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The addition of exogenous materials is a commonly reported method for promoting the anaerobic digestion (AD) of sludge. However, most exogenous materials are nano-sized and their use encounters problems relating to a need for continuous replenishment, uncontrollability and non-recyclability. Here, magnetic porous microspheres (MPMs), which can be controlled by magnetic forces, were prepared and used to enhance the methanogenesis of sludge. It was observed that the MPMs were spherical particles with diameters of approximately 100 µm and had a stable macroporous hybrid structure of magnetic cores and polymeric shells. Furthermore, the MPMs had good magnetic properties and a strong solid-liquid interfacial electron transfer ability, suggesting that MPMs are excellent carriers for methanogenic consortia. Experimental results showed that the addition of MPMs increased methane production and the proportion of methane in biogas from AD by 100.0 % and 21.2 %, respectively, indicating the MPMs notably enhanced the methanogenesis of sludge. Analyses of variations in key enzyme activities and electron transfer in sludge samples with and without MPMs in AD revealed that the MPMs significantly enhanced the activities of key enzymes involved in hydrolysis, acidification and methanation. This was achieved mainly by enhancing the extracellular electron transfer to strengthen the proton motive force on the cell membrane, which provides more energy generation for methanogenic metabolism. A careful examination of the variations in the morphology, pore structure and magnetism of the MPMs before and after AD revealed that the MPMs increased the prevalence of many highly active anaerobes, and that this did not weaken the magnetic performance. The microbial community structure and metatranscriptomic analysis further indicated that the acetotrophic methanogens (i.e., Methanosaeta) were mainly in a free state and that CO 2 -reducing methanogens (i.e., Methanolinea and Methanobacterium) mainly adhered to the MPMs. The above synergistic metabolism led to efficient methanogenesis, which indicates that the MPMs optimised the spatial ecological niche of methanogenic consortia. These findings provide an important reference for the development of magnetic porous materials promoting AD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Anaerobic digestion (AD) is a promising technique for sludge treatment and resource recovery. Metals are very important components of sludge and can have substantial effects on its complex nature and microbial activity. However, systematic reviews have not addressed how metals in sludge affect AD and how they can be regulated to improve AD. This paper comprehensively reviews the effects of metals on the AD of sludge from both abiotic and biotic perspectives. First, we introduce the contents and basic characteristics (e.g., chemical forms) of intrinsic metals in sewage sludge. Then, we summarise the main mechanism by which metals influence sludge properties and the methods for removing metals and thus improving AD. Next, we analyze the effects of both intrinsic and exogenous metals on the enzymes and microbial communities involved in anaerobic bioconversion, focusing on the types, critical concentrations and valence states of the metals. Finally, we propose ideas for future research on the roles of metals in the AD of sludge. In summary, this review systematically clarifies the roles of metals in the AD of sludge and provides a reference for improving AD by regulating these metals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The recovery of renewable bioenergy from anaerobic digestion (AD) of sludge is a promising method to alleviate the energy problem. Although methane can be effectively recovered through sludge pretreatment by cation exchange resin (CER), the simultaneous enhancement of hydrogen and methane generation from AD using CER has not been extensively investigated. Herein, the effect of CER on the sequential recovery of hydrogen and methane and the corresponding mechanisms were investigated. When CER is introduced, the maximum increases for the hydrogen and methane production are 104.7 % and 35.3 %, respectively, confirming the sequential enhancement effects of CER on the hydrogen and methane production. Analyses of the variations in the main biochemical components with and without the effect of CER demonstrate that CER promotes sludge organic solubilisation, hydrolysis, and acidification in both hydrogen- and methane-production stages. Moreover, investigations of variations in the solid-liquid interfacial thermodynamics and removal rates of main multivalent metals of sludge reveal that the ion exchange reactions between the CER and sludge in the hydrogen-production stage provide the direct driving force of effective contact between bacteria and organic particulates. Additionally, the residual effect of the CER during methane production reduces the energy barrier for mass transfer and provides a driving force for this transfer. Further analyses of the microbial community structure and metagenomics indicate that CER directly drives the enrichment of hydrogen-producing bacteria (+ 15.1 %) and key genes encoding enzymes in the hydrogen-production stage. Moreover, CER indirectly induces the enrichment of methane-producing anaerobes (e.g. Methanosaeta: + 16.7 %, Methanosarcina: + 316.5 %); enhances the bioconversion of different substrates into methyl-coenzyme M; and promotes the metabolism pathway of acetoclastic process and CO 2 reduction in the methane-production stage. This study can provide valuable insights for simultaneously enhancing the production of hydrogen and methane from AD through sequential recovery., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

As a by-product of industry, waste iron scraps (WIS) are low-cost and widely available, which was potential for the development of iron-assisted anammox. In this study, the feasibility of adding WIS to enhance the nitrogen removal of the anammox process (also called WIS-assisted anammox) was demonstrated. Results indicated that the WIS-assisted anammox reactors performed a 15-35% higher nitrogen removal efficiency than that of the control. Compared to the sludge from the control, the sludge from the WIS-assisted anammox reactors had a higher iron content (78-113 g kg -1 SS) and a better specific anammox activity (10.8-15.5 mg N g -1 VSS h -1 ). The enhanced growth of the anammox bacteria (related to Ca. Kuenenia stuttgartiensis with 99% similarity) in the WIS-assisted anammox reactors was also confirmed by high-throughput sequencing and qPCR. Furthermore, the functional genes predicted by PICRUSt2 revealed a higher level of hydroxylamine oxidoreductase (hao)-like proteins expression of the biomass from the WIS-assisted anammox reactors, implying that the hydroxylamine-related anammox pathway was promoted. Additionally, the observation of cytoplasmic nitrate reductase (narG), copper-containing nitrite reductase (nirK), and nitric oxide reductase (norB) suggested that the introduction of WIS might promote the denitrification ability. This was correlated to the lower ΔNO 3 - /ΔNH 4 + ratio observed in these WIS-assisted anammox reactors. Overall, the WIS-assisted anammox offers a sustainable nitrogen removal process for wastewater treatment with waste iron recycling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Partial nitritation is necessary for the implementation of the mainstream anammox (anaerobic ammonium oxidation) process in wastewater treatment plants. However, the difficulty in outcompeting nitrite-oxidizing bacteria (NOB) at mainstream conditions hinders the performance of partial nitritation. The present work aimed to develop a high-rate partial nitritation process for low-ammonium wastewater treatment at low temperatures by seeding aerobic granules. Experimental results suggested that both stratified structure of nitrifiers developed in the granules and sufficient residual ammonium concentration (18-35 mg N L -1 ) in the bulk liquid contributed to efficient NOB repression. With the hydraulic retention time progressively shortened from 1.0 to 0.17 h, the influent nitrogen loading rate of the partial nitritation process reached 6.8 ± 0.4 kg N m -3 d -1 even at 10-15 °C. The high concentration (7.5 gVSS L -1 ) and activity (0.48 g N g -1 VSS d -1 at 11 °C) of granular sludge made the reactor possess an overcapacity evaluated by the ratio between the actual ammonium oxidation rate of the granules and their maximum potential. The overcapacity helped the reactor to face the adverse effect of decreasing temperatures. Overall, this work indicated the great potential of applying aerobic granules to achieve high-rate partial nitritation at mainstream conditions. Moreover, anammox bacteria with a relative abundance of 2.8% was also identified in the partial nitritation granules at the end of this study, suggesting that the granules provided a habitable niche for anammox bacteria growth. Note that these results cannot fully relate to the treatment of real domestic/municipal wastewater, they are a source of important information increasing the knowledge about low temperature partial nitrification., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

High-solid sludge anaerobic digestion leads to increased organic matters in digester supernatant, which promotes heterotrophic competition and reduces anaerobic ammonium oxidation (anammox) retention. This research demonstrated effective anammox retention by hydrocycloning during a two-stage deammonification. Anammox retention was evaluated by dividing large (>0.425 mm), medium (0.25 to 0.425 mm), and small (<0.25 mm) aggregate fractions via a sieve, whereby Candidatus Kuenenia abundance in each size aggregate was measured to be 16.8%, 5.0%, and 0.9% respectively. After hydrocyclone separation, large particles took up only 1.7% to 2.7% in the overflow discharge (upper discharge from the reactor), whereas its initial proportion was 19.4%, indicating limited anammox loss. The volume ratio change of large particles to total aggregates was defined for particle breakdown evaluation. Breakdown (23% to 32% large particles) occurred mainly during pumping (influenced by pump frequency and type), while little happened in the hydrocyclone. This research provided methods to use a sieve to evaluate anammox retention by hydrocyclone during high-solid sludge anaerobic digester supernatant deammonification, and information for reducing particle breakdown, pumping selection, and separation optimization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In this study, an integrated investigation to the microbial activities, extracellular polymeric substance (EPS), microbial community and function of anammox granular sludge (AnGS) was performed.Results showed that AnGS at 0.5-1.0 mm had the highest average specific anammox activity (SAA) of 345.9 mg NH 4 + -N·gVSS -1 ·d -1 , but AnGS at 1.0-1.5 mm with higher SAA might lead to better nitrogen removal efficiency. The content of slime EPS and SAA achieved positively correlation with R 2 of 98.11%, while protein/polysaccharide ratio of slime EPS and sludge volume index achieved negatively correlation with R 2 of 99.13%. Cadidatus Broccadia and Denitratisoma were positive correlations and most abundant in AnGS 0.5-1.0 mm of 20% and AnGS 1.0-1.5 mm of 37%, respectively. AnGS at 0.5-1.0 mm exhibited higher energy metabolism which mostly contributed to produce protein. The study provides new insights into the mechanisms of AnGS about 1 mm playing more important role in nitrogen removal performance., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Effects of sludge age on volatile fatty acids (VFAs) production and Phosphorus (P) release during anaerobic acidification of waste activated sludge (WAS) were investigated. Sequencing batch reactors (SBR) fed with simulating domestic sewage were applied to produce WAS of different sludge ages, and batch tests were used for anaerobic acidification. The maximum dissolved total organic carbon, release of  PO 4 3+ -P, and accumulation of acetate (C2), propionate (C3), butyrate (C4), and valerate (C5) decreased by 56.2%, 55.8%, 52.6%, 43.7%, 82.4% and 84.8%, respectively, as the sludge age of WAS increased from 5 to 40 days. Limited degradation of protein played a dominating role in decreasing DTOC and VFAs production. Moreover, the increase in molecular weight of organics and organic nitrogen content in the supernatant after acidification suggested that the refractory protein in WAS increased as sludge age extended. Although the production of C2, C3, C4, and C5 from WAS decreased as the sludge age increased, the proportions of C2 and C3 in VFAs increased, which might be due to the declined production of C5 from protein and the faded genus Dechlorobacter. Keeping sludge age of WAS at a relatively low level (<10 days) is more appropriate for anaerobic acidification of WAS as internal carbon sources and P resource., (Copyright © 2020. Published by Elsevier B.V.)

The influence of micron-sized silica particles on the distribution of organic matters in sludge and anaerobic degradation of macromolecular organic components (MOCs) in sludge was investigated. With the addition of micron-sized particles in the influent (VS/TS decreased gradually from 90.46 ± 0.21 % to 33.36 ± 0.17 %), the protein degradation percentage was significantly promoted while the polysaccharides degradation percentage was largely inhibited, resulting in the total MOCs degradation and methane production increasing firstly (with the promotion extent within 10 %) and then declining slightly, with the peak value at VS/TS of 56.03 ± 0.21 %. The shifted degradation percentage of protein and polysaccharides were caused by the significant changed distribution of organic matters in sludge. With the addition of micron-sized silica particles, the MW of EPS and secondary structure of protein in EPS changed little, which brought about little influence. While, the promoted extracellular protein content (so that the total protein content) and declined extracellular carbohydrates content (so that the total polysaccharides content), were found to be strongly correlated to the enhanced protein degradation and inhibited polysaccharides degradation. The results suggested that large amounts of grit in sludge might not be the main reason for lower degradability of sewage sludge in China., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Biomass detachment generally occurred in granular sludge systems. However, little is known about the influence of biomass detachment on the granules performing nitritation. Here, a granular sludge reactor with high loading rates (6.8 ± 0.4 kg N·m -3 ·d -1 ) was achieved at mainstream conditions. Though the low ratio control strategy was maintained, the deterioration of nitritation performance was observed after the further increase of air supply rates to 3.4 ± 0.2 L min -1 . In parallel with that, the loss of AOB and the proliferation of NOB was observed. Additionally, with the decrease of granules size and biomass concentration, the incomplete stratification of nitrifiers in the granules was confirmed by batch tests. All these results suggested that granules abrasion under the high shear stress conditions caused the detachment of external AOB and hence resulted in the deteriorated stratified structure of nitrifiers, which subsequently contributed to the proliferation of the internal NOB and the deterioration of nitritation. These findings highlight that the granules abrasion should be well controlled in the development of high-rate nitritation process with granular sludge., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Three semi-continuous anaerobic digesters with raw sludge (R1) or thermally hydrolyzed sludge (120 °C (R2) or 160 °C (R3)) were operated to investigate the effects of thermal hydrolysis on the metabolism of amino acids during high solid anaerobic digestion with sewage sludge. Thermal hydrolysis pretreatment (THP) decomposed protein in terms of hydrolytic amino acid in raw sludge by 8.90% and 26.69% under 120 °C and 160 °C, respectively. The decomposition of amino acids during THP was the main contributor to the final enhanced amino acids degradation in sewage sludge with THP after anaerobic digestion. The dominating bacterial genera related to amino acids degradation in R2 and R3 shifted to Fastidiosipila and Proteiniphilum/Tissierella, respectively, from Gelria in R1, influencing the utilization of amino acids, especially glutamic acid, glycine and proline. Different from 120 °C, THP at 160 °C played an important role in promoting amino acid metabolism during AD through the Stickland pathway by the bacteria belonged to order Clostridiales., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study investigated the operation mode of a step-feed anoxic/oxic (A/O) process with distribution of the carbon source from the anaerobic zone in terms of the treatment effects on sewage with low carbon and high nitrogen and phosphorus. After seven phases of operation, an optimal flow distribution ratio of 75%:25% was obtained from the anaerobic zone, and the concentrations of chemical oxygen demand, ammonia nitrogen, total nitrogen, and total phosphorous in the effluent were 20.8, 0.64, 14.2, and 0.89 mg/L, respectively. The presence of an internal reflux system in the deaeration zone improved the treatment. 16S rRNA high-throughput sequencing revealed that the microbial communities in aerobic zone I(O1) of the first-step A/O sludge were different from those in aerobic zone I (O2) of the second-step A/O sludge, whereas microbial communities of the seed sludge were similar to those in O2 of the second-step A/O sludge. The richness and diversity of microbial communities in O1 of the first-step A/O sludge samples were higher than those in O2 of the second-step A/O and seed sludge. At the optimal flow distribution ratio, the microbial abundance and treatment removal efficiency were the highest.

The propane hydrate formation was proposed to have potentials in realizing free-conditioning dewatering of sewage sludge with implications to simultaneous clean water extraction and highly efficient volume reduction. Primarily, the investigation on phase equilibrium of propane hydrates found that the organic components of sewage sludge promoted the propane hydrate formation in terms of decreasing equilibrium pressure by up to 19.2%, compared with that in pure water. Further, the feasibility of hydrate-based dewatering was verified through the observation of propane hydrate formation in sewage sludge and also the quality analysis of water generated from decomposition of up-floated formed hydrates. The formation of up-floated propane hydrates extracted water molecules from sewage sludge into homogeneous crystal phase, which actually excluded sludge particles from hydrate phase and realized the reduction of water in sludge phase. The efficiency of water conversion into hydrates was determined by monitoring propane pressure, which indicated that 14 batch runs decreased the water content of sludge from 98.81wt.% to 44.3wt.% under free-conditioning conditions. The chemical oxygen demand, total nitrogen and total phosphorus of hydrate-extracted water were measured to be 21 ± 1 mg/L, 10.5 ± 0.2 mg/L and 0.4 ± 0 mg/L, respectively, which reflected the excellent separation performance and also indicated that the hydrate-extracted water can be directly discharged without further treatments. Finally, the unit energy consumption of hydrate-based dewatering process based on a continuous operation mode was calculated to be 2673.96 kW h/t dry solid of sewage sludge, which was nearly half of that in thermal drying process. Therefore, the propane hydrate-based process is believed to maximize the green operation of enhanced sludge dewatering while minimizing the energy and additional material consumption., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

High-solid anaerobic digestion of sewage sludge achieves highly efficient volatile solid reduction, and production of volatile fatty acid (VFA) and methane compared with conventional low-solid anaerobic digestion. In this study, the potential mechanisms of the better performance in high-solid anaerobic digestion of sewage sludge were investigated by using 454 high-throughput pyrosequencing and real-time PCR to analyze the microbial characteristics in sewage sludge fermentation reactors. The results obtained by 454 high-throughput pyrosequencing revealed that the phyla Chloroflexi, Bacteroidetes, and Firmicutes were the dominant functional microorganisms in high-solid and low-solid anaerobic systems. Meanwhile, the real-time PCR assays showed that high-solid anaerobic digestion significantly increased the number of total bacteria, which enhanced the hydrolysis and acidification of sewage sludge. Further study indicated that the number of total archaea (dominated by Methanosarcina) in a high-solid anaerobic fermentation reactor was also higher than that in a low-solid reactor, resulting in higher VFA consumption and methane production. Hence, the increased key bacteria and methanogenic archaea involved in sewage sludge hydrolysis, acidification, and methanogenesis resulted in the better performance of high-solid anaerobic sewage sludge fermentation.

The gaps of data for evaluating toxicity of new or overloaded organic chemicals on activated sludge system resulted in the requirements for methodology of toxicity estimation. In this study, 24 aromatic chemicals typically existed in the industrial wastewater were selected and classified into three groups of benzenes, phenols and anilines. Their toxicity on activated sludge was then investigated. Two indexes of IC(50-M) and IC(50-S) were determined respectively from the respiration rates of activated sludge with different toxicant concentration at mid-term (24h) and short-term (30min) time intervals. Experimental results showed that the group of benzenes was the most toxic, followed by the groups of phenols and anilines. The values of IC(50-M) of the tested chemicals were higher than those of IC(50-S). In addition, quantitative structure-activity relationships (QSARs) models developed from IC(50-M) were more stable and accurate than those of IC(50-S). The multiple linear models based on molecular descriptors and K(ow) presented better reliability than single linear models based on K(ow). Among these molecular descriptors, E(lumo) was the most important impact factor for evaluation of mid-term toxicity., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

The effect of pH in the range of 3.0-11.0 on anaerobic fermentation of primary sludge (PS) was investigated at room temperature. The experimental results showed that the concentrations of soluble chemical oxygen demands (SCOD), soluble protein and carbohydrate and short-chain fatty acids (SCFAs) under alkaline conditions were significantly higher than those under other pHs. At fermentation time of 5 days, the average SCFAs concentration increased from 968 to 3511mg COD/L with the increase of pH from 3.0 to 10.0. However, further increasing pH to 11.0 resulted in the decrease of SCFAs. At pH 10.0, acetic, propionic and iso-valeric acids were the three main products, and the volatile suspended solids (VSS) reduction reached 38%. It was also observed that at any pH value investigated, there were obvious ammonia and phosphorus releases during fermentation. According to this study it is obvious that alkaline pH benefited the soluble organic carbon production from PS.

[Display omitted] • A single stage PN/A process treating low strength wastewater was started rapidly. • The biofilm growth process on porous polyurethane hydrogel carrier was studied. • Biomass entrapment by interconnected micropores dominated the initial colonization. • AOB was surface-attached growth, anammox colonies proliferated as embedded growth. Media-supported biofilm is a powerful strategy for growth and enrichment of slow-growing microorganisms. In this study, a single-stage nitritation-anammox process treating low-strength wastewater was successfully started to investigate the biofilm development on porous polyurethane hydrogel carrier. Suspended biomass migration into the carrier and being entrapment by its internal interconnected micropores dominated the fast initial colonization stage. Both surface-attached growth and embedded growth of microbes occurred during the following accumulation stage. Fluorescence in situ hybridization analysis of mature biofilm indicated that ammonium-oxidizing bacteria located at the outer layers featured a surface-attached growth, while anammox microcolonies housed in the inner layers proliferated as an embedded-like growth. In this way, the growth rate of anammox bacteria (predominated by Candidatus Kuenenia) could be 0.079 d-1. The anammox potential of the biofilm reactor reached 1.65 ± 0.3 kg/m3/d within two months. This study provides novel insights into nitritation-anammox biofilm formation on the porous polyurethane hydrogel carrier. [ABSTRACT FROM AUTHOR]

Achieving mainstream nitritation with aerobic granules is attractive based on increasing evidence but generally treating artificial low-ammonium wastewater. Real municipal wastewater is much more complex in composition, the behavior of the nitritation granules would be different when treating real municipal wastewater. Herein, the response of nitritation granules to influent shift from artificial low-ammonium (35–40 mg/L) wastewater to anaerobically pre-treated municipal wastewater (MWW pre-treated) was investigated at low temperatures. Results showed that MWW pre-treated caused the outgrowth of filamentous bacteria on the granule surface and developed into finger-like structures, which in turn resulted in the decrease of the overall granular sludge settleability. Batch-tests and microbial analysis indicated the functional and microbial differentiation between the newly formed fluffy exterior and the original compact granule. The fluffy exterior was dominated by genus Flavobacterium (66.6%) and primarily functioned as COD removal, whereas the nitrifiers (mainly Nitrosomonas) were still located in the compact core and performed nitritation. Moreover, the heterotrophs-dominated fluffy exterior hindered the oxygen transfer towards nitrifiers located in the compact granule and thereby facilitated the stable NOB repression in the granule particularly at low temperatures (<10 °C). Finally, gradual recovery of the granular sludge morphology and settleability occurred after the influent reverted to synthetic low-ammonium wastewater. Overall, this work demonstrated that the feeding of MWW pre-treated only caused morphological changes of the nitritation granules, but its structural and functional stability could be maintained stably. [Display omitted] • Granule-based nitritation was maintained treating anaerobically pretreated sewage. • COD removal occurred primarily in external flocs, while nitritation in compact core. • External flocs promoted NOB repression by increasing oxygen transfer resistance. [ABSTRACT FROM AUTHOR]

Biomass segregation between granules/biofilm and flocs is widespread in anammox-based processes. The segregation of biomass allows for easy control of processes stability. The goal of this study is to understand the biomass segregation in two anoxic anammox reactors respectively operated in nitrite-limited (R NO2) and ammonium-limited (R NH4) modes treating low-strength wastewater at 20 °C. Results showed that size-based biomass segregation was developed in both reactors. But the functional and population heterogeneity was more significant in the ammonium-limited anammox reactor. The activity and abundance of anammox bacteria in large granules were significantly higher than that in flocs under the ammonium-limited conditions. The large granules played a major role in nitrogen removal in R NH4. By contrast, both large granules and small flocs contributed significantly to the nitrogen loss in the nitrite-limited anammox reactor, since a large number of anammox bacteria existed in both granules and flocs. Besides, a number of Nitrospira -like NOB were also detected in both anoxic anammox reactors, which primarily inhabited in flocs seemingly droved by the availability of oxygen. But the abundance of Nitrospira in R NH4 was much higher than that in R NO2. All these results suggested that selective flocs removal would be necessary for R NH4 to improve its anammox performance but non-essential for R NO2. The two anammox reactors shared the predominant anammox species with the closest relative to Ca. Brocadia sp. 40 (98%). Unexpectedly, the anammox species grew faster in R NH4. But the microbial diversity and evenness was much greater in R NO2 , suggesting its higher functional stability. • The ammonium-limited and nitrite-limited anammox reactors were compared. • Anammox bacteria mainly enriched in granules, NOB preferred to live in flocs. • Anammox bacteria grew faster in the ammonium-limited reactor. • The nitrite-limited reactor possessed the higher microbial diversity and evenness. [ABSTRACT FROM AUTHOR]