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Aerobic vinyl chloride metabolism in groundwater microcosms by methanotrophic and etheneotrophic bacteria Margaret Findlay, Donna F. Smoler, Samuel Fogel, and Timothy E. Mattes Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05798 • Publication Date (Web): 26 Feb 2016 Downloaded from http://pubs.acs.org on March 3, 2016
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Aerobic vinyl chloride metabolism in groundwater microcosms by methanotrophic and etheneotrophic bacteria
Authors: Margaret Findlay1, Donna F. Smoler1, Samuel Fogel1, and Timothy E. Mattes2*
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Bioremediation Consulting, c/o 55 Halcyon Rd, Newton MA, 02458, USA
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Department of Civil and Environmental Engineering, 4105 Seamans Center, University of Iowa,
Iowa City, IA, 52242, USA
*Corresponding author: Fax: (319) 335-5660 Email:
[email protected] 1
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Abstract
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Vinyl chloride (VC) is a carcinogen generated in groundwater by reductive dechlorination of
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chloroethenes. Under aerobic conditions, etheneotrophs oxidize ethene and VC, while VC-
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assimilators can use VC as their sole source of carbon and energy. Methanotrophs utilize only
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methane but can oxidize ethene to epoxyethane, and VC to chlorooxirane. Microcosms were
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constructed with groundwater from the Carver site in MA containing these three native microbial
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types. Methane, ethene, and VC were added to the microcosms singly or as mixtures. In the
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absence of VC, ethene degraded faster when methane was also present. We hypothesized that
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methanotroph oxidation of ethene to epoxyethane competed with their use of methane, and that
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epoxyethane stimulated the activity of starved etheneotrophs by inducing the enzyme alkene
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monooxygenase. We then developed separate enrichment cultures of Carver methanotrophs and
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etheneotrophs, and demonstrated that Carver methanotrophs can oxidize ethene to epoxyethane,
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and that starved Carver etheneotrophs exhibit significantly reduced lag time for ethene and VC
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utilization when epoxyethane is added. In our groundwater microcosm tests, when all three
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substrates were present, the rate of VC removal was faster than with either methane or ethene
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alone, consistent with the idea that methanotrophs stimulate etheneotroph destruction of VC.
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Introduction
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The chloroethenes tetrachloroethene (PCE) and trichloroethene (TCE) are persistent toxic
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chemicals that have been used widely as dry cleaning solvents and metal degreasers and have
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been disposed in soil and groundwater. Although they may exist as non-aqueous phase liquids
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after release into the environment, PCE and TCE are water soluble, and thus widely detected in
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groundwater1.
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Under sufficiently reducing conditions, with organic compounds and/or hydrogen as
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electron donors, a variety of anaerobic bacteria can reductively dechlorinate PCE and TCE to
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cis-dichloroethene (cDCE). If Dehalococcoides spp. are present under favorable conditions,
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cDCE can be further dechlorinated to vinyl chloride (VC) and then completely dechlorinated to
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ethene, relatively innocuous in comparison to the chloroethenes2, 3. The anaerobic process can be
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optimized by maintaining electron donor, minerals and neutral pH, and, if necessary, inoculating
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with commercial Dehalococcoides cultures4.
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Low concentrations of VC, however, can escape treatment during the anaerobic process5.
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The generation of a VC plume in groundwater is a cause for significant concern because it is
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highly mobile and is a known human carcinogen with a US EPA maximum contaminant level
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(MCL) of 2 µg/L.
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If the dilute VC plume enters a zone containing dissolved oxygen, VC may be
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biodegraded by aerobic bacteria which produce monooxygenase enzymes, allowing them to
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utilize methane and ethene as carbon and energy sources, and to fortuitously oxidize (i.e.
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cometabolize) VC to non-chlorinated products 6-10. In groundwater where chloroethenes are
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undergoing reductive dechlorination to VC and ethene, strongly reducing conditions also lead to
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the generation of significant amounts of methane. Thus, the primary substrates for down gradient
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aerobic cometabolism of VC are usually present, allowing both methanotrophs and
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etheneotrophs to participate in biodegradation of VC.
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Methanotrophs, ubiquitous aerobes that use methane as a carbon and energy source, have
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non-specific methane monooxygenases (MMOs) that incorporate a single oxygen atom into a
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variety of short chain alkanes and alkenes as well as aromatics such as benzene11. Methanotrophs
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oxidize ethene to epoxyethane, which can accumulate extracellularly12 or be further degraded by
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methanotrophs having soluble MMO (sMMO)10. Methanotrophs can also co-metabolically
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oxidize TCE, DCEs, and VC13 to short-lived chlorinated epoxides which hydrolyze and can be
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further degraded to non-chlorinated end products. Methanotrophs have been used in aerobic
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cometabolic strategies such as bioreactors 14 and aerobic in situ bioremediation 15. VC is
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oxidized to the unstable intermediate chlorooxirane, which has a half-life of about one minute
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and breaks down abiotically10.
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Etheneotrophs, aerobic bacteria that use ethene as a carbon and energy source, possess an
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alkene monooxygenase (AkMO) that oxidizes ethene2, and have been shown in groundwater
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microcosms to oxidize VC 6, 16 and cDCE 8 to non-chlorinated products. Etheneotrophs
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metabolize epoxyethane and chlorooxirane via ethene biodegradation pathway enzymes 2, 17.
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Certain etheneotrophs can also adapt to VC as a growth substrate 7, 18, 19. Although studies are
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limited, etheneotrophs appear to be widely distributed at sites contaminated with chlorinated
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solvents 19, 20.
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Degradation of VC in the presence of both methane and ethene has previously been studied
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with groundwater from a VC-contaminated site 21, which suggested that ethene can play an
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important role during aerobic natural attenuation of VC. Advancing our understanding of factors
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controlling VC oxidation in the presence of ethene, methane, methanotrophs, etheneotrophs and
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VC-assimilators is important for decision-making with respect to VC bioremediation. The
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purpose of the present study was to further investigate this scenario using groundwater from a
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VC-contaminated site in Carver, MA, to determine how the native mixed population of
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methanotrophs, etheneotrophs and VC-assimilating bacteria biodegrade VC under conditions of
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each substrate alone, and mixtures of these substrates. Additional experiments were conducted
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using enrichment cultures of each microbial type in mineral salts medium to support our
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interpretation of the interactions observed in groundwater microcosms.
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Experimental section
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Site Description. The study site in Carver, MA is located down gradient from a landfill, the
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leachate from which contained PCE and organic compounds. These organic compounds
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generated anaerobic conditions and served as electron donors for reductive dechlorination of
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PCE to its daughter products, creating a VC plume extending 4600 feet downgradient from the
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landfill into an aquifer of silt and fine sand. By 2002, a detached VC plume, 3000 feet long, 40
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feet wide and 30 feet thick, was located 50 feet below the water table, having VC concentrations
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ranging from 2 - 27 µg/L, and migrating at about 0.5 feet/day. In 2003, a full-scale
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bioremediation project was initiated to stimulate aerobic VC oxidation, by transecting the plume
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with a line of gas infusion points screened over the full depth of contamination. Over the next
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two years, additional gas infusion lines were installed down gradient along the plume. The
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system was operated to alternately inject oxygen and ethene 22. By 2008, VC in groundwater
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from wells down gradient from the second and third treatment lines was below the treatment goal
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of 2 µg/L. In 2009, although VC was still migrating into the treatment area, wells down gradient
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from the first treatment line showed significantly lower VC concentrations, and laboratory
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microcosm tests verified that site groundwater contained etheneotrophs 23.
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Chemicals. Methane (>99%) and ethene (>99.5%) were obtained from Scott Specialty Gases.
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VC (40 mg/ml in methanol) was obtained from Restek. Dilute VC Gas Stock was prepared by
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injecting 0.85 ml of VC in methanol (40 mg/ml) into a sealed 160 ml serum bottle containing
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77.5 ml deionized water. After equilibration, the headspace of this solution was the source of
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Dilute VC Gas Stock, 30 µL of which was used for feeding microcosms. As a result, potential
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gas phase methanol carry-over into microcosms was very low (~0.0008 µmole/bottle) and thus
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considered negligible. Epoxyethane (5 mg/ml in water) was purchased from AccuStandard.
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Ultra-high purity compressed air and ultra-high purity oxygen (99.99%) were obtained from
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IGOs in Watertown, MA. All other chemicals used were of reagent grade or better.
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Groundwater sampling and characterization. Groundwater from the Carver, MA site was
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collected into 1 L glass bottles according to USEPA/540/S-95/504 low-flow purge procedures.
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Samples were delivered on the same day to Bioremediation Consulting Inc. (BCI) and
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refrigerated until use. Well 63-I, located 100 feet down gradient from the fourth cross-plume line
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of air infusion points, was selected for this study because preliminary microcosm tests had
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shown that it contained all three microbial types (methanotrophs, etheneotrophs and VC
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assimilators). Well 63-I groundwater was analyzed in 2010 for Cl-, NO3-, and SO42- by capillary
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ion electrophoresis according to EPA Method 6500, for PO43- by Hach Method 8048, and for
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other elements by EPA Method 200.8. In March 2011, groundwater collected for this study from
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well 63-I was shown to contain 157 µg/L methane, 0.43 µg/L ethene, and < 2 µg/L VC using gas
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chromatography as described below.
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Groundwater characteristics and amendment of groundwater microcosms. Analysis of
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groundwater from well 63-I showed that important mineral nutrients were below detection limits,
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i.e., NO3- < 0.4 mg/L, PO43- < 0.04 mg/L, and Cu < 0.001 mg/L, while SO42-, Cl-, K+, Na+, Fe,
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and Mg were between 2 and 74 mg/L. B, Co, Mn, Se, Zn, and Ni were between 0.001 and 0.2
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mg/L. Because the microcosms would not contain sediment, which in situ would provide
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mineral nutrients required by bacteria for growth, microcosms were amended as follows. To
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avoid nitrogen limitation, 10 mg/L nitrogen as KNO3 was added, and to assure that Cu limitation
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did not occur24, a very low concentration of Cu, 0.004 mg/L, was added. To maintain pH at 6.8,
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60 mg/L PO43- was added as sodium and potassium phosphate.
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Construction of groundwater microcosms containing native microbial populations.
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Groundwater was bubbled with compressed air until the native methane had been removed to
