Anoxic sediment from a methane hydrate area (Hydrate Ridge, north-east Pacific; water depth 780 m) was incubated in a long-term laboratory experiment with semi-continuous supply of pressurized [1.4 MPa (14 atm)] methane and sulfate to attempt in vitro propagation of the indigenous consortia of archaea (ANME-2) and bacteria (DSS, Desulfosarcina/Desulfococcus cluster) to which anaerobic oxidation of methane (AOM) with sulfate has been attributed. During 24 months of incubation, the rate of AOM (measured as methane-dependent sulfide formation) increased from 20 to 230 μmol day−1 (g sediment dry weight)−1 and the number of aggregates (determined by microscopic counts) from 0.5 × 108 to 5.7 × 108 (g sediment dry weight)−1. Fluorescence in situ hybridization targeting 16S rRNA of both partners showed that the newly grown consortia contained central archaeal clusters and peripheral bacterial layers, both with the same morphology and phylogenetic affiliation as in the original sediment. The development of the AOM rate and the total consortia biovolume over time indicated that the consortia grew with a doubling time of approximately 7 months (growth rate 0.003 day−1) under the given conditions. The molar growth yield of AOM was approximately 0.6 g cell dry weight (mol CH4 oxidized)−1; according to this, only 1% of the consumed methane is channelled into synthesis of consortia biomass. Concentrations of biomarker lipids previously attributed to ANME-2 archaea (e.g. sn-2-hydroxyarchaeol, archaeol, crocetane, pentamethylicosatriene) and Desulfosarcina-like bacteria [e.g. hexadecenoic-11 acid (16:1ω5c), 11,12-methylene-hexadecanoic acid (cy17:0ω5,6)] strongly increased over time (some of them over-proportionally to consortia biovolume), suggesting that they are useful biomarkers to detect active anaerobic methanotrophic consortia in sediments.
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