Characterisation of Thiol-releasing and Lower Volatile Acidityforming Intra-genus Hybrid Yeast Strains for Sauvignon blanc Wine

A single Saccharomyces cerevisiae wine yeast strain produces a range of aroma and flavour metabolites (e.g. volatile thiols), as well as unfavourable metabolites (e.g. volatile acidity [VA]) during the alcoholic fermentation of white wine, especially Sauvignon blanc. The former contributes to the organoleptic quality of the final wine. Previous research showed that yeast derived enzymes (proteins) are involved in the release of wine quality enhancing or reducing metabolites during fermentation. Small-scale winemaking trials were initiated to evaluate protein expression and metabolite release of tropical fruit aroma wine producing S. cerevisiae hybrid yeasts. Commercial ‘thiol-releasing’ wine yeasts (TRWY) were included in winemaking trials as references. Improved hybrids were identified which showed enhanced thiol-releasing, specifically 3-mercaptohexanol (3MH), and lower VA formation during the production of Sauvignon blanc wines compared to some commercial TRWY references. It is noteworthy that the hybrid NH 56 produced wines with the second highest 3MH levels after hybrid NH 84, and lowest acetic acid of all strains included in this study. This yeast was also the only strain to have down-regulated proteins linked to amino acid biosynthesis, pentose phosphate pathway, glycolysis and fructose and galactose metabolism during the lag phase. Furthermore, differences in protein expression were reflected in the variation of metabolite release by different strains, thereby confirming that enzymes (proteins) are the final effectors for metabolite release. 

Regulatory practice and transport modelling for nitrate pollution in groundwater

This report forms the first deliverable of a project jointly funded by BGS and the Environment Agency to consider the potential for incorporating the outputs from the BGS unsaturated zone travel time work in assessing the risks to water from nitrate. This is to help to inform the nitrate vulnerable zones (NVZs) designation process. In England, the Environment Agency advises Defra on identifying areas for designation as NVZs. Over time, the designation process has developed and become more complex since the first round of designations in 1996. The designation process for groundwater initially used only public supply monitoring data and the associated source catchment area. In December 2000, the European Court of Justice held that the UK had failed to designate sufficient NVZs for the protection of all waters, not just for drinking water sources. This resulted in the development of revised methodologies for the designation of NVZs which separately address surface waters, groundwater and waters at risk of eutrophication. This was implemented in 2002. Further reviews have been carried out in 2008 and 2012 and as a result, modifications and improvements to methods have been made at each designation round. For groundwater the Environment Agency developed a numerical risk assessment procedure that uses a range of risk factors including both nitrate concentration data and nitrate-loading data to assess the risk of nitrate pollution. The loading data is based on farm census returns made to Defra and combined using the NEAP-N methodology developed by ADAS (Lord and Anthony, 2000). The overall risk assessment considers both current observed concentrations and predicted future concentrations as well as current loadings. However, this approach has a number of disadvantages including a lack of a specific term for the time of travel to the water table and emergence of pollutant both into groundwater and to groundwater discharge points that support surface water features. Instead, these issues are considered at the conceptual level in workshops with local EA hydrogeologists. A key question for Defra and the Agency is how long it will take for nitrate concentrations to peak and then stabilise at an acceptable, lower level, in response to existing and future land management control measures. This is most important for soils, for aquifers, for lakes and for groundwater-fed wetland systems that respond less quickly to changes in loading. Groundwater and lake catchment numerical models can provide first-order estimates of likely response times, but can be difficult and costly to set-up for many different situations and are difficult to apply consistently at the national scale. A previous review of nitrate vulnerable zones suggests a range of further needs: • to understand the recent developments in nitrate pollution simulation and particularly the potential to understand/characterise past nitrate loading from changing land management practices and correlate these with observed nitrate concentrations over time; • to evaluate the retention of nitrate in catchments, particularly in the unsaturated zone of soils and aquifers; • to simulate the recent and future anticipated decreases in nitrate loading by sectors within the UK; • to understand the likely time taken for nitrate concentrations to peak and then stabilise at an acceptable, lower level, in response to existing and future control measures. Without evidence of how long it may take systems to recover it is difficult to evaluate the effectiveness of existing measures or decide whether additional measures are necessary. The aim of this project is to investigate the use of new models to inform decision-making on nitrate pollution in groundwater and the potential for incorporating unsaturated zone processes in future NVZ designations. The work described here forms the first task of this project and aims to review NVZ methodology and recent designation experience. As part of this we will: • collate information from the Agency on the recent application of the methodology; • provide case study examples of designation in different time-lag settings and/or where these are not corroborated by water quality

Improved Quantitative Plant Proteomics via the Combination of Targeted and Untargeted Data Acquisition.

Quantitative proteomics strategies - which are playing important roles in the expanding field of plant molecular systems biology - are traditionally designated as either hypothesis driven or non-hypothesis driven. Many of these strategies aim to select individual peptide ions for tandem mass spectrometry (MS/MS), and to do this mixed hypothesis driven and non-hypothesis driven approaches are theoretically simple to implement. In-depth investigations into the efficacies of such approaches have, however, yet to be described. In this study, using combined samples of unlabeled and metabolically (15)N-labeled Arabidopsis thaliana proteins, we investigate the mixed use of targeted data acquisition (TDA) and data dependent acquisition (DDA) - referred to as TDA/DDA - to facilitate both hypothesis driven and non-hypothesis driven quantitative data collection in individual LC-MS/MS experiments. To investigate TDA/DDA for hypothesis driven data collection, 7 miRNA target proteins of differing size and abundance were targeted using inclusion lists comprised of 1558 m/z values, using 3 different TDA/DDA experimental designs. In samples in which targeted peptide ions were of particularly low abundance (i.e., predominantly only marginally above mass analyser detection limits), TDA/DDA produced statistically significant increases in the number of targeted peptides identified (230 ± 8 versus 80 ± 3 for DDA; p = 1.1 × 10(-3)) and quantified (35 ± 3 versus 21 ± 2 for DDA; p = 0.038) per experiment relative to the use of DDA only. These expected improvements in hypothesis driven data collection were observed alongside unexpected improvements in non-hypothesis driven data collection. Untargeted peptide ions with m/z values matching those in inclusion lists were repeatedly identified and quantified across technical replicate TDA/DDA experiments, resulting in significant increases in the percentages of proteins repeatedly quantified in TDA/DDA experiments only relative to DDA experiments only (33.0 ± 2.6% versus 8.0 ± 2.7%, respectively; p = 0.011). These results were observed together with uncompromised broad-scale MS/MS data collection in TDA/DDA experiments relative to DDA experiments. Using our observations we provide guidelines for TDA/DDA method design for quantitative plant proteomics studies, and suggest that TDA/DDA is a broadly underutilized proteomics data acquisition strategy

Quantification of nitrate storage in the vadose (unsaturated) zone: a missing component of terrestrial N budgets

National terrestrial nitrogen budgets for many developed countries have been calculated as part of the management of impacts of N on the environment, but these rarely represent the subsurface explicitly. Using estimates of vadose zone travel time and agricultural nitrate loading, we quantify, for the first time, the total mass of nitrate contained in the vadose zone of aquifers in England and Wales. This mass peaked in 2008 at 1400 kt N (800 to >1700 kt N from sensitivity analyses) which is approximately 2.5 to 6 times greater than saturated zone estimates for this period and indicates that the subsurface is an important store of reactive nitrogen. About 70% of the nitrate mass is estimated to be in the Chalk, with the remainder split between the Permo-Triassic sandstones, the Jurassic Oolitic limestones and minor aquifers. Current controls on fertiliser application mean that the vadose zone is now a nitrate source and in 2015 we estimate the net flux from the unsaturated zone to groundwater to be 72 kt N/annum. The mass of nitrate in the vadose zone should be included in future terrestrial nitrogen budgets at national and global scales to improve ecosystem management. This article is protected by copyright. All rights reserved

Characterisation of Saccharomyces cerevisiae hybrids selected for low volatile acidity formation and the production of aromatic Sauvignon blanc wine

Wine yeasts (Saccharomyces cerevisiae) vary in their ability to develop the full aroma potential of Sauvignon blanc wine due to an inability to release volatile thiols. Subsequently, the use of ‘thiolreleasing’ wine yeasts (TRWY) has increased in popularity. However, anecdotal evidence suggests that some commercially available TRWY intermittently exhibit undesirable characteristics for example, volatile acidity (VA) formation. Therefore, a trial was undertaken to select and evaluate S. cerevisiae hybrids for the production of Sauvignon blanc wine with enhanced fruity and tropical aromas, but low VA. Hybrids were characterised by clamped homogeneous electrical field (CHEF) DNA karyotyping and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) biotyping, and subsequently trialled against top commercial TRWY that is, Zymaflore VL3 and Zymaflore X5 (Laffort Oenologie), and Fermicru 4F9 (DSM Oenology) in laboratory-scale Sauvignon blanc vinifications during 2013. Most hybrids produced wines with VA levels significantly lower than those produced with Zymaflore VL3, Zymaflore X5 and Fermicru 4F9. Low VA forming hybrids also produced wines with tropical wine aroma notes. Wines produced by Fermicru 4F9 had the lowest acetic acid (the main volatile acid) of the commercial TRWY in this study. However, some hybrid yeasts produced wines with less acetic acid on average than wines produced by Fermicru 4F9. Overall, hybrids NH 6, NH 48, NH 56, NH 88 and NH 145 produced wines with enhanced tropical fruity aroma, but lower VA compared to wines produced by commercial TRWY.Keywords: Hybrid yeasts, CHEF, MALDI-TOF/TOF MS biotyping, Sauvignon blanc, tropical fruit aroma, volatile acidit

Modelling the groundwater nitrate legacy

This report details the findings of a project jointly funded by the British Geological Survey (BGS)and Defra through the Environment Agency. The overall aim of the work was to investigate the use of new models to inform decision-making on nitrate pollution in groundwater and the potential for incorporating unsaturated zone processes into the model currently used by the Environment Agency to delineate Nitrate Vulnerable Zones (NVZs). The potential application as supporting evidence for the Water Framework Directive has also been considered as nitrate pollution of groundwater remains the most significant reason for failure of WFD environmental objectives across England. The background to the nitrate legacy in groundwater and to the approaches to NVZ designation is described in Stuart et al. (2016). A series of developments to the BGS Nitrate Time Bomb (NTB) model have been made to improve a number of areas and approaches used in the first version of the model. The improvements included a spatially and temporally distributed nitrate input function, improved unsaturated zone thickness estimation, travel time attribution using a 1:250,000 geological map, estimating nitrate velocity in the unsaturated zone using groundwater recharge and aquifer properties, and introducing nitrate transport processes in low permeability superficial deposits and the saturated zones. These now allow the model to be applied at sub national scale. Using the improved model we have also made the first estimate of the mass of nitrate stored within the unsaturated zone and how this will change over time to improve UK nitrate budget estimates. The new version of the BGS NTB approach was applied in three case studies at different scales which compared its outcomes to the results from other modelling to demonstrate that the model can be benchmarked against the other nitrate modelling approaches: • For a basin-scale model of the Thames Chalk (Howden et al., 2010 & 2011). The NTB model gave comparable results to the original study back to 1925 provided that the same nitrate input function was used. Both models failed to predict nitrate concentrations in the Thames after the mid-1980s. • At the multi-borehole scale in the Permo-Triassic. A similar approach was used to the BGS model in the Eden Valley. This replicated the existing model for the area used by the Environment Agency both in terms of trend assessment and in the lack of dilution available within the aquifer block for blending purposes. • At the single borehole scale in the Chalk of the South Downs. The existing Environment and National Park model constructed by AMEC treated the unsaturated zone very similarly to the NTB model. This model provided a good fit to observed concentrations and confirmed the importance of estimating unsaturated zone delays. The assessment of modelled travel time from different areas of the catchment clearly illustrated the arable areas that would give a relatively rapid respond to changes in nitrate management. To illustrate the potential application of the BGS NTB model to support the Environment Agency’s NVZ designation methodology, areas of England were identified where unsaturated zone lags may be significant and where there is uncertainty in the NVZ designation. A major advantage of the BGS NTB model is that it covers the whole of England (and Wales) in a consistent way. A national overview of areas of designation uncertainty identified large areas of England, in particular the chalk outcrop of southern and eastern England. These were compared to areas with significant unsaturated zone travel time indicating where travel time may be contributing to designation uncertainty. The results suggest that the model may be useful both for identifying currently impacted groundwater which reflects legacy fertilizer application and also where additional designation could be needed as impacts have not yet emerged. Application of the model to support implementation of the WFD has also been considered and whilst no quantitative analysis has yet been carried out there are a number of ways that the model could be of significant benefit. For example, the model could be used to estimate when trend reversal would be expected to occur as a result of measures (at a specific location or across a groundwater body) and the time required to achieve good chemical status (alternative objective setting). A further application could be for scenario testing such as evaluating the effects of different land use/management measures as part of cost benefit analysis or considering the long term impacts of climate change through changing fertiliser use and/or recharge

Loading on a wind turbine high-speed shaft gearbox bearing : ultrasonic field measurements and predictions from a multi-body simulation

Wind turbine gearbox failures, many of which result from load-driven rolling element bearing failures, account for long downtimes and high maintenance costs. Non-invasive, in-situ measurement of bearing roller loads using ultrasound facilitates better understanding of the load history and failure mechanisms these gearbox bearings experience. In this study, ultrasonic load measurements from an instrumented high-speed shaft bearing were compared against results from a static multibody simulation. Measurements taken under steady operating conditions were found to agree well with the model. After quantification of the error in ultrasonic measurements the model enabled identification of load outliers which were attributed to transient events. Over- and under-loading instances may act to promote a variety of failure mechanisms, leading to premature bearing failure

A method for screening groundwater vulnerability from subsurface hydrocarbon extraction practices

This paper describes a new screening method for assessing groundwater vulnerability to pollution from hydrocarbon exploitation in the subsurface. The method can be used for various hydrocarbon energy sources, including conventional oil and gas, shale gas and oil, coal bed methane and underground coal gasification. Intrinsic vulnerability of potential receptors is assessed at any particular location by identifying possible geological pathways for contaminant transport. This is followed by an assessment of specific vulnerability which takes into account the nature of the subsurface hydrocarbon activity and driving heads. A confidence rating is attached to each parameter in the assessment to provide an indication of the confidence in the screening. Risk categories and associated confidence ratings are designed to aid in environmental decision making, regulation and management, highlighting where additional information is required. The method is demonstrated for conventional gas and proposed shale gas operations in northern England but can be adapted for use in any geological or hydrogeological setting and for other subsurface activities