Enzymatic Characterization of Unused Biomass Degradation Using the Clostridium cellulovorans Cellulosome

Abstract

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.