Anammox Performance Research Articles

Response of granular anammox process under mainstream conditions to continuous and transient organic loads

Even small fluctuations in the amount of organic matter in wastewater significantly affect the structure and function of anammox microbial communities under mainstream conditions with low ammonium concentrations, because a high organic C/N ratio causes heterotrophic denitrifying bacteria to outcompete anammox bacteria. This paper presents a comprehensive investigation of the effects of different levels of continuous and transient organic loads on nitrogen removal and sludge characteristics in upflow granular anammox reactors under mainstream conditions. Continuous organic load at 30 mg chemical oxygen demand (COD)/L influent improved nitrogen removal efficiency without compromising anammox performance. However, exposure to higher levels of continuous organic load (≥60 mg COD/L) caused significant inhibition of anammox activity (complete inhibition at 150 mg COD/L). The performance degradation was accompanied by the destabilization of anammox granular sludge, with a decrease in tightly bound protein and increase in loosely bound polysaccharide contents in the sludge extracellular polymeric substances. Further, DNA sequencing analysis showed that heterotrophic denitrifying Rhodocyclaceae and Pseudomonadaceae bacteria collectively accounted for 36.8–61.3% of the total bacterial reads. These bacteria emerged and outcompeted anammox Ca. Brocadiaceae (accounting for less than 8.4% of the total reads) with increasing organic load, resulting in a significant decrease in anammox activity. Meanwhile, the inhibitory effect of transient organic load (one-day shock loading) was fully reversed within one day at both 150 and 300 mg COD/L shock levels, although the latter caused a sudden fall in anammox performance. The overall results suggest that loading an appropriately low amount of organic matter can provide a means to improve the nitrogen removal performance of anammox granular sludge without sacrificing anammox activity and granule stability under mainstream conditions.

Multi-chambers of pilot-scale reactor enhanced partial nitritation performance

Nowadays, applying anammox to treat high nitrogenous side-stream wastewater has taken a step forward. However, the partial nitritation process is sensitive to the ammonium concentration and the nitrogen loading rate, which significantly influences the nitrogen removal performance. This study investigated the performance of a novel nitritation pilot-scale reactor which was divided into four chambers. The nitrite accumulation efficiency reached more than 90 % in the rural wastewater treatment process. As the reactor was divided into four chambers, the comprehensive statistical results showed that the concentration of free ammonium in the front chambers had been effectively improved. The proportion of free ammonium concentration (>0.1 mg NH3·L−1), which could inhibit the activity of nitrite oxidizing bacteria, in first chamber (PN1) was 2 times higher than in the last chamber (PN4). Meanwhile, Nitrosomonas, responsible for ammonium oxidation to nitrite, was highly enriched in the first two chambers even though the dissolved oxygen was maintained at 1.5 ± 0.3 mg·L−1. Compare to conventional reactor, the resistance of the novel reactor to volumetric shock loading has been enhanced. Even though the ammonium loading rate fluctuated greatly, the effluent was still stable and could meet the demand following the anammox process. This study demonstrated that the reactor with multi-chambers could effectively improve the nitrite accumulation efficiency in the partial nitritation process and thus provide a new perspective on the partial nitritation process in a single reactor and further promote the anammox performance in the wastewater treatment process.

A novel strategy for a reinforced anammox process with iron-modified Enteromorpha prolifera biochar

Modified biochar with higher electron transport and adsorption capabilities could significantly improve the performance of anaerobic ammonia oxidation (anammox). However, there are few related investigations on the reinforcement of anammox through iron-modified Enteromorpha prolifera biochar (IMEPB). In this study, with the addition of the IMEPB in the anammox system, the enhancing process of anammox performance was studied, the improving feasibility of anammox was evaluated, and the reinforcing mechanism of anammox was elucidated. The results showed that the optimal iron−charcoal ratio (Fe:C) and IMEPB dosage were 1:10 and 10 g L−1, respectively. Under the optimal conditions, when the nitrogen loading rate gradually increased to 0.557 (kg m−3 day−1), the nitrogen removal efficiency and nitrogen removal rate of the anammox process supplemented with IMEPB increased by 11%, and the specific anammox activity increased by 23.8%. Compared with the control, the secretion of extracellular polymeric substances (EPS) of anammox bacteria supplemented IMEPB increased by 24.4%, greatly improving the stability of the anammox system. Meanwhile, EPS secretion further promoted the microbial activity of anammox bacteria, achieving a 19% increase in the abundance of Candidatus Brocadia. These findings demonstrate the potential mechanism of IMEPB in improving anammox, provide new insights into recycling E. prolifera, and provide a novel reinforcement strategy for anammox. In the future, adding IMEPB may be a vital measure for the practical application of anammox in coastal areas.Graphical

Quantification of enhanced nitrogen removal pathways of pyrite interaction with anammox sludge system

Fe and S could improve the anammox performance by coupling with the N cycle. However, the coexistence of Fe and S complicates the metabolic process, troubling the quantification of nitrogen removal pathways. Herein, the nitrogen removal pathways were quantificationally analyzed by 15N stable isotope tracing and elaborate batch tests, and the enhancing mechanism was illuminated by physiological and metatranscriptome analyses in pyrite (FeS2)-amended anammox system. We found that anammox, denitrification and Feammox (NH4+ oxidation coupled with Fe(III) reduction) contributed to 80.22 %, 19.53 % and 0.25 % nitrogen removal in this process. With pyrite addition, Fe, S release and the extracellular polymeric substances secretion were responsible for the enhancement of nitrite and nitrate removal via chemical, autotrophic and heterotrophic denitrification, improving nitrogen removal by 14 % and 19 %. The network analyses further confirmed that the multiple nitrogen removal pathways (i.e., anammox, Feammox and denitrification) contribute to pyrite-induced nitrogen removal enhancement in the anammox system. This work enables the in-depth understanding, future design and optimization of the pyrite-amended anammox system, providing a fundamental basis for developing Fe- and S-based anammox enhancement technologies.

Elucidating the Nitrogen Removal Performance and Response Mechanisms of Anammox Under Heavy Metal Stress Using Big Data Analysis and Machine Learning

Elucidating the Nitrogen Removal Performance and Response Mechanisms of Anammox Under Heavy Metal Stress Using Big Data Analysis and Machine Learning

Use of iron-loaded biochar to alleviate anammox performance inhibition under PFOA stress conditions: Integrated analysis of sludge characteristics and metagenomics

The negative effects of perfluorooctanoic acid (PFOA) on biological nitrogen removal performance in wastewater treatment plants, are receiving increasing attention due to the widespread reporting of this issue. In this study, pomelo peel iron-loaded biochar (Fe-PBC) was added to an anammox bioreactor to alleviate the negative effects of PFOA. Results showed that the addition of Fe-PBC increased the ammonia and nitrite removal efficiencies from 77.7 ± 9.6 % and 79.5 ± 5.6 % to 94.45 ± 5.1 % and 95.9 ± 5.0 %, respectively. In addition, Fe-PBC promoted the removal of PFOA from wastewater, increasing the PFOA removal efficiency from 5.2 % to 29.2 ± 4.3 % from 100 to 200 days. The introduction of iron-loaded biochar into the anammox bioreactor increased the CO ratio by 13.64 % by 150 days. In addition, a CO fitting peak was detected in the Fe-PBC, indicating that the Fe-PBC was loaded with microorganisms. Microbial community analysis showed a decrease in the relative abundances of Proteobacteria and Nitrospirae from 31 % and 3.4 % to 16.8 % and 0.9 %, respectively, while the relative abundance of Planctomycetes increased from 26.8 % to 44.1 %. Metagenomic analysis found that the functional genes hzsB and hdh increased from 98,666 ± 11,400 and 3190 ± 460 to 119,333 ± 15,534 and 138,650 ± 11,233 copy numbers/MLSS. The increase in anammox biomass may be attributed to the presence of iron, an essential element for the synthesis of key anammox enzyme. Furthermore, iron was also associated with the enhanced extracellular electron transfer in the anammox system induced by Fe-PBC.

Promoting performance of Anammox by iron loaded sludge biochar with hydrothermal carbonization (HTC-Fe-BC) addition

Anaerobic ammonia oxidation (Anammox) treatment cannot always achieve optimal efficiency due to its sensitivity to environmental conditions. The nitrogen removal efficiency of Anammox reactors with different iron loaded sludge biochar modified by hydrothermal carbonization (HTC-Fe-BC) addition dose of 0, 2.5 and 5.0 g/L (named BC0, BC1 and BC2) were analyzed. The effects of adding HTC-Fe-BC on the performance of Anammox reactor, extracellular polymeric substances (EPS), microbial community and functional genes were investigated. The results showed that the addition of HTC-Fe-BC could reduce the environmental sensitivity of Anammox reactor, the removal efficiency of total nitrogen increased from 51.20 (BC0) to 70.83 % (BC2), and the activity of Anammox sludge improved by 49.58 %. HTC-Fe-BC increased the activity of Anammox sludge by promoting the secretion of protein, humic acids and cytochrome c in EPS and alleviated the inhibitory effect by mediating the Anammox electron transfer process. After EPS removal and enzymatic hydrolysis, the activity of Anammox sludge in BC2 decreased by 61.38% and 79.80 %, respectively, significantly higher than the reduced values of BC0. Further, the relative abundance of Candidatus_Brocadia in Anammox reactors supplemented with HTC-Fe-BC was increased by 35.56–55.56 % compared to the control group, as were the relative abundances of the key functional genes connected to N transformation. These results deepened the understanding of the potential mechanism by which HTC-Fe-BC improved the efficiency of Anammox system.

Improving anammox performance by limited filamentous bulking for wastewater treatment with organic stress

In this study, the filamentous bulking was demonstrated to improve anammox capability and anammox bacteria (AnAOB) population density under organic stress. The selective heterotrophic bacteria (HB) washout that involved in shear detachment, enmeshment and biomass washout was triggered. The microbial spatial distribution and granular detachment properties revealed that the filamentous bulking transferred the “location advantage” of HB from granules interior to surface, and endowed granular surface low shear tolerance for shear detachment, ultimately resulted in selective HB detachment. The detached filaments-mediated enmeshment provided additional selective pressure for free HB-flocs, eventually achieving the retention time differentiation between AnAOB (34 – 141 days) and HB (3 – 15 days), and a high anammox population density. Controlling dissolved oxygen level was crucial for regulating sludge bulking. Collectively, the filamentous bulking was developed as an effective anti-organic stress strategy to broaden the application of granular anammox process in actual wastewater treatment.

Unveiling the effects of soluble starch, ethanol, and sodium acetate on the interactions of functional microorganisms and nitrogen removal in a partial nitritation and anammox biofilm system

Different organic matters have different effects on the nitrogen removal performance of partial nitritation and anammox (PN/A) biofilm system. In this study, the effects of adding soluble starch, ethanol, and sodium acetate on the functional microorganisms and nitrogen removal in the PN/A biofilm system are investigated. The results show that ethanol had the strongest effects on the ammonium-oxidizing bacteria (AOB) growth, enrichment, and ammonium nitrogen removal rate, followed by soluble starch and sodium acetate. However, the suppression of anaerobic ammonium oxidation bacteria (AnAOB) and decrease in total nitrogen removal rate were affected the most by ethanol, followed by soluble starch and sodium acetate. In addition, the enrichment of nitrifying oxidizing bacteria (NOB) was the most strongly influenced by ethanol, followed by soluble starch and sodium acetate. Furthermore, the enrichment of phylum Proteobacteria was facilitated by ethanol > soluble starch > sodium acetate. The enrichment of phylum Nitrospirae and genus Nitrospira were influenced by ethanol > soluble starch > sodium acetate. However, the suppression of the phylum Planctomycetes and the genus Candidatus Brocadia were affected by ethanol > soluble starch > sodium acetate. These results show that the PN/A biofilm system is suitable for the treatment of ammonium nitrogen wastewaters containing organic salt (sodium acetate) but not for treating wastewaters containing organic alcohol (ethanol) and macromolecular organic matter (soluble starch).

Low-intensity electromagnetic field as a new engineering oriented bioaugmentation strategy for anammox granules

Improving the relative abundance of bacteria and their activity is still the basis for the efficient operation of anammox process. Here, biomagnetic effect was used to promote anammox granules. Batch test results show that the application of an electromagnetic field (EMF) with a strength of 0.09 μT increased the nitrogen removal performance of anammox by 32.44% while higher strength EMF of 0.20 and 0.25 μT inhibited the activity of anammox bacteria. Long-term experiment indicated that the addition of EMF with a strength of 0.09 μT greatly improved nitrogen removal performance of the granular sludge, especially the total nitrogen removal performance increased by 15.3%. After 120 days of reactor operation, the nitrogen loading rate was increased to 6.4 kg N/m3/d, and the total nitrogen removal rate of the reactors with and without EMF addition reached 4.92 kg N/m3/d and 4.25 kg N/m3/d, respectively. Throughout the experiment, the removal rate of NH4+-N and NO2−-N of anammox reactor with 0.09 μT EMT addition was always higher than that without EMF addition. The high-throughput sequencing analysis showed that the proportion of Candidatus Brocadia in reactors with and without EMF addition were 21.3% and 15.8%, respectively. The application of EMF with an intensity of 0.09 μT increased the relative abundance of the main anammox bacteria. 70 kos were enriched under EMF conditions, including ko00780 (Biotin metabolism), ko00540 (Lipopolysaccharide biosynthesis), ko00590 (Arachidonic acid metabolism). 51 kos like ko03030 (DNA replication) decreased after EMF addition. This study demonstrates the feasibility of EMF to promote anammox and expands the application of EMF in wastewater treatment.

Dosing with pyrite significantly increases anammox performance: Its role in the electron transfer enhancement and the functions of the Fe-N-S cycle

Anaerobic ammonium oxidation (anammox) represents an energy-efficient process for biological nitrogen removal from ammonium-rich wastewater. However, there are mechanistic issues unsolved regarding the low microbial electron transfer and undesired accumulation of nitrate in treated water, limiting its widespread engineering applications. We found that the addition of pyrite (1 g L−1 reactor), an earth-abundant iron-bearing sulfide mineral, to the anammox system significantly improved the nitrogen removal rate by 52% in long-term operation at a high substrate shock loading (3.86 kg N m−3 d−1). Two lines of evidence were presented to unravel the underlying mechanisms of the pyrite-induced enhancement. Physiochemical evidence indicated that an increase of cytochromes c and Fe-S protein was responsible for the accelerated electron transfer among metabolic enzymes. Multi-omics evidence showed that the depletion of nitrate was attributed to the Fe-N-S cycle driven by nitrate-dependent Fe(II) oxidation and S-based denitrification. This study deepens our understanding of the roles of electron transfer and the Fe-N-S cycle in anammox systems, providing a fundamental basis for the development of mediators in the anammox process for practical implications.

Segregation of effect between granules and flocs in PN/A system treating acrylic fiber wastewater: Performance and mechanism.

Nitrogen removal of petrochemical wastewater through partial nitritation/anammox (PN/A) is appealing, but its feasibility and stability under toxic inhibition remain unclear. This study started a PN/A granular sludge system in a membrane bioreactor and fed it with diluted acrylic fiber wastewater. During long-term operation, the nitritation and anammox performance remained stable at a 30% volume ratio, and declined with increasing volume ratio, resulting in deteriorated nitrogen removal. Meanwhile, the short-term inhibition batch tests further showed that ammonia oxidation bacteria (AOB) in the flocs were suppressed while anammox bacteria (AnAOB) in the granules were not affected. Further analysis indicated suppression of AnAOB over the long-term operation, which was mainly caused by the disintegration of granules as demonstrated by sludge morphology. This selective inhibition is associated with variational sludge morphology, and the distribution of functional bacteria plays an important role in the feasibility and stability of PN/A treating acrylic fiber wastewater. As above, this study demonstrated the feasibility of PN/A for acrylic fiber wastewater treatment, but wastewater dilution or pre-treatment is still required for efficient nitrogen removal.

Excellent anammox performance driven by stable partial denitrification when encountering seasonal decreasing temperature

Effluent quality deterioration caused by seasonal temperature reductions in wastewater treatment systems using partial anammox technology is a challenge that cannot be ignored. Here, relationships of denitrification and anammox under decreasing temperature were investigated in an anoxic moving bed biofilm reactor (MBBR). Compared with stable partial-denitrification (NO3– → NO2–), the NO2– reduction to N2 was considerably inhibited when the temperature decreased, conversely helping to the competition of NO2– for anammox. Namely, this transformation provided sufficient substrates for anammox bacteria. Although the TIN removal decreased slightly, anammox contribution was robustly maintained at 91.3 ± 6.6 %, even increased. High-throughput sequencing results revealed that anammox bacteria were enriched (0.56 % to 1.22 %). Moreover, qPCR results showed that increased ratio of hzsB/(nirK + nirS) further supported anammox gained an enhancement. This study demonstrated partial-denitrification/anammox process using anoxic MBBR could maintain stable autotrophic nitrogen removal contribution when encountering temperature decrease, providing a new perspective on the application of mainstream anammox.

The granular sludge membrane bioreactor: A new tool to enhance Anammox performance and alleviate membrane fouling

Anammox process has be widely concerned as a novel biological nitrogen removal technology because of reduced operating costs and environmental friendliness. However, the long start-up time and operational instability of Anammox process has hindered its wide applications. A new-type hybrid granular sludge membrane bioreactor (GSMBR) combining sludge granulation with membrane filtration was developed for accelerating start-up of Anammox process and stabilizing its operation as well as alleviating the membrane fouling. Anammox start-up period was shortened to 43 days, the maximum nitrogen removing rate and efficiency reached 0.83 kg N m−3 d−1 and 97 % respectively on 115 d. Moreover, the membrane fouling is relatively light. The maximum transmembrane pressure only reached 32 kPa on 115 d, when there were still several visible micropores and channels on the fouled membrane surface. The mature brownish-red Anammox granular sludge mostly sized at 0.5–1.0 mm. The specific population shift from norank Alphaproteobacteria to Candidatus Brocadia was found. Three Anammox species were present in the mature sludge and uncultured Candidatus Brocadia sp. was the predominated bacteria. GSMBR was proven to be a robust reactor for enhancing Anammox performance. This study was helpful to promote the engineering applications of Anammox process in treating the high-strength nitrogenous wastewaters.

Extensive data analysis and kinetic modelling of dosage and temperature dependent role of graphene oxides on anammox

Anaerobic ammonium oxidation (anammox) is carbon friendly biological nitrogen removal process, and recently more focus is given to improving the anammox activity. Because of its high adsorption and modifiability, graphene and its derivative in wastewater treatment have received much attention. However, the specific effects and mechanisms of graphene oxide (GO) and reduced graphene oxide (RGO) on anammox are still controversial. Extensive data analysis was performed to explore the effects of GO and RGO on anammox. Statistical analysis revealed that 100 mg/L GO significantly promoted the anammox process, while 200 mg/L of GO inhibited the anammox process. The promotion of anammox performance under the influence of RGO was dependent on the temperature. The Logistic model was utilized for depicting the variation of nitrogen removal efficiency under promoting dosage of graphene oxides. A neural network model-based analysis was performed to reach anammox's potential mechanisms under the influence of two graphene oxides. Spearman correlation analysis showed that GO and RGO had significant positive correlations with nitrogen removal efficiency and specific anammox activity (p < 0.01), especially for RGO. In addition, the abundance of Planctomycetes and Nitrospirae was positively correlated with the addition of graphene oxides. This work comprehensively unraveled the role of graphene oxide materials on the anammox process and provided practical directions for the enhancement of anammox.

The effects of salinity changes on anammox performance: The response rule and tolerance mechanism.

Some wastewaters contain high concentrations of ammonia coexisting with large amounts of salt, which might negatively affect the anaerobic ammonium oxidation (anammox) process. In this study, the performance of the anammox process under different saline conditions was investigated using an upflow anaerobic sludge bed-anammox system. After long-term operating for 275 days, the results indicated that the nitrogen removal efficiency remained high under the 0-40 g NaCl/L, and low salinity (15 g NaCl/L) substantially promoted specific anammox activity. Affected by the saline environment, the appearance, color, and shape of sludge notably changed, and the amount of extracellular polymeric substances gradually increased with increasing salinity, which might be one of the reasons for the strong salt tolerance of the system. Chloroflexi and Planctomycetes were the dominant strains under long-term salinity, and Brocadiaceae_g_ unclassified exhibited halophilic characteristics. The redundancy analysis results showed that the concentration of influent NH4 + -N and salinity were the main environmental factors affecting the microbial community of the system. PRACTITIONER POINTS: Provides data to support the maximum value for salinity wastewater treatment with anammox processes' tolerance of 40 g NaCl/L. EPS changes may be responsible for the response to salinity challenges and provide direction for high salinity wastewater treatment. Brocadiaceae_g_ unclassified exhibited a halophilic quality. And it can be focused on to improve treatment efficiency.

Insights into deterioration and reactivation of a mainstream anammox biofilm reactor response to C/N ratio

In-depth knowledge of the deterioration and reactivation of the anaerobic ammonium oxidation (anammox) induced by carbon-to-nitrogen (C/N) is still lacking. Herein, the anammox performance was investigated in an anaerobic sequence biofilm batch reactor fed with low-strength partial nitration effluent in the range of C/N ratio from 0.5 to 3. The anammox was hardly deteriorated at C/N lower than 1.5, while became worsen if C/N was above 2.0. The specific anammox activity (SAA) experiments showed an 85% decrease of SAA at C/N of 3.0 compared with the maximum value (C/N:0). However, anammox capacity was rapidly recovered once influent C/N was adjusted back to zero. Moreover, C/N also highly affected the composition, structure and function of extracellular polymeric substance of the anammox biofilm. High-throughput sequencing revealed a close correlation between C/N change and microbial structure shift. Finally, the potential inhibition and restoration mechanism of the C/N-dependent anammox were proposed based on metagenomic analysis. This research provides some insights into the reinstatement of a mainstream anammox biofilm process after it is interrupted by high C/N influent.

Glutamate Enhances Osmoadaptation of Anammox Bacteria under High Salinity: Genomic Analysis and Experimental Evidence.

An osmoprotectant that alleviates the bacterial osmotic stress can improve the bioreactor treatment of saline wastewater. However, proposed candidates are expensive, and osmoprotectants of anammox bacteria and their ecophysiological roles are not fully understood. In this study, a comparative analysis of 34 high-quality public metagenome-assembled genomes from anammox bacteria revealed two distinct groups of osmoadaptation. Candidatus Scalindua and Kuenenia share a close phylogenomic relation and osmoadaptation gene profile and have pathways for glutamate transport and metabolisms for enhanced osmoadaptation. The batch assay results demonstrated that the reduced Ca. Kuenenia activity in saline conditions was substantially alleviated with the addition and subsequent synergistic effects of potassium and glutamate. The operational test of two reactors demonstrated that the reduced anammox performance under brine conditions rapidly recovered by 35.7-43.1% as a result of glutamate treatment. The Ca. Kuenenia 16S rRNA and hydrazine gene expressions were upregulated significantly (p < 0.05), and the abundance increased by approximately 19.9%, with a decrease in dominant heterotrophs. These data demonstrated the effectiveness of glutamate in alleviating the osmotic stress of Ca. Kuenenia. This study provides genomic insight into group-specific osmoadaptation of anammox bacteria and can facilitate the precision management of anammox reactors under high salinity.

Saline short-term shock and rapid recovery on anammox performance

Anaerobic ammonia oxidation (anammox) is an environmental-friendly biological nitrogen removal process, which has been developed as a promising technology in industrial wastewater treatment. However, anammox nitrogen removal under high saline conditions still faces many challenges. This study investigated the performance of anammox sludge under saline short-term shock and the strategy of rapid recovery. Salinity concentration, saline exposure time, and NaCl/Na2SO4 ratio were selected as three critical factors for short-term shock. The activity inhibition of anammox sludge were tested by using response surface methodology (RSM). Our results showed that, compared with the NaCl/Na2SO4 ratio, the salinity concentration and saline exposure time were the significant factor causing the anammox inhibition. The addition of glycine betaine (GB) in moderate amounts (0.1–5 mM) was found to help anammox to resist in relative low saline shock intensities (e.g., IC25 and IC50), with the activity retention rate of 94.7%. However, glycine betaine was not worked effectively under relatively high saline shock intensities (e.g., complete inhibition condition). Microbial community analysis revealed that Brocadiaceae accounted for only about 7.6%–13.2% at inhibited conditions. Interestingly, 16S rRNA analysis showed that the abundance of activated Brocadiaceae remarkably decreased with time after high-level saline shock. This tendency was consistent with the results of qPCR targeted hzsA gene. Finally, based on quorum sensing, the anammox activity was recovered to 93.5% of original sludge by adding 30% original sludge. The study realized the rapid recovery of anammox activity under complete inhibition, promoting the development and operation of salt-tolerant anammox process.

The role of magnetite (Fe3O4) particles for enhancing the performance and granulation of anammox

In this study, two lab-scale sequencing batch reactors each with an effective volume of 2.3 L were operated as C-AMX (no carrier addition) and M-AMX (magnetite carrier added) for 147 days with synthetic wastewater at an NLR range of 0.19–0.47 kgN/m3/d. The long-term effect of magnetite on the granulation and performance of anammox bacteria in terms of nitrogen removal and other essential parameters were confirmed. In phase I (1–24 days), M-AMX took approximately 12 days to obtain a nitrogen removal rate (NRR) above 80 % of the initial input nitrogen. Although free nitrous acid inhibited the reactor at a high concentration at the onset of phase III, the NRR of M-AMX recovered about 3.7 times faster than that of C-AMX. In addition, it was confirmed that the M-AMX granules had a dense and compact structure compared to C-AMX, and the presence of the carrier promoted the development of these resilient granules. While the measured microbial stress gradually increased in C-AMX reactor, a vice versa was observed in the M-AMX reactor as granulation proceeded. Compared to other alternative iron-based carrier particles, the stable crystal structure of magnetite as a carrier created a mechanism where filamentous bacteria groups were repelled from the granulation hence the microbial stress in the M-AMX in the final phase was 61.54 % lower than that in the C-AMX. The iron rich environment created by the magnetite addition led to Ignavibacteria, (a Feammox bacteria) increasing significantly in the M-AMX bioreactor.