Anaerobic oxidation of arsenite in Mono Lake water and by a facultative, arsenite-oxidizing chemoautotroph, strain MLHE-1

Abstract

Arsenite [As(III)]-enriched anoxic bottom water from Mono Lake, California, produced arsenate [As(V)] during incubation with either nitrate or nitrite. No such oxidation occurred in killed controls or in live samples incubated without added nitrate or nitrite. A small amount of biological As(III) oxidation was observed in samples amended with Fe(III) chelated with nitrolotriacetic acid, although some chemical oxidation was also evident in killed controls. A pure culture, strain MLHE-1, that was capable of growth with As(III) as its electron donor and nitrate as its electron acceptor was isolated in a defined mineral salts medium. Cells were also able to grow in nitrate-mineral salts medium by using H(2) or sulfide as their electron donor in lieu of As(III). Arsenite-grown cells demonstrated dark (14)CO(2) fixation, and PCR was used to indicate the presence of a gene encoding ribulose-1,5-biphosphate carboxylase/oxygenase. Strain MLHE-1 is a facultative chemoautotroph, able to grow with these inorganic electron donors and nitrate as its electron acceptor, but heterotrophic growth on acetate was also observed under both aerobic and anaerobic (nitrate) conditions. Phylogenetic analysis of its 16S ribosomal DNA sequence placed strain MLHE-1 within the haloalkaliphilic Ectothiorhodospira of the gamma-PROTEOBACTERIA: Arsenite oxidation has never been reported for any members of this subgroup of the PROTEOBACTERIA:

FIG. 1.

Formation of As(V) in anoxic lake water samples amended with 1 mM As(III). Note that the time scales (x axis) vary between the four panels. (A) Samples supplemented with 5 mM NaNO₃ added as the terminal electron acceptor to both live and killed controls, plus a live control without NO₃⁻. Symbols: ○, live with nitrate; •, killed with nitrate; ▴, live without nitrate. (B) Samples amended with nitrate (○) or nitrite (□), and killed controls with nitrite (▪). (C) Live (○) and killed (•) controls amended with 5 mM Fe(III)-NTA. (D) Live (□) and killed (▪) controls amended with MnO₂. Symbols represent the means for three separately incubated water samples, and error bars indicate ± 1 standard deviation.

FIG. 2.

Scanning electron micrograph of strain MLHE-1. Bar, 5 μm.

FIG. 3.

Anaerobic growth of strain MLHE-1 with As(III) as the electron donor and nitrate as the electron acceptor. Symbols: •, As(V); ○, As(III); ▪, nitrate; □, nitrite; ▵, cells. Symbols represent the means for three separate cultures, and error bars indicate ± 1 standard deviation.

FIG. 4.

Anaerobic growth of strain MLHE-1 with nitrate as the electron acceptor and the inorganic electron donors hydrogen (A) and sulfide (B). Symbols: ▪, nitrate; □, nitrite; ▿, hydrogen; ▵, cells; ○, sulfide; •, sulfate. Symbols represent the means for three cultures, and error bars indicate ± 1 standard deviation.

FIG. 5.

Heterotrophic growth of strain MLHE-1 with acetate as the electron donor under aerobic conditions (A) and anaerobic conditions with nitrate as the electron acceptor (B). Symbols: ♦, acetate; ▴, nitrate; •, nitrite; ▪, optical density at 600 nm (O.D. 600).

FIG. 6.

Phylogenetic relationships of strain MLHE-1 based on phylogenetic analysis of its 16S rRNA gene sequence. Aerobic As(III) oxidizers, as summarized by Santini et al. (34), are in boldface, and the tree includes the chemoautotrophic strains of the α-Proteobacteria related to the genus Agrobacterium and the heterotrophic strains of the β-Proteobacteria related to the genus Alcaligenes (30). The tree is unrooted, with Halobacterium salinarum as the outgroup. Numbers adjacent to nodes indicate bootstrap support for the branch (100 iterations; values of <50 are not shown), and the bar at the bottom indicates the number of nucleotide substitutions per position.

FIG. 7.

Phylogenetic relationship between a partial sequence of the cbbL gene from strain MLHE-1 and sequences from representative isolates. The neighbor-joining tree is unrooted and is based on analysis of 744 bp of nucleotide sequence. The accession numbers of sequences used to construct the tree, the phylogenetic affiliations of the isolates (16S rRNA genes), and the BLAST (1) similarity of the MLHE-1 gene to the isolate gene are given in parentheses. The scale bar indicates an evolutionary distance of 0.1, and numbers at branch points indicate bootstrap support as in Fig. 6.

FIG. 8.

Proposed biogeochemical cycle of arsenic between its +5 and +3 oxidation states as mediated by microorganisms. Note that chemoautotrophic microorganisms occupy anaerobic niches on both the reductive and oxidative sides, using H₂ and As(III) as electron donors, respectively, to gain energy for the fixation of CO₂ into cell carbon.