Effects of Elevated Fe (III) on Anaerobic Ammonia Oxidation Biofilm Process: Inhibition and Recovery

Abstract

To investigate the treatment performance of employing the anaerobic ammonia oxidation (anammox) biofilm process to treat the iron-containing industrial wastewater with a low carbon–nitrogen ratio and obtain an optimal condition, the effects of elevated exposure to Fe (III) (ranging from 0 to 1.5 mM) on the anammox biofilm process were explored. The findings indicated that the performance of anammox nitrogen removal remained unaffected when exposed to low levels of Fe (III) (0.1 mM and 0.3 mM). However, high concentrations (higher than 0.5 mM) showed a negative effect. The semi-inhibitory concentration (IC₅₀) of Fe (III) was 1.32 mM. Additionally, under exposure to high levels of Fe (III), a remarkable accumulation of Fe (III) was observed within the anammox system. The total iron concentration increased from 30 mgFe·gVSS⁻¹ to a saturation point of approximately 300 mgFe·gVSS⁻¹. This accumulation led to a decrease in extracellular polymeric substance (EPS) from 221.4 mg·gVSS⁻¹ to 91.3 mg·gVSS⁻¹ and specific anammox activity (SAA) from 0.0482 gN·(gVSS·d)⁻¹ to 0.018 gN·(gVSS·d)⁻¹. Consequently, the activity of anammox bacteria (AnAOB) was inhibited, leading to a decline in the average total nitrogen removal efficiency (TNRE) from 86.9% to 38.3%. However, it was discovered that the inhibitory effect of continuous Fe (III) could be reversed by introducing the ferric ion complex EDTA·2Na into the system. As a result, the TNRE recovered to 60%. The findings would be useful to optimize the anammox biofilm process by adjusting the concentration of Fe (III) in the practical industrial application.

1. Introduction

Industrial wastewater refers to the contaminated water generated during industrial processes, such as manufacturing, mining, power generation, chemical production, and others. Certain industrial wastewater usually contains heavy metals, including Cd, Cr, Hg, Pb, Zn, Fe, Cu, Mn, etc. A useful method to recover heavy metals from wastewater is flotation [1,2]. However, with the increasing popularity of the biological biofilm process in contemporary wastewater purification, it is important to note that different heavy metals exhibit varying impacts on the metabolism of microorganisms. Steel processing wastewater is a typical industrial wastewater that is characterized by high production and poor biodegradability. The primary pollutants in this kind of wastewater include ammonia nitrogen (${\mathrm{N}\mathrm{H}}_4^{+}$-N), suspended solids, salts, and a significant number of iron ions [3]. Iron ions are toxic to some extent and can accumulate in the environment, leading to long-term ecological and health problems. In addition, they are detrimental to conventional biological treatment. The efficient and cost-effective denitrification of iron-containing wastewater is a pressing issue that needs to be addressed. A promising solution is the anaerobic ammonia oxidation (anammox) biofilm process, which is an innovative biological method of removing nitrogen [4]. This process shows great advantages, such as a short process flow, low energy consumption, minimal sludge production, no additional carbon sources or alkalinity requirements, and no greenhouse gas emissions [5,6,7]. Several scholars have effectively employed or investigated its potential for treating landfill leachate [8,9,10], pharmaceutical wastewater [11], sludge digester liquids [12,13], and aquaculture wastewater [14], which feature high ammonia and nitrogen concentrations. However, there is limited research investigating the potential feasibility of applying an anammox biofilm process to treat iron-containing industrial wastewater with a low carbon–nitrogen ratio at room temperature. Given the potential disruption to the steady operation of the anammox biofilm process caused by elevated levels of Fe (III) within iron-containing industrial wastewater, this study focuses on examining the effectiveness of an anammox biofilm process mediated by continuous Fe (III).

Anammox bacteria (AnAOB) are susceptible to the long-term effects of heavy metal ions. Previous reviews have revealed that AnAOB performance varies with different concentrations of metal ions [15]. Indeed, metal ions play a crucial role as micronutrients for microorganisms, ensuring their survival at low levels and acting as toxic agents at elevated concentrations [16]. For instance, trace Zn (II), Fe (II), Cu (II), Ni (II), and Mn (II) play crucial roles in microbial proteins and enzyme synthesis, electron transfer, and other essential processes, stimulating the activity of AnAOB, but high concentrations of those metals often showed damage to microorganisms [17]. Moreover, the toxicity of metal ions is not solely determined by the type of elements but also by the valence state of the metals. Chen et al. compared the impacts of Fe (III) and Fe (II) on the anammox. Their findings indicated that the continuous imposition of Fe (III) at a concentration of 30 mg·L⁻¹ exhibited a detrimental impact on the constitution of extracellular polymeric substance (EPS), which rendered AnAOB vulnerable to environmental stress. Additionally, the presence of Fe (II) at a content of 30 mg·L⁻¹ affected substrate utilization [18].

Iron is a necessary cofactor in biological activities, and most iron-related redox reactions in bacteria include Fe-S proteins and hemoglobin [19]. The reaction of iron to anammox is significant as well. Researchers have found that anammoxosome as the leading reaction site of AnAOB contains a large amount of iron protoporphyrin IX (heme) [20,21]. The heme is used by many proteins to execute a variety of tasks, including electron transfer, redox catalysis, and gas transport [22,23]. Fe (II) is required to synthesize the heme, which is involved in anammox metabolism as a c-type cytochrome [24]. Approximately 10⁷ iron-containing protein atoms were estimated in each anammox cell [19]. That is why AnAOB has a high demand for iron. To date, some researchers have examined the impact of iron ions on anammox. But most of these studies focused on the low concentration (0~0.4 mM) of Fe (II), and the minority focused on Fe (III) at the concentration range of 2.24–250 mg·L⁻¹ with various fluidized bed bioreactors [18,25,26,27]. There are almost no published studies on high levels of Fe (III) using a fixed bed bioreactor. The conclusions have been quite similar, with low concentrations stimulating anammox and high concentrations suppressing anammox [28]. For instance, Feng et al. found the maximum rate of substrate conversion rose from 2.97 kg·(m³·d)⁻¹ to 3.47 kg·(m³·d)⁻¹ when the concentration of Fe (II) exceeded 25 mg·L⁻¹ [29]. Wang et al. revealed that Fe (III) (0–0.14 mM) could remarkably improve a reactor’s nitrogen loading rate (NLR) and operation efficiency, and the TNRR was 1.54~1.94 times the control assay [27]. According to an investigation carried out by Zhang et al., prolonged exposure to a low level of Fe (III) (5–10 mg·L⁻¹) demonstrated a notable enhancement in the NRR of the anammox process. Conversely, a higher level of Fe (III) (50–100 mg·L⁻¹) aggravated the NRR of anammox by 12~27% [30]. Ahmed et al. observed that the presence of Fe (III) had a significant impact on the microbial population and functional capabilities. They also discovered a collaborative association between iron-reducing bacteria and nitrogen-fixing bacteria in a unique anaerobic biological treatment method for wastewater [31,32,33]. High Fe (III) doses were commonly detected in industrial wastewater. The iron content of actual industrial wastewater was detected regularly at 40–60 mg·L⁻¹ (0.7–1.1 mM) in our study. Studying the inhibition mechanism of high Fe (III) contents on the anammox biofilm process is significant for effectively treating industrial wastewater. However, there is still limited information available on the inhibition and recovery of the anammox biofilm process caused by high concentrations of Fe (III). Furthermore, the precise mechanism by which Fe (III) impacts the anammox biofilm process remains elusive. This research verified that high concentrations (higher than 0.5 mM) of Fe (III) showed a negative effect on the anammox reaction and the structure of the anammox biofilm by detecting the biological activity, EPS analysis, and kinetic analysis, except for a novel point regarding the total iron migration. In addition, the anammox bacteria exhibited a strong ability to resist the continuous loading of Fe (III) and could recover from severe inhibition. These discoveries would be beneficial for optimizing the anammox biofilm process by adjusting the concentration of Fe (III) in a practical industrial application and would contribute to the future study of the potential mechanism of anammox involving the mediation by a high concentration of Fe (III).

The aims of this investigation are the following: (1) study the impacts of a high amount of Fe (III) (ranging from 0 to 1.5 mM) on the anammox, (2) explore whether the inhibition caused by high concentrations of Fe (III) on anammox is reversible and analyze the mechanism of inhibition and recovery by measuring SAA, EPS, and total iron concentration, and (3) characterize the toxicity of Fe (III) on the anammox biofilm process by determining semi-inhibitory concentration (IC₅₀) and kinetics of the substrate degradation rate of anammox in a typical cycle.

2. Materials and Methods

2.1. Experimental Setup

This study employed a 2 L sequencing batch biofilm reactor (SBBR) at the laboratory scale, as indicated in Figure 1. It consisted of double-layered plexiglass (Shanghai Lei jia Scientific Instrument Co., LTD, Shanghai, China) and possessed an inner diameter of 80 mm and a height of 240 mm, sealed by a flange fixture and rubber gasket to maintain an anaerobic environment. Polyurethane foam plastic carriers modified by low-temperature plasma technology were applied to trap microorganisms. These carriers had a filling ratio of 40% and exhibited an approximate specific surface area of 4000 m²/m³. The SBBR can effectively prevent the loss of microorganisms, trap sufficient biomass, and promote the rapid start-up and long-term stable operation of the anammox process. The porous structure and well biocompatibility of Polyurethane foam plastic carriers are conducive to microbial attachment and growth.

2.2. Synthetic Wastewater

The formulation of the synthetic wastewater utilized in the SBBR experiment was acquired from previous investigations [4,34,35], as shown in

Table 1. Synthetic wastewater composition.

Compound	Concentration (mg·L−1)
(NH4)2SO4 and NaNO2	As required
KH2PO4	30
MgSO4·7H2O	300
NaHCO3	500
CaCl2·2H2O	150
Trace elements solution Ⅰ	1
Trace elements solution Ⅱ	1

. Trace elements solution Ⅰ contains EDTA·2Na 6.39 g·L⁻¹ and FeSO₄·7H₂O₅ g·L⁻¹. Trace elements solution Ⅱ contains EDTA·2Na 19.11 g·L⁻¹, H₃BO₃ 0.014 g·L⁻¹, ZnSO₄·7H₂O 0.43 g·L⁻¹, CoCl₂·6H₂O 0.24 g·L⁻¹, MnCl₂·4H₂O 0.99 g·L⁻¹, CuSO₄·5H₂O 0.25 g·L⁻¹, NiCl₂·6H₂O 0.19 g·L⁻¹, and NaMoO₄·2H₂O 0.22 g·L⁻¹.

2.3. Analytical Methods

2.3.1. Sampling and Analytical Methods

Samples of water were collected on a regular basis to measure the concentration of nitrogen and the overall iron content. As presented in

Table 2. Analysis items and detection methods.

Analysis Items	Detection Methods	Instrument
${\mathrm{N}\mathrm{H}}_4^{+}$-N	Sodium reagent spectrophotometry	UV2000 (SHIMADZU, Kyoto, Japan)
${\mathrm{N}\mathrm{O}}_2^{-}$-N	N-(1-naphthyl)-ethylenediamine spectrophotometry	UV2000 (SHIMADZU, Kyoto, Japan)
${\mathrm{N}\mathrm{O}}_3^{-}$-N	Ultraviolent spectroscopy	UV2000 (SHIMADZU, Kyoto, Japan)
pH	Glass electrode method	6309PDT, JENCO (Shanghai, China)
DO	Membrane electrode method	HQ30d, HACH (Loveland, CO, USA)
MLSS, MLVSS	Gravimetry	Analytical balance (SHIMADZU, Shanghai, China)
Total iron	o-Phenanthroline spectrophotometry	UV2000 (SHIMADZU, Kyoto, Japan)

, the levels of ${\mathrm{N}\mathrm{H}}_4^{+}$-N, ${\mathrm{N}\mathrm{O}}_2^{-}$-N, and ${\mathrm{N}\mathrm{O}}_3^{-}$-N were determined according to the guidelines stated in the Standard Methods [36]. The concentration of total nitrogen was determined by summing the contents of ${\mathrm{N}\mathrm{H}}_4^{+}$-N, ${\mathrm{N}\mathrm{O}}_2^{-}$-N, and ${\mathrm{N}\mathrm{O}}_3^{-}$-N. The pH level was measured using a portable pH meter (JENCO Model 6010, Shanghai, China), while the level of dissolved oxygen (DO) was detected using a portable DO meter (HQ 30d, HACH, Loveland, CO, USA). MLSS and MLVSS were measured by Gravimetry. The total iron was measured by o-Phenanthroline spectrophotometry.

2.3.2. Substrate Degradation Rate of Anammox Analysis

During the detection of the substrate degradation rate of the anammox (ASDR) test, liquid samples were collected at intervals of two hours at the last cycle of each phase to determine the concentration of inorganic nitrogen and further calculate TN removal load [34]. Then, drawing the TN removal load change curve along with time, substrate degradation rate parameter K was obtained through linear fitting [34]. The effect of various concentrations of Fe (III) on the substrate degradation rate of anammox was characterized by comparing the K value.

2.3.3. Specific Anammox Activity Analysis

At the last cycle of each phase (listed in Section 2.3.5), anammox biomass from the SBBR was collected to detect anammox activity. Each concentration of Fe (III) was set in triplicates; anammox biomass was immersed with a PBS buffer and centrifuged for three minutes at a rotation speed of 3000 r·min⁻¹. To obtain the active anammox sludge, the supernatant was discarded through three consecutive repetitions.

The 250 mL anaerobic serum bottle was inoculated with active anammox sludge at a concentration of 500 mg·L⁻¹, and together with the substrate, it was placed inside an incubator. The incubator maintains a constant temperature of 35 °C while being stirred at a rate of 150 r·min⁻¹. The initial contents of ${\mathrm{N}\mathrm{H}}_4^{+}$-N and ${\mathrm{N}\mathrm{O}}_2^{-}$-N in the substrate were 40 mg·L⁻¹ and 50 mg·L⁻¹, respectively. A total of 99.5% N₂ gas was purged for 10 min to maintain the DO below 0.3 mg·L⁻¹ in the serum bottle, and the pH was adjusted between 7.5 and 7.8. During the anammox activity test, water samples were collected at intervals of two hours to measure SAA [37].

Applying the nitrogen removal rate (NRR) of anammox as the study object, the inhibitory effect of elevated Fe (III) on anammox was quantitatively represented, and toxicity was evaluated by calculating the value of IC₅₀. Based on the nitrogen concentration, a modified calculation method of SAA was referred to [38] in Equation (1).

$$SAA=(C_0-C_t)\times V/\left(m\times t\right)$$

(1)

where SAA represents the specific anammox activity, mgTN·(gVSS·h)⁻¹. C₀ is the influent substrate concentration, mg·L⁻¹. C_t is the effluent substrate concentration at t hour, mg·L⁻¹. m is sludge concentration, gVSS·L⁻¹. t is reaction time (one cycle), h. V is effective volume, L.

The IC₅₀ represents the concentration of the compound being tested, which corresponds to 50% of the observed in the control assay. IC₅₀ can be calculated using Equation (2) [39].

$$IC=\left(1-SA{A}_f/SA{A}_c\right)\times 100\%$$

(2)

where IC represents the inhibition ratio, SAA_f represents the concentration of TN under a stressed condition with Fe (III), and SAA_c represents the concentration of TN in the control assay. When SAA_f corresponds to 50% compared with SAA_c, the concentration of Fe (III) was now IC₅₀.

2.3.4. Extraction and Analysis of EPS

To assess EPS in the anammox sludge gathered at the conclusion of every phase, we followed EPS extraction methods described in previous studies [40,41]. The identification of polysaccharides (PS) was achieved using the phenol-sulfuric acid technique [42], where anhydrous glucose was utilized as the reference standard. Dissolved protein (PN) was obtained through the Lowry method [43] using bovine serum protein as the standard. The total EPS content was calculated by adding the detected PS and PN.

2.3.5. Continuous Impact of Fe (III) on Anammox

• Reactor start-up

The SBBR was started up with 4000 mg·L⁻¹ of a seed AnAOB enrichment solution in the dark with HRT of 24 h and mixed by a magnetic stirrer at 80 rpm. The temperature was maintained at 35 °C during start-up, controlled by the water bath heater. To ensure that the pH level of the SBBR remained within the range of 6.5 to 7.5, the addition of 1 M HCI and 1 M NaOH was employed. To maintain the anaerobic environment at the reactor, high-purity nitrogen was exposed to influent for 15 min to remove DO (<0.3 mg·L⁻¹) after each drainage cycle. The NLR of the influent was 0.22 ± 0.01 kgN·d⁻¹, including ${\mathrm{N}\mathrm{H}}_4^{+}$-N 50 ± 5 mg·L⁻¹ and ${\mathrm{N}\mathrm{O}}_2^{-}$-N 60 ± 5 mg·L⁻¹. When the total nitrogen removal efficiency (TNRE) stabilized at 81.90%, the SBBR successfully started.

• Reactor operation strategy

When the reactor operated more than 120 d, the TNRE stabilized at 85.40% in ordinary temperature (24~27 °C), and the impact of Fe (III) was investigated on the anammox biofilm process. The reactor operation conditions referred to the start-up period. Stressed conditions with different concentrations of Fe (III) were set, and they were divided into six phases: 0 mM, 0.1 mM, 0.3 mM, 0.5 mM, 0.9 mM, and 1.5 mM, respectively. Fe (III) was introduced along with the substrate in the form of FeCl₃·6H₂O. Each phase consisted of twenty cycles, with each cycle lasting 12 h. The cycle involved a 15 min filling period followed by an 11 h reaction phase, a 30 min settling period, and finally, 15 min of decanting. At the last cycle of each phase, the determination of SAA, the determination of the anammox substrate degradation rate, and the assessment of EPS were carried out.

• Reactor recovery

After the previous six phases, the SBBR recovered for 30 days without any external addition of Fe (III). Before each cycle of filling, the pH was adjusted to 2.0 in the influent. Additionally, a solution of 100 mg·L⁻¹ EDTA·2Na was introduced into the synthetic wastewater to restore the activity of AnAOB. Finally, the pH of the feeding medium was carefully controlled within the range of 6.5 to 7.5. The HRT of the SBBR was 24 h for the first 11 days and 12 h for the last 19 days.

3. Results and Discussion

3.1. Effect of Fe (III) on the Removal of Nitrogen in the Anammox Biofilm Process

Figure 2a,b and Figure 3 show the nitrogen elimination effect of the SBBR during long-term operation under stressed conditions with various concentrations of Fe (III); the TNRE is illustrated in Figure 4.

During phase Ⅰ, without any Fe (III) added to the reactor, the average TNRE of anammox at ordinary temperature (24–27 °C) was 86.9%, and NLR was 0.22 kgN·d⁻¹ (${\mathrm{N}\mathrm{H}}_4^{+}$-N 50 mg·L⁻¹, ${\mathrm{N}\mathrm{O}}_2^{-}$-N 60 mg·L⁻¹). In the effluent, the average content of ${\mathrm{N}\mathrm{H}}_4^{+}$-N and ${\mathrm{N}\mathrm{O}}_2^{-}$-N was 0.619 mg·L⁻¹ and 1.64 mg·L⁻¹, respectively, and the content of ${\mathrm{N}\mathrm{O}}_3^{-}$-N produced was 11.99 mg·L⁻¹. The calculated value of ${\Delta \mathrm{N}\mathrm{O}}_2^{-}$-N: Δ${\mathrm{N}\mathrm{H}}_4^{+}$-N is 1.10~1.31, and the value of ${\Delta \mathrm{N}\mathrm{O}}_3^{-}$-N: ${\Delta \mathrm{N}\mathrm{H}}_4^{+}$-N is 0.13~0.28, which was in accordance with the theoretical ratio (1.32 and 0.26), indicating that the nitrogen elimination of the system was dominated by anammox.

During phases II and III, the average NRR was approximately 0.193 and 0.189 kgN·(m³·d)⁻¹ with the addition of Fe (III) (0.1 mM and 0.3 mM, respectively). In the effluent, the average contents of ${\mathrm{N}\mathrm{H}}_4^{+}$-N, ${\mathrm{N}\mathrm{O}}_2^{-}$-N, and ${\mathrm{N}\mathrm{O}}_3^{-}$-N were 3.94 mg·L⁻¹, 0.81 mg·L⁻¹, and 12.57 mg·L⁻¹, respectively. The range of ${\mathrm{N}\mathrm{O}}_2^{-}$-N: ${\Delta \mathrm{N}\mathrm{H}}_4^{+}$-N values was between 1.04 and 1.36, while the range of ${\Delta \mathrm{N}\mathrm{O}}_3^{-}$-N: ${\Delta \mathrm{N}\mathrm{H}}_4^{+}$-N values was between 0.17 and 0.31. It could be seen that the nitrogen performance varied slightly from phase Ⅰ. However, as the content of Fe (III) increased to 0.5 mM, the average TNRE of the system began to decrease to 76.3%, and the average NRR was approximately 0.169 kgN·(m³·d)⁻¹. In the effluent, the mean concentrations of ${\mathrm{N}\mathrm{H}}_4^{+}$-N, ${\mathrm{N}\mathrm{O}}_2^{-}$-N, and ${\mathrm{N}\mathrm{O}}_3^{-}$-N were 10.61 mg·L⁻¹, 4.54 mg·L⁻¹, and 11.14 mg·L⁻¹, respectively. The values of ${\Delta \mathrm{N}\mathrm{O}}_2^{-}$-N: ${\Delta \mathrm{N}\mathrm{H}}_4^{+}$-N and ${\Delta \mathrm{N}\mathrm{O}}_3^{-}$-N: ${\Delta \mathrm{N}\mathrm{H}}_4^{+}$-N were 1.39 and 0.27, respectively, and began to have a rising tendency. Meanwhile, the accumulation of nitrogen in the system compared to the previous stage suggested that Fe (III) has started to suppress the anammox reaction of the system. During phases Ⅴ and Ⅵ, the Fe (III) content further rose from 0.9 mM to 1.5 mM, the anammox performance of the system began to show obvious inhibition, the average TNRE decreased from 66.3% to 38.3%, and the mean NRR decreased from 0.148 kgN·(m³·d)⁻¹ to 0.087 kgN·(m³·d)⁻¹. The average content of ${\mathrm{N}\mathrm{H}}_4^{+}$-N and ${\mathrm{N}\mathrm{O}}_2^{-}$-N present in the effluent rose from 15.06 mg·L⁻¹ to 32.01 mg·L⁻¹ and 9.72 mg·L⁻¹ to 28.77 mg·L⁻¹, respectively. Less ${\mathrm{N}\mathrm{O}}_3^{-}$-N was produced, ranging from 12.74 to 8.69 mg·L⁻¹, noting a serious deterioration in the NRE. The increase in ${\Delta \mathrm{N}\mathrm{O}}_2^{-}$-N: ${\Delta \mathrm{N}\mathrm{H}}_4^{+}$-N and ${\Delta \mathrm{N}\mathrm{O}}_3^{-}$-N: ${\Delta \mathrm{N}\mathrm{H}}_4^{+}$-N values went up from 1.49 to 1.79 and 0.36 to 0.47, respectively, surpassing the expected ratio by a significant margin. It demonstrated that the high level of Fe (III) severely suppressed anammox, and nitration gradually dominated the system.

As previously mentioned, the NRE of anammox decreased progressively. Initially, the content of Fe (III) was relatively low (below 0.3 mM), and it did not significantly suppress the anammox. Even at a concentration of 0.1 mM, Fe (III) slightly enhanced the anammox reaction. However, when Fe (III) rose from 0.5 mM to 1.5 mM, there was obvious accumulation of ${\mathrm{N}\mathrm{H}}_4^{+}$-N and ${\mathrm{N}\mathrm{O}}_2^{-}$-N in the effluent, increasing by 68% and 84%, respectively. At Fe (III) dosages of 1.5 mM, the TN average removal rate was reduced to 38.3%. A similar conclusion was observed in other studies. The experiment by [26] showed that by adding 3.68 mg·L⁻¹ Fe (III), the NRR improved compared to the control experiment. Ref. [30] also found that when the Fe (III) doses ranged from 5 mg·L⁻¹ to 10 mg·L⁻¹, the TNRE improved by 0.05–5%, while when the Fe (III) doses ranged from 50 mg·L⁻¹ to 100 mg·L⁻¹, the TNRE decreased by 12–27% [28]. At the moment, the calculated value of ${\Delta \mathrm{N}\mathrm{O}}_2^{-}$-N: ${\Delta \mathrm{N}\mathrm{H}}_4^{+}$-N and ${\Delta \mathrm{N}\mathrm{O}}_3^{-}$-N: ${\Delta \mathrm{N}\mathrm{H}}_4^{+}$-N showed a tendency to deviate highly from the theoretical ratio (Figure 3), noting that anammox reaction in the reactor was severely damaged. Together with the degradation of nitrogen removal performance, the ${\Delta \mathrm{N}\mathrm{O}}_3^{-}$-N: ${\Delta \mathrm{N}\mathrm{H}}_4^{+}$-N reached 0.48. The result conformed with other findings [44]. One explanation for this was the microbial structure shifted and anammox coupled to iron reduction (Feammox) occurred [45].

Due to the continuous effect of a high Fe (III) exposure, the anammox reaction in the system was seriously suppressed. Then, the recovery phase was conducted. The Fe (III) dosage was stopped, and a 100 mg·L⁻¹ EDTA solution was introduced into the influent for seven consecutive days. As Figure 2a,b show, the recuperation of nitrogen performance in the anammox process occurred gradually. The TNRR increased progressively after HRT was controlled for 24 h in the first 11 days of recovery. On the sixth day after recovery, the TN removal rate enhanced to 80%, wherein the effluent exhibited ${\mathrm{N}\mathrm{H}}_4^{+}$-N, ${\mathrm{N}\mathrm{O}}_2^{-}$-N, and ${\mathrm{N}\mathrm{O}}_3^{-}$-N contents of 0 mg·L⁻¹, 1.75 mg·L⁻¹, and 13.08 mg·L⁻¹, respectively. The average values of ${\Delta \mathrm{N}\mathrm{O}}_2^{-}$-N: ${\Delta \mathrm{N}\mathrm{H}}_4^{+}$-N and ${\Delta \mathrm{N}\mathrm{O}}_3^{-}$-N: ${\Delta \mathrm{N}\mathrm{H}}_4^{+}$-N were 1.32 and 0.25, indicating that the anammox reaction in the system completely recovered. So, the activity of AnAOB in the system gradually recovered. After controlling HRT for 12 h on the 19th day, the TN removal rate decreased somewhat compared with phase Ⅰ, but it could be maintained at approximately 60%, and the NRR reached 0.126 kgN·(m³·d)⁻¹.

3.2. Kinetics of Various Fe (III) Concentrations on Periodical Substrate Degradation

To obtain a comprehensive understanding of the effects of varying amounts of Fe (III) on nitrogen removal, the periodical tests were conducted within 12 h. The effect of various dosages of Fe (III) on the SDR of anammox was demonstrated in Figure 5. The outcomes indicated that the degradation of the substrate was hindered to varying degrees as the Fe (III) dosage increased. In the low concentration of Fe (III) (lower than 0.1 mM), the contents of ${\mathrm{N}\mathrm{H}}_4^{+}$-N and ${\mathrm{N}\mathrm{O}}_2^{-}$-N in the effluent were 0 mg·L⁻¹ and 0.341 mg·L⁻¹ at 10th h of a typical cycle. This was lower than the high Fe (III) concentration (higher than 0.1 mM), and the contents of ${\mathrm{N}\mathrm{H}}_4^{+}$-N and ${\mathrm{N}\mathrm{O}}_2^{-}$-N in the effluent were 8.427 mg·L⁻¹, 12.51 mg·L⁻¹, 1.192 mg·L⁻¹, and 5.29 mg·L⁻¹ at dosages of 0.3 mM and 0.5 mM, respectively. The substrate accumulation in a typical cycle illustrated that the activity of AnAOB began to be influenced. Subsequently, when the concentration of Fe (III) rose from 0.9 to 1.5 mM, a large amount of ${\mathrm{N}\mathrm{H}}_4^{+}$-N and ${\mathrm{N}\mathrm{O}}_2^{-}$-N appeared in the reactor, and the contents of ${\mathrm{N}\mathrm{H}}_4^{+}$-N and ${\mathrm{N}\mathrm{O}}_2^{-}$-N in the effluent were 16.9 mg·L⁻¹, 30.549 mg·L⁻¹, 4.45 mg·L⁻¹, and 29.03 mg·L⁻¹, respectively. The TNRR decreased from 60.8% to 30.1%, indicating that lower Fe (III) concentration did not vary significantly with the control group in the substrate degradation under the studied condition. Nevertheless, higher Fe (III) concentration showed an apparent negative influence on substrate degradation. One explanation for this is that anammox activity may be suppressed due to the presence of a high level of Fe (III) [46].

The substrate degradation in a typical cycle followed a model of zero-order reaction kinetics. According to the linearized model between the substrate degradation of anammox changing with time at different dosages of Fe (III) in a cycle, the parameter K, representing the substrate degradation rate, was obtained by linear fitting [34], as is shown in

Table 3. Kinetic parameters of substrate degradation with various Fe (Ⅲ) addition.

Fe(Ⅲ)/mM	K	R2	Model
0	8.7734 ± 0.35	0.9920	C = C0 − K*t
0.1	8.9336 ± 0.49	0.9848
0.3	7.5216 ± 0.28	0.9933
0.5	6.6017 ± 0.32	0.9885
0.9	5.2394 ± 0.09	0.9985
1.5	2.3539 ± 0.12	0.9874

Note: * is a model of zero-order reaction kinetics, where C₀ represents the initial contents of the TN and C represents the content of the TN at t hours.

. It was clear that with increasing Fe (III) concentration, K first increased and then declined. Under a low concentration of Fe (III) (lower than 0.1 mM), K increased from 8.77 to 8.94, yet under a high concentration of Fe (III) (more than 0.1 mM), K decreased from 8.94 to 2.35, indicating that a lower Fe (III) dosage promoted the anammox reaction, while a higher content of Fe (III) inhibited it. The results were consistent with a previous study [30]. The tendency of the parameter K corresponded with the activity of the AnAOB.

3.3. Kinetics of Various Fe (III) Concentrations on Periodical Substrate Degradation

SAA is a crucial indicator of anammox nitrogen removal performance. To investigate the impact of different Fe (III) concentrations on anammox, we performed calculations for SAA, as shown in Figure 6. Compared with the control assay, SAA decreased slightly from 0.0482 gN·(gVSS·d)⁻¹ to 0.0483 gN·(gVSS·d)⁻¹ when Fe (III) dose was 0.1 mM, this responded to nitrogen removal performance mentioned above. Under various relatively high concentration Fe (III) exposure circumstances, the decline of SAA from 0.047 gN·(gVSS·d)⁻¹ to 0.018 gN·(gVSS·d)⁻¹ was observed, showing a reduction of approximately 1.6%, 12%, 23%, and 61.8% with increasing Fe (III) doses from 0.3 mM to 1.5 mM, respectively. Consequently, the presence of a continuous and relatively elevated Fe (III) load dramatically hindered the activity of AnAOB. This led to a decline in the NRR from 0.189 kgN·(m³·d)⁻¹ to 0.087 kgN·(m³·d)⁻¹ and a slowdown in the degradation of the parameter K from 8.93 to 2.35. This result corresponds with previous findings. Chen et al. determined that the enhancement of SAA was observed when low doses of Fe (III) (less than 15 mg·L⁻¹) were continuously added, whereas SAA was suppressed severely when high doses of Fe (III) (30 mg·L⁻¹) were employed [47]. J et al. conducted research on marine anammox and concluded that when Fe (III) concentration was lower than 2 mg·L⁻¹, SAA experienced a slight enhancement (8.27%). However, when the Fe (III) content reached 14 mg·L⁻¹, SAA peaked at 0.57 kg·(kg·d)⁻¹. Subsequently, SAA sharply declined by about 20% when the Fe (III) content exceeded 14 mg·L⁻¹ [25].

Figure 7 presents the measured and kinetic-fitted SAA at various Fe (III) addition levels. It is demonstrated that the maximum SAA was 0.0483 gN·(gVSS·d)⁻¹. The correlation between SAA and Fe (III) was analyzed using a logistic model. The obtained results showed a remarkably high R² value of 0.99417, indicating a strong fit between the variables. The optimal Fe (II) dosing concentration range was found to be 0~0.1 mM under the studied conditions. Additionally, the IC₅₀ value for Fe (III) was determined to be 1.32 mM.

3.4. Biofilm Extracellular Polymer Analysis

EPS is an organic polymer produced by microorganisms to ensure self-protection and promote mutual adhesion [48]. It assumes a vital function in the aggregation of AnAOB [28]. EPS is produced through the mutual polymerization of structurally identical or similar compounds, comprising a wide array of functional clusters, such as carboxyl and hydroxyl groups [49]. This gel-like substance contains a significant amount of water and possesses a negative charge, enabling microbial aggregation and supporting the growth of symbiotic microbial communities [50]. In actual sewage treatment, EPS can also create a protective layer around cells to safeguard them from unfavorable external conditions [51,52]. In terms of composition, EPS comprises a diverse range of organic compounds, primarily polysaccharides (PS) and proteins (PN), occupying approximately 70% to 80% of the total EPS.

In this study, we assessed the production of EPS when exposed to varying concentrations of Fe (III). The findings indicated that with an increase in Fe (III) dosage from 0 mM to 1.5 mM, the number of EPS produced in the system exhibited a decrease from 221.4 mg·gVSS⁻¹ to 91.3 mg·gVSS⁻¹ (depicted in Figure 8). It might be attributed to many of the active sites consisting of PN and PS, as the added Fe (III) exceeds the optimal dosage threshold and could bind positively charged Fe (III) and Fe (OH)₃ colloids in the influent, leading to functional groups of EPS changed in spatial structure and shape. Therefore, as PN and PS gradually decreased, EPS content reduced significantly [53,54]. Moreover, an increased amount of Fe (III) cations was adsorbed by the cell membrane and accumulated in the cell interior under elevated high-concentration Fe (III) exposure conditions. As a result, the structure of EPS became less compact, rendering anammox microorganisms more vulnerable to adverse external conditions, which led to the metabolism of AnAOB slowing down and the activity of AnAOB decreasing markedly [55]. Notably, much red granular anammox sludge appeared at the base of the reactor in this research, which may be related to the significant reduction in EPS and the decrease in PN/PS. Zhang et al. conducted research on how EPS influences the aggregation of microbial cells in both biofilm and suspended sludge. They discovered that EPS supported the clumping together of sludge cells that were suspended in water but had a detrimental effect on the aggregation of biofilm cells [56]. Similarly, Tang et al. provided evidence that Fe (Ⅲ) enhanced the generation of anammox granular sludge [57].

3.5. Characteristics of Total Iron Migration

By detecting total iron contents in different regions of the system, a schematic distribution diagram of total iron proportion under the influence of different Fe (III) continuous loads could be obtained, as shown in Figure 9. In general, the distribution of total iron in the anammox system can be divided into four parts [58]. Firstly, Fe (III) was enriched inside the anammox bacteria cell. Secondly, Fe (III) existed in the solution in the form of Fe (III) ions and the colloidal form of Fe (OH)₃. Thirdly, Fe (III) was adsorbed, which includes adhesion on the anammox bacteria cell membrane and bridging between the bacteria. Finally, there was a loss of total iron due to other reasons. The results indicate that as the Fe (III) concentration increased, the proportion of total iron in the bacterial cell gradually decreased. In contrast, the proportion of the total iron in the solution gradually increased. The result suggested the total iron became saturated, as it stored enough Fe (III) intracellularly. Consequently, the anammox bacteria reached a point where they were unable to adsorb additional Fe (III), leading to a gradual rise in the level of Fe (III) in the solution. As mentioned above, the main reaction site of the AnAOB was the anammoxsome, which has a high iron requirement. We deduced that the iron migration pathway was as follows: within the optimal Fe (III) dosage threshold, the Fe (III) in the solution was reduced to Fe (II), which then migrated across the anammox bacteria cell membrane into the anammoxsome, where the heme was synthesized. The heme is used by many proteins to execute a variety of tasks, including electron transfer, redox catalysis, and gas transport [22,23], and was linked to the activity of AnAOB.

The results were in response to the above results of nitrogen removal performance and SAA under elevated Fe (III) exposure. It was observed that Fe (Ⅲ) was enriched intracellularly, leading to a remarkable increase in the total iron content within the anammox sludge from 30 mgFe·gVSS⁻¹ to 300 mgFe·gVSS⁻¹. The reduction in EPS was found to be responsible for this enrichment. The anammox microorganisms were unable to withstand the adverse external conditions, which resulted in a decrease in their activity and substrate degradation rate. Consequently, the anammox reaction in the system was completely suppressed, leading to a significant decline in nitrogen removal performance. However, with the addition of ferric ion complex EDTA·2Na to the reactor, Fe (III) existed in the solution, and adhesion on the anammox bacteria cell membrane chelates with EDTA·2Na to form stable complexes. The toxic effects of Fe (III) on anammox bacteria were gradually alleviated. Thus, the TNRR of the system gradually restored to 0.126 kgN·(m³·d)⁻¹.

4. Conclusions

(1)

The effects of Fe (III) content (ranging from 0 to 1.5 mM) on the anammox biofilm process were studied. Results indicated that the average NRR slightly increased at a low concentration of Fe (III) (lower than 0.1 mM). With the addition of Fe (III) doses ranging from 0.3 mM to 1.5 mM, the average TNRE declined from 86.9% to a minimum of 38.3%, indicating severe inhibition of anammox at high levels of Fe (III). Nevertheless, when adding EDTA·2Na to the reactor, the TNRR in the system gradually restored to 0.126 kgN·(m³·d)⁻¹, and the TNRE maintained at about 60%, indicating that the anammox bacteria’s activity could be recovered. Additionally, the anammox bacteria exhibited a strong ability to resist the continuous loading of Fe (III).

(2)

The results of the kinetic fitting analysis showed that parameter K increased slightly from 8.77 to 8.93 at a low level of Fe (III) (lower than 0.1 mM) and decreased from 8.93 to 2.35 at a high level of Fe (III) (more than 0.1 mM), indicating that the lower concentrations of Fe (III) promote the anammox reaction, while higher concentrations of Fe (III) inhibited it. Additionally, the anammox reaction exhibited an IC₅₀ of 1.32 mM for Fe (III) under the conditions investigated.

(3)

Under exposure to elevated Fe (III), a notable accumulation of Fe (III) was observed in the bacteria. The total iron content in anammox sludge increased from 30 mgFe·gVSS⁻¹ to 300 mgFe·gVSS⁻¹ and gradually reached a saturation point. EPS produced by cell metabolism decreased from 221.4 mg·gVSS⁻¹ to 91.3 mg·gVSS⁻¹, indicating that the anammox biofilm was destructed gradually. The SAA of anammox bacteria declined from 0.047 gN·(gVSS·d)⁻¹ to 0.018 gN·(gVSS·d)⁻¹ as the Fe (III) doses rose from 0.3 mM to 1.5 mM. Consequently, the anammox reaction was completely inhibited in the system, leading to a significant degradation in nitrogen removal efficiency.

These discoveries expounded the potential mechanism of anammox involving the mediation by a high concentration of Fe (III) in terms of iron migration and would be beneficial for optimizing the anammox biofilm process by adjusting the concentration of Fe (III) in practical industrial application.