- Email: [email protected]
Hydroxylamine addition and real-time aeration control in sewage nitritation system for reduced start-up period and improved process stability
Bioresource Technology 294 (2019) 122183
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Hydroxylamine addition and real-time aeration control in sewage nitritation system for reduced start-up period and improved process stability Jia Lia,b, Liang Zhanga, Jie Liub, Jia Linb, Yongzhen Penga,
T
⁎
a National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, Key Laboratory of Beijing Water Quality Science and Water Environment Recovery Engineering, Beijing University of Technology, Beijing 100124, China b Beijing Capital Company Limited, Beijing 100044, 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: Hydroxylamine (NH2OH) Real-time aeration control Nitritation Sewage Nitrite-oxidizing bacteria (NOB)
Sewage nitritation is a promising process for nitrogen removal, but its practical application is limited by long start-up period and unstable operation. In this study, hydroxylamine (NH2OH) addition and real-time aeration control strategies were adopted for the promotion of sewage nitritation in a sequencing batch reactor. Initially, 4.5 mg/L NH2OH was added into reactor every 24 h to establish nitritation, increasing the nitrite accumulation ratio (NAR) from 1.7% to 93% in 19 d. In the following period, NH2OH addition was stopped and nitritation remained stable over 55 d, with NAR of 97% by real-time aeration control. The aeration duration was determined by characteristic points on pH curve. The main genera of nitrite oxidizing bacteria, Nitrobacter and Nitrospira, were both eliminated from the system, supporting the long-term stability of nitritation. Overall, NH2OH addition and real-time aeration control is an excellent strategy for establishing and maintaining effective sewage nitritation.
1. Introduction Biological nitrogen removal processes via nitrite, such as nitritationdenitritation and nitritation integrated with anaerobic ammonium oxidation (anammox), have advantages of reducing aeration energy and carbon source consumption. For partial nitritation/anammox (PN/A) processes, biological nitrogen removal could reduce aeration energy consumption by 60% and organic carbon by 100%, when compared with conventional nitrification–denitrification processes (Mulder, 2003; Wang et al., 2010). However, nitritation is rarely observed in conventional sewage treatment processes. This is because nitrite oxidizing
⁎
bacteria (NOB) activity is usually similar or higher than ammonia oxidizing bacteria (AOB) activity, resulting in the complete oxidation of ammonium to nitrate. Therefore, establishing nitritation requires specific strategies to promote AOB activity and suppress NOB activity (Lackner et al., 2014; Duan et al., 2019). Real-time aeration control has been proven to be a feasible strategy to initiate and maintain sewage nitritation, especially in sequencing batch reactor (SBR) systems (Chen et al., 2012). Real-time aeration control strategy is mainly based on water quality parameters, which indicate the end-point of ammonium oxidation. For example, the system pH value gradually decreases during the nitrification process
Corresponding author. E-mail address: [email protected] (Y. Peng).
https://doi.org/10.1016/j.biortech.2019.122183 Received 29 July 2019; Received in revised form 17 September 2019; Accepted 18 September 2019 Available online 21 September 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Bioresource Technology 294 (2019) 122183
J. Li, et al.
aerobic (time changed at different reactor operation phases), 30 min settling, 10 min decanting and idle (Fig. 1b). During the feeding period, 5 L sewage was pumped into reactor. Then, the anoxic stage was conducted for the reduction of nitrite and nitrate produced in last cycle. In anoxic stage, the SBR was aerated with an initial DO concentration of about 1 mg/L. In phase II (Day 18–45), NH2OH stock solution was injected at the beginning of the aeration stage, resulting in an initial 4.5 mg/L NH2OH concentration in the reactor. In phase III (Day 46–100), NH2OH addition was ceased and the aeration time was determined by real-time aeration control strategy, pH value was monitored on-line, with aeration stopped at the point when the “ammonia valley” appeared on pH curve. After aerobic phase, the settling, decanting and idle stages were implemented successively. The SBRs continually mixed with a mechanical stirrer at 70 rpm during reaction phases. The temperature of reactors was maintained at 25 ± 1 °C and the sludge retention time was maintained at approximately 20 d.
until ammonium is depleted. After this point, the pH value begins to increase due to the release of carbon dioxide from the reactor (Yang et al., 2007). Therefore, aeration can be stopped at the point where an “ammonia valley” is observed on the pH curve, ensuring ammonium oxidation to nitrite but avoiding further oxidation of nitrite to nitrate (Lee et al., 2013). Using the real-time aeration control strategy for treatment of low strength domestic wastewater, stable nitritation has been achieved with a nitrite accumulation ratio (NAR) above 80% (Blackburne et al., 2008). Lemaire et al. (2008) reported successful use of the real-time aeration control strategy to establish nitritation in an SBR, and the nitritation could be repeatedly and reliably achieved. So far, the application of real-time aeration control strategy in fullscale sewage nitritation systems remains limited. One of the bottlenecks is the long start-up time required to achieve nitritation. Achieving a high level of nitritation usually requires a long time period as NOB suppression and wash-out is limited in the real-time aeration control strategy (Blackburne et al., 2008). Yang et al. (2007) treated municipal wastewater in an SBR, reporting that 76 days were required to achieve nitritation using the real-time aeration control strategy. For the real-time aeration control strategy, the addition of appropriate initial inhibitors can be integrated to improve the initiation of nitritation. When appropriate inhibitor is added, such as hydroxylamine (NH2OH), the onset of nitritation is effectively promoted (Okabe et al., 2011). For example, sewage nitritation with nitrite accumulation ratio (NAR) above 95% could be achieved in 5 days with limited addition of NH2OH (Li et al., 2019). However, for the NH2OH addition strategy, the long-term maintenance of sewage nitritation was still a challenge since the microorganism gradually acclimated to NH2OH inhibition (Wang et al., 2015; Li et al., 2019). As real-time aeration control is favorable to the maintenance of stable nitritation, the combination of these strategies provides the benefits of reducing start-up periods and increasing stability of nitritation, in a convenient and economical way. However, the feasibility of the combined strategy for the initiation of sewage nitritation remains unclear. In addition, to better understand the effect of the combined strategy, variation in microbial communities also require investigation. This study proposes a combined nitritation operational strategy, which includes the initiation of nitritation via the addition of NH2OH and stability maintenance via real-time aeration control. The type of SBR was used to establish nitritation for sewage treatment, to explore the feasibility of establishing stable nitritation using NH2OH addition and a real-time aeration control strategy. The real-time aeration control strategy was performed based on the detection of characteristic point on pH curves. Furthermore, variation in nitrifying microbial community and activity were assessed during the operational period, for better understanding of the underlying mechanisms. Finally, the application potential of this novel strategy in full-scale wastewater treatment plants is discussed.
2.3. Experimental materials and analytical methods The NH2OH solution used in this study was prepared from hydroxylammonium chloride (NH2OH-HCl) supplied by Sinopharm Chemical Reagent Co., Ltd (Beijing Chian). The concentration of NH2OH was determined using the colorimetric method (Frear and Burrell, 1995). Mixed liquor samples were collected from reactors daily and filtered through disposable 0.45 mm filter units immediately. The concentrations of NH4 ± -N, NO2–-N and NO3–-N were measured using a Flow Injection Analyzer (Lachat QuikChem8500, Milwaukee, USA). MLSS and MLVSS concentrations were analyzed according to standard methods (APHA, 1998). Temperature, pH and DO levels were monitored using a handheld multi-parameter analyzer (Multi 340i, WTW company, Germany). The AOB and NOB activities during the operational period were investigated via batch tests. 2.4. DNA extraction and quantitative real-time PCR The sludge samples collected in reactor were freeze-dried by a lyophilizer (LABCONCO Co., Free Zone, USA). DNA was extracted from 0.10 to 0.20 g dry sludge samples using FastDNA™ SPIN kits for Soil (Q BIOgene Inc., Carlsbad, USA) and the DNA concentration was determined using a Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, USA). The target DNA was amplified using a Stratagene MX3005p Real-Time PCR system (Agilent Technologies, USA) using the SYBR-Green approach. qPCR assay was performed in 20 μL reaction mixtures that consisted of 7 μL of sterile water, 10 μL of SYBR Premix Ex Taq (Takara, Dalian, China), 0.4 μL of ROX Reference Dye 50, 0.3 μL of forward and reverse primer (10 μM) and 2 μL of genomic DNA.The universal bacterial 16SrRNA genes for total bacteria, the amoA functional gene of AOB and 16S rRNA genes of NOB (nib and nis for Nitrobacter and Nitrospira, respectively) were amplified. The standard curves was generated in duplicate using serial decimal dilutions of plasmid DNA and adopted when their amplification efficiency between 90% and 110% and correlation coefficient above 0.99.
2. Materials and methods 2.1. Wastewater and seed sludge
3. Results and discussion
The sewage supplied to reactors was collected from a septic tank in the residential area of Beijing University of Technology (Beijing, China). Seed sludge was collected from the Gaobeidian Wastewater Treatment Plant, which performs anaerobic-anoxic-oxic processes. The concentrations of mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solid (MLVSS) in reactors after inoculation, were approximately 2800 and 2200 mg/L, respectively.
3.1. Optimization of NH2OH dose for the establishment of nitritation Three identical reactors (SBR1, SBR2 and SBR3) were used to select the optimum NH2OH dose for establishing efficient nitrification. In all reactors, steady and complete nitrification was obtained initially with NAR less than 1%. Following this, different volumes of NH2OH stock solutions (5 g/L) were injected into each of the reactors. The NH2OH solution was dosed once per day, maintaining initial NH2OH concentrations in SBR1, SBR2 and SBR3 of 2.5, 3.5 and 4.5 mg/L, respectively. The addition of 4.5 mg/L NH2OH immediately caused nitrite accumulation, indicating the start-up of nitritation. At the end of
2.2. Reactor operation and experimental procedure The SBRs used in this study were made of polymethyl methacrylate, with a working volume capacity of 10 L (Fig. 1a). The operational cycle of the SBRs consisted of six stages: 10 min feeding, 30 min anoxic, 2
Bioresource Technology 294 (2019) 122183
J. Li, et al.
Fig. 1. Schematic representation of the SBR (a); operational mode for the SBR (b).
characteristic point on the pH curve. Nitrogen variations during a typical cycle (day 75) was shown in Fig. 3. During the aeration phase, ammonium level reduced and nitrite was continuously accumulated. A relatively stable nitritation performance was obtained during phase III with a NAR of 96.9 ± 2.2% (Fig. 2). Despite fluctuation of influent ammonium concentration from 65.9 to 80.9 mg N/L (between days 45–100), the strategy of optimized aeration time maintained a low ammonia concentration (0.9 ± 0.3 mgN/L) in effluent and an ammonium removal efficiency of 99.0 ± 0.5% was obtained (Fig. 2). A high ammonia oxidation performance using the real-time aeration control strategy has also been reported by Peng et al. (2004). These results suggest that the real-time aeration control strategy amended with NH2OH addition is feasible for the efficient start-up and maintenance of sewage nitritation. Although the addition of NH2OH reduced the start-up time for sewage nitritation, the nitritation established was not always stable during operation (Wang et al., 2015; Li et al., 2019). However, in the present study, despite the removal of NH2OH dosing, unstable nitritation performance was avoided by the use of real-time aeration control. Real-time aeration control effectively avoids excessive aeration within the system, which is of high importance for nitritation stability. It has been reported that nitrite accumulation stopped after six cycles with excessive aeration (Qian et al., 2017). Compared to the use of individual strategies, the integration of NH2OH addition and real-time aeration control is favorable for nitritation operation.
operational period, NAR increased to 94.7% in SBR3 with the addition of 4.5 mg/L NH2OH, which was higher than that of SBR1 (46.5%) and SBR2 (80.7%). Therefore, 4.5 mg/L was selected as the optimal NH2OH concentration for the establishment of nitritation in this study.
3.2. Nitrogen removal performance using the NH2OH addition and realtime aeration control strategy The NH2OH addition and real-time aeration control strategy was investigated in the SBR treating real sewage. The whole experimental period was divided into three phases. In phase I, complete nitrification of sewage was achieved. The influent ammonium concentration was maintained at an average of 70.5 ± 6.6 mg N/L and the aeration time was fixed to 240 min (Figs. 1b and 2). As shown in Fig. 2, the average ammonium, nitrite and nitrate concentrations in effluent were 0.5, 0.3 and 36.9 mg N/L, respectively. Influent ammonium was fully oxidized to nitrate during the aerobic period and steady complete nitrification was obtained. In phase II, 4.5 mg/L NH2OH was added to the SBR daily. After the addition of NH2OH, the effluent nitrate concentration declined immediately, accompanied by an increase in effluent nitrite concentration (Fig. 2). Nitritation was rapidly established in 5 d and the NAR increased to 93.3 ± 1.15% in 19 d with the addition of NH2OH once per day. The effectiveness of NH2OH addition on nitritation start-up was consistent with the findings of previous studies (Xu et al., 2012; Li et al., 2019). In phase III, NH2OH addition was ceased and the aeration time was controlled in real-time, with the aeration length determined based on 3
Bioresource Technology 294 (2019) 122183
J. Li, et al.
Fig. 2. Profiles of ammonium, nitrite, nitrate concentrations, ammonium removal efficiency and nitrite accumulation ratio (NAR) in the SBR during the experimental periods (NAR%=NO– 2-N/NO– x-N × 100, NO– x-N = NO– 2-N + NO– 3-N). The different phases of SBR was indicated by lines.
further preventing the growth of NOB (Fig. 3). Therefore, the real-time aeration control strategy effectively prevented the conversion of nitritation to nitrification. Importantly, the variation in relative abundances of Nitrospira and Nitrobacter, the two main NOB genera, were significantly different during the whole operational period. Nitrospira relative abundance decreased from 0.47 ± 0.05% to 0.15 ± 0.02% under NH2OH suppression, while Nitrobacter relative abundance only declined from 0.21 ± 0.07% to 0.13 ± 0.003% (Fig. 4b). This indicated that Nitrobacter was more tolerant to NH2OH inhibition than Nitrospira, which may be caused by differences in microbial characteristics (Vela et al., 2018). During the real-time aeration period that followed, Nitrobacter relative abundance gradually decreased from 0.13 ± 0.01% to less than 0.01%, compensating for the strong tolerance of Nitrobacter to NH2OH inhibition (Fig. 4b). The severe Nitrobacter inhibition induced using the real-time aeration control strategy has been reported in the literature previously, with Nitrobacter population decreasing by 93%, while Nitrospira population remained unchanged (Lee et al., 2013). Therefore, low abundances of Nitrobacter and Nitrospira were both achieved via the NH2OH addition and real-time aeration control strategy, enhancing the stability of sewage nitritation.
3.3. qPCR-based analysis during the start-up and maintenance of sewage nitritation For insight into the effect of combined NH2OH addition and realtime control on sewage nitritation, the activity and abundance of functional bacteria were analyzed in the SBR, including AOB and NOB (Nitrobacter and Nitrospira). With the addition of NH2OH, AOB activity increased from an initial level of 6.04 ± 0.50 mg N/(g VSS·h) on day 8–11.43 ± 0.32 mg N/(g VSS·h) on day 45, while NOB activity in the SBR declined obviously (Fig. 4a). NH2OH appeared to induce selective inhibition on AOB and NOB, which resulted in an increase of the NAR in the SBR (Fig. 2). The increase in AOB activity was due to a reduction in competition for oxygen when NOB activity was severely inhibited by NH2OH. By using the real-time aeration control strategy in phase III, AOB activity remained largely stable, although NOB activity further decreased to 0.05 ± 0.003 mg N/(g VSS·h) on day 83 (Fig. 4a). These results indicated that NH2OH addition and real-time aeration control both suppressed NOB activity. Not only AOB activity, AOB population also increased during NH2OH addition phase. The relative abundance of AOB was enriched from 0.05 ± 0.002% on day 8 to 0.16 ± 0.01% on day 45 (Fig. 4b). However, the relative abundance of NOB decreased from 0.68 ± 0.07% to 0.28 ± 0.02%, which indicated that the addition of NH2OH also caused washout of NOB in addition to NOB suppression. The inhibition on NOB might because that NH2OH inhibits the synthesis of nitrite oxidoreductase enzyme, suppressing the growth of NOB (Xu et al., 2012). With the real-time aeration control strategy, the relative abundance of NOB further declined to 0.02 ± 0.002%, showing that real-time aeration control also contributed to NOB washout from the system. When the real-time aeration control strategy was adopted in phase III, nitrite was not oxidized by NOB during the aeration phase due to out-competition by AOB. Moreover, aeration was stopped when ammonium oxidation was complete and the settling phase started,
3.4. Potential applications in sewage treatment This study proposes a promising strategy for efficient sewage nitritation. NH2OH addition induced a decrease in NOB activity and abundance, accompanied by an enrichment of AOB and the rapid startup of nitritation. Real-time aeration control was beneficial for maintaining low NOB activity and abundance, inducing stable nitritation with a NAR of 96.9 ± 2.2% between days 45–100. There are several advantages to the use of this combined strategy. Firstly, the start-up period required to establish nitritation is rapid, with nitritation achieved after 5 days of NH2OH addition (4.5 mg/L) in the present study, which was faster than other control methods reported 4
Bioresource Technology 294 (2019) 122183
J. Li, et al.
Fig. 4. Variations of nitrifying bacteria activities (a) and relative abundance of AOB and NOB (Nitrobacter and Nitrospira) (b) in the SBR during the experimental periods (relative abundance of AOB=(absolute abundance of AOB/absolute abundance of total bacteria) × 100%; relative abundance of Nitrobacter=(absolute abundance of Nitrobacter/absolute abundance of total bacteria) × 100%; relative abundance of Nitrospira=(absolute abundance of Nitrospira/absolute abundance of total bacteria) × 100%). Error bars indicate the standard deviation. Fig. 3. Variation of ammonium, nitrite and nitrate concentration in typical cycles on Day 8, Day 27 and Day 105 of SBR.
4. Conclusions This study investigated the feasibility of the NH2OH addition and real-time aeration control strategy on sewage nitritation operation. Sewage nitritation was achieved rapidly in 5 d due to daily NH2OH (4.5 mg/L) dosing. Then the nitritaion maintained at a stable level with a NAR of 97%, using a real-time aeration control strategy following the cessation of NH2OH addition. Microbial community analysis indicated that the reactor population was optimized with wash-out of NOB (Nitrobacter and Nitrospira) and enrichment of AOB. The strategy proposed in this study is a promising approach for practical application in sewage nitritation.
previously in the literature. Secondly, nitritation is stable as NOB can be effectively eliminated, including Nitrobacter and Nitrospira. It is widely accepted that Nitrobacter and Nitrospira are two prevailing NOB populations in most wastewater treatment systems (Shu et al., 2015). Therefore, this strategy is feasible for practical sewage nitritation application. The combined strategy can establish stable nitritation as numerous genera of NOB can be simultaneously suppressed and eliminated. With the use of single control strategies, stable operation is more difficult due to the gradual enrichment of specific NOB genera under certain condition. As reported in previous research, the NOB community shifted from Nitrobacter to Nitrospira under long-term low DO concentration condition, resulting in deterioration of sewage nitritation (Liu and Wang, 2013). Thus, single control strategies are not sufficient for effective nitritation operation, while combining two or more control strategies are more favorable for nitritation stability and ensuring complete washout of NOB. Recent reports show that sludge treatment with FA and FNA effectively achieved selective inhibition of Nitrospira and Nitrobacter, resulting in stable nitritation (Duan et al., 2019). In the present study, the NH2OH addition and real-time aeration control strategy exhibited excellent operational performance. Nitrospira was mostly eliminated via NH2OH inhibition and Nitrobacter was severely suppression via real-time aeration control (Fig. 4b). Therefore, the combined method proposed in this study may have high application potential and be significant for effective sewage nitritation process operation.
Acknowledgements This research was financially supported by Major Science and Technology Program for Water Pollution Control and Treatment (2017ZX07102-003), National Natural Science Foundation of China (21777005), National Natural Science Foundation of China (51978007), 111 Project (D16003) and the Funding Projects of Beijing Municipal Commission of Education. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122183. References APHA, 1998. Standard methods for the examination of water and wastewater. American Public Health Association, Washington, DC, USA.
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Okabe, S., Oshiki, M., Takahashi, Y., Satoh, H., 2011. Development of long-term stable partial nitrification and subsequent anammox process. Bioresour. Technol. 102, 6801–6807. Peng, Y.Z., Chen, Y., Peng, C.Y., Liu, M., Song, X.Q., Cui, Y.W., 2004. Nitrite accumulation by aeration controlled in sequencing batch reactors treating sewage. Water Sci. Technol. 50 (10), 35–43. Qian, W.T., Peng, Y.Z., Li, X.Y., Zhang, Q., Ma, B., 2017. The inhibitory effects of free ammonia on ammonia oxidizing bacteria and nitrite oxidizing bacteria under anaerobic condition. Bioresour. Technol. 243, 1247–1250. Shu, D., He, Y., Yue, H., Wang, Q., 2015. Microbial structures and community functions of anaerobic sludge in six full-scale wastewater treatment plants as revealed by 454 high-throughput pyrosequencing. Bioresour. Technol. 186, 163–172. Vela, J.D., Dick, G.J., Love, N.G., 2018. Sulfide inhibition of nitrite oxidation in activated sludge depends on microbial community composition. Water Res. 138, 241–249. Wang, C.C., Lee, P.H., Kumar, M., Huang, Y.T., Sung, S., Lin, J.G., 2010. Simultaneous partial nitrification, anaerobic ammonium oxidation and denitrification (SNAD) in a full-scale landfill-leachate treatment plant. J. Hazard. Mater. 175, 622–628. Wang, Y.Y., Wang, Y.W., Wei, Y.S., Chen, M.X., 2015. In-situ restoring nitrogen removal for the combined partial nitritation-anammox process deteriorated by nitrate buildup. Biochem. Eng. J. 98, 127–136. Xu, G.J., Xu, X.C., Yang, F.L., Liu, S.T., Gao, Y., 2012. Partial nitrification adjusted by hydroxylamine in aerobic granules under high DO and ambient temperature and subsequent Anammox for low C/N wastewater treatment. Chem. Eng. J. 213, 338–345. Yang, Q., Peng, Y.Z., Liu, X., Zeng, W., Mino, T., Satoh, H., 2007. Nitrogen removal via nitrite from municipal wastewater at low temperatures using real-time control to optimize nitrifying communities. Environ. Sci. Technol. 41 (23), 8159–8164.
Blackburne, R., Yuan, Z.G., Keller, J., 2008. Demonstration of nitrogen removal via nitrite in a sequencing batch reactor treating domestic wastewater. Water Res. 42, 2166–2176. Chen, Y., Li, J., Wang, C.W., Zhao, X.F., Zhao, B.H., 2012. Partial nitrification to nitrite with real-time aeration duration control in an SBR treating domestic wastewater. Adv. Mater. Res. 356–360, 1046–1049. Duan, H.R., Ye, L., Lu, X.Y., Yuan, Z.G., 2019. Overcoming nitrite oxidizing bacteria adaptation through alternating sludge treatment with free nitrous acid and free ammonia. Environ. Sci. Technol. 54 (4), 1937–1946. Frear, D.S., Burrell, R.C., 1995. Spectrophotometric method for determining hydroxylamine reductase activity in higher plants. Anal. Chem. 27, 1664–1665. Lackner, S., Gilbert, E.M., Vlaeminck, S.E., Joss, A., Horn, H., van Loosdrecht, M.C.M., 2014. Full-scale partial nitritation/anammox experiences-An application survey. Water Res. 55, 292–303. Lee, P.H., Cotter, S.F., Reyes Prieri, S.C., Attalage, D., Sung, S., 2013. pH-gradient realtime aeration control for nitritation community selection in a non-porous hollow fiber membrane biofilm reactor (MBfR) with dilute wastewater. Chemosphere 90, 2320–2325. Lemaire, R., Marcelino, M., Yuan, Z.G., 2008. Achieving the nitrite pathway using aeration phase length control and step-feed in an SBR removing nutrients from abattoir wastewater. Biotechnol. Bioeng. 100, 1228–1236. Li, J., Zhang, Q., Li, X.Y., Peng, Y.Z., 2019. Rapid start-up and stable maintenance of domestic wastewater nitritation through short-term hydroxylamine addition. Bioresour. Technol. 278, 468–472. Liu, G.Q., Wang, J.M., 2013. Long-term low DO enriches and shifts nitrifier community in activated sludge. Environ. Sci. Technol. 47 (10), 5109–5117. Mulder, A., 2003. The quest for sustainable nitrogen removal technologies. Water Sci. Technol. 48, 67–75.
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Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Hydroxylamine addition and real-time aeration control in sewage nitritation system for reduced start-up period and improved process stability Jia Lia,b, Liang Zhanga, Jie Liub, Jia Linb, Yongzhen Penga,
T
⁎
a National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, Key Laboratory of Beijing Water Quality Science and Water Environment Recovery Engineering, Beijing University of Technology, Beijing 100124, China b Beijing Capital Company Limited, Beijing 100044, 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: Hydroxylamine (NH2OH) Real-time aeration control Nitritation Sewage Nitrite-oxidizing bacteria (NOB)
Sewage nitritation is a promising process for nitrogen removal, but its practical application is limited by long start-up period and unstable operation. In this study, hydroxylamine (NH2OH) addition and real-time aeration control strategies were adopted for the promotion of sewage nitritation in a sequencing batch reactor. Initially, 4.5 mg/L NH2OH was added into reactor every 24 h to establish nitritation, increasing the nitrite accumulation ratio (NAR) from 1.7% to 93% in 19 d. In the following period, NH2OH addition was stopped and nitritation remained stable over 55 d, with NAR of 97% by real-time aeration control. The aeration duration was determined by characteristic points on pH curve. The main genera of nitrite oxidizing bacteria, Nitrobacter and Nitrospira, were both eliminated from the system, supporting the long-term stability of nitritation. Overall, NH2OH addition and real-time aeration control is an excellent strategy for establishing and maintaining effective sewage nitritation.
1. Introduction Biological nitrogen removal processes via nitrite, such as nitritationdenitritation and nitritation integrated with anaerobic ammonium oxidation (anammox), have advantages of reducing aeration energy and carbon source consumption. For partial nitritation/anammox (PN/A) processes, biological nitrogen removal could reduce aeration energy consumption by 60% and organic carbon by 100%, when compared with conventional nitrification–denitrification processes (Mulder, 2003; Wang et al., 2010). However, nitritation is rarely observed in conventional sewage treatment processes. This is because nitrite oxidizing
⁎
bacteria (NOB) activity is usually similar or higher than ammonia oxidizing bacteria (AOB) activity, resulting in the complete oxidation of ammonium to nitrate. Therefore, establishing nitritation requires specific strategies to promote AOB activity and suppress NOB activity (Lackner et al., 2014; Duan et al., 2019). Real-time aeration control has been proven to be a feasible strategy to initiate and maintain sewage nitritation, especially in sequencing batch reactor (SBR) systems (Chen et al., 2012). Real-time aeration control strategy is mainly based on water quality parameters, which indicate the end-point of ammonium oxidation. For example, the system pH value gradually decreases during the nitrification process
Corresponding author. E-mail address: [email protected] (Y. Peng).
https://doi.org/10.1016/j.biortech.2019.122183 Received 29 July 2019; Received in revised form 17 September 2019; Accepted 18 September 2019 Available online 21 September 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Bioresource Technology 294 (2019) 122183
J. Li, et al.
aerobic (time changed at different reactor operation phases), 30 min settling, 10 min decanting and idle (Fig. 1b). During the feeding period, 5 L sewage was pumped into reactor. Then, the anoxic stage was conducted for the reduction of nitrite and nitrate produced in last cycle. In anoxic stage, the SBR was aerated with an initial DO concentration of about 1 mg/L. In phase II (Day 18–45), NH2OH stock solution was injected at the beginning of the aeration stage, resulting in an initial 4.5 mg/L NH2OH concentration in the reactor. In phase III (Day 46–100), NH2OH addition was ceased and the aeration time was determined by real-time aeration control strategy, pH value was monitored on-line, with aeration stopped at the point when the “ammonia valley” appeared on pH curve. After aerobic phase, the settling, decanting and idle stages were implemented successively. The SBRs continually mixed with a mechanical stirrer at 70 rpm during reaction phases. The temperature of reactors was maintained at 25 ± 1 °C and the sludge retention time was maintained at approximately 20 d.
until ammonium is depleted. After this point, the pH value begins to increase due to the release of carbon dioxide from the reactor (Yang et al., 2007). Therefore, aeration can be stopped at the point where an “ammonia valley” is observed on the pH curve, ensuring ammonium oxidation to nitrite but avoiding further oxidation of nitrite to nitrate (Lee et al., 2013). Using the real-time aeration control strategy for treatment of low strength domestic wastewater, stable nitritation has been achieved with a nitrite accumulation ratio (NAR) above 80% (Blackburne et al., 2008). Lemaire et al. (2008) reported successful use of the real-time aeration control strategy to establish nitritation in an SBR, and the nitritation could be repeatedly and reliably achieved. So far, the application of real-time aeration control strategy in fullscale sewage nitritation systems remains limited. One of the bottlenecks is the long start-up time required to achieve nitritation. Achieving a high level of nitritation usually requires a long time period as NOB suppression and wash-out is limited in the real-time aeration control strategy (Blackburne et al., 2008). Yang et al. (2007) treated municipal wastewater in an SBR, reporting that 76 days were required to achieve nitritation using the real-time aeration control strategy. For the real-time aeration control strategy, the addition of appropriate initial inhibitors can be integrated to improve the initiation of nitritation. When appropriate inhibitor is added, such as hydroxylamine (NH2OH), the onset of nitritation is effectively promoted (Okabe et al., 2011). For example, sewage nitritation with nitrite accumulation ratio (NAR) above 95% could be achieved in 5 days with limited addition of NH2OH (Li et al., 2019). However, for the NH2OH addition strategy, the long-term maintenance of sewage nitritation was still a challenge since the microorganism gradually acclimated to NH2OH inhibition (Wang et al., 2015; Li et al., 2019). As real-time aeration control is favorable to the maintenance of stable nitritation, the combination of these strategies provides the benefits of reducing start-up periods and increasing stability of nitritation, in a convenient and economical way. However, the feasibility of the combined strategy for the initiation of sewage nitritation remains unclear. In addition, to better understand the effect of the combined strategy, variation in microbial communities also require investigation. This study proposes a combined nitritation operational strategy, which includes the initiation of nitritation via the addition of NH2OH and stability maintenance via real-time aeration control. The type of SBR was used to establish nitritation for sewage treatment, to explore the feasibility of establishing stable nitritation using NH2OH addition and a real-time aeration control strategy. The real-time aeration control strategy was performed based on the detection of characteristic point on pH curves. Furthermore, variation in nitrifying microbial community and activity were assessed during the operational period, for better understanding of the underlying mechanisms. Finally, the application potential of this novel strategy in full-scale wastewater treatment plants is discussed.
2.3. Experimental materials and analytical methods The NH2OH solution used in this study was prepared from hydroxylammonium chloride (NH2OH-HCl) supplied by Sinopharm Chemical Reagent Co., Ltd (Beijing Chian). The concentration of NH2OH was determined using the colorimetric method (Frear and Burrell, 1995). Mixed liquor samples were collected from reactors daily and filtered through disposable 0.45 mm filter units immediately. The concentrations of NH4 ± -N, NO2–-N and NO3–-N were measured using a Flow Injection Analyzer (Lachat QuikChem8500, Milwaukee, USA). MLSS and MLVSS concentrations were analyzed according to standard methods (APHA, 1998). Temperature, pH and DO levels were monitored using a handheld multi-parameter analyzer (Multi 340i, WTW company, Germany). The AOB and NOB activities during the operational period were investigated via batch tests. 2.4. DNA extraction and quantitative real-time PCR The sludge samples collected in reactor were freeze-dried by a lyophilizer (LABCONCO Co., Free Zone, USA). DNA was extracted from 0.10 to 0.20 g dry sludge samples using FastDNA™ SPIN kits for Soil (Q BIOgene Inc., Carlsbad, USA) and the DNA concentration was determined using a Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, USA). The target DNA was amplified using a Stratagene MX3005p Real-Time PCR system (Agilent Technologies, USA) using the SYBR-Green approach. qPCR assay was performed in 20 μL reaction mixtures that consisted of 7 μL of sterile water, 10 μL of SYBR Premix Ex Taq (Takara, Dalian, China), 0.4 μL of ROX Reference Dye 50, 0.3 μL of forward and reverse primer (10 μM) and 2 μL of genomic DNA.The universal bacterial 16SrRNA genes for total bacteria, the amoA functional gene of AOB and 16S rRNA genes of NOB (nib and nis for Nitrobacter and Nitrospira, respectively) were amplified. The standard curves was generated in duplicate using serial decimal dilutions of plasmid DNA and adopted when their amplification efficiency between 90% and 110% and correlation coefficient above 0.99.
2. Materials and methods 2.1. Wastewater and seed sludge
3. Results and discussion
The sewage supplied to reactors was collected from a septic tank in the residential area of Beijing University of Technology (Beijing, China). Seed sludge was collected from the Gaobeidian Wastewater Treatment Plant, which performs anaerobic-anoxic-oxic processes. The concentrations of mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solid (MLVSS) in reactors after inoculation, were approximately 2800 and 2200 mg/L, respectively.
3.1. Optimization of NH2OH dose for the establishment of nitritation Three identical reactors (SBR1, SBR2 and SBR3) were used to select the optimum NH2OH dose for establishing efficient nitrification. In all reactors, steady and complete nitrification was obtained initially with NAR less than 1%. Following this, different volumes of NH2OH stock solutions (5 g/L) were injected into each of the reactors. The NH2OH solution was dosed once per day, maintaining initial NH2OH concentrations in SBR1, SBR2 and SBR3 of 2.5, 3.5 and 4.5 mg/L, respectively. The addition of 4.5 mg/L NH2OH immediately caused nitrite accumulation, indicating the start-up of nitritation. At the end of
2.2. Reactor operation and experimental procedure The SBRs used in this study were made of polymethyl methacrylate, with a working volume capacity of 10 L (Fig. 1a). The operational cycle of the SBRs consisted of six stages: 10 min feeding, 30 min anoxic, 2
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Fig. 1. Schematic representation of the SBR (a); operational mode for the SBR (b).
characteristic point on the pH curve. Nitrogen variations during a typical cycle (day 75) was shown in Fig. 3. During the aeration phase, ammonium level reduced and nitrite was continuously accumulated. A relatively stable nitritation performance was obtained during phase III with a NAR of 96.9 ± 2.2% (Fig. 2). Despite fluctuation of influent ammonium concentration from 65.9 to 80.9 mg N/L (between days 45–100), the strategy of optimized aeration time maintained a low ammonia concentration (0.9 ± 0.3 mgN/L) in effluent and an ammonium removal efficiency of 99.0 ± 0.5% was obtained (Fig. 2). A high ammonia oxidation performance using the real-time aeration control strategy has also been reported by Peng et al. (2004). These results suggest that the real-time aeration control strategy amended with NH2OH addition is feasible for the efficient start-up and maintenance of sewage nitritation. Although the addition of NH2OH reduced the start-up time for sewage nitritation, the nitritation established was not always stable during operation (Wang et al., 2015; Li et al., 2019). However, in the present study, despite the removal of NH2OH dosing, unstable nitritation performance was avoided by the use of real-time aeration control. Real-time aeration control effectively avoids excessive aeration within the system, which is of high importance for nitritation stability. It has been reported that nitrite accumulation stopped after six cycles with excessive aeration (Qian et al., 2017). Compared to the use of individual strategies, the integration of NH2OH addition and real-time aeration control is favorable for nitritation operation.
operational period, NAR increased to 94.7% in SBR3 with the addition of 4.5 mg/L NH2OH, which was higher than that of SBR1 (46.5%) and SBR2 (80.7%). Therefore, 4.5 mg/L was selected as the optimal NH2OH concentration for the establishment of nitritation in this study.
3.2. Nitrogen removal performance using the NH2OH addition and realtime aeration control strategy The NH2OH addition and real-time aeration control strategy was investigated in the SBR treating real sewage. The whole experimental period was divided into three phases. In phase I, complete nitrification of sewage was achieved. The influent ammonium concentration was maintained at an average of 70.5 ± 6.6 mg N/L and the aeration time was fixed to 240 min (Figs. 1b and 2). As shown in Fig. 2, the average ammonium, nitrite and nitrate concentrations in effluent were 0.5, 0.3 and 36.9 mg N/L, respectively. Influent ammonium was fully oxidized to nitrate during the aerobic period and steady complete nitrification was obtained. In phase II, 4.5 mg/L NH2OH was added to the SBR daily. After the addition of NH2OH, the effluent nitrate concentration declined immediately, accompanied by an increase in effluent nitrite concentration (Fig. 2). Nitritation was rapidly established in 5 d and the NAR increased to 93.3 ± 1.15% in 19 d with the addition of NH2OH once per day. The effectiveness of NH2OH addition on nitritation start-up was consistent with the findings of previous studies (Xu et al., 2012; Li et al., 2019). In phase III, NH2OH addition was ceased and the aeration time was controlled in real-time, with the aeration length determined based on 3
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Fig. 2. Profiles of ammonium, nitrite, nitrate concentrations, ammonium removal efficiency and nitrite accumulation ratio (NAR) in the SBR during the experimental periods (NAR%=NO– 2-N/NO– x-N × 100, NO– x-N = NO– 2-N + NO– 3-N). The different phases of SBR was indicated by lines.
further preventing the growth of NOB (Fig. 3). Therefore, the real-time aeration control strategy effectively prevented the conversion of nitritation to nitrification. Importantly, the variation in relative abundances of Nitrospira and Nitrobacter, the two main NOB genera, were significantly different during the whole operational period. Nitrospira relative abundance decreased from 0.47 ± 0.05% to 0.15 ± 0.02% under NH2OH suppression, while Nitrobacter relative abundance only declined from 0.21 ± 0.07% to 0.13 ± 0.003% (Fig. 4b). This indicated that Nitrobacter was more tolerant to NH2OH inhibition than Nitrospira, which may be caused by differences in microbial characteristics (Vela et al., 2018). During the real-time aeration period that followed, Nitrobacter relative abundance gradually decreased from 0.13 ± 0.01% to less than 0.01%, compensating for the strong tolerance of Nitrobacter to NH2OH inhibition (Fig. 4b). The severe Nitrobacter inhibition induced using the real-time aeration control strategy has been reported in the literature previously, with Nitrobacter population decreasing by 93%, while Nitrospira population remained unchanged (Lee et al., 2013). Therefore, low abundances of Nitrobacter and Nitrospira were both achieved via the NH2OH addition and real-time aeration control strategy, enhancing the stability of sewage nitritation.
3.3. qPCR-based analysis during the start-up and maintenance of sewage nitritation For insight into the effect of combined NH2OH addition and realtime control on sewage nitritation, the activity and abundance of functional bacteria were analyzed in the SBR, including AOB and NOB (Nitrobacter and Nitrospira). With the addition of NH2OH, AOB activity increased from an initial level of 6.04 ± 0.50 mg N/(g VSS·h) on day 8–11.43 ± 0.32 mg N/(g VSS·h) on day 45, while NOB activity in the SBR declined obviously (Fig. 4a). NH2OH appeared to induce selective inhibition on AOB and NOB, which resulted in an increase of the NAR in the SBR (Fig. 2). The increase in AOB activity was due to a reduction in competition for oxygen when NOB activity was severely inhibited by NH2OH. By using the real-time aeration control strategy in phase III, AOB activity remained largely stable, although NOB activity further decreased to 0.05 ± 0.003 mg N/(g VSS·h) on day 83 (Fig. 4a). These results indicated that NH2OH addition and real-time aeration control both suppressed NOB activity. Not only AOB activity, AOB population also increased during NH2OH addition phase. The relative abundance of AOB was enriched from 0.05 ± 0.002% on day 8 to 0.16 ± 0.01% on day 45 (Fig. 4b). However, the relative abundance of NOB decreased from 0.68 ± 0.07% to 0.28 ± 0.02%, which indicated that the addition of NH2OH also caused washout of NOB in addition to NOB suppression. The inhibition on NOB might because that NH2OH inhibits the synthesis of nitrite oxidoreductase enzyme, suppressing the growth of NOB (Xu et al., 2012). With the real-time aeration control strategy, the relative abundance of NOB further declined to 0.02 ± 0.002%, showing that real-time aeration control also contributed to NOB washout from the system. When the real-time aeration control strategy was adopted in phase III, nitrite was not oxidized by NOB during the aeration phase due to out-competition by AOB. Moreover, aeration was stopped when ammonium oxidation was complete and the settling phase started,
3.4. Potential applications in sewage treatment This study proposes a promising strategy for efficient sewage nitritation. NH2OH addition induced a decrease in NOB activity and abundance, accompanied by an enrichment of AOB and the rapid startup of nitritation. Real-time aeration control was beneficial for maintaining low NOB activity and abundance, inducing stable nitritation with a NAR of 96.9 ± 2.2% between days 45–100. There are several advantages to the use of this combined strategy. Firstly, the start-up period required to establish nitritation is rapid, with nitritation achieved after 5 days of NH2OH addition (4.5 mg/L) in the present study, which was faster than other control methods reported 4
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Fig. 4. Variations of nitrifying bacteria activities (a) and relative abundance of AOB and NOB (Nitrobacter and Nitrospira) (b) in the SBR during the experimental periods (relative abundance of AOB=(absolute abundance of AOB/absolute abundance of total bacteria) × 100%; relative abundance of Nitrobacter=(absolute abundance of Nitrobacter/absolute abundance of total bacteria) × 100%; relative abundance of Nitrospira=(absolute abundance of Nitrospira/absolute abundance of total bacteria) × 100%). Error bars indicate the standard deviation. Fig. 3. Variation of ammonium, nitrite and nitrate concentration in typical cycles on Day 8, Day 27 and Day 105 of SBR.
4. Conclusions This study investigated the feasibility of the NH2OH addition and real-time aeration control strategy on sewage nitritation operation. Sewage nitritation was achieved rapidly in 5 d due to daily NH2OH (4.5 mg/L) dosing. Then the nitritaion maintained at a stable level with a NAR of 97%, using a real-time aeration control strategy following the cessation of NH2OH addition. Microbial community analysis indicated that the reactor population was optimized with wash-out of NOB (Nitrobacter and Nitrospira) and enrichment of AOB. The strategy proposed in this study is a promising approach for practical application in sewage nitritation.
previously in the literature. Secondly, nitritation is stable as NOB can be effectively eliminated, including Nitrobacter and Nitrospira. It is widely accepted that Nitrobacter and Nitrospira are two prevailing NOB populations in most wastewater treatment systems (Shu et al., 2015). Therefore, this strategy is feasible for practical sewage nitritation application. The combined strategy can establish stable nitritation as numerous genera of NOB can be simultaneously suppressed and eliminated. With the use of single control strategies, stable operation is more difficult due to the gradual enrichment of specific NOB genera under certain condition. As reported in previous research, the NOB community shifted from Nitrobacter to Nitrospira under long-term low DO concentration condition, resulting in deterioration of sewage nitritation (Liu and Wang, 2013). Thus, single control strategies are not sufficient for effective nitritation operation, while combining two or more control strategies are more favorable for nitritation stability and ensuring complete washout of NOB. Recent reports show that sludge treatment with FA and FNA effectively achieved selective inhibition of Nitrospira and Nitrobacter, resulting in stable nitritation (Duan et al., 2019). In the present study, the NH2OH addition and real-time aeration control strategy exhibited excellent operational performance. Nitrospira was mostly eliminated via NH2OH inhibition and Nitrobacter was severely suppression via real-time aeration control (Fig. 4b). Therefore, the combined method proposed in this study may have high application potential and be significant for effective sewage nitritation process operation.
Acknowledgements This research was financially supported by Major Science and Technology Program for Water Pollution Control and Treatment (2017ZX07102-003), National Natural Science Foundation of China (21777005), National Natural Science Foundation of China (51978007), 111 Project (D16003) and the Funding Projects of Beijing Municipal Commission of Education. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122183. References APHA, 1998. Standard methods for the examination of water and wastewater. American Public Health Association, Washington, DC, USA.
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