Using a Two-Stage Hydrogen-Based Membrane Biofilm Reactor

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Using a Two-Stage Hydrogen-Based Membrane Biofilm Reactor (MBfR) to Achieve Complete Perchlorate Reduction in the Presence of Nitrate and Sulfate He-Ping Zhao,*,†,‡ Aura Ontiveros-Valencia,‡ Youneng Tang,‡,§ Bi−O Kim,‡ Zehra Esra Ilhan,‡ Rosa Krajmalnik-Brown,‡ and Bruce Rittmann‡ †

MOE Key Lab of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Science, Zhejiang University, Hangzhou, 310058, China ‡ Swette Center for Environmental Biotechnology, Biodesign Institute at Arizona State University, P.O. Box 875701, Tempe, Arizona 85287-5701, United States § Department of Civil and Environmental Engineering, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States S Supporting Information *

ABSTRACT: We evaluated a strategy for achieving complete reduction of perchlorate (ClO4−) in the presence of much higher concentrations of sulfate (SO42−) and nitrate (NO3−) in a hydrogen-based membrane biofilm reactor (MBfR). Full ClO4− reduction was achieved by using a two-stage MBfR with controlled NO3− surface loadings to each stage. With an equivalent NO3− surface loading larger than 0.65 ± 0.04 g N/ m2-day, the lead MBfR removed about 87 ± 4% of NO3− and 30 ± 8% of ClO4−. This decreased the equivalent surface loading of NO3− to 0.34 ± 0.04−0.53 ± 0.03 g N/m2-day for the lag MBfR, in which ClO4− was reduced to nondetectable. SO42− reduction was eliminated without compromising full ClO4− reduction using a higher flow rate that gave an equivalent NO3− surface loading of 0.94 ± 0.05 g N/m2-day in the lead MBfR and 0.53 ± 0.03 g N/m2-day in the lag MBfR. Results from qPCR and pyrosequencing showed that the lead and lag MBfRs had distinctly different microbial communities when SO42− reduction took place. Denitrifying bacteria (DB), quantified using the nirS and nirK genes, dominated the biofilm in the lead MBfR, but perchlorate-reducing bacteria (PRB), quantified using the pcrA gene, became more important in the lag MBfR. The facultative anaerobic bacteria Dechloromonas, Rubrivivax, and Enterobacter were dominant genera in the lead MBfR, where their main function was to reduce NO3−. With a small NO3− surface loading and full ClO4− reduction, the dominant genera shifted to ClO4−-reducing bacteria Sphaerotilus, Rhodocyclaceae, and Rhodobacter in the lag MBfR.

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membrane biofilm reactor (MBfR),5−7 in which hydrogen gas (H2) is supplied as electron donor by diffusion through bubbleless gas-transfer membranes. In addition to ClO4−, many other electron acceptors can be reduced in the MBfR: for example, nitrate (NO3−), sulfate (SO42−), selenate, chromate, and chlorinated solvents.6−9 NO3− is found frequently as a cocontaminant with ClO4−. It has a drinking-water MCL of 10 mg N/L because it causes methemoglobinemia in infants.10 NO3− is a common electron acceptor, and its reduction can have various effects on ClO4− reduction. Some research found that NO3− inhibited ClO4− reduction, particularly when the electron donor was limiting,6,11

INTRODUCTION Perchlorate (ClO4−) is a strong oxidizing agent that has been widely used in rocket propellants, explosives, and fireworks.1 ClO4− contamination in water causes health problems by interfering with the production of thyroid hormones that are needed for pre- and postnatal growth and development, as well as for normal metabolism in adults. Although the U.S. EPA has not yet established a maximum contaminant level (MCL) for ClO4−, it has decided that it will regulate ClO4− under the Safe Drinking Water Act (SDWA),2 and some states have established cleanup levels ranging from 2 to 18 μg/L for ClO4− in drinking water.3 Biological reduction is an effective means to detoxify water contaminated with ClO4−, which can be biologically reduced stepwise to chloride (Cl−) and oxygen (O2).4 The O2 is subsequently reduced to H2O. One means to bring about biological reduction of ClO4− is with a hydrogen-based © 2013 American Chemical Society

Received: Revised: Accepted: Published: 1565

October 28, 2012 January 8, 2013 January 9, 2013 January 9, 2013 dx.doi.org/10.1021/es303823n | Environ. Sci. Technol. 2013, 47, 1565−1572

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but Nerenberg et al.12 showed that NO3− inhibition of ClO4− reduction was trivial in a H2-based MBfR when sufficient H2 was available. SO42− is another electron acceptor that often occurs with NO3− and ClO4−. SO42− is not normally considered a health concern, and no MCL has been established for SO42−.13 However, SO42− reduction usually is an unwanted process, because it produces hydrogen sulfide (H2S), a corrosive, odorous, and toxic substance;14 also, sulfide can strongly inhibit NO3− reduction.15 Thus, sulfate reduction counteracts the benefits of biological treatment of water or wastewater when denitrification and perchlorate reduction are required. It is generally accepted that NO3− reduction inhibits SO4− reduction when the donor is in limited supply, because NO3− is a more favorable electron acceptor.16 In fact, addition of NO3− is used to control SO42− reduction in various settings.17,18 For the MBfR, Tang et al.19 reported that NO3− could be fully removed in pilot-scale tests without SO42− reduction when the operating conditions, mainly including H2 pressure, pH, and NO3− surface loadings, were well controlled. Likewise, Ziv-El and Rittmann20 reported that the electron acceptors had a clear H2-utilization priority when H2 was limiting: O2 > NO3− > NO2− > ClO4−> SO42−. The effect of SO42− on ClO4− reduction is not well understood. Chung et al. reported that the reduction of ClO4− was not influenced by the presence of SO42− in an upflow packed bed reactor at high salinity.21 However, no systematic work indicates how to achieve full ClO4− reduction without incurring SO42− reduction at the same time. Clearly, interactions among perchlorate-reducing bacteria (PRB), denitrifying bacteria (DB), and sulfate-reducing bacteria (SRB) have to be understood in order to manage the reductions of the acceptors properly. These interactions should depend on two things: the availability of the electron donor and the relative abundance of PRB, DB, and SRB in the community. DB, PRB, and SRB are phylogenetically diverse. Many DB belong to the γ subclass of the Proteobacteria phylum, but they also are found in the α and β subclasses.22 The NO2− reductase genes (nirK and nirS) and N2O reductase gene (Nos) usually are used to identify and quantify DB.23,24 PRB have members in the α, β, γ, and ε subclasses of the Proteobacteria phylum.1,25 Most PRB are DB, except for a few reported strains.1,13,26,27 The pcrA gene, for the perchlorate reductase, can be used to identify and quantify perchlorate-reducing bacteria (PRB).28 Most SRB belong to ∼23 genera within the ε- and δProteobacteria, followed by the G+ SRB within the Clostridia (Desulfotomaculum, Desulfosporosinus, and Desulfosporomusa).29,30 The dsrA gene, which catalyzes the reduction of SO32− to H2S,31 can be used to identify and quantify SRB.32 Modeling an MBfR biofilm that could be active in reducing NO3− and ClO4−, Tang et al.33 explained that low NO3− surface loading (0.6 g N/m2-day) strongly inhibits ClO4− reduction. Ontiveros-Valencia et al.34 reported that, without restriction on H2 delivery, SO42− reduction always cooccurred with NO3− reduction when the NO3− surface loading was ≤0.13 g N/m2-day. Researchers have found that it is difficult to achieve full ClO4− reduction without incurring SO42‑ reduction in a single-stage MBfR, since SO42− had similar H2utilization affinity as ClO4−.20 With the aim of being able to achieve full ClO4− reduction by controlling competition from NO3− and SO42−, as well as to

more thoroughly understand the interactions among NO3−, SO42−, and ClO4− reduction when H2 is the electron donor, we set up a bench-scale, two-stage MBfR that allowed us to have different NO3− surface loadings for each stage. The goal was to enhance ClO4− reduction in the second stage without incurring SO42− reduction. We studied the reduction patterns of NO3−, SO42−, and ClO4− in the two stages when H2 availability was not limiting. We inoculated the reactor only with bacteria derived from groundwater at a pilot site. The groundwater, which was used as the influent for the lead MBfR, contained approximately 8 mg/L dissolved oxygen (DO), 100 μg/L ClO4−, 22 mg/L SO42−, and 6 mg/L NO3−−N; thus, ClO4− was present at a much smaller concentration than the other electron acceptors. We used quantitative real-time PCR (qPCR) to monitor changes in the microbial community according to specific groups: total bacteria by the16S rDNA gene, PRB by the functional gene pcrA, SRB by the functional gene dsrA, and DB by the functional genes nirK and nirS. We also analyzed changes in diversity and dominant bacteria in the H2-fed biofilm using pyrosequencing. On the basis the understanding gained from the relationships between community structure and function in this study, we are able to provide a practical guidance for achieving full perchlorate reduction without also causing sulfate reduction.

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MATERIALS AND METHODS Experimental Setup. We used the two-stage MBfR illustrated in Figure 1. Each stage was similar to that of Zhao

Figure 1. Schematic of the lead and lag MBfRs.

et al.,6 with polypropylene fibers (nonporous polypropylene fiber, 200 μm OD, 100−110 μm ID, wall thickness 50−55 μm, and produced by Teijin, Ltd., Japan) glued into a H2-supply manifold at the bottom and sealed at the top. The MBfRs were well mixed by recirculation with a peristaltic pump (Master Flex, model 7520-40, Cole-Parmer Instrument Company, U.S.A), and the H2 gauge pressure was 17.0 psig (absolute pressure, or 2.2 atm) for all experiments. Lead and lag MBfRs had 32 bundle fibers and 10 sacrificial fibers for biomass sampling. The length of each fiber was 25 cm, and the total surface area was 66 cm2. The influent feeding rates are shown in Figures 2 and 3, and NO3− surface loadings are tabulated in Table 1. 1566

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(phase 1 lead). After steady state was reached (in terms of stable effluent concentrations), we increased the flow rate to 0.42 mL/min until a new steady state was reached (from day 22 to day 46, phase 2 lead). We collected the effluent of the lead MBfR in 5-L open clear bottles; the collected effluent was exposed to the air and took on DO. On the fourth day, we inoculated the lag MBfR using the same pilot reactor flush water from the pilot site for 24 h and then feed the lag MBfR using the effluent of the lead MBfR as influent. The lag MBfR was operated at 0.28 mL/min from day 1 to day 20 (phase 1 lag), when a steady state was reached, at which time we changed the feed rate to 0.42 mL/min (phase 2 lag). We added 50 μg/L of PO4− to the well water to ensure sufficient nutrient for bacterial growth.19 We also adjusted the pH of the well water to 7.5 ± 0.2 with hydrochloric acid before feeding the reactor. Influent dissolved O2 for both MBfRs ranged between 7.8 to 8.0 mg/L.20,35 Chemical Analyses. We took liquid samples of the influent and effluent of the MBfRs with 5-mL syringes and immediately filtered them through a 0.2-μm membrane filter (LC+PVDF membrane, Pall Life Sciences Acrodisc Syringe Filters, USA). We assayed for SO 4 2− , NO 3 − , and NO 2 − using ion chromatography (Dionex ICS 3000) with an AS18 column and AG18 precolumn, an eluent concentration of 22 mM KOH, and a 1 mL/min flow rate. We assayed ClO4− using ion chromatography (Dionex ICS 2000) with an AS 16 column and AG16 precolumn, an eluent concentration of 35 mM KOH, and a 1.5 mL/min flow rate. We measured the pH of all influent and effluent samples with a pH meter (837 pH lab, Metrohm, Switzerland); the pH of the effluent was stable in the range of 7.4 to 7.8 for all stages. We measured O2 with a dissolved oxygen (DO) probe (Orion Star, USA). Flux Calculations. We calculated the NO3−, SO42−, ClO4−, and O2 removal fluxes (g/m2-day) according to eq 1

Figure 2. Concentrations and removals of NO3−, ClO4−, and SO42− in the lead MBfR. Notes: (a) Every datum of influent and effluent concentration represents 1 replicate of sample. (b) Every datum of removal percentage was calculated with (Cin − Cout)/Cin × 100, where Cin stands for the concentration of influent and Cout stands the concentration of effluent of the lead MBfR. (c) The standard deviations of removal percentages are presented in the text; the sulfate reduction percentage for phase 2 was −6 ± 6%, indicating no sulfate reduction in phase 2.

J = (S ° − S )Q / A

(1)

ClO4−,

in which S° and S are the influent and effluent SO42‑, − NO3 , or O2 concentration (g/L), Q is the influent flow rate to the MBfR (L/day), and A is the membrane surface area (m2). The H2 fluxes were calculated from the acceptor-removal fluxes and the reaction stoichiometries shown in eqs 2−5.36 ClO4 − + 0.11H+ + 0.11NO3− + 0.53CO2 + 5.48H 2 = Cl− + 5.15H 2O + 0.11C5H 7O2 N

(2)

SO4 2 − + 4.21H 2 + 0.015NO3− + 0.0752CO2 + 1.516H+ = 0.5H 2S + 0.5HS− + 4.165H 2O + 0.015C5H 7O2 N (3)

NO3−

Figure 3. Concentrations and removals of NO3−, ClO4−, and SO42− in the lag MBfR. The sulfate reduction percentage of phase 2 was −7 ± 6%.

+

+ 3.0H 2 + 0.23CO2 + H = 0.48

N2 + 0.046C5H 7O2 N + 3.4H 2O

(4)

O2 + 2.4H 2 + 0.028NO3− + 0.14CO2 + 0.028H+

Start Up and Continuous Operation. We inoculated the lead MBfR with 50 mL of flush water obtained from an MBfR pilot plant treating the same groundwater, and we initially let the flush water circulate at 150 mL/L for 24 h. We then fed the lead stage continuously with groundwater from the pilot site. Actual concentrations varied slightly over time and are presented in Results and Discussion. We set the influent feed rate at 0.28 mL/min for the lead MBfR in the first 21 days

= 0.028C5H 7O2 N + 2.3H 2O

(5)

To determine if H2 delivery was limiting, we compared the sum of the H2 fluxes in the experiments to the maximum H2 flux that can be delivered through the polypropylene fibers at the applied H2 pressure of 2.2 atm (i.e., 0.69 e− equiv/m2-day).37 Biofilm Sampling and DNA Extraction. On day 20 (phase 1), we collected biofilm samples from both MBfRs after 1567

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Table 1. Fluxes of Electron Acceptors and H2 at Steady Stated NO3−−N

O2 stages lead MBfR lag MBfR

phase phase phase phase

1 2 1 2

electron donor (H2) flux

ClO4−

SO42−

surface loading of NO3−−Nc (gN/m2-day)

flux (g/ m2-day)

electron fluxd

flux (gN/ m2-day)

electron flux

flux (g/ m2-day)

electron flux

flux (mg/ m2-day)

electron flux

maximum H2 fluxb

actual H2 flux

0.65 0.94 0.34 0.53

0.59 0.85 0.59 0.85

74 110 74 110

0.43 0.38 0.13 0.23

85 81 27 49

0.058 0 0.179 0

2.3 0 7.2 0

2.2 1.8 5.7 6.5

0.13 0.10 0.32 0.40

210 210 210 210

161 191 109 159

Standard deviations are reported in the text, but not included in the table. bThe maximum flux through the polypropylene fibers at the total H2 pressure of 2.2 atm.37 cFor the surface loading of nitrate, dissolved O2 is included by converting its flux to equivalent NO3− according to electron equivalence (i.e., 1 mg O2 is equivalent to 0.35 mg NO3−-N). Most bacteria that respire NO3− also respire O2.25,35−37 dElectron flux units: e− mequiv/m2-day. d

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both had reached steady state at the flow rate of 0.28 mL/min: ClO4−, SO42−, and NO3− concentrations in the effluent were stable (0.6 g N/m2-day) strongly inhibits ClO4− reduction, but medium NO3− loading (0.1−0.6 g N/m2-day) has no adverse effect on ClO4− removal. In our experiment, the equivalent NO3− loadings in both phases for the lead reactor were larger than 0.6 g N/m2-d (high loading), and they were between 0.1 to 0.6 g N/m2-day (medium loading) in the lag reactor. According to Tang et al.,33 high nitrate loading suppresses the perchlorate reduction because of competition between DB and PRB for space in the biofilm and for the common electron donor (H2). Our study using the two-stage MBfR system tested and experimentally supported this hypothesis. Modeling a multispecies biofilm with DB and SRB, Tang et al.51 predicted that SO42‑ reduction could be suppressed when the equivalent NO3− surface loading was 0.44 g N/m2-day, allowing DB to out-compete SRB for H2 and space in the biofilm. Our experimental findings are consistent with the model predictions of Tang et al., since our equivalent NO3surface loading of 0.53 ± 0.03 g N/m2-day suppressed SO42− reduction. Thus, the two-stage strategy allowed us to manage the competition among DB, SRB, and PRB is such a way that the lag MBfR had minimal SO42− reduction, but full ClO4− reduction. Community Structure through Functional Gene Analysis. We compare the cells/m2 calculated for each functional bacterial group with the total H2 flux in Figure 4.

Thus, the higher NO3− surface loading in the lead MBfR suppressed SRB, as well as PRB. DB, assayed by nirK (copper-based NO2− reductase) and nirS (cytochrome-based NO2− reductase), were dominant in the lead MBfR (42% of total bacteria), but decreased only slightly in the lag MBfR (36% of total bacteria); this corresponds to the equivalent NO3− flux of phase 1 being much lower in the lag MBfR (0.34 ± 0.04 g N/m2-day) compared to lead MBfR (0.65 ± 0.04 g N/m2-day). The main reason that DB remained important in the lag MBfR is that DB can reduce O2 as an electron acceptor,33 and DO was in the influent to the lag MBfR. In addition, PRB in the lag MBfR probably contained nitrite reductase genes28,52 and also were assayed as DB, while DB may have detached from lead MBfR and entered the lag MBfR. Bacterial Community Structure of the MBfR Biofilm. We used pyrosequencing to analyze the bacterial communities of the MBfR biofilms for dominant classes and genera. Figure 5-

Figure 5. Microbial community structure in the MBfRs for steady state at the flow rate of 0.28 mL/min. The top panel is the percentage distribution of all detected bacteria at the class level. The bottom panel is the distribution of the most common genera.

A, which summarizes bacterial diversity at the class level, shows that the bacterial structures of the lead and lag MBfRs were obviously different. β-Proteobacteria was by far the largest class in both MBfRs (60% in the lead MBfR and 68% in the lag MBfR). γ-Proteobacteria and δ-Proteobacteria were second and third abundant classes in the lead MBfR, while Cyanobacteria accounted for about 9% of all phylotypes at the class level. In the lag MBfR, with most NO3− already reduced and the ClO4− flux greatly increased, the abundance of α-Proteobacteria significantly increased from 2% to 22%, becoming the second most abundant class, while γ-Proteobacteria and δ-Proteobacteria significantly decreased. We summarize the relative proportions of the most abundant bacterial genera in the different reactors in Figure 5-B. Clearly, the lead and lag MBfRs had very different dominant bacterial genera as a result of the difference in the major reduction

Figure 4. Cell densities and fluxes of H2 in the lead and lag MBfRs. Standard deviations for the qPCR results are so small that they cannot be seen on the bars.

PRB (assayed with the pcrA gene) became much more enriched in the lag MBfR (4.6 × 1011 cells/m2, about 20% of total bacteria) than in the lead MBfR (4.7 × 1010 cells/m2, 2% of total bacteria). This correlates to the higher ClO4− flux and the lower equivalent NO3− surface loading in the lag MBfR (Table 1), where, DB could not out-compete PRB for space. With the SO42− flux almost 3-fold higher in the phase 1 lag MBfR, compared to the phase 1 lead MBfR (Table 1), the abundance of SRB (assayed with the dsrA gene) was more than double in the lag MBfR (26%) than in the lead MBfR (12%). 1569

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process. In the lead MBfR, where NO3− was the dominant electron acceptor, the most numerous genera were Dechloromonas (26%), Rubrivivax (16%), Enterobacter (13%), Rhodocyclaceae (11%), and Haliangium (10%). Dechloromonas (β-Proteobacteria) decreased dramatically in the lag MBfR when the NO3− flux decreased by ∼75% and the equivalent NO3− flux decreased by ∼50%, while the ClO4− and SO42‑ fluxes significantly increased. The nonsulfur purple bacteria Rubrivivax, which can reduce NO3−,53 was enriched in the lead MBfR, where NO3− was the dominant electron acceptor. In the lag MBfR, where ClO4− and SO42− became more important electron acceptors, Sphaerotilus (33%), Rhodocyclaceae (21%), Rhodobacter (19%), and Propionivibrio (6%) increased significantly, although Dechloromonas declined. Dechloromonas is reported to respire NO3−, ClO3−, and ClO4−.40 The decrease of Dechloromonas in the lag MBfR may have been due to the major decrease of NO3− entering the lag MBfR, compared to the lead MBfR. Although Haliangium, a strictly aerobic bacterium,54 was significantly depleted in the lag MBfR, the aerobic, sulfur-oxidizing bacterium Sphaerotilus was enriched in the lag MBfR, perhaps because sulfate reduction produced its electron donor;55 some strains from Sphaerotilus are able to reduce NO3− to NO2−.56 Propionivibrio became an important genus in the lag MBfR after barely being detected in the lead MBfR; this is consistent with a recent report that some strains of this genus are able to reduce ClO4−.57 Likewise, Rhodocyclaceae and Rhodobacter became important, probably because they were reported able to reduce ClO4− or chlorate.58,59 In summary, a two-stage MBfR could effectively remove ClO4− to a nondetectable level with minimal SO42‑ reduction when the acceptor surface loadings were properly managed in accord with the modeling predictions of Tang et al.33,51 An equivalent NO3− surface loading larger than 0.65 ± 0.04 g N/ m2-day in lead MBfR (phase 1) led to NO3− reduction of 87 ± 4%, but ClO4− reduction was only about 30 ± 8%; in this situation of relatively high equivalent NO3− surface loading, ClO4− reduction was inhibited by competition by DB for H2 and space in the biofilm. When the equivalent NO3− surface loading decreased to 0.34 ± 0.04−0.53 ± 0.03 g N/m2-day in the lag MBfR (both phases), ClO4 − was reduced to nondetectable levels. Furthermore, SO42− reduction was eliminated by increasing the flow rate so that the nitrate surface loading to the lag MBfR was 0.53 ± 0.03 g N/m2-day (phase 2). With denitrification responsible for at least 70% of the H2 consumption in the lead MBfR in phase 1, DB dominated the microbial community in the lead MBfR’s biofilm, followed by SRB and PRB. The facultative anaerobic bacteria Dechloromonas, Rubrivivax, and Enterobacter were dominant genera in the lead MBfR, and their main function was to reduce NO3−. With the lag MBfR receiving a smaller equivalent NO3− loading so that ClO4− was reduced to nondetectable, the dominant genera shifted to Sphaerotilus, Rhodocyclaceae, and Rhodobacter, which are able to reduce ClO4− or chlorate.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 0086-571-88982515. Fax: 0086-571-88982917. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by grant number ER-200541 from the Environmental Security Technology Certification Program (ESTCP), United States Department of Defense, via a subcontract with CDM.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

Additional material as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. 1570

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