Yeast Hardening for Cellulosic Ethanol production

1. Yeast Hardening for Cellulosic Ethanol production Bianca A. Brandt Supervisor: Prof J Gorgens Co-Supervisor: Prof WH Van Zyl Department of Process Engineering University of Stellenbosch Energy Postgraduate Conference 2013

2. Introduction • Growing global move towards sustainable green energy production • spurred by dependence on rapidly depleting finite fossil fuels • environmental and socio-economic concerns • Studies into Alternative Clean, Renewable and Sustainable energy resources: • solar-electric/thermal, hydroelectric, geothermal, tidal, wave, wind and ocean thermal power systems • furthermore, a great deal of work has gone into the development of biofuels

3. Introduction • Why Biofuels? • vehicular transportation- energy stored easier in form of combustible hydrocarbons then as electricity or heat • compatible with current distribution systems • supplement and replace fossil fuels • A range of bio-fuels are currently being investigated • Bioethanol - benchmark biofuel • production based on a proven low cost technological platform • Brazil and USA - cost effective 1st generation bioethanol • sugar and starch • 2nd generation bioethanol from lignocelluloses

4. Cellulosic Bioethanol • Bioethanol from Lignocellulose • cheap, renewable, easily available, under utilized resource • energy/fuel and suitable molecules which can replace petroleum products • Lignocellulose bioethanol production process • degradation of lignocellulose to fermentable sugars • fermentation of sugars to bioethanol • Optimum ethanol production bottle necked • suboptimal xylose utilization and release of microbial inhibitor molecules during biomass degradation Fermentation Pretreatment Hydrolysis

5. Overcoming Inhibitor toxicity • Challenge – Release of inhibitor molecules during lignocellulose degradation • furans, phenolics and weak acids • severely impact yeast fermentation efficiency • Process Optimization • feedstock, pretreatment, hydrolysis conditions • fermentation strategies • Detoxification of hydrolysate • physical (evaporation); chemical (over-liming) • biological: microbial and enzymatic approaches • Shown detoxification costs can constitute 22% of total ethanol production cost (Ding et al., 2009) • economically limited • inhibitor specific and loss of fermentable sugars

6. Overcoming Inhibitor toxicity • Sustainable cost effective bioethanol fermentation require “hardened” inhibitor resistant fermentation strains • Rational engineering approach • Genetic modification – yeast oxido-reductase detoxification genes • boost innate detoxification mechanisms of yeast • furfural, HMF, Formic acid • improved tolerance to specific inhibitor • Evolutionary engineering techniques • mutation and long term continuous cultures • simulate natural selection under selective pressure

7. Hardening yeast • Despite on-going yeast hardening strategies • Inhibitor resistant fermentation strains remain elusive and highly sought after!! • Project aim : Generate “hardened” inhibitor resistant yeast strains • Approach which combine Novel rational metabolic engineering and evolutionary engineering

8. Hardening yeast • Strain generation - Rational metabolic engineering • industrial xylose utilization base strains • Identify and select yeast detoxification genes from literature • combine specific detoxification genes with cell membrane stress response genes • Express inhibitor resistance genes in Saccharomycescerevisiae • novel gene combinations • elucidate synergistic /antagonistic combinations

9. Hardening yeast • Evolutionary engineering • long term continuous cultures - bioreactor • selective pressure – increasing concentrations of inhibitors • further enhance inhibitor resistance • evaluate fermentation efficiency in toxic hydrolysate • Novel “HARDENED” inhibitor resistant strains • Optimization of lignocellulosic bioethanol production

10. Acknowledgements Supervisors: Prof J Gorgens and Prof WH Van Zyl Department of process engineering NRF - Financial Support Thank You

11. Yeast Hardening for Cellulosic Ethanol production Bianca A. Brandt Supervisor: Prof J Gorgens Co-Supervisor: Prof WH Van Zyl Department of Process Engineering University of Stellenbosch Energy Postgraduate Conference 2013

12. Introduction • Growing global move towards sustainable green energy production • Spurred by dependence on rapidly depleting Finite Fossil fuels • Various environmental and socio-economic concerns • Studies into Alternative Clean, Renewable and Sustainable energy resources: • solar-electric/thermal, hydroelectric, geothermal, tidal, wave, wind and ocean thermal power systems • furthermore, a great deal of work has gone into the development of bio-fuels

13. Introduction • Why Biofuels? • Vehicular transportation- energy stored easier in form of combustible hydrocarbons then as electricity or heat • compatible with current distribution systems • Supplement and replace fossil fuels • A range of bio-fuels are currently being investigate • Bioethanol - benchmark biofuel • production based on a proven low cost technological platform • Brazil and USA -cost effective 1st generation bioethanol • Sugar and starch • 2nd generation bioethanol from lignocelluloses

14. Cellulosic Bioethanal • Bioethanol from Lignocellulose • cheap, renewable, easily available, under utilized resource • energy/fuel and suitable molecules which can replace petroleum products • Lignocellulose bioethanol production process • degradation of lignocellulose to fermentable sugars • fermentation of sugars to bioethanol • Optimum ethanol production bottle necked • suboptimal xylose utilization and release of microbial inhibitor molecules during biomass degradation Fermentation Pretreatment Hydrolysis

15. Overcoming inhibitor toxicity • Challenge – Release of inhibitor molecules during lignocellulose degradation • furans, phenolics and weak acids • severely impact yeast fermentation efficiency • Process Optimization • feedstock, pretreatment, hydrolysis conditions • fermentation strategies • Detoxification of hydrolysate • physical (evaporation); chemical (over-liming) • biological: microbial and enzymatic approaches • Shown detoxification costs can constitute 22% of total ethanol production cost (Ding et al., 2009) • economically limited • inhibitor specific and loss of fermentable sugars

16. Overcoming inhibitor toxicity • Sustainable cost effective bioethanol fermentation require “hardened” inhibitor resistant fermentation strains • Rational engineering approach • Genetic modification – yeast oxido-reductase detoxification genes • boost innate detoxification mechanisms of yeast • furfural, HMF, Formic acid • improved tolerance to specific inhibitor • Evolutionary engineering techniques • mutation and long term continuous cultures • simulate natural selection under selective pressure

17. Hardening yeast • Despite on-going yeast hardening strategies • Inhibitor resistant fermentation strains remain elusive and highly sought after!! • Project aim : Generate “hardened” inhibitor resistant yeast strains • Approach which combine Novel rational metabolic engineering and evolutionary engineering

18. Hardening yeast • Strain generation - Rational metabolic engineering • Industrial xylose utilization base strains • Identify and select yeast detoxification genes from literature • Combine specific detoxification genes with cell membrane stress response genes • Express inhibitor resistance genes in Saccharomyces cerevisiae • novel gene combinations • elucidate synergistic /antagonistic combinations

19. Hardening yeast • Evolutionary engineering • long term continuous cultures - bioreactor • selective pressure – increasing concentrations of inhibitors • further enhance inhibitor resistance • evaluate fermentation efficiency in toxic hydrolysate • Novel “HARDENED”inhibitor resistant strains • Optimization of lignocellulosic bioethanol production

20. Acknowledgements Supervisors: Prof J Gorgens and Prof WH Van Zyl Department of process engineering NRF - Financial Support Thank You