results. The chemical

surfactants, usually of petrochemical origin, are toxic to microbial flora at the

concentrations used and are themselves a source of pollution (Deschenes et al. 1996).

Thus, biosurfactants are ideally suited for environmental applications as they offer the

39


advantages of no environmental impact and the possibility of in situ production (Calvo et

al. 2004).

2.13.1 Hydrocarbons, polynuclear aromatic hydrocarbons (PAHs) and oils:

There are reports in literature regarding the use of biosurfactants for treating

hydrocarbon-contaminated soils (Banat 1995; Bartha 1986; Calvo et al. 2008; Herman et

al. 1997a, b). Partially purified biosurfactants can either be used in bioreactors or in situ

to emulsify and increase the solubility of hydrophobic contaminants. Ivshina et al. (1998,

2001) have reported that for bioremediation applications there is no need to purify the

biosurfactants, as crude biosurfactants achieved the desired results. Jain et al. (1992)

found that the addition of biosurfactant produced by Pseudomonas spp. enhanced the

biodegradation of tetradecane, pristane, and hexadecane. Similarly, Zhang and Miller

(1995) reported the enhanced octadecane dispersion and biodegradation in presence of

rhamnolipid surfactant produced by Pseudomonas sp. Maier and Soberon-Chavez (2000)

reported that rhamnolipid addition can enhance biodegradation of hexadecane,

octadecane, n-paraffin, and phenanthrene in liquid systems and that of tetradecane and

hydrocarbon mixtures in soils. Hua et al. (2003) studied the influence of biosurfactant

BS-UC produced by Candida antarctica on surface properties of microbial cells and

biodegradation of petroleum hydrocarbons. It was found that the addition of BS-UC

positively influenced the emulsification and the biodegradation of a variety of n-alkanes.

Further, the biosurfactant also changed the hydrophobicity of the cell surface, thereby

making BS-UC a promising choice for use in bioremediation of petroleum contaminated

sites. Bodour et al. (2004) reported that the flavolipid mixture produced by

Flavobacterium sp. strain MTN 11 was a strong and stable emulsifier even at

concentrations as low as 19 mg l-1. The authors found that the biosurfactant was an

effective solubilizing agent, and enhanced mineralization of hexadecane by two isolates

Flavobacterium sp. strain MTN 11 and P. aeruginosa ATCC 9027 by 100-fold and 2.5-

fold, respectively over a period of 8-day incubation.

Polycyclic or polynuclear aromatic hydrocarbons (PAHs) are suspected to be

carcinogens. Zhang et al. (1997) tested the effect of two rhamnolipid biosurfactants on

the dissolution and bioavailability of phenanthrene and reported an increase in both

40


solubility and degradation rate of phenanthrene. Barkay et al. (1999) used the

bioemulsifier alasan, produced by Acinetobacter radioresistens KA 53 to enhance PAH

solubility, and the degradation results showed 6.6-, 25.7- and 19.8-fold increase in the

solubilities of phenanthrene, fluoranthene and pyrene, respectively. Similar results on

PAH solubilization have been obtained with rhamnolipids produced by P. aeruginosa and

other pseudomonads (Deschenes et al. 1996; Mulligan et al. 2001; Noordman et al.

1998). Page et al. (1999) found that a biosurfactant from Rhodococcus strain H13-A was

up to 35-fold more effective than the synthetic Tween 80 in increasing the mass transfer

of PAHs into the aqueous phase. Straube et al. (2003) also evaluated the addition of P.

aeruginosa strain 64, to enhance bioremediation of PAHs and pentachlorophenol (PCP).

Bordas et al. (2005) investigated the efficacy of a rhamnolipid biosurfactants to enhance

the removal of pyrene from artificially contaminated soils. From the results, it was

observed that in the presence of biosurfactant concentrations of 2.5-5.0 g l-1, about 70%

of pyrene was recovered.

Besides the studies on biodegradation, rhamnolipid surfactants have been tested to

enhance the release of low solubility compounds from soil and other solids. In 1987, the

only commercial industrial biosurfactant in the market was emulsan, marketed by

Petroleum Fermentations (Petroferm, USA), for use in cleaning oil-contaminated vessels,

oil spills and MEOR (microbial enhanced oil recovery). The biosurfactant released three

times as much oil, as water alone from the beaches in Alaska after the Exxon Valdez

tanker spill (Harvey et al. 1990). Various biological surfactants were compared by Urum

et al. (2003) for their ability to wash a crude-oil contaminated soil. Youssef et al. (2007)

reported in situ production of biosurfactants by Bacillus strains injected into a limestone

petroleum reservoir. The authors showed that biosurfactant-mediated oil recovery is

technically feasible and will facilitate the use of computer simulations to determine the

efficacy of MEOR in different reservoirs. The potential of biosurfactants produced by B.

subtilis PT2 and P. aeruginosa SP4 in oil recovery has also been evaluated by

Pornsunthorntawee et al. (2008a). From the results, it was observed that the biosurfactant

produced by B. subtilis PT2 exhibited high oil recovery efficiency amongst the two.

41


Moreover, the authors further observed that the biosurfactants were more effective than

the chemical surfactants in the oil recovery.

Cubitto et al. (2004) assayed the effects of B. subtilis O9 biosurfactant in the

bioremediation of crude oil-polluted soils. The results indicated that 19 and 19.5 mg of

biosurfactant kg-1 of soil stimulated the growth of population involved in the crude oil

degradation and accelerated the biodegradation of the aliphatic hydrocarbons. Kumar et

al. (2007) have described the ability of Bacillus sp. strain DHT isolated from oil-

contaminated soil to degrade polyaromatic hydrocarbons and produce biosurfactant over

a wide range of temperature (30-45°C) and salt concentrations (0-10% w/v). According

to Cameotra and Singh (2008), a microbial consortium consisting of two isolates of P.

aeruginosa and Rhodococcus sp. isolated from soil contaminated with oily sludge was

able to degrade more than 98% of hydrocarbon in a medium supplemented with a

formulated nutrient mixture and a crude rhamnolipid preparation. Nikolopoulou and

Kalogerakis (2008) examined the effectiveness of lipophilic fertilizers in combination

with biosurfactants (rhamnolipids) and molasses to enhance the biodegradation of crude

oil by naturally occurring microorganisms. From the results, it was found that the use of

biosurfactants resulted in an increased removal of petroleum hydrocarbons.

2.13.2 Heavy metals:

Heavy metals (arsenic, lead, mercury, cadmium and chromium) are amongst the

most prevalent class of contaminants. Metal wastes are produced by a variety of sources

including mines, tanneries and electroplating facilities, and through the manufacture of

paints, metal pipes, batteries and munitions. A study by Tan et al. (1994) on the

formation of biosurfactant (mono-rhamnolipid produced by P. aeruginosa ATCC 9027)

and metal complexes showed that rapid and stable surfactant metal combinations were

produced. Due to anionic nature of rhamnolipids, they are able to remove metals such as

cadmium, copper, lanthanum, lead and zinc from soils, due to their complexation ability

(Herman et al. 1995; Tan et al. 1994).

Mulligan et al. (1999a, b) have used surfactin from B. subtilis to treat soil and

sediments contaminated with Zn, Cu, Cd, oil and grease. It was suggested that metal

42


desorption involved the attachment of surfactin at the solid interface and metal removal

through lowering of the interfacial tension and micellar complexation.

Ochoa-Loza et al. (2001) showed preferential complexation of metals, such as

cadmium, lead and mercury by mono-rhamnolipid RL-1 produced by P. aeruginosa.

Neilson et al. (2003) also studied lead removal by rhamnolipids. Rhamnolipids have also

been added to another metal contaminated media and mining ores, to enhance metal

extraction. Flavolipids, produced by Flavobacterium sp. strain MTN 11 were also

reported to form a complex with cadmium (Bodour et al. 2004). Dahrazma and Mulligan

(2007) evaluated the performance of rhamnolipids in a continuous flow configuration for

the removal of heavy metals (Cu, Zn, and Ni) from sediments. The authors observed that

with the addition of 1% NaOH to 0.5% rhamnolipid, the removal of copper was improved

to four times as compared to 0.5% rhamnolipid alone. Saini et al. (2008) have also

reported that viscosin, a cyclic lipopeptide produced by Pseudomonas libanensis M9-3

was able to form complexes with cadmium.

2.13.3 Biosurfactants and pesticides:

Biosurfactants capable of emulsifying pesticides have great potential to assist in

microbial degradation of the pesticides. Furthermore, the biodegradative property of

biosurfactants makes them ideal choice for environmental applications as compared to

chemical surfactants, especially pesticide removal. There are postulations on the possible

replacement of synthetic surfactants with biosurfactants in pesticide formulations and

residue clean-up (Banerjee et al. 1983; Patel and Gopinathan 1986).

Appaiah and Karanth (1991) reported that the resting cells of P. tralucida (Ptm+

strain) secreted an agent which could emulsify the insecticide hexachlorocyclohexane

(HCH). Fiebig et al. (1997) have shown that glycolipids (GL-K12) from P. cepacia

enhanced the degradation of Arochlor 1242 by mixed cultures, with almost 100%

degradation. In 2000, Veenanadig et al. reported that the biosurfactant produced by B.

subtitis was able to emulsify Fenthion, an organophosphorus pesticide and a pollutant, to

aqueous phase. Thus, the biosurfactant could be used in the cleaning operations of

containers containing Fenthion and other similar operations, where presence of Fenthion

is not desirable. Mata-Sandoval et al. (2000) compared the ability of the rhamnolipid

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mixture to solubilize the pesticides: Trifluralin, Coumaphos and Atrazine, with that of

synthetic surfactant Triton X-100. The synthetic surfactant was able to solubilize

approximately twice as much of all pesticides as the rhamnolipid. Further work by the

authors (Mata-Sandoval et al. 2001) was performed on the biodegradation of the three

pesticides in liquid cultures in the presence of rhamnolipid or Triton X-100. It was

observed from the results that Trifluralin biodegradation was enhanced in the presence of

both surfactants, while that of Atrazine decreased. Coumaphos biodegradation increased

in the presence of rhamnolipids. As the concentration of rhamnolipid increased,

biodegradation rates of Coumaphos decreased but removal was increased. During the

course of the experiment, it was observed that the concentration of rhamnolipid also

decreased indicating biodegradation of the rhamnolipid.

2.13.3.1 Hexachlorocyclohexane (HCH):

The organochlorine pesticide, γ-hexachlorocyclohexane (γ-HCH, also known as

lindane) is a persistent and recalcitrant insecticide (Phillips et al. 2005). Although its use

is restricted or completely banned, it continues to pose serious environmental and health

concerns, particularly on highly contaminated former production or dumping sites.

Owing to its toxicity, lipophilic nature and persistence in the environment, HCH has been

listed as one of the persistent organic pollutants (POPs).

The technical-grade HCH primarily consist of five isomers including: α- (60-

70%), β- (5-12%), γ- (10-15%), δ- (6-10%) and ε– (3-4%) (Kutz et al. 1991). Out of

these γ-HCH, commonly known as lindane has a significantly higher insecticidal

potential. The isomers of HCH have been reported to have toxic, carcinogenic, have a

tendency to bioaccumulate (especially β-HCH) and endocrine disrupters leading to severe

damage to reproductive and nervous systems in mammals (Singh et al. 2008). Also, there

are reports that γ-HCH has a potential to isomerize into other isomers that exhibit greater

persistence and toxicity, especially α-HCH, which possess the most carcinogenic activity

(Walker et al. 1999). The production of lindane results into the formation of

approximately six times of the other isomers, known as HCH-muck. The unsound

disposal practices of HCH-muck by the manufactures have led to the serious

contamination of the environment including air, water and soil. According to one of the

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reports, various commercial brands of drinking water samples available in Indian market

contained almost 99-141 fold higher levels of HCH-isomers than the maximal

permissible limits for drinking water i.e. 0.1 µg l-1 (Parkash et al. 2004). Thus, there is an

urgent need to develop suitable biological protocols for treatment of HCH residues to

prevent their build up in the environment.

HCH-isomers can be biologically degraded under aerobic and anaerobic

conditions. These differ not only in the spatial orientation of the chlorine atoms bound to

the aliphatic carbon ring, but also in toxicity, water solubility (and thus mobility and

bioavailability) and recalcitrance. The α- and γ-isomers are usually more rapidly

degraded than the β- and δ-isomers (Deo and Karanth 1994). Various studies reveal that

research has mainly focused on the degradation of α- and γ-HCH-isomers rather than β-

and δ-isomeric forms (Bhuyan et al. 1993; Manonmani et al. 2000). There are several

HCH-degrading strains that are reported in the literature and they mostly belong to the

genus Pseudomonas and Sphingomonas (Imai et al. 1989; Mohn et al. 2006). Some

Gram-positive HCH-degrading bacteria like Bacillus circulans and B. brevis have also

been reported that degrade all the HCH-isomers including β-HCH (Gupta et al. 2000). In

order to develop a successful HCH bioremediation field-scale protocols, more work is

required in order to understand the interaction of the HCH-degrading microorganisms

with the soil environment.

The isomers of HCH have low aqueous phase solubility, ranging from 5-10 mg l-1

(Phillips et al. 2005). In order to improve biodegradation of different HCH-isomers, there

is a need to ensure their bioavailability to efficient degraders during bioremediation

applications. There are several reports in the literature regarding the beneficial effects of

the use of surfactants/biosurfactants for bioremediation of hydrophobic organic

compounds (HOCs)-polluted soil, facilitating desorption and rendering them more

accessible to microorganisms (Mulder et al. 1998; Mulligan 2009; Singh et al. 2007;

Volkering et al. 1995). Quintero et al. (2005) evaluated the use of three surfactants viz.

Triton X100, Tween 80 and sodium dodecyl sulphate (SDS) on the soil desorption of

HCH-isomers and their anaerobic biodegradation. The authors observed that Triton X100

showed the maximum desorption of HCH-isomers; nevertheless, a remarkable inhibition

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in the HCH biodegradation was observed. In the case of Tween 80, not only a high

desorption of the isomers was observed but also there was an increase in the

biodegradation rate of β- and δ-HCH.

As there are hardly any reports regarding the effect of biosurfactants on

bioavailability and biodegradation of HCH-isomers. Thus, in light of this in the present

work, the potential of the biosurfactants to partition different isomers of HCH to aqueous

phase will be evaluated. The data obtained from this study might be helpful in designing

suitable bioremediation strategies for huge stock piles of HCH-muck and sites polluted

by reckless use/disposal of HCH-isomers.

2.14 Perspectives for biosurfactants

Considering the social and technological backgrounds, utilization of

biosurfactants, which are environment friendly and highly functional, have become more

and more important. Currently, the main factor that works against the widespread use of

biosurfactants is the economics of their production. The search for new alternative low-

cost substrates, together with the advantages of low toxicity to the environment and

excellent surface-active properties can contribute to the widespread use of these

molecules, especially in situations where the application benefits overcome the

production costs. It is predicted that by the year of 2010, biosurfactants could capture

10% of the surfactant market, reaching US$ 200 million in sales.

Although the use of cheap substrates and cost-effective recovery procedures

reduce the costs of production and recovery to some extent, the real breakthrough in

production costs can only be obtained by incorporating the hyper-producers, where they

can potentially increase the yield manifolds. Thus, future research aiming for high-level

production of biosurfactants must be focused towards the development of novel

recombinant hyper-producing strains. In the near future, we can expect the development

of many other non-pathogenic, safe, potent and high-yielding mutant and recombinant

varieties. Incorporation of these hyper-producing strains will boost the industrial

biosurfactant production process and make it possible to commercialize biosurfactants by

making the production process cheaper and safe.

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