Effect of nitrite from nitritation on biological phosphorus removal in a sequencing batch reactor treating domestic wastewater

Bioresource Technology 102 (2011) 6657–6664

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Effect of nitrite from nitritation on biological phosphorus removal in a sequencing batch reactor treating domestic wastewater Wei Zeng, Yingying Yang, Lei Li, Xiangdong Wang, Yongzhen Peng ⇑ Key Laboratory of Beijing for Water Environment Recovery, Department of Environmental Engineering, Beijing University of Technology, Beijing 100124, China

a r t i c l e

i n f o

Article history: Received 20 December 2010 Received in revised form 28 February 2011 Accepted 28 March 2011 Available online 2 April 2011 Keywords: Nitrite Nitritation Phosphate accumulating organisms (PAOs) Enhanced biological phosphorus removal (EBPR) Domestic wastewater

a b s t r a c t Although nitrite effect on enhanced biological phosphorus removal (EBPR) has been previously studied, very limited research has been undertaken about the effect of nitrite accumulation caused by nitritation on EBPR. This paper focused on nitrite effect from nitritation on EBPR in a sequencing batch reactor treating domestic wastewater. Results showed that nitrite of below 10 mg/L did not inhibit P-uptake and release; whereas EBPR deterioration was observed when nitrite accumulation reached 20 mg/L. Due to P-uptake prior to nitritation, nitrite of 20 mg/L has no effect on aerobic P-uptake. The main reason leading to EBPR deterioration was the competition of carbon source. Batch tests were conducted to investigate nitrite effect on anaerobic P-release. Under sufficient carbon source, nitrite of 30 mg/L had no impact on poly-b-hydroxyalkanoate (PHA) storage; contrarily, under insufficient carbon source, denitrifiers competing for carbon source with phosphorus accumulating organisms resulted in decrease of PHA synthesis and P-release. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Nitrogen removal via the nitrite pathway implies that the partial nitrification of ammonia to nitrite has been named nitritation, and the subsequent direct reduction of nitrite to N2 gas, denitritation (Balmelle et al., 1992; Zhu et al., 2008). The application of nitritation–denitritation could lead to a considerable saving in aeration costs and external carbon sources as compared to the complete nitrification–denitrification (Hellinga et al., 1998; Jenicek et al., 2004; Fux et al., 2006; Zeng et al., 2010). The key to achieve nitritation–denitritation is the control of oxidation of ammonia to nitrite and the build-up of nitrite (Gao et al., 2009). As a result of nitritation, nitrite concentration often reached more than 20 mg/ L, much higher than that in a conventional nitrogen removal system (Yang et al., 2007; Ma et al., 2009; Zeng et al., 2009). However, nitrite has been recognized as one inhibitor in microbial metabolism (Yarbrough et al., 1980). Previous studies have confirmed that high concentrations of nitrite inhibits microbial activities in biological wastewater treatment, such as inhibition on heterotrophic bacteria (Musvoto et al., 1999), nitrifying bacteria (Anthonisen et al., 1976; Vadivelu et al., 2006) and phosphate accumulation organisms (PAOs) (Zhou et al., 2007). Therefore, in the processes with nitrogen removal via the nitrite pathway, nitrite accumulation likely has an adverse impact on biological nutrient removal. ⇑ Corresponding author. Tel./fax: +86 10 67392627. E-mail addresses: [email protected] (W. Zeng), [email protected] (Y. Peng). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.03.091

Enhanced biological phosphorus removal (EBPR) generally with simultaneous nitrogen removal is widely implemented in wastewater treatment plants (WWTP). PAOs can grow in EBPR process with alternating anaerobic and aerobic/anoxic conditions. Under anaerobic conditions, PAOs take up carbon source such as volatile fatty acids (VFAs) and store them in the form of poly-bhydroxyalkanoates (PHA), using the energy generated from hydrolysis of polyphosphate (poly-P). Under aerobic or anoxic conditions, PAOs are able to take up excess phosphorus to form intracellular poly-P by using stored PHA as the energy source. The net removal of P can be achieved through wasting activated sludge when rich in poly-P (Oehmen et al., 2007). Previous studies demonstrated that a certain amount of nitrite could inhibit anoxic/aerobic P-uptake of PAOs, even leading to deterioration of biological P removal (Meinhold et al., 1999; Saito et al., 2004; Kuba et al., 1996; Ahn et al., 2001; Hu et al., 2003; Sin et al., 2008; Yoshida et al., 2009). Using an anaerobic/aerobic/ anoxic/aerobic SBR treating municipal wastewater, Yoshida et al. (2006) observed that nitrite exposure could inhibit aerobic phosphate uptake of PAOs and suggested that nitrite is one of the factors affecting stability of EBPR. Presently, the protonated species of nitrite, free nitrous acid (FNA) rather than nitrite is likely the actual inhibitor on the P-uptake by PAOs (Zhou et al., 2007). Zhou et al. (2007) indicated that FNA inhibits anoxic P-uptake at the low levels of 1.0  103–2.0  103 HNO2-N/L. Saito et al. (2004) reported that 0.5  103 HNO2-N/L causes a severe inhibition of aerobic P-uptake, and more than 1.5  103 HNO2-N/L results in almost complete inhibition. Moreover, as denitrifying phosphorus

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removal has been observed in many EBPR systems, it has been experimentally demonstrated that PAOs acclimated to nitrite are capable of using nitrite as sole electron acceptor to perform denitrification and phosphorus removal without inhibition (Guisasola et al., 2009; Jiang et al., 2006; Vargas et al., 2011). It should be noted that most of the research focused on the effect of nitrite/ FNA on the activity of PAOs and the inhibition levels varied in a large range at different operation modes and wastewater types. Moreover, most of these studies were undertaken with addition of nitrite at the beginning of the aerobic or anoxic phase to investigate the impact of nitrite/FNA on biological P removal. Very limited research has been conducted about the effect of nitrite accumulation caused by nitritation–denitritation on EBPR. Therefore, the impact of nitrite accumulation on biological nutrients removal in a nitritation–denitritation system should be investigated further. This study aims to (1) find an effective strategy to achieve shortcut nitrification and investigate the effect of nitrite accumulation on EBPR in an anaerobic/aerobic sequencing batch reactor (SBR) treating real domestic wastewater, (2) experimentally analyze the effect of nitrite accumulation on anaerobic P-release and aerobic P-uptake, and (3) investigate a controlling method to alleviate the nitrite inhibition, and maintain a stable EBPR performance.

into the SBR1 and SBR2. The influent characteristics are shown in Table 1. Synthetic wastewater was used in batch experiments containing per liter: 0.3–1.2 g NaAc, 180 mg MgSO47H2O, 21 mg CaCl22H2O, 3 mg peptone and 0.6 ml nutrient solution. The nutrient solution contained as shown in Smolders et al. (1994), per liter: 1.5 g FeCl36H2O, 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 ethylenediamine tetra-acetic acid (EDTA). When investigating the effect of nitrite accumulation on anaerobic metabolism of PAOs under insufficient carbon source, COD concentration in the synthetic wastewater was controlled at 200 mg/L. When investigating the effect of nitrite accumulation under sufficient carbon source, COD concentration was controlled at 800 mg/L. The seed sludge was taken from the recycling sludge of Gao bei dian wastewater treatment plant in Beijing, which utilizes a typical anaerobic–anoxic–aerobic (A2O) process to treat municipal wastewater and performs biological nutrients removal well. The wastewater composition was similar to that used in this study. After sludge acclimation for one month, a stable performance was achieved and the experiments begun. 2.3. Batch experiments

2. Methods 2.1. Reactor and operation Two lab-scale sequencing batch reactors (SBRs) fed with real domestic wastewater (composition given below) were used to carry out the experiments. SBR1 with a working volume of 11 L was operated for 180 days under anaerobic–aerobic conditions for anaerobic P-release and aerobic P-uptake. Even though in some instances the presence of nitrite in the anaerobic period makes it an actually anoxic period, we still defined this as anaerobic period to make the context consistent. Throughout the operational period, DO concentration was controlled at 0.5–1.0 mg/L, and each cycle included 2 h anaerobic duration, 1 h settling and 1 min decanting with 5.5 L supernatant discharged. To achieve partial nitrification to nitrite, aerobic duration was controlled at 3 h (day 1–30), 4 h (day 31–65) and 5 h (day 66–180), respectively. Therefore, cycle time was varying, 361 min (day 1–30), 421 min (day 31–65) and 481 min (day 66–180), respectively. The sludge retention time (SRT) in SBR1 was 20 days. SBR2 with a working volume of 7 L was operated under anaerobic–aerobic conditions. Each cycle of 7 h consisted of 2 h anaerobic period and 4 h aerobic period, followed by 1 h settling and 1 min decanting with 3.5 L supernatant removed. The SRT in SBR2 was 8 days.

2.2. Wastewater and seed sludge Both domestic wastewater and synthetic wastewater were used in this research. Domestic wastewater from a campus sewer line was pumped into a storing tank for sedimentation, and then fed

Table 1 Characteristics of the domestic wastewater. Contents (mg/L)

Average

COD NHþ 4 -N TN

195 ± 28 69 ± 10 72 ± 11 6.3 ± 2.0

PO3 4 -P

Tested sludge was taken from SBR2 at the end of aerobic stage. It was washed to remove nitrite and then divided into four parts. Each part of the sludge was put into a 1.5 L batch reactor. Four batch reactors were supplied with synthetic wastewater. The initial NO 2 -N concentration in four batch reactors was controlled at 0, 10, 20 and 30 mg N/L, respectively, by adding different amount of sodium nitrite to investigate the effect of nitrite on anaerobic phosphorus release. During the tests, pH was on-line controlled at 7.5 ± 0.05 by adding 0.5 M HCl or 0.5 M NaOH, and temperature was controlled at 25 ± 0.5 °C. 2.4. Analytical methods  3 NHþ 4 -N, NO2 -N, PO4 -P, mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) were measured according to APHA standard methods (1998). DO and pH were measured on-line using DO/pH meters (WTW Multi 340i, Germany). Volatile fatty acids (VFAs) were measured using Agilent 6890N gas chromatography (GC) with an Agilent DB-WAXetr column (30 m  1.0 lm  0.53 mm) equipped, and the injection port and the flame ionization detector (FID) were operated at 220 °C and 250 °C. Temperature program was used: maintained at 80 °C for 1 min; reached up to 160 °C by 20 °C/min and then held for 1 min; increased by 5 °C/min to 180 °C and then held for 1 min. Analysis of PHA, consisting of poly-b-hydroxybutyrate (PHB) and poly-b-hydroxyvalerate (PHV), was performed using Agilent 6890N GC with an Agilent DB-1 column (30 m  1.0 lm 0.53 mm). Weighed freeze-dried biomass, 2 ml chloroform and 2 ml methanol acidified with 3% H2SO4 were added into glass tubes, respectively, and then the tubes were heated in 100 °C for 20 h after being mixed. One milliliter of Milli-Q water was put into the tubes and mixed after cooling. After centrifugation, 1400 mL of the bottom organic phases was put into 2 mL tube, and 600 mL Milli-Q water was added and mixed. After centrifugation, 1 mL of the bottom organic phases was added into GC vial for analysis. The temperature of injector and FID detector were maintained at 200 °C and 250 °C. The temperature program was set as the following: held at 80 °C for 2 min; increased to 140 °C at the rate of 10 °C/ min, and then maintained for 1 min.The concentration of free nitrous acid (FNA, HNO2-N/L) was calculated as the formula (1) (Anthonisen et al., 1976):

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sodium acetate addition

25 Stage 1 Stage 2 Stage 3 Stage 4

20

NO -2 -N

10

3-

10

-1

15

PO4 -P (mg⋅L )

-

-1

NO2-N (mg⋅L )

80 20

PO 34 -P

60

40

NH 4 -N removal effeciency

0

5

0

20

40

60

80

100

120

140

160

180

0 200

NH4-N removal effeciency (%)

100

30

20

0

time (day) 3 Fig. 1. Variations of NO 2 -N, PO4 -P at the end of aerobic phase and NH4-N removal efficiencies in SBR1.

NO2  N

As shown in Fig. 1, in stage 1, effluent NO 2 -N concentration gradually increased from the initial 0.7 mg/L to 9 ± 1 mg/L, while a low NH4-N removal efficiency was observed due to inadequate aeration duration. During this period, effluent P-concentration was stably maintained at lower than 1 mg/L. In stage 2, nitrification was improved through extending aerobic duration, and thus NH4-N removal efficiencies increased from 27% to 58%. Meanwhile, nitrite concentration at the end of aerobic phase rapidly rose from 9.6 mg/L to 20 mg/L, and above 95% of nitrite accumulation rate  3 (NO 2 -N/NOx -N) was achieved. However, PO4 -P concentration at the end of aerobic phase rose to 5 mg/L, suggesting that EBPR in SBR1 began to deteriorate. In stage 3, 1.6 g sodium acetate (NaAc) was added at the beginning of the anaerobic phase to increase the initial COD concentration to 200 mg/L. During this stage, the average nitrite concentration in the aerobic effluent was about 20 mg/L. The PO3 4 -P concentration in the aerobic effluent continuously increased up to 12 mg/L till day 109, and then an obvious descent was observed. Even though external carbon source was added, EBPR could not recover until the 20th day. In stage 4, the NH4-N removal efficiencies and effluent NO 2 -N concentrations were maintained at 97% and 20 mg/L, respectively. During this period, effluent PO3 4 -P concentration gradually dropped and finally

ð1Þ

pH

K a  10

where Ka was calculated by fitting the temperature T (°C) to the formula e2300/(273+T). 3. Results and discussion 3.1. Nitrogen and phosphorus removal in SBR1 treating domestic wastewater The anaerobic/aerobic SBR1 was operated for a total of 180 days. The experimental period was divided into four succes3 sive stages based on the characteristic variations of NO 2 -N, PO4 P at the end of aerobic phase, i.e. stage 1 (day 1–65), stage 2 (day 66–92), stage 3 (day 93–113) and stage 4 (day 114–180). Different from the operation of stages 1 and 2, external carbon source (NaAc) was added in stages 3 and 4. Fig. 1 shows the variations of NO 2 -N, PO3 4 -P at the end of aerobic phase and NH4-N removal efficiencies in SBR1 throughout the experimental period. Fig. 2 shows the variations of phosphorus release, consumed COD in anaerobic phase and aerobic phosphorus uptake in the SBR1.

300 Stage 1

Stage 2 Stage 3

Stage 4

80 250

Phosphorus release Phosphorus uptake consumed COD anaerobically

60

200 150

40 100 20 50 0 0

20

40

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80

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140

160

180

-1

-1

Phosphorus release, Phosphorus uptake (mg ⋅L )

sodium acetate addition

consumed COD anaerobically (mg⋅L )

FNA ¼

0 200

Time (day) Fig. 2. Variations of phosphorus release, consumed COD anaerobically and aerobic phosphorus uptake in the SBR1.

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a

60

3.0

-P, NO-2-N, NH4+-N (mg⋅L-1) PO34

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50

2.5

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3.0

-

NO 2-N

1.5

+

NH 4-N FNA

20

1.0

10

0.5 0.0

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3-

PO 4 -P

30

+

NH 4-N

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1.0 10 0.5 0.0

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3.0

d

3-

1.5 15 1.0

-1

3-

3-

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0.5

5

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0 50

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t (min) NHþ 4 -N,

Fig. 3. stage 4.

NO 2 -N,

PO3 4 -P

250

3.0 70 PO 4 -P

60

2.5

-

NO 2-N +

NH 4-N

50

2.0

FNA

+

20

-

2.0

PO4 -P, NO2-N, NH4 -N (mg⋅L )

FNA

FNA×10-3(mg⋅L-1)

NH 4-N

-

+

2.5

+

0

200

3-

-

NO 2-N

-1

PO4 -P, NO2-N, NH4 -N (mg⋅L )

PO 4 -P

25

150

t (min)

35 30

1.5

FNA

t (min)

c

2.0

-

NO 2-N

40

1.5

30 1.0

FNA×10-3(mg⋅L-1)

30

2.5 FNA×10-3(mg⋅L-1)

PO4 -P

PO3-4-P, NO2- -N, NH+4-N (mg⋅L-1)

2.0

3-

40

FNA×10-3(mg⋅L-1)

40

20 0.5

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0.0

0 0

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100

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200

250

300

t (min)

and FNA profiles during aerobic phase of the SBR1 in four stages (a) day 40 of stage 1; (b) day 82 of stage 2; (c) day 100 of stage 3; (d) day 144 of

maintained at below 1 mg/L, indicating EBPR recovery in SBR1. The recovery of EBPR needed 20 days after addition of external carbon source. The above results suggested that more than 10 mg/L level of nitrite likely had an adverse impact on EBPR, leading to the EBPR deterioration. As shown in Fig. 2, the phosphorus release and uptake in four stages demonstrated the negative impact of nitrite on EBPR in SBR1. Along with the increase of nitrite in stage 2, a significant decrease of phosphorus release and uptake was observed. It should be noted that P-release and P-uptake of PAOs was gradually recovered at stage 3 when external carbon source was added to anaerobic phase. Stage 3 experienced a transition of EBPR from deterioration to recovery. One possible reason for decrease of P-release in stages 2 and 3 was denitrifying phosphorus removal occurring in anaerobic phase due to nitrite presence. The ratio of P-release/COD uptake (P/COD) was 0.53 in stage 1, and it was decreased to 0.29 and 0.23 in stages 2 and 3, then recovered to 0.49 in stage 4 with addition of external carbon source. In stage 4, P-release and P-uptake was improved to 38 mg/L and 42 mg/L on average, respectively, even though nitrite accumulation reached 20 mg/L. Based on these observations, deterioration of EBPR in SBR1 may be explained by three possible reasons. Firstly, nitrite inhibition on the aerobic phosphorus uptake led to high effluent P-concentration. Yarbrough et al. (1980) reported that nitrite inhibited active transport, oxygen uptake, and oxidative phosphorylation by a wide range of physiological types of bacteria. Weon et al. found that growth and phosphorus uptake of PAOs were both inhibited by nitrite (Weon et al., 2002). Therefore, increasing nitrite

concentrations under the aerobic conditions in SBR1 possibly inhibit the phosphorus uptake, leading to high effluent P-concentration. Secondly, nitrite inhibition on the anaerobic metabolism of PAOs seems to be one cause. Although there has been no report on nitrite toxicity to PAOs under the anaerobic conditions, possible reason is that nitrite inhibits active transport (Yarbrough et al., 1980), and thus interferes with VFA to be transferred into PAOs cells by active-transport mechanism (Smolders et al., 1994; Mino et al., 1987). The third reason was likely the competition between PAOs and denitrifiers for carbon source, which has been reported as a critical point for success of PAO against denitrifiers (Barker and Dold, 1996) and also as a critical point to obtain nitrite-DPAO cultures (Guisasola et al., 2009; Vargas et al., 2011). Since there was no anoxic phase in one SBR1 cycle, nitrite and carbon source was in existence simultaneously in anaerobic phase. That promoted the competition between PAOs and heterotrophic denitrifiers for carbon source, leading to less VFA available for PHAs synthesis of PAOs. Therefore, PHAs synthesis was reduced, which would be oxidized to produce energy for aerobic P-uptake. 3.2. Effect of nitrite accumulation on aerobic phosphorus uptake  3 Fig. 3 presents the profiles of NHþ 4 -N, NO2 -N, PO4 -P and FNA variation during aerobic phase of the SBR1 in four stages. As shown in Fig. 3(a) and (d), effluent PO3 4 -P concentration was low in both stage 1 and stage 4, although nitrite level was distinct in two stages. It should be noted that in stages 1 and 4, more than 97% of phosphorus was taken up in initial 80 min of aerobic phase; meanwhile no obvious nitrification occurred according to NHþ 4 -N

W. Zeng et al. / Bioresource Technology 102 (2011) 6657–6664

variations. In other words, phosphorus uptake was prior to nitrification. This phenomenon seems to be caused by the competition between heterotrophs and autotrophs for limiting dissolved oxygen (DO). In the initial period of aerobic phase, a large amount of heterotrophs such as PAOs outcompete a few autotrophic nitrifying bacteria and preferentially utilize DO as electron acceptor to oxidize intracellular and extracellular carbon source, leading to DO limited and lag of nitrification. As shown in Fig. 3(b) and (c), effluent PO3 4 -P concentration was high in stages 2 and 3. Although high levels of nitrite were built up at the end of aerobic phase in both stages, below 1 mg/L of nitrite was accumulated in initial 60 min of aerobic phase. Phosphorus uptake mainly occurred in the initial period of aerobic phase; however, less than 10% of phosphorus was taken up, and continued to the end of aerobic phase. Contrastively, at the same nitrite level of below 1 mg/L, more than 97% of phosphorus was taken up in initial period of stages 1 and 4. The outcomes indicated that very low phosphorus uptake in initial period of stages 2 and 3 was not caused by nitrite accumulation. PHA storage of PAOs at anaerobic conditions was possibly one reason, which should be verified by investigating the effect of nitrite accumulation on anaerobic metabolism of PAOs. In this study, FNA concentration calculated as the formula (1) was also shown in Fig. 3. As shown in Fig. 3, FNA concentration at the end of aerobic phase in stage 2–4 was 1.75  103 HNO2-N/L, 2.23  103 HNO2-N/L and 2.84  103 HNO2-N/L, respectively, which was all higher than the reported inhibition concentration of nitrite on P-uptake. However, nitrite concentration presented a gradient increase due to nitrification performing before reaching to the highest level, rather than remaining at constant high level throughout the aerobic phase. In the initial 80 min of aerobic phase, more than 97% of phosphorus was taken up; meanwhile FNA concentration was less than 0.1  103 HNO2-N/ L, which was far lower than the reported inhibition concentration of nitrite on phosphorus uptake. These outcomes indicated that nitrite produced via short cut nitrification has no effect on the aerobic phosphorus uptake and the reason causing EBPR deterioration in the SBR1 should be further investigated.

3.3. Effect of nitrite accumulation on anaerobic metabolism of PAOs   Fig. 4 shows the variations of NO 2 -N, NO3 -N and NOx -N in the influent and effluent of SBR1 anaerobic phase. As shown in Fig. 4,

sodium acetate addition

16

Stage 1

Stage 2 Stage 3

Stage 4

Concentration (mg⋅L-1)

14 12 10 8

influent NO -2 -N influent NO -3 -N influent NO -x -N effluent NO -x -N

6 4 2 0 0

20

40

60

80 100 120 time (day)

140

160

180

  Fig. 4. Variation of NO 2 -N, NO3 -N, and NOx -N in the influent and effluent of SBR1 anaerobic phase.

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during stage 1, an increase of nitrite along with a decrease of  nitrate in the influent was observed, while NO x -N (sum of NO2 N and NO -N in the influent) remained at 6.5 ± 1.5 mg/L. In stage 3 2, influent nitrate dropped to lower than 1 mg/L, meanwhile nitrite concentration continuously increased and reached up to 12 mg/L  and the influent NO x -N was mainly composed of NO2 -N. The pro  file of effluent NOx -N indicated that almost all of NOx -N (>8 mg/L) entering into anaerobic phase was reduced via denitrification in stage 2. Thus, we assumed that with increasing of NO x -N into the anaerobic phase, more carbon source would be consumed by denitrifying bacteria. In stages 3 and 4, external carbon source was added, during which nitrite concentration was higher than that in stages 1 and 2. Although nitrite concentration in stage 4 reached up to the highest level (Fig. 4), average amount of phosphorus release was obviously more than that in stages 1 and 2 without addition of external carbon source (Fig. 2). The outcome suggested that nitrite seems to have no inhibition on phosphorus release and the competition between PAOs and denitrifying heterotrophs for carbon source was possibly an important factor affecting phosphorus release of PAOs. To verify the hypothesis above, batch experiments were undertaken to investigate (1) the effect of nitrite as an inhibitor on anaerobic phosphorus release, and (2) the effect of nitrite on the competition between PAOs and denitrifying heterotrophs for carbon source. Since the activated sludge in SBR1 had undergone a long period of nitrite accumulation, PAOs in it were less sensitive to nitrite exposure. In these batch experiments, seed sludge was taken from SBR2 with the same inoculated sludge and the same domestic wastewater to treat as SBR1. SBR2 displayed a good performance of phosphorus removal (>98%) when batch experiments were carried out. 3.3.1. Effect of nitrite accumulation on anaerobic metabolism of PAOs under insufficient carbon source Fig. 5 shows the results of batch tests 1 with initial COD concentration of 200 mg/L. As shown in Fig. 5(a), VFA uptake rate at nitrite concentration of 10 mg/L, 20 mg/L and 30 mg/L was quicker than that without nitrite exposure. The increase of phosphorus and PHA concentration indicated that VFA consumption was used to store PHA by PAOs (Fig. 5(b) and (c)), based on the fact that the energy for PHA synthesis was generated by the hydrolysis of polyphosphate and release of phosphorus (Seviour et al., 2003). Moreover, the decrease of nitrite suggested that VFA consumption was also used for nitrite reduction by denitrifying heterotrophs (Fig. 5(d)). As shown in Fig. 5, in the case of 10 mg/L nitrite, when nitrite was completely reduced in the first 30 min, 52 mg/L of VFA remained in the reactor. The residual VFA was subsequently taken up to store PHA by PAOs, leading to continuous phosphorus release. At the nitrite concentration of 20 mg/L, when denitrification finished (Fig. 5(d)), VFA was exhausted (Fig. 5(a)). Thus no carbon source was available for subsequent PHA synthesis of PAOs. It should be noted that after carbon source was consumed, phosphorus release almost stopped, and then a slight decline of phosphorus concentration was observed. The decrease of PHA in Fig. 5(c) indicated that PAOs likely took up phosphorus, using the energy generated from biodegradation of PHA. Therefore, PHA driven denitrification seems to be an explanation for this difference in P profile. In the case of 30 mg/L nitrite, when carbon source was completely consumed at 50 min, 5.5 mg/L of nitrite was residual, indicating that carbon source was insufficient for denitrification. However, nitrite concentration continued to decrease after 50 min; meanwhile the decrease of phosphorus and PHA concentration was observed. This phenomenon was similar to that under 20 mg/L nitrite, but the electron acceptor in this case was more likely nitrite. The results demonstrated that a certain amount of

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a

b

40 35

NO2 -N=10mg/L

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NO2 -N=20mg/L NO2 -N=30mg/L

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15 10 5

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time (min)  Fig. 5. Variations of HAc, PO3 4 -P, PHA and NO2 -N in batch tests 1.

denitrifying phosphorus removal bacteria existed in the activated sludge. In the cases of 20 mg/L and 30 mg/L nitrite, the rapid consumption of VFA was used for denitrification and PHA synthesis. Although carbon source was insufficient for both denitrification and phosphorus release, most nitrite could still be rapidly reduced, which eliminated nitrite effect on the anaerobic phosphorus release. One reason for deterioration of EBPR in SBR1 was proposed above in this paper, i.e. nitrite promoted the competition between PAOs and denitrifiers for carbon source, leading to less VFA available for PHAs synthesis of PAOs. In fact, the results of batch tests 1 gave a support to this reason. In SBR1, with the increase of nitrite concentration in anaerobic phase, more carbon source was required for denitrification. In stages 1 and 2 of SBR1, carbon source in the influent was insufficient for both denitrification and PHA synthesis, leading to the decrease of phosphorus release. When carbon source in the influent was exhausted, DPAOs oxidized intracellular PHA using nitrite as electron acceptor to take up phosphorus, resulting in further decrease of phosphorus release and nitrite concentration (Fig. 2 and Fig. 4). That was in accordance with the outcomes of batch tests 1.

3.3.2. Effect of nitrite accumulation on anaerobic metabolism of PAOs under sufficient carbon source In order to exclude the effect of carbon source, the initial COD concentration in batch tests 2 was controlled at 800 mg/L to investigate nitrite inhibition on anaerobic metabolism of PAOs. As shown in Fig. 6(a), more than 490 mg/L of residual HAc at the

end of tests indicated that carbon source was high enough in the batch tests 2. As shown in Fig. 6, under sufficient carbon source conditions, with the increase of initial nitrite concentration, a slight decrease in the phosphorus release was observed. However, in all cases, PHA formation did not show obvious difference. The results suggested that nitrite had no inhibition on PHA formation, although it had a slight influence on phosphorus release. When enough acetate is present, there will be sufficient carbon source for the PAOs and the denitrifiers, which apparently prefer to use external acetate as carbon source instead of performing PHA-driven denitrification. Therefore, denitrifying phosphorus removal did not occur in this situation. A slight decrease of P-release along with the increase of initial nitrite concentration was possibly due to the competition of VFA uptake of PAOs with other microorganisms, such as denitrifying glycogen accumulating organism (DGAO). It should be further investigated. That could be used to explain SBR1 operation, i.e. EBPR performance was recovered after 20 days when external carbon source was added into the anaerobic phase of stage 3. In stage 3 of SBR1, 20 mg/L of nitrite was built up during the aerobic phase, whereas in the subsequent anaerobic stage the initial nitrite concentration was lower than 10 mg/L. In the batch tests 2, 30 mg/L of nitrite had no inhibition on PHA synthesis. The outcomes suggested that if carbon source was sufficient in the anaerobic phase of SBR1, nitrite had no effect on P-release and EBPR performance could be recovered. If sufficient carbon source was provided for nitrite reduction and PHA formation of PAOs, the energy produced by oxidation of PHA would be enough for

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850 800

NO- -N=0mg/L

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NO2- -N=10mg/L NO2- -N=20mg/L

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c

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8 NO- -N=0mg/L 2

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NO2- -N=10mg/L

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 Fig. 6. Variations of HAc, PO3 4 -P, PHA and NO2 -N in batch tests 2.

aerobic phosphorus uptake and thus in the subsequent cycle stored intracellular polyphosphate was hydrolyzed to supply sufficient energy for VFA uptake. Over several cycles, the amount of phosphorus uptake and release was increased, which was in accordance with the results in stage 3 of SBR1 (Fig. 2). On the other hand, if anaerobic metabolism of PAOs was inhibited by the presence of nitrite in SBR1, EBPR would deteriorate regardless of being exposed to sufficient carbon source conditions or not. However, the fact was that EBPR performance was recovered and further enhanced when sufficient carbon source was provided. Therefore, the experimental results from batch tests 1 and 2 suggested that the second reason for deterioration of EBPR in SBR1 previously proposed, i.e. nitrite inhibition on the anaerobic metabolism of PAOs, could be excluded.

4. Conclusions Nitrite accumulation resulted from nitritation was found to have an effect on EBPR in a SBR system treating domestic wastewater. Since occurrence of aerobic phosphorus uptake was prior to nitritation, nitrite accumulation has no effect on phosphorus uptake of PAOs under aerobic condition. The main reason leading to deterioration of EBPR was that under insufficient carbon source, denitrifying bacteria competing for carbon source with PAOs resulted in decrease of PHA synthesis and phosphate release. Therefore, in a nitritation–denitritation system, enough supply of carbon source could effectively alleviate the adverse impact of nitrite accumulation on EBPR.

Acknowledgements This work was financially supported by the Natural Science Foundation of China (No. 50878005), Beijing Natural Science Foundation (No. 8102005) and Fok Ying Tong Education Foundation (No. 121076).

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