Molecular Characterization of Bacterial Populations in Petroleum-Contaminated Groundwater Discharged from Underground Crude Oil Storage Cavities

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Abstract
Petroleum-contaminated groundwater discharged from underground crude oil storage cavities (cavity groundwater) harbored more than 106 microorganisms ml−1, a density 100 times higher than the densities in groundwater around the cavities (control groundwater). To characterize bacterial populations growing in the cavity groundwater, 46 PCR-amplified almost full-length 16S ribosomal DNA (rDNA) fragments were cloned and sequenced, and 28 different sequences were obtained. All of the sequences were affiliated with the Proteobacteria; 25 sequences (43 clones) were affiliated with the epsilon subclass, 2 were affiliated with the beta subclass, and 1 was affiliated with the delta subclass. Two major clusters (designated clusters 1 and 2) were found for the epsilon subclass proteobacterial clones; cluster 1 (25 clones) was most closely related to Thiomicrospira denitrificans (88% identical in nucleotide sequence), while cluster 2 (11 clones) was closely related to Arcobacterspp. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rDNA fragments showed that one band was detected most strongly in cavity groundwater profiles independent of storage oil type and season. The sequence of this major band was identical to the sequences of most of the cluster 1 clones. Fluorescence in situ hybridization (FISH) indicated that the cluster 1 population accounted for 12 to 24% of the total bacterial population. This phylotype was not detected in the control groundwater by DGGE and FISH analyses. These results indicate that the novel members of the epsilon subclass of the Proteobacteria grow as major populations in the petroleum-contaminated cavity groundwater.