The anaerobic oxidation of methane (AOM) with sulfate controls the emission of the greenhouse gas methane from the ocean floor. AOM is performed by microbial consortia of archaea (ANME) associated with partners related to sulfate-reducing bacteria. In vitro enrichments of AOM were so far only successful at temperatures p25 1C; however, energy gain for growth by AOM with sulfate is in principle also possible at higher temperatures. Sequences of 16S rRNA genes and core lipids characteristic for ANME as well as hints of in situ AOM activity were indeed reported for geothermally heated marine environments, yet no direct evidence for thermophilic growth of marine ANME consortia was obtained to date. To study possible thermophilic AOM, we investigated hydrothermally influenced sediment from the Guaymas Basin. In vitro incubations showed activity of sulfate-dependent methane oxidation between 5 and 70 1C with an apparent optimum between 45 and 60 1C. AOM was absent at temperatures X75 1C. Long-term enrichment of AOM was fastest at 50 1C, yielding a 13-fold increase of methane-dependent sulfate reduction within 250 days, equivalent to an apparent doubling time of 68 days. The enrichments were dominated by novel ANME-1 consortia, mostly associated with bacterial partners of the deltaproteobacterial HotSeep-1 cluster, a deeply branching phylogenetic group previously found in a butane-amended 60 1C-enrichment culture of Guaymas sediments. The closest relatives (Desulfurella spp.; Hippea maritima) are moderately thermophilic sulfur reducers. Results indicate that AOM and ANME archaea could be of biogeochemical relevance not only in cold to moderate but also in hot marine habitats.

SummaryThe anaerobic oxidation of methane (AOM) with sulfate as terminal electron acceptor is mediated by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Whereas three clades of ANME have been repeatedly studied with respect to phylogeny, key genes and genomic capabilities, little is known about their sulfate-reducing partner. In order to identify the partner of anaerobic methanotrophs of the ANME-2 clade, bacterial 16S rRNA gene libraries were constructed from cultures highly enriched for ANME-2a and ANME-2c in consortia with Deltaproteobacteria of the Desulfosarcina/ Desulfococcus group (DSS). Phylogenetic analysis of those and publicly available sequences from AOM sites supported the hypothesis by Knittel and colleagues that the DSS partner belongs to the diverse SEEP-SRB1 cluster. Six subclusters of SEEP-SRB1, SEEP-SRB1a to SEEP-SRB1f, were proposed and specific oligonucleotide probes were designed. Using fluorescence in situ hybridization on samples from six different AOM sites, SEEP-SRB1a was identified as sulfate-reducing partner in up to 95% of total ANME-2 consortia. SEEP-SRB1a cells exhibited a rodshaped, vibrioid, or coccoid morphology and were found to be associated with subgroups ANME-2a and ANME-2c. Moreover, SEEP-SRB1a was also detected in 8% to 23% of ANME-3 consortia in Haakon Mosby Mud Volcano sediments, previously described to be predominantly associated with SRB of the Desulfobulbus group. SEEP-SRB1a contributed to only 0.3% to 0.7% of all single cells in almost all samples indicating that these bacteria are highly adapted to a symbiotic relationship with ANME-2.

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The methane-rich, hydrothermally heated sediments of the Guaymas Basin are inhabited by thermophilic microorganisms, including anaerobic methane-oxidizing archaea (mainly ANME-1) and sulfatereducing bacteria (e.g., HotSeep-1 cluster). We studied the microbial carbon flow in ANME-1/ HotSeep-1 enrichments in stable-isotopeprobing experiments with and without methane. The relative incorporation of 13 C from either dissolved inorganic carbon or methane into lipids revealed that methane-oxidizing archaea assimilated primarily inorganic carbon. This assimilation is strongly accelerated in the presence of methane. Experiments with simultaneous amendments of both 13 C-labeled dissolved inorganic carbon and deuterated water provided further insights into production rates of individual lipids derived from members of the methane-oxidizing community as well as their carbon sources used for lipid biosynthesis. In the presence of methane, all prominent lipids carried a dual isotopic signal indicative of their origin from primarily autotrophic microbes. In the absence of methane, archaeal lipid production ceased and bacterial lipid production dropped by 90%; the lipids produced by the residual fraction of the metabolically active bacterial community predominantly carried a heterotrophic signal. Collectively our results strongly suggest that the studied ANME-1 archaea oxidize methane but assimilate inorganic carbon and should thus be classified as methane-oxidizing chemoorganoautotrophs.methanotrophy | biomarker | acetyl-CoA pathway | syntrophy M ethane is an important greenhouse gas and the most abundant hydrocarbon in marine sediments. Its upward flux to the sediment-water interface is strongly reduced by sulfate-dependent anaerobic oxidation of methane (AOM) (1, 2). AOM is performed by syntrophic associations of anaerobic methane-oxidizing archaea (ANMEs) (3, 4) and their sulfate-reducing bacterial partners (SRBs) (mainly relatives of Desulfosarcina or Desulfobulbus) (5-8). The free energy yield of the AOM net reaction is one of the lowest known for catabolic reactions under environmental conditions (ΔG ranges from -20 to -40 kJ·mol -1 ; e.g., refs. 9, 10). Consequently, activity and biomass doubling times determined under optimized laboratory conditions range from 2 to 5 mo (11) and growth yields are extremely low, around 1% relative to oxidized methane (11,12). The biomass of ANMEs and SRBs involved in AOM is usually strongly depleted in 13 C. For instance, at methane seep locations, the δ 13 C values of specific bacterial fatty acids and archaeal ether lipids range from -60 to -100‰ and -70 to -130‰, respectively (e.g., refs. 13-17). Such low values have been interpreted as evidence for the incorporation of 13 C-depleted methane into biomass (e.g., refs. 3, 4, 18).One way to identify the carbon sources of microbial biomass is to perform stable-isotope-probing (SIP) experiments (19), followed by analysis of biomolecules such as membrane lipids (lipid-SIP hereafter). Application of lipid-SIP to cold seep sediments and micro...