Characteristics of nitrous oxide (N2O) emission from intermittently aerated sequencing batch reactors (IASBRs) treating slaughterhouse wastewater at low temperature

Biochemical Engineering Journal 86 (2014) 62–68

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Regular Article

Characteristics of nitrous oxide (N2 O) emission from intermittently aerated sequencing batch reactors (IASBRs) treating slaughterhouse wastewater at low temperature Min Pan a , Xiaogang Wen b,∗∗ , Guangxue Wu c , Mingchuan Zhang d , Xinmin Zhan e,∗ a

School of Environmental Science and Technology, Xiamen University of Technology, Xiamen 361024, China Nanhu College, Jiaxing University, Jiaxing, Zhejiang Province 314001, China c Graduate School at Shenzhen, Tsinghua University, Shenzhen, Guangdong Province 518055, China d College of Resources and Civil Engineering, Northeastern University, Shenyang, Liaoning Province 110819, China e Civil Engineering, College of Engineering and Informatics, National University of Ireland, Galway, Ireland b

a r t i c l e

i n f o

Article history: Received 15 July 2013 Received in revised form 16 January 2014 Accepted 12 March 2014 Available online 20 March 2014 Keywords: Intermittent aeration Slaughterhouse wastewater Aeration rate Nitrite Dissolved oxygen

a b s t r a c t This study investigated the characteristics of nitrous oxide (N2 O) emission from intermittently aerated sequencing batch reactors (IASBRs) treating high strength slaughterhouse wastewater at 11 ◦ C, where partial nitrification followed by denitrification (PND) was achieved. N2 O generation and emission was examined at three aeration rates of 0.4, 0.6, and 0.8 L air/min in three IASBRs (SBR1, SBR2, and SBR3, respectively). The slaughterhouse wastewater contained chemical oxygen demand (COD) of 6057 ± 172.6 mg/L, total nitrogen (TN) of 576 ± 15.1 mg/L, total phosphorus (TP) of 52 ± 2.7 mg/L and suspended solids (SS) of 1843 ± 280.5 g/L. In the pseudo-steady state, the amount of N2 O emission was up to 5.7–11.0% of incoming TN. The aeration rate negatively affected N2 O emission and the ratio of N2 O emission to incoming TN was reduced by 48.2% when the aeration rate was increased from 0.4 to 0.8 L air/min. Results showed that more N2 O was generated in non-aeration periods than in aeration periods. Lower DO concentrations enhanced N2 O generation in the aeration periods (probably via nitrifier denitrification) while low DO concentrations (lower than 0.2 mg/L) did not affect N2 O generation in the non-aeration periods (probably via heterotrophic denitrification). When PHB was utilized as the organic substrate for denitrification, there was a high N2 O generation potential. It was estimated that 1.8 mg N2 O-N was generated accompanying per mg PHB consumed. © 2014 Elsevier B.V. All rights reserved.

1. Introduction N2 O is a greenhouse gas with an approximately 300-fold greater global warming potential than carbon dioxide (CO2 ) and it has 114 years steady-state lifetime [1]. When it reaches the stratosphere, it transforms into NO which is destructive for the ozone layer [2]. Agricultural soils contribute to approximately 50% of the World’s anthropogenic N2 O emissions [3] and marine ecosystems are estimated to contribute to 14% of the global N2 O emission. Direct N2 O emission from wastewater treatment plants (WWTPs) is a minor source of N2 O emission (∼0.5% of the total influent nitrogen loading rate [4]). Currently, global N2 O emission is rising at a rate of

∗ Corresponding author. Tel.: +353 091 495239; fax: +353 091 494507. ∗∗ Corresponding author. E-mail addresses: [email protected] (X. Wen), [email protected] (X. Zhan). http://dx.doi.org/10.1016/j.bej.2014.03.003 1369-703X/© 2014 Elsevier B.V. All rights reserved.

0.2–0.3% yr−1 [3], mainly due to the rising N2 O emission in WWTPs [5]. Foley et al. [6] found 0.006–0.253 kg N2 O-N per kg N denitrified in seven full-scale WWTPs, and suggested that N2 O be emitted from each reactor zone in each WWTP. Sequencing batch reactors (SBR) are widely used for wastewater treatment. Previous studies have observed that 51% of the nitrogen loading rate (NLR) was emitted from a SBR treating synthetic municipal wastewater in the form of N2 O, rather than N2 [7], and N2 O emission from a SBR treating digested pig manure was up to 15.6% of NLR [8]. Because the operation conditions in SBRs vary among aerobic, anoxic and anaerobic conditions in one SBR operation cycle, their N2 O emission could be much higher than other wastewater treatment processes where the operation conditions do not vary dramatically [9]. Hence, N2 O emission from SBR systems should be investigated. The production of N2 O is microbiological turnover of inorganic nitrogen by nitrification and denitrification [10]. In the nitrification process, it is proposed that N2 O is produced by ammonia oxidizers in two pathways: (1) reduction of nitrite (NO2 − ) under oxygen

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limited conditions, called nitrifier denitrification, in which ammonia oxidizing bacteria (AOB) oxidize NH4 + to NO2 − and then reduce NO2 − to N2 O and N2 ; and (2) chemical decomposition of NO2 − or intermediates of NH3 oxidation [11,12]. However, it has been reported that N2 O generation during chemical decomposition is not commonly found in activated sludge wastewater treatment plants [5], and nitrifier denitrification is the main contributor to N2 O generation in the nitrification process. In addition, N2 O is one of the obligatory intermediates in denitrification. Complete denitrification consists of four steps transferring nitrate (NO3 − ) to nitrogen gas (N2 ) and N2 O is an intermediate as described in Eq. (1):

temperatures. By far, no studies on N2 O emissions from slaughterhouse wastewater treatment systems at low temperature have been conducted. In this study, three laboratory-scale intermittently aerated sequencing batch reactors (IASBR) were set up to treat slaughterhouse wastewater at 11 ◦ C. The characteristics of N2 O generation and emission from the three IASBRs were studied, with the focus on the effects of the aeration rate on N2 O emission.

NO3 − → NO2 − → NO → N2 O → N2

2.1. Slaughterhouse wastewater

(1)

Incomplete denitrification is considered as the main contributor to N2 O generation [13], in which N2 O is the terminal product instead of N2 . A wide range of process conditions have been observed to affect N2 O emissions in full-scale or laboratory-scale systems, such as concentrations of dissolve oxygen (DO) and NO2 − , pH, carbon substrate availability or the ratio of COD/N, and temperature [2,5,9,14]. Limited DO is considered to have a number of effects on biological nitrogen removal (BNR), and it was observed that nitrous oxide reductase (N2 OR) was much more sensitive to oxygen than other heterotrophic reductases [14]. Thus, low DO acts as an inhibitor to the heterotrophic N2 OR and encourages N2 O generation in both nitrifier denitrification and heterotrophic denitrification processes. Tallec et al. [5] found that heterotrophic denitrification was the major contributor to N2 O emission when DO was below 0.3 mg/L; when DO was in the range of 0.4–1.1 mg/L, autotrophic nitrifier denitrification represented 60% of the N2 O production and heterotrophic denitrification accounted for 40%. The accumulation of NO2 − is a trigger for N2 O emission, and it was reported that N2 O generation occurred when the NO2 − concentration was higher than 2 mg/L [15]. However, Zeng et al. [16] found significant N2 O emission when the NO2 − concentration was lower than 2 mg/L. A low organic substrate level could also induce incomplete denitrification and N2 O emission. Itokawa et al. [11] observed that 20–30% of nitrogen loading was emitted as N2 O when the COD: N ratio was below 3.5. In addition, N2 O emission was the highest at a pH of 8.5 and the lowest at a pH of 6 [9]. Generally, huge variation of N2 O emission has been observed, 0–95% of nitrogen load from laboratory-scale WWTPs and 0–14.6% of nitrogen load from full-scale WWTPs [9]. Enhanced nutrient removal processes are considered to lead to N2 O emission [17], especially biological nitrogen removal via nitrite processes. Intermittently aerated sequencing batch reactors have been found to preferably achieve partial nitrification and efficiently remove nitrogen through partial nitrification followed by denitrification (PND) [8]. However, the characteristics of N2 O emission in an intermittent aeration pattern have not been elaborated. It is thought that N2 O emission would be high in intermittent aeration reactors due to (i) high NO2 − concentrations in the reactors because of efficient partial nitrification; and (ii) varying DO concentrations in aeration periods (from 0 to high DO levels) and in non-aeration periods (from high DO levels to 0). Therefore, it is necessary to study the characteristics of N2 O emission from intermittently aerated systems especially when treating high strength wastewater. In Ireland, there are currently 48 licensed slaughterhouses that process more than 50 tonnes of wastewater per day [18]. It contains high concentrations of organic matter, oil and grease, and nitrogenous compounds (proteins and amino acids). The annual average temperature in Ireland is 11 ◦ C, which is considered to inhibit nitrification (the optimal temperature for nitrification is 25–35 ◦ C [19]). Low temperature has been reported to encourage N2 O emission [12]. Therefore, more N2 O would be emitted when high strength slaughterhouse wastewater is treated at low

2. Materials and methods

Raw slaughterhouse wastewater was collected from the wastewater treatment plant (WWTP) of a local slaughterhouse in western Ireland and stored in the laboratory at 11 ◦ C. In the WWTP, the average organic loading rate is 0.5 g COD/(L d) and the sludge retention time (SRT) is 20–30 days. The average concentrations of SS, COD, BOD5 , TN, TP and NH4 + -N in the raw slaughterhouse wastewater over the study period were 1843 ± 280.5, 6057 ± 172.6, 4240 ± 271.3, 576 ± 15.1, 52 ± 2.7, and 567 ± 17.2 mg/L, respectively. 2.2. Laboratory-scale IASBR systems Three identical IASBR reactors (SBR1, SBR2, and SBR3) were set up in a laboratory where the temperature was controlled at 11 ◦ C. The reactors were made from 12 L transparent Plexiglas with a diameter of 194 mm and a working volume of 8.0 L. Two peristaltic pumps (323S, Walson-Marlow, UK) with three swivels were used, one feeding the wastewater into the three reactors and the other withdrawing the treated wastewater. Three mechanical stirrers with a rectangular paddle (100 mm × 80 mm) were installed to mix the liquid in the reactors. The reactors were constantly stirred during the fill, non-aeration and aeration periods, while air was supplied by air diffusers located at the bottom of reactors. The sequential operation of the IASBR systems was controlled by a programmable logic controller (PLC) (S7-CPU-224, Siemens, Germany). 2.3. Operation of the IASBR systems The IASBRs were operated in 12 h cycles. There were two cycles per day and each cycle comprised: four alternating nonaeration (60 min)/aeration (100 min), settle (70 min), and draw/idle (10 min) phases. The fill (10 min) period was included in the first non-aeration period. According to our previous studies on slaughterhouse wastewater treatment using IASBRs at ambient temperature [20,21], three aeration rates of 0.4, 0.6, and 0.8 L air/min were used in the aeration periods in the three IASBR reactors, corresponding to SBR1, SBR2, and SBR3, respectively. During the operational period (80 days), in each cycle, 400 mL of slaughterhouse wastewater was fed into the reactors. Every day 0.8 L of slaughterhouse wastewater was treated, resulting in a hydraulic retention time (HRT) of 10 days. 400 mL of mixed liquor was withdrawn from each reactor every day just before the settle phase, keeping a solid retention time (SRT) of about 20 days (if without consideration of solid loss with the effluent). The average organic loading rate and TN loading rate were 0.61 g COD/(L d) and 58 mg TN/(L d), which were close to COD and TN loading rates applied in the local slaughterhouse wastewater treatment plant. 2.4. Analytical methods NH4 + -N, NO2 − -N, NO3 − -N, and PO4 3− -P were determined with a nutrient analyser (Konelab 20, Thermo Clinical Labsystems, USA).

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TN and TP were measured with a TOC TN TP Analyser (Biotector, Ireland). COD, BOD5 , SS and VSS were examined following the standard APHA methods [22]. The DO concentrations in the reactors were monitored by a multiple probe (HI9828, Hanna, Italy). PHB contents in activated sludge biomass were analyzed by using high performance liquid chromatography (Agilent 1200, Agilent Technology, USA) with an UV index detector and an Aminer HPX-87H column (Bio-Rad, USA) according to the method adopted by Rodgers and Wu [23]. N2 O was measured by a dissolved N2 O microsensor (Unisense, Denmark), which can measure soluble N2 O concentrations in aqueous solutions in a range of 0–30 mg/L with a detection limit of 0.04 mg/L. The procedures of measurement of dissolved N2 O concentrations in the reactors and of quantification of N2 O emission and generation have been detailed by Quan et al. [13]. The formulae used to calculate N2 O emission and generation are given as follows: re = −KCN2 O rc =

dCN2 O

dt rg = rc − re

(2) (3) (4)



t2

Q = −V

re dt

(5)

t1

t2 G=V

rg dt

(6)

t1

Sg =

rg SS

(7)

where, CN2 O is the dissolved N2 O concentration, mg/L; K is the mass transfer coefficient of N2 O from the liquid phase to the air, min−1 ; re is the N2 O emission rate, mg/L min; rc is the N2 O accumulation rate at time t in the liquid phase, mg/L min; rg is the N2 O generation rate, mg/(L min); Q is the amount of N2 O emission during the period of t1 − t2 , mg; G is the amount of N2 O generation during the period of t1 − t2 , mg; V is the effective liquid volume, L; sg is the specific N2 O generation rate, mg/(g SS min); and SS is the concentration of suspended sludge in the reactors, g/L. The mass transfer coefficient K via diffusion (non-aeration periods) and air stripping (aeration periods) was obtained with clear water tests according to the method of Quan et al. [13]. K values were measured as 0.033/min (R2 = 0.95, P < 5%), 0.048/min (R2 = 0.98, P < 5%), and 0.07/min (R2 = 0.97, P < 5%) for N2 O emission via air stripping (diffusion was included) at the three aeration rates of 0.4, 0.6, and 0.8 L air/min, respectively, and 0.0029/min (R2 = 0.95, P < 5%) for N2 O emissions via diffusion during the non-aeration periods. 3. Results and discussion 3.1. N2 O generation and emission during a typical cycle in the IASBRs During the pseudo-steady state, COD was efficiently removed from slaughterhouse wastewater in the three reactors and effluent COD met the Irish emission standard. Efficient TN removal was maintained in all reactors but TN concentrations in SBR3 effluent were much higher than in SBR1 and SBR2. More than 96% of TP was effectively removed in SBR1 and SBR2; the effluent TP concentrations in SBR3 were in the range of 8.4–28.7 mg/L, with average TP removals of only 64.3%, and did not meet the discharge standard. Among the three aeration rates, the optimal aeration rate was

regarded as 0.6 L air/min, giving removal efficiencies of COD, TN and TP over 97%. Dissolved N2 O concentrations were detected in a number of SBR operational cycles. Fig. 1(A) presents dissolved N2 O concentrations during a typical cycle in IASBRs at the three aeration rates. Dissolved N2 O concentrations peaked at the end of the non-aeration periods and after aeration commenced the N2 O concentrations quickly declined, indicating that N2 O was emitted to the atmosphere through air stripping. More N2 O was generated than emitted in the non-aeration periods, causing dissolved N2 O concentrations to rise in the non-aeration periods. Using Eq. (5), the amounts of N2 O emission were calculated for the typical SBR cycles, which were up to 39.4, 32.6, and 20.6 mg, corresponding to 11.0%, 9.1%, and 5.7% of incoming TN in SBR1, SBR2, and SBR3, respectively. The ratio of the amount of N2 O-N emission to incoming TN (y, %) varied with the aeration rate (A, L air/min), and a relationship can be established: y = −13.3A + 16.6 (R2 = 0.97, P < 0.05)

(8)

The negative linear correlation between y and A, which is illustrated in Fig. S1, indicates that a lower aeration rate encouraged nitrogen conversion to N2 O gas. y was reduced by 48.2% when the aeration rate was increased from 0.4 to 0.8 L air/min. Generally, enhanced biological nitrogen removal processes, including simultaneous nitrification and denitrification (SND) and partial nitrification followed by denitrification, likely emit high amounts of N2 O [17], which can be observed in Table 1. Comparing with low nitrogen wastewater treatment systems, high strength nitrogen wastewater is a trigger for high N2 O emission. However, in high strength nitrogen wastewater treatment, the amounts of N2 O emission from the SND processes seemed to be higher than from the intermittent aeration systems (Table 1). The amount of N2 O emission obtained by Beline et al. [26] was 30% of TN in raw piggery slurry, which was much higher than the emissions in the present study where the amounts of N2 O emission were only 2.3–4.4% of TN in raw slaughterhouse wastewater (5.7–10.9% of incoming TN). One reason is different experimental conditions and influent wastewater characteristics in the two studies. Another reason might be due to the intermittent aeration strategy used in this study. Osada et al. [27] suggested that the intermittent aeration pattern could reduce the N2 O emission from 35% to 1% of the nitrogen loading. On the other hand, lower temperature was reported to enhance N2 O emission [9]. Hu et al. [12] found that, in SBR systems treating synthetic wastewater with low nitrogen concentrations, the timeweighted N2 O emission quantity was 630.4, 260.8, 218.3, 104.7, and 57.5 mg/g MLSS when temperature was at 15, 20, 25, 30, and 35 ◦ C, respectively; the time-weighted N2 O emission at 15 ◦ C was 2.9 times higher than that at 25 ◦ C. In the present study, the specific N2 O emissions were equal to 1.4, 1.4, and 0.9 mg N2 O/g MLSS (MLSS: 3.53, 3.01, and 2.77 g/L) in the three reactors at 11 ◦ C, on average. Thus, efficient mitigation of N2 O emission was achieved in this stud, possibly due to the intermittent aeration strategy applied. Using Eq. (5), the amounts of N2 O generation were calculated for the typical SBR cycles, which were up to 39.7, 32.9, and 20.6 mg, respectively. According to Eqs. (6) and (7), the specific N2 O generation rates in SBR1, SBR2 and SBR 3 can be calculated, which are shown in Fig. 1(C). In the typical cycles at the aeration rates of 0.4, 0.6, 0.8 L air/min, the average specific N2 O generation rates were 1.9, 1.6, and 1.2 ␮g N2 O/g SS min, indicating that the N2 O generation rate was reduced with the increase in the aeration rate. 3.2. N2 O generation in the aeration periods 14.2 mg, 4.2 mg, and 1.5 mg N2 O was generated during the four aeration periods in SBR1, SBR2, and SBR3, which corresponded to 3.9%, 1.1%, and 0.4% of incoming TN, respectively. An attempt was

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Fig. 1. Typical cycle profiles of dissolved N2 O concentration (A), N2 O emission rate (B) and the specific N2 O generation rate (C) in the IASBR systems at the three aeration rates (solid line: 0.4 L air/min; dash line: 0.6 L air/min; dot line: 0.8 L air/min). Table 1 N2 O emission from nitrogen removal processes. Wastewater Synthetic wastewater (230 mg/L COD, 23 mg/L NH4 + -N) Synthetic wastewater (340 mg/LCOD, 49 mg/L NH4 + -N) Synthetic wastewater (5.8–54 mg/L NH4 + -N)

Configuration

Operation

Lab-scale SBR

a

Lab-scale SBR

◦

SND ; 20–22 C; OLR, 0.55 g COD/(L d) SND; 25 ± 2 ◦ C

Lab-scale partial nitrification–Anammox reactors

Partial nitrification–Anammox; 35 ◦ C

Aerobically treated piggery slurry High-strength wastewater

Lab-scale reactor Lab-scale bioreactor

Anaerobically digested pig manure

Lab-scale IASBR

SND; 20–28 ◦ C Intermittent aeration pattern with high nitrite accumulation; ambient temperature High nitrite accumulation; 26 ± 1 ◦ C

a

N2 O emission

Reference

51% of NLR

Lemaire et al. [7]

7.05% of the removed nitrogen

Jia et al. [24]

9.6 ± 3.2% of the removed nitrogen in the Okabe et al. [25] partial nitrification process and 0.14 ± 0.09% of removed nitrogen in the Anammox process 30% of TN in raw slurry Beline et al. [26] 20–30% of influent nitrogen with influent Itokawa et al. [11] COD/N less than 3.5 15.6% of NLR

Zhang et al. [8]

SND, simultaneous nitrification and denitrification.

made to relate the N2 O generation during the aeration periods to the aeration rates, which is a controlling factor affecting the treatment cost. A negative linear correlation between the aeration rate (A, L air/min) and N2 O-N generation (G, mg) during the aeration periods was established as G = −0.04A + 0.8 (R2 = 0.90). The negative correlation confirms that a lower aeration rate encouraged N2 O generation through nitrifier denitrification [9]. At low oxygen levels nitrous oxide reductase would be inhibited leading to N2 O as the final product. Three aeration rates were applied in the three reactors, leading to different oxygen levels during the aeration periods. Fig. 2 illustrates typical profiles of DO at the three aeration rates. The mean DO concentrations in the four aeration periods in SBR1 were 0.34, 0.52, 0.53 and 0.56 mg/L, were 0.38, 0.76, 1.46 and 2.4 mg/L in the four aeration periods in SBR2, and were 0.89, 1.85, 4.86 and 5.71 mg/L in the four aeration periods in SBR3, respectively. Otte et al. [28] reported that the highest N2 O production was observed at the DO concentration of 5% air

saturation (0.38 mg/L) at 30 ◦ C. In this study, the highest N2 O generation also occurred in the aeration period when the average DO concentration was 0.56 mg/L, equal to 5.1% air saturation at 11 ◦ C. In nitrifier denitrification, most N2 O emission occurs under aerobic conditions by coupled NH4 + oxidation and NO2 − reduction (nitrifier denitrification) [29]. However, Kim et al. [30] found that the N2 O emission under nitrifying conditions did not depend on the oxidation of NO2 − by NOB but depended on the oxidation of NH4 + by AOB. Chandran et al. [31] also stated that N2 O production was essentially associated with ammonia oxidation in the aerobic conditions. In this study, N2 O generation was increased when the average DO concentrations were increased in the four aeration periods in SBR1, which can be explained by increased NH4 + -N oxidation amount in corresponding aeration periods (Fig. 3(A)). A same correlation between N2 O generation and the amount of ammonia oxidation was found in SBR2. However, higher ammonia oxidation rates in the first and second aeration periods in SBR3

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of NH4 + -N conversion to N2 O-N appeared at the average DO concentration of 0.56 mg/L; while the N2 O generation was relatively low when average DO concentrations were above 1.85 mg/L, which was same with the result reported by Tallec et al. [5]. However, it is noticed that in the three reactors, the average DO concentrations higher than 1.85 mg/L only took place during the fourth aeration period in SBR2 and the third and fourth aeration periods in SBR3, when NH4 + -N was almost completely oxidized. In low oxygen levels (<1.85 mg/L), increased DO concentration led to increased NH4 + -N oxidation rates. These findings indicate that the NH4 + -N oxidation rate could not be considered as the sole factor influencing N2 O generation and as a consequence, DO concentration was the main factor. It can be found in Fig. 3(B) that low oxygen levels enhanced N2 O generation through nitrifier denitrification. 3.3. N2 O generation in non-aeration periods

Fig. 2. Profiles of DO concentrations during a typical cycle in the laboratoryscale IASBR systems at the three aeration rates (solid line: 0.4 L air/min; dash line: 0.6 L air/min; dot line: 0.8 L air/min).

In the present study, 25.5, 28.9, and 19.1 mg of N2 O was generated during non-aeration periods (the fill, settle, and effluent draw phases were also included) in SBR1, SBR2, and SBR3, which corresponded to 7.0%, 8.0%, and 5.3% of incoming TN, respectively. The amounts of N2 O generation (probably through heterotrophic denitrification) corresponded to 64.2%, 87.3%, and 92.8% of total N2 O generation in SBR1, SBR2, and SBR3, respectively, implying that N2 O generation in a SBR operation cycle was mainly generated via heterotrophic denitrification.

Fig. 3. Profiles of NH4 + -N during a typical operational cycle (A) and dependence of N2 O generation/NH4 + -N oxidized on average DO concentration (B) (, 0.4 L air/min; , 0.6 L air/min; and , 0.8 L air/min).

failed to generate more N2 O, which can be explained by the fact that too high DO concentrations unfavoured N2 O generation [9]. The profile of the ratios of N2 O-N generation to NH4 + -N oxidation with average DO concentrations in SBR1, SBR2 and SBR3 is showed in Fig. 3(B). It can be seen that the highest value of 11.8%

Fig. 4. Profiles of NO2 − -N and TON during a typical operational cycle (A) and dependence of the amount of N2 O genration in non-aeration periods on NO2 − N reduced (B) (, variation of NO2 − -N at 0.4 L air/min; , variation of NO2 − -N at 0.6 L air/min; , variation of NO2 − -N at 0.8 L air/min; , variation of TON at 0.4 L air/min; 䊉, variation of TON at 0.6 L air/min; , variation of TON at 0.8 L air/min).

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Fig. 5. Profiles of COD (A) and PHB (mg PHB/g VSS) (B) during a typical cycle in the laboratory-scale IASBRs (B), and the correlation between PHB utilization and N2 O-N generation during corresponding non-aeration periods (C) (, 0.4 L air/min; , 0.6 L air/min; , 0.8 L air/min).

The effects of the aeration rate on denitrification in the nonaeration periods mainly result from the following factors: (i) denitrification precursors (NO2 − -N or NO3 − -N); (ii) NO2 − -N and NO3 − -N concentrations at the beginning of the non-aeration periods; and (iii) the oxygen levels in the non-aeration periods. In Fig. 2, the mean DO concentrations in the five non-aeration periods (the settle and draw phases were also included) were 0.02, 0.05, 0.08, 0.09, and 0.1 mg/L in SBR1, 0.01, 0.02, 0.03, 0.07, and 1.1 mg/L in SBR2, and 0.06, 0.1, 0.2, 3.7, and 3.35 mg/L in SBR3, respectively. When the average DO concentrations were lower than 0.2 mg/L, similar NO2 − -N reduction rates were observed; only 0.7 mg/L of NO2 − -N was reduced in the fourth non-aeration period in SBR3 with average DO of 3.7 mg/L (Fig. 4(A)). It suggests that the influences of low DO levels (<0.2 mg/L) on denitrification was negligible and only high DO levels (for instance 3.7 mg/L in the present study) disadvantaged heterotrophic denitrification, which led to N2 O generation. 63.4% of total oxidized nitrogen (TON) (82% of TON was NO2 − -N) removed was in the form of N2 O gas in the fourth nonaeration period in SBR3 with an average DO level of 3.7 mg/L. High NO2 − concentrations inhibit denitrification enzymes especially nitrous oxide reductase due to its toxic effects, which leads to N2 O generation [14]. A high NO2 − concentration causes a high turnover for the nitrite reductase [32] and therefore a relatively high nitrite reduction rate during denitrification. Since many reports have shown that N2 O emission was detected shortly after nitrite was reduced [7,14], NO2 − -N reduction rates could also be assumed to influence N2 O generation. In Fig. 4(A), N2 O levels were significantly increased shortly after the decrease in NO2 − -N and TON (NO2 − -N + NO3 − -N) concentrations during the non-aeration periods, which indicates that N2 O production was essentially associated with NO2 − -N and TON reduction in denitrification. In the

non-aeration periods in the typical cycles, among all TON reduced by means of heterotrophic denitrification, there were 75.1, 110.7, and 38.0 mg NO2 − -N reduced, corresponding to 76.8%, 65.3%, and 39.7% of TON reduction in SBR1, SBR2, and SBR3, respectively. The results explain the highest amount of N2 O generation in the non-aeration periods in SBR2 and also confirm that higher NO2 − N would encourage more N2 O generation. However, in Fig. 4(B), points of Number 1, 2, and 3, reveal low N2 O generation at high NO2 − -N reduction. The three points corresponded to the first nonaeration period in the three reactors in which the fill phase was also included. In the first non-aeration periods, because high COD concentrations in the fresh slaughterhouse wastewater was fed into the three reactors, completed denitrification occurred with sufficient organic substrate available, so NO2 − -N was completely reduced and N2 was the main end-product rather than N2 O. Thus, the amount of NO2 − -N reduction could not be considered as the sole factor influencing N2 O generation in heterotrophic denitrification. 3.4. Effect of PHB on N2 O generation Theoretically, the stoichiometric requirement for denitrification was 2.86 g COD/g N, considering the electron transmitting balance between the organic substrate and NO3 − -N [33]. However, in practice, COD/N requirements are usually higher in conventional biological nitrogen removal processes. In this study, due to high TN reduction by heterotrophic denitrifiers and formation of PHB in the activated sludge biomass during the first non-aeration period and the first aeration period (when the DO concentrations were very low) in the SBR cycle, most of incoming soluble COD was consumed in these periods (Fig. 5(A)). In Fig. 5(B), significant increase in the PHB content in activated sludge biomass can be observed in

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both SBR1 and SBR2, while a slight variation of the PHB content appeared in SBR3. It was observed that P removal was completed at the end of the second aeration period and the beginning of the third aeration period in SBR1 and SBR2 (data not shown), respectively, and the PHB contents kept decreasing after that. Although the uptake of P consumed a certain amount of PHB, there was also some PHB utilized in other ways [23], especially for heterotrophic denitrification. Rapidly 3.6 and 4.9 mg of PHB was utilized at the third and fourth non-aeration periods in SBR1 with 5.3 and 10.9 mg of N2 O-N generated, respectively; while 2.3 mg PHB was consumed in the second non-aeration period in SBR2, whereas 4.6 mg N2 O-N was generated. Thus, PHB can be utilized as an organic substrate for denitrification and more PHB consumption was found to accompany higher N2 O generation, which agreed with the finding that the PHB-driven denitrification caused more N2 O emission [24]. Third et al. [34] reported that slow degradation of PHB would preserve reducing power for denitrification, which may lead to high N2 O production. In the present study, average PHB utilized for generating N2 O (during the third and fourth non-aeration periods in SBR1, and the second, third, and fourth non-aeration periods in SBR2 and SBR3) can be roughly evaluated as 1.8 mg N2 O-N/mg PHB (R2 = 0.77, P < 0.05) (Fig. 5(C)). Further study is needed to investigate N2 O generation using PHB as the only carbon substrate. 4. Conclusions The characteristics of N2 O generation and emission were investigated in IASBRs treating slaughterhouse wastewater at 11 ◦ C. The amounts of N2 O emission corresponded to 5.7–11.0% of incoming TN. N2 O emission was successfully reduced by using the intermittent aeration pattern and 48.2% of N2 O emission was reduced when the aeration rate was increased from 0.4 to 0.8 L air/min. Lower DO encouraged N2 O generation in the aeration periods (probably via nitrifier denitrification) and low levels of DO (lower than 0.2 mg/L) did not affect N2 O generation in non-aeration periods (probably via heterotrophic denitrification). Both the amounts of NH4 + -N oxidized and NO2 − -N reduced could not be considered as the sole factors causing N2 O generation in nitrification and denitrification, respectively. PHB can be utilized as carbon substrate for denitrification; however, this would enhance N2 O generation. Acknowledgements The authors would like to thank the financial support provided by the China Scholarship Council, and the Department of Civil Engineering, National University of Ireland, Galway. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2014.03.003. References [1] IPCC, Climate Change (2007): The Physical Science Basis, Cambridge University Press, Cambridge, 2007. [2] N. Adouani, T. Lendormi, L. Limousy, O. Sire, Effect of the carbon source on N2 O emissions during biological denitrification, Resour. Conserv. Recycl. 54 (2010) 299–302. [3] IPCC, Climate Change (2001): Impacts, Adaptation, and Vulnerability, 2001. [4] IPCC, IPCC Guideline for National Greenhouse Gas Inventories, IGES, Japan, 2006. [5] G. Tallec, J. Garnier, G. Billen, M. Gousailles, Nitrous oxide emissions from denitrifying activated sludge of urban wastewater treatment plants, under anoxia and low oxygenation, Bioresour. Technol. 99 (2008) 2200–2209. [6] J. Foley, D. de Haas, Z. Yuan, P. Lant, Nitrous oxide generation in full-scale biological nutrient removal wastewater treatment plants, Water Res. 44 (2010) 831–844.

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