1

CHAPTER- 1

INTRODUCTION

This chapter deals with general description of biosurfactants. The definition of

biosurfactants and the microbes involved in production, physiology and genetic

regulation of biosurfactant production, advantages as well as disadvantages of use of

biosurfactants and application of biosurfactants in different industries are discussed in

detail. International, national and regional scenarios of biosurfactant production and

utilization are also presented in the chapter. The issues addressed in the chapter are

discussed with reviewed literatures. Finally, the background of the problem studied in

the present investigation is described briefly in the chapter followed by the objectives

carried out to study the problem.

1.1. Biosurfactants: Definition, composition and characteristics

Biosurfactants are diverse groups of surface active molecules/chemical compounds

synthesized by microorganisms (Desai and Banat 1997). These amphiphilic

compounds are produced on living surfaces, mostly on microbial cell surfaces, or

excreted extracellularly. These are amphipathic molecules having both hydrophilic and

hydrophobic domains that confer the ability to accumulate between fluid phases, thus

reducing surface and interfacial tensions at the surface and interface respectively

(Karanth et al. 1999). Most biosurfactants are either anionic or neutral and the

hydrophilic moiety can be a carbohydrate, an amino acid, a phosphate group, or some

other compounds. The hydrophobic moiety is mostly a long carbon chain fatty acid.

These molecules reduce surface and interfacial tensions in both aqueous solutions and

hydrocarbon mixtures. This property of biosurfactant makes them potential candidates

for enhancing oil recovery (Sarkar et al. 1989). Because of surface active property of

biosurfactants, micro emulsions are created in which micelle formations occur where

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hydrocarbons can solubilize in water or water in hydrocarbons (Banat 1995).

Biosurfactants enhance the emulsification of hydrocarbons, have the potential to

solubilize hydrocarbon contaminants and increase their availability for microbial

degradation. The use of chemicals for the treatment of a hydrocarbon polluted site may

contaminate the environment with their by-products, whereas biological treatment may

efficiently destroy pollutants, while being biodegradable themselves.

1.2. Producers

Quite a lot of microorganisms have been reported to produce several classes of

biosurfactants such as glycolipids, lipopeptides, phospholipids, neutral lipids or fatty

acids and polymeric biosurfactants (Cooper and Zajic 1980; Cooper 1986; Kosaric

1993). These compounds are produced during the growth of microorganisms on water

soluble and water insoluble substrates (Sheppard and Mulligan 1987; Desai et al. 1988;

Ron and Rosenberg 2001). Microorganisms utilize a variety of organic compounds as

the source of carbon and energy for their growth. When the carbon source is an

insoluble substrate like a hydrocarbon (CnHn), microorganisms facilitate their diffusion

into the cell by producing a variety of biosurfactants. Some bacteria and yeasts excrete

ionic surfactants which emulsify the CnHn substrates in the growth medium. Some

examples of this group of biosurfactants are rhamnolipids which are produced by

different Pseudomonas sp. (Burger et al. 1963; Guerra-Santos et al. 1984; Guerra-Santos

et al. 1986), or the sophorolipids which are produced by several Torulopsis sp. (Cooper

and Paddock 1983). Some other microorganisms are capable of changing the structure

of their cell wall, which they achieve by synthesizing lipopolysaccharides or nonionic

surfactants in their cell wall. Examples of this group are: Candida lipolytica and

Candida tropicalis which produce cell wall-bound lipopolysaccharides when growing

on n-alkanes (Osumi et al. 1975) and Rhodococcus erythropolis, many Mycobacterium

sp. and Arthrobacter sp. which synthesize nonionic trehalose corynomycolates

(Kretschmer et al. 1982; Ristau and Wagner 1983). There are lipopolysaccharides, such

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as emulsan, synthesized by Acinetobacter sp. (Rubinowitz et al. 1982) and lipoproteins

or lipopeptides, such as surfactin and subtilisin, produced by Bacillus subtilis (Cooper et

al. 1981). Other effective biosurfactants are mycolates and corynomycolates which are

produced by Rhodococcus sp., Corynebacteria sp., Mycobacteria sp. and Nocardia sp.

(MacDonald et al. 1981; Kretshmer et al. 1982) and ornithinlipids, which are produced

by Pseudomonas rubescens, Gluconobacter cerinus, and Thiobacillus ferroxidans

(Knoche and Shively 1972; Tahara et al. 1976).

Till now, the most commonly isolated and the best studied groups of biosurfactants

are mainly glycolipids and phospholipids in nature. Rhamnolipids are glycolipid

compounds produced mainly by Pseudomonas sp. which could reduce water surface

tension and emulsify oil (Babu et al. 1996; Deziel et al. 1999; Patel and Desai 1997;

Rahman et al. 2002). These compounds are environmental friendly since they are

biodegradable and have potential industrial and environmental applications.

1.3. Classification and chemical nature of biosurfactants

Chemically synthesized surfactants are usually classified according to the nature of their

polar groups but biosurfactants are generally categorized mainly by their chemical

composition dictated by the different molecules forming the hydrophobic and

hydrophilic moieties and microbial origin. The hydrophilic moiety may consist of amino

acids, peptides, mono-, di- or polysaccharides. The hydrophobic moiety may consist of

saturated or unsaturated fatty acids (Desai and Banat 1997). Rosenberg et al. (1999)

suggested that biosurfactants can be divided into low-molecular-mass molecules, which

efficiently lower surface and interfacial tension, and high molecular-mass polymers,

which are more effective as emulsion stabilizing agents. The major classes of low mass

surfactants include glycolipids, lipopeptides and phospholipids, whereas high mass

surfactants include polymeric and particulate surfactants like polyanionic het-

eropolysaccharides containing both polysaccharides and proteins. The yield of microbial

surfactants varies with the nutritional environment of the growing microor

most important groups of biosurfactants and some of their classes are described below.

1.3.1. Glycolipids

Glycolipds are the most known biosurfactants. They are conjugates of ca

and fatty acids. The linkage is by means of either ether or an ester group. Among the

glycolipids, the best known are rhamnolipids, trehalolipids and sophorolipids

(Muthusamy et al. 2008).

Rhamnolipids:

Rhamnolipids are the best studied

rhamnose are linked to one or two molecules of

group of one of the acids is involved in glycosidic linkage with the reducing end of the

rhamnose disaccharide, the -OH group

(Karanth et al. 1999). Jarvis and Johnson (1949) first described production of rhamnose

containing glycolipids in Pseudomonas aeruginosa

activity, the physicochemical pro

(Abalos et al. 2001; Chen 2004;

al. 2008; Abdel-Mawgoud et al. 2009; Pornsunthorntawee et al. 2009). L

rhamnosyl-β- hydroxydecanoyl

hydroxydecanoyl-β-hydroxy-

respectively. They are the principal glycolipids produced by

(Edward and Hayashi 1965).

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nts varies with the nutritional environment of the growing microorganism. The

most important groups of biosurfactants and some of their classes are described below.

lycolipds are the most known biosurfactants. They are conjugates of carbohydrates

and fatty acids. The linkage is by means of either ether or an ester group. Among the

glycolipids, the best known are rhamnolipids, trehalolipids and sophorolipids

Rhamnolipids are the best studied glycolipids in which one or two molecules of

rhamnose are linked to one or two molecules of β-hydroxydecanoic acid. While the

group of one of the acids is involved in glycosidic linkage with the reducing end of the

OH group of the second acid is involved in ester formation

(Karanth et al. 1999). Jarvis and Johnson (1949) first described production of rhamnose

Pseudomonas aeruginosa. Because of their excellent surface

activity, the physicochemical properties of RLs have received considerable interest

Chen 2004; Cohen et al. 2004; Cohen and Exerowa 2007; Hansen et

Mawgoud et al. 2009; Pornsunthorntawee et al. 2009). L-Rhamnosyl

hydroxydecanoyl–β-hydroxydecanoate and L-rhamnosyl

decanoate, referred to as di- and mono-rhamnolipids

respectively. They are the principal glycolipids produced by Pseudomonas aeruginosa

ganism. The

most important groups of biosurfactants and some of their classes are described below.

rbohydrates

and fatty acids. The linkage is by means of either ether or an ester group. Among the

glycolipids, the best known are rhamnolipids, trehalolipids and sophorolipids

one or two molecules of

hydroxydecanoic acid. While the -OH

group of one of the acids is involved in glycosidic linkage with the reducing end of the

of the second acid is involved in ester formation

(Karanth et al. 1999). Jarvis and Johnson (1949) first described production of rhamnose

. Because of their excellent surface

perties of RLs have received considerable interest

Cohen et al. 2004; Cohen and Exerowa 2007; Hansen et

Rhamnosyl-L-

rhamnosyl-β-

rhamnolipids

Pseudomonas aeruginosa

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Trehalolipids:

Several structural types of microbial trehalolipid biosurfactants have been reported.

These trehalose lipids are mainly produced by rhodococci and present interesting

physicochemical and biological properties (Lang et al. 1998). Disaccharide trehalose

linked at C-6 and C-6′ to mycolic acid is associated with most species of

Mycobacterium, Nocardia and Corynebacterium. Mycolic acids are long chain, α-

branched-β-hydroxy fatty acids. Trehalolipids from different organisms differ in the size

and structure of mycolic acid, the number of carbon atoms and the degree of

unsaturation (Asselineau and Asselineau 1978). A number of possible applications have

been proposed for these compounds. In addition, succinoyl trehalose lipids have been

found to induce differentiation of leukemia cell lines (Sudo et al. 2000) and to inhibit

protein kinase activity (Isoda et al. 1997).

Sophorolipids:

These glycolipids are mainly produced by yeast such as Torulopsis bombicola (Cooper

and Paddock 1984; Hommel et al. 1987), Torulopsis petrophilum and Torulopsis

apicola. They consist of a dimeric carbohydrate sophorose linked to a long-chain

hydroxyl fatty acid by glycosidic linkage. Generally, sophorolipids occur as a mixture of

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macrolactones and free acid form. It has been shown that the lactone form of the

sophorolipid is necessary, or at least preferable, for many applications (Hu and Ju 2001).

1.3.2. Lipopeptides and lipoproteins

A large number of cyclic lipopetides, including decapeptide antibiotics (gramicidins)

and lipopeptide antibiotics (polymyxins) are produced. These consist of a lipid attached

to a polypeptide chain. Two of them are described below-

Surfactin:

Surfactin is an important biosurfactant with superior surface activity and belongs to a

group of cyclic lipoheptapeptides containing beta-hydroxyl fatty acids and D−/L- amino

acid residues (Tang et al. 2007; Haddad et al. 2008). The cyclic lipopeptide surfactin is

produced by Bacillus sp. It is composed of a seven amino-acid ring structure coupled to

a fatty-acid chain via lactone linkage (Arima et al. 1968).

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Lichenysin:

Bacillus licheniformis produces several biosurfacants which act synergistically and

exhibit excellent temperature, pH and salt stability. These are also similar in structural

and physio-chemical properties to the surfactin (McInerney et al. 1990). The surfactants

produced by Bacillus licheniformis are capable of lowering the surface tension of water

to 27mN/m and the interfacial tension between water and n-hexadecane to 0.36 mN/m.

1.3.3. Fatty acids, phospholipids, and neutral lipids

Several bacteria and yeast produce large quantities of fatty acids and phospholipid

surfactants during growth on n-alkanes (Cirigliano and Carman 1985). The hydrophilic

and lipophilic balance (HLB) is directly related to the length of the hydrocarbon chain in

their structures. 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. Phosphatidylethanolamine produced by Rhodococcus

erythropolis grown on n-alkane causes a lowering of interfacial tension between water

and hexadecane to less than 1mN/m and a critical micelle concentration (CMC) of 30

mg/l (Kretschmer et al. 1982).

Corynomycolic acid:

Corynomycolic acids, (R1-CH (OH)-CH (R2)-COOH) are a group of surface active

compounds with varying number of carbon atoms. Substrate in the growth media

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influencs a lot in synthesizing biosurfactans with varying chain length. A mixture of

corynomycolic acids with excellent surfactant properties has been isolated from

Corynebacterium lepus. It caused significant lowering of surface tension in aqueous

solution and the interfacial tension between water and hexadecane at all values of pH

between 2 and 10 (Cooper et al. 1981).

1.3.4. Polymeric biosurfactants

Polymeric biosurfactants are high molecular weight biosurfactants. Most polymeric

biosurfactants has a backbone of three or four repeating sugars with fatty acids attached

to the sugars (Rosenberg and Ron 1997). The best studied polymeric biosurfactants are

emulsan, liposan, alasan, lipomanan and other polysaccharide-protein complexes.

Liposan is an extracellular water soluble emulsifier synthesized by Candida lipolytica

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

Emulsan:

Emulsan is a complex extracellular acylated polysacharide synthesized by the gram-

negative bacterium Acinetobacter calcoaceticus with an average molecular weight of

about 1000 KD (Kim et al. 1997) and has been extensively researched for its industrial

applications as an emulsifier (Gorkovenko et al. 1999). This molecule is composed of an

unbranched polysaccharide backbone with O-acyl and N-acyl bound fatty acid side

chains. The polysaccharide backbone consists of three aminosugers, D-galactosamine,

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D-galactosaminouronic acid and a dideoxydiaminohexose in the ratio of 1:1:1 (Panilaitis

et al. 2002). The fatty acid side chains range in length from 10 to 22 carbon atoms with

the amino groups either acetylated or covalently linked by an amide group bound to 3-

hydroxybutyric acid and can represent from 5 to 23% (w /w) of the polymer. It is is an

effective emulsifying agent for hydrocarbons in water (Zosim et al. 1982), even at a

concentration as low as 0.001 to 0.01%.

1.3.5. Particulate biosurfactants

Particulate biosurfactants are of two types, extracellular vesicles and whole microbial

cell. Extracellular membrane vesicles partition hydrocarbons to form micro-emulsions,

which play an important role in hydrocarbon uptake by microbial cells. Sometimes the

whole bacterial cell itself can work as surfactant.

Vesicles:

Acinetobacter sp. when grown on hexadecane accumulated extracellular vesicles of 20

to 50 mm diameter with a buoyant density of 1.158 g/cm3. These vesicles appear to play

10

a role in the uptake of alkanes by Acinetobacter sp. HO1-N. These vesicles with a

diameter of 20-50 nm and a buoyant density of 1.158 cubic g/cm are composed of

protein, phospholipids and lipopolysaccharide (Kappeli and Finnerty 1979). Like

Acinetobacter sp., Pseudomonas marginalis also form vesicles to work as surfactants.

Whole microbial cells:

Most hydrocarbon-degrading microorganisms, many nonhydrocarbon degraders, some

species of Cyanobacteria, and some pathogens have a strong affinity for hydrocarbon-

water and air-water interfaces. In such cases, the microbial cell itself is a surfactant

(Karanth et al. 1999).

Table 1.1. Major classes of biosurfactant, microorganisms involved in production

and economic importance

Biosurfactant

Microorganism

Economic importance References

Group Class

Glycolipids

Rhamnolipids

Pseudomonas

aeruginosa,

Pseudomonas sp., Burkholderia

glumae,

Burkholderia

plantarii,

Burkholderia

thailandensis

antimicrobial activity against Mycobacterium tuberculosis, anti-adhesive activity against

several bacterial and yeast strains isolated from voice prostheses, enhancement of

the degradation and dispersion of different classes of

hydrocarbons; emulsification of hydrocarbons and

vegetable oils; removal of metals from soil

Herman et al. 1995;

Maier et al. 2000; Sifour et al. 2007;

Whang et al. 2008;

Dubeau et al. 2009;

Hörmann et al. 2010

Trehalose lipids

Rhodococcus

erythropolis,

Nocardia

erythropolis,

Mycobacterium

sp., Arthobacter

sp.

Enhancement of the bioavailability of

hydrocarbons, antiviral activity against HSV and

influenza virus

Franzetti et al. 2010

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Sophorolipids Torulopsis

bombicola,

Torulopsis

apicola,

Torulopsis

petrophilum

Recovery of hydrocarbons from dregs and muds;

removal of heavy metals from sediments; enhancement of oil

recovery,

Whang et al. 2008

Mannosylerythritol lipid

Cellobiolipids

Candida antartica

antimicrobial, immunological and neurological properties

Wakamatsu et al. 2001

Ustilago zeae,

Ustilago maydis

_ Desai and

Banat 1997

Lipopeptides

and

lipoproteins

Surfactin/ iturin/fengycin

Bacillus subtilis,

Bacillus

licheniformis

Enhancement of the biodegradation of

hydrocarbons and chlorinated pesticides; removal of heavy metals from a contaminated soil, sediment and water;

increasing the effectiveness of phytoextraction, antimicrobial

and antifungal activities inhibition of fibrin clot

formation haemolysis and formation of ion channels in lipid membranes antitumour

activity against Ehrlich’s ascite carcinoma cells

antiviral activity against human immunodeficiency

virus 1 (HIV-1), antimicrobial activity and antifungal activity against

profound mycosis effect on the morphology and

membrane structure of yeast cells increase in the electrical conductance of biomolecular

lipid membranes non-toxic and non-pyrogenic

immunological adjuvant

Tanaka et al. 1997;

Awashti et al. 1999;

Ahimou et al. 2001

Viscosin

Pseudomonas

fluorescens

Antimicrobial activity Saini et al.

2008

Lichenysin

Bacillus

licheniformis

Enhancement of oil recovery, antibacterial activity chelating properties that might explain

the membrane-disrupting effect of lipopeptides

Sen 2008

Serrawettin Serratia

marcescens Chemorepellent

Pradelet al. 2007

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Subtilism Bacillus subtilis

Antimicrobial activity

Henry et al. 2011, Ghribi et al. 2012

Gramicidin

Brevibacterium

brevis

Antibiotic, disease control Elad and

Stewart 2007

Polymixin Bacillus polymyxa

Bactericidal and fungicidal

activity

www.cyberlipid.org/simple/simp0005.

htm

Antibiotic TA

Myxococcus

xanthus

Bactericidal activity, chemotherapeutic applications

Karanth et al. 1999;

Xiao et al. 2012

Fatty

acids/neutral

lipids/

phospholipids

Corynomycolic acid

Corynebacterium

lepus

Enhancement of bitumen recovery

Gerson et al.1978

Spiculisporic acid

Penicillium

spiculisporum

Removal of metal ions from aqueous solution; dispersion

action for hydrophilic pigments; preparation of new

emulsion-type organogels, superfine microcapsules

(vesicles or liposomes), heavy metal sequestrants

Hong et al. 1998;

Ishigami et al. 2000

Phosphati-dylethanolam

ine

Acinetobacter sp., Rhodococcus

erythropolis

Mycococcus sp.

Increasing the tolerance of bacteria to heavy metals

Appanna et al. 1995

Polymeric

surfactants

Emulsan

Acinetobacter

calcoaceticus

Stabilization of the hydrocarbon-in water

emulsions

Zosim et al. 1982

Alasan

Acinetobacter

radioresistens

Stabilization of the hydrocarbon-in water

emulsions

Toren et al. 2001

Biodispersan Acinetobacter

calcoaceticus A2

Dispersion of limestone in water

Rosenberg et al. 1988

polysaccharide protein complex

Acinebacter

calcoaceticus Bioemulsifier

Kaplan et al. 1987

Liposan Candida lipolytica

Stabilization of hydrocarbon-in-water emulsions

Cirigliano and Carman

1985; Cameron et

al. 1988

Mannoprotein Saccharomyces

cerevisiae

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Protein PA

Pseudomonas

aeruginosa

Bioemulsifier Karanth et al. 1999

Particulate

biosurfactants

Vesicles

Acinetobacter

calcoaceticus,

Pseudomonas

marginalis

Degradation and removal of hydrocarbons

Karanth et al. 1999;

Rosenberg et al. 1999

Whole

microbial cells

Cyanobacteria

1.4. Physiology of biosurfactant production

The common view attributes only one role for microbial surfactants, i.e., the growth of

microorganisms on hydrocarbons. Most publications in this field discuss biosurfactants

with respect to the growth of bacteria on water insoluble carbon sources. The models for

uptake of hydrocarbons consider the roles of dissolved molecules, contact of the cells

with large oil droplets, or contact with fine oil droplets (Hommel 1990). In addition to

the role of bacterial surfactants for growth on hydrocarbons as a carbon source, some

other functions are mentioned in two review articles.

Rosenberg (1986) suggested that the diversity of structures and functions is a

general property of microbial surfactants and clearly stated that ‘‘It is unlikely that they

all serve the same function.’’ He discussed adhesion of biosurfactants to hydrocarbons

as a special case, a function in the emulsification of water-insoluble compounds as

substrates, and a function in de-adhesion from interfaces. Furthermore, he mentioned a

role in gliding and cell-cell interaction. Haferburg et al. (1986) also made clear that the

exact physiological functions of most microbial surfactants remain unclear. They

discussed microbial surfactants mainly in terms of hydrocarbon assimilation and biocide

activity. The biocidal activity of microbial surfactants is closely related to the lipid

moiety of the molecules. However, they also suggested a possible role in gliding of

bacteria and in wetting of interfaces. In addition, biosurfactants have been shown to be

involved in cell adherence which imparts greater stability under hostile environmental

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conditions and virulence (Rosenberg and Rosenberg 1981; Rosenberg 1986), in cell

desorption to find new habitats for survival (Rosenberg and Rosenberg 1981), in

antagonistic effects toward other microbes in the environment (Lang et al. 1989; Uchida

et al. 1989a; Kitamoto et al. 1993) etc.

1.5. Biosynthesis and genetic regulation of biosurfactant production

Biosurfactants display a range of different amphiphilic structures. Biosurfactants are

made up of hydrophobic and hydrophilic moieties. For synthesis of these two moieties

two different synthetic pathways must be used: one leading to the hydrophobic and one

to the hydrophilic moiety. The hydrophobic fatty acid components- which may be a long

chain fatty acid, a hydroxyl fatty acid or alpha-alkyl-beta-hydroxy fatty acid are

synthesized by rather common pathway of lipid metabolism. The hydrophilic moieties

however exhibit a greater degree of structural complexity. This explains the wide

variety of biosynthetic pathways involved in their synthesis (Muller 2010).

Among all the biosurfactants reported till date, the molecular biosynthetic

regulation of rhamnolipid, a glycolipid type biosurfactant produced by Pseudomonas

aeruginosa and a lipopeptide biosurfactant called surfactin produced by Bacillus subtilis

were the first to be deciphered. Other biosurfactants whose molecular genetics have

been delineated in the recent years include arthrofactin from Pseudomonas sp., iturin

and lichenysin from Bacillus species, mannosylerythritol lipids (MEL) from Candida

and emulsan from Acinetobacter species (Das et al. 2008).

A putative rhamnolipid biosynthesis pathway is summarized in Fig. 1.1 (Ochsner et

al. 1996; Kanehisa and Goto 2000; Rahim et al. 2001; Soberón- Chávez et al. 2005;

Winsor et al. 2009; Müller 2011). The biosynthetic pathway can be divided into three

major steps; synthesis of the hydrophilic part, synthesis of the hydrophobic part and

synthesis of rhamnolipid from these two parts. The precursors, dTDP-L-rhamnose and

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activated 3-(3-hydroxyalkanoyloxy) alkanoate (HAA) respectively for hydrophilic and

hydrophobic parts are synthesized de novo (Burger et al. 1963). Altogether the

biosynthesis can be separated into three major parts. Finally, the rhamnolipid is

produced by the reaction of two special rhamnosyltransferases catalyzing the sequential

rhamnosyl transfer reactions from the precursors over mono- toward di-rhamnolipids.

In Pseudomonas aeruginosa, several genes have been found to be involved in

rhamnolipid biosynthesis. Ochsner et al. (1994a) discovered a 2-kb fragment capable

of restoring rhamnolipid biosynthesis while tested in a rhamnolipid deficient mutant

strain of Pseudomonas aeruginosa. The 2-kb fragment contains a single open reading

frame (rhlR) of 723 bp specifying a putative 28- kDa protein (RhlR). Disruption of the

Pseudomonas aeruginosa wild-type rhlR locus led to rhamnolipid-deficiency, thus

confirming directly that this gene is necessary for rhamnolipid biosynthesis. The rhlAB

genes encode a rhamnosyltransferase, RhlAB, which catalyzes the transfer of rhamnose

from TDP-rhamnose to β- hydroxydecanoyl-β-hydroxydecanoate (Ochsner et al.

1994b). The transcriptional activation of rhlAB appears to depend on a functional RhlR

regulatory protein. The sequence upstream of the rhlA promoter contains two inverted

repeats that define putative binding sites for the RhlR regulator. Another gene, rhlI,

which is also required for rhamnolipid synthesis, has been identified downstream of the

rhlABR gene culster. The rhlI gene production, RhlI, has been proved to be a bacterial

autoinducer (usually belongs to homoserine lactone family) synthase. The

Pseudomonas aeruginosa rhlA promoter is actived only when both the rhlR and rhlI

genes are present or when the rhlR gene alone is supplied together with synthetic

autoinducers (Ochsner and Reiser 1995). The RhlR-RhlI regulatory mechanism is

known as quorum sensing (QC). QC describes population density dependent cell to cell

communication in bacteria using diffusible signal molecules. These signal molecules

produced by bacterial cells, regulate various physiological processes important for

social behavior and pathogenesis like synthesis of rhamnolipid in Pseudomonas

aeruginosa (Dusane et al. 2010).

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Fig. 1.1. Putative rhamnolipid biosynthesis pathway according to Müller (2011), LPS,

lipopolysaccharides; PHA, polyhydroxyalkanoates; HAQ, 4-hydroxy-2- alkylquinolines; ACP, acyl

carrier protein; CoA, coenzyme A; dTDP, deoxythymidine 5’-diphosphate; NADPH/NADP+,

nicotinamide adenine dinucleotide phosphate; HAA, 3-(3- hydroxyalkanoyloxy)alkanoate; EC,

enzyme commission number; m, n = 4–8.

17

1.6. Environmental factors influence the systhesis of biosurfactant

Synthesis of biosurfactant like any other chemical reaction is influenced by a number of

environmental factors that either increase its productivity or inhibit it (Rahman and

Gakpe 2008). Literature shows that different environmental factors are required for

synthesis of biosurfactant by different microbial sp. Same conditions are not suitable for

all the microbes. For example, some bacteria synthesize maximum biosurfactant in n-

hexadecane whereas some others can not tolerate n-hexadecane. Environmental factors

such as pH, temperature, salinity, agitation and oxygen supply affect biosurfactant

production (Rahman et al. 2002; Hori et al. 2005; Raza et al. 2007). The type, quality

and quantity of biosurfactant produced are influenced by the nature of the carbon

substrate (Lang et al. 1984), the concentration of N, P, Mg, Fe, and Mn ions in the

medium, and the culture conditions (Kretschmer et al. 1982). However, it was reported

that biosurfactant production from Pseudomonas strains MEOR 171 and MEOR 172 are

not affected by temperature, pH, and Ca, Mg, concentration in the ranges found in many

oil reservoirs (Karanth et al. 1999). Interestingly, Sabra et al. (2002) recently proposed

that P. aeruginosa is producing rhamnolipids to reduce oxygen transfer rate as a means

to protect itself from oxidative stress, and it appears that this mechanism is activated by

iron deficiency (Kim et al. 2003). However, excellent rhamnolipid production is also

obtained in the absence of oxygen (Chayabutra et al. 2001). The nitrogen source can be

an important key to the regulation of biosurfactants synthesis. Arthobacter paraffineus

ATCC 19558 preferred ammonium to nitrate as inorganic nitrogen source for

biosurfactant production. A change in growth rate of the concerned microorganisms is

often sufficient to result in over production of biosurfactants (Kretschmer et al. 1982). A

change in growth rate of the concerned microorganisms is often sufficient to result in

over production of biosurfactant. Salt concentrations also affect biosurfactant production

depending on its effect on cellular activity. Production of biosurfactant by a few

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

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

(2007) reported that biosurfactant production is fully affected and influenced by the

18

nature of the carbon substrate. Diesel and crude oil were identified to be good sources of

carbon for biosurfactant production by many organisms (Hori et al. 2005). Torulopsis

petrophilum did not produce any glycolipids when grown on a single-phase medium

that contained water-soluble carbon source (Cooper et al. 1983). However, there have

been examples of the use of a water-soluble substrate for biosurfactant production by

microorganisms (Mata-sandoval et al. 2001).

1.7. Advantages of biosurfactants

When compared to synthetic surfactants, biosurfactants have several advantages

including high biodegradability, low toxicity, low irritancy and compatibility with

human skin (Banat et al. 2000; Cameotra and Makkar 2004).Therefore they are superior

to the synthetic ones. The most significant advantage of a microbial surfactant over

chemical surfactant is its ecological acceptance (Desai and Banat 1997; Karsa et al.

1999; Banat 2000). Some more advantages of biosurfactants over synthetic ones include

selectivity, specific activity at extreme temperatures, pH, salinity etc. Some of the

advantages of biosurfactants are discussed below:

1.7.1. Biodegradability

Biosurfactants are biodegradable in nature. Biodegradability is a very important issue

concerning environmental pollution. Being able to be broken down by natural processes

by bacteria, fungi or other simple organisms into more basic components, they do not

create much problem to the environment and particularly suited for environmental

applications such as bioremediation (Mulligan et al. 2005) and dispersion of oil spills.

19

1.7.2. Low toxicity

Biosurfactants do not cause serious damage/harm of the biotic ecosystem since their

toxicity level is low. Many chemical surfactants are toxic to the living beings making

them less useful for being used in different industries. Very little data are available in

the literature regarding the toxicity of microbial surfactants. They are generally

considered as low or non-toxic products and therefore, appropriate for pharmaceutical,

cosmetic and food uses. A report suggested that a synthetic anionic surfactant (Corexit)

displayed an LC50 (concentration lethal to 50% of test species) against Photobacterium

phosphoreum ten times lower than rhamnolipids. This demonstrated higher toxicity of

the chemically derived surfactant. When comparing the toxicity of six biosurfactants,

four synthetic surfactants and two commercial dispersants, it was found that most

biosurfactants degraded faster, except for a synthetic sucrose-stearate that showed

structure homology to glycolipids and was degraded more rapidly than the biogenic

glycolipids. It was also reported that biosurfactants showed higher EC50 (effective

concentration to decrease 50% of test population) values than synthetic dispersants

(Poremba et al. 1991). A biosurfactant from Pseudomonas aeruginosa was compared

with a synthetic surfactant (Marlon A-350) widely used in the industry, in terms of

toxicity and mutagenic properties. Both assays indicated higher toxicity and mutagenic

effect of the chemical-derived surfactant, whereas the biosurfactant was considered

slightly non-toxic and nonmutagenic (Flasz et al. 1998).

1.7.3. Biocompatability and digestibility

Biosurfactants are biocompatible in nature (Rosenberg et al. 1999) which means they

are well tolerated by living organisms. These when interact with living organisms do not

change bioactivity of the organisms. This property allows their application in cosmetics,

pharmaceuticals and as functional food additives.

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1.7.4. Availability of raw materials

Biosurfactants can be produced from cheap raw materials like rapeseed oil, potato

process effluents, oil refinery waste, cassava flour wastewater, curd whey and distillery

waste, sunflower oil etc. (Muthusamy et al. 2008) which are available in large

quantities. The carbon source may come from hydrocarbons, carbohydrates and/or

lipids, which may be used separately or in combination with each other.

1.7.5. Acceptable production economics

Depending on the application, biosurfactants can also be produced from industrial

wastes and by products. This is of particular interest for bulk production (e.g. for use in

petroleum related technologies) of biosurfactant which is economically acceptable. In

addition to that, a lot of biosurfactants

1.7.6. Use in environmental control

Biosurfactants can be efficiently used in handling industrial emulsions, control of oil

spills, biodegradation and detoxification of industrial effluents and in bioremediation of

contaminated soil.

1.7.7. Specificity

Biosurfactants, being complex organic molecules with specific functional groups, are

often specific in their action. This would be of particular interest in detoxification of

specific pollutants, de-emulsification of industrial emulsions, specific cosmetic,

pharmaceutical and food applications.

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1.8. Disadvantages of biosurfactants

Concerning disadvantages, one of the problems is related to large scale and cheap

production of biosurfactants. Large quantities are particularly needed in petroleum and

environmental applications, which, due to the bulk use, may be expensive. To overcome

this problem, processes should be coupled to utilization of waste substrates combating at

the same time their polluting effect, which balances the overall costs. Another problem

may be encountered in obtaining pure substances which is of particular importance in

pharmaceutical, food and cosmetic applications. Downstream processing is involved

with multiple consecutive steps. Therefore, high yields and biosurfactant concentrations

in bioreactors are essential for their facilitated recovery and purification.

1.9. Properties and Applications of biosurfactants

All surfactants are chemically synthesized. Nevertheless, in recent years, much attention

has been directed towards biosurfactants due to their broad range of functional

properties and diverse synthetic capabilities of microbes. Most important is their

environmental acceptability which allows biosurfactants for their use and possible

replacement of chemically synthesized surfactants in a great number of industrial

operations. Some of the potential applications of biosurfactants in pollution and

environmental control are microbial enhanced oil recovery, hydrocarbon degradation in

soil environment and hexachloro cyclohexane degradation, heavy metal removal from

contaminated soil and hydrocarbon in aquatic environment (Singh et al. 2007). They can

be explored for several food-processing applications also.

1.9.1. Anti-adhesive agents

A biofilm is a group of bacteria that have colonized as a surface. The biofilm not only

includes bacteria, but it also describes all the extracellular material produced at the

22

surface and any material trapped within the resulting matrix. They are potential sources

of contamination of food. Thus controlling the adherence of microorganisms to food-

contact surfaces is an essential step in providing safe and quality products to consumers.

Therefore the involvement of biosurfactants in microbial adhesion and detachment from

surfaces has become very important in quality control of food products. A surfactant

released by Streptococcus thermophilus has been used for fouling control of heat

exchanger plates in pasteurizers, as it retards the colonization of other thermophilic

strains of Streptococcus responsible for fouling. It is also reported that the

preconditioning of stainless steel surfaces with a biosurfactant obtained from

Pseudomonas fluorescens inhibits the adhesion of Listeria monocytogenes L028 strain.

The bioconditioning of surfaces through the use of microbial surfactants has been

suggested as a new strategy to reduce adhesion. Pre-treatment of silicone rubber with

Streptococcus thermophilus surfactant inhibited the adhesion of Candida albicans by

85% (Busscher et al. 1997). The biosurfactant from Lactobacillus fermentum was

reported to inhibit S. aureus infection and adherence to surgical implants (Gan et al.

2002). Surfactin decreased the amount of biofilm formation by Salmonella

typhimurium, Salmonella enterica, Escherichia coli and Proteus mirabilis in PVC plates

and vinyl urethral catheters (Mireles et al. 2001).

1.9.2. In food formulations

Apart from their obvious role as agents that decrease surface and interfacial tension,

surfactants can have several other functions in food. They are used in controlling

agglomeration of fat globules, stabilization of aerated systems, improving texture and

shelf life of starch containing products and improve consistency and texture of fat-based

products (Kachholz and Schlingmann 1987). The biosurfactants are also used in bakery

and ice cream formulations where they act by controlling consistency, retarding staling

and solubilizing flavour oils; they can be utilized as fat stabilizers and antispattering

agents during cooking of oil and fats. Improvement in dough stability, texture, volume

23

and conservation of bakery products is obtained by the addition of rhamnolipid

surfactants (Van Haesendonck and Vanzeveren 2004). The study also suggested the use

of rhamnolipids to improve the properties of butter cream and frozen confectionery

products. L-Rhamnose has considerable potential as a precursor for flavouring agents. It

is already used industrially as a precursor of high-quality flavour components like

furaneol.

1.9.3. Therapeutic and biomedical applications

1.9.3.1. Antimicrobial activity

Several biosurfactants have shown antimicrobial action against bacteria, fungi, algae

and viruses. Rhamnolipids produced by Pseudomonas aeruginosa (Itoh et al. 1971),

lipopeptides produced by Bacillus subtilis (Sandrin et al. 1990; Leenhouts et al. 1995)

and Bacillus licheniformis (Jenny et al. 1991; Fiechter 1992; Yakimov et al. 1995) and

mannosylerythritol lipids from Candida antarctica (Kitamoto et al. 1993) have all been

shown to have antimicrobial activities. There are reports that the lipopeptide iturin from

Bacillus subtilis showed potent antifungal activity (Besson et al. 1976). Inactivation of

enveloped virus such as herpes and retrovirus was observed with 80 mM of surfactin

(Vollenbroich et al. 1997). The succinoyl-trehalose lipid of Rhodococcus erythropolis

has been reported to inhibit HSV and influenza virus with a lethal dose of 10 to 30 g/ml

(Uchida et al. 1989a, b). Rhamnolipids inhibited the growth of harmful bloom algae

species, Heterosigma akashivo and Protocentrum dentatum at concentrations ranging

from 0.4 to 10.0 mg/l. Abalos et al. (2001) reported that rhamnolipid mixture obtained

from Pseudomonas aeruginosa AT10 showed inhibitory activity against the bacteria

Escherichia coli, Micrococcus luteus, Alcaligenes faecalis (32 mg/ml), Serratia

arcescens, Mycobacterium phlei (16 mg/ml) and Staphylococcus epidermidis (8 mg/ml)

and excellent antifungal properties against Aspergillus niger (16 mg/ml), Chaetonium

globosum, Enicillium crysogenum, Aureobasidium pullulans (32 mg/ml) and the

phytopathogenic Botrytis cinerea and Rhizoctonia solani (18 mg/ml). Thimon et al.

24

(1995) described another anti fungal biosurfactant, iturin, a lipopeptide produced by

Bacillus subtilis, which affects the morphology and membrane structure of yeast cells.

The reports on antibiotic effects (Neu et al. 1990) and inhibition of HIV virus growth in

white blood corpuscles have opened up new fields for their applications. Kosaric (1996)

describes possible applications as emulsifying aids for drug transport to the infection

site, for supplementing pulmonary surfactant and as adjuvants for vaccines. Respiration

failure in premature infants is caused by a deficiency in pulmonary surfactant (Tayler et

al. 1985). With the bacterial cloning of the gene for the protein molecule of the

surfactant, the fermentative production of this product for medical application is now

possible (Lang and Wullbrandt 1999). To our knowledge, commercial production of

biosurfactants for use as antimicrobial agents has not taken place yet. The involvement

of biosurfactants in microbial adhesion and desorption has also been reported. A dairy

Streptococcus thermophilus strain produced a biosurfactant which caused its own

desorption from glass, leaving a completely non adhesive coating (Busscher et al. 1990).

Pratt-Terpstra et al. (1989) reported a release of biosurfactant by an oral Streptococcus

mitis strain, which was responsible for a reduction in the adhesion of Streptococcus

mutans. Similarly Velraeds-Mar-tine et al. (1996) reported on the inhibition of adhesion

of pathogenic enteric bacteria by biosurfactant produced by a Lactobacillus strain and

later showed that the biosurfactant caused an important, dose related inhibition of the

initial deposition rate of Escherichia faecalis and other bacteria adherent on both

hydrophobic and hydrophilic substrata (Velraeds-Mar-tine et al. 1997). They also

speculated on other possible therapeutic agents through the development of anti-

adhesive biological coatings for catheter materials to delay the onset of bio film growth.

1.9.3.2. Anticancer activity

There are many reports describing anticancerous activity of biosurfactants. The

biological activities of seven microbial extracellular glycolipids, including

mannosylerythritol lipids (MEL)-A, mannosylerythritol lipids-B, polyol lipid,

25

rhamnolipid, sophorose lipid, succinoyl trehalose lipid (STL)-1 and succinoyl trehalose

lipid-3 have been investigated (Isoda et al. 1999). All these glycolipids, except

rhamnolipid, were found to induce cell differentiation instead of cell proliferation in the

human promyelocytic leukaemia cell line HL60. STL and MEL markedly increased

common differentiation characteristics in monocytes and granulocytes respectively.

Exposure of B16 cells to MEL resulted in the condensation of chromatin, DNA

fragmentation and sub-G1 arrest (the sequence of events of apoptosis). This is the first

evidence of growth arrest, apoptosis and differentiation of mouse malignant melanoma

cells which can be induced by glycolipids (Zhao et al. 1999). Reports suggested that the

sophorolipid produced by Wickerhamiella domercqiae have anticancer activity (Chen et

al. 2006). Bernheimer and Avigard (1970) demonstrated that surfactin has various

pharmacological applications such as inhibiting fibrin clot formation and hemolysis.

Sheppard et al. (1991) depicted formation of ion channels in lipid membranes by using

surfactin. It has also been reported as having an antitumor activity against Ehrlich's

ascite carcinoma cells (Kameda et al. 1974).

1.9.3.3. Immuno modulatory action

Biosurfactants show potential immuno modulatory actions. Sophorolipids are promising

modulators of the immune response. It has been previously demonstrated by Hagler et

al. (2006) that sophorolipids, (1) decreased sepsis related mortality at 36 h in vivo in a

rat model of septic peritonitis by modulation of nitric oxide, adhesion molecules and

cytokine production and (2) decreased IgE production in vitro in U266 cells possibly by

affecting plasma cell activity. The results show that sophorolipids decrease IgE

production in U266 cells by down regulating important genes involved in IgE

pathobiology in a synergistic manner. These data continue to support the utility of

sophorolipids as an anti-inflammatory agent and a novel potential therapy in diseases of

altered IgE regulation.

26

1.9.3.4. Anti-human immunodeficiency virus and sperm-immobilizing activity

The increased incidence of human immuno deficiency virus (HIV)/AIDS in women

aged 15-49 years has identified the urgent need for a female controlled, efficacious and

safe vaginal topical microbicide. To meet this challenge, sophorolipid produced by

Candida bombicola and its structural analogues have been studied for their spermicidal,

anti-HIV and cytotoxic activities (Shah et al. 2005). The sophorolipid diacetate ethyl

ester derivative is the most potent spermicidal and virucidal agent of the series of

sophorolipids studied. Its virucidal activity against HIV and sperm immobilizing

activity against human semen are similar to those of nonoxynol-9. Naruse et al. (1990)

demonstrated a significant inhibitory effect of pumilacidin (surfactin analog) on herpes

simplex virus 1 (HSV-1). They also reported an inhibitory activity against H+,

K+ATPase and protection against gastric ulcers in vivo. Itokawa et al. (1994) have

reported the potential of surfactin against human immuno deficiency virus 1 (HIV-1).

Vollenbroich et al. (1997) have reported a potential use for surfactin in the virus safety

enhancement of biotechnological and pharmaceutical products. They also suggested that

the anti viral action of surfactin is due to a physiochemical interaction between the

membrane active surfactant and the virus lipid membrane.

1.9.3.5. Stimulating agents for skin fibroblast metabolism

The fact has been established that the sophorolipids in lactone form can be used as

agents for stimulating skin dermal fibroblast cell metabolism and more particularly, as

agents for stimulating collagen neosynthesis, at a concentration of 0.01 ppm at 5% (p/p)

of dry matter in formulation. This is applicable in cosmetology and dermatology. The

purified lactone sophorolipid product is of importance in the formulation of dermis anti-

ageing, repair and restructuring products because of its effect on the stimulation of

dermis cells. By encouraging the synthesis of new collagen fibres, purified lactone

sophorolipids can be used both as a preventive measure against ageing of the skin and

used in creams for the body, and in body milks, lotions and gels for the skin (Borzeix

27

and Frederique 2003). Lipopeptides produced by Streptosporangium amethystogenes

subsp. fukuiense Al-23456 have similar natures to some biosurfactants and shown to

have the ability to induce granulocyte colony stimulating factor and granulocyte-

macrophage colony stimulating factor (Hida et al. 1995). Takizawa et al. (1995)

reported significant stimulation of the proliferation of bone marrow cells from BALB/c

female mice by Streptosporangium amethystogenes lipopeptides.

1.9.4. Biosurfactants and bioremediation

Bioremediation in general aims at providing cost effective, contaminant specific

treatments to reduce the concentration of individual or mixed environmental

contaminants (Head 1998). The process of bioremediation can be carried out by

utilizing plants, microbes and/or microbial products. Bartha (1986) estimated that

approximately 0.08-0.4% of the total worldwide production of petroleum eventually

reaches the oceans. Several oil spill accidents occurred in the last few years have

resulted in significant contamination of oceans and shoreline environments. Some

famous examples include the Amoco Cadiz oil spill in Brittany coastal waters in 1978,

the Exxon Valdez spill in the Prince William Sound in 1989 and the Haven spill of the

coast of Italy in 1991. More recent examples include the Nakhodka tanker oil spill of the

Oki Islands in the Sea of Japan, 1997, the San Jorge tanker spill on the shores of Punta

Del Este in Uruguay in 1997 and the Nissos Amorgos spill in the Maracaibo Channel in

the Gulf of Venezuela in 1997. Most recent ones include Rena oil spill occurred off the

coast of Tauranga in New Zealand on 5 October 2011 impacting the surrounding

environment of the Astrolabe Reef and the coast of Tauranga in New Zealand quite

extensively. It caused up to 2,500 barrels (400 m3) of oil spill

(http://en.wikipedia.org/wiki/Rena_oil_spill). The Yellowstone River oil spill on July 1,

2011 estimating 750-1,000 barrels of oil spill (equivalent to 42,000 gallons) into the

Yellowstone river (http://en.wikipedia.org/wiki/Yellowstone_River#2011_oil_spill).

Apart from these accidental spills, deliberate releases of oil have also caused

considerable contamination. During the Gulf War in 1991 over 105 tonnes of oil were

28

released in the Gulf waters, threatening desalination of plants and the coastal ecosystem

of the Gulf (Pearce 1993). Such incidents have intensified the efforts to develop various

chemicals, procedures and techniques for combating oil pollution both at sea and along

the shoreline. Biosurfactants are just such chemicals, and were applied to parts of the

Exxon Valdez oil spill (Harvey et al. 1990). The ability of biosurfactants to emulsify

hydrocarbon-water mixtures enhances the degradation of hydrocarbons in the

environment by increasing bioavailability of the compounds (Begley et al. 1996). The

presence of hydrocarbon degrading microorganisms in seawater renders biodegradation,

one of the most efficient methods for removing pollutants (Gutnick and Rosenberg

1977; Leahy and Colwell 1990; Atlas 1991). Most biosurfactants, in comparison to

chemical surfactants, have lower possible toxicity and shorter persistence in the

environment (Zajic et al. 1977b; Georgiou et al. 1992).

1.9.4.1. Bioremediation of soil

Growing interests in biosurfactant applications for treating hydrocarbon contaminated

soils have developed recently (Bartha 1986; Van Dyke et al. 1993b; Banat 1995).

Degradation of hydrocarbon by microbes/microbial products present in the

contaminated soil is the primary method for removing hydrocarbon pollutants from the

soil. Partially purified biosurfactants can either be used in bioreactors or in situ to

emulsify and increase the solubility of hydrophobic contaminants. Moreover, surfactant

producing microorganisms or growth limiting factors may also be added to the soil to

enhance the growth of added or indigenous microorganisms capable of producing

biosurfactants (Lang and Wagner 1993). Oberbremer et al. (1990) used a mixed soil

population to assess hydrocarbon degradation in a model oil field and reported a

statistically significant enhancement of hydrocarbon degradation when sophorose lipids

were added to a model system containing 10% soil and 1.35% hydrocarbon mixture of

tetradecane, pentadecane, hexadecane, pristane, phenyldecane and naphthalene in

mineral salt medium. In the absence of surfactant, 81% of the hydrocarbon mixture was

degraded in 114 h while, in the presence of biosurfactant, up to 90% of the hydrocarbon

29

mixture was degraded in 79 h. Biodetox (Germany) demonstrated a procedure to

decontaminate soils, industrial sludge and waste waters where the contaminated

materials are transported to a biopit and process for microbial degradation. Biodetox

also performs in situ bioreclamation for surface, deep ground and ground water

contamination (Van Dyke et al. 1991). Microorganisms are added by means of

“Biodetox foam'', which is not harmful to the environment, contains bacteria, nutrients

and biosurfactants and can be biodegraded. Jain et al. (1992) found that the addition of

Pseudomonas biosurfactant enhanced the biodegradation of tetradecane, pristane, and

hexadecane in a silt loam with 2.1% organic matter. Similarly, Zhang and Miller (1995)

reported the enhanced octadecane dispersion and lightened biodegradation by a

Pseudomonas rhamnolipid surfactant. Falatko and Novak (1992) studied biosurfactant

facilitated removal of gasoline overlaid on the top of coarse grain sand packed column.

Up to a 15 fold increase was observed in the effluent concentration of four gasoline

constituents; toluene, m-xylene, 1, 2, 4-trimethylbenzene and naphthalene, upon the

addition of biosurfactant solution (600 mg/l). Herman et al. (1997) investigated the

effects of rhamnolipid biosurfactants on in situ biodegradation of hydrocarbon

entrapped in a porous matrix and reported a mobilization of hydrocarbon entrapped

within the soil matrix at biosurfactant concentrations higher than critical micelle

concentration (CMC). Mycobacterium flavescens strain EX 91 was used for the

development of a commercial product Ekoil, which was employed in the

decontamination of an oil-polluted water system and also proved effective in the

treatment of the engine oil-contaminated wastewater of a nuclear power station

Ermolenko et al. (1997). Bai et al. (1997) used an anionic mono-rhamnolipid

biosurfactant from Pseudomonas aeruginosa to remove residual hydrocarbons from

sand columns. They could recover up to 84% of residual hydrocarbon (hexadecane)

from sand packed columns.

1.9.4.2. Marine bioremediation/dispersion

Microorganisms capable of hydrocarbon degradation have often been isolated from

aquatic environments (Brown and Braddock 1990). Chakrabarty (1985) reported that an

30

emulsifier produced by Pseudomonas aeruginosa SB30 was able to quickly disperse oil

into fine droplets and concluded that it may be useful in removing oil from

contaminated beaches. Mattei et al. (1986) studied crude oil degradation in a continuous

flow fermentor using a mixed bacterial community isolated from seawater and reported

an enhanced degradation rate of crude oil by using that bacterial community. In a

similar study on biodegradation of a mixture of hydrocarbons with Pseudomonas

aeruginosa S8, Shafeeq et al. (1989) demonstrated the presence of biosurfactants in the

culture medium. Harvey et al. (1990) tested a biosurfactant from Pseudomonas

aeruginosa for its ability to remove oil from contaminated Alaskan gravel samples

under various conditions, such as different concentrations of surfactant, time of contact,

temperature of the wash and presence or absence of gum. They reported increased oil

displacement (about 2-3 fold) with biosurfactant than with water and depicted efficiency

of biosurfactant to remove oil from surfaces. The Environmental Technology

Laboratory at the University of Alaska, Fairbanks, carried out a field trial in July 1993

in Sleepy Bay on LaTouche Island in Prince William Sound to test the effectiveness of a

biosurfactant in removing crude oil from subsurface beach material. They reported

complete removal of petroleum hydrocarbons (to the limit of 0.5 mg/kg) while semi

volatile petroleum hydrocarbons were reduced to 70% (Tumeo et al. 1994).

1.9.4.3. Bioremediation of polyaromatic hydrocarbon

Poly aromatic hydrocarbons (PAH) are the most hazardous contaminants present in soil.

Due to hydrophobicity, low aqueous solubility and strong adsorptive capacity in soil,

these compounds become less available to the soil microorganisms and hence their

biodegradation is limited to a certain extent (Mihelcic et al. 1993; Volkering et al.

1995). Only limited numbers of microorganisms are capable of degrading PAHs since

they contain four or more fused aromatic rings (Harayama 1997). Surfactants enhance

degradation of PAH by releasing the hydrocarbons sorbed to soil and/or by solubilizing

or emulsifying the hydrophobic compounds in aqueous phase (Aronstein et al. 1991).

Other investigations indicate a potential use for synthetic surfactants to enhance PAH

31

degradation by increasing microbial accessibility to insoluble substrates (Tiehm 1994).

Providenti et al. (1995) studied the effects of Pseudomonas aeruginosa UG2

biosurfactants on phenanthrene mineralization in soil slurries and detected an increase in

phenanthrene mineralization. The efficiency of biosurfactants in the remediation of soil

contaminated with metals, phenanthrene and polychlorinated biphenyls (PCBs) have

also been reported (Miller 1995a). Berg et al. (1990) described an emulsifying agent

produced by Pseudomonas aeruginosa UG2 that increased the solubility of

hexachlorobiphenyl added to soil slurries, resulting in a 31% recovery of the compound

in the aqueous phase. Churchill et al. (1995) demonstrated that rhamnolipids from

bacteria, in combination with the oleophilic fertilizer Inipol EAp-22, increased the

degradation rate of hexadecane, benzene, toluene, o- and p-cresol and naphthalene both

in aqueous phase bioreactors and in those containing soil. They also reported increased

rates of biodegradation of aliphatic and aromatic hydrocarbons by pure bacterial

cultures. A similar study was conducted by Van Dyke et al. (1993a). They surveyed

thirteen biosurfactants for the removal of hexachlorobiphenyl from soil. Out of thirteen

biosurfactants tested, seven removed hexachlorobiphenyl more efficiently compared to

the control. In their report they have mentioned that two strains of Pseudomonas

aeruginosa and one strain of Acinetobacter calcoaceticus RAG-1 produced the most

efficient biosurfactants. In an investigation of the capacity of PAH utilizing bacteria to

produce biosurfactants using naphthalene and phenanthrene, Daziel et al. (1996)

detected biosurfactant production that was responsible for an increase in the aqueous

concentration of naphthalene (31 mg/l). This indicates a potential role for biosurfactants

in increasing the solubility of such compounds. Similarly Zhang et al. (1997) tested the

effects of two rhamnolipid biosurfactants on the dissolution and bioavailability of

phenanthrene and reported increase in both solubility and degradation rate of

phenanthrene. Kanga et al. (1997) tested the glycolipid biosurfactant produced by

Rhodococcus sp. H13A and the synthetic surfactant Tween 80 for enhanced solubility of

naphthalene and methyl substituted derivatives in crude oil (representative of the PAH

content) and observed that both surfactants lowered surface tension in solutions from 72

dynes/cm to 30 dynes/cm but the biosurfactant was more efficient in increasing the

32

solubility of hydrocarbons. In a laboratory column study, Noordman et al. (1998)

applied rhamnolipid biosurfactants for the enhanced removal of phenanthrene from

phenanthrene contaminated soil eluting with an electrolyte solution containing

rhamnolipid (500 mg/l). Rhamnolipids enhanced the removal of phenanthrene (2 to 5

fold shorter time for 50% recovery and 3.5 fold for 90% recovery) compared to controls.

1.9.4.4. Remediation of metal contaminated soil

Activities like mining, manufacturing and use of synthetic products (e.g. pesticides,

paints, batteries, industrial waste, and land application of industrial or domestic sludge)

can result in heavy metal contamination of urban as well as agricultural soils. Heavy

metals also occur naturally, but rarely at toxic levels

(www.aiswcd.org/IUMPDF/appendix/u03.pdf). Potentially contaminated soils may

occur at old landfill sites (particularly those that accepted industrial wastes), old

orchards that used insecticides containing arsenic as an active ingredient, fields that had

past applications of waste water or municipal sludge, areas in or around mining waste

piles and tailings, industrial areas where chemicals may have been dumped on the

ground, or in areas downwind from industrial sites. It is well known that microbial cells

may chelate metals from solution. Little information is available however, concerning

the use of biosurfactants to chelate metals. There are several reports of

exopolysaccharide use for metal chelation (Kaplan et al. 1987; Scott and Palmer 1988;

Marques et al. 1990). Miller (1995b) reported that the addition of biosurfactant may

promote desorption of heavy metals from soils in two ways. The first is through

complexation of the free form of the metal residing in solution. This decreases the

solution-phase activity of the metal leading to desorption. The second occurs under

conditions of reduced interfacial tension; the biosurfactants accumulate at the solid-

solution interface, which may allow direct contact between the biosurfactant and the

sorbed metal. Christofi and Ivshina (2002) explained that surfactants can remove metals

from surfaces in a number of ways. First, metals in a nonionic form can complex with

biosurfactants, enhancing surface removal by Le Chatelier’s Principle (Miller 1995b).

33

Additionally, the use of anionic surfactants which contact metals can lead to their

desorption from surfaces. The surfactant–metal union would then need to be removed

from the soil matrix. Cationic surfactants can also act to reduce the association of metals

by competition for some but not all negatively charged surfaces (Christofi and Ivshina

2002). Mulligan et al. (1999a; b) used surfactin from Bacillus subtilis to treat soil and

sediments contaminated with Zn2+, Cu2+, Cd2+, oil and grease. It was found that the

heavy metals were associated with carbonate, oxides and organic fractions in the

contaminated material and that these could be removed using a combination of surfactin

and NaOH. Metal desorption involved the attachment of surfactin at the soil interface

and metal removal through lowering of the interfacial tension and micellar

complexation. Tan et al. (1994) investigated the potential of rhamnolipid biosurfactants

produced by Pseudomonas aeruginosa ATCC 9027 in the removal of metals from soils

contaminated with Cd2+ and reported 92% complexation of Cd2+ in a 0.5 mM solution of

Cd(NO3)2 using a 5 mM solution of rhamnolipid (22 mg/l). Although the use of

rhamnolipid biosurfactants in the bioremediation of metal contaminated soils has

promise, to achieve better metal removal and develop remediation technologies it is

important to understand the factors affecting rhamnolipids sorption to soil. These factors

include ionic strength, mineral composition and pore water chemistry within metal

contaminated soils (Banat et al. 2000). Future success of biosurfactant technology in

bioremediation initiatives will require targeting their use to the physical conditions and

chemical nature of the pollution affected site to maximize their efficiency and

economical viability.

1.9.5. Biosurfactants and oil storage tank cleaning

Another application of biosurfactants is oil storage tank cleaning. Surfactants have been

studied for use in reducing the viscosity of heavy oils, thereby facilitating recovery,

transportation and pipelining (Bertrand et al. 1994). A glycolipid surfactant produced by

Gram-negative, rod-shaped bacterial isolate H13A has been reported to reduce the

viscosity of heavy crude oil by 50% (Finnerty and Singer 1985). Earlier Zajic et al.

34

(1974) isolated a Pseudomonas strain which produced an emulsifying agent capable of

emulsifying heavy grade VI fuel oil. In a pilot field investigation, Banat et al. (1991)

tested the ability of biosurfactant produced by a bacterial strain (Pet 1006) to clean oil

storage tanks and to recover hydrocarbons from the emulsified sludge. Two tonnes of

biosurfactant containing whole cell culture were used to mobilize and clean 850 m3 oil

sludge. Approximately 91% (774 m3) of this sludge was recovered as re-sellable crude

oil and 76 m3 non-hydrocarbon materials remained as impurities to be manually

cleaned. The value of the recovered crude covered the cost of the cleaning operation

(US $100,000 to 150,000 per tank). Such a cleanup process is therefore economically

rewarding and less hazardous to persons involved in the process compared to

conventional processes (Lillenberg et al. 1992). It is also an environmentally sound

technology leading to less disposal of oily sludge in the natural environment. To our

knowledge however, further commercial applications of this technology have not been

carried out.

1.9.6. Biosurfactant and microbial enhanced oil recovery

An area of considerable potential for biosurfactant application is in the field of

microbial enhanced oil recovery (MEOR). Enhanced oil recovery methods were devised

to recover oil remaining in reservoirs after primary and secondary recovery procedures.

It is an important tertiary recovery technology, which utilizes microorganisms and/or

their metabolites for residual oil recovery (Banat 1995a). In MEOR, microorganisms in

reservoirs are stimulated to produce polymers and surfactants, which aid MEOR by

lowering interfacial tension at the oil-rock interface. This reduces the capillary forces

preventing oil from moving through rock pores and increases chances of recovery.

Biosurfactants can also aid oil emulsification and assist in the detachment of oil films

from rocks (Banat 1995a, b). In situ removal of oil is due to multiple effects of the

microorganisms on both environment and oil. These effects include gas and acid

production, reduction in oil viscosity, plugging by biomass accumulation, reduction in

interfacial tension by biosurfactants and degradation of large organic molecules. These

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are all factors responsible for decreasing the oil viscosity and making its recovery easier

(Jack 1988). The strategies involved in the MEOR depend on the prevalent oil reservoir

conditions, including temperature, pressure, pH, porosity, salinity and geologic makeup

of the reservoir, available nutrients and the presence of indigenous microorganisms.

These factors should be considered before devising a strategy for use in an oil well.

During the last 15-20 years, China was very active in MEOR method and today is still

active in this field and could be considered one of the leaders in this field (He et al.

2000). There are three main strategies for the use of biosurfactants in enhanced oil

recovery (EOR) or mobilization of heavy oils (Shennan and Levi 1987; Banat 1995a):

1. Production in batch or continuous culture under industrial conditions followed by

addition to the reservoir in the conventional way along with the water flood (ex situ

MEOR).

2. Production of surface-active compounds by microorganisms at the cell-oil interface

within the reservoir formation, implying penetration of metabolically active cells into

the reservoir.

3. Injection of selected nutrients into a reservoir, thus stimulating the growth of

indigenous biosurfactant producing microorganisms.

1.9.7. Biosurfactants for agricultural use

Maintaining soil health and protecting crops from various diseases are two prime issues

in agriculture. Biosurfactants have been used to deal with these issues. Low toxicity and

biodegradability of biosurfactants have made these compounds superior to their

synthetic counterparts. There are many reports demonstrating the use of biosurfactants

as biocontrol agents. Concerns about pesticide pollution have impelled global efforts to

find alternative biological control technologies. Stanghellini et al. (1996) investigated

the effects of synthetic surfactants on controlling the root rot fungal infections of

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cucumbers and peppers. They observed lysis of fungal zoospores due to some bacterial

metabolites in the nutrient solution. The metabolites were thought to be biosurfactants,

as their mode of action was similar to the synthetic surfactants. Rhamnolipid surfactant

produced by Pseudomonas aeruginosa has zoosporicidal activity against species of

Pythium, Phytophthora and Plasmopara. Biosurfactants are needed for the

hydrophilization of heavy soils to obtain good wettability and also to achieve equal

distribution of fertilizers and pesticides in the soils. Biosurfactants have also been used

in formulating poorly soluble organophosphorus pesticides. Two Bacillus strains

producing an emulsifier, possibly a glycolipopeptide, were able to form a stable

emulsion in the presence of the pesticide fenthion (Patel and Gopinathan 1986). A

biosurfactant produced by Pseudomonas aeruginosa has been reported to solubilize

toxic organic chemicals and increase the solubility and recovery of hexachlorobiphenyl

from soil slurries by 31% (Berg et al. 1990). It was found that the addition of a

biosurfactant (400 µg/ ml) produced by Bacillus subtilis MTCC 2423 enhanced the rate

of biodegradation of the chlorinated pesticide α and β-endosulfan by 30 - 40%. It also

mobilized the residual endosulfan isomers towards biodegradation. These would

otherwise have remained undegraded (Awasthi et al. 1999).

1.9.8. Biosurfactants used 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

biodispersan, an anionic polysaccharide produced 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 smaller particles. Rosenberg and Ron (1998) explained that

the anionic biodispersan 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

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coal (Kao 1984). Similarly Polman et al. (1994) tested biosurfactants for solubilization

of coal and achieved partial solubilization of lignite coal using a crude preparation of

biosurfactants from Candida bombicola.

1.10. Work on biosurfactant in North-East India

Das and Mukherjee (2005) investigated and compared biochemical as well as

pharmacological properties of biosurfactants produced by Pseudomonas aeruginosa

mucoid (M) and non-mucoid (NM) strains isolated from hydrocarbon contaminated soil

samples. Pharmacological characterization of these biosurfactants revealed that they

induced dose-dependent hemolysis and coagulation of platelet-poor plasma but were

non-detrimental to chicken lung, liver, heart and kidney tissues and documented that

biosurfactants from Pseudomonas aeruginosa M and NM strains could be exploited for

use in petroleum sectors as well as in pharmaceutical industries. Ex situ MEOR studies

of B. subtilis (DM-03 and DM-04) by using a sand-packed column showed that the two

strains were effective in oil recovery from sand pores (Das and Mukherjee 2007).

Bordoloi and Konwar (2008) isolated four strains of Pseudomonas aeruginosa (MTCC

7815, MTCC 7814, MTCC 7812, and MTCC 8165) from various oil fields in India, and

studied the oil recovery efficiency of the excreted biosurfactants, in the form of free-cell

culture broth, using a sand-packed column. The results found that the test biosurfactants

could recover oil in the range of 30-60%, depending on the investigated temperature.

Biosurfactant can make hydrocarbon complexes more mobile with the potential use in

oil recovery, pumping of crude oil and in bioremediation of crude oil contaminant.

Work on bacterial biosurfactant in enhancing solubility and metabolism of petroleum

hydrocarbons by using bacterial isolates capable of utilizing polycyclic aromatic

hydrocarbons like phenanthrene, pyrene and fluorene showed a gradual decrease of the

supplemented hydrocarbons in the culture medium with corresponding increase in

bacterial biomass and protein. The medium having the combined application of fluorine

and phenanthrene caused better biosurfactant production (0.45 g/l) and (0.38 g/l) by

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Pseudomonas aeruginosa strains MTCC7815 and MTCC7814. The biosurfactant from

MTCC7815 (41.0 µg/ml) and MTCC7812 (26l µg/ml) exhibited higher solubilization of

pyrene; whereas, MTCC8165 caused higher solubilization of phenanthrene; and that of

MTCC7812 (24.45g/l) and MTCC8163 (24.49 g/l) caused more solubilzation of

fluorene. Higher solubilization of pyrene and fluorene by the biosurfactant of

MTCC7815 and MTCC7812 respectively enhanced their metabolism causing sustained

growth. Biosurfactants were found to be lipopeptide and protein-starch-lipid complex in

nature. They differed in quantity and structure. The predominant rhamnolipids present

in biosurfactants were Rha-C8-C10 and Rha-C10-C8 (Bordoloi and Konwar 2009).

1.11. Problem studied

Petroleum industries produce large amounts of solid and semisolid wastes generated by

sedimentation processes at the bottom of crude oil and heavy black oil storage tanks, in

sludge separator unit and in biological effluent treatment plants known as oily sludge.

The composition of oily sludge varies due to the large diversity in the quality of crude

oils, differences in the processes used for oil-water separation, leakages during

industrial processes, and also mixing with the existing oily sludge. Usually, the oily

sludge contains water, sand, oils, grease, organic compounds, chemical elements, and

metals (Lima et al., 2011). The accumulation of oily residues in petroleum industry

poses a serious environmental problem. Kuriakose and Manjooran (1994) reported 70%

hydrocarbon content in the sludge generated from Cochin Refineries Ltd. at

Ambalamugal, Kerala, India which cannot be extracted by primary and secondary

extraction methods. Disposal of the oil containing sludge into pits and lagoons is

hazardous for the environment on one hand as it can pollute ground water and on the

other hand is wastage of energy. It would be very appreciable if this sludge could be

pre-treated to lessen its oil content before disposed off. Moreover, cleaning of tank

bottom sludge of crude oil storage tanks also poses a serious threat since it becomes

39

very difficult to remove the oily sludge mechanically. Once the viscosity and the

binding affinity of the different components of sludge could be reduced then it will be

easier to remove the sludge from storage tanks.

One of the most promising ways of pre-treatment of sludge to reduce its viscosity

is the use of microbe and/or microbial products because of their environmental

acceptability. A variety of microbes and their products (biosurfactants) are reported as

potential candidates for hydrocarbon recovery. Hayes et al. (1986) have demonstrated

the ability of emulsan biosurfactant to reduce the viscosity of Boscan (Venezuelan

heavy oil) from 200,000 to 100 cP, making it feasible to pump heavy oil in 26,000 miles

of commercial pipe line. Though different types of microbes have been reported to be

used in hydrocarbon recovery, Pseudomonads are the best known bacteria capable of

utilizing hydrocarbons as carbon and energy sources and producing biosurfactants

which enhance the uptake of such immiscible hydrophobic compounds (Al-Tahhan et al.

2000; Beal and Betts 2000; Rahman et al. 2002; Cameotra and Singh 2008;

Pornsunthorntawee et al. 2008). Among Pseudomonads, Pseudomonas is one of the

most often reported genera for its availability to produce biosurfactant molecules (Koch

et al. 1991; Santos et al. 2002).

Though many studies have been conducted to investigate microbial enhanced oil

recovery but not much work has been reported for recovery of hydrocarbons from

refinery sludge. Hence the purpose of this work was to evaluate an alternative process

for removal of oily sludge by using microbe/microbial product to reduce the viscosity

and promote formation of oil/water emulsions. This makes sludge pumping easier and

permits recovery of crude oil after breaking the emulsion. It could also be of great

significance of exploring native biosurfactant producing strains for hydrocarbon

recovery since native strains can be assumed to perform better in their native

environment than the exotic strains. Therefore the present experiment was conducted to

isolate biosurfactant producing strains from crude oil contaminated soil and

investigation of recovery of hydrocarbons from refinery sludge in laboratory conditions.

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As discussed previously, production of biosurfactant is greatly affected by

environmental factors. Therefore, investigation was also carried out to maximize the

production of biosurfactant by the isolated strain.

The above discussions have encouraged a lot to work on biosurfactant

emphasizing on the following objectives:

1. Isolation, screening and identification of biosurfactant producing

microorganism(s) (namely bacteria) present in hydrocarbon contaminated soil of

oil fields of upper Assam.

2. To evaluate the effect of different environmental factors on production of

biosurfactant by the efficient bacterial strains.

3. To characterize the biosurfactant

4. To study the recovery of hydrocarbons from refinery sludge.