pubs.acs.org/est Article Dual C−Br Isotope Fractionation Indicates Distinct Reductive Dehalogenation Mechanisms of 1,2-Dibromoethane in Dehalococcoides- and Dehalogenimonas-Containing Cultures Jordi Palau,* Alba Trueba-Santiso, Rong Yu, Siti Hatijah Mortan, Orfan Shouakar-Stash, David L. Freedman, Kenneth Wasmund, Daniel Hunkeler, Ernest Marco-Urrea,* and Monica Rosell Cite This: Environ. Sci. Technol. 2023, 57, 1949−1958 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Brominated organic compounds such as 1,2- dibromoethane (1,2-DBA) are highly toxic groundwater contami- nants. Multi-element compound-specific isotope analysis bears the potential to elucidate the biodegradation pathways of 1,2-DBA in the environment, which is crucial information to assess its fate in contaminated sites. This study investigates for the first time dual C− Br isotope fractionation during in vivo biodegradation of 1,2-DBA by two anaerobic enrichment cultures containing organohalide- respiring bacteria (i.e., either Dehalococcoides or Dehalogenimonas). Different εCbulk values (−1.8 ± 0.2 and −19.2 ± 3.5‰, respectively) were obtained, whereas their respective εBr bulk values were lower and similar to each other (−1.22 ± 0.08 and −1.2 ± 0.5‰), leading to distinctly different trends (ΛC−Br = Δδ13C/Δδ81Br ≈ εCbulk/εBr bulk) in a dual C−Br isotope plot (1.4 ± 0.2 and 12 ± 4, respectively). These results suggest the occurrence of different underlying reaction mechanisms during enzymatic 1,2-DBA transformation, that is, concerted dihaloelimination and nucleophilic substitution (SN2- reaction). The strongly pathway-dependent ΛC−Br values illustrate the potential of this approach to elucidate the reaction mechanism of 1,2-DBA in the field and to select appropriate εCbulk values for quantification of biodegradation. The results of this study provide valuable information for future biodegradation studies of 1,2-DBA in contaminated sites. KEYWORDS: brominated organic compounds, groundwater contamination, biodegradation, organohalide-respiring bacteria, compound-specific isotope analysis ■ INTRODUCTION 1,2-Dibromoethane (1,2-DBA), also known as ethylene by 1,2-DBA due to its use as a lead scavenger. Organohalide- respiring bacteria (OHRB) provide a potential solution to treat dibromide, is highly toxic (U.S. EPA drinking water maximum 1,2-DBA-impacted sites due to their capability to harness contaminant level, MCL, 0.05 μg/L)1 and persistent in the energy using halogenated compounds as electron acceptors.7 environment.2 It is a suspected human carcinogen that was During this organohalide respiration process, 1,2-DBA is used as a lead scavenger in gasoline, as well as a pesticide, predominantly transformed to innocuous ethene.5 Formation fumigant, solvent, and chemical intermediate.2 As a result of its of ethene can occur via dihaloelimination of 1,2-DBA, either extensive use, 1,2-DBA has been detected above its MCL in via concerted or stepwise β-elimination (Scheme 1a,b). Hydro- groundwater samples from domestic wells in the USA3 and in genolysis to bromoethane (Scheme 1c)8 and formation of about half of the contaminated underground storage tank sites small amounts of vinyl bromide (VB) via dehydrohalogenation in South Carolina (1100 sites evaluated).4,5 It is ranked 39th (Scheme 1d) 5,9 were reported in previous anaerobic (out of 275) on the 2019 substance priority list established by the U.S. Agency for Toxic Substances and Disease Registry biodegradation studies. based on a combination of its frequency, toxicity, and potential for human exposure.6 Therefore, subsurface contamination by Received: September 29, 2022 1,2-DBA is an issue of environmental concern. Revised: December 3, 2022 At contaminated sites, anoxic conditions are prevalent in Accepted: December 28, 2022 groundwater due to depletion of oxygen during degradation of Published: January 26, 2023 readily oxidizable organic contaminants such as petroleum hydrocarbons, which are often detected at field sites impacted © 2023 The Authors. Published by American Chemical Society https://doi.org/10.1021/acs.est.2c07137 1949 Environ. Sci. Technol. 2023, 57, 1949−1958 Environmental Science & Technology pubs.acs.org/est Article Scheme 1. Biodegradation Pathways of 1,2-DBAa Changes in isotope ratios during a specific transformation of a compound can be described by a modified form of the Rayleigh distillation equation R t /R 0 = f (2) The compound-specific isotope fractionation value (ε) is related to the initial isotope ratio at the beginning of a transformation process R0, the isotope ratio Rt, and the compound remaining fraction (f = Ct/C0) at a given time point. For carbon, a wide range of εCbulk (molecular-average isotope effects) values were observed during transformation of 1,2- DBA to ethene in previous laboratory studies, from −5.3 ± 0.5 to −20 ± 3‰.11,13 Such variability can reflect not only the occurrence of rate-limiting (non- or slightly isotope-fractionat- ing) steps preceding the bond cleavage, such as contaminant mass transfer16 (so called isotope masking), but also the effect of different reaction mechanisms (e.g., stepwise vs concerted dihaloelimination, Scheme 1a,b), which hampers unambiguous identification of the reaction mechanism. The situation is different if isotope analysis is conducted on two or more elements (e.g., C and Br). Combined changes in isotope ratios of both elements (e.g., Δδ13C vs Δδ81Br) generally exhibit a linear relationship with slopes (ΛC−Br = Δδ13C/Δδ81Br ≈ εCbulk/εBr bulk), reflecting the extent of C and Br isotope effects,11 which relate to the respective underlying reaction mechanisms.15,17 In contrast to single-element isotope fractionation analysis, the proportion of changes in isotope a ratios of both elements relative to each other (Δδ13C/Δδ81Br) Dots in structural formulas represent unpaired electrons. is largely insensitive toward non-degradative processes such as contaminant transport.18−20 However, Br isotope fractionation In addition to reductive dehalogenation and dehydrohaloge- data and dual C−Br isotope fractionation studies are very nation reactions, 1,2-DBA can be transformed via nucleophilic limited so far.21 substitution (SN2) to 2-bromoethanol (Scheme 1e) as an Using a multi-element CSIA approach, dual-isotope intermediate product under oxic conditions.10,11 Oxidation of fractionation patterns observed at contaminated sites can be 1,2-DBA catalyzed by a monooxygenase enzyme was also compared to the laboratory-derived Λ values in order to suggested in previous studies (Scheme 1f).12,13 Thus, the high identify degradation pathways in the field.22,23 A recent study susceptibility of 1,2-DBA to being transformed via distinct by Kuntze et al.11 reported ΛC−Br values determined during biodegradation pathways, under both anoxic and oxic debromination of 1,2-DBA to ethene by reduced corrinoids conditions, complicates the assessment of its fate in the (norpseudo-B12 and cyano-B12 types) and crude protein environment. Understanding the route of biodegradation is extracts of OHRB Sulfurospirillum multivorans. In contrast to important to evaluate the natural attenuation of 1,2-DBA at this previous study, ΛC−Br values have not yet been reported contaminated sites and to predict the potential for accumu- for in vivo biodegradation of 1,2-DBA by OHRB such as lation of toxic halogenated daughter products in the field. Dehalococcoides spp. or Dehalogenimonas spp., which is Identifying degradation pathways from analysis of daughter necessary to evaluate whether the reaction mechanisms and products in field samples is complex since it requires that initial isotope fractionation effects determined in vitro are also at contaminants and degradation products are adequately work in living organisms. In addition, previous laboratory captured by the sampling and analysis methods. Furthermore, isotope studies on biodegradation of chlorinated ethenes products can be either (i) rapidly converted (e.g., ethene or 2- showed a significant difference of εCbulk in experiments with bromoethanol10) to ubiquitous end products or (ii) formed corrinoids versus microbial cultures24 and also between (e.g., VB or ethene) from different precursors. For instance, cultures and crude extracts.16 Thus, determining C and Br ethene can be formed from brominated and chlorinated isotope fractionation and dual-element isotope slopes in in vivo ethenes via hydrogenolysis. Hence, additional tools are experiments is warranted. This information is not only necessary for better characterization of 1,2-DBA biodegrada- important for process understanding but also essential when tion in the field. using Λ and εbulk values to assess the fate and to quantify the Compound-specific isotope analysis (CSIA) is a powerful biodegradation extent of 1,2-DBA in the environment. tool to assess transformation processes of organic contami- Biodegradation of 1,2-DBA by different reductive dehaloge- nants.14,15 The ratio of the heavy to light isotopes of an nases (RDases) was reported in previous studies. The element in a sample Rsample (e.g., 13C/12C or 81Br/79Br) is trichloroethene RDase (TceA) from Dehalococcoides mccartyi expressed using the δ-notation as per mil (‰) difference from strain 195 converted 1,2-DBA to ethene and minor amounts of the isotope ratio in the respective standard Rstandard, that is VB (<1%) in enzymatic assays with the purified enzyme.9 13 81 More recently, debromination of 1,2-DBA to ethene was C or Br = R sample /R standard 1 (1) catalyzed by tetrachloroethene RDase (PceA) in enzymatic 1950 https://doi.org/10.1021/acs.est.2c07137 Environ. Sci. Technol. 2023, 57, 1949−1958 Environmental Science & Technology pubs.acs.org/est Article assays with crude protein extracts from S. multivorans.11 experiments with Dehalococcoides and Dehalogenimonas, However, in these studies, it was not investigated if 1,2-DBA respectively. Bottles were sacrificed at different extents of would support the growth of the organisms. For Dehalococ- debromination by adding concentrated phosphoric acid (300 coides, Yu et al.5 showed that the number of D. mccartyi cells in μL), as described elsewhere.23,28 For the experiments an anaerobic enrichment culture increased with 1,2-DBA performed at CU, killed controls were prepared by adding amendments, producing predominantly ethene and traces of phosphoric acid first to the bottles, followed by 1,2-DBA. For VB. Bacteria of the genus Dehalogenimonas are known for their those performed at UAB, abiotic control bottles containing the potential to grow by reductive dehalogenation with vicinal- growth medium with 1,2-DBA but without inoculum were halogenated alkanes.25 Despite this, very little information is prepared, as described for the experimental bottles. In addition, available on the biodegradation of 1,2-DBA by Dehalogenimo- live controls without 1,2-DBA were prepared to account for nas.26 the transfer of compounds from previous degradation experi- The main aim of the present study was to investigate for the ments with the inoculum. first time (i) dual C−Br isotope fractionation during in vivo Concentration and Isotopic Analyses. A detailed biodegradation of 1,2-DBA and (ii) whether distinct or similar description of analytical methods and equipment used for dual-element isotope slopes occur with different bacteria. To the isotopic and concentration analysis is available in the this end, carbon and bromine isotopic fractionation (εCbulk and Supporting Information. The concentrations of 1,2-DBA and εBr bulk) for microbial reductive dehalogenation of 1,2-DBA were daughter products (i.e., ethene and VB) were measured by determined using two laboratory cultures enriched in headspace analysis using a gas chromatograph-flame ionization Dehalococcoides and Dehalogenimonas populations, respectively. detector at CU5 and UAB29 laboratories (see the Supporting A dual C−Br isotope approach was used to characterize the Information). The concentration of 1,2-DBA in abiotic ΛC−Br value and explore the underlying reaction mechanism. controls of the experiments with Dehalococcoides (270 ± 3 The determined ΛC−Br values from in vivo experiments with μM, n = 3) and Dehalogenimonas (26 ± 2 μM, n = 2) intact cells of Dehalococcoides and Dehalogenimonas, respec- containing microcosms remained at the initial concentration, tively, were compared with those reported for 1,2-DBA indicating that compound losses through the caps during degradation by corrinoids and crude protein extracts (S. incubation were insignificant. multivorans).11 In addition, the biodegradation of 1,2-DBA by Bromine isotope measurements of 1,2-DBA were performed the Dehalogenimonas-containing culture used in this study was at Isotope Tracer Technologies Inc. (IT2), Canada, and characterized. carbon isotope ratios were analyzed at the Scientific and ■ MATERIALS AND METHODS Bacterial Cultures. Two enrichment cultures with differ- Technological Centers of the University of Barcelona (CCiT- UB), Spain. 81Br- and 13C-CSIA were performed by gas chromatography−isotope ratio mass spectrometry (GC− ent bacterial populations were used for in vivo laboratory batch IRMS). For bromine, the two most abundant fragment ions experiments with respiring cells. The Dehalogenimonas- (m/z 109 and 107) were used, which correspond to containing culture used in this study was derived from isotopologue pairs that differ by one heavy bromine isotope sediments obtained from the Besòs River (Spain); the genome ([81Br12C21H4]+ and [79Br12C21H4]+, respectively). The raw of this strain was recently sequenced and annotated, and it was δ81Br values were calibrated to the standard mean ocean denominated Dehalogenimonas alkenigignens strain BRE15M.27 bromide scale using two external laboratory standards of 1,2- The composition of this consortium is shown in the DBA (two-point calibration), which were dissolved in water Supporting Information (Figure S1). Further information and measured similarly to the samples in the same sequence. regarding enzymatic assays with cell suspensions, cell harvest- Duplicate samples and standards were analyzed as a quality ing, DNA extraction, and 16S rRNA gene amplicon sequencing control for both C and Br isotopes, and the precision (±1σ) of is provided in Supporting Information. the analysis was ≤ ±0.3‰ for both δ13C and δ81Br. The Dehalococcoides-containing enrichment culture was Evaluation of Isotope Fractionation. The compound- derived from samples collected from the Savannah River site average εbulk values were quantified by least-squares linear (USA), and previous studies have focused on its kinetics and regression of eq 2 without forcing the regression through the yields.5 Growth of Dehalococcoides occurred when the culture origin,30 and the uncertainty corresponds to the 95% used 1,2-DBA as the electron acceptor and lactate as the confidence interval (C.I.) derived from the standard deviation electron donor.5 For both cultures, details regarding bacterial of the regression slope. Like 37Cl (natural abundance of cultivation are provided in the Supporting Information. 24.22%), the heavy bromine stable isotope (81Br) also has a Isotope Fractionation Experiments with Dehalococ- high abundance (40.3%).31 A previous study32 showed that the coides and Dehalogenimonas Cultures. The chemicals and Rayleigh equation can also be applied to calculate the isotopic medium used for the preparation of microcosms, incubation fractionation of chlorine despite the higher natural abundance conditions, and sampling details are described in the of 37Cl compared to 13C, and this equation was recently used Supporting Information. Batch tests with Dehalococcoides- in Br isotope fractionation studies with different compounds and Dehalogenimonas-containing microcosms were performed (e.g., 1,2-DBA, 11 methyl bromide, 33 and brominated at Clemson University (CU), USA, and at the Universitat ethenes34). Calculation of C and Br apparent kinetic isotope Autònoma de Barcelona (UAB), Spain, respectively. Serum effects (AKIEs) from estimated εbulk values is presented in the bottles (120 mL total volume) containing 65 mL of medium Supporting Information, and their uncertainty was calculated were prepared in an anoxic chamber and sealed with Teflon- by error propagation. faced rubber septa and aluminum crimp caps to maintain anoxic conditions. Initial aqueous phase concentrations of 1,2- DBA (considering partitioning between the headspace and the ■ RESULTS AND DISCUSSION Reductive Dehalogenation of 1,2-DBA by Enrichment liquid using Henry’s law) were ∼270 and ∼25 μM for Cultures Containing Dehalogenimonas and Dehalococ- 1951 https://doi.org/10.1021/acs.est.2c07137 Environ. Sci. Technol. 2023, 57, 1949−1958 Environmental Science & Technology pubs.acs.org/est Article coides Species. Metagenomic analyses determined that the For the experiments with the Dehalococcoides-containing bacterial community in the Dehalogenimonas-containing culture culture, time-course concentration data during 1,2-DBA was dominated by the genus Dehalogenimonas (68.0%, Figure biodegradation are shown in Figure S3. Small amounts of S1) and did not detect any other OHRB belonging to the class VB (less than 0.2%, in mol/bottle basis, of the initial 1,2-DBA Dehalococcoidia. Importantly, the absence of Dehalococcoides added) were detected in experiments with the Dehalococcoides- spp. was previously corroborated in this culture by specific containing culture (Figure S3), in agreement with previous PCR assays for the genus Dehalococcoides.29 biodegradation studies of 1,2-DBA by Dehalococcoides showing Debromination of 1,2-DBA to ethene was observed after transformation of 1,2-DBA to ethene and minor amounts of transferring the Dehalogenimonas-containing culture to a VB (<1%).5,9 The amount of VB measured in this study was medium amended with 1,2-DBA (25 μM) as the sole much higher than that expected for abiotic dehydrohalogena- halogenated electron acceptor (Figure 1). After a lag phase, tion of 1,2-DBA (see Results in Supporting Information and Figure S3) and, therefore, this abiotic pathway is not a plausible explanation for the origin of VB. The product concentration pattern in Figure S3 indicates 1,2-DBA trans- formation to ethene as the main degradation pathway, as observed for Dehalogenimonas. The low level of VB that accumulated could be subsequently reduced in part to ethene according to the decreasing amounts of VB toward the end of the experiment (Figure S3). Br and C Isotope Fractionation Experiments. For both microbial cultures, bromine isotope values of 1,2-DBA showed an enrichment in 81Br over 79Br during debromination following a Rayleigh isotope fractionation trend (eq 2, Figure 2a). The determined bromine isotopic fractionation (εBr bulk) for the Dehalococcoides-containing microcosms (−1.22 ± 0.08‰) was very similar compared to those containing Dehalogenimo- nas (−1.2 ± 0.5‰). Low bromine isotopic effects were also Figure 1. Debromination of 1,2-DBA to ethene in the first generation observed during dihaloelimination of 1-bromo-2-chloroethane of Dehalogenimonas-containing culture cultivated with this halogen- by a pure culture of S. multivorans (−1.7 ± 0.5‰)35 and ated substrate. Arrows indicate additional amendments of 1,2-DBA. hydrogenolysis of tri- and dibromoethenes by crude protein Black circles correspond to micromoles of 1,2-DBA left in the extracts of S. multivorans and Desulfitobacterium hafniense PCE- microcosms, white triangles to micromoles of ethene, and green S (≤−1.3 ± 0.3‰, n = 6).34 These low bromine isotopic crosses to the total amount of 1,2-DBA and ethene. Error bars effects can be explained in part due to the lower relative mass representing the standard deviation for the values of biological difference between 81Br and 79Br (2.5%) compared, for triplicates are smaller than the symbols. example, to 37Cl and 35Cl (5.7% relative to 35Cl).36 This is consistent with the higher εCl bulk values determined in a previous the initial concentration of 1,2-DBA was depleted, and this was study during dichloroelimination of 1,2-dichloroethane (1,2- followed by consumption of additional amendments with DCA) by similar Dehalococcoides-containing (−5.1 ± 0.1‰) increasing degradation rates, suggesting microbial growth and Dehalogenimonas-containing (−12.0 ± 0.8‰) enrichment coupled to the biodegradation of 1,2-DBA. After consumption cultures as used in the present study, although in that case the of 100 μM 1,2-DBA, quantitative real-time PCR from technical values were different between them.23 triplicates of two experimental microcosms showed that For debromination of 1,2-DBA to ethene, bromine isotope Dehalogenimonas 16S rRNA gene copies doubled from 10.4 fractionation values from a previous study11 using reduced × 104 ± 2.9 × 104 (at time zero) to 22.3 × 104 ± 1.5 × 104, corrinoids (fractionation values were affected neither by suggesting again that reduction of 1,2-DBA was coupled to transport through the cell membrane nor by interactions of growth. To the best of our knowledge, this is the first evidence the target compound with other cell materials), norpseudo-B12 that the Dehalogenimonas genus can couple its growth with 1,2- (−3.9 ± 0.3‰) and cyano-B12 (−3.9 ± 0.4‰), and crude DBA dehalogenation. protein extract of S. multivorans (−2.3 ± 0.2‰) are available Degradation products of 1,2-DBA (i.e., ethene and VB) were for comparison (Table 1). These εBr bulk values are higher than analyzed with Dehalogenimonas and Dehalococcoides cultures, as those measured in our study with whole cells (around this can provide important information on the enzymatic −1.2‰). The weaker Br isotope fractionation observed in reaction mechanism. In the experiments with the Dehalogeni- the experiments with intact cells could be explained by monas-containing culture, only ethene and bromide were masking intrinsic isotopic effects (i.e., isotopic effects detected as a result of 1,2-DBA transformation, and they were associated with the chemical bond conversion) due to produced at stoichiometric amounts (Figure S2). VB was never microscale mass transfer limitations at microbial cell detected (detection limit, 0.5 μM). Reductive dehalogenation membranes.16,24 of 1,2-DBA (initial concentration of 50 μM) to ethene was For carbon, isotope ratios during debromination of 1,2-DBA corroborated using enzymatic assays with cell suspensions and by the investigated microbial cultures also showed an methyl viologen as the artificial electron donor (Table S1). In enrichment in 13C over 12C which followed a Rayleigh trend addition, VB was not transformed in enzymatic assays with cell (Figure 2b). In contrast to bromine, the εCbulk value for suspensions using the Dehalogenimonas-containing culture Dehalococcoides-containing microcosms (−1.8 ± 0.2‰) was grown with 1,2-DBA, ruling out a hypothetical formation of much smaller than that for Dehalogenimonas-containing ethene via reduction of VB as the intermediate. microcosms (−19.2 ± 3.5‰). The latter falls in the upper 1952 https://doi.org/10.1021/acs.est.2c07137 Environ. Sci. Technol. 2023, 57, 1949−1958 Environmental Science & Technology pubs.acs.org/est Article cofactors (norpseudo-B12: −16.2 ± 1.1‰ and cyano-B12: −16.9 ± 0.9‰).11 This suggests that the C−Br bond conversion is rate-determining and that the εCbulk value for the Dehalogenimonas-containing culture reflects the intrinsic isotope effect. Lower εCbulk values are generally observed for aerobic biodegradation by different bacteria (from −2.7 to −6.9‰, Figure 2b and Table S2).11,13 Nevertheless, the low εCbulk value obtained for the Dehalococcoides-containing culture in the present study was even lower than the range determined for aerobic biodegradation of 1,2-DBA (Figure 2b), represent- ing the lowest εCbulk value determined so far for anaerobic biodegradation of 1,2-DBA. In order to investigate further the observed differences of carbon isotope fractionation during reductive dehalogenation of 1,2-DBA by enrichment cultures harboring distinct bacteria, dual-element isotope slopes and estimated AKIEs are discussed below. Dual C−Br Isotope Patterns. The measured δ81Br and 13 δ C values of 1,2-DBA from the isotopic fractionation experiments with Dehalococcoides- and Dehalogenimonas- containing enrichment cultures were combined in a dual- element isotope plot, resulting in linear trends with strongly distinct slopes (ΛC−Br = Δδ13C/Δδ81Br, stated together with 95% C.I., Figure 3 and Table 1). For Dehalococcoides- containing microcosms, a much smaller ΛC−Br value (1.4 ± 0.2) than that of Dehalogenimonas-containing microcosms (12 ± 4) was observed as a result of the smaller εCbulk value (−1.8 ± 0.2 and −19.2 ± 3.5‰, respectively). The slope determined for Dehalococcoides (1.4 ± 0.2) is comparable to that obtained for experiments with crude protein extracts of S. multivorans (2.4 ± 0.2)11 in a previous study (Figure 3 and Table 1). This suggests a similar enzymatic dehalogenation mechanism and, C therefore, indicates that the low ε bulk value for the Dehalococcoides-containing culture (−1.8 ± 0.2‰), compared Figure 2. Rayleigh plots according to eq 2 of bromine (a) and carbon to the crude protein extracts of S. multivorans (−5.3 ± (b) isotopic composition of 1,2-DBA during biodegradation by 0.2‰)11 and the range determined from anaerobic biode- Dehalococcoides-containing culture (Dhc, black circles) and Dehaloge- gradation experiments (from −5.3 to −20.4‰, Figure 2b and nimonas-containing culture (Dhg, white circles); f is the fraction of Table S2),8,11,13 is probably due to masking of the intrinsic remaining 1,2-DBA. Dotted lines correspond to 95% C.I. intervals of regression parameters. The error bars for some isotope values are isotopic fractionation. This result illustrates the advantage of a smaller than the symbols. Colored areas in panel (b) represent the dual- versus a single-element isotope approach to investigate ranges of carbon isotopic fractionation of 1,2-DBA from previous biodegradation reactions. biodegradation studies conducted under aerobic (blue) and anaerobic The ΛC−Br values reported for abiotic dibromoelimination of (orange) conditions.8,11,13 1,2-DBA by reduced corrinoids (i.e., norpseudo-B12: 4.2 ± 0.2 and cyano-B12: 4.3 ± 0.4)11 were very different compared to part of the range of εCbulk values reported for anaerobic that of Dehalogenimonas (12 ± 4) but closer to those of biodegradation of 1,2-DBA by five different mixed cultures and Dehalococcoides and crude protein extracts of S. multivorans11 crude protein extracts of S. multivorans (from −5.3 to (1.4 ± 0.2 and 2.4 ± 0.2, respectively, Figure 3 and Table 1). −20.4‰, Figure 2b and Table S2)8,11,13 and is similar to Kuntze et al.11 pointed to either isotope-sensitive steps those determined for abiotic dibromoelimination by corrinoid preceding the C−Br bond cleavage (e.g., compound binding Table 1. Measured Carbon and Bromine Isotope Fractionation Values (εCbulk and εBr bulk, Respectively), Dual-Element Isotope Slopes (ΛC−Br), and Data from Previous Studies of 1,2-DBA experiment conditions εCbulk (‰) R2 εBr bulk (‰) R2 ΛC−Br R2 references A. aquaticus AD20 crude extract (oxic) −6.9 ± 0.4 0.93 −0.6 ± 0.1 0.93 10.7 ± 2.1 0.88 Kuntze et al.11 S. multivorans crude extract (anoxic) −5.3 ± 0.5 0.95 −2.3 ± 0.2 0.97 2.4 ± 0.2 0.98 Kuntze et al.11 Dehalococcoides enrichment culture (anoxic) −1.8 ± 0.2 0.94 −1.2 ± 0.1 0.98 1.4 ± 0.2 0.95 this study Dehalogenimonas enrichment culture (anoxic) −19.2 ± 3.5 0.92 −1.2 ± 0.5 0.67 12 ± 4 0.79 this study norpseudo-B12 abiotic −16.2 ± 1.1 0.98 −3.9 ± 0.3 0.98 4.2 ± 0.2 0.99 Kuntze et al.11 cyano-B12 type abiotic −16.9 ± 0.9 0.99 −3.9 ± 0.4 0.98 4.3 ± 0.4 0.96 Kuntze et al.11 Zn(0) abiotic −10.9 ± 1.1 0.97 −2.1 ± 0.3 0.96 5.3 ± 0.6 0.98 Kuntze et al.11 alkaline hydrolysis abiotic (pH 8) −29.2 ± 2.6 0.98 −1.0 ± 0.1 0.98 30.1 ± 4.2 0.98 Kuntze et al.11 Fenton oxidation abiotic −4.4 ± 0.3 0.97 not significant ∞ Kuntze et al.11 1953 https://doi.org/10.1021/acs.est.2c07137 Environ. Sci. Technol. 2023, 57, 1949−1958 Environmental Science & Technology pubs.acs.org/est Article Potential Reaction Mechanisms. The large difference between the ΛC−Br values of Dehalogenimonas-containing microcosms (12 ± 4) and Dehalococcoides-containing micro- cosms (1.4 ± 0.2) (Figure 3) might be interpreted as a result of different reaction pathways. Previous biodegradation studies of 1,2-DCA, the chlorinated analogue of 1,2-DBA, with D. mccartyi strains,38,39 and similar enrichment cultures of Dehalococcoides and Dehalogenimonas23 as used in this study, suggested a 1,2-DCA transformation to ethene via concerted dihaloelimination. In addition to concerted dihaloelimination, a recent study40 on bacterial reduction of chlorinated alkanes, using vitamin B12 as a model system for RDases, suggested for the 1,2-DCA conversion to ethene an initial SN2-reaction followed by a concerted syn-elimination (Scheme 2b). In the present study, insight was obtained from the comparison of Λ values for different transformation reactions. Figure 3. Dual C−Br isotope patterns during anaerobic biodegrada- The correlation of the isotope effects in a dual-element isotope tion of 1,2-DBA by Dehalococcoides-containing culture (Dhc, black plot exhibits characteristic trends that relate to the underlying circles) and Dehalogenimonas-containing culture (Dhg, white circles). reaction mechanisms.15,17 In previous studies with 1,2-DCA, Dotted lines indicate the 95% C.I. of the linear regression, error bars the ΛC−Cl value reported for hydrolytic dechlorination by a of Δδ13C values are smaller than the symbols, and Λ values (±95% C.I.) are given by the slope of the linear regressions. The trend lines pure culture of Ancylobacter aquaticus AD20 (7.7 ± 0.3,17 determined for 1,2-DBA transformation by reduced corrinoids assumed SN2-reaction,11,17,41Scheme 1e) was very close to that (norpseudo-B12 and cyano-B12 types), and crude protein extracts of determined in experiments with a Dehalococcoides-containing S. multivorans and A. aquaticus in a previous study are also indicated enrichment culture (6.8 ± 0.2,23 SN2-reaction40 ). The (dashed lines).11 Dehalococcoides-containing culture was similar to the one used in the present study. The ΛC−Cl value for the Dehalococcoides-containing culture was at that time associated to the enzyme) or differences in the reaction mechanism to with concerted dihaloelimination, but it was recently attributed explain the lower slope observed for the enzymatic reaction to an SN2-reaction.40 Similarly, the reported ΛC−Br value for with crude protein extracts of S. multivorans compared to those hydrolytic debromination of 1,2-DBA by haloalkane hydrolytic obtained with pure cofactors. Similarly, previous experiments37 dehalogenase (crude protein extract) of A. aquaticus AD2011 is with tetrachloroethene (PCE) showed different ΛC−Cl values within the uncertainty to that determined for the experiments between a crude extract of PceA of S. multivorans harboring with Dehalogenimonas in the present study, 11 ± 2 and 12 ± 4, different cofactors (i.e., PceA-norpseudo-B12: 2.2 ± 0.7 and respectively (Figure 3 and Table 1). Therefore, similar ΛC−Br PceA-nor-B12: 2.8 ± 0.5) and the respective purified corrinoids values of 1,2-DBA for debromination by Dehalogenimonas and (i.e., norpseudo-B12: 6.9 ± 0.7 and nor-B12: 5.0 ± 0.8). Here, hydrolytic debromination (via SN2) point to an underlying the authors suggested that the dual-element isotope slope for SN2-reaction mechanism during 1,2-DBA biodegradation with the dehalogenation of PCE by PceA could reflect preceding the Dehalogenimonas-containing culture. As shown in Scheme rate-limiting steps with small, but non-negligible isotope 2b, transformation of 1,2-DBA via nucleophilic substitution effects, during enzyme−substrate association or enzymatic (SN2), followed by a concerted syn-elimination, result in the structure.37 These results show that the characterization of formation of ethene, which is consistent with the product dual-element isotope slopes from biodegradation experiments detected in the experiments with Dehalogenimonas (Figure 1). with intact cells and from different bacteria, as done in the Isotopic effects from abiotic reactions are often considered present study for the first time with 1,2-DBA, is necessary for close to the intrinsic isotope effects. According to studies of the interpretation of multi-element isotope trends in field Zn(0)-reaction with vicinal dibromides42 and 1,2-DCA,43 a studies. Potential reasons for the significantly different ΛC−Br concerted (two-electron transfer) dibromoelimination of 1,2- values during anaerobic degradation of 1,2-DBA by Dehalo- DBA could be assumed. For 1,2-DBA, the ΛC−Br value for coccoides- and Dehalogenimonas-containing cultures are further dibromoelimination to ethene by Zn(0) from a previous study discussed below. (5.3 ± 0.6)11 was very different compared to that of Scheme 2. Suggested Reaction Mechanisms for 1,2-DBA Reductive Dehalogenation by Dehalococcoides-Containing Enrichment Culture (a) and Dehalogenimonas-Containing Enrichment Culture (b) in This Study: (a) Dihaloelimination and (b) Nucleophilic Substitution (SN2) followed by a Concerted syn-Elimination40 1954 https://doi.org/10.1021/acs.est.2c07137 Environ. Sci. Technol. 2023, 57, 1949−1958 Environmental Science & Technology pubs.acs.org/est Article Dehalogenimonas (12 ± 4) but relatively similar to those 0.6)39 and Dehalococcoides-containing culture (6.8 ± 0.2)23 obtained by the reduced corrinoids (4.2 ± 0.2 and 4.3 ± 0.4)11 were associated with an SN2-reaction and that different (Table 1). As discussed above for the reduced corrinoids, the reaction mechanisms were at work in these studies. difference between the ΛC−Br value of abiotic 1,2-DBA In accordance with these previous studies on 1,2-DCA, we transformation by Zn(0) (5.3 ± 0.6)11 and those of can infer that the different reaction mechanisms determined Dehalococcoides-containing culture and crude extract of S. for 1,2-DBA in the present study could probably be associated multivorans11 (1.4 ± 0.2 and 2.4 ± 0.2, respectively) could be with different RDases, even if they harbor a cobalamin as a explained by potential side reactions during 1,2-DBA−enzyme common cofactor. Importantly, the results of our study suggest association. Thus, this result would support a different reaction that, as observed for the degradation of 1,2-DCA by D. mechanism for 1,2-DBA debromination by the Dehalococcoides- mccartyi strain BTF08,39Dehalogenimonas could also be capable and Dehalogenimonas-containing cultures. of employing different reaction mechanisms for the biode- Additional insight can be obtained from estimated position- gradation of halogenated alkanes, that is, concerted dihaloe- specific AKIEs, provided that the chemical bond conversion in limination for 1,2-DCA23,40 and the SN2-reaction for 1,2-DBA. the enzyme reaction is rate-determining. Considering an SN2- The identification of the RDases involved in the trans- reaction scenario for 1,2-DBA transformation by Dehalogeni- formation of 1,2-DBA and 1,2-DCA by Dehalogenimonas and monas, a single C−Br bond would be cleaved in the first Dehalococcoides could shed more light on these questions with reaction step (via SN2, Scheme 2b). The pronounced εCbulk for important insights for future biodegradation studies.44,45 the Dehalogenimonas-containing culture (−19.2 ± 3.5‰, In the last decade, single-element isotope fractionation Figure 2b) reflects a C−Br bond cleavage as the rate- analysis (mainly 13C/12C) has emerged as a powerful tool to determining step. Calculated AKIEs assuming an SN2-reaction evaluate the extent of halogenated organic compounds (AKIEC = 1.040 ± 0.008 and AKIEBr = 1.002 ± 0.001, see the (bio)degradation in contaminated sites.46,47 For this approach, Supporting Information) showed a good agreement with those selection of the appropriate εCbulk value (or range of values), expected from the Streitwieser semiclassical limit model for related to the ongoing degradation pathway, is crucial to get C−Br bond breakage (KIEC = 1.042 and KIEBr = 1.002).11 accurate estimations. Determination of εbulk values is not Therefore, the estimated AKIEs for both C and Br in the possible under field conditions because changes in substrate experiments with Dehalogenimonas-containing culture are concentrations in groundwater are also related to processes consistent with those expected for an SN2-reaction. other than transformation, such as hydrodynamic dispersion or Taken together, the different lines of evidence indicate, for sorption. The redox conditions in groundwater can be useful to the first time, the occurrence of different underlying reaction constrain the εbulk values; however, this study shows that for mechanisms during enzymatic 1,2-DBA transformation to 1,2-DBA the selection of εCbulk values based only on redox data ethene (i.e., SN2-reaction and concerted dihaloelimination, could result in erroneous estimations (Figure 2b). Therefore, Scheme 2). This finding can explain, at least partially, the wide additional data is necessary to select adequate εCbulk values for range of εCbulk values observed for anaerobic biodegradation of quantification of 1,2-DBA biodegradation in the field. 1,2-DBA, from −1.8 to −20.4‰ (Figure 2b and Table The strongly pathway-dependent slopes ΛC−Br, 1.4 ± 0.2 S2).8,11,13 Our interpretation might be reinforced by the different daughter compounds detected during 1,2-DBA (concerted dihaloelimination) and 12 ± 4 (SN2-reaction), transformation by the distinct microbial cultures, that is ethene illustrate the potential of a dual C−Br isotope analysis (i) to and VB in the experiments with Dehalococcoides-containing elucidate the reaction mechanism underlying microbial culture but only ethene with Dehalogenimonas-containing reductive dehalogenation of 1,2-DBA in the environment and C culture. However, further research is necessary to elucidate (ii) to pinpoint the appropriate ε bulk value for the the source of VB. For instance, in a previous study38 of 1,2- quantification of biodegradation. The similar ΛC−Br values of DCA biodegradation by two D. mccartyi strains, the authors 1,2-DBA for reductive debromination by Dehalogenimonas and hypothesized that the traces of VC might be the result of (i) a hydrolytic debromination by A. aquaticus AD20 (crude protein subordinate malfunction of the involved RDase, (ii) a minor extract)11 (Figure 3 and Table 1) hamper the distinction of expressed additional RDase, or (iii) an abiotic reaction with these pathways in the field based only on C and Br isotope reducing agents from the mineral media. analysis. Hydrolytic debromination of 1,2-DBA to 2- Environmental Significance and Practical Implica- bromoethanol (Scheme 1e) was observed under oxic tions for the Application of CSIA. In a recent study of 1,2- conditions.10,11 Therefore, the combination of dual C−Br DCA biodegradation by the D. mccartyi strain BTF08,39 isotope data with redox conditions, product analysis (i.e., different ΛC−Cl slopes were observed for cells with distinct detection of ethene), and use of biomarkers may allow RDase inventories (i.e., 5.3 ± 0.6 and 2.0 ± 0.5 for cultures differentiation between hydrolytic and reductive debromina- with >98% TceAbtf08 and >96% VcrAbtf08, respectively). These tion of 1,2-DBA in the environment. Engineered abiotic dual C−Cl isotope fractionation trends correspond well with transformation reactions of 1,2-DBA such as Fenton oxidation those reported for biodegradation of 1,2-DCA by similar and alkaline hydrolysis (pH 8) showed much higher ΛC−Br cultures as used in the present study (i.e., 6.8 ± 0.2 and 1.89 ± values (i.e., ∞ and 30.1 ± 4.2, respectively, Table 1)11 0.02 for Dehalococcoides- and Dehalogenimonas-containing compared to the biodegradation reactions investigated in this cultures, respectively).23 In these studies,23,39 a concerted study. dihaloelimination or Co−halogen bond formation with The results of this study provide valuable information for concomitant leaving of the vicinal halogen atom was postulated future application of CSIA to investigate 1,2-DBA biode- as the underlying reaction mechanism. However, Heckel and gradation in contaminated sites, contributing to the urgent Elsner,40 based on new data from experiments of 1,2-DCA need of reducing uncertainties in the quantification of reaction with vitamin B12 (ΛC−Cl = 6.4 ± 0.2), hypothesized compound transformation. In addition, the results suggest that the high ΛC−Cl values of D. mccartyi TceAbtf08 (5.3 ± that the combination of dual C−Br isotope analysis with 1955 https://doi.org/10.1021/acs.est.2c07137 Environ. Sci. Technol. 2023, 57, 1949−1958 Environmental Science & Technology pubs.acs.org/est Article molecular biology tools might be used to characterize Monica Rosell − Grup MAiMA, SGR Mineralogia Aplicada, anaerobic biodegradation of 1,2-DBA by different bacteria. Geoquímica i Geomicrobiologia, Departament de Mineralogia, Petrologia i Geologia Aplicada, Facultat de ■ ASSOCIATED CONTENT * Supporting Information sı Ciències de la Terra, Institut de Recerca de l’Aigua (IdRA), Universitat de Barcelona (UB), Barcelona 08028, Spain; orcid.org/0000-0003-1563-8595 The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.est.2c07137. Complete contact information is available at: https://pubs.acs.org/10.1021/acs.est.2c07137 Further information about materials and methods, including DNA extraction and 16S rRNA gene Author Contributions sequencing/qPCR, activity assays, batch experiments, The manuscript was written through contributions of all C and Br isotope analysis, and calculation of AKIEs; authors. All authors have given approval to the final version of product concentration pattern during biodegradation of the manuscript. 1,2-DBA by Dehalococcoides; microbial composition of Notes the Dehalogenimonas enrichment culture; plot of ethene The authors declare no competing financial interest. production and bromide release against 1,2-DBA consumed; and comparison of literature and exper- imental carbon isotope fractionation values of 1,2-DBA ■ ACKNOWLEDGMENTS J.P. and M.R. were supported by the projects CGL2017-82331- (PDF) R (Spanish Ministry of Economy and Competitiveness) and 2017SGR 1733 (Catalan Government). A.T.-S. acknowledges ■ AUTHOR INFORMATION Corresponding Authors a predoctoral fellowship from the MINECO (BES-2014- 070817). E.M.-U. was supported by the project PID2019- 103989RB-100 (MCIN/AEI/10.13039/501100011033). 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