Dandan Zheng

Characterization of microbial communities in anaerobic bioreactors using molecular probes.

The microbial community structure of twenty-one single-phase and one two-phase full-scale anaerobic sewage sludge digesters was evaluated using oligonucleotide probes complementary to conserved tracts of the 16S rRNAs of phylogenetically defined groups of methanogens and sulfate-reducing bacteria. These probe results were interpreted in combination with results from traditional chemical analyses and metabolic activity assays. It was determined that methanogens in “healthy” mesophilic, single-phase sewage sludge digesters accounted for approximately 8–12% of the total community and thatMethanosarcinales andMethanomicrobiales constituted the majority of the total methanogen population.Methanobacteriales andMethanococcales played a relatively minor role in the digesters. Phylogenetic groups of mesophilic, Gram-negative sulfate-reducing bacteria were consistently present at significant levels:Desulfovibrio andDesulfobulbus spp. were the dominant sulfate-reducing populations,Desulfobacter andDesulfobacterium spp. were present at lower levels, andDesulfosarcina, Desulfococcus, andDesulfobotulus spp. were absent. Sulfate reduction by one or more of these populations played a significant role in all digesters evaluated in this study. In addition, sulfate-reducing bacteria played a role in favoring methanogenesis by providing their substrates. The analysis of the two-phase digester indicated that true phase separation was not accomplished: significant levels of active methanogens were present in the first phase. It was determined that the dominant populations in the second phase were different from those in the single-phase digesters.

Quantification of Methanosaeta Species in Anaerobic Bioreactors Using Genus- and Species-Specific Hybridization Probes

A bstractTo evaluate the role of Methanosaeta spp. in a variety of anaerobic environments, small-subunit rRNA targeted oligonucleotide hybridization probes were developed and experimentally characterized. The probes were designed to be genus specific for Methanosaeta and species specific for Methanosaeta concilii and Methanosaeta thermophila. The temperature of dissociation was determined for each probe. Probe specificities were determined using a diverse collection of Archaea and through an evaluation of probe nesting using samples from a variety of anaerobic bioreactors. Cell fixation and hybridization conditions for fluorescence in situ hybridizations were also evaluated. Although permeability of methanogens was variable, M. concilii cells could be permeabilized using a range of paraformaldehyde and ethanol based fixation conditions. Using the newly designed probes together with previously designed 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 bioreactors with granular sludge than in those with flocculent sludge. In lab-scale upflow anaerobic sludge blanket reactors, the levels of M. concilii rRNA were as high as 30% of the total rRNA.

The presence of humic substances and DNA in RNA extracts affects hybridization results.

ABSTRACT RNA extracts obtained from environmental samples are frequently contaminated with coextracted humic substances and DNA. It was demonstrated that the response in rRNA-targeted oligonucleotide probe hybridizations decreased as the concentrations of humic substances and DNA in RNA extracts increased. The decrease in hybridization signal in the presence of humic substances appeared to be due to saturation of the hybridization membrane with humic substances, resulting in a lower amount of target rRNA bound to the membrane. The decrease in hybridization response in the presence of low amounts of DNA may be the result of reduced rRNA target accessibility. The presence of high amounts of DNA in RNA extracts resulted in membrane saturation. Consistent with the observations for DNA contamination, the addition of poly(A) to RNA extracts, a common practice used to prepare RNA dilutions for membrane blotting, also reduced hybridization signals, likely because of reduced target accessibility and membrane saturation effects.