Anaerobic Ammonium Oxidation Process Research Articles

Enhancement of the reactivation process of long-term starved anammox granular sludge with gravel balls: Microbial succession and metabolic impact

Anaerobic ammonium oxidation (Anammox) process is an economical and energy-efficient method of wastewater nitrogen removal. However, they are highly susceptible to starvation stress caused by sudden environmental changes. Rapid reactivation of starved anammox sludge is a crucial method to address seed sludge shortages and expand practical applications. This study investigated the impact of gravel balls on the reactivation of long-term starved anammox granular sludge (628 days). The results showed that gravel balls enhanced the recovery of nitrogen removal performance in starved anammox sludge, with nitrogen removal efficiency being 19.88% higher than the control group at the end of the recovery phase. The gravel balls also increased extracellular polymeric substance (EPS) secretion, contributing to the stability of the anammox system. Furthermore, the gravel balls promoted the proliferation of anammox bacteria, with the relative abundance of anammox bacteria reaching 38.25% on the 80th day. The analyses of microbial functions indicated that gravel balls facilitated cross-feeding and co-metabolism among microbes, while enhancing quorum sensing associated with anammox bacteria, forming a multifunctional community network centered on anammox bacteria. This indicates that gravel balls can effectively accelerate the reactivation process of long-term starved anammox sludge, aiding the reutilization of long-term starved anammox sludge.

Linking ecological niche and metabolic hierarchy in low-oxygen-driven anammox systems

Dissolved oxygen (DO) is regarded as a key regulatory factor for the practical engineering applications of anaerobic ammonium oxidation (anammox) processes, however, the adaptability and metabolic mechanisms of floc anammox consortia with low-oxygen as potential substrates involved in the holistic microbial metabolic system are still elusive. This study showed that the nitrogen removal efficiency (NRE) of floc anammox consortia was 16.45% and 35.71% higher at DO of 0.2–0.5 mg·L−1 than at DO <0.2 mg·L−1 and DO of 0.5–0.8 mg·L−1, respectively. Meanwhile, the activity of anammox bacteria (AnAOB) and ammonia-oxidizing bacteria (AOB) was the highest, representing 22.64 ± 3.20 mg N·g−1 V SS·d−1 and 103.22 ± 6.47 mg N·g−1 VSS·d−1, respectively. AnAOB applied the antioxidant enzymes (CCP, T-SOD) and Cbb3-type cytochrome c oxidase (Cbb3-type CcO) for oxygen detoxification and utilization. Moreover, AOB, aerobic/anoxic heterotrophic bacteria, denitrifying bacteria, etc. defined as the core members in anammox consortia could also consume DO and adjust the NH4+-N/NO2−-N ratio for AnAOB, establishing potent microbial interactions to relieve the pressure of DO on AnAOB and improve NRE. This study reveals that anammox consortia can utilize limited DO as a substrate to promote anammox processes, which advances the practical applications of anammox processes.

Effect of plant species on wastewater treatment performance of a subsurface vertical-flow constructed wetland with step-feeding at low temperature

To improve the treatment performance of constructed wetlands under low-temperature conditions, this study investigated the effects of plant species on wastewater treatment performance at low temperature and the associated microbiological characteristics in a subsurface vertical-flow constructed wetland (VFCW) with step-feeding. The results showed that the redox microenvironment in the VFCW filter with step-feeding could be restored and optimized by planting appropriate species that can tolerate low temperature, ensuring a high nitrification performance for the system. Correspondingly, the abundance and activity of three functional microbes (namely nitrifiers, denitrifiers, and anammox bacteria) increased to different degrees in the system, eventually ensuring ideal nitrogen removal by the VFCW. Compared with the VFCW planted with Phragmites australis and Acorus gramineus, the operation performance of the VFCW planted with Iris wilsonii could be recovered at low temperature, and its chemical oxygen demand, total phosphorus, total nitrogen, and ammonium nitrate removal rates could respectively reach 95.7%, 99.2%, 93.0%, and 94.4%, respectively. Moreover, nitrogen removal in the system relied on the nitrification/denitrification and partial denitrification - anaerobic ammonium oxidation processes. Nitrosomonas, Nitrospira, Thauera, and Candidatus Brocadia were the four dominant bacterial genera in the filter layer.

Autotrophic biological nitrogen removal in anammox system at elevated temperature: EPS regulation and metagenomic analysis

Carbon reduction in wastewater treatment industry has been attracted global attention. Anaerobic ammonium oxidation (anammox) process is known as a promising and energy-efficient technology. In this study, the effects of elevated temperature on anammox system were thoroughly investigated. Analyses of effluent and microbial community indicated that long-term exposure to 45℃ led to system collapse and a huge shift in community structure. Network analysis revealed that the sudden drop in abundance of AnAOB at 45℃ (from 36.19 % to 10.71 %) made them uncompetitive with heterotrophic bacteria. The decrease of EPS concentration (a minimum of 7.22 mg·g−1-VSS at 45℃) combined with the enhancement of EPS metabolism at higher temperature suggested a shift in the role of EPS from protective function at 35℃ to utilization as a carbon source for heterotrophic bacteria at higher temperatures. Through constructing the cross-feeding relationships associated with amino acids, polysaccharides, and cofactors, we found the dependence of heterotrophs on AnAOB became weaker at high temperature, and the disturbances in interspecific relationships might be responsible for the collapse of the anammox system. This work presents significant insights into the engineering of anammox process under high temperature.

Metagenomic evidence of a novel anammox community in a cold aquifer with high nitrogen pollution

The process of anaerobic ammonium oxidation by nitrite (anammox) is a globally essential part of N cycle. To date, 8 Candidatus genera and more than 22 species of anammox bacteria have been discovered in various anthropogenic and natural habitats, including nitrogen-polluted aquifers. In this work, anammox bacteria were detected for the first time in the groundwater ecosystem with high anthropogenic nitrogen pollution (up to 1760 mg NO3−-N/L and 280 mg NH4+-N/L) and low year-round temperature (7–8 °C) in the zone of a uranium sludge repository. Further metagenomic analysis resulted in retrieval of metagenome-assembled genomes of 4 distinct anammox bacteria: a new genus named Ca. Frigussubterria, new species in Ca. Kuenenia, and two strains of a new species in Ca. Scalindua. Analysis of the genomes revealed essential genes involved in anammox metabolism. Both strains of Ca. Scalindua chemeplantae had a high copy number of genes encoding the cold shock proteins CspA/B, which can also function as an antifreeze protein (CspB). Ca. Kuenenia glazoviensis and Ca. Frigussubterria udmurtiae were abundant in less N-polluted site, while Ca. Scalindua chemeplantae inhabited both sites. Genes for urea utilization, reduction of insoluble Fe2O3 or MnO2, assimilatory sulfate reduction, reactive oxygen detoxification, nitrate reduction to ammonium, and putatively arsenate respiration were found. These findings enrich knowledge of the functional and phylogenetic diversity of anammox bacteria and improve understanding of the nitrogen cycle in polluted aquifers.

Quorum sensing mediated response mechanism of anammox consortia to anionic surfactant: Molecular simulation and molecular evidence

The widespread use of surfactants raise challenges to biological wastewater treatment. Anaerobic ammonium oxidation (anammox) process has the potential to treat wastewater containing anionic surfactants, but the response of anammox consortia at the molecular level under long-term exposure is unclear. Using high-throughput sequencing and gene quantification, combined with molecular docking, the effect of sodium dodecyl sulfonate (SDS) on anammox consortia were investigated. Levels of reactive oxygen species (ROS) might be lower than the threshold of oxidative damage, while the increase of lactate dehydrogenase (LDH) represented the cell membrane damage. Decreased abundance of functional genes (hdh, hzsA and nirS) indicated the decrease of the anammox bacterial abundance. Trace amounts of N-acyl homoserine lactone (AHL, C6-HSL, C8-HSL and C12-HSL) contained in influent could induce endogenous quorum sensing (QS), which could regulate the correlation between functional bacteria to optimize the microbial community and strengthen the resistance of anammox consortia to SDS. In addition, the proliferation of disinfectant resistance genes might increase the environmental pathogenicity of sewage discharge. This work highlights the potential response mechanism of anammox consortium to surfactants and provides a universal microbial-friendly bioenhancement strategy based on QS.

Bibliometric analysis of wastewater treatment processes based on anaerobic ammonia oxidation process

In the realm of water treatment, the anaerobic ammonia oxidation (anammox) process stands out as one of the most innovative breakthroughs. Bibliometrics is an effective method for the evaluation of scientific results and the development of research trends in a particular field of study. Employing VOSviewer and CiteSpace, this study comprehensively analyzed scholarly articles and research trends related to anammox water treatment from 2014 to 2023. The findings revealed a total of 1225 articles in this domain, exhibiting an increasing trend every year in the world, while China emerges as the foremost contributor with close collaborations with the United States, Australia, and other nations. The examination of keyword co-occurrence and cluster network showcases coupled anammox-based reactor startup and opefrational strategies, nitrogen removal studies, nitrous oxide investigations and microbial community analyses are long-term academic endeavors. Furthermore, keyword burst and temporal network analysis,the operational control conditions, driving processes and analysis of microbial macrogenes for mainstream anammox in wastewater treatment plants need more research in the future to improve the feasibility of this technology in engineering applications. Additionally, a meticulous scrutiny of the top 20 landmark and highly cited research articles over the past decade underscores a few persistent intriguing themes, such as “mainstream anammox,” “biological nitrogen removal,” and “16 S rRNA genes,” In summary, this study contributes a nuanced understanding of the contemporary landscape and development trends within the field of anammox water treatment, which will shed light on the future research efforts.

Novel insights into intrinsic mechanisms of magnetic field on long-term performance of anaerobic ammonium oxidation process

The performance of an anaerobic ammonium oxidation (anammox) reactor with the magnetic field of 40 mT was systematically investigated. The total nitrogen removal rate was enhanced by 16% compared with that of the control group. The enhancing mechanism was elucidated from the improved mass transfer efficiency, the complicated symbiotic interspecific relationship and the improved levels of functional genes. The magnetic field promoted formation of the loose anammox granular sludge and the homogeneous and well-connected porous structure to enhance the mass transfer. Consequently, Candidatus Brocadia predominated in the sludge with an increase in abundance of 13%. Network analysis showed that the positive interactions between Candidatus Brocadia and heterotrophic bacteria were strengthened, which established a more complicated stable microbial community. Moreover, the magnetic field increased the levels of hdh by 26% and hzs by 35% to promote the nitrogen metabolic process. These results provided novel insights into the magnetic field-enhanced anammox process.

Sustainable approach of biological treatment of landfill leachate by Anaerobic Ammonium Oxidation: A review

An upsurge in living standards, rising industrialization and urbanization, the protection of water environment has become a priority. Anaerobic ammonium oxidation process has drawn a lot of attention since it demonstrated substantial advantages over conventional nitrogen removal techniques, including a 100% reduction in the amount of organic carbon required, a 60% reduction in the amount of aeration needed, and a 90% reduction in the amount of sludge produced. Effective treatment of landfill leachate is extremely important as leachate is a threat to the environment. Municipal waste management is still a challenging situation in developing countries. Uncontrolled waste disposal results in greenhouse gases emissions which worsens climate change as the leachate will pollute water bodies, soil and a significant air pollution which impacts on human health will be released. This paper reviewed several published research works in Scopus dealing with the leachate treatment by Anammox process combined with some other systems and highlighted some common challenges found with the application of this new technology. Treating landfill leachate resulted in an excellent ammonium NH4 +-N removal efficiency. However, it has been highlighted that most of the research reviewed reported some limitations of the technology on a small scale such as the low start-up time affecting the growth of bacteria in the reactors and the instability of the system when pH and temperature decrease. Biological treatment, Anammox method included, offers a cost-effective, eco-friendly, and an effective solution for nitrogen removal.

Harnessing the Potential of Extracellular Polymeric Substances in Enhancing ANAMMOX Processes: Mechanisms, Strategies, and Perspectives

Anaerobic ammonium oxidation (ANAMMOX) has emerged as a promising sustainable nitrogen removal technology that offers significant advantages over conventional nitrification–denitrification processes, such as reduced energy consumption, a 60% reduction in oxygen demand, and a 90% reduction in sludge production. However, the practical application of ANAMMOX is hindered by several challenges, including the slow growth of ANAMMOX bacteria, long start-up periods, and high sensitivity to environmental disturbances. Recent studies have highlighted the crucial role of extracellular polymeric substances (EPSs) in the formation, activity, and stability of ANAMMOX biofilms and granules. An EPS is a complex mixture of high-molecular-weight polymers secreted by microorganisms, mainly composed of polysaccharides, proteins, nucleic acids, and lipids. The diverse physicochemical properties and functional groups of EPSs enable them to serve as a structural scaffold, protective barrier, sorption site, electron shuttle, and nutrient source for ANAMMOX bacteria. This review aims to provide an overview of the latest research progress on harnessing the potential of EPSs to enhance the ANAMMOX process. The characteristics, compositions, and extraction methods of ANAMMOX-derived EPSs are summarized. The mechanisms of how EPSs facilitate the enrichment, immobilization, aggregation, and adaptation of ANAMMOX bacteria are elucidated. The strategies and effects of EPS supplementation on improving the performance and robustness of ANAMMOX reactors under various stresses are critically reviewed. The challenges and future perspectives of the EPS-mediated optimization of the ANAMMOX process are also discussed. This review sheds new light on exploiting EPSs as a renewable bioresource to develop more efficient and stable ANAMMOX applications for sustainable wastewater treatment.

Regulation of nitrogen removal pathways in sulfuric autotrophic denitrification combined with ANAMMOX

The process of sulfuric autotrophic denitrification combined with anaerobic ammonia oxidation (ANAMMOX) has attracted attention because of its efficient N removal capacity, low sludge yield and low operating cost. However, the NO2−-N generated by the sulfuric autotrophic partial denitrification (SAPDN) easily continues conversion to N2 in the coupled process. Thus, ANAMMOX cannot occur in the absence of NO2−-N. In this study, the regulation of N removal pathways was studied in the combined system. The N removal performance was evaluated for treating the industrial wastewater in NO3−-N and NH4+-N with the sulfuric autotrophic denitrification combined with the ANAMMOX process. The effects of influent nitrogen load, concentration ratio of NO3−-N/NH4+-N as well as S/N mass ratio on the transformation of nitrogen removal pathway were studied. SAPDN combined with ANAMMOX became the primary nitrogen removal pathway due to regulating the high nitrogen load of 0.83–0.96 kg/(m3·d), average ratio of NO3−-N/NH4+-N ranging from 1.38 to 1.70 in the influent and a low S/N ratio of 1.5. Moreover, the microbial analysis results indicated that Thiobacillus and Candidatus Brocadia became the main functional microbes when the primary nitrogen removal pathway was SAPDN combined with ANAMMOX.

Carbon emission calculation of three biological nitrogen removal processes: A case of rare earth element tailings wastewater in Southern Jiangxi, China

Global warming has been caused by greenhouse gases (GHGs) released by human activity. Traditional sewage treatment uses much energy to achieve discharge limits in the water treatment industry, producing many GHG emissions. In this study, the emission factor approach and mass balance method are used to calculate the carbon emission equivalent of nitrification and denitrification, anaerobic ammonia oxidation, and microalgae assimilation using a rare earth tailings (REEs) wastewater example. The results show that, among the three denitrification processes, the traditional nitrification and denitrification method has the greatest carbon emission equivalent (16.23 kgCO2/kgN). Direct causes include N2O emissions, which account for 48 % of the total carbon emission equivalent. The anaerobic ammonia oxidation process produces the carbon dioxide equivalent of 0.02 kgCO2/kgN. Carbon emission comparable to microalgal assimilation is −3.23 kgCO2/kgN. The anaerobic ammonia oxidation process can thus greatly reduce carbon emissions when compared to the conventional nitrification and denitrification processes, whilst microalgae assimilation can result in negative carbon emissions.

Discrepant effects of undissolved and dissolved fractions of biochar on the nitrogen removal performance of anaerobic ammonium oxidation process

Biochar has attracted increasingly attention in the biological wastewater treatment owing to its unique environmental behavior. However, the differential effects of undissolved and dissolved fractions of biochar (hereafter named as UDB and DB, respectively) on the anaerobic ammonium oxidation (anammox) process remain unclear. The study evaluated the nitrogen removal performance of anammox system in response to UDB and DB prepared at different pyrolysis temperatures (300 °C, 550 °C and 800 °C). The DB800-amended group displayed superior nitrogen removal performance with the total nitrogen removal efficiency (TNRE) of 92.8 ± 1.4 %. Compared with CK, TNRE was changed by +5.0 % (UDB300), +0.4 % (UDB550) and −22.3 % (UDB800) for UDB-amended groups, and by −16.0 % (DB300), −9.4 % (DB550), and +9.2 % (DB800) for DB-amended groups, respectively. The differences were closely related to the physicochemical properties of UDB and DB. Particularly, dissolved organic matter released by DB were tracked at molecular level, which revealed that CHO occupied the highest proportion of 70.3 % and lignin-like and carboxylic rich alicyclic molecules were dominant in DB800. This study would facilitate the understanding of the differences between the nitrogen removal performance of UDB and DB-amended anammox, thus providing deeper insights for the comprehensive assessment of the impacts of biochar on the anammox process.

Research progress of anaerobic ammonium oxidation (Anammox) process based on integrated fixed-film activated sludge (IFAS).

The integrated fixed-film activated sludge (IFAS) process is considered one of the cutting-edge solutions to the traditional wastewater treatment challenges, allowing suspended sludge and attached biofilm to grow in the same system. In addition, the coupling of IFAS with anaerobic ammonium oxidation (Anammox) can further improve the efficiency of biological denitrification. This paper summarises the research progress of IFAS coupled with the anammox process, including partial nitrification anammox, simultaneous partial nitrification anammox and denitrification, and partial denitrification anammox technologies, and describes the factors that limit the development of related processes. The effects of dissolved oxygen, influent carbon source, sludge retention time, temperature, microbial community, and nitrite-oxidising bacteria inhibition methods on the anammox of IFAS are presented. At the same time, this paper gives an outlook on future research focus and engineering practice direction of the process.

Deciphering behaviors of 6:2 chlorinated polyfluorinated ether sulfonate (alternative-PFOS) on anammox processes: Nitrogen removal efficiency and microbial adaptability

This study investigates the behaviors and effects of F-53B, an alternative to perfluorooctane sulfonate on anaerobic ammonium oxidation (anammox) processes. Results showed that the nitrogen removal efficiency (NRE) reached 83.8 % at a F-53B concentration of 0.5 mg·L−1, while NRE decreased to 66.9 % with 5 mg·L−1 of F-53B. The defluorination rates of 17.8 % (0.5 mg·L−1) and 9.3 % (5 mg·L−1) were observed, respectively, suggesting the occurrence of F-53B degradation. The relative abundance of Ca. Kuenenia decreased from 26.1 % to 16.2 % with the F-53B concentration increasing from 0.5 mg·L−1 to 5 mg·L−1. Meanwhile, Denitratisoma was selectively enriched with a relative abundance of 40.7 % at an F-53B concentration of 0.5 mg·L−1. Ca. Kuenenia could reduce reactive oxygen species induced by F-53B to maintain the balance of oxidative stress. This study gains insight into the behaviors and metabolic mechanisms of F-53B in anammox consortia, suggesting the feasibility of anammox processes for industrial wastewater.

Insight into microbial adaptability in continuous flow anaerobic ammonium oxidation process for low-strength sewage treatment

Organic matter concentration is a critical factor influencing the adaptability of anaerobic ammonium oxidation (anammox) bacteria to low-strength sewage treatment. To address this challenge and achieve stable anammox activity, a micro-aeration partial nitrification-anammox process was developed for continuous-flow municipal sewage treatment. Under limited ammonium conditions, the effective utilization of organics in denitrification promoted the stable accumulation of nitrite and enhanced anammox activity. This, in turn, led to enhanced nitrogen removal efficiency, reaching approximately 87.7%. During the start-up phase, the protein content of extracellular polymeric substances (EPS) increased. This enhanced EPS intensified the inhibitory effect of denitrifying bacteria (DNB) on nitrite-oxidizing bacteria through competition for nitrite, thereby facilitating the proliferation of anammox bacteria (AnAOB). Additionally, several types of DNB capable of utilizing slowly biodegradable organics contributed to the adaptability of AnAOB. These findings provide valuable insights for ensuring efficient anammox performance and robust nitrogen removal in the treatment of low-strength sewage.

Analyzing the nitrogen removal performance and cold adaptation mechanism of immobilized cold-acclimation ANAMMOX granules at low temperatures.

To improve the treatment performance of anaerobic ammonium oxidation (ANAMMOX) processes at low temperatures, the immobilized cold-acclimation ANAMMOX granules (R3) were prepared and their low-temperature nitrogen removal ability as well as the cold adaptation mechanism were analyzed. The results indicated that the total inorganic nitrogen (TIN) removal efficiency of R3 was significantly higher than that of R2 (cold-acclimation granules without immobilization) and R1 (common granules), especially at 11 ± 2 and 7±2°C (68% and 54%). These were attributed to the remarkable biomass retention capacity of R3, high up to 4.3-4.9mg/gVSS even at 5-18°C. Besides, higher protein (PN) content of tightly bound extracellular polymeric substances (TB-EPS) also facilitated microbial aggregation in R3. Meanwhile, R3 granules retained higher ANAMMOX activity and heme c content at 5-25°C. The original dominant ANAMMOX genus (Candidatus Kuenenia) in R3 kept higher abundance (49%-57%) at 23 ±2 and 16 ±2°C, whereas Candidatus Brocadia became the dominant ANAMMOX genus (25%-32%) in R3 at 11 ±2 and 7±2°C. Notably, different ANAMMOX genera in R3 may adapt to cold environment by regulating the expression of cold-stress proteins (CspA, CspB, PpiD, and UspA). PRACTITIONER POINTS: Immobilized cold-acclimation ANAMMOX granules showed higher nitrogen removal efficiency at 23°C → 5°C. Immobilization method effectively retained biomass (Candidatus Kuenenia and Candidatus Brocadia). Immobilization facilitated TB-EPS release and biological aggregation in cold-acclimation granules. Expression of cold-stress proteins in immobilized cold-acclimation granules was more active.

Insights into nitrogen removal from leather wastewater by anaerobic ammonium oxidation process: Performance and microbial structure

This study aimed to assess the suitability of anaerobic ammonium oxidation (anammox) process for leather wastewater treatment using two up-flow anaerobic sludge blanket bioreactors by pumping synthetic and real wastewater (named SR and RR), respectively. Both reactors were operated steadily with an average nitrogen removal efficiency of 94–96 % at chemical oxygen demand (COD) level of 100–200 mg·L−1, which were rapidly deteriorated as the COD level increased to 300 mg·L−1 even if at the same COD to total nitrogen (TN) (COD/TN) ratio. The reactor performances could restore after pumping with synthetic wastewater, and then continued to effectively treat leather wastewater. Furthermore, the relative abundance of Candidatus Kuenenia in reactor RR was increased by 34.8 % compared with the reactor SR on day 120. The results revealed that the anammox process can be used for the leather wastewater treatment, thus broadening the engineering application of anammox-based process in industrial wastewater.

Diversity of Anammox Bacteria from Landfill Treatment Plant Sludge in Tropical Area

The anaerobic ammonium oxidation (anammox) process is known as the warm process. Tropical areas have an advantage due to their consistent temperature throughout the year. This study analyzed the diversity of anammox bacteria in the tropical area using leachate sludge from a landfill as an inoculum in a filter bioreactor (FtBR) and observed nitrogen removal performance. Ammonium and nitrite concentrations of 70, 150, and 200 mg-N/L were delivered into the reactor continuously with hydraulic retention time (HRT) of 24 h and 12 h and run for 131 days at ambient tropical temperature (25–28 °C). High performance achieved with nitrogen removal rate (NRR), nitrogen removal efficiency (NRE), and ammonium conversion efficiency (ACE) were 0.866 kg-N/m3.d, 99.19%, and 98.90%, respectively. The cultivated leachate sludge could perform an anammox process with four anammox species, Candidatus Brocadia fulgida, Candidatus Brocadia sapporoensis, Candidatus Brocadia sp uncultured, Candidatus Jettenia sp with abundance 6.52%, 13.82%, 0.77%, and 0.69%, respectively. These findings contribute to the advancement of biotechnology in wastewater treatment, particularly in tropical countries, and highlight the potential for highly cost-effective technology.

Impacts of crude glycerol on anaerobic ammonium oxidation (Anammox) process in wastewater treatment

This work investigated the impact of a waste-derived carbon source, crude glycerol (CG), on Anammox. Batch bioassays were conducted to identify inhibitory component(s) in CG, and the relationship between Anammox activity and the concentration of CG, pure glycerol, and methanol were assessed. The results showed that the half-maximal inhibitory concentration of CG and methanol are 434.5 ± 51.8 and 143.0 ± 19.6 mg chemical oxygen demand (COD) L-1, respectively, while pure glycerol at 0–2283 mg COD L-1 had no significant adverse effect on Anammox. The results suggested methanol is the major inhibitor in CG via a non-competitive inhibition mechanism. COD/total inorganic nitrogen ratio of > 1.3 was observed to cause a significant Anammox inhibition (>20 %), especially at low substrate level. These results are valuable for evaluating the feasibility of using CG for nitrogen removal in water resource recovery facilities, promoting sustainable development.