Clostridium cellulovorans Proteomic Responses to Butanol Stress

Combination of butanol-hyperproducing and hypertolerant phenotypes is essential for developing microbial strains suitable for industrial production of bio-butanol, one of the most promising liquid biofuels. Clostridium cellulovorans is among the microbial strains with the highest potential for direct production of n-butanol from lignocellulosic wastes, a process that would significantly reduce the cost of bio-butanol. However, butanol exhibits higher toxicity compared to ethanol and C. cellulovorans tolerance to this solvent is low. In the present investigation, comparative gel-free proteomics was used to study the response of C. cellulovorans to butanol challenge and understand the tolerance mechanisms activated in this condition. Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH-MS) analysis allowed identification and quantification of differentially expressed soluble proteins. The study data are available via ProteomeXchange with the identifier PXD024183. The most important response concerned modulation of protein biosynthesis, folding and degradation. Coherent with previous studies on other bacteria, several heat shock proteins (HSPs), involved in protein quality control, were up-regulated such as the chaperones GroES (Cpn10), Hsp90, and DnaJ. Globally, our data indicate that protein biosynthesis is reduced, likely not to overload HSPs. Several additional metabolic adaptations were triggered by butanol exposure such as the up-regulation of V- and F-type ATPases (involved in ATP synthesis/generation of proton motive force), enzymes involved in amino acid (e.g., arginine, lysine, methionine, and branched chain amino acids) biosynthesis and proteins involved in cell envelope re-arrangement (e.g., the products of Clocel_4136, Clocel_4137, Clocel_4144, Clocel_4162 and Clocel_4352, involved in the biosynthesis of saturated fatty acids) and a redistribution of carbon flux through fermentative pathways (acetate and formate yields were increased and decreased, respectively). Based on these experimental findings, several potential gene targets for metabolic engineering strategies aimed at improving butanol tolerance in C. cellulovorans are suggested. This includes overexpression of HSPs (e.g., GroES, Hsp90, DnaJ, ClpC), RNA chaperone Hfq, V- and F-type ATPases and a number of genes whose function in C. cellulovorans is currently unknown.

The Second-Generation Biomethane from Mandarin Orange Peel under Cocultivation with Methanogens and the Armed Clostridium cellulovorans

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.