CHAPTER 4 SCREENING, ISOLATION AND FORMULATION OF BIOSURFACTANT-PRODUCING MICROBIAL CONSORTIA

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1 CHAPTER 4 SCREENING, ISOLATION AND FORMULATION OF BIOSURFACTANT-PRODUCING MICROBIAL CONSORTIA 4.1 ABSTRACT Soil samples were collected from various points near a refinery site, petroleum depot, petrol bunk and automobile workshop for the isolation of biosurfactant-producing bacteria. Microbial specimens were isolated using the method of serial dilution. The isolated bacteria were cultured for seven days in MSM containing diesel as the sole carbon source. The isolated cultures were tested for their ability to produce biosurfactant using the following analytical methods: oil spreading technique, blood agar haemolysis test, drop collapse test, CTAB agar plate test, tilted glass slide test and determination of emulsification index and surface activity. The morphology of the colonies was studied and standard biochemical tests were performed. 16S rrna gene sequencing was performed and the sequence was compared with NCBI GenBank entries by using the BLAST algorithm. One of the main objectives of the research work was to develop a consortium of microbial isolates with the highest biodegradation efficiency. Bacillus siamensis was made part of all microbial combinations. Of the two Bacillus subtilis isolates, the better isolate, in terms of higher reduction of surface tension and better emulsification index was chosen. This isolate was Bacillus subtilis subsp. inaquosorum. In similar lines, of the two Pseudomonas aeruginosa isolates, the better was chosen. This isolate was Pseudomonas aeruginosa. Different combinations of the three isolates were formulated for development of microbial consortia. The development of microbial consortia was made solely to study the efficiency of biodegradation of selected polycyclic aromatic hydrocarbons.

2 The role of plasmid DNA in the production of biosurfactants was verified. The method of alkaline lysis was used to extract plasmid DNA from each of the bacterial isolates. The samples were loaded onto an agarose gel and subjected to agarose gel electrophoresis. When the gel was checked for the presence of bands on the agarose gel, none were observed. 4.2 INTRODUCTION Soils rich in hydrocarbons like those contaminated with petroleum spillages are a good source of microbes acclimatized to utilize the hydrocarbons for their metabolism. Many researchers have isolated a range of novel bacterial specimens that produce extracellular biosurfactants which in turn can increase the bioavailability of the hydrocarbons. Seventeen biosurfactant-producing bacterial isolates were isolated from terrestrial and marine samples contaminated with crude oil or its byproducts in a study by Batista et al., All the isolates had produced emulsions with kerosene and twelve exhibited high emulsionstabilizing capacity. Eight isolates were capable of reducing the surface tension below 40 mn/m. Five of these eight exhibited this property in cell-free filtrates. They had concluded that isolation from petroleum contaminated sites proved to be a rapid and effective manner to identify bacterial isolates with potential industrial applications. Two biosurfactant-producing Pseudomonas aeruginosa strains were isolated from Kuwaiti oil-contaminated soil, which resulted from the Gulf War (Yateem et al., 2002). When they optimized the environmental conditions that supported the growth and surfactant production, it was found that the two isolates differed in their biosurfactant-stimulating carbon source, nitrogen concentration. The biosurfactant produced by one of the strains was found to be very effective in the emulsification of crude oil.

3 A total of fifteen oil mixed soil samples were screened and nine bacterial biosurfactant producing strains were identified (Tambekar and Gadakh, 2012) as Pseudomonas aeruginosa, Aneurinibacillus miugulanus, Achromobacter insolitus, Bacillus shackletonii, Ochromobactrum intermedium, Ochromobactrum oryzae by biochemical tests and 16S rrna sequencing. Reduction in surface tension was found to be mn/m. They could form a stable emulsion with motor oil. Biosurfactant production-cum-diesel biodegradation was explored by Moliterni et al., 2012, using mixed microbial consortia from polluted sites. After designing three microbial consortia enrichment in diesel, batch experiments were done to study the effects of three variables (temperature, hydrocarbon concentration and the origin of the consortia) on the diesel biodegradation and the surface tension evolution. The three enriched consortia contained similar bacterial genera and degraded diesel with similar efficiencies (approximately 90%). All three consortia were found to be efficient biosurfactants producers. Naphthalene degrading bacterial consortium was developed by enrichment culture technique from sediment collected from the Alang-Sosiya ship breaking yard, Gujarat, India (Vilas Patel et al., 2012). 16S rrna sequencing revealed that the consortium consisted of Achromobacter sp., Pseudomonas sp., Enterobacter sp. and Pseudomonas sp.. The consortium was able to degrade 1000 ppm of naphthalene in BH medium containing peptone (0.1%) as cosubstrate. The consortium was able to utilize other aromatic and aliphatic hydrocarbons such as benzene, phenol, carbazole, petroleum oil, diesel fuel, and phenanthrene and 2-methyl naphthalene as sole carbon source. Three bacterial isolates capable of utilizing used engine-oil as a carbon source were isolated from contaminated soils using the enrichment technique (Mandri and Lin, 2007). Three

4 isolates were identified as Flavobacterium sp., Acinetobacterium calcoaceticum and Pseudomonas aeruginosa based on biochemical tests and 16S rrna sequencing. It was found that A.calcoaceticum and a consortium of the isolates were capable of utilizing 80 and 90% of used engine oil, respectively, under laboratory conditions in a 4-week period. Mohammad Abdel-Mawgoud et al., 2008, isolated Bacillus subtilis BS5, from soil which produced a promising yield of surfactin in mineral salts medium. When they tested for the presence of plasmid, no plasmid could be detected in the tested isolate, revealing that biosurfactant production by B. subtilis isolate BS5 is chromosomally mediated but not plasmidmediated. A review by Oluwafemi and Lateef, 2010 presents a deeper analysis on the role of plasmid in degradation. The degradation of many xenobiotic and hydrocarbon compounds is known to be mediated by plasmid-encoded enzymes. It has been proven that high level of plasmid involvement in the degradation of naphthalene and other 2- and 3-ring PAHs. Many of these are megaplasmids, of linear configuration, encoding part or the whole genes for the complete pathways. Padmapriya et al., 2011 worked on Proteus inconstans from which a plasmid with 1.8 kbp was isolated and was cured by acridine orange. Biodegradation and biosurfactant activities were totally inhibited. Hence, it was determined that the biosurfactant production was purely plasmid-mediated.

5 4.3 SCREENING, ISOLATION AND FORMULATION OF BIOSURFACTANT- PRODUCING MICROBIAL CONSORTIA For the isolation of biosurfactant-producing bacteria, the samples were collected from various points near a refinery site, a petroleum depot, a local petrol bunk and an automobile workshop, as outlined in Section 3.3. The microbes were isolated through serial dilution and incubated at 25 C, for 48 hr, as given in Section 3.4. The isolates are then inoculated in MSM containing 2% (v/v) filtered diesel as the sole carbon source for a duration of 7 days, as described in Section 3.5. The isolates were screened for the production of biosurfactant using a range of qualitative and quantitative tests like oil spreading technique, blood agar haemolysis test, drop collapse test, CTAB agar plate test, tilted glass slide test, emulsification index and determination of surface activity (Section 3.6). The morphology of the colonies was studied and standard biochemical tests were performed. Under the morphological aspects, the colour, shape, size, surface, edge, opacity, degree of growth and elevation of the colonies were observed and noted. 16S rrna gene sequencing was performed at Agharkar Research Institute (Pune, India) to identify the bacterial strain. PCR was carried out using the universal primers for 1.5 kb fragment amplification for eubacteria. The PCR product was then processed for cycle sequencing reaction. The sequencing output was analyzed using the DNA sequence analyzer software. The sequence was compared with NCBI GenBank entries by using the BLAST algorithm. The consortia are formulated taking into consideration their genus and specific properties. Plasmid DNA was extracted from all the isolates using the method of alkaline lysis, as outlined in Section 3.7. After the samples were subjected to agarose gel electrophoresis, the gel was observed using a UV transilluminator.

6 4.4 RESULTS AND DISCUSSION Isolation of microorganisms from the sample At the end of the incubation period, a total of twenty three different types of colonies were selected for further analysis. They were named RT1 through RT Screening for Biosurfactant Production Oil Spreading Technique Of the twenty three isolates, four samples (RT7, RT9, RT10 and RT21) significantly displaced the oil layer and started to spread in the water, showing a zone of displacement. The results of the oil displacement test showed the pattern as given in Figure 4.1. Figure 4.1 Results of Oil spread test Morikawa et al. (2000) showed that the area of displacement by a biosurfactantcontaining solution is directly proportional to the concentration of the biosurfactants tested.

7 Strains secreting a higher concentration of biosurfactant as indicated by the oil spreading test had low surface tension values. This, plus the fact that the diameter of the clear zone is proportional to the concentration of a standard biosurfactant, indicated that the oil spreading technique is a consistent method to detect biosurfactant production. All the images are shown in Appendix Blood Agar Haemolysis Test Of the twenty three isolates, fifteen strains showed clear zones around the streaks, confirming the exhibition of partial to considerable hemolytic activity. Five strains (RT3, RT7, RT10, RT19 and RT21) displayed a high degree of hemolysis. The results of the blood agar haemolysis test are shown in Figure 4.2. All the images are shown in Appendix 1. Figure 4.2 Results of Blood agar hemolysis test

8 However, not all biosurfactants have a hemolytic activity and compounds other than biosurfactants may cause hemolysis (Youssef et al., 2004). In their study, sixteen percent of the strains that lysed blood agar tested negative for biosurfactant production with the other two methods and had little reduction in surface tension (values above 60 mn/m). Thirty eight percent of the strains that did not lyse blood agar tested positive for biosurfactant production with the other two methods and had surface tension values as low as 35 mn/m Drop Collapse Test Of the twenty three isolates, six specimens (RT3, RT7, RT9, RT10, RT19 and RT21) showed appreciable effect on the glass slide. The drop collapsed completely after an hour. The results of the drop collapse test are depicted in Figure 4.3. Figure 4.3 Results of Drop collapse test

9 The use of the drop collapse method as a primary method to detect biosurfactant producers, followed by the determination of the biosurfactant concentration using the oil spreading technique, constitutes a quick and easy protocol to screen and quantify biosurfactant production (Youssef et al., 2004). Their study had concluded that the drop collapse method may not be as sensitive as the oil spreading technique in detecting low levels of biosurfactant production. All the images are shown in Appendix CTAB Agar Plate Test Of the twenty three isolates, five strains (RT3, RT7, RT9, RT16 and RT21) were surrounded by considerable bluish-green halos implying good production of extracellular biosurfactants. The results of the agar plate test are shown in Figure 4.4. All the images are shown in Appendix 1. Figure 4.4 Results of CTAB Agar plate test

10 Tilted Glass Slide Test Of the twenty three isolates, water flowed over the following seven specimens, possibly indicating the production of an extracellular biosurfactant. The seven isolates are RT3, RT7, RT9, RT10, RT16, RT19 and RT21. All the images are shown in Appendix Emulsification Index Of the twenty three isolates, nine strains (RT3, RT7, RT9, RT10, RT11, RT16, RT17, RT19 and RT21) showed a significant value of emulsification index. The emulsification indices of the culture supernatants showed the following pattern, as depicted in Figure 4.5. All the images are shown in Appendix 1. Figure 4.5 Emulsification Indices of the isolates

11 Determination of Surface Activity Of the twenty three isolates, seven strains (RT3, RT7, RT9, RT10, RT16, RT19 and RT21) showed a significant reduction of surface tension values, indicating that the microbe may be producing an extracellular biosurfactant. The surface tension values of culture supernatants are shown below in Figure 4.6. The percentage reduction of surface tension has been depicted in Figure 4.7. All the images are shown in Appendix 1. Figure 4.6 Surface activity of the isolates

12 Figure 4.7 Percentage reduction of Surface tension by the isolates Selection of Microbial Strains All the tests performed have been used in various studies for preliminary screening of biosurfactant producers. Though the tests indicate biosurfactant production to various degrees of sensitivity, the isolates that show consistent results in all the seven tests were chosen. Based on the results of the seven screening tests including the measurements of surface tension reduction, the following microbial specimens were chosen for further experimentation: RT3, RT7, RT9, RT10, RT16, RT19 and RT21.

13 4.4.4 Morphological Study of Isolates The basic morphological features of the strains were studied and summarized in Table 4.1. Table 4.1: Morphological observations of the microbial strains RT3 RT7 RT9 RT10 RT16 RT19 RT21 Colour Pale yellow Creamy Off Creamy Off Creamy Creamy white white Size 1.3 mm 1 mm 2 mm 1.1 mm 1.8 mm 1 mm 1.2 mm Shape Circular Circular Bead Circular Bead Circular Circular Microscopic Observation (shape) Rods & Cocci Rods Rods Rods Rods Rods Rods Surface Shiny & Dull & Dull & Dull & Dull Wrinkled Dull Smooth Rough Rough Rough Texture Dry Dry Dry Moist Dry Dry Dry Edge Undulate Rhizoid Entire Entire Entire Undulate Rhizoid Elevation Flat Convex Raised /Plateaux Convex Raised /Plateaux Flat Convex Opacity Translucent Translucent Opaque Opaque Opaque Translucent Translucent Degree of Growth Scant Profuse Scant Scant Scant Profuse Profuse

14 4.4.5 Biochemical Characterization of Isolates The basic biochemical tests were conducted on the strains and the results have been enlisted below. The images pertaining to the biochemical tests for all the isolates are shown in Appendix Isolate RT3 Gram Staining Reaction: Gram-negative Motility Test: Non-motile Starch Hydrolysis Test: Negative (Cannot hydrolyze starch since it cannot produce amylase & hence, no zone of clearing was observed) Casein Hydrolysis Test: Negative (Cannot hydrolyze casein since it cannot produce casesase & hence, no zone of clearing was observed) Methyl Red Voges Proskauer Test: MR-negative (Tube turns yellow colour) and VPnegative (Tube turns copper colour) Nitrate Reduction Test: Negative (Cannot produce both nitrate reductase and nitrite reductase and hence, no change of tube colour and no gas collection in Durham tube) Oxidase Test: Negative (Oxidase cytochrome is absent and hence, oxidase reagent strip remains colourless) Catalase Test: Positive (Copious amounts of bubble formation was observed) Citrate Utilization Test: Positive (Abundant growth was observed on the Simmons Citrate agar slant and the medium changes from green to blue indicating use of citrate) Indole production Test: Negative (Upper alcohol layer of the broth turns yellow) Spore-forming ability Test: Spores were not observed.

15 Isolate RT7 Gram Staining Reaction: Gram-positive Motility Test: Motile Starch Hydrolysis Test: Positive (Hydrolyzes starch since it can produce amylase & hence, a zone of clearing was observed) Casein Hydrolysis Test: Positive (Hydrolyzes casein since it can produce casesase & hence, a zone of clearing was observed) Methyl Red Voges Proskauer Test: MR-negative (Tube turns yellow colour) and VPpositive (Tube turns red colour) Nitrate Reduction Test: Positive (Produces both nitrate reductase and nitrite reductase and hence, tube changes to red colour and gas collection is observed in Durham tube) Oxidase Test: Negative (Oxidase cytochrome is absent and hence, oxidase reagent strip remains colourless) Catalase Test: Positive (Copious amounts of bubble formation was observed) Citrate Utilization Test: Positive (Abundant growth was observed on the Simmons Citrate agar slant and the medium changes from green to blue indicating use of citrate) Indole production Test: Negative (The upper alcohol layer of the broth turns yellow color) Spore-forming ability Test: Spores were observed near the terminal regions of some cells.

16 Isolate RT9 Gram Staining Reaction: Gram-negative Motility Test: Motile Starch Hydrolysis Test: Negative (Cannot hydrolyze starch since it cannot produce amylase & hence, no zone of clearing was observed) Casein Hydrolysis Test: Positive (Hydrolyzes casein since it can produce casesase & hence, a zone of clearing was observed) Methyl Red Voges Proskauer Test: MR-negative (Tube turns yellow colour) and VPnegative (Tube turns copper colour) Nitrate Reduction Test: Negative (Cannot produce both nitrate reductase and nitrite reductase and hence, no change of tube colour and no gas collection in Durham tube) Oxidase Test: Positive (Oxidase cytochrome is present and hence, oxidase reagent strip changes from colourless to purple) Catalase Test: Positive (Copious amounts of bubble formation was observed) Citrate Utilization Test: Positive (Abundant growth was observed on the Simmons Citrate agar slant and the medium changes from green to blue indicating use of citrate) Indole production Test: Negative (The upper alcohol layer of the broth turns yellow color) Spore-forming ability Test: Spores were not observed.

17 Isolate RT10 Gram Staining Reaction: Gram-positive Motility Test: Non-motile Starch Hydrolysis Test: Negative (Cannot hydrolyze starch since it cannot produce amylase & hence, no zone of clearing was observed) Casein Hydrolysis Test: Negative (Cannot hydrolyze casein since it cannot produce casesase & hence, no zone of clearing was observed) Methyl Red Voges Proskauer Test: MR-negative (Tube turns yellow colour) and VPpositive (Tube turns red colour) Nitrate Reduction Test: Negative (Cannot produce both nitrate reductase and nitrite reductase and hence, no change of tube colour and no gas collection in Durham tube) Oxidase Test: Negative (Oxidase cytochrome is absent and hence, oxidase reagent strip remains colourless) Catalase Test: Positive (Copious amounts of bubble formation was observed) Citrate Utilization Test: Negative (Growth was not observed on the Simmons Citrate agar slant and the medium remains green indicating non-use of citrate) Indole production Test: Negative (The upper alcohol layer of the broth turns yellow color) Spore-forming ability Test: Spores were not observed.

18 Isolate RT16 Gram Staining Reaction: Gram-negative Motility Test: Motile Starch Hydrolysis Test: Negative (Cannot hydrolyze starch since it cannot produce amylase & hence, no zone of clearing was observed) Casein Hydrolysis Test: Positive (Hydrolyzes casein since it can produce casesase & hence, a zone of clearing was observed) Methyl Red Voges Proskauer Test: MR-negative (Tube turns yellow colour) and VPnegative (Tube turns copper colour) Nitrate Reduction Test: Negative (Cannot produce both nitrate reductase and nitrite reductase and hence, no change of tube colour and no gas collection in Durham tube) Oxidase Test: Positive (Oxidase cytochrome is present and hence, oxidase reagent strip changes from colourless to purple) Catalase Test: Positive (Copious amounts of bubble formation was observed) Citrate Utilization Test: Positive (Abundant growth was observed on the Simmons Citrate agar slant and the medium changes from green to blue indicating use of citrate) Indole production Test: Negative (The upper alcohol layer of the broth turns yellow color) Spore-forming ability Test: Spores were not observed.

19 Isolate RT19 Gram Staining Reaction: Gram-positive Motility Test: Motile Starch Hydrolysis Test: Positive (Hydrolyzes starch since it can produce amylase & hence, a zone of clearing was observed) Casein Hydrolysis Test: Positive (Hydrolyzes casein since it can produce casesase & hence, a zone of clearing was observed) Methyl Red Voges Proskauer Test: MR-negative (Tube turns yellow colour) and VPpositive (Tube turns red colour) Nitrate Reduction Test: Positive (Produces both nitrate reductase and nitrite reductase and hence, tube changes to red colour and gas collection is observed in Durham tube) Oxidase Test: Negative (Oxidase cytochrome is absent and hence, oxidase reagent strip remains colourless) Catalase Test: Positive (Copious amounts of bubble formation was observed) Citrate Utilization Test: Positive (Abundant growth was observed on the Simmons Citrate agar slant and the medium changes from green to blue indicating use of citrate) Indole production Test: Negative (The upper alcohol layer of the broth turns yellow color) Spore-forming ability Test: Spores were observed near the terminal regions of some cells.

20 Isolate RT21 Gram Staining Reaction: Gram-positive Motility Test: Motile Starch Hydrolysis Test: Positive (Hydrolyzes starch since it can produce amylase & hence, a zone of clearing was observed) Casein Hydrolysis Test: Positive (Hydrolyzes casein since it can produce casesase & hence, a zone of clearing was observed) Methyl Red Voges Proskauer Test: MR-negative (Tube turns yellow colour) and VPpositive (Tube turns red colour) Nitrate Reduction Test: Positive (Produces both nitrate reductase and nitrite reductase and hence, tube changes to red colour and gas collection is observed in Durham tube) Oxidase Test: Negative (Oxidase cytochrome is absent and hence, oxidase reagent strip remains colourless) Catalase Test: Positive (Copious amounts of bubble formation was observed) Citrate Utilization Test: Positive (Abundant growth was observed on the Simmons Citrate agar slant and the medium changes from green to blue indicating use of citrate) Indole production Test: Negative (The upper alcohol layer of the broth turns yellow color) Spore-forming ability Test: Spores were observed near the terminal regions of some cells.

21 From the biochemical tests, the likelihood of genus of the isolated microbial specimens is listed below RT3: Inconclusive RT7: Bacillus RT9: Pseudomonas RT16: Pseudomonas RT19: Bacillus RT21: Bacillus RT10: Inconclusive S rrna Identification of the Isolates Microbial isolates were identified through 16S rrna analysis. The results have been shown in Table 4.2. Table 4.2: Results of 16S rrna identification of isolates Isolate Genus species RT 3 Acinetobacter calcoaceticus RT 7 Bacillus subtilis RT 9 Pseudomonas aeruginosa RT 10 Rhodococcus terrae RT 16 Pseudomonas aeruginosa RT 19 Bacillus siamensis RT 21 Bacillus subtilis subsp. inaquosorum The phylogenetic tree for the isolates RT9, RT19 and RT21 are shown in Fig. 4.8, 4.9 and 4.10, respectively.

22 Figure 4.8 Phylogenetic tree of Isolate RT9 Figure 4.9 Phylogenetic tree of Isolate RT19

23 Figure 4.10 Phylogenetic tree of Isolate RT Extraction of Plasmid DNA No bands were observed in all the lanes of the gel. Both high and low molecular weight plasmids were not observed in the gel. From this observation, it can be safely concluded the genes responsible for biosurfactant production is not associated with plasmids and must be located in the chromosomal DNA of the bacterial isolates, tested. Fleck et al., 2000, when studying biosurfactant production by bacteria isolated from oilpolluted sites (B.subtilis B1 and P.aeruginosa P1), obtained similar results. Both the strains did not reveal the presence of plasmids. They had also concluded that the coding genes for biosurfactant production are located in the chromosomal DNA. Likewise, no plasmids were observed by Mohammad Abdel-Mawgoud et al., 2008 from Bacillus subtilis BS5 isolated from the soil.

24 During scale-up in industrial operations, genetic instability is one of the major setbacks in hindering a microbial strain from becoming a robust producer of biosurfactant. Thus, the finding could be perceived as an advantage from industrial viewpoint. As a consequence, the genes responsible for reduced surface activity are more stable. Hence, at the production scale, minimum fluctuation would be encountered Development of Microbial Consortia Bacillus siamensis is reported for the first time for the production of biosurfactant. Hence, this isolate, RT 19, was made part of all microbial combinations. Of the two Bacillus subtilis isolates (RT7 and RT21), the better isolate, in terms of higher reduction of surface tension and better emulsification index (from preliminary screening data Figures 4.5 and 4.7) was chosen. The better isolate was Bacillus subtilis subsp. inaquosorum (RT 21). In similar lines, of the two Pseudomonas aeruginosa isolates (RT9 and RT16), the better isolate in terms of higher reduction of surface tension and better emulsification index (from preliminary screening data Figures 4.5 and 4.7) was chosen. The better isolate was Pseudomonas aeruginosa (RT 9). Another isolate was found to be Rhodococcus terrae (RT 10). It was previously decided to continue the research work to relate the effect of swarming motility behavior of the isolates on the action of biosurfactants. Due to the non-motile nature of R.terrae and minor concerns of possible pathogenicity, the isolate was dropped from the consortium. The last isolate, Acinetobacter calcoaceticus (RT 3) was also not selected based on similar reasons of non-motile character. Different combinations of Bacillus siamensis (RT 19), Bacillus subtilis subsp. inaquosorum (RT 21) and Pseudomonas aeruginosa (RT 9) were formulated for development of

25 microbial consortia. The development of microbial consortia was made solely to study the efficiency of biodegradation of selected polycyclic aromatic hydrocarbons. The following was the choice of consortia: CONS 1: RT 19 + RT 21 CONS 2: RT 19 + RT 9 CONS 3: RT 19 + RT 21 + RT CONCLUSION The bacteria were isolated from different hydrocarbon-contaminated zones such as refinery site, petroleum depot, petrol bunk and automobile workshop. Serial dilution and plating led to isolation of twenty three different bacterial isolates. The isolates were screened for the production of biosurfactants through a series of qualitative tests that followed after they were grown in MSM. The tests included oil spreading technique, blood agar haemolysis test, drop collapse test, CTAB agar plate test, tilted glass slide test, emulsification index and determination of surface activity. The results of the screening tests indicated that seven of the isolates (RT3, RT7, RT9, RT10, RT16, RT19, and RT21) showed promise in producing significant quantities of extracellular biosurfactants. The percentage reduction in surface tension of the cell-free broth after 24 hr, ranged from 49 % to 62%, among the seven isolates. Morphological features of the selected strains were observed. The standard biochemical tests were performed that included Gram Staining Reaction, Motility Test, Starch Hydrolysis Test, Casein Hydrolysis Test, Methyl Red Voges Proskauer Test, Nitrate Reduction Test, Oxidase Test, Catalase Test, Citrate Utilization Test, Indole production Test and Spore Forming

26 ability Test. From the tests, it was concluded that RT7, RT19 and RT21 were Bacilli. RT9 and RT16 were Pseudomonas isolates. Sequencing using 16S rrna technique helped in the complete identification of the seven isolates. They were identified as: RT3, RT7, RT9, RT10, RT16, RT19 and RT21 were identified as Acinetobacter calcoaceticus, Bacillus subtilis, Pseudomonas aeruginosa, Rhodococcus terrae, Pseudomonas aeruginosa, Bacillus siamensis and Bacillus subtilis subsp. inaquosorum, respectively. Bacillus siamensis (RT19) has been reported to produce biosurfactant for the first time, to the best of knowledge. Simlarly, the subsp inaquosorum of B.subtilis is also noted for the first time, in literature. Different combinations of RT9, RT19 and RT21 were formulated for development of microbial consortia. The consortium CONS-1 comprised B.siamensis and B.subtilis subsp. inaquosorum. The consortium CONS-2 was formulated with B.siamensis and P.aeruginosa. Lastly, the consortium CONS-3 had all the three isolates. After agarose gel electrophoresis of the samples, the gel, when observed using a UV transilluminator, did not reveal any bands. It could thus be concluded that the ability of the bacterial isolates to secrete biosurfactants did not arise from a gene coded on plasmid DNA. In all probability, the gene responsible for the production of biosurfactant must have been present on the bulkier chromosomal DNA.