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Impact of Inhibitors
Associated with
Lignocellulose
Hydrolysate on CBP
Yeast and Enzyme
Activity
Sizwe Mhlongo
Energy Postgraduate Conference 2013
INTRODUCTION
Figure 1: Plantation of sugar cane, wheat and maize
Agricultural
waste
from plant biomass
(sugar cane bagasse,
wheat
straw
and
maize plant) can be
converted to biofuel
Hydrolysis and
fermentation
Figure 2: Schematic representation of lignocellulose showing cellulose,
hemicellulose and lignin (Mussato and Teixeira, 2010)
PRETREATMENT
Acid catalysed steam pretreatment
Figure 3: Major components of lignocellulose biomass and hydrolysis products (Almedia et al, 2007)
HYDROLYSIS AND FERMENTATION
BiologicallyMediated
Event
Processing Configuration
(each box represents a bioreactor - not to scale)
Enzyme
O
production 2
SSCF
SSF
SHF
O2
O2
CBP
SHF:
Separate Hydrolysis and
Fermentation
Substrate
hydrolysis
SSF:
Simultaneous Saccharification
and Fermentation
Hexose
fermentation
SSCF: Simultaneous Saccharification
and co-Fermentation
Pentose
fermentation
CBP: Consolidated Bioprocessing
• Challenges in achieving CBP
• Lack of an ideal microorganism : cellulolytic and ethanologenic
phenotypes
• Bioreactor environment: Inhibitors from lignocellulose hydrolysate
HYDROLYSIS AND FERMENTATION
Recent CBP Strain developments
• Cell associated activity of S. fibuligera BGL1 in
Saccharomyces cerevisiae (Den Haan et al, 2007)
• Expression of T. reesei EG2 in S. cerevisiae (Brevnova
et al, 2011)
• Recombinant yeast strains showing high activity of
cellobiohydrolases Sc [T.e. cbh1-T. r. CBM-C] and Sc
[C.l. cbh2b] (Ilmen et al, 2011)
• However performance of these strains in an industrial
process, utilizing lignocellulose biomass is dependent
on bioreactor environment
BIOREACTOR ENVIRONMENT
Effect of inhibitors in the cell and mechanism of action
Figure 4: Schematic presentation of known mechanisms of furans, weak acids and phenolic compound
in Saccharomyces cerevisiae (Almedia et al, 2007)
TOXICITY ASSAYS
•
Maximum sub-lethal inhibitor concentration allowing
cell growth and enzyme production
• Preparation of different individual inhibitor
concentrations
• Assessment of growth profile and enzyme activity in
the presence of different inhibitors
• Determining the level of toxicity for each inhibitory
compound on yeast strain
• Expected outcomes
• Determine inhibitors that are most toxic to microbial
growth and enzyme production
• Define feed rate of inhibitors that can allow
fermentation to proceed
ENZYME-INHIBITOR RELATIOSHIP
•
Isolation and partial purification of cellulases,
assess the inhibition mechanism on enzymes
• Hydrolysis of substrate by isolated cellulase
enzymes in the presence of varying inhibitor
concentration
• Expected outcomes
• Determine enzyme-inhibitor relationship (inhibition
or deactivation)
• Identification of the most toxic inhibitors on enzyme
activity and therefore select pretreatment conditions
that limit the formation of the toxic inhibitors
• Required enzyme ratios for optimum hydrolysis
REDOX BALANCE AND GENE EXPRESSION
• Investigate the impact of
furans, weak acids and
phenolics on the redox
balance in yeast cells
• Gene expression analysis
(genes required for growth
during inhibition stress)
Figure 5: Schematic representation showing furfural and hydroxymethyl
furfural (HMF) conversion to furfuryl alcohol and furfural dimethyl alcohol
(FDM) (Liu et al, 2006).
ACKNOWLEDGEMENT
•
Supervisor and co-supervisors:
• Prof. van Zyl
• Prof. Bloom
• Dr Den Haan
•
•
NRF for funding
Stellenbosch University