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Relationships between the abundance and expression of functional genes from vinyl chloride (VC)-degrading bacteria and geochemical parameters at VC-contaminated sites yi liang, Xikun Liu, Michael A. Singletary, Kai Wang, and Timothy E. Mattes Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03521 • Publication Date (Web): 05 Oct 2017 Downloaded from http://pubs.acs.org on October 6, 2017
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Environmental Science & Technology
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Relationships between the abundance and expression of functional genes from
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vinyl chloride (VC)-degrading bacteria and geochemical parameters at VC-
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contaminated sites
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Authors:
Yi Liang1, Xikun Liu1, Michael A. Singletary2, Kai Wang3 and Timothy E. Mattes1*
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1
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University of Iowa, Iowa City, IA, 52242, USA
Department of Civil and Environmental Engineering, 4105 Seamans Center, The
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Jacksonville, FL USA
NAVFAC Southeast, EV3 Environmental Restoration Bldg. 135 Naval Air Station
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Iowa, Iowa City, Iowa 52242, USA
Department of Biostatistics, N322 College of Public Health Building, The University of
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*Corresponding author:
Fax: (319) 335-5660 Email:
[email protected] 19 20
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Abstract
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Bioremediation of vinyl chloride (VC) contamination in groundwater could be mediated
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by three major bacterial guilds - anaerobic VC-dechlorinators, methanotrophs, and
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ethene-oxidizing bacteria (etheneotrophs) via metabolic or co-metabolic pathways. We
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collected 95 groundwater samples across 6 chlorinated ethene-contaminated sites and
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searched for relationships among VC biodegradation gene abundance and expression
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and site geochemical parameters (e.g. VC concentrations). Functional genes from the
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three major VC-degrading bacterial guilds were present in 99% and expressed in 59%
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of the samples. Etheneotroph and methanotroph functional gene abundances ranged
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from 102 to 109 genes per liter of groundwater among the samples, with VC reductive
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dehalogenase gene (bvcA and vcrA) abundances reaching 108 genes per liter of
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groundwater. Etheneotroph functional genes (etnC and etnE) and VC reductive
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dehalogenase genes (bvcA and vcrA) were strongly related to VC concentrations
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(p0.05). Samples from sites with bulk VC attenuation rates > 0.08 yr-1
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contained higher levels of etheneotroph and anaerobic VC-dechlorinator functional
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genes and transcripts than those with bulk VC attenuation rates < 0.004 yr-1. We
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conclude that both etheneotrophs and anaerobic VC-dechlorinators have the potential
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to simultaneously contribute to VC biodegradation at these sites.
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Introduction
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Widespread usage of the chlorinated solvents tetrachloroethene (PCE) and
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trichloroethene (TCE) has resulted in pervasive groundwater contamination through
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spillage or otherwise inappropriate storage and disposal practices 1, 2. A naturally-
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occurring or enhanced biological anaerobic reductive dechlorination process acting on
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PCE and TCE dissolved in groundwater will often generate vinyl chloride (VC), a proven
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human carcinogen and neurotoxin 3, 4. VC often accumulates in groundwater under the
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reducing conditions necessary for PCE and TCE dechlorination 5-7, and the resulting VC
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plume presents further remediation challenges.
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Despite its tendency to accumulate in groundwater, VC is biodegradable under
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anaerobic, hypoxic (DO ~0.1 mg/L), and aerobic conditions 5, 8, 9. Under anaerobic
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conditions, VC can be reduced to ethene by certain organohalide-respiring
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microorganisms (anaerobic VC-dechlorinators) via metabolic 8 or cometabolic
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processes 10. Dehalococcoides mccartyi strains reduce VC to ethene using reductive
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dehalogenase enzymes BvcA and VcrA (encoded by genes bvcA and vcrA) 11, 12. VcrA
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also catalyzes dechlorination of TCE and DCEs 12, 13, while BvcA reduces DCEs to VC
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in addition to reducing VC to ethene11. A recently described Dehalogenimonas strain is
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also capable of VC respiration, apparently utilizing dehalogenases related to VcrA and
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BvcA14. Reductive dehalogenase genes bvcA and vcrA are commonly used indicators
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of anaerobic VC reductive dechlorination potential in groundwater environments 15-17.
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Aerobic VC co-metabolism by methanotrophs has been demonstrated under both
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laboratory and field conditions, although competition inhibition of VC oxidation by the
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presence of methane is a concern 18, 19. Methanotrophs employ soluble and particulate
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methane monooxygenases (sMMO and pMMO, respectively) to oxidize methane as a
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primary growth substrate. Both pMMO and sMMO are capable of fortuitous VC
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oxidation 20, 21. The sMMO has a broader substrate range than pMMO and is more
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efficient in degrading chlorinated ethenes20. The gene mmoX, which encodes the sMMO
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alpha subunit, and pmoA, which encodes the pMMO alpha subunit, are used as
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biomarkers of VC cometabolic potential in groundwater22, 23.
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Ethene-oxidizing bacteria (etheneotrophs) can cometabolize VC using ethene as
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the primary growth substrate 5, 24, 25. Several pure etheneotrophic strains can also utilize
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VC as sole carbon and energy source 26-29. VC oxidation (both metabolic and
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cometabolic) by etheneotrophs is initiated by alkene monooxygenase (AkMO), which
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inserts an oxygen atom into ethene, forming epoxyethane, and into VC forming
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chlorooxirane30. Etheneotrophs are also known to cometabolize cis-DCE presumably
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using AkMO 25, 31. Epoxyalkane coenzyme M transferase (EaCoMT) catalyzes the
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formation of 2-chloro-2-hydroxyethyl-CoM from chlorooxirane 30. EaCoMT also
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participates in aerobic assimilation of other short chain alkenes such as ethene and
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propene32. The genes etnC, which encodes the alpha subunit of AkMO, and etnE, which
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encodes the EaCoMT, are emerging biomarkers for etheneotroph-mediated aerobic VC
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biodegradation potential but do not distinguish between metabolic or cometabolic VC
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biodegradation pathways 33. Etheneotrophs are found in riverbed sediment microcosms
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34, 35
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samples 33, 37-39.
, a VC-contaminated vadose zone 36, and in VC-contaminated groundwater
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Relationships between anaerobic VC reductive dehalogenase genes and
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groundwater geochemical parameters have been reported 15, 16. However, VC-oxidizing
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bacteria can operate at low DO levels 40 and are found in anaerobic groundwater
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samples 41. In general, the presence and expression of genes associated with VC
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oxidation in nominally anaerobic groundwater, and the effect of geochemical
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parameters (e.g. VC and DO concentrations) on VC biodegradation gene abundance
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and expression is poorly understood.
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The purpose of this study was to estimate the abundance and expression of a
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suite of functional genes representing three major VC-degrading bacterial guilds
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(anaerobic VC-dechlorinators, methanotrophs and etheneotrophs) in multiple monitoring
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wells at several chlorinated ethene-contaminated sites. This dataset was used to
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investigate relationships between VC concentrations (among other geochemical
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parameters) and anaerobic and aerobic VC biodegradation gene abundance and
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expression in groundwater samples.
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Materials and Methods
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Site descriptions, groundwater sample collection, and geochemical parameter
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analysis
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Groundwater samples from six contaminated sites were included in this study
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(Figure S1): Site 2C in Virginia (VA Site 2C); Site 11 in Georgia (GA Site 11); Site SS-
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17 in Oklahoma (OK Site SS-17); Site 70 in California (CA Site 70); Site LF05 in Hawaii
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(HI Site LF05); Site 45 in South Carolina (SC Site 45). Different remediation strategies
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have been employed at each of the sites. At VA Site 2C, oxygen releasing compound
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(ORCTM) slurry was injected to promote aerobic VC degradation 38, 42. At GA Site 11,
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source area treatment by in situ chemical oxidation (ISCO) with Fenton’s Reagent was
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conducted 43 and emulsified vegetable oil (EVO) was injected to enhance anaerobic
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reductive dechlorination 44. At CA Site 70 and OK Site SS-17, biobarriers were installed
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along the groundwater plumes, and EVO was injected into biobarriers 45, 46. At SC Site
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45, ISCO (with permanganate), EVO and emulsified zero-valent iron (eZVI) injection
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pilot studies were conducted 47. At HI Site LF05, a bioreactor was constructed to treat a
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TCE source area with EVO injection and bioaugmentation 48.
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A total of 95 groundwater samples were collected from selected monitoring wells
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at these six sites over the course of 4 years (2013-2016) by site technicians following
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USEPA low-flow groundwater sampling procedures for chlorinated ethene and microbial
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analysis 49. Monitoring wells were purged at a flow rate
