Coupling Bioflocculation of Dehalococcoides mccartyi to High-Rate

Article pubs.acs.org/est

Coupling Bioflocculation of Dehalococcoides mccartyi to High-Rate Reductive Dehalogenation of Chlorinated Ethenes Anca G. Delgado,*,†,‡,§ Devyn Fajardo-Williams,†,‡,§ Emily Bondank,† Sofia Esquivel-Elizondo,†,‡ and Rosa Krajmalnik-Brown*,†,‡ †

Biodesign Swette Center for Environmental Biotechnology, Arizona State University, Tempe, Arizona 85287-5701, United States School of Sustainable Engineering and the Built Environment, Arizona State University, Tempe, Arizona 85287-3005, United States

‡

S Supporting Information *

ABSTRACT: Continuous bioreactors operated at low hydraulic retention times have rarely been explored for reductive dehalogenation of chlorinated ethenes. The inability to consistently develop such bioreactors affects the way growth approaches for Dehalococcoides mccartyi bioaugmentation cultures are envisioned. It also affects interpretation of results from in situ continuous treatment processes. We report bioreactor performance and dehalogenation kinetics of a D. mccartyi-containing consortium in an upflow bioreactor. When fed synthetic groundwater at 11−3.6 h HRT, the upflow bioreactor removed >99.7% of the influent trichloroethene (1.5−2.8 mM) and produced ethene as the main product. A trichloroethene removal rate of 98.51 ± 0.05 me− equiv L−1 d−1 was achieved at 3.6 h HRT. D. mccartyi cell densities were 1013 and 1012 16S rRNA gene copies L−1 in the bioflocs and planktonic culture, respectively. When challenged with a feed of natural groundwater containing various competing electron acceptors and 0.3−0.4 mM trichloroethene, trichloroethene removal was sustained at >99.6%. Electron micrographs revealed that D. mccartyi were abundant within the bioflocs, not only in multispecies structures, but also as self-aggregated microcolonies. This study provides fundamental evidence toward the feasibility of upflow bioreactors containing D. mccartyi as high-density culture production tools or as a high-rate, real-time remediation biotechnology.

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in the order of several days. However, continuous flow reactors containing D. mccartyi have seldom shown to dehalogenate PCE or TCE all the way to ethene at HRTs of days.7 Most continuous dehalogenating reactors stall at DCE and VC (see Table 1 in Delgado et al.7), despite efforts to optimize reactor operating conditions. The discrepancy between reported kinetic parameters and actual engineering efforts suggests that other parameters play a role in the metabolic activity of D. mccartyi in mixed consortia. The inability to consistently operate dehalogenating continuous reactors affects the way future biotechnologies (e.g., ex situ biological treatment) and growth approaches for bioaugmentation cultures can be designed. This inability can also affect the interpretation of results under continuous groundwater flow or during continuous or semicontinuous in situ treatment. Consequently, understanding how D. mccartyi dehalogenating bioreactors can be effectively operated in the laboratory is crucial for the optimization of biological dehalogenation approaches for chlorinated ethenes.

INTRODUCTION Since the early 2000s, in situ enhanced reductive dehalogenation has become a widely utilized, accepted remediation practice at sites contaminated with the chlorinated solvents perchloroethene (PCE) and trichloroethene (TCE). Bioaugmentation has been used at several hundred sites1 as an approach to increase reductive dehalogenation rates and to shorten time frames for cleanup and site closure. Over 100 000 L of microbial bioaugmentation consortia have been delivered to over 650 field sites in the United States as part of groundwater remediation schemes.2,3 The implementation of bioaugmentation explores the ability of the organohalide respiring bacterium Dehalococcoides mccartyi (injected in the subsurface as a consortium) to dehalogenate PCE, TCE, dichloroethene (DCE), and vinyl chloride (VC) to the nonhalogenated end-product, ethene.4 Thus, for the improvement of reductive dehalogenation in the laboratory and at field scale, it is imperative to characterize the growth and substrate utilization kinetics of D. mccartyi-containing consortia. The doubling times for D. mccartyi have been reported to be as short as 17 h,5 along with Monod constants, Ks, of 0.5−5 μM for TCE, DCE, and VC,6 suggesting that these microorganisms should be able to effectively dehalogenate PCE/TCE to ethene in continuous reactors with hydraulic retention times (HRTs) © XXXX American Chemical Society

Received: June 16, 2017 Revised: September 1, 2017 Accepted: September 5, 2017

A

DOI: 10.1021/acs.est.7b03097 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology

was constructed from a glass tube with a length of 27 cm, inner diameter of 2.17 cm, and a total volume of 100 mL. Butyl rubber stoppers were used to seal the bioreactor. These were drilled and fitted with a stainless steel line for an influent line and biofloc dispense line at the base, and effluent line at the top. Viton tubing and stainless steel connectors were used. The influent line was split with a T-connector and fed from the medium bottle and the recirculation line. The effluent line was also split with a T-connector dividing the effluent between a 1L collection bottle and the recirculation line which was reintroduced into the bioreactor by a peristaltic pump (Masterflex Easy-Load II, Cole-Parmer, U.S.A.). A 20 mL glass vial fitted with a butyl rubber stopper was connected before the medium inlet where 6 mL liquid and 14 mL gas were maintained for analyses of liquid and gas samples representative of the bioreactor. A peristaltic pump (Ismatec REGLO, ColeParmer, U.S.A.) fed medium (synthetic or natural groundwater) from a 5-L glass bottle to the influent line. The flow out of the bioreactor was by gravity. A photograph of the bioreactor on day 81 of operation is shown in Figure S1 of the Supporting Information, SI. Microbial Inocula and Operating Conditions for Biofloc Formation. Initial handling and inoculation of the bioreactor was performed in an anaerobic chamber (Coy Lab Products, U.S.A.). The bioreactor was inoculated with 20 mL (20% v v−1) ZARA-10 dehalogenating culture,19,20 1 mL of anoxic digester sludge from the Northwest Water Reclamation Plant, Mesa, Arizona, U.S.A.,21,22 and medium up to 100 mL. ZARA-10 inoculum was grown in a continuously stirred tank react (CSTR) according to our published methodology.7 ZARA-10 culture is enriched in D. mccartyi (unidentified strains and strains containing the genes tceA, vcrA, and bvcA (in a low abundance)), TCE to cis-DCE organohalide-respirers from Geobacteraceae, fermenting and homoacetogenic bacteria, and hydrogenotrophic methanogens.19,20 The anoxic digester sludge contains anaerobic bacteria along with hydrogenotrophic and acetoclastic methanogens.21,22 D. mccartyi concentration at time 0 in the bioreactor was 1011 16S rRNA gene copies L−1. The reduced anaerobic mineral medium was composed of 20 mM HEPES, 1 mM NaHCO3, salts, and trace minerals as previously described,7,8 500 μg L −1 vitamin B12, 5 mL L−1 vitamin solution for bacterial growth, 0.4 mM L-cysteine, 0.2 mM Na2S·9H2O, 0.25 μg L−1 resazurin, 15 mM methanol, and 5 mM sodium lactate. The medium pH was 7.5. Following inoculation, the bioreactor was assembled on a laboratory benchtop in a room with a constant temperature of 30 °C. The timeline and corresponding operating conditions are included in Table 1. The bioreactor was initially operated in batch mode with the recycle stream at 10 mL min−1 (2.7 cm min−1). The bioreactor was then operated continuously starting at a flow rate of 33 mL d−1 (HRT = 72 h) in order to promote biofloc formation and to determine optimal operating parameters (Table 1). Experimental Design for TCE Dehalogenation in Synthetic Groundwater and Amended Natural Groundwater. TCE dehalogenation was first tested at various HRTs using a feed of synthetic groundwater (Table 1). During synthetic groundwater phases I−III, the influent feed consisted of reduced mineral medium with 1.5−2.8 mM TCE (9−16.8 me− equiv L−1), 10 mM sodium lactate (120 me− equiv L−1), and 15 mM methanol (90 me− equiv L−1) buffered with 20 mM HEPES and 1 mM HCO3− (8 me− equiv L−1). The reducing agents were 0.2 mM Na2S·9H2O and 0.4 mM L-

Our group successfully operated continuously stirred tank reactors (CSTRs) fed with TCE using a D. mccartyi dehalogenating consortium. The CSTRs were able to achieve ≥97% dehalogenation of TCE to ethene at a 3 d HRT, leading to the highest reductive dehalogenation and growth rate of D. mccartyi thus far in a bioreactor.7 The success of our CSTR operation is in part due to limiting bicarbonate, which serves as electron acceptor for methanogens and homoacetogens competing with D. mccartyi for H2 availability.8 Dehalogenation rates, however, could be increased by over an order of magnitude in a flocculated upflow bioreactor designed to retain higher concentrations of biomass than a CSTR.9 On the one hand, we expect that using a similar medium and growth conditions that limit H2 competitors as in our CSTRs, D. mccartyi could grow effectively and perform complete dehalogenation to ethene in an upflow bioreactor. On the other hand, available literature on upflow bioreactors containing D. mccartyi shows incomplete dehalogenation of PCE/TCE,10−15 with TCE and DCE as the reported main products of dehalogenation. A possible reason for incomplete dehalogenation in past studies is the inability of D. mccartyi to effectively flocculate. In the absence of effective flocculation, D. mccartyi would be subjected to wash-out and, thus, their growth rates could not be decoupled from the HRT. Biofilms or flocs of D. mccartyi pure cultures have not yet been reported, although D. mccartyi have been grown in mixed culture biofilm reactors.16,17 To date, only two studies have documented D. mccartyi or D. mccartyi-like microorganisms in flocs with members of Bacteria and Archaea using fluorescence in situ biohybridization.12,18 In this study we present evidence on long-term operation and conversion of TCE to mainly ethene in bioflocs highly enriched in D. mccartyi. Effective bioflocculation and selective enrichment of D. mccartyi was achieved through a combination of bioreactor design, operating conditions, and medium composition (synthetic groundwater with low HCO 3 − concentrations). Reductive dehalogenation of TCE to ethene was sustained for long periods even when the bioreactor was fed with amended natural groundwater at an HRT of 3.2 h.

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MATERIALS AND METHODS Upflow Bioreactor Design. The upflow bioreactor used in this study is schematically depicted in Figure 1. The bioreactor

Figure 1. Schematic depiction of the upflow bioreactor and photographs of bioflocs formed during continuous operation. B


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Table 1. Bioreactor Operating Conditions for Establishing Bioflocs and for the Continuous Experimental Phases with Synthetic and Natural Groundwatera

a

days

operation

0−39 39−71 71−78 78−83 83−89 89−92 92−103 103−111 111−117 117−126 126−132 132−138 138−181 181−194 194−197 194−202 202−238 238−260 260−273 273−279 279−286 286−416 416−549 549−561 561−590 590−602 602−616 616−627 627−650

inoculation; batchb continuousc continuousc continuousc continuousc continuousc continuousc continuousc continuousc batchb synthetic groundwater Ic synthetic groundwater Ic synthetic groundwater Ic synthetic groundwater Ic synthetic groundwater Ic batch synthetic groundwater IIc batchb synthetic groundwater IIId synthetic groundwater IIId synthetic groundwater IIId batchb cont. batch cont. natural groundwatere natural groundwatere natural groundwatere natural groundwatere

influent flow (mL d−1)

recirculation flow (mL min−1)

33 40 44 53 69 87 116 100 100 167 222 333 667 333 222 333 667 120−200 200 200 264 393 750

10 10 12−15 18−20 20−24 25 25 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55

HRT (h)

influent TCE (mM)

TCE loading (me− equiv L−1 d−1)

72 60 54 46 37 28 21 24

1.0 1.0 1.0 1.0 1.0 1.0 2.0 2.0

2.0 2.4 2.7 3.2 4.2 5.2 13.7 11.8

24 14 11 7.2 3.6

1.5 1.5−1.8 1.8−2.8 2.5 2.5

8.8 14.7−18.2 24.2−37.8 49.4 98.7

7.2

2.5−2.0

49.4−40.7

2.3 2.3 2.3

30.5 45.8 91.6

11 7.2 3.6 20−12 12 12 9.1 6.1 3.2

1.0 1.0 0.4−0.3 0.4 0.4 0.4

7.2−12.0 12.0 4.3−3.7 6.2 10.2 19.4

Continuous operation at 3.6−3.2 h HRT is in bold. bbatch addition of 0.5−2 mM TCE. Reductive dehalogenation data shown in figures. cFigure 2. Figure 3. eFigure 4.

d

concentrations. Methane detection limit was 12 μM gas concentration. Liquid samples (1 mL) filtered through 0.2 μm polyvinylidene fluoride membrane filter (Acrodisc, Pall, U.S.A.) were used to quantify concentrations of organic acids (lactic, acetic, and propionic) and methanol with a high performance liquid chromatograph (HPLC) (LC-20AT, Shimadzu, U.S.A.) as described.20 The detection limits for organic acids were 0.02−0.08 mM. Methanol detection limit was 0.5 mM. SO42−, NO3−, and Cl− were measured using a Dionex ICS 3000 ion chromatograph (IC) according to the methods previously published.23 Total Fe and alkalinity (used to estimate HCO3− concentrations) were measured via Hach kits (Loveland, U.S.A.) as per manufacturer’s instructions. Bioflocs and planktonic cells were sampled throughout synthetic groundwater phase III (days 260−286) and natural groundwater phase (days 590−650) for microbial ecology analyses. Effluent pellets were made by centrifugation from 500 μL planktonic culture (e.g., floating cells not part of any visible aggregates) obtained from the effluent line. Pellets from bioflocs were made by collecting 500 μL sample from the base of the bioreactor. The bioflocs were then resuspended in 1 mL sterile anaerobic mineral medium, homogeneously mixed, divided equally to make triplicate pellets, and then centrifuged. DNA was extracted from these pellets with the methodology previously described.24

cysteine. Dehalogenation was also tested using natural groundwater as influent medium (Table 1). Natural groundwater was procured from the inlet of RID-92 GAC Wellhead Treatment System from the Motorola 52nd Street Superfund Site in Phoenix, Arizona, U.S.A. The natural groundwater constituents were as follows: 9.2 mM Cl−, 0.5 μM Fe, 0.43 mM NO3− (2.2 me− equiv L−1), 1.7 mM SO42− (14 me− equiv L−1), and 2.0 mM HCO3− (16 me− equiv L−1). 0.02−0.08 μM PCE, 1,1-DCE, cis-DCE, chloroform, and 1,1-dichloroethane were also present in the groundwater (data provided by Airtech Environmental Laboratories, Phoenix, AZ). Before use in experiments, the natural groundwater was buffered with 10 mM HEPES, and amended with 0.3−0.4 mM TCE (1.8−2.4 me− equiv L−1), 5 mM lactate (60 me− equiv L−1), 7.5 mM methanol (60 me− equiv L−1), 200 mg L−1 yeast extract, and 0.2 mM Na2S·9H2O. Chemical and Molecular Biological Analytical Methods. 200 μL gas samples were taken from the sampling chamber to quantify gas concentrations of chlorinated ethenes, ethene, and methane using a gas chromatograph (GC) with a flame ionization detector (FID) (GC-2010, Shimadzu, U.S.A.). The chromatographic capillary column was Rt-QS-BOND (Restek, Bellefonte, U.S.A.), and the GC temperature and flow settings were as described previously.8 The detection limits for chlorinated ethenes and ethene were 0.2−0.6 μM gas C


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the sampling chamber (Figures 1 and S1). Because the upflow bioreactor per se did not have a headspace, we converted and reported the aqueous concentrations of TCE, cis-DCE, VC, and ethene according to eq 1:

Quantitative real-time PCR (qPCR) was performed targeting the 16S rRNA gene of D. mccartyi. During synthetic groundwater phase III, the reductive dehalogenase genes of D. mccartyi, tceA, vcrA, and bvcA, and the 16S rRNA gene of Archaea were also quantified. The functional gene of homoacetogens, formyltetrahydrofolate synthase (FTHFS), was quantified at the beginning and end of the synthetic groundwater phase III. qPCR was performed using a Realplex 4S thermocycler (Eppendorf, Hauppauge, U.S.A.). The primers, probes, reagent concentrations, and PCR conditions using were those published.20,24 Six point standard curves and samples were setup in triplicate reactions. Reaction volume was 10 μL using 4 μL of 1/10 diluted DNA as template. Pipetting was performed using an automated epMotion 5070 liquid handling system (Eppendorf, U.S.A.). qPCR target detection was verified through sequencing at the Biodesign Plasmid Repository and DNA Sequencing Facility, Arizona State University. Microbial community amplicon sequencing using primers 515F and 806R for the V4 hyper-variable region of the 16S rRNA gene25 was performed using the Illumina MiSeq platform as previously described at the Microbiome Analysis Laboratory in the Swette Center for Environmental Biotechnology, Arizona State University (http://krajmalnik. environmentalbiotechnology.org/microbiome-lab.html). 26 QIIME 1.8.0 pipeline was used to process raw paired sequences.27 Sequences were quality filtered based on the following criteria: shorter than 250 bps, quality score of 25 or below, 1 or more primer mismatches, more than 6 homopolymers. Sequences were submitted to NCBI Sequence Read Archive and are available under the accession numbers: SAMN03784691, SAMN03784692, SAMN03784693, SAMN03784694, SAMN03784695, SAMN03784696, SAMN03783052, SAMN03783053, SAMN03783054, SAMN03783055, SAMN03783056, and SAMN03783005. Operational taxonomy units (OTUs) were picked based on 97% sequence similarity from the remaining sequences using the UCLUST algorithm.28 The most abundant sequence of each cluster was picked as the representative sequence of the OTU and then aligned to the Greengenes database29 using PyNAST.30 Chimera Slayer31 was used to identify chimeric sequences which were then removed. UCLUST was used to assign taxonomy based on the Greengenes database in order to construct a BIOM formatted OTU table.29 Singletons (OTUs with less than 2 sequences) were removed from the OTU table. The OTU table was rarefied at the lowest number of sequences using Qime’s default pseudorandom number generator (14 000 and 34 000 for synthetic groundwater phase III and natural groundwater phase, respectively). Scanning electron micrographs of bioflocs were taken during bioreactor operation with synthetic groundwater (days 260− 286). The bioflocs were washed with 3× phosphate-buffered saline solution. Fixation, gradual dehydration, and critical point drying in preparation for SEM imaging was achieved with our previously published methods.17 Once mounted on stubs and sputter-coated with gold−palladium, micrographs were captured by a XL-30 Environmental Scanning Electron Microscope with a Schottky Field Emission Source at the John M. Cowley Center for High Resolution Electron Microscopy, Arizona State University. Calculations. Chlorinated ethenes, ethene, and methane concentrations representative of the bioreactor constituents were determined from gas samples taken from the headspace of

Caq =

Cgas Hcc

(1)

where Caq is the aqueous phase concentration (mM), Cgas is the concentration in the gas phase (mM), and Hcc is the Henry’s law constant (Laq Lgas−1). Hcc constants for chlorinated ethenes and ethene were determined experimentally.7 During certain experimental phases, the mol sum of effluent TCE, cis-DCE, VC, and ethene was lower than what expected based on the influent TCE concentration. The effluent gas flow rate could not be measured based on the configuration of the bioreactor. The incomplete mol balance at times was likely due to increased rate of biogas production from fermentation and methanogenesis resulting in a dilution of ethene concentrations. This outcome has been reported previously by other researchers operating upflow bioreactors with chlorinated ethenes.10,12 To keep track of the mol balance, we calculated effluent mol recovery by diving the sum of effluent moles of chlorinated ethenes and ethenes by total influent TCE moles (eq 2): %effluent mol recovered =

TCEeff + DCEeff + VCeff + etheneeff × 100 TCE in

(2)

TCE loading in units of me calculated according to eq 3: TCE loading =

−

equiv Lbioreactor

−1

−1

d

[TCE in] × Q V

was

(3) −

where TCEin is the TCE influent concentration (me equiv L−1), Q is the influent flow rate (L d−1), and V is the volume of the bioreactor (L). TCE removal rates in units of me− equiv Lbioreactor−1 d−1 were calculated after at least 5 HRTs according to eq 4: TCE removal rate =

[TCE in − TCEeff ] × Q V

(4)

where TCEin and TCEeff are influent and effluent TCE concentrations (me− equiv L−1), Q is the influent flow rate (L d−1), and V is the volume of the bioreactor (L). %TCE removed was calculated from the influent and effluent concentrations (mM) (eq 5): %TCE removed =

■

[TCE in] − [TCEeff ] × 100 [TCE in]

(5)

RESULTS AND DISCUSSION Bioreactor Optimization and Bioflocculation of D. mccartyi-Containing Consortium. We investigated the coupled capability of D. mccartyi to aggregate with microbial members of the dehalogenating consortium, ZARA-10, and anoxic digester sludge and to sustain reductive dehalogenation of TCE to ethene in an upflow bioreactor. The bioreactor was transitioned to continuous mode after a period of culture acclimation and batch operation (see timeline in Table 1). Figure 2A presents the reductive dehalogenation activity during the optimization period (days 39−117). TCE was quickly reduced to cis-DCE and VC upon transition to continuous D


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Figure 3. Performance of the upflow bioreactor fed with synthetic groundwater at various HRTs. (A) Reductive dehalogenation of TCE. The average chlorinated ethenes and ethene effluent mol recovery was 91% at 11 h HRT, 76% at 7.2 h HRT, and 30% at 3.6 h HRT. (B) Consumption of lactate and methanol and coupled production of acetate, propionate, and methane. The data are concentrations of the compounds in the aqueous phase. (C) Quantification of the 16S rRNA gene of Dehalococcoides mccartyi and Archaea, and the formyltetrahydrofolate synthetase (FTHFS) gene of homoacetogens in bioflocs and planktonic culture. FTHFS was not analyzed on days 273 and 282. The data are averages with standard deviation of triplicate qPCR reactions.

Figure 2. TCE reductive dehalogenation during (A) upflow bioreactor optimization and establishment of bioflocs enriched in Dehalococcoides mccartyi and (B, C) during two experimental phases with synthetic groundwater at various HRTs. The data are concentrations of the compounds in the aqueous phase. The average chlorinated ethenes and ethene effluent mol recovery during these phases was ≥82%.

mode. Stable dehalogenation of 1 mM TCE to ethene was achieved at an HRT of 72 h after ∼5 HRTs (Figure 2A), as previously done in our CSTR work.7 Overall, conversion of TCE to ethene during the optimization period was either sustained or was recovered with decreasing HRT or increasing TCE influent concentration (Figure 2A). Substantial biomass accumulation and formation of bioflocs was also observed during this time (Figure 1). High-Rate Reductive Dehalogenation in Synthetic and Natural Groundwater. Following the optimization period, we tested the bioreactor’s ability to dehalogenate TCE at HRTs of 24−3.6 h using a feed of synthetic groundwater (Figures 2B, C and 3). We rationalized that the established bioflocs would prevent wash-out, thus, allowing reductive dehalogenation of TCE to ethene at HRTs much lower than the doubling times of D. mccartyi. Bioreactor operation at 11 and 3.6 h was replicated two times, and at 7.2 h three times (Figures 2B, C and 3A). During synthetic groundwater phases I−III, ethene was the main product of TCE reductive dehalogenation under all conditions tested. Typically, ethene accounted for 95−98% of the effluent composition at pseudo steady-state. Transient accumulation of cis-DCE and VC was correlated with increases in influent TCE concentration (Figure 2B) or with transitioning of the bioreactor from batch to continuous mode (Figure 3A). At pseudo steady-state, TCE concentrations ranged from nondetectable to 0.004 mM, while cis-DCE and VC concentrations were 0.01−0.05 mM (Figure 3A). Removal of TCE was >99.7% (Tables 2 and 3), even as the HRT was reduced from 11 to 7.2 h, and finally to 3.6 HRT. At an HRT of 3.6 h, the bioreactor reached a remarkably high removal rate of 98.51 me− equiv. L−1 d−1 (Table 3). This rate surpasses those achieved in previous upflow bioreactor studies (Table 2). This rate is also ∼15 times higher than our previously reported rate values for D. mccartyi mixed cultures grown in a CSTR7 and up to 3 orders of magnitude higher than in batch reactors from other studies.6,24

D. mccartyi concentrations ranged from 2.4 × 1013 16S rRNA gene copies L−1 (11 h HRT) to 1.6 × 1013 16S rRNA gene copies L−1 (3.6 h HRT) in the bioflocs (Figure 3C). D. mccartyi containing the reductive dehalogenase gene tceA were the most abundant, previously identified strains (avg. 1.1 × 1012 gene copies L−1, Figure S2). Noteworthy, the abundance of biofloc tceA and vcrA gene copies L−1 accounted for only up to 16% of the total abundance of the16S rRNA gene copies of D. mccartyi (Figure S2). This discrepancy strongly suggests the occurrence of D. mccartyi in ZARA-10 culture that have reductive dehalogenases other than tceA, vcrA, and bvcA; hence, they were not quantifiable with the qPCR primers utilized. Reductive dehalogenase gene concentrations lower by 1 order of magnitude than D. mccartyi 16s rRNA gene concentrations were previously noted in several studies, including in bioreactors,16 laboratory microcosms,32,33 and site groundwater material.32,33 Overall, the high D. mccartyi densities achieved when the bioreactor was fed with synthetic groundwater substantiate the high rates of reductive dehalogenation from Table 3. However, average planktonic concentrations of 2.3 × 1012 D. mccartyi 16S rRNA gene copies L−1 (Figure 3C) indicated that a significant fraction of the dehalogenation activity was also occurring in suspension. One of the driving hypothesis of this study was that bioflocs enriched in D. mccartyi and high-rate reductive dehalogenation to ethene can be achieved if hydrogenotrophic methanogenesis and homoacetogenesis are minimized. Key to the obtaining this outcome is the influent medium composition with a high concentration of TCE (up to 2.85 mM) and a lower HCO3− concentration (1 mM). Methanogens and homoacetogens were present in the ZARA-10 inoculum20 and were additionally introduced from the digester sludge inoculum. qPCR analyses E


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Environmental Science & Technology Table 2. Upflow Bioreactors Studies for Chlorinated Ethenesa

a

inoculum

chlorinated ethene

HRT

removal and primary product/s

TCE-dehalogenating culture (this study) TCE-acclimated anaerobic sludge10 acclimated anaerobic sludge11 anaerobic granular sludge12 anaerobic culture13 anaerobic acidogenic and methanogenic culture14 anaerobic digester sludge15

0.3−2.8 mM TCE 0.3 mM TCE 0.1−0.6 mM TCE 0.005−0.02 mM PCE 0.04−0.4 mM TCE 0.02−0.3 mM TCE 0.04−0.4 mM PCE

24−3.2 h 25−5 h 10 h 4−1 d 2 d-8.4 h 2 d−6 h 24−8 h

>99%, ethene 99−85%, cis-DCE 85−90%, cis-DCE 38−76%, TCE 86−90%, cis-DCE and VC 50−90%, not reported 98%, VC

The tabulated studies employed a continuous feed of chlorinated ethenes throughout bioreactor operation.

Table 3. Kinetics of TCE Removal for the Experimental Phases with Synthetic and Natural Groundwatera synthetic groundwater HRT (h) 11 7.2 3.6

natural groundwater −

−1

TCE removal rate (me equiv L

−1

d )

32.8 ± 0.10 (I); 30.5 ± 0.01 (III) 49.3 ± 0.02 (II); 45.7 ± 0.03 (III) 98.5 ± 0.05 (I); 91.9 ± 0.01 (III)

% TCE removed

HRT (h)

99.71 (I); 99.99 (III) 99.90 (II); 99.92 (III) 99.74 (I); 99.99 (III)

12 9.1 6.1 3.2

TCE removal rate (me− equiv L−1 d−1) 4.79 7.29 11.0 22.1

± ± ± ±

0.4 0.01 0.32 0.07

% TCE removed 99.73 99.93 99.71 99.62

a

Ethene was the main steady-state dehalogenation product detected in the effluent under the conditions tested. The Roman numerals signify the experimental phases with synthetic groundwater. TCE removal rates are averages with standard deviation of at least three rates at steady-state. The average SRT was ∼200 days.

during synthetic groundwater phase III revealed that methanogens and homoacetogens (1010−1011 gene copies L−1) were ∼2−2.5 orders of magnitude lower than D. mccartyi throughout bioreactor operation (Figure 3C). Homoacetogens decreased throughout continuous operation, while methanogenic Archaea showed increasing trends with time (Figure 3C). This dynamic change within the HCO3−-respiring microbial populations with the predominance of methanogens over homoacetogens at low HCO3− concentrations has been previously documented in our work.8 Overall, qPCR data further validate that the conditions provided in the bioreactor and the medium utilized were conducive to maximizing D. mccartyi growth while minimizing H2-competing microbial processes. Upon completion of the experimental phases with synthetic groundwater, the bioreactor was entered into a maintenance phase consisting of periods of batch and continuous operation (Table 1). Starting on day 590, the synthetic groundwater was replaced with a feed of amended natural groundwater (nonsterilized). The rationale for this phase was to test whether the bioflocs established would sustain TCE reductive dehalogenation in a more complex medium containing not only a population of native microorganism but also additional H2competing electron acceptors. The introduction of SO42− (1.7 mM), NO3− (0.43 mM), and additional HCO3− (2.0 mM) from the natural groundwater presented an opportunity for perturbations and/or incomplete dehalogenation of the influent TCE. The transition to natural groundwater phase starting at 12 h HRT brought no discernible decline in dehalogenation performance (Figure 4A). TCE removal was >99.6% for the duration of the experiment (Table 3) with ethene as the main product of dehalogenation in the effluent. Concomitant with the reduction of TCE to ethene, significant microbial reduction of SO42−, NO3−, and HCO3− (to methane) was occurring in the bioreactor during this phase (Figure 4C). D. mccartyi cell concentrations stabilized at 1012 and 1011 16S rRNA gene copies L−1 in bioflocs and planktonic culture, respectively, regardless of the change in HRT and consistent with the lower TCE influent concentration fed (Figure 4D). The stable TCE

Figure 4. Performance of the upflow bioreactor fed with natural groundwater at various HRTs. The natural groundwater was obtained from the Motorola 52nd St. Superfund Site, Phoenix, Arizona. (A) Reductive dehalogenation of TCE. The average chlorinated ethenes and ethene effluent mol recovery was 74% at 12 h HRT, 58% at 9.1 h HRT, 50% at 6.1 h HRT, and 47% at 3.2 h. (B) Consumption of lactate and methanol and coupled production of acetate, propionate, and methane. (C) Sulfate and nitrate reduction. The data are concentrations of the compounds in the aqueous phase. (D) Quantification of the 16S rRNA gene of Dehalococcoides mccartyi in bioflocs and planktonic culture. The data are averages with standard deviation of triplicate qPCR reactions.

dehalogenation performance of the bioreactor when challenged with natural groundwater highlights the robustness of the system. Furthermore, this experimental phase brought evidence F


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Figure 5. Relative abundance of microbial groups in bioflocs (B) and planktonic (P) culture during synthetic groundwater phase III (days 260−286) and natural groundwater phase (days 590−650). Reads of the V4 hyper-variable region of the 16S rRNA gene were clustered into operational taxonomic units. Taxonomy was assigned with uClust to Greengenes database.

that D. mccartyi are effective competitors at an HRT of 3 h, even in the presence of constituents serving as electron acceptors for H2-competing microbial processes. Dynamic Fermentation Pathways with Synthetic and Natural Groundwater. The fermentable substrates fed (influent at 10 mM lactate and 15 mM methanol) were not detected in the effluent of the bioreactor at any point during synthetic groundwater phase III (Figure 3B). Propionate and acetate were the only volatile fatty acids produced and detected in the effluent (Figure 3B). A simple electron balance between influent and effluent brings useful insight as to the availability of the electrons from lactate and methanol for H2 production (Figure S3). A total of 210 me−equiv L−1 were available from the influent, while reductive dehalogenation of influent TCE fed during this phase requires 14.4 me−equiv L−1 as H2. In the effluent, >88% of the influent lactate and methanol me−equiv were accounted as propionate and acetate at 11 and 7.2 h HRT (Figure S3), suggesting that H2 production was nearly at stoichiometric levels for TCE dehalogenation. At 3.6 h HRT, however, propionate and acetate accounted for 84.7% of the total me−equiv (Figure S3), leaving significantly more electrons available for possible H2 production and subsequent consumption by hydrogenotrophic methanogens and homoacetogens. During operation with natural groundwater, two notable changes in fermentation occurred (Figure 4B). First, residual methanol (≤1.56 mM) was measured in the bioreactor throughout continuous operation (Figure 4B). Second, the electrons from fermentation were channeled mainly to acetate (Figures 4B and S3), whereas with the synthetic groundwater feed they were mainly channeled to propionate (Figure 3B). The reasons for the shift in fermentation products coupled to the incomplete consumption of methanol are not completely clear. Reduction of SO42− and NO3− accounted for >14.3% of the me−equiv (Figure S3). Even with the additional electron sinks present, TCE dehalogenation activity to ethene by D. mccartyi was preserved and the bioreactor remained highly functional. Divergence of Bioreactor Microbial Community When Challenged with Natural Groundwater is Driven by Fermentation and Diversity of Electron Acceptors. To

understand the composition of the microbial community and its associated changes under different operational conditions and influent feeds, we performed high throughput sequencing. When fed synthetic groundwater during phase III, the microbial community profiles were similar for bioflocs and planktonic culture (Figure 5). Phylotypes most similar to microorganisms involved in lactate and methanol fermentation belonging to Firmicutes were in highest abundance (Figure 5) as previously observed in enrichment culture or bioaugmentation field studies.19,24,34,35 The organohalide-respiring Dehalococcoidaceae accounted for up to 19.5% of the total sequences. Sequences most closely related the organohalide-respiring families Geobacteraceae and Dehalobacteriaceae were also detected at abundances of ≤6.8% and ≤0.3%, respectively (Figure 5). Once the influent was changed from synthetic to natural groundwater, the microbial community diverged to a new composition (Figure 5). Phylotypes within Firmicutes and Dehalococcoidaceae decreased in relative abundance due to lower lactate, methanol, and TCE concentrations, and the introduction of various groundwater-associated components. In agreement with the chemical data showing substantial SO42− reduction, deltaproteobacterial sequences (most closely related to the sulfate-reducing genus, Desulfovibrio36) went from low abundance with synthetic groundwater up to 42% relative abundance in the bioflocs during the natural groundwater phase (Figure 5). Phylotypes involved in elemental sulfur and sulfide oxidation were also enriched (i.e., Dethiosulfovibrio37 and Desulfobulbus38), indicative of sulfur cycling within the bioreactor. The structure of the microbial communities in bioflocs and in the planktonic culture establishes the enrichment of other competing microbial processes within the bioreactor. Microcolonies of D. mccartyi Revealed within the Microbially Diverse Architecture of Bioflocs. The ability of D. mccartyi to aggregate is largely unstudied and only a few publications have documented the presence of D. mccartyi in mixed-culture bioflocs.12,18 In our study, qPCR measurements and high-throughput sequencing confirmed the presence of D. mccartyi in high abundance within bioflocs. Samples from synthetic groundwater phase III were selected for scanning electron microscopy to obtain information regarding the G


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Figure 6. Scanning electron micrographs of bioflocs during bioreactor operation with synthetic groundwater (days 260−286). Biofloc overview at (A) 150× and (B) 3500× magnification revealing morphologically diverse aggregated cells. (C) Magnified area from panel B showing a microcolony of self-aggregated cells with the characteristic morphology of Dehalococcoides mccartyi (coccoid shape, 0.4−0.6 μm in diameter, biconcave indentations on opposite flat sides of cell). A rod-shaped cell with a length of ∼2 μm is highlighted for scale reference. (D) Putative D. mccartyi cells (diameter = ∼0.5 μm, cell thickness = ∼0.1 μm). Single D. mccartyi cell shown in Figure S10. (E) Area of biofloc with cocci (2−2.5 μm diameter) organized in tetrad structures. These cocci could be Veillonella as they are known to form tetrads.39 (F) Associations of rod-shaped microorganisms (some with a length >3 μm) with coccoid-shaped D. mccartyi and a matrix of exopolymers. The full-size resolution micrographs are available in the SI.

0.4−1 μm, and a cell thickness of ≤0.2 μm.4 D. mccartyi morphology with a diameter of 0.4−0.5 μm were identified in our study in the bioflocs (Figure 6C, D). D. mccartyi pure cultures are not known to form biofilms or multicellular structures. Pili-like appendages have been identified in pure cultures of D. mccartyi40 suggesting they have the potential for attachment to cells and surfaces. In this work, putative D. mccartyi cells were found dispersed in a network of other microbes, extrapolymeric substances, and other biological structures typical of microbial aggregates (Figure 6D, F). However, Figure 6C captures what looks like a “microcolony” of self-aggregated cells with the typical morphology and size of

organizational and morphological features of bioflocs (Figure 6). Scanning electron micrographs revealed the bioflocs as aggregations of morphologically diverse microbes (Figure 6A− F). Prevalent morphologies included at least two types of rods (thinner 1−2 μm long and thicker 2−3 μm long) (Figure 6B, E, and F). Also, large cocci with a diameter of 2−2.5 and organized as tetrads were captured (likely the fermentative bacteria Veilonella39) (Figure 6E). The unique morphology of D. mccartyi facilitated their tentative identification within the bioflocs, even in such complex conglomerate of microorganisms. D. mccartyi are disc shape with biconcave indentations at opposite ends of the cell, have a diameter of H


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Environmental Science & Technology D. mccartyi. There, hundreds of putative D. mccartyi cells can be observed in a tight structural organization with appendages of unknown purpose that were previously documented by other research groups.41−43 These are the first scanning electron micrographs capturing this mode of aggregation for D. mccartyi, which was a result of bioreactor operation. Relevance of Present Study. In the present study, an upflow bioreactor was employed to facilitate the bioflocculation of a D. mccartyi-containing consortium in order to promote high-rate reductive dehalogenation of TCE to ethene at HRTs of hours. Similar efforts have been put forth in the past. The outcomes from past studies have established that (i) increased contact time (long HRTs) is required for reductive dehalogenation of chlorinated ethene in upflow bioreactors,12,13,44 (ii) HRTs of ≤1 d lead to declines in reductive dehalogenation,10,15 (iii) changes in HRT or TCE loading bring significant fluctuations in bioreactor performance,10 and (iv) D. mccartyi relative abundance is dismal within bioflocs.10 The ability of the upflow bioreactor in this study to achieve and maintain significantly more efficient dehalogenation rates and extent compared to previous works is due to key differences in the bioreactor design, startup, and operating conditions. These differences, enabled by a long SRT (on average ∼200 days), allowed for the separation of D. mccartyi growth rates and HRTs. Our bioreactor was designed with a recirculation line so that the influent and recirculation streams would meet before entering the bioreactor. Upflow bioreactors are typically limited by mass transport due to the heterogeneous distribution of biomass from influent to effluent.45 Our bioreactor configuration provided significant mixing of the influent TCE and facilitated mass transport of TCE to the bioflocculated biomass. In most previous studies, anaerobic sludge was acclimated to chlorinated ethenes (Table 2). Here, we inoculated the bioreactor with a dehalogenating culture highly enriched in D. mccartyi and a small volume of anoxic sludge to increase microbial diversity of the dehalogenating culture. The dehalogenating culture delivered D. mccartyi with the appropriate metabolic potential for complete reduction of TCE to ethene. During the optimization period and operation with synthetic groundwater, medium and conditions were purposely provided to minimize H2 competition from methanogens and homoacetogens by limiting HCO3− concentrations. This potentially directed more electrons from H2 to reductive dehalogenation and growth of D. mccartyi. Furthermore, when the bioreactor was challenged with a feed containing an array of electron acceptors and lower TCE concentrations, D. mccartyi were able to compete and maintain high dehalogenation rates. The experimental phases of the study (operation with synthetic and amended natural groundwater) provide insights into the feasibility of such bioreactor to support the growth of D. mccartyi at HRTs of 3 h. However, the very stable dehalogenation performance and the capacity of the bioreactor to maintain D. mccartyi concentrations of 1013 16S rRNA gene copies L−1 in the bioflocs at an HRT of 3 h suggest that the bioreactor can be further optimized for potentially operating at HRTs lower than those tested in this study. Nonetheless, an HRT of 3 h falls within feasible ex situ bioremediation technologies used in environmental biotechnology. The amended natural groundwater phase tested the system’s ability to maintain high TCE removal rates when applied as an ex situ remediation technology. The bioreactor operated successfully as both a high-density culture production tool and as a real-time

remediation technology. Thus, we envision a possible application in which contaminated groundwater is pumped from PCE/TCE source areas and is then treated in an upflow bioreactor where the effluent, containing high concentrations of D. mccartyi, is pumped back into the groundwater as a bioaugmentation source. D. mccartyi are the hallmark of in situ enhanced bioremediation of chlorinated ethenes for their growth-coupled ability to detoxify these compounds to a nonchlorinated form. We bring evidence supporting that D. mccartyi can bioflocculate effectively as microcolonies or in multispecies associations with other microorganisms. Aggregation of D. mccartyi is still largely unexplored within the reductive dehalogenation realm. The focus of our work was optimizing and characterizing the kinetics of D. mccartyi bioflocs in a bioreactor fed with chlorinated ethenes. However, research is warranted to determine if D. mccartyi bioflocs possibly bring advantages over planktonic cultures for bioaugmentation purposes by increasing survival to environmental conditions, resisting predation and toxicity to adverse chemicals from groundwater and sediment, and retarding microbial decay.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b03097. Figure S1, photograph of the bioreactor. Figure S2, quantification of tceA and vcrA genes. Figure S3, electron donor and electron acceptor balances. Figures S4−S10, scanning electron micrographs of bioflocs (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: + 1-4807270046; e-mail: [email protected] (A.G.D.). *Phone: + 1-4807277574; e-mail: [email protected] (R.K.-B.). ORCID

Anca G. Delgado: 0000-0002-9354-0246 Emily Bondank: 0000-0002-9577-8637 Author Contributions §

These authors contributed equally

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the National Science Foundation (NSF) CAREER award number 1053939 to R.K.-B. The authors thank Joel Peterson from Synergy Environmental for assistance with natural groundwater sampling and analyses. We acknowledge David Lowry (Electron Microscopy Laboratory, School of Life Sciences, Arizona State University) for help with SEM sample preparation and César I. Torres (School for Engineering of Matter, Transport and Energy, Arizona State University) for expertise and assistance in the SEM imaging.

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REFERENCES

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Coupling Bioflocculation of Dehalococcoides mccartyi to High-Rate

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