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Organohalide respiration with chlorinated ethenes under low pH conditions Yi Yang, Natalie L Cápiro, Tyler F Marcet, Jun Yan, Kurt D Pennell, and Frank E Löffler Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01510 • Publication Date (Web): 30 Jun 2017 Downloaded from http://pubs.acs.org on July 2, 2017
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Environmental Science & Technology
Organohalide respiration with chlorinated ethenes under low pH conditions
1 2 3
Yi Yang1,2,6, Natalie L. Cápiro7, Tyler F. Marcet7, Jun Yan1,3,6, Kurt D. Pennell7
4
and Frank E. Löffler1,2,3,4,5,6*
5 6
1
Center for Environmental Biotechnology, 2Department of Civil and Environmental Engineering,
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3
Department of Microbiology, 4 Department of Biosystems Engineering and Soil Science,
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University of Tennessee, Knoxville, TN 37996, USA; 5Biosciences Division, and 6Joint Institute
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for Biological Sciences (JIBS), Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA;
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7
11
USA
Department of Civil and Environmental Engineering, Tufts University, Medford, MA 02155,
12 13 14 15 16
*Corresponding author
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Frank E. Löffler
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University of Tennessee
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Department of Microbiology
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M409 Walters Life Sciences
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Knoxville, Tennessee 37996-0845
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Phone: (865) 974-4933
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E-mail:
[email protected] ACS Paragon Plus Environment
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Abstract
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Bioremediation at chlorinated solvent sites often leads to groundwater acidification due to
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electron donor fermentation and enhanced dechlorination activity. The microbial reductive
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dechlorination process is robust at circumneutral pH, but activity declines at groundwater pH
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values below pH 6.0. Consistent with this observation, the activity of tetrachloroethene (PCE)
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dechlorinating cultures declined at pH 6.0 and was not sustained in pH 5.5 medium, with one
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notable exception. Sulfurospirillum multivorans dechlorinated PCE to cis-1,2-dichloroethene
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(cDCE) in pH 5.5 medium and maintained this activity upon repeated transfers. Microcosms
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established with soil and aquifer materials from five distinct locations dechlorinated PCE-to-
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ethene at pH 5.5 and pH 7.2. Dechlorination to ethene was maintained following repeated
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transfers at pH 7.2, but no ethene was produced at pH 5.5, and only the transfer cultures derived
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from the Axton Cross Superfund (ACS) microcosms sustained PCE dechlorination to cDCE as a
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final product. 16S rRNA gene amplicon sequencing of pH 7.2 and pH 5.5 ACS enrichments
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revealed distinct communities, with Dehalococcoides being the dominant dechlorinator in pH 7.2
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and Sulfurospirillum in pH 5.5 cultures. PCE-to-trichloroethene- (TCE-) and PCE-to-cDCE-
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dechlorinating isolates obtained from the ACS pH 5.5 enrichment shared 98.6%, and 98.5% 16S
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rRNA gene sequence similarities to Sulfurospirillum multivorans. These findings imply that
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sustained Dehalococcoides activity cannot be expected in low pH (i.e., ≤5.5) groundwater, and
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organohalide-respiring Sulfurospirillum spp. are key contributors to in situ PCE reductive
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dechlorination under low pH conditions.
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Introduction
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Reductive dechlorination processes release hydrochloric acid, which rapidly dissociates to form
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chloride anions and hydronium ions in groundwater. Furthermore, the fermentation of
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biostimulation substrates (e.g., lactate, emulsified vegetable oil, molasses, corn cobs, newsprint,
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wood chips, microbial biomass) results in acidification due to the production of organic acids
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and protons.1 In aquifers with low buffering capacity, pH reductions can decrease rates and even
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stall microbial dechlorination processes.2 Additional undesirable secondary effects of
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acidification include increased solubility of toxic metals and metalloids, which may affect
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microbial activity and/or impair groundwater quality (i.e., exceed regulatory standards).3
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Extensive dechlorination and fermentation reactions may generate enough hydrochloric acid to
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affect the groundwater pH depending on the buffering capacity of the aquifer. For instance,
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calcite plays an important role in buffering the pH of calcareous soils, but the amount of calcite
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in aquifers varies considerably.4 A recent USGS investigation reported that groundwater in
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aquifer systems of the Northern Atlantic Coastal Plain was commonly acidic (median pH 5.3)
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due to the lack of soluble minerals (e.g., calcium carbonate) that would buffer the natural acidity
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(e.g., acidic rainfall). This observation suggests that remediation of sites impacted with
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chlorinated ethenes in this aquifer system would be challenging without pH management.5
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A common response to groundwater pH reductions following in situ biostimulation is the
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addition of alkaline chemicals or compounds with buffering capacity to increase and maintain
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the pH in a range suitable for dechlorinating bacteria. For example, additions of sodium
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bicarbonate or colloidal Mg(OH)2 have been used to manipulate groundwater pH in situ; 6, 7
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however, large amounts of bicarbonate may be required to adjust the pH of calcareous soils.
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Further complicating the issue is the fact that increased carbonate concentrations can cause
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precipitation rather than dissolution of calcite (CaCO3↓↔Ca2++CO32-).8 A low cost, self-
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regulating approach using silicate minerals, which exhibit pH dependent solubility and
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dissolution rates, was proposed to buffer groundwater, but further experimentation indicated that
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silicate minerals and their dissolution products inhibited microbial reductive dechlorination of
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chlorinated ethenes.9 Modeling approaches have been applied to estimate the aquifer buffering
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capacity required for stabilizing groundwater pH;7, 8, 10-14 however, the biogeochemical and
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hydrological complexity of in situ subsurface environments limits their widespread
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implementation. Although engineering approaches are feasible, in situ pH adjustment remains
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challenging, and all current approaches have limitations.
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An alternate solution would be to rely on microorganisms that perform reductive
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dechlorination under low pH conditions. Enhanced in situ reductive dechlorination has shown
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success as a cost-effective remedy for a variety of chlorinated pollutants.15 A number of bacterial
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isolates responsible for tetrachloroethene (PCE) reductive dechlorination have been identified
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and include members of the genera Desulfitobacterium, Sulfurospirillum, Dehalobacter,
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Desulfuromonas, Geobacter, Dehalogenimonas and Dehalococcoides (Dhc).16, 17 These
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dechlorinating bacteria were all enriched and isolated at circumneutral pH, and subsequent
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characterization revealed that maximum reductive dechlorination activity was achieved at
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circumneutral pH, and dechlorination activities of these pure cultures were severely inhibited
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below pH 6.0, and no dechlorination was reported at pH 97% sequence similarity)
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sequences (Table S3). The sequences were imported into the Geneious software environment
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(Biomatters, Auckland, New Zealand), aligned with MAFFT,32 and a 16S rRNA gene
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phylogenetic tree was constructed with Tree Builder using software’s default settings. A Zeiss
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Axio Imager.A2 was used to visualize the liquid cultures for microscopic examination.
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Analytical methods. The pH of the bulk liquid phase was measured by transferring 1 mL liquid
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aliquots from a culturing vessel into a 2-mL plastic tube. After centrifuging the tube for 30
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seconds at 18,000 x g, the pH of the supernatant was measured with a Fisher Scientific Accumet
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Glass AgCl pH electrode (Pittsburgh, PA, USA). Total chlorinated solvent mass or
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concentrations of chlorinated compounds were determined by analyzing headspace gas samples
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on an Agilent 7890A gas chromatograph (GC) equipped with an Agilent DB624 column and a
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flame ionization detector (Agilent Technologies, Santa Clara, CA, USA). Gas samples (100 µL)
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were removed from the headspace of 160-mL serum bottles using a gastight 250 µL Hamilton
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SampleLock syringe (Hamilton, Reno, NV) and then manually injected into the GC. The
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concentrations of chlorinated ethenes were calculated by normalizing the peak area values to
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those of standard curves generated by adding known amounts of chlorinated ethenes to 160-mL
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serum bottles with the same gas to liquid ratios. The total moles of polychlorinated ethenes per
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bottle were calculated according to the equation: total moles = (volume x density) / molecular
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weight. The retention times for different chlorinated ethenes and ethene were determined by
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injecting neat compounds into the GC, and used to assign peaks to PCE daughter products.33, 34
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The average PCE dechlorination rates in pH 5.5 and pH 7.2 cultures were calculated based on the
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total amount of chloride released over 11-day and 7-day incubation periods, respectively, during
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which about 90% of the initial amount of PCE had been dechlorinated. Chloride release
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calculations were based on the gas chromatographic concentration measurements of PCE, TCE
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and cDCE, and assumed that each reductive dechlorination step liberates one chlorine substituent
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as chloride.
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Sequence data. The 16S rRNA gene sequences of Sulfurospirillum sp. strain ACSTCE and strain
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ACSDCE were deposited to GenBank under accession numbers KX101071 and KX101070.
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Amplicon sequencing data of pH 7.2 and pH 5.5 ACS enrichments were deposited to GenBank
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under accession numbers SRX2547923 and SRX2547924.
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Results
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Screening dechlorinating isolates and consortium BDI for low pH reductive dechlorination
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activity. Desulfuromonas michiganensis strain BB1 (PCE→cDCE), Desulfitobacterium sp.
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strain Viet1 (PCE→TCE), Desulfitobacterium sp. strain JH1 (PCE→cDCE), Geobacter lovleyi
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strain SZ (PCE→cDCE) and consortium BDI (PCE→ethene) containing strain SZ and Dhc
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strains GT, FL2 and BAV1 dechlorinated PCE to the expected dechlorination end product ethene
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at pH 7.2, but PCE dechlorinating activity was diminished or lost following transfers in pH 6.0
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and pH 5.5 medium (Table 2). Geobacter lovleyi strain SZ dechlorinated 68.5 µmoles PCE to
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cDCE within 48 hours at pH 7.2, but dechlorination of the same amount of PCE at pH 6.0
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required more than 10 days, and no PCE dechlorination was observed at pH 5.5. Similarly,
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Desulfuromonas michiganensis strain BB1 dechlorinated PCE to cDCE within one week at pH
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7.2, but it took more than 30 days to degrade PCE to cDCE at pH 6.0. Sulfurospirillum
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multivorans was the only tested organism able to degrade PCE to cDCE at pH 5.5, 6.0 and 7.2
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within 4 days (Table S4); however, this organism could not sustain PCE dechlorination at pH 5.0.
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Dechlorination activity in microcosms and enrichment cultures. PCE to ethene reductive
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dechlorination was observed in microcosms established at pH 5.5 and pH 7.2 with samples
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collected from five sampling sites (#5, 6, 7, 11 and 16 in Table S1). Microcosms established with
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the soil sample from the ACS site (#13) degraded PCE to ethene at pH 7.2, but VC was the
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dechlorination end product at pH 5.5 (Figure 1). Microcosms established with acidic peat bog
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material from the Shady Valley location (#12) showed PCE to cDCE dechlorination at pH 5.5
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and pH 7.2. The tidal flat sample (#15) degraded PCE to TCE at pH 7.2, but not at pH 5.5. No
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PCE dechlorination activity was detected in the microcosms established with the other site
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materials tested.
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Attempts to establish stable PCE-to-ethene-dechlorinating enrichment cultures in pH 5.5
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medium were not successful for any of the ethene-producing microcosms. PCE dechlorination
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activity was lost following two consecutive transfers in pH 5.5 medium, except the ACS
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enrichment cultures, which maintained PCE-to-cDCE reductive dechlorination activity at pH 5.5
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(Figure 1). By comparison, transfer cultures derived from the ethene-producing pH 7.2 ACS
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microcosms maintained PCE-to-ethene dechlorination activity following consecutive transfers to
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pH 7.2 medium (Figure 1). At pH 4.5, PCE dechlorination to cDCE was observed in
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microcosms established with materials from the ACS site, but PCE dechlorination activity could
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not be maintained at this pH in transfer cultures (i.e., in the absence of the solid phase).
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pH effects on community structure. To investigate the changes in community structure in
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response to pH differences, and to identify the dechlorinator(s) responsible for the measured
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reductive dechlorination activity in enrichment cultures derived from the ACS microcosms that
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sustained PCE dechlorination at both pH 5.5 and pH 7.2, 16S rRNA gene amplicon sequencing
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was performed. After removing low quality reads, 69,030 sequences (17,441,054 total base pairs)
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and 103,503 sequences (26,171,881 total base pairs) were obtained from the ACS pH 5.5 and the
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pH 7.2 enrichments, respectively. A total of 172,409 sequences from the pH 5.5 and the pH 7.2
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enrichment cultures were classified into 815 operational taxonomic units (OTUs) based on a 98%
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identity cutoff (Table S2), and only 41 sequences could not be classified according to the
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SILVAngs empirical threshold ((sequence identity + alignment coverage) / 2 >= 93%).31
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Rarefaction analysis of sequences showed that the rarefaction curves did not plateau, suggesting
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that the sequencing effort did not capture the diversity of low abundance members in both the pH
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7.2 and the pH 5.5 enrichment cultures (Figure S1).
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Sequences representing the phyla Proteobacteria and Firmicutes were most abundant in the
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pH 5.5 and pH 7.2 ACS enrichment cultures, respectively (Figure 2). The phyla Proteobacteria,
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Firmicutes, and Bacteroidetes were relatively enriched and contributed 57.9%, 31.8%, and 7.2%,
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respectively, to the classified sequences in the pH 5.5 enrichment, compared with 2.8%, 59.6%,
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and 3.0%, respectively, in the pH 7.2 enrichment. At pH 7.2, sequences of the phyla Caldiserica
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(4.0%), Chloroflexi (21.9%) and Spirochaetes (4.4%) were more abundant (Figure 2). Sequences
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representing the phyla Aigarchaeota, Thaumarchaeota, Chlorobi, Lentisphaerae, Nitrospirae and
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Synergistetes were present in the pH 7.2 PCE-to-ethene-dechlorinating enrichment, but were not
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observed in the pH 5.5 enrichment, indicating their preference for circumneutral pH conditions.
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By comparison, sequences representing the phyla Euryarchaeota, Nanoarchaeota, Dictyoglomi,
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Fusobacteria and Thermotogae were detected in pH 5.5 PCE-to-cDCE-degrading ACS
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enrichment culture, but not in the pH 7.2 cultures. Furthermore, the most abundant genera in pH
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5.5 ACS enrichment cultures differed from those dominating in the pH 7.2 enrichment cultures.
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At pH 7.2, the 16S rRNA gene sequences of the genera Dhc (phylum Chloroflexi) and
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Acetobacterium (phylum Firmicutes) dominated the enrichment and accounted for 22.6% and
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57.6% of all sequences, respectively. In contrast, Desulfovibrio (phylum Proteobacteria) (33.0%),
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Sulfurospirillum (phylum Proteobacteria) (25.2%), and Megasphaera (phylum Chloroflexi)
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(19.9%) sequences dominated the pH 5.5 enrichment (Table 3).
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Low pH PCE reductive dechlorination by two Sulfurospirillum isolates. Isolation efforts
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focused on the ACS PCE-to-cDCE-dechlorinating enrichments that maintained dechlorinating
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activity for at least 10 consecutive transfers in pH 5.5 medium. Repeated dilution to extinction
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series and transferring individual colonies from the 10-4 and 10-5 semi-solid medium dilution
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tubes yielded two PCE-dechlorinating isolates. Light microscopic observations revealed uniform,
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spirillum-shaped cells, and a single 16S rRNA gene sequence type was recovered from each
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culture. Both isolates grew with PCE in defined mineral salts medium at pH 5.5, and
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dechlorinated PCE at similar rates of 28.3±2.3 µmoles Cl- released per liter and day. Isolate
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ACSTCE generated TCE as the dechlorination end product, whereas isolate ACSDCE dechlorinated
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PCE and TCE to cDCE. Approximately 30% higher dechlorination rates were measured for both
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isolates at pH 7.2 (Figure 3). Isolates ACSTCE and ACSDCE shared highly similar 16S rRNA
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genes (99.7% identity) and affiliated with the genus Sulfurospirillum within the ε-Proteobacteria
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class. Isolate ACSTCE and isolate ACSDCE shared 98.6% and 98.5% 16S rRNA gene sequence
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identity, respectively, with the characterized PCE-to-cDCE dechlorinator Sulfurospirillum
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multivorans (NR_121740.1). A phylogenetic analysis based on available Sulfurospirillum 16S
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rRNA gene sequences demonstrated that ACSTCE and ACSDCE isolates were most closely related
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to the PCE dechlorinator Sulfurospirillum sp. strain JPD-1 (AY189928.1) with 99.7% and 99.6 %
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sequence identity, respectively (Figure 4).
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Discussion
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Microbial reductive dechlorination in low pH groundwater. Successful bioremediation of
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groundwater impacted with chlorinated ethenes relies on the growth of native and/or
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bioaugmented dechlorinating microorganisms, especially Dhc. Since Dhc sustains reductive
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dechlorination activity within a narrow pH range (Table 1), adjustments to maintain a
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circumneutral pH environment may be necessary. An alternative solution is to rely on
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microorganisms capable of degrading chlorinated ethenes to ethene at low pH (i.e., pH < 6.0).
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The available Dhc cultures cannot sustain growth at pH
