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Article

Sustainable Treatment of High-Ammonia-Nitrogen Organic Wastewater via Anaerobic Ammonium Oxidation (Anammox) Combined with Effluent Recirculation/Micro-Aeration

by
Zichun Yan
1,2,3,*,
Rong Zeng
1 and
Hao Yang
1,2,3
1
School of Environmental and Municipal Engineering, Lanzhou Jiao Tong University, Lanzhou 730070, China
2
Ministry of Education Engineering Research Center of Water Resource Comprehensive Utilization in Cold and Arid Regions, Lanzhou 730070, China
3
Key Laboratory of Yellow River Water Environment of Gansu Province, Lanzhou 730070, China
*
Author to whom correspondence should be addressed.
Sustainability 2025, 17(13), 5926; https://doi.org/10.3390/su17135926
Submission received: 22 May 2025 / Revised: 19 June 2025 / Accepted: 23 June 2025 / Published: 27 June 2025
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Abstract

High-ammonia-nitrogen organic wastewater poses significant challenges to traditional nitrogen removal processes due to their high energy consumption and carbon dependency, conflicting with global sustainability goals. Anammox presents a sustainable alternative with lower energy demands, yet its application is constrained by organic matter inhibition. This study aimed to optimize nitrogen and organic matter removal in Anammox systems by comparing two strategies: effluent recirculation and micro-aeration. Anammox reactors were operated under three conditions: (1) no recirculation (control group), (2) 100–300% effluent recirculation, (3) micro-aeration at 50–150 mL/min. The effects on total nitrogen (TN) and chemical oxygen demand (COD) removal were evaluated, alongside microbial community analysis via high-throughput sequencing. The results show that micro-aeration at 100 mL/min achieved 78.9% COD and 88.3% TN removal by creating micro-anaerobic conditions for metabolic synergy. Excessive aeration (150 mL/min) inhibited Anammox, dropping TN removal to 49.7%. Recirculation enriched Planctomycetota, while micro-aeration slightly increased Planctomycetota abundance at 45 cm and enhanced Proteobacteria and Chloroflexi for denitrification. Optimal conditions—200% recirculation and 100 mL/min aeration—improve efficiency via dilution and synergistic metabolism, providing a novel comparative framework for treating high-ammonia-nitrogen organic wastewater and filling a research gap in the parallel evaluation of Anammox enhancement strategies.

1. Introduction

In recent years, high-ammonia-nitrogen organic wastewater has emerged as a severe environmental challenge, predominantly generated by industries such as animal husbandry, food processing, and chemical manufacturing [1,2]. Characterized by extremely high concentrations of ammonia nitrogen and organic pollutants [3,4], the improper discharge of such wastewater can trigger a series of environmental issues, including ecological toxicity, eutrophication of water bodies, and deterioration of soil quality [5,6].
In the treatment of high-ammonia-nitrogen organic wastewater, chemical and physical methods such as chemical precipitation and ultrafiltration can effectively remove nitrogen pollutants [7,8], but they come with relatively high costs. In contrast, biological treatment technologies, which are cost-effective, have been widely applied in the treatment of such wastewater. The traditional nitrification–denitrification biological nitrogen removal process relies on the aerobic conversion of ammonia nitrogen to nitrate nitrogen through nitrification, followed by the anaerobic conversion of nitrate nitrogen to nitrogen gas via denitrification [9]. However, this process has notable limitations: the nitrification stage demands substantial energy for aeration [10], while the denitrification stage requires additional carbon sources to facilitate nitrogen removal. Li et al. pointed out that the aeration energy demand of the aerated microbial treatment unit in wastewater treatment plants typically accounts for 50% to 70% of the total operating cost of wastewater treatment plants [11]. Similarly, Baumgartne et al. indicated that the energy required for aeration can account for 60% of the total energy demand of the plant [12]. In terms of carbon sources during the denitrification stage, previous studies have demonstrated that when the carbon-to-nitrogen ratio (C/N) is lower than 2.4, the denitrification rate significantly decreases [13]; a C/N ratio in the range of 1–3 may lead to insufficient carbon sources, inhibiting the activity of denitrification enzymes, whereas a C/N ratio of 4 results in a higher denitrification efficiency [14]. In continuous-flow reactors, the denitrification rate in the anoxic stage is optimized when the C/N ratio is between 5.4 and 7.5 [15].
In contrast, Anammox technology offers significant advantages due to its unique metabolic mechanism. Anammox bacteria can directly oxidize ammonium ions to nitrogen gas under anaerobic conditions, using nitrite ions as electron acceptors [16]. This process bypasses the energy-intensive aerobic nitrification step, greatly reducing energy consumption, and simultaneously decreases the dependence on external carbon sources, providing a more sustainable solution for treating high-ammonia-nitrogen organic wastewater [17,18].
Despite its great potential, the application of Anammox in treating high-ammonia-nitrogen organic wastewater still faces numerous challenges. The presence of organic matter in the wastewater can disrupt the Anammox process and inhibit the activity of Anammox bacteria [19,20]. To address this issue, researchers have attempted to dilute the concentration of organic matter in the influent by implementing effluent recirculation, thereby reducing its inhibitory effect on Anammox bacteria. For example, Zhang et al. introduced recirculation into the CANON process and achieved ammonia nitrogen and total nitrogen removal rates of 98.2 ± 0.8% and 77.8 ± 2.3%, respectively, when the recirculation ratio reached 200% [21]. Meng et al. found that a recirculation ratio of 35% could maintain the total nitrogen removal rate above 86.6% [22]. Liu et al. demonstrated that a recirculation ratio of 150% optimized the performance of the system when treating leachate [23]. It is worth noting that although the effluent reflux brings in dissolved oxygen (DO), it can still effectively improve the denitrification efficiency of Anammox, indicating that Anammox granular sludge has a certain tolerance to DO. For example, Fu et al. found that during the initial cultivation stage, the increase of internal reflux led to DO infiltration into the Anammox sludge, causing inhibitory effects. However, when the sludge was granulated, the influence of DO decreased [24]. In addition, Xu et al. reported that at 2.0 mg/L DO, the activity of Anammox bacteria only decreased by 18.5%. Among them, the dominant Anammox species Candidatus Kuenenia down-regulated the gene expression involved in carbon metabolism and oxidative phosphorylation, which can effectively alleviate the toxic effect of oxygen [25]. Anaerobic ammonia oxidation exhibits a certain tolerance to oxygen, which makes it possible for micro-aeration to remove nitrogen from organic wastewater. However, research on the synergistic application of micro-aeration and Anammox for treating such wastewater remains scarce.
Against this backdrop, this study aims to systematically investigate the influence of the mixing method of organic wastewater and Anammox granular sludge on the performance of Anammox reactors treating high-ammonia-nitrogen organic wastewater. By thoroughly analyzing the effects of key parameters, such as the effluent recirculation ratio and aeration intensity, on nitrogen removal efficiency and microbial community structure, this study endeavors to determine the optimal operating conditions for Anammox reactors. The research findings are expected to provide theoretical and practical guidance for the development of efficient and sustainable treatment technologies for high-ammonia-nitrogen organic wastewater.

2. Materials and Methods

2.1. Reactor Setup and Operation Strategy

The Anammox reactor was designed as a cylindrical column with an inner diameter of 10 cm, a height of 179 cm, and an effective volume of 14.05 L. To ensure a stable reaction environment, the reactor was placed inside a thermostatic incubator, where the internal temperature was precisely maintained at 32 ± 2 °C by a temperature control system. This temperature setting not only optimized the growth conditions for Anammox bacteria but also minimized the negative impact of light on their activity.
At the bottom of the reactor, a recirculation port and an aeration head were symmetrically installed, which were connected to a recirculation pump and a gas flowmeter linked to an air valve, respectively. These components enabled the adjustment of recirculation flow rate and aeration intensity, facilitating effluent recirculation or micro-aeration operations. As a result, the sludge was uniformly suspended within the reactor, preventing sedimentation. During the experiment, wastewater from the influent tank was pumped into the reactor from the bottom via a peristaltic pump. For reactors equipped with an effluent recirculation system, the outlet at the top was connected to a recirculation pipe. The treated effluent flowed into a recirculation tank through this pipe and was then pumped back to the reactor bottom, forming a closed-loop circulation. In contrast, for reactors without recirculation, the recirculation pipe served directly as the effluent discharge outlet. The detailed structure and operational process are illustrated in Figure 1.

2.2. Sludge and Synthetic Wastewater

The Anammox granular sludge was collected from a soybean wastewater treatment plant in Shandong Province, China. After 130 days of startup cultivation, the height of the sludge bed stabilized at 48 cm. The synthetic wastewater used in the experiment was formulated to mimic the influent characteristics of the Anammox unit in the treatment plant, with the following water quality parameters: ammonium nitrogen (NH4+-N) concentration of 115–125 mg/L, nitrite nitrogen (NO2−-N) concentration of 145–155 mg/L, COD concentration of 240–260 mg/L, DO concentration of 0.3–0.6 mg/L, and a pH range of 7.2–7.8. COD was configured using soybean juice anaerobically fermented for three days, and the influent pH was adjusted with HCl and NaOH. The types and proportions of trace elements added to the wastewater were configured according to previous relevant studies [26].

2.3. Analytical Methods

During the experiment, influent and effluent samples of the reactor were collected every two days for water quality analysis. The routine detection indices included NH4+-N, NO2−-N, nitrate nitrogen (NO3−-N), TN, chemical oxygen demand (COD), pH, and DO. Through the systematic analysis of these indices, the removal efficiency of nitrogen and organic pollutants by the reactor, as well as the transformation patterns of nitrogen species within the system, could be evaluated. The detection of COD was carried out in accordance with the “Determination of Chemical Oxygen Demand in Water Quality—Rapid Digestion Spectrophotometric Method” [27], while the determination methods for NH4+-N, NO2−-N, NO3−-N, and TN were consistent with those described by Babu et al. [28]. TN and COD were directly measured using a HACH DR5000 spectrophotometer (HACH, Loveland, CO, USA). NH4+-N, NO2−-N, and NO3−-N were measured using an INESA 721G visible spectrophotometer (INESA, Shanghai, China), while the pH was determined with a PHS-25 pH meter (HACH, Loveland, CO, USA), and the dissolved oxygen was measured using a HACH HQ-10 dissolved oxygen meter (HACH, Loveland, CO, USA), respectively.

3. Results and Discussion

3.1. Influence of Effluent Recirculation Stirring on the Reactor

As depicted in Stage I of Figure 2, under the condition of no organic matter addition to the influent, during the stable operation of the Anammox reactor, the average influent concentration of NH4+-N was 117.6 mg/L, which decreased to 1.2 mg/L in the effluent, resulting in an average NH4+-N removal rate of 99.0% (Figure 2b). The average influent concentration of NO2−-N was 153.2 mg/L, and the average effluent concentration was 3.1 mg/L, with a removal rate of 98.0% (Figure 2c). The average influent concentration of NO3−-N was 2.0 mg/L, while the average effluent concentration increased to 30.9 mg/L, leading to an average NO3−-N accumulation rate of 12.2% (Figure 2d). The average influent TN concentration was 272.4 mg/L, and the average effluent TN concentration decreased to 34.9 mg/L, with an average TN removal rate of 87.2% (Figure 2e).
These results indicate that after the acclimation and enrichment of microorganisms during the startup phase, anaerobic ammonium oxidation bacteria (AnAOB) exhibited high metabolic activity, enabling stable and efficient degradation of NH4+-N. Moreover, the reasonable ratio of influent NO2−-N to NH4+-N concentrations provided favorable conditions for the full progression of the Anammox reaction, thereby ensuring the efficient removal of ammonia nitrogen and the stable operation of the system.
Data from Stage II showed that after adding organic matter, the effluent COD concentration gradually decreased while the COD removal rate increased slowly. Simultaneously, the effluent concentrations of NH4+-N, NO2−-N, and TN increased, whereas the NO3−-N concentration declined. The system stabilized on the 26th day of operation. During the stable operation period, the average influent concentrations of COD, NH4+-N, NO2−-N, NO3−-N, and TN were 249.2 mg/L, 123.4 mg/L, 148.1 mg/L, 1.1 mg/L, and 270.4 mg/L, respectively; the average effluent concentrations were 125.2 mg/L, 64.5 mg/L, 68.3 mg/L, 3.2 mg/L, and 142.1 mg/L, with corresponding average removal rates of 49.8%, 47.8%, 53.9% and 47.4%. Compared with Stage I, the removal rates of NH4+-N, NO2−-N, and TN decreased by 51.2%, 44.1%, and 39.7%, respectively. These results indicate that the addition of organic matter significantly inhibited the nitrogen removal efficiency of the Anammox reactor. This inhibition might be attributed to the disruption of AnAOB’s physiological metabolism by organic matter, which hindered the normal progression of the Anammox reaction. Additionally, organic matter provided a carbon source for heterotrophic bacteria (such as denitrifying bacteria), leading to their proliferation and reducing the abundance and biomass of AnAOB in the reactor.
As shown in Stage III to V of Figure 2, after adding organic matter to the influent and implementing effluent recirculation, the effluent concentrations of COD, NH4+-N, NO2−-N, and TN decreased, while the change in NO3−-N concentration was relatively minor. Notably, when the effluent recirculation ratio reached 200%, the effluent concentrations of COD, NH4+-N, NO2−-N, and TN dropped to the lowest levels. During the stable operation period of the reactor, the average effluent concentrations of these indices were 65.3 mg/L, 6.2 mg/L, 5.9 mg/L, and 19.2 mg/L, respectively, with average removal rates increasing to 73.5%, 94.9%, 96.1%, and 93.3%. This indicates that the reactor exhibited an optimal nitrogen removal performance when the effluent recirculation ratio was controlled at approximately 200%. Similar to Jia’s findings, who concluded that the highest nitrogen removal efficiency of the system was achieved at an effluent recirculation ratio of 200% while treating high-ammonia fermentation wastewater using the CANON process [29], our research also validates this critical parameter. Although the removal rates of NH4+-N and NO2−-N were lower compared to Stage I (without organic matter addition), the TN removal rate was higher. This phenomenon can be explained as follows: While the presence of organic matter inhibited Anammox activity (leading to decreased removal rates of NH4+-N and NO2−-N), the dilution effect from recirculation reduced the organic matter concentration in the system, alleviating the inhibition on Anammox bacteria. Meanwhile, heterotrophic denitrifying bacteria utilized organic matter as a carbon source, forming a synergistic effect with Anammox bacteria—Anammox bacteria were responsible for converting part of the NH4+-N into N2, while heterotrophic denitrifiers used residual organic matter to convert NO3−-N and NO2−-N into N2, compensating for the insufficient removal of NO3−-N by the Anammox process [30]. It is worth noting that when the recirculation ratio increased to 300%, the removal rates of COD, NH4+-N, NO2−-N, and TN all decreased, primarily due to the shortened hydraulic retention time (HRT) in the reactor caused by excessive recirculation [31].
It is worth noting that during the 30th–40th days with a 100% recirculation ratio (Stage III), the COD removal rate increased (Figure 2a), accompanied by a corresponding rise in the NO2−-N removal rate (Figure 2c). This phenomenon was caused by an operational error, where the effluent pipe was not fully submerged in the recirculation tank, resulting in air exposure and an elevated DO concentration of 3.5–4.0 mg/L in the recirculation tank. Calculated based on a 100% recirculation ratio, the DO concentration in the reactor reached 1.9–2.3 mg/L, which enhanced the decomposition of organic matter by aerobic heterotrophic bacteria. Moreover, since the oxygen half-saturation coefficient of nitrite-oxidizing bacteria (NOB, such as Nitrobacter or Nitrospira) is 1.2–1.5 mg/L, the higher DO concentration stimulated the activity of NOB, accelerating the oxidation of nitrite [32]. Despite the increase in DO concentration during this period, the total nitrogen removal rate remained similar to that during the stable operation phase, indicating that the mixed microbial community in the reactor exhibited a strong oxygen tolerance, providing a basis for subsequent micro-aeration experiments.

3.2. Influence of Micro-Aeration Stirring on the Reactor

As shown in Figure 3a, when the aeration intensity increased from 50 mL/min to 100 mL/min, the effluent COD concentration decreased from 98.6 mg/L to 51.5 mg/L, and the average COD removal rate increased from 59.4% to 78.9%. However, further increasing the aeration intensity to 150 mL/min did not significantly enhance the COD removal rate. This suggests that at an aeration intensity of approximately 100 mL/min, aerobic heterotrophic bacteria can efficiently consume readily biodegradable organic matter.
As shown in Figure 3b–e, when the aeration intensity increased from 50 mL/min to 100 mL/min, the average effluent concentrations of NH4+-N, NO2−-N, and TN during the stable operation of the reactor decreased, from 22.5 mg/L, 41.5 mg/L, and 75.9 mg/L to 8.5 mg/L, 10.3 mg/L, and 33.1 mg/L, respectively. Correspondingly, the average removal rates increased from 81.5%, 70.9%, and 73.4% to 92.6%, 93.1%, and 88.3%, respectively. When the aeration intensity was further increased to 150 mL/min, the effluent concentrations of NH4+-N and NO2−-N decreased to 15.3 mg/L and 0.6 mg/L, respectively, but the concentrations of NO3−-N and TN increased significantly to 113.1 mg/L and 134.7 mg/L. Accordingly, the removal rates of NH4+-N and NO2−-N increased to 86.8% and 99.6%, respectively, while the TN removal rate dropped significantly to 49.7%. In addition, on the 58th day of the experiment, a large amount of floating sludge broke through the retaining net, resulting in severe sludge loss, which further exacerbated the decrease in the TN removal rate.
A detailed analysis of the mechanism revealed that at an aeration intensity of 100 mL/min, aerobic heterotrophic bacteria efficiently consumed the readily biodegradable organic matter in the wastewater, reducing the DO concentration in the system. This process created a suitable micro-anaerobic environment for AnAOB and denitrifying bacteria, enabling different functional microbial communities to act synergistically and enhancing the nitrogen removal efficiency. However, when the aeration intensity increased to 150 mL/min, since most of the readily biodegradable organic matter had been consumed at the 100 mL/min stage, the additional aeration led to an excess of DO that could not be fully utilized. The high DO environment activated the activity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), further increasing the removal rates of NH4+-N and NO2−-N. However, the excessively high DO concentration strongly inhibited the activity of AnAOB and denitrifying bacteria, hindering the denitrification process, increasing the accumulation of NO3−-N, and ultimately resulting in a significant decrease in the TN removal rate.

3.3. Changes in Pollutants at Different Sludge Heights

Under the conditions of an HRT of 13.7 h, influent DO concentration of 0.3–0.6 mg/L, and pH of 7.2–7.8, the pollutant concentrations at different sludge heights under the conditions of no additional mixing mode, 200% reflux ratio, and 100 mL/min mixing mode are shown in Figure 4.
As shown in Figure 4a, under the conditions of implementing effluent recirculation with a 200% recirculation ratio or aeration with an aeration intensity of 100 mL/min, the COD concentration exhibited the largest decrease in the sludge layer at 0–15 cm. In the effluent recirculation scenario, the reduction in COD at 0–15 cm was attributed to two factors: the dilution effect of recirculation lowering the COD concentration, and the consumption of COD by microorganisms in this region. For the aeration scenario, this was due to the highest sludge concentration at 0–15 cm, with the sludge concentration decreasing gradually with increasing height. Influenced by the same mechanism, NH4+-N (Figure 4b), NO2−-N (Figure 4c), and TN (Figure 4e) showed similar variation trends.
For NO3−-N (Figure 4d), the operational condition without stirring exhibited a trend similar to that with only effluent recirculation stirring. In the aeration stirring condition, however, the NO3−-N concentration increased by 3.8 mg/L in the 0–15 cm sludge layer, followed by a gradual decrease. This phenomenon was primarily caused by the increase in DO concentration at the bottom due to aeration, which enhanced the activity of aerobic bacteria such as ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), converting NH4+-N and NO2−-N into NO3−-N. As the sludge height increased, the DO concentration decreased, and the activity of denitrifying bacteria gradually intensified, converting NO3−-N into N2.

3.4. Influence of Stirring Methods on Extracellular Polymeric Substances (EPS)

As depicted in the Figure 5, for the 15 cm sample height, the protein (PN) content under an effluent reflux ratio of 200% (157.9 mg/g MLSS) is higher than that without stirring (99.6 mg/g MLSS) and with an aeration strength of 100 mL/min (89.8 mg/g MLSS). Similarly, at 45 cm, the PN content under a 200% effluent reflux ratio (150.1 mg/g MLSS) also surpasses the other two conditions (119.0 mg/g MLSS for no stirring and 126.0 mg/g MLSS for 100 mL/min aeration). In contrast, the PS (polysaccharide) content shows relatively minor variations across the three conditions at both heights.
The notable increase in PN under a 200% effluent reflux ratio arises because effluent-induced organic-matter dilution enhances anaerobic ammonium-oxidation bacterial activity, promoting PN secretion. Although 100 mL/min aeration also promotes PN secretion, the effect is less pronounced than that of a 200% effluent reflux ratio. This demonstrates that an appropriate effluent reflux ratio more effectively boosts anaerobic ammonium-oxidation bacterial activity, leading to elevated PN secretion in EPS.

3.5. Influence of Stirring Methods on Microbial Community

3.5.1. Microbial Community at the Door Level

The results of microbial community analysis (Figure 6) showed that the dominant phyla in granular sludge included Bacteroidota, Chloroflexi, Proteobacteria, and Planctomycetota. Without effluent recirculation and micro-aeration, the relative abundances of Planctomycetota in sludge samples collected at 15 cm and 45 cm from the bottom of the reactor were 13% and 15%, respectively. When the effluent recirculation ratio was set at 200%, the abundances of Planctomycetota at 5 cm and 45 cm increased to 23.7% and 17.3%, respectively. However, under micro-aeration conditions with an aeration intensity of 100 mL/min, the abundances of Planctomycetota at 15 cm and 45 cm were 14.4% and 16.9%, respectively, showing no significant difference at 15 cm but a slight increase at 45 cm compared with the control group. Notably, Planctomycetota is the major phylum of Anammox bacteria and plays a crucial role in the global carbon–nitrogen cycle [33]. These findings indicate that effluent recirculation effectively mitigates the inhibitory effect of organic matter on Anammox bacteria by diluting the organic matter concentration in wastewater, thereby promoting the enrichment of Planctomycetota (Anammox bacteria). In contrast, although micro-aeration significantly improved the nitrogen removal efficiency, it slightly increased the abundance of Planctomycetota. Therefore, it is inferred that the enhanced nitrogen removal performance under micro-aeration conditions is mainly attributed to the activation of the nitrification–denitrification process due to the increased dissolved DO concentration, rather than the direct promotion of Anammox bacteria proliferation.
Bacteroidota exhibited a strong tolerance to high concentrations of organic matter, maintaining metabolic activity and becoming the dominant microbial community under conditions where Anammox bacteria are inhibited (e.g., during the initial stage of organic matter addition) [34]. This characteristic indicates that Bacteroidota may play a crucial role in promoting the Anammox reaction through synergistic effects during the treatment of high-ammonia-nitrogen wastewater, potentially enhancing the overall nitrogen removal efficiency of the system.
Under micro-aeration conditions, the microbial community structure showed differential responses. Specifically, the abundance of Chloroflexi in sludge samples collected from the 45 cm sampling port (measured from the bottom of the sludge) increased by 5.8% compared with the non-aerated condition, while no significant change was observed at the 15 cm port. Previous research has demonstrated that Chloroflexi participates in the nitrogen cycle across various environments. Especially under anoxic or anaerobic conditions, Chloroflexi can reduce nitrate to nitrogen gas through denitrification [35]. Based on the results of this study, it is speculated that the increase in Chloroflexi abundance at the 45 cm sampling port may be related to the enhanced nitrification at the bottom of the reactor due to micro-aeration, leading to the accumulation of NO3−-N. The high concentration of NO3−-N provides substrates for the denitrification metabolism of Chloroflexi, thereby promoting its proliferation.
Furthermore, micro-aeration increased the abundance of Proteobacteria in sludge samples from the 15 cm sampling port by 8.3%. Previous studies have shown that Proteobacteria exhibits strong environmental adaptability, capable of surviving under aerobic, anoxic, and anaerobic conditions. It also possesses the functions of efficient organic matter degradation and denitrification for nitrogen removal [36]. These findings further confirm that micro-aeration enhances nitrification at the bottom of the reactor. The increased DO concentration due to aeration stimulates nitrification, generating more NO3−-N, which provides the material basis for the denitrification function of Proteobacteria and thus promotes its abundance increase.

3.5.2. Microbial Community at Genus Level

As shown in Figure 7, the addition of 200% effluent recirculation significantly altered the abundance of Anammox genera in sludge samples at different heights within the reactor. In the sludge samples collected 15 cm from the bottom, the relative abundances of Candidatus Jettenia and Candidatus Kuenenia increased by 2.4% and 7.5%, respectively. At a height of 45 cm, the abundances of Candidatus Brocadia and Candidatus Kuenenia increased by 7.5% and 3.2%, respectively. These results indicate that effluent recirculation effectively mitigated the inhibitory effect of organic matter on Anammox bacteria by diluting the influent organic concentration. Although the recirculation introduced a small amount of DO, the activity enhancement of Candidatus Brocadia, which is more sensitive to DO, was relatively limited. The proliferation of Candidatus Brocadia and Candidatus Kuenenia at 45 cm was mainly attributed to the decrease in COD and substrate concentrations.
Under micro-aeration with an intensity of 100 mL/min, the microbial community structure exhibited a distinct selective response. In the 15 cm sludge samples, the abundances of Candidatus Jettenia and Candidatus Brocadia decreased sharply from 8.1% and 3.9% to 4.9% and 0.5%, respectively, while the abundance of Candidatus Kuenenia increased from 0.4% to 8.1%. This trend suggests that Candidatus Brocadia, which has a low DO tolerance, was gradually replaced by the more oxygen-resistant Candidatus Kuenenia. This finding aligns with Xu et al.’s conclusion that Candidatus Kuenenia was the dominant Anammox species under DO-containing conditions, further validating the oxygen-tolerance characteristics of this microbial genus [25]. Notably, at 45 cm, as the concentrations of DO, substrates, and COD decreased, the abundance of Candidatus Brocadia rebounded to 2.5%, while the abundance of Candidatus Kuenenia further increased to 10.8%, whereas that of Candidatus Jettenia further declined to 1.3%, indicating that Candidatus Jettenia had weaker competitiveness compared to other genera under low-COD and low-substrate conditions.
In addition to Anammox genera, micro-aeration and high recirculation ratios promoted the enrichment of multi-functional genera. When both 200% recirculation and 100 mL/min aeration were applied, the abundance of Denitratisoma in 15 cm sludge increased from 3.1% to 8% and 16.1%, respectively. Previous studies have confirmed that Denitratisoma possesses aerobic denitrification capabilities, enabling it to convert nitrite (NO2−) into gaseous nitrogen (N2) under aerobic conditions [37], suggesting its efficient utilization of DO for denitrification within a certain DO concentration range. Moreover, micro-aeration increased the relative abundances of facultative genera: the abundance of norank f AKYH767 increased from 0.5% to 3.9%, and this genus can degrade organic matter using DO under aerobic conditions and perform denitrification under anaerobic conditions [38,39]. The abundance of Limnobacter increased from 1.1% to 2.6%, and it can flexibly switch between anaerobic respiration/fermentation and aerobic respiration depending on the environmental oxygen concentration [40].
Finally, in both the non-stirring condition and the 200% effluent recirculation stirring condition, the abundances of Nitrosomonas (AOB) and Nitrolancea (NOB) in sludge samples at 15 cm and 45 cm were both below 0.3%. By contrast, under the 100 mL/min aeration intensity condition, the abundances of Nitrosomonas and Nitrolancea in the 15 cm sludge sample increased to 1.9% and 1.6%, respectively, while those in the 45 cm sample reached 1.6% and 1.0%, respectively. Since Nitrosomonas and Nitrolancea belong to the AOB and NOB, respectively, this indicates that an aeration intensity of 100 mL/min not only enhances aerobic heterotrophic bacteria but also promotes nitrifying bacteria [41,42]. Nevertheless, as the abundance of nitrifying bacteria remains far lower than that of Anammox bacteria, the removal of NH4+-N and NO2−-N in the reactor is still dominated by the Anammox process.

4. Conclusions

This study evaluated effluent recirculation and micro-aeration in Anammox reactors for treating high-ammonia-nitrogen organic wastewater. A 200% recirculation ratio optimized TN removal (93.3%) by diluting organic inhibitors, mitigating their toxic effects on Anammox bacteria (e.g., Candidatus Kuenenia and Candidatus Brocadia) and enabling synergistic interactions with heterotrophic denitrifiers. This dilution reduced electron competition, facilitating efficient nitrogen conversion to N2. Micro-aeration at 100 mL/min improved COD (78.9%) and TN (88.3%) removal by creating micro-anaerobic niches for metabolic cooperation between aerobic degraders (e.g., Proteobacteria) and anaerobic Anammox bacteria. The controlled oxygen supply balanced organic oxidation with Anammox activity, forming a denitrification–Anammox co-occurrence gradient. Excessive aeration (150 mL/min) increased dissolved oxygen, inhibiting Anammox bacteria and favoring aerobic competitors, causing a 49.7% drop in TN removal due to nitrate accumulation.
Microbial analysis showed recirculation enriched Planctomycetota by reducing organic toxicity, while micro-aeration induced a shift to oxygen-tolerant Candidatus Kuenenia and enhanced Proteobacteria/Chloroflexi for denitrification and organic degradation. These shifts enabled simultaneous carbon and nitrogen removal via coupled Anammox–denitrification pathways. The synergistic effects of dilution and metabolic cooperation at optimal conditions (200% recirculation or 100 mL/min aeration) provide a novel framework for treating high-ammonia-nitrogen organic wastewater, addressing a research gap in parallel Anammox enhancement strategies.

5. Future Perspectives

Actual wastewater contains complex components (e.g., isoflavones, saponins), which were not evaluated in this research. Future studies should assess how these compounds affect Anammox reactor performance and microbial community structure. In addition, this study only simulated a single range of COD concentrations. Expanding the research to include multi-gradient COD conditions will help to optimize operational parameters for diverse wastewater types.

Author Contributions

Conceptualization, Z.Y. and R.Z.; methodology, Z.Y. and R.Z.; software, R.Z.; validation, R.Z.; formal analysis, R.Z.; investigation, R.Z.; resources, Z.Y. and R.Z.; data curation, R.Z.; writing—original draft preparation, R.Z.; writing—review and editing, R.Z.; visualization, Z.Y.; supervision, Z.Y.; project administration, R.Z.; funding acquisition, Z.Y. and H.Y. All authors have read and agreed to the published version of the manuscript.

Funding

Please The research was supported by the Gansu Province Ecological Civilisation Construction Key R&D Special Project (25YFFA014), the National Natural Science Foundation of China (51568034), the Department of Education of Gansu Province: Major Cultivation Project of Scientific Research Innovation Platform at Universities (2024CXPT-14), and the Open Foundation of the Key Laboratory of Yellow River Water Environment in Gansu Province (20JR2RA0002).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All valid data are reflected in the article and authorized to be used.

Acknowledgments

The authors would like to express sincere gratitude to the editors and reviewers for their valuable and constructive comments.

Conflicts of Interest

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
AnammoxAnaerobic ammonium oxidation
AOBAmmonia-oxidizing bacteria
NOBNitrite-oxidizing bacteria
AnAOBAnaerobic ammonium oxidation bacteria
HRTHydraulic retention time
TTemperature
C/NCarbon-to-nitrogen
EPSExtracellular polymeric substances
PNProtein
PSPolysaccharide
DODissolved oxygen

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Figure 1. Schematic diagram of the device.
Figure 1. Schematic diagram of the device.
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Figure 2. Effect of effluent recirculation on alleviating the inhibition of Anammox by organic matter. Notes: (a) variation in COD; (b) variation in NH4+-N; (c) variation in NO2−-N; (d) variation in NO3−-N; (e) variation in TN. Stage I: Period without organic matter addition; Stage II: Period with organic matter addition; Stage III: Period with an effluent recirculation ratio of 100% after organic matter addition; Stage IV: Period with an effluent recirculation ratio of 200% after organic matter addition; Stage V: Period with an effluent recirculation ratio of 300% after organic matter addition.
Figure 2. Effect of effluent recirculation on alleviating the inhibition of Anammox by organic matter. Notes: (a) variation in COD; (b) variation in NH4+-N; (c) variation in NO2−-N; (d) variation in NO3−-N; (e) variation in TN. Stage I: Period without organic matter addition; Stage II: Period with organic matter addition; Stage III: Period with an effluent recirculation ratio of 100% after organic matter addition; Stage IV: Period with an effluent recirculation ratio of 200% after organic matter addition; Stage V: Period with an effluent recirculation ratio of 300% after organic matter addition.
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Figure 3. Effect of micro-aeration on alleviating the inhibition of Anammox by organic matter. Notes: (a) variation in COD; (b) variation in NH4+-N; (c) variation in NO2−-N; (d) variation in NO3−-N; (e) variation in TN. Stage VI: Period with an aeration intensity of 50 mL/min after organic matter addition; Stage VII: Period with an aeration intensity of 100 mL/min after organic matter addition; Stage VIII: Period with an aeration intensity of 150 mL/min after organic matter addition.
Figure 3. Effect of micro-aeration on alleviating the inhibition of Anammox by organic matter. Notes: (a) variation in COD; (b) variation in NH4+-N; (c) variation in NO2−-N; (d) variation in NO3−-N; (e) variation in TN. Stage VI: Period with an aeration intensity of 50 mL/min after organic matter addition; Stage VII: Period with an aeration intensity of 100 mL/min after organic matter addition; Stage VIII: Period with an aeration intensity of 150 mL/min after organic matter addition.
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Figure 4. Changes in pollutants at different sludge heights: (a) variation in COD; (b) variation in NH4+-N; (c) variation in NO2−-N; (d) variation in NO3−-N; (e) variation in TN.
Figure 4. Changes in pollutants at different sludge heights: (a) variation in COD; (b) variation in NH4+-N; (c) variation in NO2−-N; (d) variation in NO3−-N; (e) variation in TN.
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Figure 5. Changes in EPS under different mixing modes.
Figure 5. Changes in EPS under different mixing modes.
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Figure 6. Microbial abundances at the phylum level. Notes: X represents the sludge samples collected from the sampling port 15 cm from the bottom of the sludge; S represents the sludge samples collected from the sampling port 45 cm from the bottom of the sludge; 1 indicates the operating condition without effluent recirculation or micro-aeration after organic matter addition; 2 indicates the operating condition with an effluent recirculation ratio of 200% after organic matter addition; 3 indicates the operating condition with an aeration intensity of 100 mL/min after organic matter addition.
Figure 6. Microbial abundances at the phylum level. Notes: X represents the sludge samples collected from the sampling port 15 cm from the bottom of the sludge; S represents the sludge samples collected from the sampling port 45 cm from the bottom of the sludge; 1 indicates the operating condition without effluent recirculation or micro-aeration after organic matter addition; 2 indicates the operating condition with an effluent recirculation ratio of 200% after organic matter addition; 3 indicates the operating condition with an aeration intensity of 100 mL/min after organic matter addition.
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Figure 7. Microbial abundances at the genus level. Notes: X represents the sludge samples collected from the sampling port 15 cm from the bottom of the sludge; S represents the sludge samples collected from the sampling port 45 cm from the bottom of the sludge; 1 indicates the operating condition without effluent recirculation or micro-aeration after organic matter addition; 2 indicates the operating condition with an effluent recirculation ratio of 200% after organic matter addition; 3 indicates the operating condition with an aeration intensity of 100 mL/min after organic matter addition.
Figure 7. Microbial abundances at the genus level. Notes: X represents the sludge samples collected from the sampling port 15 cm from the bottom of the sludge; S represents the sludge samples collected from the sampling port 45 cm from the bottom of the sludge; 1 indicates the operating condition without effluent recirculation or micro-aeration after organic matter addition; 2 indicates the operating condition with an effluent recirculation ratio of 200% after organic matter addition; 3 indicates the operating condition with an aeration intensity of 100 mL/min after organic matter addition.
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MDPI and ACS Style

Yan, Z.; Zeng, R.; Yang, H. Sustainable Treatment of High-Ammonia-Nitrogen Organic Wastewater via Anaerobic Ammonium Oxidation (Anammox) Combined with Effluent Recirculation/Micro-Aeration. Sustainability 2025, 17, 5926. https://doi.org/10.3390/su17135926

AMA Style

Yan Z, Zeng R, Yang H. Sustainable Treatment of High-Ammonia-Nitrogen Organic Wastewater via Anaerobic Ammonium Oxidation (Anammox) Combined with Effluent Recirculation/Micro-Aeration. Sustainability. 2025; 17(13):5926. https://doi.org/10.3390/su17135926

Chicago/Turabian Style

Yan, Zichun, Rong Zeng, and Hao Yang. 2025. "Sustainable Treatment of High-Ammonia-Nitrogen Organic Wastewater via Anaerobic Ammonium Oxidation (Anammox) Combined with Effluent Recirculation/Micro-Aeration" Sustainability 17, no. 13: 5926. https://doi.org/10.3390/su17135926

APA Style

Yan, Z., Zeng, R., & Yang, H. (2025). Sustainable Treatment of High-Ammonia-Nitrogen Organic Wastewater via Anaerobic Ammonium Oxidation (Anammox) Combined with Effluent Recirculation/Micro-Aeration. Sustainability, 17(13), 5926. https://doi.org/10.3390/su17135926

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