Characterization of C 1 -Metabolizing Prokaryotic Communities in Methane Seep Habitats at the Kuroshima Knoll, Southern Ryukyu Arc, by Analyzing pmoA , mmoX , mxaF , mcrA , and 16S rRNA Genes

• Published in: Applied and environmental microbiology Vol. 70; no. 12; pp. 7445 - 7455
• Main Authors: Inagaki, Fumio, Tsunogai, Urumu, Suzuki, Masae, Kosaka, Ayako, Machiyama, Hideaki, Takai, Ken, Nunoura, Takuro, Nealson, Kenneth H., Horikoshi, Koki
• Format: Journal Article
• Language: English
• Published: 01-12-2004
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.70.12.7445-7455.2004

ABSTRACT
Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (∼34% of the total prokaryotes), including both mesophilic methanogens related to the genus
Methanolobus
and ANME-2 members of the
Methanosarcinales
, as well as members of the δ-
Proteobacteria
, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (
pmoA
), soluble methane monooxygenase (
mmoX
), methanol dehydrogenese (
mxaF
), and methyl coenzyme M reductase (
mcrA
). In the MC core sediments, the most abundant gene was
mcrA
(2.5 × 10
6
copies/g [wet weight]), while the
pmoA
gene of the type I methanotrophs (5.9 × 10
6
copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in γ-
Proteobacteria
and had a high copy number of
mxaF
(7.1 × 10
6
copies/g [wet weight]) and a much lower copy number of the
pmoA
gene (3.2 × 10
2
copies/g [wet weight]). The
mmoX
gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C
1
metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.