| Summary: | Moderately thermophilic (T<sub>max</sub>, ~55 °C) methanogens are identified after extended enrichments from temperate, tropical and low-temperature environments. However, thermophilic methanogens with higher growth temperatures (T<sub>opt</sub> ≥ 60 °C) are only reported from high-temperature environments. A microcosm-based approach was used to measure the rate of methane production and methanogen community structure over a range of temperatures and salinities in sediment from a temperate estuary. We report short-term incubations (<48 h) revealing methanogens with optimal activity reaching 70 °C in a temperate estuary sediment (in situ temperature 4–5 °C). While 30 °C enrichments amended with acetate, H<sub>2</sub> or methanol selected for corresponding mesophilic trophic groups, at 60 °C, only hydrogenotrophs (genus <i>Methanothermobacter</i>) were observed. Since these methanogens are not known to be active under in situ temperatures, we conclude constant dispersal from high temperature habitats. The likely provenance of the thermophilic methanogens was studied by enrichments covering a range of temperatures and salinities. These enrichments indicated that the estuarine sediment hosted methanogens encompassing the global activity envelope of most cultured species. We suggest that estuaries are fascinating sink and source environments for microbial function study.
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