Hollow Fiber Membrane Bioreactor Research Articles

Intensification and optimization of continuous hydrogen production by dark fermentation in a new design liquid/gas hollow fiber membrane bioreactor

A liquid/gas membrane bioreactor (L/G MBR) was developed to intensify the dark fermentation process. A hollow fiber membrane module was used to combine biohydrogen production, in situ liquid–gas separation and hydrogen producing bacteria retention in a single unit. The L/G MBR was seeded once and did not require further microbial input, as consistent average hydrogen yield of 0.97 ± 0.09 mol-H2/added mol-glucose and hydrogen production rate of 106.5 ± 10.6 mL-H2/L-medium/h were reached over a year. Different biogas extraction strategies showed that efficient in situ H2 extraction is possible without sweeping gas in the lumen of the fibers, thus facilitating H2 purification in an industrial setting. Modelling predicted an optimal hydrogen yield of 1.2 mol/mol-glucose added for a glucose concentration in the feed of 13.1 g/L, close to experimental hydraulic retention time of 8–10 h with an organic loading rate of 1.4 g-glucose/L-medium/h. No washout of hydrogen-producing bacteria was observed at low HRT (2 h), suggesting the possibility of further hydrogen production rate enhancement using an optimized organic loading rate. Acetate and butyrate were the main metabolites identified. Clostridium and Enterobacter dominated in the liquid outlet. The relative abundance of Clostridium pasteurianum increased with glucose concentration in the bioreactor, as opposed to Clostridium beijerinckii which was more abundant at low glucose concentration. The original hollow fiber L/G MBR configuration enabled the testing and selection of fermentation strategies that greatly simplified the implementation of the dark fermentation process by addressing its key operational bottlenecks. Indeed, the L/G membrane surface served as a support and reservoir for the hydrogen producing bacteria across a wide range of HRT conditions.

Manufacturing T cells in hollow fiber membrane bioreactors changes their programming and enhances their potency

ABSTRACT Engineered T cell therapies have revolutionized modern oncology, however processes for manufacturing T cell therapies vary and the impact of manufacturing processes On the cell product is poorly understood. Herein, we have used a commercially available hollow fiber membrane bioreactor (HFMBR) operated in a novel mode to demonstrate that T cells can be engineered with lentiviruses, grown to very high densities, and washed and harvested in a single, small volume bioreactor that is readily amenable to automation. Manufacturing within the HFMBR dramatically changed the programming of the T cells and yielded a product with greater therapeutic potency than T cells produced using the standard manual method. This change in programming was associated with increased resistance to cryopreservation, which is beneficial as T cell products are typically cryopreserved prior to administration to the patient. Transcriptional profiling of the T cells revealed a shift toward a glycolytic metabolism, which may protect cells from oxidative stress offering an explanation for the improved resistance to cryopreservation. This study reveals that the choice of bioreactor fundamentally impacts the engineered T cell product and must be carefully considered. Furthermore, these data challenge the premise that glycolytic metabolism is detrimental to T cell therapies.

Bioaugmentation with immobilized endophytic Penicillium restrictum to improve quorum quenching activity for biofouling control in an aerobic hollow-fiber membrane bioreactor treating antibiotic-containing wastewater

The effects of bioaugmentation with immobilized Penicillium restrictum on the removal efficiency of sulfamethoxazole (SMX), erythromycin (ERY) and tetracycline (TC) antibiotics as well as membrane biofouling was studied using hollow-fiber membrane bioreactor (HF-MBR). Bioaugmentation with P. restrictum led to a significant change in the antibiotic removal efficiency and relative abundance of aerobic microbial community, most probably as a result of its quorum quenching activity. Furthermore, in addition to its role in the increase of SMX and ERY removal efficiencies and the decrease of their sorption on solid phase, bioaugmentation significantly reduced the transmembrane pressure which in turn reduced membrane clogging. The most abundant phyla in sludge and biofilm samples in the presence of P. restrictum were observed to be Proteobacteria, Bacteroidetes and Firmicutes. Differences in bacterial compositions and their specificity in biodegradation of antibiotics in different reactors showed that bacteria were specifically selected under the pressure of antibiotics and growing fungus.

Estimating glucose diffusion coefficient of membranes for tissue engineering applications using Fick’s First Law

The idea of growing artificial tissues in bioreactors such as hollow fibre membrane bioreactors (HFMBs) has started years ago and preparation of biocompatible porous membranes and scaffolds has been attempted extensively. There also have been several studies on modelling glucose transport processes in HFMBs. However, there is little information available that discusses specifically the glucose diffusivity across tissue engineering membranes or scaffolds and, importantly, its dependence on the properties of the materials (i.e., membrane and scaffold). Therefore, the objective of this study is to deduce the glucose diffusivity across different membranes. Using Fick’s law and a diffusion cell in this study, we have determined glucose diffusion coefficients for two different membranes namely cellulose nitrate (CN) and polyvinylidene fluoride (PVDF). These membranes possessed similar pore size with varying thickness and were saturated with water and cell culture medium (CCM). The diffusion experiments were conducted in a thermostated water bath at either 27 or 37 ± 1°C. It was observed that PVDF membrane with greater thickness has a lower diffusivity than CN membrane when both were saturated in CCM and water at 37°C. From the results, we derive the significance of the decrease of diffusion coefficient with increasing thickness of the membranes.

Trypsin-Free Cultivation of 3D Mini-Tissues in an Adaptive Membrane Bioreactor.

The production of large scaffold-free tissues is a key challenge in regenerative medicine. Nowadays, temperature-responsive polymers allow intact tissue harvesting without needing proteolytic enzymes. This method is limited to tissue culture plastic with limited upscaling capacity and plain process control. Here, a thermoresponsive hollow fiber membrane bioreactor is presented to produce large scaffold-free tissues. Intact tissues, rich in cell-to-cell connections and ECM, are harvested from a poly(N-vinylcaprolactam) microgel functionalized poly(ether sulfone)/poly(vinylpyrrolidone) hollow fiber membrane by a temperature shift. The harvested 3D tissues adhere in successive cultivation and exhibit high vitality for several days. The facile adsorptive coating waives the need for extensive surface treatment. The research is anticipated to be a starting point for upscaling the production of interconnected tissues enabling new opportunities in regenerative medicine, large-scale drug screening on physiological relevant tissues, and potentially opening new chances in cell-based therapies.

Hollow fibre membrane bioreactor functionalized with GO‐cryogel 3D matrix promotes liver cell anchoring and their functional maintenance

AbstractThe liver support system (bioartificial liver, BAL) is a potentially efficient treatment during reversible acute liver disease or while waiting for a liver transplant. The main challenge in implementing BAL is a bioreactor, which can fulfil the needs of hepatocyte culture, mass transfer and immunobarriers. In the present study, polymer nanocomposite hollow fibre membranes (HFMs) were fabricated using polyethersulphone (P) as a base polymer, vitamin E TPGS (T) as an additive and carboxylated carbon nanotubes (C) as a filler. These PTC HFMs were functionalized with cryogel (Cr), with or without carboxylated graphene oxide (G), known as PTC‐Cr or PTC‐CrG HFMs, respectively. These HFM substrates were characterized and evaluated for hemocompatibility, cytotoxicity and cellular functionality. Among all the HFM substrates, PTC‐CrG HFMs showed superior hemocompatibility and minimal cytotoxicity. Furthermore, a liver cell bioreactor was developed and studied using PTC‐CrG HFMs. The goat primary liver cells were seeded in a bioreactor and cultured in continuous mode. The cell functionality was better maintained in the continuous mode as compared to that in the batch culture. The three‐dimensional PTC‐CrG HFMs‐based bioreactor can be used for liver cell bioreactor, bioartificial liver system, drug testing/toxicology metabolic study and mass cell culture.

Biohydrogen production in a continuous liquid/gas hollow fiber membrane bioreactor: Efficient retention of hydrogen producing bacteria via granule and biofilm formation

The aim of this work was to develop a continuous liquid/gas membrane bioreactor (L/G MBR), i.e. a fermenting module including hollow fibers membrane for L/G separation, for biohydrogen production by dark fermentation. Originally seeded with sludge from a wastewater treatment plant, the L/G MBR underwent a complete stop for eight months. It was then operated without further reseeding. In the present experiment, performed 551 days after the last reseeding, average hydrogen yield of 1.1 ± 0.2 mol per mol glucose added and hydrogen productivity of 135 ± 22 mL/L/h were reached, with acetate and butyrate as the main metabolite products. DNA sequence analysis revealed that Clostridium beijerinckii, Clostridium pasteurianum and Enterobacter sp. were dominant in liquid outlet, in a biofilm on the surface of the hollow fibers and in microbial granules. The L/G MBR has potential for the concentration and the long-term maintenance of an active hydrogen-producing bacterial community without need for reseeding.

On modelling of glucose transport in hollow fibre membrane bioreactor for growing three‐dimensional tissue

AbstractHollow fibre membrane bioreactors (HFMBs) have been shown to overcome the diffusion limitation of nutrients (e.g., glucose) from the hollow fibres (lumens) to the porous regions of a scaffold (extracapillary space). However, direct monitoring of glucose diffusion inside the HFMBs is almost impossible because of their small size; thus, various computational modelling frameworks have been developed in the past. These models have defined that the glucose diffusivity in the cell culture medium used in the HFMBs was similar to the diffusivity in water. Similarly, other assumptions have been made that do not represent the nutrient transport processes in the HFMB accurately. In addressing these issues, a mathematical model is presented in this paper, where we employ experimentally deduced effective glucose diffusivities of tissue engineering membranes and scaffolds with and without cells along with glucose diffusivity in cell culture medium. The governing equations are non‐dimensionalized, simplified and solved numerically. The results demonstrate the roles of various dimensionless numbers (e.g., Péclet and Damköhler numbers) and non‐dimensional groups of variables on determining the glucose concentration especially in the scaffold region. The result of this study is expected to help optimize designs of HFMB as well as carry out more accurate scaling analyses.

TISSUE ENGINEERING BIOREACTORS: POTENTIAL APPLICATIONS AND SCALE UP STRATEGY

Tissue engineering bioreactors have been used in order to achieve production of artificial tissue, increasing cell proliferation capacity and yield and/or in vitro tissue/disease modelling. Although it is still discussing how to obtain functional and vascular tissue with these bioreactors, preclinical and clinical studies are ongoing. Tissue engineering bioreactors have been used as lab-scale bioreactors until now. Crucial potential application areas can be created by increasing the production capacity and bioprocess efficiency of these bioreactors. In this review, recent bioreactor technologies such as spinner flask, rotating wall/bed, hollow fiber membrane, perfusion and mechanical stimuli bioreactors are briefly presented in terms of their potential applications in medical field especially in the scope of scale-up approaches such as bubble column, stirred tank, membrane, air lift, fluidized packed and bed bioreactors.
 Peer Review History: 
 Received 1 May 2020; Revised 11 June; Accepted 6 July, Available online 15 July 2020
 UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency.
 Received file 
 
 Average Peer review marks at initial stage: 6.0/10
 Average Peer review marks at publication stage: 8.0/10
 Reviewer(s) detail:
 Name: Dr. Sally A. El-Zahaby
 Affiliation: Pharos University in Alexandria, Egypt
 E-mail: sally.elzahaby@yahoo.com
 
 Name: DR Viney Chawla
 Affiliation: University Institute of Pharmaceutical Sciences
 Baba Farid University of Health Sciences
 Sadiq Road, Faridkot-Punjab 151203
 E-mail: drvineychawla@gmail.com
 
 Comments of reviewer(s): 
 

Development of photochemical integrated submerged membrane bioreactor for textile dyeing wastewater treatment.

A pilot-scale photocatalytic membrane bioreactor (PMBR) was developed for the treatment of textile dyeing wastewater. The PMBR is made of mild steel rectangular reactor of photocatalytic unit and polyethersulphone submerged hollow fibre membrane bioreactor unit with the working volume of about 20L. For easy recovery, the tungsten oxide (WO3) and WO3/1% graphene oxide (GO)-powdered photocatalyst were made into bead and immersed in photocatalytic reactor. Graphene oxide incorporation has shown better results in decolourisation and degradation when compared with WO3 alginate alone. The incorporation of GO into WO3 minimises the recombination of photogenerated electron-hole pairs. The operating conditions such as 3h of contact time for photocatalysis reaction (WO3/1% GO), 10h hydraulic retention time for MBR and 100kPa of transmembrane pressure were optimised. Chemical oxygen demand removal efficiency of 48% was attained with photocatalysis, and the removal efficiency was further increased up to 76% when integrated with MBR. The colour removal efficiency after photocatalysis was 25% further increased up to 70% with MBR. Complete total suspended solid removal has been achieved with this hybrid system.

Analysis of elastohydrodynamics and nutrient transport through deformable porous scaffold inside a hollow fiber membrane bioreactor

Hydrodynamics and nutrient transport in a hollow fiber membrane bioreactor is studied by developing a two-dimensional mathematical model in Cartesian coordinates. In a more realistic scenario, the scaffold is considered to be elastic and deformable, which undergoes deformation with the applied pore pressure. A mixture model is used to deal with the scaffold matrix, cells, and the fluid present in the scaffold region. The method of lubrication theory is incorporated when the aspect ratio of the lumen is small. The nutrient transport in the scaffold region is assumed to be governed by advection–diffusion–reaction mass balance due to the presence of cells and by advection–diffusion in the lumen and porous membrane. Analytical solution of the coupled system is presented for a short time scale where the cell growth, death, or differentiation is neglected. The results obtained focus on the effect of various parameters on the fluid flow, solid deformation, and consumption of nutrients due to different kinds of cells. It is observed that the deformation of the scaffold matrix increases monotonically with the flow rate supplied to the bioreactor. This behavior ensures that one can adjust the fluid flux to achieve optimum deformation in favor of cell growth and avoid damage of the scaffold. Moreover, a general criterion for the distribution of adequate nutrient concentration inside the bioreactor is developed to prevent the formation of the necrosis region inside the scaffold. Accordingly, the current investigation helps to arrive at suitable parameter combinations to monitor and control an ongoing experiment for optimum cell growth.

Hysteresis effect on backwashing process in a submerged hollow fiber membrane bioreactor (MBR) applied to membrane fouling mitigation

Hysteresis effect on backwashing in a submerged MBR was investigated with dead-end hollow fiber membranes. The out-of-step changes in TMP and flux is the real hysteresis effect which is common but easily overlooked. Methods of visualization and ultrasonic spectrum analysis were implemented. The results showed that fouling layer is just the culprit of hysteresis effect. Fouling level and fiber length were determined as two key factors that affect hysteresis effect by data and model derivation. Moreover, a hysteresis evaluation index “τbw” is proposed to quantify the result of TMP vs time. The relationship between influence factors and “τbw” is interactive. A linear relationship between fouling level and “τbw” was found as well as an extreme value between fiber length and “τbw”. A lower fouling level (lower backwashing flow) and optimal backwashing duration will be helpful for an effective backwashing no matter for membrane fouling control or energy cost reduce.

Biogas purification through membrane bioreactors: Experimental study on siloxane separation and biodegradation

Sewage biogas valorization to different energy applications is hampered by the presence of volatile methyl siloxanes. Despite the high operating costs, adsorption onto activated carbon is the most implemented technology for siloxane removal from biogas. In order to purify biogas sustainably, the current work explores the diffusion of siloxanes (octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane) together with other biogas impurities (limonene, toluene and hexane) through polydimethylsiloxane membranes. Abiotic tests revealed transport efficiencies above 75% towards a clean air stream for most compounds, although the transport of the most hydrophobic pollutants was challenged when water was circulated through the shell side of the membrane. Moreover, the performance of a hollow-fiber membrane bioreactor, inoculated with anerobic active sludge, was evaluated towards biogas purification in anoxic conditions. Toluene and limonene were successfully degraded, hexane’s removal efficiency was positively correlated with the residence time, and siloxanes removal was achieved up to 21%. CO2 was detected in the outlet gas as the mineralization product as well as some byproducts from the degradation of limonene and siloxanes. The presence of 1% of O2 in the gas, as a strategy to substitute NO3−, efficiently supported high removal for volatile organic compounds and moderate for siloxanes, which would ultimately reduce the operating costs of the technology.

Modelling of hollow fiber membrane bioreactor for mammalian cell cultivation using computational hydrodynamics.

The hollow fiber membrane bioreactor (HFMB) has been investigated for the cultivation of mammalian Chinese hamster ovary cell expansion. The experiments were carried out in Petri's dishes and in the hollow fiber membrane bioreactor having 20 fibers (S2025 from FiberCell Systems). The approach to HFMB modelling which combines the model of cell growth kinetics and hydrodynamics has been proposed. The hydrodynamic model is made using ANSYS Fluent software. The mathematical model of HFMB was developed, allowing the study of the hydrodynamics into the lumen and the extracapillary spaces, the filtration through the membrane fiber with the cell expansion on outer membrane surface. The direct nutrient medium flow variant into the extracapillary space was suggested. Based on the numerical simulations, the optimal parameters were selected for daily changes in the medium flow-rate into the lumen space. The HFMB scaling up was performed for the larger size HFMB (60 fibers).

High-rate anaerobic decolorization of methyl orange from synthetic azo dye wastewater in a methane-based hollow fiber membrane bioreactor

Anaerobic biological techniques are widely used in the reductive decolorization of textile wastewater. However, the decolorization efficiency of textile wastewater by conventional anaerobic biological techniques is generally limited due to the low biomass retention capacity and short hydraulic retention time (HRT). In this study, a methane-based hollow fiber membrane bioreactor (HfMBR) was initially inoculated with an enriched anaerobic methane oxidation (AOM) culture to rapidly form an anaerobic biofilm. Then, synthetic azo dye wastewater containing methyl orange (MO) was fed into the HfMBR. MO decolorization efficiency of ∼ 100 % (HRT = 2 to 1.5 days) and maximum decolorization rate of 883 mg/L/day (HRT = 0.5 day) were obtained by the stepwise increase of the MO loading rate into the methane-based HfMBR. Scanning electron microscopy (SEM) and fluorescence in situ hybridization (FISH) analysis visually revealed that archaea clusters formed synergistic consortia with adjacent bacteria. Quantitative PCR (qPCR), phylogenetic and high-throughput sequencing analysis results further confirmed the biological consortia formation of methane-related archaea and partner bacteria, which played a synergistic role in MO decolorization. The high removal efficiency and stable microbial structure in HfMBR suggest it is a potentially effective technique for high-toxic azo dyes removal from textile wastewater.

Characterization of the bubbly flow in a hollow fiber membrane bioreactor

In membrane bioreactors (MBRs), aeration is widely used for fouling mitigation. To characterize the bubbly flow in MBRs, employing a solution that properly mimics the sludge behavior is essential. Therefore, this work investigates (i) the influence of the aerator pore size on the bubbly flow in xanthan gum solution, and (ii) the effect on the bubbly flow of using water as the continuous fluid and of the absence of the membrane module, conditions sometimes employed as model systems for MBRs. A MBR with a hollow fiber membrane module and a sintered glass plate for the aeration system was studied. Image analysis of the flow was made to assess key bubble parameters: area, circularity, Feret diameter, quantity, and velocity. The bubbly flow formed in the xanthan gum solution presented a lower turbulence level, differing greatly from the one formed in water. Nevertheless, for both fluids, the aerator with smaller pores generated a higher quantity of small bubbles, indicating a larger area of contact with the membrane surface. Such bubbles presented a higher level of size reduction with the presence of the membrane module and higher stability (higher circularity under similar velocities). This suggests that smaller bubbles should present higher antifouling activity.

Treatment of high strength industrial wastewater with membrane bioreactors for water reuse: Effect of pre-treatment with aerobic granular sludge on system performance and fouling tendency

In this study, the treatment of citrus wastewater with membrane bioreactors (MBRs) under different configurations was investigated for water reuse. In particular, one MBR and one aerobic granular sludge MBR (AGS + MBR) bench scale plants were operated for 60 days. The experimental campaign was divided into two periods. In Phase I, a conventional hollow fiber MBR was employed for the treatment of the raw high strength wastewater, whereas in Phase II a combination of in-series reactors (AGS + MBR) was adopted for the treatment of the high strength citrus wastewaterThe results demonstrated that both plant configurations enabled very high COD removal, with average values close to 99%. Respirometric batch tests revealed a considerable high metabolic activity of the biomass in both plant configurations, with higher values in the AGS + MBR. It was speculated that the MBR reactor was enriched in active biomass deriving from the erosion of the external granule layers in the upstream reactor. In terms of fouling tendency, higher resistance to filtration was observed in the AGS + MBR plant, also characterized by higher irremovable resistance increase compared to the MBR plant, that might severely affect the membrane service life.

Co-culture of Zymomonas mobilis and Scheffersomyces stipitis immobilized in polymeric membranes for fermentation of glucose and xylose to ethanol

Submerged hollow fiber membrane bioreactor (SHFMB) was fabricated and operated to co-culture Zymomonas mobilis and Scheffersomyces stipitis for fermentation of glucose and xylose mixture. Suspended Z. mobilis and S. stipitis in co-culture exhibited poor xylose fermentation due to substrate/product inhibition and catabolite repression. Immobilization in polymeric membranes was effective in alleviating these inhibitions in the SHFMB, which resulted in concomitant fermentation of glucose and xylose at initial glucose and xylose concentrations of 20–80 g L−1 and 10–40 g L−1, respectively. Selective aeration in SHFMB resulted in 100% glucose and >70% xylose fermentation within 48 h, producing 36.7 g L−1 ethanol from 90 g L−1 sugar mixture. Partial immobilization in the membranes allowed Z. mobilis to diffuse in suspension under benign condition, whereas S. stipitis remained inside membrane pores throughout the operation. Xylose fermentation in the SHFMB could be enhanced further by increasing the number of S. stipitis immobilized membranes. Cell immobilization in the SHFMB was stable during 12 consecutive batch runs over 24 days, and exhibited improved xylose fermentation due to gradual increase in S. stipitis accumulation within the membranes. These results indicate that SHFMB can be promising for co-culture of Z. mobilis and S. stipitis in fermentation of mixed sugars from lignocellulosic hydrolysate.

The Impact of Mechanically-Imposed Shear on Clogging, Fouling and Energy Demand for an Immersed Membrane Bioreactor.

The impact of the application of mechanically-imposed shear on the propensity for fouling and clogging (or “sludging”—the agglomeration of sludge solids in the membrane channel) of an immersed flat sheet (iFS) membrane bioreactor (MBR) was studied. The bench-scale test cell used contained a single flat sheet fitted with a crank and motor to allow the membrane to be oscillated (or reciprocated) vertically at a low rate (20 RPM). The membrane was challenged with sludge samples from a local MBR installation treating petroleum industry effluent, the sludge having previously been demonstrated as having a high sludging propensity. Sludging was measured by direct visual observation of membrane surface occlusion by the agglomerated solids, with fouling being notionally represented by the rate of transmembrane pressure increase. Results demonstrated membrane reciprocation to have a more beneficial impact on sludging amelioration than on suppressing fouling. Compared with the stationary membrane, sludging was reduced by an average of 45% compared with only 13% for fouling suppression at the reference flux of 15 L·m−2·h−1 applied. The specific energy demand of the mechanical shear application was calculated as being around 0.0081 kWh·m−3, significantly lower than values reported from a recent pilot scale study on a reciprocated immersed hollow fibre MBR. Whilst results appear promising in terms of energy efficiency, it is likely that the mechanical complexity of applying membrane movement would limit the practical application to low flows, and a correspondingly small number of membrane modules.

Mass transfer affects reactor performance, microbial morphology, and community succession in the methane-dependent denitrification and anaerobic ammonium oxidation co-culture

Denitrifying anaerobic methane oxidation (DAMO) combining anaerobic ammonium oxidation (Anammox) process is a novel nitrogen removal technology. However, the roles of methane transfer (gas phase) and nitrogen transfer (liquid phase) in the heterogeneous process remain unclear. In this study, granular DAMO and Anammox co-cultures were inoculated from a hollow-fiber membrane bioreactor into a sequence batch reactor (SBR). Since the methane transfer became limited in SBR, the nitrate removal rate first decreased and then increased to 10 mg/(L∙day), while the ammonium removal rate did not recover and was around 2 mg/(L∙day). The activity of DAMO archaea and Anammox bacteria decreased noticeably. Furthermore, granular aggregates dispersed into small granules and ultimately became flocs with poor settleability in SBR. The content of extracellular polymeric substances decreased, especially that of proteins and humics. DAMO archaea decreased by 94.6% and Anammox bacteria decreased by 72%. In summary, the limitation of methane transfer affected DAMO and Anammox processes more notably than nitrogen transfer, resulting in lower nitrogen removal, granule disruption, and microbial community succession.