Metagenomics of marine and mangrove sediments

319 Pages·2016·9.81 MB·English

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Thesis submitted to
Cochin University of Science and Technology
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
Under the Faculty of Science

By
HHAARRIISSRREEEE PP.. NNAAIIRR
RReegg.. NNoo.. 44883322

UUnnddeerr tthhee GGuuiiddaannccee ooff
DDrr.. SSaarriittaa GG.. BBhhaatt

MICROBIAL GENETICS LABORATORY
DEPARTMENT OF BIOTECHNOLOGY
COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
COCHIN - 682022, KERALA, INDIA.

SEPTEMBER 2015

I hereby declare that the thesis entitled “Metagenomics of marine
and mangrove sediments: Phylogenetic diversity and characterization
of amylase obtained by functional screening” is the authentic record of
research work carried out by me for my doctoral degree, under the
supervision and guidance of Dr. Sarita G. Bhat, Professor, Department of
Biotechnology, Cochin University of Science and Technology and that no
part thereof has previously formed the basis for the award of any degree,
diploma, associateship or other similar titles or recognition.

Cochin - 682022 Harisree P. Nair
23/09/ 2015

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CCOOCCHHIINN UUNNIIVVEERRSSIITTYY OOFF SSCCIIEENNCCEE AANNDD TTEECCHHNNOOLLOOGGYY
COCHIN - 682 022, KERALA, INDIA.
Ph: 0484 – 257667 |Email: [email protected] | Fax: [phone]

Dr. Sarita G. Bhat
Professor Date: 23/09/2015

This is to certify that the thesis entitled “Metagenomics of marine and
mangrove sediments: Phylogenetic diversity and characterization of
amylase obtained by functional screening” is a record of bonafide
research work done by Ms. Harisree P. Nair under my supervision and
guidance, in partial fulfilment of the requirement for the degree of Doctor
of Philosophy, under the Faculty of Sciences of Cochin University of
Science and Technology.
I certify that all the suggestions made by the doctoral committe
during her presynopsis are included in the thesis, and that no part thereof
has been presented for the award of any degree.

Dr. Sarita G. Bhat
Supervising Guide

First and foremost I thank God for all the blessings you showered on me
throughout the journey of life making my dreams come true.

I express my heartfelt and profound sense of gratitude to my research guide
and mentor, Dr. Sarita G. Bhat, for her encouragement, inspiring guidance, valuable
suggestions and wonderful backing throughout the course of my doctoral research. I
am taking this opportunity to thank you for accepting me as a Ph.D student. I really
thank you for the wonderful support and friendship throughout my research,
especially during our eight day cruise through Arabian Sea for sample collection and
also during our Indian science congress seminars in Jammu and Mumbai. I am
thanking you for giving me all freedom for designing my experiments and for allowing
me to follow my own working hours in our lab! All your suggestions and ideas
especially our Friday reports helped me a lot to finish my work on time I am truly
indebted to you for all the emotional and intellectual support at times when needed.
You are the best teacher I have ever seen and I am lucky to have you as my guide.
Thanking you once again for making my dream come true.

I express my profound gratitude to Dr. C.S. Paulose for his encouragement
and motivation throughout the research period. I thank you for all the valuable
suggestions for the review presentations during initial stages of research. I would also
like to thank you for admitting me as a Guest lecturer after my Master’s degree which
served as a stepping stone to my research career. I would also like to thank Dr. M.
Chandrasekharan, for the encouragement and valuable suggestions during his visits
to India. I specially thank Dr. Hari Krishnan K of Rajiv Gandhi Centre of
Biotechnology for introducing me to the world of metagenomics for my MSc
dissertation.

I express my gratitude to my doctoral committee members, Dr. Padma
Nambisan and Dr. Ammini Joseph for their valuable suggestions and support
throughout my research work. The valuable suggestions offered by Dr. Elyas K.K. and
Dr. E. Vijayan are gratefully acknowledged. I thank all the teachers of my school
days, graduation and post-graduation for laying my foundations especially in
biological science.

I am thankful to Cochin University for providing the financial support in the
form of Junior Research Fellowship. I also thank University Grants Commission
(UGC) for the major research project sanctioned to my guide and proving financial
assistance in the form of Junior and Senior Research Fellowship for three years. I
thank all the present and past office staffs of the Department of Biotechnology for
their prompt support and help. My special thanks to the higher authorities and
administrative staffs of Cochin University of Science & Technology for their help and
co-operation.
Words fail to express my sincere gratitude to my colleagues at the Microbial
Genetics Lab. I am deeply indebted to all my seniors in our lab for providing friendly
and motivating environment. First, I would like to thank Dr. Alphonsa Vijaya Joseph,
our Vijaya Miss for all the support, care and friendship you offered me. You are one
among my strongest pillar of support and the greatest gift I got from CUSAT! You are
such a wonderful person I have ever met and all the lovely and fun filled moments
spent with you are really cherished. Thank you for all the concerns and enquiries
about my work progress even during your busy schedule. I gratefully acknowledge Dr.
Siju M Varghese for all the advice during my research career. Thank you for teaching
me Sigma Plot and Endnote. I also extend my gratitude to Dr. Jeena Augustine for the
encouragement and support throughout my work. My special appreciation to Dr.
Raghul Subin for his support and care, and for introducing me to various
bioinformatics tools. Indeed he is my evening companion in lab during my initial
stages of work; I really thank you for the friendship and brotherly affection. All the
silly fights and joyful moments with you are really treasured.

I express my sincere gratitude to Dr. Helvin Vincent, my trainer and the
greatest friend of mine, she is moreover like an elder sister to me. Thank you Helvin
chechi for making me who I am. I really appreciate your patience in listening all my
work plans and helping and supporting me in making all my works a success. You
always found time to spend with me even after submitting your thesis, and constantly
motivated me throughout my research career by frequent enquires about my work
progress. I am grateful for all your support and care and thank you for helping me
with all calculations which made my work easy. Thank you for finding time to proof
read most of my thesis chapters with such dedication. My heartfelt thanks to Linda
Louis, my MSc teacher turned lab mate and my best buddy. I would not have stepped
into this department if you have not informed me about the Guest Lecturer vacancy. I
am really thankful for your sisterly affection, care and moral support throughout my
research. I appreciate you for all the valuable practical suggestions and for the
mental support you gave me and I know well that you are my greatest well-wisher and
all your prayers helped me a lot. My special thanks to Dr. Smitha S for all the support
and care you offered me and your guidance in my protein work need a special
mention.

I specially thank Mridula V.G, my lecturer colleague turned lab mate for all
the loving support and affection throughout my research. All the fun filled moments
with you are really cherished. I sincerely acknowledge Cikesh P.C. for his support
and care. Thank you for being a great friend and for all the joyful atmosphere created
by you. I express my gratitude to Noble K. Kurian for his endless support during the
lab hours. I am greatly indebted to you for all the suggestions you made on my work
plans and helping me in clarifying all my silly doubts. My special thanks to Laxmi M
for all her support and care.

My sincere gratitude to Rinu madhu Puthusseri or Rinu mon! My trainee
turned lab mate and moreover my younger brother. I really thank you for being my
right hand in all the experiments right from the time you joined the lab as summer
trainee. Your helping hands made my library screening easy and your assistance
during amylase assays requires special mention. I really enjoyed your companionship
and thanks for all the mental support offered by you when needed. Thanking you for
all the wonderful and joyful lunch time and tea breaks. I am deeply indebted to you for
spending almost one week in my thesis work for proof reading and helping me in
checking references. My special thanks for designing the thesis cover with such
dedication. I express my truthful appreciation to Bindiya E.S for all the support and
care throughout my research. Thank you Bindyechi for all the valuable suggestion and
for the patience in finding TLC plates and sprayer! My special thanks to Sritha K.S for
all your support from the time you joined our lab with a special mention for proof
reading my thesis. I thank Nanditha M, Tina K. J and Anu M.A for their friendship
and support.

I am thankful to Dr. Manzur Ali P.P and Dr. Sapna K for their valuable
practical suggestions in the protein work. I really thank Rekha Mol K.R for her
constant support throughout my research. Thanks for sharing all my worries during
the last stages. Special thanks to Dr. Abraham Mathew and Ramesh Sir for helpful
advices.

My sincere thanks to seniors of the Microbial Technology lab, Dr. Soorej M.
Basheer, Dr. Sreeja and Dr. Beena P. S for their suggestions, encouragement and
unconditional help during various stages of my research work. Special thanks to
Soorejettan for all the practical suggestion and support for MALDI analysis of my
protein. I am grateful to Beena chechi for your support for clarifying all my doubts
regarding DNA sequencing and helping me with NGS for my diversity studies. I thank
Dr. Roseline Alex, Dr. Karthikeyan, Dr. Manjula P, Mr. Ajith M. Thomas, Mr. Sajan
and Mr. Doles for all the friendship and support.

I am grateful to all the research scholars of Plant Biotechnology lab for the
co-operation and friendship. My special thanks to Soumya S for all the support
especially during metal ion and pH studies. My sincere thanks to Arinnina Anto for
the friendship and care with special thanks for all the help in clarifying my doubts
regarding protein modeling. I thank Kiran Lakshmi, Anala, Aiswarya, Nayana, Anuja,
Dr. Jasmine Koshy, Dr. Jikku Jose, Sudha, Aneeta Habeeba and Regi chechi for their
encouragement and support throughout the work. My thanks to all the research
scholars of the Neuroscience laboratory especially Dr. Anju T. R for the friendship,
help and co-operation.

My heartfelt thanks to contract lecturers Dr. Sreekanth, Dr Anoop and Dr.
Manjusha for their constant support and valuable suggestions during my presynopsis.
I am greatly indebed to Sreekanth Sir for all the suggestions, support and advices
offered by you. My sincere thanks to Anoopettan for all the help and support offered
by you. Even though you joined here during the final stages of my work your
contributions meant a lot. Thank you for being a great friend, and for the joyful tea
breaks.

I am grateful to the Captain and all the crew members of FORV Sagar
Sampada (Cruise #305) for all the help and support during my sample collection.
Special thanks cruise members and scholars of CMLRE, Rajeesh, Thommy, Vinu and
Jijith for the support, care and friendship offered during the cruise which helped me a
lot to overcome the sea-sickness. Special thanks to Serene Sara John, Ramya R. Nair
and Sanyo Sabu, scholars of NCAAH, CUSAT for the unconditional love and affection
toward me. Thank you all for the great companionship during our Jammu and
Mumbai trips. All the wonderful and fun filled moments spent with you are really
priceless.

Once again special thanks to Sreekanth Sir, Anoopettan, Helvin chechi, Linda
Miss, Bindiyechi, Sritha, Rinu, Akhil and Rajesh for proof reading my thesis and
helping in cross checking the references. I thankfully remember all the M.Sc. students
of the Department especially Amritha, Anjali and Akhil. I am grateful to Dr. Divya,
St.Xavier’s College for helping me with bioinformatic tool for phylogenetic diversity
analysis.

My genuine thanks to Rajesh Nair, my MSc class mate and my best friend.
Thank you for being a constant supporter since the time we met. You are good
motivator and my greatest strength. You are the one who taught me to face life with a
smiling face. Thanks for listening to all my stories especially my endless talks about
uncultured microbes! Thank you for all the encouragement, support and care and for
all the help with my thesis work from reference formatting to proof reading and you
never hesitated to make an ISD call daily to help and support me. I also thank
Aswathy N for all her support, encouragement and care in achieving my goals. Thanks
for sharing all my tensions during early stages of work and as a research student she
can understand it better. I am grateful to you for being such a loving friend
throughout my journey.

Words cannot express my gratitude to my dear parents for their love,
motivation and prayers. I am deeply indebted to them for their persistent support for
accomplishing my goals. Thank you Achan and Amma for having faith in me and
providing me complete freedom for following my destiny. Special thanks to my elder
sisters Haritha and Hani for the constant support and love since my childhood. My
heartfelt love and thanks for the three little angels of our family, Devika, Aardra and
Avanthika- my nieces! my Devutty, Appu and Avanthu. Thank you for your little
prayers and smiling face which provides me greatest relief from all the tensions and
worries. Specially mentioning Appu for her enthusiasm in selecting colors for my
phylogeny pie charts and constant enquiries about my thesis writing progress even
though she doesn’t know the spelling or meaning of it! Last things are always the best,
Vipin my better half. Thank you for being such an understanding person; you know
better who you are to me. Special thanks to my in-laws for accompanying him in my
absence.

Harisree P. Nair