Increased tolerance to commonly used antibiotics in a Pseudomonas aeruginosa ex vivo porcine keratitis model.

Introduction. Bacterial keratitis, particularly caused by Pseudomonas aeruginosa, is challenging to treat because of multi-drug tolerance, often associated with the formation of biofilms. Antibiotics in development are typically evaluated against planktonic bacteria in a culture medium, which may not accurately represent the complexity of infections in vivo.Hypothesis/Gap Statement. Developing a reliable, economic ex vivo keratitis model that replicates some complexity of tissue infections could facilitate a deeper understanding of antibiotic efficacy, thus aiding in the optimization of treatment strategies for bacterial keratitis.Methodology. Here we investigated the efficacy of three commonly used antibiotics (gentamicin, ciprofloxacin and meropenem) against Pseudomonas aeruginosa cytotoxic strain PA14 and invasive strain PA01 using an ex vivo porcine keratitis model.Results. Both strains of P. aeruginosa were susceptible to the MIC of the three tested antibiotics. However, significantly higher concentrations were necessary to inhibit bacterial growth in the minimum biofilm eradication concentration (MBEC) assay, with both strains tolerating concentrations greater than 512 mg l-1 of meropenem. When MIC and higher concentrations than MBEC (1024 mg l-1) of antibiotics were applied, ciprofloxacin exhibited the highest potency against both P. aeruginosa strains, followed by meropenem, while gentamicin showed the least potency. Despite this, none of the antibiotic concentrations used effectively cleared the infection, even after 18 h of continuous exposure.Conclusions. Further exploration of antibiotic concentrations and aligning dosing with clinical studies to validate the model is needed. Nonetheless, our ex vivo porcine keratitis model could be a valuable tool for assessing antibiotic efficacy.

Bottom-up construction and screening of algae-bacteria consortia for pollutant biodegradation.

Microbial communities have been used as important biological tools for a variety of purposes associated with agriculture, the food industry and human health. Artificial engineering of microbial communities is an emerging field of research motivated by finding stable and efficient microbial systems. However, the successful design of microbial communities with desirable functions not only requires profound understanding of microbial activities, but also needs efficient approaches to piece together the known microbial traits to give rise to more complex systems. This study demonstrates the bottom-up integration of environmentally isolated phototrophic microalgae and chemotrophic bacteria as artificial consortia to bio-degrade selected volatile organic compounds (VOCs). A high throughput screening method based on 96-well plate format was developed for discovering consortia with bioremediation potential. Screened exemplar consortia were verified for VOCs degradation performance, among these, certain robust consortia were estimated to have achieved efficiencies of 95.72% and 92.70% and near 100% removal (7 days) of benzene, toluene, and phenol, respectively, with initial concentrations of 100 mg/L. VOCs degradation by consortia was mainly attributed to certain bacteria including Rhodococcus erythropolis, and Cupriavidus metallidurans, and directly contributed to the growth of microalgae Coelastrella terrestris (R = 0.82, p < 0.001). This work revealed the potential of converting VOCs waste into algal biomass by algae-bacteria consortia constructed through a bottom-up approach. The screening method enables rapid shortlisting of consortia combinatorial scenarios without prior knowledge about the individual strains or the need for interpreting complex microbial interactions.

Integrating molecular microbial methods to improve faecal pollution management in rivers with designated bathing waters.

Rivers are at risk from a variety of pollution sources. Faecal pollution is of particular concern since it disperses pathogenic microorganisms in the aquatic environment. Currently, faecal pollution levels in rivers is monitored using faecal indicator bacteria (FIB) that do not offer information about pollution sources and associated risks. This study used a combined molecular approach, along with measurements of water quality, to gain information on pollution sources, and risk levels, in a newly designated recreational bathing site in the River Wharfe (UK). Physico-chemical parameters were monitored in situ, with water quality multiparameter monitoring sondes installed during the 2021 bathing season. The molecular approach was based on quantitative PCR (qPCR)-aided Microbial Source Tracking (MST) and 16S rRNA gene metabarcoding to obtain a fingerprint of bacterial communities and identify potential bioindicators. The analysis from the water quality sondes showed that ammonium was the main parameter determining the distribution of FIB values. Lower faecal pollution levels were detected in the main river when compared to tributaries, except for samples in the river located downstream of a wastewater treatment plant. The faecal pollution type (anthropogenic vs. zoogenic) changed the diversity and the structure of bacterial communities, giving a distinctive fingerprint that can be used to inform source. DNA-based methods showed that the presence of human-derived bacteria was associated with Escherichia coli spikes, coinciding with higher bacterial diversity and the presence of potential pathogenic bacteria mainly of the genus Mycobacterium, Aeromonas and Clostridium. Samples collected after a heavy rainfall event were associated with an increase in Bacteroidales, which are markers of faecal pollution, including Bacteroides graminisolvens, a ruminant marker associated with surface run-off from agricultural sources. The combined use of qPCR and 16S rRNA sequencing was able to identify pollution sources, and novel bacterial indicators, thereby aiding decision-making and management strategies in recreational bathing rivers.

Phage-based therapy against biofilm producers in gram-negative ESKAPE pathogens.

Persistent antibiotic use results in the rise of antimicrobial resistance with limited or no choice for multidrug-resistant (MDR) and extensively drug resistant (XDR) bacteria. This necessitates a need for alternative therapy to effectively combat clinical pathogens that are resistant to last resort antibiotics. The study investigates hospital sewage as a potential source of bacteriophages to control resistant bacterial pathogens. Eighty-one samples were screened for phages against selected clinical pathogens. Totally, 10 phages were isolated against A. baumannii, 5 phages against K. pneumoniae, and 16 phages were obtained against P. aeruginosa. The novel phages were observed to be strain-specific with complete bacterial growth inhibition of up to 6 h as monotherapy without antibiotics. Phage plus colistin combinations reduced the minimum-biofilm eradication concentration of colistin up to 16 folds. Notably, a cocktail of phages exhibited maximum efficacy with complete killing at 0.5-1 µg/ml colistin concentrations. Thus, phages specific to clinical strains have a higher edge in treating nosocomial pathogens with their proven anti-biofilm efficacy. In addition, analysis of phage genomes revealed close phylogenetic relations with phages reported from Europe, China, and other neighbouring countries. This study serves as a reference and can be extended to other antibiotics and phage types to assess optimum synergistic combinations to combat various drug resistant pathogens in the ongoing AMR crisis.

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics.

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery research less attractive. Antimicrobial drug discovery and subsequent commercialization can be made more lucrative if a fail-fast-and-fail-cheap approach can be implemented within the lead optimization stages where researchers have greater control over drug design and formulation. In this article, the setup of an ex vivo ovine wounded skin model infected with Staphylococcus aureus is described, which is simple, cost-effective, high throughput, and reproducible. The bacterial physiology in the model mimics that during infection as bacterial proliferation is dependent on the pathogen's ability to damage the tissue. The establishment of wound infection is verified by an increase in viable bacterial counts compared to the inoculum. This model can be used as a platform to test the efficacy of emerging antimicrobials in the lead optimization stage. It can be contended that the availability of this model will provide researchers developing antimicrobials with a fail-fast-and-fail-cheap model, which will help increase success rates in subsequent animal trials. The model will also facilitate the reduction and refinement of animal use for research and ultimately enable faster and more cost-effective translation of novel antimicrobials for skin and soft tissue infections to the clinic.

Absence of KpsM (Slr0977) Impairs the Secretion of Extracellular Polymeric Substances (EPS) and Impacts Carbon Fluxes in Synechocystis sp. PCC 6803.

Many cyanobacteria produce extracellular polymeric substances (EPS), composed mainly of heteropolysaccharides, that play a variety of physiological roles, being crucial for cell protection, motility, and biofilm formation. However, due to their complexity, the EPS biosynthetic pathways as well as their assembly and export mechanisms are still far from being fully understood. Here, we show that the absence of a putative EPS-related protein, KpsM (Slr0977), has a pleiotropic effect on Synechocystis sp. strain PCC 6803 physiology, with a strong impact on the export of EPS and carbon fluxes. The kpsM mutant exhibits a significant reduction of released polysaccharides and a smaller decrease of capsular polysaccharides, but it accumulates more polyhydroxybutyrate (PHB) than the wild type. In addition, this strain shows a light/cell density-dependent clumping phenotype and exhibits an altered protein secretion capacity. Furthermore, the most important structural component of pili, the protein PilA, was found to have a modified glycosylation pattern in the mutant compared to the wild type. Proteomic and transcriptomic analyses revealed significant changes in the mechanisms of energy production and conversion, namely, photosynthesis, oxidative phosphorylation, and carbon metabolism, in response to the inactivation of slr0977 Overall, this work shows for the first time that cells with impaired EPS secretion undergo transcriptomic and proteomic adjustments, highlighting the importance of EPS as a major carbon sink in cyanobacteria. The accumulation of PHB in cells of the mutant, without affecting significantly its fitness/growth rate, points to its possible use as a chassis for the production of compounds of interest.IMPORTANCE Most cyanobacteria produce extracellular polymeric substances (EPS) that fulfill different biological roles depending on the strain/environmental conditions. The interest in the cyanobacterial EPS synthesis/export pathways has been increasing, not only to optimize EPS production but also to efficiently redirect carbon flux toward the production of other compounds, allowing the implementation of industrial systems based on cyanobacterial cell factories. Here, we show that a Synechocystis kpsM (slr0977) mutant secretes less EPS than the wild type, accumulating more carbon intracellularly, as polyhydroxybutyrate. Further characterization showed a light/cell density-dependent clumping phenotype, altered protein secretion, and modified glycosylation of PilA. The proteome and transcriptome of the mutant revealed significant changes, namely, in photosynthesis and carbon metabolism. Altogether, this work provides a comprehensive overview of the impact of kpsM disruption on Synechocystis physiology, highlighting the importance of EPS as a carbon sink and showing how cells adapt when their secretion is impaired, and the redirection of the carbon fluxes.

Label-Free Quantitative Proteomics and Substrate-Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in Ex Vivo Human Skin and a Human Living Skin Equivalent Model.

We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases, and nucleases in six human skin explants and a three-dimensional living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein. Alcohol dehydrogenase 1C, the most abundant phase I XME in human skin, and glutathione S-transferase pi 1, the most abundant phase II XME in human skin, were present in similar abundance in LabSkin. Several esterases were quantified and esterase activity was confirmed in LabSkin using substrate-based mass spectrometry imaging. No cytochrome P450 (P450) activity was observed for the substrates tested, in agreement with the proteomics data, where the cognate P450s were absent in both human skin and LabSkin. Label-free protein quantification allowed insights into other related processes such as redox homeostasis and proteolysis. For example, the most abundant antioxidant enzymes were thioredoxin and peroxiredoxin-1. This systematic determination of functional equivalence between human skin and LabSkin is a key step toward the construction of a representative human in vitro skin model, which can be used as an alternative to current animal-based tests for chemical safety and for predicting dosage of topically administered drugs. SIGNIFICANCE STATEMENT: The use of label-free quantitative mass spectrometry to elucidate the abundance of xenobiotic metabolizing enzymes, transporters, redox enzymes, proteases, and nucleases in human skin enhance our understanding of the skin physiology and biotransformation of topical drugs and cosmetics. This will help to develop mathematical models to predict drug metabolism in human skin and to develop more robust in vitro engineered human skin tissue as alternatives to animal testing.

Adapting the algal microbiome for growth on domestic landfill leachate.

We aimed to improve algal growth rate on leachate by optimising the algal microbiome. An algal-bacterial consortium was enriched from landfill leachate and subjected to 24 months of adaptive laboratory evolution, increasing the growth rate of the dominant algal strain, Chlorella vulgaris, almost three-fold to 0.2 d-1. A dramatic reduction in nitrate production suggested a shift in biological utilisation of ammoniacal-N, supported by molecular 16S rRNA taxonomic analyses, where Nitrosomonas numbers were not detected in the adapted consortium. A PICRUSt approach predicted metagenomic functional content and revealed a high number of sequences belonging to bioremediation pathways, including degradation of aromatic compounds, benzoate and naphthalene, as well as pathways known to be involved in algal-bacterial symbiosis. This study enhances our understanding of beneficial mechanisms in algal-bacterial associations in complex effluents, and ultimately enables the bottom-up design of optimised algal microbiomes for exploitation within industry.

The Influence of Biofilms on Carbapenem Susceptibility and Patient Outcome in Device Associated K. pneumoniae Infections: Insights Into Phenotype vs Genome-Wide Analysis and Correlation.

Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenem-resistant K. pneumoniae are on the rise globally. The biofilm forming ability of K. pneumoniae further complicates patient management. There is still a knowledge gap on the association of biofilm formation with patient outcome and carbapenem susceptibility, which is investigated in present study. K. pneumoniae isolates from patients admitted in critical care units with catheters and ventilators were included. K. pneumoniae (n = 72) were subjected to 96-well plate biofilm formation assay followed by MBEC assay for subset of strong biofilm formers. Whole genome sequencing and a core genome phylogenetic analysis in comparison with global isolates were performed. Phenotypic analyses showed a positive correlation between biofilm formation and carbapenem resistance. Planktonic cells observed to be susceptible in vitro exhibited higher MICs in biofilm structure, hence MICs cannot be extrapolated for treatment. The biofilm forming ability had a significant association with morbidity/mortality. Infections by stronger biofilm forming pathogens significantly (p < 0.05) resulted in fewer "average days alive" for the patient (3.33 days) in comparison to those negative for biofilms (11.33 days). Phylogenetic analysis including global isolates revealed clear association of sequence types with genes for biofilm formation and carbapenem resistance. Known hypervirulent clone-ST23 with wcaG, magA, rmpA, rmpA2, and wzc with lack of mutation for hyper-capsulation might be poor biofilm formers. ST15, ST16, ST307, and ST258 (reported global high-risk clones) were wcaJ negative indicating the high potential of biofilm forming capacity. Genes wabG and treC for CPS, bcsA and pgaC for adhesins, luxS for quorum sensing were common in all clades in addition to genes for aerobactin (iutA), allantoin (allS), type I and III fimbriae (fimA, fimH, and mrkD) and pili (pilQ and ecpA). This study is the first of its kind to compare genetic features of antimicrobial resistance with a spectrum covering most of the genetic factors for K. pneumoniae biofilm. These results highlight the importance of biofilm screening to effectively manage nosocomial infections by K. pneumoniae. Further, data obtained on epidemiology and associations of biofilm and resistance genetic factors will serve to enhance our understanding on biofilm mechanisms in K. pneumoniae.

Corneal Infection Models: Tools to Investigate the Role of Biofilms in Bacterial Keratitis.

Bacterial keratitis is a corneal infection which may cause visual impairment or even loss of the infected eye. It remains a major cause of blindness in the developing world. Staphylococcus aureus and Pseudomonas aeruginosa are common causative agents and these bacterial species are known to colonise the corneal surface as biofilm populations. Biofilms are complex bacterial communities encased in an extracellular polymeric matrix and are notoriously difficult to eradicate once established. Biofilm bacteria exhibit different phenotypic characteristics from their planktonic counterparts, including an increased resistance to antibiotics and the host immune response. Therefore, understanding the role of biofilms will be essential in the development of new ophthalmic antimicrobials. A brief overview of biofilm-specific resistance mechanisms is provided, but this is a highly multifactorial and rapidly expanding field that warrants further research. Progression in this field is dependent on the development of suitable biofilm models that acknowledge the complexity of the ocular environment. Abiotic models of biofilm formation (where biofilms are studied on non-living surfaces) currently dominate the literature, but co-culture infection models are beginning to emerge. In vitro, ex vivo and in vivo corneal infection models have now been reported which use a variety of different experimental techniques and animal models. In this review, we will discuss existing corneal infection models and their application in the study of biofilms and host-pathogen interactions at the corneal surface.

Influence of phosphate dosing on biofilms development on lead in chlorinated drinking water bioreactors.

Phosphate dosing is used by water utilities to prevent plumbosolvency in water supply networks. However, there is a lack of knowledge regarding biofilm formation on lead and plastic materials when phosphate concentrations are modified in drinking water systems. In this study, biofilms were grown over lead coupons and PVC tubes in bioreactors supplied with local drinking water treated to provide different phosphate doses (below 1, 1 and 2 mg/L) over a period of 28 days. A range of commercial iron pellets (GEH104 and WARP) were tested aiming to maintain phosphate levels below the average 1 mg/L found in drinking water. Changes in biofilm community structure in response to three different phosphate treatments were characterised by Illumina sequencing of the 16S rRNA gene for bacteria and the ITS2 gene for fungi. Scanning electron microscopy was used to visualise physical differences in biofilm development in two types of materials, lead and PVC. The experimental results from the kinetics of phosphate absorption showed that the GEH104 pellets were the best option to, in the long term, reduce phosphate levels while preventing undesirable turbidity increases in drinking water. Phosphate-enrichment promoted a reduction of bacterial diversity but increased that of fungi in biofilms. Overall, higher phosphate levels selected for microorganisms with enhanced capabilities related to phosphorus metabolism and heavy metal resistance. This research brings new insights regarding the influence of different phosphate concentrations on mixed-species biofilms formation and drinking water quality, which are relevant to inform best management practices in drinking water treatment.

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis.

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

Metabolic Insights Into Infochemicals Induced Colony Formation and Flocculation in Scenedesmus subspicatus Unraveled by Quantitative Proteomics.

Microalgae can respond to natural cues from crustacean grazers, such as Daphnia, by forming colonies and aggregations called flocs. Combining microalgal biology, physiological ecology, and quantitative proteomics, we identified how infochemicals from Daphnia trigger physiological and cellular level changes in the microalga Scenedesmus subspicatus, underpinning colony formation and flocculation. We discovered that flocculation occurs at an energy-demanding 'alarm' phase, with an important role proposed in cysteine synthesis. Flocculation appeared to be initially stimulated by the production of an extracellular matrix where polysaccharides and fatty acids were present, and later sustained at an 'acclimation' stage through mitogen-activated protein kinase (MAPK) signaling cascades. Colony formation required investment into fatty acid metabolism, likely linked to separation of membranes during cell division. Higher energy demands were required at the alarm phase, which subsequently decreased at the acclimation stage, thus suggesting a trade-off between colony formation and flocculation. From an ecological and evolutionary perspective, our findings represent an improved understanding of the effect of infochemicals on microalgae-grazers interactions, and how they can therefore potentially impact on the structure of aquatic communities. Moreover, the mechanisms revealed are of interest in algal biotechnology, for exploitation in low-cost, sustainable microalgal biomass harvesting.

Characterization of Ocular Clinical Isolates of Pseudomonas aeruginosa from Non-Contact Lens Related Keratitis Patients from South India.

P. aeruginosa is the most common Gram-negative organism causing bacterial keratitis. Pseudomonas utilizes various virulence mechanisms to adhere and colonize in the host tissue. In the present study, we examined virulence factors associated with thirty-four clinical P. aeruginosa isolates collected from keratitis patients seeking care at L V Prasad Eye Institute, Hyderabad. The virulence-associated genes in all the isolates were genotyped and characteristics such as antibiotic susceptibility, biofilm formation, swarming motility, pyoverdine production and cell cytotoxicity were analyzed. All the isolates showed the presence of genes related to biofilm formation, alkaline proteases and elastases; however, there was a difference in the presence of genes related to the type III secretion system (T3SS). A higher prevalence of exoU+ genotype was noted in the drug-resistant isolates. All the isolates were capable of forming biofilms and more than 70% of the isolates showed good swarming motility. Pyoverdine production was not associated with the T3SS genotype. In the cytotoxicity assay, the presence of exoS, exoU or both resulted in higher cytotoxicity compared to the absence of both the genes. Overall, our results suggest that the T3SS profile is a good indicator of P. aeruginosa virulence characteristics and the isolates lacking the effector genes may have evolved alternate mechanisms of colonization in the host.

Publisher Correction: Deciphering the unique cellulose degradation mechanism of the ruminal bacterium Fibrobacter succinogenes S85.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Deciphering the unique cellulose degradation mechanism of the ruminal bacterium Fibrobacter succinogenes S85.

Fibrobacter succinogenes S85, isolated from the rumen of herbivores, is capable of robust lignocellulose degradation. However, the mechanism by which it achieves this is not fully elucidated. In this study, we have undertaken the most comprehensive quantitative proteomic analysis, to date, of the changes in the cell envelope protein profile of F. succinogenes S85 in response to growth on cellulose. Our results indicate that the cell envelope proteome undergoes extensive rearrangements to accommodate the cellulolytic degradation machinery, as well as associated proteins involved in adhesion to cellulose and transport and metabolism of cellulolytic products. Molecular features of the lignocellulolytic enzymes suggest that the Type IX secretion system is involved in the translocation of these enzymes to the cell envelope. Finally, we demonstrate, for the first time, that cyclic-di-GMP may play a role in mediating catabolite repression, thereby facilitating the expression of proteins involved in the adhesion to lignocellulose and subsequent lignocellulose degradation and utilisation. Understanding the fundamental aspects of lignocellulose degradation in F. succinogenes will aid the development of advanced lignocellulosic biofuels.

The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803.

Cyanobacterial alternative sigma factors are crucial players in environmental adaptation processes, which may involve bacterial responses related to maintenance of cell envelope and control of secretion pathways. Here, we show that the Group 3 alternative sigma factor F (SigF) plays a pleiotropic role in Synechocystis sp. PCC 6803 physiology, with a major impact on growth and secretion mechanisms, such as the production of extracellular polysaccharides, vesiculation and protein secretion. Although ΔsigF growth was significantly impaired, the production of released polysaccharides (RPS) increased threefold to fourfold compared with the wild-type. ΔsigF exhibits also impairment in formation of outer-membrane vesicles (OMVs) and pili, as well as several other cell envelope alterations. Similarly, the exoproteome composition of ΔsigF differs from the wild-type both in amount and type of proteins identified. Quantitative proteomics (iTRAQ) and an in silico analysis of SigF binding motifs revealed possible targets/pathways under SigF control. Besides changes in protein levels involved in secretion mechanisms, our results indicated that photosynthesis, central carbon metabolism and protein folding/degradation mechanisms are altered in ΔsigF. Overall, this work provided new evidences about the role of SigF on Synechocystis physiology and associates this regulatory element with classical and non-classical secretion pathways.

Enumeration of sulphate-reducing bacteria for assessing potential for hydrogen sulphide production in urban drainage systems.

Urban drainage structures have increasing demands which can lead to increasing hydrogen sulphide related problems forming in places where they have not previously been prevalent. This puts pressure on the methods currently used to monitor and diagnose these problems and more sophisticated methods may be needed for identifying the origin of the problems. Molecular microbiological techniques, such as quantitative polymerase chain reaction, offer a potential alternative for identifying and quantifying bacteria likely to be causing the production of hydrogen sulphide, information that, when combined with an appropriate sampling programme, can then be used to identify the potentially most effective remediation technique. The application of these methods in urban drainage systems is, however, not always simple, but good results can be achieved. In this study bacteria producing hydrogen sulphide were quantified in three small combined sewer overflow storage tanks. Bacterial counts were compared between wastewater, biofilms and sediments. Similar numbers were found in the wastewater and biofilms, with the numbers in the sediments being lower. If remediation methods for hydrogen sulphide are deemed necessary in the tanks, methods that target both the wastewater and the biofilms should therefore be considered.

Physicochemical analysis of initial adhesion and biofilm formation of Methanosarcina barkeri on polymer support material.

The retention of selective biofilms of Methanosarcina species within anaerobic digesters could reduce start-up times and enhance the efficiency of the process in treating high-strength domestic sewage. The objective of the study was to examine the effect of the surface characteristics of six common polymer support materials on the initial adhesion of the model methanogen, Methanosarcina barkeri, and to assess the potential of these support materials as selective biofilm carriers. Results from both the initial adhesion tests and extended DLVO (xDLVO) model correlated with each other, with PVC (12% surface coverage/mm(2)), PTFE (6% surface coverage/mm(2)), and PP (6% surface coverage/mm(2)), shown to be the better performing support materials for initial adhesion, as well as subsequent biofilm formation by M. barkeri after 72h. Experimental results of these three support materials showed that the type of material strongly influenced the extent of adhesion from M. barkeri (p<0.0001), and the xDLVO model was able to explain the results in these environmental conditions. Therefore, DLVO physicochemical forces were found to be influential on the initial adhesion of M. barkeri. Scanning electron microscopy suggested that production of extracellular polymeric substances (EPS) from M. barkeri could facilitate further biofilm development. This study highlights the potential of using the xDLVO model to rapidly identify suitable materials for the selective adhesion of M. barkeri, which could be beneficial in both the start-up and long-term phases of anaerobic digestion.