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

Abstract   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 × 10⁴−7.9 × 10⁶/g) of microbes in the oil-bearing strata than in the oil-free zones, where counts were uniform at approximately 1.0 × 10⁴/g. Molecular taxonomic analyses via 16S rRNA gene sequence comparisons showed that the oil-free strata were dominated by members of the γ-Proteobacteria including Pseudomonas , Stenotrophomonas and Sphingomonas , whereas the oil-bearing strata were dominated by a single species closely related to Pseudomonas stutzeri . All archaeal clones were phylogenetically affiliated with the uncultured soil group in Crenarchaeota with the exception of a single phylotype that belonged to the genus Thermococcus . 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 Pseudomonas stutzeri and neither autotrophs nor anaerobic heterotrophs could be detected in the oil-bearing strata.