Source identification of N 2 O produced during simulated wastewater treatment under different oxygen conditions using stable isotopic analysis

Nitrous oxide (N2O), a potent greenhouse gas which is important in climate change, is predicted to be the most dominant ozone depleting substance. It is mainly produced by oxidation of hydroxylamine (NH2OH) or reduction of nitrite (NO2 ) during microbiological processes such as nitrification and denitrification. Wastewater treatment plant (WWTP) is one of the anthropogenic N2O sources because inorganic and organic nitrogen compounds are converted to nitrate (NO3 , in the case of standard system) or N2 (in the case of advanced system) by bacterial nitrification and denitrification in WWTP. We investigated the N2O production mechanisms during batch experiments that simulate wastewater treatment with activated sludge under various dissolved oxygen (DO) concentrations by stable isotope analysis. About 125 mL of water was sampled from 30L incubation chamber for several times during the incubation, and concentration and isotopomer ratios of N2O and N-containing species were measured using gas chromatography/isotope ratio mass spectrometry (GC/IRMS). Ammonium (NH4 ) consumption was accompanied by increment of nitrite (NO2 ), and at the same time dissolved N2O concentration gradually increased to 4850 and 5650 nmol kg, respectively, during the four-hour incubation when DO concentrations were 0.2 and 0.5 mg L. Observed low SP values (0.2-8.9% at DO-0.2 mg L, -5.3-6.3% at DO-0.5 mg L, -1.0-8.3% at DO-0.8 mg L) in N2O and relationship of nitrogen isotope ratios between N2O and its potential substrates (NH4 , NO3 ) suggested that N2O produced under the aerobic condition derived mainly from NO2 reduction by ammonia-oxidizing bacteria (nitrifier –denitrification).


INTRODUCTION
N 2 O is a significant greenhouse gas, with an approximately 300-fold stronger effect than carbon dioxide [1].N 2 O also contributes to the destruction of the stratospheric ozone layer [2].N 2 O emission resulting from microbial activity has been detected in many man-made and natural environments, such as wastewater treatment facilities, plants, soils, and sediments.A major danger is that the proportion of N 2 O emission from wastewater treatment will increase continuously [3,4].N 2 O emission can result from the activity of different groups of microorganisms in used wastewater treatment: ammonia oxidizing bacteria (AOB, [5]), nitrite oxidizing bacteria (NOB, [6]), and denitrifying microorganisms [7].The major microbial N 2 O production pathways are nitrification, denitrification, and nitrifier-denitrification [8][9][10].N 2 O is produced as a byproduct of hydroxylamine (NH 2 OH) oxidation (Eq.( 1)), an intermediate during nitrate (NO 3 -) reduction to dinitrogen (N 2 ) (Eq. ( 2)), and as the end product of nitrifier-denitrification (Eq.( 3)).
Given the high global warming potential, N 2 O emissions are a critical component of greenhouse gas emissions reduction targets.However, contributions of individual sources to regional N 2 O budgets are difficult to estimate because of large spatial and temporal variability in fluxes [11].Therefore, it is necessary to evaluate the relative importance of ) during microbiological processes such as nitrification and denitrification.Wastewater treatment plant (WWTP) is one of the anthropogenic N 2 O sources because inorganic and organic nitrogen compounds are converted to nitrate (NO 3 -, in the case of standard system) or N 2 (in the case of advanced system) by bacterial nitrification and denitrification in WWTP.We investigated the N 2 O production mechanisms during batch experiments that simulate wastewater treatment with activated sludge under various dissolved oxygen (DO) concentrations by stable isotope analysis.About 125 mL of water was sampled from 30L incubation chamber for several times during the incubation, and concentration and isotopomer ratios of N 2 O and N-containing species were measured using gas chromatography/isotope ratio mass spectrometry (GC/IRMS).Ammonium (NH 4 + ) consumption was accompanied by increment of nitrite (NO 2 -), and at the same time dissolved N 2 O concentration gradually increased to 4850 and 5650 nmol kg -1 , respectively, during the four-hour incubation when DO concentrations were 0.2 and 0.5 mg L -1 .Observed low SP values (0.2-8.9% at DO-0.2 mg L -1 , -5.3-6.3% at DO-0.5 mg L -1 , -1.0-8.3% at DO-0.8 mg L -1 ) in N 2 O and relationship of nitrogen isotope ratios between N 2 O and its potential substrates (NH 4 + , NO 3 -) suggested that N 2 O produced under the aerobic condition derived mainly from NO 2 -reduction by ammonia-oxidizing bacteria (nitrifier -denitrification).
microbial pathways for N 2 O production and consumption mechanism based on the use of adequate application which is required to estimate qualitative and quantitative information in N 2 O global budget.Stable isotope ratios of nitrogen and oxygen are acknowledged recently as useful tools for analyzing processes contributing to N 2 O production and for detecting gross N 2 O consumption [12].With knowledge of isotopic signatures of precursor materials (ammonium, NH 4 + ; nitrate, NO 3 -), and effects of O-atom exchange between water and intermediates of nitrification or denitrification processes on the oxygen isotope ratio in N 2 O [13], the isotopomer ratios of N 2 O (δ 15 N bulk and δ 18 O, see Eqs. ( 4) and (5) in Experimental section) are useful to ascertain the N 2 O formation and decomposition processes.Aside from the bulk isotope ratios (average isotope ratios for 14 N/ 15 N and 18 O/ 16 O), the 15 N site preference (SP value, indicating the difference of 15 N/ 14 N ratio between central (α) and terminal (β) nitrogen position in the linear N 2 O molecule, i.e. β N α NO) was first found to vary significantly throughout the atmosphere by Yoshida and Toyoda [14].SP, which is independent of the substrate's isotopic signature, has unique values reflecting microbial production pathways.SPs of N 2 O produced by NH 2 OH oxidation and by NO 2 -reduction were respectively about 33‰ and 0% [15][16][17][18] that can be a robust parameter in the analysis of mechanisms.In the last years, several efforts have been made to better understand the mechanisms of N 2 O production based on the isotopomer ratios of N 2 O in complex bacterial system during wastewater treatment [19][20][21][22][23].Moreover, several parameters favoring N 2 O production were identified: low dissolved oxygen (DO) concentration, accumulation of nitrite [24,25], dynamic conditions or a low ratio of carbon to Ncompounds during heterotrophic denitrification [26].However, more studies associated with isotopic analysis are needed to understand the relative contributions from each N 2 O production pathway in wastewater treatment operated under different conditions, especially dissolved DO.It would have been essential to formulate operating strategies that minimize N 2 O emissions.The aim of this study was to elucidate the production mechanisms of N 2 O in the wastewater treatment under various reduced DO conditions.In this frame, a laboratory batch-scale experiments associated with nitrification process was performed to measure the stable isotopic signatures under key controlling factor of DO.The N 2 O production pathways were identified based on enrichment factors for each microbial process and isotopic information of substrate (NH 4 + and NO 3 -) and product (N 2 O).

Lab-scale experimental apparatus:
A laboratory-scale incubation apparatus with a working volume of 30 L capacity (Fig. 1) is filled with activated sludge.Gas phase in the incubation vessel (about 7 L) was purged continuously with N 2 gas flow of about 4 L min -1 by a flow controller.The air was applied from the vessel bottom using three flow controllers to adjust the oxygen concentrations in aerobic (nitrification) experiments.Dissolved oxygen (DO), pH, and oxidation-reduction potential (ORP) were measured using oxygen, pH, and ORP electrodes (DO-31P, HM-31P; TOA-DDK), respectively.The pH was held constant at around 7.

Batch incubation experiment:
Batch experiments were conducted during February-March in 2012 with activated sludge taken from the biological oxic tank at Analysis: The concentration of dissolved NH 4 + was measured using a coulometric ammonia meter (MT-1; Central Kagaku Corp., Tokyo, Japan, and MM-60R; DKK -TOA Corp., Tokyo, Japan).Those of NO 3 -and NO 2 were measured using an ion chromatograph equipped with a conductivity detector (DX-320; Dionex Corp., Osaka, Japan).The term isotopomer first used by Toyoda and Yoshida [27], is defined as a set of molecules that are isotopically substituted, usually with stable isotopes.However, different nomenclatures exist for molecules that have the same constitution and configuration, as summarized by Toyoda et al. [28].A new term is necessary to avoid confusion.For the present study, the N 2 O isotopomer ratios (bulk nitrogen and oxygen isotope ratios,  15 N bulk and  18 O, and intramolecular 15 N site preference, SP) were measured using an online system containing preconcentration traps, chemical traps for removing H 2 O and CO 2 , and a gas chromatographisotope ratio mass spectrometer (GC-IRMS; MAT 252, Thermo Fisher Scientific K.K., Yokohama, Japan).A complete description of this approach and the procedures used for calibration of standard N 2 O for isotopomer ratios were described by Toyoda and Yoshida [27].Isotopomer ratios are noted as d values, defined according to Eqs. ( 4) and (5): In those equations, 15 R α and 15 R β respectively represent the 15 N/ 14 N ratios at central (α) and terminal (β) nitrogen position in the linear N 2 O molecule, i.e. β N α NO. 15 R bulk and 18 R respectively denote the average values of 15 N/ 14 N and 18 O/ 16 O ratios.The subscript "standard" signifies an international standard: atmospheric N 2 for N, and Vienna Standard Mean Ocean Water (V-SMOW) for O.In addition, the 15 N site preference was calculated from isotopomer ratios [27] as shown below.
In those equations, 15 R α and 15 R β respectively represent the 15 N/ 14 N ratios at central (α) and terminal (β) nitrogen position in the linear N 2 O molecule, i.e. β N α NO. 15 R bulk and 18 R respectively denote the average values of 15 N/ 14 N and 18 O/ 16 O ratios.The subscript standard signifies an international standard: atmospheric N 2 for N, and Vienna Standard Mean Ocean Water (V-SMOW) for O.
In addition, the 15 N site preference was calculated from isotopomer ratios [27] as shown below. 15N site preference (SP) = δ 15 N a -ε 15  ) in the water during aerobic incubation under three DO conditions (0.2, 0.5, and 0.8 mg L -1 ) are presented in Figure 2(a) and 3(a-c).In all three experiments, dissolved N 2 O concentration was always greater than the concentration expected under water-atmospheric equilibrium (approximately 9 nmol kg -1 at 20°C [31], indicating that N 2 O production occurred at the range of oxygen concentrations tested in this study (Fig. 2a).N 2 O production started as soon as NH 4 + was added and decreased rapidly between 15 and 30 min.It then increased to 4849.2-5649.2 nmol kg -1 at the end of time courses with lower DO conditions of 0.2 or 0.5 mg L -1 , respectively.The magnitude of the increase in N 2 O from 60 min to 240 min was the highest in the experiment with a DO setting of 0.2 mg L -1 , about three times greater than the increase observed in the experiment with a DO setting of 0.8 mg L -1 .These results agree with those of previous studies, which also found that lower DO concentrations engender higher N 2 O emissions during nitrification [25,32].At DO setting of 0.8 mg L -1 , the N 2 O concentration was ranged around 1000 nmol kg -1 throughout the time courses after 30 min of the incubation, indicating N 2 O is produced with slightly lower amount than those of at limited DO conditions, which is consistent with findings by Zheng et al. [33] who reported that the higher DO level can minimize N 2 O production.The NH 4 + concentrations in all experiments reduced gradually while the NO 2 -and NO 3 -were increasing until the end of incubation, which confirms the presence of AOB and NOB, and indicates that no significant heterotrophic activity contributing to N 2 O production in this condition.Under low oxygen condition (0.2 mg L -1 ), NH 4 + was oxidized approximately 78.1% with high oxidation rate (4.7 mmol L -1 min -1 ) at 240 min while it was about 28.7% with low oxidation rate (1.8mmol L -1 min -1 ) at higher DO of 0.8 mg L -1 (Fig. 3a).The NO 3 -and NO 2 -concentrations were increased throughout the incubation period (Fig. 3b-c).Especially, NO 2 -concentration reached to 189.8 mmol L -1 at DO of 0.2 mg L -1 , which also agrees with the results of previous studies [4,32] showing that high -as the terminal electron acceptor, nitrifier denitrification by AOB and some strains of Nitrosospira spp., which are expected to be more abundant in activated sludge, are responsible for the high N 2 O production [26] during nitrification.The interpretations based on concentration measurements are examined further using the stable isotope analysis described below.Source-partitioning of N 2 O deduced from isotopic analysis: Isotopic analysis is useful tool for differentiating the source of N 2 O production processes such as NH 2 OH oxidation and bacterial NO 2 -reduction in several environments [18,34,35].We discuss the N 2 O production processes under steady state assuming that production processes were unchanged at t=15 min or later.This assumption is based on the time series measurements.Therefore, we can regard the isotopomer ratios after 15 min as values for N 2 O produced in this system, because isotope fractionation associated with emission of dissolved N 2 O to the gas phase is small enough compared to that related to N 2 O production [36].In this study, isotope ratios of N 2 O (δ 15 N, δ 18 O, and SP values) showed variations during the incubation and variations among the experiments with different DO concentrations (Figs.2(b)-2(d).In general, the δ 15 N value of N 2 O decreased rapidly from the beginning of incubation (-29.4-30.1‰) to t=15 min (-42.1-46.9‰)at all DO settings.Thereafter, it was becoming nearly constant ranged in -44.9-49.5% between 30 and 240 min.Not so much difference in δ 15 N was occurred between experiments (Fig. 2b).The δ 18 O value of N 2 O showed a general decrease from 16.3-18.5‰at 30 min to 10.7-14.5% at 240 min after great increase at 15 min when DO were 0.2 and 0.5 mg L -1 , respectively (Fig. 2c).Basically, N 2 O reduction (consumption) is accompanied by a simultaneous increase in the δ 15 N and δ 18 O values and a diffusive loss of N 2 O from the water to the atmosphere occurs with a marginal isotope effect.Therefore, the observed decrease in the δ 15 N and δ 18 O values of N 2 O is interpreted as an isotope effect in microbial N 2 O production during the incubation.The SP value of N 2 O showed almost constant profile ranging in 0.2-8.3‰ at all experiments (Fig. 2d).The SP is independent of the isotopic signature of the substrate and has unique values that reflect microbial production pathways [15,16,18].Therefore, we analyzed the N 2 O production processes using SP-δ 15 N bulk diagram.In Figure 4, the data obtained at t = 15 min or later are plotted in SP- 15 N bulk diagram.It is suggested that N 2 O should have been produced by NH 2 OH oxidation (nitrification) or NO 2 -reduction (nitrifier-denitrification) pathways, since this experiment was conducted under oxic conditions.It is reported that the ranges of SP of N 2 O produced by the two pathways were defined as 33 ± 4‰ for NH 2 OH oxidation and -1.0 ± 5.5‰ for NO 2 reduction according to estimations based on literature values [12].The range is shown by the vertical side of the gray boxes in Figure 4. On the other hand,  15 N of N 2 O produced by each process can be estimated from  15 N of substrate (NH 4 + or NO 2 - ) and isotopic enrichment factor for the pathway using the following equation.
The  15 N-NH 4 + is estimated from NH 4 + concentration data using Rayleigh isotope fractionation model [37], Eqn. 8, assuming that initial δ 15 N-NH 4 + and e( 15 N) for NH 4 + consumption were common to all experiment.
The range of e( 15 N) NH4 +  N2O obtained by studies incubating pure culture of nitrifying bacteria under aerobic conditions (-60 to -48‰, [19]) is assumed to represent 15   indicates that the nitrifier-denitrification by AOB was the major N 2 O production process with 70-100% of contribution at all DO concentrations which is remarkably consistent with previous results [23].Finally, we checked the occurrence of the N 2 O reduction in this condition by the correlation between  15 N bulk and d 18 O.The slope has negatively correlated (slope = -0.125,R 2 =0.09 for DO-0.2 mg L -1 , slope = -0.158,R 2 =0.09 for DO-0.5 mg L -1 , slope = -0.220,R 2 =0.377 for DO-0.8 mg L -1 ) at the time intervals and was extremely lower than those of previously reported data [39].Consequently, we assumed the contribution of N 2 O reduction is small and that the change in isotopomer ratios is negligible during the emission of N 2 O from water to gas phase.

CONCLUSION
Results obtained in this study emphasize the usefulness of SP-N 2 O together with isotopic signature of NH 4 + and NO 3 -and isotopic enrichment factor for interpretation of N 2 O production mechanisms during simulation of nitrification in a lab-scale biological wastewater treatment.Based on this information, the dependency of the relative contributions on operational parameters and biological conditions that lead to N 2 O emission by nitrification in a full scale wastewater treatment system can be controlled and in so doing mitigate emissions of an important greenhouse gas.Batch incubation experiments with activated sludge were conducted under aerobic condition to investigate the critical factors that control N 2 O production.The main findings include: The dissolved N 2 O concentration was reached up to its maximum around 5649.2nmol/kg at 240 min after substrate addition when DO concentration was set at 0.2 mg L -1 .The N 2 O production was highly dependent upon DO conditions.High accumulation of nitrite can be circumstances of N 2 O production.Most of ammonia is converted into nitrate or nitrite during the incubation.There was no heterotrophic microbial activity occurred on the basis of the presence of AOB and NOB.Nitrifier-denitrification by AOB during NH 4 + oxidation was the major contributor for N 2 O production at all experiments as implied by the isotopomer analysis (SP, d 15

Fig. 2 .
Fig. 2. Time course of concentration of N 2 O (a) and its isotopomer ratios (δ 15 N bulk , SP, d 18 O) (b-d) under aerobic condition with different DO settings

Fig. 4 .
Fig. 4. Relationships between SP and δ 15 N bulk values of dissolved N 2 O at DO settings of 0.2 mg L -1 (closed symbols), 0.5 mg L -1 (half closed symbols) and 0.8 mg L -1 (open symbols).Expected ranges of δ 15 N bulk values for the N 2 O produced by NH 2 OH oxidation and NO 2 -reduction estimated using Eqn.(7) and those of SP are shown by gray boxes at the top and bottom sides.