UNIVERSITI MALAYSIA PERLIS

ERT 314
BIOREACTOR SYSTEM

OPEN ENDED EXPERIMENT PROPOSAL

EXPERIMENT 5:

COMPARISON
ON
ENZYMATIC
ACTIVITY BETWEEN IMMOBILIZED AND
FREE CELL REACTOR FOR ETHANOL
PRODUCTION IN BATCH CULTURE

DATE OF EXPERIMENT
22ND APRIL 2015

PREPARED FOR
DR. KU SYAHIDAH BINTI KU ISMAIL

GROUP MEMBERS

(B4)

NUR AMIRAH BINTI MAHFOD

121141662
NUR ATHIRAH AWATIF ABDUL RAHMAN
121142810

PUA CHEE WEE

121142063
TAN SIEW YEN
121142363

Experiment 5
Comparison Enzymatic Activity between Immobilized and Free Cell Reactor for
Ethanol Production in Batch Culture

1.0 Objectives
1.1 To comparison enzymatic activity between immobilized and free cell reactor for
ethanol production in batch culture
1.2 To understand the principles of cell immobilization by using gel entrapment
method in alginate gel
1.3 To analyze the effect of immobilization on ethanol production

2.0 Introduction

A yeast cell-free enzyme system containing an intact fermentation assembly and

that is capable of bio-ethanol production at elevated temperatures in the absence of
living cells was developed to address the limitations associated with conventional
fermentation processes. Immobilized cells are cells attached to an inert, insoluble
material such as calcium alginate (produced by reacting a mixture of sodium alginate
solution and cells with calcium chloride). This can provide increased resistance to
changes in conditions such as pH or temperature. Cell immobilization in calcium
alginate beads and chitosan-covered calcium alginate beads allowed reuse of the beads
in eight sequential fermentation cycles of 10 h each. The immobilized cells allowed
eight 10 h sequential reuse cycles to be carried out with stable final ethanol
concentrations. In addition, there was no need to use antibiotics and no contamination
was observed. After the eighth cycle, there was a significant rupture of the beads
making them inappropriate for reuse. It has also helped to prevent the contamination of
the substrate with enzyme/protein or other compounds, which decreases purification
costs. These benefits of immobilized cells have made them highly applicable to a range
of evolving biotechnologies.

3.0 Materials And Equipments

3.1

Malt Extract

3.2

Peptone

3.3

Dextrose (D-glucose)

3.4

Yeast Agar plate

3.4

1.0% and 3.0% (w/v) sodium alginate solution

3.6

0.2 M calcium chloride solution

3.7

20g/L glucose solution

3.8

Distilled Water

3.9

Tap water

3.10

Ice

3.11

DNS

3.12

5 of 125 mL Erlenmeyer flasks

3.13

Pipette

3.14

Beaker

3.15

Autoclave

3.16

Incubator

3.17

Chemical balance

3.18

Spectrophotometer

3.19

5ml of measuring cylinder

3.20

Hot plate and magnetic stirrer

3.21

Retort Stand

3.22

Syringes

4.0 Procedures
4.1

Inoculation of yeast
1.

0.8% (w/v) malt extract, 1.0% (w/v) peptone and 2 % (w/v) dextrose
(D-glucose) is prepared for a working volume of 100ml.
This solution is put in a 100 ml Erlenmeyer flask.
The flask is cotton-plugged and covered with aluminium foil.
The medium is sterilized in batch autoclave at 1210C for 15 minutes.

2.
3.
4.

5.

After sterilization, the media is let to cool down at room temperature

before inoculation.
6. 100ml of the medium is measured and poured it into a conical flask.
7. 2 loops of yeast is scraped from agar plate and is inoculated it into the
medium and are left in the incubator to be shaken overnight.
5% (v/v) of yeast culture is pipette into the flask respectively.
The fermentation broth is incubated in incubator shaker at 150rpm.

8.
9.
4.2

Preparation of immobilized enzyme beads

1. Mix 1 ml of yeast cell broth with 9 ml of 3% (w/v) sodium alginate
solution
2. Drip the polymer solution prepared in step 4.1.1 from a height of
approximately 20 cm into the stirred calcium chloride (CaCl 2) solution
with a syringe at room temperature.
3. Leave the beads in the calcium solution to cure for 15minutes.
4. Filter the formed beads and wash thoroughly with distilled water.
5. Dry the beads using filter paper (Whatman no.1) followed by exposure to
the open air for 15 minutes before use.

4.3

Determination of ethanol produced from glucose in batch reactor for

immobilised and free cells
1. Transfer all of the gel beads prepared in Part 4.1 in a beaker
2. Add 10 ml of glucose solution in the beakers or column containing the
beads. The glucose concentration at this time is to be determined.
3. Shake the flasks at 250 rpm.
4. Measure glucose concentration every 15 min.
5. For free cells, mix 1 ml of yeast cell broth and 10 ml of glucose solution in
conical flask and repeat steps 4.2.3 4.2.4.

5.0 Expected Result

Absorbance
Type of Enzyme

Time
0

Immobilised
Enzyme

15 min

30 min

45 min

60 min

Glucose
(g/L)
Glucose
(g/L)

Free Enzyme

Expected Graph
Comparison in term of Absorbance between Immobilized Yeast Cell and Free Yeast Cell
over Time
12

Graph of Absorbance versus Time

10
8

Absorbance, nm

6
4

Immobilized Yeast Cell

2
0
0

Free Yeast Cell

2

Time, min

10

12