EE-Unit V Biosurfactants
Biosurfactants are biological surface-active agents capable of reducing interfacial tension between liquids, solids and gases, thereby allowing them to mix and disperse readily in water or other liquids. (Bio)surfactants are amphiphilic molecules consisting of a hydrophilic and a hydrophobic moiety that interacts with the phase boundary in heterogeneous systems. The non-polar “tail” is typically a hydrocarbon chain whereas the polar “head” appears in many different varieties such as carbohydrates, amino acids or phosphates.
Surfactants are used for a wide variety of applications in households, industry and agriculture. They are extensively used in cleaning applications and as a formulation aid to promote solubilisation, emulsification and dispersion of other molecules in products ranging from chemicals, cosmetics, detergents, foods, textiles and pharmaceuticals. Surfactants are molecules that intervene in nearly every product and every aspect of human daily life.
In addition to their use as a formulation aid, certain surfactants can also be used as an active compound with antimicrobial, antitumor, antiviral or immunological properties or as inducers of cell differentiation. This has resulted in a number of potential applications and related developments in biomedical sciences. Also in plant protection, apart from their general use as a formulation and dispersion aid, certain surfactants are actually the active ingredient. Biosurfactants such as rhamnolipids are known to have very high and specific antimicrobial activity against the zoospores of Phytophtora, one of the most important phytopathogenic fungi.
The large majority of the currently used surfactants are petroleum-based and are produced by chemical means. These compounds are often toxic to the environment and their use may lead to significant environmental problems, particularly in washing applications as these surfactants inevitably end up in the environment after use. The eco-toxicity, bio-accumulation and biodegradability of surfactants are therefore issues of increasing concern. Biosurfactants are an alternative, as they combine good functional properties with low environmental impact and excellent skin compatibility. Moreover, biosurfactants can be produced by fermentation from renewable resources, typically from sugars and vegetable oils.
The structure of biosurfactants is predominantly determined by the producing organism, but can to a certain extent be influenced by the culture conditions. Biosurfactants can be classified in four groups based on their chemical composition: glycolipids (1), oligopeptides and lipopeptides (2), phospholipids, fatty acids and neutral lipids (3) and polymeric biosurfactants (4). In addition to those four basic groups, there also exists biosurfactants build of carbohydrates, fatty acids and peptides, and sometimes external cell components or even whole cells show surface tension lowering properties.
The most promising group of biosurfactants are the glycolipids, this group will be discussed more in detail.
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-processing1–3, 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 reviewed4–6. Therefore, rather than describing the numerous types of biosurfactants and their properties, this article emphasizes the production of biosurfactants and their role in biodegradation of pesticides.
Microbiology
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.7–11, or the sophorolipids which are produced by several Torulopsis sp.12–14. 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-alkanes15,16; and Rhodococcus erythropolis, and many Mycobacterium sp. and Arthrobacter sp. which synthesize nonionic trehalose corynomycolates14,17–23. There are lipopolysaccharides, such as Emulsan, synthesized by Acinetobacter sp.22,23, and lipoproteins or lipopeptides, such as Surfactin and Subtilisin, produced by Bacillus subtilis24–26. Other effective BS are: (i) Mycolates and Corynomycolates which are produced by Rhodococcus sp., Corynebacteria sp.,Mycobacteria sp., and Nocardia sp.24,27,28; and (ii) ornithinlipids, which are produced by Pseudomonas rubescens, Gluconobacter cerinus, and Thiobacillus ferroxidans29–31. BS produced by various microorganisms together with their properties are listed in Table 1.
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 sub-
strates like CxHy, sulphur, etc. Exocellular surfactants are involved in cell adhesion, emulsification, dispersion, flocculation, cell aggregation, and desorption phenomena. A broad classification of BS is given in Table 2. A very brief description of each group is given below.Glycolipids
Glycolipids are the most common types of BS (ref. 32). 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:
Trehalose lipids: The serpentine growth seen in many members of the genus Mycobacterium is due to the presence of trehalose esters on the cell surface33,34. Cord factors from different species of Mycobacteria33,35–37, Corynebacteria38, Nocardia, and Brevibacteria differ in size and structure of the mycolic acid esters.
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 agents13,39,40. The sophorolipids of Torulopsis have been reported to stimulate41,42, inhibit41,43, and have no effect8 on growth of yeast on water-insoluble substrates.
Rhamnolipids: Some Pseudomonas sp. produce large quantities of a glycolipid consisting of two molecules of rhamnose and two molecules of b -hydroxydecanoic acid44,45. 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 reported46 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 limitations47. 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 conditions48.
Fatty acids
The fatty acids produced from alkanes by microbial oxidations have received maximum attention as surfactants49. 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 surfactants24,28,50.
Phospholipids
These are major components of microbial membranes. When certain CxHy-degrading bacteria51–53 or yeast54–56 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 growth57,58.
Surface active antibiotics
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 spores59,60. Mutants lacking gramicidin S germinate rapidly and do not have a lipophilic surface61. The antibacterial activity of gramicidin S is due to its high surface activity62–65.
Polymixins: These are a group of antibiotics produced by Brevibacterium polymyxa and related bacilli66. 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 enzymes67.
Surfactin (subtilysin): One of the most active biosurfactants produced by B. subtilis is a cyclic lipopeptide surfactin26,68. 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 medium24.
Antibiotic TA: Myxococcus xanthus produces antibiotic TA which inhibits peptidoglycan synthesis by interfering with polymerization of the lipid disaccharide pentapeptide69. Antibiotic TA has interesting chemotherapeutic applications70.
Polymeric microbial surfactants
Most of these are polymeric heterosaccharide containing proteins.
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 water71. This bacterium, Acinetobacter calcoaceticus, was later successfully used to clear a cargo compartment of an oil tanker during its ballast voyage22,72. The cleaning phenomenon was due to the production of an extracellular, high molecular weight emulsifying factor22, emulsan.
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 proteins73,74.
Other Acinetobacter emulsifiers: Extracellular emulsifier production is widespread in the genus Acinetobacter. In one survey75, 8 to 16 strains of A. calcaoceticus produced high amounts of emulsifier following growth on ethanol medium76,77. This extracellular fraction was extremely active in breaking (de-emulsifying) kerosene/ water emulsion stabilized by a mixture of Tween 60 and Span 60.
Polysaccharide-lipid complexes from yeast: The partially purified emulsifier, liposan, was reported to contain about 95% carbohydrate and 5% protein78. A CxHy-degrading yeast, Endomycopsis lipolytica YM, produced an unstable alkane-solubilizing factor79. Torulopsis petrophilum produced different types of surfactants depending on the growth medium39. 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.
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 threonine80.
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 benzenes79,81,82.
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, emulcyan85. 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.
Particulate surfactants
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. (refs 57, 84).
Microbial cells with high cell surface hydrophobicities: Most hydrocarbon-degrading microorganisms, many nonhydrocarbon degraders, some species of Cyanobacteria85, and some pathogens have a strong affinity for hydrocarbon-water70 and air-water86,87 interfaces. In such cases, the microbial cell itself is a surfactant.
Factors affecting biosurfactant production
Biosurfactants (BS) are amphiphilic compounds. They contain a hydrophobic and hydrophilic moiety. The polar moiety can be a carbohydrate, an amino acid, a phosphate group, or some other compound. The nonpolar moiety is mostly a long-carbon-chain fatty acid. Although the various BS possess different structures, there are some general phenomena concerning their biosynthesis. For example, BS production can be induced by hydrocarbons or other water-insoluble substrates88. This effect, described by different authors, refers to many of the interfacially active compounds. Another striking phenomena is the catabolic repression of BS synthesis by glucose and other primary metabolites. For example, in the case of Arthrobacter paraffineus, no surface-active agent could be isolated from the medium when glucose was used as the carbon source instead of hexadecane89. Similarly, a protein-like activator for n-alkane oxidation was formed by P. aeruginosa S7B1 from hydrocarbon, but not from glucose, glycerol, or palmitic acid80,81. Torulopsis petrophilum did not produce any glycolipids when grown on a single-phase medium that contained water-soluble carbon source13. When glycerol was used as substrate, rhamnolipid production by P. aeruginosa was sharply reduced by adding glucose, acetate, succinate or citrate to the medium8,10.
Olive oil mill effluent, a major pollutant of the agricultural industry in mediterranian countries, has been used as raw material for rhamnolipid biosurfactant production byPseudomonas sp. JAMM. Many microorganisms are known to synthesize different types of biosurfactants when grown on several carbon sources6,90. However, there have been examples of the use of a water-soluble substrate for biosurfactant production by microorganisms91,92. The type, quality and quantity of biosurfactant produced are influenced by the nature of the carbon substrate93, the concentration of N, P, Mg, Fe, and Mn ions in the medium9,24,94,95, and the culture conditions, such as pH, temperature, agitation and dilution rate in continous culture9,95–97.
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 reserviors. Their production, on the other hand, in many cases improves with increased salinity. Thus, they are the biosurfactants of choice for the Venezuelan oil industry and in the cosmetics, food, and pharmaceutical markets.
The nitrogen source can be an important key to the regulation of BS synthesis. Arthrobacter paraffineus ATCC 19558 preferred ammonium to nitrate as inorganic nitrogen source for BS production. Urea also result in increased BS production89. A change in growth rate of the concerned microorganisms is often sufficient to result in over production of BS (ref. 27). In some cases24, addition of multivalent cations to the culture medium can have a positive effect on BS production. Besides the regulation of BS by chemicals indicated above, some compounds like ethambutol20,98, penicillin99, chloramphenicol23, and EDTA79,100 influenced the formation of interfacially active compounds. The regulation of BS production by these compounds is either through their effect on solubilization of nonpolar hydrocarbon substrates or by increased production of water-soluble (polar) substrates. In some cases, BS synthesis is regulated by pH and temperature. For example in rhamnolipid production by Pseudomonas sp.101,102, in cellobioselipid formation by Ustilago maydis103, and in sophorolipid formation by Torulopsis bombicola42, pH played an important role, and in the case of Arthrobacter paraffineus ATCC 19558 (ref. 104), Rhodococcus crythropolis101,102, and Pseudomonas sp. DSM 2874 (refs 47, 102) temperature was important. In all these cases however the yield of BS production was temperature dependent.
Applications of biosurfactants in pollution control
The identification and characterization of microbial surfactants produced by various microorganisms have been extensively reviewed6,88,105–107. Therefore rather than describing numeric types of MS, it is proposed to examine potential applications of MS.
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 laboratory108,109. Clark et al.110, 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 cases111.
Hydrocarbon degradation
Hydrocarbon-utilizing microorganisms excrete a variety of biosurfactants. BS being natural products, are biodegradable and consequently environmentally safe. An important group of BS is mycolic acids which are the a -alkyl, b -hydroxy very long-chain fatty acids contributing to some characteristic properties of a cell such as acid fastness, hydrophobicity, adherability, and pathogenicity. Enriching waters and soils with long- and short-chain mycolic acids may be potentially hazardous. Daffe et al.112 reported trehalose polyphthienoylates as a specific glycolipid in virulent strains of Mycobacterium tuberculosis. Kaneda et al.113 reported that granuloma formation and hemopoiesis could be induced by C36–C48 mycolic acid-containing glycolipids from Nocardia rubra. Biolid extract (BE), obtained as a byproduct during the production of fodder yeast, is a dark brown heavy fluid with a characteristic odour and high interfacial activity. This product has many applications in agrochemistry, mineral flotation, and bitumen production and processing. Potentially, the product may be used as an emulsifying and dispersing agent while formulating herbicides, pesticides, and growth regulator preparations. Including phospholipids in formulations, facilitate penetration of active substances into the plant tissues114, making it possible to apply only very low concentrations of the substances115. The constituent fatty acids of biolipid extract have antiphytoviral and antifungal activities and therefore, can be applied in controlling plant diseases116. These fatty acids also increase stress tolerance of plants, leading thereby to higher yields despite physiological drought117.
Hydrocarbon degradation in the soil environment
CxHy degradation in soil has been extensively studied31,95,118–122. 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 degradation122.
Use of MS in CxHy degradation has produced variable results. In the work of Lindley and Heydeman123, 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.124 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-Harting125 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 yield126.
Biodetox (Germany) described a process to decontaminate soils, industrial sludges, and waste waters127. 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 (ref. 128). Removal of PCBs and petroleum CxHy from soil by adding surfactants to the wash water, has met with some success129.
Several strains of anaerobic bacteria produce biosurfactants130,131. 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) (ref. 106). MS can also be used to enhance solubilization of toxic organic chemicals including xenobiotics. Berg et al.132, 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 theP. 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 chlorophenols133. Thus, this emulsifier can be used
to enhance bacterial degradation of organochlorine compounds.Hydrocarbon degradation in aquatic environment
When oil is spilled in aquatic environment, the lighter hydrocarbon components volatilize while the polar hydrocarbon components dissolve in water. However, because of low solubility (< 1 ppm) of oil, most of the oil components will remain on the water surface. The primary means of hydrocarbon removal are photooxidation, evaporation, and microbial degradation. Since CxHy-degrading organisms are present in seawater, biodegradation may be one of the most efficient methods of removing pollutants95, 134. Surfactants enhance degradation by dispersing and emulsifying hydrocarbons. Microorganisms that are able to degrade CxHy have been isolated from aquatic environment. These microorganisms which exhibit emulsifying activity as well as the soil microorganisms which produced surfactants may be useful in aquatic environment. Chakrabarty136reported that an emulsifier produced by P. aeruginosa SB30 was able to quickly disperse oil into fine droplets; therefore it may be useful in removing oil from contaminated beaches135. BS produced by oil-degrading bacteria may be useful in cleaning oil tanks. When an oil tanker compartment containing oily ballast water was supplemented with urea and K2HPO4 and aerated for 4 days, the tanker was completely free of the thick layer of sludge that remained in the control tanker137. Presumably this was owing to the surfactant produced, when growth of the natural bacterial population was enhanced.
Surfactants have been studied for their use in reducing viscosity of heavy oils, thereby facilitating recovery, transportation, and pipelining138,139. Emulsan, a high MW lipopolysacharide produced by A. calcaoceticus RAG-1, has been proposed for a number of applications in the petroleum industry such as to clean oil and sludge from barges and tanks, reduce viscosity of heavy oils, enhance oil recovery, and stabilize water-in-oil emulsions in fuels140,141. Specific solubilization of various CxHy types during growth of prokaryotic organism was demonstrated by Reddy et al.79,81. The specific solubilization of CxHy was strongly inhibited by EDTA which was overcome by excess Ca++. It was concluded that specific solubilization of CxHy is an important mechanism in the microbial uptake of CxHy.
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: (i) Banarjee et al.133 on 2,4,5-tricholoacetic acid, (ii) Patel and Gopinath on Fenthion142, and (iii) Anu Appaiah and Karanth143 on alpha HCH. Very recently reports on production of microbial BS, based on preliminary studies by several groups, have appeared in posters/proceedings of symposia144–148. The noteworthy feature being the increasing interest shown by the various researchers on: (i) degradation of pesticides149–152, (ii) production and exploitation of BS for the removal of pesticides from the environment, and (iii) postulates on the possible replacement of synthetic surfactants with the biosurfactants in the pesticide formulation and clean-up153–156.
Biosurfactant and HCH degradation
Hexa-chlorocyclohexane (HCH) is still the highest ranking pesticide used in India and many other 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 mechnism of translocation of HCH to the site of destruction and degradation of alpha-HCH in bacteria is not well understood.
During the course of our work at CFTRI on the bacterial degradation of alpha-HCH, we 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 extent156, 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 thePseudomonas 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 chlorophenols157.
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 system146,147. 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 vegetables158 as well. Laboratory-scale studies have revealed that BS is very efficient in cleaning the containers where HCH residues were sticking to the wall (Figure 1). Studies using fermentor for large-scale production of this BS from Pseudomonas Ptm+ have been carried out159. A bioformulation is planned from this BS for effective removal of HCH from contaminated soils.
Other applications
By virtue of properties of biodegradability, substrate specificity, chemical and functional diversity, and rapid/ controlled inactivation, biosurfactants are gaining importance in various industires like agriculture, food, textiles, petrochemicals, etc. The potential applications of biosurfactants having desired functions and properties are listed in Table 394,160,161. The current consumption rate and estimated demand pattern for synthetic surfactants are shown in Table 4. Number of patents available on the subject are given in Table 5.
BS from some other bacterial taxa may be of public health concern. Methylrhamnolipids from Pseudomonas aeroginosa have cytotoxic effects163. Lipopolyglycans from mycoplasmas show endotoxic properties, potentially inducing procoagulant activity in human leukocytes164. The toxicity and antigenic properties of mycobacterial glycolipids, produced by pathogenic mycobacteria such as M. avium-intracellure, M. scrofulaceum, and M. fortulitum, which are habitats of water polluted with industrial and domestic residues, are well known165,166. The varied uses of BS also imply scope for MS, and the need to strengthen the research in this emerging area.
Leave a Reply
Want to join the discussion?Feel free to contribute!