Scott D. Hamilton-Brehm1, Adriane Lochner1, Richard J. Giannone2, Jennifer J. Mosher1, Robert L. Hettich2, Martin Keller1, and James G. Elkins1. (1) Bioenergy Science Center, Biosciences Division, Oak Ridge National Laboratory, P. O. Box 2008, Oak Ridge, TN 37831, (2) Bioenergy Science Center, Chemical Sciences Division, Oak Ridge National Laboratory, P. O. Box 2008, Oak Ridge, TN 37831
Cellulosic ethanol production from renewable biomass is dependent on the efficient enzymatic hydrolysis of cellulose in order to release fermentable sugars. Cellulolytic microorganisms inhabiting thermal environments are known to possess multidomain/multifunctional cellulases and hemicellulases that display increased heat-stability. Through previous discovery efforts aimed at obtaining novel organisms capable of rapid growth on lignocellulosic biomass; we isolated a new member of the genus Caldicellulosiruptor from Obsidian Pool, Yellowstone National Park, Wyoming, USA. The organism, designated Caldicellulosiruptor sp. OB47, grows optimally at 80°C and reaches cell densities >108 cells/ml on carbon sources such as cellobiose, Avicel (crystalline cellulose), xylan, pectin, filter paper, processed cardboard, and pretreated lignocellulosic biomass (switchgrass and Populus). Substrate utilization profiles, growth kinetics, and fermentation end-products were determined and compared against other Caldicellulosiruptur spp. and Clostridium thermocellum. In order to inventory the proteins involved in cellulose hydrolysis, cultures of OB47 were grown on Whatman #1 filter paper and cellular and extracellular proteins were prepared for proteomics analysis. Multidimensional liquid chromatography mass spectroscopy (LC-MS/MS) was used to identify single components of the major extracellular hydrolytic enzymes expressed by OB47. Throughout biological and technical replicates, roughly 75 non-redundant, extracellular proteins were identified by LC-MS/MS with the most abundant being glycosyl hydrolases, cellobiosidases, solute-binding proteins, as well as S-layer domain-containing proteins.
