Post on 20-Jan-2016
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
Hydrolysis and fermentation
Agricultural waste from plant biomass (sugar cane bagasse, wheat straw and maize plant) can be converted to biofuel
Figure 2: Schematic representation of lignocellulose showing cellulose, hemicellulose and lignin (Mussato and Teixeira, 2010)
Acid catalysed steam pretreatment
Figure 3: Major components of lignocellulose biomass and hydrolysis products (Almedia et al, 2007)
PRETREATMENT
• Challenges in achieving CBP• Lack of an ideal microorganism : cellulolytic and ethanologenic
phenotypes• Bioreactor environment: Inhibitors from lignocellulose hydrolysate
HYDROLYSIS AND FERMENTATION
Biologically-Mediated
Event
Enzyme production
Substratehydrolysis
Hexose fermentation
Pentose fermentation
SSF
O2
SHF
O2
SSCF
O2
CBP
Processing Configuration (each box represents a bioreactor - not to scale)
SHF: Separate Hydrolysis and Fermentation
SSF: Simultaneous Saccharification and Fermentation
SSCF: Simultaneous Saccharification and co-Fermentation
CBP: Consolidated Bioprocessing
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
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)
BIOREACTOR ENVIRONMENT
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
• 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).
REDOX BALANCE AND GENE EXPRESSION
ACKNOWLEDGEMENT
• Supervisor and co-supervisors: • Prof. van Zyl• Prof. Bloom • Dr Den Haan
• NRF for funding• Stellenbosch University