Isotopomer Analysis of Production and Consumption Mechanisms of

Dec 21, 2010 - Department of Environmental Science and Technology, Tokyo .... Japan) using an online analytical system described elsewhere (18, 20)...
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Environ. Sci. Technol. 2011, 45, 917–922

Isotopomer Analysis of Production and Consumption Mechanisms of N2O and CH4 in an Advanced Wastewater Treatment System S A K A E T O Y O D A , * ,† Y U U R I S U Z U K I , ‡ SHOHEI HATTORI,‡ KEITA YAMADA,† A Y A K O F U J I I , ‡ N A O H I R O Y O S H I D A , †,‡ R I N A K O U N O , § K O U K I M U R A Y A M A , §,⊥ AND HIROSHI SHIOMI§ Department of Environmental Chemistry and Engineering and Department of Environmental Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan, and Bureau of Sewerage, Tokyo Metropolitan Government, Tokyo 163-8001, Japan

Received August 31, 2010. Revised manuscript received November 26, 2010. Accepted November 30, 2010.

Wastewater treatment processes are believed to be anthropogenic sources of nitrous oxide (N2O) and methane (CH4). However, few studies have examined the mechanisms and controlling factors in production of these greenhouse gases in complex bacterial systems. To elucidate production and consumption mechanisms of N2O and CH4 in microbial consortia during wastewater treatment and to characterize human waste sources, we measured their concentrations and isotopomer ratios (elemental isotope ratios and site-specific N isotope ratios in asymmetric molecules of NNO) in water and gas samples collected by an advanced treatment system in Tokyo. Although the estimated emissions of N2O and CH4 from the system were found to be lower than those from the typical treatment systems reported before, water in biological reaction tanks was supersaturated with both gases. The concentration of N2O, produced mainly by nitrifier-denitrification as indicated by isotopomer ratios, was highest in the oxic tank (ca. 4000% saturation). The dissolved CH4 concentration was highest in inflow water (ca. 3000% saturation). It decreased gradually during treatment. Its carbon isotope ratio indicated that the decrease resulted from bacterial CH4 oxidation and that microbial CH4 production can occur in anaerobic and settling tanks.

Introduction Nitrous oxide (N2O) and methane (CH4) are increasingly abundant greenhouse gases. The former is also expected to be a major stratospheric ozone-depleting substances in the future (1, 2). The major N2O source is microbial nitrification and denitrification in soils and water. During nitrification, it is produced from hydroxylamine (NH2OH) as a byproduct of the oxidation of ammonium to nitrite (NO2-). During * Corresponding author e-mail: [email protected]. † Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology. ‡ Department of Environmental Science and Technology, Tokyo Institute of Technology. § Tokyo Metropolitan Government. ⊥ Present address: Water Quality Research Team, Public Works Research Institute, 1-6 Minamihara, Tsukuba 305-8516, Japan. 10.1021/es102985u

 2011 American Chemical Society

Published on Web 12/21/2010

denitrification, N2O is produced from NO2- as an intermediate during reduction of nitrate to N2. Some nitrifying bacteria reduce nitrite to N2O or N2 through a process called nitrifierdenitrification (3). Methane is mainly produced in anoxic environments such as wetlands and landfills, but it is also produced by ruminants through microbial methanogenic processes involving fermentation of easily decomposable organic matter (e.g., acetate), and by CO2 reduction with H2 (4). Wastewater treatment systems are anthropogenic sources of N2O and CH4 because they are rich in carbon and nitrogen, and they often include biological processes. Global emissions of N2O and CH4 from wastewater treatment are estimated, respectively, as about 3% and 4% of total anthropogenic emissions (1). However, these emission estimates and others related to other microbial sources present great uncertainty because microbial activities are sensitive to numerous environmental factors such as oxygen concentration, pH, temperature, and substrate availability. Therefore, it is difficult to estimate N2O and CH4 fluxes from microbial sources based on limited observations (5-7). Furthermore, production and consumption mechanisms of N2O and CH4 in specific steps of treatment processes have not attracted considerable attention. Isotopomer ratios (defined in the Experimental Section) are regarded as useful parameters to infer the origin of N2O and CH4 and their production-consumption mechanisms, and to estimate their global budget (8, 9). They are determined by isotope ratios in precursors and isotopic fractionation that is specific to each production or consumption process. Therefore, isotopomer ratios reflect qualitative information that complements the quantitative information obtained through concentration analyses. For example, N2O and CH4 of microbial origin are isotopically lighter than those molecules originating from other sources, such as thermogenic and abiotic molecules, because of large isotopic fractionation (4, 10). The remaining N2O and CH4 become isotopically heavier if bacterial consumption of N2O and CH4 proceeds (11, 12). Gas exchange between air and water entails negligible isotopic change (13, 14). Recent pure culture incubation studies have demonstrated that intramolecular 15N-site preference (SP) in N2O can differentiate two N2O formation steps: hydroxylamine oxidation and nitrite reduction (15, 16). Regarding wastewater treatment systems, however, only two reports described N2O isotopomer ratios (17, 18) and no data are available for CH4 isotope ratios. For this study, we investigated concentrations and isotopomer ratios in N2O and CH4 produced or consumed in each process of wastewater treatment to characterize this source and to elucidate their production-consumption mechanisms by comparing isotope dynamics in a complex microbial system and in a pure culture incubation system.

Experimental Section Notation. Hereinafter, concentration is expressed as C with a subscript (e.g., CN2O). We use “isotopomers” to refer to isotopically substituted molecules of N2O and CH4. The isotopomer ratio, δ, is expressed as a relative difference in heavy-to-light isotopomer ratios: δX ) (Rsample - Rstandard)/Rstandard,

(1)

where X denotes 13C, 15NR, 15Nβ, or 18O, and R denotes 13CH4/ 12 CH4, 14N15N16O/14N14N16O, 15N14N16O/14N14N16O, or 14N14N18O/ VOL. 45, NO. 3, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Simplified process diagram for the A2O treatment system. Sampling stations are shown as stars. Mean flow rate and hydraulic retention times are also shown. 14 14 16 N N O of the sample and standards (19). The δ value is conventionally expressed as permil (‰) relative to Vienna Peedee Belemnite (VPDB), atmospheric N2, and Vienna Standard Mean Ocean Water (VSMOW), respectively, for carbon, nitrogen, and oxygen. As described herein, instead of δ15NR and δ15Nβ, δ15Nbulk and site preference (SP) are used as illustrative parameters for N2O:

δ15Nbulk ) (δ15NR + δ15Nβ)/2 and

(2)

SP ) δ15NR-δ15Nβ

(3)

15

bulk

is equivalent to the convenIn those equations, δ N tional elemental isotope ratio, δ15N. The enrichment factor of a heavy isotopomer during consumption of N2O or CH4 in a closed system is defined using the ‘Rayleigh equation’: δX ) δ0X + ε(X)R × ln f,

(4)

where ε(X)R is an enrichment factor for consumption with respect to X, f is C/C0, and subscript 0 signifies an initial value. An enrichment factor is also applied to production processes. In a steady state where concentration and isotope ratios of substrate can be regarded as constant, the following relation is obtained (20): δXP ) δXS + ε(X)P,

(5)

where ε(X)P is the enrichment factor for production and subscripts S and P respectively denote the substrate and product. Study Site. The municipal wastewater treatment plant examined in this study is located in a northern area of Tokyo, Japan. The served population of approximately 160,000 generates mean wastewater flow of approximately 75,000 m3 day-1. The plant has six biological treatment systems, each including settling and reaction tanks (Figure 1). It treats municipal wastewater with an average influent BOD of approximately 100 mg L-1 with estimated suspended solids concentration of 900 mg L-l. After removal of solids in the primary settling tank, wastewater enters the reaction tanks where organic matter is biologically decomposed by the activated sludge under anaerobic and aerobic conditions. The first tank is kept anaerobic to increase the phosphateassimilation capacity of the bacteria. The second tank, the “anoxic” tank, is also kept anaerobic, but liquid returned from the third tank is combined to reduce nitrate to N2 through denitrification (return water). The third tank, the “oxic tank”, is aerated to oxidize ammonia and organic compounds. The microbe-rich liquid then passes into the secondary settling tank, where gravity separates the activated sludge from the treated wastewater. Some of the settled sludge is recycled back to the anaerobic tank continuously to maintain a proper concentration of microorganisms in the 918

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biological reaction tanks (return sludge). The treated effluent moves to the chlorine contact tank for disinfection, after which it is released into a tributary of the Arakawa River, which flows into Tokyo Bay. This system, called an “Anaerobic-Anoxic-Oxic (A2O)” system, is an advanced sewage water treatment system in Japan. Sampling. Water samples were collected at 13 stations of an A2O series (stations 1-6, Figure 1) on March 25, 2008. At stations 1, 5, and 6, surface water was sampled using a stainless steel container. In biological reaction tanks (stations 2a-4d), samples were collected at the depth of 0.5 m, 5 m (only at stations 4a-4d), and 10 m using a vacuum pump. Return sludge was sampled from a drain valve at the bottom of the secondary settling tank (RS, Figure 1). Samples for N2O and CH4 analysis were transferred into 125-mL glass vials, preserved with 5 mL of saturated HgCl2 solution, and sealed with butyl rubber stoppers and aluminum caps with special care taken to exclude air bubbles (21). They were stored at 4 °C before analysis. Samples for concentration and isotope analysis of dissolved inorganic nitrogen were filtered into plastic bottles. Samples used for ammonium and nitrate isotope analyses were pH-adjusted to pH 2 and pH 12, respectively, and kept frozen (-35 °C) until measurement. Gas samples were collected into evacuated 0.2-1 L stainless steel or glass bottles at an exhaust duct over the oxic tank (Figure 1). Analysis. The concentration of dissolved ammonium was measured using a coulometric ammonia meter (MT-1, Central Kagaku Corp., Tokyo, Japan). Those of nitrite and nitrate were measured using an ion chromatograph (DX320, Dionex Corp., Osaka, Japan). Dissolved oxygen concentration was measured using a membrane electrode (model 58, YSI/Nanotech Inc., Kawasaki, Japan). Concentration and isotopomer ratios of N2O in the water and gas samples were measured on an isotope-ratio monitoring mass spectrometer (MAT252; Thermo Fisher Scientific K.K., Yokohama, Japan) using an online analytical system described elsewhere (18, 20). To meet the dynamic range of the system, an aliquot of the water sample containing 1-5 nmol of N2O was measured gravimetrically and processed. Site-specific N isotope analysis of N2O was conducted using ion detectors that had been modified for mass analysis of the N2O fragment ions (NO+), which contained N atoms in the center positions of the N2O molecules, whereas bulk N and O isotope ratios were determined from molecular ions (19). Measurement precision was typically better than 0.4% for concentration, 0.1‰ for δ15Nbulk and δ18O, and better than 0.5‰ for δ15NR and δ15Nβ. Concentration and δ13C of CH4 in the water and gas samples were measured using an online analytical system similar to that used for N2O, except that CH4 was combusted to CO2, of which the molecular ions were analyzed to ascertain the amount and 13C/12C ratio of CH4 (22). Measurement precision levels were typically better than 5% and 0.3‰, respectively, for concentration and δ13C.

TABLE 1. Concentration and Isotopomer Ratios of N2O and CH4 in the Gas Samples Collected over an Oxic (Aeration) Tank sample

N2O (ppb)

δ15Nbulk (‰)

δ18O (‰)

SPa (‰)

CH4 (ppm)

δ13C (‰)

oxic tank 1 oxic tank 2 background 1 background 2

694.0 539.5 328.6 322.5

-4.1 -1.4 7.1 6.5

39.2 40.3 44.5 44.4

10.9 11.4 16.9 17.2

2.54 2.29 1.84 1.83

-46.9 -47.2 -47.1 -48.0

a 15

N-site preference in N2O.

The δ15N of ammonium was measured using the diffusion method (23) with an elemental analyzer (EA1110; Thermo Fisher Scientific K.K.) coupled with the isotope ratio mass spectrometer. The δ15N and δ18O of nitrate were measured using the denitrifier method (24, 25), where N2O produced by Pseudomonas aureofaciens (NBRC 3521) from the nitrate was analyzed as described above. Activated sludge was recovered from the remaining water sample after N2O isotopomer analysis by filtration. After freeze-drying, the δ13C and δ15N were determined using the EA-IRMS system (26).

decreases in the anaerobic/anoxic tanks and oxic tank, respectively, although a small increase was observed at stations 2b and 5.

Results Average flow rates of the water entering the biological tanks, return sludge, and return water are portrayed in Figure 1. The average concentration of the sludge was about 2000 mg/ L. Average temperatures of air and water were, respectively, 9.8 and 19.7 °C. Concentrations and isotopomer ratios of N2O and CH4 in the gas samples are presented in Table 1. The dissolved species were found to be distributed homogeneously with respect to depth. Therefore, the average value for each station is presented and then discussed below. The dissolved N2O concentration was higher than that expected under atmospheric equilibrium (about 9 nmol kg-1; 27) at all stations (Figure 2a). It was especially high at the exit of the primary settling tank (station 1), at the entrance of the anoxic tank (station 3a), and in the middle of the oxic tank (stations 4b-4d). The highest concentration of 888 nmol kg-1 was observed in the return sludge. The ammonium concentration decreased by about 50% between stations 1 and 2a (Figure 2a), and by 98% in the oxic tank. Nitrate concentrations increased in the oxic tank (Figure 2a) and decreased by 50% in the return sludge. The nitrite concentration was highest at station 1 and showed a smaller peak in the middle of the oxic tank (Figure 2a). It also showed an increase in the return sludge. The δ15Nbulk of N2O was highest at station 1 (Figure 2b). After the decrease at station 2a, it was nearly constant in the anaerobic and anoxic tanks. It decreased rapidly in the oxic tank and increased gradually from the minimum value of -28.7‰ at station 4c. The δ18O and SP showed similar patterns, except at station 1 (Figure 2b). Their minima were observed in the middle of anoxic tank and oxic tank, although their maxima were observed in the entrance of oxic tank and in the secondary settling tank. The δ15N of ammonium was almost constant between stations 1 and 3c, but it increased in the oxic tank and was high in the return sludge (Figure 2c). The δ15N of nitrate showed greater variation (Figure 2c). It decreased in the anaerobic and anoxic tanks except in the maximum case at station 3a, and increased in the oxic tank. It was highest in the return sludge. The δ18O of nitrate was between -4.6 and -0.9‰, except that it increased to maximum value of 8.8‰ in the anoxic tank. It was also high in the return sludge (Figure 2c). Dissolved CH4 concentrations were 0.02-10.69 µmol kg-1shigher than the levels expected under atmospheric equilibrium (about 3 nmol kg-1 (28))sat all stations (Figure 3). It was highest at station 1 and showed gradual and rapid

FIGURE 2. Concentration (a) and isotope/isotopomer ratios (b and c) of dissolved N2O and related nitrogen species at each station in the A2O system.

FIGURE 3. Concentration and carbon isotope ratio of dissolved CH4 at each station in the A2O system. VOL. 45, NO. 3, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Estimated Flux and Isotopomer Ratios of N2O and CH4 from Wastewater Treatment Processes δ15Nbulk

N2O -1

process

gd c

aeration (oxic tank) secondary settlingd discharge to riverd total

26.4 21.5 66.6 114.6

mg m

-3b

2.3 1.9 5.8 10.0

δ18O

SPa

‰

‰

‰

-13.4 -8.9 -8.3 -9.6e

34.4 59.0 43.5 44.3e

4.5 26.0 17.3 16.0e

δ13C

CH4 gd

-1

19.1 98.6 44.0 161.7

mg m

-3

1.7 8.6 3.8 14.1

‰ -45.5 -51.7 -50.8 -50.7e

a 15 N-site preference in N2O. b Emission per volume of wastewater. c Calculated from gas sample data. d Calculated from water sample data assuming that decreased concentration during secondary settling results from emission and that the water is equilibrated with the atmosphere after discharge. e Flux-weighted average.

The δ13C values of CH4, which were between -51 and -49‰ at stations 1-3c, increased rapidly to -32‰ in the oxic tank, and returned to -51‰ at station 5 (Figure 3). At station 2b, a vertical gradient was observed; the CH4 concentration and δ13C at 10 m depth were higher and lower than at 0.5 m depth, respectively (error bars in Figure 3 show data at the two depths). Average carbon and nitrogen contents of the activated sludge were, respectively, 31.5 ( 4.8 and 6.5 ( 0.8% (dry weight). Carbon and nitrogen isotope ratios were, respectively, -26.5 ( 0.9 and 5.9 ( 1.0‰. Dissolved oxygen concentrations in anaerobic, anoxic, and oxic tanks were 9-17, 0.4-0.9, and 24-43 µmol/L, respectively, although a high value (104 µmol/L) was observed at the exit of the oxic tank (station 4d).

Discussion Concentration and Estimated Flux of N2O and CH4. The observed supersaturation of N2O and CH4 indicates wastewater treatment as a potential source of their high emissions to the atmosphere. Large emissions are expected in aeration or processes in which water interface with open air. Therefore, we calculated fluxes of N2O and CH4 for three processes (Table 2). First, the flux during aeration was calculated from the concentration difference between exhaust and background air and the air flow rate. Next, the decrease in dissolved concentrations of N2O and CH4 from stations 5 to 6 was regarded as diffusive emission of the gases to the atmosphere during secondary settling. Finally, flux during the discharge was calculated assuming that the N2O and CH4 concentrations in the water discharged to the river are similar to those at station 6 and that the water reaches equilibrium with the atmosphere. Czepiel et al. (5, 6) reported N2O and CH4 emissions of 29 and 200 mg m-3, respectively, from aeration tanks. Their respective values are higher than those of this study by 1 and 2 orders of magnitude. Regarding N2O concentrations in water or gas phase in the oxic tank, values reported elsewhere (7, 17) are also about an order of magnitude higher than those of this study. The difference in N2O might be attributable to its reduction in the anaerobic/anoxic biological tanks of the advanced treatment system. Comparison of εP for a Wastewater Treatment System and a Pure Culture System. We measured nitrogen isotope ratios of ammonium, nitrate, and N2O. Therefore, we can compare εP values obtained for nitrogen metabolism in the wastewater treatment system with those obtained in previous pure culture studies. Casciotti et al. (29) reported enrichment factors during ammonia oxidation to nitrite (ε(15N)AMO) by five species of ammonia-oxidizing bacteria as ranging from -38 to -14‰. In the present study, ε(15N)AMO is estimated as -32‰, applying eq 4 to CNH4 and δ15NNH4 obtained at stations 4a and 4b. The overall enrichment factor for ammonia oxidation to nitrate (ε(15N)NH3-NO3) is unobtainable using a “pure culture” system because this transformation of N is conducted by at 920

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least two species: ammonia-oxidizing and nitrite-oxidizing bacteria. We calculate ε(15N)NH3-NO3 applying eq 5 to the observed δ15NNH4 at stations 4a and 4b and δ15N of newly produced nitrate between stations 4a and 4b and between 4b and 4c which is calculated from the observed CNO3 and δ15NNO3. The estimated values of -26 to -19‰ respectively resemble those reported for nitrification in soil (-29 to -12‰ (30)). The overall enrichment factor for ammonia oxidation to N2O (ε(15N)NH3-N2O) has been reported for nitrifying bacteria (-68 to -47‰ 10, 15) and methanotrophic bacteria (-55 to -48‰; 31). The ε(15N)NH3-N2O for the oxic tank of the wastewater treatment system examined here calculated similarly to ε(15N)NH3-NO3 is -48.4 ( 0.3‰, corresponding to the upper range of results reported in the literature. In summary, ε(15N)s for several nitrogen metabolism processes obtained in the wastewater treatment system resemble those obtained in pure culture or soil incubation studies, suggesting that, regarding nitrogen isotopes, bacterial species in the activated sludge show behavior similar to bacteria studied in laboratories. The interaction between different bacterial species does not significantly affect ε(15N) of any process. Therefore, we assume for the following discussion that the information related to δ18O, SP, and δ15Nbulk, of N2O produced by the pure culture, is applicable to wastewater treatment processes. Production and Consumption Processes of N2O. The observed variation in concentration and isotopomer ratios of N2O results from the following processes: mixing of different water masses, production or reduction of N2O by activated sludge, and N2O emission to the atmosphere. We analyze the contributions of these processes using a δ-1/C diagram. The advantages of using the diagram are that (1) water mixing or N2O addition shows a linear relation (32), (2) partly reduced N2O traces a curve determined by εR (eq 4), and (3) emissions to the atmosphere cause a horizontal shift because the values of ε for gas transfer between gaseous and aqueous phases are known to be negligibly small (14). An example for station 2a is shown in Figure 4. Closed symbols represent CN2O and δ values calculated using the values for inflow and return sludge and their flow rate. The observed data points are located at the right side in the figure. Therefore, CN2O and δ must have been changed according to the relation described above. The range of the isotopomer ratio of N2O produced by nitrifying and denitrifying bacteria can be predicted from the reported εP and the observed (or estimated) isotope ratio of the substrate (e.g., ammonium) using eq 5. This is shown by bars along the left vertical axis shown in Figure 4. Then, the data point for the mixed water would move leftward to the range indicated on the left vertical axis if N2O is added by the production. Finally, the decrease in concentration should be explained by in situ reduction and/or emission to the atmosphere. Here, an assumption was made that the reduced or emitted N2O is part of that dissolved in well-mixed water after water mixing and in situ production. This is not the case if the produced N2O is reduced

of CH4 and the isotopic constraint is not so tight. Here we estimate the ε(13C) for apparent CH4 consumption and δ13C of apparent CH4 production. At the entrance of anaerobic tank (station 2a) and between anoxic and oxic tanks (from station 3a to 4b), the CH4 concentration decreased and δ13C values increased (Figure 3). Using eq 4, ε(13C) is calculated respectively as -31 and -5.0‰. In the former case, mixing of the return sludge was incorporated into the calculation. In the latter case, ε(13C) was obtained as the slope of regression line between δ13C and lnf because a high correlation was observed (r2 ) 0.99). These values are close to the value of ε(13C) reported for microbial aerobic oxidation of CH4 (-39 to -3‰; 12, 34, 35). The small (magnitude) ε(13C) observed in anoxic-oxic tanks might result from abundant O2 supply in the oxic tank or high bacterial cell densities (12). The local increase in CH4 concentration observed at stations 2b, 3a (when the mixing of return water is considered), and 5 was accompanied by the decrease in δ13C (Figure 3). In these cases, the δ13C values for apparently produced CH4 are calculated from the mass balance for 12C and 13C. FIGURE 4. Example of process analysis at station 2a using the relation between isotopomer ratios and inverse concentration of dissolved N2O. Bars along the left vertical axis show the range of isotopomer ratios in N2O produced by bacteria, as estimated from the literature. Thin solid and broken lines indicate constraints for mixing, production, emission, and reduction of N2O. Hatched areas show the possible range for hypothetical data points after N2O production occurred. Arrows indicate an example of combination of production and emission processes if they occur sequentially. Broken curves show the relationship between isotope ratios and inverse concentration if N2O observed at the station 2a is resulted from N2O reduction. See text and Supporting Information for detailed explanation. successively inside the same bacterial cell or if the produced N2O is emitted directly to the atmosphere. However, we believe that this assumption is valid in most cases because it is known that several species of denitrifying bacteria are incapable of N2O reduction (33) and because water in the biological tanks used for wastewater treatment is well mixed by a stirrer or by aeration. Regarding Figure 4, production and emission along the arrows followed by N2O reduction along the broken curve can reconstruct observed values with respect to all three isotopomer ratios. Isotopomer ratios of the added N2O obtained from the y-intercept of the “production” arrows are, respectively, about -21 to -4, 5 to 39, and -14 to 10‰ for δ15Nbulk, δ18O, and SP. Based on SP values reported for hydroxylamine oxidation (39‰) and nitrite reduction (-14‰) (16), nitrite reduction slightly dominates over hydroxylamine oxidation at this station. The source of nitrite is expected to be oxidation of ammonia (nitrification) and reduction of nitrate (denitrification), both of which are supported from the budget of ammonium and nitrate: the observed levels of CNH3 and CNO3 were lower than those expected from simple mixing of inflow and return sludge, indicating the consumption of ammonium and nitrate by bacteria. Similar analyses at other stations (see Supporting Information) revealed that (1) N2O is mainly produced by denitrification and partly reduced in the anoxic tank, (2) hydroxylamine oxidation and nitrite reduction contribute nearly equally to the N2O production in the entrance of the oxic tank, (3) nitrite reduction (nitrifier-denitrification) is the main pathway of N2O production from the middle to the end of the oxic tank, and (4) hydroxylamine oxidation slightly dominates over nitrite reduction in the secondary settling tank and N2O is partly reduced. Production and Consumption Processes of CH4. Conducting a process analysis like that for N2O is difficult because we measured only carbon (but not hydrogen) isotope ratios

δ13Cproduced ) {CCH4(n)δ13C(n) - CCH4(n - 1)δ13C(n - 1)}/ {CCH4(n) - CCH4(n - 1)} (6) In that equation, n and n - 1 denote the target station and the preceding one. Calculated δ13Cproduced values at stations 2b, 3a, and 5 are, respectively, -65, -54, and -53‰, which are consistent with those of microbiologically produced CH4 (4). Actually, CH4 is produced by methanogens, specifically anaerobic microbes. Therefore, the apparent CH4 production suggests that especially highly anaerobic conditions were established in the water column or in suspended solids at stations 2b and 3a, where the dissolved oxygen concentration was generally low, and at station 5, where activated sludge is precipitated in the stagnant water. In conclusion, production and emission of N2O and CH4 in an advanced wastewater treatment system were less than those in a typical aerobic treatment system. Isotopomer analysis revealed that N2O production processes occurring in the bacterial consortium in activated sludge can be analyzed using isotopomer enrichment factors obtained through pure culture incubation. Furthermore, N2O is mainly produced by nitrite reduction in the aerobic reaction tank. Results also show that bacterial oxidation consumes CH4, although microbial production can occur at specific sites. Isotopomer ratios of N2O and CH4 emitted from wastewater treatment into the atmosphere were estimated for the first time ever reported, but further investigation of other treatment systems is necessary to evaluate the magnitude of human waste sources using an isotopomer mass balance.

Acknowledgments We thank the Sewerage Bureau of Tokyo Metropolitan Government for sampling and analysis, and A. Makabe, K. Inoue, and R. Shimojima for assistance with isotopic analysis. This work was supported by the Global Environment Research Fund (A-0904) of the Ministry of the Environment, Japan.

Supporting Information Available Detailed explanation for the N2O process analysis, figures for the analysis at each station, and tables summarizing observational data and input/output of the process analysis. This information is available free of charge via the Internet at http://pubs.acs.org/.

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(2) (3) (4) (5) (6) (7) (8) (9) (10) (11)

(12)

(13) (14) (15)

(16)

(17) (18) (19)

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