Article pubs.acs.org/est
Nitrate Shaped the Selenate-Reducing Microbial Community in a Hydrogen-Based Biofilm Reactor Chun-Yu Lai,† Xiaoe Yang,† Youneng Tang,‡ Bruce E. Rittmann,§ and He-Ping Zhao*,† †
Ministry of Education, Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Science, Zhejiang University, Hangzhou 310029, People’s Republic of China ‡ Department of Civil and Environmental Engineering, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States § Swette Center for Environmental Biotechnology, Biodesign Institute, Arizona State University, Post Office Box 875701, Tempe, Arizona 85287-5701, United States S Supporting Information *
ABSTRACT: To study the effect of nitrate (NO3−) on selenate (SeO42−) reduction, we tested a H2-based biofilm with a range of influent NO3− loadings. When SeO42− was the only electron acceptor (stage 1), 40% of the influent SeO42− was reduced to insoluble elemental selenium (Se0). SeO42− reduction was dramatically inhibited when NO3− was added at a surface loading larger than 1.14 g of N m−2 day−1, when H2 delivery became limiting and only 80% of the input NO3− was reduced (stage 2). In stage 3, when NO3− was again removed from the influent, SeO42− reduction was re-established and increased to 60% conversion to Se0. SeO42− reduction remained stable at 60% in stages 4 and 5, when the NO3− surface loading was re-introduced at ≤0.53 g of N m−2 day−1, allowing for complete NO3− reduction. The selenatereducing microbial community was significantly reshaped by the high NO3− surface loading in stage 2, and it remained stable through stages 3−5. In particular, the abundance of α-Proteobacteria decreased from 30% in stage 1 to less than 10% of total bacteria in stage 2. β-Proteobacteria, which represented about 55% of total bacteria in the biofilm in stage 1, increased to more than 90% of phylotypes in stage 2. Hydrogenophaga, an autotrophic denitrifier, was positively correlated with NO3− flux. Thus, introducing a NO3− loading high enough to cause H2 limitation and suppress SeO42− reduction had a long-lasting effect on the microbial community structure, which was confirmed by principal coordinate analysis, although SeO42− reduction remained intact.
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INTRODUCTION
Strategies for removing soluble forms of selenium from water include chemical precipitation, reverse osmosis, catalytic reduction, and ion exchange.8−10 Biological reduction has gained interest in the past decade because of its ability to remove SeO42−, simplicity, and low cost.11,12 One of the processes for biological reduction is the H2-based membrane biofilm reactor (MBfR), in which hydrogen gas (H2) is delivered through the walls of gas-transfer fibers to bacteria that live as a biofilm on the outside of the membranes.13 H2 serves as the electron donor to drive the respiratory reduction of a range of oxidized contaminants, including SeO42−.14−18 Bacteria that use SeO42− as a respiratory electron acceptor first reduce it to SeO32− by the action of selenate reductase (SerABC)19 or nitrate reductase (Nar and Nap).20,21 SeO32−
Selenium (Se) is associated with a variety of industries, including photoelectric devices, semiconductors, (de)colorization of glasses, and flue gas desulfurization.1,2 These industries generate a large amount of selenium-contaminated wastewater that normally contains selenate (SeO42−) and selenite (SeO32−) as the main soluble forms of Se, and they also often contain high concentrations of nitrate (NO3−) and nitrite (NO2−).2,3 Some agricultural drainage waters also contain high levels of NO3−, along with naturally occurring selenium (mostly as SeO42−) (e.g., San Joaquin Valley of California).4,5 The soluble forms of selenium in water present a health concern because of their toxicities to organisms.6 The maximum contaminant level (MCL) for drinking water is 50 μg of Se/ L of total selenium.7 In contrast, element selenium (Se0) is highly insoluble in water and, thus, has much lower toxicity. Selenide (Se2−), the most reduced form of selenium, is not toxic because it is readily oxidized to elemental selenium when exposed to air. © 2014 American Chemical Society
Received: Revised: Accepted: Published: 3395
December 3, 2013 February 14, 2014 February 28, 2014 February 28, 2014 dx.doi.org/10.1021/es4053939 | Environ. Sci. Technol. 2014, 48, 3395−3402
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can then be reduced to Se0 enzymatically by periplasmic nitrite reductase (Nir)22 or hydrogenase I.23 High levels of NO3− often occur in water contaminated by selenium.24 NO3− has a drinking water MCL of 10 mg of N/L because it causes methemoglobinemia in infants,25 and it is a surface water pollutant because of the ability to accelerate eutrophication.26 NO3− is a common electron acceptor that can have various effects on SeO42− reduction. Some researchers reported that NO3− was always reduced before SeO42− when both were present.27−29 However, Macy et al.30 found that NO3− and SeO42− were simultaneously reduced by the selenate-respiring bacterium Thauera selenatis, while van Ginkel et al. found that NO3−, NO2−, and SeO42− were simultaneously removed with high removal efficiencies.3 Moreover, Chung et al.11 reported that a small amount of NO3− improved SeO42− reduction in a MBfR when H2 was not limiting. Interactions between NO3− and SeO42− reductions may depend upon the structure of the microbial community. Although a limited number of selenate-reducing bacteria have been isolated, e.g., Desulfurispirillum indicum, Sulfurospirillum barnesii, and Enterobacter cloacae,31−34 selenate-reducing bacteria display a broad diversity and can be found among the phyla “Proteobacteria” (α, β, and γ subclasses),12,35 Deferribacteres, Firmicutes, and Actinobacteria.31,32,36,37 Some of the selenate-reducing bacteria are able to respire NO3− as an electron acceptor.31,33,35 Denitrifying bacteria (DB) are phylogenetically diverse as well. Many DB belong to the γ subclass of the Proteobacteria phylum, but they also are found in the α and β subclasses.38 Some DB can reduce SeO42− to SeO32− and further to Se0.39 Chung et al. found that Dechloromonas spp., known to respire NO3− and ClO4−, dominated the MBfR biofilm community fed with selenate.18 Stolz et al. showed that nitrate-grown Geospirillum barnesii strain SeS3 cells could reduce selenate, while selenate-grown cells could reduce nitrate, even though their cytochrome and protein contents were different.40 However, Oremland et al.41 reported that nitrate-grown strain SES-3 cells did not respire SeO42−, while selenate-grown strains did not reduce NO3−; this suggests that nitrate and selenate reductases in some bacteria might be separately inducible. The objective of this study was to elucidate interactions between NO3− and SeO42− reductions occurring simultaneously in a H2-based biofilm. We studied the reduction patterns of NO3− and SeO42− with different relative loadings of NO3− and SeO42−. We also analyzed changes in the microbial community structure associated with loading changes. On the basis of our findings, we are able to explain the effect of NO3− on SeO42− reduction and how NO3− shaped the SeO42−reducing bacterial community in the H2-based biofilm. These insights provide an explanation for the various results in past studies.
was completely mixed by water recirculation with a peristaltic pump (Master Flex, model 7520-40, Cole-Parmer Instrument Company, Vernon Hills, IL) at 100 mL/min. Startup and Continuous Operation. We inoculated the MBfR with 1 mL of diluted activated sludge obtained from Qige Wastewater Treatment Plant (WWTP) in Hangzhou (China) and enriched the community by recirculating 10 mg/L SeO42− in a mineral salt medium (described below) for 48 h. We then fed the MBfR continuously with medium containing SeO42− at a target concentration of 1 mg of Se/L (12.7 μmol/ L) throughout the experiment. To evaluate the effect of nitrate on selenate reduction, we varied the nitrate concentration in the medium, and this characterized the five successive operating stages: 0, 10, 0, 1, and 5 mg of N/L in stages 1, 2, 3, 4, and 5, respectively (0, 714, 0, 71.4, 357 μmol/L in stages 1, 2, 3, 4, and 5, respectively). Each operating stage attained a steady state, in which the effluent concentrations of all chemical species were stable for at least 1 week. Actual influent concentrations varied slightly from the targets and are presented in Figure 1 in the Results and Discussion. The influent feeding rate was 0.50 mL/min [giving a hydraulic residence time (HRT) of about 130 min], while the H2 pressure was 2.5 psig (gauge pressure or 1.17 atm) for all experiments. The medium pH was adjusted to 7.0 ± 0.2 with hydrochloric acid and contained the same mineral salts as that by Zhao et al.14 The concentration of dissolved O2 of the influent was between 7.7 and 8.0 mg/L (241−250 μmol/L) for all stages. Chemical Analyses. We took liquid samples from the MBfR with 5 mL syringes and filtered them immediately through a 0.2 μm membrane filter [liquid chromatography (LC) + polyvinylidene fluoride (PVDF) membrane, Shanghai Xinya, China]. We assayed for NO3− and NO2− using ion chromatography (Dionex ICS 2000) with an AS18 column and AG18 pre-column, an eluent concentration of 22 mM KOH, and a 1 mL/min flow rate. We centrifuged the sample at 15000g for 10 min to remove the solid element Se (Se0), and we then assayed total soluble selenium (SeO42− + SeO32−) using inductively coupled plasma−mass spectrometry (ICP− MS, Agilent 7500a, Agilent Technologies, Palo Alto, CA). We analyzed the SeO32− concentration by a fluorometric method [standard method 3500-Se E, American Public Health Association (APHA), Washington, D.C.]. The concentration of SeO42− was calculated by subtracting SeO32− from total soluble Se in the effluent. We measured the pH of all influent and effluent samples with a pH meter (Seven Easy, Mettler Toledo, Switzerland); the pH was between 7.4 and 7.7 for all stages. Flux Calculations. We calculated the NO3−, SeO42−, and O2 removal fluxes (g m−2 day−1) according to
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MATERIALS AND METHODS Experimental Setup. We used a two-column MBfR system similar to that by Zhao et al.14 The MBfR had composite hollow fibers (280 μm outer diameter and 180 μm inner diameter) manufactured by Mitsubishi Rayon (model MHF200TL, Mitsubishi, Ltd., Japan). A total of 32 bundle fibers were glued into the manifold at the bottom of the main MBfR column. A total of 10 coupon fibers (for biomass sampling) were glued into the second column. All fibers were supplied with H2 gas from one end, and the other end of each fiber was sealed. The total volume of the MBfR was 65 mL. The MBfR
J = (S 0 − S )Q / A
(1)
in which S0 and S are the influent and effluent NO3−, SeO42−, or O2 concentration (g/L), Q is the influent flow rate to the MBfR system (L/day), and A is the membrane surface area (m2). The H2 flux was calculated from the removal fluxes and reaction stoichiometry shown in eqs 2−4.42 NO3− + 3.0H 2 + 0.23CO2 + H+ = 0.48N2 + 0.046C5H 7O2 N + 3.4H 2O 3396
(2)
dx.doi.org/10.1021/es4053939 | Environ. Sci. Technol. 2014, 48, 3395−3402
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Figure 1. (A) SeO42−, SeO32−, Se0, and NO3− concentrations in the MBfR. (B) SeO42− and NO3− transformation percentages.
score lower than 25 were removed. We picked the 16S rRNA operational taxonomy units (OTUs) with uclust based on ≥97% identity.46 The taxomony to the OTUs was assigned as that by Zhao et al.47 We aligned representative sequences to the Greengenes Database using PyNast,48,49 as described previously.47 We evaluated the overall community composition using the unweighted UniFrac distance matrix50 and the relationships among samples with Cytoscape and principal coordinate analysis (PCoA).51 We calculated the alpha diversity parameters using the QIIME 1.7 pipeline and summarize the results in Table S1 of the Supporting Information as mean values with standard deviation. The Chao1 index indicates microbial richness. Shannon and Phylogenetic Diversity Whole Tree metrics indicate diversity and evenness, respectively. To reduce sample heterogeneity, we subsampled each sample 10 times at a depth of 14 882 sequences (which was the lowest number of sequences found in the sample of stage 1), took an average per sample, and then calculated the standard deviation.47 We calculated the Pearson correlation between SeO42− and NO3− fluxes and the relative abundances of the predominant in general (see Table S2 of the Supporting Information).52 We deposited all sequencing data into the National Center for Biotechnology Information (NCBI), with accession numbers SAMN02316780 for stage 1, SAMN02318096 for stage 2, SAMN02318275 for stage 3, SAMN02318287 for stage 4, and SAMN02318288 for stage 5.
SeO4 2 − + 3.38H 2 + 0.27NO3− + 0.13CO2 + 2.03H+ = Se 0 + 4.3H 2O + 0.027C5H 7O2 N
(3)
O2 + 2.4H 2 + 0.028NO3− + 0.14CO2 + 0.028H+ = 0.028C5H 7O2 N + 2.3H 2O
(4)
We compared the actual H2 flux to the maximum H2 flux that can be delivered through the composite hollow fiber at the applied H2 pressure to indicate if H2 delivery was limiting.43 Biofilm Sampling and DNA Extraction. We collected biofilm samples for all stages when the reactor reached a steady state (SeO42− and NO3− concentrations in the effluent were stable, i.e., less than 10% variation over 1 week). We cut off one ∼2 cm long section for scanning electron microscopy (SEM, Stereoscan 260) image analysis (see Figure S1 of the Supporting Information), as previously described by Chung et al.18 We cut off one ∼10 cm long section from the coupon fiber and then sealed the remaining by tying the end into a knot. We then extracted DNA using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD), as previously described by Zhao et al.14 Pyrosequencing. We used primers 341F (5′-CCTACGGGAGGCAGCAG-3′) and 1073R (5′-ACGACGTGACGACARCCATG-3′) to target the conserved V3−V6 regions of the 16S rRNA gene. We sent the DNA samples to Shanghai Majorbio Technology (Shanghai, China) to perform amplicon pyrosequencing with standard 454/GS-FLX protocols.44 We processed the pyrosequencing data using the QIIME 1.7 pipeline.45 All sequences shorter than 200 bps, having homopolymers of 6 bps and primer mismatches, and a quality
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RESULTS AND DISCUSSION Interference Effects between Nitrate and Selenate Reductions. Figure 1A shows the concentrations of measured 3397
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Calculated from eqs 2−4. bCalculated on the basis of the assumption that all selenate was reduced to elemental selenium. cMaximum H2 flux was 0.56 g of H2 m−2 day−1 (280 mmol/L).43 datm = 1 + psig/14.7. eNA = not applicable. a
40 247 42 63 155 280 280 280 280 280 38 38 38 38 38 0 208 0 21 113 1 2 3 4 5
0.039 0.014 0.090 0.100 0.083
2 1 4 4 4
NAe 0.966 NA 0.096 0.527
0.51 0.51 0.51 0.51 0.51
maximum H2 flux (mmol of H2 m−2 day−1)c,d electron donor consumed (mmol of H2 m−2 day−1) electron donor consumed (mmol of H2 m−2 day−1) flux (g of N m−2 day−1) electron donor consumed (mmol of H2 m−2 day−1)b flux stages (g of Se m−2 day−1)a
NO3−−N SeO42−
Table 1. Average Acceptor and Donor Fluxes for Each Stage of the MBfR Experiments
flux (g m−2 day−1)
O2
electron donor (H2)
NO3−, SeO42−, SeO32−, and calculated Se0 in the effluent, along with the influent concentrations of NO3− and SeO42−. Figure 1B shows the removal percentages of NO3− and transformation percentages of SeO42− to SeO32− and SeO42− to Se0. The fluxes of NO3−, SeO42−, and H2 (from eqs 2−4) are summarized in Table 1 for each stage at steady state. On the basis of comparing the H2 fluxes to the maximum H2 flux (0.56 g of H2 m−2 day−1 or 280 mmol of H2 m−2 day−1), Stages 1, 3, 4, and 5 had sufficient H2 delivery and, thus, were not limited by the electron donor, while stage 2 was H2-limited, because the actual H2 flux was close to the maximum H2 flux. In stage 1 (days 1−67), when SeO42− was the only input at a surface loading of ∼0.11 g of Se m−2 day−1 (or 4.7 mmol of H2 m−2 day−1), SeO42− was reduced to SeO32− within 3 days of SeO42− addition and, subsequently, SeO32− was reduced to Se0. Se removal increased up to day 55 and remained steady to day 67. At that time, 40% of the influent total Se was reduced to Se0, with 20% reduced to SeO32−. In stage 2 (days 67−106), when ∼10 mg/L of NO3−−N was introduced to the MBfR (NO3− surface loading of 1.14 g of N m−2 day−1 or 245 mmol of H2 m−2 day−1), Se removal dropped to less than 10% and NO3− approached 80% removal within 3 days. The effluent NO3− concentration was about 2 mg of N/L at steady state in stage 2. The high total H2 flux and incomplete NO3− reduction are clear signs that the biofilm was limited by H2 delivery in stage 2. In stage 3 (days 106−138), when the NO3− loading was returned to zero, Se reduction returned to about 60% and almost all of the Se was reduced to Se0, because the SeO32− concentration was only ∼100 μg/L in the effluent. In stage 4 (days 138−183), the NO3− surface loading of 0.10 g of N m−2 day−1 (21.5 mmol of H2 m−2 day−1) caused a slight initial decline in the removal percentage from SeO42− to Se0 but returned to 60% in 3 days. NO3− was simultaneously removed to a non-detectable level (
