BIODIESEL PRODUCTION BY TRANSESTERIFICATION USING Biodiesel production via transesterification of microalgae

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  • BIODIESEL PRODUCTION BY

    TRANSESTERIFICATION USING WHOLE-CELL

    LIPASE BIOCATALYSIS

    XIAO MAN

    NATIONAL UNIVERSITY OF SINGAPORE

    2011

  • BIODIESEL PRODUCTION BY

    TRANSESTERIFICATION USING WHOLE-CELL

    LIPASE BIOCATALYSIS

    XIAO MAN

    (M.Eng., Huazhong University of Science and Technology, China)

    A THESIS SUBMITTED FOR

    THE DEGREE OF DOCTOR OF PHILOSOPHY

    DEPARTMENT OF CIVIL AND ENVIRONMENTAL

    ENGINEERING

    NATIONAL UNIVERSITY OF SINGAPORE

    2011

  • I would like to dedicate this thesis to

    my parents and family

  • ACKNOWLEDGEMENTS

    I

    ACKNOWLEDGEMENTS

    First and foremost, I would like to extend my sincerest gratitude to my supervisor, A/P

    Jeffrey P. Obbard, from the Division of Environmental Science and Engineering,

    National University of Singapore, for his invaluable guidance, support and

    encouragement all along my study. Jeff made me realize my intended area of

    specialization in the future – renewable energy. His scientific spirit, uprightness,

    dedication and cheerfulness have been greatly inspirational to me. It is his constructive

    criticisms and numerous corrections in my manuscripts getting the thesis in present form.

    This study also benefited from valuable technical support of research group members of

    A/P Jeffrey P. Obbard. Special thanks must also go out to Dr. Sini Mathew who has been

    an excellent guider patiently assisting me through the research methodology and

    experimental techniques at the beginning of this work. I also want to sincerely thank

    Marvin Joseph Fonacier Montefr for designing and fabricating the reactor; Dr. Ma

    Lingling, Dr. Zhang Jie and Dr. Tan Jing for teaching and assisting me to operate the

    GC/MS machine; and Dr. Subramanian Karuppiah for assisting me in acquiring all the

    materials and equipment needed for my work. In addition, I would like to thank senior

    Ph.D students in our group Doan Thi Thai Yen, Probir Das and Rajesh Kumar

    Balasubramanian for their personal and technical support on microalgal cultivation and

  • ACKNOWLEDGEMENTS

    II

    harvesting. The contributions of Neo Chee Keong, Merry Wagimin, Rolavia Intan and Qi

    Chao, who have assisted me during their Final Year Project, are not forgotten as well.

    I am deeply grateful to the Ministry of Education of Singapore, the National University

    of Singapore for providing scholarship and offering the facilities for this PhD program. I

    also wish to thank Division of Environmental Science and Engineering for the financial

    support of the project. I would also like to acknowledge Mr. Sidek, Mr. Suki, Ms. Susan,

    Mr. Chandra, Ms. Hwee Bee, Mr. Michael Tan and all the staff and technical personnel

    for facilitating the administrative aspects of my research.

    Last but not least, I am deeply indebted to my family, especially my parents for their

    patient love and sustained encouragement enabled me to complete this work. In the end, I

    would like to thank my husband, Yuhao, for listening to me each time I babble my

    thoughts out, and for just being there to support me in every step of the way.

  • Table of Contents

    III

    TABLE OF CONTENTS

    ACKNOWLEDGEMENTS .............................................................................................. I

    Table of Contents ............................................................................................................ III

    SUMMARY ...................................................................................................................... X

    LIST OF FIGURES ..................................................................................................... XIII

    LIST OF TABLES ...................................................................................................... XVII

    NOMENCLATURE ..................................................................................................... XIX

    Chapter 1 INTRODUCTION AND OBJECTIVES ...................................................... 1

    1.1 Introduction ............................................................................................................................ 1

    1.2 Research Objectives ............................................................................................................... 3

    Chapter 2 LITERATURE REVIEW .............................................................................. 5

    2.1 Biodiesel Feedstock ................................................................................................................ 6

    2.2 Transesterification .................................................................................................................. 8

    2.3 Lipase as a catalyst ............................................................................................................... 10

    2.3.1 Extracellular lipase ........................................................................................................... 11

    2.3.2 Intracellular lipase ............................................................................................................ 15

  • Table of Contents

    IV

    2.4 Variables affecting the enzymatic transesterification reaction ............................................. 19

    2.4.1 Temperature ...................................................................................................................... 19

    2.4.2 Alcohol ............................................................................................................................. 19

    2.4.3 Water content.................................................................................................................... 21

    2.4.4 Glycerol ............................................................................................................................ 22

    2.4.5 Buffer molarity during lyophilization on transesterification ............................................ 22

    2.4.6 Solvent pretreatment on lipase activity ............................................................................ 23

    2.5 Enzymatic production of biodiesel in reactors ..................................................................... 24

    2.6 Biodiesel from microalgae oil-lipid...................................................................................... 26

    2.6.1 Potential of microalgae biodiesel ..................................................................................... 27

    2.6.2 Selection of microalgae strain .......................................................................................... 29

    2.6.3 Microalgal biomass production ........................................................................................ 30

    2.6.4 Transesterification of microalgal lipid to produce biodiesel ............................................ 32

    2.7 Conclusion ............................................................................................................................ 33

    Chapter 3 ISOLATION AND SCREENING OF HIGH LIPASE-PRODUCING

    FUNGAL STRAINS USED AS WHOLE-CELL BIOCATALYST FOR BIODIESEL

    PRODUCTION FROM PALM OIL ............................................................................. 35

    3.1 Introduction .......................................................................................................................... 35

    3.2 Materials and methods.......................................................................................................... 37

    3.2.1 Materials and medium ...................................................................................................... 37

    3.2.2 Isolation and screening of fungal strains .......................................................................... 37

  • Table of Contents

    V

    3.2.3 Lipase activity assay ........................................................................................................ 38

    3.2.4 Effect of physicochemical parameters on the lipase production by the isolated organism

    39

    3.2.5 Whole-cell biocatalyst preparation ................................................................................... 39

    3.2.6 Whole cell catalyzed methanolysis reaction .................................................................... 40

    3.2.7 Analytical procedure ........................................................................................................ 40

    3.3 Results and discussion .......................................................................................................... 41

    3.3.1 Isolation and preliminary identification of strain ............................................................. 41

    3.3.2 Effect of initial pH on lipase activity ............................................................................... 46

    3.3.3 Effect of carbon source on lipase activity ........................................................................ 47

    3.3.4 Immobilization of A. niger cells within BSPs .................................................................. 48

    3.3.5 Effect of water content on e

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