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The rate-limiting step of wastewater treatment is the breakdown of polymers by extracellular enzyme activity (EEA). The efficacy of EEA on biomass from full scale conventional activated sludge (AS) and modified AS with bench scale and full scale rotating biofilm reactors (RBR) was compared. The maximum amino-peptidase EEA was 394 ± 34 μmolL -1  min -1 for the bench RBR which was 11.7 and 4.5 times greater than maximum α-glucosidase and phosphatase EEA in these reactors. At full scale the RBR gave ~4.6, 13.5 and 6.3 times the EEA for amino-peptidase, α-glucosidase and phosphatase (based on enzyme V max ) compared to the highest EEA in conventional AS biomass. Controlled overloading of the bench RBRs revealed that EEA increased with OLR up to 190 g tCOD m -2 d -1 and further increases in OLR reduced the EEA. Pretreatment of wastewater by EEA in the RBR was linked to better performance of the modified activated sludge process. Maintaining high EEA of biofilms is critical for the design of high OLR wastewater treatment systems., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Turbidity in water can be caused by a range of different turbidity causing materials (TCM). Here the characteristics and attachment of bacteria to TCMs was assessed and the resultant impact on UV disinfection determined. TCMs represent potential vehicles for bacterial penetration of water treatment barriers, contamination of potable supplies and impact on subsequent human health. The TCMs under investigation were representative of those that may be present in surface and ground waters, both from the source and formed in the treatment process. The TCMs were chalk, Fe (III) hydroxide precipitate, kaolin clay, manganese dioxide and humic acids, at different turbidity levels representative of source waters (0, 0.1, 0.2, 0.4, 1, 2, and 5 NTU). Escherichia coli and Enterococcus faecalis attachment followed the order of Fe(III)>chalk, with little to no attachment seen for MnO 2 , humic acids and clay. The attachment was postulated to be due to chalk and Fe(III) particles having a more neutral surface charge resulting in elevated aggregation with bacteria compared to other TCMs. The humic acids and Fe(III) were the TCMs which influenced inactivation of E. coli and E. faecalis due to decreasing UV transmittance (UVT) with increasing TCM concentration. The presence of the Fe(III) TCM at 0.2 NTU resulted in the poorest E. coli inactivation, with 2.5 log 10 reduction at UV dose of 10mJcm -2 (k d of -0.23cm 2 mJ -1 ) compared to a 3.9 log 10 reduction in the absence of TCMs. E. faecalis had a greater resistance to UV irradiation than E. coli for all TCMs. Effective disinfection of drinking water is a priority for ensuring high public health standards. Uniform regulations for turbidity levels for waters pre-disinfection by UV light set by regulators may not always be appropriate and efficacy is dependent on the type, as well as the amount, of turbidity present in the water., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Decline-diseases are complex and becoming increasingly problematic to tree health globally. Acute Oak Decline (AOD) is characterized by necrotic stem lesions and galleries of the bark-boring beetle, Agrilus biguttatus, and represents a serious threat to oak. Although multiple novel bacterial species and Agrilus galleries are associated with AOD lesions, the causative agent(s) are unknown. The AOD pathosystem therefore provides an ideal model for a systems-based research approach to address our hypothesis that AOD lesions are caused by a polymicrobial complex. Here we show that three bacterial species, Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana, are consistently abundant in the lesion microbiome and possess virulence genes used by canonical phytopathogens that are expressed in AOD lesions. Individual and polyspecies inoculations on oak logs and trees demonstrated that B. goodwinii and G. quercinecans cause tissue necrosis and, in combination with A. biguttatus, produce the diagnostic symptoms of AOD. We have proved a polybacterial cause of AOD lesions, providing new insights into polymicrobial interactions and tree disease. This work presents a novel conceptual and methodological template for adapting Koch's postulates to address the role of microbial communities in disease.

To assess fecal pollution in coastal waters, current monitoring is reliant on culture-based enumeration of bacterial indicators, which does not account for the presence of viable but non-culturable or sediment-associated micro-organisms, preventing effective quantitative microbial risk assessment (QMRA). Seasonal variability in viable but non-culturable or sediment-associated bacteria challenge the use of fecal indicator organisms (FIOs) for water monitoring. We evaluated seasonal changes in FIOs and human enteric pathogen abundance in water and sediments from the Ribble and Conwy estuaries in the UK. Sediments possessed greater bacterial abundance than the overlying water column, however, key pathogenic species ( Shigella spp., Campylobacter jejuni, Salmonella spp., hepatitis A virus, hepatitis E virus and norovirus GI and GII) were not detected in sediments. Salmonella was detected in low levels in the Conwy water in spring/summer and norovirus GII was detected in the Ribble water in winter. The abundance of E. coli and Enterococcus spp. quantified by culture-based methods, rarely matched the abundance of these species when measured by qPCR. The discrepancy between these methods was greatest in winter at both estuaries, due to low CFU's, coupled with higher gene copies (GC). Temperature accounted for 60% the variability in bacterial abundance in water in autumn, whilst in winter salinity explained 15% of the variance. Relationships between bacterial indicators/pathogens and physicochemical variables were inconsistent in sediments, no single indicator adequately described occurrence of all bacterial indicators/pathogens. However, important variables included grain size, porosity, clay content and concentrations of Zn, K, and Al. Sediments with greater organic matter content and lower porosity harbored a greater proportion of non-culturable bacteria (including dead cells and extracellular DNA) in winter. Here, we show the link between physicochemical variables and season which govern culturability of human enteric pathogens and FIOs. Therefore, knowledge of these factors is critical for accurate microbial risk assessment. Future water quality management strategies could be improved through monitoring sediment-associated bacteria and non-culturable bacteria. This could facilitate source apportionment of human enteric pathogens and FIOs and direct remedial action to improve water quality.

We review the risk of norovirus (NoV) infection to the human population from consumption of contaminated shellfish. From a UK perspective, risk is apportioned for different vectors of NoV infection within the population. NoV spreads mainly by person-to-person contact or via unsanitary food handling. NoV also enters the coastal zone via wastewater discharges resulting in contamination of shellfish waters. Typically, NoV persists in the marine environment for several days, with its presence strongly linked to human population density, wastewater discharge rate, and efficacy of wastewater treatment. Shellfish bioaccumulate NoV and current post-harvest depuration is inefficient in its removal. While NoV can be inactivated by cooking (e.g. mussels), consumption of contaminated raw shellfish (e.g. oysters) represents a risk to human health. Consumption of contaminated food accounts for 3-11% of NoV cases in the UK (~74,000 cases/year), of which 16% are attributable to oyster consumption (11,800 cases/year). However, environmental and human factors influencing NoV infectivity remain poorly understood. Lack of standard methods for accurate quantification of infective and non-infective (damaged) NoV particles represent a major barrier, hampering identification of an appropriate lower NoV contamination limit for shellfish. Future management strategies may include shellfish quality assessment (at point of harvest or at point of supply) or harvesting controls. However, poor understanding of NoV inactivation in shellfish and the environment currently limits accurate apportionment and risk assessment for NoV and hence the identification of appropriate shellfish or environmental quality standards.

The accurate detection of pathogens in environmental matrices, such as sediment, is critical in understanding pathogen fate and behavior in the environment. In this study, we assessed the usefulness of methods for the detection and quantification of Vibrio spp. and norovirus (NoV) nucleic acids in sediment. For bacteria, a commonly used direct method using hexadecyltrimethylammonium bromide (CTAB) and phenol-chloroform-isoamyl alcohol (PCI) extraction was optimized, whereas for NoV, direct and indirect (virus elution-concentration) methods were evaluated. For quantification, commercially available quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) kits were tested alongside a digital PCR (dPCR) approach. CTAB-based extraction combined with 16 h polyethylene glycol 6000 (PEG6000) precipitation was found to be suitable for the direct extraction of high abundance bacterial and viral nucleic acids. For the indirect extraction of viral RNA, beef extract-based elution followed by PEG6000 precipitation and extraction using the NucliSENS® MiniMag® Nucleic Acid Purification System and the PowerViral® Environmental RNA/DNA Isolation Kit and qRT-PCR resulted in 83-112 and 63-69% recoveries of NoV, respectively. dPCR resulted in lower viral recoveries (47 and 9%) and ~4 orders of magnitude lower Vibrio concentrations (3.6-4.6 log 10 gc/100 g sediment) than was observed using qPCR. The use of internal controls during viral quantification revealed that the RT step was more affected by inhibitors than the amplification. The methods described here are suitable for the enumeration of viral and/or bacterial pathogens in sediment, however the use of internal controls to assess efficiency is recommended.

The long term survival of fecal indicator organisms (FIOs) and human pathogenic microorganisms in sediments is important from a water quality, human health and ecological perspective. Typically, both bacteria and viruses strongly associate with particulate matter present in freshwater, estuarine and marine environments. This association tends to be stronger in finer textured sediments and is strongly influenced by the type and quantity of clay minerals and organic matter present. Binding to particle surfaces promotes the persistence of bacteria in the environment by offering physical and chemical protection from biotic and abiotic stresses. How bacterial and viral viability and pathogenicity is influenced by surface attachment requires further study. Typically, long-term association with surfaces including sediments induces bacteria to enter a viable-but-non-culturable (VBNC) state. Inherent methodological challenges of quantifying VBNC bacteria may lead to the frequent under-reporting of their abundance in sediments. The implications of this in a quantitative risk assessment context remain unclear. Similarly, sediments can harbor significant amounts of enteric viruses, however, the factors regulating their persistence remains poorly understood. Quantification of viruses in sediment remains problematic due to our poor ability to recover intact viral particles from sediment surfaces (typically <10%), our inability to distinguish between infective and damaged (non-infective) viral particles, aggregation of viral particles, and inhibition during qPCR. This suggests that the true viral titre in sediments may be being vastly underestimated. In turn, this is limiting our ability to understand the fate and transport of viruses in sediments. Model systems (e.g., human cell culture) are also lacking for some key viruses, preventing our ability to evaluate the infectivity of viruses recovered from sediments (e.g., norovirus). The release of particle-bound bacteria and viruses into the water column during sediment resuspension also represents a risk to water quality. In conclusion, our poor process level understanding of viral/bacterial-sediment interactions combined with methodological challenges is limiting the accurate source apportionment and quantitative microbial risk assessment for pathogenic organisms associated with sediments in aquatic environments.

The impact of solids retention time (SRT) on estrone (E1), 17β-estradiol (E2), estriol (E3) and 17α-ethinylestradiol (EE2) removal in an activated sludge plant (ASP) was examined using a pilot plant to closely control operation. Exsitu analytical methods were simultaneously used to enable discrimination of the dominant mechanisms governing estrogen removal following transitions in SRT from short (3d) to medium (10d) and long (27d) SRTs which broadly represent those encountered at full-scale. Total estrogen (∑EST, i.e., sum of E1, E2, E3 and EE2) removals which account for aqueous and particulate concentrations were 70±8, 95±1 and 93±2% at 3, 10 and 27d SRTs respectively. The improved removal observed following an SRT increase from 3 to 10d was attributable to the augmented biodegradation of the natural estrogens E1 and E2. Interestingly, estrogen biodegradation per bacterial cell increased with SRT. These were 499, 1361 and 1750ng 10(12) viable cells(-1)d(-1). This indicated an improved efficiency of the same group or the development of a more responsive group of bacteria. In this study no improvement in absolute ∑EST removal was observed in the ASP when SRT increased from 10 to 27d. However, batch studies identified an augmented biomass sorption capacity for the more hydrophobic estrogens E2 and EE2 at 27d, equivalent to an order of magnitude. The lack of influence on estrogen removal during pilot plant operation can be ascribed to their distribution within activated sludge being under equilibrium. Consequently, lower wastage of excess sludge inherent of long SRT operation counteracts any improvement in sorption., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We used functional magnetic resonance imaging (fMRI) during storage of the motion aftereffect (MAE) to examine the relationship between motion perception and neural activity in the human cortical motion complex MT+ (including area MT and adjacent motion-selective cortex). MT+ responds not only to physical motion but also to illusory motion, as in the MAE when subjects who have adapted to continuous motion report that a subsequent stationary test stimulus appears to move in the opposite direction. In the phenomenon of storage, the total decay time of the MAE is extended by inserting a dark period between adaptation and test phases. That is, when the static test pattern is presented after a storage period equal in duration to the normal MAE, the illusory motion reappears for almost as long as the original effect despite the delay. We examined fMRI activation in MT+ during and after storage. Seven subjects viewed continuous motion, followed either by an undelayed stationary test (immediate MAE) or by a completely dark storage interval preceding the test (stored MAE). Like the perceptual effect, activity in MT+ dropped during the storage interval then rebounded to reach a level much higher than after the same delay without storage. Although MT+ activity was slightly enhanced during the storage period following adaptation to continuous motion (compared with a control sequence in which the adaptation grating oscillated and no MAE was perceived), this enhancement was much less than that observed during the perceptual phenomenon. These results indicate that following adaptation, activity in MT+ is pronounced only with the presentation of an appropriate visual stimulus, during which the MAE is perceived.