Caldicellulosiruptor obsidiansis sp. nov., an anaerobic, extremely thermophilic, cellulolytic bacterium isolated from Obsidian Pool, Yellowstone National Park

Abstract

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 microm long by 0.2 microm wide and grew at temperatures between 55 and 85 degrees C, with the optimum at 78 degrees 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).

FIG. 1.

Neighbor-joining tree based on 16S rRNA gene sequence comparisons of strain OB47^T and selected bacteria. Bootstrap values were based on 1,000 replicates. The scale bar represents 0.01 change per nucleotide position. T, type strain.

FIG. 2.

Specific growth rates for OB47^T at various temperatures (A) and various pHs (B). Each data point represents the mean ± standard deviation calculated from 4 replicate cultures.

FIG. 3.

Microscopy of OB47^T. (A) Scanning electron microscopy at ×5,000; (B) atomic force microscopy deflection signals at 12 μm².

FIG. 4.

Growth of OB47^T on 1.5% (wt/vol) crystalline cellulose and end product formation during fermentation. Triangles, total cellular protein accumulation; squares, hydrolysis of crystalline cellulose (Avicel); diamonds, acetate production; and circles, lactate production. Each data point represents the mean ± standard deviation calculated from triplicate samples collected from 2 independent experiments.

FIG. 5.

Growth of OB47^T on 1.0% (wt/vol) dilute acid-pretreated switchgrass and end product formation during the fermentation. Triangles, total cellular protein accumulation; squares, total mass reduction in substrate; diamonds, acetate production; and circles, lactate production. Each data point represents the mean ± standard deviation calculated from triplicate samples collected from 2 independent experiments.