Fundamentals of microbial cellulose utilization by the thermophilic bacterium Clostridium thermocellum

The thermophilic anaerobic bacterium Clostridium thermocellum has attracted researchers attention because of its potential industrial application and high cellulose hydrolysis rates. This study focuses on the physiological fundamentals of C. thermocellum ATCC 27405. Error analysis indicated that measurement of cellulase concentration by enzyme-linked-immuno-sorbent assay (ELISA) in combination with measurement of total protein allows the mass concentrations of cellulase and cells to be estimated with reasonable accuracy whereas several other combinations of analytes do not. An ELISA-based assay was developed using antibody raised against a 14-amino acid long synthetic peptide with sequence taken from the cohesin domain of the scaffoldin protein of C. thermocellum. Several lines of evidence support the validity of the newly-developed analytical protocols for determining the mass concentrations of cellulase and cells produced by C. thermocellum . Differences in the composition and expression of the C. thermocellum cellulase system were observed for cultures grown on Avicel as compared to cellobiose.; The new protocols were used to examine the bioenergetics of microbial cellulose utilization in more detail than has been undertaken previously. In addition to metabolic processes included in conventional bioenergetic models (catabolic ATP supply, growth and maintenance), several additional processes appear to be significant for growth of C. thermocellum on cellulose. A bioenergetic model was developed which evaluated the quantitative importance of three factors responsible for ATP supply (glycolysis, phosphorolytic cleavage of beta-glucosidic bonds, and acetate kinase-mediated substrate level phosphorylation), and five factors responsible for ATP consumption (substrate transport, growth, cellulase synthesis, synthesis of extracellular non-cellulase protein, and maintenance. Results indicate that cellodextrins with mean degree of polymerization ≥4 were assimilated during growth on cellulose, and that the bioenergetic cost of cellulase synthesis is several-fold smaller than the cumulative bioenergetic benefits specific to growth on cellulose.; Cellulose conversion was observed to decrease with increasing Avicel and cellobiose concentrations in continuous culture. In contrast to the conventional interpretation that ethanol and/or organic acids are the primary factors inhibiting growth, our results indicate that inhibition was primarily due to the accumulation of salts from base addition to maintain pH control. (Abstract shortened by UMI.)...