Effect of Dilution Rate on Structure of a Mesophilic Acetate-Degrading Methanogenic Community during Continuous Cultivation

Abstract

The community structures of two mesophilic acetate-degrading methanogenic consortia enriched at dilution rates of 0.025 and 0.6 d −1 were analyzed by fluorescence in situ hybridization (FISH) and phylogenetic analyses based on 16S rDNA clonal sequences and quantitative real-time polymerase chain reaction (PCR). FISH experiments with archaeal and bacterial domain-specific probes showed that archaeal cells were predominant and only a small number of bacterial cells were detected at both dilution rates. In the domain Archaea , the number of cells closely related to Methanosarcina barkeri was shown to be greater at the high dilution rate using FISH with species-specific probes. Taxonomic analyses based on rDNA clonal sequences obtained at the low and high dilution rates showed that 43% of 100 clones and 72% of 92 clones, respectively, were affiliated with the domain Archaea and the remainders at each dilution rate were affiliated with the domain Bacteria . Within the domain Archaea , all rDNA clones at both dilution rates were affiliated with the genera Methanosaeta or Methanosarcina of the aceticlastic methanogens. Within the domain Bacteria , the rDNA clones obtained at the low dilution rate were affiliated with four phyla, Firmicutes (36%), Bacteroidetes (9%), Chloroflexi (6%) and candidate division OP12 (5%). The rDNA clones obtained at the high dilution rate were affiliated with four phyla, Firmicutes (16%), Bacteroidetes (8%), Proteobacteria (1%) and candidate division OP12 (3%). Real-time quantitative PCR experiments showed that the number of rDNA sequences affiliated with the genus Methanosarcina was greater at the high dilution rate. In addition, a significant number of rDNA sequences affiliated with the genus Methanoculleus were detected only at the low dilution rate. Detection of a hydrogenotrophic methanogen at the low dilution rate suggests that the syntrophic acetate oxidation by hydrogenotrophic methanogens and acetate-oxidizing bacteria could occur at the low dilution rate.