Microbial interactions play a keystone role in rapid anaerobic ammonium oxidation sludge proliferation and biofilm formation.

Two mature anaerobic ammonium oxidation (anammox) consortia with high/low relative abundance of anammox bacteria were inoculated for the rapid sludge proliferation and biofilm formation in this study, named up-flow anaerobic sludge blanket reactor (UASB1) (high) and UASB2 (low), respectively. Results showed that the nitrogen removal efficiency of UASB2 reached 90.94% after the 120-day operation, which was 13% higher than that of UASB1. Moreover, its biomass amounts were 22.18% (biofilm) and 40.96% (flocs) higher than that of UASB1, respectively. Ca. Kuenenia possessed relative abundances of 29.32% (flocs), 27.42% (biofilm) and 31.56% (flocs), 35.20% (biofilm) in the UASB1 and UASB2, respectively. The relative abundances of genes involved in anammox transformation (hzs, nir) and carbon metabolism (fdh, lgA/B/C, acs) were higher in the UASB2, indicating that Ca. Kuenenia might produce acetate and glycogen to enhance microbial interactions. These findings emphasized the importance of microbial interactions in anammox sludge proliferation and biofilm formation.

Synergistic effects and mechanisms of plasma coupled with peracetic acid in enhancing short-chain fatty acid production from sludge: Motivation of reactive species and metabolic tuning of microbial communities.

Suitable waste activated sludge (WAS) pretreatments that boost short-chain fatty acid (SCFA) production from anaerobic fermentation are essential for carbon emission reduction and sludge resource utilization. This study established an efficient WAS pretreatment process combining atmospheric pressure plasma jet (APPJ) with peracetic acid (PAA). The maximum SCFA production (6.5-fold that of the control) largely increased under the optimal conditions (PAA dosage = 25 mg/g VSS (volatile suspended solids), energy consumption = 20.9 kWh/m3). APPJ/PAA pretreatment enhanced the production of multiple reactive species (including OH, CH3C(O)O, 1O2, ONOO-, O2-, and eaq-) and strengthened the effects of H2O2, heat, and light. This synergistically solubilized WAS and released organic substrates for SCFA-producing microbes. In addition, the enrichment of SCFA-producing bacteria and the decrease in SCFA-consuming bacteria favored SCFA accumulation. The key genes encoding for the main substrate metabolism and SCFA production in the metabolic pathway of fermentation were also enhanced.

Roles and regulation of quorum sensing in anaerobic granular sludge: Research status, challenges, and perspectives.

Anaerobic granular sludge (AnGS) has a complex and important internal microbial communication system due to its unique microbial layered structure. As a concentration-dependent communication system between bacterial cells through signal molecules, QS (quorum sensing) is widespread in AnGS and exhibits great potential to regulate microbial behaviors. Therefore, the universal functions of QS in AnGS have been systematically summarized in this paper, including the influence on the metabolic activity, physicochemical properties, and microbial community of AnGS. Subsequently, the common QS-based AnGS regulation approaches are reviewed and analyzed comprehensively. The regulation mechanism of QS in AnGS is analyzed from two systems of single bacterium and mixed bacteria. This review can provide a comprehensive understanding of QS functions in AnGS systems, and promote the practical application of QS-based strategies in optimization of AnGS treatment process.

Enhancement strategies of aerobic denitrification for efficient nitrogen removal from low carbon-to-nitrogen ratio shale oil wastewater.

The strategy of high reflux ratio and long solids retention time was adopted to realize efficient nitrogen removal from real shale oil wastewater. This was undertaken with a low chemical oxygen demand to total nitrogen (COD/TN) ratio by strengthening aerobic denitrification in an anoxic/aerobic membrane bioreactor (A/O-MBR). The TN removal load climbed from 22 to 25 g N/(kg MLSS·d) as the COD/TN ratio declined from 8 to 3. The abundance of heterotrophic nitrifying and aerobic denitrifying (HNAD) bacteria increased by 13.8 times to 42.5%, displacing anoxic denitrifying bacteria as the predominant bacteria. The abundance of genes involved in denitrification (napAB, narGHI, norBC, nosZ) increased, however the genes related to assimilatory nitrate reduction (nirA, narB, nasC) decreased. The capacity of the dominant HNAD bacteria in an A/O-MBR to efficiently utilize a carbon source is the key to efficient nitrogen removal from shale oil wastewater with a low COD/TN ratio.

Evaluation of Nanaerobic Digestion as a Mechanism to Explain Surplus Methane Production in Animal Rumina and Engineered Digesters.

Nanaerobes are a newly described class of microorganisms that use a unique cytochrome bd oxidase to achieve nanaerobic respiration at <2 µM dissolved oxygen (∼1% of atmospheric oxygen) but are not viable above this value due to the lack of other terminal oxidases. Although sharing an overlapping ecological niche with methanogenic archaea, the role of nanaerobes in methanogenic systems has not been studied so far. To explore their occurrence and significance, we re-analyzed published meta-omic datasets from animal rumina and waste-to-energy digesters, including conventional anaerobic digesters and anaerobic digesters with ultra-low oxygenation. Results show that animal rumina share broad similarities in the microbial community and system performance with oxygenated digesters, rather than with conventional anaerobic digesters, implying that trace levels of oxygen drive the efficient digestion in ruminants. The rumen system serves as an ideal model for the newly named nanaerobic digestion, as it relies on the synergistic co-occurrence of nanaerobes and methanogens for methane yield enhancement. The most abundant ruminal bacterial family Prevotellaceae contains many nanaerobes, which perform not only anaerobic fermentation but also nanaerobic respiration using cytochrome bd oxidase. These nanaerobes generally accompany hydrogenotrophic methanogens to constitute a thermodynamically and physiologically consistent framework for efficient methane generation. Our findings provide new insights into ruminal methane emissions and strategies to enhance methane generation from biomass.

Bioelectrochemical system for nitrogen removal: Fundamentals, current status, trends, and challenges.

Biological nitrogen removal (BNR) is essential for the treatment of nitrogen-containing wastewater. However, the requirement for aeration and the addition of external carbon sources, resulting in greenhouse gas emissions and additional costs, are disadvantages of the traditional BNR process. Alternative technologies have been devised to overcome these drawbacks. Bioelectrochemical nitrogen removal (BENR) has been proposed for efficient nitrogen removal, demonstrating flexibility and versatility. BENR can be performed by combining nitrification, denitrification, anaerobic ammonium oxidation (ANAMMOX), or organic carbon oxidation. Bioelectrochemical-ANAMMOX (BE-ANAMMOX) is the most promising method for nitrogen removal, as it can directly convert NH4+ to N2 and H2 in one step when the electrode is arranged as an electron acceptor. High-value-added hydrogen can potentially be recovered with efficient nitrogen removal using this concept, maximizing the benefits of BENR. Using alternative electron acceptors, such as electrodes and metal ions, for complete total nitrogen removal is a promising technology to substitute NO2- production from NH4+ oxidation by aeration. However, the requirement of electron donors for NO3- reduction, low NH4+ removal efficiency, and low competitiveness of exoelectrogenic bacteria still remain the main obstacles. The future direction for successful BENR should aim to achieve complete anaerobic NH4+ oxidation without any electron acceptor and to maximize selectivity in H2 production. Therefore, the bioelectrochemical pathways and balances between efficient nitrogen removal and high-value-added chemical production should be further studied for carbon and energy neutralities.

Construction and application of a mCherry fluorescent labeling system for Stenotrophomonas AGS-1 from aerobic granular sludge.

To elucidate the specific mechanism by which high-attachment bacteria promote aerobic granular sludge (AGS) formation, a red fluorescent protein mCherry-based biomarker system was developed in the high-attachment strain Stenotrophomonas AGS-1 from AGS. The fluorescent labeling system used plasmid-mediated mCherry expression driven by a Ptac constitutive promoter. mCherry-labeled AGS-1 had normal unimpaired growth, strong fluorescent signals, and good fluorescence imaging. Also, the mCherry labeling system had no effect on the attachment ability of AGS-1. In addition, mCherry-labeled AGS-1 maintained high plasmid stability, even after more than 100 generations. Notably, after the addition of mCherry-labeled AGS-1 into the activated sludge system, the mCherry fluorescence of the sludge system can be used as a good reflection of the relative amount of AGS-1. Moreover, the spatial distribution of mCherry-labeled AGS-1 in the sludge system could be visualized and remained clear even after 5 days by fluorescence imaging. These results revealed that the mCherry-based biomarker system would provide a valuable tool for labeling AGS-1 to monitor the spatial distribution and fate of AGS-1 in AGS, which would help to better understand the mechanism of AGS formation and facilitate the development of AGS technology.

Removal of Cr (VI) and microbial community analysis in PCB wastewater treatment based on the BESI® process.

The treatment efficiency of Chromium (Cr)-containing Printed Circuit Board (PCB) wastewater is significantly hampered by the limited physiological activity of microorganisms when activated sludge is applied. In this study, the biodegradation and electron transfer based on sulfur metabolism in the integrated (BESI®) process use sulfur as the electron acceptor to achieve sulfate reduction and sulfide oxidation, leading to efficient removal of Cr. The concentrations of total Cr and Cr(VI) in the effluent were reduced to 0.5 mg/L and 0.1 mg/L, respectively, from an initial range of 25-32 mg/L in the influent. The removal of Cr (ΔC(Cr(VI))) mainly occurred in the Sulfate Reduction (SR) reactor, which was significantly correlated with the generation of sulphide ([Formula: see text]) (R2 = 0.9987). Meantime, analysis of the microbial community showed that Cr (VI) stress increased the diversity of the bacterial community in sludge. The presence of Clostridium (52.54% and 47.78%) in SR & Sulfide Oxidation (SO) reactor, along with the Synergistaceae (31.90%) and Trichococcus (26.59%) in aerobic reactor, might contribute to the gradient degradation of COD, resulting in a removal efficiency exceeding 80% when treating an influent with a concentration of 1000 mg/L. In addition, the main precipitation components in the SR reactor were identified by scanning electron microscope, indicating that Cr has been removed from wastewater as Cr(OH)3 precipitation. This study sheds light on the potential of using the BESI® process for the real PCB wastewater treatment.

Effect of chlorine disinfectant influx on biological sewage treatment process under the COVID-19 pandemic: Performance, mechanisms and implications.

Since the onset of the COVID-19 Pandemic, large amounts of chlorine-containing disinfectants have been used to interrupt the spread of SARS-CoV-2 and residual chlorine eventually entered the hospital or municipal sewage treatment facilities. However, little is known about the effect of chlorine influx on the biological sewage treatment process. Here we investigated the effect of chlorine on the microbiome and the mechanism of microbial chlorine resistance in the activated sludge of the aerobic treatment process, using metagenomic and metatranscriptomic sequencing. We found that chlorine could negatively impact the aerobic treatment performance regarding nitrogen/COD removal with a dose-dependent effect, and the dual effects of chlorine dose and interaction time differentiated the microbial community in activated sludge. The decline of nitrogen/COD removal was attributed to the compressed activity of functional microorganisms, such as the ammonia oxidation bacteria, under chlorinated conditions, and the damage cannot be recovered in a short term. In addition, some microorganisms could survive in chlorinated conditions by up-regulating the chlorine resistance genes (CRGs) expression (approximately 1.5 times) and stimulating new CRGs expression. In particular, species Acinetobacter johnsonii could resist high concentrations of chlorine through various mechanisms, especially the overexpression of efflux pump function encoded by qac genes play a key role. Based on these results, considering the persistence of the epidemic and extensive use of chlorine disinfectants, it cannot be ignored that large amounts of residual chlorine are entering the biological treatment facility, and strictly de-chlorination measures or microbial chlorine resistance regulations before entering should be implemented.

Microbial community composition of food waste before anaerobic digestion.

Anaerobic digestion is widely used to process and recover value from food waste. Commercial food waste anaerobic digestion facilities seek improvements in process efficiency to enable higher throughput. There is limited information on the composition of microbial communities in food waste prior to digestion, limiting rational exploitation of the catalytic potential of microorganisms in pretreatment processes. To address this knowledge gap, bacterial and fungal communities in food waste samples from a commercial anaerobic digestion facility were characterised over 3 months. The abundance of 16S rRNA bacterial genes was approximately five orders of magnitude higher than the abundance of the fungal intergenic spacer (ITS) sequence, suggesting the numerical dominance of bacteria over fungi in food waste before anaerobic digestion. Evidence for the mass proliferation of bacteria in food waste during storage prior to anaerobic digestion is presented. The composition of the bacterial community shows variation over time, but lineages within the Lactobacillaceae family are consistently dominant. Nitrogen content and pH are correlated to community variation. These findings form a foundation for understanding the microbial ecology of food waste and provide opportunities to further improve the throughput of anaerobic digestion.

Hybrid microalgae-activated sludge system for carbon-efficient wastewater treatment.

Engineered microalgae-bacteria systems can play a key role in the realisation of energy-efficient carbon-neutral wastewater treatment technologies. An attempt was made to develop a hybrid microalgae-activated sludge (HMAS) system coupling carbon capture with domestic wastewater treatment. Photobioreactors internally illuminated with red light-emitting diodes (LEDs), and inoculated with mixed microbial culture, resulted in substantial savings in operational cost. System performance was evaluated at about 600 µmol/m2 s LED irradiance while treating synthetic municipal wastewater in a chemostat for about 2 months, containing about 250 mg/L soluble chemical oxygen demand (SCOD), 90 mg/L NH3-N and 10 mg/L orthophosphate. Carbon dioxide was supplied into the HMAS at 25 mL/min, 25% v/v. SCOD was efficiently removed from the wastewater (up to 70%) and bacterial oxygen requirement of >2 mg/L was met through microalgal photosynthesis. The system demonstrated its potential in achieving carbon-efficient wastewater treatment.

Evolution of aniline degradation and nitrogen removal performance in electro-enhanced sequence batch reactor under salinity stress: Sludge characteristics and microbial diversity.

To explore the influence mechanism of different concentrations of salinity on the electro-enhanced aniline biodegradation system, a control group and experimental groups (0%-NaCl, 0.5%-NaCl, 1.5%-NaCl, 2.5%-NaCl, 3.5%-NaCl) were established. The experimental results showed that the electric field strengthened the denitrification performance, while salinity had little effect on the degradation efficiency of aniline and chemical oxygen demand (COD). The removal rate of TN reached 79.6% and 74.9% in 0.5%-NaCl and 1.5%-NaCl, respectively, which were superior than 0%-NaCl. As salinity increased, the nitrogen removal effect was negatively affected. Microbial diversity analysis indicated that the microbial community structure was uniform in the control group, 0%-NaCl, and 0.5%-NaCl, with the dominant genus OLB8 ensuring the nitrogen removal performance. In contrast, in the 2.5%-NaCl and 3.5%-NaCl experimental groups, the organic degrading bacteria were still active, while nitrifiers and denitrifiers were severely damaged. In conclusion, this study suggested that low concentrations of salinity can improve the decontamination performance of the electro-enhanced aniline biodegradation system, while high concentrations of salinity could lead to the collapse of the decontamination mechanism.

Anaerobic starvation realizes partial nitrification and starts anammox bacteria self-enrichment in mainstream municipal sewage treatment in a low filling ratio sequencing batch reactor.

The initiating and stable preservation of partial nitrification (PN) and achievement of anammox bacteria self-enrichment in domestic sewage is a purposeful subject. In this article, an originality tactics of anaerobic starvation for 100 days was adopted for rapidly achieving PN in actual wastewater, the nitrite accumulation rate (NAR) improved from 4.95% to 81.73% in 18 days. After anaerobic starvation was stopped, the stable PN effect furnished enough stroma for the growth of anammox bacteria. The abundance of Candidatus Brocadia grew from 0% to 0.42% in floc sludge and 0.43% in blank biofilm, which promoted nitrogen removal effect. Anaerobic starvation continuing 74 days generated further decrease in the abundance of Nitrobacter and Nitrospira of nitrite-oxidizing bacteria (NOB), indicating that anaerobic starvation can restore the destroyed partial nitrification. In conclusion, this article furnished a low-cost method for achieving anammox bacteria self-enrichment in mainstream municipal wastewater in 10% filling ratio without chemicals addition.

Propidium monoazide - polymerase chain reaction reveals viable microbial community shifts in anaerobic membrane bioreactors treating domestic sewage at low temperature.

An anaerobic membrane bioreactor (AnMBR) treated domestic sewage at 15 °C under different hydraulic retention time (HRT) conditions (6, 12, 16, and 24 h). Propidium monoazide (PMA)-PCR excluded microorganisms without intact cell membranes, focusing on the viable microbial community in anaerobic digestion. The results showed that the 6-hour HRT had poor treatment performance: low chemical oxygen demand removal efficiency (below 80%) and high mean trans-membrane pressure and flux (15 kPa and 9.4 L/(m2 h)). Comparatively, PMA-PCR combined with next-generation sequencing improved the identification of microbial changes compared to conventional 16S rRNA gene sequencing. HRT influenced microorganisms in the hydrolysis and acid-production stages, including carbohydrate-degrading bacteria such as Bifidobacterium and Prevotella 1. Remarkably, a comparison with an AnMBR at 25 °C showed Proteobacteria to be the main cause of membrane fouling in the low-temperature AnMBR, with most operational taxonomic units negatively correlated with HRT and solids retention time.

Directed regulation of anammox communities based on exogenous siderophores for highly efficient nitrogen removal.

It is expected that the quicker domestication of anaerobic ammonia oxidation (anammox) communities and the enhancement of their nitrogen transformation capability can be achieved through targeted regulation of anammox communities. Iron cast a vital role in the growth and metabolism of anammox bacteria. Specific siderophores offer promising prospects for the targeted regulation of anammox communities by facilitating the efficient utilization of iron. Two siderophores-enterobactin and putrebactin-exclusively for Ca. Brocadia and Ca. Kuenenia were developed to specifically regulate anammox communities towards different directions, respectively. Anammox communities in the reactors evoluted targetedly towards Ca. Brocadia-dominated communities and Ca. Kuenenia-dominated communities, respectively, leading to a maximum increase in community nitrogen removal capacity by 84.64±0.55% and 210.26±0.57%, respectively, under different nitrogen concentrations. It was indicated that siderophores could regulate anammox communities by redistributing iron resources in a targeted manner based on the analyses of transcriptome and proteome. This study provides novel insights into the rational selection and utilization of exogenous siderophores as an effective implement to manipulate anammox communities and create communities with high nitrogen removal ability fleetly.

Multi-omic profiling of a novel activated sludge strain Sphingobacterium sp. WM1 reveals the mechanism of tetracycline biodegradation and its merits of potential application.

As an emerging pollutant, the antibiotic tetracycline (TC) has been consistently detected in wastewater and activated sludge. Biodegradation represents a potentially crucial pathway to dissipate TC contamination. However, few efficient TC-degrading bacteria have been isolated and a comprehensive understanding of the molecular mechanisms underlying TC degradation is still lacking. In this study, a novel TC-degrading bacterium, designated as Sphingobacterium sp. WM1, was successfully isolated from activated sludge. Strain WM1 exhibited a remarkable performance in degrading 50 mg/L TC within 1 day under co-metabolic conditions. Genomic analysis of the strain WM1 unveiled the presence of three functional tetX genes. Unraveling the complex molecular mechanisms, transcriptome analysis highlighted the role of upregulated transmembrane transport and accelerated electron transport in facilitating TC degradation. Proteomics confirmed the up-regulation of proteins involved in cellular biosynthesis/metabolism and ribosomal processes. Crucially, the tetX gene-encoding protein showed a significant upregulation, indicating its role in TC degradation. Heterologous expression of the tetX gene resulted in TC dissipation from an initial 51.9 mg/L to 4.2 mg/L within 24 h. The degradation pathway encompassed TC hydroxylation, transforming into TP461 and subsequent metabolites, which effectively depleted TC's inhibitory activity. Notably, the tetX genes in strain WM1 showed limited potential for horizontal gene transfer. Collectively, strain WM1's potent TC degradation capacity signals a promise for enhancing TC clean-up strategies.

Mechanism of antibiotic resistance development in an activated sludge system under tetracycline pressure.

The mechanism of antibiotic resistance (AR) development in an activated sludge system under tetracycline (TC) pressure was discussed and analyzed. According to the variation of macro-factors, including TC, COD, TN, TP, NH3-N, pH, heavy metals, and reactor settings, the tet genes respond accordingly. Consequently, the enrichment sites of tet genes form an invisible AR selection zone, where AR microorganisms thrive, gather, reproduce, and spread. The efflux pump genes tetA and tetB prefer anaerobic environment, while ribosome protective protein genes tetM, tetO, tetQ, tetT, and tetW were more concentrated in aerobic situations. As a corresponding micro-effect, different types of tet genes selected the corresponding dominant bacteria such as Thauera and Arthrobacter, suggesting the intrinsic relationship between tet genes and potential hosts. In summary, the macro-response and micro-effect of tet genes constitute an interactive mechanism with tet genes as the core, which is the crucial cause for the continuous development of AR. This study provides an executable strategy to control the development of AR in actual wastewater treatment plants from the perspective of macro-factors and micro-effects.

Positive responses and mechanisms of nitrifying sludge to carbon quantum dots: reactor performance, microbial behavior, and antioxidant defense.

Extensive application of carbon quantum dots (CQDs) enlarges its concentration in sewage treatment system. The response of nitrifying sludge to CQDs after long-term exposure was investigated. Results showed that CQD concentrations of 0-100 mg/L presented positive effect to enzymes involved in nitrification, accelerating NH4+-N degradation and NO2--N transformation. The oxidation rate of NO2--N was significantly improved from 3.14 to 7.91 mg/(L h) under the stress of 100 mg/L CQDs. Besides, CQDs stimulated the production of sludge biomass and kept the stability of sludge settleability. Additionally, CQDs were mainly captured by loosely bound extracellular polymeric substances, reducing aromatic-like protein. Microbes alleviated CQD stress by secreting tryptophan-like protein and polysaccharides. After few CQDs entered cells, intracellular antioxidant defense was activated. Total antioxidant capacity level was heightened at least 31%. The activities of superoxide dismutase and catalase were enhanced at relatively low and high CQD concentration levels. Hence, microbial metabolic pathways, microbial community, and nitrifying bacteria were not significantly affected by CQDs. The findings of this work provide new insight for understanding the environmental implication of CQDs in the biological treatment system.

Metatranscriptome analysis unveils the mechanisms of zero-valent iron enhancing reactivation of starvation hydrolysis acidification sludge by inducing high-level gene expression.

Hydrolysis acidification (HA) is a promising method for wastewater treatment and resource recovery. However, the extended time required for bacterial reactivation after starvation or a change in living conditions often poses a challenge to the efficient operation of the system. Although the addition of zero-valent iron (ZVI) could enhance HA performance, its effects on sludge reactivation in the HA process are not fully understood. In this study, ZVI was employed to accelerate sludge reactivation and its involved genetic mechanisms were unveiled. The results demonstrated that ZVI addition activated the sludge within 35 days with stable HA performance. Sludge characteristics revealed that ZVI improved active biomass, enzyme activity (by 11.4 % âˆ¼ 26.7 %), ETS activity (by 566 %), and cell viability, with a higher concentration of MLVSS, live cells, more microbial byproducts in EPS, and relative abundance of HA bacteria (63.41 %). Moreover, metatranscriptome analysis showed that ZVI upregulated the expression of genes related to key enzymes in carbohydrate degradation metabolism, biosynthesis of electron transfer media such as heme and ubiquinone, and biosynthesis of vital cofactors like vitamin B12 and folate during microbial growth and metabolism. These findings suggest that ZVI enhanced electron transfer, bacterial growth, and metabolism, resulting in effective starch conversion and VFAs generation. Overall, these results deepen our understanding of the mechanism by which ZVI enhanced HA sludge reactivation, providing valuable information for addressing sludge starvation issues in HA systems.

Denitrification effect and strengthening mechanism of SAD/A system at low temperature by gel-immobilization technology.

Sulfur autotrophic denitrification coupled anaerobic ammonia oxidation (SAD/A) has several advantages over other denitrification processes; for example, it does not consume the organic carbon source, has low operation costs, and produces less excess sludge; however, it has certain disadvantages as well, such as a long start-up time, easy loss of bacteria, and low microbial activity at low temperature. The use of microbial immobilization technology to embed functional bacteria provides a feasible method of resolving the above problems. In this study polyvinyl alcohol­sodium alginate was used to prepare a composite carrier for fixing anaerobic ammonia oxidizing bacteria (AAOB) and sulfur oxidizing bacteria (SOB), and the structure and morphology of the encapsulated bodies were characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Subsequently, the nitrogen removal performance of the immobilized microbial carriers in the gradient cooling process (30 °C to 10 °C) was determined, and the corresponding mechanism was discussed. The results showed that the nitrate-removal efficiencies observed with granular sludge and gel embedding were at 10 °C 21.44 % and 14.31 % lower, than those at 30 °C, respectively, whereas the ammonia-removal efficiency decreased by up to approximately three-fold. The main mechanism was the 'insulation' provided by the external gel composed of PVA and SA for the internal sludge and subsequent improvement of its low temperature resistance, while protecting AAOB and SOB from oxygen inhibition, which is conducive to enriching denitrifying bacteria. In addition, the gel does not change the internal sludge species, it can shift the dominance of specific microorganisms and improve the removal efficiency of nitrogen. In summary, the immobilization of AAOB and SOB by the gel can achieve effectively mitigate nitrogen pollution in low temperature environments, thus indicating that the SAD/A process has broad engineering application prospects.