production of clean biofuel from micro algae

82 Pages·2012·2.23 MB·English

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

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)

Department of Biotechnology, RVCE, Bengaluru i
Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

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

Department of Biotechnology, RVCE, Bengaluru ii
Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

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.

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Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

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 Biodiesel………………………………………………………………3
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

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Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

CHAPTER 3
MATERIALS AND METHODOLOGY
3.1 Materials used……………………………………………………………………16
3.2 Methodology……………………………………………………………………..17
3.2.1 Preparation of Lipase…………………………………………………………18
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.aeruginosa…………………………………………33
4.1.1 Serial dilution of soil sample and inoculation on LB agar medium……………33
4.1.2 Selective media for gram-negative bacterial growth…………………………35
4.1.3 Selective media for Pseudomonas bacteria……………………………………35
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 agar……………40
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 Lowry’s method……………………………………43

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Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

4.3 Culture and growth of B.braunii…………………………………………………44
4.3.1 Modified kuhls’ media………………………………………………………..45
4.3.2 Open pond culture…………………………………………………………….46
4.4 Screening of B.braunii……………………………………………………………48
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 Biodiesel………………56
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

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Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

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
CO : Carbon dioxide
2
HPL: Human pancreatic Lipase
MVB: Multi-Vesicular bodies
KDa: Kilo Dalton
H SO : Hydrogen sulphate
2 4
NaOH: Sodium hydroxide
NaCl: Sodium chloride
µg/ml: Micro gram per mili litre
µl: Micro litre
µm: Micrometre
nm: Nano metre
K HPO : Potassium hydrogen phosphate
2 4
MgSO : Magnesium sulphate
4

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Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

H O : Hydrogen peroxide
2 2
MR: Methyl red
EDTA: Ethylene di-ammine tetra acetic acid
PBS: Phosphate buffer solution
BSA: Bovine serum albumin
FC reagent: Folinciocalteau
OD: Optical density
KNO : Potassium nitrate
3
ZnSO :Zinc sulphate
4
Co(NO ) :Cobalt nitrate
3 2
Na .MoO :Sodium molybdate
2 4
CuSO : Copper sulphate
4
FeSO : Ferrous sulphate
4
Rpm: Revolutions per minute
FFA: Free fatty acid

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Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006

LIST OF TABLES
Sl. No TABLE NAME Page No.
1.1 Comparison of oil yield between different feedstock 5
1.2 Taxonomy of B.braunii 6
1.3 Comparison of feedstocks with oil yield and land required 7
1.4 Oil content of various microalgal species 8
3.1 Composition of Luria Bertani Agar Media 18
3.2 Composition of EMB agar medium 19
3.3 Composition of Kings’ B agar medium 20
3.4 Composition of Mac-conkey agar medium 21
3.5 Composition of Spirit Blue Agar 23
3.6 Lowry’s method for protein estimation of lipase 26
3.7 Kuhls’ media Composition - Basal media 27
3.8 Kuhls’ media Composition - Micronutrient solution 27
3.9 ASTM range of Biodiesel 30
4.1 Biochemical assay 37

4.2 Lowry’s method of Protein estimation 43
4.3 Algal Oil yield 51
Yield of Biodiesel from different edible oils based on
4.4 53
theirFFA content by Chemical Trans-esterification
Yield of Biodiesel from different edible oils by Lipase
4.5 54
mediatedTrans-esterification
4.6 Yield of Algal Biodiesel 57
Fuel properties of Biodiesel produced through Chemical
4.7 58
Trans-esterification
Fuel properties of Biodiesel produced through Lipase
4.8 58
mediated Trans-esterification

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