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Applied and Environmental Microbiology, June 2003, p. 3311-3316, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3311-3316.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Isolation of a Methanogen from Deep Marine Sediments That Contain Methane Hydrates, and Description of Methanoculleus submarinus sp. nov.

Jill A. Mikucki,1,{dagger} Yitai Liu,1 Mark Delwiche,2 Frederick S. Colwell,2 and David R. Boone1*

Biology Department, Portland State University, Portland, Oregon,1 Idaho National Environmental and Engineering Laboratory, Idaho Falls, Idaho2

Received 23 December 2002/ Accepted 4 March 2003

We isolated a methanogen from deep in the sediments of the Nankai Trough off the eastern coast of Japan. At the sampling site, the water was 950 m deep and the sediment core was collected at 247 m below the sediment surface. The isolated methanogen was named Nankai-1. Cells of Nankai-1 were nonmotile and highly irregular coccoids (average diameter, 0.8 to 2 µm) and grew with hydrogen or formate as a catabolic substrate. Cells required acetate as a carbon source. Yeast extract and peptones were not required but increased the growth rate. The cells were mesophilic, growing most rapidly at 45°C (no growth at <=10°C or >=55°C). Cells grew with a maximum specific growth rate of 2.43 day-1 at 45°C. Cells grew at pH values between 5.0 and 8.7 but did not grow at pH 4.7 or 9.0. Strain Nankai-1 grew in a wide range of salinities, from 0.1 to 1.5 M Na+. The described phenotypic characteristics of this novel isolate were consistent with the in situ environment of the Nankai Trough. This is the first report of a methanogenic isolate from methane hydrate-bearing sediments. Phylogenetic analysis of its 16S rRNA gene sequence indicated that it is most closely related to Methanoculleus marisnigri (99.1% sequence similarity), but DNA hybridization experiments indicated a DNA sequence similarity of only 49%. Strain Nankai-1 was also found to be phenotypically similar to M. marisnigri, but two major phenotypic differences were found: strain Nankai-1 does not require peptones, and it grows fastest at a much higher temperature. We propose a new species, Methanoculleus submarinus, with strain Nankai-1 as the type strain.

* Corresponding author. Mailing address: Biology Department, Portland State University, P.O. Box 751, Portland, OR 97207-0751. Phone: (503) 725-3865. Fax: (503) 725-8570. E-mail: booned{at}pdx.edu.

{dagger} Present address: Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, MT 59717.

Applied and Environmental Microbiology, June 2003, p. 3311-3316, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3311-3316.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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