Isolation, characterization, and U(VI)-reducing potential of a facultatively anaerobic, acid-resistant Bacterium from Low-pH, nitrate- and U(VI)-contaminated subsurface sediment and description of Salmonella subterranea sp. nov

Abstract

A facultatively anaerobic, acid-resistant bacterium, designated strain FRCl, was isolated from a low-pH, nitrate- and U(VI)-contaminated subsurface sediment at site FW-024 at the Natural and Accelerated Bioremediation Research Field Research Center in Oak Ridge, Tenn. Strain FRCl was enriched at pH 4.5 in minimal medium with nitrate as the electron acceptor, hydrogen as the electron donor, and acetate as the carbon source. Clones with 16S ribosomal DNA (rDNA) sequences identical to the sequence of strain FRCl were also detected in a U(VI)-reducing enrichment culture derived from the same sediment. Cells of strain FRCl were gram-negative motile regular rods 2.0 to 3.4 micro m long and 0.7 to 0.9 microm in diameter. Strain FRCl was positive for indole production, by the methyl red test, and for ornithine decarboxylase; it was negative by the Voges-Proskauer test (for acetylmethylcarbinol production), for urea hydrolysis, for arginine dihydrolase, for lysine decarboxylase, for phenylalanine deaminase, for H(2)S production, and for gelatin hydrolysis. Strain FRCl was capable of using O(2), NO(3)(-), S(2)O(3)(2-), fumarate, and malate as terminal electron acceptors and of reducing U(VI) in the cell suspension. Analysis of the 16S rDNA sequence of the isolate indicated that this strain was 96.4% similar to Salmonella bongori and 96.3% similar to Enterobacter cloacae. Physiological and phylogenetic analyses suggested that strain FRCl belongs to the genus Salmonella and represents a new species, Salmonella subterranea sp. nov.

FIG. 1.

Electron micrograph of negatively stained strain FRCl grown to the mid-exponential phase with hydrogen as the electron donor and nitrate as the electron acceptor.

FIG. 2.

Effect of temperature and pH on growth of strain FRCl. (Left) Optimal growth temperature for strain FRCl. Generation times were calculated from the slopes of the growth curves (not shown) at pH 7. (Right) Influence of pH on the growth of strain FRCl. Generation times were calculated from the slopes of the growth curves (not shown) at 30°C. All growth experiments were done with acetate as the electron donor, nitrate as the electron acceptor, and 0.02% yeast extract. The results represent the means for triplicate cultures.

FIG. 3.

Phylogenetic analysis of FRC isolate. The tree was constructed by distance analysis with Jukes-Cantor correction by using 1,422 bases for comparison. Numbers next to branches indicate bootstrap values.

FIG. 4.

Aerobic growth of strain FRCl in the presence of citrate and acetate. The results represent the means for triplicate cultures.

FIG. 5.

Cells of strain FRCl in the low-pH aerobic culture after inoculation (A) and at 47 h (B).

FIG. 6.

U(VI) reduction by the cell suspension of strain FRCl. The results represent the means for triplicate cell suspensions.