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 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

Cellobiohydrolase (Cbh) 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)

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 niger D15 Harvest enzymes Analyse paper sludge Hydrolysis trials Fermentations

Methodology Fungal Enzyme selection cellulases Strain preparation S. cerevisiae Y294[cbhI] S. cerevisiae Y294[cbhII] A. niger D15[eglA] S. cerevisiae Y294[Bgl2] Fungal cellulases Enzyme selection Strain preparation Harvest enzymes Paper sludge Select substrate Efficiency of enzyme action on a substrate may be measured in many different ways and the method chosen will depend on the ob- jectives of a particular study. From a biotechnological perspective, the main objective is to determine whether the conversion to mono- mer sugars has taken place and subsequently the percentage of car- bohydrates in the original substrate that was converted. Mostly, the yield of glucose (from cellulose) is measured and efficiency assessed in terms of glucose yield. Occasionally, the yield of other sugar mono- mers such as xylose may also be assessed. It is interesting to note, however, that different enzymes in different ratios may be required for glucose yield as compared to xylose yield 10ml working volume 2% paper sludge pH 5 30°C Hydrolysis trials Analyse Glucose yield

Optimise for different feedstocks and pretrements Enzyme cocktail development Which enzymes to produce in a CBP organism Test accessory enzymes Which enzymes to engineer 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

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

Table 1: Paper sludge analysis Percentage (on dry weight) Results Table 1: Paper sludge analysis Component Percentage (on dry weight) Cellulose 34.06 ± 0.65 Hemicellulose 14.26 ± 1.51 Lignin 27.04 ± 0.93 Ash 5.68 ± 0.12 where 0.9 is a correction factor to compensate for the addition of a water molecule during hydrolysis Figure 1: Optimised enzyme concentrations with a four enzyme cocktail, containing one: cbhI, cbhII, EgI and Bgl enzyme

Looking forward Test enzyme cocktail with a yeast strain Conclusion 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

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