Effect of micro-aeration on syntrophic and methanogenic activity in anaerobic sludge.

Micro-aeration was shown to improve anaerobic digestion (AD) processes, although oxygen is known to inhibit obligate anaerobes, such as syntrophic communities of bacteria and methanogens. The effect of micro-aeration on the activity and microbial interaction in syntrophic communities, as well as on the potential establishment of synergetic relationships with facultative anaerobic bacteria (FAB) or aerobic bacteria (AB), was investigated. Anaerobic sludge was incubated with ethanol and increasing oxygen concentrations (0-5% in the headspace). Assays with acetate or H2/CO2 (direct substrates for methanogens) were also performed. When compared with the controls (0% O2), oxygen significantly decreased substrate consumption and initial methane production rate (MPR) from acetate or H2/CO2. At 0.5% O2, MPR from these substrates was inhibited 30-40%, and close to 100% at 5% O2. With ethanol, significant inhibition (>36%) was only observed for oxygen concentrations higher than 2.5%. Oxygen was consumed in the assays, pointing to the stimulation of AB/FAB by ethanol, which helped to protect the syntrophic consortia under micro-aerobic conditions. This highlights the importance of AB/FAB in maintaining functional and resilient syntrophic communities, which is relevant for real AD systems (in which vestigial O2 amounts are frequently present), as well as for AD systems using micro-aeration as a process strategy. KEY POINTS: •Micro-aeration impacts syntrophic communities of bacteria and methanogens. •Oxygen stimulates AB/FAB, maintaining functional and resilient consortia. •Micro-aeration studies are critical for systems using micro-aeration as a process strategy.

Machine learning applied for metabolic flux-based control of micro-aerated fermentations in bioreactors.

Various bio-based processes depend on controlled micro-aerobic conditions to achieve a satisfactory product yield. However, the limiting oxygen concentration varies according to the micro-organism employed, while for industrial applications, there is no cost-effective way of measuring it at low levels. This study proposes a machine learning procedure within a metabolic flux-based control strategy (SUPERSYS_MCU) to address this issue. The control strategy used simulations of a genome-scale metabolic model to generate a surrogate model in the form of an artificial neural network, to be used in a micro-aerobic fermentation strategy (MF-ANN). The meta-model provided setpoints to the controller, allowing adjustment of the inlet air flow to control the oxygen uptake rate. The strategy was evaluated in micro-aerobic batch cultures employing industrial Saccharomyces cerevisiae yeast, with defined medium and glucose as the carbon source, as a case study. The performance of the proposed control scheme was compared with a conventional fermentation and with three previously reported micro-aeration strategies, including respiratory quotient-based control and constant air flow rate. Due to maintenance of the oxidative balance at the anaerobiosis threshold, the MF-ANN provided volumetric ethanol productivity of 4.16 g·L-1 ·h-1 and a yield of 0.48 gethanol .gsubstrate-1 , which were higher than the values achieved for the other conditions studied (maximum of 3.4 g·L-1 ·h-1 and 0.35-0.40 gethanol ·gsubstrate-1 , respectively). Due to its modular character, the MF-ANN strategy could be adapted to other micro-aerated bioprocesses.

Metabolic fluxes-oriented control of bioreactors: a novel approach to tune micro-aeration and substrate feeding in fermentations.

BACKGROUND: Fine-tuning the aeration for cultivations when oxygen-limited conditions are demanded (such as the production of vaccines, isobutanol, 2-3 butanediol, acetone, and bioethanol) is still a challenge in the area of bioreactor automation and advanced control. In this work, an innovative control strategy based on metabolic fluxes was implemented and evaluated in a case study: micro-aerated ethanol fermentation. RESULTS: The experiments were carried out in fed-batch mode, using commercial Saccharomyces cerevisiae, defined medium, and glucose as carbon source. Simulations of a genome-scale metabolic model for Saccharomyces cerevisiae were used to identify the range of oxygen and substrate fluxes that would maximize ethanol fluxes. Oxygen supply and feed flow rate were manipulated to control oxygen and substrate fluxes, as well as the respiratory quotient (RQ). The performance of the controlled cultivation was compared to two other fermentation strategies: a conventional "Brazilian fuel-ethanol plant" fermentation and a strictly anaerobic fermentation (with ultra-pure nitrogen used as the inlet gas). The cultivation carried out under the proposed control strategy showed the best average volumetric ethanol productivity (7.0 g L-1 h-1), with a final ethanol concentration of 87 g L-1 and yield of 0.46 gethanol g substrate -1 . The other fermentation strategies showed lower yields (close to 0.40 gethanol g substrate -1 ) and ethanol productivity around 4.0 g L-1 h-1. CONCLUSION: The control system based on fluxes was successfully implemented. The proposed approach could also be adapted to control several bioprocesses that require restrict aeration.

Anaerobic digestion of wastewater rich in sulfate and sulfide: effects of metallic waste addition and micro-aeration on process performance and methane production.

This work explores the effect of two metallic wastes (mining wastes, MW; fly ashes, FA) and micro-aeration (MA) on the anaerobic digestion of wastewater which is rich in sulfate and sulfide. Two initial COD concentrations (5,000 and 10,000 mg/L) were studied under both conditions in batch systems at 35 °C, with a fixed COD/SO42- ratio = 10, with 100 mg/L of S2-. It was observed that the use of MW and FA in the assays with an initial COD concentration of 10,000 mg/L resulted in a simultaneous increase in COD removal, sulfate removal, sulfide removal and methane generation, while MA only improved the COD and sulfide removals in comparison with the control system. On the contrary, the use of MW, FA or MA in systems with initial COD concentrations equal to or lower than 5,000 mg/L did not show any improvement with respect to the control system in terms of COD removal, sulfate removal or methane generation, with only sulfide removal being positively affected by MW and FA.

Simultaneous nitrification-denitrification and microbial community profile in an oxygen-limiting intermittent aeration SBBR with biodegradable carriers.

To enhance the startup and efficient simultaneous nitrification and denitrification for sewage treatment, sequencing batch biofilm reactors (SBBRs) partially coupled with rice husk were established and operated under various intermittent micro-aeration cycles (IMCs) and COD/N ratios under oxygen-limiting intermittent aeration conditions. Experimental results showed that the increase of IMCs with non-aeration/micro-aeration mode of (8 h/4 h)1 to (2 h/1 h)4 in a 12 h-cycle accelerated the startup performance and improved NH4+-N and COD removal. NH4+-N, TN and COD removal efficiencies were 98.7 ± 0.9, 89.2 ± 5.2 and 82.9 ± 6.7% at COD/N ratio of 7.6 with the highest IMCs in SBBR, respectively. Higher TN removal efficiencies of 87.2 ± 4.0 and 58.1 ± 3.5% were also achieved at lower COD/N ratio of 5.6 and 2.8, respectively. In SBBRs with various IMCs, facultative denitrifier like genus Acinetobacter and solid-phase denitrifier belonging to Comamonadaceae family were enriched. However, aerobic denitrifiers with function of heterotrophic nitrification like Paracoccus were favored to enrich under higher IMCs condition, and more anoxic denitrifiers like sulfur-based autotrophic denitrifier Thiothrix and heterotrophic denitrifiers like Pseudomonas and Methyloversatilis were observed at lower IMCs condition. Autotrophic nitrifier (Nitrosomonas and Nitrosipra) and heterotrophic nitrifiers both contributed to the efficient nitrification.

Performance evaluation of micro-aerobic hydrolysis of mixed sludge: Optimum aeration and effect on its biochemical methane potential.

This study evaluated the performance of a micro-aerobic hydrolysis of mixed sludge and its influence as a pretreatment of this waste for its subsequent anaerobic digestion. Three experimental series were carried out to evaluate the optimum micro-aeration levels in the range from 0.1 to 0.5 air volume/min.reactor volume (vvm) and operation times within the range of 24-60 h. The maximum methane yield [35 mL CH4/g volatile suspended solids (VSS) added] was obtained for an aeration level of 0.35 vvm. This methane yield value increased 114% with respect to that obtained with the non-aerated sludge. In the micro-aeration process carried out at an aeration level of 0.35 vvm, increases in soluble proteins and total sugars concentrations of 185% and 192% with respect to their initial values were found, respectively, after 48 h of aeration. At the above micro-aerobic conditions, soluble chemical oxygen demand (CODS) augmented 150%, whereas VSS content decreased until 40% of their initial respective values. Higher COD increases and VSS decreases were found at 60 h of micro-aeration, but the above parameters did not vary significantly with respect to the values found at 48 h.

Regulating microbial redox reactions towards enhanced removal of refractory organic nitrogen from wastewater.

Biotechnology for wastewater treatment is mainstream and effective depending upon microbial redox reactions to eliminate diverse contaminants and ensure aquatic ecological health. However, refractory organic nitrogen compounds (RONCs, e.g., nitro-, azo-, amide-, and N-heterocyclic compounds) with complex structures and high toxicity inhibit microbial metabolic activity and limit the transformation of organic nitrogen to inorganic nitrogen. This will eventually result in non-compliance with nitrogen discharge standards. Numerous efforts suggested that applying exogenous electron donors or acceptors, such as solid electrodes (electrostimulation) and limited oxygen (micro-aeration), could potentially regulate microbial redox reactions and catabolic pathways, and facilitate the biotransformation of RONCs. This review provides comprehensive insights into the microbial regulation mechanisms and applications of electrostimulation and micro-aeration strategies to accelerate the biotransformation of RONCs to organic amine (amination) and inorganic ammonia (ammonification), respectively. Furthermore, a promising approach involving in-situ hybrid anaerobic biological units, coupled with electrostimulation and micro-aeration, is proposed towards engineering applications. Finally, employing cutting-edge methods including multi-omics analysis, data science driven machine learning, technology-economic analysis, and life-cycle assessment would contribute to optimizing the process design and engineering implementation. This review offers a fundamental understanding and inspiration for novel research in the enhanced biotechnology towards RONCs elimination.

Effect of aeration/micro-aeration on lignocellulosic decomposition, maturity and seedling phytotoxicity during full-scale biogas residues composting.

With the accelerated construction of biogas plants, the amount of biogas residues are expanding. Composting has been widely implemented to deal with biogas residues. Aeration regulation is the main factor affecting the post-composting treatment of biogas residues as high-quality fertilizer or soil amendment. Therefore, this study aimed to investigate the impact of different aeration regulations on full-scale biogas residues compost maturity by controlling oxygen concentration under micro-aeration and aeration conditions. Results showed that micro-aerobic extended the thermophilic stage of 17 days at above 55 ℃ and facilitated the mineralization process of organic nitrogen into nitrate nitrogen to retain higher N nutrition levels compared to aerobic treatment. For biogas residues with high moisture, aeration should be regulated at different full-scale composting stages. Total organic carbon (TOC), NH4+-N, NO3--N, total potassium (TK), total phosphorus (TP) and the germination index (GI) could be used to evaluate stabilization, fertilizer efficiency and phytotoxicity of compost with frequent monitoring times. However, seedling growth trials were still necessary in full-scale composting plants when changing of composting process or biogas residues feedstock.

Micro-aeration and leachate recirculation for the acceleration of landfill stabilization: Enhanced hydrolytic acidification by facultative bacteria.

The long duration of landfill stabilization is one of the challenges faced by municipalities. In this paper, a combination of micro-aeration and leachate recirculation is used to achieve rapid degradation of organic matter in landfill waste. The results showed that the content of volatile fatty acids (VFAs) in the hydrolysis phase increased significantly and could enter the methanogenic phase quickly. Until the end of the landfill, the removal rates of chemical oxygen demand (COD), total phosphorus (TP) and ammonia nitrogen (NH4+-N) by micro-aeration and leachate recirculation reached 80.17 %, 48.30 % and 48.56 %, respectively, and the organic matter degradation rate reached 50 %. Micro-aeration and leachate recirculation enhanced the abundance of facultative hydrolytic bacteria such as Rummeliibacillus and Bacillus and the oxygen tolerance of Methanobrevibacter and Methanoculleus. Micro-aeration and leachate recirculation improved the organic matter degradation efficiency of landfill waste by promoting the growth of functional microorganisms.

Micro-aeration: an attractive strategy to facilitate anaerobic digestion.

Micro-aeration can facilitate anaerobic digestion (AD) by regulating microbial communities and promoting the growth of facultative taxa, thereby increasing methane yield and stabilizing the AD process. Additionally, micro-aeration contributes to hydrogen sulfide stripping by oxidization to produce molecular sulfur or sulfuric acid. Although micro-aeration can positively affect AD, it must be strictly regulated to maintain an overall anaerobic environment that permits anaerobic microorganisms to thrive. Even so, obligate anaerobes, especially methanogens, could suffer from oxidative stress during micro-aeration. This review describes the applications of micro-aeration in AD and examines the cutting-edge advances in how methanogens survive under oxygen stress. Moreover, barriers and corresponding solutions are proposed to move micro-aeration technology closer to application at scale.

Micro-aeration assisted with electrogenic respiration enhanced the microbial catabolism and ammonification of aromatic amines in industrial wastewater.

Improvement of refractory nitrogen-containing organics biodegradation is crucial to meet discharged nitrogen standards and guarantee aquatic ecology safety. Although electrostimulation accelerates organic nitrogen pollutants amination, it remains uncertain how to strengthen ammonification of the amination products. This study demonstrated that ammonification was remarkably facilitated under micro-aerobic conditions through the degradation of aniline, an amination product of nitrobenzene, using an electrogenic respiration system. The microbial catabolism and ammonification were significantly enhanced by exposing the bioanode to air. Based on 16S rRNA gene sequencing and GeoChip analysis, our results indicated that aerobic aniline degraders and electroactive bacteria were enriched in suspension and inner electrode biofilm, respectively. The suspension community had a significantly higher relative abundance of catechol dioxygenase genes contributing to aerobic aniline biodegradation and reactive oxygen species (ROS) scavenger genes to protect from oxygen toxicity. The inner biofilm community contained obviously higher cytochrome c genes responsible for extracellular electron transfer. Additionally, network analysis indicated the aniline degraders were positively associated with electroactive bacteria and could be the potential hosts for genes encoding for dioxygenase and cytochrome, respectively. This study provides a feasible strategy to enhance nitrogen-containing organics ammonification and offers new insights into the microbial interaction mechanisms of micro-aeration assisted with electrogenic respiration.

Enhanced landfill process based on leachate recirculation and micro-aeration: A comprehensive technical, environmental, and economic assessment.

The landfill is still the primary waste treatment method in developing countries. Due to the long stability time and long-term occupation of a large amount of land, the landfill poses a significant threat to the ecological environment and affects the process of urbanization. This study conducted a landfill simulation reactor (LSR) experiment to achieve rapid landfill stabilization through micro-aeration and leachate recirculation. More than 60 % of the degradable organic carbon in the enhanced process (LSR-IV contains 24 % of the retained carbon) can be relatively quickly converted to a gaseous state, which is nearly half higher than the degradation efficiency of the traditional process (LSR-I contains 59.3 % of the retained carbon). A comprehensive environmental assessment is developed for the enhanced process, and better environmental benefits are obtained from the whole landfill process. Compared with conventional treatment process, the enhanced process is applied to the actual landfill to analyze the economic cost. In terms of the total cost, the enhanced process cost (60.1 CNY) is about 44 % lower than the conventional landfill process cost (107.6 CNY). The enhanced process saves nearly half of the time cost and reduces the cost of land acquisition. This study can provide a reference for governmental and municipal administrations to carry out the technological transformation of traditional landfills from the aspects of technology, economy and environment.

Employing micro-aeration in anaerobic digestion of poultry litter and wheat straw: Batch kinetics and continuous performance.

In this study, different micro-aeration (MA) strategies for anaerobic digestion (AD) of poultry litter (PL) and wheat straw (WS) were examined. MA at different stages (pretreatment, middle, pretreatment plus middle, and daily) in batch AD of WS showed that daily MA had the highest increase (16.5 %) of the cumulative methane yield (CMY) compared to the control. Batch co-digestion (Co-AD) of WS and PL with daily MA obtained a furtherly improved (15.1 %) CMY of 225.44 N mL CH4/g vS added. The modified Gompertz model and Cone model were good in fitting the methane yield kinetics of MA engaged AD process (R2 greater than 0.99). Daily MA shortened the lag phase of Co-AD by 3.4 %. The sequencing batch reactor for the Co-AD of WS and PL showed an increased (21.5 %) daily methane yield when 0.5-h/d MA was employed. The results provided support for the application of micro-aeration in the AD of agricultural wastes.

Roles of micro-aeration on enhancing volatile fatty acids and lactic acid production from agricultural wastes.

Micro-aeration was proven to be an environmentally friendly strategy for efficiently enhancing volatile fatty acids (VFAs) and lactic acid (LA) production. The roles of micro-aeration on mono-digestion of swine manure (SM) for VFAs production and co-digestion of SM with corn silage (CS) for LA production were investigated, respectively. In this study, micro-aeration increased the maximum VFAs concentration by 20.3% to 35.71 g COD/L, and shortened the time to reach the maximum from 18 days to 10 days. Micro-aeration limited the conversion of LA into VFAs, leading to LA accumulation effectively to be 26.08 g COD/L. Microbial community analysis suggested that Clostridium and Terrisporobacter were always the dominant bacteria with or without micro-aeration for VFAs production, but the relative abundance increased notably during the same period. However, Bifidobacterium, which could use the higher productivity metabolism pathway, i.e., Bifidum pathway to produce LA, increased from lower than 1% to 22.9% by micro-aeration.

Upgrade the high-load anaerobic digestion and relieve acid stress through the strategy of side-stream micro-aeration: biochemical performances, microbial response and intrinsic mechanisms.

In high-load anaerobic digestion such as in kitchen waste, side-stream micro-aeration (SMA) shows excellent operational performance to direct micro-aeration (DMA). It immediately restores the acidification to stability. Methanogenic performance remained stable when organic load ratios (OLR) was further increased to 5.5 g VS/L. Enhanced enzyme activity, microbial aggregation, and proliferation of bacteria and archaea were observed in SMA. The results indicates that SMA enriched Methanosaeta (relative abundance exceeded 93%) and induced the change of the main methanogenic pathway to acetoclastic methanogenesis. Mechanisms was further explored by using metagenomic analysis, and the results show SMA avoids mass formation of ROS (reactive oxygen species) by cycling the aerated slurry, and retains benefits of trace O2 on material and energic metabolism, which poses great application potentials and deserves further investigation.

Coupling of membrane-based bubbleless micro-aeration for 2,4-dinitrophenol degradation in a hydrolysis acidification reactor.

Micro-aeration hydrolysis acidification (HA) is an effective method to enhance the removal of toxic and refractory organic matter, but the difficulty in stable dosing control of trace oxygen limits its wide application. Membrane-based bubbleless aeration has been proved as an ideal aeration method because of its higher oxygen transfer rate, more uniform mass transfer, and lower cost than HA. However, the available information on its application in HA is limited. In this study, membrane-based bubbleless micro-aeration coupled with hydrolysis acidification (MBL-MHA) was exploited to investigate the performance of 2,4-dinitrophenol (2,4-DNP) degradation via comparing it with bubble micro-aeration HA (MHA) and anaerobic HA. The results indicated that the performances in MBL-MHA and MHA were higher than those in HA during the experiment. 2,4-DNP degradation rates under redox microenvironments caused by counter-diffusion in MBL-MHA (84.43∼97.28%) were higher than those caused by co-diffusion in MHA (82.41∼94.71%) under micro-aeration of 0.5-5.0 mL air/min. The 2,4-DNP degradation pathways in MBL-MHA were nitroreduction, deamination, aromatic ring cleavage, and fermentation, while those in MHA were hydroxylation, aromatic ring cleavage, and fermentation. Reduction/oxidation-related, interspecific electron transfer-related species, and fermentative species in MBL-MHA were more abundant than that in MHA. Ultimately, more reducing/oxidizing forces formed by more redox proteins/enzymes from these rich species could enhance 2,4-DNP degradation in MBL-MHA.

Effects of micro-aeration on microbial niches and antimicrobial resistances in blackwater anaerobic digesters.

Anaerobic digestion (AD) of source-diverted blackwater (toilet flush) at ambient room temperature presents challenges for fast hydrolysis of particulate matters. This study investigated the effect of different micro-aeration dosages for blackwater AD. Sequencing batch reactors were operated at ambient room temperature (22 ± 1°C) with micro-aeration (0, 5, 10, 50, and 150 mg O2 g-1 CODfeed per cycle) and gradually reduced hydraulic retention times from 5 d to 2 d. The methanogenesis efficiencies were greater at low oxygen dosages (i.e., 0, 5, 10) while the volatile fatty acids (VFAs) accumulated more at high oxygen dosages (i.e., 50, 150). Microbial communities were significantly different under different oxygen dosages (p<0.05), with segregation of microbial ecological niches in low and high oxygen dosage communities. The low-oxygen-dosage niche (0, 5, and 10 mg g-1 CODfeed) was inhabited by fermenting and syntrophic bacteria (e.g., Cytophaga, Syntrophomonas) and methanogens (e.g., Methanobacterium, Methanolinea, Methanosaeta). The high-oxygen-dosage niche (50 and 150 mg g-1 CODfeed) had significantly (p<0.05) more facultative anaerobic bacteria (Ignavibacteriales and Cloacamonales), and aerobic bacteria (Rhodocyclales). Moreover, blackwater can be a source of antimicrobial resistance genes (ARGs), which are affected by different oxygen dosages. The ARG variation correlated with the microbial community composition (p<0.05). Low-oxygen-dosage communities contained a higher prevalence of mobile gene elements (intI1 and korB) and tetM, ermB, sul1, sul2, and blaCTX-M than the high-oxygen-dosage communities, indicating that oxygen dosage influenced the prevalence of populations carrying ARGs. These findings suggest that application of micro-aeration to AD can be used to control ARG profiles.

Enhancement of anaerobic digestion digital twin through aerobic simulation and kinetic optimization for co-digestion scenarios.

An upgraded digital twin of the Anaerobic Digestion Model 1 is proposed to enhance its industrial applicability and range of use. Through the optimization and generalization of kinetic coefficients toward co-digestion reactors simulation and insertion of new equations for the complete biokinetics modeling of H2S, the proposed model can predict more precisely the exiting biogas fractions comprehensive of H2S and O2 without any parametric adjustment. Moreover, it is proposed a new function representing the oxygen-methanogens. The model has been validated through the comparison with other literature models and with experimental data coming both from the literature and from an industrial plant. The comparisons show its flexibility and industrial applicability. Finally, an optimization of the methane content through oxygen rate adjustment is proposed, increasing CH4 content of 4%vol. The mathematical model has been built using Python™, which makes it easily spreadable and usable.

Enhanced nitrogen removal by partial nitrification-anammox process with a novel high-frequency micro-aeration (HFMA) mode: Metabolic interactions among functional bacteria.

A novel high-frequency micro-aeration (HFMA) mode with aeration frequency of 15 times/h and DO concentration lower than 0.5 mg/L was proposed. Advanced partial nitrification-anammox (PN-A) performance was achieved in a two-stage sequencing batch reactor-integrated fixed-film activated sludge reactor with the HFMA mode. When treating wastewater with carbon/nitrogen ratio of 3, the abundance of NO2--N oxidation related genes decreased, and the genes carried out NO3--N reduction and carbon source consumption were up-regulated. These variations in microbial metabolism brought more NO2--N substrate for the subsequent anammox process, and consumed part of the accumulated organic matter and NO3--N. Thus, the HFMA conditions eventually promoted the expression of anammox bacteria with NH2OH as an intermediate metabolite and the substance exchange activity of anammox bacteria. The changes in microorganisms lead to increase in the nitrite accumulation rate, nitrogen removal efficiency and abundance of anammox bacteria (16.34%, 18.71% and 5.92%, respectively).

Micro-aeration with hollow fiber membrane enhanced the nitrogen removal in constructed wetlands.

The nitrogen removal efficiency in constructed wetlands (CWs) was largely affected by the dissolved oxygen (DO). In this study, micro-aeration with different numbers of hollow fiber membrane modules (HFMEs) was adopted to increase the oxygen availability and improve the nitrogen removal efficiency in CWs under different air temperatures and different hydraulic retention time (HRT). Compared to the plant oxygen release (ROL) of wetland plants and traditional mechanical aeration, HFME increased the oxygen availability and enhanced the nitrogen removal efficiency in CWs. The COD and NH4+-N removal efficiencies increased with the increase of the HMFE. TN removal efficiency was increased by 8~16% after the application of HFME in CWs in the high-temperature stage. However, less HFME in CW-M1 realized the highest TN removal efficiency in low- and medium-temperature stages. At low temperature after 4-day HRT, the DO concentration respectively reached 6.25 mg L-1 and 3.25 mg L-1 in the upper zone and the bottom of CW-M1. The TN removal efficiencies in the upper zone of CW-M1 (60.69%) and the bottom of CW-M1 (64.98%) were all significantly higher than those in the upper zone of CK (35.98%) and the bottom of CK (39.9%). In addition, the microbial biomass and community analyses revealed that CW-M1 showed the most nitrifying bacteria and the best metabolic activity of bacteria. HEMF in CW-M1 also increased the nitrifying capacity from 0.12 to 0.46 mg kg-1 h-1. The application of HFME in CWs accelerated the nitrification process by enhancing nitrifying bacteria and less HFME realized the highest TN removal efficiency through nitrification-denitrification processes. Graphical abstract The application of hollow fiber membrane modules in CWs enhanced the pollutants (TN and COD) removal efficiency in the process of biological nitrification-denitrification and increased the number of nitrifying bacteria.