For a resolution of reducing carbon dioxide emission and increasing food production to respond to the growth of global population, production of biofuels from non-edible biomass is urgently required. Abundant orange wastes, such as peel and strained lees, are produced as by-product of orange juice, which is available non-edible biomass. However, d-limonene included in citrus fruits often inhibits yeast growth and makes the ethanol fermentation difficult. This study demonstrated that isopropanol-butanol-ethanol fermentation ability of Clostridium beijerinckii and cellulosic biomass degrading ability of C. cellulovorans were cultivated under several concentrations of limonene. As a result, C. cellulovorans was able to grow even in the medium containing 0.05% limonene (v/v) and degraded 85% of total sugar from mandarin peel and strained lees without any pretreatments. More interestingly, C. beijerinckii produced 0.046 g butanol per 1 g of dried strained lees in the culture supernatant together with C. cellulovorans.

This study was demonstrated with a coculture fermentation system using sugar beet pulp (SBP) as a carbon source combining the cellulose-degrading bacterium Clostridium cellulovorans with microbial flora of methane production (MFMP) for the direct conversion of cellulosic biomass to methane (CH 4 ). The MFMP was taken from a commercial methane fermentation plant and extremely complicated. Therefore, the MFMP was analyzed by a next-generation sequencing system and the microbiome was identified and classified based on several computer programs. As a result, Methanosarcina mazei (1.34% of total counts) and the other methanogens were found in the MFMP. Interestingly, the simultaneous utilization of hydrogen (H 2 ) and carbon dioxide (CO 2 ) for methanogenesis was observed in the coculture with Consortium of C. cellulovorans with the MFMP (CCeM) including M. mazei . Furthermore, the CCeM degraded 87.3% of SBP without any pretreatment and produced 34.0 L of CH 4 per 1 kg of dry weight of SBP. Thus, a gas metabolic shift in the fermentation pattern of C. cellulovorans was observed in the CCeM coculture. These results indicated that degradation of agricultural wastes was able to be carried out simultaneously with CH 4 production by C. cellulovorans and the MFMP.

This study demonstrates that the consortium, which consists of the microbial flora of methane production (MFMP) and Clostridium cellulovorans grown with cellulose, can perform the direct conversion of cellulosic biomass to methane. The MFMP was taken from a commercial methane fermentation tank and was extremely complicated. Therefore, C. cellulovorans grown with cellobiose could not perform high degradation ability on cellulosic biomass due to competition by various microorganisms in MFMP. Focusing on the fact that C. cellulovorans was cultivated with cellulose, which is armed with cellulosome, so that it is now armed C. cellulovorans; the direct conversion was carried out by the consortium which consisted of MFMP and the armed C. cellulovorans. As a result, the consortium of C. cellulovorans grown with cellobiose and MFMP (CCeM) could not degrade the purified cellulose and mandarin orange peel. However, MFMP and the armed C. cellulovorans reduced 78.4% of the total sugar of the purified cellulose such as MN301, and produced 6.89 mL of methane simultaneously. Furthermore, the consortium consisted of MFMP and the armed C. cellulovorans degraded mandarin orange peel without any pretreatments and produced methane that was accounting for 66.2% of the total produced gas.

The cellulolytic system of Clostridium cellulovorans mainly consisting of a cellulosome that synergistically collaborates with non-complexed enzymes was investigated using cellulosic biomass. The cellulosomes were isolated from the culture supernatants with shredded paper, rice straw and sugarcane bagasse using crystalline cellulose. Enzyme solutions, including the cellulosome fractions, were analyzed by SDS-PAGE and Western blot using an anti-CbpA antibody. As a result, C. cellulovorans was able to completely degrade shredded paper for 9 days and to be continuously cultivated by the addition of new culture medium containing shredded paper, indicating, through TLC analysis, that its degradative products were glucose and cellobiose. Regarding the rice straw and sugarcane bagasse, while the degradative activity of rice straw was most active using the cellulosome in the culture supernatant of rice straw medium, that of sugarcane bagasse was most active using the cellulosome from the supernatant of cellobiose medium. Based on these results, no alcohols were found when C. acetobutylicum was cultivated in the absence of C. cellulovorans as it cannot degrade the cellulose. While 1.5 mM of ethanol was produced with C. cellulovorans cultivation, both n-butanol (1.67 mM) and ethanol (1.89 mM) were detected with the cocultivation of C. cellulovorans and C. acetobutylicum. Regarding the enzymatic activity evaluation against rice straw and sugarcane bagasse, the rice straw cellulosome fraction was the most active when compared against rice straw. Furthermore, since we attempted to choose reaction conditions more efficiently for the degradation of sugarcane bagasse, a wet jet milling device together with L-cysteine as a reducing agent was used. As a result, we found that the degradation activity was almost twice as high with 10 mM L-cysteine compared with without it. These results will provide new insights for biomass utilization.