optimization of cultivation medium for levan...

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OPTIMIZATION OF CULTIVATION MEDIUM FOR LEVAN PRODUCTION

USING Bacillus subtilis IN SEMI-INDUSTRIAL SCALE

KHAIREDZA RAHMI BINTI A. HAMID

A dissertation submitted in partial fulfilment of the

requirements for the award of the degree of

Master of Engineering (Bioprocess)

Faculty of Chemical Engineering

University Teknologi Malaysia

JUNE 2015

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To Allah S.W.T the most gracious and merciful.

To my beloved husband, Munir; my daughters, Alya&Aqasyah, my parents &my

siblings.

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ACKNOWLEDGEMENT

I would like to express my appreciation to my supervisor, Prof. Dr. Hesham

Ali El-Enshasy for his advice, kindness, dedication and moral support. I would also

like to thank him again for his detailed and all his constructive criticism and support

throughout this study. I have wonderful journey and guidance having to work under

his supervision.

My warm thanks to the assistance of friends; RO, all Masters and PhD

student of Bioprocessing Engineering Laboratory in IBD – Mdm Roslinda, Mdm Nor

Zalina and Mr Solleh, Miss Nor Hamizah, Miss Nur Hafizah, Miss Naqiyyu Bazz,

Mr Daniel JD, Mr Amir Fuhaira, Mr Khairudin Malek, Mr Helmi, Mrs Siti Marhaida

and Mrs. Zulaiha for their help, support, and advices but not forget to mention also

their technical support while in the lab to make my work free from problem & stress.

My loving thanks to my dear husband and daughters; Munir, Alya&Aqasyah;

and not to forget my parents and parents in law. Without their encouragement and

understanding it would have been impossible for me to finish this work.

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ABSTRAK

Levan merupakan polisakarida luarsel fruktosa, dan boleh ditemui pada

tumbuhan dan produk mikrobial. Kajian tentang levan baru-baru ini menampilkan

peranannya sebagai antitumor, antioksidan, antivirus, kesan terapeutik, agen

antikanser dan tindakan fibrinolitik yang dihasilkan daripada pelbagai jenis

mikroorganisma. Kajian ini memberi tumpuan kepada mengoptimumkan media

pengkulturan Bacillus subtilis dalam kelalang goncang terhadap pertumbuhan sel and

penghasilan levan. Kesan parameter bioproses juga turut di kaji terhadap penggunaan

dalam bioreactor 16-L berskala industri. Kajian terbaru melaporkan bahawa jumlah

tertinggi levan dihasilkan daripada media menggunakan sukrosa komersial, dengan

terdapat sedikit kuantiti mendakan alkohol. Media belum di optimasikan

menghasilkan ketumpatan sel sebanyak 1.95 g L-1 dan levan sebanyak 13.5 g L-1 dari

kajian kelalang goncang, dan selepas pengoptimuman mengikut kaedah OFAT dan

statistikal dilakukan ketumpatan sel meningkat kepada 2.26 g L-1 dan levan juga

meningkat kepada 22.1 g L-1 dan 21.8 g L-1, masing-masing. Oleh itu, terdapat

peningkatan 38.07 % dalam levan yang dihasilkan daripada media dioptimumkan

secara statistik berbanding media tidak di optimumkan. Pengkulturan dalam

bioreactor 16-L telah dijalankan dikeadaan pH yang tidak dikawal dan pH dikawal

dengan menggunakan medium yang telah di optimumkan secara statistik. Formulasi

baru terdiri daripada (g L-1): sukrosa, 130.0, ekstrak yis, 6.3, K2HPO4, 5.0, dan

MgSO4.7H2O, 0.2. Hasilnya menunjukkan bahawa bioreaktor tanpa kawalan pH

adalah terbaik berbanding bioreactor dengan kawalan pH dalam mendapatkan jumlah

ketumpatan jisim sel dan penghasilan levan. Dari bioreaktor tanpa kawalan pH

menunjukkan jisim ketumpatan sel yang tertinggi iaitu 5.06 g L-1 dan levan sebanyak

26.65 g L-1. Ini adalah hampir 49.34 % lebih tinggi daripada yang di perolehi dalam

pengkulturan menggunakan kelalang goncang. Walau bagaimanapun hasilan produk

iaitu levan bagi setiap jisim sel adalah 5.64 g L-1 berbanding 11.54 g L-1 dari

bioreaktor dengan kawalan pH kerana ketumpatan selnya rendah. Kesimpulannya,

kajian ini telah mencadangkan kaedah dan strategi pengkulturan efektif untuk

menghasilkan ketumpatan sel dan pengeluaran levan yang tinggi dalam skala semi-

industri.

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ABSTRACT

Levan is extracellular polysaccharides of fructose, and it can be found in

plants and microbial products. Recent studies of levan showing characteristics as

antitumor, antioxidants, antiviral, therapeutic effects and anticancer agents with

fibrinolytic action and were produced from various types of microorganisms. This

study focused on medium optimization study of B. subtilis in the shake flask levels

on the cell growth and levan production. The effects of bioprocessing parameters was

also studied in the semi-industrial scale 16-L bioreactor. Recent studies reported that

the highest amounts of levans are produced from the medium with commercial

sucrose, with minor quantity of alcohol precipitate. The un-optimized medium

yielded the cell mass production of 1.95 g L-1 and 13.5 g L-1 of levan from the shake

flasks studies. Then, the cell mass increased to 2.26 g L-1 and the production of

levan also increased to 22.1 g L-1 and 21.8 g L-1, respectively after medium

optimization using OFAT method and statistical approach. Thus, showed that there

was 38.07% increment in levan produced from statistical optimized medium

compared to un-optimized medium. Batch cultivations in 16-L stirred tank

bioreactors were carried out under un-controlled pH and controlled pH condition by

using statistically optimized production medium generated from statistical tool. The

new formulation was composed of (g L-1): sucrose, 130.0, yeast extract, 6.3,

K2HPO4, 5.0, and MgSO4.7H2O, 0.2. The result showed that bioreactor under

uncontrolled pH was favorable compared to controlled pH condition in obtaining the

highest amount of cell mass and levan production. From the cultivation under un-

controlled pH in the 16-L bioreactor showed high cell mass production of 5.06 g L-1

and 26.65 g L-1 of levan production was produced. This was almost 49.34 % higher

than those obtained in shake flask cultivation. However the yield of levan per cell

mass was 5.64 g L-1 compared to 11.54 g L-1 from bioreactor with controlled pH due

to the low cell mass production. In conclusion, this study proposed a reliable

industrial production medium and an effective cultivation strategy for the cell mass

and levan production in semi industrial scale.

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TABLE OF CONTENTS

CHAPTER TITLE PAGE

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENTS iv

ABSTRAK v

ABSTRACT vi

TABLE OF CONTENTS vii

LIST OF TABLES xii

LIST OF FIGURES xiv

LIST OF APPENDICES xvii

LIST OF ABBREVIATIONS xviii

1 INTRODUCTION 1

1.1 Research Background 1

1.2 Problem Statement 2

1.3 Research Objectives 3

1.4 Research Scope 3

2 LITERATURE REVIEW 4

2.1 Levan 4

2.2 Structure of Levan 5

2.3 Molecular Weight of Levan 7

7

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2.4 Application of Levan 8

2.5 Levansucrase 11

2.6 Bacillus subtilis Metabolism 14

2.7 Effect of Medium Component on Levan

Production

2.7.1 Effect of Carbon Source

2.7.2 Effect of Nitrogen Source

2.7.3 Effect of Phosphate Source

16

16

18

20

2.8 Cultivation in the bioreactor 21

2.8.1 Effect of pH on Levan Production

2.9 Statistical Optimization Medium

2.9.1 Full Factorial Design

2.9.2 Fractional Factorial

2.9.2.1 Plackett-Burman

Experimental Design

2.9.3 Response Surface Methodology

(RSM)

24

26

26

27

28

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3 METHODOLOGY 32

3.1 Introduction 32

3.2 Chemical and Equipments 34

3.3 Source of Microorganism – Bacillus Subtilis

Strain B58

35

3.4 Inoculum Preparation and Fermentative

Production

3.4.1 Bacteria Cell Banking

35

35

3.5 Inoculum preparations 36

3.6 Preparation of Production of Medium 36

3.7 Medium Optimization (OFAT Method)

3.7.1 Effects of Different Concentrations of

Sucrose on the Cell Mass and Levan

Production by B. subtilis.


37

37

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Yeast Extract on the Cell Mass and Levan



K2HPO4 on the Cell Mass and Levan


37

38

3.8 The Growth Kinetics Studies of B. subtilis in

the Un-optimized and Optimized Medium for

Cell Growth and Levan Production.

38

3.9 Statistical Media Optimization

3.9.1 Full Factorial Experimental Design

3.9.2 Response Surface Methodology

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39

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3.10 Cultivation in Semi-Industrial Scale 16-L

Stirred Tank Bioreactor

3.10.1 Batch Cultivation Studies on the

effects of Controlled and Un-

Controlled pH in 16-L Bioreactor

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41

3.11 Analytical Methods

3.11.1 pH Determination

3.11.2 Cell Dry Weight and Optical Density

3.11.3 Separation of Levan Polymer

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43

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4 RESULTS & DISCUSSION 45

4.1 Introduction 45

4.2 Growth Kinetics of B. subtilis in Shake Flask

Cultivation

4.2.1 Growth Kinetics of B. subtilis in Un-

optimized Medium in Shake Flask Cultivation

4.3 One Factor At a Time (OFAT) Optimization

Study

4.3.1 Effect of Different Sucrose

Concentrations on the Cell Growth and

Levan Production by B. subtilis

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46

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4.3.2 Effect of Different Yeast Extract

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Concentration on B. subtilis Cell Growth

and Levan Production

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4.3.3 Effect of Different K2HPO4

Concentration on B. subtilis Cell Growth

and Levan Production

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4.3.4 Growth Kinetic of B. subtilis in Shake

Flask Cultivation Using Optimized

Medium (OFAT Method)

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4.4 Screening of Medium Composition using - Full

Factorial Design

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4.5 Response Surface Methodology (RSM)

4.5.1 Analysis of Variance (ANOVA) of the

Response variables for Production of

Levan

4.5.2 The effect of Interaction Factors of

Production of Levan by B. subtilis

4.5.3 Confirmation Test of Optimization Study

and the Growth Kinetics Studies of

Statistical Optimization Medium.

4.6 Growth Kinetic of B. subtilis in Shake Flask

Cultivation Using Statistical Optimized

Medium

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64

67

70

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4.7 Batch Cultivation in 16-L Stirred Tank

Bioreactor

4.7.1 Effect of Un-Controlled pH for Batch

Cultivation in 16-L Stirred Tank

Bioreactor on the Cell Growth and


4.7.2 Effect of Un-Controlled pH for Batch

Cultivation in 16-L Stirred Tank

Bioreactor on the Cell Growth and


73

74

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4.8 Growth Kinetic Comparison in the Shake Flask

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and Bioreactor Study 78

5 CONCLUSION 82

5.1 Conclusion 82

5.2 Future Work 83

REFERENCES

APPENDICES

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LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 Various applications contribute by the levan

from several series of studies conducted by many

researchers.

10

2.2 Yeast extract composition 19

2.3 General terms used in experimental design 27

3.1 Equipments used in the study 34

3.2 Chemicals and reagent used in the experiments 35

3.3 Luria Bertani agar media composition 36

3.4 Media compositions of nine different sucrose

concentrations

37

3.5 Media compositions of eight different yeast

extract concentrations.

38

3.6 Media compositions of five different K2HPO4

concentrations

38

3.7 Media compositions before and after

optimization.

39

3.8 Two-full factorial design and experimental range

of factors in cultivation of B. subtilis in

production of levan.

40

3.9

Box-Behnken experimental design of factors and

levels used.

41

4.1 Factors influenced the levan production by B.

subtilis and its range levels.

58

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4.2 Placket-Burman Experimental Design 59

4.3 Estimated effect and coeeficients for levan

production by B. subtilis.

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4.4. The Box-Behnken experimental design; the

factors and its two levels used.

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4.5 The levan production results from the Box-

Behnken experimental design.

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4.6 Estimated Regression Coefficient for Levan (g L-

1) production using coded unit.

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4.7 Estimated regression coefficient for levan

production by B. subtilis using Box-Behnken

uncoded unit.

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4.8. Estimated effect and coeeficients for levan

production by B. subtilis.

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4.9 Analysis of Variance (ANOVA) using a Box-

Behnken design for the production levan from B.

subtilis using Box-Behnken design.

73

4.10 Cultivation parameters for cultivation of Bacillus

subtilis under controlled and uncontrolled pH

condition.

78

4.11 Kinetic growth parameters of the production of

levan and cell growth during cultivation of B.

subtilis in shake flasks and 16L bioreactor.

79

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LIST OF FIGURE

FIGURE NO. TITLE PAGE

2.1 Levan β-(2,6) polyfructan chemical structure. 5

2.2 Inulin chemical structure. 7

2.3 Several types of levansucrase catalyzed reaction 13

2.4 B. subtilis bacteria after gram staining procedure. 14

2.5 B. subtilis colony and mucoid-type colony by B.

subtilis on agar plate

15

2.6 Oxidation reaction of glucose producing energy 17

2.7 The effect of different sucrose concentrations on

levan production.

17

2.8 Microbial growth curve under Batch culture

cultivation

22

3.1 General overview of the research methodology

for this study

33

4.1 Production of levan, cell dry weight, the yield

coefficient of levan based on cell mass, and pH

of B. subtilis in un-optimized medium after 27

hours cultivation at 37 ˚C and 200 rpm in the

shake flask

48

4.2 The levan production, cell dry weight and pH

obtained from the different sucrose

concentrations in shake flask cultures

cultivations at 37 ˚C after 24 hours cultivations.

50

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4.3 The cell mass and levan production and pH

obtained from the different yeast extract

concentrations in shake flask cultures at 37 ˚C

for 24 hours cultivations

53

4.4 The cell mass and levan production and pH

obtained from the different K2HPO4

concentrations in shake flask cultures at 37 ˚C

for 24 hours cultivations.

55

4.5 The growth kinetic of cell mass and levan

production, the yield coefficient of levan based

on cell mass, and changes of pH of B. subtilis in

optimized medium after 27 hours cultivation at

37 ˚C and 200 rpm in the shake flask.

57

4.6 Pareto chart of effect of sucrose, K2HPO4 and

yeast extract on levan production of B. subtilis

60

4.7. 2D Contour plots between sucrose and

dipotassium hydrogen phosphate (K2HPO4)

when response is levan.

67

4.8. 2D Contour plots between sucrose and (yeast

extract) when response is levan.

68

4.9 2D Contour plots between yeast extract and

dipotassium hydrogen phosphate (K2HPO4)

when response is levan production

69

4.10 Optimization Plot for cell mass production of

levan from B. subtilis using a Box-Behnken

design.

70

4.11 The growth kinetic of cell mass and levan

production, the yield coefficient of levan based

on cell mass, and changes of pH of B. subtilis in

statistical optimized medium after 27 hours

cultivation in the shake flask (cultivation at 37 ˚C

and 200 rpm).

71

4.12 The cell mass and levan production, changes of 72

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pH, dissolved oxygen of B. subtilis in the


cultivation in 16L bioreactor under un-controlled

pH condition (initial pH 7.0).

4.13 The cell mass and levan production, changes of

pH, dissolved oxygen of B. subtilis in the


cultivation in 16L bioreactor under controlled pH

condition (initial pH 7.0).pH 7.0)

77

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LIST OF APPENDICES

APPENDIX TITLE PAGE

A Response surface methodology optimization

data generated by Minitab 16

92

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LIST OF ABBREVIATIONS

CDW - Cell dry weight

DO - Dissolved Oxygen

OD - Optical density

OD600 - Optical density at 600 nm

sp. - Species

K2HPO4 - Di-potassium hydrogen phosphate

MgSO4.7H2O - Magnesium sulfate heptahydrate

OFAT - One Factor At Time

RSM - Response Surface Methodology

Levmax - Maximum levan production

Xmax - Maximal Dry weight

Qlev - Levan production rate

µ - Specific growth rate

Y p/x - Maximum product produce at maximum cell mass

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CHAPTER 1

INTRODUCTION

1.1 Research Background

Levan is a biopolymer of fructose with link by β(2-6) glycosidic bond. It also

have some β(2-1) linked branched. Levan can be found in plants or microbial

products but their molecular weights are different. Levan in plants are low molecular

weight compared to levan obtained from microbial products (Moosavi-Nasab et al.,

2010). Levan and inulin are two common types of fructan. Levan is polyfructose

with larger branch while inulin is much smaller branch, with β-2, 1 linkages. Levan

and inulin are totally different in terms of characteristic, where levan is water soluble

compound while inulin is robust adhesive properties than levan. Levan is different

compared to other polysaccharides that had been used as emulsifier and thickener,

because it does not swell in water.

Nowadays, the industrial production of levan is carried out by different types

of microorganisms. Levan potentials as emulsifier, stabilizer, thickener, surface-

finishing agents, carrier for flavour and fragrances had been proposed by many

research and experiments. Its value in medical, pharmaceutical, food industry has

shown to be successful through many research and journals. Regardless of many

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interesting characteristics and advantages showed by levan, the requirements of

producing it in large scale increased dramatically.

In this study the cultivation of B. subtilis in the shake flask and semi

industrial scale 16L bioreactor was investigated to study the growth kinetics of B.

subtilis and levan production in different medium composition and under different

bioprocessing parameters.

This study generally focused to optimize the production of levan that

contribute to the potentially wide application of levan in pharmaceutical and food

industry.

1.2 Problem Statement

Recent studies of levan were motivated for its role as antitumor, antioxidants,

antiviral and anti-cancer agents with fibrinolytic action. Many researchers worldwide

have published data on production of levan, but their researches are focusing on the

use of different carbon sources, yield, and comparison between different microbes

producing levan, including purification and characterization of of levan.

However, there are so little information on producing levan at higher cell

density cultivation strategy. Several studies conducted for decades in producing of

levan using various microorganisms, but most of them are facing disadvantages of

low yield of levan (Shih et al., 2010). Through optimizing medium composition in

this work, the optimized condition for levan production was studied to formulate

production medium towards industrial applications in the shake flasks levels. This

followed by study of bioprocessing condition in the semi industrial scale 16L

bioreactor that gave high impact to the cell growth and production of levan.

3

1.3 Research Objectives

This study was focused on the development of an industrial production

medium and a cultivation strategy for the cell growth and levan production by B.

subtilis in semi industrial scale bioreactor.

1.4 Research Scope

The scopes of the research were applied in order to achieve the objective of this

study which are;

(i) Medium optimization for shake flask cultivation of B. subtilis using One Factor

At Time (OFAT) approach.

(ii) Medium optimization for shake flask cultivation of B. subtilis using statistical

approach (RSM).

(iii) Study the growth kinetics and levan production before and after medium

optimization in shake flask levels.

(iv) Batch cultivation of B. subtilis in 16-L pilot scale stirred tank bioreactor to

investigate the effect of pH (controlled vs uncontrolled) on the levan production in

16-L bioreactor.

optimization of cultivation medium for levan...

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