proposal experiment 5

8/9/2019 Proposal Experiment 5

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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

1211412

NUR ATHIRAH AWATIF ABDUL RAHMAN

121142!10



PUA CHEE WEE

1211420"

TAN SIEW YEN

121142""



Experiment 5

Comparison Enzymatic Activity between Immobilized and Free Cell Reactor for

Ethanol Production in atch Culture

!"# $b%ectives

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 gel1.3 To analyze the effect of immobilization on ethanol production

&"# 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 mi"ture of sodium alginate

solution and cells with calcium chloride#. This can provide increased resistance to

changes in conditions such as p$ or temperature. %ell immobilization in calcium

alginate beads and chitosan-covered calcium alginate beads allowed reuse of the beads

in eight se&uential fermentation cycles of 1' h each. The immobilized cells allowed

eight 1' h se&uential 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

ma(ing 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.



'"# (aterials And E)uipments

3.1 *alt +"tract

3.2 ,eptone

3.3 e"trose !-glucose#

3. /east Agar plate

3. 1.'0 and 3.'0 !w)v# sodium alginate solution

3. '.2 * calcium chloride solution

3. 2'g) glucose solution

3.4 istilled 5ater

3.6 Tap water

3.1' Ice

3.11 78

3.12 9 of 129 m +rlenmeyer flas(s

3.13 ,ipette

3.1 :ea(er

3.19 Autoclave

3.1 Incubator

3.1 %hemical balance

3.14 8pectrophotometer

3.16 9ml of measuring cylinder

3.2' $ot plate and magnetic stirrer

3.21 ;etort 8tand

3.22 8yringes

*"# Procedures

*"! Inoculation of yeast

1. '.40 !w)v# malt e"tract 1.'0 !w)v# peptone and 2 0 !w)v# de"trose

!-glucose# is prepared for a wor(ing volume of 1''ml.

2. This solution is put in a 1'' ml +rlenmeyer flas(.

3. The flas( is cotton-plugged and covered with aluminium foil.

. The medium is sterilized in batch autoclave at 121'% for 19 minutes.



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

before inoculation.

. 1''ml of the medium is measured and poured it into a conical flas(.

. 2 loops of yeast is scraped from agar plate and is inoculated it into the

medium and are left in the incubator to be sha(en overnight.4. 90 !v)v# of yeast culture is pipette into the flas( respectively.

6. The fermentation broth is incubated in incubator sha(er at 19'rpm.

*"& Preparation of immobilized enzyme beads

1. *i" 1 ml of yeast cell broth with 6 ml of 30 !w)v# sodium alginate

solution

2. rip the polymer solution prepared in step .1.1 from a height of appro"imately 2' cm into the stirred calcium chloride !%a%l2# solution

with a syringe at room temperature.

3. eave the beads in the calcium solution to cure for 19minutes.

. <ilter the formed beads and wash thoroughly with distilled water.

9. ry the beads using filter paper !5hatman no.1# followed by e"posure to

the open air for 19 minutes before use.

*"' +etermination of ethanol produced from ,lucose in batch reactor for

immobilised and free cells

1. Transfer all of the gel beads prepared in ,art .1 in a bea(er

2. Add 1' ml of glucose solution in the bea(ers or column containing the

beads. The glucose concentration at this time is to be determined.

3. 8ha(e the flas(s at 29' rpm.

. *easure glucose concentration every 19 min.

9. <or free cells mi" 1 ml of yeast cell broth and 1' ml of glucose solution in

conical flas( and repeat steps .2.3 = .2..



5"# Expected Result

-ype of Enzyme -ime

Absorbance

# !5 min '# min *5 min .# min

Immobilised

Enzyme

/lucose

0,123

Free Enzyme/lucose

0,123

Expected /raph

%omparison in term of Absorbance between Immobilized /east %ell and <ree /east %ell

over Time

0 2 4 ! 10 120

2

4

!

10

12

-ime4 min

Absorbance4 nm

>raph of Absorbance versus Time

Immobilized /east %ell

<ree /east %ell

proposal experiment 5

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