1. Impact of Inhibitors Associated with Lignocellulose Hydrolysate on CBP Yeast and Enzyme Activity Sizwe Mhlongo Energy Postgraduate Conference 2013

2. 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)

3. Acid catalysed steam pretreatment Figure 3: Major components of lignocellulose biomass and hydrolysis products (Almedia et al, 2007) PRETREATMENT

4. 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 Substrate hydrolysis Hexose fermentation Pentose fermentation SSF O2O2 SHF O2O2 SSCF O2O2 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. ACKNOWLEDGEMENT Supervisor and co-supervisors: Prof. van Zyl Prof. Bloom Dr Den Haan NRF for funding Stellenbosch University