Alkaline anaerobic respiration: isolation and characterization of a novel alkaliphilic and metal-reducing bacterium

Abstract

Iron-reducing enrichments were obtained from leachate ponds at the U.S. Borax Company in Boron, Calif. Based on partial small-subunit (SSU) rRNA gene sequences (approximately 500 nucleotides), six isolates shared 98.9% nucleotide identity. As a representative, the isolate QYMF was selected for further analysis. QYMF could be grown with Fe(III)-citrate, Fe(III)-EDTA, Co(III)-EDTA, or Cr(VI) as electron acceptors, and yeast extract and lactate could serve as electron donors. Growth during iron reduction occurred over the pH range of 7.5 to 11.0 (optimum, pH 9.5), a sodium chloride range of 0 to 80 g/liter (optimum, 20 g/liter), and a temperature range of 4 to 45 degrees C (optimum, approximately 35 degrees C), and iron precipitates were formed. QYMF was a strict anaerobe that could be grown in the presence of borax, and the cells were straight rods that produced endospores. Sodium chloride and yeast extract stimulated growth. Phylogenetic analysis of the SSU rRNA gene indicated that the bacterium was a low-G+C gram-positive microorganism and had 96 and 92% nucleotide identity with Alkaliphilus transvaalensis and Alkaliphilus crotonatoxidans, respectively. The major phospholipid fatty acids were 14:1, 16:1omega7c, and 16:0, which were different from those of other alkaliphiles but similar to those of reported iron-reducing bacteria. The results demonstrated that the isolate might represent a novel metal-reducing alkaliphilic species. The name Alkaliphilus metalliredigens sp. nov. is proposed. The isolation and activity of metal-reducing bacteria from borax-contaminated leachate ponds suggest that bioremediation of metal-contaminated alkaline environments may be feasible and have implications for alkaline anaerobic respiration.

FIG. 1.

Scanning electron micrographs of isolate QYMF grown with Fe³⁺-EDTA at ×17,670 (a) and ×13,020 (b) magnifications. The arrow denotes an endospore. Bars, 1.0 μm.

FIG. 2.

Cell growth in the presence of Co(III) and 0.25 (□), 0.1 (•), 0.05 (○), or 0.01 (▾) g of yeast extract per liter determined via direct cell counts. The control with yeast extract only (▵) did not show significant growth. The medium included 2 g of borate per liter and 20 g of sodium chloride per liter, and the pH value was 9.6. The error bars indicate standard deviations of duplicates.

FIG. 3.

Cell growth in the presence of Co(III), yeast extract (0.01 g/liter), and lactate (10 mM) determined via a decrease in Co(III) (•) and an increase in cell number (▴). The controls included the following: Co(III) only (□), yeast extract only (▿), and yeast extract plus lactate (▪). The medium included 2 g of borate per liter and 20 g of sodium chloride per liter, and the pH value was 9.6. The error bars indicate standard deviations of duplicates.

FIG. 4.

Relationship between sodium chloride concentration (b) or pH (a) and growth rate when yeast extract and Fe(III)-citrate were provided as electron donor and acceptor, respectively. The borate concentration was 2 g/liter. The pH value was 9.6 for the determination of the pH optimum, and the salt concentration was 20 g/liter for the optimal pH determination. The error bars indicate standard deviations of duplicates. It should be noted that, when sodium chloride was not added, the medium still contained approximately 8 mM sodium from sodium carbonate.

FIG. 5.

Production of Fe(II) (•) and increase in cell number (▪) when cells were grown with Fe(III), yeast extract, and 20 g of sodium chloride per liter at pH 9.6. The controls included Fe(III) only (○) and yeast extract only (• with dashed line). The error bars indicate standard deviations of duplicates.

FIG. 6.

Decrease in Co(III)-EDTA (•) and increase in cell number (▪) when cells were grown with Co(III), yeast extract, and 20 g of sodium chloride per liter at pH 9.6. The controls included Co(III) only (○) and yeast extract only (□). The error bars indicate standard deviations of duplicates.

FIG. 7.

Decrease in Cr(VI) (▴) and increase in cell number (•) when cells were grown with Cr(VI), yeast extract, and 20 g of sodium chloride per liter at pH 9.6. The controls included Cr(VI) only (▪) and yeast extract only (♦). The error bars indicate standard deviations of duplicates.

FIG. 8.

Phylogenetic relationships between isolate QYMF and other alkaliphilic or metal-reducing bacteria based on nearly complete SSU rRNA gene sequences. Sequences were aligned with ClustalW, and pairwise deletions were used for neighbor joining. Bootstrap (n = 500) values below 50 are not shown, and Chrysiogenes arsenatis was used as the outgroup. GenBank accession numbers are in parentheses.