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.

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.

AHLs-mediated quorum sensing threshold and its response towards initial adhesion of wastewater biofilms.

Quorum sensing (QS) plays a crucial role during initial biofilm formation, however the QS threshold and the response of biofilm formation towards N-acyl-homoserine lactones (AHLs) remains largely unknown due to the limitation of nondestructive online methods for monitoring bacterial adherence and the complexity of QS system, which limits the application of QS signal reagents in biofilm reactors. In this study, bacterial QS threshold and its response of biofilm formation to AHLs in purely cultured Sphingomonas rubra biofilm as well as in three different wastewater biofilms #1-3 were investigated via real time cell analysis (RTCA). The main perspective was to study the biomass adherence in response to 12 different forms of AHLs at different concentrations. Results showed that bacterial adhesion was significantly improved by exogenous AHLs with the maximum increase of 2.26-, 2.36-, 2.52-, and 2.80- times biomass production in the four respective biofilms. Although the preferred form of AHL differed for various biofilms, the long-chain AHLs (12-14 carbons) resulted in an overall improvement of bacterial adhesion due to their stronger hydrophobicity and hydrolysis resistance. In addition, bacterial QS threshold of AHLs was observed to have a wide range of concentration from 10 ng/L to 10 µg/L. Meanwhile, QS response time to AHLs also showed a significant difference in different biofilms. Biofilm #2 inoculated with bulking sludge had lower QS threshold of 10 ng/L and faster response to most AHLs that is less than 6 h. Thus, considering the improvement of biofilm adhesion by AHLs, 10 ng/L of C12-HSL, 10 ng/L of C12-HSL, and 10 ng/L of C6-HSL were preferentially selected for wastewater biofilms #1-3 respectively. Unexpectedly, adding high-concentration of AHLs detected in sludges did not significantly improved the bacterial adhesion. Infact the addition of these AHLs at low concentrations or even undetected concentrations substantially improved bacterial adhesion, which could be explained by bacterial communities composition. According to the Pearson correlation analysis, 62% of the top 50 most abundant genera in bacterial communities were significantly negatively related to the response time of multiple AHLs, representing their fast QS response. The QS bacteria, Dechloromonas and Nitrospira have fast QS response for C4-HSL and C8-HSL while, Comamonadaceae has fast QS response for 3OC8-HSL, 3OC10-HSL, 3OC12-HSL, and 3OC14-HSL. In contrast, the rest 38% of the top most abundant genera, such as Ferruginibacter, Hyphomicrobium, and Terrimonas quickly responded to only one AHL, showing significant negative relationship with the response time of C6-HSL. Overall, this study provides an effective and convenient means to select appropriate AHL reagents to promote bacterial adhesion in biofilm systems. Moreover, it also suggests that exogenous AHLs may be useful in improving the settling property of bulking sludge.

In-situ monitoring of the unstable bacterial adhesion process during wastewater biofilm formation: A comprehensive study.

The initial bacterial adhesion phase is a pivotal and unstable step in the formation of biofilms. The initiation of biofilm formation is an unstable process caused by the reversible adhesion of bacteria, which is always time-consuming and yet to be elucidated. In this study, impedance-based real time cell analysis (RTCA) was employed to comprehensively investigate the initial bacterial adhesion process. Results showed that the time required for the unstable adhesion process was significantly (p < 0.05) reduced by increasing the initial concentration of bacteria, which is mainly attributed to the large deposition rate of bacteria at high concentrations. In addition, the unstable adhesion process is also regulated by shear stress, derived in this work from orbital shaking. Shear stress improves the reversibility of unstable bacterial attachment. Furthermore, attachment characteristics during the unstable phase vary between different species of bacteria (Sphingomonas rubra, Nakamurella multipartita and mixed bacteria). The S. rubra strain and mixed culture were more prone to adhere to the substratum surface during the unstable process, which was attributed to the smaller xDLVO energy barrier and motility of species in comparison with N. multipartita. Meanwhile, the molecular composition of extracellular polymeric substances (EPS) in the initial attachment phase presented a significant difference in expressed proteins, indicating the important role of proteins in EPS that strengthen bacterial adhesion. Overall, these findings suggest that during the biofilm reactor start-up process, seed sludge conditions, including the bacterial concentration, composition and hydraulics, need to be carefully considered.

Effect of salinity on mature wastewater treatment biofilm microbial community assembly and metabolite characteristics.

The response mechanism of wastewater treatment biofilms to salt stress has not yet been fully established. The aim of this study was to reveal the comprehensive biological effects of salinity on biofilm microbial community and metabonomic characteristics. The study assessed performance at a range of sodium chloride (NaCl) concentrations of 0.6, 14 and 20 g/L. Biofilm coverage rate decreased significantly with increasing NaCl concentrations. High NaCl concentrations resulted in more compact and smoother biofilm morphologies. NaCl concentrations affected bacterial community variation at the class and genus level, with Gammaproteobacteria being the most dominant Proteobacteria, exhibiting NaCl tolerance at concentrations ranging from 0 to 20 g/L. Also, NaCl sensitive or tolerant species were identified, such as Pseudomonas and Planococcus, respectively. Dominant metabolites in wastewater treatment biofilms belonging to nucleotide, lipid, vitamin, amino acid and carbohydrate metabolism pathways decreased with increasing NaCl concentrations. High concentrations of NaCl regulated cell motility, transcription and membrane transport functions. In particular, the activity of ABC transporters were up-regulated at NaCl concentrations of 0.6 g/L and down-regulated at higher salinity concentrations. In addition, transcription machinery were inhibited under the stress of 14 g/L NaCl. These findings further our understanding of the short-term adaption mechanisms of wastewater treatment biofilms to high NaCl concentration environments.