Production of Clean Biofuel from Microalgae - .Production of Clean Biofuel from Microalgae PPRN:

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  • R.V. COLLEGE OF ENGINEERING,BENGALURU-560059 (Autonomous Institution Affiliated to VTU, Belagavi)

    PROJECT REPORT

    Production of Clean Biofuel from Microalgae

    Submitted in fulfilment for the 39th

    series SPP Biofuel project

    Project Proposal Reference No. :39S_B_BE_006

    Supported & funded by

    Karnataka State Biofuel Development Board

    Submitted By

    VIDYASHREE S (USN: 1RV12BT057)

    NAVYASHRI(USN: 1RV12BT033)

    SHRAVANTHI S KUMAR(USN: 1RV12BT046)

    Under the guidance of

    Prof. Shivandappa Asst. Professor

    Department of Biotechnology

    R.V. College of Engineering, Bengaluru

    2016

  • Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

    Department of Biotechnology, RVCE, Bengaluru i

    R.V. COLLEGE OF ENGINEERING, BENGALURU-560059

    (Autonomous Institution Affiliated to VTU, Belagavi)

    DEPARTMENT OF BIOTECHNOLOGY

    CERTIFICATE

    This is to certify that the project work entitled Production of Clean Biofuel

    from Microalgae was carried out byNavyashri (1RV12BT033),Shravanthi S

    kumar (1RV12BT046) andVidyashree S (1RV12BT057), who are bonafide

    students of R.V.College of Engineering, Bengaluru in fulfilment of Biofuel

    project sponsored by Karnataka State Council for Science and Technology &

    Karnataka State Biofuel Development Board, Bengaluru under 39th

    series of

    Student Project Programme in the year 2015-2016.

    Signature of GuideSignature ofHODSignature ofPrincipal

    (Prof.Shivandappa) (Dr.Pushpa Agrawal)(Dr. K.N Subramanya)

  • Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

    Department of Biotechnology, RVCE, Bengaluru ii

    R.V. COLLEGE OF ENGINEERING, BENGALURU 560059

    (Autonomous Institution Affiliated to VTU, Belagavi)

    DEPARTMENT OF BIOTECHNOLOGY

    DECLARATION

    We, Navyashri,Shravanthi S Kumar and Vidyashree S, students of Eighth

    semester B.E.,Department of Biotechnology, R.V. College of Engineering,

    Bengaluru-560059, bearing USN: 1RV12BT033, 1RV12BT046 and

    1RV12BT057, hereby declare that the project titled Production of Clean

    Biofuel from Microalgae has been carried outby us and submitted in fulfilment

    of Biofuel project sponsored by Karnataka State Council for Science and

    Technology & Karnataka State Biofuel Development Board, Bengaluru under

    39th

    series of Student Project Programme in the year 2015-2016.

    Further we declare that the content of the dissertation has not been submitted

    previously by anybody .

    We also declare that any Intellectual property rights generated out of this project

    carried out at R.V.C.E. will be the property of R.V. College of Engineering,

    Bengaluru and we will only be the authors of the same.

    Place: Bengaluru Signature of the students

    Date:1. Navyashri

    2. Shravanthi S Kumar

    3. Vidyashree S

  • Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

    Department of Biotechnology, RVCE, Bengaluru iii

    ACKNOWLEDGEMENT

    We take this opportunity to express our sincere gratitude to the people who have been

    helpful in the successful completion of our project. We would like to show our

    greatest appreciation to professors and staff members at the Department of

    Biotechnology Engineering.

    Our debt of gratitude must go to our guide Prof.Shivandappa for his valuable

    suggestions and inspiring guidance throughout the course of this work. We would

    also like to thank Dr.Pushpa Agrawal, Professor & Head, Dept. of Biotechnology

    Engineering, Dr.K.N.Subramanya, Principal of R.V.C.E, for providing valuable

    guidance and facilities to carry out the work. We are grateful to

    Ms.Mahalakshmi,Ms.Puneetha&Ms.Smitha, project assistants for being with us,

    helping inseveral ways throughout our project.

    The study is funded by Karnataka State Biodiesel Development Board(KSBDB),

    under 39th

    series of Student Project Programme. Hence, we would like to extend our

    gratitude towards KSBDB, Govt. of Karnataka for the financial support.

    Most importantly, we are extremely grateful to our parents for their encouragement,

    quiet patience and love which has always helped us sail through obstacles and

    complete the project successfully.

  • Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

    Department of Biotechnology, RVCE, Bengaluru iv

    TABLE OF CONTENTS

    ACKNOWLEDGEMENT.iii

    ABSTRACT.iv

    TABLE OF CONTENTS.v

    ABBREVIATIONS...viii

    LIST OF TABLES...x

    LIST OF FIGURES...xii

    CHAPTER 1

    INTRODUCTION

    1.1 Biodiesel Overview..1

    1.1.1 Advantages of Biodiesel.2

    1.2 Literature survey

    1.2.1 Microalgae..3

    1.2.2 Algal Biodiesel3

    1.2.3 Microalgae as source of Biodiesel..4

    1.2.4Botryococcusbraunii....5

    1.2.5 Trans-esterification.6

    1.2.6 Enzyme Trans-esterification...6

    1.2.7Pseudomonas aeruginosa.7

    1.3 Problem outcome.7

    1.4 Objectives.9

    1.5 Brief methodology...9

    1.6 Organization of the report..10

    CHAPTER 2

    THEORY AND FUNDAMENTALS

    2.1 Motive underlying algae as the biodiesel feedstock..12

    2.2 Principles underlying high oil yield of B.braunii...................................................12

    2.3 Bacterial Lipase mediated Trans-esterification..13

    2.3.1 P.aeruginosa Lipase..13

    2.3.2 Function of P.aeruginosa lipase...14

    2.3.3 Structure and catalytic mechanism.14

    2.4 Feasibility of production of Biodiesel from algae.15

  • Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

    Department of Biotechnology, RVCE, Bengaluru v

    CHAPTER 3

    MATERIALS AND METHODOLOGY

    3.1 Materials used16

    3.2 Methodology..17

    3.2.1 Preparation of Lipase18

    3.2.1.1 Serial dilution of soil sample and inoculation on LB agar medium.18

    3.2.1.2 Inoculation on to EMB agar medium...19

    3.2.1.3Inoculation on to Kings B agar medium..20

    3.2.1.4Inoculation on to Mac-Conkey agar medium...21

    3.2.1.5 Screening of P.aeruginosa by Biochemical tests.21

    3.2.1.6 Biochemical screening of lipase by Spirit blue agar medium..23

    3.2.1.7 Extraction and purification of Lipase...24

    3.2.1.8 Quantification of purified crude Lipase...25

    3.2.2 Culture and growth of algae..26

    3.2.2.1 Media Optimization.27

    3.2.2.2 Screening of B.braunii..28

    3.2.2.3 Algal oil extraction...29

    3.2.3 Comparative studies..29

    3.2.4 Trans-esterification of Algal with lipase isolated from P.aeruginosa...30

    3.2.5 Testing of Fuel properties of Biodiesel.30

    CHAPTER 4

    RESULTS AND DISCUSSION

    4.1 Isolation and screening of P.aeruginosa33

    4.1.1 Serial dilution of soil sample and inoculation on LB agar medium33

    4.1.2 Selective media for gram-negative bacterial growth35

    4.1.3 Selective media for Pseudomonas bacteria35

    4.1.4Differential media for P.aeruginosa...36

    4.1.5 Screening of P.aeruginosaby biochemical assay..37

    4.1.6 Screening of P.aeruginosaas lipase producer by Spirit blue agar40

    4.1.7 Extraction and isolation of lipase..41

    4.1.8 Purification of lipase by dialysis method..42

    4.2 Quantification of Lipase by Lowrys method43

  • Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

    Department of Biotechnology, RVCE, Bengaluru vi

    4.3 Culture and growth of B.braunii44

    4.3.1 Modified kuhls media..45

    4.3.2 Open pond culture.46

    4.4 Screening of B.braunii48

    4.5 Algal oil extraction.49

    4.6 Comparative studies...52

    4.6.1Effect of FFA content of oil in Chemical Trans-esterification..53

    4.6.2 Effect of FFA content of oil in Lipase-mediated Trans-esterification..54

    4.7.Trans-esterification of Algal oil with lipase to produce Biodiesel56

    4.8 Testing of Fuel properties of Biodiesel..57

    CHAPTER 5

    CONCLUSION AND FUTURE SCOPE OF WORK

    5.1 Conclusion.59

    5.2 Future prospects.60

    REFERENCES.61

  • Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

    Department of Biotechnology, RVCE, Bengaluru vii

    LIST OF ABBREVIATIONS

    LB: Luria Bertani

    EMB: Eosin methylene blue

    MWCO: Molecular weight cut-off

    ASTM: American Society of testing and materials

    L ha-1

    :Litre per hectare

    ACC: Acetyl-CoA Carboxylase

    CO2: Carbon dioxide

    HPL: Human pancreatic Lipase

    MVB: Multi-Vesicular bodies

    KDa: Kilo Dalton

    H2SO4: Hydrogen sulphate

    NaOH: Sodium hydroxide

    NaCl: Sodium chloride

    g/ml: Micro gram per mili litre

    l: Micro litre

    m: Micrometre

    nm: Nano metre

    K2HPO4: Potassium hydrogen phosphate

    MgSO4: Magnesium sulphate

  • Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006