Abstract

The effects of nitrite and nitrate on the integration of ammonium oxidization and sulfate reduction were investigated in a self-designed reactor with an effective volume of 5 L. An experimental study indicated that the ammonium oxidization and sulfate reduction efficiencies were increased in the presence of nitrite and nitrate. Studies showed that a decreasing proportion of N/S in the presence of NO2 at 30 mg·L−1 would lead to high removal efficiencies of NH4+-N and SO42–-S of up to 78.13% and 46.72%, respectively. On the other hand, NO3 was produced at approximately 26.89 mg·L−1. Proteobacteria, Chloroflexi, Bacteroidetes, Chlorobi, Acidobacteria, Planctomycetes and Nitrospirae were detected in the anaerobic cycle growth reactor. Proteobacteria was identified as the dominant functional bacteria removing nitrogen in the reactor. The nitritation reaction could promote the sulfate-reducing ammonium oxidation (SRAO) process. NH4+ was converted to NO2 and other intermediates, for which the electron acceptor was SO42−. These results showed that nitrogen was converted by the nitrification process, the denitrification process, and the traditional anammox process simultaneously with the SRAO process. The sulfur-based autotrophic denitration and denitrification in the reactor were caused by the influent nitrite and nitrate.

INTRODUCTION

Industrial and domestic sewage with ammonium and sulfate are of major environmental concern (Zhao et al. 2006). High levels of ammonium lead to eutrophication of surface waters, while sulfate can leach into secondary contaminations because sulfide reduction under anaerobic conditions is hazardous to aquatic plants and affects environmental quality (Hao et al. 2014).

Sulfate reducing ammonium oxidation (SRAO) technology that achieves the simultaneous removal of ammonium and sulfate in a single bioreactor was reported by Fdz-Polanco et al. (2001a). The ammonium and sulfate were converted to elemental sulfur and dinitrogen gas without secondary pollution. The SRAO technology is a cost-effective, environmentally friendly process and has become the focus of recent research (Liu et al. 2015a). Fdz-Polanco et al. (2001a) proposed Equation (1) through Equations (2)–(4), which involve the two-step ammonium oxidizing processes. The first step is the removal of ammonium and sulfate (Equation (2)), and the second step is the anammox process, which occurs synchronously (Equation (3)).  
formula
(1)
 
formula
(2)
 
formula
(3)
 
formula
(4)

With further studies, during the anammox process the reaction between extra electron acceptors, such as NO2, SO42− and Fe3+, and ammonium was thought to be facilitated by bacteria (Chamchoi et al. 2008). The removal of ammonium observed to occur in the denitrifying process was carried out by bacteria using ammonium as an electron donor for nitrate reduction (Graaf et al. 1995). NO2 and NO3 could also act as potential electron acceptors for sulfur oxidation. Re-oxidation of elemental sulfur or sulfide to SO42− could readily take place via sulfur-utilizing denitrification (Rikmann et al. 2012). Another researcher observed that as the reaction proceeded, the SO42− concentration did not remain steady, and the NO2 concentration also decreased and promoted SO42− resynthesis via sulfur-utilizing denitritation (Liu et al. 2015b). Most other research concerning SRAO has shown that disproportionally higher removal of NO2 and NH4+ was based on the effluent (Fdz-Polanco et al. 2001b; Liu et al. 2008; Yang et al. 2009; Jing et al. 2010). At the same time, it should be noted that the effluent contained more NO3 in previous studies (Strous et al. 2002; Mahmood 2007; Sabumon 2008; Rikmann et al. 2012). Therefore, the nature of the electron acceptor was a key factor for determining the ammonium oxidation pathway in SRAO.

Strous et al. (2002) believed that some intermediates, such as NO2, NO3, S2− and S, might affect the removal efficiencies of ammonium and sulfate in the SRAO process. Liu et al. (2008) isolated and described a new autotrophic Planctomycete bacterium and thought this bacterium was an anammox bacterium. Anammox bacteria preferentially utilize nitrite and nitrate as the electron acceptor to compete with sulfate, thus showing a significant decrease in sulfate reduction (Lai & Zhou 2010; Rikmann et al. 2012). The SRAO process was heavily suppressed by increasing NO2 and NO3 concentrations.

Dissolved O2 (range: 0–0.2 mg·L−1) and NO3 present in the influent could be consumed in the SRAO process (De et al. 2016). Rikmann et al. (2014) believed that NO2 was transformed to NO3 under anaerobic conditions due to O2 entry from the tank provided in the reactor. Schrum et al. (2009) described possible involvement in the nitrate producing stage of SRAO, and the ammonium could also be oxidized to NO3. An organic compound could be used as an electron donor to couple with a subsequent heterotrophic denitrification reaction at 30° and under normal pressure:  
formula
(5)

These discoveries generated great interest in research regarding the role of nitrite and nitrate in the SRAO process. Many researchers detected different NO3 and NO2 concentrations, which made the mechanism of the SRAO process unclear and complex (Zhang et al. 2013). Few studies have focused on the metabolic pathways and microbial community changes. SRAO is a multi-step reaction that includes the production of NO2 and NO3 compounds.

This work used a self-designed anaerobic SRAO reactor, using different concentrations of nitrite and nitrate as environmental factors, to explore the performance of wastewater treatment. The anaerobic end products were tested during the experiment to evaluate the synergistic effects of nitrite and nitrate. The microbial community was analysed to study the mechanism.

MATERIALS AND METHODS

Anaerobic cycle growth bioreactor and synthetic wastewater

As shown in Figure 1, the experiment was conducted in two anaerobic cycle growth reactors. These reactors were designed independently by our laboratory. Non-woven fabric filler was used as the biological carrier to enhance the attachment performance for good adsorption characteristics. The laboratory-scale reactors were made of organic glass with an effective volume of 5 L and were located in a water bath. The flow of water was circulated clockwise and could effectively contact microorganisms. To maintain anoxic conditions, the reactor was flushed continuously with nitrogen gas and covered to protect the bacteria from light and algal growth. The temperature inside the reactor was adjusted to 35 ± 1 °C.

Figure 1

Schematic of the laboratory-scale SRAO.

Figure 1

Schematic of the laboratory-scale SRAO.

Predetermined amounts of ammonium and sulfate (using (NH4)2SO4) were added as requirements of each experiment. Trace elements, including EDTA (in mg·L−1) 5,000, ZnSO4·7H2O 430, CoCl2·6H2O 240, NaMoO4·H2O 220, NiCl2·6H2O 190, NaSeO4·10H2O 210, and H3BO4 14, were added to the water as mineral media (Sliekers et al. 2002). In addition, the synthetic wastewater trace element solution contained (in g/L) KH2PO4 0.027, CaCl2·2H2O 0.136, and MgCl2 0.2 (Zhang et al. 2019). With the addition of NaHCO3, the pH within the reactors varied between 8.1 and 8.3.

Analytical methods

The concentrations of nitrate, nitrite, and sulfate in the collected liquor samples following 0.45 μm filtration were measured by ion chromatography (ICS-1100, Thermo Fisher). HS, S2− and H2S in the liquid were analysed by Standard Methods. Temperature and pH were measured using a HQ30d portable multi-parameter measuring meter (HQ30d, USA).

Adding influencing factors nitrite and nitrate

The synthetic wastewater contained different initial concentrations of (NH4)2SO4 supplemented with NO2-N (30 mg·L−1). Table 1 summarizes the characteristics of the substrate.

Table 1

Influent concentrations for the SRAO process in the presence of nitrite

ParameterNH4+-N (mg·L−1)SO42–-S (mg·L−1)NO2-N (mg·L−1)
119.50 ± 10.43 182.69 ± 9.86 
159.86 ± 2.01 216.05 ± 11.71 33.98 ± 0.86 
109.53 ± 5.85 115.78 ± 12.76 30.02 ± 2.56 
80.23 ± 3.74 100.43 ± 8.29 27.64 ± 2.61 
ParameterNH4+-N (mg·L−1)SO42–-S (mg·L−1)NO2-N (mg·L−1)
119.50 ± 10.43 182.69 ± 9.86 
159.86 ± 2.01 216.05 ± 11.71 33.98 ± 0.86 
109.53 ± 5.85 115.78 ± 12.76 30.02 ± 2.56 
80.23 ± 3.74 100.43 ± 8.29 27.64 ± 2.61 

To explore the effects of nitrate on the removal of ammonium and sulfate, three different concentrations of nitrate, ammonium, and sulfate were used, as shown in Table 2.

Table 2

Influent concentrations for the SRAO process in the presence of nitrate

Time (days)NH4+-N (mg·L−1)SO42–-S (mg·L−1)NO3-N (mg·L−1)
1–23 120.27 ± 9.95 182.71 ± 11.26 
24–42 160.44 ± 5.03 216.03 ± 6.57 30.29 ± 1.97 
43–55 159.81 ± 2.49 216.11 ± 10.91 60.14 ± 6.53 
56–75 89.59 ± 10.74 133.00 ± 11.73 89.71 ± 8.48 
Time (days)NH4+-N (mg·L−1)SO42–-S (mg·L−1)NO3-N (mg·L−1)
1–23 120.27 ± 9.95 182.71 ± 11.26 
24–42 160.44 ± 5.03 216.03 ± 6.57 30.29 ± 1.97 
43–55 159.81 ± 2.49 216.11 ± 10.91 60.14 ± 6.53 
56–75 89.59 ± 10.74 133.00 ± 11.73 89.71 ± 8.48 

RESULTS AND DISCUSSION

Effects of nitrite on the integration of ammonium and sulfate

Performance of the reactor

The effect of nitrite on the integration of ammonium and sulfate was investigated for 97 days. A blank group without NO2 addition was also included for comparison. The SO42–-S and NH4+-N profiles, as the function of the reaction time, are shown in Figure 2(a).

Figure 2

Concentrations and removal efficiencies of NH4+-N, SO42–-S and NO2-N: (a) in the presence of a blank group without NO2; (b) with the addition of NO2-N (34 mg·L−1); (c) with the addition of NO2-N (30 mg·L−1) and (d) with the addition of NO2-N (27 mg·L−1).

Figure 2

Concentrations and removal efficiencies of NH4+-N, SO42–-S and NO2-N: (a) in the presence of a blank group without NO2; (b) with the addition of NO2-N (34 mg·L−1); (c) with the addition of NO2-N (30 mg·L−1) and (d) with the addition of NO2-N (27 mg·L−1).

From 24 to 49 days, the influent NH4+-N loading rate was 160 mg·(L·d)−1, and the SO42–-S loading rate was 216 mg·(L·d)−1; the loading rate of NO2-N was maintained at 30 mg·(L·d)−1. The hydraulic retention time (HRT) during the experiment was 24 h. The significant substance removal was initially observed after 48 days. The NH4+-N removal efficiency gradually increased from 39% to 60% with a corresponding NH4+-N removal rate of 81.46 mg·(L·d)−1. The removal efficiency of NO2-N increased, but the SO42–-S removal efficiency decreased (Figure 2(b)). In addition, complete removal of NO2-N was obtained with 48 days.

Figure 2(c) shows the high loading rate of NH4+-N and SO42–-S when the influent NO2-N was kept constant. In the blank group, the SO42–-S removal rate was approximately 70.46 mg·(L·d)−1. With the addition of NO2-N (30 mg·L−1), the concentration of SO42–-S in the effluent was higher than the influent concentration. SO42–-S removal was strongly related to the NO2-N removal level in this stage. In addition, the yellow colour of the effluent was lighter than that of the water in the previous stage. The yellow matter was determined to be S0 by using carbon as an organic solvent as described in Liu et al. (2015b). The result showed that S0 was produced during the process. This result indicated that the accumulated amounts of sulfur decreased with time during 50–74 days.

The NH4+-N loading rate gradually decreased to 80.0 mg·(L·d)−1 while maintaining an N/S of 2 (Figure 2(d)). Approximately no NH4+-N was detected in the effluent, and the removal rate of SO42–-S was 45.29 mg·(L·d)−1. The effluent NO3-N was increased to 30 mg·(L·d)−1, which was higher than the previous phase. This phenomenon continued for approximately one week and was stably maintained.

Microbial community structure in the presence of NO2

The phylogenetic relationships and classifications of the bacterial 16S rRNA sequences, with relative abundance above 1%, in the sludge samples extracted from the blank group and the group with the addition of nitrite are illustrated on a phylum basis in Figure 3(a) and 3(b). Planctomycetes (4.35% in the blank group and 3.43% in the nitrite group) provided evidence that SRAO was occurring. The increasing relative abundance of anammox bacteria (Proteobacteria (39.97% in the blank group and 29.97% in the nitrite group) and Nitrospirae (1.94% in the blank group and 2.31% in the nitrite group)) and nitrifying bacteria (Bacteroidetes (10.36% in the blank group and 13.98% in the nitrite group), Chloroflexi (11.07% in the blank group and 17.45% in the nitrite group) and Chlorobi (7.79% in the blank group and 13.35% in the nitrite group)) were investigated and could indicate the role of the observed consortium bacteria. Proteobacteria was identified as the most domain functional bacteria thought to oxidize NH4+ into NO2. The large proportion of Bacteroidetes, Chloroflexi and Chlorobi present in this reactor contributed to the oxidation of NO2 into NO3; only SO42− provided an electron acceptor during the process (Lai & Zhou 2010). Acidobacteria (5.31% in the blank group and 5.41% in the nitrite group) accounted for 5.41% with a few observations and was considered as playing a critical role in the NO2 and SO42− removal (Figure 2(d)), which was a clear indicator of the simultaneous achievement of SRAO and nitrification.

Figure 3

Charts showing the microbial community composition at the level of phylum: (sample a) the microbial community associated with the blank group; (sample b) the microbial community associated with the addition of nitrite; (sample c) the microbial community associated with the addition of nitrate.

Figure 3

Charts showing the microbial community composition at the level of phylum: (sample a) the microbial community associated with the blank group; (sample b) the microbial community associated with the addition of nitrite; (sample c) the microbial community associated with the addition of nitrate.

Sulfur-based autotrophic denitritation

The re-oxidation of elemental sulfur into sulfate could readily take place via sulfur-utilizing denitritation during the SRAO process (Rikmann et al. 2012). The loading rates of SO42–-S and NH4+-N were maintained at 110 mg·(L·d)−1 and 116.7 mg·(L·d)−1, respectively. A NO2-N loading rate up to 30 mg·(L·d)−1 was applied to the bioreactor, which changed the sulfur balance and resulted in the partial recovery of SO42− (Sun & Nemati 2012). The bioreactor for this experiment had been successfully started and run for some time, and elemental sulfur was attached to the non-woven fabric. The removal efficiency of SO42− was lower during this phase, and sulfur could only donate electrons without any possibility of accepting electrons under anaerobic conditions (Nanda et al. 2013). According to a previous report, the equation for SO42− resynthesis via sulfur-utilizing denitritation is shown as below (Equation (6)) (Liu et al. 2008). Few studies have focused on the related functional microbial communities. In the study, the presence of a high abundance of Chlorobi (7.79% in the blank group and 13.35% in the nitrite group) in the seeding sludge provided evidence in favour of this mechanism.  
formula
(6)

Effects of nitrate on the integration of ammonium and sulfate

Performance of the reactor

As shown in Figure 4(b), when the loading rate of NO3 in influent was 30 mg·(L·d)−1, the removal of NH4+-N and SO42–S in the anaerobic reactor decreased from 30% to 40%, respectively, to 11% simultaneously. The SRAO process could not be promoted with the NO3-N concentration 30 mg·L−1.

Figure 4

Concentrations and removal efficiencies of NH4+-N, SO42–-S and NO3-N: (a) in the presence of a blank group without NO3; (b) with the addition of NO3-N (30 mg·L−1); (c) with the addition of NO3-N (60 mg·L−1) and (d) with the addition of NO3-N (90 mg·L−1).

Figure 4

Concentrations and removal efficiencies of NH4+-N, SO42–-S and NO3-N: (a) in the presence of a blank group without NO3; (b) with the addition of NO3-N (30 mg·L−1); (c) with the addition of NO3-N (60 mg·L−1) and (d) with the addition of NO3-N (90 mg·L−1).

According to Figure 4(c), the NO3N loading rate increasing from 30 mg·(L·d)−1 to 60 mg·(L·d)−1 resulted in the immediate utilization of NH4+ and SO42−, although part of the NO3 was generated during SRAO. This result indicated that moderate NO3 concentration could stimulate the removal of NH4+ and SO42−.

The average NO3-N loading rate was gradually increased to 90 mg·(L·d)−1. The steady state profiles of NO3-N, NH4+-N, and SO42–-S concentrations are shown as Figure 4(d). When the feed NO3 concentration was further increased, the NH4+-N and SO42–-S removal efficiencies were greatly increased. The average NH4+-N and SO42–-S removal efficiencies were 90% and 85%, respectively. The removal rates for NH4+-N and SO42–-S reached 81.67 mg·(L·d)−1 and 113.67 mg·(L·d)−1, respectively.

Microbial community structure in the presence of NO3

Fdz-Polanco et al. (2001a) proposed a summary equation describing the SRAO process (Equation (1)) and concluded that the N/S ratio was 2:1. In cultures containing different concentrations of NO3, the N/S conversion ratios were lower than 2:1 (Table 3).

Table 3

Effects of nitrate on SRAO activity

Time (d)(NO3-N)in(NO3-N)eff(NH4+-N)in(NH4+-N)eff(SO42–-S)in(SO42–-S)effN/S
24–42 30 24.27 158.81 129.78 243.31 201.25 1.57 
43–55 60 63.55 152.82 131.78 228.5733 189.47 1.23 
56–75 85 101.61 90.29 31.56 112.09 22.64 1.50 
Time (d)(NO3-N)in(NO3-N)eff(NH4+-N)in(NH4+-N)eff(SO42–-S)in(SO42–-S)effN/S
24–42 30 24.27 158.81 129.78 243.31 201.25 1.57 
43–55 60 63.55 152.82 131.78 228.5733 189.47 1.23 
56–75 85 101.61 90.29 31.56 112.09 22.64 1.50 

All units in mg·L−1; N/S meaning is NH4+-N/SO42–-S; (in) in the presence of (influent); (eff) in the presence of (effluent).

High-throughput 16S rRNA gene sequencing technology was used to identify the microbial communities in the blank group and the group with the addition of nitrate (Figure 3(a) and 3(c)). The results showed that Proteobacteria (39.9% in the blank group and 32.86% in the nitrite group) were the most abundant, followed by Chloroflexi (11.07% in the blank group and 15.1% in the nitrite group), Bacteroidetes (10.36% in the blank group and 13.88% in the nitrite group), Chlorobi (7.79% in the blank group and 11.9%in the nitrite group), Acidobacteria (5.31% in the blank group and 5.15% in the nitrite group) and Planctomycetes (4.35% in the blank group and 3.24% in the nitrite group) in the two samples. Proteobacteria and Armatimonadetes observed in the sample could indicate that ammonium was directly oxidized to NO2 under anoxic conditions (Teeseling et al. 2015; Mi et al. 2017). Notably, the effluent contained more NO3, but NO2 was not present. Bacteroidetes, Chlorobi, and Chloroflexi were the most abundant compared with the blank group, which indicated NO2 could be oxidized to NO3 in the SRAO process (Mulder et al. 1995). NH4+ was converted to NO2 and other intermediates, in which the electron acceptor was SO42− (Xu et al. 2014). The optimum removal of NH4+ and SO42− was achieved in the system.

Sulfur-based autotrophic denitrification

Autotrophic denitrification utilizing elemental sulfur was an easy reaction for water contaminated NO3 (Huang et al. 2018). Rikmann et al. (2012) thought that successfully starting and operating SRAO for a period would accumulate sulfur. Under the condition of 30 mg·L−1 NO3-N, the effluent concentration of SO42− increased and NO3 decreased, indicating that elemental sulfur was used as an electron donor (Pokorna & Zabranska 2015). The presence of a high abundance of Chlorobi (7.79% in the blank group and 11.9% in the nitrite group) in the seeding sludge provided evidence for the oxidation of sulfur. During the process of reduction, a little NO2 was detected in the effluent. At the same time, the NO2 in the liquid began to decrease during the further reduction of the remaining sulfate. One could speculate that at this stage, the system was experiencing sulfur-based autotrophic denitrification and, with sufficient time, NO3 was reduced to NO2 and other nitrogenous compounds, possibly N2 (Yin et al. 2015). The reduction of NO3 in the presence of elemental sulfur could be represented by reaction (Equation (7)):  
formula
(7)

Impacts of the ammonium to sulfate ratio

In this study, the N/S ratios of 2:1 and 4:1 in the presence of NO2 and four levels of SO42–-S concentrations were investigated in the reactor (Figure 5). The SO42–S removal efficiency at an N/S of 4:1 was 33.2%, which was lower than that of an N/S of 2:1. Approximately 16.8 mg·L−1 of NO3 was produced when the N/S ratio was 2:1, and the NH4+-N removal efficiency was enhanced. The NO3 yield was 7.8 mg·L−1 when the N/S ratio was 4:1. Figure 5(c) and 5(d) show two different N/S ratios in the presence of NO2. At an influent NO2-N concentration of approximately 30 mg·L−1, a regular pattern that reduced the influent N/S ratio enhanced the NH4+ and SO42− removal efficiencies but accumulated more NO3.

Figure 5

Concentrations profiles of various ions with different N/S ratios. (a) The NH4+-N/SO42–-S ratios of 2:1; (b) the NH4+-N/SO42–-S ratios of 4:1; (c) the NH4+-N/SO42–-S ratios of 2:1 in the presence of nitrite; (d) the NH4+-N/SO42–-S ratios of 4:1 in the presence of nitrite.

Figure 5

Concentrations profiles of various ions with different N/S ratios. (a) The NH4+-N/SO42–-S ratios of 2:1; (b) the NH4+-N/SO42–-S ratios of 4:1; (c) the NH4+-N/SO42–-S ratios of 2:1 in the presence of nitrite; (d) the NH4+-N/SO42–-S ratios of 4:1 in the presence of nitrite.

In contrast with these two situations, with NO2 in the influent and an N/S ratio of 2:1, the NH4+ and SO42−of the conversion ratio was higher, and the NO3 formation was higher. However, the NO3 formation was lower. This result indicated that NH4+ could be oxidized into NO3 or NO2 depending on the NH4+-N/SO42–-S ratios.

The data presented in Table 4 further highlight this distinction.

Table 4

The effect of influent N/S ratio

(NH4+-N)in(NO2-N)in(SO42–-S)in(N/S)in(NH4+-N)removal(SO42–-S) removal(NO3-N)eff
83.76 112.27 71.13 38.80 13.54 
83.02 40.25 52.65 30.48 6.42 
83.23 29.16 96.64 78.13 46.72 26.89 
87.13 29.79 52.86 74.77 8.91 5.41 
(NH4+-N)in(NO2-N)in(SO42–-S)in(N/S)in(NH4+-N)removal(SO42–-S) removal(NO3-N)eff
83.76 112.27 71.13 38.80 13.54 
83.02 40.25 52.65 30.48 6.42 
83.23 29.16 96.64 78.13 46.72 26.89 
87.13 29.79 52.86 74.77 8.91 5.41 

All units in mg·L−1 except removal; N/S meaning is NH4+-N/SO42–-S; (in) in the presence of influent; (eff) in the presence of effluent.

During the SRAO process, ammonium oxidation and sulfate reduction proceeded by a three-step process, with the end-products dependent on the ratio of elemental N and S. The high-throughput 16S rRNA gene sequencing experiments clearly showed that nitrite was the intermediate of the ammonium oxidation. In the case of high influent N/S, the SRAO bacteria produced nitrite from ammonium, and subsequently, the reaction of nitrite to nitrogen gas namely, the anammox process, occurred. In the case of low influent N/S, anammox bacteria could make use of the nitrification pathway to directly produce nitrate. The appropriate ratio of N/S led to the shift in the nitrite and nitrate reduction pathways from anammox to sulfur-utilizing denitritation and denitrification, in which the electron donor was elemental sulfur. Future studies will concentrate on the mechanism of nitrite and nitrate reduction.

CONCLUSIONS

Injections of a certain amounts of nitrite and nitrate had positive effects on the SRAO process performance; the sulfate removal efficiency was 85%, and approximately no NH4+-N was detected in the effluent. Compared with the blank group, the relative abundances of microbial communities showed increased Proteobacteria, Chloroflexi, Bacteroidetes, Chlorobi, Nitrospirae Planctomycetes and Armatimonadetes after the reactor ran for 97 days. Since nitrifying bacteria and traditional anammox activity were positively impacted, and nitrification-denitrification and anammox occurred simultaneously with SRAO. This experiment was able to integrate NH4+-N, NO2-N and SO42–-S cycles to maintain optimal electron flow for the SRAO process. Sulfur-based autotrophic denitritation and denitrification occurring in the reactor were caused by nitrite and nitrate.

Studying the role of nitrite and nitrate was important for the SRAO process. Many researchers have reached different conclusions while studying the NO3 and NO2 produced during the SRAO process, which indicated the mechanism of SRAO process was unclear and complex (Zhang et al. 2013). Future work will focus on isolating the functional bacteria and reducing the production of excess nitrate and nitrite.

ACKNOWLEDGEMENTS

This study was supported by the Liaoning Provincial Department of Education General Project (Grant No. LCD2016001).

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