Enhanced Simultaneous Nitrogen and Phosphorus Removal Performance of the AGS-SBR Reactor Based on the Effects of the C/N Ratio and Microbial Community Change

Abstract

In recent years, the development of AGS technology will likely lead to a new direction in wastewater treatment development in the future. Traditional sewage treatment technology has been unable to meet the increasingly strict quality standards of wastewater treatment and limited land requirements. AGS technology may be a new method to replace traditional sewage treatment technology. However, the stable operation of AGS technology is a major obstacle to the popularization and development of this technology. The C/N ratio is an important parameter affecting the stability and simultaneous nitrogen and phosphorus removal of AGS technology. In order to enhance the nitrogen and phosphorus removal capacity of a low-load aerobic granular sludge SBR (AGS-SBR) system, changes in the morphology, EPS, and simultaneous removal of organic matter, nitrogen, and phosphorus in the AGS system were studied by regulating different C/N ratios (20, 15, 10, 5). The changes in the microbial community in the system were deeply analyzed by high-throughput sequencing technology. The results showed that different C/N ratios have a significant effect on the nitrogen removal rate of AGS but have little effect on the removal rate of organic matter and phosphorus. When the C/N ratio was reduced to 10, it was conducive to the stability of the low-load AGS-SBR system. An effective C/N ratio promoted the secretion of EPS by microorganisms, and the increase in the PN value contributed to the stability of the granular sludge, which became smooth and compact. The main functional genus in the system were norank_f__Saprospiraceae, Tetrasphaera, Ellin6067, and Pseudomonas. In addition, the simultaneous nitrogen removal performance of the system was significantly improved.

1. Introduction

Aerobic granular sludge (AGS) has the characteristics of a high sedimentation rate, high sludge load, and high biomass and has become an emerging sewage biological treatment technology as an alternative to the activated sludge method, attracting significant attention from researchers [1,2,3]. Because of the dense structure of aerobic granular sludge, the transmission of oxygen to the inside of the granules is affected, and an anaerobic-anoxic-aerobic zone is formed from the inside to the outside of the granular sludge. It has the characteristics of simultaneously removing organic matter, nitrogen, and phosphorus in the same particle [4]. Therefore, COD degradation, nitrogen, and phosphorus removal can be achieved simultaneously in the same reactor, but in the traditional activated sludge process, it usually needs to be carried out independently in multiple reactors [5]. Moreover, the high particle density of AGS leads to better settling performance, and the clarifier tank is also omitted in the process [6]. These advantages make the area of the sewage plant smaller. In addition, unlike the formation of biofilm, AGS does not need carrier plastic but forms granular sludge through flocculant secreted by its own cells under suitable culture conditions. This granular sludge can be suspended in the reactor to degrade pollutants such as COD, nitrogen, and phosphorus. This feature will reduce the environmental pollution in microplastics caused by mass-produced plastic carriers [7].

In recent years, scholars have carried out a lot of research on the production conditions, structural characteristics, influencing factors, microbial types, and wastewater treatment effects of AGS [8,9]. However, the problem of the long-term stability of the granular sludge system still exists, which leads to the poor removal efficiency of nitrogen and phosphorus in the system [10]. Most studies believed that the SBR reactor was easier to stabilize AGS than continuous flow reactors, but its nitrogen and phosphorus removal ability was average [11]. Previous researchers found that the C/N ratio plays an important role in the stable operation and nitrogen and phosphorus removal performance of granular sludge SBR systems [12]. In the SBR reactor, the stability and community changes in AGS with different C/N ratios were studied. When the C/N ratio was increased to 8, it increased the EPS content and promoted the stability of the granular sludge reactor. The effect of granular sludge microorganisms is obvious, especially nitrifying bacteria and nitrosobacteria [13]. Reducing the C/N ratio caused a significant change in the composition of the three populations of nitrifying bacteria, nitrosobacteria, and denitrifying bacteria in the granules. By reducing the C/N ratio, the activity of nitrifying bacteria was increased, and the cell hydrophobicity increased, while the extracellular polymer decreased gradually [14]. In the SBR reactor, it was found that a low C/N ratio could enhance the activity of nitrifying bacteria and denitrifying bacteria in granular sludge but weaken the reproduction rate of heterotrophic bacteria [15]. Increasing the ammonia nitrogen concentration in the AGS-SBR system can promote the growth of the autotrophic ammonia-oxidizing bacteria community. When the C/N ratio was 3, free ammonia (FA) concentration in the system inhibited the growth of heterotrophic ammonia-oxidizing bacteria and achieved stability of the AGS [16]. Previous studies have suggested that the nutrient ratio parameters of microorganisms in AGS treating high-concentration wastewater do not follow the traditional nutrient ratio (100:5:1) [17]. However, the operating parameters of optimizing nitrogen and phosphorus removal performance of the AGS-SBR system based on the C/N ratio are not uniform. Additionally, most urban sewage treatment plants use activated sludge processes to treat nitrogen and phosphorus pollutants, but there are problems such as large areas and high investment, sludge bulking, large sludge output, and chemical pollution in operation [18]. Currently, the emerging AGS technology can avoid these problems, but there are few reports about the treatment of low-load municipal sewage by this technology, and microbial ecology and its stability mechanism of granular sludge are still unclear.

Therefore, in order to deeply understand the influence of the C/N ratio on nitrogen and phosphorus removal performance of the low-load AGS-SBR system and the change of microbial community, morphological changes of aerobic granular sludge, EPS, reactor performance, and microbial community change were explored. The research results provide a theoretical basis data and important reference value for the practical popularization and application of AGS technology.

2. Materials and Methods

2.1. Experimental Setup and Operating Conditions

The test device used a cylindrical SBR reactor (Figure 1) with a height of 130 cm, an inner diameter of 9 cm, and an effective volume of 7 L. The bottom of the reactor was equipped with a microporous aeration sand head, and the aeration rate was controlled at 2.0–2.5 L·min⁻¹, which supported microbial respiration. Dissolved oxygen concentration at the end of the aeration stage was about 4.0–5.0 mg/L and provided hydraulic shear force. There were five outlets with an equal spacing of 11 cm on the side.

The experiment adopted an intermittent water aerobic operation mode, running four cycles a day. The single reaction cycle was 4 min of influent, 250 min of aeration, 2 min of sedimentation, and 4 min of drainage, and the rest of the time, it was idle. The volume ratio of water exchange in the reactor was 60%. The C/N ratios of the reactors were 20, 15, 10, and 5, respectively, and each test condition was operated for 7 days. The operation time of the SBR reactor was automatically controlled by electrical time-controlled switches, and an external circulating hot water bath was used for heat preservation.

2.2. Experimental Water and Inoculated Sludge

The raw water of the experiment was artificially simulated wastewater. In the experimental study, the water quality of the water distribution could be controlled according to the required conditions, which was easier than controlling the indicators and conducive to the progress of the research. The design influent COD concentration (glucose and sodium acetate) was about 400 mg/L, and the TP concentration was about 10 mg/L. The concentration of calcium chloride was 28 mg/L, the concentration of magnesium sulfate was 20 mg/L, trace elements were added according to the ratio of 1 mL/L, and controlling the PH value 7.0 ± 0.5 of influent in the reactor was adjusted by sodium bicarbonate. The trace elements were FeCl₃·6H₂O 1.30 g/L, MnCl₂·2H₂O 0.12 g/L, ZnSO₄·7H₂O 0.12 g/L, CoCl₂·6H₂O 0.15 g/L, CuSO₄·5H₂O 0.30 g/L, and KI 0.18 g/L.

The inoculated sludge was from AGS cultured in the laboratory. The color was golden yellow, the particle size was about 1.0–1.25 mm, the MLSS was 5.174 g/L, the water content was 96.3%, and the sedimentation rate was 13.7 m/h.

2.3. Analytical Methods

COD, NH₄⁺-N, TN, TP, MLSS, and SVI₃₀ were determined according to the standard method [19]. EPS content was mainly characterized by the sum of extracellular protein (PN) and extracellular polysaccharide (PS) content. EPS was extracted by the heat treatment method [20], and 0.5 mL of mixed liquid sludge was taken from the reactor, centrifuged at 4000 r·min⁻¹ for 5 min, and then the supernatant was poured out. After filling the volume with distilled water, it was sealed and put into a water bath at 80 °C for 30 min and then centrifuged at 10,000 r·min⁻¹ for 15 min when the temperature dropped to 4 °C. The supernatant was filtered by a 0.22 µm membrane to obtain EPS extract. PN content in EPS was determined by the Bradford method [21], and PS content in EPS was determined by the anthrone–sulfuric acid method [22]. Samples were taken regularly every day to test the concentrations of COD, NH₄⁺-N, TN and TP in the influent and the effluent. The samples of MLSS, SVI, EPS, PS and PN were taken from the last day of each operation stage for testing.

The microbial community structure was analyzed by high-throughput sequencing technique. The sequencing type of the samples was bacterial 16S rRNA. Using 338F-806R as bacterial sequencing primer, the samples with different C/N ratios were analyzed by USEARCH11-uparse algorithm, and the sequence numbers were 37460, 28915, 26884 and 30339, respectively. Granular sludge samples were taken from the last day of each operation stage, stored in a refrigerator at −80 °C, and then sent to Shanghai Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China) for analysis.

3. Results and Discussion

3.1. Changes in Properties of AGS

3.1.1. Effect of Different C/N Ratios on the External Morphology of Granular Sludge Changes

By observing the samples with different C/N ratios under an optical microscope, it was obvious (Figure 2) that when the C/N ratio was 10, the surface of the granular sludge was smoother, the structure was more compact, and there was no flocculent sludge in the reactor. When the C/N was 20 and 15, there was a small amount of flocculent sludge in the reactor, and the outer structure of the granular sludge was loose. When the C/N was 5, the structure of the granular sludge changed significantly, disintegration occurred, and the structure was fluffy. At this time, SVI₃₀ increased from 32 mL/g on the first day to 63 mL/g. Throughout the operation, when the C/N ratio decreased to 10, the MLSS value in the reactor increased slightly from 5.174g/L on the first day to 5.323 g/L, but when the C/N ratio was 5, it decreased to 5.012 g/L.

3.1.2. The Change in the C/N Ratio on EPS

The extracellular polymeric substances (EPS) content of the AGS (Figure 3) was significantly different under different C/N ratios. With a decrease in the C/N ratio to 10, the values of EPS, PN, and PS increased from 33.18 mg/g, 13.03 mg/g, and 20.15 mg/g to 65.78 mg/g, 31.66 mg/g, and 34.12 mg/g, respectively. When the C/N ratio decreased to 5, the values of EPS, PN, and PS decreased to 25.6 mg/g, 9.23 mg/g, and 16.37 mg/g, respectively, as the main components of EPS, PN, and PS play an important role in the formation and stable operation of aerobic granular sludge. Hydrogels formed by PS can maintain the stability of granular sludge through hydrogen bonding and hydrophobicity [23,24]. An increase in PN can improve the hydrophobicity of granular sludge and further promote microbial aggregation. In addition, PN/PS can be used to characterize the stability and settleability of aerobic granular sludge [25]. With the increase in the PN/PS value, the aggregation of granular sludge microorganisms was promoted, and the operation of the AGS system was more stable [26]. When the C/N was 10, the PN/PS value in the AGS system was higher than at the other ratios. This indicated that treating low-load urban sewage and controlling high nitrogen load operating conditions may be conducive to the stable operation of the AGS-SBR system [27].

3.2. Efficiency Analysis of Simultaneous Nitrogen and Phosphorus Removal in the SBR System

It can be seen from Figure 4a that different C/N ratios have little effect on COD removal. When the C/N ratio was equal to 20, the average removal rate of COD was 96.37%, and the average effluent concentration was 14.69 mg/L. When the influent ammonia nitrogen concentration was increased and the C/N ratio was equal to 15, the average COD removal rate was only increased by 0.41%, to 96.78%, and the average effluent concentration was slightly decreased by 1.64 mg/L, to 13.05 mg/L. When the influent ammonia nitrogen concentration was added to make the C/N ratio equal to 10, the removal effect of COD was slightly improved; the average removal rate was 97.31%, and the average effluent concentration was 10.85 mg/L. With the progress of the reaction, when the influent ammonia nitrogen concentration continued to increase to the C/N ratio of 5, the removal effect of COD was floating. Compared with 97.31% when the C/N ratio was 10, the decrease was 3.83%, to 93.48%. The average concentration of effluent increased from 10.85 mg/L when the C/N ratio was 10 to 26.29 mg/L and floated up by 15.44 mg/L.

It can be seen from Figure 4b that a low C/N ratio had a significant effect on the removal of ammonia nitrogen. When the C/N ratio was equal to 20, the removal effect of NH₄⁺-N was ideal; the average removal rate was 96.76%, and the average effluent concentration was 0.65 mg/L. When the C/N ratio was equal to 10, the average removal rate of NH₄⁺-N was almost unchanged compared with 98.13% when the C/N ratio was equal to 15, an increase of just 0.03%. The average concentration of NH₄⁺-N in the effluent increased from 0.50 mg/L (close to 0) when the C/N ratio was equal to 15 to only 0.24 mg/L to 0.74 mg/L. When the C/N ratio was equal to 5, the removal effect of NH₄⁺-N began to deteriorate; the removal rate decreased by 13.97% from 98.16% when the C/N ratio was equal to 10, the average removal rate decreased to 84.19%, and the effluent concentration increased from 0.74 mg/L to 12.85 mg/L, an increase of 12.11 mg/L.

It can be seen from Figure 4c that different C/N ratios had a significant effect on total nitrogen removal. When the C/N ratio was equal to 10, the removal effect of TN was ideal; the average removal rate was 88.64%, and the average effluent concentration was 4.87 mg/L. When the influent TN concentration was low (C/N = 20), the average removal rate of TN was 74.18%, and the average concentration of effluent was 5.75 mg/L. When the influent ammonia nitrogen concentration was increased, the TN removal effect was significantly improved when the C/N ratio was equal to 15, which was 10.59% higher than 74.18% when the C/N ratio was equal to 20. The effluent concentration of TN was also reduced from 5.75 mg/L to 4.44 mg/L, a reduction of 1.31 mg/L. When the influent ammonia nitrogen concentration continued to increase, when the C/N ratio was equal to 5, the TN removal effect did not further improve but decreased rapidly from 88.64% when the C/N ratio was equal to 10. The decrease was from 16.61% to 69.03%, and the effluent concentration of TN also increased to 25.83 mg/L.

It can be seen from Figure 4d that different C/N ratios had a slight effect on the TP removal rate. When the C/N ratio was in the range of 10~15, the TP removal effect was ideal. When the C/N ratio was equal to 15, the average TP removal rate was 86.19%, and the average effluent concentration was 1.45 mg/L. When the C/N ratio was equal to 10, the TP removal effect was better; the average treatment rate was 87.93%, and the average effluent concentration was 1.28 mg/L. When reducing the influent ammonia nitrogen concentration and increasing the C/N ratio to 20, the TP removal efficiency decreased slightly to 84.68% compared with a C/N ratio equal to 15, and the average effluent concentration was 1.58 mg/L. Continuing to increase the influent ammonia nitrogen concentration in the reactor to reduce the removal effect of the C/N ratio did not further improve TP. The removal effect of TP decreased by 4.32% from 87.93% over the C/N ratio was equal to 10, and the average effluent concentration increased from 1.28 mg/L to 0.42 mg/L to 1.70 mg/L.

The above test results showed that different C/N ratios have a significant effect on the nitrogen removal rate of AGS but have little effect on the removal rate of organic matter and phosphorus. When the C/N ratio is 20, the basic nutrient ratio of aerobic microorganisms can normally meet the needs of microbial reproduction and growth [28], but the nitrogen removal rate of granular sludge is not promoted. This indicated that the ability of heterotrophic bacteria in granular sludge to uptake organic substrates was greater than that of nitrifying bacteria (nitrifying bacteria, nitrosobacteria), and their activity gradually increased to become the dominant bacteria, resulting in a low nitrogen removal rate. In addition, heterotrophic bacteria compete with phosphorus-accumulating bacteria for organic matter, affecting phosphorus removal, but when granular sludge is dominated by heterotrophic bacteria the removal of organic matter is not affected. When the C/N ratio was equal to 10, the nitrogen removal rate of granular sludge was better than that at a C/N ratio equal to 15, and the removal rate of organic matter and total phosphorus was also slightly improved. This showed that the activity of nitrifying bacteria is promoted by increasing the ammonia nitrogen load [29]. Heterotrophic bacteria coexist in the same microenvironment to achieve a good balance of mutualism and symbiosis between species. Heterotrophic bacteria grow by absorbing energy from the substrate matrix, and the produced CO₂ can be used as a carbon source or energy supply for nitrifying bacteria to achieve their own synthesis and reproduction. Nitrifying bacteria are obligate autotrophic bacteria, which use inorganic carbon compounds such as CO₂, CO₃²⁻, and HCO₃⁻ as carbon and energy sources. Therefore, it does not affect the degradation of organic matter and the phosphorus uptake ability of heterotrophic bacteria and phosphorus-accumulating bacteria but promotes a better removal effect. When the C/N ratio is as low as 5, the denitrification rate is not further improved but shows a downward trend, mainly because free ammonia inhibits nitrifying bacteria (nitrosobacteria and nitrifying bacteria) [30,31]. When the concentration of free ammonia (FA) in the reactor mixture was in the range of 0.1~4 mg/L, the activity of nitrifying bacteria was inhibited. When the concentration of FA was 10~150 mg/L, the activity of nitrosobacteria and nitrifying bacteria was inhibited [32,33]. Additionally, a lack of organic matter content impacted the denitrification rate. Denitrifying bacteria are heterotrophic microorganisms that use organic matter as an electron donor to metabolize NO₂⁻ and NO₃⁻ to N₂. Heterotrophic bacteria growing in the same particle also need organic matter to provide a carbon source for metabolic synthesis. The heterotrophic bacteria in the outer layer of the granular sludge first ingest the organic matter to degrade the anabolism, and the remaining small amount of organic matter enters the inner layer of the particle to supply the denitrifying bacteria for incomplete denitrification. No external carbon source is added during the reaction process. Therefore, the denitrification process is limited, and the nitrogen removal rate is reduced. Finally, nitrate also inhibits denitrifying bacteria.

There is a certain relationship between FA and pH value. In order to further study the reaction mechanism of granular sludge denitrification under different C/N ratios, the pH value was measured by regular sampling in the reactor. The FA concentration in the reactor could be estimated by Equation (1) [34]. The daily data were recorded, and the average value of 7 days was taken to calculate the FA value. The experimental results are recorded in Table 1.

(1)$\mathrm{FA} (\mathrm{mg}/\mathrm{L}·{\mathrm{L}}^{-1})=\frac{[\mathrm{N}{\mathrm{H}}_4{}^{+}-\mathrm{N}]\times {10}^{\mathrm{p}\mathrm{H}}}{\exp [6334/(273+\mathrm{T})]+{10}^{\mathrm{p}\mathrm{H}}}$

It can be seen from Table 1 that when the C/N ratio was equal to 15 and 20, the concentration of FA was 0.441 mg/L and 0.167 mg/L, respectively. In the range of 0.1~4 mg/L, the activity of nitrifying bacteria was inhibited. When the C/N ratio was equal to 5, the concentration of FA was 10.026 mg/L, and the activity of nitrosobacteria and nitrifying bacteria was inhibited in the range of 10~150 mg/L. When the C/N ratio was equal to 10, the FA concentration of 6.016 mg/L was not in the range of free ammonia-inhibiting nitrifying bacteria. In summary, when the influent C/N ratio of the reactor was equal to 10, a better denitrification effect and removal rate of organic matter and phosphorus was achieved by the particulate sludge.

3.3. Microbial Community Change

Granular sludge is composed of many kinds of microorganisms, which can realize the synchronous removal of organic matter, nitrogen, and phosphorus pollutants [35]. Therefore, it is of great significance to deeply study the structure and function of the microbial community in the AGS-SBR system under different C/N conditions. Microbial distribution at the granular sludge gate level within the reactor was analyzed by high-throughput sequencing. Figure 5 shows that the species were similar in samples with different C/N ratios, but the relative abundance of microorganisms was significantly different. The relative abundance of Proteobacteria, Bacteroidota, Patescibacteria, and Actinobacteriota in each sample was relatively large, with accumulative proportions of 82.36%, 79.01%, 76.88%, and 83.79%, respectively. These dominant microorganisms have the ability to degrade and remove organic matter, nitrogen, and phosphorus and are also common microorganisms in activated sludge and biofilm granular sludge [36]. The relative abundance of Chloroflexi, another dominant phylum, changed significantly. With the increase of ammonia nitrogen concentration, the relative abundance of Chloroflexi increased from 4.3% at a C/N ratio of 20 to 5.69% at a C/N ratio of 10. When the C/N ratio decreased to 5, the relative abundance of this microorganism decreased to 3.41%, and the granular sludge disintegrated. This is consistent with the results of the 3.1.1 microscope observation. Previous studies have shown that Chloroflexi plays an important role in the stable structure of AGS, providing a microbial aggregation scaffold for AGS [37]. Nitrospirota was also detected in the system, which was a common phylum involved in the denitrification process. With the increase of ammonia nitrogen concentration, the abundance of this strain increased from 3.21% at a C/N ratio of 20 to 3.78% at a C/N ratio of 10, only increasing by 0.57%, but it promoted the nitrification of granular sludge to some extent. When the C/N ratio is 5, the abundance of Nitrospirota is greatly reduced to 0.12%, and the denitrification effect of the AGS-SBR system becomes worse. This is consistent with the results of 3.2. From a C/N ratio of 20 to a C/N ratio of 10, although the relative abundance of microbial populations in the system was very different, the relative stability of degrading organic matter, nitrogen, and phosphorus in the system was ensured due to the redundant function of microorganisms [27,36].

At the genus level, the top 30 species with relative abundance (others <0.1%) were analyzed. As shown in Figure 6, when the C/N ratio was 20, the dominant bacteria in the reactor was Candidatus_Competibacter, and its relative abundance was 13.8%. With the increase of ammonia nitrogen concentration, the dominant bacteria changed. When the C/N ratio was 10, norank_f__saprospiraceae became the dominant bacteria in the system, with a relative abundance of 15.31%. Candidatus_Competibacter and norank_f__Saprospiraceae belong to glycogen accumulating organisms (GAOs) [38,39]. This genus has biological phosphorus removal characteristics. Previous studies showed [40] that the norank_f__Saprospiraceae has the effect of simultaneous degradation of organic matter and denitrification. When the C/N ratio was 5, the dominant bacteria in the system became Candidatus_Competibacter again, but the content was reduced by 2.31% compared with the C/N ratio of 20. This showed that different operating parameters in the reactor had a great effect on the relative abundance of microorganisms, reflecting the selectivity of microorganisms. In addition, it was found that Tetrasphaera was enriched in the system, and its relative abundance increased from 0.22% at C/N of 20 to 2.25% at C/N of 10. This genus belonged to phosphorus accumulating organisms (PAOs), which was an important functional microorganism participating in phosphorus removal [37]. Therefore, the phosphorus removal capacity of the AGS-SBR system has been further improved. When the C/N ratio is 5, Tetrasphaera is greatly reduced, but it has little effect on the phosphorus removal performance of the system. This may be the redundant function of GAOs and PAOs microorganisms, which maintains the relative stability of the phosphorus removal capacity of the system. When the C/N ratio was 5, Tetrasphaera was greatly reduced, but the system phosphorus removal performance was less affected. This may be the redundant function of GAOs and PAOs microorganisms, maintaining the relative stability of the system‘s phosphorus removal capacity. The very important ammonia oxidizing bacteria (AOB), Nitrosomonas, was also found in the system, which is a common genus in the reactor. However, the relative abundance in this experimental sample is less, only in the range of 0.09~0.38%. However, Ellin6067 was detected in the system, which also belongs to AOB. With the increase in ammonia nitrogen concentration, this genus was enriched, and its relative abundance increased from 0.18% at a C/N ratio of 20 to 0.77% at a C/N ratio of 10, with an enrichment of only 0.59%. However, the enrichment of AOB is more likely to promote the short-cut nitrification of the system, which has an important contribution to improving the system’s performance and energy consumption [27,41]. In addition, nitrite oxidizing bacteria (NOB) and Nitrospira were also found in the samples of each stage. In addition, nitrite oxidizing bacteria (NOB), such as Nitrospira, were also found in the samples of each stage. In the stage from C/N of 20 to C/N of 10, its abundance was only enriched by 0.06%. This shows that the increase of ammonia nitrogen concentration can coordinate the relationship between nutrients in the system, and the effective C/N ratio is beneficial to the enrichment of functional microbial AOB and improves the performance of the AGS-SBR system [42]. When the concentration of ammonia nitrogen was increased to a C/N ratio of 5, AOB content did not continue to increase but decreased, and the content of NOB also decreased. This is due to the high ammonia nitrogen concentration increasing the content of FA in the system, resulting in the inhibition of AOB and NOB microbial activity. This is consistent with the results of 3.2 and similar to the research results of the ZOU team [16]. The denitrifying bacteria (DNB) in the reactor were mainly the genus of the dominant phylum Proteobacteria, such as norank_f__Saprospiraceae (in the stage from C/N of 20 to C/N of 10). Functional bacteria with denitrification and phosphorus removal performance were also detected; this was Pseudomonas [43].

4. Conclusions

The C/N ratio plays an important role in the stable operation and nitrogen and phosphorus removal performance of granular sludge SBR systems. Different C/N ratios have a significant effect on the nitrogen removal rate of AGS, while the removal rate of organic matter and phosphorus is slightly affected. An effective C/N ratio can help the various components of microbial EPS maintain a better balance in the corresponding environment, promote the stable operation of the low-load AGS-SBR reactor, and improve the simultaneous nitrogen and phosphorus removal capacity of the system. When the C/N ratio was 10, high cohesion and a compact structure between the communities in the SBR system were observed. The granular sludge in the SBR system achieved a better denitrification effect and removal rate of organic matter and phosphorus. The effective C/N relationship in the reactor is beneficial to the enrichment of nitrogen and phosphorus functional microorganisms such as AOB, NOB and PAOs, which further improves the simultaneous nitrogen and phosphorus removal performance of the AGS-SBR system and maintains the stability of the system.

Author Contributions

Validation, L.S.; Resources, L.S.; Data curation, L.S., Q.L. and Z.H.; Writing—original draft, L.S., Q.L. and Z.H.; Writing—review & editing, L.S., Y.L. and L.C.; Supervision, L.S.; Funding acquisition, L.S. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figures and Table

Figure 1. Picture of testing device.

Figure 2. Effects of different C/N ratios on the morphology of AGS.

Figure 3. Effects of different C/N ratios on the EPS of granular sludge.

Figure 4. Performance of the AGS-SBR system with different ratios of C/N. (a) COD removal rate; (b) NH4+-N removal rate; (c) TN removal rate; (d) TP removal rate.

Figure 5. Distribution of microorganisms at phylum level of AGS-SBR system under different C/N conditions.

Figure 6. Distribution of microbial community structure of AGS at genus levels under different C/N conditions.

Table 1

Estimation of the FA concentration at different pH values.

C/N Ratios	20	15	10	5
influent ammonium concentration (mg/L)	30	40	60	100
operating temperature (°C)	18	18	18	18
Mean pH	7.2 ± 0.2	7.5 ± 0.2	8.5 ± 0.2	8.5 ± 0.2
FA concentration (mg/L)	0.167	0.441	6.016	10.026

References

1. Nor-Anuar, A.; Ujang, Z.; Van Loosdrecht, M.C.M.; de Kreuk, M.K.; Olsson, G. Strength characteristics of aerobic granular sludge. Water Sci. Technol.; 2012; 65, pp. 309-316. [DOI: https://dx.doi.org/10.2166/wst.2012.837] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22233910]

2. Pronk, M.; de Kreuk, M.K.; de Bruin, B.; Kamminga, P.; Kleerebezem, R.; Van Loosdrecht, M.C.M. Full scale performance of the aerobic granular sludge process for sewage treatment. Water Res.; 2015; 84, pp. 207-217. [DOI: https://dx.doi.org/10.1016/j.watres.2015.07.011] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26233660]

3. Mohamed, S.Z.; Gopal, A. A review of mechanistic and data-driven models of aerobic granular sludge. J. Environ. Chem. Eng.; 2022; 10, 107500. [DOI: https://dx.doi.org/10.1016/j.jece.2022.107500]

4. Xu, G.; Zhang, Z.; Gao, F. Effect of COD/N ratios and DO concentrations on the NOB suppression in a multi-cycle SBR. J. Environ. Chem. Eng.; 2021; 9, 105735. [DOI: https://dx.doi.org/10.1016/j.jece.2021.105735]

5. Shameem, K.S.; Sabumon, P.C. A Review on the Stability, Sustainability, Storage and Rejuvenation of Aerobic Granular Sludge for Wastewater Treatment. Water; 2023; 15, 950. [DOI: https://dx.doi.org/10.3390/w15050950]

6. Edward, J.H.; Mario, P.; Mark, C.M. A settling model for full-scale aerobic granular sludge. Water Res.; 2020; 186, 116135. [DOI: https://dx.doi.org/10.1016/j.watres.2020.116135]

7. Sun, Z.; Zhang, J.M.; Wang, J.; Zhu, H.; Xiong, J.; Nong, G.; Luo, M.; Wang, J. Direct start-up of aerobic granular sludge system with dewatered sludge granular particles as inoculant. J. Environ. Manag.; 2023; 326, 116540. [DOI: https://dx.doi.org/10.1016/j.jenvman.2022.116540]

8. Liu, S.L.; Zhou, M.; Daigger, G.T.; Huang, J.; Song, G. Granule formation mechanism, key influencing factors, and resource recycling in aerobic granular sludge (AGS) wastewater treatment: A review. J. Environ. Manag.; 2023; 338, 117771. [DOI: https://dx.doi.org/10.1016/j.jenvman.2023.117771]

9. Silvio, L.; Antônio, R.; Paulo, I.; André, B. Aerobic granular sludge: Cultivation parameters and removal mechanisms. Bioresour. Technol.; 2018; 270, pp. 678-688. [DOI: https://dx.doi.org/10.1016/j.biortech.2018.08.130]

10. Hou, Y.; Gan, C.; Chen, R.; Chen, Y.; Yuan, S.; Chen, Y. Structural Characteristics of Aerobic Granular Sludge and Factors That Influence Its Stability: A Mini Review. Water; 2021; 13, 2726. [DOI: https://dx.doi.org/10.3390/w13192726]

11. Laila, D.A.P.; Hamzat, T.I.; Ali, Y.; Shaza, E.M.; Koji, I.; Arash, Z.; Norhayati, A. Various applications of aerobic granular sludge: A review. Environ. Technol. Innov.; 2020; 20, 101045. [DOI: https://dx.doi.org/10.1016/j.eti.2020.101045]

12. Wang, L.; Yu, X.; Xiong, W.; Li, P.; Wang, S.; Fan, A.; Su, H. Enhancing robustness of aerobic granule sludge under low C/N ratios with addition of kitchen wastewater. J. Environ. Manag.; 2020; 265, 110503. [DOI: https://dx.doi.org/10.1016/j.jenvman.2020.110503] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32421552]

13. Wang, X.C.; Chen, Z.; Shen, J.; Zhao, X.; Kang, J. Impact of carbon to nitrogen ratio on the performance of aerobic granular reactor and microbial population dynamics during aerobic sludge granulation. Bioresour. Technol.; 2019; 271, pp. 258-265. [DOI: https://dx.doi.org/10.1016/j.biortech.2018.09.119] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30278350]

14. Yang, S.F.; Tay, J.H.; Liu, Y. Effect of Substrate Nitrogen/Chemical Oxygen Demand Ratio on the Formation of Aerobic Granules. J. Environ. Eng.; 2005; 131, pp. 86-92. [DOI: https://dx.doi.org/10.1061/(ASCE)0733-9372(2005)131:1(86)]

15. Jiang, H.L.; Tay, J.H.; Liu, Y.; Tay, S.T.L. Ca²⁺ augmentation for enhancement of aerobically grown microbial granules in sludge blanket reactors. Biotechnol. Lett.; 2003; 25, pp. 95-99. [DOI: https://dx.doi.org/10.1023/A:1021967914544]

16. Zou, X.; Gao, M.; Mohammed, A.; Liu, Y. Responses of various carbon to nitrogen ratios to microbial communities, kinetics, and nitrogen metabolic pathways in aerobic granular sludge reactor. Bioresour. Technol.; 2023; 367, 128225. [DOI: https://dx.doi.org/10.1016/j.biortech.2022.128225] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36332856]

17. Hamza, R.A.; Zaghloul, M.S.; Iorhemen, O.T.; Sheng, Z.; Tay, J.H. Optimization of organics to nutrients (COD:N:P) ratio for aerobic granular sludge treating high-strength organic wastewater. Sci. Total Environ.; 2019; 650, pp. 3168-3179. [DOI: https://dx.doi.org/10.1016/j.scitotenv.2018.10.026]

18. Zou, J.; Pan, J.; Wu, S.; Qian, M.; He, Z.; Wang, B.; Li, J. Rapid control of activated sludge bulking and simultaneous acceleration of aerobic granulation by adding intact aerobic granular sludge. Sci. Total Environ.; 2019; 674, pp. 105-113. [DOI: https://dx.doi.org/10.1016/j.scitotenv.2019.04.006]

19. APHA, AWWA, & WEF. Standard Methods for Examination of Water and Wastewater; 23rd ed. American Public Health Association: Washington, DC, USA, 2012; ISBN 978-087553-013-0

20. Gaudy, A.F., Jr. Colorimetric Determination of Protein and Carbohydrate. Ind. Water Wastes; 1962; 7, pp. 17-22.

21. Fang, H.H.P.; Jia, X.S. Extraction of extracellular polymer from anaerobic sludges. Biotechnol. Technol.; 1996; 10, pp. 803-808. [DOI: https://dx.doi.org/10.1007/BF00154662]

22. Tay, J.H.; Liu, Q.S.; Liu, Y. The effects of shear force on the formation, structure and metabolism of aerobic granules. Appl. Microbiol. Biotechnol.; 2001; 57, pp. 227-233. [DOI: https://dx.doi.org/10.1007/s002530100766] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11693926]

23. Seviour, T.; Yuan, Z.; Loosdrecht, M.; Lin, Y. Aerobic sludge granulation: A tale of two polysaccharides. Water Res.; 2012; 46, pp. 4803-4813. [DOI: https://dx.doi.org/10.1016/j.watres.2012.06.018] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22776210]

24. Li, Z.W.; Lin, L.; Liu, X.; Wan, C.L.; Lee, D.J. Understanding the role of extracellular polymeric substances in the rheological properties of aerobic granular sludge. Sci. Total Environ.; 2020; 705, 135948. [DOI: https://dx.doi.org/10.1016/j.scitotenv.2019.135948] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31836231]

25. Liu, X.; Liu, J.; Deng, D.; Li, R.; Guo, C.; Ma, J.; Chen, M. Investigation of extracellular polymeric substances (EPS) in four types of sludge: Factors influencing EPS properties and sludge granulation. J. Water Process Eng.; 2021; 40, 101924. [DOI: https://dx.doi.org/10.1016/j.jwpe.2021.101924]

26. Zhao, X.; Xu, H.; Shen, J.; Yu, B.; Wang, X. Decreasing effect and mechanism of moisture content of sludge biomass by granulation process. Environ. Technol.; 2015; 37, pp. 192-201. [DOI: https://dx.doi.org/10.1080/09593330.2015.1066448] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26119981]

27. He, Q.; Zhang, W.; Zhang, S.; Wang, H. Enhanced nitrogen removal in an aerobic granular sequencing batch reactor performing simultaneous nitrification, endogenous denitrification and phosphorus removal with low superficial gas velocity. Chem. Eng. J.; 2017; 326, pp. 1223-1231. [DOI: https://dx.doi.org/10.1016/j.cej.2017.06.071]

28. Liu, Y.; Yang, S.; Tay, J. Improved stability of aerobic granules by selecting slow-growing nitrifying bacteria. J. Biotechnol.; 2004; 108, pp. 161-169. [DOI: https://dx.doi.org/10.1016/j.jbiotec.2003.11.008]

29. Kim, H.; Kim, J.; Ahn, D. Effects of carbon to nitrogen ratio on the performance and stability of aerobic granular sludge. Environ. Eng. Res.; 2021; 26, 190284. [DOI: https://dx.doi.org/10.4491/eer.2019.284]

30. Villaverde, S.; Fdz-Polanco, F.; García, P.A. Nitrifying biofilm acclimation to free ammonia in submerged biofilters: Start-up influence. Water Res.; 2000; 34, pp. 602-610. [DOI: https://dx.doi.org/10.1016/S0043-1354(99)00175-X]

31. Yang, S.F.; Tay, J.H.; Liu, Y. Inhibition of free ammonia to the formation of aerobic granules. Biochem. Eng. J.; 2004; 17, pp. 41-48. [DOI: https://dx.doi.org/10.1016/S1369-703X(03)00122-0]

32. Suthersan, S.; Ganczarczyk, J.J. Inhibition of nitrite oxidation during nitrification: Some observations. Water Pollut. Res. J. Canada; 1986; 21, pp. 257-266. [DOI: https://dx.doi.org/10.2166/wqrj.1986.020]

33. Zou, X.; Mohammed, A.; Gao, M.; Liu, Y. Mature landfill leachate treatment using granular sludge-based reactor (GSR) via nitritation/denitritation: Process startup and optimization. Sci. Total Environ.; 2022; 844, 157078. [DOI: https://dx.doi.org/10.1016/j.scitotenv.2022.157078] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35787895]

34. Ford, D.L.; Churchwell, R.L.; Kachtick, J.W. Comprehensive analysis of nitrification of chemical processing wastewaters. J. Water Pollut. Contr.; 1980; 52, pp. 2726-2746.

35. Xiong, W.; Wang, S.; Jin, Y.; Wu, Z.; Liu, D.; Su, H. Insights into nitrogen and phosphorus metabolic mechanisms of algal-bacterial aerobic granular sludge via metagenomics: Performance, microbial community and functional genes. Bioresour. Technol.; 2023; 369, 128442. [DOI: https://dx.doi.org/10.1016/j.biortech.2022.128442]

36. Jiang, C.; Peces, M.; Andersen, M.H.; Kucheryavskiy, S.; Nierychlo, M.; Yashiro, E.; Andersen, K.S.; Kirkegaard, R.H.; Hao, L.; Høgh, J. et al. Characterizing the growing microorganisms at species level in 46 anaerobic digesters at Danish wastewater treatment plants: A six-year survey on microbial community structure and key driver. Water Res.; 2021; 193, 116871. [DOI: https://dx.doi.org/10.1016/j.watres.2021.116871]

37. Larsen, P.; Nielsen, J.L.; Otzen, D.; Nielsen, P.H. Amyloid-like adhesins produced by floc-forming and filamentous bacteria in activated sludge. Appl. Environ. Microbiol.; 2008; 74, pp. 1517-1526. [DOI: https://dx.doi.org/10.1128/AEM.02274-07]

38. He, Q.; Chen, L.; Zhang, S.; Wang, L.; Liang, J.; Xia, W.; Wang, H.; Zhou, J. Simultaneous nitrification, denitrification and phosphorus removal in aerobic granular sequencing batch reactors with high aeration intensity: Impact of aeration time. Bioresour. Technol.; 2018; 263, pp. 214-222. [DOI: https://dx.doi.org/10.1016/j.biortech.2018.05.007]

39. Zhang, T.; Wang, B.; Li, X.; Zhang, Q.; Wu, L.; He, Y.; Peng, Y. Achieving partial nitrification in a continuous post-denitrification reactor treating low C/N sewage. Chem. Eng. J.; 2017; 335, pp. 330-337. [DOI: https://dx.doi.org/10.1016/j.cej.2017.09.188]

40. Liu, J.; Yuan, Y.; Li, B.; Zhang, Q.; Wu, L.; Li, X.; Peng, Y. Enhanced nitrogen and phosphorus removal from municipal wastewater in an anaerobic-aerobic-anoxic sequencing batch reactor with sludge fermentation products as carbon source. Bioresour. Technol.; 2017; 244, pp. 1158-1165. [DOI: https://dx.doi.org/10.1016/j.biortech.2017.08.055]

41. Xu, J.; Zhang, N.; Yang, G.; Xie, Z.; Yu, N.; Wang, D.; Liu, X.; Xia, J.; Tang, L. Revealing the behavior of perfluorooctane sulfonic acid in an aerobic granular sludge system: Fate and impact. Chem. Eng. J.; 2023; 457, 140478. [DOI: https://dx.doi.org/10.1016/j.cej.2022.140478]

42. Pishgar, R.; Dominic, J.A.; Sheng, Z.; Tay, J.H. Influence of operation mode and wastewater strength on aerobic granulation at pilot scale: Startup period, granular sludge characteristics, and effluent quality. Water Res.; 2019; 16, pp. 81-96. [DOI: https://dx.doi.org/10.1016/j.watres.2019.05.026] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31132565]

43. Huang, J.L.; Cui, Y.W.; Yan, J.L.; Cui, Y. Occurrence of heterotrophic nitrificationaerobic denitrification induced by decreasing salinity in a halophilic AGS SBR treating hypersaline wastewater. Chem. Eng. J.; 2022; 431, 134133. [DOI: https://dx.doi.org/10.1016/j.cej.2021.134133]