Culture-dependent and culture-independent characterization of microbial assemblages associated with high-temperature petroleum reservoirs

Recent investigations of oil reservoirs in a variety of locales have indica ted that these habitats may harbor active thermophilic prokaryotic assembla ges. In this study, we used both molecular and culture-based methods to cha racterize prokaryotic consortia associated,vith high-temperature, sulfur-ri ch oil reservoirs in California. Enrichment cultures designed for anaerobic thermophiles, both autotrophic and heterotrophic, were successful at tempe ratures ranging from 60 to 90 degrees C. Heterotrophic enrichments from all sites yielded sheathed rods (Thermotogales), pleomorphic rods resembling T hermoanaerobacter, and Thermococcus-like isolates. The predominant autotrop hic microorganisms recovered from inorganic enrichments using EI,, acetate, and CO, as energy and carbon sources were methanogens, including isolates closely related to Methanobacterium, Methanococcus, and Methanoculleus spec ies. Two 16S rRNA gene (rDNA) libraries were generated from total community DNA collected from production wellheads, using either archaeal or universa l oligonucleotide primer sets. Sequence analysis of the universal library i ndicated that a large percentage of clones were highly similar to known bac terial and archaeal isolates recovered from similar habitats. Represented g enera in rDNA done libraries included Thermoanaerobacter, Thermococcus, Des ulfothiovibrio, Aminobacterium, Acidaminococcus, Pseudomonas, Halomonas, Ac inetobacter, Sphingomonas, Methylobacterium, and Desulfomicrobium. The arch aeal library was dominated by methanogen-like rDNAs, with a lower percentag e of clones belonging to the Thermococcales. Our results strongly support t he hypothesis that sulfur-utilizing and methane-producing thermophilic micr oorganisms have a widespread distribution in oil reservoirs and the potenti al to actively participate in the biogeochemical transformation of carbon, hydrogen, and sulfur in situ.