N-214. 13C-Propionic Acid Stable Isotope Probing of an Thermophilic Methanogenic Digester

A. M. Smith1, H. M. Lappin-Scott2, S. K. Burton2, D. H. Huber1;
1West Virginia State Univ., Institute, WV, 2Univ. of Exeter, Exeter, UNITED KINGDOM.

Anaerobic digestion is known as an effective method for reduction of organic waste related pollution, however much of the bacterial diversity responsible for anaerobic digestion remains unknown. Volatile fatty acids (VFA’s) are important intermediates in the naturally occurring microbial metabolism responsible for methanogenic degradation. Degradation of VFA’s is made energetically favorable by the interspecies hydrogen transfer between syntrophic VFA oxidizers and methanogens. Accumulation of VFA’s is a sign of methanogen inhibition and therefore digester malfunction. Propionic acid is an important VFA known to accumulate in anaerobic digesters. This study utilizes stable isotope probing methods to identify propionic acid degraders in a thermophilic methanogenic digester. Three replicate 500ml thermophilic methanogenic digesters were established: two experimental reactors fed 13C-propionic acid and one control reactor fed the unlabeled substrate. All three reactors were fed three pulses of propionic acid (18mM total concentration) and were kept anaerobic at 55°C for 14 days prior to harvest. Upon harvest total genomic DNA was extracted. The 13C and 12C DNA was separated by isopycnic density gradient centrifugation in cesium trifluoroacetate (CsTFA) gradients followed by fractionation. Identification of fractions containing the 13C-labeled DNA was accomplished by real-time PCR. A bacterial 16S rDNA clone library constructed from the recovered 13C DNA was dominated by uncultured bacterial sequences classified in the phylum Firmicutes by a comparison with the RDP. These sequences are also highly similar (97-100%) to a cluster of bacterial 16S rDNA clone sequences of unknown function found to be abundant in a pilot plant thermophilic anaerobic digester treating poultry litter. The dominance of this clone group in the 13C fraction suggests that this unknown cluster of bacteria plays an important role in propionic acid degradation.