Potential Role of the Anammoxosome in the Adaptation of Anammox Bacteria to Salinity Stress.

The underlying adaptative mechanisms of anammox bacteria to salt stress are still unclear. The potential role of the anammoxosome in modulating material and energy metabolism in response to salinity stress was investigated in this study. The results showed that anammox bacteria increased membrane fluidity and decreased mechanical properties by shortening the ladderane fatty acid chain length of anammoxosome in response to salinity shock, which led to the breakdown of the proton motive force driving ATP synthesis and retarded energy metabolism activity. Afterward, the fatty acid chain length and membrane properties were recovered to enhance the energy metabolic activity. The relative transmission electron microscopy (TEM) area proportion of anammoxosome decreased from 55.9 to 38.9% under salinity stress. The 3D imaging of the anammox bacteria based on Synchrotron soft X-ray tomography showed that the reduction in the relative volume proportion of the anammoxosome and the concave surfaces was induced by salinity stress, which led to the lower energy expenditure of the material transportation and provided more binding sites for enzymes. Therefore, anammox bacteria can modulate nitrogen and energy metabolism by changing the membrane properties and morphology of the anammoxosome in response to salinity stress. This study broadens the response mechanism of anammox bacteria to salinity stress.

Biosynthetic potential of uncultured anammox community bacteria revealed through multi-omics analysis.

Microbial secondary metabolites (SMs) and their derivatives have been widely used in medicine, agriculture, and energy. Growing needs for renewable energy and the challenges posed by antibiotic resistance, cancer, and pesticides emphasize the crucial hunt for new SMs. Anaerobic ammonium-oxidation (anammox) systems harbor many uncultured or underexplored bacteria, representing potential resources for discovering novel SMs. Leveraging HiFi long-read metagenomic sequencing, 1,040 biosynthetic gene clusters (BGCs) were unearthed from the anammox microbiome with 58% being complete and showcasing rich diversity. Most of them showed distant relations to known BGCs, implying novelty. Members of the underexplored lineages (Chloroflexota and Planctomycetota) and Proteobacteria contained lots of BGCs, showcasing substantial biosynthetic potential. Metaproteomic results indicated that Planctomycetota members harbored the most active BGCs, particularly those involved in producing potential biofuel-ladderane. Overall, these findings underscore that anammox microbiomes could serve as valuable resources for mining novel BGCs and discovering new SMs for practical application.

The recovery potential and utilization pathway of chemical energy from wastewater pollutants during wastewater treatment in China.

Research on the potential for chemical energy recovery and the optimization of recovery pathways in different regions of China is still lacking. This study aimed to address this gap by evaluating the potential and optimize the utilization pathways for chemical energy recovery in various regions of China for achieving sustainable wastewater treatment. The results showed that the eastern and northeastern regions of China exhibited higher chemical energy levels under the existing operating conditions. Key factors affecting chemical energy recovery included chemical oxygen demand removal (ΔCOD), treatment scale, and specific energy consumption (µ) of wastewater treatment plants (WWTPs). Furthermore, the average improvement in the chemical energy recovery rate with an optimized utilization pathway was approximately 40% in the WWTPs. The use of the net-zero energy consumption (NZE) model proved effective in improving the chemical energy recovery potential, with an average reduction of greenhouse gas (GHG) emissions reaching next to 95% in the investigated WWTPs.

New insights into functional divergence and adaptive evolution of uncultured bacteria in anammox community by complete genome-centric analysis.

Anaerobic ammonium-oxidation (anammox) bacteria play a crucial role in global nitrogen cycling and wastewater nitrogen removal, but they share symbiotic relationships with various other microorganisms. Functional divergence and adaptive evolution of uncultured bacteria in anammox community remain underexplored. Although shotgun metagenomics based on short reads has been widely used in anammox research, metagenome-assembled genomes (MAGs) are often discontinuous and highly contaminated, which limits in-depth analyses of anammox communities. Here, for the first time, we performed Pacific Biosciences high-fidelity (HiFi) long-read sequencing on the anammox granule sludge sample from a lab-scale bioreactor, and obtained 30 accurate and complete metagenome-assembled genomes (cMAGs). These cMAGs were obtained by selecting high-quality circular contigs from initial assemblies of long reads generated by HiFi sequencing, eliminating the need for Illumina short reads, binning, and reassembly. One new anammox species affiliated with Candidatus Jettenia and three species affiliated with novel families were found in this anammox community. cMAG-centric analysis revealed functional divergence in general and nitrogen metabolism among the anammox community members, and they might adopt a cross-feeding strategy in organic matter, cofactors, and vitamins. Furthermore, we identified 63 mobile genetic elements (MGEs) and 50 putative horizontal gene transfer (HGT) events within these cMAGs. The results suggest that HGT events and MGEs related to phage and integration or excision, particularly transposons containing tnpA in anammox bacteria, might play important roles in the adaptive evolution of this anammox community. The cMAGs generated in the present study could be used to establish of a comprehensive database for anammox bacteria and associated microorganisms. These findings highlight the advantages of HiFi sequencing for the studies of complex mixed cultures and advance the understanding of anammox communities.

Motility behavior and physiological response mechanisms of aerobic denitrifier, Enterobacter cloacae strain HNR under high salt stress: Insights from individual cells to populations.

The motility behaviors at the individual-cell level and the collective physiological responsive behaviors of aerobic denitrifier, Enterobacter cloacae strain HNR under high salt stress were investigated. The results revealed that as salinity increased, electron transport activity and adenosine triphosphate content decreased from 15.75 µg O2/g/min and 593.51 mM/L to 3.27 µg O2/g/min and 5.34 mM/L, respectively, at 40 g/L, leading to a reduction in the rotation velocity and vibration amplitude of strain HNR. High salinity stress (40 g/L) down-regulated genes involved in ABC transporters (amino acids, sugars, metal ions, and inorganic ions) and activated the biofilm-related motility regulation mechanism in strain HNR, resulting in a further decrease in flagellar motility capacity and an increase in extracellular polymeric substances secretion (4.08 mg/g cell of PS and 40.03 mg/g cell of PN at 40 g/L). These responses facilitated biofilm formation and proved effective in countering elevated salt stress in strain HNR. Moreover, the genetic diversity associated with biofilm-related motility regulation in strain HNR enhanced the adaptability and stability of the strain HNR populations to salinity stress. This study enables a deeper understanding of the response mechanism of aerobic denitrifiers to high salt stress.

Fate of iron nanoparticles in anammox system: Dissolution, migration and transformation.

Iron nanoparticles (FeNPs) are commonly used in various industrial processes, leading to their release into the environment and eventual entrance into wastewater treatment plants (WWTPs). FeNPs undergo dissolution, migration, and transformation in WWTPs, which can potentially affect the stable operation of anaerobic ammonia oxidation (anammox) systems and may be discharged with wastewater or biomass. To better understand the fate of FeNPs in anammox systems, exposure experiments were conducted using anammox granular sludges (AnGS) and FeNPs. Results demonstrated that FeNPs released Fe2+ upon contact with water, with a portion being bound to functional groups in extracellular polymeric substances (EPS) and the rest entering the bacteria to form highly absorbable substances. A significant amount of FeNPs was observed to cover the surface of AnGS or aggregate and deposit at the bottom of the reactor, eventually converting into Fe3O4 and stably existing within the anammox system. The findings of this study clarify the fate of FeNPs in anammox systems and provide important insights into the stable operation of anammox systems under FeNPs exposure.

The acceleration of aerobic sludge granulation by alternating organic loading rate: Performance and mechanism.

As a highly promising treatment technology for wastewater, long start-up time is one of the bottlenecks hindering the widespread application of aerobic granular sludge (AGS). This study focused on exploring the possibility of alternating organic loading rate (OLR) in promoting AGS granulation. Under alternating OLR (3.6-14.4 kgCOD/m3·d), AGS granulation was significantly accelerated. The mean granule size under alternating load reached 234.6 µm at 17 d, while under constant OLR (7.2 kgCOD/m3·d), the mean granule size was only 179.2 µm. Moreover, the granule size maintained continuous growth even when the alternating OLR was changed to constant OLR. Alternating load significantly increased the content of extracellular polymeric substances (EPS), especially proteins (PN) in tightly bound EPS (TB-EPS), which was likely the main reason for accelerating AGS granulation. Moreover, alternating load reduced the hydrophilicity of EPS and promoted the content of proteins secondary structures that favored aggregation in TB-EPS, which were also beneficial for granulation. Microbial community results showed that alternating load might promote the enrichment of EPS producing bacteria, such as Thauera, Brevundimonas and Shinella. Meanwhile, the content of enzymes that regulated amino acids metabolism also increased under alternating load, which might be related to the increase of PN in EPS. These results further demonstrated that alternating load promoted granulation through EPS.

Potential role of quorum quenching activity of silver nanoparticles in controlling non-filamentous bulking within activated sludge process.

The effective prevention and control of non-filamentous bulking is a significant challenge. In this study, the underlying effect of quorum sensing (QS) on inducing non-filamentous bulking and the maintenance effect of silver nanoparticles (AgNPs) on sludge floc stability, aggregation and settleability based on the quorum quenching (QQ) activity during non-filamentous bulking were investigated. The results showed that the concentration of N-acyl homoserine lactone (AHL) increased significantly in the activated sludge system at a high organic load rate (OLR), triggering the AHL-mediated QS. Additionally, the triggered QS promoted exopolysaccharide secretion, reducing the surface charge and hydrophobicity of the sludge aggregates, and further deteriorating the settleability of the sludge aggregates. AgNPs, a quorum sensing inhibitor (QSI), inhibited the AHL-QS based on QQ activity under high OLR, which maintained the physicochemical properties of extracellular polymeric substances (EPS). AgNPs-QQ maintained the surface energy barrier and electrostatic barrier of sludge aggregates and the gel properties of exopolysaccharides, which is favorable for microbial aggregation. The appropriate concentrations of AgNPs (≤10 mg/L) had no negative effect on biological nutrient removal in the sequencing batch reactors (SBRs) at the high organic loading. Therefore, AgNPs effectively prevent and control non-filamentous bulking by their QQ activity in the activated sludge process. Thus, the present study provided new insights into controlling non-filamentous bulking during the activated sludge process.

Unraveling the structure and function of bacterioferritin in Candidatus Kuenenia stuttgartiensis: Iron storage sites maintain cellular iron homeostasis.

Anammox bacteria rely heavily on iron and have many iron storage sites. However, the biological significance of these iron storage sites has not been clearly defined. In this study, we explored the properties and location of iron storage sites to better understand their cellular function. To do this, the Candidatus Kuenenia stuttgartiensis iron storage protein, bacterioferritin (K.S Bfr), was successfully expressed and purified. In vitro, correctly assembled globulins were observed by transmission electron microscopy. The self-assembled K.S Bfr has active redox and can bind Fe2+ and mineralize it in the protein cavity. In vivo, engineered bacteria with K.S Bfr showed good adaptability to Fe2+, with a survival rate of 78.9% when exposed to 5 mM Fe2+, compared with only 66.0% for wild-type bacteria lacking K.S Bfr. A potential iron regulatory strategy similar to that of Anammox was identified in transcriptomic analysis of engineered bacteria. This system may be controlled by the iron uptake regulator Furto transport Fe2+ via FeoB and store excess Fe2+ in K.S Bfr to maintain cellular homeostasis. K.S Bfr has superior iron storage capacity both intracellularly and in vitro. The discovery of K.S Bfr reveals the storage location of iron-rich nanoparticles, increases our understanding of the adaptability of iron-dependent bacteria to Fe2+, and suggests possible iron regulation strategies in Anammox bacteria.

Mechanisms of survival mediated by the stringent response in Pseudomonas aeruginosa under environmental stress in drinking water systems: Nitrogen deficiency and bacterial competition.

Pseudomonas aeruginosa causes public health problems in drinking water systems. This study investigated the potential role of the stringent response in regulating the adaptive physiological metabolic behaviors of P. aeruginosa to low nitrogen stress and bacterial competition in drinking water systems. The results indicated that guanosine tetraphosphate (ppGpp) concentrations in P. aeruginosa increased to 135.5 pmol/g SS under short-term nitrogen deficiency. Meanwhile, the expression levels of the ppGpp synthesis genes (ppx, relA) and degradation gene (spoT) were upregulated by 37.0% and downregulated by 26.8%, respectively, indicating that the stringent response was triggered. The triggered stringent response inhibited the growth of P. aeruginosa and enhanced the metabolic activity of P. aeruginosa to adapt to nutrient deprivation. The interspecific competition significantly affected the regulation of the stringent response in P. aeruginosa. During short-term nitrogen deficiency, the extracellular polymeric substances concentration of P. aeruginosa decreased significantly, leading to desorption and diffusion of attached bacteria and increased ecological risks. The regulatory effect of stringent response on P. aeruginosa gradually weakened under long-term nitrogen deficiency. However, the expression of pathogenic genes (nalD/PA3310) and flagellar assembly genes (fliC) in P. aeruginosa was upregulated by the stringent response, which increased the risk of disease.

Proteomics reveals the enhancing mechanism for eliminating toxic hydroxylamine from water by nanocompartments containing hydroxylamine oxidase.

Hydroxylamine (NH2OH) is a potentially toxic pollutant when it is present in water, as it can damage both bacteria and the human body. It is still difficult to eliminate the toxic NH2OH in water. Here, we showed that the model bacterium (Escherichia coli) with nanocompartments encapsulated with hydroxylamine oxidase (HAO) can remove NH2OH from water. In addition, the removal efficiency of NH2OH by genetically modified bacteria (with HAO-nanocompartments) was 3.87 mg N L-1 h-1, and that of wild-type bacteria (without HAO-nanocompartments) was only 1.86 mg N L-1 h-1. Label-free quantitative proteomics indicated that the nanocompartments containing HAO enhanced bacterial activity by inducing the up-regulation of proteins involved in stress and stimulus responses, and decreased their intracellular NH2OH concentration. Moreover, the synthesis of proteins involved in energy metabolism, gene expression, and other processes in bacterial was enhanced under hydroxylamine stress, and these changes increased the resistance of bacterial to NH2OH. This work can aid our understanding of the toxic effects of NH2OH on bacteria as well as the development of new approaches to eliminate NH2OH in water.

Effects mechanism of bio-carrier filling rate on rotating biofilms and the reactor performance optimization method.

Benefited from the massive filling bio-carriers, the packed cage rotating biological contactors (RBCs) have better performance and application potentiality in wastewater treatment. Investigating the effects mechanism of bio-carrier filling rate is crucial for such reactors management. In this study, the pollutants removal performance, biofilms physical characteristics, and microbial communities of the biofilms under a series of bio-carrier filling rates were analyzed. The results shown, the pollutant removal rate and amount were quite different under different filling rates, and biofilms structure and microbial composition were the main factors affecting the pollutants removal performance. With the increasing filling rates, the biofilms were more mass increased (dry weight from 0.066 to 0.148 g/per carrier), thicker (from 340.30 to 850.84 µm) and lower dense (from 0.068 to 0.060 g/cm3). The microbial community composition of those biofilms was also quite different at the genus level. The effects mechanism of bio-carrier filling rate can be summarized: the filling rates affect the physical and biological characteristics of biofilms, which will further affect the microenvironment and microbial distribution in biofilms, and then determines the pollutant metabolic rate and metabolic pathway. This study will contribute to design better bio-carrier filling rate according to different wastewater treatment scenario, and promote the performance optimization of packed cage RBCs.

A new pattern of the partial nitrification and Anammox immobilized gel beads: core-shell embedded carrier.

Single-stage partial nitrification and Anammox (PN/A) is an efficient and energy-saving denitrification process for wastewater. However, its application is limited by the growth conditions of microorganisms. Therefore, we improved the PN/A by developing a novel core-shell embedded carrier. With Anammox gel as the core and Ammonia-oxidizing bacteria gel as the shell, these beads can achieve dissolved oxygen partitioning and provide a suitable environment for the growth of different bacteria. On this basis, the influence of the shape of core-shell embedded gel on nitrogen removal performance was systematically studied, and the internal morphology and pore size of gel beads were characterized. The results showed that the nitrogen removal efficiency of spherical and square gels was increased by 33.70% and 13.47%, respectively, in the batch test. Fluorescence in situ hybridization confirmed the stratified growth of ammonia-oxidizing bacteria and Anammox in carriers, and the relative abundance value of the two bacteria were 1.25:1 and 1.43:1, respectively. Although the mechanical strength of square gel beads is slightly higher than that of spherical, spherical gel is considered the most suitable gel shape due to its small pore size and poor pore connectivity, which ensures the matching of internal Anammox and external PN reaction. In the long-term experiment, the core-shell embedded beads still had the design characteristics, and the TN removal efficiency was increased by 36.25% despite occasional oxygen excess.

Cyanophycin Granule Polypeptide: a Neglected High Value-Added Biopolymer, Synthesized in Activated Sludge on a Large Scale.

Recovery of microbial synthetic polymers with high economic value and market demand in activated sludge has attracted extensive attention. This work analyzed the synthesis of cyanophycin granule peptide (CGP) in activated sludge and its adsorption capacity for heavy metals and dyes. The distribution and expression of synthetic genes for eight biopolymers in two wastewater treatment plants (WWTPs) were analyzed by metagenomics and metatranscriptomics. The results indicate that the abundance and expression level of CGP synthase (cphA) are similar to those of polyhydroxyalkanoate polymerase, implying high synthesis of CGP in activated sludges. CGP in activated sludge is mainly polymerized from aspartic acid and arginine, and its secondary structure is mainly ß-sheet. The crude yields of CGP are as high as 104 ± 26 and 76 ± 13 mg/g dry sludge in winter and in summer, respectively, comparable to those of polyhydroxyalkanoate and alginate. CGP has a stronger adsorption capacity for anionic pollutants (Cr (VI) and methyl orange) than for cationic pollutants because it is rich in guanidine groups. This study highlights prospects for recovery and application of CGP from WWTPs. IMPORTANCE The conversion of organic pollutants into bioresources by activated sludge can reduce the carbon dioxide emission of wastewater treatment plants. Identification of new high value-added biopolymers produced by activated sludge is beneficial to recover bioresources. Cyanophycin granule polypeptide (CGP), first discovered in cyanobacteria, has unique chemical and material properties suitable for industrial food, medicine, cosmetics, water treatment, and agriculture applications. Here, we revealed for the first time that activated sludge has a remarkable ability to produce CGP. These findings could further facilitate the conversion of wastewater treatment plants into resource recycling plants.

Biofilm formation during wastewater treatment: Motility and physiological response of aerobic denitrifying bacteria under ammonia stress based on surface plasmon resonance imaging.

A bacterial image analysis system based on surface plasmon resonance imaging was established to investigate the effect of bacterial motility on biofilm formation under high ammonia nitrogen at the single-cell level. The results showed that the bacterial mean rotation speed and vertical motility distance decreased with the increasing concentration of ammonia nitrogen. Ammonia nitrogen inhibited the metabolic activity of the bacteria, decreasing bacterial motility. Bacterial motility was negatively correlated with the biofilm-formation ability. The biofilm formation ability of Enterobacter cloacae strain HNR exposed to ammonia nitrogen was enhanced by reducing its movement and promoting EPS secretion. Genes related to the tricarboxylic acid cycle and oxidative phosphorylation were down-regulated, indicating inhibition of microbial energy metabolism. Genes related to bacterial secretion and lipopolysaccharide synthesis were up-regulated, facilitating the formation of biofilms and enabling the bacteria to resist ammonia nitrogen stress. This study provides new insights into the biofilm formation under ammonia stress.

The GHG mitigation opportunity of sludge management in China.

Greenhouse gas (GHG) mitigation in wastewater treatment sector is indispensable in China's carbon neutral target. As an important component of wastewater system, sludge generation is rapidly increased with the acceleration of urbanization in China. It is crucial to investigate the carbon footprint of various sludge management strategies and quantify the potential optimization of GHG reduction effect at national scale. Therefore, this study conducted a comprehensive analysis of sludge distribution and GHG profiles of various sludge systems. The overall dry sludge generation in China is 12.15 Mt, with spatial resolution at city level. Different sludge treatment options were categorized into four types: energy recovery, nutrient recovery (e.g. phosphorus and nitrogen), material valorisation (e.g. brick, biochar) and conventional disposal. With various sludge treatment options, the GHG profile of annual sludge management in China ranges from -35.86 Mt/year to 57.11 Mt/year. The best GHG mitigation can be achieved through energy recovery by co-incineration system and the greatest reduction opportunity is concentrated in highly urbanized regions, such as Yangtze River Delta, Pearl River Delta, and Beijing-Tianjin-Hebei urban agglomerations.

Encapsulins from Ca. Brocadia fulgida: An effective tool to enhance the tolerance of engineered bacteria (pET-28a-cEnc) to Zn2.

Zn2+ is largely discharged from many industries and poses a severe threat to the environment, making its remediation crucial. Encapsulins, proteinaceous nano-compartments, may protect cells against environmental stresses by sequestering toxic substances. To determine whether hemerythrin-containing encapsulins (cEnc) from anammox bacteria Ca. Brocadia fulgida can help cells deal with toxic substances such as Zn2+, we transferred cEnc into E.coli by molecular biology technologies for massive expression and then cultured them in media with increasing Zn2+ levels. The engineered bacteria (with cEnc) grew better and entered the apoptosis phase later, while wild bacteria showed poor survival. Furthermore, tandem mass tag-based quantitative proteomic analysis was used to reveal the underlying regulatory mechanism by which the genetically-engineered bacteria (with cEnc) adapted to Zn2+ stress. When Zn2+ was sequestered in cEnc as a transition, the engineered bacteria presented a complex network of regulatory systems against Zn2+-induced cytotoxicity, including functions related to ribosomes, sulfur metabolism, flagellar assembly, DNA repair, protein synthesis, and Zn2+ efflux. Our findings offer an effective and promising stress control strategy to enhance the Zn2+ tolerance of bacteria for Zn2+ remediation and provide a new application for encapsulins.

Quantitative proteomics and phosphoproteomics elucidate the molecular mechanism of nanostructured TiO2-stimulated biofilm formation.

With the increasing concerns regarding bacterial adaption to nanomaterials, it is critical to explore the main mechanism behind the adaptive morphogenesis of microorganisms. In this work, the biofilms formed from activated sludge exposed to 5 and 50 mg/L nTiO2 in the dark had increased biomass and selectively enriched pathogens. To further elaborate adaptive mechanism of biofilm formation induced by nTiO2, the protein response and protein phosphorylation modification of Escherichia coli K12 were determined using integrative systems biology analyses of proteomics and phosphoproteomics. Results identified that E. coli cultivated with nTiO2 considerably upregulated iron acquisition, and regulated protein phosphorylation states associated with of transcription and translation and biofilm formation relative to unexposed controls. Accordingly, bacteria increased siderophores and exopolysaccharide content (increased by about 57% and 231%, respectively), and enhanced resistance to transcriptional inhibitory antibiotics. Moreover, a dose of an iron chelator, i.e., deferoxamine mesylate salt, effectively retarded the biofilm development of bacteria exposed to 50 mg/L nTiO2. Overall, this work will provide a new insight for biofouling control, and contribute to an improved understanding of microbial adaption to nanomaterials.

A full-view management method based on artificial neural networks for energy and material-savings in wastewater treatment plants.

Carbon neutrality has been received extensive attention in the field of wastewater treatment. The optimal management of wastewater treatment plants (WWTPs) has great significance and urgency since the serious energy and materials waste. In this study, a full-view management method based on artificial neural networks (ANNs) for energy and material savings in WWTPs was established. More than 5 years of historical operating data from two typical plants (size 40,000 t/d and 10,000 t/d) located in Chongqing, China, were obtained, and public data in the service area of each plant were systematically collected from open channels. These abundant historical and public data were used to train two ANNs (GRA-CNN-LSTM model and PCA-BPNN model) to predict the inlets/outlets wastewater quality and quantity. The overall average prediction accuracy of inlets/outlets wastewater indicators are greater than 92.60% and 93.76%, respectively. By combining the two models, more appropriate process operation strategies can be obtained 2 weeks in advance, with more than 11.20% and 16.91% reduction of energy and material costs, respectively. This proposed method can provide full-view decision support for the optimal management of WWTPs and is also expected to support carbon emission control and carbon neutrality in the field of wastewater treatment.

New insight into filamentous sludge bulking: Potential role of AHL-mediated quorum sensing in deteriorating sludge floc stability and structure.

The microcosmic mechanisms underlying filamentous bulking remain unclear. The role of extracellular polymeric substances (EPS) governed by quorum sensing (QS) in deteriorating sludge floc stability and structure during filamentous bulking and the feasibility of using quorum quenching (QQ) to maintain sludge floc stability and structure and sludge settling were investigated in this study. The results indicated that the concentration of C6HSL increased from 22.08±3.22 ng/g VSS to 81.42±5.98 ng/g VSS during filamentous bulking. The filamentous bacteria gradually evolved the hdtS gene related to the synthesis of C6HSL with increases in the population density. Triggered QS by filamentous bacteria proliferation induced variation in the composition and structure of EPS within the sludge flocs. The proteins (PN) content of the EPS increased evidently from 40.06 ± 2.41 mg/g VSS to 110.32 ± 4.32 mg/g VSS, and the polysaccharides (PS) content slightly increased during filamentous bulking. The upregulated proteins in the EPS led to a decrease in the relative hydrophobicity of the sludge and an increase in negative surface charge. The α-helix/(ß-sheet+random coil) ratio evidently increased from 0.76 to 0.99 during filamentous bulking, revealing that the proteins were tightly structured, which prevented the exposure of inner hydrophobic groups. The total energy of the interaction (WT) between bacteria increased during sludge bulking, which resulted in the weakening of sludge aggregation. Variation in the physicochemical properties of EPS induced by QS in the filamentous bacteria markedly restrained adhesion between the filamentous bacteria and floc-forming bacteria. The production of PN in the EPS and the expression of the hdtS gene were inhibited by vanillin, which served as a QS inhibitor. The WT between bacteria with 50 mg/L of vanillin basically did not change. Filamentous bulking was significantly inhibited by the addition of vanillin. Therefore, QQ is a potential strategy for the prevention and control of filamentous bulking. This study provides new information regarding the microcosmic mechanisms of filamentous bulking.