Determination of genetic basis for biosurfactant production in distillery and curd whey wastes utilizing Pseudomonas aeruginosa strain BS2

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1 Indian Journal of Biotechnology Vol 3, January 2004, pp Determination of genetic basis for biosurfactant production in distillery and curd whey wastes utilizing Pseudomonas aeruginosa strain BS2 Kirti Dubey and Asha Juwarkar* National Environmental Engineering Research Institute, Nagpur , India Received 28 January 2002; accepted 26 June 2003 Pseudomonas aeruginosa strain BS2 has been demonstrated to have an ability to produce potent biosurfactant, an ecofriendly substitute to synthetic surfactants from distillery and whey wastes and capable of reducing the pollution load of these wastes in the range of 85-90%. To determine the basis for future identification of the genes responsible for biosurfactant production from wastes, studies on the presence of plasmid if any, its profile and role in biosurfactant production were performed. Suitable plasmid screening technique was selected because strain BS2 produced excessive slime in Luria Burnetti broth, which interfered with the migration and detection of plasmid. Among the several methods, alkaline lysis method was the most suitable which aided in recovery of slime-free cell lysate and resulted in the formation of a discrete band of plasmid in agarose gel. Plasmid profile study demonstrated that plasmid had high molecular weight of Da and possessed the genetic determinants for antibiotics (chloramphenicol, tetracycline and sulphonamide) and heavy metal salt (mercuric chloride) resistance and were used as markers in curing experiment. To determine the role of megaplasmid in biosurfactant production, curing of megaplasmid was performed at highest sublethal doses of acridine orange (100 g/ml) and mitomycin-c (15 g/ml). Results indicated that only mitomycin-c treatment resulted in 28% of cell population which turned sensitive towards marker antibiotics and heavy metal salt due to loss of megaplasmid, which was further confirmed by agarose gel electrophoresis. Comparative analysis of biosurfactant production potential of cured cells with that of wild cells in both the wastes showed that the cured cells had similar potential capability of biosurfactant production as of wild strain which illustrates that genes responsible for biosurfactant production in distillery and whey wastes utilizing strain BS2 were not plasmid borne but resided on the chromosome where they are more stable. Keywords: biosurfactant, curd whey, curing, distillery waste, plasmid, Pseudomonas aeruginosa strain BS2 Introduction Biosurfactants are the biologically synthesized surface-active agents which are beginning to acquire a status of potential performance-effective molecules in various fields due to their low toxicity, biodegradable nature and diversity. Their range of potential industrial applications include enhanced oil recovery, crude oil drilling, lubricants, surfactant-aided bioremediation of water-insoluble pollutants, cosmetics, healthcare and in food processing industries. The latest developments in biosurfactant applications and their potential role in newly emerging fields have been widely reviewed 1, 2. The economics of biosurfactant production has now started receiving considerable attention. In this context, economic strategies have been developed to use industrial effluent as no-cost substrate for biosurfactant production by Pseudomonas aeruginosa strain BS2 3,4. However, to replace chemically *Author for correspondence: Tel.: , extn 210; Fax : AAJuwarkar@rediffmail.com synthesised surfactants with biosurfactant, the understanding of physiology, genetics and biochemistry of biosurfactant producing organisms is necessary. Ochsner et al have studied the expression of the cloned rhl AB genes encoding for rhamnosyl transferase in the heterologous hosts, which catalyze final step in rhamnolipid II (L-rhamnosyl-3-hydroxy decanoyl-3 hydroxydecanoate) synthesis and found that in nitrogen-limited glucose synthetic (GS) minimal medium, the expression of biosurfactant synthesis in various hosts was different 5. This indicated that only completely defined pure substrates could be used for evaluation of biosurfactant production capabilities in heterologous host. Until now, only few significant works on genetics of industrial waste utilizing organisms have been reported. It was reported that biosurfactant producing ability of one microbe can be combined with that of others to utilize waste substrate 6. This was made possible by alteration in substrate requirements of P. aeruginosa strain by insertion of a Lac plasmid from E. coli, which made it to produce rhamnolipid from whey, a waste product from dairy industry.

2 DUBEY & JUWARKAR: GENETIC BASIS OF BIOSURFACTANT SYNTHESIS FROM WASTES 75 Production of rhamnolipids in P. aeruginosa is chromosomal encoded phenomenon when glucose, hexadecane and tetradecane are used in mineral salts medium 1. However, in the presence of industrial wastes as no-cost nutrient medium, growth of biosurfactant producing strain and biosurfactant production may be linked to plasmid or chromosome, or both. These two aspects are addressed in the present study to demonstrate the genetic basis of biosurfactant production when industrial wastes such as distillery and whey wastes are used as no-cost nutrient medium. Moreover, this study will provide a basis for future identification of the genes responsible for biosurfactant production and a target site for future genetic manipulation such as to clone, amplify, delete and transfer of genes required for isolation of stable, high expression of biosurfactant production from nocost substrate. Materials and Methods Organism and Growth Conditions for Biosurfactant Production Pseudomonas aeruginosa strain BS2, an oily sludge isolate was used for biosurfactant production from distillery and curd whey wastes. Wastes were processed prior to sterilization, then inoculated with freshly grown cells of strain BS2 and biosurfactant production was carried out in batch mode as described earlier 3. Growth of the organism in both the wastes, surface tension reduction of culture broth and biosurfactant yield were monitored. Standardization of Method for Screening of Plasmids in Biosurfactant producing strain BS2 For standardization of plasmid screening method in strain BS2, several methods were tried 7-9. The cell lysates obtained by following these methods were subjected to DNA precipitation by chilled absolute ethanol (2 volumes) and stored at -20 o C for 18 hrs. The precipitated DNA was centrifuged at 4 o C and 12,000 rpm for 10 min. The supernatant was removed by gentle aspiration and the microcentrifuge tubes were kept in an inverted position on a clean tissue paper to allow all the liquid to drain away. The dried pellets were dissolved in 20 μl TE buffer (ph 8) and stored at -20 o C before subjecting to electrophoresis. For agarose gel electrophoresis of plasmid DNA, a low salt tank buffer system consisting of Tris acetate- 40 mm, EDTA sodium salt 2 mm was used, which avoids over heating of buffer and is capable of resolving high molecular weight plasmid DNA 10. The DNA samples were suspended in loading dye made up of bromocresol purple, Tris acetate and glycerol 9. Plasmids were resolved by electrophoresis through 0.7% agarose gel and using low salt tank buffer at 10 V and 60 ma for 2 hrs until the dye reached the bottom of the gel. After completion of electrophoresis, the gel was stained in an aqueous solution of 0.5 g/ml ethidium bromide for 30 min, destained with sterile distilled water for 30 min and then visualized in Vilber Lourmat UV-transilluminator Kaiser RA-1. Determination of Molecular Weight of Plasmid DNA Escherichia coli V517 was used as a source of standard plasmid marker, which harboured eight different plasmids of known molecular weights viz., 35.8, 4.8, 3.7, 2.6, 2.0, 1.8 and 1.4 ( 10 6 Da). Fresh, 18 hrs cultures of strain BS2 and E. coli V517 in Luria broth were processed for plasmid screening by method of Brinboim and Doly 7. The molecular weight of plasmid DNA of strain BS2 was determined by its relative mobility with respect to the plasmid of known molecular weight 11. Selection of Antibiotic and Heavy Metal Salt Markers For selection of a suitable marker, strain BS2 was tested for its resistance to antibiotics (purchased from Hi media Laboratories Pvt. Ltd., Mumbai, India), viz. penicillin-g (10 units), ampicillin (10 μg), chloramphenicol (30 μg), sulphonamide (300 μg), bacitracin (10 units), methicillin (5 μg) and neomycin (30 μg) and a heavy metal salt, viz. mercuric chloride (20 μg) by standard disc assay method. Antibiotic discs were aseptically placed on the surface of Luria agar at a distance of 2 cm from each other after the plates were lawn cultured with fresh inoculum of strain BS2. Sterilized Whatman filter paper discs (5 mm in diam) were also similarly placed on lawn cultured Luria agar plates and 5 μl of membrane sterilized mercuric chloride solution (0.4%) was added onto each disc so that each was amended with 20 μg HgCl 2 (this concentration was previously determined as the minimum inhibitory concentration) for the strain BS2. The plates were incubated at 37 o C for 24 hrs and the zone size of growth inhibition was measured for evaluation of susceptibility. Determination of Highest Sublethal Concentrations of Curing Agents Acridine orange and mitomycin-c were used to cure the plasmid. Sublethal doses of these agents were determined to select optimum conditions for curing. The growth of strain BS2 was tested in Luria broth,

3 76 INDIAN J BIOTECHNOL, JANUARY 2004 separately containing acridine orange and mitomycin- C in the range of and 5-25 μg/ml, respectively. For this purpose, μl of membrane sterilized solutions of acridine orange (25 mg/10 ml distilled water) and μl of mitomycin-c solution (10 mg/10 ml phosphate buffer, ph 7) were added into the test tubes containing 10 ml of sterile Luria broth to attain the required concentrations of curing agent. The tubes were inoculated with 100 μl (0.1 OD) of 18 hrs culture. Luria broth tubes without curing agents were also inoculated with 100 μl of culture and were kept as control. The tubes were incubated in a gyro rotatory shaker at 37 o C and 150 rpm for 24 hrs in case of acridine-orange and for 48 hrs in case of mitomycin-c. The cells growing in presence of different concentrations of curing agents were enumerated by dilution and pour plate technique using Luria agar as growth medium and highest sublethal concentration of curing agent was determined. Curing of Plasmid Plasmid curing trials were carried out as per the method described by Trevors 12 using acridine orange and mitomycin-c at their highest sublethal doses, i.e. 100 μg/ml and 15 μg/ml, respectively, in Luria broth. Tetracycline (30 μg/ml), sulphonamide (300 μg/ml), chloramphenicol (30 μg/ml) and mercuric chloride (20 μg/ml) were separately amended in Luria agar and were used as markers in curing experiment. The cells which were unable to grow on the selective plates (Luria agar amended with marker antibiotics and heavy metal) were counted and their numbers were compared with those present in the master plate to determine percent curing 13. were picked from the nonselective plates (Luria agar master plates) and were preserved on nutrient agar slants at 4 o C. These cells were processed as per the method of Brinboim & Doly 7 to confirm the loss of plasmid. Biosurfactant Production from Distillery and Curd whey Wastes by Cured Cells Fresh inoculum having 10 5 c.f.u./ml of cured cells of strain BS2, raised in distillery and curd whey wastes, was separately inoculated in sterilized distillery and curd whey wastes. These wastes were processed before sterilization by earlier described method 3. The flasks were incubated at 37 o C and 150 rpm for 120 hrs. One flask of each waste was removed and after an interval of 24 hrs parameters such as biomass yield, surface tension and biosurfactant yield were determined. Analytical Methods Growth of wild and cured cells in distillery and curd whey wastes was determined in terms of c.f.u./ml by dilution and pour plate technique using Luria agar as growth medium. The surface tension of the cell free fermented wastes was determined by Du Nuoy ring detachment method by using Fisher s Autotensiomat model-21, Fisher Sci. Co., Pittsburg, Pennsylvania, USA. Biosurfactant yield was determined by method of Ramana and Karanth 14. Results and Discussion Biosurfactant Production from Distillery and Curd whey Wastes The authors have already shown that strain BS2 produced 0.91 and 0.92 g/l crystalline biosurfactant having potent surface active properties from distillery and curd whey wastes, respectively with no additional supplementation of any type of nutrient in these wastes 3. It was therefore, necessary to study the genetics of biosurfactant synthesis in strain BS2, which has in-built capacity to produce biosurfactant from industrial wastes viz. distillery and curd whey wastes. Since little is known about genetic basis of biosurfactant production from industrial wastes, studies were conducted to ascertain whether the genes encoding for biosurfactant production in P. aeruginosa reside on chromosome or plasmid DNA. Standardization of Method for Sscreening of Plasmids in Biosurfactant Producing strain BS2 Studies on standardization of method for screening plasmids in biosurfactant producing strain BS2 were carried out because during the growth of strain BS2 in Luria broth excessive slime is produced apart from the biosurfactant, which interfered with the recovery of plasmids on using different screening techniques. Among the methods tried, the method of Brinboim and Doly was the most suitable for extraction and screening of plasmid from strain BS2 because excessive slime produced by the culture was easily removed that resulted in recovery of clear cell lysate without any traces of slime 7-9. Agarose gel electrophoresis of DNA sample (obtained after ethanol precipitation of cleared cell lysate) revealed the presence of a megaplasmid in the form of a discrete band above the chromosomal band (Fig. 1). By this method, chromosomal DNA was not removed completely and was observed as a thick diffused band below the megaplasmid. However, the presence of chromosomal DNA had not interfered with the migration of megaplasmid. The method of Guerry et

4 DUBEY & JUWARKAR: GENETIC BASIS OF BIOSURFACTANT SYNTHESIS FROM WASTES 77 Fig. 1 Screening of plasmids in biosurfactant producing strain BS2 by agarose gel electrophoresis of crude cell lysates following different methods; Lanes 1&8: Standard plasmid markers of E coli V 517; Lanes 2&3: Alkaline lysis method of Brinboim & Doly; Lanes 4&5: 5M NaCl extraction method of Guerry et al; Lanes 6&7 : Small and large plasmid extraction method of Kado & Liu al 8 was not suitable for screening the high molecular weight plasmid of the strain BS2 because, on precipitation of chromosomal DNA and proteins by sodium dodecyl sulphate and high concentration of NaCl (5 M), the megaplasmid being of high molecular weight was also removed along with the precipitate. Hence, the chance of finding the high molecular weight plasmid DNA in cleared cell lysate was very low. A rapid procedure for detection and isolation of both large and small plasmids resulted in slimy cell lysate, which posed several difficulties while separation of two immiscible phases of stable emulsion formed between organic (phenol-chloroform mixture) and aqueous phases by centrifugation 9. Thus, the method of Brinboim and Doly 7 was found to be the most suitable, convenient and rapid method for screening of plasmid in strain BS2. Hence, in further studies it was used for the screening of plasmid. Molecular Weight of Megaplasmid and Selection of Marker Antibiotics and Heavy Metal Salt For further genetic analysis, megaplasmid was characterized in terms of size, i.e. molecular weight (MW), and the presence of marker genes, if any; responsible for resistance to antibiotics and heavy metal salt. Such study will be helpful in performing plasmid curing experiments. The MW of megaplasmid of strain BS2 was determined by using a standard plasmid marker E. coli V517 as a source of size reference plasmid molecules. E. coli V517 provides a convenient source of a range of covalently closed circular (CCC) plasmid molecules useful as references in agarose gel electrophoresis 11. The ethanol precipitate of cleared cell lysates of E. coli V 517 and strain BS2 when subjected to agarose gel electrophoresis revealed that E. coli V517 had eight distinct plasmid molecules of known molecular weight and strain BS2 possessed a megaplasmid of Da (Fig. 2). E. coli V517 was also used for the determination of plasmid molecular weight in 6 strains of Acinetobacter baumannii isolated from clinical samples collected from Armed Force Medical College, Pune and for the studies on plasmid profile analysis in Lactobacillus plantarum 15,16. Studies on the selection of suitable marker antibiotics and heavy metal salt were carried out because it provides basic information regarding the presence or elimination of a particular set of genetic determinant on plasmid DNA after curing. The marker gene confers to the plasmid containing host cell a distinctive resistance to some antibiotics and Fig. 2 Agarose gel electrophoresis for determination of molecular weight of plasmid in biosurfactant producing strain BS2; Lanes 1&7: Standard plasmid markers of E. coli V517; Lanes 2 to 6: Plasmid DNA of strain BS2

5 78 INDIAN J BIOTECHNOL, JANUARY 2004 heavy metal salts 17, 18. For selection of marker and its use in curing experiment, strain BS2 was tested for its resistance to various antibiotics and heavy metal salt. The culture was resistant to penicillin-g, ampicillin, chloramphenicol, tetracycline, sulphonamide, bacitracin and methicillin and also to a heavy metal salt, viz. mercuric chloride(hgcl 2 ). These results show that strain BS2 possessed genetic determinants responsible for resistance to these several antibiotics and heavy metal salt. Localization of a set of genetic determinants responsible for conferring resistances to these antibiotics and heavy metal salt on plasmid or chromosomal DNA was ascertained by curing of megaplasmid with mitomycin-c. It was observed that megaplasmid cured cells lost their resistance to several antibiotics, viz. chloramphenicol, tetracycline and sulphonamide and HgCl 2. These results demonstrate that antibiotics and heavy salt resistance was conferred by a set of genetic determinants present on megaplasmid which consisted of a marker gene and therefore, these antibiotics and HgCl 2 were selected as marker for curing experiment. To determine the genetic basis of biosurfactant synthesis from industrial wastes, the study was conducted to ascertain whether the capacity of biosurfactant production at the expense of industrial wastes as no-cost substrates in the strain BS2 reside on plasmid or chromosomal DNA. For this purpose, elimination of plasmid-encoded properties was performed by curing of plasmid DNA. The cured cells obtained were subsequently tested for their ability to produce biosurfactant from wastes. The genes which give the plasmid containing host cell a distinctive phenotypic character are many and varied, and are involved in drug resistance, bacteriocin production, resistance to UV-irradiation, enterotoxin production and possess degradative ability 18. The genes which are found on plasmid DNA are not vital for viability or growth of the cells, but confer to the cells some selective advantages which enables it to survive and grow under certain particular conditions. Since the plasmid carries no essential genes, any bacterium, which looses its plasmid, will remain viable and grow equally well under non-selective conditions where the presence of plasmid is not required. Thus, any bacterium which looses plasmid encoding for biosurfactant synthesis after curing becomes incapable of producing biosurfactant. Such cured clones can be detected by growing a dilute suspension of the culture on non-selective solid medium i.e. nutrient agar for a single colony and testing these individual colonies for the loss of plasmid by agarose gel electrophoresis, elimination of marker gene and subsequently to test their inability to produce biosurfactant. Curing of Megaplasmid by Acridine Orange and Mitomycin-C Acridine orange and mitomycin-c were separately tried for curing of megaplasmid. The highest concentration of curing agent that still allows the growth of culture (highest sublethal dose) was determined. The growth of culture at different concentrations of curing agents was assessed so that the most optimum dose for curing could be selected. On increasing the concentration of acridine orange (from μg/ml), cell count drastically reduced from 18 to c.f.u./ml indicating that concentrations within the range of μg/ml were the sublethal doses for the culture. No growth at concentration of 125 μg/ml and above were minimum inhibitory concentration of acridine orange and concentration above 125 μg/ml were lethal. From these results it was evident that 100 (g/ml) was the highest sublethal concentration which still allowed the growth of culture and this concentration was therefore, selected for curing of megaplasmid. The bacterial resistance to antibiotics and heavy metals are the common phenotypic characters, which are often plasmid encoded 12,13. In the present study, cells after treatment with highest sublethal dose of acridine orange (100 μg/ml) did not lose their resistance towards marker antibiotics such as tetracycline, chloramphenicol, and sulphonamide and heavy metal salt, HgCl 2. This indicates that acridine orange was not capable of curing megaplasmid of strain BS2. Hence, another curing agent i.e. mitomycin-c was tried. Mitomycin-C is an antibiotic produced by Streptomyces caepitosus, which has profound effect on DNA. Its mode of action on DNA is well described 19. Highest sublethal concentration of mitomycin-c was also determined and the results have shown drastic decrease in the cell count from 25to c.f.u./ml when the cells were grown in the presence of its increasing concentration (5-15 μg/ml). Concentrations between 5-15 μg/ml were sublethal and 20 μg/ml was the minimum inhibitory concentration which inhibited the formation of colonies from undiluted bacterial suspensions on nutrient agar plates. Hence, 15 μg/ml was the highest sublethal concentration, which still allowed the growth of culture, and it was selected for curing of megaplasmid. When the cells were grown in the

6 DUBEY & JUWARKAR: GENETIC BASIS OF BIOSURFACTANT SYNTHESIS FROM WASTES 79 presence of highest sublethal concentration of mitomycin-c (15 μg/ml), it was found that out of total bacterial population which were resistant to chloramphenicol, sulphonamide and tetracycline marker antibiotics and heavy metal salt, HgCl 2, 28 % were unable to grow on Luria agar, separately amended with these marker antibiotics and heavy metal salt. This is due to the loss of megaplasmid, which possessed the gene conferring resistance to marker antibiotics and heavy metal salt. This thereby shows that mitomycin-c effectively cured megaplasmid of strain BS2 (Table 1). To confirm the loss of megaplasmid, agarose gel electrophoresis of ethanolic precipitate of cleared cell lysates of wild and mitomycin-c treated (cured) cells, which lost their antibiotic and heavy metal salt marker genes was performed. Results from Fig. 3 indicate that megaplasmid was not detected in cells treated with mitomycin-c, whereas detected in the wild cells. Comparative Assessment of Biosurfactant Production by Wild and Cured Cells It has been reported that production of rhamnolipids in P. aeruginosa is chromosomal encoded phenomenon when glucose, hexadecane and tetradecane were used in mineral salts medium 1. However, in the presence of industrial wastes as nocost nutrient medium, growth of biosurfactant producing strain and biosurfactant production may be linked to plasmid or chromosome or both. There are no reports available on the demonstration of the genetic basis of biosurfactant production when industrial wastes such as distillery and whey wastes are used as no-cost nutrient medium for biosurfactant production. To assess the adaptability of cured cells in wastes and the role of plasmid in biosurfactant production process a comparative study on time course of biosurfactant production by wild and cured cells was carried out in distillery and whey wastes. Table 2 shows that after 48 hrs of incubation both wild and cured cells attained maximum cell counts of c.f.u./ml and c.f.u./ml, respectively in distillery waste and c.f.u./ml and c.f.u./ml in curd whey waste, respectively. Distillery waste is known to contain high concentrations of unfermentable compounds and toxicants such as sulphates, caramels and melanoidins, which provide adverse physico-chemical environment for microbe proliferation 20,21. In such an environment, presence of plasmid seems important to provide adaptive capabilities to the culture in order to grow under adverse physico-chemical and environmental Fig. 3 Agarose gel electrophoresis of crude lysates from wild and mitomycin-c treated cells of strain BS2; Lanes 1&6: Standard plasmid markers of E. coli V517; Lanes 2&3: Megaplasmid of wild strain; Lanes 4&5: Loss of megaplasmid in cells treated with mitomycin-c Table 1 Effect of curing agents on the susceptibility of strain BS2 to marker antibiotics and heavy metal salt Marker antibiotics and heavy metal Cell type Per cent curing (%) Wild cells Cells treated with curing agents Mitomycin-C Acridine orange Mitomycin-C Acridine orange Antibiotics R S R 28 0 Tetracycline R S R 28 0 Sulphonamide R S R 28 0 Chloramphenicol Heavy metal salt Mercuric chloride R S R 28 0 R - Resistance S - Sensitive

7 80 INDIAN J BIOTECHNOL, JANUARY 2004 Table 2 Time course of biosurfactant production by wild and cured cells of strain BS2 from distillery and whey wastes Parameters Type of waste Cell type Incubation time (hrs) Distillery waste Wild cells Biomass yield (c.f.u./ml) Whey waste Wild cells Distillery waste Wild cells Surface tension Whey waste Wild cells (mn/m) Distillery waste Wild cells Biosurfactant yield (g/l) Whey waste Wild cells Results presented are the mean values of three replicated readings conditions. However, it was noticed that in strain BS2 the presence of plasmid was not required to grow in distillery waste as the growth profile of cured cells was the same as the wild strain. Both the cell types reduced the surface tension of distillery and curd whey wastes to a minimum value of 27 mn/m after 48 hrs of incubation. The maximum yields of biosurfactant produced by wild and cured cells after 96 hrs in distillery waste were and g/l, while in whey waste the yields were and g/l, respectively. The results have shown that there was no change in the pattern of growth profile and biosurfactant production by wild and cured cells in distillery and whey wastes, indicating that after curing of megaplasmid, the cured cells still possessed the similar potential capability to grow in these wastes and produced biosurfactant as wild strain and genes responsible for biosurfactant production were not plasmid borne in strain BS2 but resided on the chromosome. However, a contrast phenomenon was observed in the case of biosurfactant producing Rhodococcus species H13A that possessed three indigenous plasmids 22. The plasmid deficient strains, As7, As50 and EIA 1 obtained after curing with acridine orange exhibited normal wild type growth kinetics at the expense of hexadecane but resulted in 83, 54 and 96 % less extracellular biosurfactant yield than the wild type strain. This indicated that, in Rhodococcus species H13A, the presence of all the three plasmids were essential for optimum production of biosurfactant. Studies on genetic analysis for emulsifier production in mineral salts medium containing glucose as carbon source showed that P. aeruginosa UG1 strain, which did not carry any plasmid and the gene(s) coding for emulsifying activity was carried on the chromosome 23. To assess the presence of plasmid, if any, in strain BS2 and its role in biosurfactant production from wastes, the present study was conducted, which demonstrated that the strain carried a megaplasmid and capability of biosurfactant production in strain BS2 was not altered after curing of megaplasmid. Furthermore, the megaplasmid has not been found to facilitate growth of strain BS2 in both the wastes although plasmids are known to confer benefit of adaptation and growth in drastic physico-chemical and environmental conditions existing in the wastes. This implies that it is beneficial for a strain to carry biosurfactant producing genes on chromosome where they were more stable because some plasmid encoded functions can be highly unstable and lost due to plasmid curing at cell segregation. Hence, the strain BS2 can be envisaged as an industrially important strain which can produce biosurfactant in the same quantum even after the loss of megaplasmid due to cell segregation and can simultaneously reduce pollution of the wastes. These observations form the basis for future identification of genes responsible for biosurfactant production and a target site for future genetic manipulations such as to clone, amplify, delete and transfer genes required for isolation of stable, high expression of biosurfactant production from industrial wastes such as distillery and whey wastes as no-cost substrates. Acknowledgement The authors thank Dr R N Singh, Director, NEERI for his kind support and encouragement They also

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