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This review discusses critically how seasonal changes might affect the community composition and dynamics of activated sludge wastewater treatment plants, and examines the factors thought more generally to control microbial community assembly, including the role of taxa–time relationships and stochastic and deterministic influences. The review also questions the differences in protocols used in these studies, which make any subsequent attempts at data comparisons problematic. These include bacterial DNA extraction and PCR methodologies, 16S rRNA sequencing and especially its depth, and subsequent statistical analyses of the data, which together often fail to reveal seasonal dynamic community shifts. Suggestions are given as to how experimental protocols need to be improved and standardized, and especially the requirement to examine bacterial populations at the species level. This review looks critically at what is known currently about seasonal influences on key members of this community, including viruses, the bacteria responsible for nitrogen and phosphorus removal and those causing bulking and foaming. The data show many of these species exhibit replicative seasonal abundances over several years, but not under all conditions, illustrating how complex these community dynamics are. Fungal and protozoal/metazoal seasonal community dynamics, less studied, are also discussed. The current data suggest that seasonal temperature fluctuations are responsible for most of the seasonal community dynamics by selectively favouring or otherwise individual populations. However, more longer term studies carried out under much stricter controlled conditions are required. [ABSTRACT FROM AUTHOR]

Droplet-based microfluidics and digital polymerase chain reaction (PCR) hold significant promise for accurately detecting and quantifying pathogens. However, existing droplet-based digital PCR (ddPCR) applications have been relying exclusively on single emulsion droplets. Single emulsion droplets may not be suitable for applications such as identifying the source and pathways of water contamination where the templates must be protected against harsh environmental conditions. In this study, we developed a core–shell particle to serve as a protective framework for DNAs, with potential applications in digital PCR. We employed a high-throughput and facile flow-focusing microfluidic device to generate liquid beads, core–shell particles with liquid cores, which provided precise control over process parameters and consequently particle characteristics. Notably, the interfacial interaction between the core and shell liquids could be adjusted without adding surfactants to either phase. As maintaining stability is essential for ensuring the accuracy of digital PCR (dPCR), we investigated parameters that affect the stability of core–shell droplets, including surfactants in the continuous phase and core density. As a proof of concept, we encapsulated a series of human faecal DNA samples in the core–shell droplets and the subsequent liquid beads. The core–shell particles ensure contamination-free encapsulation of DNA in the core. The volume of the core droplets containing the PCR mixture is only 0.12 nL. Our experimental results indicate that the liquid beads formulated using our technique can amplify the encapsulated DNA and be used for digital PCR without interfering with the fluorescence signal. We successfully demonstrated the ability to detect and quantify DNA under varying concentrations. These findings provide new insights and a step change in digital PCR that could benefit various applications, including the detection and tracking of environmental pollution. [ABSTRACT FROM AUTHOR]

Despite dramatic improvements in water treatment technologies in developed countries, waterborne viruses are still associated with many of cases of illness each year. These illnesses include gastroenteritis, meningitis, encephalitis, and respiratory infections. Importantly, outbreaks of viral disease from waters deemed compliant from bacterial indicator testing still occur, which highlights the need to monitor the virological quality of water. Human adenoviruses are often used as a viral indicator of water quality (faecal contamination), as this pathogen has high UV-resistance and is prevalent in untreated domestic wastewater all year round, unlike enteroviruses and noroviruses that are often only detected in certain seasons. Standard methods for recovering and measuring adenovirus numbers in water are lacking, and there are many variations in published methods. Since viral numbers are likely under-estimated when optimal methods are not used, a comprehensive review of these methods is both timely and important. This review critically evaluates how estimates of adenovirus numbers in water are impacted by technical manipulations, such as during adenovirus concentration and detection (including culturing and polymerase-chain reaction). An understanding of the implications of these issues is fundamental to obtaining reliable estimation of adenovirus numbers in water. Reliable estimation of HAdV numbers is critical to enable improved monitoring of the efficacy of water treatment processes, accurate quantitative microbial risk assessment, and to ensure microbiological safety of water. [ABSTRACT FROM AUTHOR]

Seventy four SNP genotypes and 54 E. coli genomes from kangaroo, Tasmanian devil, reptile, cattle, dog, horse, duck, bird, fish, rodent, human and environmental water sources were screened for the presence of the CRISPR 2.1 loci flanked by cas2 and iap genes. CRISPR 2.1 regions were found in 49% of the strains analysed. The majority of human E. coli isolates lacked the CRISPR 2.1 locus. We described 76 CRISPR 2.1 positive isolates originating from Australian animals and humans, which contained a total of 764 spacer sequences. CRISPR arrays demonstrated a long history of phage attacks especially in isolates from birds (up to 40 spacers). The most prevalent spacer (1.6%) was an ancient spacer found mainly in human, horse, duck, rodent, reptile and environmental water sources. The sequence of this spacer matched the intestinal P7 phage and the pO111 plasmid of E. coli. [ABSTRACT FROM AUTHOR]

Campylobacter is a recommended reference pathogen for the verification and validation of water recycling schemes in Australia and globally. In a larger study investigating the efficacy of pathogen removal in waste stabilization ponds (WSP), we cultivated bacteria from wastewater samples on modified charcoal–cefoperazone–deoxycholate agar (mCCDA) targeting the growth of Campylobacter. A high number of colonies characteristic of Campylobacter grew on this selective medium, but this did not correlate with qPCR data. Using primers targeting the 16S rRNA gene, and additional confirmatory tests to detect VS1, ompA, blaOXA-51-like, blaOXA-23-like genes, we tested 80 random colonies from 10 WSP samples. All 80 were identified as Acinetobacter baumannii. Wastewater grab samples taken three times over 6 months throughout the WSP system showed removal of A. baumannii in the WSP at rates similar to that of Escherichia coli. Our study suggests that mCCDA agar is not a suitable medium for isolating Campylobacter from environmental samples and that A. baumannii can be used as an indicator for removal of pathogens in WSPs. [ABSTRACT FROM AUTHOR]

The polymerase chain reaction (PCR) is a popular and well-established DNA amplification technique. Technological and engineering advancements in the field of microfluidics have fuelled the progress of polymerase chain reaction (PCR) technology in the last three decades. Advances in microfluidics-based PCR technology have significantly reduced the sample volume and thermal cycling time. Further advances led to novel and accurate techniques such as the digital PCR. However, contamination of PCR samples, lack of reusability of the microfluidic PCR platforms, complexity in instrumentation and operation remain as some of the significant drawbacks of conventional microfluidic PCR platforms. Liquid marbles, the recently emerging microfluidic platform, could potentially resolve these drawbacks. This paper reports the first liquid marble based polymerase chain reaction. We demonstrated an experimental setup for the liquid-marble based PCR with a humidity-controlled chamber and an embedded thermal cycler. A concentrated salt solution was used to control the humidity of the PCR chamber which in turn reduces the evaporation rate of the liquid marble. The successful PCR of microbial source tracking markers for faecal contamination was achieved with the system, indicating potential application in water quality monitoring. [ABSTRACT FROM AUTHOR]

Waste stabilisation ponds (WSP) are designed to treat wastewater through natural microbiological, photosynthetic, biochemical, physico-chemical and hydrodynamic processes. They are used throughout the world for wastewater treatment due to their minimal technical requirements, low cost and minimal energy consumption. It is well recognised that WSP hydrodynamics plays a crucial role influencing WSP treatment efficiency. In this study, published WSP literature is reviewed with a focus on the importance of environmental forces, pond configurations and pond loadings on WSP hydrodynamics and pond treatment efficiency. The findings are used to propose future WSP design and modelling requirements. It has been found that the interrelated effects of multiple factors such as pond length-to-width ratio, inlet/outlet configuration, temperature, solar radiation and wind on WSP treatment performance are not investigated sufficiently at present. It is suggested that a system based on numerical pond modelling and field measuring analyses be formulated to address the interacting influences of these factors on WSPs. Consequently, field studies on full-scale WSPs are required to obtain a complete dataset for validation purposes. In this regard, a combination of three-dimensional pond water temperature survey and tracer concentration mapping is suggested. Details of specific modelling components, such as sludge accumulation, turbulence mechanisms and the assumption of constant influent conditions, require further attention. As a result of this review process, a strategic evaluation framework together with the required dataset for the modelling and field work activities are proposed. This paves the way for subsequent studies on WSP hydrodynamics and treatment efficiency, thus benefitting pond design and operation processes. [ABSTRACT FROM AUTHOR]

Nanobubbles are gaseous entities suspended in bulk liquids that have widespread beneficial usage in many industries. Nanobubbles are already proving to be versatile in furthering the effectiveness of disease treatment on cellular and molecular levels. They are functionalized with biocompatible and stealth surfaces to aid in the delivery of drugs. At the same time, nanobubbles serve as imaging agents due to the echogenic properties of the gas core, which can also be utilized for controlled and targeted delivery. This review provides an overview of the biomedical applications of nanobubbles, covering their preparation and characterization methods, discussing where the research is currently focused, and how they will help shape the future of biomedicine. [ABSTRACT FROM AUTHOR]

There is a widespread need for the use of quantitative microbial risk assessment (QMRA) to determine reclaimed water quality for specific uses, however neither faecal indicator levels nor pathogen concentrations alone are adequate for assessing exposure health risk. The aim of this study was to build a conceptual model representing factors contributing to the microbiological health risks of reusing water treated in maturation ponds. This paper describes the development of an unparameterised model that provides a visual representation of theoretical constructs and variables of interest. Information was collected from the peer-reviewed literature and through consultation with experts from regulatory authorities and academic disciplines. In this paper we explore how, considering microbial risk as a modular system, following the QMRA framework enables incorporation of the many factors influencing human exposure and dose response, to better characterise likely human health impacts. By using and expanding upon the QMRA framework we deliver new insights into this important field of environmental exposures. We present a conceptual model of health risk of microbial exposure which can be used for maturation ponds and, more importantly, as a generic tool to assess health risk in diverse wastewater reuse scenarios. [ABSTRACT FROM AUTHOR]

Background Quantitative microbial risk assessment (QMRA) is the current method of choice for determining the risk to human health from exposure to microorganisms of concern. However, current approaches are often constrained by the availability of required data, and may not be able to incorporate the many varied factors that influence this risk. Systems models, based on Bayesian networks (BNs), are emerging as an effective complementary approach that overcomes these limitations. Objectives This article aims to provide a comparative evaluation of the capabilities and challenges of current QMRA methods and BN models, and a scoping review of recent published articles that adopt the latter for microbial risk assessment. Pros and cons of systems approaches in this context are distilled and discussed. Methods A search of the peer-reviewed literature revealed 15 articles describing BNs used in the context of QMRAs for foodborne and waterborne pathogens. These studies were analysed in terms of their application, uses and benefits in QMRA. Discussion The applications were notable in their diversity. BNs were used to make predictions, for scenario assessment, risk minimisation, to reduce uncertainty and to separate uncertainty and variability. Most studies focused on a segment of the exposure pathway, indicating the broad potential for the method in other QMRA steps. BNs offer a number of useful features to enhance QMRA, including transparency, and the ability to deal with poor quality data and support causal reasoning. Conclusion The method has significant untapped potential to describe the complex relationships between microbial environmental exposures and health. [ABSTRACT FROM AUTHOR]

We investigated the prevalence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in untreated hospital wastewaters and 2 sewage treatment plants (STPs). A collection of 252 ESBL-producing E. coli isolates from hospital wastewater and STPs were typed and tested for resistance to 17 antimicrobial agents and for the presence of integron-associated integrases (intI gene) and ESBL genes. Eighty-nine percent (n = 176) of the ESBL-producing E. coli strains from hospital wastewater were found in more than 1 sample (common types), with 1 common type accounting for 35% of isolates, found in all samples. These strains were also resistant to up to 9 non-β-lactam antibiotics and showed the same pattern of resistance in all samples. More than 73% of the hospital wastewater isolates possessed SHV-type ESBL as opposed to isolates from STPs that carried only CTX-M-type ESBL genes. The prevalence of the intI gene did not differ between the sources of the isolates. Certain ESBL-producing E. coli were dominant in hospital wastewaters. These strains possessed β-lactamase genes that were different from isolates found in STPs. From a public health point of view, the presence of such a high level of ESBL-producing E. coli strains in hospital wastewaters is of great importance. [ABSTRACT FROM AUTHOR]

We investigated the prevalence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in untreated hospital wastewaters and 2 sewage treatment plants (STPs). A collection of 252 ESBL-producing E. coli isolates from hospital wastewater and STPs were typed and tested for resistance to 17 antimicrobial agents and for the presence of integron-associated integrases ( intI gene) and ESBL genes. Eighty-nine percent ( n = 176) of the ESBL-producing E. coli strains from hospital wastewater were found in more than 1 sample (common types), with 1 common type accounting for 35% of isolates, found in all samples. These strains were also resistant to up to 9 non-β-lactam antibiotics and showed the same pattern of resistance in all samples. More than 73% of the hospital wastewater isolates possessed SHV-type ESBL as opposed to isolates from STPs that carried only CTX-M-type ESBL genes. The prevalence of the intI gene did not differ between the sources of the isolates. Certain ESBL-producing E. coli were dominant in hospital wastewaters. These strains possessed β-lactamase genes that were different from isolates found in STPs. From a public health point of view, the presence of such a high level of ESBL-producing E. coli strains in hospital wastewaters is of great importance. [ABSTRACT FROM AUTHOR]

Sunlight disinfection is important for treatment of wastewater within maturation ponds. This study analyses the movement of Escherichi coli within a slice of a maturation pond, being affected by stratification, sunlight attenuation and mixing driven by wind shear and natural convection using computational fluid dynamics (CFD). Since the exposure to ultraviolet light is most effective in the near-surface region of the pond, natural convective mixing mechanisms to transport the pathogens from the lower parts of the pond are critical for disinfection efficacy. Different turbulence models are considered for closure of the momentum conservation equations and compared with a laminar flow simulation and a completely stirred tank reactor (CSTR) model. The effect of turbulence and stratification is shown to be significant for thermal and velocity distributions, and predictions of E. coli die-off. Greater volume-averaged E. coli die-off was predicted by the computationally convenient CSTR model than the CFD turbulence and laminar models. The simulation results are compared with experimental data and show that complete vertical mixing occurs in a diurnal pattern aiding die-off in sunlight-attenuating water. Practical applications of the model can assist in management strategies for maturation ponds such as off-take locations/times and evaluating seasonal variations in sunlight disinfection. • A CFD model of the die-off of E. coli within a maturation pond is presented. • The model includes natural convective mixing and sunlight attenuation. • Complete vertical mixing occurs in a diurnal pattern consistent with field measurements. • Turbulence and stratification significantly influence E. coli die-off via sunlight disinfection. • The model has value for aiding pond design and operational management. [ABSTRACT FROM AUTHOR]

Characterization of microbial communities using high-throughput amplicon sequencing is an emerging approach for microbial source tracking of fecal pollution. This study used SourceTracker software to examine temporal and geographical variability of fecal bacterial community profiles to identify pollutant sources in three freshwater catchments in sub-tropical Australia. Fecal bacterial communities from 10 animal species, humans, and composite wastewater samples from six sewage treatment plants were characterized and compared to freshwater samples using Illumina amplicon sequencing of the V5–V6 regions of the 16S rRNA gene. Source contributions were calculated in SourceTracker using new fecal taxon libraries as well as previously generated libraries to determine the effects of geographic and temporal variability on source assignments. SourceTracker determined 16S rRNA bacterial communites within freshwater samples, shared taxonomic similarities to that of wastewater at low levels (typically <3%). SourceTraker also predicted occasional fecal detection of deer and flying fox sources in the water samples. No significant differences in source contributions were observed within sequences from current and previously characterized fecal samples (P ≥ 0.107). However, significant differences were observed between previously characterized and newly characterized source communities (ANOSIM P ≤ 0.001), which shared <15% community composition. Results suggest temporal instability of fecal taxon libraries among tested sources and highlight continual evaluation of community-based MST using confirmatory qPCR analyses of marker genes. Image 1 • 16S rRNA sequencing established microbial profiles of three catchments. • Sewage microbial communities showed low similarity to those of water samples. • Deer and flying fox fecal contamination were found in several samples. • Dog fecal contamination showed a greater contribution than did other sources. • Dog fecal taxon libraries showed the greatest temporal stability over the other source libraries examined. [ABSTRACT FROM AUTHOR]