Article pubs.acs.org/est
Hydroxylamine Diffusion Can Enhance N2O Emissions in Nitrifying Biofilms: A Modeling Study Fabrizio Sabba,† Cristian Picioreanu,‡ Julio Pérez,‡,§ and Robert Nerenberg*,† †
Department of Civil and Environmental Engineering and Earth Science, University of Notre Dame, 156 Fitzpatrick Hall, Notre Dame, Indiana 46556 United States ‡ Department of Biotechnology, Faculty of Applied Sciences, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands § Department of Chemical Engineering, Engineering School, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain S Supporting Information *
ABSTRACT: Wastewater treatment plants can be significant sources of nitrous oxide (N2O), a potent greenhouse gas. However, little is known about N2O emissions from biofilm processes. We adapted an existing suspended-growth mathematical model to explore N2O emissions from nitrifying biofilms. The model included N2O formation by ammoniaoxidizing bacteria (AOB) via the hydroxylamine and the nitrifier denitrification pathways. Our model suggested that N2O emissions from nitrifying biofilms could be significantly greater than from suspended growth systems under similar conditions. The main cause was the formation and diffusion of hydroxylamine, an AOB nitrification intermediate, from the aerobic to the anoxic regions of the biofilm. In the anoxic regions, hydroxylamine oxidation by AOB provided reducing equivalents used solely for nitrite reduction to N2O, since there was no competition with oxygen. For a continuous system, very high and very low dissolved oxygen (DO) concentrations resulted in lower emissions, while intermediate values led to higher emissions. Higher bulk ammonia concentrations and greater biofilm thicknesses increased emissions. The model effectively predicted N2O emissions from an actual pilot-scale granular sludge reactor for sidestream nitritation, but significantly underestimated the emissions when the NH2OH diffusion coefficient was assumed to be minimal. This numerical study suggests an unexpected and important role of hydroxylamine in N2O emission in biofilms.
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and N2O.9−12 The second is known as the hydroxylamine (NH2OH) pathway, by which NH2OH first chemically decomposes into NO and then is biologically reduced to N2O.13−15 Some research suggests that the nitrifier denitrification pathway is usually predominant, while others suggest that both pathways may be important.16,17 AOB metabolism typically consists of ammonia (NH3) conversion to NH2OH with consumption of oxygen (O2), sequential oxidation of NH2OH to NO and NO2−, and reduction of oxygen (O2) as a terminal electron acceptor (Figure 1). To explain and predict N2O emissions from nitrifying systems, researchers have developed new process models including NO and N2O formation by AOB.3,16,18−20 Detailed metabolic models have also been proposed, although these are problematic in practice because of the large number of intracellular intermediates that are difficult to measure.21,22
INTRODUCTION Wastewater treatment plants can be significant anthropogenic sources of greenhouse gases. In particular, they can emit large amounts of nitrous oxide (N2O), a potent greenhouse gas with 300 times the global-warming potential of CO2.1,2 While advances have been made in the understanding of N2O emissions from biological systems, most research has focused on suspended growth systems. However, in the wastewater field there is an increasing use of biofilm processes, such as the moving bed biofilm reactor (MBBR), integrated fixed-film activated sludge (IFAS), denitrifying filters, and granular sludge. Biofilm processes may behave differently with respect to N2O emissions because of substrate gradients and microbial stratification within the biofilm. N2O emissions can result from incomplete denitrification by heterotrophic bacteria in biological nutrient removal (BNR) systems.3−8 However, recent research has shown that ammoniaoxidizing bacteria (AOB) also can be a major source of N2O in many treatment plants, especially those with BNR. Two mechanisms have been identified for N2O formation by AOB. The first is known as the nitrifier denitrification, and consists of the sequential reduction of nitrite (NO2−) to nitric oxide (NO) © XXXX American Chemical Society
Received: September 24, 2014 Revised: December 18, 2014 Accepted: December 24, 2014
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Figure 1. Schematic of the metabolic model for AOB, adapted from Ni et al.23 Reactions: r1, NH3 oxidation to NH2OH (using reduced mediators and electrons); r2, NH2OH oxidation to NO; r3, NO oxidation to NO2−; r4, NO reduction to N2O; r5, O2 reduction; r6, NO2− reduction to N2O. Red arrows show electron-generating processes, while blue arrows indicate electron-consuming processes. Higher positioned N and O compounds reflect a higher oxidation state. Reaction stoichiometry and rates are provided in the Supporting Information, Tables S1 and S2.
Recently, Ni et al.23 developed a simplified metabolic model that explicitly included NH2OH as an intermediate in the nitrification pathway. The model lumps all reduced and oxidized intracellular electron carriers into components Mred and Mox, respectively. Monod terms for Mred and Mox are included in the rate expressions. This approach ensures a balance between electron producing and consuming processes. When electron equivalents Mred are limiting, the affinity for electron carriers determines the allocation of electrons among the competing reduction processes. Past research has addressed NO and N2O emissions from complex biofilm communities, including the impacts of AOB, nitrite-oxidizing bacteria (NOB), and heterotrophic bacteria.22,24−27 However, no previous research has systematically explored N2O emissions from a nitrifying biofilm, identified the underlying mechanisms, nor explored the effects of formation and consumption of NH2OH within the biofilm. The objectives of this study are to use numerical modeling to explore N2O emissions from AOB biofilms, using the model of Ni et al.23 as the basis, and to test the model against experimental data. We limit the model to AOB biofilms so that N2O formation mechanisms are evident. Future research should address N2O emission from more complex biofilms, where interactions among AOB, NOB, heterotrophs, anammox bacteria, and others may affect net N2O emissions from biofilms.
Rates r2 and r3 are for the sequential oxidation of NH2OH to NO and NO2−. These together provide two Mred (four e− equivalents) per mole of NH2OH oxidized to NO2−. At steady state, and assuming no net exchange of NH2OH with the bulk medium, the NH2OH oxidation rate r2 must be equal to the NH3 oxidation rate r1. Under aerobic conditions, Mox is in excess and NH2OH is the only rate-limiting variable affecting r2. Therefore, the NH2OH concentration must increase until r2 = r1. In particular, high NH2OH concentrations, relative to the NH2OH half saturation constant, would be expected for high NH3 oxidation rates. High rates are obtained when the DO and NH3 are high. Rate r4 models N2O formation via the NH2OH pathway, where NH2OH is chemically oxidized to NO, then microbially reduced to N2O. The model lumps abiotic conversion of NH2OH together with the biotic conversion. While not strictly true, it provides a small error since the maximum rates for r4 are low relative to the biotic oxidation rates (r3). Reaction r5 is the reduction of O2 as terminal electron acceptor. r5 has a higher affinity for Mred than r3 and r4, so when O2 is present, r5 outcompetes r4 and r6 (NO and NO2− reduction to N2O) for Mred. Thus, high DO will limit N2O formation because of depletion of the Mred pool via r1 and r5. Finally, r6 is the nitrifier denitrification pathway, where NO2− is reduced to form N2O using electrons from the Mred pool. This reaction rate is at its highest when Mred and NO2− are present at concentrations that are not rate-limiting. However, Mred concentrations are low in the presence of DO because of the very high oxidation rates from r5. Under anaerobic conditions and in absence of externally supplied NH2OH, Mred concentrations are also minimal because oxygen is required for NH2OH formation from NH3. Thus, under both aerobic and anoxic conditions, the rate of N2O formation via nitrifier denitrification (r6) is usually much lower than its maximum rate. The above analysis describes steady state conditions. Higher N2O emissions may result from the imbalanced rates of
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MODEL DESCRIPTION Analysis of AOB Metabolic Model. The AOB model of Ni et al.23 includes six reactions, r1−r6 (Figure 1). In r1, one mole of NH3 is oxidized to NH2OH and 1/2 mol of O2 is reduced to H2O, with the net consumption of one mole of Mred. Reaction r1 competes for O2 with reaction r5, while r1 has a much higher affinity for Mred than r5 or the other electronconsuming reactions (r4, r6). Therefore, the Mred concentration typically is not rate limiting for r1, but it can easily become rate limiting for NO, NO2−, and O2 reduction (r4, r5, and r6). B
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Figure 2. Comparison of N2O production rates, per unit reactor volume and time, as a function of bulk DO. (a) Varying biofilms thicknesses with constant total biomass; (b) varying biofilms thicknesses with constant total biofilm area.
phase and a further balance equation was solved for the gas phase concentration. As initial values, all concentrations in biofilm, bulk liquid and gas were chosen equal to the corresponding influent concentrations. The mediator concentrations in the biofilm were calculated from time-dependent balances only including the net reactions and no transport, since these compounds are immobilized in the stationary microbial phase. All model equations, process matrix, and complete list of parameters are provided in the Supporting Information (section SI 1 and Tables S1, S2 and S3). The base case conditions included a reactor hydraulic retention time of 6 h, biofilm specific surface area of 125 m2/m3, biofilm thickness 100 μm, biofilm density 50 g/L, bulk DO variable between 0.001 and 5 mg/L, and a bulk NH3 concentration of 80 mgN/L. A sensitivity analysis for the kinetic parameters in the six AOB rate expressions was carried out to determine the extent to which the N2O emissions depended on the each parameter. The model was run for N2O emissions as a function of bulk DO for the base case, while varying one parameter at a time, either increased 2-fold, or reduced by one-half. The model was implemented in COMSOL Multiphysics (v4.4, Comsol Inc., Burlington, MA). Reported steady state results were in all conditions obtained after maximum of 3 days. Reactor Setup and N2O Measurements. To evaluate the model’s ability to describe real measurements in nitritation reactors, a set of experimental data reported in literature was used. The data was taken from Pijuan and co-workers28 who characterized N2O emissions from a granular sludge reactor performing nitritation of reject water. The data was selected because the reactor was operated in continuous mode (not sequencing batch) and at steady state for different DO levels. To uncouple the potential impacts of ammonium and DO concentrations on N2O emissions, the ammonium concentration in the reactor was kept constant with a control loop based on online measurements of ammonium concentration and subsequent regulation of the inflow rate fed to the reactor. This allowed screening for a wide range of DO concentrations (i.e., 1−7.5 mgO2/L) at a fixed value of bulk ammonium concentration. Online monitoring of the N2O emissions for 3− 4 h for each of the conditions tested was used to assess the mass percentage of N2O emitted from the nitrogen oxidized. Further details related to the reactor, wastewater and measuring
NH2OH formation and consumption. NH2OH is formed as an intermediate by AOB, with the highest bulk concentrations occurring when the NH3 oxidation rate r1 is high. If the bulk NH2OH is less than the concentration needed for r2 = r1, AOB will form NH2OH faster than it can be utilized, that is, rate r1 > r2. This leads to NH2OH export to the bulk, depriving AOB of reducing equivalents. Conversely, if conditions suddenly change such that r1 < r2, the excess bulk NH2OH will be consumed at a faster rate than it is being produced, leading to a higher production rate of reducing equivalents Mred. In particular, if conditions become anoxic, O2 will no longer deplete Mred, leading to higher rates of NO2− and NO reduction via r4 and r6. This leads to a temporary spike in N2O emissions until the bulk NH2OH concentration results in r2 = r1. Transient accumulation of N2O by AOB is likely to be short-lived in conventional activated sludge, given the high concentrations of biomass. However, we argue that a similar NH2OH formation and consumption effect can occur in nitrifying biofilms under steady state conditions. This can lead to much greater total N2O emissions, compared to suspended-growth processes. Biofilm Model. The model for N2O production in biofilms is based on material balances for dissolved oxygen (O2), ammonia (NH3), hydroxylamine (NH2OH), nitrite (NO2−), nitric oxide (NO), and nitrous oxide (N2O). Time-dependent mole balances in either planar or spherical biofilms included net reaction rates for each soluble component and effective diffusion rates (50% smaller than in aqueous phase). The net component rates resulted from the process stoichiometry and kinetics of an existing AOB reaction model.23 A onedimensional stationary biofilm with fixed thickness (or radius) was assumed in all cases, without any biomass growth, attachment or detachment. AOB were considered to be uniformly distributed throughout the biofilm. Zero-flux of solutes was set at the biofilm support or granule center. The external mass transfer resistance was neglected for all solutes, that is, the concentration at the biofilm surface was equal with that in the reactor bulk liquid. With the exception of oxygen, set by aeration at a constant value, the solute concentrations in the ideally mixed bulk liquid resulted from the time-dependent balances including continuous influent and effluent, and a flux exchanged with the biofilm. For N2O, assumed to be stripped by aeration, an additional transfer term was included in the N2O balance in the liquid C
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Figure 3. Effect of the bulk dissolved oxygen concentration on N2O production, biofilm fluxes and bulk liquid concentrations. (a) N2O production rate (per L reactor volume), (b) N-compound fluxes (per m2 biofilm area), (c) NH3 and NO2− concentrations in bulk liquid, and (d) N2O, NO and NH2OH concentrations in bulk liquid. All results were obtained in the standard case conditions.
biofilms. Biofilm with “equal biomass” behaved similarly for small thicknesses (e.g., 2 and 20 μm) but differed for thicker biofilms (Figure 2a). Biofilms with “equal area” behaved similarly for large thicknesses (e.g., 100 and 200 μm) (Figure 2b). For thick biofilms, the inner portions become inactive, while the amount of active biomass close to the bulk liquid is similar. Further simulations showed that spherical biofilms with radius equal to the thickness of a planar biofilm (100 μm) display reduced N2O formation either due to deeper DO penetration into the spherical biofilm or more NH3 conversion leading to NH3 limitations (Supporting Information Figure S1a). The “equal biomass” biofilms case can roughly approximate a reactor where the granule size and numbers are varied while keeping the same total biomass amount. Indeed, results for spherical biofilms presented in Supporting Information Figure S1b show the same trends as the planar biofilms from Figure 2a. The key point is that thicker biofilms had much higher peak N2O emissions than thinner biofilms or suspended growth. Also, thicker biofilm had a broader range of DO values that resulted in high emissions. However, when the DO was sufficiently high, the N2O emissions reached the same value in the case of “equal biomass” biofilms (Figure 2a). To better explain this behavior, the next two sections explore in detail a “standard” case AOB biofilm (100 μm thickness) for two different bulk liquid DO concentrations.
strategy can be found in the Supporting Information (section SI 2).
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RESULTS We first used the model to predict N2O emissions for AOB biofilms, systematically testing the steady-state response, as a function of bulk DO, for biofilms of different thicknesses. Second, a “standard case” biofilm was analyzed in detail for two DO concentrations to reveal the underlying mechanisms leading to N2O emissions. Finally, modeled N2O emission factors were compared to those found by Pijuan and coworkers.28 Effect of Bulk DO on N2O Emissions from AOB Biofilms. The model was used to predict the effect of bulk DO on N2O emission rates per unit reactor volume, for planar biofilms. These runs were repeated for a range of biofilms thicknesses. A 2-μm thick biofilm was assumed to represent suspended growth, while the 200-μm biofilm would represent a small granule. At set different thicknesses, the model was configured to keep constant either the total biomass in the reactor by varying available biofilm area AF (Figure 2a) or keep AF constant which implicitly changes the total biomass content (Figure 2b). For the range of thicknesses tested, the N2O emission rates quickly increased with increasing DO, but plateaued at low levels for thinner biofilms and peaked at high levels for thicker D
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Figure 4. Solute concentrations and rates in the biofilm. (a) Component concentrations, (b) net component rates, (c) reaction rates, and (d) electron rates over the biofilm depth. Results are for the standard case conditions at a bulk DO of 1 mg/L. Compare these results with those obtained at DO 2 mg/L in Supporting Information Figure S4. Note that the reaction rate (c) and electron rate (d) numbering are per Figure 1 and per Supporting Information Tables S1 and S2.
Bulk Liquid Concentrations and Rates. The effects of DO on solute concentrations in bulk liquid, biofilm fluxes, and volumetric rates for the base case biofilm are provided in Figure 3. Increasing DO resulted in similar trends in N2O production (Figure 3a), flux (Figure 3b), and bulk N2O concentration (Figure 3d), all reaching a peak at 1 mg/L DO, and then decreasing to a low value above 2.5 mg/L DO. The fluxes of NH3 and NO2− reached near maximum values at around 2.5 mg/L DO, while NH2OH and NO fluxes were negligible for all DO values (Figure 3b). Supplying more DO initially increased activity within the biofilm and allowed more NH3 conversion (Figure 3c). However, further increases in DO lead to both lower bulk NH3 concentrations and greater penetration of DO into the biofilm, possibly eliminating anoxic zones. Bulk NH2OH concentrations increased almost linearly with DO and reached a peak of nearly 0.16 mg N/L at a DO of 1.3 mg/L (Figure 3d). At higher DO values, the NH2OH decreased due to the NH3 limitation on the NH3 oxidation rate r1. The influent NH3 concentration also impacted N2O emissions. Higher NH3 concentration increased N2O produc-
tion rates for the base case (Supporting Information Figure S2a). The percent of N2O formed per NH3 converted increased from less than 1% for DO values above 2 mg/L to 9% at a 0.5 mg/L, and then even more sharply to around 20% when DO approached zero (Supporting Information Figure S2b). Higher influent NH3 shifted the high percent of N2O formed toward the higher DO regions. This is explainable by the larger NH3 concentrations left in the bulk liquid due to less NH3 conversion (Supporting Information Figure S2c), which reduce the NH3 limitations within the biofilm. When the simulated bulk NH3 concentrations are controlled to a desired level by varying the flow rate Q, using the method proposed in Jemaat et al.,29 the effects of DO on N2O formation can be uncoupled from the effects of bulk NH3. When bulk NH3 is kept constant, NH3 limitation can be avoided and high N2O emissions still occur at higher DO values (Supporting Information Figure S2d). In this case, our model results for the percent of N2O formed from NH3 converted (Supporting Information Figure S2e) agree with the trends E
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Environmental Science & Technology experimentally obtained by Pijuan et al.,28 as discussed in greater detail below. Analysis of N2O Formation within the Biofilm. This section explores the mechanisms and conditions leading to the higher N2O production at DO 1 mg/L compared to 2 mg/L. For the base case conditions, with a bulk DO of 1 mg/L, the bulk NH3 and NO2− were 18 mg N/L and 55 mg N/L respectively (Figure 3c). While the DO was depleted at a biofilm depth of around 50 μm (Figure 4a), the NH3 and NO2− concentrations were relatively constant within the biofilm (x = 50 μm, Supporting Information Figure S3). The NH2OH concentration was the maximum at the outer edge of the biofilm, around 0.15 mgN/L. The net rates of formation or consumption of key N compounds and O2 are shown in Figure 4b (for clarity, the small NO rate was not displayed). In a suspended-growth system at steady state, the rate of NH3 consumption (r1) equals the rate of NH2OH oxidation (r2) and NO oxidation rate (r3). However, in a biofilm there is a gradient of NH3 oxidation rates (Figure 4c). The outer biofilm has a higher r1, which requires a higher NH2OH concentration to make r2 equal to r1. The inner biofilm, in contrast, has lower rates r1 because of DO and NH3 limitation, and requires lower NH2OH concentrations to match r2 to r1. Since r2 can exceed r1 if there is externally supplied NH2OH, the inner biofilm can provide a sink for NH2OH diffusing from the outer biofilm. The loss of NH2OH in the outer biofilm, and gain in the inner biofilm, is reflected in the positive net rates of NH2OH (formation) in the outer biofilm and negative values (consumption) in the inner biofilm (Figure 4b). At a depth of around 40 μm (x = 60 μm), the NH3 consumption rate approaches zero because of DO limitation (Figure 4b). Deeper, at 50 μm, DO also becomes rate limiting for O2 reduction as a terminal electron acceptor (r5). Since NH2OH is still available at this depth, and since O2 is not present as a sink for electrons (Figure 4a), the M red concentration increases sharply (see Supporting Information Figure S3d), increasing the rates of NO2− reduction (r6 in Figure 4c) to N2O formation (Figure 4b and Supporting Information Figure S3c). Finally, at x = 30 μm, there is almost no NH2OH left, causing the N2O formation rate to decrease significantly. A small amount of NH2OH may be exported to the bulk liquid, as the influent water does not contain any and must be in balance with the outer edge of the biofilm. However, in this case essentially all of the NH2OH diffused into the biofilm. The rates of individual processes r1−r6 are shown in Figure 4c. In the outer biofilm, r1 is higher than r2, leading to net formation of NH2OH. Interestingly, at x = 80 μm, the rate r1 of ammonia oxidation and r5 of O2 reduction are both equal to NH2OH oxidation rate r2, meaning there is a balance between NH2OH formation and demand. At this point, NO2− reduction r6 is still negligible. With increasing depth, r1 decreases more quickly than r5, allowing r6 to increase. When O2 reduction r5 approaches zero, N2O formation via NO2− (r6) peaks. Deeper, r6 decreases along with the extinction of NH2OH oxidation (rate r3). The above discussion can also be viewed in terms of electron formation and consumption rates (Figure 4d). When DO is present (outer biofilm), r1 and r5 account for essentially all the electron demand, with a negligible fraction going to NO2− and NO (r4 and r6). However, when the DO (and r1 and r5)
approaches zero, r6 gains importance and makes up for the total electron accepting rate (deeper biofilm). On the basis of this analysis, high N2O emissions can result when DO and NH3 are at high levels in the bulk, and DO is depleted within the biofilm. More specifically, N2O emissions result from a sharp gradient in the NH3 oxidation rate r1 and the existence of an anoxic zone within the biofilm. NH2OH also must be present in the anoxic region, as well as NO2−. If NH2OH does not extend beyond the aerobic zone, N2O formation will be minimal, as shown in the standard case at bulk DO 2 mg/L (Figure S4 in Supporting Information). In this case, the bulk NH3 concentration is only 3 mgN/L (from Figure 3c), leading to a smaller gradient in the rate r1 and less NH2OH formation. Also, DO penetrates almost to the base of the biofilm, so NH2OH is essentially all consumed aerobically and N2O formation is much lower. The need for a steep gradient of DO and NH3, and DO depletion within the biofilm is illustrated by decreasing the diffusivities for all compounds in the biofilm, which leads to high N2O formation rates over a large DO interval (Supporting Information Figure S5a). The NH2OH production region (high DO and NH3) is close to the anoxic region, facilitating mass transfer of NH2OH. When NH3, DO, or both are at low levels in the bulk, much lower N2O emissions will result. These results are substantiated by simulations with NH2OH diffusion is 10-times decreased (e.g., due to intracellular accumulation rather than export, as suggested by Schmidt et al.), the N2O production does not spike at low DO (Supporting Information Figure S5b). Comparison with Measured N2O Emission Factors. Model results for the conditions tested by Pijuan et al.28 were directly compared to the measured N2O emission factors, expressed as % of NH3 converted emitted as N2O (Figure 5). Bulk DO concentrations have a significant impact on N2O emission factor; higher emissions factors occur at lower DO (Figure 5). This is mainly because at lower DO concentrations the model predicts higher hydroxylamine levels in the biofilm and therefore more N2O is produced in the deeper biofilm layers. When hydroxylamine is considered to be almost
Figure 5. Comparison between measured %N2O from NH3 converted and model-calculated values in steady-state conditions. Different DO at controlled bulk 40 mg N−NH3/L, with NH2OH diffusion coefficient 50% (solid line) and 1% (dashed line) from the value in bulk liquid. The N2O emission factor was calculated as 2(QGCG,N2O + QCB,N2O)/(QG(Cin,NH3 − CB,NH3)) × 100 with parameters from Supporting Information Table S4. F
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NH2OH addition leads to faster growth rates than on NH3 alone. When supplied anoxically in the presence of NO2−, it leads to high rates of N2O formation.36−38 Our analyses were carried out for a continuous, ideally mixed reactor. For such reactors, the bulk NH3 is equal to the effluent NH3. When the NH3 loadings are low, the NH3 in the bulk may also be very low, limiting nitrifying activity and N2O emissions. This can be seen in Supporting Information Figure S2a, which explores the effects of influent NH3 concentration on N2O emissions for a range of DO values. In a sequencing batch reactor, even if the overall loadings are low, there are likely to be high NH3 concentrations at the beginning of the cycle. Therefore, SBRs are likely to have higher N2O emissions than continuous reactors treating the same influent.28 NH2OH may also be formed mixotrophically. Research has found that NH2OH can be produced anaerobically in the presence of formate.32 Future models should consider other potential NH2OH formation pathways, as it could be formed and consumed under anoxic conditions. This could have implications for nitrification in the presence of organic matter. The results found in this research are not strictly dependent on the model of Ni et al.,23 where reduced and oxidized carriers are explicitly modeled. It is sufficient for models to include NH2OH as an intermediate, and to have NH2OH-based denitrification inhibition by oxygen. Thus, maximum N2O formation rates are obtained in the presence of NH2OH and absence of oxygen. The continued presence of AOB in anoxic regions of biofilm may result from a variety of scenarios. While AOB are known to grow on NH2OH under aerobic conditions, the ability of AOB to support maintenance or even growth on NO2− has been researched and appears to be inconclusive.15,39−42 However, even if AOB cannot subsist without O2, it still is possible at the base of a biofilm to harbor AOB due to fluctuation in DO or to biofilm detachment and regrowth. Residual ammonium has been used to enhance and maintain nitritation in granular sludge reactors.28,43,44 Theoretical analysis of the effects of ammonium building-up on NOB repression focused on the advantage of a higher nitration rate,29,45 also when analyzing single stage partial nitritation/ anammox granular reactors.46 However, our study indicates that ammonium accumulation in the bulk would imply diffusion of hydroxylamine toward deeper biofilm layers. This could have implications for NOB repression. First, AOB will be able to occupy an extended biofilm region because of their capacity to oxidize hydroxylamine.36,42 Second, basal levels of hydroxylamine may inhibit selectively NOB.31,32,47−49 While these effects may help obtaining NOB repression, the hydroxylamine diffusion will also trigger N2O emissions. However, there is still a window for operational conditions in which NOB repression can be achieved while N2O emissions are minimal for DO > 3.5 mg O2/L.28 Further investigation will be required to fully understand the role of residual ammonium on NOB repression in biofilm reactors performing either nitritation or single-stage partial nitritation/anammox. In conclusion, nitrifying biofilms have distinct behavior from suspended systems and can be much more significant sources of N2O under similar conditions. Most existing models do not explicitly include NH2OH as a component, which is critical in a biofilm system. The best means to minimize N2O emission from biofilm systems are similar to those of suspended growth systems: maintain low ammonia oxidation rates. Another approach specific to biofilms would be to minimize the
nondiffusive (i.e., 1% of diffusivity in water), the model does not represent well the experimental data (Figure 5). In batch cultures of planktonic Nitrosomonas europaea, hydroxylamine leaking to the bulk liquid has been reported associated with the exponential growth phase, with NH3 in excess and DO in the range 0.5−1.5 mg/L.30 The general agreement between model predictions and experimental data strengthen the hypothesis that nitrifier denitrification in biofilms is triggered by hydroxylamine leaking out of the cells when ammonium is supplied in excess, followed by diffusion through the biofilm and its further oxidation in the anoxic zone. Therefore, even in well-aerated biofilm reactors with excess ammonium, the limited oxygen penetration can lead to a predominant role of the nitrifier denitrification pathway in the biofilm. This is in contrast to what happens with activated sludge or cells in suspension, where there is little nitrifier denitrification in aerobic conditions.6 Sensitivity Analyses. N2O emissions were generally sensitive to the kinetic parameters in reactions 1 and 2 (Supporting Information Figure S6a, b). Reactions r3 and r4, involving NO oxidation, were largely unaffected by changes in parameters (Supporting Information Figure S6c, d). The rate for r3 matched the NH2OH oxidation rate, which is consistent with the need to prevent the buildup to toxic NO. Reaction r4, NO transformation to N2O, has a very low maximum specific rate, so changes in the parameters had a small effect on N2O emissions. Reaction r5 and r6, for O2 and NO2− reduction, were only moderately sensitive to changes in kinetic parameters (Supporting Information Figure S6e, f). O2 reduction was more sensitive than the NO2− reduction, as NO2− is always present at concentrations that are not rate-limiting. Thus, the results obtained in this research are unlikely to be unique to the specific parameter values used in the simulations. All model results and conclusions strongly depend on NH2OH formation, consumption, and diffusion rates. While these parameters were adequate for our research on mechanisms and described well the data from Pijuan et al.,28 they should still be validated on more experimental data sets in order to obtain a truly predictive model.
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DISCUSSION On the basis of our modeling results, nitrifying biofilms may produce significantly more N2O than suspended growth systems under similar conditions. A key difference between suspended and biofilm processes is that in a biofilm, NH2OH can diffuse from areas with high nitrification activity to zones with lower activity, which act as a sink for NH2OH. When NH2OH penetrates into anoxic zones the Mred concentrations peak and lead to high rates of N2O formation. This can be compounded by the presence of NOB, which increases the slope of the DO gradient. Our research takes well-established principles for AOB metabolism in suspended-growth systems and applies them to a biofilm. Important premises are that (1) NH2OH is formed as an intermediate during NH3 oxidation and can diffuse into the environment, and (2) NH2OH can be used by AOB as an external substrate. It is well established that NH2OH can “leak” from AOB cells.13,15,30−35 Also, the NH2OH concentrations do not need to be high to have a stimulatory effect on N2O formation in biofilms. They only need to be high relative to their half saturation constant. It also is well established that AOB can consume NH2OH as an externally supplied substrate. G
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wastewater treatment process. Water Sci. Technol. 2004, 49 (5−6), 359−365. (6) Rassamee, V.; Sattayatewa, C.; Pagilla, K.; Chandran, K. Effect of oxic and anoxic conditions on nitrous oxide emissions from nitrification and denitrification processes. Biotechnol. Bioeng. 2011, 108 (9), 2036−2045. (7) Tallec, G.; Garnier, J.; Billen, G.; Gousailles, M. Nitrous oxide emissions from denitrifying activated sludge of urban wastewater treatment plants, under anoxia and low oxygenation. Bioresour. Technol. 2008, 99 (7), 2200−2209. (8) Desloover, J.; Vlaeminck, S. E.; Clauwaert, P.; Verstraete, W.; Boon, N. Strategies to mitigate N2O emissions from biological nitrogen removal systems. Curr. Opin. Biotechnol. 2012, 23 (3), 474− 482. (9) Kim, S.; Miyahara, M.; Fushinobu, S.; Wakagi, T.; Shoun, H. Nitrous oxide emission from nitrifying activated sludge dependent on denitrification by ammonia-oxidizing bacteria. Bioresour. Technol. 2010, 101 (11), 3958−3963. (10) Yu, R.; Kampschreur, M. J.; van Loosdrecht, M. C. M.; Chandran, K. Mechanisms and specific directionality of autotrophic nitrous oxide and nitric oxide generation during transient anoxia. Environ. Sci. Technol. 2010, 44 (4), 1313−1319. (11) Chandran, K.; Stein, L. Y.; Klotz, M. G.; van Loosdrecht, M. C. M. Nitrous oxide production by lithotrophic ammonia-oxidizing bacteria and implications for engineered nitrogen-removal systems. Biochem. Soc. Trans. 2011, 39, 1832−1837. (12) Wrage, N.; Velthof, G.; van Beusichem, M.; Oenema, O. Role of nitrifier denitrification in the production of nitrous oxide. Soil Biol. Biochem. 2001, 33 (12−13), 1723−1732. (13) Stein, L. Y. Surveying N2O-producing pathways in bacteria. Methods Enzymol. 2011, 486, 131−152. (14) Arp, D. J.; Stein, L. Y. Metabolism of inorganic N compounds by ammonia-oxidizing bacteria. Crit. Rev. Biochem. Mol. Biol. 2003, 38 (6), 471−495. (15) Wunderlin, P.; Mohn, J.; Joss, A.; Emmenegger, L.; Siegrist, H. Mechanisms of N2O production in biological wastewater treatment under nitrifying and denitrifying conditions. Water Res. 2012, 46 (4), 1027−1037. (16) Ni, B.; Yuan, Z.; Chandran, K.; Vanrolleghem, P. A.; Murthy, S. Evaluating four mathematical models for nitrous oxide production by autotrophic ammonia-oxidizing bacteria. Biotechnol. Bioeng. 2013, 110 (1), 153−163. (17) Ishii, S.; Song, Y.; Rathnayake, L.; Tumendelger, A.; Satoh, H.; Toyoda, S.; Yoshida, N.; Okabe, S. Identification of key nitrous oxide production pathways in aerobic partial nitrifying granules. Environ. Microbiol. 2014, in press. (18) Kampschreur, M. J.; Picioreanu, C.; Tan, N.; Kleerebezem, R.; Jetten, M. S. M.; van Loosdrecht, M. C. M. Unraveling the source of nitric oxide emission during nitrification. Water Environ. Res. 2007, 79 (13), 2499−2509. (19) Ni, B.; Ruscalleda, M.; Pellicer-Nacher, C.; Smets, B. F. Modeling nitrous oxide production during biological nitrogen removal via nitrification and denitrification: extensions to the general ASM models. Environ. Sci. Technol. 2011, 45 (18), 7768−7776. (20) Mampaey, K. E.; Beuckels, B.; Kampschreur, M. J.; Kleerebezem, R.; van Loosdrecht, M. C. M.; Volcke, E. I. P. Modelling nitrous and nitric oxide emissions by autotrophic ammonia-oxidizing bacteria. Environ. Technol. 2013, 34 (12), 1555−1566. (21) Perez-Garcia, O.; Villas-Boas, S. G.; Swift, S.; Chandran, K.; Singhal, N. Clarifying the regulation of NO/N2O production in Nitrosomonas europaea during anoxic−oxic transition via flux balance analysis of a metabolic network model. Water Res. 2014, 60, 267−277. (22) Schreiber, F.; Loeffler, B.; Polerecky, L.; Kuypers, M. M. M.; de Beer, D. Mechanisms of transient nitric oxide and nitrous oxide production in a complex biofilm. ISME J. 2009, 3 (11), 1301−1313. (23) Ni, B.; Peng, L.; Law, Y.; Guo, J.; Yuan, Z. Modeling of nitrous oxide production by autotrophic ammonia-oxidizing bacteria with multiple production pathways. Environ. Sci. Technol. 2014, 48 (7), 3916−3924.
gradient of NH3 oxidation rates and to avoid the formation of anoxic zones in the biofilm, potentially by maintaining high bulk DO values or keeping the biofilms thin.
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ASSOCIATED CONTENT
S Supporting Information *
Numerical model, experimental setup, stoichiometry matrix of the reaction model, reaction rates for the model of Ni et al.,23 model parameters in the base case, model parameters changed for comparison with experimental data from Pijuan et al.,28 comparison of N2O production rates, per unit reactor volume and time, as a function of bulk DO for planar and spherical biofilms of different thicknesses, effect of the set dissolved oxygen concentration at different influent ammonia concentrations, net rate of N2O production determined by the availability of reduced mediators in the biofilm, solute concentrations and rates in the biofilm at a bulk DO of 2 mg/L, effect of effective diffusivity on the N2O production rates at different dissolved oxygen concentrations, sensitivity analysis for the kinetic parameters in the AOB rate expressions r1 (a) and r2 (b), sensitivity analyses for the kinetic parameters in the AOB rate expressions r3 (c) and r4 (d), and sensitivity analysis for the kinetic parameters in the AOB rate expressions r5 (e) and r6 (f). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +1 574 631 4098. Fax: +1 574 631 9236. E-mail: [email protected]. Funding
F.S. and R.N. were supported by NSF project CBET0954918 (Nerenberg CAREER award) and WERF project U2R10. F.S. received additional support from the Bayer Corporation Fellowship. CP work was supported by a Melchor Visiting Professor grant from University of Notre Dame. JP work was supported a Marie Curie Intra European Fellowship (GreenN2, PIEF-GA-2012-326705). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge useful discussions in the manuscript preparation stage with Barth Smets and Holger Daims. REFERENCES
(1) Ahn, J. H.; Kim, S.; Park, H.; Rahm, B.; Pagilla, K.; Chandran, K. N2O Emissions from activated sludge processes, 2008−2009: Results of a national monitoring survey in the United States. Environ. Sci. Technol. 2010, 44 (12), 4505−4511. (2) IPCC. Wastewater Treatment and Discharge, 2006 IPCC Guidelines for National Greenhouse Gas Inventories, vol. 5; IPCC: Geneva, 2006. (3) Kampschreur, M. J.; van der Star, W. R. L.; Wielders, H. A.; Mulder, J. W.; Jetten, M. S. M.; van Loosdrecht, M. C. M. Dynamics of nitric oxide and nitrous oxide emission during full-scale reject water treatment. Water Res. 2008, 42 (3), 812−826. (4) Lu, H.; Chandran, K. Factors promoting emissions of nitrous oxide and nitric oxide From denitrifying Sequencing Batch Reactors operated with methanol and ethanol as electron donors. Biotechnol. Bioeng. 2010, 106 (3), 390−398. (5) Kishida, N.; Kim, J.; Kimochi, Y.; Nishimura, O.; Sasaki, H.; Sudo, R. Effect of C/N ratio on nitrous oxide emission from swine H
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in biofilm systems using biofilm modeling and Monte Carlo filtering. Water Res. 2010, 44 (6), 1995−2009. (46) Perez, J.; Lotti, T.; Kleerebezem, R.; Picioreanu, C.; van Loosdrecht, M. C. M. Outcompeting nitrite-oxidizing bacteria in single-stage nitrogen removal in sewage treatment plants: A modelbased study. Water Res. 2014, 66, 208−218. (47) Noophan, P.; Figueroa, L.; Munakata-Marr, J. Nitrite oxidation inhibition by hydroxylamine: Experimental and model evaluation. Water Sci. Technol. 2004, 50 (6), 295−304. (48) Kindaichi, T.; Okabe, S.; Satoh, H.; Watanabe, Y. Effects of hydroxylamine on microbial community structure and function of autotrophic nitrifying biofilms determined by in situ hybridization and the use of microelectrodes. Water Sci. Technol. 2004, 49 (11−12), 61− 68. (49) Harper, W. F., Jr.; Terada, A.; Poly, F.; Le Roux, X.; Kristensen, K.; Mazher, M.; Smets, B. F. The effect of hydroxylamine on the activity and aggregate structure of autotrophic nitrifying bioreactor cultures. Biotechnol. Bioeng. 2009, 102 (3), 714−724.
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Hydroxylamine Diffusion Can Enhance N2O Emissions in Nitrifying Biofilms: A Modeling Study Fabrizio Sabba,† Cristian Picioreanu,‡ Julio Pérez,‡,§ and Robert Nerenberg*,† †
Department of Civil and Environmental Engineering and Earth Science, University of Notre Dame, 156 Fitzpatrick Hall, Notre Dame, Indiana 46556 United States ‡ Department of Biotechnology, Faculty of Applied Sciences, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands § Department of Chemical Engineering, Engineering School, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain S Supporting Information *
ABSTRACT: Wastewater treatment plants can be significant sources of nitrous oxide (N2O), a potent greenhouse gas. However, little is known about N2O emissions from biofilm processes. We adapted an existing suspended-growth mathematical model to explore N2O emissions from nitrifying biofilms. The model included N2O formation by ammoniaoxidizing bacteria (AOB) via the hydroxylamine and the nitrifier denitrification pathways. Our model suggested that N2O emissions from nitrifying biofilms could be significantly greater than from suspended growth systems under similar conditions. The main cause was the formation and diffusion of hydroxylamine, an AOB nitrification intermediate, from the aerobic to the anoxic regions of the biofilm. In the anoxic regions, hydroxylamine oxidation by AOB provided reducing equivalents used solely for nitrite reduction to N2O, since there was no competition with oxygen. For a continuous system, very high and very low dissolved oxygen (DO) concentrations resulted in lower emissions, while intermediate values led to higher emissions. Higher bulk ammonia concentrations and greater biofilm thicknesses increased emissions. The model effectively predicted N2O emissions from an actual pilot-scale granular sludge reactor for sidestream nitritation, but significantly underestimated the emissions when the NH2OH diffusion coefficient was assumed to be minimal. This numerical study suggests an unexpected and important role of hydroxylamine in N2O emission in biofilms.
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and N2O.9−12 The second is known as the hydroxylamine (NH2OH) pathway, by which NH2OH first chemically decomposes into NO and then is biologically reduced to N2O.13−15 Some research suggests that the nitrifier denitrification pathway is usually predominant, while others suggest that both pathways may be important.16,17 AOB metabolism typically consists of ammonia (NH3) conversion to NH2OH with consumption of oxygen (O2), sequential oxidation of NH2OH to NO and NO2−, and reduction of oxygen (O2) as a terminal electron acceptor (Figure 1). To explain and predict N2O emissions from nitrifying systems, researchers have developed new process models including NO and N2O formation by AOB.3,16,18−20 Detailed metabolic models have also been proposed, although these are problematic in practice because of the large number of intracellular intermediates that are difficult to measure.21,22
INTRODUCTION Wastewater treatment plants can be significant anthropogenic sources of greenhouse gases. In particular, they can emit large amounts of nitrous oxide (N2O), a potent greenhouse gas with 300 times the global-warming potential of CO2.1,2 While advances have been made in the understanding of N2O emissions from biological systems, most research has focused on suspended growth systems. However, in the wastewater field there is an increasing use of biofilm processes, such as the moving bed biofilm reactor (MBBR), integrated fixed-film activated sludge (IFAS), denitrifying filters, and granular sludge. Biofilm processes may behave differently with respect to N2O emissions because of substrate gradients and microbial stratification within the biofilm. N2O emissions can result from incomplete denitrification by heterotrophic bacteria in biological nutrient removal (BNR) systems.3−8 However, recent research has shown that ammoniaoxidizing bacteria (AOB) also can be a major source of N2O in many treatment plants, especially those with BNR. Two mechanisms have been identified for N2O formation by AOB. The first is known as the nitrifier denitrification, and consists of the sequential reduction of nitrite (NO2−) to nitric oxide (NO) © XXXX American Chemical Society
Received: September 24, 2014 Revised: December 18, 2014 Accepted: December 24, 2014
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Figure 1. Schematic of the metabolic model for AOB, adapted from Ni et al.23 Reactions: r1, NH3 oxidation to NH2OH (using reduced mediators and electrons); r2, NH2OH oxidation to NO; r3, NO oxidation to NO2−; r4, NO reduction to N2O; r5, O2 reduction; r6, NO2− reduction to N2O. Red arrows show electron-generating processes, while blue arrows indicate electron-consuming processes. Higher positioned N and O compounds reflect a higher oxidation state. Reaction stoichiometry and rates are provided in the Supporting Information, Tables S1 and S2.
Recently, Ni et al.23 developed a simplified metabolic model that explicitly included NH2OH as an intermediate in the nitrification pathway. The model lumps all reduced and oxidized intracellular electron carriers into components Mred and Mox, respectively. Monod terms for Mred and Mox are included in the rate expressions. This approach ensures a balance between electron producing and consuming processes. When electron equivalents Mred are limiting, the affinity for electron carriers determines the allocation of electrons among the competing reduction processes. Past research has addressed NO and N2O emissions from complex biofilm communities, including the impacts of AOB, nitrite-oxidizing bacteria (NOB), and heterotrophic bacteria.22,24−27 However, no previous research has systematically explored N2O emissions from a nitrifying biofilm, identified the underlying mechanisms, nor explored the effects of formation and consumption of NH2OH within the biofilm. The objectives of this study are to use numerical modeling to explore N2O emissions from AOB biofilms, using the model of Ni et al.23 as the basis, and to test the model against experimental data. We limit the model to AOB biofilms so that N2O formation mechanisms are evident. Future research should address N2O emission from more complex biofilms, where interactions among AOB, NOB, heterotrophs, anammox bacteria, and others may affect net N2O emissions from biofilms.
Rates r2 and r3 are for the sequential oxidation of NH2OH to NO and NO2−. These together provide two Mred (four e− equivalents) per mole of NH2OH oxidized to NO2−. At steady state, and assuming no net exchange of NH2OH with the bulk medium, the NH2OH oxidation rate r2 must be equal to the NH3 oxidation rate r1. Under aerobic conditions, Mox is in excess and NH2OH is the only rate-limiting variable affecting r2. Therefore, the NH2OH concentration must increase until r2 = r1. In particular, high NH2OH concentrations, relative to the NH2OH half saturation constant, would be expected for high NH3 oxidation rates. High rates are obtained when the DO and NH3 are high. Rate r4 models N2O formation via the NH2OH pathway, where NH2OH is chemically oxidized to NO, then microbially reduced to N2O. The model lumps abiotic conversion of NH2OH together with the biotic conversion. While not strictly true, it provides a small error since the maximum rates for r4 are low relative to the biotic oxidation rates (r3). Reaction r5 is the reduction of O2 as terminal electron acceptor. r5 has a higher affinity for Mred than r3 and r4, so when O2 is present, r5 outcompetes r4 and r6 (NO and NO2− reduction to N2O) for Mred. Thus, high DO will limit N2O formation because of depletion of the Mred pool via r1 and r5. Finally, r6 is the nitrifier denitrification pathway, where NO2− is reduced to form N2O using electrons from the Mred pool. This reaction rate is at its highest when Mred and NO2− are present at concentrations that are not rate-limiting. However, Mred concentrations are low in the presence of DO because of the very high oxidation rates from r5. Under anaerobic conditions and in absence of externally supplied NH2OH, Mred concentrations are also minimal because oxygen is required for NH2OH formation from NH3. Thus, under both aerobic and anoxic conditions, the rate of N2O formation via nitrifier denitrification (r6) is usually much lower than its maximum rate. The above analysis describes steady state conditions. Higher N2O emissions may result from the imbalanced rates of
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MODEL DESCRIPTION Analysis of AOB Metabolic Model. The AOB model of Ni et al.23 includes six reactions, r1−r6 (Figure 1). In r1, one mole of NH3 is oxidized to NH2OH and 1/2 mol of O2 is reduced to H2O, with the net consumption of one mole of Mred. Reaction r1 competes for O2 with reaction r5, while r1 has a much higher affinity for Mred than r5 or the other electronconsuming reactions (r4, r6). Therefore, the Mred concentration typically is not rate limiting for r1, but it can easily become rate limiting for NO, NO2−, and O2 reduction (r4, r5, and r6). B
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Figure 2. Comparison of N2O production rates, per unit reactor volume and time, as a function of bulk DO. (a) Varying biofilms thicknesses with constant total biomass; (b) varying biofilms thicknesses with constant total biofilm area.
phase and a further balance equation was solved for the gas phase concentration. As initial values, all concentrations in biofilm, bulk liquid and gas were chosen equal to the corresponding influent concentrations. The mediator concentrations in the biofilm were calculated from time-dependent balances only including the net reactions and no transport, since these compounds are immobilized in the stationary microbial phase. All model equations, process matrix, and complete list of parameters are provided in the Supporting Information (section SI 1 and Tables S1, S2 and S3). The base case conditions included a reactor hydraulic retention time of 6 h, biofilm specific surface area of 125 m2/m3, biofilm thickness 100 μm, biofilm density 50 g/L, bulk DO variable between 0.001 and 5 mg/L, and a bulk NH3 concentration of 80 mgN/L. A sensitivity analysis for the kinetic parameters in the six AOB rate expressions was carried out to determine the extent to which the N2O emissions depended on the each parameter. The model was run for N2O emissions as a function of bulk DO for the base case, while varying one parameter at a time, either increased 2-fold, or reduced by one-half. The model was implemented in COMSOL Multiphysics (v4.4, Comsol Inc., Burlington, MA). Reported steady state results were in all conditions obtained after maximum of 3 days. Reactor Setup and N2O Measurements. To evaluate the model’s ability to describe real measurements in nitritation reactors, a set of experimental data reported in literature was used. The data was taken from Pijuan and co-workers28 who characterized N2O emissions from a granular sludge reactor performing nitritation of reject water. The data was selected because the reactor was operated in continuous mode (not sequencing batch) and at steady state for different DO levels. To uncouple the potential impacts of ammonium and DO concentrations on N2O emissions, the ammonium concentration in the reactor was kept constant with a control loop based on online measurements of ammonium concentration and subsequent regulation of the inflow rate fed to the reactor. This allowed screening for a wide range of DO concentrations (i.e., 1−7.5 mgO2/L) at a fixed value of bulk ammonium concentration. Online monitoring of the N2O emissions for 3− 4 h for each of the conditions tested was used to assess the mass percentage of N2O emitted from the nitrogen oxidized. Further details related to the reactor, wastewater and measuring
NH2OH formation and consumption. NH2OH is formed as an intermediate by AOB, with the highest bulk concentrations occurring when the NH3 oxidation rate r1 is high. If the bulk NH2OH is less than the concentration needed for r2 = r1, AOB will form NH2OH faster than it can be utilized, that is, rate r1 > r2. This leads to NH2OH export to the bulk, depriving AOB of reducing equivalents. Conversely, if conditions suddenly change such that r1 < r2, the excess bulk NH2OH will be consumed at a faster rate than it is being produced, leading to a higher production rate of reducing equivalents Mred. In particular, if conditions become anoxic, O2 will no longer deplete Mred, leading to higher rates of NO2− and NO reduction via r4 and r6. This leads to a temporary spike in N2O emissions until the bulk NH2OH concentration results in r2 = r1. Transient accumulation of N2O by AOB is likely to be short-lived in conventional activated sludge, given the high concentrations of biomass. However, we argue that a similar NH2OH formation and consumption effect can occur in nitrifying biofilms under steady state conditions. This can lead to much greater total N2O emissions, compared to suspended-growth processes. Biofilm Model. The model for N2O production in biofilms is based on material balances for dissolved oxygen (O2), ammonia (NH3), hydroxylamine (NH2OH), nitrite (NO2−), nitric oxide (NO), and nitrous oxide (N2O). Time-dependent mole balances in either planar or spherical biofilms included net reaction rates for each soluble component and effective diffusion rates (50% smaller than in aqueous phase). The net component rates resulted from the process stoichiometry and kinetics of an existing AOB reaction model.23 A onedimensional stationary biofilm with fixed thickness (or radius) was assumed in all cases, without any biomass growth, attachment or detachment. AOB were considered to be uniformly distributed throughout the biofilm. Zero-flux of solutes was set at the biofilm support or granule center. The external mass transfer resistance was neglected for all solutes, that is, the concentration at the biofilm surface was equal with that in the reactor bulk liquid. With the exception of oxygen, set by aeration at a constant value, the solute concentrations in the ideally mixed bulk liquid resulted from the time-dependent balances including continuous influent and effluent, and a flux exchanged with the biofilm. For N2O, assumed to be stripped by aeration, an additional transfer term was included in the N2O balance in the liquid C
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Figure 3. Effect of the bulk dissolved oxygen concentration on N2O production, biofilm fluxes and bulk liquid concentrations. (a) N2O production rate (per L reactor volume), (b) N-compound fluxes (per m2 biofilm area), (c) NH3 and NO2− concentrations in bulk liquid, and (d) N2O, NO and NH2OH concentrations in bulk liquid. All results were obtained in the standard case conditions.
biofilms. Biofilm with “equal biomass” behaved similarly for small thicknesses (e.g., 2 and 20 μm) but differed for thicker biofilms (Figure 2a). Biofilms with “equal area” behaved similarly for large thicknesses (e.g., 100 and 200 μm) (Figure 2b). For thick biofilms, the inner portions become inactive, while the amount of active biomass close to the bulk liquid is similar. Further simulations showed that spherical biofilms with radius equal to the thickness of a planar biofilm (100 μm) display reduced N2O formation either due to deeper DO penetration into the spherical biofilm or more NH3 conversion leading to NH3 limitations (Supporting Information Figure S1a). The “equal biomass” biofilms case can roughly approximate a reactor where the granule size and numbers are varied while keeping the same total biomass amount. Indeed, results for spherical biofilms presented in Supporting Information Figure S1b show the same trends as the planar biofilms from Figure 2a. The key point is that thicker biofilms had much higher peak N2O emissions than thinner biofilms or suspended growth. Also, thicker biofilm had a broader range of DO values that resulted in high emissions. However, when the DO was sufficiently high, the N2O emissions reached the same value in the case of “equal biomass” biofilms (Figure 2a). To better explain this behavior, the next two sections explore in detail a “standard” case AOB biofilm (100 μm thickness) for two different bulk liquid DO concentrations.
strategy can be found in the Supporting Information (section SI 2).
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RESULTS We first used the model to predict N2O emissions for AOB biofilms, systematically testing the steady-state response, as a function of bulk DO, for biofilms of different thicknesses. Second, a “standard case” biofilm was analyzed in detail for two DO concentrations to reveal the underlying mechanisms leading to N2O emissions. Finally, modeled N2O emission factors were compared to those found by Pijuan and coworkers.28 Effect of Bulk DO on N2O Emissions from AOB Biofilms. The model was used to predict the effect of bulk DO on N2O emission rates per unit reactor volume, for planar biofilms. These runs were repeated for a range of biofilms thicknesses. A 2-μm thick biofilm was assumed to represent suspended growth, while the 200-μm biofilm would represent a small granule. At set different thicknesses, the model was configured to keep constant either the total biomass in the reactor by varying available biofilm area AF (Figure 2a) or keep AF constant which implicitly changes the total biomass content (Figure 2b). For the range of thicknesses tested, the N2O emission rates quickly increased with increasing DO, but plateaued at low levels for thinner biofilms and peaked at high levels for thicker D
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Figure 4. Solute concentrations and rates in the biofilm. (a) Component concentrations, (b) net component rates, (c) reaction rates, and (d) electron rates over the biofilm depth. Results are for the standard case conditions at a bulk DO of 1 mg/L. Compare these results with those obtained at DO 2 mg/L in Supporting Information Figure S4. Note that the reaction rate (c) and electron rate (d) numbering are per Figure 1 and per Supporting Information Tables S1 and S2.
Bulk Liquid Concentrations and Rates. The effects of DO on solute concentrations in bulk liquid, biofilm fluxes, and volumetric rates for the base case biofilm are provided in Figure 3. Increasing DO resulted in similar trends in N2O production (Figure 3a), flux (Figure 3b), and bulk N2O concentration (Figure 3d), all reaching a peak at 1 mg/L DO, and then decreasing to a low value above 2.5 mg/L DO. The fluxes of NH3 and NO2− reached near maximum values at around 2.5 mg/L DO, while NH2OH and NO fluxes were negligible for all DO values (Figure 3b). Supplying more DO initially increased activity within the biofilm and allowed more NH3 conversion (Figure 3c). However, further increases in DO lead to both lower bulk NH3 concentrations and greater penetration of DO into the biofilm, possibly eliminating anoxic zones. Bulk NH2OH concentrations increased almost linearly with DO and reached a peak of nearly 0.16 mg N/L at a DO of 1.3 mg/L (Figure 3d). At higher DO values, the NH2OH decreased due to the NH3 limitation on the NH3 oxidation rate r1. The influent NH3 concentration also impacted N2O emissions. Higher NH3 concentration increased N2O produc-
tion rates for the base case (Supporting Information Figure S2a). The percent of N2O formed per NH3 converted increased from less than 1% for DO values above 2 mg/L to 9% at a 0.5 mg/L, and then even more sharply to around 20% when DO approached zero (Supporting Information Figure S2b). Higher influent NH3 shifted the high percent of N2O formed toward the higher DO regions. This is explainable by the larger NH3 concentrations left in the bulk liquid due to less NH3 conversion (Supporting Information Figure S2c), which reduce the NH3 limitations within the biofilm. When the simulated bulk NH3 concentrations are controlled to a desired level by varying the flow rate Q, using the method proposed in Jemaat et al.,29 the effects of DO on N2O formation can be uncoupled from the effects of bulk NH3. When bulk NH3 is kept constant, NH3 limitation can be avoided and high N2O emissions still occur at higher DO values (Supporting Information Figure S2d). In this case, our model results for the percent of N2O formed from NH3 converted (Supporting Information Figure S2e) agree with the trends E
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Environmental Science & Technology experimentally obtained by Pijuan et al.,28 as discussed in greater detail below. Analysis of N2O Formation within the Biofilm. This section explores the mechanisms and conditions leading to the higher N2O production at DO 1 mg/L compared to 2 mg/L. For the base case conditions, with a bulk DO of 1 mg/L, the bulk NH3 and NO2− were 18 mg N/L and 55 mg N/L respectively (Figure 3c). While the DO was depleted at a biofilm depth of around 50 μm (Figure 4a), the NH3 and NO2− concentrations were relatively constant within the biofilm (x = 50 μm, Supporting Information Figure S3). The NH2OH concentration was the maximum at the outer edge of the biofilm, around 0.15 mgN/L. The net rates of formation or consumption of key N compounds and O2 are shown in Figure 4b (for clarity, the small NO rate was not displayed). In a suspended-growth system at steady state, the rate of NH3 consumption (r1) equals the rate of NH2OH oxidation (r2) and NO oxidation rate (r3). However, in a biofilm there is a gradient of NH3 oxidation rates (Figure 4c). The outer biofilm has a higher r1, which requires a higher NH2OH concentration to make r2 equal to r1. The inner biofilm, in contrast, has lower rates r1 because of DO and NH3 limitation, and requires lower NH2OH concentrations to match r2 to r1. Since r2 can exceed r1 if there is externally supplied NH2OH, the inner biofilm can provide a sink for NH2OH diffusing from the outer biofilm. The loss of NH2OH in the outer biofilm, and gain in the inner biofilm, is reflected in the positive net rates of NH2OH (formation) in the outer biofilm and negative values (consumption) in the inner biofilm (Figure 4b). At a depth of around 40 μm (x = 60 μm), the NH3 consumption rate approaches zero because of DO limitation (Figure 4b). Deeper, at 50 μm, DO also becomes rate limiting for O2 reduction as a terminal electron acceptor (r5). Since NH2OH is still available at this depth, and since O2 is not present as a sink for electrons (Figure 4a), the M red concentration increases sharply (see Supporting Information Figure S3d), increasing the rates of NO2− reduction (r6 in Figure 4c) to N2O formation (Figure 4b and Supporting Information Figure S3c). Finally, at x = 30 μm, there is almost no NH2OH left, causing the N2O formation rate to decrease significantly. A small amount of NH2OH may be exported to the bulk liquid, as the influent water does not contain any and must be in balance with the outer edge of the biofilm. However, in this case essentially all of the NH2OH diffused into the biofilm. The rates of individual processes r1−r6 are shown in Figure 4c. In the outer biofilm, r1 is higher than r2, leading to net formation of NH2OH. Interestingly, at x = 80 μm, the rate r1 of ammonia oxidation and r5 of O2 reduction are both equal to NH2OH oxidation rate r2, meaning there is a balance between NH2OH formation and demand. At this point, NO2− reduction r6 is still negligible. With increasing depth, r1 decreases more quickly than r5, allowing r6 to increase. When O2 reduction r5 approaches zero, N2O formation via NO2− (r6) peaks. Deeper, r6 decreases along with the extinction of NH2OH oxidation (rate r3). The above discussion can also be viewed in terms of electron formation and consumption rates (Figure 4d). When DO is present (outer biofilm), r1 and r5 account for essentially all the electron demand, with a negligible fraction going to NO2− and NO (r4 and r6). However, when the DO (and r1 and r5)
approaches zero, r6 gains importance and makes up for the total electron accepting rate (deeper biofilm). On the basis of this analysis, high N2O emissions can result when DO and NH3 are at high levels in the bulk, and DO is depleted within the biofilm. More specifically, N2O emissions result from a sharp gradient in the NH3 oxidation rate r1 and the existence of an anoxic zone within the biofilm. NH2OH also must be present in the anoxic region, as well as NO2−. If NH2OH does not extend beyond the aerobic zone, N2O formation will be minimal, as shown in the standard case at bulk DO 2 mg/L (Figure S4 in Supporting Information). In this case, the bulk NH3 concentration is only 3 mgN/L (from Figure 3c), leading to a smaller gradient in the rate r1 and less NH2OH formation. Also, DO penetrates almost to the base of the biofilm, so NH2OH is essentially all consumed aerobically and N2O formation is much lower. The need for a steep gradient of DO and NH3, and DO depletion within the biofilm is illustrated by decreasing the diffusivities for all compounds in the biofilm, which leads to high N2O formation rates over a large DO interval (Supporting Information Figure S5a). The NH2OH production region (high DO and NH3) is close to the anoxic region, facilitating mass transfer of NH2OH. When NH3, DO, or both are at low levels in the bulk, much lower N2O emissions will result. These results are substantiated by simulations with NH2OH diffusion is 10-times decreased (e.g., due to intracellular accumulation rather than export, as suggested by Schmidt et al.), the N2O production does not spike at low DO (Supporting Information Figure S5b). Comparison with Measured N2O Emission Factors. Model results for the conditions tested by Pijuan et al.28 were directly compared to the measured N2O emission factors, expressed as % of NH3 converted emitted as N2O (Figure 5). Bulk DO concentrations have a significant impact on N2O emission factor; higher emissions factors occur at lower DO (Figure 5). This is mainly because at lower DO concentrations the model predicts higher hydroxylamine levels in the biofilm and therefore more N2O is produced in the deeper biofilm layers. When hydroxylamine is considered to be almost
Figure 5. Comparison between measured %N2O from NH3 converted and model-calculated values in steady-state conditions. Different DO at controlled bulk 40 mg N−NH3/L, with NH2OH diffusion coefficient 50% (solid line) and 1% (dashed line) from the value in bulk liquid. The N2O emission factor was calculated as 2(QGCG,N2O + QCB,N2O)/(QG(Cin,NH3 − CB,NH3)) × 100 with parameters from Supporting Information Table S4. F
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NH2OH addition leads to faster growth rates than on NH3 alone. When supplied anoxically in the presence of NO2−, it leads to high rates of N2O formation.36−38 Our analyses were carried out for a continuous, ideally mixed reactor. For such reactors, the bulk NH3 is equal to the effluent NH3. When the NH3 loadings are low, the NH3 in the bulk may also be very low, limiting nitrifying activity and N2O emissions. This can be seen in Supporting Information Figure S2a, which explores the effects of influent NH3 concentration on N2O emissions for a range of DO values. In a sequencing batch reactor, even if the overall loadings are low, there are likely to be high NH3 concentrations at the beginning of the cycle. Therefore, SBRs are likely to have higher N2O emissions than continuous reactors treating the same influent.28 NH2OH may also be formed mixotrophically. Research has found that NH2OH can be produced anaerobically in the presence of formate.32 Future models should consider other potential NH2OH formation pathways, as it could be formed and consumed under anoxic conditions. This could have implications for nitrification in the presence of organic matter. The results found in this research are not strictly dependent on the model of Ni et al.,23 where reduced and oxidized carriers are explicitly modeled. It is sufficient for models to include NH2OH as an intermediate, and to have NH2OH-based denitrification inhibition by oxygen. Thus, maximum N2O formation rates are obtained in the presence of NH2OH and absence of oxygen. The continued presence of AOB in anoxic regions of biofilm may result from a variety of scenarios. While AOB are known to grow on NH2OH under aerobic conditions, the ability of AOB to support maintenance or even growth on NO2− has been researched and appears to be inconclusive.15,39−42 However, even if AOB cannot subsist without O2, it still is possible at the base of a biofilm to harbor AOB due to fluctuation in DO or to biofilm detachment and regrowth. Residual ammonium has been used to enhance and maintain nitritation in granular sludge reactors.28,43,44 Theoretical analysis of the effects of ammonium building-up on NOB repression focused on the advantage of a higher nitration rate,29,45 also when analyzing single stage partial nitritation/ anammox granular reactors.46 However, our study indicates that ammonium accumulation in the bulk would imply diffusion of hydroxylamine toward deeper biofilm layers. This could have implications for NOB repression. First, AOB will be able to occupy an extended biofilm region because of their capacity to oxidize hydroxylamine.36,42 Second, basal levels of hydroxylamine may inhibit selectively NOB.31,32,47−49 While these effects may help obtaining NOB repression, the hydroxylamine diffusion will also trigger N2O emissions. However, there is still a window for operational conditions in which NOB repression can be achieved while N2O emissions are minimal for DO > 3.5 mg O2/L.28 Further investigation will be required to fully understand the role of residual ammonium on NOB repression in biofilm reactors performing either nitritation or single-stage partial nitritation/anammox. In conclusion, nitrifying biofilms have distinct behavior from suspended systems and can be much more significant sources of N2O under similar conditions. Most existing models do not explicitly include NH2OH as a component, which is critical in a biofilm system. The best means to minimize N2O emission from biofilm systems are similar to those of suspended growth systems: maintain low ammonia oxidation rates. Another approach specific to biofilms would be to minimize the
nondiffusive (i.e., 1% of diffusivity in water), the model does not represent well the experimental data (Figure 5). In batch cultures of planktonic Nitrosomonas europaea, hydroxylamine leaking to the bulk liquid has been reported associated with the exponential growth phase, with NH3 in excess and DO in the range 0.5−1.5 mg/L.30 The general agreement between model predictions and experimental data strengthen the hypothesis that nitrifier denitrification in biofilms is triggered by hydroxylamine leaking out of the cells when ammonium is supplied in excess, followed by diffusion through the biofilm and its further oxidation in the anoxic zone. Therefore, even in well-aerated biofilm reactors with excess ammonium, the limited oxygen penetration can lead to a predominant role of the nitrifier denitrification pathway in the biofilm. This is in contrast to what happens with activated sludge or cells in suspension, where there is little nitrifier denitrification in aerobic conditions.6 Sensitivity Analyses. N2O emissions were generally sensitive to the kinetic parameters in reactions 1 and 2 (Supporting Information Figure S6a, b). Reactions r3 and r4, involving NO oxidation, were largely unaffected by changes in parameters (Supporting Information Figure S6c, d). The rate for r3 matched the NH2OH oxidation rate, which is consistent with the need to prevent the buildup to toxic NO. Reaction r4, NO transformation to N2O, has a very low maximum specific rate, so changes in the parameters had a small effect on N2O emissions. Reaction r5 and r6, for O2 and NO2− reduction, were only moderately sensitive to changes in kinetic parameters (Supporting Information Figure S6e, f). O2 reduction was more sensitive than the NO2− reduction, as NO2− is always present at concentrations that are not rate-limiting. Thus, the results obtained in this research are unlikely to be unique to the specific parameter values used in the simulations. All model results and conclusions strongly depend on NH2OH formation, consumption, and diffusion rates. While these parameters were adequate for our research on mechanisms and described well the data from Pijuan et al.,28 they should still be validated on more experimental data sets in order to obtain a truly predictive model.
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DISCUSSION On the basis of our modeling results, nitrifying biofilms may produce significantly more N2O than suspended growth systems under similar conditions. A key difference between suspended and biofilm processes is that in a biofilm, NH2OH can diffuse from areas with high nitrification activity to zones with lower activity, which act as a sink for NH2OH. When NH2OH penetrates into anoxic zones the Mred concentrations peak and lead to high rates of N2O formation. This can be compounded by the presence of NOB, which increases the slope of the DO gradient. Our research takes well-established principles for AOB metabolism in suspended-growth systems and applies them to a biofilm. Important premises are that (1) NH2OH is formed as an intermediate during NH3 oxidation and can diffuse into the environment, and (2) NH2OH can be used by AOB as an external substrate. It is well established that NH2OH can “leak” from AOB cells.13,15,30−35 Also, the NH2OH concentrations do not need to be high to have a stimulatory effect on N2O formation in biofilms. They only need to be high relative to their half saturation constant. It also is well established that AOB can consume NH2OH as an externally supplied substrate. G
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wastewater treatment process. Water Sci. Technol. 2004, 49 (5−6), 359−365. (6) Rassamee, V.; Sattayatewa, C.; Pagilla, K.; Chandran, K. Effect of oxic and anoxic conditions on nitrous oxide emissions from nitrification and denitrification processes. Biotechnol. Bioeng. 2011, 108 (9), 2036−2045. (7) Tallec, G.; Garnier, J.; Billen, G.; Gousailles, M. Nitrous oxide emissions from denitrifying activated sludge of urban wastewater treatment plants, under anoxia and low oxygenation. Bioresour. Technol. 2008, 99 (7), 2200−2209. (8) Desloover, J.; Vlaeminck, S. E.; Clauwaert, P.; Verstraete, W.; Boon, N. Strategies to mitigate N2O emissions from biological nitrogen removal systems. Curr. Opin. Biotechnol. 2012, 23 (3), 474− 482. (9) Kim, S.; Miyahara, M.; Fushinobu, S.; Wakagi, T.; Shoun, H. Nitrous oxide emission from nitrifying activated sludge dependent on denitrification by ammonia-oxidizing bacteria. Bioresour. Technol. 2010, 101 (11), 3958−3963. (10) Yu, R.; Kampschreur, M. J.; van Loosdrecht, M. C. M.; Chandran, K. Mechanisms and specific directionality of autotrophic nitrous oxide and nitric oxide generation during transient anoxia. Environ. Sci. Technol. 2010, 44 (4), 1313−1319. (11) Chandran, K.; Stein, L. Y.; Klotz, M. G.; van Loosdrecht, M. C. M. Nitrous oxide production by lithotrophic ammonia-oxidizing bacteria and implications for engineered nitrogen-removal systems. Biochem. Soc. Trans. 2011, 39, 1832−1837. (12) Wrage, N.; Velthof, G.; van Beusichem, M.; Oenema, O. Role of nitrifier denitrification in the production of nitrous oxide. Soil Biol. Biochem. 2001, 33 (12−13), 1723−1732. (13) Stein, L. Y. Surveying N2O-producing pathways in bacteria. Methods Enzymol. 2011, 486, 131−152. (14) Arp, D. J.; Stein, L. Y. Metabolism of inorganic N compounds by ammonia-oxidizing bacteria. Crit. Rev. Biochem. Mol. Biol. 2003, 38 (6), 471−495. (15) Wunderlin, P.; Mohn, J.; Joss, A.; Emmenegger, L.; Siegrist, H. Mechanisms of N2O production in biological wastewater treatment under nitrifying and denitrifying conditions. Water Res. 2012, 46 (4), 1027−1037. (16) Ni, B.; Yuan, Z.; Chandran, K.; Vanrolleghem, P. A.; Murthy, S. Evaluating four mathematical models for nitrous oxide production by autotrophic ammonia-oxidizing bacteria. Biotechnol. Bioeng. 2013, 110 (1), 153−163. (17) Ishii, S.; Song, Y.; Rathnayake, L.; Tumendelger, A.; Satoh, H.; Toyoda, S.; Yoshida, N.; Okabe, S. Identification of key nitrous oxide production pathways in aerobic partial nitrifying granules. Environ. Microbiol. 2014, in press. (18) Kampschreur, M. J.; Picioreanu, C.; Tan, N.; Kleerebezem, R.; Jetten, M. S. M.; van Loosdrecht, M. C. M. Unraveling the source of nitric oxide emission during nitrification. Water Environ. Res. 2007, 79 (13), 2499−2509. (19) Ni, B.; Ruscalleda, M.; Pellicer-Nacher, C.; Smets, B. F. Modeling nitrous oxide production during biological nitrogen removal via nitrification and denitrification: extensions to the general ASM models. Environ. Sci. Technol. 2011, 45 (18), 7768−7776. (20) Mampaey, K. E.; Beuckels, B.; Kampschreur, M. J.; Kleerebezem, R.; van Loosdrecht, M. C. M.; Volcke, E. I. P. Modelling nitrous and nitric oxide emissions by autotrophic ammonia-oxidizing bacteria. Environ. Technol. 2013, 34 (12), 1555−1566. (21) Perez-Garcia, O.; Villas-Boas, S. G.; Swift, S.; Chandran, K.; Singhal, N. Clarifying the regulation of NO/N2O production in Nitrosomonas europaea during anoxic−oxic transition via flux balance analysis of a metabolic network model. Water Res. 2014, 60, 267−277. (22) Schreiber, F.; Loeffler, B.; Polerecky, L.; Kuypers, M. M. M.; de Beer, D. Mechanisms of transient nitric oxide and nitrous oxide production in a complex biofilm. ISME J. 2009, 3 (11), 1301−1313. (23) Ni, B.; Peng, L.; Law, Y.; Guo, J.; Yuan, Z. Modeling of nitrous oxide production by autotrophic ammonia-oxidizing bacteria with multiple production pathways. Environ. Sci. Technol. 2014, 48 (7), 3916−3924.
gradient of NH3 oxidation rates and to avoid the formation of anoxic zones in the biofilm, potentially by maintaining high bulk DO values or keeping the biofilms thin.
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ASSOCIATED CONTENT
S Supporting Information *
Numerical model, experimental setup, stoichiometry matrix of the reaction model, reaction rates for the model of Ni et al.,23 model parameters in the base case, model parameters changed for comparison with experimental data from Pijuan et al.,28 comparison of N2O production rates, per unit reactor volume and time, as a function of bulk DO for planar and spherical biofilms of different thicknesses, effect of the set dissolved oxygen concentration at different influent ammonia concentrations, net rate of N2O production determined by the availability of reduced mediators in the biofilm, solute concentrations and rates in the biofilm at a bulk DO of 2 mg/L, effect of effective diffusivity on the N2O production rates at different dissolved oxygen concentrations, sensitivity analysis for the kinetic parameters in the AOB rate expressions r1 (a) and r2 (b), sensitivity analyses for the kinetic parameters in the AOB rate expressions r3 (c) and r4 (d), and sensitivity analysis for the kinetic parameters in the AOB rate expressions r5 (e) and r6 (f). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +1 574 631 4098. Fax: +1 574 631 9236. E-mail: [email protected]. Funding
F.S. and R.N. were supported by NSF project CBET0954918 (Nerenberg CAREER award) and WERF project U2R10. F.S. received additional support from the Bayer Corporation Fellowship. CP work was supported by a Melchor Visiting Professor grant from University of Notre Dame. JP work was supported a Marie Curie Intra European Fellowship (GreenN2, PIEF-GA-2012-326705). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge useful discussions in the manuscript preparation stage with Barth Smets and Holger Daims. REFERENCES
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