Isolation of Crude Oil Degrading Marine Bacteria and Assessment for Biosurfactant Production

摘要

Ten bacterial isolates recovered from the crude oil contaminated sea water samples from a ship yard in Alang coast, Gujarat showed optimum growth in presence of crude oil. The crude oil degrading isolates were identified as species of genera Acinetobacter, Marinococcus, Micrococcus, Planococcus, Methylobacterium, Rhodococcus and Noccardia. All the ten isolates degraded 1% crude oil in Bushnell Haas broth. Highest biodegradation of crude oil was found to be 70% by the isolate M2 which belonged to genus Marinococcus. Other isolates showed biodegradation in the range of 55-30%. On addition of 1% Tween 80 biodegradation reduced in majority of cases. 70% of the bacterial isolates scored positive for biosurfactant production and emulsified toluene however emulsion stability varied drastically among the isolates. None of the biosurfactant producing bacterial isolates was hemolytic.Key words: Biodegradation, Biosurfactant, Emulsion, Hemolytic. Introduction In quantitative terms, crude oil is one of the most important organic pollutants in marine environment and it has been estimated that worldwide somewhere between 1.7- 8.8×106 tons of petroleum hydrocarbons impact marine waters and estuaries annually (1). Reports have been appearing since last three decades on the biodegradability of crude oil by bacteria which can use hydrocarbons as source of carbon and energy (2). When micro organisms grow in environment rich in hydrocarbon, they undergo many adaptations. One such adaptation is biosurfactant production which is a frequently encountered feature in hydrocarbon degrading bacteria or sometimes even a prerequisite for growth on hydrocarbons (3). Biosurfactant production helps the hydrocarbon degrading bacterium to gain better access to their hydrophobic substrates as it brings about changes like reduction of surface tension of the environment around the bacterium, reduction of interfacial tension between bacterial cell wall and hydrocarbon molecules, membrane modifications like increasing the hydrophobicity of cell wall by reducing the lipopolysaccharide content of cell wall, enhancing the dispersion of hydrocarbon by encapsulation of the hydrocarbon into micelles etc (4,5,6,7). Amongst the above changes reduction in surface and interfacial tension is a universal phenomenon displayed by almost all types of biosurfactant whereas changes like membrane modifications and emulsion formation strictly depends upon type of biosurfactant for e.g. glycolipids, lipopepetides, polymeric or particulate etc. Enhancement in hydrocarbon degradation may occur by cumulative effect of above changes.Here we present a report on isolation of crude oil degrading marine bacteria from oil contaminated sea water, their screening for crude oil degradation and biosurfactant production. Methods Collection of sampleSea water samples were collected in sterilized glass bottles from a ship yard at Alang coast, Gujarat. Isolation and screening of crude oil degrading bacteriaIsolation of crude oil degrading marine bacteria was carried out by spreading 100 μL of serially diluted seawater samples on mineral salt medium (MM2). An ethereal solution of crude oil (10% w/v) was uniformly sprayed over the surface of agar plate. The ether immediately vaporized and thin layer of oil remained on the entire surface. The crude oil was obtained from ONGC plant at Chandkheda near Ahmedabad, Gujarat. The plates were incubated at 25oC for 20 days. The bacterial isolates which appeared on MM2 plate after incubation were screened for crude oil degradation by overlay technique (8).Crude oil biodegradationCrude oil biodegradation experiment was performed by modifying the technique described by Pirnik et al. (9) by adding the inoculating cells of density 108 mL-1 to BHM with 1% crude oil added as sole carbon source. To study effect of Tween 80 on biodegradation of crude oil a similar set of experiment as described was performed with 1% Tween 80 added to all the flasks (10). The estimation of