Biofuels & Bioenergy

As the reservoir of unsustainable fossil fuels, such as coal, petroleum and natural gas, is over-utilized and continues to contribute to environmental pollution and CO2 emission, the need for appropriate alternative energy sources becomes more crucial. Bioethanol produced from dedicated crops and cellulosic waste can provide a partial answer, yet a cost-effective production method must be developed. The cellulosome system of the anaerobic thermophile, Clostridium clariflavum, comprises a large number of cellulolytic and hemicellulolytic enzymes and scaffoldins which self-assemble in a number of different cellulosome architectures for enhanced cellulosic biomass degradation. We determined the cohesin-dockerin interaction pattern of the cellulosomal system of C. clariflavum and suggested various possible cellulosome assemblies. Further on, we cultivated C. clariflavum on cellobiose-, microcrystalline cellulose- and switchgrass-containing media and isolated cell-free cellulosome complexes from each culture. Gel-filtration separation of the cellulosome samples revealed two major fractions, which were analyzed by label-free LC-MS/MS in order to identify the key players of the cellulosome assemblies therein. In addition, the catalytic activity of each cellulosome was examined on different cellulosic substrates, xylan and switchgrass. The cellulosome isolated from the microcrystalline cellulose-containing medium was the most active of all the cellulosomes that were tested and approaches the degradation capabilities of the cellulosome of the most efficient cellulose-degrading bacterium, Clostridium thermocellum. The results suggest that the expression of the cellulosome proteins is regulated by the type of substrate in the growth medium. Identification of the major cellulosomal components expressed during growth of the bacterium and their influence on its catalytic capabilities provide insight into the performance of the remarkable cellulosome of this intriguing bacterium. The findings, together with the thermophilic characteristics of the proteins, render C. clariflavum of great interest for future use in industrial cellulose-conversion processes.