Isolation of novel cellulolytic bacteria from biogas plants and characterization of their cellulolytic and hemicellulolytic potential

Biogas formation from renewable resources is presently one of the most attractive technologies for sustainable energy production. The first step in biogas fermentation, the hydrolysis of lignocellulosic biomass, is regarded as one of the rate-limiting steps in the process. Only a small fraction of these hydrolytic especially cellulose degrading bacterial species present in a biogas fermenter has been isolated in pure culture. New Isolates are pivotal providing the access to genome data and new lignocellulosic biomass degrading enzymes for various industrial applications. Therefor we tried to isolate new cellulolytic bacteria from sludge of a thermophilic biogas plant with different techniques for isolating microorganisms from biogas reactors. Out of the phylum Firmicutes we got several isolates, having high 16S rRNA sequence similarity (>99%) with the already cultured cellulolytic bacteria Clostridium cellulosi, Clostridium stercorarium, Clostridium clariflavum and Clostridium thermocellum and also one until then uncultured cellulolytic bacterium (Strain T3/55), forming a hitherto unknown subline within the family Lachnospiraceae. T3/55 was chosen for genome sequencing and for recombinant expression and characterization of biomass degrading enzymes. The cellulolytic system consists of three cellulases, one exoglucanase and two endoglucanases, which synergistically degrade cellulose. Strain T3/55 digested various cellulosic substrates. The cellulolytic activity on crystalline cellulose (Avicel) is measurable but eleven-fold lower than that from C. thermocellum (ATCC 27405). The major end products of cellulose metabolism are acetate, ethanol and propionic acid. Additionally, the hemicellulolytic activity of T3/55 culture supernatants was tested on several hemicellulosic substrates. A high hemicellulolytic activity could be detected on various xylans.