Biodegradation of Diesel oil by Rhodococcus sp. Strain SeAG1

The occurrence of diesel oil contamination in Malaysia’s environment is very common. Many come from accidental spillage during extraction and transportation. The oil contains high levels of persistent hydrocarbons which can negatively influence the ecosystems. The use of microorganisms for the biodegradation of hydrocarbons present in the diesel oil has been suggested as the best approach for the elimination of diesel oil from the environment because this method is very cost effective and safe. To date, there are very few studies on bacteria that possess the ability to survive in high concentrations of hydrocarbons, salt, heavy metals and pesticides. Therefore the isolation and characterization of a potential diesel oil-degrading bacterium from hydrocarbon contaminated sites is crucial in this research. The technique on cell immobilization was employed to evaluate the efficiency of entrapped cells as well as the effect on shielding the cells from high levels of hydrocarbons, metals and pesticides. A total of six isolates was obtained from 50 sample sites contaminated with hydrocarbon. Isolate P2C, the best diesel oil-degrading bacterium was isolated from a hydrocarbon contaminated soil from Pulau Pangkor. 16S rRNA gene analysis, show that Isolate P2C has high similarity to Rhodococcus sp., and was designated as Rhodococcus sp. strain SeAG1. This bacterium exhibited optimum growth and diesel oil degradation at 30oC in the medium containing 10% (v/v) diesel oil, and was able to degrade 64.4% of diesel oil after 30 days of incubation. The optimum nitrogen source was sodium nitrate (NaNO3) at 0.7% (w/v). The optimum pH for bacterial growth and diesel oil degradation was pH 7.5 using phosphate buffer. Rhodococcus sp. strain SeAG1 was then immobilized in gellan gum using optimized parameters. The optimized parameters for cell immobilization were; 4 mm bead size; 250 bead/100 ml for initial cell loading; 0.75 % (v/w) of gellan gum with addition of 7% (v/v) hexadecane. Degradation of diesel oil using freely-suspended cells and immobilized cell was monitored weekly by using gas chromatography equipped with flame ionization detector (GC-FID). The free cells of strain SeAG1 can resist up to 50% (v/v) diesel oil with 5.4% of degradation compared to immobilized cells can degrade 50% (v/v) of diesel oil up to 23.6%, which was the maximum concentration tested in this study within 30 days. The effects of various salinity, heavy metals and pesticides on the degradation of diesel oil were tested for both on free and immobilized cells. Based on the results obtained, immobilized cell can resist higher concentrations of diesel oil and exhibited a higher degradation rate compared to free cells. Free cells can only tolerate up to 5% (w/v) salinity, while immobilized cells can withstand up to 10% (w/v) salinity. Chromium (Cr), Argentum (Ag) and Mercury (Hg) decreased diesel oil degradation in free cells but the effect was much lower on the immobilized cells. Similar results were obtained for pesticides where 3 of the tested pesticides which is carbofuran, paraquat dichloride and atrazine. This compound slightly lowered diesel oil degradation activity by free cells and none of the tested pesticides lowered diesel oil degradation activity by immobilized cells. Therefore the immobilization method has been proven to be an effective system for the bioremediation of diesel oil compared to free cells.

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