17 th October, CENER The ABE Process: A History Lesson ABE A cetone, - - PowerPoint PPT Presentation

Clostridial Strain Development: Improving the ABE process Holly Smith Biomass for sustainable biofuels & biobased products: From lab to pilot plant 17th October, CENER

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The ABE Process: A History Lesson

ABE – Acetone, Butanol, Ethanol

• 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 1st and 2nd World Wars
• Production in Russia and South Africa into the 1980s
• Production in China in 2000s

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Central Minnesota Renewables

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• Bio-based n-butanol and acetone
• Re-purposed ethanol plant
• Capital efficient and cost competitive
• First customer shipments December 2016

Little Falls, Minnesota

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Market diversity

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Clostridium Microbes Process Development Strain Development

Research Expertise Underpinning Technology

Unique culture collection for solvent-producing bacteria and genes for optimization. Strain evolution for optimal performance in the Advanced Fermentation Process (AFP™). New technology, CLEAVE™ for genome editing and new products AFP™ for higher productivity, yields and recovery. Integration of bacteria and process.

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Our Technology

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ABE Process: Targets for Improvements

Butanol tolerance Inhibitor tolerance Faster growth rates Improved sugar uptake rates Process Improvements

• High productivity fermentation
• Continuous solvent removal

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Chemical Mutagenesis

• Generates multiple genotypes within a population
• Strains selected for an improved phenotype can often

have mutations in non-obvious gene targets

Adaptive Laboratory Evolution (ALE)

• Specific environmental pressure applied to drive

advantageous cell adaption

• “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

beneficial mutations

• Build “superior strains” by introducing single or layered

mutations in a clean genetic background

Commercial strains Wild type strains Spontaneous mutants Chemical Mutagenesis Adaptive Lab Evolution Rational Strain Improvements

Strain improvement is linked to a number of performance targets Depending on target and genetic tractability of strains, various strategies are employed for strain development

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Strain Development: Generating improved strains

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ButaNext Strain Development

Aim

• Develop next generation biobutanol using sustainable

feedstocks Challenges

• Need a high productivity, homofermentative process
• Requires tolerance to high concentrations of product

and inhibitors present in feedstocks Strategy

• Strain selection using ALE: Butanol and Inhibitor

tolerance

• Development of high productivity fermentation process

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Improving Inhibitor Tolerance

Cellulosic Feedstocks

• Inhibitors

generated during acidic thermo- chemical pre- treatment

• Organic acids
• Furfural, HMF

Adaptive Laboratory Evolution

• Sequential Batch

Reactor (SBR)

• Selective

pressure – faster growth rates in presence of inhibitors AFP™ bench scale testing

• Strains tested for

improvements in AFP™

• Fed batch

process with cellulosic hydrolysates and ISPR

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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

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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

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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.

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• 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.

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Summary

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Acknowledgements

Kimberley Baker Abdul Saqib Rachel Harper Dannielle Kydd-Sinclair Barnabas Owoh Yatin Behl Tim Davies

ButaNext partners CENER