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N domestic wastewater at low dissolved oxygen conditions
Bioresource Technology 305 (2020) 123045
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Simultaneous partial nitritation and denitritation coupled with polished anammox for advanced nitrogen removal from low C/N domestic wastewater at low dissolved oxygen conditions
T
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Wen Zhang, Yongzhen Peng , Liang Zhang, Xiyao Li, Qiong Zhang National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, Engineering Research Center of Beijing, Beijing University of Technology, Beijing 100124, PR China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Nitrogen removal Partial nitritation Denitritation Anammox Low dissolved oxygen
Simultaneous partial nitritation and denitritation (SPND) coupled with anammox was established in this study to treat domestic wastewater. Two lab-scale bioreactors, namely SPND-SBR and ANA-UASB, were used in the twostage system. In SPND-SBR, stable nitrogen removal efficiency of 51.1% was achieved with a high ammonia oxidation rate of 0.117 kg N/(m3·d). Besides, successful out-selection of nitrite-oxidizing bacteria (NOB) under low-DO of 0.1 mg/L during the steady period, resulting in an average effluent NO2−-N/NH4+-N ratio of 1.04. In ANA-UASB, the abundance of Candidatus Brocadia and Candidatus Kuenenia increased from 8.21% and 4.01% to 21.33% and 6.41% with low influent substrate contents of only 38 mg N/L. The effluent total inorganic nitrogen (TIN) was only 8.4 ± 1.1 mg N/L and the nitrogen removal efficiency reached 88.24%. Overall, the study demonstrated that the novel low-DO two-stage process for nitrogen removal is a promising technique for wastewater of low C/N ratio.
1. Introduction Inorganic nitrogen is generally removed through high-rate biological nutrient removal (BNR) process to prevent eutrophication in receiving waters. In the traditional high-rate BNR processes, most
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wastewater treatment plants (WWTPs) generally control the dissolved oxygen (DO) above 2 mg/L for a high efficient and stable nitrification, though the energy consumption due to aeration accounts for nearly half of the total power consumptions of WWTPs (Keene et al. 2017). Thus, energy-efficient aeration has received significant research attention.
Corresponding author. E-mail address: [email protected] (Y. Peng).
https://doi.org/10.1016/j.biortech.2020.123045 Received 22 December 2019; Received in revised form 15 February 2020; Accepted 18 February 2020 Available online 19 February 2020 0960-8524/ © 2020 Elsevier Ltd. All rights reserved.
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Fig 1. Schematic diagram of the novel two-stage process treating domestic sewage.
external carbon source, to produce nitrogen gas (Eq. (1)) (Strous et al., 1998; Jin et al., 2013). Partial nitritation is a critical pre-requisite step before the anammox process as the former provides nitrite to anammox reactor (Vlaeminck et al., 2009; Kartal et al., 2010). The process requires only converting 55% of NH4+-N to NO2−-N in a nitritation reactor and saves about 60% aeration energy compared with the traditional nitrification and denitrification BNR process (Siegrist et al., 2008).
Previous studies have shown that efficient nitrogen removal can be achieved with low-DO conditions, for example, simultaneous nitrification and denitrification (SND) occurred in the prolonged aeration system in the low-DO aerobic zones (Daigger and Littleton 2014). Similarly, Keene et al. (2017) observed a higher nitrogen removal in the full-scale plant due to denitrification improvements in the low-DO tanks via SND in a continuous flow pilot-scale reactor. Further, the bioelectrocoagulation system also benefited from SND performance in submerged membrane bioreactor (MBR) with limited dissolved oxygen (Li et al., 2018a,b). Yan et al. (2019) found that a lab-scale modified sequencing batch reactor (SBR) was efficient for nitrogen removal under limited DO conditions by SND via nitrite pathway. Besides, several studies confirmed that the activity of nitrite-oxidizing bacteria (NOB) (Laureni et al. 2016; Li et al., 2018a,b) was suppressed at low oxygen concentrations of 0.15–0.18 mg/L, leading to the short-cut nitrogen removal with lower demand for carbon sources. Therefore, efficient application of low-DO for the domestic wastewater for the total nitrogen removal could save the operational cost of BNR facilities (Park and Noguera, 2004; Fitzgerald et al., 2015). However, most SND processes have long hydraulic retention times (HRT) due to low ammonia oxidation rate at low-DO levels, leading to difficulty in improving volumetric process rates. The residual ammonium in the effluent increases with the sudden increase in the nitrogen loading rate, resulting in poor nitrogen removal and consequently low effluent quality not suitable for release as per stringent discharge standards for ammonia (< 2 mg N/L) (e.g. Switzerland, WPO (1998)). Moreover, the effluent contains a large amount of nitrate or nitrite due to inadequate biodegradable carbon source in domestic wastewater and insufficient denitrification in the SND process. Thus, advanced treatment techniques are necessary for secondary sewage effluents (SSE). However, the capital and operational costs are significantly higher than those of secondary treatment facilities with the same scale (Klamerth et al., 2010). In this context, the anaerobic ammonia oxidation (anammox) process provides a new approach to low-DO process. Anammox is a novel process, in which ammonium is oxidized by nitrite serving as the electron acceptor under anaerobic conditions with low aeration and no
NH+4 + 1.32 NO−2 + 0.066 HCO−3 + 0.13 H + → 1.02 N2 + 0.26 NO−3 + 0.066CH2 O0.5 N0.15 + 2.03H2 O
(1)
Thus, the effluent of low-DO process can provide the influent to the anammox zone with a suitable NO2−-N/NH4+-N ratio, leading to an improved shock resistance capacity, reduced aeration energy consumption, and a good effluent quality. Further, the polishing step can become efficient and cost-effective with the utilization of independent anammox zone followed by secondary clarifiers. However, the studies investigating the collaboration of low-DO process coupled with anammox are still limited. The objective of the present work was to investigate the nitrogen removal performance of a coupling process based on simultaneous partial nitritation, denitritation (SPND) and polished anammox for the treatment of domestic wastewater with low-DO conditions (e.g. 0.1 mg/ L) and their reliability in complying with the nitrogen discharge standards. Furthermore, the overall performance, nitrogen removal efficiencies and rates and effluent qualities (nitrogen species and COD) of this novel two-stage process were assessed along with the relative abundance, maximum specific activities and distribution of the main functional bacteria. Finally, the effect of pre-anaerobic segment on suppressing NOB was discussed and nitrogen removal potential of the full-scale mainstream application of this two-stage process were compared to the conventional systems. 2. Materials and methods 2.1. Experimental setup The schematic diagram for the two-stage process is shown in Fig. 1. 2
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The first sequence batch reactor (SBR) was used for partial nitritation and denitritation termed as SPND-SBR, while the up-flow anaerobic sludge blanket (UASB) used anammox to treat wastewater pretreated by SPND-SBR and terming as ANA-UASB. SPND-SBR and ANA-UASB were made up of polymethyl methacrylate (PMMA) and had a working volume of 10 L and 2 L, respectively. SPND-SBR was equipped with a mechanical stirrer (IKA RW20, Germany). An air-compressor (HAILEA V-20, China) was used for blast aeration with adhesion sand lump as the microporous aerator, whereas a rotameter was used to control the flow of gas and consequently the DO level in the system. ANA-UASB was wrapped with a black sponge cloth to shield the anammox granules from light. During the experiment, the temperature was not controlled and maintained as ambient.
Milwaukee, USA). Cycle measurements were performed every 2–3 days to monitor nitrogen loss and nitrite accumulation ratio (NAR) during the aerobic stage. The DO, pH and temperature were monitored using a pH/Oxi 3420i analyzer (WTW Company, Germany), while COD was analyzed using COD quick-analysis apparatus (Lian-hua Tech. Co., Ltd., 5B-1, China) and the MLSS and MLVSS were analyzed according to the standard methods (APHA, 1998). The sludge particle size distribution was measured using a Laser Particle Size Analyzer (Microtrac S3500, USA) over the 0.01–2000 μm size range. Frozen dried biomass was used to measure polyhydroxyalkanoates (PHAs) and glycogen (Gly). PHAs were determined as the sum of poly-β-hydroxybutyrate (PHB) and polyβ-hydroxyvalerate (PHV) that were measured as reported by (Oehmen et al., 2005) using the gas chromatograph (Agilent 6890 N, U.S.) with Agilent DB-1 chromatographic column. Gly was analyzed as described by (Zeng et al., 2003).
2.2. Influent and seed sludge Domestic wastewater was obtained from a septic tank at the Beijing University of Technology and stored in a wastewater tank before feeding into the SPND-SBR. The soluble COD, ammonium, nitrite, nitrate, C/N ratio and pH in sewage were 238.6 ± 53.5 mg/L, 71.4 ± 4.4 mg N/L, 0.2 ± 0.1 mg N/L, 0.3 ± 0.1 mg N/L, 3.4 ± 0.8 and 7.1–7.5, respectively. The seed sludge for SPND-SBR was ordinary nitrification sludge taken from a traditional anaerobic/anoxic/oxic activated sludge system. The anammox granular seed sludge was inoculated from side stream for treating anaerobic digestive juice. After inoculation, the mixed liquid suspended solids (MLSS) in the SPND-SBR and ANA-UASB were about 3623 and 3462 mg/L, while the mixed liquor volatile suspended solids (MLVSS) were about 3025 and 2770 mg/L, respectively.
2.4.2. Bacterial activity measurements The maximum specific activities (mg N/(gVSS·h)) of AOB and NOB were measured using the ectopic activity tests. Briefly, 500 mL sludge was extracted from SPND-SBR at the end of the aeration stage, washed thrice by distilled water and then injected into a 0.5 L wild-mouth bottle to simulate the MLSS of the mother reactor. AOB and NOB activities were tested separately under the saturated DO conditions of 7 ± 0.5 mg/L. The maximum specific activities of AnAOB were measured in the absence of DO (< 0.02 mg/L) using the anaerobic respirator (Challenge AER-208 Research Respirometer, USA). The anoxic stirring was used to remove the residual COD in the reactor before the test to avoid any disturbance to COD and to improve the accuracy. The specific experimental conditions and calculations are listed in supplementary.
2.3. Operational strategy 2.4.3. Calculations methods NAR and SND, which are indicators of the nitrite accumulation and nitrogen loss, were calculated using Eq. (2), Eq. (3):
SPND-SBR was operated in an anaerobic/aerobic mode with a short aeration time (240 min) and a low aeration rate (10–12 L/h) to achieve a suitable effluent NO2-N/NH4+-N ratio and to efficiently utilize COD to remove nitrogen. A typical cycle was operated for 6 h, comprising water intake for 5 min, oxygen deprivation for 50 min, aeration for 240 min, static settling for 60 min and water discharge for 5 min. The exchange volumetric ratio was controlled at 60% to fully utilize COD in domestic wastewater. DO was controlled at 0.1–0.8 mg/L during the aeration phase by adjusting the air flow rate. The long-term operation of SPNDSBR was divided into four phases (Phase I, II, III and IV) with the average DO in each phase being controlled at 0.8, 0.5, 0.3 and 0.1 mg/ L. HRT was maintained at 8.1 h and the sludge retention time (SRT) was kept as 30–40 d. The ANA-UASB was run in a continuous mode, where wastewater was continuously pumped into ANA-UASB at the bottom, while the upflow rate was controlled at 160 mL/min by adjusting the recirculation ratio to maintain a good fluidization condition. The ANA-UASB operation was divided into two phases. In the first phase, the reactor was started up with synthetic wastewater by gradually reducing nitrogen loading rate (NLR), while the pretreated domestic wastewater with a suitable NO2−-N/NH4+-N range was pumped into the reactor during the steady phase. HRT was generally maintained at 2 h. Non-active sludge discharge was adopted for the entire operation to ensure the growth and enrichment of anammox bacteria (AnAOB). The specific operational conditions in the two-stage system in different phases are listed in Table 1.
NO−2 − Nend ⎞ NAR = ⎛⎜ ⎟ × 100\% − − ⎝ NO2 − Nend + NO3 − Nend ⎠
(2)
NO−2 − Nend + NO−3 − Nend ⎞ SND = ⎜⎛1− ⎟ × 100\% + + ⎝ NH 4 − Nini − NH 4 − Nend ⎠
(3)
where NO2−-Nend and NO3−-Nend are the NO2−-N and NO3−-N concentrations at the end of the aerobic phase (mg N/L), respectively, while NH4+-Nini and NH4+-Nend are the NH4+-N concentrations at the beginning and end of the aerobic stage (mg N/L), respectively. CODabs, which is the indicator of the COD concentrations stored as intracellular carbon source, was calculated using Eq. (4):
CODabs (mg COD/L) = ΔCOD − (1.71ΔNO−2 − N+ 2.86ΔNO−3 −N)
(4)
where ΔCOD, ΔNO2−-N and ΔNO3−-N are the changes in COD, NO2−-N and NO3−-N concentrations in the anoxic stage of SPND-SBR (mg N/L), respectively whereas 1.71 and 2.86 are the COD concentrations required for heterotrophic denitrification of nitrite and nitrate in the anaerobic stage (mg/L), respectively. 2.4.4. Microbial diversity analysis The microbial community structure in the system was analyzed using quantitative real-time PCR (qPCR) and high-throughput sequencing data analysis. For qPCR, the abundance of AOB, Nitrospira, Nitrobacter and total bacteria were determined as described by Li et al. (2019), whereas a mixture of the amplicons was used for highthroughput sequencing on Miseq PE300 platform (Illimina, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using the standard protocols. Taxonomic classification was conducted to optimize the sequences into operational taxonomic units (OTUs) using 97% identity thresholds (setting a 0.03 distance limit) using the MOTHUR
2.4. Analytical methods 2.4.1. Conventional analysis The wastewater samples were filtered through 0.45 μm filters prior to the analysis. The concentrations of NH4+-N, NO2−-N and NO3−-N were measured using a Lachat QuikChem8000 Flow Injection Analyzer (Lachat Instruments, 3
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Table 1 Variations in the nitrogen and COD removal performance of the novel two-stage system in the four phases over a 136-day period. Phases
Time(d)
DO(mg/L)
Municipal Wastewater (influent) (mg/L) COD(mg/L)
C/N
73.0 68.9 70.0 72.4
245.2 184.4 254.5 253.8
3.7 2.7 3.7 3.3
I II III IV
1–43 44–76 77–100 101–136
0.8 0.5 0.3 0.1
Phases
Time(d)
ANA-UASB influent(mg/L)
Start-up IV
41–100 101–136
± ± ± ±
4.7 3.6 3.8 4.4
−
± ± ± ±
35.7 44.2 30.6 59.6
± ± ± ±
0.5 0.6 0.5 0.9
NH4+-N(mg-N/L)
NO2−-N(mg-N/L)
NO3−-N(mg-N/L)
COD(mg/L)
1.0 ± 1.1 13.7 ± 12.4 23.6 ± 7.6 17.8 ± 7.8
2.6 ± 4.3 14.1 ± 4.9 10.1 ± 6.0 16.7 ± 3.8
36.7 ± 8.0 9.5 ± 5.4 5.4 ± 5.1 0.7 ± 0.9
40.5 66.0 58.6 47.9
± ± ± ±
13.1 12.4 14.7 12.3
ANA-UASB effluent(mg/L)
NH4 -N(mg-N/L)
NO2 -N(mg-N/L)
NO3−-N(mg-N/L)
30.5 ± 10.4 19.4 ± 6.2
35.9 ± 10.0 17.6 ± 5.8
5.3 ± 3.8 1.0 ± 1.0
+
SPND-SBR effluent(mg/L)
NH4+-N(mg-N/L)
COD(mg/L)
NH4+-N(mg-N/L)
NO2−-N(mg-N/L)
NO3−-N(mg-N/L)
COD(mg/L)
41.1 ± 7.3 37.2 ± 7.8
1.4 ± 5.0 0.6 ± 1.3
1.0 ± 1.7 0.2 ± 0.1
29.9 ± 8.5 7.6 ± 2.0
40.8 ± 7.3 33.2 ± 7.5
inhibited in Phase III (Day 77–100) due to the temperature decrease in winter. qAOB, MAX increased sharply to 14.5 ± 0.4 mg N/(gVSS·h) in Phase IV (Day 101–136), whereas qNOB, MAX was not detected when DO was at 0.1 mg/L. These findings indicate that adopting step-wise DO reductions strategy was effective to out-select NOB. Effluent NH4+-N and NO2− -N were gradually controlled at 17.8 ± 7.7 mg N/L and 16.7 ± 3.8 mg N/L, respectively, by adjusting aeration time to 240 min at low-DO of 0.1 mg/L during the steady period (Day 101–136). Notably, the average NH4+-N conversion rate of 0.117 kg N/(m3·d) at low-DO of 0.1 mg/L is higher than that reported in previous studies (0.05–0.10 kg N/(m3·d)) conducted under higher DO (e.g. 2–3 mg/L) (Yuan and Oleszkiewicz, 2011; Gu et al., 2012). The findings indicate that the biomass gradually adapted to low oxygen level in domestic wastewater after several phases and a high-rate partial nitritation with the average effluent NO2−-N/NH4+-N ratio of around 1.04, which is close to the stoichiometric value of 1.32, was achieved in SPND-SBR (Strous et al., 1998; Jin et al., 2013).
software program. 3. Results and discussion 3.1. Performance of the mainstream SPND-SBR during long-term operation 3.1.1. COD and nitrogen removal by step-wise DO reductions SPND-SBR had an average influent COD to NH4+-N ratio of 3.36 (Fig. 2) and ran for 136 days divided into 4 phases according to the DO concentration of the aerobic stage (Table 1). The variations in influent and effluent nitrogen species are presented in Fig. 2, along with the NH4+-N and COD removal efficiencies. In the four phases, the average influent COD was 238.6 mg/L and total ammonium nitrogen (TAN) was 71.4 mg N/L. With gradual decrease in DO from 0.8 to 0.1 mg/L, the respective average nitrogen removal efficiency (NRE) and COD removal efficiency (ORE) of SPND-SBR increased from 42.83% and 64.2% to 51.11% and 81.13%, resulting in a relatively high nitrogen removal rate (NRR) of 0.089 kg N/(m3·d) and COD removal rate (ORR) of 0.494 kg/(m3·d). Therefore, SPND-SBR could efficiently remove COD and nitrogen in the domestic wastewater even when DO is reduced to 0.1 mg/L. Moreover, average ORE of SPND-SBR increased by 42.37% with the decrease of DO, indicating that heterotrophic bacteria better utilized biodegradable organic matter in the reactor under lower DO conditions.
3.1.3. Performance of SPND in SPND-SBR The average NRE and ORE of 51.1% and 81.1%, respectively, were achieved in SPND-SBR at low-DO of 0.1 mg/L and was maintained during the steady period (Day 101–136) (Fig. 2). Hence, a typical cycle on Day 121 was selected to analyze the transformation of nitrogen and COD in anaerobic/aerobic mode (anaerobic 50 min/aerobic 240 min). As shown in Fig. 3, COD decreased from 122.2 to 95.3 mg/L in the anaerobic stage (0–50 min), resulting in CODabs of 40.9 mg/L using Eq. (4). At the end of the anaerobic stage, the mixture contained 43.7 mg N/L of NH4+-N and 95.3 mg/L. During the aerobic stage, remaining soluble COD was further oxidized and decreased to 54.5 mg/L, while 31.4 mg N/L of NH4+-N was oxidized to NO2−-N (15 mg N/L) and NO3−-N (0.7 mg N/L) with the residual NH4+-N of 12.3 mg N/L. The total oxidized nitrogen (nitrite plus nitrate) was lower than the amount of NH4+-N oxidized, suggesting the nitrogen loss in the aerobic stage due to denitritation improvements in the low-DO aerobic stage via SND (Liu et al., 2017). Consequently, the discrepancy in TIN was used as a proxy for SND to estimate the degree of SND (Fig. 2c). During the 136-day operation, the average SND increased from 11.7% to 44.4% (Fig. 2c) as per Eq. (3) indicating improved nitrogen removal in SPNDSBR during the long-term operation. However, PHA and Gly decreased by 9.6 and 29.3 mg COD/gVSS in the aerobic stage, respectively, suggesting that endogenous denitritation driven by PHA or Gly contributed to the improved nitrogen removal during the aerobic stage of SPNDSBR. The particle size is critical for the micro-environment formation in activated sludge (Mosquera-Corral et al., 2005). With the decrease of DO during the long-term operation, the average sludge particle size characterized by volume (Mv) increased from 61.5 to 161.5 μm, while the percentage of granular sludge (particle size > 200 μm) increased from none to 31% (Figure could be seen in supplementary). This may be due to limited filamentous bulking under low-DO circumstances, which
3.1.2. High-rate partial nitritation at low-DO Concentrations of nitrogen species in the effluent of SPND-SBR at low DO were investigated. The temporal variation in the ammonium transformation performance and NAR in SPND-SBR are presented in Fig. 2c. NO2−-N was only 2.6 ± 4.3 mg N/L, while NO3−-N reached to 36.7 ± 7.9 mg N/L, resulting in a NAR of about 6.44% as per Eq. (2) after nitrification sludge inoculation in SPND-SBR. With the decrease of DO, the concentrations of NO2−-N increased continuously, while NO3−-N decreased from the Phase I to III (Day 1–100). In the steady period (Day 101–136), NO3−-N was less than 1.5 mg N/L, whereas NO2−-N reached to 16.7 ± 3.8 mg N/L. A high NAR (96.2 ± 4.3%) was constantly maintained, indicating the successful out-selection of NOB at the low DO of 0.1 mg/L. Meanwhile, the changes in the activity and abundance of main functional bacteria also confirmed the successful out-selection of NOB during the step-wise DO reductions. As evident in Table 2, the overall growth rate of AOB increased by 600.73%, whereas that of Nitrobacter decreased by 50.60%. The relative abundance of AOB increased from 0.008% to 0.114%, whereas that of Nitrospira decreased from 0.245% to 0.01%. Meanwhile, in Phase I (Day 1–43), qNOB, MAX (3.4 ± 0.3 mg N/ (gVSS·h)) in the reactor was higher than qAOB, MAX (2.2 ± 0.2 mg N/ (gVSS·h)) (Figure could be seen in supplementary). In Phase II (Day 44–76), DO was further reduced to 0.5 mg/L and consequently qAOB, MAX(5.8 ± 0.3 mg N/(gVSS·h)) was significantly higher than qNOB, MAX (1.5 ± 0.4 mg N/(gVSS·h)). The activities of AOB and NOB were 4
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Fig 2. Removal performance and nitrogen profiles of SPND-SBR (a) TIN removal efficiency (b) COD removal efficiency and (c) Nitrogen conversion, NAR and SND during operation.
Table 2 Variations in bacterial abundance, relative abundance and growth rate during the long-term operation of SPND-SBR. Time (d)
Gene copy number (copies/g dry sludge) AOB
0 21 62 89 125
1.16 1.98 5.62 8.11 2.45
Nitrobacter × × × × ×
8
10 108 108 108 109
1.63 1.53 2.82 7.56 3.33
× × × × ×
Growth rate (%)
Nitrospira 9
10 109 109 108 108
TB 9
3.55 × 10 1.45 4.79 × 109 1.49 1.45 × 109 9.32 4.79 × 109 2.81 2.21 × 108 2.14 Overall growth rate
× × × × ×
12
10 1012 1012 1012 1012
5
Relative abundance (%)
AOB
Nitrobacter
Nitrospira
AOB
Nitrobacter
Nitrospira
– 70.90 283.90 44.20 201.73 600.73
– −6.29 84.92 −73.22 −56.01 −50.60
– 35.03 −69.70 230.11 −95.38 100.06
0.008 0.013 0.006 0.029 0.114
0.112 0.102 0.03 0.027 0.016
0.245 0.322 0.016 0.171 0.010
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Fig 3. Variations of (a) nitrogen composition and COD (b), and PHA and glycogen in a typical cycle of SPND-SBR on Day 121.
anammox reaction (theoretical value is 7.6 ± 2.9 mg N/L as calculated by Eq. (1)). This may be due to the high DO concentrations (6–7 mg/L) in the synthetic wastewater resulting in a relatively high activity of nitrifying bacteria. During the steady period (Day 101–136), NLR further decreased to 0.456 kg N/(m3·d) after fed into the effluent in SPNDSBR at a suitable NO2−-N/NH4+-N ratio range. Influent NH4+-N and NO2−-N were completely removed within 2 h, whereas and the effluent NO3−-N gradually decreased to 7.6 ± 2 mg N/L, resulting in a high NRR of 0.406 kg N/(m3·d). Furthermore, average NRE increased from 52.1% in the start-up phase to 72.6% in the steady phase suggesting that enhanced nitrogen removal was achieved even with low influent substrate loading rate. The total effluent inorganic nitrogen was only 8.4 ± 1.1 mg N/L, which is below the threshold values prescribed by the discharge standards. The DO concentration in the effluent of ANA-UASB was only
is considered to contribute to the increase in particle size (Tian et al., 2011). The increase in the percentage of granular sludge makes conditions more favorable for SND in SPND-SBR (Mosquera-Corral et al., 2005). 3.2. Performance of ANA-UASB 3.2.1. Enhanced nitrogen removal with low influent substrates ANA-UASB was inoculated with anammox granular sludge for the start-up. As shown in Fig. 4, NLR of synthetic wastewater was gradually reduced from 0.925 to 0.555 kg N/(m3·d) to accelerate the acclimation of anammox with low influent substrates of SPND-SBR during the startup phase (Day 41–100). Influent NH4+-N and NO2−-N were completely removed, whereas effluent NO3−-N (30 ± 8.5 mg N/L) was significantly higher than that could have theoretically produced by
Fig 4. Nitrogen conversion profiles, NRE and average NLR (red dotted line) in ANA-UASB. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 6
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Proteobacteria and Chloroflexi involved in heterotrophic denitrification and chemolithotrophic denitrification (Fernandez et al., 2008; Mao et al., 2013), and some Proteobacteria were confirmed as nitrogenfixation bacteria (Wen et al., 2016), suggesting a good denitrifying effect in the reactor in addition to more COD and nitrogen removal in ANA-UASB. Bacteroidetes, which is responsible for the degradation of organic carbon, decreased by nearly half in the system (11.40% in Sample 1 and 6.10% in Sample 2) (Xu et al., 2017), suggesting limited biodegradable COD in wastewater pretreated by SPND-SBR. Additionally, the relative abundance of Nitrospira decreased from 5.18% (Sample 1) to 3.59% (Sample 2), indicating that competing nitrite of NOB decreased due to the increased activity and abundance of anammox bacteria and the oxygen-poor environment in the domestic wastewater pre-treated by SPND-SBR. Fig. 5b shows the bacterial taxonomic identification in genus level. Nitrospira, which is a NOB, dropped from 5.13% to 3.59% and Candidatus-Nitrotoga, which can convert NO2−-N to NO3−-N (Kitzinger et al., 2018), decreased from 1.67% to 1.29%. Furthermore, NOB-Nitrobacter was not detected during the high-throughput sequencing analysis. The number of NOB significantly declined due to the low-DO environment in SPND-SBR, facilitating the two-stage system to keep good performance of low effluent TIN during the steady phase in ANA-UASB. For AnAOB, Candidatus Brocadia varied from 8.21% to 21.33% in domestic wastewater, which dominated anammox in the system. Candidatus Kuenenia increased from 4.01% to 6.41%, while SM1A02 decreased slightly from 1.59% to 1.34%, indicating a preference of AnAOB for low influent substrates due to more active reactions with nucleic acids, lipid and protein synthesis at low nitrogen strength (Guo et al., 2018). Additionally, Denitratisoma, which is responsible for degrading cellular compounds and metabolites of dead microbes, increased from 2.90% to 4.02% and was also a coexist genus in the anammox reactor (Cao et al.,
0.07 ± 0.05 mg/L during the steady period, indicating that a good anoxic environment could be maintained in the reactor. An average of 29.6 mg N/L of nitrogen loss occurred in ANA-UASB with 38 mg N/L of influent TIN during the long-term operation. However, the average COD loss in ANA-UASB was 4 mg/L, which contributed to nitrogen loss only 1.1 mg N/L, while 1 mg N/L NO3−-N was used for consuming 3.71 mg COD/L in denitrification (Ma et al., 2016). Therefore, 75% of nitrogen removal was achieved by anammox process, indicating anammox rather than denitrification was the main nitrogen removal pathway since most of biodegradable COD was removed by the SPNDSBR (Table 1).
3.2.2. Microbial acclimation with low influent substrates The effect of low influent substrates on performance of anammox reactor was studied using batch experiments and high-throughput sequencing analysis. The maximum specific activity of anammox bacteria was measured based on the nitrogen gas production rate. During the start-up phase (Day 41–100), the maximum specific activity of anammox bacteria (qAMX, MAX) domesticated in synthetic wastewater was 5.7 ± 0.2 mg N/(gVSS·h) (Figure could be seen in supplementary). In the steady phase, the pre-treated domestic wastewater was fed into ANA-UASB, and qAMX, MAX (Day 101–136) further increased to 6.6 ± 0.2 mg N/(gVSS·h). For high-throughput sequencing analysis, sludge samples were collected on Day 62 (start-up phase) and Day121 (steady phase). As evident in Fig. 5a, Planctomycetes (AnAOB) on Day 121 (Sample 2) increased from 17.52% to 35.53% compared with Day 62 (Sample 1), resulting in a high anammox activity and efficient nitrogen removal via anammox pathway in ANA-UASB. However, Proteobacteria in Sample 2 decreased, while Chloroflexi increased from 26.49% to 30.44%, leading to an overall decrease of these two phyla from 50.17% to 45.37%.
Fig 5. (a) Bacterial relative abundance of phylum and (b) taxonomic classification of the major genus based on the 16S rRNA gene sequences in ANA-UASB in Phase start-up (Day 62) and Phase IV (Day 121). Relative abundance was defined as the number of sequences affiliated with that phylum or genus divided by the total number of sequences per sample. The electron microscopic image in the circle is of anammox granular sludge treating domestic wastewater.
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Table 3 Comparisons of different processes on influent quality, HRT, nitrogen removal performance and DO. Reference
This study
Ding et al. (2018)
Li et al. (2018a,b)
Morales et al., 2016
Process Influent wastewater Influent NH4+-N (mg N/L) HRT(h)/aerobic HRT (h) DO concentrations (mg/L) NRE (%)
SPND-Anammox Domestic 71.4 10.1/6.7 0.1 88.2
SNAD (suspended sludge) Domestic 60–80 9/6 0.3 86.1
SND (suspended sludge) Synthetic 40 8/8 1.2 75.1
PN/A (granular biomass) Synthetic 74 6/6 1.2 70
2016). Overall, the bacterial community identified through Illumina Miseq sequencing is favorable for the enhanced nitrogen removal in ANA-UASB.
(Guo et al., 2016). In this model, an average of 71% ammonia was oxidized to nitrite and over 22% of theoretical energy consumption for aeration can be saved.
3.3. Nitrogen removal mechanism in the two-stage process
3.4. Prospect and practical application
ANA-UASB was successfully combined with SPND-SBR after successful partial nitritation in SPND-SBR. Table 1 shows influent and effluent COD and TIN in each reactor at different phases. The nitrogen and COD removal performance was enhanced despite the fluctuation in the inflow quality (NH4+-N: 71.4 ± 4.4 mg N/L and COD: 238.6 ± 53.3 mg/L). During the steady period (Day 101–136), about 99.16% of NH4+-N in domestic wastewater was removed by the novel two-stage process. The NRE was generally above 88.24%, while effluent TIN was less than 8.4 ± 1.1 mg N/L and ORE was over 86.91%. The nitrogen removal efficiency obtained in this study was higher than that of the SND or anammox system previously reported (Table 3). The consumption of nitrogen compounds in the two-stage system (Fig. 6) was investigated as per the typical cycle on Day 121 using calculation method reported by Ma et al. (2016) as follows: For partial nitritation, the NO2−-N/NH4+-N molar ratio was 1.2:1, and 1 mg N/L of NO2−-N was used to consume 2.3 mg COD/L in denitritation. For anammox process, the stoichiometric NH4+-N/NO2−-N molar ratio of 1:1.32 produced 0.26 mol NO3−-N, while 1 mg N/L NO3−-N was used for consuming 3.71 mg COD/L in denitrification. In a typical steady phase cycle, around 36% influent TIN was removed by denitritation after mixing and around 7% TIN was removed by denitrification, while about 57% TIN was removed by anammox pathway, indicating that denitritation at low-DO of 0.1 mg/L played a stronger role and anammox was a significant nitrogen removal pathway in this two-stage process coupled SPND with anammox. Furthermore, 3.43 mg of oxygen was consumed to oxidize every 1 mg of ammonia during partial nitrification process and is only 75% of total oxygen consumed during traditional nitrification process, i.e. 4.57 mg oxygen
3.4.1. Importance of applying pre-anaerobic segment Integration of pre-anaerobic segment can inhibit NOB by slowing down its metabolism and reduce the activity of NOB (Gilbert et al., 2014; Miao et al., 2016; Liu et al., 2017). In this study, NOB was continuously suppressed through a combination of HRT control, low-DO conditions and competitive inhibition of heterotrophic bacteria (Ma et al., 2015; Cao et al., 2017) as evident by the sharp decrease in the abundance of NOB including Nitrospira and Nitrobacter during the longterm operation. In addition, the activity of NOB was completely inhibited during the last phase. The pre-anaerobic stage can remove the residual nitrite from the previous cycle and store a certain amount of intracellular carbon source to improve the nitrogen removal performance consistent with previous findings (Liu et al., 2017; Miao et al., 2018; Zhang et al., 2018). Therefore, it can be concluded that the preanaerobic segment promotes the nitrogen removal and improves the stability of the system. 3.4.2. Characteristics and potential in full-scale application As evident in the findings of this study, enhanced nitrogen removal can be achieved in a low-DO two-stage process by coupling SPND with polished anammox. There are several advantages in the low-DO twostage process. Firstly, COD in domestic wastewater can be fully utilized for nitrogen removal through step-wise reduction of DO to 0.1 mg/L in SPND-SBR, leading to the reduction of energy use and costs in BNR facilities. The aerobic HRT of the process was only 6.7 h, which was significantly lower than that of the traditional BNR process. Secondly, the treatment load can exceed the load of traditional WWTPs with the average NLR and COD loading rate (OLR) reaching to 0.17 and 0.61 kg/
Fig 6. Model based evaluation of the two-stage process. 8
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(m3·d) respectively (Lotti et al., 2015). Domestic wastewater pretreated by SPND-SBR can maintain a relatively high anammox activity and abundance with the output level of TIN being only about 8.4 ± 1.1 mg N/L (Table 1), which is below the discharge standards. Finally, aeration energy consumptions and construction costs can be reduced using polished anammox tank after sedimentation, which has a practical significance for the in-situ upgrade and reconstruction of the existing WWTPs.
Water Res. 70, 38–51. Gilbert, E.M., Agrawal, S., Brunner, F., Schwartz, T., Horn, H., Lackner, S., 2014. Response of different nitrospira species to anoxic periods depends on operational DO. Environ. Sci. Technol. 48 (5), 2934–2941. Gu, S., Wang, S., Yang, Q., Yang, P., Peng, Y., 2012. Start up partial nitrification at low temperature with a real-time control strategy based on blower frequency and pH. Bioresour. Technol. 112, 34–41. Guo, Y., Peng, Y., Wang, B., Li, B., Zhao, M., 2016. Achieving simultaneous nitrogen removal of low C/N wastewater and external sludge reutilization in a sequencing batch reactor. Chem. Eng. J. 306, 925–932. Guo, Y., Zhao, Y., Zhu, T., Li, J., Feng, Y., Zhao, H., Liu, S., 2018. A metabolomic view of how low nitrogen strength favors anammox biomass yield and nitrogen removal capability. Water Res. 143, 387–398. Jin, R.C., Xing, B.S., Yu, J.J., Qin, T.Y., Chen, S.X., 2013. The importance of the substrate ratio in the operation of the Anammox process in upflow biofilter. Ecol. Eng. 53, 130–137. Kartal, B., Kuenen, J.G., Loosdrecht, M.C.M.V., 2010. Sewage treatment with anammox. Science 328, 702–703. Keene, N.A., Reusser, S.R., Scarborough, M.J., Grooms, A.L., Seib, M., Santo Domingo, J., Noguera, D.R., 2017. Pilot plant demonstration of stable and efficient high rate biological nutrient removal with low dissolved oxygen conditions. Water Res. 121, 72–85. Kitzinger, K., Koch, H., Lüchker, S., Sedlacek, C.J., Herbold, C., Schwarz, J., Daebeler, A., Mueller, A., Lukumbuzya, M., Romano, S., Leisch, N., Karst, M.S., Kirkegaard, R., Albertsen, M., Nielsen, H.P., Wagner, M., Daims, H., 2018. Characterization of the first “Candidatus Nitrotoga” isloated reveals metabolic versatility and seperate evolution of widespread nitrite-oxidizing bacteria. mBio 9 (4). Klamerth, N., Malato, S., Maldonado, M.I., Agüera, A., Fernández-alba, A.R., 2010. Application of photo-fenten as a tertiary treatment of emerging contaminants in municipal wastewater. Environ. Sci. Technol. 44 (5), 1792–1798. Laureni, M., Falås, P., Robin, O., Wick, A., Weissbrodt, D.G., Nielsen, J.L., Ternes, T.A., Morgenroth, E., Joss, A., 2016. Mainstream partial nitritation and anammox: longterm process stability and effluent quality at low temperatures. Water Res. 101, 628–639. Li, L., Dong, Y., Qian, G., Hu, X., Ye, L., 2018a. Performance and microbial community analysis of bio-electrocoagulation on simultaneous nitrification and denitrification in submerged membrane bioreactor at limited dissolved oxygen. Bioresour. Technol. 258, 168–176. Li, J., Li, J., Gao, R., Wang, M., Yang, L., Wang, X., Zhang, L., Peng, Y., 2018b. A critical review of one-stage anammox processes for treating industrial wastewater: optimization strategies based on key functional microorganisms. Bioresour. Technol. 265, 498–505. Li, J., Peng, Y., Zhang, L., Liu, J., Wang, X., Gao, R., Pang, L., Zhou, Y., 2019. Quantify the contribution of anammox for enhanced nitrogen removal through metagenomic analysis and mass balance in an anoxic moving bed biofilm reactor. Water Res. 160, 178–187. Liu, J., Yuan, Y., Li, B., Zhang, Q., Wu, L., Li, X., Peng, Y., 2017. Enhanced nitrogen and phosphorus removal from municipal wastewater in an anaerobic-aerobic-anoxic sequencing batch reactor with sludge fermentation products as carbon source. Bioresour. Technol. 244, 1158–1165. Lotti, T., Kleerebezem, R., Hu, Z., Kartal, B., de Kreuk, M.K., van Erp Taalman Kip, C., Kruit, J., Hendrickx, T.L., van Loosdrecht, M.C., 2015. Pilot-scale evaluation of anammox-based mainstream nitrogen removal from municipal wastewater. Environ. Technol. 36 (9-12), 1167–1177. Ma, B., Bao, P., Wei, Y., Zhu, G., Yuan, Z., Peng, Y., 2015. Suppressing nitrite-oxidizing bacteria growth to achieve nitrogen removal from domestic wastewater via anammox using intermittent aeration with low dissolved oxygen. Sci. Rep. 5, 13048. Ma, B., Wang, S., Cao, S., Miao, Y., Jia, F., Du, R., Peng, Y., 2016. Biological nitrogen removal from sewage via anammox: recent advances. Bioresour. Technol. 200, 981–990. Mao, Y., Xia, Y., Zhang, T., 2013. Characterization of Thauera-dominated hydrogenoxidizing autotrophic denitrifying microbial communities by using high-throughput sequencing. Bioresour. Technol. 128, 703–710. Miao, Y., Zhang, L., Yang, Y., Peng, Y., Li, B., Wang, S., Zhang, Q., 2016. Start-up of single-stage partial nitrification-anammox process treating low-strength swage and its restoration from nitrate accumulation. Bioresour. Technol. 218, 771–779. Miao, Y., Peng, Y., Zhang, L., Li, B., Li, X., Wu, L., Wang, S., 2018. Partial nitrificationanammox (PNA) treating sewage with intermittent aeration mode: effect of influent C/N ratios. Chem. Eng. J. 334, 664–672. Morales, N., Río, Á., Vázquez-Padín, J.R., Méndez, R., Campos, J.L., Mosquera-Corral, A., 2016. The granular biomass properties and the acclimation period affect the partial nitritation/anammox process stability at a low temperature and ammonium concentration. Process Biochem. 51, 2134–2142. Mosquera-Corral, A., de Kreuk, M.K., Heijnen, J.J., van Loosdrecht, M.C., 2005. Effects of oxygen concentration on N-removal in an aerobic granular sludge reactor. Water Res. 39 (12), 2676–2686. Oehmen, A., Zeng, R.J., Yuan, Z., Keller, J., 2005. Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems. Biotechnol. Bioeng. 91 (1), 43–53. Park, H.D., Noguera, D.R., 2004. Evaluating the effect of dissolved oxygen on ammoniaoxidizing bacterial communities in activated sludge. Water Res. 38 (14–15), 3275–3286. Siegrist, H., Salzgeber, D., Eugster, J., Joss, A., 2008. Anammox brings WWTP closer to energy autarky due to increased biogas production and reduced aeration energy for N-removal. Water Sci. Technol. 57 (3), 383–388. Strous, M., Heijnen, J.J., Kuenen, J.G., Jetten, M.S.M., 1998. The sequencing batch
4. Conclusions An innovative low-DO two-stage process was established and a high TIN removal efficiency of 88.24% was achieved using the step-wise DO reductions to 0.1 mg/L. High-rate partial nitritation with a suitable effluent NO2−-N/NH4+-N ratio of around 1.04 was maintained in SPND-SBR by controlling DO concentration and aeration time, ensuring ANA-UASB had a significant nitrogen removal with high anammox activity and abundance of 6.6 ± 0.2 mg N/(gVSS·h) and 35.53%, respectively. The study investigated the feasibility and mechanism of advanced nitrogen removal by coupling partial nitritation and denitritation with polished anammox to treat low C/N (3.36) domestic wastewater. CRediT authorship contribution statement Wen Zhang: Conceptualization, Formal analysis, Investigation, Data curation, Writing - original draft. Yongzhen Peng: Supervision, Funding acquisition, Writing - review & editing. Liang Zhang: Validation, Writing - review & editing. Xiyao Li: Resources. Qiong Zhang: Methodology. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This research was financially supported by Municipal Science and Technology Project of Beijing, China (Z181100005518006), National Natural Science Foundation of China (21777005, 51978007), 111 Project (D16003) and the Funding Projects of Beijing Municipal Commission of Education, China. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2020.123045. References APHA, 1998. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC, USA. Cao, S., Du, R., Li, B., Ren, N., Peng, Y., 2016. High-throughput profiling of microbial community structures in an ANAMMOX-UASB reactor treating high-strength wastewater. Appl. Microbiol. Biotechnol. 100 (14), 6457–6467. Cao, Y., van Loosdrecht, M.C., Daigger, G.T., 2017. Mainstream partial nitritation-anammox in municipal wastewater treatment: status, bottlenecks, and further studies. Appl. Microbiol. Biotechnol. 101 (4), 1365–1383. Daigger, G.T., Littleton, H.X., 2014. Simultaneous biological nutrient removal: a state-ofthe-art review. Water Environ. Res. 86 (3), 245–257. Ding, S., Bao, P., Wang, Bo, Zhang, Q., Peng, Y., 2018. Long-term stale simultaneous partial nitrification, anammox and denitrification (SNAD) process treating real domestic sewage using suspended activated sludge. Chem. Eng. J. 339, 180–188. Fernandez, N., Sierra-Alvarez, R., Field, J.A., Amils, R., Sanz, J.L., 2008. Microbial community dynamics in a chemolithotrophic denitrification reactor inoculated with methanogenic granular sludge. Chemosphere 70 (3), 462–474. Fitzgerald, C.M., Camejo, P., Oshlag, J.Z., Noguera, D.R., 2015. Ammonia-oxidizing microbial communities in reactors with efficient nitrification at low-dissolved oxygen.
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microaerophilic sulfate and nitrate co-reduction system. Chem. Eng. J. 330, 63–70. Yan, L., Liu, S., Liu, Q., Zhang, M., Liu, Y., Wen, Y., Chen, Z., Zhang, Y., Yang, Q., 2019. Improved performance of simultaneous nitrification and denitrification via nitrite in an oxygen-limited SBR by alternating the DO. Bioresour. Technol. 275, 153–162. Yuan, Q., Oleszkiewicz, J.A., 2011. Low temperature biological phosphorus removal and partial nitrification in a pilot sequencing batch reactor system. Water Sci. Technol. 63 (12), 2802–2807. Zeng, R.J., van Loosdrecht, M.C.M., Yuan, Z., Keller, J., 2003. Metabolic model for glycogen-accumulating organisms in anaerobic/aerobic activated sludge systems. Biotechnol. Bioeng. 81 (1), 92–105. Zhang, J., Zhang, L., Miao, Y., Sun, Y., Li, X., Zhang, Q., Peng, Y., 2018. Feasibility of in situ enriching anammox bacteria in a sequencing batch biofilm reactor (SBBR) for enhancing nitrogen removal of real domestic wastewater. Chem. Eng. J. 352, 847–854.
reactor as a powerful tool for the study of slowly growing anaerobic ammoniumoxidizing microorganisms. Appl. Microbiol. Biotechnol. 50, 589–596. Tian, W.D., Li, W.G., Zhang, H., Kang, X.R., van Loosdrecht, M.C., 2011. Limited filamentous bulking in order to enhance integrated nutrient removal and effluent quality. Water Res. 45 (16), 4877–4884. Vlaeminck, S.E., Terada, A., Smets, B.F., Linden, D.V.d., Boon, N., Verstraete, W., Carballa, M., 2009. Nitrogen removal from digested black water by one-stage partial nitritation and anammox. Environ. Sci. Technol. 43 (13), 5035–5041. Wen, X., Zhou, J., Li, Y., Qing, X., He, Q., 2016. A novel process combining simultaneous partial nitrification, anammox and denitrification (SNAD) with denitrifying phosphorus removal (DPR) to treat sewage. Bioresour. Technol. 222, 309–316. WPO, 1998. Water Protection Ordinance of 28 October 1998 (814.201). UNECE, Geneva. Xu, X.J., Chen, C., Guan, X., Yuan, Y., Wang, A.-J., Lee, D.J., Zhang, Z.F., Zhang, J., Zhong, Y.J., Ren, N.Q., 2017. Performance and microbial community analysis of a
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Simultaneous partial nitritation and denitritation coupled with polished anammox for advanced nitrogen removal from low C/N domestic wastewater at low dissolved oxygen conditions
T
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Wen Zhang, Yongzhen Peng , Liang Zhang, Xiyao Li, Qiong Zhang National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, Engineering Research Center of Beijing, Beijing University of Technology, Beijing 100124, PR China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Nitrogen removal Partial nitritation Denitritation Anammox Low dissolved oxygen
Simultaneous partial nitritation and denitritation (SPND) coupled with anammox was established in this study to treat domestic wastewater. Two lab-scale bioreactors, namely SPND-SBR and ANA-UASB, were used in the twostage system. In SPND-SBR, stable nitrogen removal efficiency of 51.1% was achieved with a high ammonia oxidation rate of 0.117 kg N/(m3·d). Besides, successful out-selection of nitrite-oxidizing bacteria (NOB) under low-DO of 0.1 mg/L during the steady period, resulting in an average effluent NO2−-N/NH4+-N ratio of 1.04. In ANA-UASB, the abundance of Candidatus Brocadia and Candidatus Kuenenia increased from 8.21% and 4.01% to 21.33% and 6.41% with low influent substrate contents of only 38 mg N/L. The effluent total inorganic nitrogen (TIN) was only 8.4 ± 1.1 mg N/L and the nitrogen removal efficiency reached 88.24%. Overall, the study demonstrated that the novel low-DO two-stage process for nitrogen removal is a promising technique for wastewater of low C/N ratio.
1. Introduction Inorganic nitrogen is generally removed through high-rate biological nutrient removal (BNR) process to prevent eutrophication in receiving waters. In the traditional high-rate BNR processes, most
⁎
wastewater treatment plants (WWTPs) generally control the dissolved oxygen (DO) above 2 mg/L for a high efficient and stable nitrification, though the energy consumption due to aeration accounts for nearly half of the total power consumptions of WWTPs (Keene et al. 2017). Thus, energy-efficient aeration has received significant research attention.
Corresponding author. E-mail address: [email protected] (Y. Peng).
https://doi.org/10.1016/j.biortech.2020.123045 Received 22 December 2019; Received in revised form 15 February 2020; Accepted 18 February 2020 Available online 19 February 2020 0960-8524/ © 2020 Elsevier Ltd. All rights reserved.
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Fig 1. Schematic diagram of the novel two-stage process treating domestic sewage.
external carbon source, to produce nitrogen gas (Eq. (1)) (Strous et al., 1998; Jin et al., 2013). Partial nitritation is a critical pre-requisite step before the anammox process as the former provides nitrite to anammox reactor (Vlaeminck et al., 2009; Kartal et al., 2010). The process requires only converting 55% of NH4+-N to NO2−-N in a nitritation reactor and saves about 60% aeration energy compared with the traditional nitrification and denitrification BNR process (Siegrist et al., 2008).
Previous studies have shown that efficient nitrogen removal can be achieved with low-DO conditions, for example, simultaneous nitrification and denitrification (SND) occurred in the prolonged aeration system in the low-DO aerobic zones (Daigger and Littleton 2014). Similarly, Keene et al. (2017) observed a higher nitrogen removal in the full-scale plant due to denitrification improvements in the low-DO tanks via SND in a continuous flow pilot-scale reactor. Further, the bioelectrocoagulation system also benefited from SND performance in submerged membrane bioreactor (MBR) with limited dissolved oxygen (Li et al., 2018a,b). Yan et al. (2019) found that a lab-scale modified sequencing batch reactor (SBR) was efficient for nitrogen removal under limited DO conditions by SND via nitrite pathway. Besides, several studies confirmed that the activity of nitrite-oxidizing bacteria (NOB) (Laureni et al. 2016; Li et al., 2018a,b) was suppressed at low oxygen concentrations of 0.15–0.18 mg/L, leading to the short-cut nitrogen removal with lower demand for carbon sources. Therefore, efficient application of low-DO for the domestic wastewater for the total nitrogen removal could save the operational cost of BNR facilities (Park and Noguera, 2004; Fitzgerald et al., 2015). However, most SND processes have long hydraulic retention times (HRT) due to low ammonia oxidation rate at low-DO levels, leading to difficulty in improving volumetric process rates. The residual ammonium in the effluent increases with the sudden increase in the nitrogen loading rate, resulting in poor nitrogen removal and consequently low effluent quality not suitable for release as per stringent discharge standards for ammonia (< 2 mg N/L) (e.g. Switzerland, WPO (1998)). Moreover, the effluent contains a large amount of nitrate or nitrite due to inadequate biodegradable carbon source in domestic wastewater and insufficient denitrification in the SND process. Thus, advanced treatment techniques are necessary for secondary sewage effluents (SSE). However, the capital and operational costs are significantly higher than those of secondary treatment facilities with the same scale (Klamerth et al., 2010). In this context, the anaerobic ammonia oxidation (anammox) process provides a new approach to low-DO process. Anammox is a novel process, in which ammonium is oxidized by nitrite serving as the electron acceptor under anaerobic conditions with low aeration and no
NH+4 + 1.32 NO−2 + 0.066 HCO−3 + 0.13 H + → 1.02 N2 + 0.26 NO−3 + 0.066CH2 O0.5 N0.15 + 2.03H2 O
(1)
Thus, the effluent of low-DO process can provide the influent to the anammox zone with a suitable NO2−-N/NH4+-N ratio, leading to an improved shock resistance capacity, reduced aeration energy consumption, and a good effluent quality. Further, the polishing step can become efficient and cost-effective with the utilization of independent anammox zone followed by secondary clarifiers. However, the studies investigating the collaboration of low-DO process coupled with anammox are still limited. The objective of the present work was to investigate the nitrogen removal performance of a coupling process based on simultaneous partial nitritation, denitritation (SPND) and polished anammox for the treatment of domestic wastewater with low-DO conditions (e.g. 0.1 mg/ L) and their reliability in complying with the nitrogen discharge standards. Furthermore, the overall performance, nitrogen removal efficiencies and rates and effluent qualities (nitrogen species and COD) of this novel two-stage process were assessed along with the relative abundance, maximum specific activities and distribution of the main functional bacteria. Finally, the effect of pre-anaerobic segment on suppressing NOB was discussed and nitrogen removal potential of the full-scale mainstream application of this two-stage process were compared to the conventional systems. 2. Materials and methods 2.1. Experimental setup The schematic diagram for the two-stage process is shown in Fig. 1. 2
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The first sequence batch reactor (SBR) was used for partial nitritation and denitritation termed as SPND-SBR, while the up-flow anaerobic sludge blanket (UASB) used anammox to treat wastewater pretreated by SPND-SBR and terming as ANA-UASB. SPND-SBR and ANA-UASB were made up of polymethyl methacrylate (PMMA) and had a working volume of 10 L and 2 L, respectively. SPND-SBR was equipped with a mechanical stirrer (IKA RW20, Germany). An air-compressor (HAILEA V-20, China) was used for blast aeration with adhesion sand lump as the microporous aerator, whereas a rotameter was used to control the flow of gas and consequently the DO level in the system. ANA-UASB was wrapped with a black sponge cloth to shield the anammox granules from light. During the experiment, the temperature was not controlled and maintained as ambient.
Milwaukee, USA). Cycle measurements were performed every 2–3 days to monitor nitrogen loss and nitrite accumulation ratio (NAR) during the aerobic stage. The DO, pH and temperature were monitored using a pH/Oxi 3420i analyzer (WTW Company, Germany), while COD was analyzed using COD quick-analysis apparatus (Lian-hua Tech. Co., Ltd., 5B-1, China) and the MLSS and MLVSS were analyzed according to the standard methods (APHA, 1998). The sludge particle size distribution was measured using a Laser Particle Size Analyzer (Microtrac S3500, USA) over the 0.01–2000 μm size range. Frozen dried biomass was used to measure polyhydroxyalkanoates (PHAs) and glycogen (Gly). PHAs were determined as the sum of poly-β-hydroxybutyrate (PHB) and polyβ-hydroxyvalerate (PHV) that were measured as reported by (Oehmen et al., 2005) using the gas chromatograph (Agilent 6890 N, U.S.) with Agilent DB-1 chromatographic column. Gly was analyzed as described by (Zeng et al., 2003).
2.2. Influent and seed sludge Domestic wastewater was obtained from a septic tank at the Beijing University of Technology and stored in a wastewater tank before feeding into the SPND-SBR. The soluble COD, ammonium, nitrite, nitrate, C/N ratio and pH in sewage were 238.6 ± 53.5 mg/L, 71.4 ± 4.4 mg N/L, 0.2 ± 0.1 mg N/L, 0.3 ± 0.1 mg N/L, 3.4 ± 0.8 and 7.1–7.5, respectively. The seed sludge for SPND-SBR was ordinary nitrification sludge taken from a traditional anaerobic/anoxic/oxic activated sludge system. The anammox granular seed sludge was inoculated from side stream for treating anaerobic digestive juice. After inoculation, the mixed liquid suspended solids (MLSS) in the SPND-SBR and ANA-UASB were about 3623 and 3462 mg/L, while the mixed liquor volatile suspended solids (MLVSS) were about 3025 and 2770 mg/L, respectively.
2.4.2. Bacterial activity measurements The maximum specific activities (mg N/(gVSS·h)) of AOB and NOB were measured using the ectopic activity tests. Briefly, 500 mL sludge was extracted from SPND-SBR at the end of the aeration stage, washed thrice by distilled water and then injected into a 0.5 L wild-mouth bottle to simulate the MLSS of the mother reactor. AOB and NOB activities were tested separately under the saturated DO conditions of 7 ± 0.5 mg/L. The maximum specific activities of AnAOB were measured in the absence of DO (< 0.02 mg/L) using the anaerobic respirator (Challenge AER-208 Research Respirometer, USA). The anoxic stirring was used to remove the residual COD in the reactor before the test to avoid any disturbance to COD and to improve the accuracy. The specific experimental conditions and calculations are listed in supplementary.
2.3. Operational strategy 2.4.3. Calculations methods NAR and SND, which are indicators of the nitrite accumulation and nitrogen loss, were calculated using Eq. (2), Eq. (3):
SPND-SBR was operated in an anaerobic/aerobic mode with a short aeration time (240 min) and a low aeration rate (10–12 L/h) to achieve a suitable effluent NO2-N/NH4+-N ratio and to efficiently utilize COD to remove nitrogen. A typical cycle was operated for 6 h, comprising water intake for 5 min, oxygen deprivation for 50 min, aeration for 240 min, static settling for 60 min and water discharge for 5 min. The exchange volumetric ratio was controlled at 60% to fully utilize COD in domestic wastewater. DO was controlled at 0.1–0.8 mg/L during the aeration phase by adjusting the air flow rate. The long-term operation of SPNDSBR was divided into four phases (Phase I, II, III and IV) with the average DO in each phase being controlled at 0.8, 0.5, 0.3 and 0.1 mg/ L. HRT was maintained at 8.1 h and the sludge retention time (SRT) was kept as 30–40 d. The ANA-UASB was run in a continuous mode, where wastewater was continuously pumped into ANA-UASB at the bottom, while the upflow rate was controlled at 160 mL/min by adjusting the recirculation ratio to maintain a good fluidization condition. The ANA-UASB operation was divided into two phases. In the first phase, the reactor was started up with synthetic wastewater by gradually reducing nitrogen loading rate (NLR), while the pretreated domestic wastewater with a suitable NO2−-N/NH4+-N range was pumped into the reactor during the steady phase. HRT was generally maintained at 2 h. Non-active sludge discharge was adopted for the entire operation to ensure the growth and enrichment of anammox bacteria (AnAOB). The specific operational conditions in the two-stage system in different phases are listed in Table 1.
NO−2 − Nend ⎞ NAR = ⎛⎜ ⎟ × 100\% − − ⎝ NO2 − Nend + NO3 − Nend ⎠
(2)
NO−2 − Nend + NO−3 − Nend ⎞ SND = ⎜⎛1− ⎟ × 100\% + + ⎝ NH 4 − Nini − NH 4 − Nend ⎠
(3)
where NO2−-Nend and NO3−-Nend are the NO2−-N and NO3−-N concentrations at the end of the aerobic phase (mg N/L), respectively, while NH4+-Nini and NH4+-Nend are the NH4+-N concentrations at the beginning and end of the aerobic stage (mg N/L), respectively. CODabs, which is the indicator of the COD concentrations stored as intracellular carbon source, was calculated using Eq. (4):
CODabs (mg COD/L) = ΔCOD − (1.71ΔNO−2 − N+ 2.86ΔNO−3 −N)
(4)
where ΔCOD, ΔNO2−-N and ΔNO3−-N are the changes in COD, NO2−-N and NO3−-N concentrations in the anoxic stage of SPND-SBR (mg N/L), respectively whereas 1.71 and 2.86 are the COD concentrations required for heterotrophic denitrification of nitrite and nitrate in the anaerobic stage (mg/L), respectively. 2.4.4. Microbial diversity analysis The microbial community structure in the system was analyzed using quantitative real-time PCR (qPCR) and high-throughput sequencing data analysis. For qPCR, the abundance of AOB, Nitrospira, Nitrobacter and total bacteria were determined as described by Li et al. (2019), whereas a mixture of the amplicons was used for highthroughput sequencing on Miseq PE300 platform (Illimina, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using the standard protocols. Taxonomic classification was conducted to optimize the sequences into operational taxonomic units (OTUs) using 97% identity thresholds (setting a 0.03 distance limit) using the MOTHUR
2.4. Analytical methods 2.4.1. Conventional analysis The wastewater samples were filtered through 0.45 μm filters prior to the analysis. The concentrations of NH4+-N, NO2−-N and NO3−-N were measured using a Lachat QuikChem8000 Flow Injection Analyzer (Lachat Instruments, 3
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Table 1 Variations in the nitrogen and COD removal performance of the novel two-stage system in the four phases over a 136-day period. Phases
Time(d)
DO(mg/L)
Municipal Wastewater (influent) (mg/L) COD(mg/L)
C/N
73.0 68.9 70.0 72.4
245.2 184.4 254.5 253.8
3.7 2.7 3.7 3.3
I II III IV
1–43 44–76 77–100 101–136
0.8 0.5 0.3 0.1
Phases
Time(d)
ANA-UASB influent(mg/L)
Start-up IV
41–100 101–136
± ± ± ±
4.7 3.6 3.8 4.4
−
± ± ± ±
35.7 44.2 30.6 59.6
± ± ± ±
0.5 0.6 0.5 0.9
NH4+-N(mg-N/L)
NO2−-N(mg-N/L)
NO3−-N(mg-N/L)
COD(mg/L)
1.0 ± 1.1 13.7 ± 12.4 23.6 ± 7.6 17.8 ± 7.8
2.6 ± 4.3 14.1 ± 4.9 10.1 ± 6.0 16.7 ± 3.8
36.7 ± 8.0 9.5 ± 5.4 5.4 ± 5.1 0.7 ± 0.9
40.5 66.0 58.6 47.9
± ± ± ±
13.1 12.4 14.7 12.3
ANA-UASB effluent(mg/L)
NH4 -N(mg-N/L)
NO2 -N(mg-N/L)
NO3−-N(mg-N/L)
30.5 ± 10.4 19.4 ± 6.2
35.9 ± 10.0 17.6 ± 5.8
5.3 ± 3.8 1.0 ± 1.0
+
SPND-SBR effluent(mg/L)
NH4+-N(mg-N/L)
COD(mg/L)
NH4+-N(mg-N/L)
NO2−-N(mg-N/L)
NO3−-N(mg-N/L)
COD(mg/L)
41.1 ± 7.3 37.2 ± 7.8
1.4 ± 5.0 0.6 ± 1.3
1.0 ± 1.7 0.2 ± 0.1
29.9 ± 8.5 7.6 ± 2.0
40.8 ± 7.3 33.2 ± 7.5
inhibited in Phase III (Day 77–100) due to the temperature decrease in winter. qAOB, MAX increased sharply to 14.5 ± 0.4 mg N/(gVSS·h) in Phase IV (Day 101–136), whereas qNOB, MAX was not detected when DO was at 0.1 mg/L. These findings indicate that adopting step-wise DO reductions strategy was effective to out-select NOB. Effluent NH4+-N and NO2− -N were gradually controlled at 17.8 ± 7.7 mg N/L and 16.7 ± 3.8 mg N/L, respectively, by adjusting aeration time to 240 min at low-DO of 0.1 mg/L during the steady period (Day 101–136). Notably, the average NH4+-N conversion rate of 0.117 kg N/(m3·d) at low-DO of 0.1 mg/L is higher than that reported in previous studies (0.05–0.10 kg N/(m3·d)) conducted under higher DO (e.g. 2–3 mg/L) (Yuan and Oleszkiewicz, 2011; Gu et al., 2012). The findings indicate that the biomass gradually adapted to low oxygen level in domestic wastewater after several phases and a high-rate partial nitritation with the average effluent NO2−-N/NH4+-N ratio of around 1.04, which is close to the stoichiometric value of 1.32, was achieved in SPND-SBR (Strous et al., 1998; Jin et al., 2013).
software program. 3. Results and discussion 3.1. Performance of the mainstream SPND-SBR during long-term operation 3.1.1. COD and nitrogen removal by step-wise DO reductions SPND-SBR had an average influent COD to NH4+-N ratio of 3.36 (Fig. 2) and ran for 136 days divided into 4 phases according to the DO concentration of the aerobic stage (Table 1). The variations in influent and effluent nitrogen species are presented in Fig. 2, along with the NH4+-N and COD removal efficiencies. In the four phases, the average influent COD was 238.6 mg/L and total ammonium nitrogen (TAN) was 71.4 mg N/L. With gradual decrease in DO from 0.8 to 0.1 mg/L, the respective average nitrogen removal efficiency (NRE) and COD removal efficiency (ORE) of SPND-SBR increased from 42.83% and 64.2% to 51.11% and 81.13%, resulting in a relatively high nitrogen removal rate (NRR) of 0.089 kg N/(m3·d) and COD removal rate (ORR) of 0.494 kg/(m3·d). Therefore, SPND-SBR could efficiently remove COD and nitrogen in the domestic wastewater even when DO is reduced to 0.1 mg/L. Moreover, average ORE of SPND-SBR increased by 42.37% with the decrease of DO, indicating that heterotrophic bacteria better utilized biodegradable organic matter in the reactor under lower DO conditions.
3.1.3. Performance of SPND in SPND-SBR The average NRE and ORE of 51.1% and 81.1%, respectively, were achieved in SPND-SBR at low-DO of 0.1 mg/L and was maintained during the steady period (Day 101–136) (Fig. 2). Hence, a typical cycle on Day 121 was selected to analyze the transformation of nitrogen and COD in anaerobic/aerobic mode (anaerobic 50 min/aerobic 240 min). As shown in Fig. 3, COD decreased from 122.2 to 95.3 mg/L in the anaerobic stage (0–50 min), resulting in CODabs of 40.9 mg/L using Eq. (4). At the end of the anaerobic stage, the mixture contained 43.7 mg N/L of NH4+-N and 95.3 mg/L. During the aerobic stage, remaining soluble COD was further oxidized and decreased to 54.5 mg/L, while 31.4 mg N/L of NH4+-N was oxidized to NO2−-N (15 mg N/L) and NO3−-N (0.7 mg N/L) with the residual NH4+-N of 12.3 mg N/L. The total oxidized nitrogen (nitrite plus nitrate) was lower than the amount of NH4+-N oxidized, suggesting the nitrogen loss in the aerobic stage due to denitritation improvements in the low-DO aerobic stage via SND (Liu et al., 2017). Consequently, the discrepancy in TIN was used as a proxy for SND to estimate the degree of SND (Fig. 2c). During the 136-day operation, the average SND increased from 11.7% to 44.4% (Fig. 2c) as per Eq. (3) indicating improved nitrogen removal in SPNDSBR during the long-term operation. However, PHA and Gly decreased by 9.6 and 29.3 mg COD/gVSS in the aerobic stage, respectively, suggesting that endogenous denitritation driven by PHA or Gly contributed to the improved nitrogen removal during the aerobic stage of SPNDSBR. The particle size is critical for the micro-environment formation in activated sludge (Mosquera-Corral et al., 2005). With the decrease of DO during the long-term operation, the average sludge particle size characterized by volume (Mv) increased from 61.5 to 161.5 μm, while the percentage of granular sludge (particle size > 200 μm) increased from none to 31% (Figure could be seen in supplementary). This may be due to limited filamentous bulking under low-DO circumstances, which
3.1.2. High-rate partial nitritation at low-DO Concentrations of nitrogen species in the effluent of SPND-SBR at low DO were investigated. The temporal variation in the ammonium transformation performance and NAR in SPND-SBR are presented in Fig. 2c. NO2−-N was only 2.6 ± 4.3 mg N/L, while NO3−-N reached to 36.7 ± 7.9 mg N/L, resulting in a NAR of about 6.44% as per Eq. (2) after nitrification sludge inoculation in SPND-SBR. With the decrease of DO, the concentrations of NO2−-N increased continuously, while NO3−-N decreased from the Phase I to III (Day 1–100). In the steady period (Day 101–136), NO3−-N was less than 1.5 mg N/L, whereas NO2−-N reached to 16.7 ± 3.8 mg N/L. A high NAR (96.2 ± 4.3%) was constantly maintained, indicating the successful out-selection of NOB at the low DO of 0.1 mg/L. Meanwhile, the changes in the activity and abundance of main functional bacteria also confirmed the successful out-selection of NOB during the step-wise DO reductions. As evident in Table 2, the overall growth rate of AOB increased by 600.73%, whereas that of Nitrobacter decreased by 50.60%. The relative abundance of AOB increased from 0.008% to 0.114%, whereas that of Nitrospira decreased from 0.245% to 0.01%. Meanwhile, in Phase I (Day 1–43), qNOB, MAX (3.4 ± 0.3 mg N/ (gVSS·h)) in the reactor was higher than qAOB, MAX (2.2 ± 0.2 mg N/ (gVSS·h)) (Figure could be seen in supplementary). In Phase II (Day 44–76), DO was further reduced to 0.5 mg/L and consequently qAOB, MAX(5.8 ± 0.3 mg N/(gVSS·h)) was significantly higher than qNOB, MAX (1.5 ± 0.4 mg N/(gVSS·h)). The activities of AOB and NOB were 4
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Fig 2. Removal performance and nitrogen profiles of SPND-SBR (a) TIN removal efficiency (b) COD removal efficiency and (c) Nitrogen conversion, NAR and SND during operation.
Table 2 Variations in bacterial abundance, relative abundance and growth rate during the long-term operation of SPND-SBR. Time (d)
Gene copy number (copies/g dry sludge) AOB
0 21 62 89 125
1.16 1.98 5.62 8.11 2.45
Nitrobacter × × × × ×
8
10 108 108 108 109
1.63 1.53 2.82 7.56 3.33
× × × × ×
Growth rate (%)
Nitrospira 9
10 109 109 108 108
TB 9
3.55 × 10 1.45 4.79 × 109 1.49 1.45 × 109 9.32 4.79 × 109 2.81 2.21 × 108 2.14 Overall growth rate
× × × × ×
12
10 1012 1012 1012 1012
5
Relative abundance (%)
AOB
Nitrobacter
Nitrospira
AOB
Nitrobacter
Nitrospira
– 70.90 283.90 44.20 201.73 600.73
– −6.29 84.92 −73.22 −56.01 −50.60
– 35.03 −69.70 230.11 −95.38 100.06
0.008 0.013 0.006 0.029 0.114
0.112 0.102 0.03 0.027 0.016
0.245 0.322 0.016 0.171 0.010
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Fig 3. Variations of (a) nitrogen composition and COD (b), and PHA and glycogen in a typical cycle of SPND-SBR on Day 121.
anammox reaction (theoretical value is 7.6 ± 2.9 mg N/L as calculated by Eq. (1)). This may be due to the high DO concentrations (6–7 mg/L) in the synthetic wastewater resulting in a relatively high activity of nitrifying bacteria. During the steady period (Day 101–136), NLR further decreased to 0.456 kg N/(m3·d) after fed into the effluent in SPNDSBR at a suitable NO2−-N/NH4+-N ratio range. Influent NH4+-N and NO2−-N were completely removed within 2 h, whereas and the effluent NO3−-N gradually decreased to 7.6 ± 2 mg N/L, resulting in a high NRR of 0.406 kg N/(m3·d). Furthermore, average NRE increased from 52.1% in the start-up phase to 72.6% in the steady phase suggesting that enhanced nitrogen removal was achieved even with low influent substrate loading rate. The total effluent inorganic nitrogen was only 8.4 ± 1.1 mg N/L, which is below the threshold values prescribed by the discharge standards. The DO concentration in the effluent of ANA-UASB was only
is considered to contribute to the increase in particle size (Tian et al., 2011). The increase in the percentage of granular sludge makes conditions more favorable for SND in SPND-SBR (Mosquera-Corral et al., 2005). 3.2. Performance of ANA-UASB 3.2.1. Enhanced nitrogen removal with low influent substrates ANA-UASB was inoculated with anammox granular sludge for the start-up. As shown in Fig. 4, NLR of synthetic wastewater was gradually reduced from 0.925 to 0.555 kg N/(m3·d) to accelerate the acclimation of anammox with low influent substrates of SPND-SBR during the startup phase (Day 41–100). Influent NH4+-N and NO2−-N were completely removed, whereas effluent NO3−-N (30 ± 8.5 mg N/L) was significantly higher than that could have theoretically produced by
Fig 4. Nitrogen conversion profiles, NRE and average NLR (red dotted line) in ANA-UASB. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 6
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Proteobacteria and Chloroflexi involved in heterotrophic denitrification and chemolithotrophic denitrification (Fernandez et al., 2008; Mao et al., 2013), and some Proteobacteria were confirmed as nitrogenfixation bacteria (Wen et al., 2016), suggesting a good denitrifying effect in the reactor in addition to more COD and nitrogen removal in ANA-UASB. Bacteroidetes, which is responsible for the degradation of organic carbon, decreased by nearly half in the system (11.40% in Sample 1 and 6.10% in Sample 2) (Xu et al., 2017), suggesting limited biodegradable COD in wastewater pretreated by SPND-SBR. Additionally, the relative abundance of Nitrospira decreased from 5.18% (Sample 1) to 3.59% (Sample 2), indicating that competing nitrite of NOB decreased due to the increased activity and abundance of anammox bacteria and the oxygen-poor environment in the domestic wastewater pre-treated by SPND-SBR. Fig. 5b shows the bacterial taxonomic identification in genus level. Nitrospira, which is a NOB, dropped from 5.13% to 3.59% and Candidatus-Nitrotoga, which can convert NO2−-N to NO3−-N (Kitzinger et al., 2018), decreased from 1.67% to 1.29%. Furthermore, NOB-Nitrobacter was not detected during the high-throughput sequencing analysis. The number of NOB significantly declined due to the low-DO environment in SPND-SBR, facilitating the two-stage system to keep good performance of low effluent TIN during the steady phase in ANA-UASB. For AnAOB, Candidatus Brocadia varied from 8.21% to 21.33% in domestic wastewater, which dominated anammox in the system. Candidatus Kuenenia increased from 4.01% to 6.41%, while SM1A02 decreased slightly from 1.59% to 1.34%, indicating a preference of AnAOB for low influent substrates due to more active reactions with nucleic acids, lipid and protein synthesis at low nitrogen strength (Guo et al., 2018). Additionally, Denitratisoma, which is responsible for degrading cellular compounds and metabolites of dead microbes, increased from 2.90% to 4.02% and was also a coexist genus in the anammox reactor (Cao et al.,
0.07 ± 0.05 mg/L during the steady period, indicating that a good anoxic environment could be maintained in the reactor. An average of 29.6 mg N/L of nitrogen loss occurred in ANA-UASB with 38 mg N/L of influent TIN during the long-term operation. However, the average COD loss in ANA-UASB was 4 mg/L, which contributed to nitrogen loss only 1.1 mg N/L, while 1 mg N/L NO3−-N was used for consuming 3.71 mg COD/L in denitrification (Ma et al., 2016). Therefore, 75% of nitrogen removal was achieved by anammox process, indicating anammox rather than denitrification was the main nitrogen removal pathway since most of biodegradable COD was removed by the SPNDSBR (Table 1).
3.2.2. Microbial acclimation with low influent substrates The effect of low influent substrates on performance of anammox reactor was studied using batch experiments and high-throughput sequencing analysis. The maximum specific activity of anammox bacteria was measured based on the nitrogen gas production rate. During the start-up phase (Day 41–100), the maximum specific activity of anammox bacteria (qAMX, MAX) domesticated in synthetic wastewater was 5.7 ± 0.2 mg N/(gVSS·h) (Figure could be seen in supplementary). In the steady phase, the pre-treated domestic wastewater was fed into ANA-UASB, and qAMX, MAX (Day 101–136) further increased to 6.6 ± 0.2 mg N/(gVSS·h). For high-throughput sequencing analysis, sludge samples were collected on Day 62 (start-up phase) and Day121 (steady phase). As evident in Fig. 5a, Planctomycetes (AnAOB) on Day 121 (Sample 2) increased from 17.52% to 35.53% compared with Day 62 (Sample 1), resulting in a high anammox activity and efficient nitrogen removal via anammox pathway in ANA-UASB. However, Proteobacteria in Sample 2 decreased, while Chloroflexi increased from 26.49% to 30.44%, leading to an overall decrease of these two phyla from 50.17% to 45.37%.
Fig 5. (a) Bacterial relative abundance of phylum and (b) taxonomic classification of the major genus based on the 16S rRNA gene sequences in ANA-UASB in Phase start-up (Day 62) and Phase IV (Day 121). Relative abundance was defined as the number of sequences affiliated with that phylum or genus divided by the total number of sequences per sample. The electron microscopic image in the circle is of anammox granular sludge treating domestic wastewater.
7
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Table 3 Comparisons of different processes on influent quality, HRT, nitrogen removal performance and DO. Reference
This study
Ding et al. (2018)
Li et al. (2018a,b)
Morales et al., 2016
Process Influent wastewater Influent NH4+-N (mg N/L) HRT(h)/aerobic HRT (h) DO concentrations (mg/L) NRE (%)
SPND-Anammox Domestic 71.4 10.1/6.7 0.1 88.2
SNAD (suspended sludge) Domestic 60–80 9/6 0.3 86.1
SND (suspended sludge) Synthetic 40 8/8 1.2 75.1
PN/A (granular biomass) Synthetic 74 6/6 1.2 70
2016). Overall, the bacterial community identified through Illumina Miseq sequencing is favorable for the enhanced nitrogen removal in ANA-UASB.
(Guo et al., 2016). In this model, an average of 71% ammonia was oxidized to nitrite and over 22% of theoretical energy consumption for aeration can be saved.
3.3. Nitrogen removal mechanism in the two-stage process
3.4. Prospect and practical application
ANA-UASB was successfully combined with SPND-SBR after successful partial nitritation in SPND-SBR. Table 1 shows influent and effluent COD and TIN in each reactor at different phases. The nitrogen and COD removal performance was enhanced despite the fluctuation in the inflow quality (NH4+-N: 71.4 ± 4.4 mg N/L and COD: 238.6 ± 53.3 mg/L). During the steady period (Day 101–136), about 99.16% of NH4+-N in domestic wastewater was removed by the novel two-stage process. The NRE was generally above 88.24%, while effluent TIN was less than 8.4 ± 1.1 mg N/L and ORE was over 86.91%. The nitrogen removal efficiency obtained in this study was higher than that of the SND or anammox system previously reported (Table 3). The consumption of nitrogen compounds in the two-stage system (Fig. 6) was investigated as per the typical cycle on Day 121 using calculation method reported by Ma et al. (2016) as follows: For partial nitritation, the NO2−-N/NH4+-N molar ratio was 1.2:1, and 1 mg N/L of NO2−-N was used to consume 2.3 mg COD/L in denitritation. For anammox process, the stoichiometric NH4+-N/NO2−-N molar ratio of 1:1.32 produced 0.26 mol NO3−-N, while 1 mg N/L NO3−-N was used for consuming 3.71 mg COD/L in denitrification. In a typical steady phase cycle, around 36% influent TIN was removed by denitritation after mixing and around 7% TIN was removed by denitrification, while about 57% TIN was removed by anammox pathway, indicating that denitritation at low-DO of 0.1 mg/L played a stronger role and anammox was a significant nitrogen removal pathway in this two-stage process coupled SPND with anammox. Furthermore, 3.43 mg of oxygen was consumed to oxidize every 1 mg of ammonia during partial nitrification process and is only 75% of total oxygen consumed during traditional nitrification process, i.e. 4.57 mg oxygen
3.4.1. Importance of applying pre-anaerobic segment Integration of pre-anaerobic segment can inhibit NOB by slowing down its metabolism and reduce the activity of NOB (Gilbert et al., 2014; Miao et al., 2016; Liu et al., 2017). In this study, NOB was continuously suppressed through a combination of HRT control, low-DO conditions and competitive inhibition of heterotrophic bacteria (Ma et al., 2015; Cao et al., 2017) as evident by the sharp decrease in the abundance of NOB including Nitrospira and Nitrobacter during the longterm operation. In addition, the activity of NOB was completely inhibited during the last phase. The pre-anaerobic stage can remove the residual nitrite from the previous cycle and store a certain amount of intracellular carbon source to improve the nitrogen removal performance consistent with previous findings (Liu et al., 2017; Miao et al., 2018; Zhang et al., 2018). Therefore, it can be concluded that the preanaerobic segment promotes the nitrogen removal and improves the stability of the system. 3.4.2. Characteristics and potential in full-scale application As evident in the findings of this study, enhanced nitrogen removal can be achieved in a low-DO two-stage process by coupling SPND with polished anammox. There are several advantages in the low-DO twostage process. Firstly, COD in domestic wastewater can be fully utilized for nitrogen removal through step-wise reduction of DO to 0.1 mg/L in SPND-SBR, leading to the reduction of energy use and costs in BNR facilities. The aerobic HRT of the process was only 6.7 h, which was significantly lower than that of the traditional BNR process. Secondly, the treatment load can exceed the load of traditional WWTPs with the average NLR and COD loading rate (OLR) reaching to 0.17 and 0.61 kg/
Fig 6. Model based evaluation of the two-stage process. 8
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(m3·d) respectively (Lotti et al., 2015). Domestic wastewater pretreated by SPND-SBR can maintain a relatively high anammox activity and abundance with the output level of TIN being only about 8.4 ± 1.1 mg N/L (Table 1), which is below the discharge standards. Finally, aeration energy consumptions and construction costs can be reduced using polished anammox tank after sedimentation, which has a practical significance for the in-situ upgrade and reconstruction of the existing WWTPs.
Water Res. 70, 38–51. Gilbert, E.M., Agrawal, S., Brunner, F., Schwartz, T., Horn, H., Lackner, S., 2014. Response of different nitrospira species to anoxic periods depends on operational DO. Environ. Sci. Technol. 48 (5), 2934–2941. Gu, S., Wang, S., Yang, Q., Yang, P., Peng, Y., 2012. Start up partial nitrification at low temperature with a real-time control strategy based on blower frequency and pH. Bioresour. Technol. 112, 34–41. Guo, Y., Peng, Y., Wang, B., Li, B., Zhao, M., 2016. Achieving simultaneous nitrogen removal of low C/N wastewater and external sludge reutilization in a sequencing batch reactor. Chem. Eng. J. 306, 925–932. Guo, Y., Zhao, Y., Zhu, T., Li, J., Feng, Y., Zhao, H., Liu, S., 2018. A metabolomic view of how low nitrogen strength favors anammox biomass yield and nitrogen removal capability. Water Res. 143, 387–398. Jin, R.C., Xing, B.S., Yu, J.J., Qin, T.Y., Chen, S.X., 2013. The importance of the substrate ratio in the operation of the Anammox process in upflow biofilter. Ecol. Eng. 53, 130–137. Kartal, B., Kuenen, J.G., Loosdrecht, M.C.M.V., 2010. Sewage treatment with anammox. Science 328, 702–703. Keene, N.A., Reusser, S.R., Scarborough, M.J., Grooms, A.L., Seib, M., Santo Domingo, J., Noguera, D.R., 2017. Pilot plant demonstration of stable and efficient high rate biological nutrient removal with low dissolved oxygen conditions. Water Res. 121, 72–85. Kitzinger, K., Koch, H., Lüchker, S., Sedlacek, C.J., Herbold, C., Schwarz, J., Daebeler, A., Mueller, A., Lukumbuzya, M., Romano, S., Leisch, N., Karst, M.S., Kirkegaard, R., Albertsen, M., Nielsen, H.P., Wagner, M., Daims, H., 2018. Characterization of the first “Candidatus Nitrotoga” isloated reveals metabolic versatility and seperate evolution of widespread nitrite-oxidizing bacteria. mBio 9 (4). Klamerth, N., Malato, S., Maldonado, M.I., Agüera, A., Fernández-alba, A.R., 2010. Application of photo-fenten as a tertiary treatment of emerging contaminants in municipal wastewater. Environ. Sci. Technol. 44 (5), 1792–1798. Laureni, M., Falås, P., Robin, O., Wick, A., Weissbrodt, D.G., Nielsen, J.L., Ternes, T.A., Morgenroth, E., Joss, A., 2016. Mainstream partial nitritation and anammox: longterm process stability and effluent quality at low temperatures. Water Res. 101, 628–639. Li, L., Dong, Y., Qian, G., Hu, X., Ye, L., 2018a. Performance and microbial community analysis of bio-electrocoagulation on simultaneous nitrification and denitrification in submerged membrane bioreactor at limited dissolved oxygen. Bioresour. Technol. 258, 168–176. Li, J., Li, J., Gao, R., Wang, M., Yang, L., Wang, X., Zhang, L., Peng, Y., 2018b. A critical review of one-stage anammox processes for treating industrial wastewater: optimization strategies based on key functional microorganisms. Bioresour. Technol. 265, 498–505. Li, J., Peng, Y., Zhang, L., Liu, J., Wang, X., Gao, R., Pang, L., Zhou, Y., 2019. Quantify the contribution of anammox for enhanced nitrogen removal through metagenomic analysis and mass balance in an anoxic moving bed biofilm reactor. Water Res. 160, 178–187. Liu, J., Yuan, Y., Li, B., Zhang, Q., Wu, L., Li, X., Peng, Y., 2017. Enhanced nitrogen and phosphorus removal from municipal wastewater in an anaerobic-aerobic-anoxic sequencing batch reactor with sludge fermentation products as carbon source. Bioresour. Technol. 244, 1158–1165. Lotti, T., Kleerebezem, R., Hu, Z., Kartal, B., de Kreuk, M.K., van Erp Taalman Kip, C., Kruit, J., Hendrickx, T.L., van Loosdrecht, M.C., 2015. Pilot-scale evaluation of anammox-based mainstream nitrogen removal from municipal wastewater. Environ. Technol. 36 (9-12), 1167–1177. Ma, B., Bao, P., Wei, Y., Zhu, G., Yuan, Z., Peng, Y., 2015. Suppressing nitrite-oxidizing bacteria growth to achieve nitrogen removal from domestic wastewater via anammox using intermittent aeration with low dissolved oxygen. Sci. Rep. 5, 13048. Ma, B., Wang, S., Cao, S., Miao, Y., Jia, F., Du, R., Peng, Y., 2016. Biological nitrogen removal from sewage via anammox: recent advances. Bioresour. Technol. 200, 981–990. Mao, Y., Xia, Y., Zhang, T., 2013. Characterization of Thauera-dominated hydrogenoxidizing autotrophic denitrifying microbial communities by using high-throughput sequencing. Bioresour. Technol. 128, 703–710. Miao, Y., Zhang, L., Yang, Y., Peng, Y., Li, B., Wang, S., Zhang, Q., 2016. Start-up of single-stage partial nitrification-anammox process treating low-strength swage and its restoration from nitrate accumulation. Bioresour. Technol. 218, 771–779. Miao, Y., Peng, Y., Zhang, L., Li, B., Li, X., Wu, L., Wang, S., 2018. Partial nitrificationanammox (PNA) treating sewage with intermittent aeration mode: effect of influent C/N ratios. Chem. Eng. J. 334, 664–672. Morales, N., Río, Á., Vázquez-Padín, J.R., Méndez, R., Campos, J.L., Mosquera-Corral, A., 2016. The granular biomass properties and the acclimation period affect the partial nitritation/anammox process stability at a low temperature and ammonium concentration. Process Biochem. 51, 2134–2142. Mosquera-Corral, A., de Kreuk, M.K., Heijnen, J.J., van Loosdrecht, M.C., 2005. Effects of oxygen concentration on N-removal in an aerobic granular sludge reactor. Water Res. 39 (12), 2676–2686. Oehmen, A., Zeng, R.J., Yuan, Z., Keller, J., 2005. Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems. Biotechnol. Bioeng. 91 (1), 43–53. Park, H.D., Noguera, D.R., 2004. Evaluating the effect of dissolved oxygen on ammoniaoxidizing bacterial communities in activated sludge. Water Res. 38 (14–15), 3275–3286. Siegrist, H., Salzgeber, D., Eugster, J., Joss, A., 2008. Anammox brings WWTP closer to energy autarky due to increased biogas production and reduced aeration energy for N-removal. Water Sci. Technol. 57 (3), 383–388. Strous, M., Heijnen, J.J., Kuenen, J.G., Jetten, M.S.M., 1998. The sequencing batch
4. Conclusions An innovative low-DO two-stage process was established and a high TIN removal efficiency of 88.24% was achieved using the step-wise DO reductions to 0.1 mg/L. High-rate partial nitritation with a suitable effluent NO2−-N/NH4+-N ratio of around 1.04 was maintained in SPND-SBR by controlling DO concentration and aeration time, ensuring ANA-UASB had a significant nitrogen removal with high anammox activity and abundance of 6.6 ± 0.2 mg N/(gVSS·h) and 35.53%, respectively. The study investigated the feasibility and mechanism of advanced nitrogen removal by coupling partial nitritation and denitritation with polished anammox to treat low C/N (3.36) domestic wastewater. CRediT authorship contribution statement Wen Zhang: Conceptualization, Formal analysis, Investigation, Data curation, Writing - original draft. Yongzhen Peng: Supervision, Funding acquisition, Writing - review & editing. Liang Zhang: Validation, Writing - review & editing. Xiyao Li: Resources. Qiong Zhang: Methodology. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This research was financially supported by Municipal Science and Technology Project of Beijing, China (Z181100005518006), National Natural Science Foundation of China (21777005, 51978007), 111 Project (D16003) and the Funding Projects of Beijing Municipal Commission of Education, China. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2020.123045. References APHA, 1998. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC, USA. Cao, S., Du, R., Li, B., Ren, N., Peng, Y., 2016. High-throughput profiling of microbial community structures in an ANAMMOX-UASB reactor treating high-strength wastewater. Appl. Microbiol. Biotechnol. 100 (14), 6457–6467. Cao, Y., van Loosdrecht, M.C., Daigger, G.T., 2017. Mainstream partial nitritation-anammox in municipal wastewater treatment: status, bottlenecks, and further studies. Appl. Microbiol. Biotechnol. 101 (4), 1365–1383. Daigger, G.T., Littleton, H.X., 2014. Simultaneous biological nutrient removal: a state-ofthe-art review. Water Environ. Res. 86 (3), 245–257. Ding, S., Bao, P., Wang, Bo, Zhang, Q., Peng, Y., 2018. Long-term stale simultaneous partial nitrification, anammox and denitrification (SNAD) process treating real domestic sewage using suspended activated sludge. Chem. Eng. J. 339, 180–188. Fernandez, N., Sierra-Alvarez, R., Field, J.A., Amils, R., Sanz, J.L., 2008. Microbial community dynamics in a chemolithotrophic denitrification reactor inoculated with methanogenic granular sludge. Chemosphere 70 (3), 462–474. Fitzgerald, C.M., Camejo, P., Oshlag, J.Z., Noguera, D.R., 2015. Ammonia-oxidizing microbial communities in reactors with efficient nitrification at low-dissolved oxygen.
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W. Zhang, et al.
microaerophilic sulfate and nitrate co-reduction system. Chem. Eng. J. 330, 63–70. Yan, L., Liu, S., Liu, Q., Zhang, M., Liu, Y., Wen, Y., Chen, Z., Zhang, Y., Yang, Q., 2019. Improved performance of simultaneous nitrification and denitrification via nitrite in an oxygen-limited SBR by alternating the DO. Bioresour. Technol. 275, 153–162. Yuan, Q., Oleszkiewicz, J.A., 2011. Low temperature biological phosphorus removal and partial nitrification in a pilot sequencing batch reactor system. Water Sci. Technol. 63 (12), 2802–2807. Zeng, R.J., van Loosdrecht, M.C.M., Yuan, Z., Keller, J., 2003. Metabolic model for glycogen-accumulating organisms in anaerobic/aerobic activated sludge systems. Biotechnol. Bioeng. 81 (1), 92–105. Zhang, J., Zhang, L., Miao, Y., Sun, Y., Li, X., Zhang, Q., Peng, Y., 2018. Feasibility of in situ enriching anammox bacteria in a sequencing batch biofilm reactor (SBBR) for enhancing nitrogen removal of real domestic wastewater. Chem. Eng. J. 352, 847–854.
reactor as a powerful tool for the study of slowly growing anaerobic ammoniumoxidizing microorganisms. Appl. Microbiol. Biotechnol. 50, 589–596. Tian, W.D., Li, W.G., Zhang, H., Kang, X.R., van Loosdrecht, M.C., 2011. Limited filamentous bulking in order to enhance integrated nutrient removal and effluent quality. Water Res. 45 (16), 4877–4884. Vlaeminck, S.E., Terada, A., Smets, B.F., Linden, D.V.d., Boon, N., Verstraete, W., Carballa, M., 2009. Nitrogen removal from digested black water by one-stage partial nitritation and anammox. Environ. Sci. Technol. 43 (13), 5035–5041. Wen, X., Zhou, J., Li, Y., Qing, X., He, Q., 2016. A novel process combining simultaneous partial nitrification, anammox and denitrification (SNAD) with denitrifying phosphorus removal (DPR) to treat sewage. Bioresour. Technol. 222, 309–316. WPO, 1998. Water Protection Ordinance of 28 October 1998 (814.201). UNECE, Geneva. Xu, X.J., Chen, C., Guan, X., Yuan, Y., Wang, A.-J., Lee, D.J., Zhang, Z.F., Zhang, J., Zhong, Y.J., Ren, N.Q., 2017. Performance and microbial community analysis of a
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