Isolation,Characterizations And Construction Of High-Effective Oil-Degrading Bacterium And The Bioaugmentation On Bioremediation

Three efficient aromatic and aliphatic hydrocarbons-degrading bacteria W1, W2 and W3 were isolated from crude oil-polluted soils. They were all identified as Rhodococcus sp. according to their morphological features, physiological biochemical characterictics and 16S rDNA sequences analysis.Three isolates were all gram-positive, had spherical and rod growth cycle. W1 could form orange colony on LB agar medium after 2～3 days of incubation, whereas W2 and W3 could form apricot colony. The optimal pH value for the growth of strains W1,W2,W3 were 6.0～8.0, the optimal temperature was 30℃. The optimal NaCl concentration for the three strains was 1～4% NaCl. They could grow well when using glucose or sucrose as sole carbon sources, and peptone, beef cream or yeast extract as sole nitrogen sources.W1,W2,W3 could all grow in the mineral medium containing 500mg/L n-octadecane as sole carbon source and degrade 100%,96.9%,98.12% n-octadecane after 56h of incubation respectively. The optimal temperature, pH and NaCl concentration for degrading n-octadecane by strains W1,W2,W3 were 10～30℃, pH6.0～8.0 and 0～5%NaCl respectively. They still had good ability to degrade n-octadecane at low-temperature. W1,W2, W3 could degrade more than 85% of C₁₃～C₃₂ n-alkanes in high concentration crude oil (5g/L) . W1 could degrade more than 90% pristine and phytane in crude oil . W2 could degrade 50% pristine and phytane, while W3 could degrade 40% pristine. W1 could produce biosurfactants during growth with paraffin as sole carbon sources, resulting in decrease of surface tension of medium from 60.5 mN/m to 36.6 mN/m.Petroleum oil degradation by mixed strains was studied. There was greatly difference of petroleum oil degradation efficiency among different strains mixtures. A consortia D consisting of two species of Rhodococcus (W1,W3) and one species of Gardenia(W4) and Pesudomonas(W7) respectively had a highest degradation rate (70.3%) of petroleum oil after 5d of incubation and was used as inoculants in bioremediation of petroleum oil-polluted soil. Bioremediation results showed that total petroleum hydrocarbon (TPH) drop 50% at 25℃in 30 days. Bioremediation efficiencies were better in soil at pH6.5-9.5, 10-20℃. The degradation rates of oil by consortia D was faster in sterilized soil than in unsterilized soil. Although bioremediation efficiencies were proportional to the bacterial number inoculated, there were no obviously difference among inoculants at 10⁶,10⁷,10⁸,10⁹CFU·g^-1, all with the about 60 % removal rates of petroleum oil during 30 days.