Identifying fermenting bacteria in anoxic tidal-flat sediments by a combination of microcalorimetry and ribosome-based stable-isotope probing.

A novel approach was developed to follow the successive utilization of organic carbon under anoxic conditions by microcalorimetry, chemical analyses of fermentation products and stable-isotope probing (SIP). The fermentation of 13C-labeled glucose was monitored over 4weeks by microcalorimetry in a stimulation experiment with tidal-flat sediments. Based on characteristic heat production phases, time points were selected for quantifying fermentation products and identifying substrate-assimilating bacteria by the isolation of intact ribosomes prior to rRNA-SIP. The preisolation of ribosomes resulted in rRNA with an excellent quality. Glucose was completely consumed within 2days and was mainly fermented to acetate. Ethanol, formate, and hydrogen were detected intermittently. The amount of propionate that was built within the first 3days stayed constant. Ribosome-based SIP of fully labeled and unlabeled rRNA was used for fingerprinting the glucose-degrading species and the inactive background community. The most abundant actively degrading bacterium was related to Psychromonas macrocephali (similarity 99%) as identified by DGGE and sequencing. The disappearance of Desulfovibrio-related bands in labeled rRNA after 3days indicated that this group was active during the first degradation phase only. In summary, ribosome-based SIP in combination with microcalorimetry allows dissecting distinct phases in substrate turnover in a very sensitive manner