Under the terms of the Renewable Transport Fuel Obligation (RTFO), the UK is committed to substituting 5.75% of its gasoline consumption with bio-derived fuels by December 2010. This demand is predicted to increase in the future, particularly in response to concerns about climate change and fuel security. Current, biofuel generation in the UK is negligible and demands are met by bioethanol imports from countries such as Brazil. Bioethanol is mainly produced from 'first generation' crops (e.g. maize, wheat, sugar beet and sugar cane) which are characterised by a high non-structural carbohydrate content. The technology involved is straightforward and production has become more price competitive. The feasibility of producing biofuel from such crops in the UK is limited because of the requirement for arable land which is primarily used for food production and the high energy input involved. Production of biofuel from 'second generation' lignocellulosic crops such as grasses offers a potential alternative. Grasslands comprise up to 70% of UK agricultural land greatly exceeding the area used for food crops. Perennial ryegrass achieves similar biomass yields to other lignocellulosic crops used for biofuel production. This crop has a number of traits which are desirable in a fermentable feedstock including a readily available high water-soluble sugar content, high fibre digestibility and a low lignin content in comparison with other candidate lignocellulosic crops. Perennial grasses have low annual input requirements and contribute to the rural landscape, maintaining biodiversity and environmentally sensitive landscapes which have major attractants for the tourist industry. UK farmers have the necessary expertise involved in management of these grasses which can be harvested over a long season and stored over winter. We propose that perennial ryegrass can provide an environmentally and economically viable feedstock for the production of bioethanol and that existing biological material and technologies can be readily adapted to achieve this. The main challenges for development of a sustainable, low input process, for conversion of grasses to bioethanol will be addressed in this programme. This will include reducing the major operating costs, maximising yield and carbon cost efficiency. IGER's large selection of ryegrass germplasm will be exploited to select for appropriate varieties, in particular, high sugar perennial grasses with high digestibility (low lignin). The legume, white clover, will be included in grass swards to provide nitrogen and minimise green house gas emissions associated with artificial fertilizer. This programme will test the feasiblility of juicing on-farm to generate two separate feedstocks; a water soluble carbohdyrate (fructan) rich liquid fraction and a high dry-matter stable lignocellulosic fraction. Procedures for handling, preserving/stabilising and transporting these feedstocks will be assessed. A major aim is to maximise utilisation of the full range of sugars in perennial ryegrass for fermentation to ethanol. This will be achieved by using an appropriate combination of pre-treatments, enzymes, yeast and an ethanol producing thermophilic micro-organsim, taking advantage of TMO Renewables groundbreaking method for producing ethanol from almost any type of biomass. Fermentation conditions will be optimised to maximise ethanol production from ryegrass feedstock both at laboratory and pilot scale. The carbon and energy balance as well as the economic viability of these processes will be evaluated. Data generated by this programme will provide valuable information for accurate comparisons with other crops used in bioethanol production.

In this project, researchers from Imperial College London and the University of Bath will work together with the company TMO Renewables Ltd to (a) understand fundamental aspects of the physiology and biochemistry of the thermophilic bacterium Geobacillus thermoglucosidasius, which the company uses in its current bio-ethanol process, and (b) develop computer based metabolic models, using a combination of genome sequence information and experimental measurements, which will be useful for predicting how to make changes to the organism so that it can produce a specific end-product from a variety of different substrates. While the company has been successful in creating a strain of Geobacillus thermoglucosidasius that can produce ethanol from renewable lignocellulose and fermentable components of waste, this was done with little understanding of how the organism behaves under complex fermentation conditions. During this process, many observations have been made that are not easy to explain from our limited current knowledge of the organism. As well as a financial contribution to the project, the company will provide the genome sequence for their parent strain. This is the first (available) complete genome sequence for this species of thermophile and provides the academic researchers with a significant platform from which to make new discoveries. Building on this platform, the research team will apply recently-developed methods for model building, model validation and physiological investigation. The latter will involve the newly-developed approach of 'transcriptomics' by 'RNA -sequencing' to understand how the organism regulates its metabolism and behaviour under different physiological conditions. Direct analysis of RNA (strictly speaking, it has to be converted to DNA before sequencing) using modern methods of high-throughput sequencing is an advance on the previous approach using microarrays, because it does not rely on initial deduction of which are bona-fide gene sequences in a genome. Because the analysis is essentially blind to prior assumptions, it has revealed many unexpected features of regulation in different bacteria. Papers on the application of this method to bacteria only started appearing in 2009, and most of these either focus on methods development or pathogenic organisms. This project will give us the opportunity to look at an industrially relevant organism, addressing questions that are pertinent to industrial fuel and chemical production from biomass and ultimately testing hypotheses and strains in an industrial context. Therefore, there is a strong chance for discovering new and fundamental processes underlying the regulation of microbial growth and metabolism. One of the outputs from this project will be a set of metabolic models. In silico metabolic models can be useful for predicting how metabolic flux should be altered to achieve a specific outcome (eg enhanced growth or metabolite overproduction). So, as part of this exercise, we will use the models in a metabolic engineering programme to make a new metabolite, not normally produced by this strain. Using the model, we should be able to predict how flux through different pathways should be changed to accomplish the dual requirements of rapid growth and product formation. In addition to this, we hope to link the transcriptomic analysis to the models. Metabolic models are essentially static pictures that do not adequately incorporate the dynamic aspects of physiological regulation. By studying cells under different growth conditions, we can generate a set of 'condition-specific models' which can be linked through comparative analysis of the transcriptomic data. The team involves a systems biologist who is expert at integrating different types of data, who will explore the possibility of linking the two types of analysis in a meaningful manner.

Our aim is to develop a sustainable, integrated platform for manufacture of industrial chemicals based on biological terpenoid feedstocks to complement carbohydrate, oil and lignin-based feedstocks that will be available to sustainable chemistry-using industries of the future. Our focus will include production of aromatics and amines which are particularly challenging targets from other biofeedstocks. Transition from fossil-based feedstocks to renewable alternatives is a key challenge for the 21st Century. Major efforts are underway to address this with work currently focused on carbohydrates, fats and oils, and lignins all of which give rise to fundamental technological barriers due to the incompatibility of complex and oxygen-rich materials with conversion technologies developed for simple hydrocarbon-based petrochemical feedstocks. This often requires biological feedstocks to undergo costly and inefficient transformations and separations prior to deployment in existing supply chains. In contrast, terpenes are an abundant class of natural products based on the C5 isoprene unit. As hydrocarbons they are easily separated from aqueous environments and can be readily upgraded using existing petrochemical technologies. While terpenes have been used in limited quantities since antiquity (notably as flavours and fragrances) they have yet to be exploited systematically for the production of platform chemicals even though they represent a potentially vast resource: global biogenic production of terpenes is 10^9 t/yr. Significant volumes of useful terpenes are already available on global markets at low cost (production of turpentine oils and limonene are 330,000 and 30,000 t/yr, respectively, the former costing 0.09-0.19 Euros per L). While this is sufficient in itself to justify a viable value-added chemical platform (metrics comparable to those for lignin: 1.1m t/yr at £250-2,000 per t) such figures will be dwarfed in the near future through the large-scale (multimillion t/yr) microbial production of terpenes such as farnesene for biofuels via the engineering of isoprene metabolic pathways. This industrial biotechnology (IB) approach, developed by Amyris and others, promises large-scale and geographically flexible supplies of terpenes via fermentation of plant sugars and cellulosic waste. Thus, the exploration of new generic technologies for the chemical exploitation of terpenes is timely, not only in terms of sustainable utilization of current global resources, but also to take advantage of major developments in IB. However, key challenges to be addressed in the context of terpene-based manufacturing include: (i) development and optimization of sustainable chemical transformations; (ii) scale-up of intensive conversion processes; (iii) development of new terpene sources; and (iv) systems-level understanding of technical, environmental and economic factors associated with new terpene-based manufacturing technologies. This project will address these challenges directly in four interconnected workpackages. Outputs from the project will provide a competitive advantage for one of the UK's most successful industries. Chemistry-reliant industries contributed an equivalent of 21% GDP to the UK economy in 2007, they support 6m jobs (RSC 2010), and turnover is growing at 5% pa (UKTI, 2009). The utilization of IB is vital to sustaining competitive advantage, with the value of the UK IB market in 2025 estimated at £4b to £12b (BERR 2009). Specific to this project, the development of new integrated technologies for terpene-based manufacturing, ultimately via microbial fermentation of waste cellulose, will provide competitive advantage for UK industries through new sustainable manufacturing processes, reduced feedstock costs, security of supply and reduced environmental impact. The UK will benefit further from export of new technologies and services and from development of new skills vital to future low carbon manufacturing.

The UK government recognises that 'our economy is driven by high levels of skills and creativity' and has prioritised investment in skills as a means to recovery rapidly from the current economic downturn (HM Government: New Industry, New Jobs, 2009). Bioprocessing skills underpin the controlled culture of cells and microorganisms and the design of safe, environmentally friendly and cost-effective bio-manufacturing processes. Such skills are generic and are increasingly being applied in the chemical, pharmaceutical and regenerative medicine sectors. Recent reports, however, highlight specific skills shortages that constrain the UK's capacity to capitalise on opportunities for wealth and job creation in these areas. They emphasise the need for bioprocessing skills related to the application of 'mathematical skills... to biological sciences', in core bioprocess operations such as 'fermentation' and 'downstream processing' and, for many engineering graduates 'inadequate practical experience'. UK companies have reported specific problems in 'finding creative people to work in fermentation and downstream processing' (ABPI: Sustaining the Skills Pipeline, 2005 & 2008) and in finding individuals capable of addressing 'challenges that arise with scaling-up production using biological materials' (Industrial Biotechnology Innovation and Growth Team report: Maximising UK Opportunities from Industrial Biotechnology, 2009). Bioprocessing skills are also scarce internationally. Many UK companies have noted 'the difficulties experienced in recruiting post-graduates and graduates conversant with bioprocessing skills is widespread and is further exaggerated by the pull from overseas (Bioscience Innovation and Growth Team report: Bioscience 2015, 2003 & 2009 update). The EPSRC Industrial Doctorate Centre (IDC) in Bioprocess Engineering Leadership has a successful track record of equipping graduate scientists and engineers with the bioprocessing skills needed by UK industry. It will deliver a 'whole bioprocess' training theme based around fermentation and downstream processing skills which will benefit from access to a superbly equipped £25M bioprocess pilot plant. The programme is designed to accelerate graduates into doctoral research and to build a multidisciplinary research cohort. Many of the advanced bioprocessing modules will be delivered via our MBI Training Programme which benefits from input by some 70 industry experts annually (www.ucl.ac.uk/biochemeng/industry/mbi). Research projects will be carried out in collaboration with many of the leading UK chemical and pharmaceutical companies. The IDC will also play an important role supporting research activities within biotechnology-based small to medium size enterprises (SMEs). The need for the IDC is evidenced by the fact that the vast majority of EngD graduates progress to relevant bioindustry careers upon graduation. This proposal will enable the IDC to train the next generation of bioindustry leaders capable of exploiting rapid progress in the underpinning biological sciences. Advances in Synthetic Biology in particular now enable the rational design of biological systems to utilise sustainable sources of raw materials and for improved manufacturing efficiency. These will lead to benefits in the production of chemicals and biofuels, in the synthesis of chemical and biological pharmaceuticals and in the culture of cells for therapy. The next generation of IDC graduates will also possess a better understand of the global context in which UK companies must now operate. This will be achieved by providing each EngD researcher with international placement opportunities and new training pathways either in bioprocess enterprise and innovation or in manufacturing excellence. In this way we will provide the best UK science and engineering graduates with internationally leading research and training opportunities and so contribute to the future success of the UK bioprocess industries.

The use of fossil fuels in the energy and chemical industries is no longer tenable; they represent a finite resource and their use results in carbon dioxide emissions, which is a major cause of global warming. There is, therefore, an urgent need to find alternative sources of liquid fuels that are renewable and do not have an adverse effect on the environment. Lignocellulosic biomass is a promising substrate for biofuel production as it is not a food source, is more abundant than starch, and its use is carbon dioxide neutral. A significant limitation in the use of lignocellulosic biomass in the biofuel industry is its recalcitrance to enzyme attack. Thus, cellulose, the major polysaccharide in lignocellulosic biomass, is chemically simple but its highly crystalline structure makes it inaccessible to enzymes that act as hydrolases. Recent studies, however, have identified novel enzymes that could improve the efficiency of plant cell wall deconstruction. Thus, several reports have shown that oxidases cleave bonds in crystalline regions of cellulose, leading to increased access to hydrolase attack. Significant advances have also been made in the degradation of xylan, the major matrix polysaccharide in lignocellulosic biomass. It was widely believed that degradation of the main chain of xylan required the removal of side chains prior to attack by xylanases. It is now apparent that a cohort of xylanases not only accommodate side chains, but actually display an absolute requirement for these decorations. We have also shown that it is possible to introduce novel functionalities into the active site of biotechnologically significant arabinofuranosidases that assist in removing the side chains from xylan. The generation of such multifunctional enzymes has the potential to simplify the biocatalysts required to deconstruct plant cell walls, and thus increase the economic potential of lignocellulosic biomass as a substrate for the biofuel industry. In this project we will explore the mechanism by which cellulose oxidases, arabinoxylanases and multifunctional arabinofuranosidase/xylanases recognize their target substrates. The data will provide a blueprint for further enhancing the efficiency of the plant cell wall degrading catalytic toolbox.