Biofilm State Research Articles

Virtual Screening and Meta-Analysis Approach Identifies Factors for Inversion Stimulation (Fis) and Other Genes Responsible for Biofilm Production in Pseudomonas aeruginosa: A Corneal Pathogen

Bacterial keratitis caused by Pseudomonas aeruginosa is indeed a serious concern due to its potential to cause blindness and its resistance to antibiotics, partly attributed to biofilm formation and cytotoxicity to the cornea. The present study uses a meta-analysis of a transcriptomics dataset to identify important genes and pathways in biofilm formation of P. aeruginosa induced keratitis. By combining data from several studies, meta-analysis can enhance statistical power and robustness, enabling the identification of 83 differentially expressed candidate genes, including fis that could serve as therapeutic targets. The approach of combining meta-analysis with virtual screening and in vitro methods provides a comprehensive strategy for identifying potential target genes and pathways crucial for bacterial biofilm formation and development anti-biofilm medications against P. aeruginosa infections. The study identified 83 candidate genes that exhibited differential expression in the biofilm state, with fis proposed as an ideal target for therapy for P. aeruginosa biofilm formation. These techniques, meta-analysis, virtual screening, and invitro methods were used in combination to diagnostically identify these genes, which play a significant role in biofilms. This finding has highlighted a hallmark target list for P. aeruginosa anti-biofilm potential treatments.

Functional analysis of cyclic diguanylate-modulating proteins in Vibrio fischeri.

As bacterial symbionts transition from a motile free-living state to a sessile biofilm state, they must coordinate behavior changes suitable to each lifestyle. Cyclic diguanylate (c-di-GMP) is an intracellular signaling molecule that can regulate this transition, and it is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. Generally, c-di-GMP inhibits motility and promotes biofilm formation. While c-di-GMP and the enzymes that contribute to its metabolism have been well studied in pathogens, considerably less focus has been placed on c-di-GMP regulation in beneficial symbionts. Vibrio fischeri is the sole beneficial symbiont of the Hawaiian bobtail squid (Euprymna scolopes) light organ, and the bacterium requires both motility and biofilm formation to efficiently colonize. c-di-GMP regulates swimming motility and cellulose exopolysaccharide production in V. fischeri. The genome encodes 50 DGCs and PDEs, and while a few of these proteins have been characterized, the majority have not undergone comprehensive characterization. In this study, we use protein overexpression to systematically characterize the functional potential of all 50 V. fischeri proteins. All 28 predicted DGCs and 10 of the 14 predicted PDEs displayed at least one phenotype consistent with their predicted function, and a majority of each displayed multiple phenotypes. Finally, active site mutant analysis of proteins with the potential for both DGC and PDE activities revealed potential activities for these proteins. This work presents a systems-level functional analysis of a family of signaling proteins in a tractable animal symbiont and will inform future efforts to characterize the roles of individual proteins during lifestyle transitions.IMPORTANCECyclic diguanylate (c-di-GMP) is a critical second messenger that mediates bacterial behaviors, and Vibrio fischeri colonization of its Hawaiian bobtail squid host presents a tractable model in which to interrogate the role of c-di-GMP during animal colonization. This work provides systems-level characterization of the 50 proteins predicted to modulate c-di-GMP levels. By combining multiple assays, we generated a rich understanding of which proteins have the capacity to influence c-di-GMP levels and behaviors. Our functional approach yielded insights into how proteins with domains to both synthesize and degrade c-di-GMP may impact bacterial behaviors. Finally, we integrated published data to provide a broader picture of each of the 50 proteins analyzed. This study will inform future work to define specific pathways by which c-di-GMP regulates symbiotic behaviors and transitions.

Alkaloids solenopsins from fire ants display in vitro and in vivo activity against the yeast Candida auris

ABSTRACT The urgency surrounding Candida auris as a public health threat is highlighted by both the Center for Disease Control (CDC) and World Health Organization (WHO) that categorized this species as a priority fungal pathogen. Given the current limitations of antifungal therapy for C. auris, particularly due to its multiple resistance to the current antifungals, the identification of new drugs is of paramount importance. Some alkaloids abundant in the venom of the red invasive fire ant (Solenopsis invicta), known as solenopsins, have garnered attention as potent inhibitors of bacterial biofilms, and there are no studies demonstrating such effects against fungal pathogens. Thus, we herein investigated the antibiotic efficacy of solenopsin alkaloids against C. auris biofilms and planktonic cells. Both natural and synthetic solenopsins inhibited the growth of C. auris strains from different clades, including fluconazole and amphotericin B-resistant isolates. Such alkaloids also inhibited matrix deposition and altered cellular metabolic activity of C. auris in biofilm conditions. Mechanistically, the alkaloids compromised membrane integrity as measured by propidium iodide uptake in exposed planktonic cells. Additionally, combining the alkaloids with AMB yielded an additive antifungal effect, even against AMB-resistant strains. Finally, both extracted solenopsins and the synthetic analogues demonstrated protective effect in vivo against C. auris infection in the invertebrate model Galleria mellonella. These findings underscore the potent antifungal activities of solenopsins against C. auris and suggest their inclusion in future drug development. Furthermore, exploring derivatives of solenopsins could reveal novel compounds with therapeutic promise.

Antifungal activity’s study of the of new derivatives of pyrrolo[3,4-c]pyrazol-3-ones and pyrazol-3- carboxamides in the biofilm model of Candida spp.

The search for new antifungal drugs is current interest due to the wide spread of fungal infections. To simulate the antifungal effect of new promising compounds with high antifungal activity in planktonic culture in clinical practice, it is necessary to study their effect on the biofilm of micromycetes.Aim: To study the antifungal activity of new promising representatives of silver salts of pyrazoles and their condensed systems under conditions of biofilm formation.Material and Methods. To study the antifungal activity of silver salts I and II, the micromethod of two-fold serial dilutions was used. Activity against a typical and 14 clinical highly virulent isolates of C. albicans was studied. The study of the antimycotic activity of substances under biofilm conditions was carried out using resazurin to quantify the degree of biofilm formation. The minimum inhibitory concentrations for biofilms were calculated (sMIC50 - the concentration of the antifungal substance at which a 50% decrease in fluorescence is observed compared to the positive control).Results and Discussion. It was shown that the antifungal effect of the studied compounds in biofilm culture of clinical strains of Candida spp. is significantly lower than in planktonic culture. High antifungal activity of the silver salt of pyrazole-3- carboxamide in planktonic and biofilm cultures of resistant strains of Candida albicans, exceeding the effect of the reference drug fluconazole by 2.8–11.2 times, was revealed.Conclusion. A promising pyrazole-3-carboxamide derivative has been identified that effectively inhibits the growth of Candida albicans biofilms, which can be recommended for further study.

Hypervirulent Carbapenem-Susceptible Klebsiella pneumoniae ST412/K57 with Strong Biofilm Formation: association with gas gangrene and sepsis.

Hypervirulent Klebsiella pneumoniae (hvKp) poses a serious public health threat. Gas gangrene caused by hvKp was rarely reported, potentially resulting in a poor prognosis. In this study, we described the case of a hospitalized patient with gas gangrene and sepsis by hvKP. The carbapenem-susceptible hypervirulent Klebsiella pneumoniae (CS-hvKp) strains KPLSN and KPLSX were isolated from the knee joint pus and blood specimens of the patient for further investigations. Whole genome sequencing revealed that KPLSN and KPLSX were highly homologous (SNPs<10) and belonging to ST412/K57. The minimum inhibitory concentration and minimum bactericidal concentration under biofilm values of meropenem in KPLSN and KPLSX were significantly higher than planktonic state (>128 mg/L versus 0.25 mg/L, P<0.0001). These two strains had high biofilm formation ability, and fluorescence staining experiments results showed that they were not easily killed by meropenem in the biofilm state. KPLSN and KPLSX showed high capsular production and were confirmed with high virulence through experiments with the Galleria mellonella and BALB/c mice abdominal infection model. The persistent symptoms may be due to enhanced biofilm and capsule formation. Global ST412 strains phylogenetic analysis showed their evolution towards higher virulence and resistance. It emphasizes the critical need for judicious antibiotic use and novel therapeutic approaches to combat special infections caused by these pathogens.

Biofilm formation of Lactiplantibacillus plantarum food isolates under flow and resistance to disinfectant agents

Bacterial biofilms formed in food processing environments can be resilient against cleaning and disinfection causing recontamination and spoilage of foods. We investigated the biofilm formation of six Lactiplantibacillus plantarum food spoilage isolates (FBR1-FBR6) using WCFS1 as a reference strain, and examined the impact of benzalkonium chloride (BKC) and peracetic acid (PAA) on planktonic and biofilm cells formed under static and dynamic flow conditions. We used a custom-designed setup composed of a 48-well plate with 0.8 ml culture volumes. We quantified biofilm formation under static and dynamic flow conditions with a flow rate of 3.2 ml/h using plate counting, Crystal Violet (CV) staining, and fluorescence staining techniques. Our findings revealed significant differences in biofilm formation and disinfectant resistance among studied strains and cell types. We observed that flow promoted biofilm formation in some strains and increased the number of culturable cells within biofilms in all strains. Furthermore, biofilm cells demonstrated higher resistance to disinfectants in comparison to planktonic cells for certain strains. Interestingly, cells from dispersed under flow biofilms show higher resistance to disinfectants than cells from static biofilms. The results indicate the importance of flow conditions in influencing L. plantarum food isolates biofilm formation and disinfection resistance, which may have implications for product contamination and spoilage risks.

L-Rhamnose Globally Changes the Transcriptome of Planktonic and Biofilm Escherichia coli Cells and Modulates Biofilm Growth.

L-rhamnose, a naturally abundant sugar, plays diverse biological roles in bacteria, influencing biofilm formation and pathogenesis. This study investigates the global impact of L-rhamnose on the transcriptome and biofilm formation of PHL628 E. coli under various experimental conditions. We compared growth in planktonic and biofilm states in rich (LB) and minimal (M9) media at 28 °C and 37 °C, with varying concentrations of L-rhamnose or D-glucose as a control. Our results reveal that L-rhamnose significantly affects growth kinetics and biofilm formation, particularly reducing biofilm growth in rich media at 37 °C. Transcriptomic analysis through RNA-seq showed that L-rhamnose modulates gene expression differently depending on the temperature and media conditions, promoting a planktonic state by upregulating genes involved in rhamnose transport and metabolism and downregulating genes related to adhesion and biofilm formation. These findings highlight the nuanced role of L-rhamnose in bacterial adaptation and survival, providing insight into potential applications in controlling biofilm-associated infections and industrial biofilm management.

Development, evaluation and application of propidium monoazide (PMA) based methodologies on viable cell quantification of Pediococcus acidilactici in rice noodles products

Pediococcus acidilactici provides a special taste during dough fermentation, but its improper presence will accelerate the spoilage of rice noodles products. In this study, propidium monoazide (PMA) based methodologies on viable cells quantification of P. acidilactici were developed, evaluated and applied in rice noodles products. Prior to qPCR and RT-qPCR detection, PMA treatment was included to effectively remove the residual DNA from membrane-compromised (dead) cells. Firstly, considering the influence of food component and PMA on qPCR result determination, 4 standard curves corresponding Ct value to P. acidilactici cell number were established in 4 different rice noodle products, respectively. The 4 standard curves were subsequently applied for viable cell quantification of P. acidilactici. Secondly, mimicking the storage conditions of rice noodles products and growth states of P. acidilactici, planktonic and biofilm (early, mediate, mature) cells in 4 types of rice noodles products stored at −20 °C and 4 °C were taken into consideration to evaluate the efficiency of PMA-qPCR for viable cell quantification. Thirdly, stress response gene expression in P. acidilactici was monitored by PMA-RT-qPCR and compared with culturable and viable cell number quantification. The PMA-qPCR method with specific standard curves for 4 types of rice noodle products was applicable for viable cell quantification of P. acidilactici in planktonic and biofilm (early, mediate, and mature) states. The planktonic and biofilm cells of P. acidilactici maintained high and stable in both culturable and viable cell number in different growth states, rice noodles products, and low temperature conditions, but acquired significantly different gene expression levels. The findings provided viable cells quantification methods and an alarm for P. acidilactici contamination risk in instant rice noodles products.

Antibiotic-Polyphosphate Nanocomplexes: A Promising System for Effective Biofilm Eradication.

The eradication of bacterial biofilms poses an enormous challenge owing to the inherently low antibiotic susceptibility of the resident microbiota. The complexation of antibiotics with polyphosphate can substantially improve antimicrobial performance. Nanoparticular complexes of the model drug colistin and polyphosphate (CP-NPs) were developed and characterized in terms of their particle size and morphology, polydispersity index (PDI), zeta potential, and cytotoxicity. Enzyme-triggered monophosphate and colistin release from the CP-NPs was evaluated in the presence of alkaline phosphatase (AP). Subsequently, antimicrobial efficacy was assessed by inhibition experiments on planktonic cultures, as well as time-kill assays on biofilms formed by the model organism Micrococcus luteus. The CP-NPs exhibited a spherical morphology with particle sizes <200 nm, PDI <0.25, and negative zeta potential. They showed reduced cytotoxicity toward two human cell lines and significantly decreased hemotoxicity compared with native colistin. Release experiments with AP verified the enzymatic cleavage of polyphosphate and subsequent release of monophosphate and colistin from CP-NPs. Although CP-NPs were ineffective against planktonic M. luteus cultures, they showed major activity against bacterial biofilms, outperforming native colistin treatment. Strongly elevated AP levels in the biofilm state were identified as a potential key factor for the observed findings. Accordingly, polyphosphate-based nanocomplexes represent a promising tool to tackle bacterial biofilm.

Deciphering the influence of NaCl on social behaviour of Bacillus subtilis

Various environmental signals, such as temperature, pH, nutrient levels, salt content and the presence of other microorganisms, can influence biofilm’s development and dynamics. However, the innate mechanisms that govern at the molecular and cellular levels remain elusive. Here, we report the impact of physiologically relevant concentrations of NaCl on biofilm formation and the associated differences in an undomesticated natural isolate of Bacillus subtilis . NaCl exposure and its uptake by bacterial cells induced substantial changes in the architecture of pellicle biofilm and an upsurge in the expansion of biofilm colonies on agar surfaces. We have observed the upregulation of genes involved in motility and the downregulation of genes involved in the biosynthesis of extracellular matrix components through the transcription factor sigD, suggesting the possible underlying mechanisms. To further support these observations, we have used Δ sigD and Δ srfAC null mutants, which showed compromised NaCl-induced effects. Our results indicate that NaCl induces a lifestyle shift in B. subtilis from a sessile biofilm state to an independent unicellular motile state. Overall, we present evidence that NaCl can reprogramme gene expression and alter cellular morphology and the state of cells to adapt to motility, which facilitates the expansion of bacterial colonies.

Unveiling the role of PA0730.1 sRNA in Pseudomonas aeruginosa virulence and biofilm formation: Exploring rpoS and mucA regulation

Small RNA (sRNA) in bacteria serve as the key messengers in regulating genes associated with quorum sensing controlled bacterial virulence. This study was aimed to unveil the regulatory role of sRNA PA0730.1 on the expression of various traits of Pseudomonas aeruginosa linked to pathogenicity, with special emphasis on the growth, colony morphology, cell motility, biofilm formation, and the expression of diverse virulence factors. PA0730.1 sRNA was found to be upregulated both during planktonic stationary growth phase and at biofilm state of P. aeruginosa PAO1. PA0730.1 deleted strain showed significant growth retardation with increased doubling time. Overexpression of PA0730.1 led to enhanced motility and biofilm formation, while the ∆PA0730.1 strain displayed significant inhibition in motility and biofilm formation. Furthermore, PA0730.1 was found to regulate the synthesis of selected virulence factors of P. aeruginosa. These observations in PA0730.1+ and ∆PA0730.1 were found to be correlated with the PA0730.1-mediated repression of transcription regulators, mucA and rpoS, both at transcriptional and translational levels. The results suggest that PA0730.1 sRNA might be a promising target for developing new drug to counter P. aeruginosa pathogenesis, and could also help in RNA oligonucleotide based therapeutic research for formulating a novel therapeutant.

Cocktail Approach with Polyserotonin Nanoparticles and Peptides for Treatment of Streptococcus mutans.

Dental plaque, formed by a Streptococcus mutans biofilm, is a major contributor to cavity formation. While antimicrobial strategies exist, the growing risk of antibiotic resistance necessitates alternative therapeutic solutions. Polyserotonin nanoparticles (PSeNPs), recently recognized for their photothermal property and promising biomedical applications, open up a new avenue for antimicrobial use. Here, we introduced a UV-initiated synthetic route for PSeNPs with improved yield. Using these PSeNPs, a cocktail treatment to reduce the viability of this cavity-causing bacteria was developed. This cocktail comprises an S. mutans-targeting antimicrobial peptide (GH12), an intraspecies competence-stimulating peptide that triggers altruistic cell death in S. mutans, and laser-activated heating of PSeNPs. The "peptide + PSeNP + laser" combination effectively inhibits S. mutans growth in both planktonic and biofilm states. Moreover, the cocktail approach remains effective in reducing the viability of S. mutans in a more virulent dual-species biofilm with Candida albicans. Overall, our results reinforce the utility of a multipronged therapeutic strategy to reduce cariogenic bacteria in the complex model oral biofilm.

Lactate promotes the biofilm-to-invasive-planktonic transition in Salmonella enterica serovar Typhimurium via the de novo purine pathway.

Salmonella enterica serovar Typhimurium (S. Typhimurium) infection triggers an inflammatory response that changes the concentration of metabolites in the gut impacting the luminal environment. Some of these environmental adjustments are conducive to S. Typhimurium growth, such as the increased concentrations of nitrate and tetrathionate or the reduced levels of Clostridia-produced butyrate. We recently demonstrated that S. Typhimurium can form biofilms within the host environment and respond to nitrate as a signaling molecule, enabling it to transition between sessile and planktonic states. To investigate whether S. Typhimurium utilizes additional metabolites to regulate its behavior, our study delved into the impact of inflammatory metabolites on biofilm formation. The results revealed that lactate, the most prevalent metabolite in the inflammatory environment, impedes biofilm development by reducing intracellular c-di-GMP levels, suppressing the expression of curli and cellulose, and increasing the expression of flagellar genes. A transcriptomic analysis determined that the expression of the de novo purine pathway increases during high lactate conditions, and a transposon mutagenesis genetic screen identified that PurA and PurG, in particular, play a significant role in the inhibition of curli expression and biofilm formation. Lactate also increases the transcription of the type III secretion system genes involved in tissue invasion. Finally, we show that the pyruvate-modulated two-component system BtsSR is activated in the presence of high lactate, which suggests that lactate-derived pyruvate activates BtsSR system after being exported from the cytosol. All these findings propose that lactate is an important inflammatory metabolite used by S. Typhimurium to transition from a biofilm to a motile state and fine-tune its virulence.IMPORTANCEWhen colonizing the gut, Salmonella enterica serovar Typhimurium (S. Typhimurium) adopts a dynamic lifestyle that alternates between a virulent planktonic state and a multicellular biofilm state. The coexistence of biofilm formers and planktonic S. Typhimurium in the gut suggests the presence of regulatory mechanisms that control planktonic-to-sessile transition. The signals triggering the transition of S. Typhimurium between these two lifestyles are not fully explored. In this work, we demonstrated that in the presence of lactate, the most dominant host-derived metabolite in the inflamed gut, there is a reduction of c-di-GMP in S. Typhimurium, which subsequently inhibits biofilm formation and induces the expression of its invasion machinery, motility genes, and de novo purine metabolic pathway genes. Furthermore, high levels of lactate activate the BtsSR two-component system. Collectively, this work presents new insights toward the comprehension of host metabolism and gut microenvironment roles in the regulation of S. Typhimurium biology during infection.

Multi-omics analysis reveals genes and metabolites involved in Streptococcus suis biofilm formation

BackgroundStreptococcus suis is an important zoonotic pathogen. Biofilm formation largely explains the difficulty in preventing and controlling S. suis. However, little is known about the molecular mechanism of S. suis biofilm formation.ResultsIn this study, transcriptomic and metabolomic analyses of S. suis in biofilm and planktonic states were performed to identify key genes and metabolites involved in biofilm formation. A total of 789 differential genes and 365 differential metabolites were identified. By integrating transcriptomics and metabolomics, five main metabolic pathways were identified, including amino acid pathway, nucleotide metabolism pathway, carbon metabolism pathway, vitamin and cofactor metabolism pathway, and aminoacyl-tRNA biosynthesis metabolic pathway.ConclusionsThese results provide new insights for exploring the molecular mechanism of S. suis biofilm formation.

Green synthesis and antibacterial-antibiofilm properties of biogenic silver nanoparticles

The biosynthesis of metallic nanoparticles is gaining prominence as an alternative to traditional physicochemical methods, offering several advantages such as simplicity, non-toxicity, lower energy requirements and short reaction times leading to environmentally sustainable processes. The aims of this work were: to study the extracellular biosynthesis of silver nanoparticles (AgNPs) by Pseudomonas extremaustralis 2E-UNGS, to characterise the shape, monodispersity and size of AgNPs, to explore their antimicrobial and antibiofilm activities, and to evaluate the role of nitrate reductase activity in the biosynthesis process. The novelty of this work relies on the development of a green and sustainable method for the synthesis of stable AgNPs with optimal properties for potential applications in antimicrobial materials, especially when incorporated into polymeric matrices or used as agrochemical substitutes. Optimal conditions for the biosynthesis of spherical AgNPs were determined to be pH 7, 38 °C, 4 h of darkness and 120 rpm using stationary phase culture supernatants of P. extremaustralis 2E-UNGS. The involvement of extracellular nitrate reductase in AgNP biosynthesis was confirmed by enzymatic assays and supported by bioinformatics analysis, which identified the presence of the napA2 gene linked to the nirBD cluster. Antimicrobial assays demonstrated the inhibitory effect of AgNPs against both Gram-positive and Gram-negative bacteria, including Pseudomonas aeruginosa PA01 in both planktonic and biofilm states. In addition, the potential application of AgNPs in innovative antibacterial polymers was explored by incorporating them into polyurethane matrices either alone (PU-AgNP) or in combination with crystal violet as a photosensitizer (PU-AgNP-CV). Subsequent inoculation with a clinical isolate of Pseudomonas aeruginosa resulted in significant reductions in viable bacterial counts on both PU-AgNP-CV and PU-AgNP. Biogenic AgNPs showed antibacterial and antibiofilm properties for new antimicrobial material development.

Antibacterial activity evaluation of a novel K3-specific phage against Acinetobacter baumannii and evidence for receptor-binding domain transfer across morphologies

Acinetobacter baumannii (A. baumannii) poses a serious public health challenge due to its notorious antimicrobial resistance, particularly carbapenem-resistant A. baumannii (CRAB). In this study, we isolated a virulent phage, named P1068, from medical wastewater capable of lysing CRAB, primarily targeting the K3 capsule type. Basic characterization showed that P1068 infected the A. baumannii ZWAb014 with an optimal MOI of 1, experienced a latent period of 10 ​min and maintained stability over a temperature range of 4–37 ​°C and pH range of 3–10. Phylogenetic and average nucleotide identity analyses indicate that P1068 can be classified as a novel species within the genus Obolenskvirus of the Caudoviricetes class as per the most recent virus classification released by the International Committee on Taxonomy of Viruses (ICTV). Additionally, according to classical morphological classification, P1068 is identified as a T4-like phage (Myoviridae). Interestingly, we found that the tail fiber protein (TFP) of P1068 shares 74% coverage and 88.99% identity with the TFP of a T7-like phage (Podoviridae), AbKT21phiIII (NC_048142.1). This finding suggests that the TFP gene of phages may undergo horizontal transfer across different genera and morphologies. In vitro antimicrobial assays showed that P1068 exhibited antimicrobial activity against A. baumannii in both biofilm and planktonic states. In mouse models of intraperitoneal infection, P1068 phage protected mice from A. baumannii infection and significantly reduced bacterial loads in various tissues such as the brain, blood, lung, spleen, and liver compared to controls. In conclusion, this study demonstrates that phage P1068 might be a potential candidate for the treatment of carbapenem-resistant and biofilm-forming A. baumannii infections, and expands the understanding of horizontal transfer of phage TFP genes.

Pyocyanin stimulates membrane vesicle formation in Pseudomonas aeruginosa, but the synthesis is not required for enhanced vesiculation in biofilms

Bacterial membrane vesicles (MVs), particularly outer membrane vesicles (OMVs) in gram-negative bacteria, package their cargo for delivery to other bacteria or host cells. MV production is activated in the multicellular biofilm growth mode; however, the underlying mechanisms of enhanced MV production in biofilms are not fully understood. Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic infections due to biofilm formation, making it difficult to treat. Pyocyanin is a phenazine synthesized by P. aeruginosa, which is a blue-pigment signaling molecule and generates H2O2. Herein, we investigated the interplay among pyocyanin, H2O2, and MVs in P. aeruginosa biofilms. Pyocyanin, H2O2, and MVs were sequentially produced in P. aeruginosa static liquid biofilms, and all phenotypes were repressed in a biofilm-deficient mutant ∆pelA∆pslA, which does not synthesize extracellular polysaccharides Pel and Psl. The addition of pyocyanin increased H2O2 generation and MV formation, suggesting that pyocyanin-mediated H2O2 generation facilitated MV formation in P. aeruginosa. Because catalase repressed MV formation in the biofilm, it suggested that oxidative stress may be a pathway for the activation of MV production in P. aeruginosa biofilms. On the other hand, MV formation in biofilms was not increased in the mutant of phzM, which encodes an enzyme for pyocyanin synthesis. Thus, while additional pyocyanin promotes MV formation, several factors other than pyocyanin, such as extracellular polysaccharides, play a major role in enhanced MV formation in P. aeruginosa biofilms. Our study provides new insights into vesiculation in P. aeruginosa biofilms.

Mathematical modeling and machine learning-based optimization for enhancing biofiltration efficiency of volatile organic compounds

Biofiltration is a method of pollution management that utilizes a bioreactor containing live material to absorb and destroy pollutants biologically. In this paper, we investigate mathematical models of biofiltration for mixing volatile organic compounds (VOCs) for instance hydrophilic (methanol) and hydrophobic (α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\alpha$$\\end{document}-pinene). The system of nonlinear diffusion equations describes the Michaelis-Menten kinetics of the enzymic chemical reaction. These models represent the chemical oxidation in the gas phase and mass transmission within the air-biofilm junction. Furthermore, for the numerical study of the saturation of α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\alpha$$\\end{document}-pinene and methanol in the biofilm and gas state, we have developed an efficient supervised machine learning algorithm based on the architecture of Elman neural networks (ENN). Moreover, the Levenberg-Marquardt (LM) optimization paradigm is used to find the parameters/ neurons involved in the ENN architecture. The approximation to a solutions found by the ENN-LM technique for methanol saturation and α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\alpha$$\\end{document}-pinene under variations in different physical parameters are allegorized with the numerical results computed by state-of-the-art techniques. The graphical and statistical illustration of indications of performance relative to the terms of absolute errors, mean absolute deviations, computational complexity, and mean square error validates that our results perfectly describe the real-life situation and can further be used for problems arising in chemical engineering.

Biofilm mitigation in hybrid chemical-biological upcycling of waste polymers.

Introduction: Accumulation of plastic waste in the environment is a serious global issue. To deal with this, there is a need for improved and more efficient methods for plastic waste recycling. One approach is to depolymerize plastic using pyrolysis or chemical deconstruction followed by microbial-upcycling of the monomers into more valuable products. Microbial consortia may be able to increase stability in response to process perturbations and adapt to diverse carbon sources, but may be more likely to form biofilms that foul process equipment, increasing the challenge of harvesting the cell biomass. Methods: To better understand the relationship between bioprocess conditions, biofilm formation, and ecology within the bioreactor, in this study a previously-enriched microbial consortium (LS1_Calumet) was grown on (1) ammonium hydroxide-depolymerized polyethylene terephthalate (PET) monomers and (2) the pyrolysis products of polyethylene (PE) and polypropylene (PP). Bioreactor temperature, pH, agitation speed, and aeration were varied to determine the conditions that led to the highest production of planktonic biomass and minimal formation of biofilm. The community makeup and diversity in the planktonic and biofilm states were evaluated using 16S rRNA gene amplicon sequencing. Results: Results showed that there was very little microbial growth on the liquid product from pyrolysis under all fermentation conditions. When grown on the chemically-deconstructed PET the highest cell density (0.69g/L) with minimal biofilm formation was produced at 30°C, pH 7, 100rpm agitation, and 10sL/hr airflow. Results from 16S rRNAsequencing showed that the planktonic phase had higher observed diversity than the biofilm, and that Rhodococcus, Paracoccus, and Chelatococcus were the most abundant genera for all process conditions. Biofilm formation by Rhodococcus sp. And Paracoccus sp. Isolates was typically lower than the full microbial community and varied based on the carbon source. Discussion: Ultimately, the results indicate that biofilm formation within the bioreactor can be significantly reduced by optimizing process conditions and using pure cultures or a less diverse community, while maintaining high biomass productivity. The results of this study provide insight into methods for upcycling plastic waste and how process conditions can be used to control the formation of biofilm in bioreactors.

Estimating biofilm activity on biofilter elements in recirculating aquaculture systems (RAS) for rearing Atlantic salmon parr (Salmo salar) during operation with ozone and peracetic acid

Chemical disinfection in a recirculating aquaculture system (RAS) may affect biofilm-associated bacteria and the nitrification performance in the biofilter units. The biofilm response to chemical disinfectants in RAS remains unclear, but it can be understood using methods to quantify biofilm activity. Here, we compared the effects of two disinfection strategies, continuous ozone at 0.06 mg/L Cl2 equivalent (Ozone group) and peracetic acid (PAA) at 1 mg/L (PAA group) to control group without disinfectant, on biofilm activity on biofilter elements from nine identical experimental RAS with Atlantic salmon parr (Salmo salar) during a four-week trial. Biofilm activities on biofilter elements from the three groups were examined by measuring oxygen consumption rates (OCR) following sequential spiking with pure tap water, and tap water spiked with nitrite or ammonium, as well as oxygen release rate (kor) following hydrogen peroxide (H2O2) addition, to estimate metabolic activities of biofilm related to endogenous respiration and substrate turnover. The results show that the applied PAA dose increased the endogenous respiration activity of biofilm by 39–133%, stimulated the rate of biofilm enzymatic decomposition of H2O2 by 135%, and partially impaired the biofilm metabolism for nitrite oxidation (decrease by 36%), resulting in a significant nitrite accumulation (rise by 59%) in the cultured water, compared to control over experimental period. Ozone treatment caused an enhanced endogenous respiration activity of biofilm at the beginning of the experiment (increase by 45–74%), but dropped to control levels at the end of the experiment. The results indicate that chronic exposure to PAA can alter the metabolic state of biofilm, which can have consequences for biofilter functions, while chronic exposure to ozone improved water clarity without compromising the metabolic status of biofilm. The investigations provided insights into biofilm response to chemical disinfectant in RAS, which would benefit the optimization of an effective and safe use of disinfectants in RAS.