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study of removal btx by pseudomonas putida in bioreactor

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  • III

    STUDY OF REMOVAL BTX BY PSEUDOMONAS

    PUTIDA IN BIOREACTOR

    MOHD ASHRAF BIN ABDUL RAHMAN

    Thesis submitted in partial fulfilment of the requirements

    for the award of the degree of

    Bachelor of Chemical Engineering

    Faculty of Chemical & Natural Resources Engineering

    UNIVERSITI MALAYSIA PAHANG

    JULY 2013

    MOHD ASHRAF BIN ABDUL RAHMAN (2013)

  • VIII

    ABSTRACT

    Benzene, toluene and xylene isomers (BTX) are the major components of gasoline which it

    is used widely as solvents and feedstock by the chemical industry. It became a common

    environmental contaminant problem that represents a serious threat to groundwater resources

    and to public health, being both toxic and relatively water soluble compared to other

    petroleum constituents. The biological treatment that use microorganism such as

    Pseudomonas putida has been selected because the ability to reduce the hazard of BTX. The

    objective of this research is to remove the BTX from wastewater using P. putida in a

    bioreactor. Before starting the experiment, P. putida is prepared by the growth it inside

    nutrient broth at a suitable temperature and concentration. The bacteria are put in to ratio of

    benzene, toluene, xylene, benzene-toluene, benzene-xylene, toluene-xylene and BTX

    solution inside simple conical flask. But the only BTX sample is only running in the

    fermenter for comparing the growth of bacteria in a conical flask with the growth of bacteria

    in fermenter. The bacterial growth will be analyzed using UV-Vis Spectrophotometer while

    concentration BTX after degradation is analyzed by HPLC. From the data obtain, the growth

    rate of bacteria in toluene sample is higher than other solution. While the growth rate of

    bacteria in BTX of conical flask is higher than the growth rate of bacteria in BTX of the

    fermenter. From overall experiment, Toluene is achieved the highest percent of removal

    compare to percent removal of benzene and xylene. Thus; removal of BTX by bacteria will

    be a potential biological treatment which it can solve our environmental pollutants in the

    future.

  • IX

    ABSTRAK

    Benzena, toluena dan isomer xilena (BTX) adalah komponen utama petrol mana ia digunakan

    secara meluas sebagai pelarut dan bahan mentah oleh industri kimia. Ia menjadi masalah

    pencemaran alam sekitar yang biasa merupakan ancaman serius kepada sumber air bawah

    tanah dan kesihatan awam, yang kedua-dua toksik dan agak larut dalam air berbanding

    dengan juzuk petroleum lain. Rawatan biologi yang menggunakan mikroorganisma seperti

    Pseudomonas putida telah dipilih kerana keupayaannya untuk mengurangkan bahaya BTX.

    Objektif kajian ini adalah untuk menghapuskan BTX dari air sisa menggunakan P. putida di

    dalam bioreaktor. Sebelum memulakan eksperimen, P. putida disediakan oleh pertumbuhan

    ia di dalam sup nutrien pada suhu yang sesuai dan penumpuan. Bakteria dimasukkan ke

    dalam kepada nisbah benzena, toluena, xilena penyelesaian, benzena-toluena, benzena-

    xilena, toluena-xilena dan BTX di dalam kelalang kon yang mudah. Tetapi satu-satunya BTX

    sampel hanya berjalan dalam fermenter untuk membandingkan pertumbuhan bakteria dalam

    satu kelalang dengan pertumbuhan bakteria dalam fermenter. Pertumbuhan bakteria akan

    dianalisis dengan menggunakan spektrofotometer UV-Vis manakala BTX tumpuan selepas

    kemusnahan dianalisis oleh HPLC. Dari data yang diperolehi, kadar pertumbuhan bakteria

    dalam sampel toluene adalah lebih tinggi daripada penyelesaian lain. Walaupun kadar

    pertumbuhan bakteria dalam BTX kelalang kon adalah lebih tinggi daripada kadar

    pertumbuhan bakteria dalam BTX daripada fermenter itu. Dari eksperimen keseluruhan,

    Toluena dicapai peratus penyingkiran tertinggi berbanding dengan peratus penyingkiran

    daripada benzena dan xilena. Oleh itu, penyingkiran BTX oleh bakteria akan menjadi

    rawatan biologi yang berpotensi yang ia boleh menyelesaikan pencemaran alam sekitar kita

    pada masa hadapan.

    .

  • X

    TABLE OF CONTENTS

    .

    SUPERVISORS DECLARATION.IV

    STUDENTS DECLARATION.....V

    DEDICATION..VI

    ACKNOWLEDGEMENT...VII

    ABSTRACT...VIII

    ABSTRAKIX

    TABLE OF CONTENTS...X

    LIST OF FIGURES....XIII

    LIST OF TABLE.XV

    LIST OF ABBREVIATIONSXVI

    LIST OF ABBREVIATIONS...XVII

    1 INTRODUCTION..1

    1.1 Motivation and statement of this problem.1

    1.2 Objectives..........2

    1.3 Scope of this research2

    1.4 Main contribution of this work..............2

    1.5 Organization of this thesis.3

    2 LITERATURE REVIEW..................4

    2.1 Overview...4

    2.2 BTX...................4

    2.2.1 Benzene5

  • XI

    2.2.2 Toluene.7

    2.2.3 Xylene...........9

    2.3 Pseudomonas putida..11

    2.4. Shake Flask Culture....15

    2.4.1 Advantages.15

    2.4.1 Disadvantages.15

    2.5 Bioreactor....16

    2.5.1 Fermenter...16

    2.5.1.1 Advantages.18

    2.5.1.2 Disadvantages.18

    2.6 Summary...19

    3 METHODOLOGY.20

    3.1 Overview..20

    3.2 Preparation of culture medium.20

    3.3 Preparation of stock solution (BTX) ...22

    3.4 Preparation of Acid and Base for Fermenter.22

    3.5 Preparation Sample...23

    3.6 Monitoring Growth of Bacteria.25

    3.7 Analysis Method...25

    3.8 Summary...27

    4 RESULT AND DISCUSSION..28

    4.1 Overview.28

    4.2 Pseudomonas putida Growth Curve in Shake Flask...28

  • XII

    4.3 Pseudomonas Putida Growth Curve in Fermenter.31

    4.4 Comparison Between Growth Rate of Pseudomonas Putida in Shake Flask Culture

    with Growth Rate of Pseudomonas Putida in Fermenter.33

    4.5 Standard Curve of Benzene, Toluene and Xylene......34

    4.6 Percent Removal BTX....37

    4.7 Summary.....39

    5 CONCLUSION..40

    5.1 Conclusion..40

    5.2 Recommendation40

    6 REFERENCE.42

    7 APPEENDICES.49

  • XIII

    LIST OF FIGURES

    Figure 2.1: Major products derived from BTX (US DOE, 2012 and Anne, 2013)..4

    Figure 2.3: Benzene Structure (EPA, 1988).6

    Figure 2.5: Toluene Structure (HSDB, 2008)..8

    Figure 2.7: o-xylene, m-xylene and p-xylene structure (HSDB, 2009).10

    Figure 2.8: Pseudomonas aeruginosa (Dr. Howard Porter, 2012).13

    Figure 2.9: Pseudomonas putida (Fekete et al., 2009....14

    Figure 2.10: Shake Flask....15

    Figure 2.11 (a): Head Plate of Fermenter (Fauzan et al, 2012).17

    Figure 2.11 (b): Fermenter Body Parts (Fauzan et al, 2012).18

    Figure 3.1: Overall Process BTX Removal by P. putida in Bioreactor....20

    Figure 3.2: Preparation of Bacteria....21

    Figure 3.3: Preparation of Stock Solution of BTX.22

    Figure 3.4: Process of Preparation of 0.1 mole of Acid and Base..22

    Figure 3.6: The sample of Conical Flask is running in Incubator Shaker..24

    Figure 3.7: The Sample inside Fermenter..24

    Figure 3.8: UV-Vis Spectrometer..25

    Figure 3.9: The sample in Microbiological Centrifuge..25

    Figure 3.10: The Flow Process in make Analyze the Sample....26

    Figure 3.12: HPLC Equipment..27

    Figure 4.1: Graph of Log (OD600) Against Time for Sample in Shake Flask...28

    Figure 4.3: Graph of Log (OD600) Against Time for Sample in Fermenter..32

  • XIV

    Figure 4.5: The Graph of Growth Bacteria Against Time of Shake Flask Culture and

    Fermenter..33

    Figure 4.6: Standard Curve of Benzene35

    Figure 4.7: Standard Curve of Toluene..35

    Figure 4.8: Standard Curve of Xylene...36

  • XV

    LIST OF TABLES

    Table 2.2: Chemical and Physical Properties of benzene (Fischer Scientific, 2008).5

    Table 2.4: Chemical and Physical Properties of Toluene (HSDB, 2008)..7

    Table 2.6: Chemical and Physical Properties of m-xylene, o-xylene, and p-xylene (Reynas,

    2009).9

    Table 2.7: Type of P. putida strain and its function.....11

    Table 3.5: The Ratio of Sample Used..23

    Table 3.11: Specific Parameter of HPLC..26

    Table 4.2 : Summary of Specific Growth Rate of Sample29

    Table 4.4: Summary of Specific Growth Rate of Sample.32

    Table 4.9: Summary Comparison BTX Treatment Result36

  • XVI

    LIST OF ABBREVIATIONS

    m micrometer

    % percent

    oC degree Celsius

    atm Atmospheric pressure

    g/ml gram per milliliters

    g/l gram per liters

    hr hour

    I liter

    mg/l milligram per liters

    pH potential Hydrogen

  • XVII

    LIST OF ABBREVIATIONS

    Specific Growth rate

    ASTDR Agency for Toxic Substances and Disease Registry

    BTX Benzene, Toluene and Xylene

    DOE Department of Environment

    EPA Environment Protection Agency

    HCI Hydrochloric Acid

    HSDB Hazardous Substances Data Bank

    HPLC High Performance Liquid Chromatography

    MIDA Malaysian Investment and Development Agency

    NaOH Sodium Hydroxide

    OD Optical Density

    ppm Part per million

    rpm Rotate per minute

    US United States

    UV-Vis Ultraviolet-Visible

  • 1

    1 INTRODUCTION

    1.1 Motivation and Statement of Pr

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