Science of the Total Environment 813 (2022) 152659
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Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv
Research Paper
Trichloromethane dechlorination by a novel Dehalobacter sp. strain 8M
reveals a third contrasting C and Cl isotope fractionation pattern within
this genus
Jesica M. Soder-Walz a, Clara Torrentó b, Camelia Algora c, Kenneth Wasmund d, Pilar Cortés e, Albert Soler b,
⁎
Teresa Vicent a, Mònica Rosell b, , Ernest Marco-Urrea a
a
Departament d'Enginyeria Química, Biològica i Ambiental, Universitat Autònoma de Barcelona (UAB), Carrer de les Sitges s/n, Bellaterra, Spain
b
Grup MAiMA, Mineralogia Aplicada, Geoquímica i Geomicrobiologia, Departament de Mineralogia, Petrologia i Geologia Aplicada, Facultat de Ciències de la Terra, Institut de
Recerca de l'Aigua (IdRA), Universitat de Barcelona (UB), c/ Martí Franquès s/n, 08028 Barcelona, Spain
c
Laboratory of Environmental Microbiology, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, 14220, Praha 4, Czech Republic.
d
Division of Microbial Ecology, Centre for Microbiology and Environmental Systems Science, University of Vienna, Vienna 1010, Austria.
e
Departament de Genètica i de Microbiologia, Facultat de BioCiències, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain.
H I G H L I G H T S G R A P H I C A L A B S T R A C T
• Novel Dehalobacter strain 8M anaerobi-
cally dechlorinates TCM to DCM.
• Strain 8M shares around 99.2% 16S rRNA
gene similarity with strains CF and
UNSWDHB.
• Significantly different TCM dual CCl iso-
tope fractionation pattern of strain 8M.
• Consistent ΛC/Cl in enzymatic assays
points to enzyme binding as rate-limiting
step.
A R T I C L E I N F O A B S T R A C T
Article history: Trichloromethane (TCM) is a pollutant frequently detected in contaminated aquifers, and only four bacterial strains
Received 5 October 2021 are known to respire it. Here, we obtained a novel Dehalobacter strain capable of transforming TCM to dichlorometh-
Received in revised form 17 December 2021 ane, which was denominated Dehalobacter sp. strain 8M. Besides TCM, strain 8M also completely transformed 1,1,2-
Accepted 20 December 2021
trichloroethane to vinyl chloride and 1,2-dichloroethane. Quantitative PCR analysis for the 16S rRNA genes confirmed
Available online 24 December 2021
growth of Dehalobacter with TCM and 1,1,2-trichloroethane as electron acceptors. Carbon and chlorine isotope fractio-
Editor: Frederic Coulon nation during TCM transformation was studied in cultured cells and in enzymatic assays with cell suspensions and
crude protein extracts. TCM transformation in the three studied systems resulted in small but significant carbon
Keywords: (εC = −2.7 ± 0.1‰ for respiring cells, −3.1 ± 0.1‰ for cell suspensions, and − 4.1 ± 0.5‰ for crude protein
Trichloromethane extracts) and chlorine (εCl = −0.9 ± 0.1‰, −1.1 ± 0.1‰, and − 1.2 ± 0.2‰, respectively) isotope fractionation.
1,1,2-trichloroethane A characteristic and consistent dual CCl isotope fractionation pattern was observed for the three systems (combined
Dehalobacter ΛC/Cl = 2.8 ± 0.3). This ΛC/Cl differed significantly from previously reported values for anaerobic dechlorination of
Organohalide respiration TCM by the corrinoid cofactor vitamin B12 and other Dehalobacter strains. These findings widen our knowledge on
Isotopic fractionation
the existence of different enzyme binding mechanisms underlying TCM-dechlorination within the genus Dehalobacter
2D-CSIA
and demonstrates that dual isotope analysis could be a feasible tool to differentiate TCM degraders at field studies.
⁎ Corresponding author at: Grup MAiMA, SGR Mineralogia Aplicada, Geoquímica i Geomicrobiologia, Departament de Mineralogia, Petrologia i Geologia Aplicada, Facultat de Ciències de la
Terra, Institut de Recerca de l'Aigua (IdRA), Universitat de Barcelona (UB), c/ Martí Franquès s/n, 08028 Barcelona, Spain.
E-mail address: monica.rosell@ub.edu (M. Rosell).
http://dx.doi.org/10.1016/j.scitotenv.2021.152659
0048-9697/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
J.M. Soder-Walz et al. Science of the Total Environment 813 (2022) 152659
1. Introduction Cl), and h is the atomic mass of the heavy isotope (13 for C and 37 for
Cl). Since variations in isotope ratios are often small, the δhE values are
Trichloromethane (TCM), commonly known as chloroform (CHCl3), expressed on a per mill scale (‰).
has been extensively used in industrial applications as a solvent, cleaning The theoretical background and the mechanistic implications of appar-
agent, and precursor in the production of pesticides and fire extinguishing ent isotope effects (AKIEs, the observed or measured isotope effects) in bi-
agents, among others. When released to soil, mostly from accidental spills ological reactions for characterizing biotransformation of organohalides
or leaking storage tanks in chemical manufacturing facilities, TCM can have been recently reviewed (Ojeda et al., 2020; Nijenhuis et al., 2016).
readily accumulate at the bottom of aquifers forming a dense non- In contrast to chemical homogeneous reactions where the bond cleavage
aqueous phase liquid, where it remains relatively recalcitrant to degrada- provides the full expression of the KIE in many cases, in complex enzymatic
tion and decomposition (half-life by non-alkaline hydrolysis >1000 years) systems the AKIE can be affected by rate limitation reaction steps other than
(McCulloch, 2003). bond cleavage (such as the ones related to uptake into the cell, transport
Importantly, the US-EPA considers TCM as a probable human carcino- within the cell or binding to the enzyme) leading to masking of isotope ef-
gen (Agency, 2000), and prominently, TCM ranks 11th on the 2019 Agency fects. Multi-element CSIA, which measures two or more element isotopic
for Toxic Substances and Disease Registry Priority List of Hazardous Sub- compositions, is expected to improve differentiation of transformation
stances based on a combination of its frequency, toxicity, and potential mechanisms (Ojeda et al., 2020; Nijenhuis and Richnow, 2016). Dual ele-
for human exposure (Substance Priority List | ATSDR, n.d.). It is also ranked ment isotope slopes (Λ), which correlate isotope values of two elements rel-
32nd on the list of priority substances in the framework of water policy of ative to each other, provide a sensitive parameter to differentiate reaction
the European Union (Directive 2013/39/EU). In addition to the adverse ef- mechanisms while suppressing potential masking of intrinsic isotope effects
fects on human health, TCM is a strong inhibitor of many microbial pro- affecting in a similar degree each element of the molecule (Ojeda et al.,
cesses, including methanogenesis and organohalide respiration (Bagley 2020). However, some exceptions have been already found for different en-
et al., 2000). Therefore, when a contaminated site contains a mixture of or- zymes catalyzing the same exact reaction likely via different transition
ganochlorines including TCM, its removal is a crucial first step to proceed states or connected to enzyme-substrate binding (Rosell et al., 2012;
further with the bioremediation of the accompanying chlorinated com- Renpenning et al., 2014; Gafni et al., 2020). Therefore, an indirect assess-
pounds. This removal of TCM can be microbially mediated in oxic and an- ment of these isotope-masking effects can be performed by using cell sus-
oxic environments. A variety of primary substrates (e.g., methane, propane, pensions (concentrate of active intact cells) and/or crude protein extracts
butane, hexane, and toluene) can support aerobic cometabolism of TCM experiments, where the cell membrane is disrupted, mass transfer is absent,
(Cappelletti et al., 2012). However, the prevalence of anoxic conditions in and, therefore, the enzyme is directly available for electron transfer and re-
contaminated aquifers limits the in situ application of aerobic bioremedia- ductive dehalogenation reactions, for instance. To date, multi-element iso-
tion strategies. In anoxic environments, three main biodegradation path- tope fractionation associated with TCM degradation has been reported for
ways have been reported for TCM: i) cometabolic reductive different biotic and abiotic reaction mechanisms as summarized by Ojeda
dechlorination, ii) hydrolysis followed by oxidation to CO2, and iii) organ- et al. (2020), and this approach has already been used for identifying and
ohalide respiration (Cappelletti et al., 2012). Natural attenuation, biostim- characterizing TCM degradation at contaminated sites (Blázquez-Pallí
ulation and bioaugmentation of organohalide-respiring bacteria (OHRB) et al., 2019; Rodríguez-Fernández et al., 2018a). With regards to the respi-
are considered cost-effective and efficient approaches to transform TCM ration of TCM, a recent study showed that two closely related Dehalobacter
in contaminated sites (Jugder et al., 2016). To date, members of the genera sp. (strains CF and UNSWDHB) transform TCM to DCM with unexpected
Dehalobacter (strains CF, UNSWDHB and THM1), and Desulfitobacterium opposing dual element isotope slopes (Heckel et al., 2019). Strain CF,
(strain PR) have been described as the only OHRB capable of respiring which dechlorinates TCM by the reductive dehalogenase CfrA, exhibited
TCM, both producing DCM as the predominant transformation product identical isotope effects to those measured with the corrinoid cofactor vita-
(Ding et al., 2014; Grostern et al., 2010; Justicia-Leon et al., 2012; Lee min B12 (dual CCl slope, ΛC/Cl = 6.6 ± 0.1 and ΛC/Cl = 6.5 ± 0.2, respec-
et al., 2012). tively), indicating that they likely share the same reaction mechanism,
A major challenge facing field-scale practitioners of bioremediation is either outer-sphere-single-electron transfer (OS-SET) (Heckel et al., 2017)
the provision of definitive proof that biotransformation is occurring, or SN2 nucleophilic substitution mechanism (Heckel et al., 2019). In con-
which is often difficult to accomplish based on assessments of concentra- trast to strain CF, strain UNSWDHB, which uses the TCM reductive
tions of chemicals alone. Compound-specific isotope analysis (CSIA) is a ro- dehalogenase TmrA, exhibited an unprecedented inverse chlorine isotope
bust technique that can provide direct evidence and quantification of in situ effects leading to an opposing dual element isotope slope (ΛC/Cl =
biodegradation of contaminants (Kuntze et al., 2020; Nijenhuis et al., 2018; −1.20 ± 0.2) (Heckel et al., 2019). Cell suspension and crude protein ex-
Vogt et al., 2020). The principle of CSIA is tied to measuring the underlying tract experiments with strain UNSWDHB were both unable to unmask the
kinetic isotope effect (KIE) occurring during a transformation process intrinsic KIE of TmrA, suggesting that enzyme binding and/or mass-
whereby, in most cases, chemical bonds containing light isotopes transfer into the periplasm rather than mass transfer across the cell mem-
(e.g., 12C, 35Cl) are preferentially cleaved compared with those containing brane were rate-limiting. This inconsistency between C and Cl isotope ef-
one or more heavy isotopes (e.g., 13C, 37Cl) and, consequently, a relative en- fects within strains belonging to the same genus opens the question about
richment of the remaining contaminant mass in molecules containing the the different enzymatic reaction mechanisms underlying organohalide res-
heavier isotopes is expected (Elsner et al., 2005). These compound specific piration of TCM. Values of ΛC/Cl are not reported yet for Dehalobacter sp.
isotope effects can be evaluated according to a simplified version of the strain THM1 and Desulfitobacterium sp. strain PR.
Rayleigh equation Eq. (1): To provide insight into potential isotope-masking effects and the reac-
tion mechanisms of TCM degradation within the genus Dehalobacter, the
Rt goal of this work was to assess C and Cl isotope fractionation during anaer-
ln ¼ ε∙ ln ð f Þ (1)
R0 obic dechlorination of TCM by a novel Dehalobacter strain capable of
transforming TCM to DCM. The culture was recently obtained from ground-
where ε is the isotopic fractionation (expressed on a per mill scale, ‰), f is water and fine sediments collected from a TCM-contaminated site (named
the fraction of the remaining substrate at any given time t and Rt and R0 are “site 1” in (Blázquez-Pallí et al., 2019)). We enriched the Dehalobacter-
the abundance ratios of specific stable isotopes (e.g., 13C/12C, 37Cl/35Cl) at containing culture responsible for TCM degradation and characterized al-
any time (t) and at the beginning of the reaction, respectively. The C and Cl ternative organohalogens as electron acceptors that this enrichment used.
isotopic compositions are reported as the deviation from the corresponding Carbon and chlorine isotope fractionation during TCM dechlorination
international reference material using the conventional δhE notation, was analyzed within distinct complexity systems levels: respiring cells
where δhE = (Rsample/Rstandard) – 1, E is the considered element (C and growing in the culture medium during TCM respiration versus enzymatic
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J.M. Soder-Walz et al. Science of the Total Environment 813 (2022) 152659
assays using cell suspensions and crude protein extracts (i.e., crude protein if needed, the isotopic values of these potentially transferred compounds.
extract from lysed cells), in order to assess isotope-masking effects prior to Both controls were killed at initial and final time points of the experiment.
the enzymatic reaction.
2.3. Analytical methods
2. Experimental procedures
Volatile halogenated compounds were quantified by injecting 0.5 mL
2.1. Chemicals, inoculum source and culture medium headspace samples into an Agilent 6890 N gas chromatograph (GC),
equipped with a DB-624 column (30 m, 0.32 mm with 0.25 mm film thick-
All chemicals used were of the highest available purity and were listed ness) and a flame ionization detector, following the method of Trueba-
in SMM1. Santiso et al. (Trueba-Santiso et al., 2017). Peak areas were calculated
The parent enrichment culture originated from groundwater and sedi- using Chromeleon 6.8 Chromatography Software (Dionex Corporation).
ments from a TCM-contaminated aquifer as previously described Results are presented as nominal concentrations expressed in μM and the
(Blázquez-Pallí et al., 2019). This culture was maintained in 100 mL glass analytical uncertainty was <5%. For carbon and chlorine isotope analysis
serum bottles with 65 mL of an anoxic defined buffered medium (pH = of DCM and TCM, liquid aliquots of the sacrificed experimental bottles
7) containing 5 mM sodium acetate, 200 mg L−1 yeast extract, 22.8 μM were taken and diluted as needed in ultrapure water into 20 mL vials filled
tungsten, 24.2 μM selenium, 200 μM Na2S × 9H2O and 200 μM L-cysteine to 10 mL final volume to adjust the concentration of the pollutants for sub-
as reducing agent, 10 mM bicarbonate as a buffer, 0.25 mg L−1 resazurin as sequent isotope signature determination. Carbon isotope analyses were per-
a redox indicator, vitamins and trace elements as described previously formed by headspace solid phase microextraction (HS-SPME) coupled to
(Tschech and Pfennig, 1984). The serum bottles were sealed with Teflon- gas chromatography isotope ratio mass spectrometry (GC-IRMS), as ex-
coated butyl rubber stoppers and aluminum crimp caps. Halogenated com- plained elsewhere (Blázquez-Pallí et al., 2019) and detailed in the Supple-
pounds were added through the septum as electron acceptors and H2 was mentary Information (SMM4). Samples were analyzed in duplicate and
gassed to 0.4 bar overpressure, supplied as an available electron donor. correction by daily values of calibrated in-house TCM and DCM standards
All experimental bottles were incubated in the dark at 25 °C. Active cultures was used. Chlorine isotope analyses of TCM were performed by headspace
were transferred into fresh medium (4.3% v/v inoculum) during the expo- extraction coupled to gas chromatography quadrupole mass spectrometry
nential degradation phase of the corresponding organohalogen. Abiotic (GC-qMS), as explained elsewhere (Torrentó et al., 2017) and detailed in
controls with the above described medium and with halogenated com- the Supplementary Information (SMM4). Ten injections of each sample
pounds but without inoculum, were always included in triplicates to evalu- were performed and the two-point calibration approach was used by inter-
ate abiotic processes. The dilution-to-extinction procedure in liquid and spersing along the sequence two external working standards, also injected
semisolid medium was performed in an attempt to isolate the bacteria re- ten times each. The analytical uncertainty 1σ was ±0.7‰ for carbon
sponsible for TCM transformation and the method followed is described and ± 0.5‰ for chlorine isotope values.
in the Supporting Information (SMM2). Briefly, six consecutive serial dilu-
tion series in liquid medium were performed, recovering TCM 2.4. Evaluation of isotope data
dechlorinating activity within the 10−8 dilution tube aiming at a highest
enrichment of TCM-dechlorinating bacteria. A terminal positive tube in Carbon and chlorine isotopic fractionations (εC and εCl) of TCM dechlo-
the 10−8 dilution was used in the dilution-to-extinction approach with rination were calculated according to the Rayleigh equation (Eq. 1) using
agar shakes aiming at the isolation of single colonies (see Fig. S1 in Supple- the double logarithmic Rayleigh plot. The uncertainty for ε values was de-
mentary Information). termined from the 95% confidence interval (95% CI) of the linear regres-
sion in the Rayleigh plots. The carbon isotopic mass balance (δ13CSUM)
2.2. Stable carbon and chlorine isotope experiments was obtained according to Eq. (2), where X is the molar fraction of each
compound relative to the total molar mass of chloromethanes assuming
Three set of experiments were performed for assessing carbon and chlo- complete TCM reductive dehalogenation to DCM (Hunkeler et al., 1999;
rine isotope fractionation during TCM transformation using: (i) respiring Aeppli et al., 2010). The uncertainty of δ13CSUM values was estimated by
cells, (ii) cell suspensions, and (iii) crude protein extracts. The preparation error propagation. This carbon isotopic mass balance can be used to deter-
of cell suspensions and crude protein extracts is described in the Supporting mine if DCM is the only degradation product or if it was further degraded.
Information (SMM3). For respiring cells, 12 parallel experimental bottles Isotopic mass balance was not calculated for chlorine since DCM chlorine
with the buffered medium were inoculated as described above and fed isotope ratios were not measured.
with 500 μM TCM. Each bottle was killed at different extents of TCM deg-
radation by adding H2SO4 (final concentration in the bottle: 20 mM δ13 C SUM ð‰Þ ¼ X TCM δ13 C TCM þ X DCM δ13 C DCM ð2Þ
H2SO4). The samples were stored at 4 °C until further analysis. For cell sus-
pension and crude protein extract experiments, 12 batch experiments were Apparent kinetic isotope effects (AKIE), which characterize the isotope
performed in 20 mL vials, with 10 mL of a buffered mix solution containing effect of the atoms at the reactive position of a molecule, were calculated
potassium acetate/acetic acid (K-AC) pH 7.0, 2 mM Ti (III) citrate as a re- following Eq. (3), where n is the total number of the atoms of the considered
ducing agent and 2 mM methyl viologen as an artificial electron donor. element (E) in the target molecule, x is the number of atoms located at the
One mL of cell concentrate and 300 μL of protein extract were used for reactive site, and z the number of atoms in intramolecular isotopic compe-
cell suspension and crude protein extract experiments, respectively, and tition. For TCM AKIEC, n = x = z = 1 and for AKIECl, n = x = z = 3. The
fed with 300 μM TCM. The experimental vials were incubated at 25 °C in uncertainty of AKIE values was estimated by error propagation.
darkness, agitated at 150 rpm and killed at different times using H2SO4
(final concentration in the vial: 100 mM H2SO4). In all three scenarios, 1
AKIEE ¼ n∙z
(3)
two different type of controls were included in triplicate: (i) abiotic controls 1þ x ∙ε
containing the sterile buffered medium or buffered mix solution with TCM
and without inoculum, cell concentrate or crude protein extract. This is to Dual isotope slopes (ΛC/Cl) were calculated by ordinary linear regression
control potential abiotic transformations, volatile losses, or impurities (OLR) of measured δ13C and δ37Cl data in 2D-isotope plots (i.e., ΛC/Cl =
from the stock solution. (ii) Biotic controls consisting of sterile buffered me- Δδ13C/Δδ37Cl). The uncertainty of Δδ values was estimated by error prop-
dium or buffered mix solution plus inoculum, cell concentrate or crude pro- agation. The uncertainty of Λ was reported as the 95% CI. A comparison be-
tein extract and without TCM, to evaluate the potential transfer of TCM and tween Λ values and their uncertainties obtained in this study with the OLR
DCM from the inoculum sources to the experimental bottles and to correct, and the York (Ojeda et al., 2020; Ojeda et al., 2019) regression methods is
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J.M. Soder-Walz et al. Science of the Total Environment 813 (2022) 152659
shown in the SI. Since no systematic bias introduced by OLR was observed DNA isolation and 16S rRNA gene amplicon sequencing analysis of these
and because there has not been sufficient time for the York methods to be cultures was performed. This showed that experimental bottles inoculated
routinely adopted in 2D-CSIA studies, we therefore decided to use OLR, with colony type 2 were more enriched in Dehalobacter (20%) and
but show York results in the SI. contained fewer other taxa (Fig. S2). This culture line was thus selected
for the following studies. This enrichment culture dechlorinated TCM
2.5. DNA extraction, 16S rRNA gene amplicon sequencing,phylogenetic analyses after a lag phase of about five days and stoichiometric amounts of DCM
of 16S rRNA gene and real-time PCR (qPCR) were produced (Fig. S3). The repeated addition of TCM led to faster dechlo-
rination rates without an apparent lag phase (Fig. S3), suggesting that TCM
Details regarding cell harvesting, DNA extraction, 16S rRNA gene degradation was supporting growth of the dechlorinating bacteria.
amplicon sequencing, phylogenetic analyses of 16S rRNA gene and real- In order to assess the ability of this culture to ferment DCM, several ex-
time PCR (qPCR) are provided in the Supporting Information (SMM5). perimental bottles were established with amendments of DCM (50 μM)
after removing H2, which is known to inhibit DCM fermentation at high
2.6. Statistics analysis partial pressure (Chen et al., 2017). DCM stalled in the bottles after 120 d
of incubation, indicating that this culture was not able to ferment DCM
All measured data are presented as mean ± SD of duplicate measure- (data not shown).
ments for isotopic analysis and real-time PCR (qPCR); and triplicate mea-
surements for time-course experiments. Significant differences between
3.2. Microbial community analysis and Dehalobacter phylogeny
the dual isotope slopes for the three groups of experiments: respiring
cells, cell suspensions, and crude protein extracts, were determined using
Amplicon sequencing of 16S rRNA genes from the stable enrichment
unpaired Z score-test. Comparisons were made using GraphPad Prism ver-
cultures derived from type 2 colonies revealed a microbial consortium com-
sion 8.0.2 software (GraphPad Software). Results were considered statisti-
posed of bacterial members belonging to the genera Dehalobacter (20%;
cally significant if p < 0.05.
phylum Firmicutes), Desulfovibrio (20%; phylum Desulfobacterota, formerly
known as class Deltaproteobacteria (Waite et al., 2020; Chen et al., 2017),
3. Results
and Proteiniphilum (57%; phylum Bacteroidetes). The 16S rRNA gene se-
quence of the Dehalobacter contained in this enrichment was 99.66% simi-
3.1. Enrichment of TCM-degrading bacteria
lar to the 16S rRNA gene of Dehalobacter restrictus strain PERK23, indicating
that the strain in our culture was likely the species D. restrictus. The close
After the dilution-to-extinction in liquid and consecutively in semisolid
similarity with D. restrictus was further supported by phylogenetical analy-
agar shakes, two different morphology of colonies appeared in the 10−9
sis using publicly available 16S rRNA gene sequences of Dehalobacter and
and 10−10 dilution tubes (Fig. S1): type 1 appeared as white, small,
Desulfitobacterium strains (Fig. 1). We tentatively denominated this strain
round, and compact colonies with defined edge, and type 2 appeared as
‘Dehalobacter sp. strain 8M’ to simplify naming purposes in this study.
white colonies, bigger than type 1, with non-defined edge and a cotton ap-
pearance. We picked four colonies of type 1, and six colonies of type 2, and
they were re-inoculated back in ten individual serum bottles spiked with 3.3. Dehalogenation of alternative electron acceptors
500 μM TCM in defined liquid medium. After 28 d, four out of the ten cul-
tures exhibited degradation of TCM, one from morphological type 1 and The capability of the Dehalobacter sp. strain 8M-containing culture to
three of morphological type 2. We then selected one active culture for transform alternative halogenated compounds was assessed replacing
each type and performed four consecutive dilution to extinction series TCM with different chloroalkanes [1,1,2-trichloroethane (1,1,2-TCA,
(Fig. S1) with liquid medium that contained 500 μM TCM. After the last se- 200 μM), 1,1,1-trichloroethane (1,1,1-TCA, 50 μM) and 1,1-dichloroethane
rial dilution, cultures were established in serum bottles with defined liquid (1,1-DCA, 50 μM)]; chloroalkenes [trichloroethylene (TCE, 100 μM) and
medium and after three further transfers and stable dechlorination rates, tetrachloroethylene (PCE, 100 μM)]; chlorobenzenes [chlorobenzene
Fig. 1. Phylogenetic tree of 16S rRNA genes of Dehalobacter sp. strain 8M (blue) along with other strains of Dehalobacter and Desulfitobacterium. Strains known for respiring
TCM are marked in bold. Phylogenetic analysis was performed using the Maximum Likelihood method and the Jukes-Cantor model. Numbers adjacent to tree branches
represent percentage of branch support based on 1000 bootstrap repetitions. The scale bar shows an evolutionary distance 0.02 nucleotide substitutions per site. GenBank
accession numbers are shown in parenthesis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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J.M. Soder-Walz et al. Science of the Total Environment 813 (2022) 152659
(MCB, 100 μM) and 1,2,4-trichlorobenzene (1,2,4-TCB, 100 μM)]; and the 3.5. C and Cl isotope fractionation during TCM transformation by respiring cells,
brominated analogue tribromomethane (TBM, 50 μM) in triplicate cultures. cell suspensions and crude protein extracts
Complete biodegradation of 1,1,2-TCA was observed after 10 days of in-
cubation (Fig. S4), producing vinyl chloride (VC) and 1,2-dicloroethane In order to explore the feasibility of 2D-CSIA for assessing TCM degrada-
(1,2-DCA) as degradation products in a ratio of 1:2.3, respectively. Both tion by Dehalobacter sp. strain 8M and provide insight into the reaction
products accumulated in the culture medium stoichiometrically with no mechanism and potential isotope-masking effects within this bacterial
further biodegradation. In an attempt to determine if this Dehalobacter genus, carbon and chlorine stable isotopes were measured during TCM de-
strain exhibited a preference for either TCM or 1,1,2-TCA, both compounds chlorination.
were added simultaneously in a culture that was grown previously with First, reductive dechlorination experiments with cultured cells of this
TCM. Both compounds were concomitantly dechlorinated within 10 days. consortium amended with 500 μM TCM were conducted (“respiring cells
However, after a second amendment, TCM was dechlorinated without an experiments”). The sterile abiotic controls showed no TCM degradation
observed lag phase and 1,1,2-TCA dechlorination started after a lag phase and the isotopic composition of TCM remained unchanged over the dura-
of two days (Fig. S5). tion of the experiment (total average values of δ13C = −47.8 ± 0.1‰
The dechlorination of 1,1,1-TCA, 1,1-DCA and TBM was incomplete and δ37Cl = −5.8 ± 0.1‰, n = 3). In contrast, TCM transformation up
with the removal of ~22%, ~13% and ~ 48% of their starting concentra- to 91% was reached in the respiring cells experiments after 9 d, with a stoi-
tions after 130 days of incubation and produced non-stoichiometric chiometric release of DCM. The remaining TCM became significantly
amounts of 1,1-DCA, chloroethane (CA) and dibromomethane (DBM), re- enriched in both 13C (up to −41.7 ± 0.7‰, Fig. S7) and 35Cl isotopes
spectively. These metabolites were not detected in the abiotic controls (up to −3.7 ± 0.3‰). The C and Cl isotopic fractionation associated
(Fig. S6). with anaerobic TCM degradation by this strain was determined by the Ray-
Apart from that, no transformation of PCE, TCE, MCB and 1,2,4-TCB leigh approach as εC = −2.8 ± 0.2‰ (R2 = 0.97) and εCl = −0.9 ±
was observed after 130 days (data not shown). 0.2‰ (R2 = 0.94), respectively (Fig. 3A and B). The δ13C of the produced
DCM was initially depleted in 13C, in agreement with the normal isotope ef-
fect for TCM transformation, and shifted toward more 13C-enriched values
3.4. Growth of Dehalobacter with TCM and 1,1,2-TCA during the course of reaction (Fig. S7). The carbon isotopic mass balance (δ-
13
Csum = −48.1 ± 0.3‰ at the end of the experiment) is consistent with
To demonstrate that Dehalobacter sp. strain 8M grew with TCM and the initial TCM carbon composition (−48.3 ± 0.1‰, horizontal dashed
1,1,2-TCA as energy source, real-time PCR (qPCR) was performed for line in Fig. S7). This also supports that all TCM was transformed to DCM,
both cultures growing separately with TCM and 1,1,2-TCA as electron ac- that no other intermediates metabolites were produced, and that DCM
ceptors and using H2 as the electron donor. Non-substrate controls, was not further degraded. The 2D-isotope plot (Δδ37Cl vs. Δδ13C) showed
consisting of Dehalobacter-containing culture without the corresponding ha- a good fit to linear regression (R2 = 0.95). A slope of ΛC/Cl = 2.8 ± 0.5
logenated compound, were also analyzed at the beginning and at the end of was obtained (Fig. 3C).
the experiment. In order to assess potential isotope-masking effects of rate-limiting steps
When cultivated with TCM, Dehalobacter increased two orders of magni- involving TCM diffusive transport, the system was simplified at two differ-
tude to 1.34·107 ± 2.19·106 gene copies mL−1 after consuming 1302 ± ent complexity levels by using cell suspension (concentrate of active intact
54 μM TCM (Fig. 2), which corresponded to a growth yield of cells) and crude protein extracts (from lysed cells). Under both scenarios,
2.06·106 ± 3.36·105 cells per μmol of TCM consumed (assuming 5 16S TCM was transformed (up to 98% consumption) and DCM was concomi-
rRNA gene per genome (Kruse et al., 2013)). Similarly, Dehalobacter in- tantly produced, whereas the control experiments showed no TCM degra-
creased two orders of magnitude to 6.22·107 ± 1.00·106 gene copies dation and a constant carbon (total average values of −47.8 ± 0.2‰ for
mL−1 after consuming 1444 ± 2 μM 1,1,2-TCA, with a growth yield of cell suspensions and − 49.1 ± 0.5‰ for crude protein extracts, n = 4)
8.61·106 ± 1.39·105 cells per μmol of 1,1,2-TCA consumed. In both cul- and chlorine (−6.3 ± 0.3‰ and − 5.2 ± 0.6‰, respectively, n = 3) iso-
tures, the growth of Dehalobacter in the non-substrate controls was negligi- tope composition of TCM. Significant carbon and chlorine isotopic fractio-
ble and the accumulation of the transformation products was nation was observed for TCM dechlorination by cell suspensions (εC =
stoichiometric with the amount of electron acceptor consumed (Fig. 2). −3.1 ± 0.3‰; εCl = −1.1 ± 0.2‰) and crude protein extract (εC =
−4.1 ± 0.5‰; εCl = −1.2 ± 0.2‰) (Fig. 3A and B). The εC values were
statistically different (p < 0.01) among the three systems studied and, as ex-
pected and discussed earlier, the greatest carbon isotope effect was ob-
served in the system with the lowest ‘complexity’ and therefore less
masked (from εC = −4.1 ± 0.5‰ with crude protein extracts to
−3.1 ± 0.2‰ with cell suspensions and − 2.8 ± 0.2‰ with respiring
cells). The differences in εCl values were, however, not statistically signifi-
cant among the three systems (p > 0.05), although the trend was the
same as in carbon (from −1.2 ± 0.2‰ to −1.1 ± 0.2‰ and − 0.9 ±
0.2‰, respectively).
The carbon and chlorine apparent kinetic isotope effect (AKIEC and
AKIE Cl ) values obtained for the three systems are listed in Table S1
and ranged in both cases between 1.003 and 1.004. These values
are much lower than the theoretical Streitwieser limit for carbon
(1.057) and chlorine (1.013) KIE for CCl bond cleavage, and still
lower than the suggested as realistic value with transition states at
about 50% bond cleavage (KIEC = 1.029 and KIECl = 1.007) (Elsner
Fig. 2. Growth of Dehalobacter sp. strain 8M during the reductive dehalogenation of
et al., 2005). Therefore, the enzymatic assays (cell suspensions and
TCM (blue) and 1,1,2-TCA (green) and the concomitant accumulation of the
transformation products. Error bars indicate standard deviations for triplicate
crude protein extracts) were both unable to unmask the intrinsic KIE
bottles. Black circle and black square with an X represent the non-substrate of TCM dechlorination by Dehalobacter sp. strain 8M discarding that
control for TCM and 1,1,2-TCA culture respectively. (For interpretation of the TCM transport through the outer membrane neither interactions with
references to colour in this figure legend, the reader is referred to the web version cellular structures within the periplasmic space are important rate-
of this article.) limiting steps.
5
J.M. Soder-Walz et al. Science of the Total Environment 813 (2022) 152659
bacteria that have been shown to metabolize sugars and are expected to de-
grade oligopeptides, but their role in organohalide-contaminated systems is
unclear (Tomazetto et al., 2018). It is hypothesized that they may feed on
proteinacious material from dead cells and/or yeast extract in the cultures
(Zhao et al., 2017; Tomazetto et al., 2018).
Our results provided evidence that Dehalobacter sp. strain 8M gained en-
ergy for growth using TCM and 1,1,2-TCA as electron acceptors and is likely
the responsible for the transformation of other halogenated compounds as
1,1,1-TCA, 1,1-DCA and TBM. To date, three strains belonging to
Dehalobacter (strains CF, THM1 and UNSWDHB) and Desulfitobacterium
sp. strain PR are described to respire TCM (Ding et al., 2014; Grostern
et al., 2010; Zhao et al., 2017; Wong et al., 2016). All these strains trans-
form TCM to DCM, except Desulfitobacterium sp. strain PR that also pro-
duces trace amounts of chloromethane (Ding et al., 2014). The 16S rRNA
gene sequence of the strain enriched here, named “Dehalobacter sp. strain
8M”, shared 99.66% similarity with the 16S rRNA gene of Dehalobacter
restrictus strain PER-K23. The similarity with Dehalobacter strain CF, strain
THM1, strain UNSWDHB is 99.21%, 96.81%, and 99.16%, respectively,
which indicates that the Dehalobacter strain in our culture likely belongs
to the species D. restrictus. Phylogenetic analysis of the 16S rRNA genes sug-
gests that Dehalobacter sp. strain 8M is grouped into a cluster with
D. restrictus strain PER-K23, strain E1 and strain TeCB1, of which, none of
them are known to respire TCM. D. restrictus strain PER-K23 is able to use
PCE and TCE as electron acceptor and produce cis-1,2-dichloroethene
(Holliger et al., 1998), strain E1 grows by dechlorination of β-
hexachlorocyclohexane to benzene and chlorobenzene (van Doesburg
et al., 2005), and strain TeCB1 grows by dechlorination of 1,2,4,5-
tetrachlorobenzene and 1,2,4-trichlorobenzene (Alfán-Guzmán et al.,
2017). Although strain 8M and strain UNSWDHB share the ability to trans-
form TCM, 1,1,2-TCA and, at a slower rate, 1,1,1-TCA, these two strains dif-
fer distinctively in the ratio of vinyl chloride to 1,2-DCA produced from
1,1,2-TCA dechlorination, consisting in 1:1.7 for strain UNSWDHB (Wong
et al., 2016) and 1:2.3 for strain 8M. The strong activity of Dehalobacter
strain CF, strain THM1 and Desulfitobacterium sp. strain PR toward 1,1,1-
TCA, which is transformed by the TCM-reductive dehalogenases CfrA,
ThmA and CtrA, respectively, contrasts with the incomplete and slow deg-
Fig. 3. Double logarithmic Rayleigh plot for carbon (A) and chlorine (B) isotope radation of 1,1,1-TCA by Dehalobacter strains UNSWDHB (Ding et al., 2014;
data and dual carbon and chlorine isotope plot (C) for the three studied Zhao et al., 2017; Wong et al., 2016; Tang and Edwards, 2013) and 8M.
experiment systems (respiring cells, cell suspensions and crude protein extracts) This finding provides additional supporting evidence that substrate speci-
during dechlorination of TCM by the Dehalobacter sp. strain 8M-containing ficity of reductive dehalogenases is not necessarily correlated with the
culture. Dotted lines indicate 95% confidence intervals. The error bars structural similarity of the electron acceptor or the phylogenetic affiliation
represented the uncertainty calculated by error propagation including
of organisms. The inefficient degradation of 1,1,1-TCA, 1,1-DCA and TBM
uncertainties in concentration and isotope measurements.
might point to a cometabolic degradation of these substrates.
Carbon and chlorine isotope effects were analyzed to pinpoint the reac-
As mentioned above, respiring cells displayed a slope of ΛC/Cl = 2.8 ± tion mechanism of TCM dechlorination in Dehalobacter sp. strain 8M in the
0.5, for the cell suspensions and crude protein extracts experiments the dual light of a recent study that addressed this question with two other
element isotope plot showed a slope of ΛC/Cl = 2.5 ± 0.3 and ΛC/Cl = Dehalobacter strains (strain CF and UNSWDHB) (Heckel et al., 2019). Inter-
2.7 ± 0.5 respectively (Fig. 3C). Consistently, there were not significant estingly, the results indicate that all three Dehalobacter strains exhibit dis-
differences among the slopes for the three systems (p > 0.05). Thus, the tinctive carbon and chlorine isotope fractionation patterns (Table S1). In
slope obtained after integrating the three data sets in one linear regression both strains UNSWDHB and 8M, a small and indistinguishable (p > 0.05)
was ΛC/Cl = 2.8 ± 0.3 (R2 = 0.94) (Fig. 3C). carbon isotope effect is observed for TCM dechlorination in respiring cells
experiments (εC = −3.1 ± 0.5‰ and − 2.8 ± 0.2‰, respectively),
4. Discussion whereas degradation by strain CF resulted in a much more pronounced car-
bon isotope fractionation (εC = −28 ± 2‰). Resulting εCl values are sig-
In this study, we enriched a Dehalobacter-containing culture capable of nificantly different (p < 0.001) among these three strains, with a normal
dechlorinating TCM, 1,1,2-TCA, 1,1,1-TCA, 1,1-DCA and TBM. The result- chlorine isotope effect for strains CF and 8M (εCl = −4.2 ± 0.2‰
ing culture was composed of the genera Dehalobacter (20%), Desulfovibrio and − 0.9 ± 0.2‰, respectively), versus the unexpected inverse chlorine
(20%), and Proteiniphilum (57%). Species of the genus Desulfovibrio have isotope effect observed previously for strain UNSWDHB (εCl = +2.5 ±
been frequently detected accompanying organohalide-respiring bacteria, 0.3‰). For strains UNSWDHB and 8M, however, the carbon and chlorine
including Dehalobacter co-cultures that transform TCM (Waite et al., kinetic isotope effect of the CCl bond cleavage is masked (Table S1). Exper-
2020). Previous studies showed that growth of Desulfovibrio populations iments with cell suspensions and crude protein extracts in strain UNSWDHB
displayed no correlation with TCM dechlorination (Zhao et al., 2017) and resulted in the same small carbon isotopic fractionation as with respiring
that interspecies interactions involving the presence of members of cells, whereas the inverse chlorine isotope effect increased with the increas-
Desulfovibrio may benefit the degrading microbial community by mediating ing complexity of the system (respiring cells > cell suspensions > crude pro-
acetate and H2 sources for dechlorinating bacteria (Trueba-Santiso et al., tein extracts), leading to significantly different 2D-isotope slopes (Heckel
2020; Wang et al., 2019). Members of Proteiniphilum are obligate anaerobic et al., 2019). This observation suggested to the authors that contribution
6
J.M. Soder-Walz et al. Science of the Total Environment 813 (2022) 152659
to the inverse chlorine isotope effect was probably due to rate-limiting steps confirming its catalyzing effect. However, active microbial community
during transport prior to CCl bond cleavage. However, the membrane- through 16S rRNA MiSeq high-throughput sequencing revealed that up to
associated and periplasm-oriented location of the corresponding TCM re- now well-known TCM degraders, Dehalobacter and Desulfitobacterium, were
ductive dehalogenase is expected to be the same in all Gram-negative below 0.1% of relative abundance and the cfrA gene was not detected. The
Dehalobacter (Jugder et al., 2017), including our strain 8M. The authors distinct value of ΛC/Cl obtained in the present study for strain 8M (ΛC/Cl =
discarded the nondirected intermolecular interactions and therefore other 2.8 ± 0.3) compared to the value obtained in the abovementioned contam-
uncharacterized interactions of TCM with cellular structures were adduced inated site, may indicate that an important contribution of a Dehalobacter sp.
to contribute to the unprecedented inverse isotope effects (Heckel et al., strain similar to 8M in that field site is very unlikely.
2019). In our case, however, the similar decrease of the carbon and chlorine Further identification and localization of the TCM reductive
isotope effect with higher complexity of the system of strain 8M, resulting dehalogenase in Dehalobacter sp. strain 8M and its amino acid similarity
in identical ΛC/Cl values for the three systems (p > 0.05) overruled with TmrA (TCM reductive dehalogenase in strain UNSWDHB) and CfrA
membrane-induced masking effects. This result is important as it is differ- (strain CF) will help to understand the distinct isotope fractionation ob-
ent from other gram-negative bacteria degrading PCE (Renpenning et al., served in this study and to look for these useful complementary target func-
2015) or atrazine (Ehrl et al., 2018) where mass transfer through the cell tional and phylogenetic markers in future field studies.
envelope was identified as a relevant rate-limiting step causing masking ef-
fects. However, taking into account that AKIEs of crude protein extracts of 5. Environmental significance
8M (without this barrier) were unable to unmask the intrinsic KIE, partially
rate-limiting enzyme binding previous to the CCl cleavage can be deduced. The widespread presence of TCM in groundwater has led to increased
The dual element isotope approach illustrates thus remarkable different public concern and the need for tools and strategies to achieve its efficient re-
(p < 0.0001) slopes (ΛC/Cl) within the strains that have been previously re- mediation. The present study demonstrates that effective reductive dechlori-
ported to respire TCM (strains CF and UNSWDHB) compared to nation of TCM by a novel Dehalobacter strain is accompanied by a significant
Dehalobacter sp. strain 8M (Table S1 and Fig. 4). In Fig. 4, in addition, char- carbon and chlorine isotope fractionation, which can serve as both quantita-
acteristic slopes of other TCM degradation processes are presented based on tive and qualitative indicators of anaerobic biodegradation in TCM-impacted
carbon and chlorine isotope fractionation that are also mentioned in groundwaters. But more importantly, dual CCl isotope fractionation pattern
Table S1. during TCM degradation by this novel Dehalobacter differs from the fractiona-
In the case of strain CF, the ΛC/Cl value (6.6 ± 0.1) was undistinguishable tion pattern reported for Dehalobacter strains CF and UNSWDHB, which
to that obtained for abiotic TCM dechlorination by vitamin B12 (6.5 ± 0.2) would clearly allow to distinguish its contribution to TCM degradation in a
(Heckel et al., 2019). This was in accordance with an expected common reac- field site. In this scenario, when ΛC/Cl obtained from site-specific isotopic
tion mechanism (likely either outer-sphere-single-electron transfer (OS-SET) data coincides with ΛC/Cl from reference laboratory experiments, the deter-
or SN2 nucleophilic substitution mechanism) for TCM dechlorination be- mined carbon and chlorine isotopic fractionation values are valuable to quan-
tween the reductive dehalogenase of Dehalobacter and the cobamide cofactor tify the degree of in situ biodegradation (%) between the source and a
located within this enzyme (Heckel et al., 2019). A similar ΛC/Cl value (7 ± monitoring point (or with time) in the field using the modified Rayleigh
1) was also obtained from biotic field-derived anoxic microcosms (from a dif- equation as recommended by the US EPA guide (Hunkeler et al., 2008). To
ferent contaminated site close to Barcelona, Spain) amended with vitamin this regard, taking into account that differences in isotopic compositions in
B12 as catalyzer. In that case, pure abiotic reaction with B12 was discarded the field for both carbon and chlorine (Δδ13C, Δδ37Cl) must be >2‰ for
in killed controls (Rodríguez-Fernández et al., 2018b), but biodegradation being considered significant (Hunkeler et al., 2008), TCM biodegradation
of TCM did not take place in the live microcosms without this vitamin, from 51% and 89%, respectively would be detectable for strain 8M. In fact,
in a previous study in the site from where strain 8M was enriched
(Blázquez-Pallí et al., 2019), up to 11 ± 7% TCM degradation was estimated
considering the εC reported in literature for Dehalobacter sp. strain CF (εC =
−28‰, (Heckel et al., 2019)). Based on the results of the present work,
the extent of biotransformation was very likely underestimated; applying
the εC calculated here for strain 8M (−2.8 ± 0.2‰) this estimation goes
up to 69 ± 2%. This fact emphasizes the importance of using site-specific iso-
topic fractionation values derived from microcosm experiments prepared
with soil and/or groundwater from the contaminated site instead of litera-
ture values. Moreover, strain 8M provides a distinct (p < 0.0001) ΛC/Cl
from all the so far described abiotic treatments for TCM remediation (see
Fig. 4), which makes this strain a perfect candidate for bioaugmentation
treatments because biotransformation of TCM by strain 8M can be distin-
guished from abiotic transformation and can be reliably quantified by dual-
isotopic assessment. The present study data provides key information for
practitioners to support the interpretation of stable isotope analyses derived
from chloromethanes polluted sites.
Fig. 4. Dual CCl trends during TCM degradation by different strains and different CRediT authorship contribution statement
degradation processes (see references in Table S1). Legend: oxidation with
persulfate (PS) in red; alkaline hydrolysis (AH) in orange; hydrogenolysis plus Jesica M. Soder-Walz: Investigation, Writing – original draft, Formal
reductive elimination by Fe(0) in brown; biodegradation on field-derived anoxic analysis, Visualization. Clara Torrentó: Methodology, Validation, Writing
microcosms amended with vitamin B12 (Bio B12) in dark green; abiotic
– review & editing. Camelia Algora: Methodology, Writing – review &
dechlorination by vitamin B12 (B12) in soft green; outer-sphere single electron
editing. Kenneth Wasmund: Methodology, Writing – review & editing.
transfer (OS-SET) in gray; dehalogenation by Dehalobacter restrictus strain CF
(strain CF) in black, D. restrictus strain UNSWDHB (strain UNSWDHB) in pink, and
Pilar Cortés: Methodology, Validation. Albert Soler: Resources. Teresa
Dehalobacter sp. strain 8M (strain 8M) in blue. Results from cell suspensions and Vicent: Funding acquisition, Project administration, Writing – review &
crude protein extract experiments for strains UNSWDHB and 8M are also shown. editing. Mònica Rosell: Methodology, Validation, Supervision, Writing –
(For interpretation of the references to colour in this figure legend, the reader is review & editing. Ernest Marco-Urrea: Supervision, Conceptualization,
referred to the web version of this article.) Writing – review & editing
7
J.M. Soder-Walz et al. Science of the Total Environment 813 (2022) 152659
Declaration of Competing Interest Nijenhuis, I., Richnow, H.H., 2016. Stable isotope fractionation concepts for characterizing
biotransformation of organohalides. Curr. Opin. Biotechnol. 41, 108–113. https://doi.
org/10.1016/j.copbio.2016.06.002.
The authors declare that they have no known competing financial inter- Rosell, M., Gonzalez-Olmos, R., Rohwerder, T., Rusevova, K., Georgi, A., Kopinke, F.D., et al.,
ests or personal relationships that could have appeared to influence the 2012. Critical evaluation of the 2D-CSIA scheme for distinguishing fuel oxygenate degra-
dation reaction mechanisms. Environ Sci Technol. 46 (9), 4757–4766.
work reported in this paper. Renpenning, J., Keller, S., Cretnik, S., Shouakar-Stash, O., Elsner, M., Schubert, T., et al., 2014.
Combined C and cl isotope effects indicate differences between corrinoids and enzyme
Acknowledgements (Sulfurospirillum multivorans PceA) in reductive dehalogenation of tetrachloroethene,
but not trichloroethene. Environ Sci Technol. 48 (20), 11837–11845.
Gafni, A., Gelman, F., Ronen, Z., Bernstein, A., 2020. Variable carbon and chlorine isotope
This work has been supported by the Spanish Ministry of Economy and fractionation in TCE co-metabolic oxidation. Chemosphere 242, 125130. https://doi.
Competitiveness State Research Agency (CTM2016-75587-C2-1-R, org/10.1016/j.chemosphere.2019.125130 Available from:.
Blázquez-Pallí, N., Shouakar-Stash, O., Palau, J., Trueba-Santiso, A., Varias, J., Bosch, M.,
PID2019-103989RB-100 and CGL2017-87216-C4-1-R projects) cofinanced
Soler, A., Vicent, T., Marco-Urrea, E., Rosell, M., 2019. Use of dual element isotope anal-
by the European Union through the European Regional Development Fund ysis and microcosm studies to determine the origin and potential anaerobic biodegrada-
(ERDF). This work was partly supported by the Generalitat de Catalunya tion of dichloromethane in two multi-contaminated aquifers. Sci. Total Environ. 696,
(Consolidate Research Groups 2017-SGR-14 and 2017-SGR-1733). We thank 134066. https://doi.org/10.1016/j.scitotenv.2019.134066.
Rodríguez-Fernández, D., Torrentó, C., Palau, J., Marchesi, M., Soler, A., Hunkeler, D.,
Angie Steffany Albarracín and Natàlia Blázquez Pallí for their collaboration Domènech, C., Rosell, M., 2018. Unravelling long-term source removal effects and chlo-
in this project and CCiT-UB for the technical support. rinated methanes natural attenuation processes by C and cl stable isotopic patterns at a
complex field site. Sci. Total Environ. 645, 286–296. https://doi.org/10.1016/j.
scitotenv.2018.07.130.
Appendix A. Supplementary data Heckel, B., Phillips, E., Edwards, E., Sherwood Lollar, B., Elsner, M., Manefield, M.J., Lee, M.,
2019. Reductive dehalogenation of trichloromethane by two different dehalobacter
Supplementary data to this article can be found online at https://doi. restrictus strains reveal opposing dual element isotope effects. Environ. Sci. Technol. 53
(5), 2332–2343. https://doi.org/10.1021/acs.est.8b03717.
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