Diversity and abundance of bacteria in an underground oil-storage cavity

DNA was extracted from the groundwater obtained from an oil-storage cavity situated at Kuji in Iwate, Japan, and 16S rRNA gene (16S rDNA) fragments were amplified by PCR using combinations of universal and Bacteria-specific primers. The sequence analysis of 154 clones produced 31 different bacterial sequence types (a unique clone or group of clones with sequence similarity of > 98). Major sequence types were related to Desulfotomaculum, Acetobacterium, Desulfovibrio, Desulfobacula, Zoogloea and Thiomicrospira denitrificans. The abundance in the groundwater of bacterial populations represented by these major sequence types was assessed by quantitative competitive PCR using specific primers, showing that five rDNA types except for that related to Desulfobacula shared significant proportions (more than 1%) of the total bacterial rDNA.Bacteria inhabiting the oil-storage cavity were unexpectedly diverse. A phylogenetic affiliation of cloned 16S rDNA sequences suggests that bacteria exhibiting different types of energy metabolism coexist in the cavity.Underground cavities have been used for long-term storage of crude oil in several countries, and one of such facilities is situated at Kuji in Iwate, Japan. These cavities have been constructed in groundwater-rich rocky strata, where high groundwater pressure confines the stored oil in the cavities [1]. Consequently, groundwater migrates into and accumulates at the bottom of a cavity (cavity groundwater), and this cavity groundwater is discharged to maintain the oil storage capacity of the cavity (this system has been detailed in our previous study [1]). Our previous study [1] has also shown active growth of microorganisms in groundwater accumulating at the bottom of the cavities; the total count of microorganisms in the cavity groundwater was constantly more than 106 cells per ml (densities 100 times higher than those in groundwater around the cavities). This habitat can be characterized by (i) immediate contact with a larg