1. 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

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. 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)

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 niger D15
Harvest enzymes
Analyse paper sludge
Hydrolysis trials
Fermentations

5. 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

6. 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

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. 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

9. 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

10. 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