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Methane (CH4) has attracted attention as not only synthetic natural gas, but also one of the hy-drogen carriers in terms of energy density. On the other hand, there exist bacterial ecosystems in nature that can decompose organic compounds to produce CH4 and CO2. In this study, Clostridi-um cellulovorans was first cultivated with pig manure (PM) as an unused biomass. Regarding the measurement of organic acids by high-performance liquid chromatography (HPLC), acetate and butyrate were increased in the C. cellulovorans medium containing 0.5% PM, while formate and lactate were decreased in it. Next, in comparison with carbon sources such as glucose, cellobiose, and acetate, cocultivation of C. cellulovorans and Methanosarcina mazei or microbial flora of me-thane production (MFMP) was performed in the C. cellulovorans medium. These results revealed that 0.5% acetate as the sole carbon source produced CH4 only by cocultivating C. cellulovorans and MFMP. Furthermore, MFMP was only cultivated with 1% acetate or 1% methane as a carbon source after precultivated with 0.5% glucose medium for 12 h. As a result, methane productivity of MFMP with 1% methanol medium was approximately eight times higher than that with 1% acetate medium. Finally, next-generation sequencing (NGS) analysis of MFMP after cultivation with 1% acetate or 1% methane was carried out. Interestingly, Methanofollis (0.211%) belonging to H2/CO2 -using methanogens (CO2 reduction pathway) was dominant in the 1% acetate medium for 72 h cultivation, whereas Methanosarcina siciliae (1.178%), M. barkeri (0.571%), and Methano-follis (0.490%) were major species in 1% methanol medium for 72 h cultivation. Since Methano-sarcina spp. are belonging to acetoclasts (acetoclastic pathway), methanol could promote to grow Methanosarcina spp. rather than acetate. Therefore, it seemed Methanosarcina spp. may play a key methanogenesis in MFMP. Thus, these results will provide important information for low cost biomethane production.

Sugar beet pulp (SBP) is the residue of beet sugar processing and is a promising feedstock for bio-fuels and chemicals production. The aim of this study was to develop the co-culture system of two mesophilic Clostridia, Clostridium cellulovorans and Clostridium beijerinckii, which allows simultaneous saccharification and fermentation of SBP to useful chemicals. Clostridium cellulovorans degraded SBP without pre-treatment and addition of exogenous enzymes, but could not utilize the arabinose produced by the arabinan degradation and Clostridium beijerinckii fermented this arabinose to produce organic acids and alcohols. Approximately 4.270 g/L of butyric acid and 0.276 g/L of 1-butanol from 20 g/L of SBP were obtained. These results indicated that co-culture of these two clostridia strains provides a technically feasible and more simplified way for producing useful chemicals directly from SBP., 本研究では，砂糖の製造工程における副生物であるシュガービートパルプ（SBP）からの有用物質生産を目指し，2種のClostridium 属中温細菌，すなわちセルロースおよびアラビナン，ペクチン等に対する優れた分解能を有するC. cellulovorans，およびアセトン，ブタノール，エタノール発酵細菌であるC. beijerinckii の共培養によりSBP の直接的発酵を行った。その結果，C. cellulovorans は，SBP のアラビナンを分解し，自らは資化できないアラビノースを生成し，これをC. beijerinckii が資化，増殖し，有機酸やアルコールを生産することが明らかになった。このことから，C. cellulovoransとC. beijerinckii は異なる特性，すなわち，バイオマス分解能力および発酵特性，糖利用性を持ち，互いに補完し合えることが示された。これらのことから，C. cellulovorans およびC. beijerinckii の共培養系が，SBP からの有用物質生産に有用であることが示された。

The regional issues of population decrease and a super-aging society are a serious bottleneck in Japan.Therefore, new industries by innovation expect to foster new jobs, resulting that both of them lead toregional revitalization. Because Mie prefecture has huge petrochemical industrial complexes, technologyplatforms and research institutions in addition to biomass sources, this area is suitable for challenging thebiorefinery in Japan. We used Clostridium cellulovorans that completely degrades biomass for biorefiney.As a result, we have so far succeeded to produce not only biofuels but also chemical compounds frommandarin lees. Therefore, we established a bio-venture company "EcoBioful Co. Ltd." in order to promotethose efforts. Furthermore, the networks among regional companies and academia having excellenttechnologies are tightly cooperated. In future, biorefinery as a new industry will contribute the creation ofnew industries and regional vitalization with developing regional innovation.

Among Clostridia, an extracellular enzyme complex called the cellulosomes produced by Clostridium species are particularly designed for efficient degradation of plant cell wall polysaccharides. Clostridium cellulovorans 743B, an anaerobic and mesophilic bacterium, produces the cellulosomes on the cell surface and free extracellular enzymes called noncellulosomes. Recently, we have reported the whole genome sequence of C. cellulovorans comprising 4,220 predicted genes in 5.10Mbp. As a result, the genome size of C. cellulovorans was about 1Mbp larger than that of other cellulosome-producing clostridia, mesophilic C. cellulolyticum and thermophilic C. thermocellum. A total of 57 cellulosomal genes were found in the C. cellulovorans genome, in addition to two novel genes encoding scaffolding proteins CbpB and CbpC. C. cellulovorans genome included not only cellulosomal genes but also a large number of genes encoding noncellulosomal enzymes, the genome expansion of C. cellulovorans included genes more related to degradation of polysaccharides, such as hemicelluloses and pectins, than to cellulose., Clostridia の中にはセルロソームと呼ばれる細胞外に酵素複合体を形成する Clostridium 属が存在し、それらは植物細胞壁を効率よく分解する。Clostridium cellulovorans 743B は, 嫌気性中温菌であり, 細胞表層にセルロソームと細胞外にフリーな酵素であるノンセルロソームを生産する。近年, 我々は, C. cellulovorans の全ゲノム配列を決定し, 予想される遺伝子は 4,220 あり, そのゲノムサイズは 5.1Mbp であった。中温菌である C. cellulolyticum や高温菌である C. thermocellum と比較すると C. cellulovorans は約 1Mbp ゲノムサイズが大きいことが分かった。C. cellulovorans のゲノム中にセルロソーマルな遺伝子は合計 57 個あり, 新たに 2 つの骨格タンパク質をコードする遺伝子である CbpB と CbpC が見つかった。C. cellulovorans のゲノムは, セルロソーマルな遺伝子だけでなく, 多数のノンセルロソーマルな酵素をコードする遺伝子が含まれており, セルロースを分解する酵素よりもヘミセルロースやペクチンを分解する酵素の方が多く含まれていた。

The beta-agarase-d gene (agaD) from a marine bacterium, Vibrio sp. strain PO-303, was cloned and expressed in Escherichia coli. The gene consists of 1,362 bp and encodes a protein of 453 amino acids with a predicted molecular weight of 50,824. The full length of agarase-d consists of a signal peptide, a glycoside hydrolase family 16 catalytic module (CM), and a carbohydrate binding module (CBM). The full length of agarase-d without the signal peptide (rAgaDDeltafull), the catalytic module (rAgaDCM), or the CBM (rAgaDCBM) was expressed in E. coli as recombinant proteins. rAgaDCM exhibited higher enzyme activity (63.6 units/mg) than rAgaDDeltafull (1.20 units/mg) against agarose. rAgaDCM hydrolyzed agar and porphyran to several oligosaccharides and acted on neoagarohexaose to produce neoagarotetraose and neoagarobiose, but did not act on neoagarotetraose. rAgaDCBM bound to agarose.