isolation and screening of efficient algae for biofuel production

107 Pages·2013·2.15 MB·English

ISOLATION AND SCREENING OF EFFICIENT
ALGAE FOR BIOFUEL PRODUCTION

Thesis

Submitted to the Punjab Agricultural University
in partial fulfilment of the requirements
for the degree of

MASTER OF SCIENCE
in
MICROBIOLOGY
(Minor Subject: Biochemistry)

By

Jyoti Rai
(L-2011-BS-241-M)

Department of Microbiology
College of Basic Sciences and Humanities

©PUNJAB AGRICULTURAL UNIVERSITY
LUDHIANA -141004

2013
CERTIFICATE-I
This is to certify that the thesis entitled, “Isolation and screening of efficient algae
for biofuel production” submitted for the degree of Master of Science, in the subject of
Microbiology (Minor subject: Biochemistry) of Punjab Agricultural University, Ludhiana, is
a bonafide research work carried out by Jyoti Rai (L-2011-BS-241-M) under my supervision
and that no part of this thesis has been submitted for any other degree.
The assistance and help received during the course of investigation have been fully
acknowledged.

_________________________
Major advisor
Dr. (Mrs.) S. K. Gosal
Professor
Deptt. of Microbiology
Punjab Agricultural University
Ludhiana- 141004

2
CERTIFICATE-II

This is to certify that the thesis entitled, “Isolation and screening of efficient algae
for biofuel production”submitted by Jyoti Rai (L-2011-BS-241-M) to the Punjab
Agricultural University, Ludhiana, in partial fulfillment of the requirements for the degree of
Master of Science, in the subject of Microbiology (Minor subject: Biochemistry) has been
approved by the Student‟s Advisory Committee along with Head of the Department after an
oral examination of the same.

____________________ _________________
Dr. (Mrs.) S. K. Gosal Dr. R.P Gupta
Major Advisor External Examiner
Dean Academics
BIS Group of Colleges

_________________________
Dr. (Mrs.) Param Pal Sahota
Head of the Department

________________________
Dr. Gursharan Singh
Dean Post graduate studies

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ACKNOWLEDGMENT

At the very outset, I owe it all to “Almighty God”, the sovereign, for granting me the
wisdom, health and strength to undertake this research task and enabling me to its
completion. I genuflect my head with reverence to Waheguru, by whose grace and blessings,
I have been able to present this humble contribution to the field of „Microbiology‟.
The words “one who directs the path of progress is angelic” are inadequate to
express my deep sense indebtedness and gratitude to my Major Advisor Dr. (Mrs.) S. K.
Gosal, Professor, Department of Microbiology, for her resolute guidance, unwavering
encouragement, abiding interest, constructive criticism, tremendous enthusiasm and
meticulous supervision through the period of my research. She has been the torchbearer and
pathfinder in completing my research work. She has been an excellent guide. Her advices will
play a crucial role in fulfilling my dreams in future.
Words are inadequate in the available lexicon to avouch the excellent co-operation
given by Dr (Mrs.) Param Pal Sahota, Senior Microbiologist cum Head of Department of
Microbiology. I am highly grateful to the members of my advisory committee viz. Dr. (Mrs.)
Poonam Sharma, Senior Microbiologist, Department of Microbiology, Dr. (Mrs.) Sucheta
Sharma, Associate Professor, Department of Biochemistry, Dr. (Mr.) Sohan Singh Walia,
Agronomist, Department of Agronomy and Dr. (Mr.) H.S. Sodhi, Senior Mycologist,
Department of Microbiology for their constant support and valuable suggestions during the
course of investigations and preparation of this manuscript.
I take in the inadequacy of words at my command to pay my sublime obeisance to my
parents, “Mr. Harjeet Singh Rai” and “Mrs. Gurdeep Kaur Rai” whose love, affection,
moral support, guidance, sacrifices, blessings and encouragement enabled me to complete
another task of my life. I have deep feelings in my heart for my loving brother “Gureet
Singh Rai” whose great help and affection is unparalleled boon to me. I am greatly thankful
to my Fuffar ji, “Dr. (Mr.) Harnek Singh Garcha” and Bhua ji, “Dr. (Mrs.) Seema Garcha”
for their timely help, judicious and valuable guidance and for being their every time I needed,
during my tenure of work.
The thesis would not have come to a successful completion without the help and
support of kind people around me. My vocabulary falls short to thank and appreciate Gaurav
bhaiya, Amita didi, Poonam didi and Sukhdeep didi. Words are short to express my deep
sense of gratitude towards my friends Jupinder and Harleen for their support,
encouragement, memorable company, ever willing help, and my juniors Aman and Jaspreet
for providing me few moments of refreshment in between the exhausting hours to shed the
workload which kept me energetic during this period and made my stay at the university
memorable.
I am thankful to the members of Lab for their cooperation in the lab and timely help
at the hours of need.
Cordial thanks to all kith and kins who helped me one way or the other. All may not
be mentioned but none is forgotten. Needless to say, errors and omissions if any are mine.

(Jyoti Rai)
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Title of the thesis : Isolation and screening of efficient algae for biofuel
production
Name of the student : Jyoti Rai
and Admission No. (L-2011-BS-241-M)
Name and Designation of : Dr. (Mrs.) S. K. Gosal
Major Advisor : Professor
Major Subject : Microbiology
Minor Subject : Biochemistry
Degree to be awarded : M.Sc
Year of award of degree : 2013
Number of pages : 59+Appendices+Vita
Name of the university : Punjab Agricultural University, Ludhiana, Punjab,
India.

ABSTRACT
A total of 20 different algal isolates were isolated from stagnant water samples
collected from different districts of Punjab, Rajasthan and Himachal Pradesh. On the basis of
morphological characterization, these algal isolates were tentatively identified as Chlorella,
Oedogonium, Aphanocapsa, Phormidium, Spirogyra, Anabeana, Scenedesmus, Tolypothrix,
Anacystis, Microcystis, Volvox and Spirulina. Data analysis revealed that the isolate JA8 had
highest growth rate followed by JA12 after 16 days of incubation. Increase in pH of the
medium was recorded with the growth of algal isolates and increase in pH in different algal
isolates ranged from 7.0 (initial) to 10.6 (final) during algal growth after 16 days of
incubation. The highest increase in pH was recorded in the JA18 (10.6) which were
significantly higher than control (7.0). The supplementation of urea to the growth medium did
not result in much increase in pH as compared to the medium which was not supplemented
with urea. The increase in pH with urea supplementation ranged from 7.0 to 8.2 whereas
without urea supplementation the pH increased upto 10.6. The isolate JA11 showed highest
fresh weight (16.284 g/l) and dry weight (7.211 g/l) of biomass after one month of incubation.
All the 20 isolates, were screened for their lipid content and the highest lipid content was
recorded in the isolate JA8 (5.60 %) followed by JA9 (5.25 %). The addition of glucose (10
g/l) to the growth medium resulted in increase in lipid content in the isolate JA8 (5.60 % to
7.52 %) and JA9 (5.25% to 7.15%). Saponification and acid value of isolates was analyzed
using five best screened isolates based on their lipid content. The isolate JA8 showed highest
saponification (169.69 mg/g of oil) and acid value (4.75 mg/g of oil) followed by the isolate
JA9. The fatty acid composition of three algal isolates JA8, JA9 and JA15 was determined
with Gas liquid Chromatography. The isolate JA8 contained highest proportion of stearic acid
(41.60), followed by pentadecanoic acid (38.32) and oleic acid (19.01). Isolate JA9 and JA15
contained saturated fatty acids only. Hence, the results revealed that the isolate JA8 may be
considered best among the isolates isolated making it suitable for biofuel production.
However, the lipid content of the isolates is not much promising. So, there is need to explore
the possibility to find algal isolate which is having high lipid content for biofuel production.
Key words: Algae, Acid value, Biofuel, Fatty Acid, Lipid content, Morphological characterization

_____________________ ____________________
Signature of Major Advisor Signature of the Student

5
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6
CONTENTS
CHAPTER TOPIC PAGE NO.
I INTRODUCTION 1-4
II REVIEW OF LITERATURE 5-17
III MATERIAL AND METHODS 18-22
IV RESULTS AND DISCUSSION 23-46
V SUMMARY 47-49
REFERENCES 50-59
APPENDICES i-xxiii
VITA

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LIST OF TABLES
TABLE TITLE PAGE
NO. NO.
2.1 Oil content of various algae 13
4.1 Algal sample collection sites from different regions 23
4.2 Morphological characterization of different algal isolates 25
4.3 Growth kinetics of different algal isolates with time 27
4.4 Effect of growth of different algal isolates on pH of medium with time 29
4.5 Effect of urea supplementation on the pH of medium for different algal 37
isolates
4.6 Biomass different algal isolates after one month 38
4.7 Total lipid content of different algal isolates 40
4.8 Total lipid content of algal isolates with addition of glucose 42
4.9 Acid and Saponification value of algal oil 43
4.10 Fatty acid composition (% of total fatty acids) of algal isolates 44

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LIST OF FIGURES
FIGURE TITLE PAGE
NO. NO.
2.1 Transesterification reaction 16
4.1 Effect of growth of different algal isolates on pH of the medium 30-34
4.2 Dry weight of different algal isolates after one month 39
4.3 Lipid content of different algal isolates 41
4.4 Chromatographs showing fatty acid peaks of algal isolates 45

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LIST OF PLATES
PLATE
TITLE
NO.
1 Isolation of algae
(A) Spreading of algae
(B) Isolated colonies of algae
2 Purification of algae
(A) Streaking of algal culture on beneck‟s medium
(B) Maintenance of algal isolates on the slants
3 Morphological view of algal Isolates under the compound light microscope (45X)
(A) JA1 - Spirogyra
(a) Microscopic view of algae
(b) Algae in the flask
(B) JA2 - Tolypothrix
(a) Microscopic view of algae
(b) Algae in the flask
(C) JA3 - Aphanocapsa
(a) Microscopic view of algae
(b) Algae in the flask
(D) JA4 - Chlorella
(a) Microscopic view of algae
(b) Algae in the flask
(E) JA5 - Phorimidium
(a) Microscopic view of algae
(b) Algae in the flask
(F) JA6 – Chlorella
(a) Microscopic view of algae
(b) Algae in the flask
(G) JA7- Spirogyra
(a) Microscopic view of algae
(b) Algae in the flask
(H) JA8 - Chlorella
(a) Microscopic view of algae
(b) Algae in the flask
(I) JA9 - Chlorella
(a) Microscopic view of algae
(b) Algae in the flask

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