Evaluation of consolidated bioprocessing as a strategy for production of fuels and chemicals from lignocellulose

There are a number of reasons why it is imperative to look at alternatives to fossil fuels as sources of energy and chemicals. Of immediate concern is the major contribution that the burning of fossil fuels is making to greenhouse gas accumulation. While power stations might address this through carbon capture, this is not feasible for distributed systems such as transport and chemicals. Therefore, there is a strong drive to find renewable sources of liquid fuels and chemicals. Currently, this is being addressed using glucose obtained from maize or wheat starch, which is having a negative impact on the price of starch for food. We urgently need to find ways to use the more abundant carbohydrates found in woody material and grasses (lignocellulose), either purpose grown or as wastes. However, this is currently uneconomic due mainly to the cost of releasing the useful carbohydrate from woods and grasses (pre-treatment). This project will address the economics of pre-treatment by exploring the benefits of an approach called 'Consolidated Bioprocessing' (CBP). Current methods rely on acids or alkalis to break down either the lignin and/or some of the long chains (polymers) of carbohydrates found in woody material, followed by treatment with enzyme cocktails, generally referred to as cellulases, but typically containing a complex mixture of enzyme activities, to produce monomeric carbohydrates. These cellulases are typically bought from one of the two major enzyme producers and add a significant cost and greenhouse gas contribution to the process. The concept of CBP envisages that the micro-organism that produces the desired end-product also produces some/all of the enzymes necessary to break down the carbohydrate polymers, so reducing these additional costs. In order to be able to investigate the benefits and disadvantages of CBP within the timescale of the programme we intend to work with a group of bacteria known as Geobacillus spp which grow best in the 50-70oC temperature range and already have the ability to use some of the intermediates produced from polymer degradation. In this way we should only have to engineer in a limited array of novel activities, and we already have the genetic tools and organisms containing the relevant additional enzymatic activities that we require. The project will focus on using miscanthus, a high-yielding grass under consideration in the USA and Europe as a purpose-grown lignocellulosic feedstock. This will be pre-treated by two established methods that leave the polymeric carbohydrates largely intact but disrupt the lignin. Having constructed suitable bacterial strains for CBP the intention is to generate information that allows us to judge the practically and economics of the process when operated at a large scale. This will be done largely by generating models, based on information gathered from small scale operations, and applying appropriate scaling-up criteria. Factors such as the consequence of including significant amounts of solids in the fermentation process and the 'cost' to the organism of producing extra enzymes will be examined. Because CBP is still largely a concept, there are a number of areas of process development which may impact overall practicality and economics which will need to be addressed during the project. By doing this we intend to produce the first genuine assessment of the practicalities of CBP and guidelines on areas that may need further improvement.

To improve the greenhouse gas balance associated with using renewables for fuels and chemicals and avoid the food versus fuel conflict, we must move to using lignocellulose (LC) from crop wastes or purpose grown feedstocks. Although the economics have improved in recent years, the costs associated with recovering fermentable carbohydrate from LC need to be improved. A typical pretreatment involves physico-chemical disruption followed by enzymatic hydrolysis of the carbohydrate polymers. Conceptually, a more economic process would replace much or all of the externally sourced enzymes with enzymes produced by the fermenting organism. This is the basis of 'Consolidated Bioprocessing' (CBP). Currently CBP is little more than a concept. In this project we intend to use the thermophilic facultative anaerobe Geobacillus thermoglucosidasius to build a CBP platform, using miscanthus as a substrate, in order to evaluate the benefits/potential problems associated with CBP. G. thermoglucosidasius is a good platform for CBP as it already metabolises cellobiose and short chain xylans. We have isolated other Geobacillus spp which are cellulolytic, xylanolytic and pectinolytic and have the genetic tools to add the relevant catabolic capabilities to this host. Miscanthus is a high yielding grass which is under consideration as an 'energy crop' in Europe and the USA. We will pre-treat miscanthus with 2 different ammonia based pre-treatments which largely attack the lignin and use these to gain process data on the effectiveness of different fermentation scenarios, measuring a range of different process parameters to enable effective modelling. These will include biological factors such as growth, metabolite and enzyme productivity (linked to transcription, translation, secretion and sequestration), rates and nature (progression) of utilisation of complex substrates. Different processing regimes (batch and fed batch) will be considered and studies of fluid dynamics made throughout.

Who will benefit from this research? This is a full proposal invited on the basis of an outline submission to the IBTI club. As an industry club, the immediate beneficiaries of the research will be the club members who have contributed to its foundation, some of whom have a direct interest in the use of thermophilic bacteria for industrial processes. In the longer term, a second group of beneficiaries will be UK farmers. The project is based on the use of lignocellulosic biomass which is already being grown on a small scale in the UK but, if this project is successful, could become major crops for renewable production of bulk chemicals. It is also reasonable to suggest that the benefits to UK Industry and farmers ultimately benefit UK government and that by targeting fossil fuel replacement there will be a wider environmental benefit for all of us. How will they benefit? Industrial club members of IBTI will benefit via a number of routes. Firstly, club members have first refusal on the right to license an IP arising from the programme. Secondly, they will get the opportunity to see documents prepared for publication in advance of submission. In this way they will get a unique opportunity to secure any unforeseen IP contained in the work. Finally, by attending dissemination meetings (see below) they will gain access to early stage results in the research programme, which could assist their own research. This benefit could be realised within the next 5 years. UK farmers will benefit through the development of industrial processes which require feedstocks, and subsequent long term supply contracts. This would be subsequent to scale-up of the industrial processes, but potentially in a 5-7 year timeframe. The global development of biorefineries which supplant fossil fuel usage, will ultimately benefit us all by reduction of net CO2 emissions. The timescale of this depends on other factors, outside of our control. PDRAs on the project will gain valuable experience on working on future feedstocks. CBP is a fledgling research topic, so there is every possibility that their skills gained during this project will subsequently be in demand. What will be done to ensure that they have the opportunity to benefit? IBTI intends to run meetings at 6 monthly intervals (similar to BRIC) where the results of IBTI funded research will be presented. Assuming that this is done under an agreement of confidentiality, this will provide early access for IBTI club Industrial members to arising results. IBTI club members will also get sight of any documents planned for publication, 4 weeks prior to submission. We will also use the forum of IBTI meetings to explore the opportunity to add value to this work by internal collaboration with IBTI funded groups, and use our links in BSBEC to look for further synergies, given the overlap between biofuel production and chemicals from biomass. Any IP arising from work done within the programme will be secured by Imperial Innovations. Funding through IBTI will create a contractual obligation to offer licences to IBTI club companies in the first instance and, given their remit, Imperial Innovations will actively pursue the possibility of licensing the technology as rapidly as possible. The PI and CoIs all have experience of working with Industry and Imperial Innovations (see Pt1A of the proposal).