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Improving nitrogen removal in biological aeration filter for domestic sewage treatment via adjusting microbial community structure
Bioresource Technology 293 (2019) 122006
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Improving nitrogen removal in biological aeration filter for domestic sewage treatment via adjusting microbial community structure
T
Bin Cuia, Qing Yanga, , Yanping Zhangb, Xiuhong Liua, Wenjun Wua, Jianmin Lia ⁎
a
National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, Beijing University of Technology, Beijing 100124, PR China Key Laboratory of Cleaner Production and Integrated Resource Utilization of China National Light Industry, Beijing Technology and Business University, Beijing 100048, PR China
b
GRAPHICAL ABSTRACT
ARTICLE INFO
ABSTRACT
Keywords: Biological aeration filter Backwash Biofilm Nitrogen removal Microbial community structure
The rapid growth of nitrite-oxidizing bacteria (NOB) in reactor prevents the application of anaerobic ammonium oxidation (anammox) technology to main-stream wastewater treatment. How to eliminate NOB and reserve anaerobic ammonium oxidation bacteria (AnAOB) simultaneously becomes the biggest challenge. In this study two coupled biological aeration filters (BAFs) were built up to treat domestic sewage. In BAF1 nitrogen removal concentration was 21.4 mg/L via heterotrophic denitrification pathway. Backwash was conducted to BAF2 to improve nitrogen removal performance. After backwash Nitrospira proportion declined from 10.8% to 2.1%, while Candidatus Kuenenia percentage increased from 5.6% to 10.2%. Nitrogen removal concentration improved from 8.6 mg/L to 22.8 mg/L via anammox pathway in BAF2, and total nitrogen removal concentration reached to 44.2 mg/L in two coupled BAFs during aeration process. These findings could provide a new strategy for the application of anammox technology to main-stream wastewater treatment.
1. Introduction Partial nitrification-anammox process is considered as the most energy-efficient environmental biotechnology for nitrogen removal from wastewater (Ding et al., 2018a,b). More than one hundred fullscale anammox installations have been built around the world by 2014. However, most of these are applied to treat high strength ammonium wastewater, such as landfill leachate, sludge digestion liquid and industrial wastewater (Fux and Siegrist, 2004; Li et al., 2018a,b). Because
⁎
the growth of NOB could be inhibited by the free ammonia (FA) and free nitrous acid (FNA) in the wastewaters with high nitrogen concentrations (Qian et al., 2017; Ma et al., 2017). How to achieve nitrogen removal via anammox process for mainstream wastewater treatment has become the hot topic all over the world (Ma et al., 2016; Zhang and Liu, 2014). It is unfeasible to keep long-term and stable partial nitrification process that prevents the application of two-stage partial nitrification-anammox process to main-stream wastewater treatment. So many studies focused on achieving completely autotrophic nitrogen
Corresponding author. E-mail address: [email protected] (Q. Yang).
https://doi.org/10.1016/j.biortech.2019.122006 Received 13 April 2019; Received in revised form 9 August 2019; Accepted 10 August 2019 Available online 13 August 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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removal in a reactor that relied on the interactions of various functional bacteria (Chen et al., 2019; Ding et al., 2018a,b). The growth of AnAOB depends on the nitrite from AOB, and they also compete for ammonia simultaneously, moreover NOB and AnAOB also compete for nitrite. So it is crucial to control different metabolism activities of multiple microorganisms for achieving advanced nitrogen removal. Many operation strategies were tried to improve nitrogen removal, including controlling low dissolved oxygen (DO) concentration, alternating anoxic and aerobic conditions, controlling aeration time (Laureni et al., 2016; Ma et al., 2013; Reino and Carrera, 2017). Partial nitrification-anammox process for domestic sewage treatment with intermittent aeration mode was achieved, but the seeding sludge was obtained from a partial nitrification reactor immediately (Miao et al., 2018). Simultaneous partial nitrification, anammox and denitrification process for domestic sewage treatment was also reported, but high ammonia wastewater was treated to inhibit NOB at the initial phase (Ding et al., 2018a,b). For the above studies, most of operation strategies were so complex that they were difficult to implement in engineering. Thus it is essential to develop feasible and manageable operation methods for the application of anammox technology in mainstream wastewater treatment. The long generation time of AnAOB results in the low sludge output, while it also brings about the problem that it is difficult to enrich enough biomass (Jia et al., 2017). It is vital to maintain sufficient AnAOB in reactor, because of the large treatment volume of municipal wastewater. For conventional activated sludge system, it is difficult to reserve enough anammox biomass due to the periodical sludge discharge. The most feasible and effective method to improve the AnAOB accommodation capacity are granulation and biofilm formation (Chen et al., 2019, 2018). Granular sludge is suitable for high nitrogen concentration wastewater treatment (Bagchi et al., 2016; Lin and Wang, 2017), while biofilm process is widely used in municipal wastewater treatment. Gradient distribution of various substrates leads to the stratified structure of biofilm, which is helpful for the enrichment of AnAOB and AOB simultaneously. Therefore, biofilm reactor has great potential in achieving advanced nitrogen removal via anammox pathway. BAF is a conventional biofilm reactor, it is widely applied for organic matter oxidation and nitrification process in wastewater treatment plant. BAF combines the advantages of small footprint, great capacity of treatment volume and strong resistance to impact load (AbouElela et al., 2015; Liu et al., 2017). Furthermore, the biofilm is immobilized in BAF, and AnAOB can be reserved in system efficiently. In this paper, two BAFs treating domestic sewage were built up in laboratory aimed to: (a) optimize the running conditions of two BAFs to improve nitrogen removal, (b) determine an available operation strategy for BAF2 to improve nitrogen removal via anammox pathway, (c) analyze the mechanism of improving nitrogen removal.
Table 1 Operation parameters of BAF1 and BAF2. Operation parameters
Backwash parameters
Time(d)
Gas and water ratio
Backwash Mode
Backwash strength (L/(m2·s))
Time (min)
BAF1
1–61 62–190 191–250
10:1 5:1 8:1
Gas Gas + Water Water
21.6 21.6 + 10.8 10.8
3 4 6
BAF2
1–26 27–146 147–216
5:1 1.5:1 3:1
Gas Gas + Water Water
14.4 14.4 + 7.22 5.42
2 3 3
2.2. Reactors setup and operation Two same BAFs were built up with a working volume of 18.4 L in this study. Volcanic filter media with particle size 3–5 mm was packed at a bed depth of 70 cm. Supporting layer was under the filter media layer with the height of 10 cm. BAF1 was supplied with domestic sewage and its effluent was fed to BAF2. Hydraulic retention time of two BAFs was 90 min, and temperature was controlled at 25 °C. BAF1 was backwashed 18 days intervals approximately. The detailed operation parameters of BAF1 and BAF2 were listed in Table 1. 2.3. Analytical methods All samples were filtered using 0.45 um syringe filter prior to analyze. NH4+-N, NO2–-N, and NO3–-N were analyzed according to standard method (APHA, 2017). COD was measured by COD quick-analysis apparatus (Lian-hua Tech. Co., Ltd, 5B-1, China). Dissolved oxygen (DO) concentration was detected by DO probe (WTW3420, Germany). MLVSS was measured by weight method. The gaseous N2O was collected and measured by gas chromatography with electron capture detector (Agilent 7890, U.S.). 2.4. DNA extraction and Real-time quantitative polymerase chain reaction (QPCR) Biofilm samples were collected and dried by freeze dryer (Labconco, USA) at the different periods of BAF. Genomic DNA was extracted from the dried biofilm sample using the Fast DNA SPIN Kit for Soil (MP Biomedicals, Solon, OH) according to the manufacturer’s instruction. DNA concentration was determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Schwerte, Germany). Real-time quantitative PCR were conducted using the fluorescent dye SYBR-Green approach in a MX3000P Real-Time PCR system (Agilent Technologies, USA). The abundances of functional genes for ammonia-oxidizing archaea (AOA-amoA), AOB (AOB-amoA), AnAOB (AnAOB-hzsB), and DNB (DNB-nirK, DNB-nirS), 16s rRNA genes for Nitrobacter (NOB-16s) and Nitrospira (NOS-16s) were quantified. The standard curve was conducted using ten-fold serial dilutions of the corresponding genes with plasmid DNAs of known concentrations. The results with amplification efficiency in a range from 90–110% and correlation coefficient above 0.95 were employed. Finally, the copy numbers of unit mass dry sludge for each of these genes were calculated.
2. Materials and methods 2.1. Characteristics of domestic sewage and seeding sludge The raw domestic sewage used in this study was taken from a septic tank in the residential area of Beijing University of Technology (Beijing, China). The main characteristics of the wastewater were: COD = 182 ± 32 mg/L; NH4+-N = 60 ± 13 mg/L; NO2––N = 0.32 ± 0.23 mg/L; NO3–-N = 0.82 ± 0.61 mg/L. The seeding sludge was the returned sludge of an A/A/O process from a Municipal Wastewater Treatment Plant in Beijing, China. The biofilm of the two BAFs were both cultivated by the activated sludge. On the 40th day, 300 mL anammox biofilm with MLSS of 10200 mg/L was seeded into BAF2. The anammox biofilm was collected from an anammox biofilter in our laboratory that has been running for more than two years steadily.
2.5. High-throughput sequencing After detecting the concentrations of genomic DNA, PCR was conducted to amplify the genes of 16s rRNA based the primers of 338F and 806R as we used before (Cui et al., 2017). The sequencing was carried out on Illumina HiSeq 2500 platform (Illimina, USA) at Biomarker technologies CO., LTD(Beijing, China). The high quality reads were attained after the optimization of the raw reads, and the high quality reads were divided into different Operational Taxonomic Units (OUT) 2
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at 97% sequence similarity. The microbial compositions were annotated on Silva database. 2.6. Microbial specific activity studies
(NO2
Neff )
(NO3
TINRE = TIN /[(NH4+
(NH4+
Ninf ) + (NO3
Ninf )]
(8)
0.26) × 1.32
Neff ) (NO3
NOBbio = 113/14 ×
Ninf )
(NH4+
NAnAOB ) × 0.26
NH4+
NAOB × Q × 0.01
NO2
NNOB × Q × 0.003
1
55
AnAOBbio = 24.1/14 ×
NH4+
NAnAOB × Q × 0.066
1
(10) (11) (12)
Q was the treatment volume of BAF2, it was 43.2L/d. 3. Results and discussion 3.1. Performances of BAF1 and BAF2 3.1.1. Performance of BAF1 under different gas and water ratios BAF1 was started-up at gas and water ratio of 10:1, after that the gas and water ratio was controlled at 5:1 and 8:1, respectively. Organic matter oxidation, nitrification and denitrification reactions occurred in BAF1, which resulted in part of total inorganic nitrogen (TIN) being removed. The COD removal efficiency was higher than 70%, remaining COD was less than 50 mg/L, and no nitrite was detected in effluent (Fig. 1). When gas and water ratio was controlled at 10:1, average NH4+-N removal efficiency was 36.4% and 13.1 mg/L NO3–-N was produced in effluent. In order to reduce energy consumption, the gas and water ratio was reduced to 5:1 from the 62th day. Effluent NO3–-N concentration decreased greatly, but the improving denitrification activity resulted in the ΔTIN increasing to 13.1 mg/L. When gas and water ratio was adjusted to 8:1 on the 191th day, average effluent NO3–-N was only 4.3 mg/L, and the concentration of removal nitrogen reached to 21.4 mg/L. Therefore, gas and water ratio of 8:1 was the proper condition for BAF1 to achieve ammonia oxidation and nitrogen removal.
(2)
where NH4+-Ninf, NO2–-Ninf and NO3–-Ninf were the influent NH4+-N, NO2–-N, and NO3–-N concentration (mg/L); NH4+-Neff, NO2–-Neff and NO3–-Neff were the effluent NH4+-N, NO2–-N and NO3–-N concentration (mg/L), respectively.
3.1.2. Performance of BAF2 under different conditions BAF2 was fed with domestic sewage, gas and water ratio was
2.7.2. Calculations of nitrogen concentration transformed by AOB, NOB and AnAOB Microbial metabolism equations of AOB, NOB and AnAOB were listed as Eqs. (3–5). The concentration of NH4+-N degraded by AnAOB: NH4+ NAnAOB , the concentration of NH4+-N degraded by AOB: NH4+ NAOB , the concentration of NO2–-N degraded by AnAOB: NO2 NAnAOB , and the concentration of NO2–-N degraded by NOB: NO2 NNOB were calculated by the Eqs. (6–9), respectively.
NH4+ + 1.382O2 + 0.09HCO3
NNOB = (NO3
(7)
NAnAOB )
55
(1)
Ninf ) + (NO2
NO2
(NH4+
Neff )
1
Neff )
Neff
NAnAOB = TIN /(1 + 1.32
55
TIN Ninf )
NO2
AOBbio = 113/14 ×
2.7.1. Calculations of wastewater quality for effluent The concentration of removed total inorganic nitrogen (ΔTIN) and total inorganic nitrogen removal efficiency (TINRE) were calculated using Eqs. (1) and (2), respectively.
Ninf ) + (NO3
(NH4+
Ninf )
2.7.3. Calculations of growth biomass The growth biomass of AOB, NOB and AnAOB were AOBbio, NOBbio, and AnAOBbio, respectively, and the calculation equations were as following (10–12):
2.7. Calculations
Ninf )+(NO2
NAOB = (NH4+
(9)
Batch tests were conducted to investigate the specific nitrogen conversion rates of biofilm samples before and after backwash for BAF2. Before and after backwash 40 mL filter media was collected from BAF2, respectively. The filter media was washed with PBS thrice times before reaction. For each sample, three reaction activities were tested, including aerobic NH4+-N oxidation, aerobic NO2–-N oxidation, anaerobic NH4+-N and NO2–-N oxidation. The batch tests were carried out in a 500 mL reactor as we used before (Cui et al., 2017). For the reaction medium, it contained (per liter): 11.1 mg KH2PO4, 6 mg MgSO4·7H2O, 3 mg CaCl2·2H2O and 1 mL trace element stock solution. The trace element stock solution contained (per liter): 1.5 g FeCl3·6H2O, 0.03 g CuSO4·5H2O, 0.12 g MnCl2·4H2O, 0.06 g Na2MoO4·2H2O, 0.12 g ZnSO4·7H2O, 0.15 g CoCl2·6H2O, 0.18 g KI, 0.15 g H3BO3, and 10 g ethylenediaminetetraacetic acid (EDTA). NaHCO3 was used to supply alkalinity and adjust pH to 7.3–7.5. Before each test, nitrogen gas was pumped into medium to reduce DO concentration, for aerobic reaction DO was controlled at 2–3 mg/L, for anaerobic reaction, DO was reduced to lower than 0.1 mg/L. During the reaction process, pH and DO probes (WTW3420, Germany) were used to monitor online. Water samples were collected at intervals for NH4+-N, NO2–-N and NO3–-N measurement. After each batch test, the biomass on the filter media was measured for the calculation of specific nitrogen degradation rates.
= (NH4+
NH4+
0.982NO2 + 1.036H2 O + 0.018C5 H7 O2 (3)
N + 1.89H+
NO2 + 0.003NH4+ + 0.485O2 + 0.015HCO3 + 0.012H+ (4)
NO3 + 0.009H2 O + 0.003C5 H7 O2 N NH4+ + 1.32NO2 + 0.66HCO3 + 0.13H+
1.02N2 + 0.066CH2 O0.5
N0.15 + 0.26NO3 + 2.03H+ NH4+
NAnAOB = TIN /(1 + 1.32
0.26)
(5)
Fig. 1. Variations of COD, NH4+-N, NO3–-N, ΔTIN and TINRE in influent and effluent of BAF1 under different gas and water ratios.
(6) 3
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DO concentration led to the better performance in BAF2. Recently similar result was also discovered in activated sludge system that high DO was benefit for achieving partial nitrification (Bao et al., 2017). When influent NH4+-N concentration declined, high DO concentration led to high AOB reaction rate and more NO2––N was captured by AnAOB to achieve autotrophic nitrogen removal (Regmi et al., 2014; Guo et al., 2010). So gas and water ratio was operated at 3:1 to control DO concentration of 2.0 mg/L in the following running. Furthermore, avoiding the inhibition of high DO concentration to AnAOB activity, different strategies were adopted to optimize the activities of various functional microorganisms and achieve great nitrogen removal performance. Aerobic and anaerobic interval model was often used in activated sludge system to promote NO2––N accumulation (Gilbert et al., 2014; Miao et al., 2018), and anaerobic condition is also helpful for anammox reaction. So BAF2 was operated in an aerobic-anaerobic alternating mode that was aerobic for the first 7 days and anaerobic for the next 7 days. In aerobic period, gas and water ratio was controlled at 3:1. In anaerobic period, BAF1 effluent adding NaNO2 was fed to BAF2. During the 60 days, nitrogen removal concentration reached to 14.5 mg/L in aerobic condition. But the nitrogen removal performance was not further improved when anaerobic period increased from 7 days to 15 days. Nitrogen removal performance was not improved further via optimizing microbial activity. It was speculated that NOB abundance was much higher than AnAOB after 173 days. So BAF2 was backwashed on the 174th day, the backwash strength was controlled much weaker than that of BAF1 to avoid the excessive shedding of biofilm. After backwash gas and water ratio was still kept at 3:1. The nitrogen removal concentration and TINRE reached to 26.1 mg/L and 48.9%, respectively, and no NH4+-N was detected in effluent. On the 206th day nitrogen removal concentration of BAF2 began to decline, so it was backwashed again on the 214th day. After that nitrogen removal ability revived again, indicating backwash could promote nitrogen removal by adjusting microbial community structure.
Fig. 2. Variations of COD, NH4+-N, NO3–-N, ΔTIN and TINRE in influent and effluent of BAF2 under different conditions.
controlled at 5:1 during the initial period (Fig. 2). After 26 days it was coupled with BAF1, gas and water ratio was adjusted to 1.5:1. From the 30th day nitrification activity improved gradually, but TINRE was always less than 15%. On the 97th day TINRE increased to 80% sharply, and only 3.5 mg/L NO3–-N remained in effluent. While excellent nitrogen removal performance only kept for 20 days. After 117 days, ΔTIN declined to 8.6 mg/L, and remaining NO3–-N increased to 22.1 mg/L in effluent. The cause of changing nitrogen removal performance in BAF2 was analyzed further. Since constant gas and water ratio was kept, the fluctuation of domestic sewage resulted in DO concentration changing in BAF2. As Fig. 3 showed that on the 93th day influent NH4+-N was 27.9 mg/L, along the filter height effluent NH4+-N and TIN both declined gradually. On the 100th day influent NH4+-N declined to 20.2 mg/L, most of nitrogen was degraded at the bottom of 40 cm filter media, and average DO concentration was 2.38 mg/L. On the 120th day influent NH4+-N went up to 33.8 mg/L, average DO concentration dropped to 1.20 mg/L, and only 6.31 mg/L nitrogen was removed. According to the variations of DO concentration and effluent quality, it could be found that the higher
3.2. Analysis of nitrogen transformation Only little COD degradation indicated that heterotrophic denitrification reaction was weak in BAF2, and anammox reaction was the
Fig. 3. Variations of effluent NH4+-N, NO3–-N, TIN, COD and DO concentration along BAF2 height. 4
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main nitrogen removal pathway. The functional bacteria involving to nitrogen transformation mainly included AOB, NOB and AnAOB in BAF2. Excellent performance of nitrogen removal depended on the complex interaction mechanisms among these bacteria. AOB and NOB collaborated with each other to complete nitrification reaction. The growth of AnAOB relied on the NO2–-N generating from AOB, but it also competed with AOB for NH4+-N. Furthermore, AnAOB competed for NO2–-N with NOB. The concentrations of nitrogen utilized by AOB, NOB and AnAOB were calculated according to stoichiometric reaction equation and removal TIN in BAF2. From the 60th day to the 96th day, little nitrogen was degraded by AnAOB, AOB and NOB completed nitrification process. Since the declining of influent NH4+-N, DO concentration increased in BAF2. High DO concentration improved AOB oxidation rate greatly, which led to the activity difference between AOB and NOB (Regmi et al., 2014; Guo et al., 2010). More NO2–-N was utilized by AnAOB in biofilm interior. Nitrogen transformation was compared in BAF2 under two control strategies, after backwash the removal concentration of NH4+-N and NO2–-N by AnAOB were much higher than that in aerobic and anaerobic interval period. It indicated that backwash played crucial role in improving nitrogen removal ability of BAF2 via optimizing microbial community structure. Balance analysis of nitrogen was calculated under different operation periods for BAF2 (Fig. 4). Gaseous N2O was detected in the three periods. From the 118th day to the 147th day, nitrification was the main nitrogen transformation pathway, 0.032 mg N2O-N was generated for per liter wastewater treatment and 15.51% of the influent nitrogen was transformed into biomass and N2. In aerobic-anaerobic alternating period, the proportion of remaining NH4+-N in effluent reduced, N2O-N emission decreased to 0.07%, and TINRE increased to 38.69%. Anaerobic operation was benefit for improving nitrogen removal. After backwash some biofilm was washed out from BAF2, the proportion of nitrogen converting to biomass and N2 further increased to 52.87%, and the percent of N2O-N was only 0.01%. Above results illustrated that backwash not only improved anammox ability to promote nitrogen removal, but it also reduced N2O emission from nitrifier denitrification and hydroxylamine oxidation pathways.
Fig. 5. Variations of gene abundances in BAF2 at different periods (Seed, 110th, Be-wash, Wash and Af-wash represented sample were collected from the seed sludge, the 110th day, before backwash, shedding biofilm during backwash and after backwash, respectively).
backwash were also measured via QPCR. The similar changing tendency suggested the efficient elimination of Nitrobacter and Nitrospira from BAF2 through backwash. Autotrophic biofilm formed via the aggregation of AOB, NOB and AnAOB in BAF2. Since the different requirements of oxygen, AOB and NOB tended to aggregate in the outer layer of the biofilm that was benefit for the capture of oxygen and substances. AnAOB tended to enrich in the internal anoxic or anaerobic area that was close to the surface of filter media (Miao et al., 2016). In the autotrophic biofilm system, part of NO2–-N generated from AOB was utilized by NOB immediately, the other spread into the interior of biofilm and was captured by AnAOB to release N2. The depth of biofilm increased gradually with the running of BAF2, and the resistance of substance transfer also enhanced at the same time, which could reduce the ability of AnAOB to compete for NH4+-N and NO2–-N with AOB and NOB, respectively. Finally, NO3–-N and TIN concentration increased in effluent. So it was necessary to backwash regularly for BAF2. On the one hand, AOB and NOB existed on the outer layer of biofilm could slough off first under the actions of hydraulic forces (Lawson et al., 2017). On the other hand, hydraulic shear could weaken the biofilm depth, and promote substances transfer into biofilm interior (Dror-Ehre et al., 2010). During the 40 days between twice backwashes of BAF2, according to the stoichiometric reaction equations it was calculated that the growth biomass of AOB, NOB and AnAOB were 12.209, 0.983 and 3.388 g, respectively. The proliferative biomass of AOB was higher than NOB and AnAOB obviously, which accounted for massive NH4+-N was utilized by AOB. Backwash contributed to optimizing the abundance of various functional bacteria in BAF2.
3.3. Mechanisms of microbial community adjustment 3.3.1. Abundance of functional microorganism The variations of functional microorganism abundance in different periods of BAF2 were showed in Fig. 5. The abundance of nirK and nirS were the highest in seeding sludge, indicating much denitrifier was enriched. However, because of little biodegradable organic matter, the abundance of denitrifier decreased greatly on the 110th day. In seeding sludge the abundance of AOB, NOB and AnAOB was less than 106 copies/mg dry sludge, but they were all enriched on the 110th day. On the 175th day before backwash, the abundances of AOB, Nitrobacter and Nitrospira all reached to 107 copies/mg dry sludge. After backwash the abundance of remaining AnAOB increased, while the abundances of AOB, Nitrobacter and Nitrospira decreased obviously. Furthermore, the variations of functional microorganism abundances during the second
Fig. 4. Transformation forms and proportions of nitrogen for BAF2 at different periods. 5
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Fig. 6. Microbial community structures in genus level for BAF2 before and after backwash (Be-wash, Wash and Af-wash represented sample were collected from before backwash, shedding biofilm during backwash and after backwash, respectively).
3.3.2. Microbial community structures After backwash the microbial community structure of BAF2 changed obviously (Fig. 6). Before backwash, the dominant microorganism at genus level included norank_f_Anaerolineaceae, Nitrospira, Candidatus Kuenenia, and Candidatus Brocadia. After backwash the proportions of some nitrogen transformation bacteria varied obviously, the percent of Nitrospira decreased greatly, but Candidatus Kuenenia and Candidatus Brocadia increased clearly. For AOB, its ratio declined from 0.95% to 0.21%. For the microbial structure of the removed biofilm, it included Nitrospira, Denitratisoma and norank_f_Anaerolineaceae mainly. The proportion of AnAOB in the sloughing biofilm was much lower than that remaining in BAF2, while the ratio of AOB was much higher correspondingly. Microbial community structures of the three samples illustrated that AnAOB aggregated in the inner layer of the biofilm, AOB and NOB distributed in the outer layer. Although much microorganism was eliminated from BAF2 during backwash, sufficient AnAOB was reserved in system that facilitated nitrogen removal.
backwash can influence the composition of microorganism. After backwash the thinner biofilm was benefit to substance transfer, and the declining abundances of AOB and NOB was favorable to nitrogen remove via anammox pathway. In this study, it was crucial to inhibit NOB activity to facilitate autotrophic nitrogen removal in BAF2. According to the influent quality, different gas and water ratios were controlled to achieve high DO concentration. Improving AOB reaction rate with high DO concentration caused more NO2–-N was utilized by AnAOB, remaining nitrogen declined in effluent. However, NOB abundance kept increasing in BAF2 even with high DO concentration. Based on the microbial hierarchy of the biofilm, backwash was operated periodically to wash out much Nitrospira and Nitrobacter from BAF2. Declining NOB abundance caused nitrite oxidation rate reducing, and effluent TIN was also reduced. Nitrogen removal performance improved greatly. Therefore, it is an efficient and manageable strategy to optimize the microbial community structure via backwash to promote autotrophic nitrogen removal in BAF2.
3.3.3. Reaction activities of BAF2 before and after backwash The reaction activities of AOB, NOB and AnAOB under aerobic and anaerobic conditions were measured in batch tests. The results showed that the activities of aerobic ammonia oxidation, nitrite oxidation, and anaerobic ammonium oxidation all declined, which was resulted from the shedding of biofilm (Table 2). Because the less AnAOB was eliminated from BAF2, the anaerobic ammonium oxidation rate did not decline much. In addition, the ratio of specific degradation rate of TIN and NH4+-N under aeration condition increased clearly after backwash, which indicated AnAOB could compete out NOB for NO2–-N after backwash. The discrepancy of reaction rates among several functional microorganisms was the most crucial factor influencing the performance of autotrophic nitrogen removal process. Backwash was only suit to the reactor that biofilm and filter media are both fixed, and
3.4. Advantages of improving nitrogen removal from BAF For domestic sewage when NH4+-N concentration reached to 70 mg/L, the ratio of gas and water of BAF1 and BAF2 was controlled at 8:1 and 3:1, nitrogen removal concentration reached to 44.2 mg/L in this two coupled BAFs process, and effluent COD concentration was less than 50 mg/L. The carbon source from domestic sewage was used for denitrification to remove 21.4 mg/L nitrogen in BAF1, and 22.8 mg/L nitrogen was removed through anammox pathway in BAF2. Moreover, comparing with nitrification process greenhouse gas emission declined greatly. Traditional A/O or A/A/O process for biological nitrogen removal depends on the nitrification and denitrification technology, all the influent NH4+-N is oxidized completely, and its effluent remaining 20 ∼ 25 mg/L NO3–-N needs tertiary treatment generally (Liu et al., 2016). However, part of influent NH4+-N was not oxidized into NO2–-N or NO3–-N during aeration process in this study, more than 30% of the influent nitrogen was removed by anammox pathway. Recently, some research also focused on improving nitrogen removal during aeration process. Improving nitrogen removal during partial nitrification process was also reported in SBR system (Zhou et al., 2018). The TINRE could reach to 45% with 4.5 h aeration, but the removal nitrogen concentration was only 12 mg/L. While in this study, the hydraulic retention time was only 3 h for two coupled BAFs, and the treatment capacity of BAF was higher than SBR obviously. Now partial nitrification was
Table 2 Activities of functional microorganisms in BAF2 before and after backwash. Reaction Conditions
Types of specific degradation rate
Before backwash (mg N/(g VSS h))
After backwash (mg N/(g VSS h))
Aerobic NH4+-N
NH4+-N TIN
22.02 ± 1.44 13.62 ± 0.89
5.85 ± 0.15 4.16 ± 0.10
Aerobic NO2–-N
NO2–-N
14.06 ± 0.92
0.99 ± 0.02
Anaerobic NH4+-N and NO2–-N
NH4+-N NO2–-N TIN
6.48 ± 0.86 8.01 ± 0.62 13.91 ± 0.64
4.86 ± 0.62 5.64 ± 0.41 8.14 ± 0.21
6
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achieved widely by the means of on-line controlling (Yang et al., 2007), but it is still a challenge to keep long-term stable. In this paper, periodical backwash was adopted to optimize the activities of various microorganisms, and stable nitrogen removal performance was achieved in two coupled BAFs. When the concentration of NH4+-N in domestic sewage was less than 40 mg/L, advanced nitrogen removal could be achieved in this two coupled BAFs process, effluent TIN was less than 10 mg/L. When NH4+N concentration reached to 60 mg/L, tertiary treatment was required for the remaining NO3–-N in effluent. Comparing with the conventional nitrification and denitrificaton process, this process showed typical advantages: ①More nitrogen was removed during aeration process; ②Saving energy consumption for aeration; ③No sludge backflow; ④Low effluent turbidity, without secondary sedimentation tank; ⑤High treatment capacity; ⑥Less greenhouse gas N2O emission.
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Response of different nitrospira species to anoxic periods depends on operational DO. Environ. Sci. Technol. 48 (5), 2934–2941. Guo, J., Peng, Y., Huang, H., Wang, S., Ge, S., Zhang, J., Wang, Z., 2010. Short- and longterm effects of temperature on partial nitrification in a sequencing batch reactor treating domestic wastewater. J. Hazard. Mater. 179, 471–479. Jia, F., Yang, Q., Liu, X., Li, X., Li, B., Zhang, L., Peng, Y., 2017. Stratification of extracellular polymeric substances (EPS) for aggregated anammox microorganisms. Environ. Sci. Technol. 51 (6), 3260–3268. 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. Lawson, C.E., Wu, S., Bhattacharjee, A.S., Hamilton, J.J., McMahon, K.D., Goel, R., Noguera, D.R., 2017. Metabolic network analysis reveals microbial community interactions in anammox granules. Nat. Commun. 15416, 1–12. Li, J., Li, J., Gao, R., Wang, M., Yang, L., Wang, X., Zhang, L., Peng, Y., 2018a. A critical review of one-stage anammox processes for treating industrial wastewater: optimization strategies based on key functional microorganisms. Bioresource Technol. 265, 498–505. Li, Z., Kechen, X., Yongzhen, P., 2018b. Composition characterization and transformation mechanism of refractory dissolved organic matter from an ANAMMOX reactor fed with mature landfill leachate. Bioresource Technol. 250, 413–421. Lin, X., Wang, Y., 2017. Microstructure of anammox granules and mechanisms endowing their intensity revealed by microscopic inspection and rheometry. Water Res. 120, 22–31. Liu, H., Zhu, L., Tian, X., Yin, Y., 2017. Seasonal variation of bacterial community in biological aerated filter for ammonia removal in drinking water treatment. Water Res. 123, 668–677. Liu, X., Wang, H., Long, F., Qi, L., Fan, H., 2016. Optimizing and real-time control of biofilm formation, growth and renewal in denitrifying biofilter. Bioresource Technol. 209, 326–332. Ma, B., Peng, Y., Zhang, S., Wang, J., Gan, Y., Chang, J., Wang, S., Wang, S., Zhu, G., 2013. Performance of anammox UASB reactor treating low strength wastewater under moderate and low temperatures. Bioresource Technol. 129, 606–611. Ma, B., Wang, S., Cao, S., Miao, Y., Jia, F., Du, R., Peng, Y., 2016. Biological nitrogen removal from sewage via anammox: Recent advances. Bioresource Technol. 200, 981–990. Ma, B., Yang, L., Wang, Q., Yuan, Z., Wang, Y., Peng, Y., 2017. Inactivation and adaptation of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria when exposed to free nitrous acid. Bioresource Technol. 245, 1266–1270. Miao, L., Wang, S., Cao, T., Peng, Y., Zhang, M., Liu, Z., 2016. Advanced nitrogen removal from landfill leachate via Anammox system based on Sequencing Biofilm Batch Reactor (SBBR): Effective protection of biofilm. Bioresource Technol. 220, 8–16. 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. Qian, W., Peng, Y., Li, X., Zhang, Q., Ma, B., 2017. The inhibitory effects of free ammonia on ammonia oxidizing bacteria and nitrite oxidizing bacteria under anaerobic condition. Bioresource Technol. 243, 1247–1250. Regmi, P., Miller, M.W., Holgate, B., Bunce, R., Park, H., Chandran, K., Wett, B., Murthy, S., Bott, C.B., 2014. Control of aeration, aerobic SRT and COD input for mainstream nitritation/denitritation. Water Res. 57, 162–171. Reino, C., Carrera, J., 2017. Low-strength wastewater treatment in an anammox UASB reactor: Effect of the liquid upflow velocity. Chem. Eng. J. 313, 217–225. Yang, Q., Peng, Y., 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. Zhang, Z., Liu, S., 2014. Hot topics and application trends of the anammox biotechnology: a review by bibliometric analysis. Springerplus 220, 1–8. Zhou, X., Liu, X., Huang, S., Cui, B., Liu, Z., Yang, Q., 2018. Total inorganic nitrogen removal during the partial/complete nitrification for treating domestic wastewater: removal pathways and main influencing factors. Bioresource Technol. 256, 285–294.
4. Conclusions Gas and water ratio of 8:1 was proper for BAF1 to remove nitrogen, and 2.0 mg/L DO was benefit for nitrogen removal from BAF2. Backwash could wash out abundant AOB and NOB that distributed on the outer layer of biofilm and improve the proportion of AnAOB in BAF, the declining reaction activity of NOB promoted nitrogen removal via anammox pathway. For domestic sewage treatment, nitrogen removal concentration reached to 44.2 mg/L during aeration process in the two coupled BAFs, which also could reduce energy consumption and N2O emission simultaneously.
• • •
Acknowledgements This research was supported by Beijing Natural Science Foundation (grant numbers 8182012), and the Open Research Fund Program of Key Laboratory of Cleaner Production and Integrated Resource Utilization of China National Light Industry. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122006. References Abou-Elela, S.I., Fawzy, M.E., El-Gendy, A.S., 2015. Potential of using biological aerated filter as a post treatment for municipal wastewater. Ecol. Eng. 84, 53–57. APHA, 2017. Standard Methods for the Examination of Water and Wastewater, twentythird ed. American Public Health Association, Washington DC, USA. Bagchi, S., Lamendella, R., Strutt, S., Van Loosdrecht, M.C.M., Saikaly, P.E., 2016. Metatranscriptomics reveals the molecular mechanism of large granule formation in granular anammox reactor. Sci. Rep. 6, 1–10. Bao, P., Wang, S., Ma, B., Zhang, Q., Peng, Y., 2017. Achieving partial nitrification by inhibiting the activity of Nitrospira-like bacteria under high-DO conditions in an intermittent aeration reactor. J. Environ. Sci.-China. 56, 71–78. Chen, R., Ji, J., Chen, Y., Takemura, Y., Liu, Y., Kubota, K., Ma, H., Li, Y., 2019. Successful operation performance and syntrophic micro-granule in partial nitritation and anammox reactor treating low-strength ammonia wastewater. Water Res. 155, 288–299. Chen, R., Takemura, Y., Liu, Y., Ji, J., Sakuma, S., Kubota, K., Ma, H., Li, Y., 2018. Using Partial Nitrification and Anammox To Remove Nitrogen from Low-Strength Wastewater by Co-immobilizing Biofilm inside a Moving Bed Bioreactor. ACS Sustain. Chem. Eng. 7 (1), 1353–1361. Cui, B., Liu, X., Yang, Q., Li, J., Zhou, X., Peng, Y., 2017. Achieving partial denitrification through control of biofilm structure during biofilm growth in denitrifying biofilter. Bioresource Technol. 238, 223–231.
7
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Improving nitrogen removal in biological aeration filter for domestic sewage treatment via adjusting microbial community structure
T
Bin Cuia, Qing Yanga, , Yanping Zhangb, Xiuhong Liua, Wenjun Wua, Jianmin Lia ⁎
a
National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, Beijing University of Technology, Beijing 100124, PR China Key Laboratory of Cleaner Production and Integrated Resource Utilization of China National Light Industry, Beijing Technology and Business University, Beijing 100048, PR China
b
GRAPHICAL ABSTRACT
ARTICLE INFO
ABSTRACT
Keywords: Biological aeration filter Backwash Biofilm Nitrogen removal Microbial community structure
The rapid growth of nitrite-oxidizing bacteria (NOB) in reactor prevents the application of anaerobic ammonium oxidation (anammox) technology to main-stream wastewater treatment. How to eliminate NOB and reserve anaerobic ammonium oxidation bacteria (AnAOB) simultaneously becomes the biggest challenge. In this study two coupled biological aeration filters (BAFs) were built up to treat domestic sewage. In BAF1 nitrogen removal concentration was 21.4 mg/L via heterotrophic denitrification pathway. Backwash was conducted to BAF2 to improve nitrogen removal performance. After backwash Nitrospira proportion declined from 10.8% to 2.1%, while Candidatus Kuenenia percentage increased from 5.6% to 10.2%. Nitrogen removal concentration improved from 8.6 mg/L to 22.8 mg/L via anammox pathway in BAF2, and total nitrogen removal concentration reached to 44.2 mg/L in two coupled BAFs during aeration process. These findings could provide a new strategy for the application of anammox technology to main-stream wastewater treatment.
1. Introduction Partial nitrification-anammox process is considered as the most energy-efficient environmental biotechnology for nitrogen removal from wastewater (Ding et al., 2018a,b). More than one hundred fullscale anammox installations have been built around the world by 2014. However, most of these are applied to treat high strength ammonium wastewater, such as landfill leachate, sludge digestion liquid and industrial wastewater (Fux and Siegrist, 2004; Li et al., 2018a,b). Because
⁎
the growth of NOB could be inhibited by the free ammonia (FA) and free nitrous acid (FNA) in the wastewaters with high nitrogen concentrations (Qian et al., 2017; Ma et al., 2017). How to achieve nitrogen removal via anammox process for mainstream wastewater treatment has become the hot topic all over the world (Ma et al., 2016; Zhang and Liu, 2014). It is unfeasible to keep long-term and stable partial nitrification process that prevents the application of two-stage partial nitrification-anammox process to main-stream wastewater treatment. So many studies focused on achieving completely autotrophic nitrogen
Corresponding author. E-mail address: [email protected] (Q. Yang).
https://doi.org/10.1016/j.biortech.2019.122006 Received 13 April 2019; Received in revised form 9 August 2019; Accepted 10 August 2019 Available online 13 August 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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removal in a reactor that relied on the interactions of various functional bacteria (Chen et al., 2019; Ding et al., 2018a,b). The growth of AnAOB depends on the nitrite from AOB, and they also compete for ammonia simultaneously, moreover NOB and AnAOB also compete for nitrite. So it is crucial to control different metabolism activities of multiple microorganisms for achieving advanced nitrogen removal. Many operation strategies were tried to improve nitrogen removal, including controlling low dissolved oxygen (DO) concentration, alternating anoxic and aerobic conditions, controlling aeration time (Laureni et al., 2016; Ma et al., 2013; Reino and Carrera, 2017). Partial nitrification-anammox process for domestic sewage treatment with intermittent aeration mode was achieved, but the seeding sludge was obtained from a partial nitrification reactor immediately (Miao et al., 2018). Simultaneous partial nitrification, anammox and denitrification process for domestic sewage treatment was also reported, but high ammonia wastewater was treated to inhibit NOB at the initial phase (Ding et al., 2018a,b). For the above studies, most of operation strategies were so complex that they were difficult to implement in engineering. Thus it is essential to develop feasible and manageable operation methods for the application of anammox technology in mainstream wastewater treatment. The long generation time of AnAOB results in the low sludge output, while it also brings about the problem that it is difficult to enrich enough biomass (Jia et al., 2017). It is vital to maintain sufficient AnAOB in reactor, because of the large treatment volume of municipal wastewater. For conventional activated sludge system, it is difficult to reserve enough anammox biomass due to the periodical sludge discharge. The most feasible and effective method to improve the AnAOB accommodation capacity are granulation and biofilm formation (Chen et al., 2019, 2018). Granular sludge is suitable for high nitrogen concentration wastewater treatment (Bagchi et al., 2016; Lin and Wang, 2017), while biofilm process is widely used in municipal wastewater treatment. Gradient distribution of various substrates leads to the stratified structure of biofilm, which is helpful for the enrichment of AnAOB and AOB simultaneously. Therefore, biofilm reactor has great potential in achieving advanced nitrogen removal via anammox pathway. BAF is a conventional biofilm reactor, it is widely applied for organic matter oxidation and nitrification process in wastewater treatment plant. BAF combines the advantages of small footprint, great capacity of treatment volume and strong resistance to impact load (AbouElela et al., 2015; Liu et al., 2017). Furthermore, the biofilm is immobilized in BAF, and AnAOB can be reserved in system efficiently. In this paper, two BAFs treating domestic sewage were built up in laboratory aimed to: (a) optimize the running conditions of two BAFs to improve nitrogen removal, (b) determine an available operation strategy for BAF2 to improve nitrogen removal via anammox pathway, (c) analyze the mechanism of improving nitrogen removal.
Table 1 Operation parameters of BAF1 and BAF2. Operation parameters
Backwash parameters
Time(d)
Gas and water ratio
Backwash Mode
Backwash strength (L/(m2·s))
Time (min)
BAF1
1–61 62–190 191–250
10:1 5:1 8:1
Gas Gas + Water Water
21.6 21.6 + 10.8 10.8
3 4 6
BAF2
1–26 27–146 147–216
5:1 1.5:1 3:1
Gas Gas + Water Water
14.4 14.4 + 7.22 5.42
2 3 3
2.2. Reactors setup and operation Two same BAFs were built up with a working volume of 18.4 L in this study. Volcanic filter media with particle size 3–5 mm was packed at a bed depth of 70 cm. Supporting layer was under the filter media layer with the height of 10 cm. BAF1 was supplied with domestic sewage and its effluent was fed to BAF2. Hydraulic retention time of two BAFs was 90 min, and temperature was controlled at 25 °C. BAF1 was backwashed 18 days intervals approximately. The detailed operation parameters of BAF1 and BAF2 were listed in Table 1. 2.3. Analytical methods All samples were filtered using 0.45 um syringe filter prior to analyze. NH4+-N, NO2–-N, and NO3–-N were analyzed according to standard method (APHA, 2017). COD was measured by COD quick-analysis apparatus (Lian-hua Tech. Co., Ltd, 5B-1, China). Dissolved oxygen (DO) concentration was detected by DO probe (WTW3420, Germany). MLVSS was measured by weight method. The gaseous N2O was collected and measured by gas chromatography with electron capture detector (Agilent 7890, U.S.). 2.4. DNA extraction and Real-time quantitative polymerase chain reaction (QPCR) Biofilm samples were collected and dried by freeze dryer (Labconco, USA) at the different periods of BAF. Genomic DNA was extracted from the dried biofilm sample using the Fast DNA SPIN Kit for Soil (MP Biomedicals, Solon, OH) according to the manufacturer’s instruction. DNA concentration was determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Schwerte, Germany). Real-time quantitative PCR were conducted using the fluorescent dye SYBR-Green approach in a MX3000P Real-Time PCR system (Agilent Technologies, USA). The abundances of functional genes for ammonia-oxidizing archaea (AOA-amoA), AOB (AOB-amoA), AnAOB (AnAOB-hzsB), and DNB (DNB-nirK, DNB-nirS), 16s rRNA genes for Nitrobacter (NOB-16s) and Nitrospira (NOS-16s) were quantified. The standard curve was conducted using ten-fold serial dilutions of the corresponding genes with plasmid DNAs of known concentrations. The results with amplification efficiency in a range from 90–110% and correlation coefficient above 0.95 were employed. Finally, the copy numbers of unit mass dry sludge for each of these genes were calculated.
2. Materials and methods 2.1. Characteristics of domestic sewage and seeding sludge The raw domestic sewage used in this study was taken from a septic tank in the residential area of Beijing University of Technology (Beijing, China). The main characteristics of the wastewater were: COD = 182 ± 32 mg/L; NH4+-N = 60 ± 13 mg/L; NO2––N = 0.32 ± 0.23 mg/L; NO3–-N = 0.82 ± 0.61 mg/L. The seeding sludge was the returned sludge of an A/A/O process from a Municipal Wastewater Treatment Plant in Beijing, China. The biofilm of the two BAFs were both cultivated by the activated sludge. On the 40th day, 300 mL anammox biofilm with MLSS of 10200 mg/L was seeded into BAF2. The anammox biofilm was collected from an anammox biofilter in our laboratory that has been running for more than two years steadily.
2.5. High-throughput sequencing After detecting the concentrations of genomic DNA, PCR was conducted to amplify the genes of 16s rRNA based the primers of 338F and 806R as we used before (Cui et al., 2017). The sequencing was carried out on Illumina HiSeq 2500 platform (Illimina, USA) at Biomarker technologies CO., LTD(Beijing, China). The high quality reads were attained after the optimization of the raw reads, and the high quality reads were divided into different Operational Taxonomic Units (OUT) 2
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at 97% sequence similarity. The microbial compositions were annotated on Silva database. 2.6. Microbial specific activity studies
(NO2
Neff )
(NO3
TINRE = TIN /[(NH4+
(NH4+
Ninf ) + (NO3
Ninf )]
(8)
0.26) × 1.32
Neff ) (NO3
NOBbio = 113/14 ×
Ninf )
(NH4+
NAnAOB ) × 0.26
NH4+
NAOB × Q × 0.01
NO2
NNOB × Q × 0.003
1
55
AnAOBbio = 24.1/14 ×
NH4+
NAnAOB × Q × 0.066
1
(10) (11) (12)
Q was the treatment volume of BAF2, it was 43.2L/d. 3. Results and discussion 3.1. Performances of BAF1 and BAF2 3.1.1. Performance of BAF1 under different gas and water ratios BAF1 was started-up at gas and water ratio of 10:1, after that the gas and water ratio was controlled at 5:1 and 8:1, respectively. Organic matter oxidation, nitrification and denitrification reactions occurred in BAF1, which resulted in part of total inorganic nitrogen (TIN) being removed. The COD removal efficiency was higher than 70%, remaining COD was less than 50 mg/L, and no nitrite was detected in effluent (Fig. 1). When gas and water ratio was controlled at 10:1, average NH4+-N removal efficiency was 36.4% and 13.1 mg/L NO3–-N was produced in effluent. In order to reduce energy consumption, the gas and water ratio was reduced to 5:1 from the 62th day. Effluent NO3–-N concentration decreased greatly, but the improving denitrification activity resulted in the ΔTIN increasing to 13.1 mg/L. When gas and water ratio was adjusted to 8:1 on the 191th day, average effluent NO3–-N was only 4.3 mg/L, and the concentration of removal nitrogen reached to 21.4 mg/L. Therefore, gas and water ratio of 8:1 was the proper condition for BAF1 to achieve ammonia oxidation and nitrogen removal.
(2)
where NH4+-Ninf, NO2–-Ninf and NO3–-Ninf were the influent NH4+-N, NO2–-N, and NO3–-N concentration (mg/L); NH4+-Neff, NO2–-Neff and NO3–-Neff were the effluent NH4+-N, NO2–-N and NO3–-N concentration (mg/L), respectively.
3.1.2. Performance of BAF2 under different conditions BAF2 was fed with domestic sewage, gas and water ratio was
2.7.2. Calculations of nitrogen concentration transformed by AOB, NOB and AnAOB Microbial metabolism equations of AOB, NOB and AnAOB were listed as Eqs. (3–5). The concentration of NH4+-N degraded by AnAOB: NH4+ NAnAOB , the concentration of NH4+-N degraded by AOB: NH4+ NAOB , the concentration of NO2–-N degraded by AnAOB: NO2 NAnAOB , and the concentration of NO2–-N degraded by NOB: NO2 NNOB were calculated by the Eqs. (6–9), respectively.
NH4+ + 1.382O2 + 0.09HCO3
NNOB = (NO3
(7)
NAnAOB )
55
(1)
Ninf ) + (NO2
NO2
(NH4+
Neff )
1
Neff )
Neff
NAnAOB = TIN /(1 + 1.32
55
TIN Ninf )
NO2
AOBbio = 113/14 ×
2.7.1. Calculations of wastewater quality for effluent The concentration of removed total inorganic nitrogen (ΔTIN) and total inorganic nitrogen removal efficiency (TINRE) were calculated using Eqs. (1) and (2), respectively.
Ninf ) + (NO3
(NH4+
Ninf )
2.7.3. Calculations of growth biomass The growth biomass of AOB, NOB and AnAOB were AOBbio, NOBbio, and AnAOBbio, respectively, and the calculation equations were as following (10–12):
2.7. Calculations
Ninf )+(NO2
NAOB = (NH4+
(9)
Batch tests were conducted to investigate the specific nitrogen conversion rates of biofilm samples before and after backwash for BAF2. Before and after backwash 40 mL filter media was collected from BAF2, respectively. The filter media was washed with PBS thrice times before reaction. For each sample, three reaction activities were tested, including aerobic NH4+-N oxidation, aerobic NO2–-N oxidation, anaerobic NH4+-N and NO2–-N oxidation. The batch tests were carried out in a 500 mL reactor as we used before (Cui et al., 2017). For the reaction medium, it contained (per liter): 11.1 mg KH2PO4, 6 mg MgSO4·7H2O, 3 mg CaCl2·2H2O and 1 mL trace element stock solution. The trace element stock solution contained (per liter): 1.5 g FeCl3·6H2O, 0.03 g CuSO4·5H2O, 0.12 g MnCl2·4H2O, 0.06 g Na2MoO4·2H2O, 0.12 g ZnSO4·7H2O, 0.15 g CoCl2·6H2O, 0.18 g KI, 0.15 g H3BO3, and 10 g ethylenediaminetetraacetic acid (EDTA). NaHCO3 was used to supply alkalinity and adjust pH to 7.3–7.5. Before each test, nitrogen gas was pumped into medium to reduce DO concentration, for aerobic reaction DO was controlled at 2–3 mg/L, for anaerobic reaction, DO was reduced to lower than 0.1 mg/L. During the reaction process, pH and DO probes (WTW3420, Germany) were used to monitor online. Water samples were collected at intervals for NH4+-N, NO2–-N and NO3–-N measurement. After each batch test, the biomass on the filter media was measured for the calculation of specific nitrogen degradation rates.
= (NH4+
NH4+
0.982NO2 + 1.036H2 O + 0.018C5 H7 O2 (3)
N + 1.89H+
NO2 + 0.003NH4+ + 0.485O2 + 0.015HCO3 + 0.012H+ (4)
NO3 + 0.009H2 O + 0.003C5 H7 O2 N NH4+ + 1.32NO2 + 0.66HCO3 + 0.13H+
1.02N2 + 0.066CH2 O0.5
N0.15 + 0.26NO3 + 2.03H+ NH4+
NAnAOB = TIN /(1 + 1.32
0.26)
(5)
Fig. 1. Variations of COD, NH4+-N, NO3–-N, ΔTIN and TINRE in influent and effluent of BAF1 under different gas and water ratios.
(6) 3
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DO concentration led to the better performance in BAF2. Recently similar result was also discovered in activated sludge system that high DO was benefit for achieving partial nitrification (Bao et al., 2017). When influent NH4+-N concentration declined, high DO concentration led to high AOB reaction rate and more NO2––N was captured by AnAOB to achieve autotrophic nitrogen removal (Regmi et al., 2014; Guo et al., 2010). So gas and water ratio was operated at 3:1 to control DO concentration of 2.0 mg/L in the following running. Furthermore, avoiding the inhibition of high DO concentration to AnAOB activity, different strategies were adopted to optimize the activities of various functional microorganisms and achieve great nitrogen removal performance. Aerobic and anaerobic interval model was often used in activated sludge system to promote NO2––N accumulation (Gilbert et al., 2014; Miao et al., 2018), and anaerobic condition is also helpful for anammox reaction. So BAF2 was operated in an aerobic-anaerobic alternating mode that was aerobic for the first 7 days and anaerobic for the next 7 days. In aerobic period, gas and water ratio was controlled at 3:1. In anaerobic period, BAF1 effluent adding NaNO2 was fed to BAF2. During the 60 days, nitrogen removal concentration reached to 14.5 mg/L in aerobic condition. But the nitrogen removal performance was not further improved when anaerobic period increased from 7 days to 15 days. Nitrogen removal performance was not improved further via optimizing microbial activity. It was speculated that NOB abundance was much higher than AnAOB after 173 days. So BAF2 was backwashed on the 174th day, the backwash strength was controlled much weaker than that of BAF1 to avoid the excessive shedding of biofilm. After backwash gas and water ratio was still kept at 3:1. The nitrogen removal concentration and TINRE reached to 26.1 mg/L and 48.9%, respectively, and no NH4+-N was detected in effluent. On the 206th day nitrogen removal concentration of BAF2 began to decline, so it was backwashed again on the 214th day. After that nitrogen removal ability revived again, indicating backwash could promote nitrogen removal by adjusting microbial community structure.
Fig. 2. Variations of COD, NH4+-N, NO3–-N, ΔTIN and TINRE in influent and effluent of BAF2 under different conditions.
controlled at 5:1 during the initial period (Fig. 2). After 26 days it was coupled with BAF1, gas and water ratio was adjusted to 1.5:1. From the 30th day nitrification activity improved gradually, but TINRE was always less than 15%. On the 97th day TINRE increased to 80% sharply, and only 3.5 mg/L NO3–-N remained in effluent. While excellent nitrogen removal performance only kept for 20 days. After 117 days, ΔTIN declined to 8.6 mg/L, and remaining NO3–-N increased to 22.1 mg/L in effluent. The cause of changing nitrogen removal performance in BAF2 was analyzed further. Since constant gas and water ratio was kept, the fluctuation of domestic sewage resulted in DO concentration changing in BAF2. As Fig. 3 showed that on the 93th day influent NH4+-N was 27.9 mg/L, along the filter height effluent NH4+-N and TIN both declined gradually. On the 100th day influent NH4+-N declined to 20.2 mg/L, most of nitrogen was degraded at the bottom of 40 cm filter media, and average DO concentration was 2.38 mg/L. On the 120th day influent NH4+-N went up to 33.8 mg/L, average DO concentration dropped to 1.20 mg/L, and only 6.31 mg/L nitrogen was removed. According to the variations of DO concentration and effluent quality, it could be found that the higher
3.2. Analysis of nitrogen transformation Only little COD degradation indicated that heterotrophic denitrification reaction was weak in BAF2, and anammox reaction was the
Fig. 3. Variations of effluent NH4+-N, NO3–-N, TIN, COD and DO concentration along BAF2 height. 4
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main nitrogen removal pathway. The functional bacteria involving to nitrogen transformation mainly included AOB, NOB and AnAOB in BAF2. Excellent performance of nitrogen removal depended on the complex interaction mechanisms among these bacteria. AOB and NOB collaborated with each other to complete nitrification reaction. The growth of AnAOB relied on the NO2–-N generating from AOB, but it also competed with AOB for NH4+-N. Furthermore, AnAOB competed for NO2–-N with NOB. The concentrations of nitrogen utilized by AOB, NOB and AnAOB were calculated according to stoichiometric reaction equation and removal TIN in BAF2. From the 60th day to the 96th day, little nitrogen was degraded by AnAOB, AOB and NOB completed nitrification process. Since the declining of influent NH4+-N, DO concentration increased in BAF2. High DO concentration improved AOB oxidation rate greatly, which led to the activity difference between AOB and NOB (Regmi et al., 2014; Guo et al., 2010). More NO2–-N was utilized by AnAOB in biofilm interior. Nitrogen transformation was compared in BAF2 under two control strategies, after backwash the removal concentration of NH4+-N and NO2–-N by AnAOB were much higher than that in aerobic and anaerobic interval period. It indicated that backwash played crucial role in improving nitrogen removal ability of BAF2 via optimizing microbial community structure. Balance analysis of nitrogen was calculated under different operation periods for BAF2 (Fig. 4). Gaseous N2O was detected in the three periods. From the 118th day to the 147th day, nitrification was the main nitrogen transformation pathway, 0.032 mg N2O-N was generated for per liter wastewater treatment and 15.51% of the influent nitrogen was transformed into biomass and N2. In aerobic-anaerobic alternating period, the proportion of remaining NH4+-N in effluent reduced, N2O-N emission decreased to 0.07%, and TINRE increased to 38.69%. Anaerobic operation was benefit for improving nitrogen removal. After backwash some biofilm was washed out from BAF2, the proportion of nitrogen converting to biomass and N2 further increased to 52.87%, and the percent of N2O-N was only 0.01%. Above results illustrated that backwash not only improved anammox ability to promote nitrogen removal, but it also reduced N2O emission from nitrifier denitrification and hydroxylamine oxidation pathways.
Fig. 5. Variations of gene abundances in BAF2 at different periods (Seed, 110th, Be-wash, Wash and Af-wash represented sample were collected from the seed sludge, the 110th day, before backwash, shedding biofilm during backwash and after backwash, respectively).
backwash were also measured via QPCR. The similar changing tendency suggested the efficient elimination of Nitrobacter and Nitrospira from BAF2 through backwash. Autotrophic biofilm formed via the aggregation of AOB, NOB and AnAOB in BAF2. Since the different requirements of oxygen, AOB and NOB tended to aggregate in the outer layer of the biofilm that was benefit for the capture of oxygen and substances. AnAOB tended to enrich in the internal anoxic or anaerobic area that was close to the surface of filter media (Miao et al., 2016). In the autotrophic biofilm system, part of NO2–-N generated from AOB was utilized by NOB immediately, the other spread into the interior of biofilm and was captured by AnAOB to release N2. The depth of biofilm increased gradually with the running of BAF2, and the resistance of substance transfer also enhanced at the same time, which could reduce the ability of AnAOB to compete for NH4+-N and NO2–-N with AOB and NOB, respectively. Finally, NO3–-N and TIN concentration increased in effluent. So it was necessary to backwash regularly for BAF2. On the one hand, AOB and NOB existed on the outer layer of biofilm could slough off first under the actions of hydraulic forces (Lawson et al., 2017). On the other hand, hydraulic shear could weaken the biofilm depth, and promote substances transfer into biofilm interior (Dror-Ehre et al., 2010). During the 40 days between twice backwashes of BAF2, according to the stoichiometric reaction equations it was calculated that the growth biomass of AOB, NOB and AnAOB were 12.209, 0.983 and 3.388 g, respectively. The proliferative biomass of AOB was higher than NOB and AnAOB obviously, which accounted for massive NH4+-N was utilized by AOB. Backwash contributed to optimizing the abundance of various functional bacteria in BAF2.
3.3. Mechanisms of microbial community adjustment 3.3.1. Abundance of functional microorganism The variations of functional microorganism abundance in different periods of BAF2 were showed in Fig. 5. The abundance of nirK and nirS were the highest in seeding sludge, indicating much denitrifier was enriched. However, because of little biodegradable organic matter, the abundance of denitrifier decreased greatly on the 110th day. In seeding sludge the abundance of AOB, NOB and AnAOB was less than 106 copies/mg dry sludge, but they were all enriched on the 110th day. On the 175th day before backwash, the abundances of AOB, Nitrobacter and Nitrospira all reached to 107 copies/mg dry sludge. After backwash the abundance of remaining AnAOB increased, while the abundances of AOB, Nitrobacter and Nitrospira decreased obviously. Furthermore, the variations of functional microorganism abundances during the second
Fig. 4. Transformation forms and proportions of nitrogen for BAF2 at different periods. 5
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Fig. 6. Microbial community structures in genus level for BAF2 before and after backwash (Be-wash, Wash and Af-wash represented sample were collected from before backwash, shedding biofilm during backwash and after backwash, respectively).
3.3.2. Microbial community structures After backwash the microbial community structure of BAF2 changed obviously (Fig. 6). Before backwash, the dominant microorganism at genus level included norank_f_Anaerolineaceae, Nitrospira, Candidatus Kuenenia, and Candidatus Brocadia. After backwash the proportions of some nitrogen transformation bacteria varied obviously, the percent of Nitrospira decreased greatly, but Candidatus Kuenenia and Candidatus Brocadia increased clearly. For AOB, its ratio declined from 0.95% to 0.21%. For the microbial structure of the removed biofilm, it included Nitrospira, Denitratisoma and norank_f_Anaerolineaceae mainly. The proportion of AnAOB in the sloughing biofilm was much lower than that remaining in BAF2, while the ratio of AOB was much higher correspondingly. Microbial community structures of the three samples illustrated that AnAOB aggregated in the inner layer of the biofilm, AOB and NOB distributed in the outer layer. Although much microorganism was eliminated from BAF2 during backwash, sufficient AnAOB was reserved in system that facilitated nitrogen removal.
backwash can influence the composition of microorganism. After backwash the thinner biofilm was benefit to substance transfer, and the declining abundances of AOB and NOB was favorable to nitrogen remove via anammox pathway. In this study, it was crucial to inhibit NOB activity to facilitate autotrophic nitrogen removal in BAF2. According to the influent quality, different gas and water ratios were controlled to achieve high DO concentration. Improving AOB reaction rate with high DO concentration caused more NO2–-N was utilized by AnAOB, remaining nitrogen declined in effluent. However, NOB abundance kept increasing in BAF2 even with high DO concentration. Based on the microbial hierarchy of the biofilm, backwash was operated periodically to wash out much Nitrospira and Nitrobacter from BAF2. Declining NOB abundance caused nitrite oxidation rate reducing, and effluent TIN was also reduced. Nitrogen removal performance improved greatly. Therefore, it is an efficient and manageable strategy to optimize the microbial community structure via backwash to promote autotrophic nitrogen removal in BAF2.
3.3.3. Reaction activities of BAF2 before and after backwash The reaction activities of AOB, NOB and AnAOB under aerobic and anaerobic conditions were measured in batch tests. The results showed that the activities of aerobic ammonia oxidation, nitrite oxidation, and anaerobic ammonium oxidation all declined, which was resulted from the shedding of biofilm (Table 2). Because the less AnAOB was eliminated from BAF2, the anaerobic ammonium oxidation rate did not decline much. In addition, the ratio of specific degradation rate of TIN and NH4+-N under aeration condition increased clearly after backwash, which indicated AnAOB could compete out NOB for NO2–-N after backwash. The discrepancy of reaction rates among several functional microorganisms was the most crucial factor influencing the performance of autotrophic nitrogen removal process. Backwash was only suit to the reactor that biofilm and filter media are both fixed, and
3.4. Advantages of improving nitrogen removal from BAF For domestic sewage when NH4+-N concentration reached to 70 mg/L, the ratio of gas and water of BAF1 and BAF2 was controlled at 8:1 and 3:1, nitrogen removal concentration reached to 44.2 mg/L in this two coupled BAFs process, and effluent COD concentration was less than 50 mg/L. The carbon source from domestic sewage was used for denitrification to remove 21.4 mg/L nitrogen in BAF1, and 22.8 mg/L nitrogen was removed through anammox pathway in BAF2. Moreover, comparing with nitrification process greenhouse gas emission declined greatly. Traditional A/O or A/A/O process for biological nitrogen removal depends on the nitrification and denitrification technology, all the influent NH4+-N is oxidized completely, and its effluent remaining 20 ∼ 25 mg/L NO3–-N needs tertiary treatment generally (Liu et al., 2016). However, part of influent NH4+-N was not oxidized into NO2–-N or NO3–-N during aeration process in this study, more than 30% of the influent nitrogen was removed by anammox pathway. Recently, some research also focused on improving nitrogen removal during aeration process. Improving nitrogen removal during partial nitrification process was also reported in SBR system (Zhou et al., 2018). The TINRE could reach to 45% with 4.5 h aeration, but the removal nitrogen concentration was only 12 mg/L. While in this study, the hydraulic retention time was only 3 h for two coupled BAFs, and the treatment capacity of BAF was higher than SBR obviously. Now partial nitrification was
Table 2 Activities of functional microorganisms in BAF2 before and after backwash. Reaction Conditions
Types of specific degradation rate
Before backwash (mg N/(g VSS h))
After backwash (mg N/(g VSS h))
Aerobic NH4+-N
NH4+-N TIN
22.02 ± 1.44 13.62 ± 0.89
5.85 ± 0.15 4.16 ± 0.10
Aerobic NO2–-N
NO2–-N
14.06 ± 0.92
0.99 ± 0.02
Anaerobic NH4+-N and NO2–-N
NH4+-N NO2–-N TIN
6.48 ± 0.86 8.01 ± 0.62 13.91 ± 0.64
4.86 ± 0.62 5.64 ± 0.41 8.14 ± 0.21
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achieved widely by the means of on-line controlling (Yang et al., 2007), but it is still a challenge to keep long-term stable. In this paper, periodical backwash was adopted to optimize the activities of various microorganisms, and stable nitrogen removal performance was achieved in two coupled BAFs. When the concentration of NH4+-N in domestic sewage was less than 40 mg/L, advanced nitrogen removal could be achieved in this two coupled BAFs process, effluent TIN was less than 10 mg/L. When NH4+N concentration reached to 60 mg/L, tertiary treatment was required for the remaining NO3–-N in effluent. Comparing with the conventional nitrification and denitrificaton process, this process showed typical advantages: ①More nitrogen was removed during aeration process; ②Saving energy consumption for aeration; ③No sludge backflow; ④Low effluent turbidity, without secondary sedimentation tank; ⑤High treatment capacity; ⑥Less greenhouse gas N2O emission.
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4. Conclusions Gas and water ratio of 8:1 was proper for BAF1 to remove nitrogen, and 2.0 mg/L DO was benefit for nitrogen removal from BAF2. Backwash could wash out abundant AOB and NOB that distributed on the outer layer of biofilm and improve the proportion of AnAOB in BAF, the declining reaction activity of NOB promoted nitrogen removal via anammox pathway. For domestic sewage treatment, nitrogen removal concentration reached to 44.2 mg/L during aeration process in the two coupled BAFs, which also could reduce energy consumption and N2O emission simultaneously.
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Acknowledgements This research was supported by Beijing Natural Science Foundation (grant numbers 8182012), and the Open Research Fund Program of Key Laboratory of Cleaner Production and Integrated Resource Utilization of China National Light Industry. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122006. References Abou-Elela, S.I., Fawzy, M.E., El-Gendy, A.S., 2015. Potential of using biological aerated filter as a post treatment for municipal wastewater. Ecol. Eng. 84, 53–57. APHA, 2017. Standard Methods for the Examination of Water and Wastewater, twentythird ed. American Public Health Association, Washington DC, USA. Bagchi, S., Lamendella, R., Strutt, S., Van Loosdrecht, M.C.M., Saikaly, P.E., 2016. Metatranscriptomics reveals the molecular mechanism of large granule formation in granular anammox reactor. Sci. Rep. 6, 1–10. Bao, P., Wang, S., Ma, B., Zhang, Q., Peng, Y., 2017. Achieving partial nitrification by inhibiting the activity of Nitrospira-like bacteria under high-DO conditions in an intermittent aeration reactor. J. Environ. Sci.-China. 56, 71–78. Chen, R., Ji, J., Chen, Y., Takemura, Y., Liu, Y., Kubota, K., Ma, H., Li, Y., 2019. Successful operation performance and syntrophic micro-granule in partial nitritation and anammox reactor treating low-strength ammonia wastewater. Water Res. 155, 288–299. Chen, R., Takemura, Y., Liu, Y., Ji, J., Sakuma, S., Kubota, K., Ma, H., Li, Y., 2018. Using Partial Nitrification and Anammox To Remove Nitrogen from Low-Strength Wastewater by Co-immobilizing Biofilm inside a Moving Bed Bioreactor. ACS Sustain. Chem. Eng. 7 (1), 1353–1361. Cui, B., Liu, X., Yang, Q., Li, J., Zhou, X., Peng, Y., 2017. Achieving partial denitrification through control of biofilm structure during biofilm growth in denitrifying biofilter. Bioresource Technol. 238, 223–231.
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