Effect of PHB and oxygen uptake rate on nitrous oxide emission during simultaneous nitrification denitrification process

Bioresource Technology 113 (2012) 232–238

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Effect of PHB and oxygen uptake rate on nitrous oxide emission during simultaneous nitrification denitrification process Wenlin Jia a, Jian Zhang a,⇑, Huijun Xie b, Yujie Yan a, Jinhe Wang a,c, Yongxin Zhao a, Xiaoli Xu a a

Shandong Provincial Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, Jinan, China Environmental Research Institute, Shandong University, Jinan, China c School of Municipal and Environmental Engineering, Shandong Jianzhu University, Jinan, China b

a r t i c l e

i n f o

Article history: Received 2 September 2011 Received in revised form 24 October 2011 Accepted 25 October 2011 Available online 10 November 2011 Keywords: N2O emission SND PHB Oxygen uptake rate

a b s t r a c t Simultaneous nitrification denitrification (SND) process was achieved in a SBR system to evaluate the impacts of intracellular carbon source PHB and oxygen uptake rate (OUR) on nitrous oxide (N2O) emission. Compared with the sequential nitrification and denitrification (SQND) process, SND process significantly improved the nitrogen removal. N2O emission during SND process was much higher than the SQND process. The amount of N2O emission was 26.85 mg N per cycle in SND process, which was almost four times higher than that in SQND process. About 7.05% of the removed nitrogen during SND process was converted to N2O-N. N2O emission had great relations with the OUR and the OUR could reflect the N2O emission trend more exactly than the DO concentration. At the aerobic stage of SND, the simultaneous denitrification could carried out using PHB as the carbon source and N2O emission increased because of the slow degradation of PHB. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction In order to protect the water from eutrophication, many countries enforce the removal of nutrients through biological treatment of wastewater. The effluent quality standards are more stringent. So many modifications and novel processes have been developed and implemented for nitrogen removal from wastewater (Hu et al., 2011a). In biological nitrogen removal, inorganic nitrogen in the form of ammonium is removed through aerobic, autotrophic nitrification followed by anoxic, heterotrophic denitrification (Meyer et al., 2005). However, some heterotrophic nitrifiers have  been reported to denitrify nitrite (NO 2 ) and nitrate (NO3 ) aerobically (Zart and Eberhard, 1998). Several literatures have illustrated that nitrification and denitrification can occur simultaneously at low oxygen level (Yoo et al., 1999; Li et al., 2007). This is often referred to as simultaneous nitrification and denitrification (SND) process. The SND process represents a significant advantage over the conventional separated nitrification and denitrification processes (Chiu et al., 2007). It is considered that the biological treatment process of domestic wastewater is an important source of greenhouse gas, like CH4 and N2O. N2O is an important greenhouse gas, having an atmospheric lifetime of about 114 years, a global warming potential of 298 relative to CO2 over a 100 year time horizon, and is responsible ⇑ Corresponding author. Tel./fax: +86 531 88363015. E-mail address: [email protected] (J. Zhang). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.10.095

for about 6% of anticipated global warming (IPCC, 2007). N2O also contributes to the depletion of stratospheric ozone, because of the stratospheric reaction with atomic oxygen to nitric oxide (NO) (Mosier, 1998). Therefore, even low amounts of N2O emission are unwanted. Nowadays, the concentration of atmospheric N2O is estimated to be approximately 319 ppbv, which is approximately 16% higher than that during the preindustrial era, and it is increasing at a rate of 0.3% year1 (IPCC, 2007). During the microbial transformations of nitrogenous compounds, N2O can be produced during nitrification, denitrification, dissimilatory reduction of þ NO 3 —NH4 and chemo-denitrification (Wu et al., 2009a). Studies show that the heterotrophic nitrifying bacteria are often able to denitrify under aerobic conditions and N2O is produced as an intermediate in this process. Heterotrophic nitrifiers produce much more N2O per cell than autotrophic nitrifiers, and it might produce significant amount of N2O under certain sets of circumstances (Wrage et al., 2001). N2O emission during the biological treatment is affected by many factors, such as COD/N ratio (Hanaki et al., 1992; Wu et al., 2009a), pH (Thoern and Soerensson, 1996), carbon content (Wu et al., 2009b), nitrite concentration (Tallec et al., 2006), dissolved oxygen (Tallec et al., 2006) and so on. The dissolved oxygen (DO) concentration is considered as a very important parameter controlling N2O emission. In oxygen limiting conditions, autotrophic ammonia oxidizers use nitrite as the terminal electron acceptor to save oxygen for the oxygenation reaction (Hu et al., 2011b). Usually, the SND process occurred at a DO concentration lower

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than 0.5 mg/L (Chiu et al., 2007). This suggests that some heterotrophic nitrifiers have the ability to denitrify under low oxygen conditions to affect a SND process, and the N2O emission during the SND process may be significant. Previous literatures studied the effect of DO concentration on N2O emission (Tallec et al., 2006; Kampschreur et al., 2008; Hu et al., 2009). However, they only studied the trend of N2O emission with DO concentration. Dissolved oxygen cannot describe the microbial condition of the sludge accurately and directly, especially at low oxygen level. The oxygen uptake rate (OUR) is a parameter that can be used to evaluate the rate at which metabolic processes take place in active sludge treatment processes. So according to the OUR, we could study the relation of N2O emission and microbial activity. Another possible factor influencing the N2O emission is the consumption of intracellular storage compounds, e.g. poly-hydroxybutyrate (PHB) and glycogen (Kampschreur et al., 2009). Glycogen accumulating organisms (GAO) and phosphate accumulating organisms (PAO) in the SND system both employ a special mechanism to store organic carbon during anaerobic periods, involving storage compounds, which are finally degraded via their internal PHB pool. The denitrification need carbon source to proceed when treating low C/N ratio wastewater and the microbes can carry out denitrification using their stored carbon compounds. PHB plays an essential ecological role in several wastewater treatment processes, so it is a general factor related to N2O emission (Kampschreur et al., 2009). However, few studies focus on this point, and the relation between the growth of PHB and N2O emission have not been well investigated. In this study, the SND process was achieved using the SBR system. The contaminant removal performance and N2O emission were evaluated, as well as OUR and PHB content. The aim of this paper was to (1) investigate the N2O emission rate and amount during the SND process; (2) evaluate the impact of PHB consumption on N2O emission and, (3) study the relation between OUR and N2O emission during SND process. 2. Methods

2.2. Reactor setup and operation The experiments were conducted in two gastight sequencing batch reactors (SBRs), constructed using transparent, rigid plexiglas cylinders, with an effective volume of 15 L each. The schematic diagram was illustrated in Fig. 1. Biomass was enriched in the SBRs seeding with sludge from the Second Wastewater Treatment Plant of Everbright Water (Jinan) Ltd. (Jinan, China). Both the two SBRs were operated with a cycle time of 6 h consisting of a 6 min feeding, 90 min anaerobic reaction and 180 min aerated period, followed by 70 min settling and 14 min decant. The SBRs were operated at 25 ± 2 °C. At the feeding period 7.5 L of synthetic wastewater was feed into each reactor using peristaltic pump. The electric agitator with a rectangular paddle was used to keep the suspension of the sludge at the anaerobic stage. At the aeration stage, air pump was used to supply air through the diffuser located at the bottom of the reactor. The difference between the two reactors was the aeration rate. In one SBR, the DO concentration at the aeration stage was maintained 0.35–0.80 mg/L by on/off control of air pump to achieve SND process. As a contrast, another SBR was operated with an aeration rate of 7.5 Lair/(Lreactor h) at the aeration stage to mimic the actual wastewater treatment plant to achieve the sequential nitrification and denitrification (SQND) process. After settling, 7.5 L of supernatant was removed, resulting in a hydraulic retention time (HRT) of 12 h. The mixed liquor suspended solid (MLSS) was maintained at approximately 3000 mg/L and certain amount of excess sludge was disposed at the end of aerobic phase to control the SRT at approximately 15 days. The effluent was analyzed every 5 days to evaluate the operation conditions of the reactors at first. After running for about 4 months, the effluent of the two reactors was stable and the SND process was achieved. Then the COD and nutrients removal performances were evaluated every 2 days. Meanwhile on day 130, the N2O emission during one cycle was also measured by collecting the off-gases at intervals of 15 min. At the same time, liquid phase and sludge samples were taken to measure the water quality and PHB content.

2.1. Synthetic wastewater Synthetic wastewater was used in this study. The wastewater contained, per liter: 260.2 mg C6H12O6H2O; 260.2 mg CH3COONa3H2O; 191 mg NH4Cl; 200 mg NaHCO3; 11 mg KH2PO4; 18 mg K2HPO43H2O; 10 mg MgSO47H2O; 10 mg FeSO47H2O; 10 mg CaCl22H2O and 1 mL nutrient solution. One liter of nutrient solution contained: 0.15 g H3BO3; 0.03 g CuSO45H2O; 0.18 g KI; 0.12 g MnCl24H2O; 0.06 g Na2MoO42H2O; 0.12 g ZnSO47H2O; 0.15 g CoCl26H2O and 10 g ethylene diamine tetraacetic acid (Zeng et al., 2003). The influent characters were shown in Table 1.

Gas flowmeter

Electric agitator

Gas sampling pump

Air Gas sampling bag ORP/pH meter DO meter

Liquid sampling port Table 1 Mean contaminant concentrations with standard deviations (in brackets) and removal efficiencies (%) in each system.

COD (mg/L) COD removal (%) TP (mg/L) TP removal (%) NHþ 4 (mg/L) NHþ 4 removal (%) TN (mg/L) TN removal (%) NO 3 NO 2 pH ND: not detected.

Influent

SND

SQND

342.24 (36.32)

28.68 (7.08) 91.62 0.28 (0.20) 91.08 0.97 (0.49) 98.03 7.63 (1.96) 85.06 6.74 (1.63) 0.06 (0.10) 7.15 (0.23)

29.58 (12.74) 91.36 0.44 (0.29) 85.99 1.05 (1.04) 97.87 17.22 (3.64) 66.29 16.46 (1.22) 0.03 (0.01) 7.30(0.32)

3.14 (0.51) 49.31 (5.71) 51.08 (5.99) 1.39 (1.08) ND 7.21 (0.14)

Electron magnetic valve Effluent port

Temperature controller

Gas diffuser

Peristaltic pump

Water tank

Fig. 1. Schematic description of the experiment system.

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2.3. Batch experiments In order to evaluate the effect of PHB on N2O emission during SND process, the following batch tests were conducted. A total of 2 L of the mixed liquor was taken from the SND reactor at the end of anaerobic stage, settled for 10 min to remove the supernatant, washed three times with 0.9% NaCl solution to remove the soluble compounds, such as ammonium and phosphorus (Zhu and Chen, 2011), and then divided equally into two reactors (reactor-1 and 2) with a volume of 1.5 L each. Then solution A contained KNO3, KH2PO4 and K2HPO4 was supplied to reactor-1 to get NO 3 -N and TP concentrations of 25 mg/L and 15 mg/L, respectively (Run 1). Reactor-2 was supplied by solution A and solution B (consisted by glucose and sodium acetate) to get the final concentrations of 200 mg/L, 25 mg/L and 15 mg/L for COD, NO 3 -N and TP, respectively (Run 2). Before the solutions were supplied to reactor-2, the sludge was sparged with air at a rate of 0.5 L/min for 5 h to exhaust the internal carbon source completely. The final volume of the mixed liquor was 1 L in both reactors. The two reactors were stirred by magnetic agitator under low DO (0.35–0.80 mg/L) conditions for 3 h. N2O concentration was measured using N2O microsensor (Unisense, Denmark). The experiments carried out four times from day 130 on. 2.4. Analytical methods   COD, NHþ 4 -N, NO3 -N, NO2 -N, TN, TP and MLSS were measured according to the standard methods (APHA, 2001). Dissolved oxygen was measured with a DO meter (HQ30d53LDO™, USA) and pH was measured with a pH meter (PHS-3C, China). The N2O concentration was determined by the gas chromatography (SP-3410, China) with an electron capture detector (ECD) and a Poropak Q column (Wu et al., 2009a). PHB was measured using the gas chromatography according to the method described by Jiang et al. (2009). The on-line oxygen data were used to calculate the oxygen uptake rate (OUR) during the aerobic period using the method described by Meyer et al. (2005). The N2O emission rate and emission quantity were calculated through the equations described by Hu et al. (2011a).

3. Results and discussion 3.1. Contaminant removal performance and characteristic Table 1 showed the contaminant removal performance of the two SBRs. The COD removal rates during SND and SQND processes were similar and high, both around 91%. This was because the feed water used was composed of glucose and sodium acetate, which were easily degradable substrates and could be consumed quickly during the anaerobic phase. In both reactors nearly all the NHþ 4 -N in the influent was removed because of the complete nitrification and the removal rate reached to about 98%. However, TN removal in two reactors was different. The SND process enhanced the removal of TN and its removal rate reached to 85.06%, which was much higher than that of SQND process. This was because the SND process could remove nitrogen by simultaneous denitrification at the aerobic stage under low dissolved oxygen condition. So the accumulative NO 3 -N concentration during the SND process was significantly lower than the SQND process. And nearly no nitrite was in effluent in both reactors. The SND process had slightly higher TP removal rate than the SQND process. The reason may be that the denitrifying phosphorus removal occurred in the SND reactor. It was known that phosphorus removal was based on the ability of polyphosphate-accumulating organisms (PAOs) to uptake P and accumulate it intracellularly as polyphosphate when

exposed to alternating anaerobic and aerobic conditions (Comeau et al., 1986). However, it has been found that PAOs capable of denitrification (DPAOs) could perform P uptake with NO x as electron acceptor (Kuba et al., 1993; Meinhold et al., 1999). In the SND reactor, carbon was taken up by PAOs in the initial anaerobic period and stored as PHB. In the following aerobic period, the presence of adjacent aerobic and anoxic microzones in microbial aggregates provided conditions for simultaneous nitrification and denitrification. Theoretically, denitrification with simultaneous P uptake is carried out by PAOs using PHB stored in the previous anaerobic period as the carbon source (Meyer et al., 2005). The carbon source could be used for P uptake and N denitrification simultaneously, so the SND process removed more phosphorus. Fig. 2 showed the time profiles of DO concentration, pH value, COD and nutrients concentrations in a typical cycle of different reactor. During the SND process, the DO concentration was maintained between 0.35 mg/L and 0.85 mg/L at the aerobic stage. The DO concentration increased rapidly at the beginning of aeration because the OUR of the activated sludge was lower than the oxygen supply rate (OSR) (Hu et al., 2011a). Then after about 10 min, it reached the ‘‘DO plateau’’ and maintained at this level till to the end. The pH value increased during the anaerobic period because the denitrification process increased the alkalinity. Then the pH value decreased at aerobic stage for the consumption for alkalinity during nitrification process. COD decreased rapidly at the beginning of anaerobic period to supply carbon source for the denitrification process and it mainly occurred in the first 30 min. During the anaerobic period, TP concentration increased for the release of phosphorus by PAOs and then at the aerobic stage the PAOs uptook more P. The ammonium removal occurred mainly at the aerobic stage for nitrification. And no nitrite was accumulated during the whole period. At the first hour of aeration, there was no nitrate accumulated for the simultaneous denitrification and at last the nitrate concentration was only 6.88 mg N/L. In the SQND reactor, the DO concentration kept increasing at the beginning of aerobic stage for the higher aeration rate. After about one and a half hours of aeration, the DO concentration reached to the peak, indicating the completion of nitrification. And the pH value had similar trend with that in SND reactor. When the nitrification process completed, the pH value increased again because of the aeration. Also the COD removal occurred mainly at the beginning of anaerobic period. The ammonium concentration decreased to the lowest at about 180 min, showing that the nitrification process completed. The nitrite concentration increased firstly and reached to 1.66 mg N/L at 150 min. Then it decreased gradually. And the nitrate concentration increased as soon as the aeration started. During the anaerobic period the amount of released phosphorus in SQND reactor was lower than that in SND reactor. That may be one reason of the low removal rate of TP during the SQND process. 3.2. N2O emission characteristics and OUR variation The N2O emission rates and OUR variation during one cycle were shown in Fig. 3. At the anaerobic stage, N2O emission rates were very low both in SND and SQND reactors. And at the aerobic stage, N2O emission characteristics were different. In the SND reactor, the emission rate increased rapidly at the beginning of aeration and reached to the peak value of 4.45 lg/min/g MLSS at 150 min. Then the emission rate maintained between 3.80 and 4.26 lg/min/g MLSS. However, a transient accumulation of N2O was observed in SQND reactor. The emission rate increased rapidly at first and reached to the highest at the same time as NHþ 4 -N was depleted. Then the emission rate decreased drastically and maintained around 0 at last for the completion of nitrification process at 150 min. Significant positive correlation between N2O emission rate and NO 2 -N concentration (r2 = 0.727, p < 0.05) was found during the SQND process.

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Fig. 2. Temporal changes of the contaminant concentration and DO and pH values during one batch cycle in two reactors. (a) SND reactor and (b) SQND reactor.

Previous studies showed that a higher N2O emission rate was observed when NO 2 -N was accumulated (Tallec et al., 2008; Rajagopal and Béline, 2011). Nitrite can be used as a terminal electron acceptor to produce nitrous oxide by an ammonia oxidizer. Table 2 showed the N2O emission amount in different reactors. It was found that the main N2O emission occurred at aerobic stage and the emission amount in SND reactor was about four times higher than in SQND reactor. The N2O-N produced during SND process account for 7.05% of the total removed nitrogen, which was more than three times higher than in SQND reactor. N2O produces mainly through the nitrate ammonification and denitrification processes during the anaerobic phase. Smith and Zimmerman (1981) have reported that nitrate can be converted into ammonia in a dissimilatory process called nitrate ammonification, with about 5–10% and up to 34% of nitrogen lost as N2O, inevitably. And N2O was produced as an intermediate during denitrification. However, the residual nitrate, anaerobic condition and abundant carbon source supplied suitable conditions for complete denitrification process, causing the rapid reduction of N2O to N2, thus reducing the production of N2O. At the aerobic stage, SND produced more N2O mainly by two reasons. First, at low oxygen level the denitrification process could proceed at the aerobic stage, using the residual influent carbon or intracellular storage compounds as carbon source. And the presence of oxygen could inhibit the activity of nitrous oxide reductase,

causing the accumulation of N2O (von Schulthess et al., 1994). Second, the nitrifier denitrification during simultaneous nitrification denitrification process played an important role during the production of N2O. Nitrifier denitrification is the pathway of nitrification in which ammonium is oxidized by autotrophic nitrifiers to nitrite followed by the reduction of nitrite to nitric oxide, nitrous oxide and molecular nitrogen (Poth and Focht, 1985). Usually low oxygen conditions coupled with low organic carbon favor this way and it contributes up to 30% of the total N2O production (Wrage et al., 2001). It was found that the oxygen level affected the production of N2O significantly during the SND process. The oxygen uptake rate (OUR) reflects the microbial activity and oxygen utilization conditions in the reactor. The OUR of the sludge was evaluated and shown in Fig. 3. At the beginning of aerobic stage, the OURs were higher in both SND and SQND reactors. This was because the medium in the reactors was sufficient and the activities of microbes were higher. During this period, N2O emission rate increased rapidly. In SND reactor, the OUR maintained a high level for the low aeration rate at the beginning of aeration. During this period, N2O emission rate was also high. And after 210 min, the OUR decreased gradually for the consumption of medium and completion of process. And the N2O emission rate also decreased. In SQND reactor, the OUR decreased rapidly after 150 min, for the completion of process. The

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Fig. 3. Time profiles of N2O emission, PHB content and OUR during the reaction period in the SBRs. (a) SND reactor and (b) SQND reactor.

3.3. Effect of PHB on N2O emission

Table 2 N2O emission amount per cycle in different reactors.

a

Reactor

N2O emission at anaerobic stage (mg)

N2O emission at aerobic stage (mg)

Total N2O emission (mg)

N2O-N conversion ratea (%)

SND SQND

0.23 0.51

26.62 6.38

26.85 6.90

7.05 2.12

N2O conversion rate = (total N2O-N emission)/(TN removed)  100%.

OUR during SND process was higher than during SQND process, showing that the microbial activity was high. The oxygen was consumed mainly by nitrification, organic matters degradation and growth of microbe during the aerobic stage. In SND reactor, the microbial activity proceeded during the whole cycle. Nitrifier denitrification and degradation of PHB main occurred in the reactor, leading to the high oxygen uptake. Both these processes lead high N2O emission. In contrast, the OUR in the SQND reactor decreased for the completion of nitrification and no denitrification process occurred. So it produced less N2O than the SND reactor. The N2O emission has great relations with the OUR. The OUR could reflects the N2O emission trend more exactly than the DO concentration.

The temporal changes of PHB in two reactors were shown in Fig. 3. At the anaerobic stage, the carbon source, glucose and sodium acetate, was consumed and stored as PHB, which was accompanied by release of phosphorus. Then at the aerobic stage, PHB was oxidized and decreased in both reactors with the taken up of phosphorus. However, the change of PHB during SND process was more drastic than during SQND process. In the SQND reactor, the peak value of PHB content was only 0.063 g/g SS. The content decreased rapidly at the beginning of aeration and after about one and a half hours it maintained at 0.044 g/g SS and changed little. It was found in Fig. 2(b) that P in the SQND reactor was almost removed completely at 180 min. So it was concluded that the consumption of PHB during SQND process was mainly caused by the uptake of P. In the SND reactor, the PHB increased to 0.078 g/g SS at the end of anaerobic stage. And then it decreased rapidly at the beginning of aerobic stage and the rate was higher than in SQND reactor. At last the content was only 0.03 g/g SS. The PHB content decreased continuously after the removal of P completed. Although the uptake of P consumed a certain amount of PHB, there was also some PHB consumed in other ways during SND process. At the beginning of aerobic stage, the carbon influent was almost consumed out, and COD/N ratio was very low (only 1.5). However,

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during SND process, leading to high N2O emission. In SND process, the denitrification could carry out using PHB as the carbon source at low oxygenation for the lack of carbon and it could cause more N2O emission.

Acknowledgements This work was supported by National Natural Science Foundation of China (No. 21177075), Program for New Century Excellent Talents in University (No. NCET-10-0554), National Natural Science Foundation of China (No. 21007032) and Independent Innovation Foundation of Shandong University (No. 2009JQ009).

References

Fig. 4. N2O profiles during two batch experiments with (Run 2) and without (Run 1) external carbon addition.

the denitrification proceeded for the low oxygen, i.e. simultaneous denitrification. So the PHB was needed and used as carbon source for denitrification process. It was found that the consumption of PHB mainly occurred at the first hour of aeration (Fig. 3(a)). During this period the simultaneous denitrification process was strong (Fig. 2(a)) and no nitrate was accumulated. In order to study the N2O production of PHB-driven denitrification, batch experiments were conducted and the results were shown in Fig 4. About 35% and 52% of nitrate was removed through denitrification in Run 1 and Run 2. There was no carbon influent in Run 1, so the denitrification was driven by intracellular PHB. And in Run 2, the COD removal rate was 80.74%. The internal carbon was exhausted through the aeration before the experiment, so the denitrification proceeded using the external carbon. It was shown that N2O emission varied between the two different kinds of denitrification. The PHB-driven denitrification caused more N2O emission. Some studies also suggested that the consumption of PHB caused more production of N2O (Meyer et al., 2005; Kampschreur et al., 2009). The PHB degraded slowly, so it could produce competition for electrons between denitrifying enzymes. And the activity of nitric oxide reductase would be higher than the nitrous oxide reductase, causing a higher NO reduction rate compared to the N2O reduction rate (Kampschreur et al., 2009). That was an important reason for the high N2O production during SND process. Pervious study showed that increased PHB accumulation in the biomass could be one strategy for stimulating denitrification towards the end of the reactor cycle to enhance the nitrogen removal (Meyer et al., 2005). However, the above results suggested that this method may increase the production of N2O. Step-feed and carbon source addition schemes were also studied to enhance nitrogen removal as well as reduce N2O emission (Yang et al., 2009; Li et al., 2010). This method could change the carbon source for denitrification from intracellular PHB to external carbon and increase the C/N ratio to achieve the control of N2O.

4. Conclusion Although SND process enhanced the removal of nutrients, it also increased production of N2O. The amount of N2O-N emission during SND process was almost four times higher than that during SQND process and 7.05% of the removed nitrogen was converted to N2ON. The higher OUR in SND reactor than in SQND reactor showed that nitrifier denitrification and degradation of PHB processes may occur

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