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With the advent of the SARS-CoV-2 pandemic, Wastewater-Based Epidemiology (WBE) has been applied to track community infection in cities worldwide and has proven succesful as an early warning system for identification of hotspots and changingprevalence of infections (both symptomatic and asymptomatic) at a city or sub-city level. Wastewater is only one of environmental compartments that requires consideration. In this manuscript, we have critically evaluated the knowledge-base and preparedness for building early warning systems in a rapidly urbanising world, with particular attention to Africa, which experiences rapid population growth and urbanisation. We have proposed a Digital Urban Environment Fingerprinting Platform (DUEF) – a new approach in hazard forecasting and early-warning systems for global health risks and an extension to the existing concept of smart cities. The urban environment (especially wastewater) contains a complex mixture of substances including toxic chemicals, infectious biological agents and human excretion products. DUEF assumes that these specific endo- and exogenous residues, anonymously pooled by communities’ wastewater, are indicative of community-wide exposure and the resulting effects. DUEF postulates that the measurement of the substances continuously and anonymously pooled by the receiving environment (sewage, surface water, soils and air), can provide near real-time dynamic information about the quantity and type of physical, biological or chemical stressors to which the surveyed systems are exposed, and can create a risk profile on the potential effects of these exposures. Successful development and utilisation of a DUEF globally requires a tiered approach including: Stage I: network building, capacity building, stakeholder engagement as well as a conceptual model, followed by Stage II: DUEF development, Stage III: implementation, and Stage IV: management and utilization. We have identified four key pillars required for the establishment of a DUEF framework: (1) Environmental fingerprints, (2) Socioeconomic fingerprints, (3) Statistics and modelling and (4) Information systems. This manuscript critically evaluates the current knowledge base within each pillar and provides recommendations for further developments with an aim of laying grounds for successful development of global DUEF platforms.

Riverine fungi have the capacity for both pathogenicity, pertinent for countries with elevated immunosuppressed individuals, and bioremediation potential. The purpose was (i) to screen for the presence of clinically relevant riverine fungi and associations with anthropogenic influence, and (ii) the acclimatisation of environmental communities toward potential bioremediation application. Communities were harvested from polluted rivers in Stellenbosch, South Africa, and mycobiomes characterised by high-throughput amplicon sequencing. The remainder of the biomass was inoculated into continuous bioreactors with filtered river water or sterile minimal medium. Seven weeks later, the mycobiomes were re-sequenced. At least nine clinically relevant species were detected, including agents of mycoses belonging to the genus Candida . The occurrence of genera that harbour opportunisticstrains was significantly higher ( P  = 0.04) at more polluted sites. Moreover, positive correlations occured between some genera and pollution indices, demonstrating the potential of fungi for addition to water quality indicators. Despite biomass increase, almost all pathogens were undetectable after seven weeks, demonstrating less resilience in conditions mimicking rivers. Thus, when screening riverine biomes for bioremediation potential, ambient reactors select against human pathogens. This indicates a transient introduction of allochthonous opportunistic species into rivers due to insufficient sanitation, and the potential of bioremediation strategies that selects for environmental rather than pathogenic traits., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

There has been a significant increase in antimicrobial agents (AAs) usage, globally - however the relative consumption is unevenly distributed between nations. Inappropriate use of antibiotics can harbour inherent antimicrobial resistance (AMR); therefore, it is important to understand and monitor community-wide prescribing and consumption behaviours throughout different communities around the world. Wastewater-Based Epidemiology (WBE) is a novel tool enabling low cost and large scale studies focussed on AA usage patterns. The back-calculation of community antimicrobial intake was performed from quantities measured in municipal wastewater and informal settlement discharge in the city of Stellenbosch, utilising WBE. Seventeen antimicrobials, and their human metabolites, were evaluated, in concordance with prescription records corresponding to the catchment region. The proportional excretion, biological/chemical stability, and method recovery of each analyte were all crucial factors in the efficacy of the calculation. Mass per day measurements were normalised to the catchment area via population estimates. Municipal wastewater treatment plant population estimates were used to normalise the wastewater samples and prescription data (mg/day/1000 inhabitants). Population estimates for the informal settlements were less accurate due to a lack of reliable sources that were relevant to the sampling time period. Both mass loads and normalised loads suggested higher than average usage throughout the settlements, relative to municipal wastewater. This was seen most prominently in emtricitabine and lamivudine; but also, sulfamethoxazole, trimethoprim, sulfadiazine, clindamycin, ciprofloxacin, ofloxacin, and doxycycline. Urban water fingerprinting (UWF) data triangulation with prescription datasets showed good correlations for several antimicrobial agents (AAs) (e.g., clindamycin, clarithromycin, ofloxacin, and doxycycline). It also revealed discrepancies in usage for some compounds (e.g., tetracycline and sulfapyridine). This might be linked with a lack of pharma compliance in prescription datasets; erroneous association of prescription boundaries with the sewerage catchment; and/or uncertainties within the sewerage catchment (e.g., population estimations). The UWF tool provided a comprehensive overview of multiclass AAs usage, both prescription and over-the counter. For example, tetracycline was not reported in available prescription statistics, but was detected at an average of 18.4 mg/day/1000inh; and no antiviral prescriptions were obtained, but emtricitabine and lamivudine were quantified at 2415.4 and 144.4 mg/day/1000inh, respectively. A lack of clarity regarding prescriptions and a lack of inclusion of several critical (often over-the-counter) medications in public health databases makes WBE a useful and comprehensive epidemiology tool for tracking pharma usage within a catchment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

River water is an essential human resource that may be contaminated with hazardous microorganisms. However, the risk of yeast infection through river water exposure is unclear because it is highly dependant on individual susceptibility and has therefore not been well-studied, to date. To evaluate this undefined risk, we analysed the fungal communities in less polluted (LP) and highly polluted (HP) river water, as determined using principal coordinate analysis of pollution indicators. We enumerated culturable yeasts using a thermally selective isolation procedure (37 °C) and thus promoted the growth of potentially opportunistic species. Yeast species identified as clinically relevant were then tested for antifungal resistance. In addition, we propose a quantitative microbial risk assessment (QMRA) framework to quantitatively assess the potential risk of yeast infection. Our results indicated that pollution levels significantly altered fungal communities (p = 0.007) and that genera representing opportunistic and pathogenic members were significantly more abundant in HP waters (p = 0.038). Additionally, the yeast species Candida glabrata and Clavispora lusitaniae positively correlated with other pollution indicators, demonstrating the species' indicator potential. Our QMRA results further indicate that higher risk of infection is associated with increased water pollution levels (considering both physicochemical and bacterial indicators). Furthermore, yeast species with higher pathogenic potential present an increased risk of infection despite lower observed concentrations in the river water. Interestingly, the bloom of Meyerozyma guilliermondii during the wet season suggests that other environmental factors, such as dissolved oxygen levels and water turbulence, might affect growth characteristics of yeasts in river water, which consequently affects the distribution of annual infection risks. The presence of antifungal resistant yeasts, observed in this study, could further contribute to variation in risk distribution. Research on the ecophysiology of yeasts in these environments is therefore necessary to ameliorate the uncertainty and sensitivity of the proposed QMRA model. In addition to the vital knowledge on opportunistic and pathogenic yeast occurrence in river water and their observed association with pollution, this study provides valuable methods and insights to initiate future QMRAs of yeast infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alf Botha reports financial support was provided by Water Research Commission. Joao Brandao reports financial support was provided by University of Aveiro Centre for Environmental and Marine Studies. Joao Brandao reports financial support was provided by University of Tras-os-Montes and Alto Douro Centre for the Research and Technology of Agro-Environmental and Biological Sciences. Heidi Christa Steffen reports financial support was provided by National Research Foundation., (Copyright © 2023. Published by Elsevier Ltd.)

Chemical pollution (including chemicals of emerging concern - CECs) continues to gain increasing attention as a global threat to human health and the environment, with numerous reports on the adverse and sometimes devastating effects upon ecosystems the presence of these chemicals can have. Whilst many studies have investigated presence of CECs in aquatic environments, these studies have been often focused on higher income countries, leaving significant knowledge gaps for many low-middle income countries. This study proposes a new integrated powerless, in-situ multi-mode extraction (iMME) sampler for the analysis of chemicals (105 CECs) and biological (5 genes) markers in water in contrasting settings: an urbanized Avon River in the UK and remote Olifants River in Kruger National Park in South Africa. The overarching goal was to develop a sampling device that maintains integrity of a diverse range of analytes via analyte immobilization using polymeric and glass fibre materials, without access to power supply or cold chain (continuous chilled storage) for sample transportation. Chemical analysis was achieved using ultra-performance liquid chromatography coupled with tandem mass spectrometry. Several mobile CECs showed low stability in river water, at room temperature and typical 24 h sampling/transport time. It is therefore recommended that, in the absence of cooling, environmental water samples are spiked with internal standards on site, immediately after collection and analyte immobilization option is considered, in order to allow fully quantitative analysis. iMME has proven effective in immobilization, concentration and increased stability of CECs at room temperature (and at least 7 days storage) allowing for sample collection at remote locations. The results from the River Avon and Olifants River sampling indicate that the pristine environment of Olifants catchment is largely unaffected by CECs common in the urbanized River Avon in the UK with a few exceptions: lifestyle chemicals (e.g., caffeine, nicotine and their metabolites), paracetamol and UV filters due to tourism and carbamazepine due to its persistent nature. iMME equipped with an additional gene extraction capability provides an exciting new opportunity of comprehensive biochemical profiling of aqueous samples with one powerless in-situ device. Further work is required to provide full integration of the device and comprehensive assessment of performance in both chemical and biological targets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic capacity is limited in defined communities, posing a challenge in tracking and tracing new infections. Monitoring student residences, which are considered infection hotspots, with targeted wastewater surveillance is crucial. This study evaluated the efficacy of SARS-CoV-2 targeted wastewater surveillance for outbreak mitigation at Stellenbosch University's student residences in South Africa. Using torpedo-style passive sampling devices, wastewater samples were collected biweekly from manholes at twelve Stellenbosch University Tygerberg (SUT) campus and Stellenbosch University-Main (SUM) campus student residences. The surveillance led to an early warning detection of SARS-CoV-2 presence on campus, followed by an informed management strategy leading to restriction of student activities on campus and a delay in the onset of the third wave that was experienced throughout the country. Moreover, the study highlighted the extent of possible infections at defined locations even when a low number of confirmed coronavirus disease 2019 (COVID-19) cases were reported. The study also tracked the surge of the Delta and Omicron variants in the student residences using the Thermo Fisher TaqMan® RT-qPCR genotyping assay., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

With increasing emergence of antimicrobial resistant bacteria (ARB) and the risk this poses to public health, there are growing concerns regarding water pollution contributing to the spread of antimicrobial resistance (AMR) through inadequate amenities and the rapid rate of urbanization. In this study, the impact of different anthropogenic factors on the prevalence of AMR in the urban water cycle in Stellenbosch, South Africa (SA) was examined. Carbapenem, colistin, gentamicin and sulfamethoxazole resistant Gram-negative bacteria were recovered by selectively culturing aqueous, biofilm and sediment samples from sites impacted to varying degrees by informal settlements, residential, industrial, and agricultural activities, as well as a municipal wastewater treatment works (WWTW). A metagenomic approach determined community profiles and dominant AMR genes at various sites, while carbapenem resistant colonies were characterized using whole genome sequencing (WGS). Isolates recovered from agricultural sites exhibited relatively high levels of resistance to carbapenems and colistin, whereas sites impacted by domestic run-off had a higher prevalence of resistance to gentamicin and sulfamethoxazole, corresponding to usage data in SA. Similar microbial taxa were identified in raw sewage, sites downstream of informal settlements, and industrial areas that have limited waste removal infrastructure while WWTW were seen to reduce the prevalence of ARB in treated wastewater when operating efficiently. The results indicate the multiple complex drivers underpinning environmental dissemination of AMR and suggest that WWTW assist in removing AMR from the environment, reinforcing the necessity of adequate waste removal infrastructure and antibiotic stewardship measures to mitigate AMR transmission. IMPORTANCE The results from this study are of importance as they fill a gap in the data available on environmental AMR in South Africa to date. This study was done in parallel with co-investigators focusing on the prevalence of various antimicrobials at the same sites selected in our study, verifying that the sites that are influenced by informal settlements and WWTW influent had higher concentrations of antimicrobials and antimicrobial metabolites. The various locations of the sample sites selected, the frequency of the samples collected over a year, and the different types of samples collected at each site all contribute to informing how AMR in the environment might be affected by anthropogenic activity.

Antimicrobial agent (AA) usage, excretion, and persistence are all important factors in association with the occurrence and dissemination of antimicrobial resistance. Urban water profiling was utilised in the Eerste River catchment (South Africa) to establish AA usage in a region where comprehensive prescription records were not readily available and where portions of the community did not have sufficient access to sanitation. This technique enabled the environmental exposure to be quantified throughout the catchment area and the identification of contamination hotspots. Monitoring occurred over a 11-month period. 812 samples were processed using UPLC-MS/MS for the quantitation of 56 antimicrobials and 26 of their metabolites. Spatiotemporal trends were established, with consideration to community behaviour, seasonal changes, and physiochemical properties of the analytes. The Eerste River samples collected upstream from the town of Stellenbosch had the lowest AA loads (<4 g/day), unafflicted by industrial presence and with only small impact from farming activities. This was followed by sites downstream from a wastewater treatment plant (serving 178 K people). The measurement of low AA loads (influent: 500-800 g/day and effluent 50-100 g/day), indicates a high efficiency of wastewater treatment, allowing for an effective reduction of AA and a lower environmental burden. This is compared to river sites that receive untreated waste from communities in informal settlements (6-12 K people) that are not connected to the sewer infrastructure (with AA levels accounting for 100-600 g/day). Temporal trends exhibited reduced daily loads during the summer to early autumn (Nov-May). This is likely due to seasonal patterns in community health and/or notable changes in rainfall and temperatures at the sampling locations throughout the year. However, weather patterns are also important to consider - particularly for the river sites. South Africa has notable rainfall and temperature seasonality. Antiretrovirals (ARV), emtricitabine and lamivudine, were the most prevalent drugs throughout the monitoring campaign, followed by tuberculosis drugs and sulfonamides. ARVs were, however, effectively reduced via wastewater treatment processes (>97%). This was also the case for beta-lactams, nitrofurantoin, and trimethoprim. The treatment efficacy for other drugs was more variable, that did not appear to have temporal significance., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

With the advent of the SARS-CoV-2 pandemic, Wastewater-Based Epidemiology (WBE) has been applied to track community infection in cities worldwide and has proven succesful as an early warning system for identification of hotspots and changingprevalence of infections (both symptomatic and asymptomatic) at a city or sub-city level. Wastewater is only one of environmental compartments that requires consideration. In this manuscript, we have critically evaluated the knowledge-base and preparedness for building early warning systems in a rapidly urbanising world, with particular attention to Africa, which experiences rapid population growth and urbanisation. We have proposed a Digital Urban Environment Fingerprinting Platform (DUEF) - a new approach in hazard forecasting and early-warning systems for global health risks and an extension to the existing concept of smart cities. The urban environment (especially wastewater) contains a complex mixture of substances including toxic chemicals, infectious biological agents and human excretion products. DUEF assumes that these specific endo- and exogenous residues, anonymously pooled by communities' wastewater, are indicative of community-wide exposure and the resulting effects. DUEF postulates that the measurement of the substances continuously and anonymously pooled by the receiving environment (sewage, surface water, soils and air), can provide near real-time dynamic information about the quantity and type of physical, biological or chemical stressors to which the surveyed systems are exposed, and can create a risk profile on the potential effects of these exposures. Successful development and utilisation of a DUEF globally requires a tiered approach including: Stage I: network building, capacity building, stakeholder engagement as well as a conceptual model, followed by Stage II: DUEF development, Stage III: implementation, and Stage IV: management and utilization. We have identified four key pillars required for the establishment of a DUEF framework: (1) Environmental fingerprints, (2) Socioeconomic fingerprints, (3) Statistics and modelling and (4) Information systems. This manuscript critically evaluates the current knowledge base within each pillar and provides recommendations for further developments with an aim of laying grounds for successful development of global DUEF platforms., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The oxidation of ammonia by autotrophic bacteria is a central part of the nitrogen cycle and a fundamental aspect of biological nutrient removal (BNR) during wastewater treatment. Autotrophic ammonia oxidation produces protons and results in net-CO 2 production due to the neutralizing effect of bicarbonate alkalinity. Attention must be paid to the propensity for this produced CO 2 to be transferred to the atmosphere where it can act as a greenhouse gas (GHG). In the context of BNR systems, bicarbonate-derived CO 2 emissions should be considered distinct from the biogenic CO 2 that arises from cellular respiration, though this distinction is not made in current GHG accounting practices. The aim of this study was to evaluate the performance of two experimental systems operated under autotrophic mode and buffered with bicarbonate, to investigate the relationship between ammonia removal and gaseous CO 2 emissions. The first system consisted of continuously aerated lab-scale batch reactors, which were effective in demonstrating the important link between ammonia oxidizer activity, pH, and gaseous CO 2 production. Depletion of the buffer system always led to a rapid decline in system pH and cessation of CO 2 emissions when the pH fell below 7.0. The second system was a tubular continuous-flow biofilm reactor which permitted comparison of ammonia removal and CO 2 emission rates. A linear relationship between ammonia removal and CO 2 emissions was demonstrated and the quantified CO 2 production was relatively close to that which was predicted based on the stoichiometry of nitrification, with this CO 2 being detected in the gas phase. It was apparent that this system offered minimal resistance to the mass transfer of CO 2 from the liquid to gas, which is an important factor that determines how much of the bicarbonate-derived CO 2 may contribute to greenhouse gas emissions in engineered systems such as those used for BNR., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Ecological restorations of reservoirs are implemented worldwide; however, minimal successes are reported and understood for warmer African lakes like Swakoppoort Dam, Namibia. The objectives of the study were (a) to establish the effectiveness of the two control measures in reducing cyanobacteria growths in comparison with untreated control areas and (b) to compare the results generated before and after control measures with the reference Von Bach Dam. During Phoslock ® treatment, the average cyanobacteria cells and total phosphate (TP) were 90,521 cells/ml and 0.3 mg/L in the treated area and 55,338 cells/ml and 0.1 mg/L in the control area. During Solar Powered Circulation (SPC) treatment, the average cyanobacteria cells were on average 906,420 cells/ml in the treated areas and 121,891 cells/ml in the control area. The TP on average was 0.3 mg/L during SPC treatment, while during the combined treatment, the average cyanobacteria cells, TP, and total nitrogen (TN) were 18,387,226 cells/ml, 0.27 mg/L, and 2.41 mg/L before and 22,836,511 cells/ml, 0.42 mg/L, and 1.50 mg/L after treatment. This was higher compared to the reference site. PCA triplot indicates no grouping pattern, and the repeated-measures mixed model analyses indicate that treatment had no significant effect on cyanobacteria cells. It was evident that the two control measures were ineffective in reducing cyanobacterial cells. PRACTITIONER POINTS: Key findings of the article: Two phytoplankton control measures were found ineffective to reduce the cyanobacterial cell numbers. High cell numbers of cyanobacteria were recorded at the treatment areas compared to untreated control areas during both treatments. The combined effect of the two control measures was ineffective as more cyanobacterial cells were recorded during the treatment. During control measure treatment, the Swakoppoort Dam was hypertrophic, which could be due to a malfunctioned WWTP upstream. The inefficiency of the control measures could be due to small treatment area, higher nutrients, or treatment period. The implications of the results to water/wastewater practice: The selection of appropriate mitigation measures considering treatment area for dams with high nutrient situated in warmer arid environments. There is a need to understand the trophic relationships, climatic conditions, and the sources of the internal and external nutrients to manage water quality. Focus on point and non-point sources of nutrients as the root causes of the degradation of Swakoppoort Dam water., (© 2021 Water Environment Federation.)

As the effects of climate change become increasingly evident, the need for effective CO2 management is clear. Microalgae are well-suited for CO2 sequestration, given their ability to rapidly uptake and fix CO2. They also readily assimilate inorganic nutrients and produce a biomass with inherent commercial value, leading to a paradigm in which CO2-sequestration, enhanced wastewater treatment, and biomass generation could be effectively combined. Natural non-axenic phototrophic cultures comprising both autotrophic and heterotrophic fractions are particularly attractive in this endeavour, given their increased robustness and innate O2-CO2 exchange. In this study, the interplay between CO2-consuming autotrophy and CO2-producing heterotrophy in a non-axenic phototrophic biofilm was examined. When the biofilm was cultivated under autotrophic conditions (i.e. no organic carbon), it grew autotrophically and exhibited CO2 uptake. After amending its growth medium with organic carbon (0.25 g/L glucose and 0.28 g/L sodium acetate), the biofilm rapidly toggled from net-autotrophic to net-heterotrophic growth, reaching a CO2 production rate of 60 μmol/h after 31 hours. When the organic carbon sources were provided at a lower concentration (0.125 g/L glucose and 0.14 g/L sodium acetate), the biofilm exhibited distinct, longitudinally discrete regions of heterotrophic and autotrophic metabolism in the proximal and distal halves of the biofilm respectively, within 4 hours of carbon amendment. Interestingly, this upstream and downstream partitioning of heterotrophic and autotrophic metabolism appeared to be reversible, as the position of these regions began to flip once the direction of medium flow (and hence nutrient availability) was reversed. The insight generated here can inform new and important research questions and contribute to efforts aimed at scaling and industrializing algal growth systems, where the ability to understand, predict, and optimize biofilm growth and activity is critical., Competing Interests: The authors have declared that no competing interests exist.

Recent studies have shown that the detection of SARS-CoV-2 genetic material in wastewater may provide the basis for a surveillance system to track the environmental dissemination of this virus in communities. An effective wastewater-based epidemiology (WBE) system may prove critical in South Africa (SA), where health systems infrastructure, testing capacity, personal protective equipment and human resource capacity are constrained. In this proof-of-concept study, we investigated the potential of SARS-CoV-2 RNA surveillance in untreated wastewater as the basis for a system to monitor COVID-19 prevalence in the population, an early warning system for increased transmission, and a monitoring system to assess the effectiveness of interventions. The laboratory confirmed the presence (qualitative analysis) and determined the RNA copy number of SARS-CoV-2 viral RNA by reverse transcription polymerase chain reaction (quantitative) analysis from 24-hour composite samples collected on 18 June 2020 from five wastewater treatment plants in Western Cape Province, SA. The study has shown that a WBE system for monitoring the status and trends of COVID-19 mass infection in SA is viable, and its development and implementation may facilitate the rapid identification of hotspots for evidence-informed interventions.

Climate change brought about by anthropogenic CO 2 emissions has created a critical need for effective CO 2 management solutions. Microalgae are well suited to contribute to efforts aimed at addressing this challenge, given their ability to rapidly sequester CO 2 coupled with the commercial value of their biomass. Recently, microalgal biofilms have garnered significant attention over the more conventional suspended algal growth systems, since they allow for easier and cheaper biomass harvesting, among other key benefits. However, the path to cost-effectiveness and scaling up is hindered by a need for new tools and methodologies which can help evaluate, and in turn optimize, algal biofilm growth. Presented here is a novel system which facilitates the real-time in situ monitoring of algal biofilm CO 2 sequestration. Utilizing a CO 2 -permeable membrane and a tube-within-a-tube design, the CO 2 sequestration monitoring system (CSMS) was able to reliably detect slight changes in algal biofilm CO 2 uptake brought about by light-dark cycling, light intensity shifts, and varying amounts of phototrophic biomass. This work presents an approach to advance our understanding of carbon flux in algal biofilms, and a base for potentially useful innovations to optimize, and eventually realize, algae biofilm-based CO 2 sequestration.

With the risks associated with healthcare-associated infections and the rise of antibiotic resistant microorganisms, there is an important need to control the proliferation of these factors in hospitals, retirement homes and other institutions. This work explores the development and application of a novel class of sulfonamide-based quaternary ammonium antimicrobial coatings, anchored to commercially and clinically relevant material surfaces. Synthesized in high yields (60-97%), benzophenone-anchored antimicrobials were spray-coated and UV grafted onto plastic surfaces, while silane-anchored variants were adhered to select textiles via dip-coating. Surface modified samples were characterised by advancing contact angle, anionic dye staining, X-ray photoelectron spectroscopy and atomic force microscopy. After verifying coating quality through the above characterization methods, microbiological testing was performed on batch samples in conditions that simulate the natural inoculation of surfaces and objects (solid/air) and water containers (solid/liquid). Using the previously established Large Drop Inoculum (LDI) protocol at solid/air interfaces, all treated samples showed a full reduction (10 5 -10 7 CFU) of viable Arthrobacter sp., S. aureus , and E. coli after 3 h of contact time. Additional testing of the walls of plastic LDPE vials treated with a UV-cured sulfonamide antimicrobial at a solid/liquid interface using the newly developed Large Reservoir Inoculum (LRI) protocol under static conditions revealed a complete kill (>10 6 reduction) of Gram-positive Arthrobacter sp., and a partial kill (>10 4 reduction) of Gram-negative E. coli within 24-48 h of contact., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

A series of UV active benzophenone ([C 6 H 5 COC 6 H 4 -O-(CH 2 ) n -N + Me 2 R][X - ]; 4, R = C 12 H 25 , n = 3, X - = Br - ; 5a-c, R = C 18 H 37 , n = 3, X - = Cl - , Br - , I - ; 6a-c, R = C 18 H 37 , n = 4, X - = Cl - , Br - , I - ; 7a-c, R = C 18 H 37 , n = 6, X - = Cl - , Br - , I - ) terminated C 12 and C 18 quaternary ammonium salts (QACs) were prepared by thermal or microwave-driven Menshutkin protocols of the appropriate benzophenone alkyl halide (1a-c, 2a-c, 3a-c) with the corresponding dodecyl- or octadecyl N,N-dimethylamine. All new compounds were characterized by NMR spectroscopy, HRMS spectrometry, and, in one instance (4), by single-crystal X-ray crystallography. Representative C 12 and C 18 benzophenone QACs were formulated into 1% (w/v) water or water/ethanol-based aerosol spray coatings and then UV-cured onto plastic substrates (polypropylene, polyethylene, polystyrene, polyvinyl chloride, and polyether ether ketone) with exposure to low to moderate doses of UV (20-30 J cm -2 ). Confirmation as to the presence of the coatings was detected by advancing water contact angle measurements, which revealed a more hydrophilic surface after coating. Further confirmation was gained by X-ray photoelectron spectroscopy analysis, time of flight secondary ion mass spectrometry, and bromophenol blue staining, all of which showed the presence of the attached quaternary ammonium molecule. Analysis of surfaces treated with the C 18 benzophenone 5b by atomic force microscopy and surface profilometry revealed a coating thickness of ∼350 nm. The treated samples along with controls were then evaluated for their antimicrobial efficacy against Gram-positive (Arthrobacter sp., Listeria monocytogenes) and Gram-negative (Pseudomonas aeruginosa) bacteria at a solid/air interface using the large drop inoculum protocol; this technique gave no evidence for cell adhesion after a 3 h time frame. These antimicrobial materials show promise for their use as coatings on plastic biomedical devices with the aim of preventing biofilm formation and preventing the spread of hospital acquired infections.

This study evaluated the effect of combining antibiotics with ultrasound and microbubbles (USMB) toward the eradication of biofilms. Pseudomonas aeruginosa PAO1 biofilms were treated with the antibiotics gentamicin sulfate or streptomycin sulfate, or a combination of USMB with the respective antibiotics. Biofilm structure was quantified using confocal laser scanning microscopy with COMSTAT analysis, while activity was measured as whole-biofilm CO2 production in a continuous-flow biofilm model. The combined antibiotic-USMB treatment significantly impacted biofilm biomass, thickness and surface roughness compared to antibiotics alone (p<0.05). USMB exposure caused the formation of craters (5-20μm in diameter) in the biofilms, and when combined with gentamicin, activity was significantly lower, compared to gentamicin, USMB or untreated controls, respectively. Interestingly, the CO2 production rate following combined streptomycin-USMB treatment was higher than after streptomycin alone, but significantly lower than USMB alone and untreated control. These results show strong evidence of a synergistic effect between antibiotics and USMB, although the varied response to different antibiotics emphasize the need to optimize the USMB exposure conditions to maximize this synergism and ultimately transfer this technology into clinical or industrial practice., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Time-lapse videos of growing biofilms were analyzed using a background subtraction method, which removed camouflaging effects from the heterogeneous field of view to reveal evidence of streamer formation from optically dense biofilm segments. In addition, quantitative measurements of biofilm velocity and optical density, combined with mathematical modeling, demonstrated that streamer formation occurred from mature, high-viscosity biofilms. We propose a streamer formation mechanism by sudden partial detachment, as opposed to continuous elongation as observed in other microfluidic studies. Additionally, streamer formation occurred in straight microchannels, as opposed to serpentine or pseudo-porous channels, as previously reported.

The dynamic interaction of bacteria within bed sediment and suspended sediment (i.e., floc) in a wave-dominated beach environment was assessed using a laboratory wave flume. The influence of shear stress (wave energy) on bacterial concentrations and on the partitioning and transport of unattached and floc-associated bacteria was investigated. The study showed that increasing wave energy (0.60 and 5.35 N/s) resulted in a 0.5 to 1.5 log increase in unattached cells of the test bacterium Pseudomonas sp. strain CTO7::gfp-2 in the water column. There was a positive correlation between the bacterial concentrations in water and the total suspended solids, with the latter increasing from values of near 0 to up to 200 mg/L over the same wave energy increase. The median equivalent spherical diameter of flocs in suspension also increased by an order of magnitude in all experimental trials. Under both low (0.60 N/s) and high (5.35 N/s) energy regime, bacteria were shown to preferentially associate with flocs upon cessation of wave activity. The results suggest that collecting water samples during periods of low wave action for the purpose of monitoring the microbiological quality of water may underestimate bacterial concentrations partly because of an inability to account for the effect of shear stress on the erosion and mobilization of bacteria from bed sediment to the water column. This highlights the need to develop a more comprehensive beach analysis strategy that not only addresses presently uncharacterized shores and sediments but also recognizes the importance of eroded flocs as a vector for the transport of bacteria in aquatic environments.

We present a microfluidic device capable of patterning linear biofilm formations using a flow templating approach. We describe the design considerations and fabrication methodology of a two level flow-templating micro-bioreactor (FT-μBR), which generates a biofilm growth stream surrounded on 3 sides by a growth inhibiting confinement stream. Through a combination of experiments and simulations we comprehensively evaluate and exploit control parameters to manipulate the biofilm growth template stream dimensions. The FT-μBR is then used to grow biofilm patterns with controllable dimensions. A proof-of-principle study using the device demonstrates its utility in conducting biofilm growth rate measurements under different shear stress environments. This opens the way for quantitative studies into the effects of the local shear environment on biofilm properties and for the synthesis of a new generation of functional biomaterials with controllable properties.

A series of H 2 O and/or EtOH soluble, self-assembling quaternary ammonium salts containing a dansyl (DNS) fluorescent moiety suitable for attachment to both porous ([DNS-NH-(CH 2 ) 3 -NMe 2 -R + ][X - ] (2; R = -Si(OMe) 3 , X - = Cl - ) and non-porous (3a; R = -PO(OEt) 2 , 3b; -PO(Oi-Pr) 2 , 3c; -(PO(OH) 2 , X - = Br - ), 4; ([DNS-NH-(CH 2 ) 3 -NMe 2 -(CH 2 ) 3 -NH(CH 2 PO(OEt) 2 ) 2 + ][Br - ], 5; R = -((CH 2 ) 3 SCOCH 3 , X - = Cl - ), 6a; (R = -(CH 2 ) n O(C 6 H 4 )CO(C 6 H 5 ), n = 3, X - = Br - , 6b; n = 3, X - = Cl - , 6c; n = 3, X - = I - , 6d; n = 4, X - = Br - , 6e; n = 4, X - = I - , 6f; n = 6, X - = Br - , 6g; n = 6, X - = Cl - , 6h; n = 6, X - = I - ), 7; (R = -CH 2 -CH[double bond, length as m-dash]CH 2 , X - = Br - ) surfaces were prepared from the precursor dansyl amine (1; DNS-NH-(CH 2 ) 3 -NMe 2 ). Compounds 1-7 were characterized by NMR ( 1 H, 13 C, 29 Si (2), 31 P (3a-c, 4)) spectroscopy, HRMS, UV-Vis and fluorescence spectroscopy. Additional characterization of compounds 1 and 7 were carried out by X-ray structure determinations. Physical attachment of compound 2 to cotton surfaces after immersion in solutions containing fluorescent dyes was verified by exposure to UV light and by complexation with bromo-phenol blue that rendered the surfaces visibly blue in colour. Phosphorus containing dansyl fluorescent dye, 3c, was attached to a stainless steel surface by exposure to an aqueous solution containing this dye, resulting in the formation of a self-assembled fluorescent monolayer. UV cure of plastic surfaces (polypropylene, silicon medical tubing) coated with compound 6a resulted in the covalent attachment of the dyes.

The importance of bacterial adherence has been acknowledged in microbial lignocellulose conversion studies; however, few reports have described the function and structure of biofilms supported by cellulosic substrates. We investigated the organization, dynamic formation, and carbon flow associated with biofilms of the obligately anaerobic cellulolytic bacterium Clostridium thermocellum 27405. Using noninvasive, in situ fluorescence imaging, we showed biofilms capable of near complete substrate conversion with a characteristic monolayered cell structure without an extracellular polymeric matrix typically seen in biofilms. Cell division at the interface and terminal endospores appeared throughout all stages of biofilm growth. Using continuous-flow reactors with a rate of dilution (2 h(-1)) 12-fold higher than the bacterium's maximum growth rate, we compared biofilm activity under low (44 g/liter) and high (202 g/liter) initial cellulose loading. The average hydrolysis rate was over 3-fold higher in the latter case, while the proportions of oligomeric cellulose hydrolysis products lost from the biofilm were 13.7% and 29.1% of the total substrate carbon hydrolyzed, respectively. Fermentative catabolism was comparable between the two cellulose loadings, with ca. 4% of metabolized sugar carbon being utilized for cell production, while 75.4% and 66.7% of the two cellulose loadings, respectively, were converted to primary carbon metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). However, there was a notable difference in the ethanol-to-acetic acid ratio (g/g), measured to be 0.91 for the low cellulose loading and 0.41 for the high cellulose loading. The results suggest that substrate availability for cell attachment rather than biofilm colonization rates govern the efficiency of cellulose conversion.

Background: Pseudomonas aeruginosa has been increasingly recognized for its ability to cause significant hospital-associated outbreaks, particularly since the emergence of multidrug-resistant strains. Biofilm formation allows the pathogen to persist in environmental reservoirs. Thus, multiple hospital room design elements, including sink placement and design, can impact nosocomial transmission of P. aeruginosa and other pathogens., Methods: From December 2004 through March 2006, 36 patients exposed to the intensive care unit or transplant units of a tertiary care hospital were infected with a multidrug-resistant strain of P. aeruginosa. All phenotypically similar isolates were examined for genetic relatedness by means of pulsed-field gel electrophoresis. Clinical characteristics of the affected patients were collected, and a detailed epidemiological and environmental investigation of potential sources was carried out., Results: Seventeen of the infected patients died within 3 months; for 12 (71%) of these patients, infection with the outbreak organism contributed to or directly caused death. The source of the outbreak was traced to hand hygiene sink drains, where biofilms containing viable organisms were found. Testing by use of a commercial fluorescent marker demonstrated that when the sink was used for handwashing, drain contents splashed at least 1 meter from the sink. Various attempts were made to disinfect the drains, but it was only when the sinks were renovated to prevent splashing onto surrounding areas that the outbreak was terminated., Conclusion: This report highlights the importance of biofilms and of sink and patient room design in the propagation of an outbreak and suggests some strategies to reduce the risks associated with hospital sinks.

Biofilm cells differ phenotypically from their free-floating counterparts. Differential growth rates in biofilms are often referred to, particularly in response to limited diffusion of oxygen and nutrients. We observed growth rates of attached Pseudomonas sp. strain CT07 cells that were notably higher than the maximum specific growth rate measured in batch culture. Despite dilution rates in continuous flow cells that exceeded the maximum planktonic specific growth rate by 58 times, sampling of the effluent revealed >10(9) cells ml(-1), suggesting that biofilms function as a source of planktonic cells through high cell yield and detachment. Further investigation demonstrated considerable planktonic cell yield from biofilms as young as 6 h, indicating that detachment is not limited to established biofilms. These biofilm-detached cells were more sensitive to a commercial biocide than associated biofilm- and chemostat-cultivated populations, implying that detached biofilm cells exhibit a character that is distinct from that of attached and planktonic cell populations.

Winery wastewaters are characterised by large seasonal fluctuations in volume and composition and are often discarded with little or no treatment. A rotating biological contactor (RBC) was used to investigate microorganisms associated with the biological treatment of winery wastewater. Extensive biofilms developed on the RBC discs and contained a number of yeast and bacterial species that displayed a dynamic population shift during the evaluation period. This suggested that the naturally occurring microorganisms were able to form a stable biofilm and also reduce the chemical oxygen demand (COD) of winery wastewater (on average 43% with a retention time of 1h). One of the yeast isolates, MEA 5, was able to reduce the COD of synthetic wastewater by 95% and 46% within 24h under aerated and non-aerated conditions, respectively. The yeast isolates could therefore play an important role in the degradation of organic compounds under aerobic conditions, such as those associated with an RBC.