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     1. The importance and the relevance of scientific content

     1.1. The relevance of the thematic area

     Research in the area of biosurfactants has expanded quite a lot in recent years due to its potential use in different areas, such as agriculture, food industry, pharmaceutics, the oil industry, petrochemistry and paper industry. The development of this line of research is of paramount importance, mainly in view of the present concern with protection of the environment. Therefore, the most significant advantage of a microbial surfactant over chemical surfactants is its ecological acceptance because it is biodegradable and nontoxic to natural environment.

     Biosurfactants (Microbial Surface Active Agents) have become recently an important product of biotechnology for industrial and medical applications. The reason for their popularity, as high value microbial products, is primarily in their specific action, low toxicity, relative ease of preparation and widespread applicability. They can be used as emulsifiers, de-emulsifiers, wetting agents, spreading agents, foaming agents, functional food ingredients and detergents in various industrial sectors such as: Petroleum and Petrochemicals, Organic Chemicals, Foods and Beverages, Cosmetics and Pharmaceuticals, Mining and Metallurgy, Agrochemicals and Fertilizers, Environmental Control and Management, and many others.

     Many bacteria, yeasts and fungi produce biochemicals or macromolecules that possess surface-active properties. Biosurfactants have expansive commercial potential; they have been indicated in a myriad of food and non-food applications, such as bioremediation, enhanced oil recovery, food thickening, herbicide and pesticide formulations, consumer products (e.g., detergents and cosmetics) manufacturing, lubricant formulations, and inhibition of microbial growth. Sophorolipids, a family of glycolipids secreted by several Candida or related yeast species, consist of a disaccharide linked to a hydroxy fatty acid via a glycosidic bond. Sophorolipids are a group of exciting emerging biosurfactants with many new product and application potentials (Solaiman, 2004).

     1.2. The scientific importance of the theme

     The scientific importance of the proposed theme can be underline by the following applications of biosurfactants in pollution control and in plant growth promoting and control against pathogens:

    1.2.1. The biosurfactants rol in the mechanisms of control phytopathogenic fungi.

    Relatively little is known about the exact mechanisms used by Bacillus subtilis in its behavior as a biocontrol agent on plants. Some studies were done in order to develop of a sensitive plant infection model demonstrating that the bacterial pathogen Pseudomonas syringae pv tomato DC3000 is capable of infecting Arabidopsis roots both in vitro and in soil. Using this infection model, was demonstrated the biocontrol ability of a wild-type B. subtilis strain 6051 against P. syringae. Arabidopsis root surfaces treated with B. subtilis were analyzed with confocal scanning laser microscopy to reveal a three-dimensional B. subtilis biofilm. It is known that formation of biofilms by B. subtilis is a complex process that includes secretion of surfactin, a lipopeptide antimicrobial agent. To determine the role of surfactin in biocontrol by B. subtilis, was tested a mutant strain, M1, with a deletion in a surfactin synthase gene and, thus, deficient in surfactin production. B. subtilis M1 was ineffective as a biocontrol agent against P. syringae infectivity in Arabidopsis and also failed to form robust biofilms on either roots or inert surfaces. The antibacterial activity of surfactin against P. syringae was determined in both broth and agar cultures and also by live-dead staining methods. Although the minimum inhibitory concentrations determined were relatively high (25 µg mL-1), the levels of the lipopeptide in roots colonized by B. subtilis are likely to be sufficient to kill P. syringae. These results collectively indicate that upon root colonization, B. subtilis 6051 forms a stable, extensive biofilm and secretes surfactin, which act together to protect plants against attack by pathogenic bacteria (Bais, 2003).

     Beneficial plant rhizobacteria (PR) are associated with the surfaces of plant roots and may increase plant yield by mechanisms that impart improved mineral nutrient uptake, disease suppression, or phytohormone production (Kloepper et al., 1991; Lutenberg et al., 1991; Costacurta and Vanderleyden, 1995; Defago and Keel, 1995). An important trait of PR is their ability to effectively colonize the rhizosphere and maintain a stable relationship with the surface of plant roots (Lutenberg and Dekkers, 1999). PR may also interact with a variety of soil microorganisms that are normally present in the rhizosphere, in some cases acting as a biocontrol agent against pathogenic bacteria (Pinton et al., 2001). Interestingly, poor root colonization by PR may result in decreased biocontrol activity (Schippers et al., 1987). One beneficial rhizobacterium is Bacillus subtilis, which is ubiquitous in soil, can promote plant growth, protect against fungal pathogen attack (Utkhede and Smith, 1992; Asaka and Shoda, 1996; Emmert and Handelsman, 1999), and play a role in the degradation of organic polymers in the soil (Emmert and Handelsman, 1999).

     Commercial strains of B. subtilis have been marketed as biocontrol agents for fungal diseases of crops (Emmert and Handelsman, 1999; Warrior et al., 2002). The commercial biofungicide, Serenade, which contains a B. subtilis strain, is reported to be effective against a variety of pathogenic bacteria, including Erwina, Pseudomonas, and Xanthomonas strains (http://www.agraquest.com/. The mechanism of this antibacterial effect is uncertain, although it is known that B. subtilis can produce a variety of antibacterial agents, including a broad spectrum of lipopeptides, such as surfactin, that are potent biosurfactants (Zuber et al., 1993; Peypoux et al., 1999).

     Hydrophobin. Mycelial fungi secrete small, cysteine-rich, proteins, called hydrophobins, that self-assemble at hydrophilic-hydrophobic interfaces into amphipathic membranes, highly insoluble in case of Class I hydrophobins. By self-assembly at the culture medium-air interface they greatly lower the surface tension enabling emergent structures to grow into the air. By self-assembly at the interface between the hydrophilic cell wall and the air or any other hydrophobic environment, these emergent structures are coated with a hydrophobin membrane. These properties allow hydrophobins to fulfil a broad spectrum of functions in fungal development. They are involved in formation of aerial (reproductive) structures, in aerial dispersion of spores, and they line air channels within fruiting bodies with a hydrophobic coating, probably serving gas exchange. Hydrophobins also mediate hyphal attachment to hydrophobic surfaces such as those of plants. Moreover, they appear involved in complex interhyphal interactions, and in interactions with algae in lichens. Their resistance towards chemical and enzymatic treatments suggests that assembled hydrophobins also protect fungal emergent structures against adverse environmental conditions.

      Class I and class II hydrophobins are small secreted fungal proteins that self-assemble at hydrophilic-hydrophobic interfaces into amphipathic films. Apart from eight conserved cysteine residues, the amino acid sequences between and within both classes have diverged considerably, and this is reflected in the biophysical properties of these proteins. For instance, assemblages of class I hydrophobins are highly insoluble, while those of class II hydrophobins readily dissolve in a variety of solvents. The properties of hydrophobins make them interesting candidates for use in a wide range of medical and technical applications. Each application has its own requirements, which may be met by using specific natural variants of hydrophobins or by modifying hydrophobins chemically or genetically. Applications also require high production systems for hydrophobins. In this respect, filamentous fungi that naturally secrete hydrophobins into the medium seem to be the hosts of choice.

     1.2.2.Microbial enhanced oil recovery. An area of considerable potential for BS application is microbial enhanced oil recovery (MEOR). In MEOR, microorganisms in reservoir are stimulated to produce polymers and surfactants which aid MEOR by lowering interfacial tension at the oil–rock interface. To produce MS in situ, microorganisms in the reservoir are usually provided with low-cost substrates, such as molasses and inorganic nutrients, to promote growth and surfactant production. To be useful for MEOR in situ, bacteria must be able to grow under extreme conditions encountered in oil reservoirs such as high temperature, pressure, salinity, and low oxygen level. Several aerobic and anaerobic thermophiles tolerant of pressure and moderate salinity have been isolated which are able to mobilize crude oil in the laboratory. Clark et al., based on a computer search estimated that about 27% of oil reservoirs in USA are amenable to microbial growth and MEOR. The effectiveness of MEOR has been reported in field studies carried out in US, Czechoslovakia, Romania, USSR, Hungary, Poland, and The Netherlands. Significant increase in oil recovery was noted in some cases.

     1.2.3. Hydrocarbon degradation in the soil environment. CxHy degradation in soil has been extensively studied. Degradation is dependent on presence in soil of hydrocarbon-degrading species of microorganisms, hydrocarbon composition, oxygen availability, water, temperature, pH, and inorganic nutrients. The physical state of CxHy can also affect biodegradation. Addition of synthetic surfactants or MS resulted in increased mobility and solubility of CxHy, which is essential for effective microbial degradation. Use of MS in CxHy degradation has produced variable results. In the work of Lindley and Heydeman, the fungus Cladosporium resiuae, grown on alkane mixtures, produced extracellular fatty acids and phospholipids, mainly dodecanoic acid and phosphatidylcholine. Supplement of the growth medium with phosphatidylcholine enhanced the alkane degradation rate by 30%. Foght et al. reported that the emulsifier, Emulsan, stimulated aromatic mineralization by pure bacterial cultures, but inhibited the degradation process when mixed cultures were used. Oberbremer and Muller-Harting used mixed soil population to assess CxHy degradation in model oil. Naphthalene was utilized in the first phase of CxHy degradation; other oil components were degraded during the second phase after the surfactants produced by concerned microorganisms lowered the interfacial tension. Addition of biosurfactants, such as some sophorolipids, increased both the extent of degradation and final biomass yield. Biodetox (Germany) described a process to decontaminate soils, industrial sludges, and waste waters. They also described in situ bioreclamation of contaminated surface, deep ground and ground water. Microorganisms were added by means of a biodetox foam that contained bacteria, nutrients and surfactants; and was biodegradable. Another method to remove oil contaminants is to add BS into contaminated soil to increase CxHy mobility. The emulsified CxHy could then be recovered by using a production well, and subsequently degrading above ground in a bioreactor. In situ washing of soil was studied using two synthetic surfactants, Adsee 799 and Hyonic NP-90. Removal of PCBs and petroleum CxHy from soil by adding surfactants to the wash water, has met with some success. Several strains of anaerobic bacteria produce biosurfactants. However, the observed reduction in surface tension (45 to 50 mN/m) was not as large as the observed reduction in surface tension by anaerobic organisms (27 to 50 mN/m). MS can also be used to enhance solubilization of toxic organic chemicals including xenobiotics. Berg et al., using the surfactant from Pseudomonas aeruginosa UG2, reported an increase in the solubility of hexachlorobiphenyl added to soil slurries, which resulted in a 31% recovery of the compound in the aqueous phase. This was about 3-times higher than that solubilized by the chemical surfactant sodium ligninsulfonate (9.3%). When the P. aeruginosa bioemulsifier and sodium ligninsulphonate were used together, additive effect on solubilization (41.5%) was observed. Pseudomonas ceparia AC 1100 produced an emulsifier that formed a stable suspension with 2,4,5-T, and also exhibited some emulsifying activity against chlorophenols. Thus, this emulsifier can be used to enhance bacterial degradation of organochlorine compounds.

    1.2.4. Pesticide-specific biosurfactants. Due to biodegradative property of biosurfactants, they are ideally suited for environmental applications, specially for removal of the pesticides—an important step in bioremediation. Survey of the literature reveals that application of biosurfactants in the field of pesticides is still in its infancy compared to the field of hydrocarbons. In India, a number of laboratories have initiated studies on BS. Some of the earlier works are by: (a) Banarjee et al. on 2,4,5-tricholoacetic acid, (b) Patel and Gopinath on Fenthion, and (c) Anu Appaiah and Karanth on alpha HCH. Very recently reports on production of microbial BS, based on preliminary studies by several groups, have appeared in posters/proceedings of symposia. The noteworthy feature being the increasing interest shown by the various researchers on: (a) degradation of pesticides, (b) production and exploitation of BS for the removal of pesticides from the environment, and (c) postulates on the possible replacement of synthetic surfactants with the biosurfactants in the pesticide formulation and clean-up. Hexa-chlorocyclohexane (HCH) is still the highest ranking pesticide used in many countries. Of the eight known isomers of HCH, the alpha-form constitutes more than 70% of the technical product, which is not only noninsecticidal but also a suspected carcinogen. The use of technical HCH, which is a mixture of isomers, will continue in the Indian market because of their all-time availability with good insecticidal efficiency and at a price which is 10–12 times less than that of the pure gamma HCH (Lindane). It is pertinent to note that the environment burden of already-dumped HCH continues to pose threat to all forms of ‘life’. The poor solubility is one of the limiting factors in the microbial degradation of alpha-HCH. Presence of six chlorines in the molecule is another factor that renders HCH lipophilic and persistent in the biosphere. Even though several reports are available on biodegradation of specific isomers of HCH in animals, plants, soil and microbial systems, literature on metabolism of alpha-HCH by microorganisms is limited. Furthermore, the exact mechanism of translocation of HCH to the site of destruction and degradation of alpha-HCH in bacteria is not well understood. During the course of work at CFTRI on the bacterial degradation of alpha-HCH, they isolated several bacterial strains capable of degrading HCH. One of the strains efficient in HCH degradation was characterized as Pseudomonas Ptm+ strain. The CFTRI isolate produced extracellular biosurfactant in a mineral medium containing HCH. While this BS emulsified the solid organochlorine-HCH to a higher extent, it emulsified other organochlorines such as DDT and cyclodienes to a lesser extent, implying thereby the specificity of the BS in dispersing HCH. It was also demonstrated that the peak in production of the emulsifier appeared before the onset of HCH degradation by the Pseudomonas growing in liquid culture. The role of biosurfactant in the HCH degradation was ascertained using partially purified BS. The extracellular BS was a macro-molecule containing lipid, carbohydrate, and protein moieties. The carbohydrate part was identified as rhamnose by different analytical methods. The rhamnose part of the BS was stable and was necessary for the BS activity. Careful investigations revealed that the protein fraction represented the proximal enzymes of HCH metabolism. In the presence of BS, HCH was converted through the involvement of isomerase and dechlorinase to tertachlorohexenes and then to chlorophenols. The BS acted by increasing the surface area of HCH, which accelerated this transformation. Hence, it is evident that extracellular BS has a definite role in HCH degradation by CFTRI strain of Pseudomonas Ptm+. Production of BS for Fenthion, a liqiud OP insecticide, has also received attention. Bacillus subtilis excreted the BS both in liqiud as well as in solid state fermentation system. The microbial surfactant produced by these two organisms also shows properties of a good cleansing agent for dislodging the pesticides from used containers, mixing tanks, cargo docks, etc. Attempts have also been made to standardize parameters for BS production both in liquid and solid state fermentations. A limited number of scale-up studies indicate good scope for expolitation of BS in industries. In a separate study, it has been shown that addition of BS from Pseudomonas Ptm+ strain facilitied 250-fold increase in dispersion of HCH in water. Addition of either this organism or BS dislodged surface-borne HCH residues from many types of fruits, seeds and vegetables as well. Laboratory-scale studies have revealed that BS is very efficient in cleaning the containers where HCH residues were sticking to the wall. Studies using fermentor for large-scale production of this BS from Pseudomonas Ptm+ have been carried out. A bioformulation is planned from this BS for effective removal of HCH from contaminated soils.

     1.2.5. The significance of biosurfactant for cell architecture. The importance of biosurfactants to cellular architecture has recently been reported for a number of bacteria. These include the role of surfactin in Bacillus subtilis fruiting body formation, the role of rhamnolipid in Pseudomonas aeruginosa biofilm formation, and the role of streptofactin in the formation of Streptomyces tendae aerial mycelia. In terms of structure, biosurfactants are amphiphilic molecules, containing distinct polar and nonpolar moieties that confer the ability to accumulate at surfaces and interfaces, hence the term surface active.

     1.3. The description of the actual knowledge stage

     1.3.1. Microbial production of biosurfactant and their importance. A large variety of microorganisms produce potent surface-active agents, biosurfactants, which vary in their chemical properties and molecular size. While the low molecular weight surfactants are often glycolipids, the high molecular weight surfactants are generally either polyanionic heteropolysaccharides containing covalently-linked hydrophobic side chains or complexes containing both polysaccharides and proteins. The yield of the biosurfactant greatly depends on the nutritional environment of the growing organism. The enormous diversity of biosurfactants makes them an interesting group of materials for application in many areas such as agriculture, public health, food, health care, waste utilization, and environmental pollution control such as in degradation of hydrocarbons present in soil.

     Biosurfactants (BS) are amphiphilic compounds produced on living surfaces, mostly microbial cell surfaces, or excreted extracellularly and contain hydrophobic and hydrophilic moieties that reduce surface tension (ST) and interfacial tensions between individual molecules at the surface and interface, respectively. Since BS and bioemulsifiers both exhibit emulsification properties, bioemulsifiers are often categorized with BS, although emulsifiers may not lower surface tension. A biosurfactant may have one of the following structures: mycolic acid, glycolipids, polysaccharide–lipid complex, lipoprotein or lipopeptide, phospholipid, or the microbial cell surface itself.

     Considerable attention has been given in the past to the production of surface-active molecules of biological origin because of their potential utilization in food-processing, pharmacology, and oil industry. Although the type and amount of the microbial surfactants produced depend primarily on the producer organism, factors like carbon and nitrogen, trace elements, temperature, and aeration also affect their production by the organism.

     Hydrophobic pollutants present in petroleum hydrocarbons, and soil and water environment require solubilization before being degraded by microbial cells. Mineralization is governed by desorption of hydrocarbons from soil. Surfactants can increase the surface area of hydrophobic materials, such as pesticides in soil and water environment, thereby increasing their water solubility. Hence, the presence of surfactants may increase microbial degradation of pollutants. Use of biosurfactants for degradation of pesticides in soil and water environment has gained importance only recently. The identification and characterization of biosurfactant produced by various microorganisms have been extensively reviewed.

     1.3.2. Microorganisms. 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 (CxHy), microorganisms facilitate their diffusion into the cell by producing a variety of substances, the biosurfactants. Some bacteria and yeasts excrete ionic surfactants which emulsify the CxHy substrate in the growth medium. Some examples of this group of BS are rhamnolipids which are produced by different Pseudomonas sp., or the sophorolipids which are produced by several Torulopsis sp. 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 C. tropicalis which produce cell wall-bound lipopolysaccharides when growing on n-alkanes; and Rhodococcus erythropolis, and many Mycobacterium sp. and Arthrobacter sp. which synthesize nonionic trehalose corynomycolates. There are lipopolysaccharides, such as Emulsan, synthesized by Acinetobacter sp., and lipoproteins or lipopeptides, such as Surfactin and Subtilisin, produced by Bacillus subtilis. Other effective BS are: (a) Mycolates and Corynomycolates which are produced by Rhodococcus sp., Corynebacteria sp., Mycobacteria sp., and Nocardia sp.; and (b) ornithinlipids, which are produced by Pseudomonas rubescens, Gluconobacter cerinus, and Thiobacillus ferroxidans.

     1.3.3. Classification and chemical nature of biosurfactants. The microbial surfactants (MS) are complex molecules covering a wide range of chemical types including peptides, fatty acids, phospholipids, glycolipids, antibiotics, lipopeptides, etc. Microorganisms also produce surfactants that are in some cases combination of many chemical types: referred to as the polymeric microbial surfactants (PMS). Many MS have been purified and their structures elucidated. While the high molecular weight MS are generally polyanionic heteropolysaccharides containing both polysaccharides and proteins, the low molecular weight MS are often glycolipids. The yield of MS varies with the nutritional environment of the growing microorganism. Intact microbial cells that have high cell surface hydrophobicity are themselves surfactants. In some cases, surfactants themselves play a natural role in growth of microbial cells on water-insoluble substrates like CxHy, sulphur, etc. Exocellular surfactants are involved in cell adhesion, emulsification, dispersion, flocculation, cell aggregation, and desorption phenomena. A very brief description of each group is given below:

1.3.3.1.Glycolipids are the most common types of BS. The constituent mono-, di-, tri- and tetrasaccharides include glucose, mannose, galactose, glucuronic acid, rhamnose, and galactose sulphate. The fatty acid component usually has a composition similar to that of the phospholipids of the same microorganism. The glycolipids can be categorized as:

a)Trehalose lipids: The serpentine growth seen in many members of the genus Mycobacterium is due to the presence of trehalose esters on the cell surface. Cord factors from different species of Mycobacteria, Corynebacteria, Nocardia, and Brevibacteria differ in size and structure of the mycolic acid esters.

b)Sophorolipids: These are produced by different strains of the yeast, Torulopsis. The sugar unit is the disaccharide sophorose which consists of two b -1,2-linked glucose units. The 6 and 6¢ hydroxy groups are generally acetylated. The sophorolipids reduce surface tensions between individual molecules at the surface, although they are effective emulsifying agents. The sophorolipids of Torulopsis have been reported to stimulate, inhibit, and have no effect on growth of yeast on water-insoluble substrates.

 c)Rhamnolipids: Some Pseudomonas sp. produce large quantities of a glycolipid consisting of two molecules of rhamnose and two molecules of b -hydroxydecanoic acid. While the OH group of one of the acids is involved in glycosidic linkage with the reducing end of the rhamnose disaccharide, the OH group of the second acids is involved in ester formation. Since one of the carboxylic acid is free, the rhamnolipids are anions above pH 4.0. Rhamnolipids are reported to lower surface tension, emulsify CxHy, and stimulate growth of Pseudomonas on n-hexadecane. Formation of rhamnolipids by Pseudomonas sp. MVB was greatly increased by nitrogen limitations. The pure rhamnolipid lowered the interfacial tension against n-hexadecane in water to about 1 mN/m and had a critical micellar concentration (cmc) of 10 to 30 mg/l depending on the pH and salt conditions.

1.3.3.2. Fatty acids produced from alkanes by microbial oxidations have received maximum attention as surfactants. Besides the straight-chain acids, microorganisms produce complex fatty acids containing OH groups and alkyl branches. Some of these complex acids, for example corynomucolic acids, are surfactants.

1.3.3.3. Phospholipids are major components of microbial membranes. When certain CxHy-degrading bacteria or yeast are grown on alkane substrates, the level of phospholipids increases greatly. Phospholipids from hexadecane-grown Acinetobacter sp. have potent surfactant properties. Phospholipids produced by Thiobacillus thiooxidans have been reported to be responsible for wetting elemental sulphur, which is necessary for growth.

 1.3.3.4. Surface active antibiotics

a)Gramicidin S: Many bacteria produce a cyclosymmetric decapeptide antibiotic, gramicidin S. Spore preparations of Brevibacterium brevis contain large amounts of gramicidin S bound strongly to the outer surface of the spores. Mutants lacking gramicidin S germinate rapidly and do not have a lipophilic surface. The antibacterial activity of gramicidin S is due to its high surface activity.

b) Polymixins: These are a group of antibiotics produced by Brevibacterium polymyxa and related bacilli. Polymixin B is a decapeptide in which amino acids 3 through 10 form a cyclic octapeptide. A branched chain fatty acid is connected to the terminal 2,4-diaminobutyric acid (DAB). Polymixins are able to solubilize certain membrane enzymes.

c)Surfactin (subtilysin): One of the most active biosurfactants produced by B. subtilis is a cyclic lipopeptide surfactin. The yield of surfactin produced by B. subtilis can be improved to around 0.8 g/l by continuously removing the surfactant by foam fractionation and addition of either iron or manganese salts to the growth medium.

d)Antibiotic TA: Myxococcus xanthus produces antibiotic TA which inhibits peptidoglycan synthesis by interfering with polymerization of the lipid disaccharide pentapeptide. Antibiotic TA has interesting chemotherapeutic applications.

1.3.3.5. Polymeric microbial surfactants Most of these are polymeric heterosaccharide containing proteins.

 a) Acinetobacter calcoaceticus RAG-1 (ATCC 31012) emulsan: A bacterium, RAG-1, was isolated during an investigation of a factor that limited the degradation of crude oil in sea water. This bacterium efficiently emulsified CxHy in water. This bacterium, Acinetobacter calcoaceticus, was later successfully used to clear a cargo compartment of an oil tanker during its ballast voyage. The cleaning phenomenon was due to the production of an extracellular, high molecular weight emulsifying factor, emulsan.

b)The polysaccharide protein complex of Acinetobacter calcoaceticus BD413: A mutant of A. calcoaceticus BD4, excreted large amounts of polysaccharide together with proteins. The emulsifying activity required the presence of both polysaccharide and proteins.

c)Other Acinetobacter emulsifiers: Extracellular emulsifier production is widespread in the genus Acinetobacter. In one survey, 8 to 16 strains of A. calcaoceticus produced high amounts of emulsifier following growth on ethanol medium. This extracellular fraction was extremely active in breaking (de-emulsifying) kerosene/ water emulsion stabilized by a mixture of Tween 60 and Span 60.

d)Polysaccharide-lipid complexes from yeast: The partially purified emulsifier, liposan, was reported to contain about 95% carbohydrate and 5% protein. A CxHy-degrading yeast, Endomycopsis lipolytica YM, produced an unstable alkane-solubilizing factor. Torulopsis petrophilum produced different types of surfactants depending on the growth medium. On water-insoluble substrates, the yeast produced glycolipids which were incapable of stabilizing emulsions. When glucose was the substrate, the yeast produced a potent emulsifier.

e)Emulsifying protein (PA) from Pseudomonas aeruginosa: The bacterium P. aeruginosa has been observed to excrete a protein emulsifier. This protein PA is produced from long-chain n-alkanes, 1-hexadecane, and acetyl alcohol substrates; but not from glucose, glycerol or palmitic acid. The protein has a MW of 14,000 Da and is rich in serine and threonine.

 f)Surfactants from Pseudomonas PG-1: Pseudomonas PG-1 is an extremely efficient hydrocarbon-solubilizing bacterium. It utilizes a wide range of CxHy including gaseous volatile and liquid alkanes, alkenes, pristane, and alkyl benzenes.

 g)Bioflocculant and emulcyan from the filamentous Cyanobacterium phormidium J-1: The change in cell surface hydrophobicity of Cyanobacterium phormidium was correlated with the production of an emulsifying agent, emulcyan. The partially purified emulcyan has a MW greater than 10,000 Da and contains carbohydrate, protein and fatty acid esters. Addition of emulcyan to adherent hydrophobic cells resulted in their becomeing hydrophilic and detach from hexadecane droplets or phenyl sepharose beads.

6. Particulate surfactants

a)Extracellular vesicles from Acinetobacter sp. H01-N: 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 a role in the uptake of alkanes by Acinetobacter sp. HO1-N.

b)Microbial cells with high cell surface hydrophobicities: 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.

 

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