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