Detection of active, potentially acetate-oxidizing syntrophs in an anaerobic digester by flux measurement and formyltetrahydrofolate synthetase (FTHFS) expression profiling.

Syntrophic oxidation of acetate, so-called reversed reductive acetogenesis, is one of the most important degradation steps in anaerobic digesters. However, little is known about the genetic diversity of the micro-organisms involved. Here we investigated the activity and composition of potentially acetate-oxidizing syntrophs using a combinatorial approach of flux measurement and transcriptional profiling of the formyltetrahydrofolate synthetase (FTHFS) gene, an ecological biomarker for reductive acetogenesis. During the operation of a thermophilic anaerobic digester, volatile fatty acids were mostly depleted, suggesting a high turnover rate for dissolved H(2), and hydrogenotrophic methanogens were the dominant archaeal members. Batch cultivation of the digester microbiota with (13)C-labelled acetate indicated that syntrophic oxidation accounted for 13.1-21.3 % of methane production from acetate. FTHFS genes were transcribed in the absence of carbon monoxide, methoxylated compounds and inorganic electron acceptors other than CO(2), which is implicated in the activity of reversed reductive acetogenesis; however, expression itself does not distinguish whether biosynthesis or biodegradation is functioning. The mRNA- and DNA-based terminal RFLP and clone library analyses indicated that, out of nine FTHFS phylotypes detected, the FTHFS genes from the novel phylotypes I-IV in addition to the known syntroph Thermacetogenium phaeum (i.e. phylotype V) were specifically expressed. These transcripts arose from phylogenetically presumed homoacetogens. The results of this study demonstrate that hitherto unidentified phylotypes of homoacetogens are responsible for syntrophic acetate oxidation in an anaerobic digester.