Investigation of dissolved N 2 O production processes during wastewater treatment system in Ulaanbaatar

Nitrous oxide (N2O) is an increasing greenhouse gas in the troposphere and a potential destroyer of stratospheric ozone layer. 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 denitrifcation processes in WWTP. These major processes can be distinguished by isotopocule analysis. In order to reveal production mechanisms of N2O in a standard wastewater treatment, we made water sampling at the central WWTP in Ulaanbaatar. The water samples collected from seven stations including biological reaction tanks were measured for concentration and isotopocule ratios of dissolved N2O and other inorganic nitrogen. Dissolved N2O concentration was extremely higher than that expected under atmospheric equilibrium (about 9 nmol/l) at all stations, indicating that this system is a potential source of N2O. It showed a gradual increase with the progress of biological reaction and the highest concentration (335.7 nmol/l) was observed at station N5-4 of the aeration tank when the DO was 5.7 mg/l. Nitrification by nitrifying bacteria could actively occur by the concentration of NH4 decreased whereas NO2 and NO3 showed a temporal and monotonic increase, respectively, under high DO concentration. Although the reported values of site preference (SP) of N2O, the difference in 15N/14N ratio between central (α) and terminal (β) nitrogen, produced via NO2 reduction (SP(ND)), including both nitrifier and denitrifier denitrification, and NH2OH oxidation (SP(HO)) ranged from -10.7‰ to 0‰ and 31.4‰ to 36.3‰, respectively, the observed SP at aeration tank was close to SP(ND) rather than SP(HO). It was ranged from 0.4‰ to 13.3‰ when N2O concentration was high, implying that the NO2 reduction made a greater contribution to N2O production. Slightly elevated SP (13.3‰) only at station N5-1 was derived from the mixing of N2O produced via NH2OH oxidation and the maximal contribution of this pathway was estimated to be about 40%. In other words, the contribution of NO2 reduction was more than 60%.


INTRODUCTION
Nitrous oxide (N 2 O) plays an important role as a greenhouse gas and catalyst in the destruction of ozone in the stratosphere.Concentrations of N 2 O in the atmosphere are increasing at the rate of about 0.3% per year [1].Atmospheric N 2 O is derived from natural and anthropogenic sources, but anthropogenic sources are the only ones that are likely to be reduced through improvements in technology.Several anthropogenic sources of N 2 O to the atmosphere have been identified, including wastewater treatment plants and agricultural soils [2,3].Biological nitrogen removal processes in wastewater treatment system is leading to produce N 2 O because inorganic and organic nitrogen compounds are converted to nitrate or N 2 by bacterial nitrification and denitrifcation processes [4].Nitrification consists of the aerobic oxidation of ammonia (NH 4 + ) to nitrate (NO 3 -) via nitrite (NO 2 -), carried out in a two-stepped reaction by ammonia-oxidizing bacteria *corresponding author: e-mail: azzaya@icct.mas.ac.mnDOI: http://dx.doi.org/10.5564/mjc.v17i43.742 that oxidize NH 4 + to NO 2 -, and by nitrite-oxidizing bacteria that oxidize NO 2 -to NO 3 -.Denitrification is the multi-stepped, anoxic reduction of nitrate (NO 3 -) to dinitrogen gas (N 2 ) by heterotrophic microorganisms.In Ulaanbaatar, the capital of Mongolia, about one-third of domestic and commercial wastewater is treated by sewer systems with aerobic treatment.Because, most of the population lives in houses, gers using latrine while the 30% of rest inhabitants are living in apartment connected with central sewer system [5].National estimates of wastewater N 2 O emission rates, however, are poorly constrained by fractions of the housing category and wastewater handling method based on population and housing census.For an assessment of the anthropogenic influence on emission rates and climate change projections of wastewater N 2 O emissions, a more process-based understanding of the relevance of varying environmental conditions for N 2 O production rates is needed.Furthermore, the production processes of N 2 O have been the subject of study for many years.Recently, a high-precision analytical technique for determining intramolecular 15 N-site preference in and asymmetric molecules of N 2 O was developed [6].Since N 2 O has two N atoms within the molecule (central and outer N), distribution of a stable isotope, 15 N, results in the distribution of three isotopomers, such as 15 N 15 NO, 15 N 14 NO, and 14 N 15 NO.By using this newly developed innovative technique, the latter two types of molecules, which exist abundantly in the environment, can be individually measured.The difference in δ 15 N between δ 15 N α and δ 15 N β is the so-called site preference (SP = δ 15 N α -δ 15 N β , where 15 N α and 15 N β represent the 15 N/ 14 N ratios at the center and end sites of the nitrogen atoms, respectively).The SP enabled us to identify the source and sinks of N 2 O in the environment [6,7].Using this technique, Sutka et al. [8][9][10] found that the SP for N 2 O from hydroxylamine oxidation (~33‰) and nitrite reduction (~0‰) differs in a pure culture study and noted that this difference can be used to distinguish the relative contributions of nitrification and denitrification sources to N 2 O emissions.There have still been only several reported studies which applied this measurement technique to field N 2 O samples [11,12] or referred to the relative contributions of nitrification and denitrification.
To our knowledge, the present study is the first to (1) apply the stable isotopic analysis to the determination of N 2 O sources during the case of WWTS in Mongolia and to (2) understand the actual contributions of nitrification and denitrification to N 2 O production.

EXPERIMENTAL
Study site: Municipal wastewater treatment plant (WWTP) located in Ulaanbaatar was studied in this investigation.The plant, which receives wastewater generated by onethird of total inhabitants of 1.3 million, has conventional activated sludge (CAS) treatment system.The system comprises two settlings and a series of biological reaction tanks.Heavy solids are removed from wastewater in the primary clarifier.Then water enters biological reaction tanks to decompose organic matter by activated sludge under aerobic conditions.Subsequently, the microbe-rich liquid flows into the secondary clarifier where activated sludge is separated from treated wastewater by gravity.Some of the settled sludge is continuously recycled back to the aeration tanks to maintain a proper concentration of microorganisms and, the water is moved to disinfection section.Finally, treated effluent is directly discharged into Tuul river, which flow near to the city (Figure 1).Sample collection: Sampling period was in July 2015 in the CWWTP.For dissolved N 2 O (DN 2 O) analysis, water samples were collected from seven points in each treatment systems: exit of the primary settling, sections N1-1, N1-4, N5-1, N5-4, secondary settling and return sludge tunnel.The water temperatures during the sampling were ranged in 20.0-23.0°C.Samples for isotopic analyses were transferred into 115 ml glass vials without a headspace, sterilized with 5 ml of saturated HgCl 2 , and sealed with butyl rubber stoppers and aluminum caps.They were stored at 4°C until analysis.Those for concentration and isotope analysis of NH 4 + was filtered into 50 ml plastic bottles and kept in a freezer at -35°C until measurement.-and NO 2 -were measured using a portable spectrophotometer (DR 2800 TM ; Hach company, Colorado, US).Analysis of isotopocule ratios: For analysis of isotopocule ratios of DN 2 O, samples were prepared by injecting 60 ml of ultrapure helium (He) and subsequent equilibrating of liquid and gas phase at constant temperature (20°C).Then, gas was taken from the headspace into 115 ml glass bottles, which had been flushed with N 2 .Analyses were performed using a Delta XP isotope ratio mass spectrophotometer (IRMS, MAT 251, Thermo-Finnigan, Bremen, Germany) allowing simultaneous detection of m/z 30, 31, 44, 45 and 46.The notation of the isotopocule ratios is the following:

Analysis of dissolved gases and inorganic N species:
where 15 R α and 15 R β represent the 15 N/ 14 N ratios of α and β N atoms, respectively. 15R bulk and 18 R denote average isotope ratios for 15 N/ 14 N and 18 O/ 16 O, respectively.Subscripts "sample" and "std", respectively, signify isotope ratios for the sample and the standard, atmospheric N 2 for N and Vienna Standard Mean Ocean Water (VSMOW) for O.The 15 N site preference (hereinafter, SP) was also defined as an illustrative parameter of the intramolecular distribution of 15  Site-specific N isotope analysis in NO was conducted using ion detectors modified for mass analysis of the N 2 O fragment ions (NO + ), which contained N atoms in the a position of the N 2 O molecules, whereas bulk (average) N and O isotope ratios were determined from molecular ions (N 2 O + ) [5].
Assuming that the N 2 O reduction process is to be negligible, the contributions of NO The δ 15 N value of NH 4 + was measured using the diffusion method [13] and analyzed by an EA1110 elemental analyzer (Thermo Fisher Scientific K.K.) coupled with the IRMS.+ oxidation was occurred in this system by the effect of ammonia-oxidizing bacteria.However, it is clear that the process of NH 4 + oxidation into NO 3 -was not completely conducted as seen from the fraction of NH 4 + conversion rate.This problem can be caused by either treatment capacity of the plant or old facilities applying for operation.The NO 3 concentration was highly fluctuated throughout treatment, and the highest concentration (139.3 μmol/l) was at N1-4.We found that total DIN compounds were removed about 30.1% and those removed N fraction probably converted into other gaseous forms based on mass balance estimation (Figure 2a).The DO concentration at primary settling (PS) and beginning of aerobic tank (N1-1) was initially low, however, it increased gradually from station N1-4, indicating that more oxygen was consumed for the oxidation of NH 4 + and organics (Figure 2b).The highest DN 2 O concentration (335.7nmol/l) was observed at station N5-1 following great decline until secondary settling (SS) in biological reaction basin.This high value can be linked to relatively lower DO concentration compared to DO at other stations.Generally, the DN 2 O concentration at all stations was always higher than the value expected under Mongolian Journal of Chemistry 17 (43) water-atmosphere equilibrium (about 9 nmol/kg at 20°C, showed by red line in Figure 2b [14]) which means that this treatment process is potential source of N 2 O emitted to the atmosphere.An operating parameter believed to play a critical role in influencing emissions is DO [15].An insufficient supply of oxygen in a nitrifying process can lead to incomplete nitrification, whereby autotrophic ammoniaoxidising bacteria (AOB) reduce NO 2 -to N 2 O, instead of oxidation to NO 3 -.While the presence of oxygen can inhibit denitrification enzymes, particularly N 2 O reductase, which converts N 2 O to dinitrogen gas (N 2 ) in denitrification process.Therefore, DO may be key in determining the metabolic mechanisms that trigger N 2 O production, from either nitrifying or denitrifying microorganisms, depending on whether conditions are aerobic and/or anoxic [16].High N 2 O observed at biological reaction tanks under sufficient DO, therefore, probably due to not favourable environment for denitrifying enzymes locally existed in activated sludge.We found that water in biological tanks was supersaturated with DN 2 O between 2144 and 3760% which suggests CAS system of this plant is a potential source of N 2 O emitted to the atmosphere.Source identifying of N 2 O deduced from stable isotope ratios: N 2 O has a strong greenhouse effect, and its emissions must be mitigated.To devise a strategy for mitigation, it is necessary to understand its sources in detail.Stable isotopic analysis is promising tool to differentiate the main N 2 O production processes such as NH 2 OH oxidation and bacterial NO 2 -reduction in several environments.In this study, isotope ratios of N 2 O (δ 15 N, δ 18 O, and SP values) showed slight variations during the treatment process (Figure 3).In general, the δ 15 N value of N 2 O was slightly increased from the PS (+16.8‰) to station N5-4 (+26.9‰)except for sudden decline at station N5-1.At the return sludge (RS), it was the lowest around 11.9‰, however, all the values at all stations were higher than that of atmospheric δ ) is used to interpret the relation between SP and δ 15 N bulk (Figure 4, Eq.7).

Concentrations of dissolved inorganic nitrogen (DIN) compounds and DN
The N 2 O produced by NO 2 − reduction (nitrifierdenitrification) is assumed to have SP of -13.6‰ to +5.0‰ [8][9][10]19] and the δ 15 N bulk of +10.7‰ to +38.9‰ according to the measured δ 15 N NH4+ and the expected isotopic fractionation during NO 2 − reduction to N 2 O (-76 to -11‰ [20]).In contrast, N 2 O produced by NH 2 OH oxidation (nitrification) is supposed to be SP of +27.2‰− +35.6‰ and the expected isotopic fractionation during NO 2 − reduction to N 2 O (-76 to -11‰ [20]).Note that the δ 15 N value of substrate was identical as NO 2 -reduction process.Based on this mapping approach, most of data was fallen near to NO 2 -reduction suggesting that the nitrifierdenitrification was dominant pathway to N 2 O production.This is agreed with previous results that found nitrifierdenitrification was a key process for N 2 O production in aerobic tank of CAS system applied in Japanese WWTP [11,12].The contribution of this pathway is estimated as 87-99% at stations N1-1, N1-4 and N5-4, respectively.At station N5-1 and SS, data were observed at middle of two boxes demonstrated that N 2 O produced by two microbial processes. Itmeans that NH 2 OH oxidation and NO 2 reduction were partly produced to N 2 O. NH 2 OH oxidation was contributed to N 2 O production about 40 and 53%.
Fig. 1.Aerial view of the Central Wastewater Treatment Plant (CWWTP) in Ulaanbaatar with the different unit processes and sampling points for dissolved N 2 O

2 O: 4 +
The distribution of NH 2 O and dissolved oxygen (DO) in the water from different sampling points at CWWTP are presented, respectively, in Figure 2. The concentration of NH 4 + at station PS was gradually decreased from 3464.3 to 2314.3 μmol/l at the end of aeration tank (N5-4) which implies NH 4

Fig. 2 .
Fig. 2. Concentration profiles of DIN (a), DN2O and DO (b) in conventionalactivated sludge system at CWWTP.Red line in right figure indicates water-atmospheric equilibrium for dissolved N 2 O

Fig. 4 .
Fig. 4. Schematic diagram portraying N 2 O production processes in CWWTP.The range of δ 15 N NH4+ is shown as horizontal side of colored-dashed box at up and bottom in the graph Therefore, the observed decrease in the δ15N and δ 18 O values of N 2 O at station N5-1 is interpreted as an isotope effect in microbial N 2 O production during the treatment process.The SP value, which is unique tool for differentiating the microbial processes, was ranged from 0.4‰ at N1-1 to 13.3‰ at N5-1 with high DN 2 O concentration.Exceptions were observed at station N5-1 and SS that can be partly produced by NH 2 OH oxidation.Production and consumption of N 2 O can be revealed using δ 15 N bulk -SP mapping approach, in which the δ 15 N of substrate (NH 4 + [18]value.The δ18O values were within +70 to +80‰ that is nearly two-fold greater than atmospheric value.Basically, N 2 O reduction is accompanied by a simultaneous increase in δ 15 N and δ18O values[17]and a diffusive loss of N 2 O from the water to the atmosphere occurs with a marginal isotope effect[18].