marine actinomycetes as source of antimicrobial compounds and as probiotics and single cell

283 Pages·2013·3.49 MB·English

by  Biji Mathew

MARINE ACTINOMYCETES AS SOURCE OF
ANTIMICROBIAL COMPOUNDS AND AS PROBIOTICS
AND SINGLE CELL PROTEIN FOR APPLICATION
IN PENAEID PRAWN CULTURE SYSTEMS
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Cochin University of Science and Technology
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DOCTOR 01= PHILOY
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MICROBIOLOGY
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UNDER THE FACULTY OF MARINE SCIENCES
By
BIJ I MATHEW
DIVISION OF MARINE BIOLOGY, MICROBIOLOGY
AND BIOCHEMISTRY
SCHOOL OF MARINE SCIENCES
COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
COCHIN - 682 016
2003
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DECLARATION
I hereby do declare that the thesis entitled "Marine Actinomycetes as
Source of Antimicrobial Compounds and as Probiotics and Single Cell
Protein for Application in Penaeid Prawn Culture Systems”, is a
genuine record of research work done by ine under the supervision of Dr.
Rosamma Philip, Senior Lecturer, School of Marine Sciences, Cochin
University of Science and Technology, Cochin — 682016, and that no part of
this work has previously formed the basis for the award of any degree, diploma
associate ship, fellowship or other similar title of any University or Institution
Cochin- 682016
February 2003 Biji Mathew
('lertit'imte
This is to certify that the thesis entitled "Marine Actinomycetes as
Source of Antimicrobial Compounds and as Probiotics and Single Cell
Protein for Application in Penaeid Prawn Culture Systems” an
authentic record of research work carried out by Ms. Biji Mathew under my
supervision and guidance in the school of Marine Sciences, Cochin University
of Science and Teclmology in partial fulfillment of the requirements for the
degree of Doctor of Philosophy and no part thereof has been presented before for
the award of any other degree, diploma , or rissociateship in any university.
i
Dr.R0samma Philip i
(Supervising Teacher) &‘_ "1 ,
Dr.Rosamma Philip
Senior lecturer,
Dept. Marine Biology, Microbiology and Biochemistry,
School of Marine Sciences,
Cochin University of Science and Technology,
Cochin - 682 O16
Cochin — 16
Februaw 2003
"1:
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ACKNOWLEDGEMENT
For whatever merit this book may posses I am indebted beyond measure to my guide,
Dr. Rosannna Philip, Senior Lecturer, Department of Marine Biology, Microbiology and
Biochemistry, Cochin Llniversity of Science and Technology (CUSAT). My interest in this
subject was initiated by her. Her inspiring guidance, constant encouragement, critical
evaluation and unfailing moral support have always instilled in me the energy and strength to
face and overcome several hurdles on the way during this research programme.
I am extremely thankful to Dr. (Prof. ) A.L.N. Shanna, Former Head. Dept. of Zoology,
Regional College of Education, Bhubaneswar, who was the master force behind my venture
into the research field.
I am indebted to Dr. (Prof) R. Damodaran, Dean, Faculty of Marine Sciences, for
introducing me to Dr. Rosamma Philip which paved the way for the successful completion of
the work. I also thank him for his valuable suggestions during the various phases of the work.
I am grateful to Dr. (Prof) Babu Philip, Head, Department of Marine Biology,
Microbiology and Biochemistry and Prof (Dr.) N.R. Menon, Honorary Director, C-IMCOZ,
School of Marine Sciences, Cochin University of Science and Technology, for their valuable
help and support during the period ofm y research work.
My thanks are due to Dr. (Prof). P.G. Kurup, Director, School of Marine Sciences,
CLISAT for allowing me to utilize the facilities of the School of Marine Sciences.
I wish to record my sincere thanks to Dr. I. S. Bright Singh, Reader, School of
Environmental Studies, CLISAT for dedicating his valuable time for analyzing my work and
giving excellent scientific touches to this study. I also thank him for the facilities that he kindly
made available to me all through my work.
I gratefully acknowledge Dr. /1. V. Saranuna, Reader, Department of Marine Biology,
Microbiology and Biochemistry. CLISAT for all the help and valuable suggestions rendered
throughout the period of study.
I express my sincere thanks to all other teachers and oflice slay‘ of the Department of
Marine Biology, Microbiology and Biochemistry, C LISAT.
I am extremely thankful to Dr. (Prof) (Rid). Madhava Pillai, Department of Chemistry
C LISAT, Dr. Mu raleedhararn Nair, Lecturer, Department of Chemical Oceanography for their
valuable suggestions in this work.
The assistance received from the Librarians of Cochin University of Science and
Technology, Central Institute of I-"isheries Technology, Cochin; Central Marine Fisheries
Research Institute, Cochin ; Central Food Technology Research Institute, Mysore ; National
Chemical Laboratory, Pune and Indian Institute of Science, Bangalore are gratefully
acknowledged.
I record my sincere gratitude to Dr. lbinu Saud, Reader, School of Chemical
Sciences,Mahatma Gandhi University for providing me an opportunity to undergo a short
term training in chromatography techniques in his Department. I gratefully acknowledge
Mrs. Chithra Gopinath and Mrs. Shalini Thomas of School of Chemical Sciences,
M.G. Llniversity for their whole hearted assistance. I extend my thanks to Mr. Tom Thomas,
Biocon India Ltd, Bangalore for his valuable suggestions.
Thanks are due to Regional shrimp Hatchery, Azhikode, Matsyafed Hatchery, Ponnani
for providing me with shrimps for my research work. Department of Ocean Development is
gratefitlly acknowledged for providing the facilities for sample procurement.
I thank Cochin University of Science and Technology for providing me fellowship
during the tenure of the study and AICTE, Govt. of India for necessary financial support to
carry out this work.
I deeply appreciate the extreme sincerity. assistance and care rendered by
Ms. Manjusha. M, Mr. Jayaprakash. N. S, Mr. Somnath Pai, Dr. Renjith. S, Mr. Arias Abdul
Aziz, Mr. Rejish I<umar, Mr. Sudheer. N.S, Mr. Shreedharan.K and Ms. Preetha. R of School
of Environment studies.
I express my whole hearted gratitude to Mrs. Beatrice Amar and Mr. Sajeevan. T. P for
their unfailing support and valuable suggestions. My special thanks are due to Mr. Selvan,
Ms. Annies losepii and Mr. B. Hari for their whole hearted assistance on all the occasions.
Mere wards of thanks will be inadequate for the timely help, support, love and concern
rendered by all my friends Mrs. Minimal K. G, Mrs. Meera Venugopal, Ms. Maya Paul. T,
Ms. Sincy Ioseph, Ms. Leksluni. G. Nair, Mrs.Newby Sabu, Mrs. Sarlin Poly Mrs. Bindu
Abraham, Mr. Sreevalsam Gopinath and Mr. I-larish.
I acknowledge with thanks the timely assistance received from Dr. Nandini Menon.,
Mr. Arun. All and all my friends in the Dept.of Marine Biology.
Words are still before, the deep, dedicated and unparallel fiiendship of my hostel mates
Mrs. Sini Anilkumar, Ms. Sandhya. K and Ms. Roopa Rani. Mrs. Alka lain, Ms. Deepti
Sharma, Mrs. Sangeeta Sukdev, Mrs. Parvathy Ramesh and Mrs Sujatha Ramesh are fondly
remembered for their encouragement and moral support.
The love and care of my pa rents and my parents- i n-law have been the motivating factor
in all my endeavors. The moral support given to me by my brothers and sisters has always
helped me in coping with the hardships during the tenure of this work. I take this opportunity
to express my love and gratitude to them.
Finally, I must pay tribute to my husband, Mr. C. Aneesh, who has always been an
aflectionate, understanding and patient companion with me. Without his encouragement, this work
would not have reached completion. I dedicate this work to the boundless love of Aneesh and my
little daughter Auagha as a small compensation for the time which I had not spent with them.
CONTENTS
1. GENERAL INTRODUCTION
2. ISOLATION OF ACTINOMYCETES FROM MARINE ENVIRONMENT
2.1 Introduction
.-2.1.1 Soil Actinomycetes
2.1.2 Actinomycetes in marine environment
2.1.3 Selective isolation of actinomycetes
2.1.3.1 Composition of the isolation media
2.1.3.2 Inhibitory agents used in isolation media
2.1.3.3 Pre-treatment of the samples
2.1.3.4 Incubation time
2.1.4 Prcscwution 01' Isolates
2.2 Materials and Methods
2.2.1 Samples
2.2.2 Pre-treatment of the samples
2.2.3 Sample Preparation
2.2.4 Media used .
2.2.5 Antibiotics and Other Inhibitory Agents Used
2.2.6 Plating and Incubation
2.2.7 Isolation and Purification
2.2.8 Preservation
2.3 Results
2.3.1 Isolation of Marine actinomycetes
2.4 Discussion
3. ACTINOMYCETES AS A SOURCE OF ANTIBACTERIAL
COMPOUNDS: SCREENING FOR ANTAGONISTIC ACTIVITY
AND SELECTION OF STRAINS.
3.1 Introduction
3.1.1 Antibiotics and secondary metabolites
3.1.2 Actinomycetes as a source of antimicrobial compounds
3.1.3 Screening actinomycetes for antimicrobial property
3.1.3.1 Methods of screening
3.2 Materials And Methods
3.2.1 Microorganisms
3.2.2 Testing Bioactivity
3.2.3 Testing antagonistic activity of selected strains of
actinomycetes against natural flora
3.2.4 Testing sensitivity of bacterial prawn pathogens to
commercially available antibiotics
3.3 Results
3.3.1 Screening of actinomycetes for inhibitory property
3.3.2 Comparison of the antibiogram of commercially
available antibiotics and the selected actinomycete
isolates against Vibrios
3.3.3 Inhibition of selected actinomycetes to natural flora
3.4 Discussion
4. SELECTION OF FERMENTATION MEDIA AND
PROCESS OPTIMIZATION 6'7
4.1 Introduction 68
4.1.1 Fermentation media 68
4.1.1.1 Nutritional factors in medium formulation 69
4.1.1.2 Seed media and Production Medium 71
4.1.2 Fermentation process optimization 71
4.1.2.1 Effect of nitrogen sources 72
2.2 Effect of glucose concentration 73
3 Effect of phosphate concentration 73
4 Fatty acids and amino acids 75
5 Effect of Sodium, Potassium and
other metal ions 75
.2.6 Effect of aeration 76
.1.2.7 Effect of NaCl concentration 76
4.1.2.8 Effect of medium with alkaline pH 77
4.1.2.9 Effect of temperature 77
4.2 Materials And Methods 78
4.2.1 Selection of Fermentation Media 78
4.2.2 Process Optimization 82
4.2.2.1 Parameters 82
4.2.2.2 Media preparation {Fermentation media) 82
4.2.2.3 Inoculum 83
4.2.2.4 Inoculation and incubation. 83
4.2.2.5 Determination of growth 83
4.2.2.6 Estimation of antibiotic production 84
4.3 Results 84
4.3.1 Comparison of various media for antibiotic production 84
4.3.2 Optimal conditions for growth and antibiotic
production by the selected actinomycetes. 89
4.4 Discussion 94
4.4.1 Selection of medium 94
4.2.2 Process Optimization 96
5. CHARACTERIZATION OF ACTINOMYCETES AND
EXTRACTION OF BIOACTIVE COMPOUNDS 99
5.1 Introduction 100
5.1.1 Characterization of actinomycetes 100
5.1.2 Extraction of Bioactive compounds 101
5. 1.2. 1 Solvent Extraction 102
5.1.2.2. Solid Phase Extraction 104
5.1.2.3. Thin Layer Chromatography [TLC] 104
5.1.4 Characterization of bioactive compounds 108
5.2 Materials And Methods 110
5.2.1 Taxonomic studies 110
5.2.2 Morphological studies 110
5.2.3 Fermentation 114
5.2.4 Chemical Screening for bioactive compounds 116
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5.2.5 Antibacterial assay 117
5.2.6 Solvent extraction 117
5.2.7 Detection of the bioactive compounds using
Thin Layer Chromatography (TLC) 118
5.2.7.1 Preparation of TLC plates 118
5.2.7.2 TLC of whole broth 118
5.2.7.3 TLC of solvent extracts 119
5.2.8 Preparative TLC 119
5.2.9 Compound Isolation 120
5.3 Results 121
5.3.1 Morphological characteristics 121
5.3.2 Chemical Screening 124
5.3.3 Bioactivity of whole fermentation broth and the
various solvent extracts of the fermentation broth 124
5.3.4 Thin Layer chromatography for bioactive compounds 125
5.3.4.1 TLC of fermentation broth 125
5.3.4.2 The TLC of solvent (ethyl acetate)
extract of B361and B451 127
5.3.5 PTLC of Ethyl acetate extract of B45lE 128
5.4 Discussion 129
5.4.1 Thin layer chromatography 131
5.4.2 Solvent extract of fermentation broth 132
6. MARINE ACTINOMYCETES AS SINGLE CELL PROTEIN FOR
APPLICATION IN PENAEID PRAWN CULTURE SYSTEMS 133
6.1 Introduction 134
6.1.1 Single Cell Protein 134
6.1.2 Diseases in Aquaculture and Prevention 137
6.2 Materials And Methods 140
6.2.1 Microorganisms used 140
6.2.2 Medium used 140
6.2.3 Inoculation and Incubation 140
6.2.4 Separation of Actinomycete Biomass 141
6.2.5 Proximate composition of the experimental diets,
actinomycete biomass and prawn flesh 141
6.2.6 Feeding experiment with penaeid prawns using
actinomycete biomass as SCP 141
- 6.2.6.1 Feeding Experiment I with Penaeus
rnonodon post larvae 141
6.2.6.2 Feeding Experiment II with Penaeus
monodon juveniles Experimental Animals 146
6.3 Results 148
6.3.1 Feeding experiment I 148
6.3. 1.1 Biogrowth parameters 149
6.3.1.2 Post challenge survival 149
6.3.1.3 Statistical analysis 154
6.3.2. Feeding Experiment II 154
6.3.2.1 Proximate composition of actinomycete
biomass (SCP], feed and prawn flesh. 154
6.3.2.1 Biogrowth parameters 157
6.3.2.2 Post Challenge Survival 165
6.3.2.3 Statistical analysis 167
6.4 Discussion 167
7. ACTINOMYCETES AS PROBIOTIC FOR PENAEID
PRAWNS CULTURE SYSTEM 173
7.1 Introduction 174
7.1.1 Selection of probionts 176
7.1.2 Mode of application of probiotics 177
7.1.3 Mechanism of action of probiotics 177
7.1.4 Screening and pre-selection of the putative probiotics 182
7.1.5 Probiotic studies in the larval cultures of shrimp 182
7.2 Materials And Methods 184
7.2.1 Actinomycete Strains 184
7.2.2 Testing I-lydrolytic enzyme production 186
7.2.3 Testing antagonistic property against pathogenic vibrios 186
7.2.4 Testing antagonistic activity against Natural flora 186
7.2.5 Screening for pathogenicity 187
7.2.5.1 Testing hemolytic property 187
7.2.5.2 Testing pathogenicity to penaeid prawns 188
7.2.5.4 Testing pathogenicity of Actinomycetes, if any 189
7.2.6 Testing the efficacy of selected
actinomycetes as probiotics 189
7.2.6.1 Animals used 198
7.2.6.2 Design of experiment 189
7.2.6.3 Analysis of water quality 190
7.3 Results 191
7.3.1 Hydrolytic enzyme production by
the various actinomycetes 191
7.3.2 Inhibition of pathogenic vibrios (non
luminescent and luminescent) by actinomycetes 192
7.3.3 Inhibition of Natural Flora 193
7.3.4 1-Iaernolytic property on blood agar 194
7.3.5 Pathogenicity test in vivo 195
7.3.6 Testing the Efficacy of the selected strains
as a probiotic in a bioassay system 197
7.3.6.1 Treatment 1. [Rearing tanks with water.) 197
7.3.6.2 Treatment 2 202
7.4 Discussion 207
7.4.1 Efficacy as a probiotic in aquaculture 210
SUMMARY AND CONCLUSION 213
REFERENCES 219
APPENDIX 265