O R I G I N a L a R T I C L E Open Access

The contaminant concentrations over which type strains of the species Dehalogenimonas alkenigignens and Dehalogenimonas lykanthroporepellens were able to reductively dechlorinate 1,2-dichloroethane (1,2-DCA), 1,2-dichloropropane (1,2-DCP), and 1,1,2-trichloroethane (1,1,2-TCA) were evaluated. Although initially isolated from an environment with much lower halogenated solvent concentrations, D. alkenigignens IP3-3 T was found to reductively dehalogenate chlorinated alkanes at concentrations comparable to D. lykanthroporepellens BL-DC-9 T. Both species dechlorinated 1,2-DCA, 1,2-DCP, and 1,1,2-TCA present at initial concentrations at least as high as 8.7, 4.0, and 3.5 mM, respectively. The ability of Dehalogenimonas spp. to carry out anaerobic reductive dechlorination even in the presence of high concentrations of chlorinated aliphatic alkanes has important implications for remediation of contaminated soil and groundwater.


Introduction
In industry, polychlorinated ethanes and propanes are used as solvents, degreasing agents, and paint removers; they are also globally produced on a massive scale as intermediates during production of other industrially important chemicals Field and Sierra-Alvarez, 2004). Due to spills and past disposal methods, these chlorinated organic compounds are prevalent groundwater and soil contaminants. For example, 1,2-dichloroethane (1,2-DCA) is present in at least 570 current or former Superfund sites (ATSDR 2001), and 1,2-dichloropropane (1,2-DCP) is present at more than 100 Superfund sites (Fletcher et al., 2009). The prevalence of these polychlorinated alkanes as environmental contaminants is of concern because of their known or suspected toxicity and/or carcinogenicity (ATSDR, 2001;1989).
Previously reported studies of Dehalogenimonas strains were conducted only at initial chlorinated solvent concentrations of 0.5 mM (Bowman et al., 2012;Yan et al., 2009a). Research reported here was aimed at evaluating the solvent concentration ranges over which D. lykanthroporepellens and D. alkenigignens can reductively dechlorinate 1,2-DCA, 1,2-DCP, and 1,1,2-TCA in order to assess their suitability for bioremediation of high contaminant concentrations.
Each serum bottle received 0.3 mL inoculum (3% v/v) of D. alkenigignens strain IP3-3 T (=JCM 17062 T =NRRL B-59545 T ) or D. lykanthroporepellens strain BL-DC-9 T (=JCM 15061 T = ATCC BAA-1523 T ) previously grown on 1,2-DCP. Incubation was in the dark at 30 o C without shaking. Triplicate bottles were sacrificed at time zero and after eight weeks incubation for analysis of chlorinated solvents and potential degradation products. To account for potential abiotic reactions, triplicate negative controls prepared in the same manner as inoculated bottles but without bacterial addition were incubated under identical conditions. Chlorinated solvents and degradation products were measured using an HP model 6890 gas chromatograph (GC) equipped with a flame ionization detector and GS-GasPro capillary column (60 m × 0.32 mm I.D., J&W P/N 113-4362) as described previously (Yan et al., 2009a). Gas headspace samples collected in 100 μL gastight glass syringes (Hamilton, Baton Rouge, LA) were introduced to the GC via direct injection. Aqueous samples (500 μL) were introduced to the GC via a Tekmar 2016/3000 purge and trap autosampler and concentrator. Both gas-headspace and aqueous-phase aliquots were analyzed for each sample bottle.
Hydrogen concentrations in the gas headspace were measured using an SRI Instruments model 310 gas chromatograph (Torrence, CA) equipped with a thermal conductivity detector and molecular sieve column (Alltech Molesieve 5A 80/100) as described previously (van Ginkel et al., 2001).

Results
The quantity of the dechlorination product determined at the end of the eight week incubation period as a function of initial aqueous-phase 1,2-DCA, 1,-DCP, and 1,1,2-TCA is shown in Figures 1, 2 and 3 respectively.
The production of ethene ( Figure 1) coupled with 1,2-DCA disappearance in the inoculated bottles is consistent with the 1,2-DCA dihaloelimination degradation pathway reported previously for D. alkenigignens IP3-3 T and D. lykantroporepellens BL-DC-9 T in tests conducted with initial 1,2-DCA concentrations of 0.5 mM in serum bottles containing H 2 at an initial concentration of 10% v/v (as opposed to the 80% v/v employed in the present study) (Bowman et al., 2012;Yan et al., 2009a). Trace levels of 1-chloroethane (<0.3 μmol/bottle) were detected at comparable levels in both inoculated bottles and in uninoculated abiotic controls (data not shown) and small amounts of ethene (<0.7 μmol/bottle) were detected in abiotic negative controls ( Figure 1) indicating that some abiotic 1,2-DCA transformation occurred in the anaerobic medium employed here, but the amount was negligible. The sum of parent compound (i.e., 1,2-DCA) plus daughter product (i.e., ethene and 1-chloroethane) in replicate serum bottles inoculated with the bacterial strains ranged from 74-107% of the mass determined in abiotic negative controls (average 89%). Dechlorination was essentially complete (< 1% of the starting 1,2-DCA remaining) at the end of the eight week incubation period for serum bottles supplemented with 1,2-DCA at initial concentrations less than 3.16 ± 0.05 mM and 1.48 ± 0.03 mM (mean ± standard deviation) for D. alkenigignens IP3-3 T and D. lykanthroporepellens BL-DC-9 T , respectively (Figure 1). At higher initial 1,2-DCA concentrations (at and to the right of concentrations denoted by arrows in Figure 1), untransformed 1,2-DCA remained at the end of the eight week incubation in amounts increasing with increasing initial 1,2-DCA concentration.
The quantity of ethene observed increased with increasing initial 1,2-DCA concentration in the range of 0.5 to approximately 4 mM (maximum ethene observed in bottles containing initial 1,2-DCA concentrations of 4.03 ± 0.09 and 4.08 ± 0.16 mM for D. alkenigignens IP3-3 T and D. lykanthroporepellens BL-DC-9 T , respectively) and then decreased at higher initial 1,2-DCA concentrations. The decrease in ethene production as 1,2-DCA concentrations increased indicates that sufficiently high 1,2-DCA concentrations can inhibit dechlorination activity of both Dehalogenimonas spp. Biologically mediated 1,2-DCA reductive dechlorination, however, was observed in serum bottles with initial 1,2-DCA concentrations as high as 9.81 ± 0.98 and 8.69 ± 0.26 mM for D. alkenigignens IP3-3 T and D. lykanthroporepellens BL-DC-9 T , respectively. At higher initial 1,2-DCA concentrations, small amounts of ethene were also detected, but in amounts that were not statistically different from abiotic negative controls at a confidence level of 95%.
The production of propene ( Figure 2) coupled with 1,2-DCP dechlorination in the inoculated bottles is consistent with the previously reported tests conducted with 0.5 mM 1,2-DCP in serum bottles with 10% v/v H 2 in the gas headspace (Bowman et al., 2012;Yan et al., 2009a). Trace levels of 1-chloropropane (<0.03 μmol/ bottle) were detected in inoculated bottles and uninoculated abiotic controls (data not shown), and propene was detected in relatively minute quantities (<0.13 μmol/bottle) in abiotic negative controls (Figure 2), indicating small amounts of abiotic 1,2-DCP transformation. The sum of parent chlorinated solvent (i.e., 1,2-DCP) and daughter products (i.e., propene and 1-chloropropane) in replicate bottles inoculated with the bacterial strains ranged from 74-131% of the mass determined in abiotic negative controls (average 95%). When provided with 1,2-DCP at initial aqueous-phase concentrations less than 3.19 ± 0.20 mM and 2.14 ± 0.12 mM, dechlorination  of 1,2-DCP to a final product of propene was essentially complete in bottles inoculated with D. alkenigignens IP3-3 T and D. lykanthroporepellens BL-DC-9 T , respectively, with <1% of the starting 1,2-DCP remaining at the end of the eight week incubation period (Figure 2). At higher initial 1,2-DCP concentrations (denoted by arrows in Figure 2), 1,2-DCP remained at the end of eight weeks in amounts increasing with increasing initial 1,2-DCP concentration.
Similar to what was observed with 1,2-DCA, the quantity of propene formed from 1,2-DCP dechlorination increased at initial 1,2-DCP concentrations ranging from 0.5 to roughly 3 mM (maximum propene was observed in bottles containing initial 1,2-DCP concentrations of 3.21 ± 0.46 and 3.08 ± 0.05 mM for D. alkenigignens IP3-3 T and D. lykanthroporepellens BL-DC-9 T , respectively) and then decreased at higher initial 1,2-DCP concentrations. This indicates that beyond a certain threshold, as was observed with 1,2-DCA, 1,2-DCP became inhibitory to dechlorination activity. Nevertheless, 1,2-DCP reductive dechlorination was observed in serum bottles with initial 1,2-DCP concentrations as high as 5.05 ± 0.29 and 4.02 ± 0.09 mM for D. alkenigignens IP3-3 T and D. lykanthroporepellens BL-DC-9 T , respectively. At higher initial 1,2-DCP concentrations, propene was also detected, but in amounts that were not statistically different from abiotic negative controls at a 95% confidence level.
In contrast to the relatively high concentrations of 1,2-DCP that were dechlorinated by Dehalogenimonas spp. in the present study, (Löffler et al. 1997) reported that 1,2-DCP dechlorination by an undefined mixed culture derived from Red Cedar Creek sediment (Michigan, USA) was completely inhibited when 1,2-DCP was supplied in amounts corresponding to an aqueous phase concentration of roughly 0.9 mM or higher. D. alkenigignens IP3-3 T and D. lykanthroporepellens BL-DC-9 T may be better suited to degradation of higher 1,2-DCP concentrations than other microbial populations studied previously.
Similar to what was observed with 1,2-DCA and 1,2-DCP, the quantity of vinyl chloride formed from 1,1,2-TCA dechlorination increased at initial 1,1,2-TCA concentrations ranging from 0.5 to roughly 2 mM and then decreased at higher initial 1,1,2-TCA concentrations  Figure 3 Experimentally measured vinyl chloride production as a function of initial aqueous-phase 1,1,2-TCA concentration after eight-weeks incubation of D. alkenigignens IP3-3 T (left) and D. lykanthroporepellens BL-DC-9 T (right). Filled symbols indicate average of replicate bottles inoculated with bacterial strains. Open symbols indicate average of replicate uninoculated negative control bottles. Bars represent one standard deviation. Arrows denote concentration at and above which >1% of the starting 1,1,2-TCA remained at the end of the incubation period.
Hydrogen (H 2 ) remained at relatively high concentrations (>62%, v/v) in the gas headspace at the end of the eight-week incubation period for all chlorinated solvent concentrations tested for both strains, indicating that it was not stoichiometrically limiting.
Although reports of pure cultures' abilities to dehalogenate high concentrations of chlorinated alkanes are generally lacking in the literature, Marzorati et al. (2007) reported an enrichment culture referred to as 6VS (originating from groundwater in Italy where there was 1,2-DCA contamination for more than 30 years) that repeatedly dechlorinated 8 mM 1,2-DCA. Also, Grostern and Edwards (2009) described an enrichment culture, including Dehalobacter sp. and an Acetobacterium sp., capable of dechlorinating 2 mM 1,2-DCA. Though not previously evaluated for chlorinated ethanes or propanes, previous research on chlorinated ethenes has shown that microbial populations reductively dechlorinating chlorinated aliphatic alkenes, particularly perchloroethene (PCE) and trichloroethene (TCE) can maintain their activity and increase contaminant dissolution rates (Cope and Hughes, 2001;Dennis et al., 2003;Sleep et al., 2006;Yang and McCarty 2002).
The ability of Dehalogenimonas spp. to reductively dechlorinate high concentrations of halogenated alkanes has important implications for cleanup of contaminated soil and groundwater. Abiotic transformation of these chemicals in the environment is generally quite slow. For example, the environmental half-life of 1,2-DCA from abiotic transformation in water was estimated to be 50 years (Vogel et al., 1987). Unlike chlorinated ethenes (e.g., tetrachloroethene and trichloroethene), several of the polychlorinated ethanes and propanes, 1,2-DCA in particular, are resistant to transformation by zero-valent iron (Sarathy et al., 2010;Song and Carraway, 2005), limiting physicochemical remediation approaches for cleanup. The fact that Dehalogenimonas spp. are able to perform reductive dechlorination even in the presence of high concentrations of chlorinated compounds suggests that they may provide an important role in bioremediation.

Competing interests
The authors declare that they have no competing interests.