Free ammonia (FA), a form of ammonia nitrogen (NH₄⁺—N), is widely present in municipal/industrial wastewater. Commonly, Eq. (1) is used to calculate the concentration of FA^[1].

There are positive relationships between the concentration of FA and NH₄⁺—N, and the pH and T (temperature) based on Eq.(1).

FA has a significant effect on the nitrogen removal performance in the biological nutrient removal (BNR) pathway, because FA can affect the activity of nitrifying bacteria (including ammonia- oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB))^[1-4]. The earliest inhibition threshold levels were reported by Anthonisen et al.^[1], who demonstrated that AOB and NOB are inhibited by 10~150 mgFA/L and 0.1~1.0 mgFA/L, respectively. Considerable research has focused on the inhibition of AOB and NOB activity^[5-8]. These studies mainly explored how to achieve effective partial nitrification during aerobic nitrification using the gap in the inhibitory concentration of FA between AOB and NOB. Nitrification, a two-step microbial process, is the oxidation of ammonia to nitrite by AOB and of nitrite to nitrate by NOB^[9-12]. Partial nitrification is usually achieved by using the difference in activity between AOB and NOB, so ammonia is only oxidized to nitrite^[13-15]. Over several decades, much research has been performed on stabilizing partial nitrification in BNR systems to remove nitrogen from high ammonia nitrogen wastewater by adjusting the concentration of FA to a specific level that inhibits NOB rather than AOB^[16-18].

It is widely acknowledged that the nitrogen removal performance in BNR systems is affected by the activity of nitrifying bacteria^[19-20]. From the perspective of the biological inhibition mechanism, the relative abundance and structure of nitrifying bacteria in the sewage treatment system is significantly influenced by the concentration of FA. Recently, Illumina high-throughput sequencing (HTS) technology was used to gain an in-depth understanding of characteristics of the microbial community in the water treatment system. During past years, much literature focused on the relative abundance and the structure of the nitrifying bacteria caused by FA in the BNR process^[21-23]. These studies revealed that the relative abundance and structure of nitrifying bacteria are significantly affected at 2.9~50.1 mgFA/L.

Although exploration of the effects of FA on nitrifying bacteria during aerobic nitrification is increasingly successful, there is still a technical problem in that there is no consistent threshold level at which FA inhibits AOB and NOB. Thus, it is essential and urgent to investigate a consistent level at which FA inhibits AOB and NOB in biological systems. Most reported studies focused on the nitrifying bacteria in wastewater treatment bioreactors with a random FA concentration range (the concentration of FA depends on the quality of the wastewater). Few studies investigated nitrifying bacteria in the bioreactor under the precisely controlled concentration of FA. In addition, although previous studies revealed that FA has adverse effects on the microbial activity and stability of sludge, detailed information on the mechanisms by which it accomplished this was limited to various sludge characteristics and the structure of the microbial community caused by the concentration of FA. To eliminate these drawbacks, it is indispensable to systematically assess the variety and structure of nitrifying bacteria of relative abundance under different concentrations of FA during aerobic nitrification.

During our experiment, we filled four parallel sequencing batch reactors (SBRs) with precisely controlled FA concentrations of 0.5, 5, 10 and 15 mg/L. Activated sludge (AS) was used to tame microorganisms exposed to a specific concentration of FA in the SBRs. First, the long-term nitrogen removal performance of the SBRs was investigated. A method was explored to successfully achieve rapid and stable partial nitrification in the SBR at a high concentration of FA. The variety and structure of AOB and NOB of relative abundance in the SBRs with different concentrations of FA and the related mechanisms were examined in detail using high-throughput sequencing of the 16S rRNA gene. The information obtained has implications for the biological mechanism of nitrogen removal and the mechanism by which FA inhibits nitrifying bacteria.

1 Material and methods

1.1 Batch experiments design and operation

Four 4 L parallel SBRs with 0.5, 5, 10 and 15 mg/L, concentrations of FA were operated to enrich the microbial community. The concentration of ammonia, the temperature and the pH were adjusted to obtain different concentrations of FA. Every cycle of the four SBRs consisted of 5 min filling, aerobic reaction, anoxic reaction, settling, 5 min decanting, and an idling period. The aerobic reaction, anoxic reaction, settling and idling period were flexible because of the different initial concentrations of FA.

The nitrification and denitrification were performed by adjusting the ORP, pH and DO. The pH was adjusted by the addition of 0.1 mol/L HCl and 0.4 mol/L NaOH. The temperature control system was used to control the temperature in the SBR. The DO concentrations in the four SBRs were maintained at 1.0~2.5 mg/L by an air compressor during aeration and by continuous stirring at 150 r/min by a mechanical stirrer rotating during the anoxic reaction. Table 1 summarizes the reactor operation.

表 1

Table 1 Operational conditions with variable concentrations of FA in four SBRs FA/(mg·L-1) NH4+—N/(mg·L-1) The running time of each stage of SBR/min Operational parameters Cycle Filling Aeration Hypoxia Setting Decantation MLSS/(mg·L-1) Temperature/℃ pH DO/(mg·L-1) 0.5 040 620 5 270 300 40 5 3 900 20±2.0 7.5±0.2 1.0~2.5 5 090 710 5 300 360 40 5 4 400 25±2.0 8.0±0.2 1.0~2.5 10 130 810 5 360 420 20 5 4 500 30±2.0 8.0±0.2 1.0~2.5 15 055 570 5 240 300 20 5 4 400 35±2.0 8.5±0.2 1.0~2.5

Table 1 Operational conditions with variable concentrations of FA in four SBRs

Activated sludge with a mixed liquor suspended solid (MLSS) of 3 000 mg/L was collected at a local domestic sewage treatment plant (WWTP) in Lanzhou, Gansu to start up the batch reactors.

The four SBRs were operated for 250 days under the above mentioned concentrations of FA. After sustained long-term steady treatment by the reactors, four acclimated activated sludge samples, S_0.5, S₅, S₁₀ and S₁₅, were obtained. On day 240, 12 samples were collected from the four SBRs (3 samples from each SBR). The collected samples were immediately mixed with absolute ethanol at a 1 :1 volume ratio, and placed in a refrigerator, where they were maintained at -20 ℃ for DNA extraction.

1.2 Synthetic media

Nutrients and trace elements in the synthetic media support microbial growth in the reactors. The synthetic media contains (adapted from Kuai et al.^[24]). 115 mg/L NH₄Cl, 385 mg/L CH₃COONa, 26 mg/L of K₂HPO₄ and KH₂PO₄, and 2 mL of trace elements solution. 5.07 mg MgSO₄·7H₂O, 1.26 mg Na₂MoO·2H₂O, 2.49 mg FeSO₄·7H₂O, 0.41 mg CoCl₂·6H₂O, 0.44 mg ZnSO₄·7H₂O, 0.31 mg MnSO₄·4H₂O, 0.43 mg CaSO₄·2H₂O, 0.25 mg CuSO₄, 1.88 mg EDTA and 0.25 mg NaCl were contained per liter in the trace elements solution.

1.3 Analytical measurements

The concentrations of NH₄⁺—N, NO₂^-—N, NO₃^-—N and COD were measured every 30 minutes to determine the performance of the reactor. These parameters were monitored simultaneously in both influent and effluent throughout the experiment. Standard methods (APHA^[25])were used to measure NH₄⁺—N, NO₃^-—N, NO₂^-—N, COD and MLVSS. The pH/oxi 340 analyzer (WTW, Germany) was used to measure the pH, DO and temperature.

1.4 Calculations

The concentration of FA was calculated by Eq.(1).

The ammonia oxidation rate (AOR) was calculated by Eq.(2).

$ \begin{array}{l} {\rm{AOR}}\left( {{\rm{mgN}}/{\rm{gVSS}}\cdot{\rm{min}}} \right) = \\ \frac{{\left[ {{\rm{NH}}_4^ + - {{\rm{N}}_{{\rm{inf}}}}} \right] - \left[ {{\rm{NH}}_4^ + - {{\rm{N}}_{{\rm{eff}}}}} \right]}}{{{\rm{MLVSS}} \times t}} \end{array} $

(2)

The nitrite oxidation rate (NOR) was calculated by Eq. (3).

$ \begin{array}{l} {\rm{NOR}}\left( {{\rm{mgN}}/{\rm{gVSS}}\cdot{\rm{min}}} \right) = \\ \frac{{\left[ {{\rm{NO}}_2^ - - {{\rm{N}}_{{\rm{eff}}}}} \right] - \left[ {{\rm{NO}}_2^ - - {{\rm{N}}_{{\rm{inf}}}}} \right]}}{{{\rm{MLVSS}} \times t}} \end{array} $

(3)

The nitrite accumulation rate (NAR) was calculated by Eq. (4).

$ {\rm{NAR}}\left( \% \right) = \frac{{{\rm{NO}}_2^ - - {{\rm{N}}_{{\rm{eff}}}}}}{{\left[ {{\rm{NO}}_2^ - - {{\rm{N}}_{{\rm{eff}}}}} \right] + \left[ {{\rm{NO}}_3^ - - {{\rm{N}}_{{\rm{eff}}}}} \right]}} \times 100\% $

(4)

Where NH₄⁺—N_inf and NH₄⁺—N_eff are the ammonia concentrations (mg/L) in the influent and the effluent, respectively; NO₂^-—N_inf and NO₂^-—N_eff are the nitrite concentrations (mg/L) in the influent and the effluent, respectively; NO₃^-—N_eff is the nitrate concentration in the effluent (mg/L); T is the temperature (℃); t is the running time (min); and MLVSS is the mixed liquid volatile suspended solids (mg/L).

1.5 DNA extraction steps, PCR quantification and high-throughput sequencing

First, a 10 mL AS sample fixed with absolute ethanol was centrifuged at 12 000 r/min for 10 min. The obtained sediment was used for subsequent DNA extraction. We followed the manual steps to extract DNA with FastDNA^TM Spin Kit for Soil (MP Biomedicals, USA). Agarose gel electrophoresis was used to assess the quality of the DNA and a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) was used to measure the concentration of DNA. The extracted DNA samples were stored in the refrigerator at -80 ℃. The V3-V4 hypervariable region of the 16S rRNA gene was targeted for PCR using the 338F (5′-ACTCCTACGGGCAGCA-3′) forward primer and the 806R (5′-GGACTACHVGGGTWTCTAAT-3′) reverse primer. The thermocycler was operated with an initial denaturation at 98 ℃ and 2 min, followed by 25 cycles of denaturation at 98 ℃ for 15 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 30 s, and a final elongation at 72 ℃ for 5 min.

Agencourt AMPure Beads (Beckman Coulter, Indianapolis, IN) and PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA) were used to purify and quantify the PCR amplicons, respectively. The amplicons were combined in equal amounts and double-ended 2×300 bp sequencing was performed using the Illumina MiSeq platform and MiSeq kit V3 from the Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China). QIMME software was used to classify and quantify the effective sequences and the sequence with a similarity threshold higher than 97% was classified as the same operational taxonomic unit (OTU).

2 Result and discussion

2.1 Nitrogen removal performance in SBRs

We launched a study to explore the long-term nitrogen removal performance caused by variable concentrations of FA in SBRs. Fig. 1 shows the NH₄⁺—N concentrations in the influent and effluent and the NH₄⁺—N removal efficiency in the four SBRs throughout the experiment. S_0.5~S₁₅ was fed in the influent with average NH₄⁺—N concentrations of 40, 90, 130 and 55 mg/L, respectively. Low NH₄⁺—N concentrations of 0.7, 1.1, 1.2 and 0.7 mg/L in S_0.5~S₁₅, respectively, were found in the effluent in the four SBRs throughout the experiment. A high ammonia removal efficiency of 98.1%, 98.8%, 99.0% and 98.8% in S_0.5~S₁₅ (averaging 98.7%), respectively, was obtained at the variable FA concentrations. It is clear that the variable concentration of FA in the SBRs had an insignificant impact on the NH₄⁺—N removal efficiency. The result confirmed the discovery of Vadivelu et al.^[2] that the activity of ammonia-oxidizing bacteria (AOB) is minimally affected when the concentration of FA is lower than 16 mg/L. However, our findings directly contradicted those of Cao et al.^[22], who found a downward trend in the removal efficiency of NH₄⁺—N (99.5% to 38.8%) with the increased concentration of FA (2.9~19.5 mg/L).

图 1

Fig. 1 The influent and effluent concentration of NH4+—N and NH4+—N removal efficiency in SBRs during the operational period

The biological reaction rate is strongly affected by the concentration of FA. Past studies proved that AOR and NOR show a sharp downward trend when the concentration of FA is higher than 25 mg/L^[26-28]. In our study, we also found that variation in the concentration of FA significantly affects the AOR and NOR during nitrification throughout the experiments. A considerable increase in AOR occurred from 0.038 mgN/gVSS·min at 0.5 mg/L (S_0.5) to a maximum of 0.093 mgN/gVSS·min at 10 mg/L (S₁₀), and then slightly declined to 0.073 mgN/gVSS·min at 15 mgFA/L (S₁₅). However, the NOR was maintained at 0.038±0.009 in S_0.5 and S₅ and 0.012±0.004 in S₁₀ and S₁₅ (Fig. 2). The AOR was still maintained at a high level of 0.073-0.093 mgN/gVSS·min even though the concentration of FA was higher than 10 mg/L, indicating that stable and efficient NH₄⁺—N removal was achieved.

图 2

Fig. 2 Effect of concentration of FA on AOR and NAR

A high level of NH₄⁺—N removal efficiency (above 98%) was observed in the four SBRs, demonstrating that NH₄⁺—N is removed, to a great extent, during nitrification and denitrification in the SBR. Therefore, the SBR is a useful reactor to remove NH₄⁺—N from high nitrogen wastewater.

2.2 Achieving a nitrate pathway in S_0.5 and S₅ and a nitrate pathway in S₁₀ and S₁₅ for nitrogen removal

2.2.1 Achieving a nitrate pathway in S_0.5 and S₅ at a lower concentration of FA

In S_0.5 and S₅, the SBRs were operated for 250 days by adjusting the pH, DO and temperature to switch between aerobic and anaerobic. In the nitrified effluent, the nitrite concentrations were 0.38 mg/L and 0.51 mg/L, the nitrate concentrations were 28.2 mg/L and 66.5 mg/L, and NAR and the accumulation rate of nitrate were 0.57% and 0.50%, 98.8% and 99.1% in the S_0.5 and S₅, respectively (Fig. 3). Extremely low NAR and nitrate accumulation indicated that complete nitrification was obtained in S_0.5 and S₅. Simultaneously, the results indicated that the NH₄⁺—N removal efficiency was not affected by the rise of FA concentration (0.5 to 5 mg/L) and full nitrification was achieved in both S_0.5 and S₅. The results showed that nitrifying bacteria could quickly adapt to specific concentrations of FA.

图 3

Fig. 3 Nitrogen removal performance in SBRs along the operational period

The concentrations of FA in S_0.5 and S₅ were 0.5 and 5 mg/L, that were both within and also much higher than the inhibition threshold of FA on NOB (begin at 0.1~1.0 mg/L) reported by Anthonisen et al.^[1]. However, obvious nitrite accumulation was not found in S_0.5 and S₅ (Fig. 3(a)), showing that NOB activity could not be inhibited significantly when the FA is lower than 5 mg/L. Our research fully confirmed the conclusion of Vadivivelu et al.^[2], who also found no significant decrease of NOB activity from 0 to 4 mgFA/L, indicating that a low concentration of FA has a small inhibitory effect on NOB.

In addition, nitrite accumulation did not occur in S_0.5 and S₅ even though we strictly controlled the process in each SBR cycle within 250 days. Our finding was consistent with Cao et al.^[22]. There was no obvious nitrite accumulation by applying process control after 29 cycles at 2.9 and 5.6 mgFA/L, but they also achieved high total nitrogen removal efficiency averaging over 99%. However, Vlaeminck et al.^[13] obtained stable nitrite accumulation and high nitrogen removal efficiency when the concentration of FA was higher than 3 mg/L, meaning that NOB suffered from strong suppression.

2.2.2 Initiating the nitrite pathway at a high concentration of FA in S₁₀ and S₁₅

In the nitrified effluent, a remarkable decline took place in the NO₃^--N concentration from 80.8 to 1.0 mg/L and 47.2 to 1.1 mg/L in S₁₀ and S₁₅, respectively (Fig. 4). However, the NO₂^--N concentration increased considerably, from 0.07 to 81.2 mg/L and 0.18 to 48.3 mg/L in S₁₀ and S₁₅, respectively, indicating the accumulation of nitrite in S₁₀ and S₁₅. The NAR dramatically went up to 90.3% on day 79 in S₁₀ and 90.5% on day 139 in S₁₅, and the nitrate accumulation rate decreased to 1.1% on day 79 in S₁₀ and 2.3% on day 139 in S₁₅, respectively (Fig. 4). The increase of NAR with the decline of nitrate accumulation showed that NOB activity was greatly inhibited and partial nitrification was successfully achieved. AOB gradually became the dominant bacteria, because NOB was inhibited, forming a new nitrifying bacteria structure. S₁₀ and S₁₅ successfully achieved partial nitrification at a high concentration of FA.

图 4

Fig. 4 Nitrogen removal performance in SBRs along the operational period

The nitrite pathway was successfully established at 10 mgFA/L and 15 mgFA/L, and S₁₀ achieved the nitrite pathway faster than S₁₅ (Fig. 4). NOB activity was strongly inhibited in S₁₅ and S₁₀, while AOB activity was reduced more in S₁₅ than S₁₀, leading to the decline of the oxidation rate of the ammonia, and increasing the realization time of the nitrite pathway. Both the continuous ammonia oxidation process and the nitrite accumulation indicated that AOB activity wasn't greatly affected by 10 and 15 mgFA/L, which confirmed that AOB is more tolerant of FA than NOB.

Although several authors analyzed the inhibitory thresholds of FA on AOB and NOB, a consistent inhibitory threshold was not obtained. For example, the first inhibition threshold level of FA on AOB and NOB reported by Anthonisen et al.^[1] was 10~15 mg/L and 0.1~1.0 mg/L, respectively. Kim et al.^[4] suggested that only NOB was inhibited, while AOB could still oxidize ammonium to nitrite at 14~17 mgFA/L. Van Hulle et al.^[27]reported that AOB activity was not inhibited in SHARON reactors at 70-300 mgFA/L.

Vadivelu et al.^[2] found that the inhibitory effect of FA on NOB started from 1 mg/L and stopped growing when it was higher than 6 mg/L. In comparison, Vadivelu et al.^[3] revealed that the inhibitory effect of FA on AOB started at 16 mg/L. Our research and previous studies showed that FA concentrations at S₁₀ and S₁₅ can strongly inhibit NOB activity, but have little effect on AOB activity.

2.2.3 Maintaining the nitrite pathway at a high concentration of FA in S₁₀ and S₁₅

During the period the nitrite pathway was maintained (S₁₀ on day 80~250 and S₁₅ on day 140~250), the main product in the nitrification was nitrite (81.2 and 48.3 mg/L), the nitrate concentration was still maintained at the bottom level (1.0 and 1.1 mg/L) and the NARs remained stable at 98.8% for 170 days and 98.2% for 110 days in S₁₀ and S₁₅, respectively (Fig. 4). Therefore, we can conclude that 10 mgFA/L and 15 mgFA/L can continue to inhibit NOB activity, and the nitrite conversion process was strongly inhibited.

In this study, the selective inhibition at high concentration of FA combined with process control is a basic method to achieve and stabilize the nitrite pathway in the treatment of synthetic wastewater in the SBR.

2.3 Confirmation of the dominant nitrifying bacterial population

Nitrifying bacteria plays a crucial role in nitrogen removal in the SBR. Therefore, we analyzed the relative abundance and structure of the nitrifying bacteria to better understand the microbial role in nitrification using high-throughput sequencing. Considerable studies have reported that five kinds of AOB (Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus and Nitrosovibrio) and four kinds of NOB (Nitrococcus, Nitrospira, Nitrobacter and Nitrospira) were widely found in the sewage treatment system^[29-32]. In this work, we found two types of nitrifying bacteria (Nitrosomonas (AOB) and Nitrospira (NOB)), which were regarded as the dominant nitrifying bacteria in WWTPs, as reported. A widespread and reliable consensus that NOB is even more sensitive to FA inhibition than AOB was reached and applied in numerous studies^{[6, 13, 23]}.

As depicted in Fig. 5(a), the relative abundance of AOB and NOB suffered from a significant change by the variation in the concentration of FA, influencing the nitrogen removal. Surprisingly, we found that AOB activity was not inhibited, but enhanced at S₁₀ with the increasing concentration of FA. AOB first increased sharply from 0.13% in S_0.5 to 3.17% in S₁₀ and then decreased to 0.60 in S₁₅, but NOB linearly decreased from 6.14% in S_0.5 to 0.96% in S₁₅ and had a significant negative relationship with concentrations of FA (y=-0.3x+5.3, R²=0.72). The reason for the entirely different trend could be that FA can serve as a matrix for AOB to increase its relative abundance when the concentration of FA is lower than 10 mg/L, but FA reached the threshold for inhibiting AOB and made its abundance decrease when the concentration of FA was higher than 10 mg/L. However, the inhibition threshold of NOB was 0.5 mgFA/L. Thus, there was a gradual decline in the abundance of NOB with the increasing concentration of FA. The results were in agreement with AOR and NAR under the four FA treatments and confirmed that NOB was even more sensitive to FA inhibition than AOB.

图 5

Fig. 5 Distribution and relative abundance of AOB and NOB, AOB/NOB and NAR in SBRs

Furthermore, it was clear that that NOB Nitrospira was the dominant nitrifier (AOB/NOB < 1) at FA below 5 mg/L, and led to complete oxidation of ammonia to nitrate. But AOB Nitrosomonas was the predominant nitrifier (AOB/NOB=2.03) at 10 mg FA/L, and resulted in sufficient oxidation of ammonia to nitrite. A stable nitrite pathway was maintained at 15 mg FA/L with higher AOR (0.073 mgN/gVSS·min) than NOR (0.017 mgN/gVSS·min) although the relative abundance of NOB Nitrospira (0.95%) was higher than AOB Nitrosomonas (0.6%) (AOB/NOB=0.63) (Fig. 5(a) and (b)). In other words, a higher abundance of NOB Nitrospira had lower activity of utilizing the nitrite substrate, indicating that the NOB activity was strongly inhibited in S₁₅. However, AOB Nitrosomonas exhibited the opposite trend. For example, the lower abundance had a higher microbial utilization capacity of the ammonia substrate. Thus, it was still able to make the AOR higher than the

NOR when the AOB abundance was greater than that of the NOB and remain stable during the ammoxidation process. Similar inhibitory threshold levels of FA on NOB were reported by Sun et al.^[32], who obtained a nitrite pathway of over 90% at 16.3 mgFA/L in the UASB-SBR system treating landfill leachate and intense oppression occurred of FA on NOB activity.

Based on the results, we can draw the conclusion that FA can affect not only the relative abundance of AOB and NOB, but also the NOB activity. However, AOR decreases with AOB abundance. Thus, we cannot conclude that AOB activity is inhibited at 15 mgFA/L. Moreover, by adjusting the concentration of FA, promoting AOB but suppressing NOB, stable short-range nitrification was successfully achieved.

3 Conclusions

The SBR is an efficient and stable reactor to remove ammonia from synthetic wastewater. Stable partial nitrification was successfully achieved in the SBR at high concentrations of FA (10 and 15 mgFA/L). Although AOB and NOB coexist in the four systems, AOB is still the main nitrifying bacteria. This finding emphasizes the importance of cultivating the appropriate bacteria to achieve short-range nitrification. FA can affect not only the relative abundance of AOB and NOB but also the activity of NOB. Furthermore, we found that a lower abundance of AOB had a higher microbial utilization capacity of ammonia substrate at 15 mgFA/L.

Acknowledgements: The authors would like to acknowledge the financial support from the National Natural Science Foundation of China (Grant No. 51668031).

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