Abstract
The metabolic pathways of methane formation vary with environmental conditions, but whether this can also be linked to changes in the active archaeal community structure remains uncertain. Here, we show that the suppression of aceticlastic methanogenesis by methyl fluoride (CH3F) caused surprisingly little differences in community composition of active methanogenic archaea from a rice field soil. By measuring the natural abundances of carbon isotopes we found that the effective dose for a 90% inhibition of aceticlastic methanogenesis in anoxic paddy soil incubations was <0.75% CH3F (v/v). The construction of clone libraries as well as t-RFLP analysis revealed that the active community, as indicated by mcrA transcripts (encoding the α subunit of methyl-coenzyme M reductase, a key enzyme for methanogenesis), remained stable over a wide range of CH3F concentrations and represented only a subset of the methanogenic community. More precisely, Methanocellaceae were of minor importance, but Methanosarcinaceae dominated the active population, even when CH3F inhibition only allowed for aceticlastic methanogenesis. In addition, we detected mcrA gene fragments of a so far unrecognised phylogenetic cluster. Transcription of this phylotype at methyl fluoride concentrations suppressing aceticlastic methanogenesis suggests that the respective organisms perform hydrogenotrophic methanogenesis. Hence, the application of CH3F combined with transcript analysis is not only a useful tool to measure and assign in situ acetate usage, but also to explore substrate usage by as yet uncultivated methanogens.
Highlights
Methanogenesis is the dominating terminal process in anoxic freshwater habitats like sediments and flooded soils
The amount of acetate-derived methanogenesis can be assessed with CH3F, a specific inhibitor for aceticlastic methanogenesis
When applied for the first time in microbial ecology, CH3F was assumed to be a specific inhibitor for methane oxidation and ammonium oxidation [3,4]
Summary
Methanogenesis is the dominating terminal process in anoxic freshwater habitats like sediments and flooded soils. The fraction of methane produced via acetate is an important variable in understanding what controls mineralization in anoxic environments. The amount of acetate-derived methanogenesis can be assessed with CH3F (methyl fluoride, fluoromethane), a specific inhibitor for aceticlastic methanogenesis. When applied for the first time in microbial ecology, CH3F was assumed to be a specific inhibitor for methane oxidation and ammonium oxidation [3,4]. In anoxic incubations treated with CH3F, approximately as much acetate accumulates as methane is lacking compared to untreated controls. Selectivity of CH3F for suppression of aceticlastic methanogenesis was further validated in pure culture studies demonstrating that 1% v/v inhibited growth of and methanogenesis by pure cultures of aceticlastic Methanosaeta and Methanosarcina. Homoacetogenic, sulfate reducing and fermentative bacteria, and a methanogenic mixed culture based on hydrogen syntrophy, were not inhibited [7]. Some populations may show a behavior different from that found in pure cultures
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