| Summary: | Consolidated bioprocessing, a method that involves cellulase production, substrate hydrolysis, and fermentation all in one step, requires lower energy input and aims at achieving reduced biofuel production costs than traditional processes. It is an economically appealing strategy for the efficient production of biofuels such as ethanol or H2. At present, the yields of fermentative hydrogen and ethanol production are less than the theoretical maximum and vary between anaerobic Clostridia due to the presence of highly branched metabolic pathways. With the recent advancements in ‘Omic technologies, the selected cellulolytic species, in this case, C. termitidis, was extensively studied to identify the key enzymes that are involved in hydrogen and ethanol synthesis pathways in both the genome and proteome under different culture conditions. Metabolic characterization involving growth and end-product synthesis patterns were performed on 2 g L-1 cellobiose and α-cellulose under batch conditions to determine its metabolic potential for hydrogen and/or ethanol production. Initial characterization has shown the ability of C. termitidis to produce hydrogen, ethanol, and various other end-products on the two susbtrates. Continous N2 sparging in the pH-controlled bioreactors with cellobiose and α-cellulose showed a consistent increase in the H2 synthesis and lowered ethanol production compared to batch studies, with the H2 yields of 1.03 and 1.34 mol product per mol hexose equivalent added, respectively. Shotgun 2-D proteome analyses were performed to compare cellulose versus cellobiose grown cultures across exponential and stationary phases of growth. Most of the glycolytic proteins were detected in the proteome with some exceptions and no significant change was observed across both growth conditions. Hydrogen synthesis was regulatd via PFOR and ferredoxin-dependent hydrogenase, where as ethanol synthesis was regulated primarily via bifunctional AdhE activity. Proteomic analyses of C. termitidis cultured on hexose sugars in the absence of xylose suggested possible sequential utilization of xylose and cellobiose for the first time. Putative proteins consistent with xylose fermentation were observed at high levels. The hypothesis that C. termitidis can sequentially utilize xylose and cellobiose was further validated using batch fermentations tests on pure (xylose, cellobiose, xylan) and mixed substrates (xylose + cellobiose).
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