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
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
Pretreatment
Hydrolysis
Fermentation
• Optimum ethanol production bottle necked
– suboptimal xylose utilization and release of microbial inhibitor
molecules during biomass degradation
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. Brandt
Supervisor: Prof J Gorgens
Co-Supervisor: Prof WH Van Zyl
Department 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
Pretreatment
Hydrolysis
Fermentation
• Optimum ethanol production bottle necked
– suboptimal xylose utilization and release of microbial inhibitor
molecules during biomass degradation
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