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SEPARATION OF FRUCTOSYLTRANSFERASE USING

ULTRAFILTRATION MEMBRANE:

EFFECT OF pH AND IONIC STRENGTH ON FLUX AND REJECTION

MOHD KHAIRUL AFIZAN BIN HARUN

A thesis submitted in fulfillment of the

requirement for the award of the degree of

Bachelor of Chemical Engineering

Faculty of Chemical and Natural Resources Engineering

Universiti Malaysia Pahang

APRIL 2009

I declare that this thesis entitled “Separation of Fructosyltransferase Using

Ultrafiltration Membrane: Effect of pH and Ionic Strength on Flux and Rejection” is

the result of my own research except as cited in the references. The thesis has not

been accepted for any degree and is not concurrently submitted in candidature of any

other degree.

Signature : ..................................................

Name of Candidate : MOHD KHAIRUL AFIZAN BIN HARUN

Date : 2 APRIL 2009

iii

Special Dedication of This Grateful Feeling to My

Beloved parents;

Hj. Harun bin Jusoh & Hajjah Aminah binti Hj. Othman

Loving brother and sisters;

Nor Ashikin Hj. Harun

Samseema Hj Harun

Suraya Hj. Harun

Mohd Reduan Hj. Harun

Suzana Hj. Harun

Siti Khatijah Hj. Harun

Noor Nabila Huda Hj. Harun

Nor Bashirah Hj. Harun

iv

ACKNOWLEDGEMENT

I would to express gratitude to all who gave me the possibility to complete this

Undergraduate Research Project (PSM). I want to thank the first and foremost, my

sincere appreciation to my Undergraduate Research Project supervisor, Dr Mimi

Sakinah Binti Abdul Munaim, for guiding and encouraging me throughout this

experiment. Thanks a lot for giving me a professional training, advice and suggestion

to bring this Undergraduate Research Project to its final form. Without her support

and interest, this PSM would not have been the same as presented here.

I am grateful to the staff of Technical Unit, Faculty of Chemical & Natural

Resources Engineering of Universiti Malaysia Pahang as Mr. Zainal bin Gimban and

En. Abd Razak bin Abd Hamid for their cheerfulness and professionalism in handling

their work.

Special appreciation for Miss Kamariah bt Mat Peah from Faculty of Civil &

Earth Resources as her interest to help for using Total Orgnanic Carbon.

In particular, my sincere thankful is also extends to all my colleagues as Nor

Diyana binti Abu Bakar Sidek and others masters student who have provided

assistance at various occasions. Their views and tips are useful indeed. Unfortunately,

it is not possible to list all of them in this limited space.

And last, but not least I thank my mother’s and other family members for their

continuous support while completing this PSM.

v

ABSTRACT

There are various methods to separate between macro molecule and non

dissolved particle in chemical process. One of the methods is separation process using

ultrafiltarion membrane. In filtration process, the macromolecules such as enzyme

will be retained on the membrane surface. This experiment is study about fouling

characteristic occur in an industry. The fouled membrane surface problem gives the

high cost operation and reduces the quality of production. Therefore, the main

objectives for this experiment are to determine the effect of pH and ionic strength on

membrane flux and rejection during fructosyltransferase (FTase) separation. The 50

kDa molecular weight cut off (MWCO) of ultrafiltration membrane was used during

this experiment. Cross flow filtration was used to run this experiment in the lab scale.

Total organic carbon (TOC) was used to analysis the concentration of sample.

Potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate

(K2HPO4) buffer solution range pH 5 to pH 8 was applied to find the effect of pH and

various molarities of NaCl (0.5M to 2.0M) was used to find the effect of ionic

strength in ultrafiltration membrane. The experimental result shows that the optimum

pH and ionic strength was 8.0 and 0.5M, respectively, in order to separate the FTase

solution using ultrafiltration membrane.

vi

ABSTRAK

Pelbagai langkah dan teknik digunakan bagi pemisahan antara molekul macro

dan bahan tidak terlarut dalam prosess kimia. Salah satu kaedah yang digunakan ialah

proses pemisahan dengan menggunakan penapis ultra. Dalam proses pengasingan atau

penapisan ini, molekul macro seperti enzim akan tertahan di permukaan penapis.

Dalam eksperimen ini, kajian dijalankan bagi mengenal pasti ciri – ciri bahan yang

menyebabkan berlaku penyumbatan penapis dalam industri kerana ia akan memberi

kesan negatif seperti peningkatan kos operasi dan mengurangkan kualiti produk.

Objektif utama kajian ini adalah bagi mengenal pasti, kesan pH dan kekuatan ionik

kepada fluks dan bahan tertahan dengan menggunakan enzim fructosyltransferase

(FTase). Saiz penapis yang digunakan dalam eksperimen ini ialah 50 kDa. Manakala

penapis aliran songsang yang digunakan adalah berskala kecil. Total organic content

(TOC) digunakan bagi menganalisa kepekatan sampel. Kalium dihodrogen Phospahte

(KH2PO4) dan diKalium hydrogen phosphate (K2HPO4) digunakan sebagai larutan

penimbal dengan skala antara pH 5 sehingga pH 8, manakala 0.5M sehingga 2.0M

Natrium Klorida (NaCl) digunakan bagi menganalisa kekuatan ionik larutan tersebut.

Keputusan daripada kajian ini menunjukan, pH 8 dan 0.5M merupakan larutan yang

optimum untuk digunakan dalam proses penapisan dengan menggunakan penapis

ultra.

TABLE OF CONTENTS

CHAPTER TITLE PAGE

TITLE PAGE i

DECLARATION ii

ACKNOWLEDGEMENT iii

ABSTRACT v

ABSTRAK vi

LIST OF TABLES xi

LIST OF FIGURES xvi

1 INTRODUCTION

1.1 Background of Study 1

1.2 Problem Statement 2

1.3 Objective 3

1.4 Scope of Study 3

1.5 Significant of Study 4

2 LITERATURE REVIEW

2.1 Enzyme of Fructosyltransferase 6

2.2 Definition of Membrane 10

2.2.1 Driving force in membrane separation process 10

2.2.2 Transmembrane pressure 10

2.3 Membrane Structure 11

2.3.1 Porous Membrane 11

2.3.2 Non-Porous Membrane 12

2.3.3 Carrier Membrane 12

2.4 Membrane Type 13

2.5 Flowsheet 14

CHAPTER TITLE PAGE

2.6 Process Operation 15

2.7 Membrane Module Type and Their Characteristic 16

2.7.1 Plate and Frame 16

2.7.2 Spiral – Wound Module 17

2.7.3 Hollow Fiber, Capillary and Tubular 18

2.8 Unltrafiltration Membrane 19

2.8.1 Asymmetric Membrane 19

2.8.2 Porous Membrane 19

2.8.3 Types of Flow Ultrafiltration Process 20

2.8.4 Protein Separation Mechanisme 20

2.8.5 Factor Affecting Ultrafiltration Membrane 21

2.8.5.1 Temperature 21

2.8.5.2 Ratio of Concentration 21

2.8.5.3 Viscosity and Volume Flow Rate 21

2.9 Fouling 22

2.9.1 Definition of Fouling 22

2.9.2 Particles, Biofouling and Scaling 23

2.9.3 Predict Fouling 23

2.9.4 Membrane Fouling Control 24

2.9.4.1 Silt Density Index (SDI) 24

2.10 Limitations 25

3 METHODOLOGY

3.1 Overall Methodology 27

3.2 List of Apparatus 27

3.2.1 Ultrafiltrtion System 29

3.2.2 Membrane Type 30

3.3 List of Chemical 30

3.4 Preparation of Solution 31

3.4.1 Preparation of Buffer Solution 31

CHAPTER TITLE PAGE

3.5 Separation of FTase using Ultrafiltration Membrane

for Effect of pH

32

3.6 Flux Analysis 33

3.7 Protein Rejection Analysis 34

3.8 Total Organic Carbon (TOC) Analysis 34

3.9 Separation of FTase using Ultrafiltration Membrane

for Inoic Strength

35

4 RESULT AND DISCUSSION

4.1 Effect of pH on Flux during Membrane Separation 37

4.1.1 FTase Flux at pH 5 using Ultrafiltration

Membrane

37


Membrane

38


Membrane

39


Membrane

41

4.1.5 Overall Flux Analysis during FTase Separation

at Different pH Solution

42

4.2 Effect of pH on Membrane Rejection 45

4.2.1 Rejection Analysis of FTase at Different pH

Solution

45

4.3 Effect of Ionic Strength on Membrane Flux 47

4.3.1 Flux Decline during FTase Separation at 0.5 M

NaCl

47


NaCl

48


NaCl

50

CHAPTER TITLE PAGE


NaCl

51

4.3.5 Overall Inonic Strength Analysis during FTase

Separation.

53

5 CONCLUSIONS AND RECOMENDATION

5.1 Conclusions 56

5.2 Recommendation 57

REFERENCES 58

APPENDIX A 61

APPENDIX B 74

APPENDIX C 95

xi

LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 Membrane Materials for Various Applications 13

2.2 Membrane Separation Processes with its Various

Characteristics

15

2.3 Plate and Frame 16

2.4 Spiral –Wound Modules 17

2.5 Hollow-Fiber, Capillary and Tubular 18

3.1 Preparation of Buffer Solution 31

3.2 Recommendation Cleanign Conditions 33

4.1 % of Rejection at Different pH Solution 46

A.1 Flux Decline during FTAse Separation at pH 5 62




A.5 Volume of Flux for every pH 66

A.6 Flux for every pH 67

A.7 Flux Volume for 0.5M NaCl 68




A.11 Volume of Flux for every Mole 72

A.12 Flux for every Mole 73

B Table from TOC 74

xvi

LIST OF FIGURES

FIGURES NO. TITLE PAGE

1.1 KvickTM Lab Cross-Flow System Units 4

2.1 Process flowsheet of industrial production of FTase. 9

2.2 Porous Membrane (separation of smaller species) 11

2.3 Non-porous membrane 12

2.4 Carriers membrane 13

2.5 Parallel Flow 14

2.6 Series Flow 14

2.7 Two Stage Flow 14

2.8 Plate and Frame Schematic 16

2.9 Spiral-Wound Schematic 17

2.10 Bore Feed Schematic 18

2.11 Shell Feed Schematic 19

2.12 Rtotal in membrane 23

3.1 Methodology for Effect of pH 27

3.2 Methodology for Effect of Ionic Strength 28

3.3 Cross flow Ultrafiltration Membrane 29

3.4 Polyethersulfone membrane 30

3.5 Total Organic Carbon 34

4.1 Flux during FTase separation at pH 5 37

4.2 Volume during FTase separation at pH 5 37






ix

FIGURES NO. TITLE PAGE


4.9 Overall Flux Analysis during FTase separation at

different pH

43

4.10 Overall Volume Analysis during FTase separation at

different pH

43

4.11 Flux on 10 minute on different pH 44

4.12 Volume on 10 minute on different pH 44

4.13 Analysis of Rejection at Different pH Solution 46

4.14 Ionic Strength during FTase separation at 0.5M 47

4.15 Volume of Flux during FTase separation at 0.5M 48







4.22 Overall analysis of Flux during FTase separation at

different mole solution.

54

4.23 Overall analysis of volume during FTase separation at

different mole solution.

54

4.24 Flux on 10 minute at different mole 55

4.25 Volume on 10 minutes at different mole 55

C.1 Cross Flow Filtration 96

C.2 Apparatus of Experiment 96

C.3 Apparatus of Experiment 97

C.4 Sample of Experiment 97

C.5 Sample of Experiment 98

C.6 Total Organic Carbon 98

CHAPTER 1

INTRODUCTION

1.1 Background of Study

Excellent water quality produced by membrane filtration has made this

advanced technology a promising process in providing better drinking water for water

supply. Membrane filtration processes involving microfiltration (MF), ultrafiltration

(UF), nanofiltration (NF) and reverse osmosis (RO) in potable water production have

increased rapidly for the past decade and would potentially replace the conventional

treatment process trains which consist of ozonation–precipitation– coagulation–

flocculation–chlorination–gravel filtration (Clever et al., 2000)

Recently, membrane separation involves partially separating a feed containing

a mixture of two or more components by use of a semi permeable barrier (the

membrane) through which one or more of the species moves faster than another or

other species. As shown in Figure 1.1, the basic process of the membrane separation

involves a feed mixture separated into a retentate (part of the feed that does not pass

through the membrane, or retained) and a permeate (part of the feed that passes

through the membrane). Although the majority of time the feed, retentate, and

permeate are usually liquid or gas, they may also be solid. The optional sweep is a

liquid or gas, used to help remove the permeate. (Ali et al., 2003)

2

Fructosyltransferase is an enzyme transforming sucrose into

fructooligosaccharides (FOS). FOS is fructose oligomers with a terminal glucosyl unit

and with a general formula GFn, where typical values of n are 2–4. FOS is classified

as prebiotics and has numerous beneficial properties for human health (Yun, 1996).

They are widely utilized in food and pharmaceutical industries. Although

FTase was found in many higher plants and microorganisms, the most important

industrial sources are strains of Aspergillus niger, Aspergillus japonicus and

Aureobasidium pullulans (Yun, 1996).

In spite of the utilization of FTase in the industrial production of FOS and

numerous scientific investigations, the only commercially available source of FTase is

Pectinex SP-L, a pectinolytic and cellulolytic preparation designated for fruit juice

processing.

1.2 Problem Statement

Many bioproducts are enzyms and there is a great demand for their separation.

Conventional techniques such as precipitation, crystallization and centrifugation can

suffer from poor selectivity of separation. The high-resolution separation techniques

such as chromatography, affinity separation and electrophoresis have a very low

throughput and produce small quantities of very pure proteins; to produce larger

amounts of proteins using these methods is expensive. (Yunos and Field, 2007)

One of the critical issues in the development of effective whey ultrafiltration

processes is the decline in system performance due to enzyme fouling, which limits

the economic efficiency of the processing operation. Membrane fouling is generally

characterized as a reduction of permeate flux through the membrane as a result of

increased flow resistance due to pore blocking and cake formation. Several

approaches have been proposed to reduce such membrane fouling and to improve the

3

membrane cleaning efficiency. Such methods include intermittent back flushing, flow

pulsation and electrical field inducement. (Muthukumaran et al., 2007)

1.3 Objectives

The objectives of this research are:

a) To determine the effect of pH on membrane flux and rejection during

Fructosyltransferase separation.

b) To determine the effect of ionic strength on membrane flux and rejection

during Fructosyltransferase separation.

c) To determine the optimum condition of pH and ionic strength for

Fructosyltransferase separation.

1.4 Scope of Study

There are few purposes doing this research. The purposes are:

i. The membrane will be used is which have 50kDA number of molecular cut

off.

ii. The protein that is used is Fructosyltransferase (FTase)

iii. KvickTM

Lab Cross-Flow System Unit was used in order to separate the

solution of DI water and FTase.

iv. The FTase solution will be prepared in sample which is pH 5 to pH 8.

v. The buffer solution will be prepared around 0.5M to 2.0M

vi. Total Organic Carbon will be used to measured the carbon in feed and

permeate

Figure 1.1

1.5 Significant of Study

By doing this research, it is hoped can add values of FTase and membrane

ultrafiltration. The main problem to solve in this experiment is to produce maximum

the production of FTase using ultrafiltartion membrane. If the common industry used

the others membrane like

produce the FTase, this experiment hope get better result if using the ultrafiltration

membrane system.

Total Organic Carbon will be used to measured the carbon in feed and

1.1 KvickTM Lab Cross-Flow System Units

Significant of Study




rane like chromatography, affinity separation and electrophoresis


4

Total Organic Carbon will be used to measured the carbon in feed and




chromatography, affinity separation and electrophoresis to


5

Normally during the separation process between FTase and solution, FTase

fouling will occur, this because the molecular weight of FTase not suitable with the

pore size of membrane. The important thing here is use the different value of

molecular weight and pore size.

This research also suggests using the continuous system. Hence it can reduce

the cost of operation. The price which is use as a raw material to produce

fructoligoscaride (FOS) is expensive; the continuous system is preferable due to this

problem.

CHAPTER 2

LITERATURE REVIEW

2.1 Enzyme of Fructosyltransferase

Fructosyltransferase (FTase) is an enzyme that catalyzes the transformation of

sucrose into fructooligosaccharides (FOS), which are important prebiotic compounds

having a broad application in food and pharmaceutical industries. Fructosyltransferase

catalyzes the transfer of fructosyl moieties where a donor or acceptor of these

moieties can be sucrose or fructooligosaccharides. In the industrial production of

fructooligosaccharides, the cells with the FTase activity are produced by aerobic

cultivation of fungi such as Aspergillus niger, Aspergillus japonicas, or

Aureobasidium pullulans. They are applied for the biocatalytic process in

immobilized form. (Vankova, Antosova, and Polakovic, 2005)

In our laboratory, we have dealt with the development and optimization of the

process of cultivation of the cells of A. pullulans with the FTase activity. The

increasing interest in prebiotic compounds opens also possibilities for small-scale use

of FTase. Isolated enzyme could be a suitable form for such purposes. For that reason,

we have also recently dealt with the downstream processing of FTase from the broth

7

obtained at the cultivation of A. pullulans. The obtained data can be used for the

design of the production process of FTase and analysis of its economic efficiency.

(Vankova, Antosova, and Polakovic, 2005)

The overall production of FTase depended strongly on the initial sucrose

concentration. This effect was the most notable where the production of FTase was

stopped after two days. The relative increment of the total FTase activity between the

2nd and 4th day was much lower in comparison with that between the 1st and 2nd day.

Such a drop of the enzyme production rate was not observed in the cultivations with

the initial sucrose concentration where the total enzyme activity reached the value in

the fourth day. (Antosova et al., 2002). Suppression of FTase production by

increasing sucrose concentration was observed, which is contrary to the results found

the largest amount of enzyme produced of sucrose after two cultivation days.

(Hayashi et al., 1991)

The FTase activity of cells represented approximately 60 to 70 % of the total

activity since the second cultivation day and the ratio of activities of cells and

activities in cultivation medium was 1.3 to 1.6 independently of the sucrose

concentration. The ratio of the cell to cultivation medium activities depends on the

content of magnesium sulfate in the production medium. The addition of magnesium

sulfate to the medium at the content of 0.2 % increased this ratio to the value of about

1.2 which was almost constant during the entire cultivation period. From this point of

view, the value of the ratio of 1.3:1.6 obtained by us at 0.05 % MgSO4 is noteworthy

(Hayashi et al., 1991).

The specific cell activity with respect to dry cell mass is a crucial factor for the

control of a cultivation run if whole cells, either free or immobilized, are used as

biocatalysts. Its value reached the maximum already in the rest day at S0 = 50 g dm−3

or in the second day at S0 = 200 g dm−3

and 350 g dm−3

. The maximum value of 8860

U g−1

was reached again in the cultivation with initial sucrose concentration of 350 g

dm−3

. As it has been mentioned above, the initial sucrose concentration influenced the

8

amount of produced FTase whereas the cell mass produced after four cultivation days

was unelected. This result suggests that the FTase production was promoted by high

sucrose concentrations. Although other authors used different activity assay

conditions and the absolute values are not fully comparable, the FTase activities of

AP CCY 27-1-1194 are of the same order of magnitude as those published for highly

active production strains, which suggests a potential of our strain for industrial

production of fructosyltransferase (Hayashi et al., 1991).

The design and scheduling of industrial biotechnological process is often

simplified by specialized computer-aided software such as Aspen Batch Plus or

SuperPro-Designer. These were applied in several studies of scale-up, optimal plant

design, and analysis of investment and operating costs of pilot and industrial

production of proteins. The examples include the production of insulin, tissue

plasminogen activator, β-galactosidase, heparinase, or growth hormone. (Vankova,

Antosova and Polakovic, 2005)

FTase of A. pullulans occurs in the periplasmic space of cells and so the part

of the enzyme is easily released to the cultivation medium. Therefore, the recovery of

the enzyme was considered from both the harvested cells and cultivation medium.

(Vankova, Antosova and Polakovic, 2005)

9

Figure 2.1 Process flowsheet of industrial production of FTase.

10

2.2 Definition of Membrane

Membrane can be define as a thin barrier which is allow passage of particle

with a certain size, particular physical or chemical properties (Ghosh, 2003). A

membrane can be dividing into types which are cell membrane and synthetic

membrane. The cell membrane is a semi permeable lipid bilayer which can be found

in all cells (Ghosh, 2003). Meanwhile, the synthetic membrane is a membrane that

being prepared for separation task in laboratory and industry. Their active part, which

permits selective transport of material, usually consists of polymer or ceramics,

seldom glass or material (Ghosh, 2003). Membrane can be prepare in variety forms

like flat sheets, tubes, capillary and hollow fibres. Membrane is built in membrane

modules like plate and frame, spiral-wound module, hollow fibre module or tube-in-

shell module (Ghosh, 2006).

2.2.1 Driving force in membrane separation process

Different driving force does include in membrane separation process. Some of

this are being applied when to transport solute and solvent molecules through

membranes. The forces include transmembrane pressure, concentration or

electrochemical gradient, osmotic pressure and electric field (Ghosh, 2003)

2.2.2 Transmembrane pressure

The transmembrane pressure is the main applied driving force (Ghosh, 2003).

Due to this applied driving force, the bulk liquid medium which is the solvent is

forced through the pores. The solvent molecules carry the solute molecules towards

the membrane and in certain case through membrane. Solute molecules might be fully

11

transmitted, partially transmitted or totally retained (or rejected) by membrane

(Ghosh, 2003).

2.3 Membrane Structure

Because the membrane must allow certain constituents to pass through, they must

have a high permeability to certain types of molecules. Membrane structures consist

of the following three basic types:

2.3.1 Porous Membranes

Porous membranes are used in microfiltration and ultrafiltration. The

dimension of the pores (0.1~10um) mainly determines the separation characteristics.

High selectivity can be obtained when the size of the solute is large relative to the

pore size in the membrane. Microporous membranes are similar to porous membranes

and differ in regards to pore dimension (50~500 Angstrom).

Figure 2.2 Porous Membrane (separation of smaller species)

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