N domestic wastewater at low temperature

Bioresource Technology 219 (2016) 420–429

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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Performance of partial denitrification (PD)-ANAMMOX process in simultaneously treating nitrate and low C/N domestic wastewater at low temperature Rui Du a, Shenbin Cao b, Shuying Wang a, Meng Niu a, Yongzhen Peng a,⇑ a National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, Engineering Research Center of Beijing, Beijing University of Technology, Beijing 100124, China b State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China

h i g h l i g h t s
A novel PD-ANAMMOX was developed for treating nitrate and domestic wastewater. 

+


NO3 -N, NH4 -N and COD removal efficiencies achieved 96.7%, 99.5% and 69.6%.
The nitrogen removal increased when the ANAMMOX influent contained more nitrate.
ANAMMOX maintained as the dominant pathway with over 70% total nitrogen removal.
Candidatus Jettenia species, present at 2.7%, dominated the ANAMMOX performance.

a r t i c l e

i n f o

Article history: Received 9 July 2016 Received in revised form 22 July 2016 Accepted 24 July 2016 Available online 26 July 2016 Keywords: Partial denitrification ANAMMOX Nitrate Domestic wastewater Low temperature High-throughput sequencing analysis

a b s t r a c t 1 The simultaneous treatment of nitrate (NO ) and domestic wastewater (ammonia (NH+43 -N  50 mg L N)  60.6 mg L1, COD  166.3 mg L1) via a novel partial denitrification (PD)-ANaerobic AMMonium OXidation (ANAMMOX) process was investigated at low temperature (12.9  15.1 °C). Results showed + that desirable performance was achieved with average NO 3 -N, NH4-N and COD removal efficiencies of 89.5%, 97.6% and 78.7%, respectively. However, deteriorated sludge settleability in PD reactor was observed during operation, which bulked with serious sludge wash-out, leading to excess NO 3 -N remaining in PD effluent. Fortunately, a satisfactory nitrogen removal was still achieved due to the occurrence of partial denitrification in ANAMMOX reactor. This was demonstrated by high-throughput sequencing, which revealed that the high nitrite (NO 2 -N) production denitrifying bacteria of Thauera was detected (6.1%). ANAMMOX (above 70%) maintained the dominant pathway for nitrogen removal, and Candidatus Jettenia was identified as the major ANAMMOX species accounted for 2.7%. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Nitrogen contamination such as nitrate in water body can create serious problems, such as eutrophication of rivers, deterioration of water quality and potential hazard to human and animal health (Ghafari et al., 2008). Since it is very soluble in water and easily transported to the groundwater and surface water once discharged, nitrate contamination has become an increasingly serious environmental problem worldwide. Recently, many countries have enforced stringent wastewater discharge standards, as the removal of nitrogen and organic matter from wastewater was of supreme

⇑ Corresponding author. E-mail address: [email protected] (Y. Peng). http://dx.doi.org/10.1016/j.biortech.2016.07.101 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.

importance to prevent water pollution. The effluent of municipal wastewater treatment plants (WWTPs) must meet the class-A discharge standards in China, which the COD, NH+4-N and TN-N concentration was lower than 50 mg L1, 5 mg L1 and 15 mg L1 (GB18918-2002), respectively. However, the traditional nitrification/denitrification process (Eq. (1) and (2)) for wastewater nitrogen removal still possesses some drawbacks, such as external carbon sources requirement to provide electron donor in treating wastewater with low carbon to nitrogen ratio (C/N), large amount of electric energy consumption for aeration, high amounts of sludge production due to the short doubling time of denitrifying bacteria and the greenhouse gases emission in nitrification and denitrification process.

NHþ4 þ 2O2 ! NO3 þ H2 O þ 2Hþ

ð1Þ

R. Du et al. / Bioresource Technology 219 (2016) 420–429

1:6NO3 þ CH3 COO þ Hþ ! 0:8N2 þ 2CO2 þ 1:2H2 O þ 1:6OH

ð2Þ

NO 2 -N

The discovered ANAMMOX process, which uses as electron acceptor oxidizing NH+4-N to nitrogen gas (N2) (Eq. (3)) (Strous et al., 1998), has attracted much attention in recent years for its significant advantages compared to traditional nitrogen removal process, such as reducing the need for organic carbon by 100%, aeration requirements by about 60% and sludge production by about 90%. It has been widely studied for its potential engineering application as an alternate for nitrogen removal (Van Hulle et al., 2010; Ma et al., 2016, 2011; Zhang et al., 2015). Nevertheless, the NO 2 -N, which is an essential substrate for ANAMMOX, was hardly produced efficiently by short-cut nitrification (NH+4-N ? NO 2 -N), especially for the low NH+4-N wastewater (i.e. domestic wastewater) (Ge et al., 2015). In addition, there would be 11% nitrogen production as NO 3 -N in the ANAMMOX process theoretically, which could lead to excess nitrogen remaining in the effluent and could not discharged immediately.

NHþ4 þ 1:32NO2 þ 0:066HCO3 þ 0:13Hþ ! 1:02N2 þ 0:26NO3 þ 0:066CH2 O0:5 N0:15 þ 2:03H2 O

ð3Þ

NO 2 -N

The accumulation in denitrification process has drawn much attention as it could serve as another way to supply electron donor for ANAMMOX (Gong et al., 2013; Waki et al., 2013). Recently our laboratory cultivated a high NO 2 -N accumulation  denitrification sludge with the NO 3 -N-to-NO2 -N transformation ratio (NTR) above 80%, and maintained stably in the long-term operation (Cao et al., 2013). This provided a potential alternative for the NO 3 -N containing wastewater treatment with a more efficient and cost-saving way, such as the effluent of the second class treatment in WWTPs, the effluent of ANAMMOX process for highammonia wastewater treatment and some industrial wastewater with excess NO 3 -N contained. The partial denitrification has been applied in treating the ANAMMOX effluent with excess NO 3 -N, and a nitrogen removal efficiency (NRE) as high as 94.1% was obtained (Du et al., 2015b), demonstrated that the partial denitrification  (NO 3 -N ? NO2 -N) could be realized. Therefore, as regards the  NO3 -N wastewater and domestic wastewater, the partial denitrification combined with ANAMMOX could be adopted to achieve the nitrogen removal, and this process would manifest huge advantages, such as low carbon demand, no aeration consumption, low sludge production and greenhouse gases emission (Du et al., 2016). A novel PD-ANAMMOX process was established in this study for simultaneously treating NO 3 -N and domestic wastewater. The NO 3 -N was firstly fed to PD reactor along with the external carbon source for NO 2 -N production, then mixed with domestic wastewater and removed with the NH+4-N in ANAMMOX reactor. The NO 3 -N generated by ANAMMOX (and/or residual in PD effluent) was expected to be reduced in situ with the carbon source involved in domestic wastewater, consequently achieved the organic matter removal of domestic wastewater as well as the advanced nitrogen removal. The long-term performance of the PD-ANAMMOX process at relatively low temperature was investigated. Additionally, the microbial community structure was explored via highthroughput sequencing analysis. 2. Materials and methods

421

10 cm (internal diameter)  70 cm (height); working volume: 5 L) was used to perform partial denitrification for NO 2 -N production. A row of outlets were set up for taking sample, feeding NO 3 -N as well as organic matter. Since the substrate (NO 3 -N and organic matter) was continuously fed to the PD reactor, a three-phase separator refitted by a funnel was installed in the top in order to prevent the sludge wash-out. And a cantilever agitator was used with a low working rate for maintaining a relatively mixing at the bottom of reactor. During the whole operation process, the PD reactor was operated with a constant Hydraulic Retention Time (HRT) of 30 min. An upflow anaerobic sludge blanket (UASB) (dimension: 6 cm (internal diameter)  110 cm (height), working volume: 3.2 L) was used for ANAMMOX process. It was constructed of plexiglas and covered completely with black sponge to prevent penetration of light. An internal reflux pump controlled by peristaltic pump was installed in order to enhance the fluidization. The influent of ANAMMOX reactor was the effluent of PD reactor and domestic wastewater. In order to avoid inhibition of excess organic matter addition on ANAMMOX bacteria, NH+4-N solution (NH4Cl) was added to domestic wastewater to achieve a low C/N at initial period. The NH+4-N solution addition was decreasing gradually with improving influent C/N, and finally pure domestic wastewater without NH+4-N solution addition was fed. In phase 1, the NH+4-N of domestic wastewater was around 165.8 mg L1 with the feeding rate of 0.36 L h1 (Table 1), 113.3 mg L1 with the feeding rate of 0.54 L h1 in phase 2, 83.5 mg L1 with the feeding rate of 0.72 L h1 in phase 3, and pure domestic wastewater (NH+4N  60.6 mg L1) was fed in phase 4 with the rate of 1.08 L h1. During the whole operation process, the PD effluent was fed to ANAMMOX reactor with a constant rate of 3.6 L h1. 2.2. Biomass and feeding media The seeding partial denitrification sludge was present with granule, and it showed good settling property with the sludge volume index (SVI) around 50 mL g MLSS1 (Mixed Liquor Suspended  Solids). An ideal NO 2 -N production was displayed with the NO3 -Nto-NO -N transformation ratio (NTR) of above 80%. The MLSS and 2 mixed liquor volatile suspended solid (MLVSS) after inoculation were approximately 4.4 g L1 and 2.4 g L1, respectively. The ANAMMOX UASB had run for 400-days feeding with synthetic wastewater (Cao et al., 2016). The ANAMMOX biomass was also present with granule and had nitrogen removal rate (NRR) of 1.86 kg N m3 d1 before this study. The synthetic NO 3 -N wastewater (NaNO3) was prepared to feed 1 to PD reactor with constant NO . The 3 -N concentration of 50 mg L 1 compositions are listed as follow (L ): 0.05 g KH2PO4, 0.40 g CaCl22H2O, 0.10 g MgSO47H2O, 1.0 mL trace element solution A and B according to (Du et al., 2014). The acetate sodium solution was also prepared and pumped into the PD reactor individually at a constant flowing rate. The influent COD/NO 3 -N was controlled at 3.0 in order to maintain high NO 2 -N production according to our previous study (Cao et al., 2013). The raw domestic wastewater used in this study was taken from a septic tank in the residential area of Beijing University of Technology (Beijing, China). The main characteristics of the wastewater were: COD, 141.8  205.1 mg L1; NH+4-N, 58.2  65.1 mg L1; NO 21 1 N < 0.5 mg L1; NO ; PO3 . 3 -N < 1 mg L 4 -P: 5.1  7.9 mg L

2.1. Experimental set-up and operation 2.3. Analytical methods The schematic diagram of PD-ANAMMOX process was exhibited in Fig. 1. The NO 3 -N wastewater was fed to PD reactor along with the carbon source for NO 2 -N production, then its effluent was mixed with domestic wastewater and fed to ANAMMOX reactor. A columnar reactor with high height/ diameter ratio (dimension:

The influent and effluent samples were collected on a daily basis and were analyzed immediately. The parameters including  NH+4-N, NO 2 -N, NO3 -N were measured with Lachat Quik Chem 8500 Flow Injection Analyzer (Lachat Instruments, Milwaukee,

422

R. Du et al. / Bioresource Technology 219 (2016) 420–429

Fig. 1. Schematic diagram of the PD-ANAMMOX process for simultaneously treating nitrate and domestic wastewater.

Table 1 Performance and operational conditions of PD reactor in different phases. Phase (day)

1 2 3 4

(1  22) (23  41) (42  68) (69  105)

Temp. (°C)

15.1 13.1 12.9 12.9

Partial-Denitrification reactor (mg L1)

Domestic wastewater (+Synthetic NH+4-N)

NTR (%)

Feeding rate (L h1)

COD (mg L1)

NH+4-N (mg L1)

Inf. NO 3 -N

Eff. NO 2 -N

Eff. NO 3 -N

Eff.COD

0.36 0.54 0.72 1.08

177.7 166.8 164.2 156.6

165.8 113.3 83.5 60.6

50.5 50.2 50.1 50.5

22.3 22.5 20.9 14.4

10.9 11.4 10.9 21.3

38.3 37.7 39.3 54.9

USA), and chemical oxygen demand (COD) was analyzed using COD quick-analysis apparatus (Lian-hua Tech. Co., Ltd., 5B-1, China). The temperature was measured by pH probe (WTW 340i, WTW Company). The MLSS and MLVSS of activated sludge were measured according to the Standard Methods (APHA, 1998). 2.4. Calculations

CODmix ¼ VDW  CODinf: =ðVPD þ VDW Þ

55.1 57.5 51.3 48.1

ð9Þ

 where NH+4-N mix, NO 2 -Nmix, NO3 -Nmix, TNmix and CODmix represented the concentration of mixture of PD effluent and domestic wastewater before introduced to ANAMMOX reactor; VDW was the feeding rate of domestic wastewater, and VPD was the feeding rate of PD effluent (L h1); NH+4-Ninf., and CODinf. was the NH+4-N and COD concentration of domestic wastewater, respectively (mg L1).

2.4.1. NTR

NTRð%Þ ¼ NO2 -NPD-eff: =ðNO3 -Ninf:  NO3 -NPD-eff: Þ  100%

ð4Þ

  where NO 3 -Ninf., NO2 -NPD-eff. and NO3 -NPD-eff. was the influent   NO 3 -N, effluent NO2 -N, and effluent NO3 -N concentration of PD reactor, respectively.

2.4.2. Nitrogen and COD concentration of ANAMMOX influent (mixture of PD effluent and domestic wastewater)

2.4.3. Stoichiometric ratios of ANAMMOX process + The substrate ratio of influent NO 2 -N to NH4-N concentration in ANAMMOX reactor (RS-Inf.):

RS-Inf: ¼ NO2 -Nmix =NHþ4 -Nmix NO 2 -N

The ratio of MMOX reactor (RS);

ð10Þ

consumption to

NH+4-N

removal in ANA-

RS ¼ ðNO2 -Nmix  NO2 -Neff: Þ=ðNHþ4 -Nmix  NHþ4 -Neff: Þ NHþ4 -Nmix

¼ VDW 

NHþ4 -Ninf: =ðVPD

þ VDW Þ

ð5Þ

NO2 -Nmix ¼ VPD  NO2 PD-eff: =ðVPD þ VDW Þ

ð6Þ

NO3 -Nmix ¼ VPD  NO3 -NPD-eff: =ðVPD þ VDW Þ

ð7Þ

TNmix ¼ NHþ4 -Nmix þ NO2 -Nmix þ NO3 -Nmix

ð8Þ

NO 3 -N

The ratio of MMOX reactor (RP);

production to

NH+4-N

ð11Þ

consumption in ANA-

RP ¼ ðNO3 -Nmix  NO3 -Neff: Þ=ðNHþ4 -Nmix  NHþ4 -Neff: Þ

ð12Þ

 where NH+4-Neff., NO 2 -Neff. and NO3 -Neff. were the effluent nitrogen concentration of ANAMMOX reactor (mg L1).

R. Du et al. / Bioresource Technology 219 (2016) 420–429

2.4.4. Percentages of ANAMMOX and complete denitrification contribution on TN removal in ANAMMOX reactor Without regard to the biomass synthesis, the nitrogen removal in ANAMMOX reactor was mainly via ANAMMOX and complete denitrification. The percentage of ANAMMOX and denitrification contribution on TN removal could be calculated in Eq. (8):

ANAMMOX percentage ¼ ðNHþ4 -Nmix  NHþ4 -Neff: Þ  ð1 þ 1:32  0:26Þ  100%=ðTNmix  TNeff: Þ

ð13Þ

Denitrification percentage ¼ 100%  ANAMMOX percentageð%Þ ð14Þ 2.5. High-throughput sequencing analysis The sludge sample in ANAMMOX-UASB reactor was collected on day 102 for investigating the microbial community structures. The DNA was extracted using the Fast DNA Kit for Soil (BIO101, Vista, CA) according to the manufacturer’s instruction, and measured at 260 and 280 nm wavelengths using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, DE, USA). The V3 and V4 regions of bacterial 16S rRNA genes were amplified using bacterial primers 338F (50 -ACTCCTACGGGAGGCAGCA-30 ) and 806R (50 - GGACTACHVGGGTWTCTAAT-30 ) (Cao et al., 2016). The PCR reaction mixture contained 4 lL 5  FastPfu buffer, 2 lL dNTPs (2.5 mM), 0.8 lL of formar primer(5 lM), 0.8 lL of reverse primer (5 lM), 0.4 lL FastPfu Polymerase, 10 ng of template DNA, and added Milli-Q water to 20 lL. The PCR thermal programs consisted of an initial denaturation at 95 °C for 3 min, followed by 27 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s, followed by a final extension at 72 °C for 10 min. The PCR products for sequencing were carried out on Illumina Miseq PE300 platform (Illimina, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Taxonomic classification was carried out to determine the optimized sequences into operational taxonomic units (OTUs) using 97% identity thresholds (i.e., 3% dissimilarity levels) by the MOTHUR software program. The OTU numbers were counted for the sample as the species richness, and rarefaction curves and Shannon-Wiener were generated. The generated raw sequences of the sludge sample were assigned by Silva to trim off the adapters and barcodes. 3. Results and discussion 3.1. Simultaneous treatment of NO 3 -N and domestic wastewater at low temperature The PD-ANAMMOX process was operated for 105 days, at the low temperature varied from 18.3 °Cto 10.6 °C. Based on the different feeding rate of domestic wastewater, four phases were divided during the whole operation, and the performance in simultaneously treating NO 3 -N and domestic wastewater was investigated. 3.1.1. NO 3 -N removal performance The efficiency of NO 3 -N removal was investigated with the 1 influent NO during the whole operation 3 -N around 50 mg L (Fig. 2a). The domestic wastewater feeding rate kept at 0.36 L h1 in phase 1. It was observed that a satisfactory NO 3 -N removal was achieved after the combined process started up, with the efflu1 ent NO and the removal efficiency of 83.1% in 3 -N of 8.9 mg L average (Table 1). Moreover, the effluent NO 3 -N reduced to 6.7 mg L1 and 4.0 mg L1 when the feeding rate of domestic

423

wastewater increased to 0.54 L h1 in phase 2 and to 0.72 L h1 in phase 3, corresponding to the removal efficiency declined to 85.9% and 89.5%, respectively. This was mainly due to the increase of carbon source involved in domestic wastewater introduced to ANAMMOX reactor, which enhancing the complete denitrification of NO 3 -N produced by ANAMMOX and/or the residual from partial denitrification. However, a deteriorated NO 3 -N removal was 1 observed on day 53 with the effluent NO , 3 -N up to 39.4 mg L which was ascribed to the pump trouble and failing organic matter addition for partial-denitrification, leading to the ANAMMOX influ ent containing little NO 2 -N but only NO3 -N. Furthermore, the pure domestic wastewater without any NH+4N addition was fed to the ANAMMOX reactor in phase 4, as well as the influent feeding rate increased to 1.08 L h1 in order to provide sufficient NH+4-N for ANAMMOX process. It was expected that the effluent NO 3 -N concentration would be further decreased in this phase for the increase of organic matter introduced. Neverthe1 less, the average effluent NO , which was slight 3 -N was 5.7 mg L higher than that in phase 3 (Fig. 3). This was resulted from the deteriorated performance of partial denitrification reactor, in 1 which the effluent NO , but the NO 2 -N was only 14.4 mg L 3 -N 1 was as high as 21.3 mg L (Table 2). It could be speculated that the NO 3 -N removal efficiency would be further improved by elevating feeding rate of domestic wastewater under the undesirable partial denitrification condition. 3.1.2. Removal of NH+4-N and COD in domestic wastewater The domestic wastewater was mixed with the effluent of partial denitrification and fed to UASB, thus the removal of NH+4-N was mainly via ANAMMOX pathway. It was found that a low effluent NH+4-N concentration was observed after the combined process established due to the high ANAMMOX activity in UASB reactor. In phase 1, the influent NH+4-N was 165.8 mg L1 with feeding rate of 0.36 L h1, corresponding to the average effluent NH+4-N of 2.8 mg L1 with the removal efficiency as high as 98.1%. With the domestic wastewater feeding rate increasing gradually, the NH+4N removal performance maintained stably. The mean effluent NH+4-N of 4.3 mg L1, 1.7 mg L1 and 2.0 mg L1 were obtained during phase 2, 3 and 4, respectively, corresponding to the removal efficiency of 95.4%, 97.6% and 96.9% were achieved (Table 1). This clearly indicated that the elevated strength of organic matter had little negative influence on ANAMMOX. Additionally, compared with the traditional nitrification/denitrification process, the aerobic energy consumption and cost for NH+4-N oxidization could be saved by 100%. On the other hand, the COD concentration of domestic wastewater varied from 141.8 mg L1 to 205.1 mg L1 with the mean value of 166.3 mg L1 (Table 1). This organic matter was expected to be consumed as electron donor of the potential denitrification process in ANAMMOX reactor. In phase 1, the mean influent COD/NH+4-N was around 1.07 and the feeding rate was 0.36 L h1, meaning that the denitrification reaction could be performed within HRT of approximately 8.9 h. As a result, ideal COD removal performance with effluent concentration of 44.7 mg L1 (corresponding to the removal efficiency of 74.1%) was obtained in this phase (Table 2). With the domestic wastewater feeding rate increasing, the COD loading rate was correspondingly rising but the HRT for denitrification reaction was reduced gradually. Notably, the effluent COD was still maintained at a low level, the mean removal efficiency of 75.1% and 78.7% achieved in phase 2 (COD/NH+4-N of 1.48 and HRT of 5.9 h) and phase 3 (COD/NH+4-N of 1.97 and HRT of 4.4 h), corresponding to the effluent COD of 36.6 mg L1 and 36.7 mg L1 (Table 2). Moreover, pure domestic wastewater was fed to the ANAMMOX reactor in phase 4 with flow rate of 1.08 L h1, consequently the HRT was shortened to 3.0 h and the COD loading was improved to COD/NH+4-N of 2.59. It was observed that the effluent

0 30

-

-

Inf.NO3 -N

(c)

Phase 1

20

45 60 Time (day) Eff.NO3 -N

Phase 2

75

90

Phase 4

40

15

30 TN=15 mg/L

10

20 5

10

0 0

15

30 Eff.TN

45 60 Time (day)

75

90

-1

Feeding rate (L h )

1.0 80 0.5

15 +

Inf.NH4 -N

(d)

50

100

120

0

Removal efficiency

Phase 3

2.0 1.5

0

0 105

Phase 1

300

30

45 60 Time (day) +

90

Feeding rate

Eff.NH4 -N Phase 2

75

Phase 4

Phase 3

0.0 105

4

Temperature

2

100 1 50 0 0

15

Inf.COD

30

45 60 Time (day)

Eff.COD

75

90 +

COD/NH4 -N

40

0

100 80

3

0 105

60

Removal efficiency

200 150

80

20

250 -1

15

160

40

COD (mg L )

0

Phase 4

Phase 3

0 105

60 40 20

COD removal efficiency (%)

20

Phase 2

+

20

-

40

Phase 1

COD/NH4 -N

60 40

-1

80

60

(b) 200

+

100

NH4 -N (mg L )

Phase 4

Phase 3

-1

Phase 2

Effluent TN (mg L )

Phase 1

-

-1

NO3 -N (mg L )

(a) 80

NH4+-N removal efficiency (%)

R. Du et al. / Bioresource Technology 219 (2016) 420–429

NO3 -N removal efficiency (%)

424

0

Removal efficiency

  Fig. 2. Performance of PD-ANAMMOX process for simultaneous treatment of NO 3 -N and domestic wastewater in long-term operation: (a) influent NO3 -N, effluent NO3 -N as + + + well as NO 3 -N removal efficiency; (b) influent NH4-N, effluent NH4-N, NH4-N removal efficiency as well as feeding rate of domestic wastewater; (c) effluent TN and temperature; (d) influent COD, effluent COD, COD removal efficiency as well as the COD/NH+4-N of domestic wastewater.

COD was 37.7 mg L1 with the removal efficiency of 75.7%. Since the COD loading was much higher compared to previous phases, an excellent COD removal performance in this phase was mainly attributed to the excess NO 3 -N fed to the ANAMMOX reactor, which increasing the consumption of organic matter. On the other hand, since the NH+4-N was mainly removed via autotrophic ANAMMOX and NO 3 -N production in nitrification was left out, the organic carbon required for denitrification could be saved compared with traditional process. The above results clearly suggested that the increasing denitrification for COD removal would be taken place with elevated COD loading, thus the COD in domestic wastewater could be removed effectively at low temperature by the PD-ANAMMOX process.

3.1.3. TN removal performance In fact, the effluent TN was closely associated with the NO 3 -N + removal efficiency, since NO 2 -N and NH4-N in effluent was always at a low level. During the long-term operation, the mean effluent TN of 13.5 mg L1 was obtained in phase 1, and it declined to 11.5 mg L1 and 7.1 mg L1 in phase 2 and 3 (Fig. 2c), respectively. But it slightly rose in phase 4 with 7.7 mg L1 on average, which was caused by the excess NO 3 -N fed to ANAMMOX reactor for the deteriorated partial denitrification. The above results showed that the effluent COD, NH+4-N and TN all reached the discharge standards (COD, NH+4-N and TN limitation of 50 mg L1, 5 mg L1 and 15 mg L1, respectively) despite of low temperature during the whole operation, which further demonstrated the advantages of applying PD-ANAMMOX process for simultaneously treating NO 3 -N and domestic wastewater.

3.2. NO 2 -N production in PD reactor During the whole operation process, the influent NO 3 -N was kept around 50 mg L1 and the external carbon source addition  was maintained at COD/NO 3 -N of 3.0 for the optimal NO2 -N pro duction. It was observed that the effluent NO2 -N varied from 18.2 mg L1 to 27.0 mg L1 with a mean value of 22.3 mg L1 in 1 phase 1, corresponding to approximately NO 3 -N of 10.9 mg L 1 as well as COD of 38.3 mg L residual in effluent (Fig. 3a and Table 1). Based on our previous study conducted with batch feeding operation (Du et al., 2015a), the HRT of 30 min was enough for 1 partial denitrification at the initial NO with the 3 -N of 50 mg L MLSS  4000 mg L1. In this study, the incomplete NO 3 -N reduction occurred was mainly caused by the continuously feeding of NO 3 -N and COD, which leaded to an inadequate substrates transfer of the biomass (NO 3 -N and organic matter). Consequently, the NTR of 55.1% on average was obtained in this phase, which was lower than our previous study (approximately 80%) operated with batch feeding (Cao et al., 2013; Du et al., 2015a). Moreover, the partial denitrification performance little changed  in phase 2, with average effluent NO 2 -N, NO3 -N as well as COD in 1 1 PD reactor of 22.5 mg L , 11.4 mg L and 37.7 mg L1, respectively. Similar results was observed in phase 3 with effluent 1 1 NO , NO and COD of 2 -N of 20.9 mg L 3 -N of 10.9 mg L 1 39.3 mg L , which was also closely to that in phase 1. Neverthe less, NO 2 -N concentration declined but NO3 -N and COD in effluent increased in phase 4, with the mean value of 14.4 mg L1 for NO 21 N, 21.3 mg L1 for NO for COD, respectively, 3 -N and 54.9 mg L indicated that the performance of partial denitrification was deteriorated in this phase.

425

R. Du et al. / Bioresource Technology 219 (2016) 420–429

100

50

80

60 -1

COD (mg L )

40 60 30 40 20

15

30

-

45 60 Time (day) -

-

Eff.NO2 -N

Eff.NO3 -N

90

0 105

(b)

7.5

100

6.0

80

4.5

60

3.0

40 20

1.5

0

0.0 0

15

30

MLSS

45 60 Time (day)

75

300 250 200 150 100 50 0

105

Effluent MLSS

MLVSS/MLSS

MLVSS

90

0

NTR

Eff.COD

MLSS and MLVSS (g L )

Inf.NO3 -N

75

-1

0

40

20

20

10 0

-1

80

NTR (%)

Phase 4

Phase 3

-1

Phase 2

Effluent MLSS (mg L ) and SVI (mL gMLSS )

Phase 1

60

MLVSS / MLSS (%)

-1

Nitrogen concentration (mg L )

(a)

SVI

  Fig. 3. Long-term performance of partial denitrification reactor with continuously feeding: (a) variation of influent NO 3 -N, effluent NO2 -N, effluent NO3 -N, effluent COD as well as NTR; and (b) profiles of MLSS, MLVSS, MLVSS/MLSS, effluent MLSS as well as SVI value.

Table 2 Performance and operational conditions of ANAMMOX reactor in different phases. Phase (day)

1 2 3 4

(1  22) (23  41) (42  68) (69  105)

Temp. (°C)

15.1 13.1 12.9 12.9

Effluent of ANAMMOX UASB reactor (mg L1) NH+4-N

NO 2 -N

NO 3 -N

TN

COD

3.5 3.2 1.7 1.2

1.1 0.4 1.5 0.8

8.9 7.9 4.0 5.7

13.5 11.5 7.1 7.7

44.7 44.1 34.9 37.7

The performance of partial denitrification was closely related to the reaction time, sludge amount and activity. Since the HRT was not changed and the temperature was close to the previous phases, the deteriorated NO 2 -N production was assumed to be caused by the reduced biomass (MLSS) in reactor, as shown in Fig. 3b. The initial MLSS was 4.38 g L1, and it maintained relatively stable amount in phase 2 and phase 3 (4.13  3.77 g L1). However, a sharply decline was observed in phase 4, resulting in a MLSS as low as 1.48 g L1 on day 102. Furthermore, the MLSS in the effluent of partial denitrification reactor was also measured. It varied from 26.4 mg L1 to 45.9 mg L1 in phase 1  3 (Fig. 3b), but rose up to 61.5 mg L1 on day 75 and 112.4 mg L1 on day 102. This suggested that a serious biomass washout was taken place in phase 4, resulting in the sludge losing and MLSS decrease in the PD reactor. The seeding partial denitrification sludge was present with granule (Fig. S1a) and manifested a good settling property. However, the sludge settleability deteriorated during the operation.

NO 3 -N removal efficiency (%)

NH+4-N removal efficiency (%)

COD removal efficiency (%)

83.1 84.1 89.5 88.6

98.1 97.1 97.6 98.1

74.1 75.1 78.7 75.7

The SVI was an important indicator to evaluate the sludge settling property and it was measured during the operation. It was shown with increasing SVI, which varied from 56.3 mL g MLSS1 in initial to 265.4 mL g MLSS1 on day 102 (Fig. 3b). The sludge sample was taken on day 94, and presented with loose appearance, roughvague edge and larger diameter compared with that in phase 1. Previous studies had reported that a sludge bulking would be happened when SVI exceeding 150 mL g MLSS1 (Peng et al., 2003). Thus, it was speculated that the sludge bulking was occurred with the continuous feeding of substrates for partial denitrification. Since no filamentous microorganisms were observed under the microscope, the sludge bulking was identified as viscous bulking (also referred to as non-filamentous bulking). The viscous bulking is characterized by the production of excessive amounts of extracellular polymeric substances (EPS) (Peng et al., 2003), which could be manifested by the sludge stickyappearance (Fig. S1b). This was also revealed by the increasing MLVSS to MLSS ratio (MLVSS/MLSS) from 54.4% in initial to 85.7%

R. Du et al. / Bioresource Technology 219 (2016) 420–429

Phase 1

Phase 2

Phase 3

Phase 4

25 20 15 10 5 0

0

15

30

45 60 75 Time (day) NO 2--N mix

NH 4+-N mix

(b)

Phase 1

1.8

Phase 2

90

105

NO 3--N mix Phase 4

Phase 3

5

1.5 Rs-Inf.

3.3. Characteristic of nitrogen removal by ANAMMOX in UASB reactor 3.3.1. The stoichiometric ratios assessment of ANAMMOX process Stoichiometry was the calculation of quantitative relationships of the reactants and products in a balanced chemical reaction, which was an important indicator to evaluate the reaction pro+ ceeding. The stoichiometric ratios of NO 2 -N reduction to NH4-N + consumption (RS) as well as the NO -N production to NH -N con3 4 sumption (RP) were calculated during the whole operation (Table 3). + In phase 1, the influent ratio of NO 2 -N to NH4-N (RS-Inf.) was around 1.35 (Fig. 4b), which was close to the optimal ratio of 1.32 for ANAMMOX. The RS of 1.68 as well as RP of 0.10 on average were obtained during this operational period, which were higher than the theoretical values of 1.32 and 0.26, respectively (Kuenen, 2008). This indicated the occurence of heterotrophic denitrification since the organic matter involved in domestic wastewater was introduced to ANAMMOX reactor. Additionally, enhanced denitrification for NO 3 -N removal was observed with increasing domestic wastewater feeding rate, corresponding to the RP of 0.17, 0.45 and 0.84 in phase 2 (0.54 L h1), 3 (0.72 L h1) and 4 (1.08 L h1), respectively. Whereas, the RS declined to 1.67, 1.27 and 0.81 as a result of the decrease of RS-Inf., which were 1.33, 1.25 and 0.80 in phase 2, 3 and 4, respectively. Previous study indi+ cated stoichiometric molar ratios of NO 2 -N to NH4-N conversion varied in the range of 0.5  4.0, based on the different operation conditions and reactor configuration (Ahn, 2006), and the influent + substrate ratio of NO 2 -N to NH4-N was shown to be a key factor in the ANAMMOX process (Jin et al., 2013). The percentages of ANAMMOX and denitrification contributing to TN removal could be regarded to evaluate the system stability. Notably, it showed that the nitrogen was removed mainly via ANAMMOX process, which accounted for over 70% in most case (Fig. 4c). The mean percentage of ANAMMOX was 75.1% in phase 1, and 72.8% in phase 2, corresponding to 24.9% and 27.2% for

4

1.32

1.2

3

0.9

2

0.6

-0.26

Rs and Rp

Nitrogen concentration (mg L

(a)

)

on day 102 (Fig. 3b). It was supposed to be caused by the elevated production amounts of EPS. Previous studies had been reported that the quantity of EPS production was dependent on a number of factors, including microbial species composition, growth phase, the type of limiting substrate (carbon, nitrogen and phosphorous), culture temperature and hydrodynamic shear force (Liu et al., 2004). It was reported that the microorganisms would produce more EPS for protection from environmental stress for surviving at the inhibitory conditions, such as at low temperature (Knoop and Kunst, 1998). It was necessary to investigate the detailed reasons for the excess EPS production in future in order to achieve good solid–liquid separation and excellent performance of partial denitrification.

-1

426

1

0.3 0 0.0

0

15

30

45 60 Time (day) Rs

Rs-Inf.

(c)

Phase 1

100

75

Phase 2

90

105

Rp Phase 4

Phase 3

Percentage (%)

80 60 40 20 0

0

15

30

45 60 Time (day)

ANAMMOX

75

90

105

Denitrification

Fig. 4. Variation of (a) influent as well as NO 3 -N of ANAMMOX reactor by mixing PD effluent and domestic wastewater; (b) RS-Inf., RS as well as RP; and (c) percentage of ANAMMOX and denitrification contribution on TN removal in ANAMMOX-UASB reactor during the long-term operation. NH+4-N,

NO 2 -N

complete denitrification. It was speculated that an increasing denitrification for nitrogen removal would be taken place in phase 3 and 4 due to the improvement of carbon loading rate. However, the denitrification percentages declined slightly with the mean value of 23.6% in phase 3 and 21.9% in phase 4. Considering the drastic increase of RP (Table 3), it clearly indicated that part of

Table 3 The mean influent nitrogen concentration, C/NO x -N, stoichiometric ratios, and percentage of ANAMMOX as well as denitrification on TN removal in ANAMMOX UASB reactor in different phases. Phase (day)

1(1  22) 2(23  41) 3(42  68) 4(69  105) a b c d e f

Nitrogen concentration (mg L1)

a

RS-Inf.

NH+4-N mix

NO 2 -N mix

NO 3 -N mix

15.1 14.8 13.9 14.0

20.3 19.5 17.4 11.1

9.9 9.9 9.1 16.4

b

RS 1.35 1.33 1.25 0.80

C/NO X -N

ANAMMOX c

1.68 1.67 1.27 0.81

RP

d

0.10 0.17 0.45 0.84

0.54 0.74 1.06 1.33

e

Percentages (%)

f

ANAMMOX

Denitrification

75.1 72.8 76.4 78.1

24.9 27.2 23.6 21.9

The nitrogen concentration referred to ANAMMOX influent after mixture between PD effluent and domestic wastewater, it were calculated as shown in Eqs. (5)–(7). + RS-Inf.: the substrate ratio of influent NO 2 -N and NH4-N in ANAMMOX reactor. + RS: the ratio of NO 2 -N conversion to NH4-N consumption (RS) in ANAMMOX reactor. + RP: the ratio of NO 3 -N production to NH4-N consumption (RP) in ANAMMOX reactor.    C/NO X -N referred to the ratio of influent COD to NOX -N (NO2 -N + NO3 -N) in the mixture of PD effluent and domestic wastewater. The percentages referred to the contribution of ANAMMOX and complete denitrification on TN removal in ANAMMOX UASB reactor, calculated as shown in Eqs. (13), (14).

R. Du et al. / Bioresource Technology 219 (2016) 420–429  NO 3 -N in the ANAMMOX reactor was reduced to NO2 -N (partial denitrification process), then removed via ANAMMOX promptly. In other words, though the heterotrophic denitrification had trend to be improved due to the increasing feeding rate of domestic wastewater, it did not enhance the complete denitrification (NO 3 -N ? N2) for nitrogen removal, but the partial denitrification for NO 2 -N production. According to the results above, the occurrence of partial denitrification was mainly resulted from the poor settleability of the sludge in PD reactor and introducing certain amount of partial denitrification bacteria to ANAMMOX reactor, which could utilize the organic matter involved in domestic wastewater. The presence of organic matter was generally believed to have adverse effect on ANAMMOX process, as the heterotrophic denitrifying bacteria grew with much higher rate and cellular synthesis growth yield compared to autotrophic ANAMMOX bacteria (Kumar and Lin, 2010). Tang et al. (2010) had reported that the ANAMMOX bacterial growth was significantly suppressed by denitrifying communities at influent COD/NO 2 -N of 2.92, and it was found that the organic matter concentration over 300 mg COD L1 (C/N of 2) would inactivate or eradicate ANAMMOX communities (Chamchoi et al., 2008). Nevertheless, in this study, the ANAMMOX bacteria and denitrifiers could coexist harmoniously and achieve high nitrogen removal efficiency. This was mainly ascribed to the relatively low organic matter supply. The mean influent C/NO x -N   (NO x -N = NO2 -N + NO3 -N) of ANAMMOX-UASB reactor was 0.54 in phase 1, and 0.74, 1.06 and 1.33 for phase 2, 3 and 4, respectively, which was lower than the reported value. These results were consistent with previous study (Du et al., 2014), which

427

revealed that the ANAMMOX bacteria and denitrifiers could coexist at C/N of 2. Ni et al. (2012) found that low COD concentration (C/N below 3.0) did not affect ANAMMOX significantly but improved the total nitrogen removal via denitrification. Moreover, organic matter in the ANAMMOX reactor could also be used in partial denitrification process for NO 2 -N production, and the residual carbon source was supplied for complete denitrification. According to the results above, the stable partial denitrification with high NO 2 -N production was a key point for achieving an excellent nitrogen removal from NO 3 -N and domestic wastewater. While the present study indicated that desirable nitrogen removal performance could still be achieved when partial denitrification deteriorated occurred with high effluent NO 3 -N due to the partial denitrification occurrence in ANAMMOX reactor. Thus, certain amount of partial denitrification sludge could be introduced to ANAMMOX reactor when treating the high-strengthen wastewater with high NO 3 -N production. Furthermore, it was of supreme importance to maintain the preponderant position of ANAMMOX process for TN removal in practical application, so that a sustainable and stable nitrogen removal would be achieved from NO 3 -N and domestic wastewater in long-term operation. 3.3.2. Microbial community structure in UASB reactor The sludge sample in ANAMMOX-UASB reactor was taken on day 102 in order to investigate the microbial community structure via Illumina high-throughput sequencing analysis. The effective sequences of approximately 26,478 were obtained, with an average length of 443 bp after removing low-quality sequences. There were approximately 582 OTUs generated with the Shannon

Fig. 5. (a) The sample rarefaction and Shannon curves; (b) distribution of bacterial community at phylum level; and (c) abundance at genus level in the ANAMMOX reactor on day 102.

428

R. Du et al. / Bioresource Technology 219 (2016) 420–429

indexes of 4.94 (Fig. 5a). The diversity and richness was noticeably lower than that of the municipal wastewater treatment systems, in which OTUs generally exceeded to 3000 and Shannon indexes over 6.0 (Zhang et al., 2012). While, it was higher than the ANAMMOX reactor treating synthetic wastewater, in which the OTUs varied from 291 to 309 and Shannon indexes was in the range of 3.49  3.92 (Cao et al., 2016). This was mainly related with the introduced real domestic wastewater, which enhanced the growth of heterotrophic bacteria in the ANAMMOX system. There six major phylum were detected, and the most predominant phylum was Proteobacteria accounted for as high as 51.5%, followed by Bacteroidetes (10.6%), Chloroflexi (10.0%), Chlorobi (7.5%), Acidobacteria (7.3%) and Planctomycetes (5.8%). The above phylum was also found in granular sludge bed reactor via autotrophic nitrogen removal process (Wang et al., 2012). This was similar to the previous study for treating synthetic wastewater through ANAMMOX process (Cao et al., 2016). The high-scale of phyla Proteobacteria mainly contained twelve genera, in which the Thauera was detected with 6.1% (Fig. 5c). Genera Thauera had been identified with a dominant bacteria in the partial denitrification reactor in previous study (Du et al., 2015a), and it was supposed to be responsible for the high NO 2 -N accumulation. Additionally, since it was not detected in the sole ANAMMOX reactor in our previous study (Cao et al., 2016), the Thauera genera existed in this study was likely introduced from the effluent of partial denitrification, and surviving with the carbon source from domestic wastewater. Therefore, it demonstrated the partial denitrification process performed in the ANAMMOX reactor. This could also explain for the high proportion of ANAMMOX contributing on TN removal in phase 4, since the NO 2 -N was generated from the NO 3 -N reduction via partial denitrification, as described above. In addition, previous study found the heterotrophic denitrifying bacteria of Denitratisoma genus (also belonging to Proteobacteria phylua) was dominant with percentage of 23.6% in ANAMMOX reactor treating synthetic wastewater (Cao et al., 2016), while it was not found in this study. The functional microorganism in the UASB reactor was the ANAMMOX bacteria belonging to phylum Planctomycetes, which performed the main nitrogen removal from wastewater. Three genera of ANAMMOX bacteria: Candidatus Jettenia, Candidatus Brocadia and Candidatus Kuenenia, were detected in the UASB reactor. This was in accordance with previous studies, which reported these genera were widespread in freshwater ecosystems (Zhu et al., 2013) and the bioreactors (Wang et al., 2015). Candidatus Jettenia was the major ANAMMOX species with the percentage of 2.01% in this study. The proportions of Candidatus Brocadia and Candidatus Kuenenia were 0.94% and 0.22%, respectively. This was different with most of other studies and our previous work, which Candidatus Brocadia (or Candidatus Kuenenia) was identified as the predominant ANAMMOX species. Nevertheless, study by Liang et al. (2015) revealed the profitable effect of both acetate and propionate on the survival of Candidatus Jettenia, in which the predominant species in ANAMMOX granular sludge was transformed to Candidatus Jettenia from the Candidatus Kuenenia after the addition of acetate and propionate. It was also reported by Huang et al. (2014), who found the Candidatus Jettenia were capable of growing at the present of low-concentration acetate and propionate with a low growth rate. It was assumed that the organic matter involved in the raw domestic wastewater had an important effect on microbial structure of the ANAMMOX system.

4. Conclusions The novel PD-ANAMMOX process was established successfully for simultaneous treatment of NO 3 -N and low C/N domestic

+ wastewater at low temperature, with the NO 3 -N, NH4-N and COD removal efficiency of 89.5%, 97.6% and 78.7%, respectively. Desirable performance still achieved with effluent TN below 8 mg L1 when partial denitrification deteriorated with excess NO 3 -N remained due to the partial denitrification occurred in ANAMMOX reactor. Low C/N did not cause the predominant of complete denitrification, and ANAMMOX maintained as the dominant pathway for nitrogen removal (over 70%). The Candidatus Jettenia was the major ANAMMOX species with percentage of 2.7%.

Acknowledgements This research was financially supported by Natural Science Foundation of China (51478013) and the Funding Projects of Beijing Municipal Commission of Education.

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