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 2 nd 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 Pretreatment Fermentation 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 Saccharomyces cerevisiae –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

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 Pretreatment Fermentation 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