BIODEGRADATION OF CRUDE OIL USING GENETICALLY MODIFIED STRAINS OF BACILLUS CEREUS AND PSEUDOMONAS AERUGINOSA ISOLATED FROM KADUNA REFINERY EFFLUENT
BIODEGRADATION OF CRUDE OIL USING GENETICALLY MODIFIED STRAINS OF BACILLUS CEREUS AND PSEUDOMONAS AERUGINOSA ISOLATED FROM KADUNA REFINERY EFFLUENT
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Date
2018-03
Authors
CHONOKO, GARBA UMAR
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Abstract
Extensive hydrocarbon extraction and refining activities often result in environmental pollution that has undesired consequences on the biotic and abiotic components of the ecosystem. Biodegradation of hydrocarbons in contaminated systems could be achieved by either physicochemical or biological methods. The present work was undertaken to isolate, identify and assess the hydrocarbon degrading capacity of bacteria associated with effluent samples collected from the Refining and Petrochemical Company at Kaduna. Physicochemical properties of the raw refinery effluents analysed showed that temperature (270C) and pH (6. 61) were within normal range but biochemical oxygen demand, chemical oxygen demand as well as oil and grease had by far exceeded the Federal Environmental Protection Agency maximum permissible limits. The higher values recorded in the raw effluent samples showed that BOD was 300mg/l, COD 400mg/l, oil and grease 540mg/l as against the FEPA limits of 50, 40 and 10mg/l for BOD, COD, Oil and greases respectively. Physicochemical properties of the crude oil showed that the crude oil obtained from Nigeria had low sulphur contents and is predominantly of the light crude oil category. The effluent samples were further analysed using standard microbiological techniques. Bacillus cereus and Pseudomonas aeruginosa were isolated from the refinery effluents. A total of 41 and 21 strains of Bacillus cereus and Pseudomonas aeruginosa respectively were isolated and identified on the basis of their biochemical characteristics from three sites (i.e untreated wastewater channel, waste oil retention pond and outfall of the effluent discharge outside of the refinery). Biochemical characterization confirmed 41 and 21isolates of Bacillus cereus and Pseudomonas aeruginosa respectively. Further identification of the isolates using Microgen kit revealed that only 17(42%) of the 41 and 9 (43%) of the 21 isolates were actually B. cereus and P. aeruginosa respectively. The organisms were assessed to determine their biodegrading capacities on crude oil as the sole carbon source using minimal salt medium. The increase in bacterial count (increase in cell number) was used as index of biodegradation. The test on the degrading activity of the isolates on crude oil from effluent samples revealed that Bacillus cereus and Pseudomonas aeruginosa were the potent degraders of crude oil. The wild strains of Bacillus cereus and Pseudomonas aeruginosa were UV-irradiated for 30minutes and subjected to nitrous acid treatment. The result of mutagenesis showed that B. cereus and P. aeruginosa exhibited comparable mean survival rates of 54.74 and 54.29% respectively following 30minutes exposure to UV-radiation. The growth rate of the two bacteria on crude oil supplemented medium was monitored at varying concentrations of crude oil (0.5, 1, 1.5 and 2%) over a period of eight weeks. Crude oil concentrations of 0.5, 1 and 1.5% were more effective for biodegradation compared to 2%. Petroleum product degradation increased from 96.59% for parent B. cereus to 99.60% for UV-irradiated nitrous acid treated B. cereus, and from 97.58% for parent P. aeruginosa to 99.80% for UV-irradiated nitrous acid treated P. aeruginosa. Higher crude oil degradation potential was observed for second stage mutants of P. aeruginosa than B. cereus. In this study, Pseudomonas aeruginosa exposed to UV-radiation and nitrous acid mutagenesis exhibited significantly higher potential for crude oil degradation and was thus selected as the best performing mutant strain for degradation. The ability of these isolates to degrade crude oil is clear evidence that their genome harbours the relevant degrading genes.
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These bacteria were screened for the presence of the hydrocarbon degrading enzymes (catechol 2, 3 dioxygenase and phenol hydroxylase) by DNA isolation and PCR amplification of gene using specific primers. The organism with catechol 2, 3 dioxygenase enzymes was identified as Bacillus cereus, with 216bp amplification using C23O specific primers. The organism with phenol hydroxylase (Pheh) gene was identified as Pseudomonas aeruginosa with 870bp amplification using Pheh specific primers. Thus, modification of the organisms with UV-irradiation for 30 min followed by nitrous acid treatment resulted in their increased petroleum product degradation ability and could therefore be used for biodegradation of hydrocarbons in petroleum products polluted environments.
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A DISSERTATION SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES, AHMADU BELLO UNIVERSITY, ZARIA, NIGERIA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF DEGREE OF DOCTOR OF PHYLOSOPHY INMICROBIOLOGY DEPARTMENT OF MICROBIOLOGY, FACULTY OF LIFE SCIENCES
AHMADU BELLO UNIVERSITY, ZARIA, NIGERIA
Keywords
BIODEGRADATION,, CRUDE OIL USING, GENETICALLY MODIFIED STRAINS,, BACILLUS CEREUS,, PSEUDOMONAS AERUGINOSA,, ISOLATED,, KADUNA,, REFINERY EFFLUENT,