Anaerobic Bacterial Degradation for the Effective Utilization of Biomass

Biomass is originally photosynthesized from inorgainic compounds such as $CO_2$, minerals, water and solar energy. Recent studies have shown that anaerobic bacteria have the ability to convert recalcitrant biomass such as cellullosic or chitinoic materials to useful compounds. The biomass containing agricultural waste, unutilized wood and other garbage is expected to utilize as feed, food and fuel by microbial degradation and other metabolic functions. In this study we isolated several anaerobic, cellulolytic and chitinolytic bacteria from rumen fluid, compost and soil to study their related enzymes and genes. The anaerobic and cellulolytic bacteria, Clostridium thermocellum, Clostridium stercorarium, and Clostridium josui, were isolated from compost and the chitinolytic Clostridium paraputrificum from beach soil and Ruminococcus albus was isolated from cow rumen. After isolation, novel cellulase and xylanase genes from these anaerobes were cloned and expressed in Escherichia coli. The properties of the cloned enzymes showed that some of them were the components of the enzyme (cellulase) complex, i.e., cellulosome, which is known to form complexes by binding cohesin domains on the cellulase integrating protein (Cip: or core protein) and dockerin domains on the enzymes. Several dockerin and cohesin polypeptides were independently produced by E. coli and their binding properties were specified with BIAcore by measuring surface plasmon resonance. Three pairs of cohesin-dockerin with differing binding specificities were selected. Two of their genes encoding their respective cohesin polypeptides were combined to one gene and expressed in E. coli as a chimeric core protein, on which two dockerin-dehydrogenase chimeras, the dockerin-formaldehyde dehydrogenase and the dockerin-NADH dehydrogenase are planning to bind for catalyzing $CO_2$ reduction to formic acid by feeding NADH. This reaction may represent a novel strategy for the reduction of the green house gases. Enzymes from the anaerobes were also expressed in tobacco and rice plants. The activity of a xylanase from C. stercorarium was detected in leaves, stems, and rice grain under the control of CaMV35S promoter. The digestibility of transgenic rice leaves in goat rumen was slightly accelerated. C. paraputrificum was found to solubilize shrimp shells and chitin to generate hydrogen gas. Hydrogen productivity (1.7 mol $H_2/mol$ glucos) of the organism was improved up to 1.8 times by additional expression of the own hydrogenase gene in C. paraputrficum using a modified vector of Clostridiu, perfringens. The hydrygen producing microflora from soil, garbage and dried pelletted garbage, known as refuse derived fuel(RDF), were also found to be effective in converting biomass waste to hydrogen gas.