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

Lead Research Organisation: University of Nottingham
Department Name: Sch of Molecular Medical Sciences

Abstract

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.

Technical Summary

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

Planned Impact

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.

Publications

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Cragg SM (2015) Lignocellulose degradation mechanisms across the Tree of Life. in Current opinion in chemical biology

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Labourel A (2016) The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans. in The Journal of biological chemistry

 
Description NEW KNOWLEDGE GENERATED

- Metagenomics and metatranscriptomics have significantly advanced our understanding of complex microbial communities containing numerous unculturable species (York).

- A greater knowledge of how aerobic bacterium degrades crystalline cellulose (Newcastle, NEW). Specifically the data show how lytic polysaccharide monooxygeneases (LPMOs) contribute to the process, while genetic null mutations show that respiratory proteins play a critical role in the process.

- A genome scale metabolic model for Geobacillus thermoglucosidasius C56-YS93 has been constructed (Oxford Brookes, OBM) that is able to produce all biomass components in experimentally observed proportions and is free from imbalances in terms of carbon, nitrogen, phosphor and sulphur. The model has been validated by comparing growth phenotype on carbon sources for which experimental data could be obtained, with good agreement.

- We (Nottingham, UoN) have refined and optimised our gene tools and used them to recreate the commercial production strain (TM242) of Rebio Ltd on several independent occasions, but have been unable to equal its productivity. Extension high throughput sequencing has shown that the cause is most likely the predilection of the strain to acquire SNPs. During this process we have discovered a considerable number of errors in the 'published' genome sequence.

NEW OR IMPROVED RESEARCH METHODS OR SKILLS DEVELOPED

- The York group have developed a technique for targeting and enriching the extracellular proteome by tagging prior to stringent total extraction. The methodology combined with mass spectrometry-based proteomics and metatranscriptomics has enabled the identification of unique metaexoproteome pools from complex lignocellulose degrading communities

IMPORTANT NEW RESEARCH RESOURCES IDENTIFIED
- Bespoke LPMO enzymes fused to heterologous CBMs

- Two novel fluorescent reports have been identified as suitable for reporter gene studies in Geobacillus thermoglucosidasius

IMPORTANT NEW RESEARCH QUESTIONS OPENED UP

- The identification of large numbers of uncharacterized proteins offers an invaluable opportunity to expand our knowledge of lignocellulose degradation, with the potential to mine for new commercially valuable biomass processing enzymes. In addition, this protein-labelling approach could be applied to a variety of complex microbial ecosystems to provide details on major metabolic players and the function and contribution of the exoproteome in those communities.

SIGNIFICANT NEGATIVE RESULTS AND/OR RESEARCH PATHS CLOSED OFF

- The use of the enzymes developed by Newcastle have not been effective if degrading the target substrate of this project, rice straw. Further enzyme combinations need to be explored.

PARTICULARLY NOTEWORTHY NEW RESEARCH NETWORKS/COLLABORATIONS/PARTNERSHIPS, OR COMBINATIONS OF THESE

- Collaboration with Gardner in Maryland USA introducing genetics into our biochemical studies
Exploitation Route Further refinement of the data is needed before they may be exploited by others.
Sectors Chemicals,Manufacturing, including Industrial Biotechology

URL http://www.ricefuel.net
 
Description The development of second generation, lignocellulose-based, biofuels is a global issue that is highly relevant to the economies of the UK and India. RICEFUEL focuses on rice straw as the source of biomass. It 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. There have been attempts to use rice straw as a feedstock for power stations, but this has not been successful as the cereal contains a high concentration of silicates, which led to high levels of ash and the fouling of furnace linings. 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, methane and leads to large scale generation of tropospheric ozone. The impacts of burning agricultural residues has recently been identified by the UN Environment Programme as one the priority areas that need to be dealt with in order to help slow global warming (particularly black carbon and methane emissions). It is evident, therefore, that rice straw represents an excellent substrate for the biofuel sector. The RICEFUEL proposal draws on the key strengths of the two research communities, and in particular INDIA's expertise/skills in rice straw pre-treatment and hydrolysis, process development and scale-up combined with UK skills/expertise in enzyme characterisation, systems/synthetic biology and metabolic engineering. The project has cemented the growing collaboration between the two communities to the benefit of both parties, and provided a natural extension to outputs from the BBSRC Sustainable Bioenergy Centre (BSBEC). A number of bids have been made to the Newton Bhabha PhD exchange scheme to help strengthen the collaboration, two successfully leading to 3 month secondments of two students from Nottingham to ICGEB in New Delhi. The scientific outputs of the project have not yet realised their potential, but are providing the grandwork for future bids to the ongoing Newton funding stream.
First Year Of Impact 2014
Sector Chemicals,Communities and Social Services/Policy,Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description BBSRC CASE DNA2.0
Amount £100,126 (GBP)
Funding ID BB/L016478/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description FP7 Marie Curie IIF, GEO-HPA (ESR 625585): Development of a Sustainable Route to the Important Platform Chemical 3-Hydroxypropanoic Acid Using Synthetic Biology and a Geobacillus Chassis
Amount € 309,235 (EUR)
Funding ID PIIF-GA-2013-625585 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 01/2014 
End 12/2016
 
Description IB Catalyst Round 4 Early Stage Translation
Amount £514,000 (GBP)
Funding ID BB/N022718/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2016 
End 04/2021
 
Description Mexican CONACYT Fellowship
Amount $25,000 (USD)
Funding ID 250294 
Organisation National Council on Science and Technology (CONACYT) 
Sector Public
Country Mexico
Start 07/2015 
End 06/2016
 
Description SBRC & C1net Outreach Activity at The Secret Science Show, Nottingham 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact "European Researchers' Night" celebrated its 10th anniversary this year. Led by an EU initiative, and taking place simultaneously in 280 cities across Europe on 25 September, this event gave anybody and everybody the opportunity to become a scientist for a day.
Nottingham's contribution was the much anticipated "Secret Science Show" at Wollaton Hall, and taking part were C1net members Louise Dynes and Dr Vinod Kumar. Together they gave a presentation and interactive demonstration on 'the power of microbes' explaining how microbes can turn unwanted substances into useful ones such as plastics and fuels. Dr Kumar also explained and demonstrated how a bioreactor worked. The audience of 86, with an ages range from 6 to 60 participated enthusiastically in the Q&A session and left with the message that only a very small percentage of bacteria are bad and that many are useful .
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Year(s) Of Engagement Activity 2015
URL http://englandevents.co.uk/nottingham-the-secret-science-show/407457