Monitoring bioaerosol and odour emissions from composting facilities - WR1121

Government policy requires that valuable resources should be recovered and recycled from biodegradable waste. A successful and growing organics recycling industry delivers this policy with composting being one of the principal technologies deployed to process suitable feedstock such as garden and food waste. Composting inevitably generates bioaerosols – particulate matter comprising cells or cellular components that are released into the air as a result of disturbance of composting feedstock or the processing of final product. Exposure to bioaerosols has the potential to be harmful to human and animal health. The Environment Agency adopts a precautionary and risk-based approach to the regulation of composting facilities which was developed on the basis of research by Wheeler et al. (2001) and which has been updated as new evidence has become available. The Environment Agency also requires site operators to monitor bioaerosols around their facilities using methods specified in a standard protocol which relies upon classical microbiology methods which are tried and tested but which are labour-intensive, slow and offer only a snapshot view of a highly dynamic system. A recent IOM review commissioned by Defra (Searl, 2009) on exposure-response relationships for bioaerosol emissions from waste treatment processes identified significant gaps in knowledge of exposure to bioaerosols and recommended that more research was needed into alternatives to viable microbial monitoring such as priority biomarkers (notably endotoxin) and potential surrogates such as particulate matter. The IOM review also concluded that there is a lack of information to support the development of appropriate stand-off distances. The overall aim of this project was to provide evidence on bioaerosol production, dispersion and potential exposures from composting facilities in support of future developments in policy and regulation of biowaste facilities. The objectives were: (i) to undertake a comprehensive set of standard and novel bioaerosol measurements at representative composting sites to assess comparability between different methods and also to measure spatial and temporal variations; and (ii) to determine the odour emissions and then compare these with bioaerosol emissions to see if odour is a marker of significant bioaerosol exposure. Standard (AfOR, 2009) and novel (CEN filter method, endotoxin, glucan, qPCR, real-time particulates) bioaerosols measurements were taken on a minimum of three to a maximum of six occasions over a twelve month period at four different composting facilities in England. The composting facilities were selected to represent sites of varying sizes (tonnages) and to allow a comparison of bioaerosol concentrations at standard open windrow sites versus a fully-contained site. Additional supporting information was collected including meteorological data at the time of sampling, observation of site operations and measurements of odour at one of the sites. Supporting bioaerosol and odour dispersion modelling was conducted at the site where the odour measurements were made. The spatial trend of bioaerosol concentrations described by Wheeler et al., (1991) and upon which EA regulatory policy is based was broadly corroborated by this dataset. Excursions above the EA acceptable levels at or beyond 250m from source were rare. Bioaerosol concentrations at the enclosed site were generally lower than at the open windrow sites. There was no evidence of a seasonal pattern in bioaerosol concentrations at any of the sites whereas between-sampling day variations were apparent. The cause(s) of these variations were not identified. No consistent relationship was observed between the concentration of bioaerosols measured by the two AfOR standard methods. The two methods displayed certain strengths and weakness in different situations. The IOM sampling device proved to be better suited to situations where high bioaerosol concentrations were encountered (close to source); the Andersen proving to be more effective in the lower concentration range typically found upwind of a site or at distance downwind from source. The higher volume filtration device tested in this project (referred to as the CEN method) produced data that did not consistently match either of the AfOR standard methods. This device demonstrated greater sensitivity than the IOM filter method but suffered drawbacks associated with its weight and a lack of ease of use in the field. Endotoxin concentrations were normally below the level recommended by the Dutch Expert Committee on Occupational Safety but occasional exceedances of this standard were detected at the larger open windrow sites. The majority of glucan measurements were below a widely referred to 10ng/m3 threshold. Significantly elevated concentrations were detected at one of the larger open windrow sites. The dynamic range of the qPCR method is wider (4-5-log) than either of the AfOR and the CEN methods. It is also quicker to carry out and has the potential for automation. The results from the qPCR method are mainly higher than standard AfOR methods, as the method does not distinguish viable and non-viable spores. The spatial distribution of Aspergillus fumigatus spores (by qPCR) along sampling transects, gives similar results compared to AfOR (and CEN) methods. Real time particle detection showed that both TSP and PM10 are correlated to Aspergillus fumigatus spore concentration. No consistent relationship was observed between odour and bioaerosol concentrations (although this was a limited dataset). The envelope of modelled (back-extrapolated) bioaerosol emission rates straddles several orders of magnitude. Distinguishing the influences of meteorological conditions on this variability was not possible. It was not possible to predict bioaerosol or odour emission rates with confidence. This continues to hamper confidence in modelling of odours and bioaerosols from open windrow facilities. The findings of this research have implications for the current standard monitoring protocol which should be reviewed accordingly. The findings of this multi-site survey accord with existing regulatory policy and are supportive of the general trend towards enclosed facilities. Notwithstanding this, continuing research is needed to enhance the database on emission from bioaerosol and odour abatement technologies (e.g. biofilters); to determine the cause(s) of occasional bioaerosol peaks from open facilities; to improve exposure assessments through better modelling protocols; and to link enhanced exposure information to future health impact studies

Cicada-inspired cell-instructive nanopatterned arrays

Biocompatible surfaces hold key to a variety of biomedical problems that are directly related to the competition between host-tissue cell integration and bacterial colonisation. A saving solution to this is seen in the ability of cells to uniquely respond to physical cues on such surfaces thus prompting the search for cell-instructive nanoscale patterns. Here we introduce a generic rationale engineered into biocompatible, titanium, substrates to differentiate cell responses. The rationale is inspired by cicada wing surfaces that display bactericidal nanopillar patterns. The surfaces engineered in this study are titania (TiO(2)) nanowire arrays that are selectively bactericidal against motile bacteria, while capable of guiding mammalian cell proliferation according to the type of the array. The concept holds promise for clinically relevant materials capable of differential physico-mechanical responses to cellular adhesion

A de novo virus-like topology for synthetic virions

A de novo ultra-small topology of viral assembly is reported. The design is a tri-faceted coiled-coil peptide helix, which self-assembles into monodisperse, anionic virions able to encapsulate and transfer both RNA and DNA into human cells. Unlike existing artificial systems, the virions share the same physical characteristics of viruses being anionic, non-aggregating, abundant, hollow and uniform in size, while effectively mediating gene silencing and transgene expression. These are the smallest virions reported to date with the ability to adapt and transfer small and large nucleic acids thus offering a promising solution for engineering bespoke artificial viruses with desired functions

Structurally plastic peptide capsules for synthetic antimicrobial viruses

A conceptual design for artificial antimicrobial viruses is described.</p

Probing label-free intracellular quantification of free peptide by MALDI-ToF mass spectrometry

Cell-penetrating peptides are promising reagents for gene and drug delivery. They can efficiently traverse the plasma membrane and deliver various cargo materials ranging from genes to nanoparticles. The functional efficiency of cargo often depends on the completeness of intracellular peptide uptake, which can be measured, but its quantification remains largely inconclusive. Existing approaches rely on the use of radioactive and fluorescent labels or tags which allow colorimetric, fluorescent or spectrometric detection, but lack the ability to detect free peptide. Herein we describe a generic label- and tag-free method to measure the concentration of internalised peptide by matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Quantification is preceded by two-dimensional chromatography and is performed at benign temperatures for the lysates of human dermal fibroblasts transfected with cell penetrating peptides in free form. Isotopically labelled peptides of the same structure are used as internal standards to enable accurate determination of concentration of the recovered free peptide. The method offers a minimalistic approach for intracellular quantification, which can be used as a correlative measure for fluorescence-based imaging methods.</p

DNA Origami Inside-Out Viruses

A synthetic topology for everted viruses is reported. The topology is a single-stranded virion DNA assembled into a hollow cube with exterior decorated with HIV-Tat transduction domains. The cube incorporates a pH-responsive lid allowing for the controlled encapsulation of functional proteins and their transfer and release into live cells. Unlike viruses, which are protein shells with a [3,5]-fold rotational symmetry that encase nucleic acids, these cubes are [3, 4]-fold DNA boxes encapsulating proteins. Like viruses, such everted DNA-built viruses are monodisperse nanoscale assemblies that infect human cells with a specialist cargo. The design offers a bespoke bottom-up platform for engineering nonpolyhedral, nonprotein synthetic viruses

Differentially Instructive Extracellular Protein Micro-nets

An ability to construct biological matter from the molecule up holds promise for applications ranging from smart materials to integrated biophysical models for synthetic biology. Biomolecular self-assembly is an efficient strategy for biomaterial construction which can be programmed to support desired function. A challenge remains in replicating the strategy synthetically, that is at will, and differentially, that is for a specific function at a given length scale. Here we introduce a self-assembly topology enabling a net-like architectural mimetic of native extracellular matrices capable of differential responses to cell adhesionenhanced mammalian cell attachment and proliferation, and enhanced resistance to bacterial colonizationat the native sub-millimeter length scales. The biological performance of such protein micro-nets directly correlates with their morphological and chemical properties, offering thus an application model for differential extracellular matrices

Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing

International audienceTwo-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750–1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image co-registration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development. Multiphoton microscopy is a powerful tool for label-free and non-invasive functional imaging in small organisms and tissues 1, 2. Pulsed near infrared excitation light allows in-depth imaging based on contrasts such as endog-enous fluorescence 2 , second harmonic generation (SHG) 3 and third harmonic generation (THG) 4. Endogenous fluorescence in living tissues arises from several intrinsic biomarkers that play important roles in physiological processes 2. The primary intracellular sources are NAD(P)H and FAD, the two major cofactors of redox reactions in the cell and central regulators of energy production and metabolism 5, 6. Their fluorescence reports on the metabolic activity of cells allowing tissue physiology and processes such as stem cell differentiation, cancer development and neurodegenerative diseases to being non-invasively monitored 7–12. The fluorescence lifetimes of NADH and FAD are different upon binding to the protein during the electron transport chain. FLIM provides sensitive measurements of the free and protein-bound NAD(P)H ratio and of the redox states (NADH/NAD +) of cells, and can be used to distinguish glycolytic and oxidative phosphorylation metabolic states 13–17. Monitoring lifetime of free and protein-bound FAD has also been exploited to quantify redox ratio FAD/FADH 2 , and used as a biomarker of precancerous epithelial cells 12. It is well established that retinoids play a crucial role in stem cell differentiation and embryo development 18, 19 and their concentration and gradients have been detected in vivo during zebrafish development 9, 20. Other intrinsic fluorophores such as porphyrin, collagen, elastin, keratin

Physicochemical and biological assays for quality control of biopharmaceuticals: Interferon alfa-2 case study

A selection of physicochemical and biological assays were investigated for their utility in detecting changes in preparations of Interferon alfa-2a and Interferon alfa-2b (IFN-α2a, IFN-α2b), which had been subjected to stressed conditions, in order to create models of biopharmaceutical products containing product-related impurities. The stress treatments, which included oxidation of methionine residues and storage at elevated temperatures for different periods of time, were designed to induce various degrees of degradation, aggregation or oxidation of the interferon. Biological activity of the stressed preparations was assessed in three different in vitro cell-based bioassay systems: a late-stage anti-proliferative assay and early-stage assays measuring reporter gene activation or endogenous gene expression by quantitative real time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Relevant physicochemical methods such as SDS-PAGE, reverse phase (RP) chromatography, size-exclusion chromatography (SEC) and dynamic light scattering (DLS), proved their complementarity in detecting structural changes in the stressed preparations which were reflected by reductions in biological activity