Rational Engineering of Advanced Clostridia for Transformational Improvements in Fermentation (REACTIF)

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

Abstract

CONTEXT

n-Butanol is a valuable platform chemical with a large market (>3 M tonne/yr). It is used as a chemical precursor for a variety of polymers, plastics and solvents with a global market worth ~$8 billion). Average growth is about 3.2% with demand concentrated in N. America (28%), Western Europe (23%) and North East Asia (35%). Virtually all n-butanol today is synthetic and produced from propylene (derived from oil).
The fermentation route to butanol is an old industrial process that was developed in the UK in 1912 and spread quickly to the N. America, S.Africa, Japan and Russia. It is reliant on a bacteria called Clostridia, which ferments sugar-rich liquors and converting them to butanol, in a similar way to which yeasts is employed in brewing to make ethanol. The commercial butanol fermentation process became uneconomic in the mid 1950's when it could no longer compete with petro-derived equivalents from low cost oil and gradually disappeared.

More recently, the fermentation has been re-commercialized in China with at least 6 new plants and capacity for >300,000 T solvent/yr. However, these plants are struggling because the technology is not sufficiently advanced, the bacterial strains produce low amounts of butanol (due to butanol toxicity) and they rely on expensive starch-rich plant material as the source of sugar for the fermentation. Aside from the prohibitive cost of such feedstocks, their use for this purpose conflicts with a possible use as a food for human consumption. There is, therefore, a need to derive the sugar used from non-food plant derived material, the so-called lignocellulose component. Lignocellulose is the most abundant source of carbon on the plant. However, it is particularly recalcitrant to breakdown, and the treatments required (acid, alkali or steam explosion) to its conversion into sugar frequently leads to the formation of by-products that inhibit bacterial growth, so-called 'inhibitors'.

AIMS & OBJECTIVES

Today, advances in fermentation technology and the utilization of cheaper feedstocks, together with novel gene-based methods for strain improvement have the potential to transform fermentation process economics. We have assembled an exceptional group of academic and industrial partners, with specific expertise in the Clostridia fermentation. The industrial partners bridge the commercial supply chain (from feedstock to butanol production) whilst the academic groups focus on delivery of advanced microbes and superior fermentation performance using an array of advanced molecular biology techniques. Our objectives are to:-

1. identify and characterise tolerance traits (to high butanol titres and lignocellulose-derived inhibitors), both in historical strains that were used commercially over four decades and in current production strains following the implementation of novel directed evolution strategies;
2. transfer the identified alleles into current, robust production strains, together with rationale metabolic engineering to improve product titres, and;
3. assessment of strain(s) performance on cellulosic feedstocks at lab and pilot scale

APPLICATIONS AND BENEFITS

The project will deliver advanced Clostridia strains that offer a transformational change to fermentation performance and process economics of butanol production. Advanced strains not only improve fermentation performance but they will enable plant owners to switch to lower cost and more environmentally friendly cellulosic feedstocks. The production of butanol from renewable plant biomass as opposed to fossil fuels will provide a net reduction in greenhouse gas emission, contributing to a reduction in global warming and reducing the effects of environmental pollution on human health.

Biobutanol has the potential to directly replace all synthetic butanol addressing a specific market need in the chemical industry for cost efficient renewable chemical replacements, particularly in Europe & N. America

Technical Summary

The clostridial-based butanol fermentation is an old industrial process, developed in 1912. The process became uneconomic in the mid 1950's when it could no longer compete with the petrochemical industry. Although recently revived in China, many of the plants are struggling because the technology is not sufficiently advanced and is reliant on expensive and non-sustainable starchy feedstocks.

This project seeks to rectify this deficiency through the development of improved commercial strains able to achieve high butanol productivity and capable of fermenting non-food, cellulosic feedstocks. Strains will exhibit greater tolerance to commercial process conditions, feedstock inhibitors and butanol end titres and offer a transformational change to fermentation performance and process economics. Results will include:-

- Reproducible feedstock & defined process conditions
- Advanced methods for strain selection (transposon mediated and continuous culture)
- The identification and characterisation of beneficial tolerance traits
- Advanced GBL production strains proven at pilot scale (and ready to commercialize)
- Novel integrated process design for hydrolysis/fermentation
- Cost models (and cost savings) & calculations for energy & GHG emissions

The Consortium has an excellent balance between industry and academia that fills the entire supply chain (from feedstock to product). Three world class academic groups (University of Nottingham, Ulm & Gottingen) are complemented and supported by two industrial partners (Weyland & Green Biologics) who will provide significant technical input, financial support, commercial steerage and market opportunities. GBL is a world leader in commercialization activities for biobutanol fermentation and Weyland is a world leading developer of sugars from cellulosic feedstocks. Together the industrial partners will deliver an integrated process solution for producing butanol from a cellulosic feedstock and demonstrate at pilot-scale.

Planned Impact

WHO WILL BENEFIT?

The overall aim of this project is to enhance and extend the capabilities of solventogenic bacteria in terms of fuel and chemical production from renewable and sustainable resources. As this is an Industrial Partnership, the primary beneficies other than the Academic groups involved (University of Nottingham, Ulm and Goettingen) is GBL and Weyland. They will directly commercialise all useful strains that emerge from the project and will have first refusal on any foreground intellectual property that arises.

All parties have extensive global networks of existing commercial contacts and strategic partners. For example, GBL have partnerships with Guangxi Jinyuan Biochemical and Lianyungang Union of Chemicals in China. Nottingham have partnerships/ collaborations with EBI, Qteros, Lanxess and Genencor (N America), Evonik, Universities of Munich, Ulm and Berlin (Germany), TMO Renewables Ltd, Invista and Unilever (UK), Metabolic Explorer Ltd, INRA and CNRS (France), Chinese Academy of Sciences (Shanghai and Tsinghau, China), the Mumbai Institute of Chemical Technology (India) and LanzaTech (New Zealand). Working together, the consortium will seek to maximise these links for the benefit of all partner organisations involved.

The successful commercialization of anticipated outputs will have a rapid and global impact for both humanity and the environment. It will reduce greenhouse gas emissions and environmental pollution, provide an alternative to the use of food or farm resources for the production of low cost low carbon fuels and chemicals. It is therefore of benefit to society, ultimately impacting on health and well-being.


HOW WILL THEY BENEFIT?

Project outcomes will and allow improved fermentation process economics and product diversity, thus encouraging more rapid and wide spread adoption of clostridia-based butanol fermentation as a process to produce high volumes of low cost butanol as a chemical commodity and potentially as a biofuel. The partnership are anticipated to directly benefit from the outputs of the project through their commercial adoption via the pipeline and partnerships established by GBL and Weyland to both scale-up and commercially produce fuel and chemical products by clostridia fermentation. Additionally the partnership intends to explore strategic licensing deals with third party organisations. These will take the form of up front and milestone payments as well as ongoing royalty streams.

The successful scale-up and commercialization of processes will assist the UK and Europe in meeting challenging 'greenhouse' gas reduction targets, and contribute to indirectly to food security. The generation of butanol from cellulosic feedstocks will additionally impact on reducing reliance on fossil reserves, and therefore increase national fuel security.

Publications

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De Tissera S (2019) Syngas Biorefinery and Syngas Utilization. in Advances in biochemical engineering/biotechnology

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Poehlein A (2017) Microbial solvent formation revisited by comparative genome analysis. in Biotechnology for biofuels

 
Description PROJECT OBJECTIVES (problem to be solved)
Develop advanced clostridial production strains which can convert non-food cellulosic feedstocks to acetone and n-butanol.

General project approach
- Identification of genetic traits enriched in strains used commercially for butanol production over 40 years, but also new traits generated by adaptive laboratory evolution.
- Transfer of genetic traits into production strains.

CONSORTIUM TASKS UNDERTAKEN
- Screening of cellulosic hydrolysate from Weyland and Borregaard.
- Evaluation of the performance of historical production strains on sucrose.
- Selection of mutants for faster utilization of xylose, co-utilization of xylose and glucose, and butanol tolerance.
- Sequencing of up to 28 different wild-type solvent producing clostridia strains and several evolved strains with new traits.
- Transcriptome analysis of solventogenesis in solventogenic clostridia and detection of possible anti-sense RNA regulation and small non-coding RNA.

TECHNICAL OVERVIEW
- Development of a genetic modification protocol for a previously non-genetic tractable butanol production strains.
- Re-engineering of evolved traits into clean strain backgrounds.

WHAT HAS BEEN ACHIEVED TO DATE

- Traits identified which allows better conversion of sugars to butanol.
- Adaptive laboratory evolution protocol, which only introduces few and beneficial mutations.
- Better genetic understanding of Clostridial solvent producers.
- Targeted genetic modification protocol developed for an industrial production strain, which was previously un-tractable.
- Identification of a clostridial compatible hydrolysis process for lignocellulosic feedstocks.

WHAT ARE THE PLANS FOR FUTURE (ANY FOLLOW-UP PROJECTS?)
- Start-up of commercial bio-butanol production in the USA
- ButaNext: Next Generation Bio-butanol (Horizon 2020 LCE11)
- W2Bu: Cost Competitive Conversion of Municipal Solid Waste to Advanced biofuels (ERA-Net + BESTF)
- BIOFOREVER: BIO-based products from FORestry via Economically Viable European Routes (Horizon 2020)
- MAXBIO - Maximizing conversion yields in Biorefining (IB CATALYST)

PROJECT OUTCOME

Implementation and exploitation of results

- De-risking the cellulosic butanol production by initiating sugar to butanol at CMR.

So far 3 publications has been published, an additional publication accepted and one is in active preparation.

Green Biologics is taken part in a University of Nottingham led Marie Sklodowska Curie Innovative Training Networks (ETN) - CLOSPORE (2015-2019)
Exploitation Route Our findings are of value to others attempting to commercialise processes based on clostridial chassis, such as LanzaTech and ZuvaSyntha
Sectors Agriculture, Food and Drink,Chemicals,Energy,Environment,Manufacturing, including Industrial Biotechology

 
Description Microbial formation of acetone, isopropanol, and butanol is largely restricted to bacteria belonging to the genus Clostridium. This ability has been industrially exploited over the last 100 years. The solvents are important feedstocks for the chemical and biofuel industry. However, biological synthesis suffers from high substrate costs and competition from chemical synthesis supported by the low price of crude oil. To render the biotechnological production economically viable again, improvements in microbial and fermentation performance are necessary. However, no comprehensive comparisons of respective species and strains used and their specific abilities exist today. The genomes of a total 30 saccharolytic Clostridium strains, representative of the species Clostridium acetobutylicum, C. aurantibutyricum, C. beijerinckii, C. diolis, C. felsineum, C. pasteurianum, C. puniceum, C. roseum, C. saccharobutylicum, and C. saccharoperbutylacetonicum, have been determined; 10 of them completely, and compared to 14 published genomes of other solvent-forming clostridia. Two major groups could be differentiated and several misclassified species were detected. Our findings represent a comprehensive study of phylogeny and taxonomy of clostridial solvent producers that highlights differences in energy conservation mechanisms and substrate utilization between strains, and allow for the first time a direct comparison of sequentially selected industrial strains at the genetic level. Detailed data mining is now possible, supporting the identification of new engineering targets for improved solvent production.
First Year Of Impact 2017
Sector Chemicals,Energy,Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description HORIZON2020 Marie Curie ETN, CLOSPORE
Amount € 3,946,605 (EUR)
Funding ID 642068 
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 03/2015 
End 02/2018
 
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 250293 
Organisation National Council on Science and Technology (CONACYT) 
Sector Public
Country Mexico
Start 07/2015 
End 06/2016