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Isolation of a Bacterium That Reductively Dechlorinates Tetrachloroethene to Ethene

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Science  06 Jun 1997:
Vol. 276, Issue 5318, pp. 1568-1571
DOI: 10.1126/science.276.5318.1568

Abstract

Tetrachloroethene is a prominent groundwater pollutant that can be reductively dechlorinated by mixed anaerobic microbial populations to the nontoxic product ethene. Strain 195, a coccoid bacterium that dechlorinates tetrachloroethene to ethene, was isolated and characterized. Growth of strain 195 with H2 and tetrachloroethene as the electron donor and acceptor pair required extracts from mixed microbial cultures. Growth of strain 195 was resistant to ampicillin and vancomycin; its cell wall did not react with a peptidoglycan-specific lectin and its ultrastructure resembled S-layers of Archaea. Analysis of the 16S ribosomal DNA sequence of strain 195 indicated that it is a eubacterium without close affiliation to any known groups.

The solvent tetrachloroethene [perchloroethylene (PCE)] is a common groundwater pollutant (1,2) that is highly toxic and is suspected to be a human carcinogen. It is nonbiodegradable by aerobes but can be reductively dechlorinated by natural microbial communities and mixed microbial enrichment cultures under anaerobic conditions according to the reaction sequence shown in Fig. 1 (3). The formation of nontoxic products such as ethene (ETH) (4) and ethane (5) indicates the potential for complete anaerobic detoxification of chloroethenes in situ.

Figure 1

Reductive dechlorination of chloroethenes. TCE, trichloroethene; DCEs, dichloroethene isomers (represented bycis-DCE); 2H, electron pair derived from the electron donor.

Slow reductive dechlorination of chloroethenes and other haloorganic compounds can be carried out by a cometabolic process by organisms rich in reduced transition-metal cofactors such as methanogens and acetogens (6, 7). However, some organisms can use haloorganic compounds as electron acceptors for energy conservation and growth (sometimes called dehalorespiration) (8), and several anaerobes that grow by dechlorinating PCE partially tocis-dichloroethene (cis-DCE) have been described recently (9).

We have studied a set of enrichment cultures that dechlorinate PCE to ETH, using methanol, H2, or butyrate as electron donors (10-13). A partially purified culture was derived from a 10−6 dilution of a methanol-PCE culture inoculated into medium with H2 and PCE (14). This culture contained no methanogens or acetogens and could be transferred indefinitely into H2-PCE medium supplemented with a mixture called ABSS [2 mM acetate, 0.05 mg of vitamin B12 per liter, and 25% (v/v) anaerobic digester sludge supernatant], but it could not be transferred if either H2 or PCE was omitted. The amount of H2 consumed was stoichiometric to the chlorine eliminated through reductive dechlorination of PCE. The two main morphotypes in this culture were an irregular coccus and a short rod.

PCE dechlorination with H2 as the electron donor was resistant to vancomycin (100 mg/liter), an inhibitor of eubacterial peptidoglycan cell wall synthesis (11). Therefore, we transferred the partially purified H2-PCE culture [2% (v/v) inoculum] to medium containing vancomycin (100 mg/liter) or ampicillin (another peptidoglycan-synthesis inhibitor) at up to 3 g/liter. PCE was dechlorinated and only the coccoid morphotype was present under these conditions (15). No PCE dechlorination was detected in cultures transferred to medium containing tetracycline (a eubacterial protein-synthesis inhibitor) at 20 mg/liter.

Ampicillin- or vancomycin-treated cultures could not be transferred a second time into antibiotic-containing medium. On the basis of previous results (11), it was likely that other organisms present in the culture were producing a nutrient or nutrients required by the PCE dechlorinator and not present in ABSS. Therefore, we amended the ABSS medium of second-generation ampicillin-treated cultures with either the filter-sterilized supernatant or sonicated pellet fractions of mixed cultures (Table 1). Both culture supplementations stimulated dechlorination, with the pellet fraction extract being more stimulatory. Moreover, only cultures amended with the pellet extract could be transferred again. These results indicate that one or more cell components of contaminating organisms were responsible for the stimulation.

Table 1

Effect of nutrient addition on product formation from PCE by cultures transferred a second time into ABSS-supplemented medium containing ampicillin (0.3 g/liter). Products were measured 23 days after transfer. Values less than 1 μmol/liter are denoted as 0.

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Other potential nutrient amendments were examined for their effect on dechlorination (Table 1). Cholesterol and horse serum, two nutrients that are required by many mycoplasmas (eubacteria lacking a cell wall), did not stimulate dechlorination, whereas yeast extract and a mixture of volatile fatty acids, which are required by certain anaerobes, had a slight stimulatory effect. Casamino acids (Difco Laboratories, Detroit, Michigan) had a stimulatory effect similar to that produced by the supernatant fraction of mixed cultures. Nevertheless, none of these amendments allowed further transfer. Growth stimulation was not conferred by cell extracts of the Gram-negative aerobic eubacteriumEscherichia coli or of the Gram-positive anaerobic eubacterium Clostridium pasteurianum strain W5, which indicates that the growth factor is not ubiquitous in bacteria.

Using a growth medium supplemented with filter-sterilized extract from mixed H2-PCE cultures and ABSS (14), we isolated the PCE-dechlorinating organism (strain 195) by a 10−7dilution to liquid H2-PCE medium containing ampicillin (0.3 g/liter). These cultures, when transferred several times in the absence of ampicillin, showed no morphotypes other than irregular cocci. No visible growth was detected by tests for contamination (with a sensitivity of about 10 organisms per milliliter) with basal growth medium amended with lactate, sulfate, or thiosulfate to detect sulfate reducers, yeast extract (0.2 g/liter) to detect fermentative heterotrophs, or Brewer’s thioglycollate medium (Difco) to detect fermentative heterotrophs.

Growth of strain 195 on H2 and PCE was measured by direct microscopic cell counts and cell protein during metabolism of PCE to vinyl chloride (VC) and ETH (Fig. 2, A and B). Cultures continued to grow until day 5, with a doubling time of about 19.2 hours. After day 5, growth ceased but PCE dechlorination continued, which suggests uncoupling of growth and dechlorination. Cultures receiving H2 but not PCE showed only slight growth, and PCE dechlorination products were not detected in uninoculated cultures. The amount of VC and ETH produced represented more than 90% of the PCE added to inoculated cultures. The protein yield for days 1 through 5 was 4.8 ± 0.3 g of protein per mole of chloride released; a specific activity of 69.0 ± 10.5 nmol of chloride released per minute per milligram of protein was determined.

Figure 2

(A) Growth of strain 195 on H2-PCE as measured by particulate protein or cell counts. Growth medium was supplemented with ABSS and 5% (v/v) extract from a mixed culture. Protein was quantified with the NanoOrange kit (Molecular Probes, Eugene, OR); cells were counted in a Petroff Hauser counting chamber with the use of an epifluorescence microscope after staining with acridine orange (4 mg/liter, final concentration). (B) PCE utilization and product formation by the same cultures as in (A). Data points represent means ± SD, unless values are smaller than the symbols, from triplicate tubes. (C) Conversion of PCE to ETH (about 120 μmol/liter) by a pure culture of strain 195. The culture received several doses of PCE followed by flushing with N2 and CO2, which removed all ETHs except for about 50 μmol of VC per liter. PCE was underestimated in early data points because of its slow dissolution into the aqueous phase (13).

Analysis of the conversion of PCE to ETH by a culture of strain 195 that had received five previous doses of PCE showed that PCE was metabolized to VC at a rate of 40 μmol per hour per liter of culture medium, with little buildup of intermediates (Fig. 2C). VC dechlorination to ETH commenced after PCE depletion and could be fit by first-order kinetics with a half-life of about 80 hours for the first 300 hours and of about 150 hours thereafter. This indicated a decay with time in the ability of the culture to metabolize VC. These results resemble those for the mixed methanol-PCE culture from which it was derived (13), except that the mixed culture dechlorinated VC more rapidly relative to PCE. If strain 195 is responsible for VC dechlorination in those mixed cultures, then some factor, perhaps nutritional, limits the rate of VC dechlorination in the pure culture. It is also possible that there is another organism or strain present in the mixed culture that is capable of more rapid VC metabolism.

Physiological characterization of strain 195 revealed that it required H2 for PCE reduction and that it grew only when both H2 and PCE were present (Fig. 2). Potential electron donors that supported neither PCE dechlorination, nor growth in the absence of PCE, were methanol, pyruvate, lactate, ethanol, formate, glucose, and yeast extract. Potential electron acceptors that did not support growth or were not reduced when H2 was provided as the electron donor included sulfate, sulfite, thiosulfate, nitrate, nitrite, fumarate, and oxygen (2 or 21%). The culture could reductively dechlorinate 1,2-dichloroethane and 1,2-dibromoethane to ETH, as did the original enrichment culture (13).

Electron microscopic examination (16) of strain 195 (Fig. 3, A and B) revealed small, irregular coccoid cells with an unusual cell wall ultrastructure that resembled the S-layer protein subunit type of cell walls found in many Archaea (17). To test for the presence of a peptidoglycan cell wall, we used fluorescently labeled wheat germ agglutinin, which specifically binds to N -acetylglucosamine and N -acetylneuraminic acid (18). This stain bound to whole cells of the Gram-positive eubacterium C. pasteurianumWF (15) and to cell wall preparations of the Gram-negative eubacterium E. coli DH5α (Fig. 3, C and D) (18). No binding was detected for whole cells (15) or for cell wall preparations (Fig. 3, E and F) of strain 195.

Figure 3

Thin-section electron micrographs of coccoid (A) and flattened (B) cells of strain 195 stained with uranyl acetate. Scale bar, 0.2 μm. Phase-contrast (C) and epifluorescence (D) micrographs of cell wall preparations of E. coli DH5α stained with fluorescein-labeled wheat germ agglutinin (100 mg/liter) (Molecular Probes) (18). Phase-contrast (E) and epifluorescence (F) micrographs of cell wall preparations of strain 195 stained with wheat germ agglutinin. Cell wall samples for (C) through (F) were prepared by lysing the cells in boiling 4% SDS in 25 mM phosphate buffer (pH 7) (20) followed by heat fixation to a microscope slide and washing with distilled water to remove SDS and other chemicals before staining. Scale bar in (C) [for (C) through (F)], 5 μm.

The phylogenetic position of strain 195 was determined on the basis of its 16S ribosomal DNA sequence (Fig.4). The PCE dechlorinator grouped within the eubacteria in all analyses but did not cluster within any of the known phylogenetic lines. Although the maximum likelihood analysis presented places strain 195 on a branch that includes cyanobacteria and planctomycetes, DNA distance analyses placed it closer toClostridium butyricum and its relatives (19) but with little affiliation for other members of the Gram-positive branch. Thus, its relationship with the presently described eubacterial branches is unclear at this time. It is clearly distinct from other recent isolates that reduce PCE to cis-DCE (9), which are affiliated with the ɛ and γ branches of the Proteobacteria or with the Gram-positive sulfate-reducing bacteria. Because strain 195 does not appear to belong to any presently known genus or species, we suggest naming it Dehalococcoides ethenogenes strain 195, pending a more thorough taxonomic description.

Figure 4

Unrooted phylogenetic tree generated for the 16S ribosomal DNA sequence (GenBank database number AF004928) from strain 195 with the use of the SUGGEST TREE maximum-likelihood program provided by the Ribosome Database Project (RDP) (21). DNA was extracted from strain 195 as described (22). The sequence was amplified as a polymerase chain reaction product with the use of primers 27f and 1522r under standard conditions (23), followed by cloning with the Invitrogen (San Diego, California) TA cloning kit and sequencing with an Applied Biosystems model 373 analyzer operated by the Cornell Biotechnology Institute. Eight sequencing primers were used (23), including two against the vector, resulting in only a single ambiguous base in the entire sequence. For simplicity, some organisms included in the original analyses have been deleted from the figure. Other analyses of these sequences were performed by manually aligning the sequence of strain 195 to other prealigned sequences from the RDP, followed by the use of the PHYLIP 3.5c package (24), including DNAML (maximum-likelihood analysis), and DNADIST (Kimura model) coupled to either FITCH or NEIGHBOR.

In summary, we isolated an organism that is capable of respiratory reductive dechlorination of PCE completely to ETH with H2as an electron donor. Previous isolates reduce PCE only as far ascis-DCE. It is of interest that at many PCE-contaminated sites, dechlorination proceeds only as far as cis-DCE, whereas at other sites VC and ETH are produced (3). It is not clear whether incomplete dechlorination at a given site is due to suboptimal physiochemical conditions, deficiencies in electron donors or nutrients present, or a lack of appropriate organisms.

  • * To whom correspondence should be addressed. E-mail: shz1{at}cornell.edu

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