Yeast Hardening for

Cellulosic Ethanol

productionBianca A. BrandtSupervisor: Prof J Gorgens

Co-Supervisor: Prof WH Van ZylDepartment of Process

Engineering University of Stellenbosch

Energy Postgraduate Conference 2013

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

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

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 FermentationHydrolysis

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

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

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

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

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

Acknowledgements

Supervisors: Prof J Gorgens and Prof WH Van Zyl

Department of process engineering

NRF - Financial Support

Yeast Hardening for Cellulosic Ethanol

production

Bianca A. BrandtSupervisor: Prof J Gorgens

Co-Supervisor: Prof WH Van ZylDepartment of Process Engineering

University of Stellenbosch

Energy Postgraduate Conference 2013

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

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

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 FermentationHydrolysis

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

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

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

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

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

AcknowledgementsSupervisors: Prof J Gorgens and Prof WH Van Zyl

Department of process engineering

NRF - Financial Support