Thermoanaerobacteriaceae oxidize acetate in methanogenic rice field soil at 50°C

P>Rice field soils contain a thermophilic microbial community. Incubation of Italian rice field soil at 50 degrees C resulted in transient accumulation of acetate, but the microorganisms responsible for methane production from acetate are unknown. Without addition of exogenous acetate, the delta 13C of CH4 and CO2 indicated that CH4 was exclusively produced by hydrogenotrophic methanogenesis. When exogenous acetate was added, acetoclastic methanogenesis apparently also operated. Nevertheless, addition of [2-13C]acetate (99% 13C) resulted in the production not only of 13C-labelled CH4 but also of CO2, which contained up to 27% 13C, demonstrating that the methyl group of acetate was also oxidized. Part of the 13C-labelled acetate was also converted to propionate which contained up to 14% 13C. The microorganisms capable of assimilating acetate at 50 degrees C were targeted by stable isotope probing (SIP) of ribosomal RNA and rRNA genes using [U-13C] acetate. Using quantitative PCR, 13C-labelled bacterial ribosomal RNA and DNA was detected after 21 and 32 days of incubation with [U-13C]acetate respectively. In the heavy fractions of the 13C treatment, terminal restriction fragments (T-RFs) of 140, 120 and 171 bp length predominated. Cloning and sequencing of 16S rRNA showed that these T-RFs were affiliated with the bacterial genera Thermacetogenium and Symbiobacterium and with members of the Thermoanaerobacteriaceae. Similar experiments targeting archaeal RNA and DNA showed that Methanocellales were the dominant methanogens being consistent with the operation of syntrophic bacterial acetate oxidation coupled to hydrogenotrophic methanogenesis. After 17 days, however, Methanosarcinacea increasingly contributed to the synthesis of rRNA from [U-13C]acetate indicating that acetoclastic methanogens were also active in methanogenic Italian rice field soil under thermal conditions.