Caldicellulosiruptor obsidiansis sp. nov., an Anaerobic,Extremely Thermophilic,Cellulolytic Bacterium Isolated from Obsidian Pool,Yellowstone National Park

A novel, obligately anaerobic, extremely thermophilic, cellulolytic bacterium, designated OB47^T, was isolated from Obsidian Pool, Yellowstone National Park, WY. The isolate was a nonmotile, non-spore-forming, Gram-positive rod approximately 2 μm long by 0.2 μm wide and grew at temperatures between 55 and 85°C, with the optimum at 78°C. The pH range for growth was 6.0 to 8.0, with values of near 7.0 being optimal. Growth on cellobiose produced the fastest specific growth rate at 0.75 h⁻¹. The organism also displayed fermentative growth on glucose, maltose, arabinose, fructose, starch, lactose, mannose, sucrose, galactose, xylose, arabinogalactan, Avicel, xylan, filter paper, processed cardboard, pectin, dilute acid-pretreated switchgrass, and Populus. OB47^T was unable to grow on mannitol, fucose, lignin, Gelrite, acetate, glycerol, ribose, sorbitol, carboxymethylcellulose, and casein. Yeast extract stimulated growth, and thiosulfate, sulfate, nitrate, and sulfur were not reduced. Fermentation end products were mainly acetate, H₂, and CO₂, although lactate and ethanol were produced in 5-liter batch fermentations. The G+C content of the DNA was 35 mol%, and sequence analysis of the small subunit rRNA gene placed OB47^T within the genus Caldicellulosiruptor. Based on its phylogenetic and phenotypic properties, the isolate is proposed to be designated Caldicellulosiruptor obsidiansis sp. nov. and OB47 is the type strain (ATCC BAA-2073).Cellulosic biomass will likely serve as an important source of stored renewable energy in the future. However, improvements in overcoming the recalcitrance of lignocellulosic materials to enzymatic hydrolysis must be made in order to efficiently convert biomass to liquid fuels (23, 27). Members of the genera Caldicellulosiruptor and Anaerocellum are obligatory anaerobic, extreme thermophiles within the Firmicutes and are known to express heat-stable extracellular enzyme systems for breaking down biomass (4). In addition, both hexose and pentose sugars can be utilized for fermentation (12, 16, 19, 28). Given these properties, recent studies have focused on the use of extreme thermophiles for biomass conversion to fuels, including Caldicellulosiruptor saccharolyticus as a biocatalyst for hydrogen production from biomass (10, 26) and A. thermophilum (also known as Caldicellulosiruptor bescii), which has been evaluated for growth on plant biomass without physical or chemical pretreatment (28, 29).A number of isolated strains of Caldicellulosiruptor have been described thus far, with several organisms originating from Icelandic hot springs (3, 14, 17, 18); the geothermal region of Kamchatka (15, 24); thermal features in New Zealand (19); and solar-heated freshwater ponds in Owens Valley, CA (9). The environmental parameters for growth appear to be fairly uniform for these organisms which prefer circumneutral to slightly alkaline pH and temperatures ranging from 60 to 83°C. None of the described isolates form spores, and all strains are heterotrophic obligate anaerobes which utilize a broad range of carbohydrates for fermentative growth. Complete genome sequences are available for C. saccharolyticus (26) and Anaerocellum thermophilum (28).Thus far, no species of Caldicellulosiruptor have been isolated and characterized from the major geothermal formations within Yellowstone National Park (YNP), WY. Recent enrichment and isolation attempts revealed a high abundance of cellulolytic organisms from hot springs within the Mud Volcano region of YNP. Using a high-throughput isolation method based on flow cytometry, a total of 53 isolates of Caldicellulosiruptor, based on small subunit rRNA analysis, were isolated from Obsidian Pool. Secondary screening of these strains provided an organism with rapid growth kinetics on pretreated biomass substrates as well as crystalline cellulose and xylan at 80°C. Based on small subunit rRNA analysis, genomic sequence comparisons, and phenotypic properties, we propose this organism as Caldicellulosiruptor obsidiansis OB47^T sp. nov., which is named in reference to the location from which it was isolated, Obsidian Pool, YNP.