marine yeast isolates from arabian sea and bay of bengal

287 Pages·2013·6.31 MB·English

by  Reema Kuriakose

MARINE YEAST ISOLATES FROM ARABIAN SEA AND BAY OF 
BENGAL: BIOCHEMICAL, MOLECULAR AND PHYLOGENETIC 
ANALYSIS 

THESIS 
Submitted to the
COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY 
In partial fulfillment of the degree of

DOCTOR OF PHILOSOPHY 
IN 
MICROBIOLOGY

UNDER THE FACULTY OF MARINE SCIENCES 
By
REEMA KURIAKOSE 

DEPT. OF MARINE BIOLOGY, MICROBIOLOGY AND BIOCHEMISTRY 
SCHOOL OF MARINE SCIENCES COCHIN UNIVERSITY OF SCIENCE AND 
TECHNOLOGY 
KOCHI‐682016 
NOVEMBER, 2012
Dedicated to the Lords Divine Mercy
Declaration

I hereby do declare that the thesis entitled “Marine yeast isolates from Arabian
Sea and Bay of Bengal :Biochemical, Molecular and Phylogenetic Analysis”is an authentic
record of research work done by me under the supervision and guidance of Dr. Rosamma
Philip, Assistant Professor, Department of Marine Biology, Microbiology and Biochemistry,
School of Marine Sciences, Cochin University of Science and Technology, for the degree of
Doctor of Philosophy in Marine Biology and that no part of this work has been presented
before for the award of any other degree, diploma or associateship in any University.

Kochi-682016
November,2012. Reema Kuriakose
This is to certify that the thesis entitled “Marine yeast isolates from Arabian Sea and Bay
of Bengal :Biochemical, Molecular and Phylogenetic Analysis is an authentic record of
research work carried out by Ms. Reema Kuriakose under my supervision and guidance in the
Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences,
Cochin University of Science and Technology, in partial fulfillment of the requirements for the
degree of Doctor of Philosophy in Microbiology and that no part thereof has been presented
before for the award of any other degree, diploma or associate ship in any University.

Kochi-682 016 Dr. Rosamma Philip
November,2012 (Supervising Guide)
Assistant Professor
Department of Marine Biology,
Microbiology and Biochemistry
School of Marine Sciences
Cochin University of Science and Technology
Kochi-682 016
Acknowledgements

“I will always thank the Lord . I will praise him for what he has done.”

I always believe that it was divine mercy who lead to me to the guide of my
dreams Dr. Rosamma Philip. At the outset let me place on record my deep
appreciation and gratitude to my mentor and guide Dr. Rosamma Philip, who has
been my strong pillar of support throughout my work. Her genuine and deep
interest in research, her alertness and perseverance was a driving force in
completion of this work on time. Her amiable nature, pleasing manners, coupled
with her empathetic ears, rare blend of qualities has made her unique and special
to each and every one of her research scholars.

I gratefully acknowledge Dr. A. A. Mohamed Hatha, Head, Department of Marine
Biology, Microbiology and Biochemistry and acting Head Dr. S. Bijoy Nandan,
for providing all help and facilities in the department.

My sincere thanks to Dr.H.S. Ram Mohan, former Director and Dean, and Dr,
Sajan K, Dean, School of Marine sciences for providing all facilities for research.
I am thankful to all the teachers in the Department of Marine Biology, especially
Dr. Babu Philp for the patience with which he cleared my doubts, Dr.
A.V.Saramma and Dr. Aneykutty Joseph for their friendly nature , valuable
advice, suggestion and support. I also thank the Non -teaching staff of the
department for their help and good wishes .The help provided by the library staff
specially Mr. Manuel is also acknowledged.
My heartfelt gratitude to Dr. I.S.Bright Singh, Co -ordintor, National centre for
aquatic animal health for his encouragement, help and facilities provided
throughout my work period.
I am much obliged to Rev Sr. Christaballe, Former Principal, CSST, Rev Dr. Sr.
Treesa, Principal, Rev.Sr.Tessa CSST, Provincial and manager and the
management of St.Teresa’s College, Ernakulam for their encouragement and
support throughout the tenure my research.
I am indebted to Rev Sr. Francis Ann, Former Provincial , CSST and my
colleagues of the Department of zoology, Smt. Gladys Francis, H.O.D , Dr. Meera
Jan Abraham, Dr. Soja Louis ,Dr Simi Joseph, and Ms. Niji Philomina for their
invaluable friendship and timely help whenever needed. I specially thank and
appreciate Dr Simi Joseph P for the systematic way and for her patience while
assisting me in computer works.

I acknowledge Mr. V Ravindranathan Former Director CMLRE, Ministry of
earth sciences and Dr V.N Sanjeevan , Director CMLRE, Ministry of Earth
sciences for the facilities provided to collect samples during the cruise of FORV
Sagar Sampada for this study.

I am indebted to University Grants Commission for providing the
fellowship for the fulfillment of academic requisites at different stages of
the study.

I record my sincere gratitude to my Senior researchers Dr. Sreedevi.N.Kutty, Dr.
Selven Subramanian, Dr. Lakshmi G. Nair, Mr. Neil Scolastin Correya ,Dr.
Prabhakaran ,Ms.Sini P J and Ms. Smitha S.L.

I would also like to specially mention three of my very dear students Dr. Swapna
P. Antony, Dr Manjusha K and Ms. Divya T Babu who turned out to be my
teachers during the process of my research. I thank them for their patience,
constant help and support which went a long way in helping me to complete this
thesis.

Mere word of thanks will be inadequate for the timely help , love and sisterly
concern rendered by my friends Ms. Deepthi Augustine and Ms.Divya P.S. I also
appreciate the sincere friendship and concern of Mr. Shaji E.M, Dr. Venu, and
Dr. Rasheed.

My immense thanks to Mr. Naveen Sathyan who appeared like a divine
messenger whenever trouble shoots up. My special thanks to Mr.Anil Kumar.P.R ,
Ms. Jini Jacob and Ms. Chaithanya E.R. for the invaluable help rendered
whenever needed.

I deeply appreciate the extreme sincerity and care with which Ms Sunitha
Poulose of NCAAH taught me the basics of assay techniques. I also sincerely
appreciate Ms Priyaja P, Ms. Vrinda S, Ms Deepa G D and Ms. Sreelakshmy G
of NCAAH for their timely assistance provided throughout the research.

My heart felt gratitude to all the research fellows of the Department Marine
Biology especially, Ms .Nifty John, Ms. Remya K.D, Ms. Jimly. C .Jacob , Ms.
Emilda Rosemine , Ms. Nima job, Ms. Wilsy Wilson, Mr. Anit M Thomas , Mr.
Subhankar Gosh , Mr. Harishankar H.E, Ms Susanne , Ms Jisha Jose, Ms. Jisha
V.K, Ms Neethu . C.S, Ms Reshma, Deborah, and Dr. Mujeeb Rahman

I appreciate with thanks the invaluable assistance received from Ms. Thresy
Kurian , Mr. Xavier Kurian , Mr. Babish Jose, Mr.Akshay Kurien and Ashish
Kurien in various stages of this work.
My special thanks should go to my brother and sister Mr. Mathew Kuriakose and
Sheena Shaju and their family whose support has always given me dependable
measure of strength . My special thanks are due to my in laws Mr. Joseph
Mathew, Dr. Ajith Joseph, Mrs. Rosemary Johnny, Ms.Anitha Jose, Mr. Jose
Monipilly for their prayers, support and co operation throughout my work.
I feel profound sense of gratitude for my late father Prof. C. M. Kuriakose and
mother Mrs. Rosamma Kuriakose. It was my father who has sown the seeds of
ambition to do research in me and my mother’s prayers always supported me.
The love and moral support my family members and in laws have helped me in
coping with the hard ships during the tenure of this work.

Finally, I must pay tribute to my husband, Mr. Anil Joseph whose undaunted
support and encouragement helped me to complete this work. I appreciate the
boundless love and sacrifice of my daughter Nikita Anil who has adjusted
amazingly well in my absence.

I sincerely thank all those who have helped me in one way or other way in the
fulfillment of this cherished ambition.
Reema Kuriakose

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TABLE OF CONTENTS
Chapter - I
GENERAL INTRODUCTION 1-6

Chapter - II
ISOLATION AND IDENTIFICATION OF MARINE YEASTS 7-52
2.1. Introduction ..................................................................................7
2.1.1. Distribution of yeasts in the aquatic habitats: 8
2.1.2. Isolation and Maintenance of Yeasts: 22
2.1.3. Identification of the isolates 25
2.1.4. Hydrolytic Enzyme production 27

2.2. Materials and Methods 29
2.2.1a. Sample collection. 29
2.2.1b. Isolation of marine yeasts: 30
2.2.2. Identification of the Isolates: 31
2.2.3. Hydrolytic Enzyme production: 35
2.2.4. Utilisation of different sugars: 37
2.2.5. Growth assessment of the isolates at
different temperatures: 38

2.3. Results: 38
2.3.1. Generic Composition: 38
2.3.2. Hydrolytic Enzymes: 38
2.3.3. Ability to ferment different sugars: 40
2.3.4. Growth of marine yeast isolates at
different temperatures 44

2.4. Discussion 47
Chapter - III
NON PIGMENTED YEASTS : MOLECULAR IDENTIFICATION AND
PHYSIOLOGICAL CHARACTERIZATION 53-108
3.1. Introduction 53
3.1.1 Molecular Identification of Yeasts 55
3.1.2 Identification Methods 55

3.2 Materials and Methods: 74
3.2.1. Yeast Strains: 74
3.2.2. Molecular Identification 74
3.2.2.1 Yeast genomic DNA isolation: 74
3.2.2.2 Amplification of ITS sequence 75
3.2.2.3 Restriction endonuclease digestion and analysis: 76
3.2.2.4 PCR clean up and Sequencing 77
3.2.2.5 ITS gene sequence similarity & identification: 78
3.2.3 Physiological Characterization of the strains 78

3.3 Results 80
3.3.1 RFLP Pattern of Yeast ITS Amplicons 80
3.3.2 Physiological characterization 96

3.4. Discussion 102