PRODUCTION OF MICROBIAL BIOSURFACTANT USING COST-EFFECTIVE RESOURCES

Abstract

Biosurfactants are structurally diverse group of surface active compounds produced by a wide variety of microorganisms. They have unique amphiphatic properties derived from their complex structures, which include a hydrophilic moiety and a hydrophobic portion. In the present investigation focus was on the isolation of bacterial strain for utilizing cost-effective substrate for biosurfactant production. A total of 340 bacterial isolates were obtained from hydrocarbon contaminated sites viz. oil sludge samples, fuel filling stations, ware houses and phenol waste water of which 26 bacterial isolates (DSVP1- DSVP26) were selected on basis of their ability to grow on hydrocarbons (dodecane, hexadecane, pristane, toluene and fluoranthene). Screening of these bacterial isolates was done for biosurfactant production using drop collapse assay, emulsification assay, hemolytic assay and surface tension reducing ability. Of these five bacterial isolates DSVP2, DSVP9, DSVP11, DSVP18 and DSVP23 were selected as potent biosurfactant producers. Cell surface hydrophobicity tests like hydrocarbon interaction chromatography (HIC), salt aggregation test (SAT), bacterial adherence to hydrocarbons (BATH) and replica plate (RP), confirmed biosurfactant producing ability of these bacterial isolates. These five selected bacterial isolates were further exploited for their ability to produce biosurfactant by utilizing cost effective raw materials (cotton seed hull, tea leaves, wheat bran, corn starch, rice straw, wheat straw, bagasse, ground nut kernel, potato peel, apple peel, cotton seed, molasses, bamboo wood saw dust and gram husk). Amongst above isolates DSVP23 was chosen to be the best candidates for biosurfactant production effectively utilizes cotton seed hull as substrate in minimal salt medium (MSM). The isolated strain was identified by morphological, biochemical and molecular biology technique using the taxonomic scheme of Bergey's manual of determinative bacteriology and 16S rRNA. Using the Basic Local Alignment Search Tool (BLAST) available in the National Center for Biotechnology Information (NCBI) database, homology of 16S ribosomal RNA (16S rRNA) gene sequence obtained for strain DSVP23 depicted it to be of B. subtilis (Gene bank accession no. EU679368). Biosurfactant from cell free broth of B. subtilis DSVP23 isolated using acid precipitation was further extracted by dichloromethane. The surfactant obtained showed reduction in surface-tension value of water from 72mN/m to 28mN/m. The critical micelle concentration of the surfactant was 30 mg/1. The surface tension and emulsification capacity remained unaltered within a widepH (2-12), temperature (4-80°C) ranges and underNaCl (2- 10%) concentrations. Biosurfactant was characterized using thin layer chromatography (TLC) and Fourier transforms infrared (FTIR). FTIR spectra showed strong absorption bands of peptides at 3343 cm"1, 1641 cm"1, 1518 cm"1 resulting from N-H stretching, CO stretching and combined C-N stretching mode respectively. 1368 cm"1, 1451 cm"1 and 2960 cm"1, bands are predominant and indicate aliphatic chains (CH2 ,CH3) of sample. The intense band at 1641 cm" corresponds to -CO-NH-R group indicated biosurfactant to be lipopeptide in nature. The biosurfactant was further analysed using HPLC. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and nuclear magnetic resonance (NMR) revealed that the major constituent of lipopeptide being leucine and isoleucine. The presence of a lactone ring in the surfactant was indicated by NMR spectra, which detected an ester carbonyl group, and was supported by the fact that the peptide has a blocked N terminus. The mass spectrum of this sample showed an isolated peak around mlz 1044.6. A high yield of biosurfactant was obtained from a culture of B. subtilis DSVP23 using 2% cotton seed hull supplemented with sucrose (2%) as a carbon source in MSM. Ammonium nitrate (4.5g/l) and sodium nitrate (4.0g/l) were among the best nitrogen sources for biosurfactant production when incubated at 37°C temperature with 0.5 vvm and 180 rpm of aeration rate and agitation speed respectively. The addition of various metal supplements (manganese, magnesium, calcium, iron, and trace elements) greatly affected growth and biosurfactant production. A high yield of biosurfactant (4.0-4.5 g/1) was elucidated upon addition of 30 mg/1, MnS04and 5mM FeS04. Amino acids (0.1% w/v), such as aspartic acid, glutamic acid, lysine and valine increased the final yield of biosurfactant. B. subtilis mutants were obtained using EMS (3%) treatment producing high level of the lipopeptide biosurfactant to that of wild type DSVP23. Mutant was selected using reduction in surface tension value and zone of hemolysis formed compared to that of wild type. EMS treatment resulted in 8 mutant colonies, showing a 2 fold increase in biosurfactant production to that of wild type DSVP23. Comparative analysis of biosurfactant production mutant {DVM4) and wild type (DSVP23) to utilize cheap raw materials was assessed using fermentation. The mutant, designated B. subtilis DVM4, was capable of producing lipopeptide biosurfactant at concentrations up to 6.0g/l compared to that of 4.0g/l of wild type (DSVP23). The application of isolated biosurfactant in enhanced oil recovery (EOR) was evaluated using the sand pack technique. Recovery of 76% kerosene oil, 72% motor oil, 68% n-paraffin 70% crude oil and 62% mobile oil was obtained by using 0.5 % of aqueous solution of biosurfactant in sand pack column. Biosurfactant produced by B. subtilis DSVP23 was found to be an effective emulsifier when tested against different hydrocarbons kerosene oil (78%), hexadecane (72%), toluene (72%), motor oil (70%), dodecane (68%), tetradecane (64%), and hexane (64%). Thus signifies its potential application in oil spill management. The biosurfactant obtained using cotton seed hull in MSM as cost effective substrate showed profoundly distinct antibacterial activity toward test organisms namely S. aureus, E.coli, P. aeruginosa and B. cereus with a zone of inhibition 18mm, 13.8mm, 12.1mm and 11.2mm respectively. Also biosurfactants displayed a maximum antifungal activity against C. albicans followed by R. solani, F. oxysporum and T. viride with a zone of inhibition of 15.2mm, 11.3mm, 10mm and 12.5mm respectively. The potential of lipopeptide biosurfactant in inhibiting biofilm adhesion of bacteria and fungi was demonstrated by using the MTP assay. In particular, Candida albicans and Staphylococcus aureus biofilm formation was decreased to 78% and 72% respectively at biosurfactant concentration 10.5 fig/ml and 12 ug/ml. Microscopic studies further explored morphological alterations in biofilms upon biosurfactant treatment. Visualization of biofilms of both yeast and bacterial biofilm ultrastructure by SEM revealed that major damage to the biofilm constituents was caused by lipopeptide biosurfactant. Reduction in viable cell count was also checked using fluorescent viability test using fluorescent dyes (FDA and EtBr). AFM studies revealed topographic images of S. aureus and C. albicans biofilms. Images revealed that cell disintegration occurred upon lipopeptide biosurfactant treatment. Biodegradation potential of microbes in degrading mixed substrates (i.e. oily sludge) vis-D-vis pure hydrocarbons like pristane and fluoranthene was also investigated. Degradation of different substrates was assessed by monitoring dry cell biomass. pH, reduction in surface tension, biosurfactant production and gas chromatographic profiles. Our data using capillary gas chromatographic analysis revealed that B. subtilis effectively degrade oily sludge components aliphatic hydrocarbons and aromatic hydrocarbons after 7 days of incubation.