This document summarizes research on converting sugar beet pulp waste into valuable products. Small-scale batch studies showed cell lysate had higher arabinofuranosidase enzyme activity than whole cells or purified enzyme. Larger-scale studies in an agitated continuous reactor achieved higher arabinose yields than batch reactions, though conditions require optimization. Future work includes testing other enzyme forms in the continuous reactor, increasing enzyme expression and concentration, and investigating enzyme retention strategies.

1. Sweet, Sweet Waste
Turning sugar production waste
materials into value added products
Group 4
Patcha Chaniwatana, Yau Kiat Lee, Mohit
Santilal, Evangelia Argentou

2. Outline
• Introduction and Background
• Material and Methods
• Results
– Enzyme Activity
– Microwell (4 mL) Batch Study
– Large scale (200 mL) Batch Study
– Continuous Reaction
• Conclusion
• Future Work

3. Introduction

4. Pretreatment of
Sugar beet pulp
Breakdown of
arabinans and
pectins
Biotransformation
of sugars
Introduction
Glucose
(Bioethanol)
L- Arabinose
Cellulose
Polygalacturonic
acid
Arabinans
Galacturonic acid
Sugar Beet Pulp

5. Introduction
• Objective:
To investigate the continuous
breakdown of arabinans to
arabinose via
arabinofuranosidase
Run small scale and batch
study to optimise the scale-
up using the agitated cell
reactor (ACS)
• Pectin Sugars are difficult to use in fermentations
• They can, however, be used as building blocks for fine chemicals production

6. Materials and Methods
• Use pre-treated sugar beet pulp (SBP) as raw material
• Preparation of 3 enzymatic forms
whole cell cell lysate purified enzyme
• Arabinofuranosidase activity assay
– Detect nitrophenol at 405 nm
• Small-scale study on 24-well plate in a thermomixer
• Ion chromatography system (ICS)
• Effect of agitation, time and enzyme concentration on hydrolysis.
• Pump Calibration – flow rate
• Scale-up via the agitated continuous reactor (ACR)
and shake flasks.
Ultrasonic cell
disintegration Ni-NTA column

7. Enzyme Activity at Small Scale
454.94
14.99 20.35
0.00
Whole cell Cell Lysate Purified Enzyme Blank
EnzymeactivityU/ml
Treatment of Sample
Arabinofuranosidase activity assay
Whole cell Blank

8. Chromatograms
Standard
Sample
Rhamnose
Arabinose Galactose
Glucose
Arabinose

9. 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 10 20 30 40 50 60
Concentrationofarabinose(g/L)
Time (minutes )
Non-agitated enzymatic hydrolysis at high and low (1:10)
concentration of arabinosefuranosidase at 50 °C
Whole cells-Low Whole cells-High Cell lysate -Low Cell lysate -High
Purified enzyme-High Purified enzyme-Low Control
ICS Analysis
Cell lysate – [high]
• High concentration of cell lysate (1 in 40) gives best results
• Reaction plateaus after 30 minutes

10. ICS Analysis
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 10 20 30 40 50 60
Concentrationofarabinose(g/L)
Time (minutes)
Agitated (750rpm) enzymatic hydrolysis at high and low (1:10)
concentration of arabinosefuranosidase at 50 °C
Whole cells-Low Whole cells-High Cell lysate -Low
Cell lysate -High Purified enzyme-Low Purified enzyme-High
Control
Cell lysate -high
• Similar trend to previous experiment
• However, Lower concentration for all of the conditions
• Possibly due to the use of different Thermomixers

11. Condition Decided for Scale Up
• Flask
– Cell lysate
– 50oC
– Agitation and no agitation
– 30min
– Enzyme concentration (low)
• ACR
– Cell lysate and whole cell
– 50oC
– Agitation
– 30min
– Enzyme concentration (low)
Whole
cells
Arabinan Arabinose
Cell lysate
Purified Enzyme
C
O
S
T

12. Agitated Cell Reactor (ACR)
• Continuous stirred reactor
• Metal reactor block with 10 interconnected cells
(10ml)
• Block shakes – agitator present in all cells, causing
mixing the reactor

13. ACR in real life
ACR in Real Life
10 inter connected cells
Temperature probes
Process outlet
Process inlet
Heated waterHeated plate

14. Results - 24 Well Plate
• Diluting the enzyme resulted in a drop in arabinan hydrolysis
• Cell lysate performed better than whole cells

15. Comparison across scales
• ACR achieves higher arabinose concentration
• At Large batch scale (200 mL) agitation has an effect
• Whole cells perform better in continuous – possible due to shear and lysis

16. Comparison of Yields
• Whole cells has higher
yields
• Maximum yield ~ 13%
• Continuous has higher yields
• Conditions need to be optimized

17. Conclusion
Summary
• Continuous mode achieves higher yields (could be
higher)
• Ideal enzymatic form needs to be identified for ACR
• Agitation shown to be important at larger scale
Future work
 Analytics are lengthy, so investigating reproducibility of
results is necessary
 Higher enzyme concentration
 Test other enzyme forms in ACR (purified enzyme)
 Higher expression of enzyme in the cell
 Investigate enzyme retention strategies

18. References
• British Sugar. (2010). How our factory operates.
Retrieved August 06, 2015, from
http://www.britishsugarlearningzone.com/how-our-
factory-operates/
• Jones, E., McClean, K., Housden, S., Gasparini, G., &
Archer, I. (2012). Biocatalytic oxidase: Batch to
continuous. Chemical Engineering Research and
Design, 90(6), 726–731.
doi:10.1016/j.cherd.2012.01.018