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

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Presentation on theme: "The Development of Recombinant Fungal Enzyme Cocktails for the Hydrolysis of Cellulosic Waste Products Rosemary Dobson University of Stellenbosch Energy."— Presentation transcript:

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

3 β-glucosidase (Bgl) Cellobiohydrolase (Cbh) Endoglucanase (Eg) Glucose Cellobiose 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 Cellulases

4 Develop a recombinant fungal enzyme cocktail for effective hydrolysis of paper sludge Select core enzymes (cellulases) Express enzymes in Saccharomyces cerevisiae Y294 and Aspergillus niger D15 Harvest enzymes Analyse paper sludge Hydrolysis trials Fermentations Aims and objectives

5 Methodology Enzyme selection Strain preparation Harvest enzymes Select substrate Hydrolysis trials Glucose yield Fungal cellulases Paper sludge Analyse S. cerevisiae Y294[cbhI] S. cerevisiae Y294[cbhII] A. niger D15[eglA] S. cerevisiae Y294[Bgl2] 10ml working volume 2% paper sludge pH 5 30°C

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

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 ComponentPercentage (on dry weight) Cellulose34.06 ± 0.65 Hemicellulose14.26 ± 1.51 Lignin27.04 ± 0.93 Ash5.68 ± 0.12 Table 1: Paper sludge analysis Figure 1: Optimised enzyme concentrations with a four enzyme cocktail, containing one: cbhI, cbhII, EgI and 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 Substrate Enzymatic saccarification Sugars Microbial fermentation Biofuels

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


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