We continuously fed an anaerobic chemostat with synthetic wastewater containing glucose as the sole source of carbon and energy to study the effects of temperature on the microbial community under hyperthermophilic (65-80°C) conditions. Methane was produced normally up to 77.5°C at a dilution rate of 0.025 d -1. However, the concentration of microorganisms and the rate of gas production decreased with increasing operation temperature. The microbial community in the chemostat at various temperatures was analyzed based on the 16S rRNA gene using molecular biological techniques including clone library analysis and denaturing gradient gel electrophoresis (DGGE). Aceticlastic methanogens related to Methanosarcina thermophila were detected at 65°C and hydrogenotrophilic methanogens related to Methanothermobacter thermautotrophicus were the dominant methanogens between 70°C to 77.5°C. Bacteria related to Clostridium stercorarium and Thermoanaerobacter subterraneus comprised the dominant glucose-fermenting bacteria at temperatures of 65°C and above, respectively. Bacteria related to Thermacetogenium phaeum and to Tepidiphilus margaritifer and Petrobacter succinatimandens were the dominant acetate-oxidizing bacteria at 70°C and at 75-77.5°C, respectively. The results suggested that, at temperatures of 70°C and above, methane production via the aceticlastic pathway was negligible and indirect methanogenesis from acetate was dominant. Since acetate oxidation is a rate limiting step and a higher temperature favors the hydrolysis and acid formation, a two stage fermentation process, acidogenic and methanogenic fermentation stages operated under different temperatures, should be more suitable for the thermophilic anaerobic treatment at temperatures above 65°C.

Distillery wastewater from awamori making was anaerobically treated for one year using thermophilic upflow anaerobic filter (UAF) reactors packed with pyridinium group-containing nonwoven fabric material. The microbial structure and spatial distribution of microorganisms on the support material were characterized using molecular biological methods. The reactor steadily achieved a high TOC loading rate of 18 g/l/d with approximately 80% TOC removal efficiency when nondiluted wastewater was fed. The maximum TOC loading rate increased to 36 g/l/d when treating thrice-diluted wastewater. However, the TOC removal efficiency and gas evolution rate decreased compared with that when non-diluted wastewater was used. Methanogens closely related to Methanosarcina thermophila and Methanoculleus bourgensis and bacteria in the phyla Firmicutes and Bacteroidetes were predominant methanogens and bacteria in the thermophilic UFA reactor, as indicated by 16S rRNA gene clone analysis. Fluorescence in situ hybridization (FISH) results showed that a large quantity of bacterial cells adhered throughout the whole support, and Methanosarcina-like methanogens existed mainly in the relative outside region while Methanoculleus cells were located in the relative inner part of the support. The support material used proved to be an excellent carrier for microorganisms, and a UAF reactor using this kind of support can be used for high-rate treatment of awamori/shochu distillery wastewater.

An ethanol production process using the flocculating yeast Saccharomyces cerevisiae strain KF-7 with acid hydrolysate of wood biomass as feed was investigated. Corn steep liquor (CSL) supplemented with KH2PO4, MgSO4, and CaCl2 was used as the source of nitrogen and mineral nutrients. Using a tower-type reactor (working volume (WV), 0.45 L), continuous fermentation with a high ethanol productivity of over 20 g/(L h) was achieved at a dilution rate of 0.3h-1 with a pH of 4.5 at 35 8C. A continuous fermentation process using two tower-type reactors (WV, 4.5 Leach) connected in series was then constructed to decrease the concentration of residual sugars (except for xylose). The contamination of bacteria was effectively repressed and an ethanol productivity of over 12.6 g/(L h) was achieved at an overall dilution rate of 0.2h-1 with a pH of 4.0 at 35 8C. Strain KF-7 was thus demonstrated to be suitable for the production of ethanol from acid hydrolysate of wood biomass.

Two mesophilic anaerobic chemostats, one without added Ni2+ and Co2+ (chemostat 1) and the other with added Ni2+ and Co2+ (chemostat 2), were supplied with synthetic wastewater containing bovine serum albumin (BSA) as the sole carbon and energy source in order to study the capacity of protein degradation, microbial community structure and the effects of the addition of trace metals. Volatile fatty acids and ammonia were the main products of chemostat 1, while methane, CO2 and ammonia were the main products of chemostat 2, and critical dilution rates of 0.15 d-1 and 0.08 d-1 were obtained, respectively. Fluorescence in situ hybridization (FISH) with archaeal and bacterial domain-specific probes showed that archaeal cells were very limited in chemostat 1 while large populations of several types of archaeal cells were present in chemostat 2. Phylogenetic analyses based on 16S rRNA gene clonal sequences, DGGE, and quantitative real-time polymerase chain reaction (PCR) showed that, within the domain Archaea, methanogens affiliated with the genera Methanosaeta and Methanoculleus were predominant in chemostat 2. Within the domain Bacteria, rRNA genes obtained from chemostat 1 were affiliated with the three phyla; Firmicutes (43%), Bacteroidetes (50%) and Proteobacteria (7%). A total of 56% of rRNA genes obtained from chemostat 2 was affiliated with the three phyla, Firmicutes (32%), Bacteroidetes (11%) and Proteobacteria (13%) while 44% of rRNA genes remained unclassified. Phylogenetically distinct clones were obtained in these two chemostats, suggesting that different protein degradation pathways were dominant in the two chemostats: coupled degradation of amino acids via the Stickland reaction in chemostat 1 and uncoupled degradation of amino acids via syntrophic association of amino acid degraders and hydrogenotrophic methanogens in chemostat 2.

We demonstrated previously that micro-aeration allows construction of an effective thermophilic methanefermentation system for treatment of municipal solid waste (MSW) without production of H2S. In the present study, we compared the microbial communities in a thermophilic MSW digester without aeration and with micro-aeration by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), phylogenetic analysis of libraries of 16S rRNA gene clones and quantitative real-time PCR. Moreover, we studied the activity of sulfatereducing bacteria (SRB) by analysis of the transcription of the gene for dissimilatory sulfite reductase (dsr). Experiments using FISH revealed that microorganisms belonging to the domain Bacteria dominated in the digester both without aeration and with micro-aeration. Phylogenetic analysis based on 16S rRNA gene and analysis of bacteria by DGGE did not reveal any obvious difference within the microbial communities under the two aeration conditions, and bacteria affiliated with the phylum Firmicutes were dominant. In Archaea, the population of Methanosarcina decreased while the population of Methanoculleus increased as a result of micro-aerations as revealed by the analysis of 16S rRNA gene clones and quantitative real-time PCR. Reverse transcription and PCR (RT-PCR) demonstrated the transcription of dsrA not only in the absence of aeration but also in the presence of micro-aeration, even under onditions where no H2S was detected in the biogas. In conclusion, micro-aeration has no obvious effects on the phylogenetic diversity of microorganisms. Furthermore, the activity of SRBs in the digester was not repressed even though the concentration of H2S in the biogas was very low under the micro-aeration conditions.