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Rosemary Dobson University of Stellenbosch

The Development of Recombinant Fungal Enzyme Cocktails for the Hydrolysis of Cellulosic Waste Products. Rosemary Dobson University of Stellenbosch. Energy Postgraduate Conference 2013. Introduction. Alternative fuels Renewable resources = sustainable

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Rosemary Dobson University of Stellenbosch

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  1. The Development of Recombinant Fungal Enzyme Cocktails for the Hydrolysis of Cellulosic Waste Products Rosemary Dobson University of Stellenbosch Energy Postgraduate Conference 2013

  2. Introduction • Alternative fuels • Renewable resources = sustainable • Bioconversion of lignocellulosic biomass • Abundant resource • Hydrolysis = major bottleneck • Enzymatic hydrolysis • Current enzyme mixtures not sufficient • Cellulases are expensive http://www.ijbs.com/v05p0578.htm

  3. Cellulases • Hydrolysis of cellulosic biomass • Mixtures of hydrolytic enzymes • Collectively known as cellulases • T. reesei & A. niger produce large amounts of extracellular cellulolytic enzymes • Synergistic manner Cellobiohydrolase (Cbh) Glucose Endoglucanase (Eg) Cellobiose β-glucosidase (Bgl)

  4. Aims and objectives • Develop a recombinant fungal enzyme cocktail for effective hydrolysis of paper sludge • Select core enzymes (cellulases) • Express enzymes in Saccharomyces cerevisiae Y294 and Aspergillus nigerD15 • Harvest enzymes • Analyse paper sludge • Hydrolysis trials • Fermentations

  5. Methodology S. cerevisiae Y294[cbhI] S. cerevisiae Y294[cbhII] A. nigerD15[eglA] S. cerevisiae Y294[Bgl2] Fungal cellulases Enzyme selection Strain preparation Harvest enzymes Paper sludge Select substrate 10ml working volume 2% paper sludge pH 5 30°C Hydrolysis trials Analyse Glucose yield

  6. Enzyme cocktail development Which enzymes to produce in a CBP organism Which enzymes to engineer Test accessory enzymes Which enzymes to pursue by bioprospecting Identify non-essential enzymes Core Enzymes Test new enzymes Try different ratios of enzymes Optimise for different feedstocks and pretrements

  7. Paper sludge • Solid waste material • Non recyclable paper fibres • Attractive biomass for enzymatic hydrolysis • Susceptible to enzymatic digestion • Negative feedstock cost • Integration of processes into an existing industrial infrastructure • No pre-treatment needed Bayer, Lamed, & Himmel, 2007

  8. Results Table 1: Paper sludge analysis Figure 1: Optimised enzyme concentrations with a four enzyme cocktail, containing one: cbhI, cbhII, EgIand Bgl enzyme

  9. Looking forward • Test enzyme cocktail with a yeast strain • Analyse for ethanol (HPLC) • Conclusion • First report to use enzymes from recombinant strains for the hydrolysis of paper sludge • Important to optimise enzyme cocktails Enzymatic saccarification Microbial fermentation Substrate Sugars Biofuels

  10. Acknowledgements • Supervisors: • Prof W.H. van Zyl • Dr S Rose • Bloom and van Zyl lab • Funding: • NRF • University of Stellenbosch

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