Pilot-scale investigation of performance and microbial community in a novel system combining fixed and suspended activated sludge.

The conventional activated sludge (CAS) process is a widely used method for wastewater treatment due to its effectiveness and affordability. However, it can be prone to sludge abnormalities such as sludge bulking/foaming and sludge loss, which can lead to a decrease in treatment efficiency. To address these issues, a novel bag-based fixed activated sludge (BBFAS) system utilizing mesh bags to contain the sludge was developed for low carbon/nitrogen ratio wastewater treatment. Pilot-scale experiments demonstrated that the BBFAS system could successfully avoid the sludge abnormalities. Moreover, it was not affected by mass transfer resistance and exhibited significantly higher nitrogen removal efficiency, surpassing that of the CAS system by up to 78%. Additionally, the BBFAS system demonstrated comparable organic matter removal efficiency to CAS system. 16S rRNA gene high-throughput sequencing revealed that the bacterial community structure within the BBFAS system was significantly different from that of the CAS system. The bacteria associated with ammonium removal were more abundant in the BBFAS system than in the CAS system. The abundance of Nitrospira in the BBFAS could reach up to 6% and significantly higher than that in the CAS system, and they were likely responsible for both ammonia-oxidizing and nitrite-oxidizing functions. Clear stratification of microbial communities was observed from the outer to inner layers of the bag components due to the gradients of dissolved oxygen and other substrates. Overall, this study presents a promising approach for avoiding activated sludge abnormalities while maintaining high pollutant removal performance.

New insight into soluble extracellular metabolites during sludge bulking process based on excitation-emission matrix spectroscopy and ultrahigh-performance liquid chromatography-mass spectrometry.

Soluble extracellular metabolites (SEM) produced by microorganisms might significantly change during sludge bulking, which is a major operational problem caused by the excessive growth of filamentous bacteria. However, knowledge remains limited about the dynamics and potential role of SEM in the bulking of sludge. In this study, filamentous bulking was simulated in a laboratory-scale reactor and changes to SEM characteristics during the bulking process were investigated using excitation-emission matrix spectroscopy and ultrahigh-performance liquid chromatography-mass spectrometry. SEM components changed significantly at different phases of sludge bulking. Changes in SEM were closely correlated with the structure of the bacterial community. Based on the EEM profiles, significant increases in fulvic acid-like and humic acid-like substances in SEM were observed with the development of filamentous bulking. The degree of humification in SEM showed a clear increasing trend. Untargeted extracellular metabolomic analysis showed that the intensity of berberine and isorhamnetin in SEM increased significantly during the bulking phase, which might synergistically facilitate the development of filamentous bulking.

Sludge bulking monitoring in industrial wastewater treatment plants through graphical methods: A dynamic graph embedding and Bayesian networks approach.

Sludge bulking is a prevalent issue in wastewater treatment plants (WWTPs) that negatively impacts effluent quality by hindering the normal functioning of treatment processes. To tackle this problem, we propose a novel graph-based monitoring framework that employs advanced graph-based techniques to detect and diagnose sludge bulking events. The proposed framework utilizes historical datasets under normal operating conditions to extract pertinent features and causal relationships between process variables. This enables operators to trigger alarms and diagnose the root cause of the bulking event. Sludge bulking detection is carried out using the dynamic graph embedding (DGE) method, which identifies similarities among process variables in both temporal and neighborhood dependencies. Consequently, the dynamic Bayesian network (DBN) computes the prior and posterior probabilities of a belief, updated at each time step. Variations in these probabilities indicate the potential root cause of the sludge bulking event. The results demonstrate that the DGE outperforms other linear and non-linear feature extraction methods, achieving a detection rate of 99%, zero false alarms, and less than one percent incorrect detections. Additionally, the DBN-based diagnostic method accurately identified the majority of sludge bulking root causes, primarily those resulting from sudden drops in COD concentration, with an accuracy of 98% an improvement of 11% over state-of-the-art techniques.

Recent advances in understanding the ecology of the filamentous bacteria responsible for activated sludge bulking.

Activated sludge bulking caused by filamentous bacteria is still a problem in wastewater treatment plants around the world. Bulking is a microbiological problem, and so its solution on species-specific basis is likely to be reached only after their ecology, physiology and metabolism is better understood. Culture-independent molecular methods have provided much useful information about this group of organisms, and in this review, the methods employed and the information they provide are critically assessed. Their application to understanding bulking caused by the most frequently seen filament in Japan, 'Ca. Kouleothrix', is used here as an example of how these techniques might be used to develop control strategies. Whole genome sequences are now available for some of filamentous bacteria responsible for bulking, and so it is possible to understand why these filaments might thrive in activated sludge plants, and provide clues as to how eventually they might be controlled specifically.

Viscosity dynamics and the production of extracellular polymeric substances and soluble microbial products during anaerobic digestion of pulp and paper mill wastewater sludges.

The production processes of the pulp and paper industry often run in campaigns, leading to large variations in the composition of wastewaters and waste sludges. During anaerobic digestion (AD) of these wastes, the viscosity or the production of extracellular polymeric substances (EPS) and soluble microbial products (SMP) may be affected, with the risk of foam formation, inefficient digester mixing or poor sludge dewaterability. The aim of this study was to investigate how viscosity and production of EPS and SMP during long-term AD of pulp and paper mill sludge is affected by changes in organic loading rate (OLR) and hydraulic retention time (HRT). Two mesophilic lab-scale continuous stirred tank reactors (CSTRs) were operated for 800 days (R1 and R2), initially digesting only fibre sludge, then co-digesting fibre sludge and activated sludge. The HRT was lowered, followed by an increase in the OLR. Reactor fluids were sampled once a month for rheological characterization and analysis of EPS and SMP. The production of the protein fraction of SMP was positively correlated to the OLR, implicating reduced effluent qualities at high OLR. EPS formation correlated with the magnesium content, and during sulphate deficiency, the production of EPS and SMP increased. At high levels of EPS and SMP, there was an increase in viscosity of the anaerobic sludges, and dewatering efficiency was reduced. In addition, increased viscosity and/or the production of EPS and SMP were important factors in sludge bulking and foam formation in the CSTRs. Sludge bulking was avoided by more frequent stirring.

Soft Sensor Application in Identification of the Activated Sludge Bulking Considering the Technological and Economical Aspects of Smart Systems Functioning.

The paper presented the methodology for the construction of a soft sensor used for activated sludge bulking identification. Devising such solutions fits within the current trends and development of a smart system and infrastructure within smart cities. In order to optimize the selection of the data-mining method depending on the data collected within a wastewater treatment plant (WWTP), a number of methods were considered, including: artificial neural networks, support vector machines, random forests, boosted trees, and logistic regression. The analysis conducted sought the combinations of independent variables for which the devised soft sensor is characterized with high accuracy and at a relatively low cost of determination. With the measurement results pertaining to the quantity and quality of wastewater as well as the temperature in the activated sludge chambers, a good fit can be achieved with the boosted trees method. In order to simplify the selection of an optimal method for the identification of activated sludge bulking depending on the model requirements and the data collected within the WWTP, an original system of weight estimation was proposed, enabling a reduction in the number of independent variables in a model-quantity and quality of wastewater, operational parameters, and the cost of conducting measurements.

Impact of sludge bulking on receiving environment using quantitative microbial risk assessment (QMRA)-based management for full-scale wastewater treatment plants.

During sludge bulking in wastewater treatment plants (WWTPs), high amounts of potentially pathogenic bacteria would release into the environment, causing various human-health risks. This is the first study attempting to assess the microbial infections associated with the reuse of WWTP effluents under various bulking conditions. Three common waterborne pathogens, viz., E. coli O157:H7, Salmonella, and Mycobacterium, were quantified from full-scale WWTPs using DNA extraction and qPCR at different sludge volume indices (SVIs). The detected pathogens were incorporated into a quantitative microbial risk assessment (QMRA) model to determine the applicability of WWTP discharge for recreational (bathing) activities and agricultural practices. The QMRA exposures were children, women, and men during swimming, and farmers and vegetable consumers during irrigation. Bacterial abundance in the treated wastewater increased in response to SVIs, and the QMRA values at all bulking events exceeded the tolerable risk of one case of infection per 10,000 people per year. Hence, various disinfection scenarios (chlorination, ultraviolet, and ozonation) were hypothetically tested to control the risks associated with pathogenic bacteria, allowing for safe disposal and reuse of the treated effluent. The ultraviolet application provided the highest ability to inactivate the pathogenic bacteria, except for the case of children exposed to Salmonella infection during swimming. The reduction of Mycobacterium infection risks with either chlorination or ozonation showed inefficient results. This study would be helpful for the management of human health risks associated with effluent wastewater containing pathogens, i.e., particularly concerning the case of sludge bulking.

The impact of powdered keramsite on activated sludge and wastewater treatment in a sequencing batch reactor.

As the efficiency of biological wastewater treatment in activated sludge systems can be increased if powdered mineral materials are added, the work described here investigated the effect of powdered keramsite on activated sludge and wastewater treatment in a sequencing batch reactor on the laboratory scale. Specifically, experiments were carried out on two sequencing batch reactors, with the reference system being the classical SBR with conventional activated sludge, while the second system involved a K-SBR (Keramsite - Sequencing Batch Reactor) with powdered keramsite added with a view to enhancing the effectiveness of the activated sludge. The concentration of powdered keramsite in the latter reactor was maintained at 0.75 g/L. The results confirmed the greater efficiency of biological wastewater treatment processes where activated sludge was supported by the keramsite.

Phage-host associations in a full-scale activated sludge plant during sludge bulking.

Sludge bulking, a notorious microbial issue in activated sludge plants, is always accompanied by dramatic changes in the bacterial community. Despite large numbers of phages in sludge systems, their responses to sludge bulking and phage-host associations during bulking are unknown. In this study, high-throughput sequencing of viral metagenomes and bacterial 16S rRNA genes were employed to characterize viral and bacterial communities in a sludge plant under different sludge conditions (sludge volume index (SVI) of 180, 132, and 73 ml/g). Bulking sludges (SVI > 125 ml/g) taken about 10 months apart exhibited similar bacterial and viral composition. This reflects ecological resilience of the sludge microbial community and indicates that changes in viral and bacterial populations correlate closely with each other. Overgrowth of "Candidatus Microthrix parvicella" led to filamentous bulking, but few corresponding viral genotypes were identified. In contrast, sludge viromes were characterized by numerous contigs associated with "Candidatus Accumulibacter phosphatis," suggesting an abundance of corresponding phages in the sludge viral community. Notably, while nitrifiers (mainly Nitrosomonadaceae and Nitrospiraceae) declined significantly along with sludge bulking, their corresponding viral contigs were identified more frequently and with greater abundance in the bulking viromes, implying that phage-mediated lysis might contribute to the loss of autotrophic nitrifiers under bulking conditions.

Impact of nutrient deficiency on biological sewage treatment - Perspectives towards urine source segregation.

Human urine contains 9 g/L of nitrogen (N) and 0.7 g/L of phosphorus (P). The recovery of N and P from urine helps close the nutrient loop and increase resource circularity in the sewage treatment sector. Urine contributes an average of 80 % N and 50 % P in sewage, whereby urine source segregation could reduce the burden of nutrient removal in sewage treatment plants (STPs) but result in N and P deficiency and unintended negative consequences. This review examines the potential impacts of N and P deficiency on the removal of organic carbon and nutrients, sludge characteristics and greenhouse gas emissions in activated sludge processes. The details of how these impacts affect the operation of STPs were also included. This review helps foresee operational challenges that established STPs may face when dealing with nutrient-deficient sewage in a future where source separation of urine is the norm. The findings indicate that the requirement of nitrification-denitrification and biological P removal processes could shrink at urine segregation above 80 % and 100 %, respectively. Organic carbon, N and biological P removal processes can be severely affected under full urine segregation. The decrease in solid retention time due to urine segregation increases treatment capacity up to 48 %. Sludge flocculation and settleability would deteriorate due to changes in extracellular polymeric substances and induce various forms of bulking. Beneficially, N deficiency reduces nitrous oxide emissions. These findings emphasise the importance of considering and preparing for impacts caused by urine source segregation-induced nutrient deficiency in sewage treatment processes.

Effects of enhanced biological phosphorus removal on rapid control of sludge bulking and fast formation of aerobic granular sludge.

This study investigated the effects of enhanced biological phosphorus removal (EBPR) on rapid sludge bulking control and fast aerobic granular sludge (AGS) formation by adding 20 % of EBPR activated sludge to the bulking activated sludge (BAS) reactor. The results indicate that activating EBPR activity swiftly improved BAS settleability within 16 days, thus resolving sludge bulking issues. Subsequently, a settling time-based selection was employed, resulting in the BAS granulation within another 16 days. The rapid achievement of EBPR activity improved the BAS settleability and facilitated the formation of sludge aggregates, thereby expediting BAS granulation. Inhibition of filamentous bacteria and enrichment of slow-growing organisms contributed to both sludge bulking control and aerobic granulation. Furthermore, the increase in proteins/polysaccharides ratio facilitated the granulation process. Additionally, total nitrogen removal increased from 59.4 % to 71.7 % because of the mature AGS formation. This study provided an approach to simultaneously control sludge bulking and promote aerobic granulation.

Filamentous bacteria-induced sludge bulking can alter antibiotic resistance gene profiles and increase potential risks in wastewater treatment systems.

Sludge bulking caused by filamentous bacteria is a prevalent issue in wastewater treatment systems. While previous studies have primarily concentrated on controlling sludge bulking, the biological risks associated with it have been overlooked. This study demonstrates that excessive growth of filamentous bacteria during sludge bulking can significantly increase the abundance of antibiotic resistance genes (ARGs) in activated sludge. Through metagenomic analysis, we identified specific ARGs carried by filamentous bacteria, such as Sphaerotilus and Thiothrix, which are responsible for bulking. Additionally, by examining over 1,000 filamentous bacterial genomes, we discovered a diverse array of ARGs across different filamentous bacteria derived from wastewater treatment systems. Our findings indicate that 74.84% of the filamentous bacteria harbor at least one ARG, with the occurrence frequency of ARGs in these bacteria being approximately 1.5 times higher than that in the overall bacterial population in activated sludge. Furthermore, genomic and metagenomic analyses have shown that the ARGs in filamentous bacteria are closely linked to mobile genetic elements and are frequently found in potentially pathogenic bacteria, highlighting potential risks posed by these filamentous bacteria. These insights enhance our understanding of ARGs in activated sludge and underscore the importance of risk management in wastewater treatment systems.

Effects of silica fume powder modified by oleic acid on the settleability of bulking sludge.

Modified silica fume powder with oleic acid through coupling agent was prepared based on the in situ utilizing long-chain fatty acids (LCFA) properties of Microthrix parvicella (M. parvicella) in the activated sludge system. The modification was confirmed by XRD and infrared spectrum. The contact angle analysis showed that the modification gave the silica fume powder a hydrophobic surface. The modified silica fume powder had a good combination with M. parvicella from the SEM and Gram staining measurements. The addition of modified silica powder has a certain effect on the settling capacity of sludge, but has little effect on the sludge treatment capacity, while the SVI dropped from 400.1 to 100.0 mL/g. These suggested that the modified silica fume powder could be used as an excellent weight-increasing agent to inhibit sludge bulking.

A sludge bulking wastewater treatment plant with an oxidation ditch-denitrification filter in a cold region: bacterial community composition and antibiotic resistance genes.

Bacterial community structure of activated sludge directly affects the stable operation of WWTPS, and these bacterial communities may carry a variety of antibiotic resistance genes (ARGs), which is a threat to the public health. This study employed 16S rRNA gene sequencing and metagenomic sequencing to investigate the bacterial community composition and the ARGs in a sludge bulking oxidation ditch-denitrification filter WWTP in a cold region. The results showed that Trichococcus (20.34%), Blautia (7.72%), and Faecalibacterium (3.64%) were the main bacterial genera in the influent. The relative abundances of norank_f_Saprospiraceae and Candidatus_Microthrix reached 10.24% and 8.40%, respectively, in bulking sludge, and those of norank_f_Saprospiraceae and Candidatus_Microthrix decreased to 6.56 and 7.10% after the anaerobic tank, indicating that the anaerobic tank had an inhibitory effect on filamentous bacteria. After 20 mJ/cm2 UV disinfection, about 540 bacterial genera, such as Romboutsia (7.99%), Rhodoferax (7.98%), and Thermomonas (4.13%), could still be detected in the effluent. The ARGs were 345.11 ppm in the influent and 11.20 ppm in the effluent; 17 subtypes, such as sul1, msrE, aadA5, ErmF, and tet(A), could be detected throughout the entire process. These ARG subtypes were persistent ARGs with a high health risk. Network analysis indicated that the changes in filamentous bacteria norank_f_Saprospiraceae abundance mainly contributed to the abundance shift of MexB, and Acinetobacter mainly increased the abundance of drfA1. These results above will provide theoretical support for the sludge bulking and ARGs controls of WWTPs in cold regions.

Metabolomic pathway regulation for prevention and control of granule sludge bulking in thiosulfate-driven denitrification.

The performance of thiosulfate-driven denitrification (TDD) granule reactor and the mechanism of granule sludge bulking were investigated in this study. The results showed that TDD granule bulking occurred under 12 kgNm-3d-1 of nitrogen loading rate (NLR). The higher NLR promoted accumulation of intermediates in the carbon fixation pathway, including citrate, oxaloacetate, oxoglutarate and fumarate. The carbon fixation improved amino acids biosynthesis, which increased proteins (PN) in extracellular polymers (EPS) to 134.6 ± 11.8 mg/gVSS. The excessive PN altered the content, components and chemical groups of EPS, leading to change of granule structure and decline in settling property, permeability and nitrogen removal. By adopting the strategy of intermittently reducing NLR, excess amino acids in sulfur-oxidizing bacteria was consumed through microbial growth-related metabolism instead of EPS synthesis. Therefore, the nitrogen removal rate increased to 10.23 kg-Nm-3d-1 and maintained stable in the long term. The EPS contents decreased from 168.8 ± 13.5 mg/gVSS to 93 ± 11.5 mg/gVSS and the SVI5 decreased from 66 ± 3.5 ml/g to 25 ± 1.5 ml/g. These findings provide an effective strategy to prevent granule bulking and guide practical application of TDD process.

Study on the inhibition of sludge bulking in A2/O coupled system at low temperature by segmented inlet water mode.

This study aimed to propose a segmented influent method to inhibit sludge bulking. The sludge bulking phenomenon was observed in a A2/O coupled system treating municipal wastewater under low temperature (15 ± 0.5)°C. Adopting the segmented inlet water process, the distribution ratio of the inlet flow in the anaerobic zone and the aerobic zone were 2:1 and 1:1, the sludge bulking phenomenon was suppressed. The sludge loading rate (F/M) analysis showed that the F/M of the anaerobic zone with single-point inflow was only 0.15 kg COD·(kg MLSS·d)-1, which was prone to induce sludge bulking. However, the F/M concentration gradient of the system under segmented inlet water conditions was obvious, which could inhibit the sludge bulking caused by low F/M. The effluent removal results showed that the system had high removal rates of COD, NH4+-N, TN, and TP at a flow distribution ratio of 2:1, with average removal rates of 88.85% ± 2.94%, 91.26% ± 6.68%, 76.60% ± 5.60%, and 96.80% ± 2.17%, respectively. This study confirmed that the segment inlet method inhibited sludge bulking, while the flow distribution ratio of 2:1 also ensured efficient pollutant removal capacity of the system.

Non-filamentous sludge bulking induced by exopolysaccharide variation in structure and properties during aerobic granulation.

The forming mechanism of non-filamentous sludge bulking during aerobic granulation were investigated basing on three feeding strategies (R1 direct aeration after fast feeding, R2 anaerobic stirring after fast feeding and R3 anaerobic plug-flow slow feeding). Results showed that strong selection stress (shortening settling time) led to a sharp flocs washout and the subsequent increase of food to microorganisms (F/M) in R1 and R3 reactors, but not found in R2 due to the different strategies of feeding modes. With the increase of F/M, zeta potential and hydrophobicity of sludge surfaces significantly decreased and thus leading to an enhanced repulsive force and energy barriers for sludge aggregation. Particularly, when F/M exceeded 1.2 kgCOD/(kgMLSS·d), non-filamentous sludge bulking was ultimately triggered in R1 and R3. Further analysis showed that massive extracellular exopolysaccharide (PS) accumulated on the surfaces of non-filamentous bulking sludge due to the increased abundance of the microorganisms related to PS secretion during sludge bulking. In addition, significantly increased intracellular second messenger (c-di-GMP), a key substance regulating PS biosynthesis, was confirmed via its concentration determination as well as microbial function prediction analysis, which played a critical role in sludge bulking. Combing with the systematic detection from surface plasmon resonance system, rheometer and size-exclusion chromatography-multiangle laser light detection-refractive index system, higher molecular weight, compact conformation, higher viscosity and higher hydrophilicity was determined in sludge bulking PS relative to PS extracted from non-filamentous bulking sludge. Clearly, the changes of PS (content, structures and properties) driven by c-di-GMP are the dominant mechanism for the formation of non-filamentous sludge bulking during aerobic granulation. This work could provide theoretical support for successful start-up and application of aerobic granular sludge technology.

[Aerobic Granulation Stability and Microbial Diversity of Filamentous Bulking Sludge].

Filamentous bacteria, as one of the common bacteria types in wastewater biological treatment, are considered to be the main factor to induce sludge bulking. However, because of its special filamentous shape, it plays a crucial role in the formation of sludge particles. Taking filamentous bulking sludge as the research object, the effect of filamentous bacteria on the sludge granulation process and maintaining the stability of sludge granules was studied, and the microbial diversity of the sludge system was analyzed. Filamentous bulking sludge (SVI=241.56 mL·g-1) and flocculated sludge (SVI=64.22 mL·g-1) were respectively inoculated to carry out granulation culture. The results showed that the time of particle appearance of bulking sludge and flocculated sludge was 20 days and 40 days, respectively; the mature particle sizes were 650 µm and 700 µm, respectively; and the granulation time of bulking sludge was only half that of flocculated sludge. After adding the anoxic zone, the granules were broken to differing degrees, but the SV30/SV5 value of mature granules recovered to 1 after short-term fluctuation, and the stability of the mature granules was stronger. The analysis of microbial community structure showed that the relative abundance of norank_o__Saccharimonadales, unclassified_o__Saccharimonadales, and unclassified_f__Saccharimonadaceae increased from 0.05%, 0.01%, and 0.01% to 4.09%, 3.15%, and 1.12%. The existence and accumulations of these hydrophobic bacteria were important for rapid granulation. The removal rates of COD, NH4+-N, and TN were 94%, 99%, and 35% and 92%, 97%, and 30%, respectively, in SBR1 of bulking sludge and SBR2 of flocculated sludge, and the removal rates of TP were 60% and 30%, respectively.

Regulation of exogenous acyl homoserine lactones on sludge settling performance: Monitoring via ultrasonic time-domain reflectometry.

Despite extensive studies, a comprehensive solution for sludge bulking has not yet been found. This study improves the sludge settling performance via quorum sensing (QS) by adding exogenous acyl homoserine lactones (AHLs). First, a novel approach based on ultrasonic time-domain reflectometry, which can automatically and in-situ assess a sludge volume index (SVI), was developed using the displacement in the ultrasonic spectra as a feasible indicator (R2 = 0.98, p < 0.01). Next, the effects of typical AHLs, i.e., 3OC6-HSL, C12-HSL, and 3OC14-HSL, on sludge settling properties were investigated. Results indicated that the three AHLs significantly promoted the sludge settleability by 1.90, 2.03, and 1.62 times, respectively. The regulation mechanisms were investigated from the perspective of sludge physicochemical properties and biological community interactions. The draining degree of water to extracellular polymeric substances (EPS) significantly increased (p < 0.05) with all three AHLs. Meanwhile, the hydrophobic tryptophan content increased with the addition of 3OC6-HSL and C12-HSL. Hence, EPS hydrophobicity was promoted, which is conducive to microbial aggregation. In addition, molecular ecological networks of activated sludge (AS) indicated that bacterial community structures were more complex and species interactions were more intense when adding 3OC6-HSL and C12-HSL. Meanwhile, additional keystones were identified, with the proportion of QS species increasing by 63.6% and 22.2%, respectively. Exogenous 3OC6-HSL eventually decreased the gross relative abundance of filamentous bacteria by 2.37%. Overall, appropriate AHLs could enhance community stability and microbial cooperation by strengthening the communication hub role of QS species, thereby suppressing the overgrowth of filamentous bacteria and improving the sludge settleability. This study provides an effective strategy to determine the appropriate AHL to rapidly eliminate filamentous bulking problems.

Transboundary intercellular communications between Penicillium and bacterial communities during sludge bulking: Inspirations on quenching fungal dominance.

Fungal bulking is caused by the evolution toward a fungi-dominant unbalanced sludge system, which is indeed the phenomenon of fungi competing against bacterial cells. We hypothesized that the cross-kingdom intercellular communication between fungi and bacteria was internal driving force that stimulated fungal bulking. In this study, we identified three signal molecules related to Penicillium fungi bulking under low-pH stress in an activated sludge reactor, which inspired us to propose a sludge bulking prevention strategy using the quorum quenching theory. When pH dropped from 7.0 to 4.5, the abundance of Penicillium increased from 12.5% to 44.8%. However, some functional bacterial genera, such as Nitrosomonas and Sphingopyxis, were washed out from the sludge. The production of quorum-sensing (QS) molecules N-Heptanoyl-L-homoserine lactone (C7-HSL), N-Dodecanoyl-L-homoserine lactone (C12-HSL), and N-Tetradecanoyl-L-homoserine lactone (C14-HSL) was regulated with sludge bulking; especially the response of the latter two was significantly negative to Penicillium blooming (P < 0.05). To test their roles, trace commercial C12-HSL and C14-HSL were added to Penicillium culture, successfully causing 8.3% and 30.2% inhibition of mycelial formation, respectively. They also contributed to the improvement of activated sludge settleability by 6.1% and 39.7%, respectively (represented by sludge volume index). The transcriptome technique further revealed the regulation of the expression of genes in |logFC| >1, involving signal transduction, mycelium synthesis, and metabolic pathways. Our study provided an innovative strategy for controlling fungal bulking from the perspective of microbial transboundary informatics.