Multitasking functions of bacterial extracellular DNA in biofilms.

Bacterial biofilms are intricate ecosystems of microbial communities that adhere to various surfaces and are enveloped by an extracellular matrix composed of polymeric substances. Within the context of bacterial biofilms, extracellular DNA (eDNA) originates from cell lysis or is actively secreted, where it exerts a significant influence on the formation, stability, and resistance of biofilms to environmental stressors. The exploration of eDNA within bacterial biofilms holds paramount importance in research, with far-reaching implications for both human health and the environment. An enhanced understanding of the functions of eDNA in biofilm formation and antibiotic resistance could inspire the development of strategies to combat biofilm-related infections and improve the management of antibiotic resistance. This comprehensive review encapsulates the latest discoveries concerning eDNA, encompassing its origins, functions within bacterial biofilms, and significance in bacterial pathogenesis.

The roles of extracellular polymeric substances of Pandoraea sp. XY-2 in the removal of tetracycline.

In this study, the roles of extracellular polymeric substances (EPSs) excreted by Pandoraea sp. XY-2 in the removal of tetracycline (TC) were investigated. In the early stage, TC in the solution was mainly removed by the adsorption of EPSs, which accounted for 20% of TC. Thereafter, large amount of TC was transported into the intracellular and biodegraded. EPSs was extracted and the contents of polyprotein and polysaccharides reached their maximum values (30.84 mg/g and 11.15 mg/g) in the first four days. Fourier transform infrared spectroscopy analysis revealed that hydroxyl, methylidyne, methylene and amide I groups in EPSs participated in the adsorption of TC. Furthermore, three-dimensional excitation-emission matrix fluorescence spectroscopy analysis revealed that TC caused the quenching of EPSs fluorescent groups. The quenching mechanism was attributed to static quenching and protein-like substances in EPSs from Pandoraea sp. XY-2 dominated the TC adsorption process. Bioinformatic analysis of Pandoraea sp. XY-2 genome identified multiple genes involved in exopolysaccharide synthesis and EPSs formation. The insights gained in this study might provide a better understanding about the adsorption process of EPSs in tetracycline-contaminated environment.

Quorum sensing: an emerging link between temperature and membrane biofouling in membrane bioreactors.

Lab-scale membrane bioreactors (MBRs) were investigated at 12, 18, and 25 °C to identify the correlation between quorum sensing (QS) and biofouling at different temperatures. The lower the reactor temperature, the more severe the membrane biofouling measured in terms of the transmembrane pressure (TMP) during filtration. More extracellular polymeric substances (EPSs) that cause biofouling were produced at 18 °C than at 25 °C, particularly polysaccharides, closely associated with QS via the production of N-acyl homoserine lactone (AHL). However, at 12 °C, AHL production decreased, but the release of EPSs due to deflocculation increased the soluble EPS concentration. To confirm the temperature effect related to QS, bacteria producing AHL were isolated from MBR sludge and identified as Aeromonas sp., Leclercia sp., and Enterobacter sp. through a 16S rDNA sequencing analysis. Batch assays at 18 and 25 °C showed that there was a positive correlation between QS through AHL and biofilm formation in that temperature range.

Auto-aggregation properties of a novel aerobic denitrifier Enterobacter sp. strain FL.

Enterobacter sp. strain FL was newly isolated from activated sludge and exhibited significant capability of auto-aggregation as well as aerobic denitrification. The removal efficiencies of NO3--N, total nitrogen (TN), and TOC by strain FL in batch culture reached 94.6, 63.9, and 72.5% in 24 h, respectively. The production of N2O and N2 in the presence of oxygen demonstrated the occurrence of aerobic denitrification. The auto-aggregation index of strain FL reached 54.3%, suggesting a high tendency that the cells would agglomerate into aggregates. The production of extracellular polymeric substances (EPSs), which were mainly composed of proteins followed by polysaccharides, was considered to be related to the cell aggregation according to Fourier transform infrared (FT-IR) and confocal laser scanning microscopy (CLSM). The proteins in EPS were evenly and tightly combined to cells and altered the protein secondary structures of cell surface from random coils to ß-sheets and three-turn helices. The alteration of protein secondary structures of cell surface caused by the proteins in EPS might play a dominant role in the auto-aggregation of strain FL. To further assess the feasibility of strain FL for synthetic wastewater treatment, a sequencing batch reactor (SBR), solely inoculated with strain FL, was conducted. During the 16 running cycles, the removal efficiency of NO3--N was 90.2-99.7% and the auto-aggregation index was stabilized at 35.0-41.5%. The EPS promoted the biomass of strain FL to aggregate in the SBR.

Fate of aerobic granular sludge in the long-term: The role of EPSs on the clogging of granular sludge porosity.

This work aims to investigate the stability of aerobic granular sludge in the long term, focusing on the clogging of the granular sludge porosity exerted by the extracellular polymeric substances (EPSs). The effects of different cycle lengths (short and long-term cycle) on the granular sludge stability were investigated. Results obtained outlined that during the short duration cycle, the formation and breakage of the aerobic granules were continuously observed. During this period, the excess of EPS production contributed to the clogging of the granules porosity, causing their breakage in the long run. During the long-duration cycle, the extended famine period entailed a greater EPSs consumption by bacteria, thus limiting the clogging of the porosity, and allowed obtaining stable aerobic granules. Reported results demonstrated that an excess in EPSs content could be detrimental to the stability of aerobic granular sludge in the long-term.

Antimicrobial Proteins: Structure, Molecular Action, and Therapeutic Potential.

Second- and third-line treatments of patients with antibiotic-resistant infections can have serious side effects, such as organ failure with prolonged care and recovery. As clinical practices such as cancer therapies, chronic disease treatment, and organ transplantation rely on the ability of available antibiotics to fight infection, the increased resistance of microbial pathogens presents a multifaceted, serious public health concern worldwide. The pipeline of traditional antibiotics is exhausted and unable to overcome the continuously developing multi-drug resistance. To that end, the widely observed limitation of clinically utilized antibiotics has prompted researchers to find a clinically relevant alternate antimicrobial strategy. In recent decades, the discovery of antimicrobial peptides (AMPs) as an excellent candidate to overcome antibiotic resistance has received further attention, particularly from scientists, health professionals, and the pharmaceutical industry. Effective AMPs are characterized by a broad spectrum of antimicrobial activities, high pathogen specificity, and low toxicity. In addition to their antimicrobial activity, AMPs have been found to be involved in a variety of biological functions, including immune regulation, angiogenesis, wound healing, and antitumor activity. This review provides a current overview of the structure, molecular action, and therapeutic potential of AMPs.

The Role of Extracellular Polymeric Substances in the Toxicity Response of Anaerobic Granule Sludge to Different Metal Oxide Nanoparticles.

Wastewater treatment plants (WWTP) are regarded as the last barriers for the release of incompletely separated and recycled nanoparticles (NPs) into the environment. Despite the importance and ubiquity of microbial extracellular polymeric substances (EPSs) in the complex wastewater matrix, the interaction between NPs and EPSs of anaerobic microflora involved in wastewater treatment and the resultant impact on the biomass metabolomics are unclear. Thus, the impacts of different metal oxide (TiO2, ZnO, and CuO) NPs on functional bacteria in anaerobic granular sludge (AGS) and the possible toxicity mechanisms were investigated. In particular, the binding quality, enhanced resistance mechanism, and chemical fractional contribution of EPSs from AGS against the nanotoxicity of different NPs was assessed. The results showed that CuO NPs caused the most severe inhibition towards acetoclastic and hydrogenotrophic methanogens, followed by ZnO NPs, whereas TiO2 NPs caused no inhibition to methanogenesis. Excessive EPS production, especially the protein-like substances, was an effective strategy for reducing certain NPs' toxicity by immobilizing NPs away from AGS cells, whereas the metabolism restriction on inner microorganisms of AGS induced by CuO NPs can deteriorate the protective role of EPS, indicating that the roles of EPS may not be amenable to generalizations. Further investigations with lactate dehydrogenase (LDH) and reactive oxygen species (ROS) assays indicated that there are greatly essential differences between the toxicity mechanisms of metal NPs to AGS, which varied depending on the NPs' type and dosage. In addition, dynamic changes in the responses of EPS content to different NPs can result in a significant shift in methanogenic and acidogenic microbial communities. Thus, the production and composition of EPSs will be a key factor in determining the fate and potential effect of NPs in the complex biological matrix. In conclusion, this study broadens the understanding of the inhibition mechanisms of metal oxide NPs on the AGS process, and the influence of EPSs on the fate, behavior, and toxicity of NPs.

Salmonella Biofilms Tolerate Hydrogen Peroxide by a Combination of Extracellular Polymeric Substance Barrier Function and Catalase Enzymes.

The ability of Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) to cause chronic gallbladder infections is dependent on biofilm growth on cholesterol gallstones. Non-typhoidal Salmonella (e.g. S. Typhimurium) also utilize the biofilm state to persist in the host and the environment. How the pathogen maintains recalcitrance to the host response, and oxidative stress in particular, during chronic infection is poorly understood. Previous experiments demonstrated that S. Typhi and S. Typhimurium biofilms are tolerant to hydrogen peroxide (H2O2), but that mutations in the biofilm extracellular polymeric substances (EPSs) O antigen capsule, colanic acid, or Vi antigen reduce tolerance. Here, biofilm-mediated tolerance to oxidative stress was investigated using a combination of EPS and catalase mutants, as catalases are important detoxifiers of H2O2. Using co-cultured biofilms of wild-type (WT) bacteria with EPS mutants, it was demonstrated that colanic acid in S. Typhimurium and Vi antigen in S. Typhi have a community function and protect all biofilm-resident bacteria rather than to only protect the individual cells producing the EPSs. However, the H2O2 tolerance deficiency of a O antigen capsule mutant was unable to be compensated for by co-culture with WT bacteria. For curli fimbriae, both WT and mutant strains are tolerant to H2O2 though unexpectedly, co-cultured WT/mutant biofilms challenged with H2O2 resulted in sensitization of both strains, suggesting a more nuanced oxidative resistance alteration in these co-cultures. Three catalase mutant (katE, katG and a putative catalase) biofilms were also examined, demonstrating significant reductions in biofilm H2O2 tolerance for the katE and katG mutants. Biofilm co-culture experiments demonstrated that catalases exhibit a community function. We further hypothesized that biofilms are tolerant to H2O2 because the physical barrier formed by EPSs slows penetration of H2O2 into the biofilm to a rate that can be mitigated by intra-biofilm catalases. Compared to WT, EPS-deficient biofilms have a heighted response even to low-dose (2.5 mM) H2O2 challenge, confirming that resident bacteria of EPS-deficient biofilms are under greater stress and have limited protection from H2O2. Thus, these data provide an explanation for how Salmonella achieves tolerance to H2O2 by a combination of an EPS-mediated barrier and enzymatic detoxification.

Acid soluble extracellular matrix confers structural stability to marine Bacillus haynesii pellicle biofilms.

In natural and engineered settings, bacteria predominantly thrive in biofilms, which are complex microbial communities embedded in a self-produced extracellular polymeric substances (EPSs) matrix. Pellicles are complex macroscopic biofilms floating at air-water interface. Though pellicle formation has been studied in detail in Bacillus subtilis, a soil bacterium, it is not reported in aquatic bacteria, which may use pellicle-growth as survival-strategy. This study shows that Bacillus haynesii isolated from a marine environment forms robust pellicle biofilms at air-water interface. B. haynesii pellicles showed complex architecture, involving dense cell-aggregates with interconnecting thread-like structures in an extracellular matrix. In situ staining by Alcian blue, Concanavalin A and ThioflavinT (ThT), respectively, localized acidic polymers, glycoconjugates and amyloid-like fibers in the pellicle. The pellicle was rigid and not disrupted by common EPS extraction protocols. Hence, a set of reagents and conditions were evaluated for solubilizing the EPS and pellicle. Acetic acid was able to effectively solubilize the structural EPS and pellicle structure. Acid soluble structural EPS contained chemical signatures for both proteins and carbohydrates, as revealed by elemental analysis, Fourier Transform Infrared Spectroscopy and Raman Spectroscopy. Ex situ staining of acid soluble EPS by ThT showed recovery of amyloid-forming proteins from pellicle. Results show that structural stability of the pellicle is mainly conferred by amyloid-like fibers of the EPS matrix. The robust pellicle-growth reported here may represent a survival-strategy in the aquatic bacterium. The findings reported here can support future research on biofilm structure, EPS matrix and its formation, which are critical for understanding how microbes thrive in natural and engineered settings.

Biofilm Matrixome: Extracellular Components in Structured Microbial Communities.

Biofilms consist of microbial communities embedded in a 3D extracellular matrix. The matrix is composed of a complex array of extracellular polymeric substances (EPS) that contribute to the unique attributes of biofilm lifestyle and virulence. This ensemble of chemically and functionally diverse biomolecules is termed the 'matrixome'. The composition and mechanisms of EPS matrix formation, and its role in biofilm biology, function, and microenvironment are being revealed. This perspective article highlights recent advances about the multifaceted role of the 'matrixome' in the development, physical-chemical properties, and virulence of biofilms. We emphasize that targeting biofilm-specific conditions such as the matrixome could lead to precise and effective antibiofilm approaches. We also discuss the limited knowledge in the context of polymicrobial biofilms, and the need for more in-depth analyses of the EPS matrix in mixed communities that are associated with many human infectious diseases.

[Comparison of Extraction Methods of Extracellular Polymeric Substances from Activated Sludge].

Extracellular polymeric substances (EPSs) are biosynthetic polymers of microbial origin in the sludge activation process and crucially affect the properties of sludge in biological wastewater treatment reactors, such as the formation of sludge flocs, stabilization of sludge structure, and protection of microbes against noxious environmental conditions. However, the EPS extraction efficiency differs significantly according to the extraction method used. In this study, soluble EPSs and loosely bound EPSs can be extracted by centrifugation first and tightly bound EPSs in activated sludge require additional eight treatments for extraction, respectively. Three physical methods (centrifugation, sonication, and heating) and five chemical methods (cation exchange resin, NaOH, formaldehyde+NaOH, EDTA, and formaldehyde+EDTA) were tested, and the content and composition of TB-EPS were analyzed. Meanwhile, the functional groups and elements in TB-EPS were investigated. Results showed that the heating method did not introduce exogenous substances during the EPS extraction process and that the destruction of cells from this method was relatively slight. Heating was shown to be a gentle and efficient method in this study. The three-dimensional excitation and emission matrix (EEM) fluorescence spectroscopy showed that the cation exchange resin method had good extraction effect on humic-like and protein-like substances. As to fulvic-acid-like substances, NaOH was better than the other seven methods. Infrared spectroscopy showed that no notable difference appeared in the functional groups of the TB-EPS extracted by physical methods, whereas chemical methods induced big differences and showed particular bands that did not appear in the TB-EPS extracted by physical methods. Overall, the amounts of TB-EPS elements extracted using chemical methods were greater than those extracted using physical methods. In conclusion, a method must be selected and established for each case, taking into consideration the experimental purpose, and the most appropriate method should be chosen carefully.

A novel membrane bioreactor inoculated with symbiotic sludge bacteria and algae: Performance and microbial community analysis.

This study combined sludge MBR technology with algae to establish an effective wastewater treatment and low membrane fouling system (ASB-MBR). Compared with control-MBR (C-MBR), the amelioration of microbial activity and the improvement of sludge properties and system environment were achieved after introducing algae resulting in high nutrients removal in the combined system. Further statistical analysis revealed that the symbiosis of algae and sludge displayed more remarkable impacts on nutrients removal than either of them. Additionally, membrane permeability was improved in ASB-MBR with respect to the decreased concentration, the changed of characteristics and the broken particular functional groups of extracellular polymeric substances (EPSs). Moreover, the algae inoculation reduced sludge diversity and shifted sludge community structure. Meantime, the stimulated bacteria selectively excite algal members that would benefit for the formation of algal-bacterial consortia. Consequently, the stimulated or inhibited of some species might be responsible for the performance of ASB-MBR.

High Correlation Between Structure Development and Chemical Variation During Biofilm Formation by Vibrio parahaemolyticus.

The complex three-dimensional structure of biofilms is supported by extracellular polymeric substances (EPSs) and additional insight on chemical variations in EPS and biofilm structure development will inform strategies for control of biofilms. Vibrio parahaemolyticus VPS36 biofilm development was studied using confocal laser scanning microscopy (CLSM) and Raman spectroscopy (RM). The structural parameters of the biofilm (biovolume, mean thickness, and porosity) were characterized by CLSM and the results showed that VPS36 biofilm formed dense structures after 48 h incubation. There were concurrent variations in carbohydrates and nucleic acids contents in the EPS as evidenced by RM. The Raman intensities of the chemical component in EPS, measured using Pearson's correlation coefficient, were positively correlated with biovolume and mean thickness, and negatively correlated with porosity. The Raman intensity for carbohydrates correlated closely with mean thickness (p-value < 0.01) and the Raman intensity for nucleic acid correlated closely with porosity (p-value < 0.01). Additional evidence for these correlations were confirmed using scanning electron microscopic (SEM) and crystal violet staining.

Different bacterial species and their extracellular polymeric substances (EPSs) significantly affected reverse osmosis (RO) membrane fouling potentials in wastewater reclamation.

Biofouling represents the "Achilles' heel" for reverse osmosis (RO) processes due to the growth of bacteria and their production of extracellular polymeric substances (EPSs). Although the microbial community structure on the RO membrane has been analysed previously, the bacterial species with a high potential of causing RO membrane fouling have not yet been identified clearly. The key components in EPSs causing RO membrane fouling have not been revealed either. In this study, seven different bacterial species were isolated from fouled RO membranes, and their EPSs were analysed in terms of the content of polysaccharides and proteins, fluorescence characteristics and molecular weight (MW) distributions. The membrane fouling potentials of these bacterial species and EPSs were evaluated based on normalized flux decline. Generally, under the same growth conditions, bacterial species with higher EPS concentrations, rather than higher cell numbers, resulted in more severe flux decline. The flux decline showed an apparent positive correlation with the EPS concentration, indicating that the concentration of EPS rather than the bacterial number mainly contributed to biofouling. Furthermore, it was found that the MW distribution was the key factor affecting the RO membrane fouling potential of EPSs from different bacterial species. With the increase in the percentage of the high-MW fraction (>10 kDa) in the EPSs from 12.6% to 74.4%, the normalized flux decline increased from 0.4 to 0.59. The components in EPSs with a MW over 10 kDa were also separated by the ultrafiltration membrane and were proven to have a higher membrane fouling potential.

Assessment of microbial products in the biosorption process of Cu(II) onto aerobic granular sludge: Extracellular polymeric substances contribution and soluble microbial products release.

In the present study, the responses of microbial products in the biosorption process of Cu(II) onto aerobic granular sludge were evaluated by using batch and spectroscopic approaches. Batch experimental data showed that extracellular polymeric substances (EPSs) contributed to Cu(II) removal from an aqueous solution, especially when treating low metal concentrations, whereas soluble microbial products (SMPs) were released under the metal stress during biosorption process. A three-dimensional excitation-emission matrix (3D-EEM) identified four main fluorescence peaks in the EPS, i.e., tryptophan protein-like, aromatic protein-like, humic-like and fulvic acid-like substances, and their fluorescence intensities decreased gradually in the presence of Cu(II) during the sorption process. Particularly, tryptophan protein-like substances quenched the Cu(II) binding to a much higher extent through a static quenching process with less than one class of binding sites. According to the synchronous fluorescence spectra, the whole fluorescence intensity of released SMP samples expressed an increased trend with different degrees along with contact time. Two-dimensional correlation spectroscopy (2D-COS) suggested that the fulvic-like fluorescence fraction might be more susceptible to metal exposure than other fractions. The result of molecular weight distribution demonstrated that the SMPs released from the biosorption process differed significantly according to contact time. The result obtained could provide new insights into the responses of microbial products from aerobic granular sludge with heavy metal treatment.