Anammox System Research Articles

Effect of linear anionic surfactant (sodium dodecyl sulfate) dosage on nitrogen removal performance of an anammox reactor

AbstractThe impact of sodium dodecyl sulfate (SDS, as model linear anionic surfactant) dosage on overall nitrogen removal performance of anammox reactor alongside its microbial population and sludge properties was investigated. In this study (day 136), an anmmox sequencing batch biofilm reactor was subjected to gradual dosage of SDS from 0 to 20 mg L−1. Intriguingly, results revealed that SDS at ≥7.5 mg L−1 prompted sludge disintegration, evidenced by increased protein and polysaccharide content in the effluent. Nevertheless, reactor's average total nitrogen removal efficiency slightly improved from 83.12% (0 mg L−1) to 86.3% (20 mg L−1). The 16S rRNA gene sequencing revealed that SDS dosing significantly suppressed the unwanted and unavoidable nitrite oxidizing bacteria (NOBs) in the reactor as the abundance of genus Nitrospira declined from 40.68% (day 1) to 19.15% (day 136). The abundance of anammox genus Candidatus Kuenenia significantly improved from 1.86% (day 1) to 40.02% (day 136) as a result of NOB suppression. This study revealed that low concentration of surfactants in wastewater does not affect the anammox bacteria in a biofilm reactor. Furthermore, adding low concentrations of SDS (≥7.5 to 20 mg L−1) to wastewater may effectively suppress notorious NOBs in biofilm‐based anammox systems.

Long-term effect of microplastics on anammox systems: From macro efficiency to micro metabolic mechanisms and antibiotic resistance genes proliferation

Wastewater treatment plants (WWTPs) are sinks for various types of microplastics (MPs), which have an undeniable impact on anaerobic ammonia oxidation (anammox) systems, yet their differential effects remain elusive. In this study, the long-term effects of non-biodegradable polyvinyl chloride (PVC) MPs and biodegradable polylactic acid (PLA) MPs in anammox systems were investigated from macro efficiency, micro metabolic mechanism, and antibiotic resistance gene proliferation. Results showed that the introduced 0.5 mg L−1 PVC MPs caused a 20.64 % decrease in nitrogen removal efficiency (NRE) compared to the control one, while PLA MPs resulted in a 10.73 % increase in NRE. Accordingly, the relative abundance of Ca. Kuenenia with the introduced PLA MPs was 16.68 % higher than the control group, while it was 10.89 % lower with the introduced PVC MPs. Furthermore, MPs inhibit the reactor performance by inducing intracellular reactive oxygen species (ROS) production in anammox cells. However, PLA MPs, as a slow biodegradable organic, have a reversible negative effect on ROS induction, which further improves nitrogen removal performance and sludge proliferation through dissimilatory nitrate reduction to ammonium (DNRA) and microbial interactions. Nevertheless, this also poses an increased risk of antibiotic resistance genes (ARGs) spread and proliferation in anammox sludge. This study clarified the comprehensive effects of different MPs types in anammox processes, highlighting the feasibility and potential risks in the engineering applications of anammox processes for MPs-contained wastewater treatment.

Metagenomic insights into the mechanism for the rapid enrichment and high stability of Candidatus Brocadia facilitated by Fe(Ⅲ)

The rapid enrichment of anammox bacteria and its fragile resistance to adverse environment are the critical problems facing of anammox processes. As an abundant component in anammox bacteria, iron has been proved to promote the activity and growth of anammox bacteria in the mature anammox systems, but the functional and metabolic profiles in Fe(III) enhanced emerging anammox systems have not been evaluated. Results indicated that the relative abundance of functional genes involved in oxidative phosphorylation, nitrogen metabolism, cofactors synthesis, and extracellular polymers synthesis pathways was significantly promoted in the system added with 5 mg/L Fe(III) (R5). These enhanced pathways were crucial to energy generation, nitrogen removal, cell activity and proliferation, and microbial self-defense, thereby accelerating the enrichment of anammox bacteria Ca. Brocadia and facilitating their resistance to adverse environments. Microbial community analysis showed that the proportion of Ca. Brocadia in R5 also increased to 64.42 %. Hence, R5 could adapt rapidly to the increased nitrogen loading rate and increase the nitrogen removal rate by 108 % compared to the system without Fe(III) addition. However, the addition of 10 and 20 mg/L Fe(III) showed inhibitory effects on the growth and activity of anammox bacteria, which exhibited the lower relative abundance of Ca. Brocadia and unstable or even collapsed nitrogen removal performance. This study not only clarified the concentration range of Fe(III) that promoted and inhibited the enrichment of anammox bacteria, but also deepened our understanding of the functional and metabolic mechanisms underlying enhanced enrichment of anammox bacteria by Fe(III), providing a potential strategy to hasten the start-up of anammox from conventional activated sludge.

Controlling Dissolved Oxygen in Electrochemical Anammox Systems through Sodium Sulfite with Nitrogen Stripping

Controlling Dissolved Oxygen in Electrochemical Anammox Systems through Sodium Sulfite with Nitrogen Stripping

Efficient photocatalytic reduction of nitrate byproducts during anammox process by novel extracellular polymeric substances-embedded NH2-MIL-101(Fe) photocatalysts

Anaerobic ammonium oxidation (anammox) is an efficient nitrogen removal process; however, nitrate byproducts hampered its development. In this study, extracellular polymeric substances (EPS) were embedded into NH2-MIL-101(Fe), creating NH2-MIL-101(Fe)@EPS to reduce nitrate. Results revealed that chemical nitrate reduction efficiency of NH2-MIL-101(Fe)@EPS surpassed that of NH2-MIL-101(Fe) by 17.3 %. After adding 0.5 g/L NH2-MIL-101(Fe)@EPS within the anammox process, nitrate removal efficiency reached63.9 %, consequently elevating the total nitrogen removal efficiency to 92.4 %. 16S rRNA sequencing results elucidated the predominant role of Candidatus Brocadia within NH2-MIL-101(Fe)@EPS-anammox system. Concurrently, sufficient photogenerated electrons were transferred to microorganisms, promoting the growth of Desnitratisoma and OLB17. Additionally, photogenerated electrons activated flavin and Complex III, thereby up-regulating crucial genes involved in intra/extracellular electron transfer. Subsequently, denitrification and dissimilatory nitrate reduction to ammonium were activated to reduce nitrate. In summary, this study achieved a notable rate of photocatalytic nitrate reduction within anammox process through the NH2-MIL-101(Fe)@EPS photocatalysts.

Efficient nitrogen removal from real municipal wastewater and mature landfill leachate using partial nitrification-simultaneous anammox and partial denitrification process

Anaerobic ammonia oxidation (anammox) of municipal wastewater is a research focus, especially the combined treatment with mature landfill leachate is a current research hotspot. In this study, municipal wastewater was treated by partial nitrification via sequencing batch reactor (SBR), and its effluent and mature landfill leachate were then mixed into an up-flow anaerobic sludge blanket (UASB) for simultaneous anammox and partial denitrification reaction. Through partial nitrification, a high nitrite accumulation rate (93.0 ± 3.8 %) was achieved by low dissolved oxygen (0.5–1.6 mg/L) and controlled aerobic time (3.5 h) in SBR. The UASB system was responsible for 78.8 ± 2.1 % nitrogen removal of the entire system with a hydraulic reaction time (HRT) of 3.8 h, accompanied by the anammox contribution up to 89.4 ± 6.0 %. The overall partial nitrification-simultaneous anammox and partial denitrification (PN-SAPD) system was controlled at a total COD/TIN of 2.8 ± 0.3 and a total HRT of only 10.2 h, achieving the nitrogen removal efficiency and effluent TIN were 95.2 ± 2.2 % and 3.4 ± 1.5 mg/L, respectively. The qPCR results showed functional genes (hzsA(B), hdh) associated with anaerobic ammonia-oxidizing bacteria (AnAOB), whose high gene copy abundance and transcription expression ensured the removal of major nitrogen from municipal wastewater and mature landfill leachate. 16S amplicon sequencing showed that the Ca. Brocadia (9.72–12.6 %) was further enrichment after sodium acetate was added, and the transcription expression of Thauera (0.5–7.0 %) caused nitrate to nitrite. The high abundance of related enzymes (hao, hzs, hdh, narGHI) involved in anammox and partial denitrification processes were found in the macrogenomic sequencing, and only Ca. Brocadia was involved in multi-pathway nitrogen metabolism in AnAOB. Based on the efficient nitrogen removal by AnAOB and denitrifying bacteria, this modified PN-SAPD process provides a new option for the co-treatment of mature landfill leachate in municipal wastewater treatment plants.

Temporal differentiation in the adaptation of functional bacteria to low-temperature stress in partial denitrification and anammox system

Despite reliable nitrite supply through partial denitrification, the adaptation of denitrifying bacteria to low temperatures remains elusive in partial denitrification and anammox (PDA) systems. Here, temporal differentiations of the structure, activity, and relevant cold-adaptation mechanism of functional bacteria were investigated in a lab-scale PDA bioreactor at decreased temperature. Although distinct denitrifying bacteria dominated after low-temperature stress, both short- and long-term stresses exerted differential selectivity towards the species with close phylogenetic distance. Species Azonexus sp.149 showed high superiority over Azonexus sp.384 under short-term stress, and long-term stress improved the adaptation of Aquabacterium sp.93 instead of Aquabacterium sp.184. The elevated transcription of nitrite reductase genes suggested that several denitrifying bacteria (e.g., Azonexus sp.149) could compete with anammox bacteria for nitrite. Species Rivicola pingtungensis and Azonexus sp.149 could adapt through various adaptation pathways, such as the two-component system, cold shock protein (CSP), membrane alternation, and electron transport chain. By contrast, species Zoogloea sp.273 and Aquabacterium sp.93 mainly depended on the CSP and oxidative stress response. This study largely deepens our understanding of the performance deterioration in PDA systems during cold shock and provides several references for efficient adaptation to seasonal temperature fluctuation.

Increasing carbon to nitrogen ratio promoted anaerobic ammonia-oxidizing bacterial enrichment and advanced nitrogen removal in mainstream anammox system

The effects of fluctuating organic carbon to nitrogen (C/N) ratios on mainstream simultaneous partial nitrification, anammox, and denitrification (SNAD) process were studied over 376-day period. The nitrogen removal efficiency decreased from 85.0 ± 6.6 % to 75.8 ± 2.8 % as C/N ratio decreased (3.4 → 1.7), but increased to 82.0 ± 1.9 % when C/N ratio raised to 2.9 and to 78.4 ± 3.0 % when C/N ratio decreased again (2.9 → 2.1), indicating that high C/N ratios promoted nitrogen removal. As C/N ratio raised (1.7 → 2.9), anaerobic ammonia-oxidizing bacteria (AnAOB) abundance increased from 1.3 × 109 to 2.0 × 109 copies/L, which explained the improved nitrogen removal. With an elevated C/N ratio, partial nitrification and endogenous partial denitrification reactions were enhanced, providing more nitrite for AnAOB. Additionally, the aerobic_chemoheterotrophy function and particle sizes increased, forming more stable anoxic microenvironment for AnAOB. Overall, increasing C/N ratio promoted the stability of mainstream SNAD.

Anammox activity improved significantly by the cross-fed NO from ammonia-oxidizing bacteria and denitrifying bacteria to anammox bacteria

Nitric oxide (NO) has been suggested as an obligate intermediate in anaerobic ammonium oxidation (anammox), nitrification and denitrification. At the same time, ammonia-oxidizing bacteria (AOB) and denitrifying bacteria (DNB) are always existed in anammox flora, so what is the role of NO produced from AOB and DNB? Could it accelerate nitrogen removal via the anammox pathway with NO as an electron acceptor? To investigate this hypothesis, nitrogen transforming of an anammox biofilter was analyzed, functional gene expression of anammox bacteria (AnAOB), AOB and DNB were compared, and NO source was verified. For anammox biofilter, anammox contributed to 91.3 % nitrogen removal with only 14.4 % of AnAOB being enriched, while DNB was dominant. Meta-omics analysis and batch test results indicated that AOB could provide NO to AnAOB, and DNB also produced NO via up-regulating nirS/K and down-regulating nor. The activation of the anammox pathway of NH4++NO→N2 caused the downregulation of nirS and nxr in Ca. Kuenenia stuttgartiensis. Additionally, changes in nitrogen transforming pathways affected the electron generation and transport, limiting the carbon metabolism of AnAOB. This study provided new insights into improving nitrogen removal of the anammox system.

The start-up of anammox system inoculated with sludge overwhelmingly dominated by Candidatus Competibacter: System performance and microbial community succession

In situ start-up of the anaerobic ammonia oxidation (anammox) process using ordinary sludge is quite time-consuming, and the existing denitrifying bacteria can compete for nitrite during the initial start-up of the reactor, resulting in inadequacy of substrates. In this study, anaerobic sludge with the relative abundance of Candidatus Competibacter up to 40.27 % was used as a seed, and sufficient nitrite was provided to maintain the levels of electron acceptor for denitrifiers during the initial start-up of the reactor. After 176 days of operation, the anammox reactor achieved removal efficiencies of ammonia and nitrite at 93.75 % and 99.88 %, respectively, and the total nitrogen removal was 73.16 %. The refined ratios of influent nitrite to ammonia in the range of 1.33–2.66 were found to minimize the effect of the large consumption of nitrite by denitrifying bacteria. High-throughput sequencing results showed that Candidatus Brocadia was the only anammox genus obtained, which accounted for 20.31 % at the end of operation. Functional bacteria for denitrification, assumed to be the Candidatus Competibacter, were decreased to 0.88 %. The predicted functions confirmed the dominance of nitrate reductase gene napA, which was positively correlated with Candidatus Competibacter and Thermovirga (Spearman correlation, p < 0.05). Therefore, the speculated molecular mechanism was the Thermovirga-dominated syntrophic metabolism coupled with the endogenous denitrification process of Candidatus Competibacter. This study revealed the microbial ecology and evolution responsible for nitrogen removal during the rapid start-up of anammox, and provided an extra line of guidance for the start-up of anammox reactors inoculated with denitrifying sludge.

Exploring the potential of a novel alternating current stimulated iron‑carbon anammox process: A new horizon for nitrogen removal

This study explored a novel alternating current (AC) stimulation approach to enhance the nitrogen removal efficiency of an iron‑carbon based anammox (FeC anammox) system. In the preliminary experiment, the TN removal efficiency of the AC stimulated system was 8.06 % higher than that of a DC simulated system in same current densities of 0.25 mA/cm2. Gene expression analysis revealed that the AC-stimulated system, where, compared with the anammox system alone, the expression of HZS, HDH, NarG, NirS, NorB and NosZ increased by 1.81, 2.50, 1.64, 0.23, 1.15 and 1.27 times, respectively. In the continuous experiment, the TN removal rate increased from 60.13 % to 84.34 % after AC stimulation, and the working time of the FeC materials increased to 20 days. An analysis of the mechanism revealed that the parallel connection between the capacitive reactance and filler resistance in AC might reduce the internal resistance of the system, thereby improving the actual current density received by local microorganisms, and achieving a better strengthening effect.

Robust rehabilitation of anammox system by granular activated carbon under long-term starvation stress: Microbiota restoration and metabolic reinforcement

This article investigates the buffering capacity and recovery-enhancing ability of granular activated carbon (GAC) in a starved (influent total nitrogen: 20 mg/L) anaerobic ammonium oxidation (anammox) reactor. The findings revealed that anammox aggregated and sustained basal metabolism with shorter performance recovery lag (6 days) and better nitrogen removal efficiency (84.9 %) due to weak electron-repulsion and abundance redox-active groups on GAC’s surface. GAC-supported enhanced extracellular polymeric substance secretion aided anammox in resisting starvation. GAC also facilitated anammox bacterial proliferation and expedited the restoration of anammox microbial community from a starved state to its initial-level. Metabolic function analyses unveiled that GAC improved the expression of genes involved in amino acid metabolism and sugar-nucleotide biosynthesis while promoted microbial cross-feeding, ultimately indicating the superior potential of GAC in stimulating more diverse metabolic networks in nutrient-depleted anammox consortia. This research sheds light on the microbial and metabolic mechanisms underlying GAC-mediated anammox system in low-substrate habitats.

Stimulating extracellular polymeric substances production in integrated fixed-film activated sludge reactor for advanced nitrogen removal from mature landfill leachate via one-stage double anammox

Introducing carbon sources to achieve nitrogen removal from mature landfill leachate not only increases the costs and carbon emissions but also inhibits the activity of autotrophic bacteria. Thus, this study constructed a double anammox system that combines partial nitrification-anammox (PNA) and endogenous partial denitrification-anammox (EPDA) within an integrated fixed-film activated sludge (IFAS) reactor. In this system, PNA primarily contributes to nitrogen removal pathways, achieving a nitrite accumulation rate of 98.23%. The production of extracellular polymer substances (EPS) in the IFAS reactor is stimulated by introducing co-fermentation liquid. Through the utilization of EPS, the system effectively achieves EPDA with the nitrite transformation rate of 97.20%. Under the intermittent aeration operation strategy, EPDA combined with PNA and anammox in the oxic and anoxic stages enhanced the nitrogen removal efficiency of the system to 99.70 ± 0.12%. The functional genus Candidatus kuenenia became enriched in biofilm sludge, while Thauera and Nitrosomonas predominated in floc sludge.

Successful establishment of Anammox by cultivating residual sludge and its response to elevated nitrogen loading

Anaerobic ammonia oxidation (Anammox) process was one of the most economical nitrogen removal technology, however, the slow growth rate and the shortage of sufficient seed sludge limit its application. In this study, Anammox was successfully established using residual sludge as seed sludge, and the variation in its nitrogen removal, stability, and microbial characteristics in response to the elevated ammonia and nitrite were explored. The sludge exhibited Anammox activity on day 34, and the system was successfully established on day 67, while the hzo gene expression increased from 1.1 * 104 to 9.4 * 105 copies/mL. The established Anammox system showed good resistance to the high nitrogen shock (200–1000 mg/L), and the relative abundance of Candidatus_Kuenenia notably increased. However, when the total nitrogen in influent reached 1200 mg/L, the nitrogen removal of the Anammox system was suppressed. And Candidatus_Kuenenia abundance significantly decreased while Arenimonas increased. Residual sludge could be adopted for establishing Anammox system, and the system could be used for efficiently treating wastewater with nitrogen lower than 1000 mg/L. The result in the present study has significance to the application of Anammox process and deeply save the economic cost of seed sludge since residual sludge was widely available.

Responses of microbial interactions and functional genes to sulfamethoxazole in anammox consortia

Sulfamethoxazole (SMX) has been widely detected in various environments and its potential environmental risks have caused great concerns. However, the impact mechanism of SMX on microbial interactions among anammox consortia remain unknown. A long-term exposure experiments (140 d) was carried out to systematically examine the influence of SMX (0–1000 μg/L) on the anammox system, especially microbial network dynamics and variations of key metabolic genes. Results showed that anammox system could adapt to SMX below 500 μg/L and maintain a high nitrogen removal efficiency (NRE) of 85.35 ± 2.42%, while 1000 μg/L SMX significantly decreased the abundance of functional microbes and deteriorated denitrification performance with NRE dropped to 36.92 ± 15.01%. Co-occurrence network analysis indicated that 1000 μg/L SMX decreased the interactions between Proteobacteria and Chloroflexi and limited AnAOB from playing an important role as central nodes in the subnetwork of Planctomycetes. Metagenomics analysis found that genes associated with nitrogen removal (i.e., hdh, hzs, nirS, and hao) showed lower expression level after addition of SMX, while SMX-related ARGs (sul1 and sul2) increased by 1.22 and 2.68 times. This study provided us a relatively comprehensive perspective in response of microbial interactions and metabolic activity to various SMX concentrations.

Fate of iron nanoparticles in anammox system: Dissolution, migration and transformation

Iron nanoparticles (FeNPs) are commonly used in various industrial processes, leading to their release into the environment and eventual entrance into wastewater treatment plants (WWTPs). FeNPs undergo dissolution, migration, and transformation in WWTPs, which can potentially affect the stable operation of anaerobic ammonia oxidation (anammox) systems and may be discharged with wastewater or biomass. To better understand the fate of FeNPs in anammox systems, exposure experiments were conducted using anammox granular sludges (AnGS) and FeNPs. Results demonstrated that FeNPs released Fe2+ upon contact with water, with a portion being bound to functional groups in extracellular polymeric substances (EPS) and the rest entering the bacteria to form highly absorbable substances. A significant amount of FeNPs was observed to cover the surface of AnGS or aggregate and deposit at the bottom of the reactor, eventually converting into Fe3O4 and stably existing within the anammox system. The findings of this study clarify the fate of FeNPs in anammox systems and provide important insights into the stable operation of anammox systems under FeNPs exposure.

Response of anammox to long-term stress of Ni(II): Nitrogen removal, microbial products and microbial community

Anaerobic ammonium oxidation (anammox) has been successfully applied to the practical wastewater treatment due to a high nitrogen removal performance, low sludge yield and low operating cost. Heavy metals in wastewater have received extensive attention because of their non-degradability and biotoxicity. In order to apply anammox system to treat wastewater containing heavy metals and explore the anammox system recovery to complex engineering environments, Ni(II) was used as representative substance to investigate its long-term effects on the anammox process in continuous flow reactors. The self-recovery ability of the anammox system after long-term exposure to Ni(II) was also investigated. The results showed that low concentrations of Ni(II) (≤ 5 mg/L) had a slight inhibitory effect on the anammox system, while high concentrations of Ni(II) (≥ 10 mg/L) could lead to the deterioration of the anammox system. Extracellular polymeric substance (EPS) content increased with increasing Ni(II) concentration. High-throughput sequencing results showed that Ni(II) severely impacted the microbial community of the anammox system, especially Candidatus Kuenenia. The abundance of Candidatus Kuenenia decreased from 32.11% to 13.99% with increasing Ni(II) concentration from 0 mg/L to 15 mg/L. After stopping Ni(II) addition, EPS and functional bacterial abundance recovered to some extent, and nitrogen removal efficiency (NRE) only returned to 44.53% of normal levels. The self-recovery of the anammox system was a longer process.

Deciphering the partial denitrification function of companion bacteria in mixotrophic anammox systems under different carbon/nitrogen ratios

Although partial denitrification coupled with the anammox process has received considerable attention, the partial denitrification function of companion bacteria in mixotrophic anammox systems remains experimentally investigated. Herein, a partial denitrification process was realized in mixotrophic anammox systems with a COD/TN ratio of 0.75. Furthermore, the experimental verification results revealed that nitrate and ammonia were eliminated concurrently, showing that partial denitrifying bacteria were present in the mixotrophic anammox systems. Denitratisoma and Norank Xanthobacteraceae were the dominant genera of companion bacteria containing functional genes related to partial denitrification. Especially, Denitratisoma was a companion bifunctional bacterium for Candidatus Kuenenia shifted between complete and partial denitrification at different COD/TN ratios. Moreover, nir gene abundance in the mixotrophic anammox system increased by 135.4 % compared to that in the autotrophic anammox system. In contrast, nor gene abundance decreased by 28.00 %, indicating that NO might be used as a medium for cross-feeding anammox and partial denitrification. This study provides a more thorough understanding of companion bacteria in mixotrophic anammox systems.

Achieving intensified nitrogen removal and vivianite recovery from sludge digester liquor in two-stage partial nitrification-electrolysis anammox system

Achieving intensified nitrogen removal and vivianite recovery from sludge digester liquor in two-stage partial nitrification-electrolysis anammox system

Metagenomics insight into the long-term effect of ferrous ions on the mainstream anammox system

Anaerobic ammonium oxidation (anammox) bacteria have a high requirement for iron for their growth and metabolism. However, it remains unclear whether iron supplementation can sustain the stability of mainstream anammox systems at varying temperatures. Here, we investigated the long-term effects of Fe2+ on the mainstream anammox systems. Our findings revealed that the nitrogen removal efficiency (NRE) of the anammox system supplemented with 5 mg/L Fe2+ decreased from 76.5 ± 0.76% at 35 °C to 39.0 ± 9.9% at 25 °C. Notably, higher dosages of Fe2+ (15 mg/L and 30 mg/L) inhibited the anammox system, resulting in NREs of 15.9 ± 8.1% and 2.5 ± 1.1% at 25 °C, respectively. The results of microbial communities and function profiles suggested that the high Fe2+ dosage seriously affected the iron assimilation and utilization in the mainstream anammox system. This was evident from the decreased abundance of genes associated with Fe(II) transport and uptake, which in turn hindered the biosynthesis of intracellular iron-cofactors, resulting in decrease in the absolute abundance of Candidatus Brocadia, a key anammox bacterium, as well as a decline in NRE. Furthermore, our results showed that the anammox process was more susceptible to iron supplementation at 25 °C compared to 35 °C, which may be due to the oxidative stress reactions induced by combined lowered temperature and a high Fe2+ dosage. Overall, these findings offer a deeper understanding of the effect of iron in mainstream anammox systems, which can contribute to improved stability maintenance and effectiveness of anammox processes.