Growth and activity of ANME clades with different sulfate and sulfide concentrations in presence of methane

Extensive geochemical data showed that significant methane oxidation activity exists in marine sediments. The organisms responsible for this activity are anaerobic methane-oxidizing archaea (ANME) that occur in consortia with sulfate-reducing bacteria. A distinct zonation of different clades of ANME (ANME-1, ANME-2a/b and ANME-2c) exists in marine sediments, which could be related to the localized concentrations of methane, sulfate and sulfide. In order to test this hypothesis we performed long-term incubation of marine sediments under defined conditions with methane as a headspace gas: low or high sulfate (?4 and ?21 mM, respectively) in combination with low or high sulfide (?0.1 and ?4 mM, respectively) concentrations. Control incubations were also performed, with only methane, high sulfate or high sulfide. Methane oxidation was monitored and growth of subtypes ANME-1, ANME-2a/b, and ANME-2c assessed using qPCR analysis. A preliminary archaeal community analysis was performed to gain insight into the ecological and taxonomic diversity. Almost all of the incubations with methane had methane oxidation activity, with the exception of the incubations with combined low sulfate and high sulfide concentrations. Sulfide inhibition occurred only with low sulfate concentrations, which could be due to the lower Gibbs free energy available as well as sulfide toxicity. ANME-2a/b appear to mainly grow in incubations which had high sulfate levels and methane oxidation activity, whereas ANME-1 did not show this distinction. ANME-2c only grew in incubations with only sulfate addition. These findings are consistent with previously published in situ profiling analysis of ANME subclusters in different marine sediments. Interestingly, since all ANME subtypes also grew in incubations with only methane or sulfate addition, ANME may also be able to perform anaerobic methane oxidation under substrate limited conditions or alternatively perform additional metabolic processes.We want to thank all reviewers for constructive comments, Joan Edwards (Laboratory of Microbiology, WUR) for extensive proof-reading, Bartholomeus van den Bogert (Laboratory of Microbiology, WUR) for help with the MiSeq sequencing and Diego A. Suarez-Zuluaga (Environmental Technology, WUR) for help with carbon dioxide calculations. This research is supported by the Dutch Technology Foundation STW (project 10711), which is part of the Netherlands Organization for Scientific Research (NWO), and which is partly funded by the Ministry of Economic Affairs. Research of AJMS is supported by ERC grant (project 323009) and the Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO)