Rapid Start-Up and Stable Maintenance of Partial Nitrification Process Through Different Inhibitor Addition and Real-Time Aeration Control

Abstract

This study investigated the rapid start-up of partial nitrification through different inhibitor addition, including hydroxylamine (NH₂OH), potassium chlorate (KClO₃), and formic acid (HCOOH). Then, the performances of the three inhibitors on the rapid recovery of partial nitrification once increasing the influent ammonium sharply were also compared. Finally, real-time aeration control strategy was adopted to maintain partial nitrification during a period. In the present work, batch experiments were used to confirm the optimum dosage of each inhibitor in a similar adding range first. The results showed that nitrite-oxidizing bacteria (NOB) were more vulnerable to NH₂OH and KClO₃ than HCOOH. Second, the rapid start-up of partial nitrification in a sequencing batch reactor was evaluated. It finished in 22 days without any inhibitor. However, only 12 days were needed when adding 0.15 mM NH₂OH. It used 14 days when adding 0.5 mM KClO₃ and 17 days under 0.5 mM HCOOH addition, respectively. The partial nitrification, which was destroyed by the high influent ammonium, was easily and rapidly recovered using the inhibitors. The nitrite accumulation rate was still more than 80% using the real-time aeration control strategy when ceasing the inhibitor addition. Furthermore, high-throughput sequencing analysis revealed that the main genera of NOB, Nitrospira, was suppressed by the three inhibitors; however, a difference in bacterial community structure was found due to the different influence mechanisms. Overall, inhibitor addition and real-time aeration control is an excellent strategy for establishing, recovering, and maintaining effective partial nitrification.

Introduction

The conventional biological nitrogen removal technology is based on the nitrification and denitrification processes, which require high energy for aeration and external biodegradable chemical oxygen demand for denitrification (Gabarró et al., 2012; Ma et al., 2016; Wang et al., 2019). Considering the processing cost, many novel processes such as the partial nitrification-anammox (PNA) process have recently been developed (Qian et al., 2013; Miao et al., 2018). The PNA process can reduce at least 60% of energy for aeration and save up to 100% of the organic matter requirement. However, the anammox process requires both ammonium and nitrite in a molar ratio of 1:1.32 as reaction substrates (Qian et al., 2013). Unfortunately in most wastewater, ammonium is the main nitrogen compound, and therefore, it needs a pretreatment for the achievement of the suitable molar ratio. Thus, partial nitrification, which converts 50–57% of influent ammonium to nitrite and prevents the production of nitrate, plays an important role in the PNA process (Wang et al., 2018; Yang et al., 2018).

However, the application of partial nitrification is rarely observed in conventional wastewater treatment processes. This is because nitrite-oxidizing bacteria (NOB) activity is usually similar or higher than ammonia-oxidizing bacteria (AOB) activity. As a result, ammonium can be easily oxidized to nitrate completely not to nitrite (Peng and Zhu, 2006; Miao et al., 2018). Thus, the key to start-up and maintain partial nitrification is to accumulate AOB and inhibit or washout NOB (Lackner et al., 2014; Li et al., 2019a). There is a growing effort toward addressing the problem, for instance, low dissolved oxygen (DO) (Chuang et al., 2007; Blackburne et al., 2008; Guo et al., 2009), high free ammonia (FA) and free nitrous acid (FNA) (Guo et al., 2009a; Park and Bae, 2009; Qiao et al., 2010, 2013), low sludge retention time (SRT) (Hellinga et al., 1998; Guo et al., 2009b, 2009c), and real-time control (Regmi et al., 2014) have been reported to achieve partial nitrification successfully. However, there are some drawbacks to these strategies. For example, Hulle et al. (2010) found that low DO effectively inhibited the NOB activity, but it also limited the reaction rate of partial nitrification. Moreover, low DO caused the excessive growth of filamentous organisms, leading to the unqualified sedimentation performance of sludge (Bao et al., 2009). Wang et al. (2014, 2017) illustrated a strategy for achieving partial nitrification, which involves using high FA and FNA as inhibitors. However, they also found that the strategy increased the input of additional nitrogen load, and the cumulative nitrite concentration in the common wastewater treatment would seldom reach the NOB inhibition threshold. Moreover, Bae et al. (2002) and Duan et al. (2019) reported that partial nitrification could deteriorate during long-term operation in the case of high FA. Ma et al. (2017) observed that NOB showed capable of tolerating increasing levels of FNA, causing the failure of the FNA established partial nitrification system. Yuan and Oleszkiewicz (2011) indicated that low SRT led to the low net biomass production of functional bacteria because of continuous biomass washout. In a word, achieving stable partial nitrification is a challenge through the normal control parameters. Effective and economical strategies for the start-up and maintenance of partial nitrification are of significance for the application of novel biological nitrogen removal processes.

Recently, the addition of some inhibitors for realizing partial nitrification has received much attention. As an intermediate of nitrification, hydroxylamine (NH₂OH) showed an irreversible inhibitory effect on the growth of NOB but not AOB (Van et al., 2008; Xu et al., 2012). Li et al. (2019b) investigated the partial nitrification for domestic wastewater through the short-term addition of 5 mg/L NH₂OH. The results showed that the process was quickly established in 5 days. Hynes and Knowles (1983) found that potassium chlorate (KClO₃) selectively inhibited NOB but not AOB. Xu et al. (2011) reported that the partial nitrification was successfully achieved with the addition of 5 mM KClO₃ in the aerobic granule system. Formic acid (HCOOH) is an environmental-friendly organic acid and can hinder full nitrification (Eilersen et al., 1994). Wang et al. (2020a) evaluated the feasibility of achieving stable partial nitrification by adding 30 mM HCOOH. During 27 days of operation, the nitrite accumulation rate (NAR) was rapidly raised and maintained at ∼90%. In brief, the above inhibitors have been successfully proposed, while there is no consensus on the most effective inhibitor today, making it necessary to develop an effective and environmental-friendly inhibitor, which was easy to be decomposed by microorganisms and would not bring any toxicity to the environment or the bacteria in the subsequent wastewater treatment processes. It is interesting to compare the performances of different inhibitors in the start-up of partial nitrification when adding a similar dosage. Indeed, the inhibitor addition occurred in intermittent and continuous modes (Xu et al., 2011; Li et al., 2019a; Wang et al., 2020b). Until now, limited research has been done on the low dose concentration, as well as intermittent modes, of the three compounds on the recovery of partial nitrification once destroyed due to the changes of environmental conditions. Thus, data are essential in evaluating the feasibility and application of the inhibitor for achieving stable partial nitrification.

Previous studies found that the deterioration of partial nitrification appeared without continuous inhibitor addition. For example, Wang et al. (2015) illustrated that the nitrate buildup occurred within just 11 days after NH₂OH dosing was stopped since the microorganism gradually acclimated to NH₂OH inhibition. It is necessary to develop an efficient and cost-effective method to maintain partial nitrification once ceasing the addition of inhibitor. Real-time aeration control has been proven to be a feasible strategy to maintain stable partial nitrification of low strength domestic wastewater (Blackburne et al., 2008). They also pointed out the drawback of real-time aeration control: it usually needed a long period to initiate partial nitrification. Yang et al. (2007) reported that 76 days were required to achieve partial nitrification when using real-time aeration control strategy. Interestingly, the addition of appropriate inhibitors can enhance the start-up of partial nitrification rapidly, and the real-time aeration control can maintain partial nitrification steadily. The combination of these strategies seems to be a convenient and economical method for the application of partial nitrification.

The aims of this study were to (1) compare the performances for achieving partial nitrification by adding different inhibitors in a similar dosage range, including NH₂OH, KClO₃, and HCOOH; (2) study the feasibility of using the three inhibitors for recovering partial nitrification once destroyed due to the increase of influent ammonium; and (3) explore the potential of maintaining partial nitrification by real-time aeration control when ceasing the inhibitor addition. Batch experiments were established to explore the optimal conditions for inhibitors. Furthermore, the nitrogen removal performances of sequencing batch reactor (SBR) under the optimal inhibitor addition were evaluated during a long-term experiment. Then, variation in the nitrifying microbial community was assessed through the high-throughput sequencing analysis.

Materials and Methods

Seed sludge and synthetic wastewater

The activated sludge was taken from the Tuandao wastewater treatment plant in Qingdao, China, which used the Anaerobic–Anoxic–Oxic (A/A/O) biological process to treat the domestic wastewater. The concentrations of mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solid (MLVSS) in reactors after inoculation were ∼3,000 and 2,000 mg/L, respectively. NH₂OH, KClO₃, HCOOH, and other chemicals were reagent grade (AR) and supplied from Sinopharm Chemical Reagent Co., Ltd.

The synthetic wastewater used in the experiment was modified from a previous study (Wang et al., 2017), which comprised ∼229.25 mg/L of NH₄Cl (60 mg/L $N H_{4}^{+} - N$ ), 1,500 mg/L of KHCO₃, 42 mg/L of KH₂PO₄, 39 mg/L of K₂HPO₄·3H₂O, 25 mg/L of MgCl₂·6H₂O, 20 mg/L of CaCl₂·2H₂O, 1 mg/L of FeCl₃·6H₂O, 0.06 mg/L of CuSO₄·5H₂O, 0.06 mg/L of MnCl₂·4H₂O, 0.19 mg/L of Na₂MoO₄·2H₂O, 0.05 mg/L of ZnSO₄·7H₂O, 0.13 mg/L of CoCl₂·6H₂O, 0.05 mg/L of KI, 0.06 mg/L of H₃BO₃, 0.04 mg/L of NiCl₂·6H₂O, and 1 mg/L of EDTA. The initial pH of the synthetic wastewater was adjusted to 7.9 ± 0.1 by sodium bicarbonate.

Batch experiment

Different dosage of NH₂OH, KClO₃, and HCOOH was used in the experiments, which were carried out in 500 mL glass bottles. According to the previous research (Li et al., 2019a, 2019b), the dosage of NH₂OH was chosen from 0 to 0.3 mM. Previous research (Lees and Simpson, 1957) indicated that 1 to 10 mM KClO₃ blocked the oxidation of nitrite completely. Xu et al. (2011) used 1 mM KClO₃ for the selective inhibition of nitrite oxidation in the SBR. To compare the effect of the three inhibitors in the case of a similar dosage, the adding range of KClO₃ and HCOOH was both from 0 to 1 mM in the present work. During operation, the initial ammonium was 60 mg/L, and the initial pH was adjusted to 8.0. The DO concentration was 2 mg/L. The temperature was maintained at 25°C using a thermostat water bath. After 15 cycles, the samples and sludge were collected at predetermined intervals and analyzed through the methods mentioned in Analytical methods.

Long-term experiment

The long-term experiments were conducted in four SBRs with a working volume of 2 L. It was divided into three phases. In phase I (day 0–22), the inhibitor was added every day and the influent ammonium was about 60 mg/L. The operational cycle of the SBRs consisted of 10 h, and the aeration time was 8 h. In phase II (days 23–35), the inhibitor was added every three days, and the influent ammonium increased to 150 mg/L. The operational cycle was also 10 h, while the aeration time was reduced to 6 h. In phase III (days 36–40), the addition of inhibitor was ceased. The aeration time was determined by a real-time aeration control strategy. The dosage of each inhibitor used in the experiments was the optimum concentration determined in Batch experiment. The pH of the SBRs was between 7.9 and 8.0, and DO was controlled at about 2 mg/L during the aeration. All reactors were operated at a temperature of 25°C, and no sludge was discharged during the whole process.

Analytical measurements

The parameters, including MLSS, MLVSS, $N H_{4}^{+} - N$ , $N O_{2}^{-} - N$ , and $N O_{3}^{-} - N$ , were measured based on the standard methods (APHA et al., 2012). All samples were filtered through 0.45 μm polyethersulfone filters (ANPEL Scientific Instrument Co., Ltd., Shanghai). DO and pH were monitored by DO and pH probes (JPSJ-605F and PHS-3F; Shanghai Electronic Scientific Instrument Co., Ltd.).

High-throughput sequencing analysis

The sludge samples collected in the reactors were freeze-dried by a lyophilizer (Labconco Co., Free Zone, USA). DNA was extracted from 0.10 to 0.20 g dry sludge samples using FastDNA™ SPIN Kits for Soil (M5635-02; Omega, USA), and the DNA concentration was determined using a Qubit2.0 DNA Detection Kit (Q10212; Life, USA). The universal bacterial 16SrRNA genes for total bacteria were amplified. The standard curves were generated in duplicate using serial decimal dilutions of plasmid DNA and adopted when their amplification efficiency was between 90% and 110% and correlation coefficient above 0.99.

Analytical methods

The ammonium removal efficiency (ARE) and NAR were estimated with Equations (1) and (2) (Miao et al., 2018; Li et al., 2019b; Wang et al., 2020a).

Where $I n f . N H_{4}^{+} - N$ and $I n f . N O_{2}^{-} - N$ represented the amount of $N H_{4}^{+} - N$ and $N O_{2}^{-} - N$ in the influent (mg/L), and $E f f . N H_{4}^{+} - N$ and $E f f . N O_{2}^{-} - N$ represented the amount of $N H_{4}^{+} - N$ and $N O_{2}^{-} - N$ in the effluent (mg/L) at the end of the aeration phase.

Previous studies used the biomass-specific ammonium and nitrite oxidation rates to evaluate the activities of AOB and NOB, respectively (Wang et al., 2017; Cui et al., 2020). In the present work, the biomass-specific ammonium and nitrite oxidation rates were calculated using the following formula [Eqs. (3) and (4)]. All experiments were performed in duplicate. One-way analysis of variance (ANOVA) was used to assess the significance of results with p < 0.05.

Results and Discussion

Effect of different inhibitors in batch experiments

The identical reactors were used to select the optimum inhibitor dosage for establishing efficient partial nitrification. In all reactors, steady and complete nitrification was obtained initially with a NAR of less than 1%. Following this, different volumes of inhibitors (as shown in the Batch experiments) were injected into each of the reactors per cycle.

The effect of NH₂OH is shown in Fig. 1a and b. After 15 cycles, ARE without NH₂OH addition was 97.87% within 10 h, while it increased to 99.83% when adding 0.15 mM NH₂OH. However, it decreased to 82.98% when 0.3 mM NH₂OH was used (Fig. 1a). For NAR, it was 38% with no inhibitor, suggesting the accumulation of nitrite. Suthersan and Ganczarczyk (1986) also found that when the pH value of wastewater was controlled above 8.0, the accumulation of nitrite could be realized. When adding 0.15 mM NH₂OH, the maximum value of NAR was 81.5%, while the value decreased to 57.8% as increasing the dosage to 1 mM. The results indicated that NH₂OH could not only enhance the removal of $N H_{4}^{+} - N$ but also stimulate the accumulation of nitrite in nitrification (Villaverde et al., 2000); however, a high dosage of NH₂OH not only prevented the $N O_{2}^{-} - N$ oxidation but also reduced the $N H_{4}^{+} - N$ oxidation performance. As a result, the optimum dosage of NH₂OH was 0.15 mM.

FIG. 1.

The results of ARE and NAR at various concentrations of inhibitors: (a, c, d) are the ARE of NH₂OH, KClO₃, and HCOOH, respectively; (b, e, f) are the NAR of NH₂OH, KClO₃, and HCOOH, respectively. ARE, ammonium removal efficiency; HCOOH, formic acid; KClO₃, potassium chlorate; NAR, nitrite accumulation rate; NH₂OH, hydroxylamine.

As shown in Fig. 1c, ARE was 97.1, 99.2, 78.1, and 78.9% with the addition of 0.2, 0.5, 0.8, and 1 mM KClO₃, respectively. NAR increased from 38% to 66% by increasing the dosage to 0.5 mM, while it decreased to 44.65% when adding 1 mM KClO₃ (Fig. 1e). The result was consistent with that obtained by Lees and Simpson (1957), who indicated that low dosage KClO₃ had no effect on AOB, but could block the oxidation of nitrite. We also found that high KClO₃ would suppress the performance of $N O_{2}^{-} - N$ oxidation. Similarly, Hynes and Knowles (1983) found that the oxidations of both $N H_{4}^{+} - N$ and $N O_{2}^{-} - N$ were inhibited in the presence of 10 mM chlorate after a 2-h lag period. They found that Nitrobacter winogradskyi could reduce ${C l O}_{3}^{-}$ to ${C l O}_{2}^{-}$ and the undetected ${C l O}_{2}^{-}$ inhibited the mixed Nitrosomonas europaea and N. winogradskyi cell suspensions, resulting in the deterioration of ARE and NAR. Thus, 0.5 mM KClO₃ was chosen in the following experiments.

The results of exploring the optimal dosage of HCOOH are shown in Fig. 1c–f. ARE increased to 98.36% when adding 0.5 mM HCOOH, while it decreased to 42.55% with 1 mM addition. NAR increased from 38% to 63.9% when adding HCOOH from 0 to 0.5 mM. However, as continuously increasing the dosage to 1 mM, it decreased sharply to 42.7%. We measured the pH of the system and found it changed to acidic by adding more HCOOH. Law et al. (2011) indicated that low pH inhibited the activity of nitrifying bacteria obviously, resulting in the reduction of both $N H_{4}^{+} - N$ and $N O_{2}^{-} - N$ oxidation performances. Hence, the dosage of HCOOH was chosen as 0.5 mM.

The activity of AOB and NOB in the four systems after 15 cycles was compared in the present work. As shown in Supplementary Fig. S1, the activity of AOB without any inhibitor was 2.934 $m g N H_{4}^{+} - N ∕ (g M L V S S \cdot h)$ , while it was 2.994, 2.976, and 2.949 $m g N H_{4}^{+} - N ∕ (g M L V S S \cdot h)$ under the addition of 0.15 mM NH₂OH, 0.5 mM KClO₃, and 0.5 mM HCOOH, respectively. For the activity of NOB, it was 0.35 $m g N O_{3}^{-} - N ∕ (g M L V S S \cdot h)$ without any inhibitor; however, it was 0.12, 0.07, and 0.25 $m g N O_{3}^{-} - N ∕ (g M L V S S \cdot h)$ when adding NH₂OH, KClO₃, and HCOOH, respectively. ANOVA revealed that the three inhibitors had no effect on the activity of AOB (p > 0.05), but induced a significant inhibition on nitrite oxidation (p < 0.05). Compared to HCOOH, NOB was more sensitive to NH₂OH and KClO₃. The phenomenon can be explained by the following reasons: Kindaichi et al. (2004) found that an average consumption rate of ${N H}_{4}^{+}$ was about four times higher than an average production rate of $N O_{3}^{-}$ , indicating that the activity of NOB was inhibited by the addition of NH₂OH. Furthermore, Li et al. (2019b) found that the NOB population continuously decreased even after the discontinuation of NH₂OH addition. They thought the reason was the inhibition of the synthesis of nitrite oxidoreductase enzyme by NH₂OH. Xu et al. (2012) also found a similar phenomenon. They found that the start-up of the partial nitrification process by NH₂OH addition was attributed to the toxic inhibition of NOB activity and the inhibition of the NOB growth. For KClO₃, Xu et al. (2011) found that KClO₃ could be decomposed by nitrate reductase (N. winogradskyi) because of its similar structure as nitrate. The $C {l O}_{3}^{-}$ was reduced to $C {l O}_{2}^{-}$ , and $C {l O}_{2}^{-}$ could inhibit the mixed N. europaea (AOB) and N. winogradskyi (NOB) cell, leading to the inhibition of NOB activity. For the activity of AOB and NOB in the HCOOH-adding system, Wang et al. (2020b) showed that competitive inhibition contributed more to the activity inhibition during exposure to low concentrations of HCOOH. In the presence of a high concentration of HCOOH, AOB could still maintain high activity because of its high noncompetitive inhibition constant. In addition, Wang et al. (2020a) used reverse transcription quantitative polymerase chain reaction assays to analyze the transcriptions of nitrification genes in the HCOOH-adding SBR. They found that the transcription of nxrB, which encodes the protein responsible for nitrite oxidation, was significantly reduced by ∼4.5-fold after being in the presence of HCOOH. The inhibition of nxrB gene transcription resulted in inhibition of NOB growth.

Long-term operating performance in the presence of different inhibitors

A long-term experiment of SBR in the presence of different inhibitors was divided into three phases. In phase I (0–22 days), the ammonium concentration of effluent was 13.5 mg/L after 22 days without any inhibitor. A measure of 25.4 mg/L nitrite and 25.5 mg/L nitrate was also detected in the effluent (Fig. 2a). In addition, Fig. 3a showed that ARE reached above 81.6% throughout the acclimation period. Simultaneously, NAR increased gradually and reached above 42.3%, suggesting the start-up of partial nitrification (Fig. 3b). When adding 0.15 mM NH₂OH, 11.8 mg/L of ammonium, 29.9 mg/L of nitrite, and 15.5 mg/L of nitrate were detected in the effluent on day 12 (Fig. 2b). Meanwhile, ARE was 80.4% and NAR was 61.9%, respectively. When 0.5 mM KClO₃ was added for 14 days, ARE was 80.7% and NAR was 52.85%. The start-up needed 17 days for 0.5 mM HCOOH addition. It was 80% of ARE and 53.27% of NAR, respectively. These results illustrated that the addition of an inhibitor could reduce the start-up time of partial nitrification significantly.

FIG. 2.

Profiles of ammonium, nitrite, and nitrate concentrations during the experimental. The different phases were indicated by lines: (a) no inhibitor; (b) adding 0.15 mM/L NH₂OH; (c) adding 0.5 mM/L KClO₃; (d) adding 0.5 mM/L HCOOH.

FIG. 3.

The results of (a) ARE and (b) NAR using different inhibitors.

In phase II (23–35 days), the influent ammonium increased to around 150 mg/L, and the aeration time was reduced to 6 h. The partial nitrification was destroyed once increasing the influent ammonium initially. ARE reduced to 5.23% (without any inhibitor), 23.4% (NH₂OH), 15.8% (KClO₃), and 8.13% (HCOOH), respectively. For NAR, it was 13.25%, 57.41%, 46%, and 54.26%, respectively (Fig. 3). However, the results showed that a long-term stimulation by inhibitor improved the steady operation of partial nitrification. Furthermore, the ammonium was removed thoroughly, and NAR returned to 68.7% with the NH₂OH addition on day 35. It is clear that the usage of inhibitor was to the advantage of the recovery of partial nitrification in a short time.

However, Li et al. (2019a) found that the deterioration of partial nitrification appeared during a long-term operation without continuous NH₂OH addition. Real-time aeration control strategy is developed to effectively avoid excessive aeration, which is favorable to the maintenance of stable partial nitrification (Qian et al., 2017). In phase III (36–40 days), we ceased the inhibitor addition and the aeration time was controlled in real time, which was determined based on the performances of ARE and NAR in a cycle. As shown in Fig. 4, ARE in the absence of inhibitor was 100% after 300 min, while it reached 100% within 180 min under the NH₂OH addition. It needed 240 min to remove ammonium thoroughly for both KClO₃ and HCOOH addition. Meanwhile, NAR was only 37.25% without any inhibitor, while it was 51.9% for NH₂OH addition, 50.93% for KClO₃ addition, and 50.9% for HCOOH addition, indicating the occurrence of partial nitrification. As a result, the aeration time was 300 min for no inhibitor addition, while it was 180, 240, and 240 min for the three inhibitors, respectively. Nitrogen variations under real-time control are also shown in Fig. 2. Ammonium level was reduced, and nitrite was continuously accumulated. ARE was more than 90% (Fig. 3a), and a relatively stable partial nitrification performance was obtained to more than 60% (Fig. 3b) under the three inhibitor addition.

FIG. 4.

The results of (a) ARE and (b) NAR using different inhibitors at different times.

Figure 5 showed the variations of nitrifying bacteria activities during the experimental period. As shown in Fig. 5a, the initial activity of AOB activity was 0.38 $m g N H_{4}^{+} - N ∕ (g M L V S S \cdot h)$ (without any inhibitor), 0.24 $m g N H_{4}^{+} - N ∕ (g M L V S S \cdot h)$ (0.15 mM NH₂OH), 0.23 $m g N H_{4}^{+} - N ∕ (g M L V S S \cdot h)$ (0.5 mM KClO₃), and 0.36 $m g N H_{4}^{+} - N ∕ (g M L V S S \cdot h)$ (0.5 mM HCOOH), respectively. At the end of phase I (day 22), it increased to 2.45, 2.98, 2.95, and 2.76 $m g N H_{4}^{+} - N ∕ (g M L V S S \cdot h)$ , respectively. When the influent ammonium increased to 150 mg/L, it decreased sharply to 0.4, 1.75, 0.18, and 0.61 $m g N H_{4}^{+} - N ∕ (g M L V S S \cdot h)$ on day 23. However, it returned to 5.75, 7.3, 7.5, and 7.04 $m g N H_{4}^{+} - N ∕ (g M L V S S \cdot h)$ on day 35, respectively. Using the real-time aeration control strategy in phase III, AOB activity was obtained to 7.02, 7.5, 7.5, and 7.4 $m g N ∕ (g M L V S S \cdot h)$ , respectively. For NOB activity (Fig. 5b), the addition of inhibitor could limit the activity available, and the real-time aeration control strategy seemed to be beneficial to remain the suppression. Li et al. (2019a) found that the increase in AOB activity was due to a reduction in competition for oxygen when NOB activity was inhibited by NH₂OH. Xu et al. (2011) and Wang et al. (2020a) also found a similar phenomenon under the KClO₃ and HCOOH addition.

FIG. 5.

Variations of nitrifying bacteria activities (a) AOB and (b) NOB during the experimental periods. AOB, ammonia-oxidizing bacteria; NOB, nitrite-oxidizing bacteria.

Effect of inhibitor on microbial community

To understand the changes in active bacterial diversity and structure of functional bacteria, high-throughput sequencing was used to test the activated sludge samples of day 35. The Good's coverage values were above 98% in all samples, suggesting that the microbial sequencing matched the requirements of analysis. The difference analysis of operational taxonomic units (OTUs) was presented through the Venn diagram (Fig. 6). A total of 3,496 OTUs were detected, whereas only 26.2% (916 OTUs) species were shared by the four systems, indicating that the active microbial community structure was different in each system. Figure 7a showed the main genus with relative abundances of more than 1%. The results indicated that the active microbial community without any inhibitor was similar to that with HCOOH addition, while it was quite different from that under NH₂OH or KClO₃ addition. Comamonas, a typical bacillus, was found much more in the NH₂OH or KClO₃ system than in the HCOOH system. However, plenty of Rhodobacteraceae, which are common heterotrophic nitrifying bacteria, was found in the HCOOH system, suggesting that the effect of HCOOH acted as a carbon source. Figure 7b showed the AOB and NOB activity levels. Nitrosomonas was the main genera of AOB, and the dominant NOB genus Nitrospira was detected. The relative abundance of AOB in the absence of an inhibitor was 1.58%, while it was 3.46% (0.15 mM NH₂OH), 2.8% (0.5 mM KClO₃), and 2.43% (0.5 mM HCOOH), respectively. However, NOB represented 1.03%, 0.57%, 0.4%, and 0.78% of the microbial communities, respectively. The results indicated that not only AOB activity, the AOB population also increased when using the inhibitors, while the NOB population was also inhibited by the inhibitors.

FIG. 6.

Shifts in community structure as illustrated by Venn.

FIG. 7.

The composition of (a) taxonomy at genus levels and (b) relative abundance of AOB and NOB (Nitrosomonas and Nitrospirae) using different inhibitors on day 35.

Conclusion

This study investigated the feasibility of inhibitor addition on the start-up and real-time aeration control strategy on the maintenance of partial nitrification. First, the performances of different dosage inhibitors on the activity of AOB and NOB were compared. The results showed that NOB was more sensitive to NH₂OH and KClO₃ than HCOOH. Second, based on the results of long term experiments, the addition of each inhibitor was a favor for the rapid start-up and recovery of partial nitrification. NH₂OH performed better than the other two inhibitors in a similar range. When using real-time aeration control strategy after ceasing the inhibitor addition, a stable level with a NAR of 80% was still achieved. The results of high-throughput sequencing revealed that the inhibitor selectively suppressed the transcription and growth of NOB. In addition, different inhibitors caused a difference in bacterial community structure. The method herein may have important scientific and practical significance for the practical application in partial nitrification.

Footnotes

Author Disclosure Statement

No potential conflict of interest was reported by authors.

Funding Information

This study was financially supported by the National Natural Science Foundation of China (Grant No. 51408295), Key Research and Development Project of Shandong Province (Grant Nos. 2017GSF217013 and 2018GSF117007), and Natural Science Foundation of Shandong Province (Grant No. ZR2019BEE047).

Supplementary Material

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