Quantification of Methanosaeta species in anaerobic bioreactors using genus- and species-specific hybridization probes

To evaluate the role of Methanosaeta spp. in a variety of anaerobic environ ments, small-subunit rRNA targeted oligonucleotide hybridization probes wer e developed and experimentally characterized. The probes were designed to b e genus specific for Methanosaeta and species specific for Methanosaeta coc ilii and Methanosaeta thermophila. The temperature of dissociation was dete rmined for each probe. Probe specificities were determined using a diverse collection of Archaea and through an evaluation of probe nesting using samp les from a variety of anaerobic bioreactors. Cell fixation and hybridizatio n conditions for fluorescence in situ hybridizations were also evaluated. A lthough permeability of methanogens was variable, M. concilii cells could b e permeabilized using a range of paraformaldehyde and ethanol based fixatio n conditions. Using the newly designed probes together with previously desi gned probes for methanogens, it was determined that Methanosaeta spp. were the dominant aceticlastic methanogens in a variety of anaerobic bioreactors when acetate concentrations were low. Their levels were higher in bioreact ors with granular sludge than in those with flocculent sludge. In lab-scale upflow anaerobic sludge blanket reactors, the levels of M, concilii rRNA w ere as high as 30% of the total rRNA.