RICEFUEL: Engineering enzymes, bacteria and bioconversion processes for advanced biofuels from waste grain straw

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 emission, 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 enzymatic attack, and the lack of microbial strains capable of fermenting lignocellulosic-derived sugars into non-ethanol advanced biofuels, such as butanol and alkanes. The development of second generation, lignocellulose-based, biofuels is a global issue that is highly relevant to the economies of the UK and India. The research programme will focus on rice straw as the source of biomass. Our selection of this cereal waste product is based on several criteria: rice is the third biggest crop grown in the world and the major staple crop for most tropical nations. The case for developing biorefining technologies to generate fuels and chemicals is particularly compelling. Rice straw is produced in large quantities because tropical agriculture allows 2-4 crops per year to be produced. The use of rice straw in agriculture is particularly problematic as it is even less digestible than other cereal straws, and this severely limits its use as an animal feed, and this restricts its use to construction materials, animal bedding and cooking fires. Because of this rice straw, in the order of hundreds of millions of tons per year, are burned on the field in South and Southeast Asia alone to facilitate its disposal. Not only is straw burning a waste of a potentially valuable resource, but the process causes large scale emissions of black carbon, CO2 and methane. It is evident, therefore, that rice straw represents an excellent substrate for the biofuel sector. In this research programme we will 1) develop an enzyme cocktail optimised for rice straw deconstruction and (2) develop strains of Geobacillus, a bacterium whose metabolism can easily be modified, to produced advanced biofuels such as alkanes. These two streams of the programme we then be incorporated into a single process capable of producing high utility biofuels from rice straw.

Our overall aim is the pilot-scale development of a microbial based process able to convert rice straw-derived sugars into the advanced biofuels butanol and alkanes. This will be accomplished by implementing three workpackages (WPs). WP1 will develop novel enzymes with elevated activity against rice straw. The WP will deploy two strategies. The first strategy will mine rice straw composting microbial communities and insect rice stem borer symbionts for novel enzymes that display elevated activity against rice straw. The mining of the straw composts will use proteomics to identify protein targets, informed by transcriptomic data. The rice straw enzymes from the insect will be identified from genomic and transcriptomic data of microbes that degrade rice straw. The second approach will evaluate the capacity of natural and engineered arabinoxylan degrading enzymes against rice straw. WP2 will develop engineered bacterial strains that produce advanced biofuels (alkanes) from rice straw. In the WP2 In Silico Design will be used to model metabolic engineering strategies to produce alkanes and other biofuels from Clostridium acetobutylicum and Geobacillus. Based on the models, metabolic pathways will be constructed using BioBrick technologies that will be optimized through iterative hypothesis and testing through transcriptome, proteome and metabolome profiling. WP2 will also develop partial consolidated bioprocessing systems based on the Geobacillus strains. In WP3 the enzymes and microbial strains developed in WP1 and WP2, respectively, will be used to develop production systems that ferment pretreated rice straw into sugars, which are then fermented into advanced biofuels by Geobacillus and C. acetobutylicum

The proposed research programme has the potential to inform novel enzymatic and microbial strategies that improve the conversion of plant biomass into advanced biofuels such as butanol and alkanes. Currently the major economic limitation to the use of lignocellulosic biomass in biofuel production is the cost of the enzymatic treatments used to generate the monosaccharides. By generating novel glycoside hydrolases with improved activities against cell walls, this project may reduce both enzyme inputs into the process, and thus increase its economic viability. Similar to enzyme development, there is also a need to deploy synthetic biology to generate novel production strains capable of fermenting sugars into molecules that have wider utility in the biofuel sector than ethanol. Specifically this project is important to companies that are using plant biomass for industrial fermentations, such as bioethanol production. The importance of this research programme is illustrated by the fact that this project will interact with TMO Renewables, a leading player in the U.K. bioenergy industry. If successful we anticipate that that within the 3 -year programme the enzymes and microbial strains developed will be protected and commercialized, likely through licences with leading enzyme companies and through the development of Consolidated Bioprocessing Systems with TMO Renewables.

Increased employment: The research has the potential to deliver green jobs in the UK and further afield: The development of enzyme systems that contribute to the efficient deconstruction of lignocellulosic biomass will increase the take up of the technology, promoting growth within the clean technology sector. Furthermore the project will assist in addressing the shortage in industry of people able to construct and analyze genome-scale metabolic models from genome sequences.

Benefit to the environment: A primary driver for the move from fossil fuels to fuels and chemicals from waste or renewable sources of lignocellulose, is the production of greenhouse gas (GHG) emissions. An efficiently operated biorefinery using lignocellulose should be able to deliver an 80 % reduction in GHG emissions compared to its fossil fuel equivalent (based on ethanol production). This project will assist in reaching national and international targets for use of renewables and mitigation of climate change.

International collaboration: In the project there will be extensive collaboration between the Indian and UK partners. During the programme, not only with the groups meet regularly but there are clear pathways to the transfer of both technology and approaches between the two countries. These interactions will be cemented by PDRAs from the UK working in India and vice versa.