Significant progress has been made previously in the research and development of graphene oxide (GO) membranes for water purification, but their biofouling behavior remains poorly understood. In this study, we investigated the biofilm formation and biofouling of GO membranes with different surface microstructures in the context of filtering natural surface water and for an extended operation period (110 days). The results showed that the relatively hydrophilic and smooth Fe(OH)3/GO membrane shaped a thin and spatially heterogeneous biofilm with high stable flux. However, the ability to simultaneously mitigate biofilm formation and reduce biofouling was not observed in the weakly hydrophilic and wrinkled Fe/GO and H-Fe(OH)3/GO membranes. Microbial analyses revealed that the hydrophilicity and roughness distinguished the bacterial communities and metabolic functions. The organic matter-degrading and predatory bacteria were more adapted to hydrophilic and smooth GO surfaces. These functional taxa were involved in the degradation of extracellular polymeric substances (EPS), and improved biofilm heterogeneity. In contrast, the weakly hydrophilic and wrinkled GO surfaces had reduced biodiversity, while unexpectedly boosting the proliferation of EPS-secreting bacteria, resulting in increased biofilm formation and aggravated biofouling. Moreover, all GO membranes achieved sustainable water purification during the entire operating period.

Staphylococcus aureus forms biofilms consisting of cells embedded in a matrix made of proteins, polysaccharides, lipids, and extracellular DNA (eDNA). Biofilm-associated infections are difficult to treat and can promote antibiotic resistance, resulting in negative healthcare outcomes. eDNA within the matrix contributes to the stability, growth, and immune-evasive properties of S. aureus biofilms. eDNA is released by autolysis, which is mediated by murein hydrolases that access the cell wall via membrane pores formed by holin-like proteins. The eDNA content of S. aureus biofilms varies among individual strains and is influenced by environmental conditions, including the presence of antibiotics. eDNA plays an important role in biofilm development and structure by acting as an electrostatic net that facilitates protein-cell and cell-cell interactions. Because of eDNA\'s structural importance in biofilms and its ubiquitous presence among S. aureus isolates, it is a potential target for therapeutics. Treatment of biofilms with DNase can eradicate or drastically reduce them in size. Additionally, antibodies that target DNABII proteins, which bind to and stabilize eDNA, can also disperse biofilms. This review discusses the recent literature on the release, structure, and function of eDNA in S. aureus biofilms, in addition to a discussion of potential avenues for targeting eDNA for biofilm eradication.

UNASSIGNED: Periodontal diseases are known to be associated with polymicrobial biofilms and inflammasome activation. A deeper understanding of the subgingival cytological (micro) landscape, the role of extracellular DNA (eDNA) during periodontitis, and contribution of the host immune eDNA to inflammasome persistence, may improve our understanding of the mechanisms underlaying severe forms of periodontitis.
UNASSIGNED: In this work, subgingival biolfilms developing on biologically neutral polyethylene terephthalate films placed in gingival cavities of patients with chronic periodontitis were investigated by confocal laser scanning microscopy (CLSM). This allowed examination of realistic cytological landscapes and visualization of extracellular polymeric substances (EPS) including amyloids, total proteins, carbohydrates and eDNA, as well as comparison with several single-strain in vitro model biofilms produced by oral pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus gordonii, S. sanguinis and S. mitis. Fluorescence in situ hybridization (FISH) analysis was also used to identify eDNA derived from eubacteria, streptococci and members of the Bacteroides-Porphyromonas-Prevotella (BPP) group associated with periodontitis.
UNASSIGNED: Analysis of subgingival biofilm EPS revealed low levels of amyloids and high levels of eDNA which appears to be the main matrix component. However, bacterial eDNA contributed less than a third of the total eDNA observed, suggesting that host-derived eDNA released in neutrophil extracellular traps may be of more importance in the development of biofilms causing periodontitis.
UNASSIGNED: eDNA derived from host immunocompetent cells activated at the onset of periodontitis may therefore be a major driver of bacterial persistence and pathogenesis.

Geobacter sulfurreducens DL1 is a metal-reducing dissimilatory bacterium frequently used to produce electricity in bioelectrochemical systems (BES). The biofilm formed on electrodes is one of the most important factors for efficient electron transfer; this is possible due to the production of type IV pili and c-type cytochromes that allow it to carry out extracellular electron transfer (EET) to final acceptors. In this study, we analyzed the biofilm formed on different support materials (glass, hematite (Fe2O3) on glass, fluorine-doped tin oxide (FTO) semiconductor glass, Fe2O3 on FTO, graphite, and stainless steel) by G. sulfurreducens DL1 (WT) and GSU1771-deficient strain mutant (Δgsu1771). GSU1771 is a transcriptional regulator that controls the expression of several genes involved in electron transfer. Different approaches and experimental tests were carried out with the biofilms grown on the different support materials including structure analysis by confocal laser scanning microscopy (CLSM), characterization of electrochemical activity, and quantification of relative gene expression by RT-qPCR. The gene expression of selected genes involved in EET was analyzed, observing an overexpression of pgcA, omcS, omcM, and omcF from Δgsu1771 biofilms compared to those from WT, also the overexpression of the epsH gene, which is involved in exopolysaccharide synthesis. Although we observed that for the Δgsu1771 mutant strain, the associated redox processes are similar to the WT strain, and more current is produced, we think that this could be associated with a higher relative expression of certain genes involved in EET and in the production of exopolysaccharides despite the chemical environment where the biofilm develops. This study supports that G. sulfurreducens is capable of adapting to the electrochemical environment where it grows.

UNASSIGNED: Biofilms, which are created by most microorganisms, are known for their widely developed drug resistance, even more than planktonic forms of microorganisms. The aim of the study was to assess the effectiveness of agents composed of farnesol and nanoparticles (silver, gold, copper, and zinc oxide) in the degradation of biofilms produced by pathogenic microorganisms.
UNASSIGNED: Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans were used to create the biofilm structure. Colloidal suspensions of silver, gold, copper, and zinc oxide (Ag, Au, Cu, ZnO) with the addition of farnesol (F) were used as the treatment factor. The size distribution of those composites was analyzed, their zeta potential was measured, and their structure was visualized by transmission electron microscopy. The viability of the microorganism strains was assessed by an XTT assay, the ability to form biofilms was analyzed by confocal microscopy, and the changes in biofilm structure were evaluated by scanning electron microscopy. The general toxicity toward the HFFF2 cell line was determined by a neutral red assay and a human inflammation antibody array.
UNASSIGNED: The link between the two components (farnesol and nanoparticles) caused mutual stability of both components. Planktonic forms of the microorganisms were the most sensitive when exposed to AgF and CuF; however, the biofilm structure of all microorganism strains was the most disrupted (both inhibition of formation and changes within the structure) after AgF treatment. Composites were not toxic toward the HFFF2 cell line, although the expression of several cytokines was higher than in the not-treated group.
UNASSIGNED: The in vitro studies demonstrated antibiofilm properties of composites based on farnesol and nanoparticles. The greatest changes in biofilm structure were triggered by AgF, causing an alteration in the biofilm formation process as well as in the biofilm structure.

Industrial biocides aim to keep water systems microbiologically controlled and to minimize biofouling. However, the resulting dead cells are usually not removed from the water streams and can influence the growth of the remaining live cells in planktonic and sessile states. This study aims to understand the effect of dead Pseudomonas fluorescens cells killed by industrial biocides-benzalkonium chloride (BAC) and 2,2-dibromo-3-nitrilopropionamide (DBNPA)-on biofilm formation. Additionally, the effect of different dead/live cell ratios (50.00% and 99.99%) was studied. The inoculum was recirculated in a Parallel Plate Flow Cell (PPFC). The overall results indicate that dead cells greatly affect biofilm properties. Inoculum with DBNPA-dead cells led to more active (higher ATP content and metabolic activity) and thicker biofilm layers in comparison to BAC-dead cells, which seems to be linked to the mechanism of action by which the cells were killed. Furthermore, higher dead cell ratios (99.99%) in the inoculum led to more active (higher culturability, metabolic activity and ATP content) and cohesive/compact and uniformly distributed biofilms in comparison with the 50.00% dead cell ratio. The design of future disinfection strategies must consider the contribution of dead cells to the biofilm build-up, as they might negatively affect water system operations.

In industry, treatments against biofilms need to be optimized and, in the wastewater treatment field, biofilm composition needs to be controlled. Therefore, describing the biochemical and physical structures of biofilms is now required to better understand the influence of operating parameters and treatment on biofilms. The present study aims to investigate how growth conditions influence EPS composition, biofilm physical properties and volume detachment using a 1D biofilm model. Two types of EPS are considered in the present model, proteins and polysaccharides. The main hypotheses are that: (i) the production of polysaccharides occurs mainly under strong nutrient limitation(s) while the production of proteins is coupled to both the substrate uptake rate and the lysis process; (ii) the local biofilm porosity depends on the local biofilm composition. Both volume and surface detachment occur in biofilms and volume detachment extent depends on the biofilm local cohesion and thus on the local composition of biofilms for a given shear stress. The model is based on experimental trends and aims to represent these observations on the basis of biochemical and physical processes. Four case studies covering a wide range of contrasting growth conditions such as different COD/N ratios, applied SOLR and shear stresses are investigated. The model predicts how the biochemical and physical biofilm structures change as a result of contrasting growth conditions. More precisely simulation results are in good agreement with the main experimental observations reported in the literature, such as: (i) a strong nitrogen limitation of growth induces an important accumulation of polysaccharides leading to a more porous and homogenous biofilm, (ii) a high applied surface organic loading load allows to obtain a high biofilm thickness, (iii) a strong shear stress applied during the biofilm growth leads to a reduction of the biofilm thickness and to a consolidation of the biofilm structure. Overall, this model represents a relevant decision tool for the selection of appropriate enzymatic treatments in the context of negative biofilm control. From our results, it appears that protease based treatments should be more appropriate for biofilms developed under low COD/N ratios (about 20 gCOD/gN) whereas both glucosidases and proteases based treatments should be more appropriate for biofilms developed under high COD/N ratio (about 70 gCOD/gN). In addition, the model could be useful for other applications such as resource recovery in biofilms or granules, and help to better understand biological membrane fouling.

Extracellular DNA (eDNA) is a major component of bacterial biofilms. In this study, we performed a three-dimensional analysis of Leptospira biofilm using advanced imaging by confocal laser scanning microscopy (CLSM) and multi-parameter analysis by COMSTAT 2 software, with quantification of Leptospira and eDNA fluorescence. To investigate the role of eDNA in Leptospira biofilm, we treated Leptospira biflexa biofilms with DNase I enzyme (DNase), which digested eDNA, and compared DNase treated biofilms and controls. There was a significant reduction of the biomass of biofilms treated with DNase, by spectrophotometry and COMSTAT analysis. The multiparameter analysis evidenced for DNase-treated biofilms a significant decrease in the surface area and the average thickness; opposing to a significant augmentation of the surface/biovolume ratio and the roughness coefficient (Ra*), when compared to controls. We analyzed the parameters of DNase-treated biofilms by Pearson\'s correlation coefficient and found significant positive correlations between biomass and average thickness; biomass and surface area; surface area and average thickness. On the other hand, there were significant negative correlations between Ra* and biomass; Ra* and average thickness; Ra* and surface area. These findings suggest that eDNA digestion results in biofilm instability and alteration of the three-dimensional architecture, justifying the negative correlation between Ra* and the above-mentioned parameters. In conclusion, our study showed that eDNA digestion produced a massive structural loss, instability, and dramatic changes in the three-dimensional architecture of Leptospira biflexa biofilm. These findings contribute to a better understanding of the role of eDNA and highlight the importance of eDNA as a key component in Leptospira biofilms.

The occurrence of viable but non-culturable (VBNC) bacteria in drinking water may result in significant underestimation of viable cell counts detected by culture-based method, thus raising microbiological safety concern. Chlorine disinfection has been widely used in drinking water treatment to ensure microbiological safety. However, the effect of residual chlorine on inducing bacteria in biofilms into a VBNC state remains unclear. We determined cell numbers of Pseudomonas fluorescence in different physiological states (culturable, viable, dead) by heterotrophic plate count method and flow cytometer in a flow cell system under 0, 0.1, 0.5, 1.0 mg/L chlorine treatment. Numbers of culturable cells were 4.66 ± 0.47 Log10, 2.82 ± 0.76 Log10, 2.30 ± 1.23 Log10 (CFU/112.5 mm3) in each chlorine treatment group. However, viable cell numbers remained at 6.32 ± 0.05 Log10, 6.11 ± 0.24 Log10, 5.08 ± 0.81 Log10 (cells/112.5 mm3). Significant difference between numbers of viable and culturable cells demonstrated chlorine could induce bacteria in biofilms into a VBNC state. In this study, flow cells combination with Optical Coherence Tomography (OCT) were applied to construct an Automated experimental Platform for replicate Biofilm cultivation and structural Monitoring (APBM) system. The OCT imaging results demonstrated that changes of biofilm structure under chlorine treatment were closely related to their inherent characteristics. Biofilms with low thickness and high roughness coefficient or porosity were easier to be removed from the substratum. Biofilm with high rigid properties were more resistant to chlorine treatment. Even though >95 % bacteria in biofilms entered a VBNC state, the biofilm physical structure was still remained. This study revealed the possibility of bacteria to enter a VBNC state in drinking water biofilms and changes of biofilm structure with different characteristics under chlorine treatment, which provide reference for biofilms control in drinking water distribution systems.

Simultaneous nitrogen and phosphorus removal (SNPR) biofilm system is an effective wastewater treatment process. However, the understanding on the mechanism of functional microorganisms driving SNPR is still limited, especially the role of complete ammonia oxidation (comammox) Nitrospira and glycogen-accumulating organisms (GAO). In this study, a sequencing batch biofilm reactor (SBBR) performing SNPR was operated for 249 d. Based on the 16S rRNA gene, comammox amoA amplicon sequencing, metagenomics and batch experiment, we found that comammox Nitrospira was the main ammonia-oxidizing microorganisms (AOM) and provided nitrite for anaerobic ammonia oxidation (anammox) bacteria (AnAOB). Besides, GAO was dominated by the bacteria of genus Defluviicoccus and played a primary role in reducing nitrate rather than nitrite. Fluorescent in situ hybridization (FISH) analysis confirmed that Nitrospira was enriched in the inner layer of the biofilm. Thus, we put forward a novel insight into the mechanism of SNPR biofilm system. Comammox Nitrospira was responsible for nitrite and nitrate production in the inner biofilm, and AnAOB consumed the produced nitrite during the anammox process. While GAO reduced nitrate to nitrite and polyphosphate-accumulating organisms (PAO) converted nitrite to dinitrogen via denitrifying phosphorus removal in the outer biofilm. These findings provide a new understanding in SNPR biofilm system.