Isolation of bacteria from contaminated soil and their biodegradation potential

Industrialization is accompanied by inevitable complications among which pollutions and global warming are of utmost importance. Oil spills are an important part of pollution and can be very hard to clean up, requiring weeks to even years. Among various different remediation methods, bioremediation is considered one of the best. Fundamental concern of this work was enhancing biodegradation capability of potential indigenous tropical soil bacteria without genetic mutations, changing soil mineral ratios or soil microbial community. Thus, in a new approach suitable bacterial isolates adapted sequentially to higher concentrations of crude oil at pH and temperature values simulating their natural habitat. Their biodegradation ability in terms of n-alkane removal analyzed both in liquid medium and soil within 28 days. In doing so, soil bacteria were isolated, enriched and selected based on their ability to grow on 5% (v/v) crude oil, leading to selection of 8 isolates. Later a simple mathematical model for their growth was calculated to monitor their growth easily and accurately. At later stages isolates were adapted sequentially to higher concentration of crude oil, i.e. 10, 20 and 50% (v/v) at the pH and temperature values similar to their natural habitat. Their growth monitored using their growth equations and exponential growth rate and doubling time of each isolate at each concentration was calculated. Only 5 out of 8 isolates could survive 50% (v/v) crude oil and the other 3 showed negative values for exponential growth indicateing lack of growth at 50% (v/v). To identify the bacteria, biochemical, morphological and molecular identification techniques were conducted; Top 5 oil degraders were identified as P. putida, A. lwoffi, A. hydrophila, P. stutzeri, and A. johnsonii by 16S rRNA sequencing; The other 3 isolates identified morphologically and biochemically only, and found to be 1 isolate from Rhodococcus spp. and 2 from Bacillus spp. Based on gas chromatography-mass spectrometry analysis adapted isolates and their consortium proved to be more efficient in n-alkane degradation as removal of C8-C33 was enhanced up to 93% in liquid cultures and 70% in artificially polluted soil after 28 days. P. putida, A. lwoffi, A. hydrophila, P. stutzeri,and A. johnsonii removed 74%, 56%, 76%, 61%, and 67%, respectively from a liquid medium amended with 50% (v/v) crude oil. A comparison between degradation ability of 5 bacterial mixture in soil before and after sequential adaptation showed that their biodegradation ability was enhanced up to 41% without any exposure to biological mutation or addition of nutrients. In addition ratios of n17 to pristane and n18 to phytane before and after degradation supported the promising bioremediation ability of the consortiums and that removal was indeed happened as a result of their activity. In general result suggests preconditioning bacterial isolates to higher oil concentration can significantly enhance their biodegradation ability especially when the isolates are applied as a consortium