Bioresource Technology 162 (2014) 316–322

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Advanced nitrogen removal with simultaneous Anammox and denitrification in sequencing batch reactor Rui Du, Yongzhen Peng ⇑, Shenbin Cao, Chengcheng Wu, Dongchen Weng, Shuying Wang, Jianzhong He Key Laboratory of Beijing for Water Quality Science and Water Environment Recovery Engineering, Beijing University of Technology, Beijing 100124, China

h i g h l i g h t s  A simultaneous Anammox and denitrification was achieved in the SBR system.  The Anammox biomass aggregated as wall growth in SBR.  The maximum nitrogen removal efficiency of 97.47% was realized at C/N of 2.  The Anammox activity recovered rapidly after suppression of high organic matter concentration.

a r t i c l e

i n f o

Article history: Received 20 January 2014 Received in revised form 5 March 2014 Accepted 8 March 2014 Available online 20 March 2014 Keywords: Anammox Denitrification Organic matter Sequencing batch reactor Nitrogen removal

a b s t r a c t In this study, a sequencing batch reactor (SBR) was used to achieve advanced nitrogen removal by simultaneous Anammox and denitrification processes. During the entire experiment, the Anammox microorganisms aggregated in the reactor as wall growth. Nitrogen removal was improved due to the reduction of nitrate, and the maximum total nitrogen (TN, including ammonia, nitrite and nitrate nitrogen) removal efficiency of 97.47% was obtained at C/N of 2. However, the sequentially increased organic matter resulted in a poor TN removal performance due to the suppression of Anammox. Fortunately, the Anammox activity completely resumed quickly after stopping dosing organic matter. PCR analysis results revealed that the Anammox bacteria gene copy number was not significantly reduced during the inhibition, which might explain the quick recover. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Recently, the anaerobic ammonium oxidation (Anammox) process has received much attention for its economy and high efficiency in removing nitrogen in the wastewater. In this process, as shown in Eq. (1), the ammonium (NH+4-N) was oxidized with nitrite (NO 2 -N) as electron acceptor to dinitrogen gas (N2) under anaerobic condition by the Anammox bacteria, which eliminated the requirements of aeration and exogenous carbon sources compared to the traditional nitrification–denitrification nitrogen removal process (Strous et al., 1998). The discovery of the Anammox process opened an alternative way to treat the wastewater and it had been successfully applied in treating certain ammonium-rich wastewater such as landfill leachate (Ruscalleda et al., 2008), digester liquor (Furukawa et al., 2009), pig manure effluents (Molinuevo et al., 2009) and turtle breeding wastewater (Chen et al., 2013). ⇑ Corresponding author. Tel./fax: +86 10 67392627. E-mail address: [email protected] (Y. Peng). http://dx.doi.org/10.1016/j.biortech.2014.03.041 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

NHþ4 þ 1:32NO2 þ 0:066HCO3 þ 0:13Hþ ! 1:02N2 þ 0:26NO3 þ 0:066CH2 O2:5 N0:15 þ 2:03H2 O

ð1Þ

Nevertheless, because the Anammox bacteria had an extremely low growth rate with a double time of approximately two weeks (Strous et al., 1998), efficient biomass retention in the reactor must be achieved. Thus far, a variety of reactors had been investigated to develop Anammox, such as expanded granular sludge bed (EGSB) reactor (Wang and Kang, 2005), up-flow anaerobic sludge blanket (UASB) reactor (Chamchoi et al., 2008; Molinuevo et al., 2009; Tang et al., 2011), sequencing batch reactor (SBR) (Strous et al., 1998; Van Dongen et al., 2001; Wang et al., 2011), and gas-lift reactor (Dapena-Mora et al., 2004). Among them, SBR was found to be a suitable system for the enrichment of Anammox bacteria as it showed advantageous in the simplicity, efficient biomass retention, as well as stability and reliability for a long period of operation (Strous et al., 1998). However, only part of the liquid was withdrawn in the SBR system, which led to the by-product nitrate (NO 3 -N) accumulation gradually and the TN concentration could

R. Du et al. / Bioresource Technology 162 (2014) 316–322

not meet the discharge standard. Usually, 11% of total nitrogen could be converted to NO 3 -N in Anammox process, as shown in Eq. (1). Furthermore, the NO 3 -N was always inevitable in the influent of Anammox in practice, since stable operation of the partial nitrification process (a common pretreatment process before Anammox) was difficult to realize under normal conditions (Jin et al., 2013; Okabe et al., 2011; Cho et al., 2011), and the nitrite accumulation rate in this process could not achieve 100% for long time even under the optimized conditions (Yang et al., 2007; Ma et al., 2011), which aggravated the above mentioned phenomenon. As to this problem, a denitrification process was expected in the Anammox SBR system for the NO 3 -N reduction by supplying organic carbon source. Experiments had been conducted to investigate the relationship between denitrification and Anammox under the presence of organic matter. Tang et al. (2010) had reported that the Anammox bacterial growth was significantly suppressed by denitrifying communities at an influent COD/NO 2 -N of 2.92; Chamchoi et al. (2008) found that the COD concentration was a control variable for process selection between Anammox reaction and denitrification; and Ni et al. (2012) demonstrated that low organic matter concentration did not affect ammonia and nitrite removal significantly but improved the total nitrogen removal via denitrifiers with the non-fat dry milk as carbon source. However, to the best of our knowledge, all of these focused on the impact of organic matter on the Anammox process and were conducted in a UASB reactor with Anammox granule sludge. Limited information was available on the enhancement of nitrogen removal in an Anammox system combined with denitrification in the SBR. Therefore, this study was performed to evaluate the combination of heterotrophic denitrification and Anammox in a single reactor to improve nitrogen removal efficiency of Anammox process. In this study, an Anammox system was established in a SBR, then starch and peptone which simulated the organic matter in real wastewater was added to create an influent COD to nitrite ratio (C/N) of 1, 2 and 4 for nitrogen removal investigation. The specific objective was to achieve further nitrogen removal with simultaneous Anammox and denitrification process in the SBR. Moreover, the recovery of the system was also evaluated after suppression under high organic matter conditions.

2. Methods 2.1. Seed sludge and synthetic wastewater The Anammox biomass was derived from an ongoing lab-scale SBR with working volume of 12 L. The Anammox seed sludge was reddish and small granules with a diameter of 0.2–0.5 mm. The original SBR was operated for 2 years with influent NH+4-N and NO 2 -N concentrations of 30 mg/L and 40 mg/L, respectively. The mixed liquor suspended solid (MLSS) and mixed liquor volatile suspended solid (MLVSS) of seed sludge was about 1.08 g/L and 0.79 g/L, respectively. The experimental SBR was inoculated with 1.8 L seed sludge, and the MLVSS after inoculation was 0.50 g/L. Synthetic wastewater was used as the feeding, which contained NH+4-N, NO 2 -N and trace element solution. The composition of synthetic wastewater was (/L): 0.11 g NH4Cl, 0.20 g NaNO2, 0.03 g NaNO3, 0.03 g KH2PO4, 0.14 g CaCl22H2O, 0.5 g KHCO3, 0.14 g MgSO47H2O, 1 ml trace element solution I and II according to van de Graaf et al. (1996). Trace element solution I contained (/L): 5 g EDTA, 5 g FeSO4; and trace element solution I1 contained (/L): 15 g EDTA, 0.43 g ZnSO47H2O, 0.24 g CoCl26H2O, 0.99 g MnC124H2O, 0.25 g CuSO45H2O, 0.22 g NaMoO42H2O, 0.19 g NiC126H2O, 0.21 g NaSeO410H2O, 0.014 g H3BO4. A small fraction of NO 3 -N (5 mg/L) was introduced in the influent by adding NaNO3 in order to simulate the effluent of partial nitrification (the

317

common influent of Anammox). The mixture of starch and peptone as the organic carbon source was mixed by equivalent COD ratio of 1:1 (according to the COD measured with certain mass concentration of separate solution). Starch and peptone was complex organic substrate which could be representative to the biodegradable organics in wastewater and used to simulate the COD in real wastewater. 2.2. Reactor and operation A SBR reactor with a working volume of 2.8 L (120 mm in diameter and 250 mm in height) was used. The SBR was operated with a fixed temperature at 30 °C ± 0.5 °C by an electric heater. An influent of 2.0 L was introduced using a peristaltic pump, a magnetic mixing was kept during the feeding and reaction stages to make the contents homogenous and reacting smoothly. The reactor was covered completely with a black PVC material (5 mm thickness) to prevent penetration of light (van der Star et al., 2007). The Anammox SBR was operated in a 12 h-cycle. The operation stage of each cycle included four periods: 10 min feeding period, 11 h anaerobic reaction with a magnetic mixing (250 rpm), followed by 40 min settling, and 10 min discharging of a 2.0 L effluent. During the experiment with different C/N, 8 mL, 16 mL and 32 mL (10 g COD/L) mixed solution of starch and peptone was added into the reactor manually at the end of the feeding period to result in an influent C/N of 1, 2 and 4. 2.3. Analytical methods The influent and effluent samples were collected on a daily basis and were analyzed immediately. The analyzed parameters  including NH+4-N, NO 2 -N, NO3 -N were measured with a Lachat Quik Chem 8500 Flow Injection Analyzer (Lachat Instruments, Milwaukee, USA), and chemical oxygen demand (COD) was analyzed using a COD quick-analysis apparatus (Lian-hua Tech. Co., Ltd, 5B-1, China). MLSS and MLVSS of activated sludge were measured according to the Standard Methods (APHA, 1998). 2.4. Microscopic observation Morphology characteristics of the biomass specimens were observed using scanning electron microscopy (SEM) of model Hitachi S-4300 (Japan). The samples for SEM were obtained from the SBR reactor using a sterile blade, then prepared by fixing with 2.5% glutaraldehyde for 2 h. After that, the specimens were rinsed 3 times in the 0.1 M phosphate buffer at pH 7.2 for 10 min/each, followed by dehydration with a gradient series of ethanol: 50%, 70%, 80%, 90% with 10 min per concentration, then 100% for 3 times with 15 min/each, and dried at critical point. At last, the specimens were coated with gold and observed under the microscope. 2.5. DNA extraction The activated sludge samples of the Anammox SBR were collected during the period 1, 3, 4 and 5 (see Table 1), respectively. DNA was extracted from 0.10 to 0.20 g dry sludge sample using the FastDNA SPIN Kit for Soil (Bio 101, Vista, CA). DNA concentrations were measured with a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA). 2.6. Quantitative real-time PCR Abundances of all bacteria and Anammox were determined by real-time PCR using an MX3000P Real-Time PCR system (Stratagene, La Jolla, CA) with fluorescent SYBR-Green. The real-time PCR was carried out with primers 341f-543r for all bacteria (Koike et al.,

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Table 1 Performance of simultaneous Anammox and denitrification SBR at different phases. Phase

1 2 3 4 5 a

Time (d)

1–54 55–90 91–138 139–153 154–180

C/N

a

– 1 2 4 –a

Effluent nitrogen concentration (mg/L)

Nitrogen removal efficiency (%)

NH+4-N

NO 2 -N

NO 3 -N

NH+4-N

NO 2 -N

TN

1.27 ± 1.08 0.81 ± 0.42 1.81 ± 0.81 21.17 ± 1.68 0.85 ± 0.33

0.78 ± 0.48 0.50 ± 0.46 0.69 ± 0.61 0.17 ± 0.37 0.27 ± 0.31

18.05 ± 1.70 13.95 ± 1.69 2.51 ± 1.26 0.37 ± 0.23 16.79 ± 1.61

95.42 ± 0.74 96.98 ± 1.72 93.14 ± 2.27 24.39 ± 7.31 97.07 ± 1.13

97.88 ± 0.93 98.7 ± 1.40 98.17 ± 1.63 99.56 ± 0.97 99.27 ± 0.85

71.4 ± 2.05 77.47 ± 2.68 93.14 ± 2.47 70.16 ± 2.46 74.84 ± 1.92

Phase without organic matter addition.

2007) and Anammox primers were Amx368f-Amx820r (Schmid et al., 2005) based on 16S rRNA genes. The amplification was performed in 20 lL reaction mixtures, consisting of 10 lL of SYBR Green exTaq (Takara, Dalian, China), 0.4 lL of ROX Reference Dye50, 0.2 lL of each primer (10 mmol/L), and 2 lL of DNA template (1–10 ng). The Anammox program consisted of the following steps: 3 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 30 s at 59 °C, and 30 s at 72 °C. The standard curves for Anammox gene copies were constructed from a series of 10-fold dilutions (from 102 to 108).

Thus, denitrification was expected to reduce the NO 3 -N to N2 in a single reactor, namely simultaneous Anammox and denitrification. However, because the presence of organic matter might result in excessive growth of heterotrophic denitrification bacteria and inactivating or eradicating Anammox communities in the end (Tang et al., 2010; Chamchoi et al., 2008), different initial C/N experiment was conducted to investigate nitrogen removal performance and the relationship between Anammox bacteria and denitrification communities in the SBR system.

3. Results and discussion

3.2.1. Nitrogen removal performance at C/N of 1 The organic matter was supplied on day 55 with the C/N of 1 in phase 2 (on days 55–90). As shown in Fig. 1, there was no pronounced change in the NH+4-N and NO 2 -N removal process. However, the average effluent NO 3 -N concentration declined to 13.95 mg/L, which was approximately 28% lower than that in phase 1 (18.05 mg/L). This indicated the coexistence of Anammox and heterotrophic denitrification in the SBR. The average TN removal efficiency was improved to 77.47%, indicating that small amount of organic matter could enhance TN removal. This was in accordance with the result of Ni et al. (2012), who found that low organic matter concentration in a granular Anammox system did not affect NH+4-N and NO 2 -N removal significantly but improved the TN removal via denitrifiers. The nitrogen consumption and percentages of different routes on nitrogen removal were calculated. The possible processes taken in the SBR included Anammox, heterotrophic denitrification and fermentation according to earlier researches (Ahn et al., 2004; Chamchoi et al., 2008). In these processes, the Anammox caused NH+4-N and NO 2 -N reduction (Eq. (1)). The fermentation resulted in a fraction of COD and NH+4-N consumption (Eq. (2)) due to the biomass synthesis. Butyric acid was the main organic acid of fermentation process in the Anammox system according to previous studies (Ahn et al., 2004; Chamchoi et al., 2008), which was used as the electron donor for denitrification 1 (Eq. (3)) and denitrification 2 (Eq. (4)). Both of the NO 3 -N produced in the Anammox process and remained from the previous cycle were taken into account in denitrification 1 process. The nitrogen removal by different routes could be calculated by the COD balance between measured COD and consumptions of NO denitrification, NO 3 -N 2 -N denitrification and fermentation processes (Ahn et al., 2004; Chamchoi et al., 2008). Table 2 showed the results of calculation based on the experimental results and the above-mentioned Eqs. (1)–(4). During phase 2, the average nitrogen removal percentage by Anammox route was 87.95%, the heterotrophic denitrification contribution was only 8.87%, indicating the dominant role of Anammox in nitrogen removal (Fig. 2). Other ways accounted for 3.17%, such as ammonia volatilization, sulfur-based autotrophic denitrification, hydrogen autotrophic denitrification, methane oxidation coupled to denitrification, and so on. (Chen et al., 2013). Fermentation:

3.1. Nitrogen removal by Anammox process solely Experiment was carried out without addition of organic matter  in phase 1 (days 1–54). The profiles of NH+4-N, NO 2 -N, and NO3 -N in the influent and effluent, as well as nitrogen removal efficiency + were presented in Fig. 1. The influent NO 2 -N and NH4-N was kept at 40 mg/L and 30 mg/L, respectively, which was in accordance + with the optimal stoichiometric ratio (NO 2 -N/NH4-N = 1.32) for Anammox (Strous et al., 1998). It could be seen that the NH+4-N removal efficiency and NO 2 -N removal efficiency was increasing gradually. This indicated the adaptation of seed sludge in the new conditions. Table 1 presented the mean values of data under stable conditions. During days 31–54, the average NH+4-N and NO 2 -N removal efficiency was 95.42% and 97.88%, respectively. However, the overshooting of NO 3 -N in the effluent was observed, which represented more than 20% influent TN. It was caused by two factors: the production of NO 3 -N in Anammox process; and the accumulation of undischarged NO 3 -N from the previous cycle in the SBR. For this reason, the TN removal efficiency was only 71.40% in this phase. It should be noted that the Anammox granule seeds disappeared. Moreover, the reddish microbes attached on the internal wall and aggregated as wall growth gradually in the operation process. The SEM was used to visualize the surface of aggregated biomass (Fig. S1(a)). The sludge sample was taken from the internal wall in the middle of the reactor on day 50. The dominant microorganisms in the sludge were dense coccoid cells which were supposed to be Anammox bacteria (Jetten et al., 1998; Kuenen, 2008). The structure was different from the previous studies of Anammox granules which had smooth surface and consisted of spherical bacteria co-existing with filamentous and short-rod bacteria (Wang and Kang, 2005; Chamchoi and Nitisoavut, 2007; Cho et al., 2010). Real-time PCR quantified the sludge sample on day 50, showing that the Anammox gene copy number was as high as 1.38  109 copies/g dry sludge. This result was in accordance with the high activity of the Anammox bacteria, which indicated its dominance in the microbial community. 3.2. Nitrogen removal by simultaneous Anammox and denitrification From the results above, the excess NO 3 -N accumulated in the SBR led to the TN concentration exceeded the discharge standard.

C6 H12 O6 þ 0:2 NHþ4 þ 0:2 HCO3 ! 0:2 C5 H7 O2 N þ CH3 CH2 CH2 COOH þ 1:8 H2 O þ 1:2 CO2

ð2Þ

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phase 2

phase 1

50

phase 3

phase 4

phase 5

100

30

60

NH4+-N concentration (mg/L)

80

20

influent NH4+-N

40

effluent NH4+-N NH4+-N removal efficiency

10

20

0

NH4+-N removal efficiency (%)

a 40

0 0

20

40

60

80

100

120

140

160

180

50

100

40

80

30

60

influent NO2--N effluent NO2--N

20

40

NO2--N removal efficiency

10

20

0

NO2- -N removal efficiency (%)

NO2- -N concentration (mg/L)

b

0 0

20

40

60

80

100

120

140

160

180 100

50

40

80

influent NO3--N

30

60

effluent NO3--N TN removal efficiency

20

40

10

20

0

TN removal efficiency(%)

NO-3-N concentration (mg/L)

C

0 0

20

40

60

80

100

120

140

160

180

time (d) Fig. 1. Profiles of nitrogen concentration and removal efficiency during phase 1 (without organic matter), phase 2 (C/N = 1), phase 3 (C/N = 2), phase 4 (C/N = 4) and phase 5  (recovery stage) of NH+4-N, NO 2 -N, NO3 -N and TN.

Table 2 Average consumption and percentages of different routes for nitrogen removal at different C/N. Consumption

Removal route

Phase 2

Phase 3

Phase 4

NH+4-N removed (mg/L)

Anammox Fermentation Anammox Denitrification Denitrification

24.26 1.77 32.03 3.74 1.37 56.86 87.95 8.87 3.17

25.67 1.16 33.89 2.93 11.58 68.56 77.15 21.15 1.70

5.18 1.72 6.84 30.01 8.75 51.14 27.47 68.91 3.62

NO 2 -N removed (mg/L) NO 3 -N removed (mg/L) Total nitrogen removed (mg/L) Average percentages of nitrogen removal routes (%)

Anammox Denitrification Others

R. Du et al. / Bioresource Technology 162 (2014) 316–322

80

60

40

20

(a) C/N=2

+

NH4 -N -

NO2 -N

30

60

-

NO3 -N COD

20

50

10

40

0

0 54

63

72

81

90

99

108

117

126

135

144

NO3 þ 0:29 CH3 CH2 CH2 COOH þ H2 CO3 ð3Þ

Denitrification 2:

nitrogen concentration (mg /L)

Denitrification 1:

4

6

8

10

12 120

(b) C/N=4 30 90 20 60 10

0

30 0

þ 0:19 CH3 CH2 CH2 COOH þ H2 CO3

! 0:037 C5 H7 O2 N þ HCO3 þ 1:14 H2 O þ 0:585 CO2 þ 0:481 N2

2

40

Fig. 2. Evolution of Anammox and denitrification contribution to total nitrogen removal in the SBR.

! 0:034 C5 H7 O2 N þ HCO3 þ 1:54 H2 O þ 0:986 CO2 þ 0:483 N2

30 0

153

time (d)

NO2

70

40

phase 4

TN removal

ð4Þ

COD (mg/L)

Anammox Denitrification Others

nitrogen concentration (mg /L)

percentage of nitrogen removal routes (%)

phase 3

phase 2

100

COD (mg/L)

320

2

4

6

8

10

12

time (h) Fig. 3. Variations of nitrogen compound concentration in typical cycle under (a) C/N of 2 and (b) C/N of 4.

3.2.2. Nitrogen removal performance at C/N of 2 The C/N ratio was increased to 2 in phase 3 (days 91–138). The NH+4-N and NO–2-N removal were not influenced significantly according to Fig. 1, but the effluent NO 3 -N decreased sharply to 2.51 mg/L in average – much lower than that in phase 1 and phase 2. Concurrently, TN removal efficiency was up to 97.47% on day 125. As Fig. 2 showed that the denitrification efficiency was increased gradually and there was 21.15% of nitrogen removed by heterotrophic denitrification during days 110–138. Obviously, the high TN removal efficiency was attributed to the enhanced heterotrophic denitrification (NO 3 -N reduction) with sufficient organic matter. It could be seen that the effluent nutrients kept stable during days 110–138. This indicated that the Anammox and heterotrophic denitrification bacteria could coexist and achieve nitrogen removal collaboratively. Fig. 3(a) showed the variation of nitrogen compound with time in one typical cycle. It could be seen that the NH+4-N was almost linearly decreasing during the reaction. Meanwhile, a small fraction of NO 3 -N in the influent was decreasing gradually in correspondence to COD consumed. On the other hand, the NO 2 -N existed in the system over the experiment, which was necessary for Anammox to avoid inhibited by the heterotrophic denitrification in presence of organic matter. This was supported by Tang et al. (2010) who concluded that the poor competitive ability for electron acceptor (nitrite) compared to heterotrophic denitrifiers resulted in the Anammox bacteria starved and eliminated in the end. It was confirmed by the PCR result that the Anammox copy number was 1.22  109 copies/g dry sludge on day 135, which revealed that the abundance of Anammox bacteria had no significant reduction in the system, though obvious denitrification performance occurred. From the above observation, the Anammox microorganisms could collaborate well with the denitrifying bacteria at low C/N (C/N < 2.0). Chen et al. (2008) had found the best C/N was 1:2 in non-woven rotating biological contactor reactor for Anammox and C/N of 3:4 resulted in inhibition of Anammox activity. Gao

et al. (2012) found the highest total nitrogen removal + performances in a COD to NO 2 -N to NH4-N ratio of 0.6:1.26:1 in a fixed-bed bioreactor. However, these C/N thresholds in the Anammox system were lower than the value in this study. This was probably related to different growing conditions for Anammox microorganisms, such as the reactor configuration, operation and type of organic matter.

3.2.3. Nitrogen removal performance at C/N of 4 It could be seen from the above results that there was no significant effect on Anammox when the C/N of 1 and 2 were applied, and the simultaneous Anammox and denitrification for nitrogen removal could be achieved. However, the increase of organic matter concentration often encountered in practice during influent fluctuation. Thus, the nitrogen removal performance with higher C/N ratio was further investigated. The amount of organic matter increased to a C/N ratio of 4 on days 139–153 (phase 4). As predicted, the Anammox was significantly suppressed, illustrated by the poor NH+4-N removal efficiency (below 20% rapidly). Consequently, the TN removal efficiency dropped to below 70%. Furthermore, the average nitrogen removal percentage via Anammox route was only 27.47%, while heterotrophic denitrification contribution was up to 68.91% as indicated in Fig. 2. The results suggested that the denitrification prevailed in the reactor and the simultaneous Anammox and denitrification system for nitrogen removal was disturbed. This was due to that the NO 2 -N removing Anammox bacteria could not compete with the heterotrophic denitrifiers at high C/N (Fig. 3(b)), because the denitrification bacteria had a higher growth yield (yield coefficient of heterotrophs: Y = 0.3 g VSS/g NH+4-N) (Rittmann and McCarty, 2001) compared to Anammox bacteria (Y = 0.066 ± 0.01 g VSS/g NH+4-N) (Strous et al., 1998). Similar findings had been reported that high organic matter concentrations resulted in lower TN removal in wastewater treatment by Anammox

R. Du et al. / Bioresource Technology 162 (2014) 316–322

process (Chamchoi et al., 2008; Güven et al., 2005; Waki et al., 2007). In addition, it should be pointed out that the change in the physiological characteristics of the sludge was observed. The color of the Anammox sludge altered to black after high organic matter was supplied, which was consistent with the previous studies (Molinuevo et al., 2009; Chen et al., 2013). This was mainly attributed to the increase of denitrifiers in the system. It was confirmed by the results of real-time PCR that the percentage of Anammox bacteria was reduced after the 15-day operation at high organic matter. Moreover, the SEM photographs of sludge sample collected on day 153 showed that there were other microbes like filamentous and short-rod bacteria co-existed with coccoid bacteria (Fig. S1(b)), which may be the denitrification bacteria. 3.3. Recovery of Anammox activity without organic matter Because the Anammox was suppressed seriously under high organic matter concentration (Tang et al., 2010; Ni et al., 2012), the operational strategy in phase 5 (days 154–180) was to stop feeding with organic matter. As shown in Fig. 1, the effluent NH+4-N decreased rapidly, corresponding with the increase of NH+4-N removal efficiency. The maximum NH+4-N removal efficiency was achieved 96.9% on day 158. But the effluent NO 3 -N increased. On the other hand, the color of the sludge returned to red gradually in this phase. These clearly indicated that the Anammox activity had recovered rapidly after being suppressed for over two weeks. Taking into account the low biomass yielding rate, the quick recovery of Anammox activity in this study might be caused by the efficient retention of Anammox biomass for its wall growth in SBR. The quantitative real-time PCR analysis revealed that, after two weeks feeding with C/N of 4, the gene copy number of Anammox decreased slightly (1.19  109 copies/g dry sludge compared to 1.22  109 copies/g dry sludge at C/N of 2). It was deemed that the distribution of biomass over the reactor by wall growth could be efficient for Anammox biomass conservation. Overall, it could be concluded that the system had the capacity of resisting the shock of high organic matter concentration. Therefore, the simultaneous Anammox and denitrification SBR system could be applied for nitrogen removal in treating low C/N wastewater, even though the high organic matter occurred unconsciously at some time. Nevertheless, some unexpected factors may affect the stability of the system in treating the actual wastewater, which should be further investigated in the future. 4. Conclusions This study demonstrated that advanced nitrogen removal could be achieved by simultaneous Anammox and denitrification under low organic matter in SBR. As high as 97.47% of maximum TN removal efficiency was obtained at a C/N ratio of 2. Nevertheless, Anammox activity was inhibited under high organic matter and resulted in the TN removal efficiency dropped. Moreover, the Anammox could recover quickly after suppressed, and the quantitative real-time PCR results indicated that the Anammox biomass accumulated as wall growth in SBR could contribute to biomass retention. Acknowledgements This research was financially supported by Natural Science Foundation of China (21177005) and Specialized Research Fund for the Doctoral Program of Higher Education (20111103130002).

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