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Sand filtration is a cost-effective means of reducing microbial pathogens in drinking-water treatment. Our understanding of pathogen removal by sand filtration relies largely on studies of process microbial indicators, and comparative data from pathogens are sparse. In this study, we examined the reductions of norovirus, echovirus, adenovirus, bacteriophage MS2 and PRD1, Campylobacter jejuni, and Escherichia coli during water filtration through alluvial sand. Duplicate experiments were conducted using 2 sand columns (50 cm long, 10 cm diameter) and municipal tap water sourced from chlorine-free untreated groundwater (pH 8.0, 1.47 mM) at filtration rates of 1.1-1.3 m/day. The results were analysed using colloid filtration theory and the HYDRUS-1D 2-site attachment-detachment model. The average log10 reduction values (LRVs) of the normalised dimensionless peak concentrations (Cmax/C0) over 0.5 m were: MS2: 0.28; E. coli: 0.76; C. jejuni: 0.78; PRD1: 2.00; echovirus: 2.20; norovirus: 2.35; and adenovirus: 2.79. The relative reductions largely corresponded to the organisms' isoelectric points rather than their particle sizes or hydrophobicities. MS2 underestimated virus reductions by 1.7-2.5 log, and the LRVs, mass recoveries relative to bromide, collision efficiencies, and attachment and detachment rates differed mostly by ∼1 order of magnitude. Conversely, PRD1 reductions were comparable with those of all 3 viruses tested, and its parameter values were mostly within the same orders of magnitude. E. coli seemed an adequate process indicator for C. jejuni with similar reductions. Comparative data describing pathogen and indicator reductions in alluvial sand have important implications for sand filter design, risk assessments of drinking-water supplies from riverbank filtration and the determination of safe setback distances for drinking-water supply wells.

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Synthetic DNA tracers are gaining interest as tools for tracking contamination pathways and hydraulic connections in surface water and groundwater systems. However, few quantitative data exist that describe DNA tracer degradation and adsorption in environmental matrices. We undertook laboratory experiments to quantify the degradation of multiple double-stranded DNA tracers in stream water, groundwater, and domestic and dairy-shed effluent, and adsorption to stream sediments, soils, coastal sand aquifer media and alluvial sandy gravel aquifer media. Faster DNA tracer degradation seemed to be associated with high bacterial concentrations in the liquid phase. Overall, the degradation of the 352 base pair (bp) DNA tracers in the aqueous phase was significantly (P = 0.018) slower than that of the 302 bp DNA tracers. Although the tracers' internal amplicon lengths were similar, the longer non-amplified flanking regions of the 352 bp tracers may better protect them from environmental degradation. Thermodynamic analysis suggests that longer flanking regions contribute to greater tracer stability. This finding may explain our previous field observations that 352 bp tracer mass reductions were often lower than 302 bp tracer mass reductions. The 2 sets of DNA tracers did not differ significantly regarding their adsorption to stream sediment-stream water or aquifer media-groundwater mixtures (P > 0.067), but the 352 bp tracers showed significantly less adsorption to soil-effluent mixtures than the 302 bp tracers (P = 0.005). The DNA tracers' adsorption to soil-effluent mixtures was comparatively less than their adsorption to the aquifer media-groundwater and stream sediment-stream water mixtures, suggesting that DNA tracers may compete with like-charged organic matter for adsorption sites. These findings provide insights into the fate of DNA tracers in the environment. The DNA tracers' degradation rate constants determined in this study for a range of environmental conditions could assist the design of future field investigations.

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A high-throughput non-viral intracellular delivery platform is introduced for the transfection of large cargos with dosage-control. This platform, termed Acoustic-Electric Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with poration of the cell membranes via mechanical shear followed by the modulated expansion of these nanopores via electric field. Furthermore, AESOP utilizes acoustic microstreaming vortices wherein up to millions of cells are trapped and mixed uniformly with exogenous cargos, enabling the delivery of cargos into cells with targeted dosages. Intracellular delivery of a wide range of molecule sizes (<1 kDa to 2 MDa) with high efficiency (>90%), cell viability (>80%), and uniform dosages (<60% coefficient of variation (CV)) simultaneously into 1 million cells min-1 per single chip is demonstrated. AESOP is successfully applied to two gene editing applications that require the delivery of large plasmids: i) enhanced green fluorescent protein (eGFP) plasmid (6.1 kbp) transfection, and ii) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and single guide RNA (sgRNA). Compared to alternative platforms, this platform offers dosage-controlled intracellular delivery of large plasmids simultaneously to large populations of cells while maintaining cell viability at comparable delivery efficiencies.

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Investigating contamination pathways and hydraulic connections in complex hydrological systems will benefit greatly from multi-tracer approaches. The use of non-toxic synthetic DNA tracers is promising, because unlimited numbers of tracers, each with a unique DNA identifier, could be used concurrently and detected at extremely low concentrations. This study aimed to develop multiple synthetic DNA tracers as free molecules and encapsulated within microparticles of biocompatible and biodegradable alginate and chitosan, and to validate their field utility in different systems. Experiments encompassing a wide range of conditions and flow rates (19 cm/day-39 km/day) were conducted in a stream, an alluvial gravel aquifer, a fine coastal sand aquifer, and in lysimeters containing undisturbed silt loam over gravels. The DNA tracers were identifiable in all field conditions investigated, and they were directly detectable in the stream at a distance of at least 1 km. The DNA tracers showed promise at tracking fast-flowing water in the stream, gravel aquifer and permeable soils, but were unsatisfactory at tracking slow-moving groundwater in the fine sand aquifer. In the surface water experiments, the microencapsulated DNA tracers' concentrations and mass recoveries were 1-3 orders of magnitude greater than those of the free DNA tracers, because encapsulation protected them from environmental stressors and they were more negatively charged. The opposite was observed in the gravel aquifer, probably due to microparticle filtration by the aquifer media. Although these new DNA tracers showed promise in proof-of-concept field validations, further work is needed before they can be used for large-scale investigations.

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An important policy consideration for integrated land and water management is to understand the spatial distribution of nitrate attenuation in the groundwater system, for which redox condition is the key indicator. This paper proposes a methodology to accommodate the computational demands of large datasets, and presents national-scale predictions of groundwater redox class for New Zealand. Our approach applies statistical learning methods to relate the redox class determined on groundwater samples to spatially varying attributes. The trained model uses these spatial variables to predict redox status in areas without sample data. We assembled the groundwater sample data from regional authority databases, and assigned each sample a redox class. A key achievement was to overcome the influence of sample selection bias on model training via oversampling. We removed additional bias imposed by imbalances in the predictor variables by applying a conditional inference random forest classifier. The unbiased trained model uses eight predictors, and achieves a high validation performance (accuracy 0.81, kappa 0.71), providing good confidence in model predictions. National maps are provided for redox class and probability at specified depths. Feature importance rankings indicate that reducing conditions are associated with poorly-drained soils, and to a lesser extent, high hydrological variability, low elevation, and low-permeability lithology. These conditions are common in New Zealand's coastal and lowland plains, where artificial drainage is required to make land suitable for production. The spatial extent of reduced groundwater increases with depth, suggesting a shallow influence of soil infiltration or mobile organic carbon, and a deeper influence of lithological electron donors. Our model provides unbiased predictions at a scale relevant for environmental policy development and legislation. Identifying where the ecosystem service provided by denitrification can be utilised will enable spatially targeted interventions that can achieve the desired environmental outcome in a more cost-effective manner than non-targeted interventions.

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With the intensification of human activities, fresh water resources are increasingly being exposed to contamination from effluent disposal to land. Thus, there is a greater need to identify the sources and pathways of water contamination to enable the development of better mitigation strategies. To track discharges of domestic effluent into soil and groundwater, 10 synthetic double-stranded DNA (dsDNA)3 tracers were developed in this study. Laboratory column experiment and field groundwater and soil lysimeter studies were carried out spiking DNA with oxidation-pond domestic effluent. The selected DNA tracers were compared with a non-reactive bromide (Br) tracer with respect to their relative mass recoveries, speeds of travel and dispersions using the method of temporal moments. In intact stony soil and gravel aquifer media, the dsDNA tracers typically showed earlier breakthrough and less dispersion than the Br tracer, and underwent mass reduction. This suggests that the dsDNA tracers were predominantly transported through the network of larger pores or preferential flow paths. Effluent tracking experiments in soil and groundwater demonstrated that the dsDNA tracers were readily detectable in effluent-contaminated soil and groundwater using quantitative polymerase chain reaction. DNA tracer spiked in the effluent at quantities of 36µg was detected in groundwater 37m down-gradient at a concentration 3-orders of magnitude above the detection limit. It is anticipated it could be detected at far greater distances. Our findings suggest that synthetic dsDNA tracers are promising for tracking effluent discharges in soils and groundwater but further studies are needed to investigate DNA-effluent interaction and the impact of subsurface environmental conditions on DNA attenuation. With further validation, synthetic dsDNA tracers, especially when multiple DNA tracers are used concurrently, can be an effective new tool to track effluent discharge in soils and groundwater, providing spatial estimation on the presence or absence of contamination sources and pathways.

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Phosphorous (P) leaching (e.g., from effluents, fertilizers) and transport in highly permeable subsurface media can be an important pathway that contributes to eutrophication of receiving surface waters as groundwater recharges the base-flow of surface waters. Here we investigated attenuation and transport of orthophosphate-P in gravel aquifer and vadose zone media in the presence and absence of model colloids (Escherichia coli, kaolinite, goethite). Experiments were conducted using repacked aquifer media in a large column (2m long, 0.19m in diameter) and intact cores (0.4m long, 0.24m in diameter) of vadose zone media under typical field flow rates. In the absence of the model colloids, P was readily traveled through the aquifer media with little attenuation (up to 100% recovery) and retardation, and P adsorption was highly reversible. Conversely, addition of the model colloids generally resulted in reduced P concentration and mass recovery (down to 28% recovery), and increased retardation and adsorption irreversibility in both aquifer and vadose zone media. The degree of colloid-assisted P attenuation was most significant in the presence of fine material and Fe-containing colloids at low flow rate but was least significant in the presence of coarse gravels and E. coli at high flow rate. Based on the experimental results, setback distances of 49-53m were estimated to allow a reduction of P concentrations in groundwater to acceptable levels in the receiving water. These estimates were consistent with field observations in the same aquifer media. Colloid-assisted P attenuation can be utilized to develop mitigation strategies to better manage effluent applications in gravelly soils. To efficiently retain P within soil matrix and reduce P leaching to groundwater, it is recommended to select soils that are rich in iron oxides, to periodically disturb soil preferential flow paths by tillage, and to apply a low irrigation rate.

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This paper presents a rapid, simple, and low-cost fabrication method to prepare solvent resistant and biocompatible microfluidic devices with three-dimensional geometries. The devices were fabricated in thiolene and replicated from PDMS master with high molding fidelity. Good chemical compatibility for organic solvents allows volatile chemicals in synthesis and analysis applications. The surface can be processed to be hydrophobic or hydrophilic for water-in-oil and oil-in-water emulsions. Monodisperse organic solvent droplet generation is demonstrated to be reproducible in thiolene microchannels without swelling. The thiolene surface prevents cell adhesion but normal cell growth and adhesion on glass substrates is not affected by the adjacent thiolene patterns.

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The laboratory implementation of a fault detection and localization method based on inversion of dynamic surface displacements measured by a scanned laser Doppler vibrometer (SLDV) was investigated. The technique uses flexural wave and generalized force inversion algorithms which have previously been demonstrated using simulated noise-free vibration data generated for thick plates with a finite element model. Here these inversion algorithms to SLDV measurements made in the laboratory on a thin nickel plate and a thin carbon fiber composite plate, both having attached reinforcing ribs with intentional de-bonding of the rib/plate interface at a specific location on each structure are applied. The inverted displacement maps clearly detect and locate the detachment, whereas direct observation of the surface displacements does not. It is shown that the technique is relatively robust to the choice of frequency and to the presence of noise.

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Cells have been encapsulated inside lipid vesicles by using a new microfluidic lipid vesicle formulation technique. Lipid vesicles are formulated within minutes without using toxic lipid solvents. The encapsulation efficiency inside the vesicles is controlled by the microfluidic flows. Green fluorescent proteins (GFP), carcinoma cells, and bead encapsulated vesicles have mean diameters of 27.2 mum, 62.4 mum, and 55.9 mum, respectively. The variations of vesicle sizes are approximately 20% for the GFP and cell encapsulated vesicles and approximately 10% for the bead encapsulated vesicles.

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A multifunctional and high-efficiency microfluidic device for droplet generation and fusion is presented. Through unique design of the micro-channels, the device is able to alternately generate droplets, generating droplet ratios ranging from 1 ratio 5 to 5 ratio 1, and fuse droplets, enabling precise chemical reactions in several picoliters on a single chip. The controlled fusion is managed by passive control based on the channel geometry and liquid phase flow. The synthesis of CdS nanoparticles utilizing each fused droplet as a microreactor for rapid and efficient mixing of reagents is demonstrated in this paper. Following alternating droplet generation, the channel geometry allows the exclusive fusion of alternate droplets with concomitant rapid mixing and produces supersaturated solution of Cd2+ and S2- ions to form CdS nanoparticles in each fused droplet. The spectroscopic properties of the CdS nanoparticles produced by this method are compared with CdS prepared by bulk mixing.

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Emulsions are widely used to produce sol-gel, drugs, synthetic materials, and food products. Recent advancements in microfluidic droplet emulsion technology has enabled the precise sampling and processing of small volumes of fluids (picoliter to femtoliter) by the controlled viscous shearing in microchannels. However the generation of monodispersed droplets smaller than 1 microm without surfactants has been difficult to achieve. Normally, the generation of satellite droplets along with parent droplets is undesirable and makes it difficult to control volume and purity of samples in droplets. In this paper, however, several methods are presented to passively filter out satellite droplets from the generation of parent droplets and use these satellite droplets as the source for monodispersed production of submicron emulsions. A passive satellite droplet filtration system and a dynamic satellite droplet separation system are demonstrated. Satellite droplets are filtered from parent droplets with a two-layer channel geometry. This design allows the creation and collection of droplets that are less than 100 nm in diameter. In the dynamic separation system, satellite droplets of defined sizes can be selectively separated into different collecting zones. The separation of the satellite droplets into different collecting zones correlates with the cross channel position of the satellite droplets during the breakup of the liquid thread. The delay time for droplets to switch between the different alternating collecting zones is nominally 1 min and is proportional to the ratio of the oil shear flows. With our droplet generation system, monodispersed satellite droplets with an average radius of 2.23 +/- 0.11 microm, and bidispersed secondary and tertiary satellite droplets with radii of 1.55 +/- 0.07 microm and 372 +/- 46 nm respectively, have been dynamically separated and collected.

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Passive microfluidic channel geometries for control of droplet fission, fusion and sorting are designed, fabricated, and tested. In droplet fission, the inlet width of the bifurcating junction is used to control the range of breakable droplet sizes and the relative resistances of the daughter channels were used to control the volume of the daughter droplets. Droplet fission is shown to produce concentration differences in the daughter droplets generated from a primary drop with an incompletely mixed chemical gradient, and for droplets in each of the bifurcated channels, droplets were found to be monodispersed with a less than 2% variation in size. Droplet fusion is demonstrated using a flow rectifying design that can fuse multiple droplets of same or different sizes generated at various frequencies. Droplet sorting is achieved using a bifurcating flow design that allows droplets to be separated base on their sizes by controlling the widths of the daughter channels. Using this sorting design, submicron satellite droplets are separated from the larger droplets.

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