Identification of extracellular polymeric substances layer barrier in chloroquine phosphate-disturbed anammox consortia and mechanism dissection on cytotoxic behavior by computational chemistry.

The over-dosing use of chloroquine phosphate (CQ) poses severe threats to human beings and ecosystem due to the high persistence and biotoxicity. The discharge of CQ into wastewater would affect the biomass activity and process stability during the biological processes, e.g., anammox. However, the response mechanism of anammox consortia to CQ remain unknown. In this study, the accurate role of extracellular polymeric substances barrier in attenuating the negative effects of CQ, and the mechanism on cytotoxic behavior were dissected by molecular spectroscopy and computational chemistry. Low concentrations (≤6.0 mg/L) of CQ hardly affected the nitrogen removal performance due to the adaptive evolution of EPS barrier and anammox bacteria. Compact protein of EPS barrier can bind more CQ (0.24 mg) by hydrogen bond and van der Waals force, among which O-H and amide II region respond CQ binding preferentially. Importantly, EPS contributes to the microbiota reshape with selectively enriching Candidatus_Kuenenia for self-protection. Furthermore, the macroscopical cytotoxic behavior was dissected at a molecular level by CQ fate/distribution and computational chemistry, suggesting that the toxicity was ascribed to attack of CQ on functional proteins of anammox bacteria with atom N17 (f-=0.1209) and C2 (f+=0.1034) as the most active electrophilic and nucleophilic sites. This work would shed the light on the fate and risk of non-antibiotics in anammox process.

Dissolved organic matter, calcium ion and extracellular polymeric substances on living associated bacteria of Microcystis colony are crucial for unicellular Microcystis to efficiently form colonies.

Microcystis typically forms colonies under natural conditions, which contributes to occurrence and prevalence of algal blooms. The colonies consist of Microcystis and associated bacteria (AB), embedded in extracellular polymeric substances (EPS). Previous studies indicate that AB can induce Microcystis to form colonies, however the efficiency is generally low and results in a uniform morphotype. In this study, by using filtrated natural water, several AB strains induced unicellular M. aeruginosa to form colonies resembling several Microcystis morphotypes. The mechanisms were investigated with Methylobacterium sp. Z5. Ca2+ was necessary for Z5 to induce Microcystis to form colonies, while dissolved organic matters (DOM) facilitated AB to agglomerate Microcystis to form large colonies. EPS of living Z5, mainly the aromatic protein components, played a key role in colony induction. Z5 initially aggregated Microcystis via the bridging effects of Ca2+ and DOM, followed by the induction of EPS synthesis and secretion in Microcystis. In this process, the colony forming mode shifted from cell adhesion to a combination of cell adhesion and cell division. Intriguingly, Z5 drove the genomic rearrangement of Microcystis by upregulating some transposase genes. This study unveiled a novel mechanism about Microcystis colony formation and identified a new driver of Microcystis genomic evolution.

Sustained oxidation of Tea-Fe(III)/H2O2 simultaneously achieves sludge reduction and carbamazepine removal: The crucial role of EPS regulation.

Establishing an economic and sustained Fenton oxidation system to enhance sludge dewaterability and carbamazepine (CBZ) removal rate is a crucial path to simultaneously achieve sludge reduction and harmless. Leveraging the principles akin to "tea making", we harnessed tea waste to continually release tea polyphenols (TP), thus effectively maintaining high level of oxidation efficiency through the sustained Fenton reaction. The results illustrated that the incorporation of tea waste yielded more favorable outcomes in terms of water content reduction and CBZ removal compared to direct TP addition within the Fe(III)/hydrogen peroxide (H2O2) system. Concomitantly, this process mainly generated hydroxyl radical (•OH) via three oxidation pathways, effectively altering the properties of extracellular polymeric substances (EPS) and promoting the degradation of CBZ from the sludge mixture. The interval addition of Fe(III) and H2O2 heightened extracellular oxidation efficacy, promoting the desorption and removal of CBZ. The degradation of EPS prompted the transformation of bound water to free water, while the formation of larger channels drove the discharge of water. This work achieved the concept of treating waste with waste through using tea waste to treat sludge, meanwhile, can provide ideas for subsequent sludge harmless disposal.

The extracellular polymeric substances (EPS) accumulation of Spirulina platensis responding to Cadmium (Cd2+) exposure.

Spirulina platensis can secrete extracellular polymeric substances (EPS) helping to protect damage from stress environment, such as cadmium (Cd2+) exposure. However, the responding mechanism of S. platensis and the secreted EPS to exposure of Cd2+ is still unclear. This research focuses on the effects of Cd2+ on the composition and structure of the EPS and the response mechanism of EPS secretion from S. platensis for Cd2+ exposure. S. platensis can produce 261.37 mg·g-1 EPS when exposing to 20 mg·L-1 CdCl2, which was 2.5 times higher than the control group. The S. platensis EPS with and without Cd2+ treatment presented similar and stable irregularly fibrous structure. The monosaccharides composition of EPS in Cd2+ treated group are similar with control group but with different monosaccharides molar ratios, especially for Rha, Gal, Glc and Glc-UA. And the Cd2+ treatment resulted in a remarkable decline of humic acid and fulvic acid content. The antioxidant ability of S. platensis EPS increased significantly when exposed to 20 mg·L-1 CdCl2, which could be helpful for S. platensis protecting damage from high concentration of Cd2+. The transcriptome analysis showed that sulfur related metabolic pathways were up-regulated significantly, which promoted the synthesis of sulfur-containing amino acids and the secretion of large amounts of EPS.

Investigating Biofilms: Advanced Methods for Comprehending Microbial Behavior and Antibiotic Resistance.

Biofilms, which consist of microorganisms enclosed in an extracellular polymeric material (EPS), hold immense importance in the fields of environmental research, industry, and medicine. They play a significant role in ecosystem dynamics and stability, but they also pose issues such as biofouling, corrosion, and pollution. Biofilms in medical environments are linked to persistent infections and elevated healthcare expenses. The EPS matrix plays a crucial role in maintaining the structural integrity and antibiotic resistance of these structures. The research primarily investigates the role of the EPS matrix in facilitating horizontal gene transfer among biofilm communities, with a particular emphasis on EPS and its impact on this process. The process is recognized as a pivotal mechanism in the emergence of antibiotic resistance, underscoring the crucial function of EPS in the dynamics of biofilms. The analysis also highlights the significant financial constraints caused by biofilms in several industries. Biofilm-associated infections in the healthcare sector result in escalated treatment expenses and extended hospitalization periods. In an industrial context, biofilms have a role in increasing maintenance expenses and product contamination, emphasizing the need for efficient management solutions. This review presents the most recent progress in biofilm research, emphasizing the utilization of sophisticated imaging tools and molecular methodologies. In addition to conventional imaging techniques, the research explores the utilization of sophisticated molecular tools, such as DNA and RNA sequencing, in conjunction with proteomics. These approaches are essential for assessing the genetic and metabolic mechanisms that regulate biofilm development and antibiotic resistance. The review underscores the significance of employing an interdisciplinary methodology in the study of biofilms. By incorporating a range of approaches, such as sophisticated imaging and molecular analysis, a comprehensive understanding of biofilm dynamics may be achieved. This approach also opens up possibilities for developing novel solutions to address the negative impacts of biofilms on health, industry, and the environment.

Differentiation and quantification of extracellular polymeric substances from microalgae and bacteria in the mixed culture.

Extracellular polymeric substances (EPS) play significant roles in the formation, function, and interactions of microalgal-bacteria consortia. Understanding the key roles of EPS depends on reliable extraction and quantification methods, but differentiating of EPS from microalgae versus bacteria is challenging. In this work, cation exchange resin (CER) and thermal treatments were applied for total EPS extraction from microalgal-bacteria mixed culture (MBMC), flow cytometry combined with SYTOX Green staining was applied to evaluate cell disruption during EPS extraction, and auto-fluorescence-based cell sorting (AFCS) was used to separate microalgae and bacteria in the MBMC. Thermal extraction achieved much higher EPS yield than CER, but higher temperature and longer time reduced cell activity and disrupted the cells. The highest EPS yield with minimal loss of cell activity and cell disruption was achieved using thermal extraction at 55℃ for 30 min, and this protocol gave good results for MBMC with different microalgae:bacteria (M:B) mass ratios. AFCS combined with thermal treatment achieved the most-efficient biomass differentiation and low EPS loss (<4.5 %) for the entire range of M:B ratios. EPS concentrations in bacteria were larger than in microalgae: 42.8 ± 0.4 mg COD/g TSS versus 9.19 ± 0.38 mg COD/g TSS. These findings document sensitive and accurate methods to extract and quantify EPS from microalgal-bacteria aggregates.

Bioprospecting of Chlamydomonas reinhardtii for boosting biofuel-related products production based on novel aggregation-induced emission active extracellular polymeric substances nanoprobes.

Biofuel production from microalgae has been greatly restricted by low biomass productivity and long-term photosynthetic efficacy. Here, a novel strategy for selecting high-growing, stress-resistant algal strains with high photosynthetic capacity was proposed based on biocompatible extracellular polymeric substances (EPS) probes with aggregation-induced emission (AIE) properties. Specifically, AIE active EPS probes were synthesized for in-situ long-term monitoring of the EPS productivity at different algal growth stages. By coupling the AIE-based fluorescent techniques, algal cells were classified into four diverse populations based on their chlorophyll and EPS signals. Mechanistic studies on the sorted algal cells revealed their remarkable stress resistance and high expression of cell division, biopolymer production and photosynthesis-related genes. The sorted and subcultured algal cells consistently exhibited relatively higher growth rates and photosynthetic capacities, resulting in an increased (1.2 to 1.8-fold) algal biomass production, chlorophyll, and lipids. This study can potentially open new strategies to boost microalgal-based biofuel production.

Coupling of gene regulation and carrier modification manipulates bacterial biofilms as robust living catalysts.

The biofilm of an engineered strain is limited by slow growth and low yield, resulting in an unsatisfactory ability to resist external stress and promote catalytic efficiency. Here, biofilms used as robust living catalysts were manipulated through dual functionalized gene regulation and carrier modification strategies. The results showed that gene overexpression regulates the autoinducer-2 activity, extracellular polymeric substance content and colony behavior of Escherichia coli, and the biofilm yield of csgD overexpressed strains increased by 79.35 % compared to that of the wild type strains (p < 0.05). In addition, the hydrophilicity of polyurethane fibres modified with potassium dichromate increased significantly, and biofilm adhesion increased by 105.80 %. Finally, the isoquercitrin yield in the catalytic reaction of the biofilm reinforced by the csgD overexpression strain and the modified carrier was 247.85 % higher than that of the untreated group. Overall, this study has developed engineered strains biofilm with special functions, providing possibilities for catalytic applications.

Iron-mediated DAMO-anammox process: Revealing the mechanism of electron transfer.

The nitrate denitrifying anaerobic methane oxidation-anaerobic ammonia oxidation (DAMO-anammox) can accomplish nitrogen removal and methane (CH4) reduction. This process greatly contributes to carbon emission mitigation and carbon neutrality. In this study, we investigated the electron transfer process of functional microorganisms in the iron-mediated DAMO-anammox system. Fe3+ could be bound to several functional groups (-CH3, COO-, -CH) in extracellular polymeric substance (EPS), and the functional groups bound were different at different iron concentration. Fe3+ underwent reduction reactions to produce Fe2+. Most Fe3+ and Fe2+ react with microorganisms and formed chelates with EPS. Three-dimensional fluorescence spectra showed that Fe3+ affected the secretion of tyrosine and tryptophan, which were essential for cytochrome synthesis. The presence of Fe3+ accelerated c-type cytochrome-mediated extracellular electron transfer (EET), and when more Fe3+ existed, the more cytochrome C expressed. DAMO archaea (M. nitroreducens) in the system exhibited a high positive correlation with the functional genes (resa and ccda) for cytochrome c synthesis. Some denitrifying microorganisms showed positive correlations with the abundance of riboflavin. This finding showed that riboflavin secreted by functional microorganisms acted as an electron shuttle. In addition, DAMO archaea were positively correlated with the hair synthesis gene pily1, which indicated that direct interspecies electron transfer (DIET) may exist in the iron-mediated DAMO-anammox system.

Effects of temperature on nitrifying membrane-aerated biofilms: An experimental and modeling study.

Temperature is known to have an important effect on the morphology and removal fluxes of conventional, co-diffusional biofilms. However, much less is known about the effects of temperature on membrane-aerated biofilm reactors (MABRs). Experiments and modeling were used to determine the effects of temperature on the removal fluxes, biofilm thickness and morphology, and biofilm microbial community structure of nitrifying MABRs. Steady state tests were carried out at 10 °C and 30 °C. MABRs grown at 30 °C had higher ammonium removal fluxes (5.5 ± 0.9 g-N/m2/day at 20 mgN/L) than those grown at 10 °C (3.4 ± 0.2 g-N/m2/day at 20 mgN/L). The 30 °C biofilms were thinner and rougher, with a lower protein to polysaccharides ratio (PN/PS) in their extracellular polymeric substance (EPS) matrix and greater amounts of biofilm detachment. Based on fluorescent in-situ hybridization (FISH), there was a higher relative abundance of nitrifying bacteria at 30 °C than at 10 °C, and the ratio of AOB to total nitrifiers (AOB + NOB) was higher at 30 °C (95.1 ± 2.3%) than at 10 °C (77.2 ± 8.6 %). Anammox bacteria were more abundant at 30 °C (16.6 ± 3.7 %) than at 10 °C (6.5 ± 2.4 %). Modeling suggested that higher temperatures increase ammonium oxidation fluxes when the biofilm is limited by ammonium. However, fluxes decrease when oxygen becomes limited, i.e., when the bulk ammonium concentrations are high, due to decreased oxygen solubility. Consistent with the experimental results, the model predicted that the percentage of AOB to total nitrifiers at 30 °C was higher than at 10 °C. To investigate the effects of temperature on biofilm diffusivity and O2 solubility, without longer-term changes in the microbial community, MABR biofilms were grown to steady state at 20 °C, then the temperature changed to 10 °C or 30 °C overnight. Higher ammonium oxidation fluxes were obtained at higher temperatures: 1.91 ± 0.24 g-N/m2/day at 10 °C and 3.19 ± 0.40 g-N/m2/day at 30 °C. Overall, this work provides detailed insights into the effect of temperature on nitrifying MABRs, which can be used to better understand MABR behavior and manage MABR reactors.

Bacillus subtilis Matrix Protein TasA is Interfacially Active, but BslA Dominates Interfacial Film Properties.

Microbial growth often occurs within multicellular communities called biofilms, where cells are enveloped by a protective extracellular matrix. Bacillus subtilis serves as a model organism for biofilm research and produces two crucial secreted proteins, BslA and TasA, vital for biofilm matrix formation. BslA exhibits surface-active properties, spontaneously self-assembling at hydrophobic/hydrophilic interfaces to form an elastic protein film, which renders B. subtilis biofilm surfaces water-repellent. TasA is traditionally considered a fiber-forming protein with multiple matrix-related functions. In our current study, we investigate whether TasA also possesses interfacial properties and whether it has any impact on BslA's ability to form an interfacial protein film. Our research demonstrates that TasA indeed exhibits interfacial activity, partitioning to hydrophobic/hydrophilic interfaces, stabilizing emulsions, and forming an interfacial protein film. Interestingly, TasA undergoes interface-induced restructuring similar to BslA, showing an increase in ß-strand secondary structure. Unlike BslA, TasA rapidly reaches the interface and forms nonelastic films that rapidly relax under pressure. Through mixed protein pendant drop experiments, we assess the influence of TasA on BslA film formation, revealing that TasA and other surface-active molecules can compete for interface space, potentially preventing BslA from forming a stable elastic film. This raises a critical question: how does BslA self-assemble to form the hydrophobic "raincoat" observed in biofilms in the presence of other potentially surface-active species? We propose a model wherein surface-active molecules, including TasA, initially compete with BslA for interface space. However, under lateral compression or pressure, BslA retains its position, expelling other molecules into the bulk. This resilience at the interface may result from structural rearrangements and lateral interactions between BslA subunits. This combined mechanism likely explains BslA's role in forming a stable film integral to B. subtilis biofilm hydrophobicity.

Enhancing methane production and interspecies electron transfer of anaerobic granular sludge by the immobilization of magnetic biochar.

Supplementation of conductive materials has been proved to be a promising approach for enhancing microbial interspecies electron transfer (IET) in anaerobic digestion systems. In this study, magnetic bamboo-based biochar was prepared at temperatures of 400-800 °C via a ball milling/carbonization method, and it immobilized in mature anaerobic granular sludge (AGS) aimed to enhance methane production by improving the IET process between syntrophic microbial communities in the AGS. Results showed that the AGS with magnetic biochar immobilization demonstrated increased glucotrophic and acetotrophic methane production by 69.54-77.56 % and 39.96-54.92 %, respectively. Magnetic biochar prepared at 800 °C with a relatively higher Fe content (0.37 g/g magnetic biochar) displayed a stronger electron charge/discharge capacity (36.66 F/g), and its immobilization into AGS promoted methane production most. The conductivity of AGS increased by 52.13-87.32 % after incorporating magnetic biochar. Furthermore, the extracellular polymeric substance (EPS) of AGS showed an increased capacitance and decreased electron transfer resistance possibly due to the binding of magnetic biochar and more riboflavin secretion in EPS, which could contribute to the accelerated IET process in the inner AGS. In addition, the immobilization of magnetic biochar could promote the production of volatile fatty acids by 15.36-22.50 %. All these improvements may jointly lead to the enhanced methane production capacity of AGS. This study provided a fundamental understanding of the role of incorporated magnetic biochar in AGS in promoting anaerobic digestion performance.

Lumen air pressure regulated multifunctional microbiotas in membrane-aerated biofilm reactors for simultaneous nitrogen removal and antibiotic elimination from aquaculture wastewater.

In this study, two membrane-aerated biofilm reactors (MABRs) were constructed: one solely utilizing biofilm and another hybrid MABR (HMABR) incorporating both suspended-sludge and biofilm to treat low C/N aquaculture wastewater under varying lumen air pressure (LAP). Both HMABR and MABR demonstrated superior nitrogen removal than conventional aeration reactors. Reducing LAP from 10 kPa to 2 kPa could enhance denitrification processes without severely compromising nitrification, resulting in an increase in total inorganic nitrogen (TIN) removal from 50.2±3.1 % to 71.6±1.0 %. The HMABR exhibited better denitrification efficacy than MABR, underscoring its potential for advanced nitrogen removal applications. A decline in LAP led to decreased extracellular polymeric substance (EPS) production, which could potentially augment reactor performance by minimizing mass transfer resistance while maintaining microbial matrix stability and function. Gene-centric metagenomics analysis revealed decreasing LAP impacted nitrogen metabolic potentials and electron flow pathways. The enrichment of napAB at higher LAP and the presence of complete ammonia oxidation (Comammox) Nitrospira at lower LAP indicated aerobic denitrification and Comammox processes in nitrogen removal. Multifunctional microbial communities developed under LAP regulation, diversifying the mechanisms for simultaneous nitrification-denitrification. Increased denitrifying gene pool (narGHI, nirK, norB) and enzymatic activity at a low LAP can amplify denitrification by promoting denitrifying genes and electron flow towards denitrifying enzymes. Sulfamethoxazole (SMX) was simultaneously removed with efficiency up to 80.2 ± 3.7 %, mainly via biodegradation, while antibiotic resistome and mobilome were propagated. Collectively, these findings could improve our understanding of nitrogen and antibiotic removal mechanisms under LAP regulation, offering valuable insights for the effective design and operation of MABR systems in aquaculture wastewater treatment.

Electron syntrophy between mixed hydrogenogens and Geobacter metallireducens boosted dark hydrogen fermentation: Clarifying roles of electroactive extracellular polymeric substances.

To modulate the electron transfer behavior of hydrogen-producing bacteria (HPB) for enhanced hydrogen production, Geobacter metallireducens culture (GM) was introduced as an electron syntrophy partner and redox balance regulator in dark fermentation systems with hydrogen-producing sludge (HPS) as inoculum. The highest hydrogen yield was 306.5 mL/g-COD at the GM/HPS volatile solids ratio of 0.08, which was 65.2 % higher than the HPS group. The multi-layered extracellular polymeric substances (EPS) of GM played a significant role in promoting hydrogen production, with c-type cytochromes probably serving as electroactive functional components. The addition of GM significantly improved the NADH/NAD+ ratio, electron transport system activity, hydrogenase activity, and electrochemical properties of HPS. Furthermore, the microbial community structure and metabolic functions were optimized due to the potential syntrophic interaction between Clostridium sensu stricto (dominant HPB) and Geobacter, thus promoting hydrogen production. This study provided novel insights into the interactions among exoelectrogens, electroactive EPS, and mixed HPB.

Influences of extracellular polymeric substances (EPS) recovered from waste sludge on the ability of Jiaozhou Bay to self-remediate of diesel-polluted seawater.

The introduction of EPS recovered from waste sludge may have an impact on the process of microbial remediation of oil-contaminated seawater. This study investigated the effect of EPS on the self-remediation capacity of diesel-polluted seawater in Jiaozhou Bay. Hydrocarbon attenuation and microbial activity were monitored in seawater collected from five islands after diesel and N, P addition, with and without EPS, incubated under aerobic conditions. Compared to seawater without EPS, degradation of TPH (total petroleum hydrocarbon) doubled and improved degradation of non-volatile (C16-C24) hydrocarbons to some extent in EPS-added seawater. The introduction of EPS led to changes in microbiota richness and diversity, significantly stimulating the growth of Proteobacteria and Firmicutes phyla or Bacillus and Pseudomonas genera. RT-qPCR analysis indicated EPS caused higher increases in cytochrome P450 gene copies than alkB. Prediction of alkane decay genes from 16S rRNA sequencing data revealed that EPS addition obviously promoted genes related to ethanol dehydrogenation function in the microbial community. Additionally, EPS enhanced the enzymatic activities of alkane hydroxylase, ethanol dehydrogenase, phosphatase and lipase, but increased protease and catalase inconspicuously. The above outlook that environmental sustainability of EPS from waste sludge for diesel-contaminated seawater remediation may provide new perspectives for oil spill bioremediation.

Polysaccharide preferred minority-dominant community assembly and exoenzyme enrichment in transparent exopolymer particles: Implication for global carbon cycle in water.

The ubiquitous transparent exopolymer particles (TEPs) are an important organic carbon pool and an ideal microhabitat for bacteria in aquatic environments. They play a crucial role in the global carbon cycle. Organic matter transformation and carbon turnover in TEPs strongly depend on the assembly of their associated bacterial communities and enzyme activity. However, the mechanisms of bacterial community assembly and their potential effects on the organic carbon cycle in TEPs are still unclear. In this study, we comparatively explored the community assembly of TEP-associated bacteria and bacterioplankton from surface freshwater using metagenomics. It was found that the bacterial community assembly in TEPs followed a minority-dominant rule and was governed by homogeneous selection. Pseudomonadota and Actinomycetota, which are responsible for polysaccharide degradation, serve as taxon-specific biomarkers among the abundant and diverse bacteria in TEPs. The network of TEP-associated bacteria displayed stronger robustness than that of bacterioplankton. Bin 76 (majorly Acinetobacter) was the overwhelmingly dominant taxa in TEPs, whereas there was no clearly dominant taxa in TEP-free water. Exoenzyme analysis showed that 64 out of 71 identified polysaccharide hydrolases were markedly linked with the dominant bin 76 in TEPs, while no such linkage was observed for bacterioplankton. Generally, Acinetobacter, which is capable of utilizing polysaccharides, is preferred to be assembled in TEPs together with high polysaccharide hydrolase activity. This may significantly accelerate the turnover of organic carbon in the giant global TEP pool. These findings are important for a deep understanding of the carbon cycle in water.

Outer membrane vesicles and the outer membrane protein OmpU govern Vibrio cholerae biofilm matrix assembly.

Biofilms are matrix-encased microbial communities that increase the environmental fitness and infectivity of many human pathogens including Vibrio cholerae. Biofilm matrix assembly is essential for biofilm formation and function. Known components of the V. cholerae biofilm matrix are the polysaccharide Vibrio polysaccharide (VPS), matrix proteins RbmA, RbmC, Bap1, and extracellular DNA, but the majority of the protein composition is uncharacterized. This study comprehensively analyzed the biofilm matrix proteome and revealed the presence of outer membrane proteins (OMPs). Outer membrane vesicles (OMVs) were also present in the V. cholerae biofilm matrix and were associated with OMPs and many biofilm matrix proteins suggesting that they participate in biofilm matrix assembly. Consistent with this, OMVs had the capability to alter biofilm structural properties depending on their composition. OmpU was the most prevalent OMP in the matrix, and its absence altered biofilm architecture by increasing VPS production. Single-cell force spectroscopy revealed that proteins critical for biofilm formation, OmpU, the matrix proteins RbmA, RbmC, Bap1, and VPS contribute to cell-surface adhesion forces at differing efficiency, with VPS showing the highest efficiency whereas Bap1 showing the lowest efficiency. Our findings provide new insights into the molecular mechanisms underlying biofilm matrix assembly in V. cholerae, which may provide new opportunities to develop inhibitors that specifically alter biofilm matrix properties and, thus, affect either the environmental survival or pathogenesis of V. cholerae.IMPORTANCECholera remains a major public health concern. Vibrio cholerae, the causative agent of cholera, forms biofilms, which are critical for its transmission, infectivity, and environmental persistence. While we know that the V. cholerae biofilm matrix contains exopolysaccharide, matrix proteins, and extracellular DNA, we do not have a comprehensive understanding of the majority of biofilm matrix components. Here, we discover outer membrane vesicles (OMVs) within the biofilm matrix of V. cholerae. Proteomic analysis of the matrix and matrix-associated OMVs showed that OMVs carry key matrix proteins and Vibrio polysaccharide (VPS) to help build biofilms. We also characterize the role of the highly abundant outer membrane protein OmpU in biofilm formation and show that it impacts biofilm architecture in a VPS-dependent manner. Understanding V. cholerae biofilm formation is important for developing a better prevention and treatment strategy framework.

Mediating the performance of anaerobic granular sludge by exogenous flavin supplementation: Flavin binding capacity and behavior, interspecies electron transfer, and methane production.

Although flavins are known as effective electron mediators, the binding capacity of exogenous flavins by anaerobic granular sludge (AGS) and their role in interspecies electron transfer (IET) remains unknown. In this study, AGS was mediated by using three exogenous flavins of riboflavin (RF), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Results showed that the total amounts of flavins associated with extracellular polymeric substance (EPS) of AGS increased by 2.03-2.42 and 3.83-4.94 folds, after exposure to 50 and 200 µM of exogenous flavins, respectively. A large portion of FMN and FAD was transformed into RF by AGS. Exogenous flavin mediation also stimulated the production of EPS and cytochrome c (c-Cyts) as well as cytochrome-bound flavins. The increased abundance of these electron mediators led to a reduced electrochemical impedance of EPS and improved extracellular electron transfer capacity. The methane production of AGS after mediation with exogenous RF, FMN, and FAD increased by 19.03-31.71%, 22.86-26.04%, and 28.51-33.44%, respectively. This study sheds new light on the role of exogenous flavins in promoting the IET process of a complex microbial aggregate of AGS.

Ascorbic acid reduction pretreatment enhancing metal regulation to improve methane production from anaerobic digestion of waste activated sludge.

Conversion of waste activated sludge (WAS) to methane by anaerobic digestion (AD) is often limited by the slow rate of hydrolysis, and the presence of metal ions in sludge is regarded as a critical factor hindering sludge hydrolysis. This study developed a novel strategy to remove Fe from WAS by using ascorbic acid (VC) as a reducing agent under acidic conditions. The feasibility of reduction pretreatment in improving methane production of AD and its intrinsic mechanism were investigated. Results indicate that, under VC doses of 100 mmol/L and pH of 3.50, pretreatment removed 47.60 % of Fe, 59.88 % of Ca, and 51.86 % of Mg contained in the sludge. The removal of metal ions facilitated the disruption of sludge flocculation structure and extracellular polymeric substance (EPS) layers, leading to a 14.78 % increase in cell lysis and a decrease in fractal dimension values to 2.08. Batch AD experiments showed that VC pretreatment improved methane production, with an optimized net methane yield of 190.22 mL/g·VS, an increase of 134.75 % compared to raw WAS. The pretreatment affected the interfacial interaction energy of the sludge, leading to a transformation in the sludge surfaces from hydrophilic to hydrophobic, reducing the interaction between sludge molecules and increasing the number of binding sites available for enzymatic reactions. According to a study of microbial communities, it was found that VC pretreatment caused an increase in the presence of essential functional microbes responsible for hydrolysis, acidification, and methanation. This increase in acetoclastic and hydrogenotrophic methanogens resulted in a substantial enhancement in methane production. These results can be used to develop better pretreatment methods to enhance AD performance.

Influence of extracellular polymeric substances on arsenic bioaccumulation and biotransformation in biofilms.

It is well recognized that biofilms can biosorb and biotransform heavy metals in aquatic environments. However, the effects of extracellular polymeric substance (EPS) on inorganic arsenic (As) bioaccumulation and biotransformation in biofilms are still unrevealed and need to be investigated. In order to explore the above scientific issues, the As accumulation and speciation in EPS-containing or EPS-free biofilms and growth medium under As(V)/As(III) exposure conditions were measured. After the removal of EPS, the amount of As uptake (Asup) and As adsorption (Asad) in biofilms were significantly reduced, no matter whether exposed to As(V) or As(III). FTIR analysis further suggested that the interaction between these functional groups with As was limited after the removal of EPS. In the EPS-containing biofilms, the Asad was mainly As(V) with low toxicity. However, after the removal of EPS, the Asad was mainly As(III) with high fluidity, and no methylated As was found. Moreover, the removal of EPS inhibited As(III) oxidation and methylation by biofilms, resulting in the decrease of As(V) and methylated As in the growth medium. The findings of this study emphasized the essential impact of EPS on the biosorption and biotransformation of As in biofilms. This study provides a unique understanding of the role of biofilms in As biogeochemical cycle, and water quality purification function in water environments.