Application of real-time PCR, DGGE fingerprinting, and culture-based method to evaluate the effectiveness of intrinsic bioremediation on the control of petroleum-hydrocarbon plume

Real-time polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and the culture-based method were applied in the intrinsic bioremediation study at a petroleum-hydrocarbon contaminated site. The genes of phenol hydroxylase (PHE), ring-hydroxylating toluene monooxygenase (RMO), naphthalene dioxygenase (NAH), toluene monooxygenase (TOL), toluene dioxygenase (TOD), and biphenyl dioxygenase (BPH4) were quantified by real-time PCR. Results show that PHE gene was detected in groundwater contaminated with benzene, toluene, ethylbenzene, xylene isomers (BTEX) and methyl tert-butyl ether (MTBE), and this indicates that intrinsic bioremediation occurred at this contaminated site. Results from DGGE analyses reveal that the petroleum-hydrocarbon plume caused the variation in microbial communities. In this study, MTBE degraders including Pseudomonas sp. NKNU01, Bacillus sp. NKNU01, Klebsiella sp. NKNU01, Enterobacter sp. NKNU01, and Enterobacter sp. NKNU02 were isolated from the contaminated groundwater using the cultured-based method. Results from MTBE biodegradation experiment show that the isolated bacteria were affected by propane. This indicates that propane may influence the metabolic pathway of MTBE by these bacteria. Knowledge and comprehension obtained from this study will be helpful in evaluating the occurrence and effectiveness of intrinsic bioremediation on the remediation of petroleum-hydrocarbon contaminated groundwater.