Monday, May 4, 2009 - 1:30 PM
6-02
Cellulolytic Extreme Thermophiles and Hyperthermophiles from Terrestrial Hot Springs
Joel E. Graham1, Priya Jayachandran2, Melinda Clark3, Harvey W. Blanch2, Douglas S. Clark2, and Frank T. Robb1. (1) Center of Marine Biotechnology, University of Maryland, 701 E. Pratt Street, Baltimore, MD 21202, (2) Chemical Engineering, University of California, Berkeley, Berkeley, CA 94720, (3) Energy Biosciences Institute, 130 Calvin Hall, Berkeley, CA 94720
Extremely thermophilic microorganisms are good candidates to provide highly stable, active enzymes for cellulose deconstruction. Elevated operating temperatures may also be beneficial in fermentations producing biofuels. To that end, cellulose-degrading microorganisms from hot springs in Nevada and Northern California were enriched on anaerobic medium containing ground (80 mm diameter particles) Miscanthus sinensis as the sole carbon source. Enrichments were performed at temperatures between 70°C and 90°C. Organisms growing in the enrichments were identified by 16S RNA clone libraries generated from PCR amplification of 16S coding sequences from DNA extracted from the enrichments, or 454 sequencing with bar-coded primers from the same DNA. Enrichments at 70°C contained a variety of thermophilic bacteria related to strains known to be cellulolytic as well as some that were not closely related to any characterized strain and may represent new genera of cellulolytic organisms. In order to verify that cellulolytic organisms were present in miscanthus enrichments, secondary enrichments were made using Whatman #1 and #3 filter paper as the carbon source and a portion of the primary enrichments as the inoculum. At 90°C the dominant microorganisms present in miscanthus or filter paper enrichments were archaeal, establishing a role for archaea as cellulose degrading organisms and as potential targets for the identification of new cellulases. Cellulase assays were performed to determine the activity of free and bound cellulases in each of the enrichment cultures. The media constituents were refined to promote accelerated cellulose degradation, and pure cultures, including archaeal species, were isolated from the enrichments.