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Summary To assess and study the heterogeneity of δ13C values for seep microorganisms of the Eel River Basin, we studied two principally different sample sets: sediments from push cores and artificial surfaces colonized over a 14 month in situ incubation. In a single sediment core, the δ13C compositions of methane seep‐associated microorganisms were measured and the relative activity of several metabolisms was determined using radiotracers. We observed a large range of archaeal δ13C values (> 50‰) in this microbial community. The δ13C of ANME‐1 rods ranged from −24‰ to −87‰. The δ13C of ANME‐2 sarcina ranged from −18‰ to −75‰. Initial measurements of shell aggregates were as heavy as −19.5‰ with none observed to be lighter than −57‰. Subsequent measurements on shell aggregates trended lighter reaching values as 13C‐depleted as −73‰. The observed isotopic trends found for mixed aggregates were similar to those found for shell aggregates in that the initial measurements were often enriched and the subsequent analyses were more 13C‐depleted (with values as light as −56‰). The isotopic heterogeneity and trends observed within taxonomic groups suggest that ANME‐1 and ANME‐2 sarcina are capable of both methanogenesis and methanotrophy. In situ microbial growth was investigated by incubating a series of slides and silicon (Si) wafers for 14 months in seep sediment. The experiment showed ubiquitous growth of bacterial filaments (mean δ13C = −38 ± 3‰), suggesting that this bacterial morphotype was capable of rapid colonization and growth.

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[1] Our understanding of the metabolic pathways involved in the anaerobic oxidation of methane (AOM) is challenged by not having an isolated anaerobic methanotroph. Fortunately, trace methane oxidation (TMO) is carried out by isolated organisms closely related to anaerobic methanotrophs. As TMO likely shares metabolic similarities with the AOM metabolism, studies of TMO in culture experiments can serve to enhance our understanding of AOM. Here we explored TMO in cultures of Methanosarcina acetivorans grown separately on acetate and carbon monoxide. We observed no methane oxidation to carbon dioxide in the cultures grown on acetate, but did observe methane oxidation to acetate (1.1 Â 10 À6 ± 3.5 Â 10 À8 moles CH 4 oxidized, 0.10% of the CH 4 produced). Methane was exclusively converted to the methyl position. Because AOM lipids exhibit strong 13 C depletion in nature, our results imply acetate does not play a major role as an AOM intermediate. The carbon monoxide cultures showed little or no methane conversion to carbon dioxide (1.9 Â 10 À7 ± 2.0 Â 10 À7 moles CH 4 oxidized), moderate methane oxidation to acetate (1.8 Â 10 À7 ± 7.3 Â 10 À8 moles CH 4 oxidized, 0.14% of biogenic CH 4 production), and high methane conversion to the methyl groups of product methyl sulfides (4.0 Â 10 À6 ± 7.7 Â 10 À7 moles CH 4 oxidized, 3.1% of biogenic CH 4 production). Taken together, our results identify methyl sulfides as more likely intermediates in natural AOM than acetate.

Anaerobic oxidation of methane (AOM) impacts carbon cycling by acting as a methane sink and by sequestering inorganic carbon via AOM-induced carbonate precipitation. These precipitates commonly take the form of carbonate nodules that form within methane seep sediments. The timing and sequence of nodule formation within methane seep sediments are not well understood. Further, the microbial diversity associated with sediment-hosted nodules has not been well characterized and the degree to which nodules reflect the microbial assemblage in surrounding sediments is unknown. Here, we conducted a comparative study of microbial assemblages in methane-derived authigenic carbonate nodules and their host sediments using molecular, mineralogical, and geochemical methods. Analysis of 16S rRNA gene diversity from paired carbonate nodules and sediments revealed that both sample types contained methanotrophic archaea (ANME-1 and ANME-2) and syntrophic sulfate-reducing bacteria (Desulfobacteraceae and Desulfobulbaceae), as well as other microbial community members. The combination of geochemical and molecular data from Eel River Basin and Hydrate Ridge suggested that some nodules formed in situ and captured the local sediment-hosted microbial community, while other nodules may have been translocated or may represent a record of conditions prior to the contemporary environment. Taken together, this comparative analysis offers clues to the formation regimes and mechanisms of sediment-hosted carbonate nodules.