Sweet, Sweet Waste

Turning sugar production waste materials into value added products

Group 4

Patcha Chaniwatana, Yau Kiat Lee, Mohit Santilal, Evangelia Argentou

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

Introduction

Pretreatment of Sugar beet pulp

Breakdown of arabinans and

pectins

Biotransformation of sugars

Introduction

Glucose (Bioethanol)

L- ArabinoseCellulose

Polygalacturonic acid

Arabinans

Galacturonic acid

Sugar Beet Pulp

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

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

Enzyme Activity at Small Scale

Whole cell Cell Lysate Purified Enzyme Blank

454.94

14.99 20.35

0.00

Arabinofuranosidase activity assay

Treatment of Sample

Enzy

me

activ

ity U

/ml

Whole cell Blank

Chromatograms

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0-20.0-12.50.012.525.037.550.062.575.087.5100.0112.5125.0137.5150.0160.0 1 - Rha - 6.0252 - Ara - 7.592

3 - Gal - 9.6674 - Glu - 10.050

min

nCNeutral Sugars Large Scale #5 0.02 ED_1

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0-100102030405060708090100

1 - Rha - 5.892

2 - Ara - 7.400

3 - Gal - 9.4504 - Glu - 9.8345 - 10.4426 - 12.825

min

nCNeutral Sugars Large Scale #23 15 ED_1

Standard

Sample

Rhamnose

Arabinose Galactose

Glucose

Arabinose

0 10 20 30 40 50 600

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Non-agitated enzymatic hydrolysis at high and low (1:10) concen-tration of arabinosefuranosidase at 50 °C

Whole cells-Low Whole cells-High Cell lysate -Low Cell lysate -High Purified enzyme-High

Purified enzyme-Low Control

Time (minutes )Con

cent

ratio

n of

ara

bino

se (g

/L)

ICS Analysis

Cell lysate – [high]

• High concentration of cell lysate (1 in 40) gives best results• Reaction plateaus after 30 minutes

ICS Analysis

0 10 20 30 40 50 600

0.1

0.2

0.3

0.4

0.5

0.6

0.7

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 -HighPurified enzyme-Low Purified enzyme-High Control

Time (minutes)

Con

cent

ratio

n of

ara

bino

se (g

/L)

Cell lysate -high

• Similar trend to previous experiment• However, Lower concentration for all of the conditions• Possibly due to the use of different Thermomixers

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

COST

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

ACR in real life

ACR in Real Life

10 inter connected cells

Temperature probes

Process outlet Process inlet

Heated waterHeated plate

Results - 24 Well Plate

• Diluting the enzyme resulted in a drop in arabinan hydrolysis• Cell lysate performed better than whole cells

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

Comparison of Yields

• Whole cells has higher yields

• Maximum yield ~ 13%

• Continuous has higher yields • Conditions need to be optimized

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 scaleFuture 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

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