Fe²⁺-Coupled Organic-Substrate-Enhanced Nitrogen Removal in Two-Stage Anammox Biofilm Reactors

Abstract

Anammox is a novel and energy-efficient biological nitrogen removal technology. Enhancing its performance in treating low-strength nitrogen wastewater is essential for expanding its practical applications. In response to challenges such as low nitrogen removal efficiency (NRE), poor operational stability, limited environmental resistance, and the interference of organic compounds commonly found in real wastewater, this study developed a two-stage upflow anammox biofilm reactor system (R1 and R2) enhanced by an Fe²⁺-coupled organic substrate strategy for deep nitrogen removal under low-nitrogen conditions. Results showed that sodium acetate at a chemical oxygen demand (COD) concentration of 40 mg/L provided the greatest enhancement to anammox activity, achieving an average total nitrogen removal efficiency (NRE) of 90.02%. However, the reactor performance was significantly inhibited under higher COD conditions (e.g., COD = 60 mg/L). Under an influent Fe²⁺ concentration of 10 mg/L, the reactors’ NRE increased and then decreased as the COD concentration rose from 0 to 100 mg/L, resulting in the highest efficiency being achieved at an average NRE of 94.11%, observed under 10 mg/L Fe²⁺ coupled with 60 mg/L of COD in the two-stage anammox system. Scanning electron microscopy revealed that the co-addition of Fe²⁺ and organic substrates led to the formation of granular protrusions and pores on the sludge surface, which favored the structural stability of the biomass. At a COD level of 40 mg/L, the contents of extracellular polymeric substances and heme c in anammox biofilm were significantly higher compared to the addition of 10 mg/L Fe²⁺ alone, whereas excessive COD inhibited both indicators. These findings suggest that moderate levels of Fe²⁺ coupled with organic matter can promote anammox activity for deep nitrogen removal, while excessive organics have inhibitory effects. This study provides theoretical support for enhancing nitrogen removal from low-strength wastewater using Fe²⁺ and organic-substrate-assisted anammox processes.

1. Introduction

Anammox has emerged as one of the most cost-effective and energy-efficient biological nitrogen removal technologies in the field of wastewater treatment. Within less than a decade, the process has evolved from its initial discovery to laboratory-scale studies and large-scale engineering applications. As scientific interest in anammox continues to grow, increasing numbers of full-scale systems have been implemented worldwide. To date, anammox has been primarily applied to high-strength ammonium wastewater such as sludge digester effluent [1], pharmaceutical wastewater [2], landfill leachate [3], and livestock wastewater [4], achieving impressive nitrogen removal performance. Although anammox has been successfully employed for high-ammonium, low-C/N-ratio wastewater, its application in low-strength nitrogen wastewater remains limited. This is largely attributed to the slow growth rate, long doubling time, and low environmental resilience of anaerobic ammonium-oxidizing bacteria (AnAOB), the core functional group in the anammox process. Therefore, enhancing the activity of AnAOB and improving their tolerance to environmental conditions is essential for promoting the practical application of anammox technology in the advanced treatment of low-strength wastewater.

Enhancing the activity of AnAOB is critical for treating low-strength nitrogen wastewater and is a fundamental challenge in the practical application of this technology. A growing body of research has demonstrated that AnAOB activity can be enhanced through external energy sources such as electric fields [5], magnetic fields [6], and ultrasound [7], as well as by supplementing with essential trace elements like copper, manganese, zinc, and iron [8]. Among these, iron plays a particularly important role as it is involved in nearly all major metabolic pathways in microbial cells, including electron transport, the tricarboxylic acid (TCA) cycle, and the biosynthesis of amino acids and pyrimidines [9]. During the anammox reaction, AnAOB synthesize heme-containing enzymes such as cytochrome c, hydrazine synthase, and hydrazine dehydrogenase, which account for over 20% of the total cellular protein content. Iron, as a critical structural component of these enzymes, is thus essential for microbial metabolism and the proliferation of AnAOB [10]. Numerous studies have shown that suitable concentrations of iron promote the accumulation of intracellular iron and the synthesis of cytochrome c in AnAOB [11]. In addition, iron’s flocculation effect can stimulate the secretion of EPS, which aids in the formation of microbial aggregates and further enhances the nitrogen removal performance in anammox systems [12]. For instance, Fe²⁺ concentrations in the range of 1–5 mg/L significantly boosted AnAOB activity, whereas higher levels (10–30 mg/L) led to various degrees of inhibition, resulting in reduced total nitrogen removal efficiency (NRE) [13]. The effect of sponge iron (SI) addition on nitrogen removal performance under low-nitrogen-loading-rate (NLR) conditions showed that it effectively stimulated specific metabolic pathways and microbial activities related to nitrogen transformation [14], with the removal efficiency of total nitrogen (TN), ammonium–nitrogen (NH₄⁺-N), and nitrite–nitrogen (NO₂⁻-N) reaching peak values of 92.63%, 91.6%, and 95.6% under 20 g/L of SI, respectively. Similarly, the addition of 3 g/L iron-loaded diatomaceous earth (Fe-DE) at an Fe:DE ratio of 1:20 significantly enhanced the performance of the mainstream anammox process by shortening the start-up period from 29 days to 17 days and increasing the nitrogen removal rate (NRR) from 0.234 kg N/(m³·d) to 0.437 kg N/(m³·d) [15]. These findings confirm that iron is a key factor influencing AnAOB activity and the nitrogen removal performance of an anammox system. However, few studies have examined the role of iron under low-strength nitrogen conditions. Therefore, it is essential to investigate the influence of iron on AnAOB activity and NRE in the treatment of low-strength wastewater. Enhancing AnAOB performance and environmental resilience under such conditions is expected to promote the broader application of anammox technology in advanced effluent treatment systems.

Organic substrates significantly influence the NRE of anammox systems. Among the various carbon sources, sodium acetate stands out due to its wide availability, low sludge yield, and minimal dosing requirement. The effects of different types and concentrations of organic substrates on anammox demonstrated that the addition of glucose, acetic acid, and sodium acetate within the concentration range of 20 to 250 mg COD/L promoted nitrite removal [16]. Similarly, the introduction of a moderate amount of acetate (with COD levels below 100 mg/L) into the anammox system shortened the start-up period, enhanced nitrogen removal performance, and facilitated the proliferation of all types of anammox bacteria [17]. However, limited research has focused on the inhibitory concentrations of organics and the associated sludge characteristics under long-term conditions, especially in the context of low-strength nitrogen effluents. Given the potential of Fe²⁺ to stimulate AnAOB activity, investigating the feasibility of combining Fe²⁺ with organic substrates under low-nitrogen conditions may help to identify more efficient, cost-effective, and practical strategies for enhancing anammox performance. This coupling approach holds promise for improving both the metabolic activity of functional microorganisms and the overall system stability in the advanced treatment of low-strength wastewater.

To this end, a two-stage upflow anammox biofilm reactor was constructed to treat low-strength nitrogen wastewater. The effects of varying concentrations of sodium acetate, Fe²⁺, and their combined application on nitrogen removal performance were systematically investigated. Furthermore, the influence of Fe²⁺–sodium acetate coupling on biofilm characteristics, EPS, and intracellular heme c content within the anammox system was analyzed. This study aimed to clarify the enhancement potential and regulatory mechanisms of Fe²⁺–sodium acetate coupling in improving NRE in anammox biofilm reactors. The findings are expected to provide a theoretical basis and practical guidance for the stable and efficient application of anammox technology in the advanced treatment of low-strength effluents, thereby promoting its broader adoption in tertiary wastewater treatment.

2. Materials and Methods

2.1. Reactor Configuration and Operational Strategy

Two identical upflow biofilm reactors were used in this study to form a two-stage anammox system. The schematic diagram of the experimental setup is shown in Figure 1. Both anammox reactors (R1 and R2) were constructed from transparent acrylic plexiglass, with internal dimensions of 30 cm in length and width and 70 cm in height. The working water height was maintained at 60 cm, resulting in an effective volume of 54 L. Each reactor was filled with evenly distributed fiber-type carriers to provide support for biofilm attachment. To maintain optimal conditions for AnAOB growth, water temperature was controlled at 31.0 ± 2.0 °C using thermostatic heating rods. In addition, to prevent the growth of phototrophic bacteria capable of oxygen generation, the reactors and the influent storage bottles for Fe²⁺ and organic substrates were wrapped with black shading plastic film to block light exposure. During operation, the effluent from R1 was fed directly into R2 in a sequential mode using a gravity-driven flow setup, utilizing the height difference between reactors. The influent flow rate for both reactors was precisely regulated using peristaltic pumps. The hydraulic retention time (HRT) was controlled at 5.8 h during the whole experiment.

The whole study was divided into three phases with constant influent ammonium–nitrogen (NH₄-N) and nitrite–nitrogen (NO₂-N) concentrations of 65 mg/L and 85 mg/L, respectively. In phase I, sodium acetate was chosen as carbon source added to supply COD at 0, 10, 20, 40, and 60 mg/L, where the reactors were operated for 20 days under each COD condition. Phase II focused on the effect of Fe²⁺ on nitrogen removal, which was divided into four individual stages with Fe²⁺ concentrations of 0, 5, 10, and 15 mg/L operated for 20 days, respectively. After stopping Fe²⁺ addition for 20 days, the reactors were operated under the condition of sodium acetate coupled with Fe²⁺ addition in phase III, in which the Fe²⁺ concentration was constant at 10 mg/L and the COD concentration varied, with 0, 20, 40, 60, and 100 mg/L divided into five stages which were operated for 20 days.

2.2. Sludge Inoculation and Synthetic Wastewater Feeding

The seed sludge used in this study was obtained from a laboratory-scale anammox reactor that had been stably operated by our research group over the long term. The mixed liquor volatile suspended solids (MLVSS) concentration of the inoculated sludge was 4240 mg/L, and the total NRR was 0.7 kg N·m⁻³·d⁻¹. The sludge exhibited a characteristic brick-red color, indicating the presence of active anammox biomass.

Synthetic wastewater was used throughout the experiment, with the influent pH maintained at 7.50 ± 0.50. NH₄⁺-N and NO₂⁻-N were supplied by NH₄Cl and NaNO₂, respectively, and their concentrations were kept constant at 65 mg/L and 85 mg/L during all experimental phases. Alkalinity was provided by NaHCO₃ at a concentration of 400 mg/L. Fe²⁺ and the organic substrate were supplied using FeSO₄·7H₂O and sodium acetate, respectively, and their concentrations were adjusted according to the specific experimental requirements. Other essential nutrients and trace elements required for microbial growth were added, as listed in

Table 1. Composition of nutrients and trace elements.

Content	Concentration (mg/L)
KH2PO4	68
MgSO4·7H2O	150
CaCl2	68

[13].

2.3. Water Quality Analysis and Calculations

The routine monitoring parameters in this study included the concentrations of NH₄⁺-N, NO₂⁻-N, and nitrate–nitrogen (NO₃⁻-N), as well as pH, dissolved oxygen (DO), mixed liquor suspended solids (MLSS), and MLVSS. NH₄⁺-N was determined using Nessle’s reagent spectrophotometric method, NO₂⁻-N by the N-(1-naphthyl)-ethylenediamine spectrophotometric method, and NO₃⁻-N by ultraviolet spectrophotometry. pH was measured using a glass electrode, and DO was monitored with a portable HACH-HQ30d DO meter. MLSS and MLVSS were both measured gravimetrically. The total nitrogen (TN) concentration was calculated as the sum of NH₄⁺-N, NO₂⁻-N, and NO₃⁻-N concentrations.

In this study, the NLR, NRR, and total NRE were calculated according to Equations (1), (2), and (3), respectively:

$$\mathrm{N}\mathrm{L}\mathrm{R}(\mathrm{k}\mathrm{g}\mathrm{N}·{\mathrm{m}}^{-3}·{\mathrm{d}}^{-1})={\displaystyle \frac{\mathrm{T}{\mathrm{N}}_{\mathrm{i}\mathrm{n}\mathrm{f}}}{\mathrm{H}\mathrm{R}\mathrm{T}\times 1000}}\times 24$$

(1)

$$\mathrm{N}\mathrm{R}\mathrm{R}(\mathrm{k}\mathrm{g}\mathrm{N}·{\mathrm{m}}^{-3}·{\mathrm{d}}^{-1})={\displaystyle \frac{\mathrm{T}{\mathrm{N}}_{\mathrm{i}\mathrm{n}\mathrm{f}}-{\mathrm{T}\mathrm{N}}_{\mathrm{e}\mathrm{f}\mathrm{f}}}{\mathrm{H}\mathrm{R}\mathrm{T}\times 1000}}\times 24$$

(2)

$$\mathrm{N}\mathrm{R}\mathrm{E}\left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{T}{\mathrm{N}}_{\mathrm{i}\mathrm{n}\mathrm{f}}-{\mathrm{T}\mathrm{N}}_{\mathrm{e}\mathrm{f}\mathrm{f}}}{\mathrm{T}{\mathrm{N}}_{\mathrm{i}\mathrm{n}\mathrm{f}}}}\times 100$$

(3)

where TN_inf and TN_eff represent the influent and effluent TN concentrations (mg/L), respectively, and HRT denotes the hydraulic retention time of the reactor (h).

2.4. Extraction and Analysis of Extracellular Polymeric Substance (EPS)

To extract EPS, 5 mL of homogenized biofilm was first collected from the reactor and centrifuged at 8000 r/min for 15 min. The supernatant was discarded, and 10 mL of phosphate-buffered saline (PBS) solution was added to the pellet. The mixture was then subjected to ultrasonication at 40 kHz and 120 W for 3 min, followed by water bath heating at 80 °C for 30 min. After heating, the sample was centrifuged again at 8000 r/min for 15 min. The resulting supernatant was filtered through a 0.45 μm microporous membrane to obtain the EPS extract. The extracted EPS was subsequently analyzed for protein (PN) and polysaccharide (PS) content according to the method described by reference [18].

2.5. Extraction and Determination of Heme C

To extract heme c, 5 mL of homogenized sludge was collected from the reactor and suspended in 10 mL of PBS solution (10 mmol/L, pH 7.50). The sludge suspension was then ultrasonicated in an ice bath for 5 min. After sonication, the mixture was centrifuged at 12,000 r/min at 4 °C for 15 min. The resulting supernatant was filtered through a 0.45 μm microporous membrane. The concentration of heme c was then determined using the pyridine–NaOH spectrophotometric method, following the procedure described in reference [19].

2.6. Scanning Electron Microscopy (SEM)

SEM (Merlin, Zeiss, Oberkochen, Germany) was employed to analyze the morphological changes in anammox biofilms in the two-stage reactors before and after the addition of Fe²⁺ coupled with organic substrates.

2.7. Data Analysis and Visualization

Data analysis and graphical visualization in this study were performed using Excel (Microsoft Office 2020) and Origin 2018 software.

3. Results and Discussion

3.1. Effect of Organic Substrate on the Performance of the Two-Stage Anammox System

3.1.1. Effect of Organic Substrate Concentration on Nitrogen Removal

The variations in effluent nitrogen species concentrations, TN, NRE, and NRR under different organic substrate concentrations are shown in Figure 2a,b. As illustrated in Figure 2a, during the initial phase without organic substrate addition (days 1–21), effluent nitrogen concentrations were relatively high due to the sludge removal and cleaning operations conducted in the R1 reactor, which temporarily disrupted the anammox process. With continued operation and biofilm adaptation, the nitrogen concentrations gradually decreased and stabilized. By the end of this phase, the effluent concentrations of NH₄⁺-N, NO₂⁻-N, and NO₃⁻-N of R2 were 2.31 mg/L, 2.19 mg/L, and 26.08 mg/L, respectively. As shown in Figure 2b, under the same conditions, the average effluent TN concentrations of R1 and R2 were 51.70 mg/L and 31.82 mg/L, corresponding to NRRs of 0.41 kg N·m⁻³·d⁻¹ and 0.08 kg N·m⁻³·d⁻¹, respectively, and the overall NRE after two-stage treatment was 78.79%. When the COD was increased to 10 mg/L and 20 mg/L, no significant changes were observed in the effluent NH₄⁺-N and NO₂⁻-N concentrations for both R1 and R2; however, the NO₃⁻-N concentration showed a noticeable decrease, indicating that low concentrations of organic substrates had no significant impact on anammox performance, but may have promoted heterotrophic denitrification and contributed to partial NO₃⁻-N reduction. As shown in Figure 2b, the addition of COD at 20 mg/L reduced the average effluent TN concentrations in R1 and R2 to 42.56 mg/L and 23.06 mg/L, respectively, corresponding to NRRs of 0.44 kg N·m⁻³·d⁻¹ and 0.08 kg N·m⁻³·d⁻¹, while the overall NRE after two-stage treatment increased to 84.63%. During days 64–83, when COD was further increased to 40 mg/L, although a sharp rise in effluent nitrogen concentration was observed in the first three days, a slight decrease followed thereafter. Under this condition, the average effluent concentrations of NH₄⁺-N and NO₂⁻-N in R1 decreased from 12.00 mg/L and 13.86 mg/L (at 20 mg/L COD) to 11.35 mg/L and 12.59 mg/L, respectively, which may suggest that anammox activity was moderately enhanced at this COD level. In addition, as the COD concentration increased, the effluent NO₃⁻-N concentration in the two-stage anammox system decreased progressively, with the average NO₃⁻-N concentration in the R2 effluent dropping to 10.74 mg/L at a COD of 40 mg/L. According to Figure 2b, under this condition, the average TN concentrations in the effluents of R1 and R2 decreased to 32.99 mg/L and 14.96 mg/L, respectively, corresponding to NRRs of 0.48 kg N·m⁻³·d⁻¹ and 0.07 kg N·m⁻³·d⁻¹, while the overall NRE reached as high as 90.02%. The effluent TN concentration of R2 met the Chinese Class A discharge standard (TN ≤ 15 mg/L), indicating that AnAOB and other heterotrophic denitrifying bacteria were able to coexist effectively at a COD of 40 mg/L in the R1 reactor, thereby enhancing the anammox process. Meanwhile, the corresponding NRR in R1 increased with the COD level, suggesting improved nitrogen removal performance. For the R2 reactor, the NRR remained stable in the range of 0.07–0.08 kg N·m⁻³·d⁻¹, indicating that R2 provided a consistent performance for further nitrogen removal and that the NRE of R2 was closely related to the substrate concentration in the effluent from R1. During days 84–97, when the COD was increased to 60 mg/L, the effluent quality of the two-stage anammox system deteriorated rapidly, as evidenced by gradually rising concentrations of NH₄⁺-N and NO₂⁻-N in the effluents. After stabilization, the effluent concentrations of NH₄⁺-N and NO₂⁻-N in R1 reached approximately 28 mg/L and 29 mg/L, respectively. Due to the limited treatment capacity of the R2 reactor under long-term low-load operation, the stabilized effluent NH₄⁺-N and NO₂⁻-N concentrations in R2 also increased to about 11 mg/L and 7 mg/L, respectively. On day 97, the NO₃⁻-N concentrations in the effluents of R1 and R2 further decreased to 2.49 mg/L and 4.26 mg/L, respectively. However, due to the substantial increases in NH₄⁺-N and NO₂⁻-N concentrations, the effluent TN levels rose to 58.63 mg/L and 24.15 mg/L, and the overall NRE dropped to 83.9%. Analysis of nitrogen removal performance under different COD concentrations revealed a trend consistent with previous studies [16,20,21], indicating that low COD concentrations have negligible effects on anammox systems, while high COD concentrations significantly inhibit anammox activity. The COD concentration that provides the most effective stimulation of anammox performance generally lies between these two extremes.

3.1.2. Analysis of pH Variations and Nitrogen Stoichiometric Ratios

The variations in pH within the two-stage anammox reactors under different organic substrate concentrations are shown in Figure 3. The influent pH remained relatively stable in the range of 7.40–7.60 throughout the experiment. With an increasing influent COD concentration, the effluent pH values of both R1 and R2 exhibited an upward trend, which can be attributed to the combined effects of the anammox process performed by AnAOB and the denitrification carried out by heterotrophic bacteria. The average effluent pH of R1 and R2 increased from 7.92 and 8.02 (without organic addition) to 8.28 and 8.38 at a COD of 40 mg/L, respectively. At this concentration, the pH of the R1 effluent still fell within the optimal pH range for AnAOB growth (6.70–8.30); however, after undergoing further anammox reaction and alkalinity production in R2, the effluent pH exceeded 8.30. The combined impact of pH and nitrite stress exerted a synergistically inhibitory effect on anammox activity, accompanied with complete inhibition, which occurred under both acidic (pH 6.7) and alkaline (pH 8.5) conditions, and restoration, which occurred upon readjustment to the optimal pH range of 7.5 ± 0.2 [22]. It has been reported that the general enrichment range of AnAOB is between pH 6.50–8.50 [23], with optimal growth occurring at pH 6.50–9.00 [24]. Therefore, the optimal pH range may vary across different anammox systems. In the two-stage anammox system used in this study, the relatively low influent substrate concentration may allow pH to influence the concentrations of free ammonia (FA) and free nitrous acid (FNA). A moderately elevated pH helps regulate FA and FNA levels within a range suitable for AnAOB growth, suggesting that AnAOB in this system may possess a higher pH tolerance. This explains why the anammox performance in R1 was enhanced at 40 mg/L COD and why the highest NRE was observed after two-stage treatment. However, when the COD concentration was further increased to 60 mg/L, the pH in both reactors rose sharply, with the average effluent pH values of R1 and R2 reaching 8.69 and 8.84, respectively, which far exceeded the optimal range for AnAOB growth and consequently resulted in the significant inhibition of the anammox process.

The changes in stoichiometric ratios in the two-stage anammox reactors under different organic substrate concentrations are shown in Figure 4. In R1, the ΔNO₂⁻-N/ΔNH₄⁺-N ratio under COD concentrations of 10, 20, and 40 mg/L was close to the theoretical value of 1.32, indicating that the anammox process was not significantly affected within this COD range. However, when the COD concentration was increased to 60 mg/L, the ΔNO₂⁻-N/ΔNH₄⁺-N ratio gradually increased from 1.37 to 1.57. This could be attributed to the excessive organic substrate promoting the proliferation of denitrifying bacteria in R1, which competed with AnAOB for NO₂⁻-N, resulting in greater NO₂⁻-N consumption than theoretically expected. In addition, under the condition without COD addition, the average ΔNO₃⁻-N/ΔNH₄⁺-N ratio in R1 was 0.45, significantly higher than the theoretical value of 0.26. This may have been caused by relatively high DO levels in the influent, leading to elevated NO₃⁻-N concentrations in the effluent. As the COD concentration increased, the average ΔNO₃⁻-N/ΔNH₄⁺-N ratio in R1 gradually decreased, reaching 0.09 at a COD of 60 mg/L, likely due to enhanced heterotrophic denitrification that consumed NO₃⁻-N as an electron acceptor in the presence of organic carbon. In R2, the ΔNO₂⁻-N/ΔNH₄⁺-N ratio remained relatively stable at approximately 1.26, slightly below the theoretical value, and was not significantly influenced by the increase in influent COD, indicating the good stability and shock resistance of the R2 reactor. Furthermore, the ΔNO₃⁻-N/ΔNH₄⁺-N ratio in R2 under 10 mg/L COD and under conditions without COD addition was similar and fluctuated around the theoretical value of 0.26. However, as the COD concentration increased, the average ΔNO₃⁻-N/ΔNH₄⁺-N ratio in R2 decreased to 0.10 at 60 mg/L COD. This suggests that while 10 mg/L COD had no significant impact on R2, the residual COD from R1 at higher concentrations entered R2 and stimulated the growth of denitrifying bacteria, thereby enhancing denitrification and resulting in lower NO₃⁻-N production compared to theoretical expectations.

3.2. Effects of Fe²⁺ Coupled with Organic Substrates on the Two-Stage Anammox System

3.2.1. Effect of Fe²⁺ on Nitrogen Removal

The performance of the two-stage anammox system under different Fe²⁺ concentrations is illustrated in Figure 5. During the initial phase without Fe²⁺ addition (days 1–14), the average effluent TN concentrations of R1 and R2 were 71.89 mg/L and 49.30 mg/L, respectively, resulting in an overall NRE of 67.13% after two-stage treatment. Upon the introduction of Fe²⁺ at varying concentrations (days 15–84), a notable decrease in nitrogen concentrations was observed in both reactors compared to the control phase. As shown in Figure 5b, when 5 mg/L Fe²⁺ was initially introduced on day 15, a temporary increase in the nitrogen concentrations occurred, with TN in the R1 effluent rising sharply to 82.75 mg/L, likely due to microbial adaptation. However, the NRE gradually improved over time. By the end of the 5 mg/L Fe²⁺ phase (day 41), TN concentrations in the effluents of R1 and R2 had decreased to 46.52 mg/L and 32.48 mg/L, respectively, and the NRE increased to 78.35%. When the Fe²⁺ concentration was increased to 10 mg/L (days 42–62), the nitrogen concentrations in both R1 and R2 were further reduced compared to the 5 mg/L phase. On day 62, the TN concentrations in the effluents of R1 and R2 were 43.77 mg/L and 28.24 mg/L, respectively, with the overall NRE reaching 81.17%, demonstrating the improved nitrogen removal performance of the two-stage anammox system. As shown in Figure 5a, although the effluent nitrogen concentrations from R1 exhibited considerable fluctuations immediately after Fe²⁺ addition at initial concentrations of 5 and 10 mg/L, the nitrogen species in the effluent of R2 remained relatively stable. With continued reactor operation, the effluent nitrogen concentrations from both R1 and R2 progressively declined. Following the two-stage anammox process, NH₄⁺-N and NO₂⁻-N in the R2 effluent were consistently reduced to below 5 mg/L. Moreover, the NO₃⁻-N concentration in the R2 effluent decreased with increasing Fe²⁺ concentration, with the average value dropping from 26.71 mg/L at 0 mg/L Fe²⁺ to 24.29 mg/L at 10 mg/L Fe²⁺. This contributed to a lower TN concentration and a higher NRE under the 10 mg/L Fe²⁺ condition compared to that at 5 mg/L. These results suggest that, after the partial consumption of Fe²⁺ in R1, the residual Fe²⁺ entering R2 could further enhance AnAOB activity and promote nitrogen removal performance. This also indicates that the two-stage biofilm-based anammox system operated in series exhibits a certain degree of resistance to Fe²⁺ perturbation.

3.2.2. Effect of Fe²⁺ Coupled with Organic Substrates on Nitrogen Removal

Figure 6a presents the variations in the effluent concentrations of NH₄⁺-N, NO₂⁻-N, and NO₃⁻-N in R1 and R2 across different operational stages. When the influent transitioned from a condition without Fe²⁺ and COD addition to one supplemented with 10 mg/L Fe²⁺, a noticeable decrease in the average effluent concentrations of the three nitrogen species in R1 was observed, confirming that an appropriate concentration of Fe²⁺ could enhance AnAOB activity. Additionally, the presence of Fe²⁺ likely facilitated nitrogen-dependent ferrous oxidation (NDFO), thereby improving overall NRE. After day 13, with Fe²⁺ maintained at 10 mg/L and COD concentrations gradually being increased, the combination of 10 mg/L Fe²⁺ and 10 mg/L COD had no significant effect on NH₄⁺-N and NO₂⁻-N concentrations in the effluents of R1 and R2. During days 40–54, when COD was elevated to 40 mg/L, a further reduction in all three nitrogen species was observed in both reactors. The average effluent concentrations of NH₄⁺-N, NO₂⁻-N, and NO₃⁻-N after R2 treatment were 1.53 mg/L, 0.69 mg/L, and 9.30 mg/L, respectively, suggesting that this condition enhanced the anammox reaction rate and promoted the coexistence of AnAOB with denitrifying bacteria. As the COD was further increased to 60 and 80 mg/L, effluent NH₄⁺-N and NO₂⁻-N concentrations in R1 began to rise, indicating a degree of inhibition of the anammox process due to the organic carbon shock; however, following R2 treatment, NH₄⁺-N and NO₂⁻-N concentrations remained below 5 mg/L. When COD was further increased to 100 mg/L, a sharp increase in NH₄⁺-N and NO₂⁻-N was observed in R1 on day 83, reaching 21.94 mg/L and 36.57 mg/L, respectively. Over time, NH₄⁺-N concentrations continued to rise, while NO₂⁻-N gradually declined, eventually stabilizing at approximately 35 mg/L and 23 mg/L. A similar trend was observed in R2, where final effluent NH₄⁺-N and NO₂⁻-N concentrations stabilized around 20 mg/L and 4 mg/L, respectively. In addition, with increasing COD concentrations, the effluent NO₃⁻-N concentrations in both reactors decreased significantly. Specifically, the average NO₃⁻-N concentration in R2 decreased from 23.37 mg/L under 10 mg/L Fe²⁺ alone to 0.57 mg/L when 100 mg/L COD was coupled with Fe²⁺ addition, indicating that the enhancement of heterotrophic denitrification was promoted under higher COD levels.

Figure 6b illustrates the variations in effluent TN concentrations, NRE, and NRRs in R1 and R2 across different operational stages. It is evident that the nitrogen removal performance of the two-stage anammox system varied under 10 mg/L Fe²⁺ coupled with different COD concentrations. Under the condition of Fe²⁺ combined with 40 mg/L COD, the TN concentrations in the effluents of R1 and R2 decreased significantly compared to previous stages, reaching 25.39 mg/L and 10.33 mg/L, respectively, on day 54. The corresponding NRRs were 0.51 kg N·m⁻³·d⁻¹ and 0.06 kg N·m⁻³·d⁻¹, and the overall NRE after two-stage treatment reached 93.11%, indicating excellent nitrogen removal performance. Under the conditions of Fe²⁺ coupled with 60 and 80 mg/L COD, although TN in R1 gradually increased (e.g., reaching 35.04 mg/L on day 82), R2 effluent TN continued to decline, with the lowest value at 7.72 mg/L. This phenomenon could be attributed to two factors: on one hand, the TN level in R1 was not excessively high, and the TN loading to R2 remained within its treatment capacity. The downstream reactor compensated the upstream inhibition and improved the overall nitrogen removal efficiency in this two-stage biofilm system. On the other hand, the residual low-concentration COD from the R1 effluent might not have caused inhibition in R2, and even improved the total nitrogen removal of the two-stage anammox system through denitrification. Meanwhile, the residual Fe²⁺ also might have further promoted the activity of AnAOB in R2. As a result, NRRs in R2 increased during these two stages, and the average NRE after two-stage treatment were 94.11% and 93.72%, respectively, with final effluent TN concentrations meeting the Chinese Class A discharge standard. However, when Fe²⁺ was coupled with 100 mg/L COD, the anammox process was notably inhibited, with TN concentrations in the effluents of R1 and R2 rising to 58.94 mg/L and 28.05 mg/L, respectively, on day 96. The corresponding NRRs were 0.38 kg N·m⁻³·d⁻¹ and 0.13 kg N·m⁻³·d⁻¹, and the overall NRE declined to 82.63%. Combined with Figure 6a, it is evident that NH₄⁺-N was the dominant component in TN at this stage. The observed increase in R2’s NRR with rising COD could be attributed to enhanced heterotrophic denitrification driven by the higher COD concentration in the R2 influent, resulting in lower effluent NO₂⁻-N and NO₃⁻-N concentrations.

3.2.3. Analysis of pH Dynamics and Nitrogen Stoichiometry

The pH variations in the two-stage anammox reactors under Fe²⁺ coupled with different concentrations of organic substrates are illustrated in Figure 7. Throughout the experimental period, the influent pH remained consistently stable at approximately 7.50. Under the condition of 10 mg/L Fe²⁺ addition, a gradual increase in effluent pH was observed as the influent COD concentration increased. When Fe²⁺ was coupled with 80 mg/L COD, the average effluent pH values in R1 and R2 reached 8.21 and 8.32, respectively. Compared to the condition involving the addition of organic substrates alone (as discussed in Section 3.1.2), the pH increase in both reactors occurred at a noticeably slower rate. This difference was likely attributable to the presence of Fe²⁺, which may have facilitated acid-producing reactions such as nitrogen-dependent ferrous oxidation (NDFO), thereby buffering the pH rise induced by increasing COD levels and enhancing the reactors’ tolerance to organic carbon loading. During days 82–96, when the system operated under 10 mg/L Fe²⁺ coupled with 100 mg/L COD, a sharp rise in effluent pH was observed, with the pH values in both R1 and R2 exceeding the optimal range for AnAOB activity.

The changes in nitrogen stoichiometric ratios in the two-stage anammox reactors under Fe²⁺ coupled with varying concentrations of organic substrates are shown in Figure 8. During days 1–82, the ΔNO₂⁻-N/ΔNH₄⁺-N ratio in R1 remained around 1.32, while the ratio in R2 was slightly below 1.32, indicating that Fe²⁺ coupled with COD concentrations up to 80 mg/L had no significant inhibitory effect on the anammox process in either reactor. However, when Fe²⁺ was combined with 100 mg/L COD, both R1 and R2 exhibited substantial fluctuations in ΔNO₂⁻-N/ΔNH₄⁺-N ratios, which eventually stabilized at approximately 2.1 and 1.4, respectively—both notably higher than the theoretical value of 1.32. This deviation suggests that the high influent organic loading led to varying degrees of anammox inhibition in both reactors, with system performance rapidly deteriorating. As a result, excessive NO₂⁻-N was consumed via the combined effects of Fe²⁺ and heterotrophic denitrifying bacteria. In addition, a continuous decline in ΔNO₃⁻-N/ΔNH₄⁺-N ratios was observed in both R1 and R2 with increasing COD concentrations, eventually approaching zero. This trend was consistent with the patterns observed under varying COD concentrations without Fe²⁺ addition.

3.3. Effects of Fe²⁺ Coupled with Organic Substrates on Anammox Biofilm Morphology

Figure 9 shows the SEM images of anammox biofilm samples taken from R1 and R2 on day 96 after treatment under the condition of Fe²⁺ coupled with an organic substrate (sodium acetate). Figure 9a and Figure 9b present SEM images of R1 biofilm at magnifications of 2k and 20k, respectively. The surface of the sludge appears irregular and relatively coarse; a magnified view of a representative region reveals a densely packed anammox microbial structure with a large number of spherical granular attachments. Figure 9c,d depict the SEM images of different regions of the R2 biofilm at 2k magnification. Similarly to R1, the R2 biofilm surface exhibits abundant protruding particles and porous features. This type of morphology may contribute to the enhanced tolerance of anammox bacteria to environmental fluctuations and their improved resistance to operational shocks. Moreover, the presence of pores is considered critical for sustaining microbial metabolism within the biofilm. Overall, the SEM analysis suggests that the structural morphology of the biofilm tends to stabilize under the combined influence of Fe²⁺ and sodium acetate, indicating that this coupling strategy supports the formation of a robust anammox biofilm structure.

3.4. EPS Variation

During the third experimental stage where 10 mg/L Fe²⁺ was coupled with 0, 40, and 100 mg/L COD, the contents of PN and PS in the anammox biofilm from R1 and R2 were analyzed on days 26, 54, and 96, respectively. The results are presented in Figure 10, which shows the variations in PN and PS concentrations in the sludge under different COD conditions with constant Fe²⁺ input. As observed, under the condition of 10 mg/L Fe²⁺ coupled with 40 mg/L COD, both reactors exhibited notable increases in PN, PS, and total EPS contents compared to the control group (Fe²⁺ only). In R1, the PN, PS, and EPS concentrations reached 238.17 mg/g, 48.76 mg/g, and 286.93 mg/g, representing increases of 41%, 26%, and 38.17%, respectively. In R2, the corresponding values were 186.94 mg/g, 43.15 mg/g, and 230.09 mg/g, with increases of 30%, 23%, and 28.25%, respectively. These findings suggest that the presence of organic carbon significantly stimulated microbial EPS secretion, and that the co-addition of Fe²⁺ and a moderate COD concentration could enhance anammox activity while promoting EPS production. Increasing COD concentrations initially led to the elevated production of EPS, while excessively high COD levels resulted in a decline in EPS content [25]. However, under the condition of 10 mg/L Fe²⁺ coupled with 100 mg/L COD, PN and PS concentrations in both R1 and R2 decreased markedly. This may be due to the inhibitory effect of excessive organic carbon on anammox activity, which leads to a reduction in EPS and its components, potentially resulting in system destabilization or even collapse [26]. Furthermore, under constant Fe²⁺ dosing, the PN/PS ratio in both reactors showed a rise-and-fall trend as COD increased from 0 to 40 and then to 100 mg/L. In R1, the variation in PN/PS was more pronounced, whereas in R2, the decline was less significant, indicating that R2 was less affected by organic shock and maintained a more stable nitrogen removal performance.

3.5. Variation in Cytochrome C Content

Figure 11 presents the variation in cytochrome c content in the two-stage anammox reactors under the condition of 10 mg/L Fe²⁺ coupled with 0, 40, and 100 mg/L COD. As shown in the figure, when 10 mg/L Fe²⁺ was coupled with 40 mg/L COD, the cytochrome c content in the sludge of R1 and R2 reached 13.93 ± 0.82 μmol/g and 5.22 ± 0.32 μmol/g, respectively, representing increases of 51.25% and 66.24% compared to the condition with Fe²⁺ addition alone. In contrast, under the condition of 10 mg/L Fe²⁺ coupled with 100 mg/L COD, the cytochrome c content in R1 sharply declined to 5.87 ± 0.57 μmol/g, whereas R2 exhibited a relatively smaller decrease. These results indicate that the co-addition of Fe²⁺ and a moderate concentration of COD significantly enhanced the cytochrome c content in anammox biofilm, likely promoting AnAOB activity. However, excessive COD loading exerted an inhibitory effect on AnAOB, leading to a substantial reduction in cytochrome c levels. Currently, limited studies have focused on the impact of organic substrates or Fe²⁺-coupled organics on cytochrome c production in anammox systems. Previous research has suggested that certain AnAOB are capable of utilizing organic compounds as auxiliary energy sources [27]. Thus, the moderate presence of organics may enhance the activity of key enzymes and facilitate AnAOB metabolism, potentially stimulating cytochrome c biosynthesis. It is hypothesized that organic substrates might serve as nutrients that are directly assimilated by AnAOB to promote cytochrome c formation. However, under high COD concentrations, the increased activity of heterotrophic denitrifiers may have outcompeted AnAOB, resulting in suppressed anammox activity and decreased cytochrome c synthesis.

4. Conclusions

(1)

Low concentrations of sodium acetate (10–20 mg/L COD) had no inhibitory effect on nitrogen removal, while 40 mg/L COD significantly enhanced anammox performance, achieving an average NRE of 90.02%. In contrast, 60 mg/L COD led to the significant inhibition of the anammox process.

(2)

The optimal influent Fe²⁺ concentration was determined to be 10 mg/L for the enhancement of the nitrogen removal performance in a two-stage anammox system. Under this Fe²⁺ addition, the system showed enhanced NRE with increasing COD up to 40 mg/L. Under the condition of 10 mg/L Fe²⁺ coupled with 60 mg/L COD, the two-stage anammox system achieved the highest NRE, with an average NRE of 94.11%. When COD was further increased to 100 mg/L, denitrification was intensified while anammox activity in R1 was severely inhibited, resulting in a decline in the overall nitrogen removal performance.

(3)

At a COD concentration of 40 mg/L, both EPS and cytochrome c contents in anammox biofilm were significantly elevated compared to the condition with 10 mg/L Fe²⁺ alone. However, higher COD concentrations resulted in a decline in these indicators. SEM analysis further revealed that under the influence of Fe²⁺ coupled with organic substrates, the sludge surface exhibited abundant granular protrusions and porous structures, which contributed to the morphological stability of the biofilm. These findings provide insight into the regulatory role of Fe²⁺ and organic substrates on anammox technology and offer a theoretical basis for its application in low-strength nitrogen wastewater treatment.

(4)

This study demonstrated that an Fe²⁺-coupled organic substrate strategy could significantly enhance anammox activity and nitrogen removal efficiency under low-strength nitrogen conditions, with optimal performance observed at moderate COD levels. However, the observed inhibitory effects at higher COD concentrations underscored a key limitation of this approach when applied to real wastewater, which often exhibits fluctuating and complex compositions. In real-world treatment settings, the variability in COD levels and the presence of diverse organic compounds may reduce system stability and efficiency. Therefore, while the proposed strategy shows promise for enhancing anammox-based nitrogen removal, it is essential to further investigate its robustness across a broader range of wastewater characteristics, including varying COD/N ratios, organic compositions, and potential inhibitory substances. Future research should focus on optimizing operational parameters and pretreatment strategies to mitigate the effects of excessive or inhibitory organics, thereby ensuring reliable application in full-scale wastewater treatment systems.