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Modeling the Effect of External Carbon Source Addition under Different Electron Acceptor Conditions in Biological Nutrient Removal Activated Sludge Systems Xiang Hu,† Kamil Wisniewski,*,‡ Krzysztof Czerwionka,‡ Qi Zhou,† Li Xie,† and Jacek Makinia‡ †

State Key Laboratory of Pollution Control and Resources Reuse, Tongji University, 1239 Siping Road, Shanghai 200092, China Faculty of Civil and Environmental Engineering, Gdansk University of Technology, ul. Narutowicza 11/12, 80-233 Gdansk, Poland

‡

S Supporting Information *

ABSTRACT: The aim of this study was to expand the International Water Association Activated Sludge Model No. 2d (ASM2d) to predict the aerobic/anoxic behavior of polyphosphate accumulating organisms (PAOs) and “ordinary” heterotrophs in the presence of different external carbon sources and electron acceptors. The following new aspects were considered: (1) a new type of the readily biodegradable substrate, not available for the anaerobic activity of PAOs, (2) nitrite as an electron acceptor, and (3) acclimation of “ordinary” heterotrophs to the new external substrate via enzyme synthesis. The expanded model incorporated 30 new or modified process rate equations. The model was evaluated against data from several, especially designed laboratory experiments which focused on the combined effects of different types of external carbon sources (acetate, ethanol and fusel oil) and electron acceptors (dissolved oxygen, nitrate and nitrite) on the behavior of PAOs and “ordinary” heterotrophs. With the proposed expansions, it was possible to improve some deficiencies of the ASM2d in predicting the behavior of biological nutrient removal (BNR) systems with the addition of external carbon sources, including the effect of acclimation to the new carbon source.

1. INTRODUCTION The International Water Association (IWA) Activated Sludge Model No. 2d (ASM2d), developed over 15 years ago, helped promote the implementation of activated sludge systems for combined N and P removal.10 That model included, however, some simplifying assumptions which could not be valid under specific operating conditions, such as the presence of readily biodegradable organic compounds under aerobic/anoxic conditions or the presence of nitrite under anoxic conditions. Hence, appropriate expansions in the ASM2d are still needed for achieving a proper description of both biological nitrogen and phosphorus removal, and increasing the reliability of wastewater treatment design and analysis based on simulations. Supplemental readily biodegradable organic compounds (external carbon sources), for example, methane, ethanol, acetic acid, sodium acetate, glucose etc., are added in anoxic zones of the biological nutrient removal (BNR) systems to enhance denitrification and improve the overall nitrogen removal efficiency. The external carbon sources also interact with the enhanced biological phosphorus removal (EBPR) process which is carried out by polyphosphate accumulating organisms (PAOs) under sequential anaerobic and aerobic (or anoxic) conditions. The ASM2d and other common mathematical models incorporating the EBPR process assume © XXXX American Chemical Society

that PAOs grow exclusively on the stored polyhydroxyalkanoates (PHA) under aerobic/anoxic conditions, even though PAOs are also known to grow at the expense of soluble substrate (e.g., fermentation products, SA).4,12,22,27 This possibility has been ignored in the models because “it is unlikely that such substrates ever become available under aerobic or anoxic conditions in a BNR plant”10 However, for the BNR systems with external carbon addition, such a simplification becomes invalid as the readily biodegradable compounds become available for direct utilization by PAOs under anoxic (and possibly aerobic) conditions. Furthermore, in the existing models for EBPR, the readily biodegradable organic compounds are divided into only two groups, which become fully available for PAOs either directly (fermentation products, SA) or via fermentation (“complex” substrate, SF). In practice, however, there are compounds (e.g., ethanol) that are known to be readily biodegradable but they are not utilized by PAOs under anaerobic conditions.20,21,24 Satoh et al.24 proposed a modified conceptual model for Received: October 5, 2015 Revised: December 21, 2015 Accepted: January 19, 2016

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DOI: 10.1021/acs.est.5b04849 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology anaerobic COD metabolisms that assumes the presence of soluble substrate, which is not utilized by PAOs but it becomes available for “ordinary” heterotrophs in the presence of electron acceptors (oxygen or nitrate). Based on a similar concept, Swinarski et al.28 developed a new model as an expansion of ASM2d to predict the effect of adding a readily biodegradable substrate (SA,1) which is not available for PAOs under anaerobic conditions, but can be consumed by PAOs in a similar way to the “ordinary” heterotrophs under anoxic and aerobic conditions. Another important limitation of the above-mentioned models was the lack of nitrite (SNO2) as a model component. Currently, nitrite is considered a key intermediate to describe accurately the nitrification and denitrification processes, especially in some sidestream processes.25 Different approaches have recently been proposed for modeling nitrification and denitrification as two-step processes.5,8,26,29 As a consequence, modeling the nitrite behavior becomes essential for the description of other processes, including P uptake.25 Several studies with PAOs have confirmed that elevated concentrations of nitrite would negatively affect the activities of PAOs under either anoxic or aerobic conditions, although the different threshold values of inhibition were reported.11,17,23,26 Extended activated sludge models, including two-stage nitrification, twostage denitrification and EBPR were developed to successfully predict the behavior of nitrite and nitrate.18 The aim of this study was to develop further a comprehensive ASM2d-based model considering (1) nitrite as an electron acceptor, (2) acclimation of “ordinary” heterotrophs to the new external substrate via enzyme synthesisdecay, and (3) modification of the process rate expression for storage of PHA. The expanded model was evaluated against several, especially designed laboratory experiments which focused on the combined effect of different types of external carbon sources (acetate (SA), ethanol and fusel oil (SA1)), and electron acceptors (dissolved oxygen (DO) (SO), nitrate (SNO3), and nitrite (SNO2)) on the behaviors of both PAOs and “ordinary” heterotrophs.

Figure 1. Conceptual model of the expanded ASM2d (a) in the absence of electron acceptor (anaerobic conditions) and (b) in the presence of electron acceptor (aerobic/anoxic conditions).

2.1.2. Storage of Polyphosphate (XPP) Related to SA and SA,1. In the original ASM2d, it is assumed that the energy for storage of XPP by PAOs (XPAO) can only be obtained from the aerobic or anoxic respiration of PHA (XPHA). In the expanded model, the effects of external carbon addition (SA, SA,1) are taken into consideration with respect to storage of XPP by PAOs (XPAO). It is assumed that this process is combined with utilization of the external substrates SA and SA,1 under aerobic or anoxic conditions (processes ρ12−ρ17 in SI Table S2). The processes related to utilization of SA (ρ12−ρ15) may occur simultaneously with storage of XPHA (ρ8). 2.1.3. Reduction of SNO2 and SNO3 Related to Anoxic Growth of XH and XPAO. The expanded model incorporates nitrite (SNO2) as a new state variable and thus denitrification is considered as a two-step process, that is, sequential reduction of SNO3 to SNO2 and eventually to molecular nitrogen gas (SN2) by both “ordinary” heterotrophs (XH) and PAOs (XPAO). It is assumed that different carbon sources can be used for reduction of SNO3 and SNO2 by XH (substrates SF, SA, and SA,1) and XPAO (substrates XPHA, SA, and SA,1). This concept is graphically illustrated in Figure 2. Furthermore, no switching function between NO3−N and NO2−N has been assumed in the expanded model. 2.1.4. Synthesis and Decay of the Denitrification Enzymes. The processes of synthesis and decay of denitrification enzymes were incorporated in the expanded model as presented by Wild et al.31 This approach allows for simulation of the effect of cell saturation with denitrifying enzymes and improve predictions of denitrification activity during long-term acclimation to the

2. MATERIALS AND METHODS 2.1. Conceptual and Mathematical Models. The complete stoichiometric matrix and 30 new process rate equations of the expanded model are summarized in Tables S1 and S2 in the Supporting Information (SI) . In comparison with the original ASM2d, the expanded model accounts for the following new aspects of N and P removal processes: 2.1.1. Growth of “Ordinary” Heterotrophs and PAOs on a New Kind of Substrate (S A,1 ). The expanded model incorporates a new component (SA,1) which is termed ‘‘external readily biodegradable substrate’’ to differentiate it from the original ASM2d ‘“fermentation products”’ (assumed to be acetate), SA, and to denote that this kind of substrate is not available for PAOs under anaerobic conditions but can be consumed by PAOs under anoxic and aerobic conditions.28 Therefore, for the wastewater characterization, the SA,1-type substrate can be differentiated experimentally from SA based on results of the two-phase anaerobic-anoxic (or aerobic) experiments. The new substrate is used for growth of “ordinary” heterotrophs (XH) and PAOs (XPAO) under different electron acceptor conditions (SO, SNO3, and SNO2). A conceptual model of the ASM2d extension considering the SA,1 substrate in the absence and presence of electron acceptors is illustrated in Figure 1. B


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Figure 2. (a) One-step denitrification mechanism in the original ASM2d and (b) two-sep denitrification mechanism in the expanded ASM2d.

injected at the beginning and the test lasted 4 h. The DO concentration was kept at 6 g O2/m3 throughout the test to avoid oxygen limitation. In the one-phase anoxic experiments, a source of nitrate (KNO3)/nitrite (KNO2) and the single external carbon source were injected at the beginning and the test lasted 4 h. In the two-phase tests, process biomass and sodium acetate (CH3COONa) were mixed together and kept first under anaerobic conditions for 2.5 h. The anaerobic phase was followed by the anoxic or aerobic phase lasting 4.5 h. At the beginning of the anoxic phase, a source of nitrate (KNO3)/ nitrite (KNO2) and the single external carbon source were injected. At the beginning of the aerobic phase, the single external carbon source was injected and the aeration system was turned on. The DO concentration was kept at 6 g O2/m3 throughout the phase. At the beginning of all the aerobic phases in both one- and two-phase batch experiments, nitrification inhibitor (ATU) was added in the amount of 10 g/m3. Furthermore, oxygen uptake rates (OURs) were also measured in a cyclic (3 min) mode. Because PHA concentrations inside the PAO cells were not measured directly, the effect of storage compounds was indirectly reflected in the OUR results. The reactors for batch experiments were equipped with electrodes and probes (WTW, Germany) for continuous monitoring of pH (SenTix 21), oxidation−reduction potential (SenTix ORP), temperature and DO (CellOx 325). Samples of the mixed liquor for analysis were frequently (every 5−60 min) withdrawn from the batch reactors. 2.4. Acclimation Experiment in a Bench-Scale Pilot Plant. In terms of the denitrification and EBPR kinetics, the properties of ethanol and fusel oil were found to be very similar.15,28 Therefore, in simulations of the long-term acclimation experiments, fusel oil was treated as the SA,1 type of the substrate (the same as ethanol). The acclimation of biomass to fusel oil was investigated in a bench-scale Johannesburg (JHB) system (V = 30 dm3) fed with the settled wastewater from the Gdansk WWTP. The system was operated for over 100 days between the months of March and June. The process temperature was gradually increasing from 13 to 20 °C. Fusel oil with the total COD concentration of 1 690 000 g COD/m3 was added to the anoxic compartment in the total amount of 1.5 cm3/d. The mixed liquor recirculation (MLR) was first set to 500% of the influent flow rate and increased to 600% in the final stage of the experiment (on day 93). The solids retention time (SRT) was kept at a constant level of 20 days. The set point for the DO concentration in the aerobic compartment was set to 2 g O2/m3. The performance of the pilot system over the entire study period was evaluated in two ways. First, the concentrations of principal parameters (TN, NH4−N, NO3−N, NO2−N, TP, PO4−P, and COD) were determined on a regular basis (twice a week) in both influent and effluent grab samples. Some parameters (NH4−N, NO3−

new external substrate (SA,1). Due to the limited information on kinetic parameters for the synthesis and decay process rates, it is assumed that the expressions for nitrate reductase and nitrite reductase are identical. The anoxic growth process rates (processes ρ2−ρ7 in SI Table S2) have to be multiplied by the degree of the cell saturation with denitrification enzymes (Esat) which is proportional to the actual amount of enzymes divided by the biomass concentration of “ordinary” heterotrophs (XH). Furthermore, the following modifications/expansions of the process rates with respect to the original ASM2d were also included in the expanded model (the model limitations are listed in section “Model limitations” in SI): (1) The inhibitory effect of DO (SO) and nitrite (SNO2) on storage of PHA (XPHA) by PAOs (XPAO) under aerobic and anoxic conditions, respectively. The DO and nitrite inhibition on phosphorus release were expressed using simple/reversible inhibition terms: K IOPHA/(SO + KIOPHA) and KINO2,PAO/(SNO2 + KINO2,PAO), where KIOPHA stands for the inhibition concentration of DO on PAOs and KINO2,PAO stands for the inhibition concentration of nitrite on PAOs (process ρ8 in SI Table S2). (2) XPHA storage inhibition coefficient. The storage of XPHA declines if the PHA content of PAO biomass (XPHA/ XPAO) becomes abundant and reaches the maximum value of KMAX1. This approach is analogous to modeling the saturation of XPP storage in the ASM2d (process ρ8 in SI Table S2). (3) Anoxic hydrolysis of slowly biodegradable substrate (XS) is possible with two electron acceptors, including SNO3 and SNO2 (processes ρ27 and ρ28 in SI Table S2). 2.2. Organization of the Modeling Study Procedure. GPS-X ver. 6.3.3. (Hydromantis, Canada) was used as a simulator environment. The newly developed model was implemented using a special utility called “Model Developer”. The modeling study procedure consisting of six consecutive steps is described in details in section “Organization of the modeling study procedure” in the SI. 2.3. Batch Experiments with Nonacclimated Biomass. Two types of batch experiments, including one-phase experiments under aerobic or anoxic conditions and two-phase experiments (anaerobic/anoxic or anaerobic/aerobic), were carried out with different external carbon sources (acetate and ethanol) and electron acceptors (DO, nitrate, and nitrite). The nonacclimated process biomass used in the batch experiments originated from a large BNR wastewater treatment plant (600 000 PE, Modified University of Cape Town (MUCT) process configuration) located in the city of Gdansk (northern Poland). In the one-phase aerobic experiments, the single external carbon source and phosphorus (NaH2PO4/Na2HPO4) were C


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Figure 3. Measured data vs model predictions with the expanded ASM2d: (a,b) PO4−P in the aerobic experiment with acetate (simulations of the ASM2d included) and ethanol, (c,-d) COD and OUR in the aerobic experiment with acetate and ethanol, (e,f) PO4−P in the anaerobic/aerobic experiment with acetate (simulations of the ASM2d included) and ethanol, (g,h) COD and OUR in the anaerobic/aerobic experiment with acetate and ethanol.

N, and PO4−P) were determined once per week inside the reactor (anaerobic, anoxic, and aerobic compartments). Second, conventional nitrate utilization rates (NUR) measurements were carried out on a regular basis (every 1−3 weeks) with the process biomass during the operation of the system.

2.5. Analytical Methods. Prior to analysis, the samples of mixed liquor were filtered under vacuum pressure through a 1.2 μm pore size Millipore (Billerica, MA). Concentrations of PO4−P, NO3−N, NO2−N, and COD were determined using Xion 500 spectrophotometer (Dr Lange GmbH, Berlin, D


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Figure 4. Measured data vs model predictions with the expanded ASM2d in the anoxic experiment with different external carbon sources and different electron acceptors: (a,b) acetate, NO3−N and NO2−N, (c,d) ethanol, NO3−N and NO2−N.

Germany) following the Standard Methods.3 The mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) were measured by the gravimetric method according to the Polish Standards (PN-72/C-04559). Free nitrous acid (FNA) concentrations were calculated from the following formula:2 FNA = S NO2/(Ka × 10 pH)

ethanol addition (Figure 3b). Furthermore, results from the two-phase experiments revealed that no PO4−P release in the aerobic phase was observed with either acetate (Figure 3e) or ethanol (Figure 3f). Provided that the SA substrate is available for PAOs and PO4−P is not completely released from the PAOs cells, the inconsistent PO4−P behaviors under aerobic conditions, that is, release in the one-phase experiments vs uptake in two-phase experiments, may be explained by different amounts of the stored PHA inside the PAO cells. In OUR measurements with the acetate addition, there were significant initial OUR increases in both one- and two-phase experiments (Figure 3c and g) which indicate the aerobic utilization of SA by “ordinary” heterotrophs and PAOs. The increased OURs well corresponded to the utilization of COD (storage of acetate as PHA by PAO) and phosphate release (in the one-phase experiments) and the utilization of COD (in the two-phase experiments). For comparison, in the case of ethanol addition, the OUR values were remaining almost constant during the entire aerobic phase of both one- and two-phase experiments (Figure 3d and h). This may be attributed to the specific SA,1 characteristics. That substrate was incompletely utilized at a slower rate by the nonacclimated “ordinary” heterotrophs during the entire experiment. When simulating the batch experiments with the original ASM2d, which assumes growth of PAOs on XPHA only and ignores the possibility of aerobic growth of PAOs on SA, consistent predictions were obtained for the COD and OUR behaviors in both one- and two-phase experiments (Figure 3c

(1)

where Ka = e−2300/(273+T(°C)).

3. RESULTS AND DISCUSSION 3.1. Parameter Estimation, Confidence Intervals and Correlation Matrix. Results of parameter estimation are discussed in details in section “Parameter estimation, confidence intervals and correlation matrix” in SI. Table S4 contains a list of the adjusted parameters. Correlations between the parameters estimated in step 3.1 and step 3.2−3.4 are shown in SI Table S6 and Table S7, respectively. 3.2. Simulation of the Aerobic and Anaerobic/Aerobic Batch Experiments. Figure 3 shows the aerobic behaviors of PO 4 −P, COD and OUR in the one- and two-phase experiments with the SA substrate (acetate) and SA,1 substrate (ethanol) as the external carbon sources. In the one-phase aerobic experiment with the acetate addition, PO4−P was released in the initial 45 min of the experiment until acetate was depleted in the reactor (Figure 3a). In contrast, no PO4−P release was observed in the one-phase experiment with the E


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Figure 5. Measured data vs model predictions with the expanded ASM2d in anaerobic/anoxic experiments with different external carbon sources and different electron acceptors: (a,b) no external carbon addition, NO3−N and NO2−N, (c,d) acetate, NO3−N and NO2−N, (e,f) ethanol, NO3−N, and NO2−N.

and g) as well as the PO4−P behavior in the one-phase experiment (Figure 3a). However, the PO4−P behavior in the two-phase experiment could not be described accurately (Figure 3e). These results suggest that the ASM2d requires appropriate extensions to better describe the effect of external carbon addition on the aerobic behavior of PAOs. For this purpose, the process rate for storage of PHA (XPHA) by PAO was extended by the inhibitory effect of DO (SO), nitrite (SNO2) and inhibition term of PHA storage (XPHA) (process ρ8 in SI Table S2). This approach allowed to reasonably predict the behavior of PO4−P, COD, and OUR in both one- and two-

phase experiments (Figure 3a, c, e, and g) after estimating the kinetic and stoichiometric parameters as shown in SI Table S4. In the experiments with ethanol as the SA,1 substrate, model predictions of the expanded ASM2d also matched accurately the experimental data (Figure 3b, d, f, and h) by adjusting two kinetic and one stoichiometric parameters, that isthe heterotrophic yield coefficient of XH for SA,1 (YH1), maximum growth rate of XH on SA1 (μH1) and rate constant for storage of XPP (qPPSA1) (step 3.2 in SI Table S4). Values of the kinetic and stoichiometric parameters adjusted in the expanded ASM2d are listed in SI Table S5 and compared with the ASM2d defaults and calibrated values reported by F


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Figure 6. (a) Measured NUR data vs model predictions with the original ASM2d and with the expanded ASM2d, (b) model predictions of the ESAT coefficient and concentration of the “ordinary” heterotrophs (XH) with the expanded ASM2d, (c) measured data vs model predictions with the expanded ASM2d for NO3−N concentrations in the anoxic and aerobic compartments, (d) measured data vs model predictions with the expanded ASM2d for PO4−P concentrations in the anoxic and anaerobic compartments.

Swiniarski et al.28 It should be noted that values of the ASM2d parameters were earlier adjusted during a dynamic calibration of the full-scale MUCT bioreactor performance and batch experiments with the real (settled) wastewater.28 In the present study, these parameters were used in the initial simulations and were only slightly modified during calibration of the expanded ASM2d. The remaining kinetic and stoichiometric parameters in the expanded model were adopted from the study of Swinarski et al.28

3.3. Simulation of the Anoxic and Anaerobic/Anoxic Batch Experiments. Figure 4 shows the anoxic behaviors of PO4−P, COD, NO3−N, and NO2−N in the one-phase experiments with acetate and ethanol as the external carbon sources. In the experiment with the acetate addition, regardless of the electron acceptor (SNO3 or SNO2), PO4−P was released to indicate the anoxic consumption of SA by PAOs until the SA substrate was depleted in the reactor (Figure 4a and b). This suggests that PAOs are capable of uptaking acetate under G


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Environmental Science & Technology anoxic conditions (with either SNO3 or SNO2), linking this consumption to PO4−P release, PHA formation and glycogen degradation.1,9,19 In contrast, no significant PO4−P release was observed in the experiments with the ethanol addition (Figure 4c and d). In the two-phase experiments, when nitrate was used as the electron acceptor in the anoxic phase, the addition of acetate resulted in PO4−P release until acetate was depleted in the reactor (Figure 5c). For comparison, the ethanol addition resulted in the opposite PO4−P behavior, that isits uptake (Figure 5e), which was similar to the experiment without the addition of any external carbon (Figure 5a). In the experiments with nitrite, negligible PO4−P release or uptake was observed regardless of the absence or presence of the external carbon (Figure 5b, d, and f). These results suggest that the nonacclimated PAOs are not able to use nitrite under anoxic conditions with the examined initial NO2−N concentration of approximately 25 g N/m3. In the literature, high concentrations of nitrite have indeed been reported to be a severe inhibitor of the anoxic phosphate uptake by PAOs.17,23 However, some recent studies reported that the protonated species of nitrite, FNA, rather than the nitrite itself, is likely the actual inhibitor on bacteria in BNR systems. This also refers to PAOs responsible for the anoxic phosphorus uptake, depending on the concentration of nitrite, operating pH and temperature.32,33 Figure S2 (in SI) shows the FNA profiles measured in twophase anaerobic-anoxic experiments. At 20 °C and initial pH of 7.2−7.4, the calculated maximum FNA concentrations for the experiments with acetate and ethanol were in the range of 0.0019−0.0035 g FNA/m3 and 0.0017−0.0028 g FNA/m3, respectively. Zhou et al.32 reported that the anoxic P-uptake activity of PAO was inhibited by FNA at concentrations in the range of 0.001−0.002 g FNA/m3, which is substantially lower compared to those in the present study. With the calibrated expanded ASM2d model, the observed principal processes (PO4−P release, anoxic PO4−P uptake and NO3−N/NO2−N utilization) could accurately be predicted in both one- and two-phase experiments with different types of electron donors (SA and SA,1) and electron acceptors (SNO3 and SNO2). The assumed approach of anoxic XPP storage and twostep denitrification, separated for the different carbon sources (Figure 2b), resulted in a significant number of additional anoxic processes in the expanded model. The anoxic reduction factors for “ordinary” heterotrophs (ηNO2,H, ηNO3,H, ηNO2,H1, ηNO3,H1) were adjusted to calibrate the observed NURs in both one- and two-phase experiments (steps 3.3 and 3.4 in SI Table S4). The values for ηNO3,H, and ηNO3,H1 were changed in comparison with the calibrated ASM2d by Swinarski et al.28 and set to 0.35, and 0.21, respectively (in SI Table S5). Additionally, the new parameters such as ηNO2,H, ηNO2,H1 in the expanded ASM2d were set to 0.32, 0.06, respectively. It should be noted that all the anoxic reduction factors for PAO related to SNO2 had to be set to 0 to describe the negligible PO4−P uptake observed for SNO2 as the electron acceptor. Furthermore, the anoxic reduction factor for the PAO growth on S A1 (ηNO3,PAOSA1) remained unchanged in comparison to the study of Swiniarski et al.28 The calibrated value of this coefficient (η NO3,PAOSA1 = 0.6) was the same as the corresponding coefficients related to PHA (ηNO3,PAO = 0.6) and acetate (ηNO3,PAOSA = 0.6). 3.4. Simulation of the Long-Term Performance of the Bench-Scale Pilot Plant. When fusel oil was used in the acclimation experiments in the bench-scale JHB system, an

acclimation period of approximately 50 days was required to achieve the maximum NURs (Figure 6a), which is similar to the reported acclimation periods of 30 days from Puig et al.21 and 50 days from Wang et al.30 The observed NURs were apparently increasing from the initial value of less than 2 g N/(kg VSS·h) and reached the maximum of over 6 g N/(kg VSS·h) on day 50 and then stabilized. This increase could be attributed to both acclimation to the SA,1 substrate and gradually increasing process temperature from 13 to 20 °C. Significantly lower NURs (up to 2 g N/(kg VSS·h)) were obtained in the parallel experiments with the nonacclimated biomass from the full-scale bioreactor (data not shown). A consequence of adding fusel oil to the bench-scale JHB system was the improved performance in terms of nitrogen removal. The effluent TN concentrations decreased from the initial value of 17.9 g N/m3 to approximately 10 g N/m3 after 30 days and then remained stable to the end of the acclimation experiment. The primary contribution originated from the improved denitrification efficiency and decreasing NO3−N concentrations in the aerobic zone (Figure 6c). Effluent concentrations of the other inorganic nitrogen compounds were relatively low during the entire experiment and ranged from 0.05 to 0.60 g N/m3 (average 0.28 g N/m3) and 0.04 to 1.28 g N/m3 (average 0.29 g N/m3) for nitrite and ammonia, respectively. The addition of fusel oil also improved the EBPR performance. From 0 to 45 days of the acclimation experiment, PO4−P concentrations in the aerobic compartment were in the range of 0.24−0.71 g P/m3 with the average PO4−P concentration of 0.40 g P/m3. Subsequently, the PO4−P concentrations in the aerobic compartment were decreasing and remained in the range of 0.09−0.41 g P/m3 with the average PO4−P concentration of 0.29 g P/m3. Due to the acclimation period required, the expanded ASM2d had to include synthesis and decay of the denitrification enzyme in order to improve predictions of the NURs data during the long-term operation of the system. Without the proposed extension, the predicted NURs are underestimated compared to the measured NURs with the acclimated biomass (Figure 6a). The processes of synthesis and decay of the denitrification enzyme were simulated with the suggested values for the kinetic parameters by Wild et al.31(SI Table S5). A different stage of acclimation was reflected by variations in the value of Esat coefficient, which was calculated as a state variable based on eqs 29−30 in Table S1. The Esat value for the process biomass was gradually increasing in the initial operating period and reached the value of 0.50 on day 8. The maximum NUR was obtained on day 50 and corresponded well to the Esat value (0.84), which was very close to the maximum Esat,max = 0.88 reported by Wild et al.31 During the entire experiment, the MLVSS concentration was maintained at 2.4−3.7 g/m3 and the estimated contribution of XH biomass to MLVSS was in the range of 31−38% (Figure 6b). The expanded ASM2d, including synthesis and decay of the denitrification enzyme, was also used for predicting the longterm performance of the bench-scale BNR pilot system with addition of the SA1 substrate. Figure 6c,d shows the measured data vs model predictions of NO3−N and PO4−P in the sampling points inside the JHB reactor. The average measured concentrations of NO3−N were 0.5 g N/m3 and 8.4 g N/m3, respectively, in the anoxic and aerobic compartments. The average measured concentrations of PO4−P were 30.6 g P/m3 and 11.4 g P/m3 in the anaerobic and anoxic compartments, respectively. The absolute prediction errors of the average H



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concentrations were relatively small and varied in the range from 0.14 g N/m3 (aerobic NO3 −N) to 0.96 g P/m3 (anaerobic PO4−P). 3.5. the Implications of the Expanded Model for the Operation of Full-Scale Activated Sludge Systems. The expanded model improves some deficiencies of the original ASM2d which are important for modeling the operation of the full-scale activated sludge systems with dosing external carbon for enhancing denitrification. Several new kinetic and stoichiometric coefficients were estimated under laboratory conditions, however, these coefficients still need to be validated in full-scale WWTPs. In the previous study of Swinarski et al.,28 the concept of a new readily biodegradable substrate (not available for the PAO metabolism) was introduced and tested. The present study focused on two other aspects of modeling which are important in full-scale applications, including the acclimation by the “ordinary” heterotrophs to the new type of external substrate and the effects of NO2−N as an electron acceptor with different types of organic substrates. The acclimation of the microbial population to a new carbon source, other than SF or SA types considered in the original ASM2d, often requires a transition period, which may involve microbial community structure change and/or specific enzymes production needed to utilize the carbon alternative. The length of the acclimation period varies, depending on the used carbon source, microbial community composition, process configuration as well as environmental conditions. The typical acclimation periods range from 1 day to several weeks.7 For example, the reported acclimation periods for methanol varied from a few days up to 90 days. In the present study, the acclimation period of a few weeks was accurately predicted for fusel oil applied in the pilot scale system. Full-scale trials with fusel oil suggested that a similar acclimation period would be required to reach the maximum denitrification capability of process biomass.16 Furthermore, the expanded model includes 9 new processes with nitrite (NO2−N) as an electron acceptor. NO2−N is a common intermediate in nitrogen conversions and its accumulation in full-scale WWTPs has been reported in literature for decades. The mechanisms of NO2−N formation are complex with many competing and parallel conversions of production and consumption for the compound. Foley et al.6 noted that NO2−N is simultaneously a product, a substrate and an inhibitor, which can be formed and utilized by several different types of microorganisms under both aerobic and anoxic conditions. Under normal operating conditions, NO2−N accumulation is undesired in BNR WWTPs, however, during unstable operation of municipal WWTPs, for example, due to insufficient DO concentration, low temperature, short SRT or the presence of inhibitory compounds, NO2−N can play an essential role in the nitrogen conversions.25 Furthermore, it has been reported that NO2−N could inhibit P release and uptake by PAO. The accumulation of NO2−N has also been shown to trigger the emission of nitrous oxide (N 2 O) during denitrification.6,13 Depending on the specific needs, further model expansions may involve some new processes which are currently of the special interest in the area of biological nutrient removal, e.g. two-stage nitrification and anammox for mainstream deammonification, three- or four-stage denitrification for N2 O production and emission, and glycogen accumulating organisms (GAO) metabolism for competition between GAO and PAO.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b04849.

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Additional information, tables and figures (PDF)

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Corresponding Author

*Phone: +48 58 347-23-03; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study has been financially supported by European Regional Development Fund within the framework of the Innovative Economy Operational Programme 2007-2013 under the project no. UDA-POIG.01.03.01-22-140/09-04. During the time of the study at Gdansk University of Technology, X.H. was a researcher supported by the project “CARbon BALAncing for nutrient control in wastewater treatment (CARBALA)” (PIRSES-GA-2011-295176) under the International Research Staff Exchange Scheme FP7-PEOPLE-2011IRSES.

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