Project description:R. cellulolyticum strains (the parent strain, ΔccpA, ΔccpB and Δcph) were cultivated in defined VM medium with cellulose (10 g/L).Since ΔccpA and Δcph could not grow on cellulose, we performed an incubation experiment to understand how ΔccpA and Δcph respond to cellulose. Specifically, the parent strain, ΔccpA and Δcph were grown to an OD600 of 0.5-0.6 in 50 ml defined VM media with cellobiose as the carbon source. Each strain had three biological replicates. Bacterial cells from each biological replicate were then collected by centrifugation at 4000 g and washed twice with the defined VM medium (no carbon added). Finally, washed cells from each biological replicate were inoculated into the defined VM medium with 10 g/L cellulose. During shaking incubation at 34°C, samples were collected at five time points (0,1, 3, 6, 12 hours). After centrifugation at 4°C, 5000×g for 10 min, cell pellets were immediately flash frozen with liquid nitrogen and then stored at -80°C for further use. For ΔccpB, ΔccpB and the parent strain were cultivated with six biological replicates and collected at mid-exponential growth phase.
Project description:Genome-wide maps of primary and processed start-sites of transcripts revealed mechanism controlling in vivo stoichiometry of protein complex in bacteria
Project description:Biofuel production from lignocellulosic waste and residues is a promising option to mitigate the environmental costs associated to energy production. However, the difficulty to cost-effectively overcome lignocellulose recalcitrance hampers a widespread application of such bioprocesses. Through an integrated approach, we focused on the factors affecting cellulose reactivity and their impact on downstream fermentation. Three cellulosic manufactured materials were characterized in details: facial tissue, Whatman paper, cotton pads. The model mesophilic cellulolytic bacterium Clostridium cellulolyticum was used to study colonization and metabolic patterns during fermentation of these materials. Facial tissue was extensively colonized and exhibited the fastest degradation and the highest ethanol-to-acetate ratio. Comparing facial tissue fermentation to Whatman paper fermentation by label-free quantitative shotgun proteomics and statistical analyses, 187 proteins showed a different behavior; higher concentration levels were detected for many enzymes from the carbohydrate central metabolic pathway; distinct patterns of expression levels were observed for carbohydratases degrading cellulose and hemicellulose. Overall, lower degrees of polymerization, lower crystallinity index, and the presence of hemicelluloses could explain the higher biological reactivity and bioethanol production yields.
