Accepted Manuscript
The effect of dissolved oxygen on N2O production by ammonia-oxidizing bacteria in an enriched nitrifying sludge Lai Peng, Bing-Jie Ni, Dirk Erler, Liu Ye, Zhiguo Yuan PII: DOI: Reference: To appear in: S0043-1354(14)00576-4 10.1016/j.watres.2014.08.009 WR 10816 Water Research

Received Date: 13 June 2014 Revised Date: 4 August 2014 Accepted Date: 8 August 2014

Please cite this article as: Peng, L., Ni, B.-J., Erler, D., Ye, L., Yuan, Z., The effect of dissolved oxygen on N2O production by ammonia-oxidizing bacteria in an enriched nitrifying sludge, Water Research (2014), doi: 10.1016/j.watres.2014.08.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract (for review)
Specific N2O Production Rate
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Contribution of Different Pathways to N2O Production (%)

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Contribution of NH2OH oxidation pathway (based on site preference data) Contribution of AOB denitrification pathway (based on site preference data) Contribution of NH2OH oxidation pathway (model predictions) Contribution of AOB denitrification pathway (model predictions)

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Highlights     We investigated the effect of DO on N2O production by AOB. As DO increased, N2O production increased, but its emission factor decreased. The site preference and a model were involved to investigate N2O pathways.

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DO plays a pivotal role in affecting the relative contributions of N2O pathways.

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1 2 3 4 5 6 7 8 9 10 11 12 13 a The effect of dissolved oxygen on N2O production by ammonia-oxidizing bacteria in an enriched nitrifying sludge

Lai Penga, Bing-Jie Nia, Dirk Erlerb, Liu Yea,c, Zhiguo Yuana*

Advanced Water Management Centre, The University of Queensland, St. Lucia,

Brisbane, QLD 4072, Australia b Centre for Coastal Biogeochemistry Research, Southern Cross University School of Chemical Engineering, The University of Queensland, St. Lucia, Brisbane,

c

QLD 4072, Australia

*Corresponding author:

Zhiguo Yuan, P + 61 7 3365 4374; F +61 7 3365 4726; E-mail zhiguo@awmc.uq.edu.au

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emission factor (the ratio between N2O nitrogen emitted and the ammonium nitrogen converted) decreased from 10.6 ± 1.7% (n=3) at DO = 0.2 mg O2/L to 2.4 ± 0.1% (n=3) at DO = 3.0 mg O2/L. The site preference measurements indicated that both the AOB denitrification and hydroxylamine (NH2OH) oxidation pathways contributed to N2O production, and DO had an important effect on the relative contributions of the

two pathways. This finding is supported by analysis of the process data using an N2O model describing both pathways. As DO increased from 0.2 to 3.0 mg O2/L, the contribution of AOB denitrification decreased from 92% − 95% to 66% − 73%, accompanied by a corresponding increase in the contribution by the NH2OH oxidation pathway.

Keywords: Dissolved oxygen; Nitrous oxide; Ammonia-oxidizing bacteria; Pathways; Site preference; Model

1. Introduction

Nitrous oxide (N2O) is not only a potent greenhouse gas with approximately 300 times global warming potential of carbon dioxide (CO2), but also a major sink for stratospheric ozone (IPCC, 2007; Ravishankara et al., 2009). Wastewater treatment

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systems are a recognised source of N2O (Law et al., 2012b). During biological

wastewater treatment, N2O is mainly generated from biological nitrogen removal

(BNR), which involves both nitrification and denitrification (Tchobanoglous et al., 2003; Kampschreur et al., 2009). Recently, ammonia-oxidizing bacteria (AOB) are identified as the major contributor to N2O production in wastewater treatment plants

(Kampschreur et al., 2007; Yu et al., 2010; Law et al., 2012b). However, the mechanisms of N2O production by AOB are still not fully understood. According to

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the current understanding, there are two main pathways involved in N2O production by AOB: (i) the reduction of nitrite (NO2-) to N2O via nitric oxide (NO), known as nitrifier or AOB denitrification (Kim et al., 2010) and (ii) N2O as a side product during incomplete oxidation of hydroxylamine (NH2OH) to NO2- (Stein, 2011; Chandran et al., 2011; Law et al., 2012a).

Key factors affecting N2O production during nitrification include the ammonium (NH4+) loading rate, the pH, NO2- or free nitrous acid (FNA) levels, and the dissolved oxygen (DO) concentration. It is reported that N2O production increases upon

increasing nitrogen load during aerobic ammonium oxidation (Tallec et al., 2006; Kampschreur et al., 2007; Yang et al., 2009; Kim et al., 2010; Law et al., 2012a). The influence of pH on N2O production has been investigated in several studies with the use of different cultures. The maximum N2O production during nitrification is generally observed at pH of 8.0 – 8.5 (Hynes and Knowles, 1984; Law et al., 2011). Positive correlation between N2O production and NO2- or FNA concentration has also been widely reported for both full-scale and lab-scale sludges (Shiskowski and Mavinic, 2006; Tallec et al., 2006; Foley et al., 2010; Kim et al., 2010). However, Law et al. (2013a) observed an inhibitory effect of high NO2- concentration (over 50 mg NO2--N/L) on N2O production by AOB in a nitritation system treating anaerobic

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sludge digestion liquor.

DO is a very important factor affecting N2O emission in nitrification. However,

contradictory observations have been reported in literature. (Table 1). For example, Kampschreur et al. (2007) reported that N2O production increased with the decrease of DO concentration and suggested that the decreasing DO to oxygen limiting

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conditions could prompt N2O production from AOB denitrification. Goreau et al. (1980) observed a similar dependency of N2O production on DO concentration. In contrast, Law et al. (2012a) found that N2O production increased with increasing DO levels. The high level of NO2- (around 500 mg N/L) was later suggested to suppress the AOB denitrification pathway (Law et al., 2013). Therefore, the observation likely

reflected the effect of DO on N2O production via the NH2OH oxidation pathway (Ni et al., 2014). Further complicating the observation, Tallec et al. (2006) and Yang et al.

(2009) observed a maximum of N2O production at a DO concentration of 1.0 mg O2/L. Tallec et al. (2006) further pointed out that the AOB denitrification, rather than heterotrophic denitrificaiton, was the main contributor of N2O production in the DO range from 0.1 to 2.0 mg O2/L.

Two factors that may have influenced the results of the previous studies investigating the effect of DO on N2O production. In several studies (e.g., Tallec et al. (2006), Yang et al. (2009) and Wunderlin et al. (2012)), activated sludge comprising a large amount of heterotrophic biomass in addition to AOB and nitrite-oxidizing bacteria (NOB) was used. It is known that heterotrophic bacteria are able to produce and consume N2O, which is influenced by the DO concentration (Law et al., 2012b). In such cases, it is difficult to isolate the effect of DO on N2O production by AOB from the DO effect on

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N2O production/consumption by heterotrophic bacteria. Another factor is the effect of the accumulated nitrite (Tallec et al., 2006; Foley et al., 2010; Kim et al., 2010).

Variation in DO affects the activities of both AOB and NOB, and nitrite accumulation

may occur particularly under low DO conditions (Guisasola et al., 2005). The variation in nitrite accumulation would incur an independent effect on N2O production by AOB, which cannot be easily separated from that of DO.

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101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 2. Materials and methods The aim of this study is to further clarify the effect of DO on N2O production by AOB. An enriched nitrifying culture comprising primarily AOB and NOB was used. While the presence of a relatively small amount of heterotrophic bacteria is expected (growing on AOB and NOB cell lysate), their effects on N2O production were

identified through control tests. To minimize nitrite accumulation under varying DO conditions, we added additional NOB, enriched in a separate reactor, to the nitrifying

sludge during all experiments. To reveal the effects of DO on each of the two known pathways, isotopic measurements of site preference (SP) and bulk
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(δ15NN2Obulk) were used to identify the contribution of each pathway to the overall N2O production. Furthermore, a recently proposed N2O model incorporating both pathways was employed to analyze the experimental data, in order to gain independent evidence of the relative contributions by the pathways (Ni et al., 2014).

2.1. Culture enrichment and reactor operation Two lab-scale sequencing batch reactors (SBRs) were operated in the laboratory at room temperature (22.0 – 23.0 ºC) seeded with sludge from a domestic wastewater treatment plant in Brisbane, Australia. One was fed with ammonium with the aim to

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obtain an enriched culture of AOB and NOB, and the other with nitrite to obtained an

enriched culture of NOB. Both reactors had working volumes of 8 L, and were

operated with a cycle time of 6 hr consisting of 260 min aerobic feeding, 20 min

aeration, 1 min wasting, 60 min settling and 19 min decanting periods. During each cycle, 2 L of synthetic wastewater (compositions are described below) was fed to the reactors, resulting in a hydraulic retention time (HRT) of 24 hr. Compressed air was

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supplied to the reactors during the feeding and aerobic phases. DO in both reactors were continuously monitored online using miniCHEM-DO2 meters and controlled between 2.5 and 3.0 mg O2/L with a programmed logic controller (PLC). pH in the two reactors were measured with miniCHEM-pH meters. For the AOB + NOB culture, pH was controlled at 7.5 by dosing 1 M NaHCO3, and for the NOB culture,

the pH was not controlled but was stable in the range of 7.0 – 7.3. The solids retention time (SRT) was kept at 15 days for both reactors by wasting 130 mL of sludge during the 1-min wasting period.

The synthetic wastewater for the AOB + NOB culture comprised per liter (adapted from Kuai and Verstraete (1998)): 5.63 g of NH4HCO3 (1 g NH4+-N), 5.99 g of NaHCO3, 0.064 g of each of KH2PO4 and K2HPO4 and 2 mL of a trace element stock solution. The trace element stock solution contained: 1.25 g/L EDTA, 0.55 g/L ZnSO4·7H2O, 0.40 g/L CoCl2·6H2O, 1.275 g/L MnCl2·4H2O, 0.40 g/L CuSO4·5H2O, 0.05 g/L Na2MoO4·2H2O, 1.375 g/L CaCl2·2H2O, 1.25 g/L FeCl3·6H2O and 44.4 g/L MgSO4·7H2O. The synthetic wastewater for the NOB culture comprised per liter (adapted from Kuai and Verstraete (1998)): 4.93 g of NaNO2 (1 g NO2--N), 0.4 g of NaHCO3, 1 g of each of KH2PO4 and K2HPO4 and 2 ml of a stock solution containing trace elements, as described above.

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At the time the batch tests described in the next section were conducted, the two

reactors were both in steady state for more than 5 months, with 100% conversion of

NH4+ to NO3- by the AOB + NOB culture and of NO2- to NO3- by the NOB culture at the end of each cycle. The mixed liquor volatile suspended solids (MLVSS) concentrations in both reactors were also stable at 1480 ± 28 (n=8) and 570 ± 43

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(n=6) mg/L, respectively. Characterization of the biomass compositions using fluorescence in situ hybridization (FISH) indicated that (1) for the enriched AOB + NOB culture, 46 ± 6% of the EUBMix probe targeted cells bound to the NSO1225 probe, which covers ammonia-oxidising beta-proteobacteria comprising the Nitrosospira, Nitrosococcus and Nitrosomonas genera; 38 ± 5% of the EUBMix

probe targeted cells bound to the Ntspa662 probe, specific for the Nitrospira genera

(nitrite oxidizers); All the NIT3 probes applied did not give any signals, suggesting the absence or very low abundance of the Nitrobacter genera (nitrite oxidizers); (2) for the enriched NOB culture, 75 ± 8% of the EUBMix probe targeted cells bound to the Ntspa662 probe, specific for the Nitrospira genera; 1.2 ± 0.5% of the EUBMix probe targeted cells bound to the NIT3 probes, specific for the Nitrobacter genera. FISH was performed according to the procedure as previously described (Law et al., 2011). The biovolume fraction of the bacteria of interest was determined by analysing 20 FISH images for each reactor using DAIME version 1.3 (Daims et al., 2001). Reported values are mean percentages with standard deviations.

2.2. Experimental design

Eight sets of batch tests were carried out, with key experimental conditions summarized in Table 2. All tests were performed in triplicate. For each test, 0.133 L

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and 0.3 L mixed liquor was withdrawn from the AOB + NOB and the NOB reactors,

respectively. They were mixed in a 2-L beaker on a magnetic stirrer and diluted to 1 L

by adding 0.567 L decant from the AOB + NOB reactor, before being used for the

experiments. Before each test, 50 mL mixed liquor samples were taken from the AOB + NOB reactor to determine the mixed liquor suspended solids (MLSS) concentration and its volatile fraction (MLVSS) (all in triplicate). The MLVSS concentration of the

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AOB + NOB sludge in each batch test was calculated based on these measured concentrations and the dilution ratio.

All batch tests were carried out at room temperature (22.0 – 23.0 ºC) in a 1.3 L reactor with a sealable lid. DO and pH in all tests were continuously monitored online using a

miniCHEM-DO2 sensor and a miniCHEM-pH metre, respectively. pH was controlled at 7.5 using a PLC by dosing 1 M NaHCO3 or 1 M HCl. In all tests, DO concentration

was manually controlled at the designed level (Table 2) during the entire experiment with a gas mixture of N2 and air. The N2 flow and air flow were adjusted using two mass flow controllers (Smart- Trak 50 series- 1 L/min and 5 L/min, Sierra). The total gas flow rate was controlled constantly at 0.5 L/min. For each change in DO concentration, the change in the air flow rate was compensated for by an equivalent change in the N2 flow rate.

Each test consisted of two phases, namely a control phase and an experimental phase. The same DO level (Table 2) was applied to both phases. In the control phase lasting for 20 min, no NH4+ was added and N2O production can therefore be attributed to heterotrophic activity. In the following experimental phase, which lasted for 90 min, the NH4+ concentrations in all tests were controlled at 18 ± 2 mg NH4+-N/L through

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manually adding a stock solution of 33.8 g/L NH4HCO3 and 36 g/L NaHCO3 with

intervals of 15 – 30 min, with amounts determined using the method described in the

Supplementary Information. During each test, mixed liquor samples were taken every

30 min for NH4+, NO2- and NO3- analyses using a syringe and immediately filtered through disposable Millipore filters (0.22 µm pore size).

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In order to evaluate the possible N2O consumption by heterotrophic bacteria, three N2O consumption tests were also conducted at different DO levels of 0.5, 1.5 and 3.0 mg O2/L using the same mixture of the two cultures as described above. Aeration was stopped after DO reached the designed levels. Then 1 mL of saturated N2O solution was added into the batch reactor. The headspace was removed by adding the same

mixture of the two cultures. After that the batch reactor was fully sealed. The N2O concentration was then monitored online for 30 min using a N2O microsensor (to be

further described). The N2O consumption rate was determined from the measured N2O concentration profile.

2.3. On-line N2O monitoring

N2O concentration in the gas phase of batch reactor was measured with a URAS 26 infrared photometer (Advance Optima Continuous Gas Analyser AO2020 series, ABB), with a measuring range of 0 – 100 ppmv and detection limit of 1.0 ppmv. Data were logged every 30 s. To prevent moisture from entering the analyser, a moisture filter was installed at the gas inlet of the analyser. A t-shaped tubing joint was fitted on to the gas sampling tube connecting the gas outlet of the reactor and the gas analyser. This allowed the excess gas flow from the reactor to escape from the system, maintaining atmospheric pressure in the reactor. The sampling pump of the analyser

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was adjusted to be lower than the total gas flow rate in the reactor at all time. The

N2O analyser was calibrated periodically as per manufacturer’s instruction and no

signal drift was detected.

N2O concentration in liquid phase was measured using a N2O microsensor (N2O-100 with a detection limit of 0.0028 mg N2O-N/L, Unisense A/S. Aarhus, Denmark), with

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data logged every 10 seconds. A two-point calibration was performed before each batch test with deionised water as zero and a N2O stock solution with a dissolved N2O concentration of 7.235 mg N/L. The signal of the sensor has been shown to be linear within the range of 0-14 mg N2O-N/L in water (Andersen et al., 2001).

2.4. Calculations

Biomass specific ammonia oxidation rate (AOR), biomass specific N2O production rate (N2OR) and the ratio between N2O nitrogen emitted and the ammonium nitrogen converted (N2O emission factor) were determined for each batch test. Due to the fact

that the NH4+ concentration was kept approximately constant during each batch test by periodical addition of NH4+HCO3, the converted NH4+ was calculated based on the amounts of NH4+ added and the measured NH4+ concentration profile. N2OR was calculated by multiplying the measured gas phase N2O concentration and the known gas flow rate. The average N2OR over each testing period (with constant conditions applied) was calculated by averaging the measured N2OR over the period (relatively constant in all cases). The biomass-specific N2OR (mg N2O-N/hr/g VSS) and biomass-specific AOR (mg NH4+-N/hr/g VSS) were calculated by normalising the N2OR and AOR data with the MLVSS concentration of the AOB + NOB sludge. The N2O emission factor was calculated based on the ratio between the total N2O emitted

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(mg N2O-N) and the total NH4+ converted (mg NH4+-N) during each batch test. In all the plotted graphs, error bars show the standard deviation calculated from triplicate

tests.

2.5. Isotopic measurement

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200 mL mixed liquor samples were taken in triplicate at the end of one batch test at each DO level for isotopic measurement. The samples were placed in 200 mL bottles with 150 µL saturated mercury chloride and sealed. Bulk isotope and isotopomer signatures were measured with cavity ring down spectroscopy (G5101-i Picarro Inc., Santa Clara, CA. USA). A 50 mL of sample was replaced by 50 mL of zero air (i.e. N2O free synthetic air, Air Liquide, Aust.) in bottles containing the wastewater

samples. After 24 hr on a shaker table (100 rpm) the equilibrated headspace gas was transferred into a 500 mL gas sampling bag (CaliBond 5) using a gas tight syringe. The headspace in each bottle was replaced with tap water that had been bubbled with zero air for 0.5 hr. The headspace gas in the sample bags was diluted with appropriate amounts of zero air to give a final N2O concentration between 0.5 and 1.5 ppmv.

The prepared headspace samples were attached to the G5101-I inlet. Prior to entering the instrument the gas samples were passed through a series of filters. These included water (Drierite), CO2 (Ascarite), CO (Pt granules) and H2S (Cu filings) traps. The G5101-i is sensitive to variable N2O and O2 concentrations and corrections were applied post analysis for these parameters. The G5101-i has a precision of ± 2‰ for alpha and beta isotopomers. The bulk 15N-N2O was given as the average of the alpha and beta values. The site preference (SP) was calculated as the difference between

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alpha and beta isotopomer signatures.

The contributions of the two pathways to the total N2OR were estimated using

Equation 1: (1) adapted from Wunderlin et al. (2013) where: FND = the fraction of N2OR from AOB denitrification, FNO = the fraction of

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N2OR from NH2OH oxidation, SPtot = measured SP value at varying DO levels, 28.5‰ = the SP value of N2O produced from NH2OH oxidation (Wunderlin et al., 2013), and -2‰ = the SP value of N2O produced from AOB denitrification (Wunderlin et al., 2013).

2.6. Chemical analysis

The NH4+, NO3- and NO2- concentrations were analysed using a Lachat QuikChem8000 Flow Injection Analyser (Lachat Instrument, Milwaukee). The MLSS

concentration and its volatile fraction (MLVSS) were analysed in triplicate according to the standard methods (APHA1998).

2.7. Model-based estimation of N2O production by the two pathways A previously proposed N2O model incorporating both the NH2OH oxidation and AOB denitrification pathways was employed to interpret the experimental data. The key feature of the model is that the model links the oxidation and reduction processes through a pool of electron carriers (Ni et al., 2014). The stoichiometry and kinetics of the two-pathway N2O model, as well as the parameter values used, are summarized in Table S1 and S2 (refer to Supplementary Information). Two model parameters, namely the maximum ammonia oxidation rate ( ) and the oxygen affinity

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constant for ammonia oxidation (

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AOR data from the eight sets of batch tests. The maximum oxygen reduction rate ), maximum nitrite reduction rate and maximum NO reduction rate

(

(

), which are the key parameters governing the N2O production via the two

pathways, were estimated using the experimental N2OR data from eight sets of batch tests. All other parameters were adapted from the literature.

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300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 3. Results 3.1. N2O production in the batch tests Parameter estimation and parameter uncertainty evaluation were done according to Batstone et al. (2003). The standard errors and 95% confidence intervals of individual parameter estimates were calculated from the mean square fitting errors and the sensitivity of the model to the parameters. The determined F-values were used for parameter combinations and degrees of freedom in all cases. A modified version of AQUASIM 2.1d was used to determine the parameter surfaces (Ge et al., 2010).

As an example, Figure 1 shows the dissolved and gaseous N2O profiles along with the DO, NH4+, NO2- and NO3- profiles in a batch test with DO at 0.5 mg O2/L. The profiles of all these variables in all other tests displayed very similar trends (Figure S1). During the 20 min control phase without NH4+ addition, N2O concentrations in both the liquid and gas phases remained constant at zero, indicating no N2O production from heterotrophic denitrification, likely due to the lack of readily degradable carbon sources. Moreover, the results of the N2O consumption tests (Figure S2) indicated that the ordinary heterotrophic organisms (OHO) consumed N2O at negligible rates, also likely due to the lack of readily degradable carbon

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sources (Details are shown in Supplementary Information). Therefore, the effect of

heterotrophic denitrification on N2O production during the entire test was negligible. After the addition of NH4+, both liquid and gaseous N2O concentrations increased

sharply and reached steady state within 10 min. N2OR in the pseudo steady state was determined as 1.09 mg N/hr/g VSS for this test. The NH4+ concentration was relatively constant at 18 mg N/L due to periodic addition of NH4+. The NO2-

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concentration was around 0.5 mg N/L during the entire test. NO3- concentration was around 1000 mg N/L, a level that was also observed in the parent reactors. AOR in this test was determined from the NH4+ profile and the amount added, as 19.3 mg N/hr/g VSS. The N2O emission factor was calculated to be 5.4% for this test.

In batch tests, with the increase of DO concentration from 0 to 3.0 mg O2/L, AOR

increased from 0 to 74 ± 1.6 (n=3) mg N/hr/g VSS (Figure 2a), while its increasing rate decreased. The N2OR increased quickly with the increasing DO concentration from 0 to 1.0 mg O2/L, but stabilized at approximately 1.9 mg N/hr/g VSS for DO concentrations in the range of 1.0 – 3.0 mg O2/L (Figure 2b). The correlation between N2OR and AOR was positive when the AOR was below 40 mg N/hr/g VSS, above which the N2OR became independent of the AOR (Figure 2c). In contrast, a negative correlation between the N2O emission factor and the DO concentration was detected (Figure 2d). The highest N2O emission factor (10.6 ± 1.7%, n=3) was achieved at the lowest non-zero DO concentration used in this study (0.2 mg O2/L) and it dropped sharply to 5.2% when the DO concentration increased to 0.5 mg O2/L. The emission

factor further decreased with the increase of DO, albeit at a lower rate. The lowest N2O emission factor of 2.4 ± 0.1% (n=3) was observed at the highest DO level of 3.0 mg O2/L.

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3.2. Model-based analysis of the batch test data to estimate the contributing

pathways

The calibration of the N2O model involved optimizing key parameter values for the N2O production via the two pathways by fitting simulation results to the eight sets of batch data under various DO conditions. The obtained values of the five key

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parameters (

,

,

,

and

) are comparable with those

reported in Ni et al. (2014) based on the two-step calibration procedure. The 95% confidence regions for all the parameter pairs were bound by small ellipsoids having mean values for the parameter estimates approximately at the center, indicating good identifiability of these five estimated parameters (Figure S4).

The model simulated N2OR fitted well with the batch test measured N2OR at all DO concentrations (Figure 3). The simulated N2OR consists of two parts, namely N2OR via the AOB denitrification pathway and N2OR via the NH2OH oxidation pathway. The model-based data analysis indicated that N2OR from the AOB denitrification pathway increased as DO increased from 0 to 1.0 mg O2/L and then remained almost constant in the DO concentration range of 1.0 to 3.0 mg O2/L. The model-predicted N2OR from the NH2OH oxidation pathway increased with DO increase in the entire DO concentration range studied. At all DO levels, N2OR from AOB denitrification dominated over that from NH2OH oxidation (Figure 3).

3.3. Isotopic analysis to identify the contributing pathways The δ15Nbulk and the SPs of the produced N2O during batch tests under varying DO levels were determined (Figure 4). The δ15Nbulk of N2O increased from -67.8‰ to -

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36.9‰ with the increase of DO level from 0.2 to 3.0 mg O2/L (Figure 4a). The SP

results also showed a clearly increasing trend (from -0.5‰ to 6.4‰) as DO increased

from 0.2 to 3.0 mg O2/L (Figure 4b). In the lower DO (0.2 – 0.5 mg O2/L) and higher

DO (1.5 – 3.0 mg O2/L) ranges, the SP values increased gradually with increased DO concentration. A rapid SP value increase was observed when the DO increased from 0.5 to 1.5 mg O2/L. The lowest SP value (-3.4‰) observed at DO level of 1.0 mg

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O2/L is likely an outliner possibly due to improper preservation of the samples on the day, and is excluded from further data analysis.

From the SP data the contributions of the AOB denitrification and the NH2OH oxidation pathways to the overall N2O production at all DO levels was estimated (Equation 1). It was seen that the contribution of the AOB denitrification pathway

decreased from 95% to 73% as DO increased from 0.2 to 3.0 mg O2/L, while the contribution of the NH2OH oxidation pathway increased from 5% to 27% upon increasing DO concentrations (Figure 5). These results are consistent with the modelpredicted contributions of the two pathways as shown in Figure 3 (Figure 5). The

model predicted that the contribution of the AOB denitrification pathway decreased from 92% to 66% when DO concentration increased from 0.2 to 3.0 mg O2/L, while the contribution of the NH2OH oxidation to N2OR increased from 8% to 34% with the DO increase.

4. Discussion

4.1. The effect of DO concentration on N2O production by AOB The effect of DO concentration on N2O production by AOB in wastewater treatment has not been fully elucidated, due to the interfering factors such as the accumulation

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of nitrite when DO was varied and also the presence of N2O producing/consuming

heterotrophic bacteria (refer to Introduction). As summarized in Table 1, such

interferences have likely resulted in inconsistent observations.

The lowest NO2- accumulation in the preliminary batch tests using the enriched AOB+NOB culture alone was above 3 mg N/L due to the unbalanced microbial

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activity between AOB and NOB. However, in the eight sets of batch tests with addition of NOB culture, N2O production was investigated under the conditions of low NO2- accumulation (< 2.0 mg N/L in all tests, and < 1.5 mg N/L in most cases). Further, the NO2- profiles in each set of batch test showed very similarly increasing trends (with the exception of Figure S1a with DO = 0 mg O2/L). The consistency in the nitrite profiles in all tests implies that, the effect of the low level accumulation of

nitrite on N2O production, if any, would have been similar in all tests. Therefore, the

differences in N2O production in different tests can be attributed to DO variation. It should be highlighted that, as nitrite is a product of ammonium oxidation, it is not possible to completely eliminate nitrite during ammonium oxidation. Even in the absence of nitrite accumulation in the bulk, some accumulation of nitrite in bacterial cells is expected.

Under the conditions of low NO2- accumulation, minimum interference by heterotrophic bacteria, and controlled NH4+ and pH levels, we were able to successfully isolate the effect of DO as the primary varying factor for the first time. The results of this work revealed that N2OR increased as DO concentration increased from 0 to 3.0 mg O2/L, while the N2O emission factor decreased upon increasing DO (Figure 2b and 2d). Some full-scale and lab-scale studies have also observed this

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increase of N2OR against increasing DO (Foley et al., 2010; Ahn et al., 2010; Law et al., 2012a; Ni et al., 2013). In a similar nitrifying culture study by Kampschreur et al.

(2007) the emission factor was 2.8% at a DO of 4.6 mg O2/L, which is consistent with our result of 2.4% at a DO of 3.0 mg O2/L. Other studies have also detected a negative

correlation between the DO and the N2O emission factor in batch tests using pure and enriched cultures (Goreau et al., 1980; Zheng et al., 1994).

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425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 Our findings could partially offer an explanation of those inconsistent observations regarding the effects of DO (Tallec et al., 2006; Yang et al., 2009; Law et al., 2012a). In comparison to the cultures used in this study and in Kampschreur et al. (2007), those used in Tallec et al. (2006), Yang et al. (2009) and Wunderlin et al. (2012) most certainly contained a substantial amount of heterotrophic biomass due to the use of

real wastewater. Contrary to what we observed in this study that heterotrophic bacteria had a negligible involvement in N2O production and consumption,

heterotrophic bacteria in their sludges likely contributed to both N2O production and consumption. As DO concentration is known to influence both N2O production and consumption by heterotrophic bacteria (Law et al., 2012b), the effects of DO on N2O production observed in some of the previous studies may not be solely associated with AOB. The enriched AOB culture in the study of Law et al. (2012a) was adapted to higher temperature (33 ºC) and high levels (500 mg N/L) of NH4+ and NO2-. The observed N2OR dependency on DO may be quite unique for such conditions. Indeed, Law et al. (2013) demonstrated that AOB denitrification was likely completely suppressed by nitrite, and therefore the DO effect on N2OR was likely linked to its effect on the NH2OH oxidation pathway only.

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4.2. Dependency of AOB N2O production pathways on DO levels Isotopic techniques have been used as an important tool for source-identification of

N2O production in wastewater treatment (Decock and Six, 2013; Wunderlin et al.,

2013). Unlike δ15N, which depends on isotopic composition of substrates, SP has a major advantage of being independent of the precursors (Toyoda et al., 2002). The SP values measured in this study varied between -0.5‰ and 6.4‰ rising with DO

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increasing levels (Figure 4b). This is consistent with other reported SP values during NH4+ oxidation (e.g. -5.8‰ to 5.6‰) (Wunderlin et al., 2013). Previous studies have shown a large variation of SP values between the AOB denitrification pathway (−10.7 ± 2.9‰ to 0.1 ± 1.7‰) and the NH2OH oxidation pathway (30.8 ± 5.9‰ to 36.3 ± 2.4‰) (Sutka et al., 2006; Frame and Casciotti, 2010; Wunderlin et al., 2013). The

observed SP values in our study were well between these two ranges, indicating that the N2O production from all batch tests resulted from a combination of the two N2O production pathways. The large difference of the SP values associated with the N2O produced by AOB denitrification and NH2OH oxidation also provides an opportunity

to identify the relative contribution of each pathway. Based on the SP analysis (Equation 1), it is revealed that AOB dentrification was the dominant pathway (73% – 95%) over the NH2OH oxidation pathway (5% – 27%) for the AOB culture studied in this work (Figure 5). This finding is consistent with some previous studies of soil and mixed cultures (Ostrom et al., 2010; Toyoda et al., 2010; Wunderlin et al., 2013).

The SP results (Figure 5) also demonstrated that at higher DO the relative contribution of the NH2OH oxidation pathway increased. These results are in good agreement with the prediction by a mathematical model that integrated the AOB denitrification and the NH2OH oxidation pathways (Figure 3, Figure 5). In addition,

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results of linear regression showed that the effect of NO2- on N2O production occurred

at these low NO2- accumulation levels (Figure S5) was not significant (R2 = 0.29),

compared to the effect of DO (R2 = 0.81). Collectively these demonstrate that DO is an important factor regulating the relative contributions of the two pathways. The

model-based data analysis further revealed that N2OR due to the NH2OH oxidation pathway increased almost linearly (R2=0.92) with DO concentration (Figure 3), which

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is in agreement with the results of Law et al. (2013). With the AOB denitrification pathway largely suppressed (Law et al., 2013; Ni et al., 2014), it is reported that the enriched AOB culture produces N2O at a rate almost proportional to DO concentration.

In other studies, it is reported that AOB denitrification is promoted at oxygen limiting conditions (Tallec et al., 2006; Kampschreur et al., 2007). However, the model based data analysis in this study suggested that the biomass-specific N2O production rate from AOB denitrification increased as DO concentration increased. One explanation is that, as nitrite was quickly oxidised to nitrate at all DO levels by the enhanced NOB population used in this study, the possible stimulating effect of NO2- accumulation, which often happens particularly at low DO levels, on N2O production by AOB would thus be diminished. It has indeed been frequently reported that nitrite accumulation stimulates N2O production (Tallec et al., 2006; Foley et al., 2010; Kim et al., 2010).

Our observation that N2OR from AOB denitrification increased upon increasing DO likely resulted from increased electron flow during the higher levels of ammonia and hence NH2OH oxidation. It is probable that the electron flux to AOB denitrification also increased as a result. This hypothesis should be understood in the context that lower DO concentrations did lead to higher N2O emission factors (Figure 2d),

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implying that the fraction of electrons distributed to AOB denitrification decreased

with increasing DO.

5. Conclusions

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In this work, the effect of DO on the N2O production by an enriched nitrifying culture was investigated under the condition of low nitrite accumulation (< 1.5 mg NO2-N/L). The main conclusions are: • The N2O production rate was found to increase with increased DO concentration in the studied range of 0 – 3.0 mg O2/L, while the N2O emission factor decreased substantially with increased DO. •

The site preference measurements indicated that both the AOB denitrification and NH2OH oxidation pathways contributed to N2O production.

•

The experimental observations were well described by a two-pathway N2O model that including both the AOB denitrification and NH2OH oxidation pathways.

•

Both the isotope measurement and the modeling results suggested that the AOB denitrification pathway dominates (66% – 95%) over the NH2OH

at low NO2- concentrations (< 1.5 mg NO2--N/L) with DO playing a pivotal role in affecting their relative contributions.

Acknowledgements

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We acknowledge the Australian Research Council (ARC) for funding support through

Project LP0991765 and DP0987204. Mr. Lai Peng acknowledges the scholarship support from China Scholarship Council. Dr Bing-Jie Ni acknowledges the support of

the Australian Research Council Discovery Early Career Researcher Award

DE130100451.

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List of tables and figures

Table 1. Summary of previous studies on the effect of DO on N2O production by nitrifying cultures.

Table 2. Experimental conditions applied in the batch tests.

 Figure 1. Levels of gas phase N2O (—), liquid phase N2O (), NH4+ ( ), NO2- ( ) and NO3- ( ) in a batch test with DO (—) at 0.5 mg O2/L and pH at 7.5. Figure 2. Relationships between (a) AOR and DO, (b) N2OR and DO, (c) N2OR and AOR and (d) the N2O emission factor and DO detected in batch tests (error bars in all plots are standard deviation, n=3). Figure 3. The experimentally observed and model predicted correlations between

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N2OR and DO.

Figure 4. δ15Nbulk and SP of N2O produced during batch tests under varying DO levels (error bars in all plots are standard deviation, n=3).

Figure 5. The site estimated relative contributions of the two pathways for N2O production under varying DO concentrations.

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Table 1. Summary of previous studies on the effect of DO on N2O production by nitrifying cultures.

Goreau et al. (1980)

A pure culture of AOB; DO: 0.18, 0.35, 1.8, 3.5 and 7 mg O2/L; NO2-: 3.5-37.8 mg N/L

condition; NO2-: average at 14.4 mg N/L Kester et al. (1997) Tallec et al. (2006) Nitrosomonas europaea; DO: 0-7 mg O2/L; NO2-:109-123 mg N/L

SC

Poth and Focht (1985)

Nitrosomonas europaea are grown under well-aerated condition and static

Full-scale activated sludge; DO was controlled at levels from 0.1-6.2 mg O2/L; NH4+: 0-20 mg N/L

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Enriched nitrifying culture; DO: imposition of transient anoxia; NO2-: around 20

Yang et al. (2009)

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effluent were 2.2±1.1 and 240±41 mg N/L, respectively.

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A pure culture of AOB; DO: imposition of transient anoxia; NH4+ and NO2- in the

fully aerobic; (2) anoxic-aerobic with high DO; (3) anoxic-aerobic with low DO; (4) intermittent aeration; NO2-: 0-11 mg N/L Wunderlin et al. (2012) Pilot-scale activated sludge treating domestic wastewater; DO was controlled at N2O production was dynamic and variable, but not oxygen-dependent.

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Rassamee et al. (2011)

Lab-scale activated sludge treating municipal wastewater; four DO conditions: (1)

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Reference

Experimental Conditions

Remarks

Both the N2O production rate and the emission factor increased when DO was reduced. Nitrosomonas europaea produced N2O only under oxygen limiting

Highest N2O production occurred at 0.087 mg O2/L. Highest N2O emission was observed at 1.0 mg O2/L.

N2O emission increased with decreasing oxygen.

Highest N2O production was observed at 1.0 mg O2/L.

Recovery from anoxia to aerobic conditions led to high N2O

Changes in aeration induced higher N2O production.

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different levels from 0.6-3.1 mg O2/L; four series of batch tests with different

(4) NH2OH (10 mg N/L). Law et al. (2012a)

Enriched AOB culture treating anaerobic sludge digestion liquor; DO: 0.55, 1.25, 1.8 and 2.4 mg O2/L; NO2-: 500±50 mg N/L; NH4+: 500±50 and 50±5 mg N/L.

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additions of (1) NH4+ (25 mg N/L); (2) NO2- (15 mg N/L); (3) NO3- (20 mg N/L);

An exponential relationship between the N2O production and ammonia oxidation rate was found.

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Table 2. Experimental conditions applied in the batch tests. Ammonium Test series pH (mg N/L) I II III IV V VI VII VIII 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 18±2 18±2 18±2 18±2 18±2 18±2 18±2 18±2 3.0 DO (mg O2/L)

2.0 1.5 1.0 0.5 0.2 0

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Liquid Phase N2O (mg N2O-N/L)

NH4 /NO2 /NO3 (mg N/L)

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2.8 mg NH4 -N/L 2.8 mg NH -N/L 4 2.8 mg + NH4 -N/L

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Supplementary Information

The effect of dissolved oxygen on N2O production by ammonia-oxidizing bacteria in an enriched nitrifying sludge

Lai Penga, Bing-Jie Nia, Dirk Erlerb, Liu Yea,c, Zhiguo Yuana*

Advanced Water Management Centre, The University of Queensland, St. Lucia,

Brisbane, QLD 4072, Australia b Centre for Coastal Biogeochemistry Research, Southern Cross University School of Chemical Engineering, The University of Queensland, St. Lucia, Brisbane,

c

QLD 4072, Australia

zhiguo@awmc.uq.edu.au

The following are included as supplementary information for this paper: number of pages: 10

number of figures: 5 number of tables: 2

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Zhiguo Yuan, P + 61 7 3365 4374; F +61 7 3365 4726; E-mail

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*Corresponding author:

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ACCEPTED MANUSCRIPT
KLa test. An experiment for determining volumetric oxygen mass transfer coefficient (KLa) was conducted before each batch test. The calculated KLa was used to calculate the oxygen uptake rate (OUR) based on the on-line measured DO signals and the ammonia oxidation rate (AOR) in order to manually adding NH4+ into the reactor to

detailed experimental procedures: (1) 0.5 L/min nitrogen was sparged into batch reactor until DO reach zero; (2) 0.5 L/min air was then sparged until the reading of

based on Equation S1. The calculated KLa was used to calculate OUR and AOR

(S1)

Where KLa = volumetric mass transfer coefficient, T-1

Cts = saturated oxygen concentration in liquid bulk phase at time t

Where AOR = ammonia oxidation rate OUR = oxygen uptake rate

4.33 = constant, g O2/g N (Wezernak and Gannon 1967) 8.69 = oxygen concentration in equilibrium with gas as given by Henry’s Law, mg/L (Tchobanoglous et al., 2003) Qair = air flow rate, L/min Qtotal = total flow rate (air + nitrogen), L/min Ct = oxygen concentration in liquid bulk phase at time t

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(S2)

(Equation S2).

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DO meter is stable; (3) Experimental data of DO was input into a designed model

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keep NH4+ concentration constant during all the batch tests. The following is the

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N2O consumption test. The ratios of N2O consumption rate to N2O production rate of batch tests at each DO level are summarized as: 5.33% (DO of 3.0 mg O2/L), 6.52% (DO of 1.5 mg O2/L) and 7.74% (DO of 0.5 mg O2/L) (Figure S1). Therefore, in comparison to the N2O production rates of AOB, the consumption rates are negligible.

Additional References

Wezernak, C.T., Gannon, J.J., 1967. Oxygen-nitrogen relationships in autotrophic nitrification. Applied Microbiology 15, 1211-1214.

Tchobanoglous, G., Burton, F., Stensel, H.D., 2003. Wastewater Engineering: Treatment and Reuse, fourth ed. Metcalf & Eddy Inc. McGraw Hill Education: New York.

Ni, B.J., Peng, L., Law, Y., Guo, J., Yuan, Z., 2014. Modeling of nitrous oxide production by autotrophic ammonia-oxidizing bacteria with multiple production pathways. Environmental Science & Technology 48, 3916-3924.

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Table S1. N2O production model involving both the NH2OH oxidation and the AOB denitrification pathways
Variable Process SO2 mmol/L SNH3 mmol/L SNH2OH mmol/L SNO2 mmol/L SNO mmol/L SN2O mmol/L SMox mmol/L SMred mmol/L Kinetic rate expressions

SC rNH 3,ox

1-R1

-1

-1

1

1

-1

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-3/2 3/2 -1/2 1/2 1/2 1/2 -1/2

2-R2

-1

1

rNH 2OH ,ox

3-R3

1

-1

rNO ,ox

4-R4

-1

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rNO ,red

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5-R5

-1/2

1

-1

rO 2,red

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6-R6

-1

1/2

1

-1

rNO 2,red

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7

S Mred

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SO2 SO2 K O 2, NH 3 S NO K NO ,ox S NO S NO K NO ,red SO2 K O 2,red SO2 S NO

S NH 3 S Mred X AOB K NH 3 S NH 3 K Mred,1 S Mred

S NH 2OH S Mox X AOB K NH 2OH S NH 2OH K Mox S Mox
S Mox X AOB K Mox S Mox S Mred X AOB K Mred, 2 S Mred S Mred X AOB K Mred,3 S Mred

S NO 2 S Mred X AOB K NO 2 S NO 2 K Mred, 4 S Mred

S Mox

Ctot

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Table S2. Kinetic and stoichiometric parameters of the N2O model
Parameter Definition Maximum ammonia oxidation rate Values 17.70 Unit mmol/(g VSS*h) Source Estimated

rNH 3,ox rNH 2 OH ,ox rNO ,ox rO 2 , red

Maximum NH2OH oxidation rate

22.86

mmol/(g VSS*h)

Ni et al. (2014)

Maximum NO oxidation rate

22.86

RI PT mmol/(g VSS*h) mmol/(g VSS*h) mmol/(g VSS*h) mmol/(g VSS*h) mmol O2/L mmol N/L mmol N/L mmol N/L mmol O2/L mmol N/L mmol N/L mmol/g VSS mmol/g VSS mmol/g VSS mmol/g VSS mmol/g VSS mmol/g VSS

Ni et al. (2014)

Maximum oxygen reduction rate

38.5

Estimated

rNO 2,red rNO ,red K O 2, NH 3
K NH 3
K NH 2OH

Maximum nitrite reduction rate

3.92

Estimated

Oxygen affinity constant for ammonia oxidation

SC
4.7×10-2 1.7×10-1 5×10-2 6×10-4

Maximum NO reduction rate

1.2×10-2

Estimated

Estimated

Ammonia affinity constant for ammonia oxidation NH2OH affinity constant for NH2OH oxidation

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K NO ,ox K O 2,red
K NO 2

NO affinity constant for NO oxidation

Ni et al. (2014)

Oxygen affinity constant for oxygen reduction

1.9×10-3 1×10-2 6×10-4

Ni et al. (2014)

K Mox

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K NO , red

NO affinity constant for NO reduction

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Nitrite affinity constant for nitrite reduction

Ni et al. (2014)

Ni et al. (2014)

SMox affinity constant for NH2OH and NO oxidation

1×10-2×Ctot 1×10-3×Ctot

Ni et al. (2014)

SMred affinity constant for ammonia oxidation

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K Mred,1 K Mred, 2 K Mred,3 K Mred, 4
C tot

Ni et al. (2014)

SMred affinity constant for NO reduction

1×10-3×Ctot

Ni et al. (2014)

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SMred affinity constant for oxygen reduction

6.9×10-2

Ni et al. (2014)

SMred affinity constant for nitrite reduction

1.9×10-1 1×10-2

Ni et al. (2014)

The sum of SMred and SMox, which is a constant

Ni et al. (2014)

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N2OR (mg N/hr/g VSS)

2.0 1.5 1.0 0.5 0.0

(a)

20 15 10 5 0

N2OR (mg N/hr/g VSS)

2.0 1.5 1.0 0.5 0.0

3 2 1 0

(b)

20 15 10 5 0

NH4 and NO2 (mg N/L)

NH4 and NO2 (mg N/L)

3 2 1 0

DO (mg O2/L)

DO (mg O2/L)

+

+ -

-

0.0 0.2 0.4 0.6 0.8 1.0
Time (hour)

0.0
N2OR (mg N/hr/g VSS)

0.5

1.0
Time (hour)

1.5

2.0

N2OR (mg N/hr/g VSS)

2.0 1.5 1.0 0.5 0.0

(c)

20 15 10 5 0

2 1 0

1.0 0.5 0.0 0.0 0.5

0.0
N2OR (mg N/hr/g VSS)

0.5

1.0
Time (hour)

1.5

2.0 20 15 10 5 0

1.0

Time (hour)

2.0 1.5 1.0 0.5 0.0

N2OR (mg N/hr/g VSS)

(e)

2.0 1.5 1.0 0.5 0.0

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0.0 0.5 1.0 1.5 2.0
Time (hour)

3 2 1 0

(f)

0.0
N2OR (mg N/hr/g VSS)

0.5

Time (hour)

1.0

1.5

2.0

N2OR (mg N/hr/g VSS)

2.0 1.5 1.0 0.5 0.0

(g)

20 15 10 5 0

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-

(h)

0.0

0.5

1.0

1.5

2.0

0.0

0.5

1.0
+

Time (hour)

Time (hour)

DO concentrations (—) of (a) 0 mg O2/L, (b) 0.2 mg O2/L, (c) 0.5 mg O2/L, (d) 1.0 mg O2/L, (e) 1.5 mg O2/L, (f) 2.0 mg O2/L, (g) 2.5 mg O2/L, and (h) 3.0 mg O2/L (error bars in all plots are standard deviation, n=3).

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set of batch test with NO3- concentrations of approximate 1000 mg N/L and at varying

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Figure S1. The average gaseous N2O (—), NO2 () and NH4 () profiles in each

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10 5 0 2 1 0
-

NH4 and NO2 (mg N/L)

2.0 1.5

3

(d)

20 15

NH4 and NO2 (mg N/L)

3

DO (mg O2/L)

DO (mg O2/L)

+ + + -

+

1.5

2.0

NH4 and NO2 (mg N/L)

20 15 10 5 0

NH4 and NO2 (mg N/L) NH4 and NO2 (mg N/L)

3 2 1 0

DO (mg O2/L) DO (mg O2/L)

DO (mg O2/L)

+ + -

20 15 10 5 0 1.5 2.0

NH4 and NO2 (mg N/L)

3 2 1 0

DO (mg O2/L)

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4 0.6 0.5

Liquid Phase (mg N2O-N/L)

DO Concentration (mg O2/L)

3 0.4 2 0.3

N O Concentration in

2

DO Liquid Phase N2O
1

0.2 0.1

0 0.0 4

0.1

0.2 0.3 Time (hour)

0.4

0.0 0.5

0.6 0.5 0.4 0.3 0.2 0.1

DO Concentration (mg O2/L)

3

DO Liquid Phase N2O

2

1

0 0.0 4

0.1

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DO Liquid Phase N2O
0.1 0.2 0.3 Time (hour) 0.4

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0.2 0.3 Time (hour) 0.4

DO Concentration (mg O2/L)

3

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2

1

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0 0.0

Figure S2. N2O (in liquid phase) and DO concentrations of three N2O consumption tests.

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Liquid Phase (mg N2O-N/L) N O Concentration in
2

0.0 0.5 0.6 0.5

0.4 0.3 0.2 0.1 0.0 0.5

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Liquid Phase (mg N2O-N/L) N2O Concentration in

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2.0
Measured N2OR

Specific N2O Production Rate

Model-predicted N2OR

1.5

Model-predicted N2OR by NH2OH oxidation pathway Model-predicted N2OR by AOB denitrification pathway

(mg N/hr/g VSS)

1.0

0.5

0.0 0.0

0.5 Time (hour)

1.0

Figure S3. The experimentally observed and model predicted dynamic N2O profiles

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of a batch test with DO at 0.5 mg O2/L and pH at 7.5.

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1.5

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18.4 18.2 (a)

0.0130 (b)

(mmol/hr/g VSS)

rNO,red (mmol/hr/g VSS)

18.0 17.8 17.6 17.4 17.2 17.0 0.042

0.0125

0.0120

NH3,ox

0.0115

r

0.044

0.046

0.048

0.050

0.052

0.0110 3.850
39.0

3.875

3.900

KO2,NH3 (mmol/L)
39.0 (c) 38.8

rNO2,red (mmol/hr/g VSS)

rO2,red (mmol/hr/g VSS)

rO2,red (mmol/hr/g VSS)

38.8

38.4

38.4

38.0 3.850

3.875

3.900

3.925

3.950

3.975

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4.000

38.2

38.2

38.0 0.0110

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0.0115 0.0120 0.0125 0.0130

38.6

38.6

rNO2,red (mmol/hr/g VSS)

rNO,red (mmol/hr/g VSS)

Figure S4. 95% confidence regions for the parameter combinations among the key model parameters for the N2O production processes by AOB with the best fits in the

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; (c)

vs.

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center, as well as their standard errors: (a) ; (d) vs. .

vs.

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3.925 3.950 3.975
(d)

4.000

; (b)

vs.

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100

Contribution of AOB Denitrification Pathway to N2O Production (%)

90

80

R = 0.2916

2

70

SP prediction Model prediction Linear fit

NO2- concentration.

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Figure S5. The correlations between the contribution of AOB denitrification and

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60 0.75

1.00 1.25 1.50 NO2 Concentration (mg N/L)

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1.75