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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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Environmental Science & Technology

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

2013

ACS Paragon Plus Environment

2014

2015


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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

312

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

313

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

314

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

315

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

316

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

317

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

318

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

319

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

320

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

321

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

322

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

323

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

324

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

325

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

326

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

327

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

328

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

329

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

330

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

331

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

332

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

333

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

334

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

335

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

336

3.4 Implications for Contaminated Sites

337

Our results underscore the importance of electron donor/chloroethene stoichiometry

338

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

339

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

340

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



341

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

342

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

343

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

344

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

345

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

346

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

347

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

348

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

349

removal.

350

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

351

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

352

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

353

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

354

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

355

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

356

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

357

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

358

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

359

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

360

dispersal and growth of multiple Dehalococcoides respiring organisms following a discrete

361

biostimulation event. Therefore, asymmetrical growth rates among different Dehalococcoides

362

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

363

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

364

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

365

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

366

ACKNOWLEDGMENTS

367

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

368

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

369

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

370

sequences data.


Page 14 of 24

Page 15 of 24


371 372 373

Supporting Material

374

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

375

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

376

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

377

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

378

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

379

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

380

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

381

donor.

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

382 383 384



385 386 387

Page 16 of 24

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


Page 17 of 24

390


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%

Survival of Vinyl Chloride Respiring - ACS Publications - American

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