Clostridial Strain Development: Improving the ABE process Holly Smith Biomass for sustainable biofuels & biobased products: From lab to pilot plant 17 th October, CENER
The ABE Process: A History Lesson ABE – A cetone, B utanol, E thanol • A long history of solvents produced by fermentation – Bio-butanol (Pasteur) 1861 – Bio-acetone 1905 • The Weizmann process for ABE patented in 1915 • First ABE plant (UK) 1916 Deployment globally between the 1 st and 2 nd World Wars • • Production in Russia and South Africa into the 1980s • Production in China in 2000s Private and confidential 2
Central Minnesota Renewables • Bio-based n-butanol and acetone • Re-purposed ethanol plant • Capital efficient and cost competitive • First customer shipments December 2016 Little Falls, Minnesota Private and confidential 3
Market diversity Private and confidential 4
Our Technology Research Expertise Underpinning Technology Unique culture collection for solvent-producing Clostridium Microbes bacteria and genes for optimization. Strain evolution for optimal performance in the Advanced Fermentation Process (AFP ™ ). Strain Development New technology, CLEAVE™ for genome editing and new products AFP ™ for higher productivity, yields and recovery. Process Development Integration of bacteria and process. Private and confidential 5
ABE Process : Targets for Improvements Process Improvements Butanol tolerance • High productivity fermentation • Continuous solvent removal Inhibitor tolerance Faster growth rates Improved sugar uptake rates Private and confidential 6
Strain Development : Generating improved strains Strain improvement is linked to a number of Depending on target and genetic tractability of strains, performance targets various strategies are employed for strain development Chemical Mutagenesis Wild type • Generates multiple genotypes within a population strains • Strains selected for an improved phenotype can often have mutations in non-obvious gene targets Rational Strain Spontaneous Adaptive Laboratory Evolution (ALE) Improvements mutants • Specific environmental pressure applied to drive advantageous cell adaption Commercial strains • “Natural” selection means the resulting cultures are generally healthy and robust for scale up process Rational Strain Improvement • NGS – sequence mutant strain libraries with potentially Adaptive Lab Chemical beneficial mutations Evolution Mutagenesis • Build “superior strains” by introducing single or layered mutations in a clean genetic background Private and confidential 7
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ButaNext Strain Development • Develop next generation biobutanol using sustainable feedstocks Aim • Need a high productivity, homofermentative process • Requires tolerance to high concentrations of product Challenges and inhibitors present in feedstocks • Strain selection using ALE: Butanol and Inhibitor tolerance Strategy • Development of high productivity fermentation process 9
Improving Inhibitor Tolerance Cellulosic Adaptive Laboratory AFP™ bench scale Feedstocks Evolution testing • Inhibitors • Sequential Batch • Strains tested for generated during Reactor (SBR) improvements in acidic thermo- AFP™ • Selective chemical pre- pressure – faster • Fed batch treatment growth rates in process with • Organic acids presence of cellulosic inhibitors hydrolysates and • Furfural, HMF ISPR 10
Sequential Batch Reactor • Automated system whereby culture from the previous batch is used to seed fresh medium • Monitor system by on-line pH measurements and microscopy • Selecting for faster growth rates on C6/C5 blended representative lignocellulosic feedstock 11
Inhibitor tolerant SBR strain testing • Results shown are normalised to the control strain – values >1 demonstrates improvement. • Strains from the SBR were tested in AFP™ using the same representative cellulosic feedstock • Testing in a fed batch process with feed containing high concentration of a C6/C5 blend of sugars • Strains 1 and 2 showed significant improvements in total ABE productivity and sugar uptake rate compared to the parent control strains • Indicative of tolerance to feedstock inhibitors 12
AFP™ testing using ButaNext Feedstocks • Hydrolysates of miscanthus and wheat straw feedstocks were provided by CENER and tested in GBL’s AFP™ systems • Testing blend of C6/C5 sugars, with an initial batch of approx. 15 g/L sugars to reduce inhibitor effect • Aimed to keep glucose concentration below 1 g/L during fed batch phase to prevent xylose build-up • Results shown are normalised to the control strain – productivity >1 demonstrates improvement • Strain 1 showed significant improvements over the parental strain with miscanthus feedstock • Slightly diminished performance using wheat straw hydrolysate. • Tests performed using standard conditions for AFP™ • Optimisation of AFP™ conditions may improve metrics further. 13
Summary • Green Biologics – Established business with operating commercial plant − Targeting specialty chemical market with high value applications − Development of new processes important to drive innovation and commercialisation of biofuels. • Improving economics of biobutanol as a next generation biofuel – Cost effective feedstocks – Strain development & high productivity fermentation important for economics of process – Adaptive laboratory evolution is a powerful tool for selection of robust strains with improved tolerance to feedstock inhibitors. Private and confidential 14
Acknowledgements ButaNext partners CENER Kimberley Baker Abdul Saqib Rachel Harper Dannielle Kydd-Sinclair Barnabas Owoh Yatin Behl Tim Davies Private and confidential 15
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