Identification of acetate-assimilating microorganisms under methanogenic conditions in anoxic rice field soil by comparative stable isotope probing of RNA

Acetate is the most abundant intermediate of organic matter degradation in anoxic rice field soil and is converted to CH4 and/or CO2. Aceticlastic methanogens are the primary microorganisms dissimilating acetate in the absence of sulfate and reducible ferric iron. In contrast, very little is known about bacteria capable of assimilating acetate under methanogenic conditions. Here, we identified active acetate-assimilating microorganisms by using a combined approach of frequent label application at a low concentration and comparative RNA-stable isotope probing with C-13-labeled and unlabeled acetate. Rice field soil was incubated anaerobically at 25 degrees C for 12 days, during which C-13-labeled acetate was added at a concentration of 500 mu M every 3 days. C-13-labeled CH4 and CO2 were produced from the beginning of the incubation and accounted for about 60% of the supplied acetate C-13. RNA was extracted from the cells in each sample taken and separated by isopycnic centrifugation according to molecular weight. Bacterial and archaeal populations in each density fraction were screened by reverse transcription-PCR-mediated terminal restriction fragment polymorphism analysis. No differences in the bacterial populations were observed throughout the density fractions of the unlabeled treatment. However, in the heavy fractions of the C-13 treatment, terminal restriction fragments (T-RFs) of 161 by and 129 by in length predominated. These T-RFs were identified by cloning and sequencing of 16S rRNA as from a Geobacter sp. and an Anaeromyxobacter sp., respectively. Apparently these bacteria, which are known as dissimilatory iron reducers, were able to assimilate acetate under methanogenic conditions, i.e., when CO2 was the predominant electron acceptor. We hypothesize that ferric iron minerals with low bioavailability might have served as electron acceptors for Geobacter spp. and Anaeromyxobacter spp. under these conditions.