MATERIALS AND METHODS

Transcription

1 MATERIALS AND METHODS 3.1 Pesticides and chemicals Lindane/γ-Hexachlorocyclohexane (γ-hch, 99% pure) was purchased from Sigma Chemical Co., USA. HCH-muck was a generous gift from Kanoria Chemicals and Industries Ltd. (U.P., India). Alufolein kiesel gel 60 F 254 plates were purchased from Merck (Darmstadt, Germany). Acetone and ethyl acetate (purity >99.0%) were purchased from Thomas Baker, Mumbai, India. The media components were purchased from HiMedia Lab., India. All other solvents and reagents used were of highest available purity. The oligonucleotide primers for polymerase chain reaction (PCR) were supplied by Sigma-Genosys (USA) and PCR reaction-mix was purchased from Genei, Bangalore, India. 3.2 Media used Medium used for growth and storage of isolates: The cultures were grown in nutrient broth and were stored on nutrient agar slants at 4 C. The stored microbial strains were sub-cultured every month. The cells were also maintained as 20% (v/v) glycerol stock in nutrient broth at -80 C. The composition of the nutrient agar medium used is as given below: Components Quantity (g l -1 ) Peptone Beef extract NaCl Agar-agar Distilled water to make ml ph Biosurfactant production medium: Mineral salt medium (MSM) of the following composition was used for biosurfactant production studies, unless specified otherwise. 47

2 Components Quantity (g l -1 ) Na 2 HPO 4 (anhydrous) (NH 4 ) 2 SO KH 2 PO MgSO 4.7H 2 O Fe(NH 4 )Citrate CaCl 2.2H 2 O Yeast extract - as per requirement Glucose - as per requirement Trace element solution ml l -1 Distilled water to make ml ph Composition of trace element solution: Components Quantity (mg l -1 ) ZnSO 4.7H 2 O MnCl 2.4H 2 O CoCl 2.6H 2 O NiCl 2.6H 2 O Na 2 MoO 4.2H 2 O H 3 BO CuCl 2.2H 2 O Distilled water to make ml The stock solutions of yeast extract (25.0% w/v) and glucose (50.0% w/v) were sterilized separately and added to MSM as per the requirement. Similarly, stock solutions of other sugars (fructose, mannitol and sucrose) and organic extracts/supplements (beef extract, malt extract, peptone, soy peptone and tryptone) used in optimization studies were prepared and used as per the requirement of a particular experiment Chloride-free medium: Chloride-free mineral salt medium (CFMSM) of the following composition was used to assay the release of free chloride-ion during HCH-biodegradation. 48

3 Components Quantity (g l -1 ) Na 2 HPO 4 (anhydrous) (NH 4 ) 2 SO KH 2 PO MgSO 4.7H 2 O Fe(NH 4 )Citrate Ca(NO 3 ) Distilled water to make ml ph Isolation of microbial strains Isolation of biosurfactant-producing microorganisms: The microbial populations in soil samples collected from sites contaminated with petrochemicals (petrol stations, motor workshops, etc.) were isolated. The soil samples were collected in plastic bags from just below the surface, appropriately labeled and stored at 4 C. To a 9 ml water blank, 1 g of soil sample was added and contents of the test tube were vortexed and the suspension was used as inoculum (10% v/v) to inoculate 50 ml MSM in 250 ml Erlenmeyer flask supplemented with 0.1% (w/v) yeast extract. Different supplements viz. n-hexadecane, diesel-petrol mix (1:1), mobile oil and vegetable oil were added at the rate of 2.0% (v/v) to the respective flasks for enrichment of microbial populations present in the soil samples. The flasks were incubated at 30 C on an orbital shaker at 100 rpm. The cell suspension was used as 10% (v/v) inoculum in fresh MSM medium, containing respective the carbon sources to further enrich the microbial populations, after every 15 days for a period of two months. At each transfer, 1 ml of sample was withdrawn and serially diluted and 0.1 ml of the appropriately diluted culture broth was spread plated onto MSM agar plates supplemented with 0.1% (w/v) yeast extract and 2.0% (v/v) of the respective carbon sources, separately. The plates were incubated at 30 C and observed for growth. Morphologically distinct bacterial colonies were isolated and purified. 49

4 The enrichment of microbial population on molasses and whey was carried out using 1/100 diluted nutrient broth. Five grams of the soil sample was added to 250 ml of Erlenmeyer flask containing 50 ml of nutrient broth (N/100) and incubated on a rotary shaker at 30 C and 100 rpm for 24 h. 5 ml of culture was transferred to 50 ml MSM supplemented with 0.01% (w/v) yeast extract and carbon sources viz. molasses (2.0% w/v) and whey (1.0% v/v), respectively. Finally, 0.1 ml of culture was plated onto nutrient agar plates and incubated at 30 C for 24 h. After incubation, plates were enumerated and morphologically distinct bacteria were screened for biosurfactant production. The microbial populations present in spoiled curd and cheese samples were screened for their culture diversity in MSM supplemented with n-hexadecane (1.0% v/v), casein (1.0% w/v) and tributyrin (1.0% v/v), separately. The enriched samples were plated onto nutrient agar plates and morphologically distinct isolates were selected for further studies. The isolates were stored on nutrient agar slants and stored at 4 C. Regular subculturing was carried out on fresh medium at a regular interval of 15 days. The cells were also maintained as 20% (v/v) glycerol stock at -80 C after growing in nutrient broth Isolation of HCH-degrading microbes: The microbial populations present in soil samples collected from sites polluted with organochlorine pesticides, in and around the city of Amritsar, were enriched in the presence of γ-hch. Ten gram (10 g) of soil was used to inoculate 90 ml MSM supplemented with 0.1% (w/v) glucose, 0.01% (w/v) yeast extract and 0.01 g of γ-hch. The flasks were incubated at 30 C and 100 rpm. The process of enrichment was continued for two months, using the enriched culture as inoculum (10% v/v) to fresh MSM supplemented with the components mentioned above. At each transfer, the samples were plated onto MSM plates containing glucose (0.1% w/v), yeast extract (0.01% w/v) and 100 ppm γ-hch. The plates were incubated at 30 C and observed for the microbial growth. The morphologically distinct colonies were purified by several transfers on the same medium. 50

5 3.4 Screening for potential biosurfactant-producing strains Blood agar method: The isolates were screened for their ability to produce surface-active molecules on blood agar plates. The overnight-activated cells in 20 ml nutrient broth were point inoculated onto the nutrient agar plates supplemented with 5.0% (v/v) sheep blood and incubated at 30 C. The plates were observed at a regular interval of 24 h up to 7 days for a clear zone around the colony due to hemolysis of red blood cells Measurement of surface tension (mn m -1 ): The microbial strains were checked for biosurfactant production in 250 ml Erlenmeyer flasks, under shake flask conditions. The activation of the respective isolates was carried out by inoculating a single colony to nutrient broth and the flasks were incubated overnight at 30 C and 100 rpm. The activated cells were used as inoculum (5.0% v/v) in 50 ml MSM supplemented with glucose (2.0% w/v) and yeast extract (0.25% w/v). The flasks were incubated at 30 C and 100 rpm. The samples (20 ml) removed at a regular interval of 24 h were centrifuged (10,000 rpm, 5 min) and surface tension (mn m -1 ) of cell-free supernatant (20 ml) was measured using CSC-duNouy tensiometer (CSC, Fairfax, USA) that employs the ring method (Chopineau et al. 1988). The platinum ring was rinsed twice with acetone and water after each measurement Assay of emulsification activity and emulsion stability: The emulsification activity of the cell-free supernatant of different isolates was studied by the method given by Cirigliano and Carman (1984). The crude biosurfactant sample (800 µl) was diluted with distilled water to a final volume of 4 ml, followed by addition of 1 ml n-hexadecane. The mixture was mixed vigorously on a vortex mixer for 2 min. The resulting emulsion was allowed to stand for 10 min at room temperature after which its turbidity was measured at 540 nm. The absorbance was multiplied by the dilution factor and expressed as the emulsification activity. The stability of the emulsion was determined by reading the turbidity of emulsion formed at a regular interval of 10 min up to 50 min (Cirigliano and Carman 1985). 51

6 3.4.4 Screening of isolated strains on cetyltrimethylammonium bromide-methylene blue agar plates (CTAB-MB agar): The production of anionic biosurfactants by the isolated strains was detected by using the method described by Seigmund and Wagner (1991). The activated cell suspension of the respective cultures, grown in nutrient broth was spotted on the surface of a cetyltrimethylammonium bromide (CTAB)-methylene blue agar plate. The CTAB medium was prepared by adding 0.2 g l -1 of CTAB and g l -1 of methylene blue to MSM containing 2.0% (w/v) glucose and 0.25% (w/v) yeast extract. The plates were incubated at 30 C and were observed for a dark blue halo around the colony indicating the formation of a cationic CTAB-methylene blue complex and to the presence of anionic glycolipid biosurfactants. 3.5 Identification of selected cultures The bacterial isolates showing positive results in the above mentioned screening methods were selected for further studies. The cultures were identified as Pseudomonas aeruginosa (WH-2), Stenotrophomonas maltophilia (WH-13), P. aeruginosa (WH-15), Ochrobactrum anthropi (MO-3), Bacillus subtilis (MOL-1), Paenibacillus lentimorbus (MOL-8), Micrococcus lylae (CHE-1) and Bacillus sp. (CHE-2) by Microbial Type Culture Collection (MTCC) and Gene bank, Institute of Microbial Technology (IMTECH), Chandigarh, India. The strains B. subtilis MTCC 1427 (Makkar and Cameotra 1998) and Sphingomonas sp. MTCC 8061 (Manickam et al. 2008) were used as reference strains for producing lipopeptide-based surfactants and for HCHbiodegradation studies, respectively during the course of this study. 3.6 PCR-based functional characterization of the isolates The presence of genes coding for glycolipid and cyclic lipopeptide biosurfactants and their regulatory genes was detected by PCR using consensus primers specific for these loci viz. rhl and kpd locus of Pseudomonas sp. coding for rhamnolipid production, and for the sfp locus of Bacillus sp. coding for surfactin production by the selected isolates. 52

7 3.6.1 Preparation of cell lysate: The biosurfactant-producing strains were grown overnight in nutrient broth medium. 1.5 ml of the overnight grown cells of respective cultures were harvested by centrifugation at 9000 rpm for 2 min. Pellet was washed with sterilized-double distilled water (SDDW) and was suspended in 25.0 µl of 0.5 N NaOH. After incubating it at room temperature for 30 min, 25.0 µl Tris-Cl (1 M) and 445 µl SDDW was added. The contents of the tube were gently shaken and stored in freezer for further use in their respective PCR reactions as below PCR protocol: The PCR reactions were carried using a MasterCycler Personal PCR (Eppendorf, Germany). The PCR reaction mix of 50 µl contained 25.0 µl PCR mix (Genei, Bangalore, India), 2.5 µl DMSO, 0.5 µl of each primer and 5.0 µl of appropriately diluted template DNA. The PCR protocols as described below were used for respective primers. The PCR products were visualized by electrophoresis using a 1.0% (w/v) agarose gel containing 1 µg ml -1 of ethidium bromide and scanned in Gel Doc (Syngene) rhl locus: The PCR-amplification was carried out using rhl-f (5'- CGGCGCCTGGGCTTCGATTAC- 3') and rhl-r (5'- CGTTCGCGATGGCTCAGGC AG- 3'). The cycling program used was: 95 C for 2 min, denaturation at 95 C for 1 min, annealing at 60 C for 45 s and extension at 72 C for 45 s, for a total of 30 cycles followed by 72 C for 1:30 min as a final extension step. Following amplification, the PCR products were observed for 445 bp PCR product of rhl locus Detection of rhlb gene using kpd locus: The presence of rhlb gene in the DNA extracts was checked by using the forward primer kpd-f (5' -GCCCACGACCAGTTCGAC- 3') and the reverse primer kpd-r (5' - CATCCCCCTCCCATGAC- 3'). The PCR-amplification program was 94 C for 2 min followed by 30 cycles of 94 C for 15 s, 53 C for 15 s, 72 C for 15 s, and finally to a final extension of 72 C for 2 min. The presence of 226 bp PCR product indicates that the respective isolate is positive for the locus. 53

8 sfp locus: The forward primer sfp-f (5' ATGAAGATTTACGGAATTTA- 3') and reverse primer sfp-r (5' -TTATAAAAGCTCTTCGTACG- 3') was used for amplification of the regulatory gene sfp. The PCR protocol was as follows: initial denaturation at 95 C for 2 min was followed by cycling events of 95 C for 1 min, annealing at 46 C for 30 s and extension at 72 C for 1 min, for a total of 24 cycles. It was followed by a final extension step at 72 C for 2 min. A PCR product of 675 bp in size will be observed in surfactinpositive isolates. 3.7 Biosurfactant production by selected strains The bacterial strains WH-2, WH-13, WH-15, MO-3, MOL-1, MOL-8, CHE-1 and CHE-2 were further screened for their biosurfactant production potential. The cells of respective isolates were activated for an overnight in nutrient broth at 30 C and 100 rpm. The activated cells were centrifuged (Sigma, model 3K30) at 10,000 rpm for 10 min, washed and resuspended in 0.8% (w/v) saline. The cell suspension, corresponding to 0.3 OD 600 was used as inoculum, unless specified otherwise. The isolates were inoculated to MSM supplemented with glucose (2.0% w/v) and yeast extract (0.25% w/v). The flasks were incubated at 30 C and 100 rpm for 96 h. The medium was centrifuged at 10,000 rpm for 10 min to get a crude biosurfactant extract (CBE), which was used for further studies. The growth and biosurfactant production profile of each strain was evaluated by following the respective parameters as described below, at a regular interval of 24 h Cell growth: Cellular growth at the respective time intervals was measured by following optical density of the culture at 600 nm using UV-Visible Spectrophotometer (UV-1601, Shimadzu) Determination of biomass: The gram dry weight per liter was determined by taking 1.5 ml sample of the culture broth from the growth medium, at a specific time interval of 24 h. The cells were separated by centrifugation at 10,000 rpm for 10 min and washed. The cell pellet was dried at 100 C. The reading was taken every 24 h till a constant weight was achieved. 54

9 3.7.3 Surface tension (mn m -1 ): The surface tension of cell-free supernatant at each sampling was determined as described in Section Determination of biosurfactant concentration: For rhamnose containing biosurfactants: The orcinol assay (Chandrasekaran and Bemiller 1980) was used to assess the amount of rhamnolipids in the sample as per the following protocol: 333 µl of the culture supernatant was extracted thrice with 1 ml of diethyl ether. The ether fractions were pooled and evaporated to dryness and redissolved in 0.5 ml of water. To 100 µl of each sample in a 5 ml test tube, 900 µl of a solution containing 0.19% (w/v) orcinol was added and heated at 80 C for 30 min. The samples were cooled for 15 min at room temperature and the absorbance (at 421 nm) was measured. The concentration of rhamnolipid was determined from standard curve of rhamnose between 0 and 50 µg ml -1. Rhamnolipid content was determined by multiplying rhamnose concentration by 3 (Itoh et al. 1971) For non-rhamnose containing biosurfactants: The concentration of the non-rhamnose based surface-active molecules was determined using dry weight method (Moran et al. 2002). The pellets obtained after acid precipitation of the cell-free supernatant (as described in Section 3.8) were extracted twice with a mixture of chloroform: methanol (2:1 v/v). Organic phase was concentrated and pooled in a weighed 1.5 ml microcentrifuge tube. The tubes were weighed again after the solvent was evaporated, to determine the weight of surface-active components produced by each isolate Specific productivity (Yp/x) determination: The yield of biosurfactant on biomass (Yp/x, g rhamnolipid g -1 dry cell mass) was also evaluated as below. Yp/x = A/B-C where, A = Total biosurfactant produced (g l -1 ) B = Biomass at the time of harvesting (g l -1 ) C = Biomass at 0 h (g l -1 ) 55

10 3.8 Extraction of biosurfactants The cells of respective isolates were grown in the medium as described in Section 3.7 and ph of the cell-free supernatant was lowered to 2.0 with 5 N HCl. The flask was incubated for 48 h at 4 C to allow precipitation. The precipitated surface-active components were separated by centrifugation at 10,000 rpm for 10 min. The pellet was washed with sterile distilled water, air dried and dissolved in 5 ml of alkaline water. The precipitate was extracted thrice with equal volume of ethyl acetate and chloroform: methanol (2:1) for rhamnose-containing biosurfactants and non-rhamnose type of biosurfactants, respectively. The pooled organic extracts were dried using rotary vacuum evaporator (Büchi, Switzerland) for further analysis. 3.9 Screening of HCH-degrading microbial strains The isolates were screened for their HCH-degradation potential in 50 ml nutrient broth supplemented with γ-hch at a rate of 100 µg ml -1. The medium was inoculated with 10% (v/v) of the overnight-activated isolates, separately. The flasks were incubated at 30 C and 100 rpm for 96 h. The medium was extracted thrice with chloroform. Organic phase was pooled and concentrated using rotary vacuum evaporator (Büchi, Switzerland). The concentrated organic extracts were analyzed by thin-layer chromatography (TLC) using the ethyl acetate: hexane (1:9) solvent system and chromatograms were visualized both under UV light and iodine vapors. HCHdegradation by the isolates was also monitored by chloride-ion release using mercuric thiocyanate method (Bergmann and Sanik 1957, Appendix-1) Biodegradation of HCH-muck in the presence of crude biosurfactant extracts The effect of absence and presence of 5.0% (v/v) crude biosurfactant extracts (CBEs) of different surfactant-producing strains viz. P. aeruginosa (WH-2), Paenibacillus lentimorbus (MOL-8), and Bacillus sp. (CHE-2) on the biodegradation of HCH-muck by B. licheniformis HA-12 and B. alcalophilus HA-13 was evaluated in 250 ml Erlenmeyer flasks containing 50 ml chloride-free mineral salt medium (CFMSM). The cells of HA-12 and HA-13 were activated in CFMSM at 30 C and 100 rpm in the presence of 20 ppm HCH-muck. Cells were harvested by centrifugation at 10,000 rpm for 56

11 10 min, washed with CFMSM and resuspended in 10 ml of the same medium. The flasks containing 5.0% (v/v) of the respective CBEs supplemented with 20 ppm HCH-muck were inoculated with 500 µl of the activated culture and incubated at 30 C (100 rpm) for 48 h. The control flask having MSM without any CBE supplement was also inoculated. The samples of 0 h and 48 h were evaluated for growth by taking OD 600. The release of chloride-ion into growth medium was monitored using Ion meter (Orion 720) fitted with chloride-ion specific electrode Optimization of process parameters for biosurfactant production by P. aeruginosa WH-2 The effect of different medium components and physico-chemical parameters on growth and biosurfactant production potential of P. aeruginosa WH-2 was followed by using the conventional one-factor at a time approach. The parameter supporting best biosurfactant yield at each stage was selected for further studies. The optimization studies were conducted in MSM (ph 7.0 ± 0.2) supplemented with appropriate carbon and nitrogen sources. The incubation was carried out at 30 C and 100 rpm for 96 h. The various parameters viz. cell biomass, ph change, reduction in surface tension, biosurfactant concentration and specific productivity, were determined as described in Section Optimization of medium components for biosurfactant production The effect of different carbon and nitrogen (organic/inorganic) sources on biosurfactant production by WH-2 was studied. Further, the influence of different levels of phosphate ions, magnesium ions and iron (Fe 3+ ) on the overall biosurfactant yield was assessed. The effect of other trace elements viz. Zn 2+, Mn 2+, Co 2+, Ni 2+ and Cu 2+ on surfactant production by WH-2 was also examined Effect of carbon sources on biosurfactant production: The effect of different carbon sources viz. glucose, fructose, mannitol, sucrose, sodium citrate, sodium acetate, ethanol, oleic acid and n-hexadecane on biosurfactant production efficiency of WH-2 was evaluated. The MSM was supplemented with 2.0% (w/v) of the respective carbon source and 0.25% (w/v) yeast extract. The respective 57

12 carbon sources were added to growth medium from their sterilized stock solutions into the medium after sterilization. Fructose, found to be the most suitable carbon source out of the tested carbon sources, was used for further studies. The effect of different concentrations of fructose viz. 0.5, 1.0, 2.0, 3.0 and 4.0 (% w/v) on biosurfactant production efficiency of WH-2 was studied. The WH-2 cells grown in MSM supplemented with 2.0% (w/v) fructose supported the best biosurfactant potential of the strain and this concentration was used for further studies, unless specified otherwise Effect of nitrogen sources on biosurfactant production: The effect of different organic and inorganic nitrogen sources on biosurfactant production efficiency of WH-2 was studied Effect of organic nitrogen supplements on biosurfactant production: The potential of different organic supplements viz. yeast extract, beef extract, malt extract, peptone, soy peptone and tryptone to support biosurfactant production by WH-2 was studied. The MSM was supplemented with 2.0% (w/v) fructose and 0.25% (w/v) of different organic nitrogen supplements. The medium supplemented with tryptone (0.25% w/v) supported best biosurfactant yield. Thus, tryptone was selected for further studies. The effect of different concentrations ( % w/v) of tryptone on biosurfactant production by WH-2 was studied, in order to determine its optimal concentration. The cells grown in presence of 0.25% (w/v) tryptone supported maximum biosurfactant yield and was used for further studies, unless specified otherwise Effect of inorganic nitrogen sources on biosurfactant production: Different inorganic nitrogen sources viz. ammonium sulphate, ammonium chloride, ammonium nitrate, ammonium dihydrogen orthophosphate, sodium nitrate, potassium nitrate and ammonium acetate were supplemented to MSM at a rate of 0.10% (w/v), to evaluate their effect on biosurfactant production efficiency of WH-2. Out of these, ammonium sulphate was found to support the best biosurfactant production. 58

13 The cells of WH-2 were grown in MSM supplemented with different concentrations of (NH 4 ) 2 SO 4 viz. 0.05, 0.10 and 0.20 (% w/v). MSM supplemented with 0.10% (w/v) of (NH 4 ) 2 SO 4 supported maximum biosurfactant production by the strain Effect of Na +, K + and phosphate ions on biosurfactant production: The effect of different levels of Na +, K + and phosphate (PO 3-4 ) ions in MSM on the potential of biosurfactant production by cells of WH-2 was evaluated. The biosurfactant production potential of WH-2 cells was studied using MSM supplemented with different concentrations of Na 2 HPO 4 (anhydrous, g l -1 ) and KH 2 PO 4 ( g l -1 ) Effect of magnesium ions on biosurfactant production: The effect of varying concentrations of magnesium ion on the biosurfactant production efficiency of WH-2 was studied by supplementing MSM with different concentrations of MgSO 4.7H 2 O in the range of g l Effect of iron on biosurfactant production: The effect of iron (Fe 3+ ) supplemented in growth medium on biosurfactant production efficiency of WH-2 was studied by varying the concentration of ferric ammonium citrate (FAC), a constituent of MSM. The medium supplemented with varying concentrations of FAC viz. 0.0, 0.01 and 0.02 (g l -1 ) was inoculated with activated suspension of WH-2 cells Effect of trace elements on biosurfactant production: The effect of different metal ions viz. Zn 2+, Mn 2+, Co 2+, Ni 2+ and Cu 2+ on biosurfactant production efficiency of WH-2 was studied by supplementing MSM with trace element solution (TES) at a rate of 1.0 and 2.0 (% v/v). The MSM without TES supplement was taken as a control Optimization of physico-chemical parameters for biosurfactant production by WH Effect of incubation temperature on biosurfactant production: The effect of incubation temperature, in the range of 25 C to 40 C, on the biosurfactant production was studied. The flasks were incubated at 100 rpm for 96 h. 59

14 Effect of inoculum level on biosurfactant production: The effect of initial inoculum level on biosurfactant production was assessed. The cells grown for 18 h in nutrient broth (ph 7.0) were used as inoculum. The cells were centrifuged at 10,000 rpm for 10 min, washed with saline (0.8% w/v) and suspended in the same. The OD 600 of the cell suspension was determined and appropriate volume was used to inoculate MSM to achieve an initial OD in the range of (OD 600 ). The inoculum level of 0.3 OD 600 supporting maximum biosurfactant production by WH-2 cells was selected for further studies Effect of inoculum age on biosurfactant production: The effect of age of inoculum on the biosurfactant yield of the WH-2 was studied by using cells grown for 12, 24, 36, 48, 60 and 72 h as inoculum, separately. The harvested cells as per protocol described in Section were used to inoculate MSM so as to attain an initial OD 600 level of Effect of initial ph on biosurfactant production: The effect of initial ph of the MSM on biosurfactant production potential of WH- 2 cells was studied. The ph of MSM was adjusted in the range of 5.0 to 8.5 using either 0.5 N NaOH or 1 N HCl prior to sterilization. The medium was inoculated with activated cell suspension and incubation was carried out at 30 C and 100 rpm for 96 h Effect of static and shaking conditions on biosurfactant production: The effect of incubation under static and shaking (50, 100, 150 and 200 rpm) conditions on biosurfactant production by WH-2 was studied. The flasks were incubated at 30 C Biosurfactant production by P. aeruginosa WH-2 under optimized conditions: The biosurfactant production under optimized conditions was carried out in 250 ml Erlenmeyer flasks having 50 ml of MSM. The MSM (ph 7.0) was supplemented with 2.0 % (w/v) fructose and 0.25 % (w/v) tryptone, and was inoculated with activated (24 h) cell suspension to get a final OD 600 of 0.3. The flasks were incubated at 30ºC in an environmental incubator shaker at 100 rpm. The surface-active properties of the 60

15 biosurfactant were determined at a regular interval of 24 h of incubation (Section 3.7). The study was conducted in three independent experiments and the data reported is the average of three observations Biosurfactant production by P. aeruginosa WH-2 on alternative/low-cost medium supplements The cane molasses, a by-product from sugar industry, was used as a carbon source in MSM to evaluate biosurfactant production ability of strain WH Effect of molasses concentration on biosurfactant production: The MSM was supplemented with different concentrations of cane molasses ( % v/v) equivalent to % (w/v) reducing sugars. The initial reducing sugar level in cane molasses was determined by standard DNS (dinitrosalicylic acid) method (Miller 1959). The flasks were inoculated with activated WH-2 cells corresponding to 0.3 OD 600 and incubated at 30 C and 100 rpm in an orbital environmental shaker for 96 h. The biosurfactant production was calculated as per method described in Section The concentration supporting the maximum biosurfactant yield was used for further experiments Biosurfactant production by P. aeruginosa WH-2 using molasses as a sole source of carbon: The production of biosurfactant was studied by growing cells of P. aeruginosa WH-2 in MSM supplemented with 4.0% v/v (equivalent to 1.6% w/v of reducing sugars) of cane molasses as sole carbon and energy source. The abiotic control having the same media composition but without inoculum, was also incubated under same conditions. The samples were drawn at a regular interval of 24 h till 120 h and analyzed for biosurfactant production (Section 3.7). The study was conducted in three independent experiments and the data reported is the average of three readings Statistical model studies Response surface methodology (RSM) using the Box-Behnken design (Box and Behnken 1960) of experiments was used to develop a mathematical correlation between 61

16 three independent variables, found to be significantly affecting the biosurfactant production by P. aeruginosa WH-2 during initial optimization studies Response Surface Methodology (RSM) using Box Behnken design of experiments for fructose- and molasses-supplemented medium: Based on one-factor at a time approach experiments, three independent variables affecting the biosurfactant production by WH-2 were chosen for optimization by Response Surface Methodology (RSM) using Box Behnken design of experiments. This was used to develop a mathematical correlation between three independent variables on the production of biosurfactants by P. aeruginosa WH-2 (Table 3.1), based on fructose and cane molasses as a carbon source, separately. The three independent variables, fructose concentration (X 1 ), ph (X 2 ) and inoculum level (X 3 ) were chosen to study their effect on biosurfactant production by WH-2 in fructose-supplemented medium. On the other hand, molasses concentration (X 1 ), cornsteep liquor (CSL) concentration (X 2 ) and phosphate (Na 2 HPO 4 ) concentration (X 3 ) were chosen to study the effect of these variables on biosurfactant production in molasses-supplemented medium. The model was studied within a range of variables designated as low (-1), middle (0) and high (+1) concentration (Table 3.2). The experimental design included 17 flasks with five replicates having all the three variables at their central coded values (Table 3.3). The mathematical relationship of response G (rhamnolipid concentration) and variable X was approximated by the quadratic model equation G = β 0 + β 1 X 1 + β 2 X 2 + β 3 X 3 + β 11 X 1 + β 22 X 2 + β 33 X 3 + β 12 X 1 X 2 + β 13 X 1 X 3 + β 23 X 2 X 3 where G is the predicted response, β 0 intercept, β 1, β 2 and β 3, linear coefficients, β 11,β 22, β 33, squared coefficients and β 12, β 13, β 23, interaction coefficients. The MINITAB statistical software (MINITAB Inc., version 11.12, PA, USA) was used to analyze parameters and to generate response surface graphs Extraction and purification of WH-2 biosurfactant Extraction of biosurfactant The biosurfactant produced by cells of WH-2 grown in MSM was isolated from cell-free supernatant. The suitable method of recovery for biosurfactant was optimized by 62

17 using different precipitation and extraction protocols reported in literature with suitable variations. The effectiveness of the recovery processes was determined by comparing surface tension and emulsification activity, for γ-hch and n-hexadecane of the cell-free supernatant, before and after the recovery of biosurfactant Acid precipitation: The recovery of anionic surface-active components was carried out by acid precipitation at ph 2.0. For this, the effect of different strengths of acid viz. 1 N HCl, 5 N HCl and concentrated HCl was studied. After adjusting the ph of cell-free supernatant to 2.0, the flasks were incubated overnight at 4 C to allow complete precipitation of the surface-active components. The precipitates were pelleted out by centrifugation at 10,000 rpm for 10 min in a pre-weighed centrifuge tube. After centrifugation, the pellet was washed with distilled water and re-centrifuged. The pellet was dried and the tube was weighed again to determine the yield Solvent extraction: The cell-free supernatant of WH-2 was extracted thrice with different solvent combinations i.e. chloroform, chloroform: methanol (1:1), chloroform: methanol (2:1), chloroform: methanol (1:2), hexane and ethyl acetate. The pooled samples were concentrated using rotavapor (Büchi, Switzerland) Column chromatography for purification of surfactant preparation The ethyl acetate-extracted WH-2 surfactant was further purified by column chromatography. A column 28 cm (length) x 1.3 cm (diameter) was used for preparing a bed of silica gel ( mesh)-chloroform slurry. The WH-2 biosurfactant sample (1 g of dry weight) was dissolved in 2 ml of chloroform and was loaded carefully on the silica bed with Pasteur pipette. The column was washed with 100% chloroform until neutral lipids were completely eluted. The column was further developed with chloroform: methanol mobile phase starting at an initial gradient of 50:3 (v/v, 200 ml), followed by 50:5 (v/v, 100 ml); 50:50 (v/v, 100 ml) and then 100% methanol (100 ml) at a flow rate of 1 ml min -1. The presence of surface-active molecules in every 20 ml fraction collected was checked by thin-layer chromatography using pre-formed silica gel plates (Merck). 63

18 The mobile phase used for developing the thin-layer chromatogram was chloroform: methanol: water (65:25:4, v/v). The plates were sprayed with anthrone-sulphuric acid reagent (Appendix-1) and heated at 105 C for 10 min to develop the spots. The fractions having similar compounds were pooled and dried under vacuum with a rotavapor. Rhamnolipids were quantified by weighing after drying in oven until a constant weight was achieved Determination of surfactant composition Thin-layer chromatography (TLC): The different biosurfactant types were separated by analytical thin-layer chromatography (TLC) carried out on silica gel plates 60 F 254 (Merck, Darmstadt, Germany) using the solvent system: chloroform: methanol: water = 65:25:4 (v/v/v). The nature of biosurfactant was determined by using different spraying reagents viz. anthrone, α-naphthol and diphenylamine reagent (for carbohydrates), rhodamine 6G (for lipids), hydroxylamine-ferric chloride (for esterified fatty acids) and ninhydrin (for amino acids). The composition and method of preparation of these reagents is given in Appendix Saponification and acid hydrolysis: The biosurfactant produced by P. aeruginosa WH-2 (500 mg) was dissolved in diethyl ether (10 ml) and treated with 0.5 N NaOH in 90% ethanol (15 ml) for 12 h at 60 C for hydrolysis of the ester bond. After saponification, water (10 ml) was added and mixed with the hydrolysate. The resulting ether-extractable and water-soluble fractions were separated. The water-soluble fraction was hydrolyzed with 1 ml of 2 N H 2 SO 4 in a screw-capped tube at 90 C for 4 h. After the hydrolysis, CaCO 3 (0.5 g) was added to neutralize the acid and the resulting solution was used for further chromatographic analysis using the solvent system and spraying reagents as discussed in Section H Nuclear Magnetic resonance (NMR) analysis: The biosurfactant sample was dried in vacuum to remove the traces of organic solvents used for extraction. The dried sample was then dissolved in 1 ml of a mixture of deutrated chloroform (CDCl 3 ) and deutrated methanol (CD 3 OD) in the ratio of 70:30 (v/v) and pipetted into the NMR cell (Wilmad, USA). 1 H-spectra of biosurfactant sample 64

19 of P. aeruginosa strain WH-2 was recorded on AMX 300 NMR spectrophotometer (Bruker, Germany, 300 MHz) locked to the major deuterium resonance of Me 4 Si (TMS) in deutrated solvents, without spinning. Chemical shifts were recorded as δ values relative to TMS Physico-chemical properties of WH-2 biosurfactant preparation Critical micelle concentration (CMC) and γ CMC determination: To determine the CMC of partially-purified as well as purified WH-2 biosurfactant, different concentrations (0-1.0 g l -1 ) of the biosurfactant were prepared in alkaline water. Surface tension was measured with a tensiometer as described in Section The surface tension vs concentration graphs were plotted using Sigma plot to determine CMC and the surface tension at this point was designated as γ CMC. The CMC was expressed as mg l ph stability: The ph of partially-purified biosurfactant solution (200 mg l -1 ) was adjusted to various levels in the range of 2-12 at room temperature using 0.5 N HCl/NaOH. The solutions were incubated at room temperature for 24 h followed by evaluation of surfaceactive properties viz. surface tension, ability to emulsify n-hexadecane, and stability of emulsion formed. The emulsification activity for γ-hch was determined as described by Appaiah and Karanth (1991). 20 mg of γ-hch (0.4% w/v) as acetone solution (0.2 ml) was added to 5.0 ml of crude biosurfactant extract (CBE) and vortexed for 1 min. The colloidal supernatant was carefully decanted after allowing the excess pesticide to settle for 2 h at ambient conditions. The emulsion thus formed was read at 660 nm against distilled water as a blank. The MSM treated in a similar way served as a control Thermal stability: The thermal stability of biosurfactant was evaluated by incubating 50 mg l -1 solution of the partially-purified biosurfactant at 100 C for 4 h. The samples withdrawn at regular intervals of 20 min were used to determine different surface-active properties viz. surface tension, emulsification activities for γ-hch and n-hexadecane, and stability of emulsion formed. 65

20 Resistance to salts: The ability of biosurfactant produced by WH-2 to retain its activity in the presence of salts was studied by adding salts such as NaCl, CaCl 2 and MgSO 4 ranging from % (w/v) to 50 mg l -1 solution of the partially-purified biosurfactant preparation in alkaline water. The effect of salts was assessed by measuring surface tension and emulsification activity of the respective biosurfactant solutions Emulsification index (E 24 ): The emulsification index i.e. the ability of the biosurfactant preparation to emulsify non-water soluble hydrophobic compounds, was also determined. For this, 6 ml of substrate (diesel oil, n-hexadecane, kerosene oil, vegetable oil, and petrol) was added to 4 ml of the crude biosurfactant extract in a graduated tube and vortexed at full speed for 2 min. The height of the emulsion formed was noted and it was allowed to stand for 24 h. After 24 h, the height of the emulsion was again noted and from the data E 24 was determined (Cooper and Goldenberg 1987). The emulsion index (E 24 ) is the height of the emulsion layer (H EL ), divided by total height (H S ), and multiplied by 100. Thus, E 24 (%) = (H EL / H S ) x Comparison of surface-active properties of the biosurfactant with sodium dodecyl sulphate (SDS) The emulsification potential of the biosurfactant preparation of WH-2 was compared with a commonly used anionic surfactant, SDS. The emulsification assay of SDS was carried out at various conditions of ph (Section ), temperature (Section ) and salt (Section ). The emulsification activities for γ-hch (Section ) and n-hexadecane (Section 3.4.3), and emulsion stability (Section 3.4.3) were evaluated Fermenter studies Fermenter: Scale-up studies for the production of rhamnolipids by P. aeruginosa WH-2 were carried out in a 3 l stirred-type bioreactor (STB, Büchi, Switzerland). Agitation was 66

21 provided by six flat-blade impellers and the filtered air was introduced through a sparger at 5 l min -1. Dissolved oxygen in the broth was measured with a galvanic oxygen electrode. The temperature of fermenter was maintained at 30 ± 2 C using a BioRad (USA) cooling system. The fermenter was fitted with an outlet for continuous removal of foam formed due to biosurfactant production in the growth medium Medium used: The MSM (2 l) was sterilized in situ in the glass vessel and was allowed to cool down to room temperature. The medium was supplemented with fructose (2.0% w/v), tryptone (0.05% w/v), MgSO 4.7H 2 O (10.0% w/v) and CaCl 2.2H 2 O (1.0% w/v) from their respective sterilized stock solutions Inoculum development: A single colony of an overnight-activated culture on nutrient agar plate was aseptically transferred into 500 ml nutrient broth. The culture was grown for 24 h at 30 C and 100 rpm on a rotary shaker. The cells were harvested (10,000 rpm, 10 min) and washed with 0.8% (w/v) saline to remove medium components. The pellet was suspended in the saline and was used to inoculate the medium in fermenter to achieve a final OD 600 of 0.3. The culture broth samples were collected from the fermenter at regular intervals of time for further analysis including OD 600, ph of medium, the concentration of reducing sugars and surface tension of the centrifuged fermentation broth Substrate consumption: Total reducing sugars in the growth medium were determined colorimetrically by dinitrosalicylic acid (DNS) method. 3.0 ml of DNS solution (Appendix-1) was added to 1.0 ml of an appropriately diluted sample. The mixture was heated in a water bath at 100 C for 10 min. The solution was then cooled at room temperature and absorbance was measured at 540 nm. The concentration of sugars in the medium was calculated from the standard curve for fructose in the range of µg ml Foam collection: The foam formed after the initiation of biosurfactant production was collected in a sterilized flask through an outlet attachment. The flask was kept at 4 C to allow 67

22 the collected foam to settle down. The liquid obtained after settling down of foam was centrifuged and used for surface tension measurement and acid precipitation of surface-active molecules as per protocols described in Sections and 3.8 respectively Role of biosurfactant in bioremediation The ability of WH-2 rhamnolipids to improve the partitioning of HCH-muck to aqueous phase was studied. The efficacy of partitioning of the biosurfactant solutions with concentrations equivalent to CMC (38 mg l -1 ), above CMC (60 mg l -1 ) and below CMC (20 mg l -1 ) was evaluated Aqueous phase partitioning of HCH-muck by WH-2 biosurfactant The aqueous phase partitioning of HCH was evaluated in presence and absence of purified WH-2 biosurfactant in a glass vial (15 ml). For each experiment, 20 µg HCH ml - 1 was carefully added to the bottom of a screw cap vial. After evaporation of solvent, 5 ml MSM supplemented with appropriate concentration of biosurfactant was added to the vial. Two vials in duplicate were used for each biosurfactant concentrations i.e. at CMC, above CMC, below CMC, and in absence of surfactant. The vials were properly screwed and placed on a rotary shaker at 150 rpm and 30 C for 24 h. The aqueous phase was removed from the vials after 24 h incubation and was extracted thrice with acetone: hexane (20:80) mix. Further, the vials were rinsed with organic solvent mixture to extract the residual HCH-muck Analytical methods: The respective pooled organic phase from both the treatments were concentrated separately using rotavapor and dissolved in a final volume of 1.0 ml. Samples were analyzed using a gas chromatograph (Nucon, Model-5765) fitted with a fused silica capillary column BPX608 (Agilent) 25 m x 0.32 mm (i.d.) x 0.43 mm (o.d.), using electron capture detector (ECD). The injector, detector and column were maintained at 250, 270 and 240 C, respectively. The flow of carrier gas (Nitrogen) was 25 ml min -1. One micro liter (µl) of the sample was injected to the column using a 5 µl glass syringe. 68

23 Effect of WH-2 biosurfactant on growth of HCH-degrading strains The biosurfactants are known to inhibit the growth of other microorganisms. In light of this, the effect of different concentrations of the biosurfactant preparation of WH-2 on the overall growth of HCH-degrading strain Sphingomonas sp. MTCC 8061 was checked. The cells of Sphingomonas sp. were grown in MSM supplemented with both glucose (0.5 g l -1 ) and the respective concentrations of purified biosurfactant (at CMC, above CMC and below CMC). The controls having MSM supplemented with glucose and respective concentrations of biosurfactant, separately were also simultaneously studied. The flasks were incubated at 30 C and 100 rpm for 48 h. The growth was determined at different intervals of time using Shimadzu UV1601 PC spectrophotometer Biodegradation of HCH-isomers by Sphingomonas sp. in the presence of purified WH-2 surfactant The ability of Sphingomonas sp. MTCC 8061 to degrade HCH in the absence and presence of purified WH-2 surfactant was studied Inoculum development: The cells were grown at 30 C and 100 rpm for 48 h in MSM supplemented with sodium benzoate (1% w/v). The cells were centrifuged at 10,000 rpm and 10 min, washed twice using sterilized MSM and resuspended in the same medium to an optical density of Biodegradation studies: The studies were carried out in 15 ml screw cap vials. The initial concentration of HCH used was 20 µg ml -1 added from a stock solution (2000 µg ml -1 ) in acetone. The solvent was allowed to evaporate overnight at 30 C and 4.5 ml of filter-sterilized biosurfactant solution was added to MSM at the desired concentration (below CMC: 20 mg l -1 and at CMC: 38 mg l -1 ). Finally, each vial was inoculated with 0.5 ml of the activated cell suspension and incubated on a rotary shaker at 150 rpm and 30 C for 24 h. The entire contents of the vials were extracted by adding acetone: hexane mix (20:80). 69

24 Organic phase was concentrated using rotary vacuum evaporator (Büchi, Switzerland). The dried extracts were dissolved in 1 ml of acetone and were analyzed for the residual HCH-congeners using ECD (Section ). The degradation for each congener in HCH-muck was quantified by comparing the peak area with the cell-free control incubated along with the biotic samples and was represented as percent degradation achieved. 70