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Applied and Environmental Microbiology, August 2002, p. 3899-3907, Vol. 68, No. 8
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.8.3899-3907.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Diversity, Abundance, and Activity of Archaeal Populations in Oil-Contaminated Groundwater Accumulated at the Bottom of an Underground Crude Oil Storage Cavity

Kazuya Watanabe,* Yumiko Kodama, Natsuko Hamamura, and Nobuo Kaku

Marine Biotechnology Institute, Heita, Kamaishi City, Iwate 026-0001, Japan

Received 14 February 2002/ Accepted 16 May 2002

Fluorescence in situ hybridization has shown that cells labeled with an Archaea-specific probe (ARCH915) accounted for approximately 10% of the total cell count in oil-contaminated groundwater accumulated at the bottom of an underground crude oil storage cavity. Although chemical analyses have revealed vigorous consumption of nitrate in cavity groundwater, the present study found that the methane production rate was higher than the nitrate consumption rate. To characterize the likely archaeal populations responsible for methane production in this system, fragments of 16S ribosomal DNA (rDNA) were amplified by PCR using eight different combinations of universal and Archaea-specific primers. Sequence analysis of 324 clones produced 23 different archaeal sequence types, all of which were affiliated with the kingdom Euryarchaeota. Among them, five sequence types (KuA1, KuA6, KuA12, KuA16, and KuA22) were obtained in abundance. KuA1 and KuA6 were closely related to the known methanogens Methanosaeta concilii (99% identical) and Methanomethylovorans hollandica (98%), respectively. Although no closely related organism was found for KuA12, it could be affiliated with the family Methanomicrobiaceae. KuA16 and KuA22 showed substantial homology only to some environmental clones. Both of these branched deeply in the Euryarchaeota, and may represent novel orders. Quantitative competitive PCR showed that KuA12 was the most abundant, accounting for ~50% of the total archaeal rDNA copies detected. KuA1 and KuA16 also constituted significant proportions of the total archaeal rDNA copies (7 and 17%, respectively). These results suggest that limited species of novel archaea were enriched in the oil storage cavity. An estimate of specific methane production rates suggests that they were active methanogens.

* Corresponding author. Mailing address: Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan. Phone: 81 193 26 5781. Fax: 81 193 26 6592. E-mail: kazuya.watanabe{at}mbio.jp.

Applied and Environmental Microbiology, August 2002, p. 3899-3907, Vol. 68, No. 8
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.8.3899-3907.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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