The Development of Recombinant Fungal Enzyme Cocktails for

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 • Bioconversion of lignocellulosic biomass • Abundant resource • Hydrolysis = major bottleneck • Enzymatic hydrolysis • Current enzyme mixtures not sufficient • Cellulases are expensive http: //www. ijbs. com/v 05 p 0578. htm

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

Aims and objectives • Develop a recombinant fungal enzyme cocktail for effective hydrolysis of paper sludge • Select core enzymes (cellulases) • Express enzymes in Saccharomyces cerevisiae Y 294 and Aspergillus niger D 15 • Harvest enzymes • Analyse paper sludge • Hydrolysis trials • Fermentations

Methodology Fungal cellulases Enzyme selection S. cerevisiae Y 294[cbh. I] S. cerevisiae Y 294[cbh. II] A. niger D 15[egl. A] S. cerevisiae Y 294[Bgl 2] Strain preparation Harvest enzymes Select substrate 10 ml working volume 2% paper sludge p. H 5 30°C Paper sludge Hydrolysis trials Glucose yield Analyse

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

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

Results Table 1: Paper sludge analysis Component 40 Percentage (on dry weight) 35 Cocktail (double concentration) Cellulose 34. 06 ± 0. 65 Hemicellulose 14. 26 ± 1. 51 Lignin 27. 04 ± 0. 93 Ash 5. 68 ± 0. 12 Ds glucan (%) 30 Complete cocktail (4 Enzymes) 25 Cocktail (without cbh. I) 20 Cocktail (without cbh. II) 15 10 Cocktail (without Eg. I) 5 Cocktail (without Bgl) 0 Hours Figure 1: Optimised enzyme concentrations with a four enzyme cocktail, containing one: cbh. I, cbh. II, Eg. I and Bgl enzyme

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

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