INTRODUCTION

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Trichloroacetic acid (TCA) is an industrial chemical that is found in nature mainly through its use as a herbicide against perennial weeds. Decomposition of TCA in soil seems to be slow (9) and is mainly microbially mediated. However, little is known about the microorganisms and processes involved. In the experiments of Hirsch and Alexander (7) a Pseudomonas isolate, actively degrading 2,2-dichloropropionic acid, also showed activity on TCA. Jensen (8) and Lode (10) reported on Pseudomonas, Arthrobacter, and Agrobacterium species and on an Arthrobacter strain, respectively, that were all able to split off chlorine from TCA in a dissimilatory process. In a recent study by Yu and Welander (19), the first aerobic bacterium able to grow on TCA as the sole carbon and energy source was characterized. The isolate belonged to the  subgroup of proteobacteria and could not grow on mono- and dichloroacetic acid (MCA and DCA). Although aerobic TCA decomposition has been recognized for a few decades, its anaerobic degradation has not been demonstrated. We now report on the isolation of a bacterium capable of anaerobic growth via reductive dehalogenation of TCA, and we propose that a sulfur-sulfide redox cycle is involved in this process.

	MATERIALS AND METHODS

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Isolation and maintenance of strain K1. Anaerobic medium was prepared under oxygen-free N₂-CO₂ (80:20) as described by Cole et al. (3) and consisted of a basal salts medium amended with vitamins and a Na₂SeO₃-Na₂WO₄ solution (3). As a reductant, 1 mM Na₂S was added to the medium after it had been boiled and cooled to room temperature. The pH was adjusted to 7.2 by varying the flow of CO₂. Except for acetate, which was added before boiling the medium, electron donors, electron acceptors, and vitamins were added from sterile anoxic stock solutions after autoclaving.

Identification of strain K1. Strain K1 was selected for further study. To confirm its purity, 1% inocula were transferred to fresh anaerobic medium with different electron donor-electron acceptor combinations that supported growth of K1. These were acetate-sulfur, acetate-TCA, or acetate-fumarate (see Results). After growth at 25°C, cells were harvested, washed twice with 1 M NaCl, and resuspended in 50 µl of MilliQ water. Aliquots were then subjected to BOX-PCR according to the method of Rademaker et al. (15), and the genomic fingerprints were compared.

Substrate use by strain K1. To evaluate the electron donor and electron acceptor specificity of strain K1, duplicate cultures were grown in 160-ml serum bottles containing 100 ml of anaerobic medium. All cultures were inoculated with a 1% transfer from a liquid culture grown on acetate-TCA. Uninoculated controls and references without electron donors or acceptors were included, and incubations were performed quiescently at 25°C. Different substrates were tested for their potential to support 1 mM TCA dechlorination by strain K1. The following compounds were selected: acetoin (10 mM), glycerol (10 mM), ethylene glycol (10 mM), ethanol (10 mM), butyrate (1 mM), propionate (1 mM), acetate (1 and 2.5 mM), formate (5 mM), lactate (10 mM), thiosulfate (5 mM), ferric citrate (5 mM), and H₂ (10% in the headspace). At regular time intervals, samples were taken and centrifuged, and the supernatant was monitored for TCA disappearance by HPLC analysis.

TCA dechlorination by strain K1. To determine whether the enzymes necessary for TCA dechlorination are constitutively expressed or induced by TCA, an induction experiment was performed. Cultures were grown in parallel on acetate-sulfur and on acetate-TCA-sulfur media. After conversion of TCA to DCA in the latter culture, both sets were fed 0.5 or 1 mM TCA. They were sampled over several hours, and the culture supernatants were monitored for TCA dechlorination.

Analytical methods. The disappearance of TCA and other haloalkanoates was monitored by HPLC analysis (Hewlett-Packard HPLC 1050; Microsorb MV Amino column [25 cm]; 60:40 25 mM phosphate buffer [pH 3.8]-acetonitrile eluents; flow rate, 1 ml · min¹; UV detection at 210 nm). Trichloroethane and trichloroethene were measured in headspace samples by gas chromatography (GC) (11), and nonhalogenated organic acids were measured by HPLC (12). Sample volumes for GC and HPLC analysis were 200 and 50 µl, respectively. HPLC analyses were performed on the supernatants of centrifuged culture samples (5 min, 10,000 × g).

	RESULTS

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Isolation and identification of TCA-degrading organisms. As anaerobic enrichments of soil samples were repeatedly transferred to fresh medium, soil-free cultures were obtained with stable TCA-degrading properties. Serial 1:10 dilutions to extinction finally yielded pure cultures which consisted of strictly anaerobic, gram-negative short curved nonmotile rods. Although the liquid cultures never appeared turbid, cells were present in high densities. Tenfold dilutions from a 100-ml culture into balchtubes containing 9 ml of acetate-TCA amended medium still showed TCA dechlorination at dilutions up to 10⁸. Vitamins were not required for growth on acetate-TCA in the basal salts medium, and no growth was ever detected on complex media, on solid media, and in aerobic conditions.

View larger version (49K): [in this window] [in a new window]   FIG. 1.   Phylogenetic tree based on the 16S rRNA sequences of strain K1 (T. thiogenes) and representative bacteria.

Substrate use by strain K1. Strain K1 was isolated on 2.5 mM acetate-1 mM TCA amended anaerobic medium. As alternative electron donors, substrates were selected which supported the growth of related Pelobacter species. In the experimental conditions used, only acetate and acetoin amendment led to TCA disappearance, and TCA dechlorination always stopped at the level of DCA. As for alternative electron acceptors, none of the halogenated compounds other than TCA was transformed by strain K1 in the presence of acetate as an electron donor. However, acetate consumption was observed in the presence of sulfur or fumarate as the electron acceptor. In the latter case, succinate was detected as the product of fumarate reduction.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Transformations during growth of strain K1 in basal salts medium with 2.5 mM acetate and 1 mM TCA. Symbols: , acetate; , TCA; , DCA.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Dechlorination of subsequent 1 mM TCA pulses by strain K1 in a chloride-free medium. Symbols: , TCA additions; , TCA; , DCA; , MCA; ×, chloride.

TCA dechlorination by strain K1. From resting cell assays, it was clear that filtered supernatant of spent medium did not show any TCA dechlorination activity. Reduction of TCA required cells and an electron donor, in casu acetate. However, TCA dechlorination was faster when cells were resuspended in supernatant than when resuspended in fresh medium. In the cell-free controls, no TCA disappearance was observed.

View larger version (62K): [in this window] [in a new window]   FIG. 4.   Microscopic image of strain K1 grown in basal salts medium with acetate and TCA after conversion of the first TCA pulse (left) and 1 h after the addition of a second TCA pulse (right). The refractile spheres are sulfur granules.

View larger version (20K): [in this window] [in a new window]   FIG. 5.   Concentration profiles after the addition of a second 1 mM TCA pulse to basal salts medium amended with acetate. Sulfide was the reducing agent in the medium.

	DISCUSSION

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The ability of TCA to support the growth of microorganisms had been questioned until Yu and Welander (19) demonstrated the existence of aerobic bacteria capable of using TCA as the sole source of carbon and energy. In contrast, TCA degradation in anaerobic conditions has not yet been described. Only one report was found which mentions TCA disappearance in a reactor with an immobilized methanogenic mixed culture, but the authors ascribe TCA transformation to a combination of chemical and biological reactions (4). The isolation of a bacterium able to grow via reductive TCA dechlorination clearly establishes the existence of an anaerobic TCA removal process. Anaerobic soil enrichments on acetate-TCA yielded two similar isolates, of which strain K1 was further characterized.

The new isolate appears to gain energy from the dechlorination reaction. Numerous 1% serial transfers into anaerobic medium amended with acetate and TCA consistently led to TCA disappearance and DCA production, along with an increase in cell number to densities of at least 10⁸ · ml¹. By varying the molar ratio of acetate to TCA in the medium, the stoichiometry of TCA was indicated to be as follows: CH₃COOH + 4 CCl₃COOH + 2 H₂O = 2 CO₂ + 4 HCl + 4 CHCl₂COOH. The growth of strain K1 in chloride-free medium and the measurement of chloride release confirmed the proposed stoichiometry (Fig. 3). Why chloride concentrations decreased after conversion of the first TCA pulse, is unclear, but is probably attributable to experimental error. In any case, four independent experiments demonstrated the presence of equimolar amounts of DCA and chloride, which strongly suggests that TCA dechlorination stops at the level of DCA. In one instance only, a gradual MCA accumulation was evidenced by HPLC and GC-MS analysis. Although MCA production could never be reproduced, strain K1 may have the capability to dechlorinate TCA to MCA under certain conditions. Further research will be necessary to elucidate which factors are responsible for a limited dehalogenation and/or which factors can trigger a twofold dechlorination. In further support of this suggestion, we observed that in some soil microcosms TCA was converted to DCA when acetate was provided as an electron donor, while TCA was completely dehalogenated in the presence of other electron donors. This was, however, not the case with strain K1. Whether TCA dechlorination proceeds to DCA or MCA does not alter the fact that strain K1 most probably gains energy by using TCA as a respiratory electron acceptor.

In addition to the fact that it is an unusual and constitutive reaction, TCA dehalogenation also seems to have high substrate specificity. Indeed, none of the alternative halogenated compounds tested was transformed by strain K1. Only elemental sulfur or fumarate could serve as electron acceptor for acetate oxidation. The results for ferric iron reduction were not consistent. Therefore, it appears that strain K1 has an extremely narrow electron donor and acceptor specificity. Its ability to use sulfur as an electron acceptor is not surprising in view of its phylogeny. Based on its 16S rRNA sequence, it is placed among a group of microorganisms in the  subdivision of the proteobacteria, which are known ferric iron or elemental sulfur reducers.

In strain K1, sulfur reduction seems to be connected to TCA dechlorination in a novel redox cycle. In all experiments, respiking of TCA to acetate-TCA-grown cultures induced the transient formation of a precipitate in the medium. This phenomenon apparently depended on the physiological state of the microorganisms and on the medium composition. No precipitation was seen when the time lag between TCA depletion and readdition was too long. Neither did the medium turn turbid upon a second TCA addition, when the reductant sulfide was replaced by L-cysteine. Stoichiometry experiments confirmed that TCA respiking led to the temporary oxidation of sulfide to sulfur followed by its reduction to sulfide at the time TCAA was nearly exhausted (Fig. 5).

To explain these experimental observations, we hypothesize that a sulfur-sulfide redox cycle is involved in TCA conversion. As visualized in Fig. 6, electrons are transferred in a first step from acetate to sulfur, yielding sulfide, CO₂, and energy. The sulfide-reducing equivalents are then used in TCA dehalogenation to DCA, thereby closing the sulfur-sulfide cycle. The observed temporary appearance of sulfur in the medium implies that the TCA-sulfide reaction is faster than the sulfur-acetate reaction. The following observations are all in accordance with the proposed mechanism. Strain K1 is a sulfur reducer and oxidizes acetate in an acetate-sulfur medium, either in the absence or presence of sulfide. When incubated with acetate and sulfide, no sulfur production or acetate consumption takes place, but when strain K1 was incubated with sulfide and TCA, partial TCAA dechlorination was noticed. Although it is not yet known how electrons are being transferred from sulfide to TCA, evidence suggests that a cell-associated catalyst and a reductant (acetate) are required. Resting-cell experiments clearly showed that the process of TCA dechlorination required actively metabolizing cells, since cells in the buffer did not mediate the reaction. The fact that the dechlorination rate was higher when cells were returned to spent medium than when resuspended in fresh medium may implicate that a secreted electron shuttle is also involved.

View larger version (7K): [in this window] [in a new window]   FIG. 6.   Proposed sulfur-sulfide redox cycle as electron transmitter between acetate and TCA.

Other evidence suggests that the sulfur-sulfide cycle is not essential nor exclusive for TCA dechlorination. First, the sulfur-sulfide redox cycle was also found to mediate electron transfer from acetate to fumarate, since respiking of fumarate also led to sulfur precipitation. Second, parallel incubations of strain K1 on acetate-TCA in a medium with either sulfide or cystein as a reductant, showed a similar TCA dechlorination behavior. Up to nine TCA additions were dehalogenated at the same rate, but transient turbidity due to sulfur was evident only in the sulfide-amended medium. Subculturing in cysteine-reduced medium did not affect dechlorinating activity, and in the absence of acetate no TCA conversion took place. Hence, cysteine probably had a similar electron shuttling function as proposed for sulfide. In spite of the fact that the sulfur-sulfide cycle is not essential for TCA dechlorination, it remains to be investigated how widespread this type of redox cycle is, particularly among sulfur-reducing bacteria, since they may present an as-yet-unknown potential for dechlorination activity in nature.

In summary, we have isolated a novel bacterium growing via reductive dehalogenation of TCA and also capable of sulfur reduction. Its most interesting feature is the involvement of a sulfur-sulfide redox cycle in TCA dehalogenation. The difference in 16S rRNA sequence from the closest strains in the phylogenetic tree (6%) and its unique physiological characteristics demonstrate that strain K1 represents a new genus. The name Trichlorobacter thiogenes is proposed, which emphasizes its capability to reductively dechlorinate TCA and its sulfur cycling activity.

Description of Trichlorobacter gen. nov. Trichlorobacter (Tri.chlor.o.bac'ter) L. num. tria three; Gr. n. chloros chlorine; M.L. masc. n. bacter the equivalent of Gr. neut. n. bactrum a rod, M. L. masc. n. Trichlorobacter because it is a TCA-dechlorinating rod.

Description of T. thiogenes sp. nov. T. thiogenes (thi.og'en.es) Gr. n. theion sulfur; M. L. n. genes production; M. L. n. thiogenes because it produces sulfur. T. thiogenes is a strictly anaerobic bacterium with rod-shaped cells. Cells are gram negative, short, curved and nonmotile. T. thiogenes grows by oxidizing acetate and acetoin with the concomitant reduction of TCA. Other halogenated compounds are not used as an electron acceptor. T. thiogenes can also conserve energy for growth by the reduction of sulfur and fumarate. It does not grow on complex and solidified media.