A forced aeration system for microbial culture of multiple shaken vessels suppresses volatilization.

Shake-flask culture, an aerobic submerged culture, has been used in various applications involving cell cultivation. However, it is not designed for forced aeration. Hence, this study aimed to develop a small-scale submerged shaking culture system enabling forced aeration into the medium. A forced aeration control system for multiple vessels allows shaking, suppresses volatilization, and is attachable externally to existing shaking tables. Using a specially developed plug, medium volatilization was reduced to less than 10%, even after 45 h of continuous aeration (~ 60 mL/min of dry air) in a 50 mL working volume. Escherichia coli IFO3301 cultivation with aeration was completed within a shorter period than that without aeration, with a 35% reduction in the time-to-reach maximum bacterial concentration (26.5 g-dry cell/L) and a 1.25-fold increase in maximum concentration. The maximum bacterial concentration achieved with aeration was identical to that obtained using the Erlenmeyer flask, with a 65% reduction in the time required to reach it.

Towards bioprocess engineering of cable bacteria: Establishment of a synthetic sediment.

Cable bacteria, characterized by their multicellular filamentous growth, are prevalent in both freshwater and marine sediments. They possess the unique ability to transport electrons over distances of centimeters. Coupled with their capacity to fix CO2 and their record-breaking conductivity for biological materials, these bacteria present promising prospects for bioprocess engineering, including potential electrochemical applications. However, the cultivation of cable bacteria has been limited to their natural sediment, constraining their utility in production processes. To address this, our study designs synthetic sediment, drawing on ion exchange chromatography data from natural sediments and existing literature on the requirements of cable bacteria. We examined the effects of varying bentonite concentrations on water retention and the impacts of different sands. For the first time, we cultivated cable bacteria on synthetic sediment, specifically the freshwater strain Electronema aureum GS. This cultivation was conducted over 10 weeks in a specially developed sediment bioreactor, resulting in an increased density of cable bacteria in the sediment and growth up to a depth of 5 cm. The creation of this synthetic sediment paves the way for the reproducible cultivation of cable bacteria. It also opens up possibilities for future process scale-up using readily available components. This advancement holds significant implications for the broader field of bioprocess engineering.

Comammox Nitrospira cooperate with anammox bacteria in a partial nitritation-anammox membrane bioreactor treating low-strength ammonium wastewater at high loadings.

Research has revealed that comammox Nitrospira and anammox bacteria engage in dynamic interactions in partial nitritation-anammox reactors, where they compete for ammonium and nitrite or comammox Nitrospria supply nitrite to anammox bacteria. However, two gaps in the literature are present: the know-how to manipulate the interactions to foster a stable and symbiotic relationship and the assessment of how effective this partnership is for treating low-strength ammonium wastewater at high hydraulic loads. In this study, we employed a membrane bioreactor designed to treat synthetic ammonium wastewater at a concentration of 60 mg N/L, reaching a peak loading of 0.36 g N/L/day by gradually reducing the hydraulic retention time to 4 hr. Throughout the experiment, the reactor achieved an approximately 80 % nitrogen removal rate through strategically adjusting intermittent aeration at every stage. Notably, the genera Ca. Kuenena, Nitrosomonas, and Nitrospira collectively constituted approximately 40 % of the microbial community. Under superior intermittent aeration conditions, the expression of comammox amoA was consistently higher than that of Nitrospira nxrB and AOB amoA in the biofilm, despite the higher abundance of Nitrosomonas than comammox Nitrospira, implying that the biofilm environment is favorable for fostering cooperation between comammox and anammox bacteria. We then assessed the in situ activity of comammox Nitrospira in the reactor by selectively suppressing Nitrosomonas using 1-octyne, thereby confirming that comammox Nitrospira played the primary role in facilitating the nitritation (33.1 % of input ammonium) rather than complete nitrification (7.3 % of input ammonium). Kinetic analysis revealed a specific ammonia-oxidizing rate 5.3 times higher than the nitrite-oxidizing rate in the genus Nitrospira, underscoring their critical role in supplying nitrite. These findings provide novel insights into the cooperative interplay between comammox Nitrospira and anammox bacteria, potentially reshaping the management of nitrogen cycling in engineered environments, and aiding the development of microbial ecology-driven wastewater treatment technologies.

Performance and microbial community composition of full-scale high-rate cascade sludge digestion system via pie-shaped reactor configuration.

A full-scale high-rate cascade anaerobic digestion (CAD) system was evaluated for its ability to enhance enzymatic sludge hydrolysis. The system included a newly built digester, innovatively divided into three pie-shaped compartments (500 m3 each), followed by an existing, larger digester (1500 m3). The system treated a mixture of waste activated sludge and primary sludge, achieving a stable total chemical oxygen demand reduction efficiency (56.1 ± 6.8 %), and enhanced sludge hydrolytic enzyme activities at a 14.5-day total solids retention time (SRT). High-throughput sequencing data revealed a consistent microbial community across reactors, dominated by consortia that govern hydrolysis and acidogenesis. Despite relatively short SRTs in the initial reactors of the CAD system, acetoclastic methanogens belonging to Methanosaeta became the most abundant archaea. ‬‬‬‬‬‬‬‬‬‬‬‬‬ This study proves that the CAD system achieves stable sludge reduction, accelerates enzymatic hydrolysis at full-scale, and paves the way for its industrialization in municipal waste sewage sludge treatment.

Enhanced anaerobic digestion performance and sludge filterability by membrane microaeration for anaerobic membrane bioreactor application.

Slow dissolution/hydrolysis of insoluble/macromolecular organics and poor sludge filterability restrict the application potential of anaerobic membrane bioreactor (AnMBR). Bubble-free membrane microaeration was firstly proposed to overcome these obstacles in this study. The batch anaerobic digestion tests feeding insoluble starch and soluble peptone with and without microaeration showed that microaeration led to a 65.7-144.8% increase in methane production and increased critical flux of microfiltration membrane via driving the formation of large sludge flocs and the resultant improvement of sludge settleability. The metagenomic and bioinformatic analyses showed that microaeration significantly enriched the functional genes and bacteria for polysaccharide and protein hydrolysis, microaeration showed little negative effects on the functional genes involved in anaerobic metabolisms, and substrate transfer from starch to peptone significantly affected the functional genes and microbial community. This study demonstrates the dual synergism of microaeration to enhance the dissolution/hydrolysis/acidification of insoluble/macromolecular organics and sludge filterability for AnMBR application.

Microbiome structure and function in parallel full-scale aerobic granular sludge and activated sludge processes.

Aerobic granular sludge (AGS) and conventional activated sludge (CAS) are two different biological wastewater treatment processes. AGS consists of self-immobilised microorganisms that are transformed into spherical biofilms, whereas CAS has floccular sludge of lower density. In this study, we investigated the treatment performance and microbiome dynamics of two full-scale AGS reactors and a parallel CAS system at a municipal WWTP in Sweden. Both systems produced low effluent concentrations, with some fluctuations in phosphate and nitrate mainly due to variations in organic substrate availability. The microbial diversity was slightly higher in the AGS, with different dynamics in the microbiome over time. Seasonal periodicity was observed in both sludge types, with a larger shift in the CAS microbiome compared to the AGS. Groups important for reactor function, such as ammonia-oxidising bacteria (AOB), nitrite-oxidising bacteria (NOB), polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs), followed similar trends in both systems, with higher relative abundances of PAOs and GAOs in the AGS. However, microbial composition and dynamics differed between the two systems at the genus level. For instance, among PAOs, Tetrasphaera was more prevalent in the AGS, while Dechloromonas was more common in the CAS. Among NOB, Ca. Nitrotoga had a higher relative abundance in the AGS, while Nitrospira was the main nitrifier in the CAS. Furthermore, network analysis revealed the clustering of the various genera within the guilds to modules with different temporal patterns, suggesting functional redundancy in both AGS and CAS. KEY POINTS: • Microbial community succession in parallel full-scale aerobic granular sludge (AGS) and conventional activated sludge (CAS) processes. • Higher periodicity in microbial community structure in CAS compared to in AGS. • Similar functional groups between AGS and CAS but different composition and dynamics at genus level.

Occurrence, seasonal variations, and fate of household and personal care chemicals in a wastewater treatment plant with Bacillus bioreactor process.

Household and personal care chemicals (HPCCs) constitute a significant component of everyday products, with their global usage on the rise. HPCCs are eventually discharged into municipal wastewater treatment plants (WWTPs). However, the behaviors of HPCCs inside the Bacillus Bioreactor (BBR) process, including their prevalence, fate, and elimination mechanisms, remain underexplored. Addressing this gap, our study delves into samples collected from a BBR process at a significant WWTP in the northeast of China. Our results spotlight the dominance of linear alkylbenzene sulfonates (LASs) in the influent with concentrations ranging between 238 and 789 µg/L, much higher than the other HPCC concentrations, and remained dominant in the subsequent treatment units. After treatment using the BBR process, the concentrations of HPCCs in the effluent were diminished. Examination of different treatment units underscores the grit chamber removed over 60% of higher-concentration HPCCs, while the performance of the (RBC) tank needs to be improved. Except for the ultraviolet radiation (UV)-filters, seasonal variations exert minimal impact on the concentrations and removal efficiencies of other HPCCs in the BBR process. According to the mass balance analysis, the important mechanisms for HPCC removal were biodegradation and sludge adsorption. Also, the octocrylene (OCT) concerns raised by the environmental risk assessment of the HPCCs residuals in the final effluent, indicate a moderate risk to the surrounding aquatic environment (0.1 < RQ < 1), whereas other HPCCs have a lower risk level (RQ < 0.1). Overall, the research offers new perspectives on the fate and elimination mechanisms of HPCCs throughout the BBR process.

Microbial interactions facilitating efficient methane driven denitrification via in-situ utilization of short chain fatty acids.

High nitrate pollution in agriculture and industry poses a challenge to emerging methane oxidation coupled denitrification. In this study, an efficient nitrate removal efficiency of 100 % was achieved at an influent loading rate of 400 mg-N/L·d, accompanied by the production of short chain fatty acids (SCFAs) with a maximum value of 80.9 mg/L. Batch tests confirmed that methane was initially converted to acetate, which then served as a carbon source for denitrification. Microbial community characterization revealed the dominance of heterotrophic denitrifiers, including Simplicispira (22.8 %), Stappia (4.9 %), and the high­nitrogen-tolerant heterotrophic denitrifier Diaphorobacter (19.0 %), at the nitrate removal rate of 400 mg-N/L·d. Notably, the low abundance of methanotrophs ranging from 0.24 % to 3.75 % across all operational stages does not fully align with the abundance of pmoA genes, suggesting the presence of other functional microorganisms capable of methane oxidation and SCFAs production. These findings could facilitate highly efficient denitrification driven by methane and contributed to the development of denitrification using methane as an electron donor.

Archaeal community composition as key driver of H2 consumption rates at the start-up of the biomethanation process.

The performance of hydrogen consumption by various inocula derived from mesophilic anaerobic digestion plants was evaluated under ex situ biomethanation. A panel of 11 mesophilic inocula was operated at a concentration of 15 gVS.L-1 at a temperature of 35 °C in batch system with two successive injections of H2:CO2 (4:1 mol:mol). Hydrogen consumption and methane production rates were monitored from 44 h to 72 h. Hydrogen consumption kinetics varies significantly based on the inoculum origin, with no accumulation of volatile fatty acids. Microbial community analyses revealed that microbial indicators such as the increase in Methanosarcina sp. abundance and the increase of the Archaea/Bacteria ratio were associated to high initial hydrogen consumption rates. The improvement in the hydrogen consumption rate between the two injections was correlated with the enrichment in hydrogenotrophic methanogens. This work provides new insights into the early response of microbial communities to hydrogen injection and on the microbial structures that may favor their adaptation to the biomethanation process.

Insight into effect of polyethylene microplastic on nitrogen removal in moving bed biofilm reactor: Focusing on microbial community and species interactions.

Microplastics (MPs) pollution has emerged as a global concern, and wastewater treatment plants (WWTPs) are one of the potential sources of MPs in the environment. However, the effect of polyethylene MPs (PE) on nitrogen (N) removal in moving bed biofilm reactor (MBBR) remains unclear. We hypothesized that PE would affect N removal in MBBR by influencing its microbial community. In this study, we investigated the impacts of different PE concentrations (100, 500, and 1000 µg/L) on N removal, enzyme activities, and microbial community in MBBR. Folin-phenol and anthrone colorimetric methods, oxidative stress and enzyme activity tests, and high-throughput sequencing combined with bioinformation analysis were used to decipher the potential mechanisms. The results demonstrated that 1000 µg/L PE had the greatest effect on NH4+-N and TN removal, with a decrease of 33.5 % and 35.2 %, and nitrifying and denitrifying enzyme activities were restrained by 29.5-39.6 % and 24.6-47.4 %. Polysaccharide and protein contents were enhanced by PE, except for 1000 µg/L PE, which decreased protein content by 65.4 mg/g VSS. The positive links of species interactions under 1000 µg/L PE exposure was 52.07 %, higher than under 500 µg/L (51.05 %) and 100 µg/L PE (50.35 %). Relative abundance of some metabolism pathways like carbohydrate metabolism and energy metabolism were restrained by 0.07-0.11 % and 0.27-0.4 %. Moreover, the total abundance of nitrification and denitrification genes both decreased under PE exposure. Overall, PE reduced N removal by affecting microbial community structure and species interactions, inhibiting some key metabolic pathways, and suppressing key enzyme activity and functional gene abundance. This paper provides new insights into assessing the risk of MPs to WWTPs, contributing to ensuring the health of aquatic ecosystems.

Unveiling the potential of specific growth rate control in fed-batch fermentation: bridging the gap between product quantity and quality.

During the epoch of sustainable development, leveraging cellular systems for production of diverse chemicals via fermentation has garnered attention. Industrial fermentation, extending beyond strain efficiency and optimal conditions, necessitates a profound understanding of microorganism growth characteristics. Specific growth rate (SGR) is designated as a key variable due to its influence on cellular physiology, product synthesis rates and end-product quality. Despite its significance, the lack of real-time measurements and robust control systems hampers SGR control strategy implementation. The narrative in this contribution delves into the challenges associated with the SGR control and presents perspectives on various control strategies, integration of soft-sensors for real-time measurement and control of SGR. The discussion highlights practical and simple SGR control schemes, suggesting their seamless integration into industrial fermenters. Recommendations provided aim to propose new algorithms accommodating mechanistic and data-driven modelling for enhanced progress in industrial fermentation in the context of sustainable bioprocessing.

Evaluation of different glycerol fed-batch strategies in a lab-scale bioreactor for the improved production of a novel engineered ß-fructofuranosidase enzyme in Pichia pastoris.

The ß-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the ß-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the ß-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.

Distinguished denitrifying phosphorus removal in the high-rate anoxic/microaerobic system for sewage treatment.

This study re-evaluated the role of anoxic and anaerobic zones during the enhanced biological phosphorus (P) removal process by investigating the potential effect of introducing an anoxic zone into a high-rate microaerobic activated sludge (MAS) system (1.60-1.70 kg chemical oxygen demand (COD) m-3 d-1), i.e., a high-rate anoxic/microaerobic (A/M) system for sewage treatment. In the absence of a pre-anaerobic zone, introducing an anoxic zone considerably reduced effluent NOx--N concentrations (7.2 vs. 1.5 mg L-1) and remarkably enhanced total nitrogen (75% vs. 89%) and total P (18% vs. 60%) removal and sludge P content (1.48% vs. 1.77% (dry weight)) due to further anoxic denitrifying P removal in the anoxic zone (besides simultaneous nitrification and denitrification in the microaerobic zone). High-throughput pyrosequencing demonstrated the niche differentiation of different polyphosphate accumulating organism (PAO) clades (including denitrifying PAO [DPAO] and non-DPAO) in both systems. Introducing an anoxic zone considerably reduced the total PAO abundance in sludge samples by 42% and modified the PAO community structure, including 17-19 detected genera. The change was solely confined to non-DPAOs, as no obvious change in total abundance or community structure of DPAOs including 7 detected genera was observed. Additionally, introducing an anoxic zone increased the abundance of ammonia-oxidizing bacteria by 39%. The high-rate A/M process provided less aeration, higher treatment capacity, a lower COD requirement, and a 75% decrease in the production of waste sludge than the conventional biological nutrient removal process.

Assessing the efficiency and microbial diversity of H2S-removing biotrickling filters at various pH conditions.

This study aimed to investigate the operation of three parallel biotrickling filters (BTFs) in removing H2S at different pH conditions (haloalkaliphilic, neutrophilic, and acidophilic) and their associated microbial population in the biodesulfurization process. BTF columns were inoculated with enriched inoculum and experiments were performed by gradually reducing Empty Bed Retention Time (EBRT) and increasing inlet concentration in which the maximum removal efficiency and maximum elimination capacity in EBRT 60 s reached their maximum level in haloalkaline condition (91% and 179.5 g S-H2S m-3 h-1). For visualizing the attached microbial biofilms on pall rings, Scanning Electron Microscopy (SEM) was used and microbial community structure analysis by NGS showed that the most abundant phyla in haBTF, nBTF, and aBTF belong to Gammaproteobacteria, Betaproteobacteria, and Acidithiobacillia, respectively. Shannon and Simpson indexes evaluation showed a lower diversity of bacteria in the aBTF reactor than that of nBTF and haBTF and beta analysis indicated a different composition of bacteria in haBTF compared to the other two filters. These results indicated that the proper performance of BTF under haloalkaliphilic conditions is the most effective way for H2S removal from air pollutants of different industries.

Stochastic processes shape microbial community assembly in biofilters: Hidden role of rare taxa.

Stochastic and deterministic processes are the major themes governing microbial community assembly; however, their roles in bioreactors are poorly understood. Herein, the mechanisms underlying microbial assembly and the effect of rare taxa were studied in biofilters. Phylogenetic tree analysis revealed differences in microbial communities at various stages. Null model analysis showed that stochastic processes shaped the community assembly, and deterministic processes emerged only in the inoculated activated sludge after domestication. This finding indicates the dominant role of stochastic factors (biofilm formation, accumulation, and aging). The Sloan neutral model corroborated the advantages of stochastic processes and mainly attributed these advantages to rare taxa. Cooccurrence networks revealed the importance of rare taxa, which accounted for more than 85% of the keystones. Overall, these results provide good foundations for understanding community assembly, especially the role of rare taxa, and offer theoretical support for future community design and reactor regulation.

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.

Insights into the response mechanisms of activated sludge system under long-term dexamethasone stress.

Dexamethasone (DEX) is a hormone drug that is often detected in wastewater treatment plants, but its impact on activated sludge systems is unknown. This study explored the long-term effects of DEX on nutrient removal, microbial activities, microbial assembly, and microbial interactions in the activated sludge system. During the 90-day DEX exposure experiment, both chemical oxygen demand and total nitrogen removal efficiencies were initially inhibited and then recovered. Microbial activities, i.e., specific oxygen uptake rate and denitrification, did not differ significantly from that of the control reactor (p > 0.05), possibly due to the secretion of extracellular polymers that act as a protective barrier against excess reactive oxygen species induced by DEX. This barrier protects cell membrane integrity and ensures stable treatment performance. Analysis of microbial assembly identified the drift of stochastic processes (from 92.7 % to 51.8 %) and homogeneous selection of deterministic processes (from 1.6 % to 38.7 %) as the main driving forces of microbial community structure succession under long-term DEX stress. Although long-term exposure to 1000 µg/L DEX did not significantly increase the abundance levels of functional bacteria (Nitrosomonas and 996-1) and key genes (AmoCAB and Hao), the ammonia oxidation capacity of the activated sludge system was enhanced. Analysis of microbial interactions indicated that streamlining of functional subnetworks and increased cooperation were the primary reasons. This is the first study to explore the long-term effects of DEX on activated sludge and provide insights into microbial interaction and assembly. Moreover, the findings of this study broaden our knowledge and lay an experimental foundation for reducing risks associated with hormone drugs.

Enhancing Phycocyanobilin Production Efficiency in Engineered Corynebacterium glutamicum: Strategies and Potential Application.

Phycocyanobilin, an algae-originated light-harvesting pigment known for its antioxidant properties, has gained attention as it plays important roles in the food and medication industries and has surged in demand owing to its low-yield extraction from natural resources. In this study, engineered Corynebacterium glutamicum was developed to achieve high PCB production, and three strategies were proposed: reinforcement of the heme biosynthesis pathway with the introduction of two PCB-related enzymes, strengthening of the pentose phosphate pathway to generate an efficient cycle of NADPH, and fed-batch fermentation to maximize PCB production. Each approach increased PCB synthesis, and the final engineered strain successfully produced 78.19 mg/L in a flask and 259.63 mg/L in a 5 L bioreactor, representing the highest bacterial production of PCB reported to date, to our knowledge. The strategies applied in this study will be useful for the synthesis of PCB derivatives and can be applied in the food and pharmaceutical industries.

Molecular insight into the allocation of organic carbon to heterotrophic bacteria: Carbon metabolism and the involvement in nitrogen and phosphorus removal.

Carbon metabolism and nutrient removal are crucial for biological wastewater treatment. This study focuses on analyzing carbon allocation and utilization by heterotrophic bacteria in response to increasing COD concentration in the influent. The study also assesses the effect of denitrification and biological phosphorus removal, particularly in combination with anaerobic ammonia oxidation (anammox). The experiment was conducted in a SBR operating under anaerobic/anoxic/oxic conditions. As COD concentration in the influent increased from 100 to 275 mg/L, intracellular COD accounted for 95.72 % of the COD removed. By regulating the NO3- concentration in the anoxic stage from 10 to 30 mg/L, the nitrite accumulation rate reached 69.46 %, which could serve as an electron acceptor for anammox. Most genes related to the tricarboxylic acid (TCA) cycle declined, while the genes involved in the glyoxylate cycle, gluconeogenesis, PHA synthesis increased. This suggests that glycogen accumulation and carbon storage, rather than direct carbon oxidation, was the dominant pathway for carbon metabolism. However, the genes responsible for the reduction of NO2--N (nirK) and NO (nosB) decreased, contributing to NO2- accumulation. The study also employed metagenomic analysis to reveal microbial interactions. The enrichment of specific bacterial species, including Dechloromonas sp. (D2.bin.10), Ca. Competibacteraceae bacterium (D9.bin.8), Ca. Desulfobacillus denitrificans (D6.bin.17), and Ignavibacteriae bacterium (D3.bin.9), played a collaborative role in facilitating nutrient removal and promoting the combination with anammox.

Influence of temperature on performance and mechanism of advanced synergistic nitrogen removal in lab-scale denitrifying filter with biogenic manganese oxides.

Temperature is a significant operational parameter of denitrifying filter (DF), which affects the microbial activity and the pollutants removal efficiency. This study investigated the influence of temperature on performance of advanced synergistic nitrogen removal (ASNR) of partial-denitrification anammox (PDA) and denitrification, consuming the hydrolytic and oxidation products of refractory organics in the actual secondary effluent (SE) as carbon source. When the test water temperature (TWT) was around 25, 20, 15 and 10 °C, the filtered effluent total nitrogen (TN) was 1.47, 1.70, 2.79 and 5.52 mg/L with the removal rate of 93.38%, 92.25%, 87.33% and 74.87%, and the effluent CODcr was 8.12, 8.45, 10.86 and 12.29 mg/L with the removal rate of 72.41%, 66.17%, 57.35% and 51.87%, respectively. The contribution rate of PDA to TN removal was 60.44%∼66.48%, and 0.77-0.96 mg chemical oxygen demand (CODcr) was actually consumed to remove 1 mg TN. The identified functional bacteria, such as anammox bacteria, manganese oxidizing bacteria (MnOB), hydrolytic bacteria and denitrifying bacteria, demonstrated that TN was removed by the ASNR, and the variation of the functional bacteria along the DF layer revealed the mechanism of the TWT affecting the efficiency of the ASNR. This technique presented a strong adaptability to the variation of the TWT, therefore, it has broad application prospect and superlative application value in advanced nitrogen removal of municipal wastewater.