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Applied and Environmental Microbiology, February 2000, p. 700-711, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Culture-Dependent and Culture-Independent Characterization of Microbial Assemblages Associated with High-Temperature Petroleum Reservoirs

V. J. Orphan,1,2,* L. T. Taylor,2 D. Hafenbradl,3 and E. F. Delong2,*

Marine Science Institute, University of California, Santa Barbara, California 931061; Monterey Bay Aquarium Research Institute, Moss Landing, California 950392; and Diversa Corporation, San Diego, California 921213

Received 13 September 1999/Accepted 25 November 1999

Recent investigations of oil reservoirs in a variety of locales have indicated that these habitats may harbor active thermophilic prokaryotic assemblages. In this study, we used both molecular and culture-based methods to characterize prokaryotic consortia associated with high-temperature, sulfur-rich oil reservoirs in California. Enrichment cultures designed for anaerobic thermophiles, both autotrophic and heterotrophic, were successful at temperatures ranging from 60 to 90°C. Heterotrophic enrichments from all sites yielded sheathed rods (Thermotogales), pleomorphic rods resembling Thermoanaerobacter, and Thermococcus-like isolates. The predominant autotrophic microorganisms recovered from inorganic enrichments using H2, acetate, and CO2 as energy and carbon sources were methanogens, including isolates closely related to Methanobacterium, Methanococcus, and Methanoculleus species. Two 16S rRNA gene (rDNA) libraries were generated from total community DNA collected from production wellheads, using either archaeal or universal oligonucleotide primer sets. Sequence analysis of the universal library indicated that a large percentage of clones were highly similar to known bacterial and archaeal isolates recovered from similar habitats. Represented genera in rDNA clone libraries included Thermoanaerobacter, Thermococcus, Desulfothiovibrio, Aminobacterium, Acidaminococcus, Pseudomonas, Halomonas, Acinetobacter, Sphingomonas, Methylobacterium, and Desulfomicrobium. The archaeal library was dominated by methanogen-like rDNAs, with a lower percentage of clones belonging to the Thermococcales. Our results strongly support the hypothesis that sulfur-utilizing and methane-producing thermophilic microorganisms have a widespread distribution in oil reservoirs and the potential to actively participate in the biogeochemical transformation of carbon, hydrogen, and sulfur in situ.

* Corresponding authors. Mailing address: Monterey Bay Aquarium Research Institute, P.O. Box 628, 7700 Sandholdt Rd., Moss Landing, CA 95039. Phone: (831) 775-1843. Fax: (831) 775-1645. E-mail: orphan{at}mbari.org or delong{at}mbari.org.

Applied and Environmental Microbiology, February 2000, p. 700-711, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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