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.

A process for producing ethanol from kitchen waste was developed in this study. The process consists of freshness preservation of the waste, saccharification of the sugars in the waste, continuous ethanol fermentation of the saccharified liquid, and anaerobic treatment of the saccharification residue and the stillage. Spraying lactic acid bacteria (LCB) on the kitchen waste kept the waste fresh for over 1 week. High glucose recovery (85.5%) from LCB-sprayed waste was achieved after saccharification using Nagase N-40 glucoamylase. The resulting saccharified liquid was used directly for ethanol fermentation, without the addition of any nutrients. High ethanol productivity (24.0gl-1•h-1) was obtained when the flocculating yeast strain KF-7 was used in a continuous ethanol fermentation process at a dilution rate of 0.8h 1. The saccharification residue was mixed with stillage and treated in a thermophilic anaerobic continuous stirred tank reactor (CSTR); a VTS loading rate of 6 g l-1•d-1 with 72% VTS digestion efficiency was achieved. Using this process, 30.9g ethanol, and 65.2 l biogas with 50% methane, was produced from 1 kg of kitchen waste containing 118.0 g total sugar. Thus, energy in kitchen waste can be converted to ethanol and methane, which can then be used as fuels, while simultaneously treating kitchen waste.

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.

We constructed two mesophilic anaerobic chemostats that were continuously fed with synthetic wastewater containing butyrate as the sole source of carbon and energy. Steady-state conditions were achieved at dilution rates between 0.025 and 0.7 day-1. Butyrate, fed into the chemostat, was almost completely mineralized to CH4 and CO2 at dilution rates below 0.5 day-1. The butyratedegrading methanogenic communities in the chemostats at dilution rates between 0.025 and 0.7 day-1 were monitored based on the 16S rRNA gene, using molecular biological techniques including clone library analysis, denaturing gradient gel electrophoresis, and quantitative real-time polymerase chain reaction. The aceticlastic methanogen Methanosaeta and the hydrogenotrophic methanogen Methanoculleus dominated in methanogens at low dilution rates, whereas the aceticlastic methanogen Methanosaeta, Methanosarcina, the hydrogenotrophic methanogen Methanoculleus, and Methanospirillum dominated at high dilution rates. Bacteria affiliated with the family Syntrophaceae in the phylum Proteobacteria predominated at the low dilution rate of 0.025 day-1, whereas bacteria affiliated with the phylum Firmicutes and Candidate division OP3 predominated at high dilution rates. A significant quantity of bacteria closely related to the genus Syntrophomonas was detected at high dilution rates. Dilution rate showed an apparent effect on archaeal and bacterial communities in the butyrate-fed chemostats.

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.