The Development of Recombinant Fungal Enzyme Cocktails for the

Hydrolysis of Cellulosic Waste Products

Rosemary DobsonUniversity 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

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

• 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

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 volume2% paper sludge

pH 530°C

Core Enzyme

s

Optimise for different

feedstocks and pretrements

Test new enzymes

Test accessor

y 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

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 neededBayer, Lamed, & Himmel, 2007

Results

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

Table 1: Paper sludge analysis

Figure 1: Optimised enzyme concentrations with a four enzyme cocktail, containing one: cbhI, cbhII, EgI and Bgl enzyme𝐷𝑠𝑔𝑙𝑢𝑐𝑎𝑛 = ሾ𝑔𝑙𝑢𝑐𝑜𝑠𝑒ሿ 𝑥 0.9 [𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒] 𝑥 100%

24 48 72 96 120 1440

5

10

15

20

25

30

35

40

Cocktail (double concen-tration)

Complete cocktail (4 Enzymes)

Cocktail (without cbhI)

Cocktail (without cbhII)

Cocktail (without EgI)

Cocktail (without Bgl)

Hours

Ds g

luca

n (%

)

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

• Supervisors:– Prof W.H. van Zyl– Dr S Rose

• Bloom and van Zyl lab• Funding:

– NRF– University of Stellenbosch

Acknowledgements