The objectives of this research are to: (1) screen and identify gasoline oxygenate (i.e., methyl tert butyl ether (MTBE)) degrading microorganisms from contaminated sites by denaturing gradient gel electrophoresis (DGGE), detect and quantify bacteria that are closely associated with degradation of MTBE by quantitative real-time polymerase chain reaction (PCR), (2) conduct feasibility study of using earthworm to remediate diesel contaminated soil and study effectiveness of ex-situ treatment of diesel contaminated soils at presence of earthworm for cleanup of diesel contaminated soils. The 16S rDNA-denaturing gradient gel electrophoresis (DGGE) was performed to identify the petroleum-hydrocarbons-degrading bacteria in the contaminated ground water. Realtme PCR was performed to provide biodegradation ability by analyzing six genes (phe, rmo, nah, tod, tol and bph4) connected with BTEX (benzene, toluene, ethylbenzene, xylenes) degradation. It has proved difficult to bacteria using MTBE as sole cabon and energy source, so a methyl tertiary butyl ether (MTBE)-metabolizing bacteria were cultured, and then identified with their separate 16S rDNA sequences. All of these sequences were blasted with NCBI database. The significance of this study is to enhance the efficacy of petroleum bioremediation with the establishment of databank of petroleum-hydrocarbons-degrading bacteria. Results for DGGE showed microbial community reduce as the pollution increase and Burkholderia and Geobacteraceae are the bacteria could degradate petroleum hydrocarbons. PHE gene was detected by realtme PCR in petroleum hydrocarbons-impacted wells could indicate the ability for biodegradation. Five bacteria (Pseudomonas sp. NKNU01, Bacillus sp. NKNU01, Klebsiella sp. NKNU01, Enterobacter sp. NKNU01 and Enterobacter sp. NKNU02) were cultured, and then proceeded with MTBE-metabolizing by single bacteria and mix culture. Results for MTBE-metabolizing show the degradation rate and degradation percent for mix culture (32%) was higher than single bacteria (Pseudomonas sp. NKNU01, 16%; Bacillus sp. NKNU01, 15%; Klebsiella sp. NKNU01, 20%; Enterobacter sp. NKNU01, 20% and Enterobacter sp. NKNU02, 25%) during three weeks. In order to assess applicability of using earthworms to degrade polynuclear aromatic hydrocarbons (PAHs), batch experiments were conducted. Two species of earthworm (Eisenia fetida and Perionyx excavatu) were adopted to degrade diesel-contaminated soils for exposure time of 120 days. The total PAHs concentration is around 25, 50, and 100 mg/kg in diesel contaminated soils, respectively. Removal efficiency of PAHs was evaluated after 120 day incubation. The results indicated that removal efficiency of PAHs was compatible among two species. After 60 day exposure, total PAH was reduced to less than 10% in soils of all contaminant levels. The results of control sets illustrated that PAHs may also be degraded by microorganisms in contaminated soils. Bioaccumulation of PAHs in earthworm tissues was not observed in this study. It is believed that microorganisms can assist the degradation of PAHs. Given the proper conditions such as proper humidy, nutrients, to allow earthworms to survive in the contaminated, earthworm can be used for bioremediation of contaminated soil. This study focused on tha potential options of bioremediation. Bacteria community in petroleum hydrocarbon contaminated site was observed and potential MTBE-metabolizing bacteria was evaluated. Applicability of earthworms for remediating PAHs was feasible, in particular, for enhanced bioremediation. As a result, bioremediation technologies will be recommended for cleanup of petroleum contaminated soils depending on their contamination levels, composition of contaminants, soil characteristics and climate.

Petroleum hydrocarbon contamination, bioremediation, methyl tert butyl ether(MTBE), polynuclear aromatic hydrocarbons(PAHs), MTBE-degrading bacteria, earthworm