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Effect of H2S on N2O Reduction and Accumulation during Denitrification by Methanol Utilizing Denitrifiers Yuting Pan,† Liu Ye,†,‡ and Zhiguo Yuan*,† †

Advanced Water Management Centre (AWMC) and ‡School of Chemical Engineering, The University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia S Supporting Information *

ABSTRACT: Sulfide is produced in sewer networks, and previous studies suggest that sulfide in sewage could alter the activity of heterotrophic denitrification and lead to N2O accumulation during biological wastewater treatment. However, the details of this phenomenon are poorly understood. In this study, the potential inhibitory effects of sulfide on nitrate, nitrite, and N2O reduction were assessed with a methanolutilizing denitrifying culture both prior to and after its exposure and adaptation to sulfide. Hydrogen sulfide was found to be strongly inhibitory to N2O reduction, with 50% inhibition observed at H2S concentrations of 0.04 mg H2S−S/L and 0.1 mg H2S−S/L for the unadapted and adapted cultures, respectively. In comparison, both nitrate and nitrite reduction was more tolerant to H2S. A 50% inhibition of nitrite reduction was observed at approximately 2.0 mg H2S−S/L for both unadapted and adapted cultures, while no inhibition of nitrate reduction occurred at the highest H2S concentrations applied (2.0 mg H2S− S/L) to either culture. N2O accumulation was observed during nitrate and nitrite reduction by the adapted culture when H2S concentrations were above 0.5 and 0.2 mg H2S−S/L, respectively. Additionally, we reveal that hydrogen sulfide (H2S), rather than sulfide, was likely the true inhibitor of N2O reduction, and the inhibitory effect was reversible. These findings suggest that sulfide management in sewers could potentially have a significant impact on N2O emission from wastewater treatment plants.

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aerobic phase.12,13 For example, an N2O accumulation of 0.1 mg N2O−N/L at the end of the anoxic reactor in a modified Ludzack−Ettinger (MLE) system could lead to an emission factor of approximately 1%, calculated by assuming an influent total nitrogen concentration of 50 mgN/L, an internal recirculation flow rate three times that of the influent flow rate, and a returned activated sludge flow rate equaling the influent flow rate (0.1(1 + 3 + 1)/50 = 1%). de Haas and Hartley estimated that an emission factor of 1% would add substantially to the carbon footprint of a biological nutrient removal plant.8 Nitrous oxide is an obligatory intermediate of denitrification. Complete heterotrophic denitrification consists of sequential reductive reactions from nitrate (NO3−) to nitrite (NO2−), nitric oxide (NO), nitrous oxide (N2O), and finally to nitrogen gas (N2). Regarding environmental factors leading to N2O accumulation during denitrification in WWTPs, research has focused on parameters such as dissolved oxygen (DO),7,14−17 carbon source availability,7,15,18−23 pH,20,24,25 and nitrite or free nitrous oxide acid (FNA).7,15,21,26,27 Much less attention has been paid to sulfide, which is often a constituent of sewage. Sulfide is produced biologically in sewer pipes and could be

INTRODUCTION With increasing awareness and concern about the greenhouse gas effect of nitrous oxide (N2O) emissions, substantial efforts have been made to quantify and understand the mechanisms of N2O emission from wastewater treatment plants (WWTPs).1−5 Studies suggest that nitrous oxide emissions could contribute significantly, and in some cases could even dominate, the carbon footprint of WWTPs.6−9 In 2009, Kampschreur et al.10 reviewed previously reported N2O emission factors (fraction of influent nitrogen that is emitted as N2O) from WWTPs and found that the factors varied in the range of 0−14.6% in fullscale wastewater treatment plants. More recent studies showed that N2O emission could be highly dynamic;3,11 therefore, online and continuous monitoring of N2O is crucial for accurate quantification of N2O emissions from wastewater treatment systems.12 A national survey in the United States involving online monitoring of N2O emissions from 12 WWTPs revealed a narrower emission factor range of 0.01− 1.8%.1 N2O is mainly produced through biological nitrification and denitrification. It was recently revealed that nitrification is the primary source of N2O emitted from WWTPs, as the N2O gas, while being produced, is readily stripped from the wastewater due to the presence of aeration.1,10,12 However, N2O production by denitrification cannot be ignored. Even low levels of N2O accumulation during denitrification could lead to significant N2O emission if it is stripped in the subsequent © 2013 American Chemical Society

Received: Revised: Accepted: Published: 8408

April 17, 2013 June 23, 2013 June 26, 2013 June 26, 2013 dx.doi.org/10.1021/es401632r | Environ. Sci. Technol. 2013, 47, 8408−8415

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during denitrification and thus improve settling), 6 min sludge wasting, 60 min settling, and 5 min decanting periods. Before settling, 200 mL of mixed liquor was wasted to keep the solids retention time (SRT) at approximately 10 days. A 2 L amount of synthetic wastewater was slowly pumped into the reactor in the 40 min feed period, and 1.8 L of supernatant was discharged after settling, resulting in a hydraulic retention time (HRT) of 24 h (8 L/(1.8 L/cycle + 0.2 L/cycle) × 6 h/cycle = 24 h). The SBR operation was automatically controlled by a programmable logic controller (PLC). The dissolved oxygen (DO) concentration and pH were continuously monitored online using a miniCHEM-DO2 and a pH probe. DO concentration in the reactor was always below the detection limit (0.01 mg/L). pH was automatically controlled at 7.2 ± 0.1 by addition of a 0.5 M hydrochloric acid stock solution. Liquidphase N2O in the reactor was also periodically measured using a microsensor. Cycle studies were performed weekly throughout the entire course of the study by measuring the ammonium, nitrate, nitrite, phosphate, and methanol concentrations throughout the 6 h cycle with a sampling interval of 30 min. The mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) concentrations were measured every week. The system was operated in pseudosteady state when the batch experiments were carried out. In pseudosteady state, the MLVSS concentration in SBR1 was stable at approximately 1.0 g/L and removal of nitrate and methanol from the synthetic wastewater were both above 99%. The synthetic wastewater feed contained nitrate at 200 mg N/ L, methanol at 800 mg/L (measured as chemical oxygen demand or COD), as well as other nutrients and trace elements as previously described.25 SBR2 Setup and Operation (Sulfide-Adapted Culture). To adapt the denitrifying culture to sulfide, another 4 L reactor (SBR2) was inoculated with sludge from SBR1. The operational conditions of SBR2, including temperature, operational phases, and cycle time, wastewater feed, DO, and pH were identical to those of SBR1. Before settling, 100 mL of mixed liquor was wasted to maintain the SRT at approximately 10 days. A 1 L amount of synthetic wastewater described above and 0.1 L of sodium sulfide solution was slowly pumped into the reactor during the 40 min feed period, and 1 L of supernatant was discharged after the settling period, resulting in a hydraulic retention time (HRT) of 22 h. The dissolved sulfide solution was prepared freshly every second day by adding sodium sulfide into oxygen-free water and stored in a gastight bottle sealed with silicone grease. A gas bag filled with nitrogen gas was connected to the bottle to replace the volume of sodium sulfide solution fed to the reactor. The total dissolved sulfide concentration in the influent was increased each week to be 1.0, 2.0, 4.0, and finally 8.0 mg S/L. Following that the total sulfide concentration in the influent was maintained at 8.0 mg S/L for over 6 months. The MLVSS concentration was stable at 1.0 g/L. During operation of SBR1, removal of nitrate and methanol from the synthetic wastewater was both above 99%. Batch Experiments. Batch Reactor Setup and Operation. A 300 mL sealable gastight reactor was used for all tests. To perform online monitoring, the batch reactor was fitted with a N2O microsensor, a H2S microsensor, and a pH probe. At the beginning of each test, the reactor was filled with fresh mixed liquor withdrawn from either SBR1 or SBR2 at the end of their anoxic reaction phase and then sparged with N2 gas for 5 min to ensure anoxic conditions for denitrification. The reactor was completely filled and sealed (no gas headspace) and fitted with

introduced to the denitrification tank via the influent wastewater. In sewage, dissolved sulfide concentrations of over 10 mg S/L have been reported.28,29 While some of the sulfide may be removed during the pretreatment of sewage, such as the screening and aerated grit removal processes, it is expected that a significant fraction of sulfide present in raw sewage would be fed into the biological treatment reactor. Sulfide is known to affect microbial activity in general since it is usually toxic to bacteria. Currently, there are very limited studies investigating the effect of sulfide on denitrification during wastewater treatment. However, some studies on bacterial pure cultures isolated from soil or marine sediments have reported inhibitory effects of sulfide on N2O reduction during denitrification. Tam and Knowles30 reported that high levels of sodium sulfide (256 mg S/L) caused marked inhibition of N2O reduction in cell suspensions of Pseudomonas aeruginosa and in soil inoculated with P. aeruginosa. Another study by Sørensen et al. reported strong inhibition of N2O reduction at a sulfide concentration of 9.6 mg S/L in P. f luorescens isolated from soil.31 Further study by Sørensen et al.32 found that sulfide levels as low as 0.16 mg S/L caused 80% inhibition of N2O reduction. However, the effects of sulfide on nitrate and nitrite reduction were not investigated in these studies. Research assessing the effect of sulfide on N2O reduction and accumulation during the wastewater treatment process is very limited. One study conducted with denitrifying activated sludge found that N2O accumulation to above 5 mg N/L when nitrate, acetate, and sulfide were slowly loaded into the reactor.33 However, only two fed-batch tests (at pH 7.5 and 8.0) were carried out in this study to evaluate the effect of sulfide on N2O accumulation, with sulfide concentration changing throughout each test. Such an approach, while demonstrating an effect of sulfide on N2O accumulation, does not reveal the detailed, specific effects of sulfide on N2O reduction or on nitrate or nitrite reduction. Thus, details of the effect of sulfide on biological denitrification by activated sludge and the mechanisms involved are yet to be fully revealed. This study aims to improve the understanding of the effects of sulfide on N2O reduction and accumulation during biological denitrification in the wastewater treatment process. Experiments were designed to establish a quantitative relationship between the N2O reduction rate and sulfide concentration in the sludge. Recovery of the N2O reductase activity, after removal of sulfide, was also investigated. In addition, the effects of sulfide on the other denitrification steps, namely, nitrate and nitrite reduction, were studied. A methanol-utilizing denitrifying sludge was used to study the inhibitory effects of sulfide both before and after adaptation of the sludge to sulfide in the wastewater feed.

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MATERIALS AND METHODS Sludge Source. The sludge used in this study was withdrawn from two laboratory-scale sequencing batch reactors (called SBR1 and SBR 2). A sulfide-unadapted methanolutilizing denitrifying culture was enriched in SBR1. A sulfideadapted methanol-utilizing denitrifying culture was enriched in SBR2. SBR1 Setup and Operation (Sulfide-Unadapted Culture). SBR1 (8L), seeded with sludge from a domestic WWTP in Brisbane, Australia, was operated at room temperature (22.0−23.0 °C) with a cycle time of 6 h. The cycle consisted of 40 min anoxic feed and reaction, 240 min anoxic reaction, 9 min aeration (to strip the nitrogen gas produced 8409

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operated at pseudosteady state the total sulfide concentration of the influent was 8.0 mg S/L. These five tests were carried out according to the experimental conditions of tests 2, 4, 6, 8, and 13 listed in Table 1. Two steps were conducted in each of the Set I batch tests, following initial manual addition of 1.0 M HCL solution to adjust the reactor pH to its set point. In the first step, Step I, the N2O reduction rate in the absence of sulfide is assessed. N2O and methanol were added to the reactor to achieve initial concentrations of 10 mg N/L and 150 mg COD/L, respectively. The N2O profile was continuously monitored online using a microsensor. After depletion of N2O in Step I, Step II was initiated by addition of sulfide to achieve initial concentrations of between 0 and 10 mg S/L (Table 1), followed by addition of N2O to a concentration of 10 mg N/L in the batch reactor. No extra methanol was added in Step II, since it was still in excess after Step I . Reduction of N2O, with sulfide present, was monitored for periods of 20−60 min. Mixed liquor samples were taken at the beginning and end of Step II to determine the sulfide concentration. The MLVSS concentration was measured at the end of each test in triplicate. The N2O reduction rate in both the control phase (Step I) and the experimental phase (Step II) was determined through linear regression of the linear part of the measured N2O profiles. The N2O reductase activity in the presence of sulfide (Step II) was expressed as the percentage of that measured in the control phase (Step I). Set II Batch Tests: Recovery of N2O Reductase Activity from Sulfide Inhibition. Two batch tests were performed with sludge from SBR1 to investigate the recovery of N2O reductase activity after removal of sulfide. In both tests, pH was controlled at 7.0. Three steps were involved in each test of this set. The first two steps were the same as Steps I and II in Set I batch tests, in which the N2O reduction rates without and with sulfide were determined. The initial dissolved sulfide concentration in Step II was 0.4 and 3.0 mg S/L, respectively. After depletion of N2O in Step II, the sludge was washed three times using sterilized decant from SBR1 to remove sulfide. MLVSS concentrations were measured in triplicate both before and after the wash step. To initiate Step III, N2O and methanol were added to the mixed liquor to achieve initial concentrations of 10 mg N/L and 150 mg COD/L, respectively (the same concentrations used in Step I), and then N2O concentrations were monitored during Step III. The N2O reduction rates in Steps I, II, and III were determined through linear regression of the linear part of the measured N2O profiles. The biomassspecific N2O reductase activity in Steps II (in the presence of sulfide) and III (after sulfide removal) was then expressed as the percentage of that measured in Step I (control). Set III Batch Tests: Nitrate and Nitrite Reduction in the Presence of Different Levels of Sulfide. A total of 20 batch tests were carried out to investigate the effect of different sulfide levels on nitrate and nitrite reduction by the SBR1 and SBR 2 sludges. pH was controlled at 7.5 in all tests. At the beginning of each test nitrate or nitrite was added into the mixed liquor to achieve an initial concentration of 30 mg N/L, and methanol was added at an initial concentration of 150 mg COD/L. Following that sulfide was added to achieve initial sulfide concentrations of 0, 0.5, 3.0, 6.0, or 8.0 mg S/L (no sulfide addition in the case of 0 mg S/L). An additional test was carried out with sludge from SBR2 by adding nitrate and sulfide to achieve initial concentrations of 30 mgN/L and 8 mg S/L, respectively, without methanol addition, to assess the rate of

a 15 mL reservoir connected to the lid of the reactor. The reservoir (a syringe filled with the same mixed liquor as in the reactor) was used to balance the pressure inside the reactor during the liquid-phase sampling in the tests. The piston of the syringe prevented mixed liquor in the syringe from getting in contact with air, and thus, anoxic conditions were maintained. The reactor had a solution injection/liquid sampling point equipped with a butyl rubber stopper. The reactor was well mixed throughout all tests by use of a magnetic stirrer. For addition of N2O to the batch experiments a stock solution was freshly prepared each time by sparging Milli-Q water with 100% N2O gas for 5 min.26 A sulfide stock solution was prepared by adding 0.38 mg of Na2S·9H2O in 100 mL of oxygen-free Milli-Q water, resulting in a total dissolved sulfide concentration of 0.5 g S/L. The stock solution was kept in a 100 mL serum bottle sealed with a butyl rubber stopper and a metal cap and stored at 4 °C. Set I Batch Tests: N2O Reduction at Different pH in the Presence of Different Levels of Sulfide. Batch tests were performed with sludge from both SBR1 and SBR2 to investigate the effect of sulfide on N2O reduction. Fourteen tests were performed on sludge from SBR1, these tests having variations of pH between 7.0 and 8.5 and variations of initial sulfide concentrations between 0.1 and 10.0 mg S/L (Table 1). Table 1. Experimental Conditions Applied in H2S Inhibition Batch Tests test

pHa

dissolved sulfide (mg S/L)b

H2S (mg S/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

7.0 7.0 7.0 7.0 7.5 7.5 7.5 8.0 8.0 8.5 8.5 8.5 8.5 8.5

0.1 2.0 3.0 4.0 0.1 2.0 4.0 2.0 7.0 0.5 1.0 2.0 4.0 10.0

0.05 1.00 1.50 2.00 0.024 0.48 0.96 0.18 0.64 0.02 0.03 0.06 0.12 0.31

a

Operational pH of wastewater treatment plants is typically between 7.0 and 8.0. A relatively broad pH range of 7.0−8.5 has thus been chosen in this study to investigate the effect of H2S on N2O reduction during heterotrophic denitrification bDissolved sulfide levels were chosen based on the sulfide levels reported for raw sewage by also considering its possible dilution in the bioreactor.28

The H2S concentration can be calculated based on the relationship [H2S]/[total dissolved sulfide] = 1/(l0pH‑pK1 + l) with pK1 = 7.01 at 22.5 °C (pK1 = 2.151 + 1437/T, T in degrees Kelvin).34 This resulted in initial hydrogen sulfide (H2S) concentrations between 0.02 and 2.0 mg S/L in the batch tests (Table 1). pH was controlled at the preselected set point throughout each test. Nine batch tests were performed on sludge from SBR2. Among them, four batch tests were performed during the period when the influent total dissolved sulfide concentration was increased from 1.0 to 8.0 mg S/L. These four tests were carried out according to the experimental conditions of test 5 shown in Table 1. Five more tests were performed with sludge from SBR2; when the reactor was 8410

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nitrate reduction coupled to sulfide oxidation. Mixed liquor samples were taken during the batch tests every 5−10 min for nitrate, nitrite, sulfide, and methanol analysis. Dissolved N2O and H2S were continuously monitored using microsensors. MLVSS concentration was measured at the end of each test in triplicate. Nitrate and nitrite reduction rates were determined through linear regression of the linear part of the measured nitrate or nitrite profiles. In all tests, the nitrate or nitrite reductase activity in the presence of sulfide was expressed as the percentage of that measured without sulfide. Sampling and Analytical Methods. Mixed liquor samples were taken using a syringe and immediately filtered through disposable Millipore filter units (0.22 μm pore size) for analyses of nitrate, nitrite, sulfide, and methanol. Nitrate and nitrite concentrations were analyzed using a Lachat QuikChem8000 Flow Injection Analyzer (Lachat Instrument, Milwaukee, WI). Methanol was analyzed by gas chromatography (Perkin-Elmer Autosystem). For analyses of dissolved inorganic sulfur species (sulfide, sulfite, thiosulfate, and sulfate) 1.5 mL of wastewater was filtered (0.22 μm membrane) into 0.5 mL of a sulfide antioxidant preserving solution.35 Then these samples were analyzed within 24 h by ion chromatography (IC) using UV and conductivity detectors (Dionex ICS-2000). MLSS and MLVSS were measured according to Standard Methods.36 N2O in the liquid phase was measured online using a N2O microsensor (N2O-100, Unisense A/S, Aarhus, Denmark) with a detection limit of 0.0028 mg N/L in water and a responding time of less than 10 s. H2S in the liquid phase was measured online using a H2S microsensor (H2S-500, Unisense A/S, Aarhus, Denmark) with a detection limit of 0.0096 mg H2S−S/L in water and a responding time of less than 20 s. Sensors were calibrated before each test.

Figure 1. N2O reduction activities detected in sludge from SBR1 (a− c) and SBR2 (d): (a) pH levels (●) 7.0, (○) 7.5, (□) 8.0, (▲) 8.5; (b) total dissolved sulfide levels (×) 0 (no sulfide added, each of them is used as a reference for each pH level), (●) 0.1, (○ ) 0.5, (■) 1.0, (□) 2.0, (▲) 3.0, (△) 4.0, (◊) 7.0, (◆) 10.0. (c) ○-different H2S levels in all the tests; (d) (●) tests performed during the adaptation period, (○) tests performed after full adaptation. () Regression lines in c and d were obtained by fitting the noncompetitive inhibition model with the experimental data using SigmaPlot 12 (Systat Software Inc., USA).

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correlation was observed (Figure 1c). N2O reductase activity decreased sharply with increasing H2S concentration. N2O reductase activity was reduced to 50% of the original activity at H2S concentrations of 0.04 mg S/L and almost completely inhibited at 1.5 mg S/L of H2S (equivalent to 3.0 mg S/L of sulfide at pH 7.0). Most previous studies measuring inhibition of N2O reductase activity performed the tests at a constant pH, and the form of sulfide was not considered.30−32,37,38 Thus, the terms sulfide and H2S were used ambiguously to designate the total sulfide. In contrast, Schonharting et al.33 proposed that H2S was likely to be the direct inhibitor, although this was based on only two fed-batch tests on activated sludge. In our study, 14 tests were conducted by verifying the sulfide concentration and pH levels. Consequently, our results convincingly show that the level of inhibition on N2O reduction is strongly correlated with the H2S concentration rather than the sulfide. Usually, in the presence of a competitive inhibitor, the maximum enzyme activity can still be reached if sufficient substrate is available.39,40 However, in all Set I batch tests, although the initial N2O concentration was substantially in excess, the N2O reduction rate measured in Step II (in the presence of H2S) was always lower than that in Step I (in the absence of H2S), which suggested that inhibition was not competitive inhibition. In contrast, a noncompetitive-type inhibition model39,40 was found to describe the experimental data well, which suggests that H2S inhibition on N2O reduction was likely due to noncompetitive inhibition. Inhibition of N2O reductase activity detected here at different H2S levels is likely relevant to wastewater treatment. Reported total sulfide concentrations in sewage could reach higher than 10 mg S/L.28 Consequently, it is highly possible that the N2O reductase activity could be inhibited during denitrification if the

RESULTS AND DISCUSSION Effect of Sulfide on N2O Reduction. N2O Reductase Activity Correlates with H2S Concentration. In the Set I batch tests N2O reductase activity was determined at different sulfide concentrations (Figure 1). An example of the raw data used for generation of Figure 1 is presented in Figure S1, Supporting Information. On sludge from SBR1 (culture unadapted to sulfide) there was a negative impact of sulfide on N2O reduction (Figure 1a). Generally, as the sulfide concentration went higher, the N2O reductase activity reduced. However, this relationship varied also depending on pH. For example, at 2.0 mg S/L sulfide the N2O reductase activity varied between 7% at pH 7.0 and 35% at pH 8.5. Similarly, at other sulfide concentrations, higher inhibition of the acitivity occurred at lower pH values, thus indicating that pH is important for N2O reductase activity. However, similar to the effect of total dissolved sulfide concentrations there is no clear relationship between the N2O reductase activity and the pH levels (Figure 1b). At the same pH level, less activity was observed at higher sulfide concentrations. These observations suggest that sulfide and pH jointly cause the inhibitory effect on N2O reduction. Dissolved sulfide exists in three forms in water, H2S, HS−, and S2−, and the coexisting abundance of these forms is pH dependent. In this study, since the pH ranges from 7.0 to 8.5, the majority of the total dissolved sulfide species existed in was were H2S and HS− while S2− was negligible (always less than 0.001% of the total dissolved sulfide). When the N2O reductase activity was plotted against the HS− concentration, no clear correlation was observed. However, when the N2O reductase activity was plotted against H2S concentrations, a strong 8411

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therefore do not support the above hypothesis. Overall, it is still inclusive how bacterial N2O reduction is inhibited by H2S. Further investigation is required. Effect of H2S on N2O Accumulation. Effect of H2S on Nitrate and Nitrite Reduction. The effects of H2S on nitrate and nitrite reduction were studied in Set III batch tests. For the sludge unadapted to sulfide, the nitrate reductase activity remained unchanged with the increasing H2S concentration from 0 to 2.0 mg S/L (Figure 3). However, the nitrite reductase

sulfide generated in sewer networks enters the denitrification tank in wastewater treatment plants. Adaptation Ability of N2O Reduction to H2S Inhibition. It is known that bacteria in wastewater can adapt to changing conditions depending on the pollutant composition of the sewage.41 However, presently it is not known if bacteria adapted to sulfide could have higher resistance to H2S in terms of their N2O reduction capability. Consequently, to answer this question we performed Set I batch tests with sludge from SBR 2 during the adaption period and after receiving sulfide in the feed for 6 months. It can be seen that H2S still has a very strong inhibitory effect on the N2O reductase activity of the adapted culture (Figure 1d). However, the adapted culture appears to be slightly more tolerant. The N2O reductase activity reduced to 50% of the original activity at an H2S concentration of 0.1 mg S/L. The slightly higher tolerance could be related to the ability of sulfide-adapted sludge to remove sulfide, thus likely creating lower H2S concentrations inside of flocs, rather than due to physiological changes of denitrifying bacteria. The detailed mechanism requires further investigation. Recovery Ability of N2O Reductase Activity after Removal of H2S. Recovery of N2O reductase activity after removal of H2S was studied in Set II batch tests. It was seen that the N2O reductase activity of the culture almost fully recovered after removal of sulfide at two different H2S concentrations studied (0.2 and 1.5 mgH2S−S/L) (Figure 2). The results suggest that

Figure 3. Nitrate and nitrite reduction activities of the sulfide-adapted and sulfide-unadapted sludges under various initial H2S concentrations.

activity gradually reduced by 40% when the H2S concentration increased from 0 to 2.0 mg S/L. In comparison, the N2O reductase activity reduced to negligible levels at a H2S concentration of 2.0 mg S/L (Figure 1c). These results suggest that nitrate, nitrite, and N2O reduction have different tolerance to H2S, with nitrate reduction having the highest tolerance to H2S. In fact, accumulation of nitrite and/or N2O is often reported during nitrate reduction when sulfide is present,33,38,43 indirectly suggesting that N2O or nitrite reduction is less tolerant to sulfide inhibition compared with nitrate reduction. However, in a study on Desulfovibrio desulf uricans, which reduces nitrate to ammonia, it was found that 70% inhibition of the nitrate reduction rate occurred when the organism was exposed to a total dissolved sulfide concentration of 4.0 mg S/L at pH 7 (equivalent to ∼2.0 mg S/L of H2S).44 Differences in the inhibition detected suggest the effect of sulfide on nitrate reduction is likely depending on the types of organism or the denitrification pathways, and further studies are required to clarify this mechanism. The effects of H2S on nitrate and nitrite reduction were also studied with the sulfide-adapted sludge. The nitrate reductase activity was observed to increase by around 40% when the initial H2S concentration increased from 0 to 0.75 mg S/L (Figure 3). This increase could be due to the contribution of chemolithotrophic denitrification.45 Addition of sodium sulfide in the influent for SBR2 might enrich denitrifiers that couple sulfide oxidation to reduction of nitrate for energy conservation. This was examined in our batch test when only sulfide and nitrate were added without methanol. The nitrate reduction rate measured was around 40% of that when methanol and nitrate was added (without sulfide). This observation supports the hypothesis that sulfide oxidizers using nitrate as the electron acceptor caused increased nitrate reduction activity in the

Figure 2. Recovery ability of N2O reduction of the sludge from H2S inhibition. Activity was detected prior to H2S exposure, during exposure, and after removal of H2S.

inhibition is reversible. The reversible nature of H2S-caused inhibition on N2O reductase activity has previously been observed in pure cultures of Pseudomonas f luorescens and P. aeruginosa.32,38 Thus, it seems the H2S inhibition on N2O reduction is generally reversible. Mechanisms for H2S Caused Inhibition on N2O Reduction. The active form of bacterial N2O reduction contains 12 copper ions; consequently, low copper supply could interfere with the activity of the enzyme.42 As sulfide reacts strongly to form metal sulfides, this could be the reason for the detected sulfide inhibition on N2O reduction. Bartacek et al.38 proposed that sulfide inhibition was due to sulfide precipitation of copper needed for nitrous oxide reductase activity. It is known that N2O reduction is located in the cell periplasm,42 which is accessible by sulfide in all its forms (S2−, HS−, and H2S). Our results show that H2S, rather than total dissolved sulfide, is likely the direct inhibitor to N2O reduction. Our results 8412

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Figure 4. Nitrate, nitrite, N2O, and H2S profiles measured in one batch test with sulfide-adapted sludge (a) at pH 7.5, in one batch test with sulfideunadapted sludge (b) at pH 7.5, and one typical cycle of SBR2 (c) at pH 7.2 ± 0.1.

N2O reduction rate at this H2S level would be still higher than the corresponding nitrate reduction rate. This could be verified by a simple calculation. The biomass-specific N2O reduction rate at pH 7.5 without sulfide was around 320 mgN/L (determined in Set I batch test), while the corresponding nitrate reduction rate was much lower, at 37 mgN/L (determined in Set III batch tests). On the basis of the model generated in Figure 1, the N2O reduction rates of both cultures should be higher than 90 mgN/L at a H2S concentration of 0.12 mg S/L, which is higher than the corresponding nitrate reduction rates. Thus, N2O accumulation was not observed in this test. Typical nitrate, nitrite, N2O, and H2S profiles in a normal SBR2 cycle are shown in Figure 4c. The nitrate, nitrite, H2S, and N2O concentrations were all negligible during the entire cycle. This was due to the continuous slow feeding applied to SBR2, with a feeding period lasting for 40 min. Thus, sulfide was consumed while it was fed before it could reach a level leading to N2O accumulation. Implications for Operation of Wastewater Treatment Plants. In this study, using both sulfide-adapted and sulfideunadapted methanol-utilizing denitrifiers, we found that N2O accumulation could occur in the presence of very low levels of H2S, e.g., 0.5 mg S/L (Figure 4c). N2O could accumulate to high concentrations (e.g., 5 mgN/L) as shown in Figure 4a, which would result in significant N2O emissions if leaked to the aerobic zone/period and stripped there. Similar amounts of N2O accumulation at similar H2S levels was also reported by Schonharting et al.33 when an acetate-utilizing denitrifier was studied. The levels of dissolved sulfide/H2S used in the feed in both studies are within the levels normally observed in raw sewage.28,29 In addition, both methanol and acetate are the most commonly used externally added carbon sources in

adapted culture. For nitrite reduction, H2S had a similar inhibitory effect as on the unadapted sludge. N2O Accumulation during Nitrate and Nitrite Reduction. N2O accumulation was observed in the Set III batch tests during nitrate and nitrite reduction when initial H 2S concentrations were above 0.5 and 0.2 mg H2S−S/L, respectively, for both sulfide-adapted and sulfide-unadapted sludge. As an example, Figure 4a shows typical nitrate, nitrite, N2O, and H2S profiles in one batch test with sulfide-adapted sludge. N2O began to accumulate at the beginning of the test when nitrate and sulfide were added. The N2O accumulation rate gradually decreased as the concentration of sulfide was reduced. When the total dissolved sulfide was depleted, the accumulated N2O reached the highest point, around 4.8 mgN/ L. After that the N2O concentration began to decline and was completely removed in the next 20 min. This also shows that the H2S inhibition on N2O reduction is reversible. The profile of nitrite accumulation shows a similar trend as that of N2O. However, nitrite only accumulated to 1.5 mgN/L before it began to decrease. It was also apparent that the nitrate reduction rate reduced slightly after depletion of H2S. In comparison, Figure 4b shows typical nitrate, nitrite, N2O, and H2S profiles in one batch test with sulfide-unadapted sludge. N2O also began to accumulate at the beginning of the test when nitrate and sulfide were added. However, since the H2S consumption rate was much slower with sulfide-unadapted sludge compared with that of the sulfide-adapted sludge, N2O accumulated to a much higher level at 12.0 mgN/L in Figure 4b compared with at 4.8 mgN/L in Figure 4a. Complete removal of N2O was observed at around 100 min. N2O accumulation was not observed in tests with an initial H2S concentration of 0.12 mg S/L during nitrate reduction. This could be explained by the fact that the biomass-specific 8413

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wastewater treatment for biological nitrogen removal.46 Thus, there is a high risk of N2O accumulation during denitrification should sulfide be present. However, our results also indicate that if N2O accumulation does occur this can be temporary and could be alleviated by increasing the length of the anoxic period. The comparison between Figure 4a and Figure 4c indicates that changing the feeding pattern could be a potential method to mitigate N2O emission. By slowly feeding influent into anoxic zones, H2S would be consumed as it is loaded; thus, this could reduce the risk of N2O accumulation.

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ASSOCIATED CONTENT

S Supporting Information *

An example of the raw data used for the generation of Figure 1 is presented in Figure S1 in Supporting Information. This information is available free of charge via the Internet at http:// pubs.acs.org/

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was funded by the Australian Research Council, Western Australia Water Corp., and Melbourne Water Corp. through project LP0991765. Y.P. acknowledges UQ Research Scholarship (UQRS) support.

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REFERENCES

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