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Microbial Dehalorespiration with 1,1,1-Trichloroethane

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Science  01 Nov 2002:
Vol. 298, Issue 5595, pp. 1023-1025
DOI: 10.1126/science.1074675

Abstract

1,1,1-Trichloroethane (TCA) is a ubiquitous environmental pollutant because of its widespread use as an industrial solvent, its improper disposal, and its substantial emission to the atmosphere. We report the isolation of an anaerobic bacterium, strain TCA1, that reductively dechlorinates TCA to 1,1-dichloroethane and chloroethane. Strain TCA1 required H2 as an electron donor and TCA as an electron acceptor for growth, indicating that dechlorination is a respiratory process. Phylogenetic analysis indicated that strain TCA1 is related to gram-positive bacteria with low DNA G+C content and that its closest relative is Dehalobacter restrictus, an obligate H2-oxidizing, chloroethene-respiring bacterium.

TCA is a synthetic organic solvent widely used in industrial processes and is a major environmental pollutant commonly found in soil (1), groundwater (2), and the atmosphere (3). TCA is present in at least 696 of the 1430 National Priorities List sites identified by the U.S. Environmental Protection Agency (EPA) (1). Because of TCA's adverse effects on human health, the EPA has set a maximum contaminant level of 200 μg/liter in drinking water (4). TCA is also listed as an ozone-depleting substance by the United Nations Environment Programme (5). Even when released to soil or leached to groundwater, the primary environmental fate of TCA is volatilization to the atmosphere, where it interacts with ozone and contributes to the erosion of the ozone layer (1, 5). TCA is often a co-contaminant in aquifers with chlorinated ethenes, especially tetrachloroethene (PCE) and trichloroethene (TCE), because they have similar industrial uses. Although in situ bioremediation processes for the chloroethenes are known (6), TCA remediation remains problematic and can prevent site restoration.

TCA undergoes slow abiotic degradation to acetic acid and 1,1-dichloroethene, an EPA priority pollutant (7). Biotransformation of TCA has been observed under aerobic and anaerobic conditions only in cometabolic processes (8–11). A growth-linked, or dehalorespiratory, process would be more effective for in situ bioremediation of TCA- contaminated sites, because reaction rates would be faster and natural selection would ensure growth in situ.

Although bacterial growth by dehalorespiration of chloroethenes, chlorobenzenes, 3-chlorobenzoate, and 2-chlorophenol has been well documented (12–15), bacterial growth by reductive dechlorination of TCA has not been reported until now. We describe the isolation of a bacterium capable of energy conservation for growth through the reductive dechlorination of TCA.

Isolation was initiated from a sediment microcosm that reductively dechlorinated TCA (16). Single colonies were subcultured and dechlorination activity was maintained in deep agarose shake cultures until a pure culture was obtained. The isolate is designated strain TCA1 and is a motile, short rod with a diameter of 0.4 to 0.6 μm and a length of 1.0 to 2.0 μm (Fig. 1). Cells stain gram-negative. No spores were observed in starved cultures. Although strain TCA1 did not grow or dechlorinate in the presence of oxygen, exposure of the active culture to aerobic conditions for up to 3 days did not result in the loss of dechlorinating activity (16). Dechlorination of TCA was sequential with the accumulation of 1,1-dichloroethane (DCA) before conversion to chloroethane (CA) (Fig. 2). Dechlorination of TCA occurred at a faster rate than that of DCA. The temperature range for reductive dechlorination was from 12° to 30°C with the optimum at 25°C. No dechlorination occurred at 37°C. Growth yield from reductive dechlorination was 5.60 ± 1.26 g (dry weight) (mean ± SD, n = 3 cultures) of cells per mole of chloride released. TCA, H2, and acetate were essential for growth of strain TCA1. Formate could replace H2 as an electron donor, which resulted in a similar dechlorination rate. Acetate alone did not support TCA dechlorination, suggesting that it was used only as a carbon source and not as an electron donor for reductive dechlorination. Because H2 oxidation does not support substrate-level phosphorylation, growth in a defined medium with TCA as the electron acceptor and H2 as the electron donor indicated that strain TCA1 conserves energy in a respiratory process.

Figure 1

Scanning electron micrograph of strain TCA1. The rod-shaped morphology and dividing cells are shown. Scale bar, 1 μm.

Figure 2

Stoichiometry of the dechlorination of TCA to DCA and CA by strain TCA1. Samples were incubated at 25°C and analyzed every 3 days over a 2-month period for depletion of TCA and production of DCA and CA. Error bars (shown if larger than the symbols) represent standard deviations of triplicate cultures.

Strain TCA1 did not dechlorinate 1,1,1,2-tetrachloroethane, 1,1,2-trichloroethane, 1,2-dichloroethane, 1,2-dichloropropane, PCE, or TCE when they were added as potential electron acceptors. No growth occurred in liquid media amended with 4 mM H2 as an electron donor, 5 mM acetate as a carbon source, and either 5 mM sulfate, sulfite, thiosulfate, nitrate, or fumarate as an electron acceptor. Strain TCA1 did not use lactate, pyruvate, propionate, fumarate, butyrate, benzoate, phenol, glucose, ethanol, or methanol as electron donors. No fermentative growth was observed. Sulfite or thiosulfate at 5 mM concentration completely inhibited TCA dechlorination, whereas 5 mM sulfate, nitrate, or fumarate had no effect. Because these compounds did not serve as electron acceptors for strain TCA1, inhibition was not due to competition for electron donors. Instead, the reactive sulfur oxyanions may directly inhibit the components of the electron transport chain or even the reductive dehalogenase, as observed in Desulfomonile tiedjei(17).

Comparative analysis of the nearly full-length 16S ribosomal DNA (rDNA) sequence of strain TCA1 and available 16S rDNA sequences revealed that strain TCA1 is related to gram-positive bacteria with low G+C content in their DNA. Strain TCA1,Dehalobacter restrictus (18, 19), and three clones from trichlorobenzene- and 1,2-dichloropropane–dechlorinating consortia (20,21) form a phylogenetic cluster (Fig. 3) defined by 16S rDNA sequence similarities of 97% and higher. The closest relative of strain TCA1 is D. restrictus strain TEA, with a sequence similarity of 99%. D. restrictus strains PER-K23 and TEA are strict anaerobes capable of coupling PCE and TCE dechlorination to H2 oxidation for growth in a respiratory process. However, we did not detect TCA dechlorination by strain PER-K23. These bacterial strains seem to be obligate H2-oxidizing dechlorinators that only grow by reductive dechlorination of specific chlorinated ethanes or ethenes in anaerobic respiration. The physiology, morphology, and 16S rDNA sequence of strain TCA1 suggest that it is a Dehalobacter and perhaps represents a new species based on its unique features of TCA dechlorination and formate oxidation. The isolation of strain TCA1 further suggests an important role for Dehalobacter species in polluted anoxic environments.

Figure 3

16SrDNA–based phylogenetic tree of strain TCA1 and representative bacteria. The sequence of strain TCA1 was aligned against the most similar sequence in the Ribosomal Database Project (25) and GenBank using the automated aligner of the ARB software package (26). The alignment was corrected manually based on both primary and secondary structural considerations. The tree was generated by a maximum-likelihood method with the program fastDNAml (27), with 1375 nucleotide positions unambiguously aligned. Values in each node are the percentage of 100 bootstrap trees. Scale bar, 0.1 substitutions per nucleotide position. T, type strain.

To determine the potential of strain TCA1 to attenuate TCA in the natural environment, we bioaugmented anoxic aquifer sediments from Bachman and Schoolcraft (both in Michigan, USA) contaminant plumes. Both sites are contaminated with PCE and daughter products, and the Schoolcraft plume G site is also contaminated with TCA, DCA, and chromium. TCA was completely converted to CA within 2 months in both aquifer sediment samples amended with strain TCA1, whereas no dechlorination was observed in samples without the inoculum. These results suggest that bioaugmentation with strain TCA1 could ensure and speed the degradation of TCA, especially if naturally occurring populations are patchy or absent.

CA, rather than ethane, appears to be the terminal TCA product from our culture, and studies have shown that both DCA and CA can be degraded under aerobic conditions (22, 23). Because the aerobic transformation of DCA is much slower than that of CA (24), complete conversion to CA would result in more reliable removal of chloroethanes on the aerobic fringes of a plume.

The discovery of an anaerobic dehalorespiring Dehalobacterthat couples reductive dechlorination of TCA to growth not only may lead to a better understanding of the physiology, phylogeny, and biochemistry of dehalorespiring bacteria, but also suggests a strategy for bioremediation of TCA in soils and ground water, thereby aiding in the attenuation of this ozone-depleting compound.

Supporting Online Material

www.sciencemag.org/cgi/content/full/298/5595/1023/DC1

Materials and Methods

  • * To whom correspondence should be addressed. E-mail: tiedjej{at}msu.edu

REFERENCES AND NOTES

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