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

Lead Research Organisation: University of Bath
Department Name: Biology and Biochemistry

Abstract

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.

Technical Summary

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

Planned Impact

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

Publications

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Calverley J (2020) Hot Microbubble Air Stripping of Dilute Ethanol-Water Mixtures in Industrial & Engineering Chemistry Research

 
Description Chemicals, such as ethanol (a biofuel) or lactic acid (a precursor for bioplastics) can be made by fermentation of sugars derived from plant material. The most abundant plant material on earth is lignocellulose, but processes to obtain the fermentable cellulose and hemi-cellulose components are relatively expensive and need to be improved. Typically these processes involve a physico-chemical pretreatment (eg high temperature and pressure water or steam, with acid or alkali) followed by enzyme hydrolysis to make the individual (monomer) components of cellulose and hemi-cellulose. These are then fermented by natural or engineered yeast or bacteria.
Some organisms can degrade cellulose and hemicellulose, but this is very slow; however breaking these down to monomers uses a lot of enzymes. We are trying to demonstrate that, with the right combination of yeast/bacteria, enzymes and pretreatment methods you can develop a process which requires a lot less enzyme because the fermenting organism can ferment larger "chunks of sugars" (oligomers) as quickly as monomers ie the organism does more of the work.
Starting with sugar cane straw (a field residue) in Brazil and MIscanthus (a rapidly growing grass that produces bamboo-like canes) in the UK we have successfully developed physico-chemical and enzyme pretreatment methods that generate oligosaccharides and developed strains of yeast and the thermophilic bacteria Parageobacillus thermoglucosidasius which ferment these oligosaccharides to produce ethanol. Initially developed as individual steps, in the latter stages of the project we have demonstrated an end-to-end process at small scale to allow cost predictions to be made.
The process has a some useful added value as a biorefinery process as xylo-oligosaccharides and cello-oligosaccharides of the right size range are useful as nutritional supplements (stimulating the growth of "good" gut bacteria), so this higher value stream could benefit the economics of the whole process.
Exploitation Route Xylo-oligosaccharides and cello-oligosaccharides also have value in dietary applications.
Sectors Agriculture

Food and Drink

Energy

Manufacturing

including Industrial Biotechology

 
Description The groups at IBERS (Aberystwyth) have followed up on possible dietary uses of Miscanthus-derived xylo-oligosaccharides, by process scale-up and provision of samples to AB-Agri for evaluation
First Year Of Impact 2021
Sector Agriculture, Food and Drink,Digital/Communication/Information Technologies (including Software),Energy,Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description BEACON+ Biorefing Centre East Wales funded through ERDF via Welsh European Funding Office
Amount £443,888 (GBP)
Funding ID 82163 
Organisation Welsh Assembly 
Sector Public
Country United Kingdom
Start 01/2019 
End 12/2021
 
Description BEACON+ Biorefing Centre West Wales & the Valleys Extension funded through ERDF via Welsh European Funding Office
Amount £3,782,889 (GBP)
Funding ID 80851 
Organisation Welsh Assembly 
Sector Public
Country United Kingdom
Start 01/2019 
End 12/2021
 
Description European regional development fund
Amount € 2,884,534 (EUR)
Organisation Welsh Assembly 
Sector Public
Country United Kingdom
Start 03/2018 
End 03/2023
 
Title Genome scale metabolic model of Parageobacillus thermoglucosidasius 
Description Parageobacillus thermoglucosidasius represents a thermophilic, facultative anaerobic bacterial chassis, with several desirable traits for metabolic engineering and industrial production. To further optimize strain productivity, a systems level understanding of its metabolism is needed, which can be facilitated by a genome-scale metabolic model. Here, we present p-thermo, the most complete, curated and validated genome-scale model (to date) of Parageobacillus thermoglucosidasius NCIMB 11955. It spans a total of 890 metabolites, 1175 reactions and 917 metabolic genes, forming an extensive knowledge base for P. thermoglucosidasius NCIMB 11955 metabolism. The model accurately predicts aerobic utilization of 22 carbon sources, and the predictive quality of internal fluxes was validated with previously published 13C-fluxomics data. In an application case, p-thermo was used to facilitate more in-depth analysis of reported metabolic engineering efforts, giving additional insight into fermentative metabolism. Finally, p-thermo was used to resolve a previously uncharacterised bottleneck in anaerobic metabolism, by identifying the minimal required supplemented nutrients (thiamin, biotin and iron(III)) needed to sustain anaerobic growth. This highlights the usefulness of p-thermo for guiding the generation of experimental hypotheses and for facilitating data-driven metabolic engineering, expanding the use of P. thermoglucosidasius as a high yield production platform. 
Type Of Material Computer model/algorithm 
Year Produced 2021 
Provided To Others? Yes  
Impact We applied the GSMM to examine problems associated with anaerobic metabolism of P. thermoglucosidasius, which had hitherto required addition of a small amount of oxygen . By narrowing this down to a small number of possibilities, experimental analysis identified a single component required for anaerobic growth which was missing from the medium routinely used. This discovery now allows the fully anaerobic growth of this organism which is a major benefit for industrial exploitation. 
URL https://github.com/biosustain/p-thermo/releases/v1.0
 
Description Assessment of miscanthus pellets by DRAX 
Organisation Drax
Country United Kingdom 
Sector Private 
PI Contribution Processed miscanthus in our pilot scale facility to produce fibre and xylo-oligosaccharides. The fibre were then pelleted.
Collaborator Contribution The pellets were analysed for their calorific value, ash content, and suitability for use a feedstock for power generation.
Impact Data on combustion potential of the material.
Start Year 2023
 
Description Assessment of pellets by TATA 
Organisation TATA Steel
Country India 
Sector Private 
PI Contribution Processing of miscanthus to remove xylan and produce pellets from the remaining fibre
Collaborator Contribution Assess the material as a potential replacement for coal in steel production
Impact Outputs included the thermal properties of the miscanthus pellets and their suitability for burning in coke production.
Start Year 2023
 
Description Steam Explosion Pretreatment of Miscanthus, Birch and Wheat Straw to Improve Combustion Characteristics for Steel Production. Life cycle assessment and land take of MIscanthus to displace 200k tonnes of coke. 
Organisation TATA Steel
Department Tata Limited UK
Country United Kingdom 
Sector Private 
PI Contribution Advised on biomass pretreatment conditions to remove low calorific value Hemicellulose for the biomass feedstock performed biomass pretreatment and analysis. Commissioned a life cycle assessment study
Collaborator Contribution Performed additional pretreatment and analysis of biomass feedstock and assessed for quality characteristics suitable for steel production
Impact This is a multidisciplinary collaboration involving plant scientists
Start Year 2023
 
Description UK-Brazil 
Organisation State University of Campinas
Country Brazil 
Sector Academic/University 
PI Contribution This is a joint BBSRC-FAPESP project linking the work of a UK consortium (Bath, Aberystwyth, Imperial College) with a Brazilian consortium (Unicamp, Sao Paulo, CTBE) led by Unicamp
Collaborator Contribution Teams in the UK are working on complimentary feedstocks and strain engineering approaches in order to demonstrate a new paradigm for conversion of lignocellulosic substrates to fuels and chemicals.
Impact Work in progress
Start Year 2017
 
Description 4th LBNet conference BBSRC-FAPASP 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation of recent key finding on the optimisation of pre-treatments for the production of oligomeric arabinoxyland and cellulose components fractions from lignocellulosic biomass.
Year(s) Of Engagement Activity 2019
 
Description AIEC opening 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Opening of AU new Biorefining centre. Presentations were made on current biorefining projects along with equipment available to academics and industry.
Year(s) Of Engagement Activity 2019
 
Description Attending the 15th anniversary of UKRI India and discussions with the British High Commission on the use of Biomass in steel and sustainable Fuel production 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Discussed the potential for the BHC to host a workshop with Tata steel the Office of the Principle Scientifica Adviser to the Inidan Governement, BBSRC, other industrial pertners ICRISAT and IBERS
Year(s) Of Engagement Activity 2023
 
Description Azerbaijan Ambassador visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Presentation on biorefining research and potential opportunities for international collaboration.
Year(s) Of Engagement Activity 2018
 
Description Beer and cider special interest group 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Presentation on capability and research projects
Year(s) Of Engagement Activity 2018
 
Description Ben Lake MP visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Presentation on our biorefining research
Year(s) Of Engagement Activity 2018
 
Description Bryant and Gallagher visit to Dr Mohanty Office of the Principle Scientitfical Adviser to the Indian Government 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact meeting to developing a workshop on the application of Agritech solutions focussed on the use of biomass (wheat/rice straw) to displace coal and produce the high-value sweetener xylitol at demonstration scale.
Outcome, an on-line meeting to develop the workshop on biorefining to improve air quality reduce carbon emmissions and increase farmer income in India.
Year(s) Of Engagement Activity 2023
 
Description Consultative Group Meeting on Converting Agri-biomass to Bioenergy and associated products of economic importance". 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact An online meeting was held of approximately 20 participants from TATA Steel India, Tata Steel UK the BBSRC and Dr Mohanty Office of the Princip[le Scientific Advoser to the Indian Government, ARCITREKBio Ltd and Invest India to discuss biorefining Agri-biomass to Bioenergy and associated products of economic importance particularly in relation to cereal straws in the Haryana region and their application as alternative feedstock for steel production.
Year(s) Of Engagement Activity 2023
 
Description Drax discussions on biorefinig and biomass pretreatment 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Initila meeting between IBERS staff and Drax management on areasof mutual interest such as biomass supply chainse bioenergy productionand high value biorefining. This was progressed to an on line meeting invloving key personel at the DRAX biorefinery in the US and an MOU is being establised with Aberystwyth University.
Year(s) Of Engagement Activity 2023
 
Description Guto Bebb MP visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Discussions regarding the importance of EU funding and the implications of Brexit on our research in the area of Industrial Biotechnology. Discussions also in the area of existing and future 'non-EU' funded research.
Year(s) Of Engagement Activity 2017
 
Description IBERS Centenary Celebrations 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Celebration of IBERS 100 years in plant breeding. Presentations given on biorefining projects and available equipment.
Year(s) Of Engagement Activity 2019
 
Description Meeting with Mr T.V. Narendran, Global CEO of TATA Steel 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Mr TV Narendran requested an appointment to discuss how Welsh Universities can help TSUK in the planned transition to electric arc furnaces. Under discussion was the potential use of biomass to displace coke that will still be required in EAF and how this could be of benefit to the local community in terms of new jobs. More broadly, this could have major impact in India where biomass is routinely burnt in the fields and blights the lives of millions through the poor air quality that results. Translation of biorefining technology developed in the UK to India could have major benfits for net-zero on a planetary scale.
Year(s) Of Engagement Activity 2024
 
Description Newtown rotary Club Visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Presented current and post research in the area of biorefining.
Year(s) Of Engagement Activity 2019
 
Description Reed Panel visit 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Discussions with the Reed panel on the positioning of the university with regard to international collaborations in the area of Industrial Biotechnology.
Year(s) Of Engagement Activity 2017
 
Description Regional Stakeholder Partnership for Land-based Goods 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Presentation of production of platform chemicals/fuels/fine chemicals and potential health care products as well as integrated biorefining from a range of land-based feedstock.
Year(s) Of Engagement Activity 2018
 
Description Society for Dairy Technology Visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Discussions with representatives from the dairy industry to discuss alternative products, including oligomers as pre-biotics, processing routes and valorisation of waste streams.
Year(s) Of Engagement Activity 2019
 
Description Symposium at Unicamp (Brazil) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was a showcase of the work of 2 BBSRC-FAPESP projects held before 1:1 meetings between the projects. Representatives of key industries in the bioenergy and bio-materials sectors were invited.
Year(s) Of Engagement Activity 2019
 
Description Taiwan Ambassador visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Presentation on current research activities and opportunity to collaboration and/or student exchanges
Year(s) Of Engagement Activity 2018
 
Description Visit to Universiti Teknologi Malaysia (UTM) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Major discussions with Staff at both of the UTM campuses on areas of collaboration in Industrial biotechnology, bio-circular economy, engagement with Business, possible studentships and available equipment.
Year(s) Of Engagement Activity 2019
 
Description Welsh Gov. innovation team 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Policymakers/politicians
Results and Impact Presentation and discussion with Welsh Government Innovation Team, current projects, research strategy and future directions in the area of Industrial Biotechnology. This included our strategy for interacting with the business community, both in Wales and beyond.
Year(s) Of Engagement Activity 2017
 
Description Workshop in Brazil (Unicamp) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Use the opportunity to present the philosophy of NiBBs, with a focus on P2P to Brazilian delegates at a workshop hosted by Unicamp (Campinas)
Year(s) Of Engagement Activity 2015