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

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