Performance Optimization And Microbial Community Succession in A Continuous-Flow Anoxic MBBR-AAO Process For Enhanced Nitrogen And Phosphorus Removal From Municipal Sewage

Performance Optimization and Microbial Community Succession of Continuous-Flow Anoxic MBBR-AAO Process

In recent years, the advanced treatment of urban sewage and the realization of resource recycling have become hot topics in the field of water environment. However, the traditional nitrogen and phosphorus removal processes widely adopted by wastewater treatment plants not only result in excessive waste of resources but also increase operating costs [1]. Moreover, the gradual decrease in the carbon-to-nitrogen ratio (C/N) of urban sewage and the differences in the living environments of different functional microbial communities have become important limiting factors for water treatment technologies.

The sludge-film hybrid MBBR process combines the activated sludge process with the suspended carrier biofilm process to achieve enhanced enrichment of functional microorganisms, solving the problems of large land occupation and poor low-temperature tolerance of the traditional activated sludge process [2]. In 2008, Wuxi Lucun Wastewater Treatment Plant in Jiangsu Province, as the first wastewater treatment plant in China to carry out the upgrading and reconstruction to Class IA standards, successfully enhanced the treatment effect by adding suspended carriers to the sludge system [3]; Hu Youbiao et al. [4] investigated the effect of temperature on the removal of ammonia nitrogen and organic matter in MBBR and activated sludge, and the results showed that temperature had a smaller impact on MBBR but a greater impact on activated sludge; Zhang Ming et al. [5] used the A²O-MBBR process to treat rural domestic sewage, achieving high removal rates of COD, ammonia nitrogen, TP, and TN; Zhou Jiazhong et al. [2] found through small-scale experiments that DO, temperature were positively correlated with the sludge-film hybrid MBBR system, while the influent C/N ratio was negatively correlated.

The anoxic MBBR (AM-MBBR) process can realize simultaneous denitrification and phosphorus removal in the anoxic tank, which is also the denitrifying phosphorus removal (DPR) process. Compared with traditional wastewater treatment processes, the DPR process can save organic carbon sources and reduce oxygen consumption. Zhang Yongsheng [6] et al. developed a continuous-flow biofilm reactor, and the results showed that at a temperature of 20 ℃, a DO concentration of 5.5 mg/L, a load of 2.2 kg/(m³·d), and an intermittent aeration condition of anaerobic 3 h/aerobic 6 h, the average concentrations of COD and phosphorus in the effluent were 76 mg/L and 0.67 mg/L, with removal rates of 72.9% and 78.5%, respectively.

However, for the sludge-film hybrid AM-AAO system, there is a complex relationship between suspended flocculent sludge and attached biofilm. Previous studies have focused on engineering practices such as bidding and reconstruction of wastewater treatment plants, but there are few studies on synchronous nitrification and DPR to enhance nitrogen and phosphorus removal in continuous-flow sludge-film hybrid AM-AAO systems, and the stability of pollutant removal performance of this process through DPR technology is also one of the difficulties.

This study optimized the start-up and operation strategies of continuous-flow (AAO) and continuous-flow sludge-film hybrid (AM-AAO) processes, focusing on investigating the effects of aeration rate, filler dosage, hydraulic retention time (HRT), nitrification liquid reflux ratio, influent C/N ratio, and temperature on the long-term nitrogen and phosphorus removal performance of the AM-MBBR process and the denitrifying phosphorus removal efficiency in the anoxic tank. At the same time, the succession of microbial communities and the change rules of functional microbial communities in activated sludge and biofilm were studied.

1 Materials and Methods

1.1 Experimental Device and Operating Parameters

A continuous-flow AAO reaction device (Figure 1) was used in this study. It was made of organic glass, with a total of 7 compartments, each with a size of 10 cm × 10 cm × 40 cm; the working volume was 21 L, and the volume ratio of each reaction tank was anaerobic: anoxic: aerobic = 2:2:3. Mechanical stirring was adopted in the anaerobic and anoxic tanks; the aerobic tank used aeration sand heads as micro-porous aerators and external force for sludge-water mixing, and the aeration rate was controlled by a gas flow meter. The DO concentration in the aerobic tank of the reactor was controlled at 2~3 mg/L; the secondary sedimentation tank was a cylinder with a working volume of about 40 L; the sludge retention time (SRT) was 40 d, and the sludge reflux ratio was 50%. The reactor operated for a total of 263 d (divided into 6 operation stages), and polyethylene fillers were added to the anoxic tank starting from the 159th day to operate in the AM-AAO mode. The specific operating conditions are shown in Table 1.

(Figure 1 Schematic diagram of AM-AAO process equipment: The figure includes a water inlet bucket, peristaltic pump, anaerobic tank, anoxic tank, aerobic tank, sedimentation tank, water outlet bucket, as well as internal reflux, sludge reflux pipelines, and drain valves)

Table 1 Process system type and operating parameters

1.2 Inoculated Sludge and Influent Water Quality

The inoculated sludge in this experiment was taken from the excess sludge discharged from the secondary sedimentation tank of a wastewater treatment plant. After inoculation, the sludge concentration (MLSS) in the reactor was 2.3 g/L, and the sludge volatile solids (MLVSS) was 2.1 g/L.

The influent of the reactor was actual domestic sewage from restaurants, which was added to the reactor after filtering impurities through a filter screen. Its pollutants included NH₄⁺-N (35.0456.54 mg/L), NO₂⁻-N (00.42 mg/L), NO₃⁻-N (00.05 mg/L), COD (362.1605.1 mg/L), and PO₄³⁻-P (1~5.08 mg/L).

1.3 Detection Items and Analysis Methods

1.3.1 Routine Detection Methods

Sludge-water samples were collected from the influent, anaerobic tank, anoxic tank, aerobic tank, sedimentation tank, and effluent, and filtered with 0.45 μm filter paper. NH₄⁺-N was determined by Nessler's spectrophotometer; NO₂⁻-N was determined by N-(1-naphthyl) ethylenediamine photometry; NO₃⁻-N was determined by ultraviolet spectrophotometry; COD was determined by Lianhua 5B-3A COD multi-parameter rapid detector; pH/DO and temperature were determined by WTW Multi3620 detector; MLSS was determined by gravimetric method; MLVSS was determined by muffle furnace combustion weight loss method [7].

1.3.2 Extraction and Detection of Extracellular Polymeric Substances

Extracellular polymeric substances (EPS) are considered to be composed of polysaccharides (PS), proteins (PN), and humic acids (HA). Three types of EPS, namely soluble extracellular polymeric substances (S-EPS), loosely bound extracellular polymeric substances (LB-EPS), and tightly bound extracellular polymeric substances (TB-EPS), were separated and extracted. The determination method of PS was sulfuric acid-anthrone method, and the determination methods of PN and HA were modified Folin-Lowry method [7].

1.3.3 Calculation Method of Pollutant Removal Rate

The pollutant removal rate (SRE) was used to characterize the overall pollutant removal of the AM-AAO process system. Among them, Sinf and Seff are the pollutant concentrations of the influent and effluent, respectively, which can represent the mass concentrations of pollutants such as NH₄⁺-N, NO₂⁻-N, NO₃⁻-N, COD, and PO₄³⁻-P in the influent and effluent, mg/L.

1.3.4 High-Throughput Sequencing Method

Illumina high-throughput sequencing method was used. Sludge samples from the anaerobic tank, anoxic tank, and aerobic tank on days 1, 110, 194, and 237 were collected and named as group D01 (D01_A1, D01_A2, D01_O), group D110 (D110_A1, D110_A2, D110_O), group D194 (D194_A1, D194_A2, D194_O), and group D237 (D237_A1, D237_A2, D237_O), respectively; biofilm sludge samples on days 194 and 237 were collected and named as M194 and M237, respectively. A total of 14 sludge samples were analyzed for changes in microbial communities. DNA was extracted using Fast DNA SPIN kit (MP Biomedicals, Santa Ana, CA, USA). The V3-V4 region of the bacterial 16S rRNA gene was amplified with 338F/806R primers. The purified amplicons were sequenced on the Illumina MiSeq PE300 platform (Illumina, USA) by Shanghai Majorbio Biomedical Technology Co., Ltd. (Shanghai, China) [7].

2 Results and Discussion

2.1 Long-Term Pollutant Removal Rules in AAO and AM-AAO Processes

The long-term pollutant removal during the operation of the continuous-flow AAO process (Stages 13) and the AM-AAO process with suspended polyethylene fillers added (Stages 46) is shown in Figure 2.

In Stage 1 (1~45 d), the PO₄³⁻-P release amount (PRA) in the anaerobic tank, PO₄³⁻-P uptake amount in the anoxic tank (PUAA), and PO₄³⁻-P uptake amount in the aerobic tank (PUAO) were 66.06 mg, 14.22 mg, and 87.81 mg, respectively, and the phosphorus uptake process was mainly achieved in the aerobic tank. The removal rates of NH₄⁺-N and total inorganic nitrogen (TIN) were 92.85% and 86.37%, respectively, ensuring the denitrification effect. After fine-tuning the aeration (DO=2~3 mg/L), the NH₄⁺-N removal effect increased to 98.68%, and the effluent TIN concentration and removal rate were 1.75 mg/L and 95.75%, respectively, indicating that proper adjustment of DO is conducive to nitrification and denitrification processes; the COD removal effect in the anaerobic tank weakened (91.60%). In addition, the fine-tuning of DO had no effect on the effluent PO₄³⁻-P, with an average of 0.47 mg/L, which is consistent with the conclusion of Yang Sijing et al. [8].

In Stage 2 (46~120 d), after adjusting HRT=8 h, the COD removal performance fluctuated slightly; the maximum values of PRA, PUAA, and PUAO reached 148.01 mg, 81.95 mg, and 114.15 mg, indicating that the increase in influent flow did not affect phosphorus removal, and maintained high NH₄⁺-N and TIN removal performance. On day 72, the nitrification liquid reflux ratio was increased to 300% and 400%. The increase in reflux ratio decreased the TIN removal effect, with removal rates of 80.37% (300%) and 68.68% (400%), respectively. From day 108 to 120, the nitrification liquid reflux ratio was determined to be 250%. The COD removal amount in the anaerobic tank at a nitrification liquid reflux ratio of 250% (127.1 mg/L) was higher than or equal to that of others (86.2 mg/L, 124.7 mg/L, and 128.0 mg/L for 200%, 300%, and 400%, respectively); the effluent phosphorus concentrations corresponding to different reflux ratios were 0.52 mg/L, 0.35 mg/L, and 0.06 mg/L, indicating that increasing the nitrification liquid reflux ratio within a certain range can promote phosphorus removal. In addition, the reflux ratio of 250% had good denitrification performance, with a TIN removal rate of 86.86%.

In Stage 3 (121~158 d), the nitrification liquid reflux ratio was fixed at 250%. On day 131, the influent flow was increased to 5 L/h, the COD and phosphorus removal effects decreased, and the effluent concentrations were 73.3 mg/L and 3.92 mg/L, respectively, indicating that the increase in influent flow resulted in more COD being discharged without treatment. In addition, the maximum removal rates of NH₄⁺-N and TIN were 93.82% and 79.12%, respectively, among which NO₃⁻-N became the main pollutant in the effluent (4.70 mg/L). On day 139, the influent flow was reduced to 4 L/h, the effluent COD and removal rate were 55.7 mg/L and 85.97%, respectively, which was higher than the carbon removal performance at HRT=5.6 h, indicating that the reduction of HRT may lead to a decrease in COD removal effect. In addition, the maximum removal rates of NH₄⁺-N and TIN were 100% and 97.41%, indicating that the adjustment of HRT promoted nitrification and denitrification, but excessively short HRT may lead to a decrease in denitrification effect. Therefore, when HRT=7 h, it is sufficient for the reactions in each tank to proceed fully, and a significant increase in HRT has little promoting effect on the denitrification effect.

On day 159, 20% suspended polyethylene fillers were added to the anoxic tank of the AAO process. In Stage 4 (159~209 d), the COD and PO₄³⁻-P removal performances were enhanced. Starting from day 172, the influent NH₄⁺-N concentration was increased to 64.17 mg/L (C/N=8.59), the effluent COD and removal rate were 77.7 mg/L and 86.06%, respectively. The reason may be that the biofilm grew slowly, and the activated sludge made the main contribution to the removal of most COD; the suspended fillers increased the PO₄³⁻-P removal rate by 1.18%. However, the increase in influent NH₄⁺-N in the anoxic tank led to the need for more carbon sources for the denitrification process of NO₃⁻-N, which was not conducive to the phosphorus release and uptake of PAOs; at the same time, this operation did not completely reduce NO₃⁻-N, and the minimum effluent concentration was 7.30 mg/L. On day 185, changing HRT to 5.6 h, it was found that the COD removal effect fluctuated slightly, with a removal rate of 86.05%; the effluent PO₄³⁻-P concentration increased by 0.05 mg/L, accompanied by an increase in PUAA (from 13.02 mg to 18.90 mg), indicating that the sludge and biofilm synergistically exerted a certain phosphorus removal efficiency. In addition, the effluent NH₄⁺-N, NO₃⁻-N, and TIN concentrations were 10.23 mg/L, 6.52 mg/L, and 16.82 mg/L, respectively, indicating that the reduction of HRT would lead to a decrease in the removal effects of NH₄⁺-N and TIN. On day 195, HRT was adjusted back to 7 h, and at this time, the pollutant content in the effluent decreased, and the nitrogen and phosphorus removal and organic matter removal performances of the system gradually recovered.

In Stage 5 (210~240 d), the influent NH₄⁺-N concentration was increased to 84.06 mg/L (C/N=6.28), and the activated sludge still made the main contribution to the removal of organic matter. The increase in NH₄⁺-N had little effect on COD removal. The proportion of COD absorbed in the anaerobic tank was 68.02%, and most of the organic matter was absorbed by PAOs in the anaerobic tank and synthesized into internal carbon sources (PHAs), and the anaerobic phosphorus release was fully completed [9]. The maximum PRA was 72.75 mg, and PUAA and PUAO were 35.82 mg/L and 48.20 mg/L, respectively, but the main contribution to phosphorus uptake still came from the aerobic tank. On day 221, the filling ratio was increased to 30%, and the effluent NH₄⁺-N and TIN concentrations were reduced by 4.49 mg/L and 5.16 mg/L, respectively; among them, NH₄⁺-N and NO₃⁻-N accounted for 70.11% and 28.75% of the effluent TIN, respectively. On day 231, the influent NH₄⁺-N concentration was adjusted to 66.34 mg/L, and the pollutant removal performance of the system was basically stable.

In Stage 6 (241~263 d), the reactor temperature was regulated to explore its effect on pollutant removal. On day 241, the temperature was reduced to 18 ℃, the COD removal rate decreased to 84.37%, but the COD change rule did not change due to the temperature decrease. The removal proportion in the anaerobic tank was the highest, 62.02%, the denitrifying phosphorus removal process in the anoxic tank consumed 26.72% of COD, the NO₃⁻-N concentration in the effluent of the aerobic tank was 10.44 mg/L, and 8.50 mg/L of NH₄⁺-N remained; in addition, PRA was less affected by temperature, but the phosphorus uptake performance of the anoxic tank decreased, with PUAA only 19.77 mg, and phosphorus was removed by 3.94 mg/L in the aerobic tank. Most psychrophilic PAOs carried out aerobic phosphorus uptake process [10]. When the temperature was further reduced to 13 ℃, the removal rates of NH₄⁺-N and TIN decreased by 6.38% and 6.25%, respectively; at the same time, PUAA and PUAO decreased by 7.77 mg and 15.00 mg, respectively, which may be related to the decrease in microbial activity and growth and metabolism capacity caused by the temperature decrease. Jin Yu [11] found that when the temperature is lower than 14 ℃, it is difficult to guarantee the effluent pollutant concentration of the system.

(Figure 2 Removal of pollutants in AAO and AM-AAO processes during long-term operation: Including (c) Curves of NH₄⁺-N concentration and removal rate changing with operation days, (d) Curves of NOₓ⁻-N concentration changing with operation days, (e) Curves of TIN removal rate changing with operation days. The horizontal axis is the operation days (0~260 d), and the vertical axes are ρ (NH₄⁺-N)/(mg·L⁻¹), ρ (NO₃⁻-N)/(mg·L⁻¹), and removal rate/%, respectively. Each operation stage is marked on the curves)

2.2 Pollutant Change Rules in Typical Cycles of AAO and AM-AAO Processes

To further explore the pollutant removal mechanism of AAO and AM-AAO processes, the pollutant concentration changes in typical cycles of different operation stages were analyzed, as shown in Figure 3.

On day 42 (Stage 1), the AAO process had good denitrification and phosphorus removal performance. However, the high influent COD did not improve the phosphorus release performance, and the PRA was 9.13 mg/L at this time. In addition, NH₄⁺-N was consumed in advance when entering the anoxic tank; then, the anoxic tank reduced the generated NO₃⁻-N to N₂; however, the aerobic tank only removed 3.52 mg/L of NH₄⁺-N, which may be due to the long HRT in Stage 1 leading to an increase in DO returned to the anoxic tank, and most of the NH₄⁺-N had completed nitrification in the anoxic tank, resulting in a low concentration entering the aerobic tank.

On day 118 (Stage 2), with the decrease of influent COD, the phosphorus release and denitrification performances deteriorated. The phosphorus release concentration in the anaerobic tank was 5.91 mg/L, and the NO₃⁻-N concentration in the effluent of the aerobic tank was 8.20 mg/L. The PO₄³⁻-P concentration in the anoxic tank decreased to 2.78 mg/L, indicating that PO₄³⁻-P was removed in the anoxic tank. In addition, the nitrification liquid reflux ratio was fixed at 250% at this time. Compared with the reflux ratios of 300% and 400%, the nitrogen and phosphorus removal and organic matter removal performances of the process were enhanced, indicating that increasing the nitrification liquid reflux within a certain range can enhance the pollutant removal effect.

On day 207 (Stage 4), after adjusting the influent NH₄⁺-N and HRT in the AM-AAO process, the COD removal rate was 86.15%; the aerobic tank removed 13.34 mg/L of NH₄⁺-N, the remaining TIN concentration was 7.51 mg/L, and 4.39 mg/L of NO₃⁻-N was produced, and NO₃⁻-N became the dominant pollutant in the effluent. There was no significant difference in the phosphorus removal contribution between the anoxic tank and the aerobic tank. In addition, increasing the influent NH₄⁺-N did not affect the nitrification, but the increase in the influent TIN concentration decreased the denitrification performance of the AM-AAO process, thereby affecting the TIN removal.

On day 262 (Stage 6), the reactor temperature was 13 ℃, and the COD removal rate was 83.67% at this time. At the same time, 6.95 mg/L of phosphorus was released in the anaerobic tank; 20.22 mg/L of NH₄⁺-N was consumed by the anoxic tank and denitrification was carried out, and the NO₃⁻-N concentration in the effluent of the anoxic tank was 5.07 mg/L; the aerobic tank had a TIN loss of 1.32 mg/L; the TIN removal rate was 77.00%, and the effluent TIN contained 11.24 mg/L of NH₄⁺-N, indicating that the low temperature reduced the activity of nitrifying bacteria and denitrifying bacteria, resulting in the incomplete removal of pollutants in the sewage. In addition, PRA decreased to 6.95 mg/L, and the phosphorus uptake performances of the anoxic tank and aerobic tank decreased to 2.41 mg/L and 3.61 mg/L, respectively, indicating that the decrease in reactor temperature inhibited the phosphorus removal performance of PAOs, leading to the decrease of PRA in the anaerobic tank and the high effluent phosphorus concentration.

(Figure 3 Contaminant changes in typical cycles: Including (a) Day 42 of AAO process, (b) Day 118 of AAO process, (c) Day 207 of AM-AAO process, (d) Pollutant concentration change curves on day 262 of the AM-AAO process. The horizontal axis is the reaction process, and the vertical axis is the concentration (mg/L) of each pollutant (COD, NH₄⁺-N, NO₃⁻-N, PO₄³⁻-P))

2.3 Changes in Composition and Content of Extracellular Polymeric Substances (EPS) in AAO and AM-AAO Processes

During the experiment, the changes in the composition and content of EPS on day 101 (AAO process) and day 255 (AM-AAO process) were determined and analyzed, as shown in Figure 4. Overall, the total EPS content on days 101 and 255 can be attributed to the increase in TB-EPS content, and PN and PS accounted for the main part of TB-EPS; on day 101, the total EPS content in the anaerobic tank, anoxic tank, and aerobic tank showed an increasing trend (0.12 mg/gVSS, 0.29 mg/gVSS, and 0.37 mg/gVSS, respectively); among them, the EPS content increased significantly during the nitrification stage, which may be due to the active metabolism of internal microorganisms when the system was operated under high carbon-to-nitrogen ratio (C/N=5.9) conditions [12]. However, TB-EPS played a positive role in the formation of sludge flocs, while S-EPS and LB-EPS had negative effects [8]; in this experiment, the contents of S-EPS and LB-EPS were relatively low, which created conditions for sludge growth; in the continuous-flow sludge-film hybrid system, the role of flocculent sludge is irreplaceable [2].

In addition, the change rules of PN/PS in different layers of sludge in each reaction tank were different. The PN in each reaction tank was always higher than PS. On day 101, the PN/PS ratios in S-EPS, LB-EPS, and TB-EPS of sludge were 0.06, 1.62, and 2.67, respectively, while on day 255, they were 0.03, 1.30, and 3.27, indicating that the PN/PS ratio showed an increasing trend from the outer layer to the inner layer of sludge cells. However, when the reactor temperature was reduced to 13 ℃, the total EPS content in the three tanks showed an increasing trend (0.28 mg/gVSS, 0.41 mg/gVSS, and 0.63 mg/gVSS, respectively). The reason may be that microorganisms unable to adapt to low temperature died or autolyzed, and these dead microorganisms released EPS, leading to an increase in the EPS content of sludge, or low temperature induced some psychrophilic microorganisms to secrete more EPS to adapt to the temperature decrease in the reactor [13].

(Figure 4 Changes in EPS content and composition on Day 101 (AAO process) and Day 255 (AM-AAO process): The left side is the AAO process, and the right side is the AM-AAO process. The horizontal axis is the reaction tank (end of anaerobic, end of anoxic, end of aerobic) and EPS type (S, LB, TB). The left vertical axis is the EPS content (mg·gVSS⁻¹), and the right vertical axis is the PN/PS ratio. It includes histograms of PN, PS, and total EPS contents and a line chart of PN/PS ratio)

2.4 Microbial Diversity and Population Dynamic Community Succession Rules

The high-throughput sequencing results showed that the number of sequences of the 14 sludge samples was 1,027,419, and the number of OTU sequences of each sample is shown in Table 2. The Coverage of the samples was above 0.995, indicating that the sequencing results had high accuracy. Group D01 described the initial microbial community structure, with a high Ace index, indicating that the sludge had high microbial species richness at the start-up of the system. With the transformation of the system from AAO to AM-AAO process, the Ace index decreased, and the richness of the microbial community in the AM-AAO system decreased. In addition, the Simpson index decreased, indicating that the diversity of the microbial community decreased. According to the change of Ace index, the total number of species in the microbial community of the anoxic tank biofilm showed a decreasing trend; the decrease of Shannon index proved that the diversity of the microbial community in the biofilm decreased.

Table 2 Variation of microbial diversity index

The main phyla with relative abundance >10% in the 14 samples were analyzed (Figure 5a). The dominant phyla in group D01 were Actinobacteriota (25.76%32.90%), Proteobacteria (21.98%27.16%), Bacteroidota (15.50%18.36%), and Firmicutes (10.37%13.77%); however, the relative abundances of Actinobacteriota (16.89%19.16%) and Firmicutes (3.83%6.52%) in group D110 decreased, and the relative abundance of Proteobacteria increased (32.96%~40.75%). In the AM-AAO process system, Actinobacteriota decreased rapidly, even to less than 3% in group D237, while Proteobacteria (33.72%43.54%), Bacteroidota (17.40%24.19%), and Chloroflexi (12.46%~12.77%) have become the phyla with relatively high abundances. In addition, in sample M194, the phyla with relative abundance >10% were Proteobacteria (35.26%) and Bacteroidota (30.61%), indicating that the microbial community structure of the biofilm was similar to that of activated sludge. In sample M237, the relative abundance of Firmicutes decreased to less than 2%, and the abundance of Acidobacteriota (5.33%) increased.

By creating a heat map (Figure 5b), the 14 samples were compared at the genus level (relative abundance >3%). It was found that the dominant genera in group D01 were Candidatus_Microthrix (11.32%20.65%), norank_f__norank_o__norank_c__SJA-28 (3.97%6.36%), Trichococcus (6.99%9.95%), and Ornithinibacter (3.99%6.41%); after the system was operated in the AM-AAO process, the relative abundance of Candidatus_Microthrix dropped sharply to 0.02% (group D237); while norank_f__norank_o__norank_c__SJA-28 showed a trend of first increasing and then decreasing (group D237, 1.91%2.91%). When the process was stably operated, Azospira became one of the relatively dominant genera (group D237, 7.37%18.41%). In addition, the biofilm genera were basically similar to the sludge, and the relative abundances of norank_f__norank_o__Run-SP154 in M194 and M237 were 6.61%~7.66% and 7.43%, respectively.

A total of 12 genera and 1 family of ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), glycogen-accumulating organisms (GAOs), and phosphorus-accumulating organisms (PAOs) in the system were selected for analysis (Table 3). It was found that in group D01, Nitrosomonas (0.02%0.03%), Ellin6067 (0.01%0.02%), and Nitrospira (0.04%0.07%) may ensure the oxidation performance of NH₄⁺-N. The decrease of Nitrosomonas and Nitrospira in group D110 may be caused by the high internal reflux ratio, but Ellin6067 (0.01%0.02%) was not disturbed. In group D194, the system was operated in the AM-AAO process, and the reduction of HRT washed out NOB and some AOB. The increase in influent ammonia nitrogen may be the reason for the increase in the relative abundances of the above three genera in group D237 (Figure 5b). In addition, AOB (Nitrosomonas and Ellin6067, 0.03%0.07%) and NOB (Nitrospira, 0.01%0.02%) in sample M237 showed a slight increase, indicating that the biofilm assisted the sludge system to achieve the denitrification process.

There were a wide range of PAOs in group D01, including Acinetobacter, Candidatus_Accumulibacter, Candidatus_Microthrix, Defluviimonas, Pseudomonas, and Tetrasphaera. The changes of Candidatus_Microthrix (10.93%~11.88%) and PAOs with relative abundance <5% in group D110 may be the reasons for the decrease of PRA in Stage 2. In group D194, the relative abundances of Candidatus_Microthrix and Tetrasphaera decreased to 0.711.14 and 0.31%0.39% [14]. In group D237, Candidatus_Microthrix was almost eliminated (0.02%), and the PAOs that replaced it to exert phosphorus removal function were Defluviimonas (0.70%1.07%) and Dechloromonas (0.95%1.06%); in addition, the Comamonadaceae family has also been confirmed to have phosphorus removal performance [8], and the relative abundance of Comamonadaceae in the anaerobic tank or anoxic tank was relatively high, about twice that of the aerobic tank. In addition, Candidatus_Competibacter and Defluviicoccus were the dominant genera of GAOs in all samples, but the abundances of the two genera in group D01 were <1%. In the remaining samples, the growth of Defluviicoccus lagged behind that of Candidatus_Competibacter. In group D237, the abundances of the two genera were 2.96%~3.89% and 0.54%~0.57%, respectively. GAOs are considered to compete with PAOs for organic matter, thereby causing the deterioration of biological phosphorus removal performance, but recent studies have found that GAOs can carry out endogenous denitrification to achieve denitrification (the average TIN removal rate was 83.08% when the system was stable) [7].

(Figure 5 Microbial community composition: (a) Bar chart of relative abundance at the phylum level. The horizontal axis is the sample, and the vertical axis is the relative abundance/%. It includes major phyla such as Actinobacteriota and Proteobacteria; (b) Heat map of relative abundance at the genus level. The horizontal axis is the sample, and the vertical axis is the dominant genera. The depth of color indicates the level of relative abundance)

Table 3 Abundance of functional groups in 14 biological samples

3 Conclusions

Using actual sewage as the treatment object, the operating conditions of the AM-AAO process were optimized. It was found that when the process was operated under the conditions of HRT=7 h, temperature about 25 ℃, internal reflux=250%, SRT=40 d, sludge reflux=50%, and anoxic tank filler filling rate=30%, the pollutant removal effect was the best. The maximum NH₄⁺-N removal rate was 98.57%; the effluent NO₃⁻-N concentration, PO₄³⁻-P concentration, TIN removal rate, and COD removal rate were 6.64 mg/L, 0.42 mg/L, 83.08%, and 86.16%, respectively.

The anaerobic tank performed good organic matter removal and phosphorus release processes, with 64.51% of COD removed and 9.77 mg/L of phosphorus released at the same time; the anoxic tank performed good denitrifying phosphorus removal reactions; the aerobic tank performed complete nitrification and phosphorus uptake processes, with the NH₄⁺-N removal rate and PUAO being 97.85% and 59.12 mg, respectively.

When the AM-AAO process was stably operated, the increase of AOB (Ellin6067 and Nitrosomonas, 0.02%~0.04% → 0.04%0.12%) and NOB (Nitrospira, 00.01% → 0.02%0.04%) ensured the sufficient progress of nitrification, and the NH₄⁺-N removal rate increased by 8.35%; GAOs (Candidatus_Competibacter and Defluviicoccus, 1.31%1.61% → 3.49%4.46%) dominated the endogenous denitrification process; the growth of PAOs (Defluviimonas, Dechloromonas, and Comamonadaceae family, 3.29%8.67% → 3.79%~9.35%) was the reason for maintaining good phosphorus removal performance; in addition, the microbial community structure of the anoxic tank biofilm was basically similar to that of activated sludge, which jointly guaranteed the nitrogen and phosphorus removal performance of the system.