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Survival of Vinyl Chloride Respiring Dehalococcoides mccartyi Under Long-Term Electron Donor Limitation Koshlan Mayer-Blackwell, Mohammad F Azizian, Jennifer K. Green, Alfred M. Spormann, and Lewis Semprini Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05050 • Publication Date (Web): 21 Dec 2016 Downloaded from http://pubs.acs.org on January 3, 2017

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Biodegradation Rate

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7-Year Electron Donor Limitation

TCE VC

3-Month Electron Donor Stimulation

TCE

TCE VC

Dehalococcoides mccartyi sp. Population Fraction

VC

D.mccartyi (tceA)

1:1 10:1 D.mccartyi (vcrA)

100:1 2009

2010

2011

2012

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Title: Survival of Vinyl Chloride Respiring Dehalococcoides mccartyi Under Long-Term

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Electron Donor Limitation

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Authors: Koshlan Mayer-Blackwell†, Mohammad F. Azizian‡, Jennifer K. Green‡, Alfred M.

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Spormann †§⊥, Lewis Semprini‡*

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†Civil and Environmental Engineering, §Geological and Environmental Sciences, and

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⊥Chemical Engineering, Stanford University, Stanford, California 94305, United States

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‡Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis,

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Oregon 97331, United States

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*Corresponding Author

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Abstract: In anoxic groundwater aquifers, the long-term survival of Dehalococcoides mccartyi

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populations expressing the gene vcrA (or bvcA) encoding reductive vinyl chloride dehalogenases

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are important to achieve complete dechlorination of tetrachloroethene (PCE) and trichloroethene

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(TCE) to non-chlorinated ethene. The absence or inactivity of vcrA-containing Dehalococcoides

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results in the accumulation of the harmful chlorinated intermediates dichloroethene (DCE) and

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vinyl chloride (VC). While vcrA-containing Dehalococcoides subpopulations depend on

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synergistic interaction with other organohalide-respiring populations generating their metabolic

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electron acceptors (DCE and VC), their survival requires successful competition for electron

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donor within the entire organohalide-respiring microbial community. To understand this dualism

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of synergy and competition under growth conditions relevant in contaminated aquifers, we

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investigated Dehalococcoides-level population structure when subjected to a change in ratio of

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electron donor to chlorinated electron acceptor in continuously stirred reactors (CSTRs) operated

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over seven years. When the electron donor formate was supplied in stoichiometric excess to

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TCE, both tceA-containing and vcrA-containing Dehalococcoides populations persisted, and

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near-complete dechlorination to ethene was stably maintained. When the electron donor formate

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was supplied at sub-stoichiometric concentrations, the interactions between tceA-containing and

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vcrA-containing populations shifted towards direct competition for the same limiting catabolic

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electron donor substrate with subsequent niche exclusion of the vcrA-containing population.

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After more than 2000 days of operation under electron donor limitation, increasing the electron

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donor to TCE ratio facilitated a recovery of the vcrA-containing Dehalococoides population to its

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original frequency. We demonstrate that electron donor scarcity alone, in the absence of

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competing metabolic processes or inhibitory dechlorination intermediate products, is sufficient to

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alter Dehalococcoides population structure. These results underscore the importance of electron

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donor/chloroethene stoichiometry in maintaining balanced functional performance within

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consortia comprised of multiple Dehalococcoides mccartyi subpopulations, even when other

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competing electron acceptor processes are absent.

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1. Introduction

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In situ bioremediation using organohalide-respiring bacteria is a viable and often cost-

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effective strategy for the removal of chlorinated solvents – such as trichloroethene (TCE) and

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tetrachloroethene (PCE) – from anoxic contaminated groundwater.1 Complete reductive

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dehalogenation of PCE and TCE to ethene occurs in a stepwise fashion via dichloroethene

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(DCE) and vinyl chloride (VC) as intermediates. There is apparent metabolic specialization

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among organohalide-respiring microorganisms and Dehalococcoides mccartyi strains to catalyze

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specific dehalogenation steps in the overall chloroethene dehalogenation pathway. Thus, the

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appropriate assembly of microorganisms – collectively containing the full inventory of genes

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necessary for the complete dechlorination of PCE or TCE – is a prerequisite for preventing the in

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situ buildup of toxic intermediates DCE or VC.

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Complete dechlorination of PCE or TCE in situ depends not only on the initial bacterial

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community composition, but also on the long-term survival of obligate organohalide-respiring

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Dehalococcoides mccartyi sp. expressing the genes encoding VcrA (or BvcA) reductive

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dehalogenases catalyzing VC reduction to ethene.2-7 Based on evidence from isolate studies,

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vcrA-containing Dehalococcoides are niche-specialized and typically lack those reductive

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dehalogenases required to efficiently dechlorinate PCE or TCE. Therefore, while vcrA-

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containing Dehalococcoides populations depend on other organohalide-respiring populations for

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a supply of metabolic electron acceptors (DCE and VC), their survival involves successful

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competition, within the entire organohalide-respiring population, for molecular hydrogen as an

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electron donor. Studying such competition among Dehalococcoides subpopulations in batch

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growth experiments has limitations, as these growth conditions do not represent the persistent

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slow flux of electron donor that may limit bacteria within subsurface plume environments.

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We, therefore, investigated Dehalococcoides level population structure and dynamics in

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continuously stirred reactors (CSTRs), operated as chemostats, as we varied the ratio of electron

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donor to chlorinated electron acceptor. A chemostat is a continuous-growth laboratory model

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system that enables direct competition among microorganism at low substrate concentrations. It

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captures an essential feature of plume conditions where microorganisms may experience low but

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steady substrate concentrations and prolonged electron donor limitation. However, mineral

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surfaces for attached microbial growth, which may also influence population structure in

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subsurface habitats, are missing from a chemostat. Despite this limitation, the chemostat

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uniquely permits precise control of growth conditions and reproducible non-destructive sampling

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of a microbial community for molecular analysis and kinetic batch testing. For this experiment,

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we operated four CSTRs, as chemostats, over a period of seven years at a mean residence time of

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50 days.

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We studied the outcome of competition for electron donor in four CSTRs, catalyzing the

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transformation of TCE to ethene (Table 1) under two growth conditions (Table 2). Formate was

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provided as a soluble electron donor which can be converted to hydrogen via the action of

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syntrophic bacteria in the presence of hydrogen consuming bacteria.8,9 Formate was chosen

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based on its stoichiometric conversion to hydrogen and the production of bicarbonate, which

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serves as a buffer for the acid released via dechlorination,10 thus providing for excellent pH

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control. The ability to reduce the acidity produced by reductive dechlorination is especially

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important when high concentrations of PCE and TCE, associated with DNAPL contamination,

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are being considered.11 In a previous column study of TCE degredation – in the presence of

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sulfate, Fe(III), and Mn(IV) – formate was found to a more effective substrate than lactate and

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propionate.12 Thus, we selected formate as the electron donor substrate to use in our chemostat

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studies.

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We considered the stoichiometry of electron donor and electron acceptor on an electron

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equivalent (eeq) basis. Under the first growth condition maintained in reactors EV5L and VS5L,

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the concentration of the electron donor formate (45 mM formate, 90 mM eeq) was provided in

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considerable stoichiometric excess to the concentration of available chlorinated electron

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acceptors (9-10 mMol TCE, requiring 27-30 mM eeq for complete dehalogenation). In contrast,

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in the second growth condition established in reactors EV2L and VS2L, the concentration of

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formate was diminished to 25 mM (50 mM eeq), which is less electron donor required for both

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the complete reductive dechlorination of TCE, DCE, and VC to ethene (27-39 mM eeq) and the

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synthesis of cellular biomass (24- 25 mM eeq), given the reported Fs = 0.46 for Dehalococcoides

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mccartyi 195.13 In remediation practice, other fermentable electron donors (e.g. emulsified

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soybean oil or lactate) are commonly used as a means of slowly delivering hydrogen to

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organohalide-respiring bacteria. Therefore, when these substrates are used instead of sodium

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formate (the electron donor used in this study), stoichiometric analysis should also consider the

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percentage of the primary electron donor converted to hydrogen and the concentration-dependent

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microbiological diversion of hydrogen to methanogenesis, acetogenesis, and metal reduction.14

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2. Materials and Methods

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2.1 Reactor Operation

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The design and operation of the CSTR system were described previously by Berggren et

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al. (2013).15 Reactors were inoculated with mixed cultures enriched from aquifer material

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obtained from the Evanite contaminated sites in Corvallis, OR, USA and a former DuPont

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facility in Victoria, Texas, USA.16,17 Reactors EV5L and VS5L were maintained under formate-

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excess conditions for their entire operation. Reactors EV2L and VS2L were initially maintained

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under formate-excess for the first 180 days of operation before decreasing influent formate to

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stoichiometric formate-limiting conditions for 2500 and 2200 days, respectively. Reactors EV2L

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and VS2L were then switched back to formate-excess conditions. Chemical analysis included

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frequent monitoring of the CAHs, ethene, hydrogen, and acetate concentrations in the reactors

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and batch vial fluid. Details pertaining to the analytical methods are provided by Azizian et al.

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(2008, 2010) and are summarized in the Supporting Information.12,18 During the final transition

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of reactors EV2L and VS2L from formate-deficit to formate-excess conditions, reactor material

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was sampled weekly to determine the ratio of Dehalococcoides mccartyi populations.

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2.2 Molecular Analysis

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DNA was isolated from centrifuged cell pellets (16,000 g and stored at -80 °C) by

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methods described in Mayer-Blackwell et al. (2014).19 Universal bacterial primers, targeting the

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V2 and V3 variable regions of the 16S rRNA gene, were selected to include Dehalococcoides

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single nucleotide polymorphisms at this gene locus, which we amplified with sample-specific

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molecular barcodes by the protocol reported by Mayer Blackwell et al. (2016).20 Amplicons were

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sequenced en mass using an Illumina MiSeq DNA sequencing instrument, producing on average

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32,000 unique reads per sample. DNA reads were denoised and the relative frequency of each

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unique sequence variant [used here to define operational taxonomic units (OTUs)] in each

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sample estimated using DADA2.21 The association of distinct OTUs with functional genes (i.e.

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vcrA and tceA) was determined by time-series correlation with qPCR targets using primers

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developed by Mayer-Blackwell et al. (2014).19

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2.3. Kinetic Tests Consortium-level kinetic parameters were measured in cell suspensions derived from the

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continuously operating reactors by methods described in detail by Breggren et al. (2013) and

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Green (2016).22,23 Briefly, the consortium’s maximum degradation rates at a given point during

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the reactors’ operation was determined in triplicate batch cell suspension assays. 125-mL clear

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borosilicate glass media bottles (Wheaton, Millville, NJ) with chlorobutyl rubber septa screen

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caps (Wheaton, Millville, NJ) were made anoxic in a glovebox atmosphere (95:5 N2:H2) before a

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50-mL chemostat sample was transferred to each vial. The headspace of each sample was purged

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of residual chlorinated aliphatic hydrocarbons (CAHs) and ethene from the parent CSTRs with

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anoxic gas (75:25 N2:CO2) for approximately 15 minutes. Approximately 8 µmol of TCE and

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100 µmol (eeq) of formate were added via anoxic stock solutions to each batch vial. Headspace

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concentrations of chloroethenes and ethene were monitored over time using an HP 6890 Series

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gas chromatograph (GC, Hewlett-Packard, Palo Alto, CA) fitted with a flame ionization detector

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(FID) utilizing helium as the carrier gas (15 mL/min) and a 30 m-0.53 mm GS-Q column

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(Agilent Technologies, Santa Clara, CA). The method for determining kinetic parameters –

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based on error-minimizing fit to a multi-Monod model considering each dechlorination step – is

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described in detail by Bergren et al. (2013) and Green (2016).22,23

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3 Results and Discussion

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3.1 Long-Term Dehalococcoides-Population Structure Under Formate Excess and Formate

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Limiting Conditions

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We studied four TCE-dehalogenating CSTRs dominated by Dehalococcoides mccartyi

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bacteria under two growth conditions (Table 2). One set of reactors (EV5L and VS5L) were

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maintained under the constant electron donor excess condition (45 mM influent formate) for the

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entire duration of the seven-year experiment, while a second set of reactors (EV2L and VS2L)

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were exposed to prolonged electron donor limitation (25 mM influent formate). Population

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structure, as determined and quantified at the 16S rRNA gene level, showed that two distinct

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Dehalococcoides ribosomal sequence variants (OTUs) accounted for between 90-99% of the

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entire bacterial population in each reactor under all conditions studied (Figure 1, Figure S1,

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Supporting Information). Dehalococcoides mccartyi’s small (0.5 µm diameter) cell size implies

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that its fractional representation at the DNA level is likely greater than in the corresponding

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fraction of the total consortium’s biomass. A Desulfuromonadales OTU with high nucleotide

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similarity to isolated members of the Geobacter genus was the third most abundant ribosomal

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sequence variant, accounting for an overall population frequency ranging from 1 to 10 % across

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the duration of the experiment. (The population fraction represented by Desulfuromonadales

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OTU is shown in red in Figure S1, Supporting Information). Other OTUs including ribosomal

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sequences associated with the bacterial classes Selenomondales and Spirochaetes were

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consistently detected in all four reactors under both growth conditions, usually at less than 1% of

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the overall population (Figure S1, Supporting Information). Our molecular approach, based on

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the amplification and enumeration of bacterial 16S gene variants, does not measure the possible

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fraction of archaea in the reactors. However, methane – the primary expected metabolic end

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product if methanogenic archaea were abundant – was not detected in the reactors’ headspace.

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Figure 1 presents key molecular and chemical data obtained from the four reactors

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studied. Two reactors (EV2L and VS2L) were exposed to electron donor limiting conditions (25

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mM influent formate) at the onset of the experiment, while the parent reactors (EV5L and VS5L)

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were fed influent containing 45 mM formate. Figure 1A and Figure 1B show the population

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structure response in reactors (EV2L and VS2L) exposed to electron donor limitation, which can

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be directly compared to Figure 1E and Figure 1F showing the relatively stable Dehalococcoides

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population structure present in the parent reactors (EV5L and VS5L) maintained under electron

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donor excess. Figure 1C and Figure 1D, which can be compared to Figure 1G and Figure 1H,

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show the major differences in the steady state concentrations of the hydrogen as well acetate

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resulting from the different influent electron donor concentrations supplied to each reactor set.

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The dashed lines in Figure 1 – in panels A, B, C, and D – show the point in the experiments at

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which the concentration of formate in EV2L and VS2L influent was increased from 25mM to 45

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mM. For continuity, the same dashed line is presented in Figure 2, which presents an expanded

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view of this transition period. Figure 2 also displays the perturbation’s influence on

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concentrations of TCE, DCE, VC and ethene in the reactors.

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Under the formate-excess growth condition, where the flux of electron donor (45 mM, 90

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mM eeq) exceeded the flux of chloroethene electron accepting equivalents (27-30 mM eeq), we

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observed: (i) steady state aqueous hydrogen concentration between 20-40 nM (Figure 1G and

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Figure 1H), (ii) persistent co-existence of OTUs representing the vcrA-containing and tceA-

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containing Dehalococcoides mccartyi populations (Figure 1E and Figure 1F), and (iii) 20-25 %

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of total influent reducing equivalents consumed by homoacetogenesis. In both the EV5L and

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VS5L chemostats, TCE was transformed to ethene (~99%) and VC (~1%). Under the formate-

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limiting conditions, imposed on EV2L and VS2L, the steady-state hydrogen concentration

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dropped to 1-2 nM and acetate dropped below its detection limit (20 uM) (Figure 1C and 1D). A

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reduction in the frequency of Selenomondales and Spirochaetes OTUs occurred in the 1000 days

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following the onset of electron donor limitation (Figure S1, Supporting Information). Most

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notably, the frequency of OTU representing the tceA-containing Dehalococcoides mccartyi

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population increased, numerically dominating the vcrA-Dehalococcoides population. After 2000

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days of reactor operation, greater than 99 % of total Dehalococcoides were the tceA-containing

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population in both reactors EV2L and VS2L (Figure 1A and Figure 1B). The change in non-

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Dehalococcoides population members is shown in the Supporting Information, Figure S1. Under

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the formate-limiting conditions in EV2L and VS2L, TCE was transformed to ethene (~50%) and

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VC (~50%) as shown in Figure 2E and Figure 2F.

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After approximately 2500 days of operation of the EV2L and VS2L reactors under

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formate-limiting condition, the OTU frequencies associated with the tceA-containing

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Dehalococcoides population exceeded that of vcrA-associated OTU by more than three orders of

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magnitude (Figure 1). After 2500 days, the vcrA-containing population had decreased in the

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EV2L reactor to non-detectable levels, equivalent to a frequency of less than ~ 0.0001 of the

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population at the DNA level (Figure 1A). After 2200 days, the vcrA-containing population

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decreased in the VS2L reactor to less than 0.001 of the population at the DNA level (Figure 1B).

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By contrast, in control reactors EV5L and VS5L, the long-running parent reactors of EV2L and

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VS2L that had been maintained under constant formate-excess conditions, the vcrA-containing

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Dehalococcoides population frequency remained above 0.1 of the total community, at the DNA

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level, for the entire period of the seven-year experiment (Figure 1E and Figure 1F).

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We attribute the decrease in vcrA-containing Dehalococcoides under formate limiting

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conditions to the ecological process of competitive exclusion. When electron donor becomes the

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environment’s limiting catabolic resource, the non-vcrA Dehalococcoides subpopulation exhibits

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higher relative fitness than in the non-limited environment. The gradual competitive exclusion

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observed suggests a possible mechanism underlying the fitness difference between

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Dehalococcoides subpopulations other than a strict hydrogen threshold difference between

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subpopulations, which is not well supported by the data. Its stands to reason that if the tceA-

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population had a lower hydrogen threshold than the vcrA-population, steady-state hydrogen

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levels would have been depressed below the higher threshold concentration, causing the abrupt

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loss of one but not the other population. Instead, the fitness advantage manifest by the tceA

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population is more likely explained by differences in effective dechlorination rates, since these

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effectively govern the rate at which each Dehalococcoides subpopulation can consume electron

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donor and thus control the relative rate of ATP synthesis in each population. If the Kmax,TCE,tceA

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(the maximum rate of TCE dechlorination by the tceA population) is greater than the Kmax,VC,vcrA

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and the Kmax,DCE,tceA is greater or equal to the Kmax,DCE,vcrA, then the tceA strain would be predicted

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to consume a larger fraction of environment’s limiting resource, resulting in the decline of the

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vcrA-containing population under stoichiometric electron donor deficit conditions. Based on this

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differential kinetic hypothesis, ecological exclusion need not be rapid.

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After seven years of operation, we increased the electron donor concentration in the

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EV2L and VS2L reactors from 50 mM eeq formate back to the conditions of the parent reactors

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– 90 mM eeq formate. Figure 2 shows the relative abundance of the vcrA and tceA-containing

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Dehalococcoides populations shifting dramatically. The frequency of the vcrA-population

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increased from less than 0.001 in both reactors to 0.38 in reactor EV2L and 0.57 in reactor

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VS2L. The near convergence in the frequency of the vcrA and tceA populations occurred within

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two CSTR residence times (100 days). In both reactors, the increase in influent formate

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concentration did not have a major impact on the frequency of the Desulfuromonadales OTU.

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With respect to other potential homoacetogenic populations in reactor EV2L, we observed a

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transient increase in the frequency in Selenomondales OTU 3, peaking 77 days following the

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onset of formate stimulation (EV2L, day 2684). In reactor VS2L, Spirochaetes OTU 9

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temporarily increased between the samples taken at operational days 2376 and 2406 following

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the onset of formate stimulation (VS2L,day 2380), but the aggregate frequency of

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Selenomondales and Spirochaetes OTUs did not return to the level observed at the onset of the

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formate-deficit condition (Figure S1, Supporting Information).

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In both reactor EV2L and VS2L, hydrogen rapidly increased from < 2 nM to steady state levels between 20-40 nM within four days after formate-excess conditions were restored.

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Acetate, which previously had not been observed above the limit of detection (20 µM), gradually

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increased to more than 3 mM within 100 days after formate-excess conditions were restored

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(Figure 2C and Figure 2D). The acetate concentration of 3 mM is in the range of that produced in

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the EV5L and VS5L chemostats (Figure 1G and Figure 1H). Within 100 days following the

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increase in the influent formate concentration fed to the EV2L and VS2L reactors, the fraction of

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non-Dehalococcoides microbes as well as their total biomass had not recovered to those levels

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observed at the beginning of the study (Figure S1, Supporting Information). The residual VC

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concentration (4 mM) in the reactor remaining at the onset of increase in influent formate

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concentrations may have biased the assemblage of the re-stimulated consortium towards

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organohalide-respiring organisms. The production of methane was not observed after restoring

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the reactors back to the 45 mM influent formate condition.

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3.2 Vinyl Chloride Dechlorination Kinetics After Long-Term Electron Donor Limitation

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According to the consensus view of organohalide-respiration, specific VC reductases, not

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present in all strains of Dehalococcoides mccartyi, are necessary to efficiently catalyze complete

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chloroethene degradation to ethene. Other strains of Dehalococcoides mccartyi may contain

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reductive dehalogenases with non-specific, and perhaps cometabolic, VC activity.24,25 In our

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reactor study, constant VC dechlorination did indeed proceed even as the vcrA-containing

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Dehalococcoides population was greatly depleted from the EV2L and VS2L reactor consortia

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during formate-limiting conditions. In the reactors during formate-limitation, the steady-state rate

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of VC dechlorination was approximately 80 µmol L-1d-1. Notably, this VC dechlorination rate

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was sustained under high steady-state VC concentrations in excess of 4 mM, favorable for co-

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metabolic or non-specific VC dechlorination by reductive dehalogenases with low affinity for

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VC as a halogenated substrate. VC transformation Ks values of 0.29 mM and 0.602 mM have

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been reported for Dehalococcoides cultures when the cometabolic transformation of VC was

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implied.14,26 Thus, 4 mM VC in the chemostat is in the range where significant transformation

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via cometabolism is expected. At these high concentrations, and given reported KS,VC values,

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the maximum rate of VC dechlorination would be expected; however, other factors such as the

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low concentration of hydrogen contribute to the overall rates observed. To elucidate the

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functional consequences of depleting the vcrA-subpopulation from the dechlorinating

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community, we measured consortium level dechlorination kinetics after exposure to the long

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period of electron donor deficit. To enable kinetic studies over a range of substrate

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concentrations, we transferring reactor biomass to batch reaction vials which we spiked with

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TCE and formate. Although we had observed an appreciable steady-state rate of VC

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dechlorination (80 µmol L-1d-1) at millimolar VC concentrations (4 mM, 250 ppm) in the EV2L

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and VS2L, batch kinetic tests revealed that the depletion of the vcrA-containing Dehalococcoides

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population greatly reduced these consortia’s capacity for VC reduction at sub-millimolar

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concentrations. This was indicated in batch depletion studies where VC was present at an

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aqueous concentration of 50 µM – 1 µM (3.2 ppm - 62.5 ppb). Here, the maximum observed rate

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of VC dechlorination by the vcrA-depleted consortium, after TCE and DCE had been fully

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consumed, was 5 µmol L-1 d-1 (95 % CI [2,8] µmol L-1 d-1). By comparison – at similar low µM

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concentrations – the maximum observed rates of TCE and DCE dechlorination in these batch

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depletion assays were more than two orders of magnitude greater than those observed for VC.

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The observed dechlorination rates are presented in Table S1, Supporting Information. Notably,

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the slow rate of VC dechlorination manifest in the vcrA-depleted consortia at micromolar VC

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concentrations would alone be kinetically insufficient to support a tceA-Dehalococcoides

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population’s net growth given natural decay rates.27

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3.3 Vinyl Chloride Dechlorination Kinetics after Electron Donor Stimulation

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When the EV2L and VS2L reactors were returned to formate-excess conditions, the rates

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of VC dechlorination in subsequent batch depletion assays increased after a lag period of 30

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days. A strong positive correlation was found between the consortium’s maximum VC

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dechlorination rate in batch assays and the abundance of the vcrA-containing Dehalococcoides

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mccartyi population in the reactor at the time the batch assay was conducted (Figure 3).

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Additionally, the consortium’s affinity for VC dechlorination at low concentrations increased

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following the return to formate excess-conditions. Figure 4 shows a direct comparison of batch

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kinetics observed with reactor material sampled before (Figure 4A) and after (Figure 4B) the

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reactors’ return to growth under elevated concentrations of electron donor. Once the vcrA-

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containing population exceeded 25 % of the total bacteria community, on the DNA level, high

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rates of VC dechlorination (> 200 - 600 µmol L-1 d-1) were observed even at low (< 25 µM, 1.5

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ppm) VC concentrations (Figure 4, Table S1, Supporting Information).

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Close examination of the temporal changes in the VC concentrations in the chemostats

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reveals two time periods after the influent formate concentration was increased where different

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VC transformation processes occurred (Figure 2). First, an immediate decrease in effluent VC

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was observed after increasing the influent formate concentration, followed by a plateau before a

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second acceleration in rate was observed 30 days later (Figure 2). This pattern is mirrored by a

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step-wise increase in ethene concentrations. A log plot of VC concentrations versus time well

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illustrates these discrete shifts in the CSTRs’ rate of VC dechlorination (Figure S3, Supporting

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Information). The first decrease in VC concentration likely resulted from enhanced VC

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transformation by the tceA-enriched population dominant in the chemostat at the time of the

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formate shift. This response occurred with the addition of higher influent formate and the rapid

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increase in hydrogen concentrations from ~ 1 nM to 30 nM. A previous study estimated the Ks

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values for hydrogen in Dehalococcoides mccartyi strain VS to be 5 nM.28 Thus if the

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Dehalococcoides mccartyi in the CSTRs under investigation here had similar affinity for

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hydrogen, an immediate increase in overall dechlorination rate would be expected following the

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observed increase in hydrogen concentration after relieving formate-limitation. The second

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decrease in VC concentrations starting at 2740 and 2440 days in EV2L and VS2L chemostats,

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respectively, coincides with the increased frequency in vcrA-population.

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In the reactor experiments, the recovery of the vcrA-population and dechlorination of VC

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to low micromolar concentrations occurred in less than two reactor residence times. The resilient

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population response was likely hastened by the 4 mM residual VC remaining in the reactor when

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the influent formate concentration was increased from 25 mM to 45 mM. In situ recovery times

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of the vcrA-containing populations in a groundwater contaminant plume, without a large

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reservoir of VC from stalled TCE dechlorination, would likely be slower. Nevertheless, the

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apparent difference in affinity for VC at low concentrations observed in the balanced tceA/vcrA

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consortium versus the tceA-dominated consortium (Figure 4 and Table S1, Supporting

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Information) indicates that the vcrA-population would rebound in the presence of excess electron

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donor even in cases where VC is present at lower steady state concentrations.

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3.4 Implications for Contaminated Sites

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Our results underscore the importance of electron donor/chloroethene stoichiometry

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whether formate or more commonly used substrates (e.g., lactate or emulsified vegetable oil) are

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used. Electron donor competition is a well-recognized determinant of dechlorination efficiency

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in the environment. A number of previous investigations have shown that electron donor

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limitation, often exacerbated by the presence of competing terminal electron accepting processes

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(e.g. metal reduction, methanogenesis, or sulfate reduction), can diminish rates of dechlorination

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and survival of Dehalococcoides.14,17,22,29-31 In this study, we demonstrated that electron donor

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scarcity alone, in the absence of competing metabolic processes or inhibitory concentrations of

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intermediate dechlorination products, is sufficient to greatly alter intra-Dehalococcoides

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population structure. Where the flux of available hydrogen is in stoichiometric deficit to TCE

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input, our results suggest that tceA-containing Dehalococcoides, if present, will likely

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outcompete vcrA-containing populations, increasing the risk of incomplete chloroethene

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removal.

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As greater attention is being paid to catabolic food webs, cross-feeding, and competition

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within organohalide respiring consortia,32,33 the sensitive ecological balance between closely

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related Dehalococcoides mccartyi populations should not be overlooked. In this study, we

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showed directly through time-series community characterization paired with consortium level

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kinetic tests that these intra-species population shifts have significant functional consequences.

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Even though our results suggest appreciable VC dechlorination may still proceed after the

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depletion of the VC-specialized Dehalococcoides population, the survival of a vcrA-population is

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likely crucial for low-concentration VC removal to stringent cleanup levels (MCLVC: 2 ppb, 30

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nM in the United States) mandated when groundwater is to be restored to drinking water

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standards. Our results provide a cautionary perspective on remediation forecasts that assume

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dispersal and growth of multiple Dehalococcoides respiring organisms following a discrete

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biostimulation event. Therefore, asymmetrical growth rates among different Dehalococcoides

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subpopulations during electron donor limitation should be included in remediation models.

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However, this study is encouraging for the design of multi-phase biostimulation efforts given the

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observed long-survival times and robust recovery of the depleted vcrA-containing

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Dehalococcoides populations after more than six years of electron donor-limitation.

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ACKNOWLEDGMENTS

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This work was funded by an NSF Grant (MCB-1330832). KMB was additionally

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supported by an NSF Graduate Research Fellowship (2011103493). We wish to thank Benjamin

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Callahan for assistance in denoising and variant detection performed on the DNA amplicon

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sequences data.

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Supporting Material

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assays, after increasing the influent concentration formate from 25 mM to 45 mM in reactors

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EV2L and VS2L. Supporting Figure S1 displays the bacterial population structure during

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prolonged exposure to electron donor limitation and electron donor stimulation in reactor EV2L.

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Supporting Figure S2 displays the bacterial population structure, hydrogen levels, and

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dechlorination products in EV2L and VS2L reactors upon electron donor stimulation.

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Supporting Figure S3 shows the log-concentration of VC and ethene in reactors EV2L and

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VS2L, emphasizing two discrete changes in VC rate following the increase in influent electron

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donor.

Supporting Table S1 reports the dechlorination kinetics measured in batch cell suspension

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Tables and Figures Table 1: CSTRs in this study Reactor

Electron Acceptor

Electron Donor

Formate-Excess Conc.

EV5L

Conc.

Formate-Limiting Conc.

Compound

(mM)

Compound

(mM)

Days

(mM)

Days

TCE

8.5

Formate

45

1-3000

-

-

1-180; 2684EV2L

TCE

8.5

Formate

45

2830

25

181-2684

VS5L

TCE

8.5

Formate

45

1-2500

-

-

25

181-2380

1-180; 2380VS2L

TCE

8.5

Formate

45

2600

388 389

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Table 2: CSTR growth conditions

Formate-Excess

Formate-Deficit

45 mM

25 mM

(90 eeq)

(50 eeq)

8-9 mM

8-9 mM

(48-54 eeq)

(48-54 eeq)

Residence time

50 days

50 days

Equilibrium hydrogen (aq)

~ 40 nM

~ 2 nM

30%