Culture‐dependent and independent analyses of subsurface microbial communities in oil‐bearing strata of the Sagara oil reservoir

<jats:p><jats:bold>Abstract </jats:bold> Culture‐dependent and independent methods were used to evaluate the microbial communities in cores collected at depths up to 200 m in oil‐bearing and oil‐free strata near the Sagara oil reservoir near Shizuoka, Japan. Direct microbial counts revealed much higher numbers (2.2 × 10<jats:sup>4</jats:sup>−7.9 × 10<jats:sup>6</jats:sup>/g) of microbes in the oil‐bearing strata than in the oil‐free zones, where counts were uniform at approximately 1.0 × 10<jats:sup>4</jats:sup>/g. Molecular taxonomic analyses via 16S rRNA gene sequence comparisons showed that the oil‐free strata were dominated by members of the <jats:italic>γ‐Proteobacteria</jats:italic> including <jats:italic>Pseudomonas</jats:italic>, <jats:italic>Stenotrophomonas</jats:italic> and <jats:italic>Sphingomonas</jats:italic>, whereas the oil‐bearing strata were dominated by a single species closely related to <jats:italic>Pseudomonas stutzeri</jats:italic>. All archaeal clones were phylogenetically affiliated with the uncultured soil group in <jats:italic>Crenarchaeota</jats:italic> with the exception of a single phylotype that belonged to the genus <jats:italic>Thermococcus</jats:italic>. Culture‐dependent analysis was carried out by most‐probable‐number culturing as well as direct plating to determine viable cell counts, using both complex organic substrates or native oil and autotrophic media. Both culture‐dependent and independent methods revealed the abundant cultivable member was the aerobic oil‐degrading bacterium <jats:italic>Pseudomonas stutzeri</jats:italic> and neither autotrophs nor anaerobic heterotrophs could be detected in the oil‐bearing strata.</jats:p>