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Summary

Anaerobic oxidation of methane (AOM) is an important methane sink in the ocean but the microbes responsible for AOM are as yet resilient to cultivation. Here we describe the microbial analysis of an enrichment obtained in a novel submerged-membrane bioreactor system and capable of high-rate AOM (286 μmol  gdry weight−1 day−1) coupled to sulfate reduction. By constructing a clone library with subsequent sequencing and fluorescent in situ hybridization, we showed that the responsible methanotrophs belong to the ANME-2a subgroup of anaerobic methanotrophic archaea, and that sulfate reduction is most likely performed by sulfate-reducing bacteria commonly found in association with other ANME-related archaea in marine sediments. Another relevant portion of the bacterial sequences can be clustered within the order of Flavobacteriales but their role remains to be elucidated. Fluorescent in situ hybridization analyses showed that the ANME-2a cells occur as single cells without close contact to the bacterial syntrophic partner. Incubation with 13C-labelled methane showed substantial incorporation of 13C label in the bacterial C16 fatty acids (bacterial; 20%, 44% and 49%) and in archaeal lipids, archaeol and hydroxyl-archaeol (21% and 20% respectively). The obtained data confirm that both archaea and bacteria are responsible for the anaerobic methane oxidation in a bioreactor enrichment inoculated with Eckernförde bay sediment.