An arsenite-oxidizing Hydrogenobaculum strain was isolated from a geothermal spring in Yellowstone National Park, Wyo., that was previously shown to contain microbial populations engaged in arsenite oxidation. The isolate was sensitive to both arsenite and arsenate and behaved as an obligate chemolithoautotroph that used H 2 as its sole energy source and had an optimum temperature of 55 to 60°C and an optimum pH of 3.0. The arsenite oxidation in this organism displayed saturation kinetics and was strongly inhibited by H 2 S.Arsenite [As(III)] is often the predominant valence of inorganic arsenic in geothermal source waters, although arsenate [As(V)] can also be present, with As(V)/As(III) ratios varying among different springs due to mixing with meteoric surface waters prior to discharge (3,12,19). However, subsequent to discharge, As(V)/As(III) ratios in the spring water can also be significantly influenced by redox transformations (10, 12), which are well documented for microorganisms (2,4,5,6,10,11,16,17). As(V) reduction is widespread among prokaryotes, occurring when As(V) is utilized as an electron acceptor for anaerobic or microaerobic respiration (13) or as part of a detoxification strategy (8). As(III) oxidation has likewise been observed in various organisms, where it has also been viewed as an apparent detoxification mechanism (1, 14, 15) or as a source of energy to support chemolithoautotrophic growth (1, 17).We previously documented rapid microbial oxidation of As(III) in an acid-sulfate-chloride-type geothermal spring in Norris Geyser Basin, Yellowstone National Park (9). This shallow spring is fed by a nearly constant geothermal source water (63°C, pH 3.1) containing ϳ35 M As(III). The prokaryote microbial community in this spring forms visually and chemically distinguishable mats. A filamentous yellow microbial mat containing visible amounts of S 0 (63 to 60°C) is present 0 to ϳ3.5 m from the spring source and changes to a brown, Fe(III) oxyhydroxide filamentous microbial mat (51 to 55°C) at ϳ3.5 to 5 m from the spring source (9). Chemical analysis of the aqueous and solid phases documented high rates of As(III) oxidation in the brown mat region, and the role of microorganisms in As(III) oxidation was confirmed in assays that showed no As(III) oxidation in the formaldehyde-killed samples (9). The PCR-generated 16S ribosomal DNA clone libraries representing the yellow and brown mat regions were dominated by Hydrogenobaculum-and Desulfurella-like sequences (7). However, since the phylogenetic data could not predict which population(s) was involved in the As(III) oxidation, the present study was conducted to initiate isolation and characterization of the As(III)-oxidizing microorganism(s) in this spring for use in modeling important and dominant biogeochemical features found in this spring type.Sampling, enrichment, and isolation. Brown microbial mat material was aseptically sampled and transferred to sterile 70-ml serum bottles and submerged with 35 ml of spring water sampled from above the mat. Th...