Effect of Nitrate and Perchlorate on Selenate Reduction in a Sequencing Batch Reactor

Abstract

Selenate removal from a water body is being vigorously debated owing to severe health impact, but inhibitions of coexisting anions have been reported. To suggest a viable treatment option, this study investigates the effect of nitrate and perchlorate on selenate reduction in a laboratory-scale sequencing batch reactor. The experimental design tests how competing electron acceptors (NO₃⁻ and ClO₄⁻) and electron donor (acetate) limitations affect selenate reduction in the reactor. Results show that the reactor achieves almost complete selenate reduction within the initial concentration ranges of 0.1–1 mM by enriching selenate-reducing bacteria with appropriate temperature (30 °C) and acclimation period (50 days). We monitored simultaneous selenate and nitrate reduction in the reactor without specific inhibition due to a difference in microbial growth strategy related to electron donor status. Lack of perchlorate-reducing bacteria makes perchlorate addition (0.2 mM) not to be closely associated with dissimilative perchlorate reduction. These results provide information that can help us to understand the effect of competing electron acceptors on selenate reduction and the kinetics of potential parallel reactions in the reactor.

1. Introduction

Selenium (Se) is an essential micronutrient but can cause adverse health effects (e.g. hair loss, fingernail loss, numbness in fingers or toes, and circulatory problems) with long-term and heavy exposure [1,2]. Since Se in water originates from not only geological sources such as weathering of seleniferous soils/rocks but also anthropogenic processes such as mining, fossil fuel combustion, and other industrial activities [3], the World Health Organization has set a provisional total Se guideline of 40 μg/L in drinking water [4]. The United States Environmental Protection Agency permits the maximum concentration limit (MCL) of total Se as 50 μg/L and the regulations of national primary drinking water as 5 μg/L [2]. Likewise, the Korean Ministry of Environment is reducing the MCL to 10 μgSe/L in drinking water [5].

Se has four oxidation states (−II, 0, IV, VI) and forms several organic complexes [6]. In surface water, most Se primarily exists either selenate (SeO₄²⁻) or selenite (SeO₃²⁻). Both oxyanions are toxic to living organisms thus various treatment technologies have been investigated to remove Se from water [7]. Although physicochemical technologies effectively separate Se from the water supplied for domestic and industrial use, eventual post-treatments for the byproducts are required and technical limitations are still existing [8]. Fortunately, biological treatment can reduce selenate and selenite to insoluble elemental Se (Se⁰) via anaerobic microbial metabolisms [6,9]. From a wide variety of environments, selenate- or selenite-reducing bacteria have been isolated [10,11].

When selenate or selenite coexist with other anions such as nitrate (NO₃⁻), sulfate (SO₄²⁻), and perchlorate (ClO₄⁻), biological Se reduction can be inhibited by the electron scavenging of denitrifying bacteria, sulfate-reducing bacteria, or perchlorate-reducing bacteria because most selenate-reducing bacteria are heterotrophic facultative anaerobes which compete for electron donors under anoxic or anaerobic conditions. Another limiting factor might be the drastic change of selenate in the water body due to irrigated agricultural drainage [11], sedimentary soil erosion [3], surface mining [12], coal-fired power plants [13], and so on.

Most biological selenate reductions are targeted for either pure culture or up-flow anaerobic sludge blanket process [14]. Relatively little reports are available about the simultaneous reduction of selenate in a mixed culture when competing anions exist [14,15,16,17]. This study, therefore, investigates the feasibility of simultaneous selenate, nitrate, and perchlorate reduction in a sequencing batch reactor (SBR) and evaluate the inhibitory effects of nitrate and perchlorate on biological selenate reduction.

2. Materials and Methods

2.1. Selective Enrichment of Selenate-Reducing Bacteria

To selectively enrich selenate-reducing bacteria, bench-scale SBRs were semi-continuously operated in parallel for more than one and a half months. Seed sludge was activated sludge taken from a local municipal wastewater treatment plant with a treatment capacity of 30,000 m³/d in the northern part of I-city, Korea. Using selenate as a sole electron acceptor, the enrichment period was kept under anoxic conditions. To support selective pressure on selenate-reducing bacteria, the temperature was controlled to 30 °C by aquarium heaters following previous literature [18].

2.2. Operating Condition of SBRs

Figure 1 shows the schematic diagram of the triplicate SBRs. The working volume of each SBR was 5 L. To verify the proper temperature condition (25 °C and 30 °C), SBRs were continuously monitored for more than 200 h until complete selenate reduction at the first batch. And then all the reactors were operated with 24 h sequence with the optimal temperature condition using the pre-acclimated biomass for 30 °C. Each SBR was completely mixed for 23 h. And then, an hour of settling period followed by rapid draw sequence of the upper liquid (2.5 L) and fill sequence with fresh feed solution. The feed solution contains selenate, acetate (CH₃COO⁻), buffer, and essential minerals: 50 mg/L of SeO₄²⁻, 200 mg/L of CH₃COO⁻, 46 mg/L of (NH₄)₂SO₄, 13.7 mg/L of K₂HPO₄, 84 mg/L of NaHCO₃, 51.3 mg/L of MgSO₄∙7H₂O, 43 mg/L of CaSO₄∙2H₂O, and 2.5 mg/L of FeSO₄∙7H₂O. Other micronutrients were available from inoculum and endogenous cell decay. Acetate was a sole carbon source (electron donor). To test the effects of nitrate and perchlorate on selenate reduction, we designed the experiments as shown in

Table 1. Operating conditions of SBRs according to experimental design.

Division	Initial Concentration of Target Contaminants				C: N (CH3COO−-C: NO3-N)
	Selenate (mM SeO42−)	Nitrate (mM NO3−)	Perchlorate (mM ClO4−)	Acetate (mM CH3COO−)
Phase 0 a	0.35	0.00	0.0	3.4	N.A.
Phase 1	0.1	1.0	0.0	3.4	6.7: 1
Phase 2	1.0	1.0	0.0	5.1	11.1: 1
Phase 3	0.1	1.0	0.0	0.6	1.2: 1
Phase 4	1.0	1.0	0.0	0.9	2.3: 1
Phase 5	0.1	1.0	0.2	3.4	5.8: 1

^a Initial acclimation period.

.

2.3. Analytical Methods

Influent and effluent liquid samples were filtered using a 0.2 μm syringe filter (Whatman, GE Healthcare Life Sciences, Marlborough, MA, USA) and kept in a refrigerator at 4 °C before analysis. Selenate was determined by using an ion chromatograph (Dionex ICX-1100, Dionex, Sunnyvale, CA, USA) equipped with an IonPac AS15 analytical column and AG15 guard column. The used eluent was a 36.5 mM NaOH solution (Daejung Chemicals, Siheung, Korea). The volume of the used sample loop for selenate determination was 100 μL. For perchlorate determination, we used the same ion chromatograph equipped with IonPac AS16 analytical column and AG16 guard column (Dionex, Thermo Fisher Scientific, Waltham, MA, USA). In this case, we used the sample loop volume of 1000 μL with the same 50 mM NaOH eluent. Nitrate and acetate concentrations were monitored by using an IonPac AS9-HC analytical and AG9-HC guard column with 9 mM Na₂CO₃ eluent and a 25 μL sample loop. The detection limits for selenate and perchlorate were 5 μg/L each. And those of acetate and nitrate were 0.5 mg/L. All the regressions for experimental data were conducted by Sigmaplot software (Systat Software Inc., San Jose, CA, USA) based on the assumption of first-order removal [19].

3. Results and Discussion

3.1. Appropriate Temperature for Selenate-Reducing Bacteria Acclimation in SBRs

To increase the activity of selenate-reducing bacteria in the seed sludge, initial acclimation (phase 0) was conducted for about 50 days using two sets of triplicate SBRs. Figure 2a shows the variations of selenate concentrations at the very first batch of the SBRs. During nine days of phase 0, only 27% of initial selenate (0.72 mM SeO₄²⁻) was reduced on average at the SBRs of 25 °C. However, in the SBRs at 30 °C selenate was reduced to below detection level after nine days. This result indicates that 30 °C, higher than room temperature, is more appropriate for the growth of selenate-reducing bacteria, which is consistent with previous literature [14,20,21]. With the revealed temperature condition, all the SBRs enriched selenate-reducing bacteria at 30 °C for the rest of phase 0 for further experiments.

At the end of phase 0, monitoring results indicate that SBRs could reduce selenate (0.9 mM) to below detection level in less than four hours. This enhancement indicates that phase 0 must have made the selenate-reducing bacteria successfully acclimated to start instantaneous selenate reduction right after fill-sequence without lag-period. Figure 2b demonstrates that enriched microorganisms actively reduce selenate to Se⁰ biologically at the last batch of phase 0, consistent with the literature [6,14,22,23]. Regression indicates that the observed selenate reduction rate was revealed as rapid as 0.96 h⁻¹.

3.2. Effect of Nitrate on Selenate Reduction

At phase 1 and phase 2, this study tests the effect of most probable electron-competing anion, nitrate, on selenate reduction (Table 1). We artificially constitute low (0.1 mM, phase 1) and high (1 mM, phase 2) selenate conditions for better interpretation. Figure 3 illustrates the dynamics of average (n = 3) selenate and nitrate in a whole sequence of SBRs at a steady state. When 3.8 mM CH₃COO⁻ was added to 0.1 mM SeO₄²⁻ (phase 1) as an excess electron donor in the presence of 0.96 mM NO₃⁻-N (approximately 1:10 of influent SeO₄²⁻: NO₃⁻ mole ratio), selenate and nitrate were simultaneously reduced to below detection level within six hours in SBRs (Figure 3a). In the case of phase 2, nitrate was completely reduced to below detection level, whereas a small amount of selenate was detected (0.02 mM, 98% reduction) after six hours in SBRs (Figure 3b). Close to the end of the sequence, the selenate concentration decreased to below detection level.

Within the ratio of SeO₄²⁻: NO₃⁻ between 1:1 and 1:10 tested in this study, both selenate and nitrate could be simultaneously reduced without significant inhibition. The selenate reduction rate was maintained at 0.55–0.57 h⁻¹ regardless of initial concentration. This result indicates that selective enrichment and long acclimation (>30 days) could make selenate-reducing bacteria endure competitive inhibition, described previously [24]. In addition, it was noticed that the denitrification rate was not interrelated with the selenate concentration and kept the rate as 0.88 h⁻¹ almost constantly, which supports simultaneous selenate and nitrate reduction under excess electron donor condition.

3.3. Effect of External Carbon Limitation on Selenate Reduction

Two sets of experiments were performed to investigate the effect of carbon source limitation on simultaneous selenate and nitrate reduction in the SBRs under low (0.1 mM, phase 3) and high selenate (1 mM, phase 4) conditions. Acetate concentration was limited to 0.8 mM for phase 3 when the initial SeO₄²⁻ concentration was 0.1 mM. Keeping the nitrate concentration as 1.0 mM results in the decrease of C:N ratio from 6.7:1 to 1.2:1 compared to phase 1. Phase 4 was conducted with 1 mM of SeO₄²⁻ reducing C:N ratio from 11.1:1 (phase 2) to 2.3:1 (phase 4). Phase 3 and phase 4 were directly comparable to phase 1 and phase 2, respectively. Figure 4a,b demonstrate the variations of selenate, nitrate, and acetate concentrations in SBRs at phase 3 and 4, respectively, as described in Table 1.

Figure 4a (phase 3) shows that all the selenate was reduced instantaneously within two hours but the accompanying nitrate reduction significantly decelerates when the acetate was depleted at around 3 h. Figure 4b (phase 4) illustrates that nitrate reduction similarly stops when the acetate was depleted, but selenate reduction gradually progressed further despite the depletion of external carbon sources. This result indicates that denitrifying bacteria are more sensitive to electron donor compared to selenate-reducing bacteria. The increase of acetate concentration from 0.7 mM to 1.2 mM enhanced the nitrate reduction rate about 80% (from 0.79 h⁻¹ to 1.42 h⁻¹) at phase 4 but the nitrate reduction rate drastically ceased as the carbon source depleted. Selenate reduction rate was also decreased by 27.4% (from 0.95 h⁻¹ to 0.69 h⁻¹) possibly owing to inhibition associated with carbon source competition.

When the selenate and nitrates are coexisting, selenate-reducing bacteria might present the ability to compete successfully for limited carbon resources like K-strategist microorganisms [25] while nitrate-reducing bacteria exploit relative offspring trends like r-strategist microorganisms [26] in this study. This result suggests that selenate-reducing bacteria has a more competitive advantage over withstanding harsh carbon-limiting condition than nitrate-reducing bacteria.

3.4. Nitrate and Perchlorate Effect on Selenate Reduction in SBRs

To investigate the effect of another oxyanion, perchlorate, on the simultaneous selenate reduction, SBRs were operated with a feed solution containing selenate (0.1 mM), nitrate (1.0 mM), and perchlorate (0.15 mM) with an excess amount of external carbon source (3.4 mM).

Figure 5 demonstrates that selenate and nitrate reduction are not affected by perchlorate significantly. It was observed that 38% of perchlorate (reduction rate of 0.02 h⁻¹) can be reduced together with selenate and nitrate in the SBRs during 24 h of a sequence. This result indicates that dissimilatory perchlorate-reducing bacteria can grow together with selenate- and nitrate-reducing bacteria under anaerobic conditions if the carbon source (electron donor) is not limiting [27]. In this study, an insufficient population of perchlorate-reducing bacteria might have prevented the perchlorate from being a competitive inhibitor of selenate or nitrate reduction under excess electron donor conditions. Owing to perchlorate, the reduction rate of nitrate was significantly reduced from 0.9~1.4 h⁻¹(excess electron donor condition) to 0.5 h⁻¹ at phase 5. However, that of selenate did not decline but maintained to around 0.7–1.3 h⁻¹, which evidences the ability of selenate-reducing bacteria to endure harmful perchlorate as well as electron donor competition without significant inhibition. This result also means that selenate-reducing bacteria can be dominantly enriched from activated sludge within a reasonable period of time if the carbon source is not limiting.

4. Conclusions

This research provides information about how competing anions, nitrate, and perchlorate, affect selenate reduction in SBRs which are seeded with activated sludge. Based on the observed data from this research, the following conclusions are drawn as below:

(1) SBRs can rapidly enrich selenate-reducing bacteria from the activated sludge by using the selective pressure of temperature (30 °C) and sufficient acclimation period of >40 days.

(2) Complete selenate and nitrate reduction can be accomplished simultaneously in anaerobic SBRs by supplying the excess amount of electron donor. Limitation of electron donor may decrease the activity of nitrate-reducing bacteria instantaneously while selenate-reducing bacteria responds slowly using the limited resources more efficiently.

(3) Coexistence of perchlorate in the feed did not affect selenate reduction significantly owing to the shortage of dissimilatory perchlorate reducing bacteria. However, together with selenate and nitrate, 38% of perchlorate could be reduced without acclimation when electron donor is not limited.

Overall, these results evidence that selenate-reducing bacteria are capable of enduring competitions associated with other oxyanions reduction and electron donor without significant inhibition after appropriate acclimation. This study may contribute to understanding biological Se reduction better in relation to competing anions and electron donor conditions.