[16- FAPESP-BE] An integrated approach to explore a novel paradigm for biofuel production from lignocellulosic feedstocks

Climate change is being driven, at least partly, by the burning of fossil fuels and consequent CO2 release into the environment. To mitigate this we need to produce more fuels/chemicals from renewable resources. One globally relevant abundant resource is lignocellulose (present in wood, straw, grasses and in many waste streams) and efforts are being made to exploit this efficiently. However, current processes have inherent inefficiencies due to the limitations of yeast, the most common organism used in biofuel fermentations. Yeasts are good at converting simple sugars such as glucose and sucrose to ethanol, but natural strains cannot metabolise xylose, which is abundant in lignocellulose, or longer chains of sugars (oligosaccharides). This means that for yeast fermentations it is necessary to break down the lignocellulose to simple monomeric sugars for them to be utilised effectively. This approach generally requires harsh physico-chemical pre-treatment methods which, increase the energy demand of the process and produce compounds that can inhibit the subsequent fermentation. Thus it is often necessary to remove these inhibitors, which adds expense to the process. In this project we intend to demonstrate that it is more sensible (logical and economic) not to pre-treat lignocellulose so harshly, and have a more "holistic" approach to the process: delivering the desired products whilst minimising overall process energy and cost by working on the optimisation of generating partial breakdown products and ensuring that the subsequent fermentation organism is able to convert these directly to product.
The most commonly employed class of fermentation organisms - yeasts - will be engineered to be able to convert the oligomeric sugars directly. However, there is a class of organisms - Geobacillus - that have been quite extensively studied by one of the UK groups, which already naturally has the propensity to utilise oligomeric sugars and can also be readily engineered to optimise key metabolic pathways. Therefore, in this project we will use a representative of this group of bacteria to compare performance with the engineered yeast.
We also propose to consider three different lignocellulosic feedstocks in this study, all of which have the potential to be used for sustainable fuels and chemicals production: Brazilian cane straw - which is current left in the fields after harvesting, Miscanthus - which is grown in the UK for burning in power stations (co-firing) and has a lot of similarities to cane straw, and Eucalyptus forestry residues, which are abundant in Brazil and represent a different type of opportunity and material to evaluate. Some of the team involved will focus on developing methods to convert these to oligosaccharides that can be taken up by these new strains. This will be a combination of less severe (than currently) pre-treatment and the use of selected enzymes to produce the oligo-saccharides required. Another part of the team will focus on producing the enzymes required for these conversions to oligosaccharides, while a third group will engineer the yeast strains to use oligosaccharides of both xylose and glucose.
To increase the energy efficiency of the feedstocks in the new lignocelulose mills we are going to recover chemicals and biogas from the liquid effluents, vinasse and hemicellulose hydrolysates, by integrating anaerobic digestion (AD) to the process. AD with mixed culture fermentation will improve the energy ratio bringing biogas production and fertilizers as products.
Underpinning all this is the need to ensure that the outputs of this work remains relevant to the industry processes that they potentially feed into. Therefore we have a team of LCA experts ensuring that feedstock/ product choice is appropriate, that the proposed process optimisation approaches are delivering a positive impact on process performance and pinpointing where further changes/modifications could be made.

Current biofuel production focusses on ethanol using carbohydrates generated from first generation feedstocks (sucrose from cane/beet and glucose from corn/wheat). Yeasts use these simple substrates and naturally produce ethanol; in Brazil the fermentation tanks are open to invasion and natural selection of wild yeast strains. To generate fermentation products other than ethanol this strategy is unfeasible; indeed protection from wild yeast is essential to avoid take-over by ethanol producers. This can be achieved by maintaining absolute asepsis (which is expensive to maintain) or devising conditions in which wild yeast will not grow. We propose a strategy for the latter which incorporates the desire to move towards second generation (lignocellulose based) feedstocks. The carbohydrates in lignocellulose are polymeric and also more diverse, including pentose sugars that some yeast strains cannot naturally ferment, although engineered strains have been developed for this. However, wild yeasts cannot ferment polymeric or oligomeric carbohydrates, so current second generation processes require conversion of the polymers to monomers. The experience of the UK PI with thermophilic Geobacillus (bacteria) shows that organisms that use oligomers directly are a) metabolically efficient and b) can outcompete pure monomer utilisers (as the monomers are not produced). So, at the heart of this project is a combined strategy of developing lignocellulose pretreatment methods (physical and enzymatic) that produce mainly oligomers and developing/utilising strains that are capable of utilising oligomers. For this we will create strains of oligomer utilising yeasts and compare the fermentation characteristics of these to Geobacillus spp. To use a non-ethanol producing system we will engineer yeast and Geobacillus to produce isobutanol. This has been described for yeast, and a strategy is available for Geobacillus spp.
As part of the programme we will also investigate AD of the residue

Wider Academic Community: Although this project has an industrial goal, to achieve this requires fundamental advances in biomass processing technology and development of the process organisms Saccharomyces cerevisiae and Geobacillus spp. These advances will benefit the wider community of research microbiologists using these hosts as well as those working in the wider field of metabolic engineering and bioenergy research.
Commercial Private Sector: The focus of this project is to develop more efficient and economic processes to generate novel biofuels (and other chemicals). We will be engaging an academic forum and advisory group in order to translate this research into practice as rapidly as possible. They will also provide data for the project and help define useful boundaries and targets for process parameters. Towards the end of the programme we will hold a workshop/meeting targeted at an industrial user group in order to bring them up to date with the field and put our work into context.
More broadly, the results of this programme should benefit all companies operating in the area of chemicals from renewables. Primarily, this will be through furthering our understanding of substrate utilisation and catabolite regulation in two different potential host organisms. The project intends to develop and demonstrate a novel paradigm for biofuel fermentation, which addresses the challenge of moving from ethanol fermentations to novel products. This has the potential to generate a step change in technology and approach to the production of chemicals from biomass.
National and International Perspective:
Climate change: A primary driver for the move from fossil fuels to fuels and chemicals from waste, or sustainably derived renewables, is the reduction in greenhouse gas (GHG) emissions. An efficiently operated biorefinery using cellulosic substrates should be able to deliver an 80% reduction in GHG emissions compared to its fossil fuel equivalent (based on ethanol production). This will help meet national and international targets for use of renewables and mitigation of climate change.
Green jobs: The successful delivery of this project will have an impact on delivering green jobs within the UK and further afield - a more diversified, and hence valuable, technology platform will be more attractive to new customers and take up of the technology will be greater, promoting growth within the Cleantech sector. For the PDRAs, the possibility of working closely with an international, industry focussed consortium will give them an excellent perspective of both academic and industrial research environments, which should be invaluable for their future employment prospects