Sludge Community Research Articles

Impact of Silver and Copper Oxide Nanoparticles on Anaerobic Digestion of Sludge and Bacterial Community Structure

The effect of metal nanoparticles on the anaerobic digestion of sludge and the sludge bacterial community are still not well-understood, and both improvements and inhibitions have been reported. This study investigated the impact of 2, 10, and 30 mg/g TS silver and copper oxide nanoparticles (AgNPs and CuONPs) on the mesophilic anaerobic digestion of sludge and the bacterial community structure. The reactors were monitored for changes in tCOD, sCOD, TS, VS, biogas generation, and cell viability. Also, the relative abundance and taxonomic distribution of the bacterial communities were analyzed at the phylum and genus levels, including the genera involved in anaerobic digestion. Both AgNPs and CuONPs exhibited some inhibition on anaerobic digestion of sludge and biogas generation, and the inhibition was more evident at higher concentrations. CuONPs had a stronger inhibitory effect compared to AgNPs. After the introduction of AgNPs and CuONPs, cell viability initially decreased over the first two weeks but recovered after that. At high concentrations, AgNPs and CuONPs decreased the overall bacterial diversity, and inhibited the dominant bacterial species, allowing those in less abundance to flourish. The relative abundance of the bacteria responsible for hydrolysis and acidogenesis increased and the relative abundance of acetogenic bacteria decreased with higher AgNP and CuONP concentrations. The majority of the parameters measured for monitoring the anaerobic digestion performance and bacterial community were not statistically significant at 2 mg/g TS of AgNPs and CuONPs, which represents naturally present concentrations in wastewater sludge that are below the USEPA ceiling concentration limits.

Total-solids-controlled microbial response and volatile fatty acids production in sludge and chicken manure co-fermentation.

With the aim of exploring the association between microbial response and volatile fatty acids (VFAs) production in sludge and chicken manure co-fermentation with total solids (TS) controlled, four fermentation experimental groups (TS=20, 40, 60, and 80g/L) were established in this study. The results demonstrated that the yield of VFAs reached the peak (530.08mg COD/g VSS) at the 40g-TS group. For microbial characteristics, Firmicutes, Bacteroidota, Spirochaetota, and Proteobacteria were the main dominant phyla in each experimental group. Meanwhile, it could be proven that the enrichment of functional strains had a significant effect on the production and accumulation of VFAs at the 40g-TS group through α analysis and microbial community structure analysis. In addition, Bacteroidota was predicted to be the main producer of VFAs in the experimental co-fermentation systems through the Faprotax function prediction. This study revealed the effects of different TS concentrations on microbial communities in sludge and chicken manure co-fermentation, and investigated the relationship between microbial community and VFAs production.

Intensification of Wastewater Treatment at Small Sewage Treatment Plants

The ways of intensification of domestic wastewater treatment processes at small wastewater treatment plants with capacity of 40 m3/day due to modernization of process scheme and application of bioaugmentation technology are considered. The directed selection of community with the introduction of fungal culture Penicillium brasilanum, which presumably plays the role of "biological framework" in the formation of aerobic sludge granule, was carried out. It is found that the isolated fungal culture contributes to the improvement of sedimentation properties. It is concluded that the introduction of fungal culture into the activated sludge community of wastewater treatment plants reduces the sludge index.

Choice of activated sludge microorganisms under phosphate deficiency: Competition for inorganic phosphate or change to organophosphorus metabolism

Phosphorus (P) plays a crucial role in the microbial processes of wastewater treatment. However, the current understanding of P metabolic pathways in wastewater is incomplete, and the mechanisms of microbial P metabolism under P deficient conditions remain unclear. This study explores the succession patterns of activated sludge bacterial communities and their potential P utilization strategies under P-deficient conditions. The results indicate that changes in phosphorus concentration and composition drive microbial community succession. Under P deficiency, microorganisms initially focus on inorganic phosphorus (IP) metabolism, prioritizing competition for IP. Subsequently, the bacterial community shifts to predominantly utilizing organic phosphorus (OP), with over 94% of genomes carrying OP metabolism genes. The OP in the system primarily consists of phosphonates and phosphoester. Consequently, distinct bacterial responses to phosphoester and phosphonates were observed, with the bacterial community transitioning from low modular to highly modular communities with differentiated niches. Phosphoester and phosphonate metabolism emerged as the two main OP metabolic pathways within the community. By reconstructing the P metabolic pathways of dominant genera, including Pannonibacter (Proteobacteria) and f_Saprospiraceae (Bacteroidota), metabolic models of phosphonate and phosphoester were developed in a wastewater treatment system for the first time. This study provides new insights into biological P metabolism in wastewater treatment, expanding our understanding of P metabolic pathways in wastewater.

Comparative Investigation of the Anammox Process Using Free-Floating Carriers of Activated Sludge-Attached Biocenosis

For ammonium removal from wastewater, anammox technologies are among the most efficient and rapidly developing ones. Due to the low growth rate of anammox bacteria and their sensitivity to various inhibitors, technologies using attached biocenosis carriers (ABCs) provide for reliable operation. The goal of the present work was to investigate a new ABC type, ETEK biochips based on a nonwoven fibrous material. The work involved the techniques of materials science (design of a new ABC type) and physical modeling of the anammox process (in a laboratory bioreactor), as well as electron microscopy and molecular profiling of activated sludge communities. Comparison of the ETEK biochips with the ABCs of foamed polyethylene BF33 and Mutag revealed more rapid accumulation (5-fold) of the activated sludge biomass on ETEK biochips upon reactor launching, as well as comparable buoyancy and reactor productivity regarding N removal. The specific rate of nitrogen removal obtained with ETEK biochips considerably exceeded that for foamed polyethylene with a filler: 1.5–3 times higher per chip and 1.5 times higher per activated sludge biomass unit. The studied ABC shared the same issue of floating to the surface due to the active formation of gas (N2). The algorithm for calculating the downward flows in bioreactors with rapidly surfacing ABC is proposed, and a new hydrodynamic type of a bioreactor (with hybrid hydrodynamics) is described, a moving bed–sequencing batch reactor (MB-SBR).

The effects of small plastic particles on antibiotic resistance gene transfer revealed by single cell and community level analysis

Small plastic particles with sizes comparable to bacterial cells, widely exist in environment. However, their effects on antibiotic resistance gene (ARG) dissemination remain unclear. Using polystyrene (PS) particles (0.2 µm, 2 µm, 5 µm, 10 µm, 15 µm, 20 µm) as models, conjugative transfer of ARGs between the donor E. coli and different recipients (E. coli or sludge bacterial community) was investigated. Compared to the pure strain, the sludge bacterial community exposed to PS particles showed higher transfer frequencies (1.67 to 14.31 times the blank control). The transfer frequencies first decreased and then increased with particle size, and plastics similar in size to bacteria (e.g., 2 µm) appear to be a transitional zone with minimal impact on ARG transmission. Furthermore, using microfluidics, in-situ observation at single cell level found that 2 µm plastics can act as barriers between donor and recipient bacteria inhibiting growth, but conjugation events mostly occurred around them. Conversely, nanoplastics (e.g., 0.2 µm) and larger microplastics (e.g., 20 µm) significantly promote conjugation, mainly due to increased reactive oxygen species production and cell membrane permeability, or facilitating bacterial adhesion and biofilm formation, respectively. This study aids in assessing environmental risks of small plastic particles on ARG dissemination.

Contributions of the bacterial communities to the microcystin degradation and nutrient transformations during aerobic composting of algal sludge

Aerobic composting is a useful method for managing and disposing of salvaged algal sludge. To optimize the composting process and improve compost quality, it is necessary to understand the functions and responses of microbial communities therein. This work studied the degradation process of organic matter and the assemblage of bacterial communities in algal sludge composting via 16S rRNA amplicon sequencing. The results showed that 77.08% of the microcystin was degraded during the thermophilic stage of composting, which was the main period for microcystin degradation. Bacterial community composition and diversity changed significantly during the composting, and gradually stabilized as the compost matured. Different composting stages may be dominated by different module groups separately, as shown in the co-occurrence networks of composting bacterial communities. In the networks, all bacteria associated with microcystin degradation were identified as connectors between different module groups. The algal sludge composting process was driven primarily by deterministic processes, and the main driving forces for bacterial community assembly were temperature, dissolved organic carbon, ammonium, and microcystin. At last, by applying the structural equation modeling method, the bacterial communities under influences of physiochemical properties were proved as the main mediators for the microcystin degradation. This study provides valuable insights into the optimization of bacterial communities in composting to improve the efficiency of microcystin degradation and the quality of the compost product.

Combination of a membrane bioreactor with a rotating biological contactor holding several diverse metazoans can reduce excess sludge with fouling mitigation

In a membrane bioreactor (MBR) system, in situ sludge reduction techniques induce membrane fouling. To address this challenge, we incorporated a rotating mesh carrier, which can adsorb organic matter and provide a habitat for metazoans, into the anoxic tank of a conventional anoxic/oxic-MBR (A/O-MBR) system, termed rotating biological contactor-MBR (RBC-MBR), and evaluated treatment performance. Over 151 days, lab-scale RBC-MBR and A/O-MBR were used to treat municipal sewage. Both reactors showed similar COD and NH4+ removal rates. However, RBC-MBR reduced excess sludge by approximately 45 % compared with A/O-MBR. Microscopic observation and 18S rRNA gene-based microbial analysis revealed the persistence of microfauna and metazoans (oligochaetes, nematodes, and rotifers) in RBC, which are typically absent in activated sludge. Additionally, the metazoan's population in the RBC-MBR membrane tank was two-fold that of A/O-MBR, indicating enhanced sludge reduction through predation. Despite these reductions, the increase in transmembrane pressure was similar between RBC-MBR and A/O-MBR, suggesting that sludge holding by RBC mesh media degrade fouling substances, such as proteins and polysaccharides and improves sludge filterability, resulting in membrane fouling mitigation. Microbial communities in both reactors were similar, indicating that the installation of RBC did not alter the microbial community of sludge. Network analysis suggested potential symbiotic or prey–predator relationships between bacteria and metazoans. This study reveals that RBC-MBR effectively reduced the excess sludge while mitigating membrane fouling, highlighting one of the promising technology for applying metazoan predation into MBR.

Activated sludge microbial community assembly: the role of influent microbial community immigration.

Wastewater treatment plants (WWTPs) are host to diverse microbial communities and receive a constant influx of microbes from influent wastewater. However, the impact of immigrants on the structure and activities of the activated sludge (AS) microbial community remains unclear. To gain insight on this phenomenon known as perpetual community coalescence, the current study utilized controlled manipulative experiments that decoupled the influent wastewater composition from the microbial populations to reveal the fundamental mechanisms involved in immigration between sewers and AS-WWTP. The immigration dynamics of heterotrophs were analyzed by harvesting wastewater biomass solids from three different sewer systems and adding to synthetic wastewater. Immigrating influent populations were observed to contribute up to 14% of the sequencing reads in the AS. By modeling the net growth rate of taxa, it was revealed that immigrants primarily exhibited low or negative net growth rates. By developing a protocol to reproducibly grow AS-WWTP communities in the lab, we have laid down the foundational principles for the testing of operational factors creating community variations with low noise and appropriate replication. Understanding the processes that drive microbial community diversity and assembly is a key question in microbial ecology. In the future, this knowledge can be used to manipulate the structure of microbial communities and improve system performance in WWTPs.IMPORTANCEIn biological wastewater treatment processes, the microbial community composition is essential in the performance and stability of the system. This study developed a reproducible protocol to investigate the impact of influent immigration (or perpetual coalescence of the sewer and activated sludge communities) with appropriate reproducibility and controls, allowing intrinsic definitions of core and immigrant populations to be established. The method developed herein will allow sequential manipulative experiments to be performed to test specific hypothesis and optimize wastewater treatment processes to meet new treatment goals.

Operation parameters and temperature affected sludge microbial metabolisms: An integrated perspective considering extracellular polymeric substances, soluble microbial products, biomass quantities, and community shifts

Effluent soluble microbial products (SMP) and extracellular polymeric substances (EPS) are significant organics that pose challenges to advanced treatment processes. However, their production, transformation, and decomposition remain unclear due to their heterogeneity and the combined effects of environmental and operational factors. In this work, we investigated the impact of solids retention time (SRT), hydraulic retention time (HRT), and temperature on the changes in effluent SMP, with the consideration of the co-variation of EPS, sludge biomass, and community structures. Results show that longer SRT increased the biomass and relative abundance of functional microorganisms such as Myxococcota, Actinobacteria, and Terrimonas, which hindered EPS-to-SMP turnover and/or facilitated SMP consumption. This resulted in the accumulation of EPS and lower SMP concentrations at the beginning of the SRT adjustment. Both longer and shorter HRT (12 h and 8 h) led to increased SMP concentration, with the shorter HRT nearly doubling it (from approximately 6 to 12 mg/L), especially in terms of its protein and polysaccharide contents. Lower temperatures increased the SMP concentration and the relative abundance of Proteobacteria (including Zoogloea, the most dominant phylum and genus, relative abundance from 15.7 % to 61.1 %) while decreasing fluorescent EPS components, indicating the key role of Proteobacteria in SMP production and fluorescent EPS-to-SMP transformation. The results provided key insights into how changes in operational/environmental parameters impact sludge-EPS-SMP interactions, which could benefit the model development and operational optimization of activated sludge systems. This study also highlighted the important role of the sludge community in the EPS/SMP dynamics.

Acute inhibitory effects of tire wear particles on the removal of biological phosphorus：The critical role of aging in improving environmentally persistent free radicals

A comparative study explored how photoaging, ozonation aging, and Fenton aging affect tire wear particles (TWPs) and their phosphorus (P) removal in activated sludge. Aging altered TWPs' properties, increasing surface roughness, porosity, and generating more small particles, especially environmental persistent free radicals (EPFRs) in ozonation and Fenton aging. Post-aging TWPs (50 mg/L) inhibited sludge P removal significantly (p < 0.05), with rates of 44.3% and 59.6% for ozonation and Fenton aging, respectively. In addition, the metabolites involved in P cycling (poly-β-hydroxyalkanoates: PHA and glycogen) and essential enzymes (Exopolyphosphatase: PPX and Polyphosphate kinase: PPK) were significantly inhibited (p < 0.05). Moreover, TWPs led to a decrease in microbial cells within the sludge and altered the community structure, a situation exacerbated by the aging of TWPs. P-removing bacteria decreased (e.g., Burkholderia, Candidatus), while extracellular polymeric substance-secreting bacteria increased (e.g., Pseudomonas, Novosphingobium). Pearson correlation analysis highlighted EPFRs' role in TWPs' acute toxicity to microbial cells, yet, emphasizing particle size's impact on the sludge system's purification and community structure.

Temperature-Driven Activated Sludge Bacterial Community Assembly and Carbon Transformation Potential: A Case Study of Industrial Plants in the Yangtze River Delta.

Temperature plays a critical role in the efficiency and stability of industrial wastewater treatment plants (WWTPs). This study focuses on the effects of temperature on activated sludge (AS) communities within the A2O process of 19 industrial WWTPs in the Yangtze River Delta, a key industrial region in China. The investigation aims to understand how temperature influences AS community composition, functional assembly, and carbon transformation processes, including CO2 emission potential. Our findings reveal that increased operating temperatures lead to a decrease in alpha diversity, simplifying community structure and increasing modularity. Dominant species become more prevalent, with significant decreases in the relative abundance of Chloroflexi and Actinobacteria, and increases in Bacteroidetes and Firmicutes. Moreover, higher temperatures enhance the overall carbon conversion potential of AS, particularly boosting CO2 absorption in anaerobic conditions as the potential for CO2 emission during glycolysis and TCA cycles grows and diminishes, respectively. The study highlights that temperature is a major factor affecting microbial community characteristics and CO2 fluxes, with more pronounced effects observed in anaerobic sludge. This study provides valuable insights for maintaining stable A2O system operations, understanding carbon footprints, and improving COD removal efficiency in industrial WWTPs.

Influence of the oxidative phosphorylation uncoupler m-chlorophenol on activated sludge production, nutrients removal efficiency and bacterial community structure of a membrane reactor system

In this work, the effects of the metabolic uncoupler meta-chlorophenol (m-CP) on excess sludge production and performance of an MBR system, as well as on activated sludge communities, were investigated. MLVSS were significantly reduced from 8410 mg/L to 5175 mg/L during the first week of the addition of 125 mg/L m-CP, while, thereafter, biosolids were restored to the level prior to uncoupler’s addition. Adding m-CP resulted in more severe effects on nitrifiers rather than organoheterotrophs. Both alkalinity, dissolved oxygen (DO), electrical conductivity (EC) and pH data evidenced the adverse effect of m-CP on the nitrification process. PO43--P concentration drastically raised immediately after m-CP addition from 2.48 ± 0.35 mg/L to 11.84 mg/L, possibly due to the uncoupling of P-release and the hydrolysis of phosphorus reserves. Diversity was highly affected by the addition of m-CP, resulting in the significant decrease of Chao1, Shannon and Fisher indices. By establishing stable operating conditions after m-CP addition, Pseudomonas, Arcanobacterium, Trichococcus, Kocuria and Actinobaculum raised from almost undetectable levels (0.12 % of the total relative abundance) to 35.9 %, 17.8 %, 15.1 %, 11.5 % and 5.7 %, respectively, indicating a massive shift in bacterial community structure, while no nitrifying taxa were detected after m-CP addition.

Short-term electrochemical stimulation induces sustained enhancement of extracellular pollutant degradation in anaerobic granular sludge

Electroactive bacteria (EAB) are advantageous for degrading the pollutants extracellularly, but the enrichment primarily restricts on the electrode surface of bioelectrochemical system and is challenging to scale up. To address this, a new strategy was proposed to electrochemically stimulate the anaerobic granular sludge (AGS) and employ the harvested electroactive granular sludge (EGS) to an electrode-free bioreactor for pollutant removal. Fast establishment of EGS was achieved after 7 days' electrochemical stimulation, with a maximum current output of 112.5 A/m3 achieved and the abundance of electroactive bacteria-like genera increased from 5% to around 20%. The electric conductivity of EGS improved by 1-fold, while the increased extracellular protein and humic acid substance but unchanged polysaccharide content in the extracellular polymeric substance suggested a more robust redox network. After assembling in a sequential batch reactor, the methylene orange (MO) and Cr(VI) removal rate by EGS was enhanced by 65–80% and 38–55%, respectively. Better oxygen tolerance was also observed. The comparative study with exogenous riboflavin and respiration inhibitors indicated the complete extracellular reduction achieved by EGS while almost half of MO was metabolically degraded by AGS, which was kinetically sluggish. More importantly, there is no performance and electroactivity decay in a 60 days' batch operation. Meanwhile, the characterization of microbial community revealed the further increased abundance of EAB-like genera (∼28%) but Acinetobacter became dominant (∼15%), suggesting the competition of EAB species in EGS. In brief, a simple 7-day electrochemical stimulation imposed diverse and sustained benefits on the performance and microbial community of granular sludge, which may inspire the application of EAB and their extracellular degradation capacity in real wastewater treatment.

Methylotrophic methanogenesis induced by ammonia nitrogen in an anaerobic digestion system

This lab-scale study aimed to investigate the effect of total ammonia nitrogen (TAN) stress on the methanogenic activity and the taxonomic and functional profiles of the microbial community of anaerobic sludge (AS) from a full-scale bioreactor. The AS was subjected to a stepwise increase in TAN every 14 days at concentrations of 1, 2, 2.5, 3, 3.5, and 4g TAN/L (Acclimated-AS or AAS). This acclimation stage was followed by an ammonia stress stage (4g/L). A blank-AS (BAS) was maintained without TAN during the acclimation stage. In the second stress stage (ST), the BAS was divided into two new treatments: a control (BAS') and one that received a shock load of TAN of 4g/L (SBAS'). Methane production was measured, and a metagenomic analysis was conducted to describe the microbial community. A decrease in the relative abundance of Methanothrix soehngenii of 16% was related to a decrease of 23% in the methanogenic capacity of AAS when comparing with the final stage of BAS. However, recovery was observed at 3.5g TAN/L, and a shift to methylotrophic metabolism occurred, indicated by a 4-fold increase in abundance of Methanosarcina mazei. The functional analysis of sludge metagenomes indicated that no statistical differences (p>0.05, RM ANOVA) were found in the relative abundance of methanogenic genes that initiate acetoclastic and hydrogenotrophic pathways (acetyl-CoA synthetase, ACSS; acetate kinase, ackA; phosphate acetyltransferase, pta; and formylmethanofuran dehydrogenase subunit A, fwdA) into the BAS and AAS during the acclimation phase. The same was observed between groups of genes associated with methanogenesis from methylated compounds. In contrast, statistical differences (p<0.05, one-way ANOVA) in the relative abundance of these genes were recorded during ST. The functional profiles of the genes involved in acetoclastic, hydrogenotrophic, and methylotrophic methanogenic pathways were brought to light for acclimatation and stress experimental stages. TAN inhibited methanogenic activity and acetoclastic metabolism. The gradual acclimatization to TAN leads to metabolic and taxonomic changes that allow for the subsequent recovery of methanogenic functionality. The study highlights the importance of adequate management of anaerobic bioprocesses with high nitrogen loads to maintain the methanogenic functionality of the microbial community.

Effect of Thermal Hydrolysis Pretreatment Time on Microbial Community Structure in Sludge Anaerobic Digestion System

To evaluate the effect of thermal hydrolysis pretreatment time on the sludge anaerobic digestion system of wastewater treatment plants (WWTPs) in Daxing district, Beijing, the structure and diversity of microbial communities in primary sludge and an activated sludge anaerobic digestion system with different thermal hydrolysis pretreatment times (15 min, 30 min, and 45 min) were analyzed using Illumina MiSeq high-throughput sequencing. The results showed that the dominant groups of digested sludge were mainly distributed in Firmicutes, Cloacimonadota, Chloroflexi, and Synergistota, with W5 being the most common genus. The sum of relative abundance of the dominant phylum was greater than 60%, and W5 accounted for 20.8%-54.5%, showing a high abundance of a few dominant species. During the anaerobic digestion of thermo-hydrolyzed sludge, the relative abundance of acetogenic methanogens decreased due to high levels of volatile fatty acids (VFAs) and ammonia nitrogen (NH4+-N) concentrations, which suggested that the hydrogenophilic methanogenic pathway was more than that of the acetogenic methanogenic pathway. Correlation analysis showed that the soluble protein and pH of thermo-hydrolyzed sludge, NH4+-N of digested sludge, and thermal hydrolysis pretreatment time were the four main environmental factors affecting microbial community structure, and NH4+-N of digested sludge had the largest negative correlation with methanogens. The thermal hydrolysis pretreatment time was negatively correlated with both the Chao index and Shannon index, so longer thermal hydrolysis pretreatment time was not conducive to microbial flora during anaerobic digestion.

Wastewater treatment bacteria show differential preference for colonizing natural biopolymers

Most reduced organic matter entering activated sludge systems is particulate (1–100-µm diameter) or colloidal (0.001–1-µm diameter), yet little is known about colonization of particulate organic matter by activated sludge bacteria. In this study, colonization of biopolymers (chitin, keratin, lignocellulose, lignin, and cellulose) by activated sludge bacteria was compared with colonization of glass beads in the presence and absence of regular nutrient amendment (acetate and ammonia). Scanning electron microscopy and quantitative PCR revealed chitin and cellulose were most readily colonized followed by lignin and lignocellulose, while keratin and glass beads were relatively resistant to colonization. Bacterial community profiles on particles compared to sludge confirmed that specific bacterial phylotypes preferentially colonize different biopolymers. Nitrifying bacteria proved adept at colonizing particles, achieving higher relative abundance on particles compared to bulk sludge. Denitrifying bacteria showed similar or lower relative abundance on particles compared to sludge.Key points• Some activated sludge bacteria colonize natural biopolymers more readily than others.• Nitrifying bacteria are overrepresented in natural biopolymer biofilm communities.• Biopolymers in wastewater likely influence activated sludge community composition.

Effects of sludge retention time on sludge reduction by ultrasound treatment: Sludge characteristics, microbial community, and metabolism

Both ultrasound and sludge retention time (SRT) enable the in-situ sludge reduction during wastewater treatment, but the influence of SRT on ultrasonic lysis – cryptic growth is unclear. This paper researched the influence of different SRTs on sludge lysis - cryptic growth using a sequential bio-reactor (SBR), then explained in details the changes of microorganisms in the SBR. The best SRT for sludge reduction was 30 d, and 47.29% reduction in sludge was achieved. The different SRTs changed the organic matter removal in the wastewater, and the removal rate decreased when SRT exceeded 60 d. The size of the sludge particles varied depending on the SRT, with the smallest size at SRT of 10d being 45.6 μm and the largest size at SRT of 90d being 110.0 μm. SEM showed that the sludge surface changed rough at longer SRT. FTIR and XPS showed notable effect in sludge functional group strength at SRT of 30 d. Extracellular polymeric substance (EPS) reduced the most at SRT of 30 d. The microbial communities of sludge varied with the SRT, and the unique main genus at SRT of 5, 15, 30 and 90 d were C10-SB1A, Lactococcus, Propioniciclava, Lactococcus, respectively. Furthermore, the SRT changed relative abundance of enzymes concerned with metabolism of carbon, nitrogen, and phosphorus. Similarly, SRT changed the metabolic rate, and the metabolic rate of carbon, nitrogen and phosphorus was best at SRT of 30 d.

Enhanced AHL-mediated quorum sensing accelerates the start-up of biofilm reactors by elevating the fitness of fast-growing bacteria in sludge and biofilm communities

Quorum sensing (QS)-based manipulations emerge as a promising solution for biofilm reactors to overcome challenges from inefficient biofilm formation and lengthy start-ups. However, the ecological mechanisms underlying how QS regulates microbial behaviors and community assembly remain elusive. Herein, by introducing different levels of N-acyl-homoserine lactones, we manipulated the strength of QS during the start-up of moving bed biofilm reactors and compared the dynamics of bacterial communities. We found that enhanced QS elevated the fitness of fast-growing bacteria with high ribosomal RNA operon (rrn) copy numbers in their genomes in both the sludge and biofilm communities. This led to notably increased extracellular substance production, as evidenced by strong positive correlations between community-level rrn copy numbers and extracellular proteins and polysaccharides (Pearson's r = 0.529−0.830, P < 0.001). Network analyses demonstrated that enhanced QS significantly promoted the ecological interactions among taxa, particularly cooperative interactions. Bacterial taxa with higher network degrees were more strongly correlated with extracellular substances, suggesting their crucial roles as public goods in regulating bacterial interactions and shaping network structures. However, the assembly of more cooperative communities in QS-enhanced reactors came at the cost of decreased network stability and modularity. Null model and dissimilarity-overlap curve analysis revealed that enhanced QS strengthened stochastic processes in community assembly and rendered the universal population dynamics more convergent. Additionally, these shaping effects were consistent for both the sludge and biofilm communities, underpinning the planktonic-to-biofilm transition. This work highlights that QS manipulations efficiently drive community assembly and confer specialized functional traits to communities by recruiting taxa with specific life strategies and regulating interspecific interactions. These ecological insights deepen our understanding of the rules governing microbial societies and provide guidance for managing engineering ecosystems.

Quorum sensing bacteria improve microbial networks stability and complexity in wastewater treatment plants

Quorum-sensing bacteria (QSB) are crucial factors for microbial communication, yet their ecological role in wastewater treatment plants (WWTPs) remains unclear. Here, we developed a method to identify QSB by comparing 16S rRNA gene sequences. QSB in 388 activated sludge samples collected from 130 WWTPs across China primarily were identified as rare taxa and conditionally rare taxa. A co-occurrence network shared by all sludge communities revealed that QSB exhibited higher average clustering coefficient (0.46) than non-QSB (0.15). Individual sludge networks demonstrated that quorum sensing microbiomes were positively correlated with network robustness and network complexity, including average clustering coefficient and link density. We confirmed that QSB keystones and QSB nodes have a positive impact on network complexity by influencing network modularity through a structural equation model. Meanwhile, QSB communities directly contributed to maintaining network robustness (r = 0.29, P < 0.05). Hence, QSB play an important role in promoting network complexity and stability. Furthermore, QSB communities were positively associated with the functional composition of activated sludge communities (r = 0.33, P < 0.01), especially the denitrification capacity (r = 0.45, P < 0.001). Overall, we elucidated the ecological significance of QSB and provided support for QS-based regulation of activated sludge microbial communities.