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Dual carbon-chlorine isotope analysis indicates distinct anaerobic dichloromethane degradation pathways in two members of the Peptococcaceae Gao Chen, Orfan Shouakar-Stash, Elizabeth Phillips, Shandra D. Justicia-Leon, Tetyana Gilevska, Barbara Sherwood Lollar, Elizabeth Erin Mack, Edward S. Seger, and Frank E Löffler Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01583 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018
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Environmental Science & Technology
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Dual carbon-chlorine isotope analysis indicates distinct anaerobic dichloromethane
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degradation pathways in two members of the Peptococcaceae
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Gao Chen,† Orfan Shouakar-Stash,§,‡,Ω Elizabeth Phillips,# Shandra D. Justicia-Leon,◊,ǁ Tetyana
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Gilevska,# Barbara Sherwood Lollar,#,* E. Erin Mack,⊥ Edward S. Seger,∆ and Frank E.
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Löffler†,∏,∇,*
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†
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and Department of Microbiology, University of Tennessee, Knoxville, TN 37996, USA
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§
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‡
Center for Environmental Biotechnology, Department of Civil and Environmental Engineering,
Isotope Tracer Technologies Inc. (IT2), Waterloo, Ontario N2V 1Z5, Canada Department of Earth and Environmental Sciences, University of Waterloo, Waterloo, Ontario
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N2L 3G1, Canada
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Ω
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#
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◊
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⊥
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DE 19805, USA
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∆
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∏
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Biological Sciences (JIBS) and Biosciences Division, Oak Ridge National Laboratory, Oak
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Ridge, TN 37831, USA
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∇
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TN 37996, USA
School of Engineering, University of Guelph, Guelph, Ontario N1G 2W1, Canada
Department of Earth Sciences, University of Toronto, Toronto, Ontario, M5S 3B1, Canada
School of Biology, Georgia Institute of Technology, Atlanta, GA 30332, USA DuPont Corporate Remediation Group, E. I. DuPont de Nemours and Company, Wilmington,
The Chemours Company, Wilmington, DE 19899, USA University of Tennessee and Oak Ridge National Laboratory (UT-ORNL) Joint Institute for
Department of Biosystems Engineering and Soil Science, University of Tennessee, Knoxville,
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*Corresponding authors
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Frank E. Löffler
Barbara Sherwood Lollar
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University of Tennessee
University of Toronto
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Department of Microbiology
Department of Earth Sciences
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M409 Walters Life Science Bldg.
22 Russell St.
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Knoxville, TN 37996, USA
Toronto, ON M5S 3B1, Canada
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Phone: (865) 974-4933
Phone: (416) 978-0770
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Fax: (865) 974-4007
Fax: (416) 978-3938
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E-mail:
[email protected] E-mail:
[email protected] Page 2 of 34
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Running title: Carbon and chlorine fractionation during anaerobic DCM degradation
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Keywords: dichloromethane, dual C-Cl isotope fractionation, anaerobic degradation, pathways,
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Peptococcaceae family
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ǁ
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00968
Current address: 48 City View Plaza I, Suite 401, Road 165 Km 1.2, Guaynabo, Puerto Rico,
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Abstract
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Dichloromethane (DCM) is a probable human carcinogen and frequent groundwater
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contaminant, and contributes to stratospheric ozone layer depletion. DCM is degraded by
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aerobes harboring glutathione-dependent DCM dehalogenases; however, DCM contamination
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occurs in oxygen-deprived environments and much less is known about anaerobic DCM
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metabolism. Some members of the Peptococcaceae family convert DCM to environmentally
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benign products including acetate, formate, hydrogen (H2), and inorganic chloride under strictly
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anoxic conditions. The current study applied stable carbon and chlorine isotope fractionation
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measurements to the axenic culture Dehalobacterium formicoaceticum and to the consortium
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RM comprising the DCM degrader Candidatus Dichloromethanomonas elyunquensis.
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Degradation-associated carbon and chlorine isotope enrichment factors (εC and εCl) of –42.4 ±
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0.7‰ and –5.3 ± 0.1‰, respectively, were measured in D. formicoaceticum cultures. A similar
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εCl of –5.2 ± 0.1‰, but a substantially lower εC of –18.3 ± 0.2‰, were determined for Ca.
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Dichloromethanomonas elyunquensis. The εC and εCl values resulted in distinctly different dual
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element C-Cl isotope correlations (ΛC/Cl = ∆δ13C / ∆δ37Cl) of 7.89 ± 0.12 and 3.40 ± 0.03 for D.
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formicoaceticum and Ca. Dichloromethanomonas elyunquensis, respectively. The distinct ΛC/Cl
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values obtained for the two cultures imply mechanistically distinct C-Cl bond cleavage reactions,
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suggesting that members of Peptococcaceae employ different pathways to metabolize DCM.
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These findings emphasize the utility of dual carbon-chlorine isotope analysis to pinpoint DCM
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degradation mechanisms, and to provide an additional line of evidence that detoxification is
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occurring at DCM-contaminated sites.
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Introduction
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Substantial amounts of dichloromethane (DCM) are produced naturally in the oceans, wetlands,
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and soils, or through volcanic activity and emissions; however, the majority of DCM
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contamination in aquifers is due to anthropogenic activities.1 DCM is an excellent solvent, and
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has been widely used as paint remover, dry-cleaning solvent, and degreasing agent in
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electronics, manufacturing, and machine maintenance industries.2 As a legacy of extensive
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usage, as well as improper handling and storage, DCM is commonly detected in subsurface
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environments, and in groundwater and drinking-water supply wells in the United States (U.S.).3
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DCM was measured in groundwater samples taken throughout the contiguous U.S. in
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concentrations ranging from 0.02 to 100 µg L–1 with a detection frequency of 3%.4 The U.S.
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Environmental Protection Agency (EPA) catalogs DCM as a probable human carcinogen and
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has classified DCM as a priority contaminant with a maximum contaminant level (MCL) in
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drinking water of 5 µg L–1 (ppb).5,6 In addition, DCM causes ozone layer depletion and is an
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increasing threat to stratospheric ozone.7
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DCM degradation under oxic and nitrate-reducing conditions is mediated by aerobes and
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facultative bacteria containing glutathione-dependent DCM dehalogenases and leads to
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complete detoxification to carbon dioxide (CO2) and inorganic chloride (Cl–).8-10 In contrast, the
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details of DCM degradation under strictly anoxic conditions have remained elusive because of
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the limited number of cultures and the fastidious growth of these bacteria. Dehalobacterium
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formicoaceticum, affiliated with the Peptococcaceae family, is so far the only pure culture
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utilizing DCM as a growth substrate under strictly anoxic conditions, generating acetate,
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formate, and Cl– according to Eq. 1.11,12
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3 CH2Cl2 + 4 H2O + CO2 → 2 HCOO– + CH3COO– + 9 H+ + 6 Cl–
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(1)
Recently, a few mixed cultures harboring bacteria affiliated with the Peptococcaceae
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family, e.g., Dehalobacter sp., and/or Dehalobacterium sp., were reported to degrade DCM
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anaerobically, generating acetate as the major degradation product.13-17 Consortium RM was
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derived from pristine Rio Mameyes River sediment collected near the El Yunque National Forest
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in Puerto Rico,15 and the DCM-degrading bacterium in the mixed culture has been identified as
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Candidatus Dichloromethanomonas elyunquensis, representing a new genus and species within
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the Peptococcaceae family, which is able to metabolize DCM to acetate, H2, CO2, and Cl–
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according to Eq. 2.16,18
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3 CH2Cl2 + 4 H2O → 2 H2 + CO2 + CH3COO– + 7 H+ + 6 Cl–
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(2)
Physiological and biochemical studies of D. formicoaceticum and Ca.
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Dichloromethanomonas elyunquensis suggested that DCM is degraded via a fermentation
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pathway under strictly anoxic conditions (Eq. 1 and Eq. 2)12,15,18 After removal of the chlorine
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substituents, DCM is transformed to methylene-tetrahydrofolate (CH2=H4folate), funneled into
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the Wood-Ljungdahl pathway (i.e., reductive acetyl-CoA pathway) and disproportionated. Two
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thirds of CH2=H4folate is oxidized to formate or H2/CO2 generating reducing equivalents, and the
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remaining third, in combination with CO2, is reduced to acetate (Eq. 1 and Eq. 2).12,18 For both
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bacterial cultures, the removal of the chlorine substituents has not been resolved.
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Practitioners confront challenges both in providing definitive evidence that
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bioremediation will lead to detoxification, and in delineating biodegradation mechanisms so that
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optimization of remedial strategies is possible. Decreasing contaminant concentrations
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generally do not provide sufficient evidence that cleanup will be achieved because physical
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processes such as sorption or dilution, rather than degradation, may be the driver behind the
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observations. Compound specific isotope analysis (CSIA) provides an effective means of
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directly assessing transformation of organic contaminants in situ and has received increasing
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attention in recent years.19,20 CSIA measures stable isotope ratios of carbon and/or other
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elements of contaminants dissolved in groundwater at environmentally relevant concentrations,
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i.e., parts per million (ppm, mg L–1) to parts per billion (ppb, µg L–1). CSIA is in principal based
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on kinetic isotope effects (KIEs) of biochemical reactions, viz. chemical bonds containing
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exclusively light isotopes (e.g., 12C, 35Cl) are preferentially cleaved compared with those
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containing one or more heavy isotopes (e.g., 13C, 37Cl), resulting in a relative enrichment in
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molecules containing the heavier isotope in the remaining contaminant mass – a process called
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isotopic fractionation.21 CSIA has been successfully implemented to evaluate and monitor
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degradation of many organic contaminants, including hydrocarbons,22-24, organohalogens,25-28
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and pesticides,29,30 in both laboratory and in situ studies. KIEs can be useful in elucidating
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degradation reaction mechanisms, and this approach has been successfully applied to identify
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characteristic fractionation factors associated with different degradation pathways.19
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Previous studies explored stable carbon isotope fractionation during DCM degradation
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by methylotrophic bacteria under oxic and nitrate-reducing conditions.31-33 This prior work31-33
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demonstrated a significant stable carbon isotope fractionation during DCM degradation, with εC
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values ranging from −22 to −66.3‰ under oxic conditions and −26 to −61.0‰ under nitrate-
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reducing conditions. Notably, in all of the aerobic and facultative anaerobic bacterial strains
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tested, DCM degradation is carried out by glutathione-dependent dehalogenases catalyzing
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nucleophilic substitution reactions.9,10,32 Limited information is available about the carbon isotope
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fractionation associated with DCM degradation performed by strict anaerobes. Carbon isotope
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enrichment factors (εC) of −15.5 ± 1.5‰ and −27 ± 2‰ were reported during anaerobic DCM
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degradation in mixed cultures containing Dehalobacter14 and Dehalobacterium17 populations,
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respectively. However, single element isotope effects can be masked by the bioavailability of
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the electron donor or the electron acceptor,34-36 mass transfer limitations,37,38 uptake and
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transport of a substrate across the cell membrane,39,40 microbial cell densities,41 and/or
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preceding enzymatic reactions42. Such other rate-limiting steps can lead to observed apparent
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kinetic isotope effect (AKIE) values that are smaller than those predicted based on the
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associated bond cleavage. A recent study suggested that the concentration of dissolved organic
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carbon and toxic effects of DCM and other contaminants may affect the carbon isotope
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fractionation associated with aerobic DCM degradation.33 In such cases, two-dimensional or
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dual isotope analysis - the stable isotope fractionation of two elements (e.g., 13C and 37Cl)
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involved in the reaction - is helpful, as masking typically affects both elements, and while the
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single element fractionation factors may be masked, the ratio of fractionation factors, expressed
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as the lambda value (ΛC/Cl = ∆δ13C/ ∆δ37Cl), is independent of such complicating effects.43,44
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Only a single study has applied carbon and chlorine dual isotope analysis to investigate
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DCM biodegradation by the aerobic bacterium, Hyphomicrobium sp. strain MC8b.31 In order for
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this approach to be successfully applied to monitor anaerobic biodegradation of DCM in the
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field, reference dual isotope studies under anoxic conditions are needed. To explore the
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possible application of CSIA as a tool for monitoring DCM degradation under strictly anoxic
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conditions and for probing possible mechanism(s) of C-Cl bond cleavage, carbon and chlorine
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stable isotopes were measured during growth of D. formicoacetium and Ca.
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Dichloromethanomonas elyunquensis in defined anoxic medium supplied with DCM as the sole
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energy source.
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Material and Methods
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Cultures, cultivation conditions, and experimental setup. The DCM-degrading axenic
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culture D. formicoaceticum was obtained from the American Type Culture Collection (ATCC
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700118).11,12 Consortium RM comprising Ca. Dichloromethanomonas elyunquensis was derived
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from freshwater sediments collected from pristine Rio Mameyes in Puerto Rico.15,16 Both
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cultures were grown with DCM as the growth substrate in defined, strictly anoxic mineral salt
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medium containing 30 mM of sodium bicarbonate (NaHCO3) (pH 7.3) reduced with 0.2 mM
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sodium sulfide (Na2S) and 0.2 mM L-cysteine.45
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The experiments were conducted in 300-mL Duran glass vessels (Chemglass, Vineland,
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NJ, US) containing 200 mL of medium sealed with black butyl rubber stoppers (Bellco Glass
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Inc., Vineland, NJ, US) under a headspace of N2/CO2 (80/20, vol/vol). Culture vessels were
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amended with 25 µL neat DCM (total amount ca. 400 µmoles; DCM aqueous concentration ca.
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2 mM) using a 10-µL Hamilton microliter syringe (Reno, NV, US) prior to inoculation (5%,
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vol/vol) from a DCM-grown culture, i.e., D. formicoaceticum or Ca. Dichloromethanomonas
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elyunquensis. Six replicate vessels were prepared for each culture. Controls consisted of
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duplicate vessels that received 10 mL of autoclaved inocula, and duplicate vessels amended
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with DCM but not receiving inocula. All culture vessels were incubated at 30°C in the dark with
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the stoppers up without agitation. Growth was assessed by monitoring DCM consumption.
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During the incubation, 8 mL aqueous samples were periodically withdrawn using N2-flushed
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glass syringes and transferred to closed glass serum vials (vol. = ca. 7.5 mL) (Supelco,
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Bellefonte, PA, US) without any headspace. A volume of 10 µL saturated mercuric chloride
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(HgCl2) solution was added to the serum vials to inhibit microbial activity and mixed thoroughly
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following the addition of culture samples. All samples were stored at 4°C in the dark until carbon
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and chlorine stable isotope analysis.
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DCM concentration analysis and DCM degradation rate calculations. DCM headspace
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concentrations were monitored by injecting 0.1 mL headspace samples into an Agilent 7890A
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gas chromatograph (GC) (Santa Clara, CA, US) equipped with a DB-624 column (60 m length,
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0.32 mm i.d., 1.8 µm film thickness) and a flame ionization detector (FID). The GC inlet was
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maintained at 200°C, the GC oven temperature was kept at 60°C for 2 minutes followed by an
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increase to 200°C at a ramping rate of 25°C min–1, and the FID detector was operated at 300°C.
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DCM calibration curves were prepared by adding DCM (ca. 8 - 800 µmoles) to 300-mL culture
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vessels containing 200 mL of medium. The uncertainty for DCM concentration measurements
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using GC/FID was
