Protocols in Actinobacterial Research [1st ed.] 9781071607275, 9781071607282

Table of contents :
Front Matter ....Pages i-xxxi
Sample Collection, Isolation, and Diversity of Actinobacteria (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 1-24
Dereplication and Ex Situ Conservation of Actinobacteria (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 25-37
Characterization and Identification of Actinobacteria (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 39-64
Screening of Actinobacteria for Biological Activities (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 65-112
Production of Bioproducts from Actinobacteria (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 113-128
Eliciting Cryptic Metabolite Production from Actinobacteria (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 129-137
Metabolite Profiling of Actinobacterial Metabolites (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 139-146
Bioassay-Guided Isolation and Characterization of Metabolites from Actinobacteria (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 147-163
Evaluation of Actinobacteria for Environmental Applications (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 165-174
Evaluation of Actinobacteria for Agricultural Applications (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 175-180
Evaluation of Actinobacteria for Aquaculture Applications (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 181-188
Evaluation of Actinobacteria for Nanoparticle Synthesis (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 189-195
Determination of Cytotoxicity of Actinobacterial Extracts and Metabolites (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 197-203
Evaluation of Actinobacteria for Bioenergy Applications (Ramasamy Balagurunathan, Manikkam Radhakrishnan, Thangavel Shanmugasundaram, Venugopal Gopikrishnan, Joseph Jerrine)....Pages 205-210
Back Matter ....Pages 211-225

Citation preview

Ramasamy Balagurunathan Manikkam Radhakrishnan Thangavel Shanmugasundaram Venugopal Gopikrishnan Joseph Jerrine

Protocols in Actinobacterial Research

SPRINGER PROTOCOLS HANDBOOKS

For further volumes: http://www.springer.com/series/8623

Springer Protocols Handbooks collects a diverse range of step-by-step laboratory methods and protocols from across the life and biomedical sciences. Each protocol is provided in the Springer Protocol format: readily-reproducible in a step-by-step fashion. Each protocol opens with an introductory overview, a list of the materials and reagents needed to complete the experiment, and is followed by a detailed procedure supported by a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. With a focus on large comprehensive protocol collections and an international authorship, Springer Protocols Handbooks are a valuable addition to the laboratory.

Protocols in Actinobacterial Research Ramasamy Balagurunathan Department of Microbiology, Periyar University, Salem, Tamil Nadu, India

Manikkam Radhakrishnan Centre for Drug Discovery & Development, Sathyabama Institute of Science & Technology, Chennai, Tamil Nadu, India

Thangavel Shanmugasundaram DRDO – BU Centre for Life Sciences, Bharathiar University, Coimbatore, Tamil Nadu, India

Venugopal Gopikrishnan Centre for Drug Discovery & Development, Sathyabama Institute of Science & Technology, Chennai, Tamil Nadu, India

Joseph Jerrine Centre for Drug Discovery & Development, Sathyabama Institute of Science & Technology, Chennai, Tamil Nadu, India

Ramasamy Balagurunathan Department of Microbiology Periyar University Salem, Tamil Nadu, India

Manikkam Radhakrishnan Centre for Drug Discovery & Development Sathyabama Institute of Science & Technology Chennai, Tamil Nadu, India

Thangavel Shanmugasundaram DRDO – BU Centre for Life Sciences Bharathiar University Coimbatore, Tamil Nadu, India

Venugopal Gopikrishnan Centre for Drug Discovery & Development Sathyabama Institute of Science & Technology Chennai, Tamil Nadu, India

Joseph Jerrine Centre for Drug Discovery & Development Sathyabama Institute of Science & Technology Chennai, Tamil Nadu, India

ISSN 1949-2448 ISSN 1949-2456 (electronic) Springer Protocols Handbooks ISBN 978-1-0716-0727-5 ISBN 978-1-0716-0728-2 (eBook) https://doi.org/10.1007/978-1-0716-0728-2 © Springer Science+Business Media, LLC, part of Springer Nature 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

DEDICATED To The Almighty, Actinobacteria, and all our Teachers

Foreword I am pleased to write this foreword to this protocol series titled Protocols in Actinobacterial Research. The authors of this protocol series have continuously pursued research on actinobacteria for the past 10–20 years in India. They are known to me for several decades, and I am aware of their passion in this area. They have also been my collaborators in the area of actinobacteria for anti-TB drug discovery. Actinobacteria are one of the largest phylum in the bacterial domain, which have greater ecological and industrial significance. They have the bioprospecting potential due to their extreme diversity, taxonomy, ecology, metabolic potential, and diverse applications. Working constantly on this group of bacteria is a cumbersome and time-consuming task due to their slow growing nature and complexity. Further, the protocols or manuals available for routine microbial experiments are not suitable for actinobacteria due to the morphology, metabolic potential, and genetic makeup differences between them. The efforts taken up by the authors as actinobacteriologists in bringing this Protocols in Actinobacterial Research are a timely initiative. The content of the protocol series is very clear and reproducible. Importantly, the cited references evidenced that almost around 80% of the experimental protocols have emanated from their own research on actinobacteria. Hence, I strongly believe that this will be a useful and thought-provoking resource for young researchers, scholars, and students pursuing and intended to start actinobacterial research. This protocol series is an outcome of two decades of mentor–student relationship and their sincere work. I deeply appreciate the mentor and senior author Prof. R. Balagurunathan and his students and coauthors of this actinobacterial protocol series Dr. M. Radhakrishnan, Dr. V. Gopikrishnan, Dr. T. Shanmugasundaram, and Dr. Jerrine Joseph for their dedicated and untiring efforts as a team to bring this protocol series on actinobacterial research. I also urge that the authors continue their research pursuit as well as other such noble initiatives to empower students and scholars. All the best. Department of Biotechnology, Bioengineering & Drug Design Lab, Bhupat & Jyoti Mehta School of BioSciences, IIT Madras, Chennai, India

vii

Mukesh Doble

Preface The compilation of this manual is based on the exhaustive research carried out over the last several decades on this unique microbe called Actinobacteria. The contents have been carefully planned so as to be a user-friendly protocol guideline for researchers and scientists working on this organism. Indeed, it will be very useful to understand and carry out the experiments with ease. This bacteria has the tremendous potential to provide several products with biomedical and industrial significance. It is due to its survival mechanisms under different extreme terrains. Hence, this manual on actinobacteria is exclusively dedicated to the microbe due to its multifaceted multifunctional potential. The versatility of the microbe is a challenge for isolation and screening in laboratory conditions. Hence, several parameters and conditions need to be optimized and standardized. All of which have been taken into consideration and explained in detail in the notes part when and where necessary. Utmost care has been taken to make this manual user-friendly to the targeted audience of researchers. This manual is unique and first in a protocol series for actinobacteria. This manual will be an asset to labs working on actinobacteria owing to its lucidity and its content designed in an effective and concise manner. We hope this manual will be very useful and the reader will be benefited through this manual, which will facilitate the exploration of the microbe systematically to be able to generate several components with promising applications. Salem, Tamil Nadu, India Chennai, Tamil Nadu, India Coimbatore, Tamil Nadu, India Chennai, Tamil Nadu, India Chennai, Tamil Nadu, India

R. Balagurunathan M. Radhakrishnan T. Shanmugasundaram V. Gopikrishnan Jerrine Joseph

ix

Acknowledgments Our long thrust in the field of actinobacteriology has been quenched with the blessings of Almighty by helping us write this protocol series titled Protocols in Actinobacterial Research. This manual would not be possible without the invaluable help and support of our mentors, professors, scientific collaborators, research scholars, and students. We would like to take this opportunity to express our sincere gratitude to all those who have accompanied us on the research journey on actinobacteria. We have made efforts to prepare this manual. We would like to express our heartfelt thanks to our host institutes—Periyar University, Salem, Tamil Nadu, DRDO-BU Centre for Life Sciences, Coimbatore and Sathyabama Institute of Science and Technology, Chennai, Tamil Nadu—for providing us all the research facilities, scientific freedom, and adequate time to work on actinobacteria. We are highly indebted to the higher authorities of our institutes for their constant support and encouragement. We would like to express our special gratitude and thanks to our mentors Dr. Vanaja Kumar, Dr. K. Kathiresan, and Dr. T. Balasubramanian for their valuable suggestions and mentoring to work on actinobacteria. We thank our collaborators for their detailed and constructive comments and suggestions for the enrichment of the study, and it has always been illuminating. The strong sense of community within the actinobacteriology group has been an important part of our time at Lab, and we would like to express our appreciation to all of our group members. We have worked with a brilliant set of colleagues in lab, and we thank them for the untiring assistance and constant encouragement throughout our research. We would like to express our sincere gratitude toward our research scholars Mr. K. Manigundan and Mr. A. Vignesh for their kind co-operation and assistance, which helped us in completion of this manual. A very special gratitude goes out to funding agencies like UGC, DST, DBT, ICMR, MoES, and National Centre for Polar and Ocean Research for helping and providing the research grant, which allowed us to work in depth on actinobacteria from rare ecosystems in India. We are fortunate to have been surrounded by a wonderful set of people with as much exceptional scientific caliber and grace as one could ever hope for. They have cultivated our growth in embarking on an exciting new journey and shaped our vision of doing science. Our thanks and appreciations also go to our colleagues in developing the manual and people who have willingly helped us out with their abilities.

xi

Contents Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Foreword. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi About the Authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxv Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxvii Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxxi

1 Sample Collection, Isolation, and Diversity of Actinobacteria. . . . . . . . . . . . . . . . . 1 General Guidelines for Sample Collection for the Isolation of Actinobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 General Instructions for Sample Collection . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Sample Pretreatment and Media Selection for the Selective Isolation of Actinobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Isolation and Enumeration of Actinobacteria from Soil Sample . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Isolation of Actinobacteria from Sediments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Isolation of Actinobacteria (Endophytic) from Plants . . . . . . . . . . . . . . . . . . . . 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Isolation of Actinobacteria (Endosymbiotic) from Fish. . . . . . . . . . . . . . . . . . . 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Isolation of Actinobacteria from Insect Gut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xiii

1 1 1 2 3 3 3 4 4 4 5 5 6 7 8 8 8 9 9 12 13 13 13 13 14 15 15 15 16 17 17 17 18

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Contents

7.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Recovery and Purification of Actinobacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Metagenomic Analysis of Uncultured Actinobacterial Diversity . . . . . . . . . . . 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

18 19 19 19 19 20 20 20 20 21 21 23

2 Dereplication and Ex Situ Conservation of Actinobacteria . . . . . . . . . . . . . . . . . . . 1 Dereplication of Actinobacterial Cultures Based on Color Grouping and Micromorphology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Dereplication of Actinobacterial Cultures Based on FT-IR Analysis. . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Dereplication of Actinobacterial Cultures by RFLP Analysis . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Dereplication of Actinobacterial Cultures by Random Amplified Polymorphic DNA: Polymerase Chain Reaction (RAPD-PCR) Analysis . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Ex Situ Conservation of Actinobacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

25

31 31 32 32 33 33 33 34 36 37

3 Characterization and Identification of Actinobacteria . . . . . . . . . . . . . . . . . . . . . . . . 1 Micromorphological Characterization of Actinobacteria. . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Scanning Electron Microscopic Studies on Actinobacteria . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

39 39 39 40 40 41 41 41

25 25 26 26 26 27 27 28 28 28 29 29 29 30

Contents

2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Cultural Characterization of Actinobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Physiological Characterization of Actinobacteria . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Analysis of Actinobacterial Cell Wall for Amino Acids and Sugars . . . . . . . . . 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Analysis of Actinobacterial Cell Wall for Polar Lipids. . . . . . . . . . . . . . . . . . . . . 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Detection of Actinobacterial Menaquinones in Actinobacterial Cell Wall. . . 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Detection of Mycolic Acid in Actinobacterial Cell Wall. . . . . . . . . . . . . . . . . . . 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Isolation of Chromosomal DNA from Actinobacteria. . . . . . . . . . . . . . . . . . . . 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 PCR Amplification of 16s rRNA Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Separation of Actinobacterial DNA by Agarose Gel Electrophoresis . . . . . . . 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Sequencing of Actinobacterial 16S rRNA Gene . . . . . . . . . . . . . . . . . . . . . . . . . 12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Phylogenetic Tree Construction and GenBank Submission of Actinobacterial 16S rRNA Gene Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4 Screening of Actinobacteria for Biological Activities . . . . . . . . . . . . . . . . . . . . . . . . . 1 Screening of Actinobacteria for Antimicrobial Activity by Agar Plug Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Screening of Actinobacteria for Antimicrobial Activity by Agar Diffusion Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Screening of Actinobacterial Extracts for Antimicrobial Activity by Microbroth Dilution Method and Determination of Minimal Inhibitory Concentration (MIC). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Screening of Actinobacterial Cultures for Antimycobacterial Activity Using Mycobacterium smegmatis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Screening of Actinobacterial Extracts/Compounds for Antimycobacterial Activity by Luciferase Reporter Phage (LRP) Assay . . . . . . . . . . . . . . . . . . . . . . 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Screening of Actinobacteria for Anti-TB Activity by Microplate AlamarBlue Assay (MABA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Screening of Actinobacteria for Anti-TB Activity by Agar Dilution Assay . . 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Screening for Anticancer Activity: 3-(4,5-Dimethylthiazol-2-yl)2,5-Diphenyltetrazolium (MTT) Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Screening for Anticancer Activity: Dual Staining Method. . . . . . . . . . . . . . . . . 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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9.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Screening for Anticancer Activity: Neutral Red Uptake Assay . . . . . . . . . . . . . 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Screening for Anticancer Activity: Lactic Acid Dehydrogenase Assay . . . . . . 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Screening for Anticancer Activity: Trypan Blue Exclusion Assay. . . . . . . . . . . 12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Screening for Anticancer Activity: DNA Fragmentation Assay . . . . . . . . . . . . 13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Screening for Antioxidant Activity: Diphenylpicrylhydrazine (DPPH) Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Screening for Antioxidant Activity: Nitric Oxide Scavenging Assay . . . . . . . . 15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Screening for Antioxidant Activity: Metal Chelating Assay. . . . . . . . . . . . . . . . 16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Screening for Antioxidant Activity: Total Antioxidant Assay . . . . . . . . . . . . . . 17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Screening for Antioxidant Activity: Hydrogen Peroxide Scavenging Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Screening of Microbes for the Production of Pigment (Melanin) . . . . . . . . . 19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Screening of Microbes for the Production of Enzymes. . . . . . . . . . . . . . . . . . . 20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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20.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Screening of Actinobacteria for Biosurfactant Production . . . . . . . . . . . . . . . . 21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Screening of Actinobacteria for Enzyme Inhibitor Activity . . . . . . . . . . . . . . . 22.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Screening of Actinobacteria for Quorum Sensing Inhibition. . . . . . . . . . . . . . 23.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Screening of Actinobacterial Extracts for Antibiofilm Activity. . . . . . . . . . . . . 24.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Screening of Actinobacteria for Siderophore Production . . . . . . . . . . . . . . . . . 25.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Screening of Actinobacteria for Mosquitocidal Activity. . . . . . . . . . . . . . . . . . . 26.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.2 Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Screening for Antiviral Activity: MTT Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

95 95 96 96 97 97 98 99 99 100 100 101 101 101 101 102 103 103 104 104 105 105 105 106 106 106 107 107 108 108 109 109 110

5 Production of Bioproducts from Actinobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Production and Extraction of Antibiotic Compounds from Actinobacteria by Agar Surface Fermentation Method/Process . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Production of Antibiotic (Pigment) Compounds from Actinobacteria by Solid-State Fermentation Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Production of Antibiotic Compounds from Actinobacteria by Submerged Fermentation: Shake Flask Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

113 113 113 114 114 114 114 115 115 116 116 116 116 117

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Production of Antibiotic Compounds from Actinobacteria by Submerged Fermentation: Lab Fermentor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Extraction of Bioactive Compounds by Liquid–Liquid Extraction. . . . . . . . . 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Production of Bioactive Pigment from Actinobacteria Using Immobilized Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Optimization of Antibiotic Production from Streptomyces Species: Classical One-Factor-at-a-Time (OFAT) Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Determination of Bacterial Growth Kinetics by Batch and Fed-Batch Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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6 Eliciting Cryptic Metabolite Production from Actinobacteria. . . . . . . . . . . . . . . . . 1 Effect of One Strain Many Compounds (OSMAC) Approach on Bioactive Metabolite Production from Actinobacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Effect of Elicitors on Inducing Cryptic Metabolite Production from Actinobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Inducing Cryptic Metabolite Production from Actinobacteria by Cocultivation Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

117 117 118 118 119 119 119 120 120 121 121 121 122 122 123 123 123 124 124 124 126 126 127 127 129 129 129 130 131 132 133 133 133 133 135 135 135 135 135 136 136

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7 Metabolite Profiling of Actinobacterial Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . 1 Metabolite Profiling of Actinobacterial Extracts Using High-Performance Thin-Layer Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Metabolite Profiling of Actinobacterial Extracts by UV-Visible Spectrophotometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Metabolite Profiling of Actinobacterial Extracts by Gas Chromatography Mass Spectrometry (GC-MS) . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Metabolite Profiling of Actinobacterial Extracts by Liquid Chromatography–Mass Spectrometry (LC-MS) . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Bioassay-Guided Isolation and Characterization of Metabolites from Actinobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Purification of Antibiotic Compound by Thin-Layer Chromatography (TLC) and Bioautography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Purification of Bioactive Metabolites from Actinobacterial Extract by Column Chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Separation of Bioactive Metabolites from Actinobacterial Extracts Through HPLC Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Characterization of Bioactive Metabolites from Actinobacteria Physicochemical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

139 139 139 140 140 141 141 141 142 142 143 143 143 144 145 145 145 145 146 147 147 147 148 148 149 150 150 151 151 152 152 152 153 153 155 155 155 155 156

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Fourier Transform–Infrared Spectroscopy (FT–IR) Analysis . . . . . . . . . . . . . . 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Nuclear Magnetic Resonance (NMR) Spectroscopic Analysis of Actinobacterial Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 X-Ray Diffraction (XRD) Analysis of Actinobacterial Metabolites . . . . . . . . . 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

158 158 158 158

9 Evaluation of Actinobacteria for Environmental Applications . . . . . . . . . . . . . . . . . 1 Screening of Actinobacteria for Heavy Metal Resistance/Accumulation . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Biodegradation of Pesticides by Actinobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Isolation of Dye-Decolorizing Actinobacteria by Using Dilution Plate Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Screening the Actinobacterial Compounds for Anticorrosion Activity . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Screening the Actinobacterial Compounds for Antifouling Activity. . . . . . . . 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Screening the Actinobacterial Compounds for CO2 Sequestration . . . . . . . . 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

165 165 165 166 166 166 167 167 167 167 167

159 159 160 160 160 161 161 162 162

168 168 168 169 169 169 169 169 170 170 171 171 171 171 172 173 173 173 173 174 174

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Evaluation of Actinobacteria for Agricultural Applications . . . . . . . . . . . . . . . . . . . 1 Screening of Actinobacterial Metabolites for Anti-phytopathogenic Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Screening of Actinobacterial Isolates for Its Plant Growth-Promoting Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evaluation of Actinobacteria for Aquaculture Applications . . . . . . . . . . . . . . . . . . . 1 Preexperimental Screening of Actinobacterial Cultures for Aquaculture Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Experimental Screening of Actinobacterial Cultures for Aquaculture Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evaluation of Actinobacteria for Nanoparticle Synthesis . . . . . . . . . . . . . . . . . . . . . 1 Screening of Actinobacterial Strains for Nanoparticle Biosynthesis . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Characterization of Biosynthesized Nanoparticles by UV Spectral Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Characterization of Nanoparticles by Fourier Transform–Infrared Spectroscopic (FT–IR) Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 X-Ray Diffraction (XRD) Analysis of Nanoparticles. . . . . . . . . . . . . . . . . . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

175 175 175 176 176 176 177 177 177 178 180 181 181 181 182 182 184 184 184 184 185 188 189 189 189 190 190 192 192 192 192 192 193 193 193 194 194 194 194 195 195

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Determination of Cytotoxicity of Actinobacterial Extracts and Metabolites . . . . 1 Determination of Cytotoxicity of Actinobacterial Extracts and Metabolites Using Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Determination of Cytotoxicity of Actinobacterial Extracts and Metabolites Using Ethidium Bromide/Acridine Orange Stain (EtBr/AO) Apoptotic Dual Staining Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Determination of Cytotoxicity of Actinobacterial Extracts and Metabolites Using Zebrafish Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Determination of Cytotoxicity of Actinobacterial Extracts and Metabolites Using Animal Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Evaluation of Actinobacteria for Bioenergy Applications . . . . . . . . . . . . . . . . . . . . . 1 Screening of Actinobacteria for Biohydrogen Production . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Evaluation of Actinobacteria for Bioelectricity Production. . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Production of Polyhydroxy Butyrate (PHB) from Actinobacteria . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

205 205 205 206 206 207 207 207 208 208 208 209 209 210

Media Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

211 219

About the Authors RAMASAMY BALAGURUNATHAN has been working for the past three decades exclusively in actinobacteria research for production of bioactive compounds. He has obtained his Doctoral degree in Marine Biology from Annamalai University for his research on bioactive compounds from marine Streptomyces. He pursued his post doctoral research on rare actinobacteria in reputed research Institutes in China and Malaysia. In 2019, he has obtained Doctor of Science degree in Microbiology from Periyar University for his research contribution on Bioprospecting of actinobacteria from rare ecosystems in India. He started his teaching career in 2000. He has handled eleven research projects on actinobacteria worth around Rs. 2.5 Crore. His research team discovered novel anti–TB and anti–HIV molecule from marine Streptomyces sp. R2 and named as “TRANSITMYCIN”. He is the Co-inventor in the US & Indian patent filed on Transitmycin.He has isolated Leptomycin—an antileptospiral compound from marine actinobacteria. With the goal of developing a centre for collection of actinobacteria from less explored habitats such as marine, desert, forest and mountain ecosystems, presently his team has isolated and are maintaining more than 500 actinobacteria isolated from various extreme environments including Thar desert, Himalayas, western Ghats, rubber forest, Andaman & Nicobar islands, which are the sources for production of antibiotics and other high value products. He has authored 145 publications including research articles, reviews, books, book chapters and magazine articles around the single focused aspect of actinobacteria for various applications like bioactive compound and bio-nanotechnology. He has an h-index 21 and i10-index 38. He has guided 80 M.Sc., 25 M.Phil. Projects, 13 Ph.D thesis awarded and is guiding 6 Ph.D., scholars and 1 Post-doctoral fellow. MANIKKAM RADHAKRISHNAN is a microbiologist pursuing research on Ecology, Systematics and Bioprospecting of actinobacteria from under studied ecosystems across India for the past two decades. He obtained his Doctoral degree in Microbiology from National Institute for Research in Tuberculosis under University of Madras on “Antitubercular metabolites from Desert soil Streptomyces”. After his Post graduation, he started his career as Assistant Professor in Microbiology. He is passionate about research and joined at Centre for Drug Discovery and Development, Sathyabama Institute of Science and Technology in the year 2013. He set up a consortium of >500 actinobacterial cultures under his headship in Bioprospecting division. He has isolated a novel chromopeptide molecule, Transitmycin which is effective against TB and HIV, from a novel marine Streptomyces species R2 isolated from Coral reef ecosystem of South India. At present he is working on Diversity and Taxonomy of Psychrophilic and psychrotolerant actinobacteria from Indian sector of Southern Ocean (Antarctic). He has participated as the scientific member (Microbiology) in the 11th Indian Scientific Expedition to Southern Ocean. He has authored 110 publications including research articles, reviews, book chapters and magazine articles and has also three patents on actinobacteria to his credit. His vision aims that this gold mine of Actinobacterial resource should be best exploited for the welfare of humans, animals, aquaculture and environmental applications since it has proven therapeutic potential and translational scope.

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About the Authors

THANGAVEL SHANMUGASUNDARAM has completed his Masters in Microbiology with gold medal from Periyar University. He has received prestigious INSPIRE fellowship from DST to pursue his doctoral degree on Exploring magnesite soil actinobacteria for metal and metal alloy nanoparticles for its biomedical and environmental applications. He also received Best Ph.D Thesis award for his meritorious Doctoral thesis. He has published 31 research articles on actinobacteria in high impact. Based on his outstanding publications on actinobacteria and nanotechnology he received DST-National Post doctoral fellowship to work on actinobacteria and its enzymes for nanoparticle synthesis and applications at the DRDO Bharathiar University Centre for Life Sciences, He has a great scientific acumen and aptitude and dedicated research in the field Actinobacteriology and Cancer biology for a decade. VENUGOPAL GOPIKRISHNAN is working in the field of actinobacterial research for the past 8 years. He obtained his Doctoral degree from Actinobacterial Research Lab, Department of Microbiology, Periyar University. During his doctoral degree, he isolated about 200 actinobacterial cultures from marine and mangrove ecosystems of South India. He has identified Quercetin and Taxifolin two active metabolites from two potential Streptomyces sp. PE7 and PM33 which have showed promising antifouling and antimicrobial activity. The active molecules have evidenced good antimicrobial, anti TB and anti-cancer activities. He started his career as scientist in Bioprospecting division of Centre for Drug Discovery and Development at Sathyabama Institute of Science and Technology. At present he is continuing his research on exploring the fish gut associated actinobacteria for quorum sensing inhibitors and antibiofilm compounds. He has published 29 articles including research articles, reviews, book chapters and magazine articles. His aim is to explore the host or symbiotic actinobacteria marine and terrestrial organisms for bioprospecting purposes. JOSEPH JERRINE has completed her under graduation with gold medal from St. Ann’s College, Visakhapatnam, Andhra University majoring in Biochemistry, Botany and Chemistry. She has done her Masters in Life sciences from Pondicherry Central University. She also cleared GATE 2000 with 98.6 percentile and UGC-NET. She obtained the Doctor of Philosophy from University of Madras in 2011 for her research on Human Pappiloma Virus (HPV) 16 & 18 with respect to cervical cancer. She is the recipient of the ICMR—Senior Research Fellowship for “Establishment of National Database on TB”. Further, she has been awarded ICMR-Post Doctoral Fellowship for “Anti TB metabolites from marine Streptomycetes”. She has published 40 research articles on actinobacteria and plants with reference to anti TB and other biomedical activities, cancer and asthma. She is recipient of three research projects from central and state funding agencies to work on actinobacteria with special reference to anti TB metabolites. Presently she is working as Scientist D and heading the Centre for Drug Discovery and Development, Sathyabama Institute of Science and Technology, TamilNadu with focused research on bioprospecting of actinobacteria and plants from rare ecosystems in India with reference to anti TB and anti cancer molecules.

Abbreviations AG AGR AMC APCI BLAST BSL3 CaCl2 CaCO3 CAD CAS CAS CDCl3 CFS CFU CMC CO2 CTAB DAT DC ddNTPs dl-DAP dNTPs dNTPs DMF DMSO DNA DNS DPPH dsDNA DST ECM EDTA EGA-FTIR EPSs ESI EtBr/AO EtOH FCE FCR FID FT-IR GC-MS GI GOD and POD

Arabinogalactan Absolute growth rate Aerial mass color Atmospheric pressure chemical ionization Basic local alignment search tool Biological safety level 3 Calcium chloride Calcium carbonate Caspase-activated DNase Chrome azurol sulfonate Chrome azurol S Deuterated chloroform Cell-free supernatant Colony forming unit Carboxymethyl cellulose Carbon dioxide Cetyltrimethylammonium bromide Days after transplanting Direct current Dideoxynucleotide triphosphates 2,6-diaminopimelic acid Deoxyribonucleotide triphosphates Deoxynucleotide triphosphates Dimethylformamide Dimethyl sulfoxide Deoxyribonucleic acid Dinitrosalicylic acid 1,1-diphenyl-2-picrylhydrazyl Double-stranded DNA Drug susceptibility testing Extracellular matrix Ethylenediaminetetraacetic acid Evolved gas analysis—Fourier transform infrared spectroscopy Extracellular polymeric substances Electrospray ionization Ethidium bromide/acridine orange Ethanol Feed conversion efficiency Feed conversion ratio Free inductive decay Fourier transform infrared spectroscopy Gas chromatography mass spectrometry Gastrointestinal Glucose oxidase and peroxidase

xxvii

xxviii

Abbreviations

H2O2 HBSS HCl HPLC HPTLC HV agar IAA IL-10 INT iPrOH ISP2 KBr KH2PO4 KNO3 LB LCL LC-MS LDH LED LJ LRP MABA MAs MCT MDR MeCN MEGA MFC MIC MgCl2 MgSO4 MHA MHB MPO MTT NaCl NADH NaHCO3 NaOH NCBI NJ NMR NO OFAT OSMAC OTR OTUs PBS

Hydrogen peroxide Hank’s balanced salt solution Hydrochloric acid High-performance liquid chromatography High-performance thin layer chromatography Humic acid-vitamin agar Indole acetic acid Interleukin-10 Iodonitrotetrazolium chloride Isopropyl alcohol International Streptomyces Project 2 Potassium bromide Potassium dihydrogen phosphate Potassium nitrate Luria-Bertani Lower confidence limit Liquid chromatography-mass spectrometry Lactate dehydrogenase Light-emitting diode Lowenstein-Jensen Luciferase reporter phage Microplate Alamar blue assay Mycolic acids Mercury–Cadmium–Telluride Multidrug resistant Acetonitrile Molecular Evolutionary Genetics Analysis Microbial fuel cells Minimum inhibitory concentration Magnesium chloride Magnesium sulfate Mueller Hinton Agar Mueller Hinton Broth Myeloperoxidase 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Sodium chloride Nicotinamide adenine dinucleotide Sodium bicarbonate Sodium hydroxide National Center for Biotechnology Information Neighbor joining Nuclear magnetic resonance Nitric oxide One-factor-at-a-time One strain many compounds Oxygen transfer rate Operational taxonomic units Phosphate buffered saline

Abbreviations

PCR PGP p-NPA PPG QIIME QS RAPD Rf RFLP RGR RLU RNA ROS RPM rRNA RSB RSP SAC SCA SDA SDS SEM SGR SP SPSS SSF TAC TAE TBE TE TEM TGA TIF TLC TSA TSB TTC UCL UPGMA UV XRD XTT

YEME ZOI 1 H 13 C 31 P

xxix

Polymerase chain reaction Plant growth promotion Para-nitro phenyl acetate Polypropylene glycol Quantitative Insights Into Microbial Ecology Quorum sensing Random amplified polymorphic DNA Retention factor Restriction fragment length polymorphism Relative growth rate Relative light unit Ribonucleic acid Reactive oxygen species Revolutions per minute Ribosomal RNA Resuspension buffer Reverse side pigment Surface active compounds Starch casein agar Sabouraud dextrose agar Sodium dodecyl sulfate Scanning electron microscopy Specific growth rate Soluble pigment Statistical Package of Social Sciences Software Solid-state fermentation Total antioxidant capacity Tris acetic acid-EDTA Tris-borate-EDTA Tris EDTA Transmission electron microscopy Thermal gravimetric analysis Tagged image format Thin layer chromatography Trypticase soy agar Tryptic soy broth 2,3,5-Triphenyl tetrazolium chloride Upper confidence limit Unweighted pair group method using arithmetic averages Ultra violet X-ray diffraction 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2Htetrazoliumhydroxide Yeast extract and malt extract Zone of inhibition Hydrogen-1 Carbon-13 Phosphorus-31

Symbols μg ppm PSI θ λ ml  C % U g kg cm min w/v v/v M mM OD M/Z

Microgram Parts-per million Pounds per square inch Theta Lambda Milliliter Celsius Percentage Units Gram Kelogram Centimeter Minute Weight/volume Volume/volume Molar Millimolar Optical density Mass charge ratio

xxxi

Chapter 1 Sample Collection, Isolation, and Diversity of Actinobacteria Abstract Actinobacteria are one of the largest bacterial phyla in the domain bacteria. The results of culturable and unculturable (metagenomic) studies revealed that the members of this phylum are widely distributed in wide range of living and nonliving sources in diverse ecosystems including the extreme one. Pretreatment is very important for the selective isolation of actinobacteria members, which can grow slower than other eubacteria and fungi. In general, the pretreatment approaches select targeted actinobacterial genera by inhibiting or eliminating the unwanted bacteria and fungi. In addition, actinobacterial spores are more resistant to desiccation than most other bacteria. However, it is not possible to recommend single isolation procedure for the isolation of different kinds of actinobacteria from the multiplicity of environments. Selective pretreatment procedures will be followed at different levels such as pretreatment of samples, isolation media, and incubation conditions. Metagenomic studies pave the way for knowing the distribution of different actinobacterial genera including the yet-to-be-cultured rare actinobacterial genera in different ecosystems. Based on the metagenomic data, different rare actinobacterial genera will be isolated from such sources by adopting suitable pretreatment methods, isolation media, and culture conditions. This chapter describes the general guidelines, suitable pretreatment methods, and isolation protocols for the selective isolation of actinobacteria. Methods for the determination of uncultured actinobacterial diversity will also be discussed. Keywords Sample collection, Pretreatment, Selective isolation, Pyrosequencing

1 1.1

General Guidelines for Sample Collection for the Isolation of Actinobacteria Introduction

Actinobacteria are the group of Gram-positive bacteria with high guanine and cytosine (G + C) content in their DNA, and they are one of the largest bacterial phyla. They are the largest group of microorganisms being widely distributed in various terrestrial and aquatic ecosystems. Mostly they are saprophytic in nature with the ability to utilize a wide range of organic materials, and they are able to grow normally at pH 7 and temperatures of 28–30
C. Usually they are slow growing in nature [1]. Members of the phylum especially from the order Actinomycetales are being well explored as a source of antibiotics. The genus Streptomyces is very commonly present in normal as well as extreme ecosystems with the unprecedented ability to produce bioactive secondary metabolites. Pretreatment of soil/sediment samples by physical agents like heat or chemicals like phenol and supplementing the isolation medium

Ramasamy Balagurunathan et al., Protocols in Actinobacterial Research, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0728-2_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020

1

2

Sample Collection, Isolation, and Diversity of Actinobacteria

with antibacterial and antifungal antibiotics will facilitate the isolation of actinobacteria. Several years ago, soil is recognized as a richest source for actinobacteria; notably several antibiotics producing Streptomyces are isolated from them. To expand the chances of isolating novel actinobacteria, researchers started investigating various other living and nonliving sources from under studied ecosystems. In recent years, there are several novel actinobacterial genera, and species were reported from living and nonliving sources of extreme environments. The method to be followed may vary for collecting different samples from different locations. As several actinobacterial genera like Streptomyces, Micromonospora, and Streptoverticillium are reported to produce spores, they can withstand dry conditions. Hence, in most of the cases, dried soil or sediment samples are used for the isolation of actinobacteria. But care should be taken while isolating non-sporulating actinobacteria like Arthrobacter, Bifidobacterium, and Corynebacterium. However, the proper collection and processing of samples is the prerequisite to study the diversity and isolation of actinobacteria. It is advisable to examine samples from diverse habitats to isolate, rare, novel, and extremophilic actinobacteria [2]. Some general rules for applying ecological approaches to microbial isolation are as follows:

1.2 General Instructions for Sample Collection

l

Groups of microbes that are to be isolated, i.e., actinobacteria, bacteria, fungi, and algae.

l

The ecosystem or habitat from which the samples are to be collected.

l

Samples to be collected like soil, sediment, plants, water, insects, etc.

l

The environmental parameters to be considered and measured such as pH, salinity, temperature, and soil composition.

l

Natural substrates available in that ecosystem, e.g., lignin and cellulose in forest soil and chitin in salt marshes.

l

Designing of isolation techniques based on the ecological data, i.e., diluents, substrates, natural extracts, and incubation conditions.

l

Modify known isolation procedures as required by the ecological methods as controls.

l

Using specific procedures for microbial groups to be isolated.

l

Samples should be collected as aseptically as possible using sterile spatula, soil profile sampler, scalpels, gloves, plastic bottles, and plastic bags.

l

Samples should be representatives of a site, e.g., a particular soil type and its horizons, marine sediments, mud, plant surface, and parts or water column.

Sample Pretreatment and Media Selection for the Selective Isolation of. . .

1.3

Notes

3

l

Samples should be fully labeled with description and date.

l

Once the samples are brought into the lab, they should be examined immediately or stored overnight at 4
C.

l

Ecological parameters such as temperature, pH, salinity, and even substrate concentration of the sampling location should be noted.

l

Most of these parameters are measured in the field or laboratory

l

Care should be taken while collecting the live samples like marine organisms, insects, and animals.

l

Get necessary approval or clearance from respective authorities before collecting the endangered samples as well as samples from restricted places like the National Biosphere Reserve.

l

Care must be taken while transporting the samples to the laboratory.

l

Authentication of living samples with the experts before processing must be done.

l

While preparing the isolation media, incorporation of natural extracts like plant extracts or the environmental physical parameters in the media can influence the numbers and variety of actinobacteria isolated in the laboratory.

2 Sample Pretreatment and Media Selection for the Selective Isolation of Actinobacteria 2.1

Introduction

Pretreatment of samples and selection of suitable media are the prerequisite for the successful isolation of actinobacteria. There are two common pretreatment methods used individually or applied in combination. One is the physical pretreatment method which uses physical processes like heat or centrifugation, and another is chemical pretreatment method which uses any toxic chemicals. The basic principle behind all the approaches is retarding the growth of fungal and bacteria and/or enhancing the growth of actinobacteria. Based on experience and evidence, it was noted that the combination of both physical and chemical pretreatment of samples and the use of suitable media only lead to the successful isolation of actinobacteria [1]. The use of specific antibiotics to select for or against desired microbes becomes one of the most important selective approaches for the isolation of different actinobacterial genera. The antifungal antibiotics like cycloheximide and nystatin are used at approximately 50 μg/ml to eliminate the outgrowth of any fungal colonies. In addition, several antibacterial antibiotics are used for the selective isolation of specific actinobacterial genera. The basic principle

4

Sample Collection, Isolation, and Diversity of Actinobacteria

behind this is the antibiotic to be used which is produced by the specific actinobacterial genera to be isolated. In general, any antibiotic-producing actinobacteria possess the resistant genes for the antibiotics they produce. Sometimes, the use of the same antibiotic at different concentrations may result in the isolation of different actinobacterial genera [2]. 2.2

2.3

Materials

Methods

2.3.1 Pretreatment of Sample

2.3.2 Pretreatment of Isolation Media

l

Soil sample.

l

Glasswares.

l

Hot air oven.

l

Centrifuge.

l

Distilled water.

l

Electronic balance.

l

Pretreatment chemicals (phenol, SDS, sodium hypochlorite, chloramine T).

l

Measure required quantity of soil or sediment sample in a clean glass dish.

l

Subject the sample for any of the following pretreatment methods (see Table 1).

l

Prepare the isolation agar media by supplementing with the following antibiotics.

l

Dilute all the antibiotics in a small volume to sterile water or DMSO.

l

2.4

Notes

Mix the antibiotic solution well and filter using 0.22 μm syringe filter.

l

Add the filtered antibiotic solution into sterilized isolation agar media in conical flask under aseptic condition (see Table 2).

l

Pour the agar media into sterile petri plates and allow for solidification.

l

Care should be taken while handling the pretreatment chemicals like phenol, sodium hypochlorite, SDS, etc.

l

Do not add any antibiotics directly into the media before autoclaving.

l

Dissolve the antibiotics using solvents like water, DMSO, and DMF based on their solubility.

l

Check the expiry date of the antibiotics before using.

Isolation and Enumeration of Actinobacteria from Soil Sample

5

Table 1 Pretreatment methods Method

Comments

Physical methods Drying at room temperature

Eliminate most of the Gram-negative bacteria that form mucoid spreading colonies on isolation agar plates

Alternate drying and wetting for Enrich sporangium-forming actinobacteria like Streptosporangium, each for 24 h and eliminate unwanted competitors To isolate Micromonospora Differential centrifugation Cesium chloride density gradient centrifugation Heat method—moist heat 45
C for 1 h

To isolate Micromonospora

50
C for 10 min

To isolate Rhodococcus




70 C for 30 min

Elimination of spreading bacterial colonies on isolation agar plates

Dry heat 120
C for 1 h

To isolate Micromonospora

55
C for 10 min

Facilitate isolation of actinobacteria, and regard the growth of fastgrowing bacteria and fungi

Chemical methods Phenol treatment 1.5% (w/v) at Facilitate isolation of Streptomyces 30
C for 30 min Sodium dodecyl sulphate (SDS)

To isolate Rhodococcus

Sodium hypochlorite and osmium tetroxide

To isolate Frankia from Root nodules

Chloramine T (1%)

Micromonospora and Streptomyces

Chloramine T 1% + HV agar

Herbidospora, Microbispora, Microtetraspora, and Streptosporangium

3 3.1

Isolation and Enumeration of Actinobacteria from Soil Sample Introduction

Soil is the primary and well-investigated reservoir of most actinobacterial genera. Streptomyces tend to predominate, but other genera like Actinomadura, Nocardia, Rhodococcus, Arthrobacter, and Micromonospora are common in most soils. Serial dilution of the pretreated sample and its spread plating on isolation agar are normally followed for the isolation of actinobacteria from any samples. Spread plate technique is a method employed to plate a liquid sample for the purpose of isolating or counting the bacteria or fungi present in that sample [3]. A perfect spread plate technique will result in visible and isolated colonies of bacteria that are evenly

6

Sample Collection, Isolation, and Diversity of Actinobacteria

Table 2 Antibiotics used for pretreatment Antibiotics

Selection for

Gentamicin (2–5 μg/ml)

Streptosporangium and Actinomadura

Gentamicin (10 μg/ml)

Micromonospora

Kanamycin (25 μg/ml)

Actinomadura

Nalidixic acid (20 μg/ml) Penicillin G (10 U/ml) Potassium tellurite (0.005%)

Rhodococcus

Novobiocin (10–15 μg/ml)

Actinoplanes

Novobiocin (100 μg/ml)

Thermoactinomyces

Novobiocin (25 μg/ml) Streptomycin (25 μg/ml)

Glycomyces

Penicillin G (5–10 μg/ml) Nalidixic acid (5 μg/ml)

Saccharothrix

Rifampicin (25 μg/ml)

Actinomadura

Tunicamycin (20 μg/ml) Nalidixic acid (30 μg/ml)

Micromonospora

Vancomycin (1–10 μg/ml) Polymyxin B (5 U/ml)

Amycolatopsis

distributed in the plate and are countable. The technique is most commonly applied for microbial testing of foods or any other samples or to isolate and identify a variety of microbial flora present in the environmental samples, e.g., soil. In this method, the substance to be tested, if not in liquid form, is grinded and dissolved in suitable liquid medium. The sample is then diluted in tenfold serial dilutions and plated in appropriate medium. Following incubation the number of colonies present in the plate is counted. Assuming that each organism gives a single colony, the number of total bacteria present in a sample is calculated [4]. 3.2

Materials

l

Soil sample.

l

Conical flask—250 ml.

l

Test tubes—15 ml.

l

Micropipettes.

l

Starch casein agar plates.

l

L-rod.

l

Incubator.

l

ISP2 agar plates and slants.

l

Glycerol broth (20%).

Isolation and Enumeration of Actinobacteria from Soil Sample

3.3

Methods

7

l

Select the sampling location like plant rhizosphere or agricultural land.

l

Record the information such as sampling site, district, state, any specific ecosystem, and its latitude and longitude of the study area.

l

Remove any leaf litter or any other contaminants present over the soil surface.

l

Collect the required quantity of soil (50–500 g) from 8 to 10 cm depth using sterile spatula.

l

Transfer the samples to sterile glass bottles with rubber stoppers, or collect the samples in a clean plastic cover.

l

Transport the samples to the laboratory, and process immediately, or store under refrigerated conditions

l

Dry the samples at room temperature for 2–3 days,

l

Take 90 ml distilled water blank in a 500 ml conical flask and each 9 ml distilled water in five test tubes (15 ml size).

l

Sterilize the water blanks by autoclaving at 121
C for 15 min.

l

Measure 10 g of air-dried soil, and pretreat by keeping it in hot air oven at 55
C for 10 min.

l

Add the pretreated sample into 90 ml sterile distilled water blank (101 dilution), and keep it in rotary shaker with 90 rpm speed for 30 min.

l

l l

l

l

Transfer 1 ml of diluted sample from 101 dilution into 9 ml of sterile distilled water, and mix well (102 dilution). Continue the serial dilution up to 106 dilutions. Prepare starch casein nitrate agar medium and sterilize by autoclaving at 121
C for 15 min. Measure required quantity of nalidixic acid (20 μg/ml) and nystatin (50 μg/ml), and mix with 1 ml of sterile distilled water. Sterilize the antibiotic solution by filtration using 0.22 μm syringe filter.

l

Add the filter-sterilized antibiotic solution into starch casein nitrate agar medium.

l

Pour the antibiotic-supplemented medium onto sterile petri plates under aseptic conditions, and allow to solidify.

l

l l

Transfer each 100 μl of aliquot from 103 to 106 dilutions onto starch casein agar plates, and spread using sterile L-shaped glass rod. Incubate all the plates at 28
C for 2–6 weeks. Observe all the plates for powdery or leathery colonies and enumerate.

8

Sample Collection, Isolation, and Diversity of Actinobacteria

Table 3 Dilution factor and total number of actinobacterial colonies Dilution factor 10

Number of actinobacterial colonies

1

102 103 104 105 106

l

Recover the colonies using ISP2 agar plates and incubate at 28
C for 7–14 days.

l

Subculture the morphologically different actinobacterial colonies using ISP2 agar slants as well as in 20% glycerol broth.

Total number of actinobacterial population in the given sample (see Table 3) calculated using following formula (CFU): CFU ¼

3.4

4 4.1

Notes

No: of colonies counted  Dilution f actor Volume of sample ðorÞ inoculum

l

Adjusting the pH of the isolation agar medium as same as or near to that of the soil pH will facilitate the enhanced recovery of actinobacterial colonies. Sometimes the samples may be heat treated for 6 min to 30 min to recover rare actinobacterial genera.

l

In addition to or instead of starch casein nitrate agar, other media like humic acid vitamin agar and chitin agar may also be used for the isolation of actinobacteria.

l

Instead of recovering actinobacterial colonies at the end of incubation, recover the colonies in parallel during incubation. This will save/rescue the actinobacterial colonies from other fast-growing bacterial and fungal contaminants.

Isolation of Actinobacteria from Sediments Introduction

Actinobacteria are ubiquitous in the marine environment, playing an important ecological role in the nutrient recycling and production of novel natural products with pharmaceutical applications. The discovery of novel secondary metabolites from marine

Isolation of Actinobacteria from Sediments

9

actinobacteria has recently surpassed that of their terrestrial counterparts. Studies on unique marine actinobacterial genera like Salinispora and Verrucosispora, which produce salinosporamide and abyssomicin, respectively, suggest that actinobacteria add an important facet to marine drug discovery research. Moreover, marinederived antibiotics are more efficient in fighting infections because the terrestrial bacteria have not had an opportunity to develop resistance to them [5]. Actinobacteria are widely distributed in various living and nonliving marine sources. Culturable study revealed that marine sediments are the richest source for the isolation of various actinobacterial genera. There are several novel actinobacterial genera, and species are isolated from diverse marine samples such as sediments, water, coral, sponges, mangroves, seaweed, and fishes. There are several isolation strategies that have been reported for the isolation of actinobacteria from different marine samples. But there is no single isolation approach suitable to isolate different actinobacterial genera from different marine samples. Different pretreatment methods and isolation media with different compositions are used for the isolation of novel rare actinobacterial genera. All the media used for the isolation of actinobacteria are invariably prepared using 50 or 100% natural filtered sea water or artificial sea water which facilitate the recovery of marine actinobacteria [6]. 4.2

4.3

Materials

Methods

l

Sediment sample.

l

Conical flask—250 ml.

l

Test tubes—15 ml.

l

Micropipettes.

l

Starch casein agar, Kuster’s agar, and Actinomycetes isolation agar plates.

l

L-rod.

l

Incubator.

l

ISP2 agar plates and slants.

l

Glycerol broth (20%).

l

Select the sampling locations like mangrove, estuarine, or intertidal regions in the marine ecosystem.

l

Collect approximately 50–500 g of the sediment samples at 4 cm below the seawater level.

l

Transfer the samples into a clean plastic bag.

l

Keep the samples in a cold box containing ice and transfer to laboratory.

4.3.1 Sediment Samples

10

Sample Collection, Isolation, and Diversity of Actinobacteria

4.3.2 Water Sample

4.3.3 Isolation of Actinobacteria from Marine Sediments Stamping Method

l

Select the marine region like estuarine, mangroves, or intertidal regions for water sample collection.

l

Collect the samples at subsurface level at about 10–15 cm depth by dipping presterilized glass bottles.

l

Tightly fasten the stopper and label.

l

Transport the sample to the lab for immediate processing, or store under refrigerated conditions.

l

Place 10 g of wet sediment sample aseptically into sterile aluminum dish, and dry in a laminar flow cabinet for 24 h.

l

Grind the sample lightly with pestle.

l

Prepare starch casein agar plates using 50 or 100% natural or artificial seawater.

l

Supplement the agar medium with antibiotics as described in Subheading 3.

l

Press the sample into a sterile foam plug with 14 mm in diameter, and inoculate into starch casein nitrate agar plates by stamping.

l

Stamp eight to nine times in a circular fashion to give a serial dilution effect.

l

4.3.4 Heat Shock and Dilution Method

l

Consider any colonies with a tough, leathery, texture dry, or folded appearance and branching filaments with or without aerial hyphae.

l

Enumerate and recover the actinobacterial colonies using ISP2 agar or oatmeal agar plates.

l

Take 1 g of sediment and add into 4 ml of sterile seawater.

l

Heat for 6 min at 55
C and shake vigorously.

l

l

Serial Dilution and Plating Method

Incubate the plates at 28
C for 2–6 weeks.

Dilute the sample at 1:4 ratio using sterile seawater up to 106 dilutions. Inoculate 50 μl of aliquot from each dilution on to agar-based isolation media, and spread with a sterile L-shaped glass rod.

l

Prepare all the isolation media like starch casein nitrate agar, Kuster’s agar, and Actinomycetes isolation agar by supplementing with media with filter-sterilized cycloheximide (50 μg/ml) and nalidixic acid (20 μg/ml) to reduce the growth of fungal and fast-growing bacteria while selecting for actinobacteria.

l

Collect the sediment sample from the respective marine location.

l

Take 90 ml of filtered seawater in 500 ml conical flask and each 9 ml of filtered seawater in five test tubes, and sterilize by autoclaving at 121
C for 15 min.

Isolation of Actinobacteria from Sediments l

l

l

l

l l

l

l l

l

11

Weigh 10 g of sediment sample and dry in hot air oven at 55
C for 10 min. Add the sediment sample in to 90 ml of sterile seawater, and keep it in a shaker at 95 rpm for 30 min Transfer 1 ml of diluted sample from 101 dilution to 9 ml sterile seawater bank, and mix well. Continue the dilution process up to 106 dilutions and mix all the tubes well. Prepare different isolation agar plates as described above. Transfer 100 μl of aliquot from all the dilutions into isolation agar plates, and spread using sterile L-glass rod. Do the plating in triplicate and keep one uninoculated agar plate as control. Incubate all the plates at 28
C for 1–3 months. Enumerate and purify colonies with suspected actinobacterial morphology using the same media. Preserve the purified isolates at 80
C in 20% glycerol (v/v) broth as well as at 4
C in ISP2 agar slants.

Isolation of Actinobacteria from Marine Waters

l

Prepare starch casein nitrate agar, Kuster’s agar, and Actinomycetes isolation agar plates for the isolation of actinobacteria as described above.

Direct Spread Plate Method

l

Take 1 ml of collected water sample without dilution.

l

Spread the water sample on different isolation agar plates such as starch casein agar, Kuster’s agar (glycerol: 10 g, casein: 0.3 g, KNO3: 2 g, K2HPO4: 2 g, soluble starch: 0.5 g, asparagine: 0.1 g, FeSO4.7H2O: 0.01 g, CaCO3: 0.02 g, MgSO4.7H2O: 0.05 g, agar: 15 g, filtered sea water: 1000 ml and pH: 7.0  0.1), etc

l

Keep the plates at laminar air flow cabinet for 5–10 min to allow the water from agar surface to dry.

l l

l

Filtration and Plating Technique

l

l

Incubate all the isolation agar plates at 28
C for 1–3 months. Observe the plates for colonies with actinobacterial morphology and subculture on YEME agar/oatmeal agar plates. Preserve the cultures at 80
C in 20% (v/v) sterile glycerol broth as well as in YEME agar slants under refrigerated conditions. Collect the required quantity of water sample (usually 1–5 l) from a specific aquatic ecosystem. Filter the water sample through 0.22 μm sterile membrane filter.

12

Sample Collection, Isolation, and Diversity of Actinobacteria

Table 4 Dilution factor and total number of actinobacterial colonies Dilution factor 10

Number of actinobacterial colonies

1

102 103 104 105 106

l

Remove the filter paper from the filtration unit, and immerse in 9 ml sterile seawater, and mix gently.

l

Take 1 ml of seawater loaded with microbial community, and serially dilute up to 106 dilutions using each 9 ml of sterile seawater.

l

Inoculate 1 ml of diluted sample from each dilution onto different isolation agar plates in triplicate.

l l

l

Incubate all the plates at 28
C for 1–3 months. Recover and subculture the colonies with suspected actinobacterial morphology using the same media. Preserve the cultures at 80
C using 20% (v/v) sterile glycerol broth or in YEME agar slants under refrigerator conditions.

Total number of actinobacterial population in the given sample (see Table 4) calculated using following formula: CFU ¼

No: of colonies counted  Dilution f actor Volume of sample ðorÞ inoculum

The total actinobacterial population present in the given sediment sample is estimated as ____ CFU/g of sediment. The total actinobacterial population present in the given marine water sample is estimated as ____ CFU/ml of water. 4.4

Notes

l

Always prepare all the media to be used for marine actinobacterial isolation, recovery, or preservation using 50% or 100% natural or artificial sea water.

l

Plate the sample from each dilution in triplicate, and always keep one uninoculated agar plate as control.

Isolation of Actinobacteria (Endophytic) from Plants

5

13

Isolation of Actinobacteria (Endophytic) from Plants

5.1

Introduction

Actinobacteria residing in healthy plant tissues without causing any ill effect to their host plants are termed as endophytic actinobacteria. Endophytic actinobacteria have particularly attracted significant attention, with increasing documentation of isolates from a wide range of plants, including various crop plants like wheat, rice, banana, apple, and tea plants, in addition to medicinal plants. Streptomyces, Micromonospora, Micrococcus, Pseudonocardia, and Microbacterium are the most predominant endophytic actinobacterial genera isolated from several medicinal plants. Several endophytic actinobacteria are reported to produce novel bioactive compounds with antimicrobial and antitumor properties, besides showing a good potential for pharmaceutical development. The method to be used for the isolation of endophytic actinobacteria is an important factor that affects the acquisition and diversity of pure cultures [7]. Different methods have been used for the isolation of endophytic actinobacteria in which most of them start with the collection of plant parts such as root, stem, bark, or leaves followed by three to five step surface sterilization process. The collected samples are washed in running tap water to remove adhered epiphytes, soil debris, or dust particles on the surface followed by surface sterilization using one or more surface-sterilizing agents like ethanol, sodium hypochlorite, etc. All the samples are finally rinsed in sterile distilled water. The pretreated samples will be either cut into pieces with 1 cm size and placed over isolation agar plates or crushed using mortar and pestle followed by serial dilution and plating [7].

5.2

Materials

l

Plant samples.

l

Pretreatment solutions [1% Tween 20, 70% ethanol, 6% sodium hypochlorite).

l

Sterile distilled water.

l

Glass wares.

l

Mortar and pestle.

l

Isolation agar media (starch casein agar, chitin agar, cellulose agar, plant extract agar).

l

Phosphate buffer pH 7.

l

Select the plants for the isolation of endophytic actinobacteria based on their ethnobotanical history.

l

Choose the healthy, and collect the infection-free plant parts in a sterile container or plastic bags by handpicking or using a small knife.

5.3

Methods

14

Sample Collection, Isolation, and Diversity of Actinobacteria l

Transport the plant samples to the laboratory immediately for further processing, or store the samples under refrigerator condition until further processing.

l

Wash the samples thoroughly with running tap water, and sonicate for 20 s to dislodge any soil and organic matter.

l

Wash 1–2 g of samples in sterile water and dry on a paper towel.

l

Immerse the sample in sterile 0.1 % Tween 20 for 5 min, in 70% ethanol for 5 min, and in sodium hypochlorite solution (6% available chloride, freshly prepared) for 5 min, followed by washing in sterile water five times to remove the chemicals.

l

Finally soak the pretreated sample in sterile 10% (w/v) NaHCO3 for 10 min to retard the growth of endophytic fungi, followed by washing twice in sterile water.

l

Crush the sample using sterile mortar and pestle by adding 5 ml phosphate buffer, pH 7.

l

Serially dilute 1 ml of crushed plant sample up to 106 dilutions using each 9 ml of sterile water blank.

l

Prepare different isolation agar media such as chitin agar, cellulose agar, starch casein agar, and plant extract agar, and sterilize by autoclaving at 121
C for 15 min.

l

Supplement the medium (after sterilization) with filter-sterilized antibiotics, cycloheximide (50 μg/ml) and nalidixic acid (20 μg/ml), in order to retard the growth of fungi and fastgrowing bacteria, respectively.

l

l

l

Transfer 100 μl of aliquot from all the dilutions onto isolation agar plates, and incubate at 28
C for 1–2 months. Observe the plates from the first week onward for the appearance of colonies with actinobacterial morphology. Gradually recover the actinobacterial colonies with >1 mm in size, and leave the small, slow-growing, pinpointed colonies to develop further.

Total number of actinobacterial population in the given sample (see Table 4) calculated using following formula: CFU ¼

No: of colonies counted  Dilution f actor Volume of sample ðorÞ inoculum

The total actinobacterial population present in the given plant sample is estimated as ____ CFU/g of plant sample. 5.4

Notes

l

The diversity, distribution and isolation of endophytic actinobacteria depends on various factors such as host plant species, age, type of tissue, geography and habitant distribution,

Isolation of Actinobacteria (Endosymbiotic) from Fish

15

sampling season, surface sterilants, selective media, and culture conditions

6

l

In general, maximum endophytic actinobacterial populations have been recovered from roots followed by stem and least in leaves. Far greater diversity of actinobacteria is conferred in the woody plants when in comparison to herbaceous plants.

l

Addition of or replacement of medium components with plant extract will facilitate the recovery of endophytic actinobacteria. However, the plant extract should be prepared using sterile water and supplemented to the isolation agar medium after sterilization. Autoclaving of plant extract leads to destruction of their chemical components.

Isolation of Actinobacteria (Endosymbiotic) from Fish

6.1

Introduction

The gastrointestinal (GI) tract of a fish is a complex ecosystem that harbors an estimated 107–108 colony-forming units (CFU)/g. The activity and composition of the GI microbiome are affected by host genome, lifestyle, and dietary preferences. During the co-development of the GI microbiome and the host, the microbial community plays an important role in host physiology, nutrition, and health. Several special groups, such as probiotics, pathogens, and cellulose-decomposing bacteria, were also isolated from various freshwater or marine fish species. Additionally, certain authors demonstrated that the fish gut microbiome could be used as a new source for the discovery of natural products [8]. Although Actinobacteria are prolific producers of secondary metabolites, their role in the gut is poorly understood. Commensal actinobacteria of the genus Bifidobacterium have been shown to regulate interlukin-10 (IL-10) production in healthy hosts. There are very limited studies which reported the bioactive potentials of fish gut-associated Actinobacteria. Isolation of actinobacteria from fish gut is important for its bioprospecting. Isolation of actinobacteria from fish gut starts from surface sterilization of fish skin followed by dissection and removal of gut content. The gut content is to be crushed, diluted, and plated on suitable isolation media for the isolation of actinobacterial colonies [8].

6.2

Materials

l

Fish sample.

l

Dissection tools.

l

Seawater blanks.

l

Isolation agar plates (chitin agar, starch casein nitrate agar, Kuster’s agar).

l

Mortar and pestle.

16

6.3

Sample Collection, Isolation, and Diversity of Actinobacteria

Methods

l

70% ethanol.

l

Collect the fish samples of your interest from the local coastal area, and transport immediately to the laboratory in ice box, or store at 20
C until further study.

l

Confirm their taxonomy with the help of an aquaculturist or marine biologist.

l

Treat the surfaces of all instruments, surfaces, and the exterior of each fish with 70% EtOH.

l

Flame sterilizes all the dissecting tools prior to dissection.

l

Make an incision on the ventral side of each fish at the anus, and extend anteriorly to the isthmus.

l

Make the second cut dorsally through the operculum.

l

Separate the stomach, pyloric caeca, and intestines from the body cavity, and sterilize with 70% EtOH.

l

Transfer the contents of the digestive tract to a sterile 10 ml Falcon tube at discrete portions of the gut, the posterior portion of the intestine, the mid-intestine, the stomach, and the pyloric caeca, as appropriate for each specimen.

l

Transfer the fish intestine contents to sterile Falcon tubes, and add 1 ml of sterile Milli-Q water.

l

Vortex the samples for 1 min.

l

Prepare different agar media plates such as actinomycete isolation agar, starch casein agar, Kuster’s agar, and glucose asparagine agar using sterile seawater supplementing with filtersterilized cycloheximide (50 μg/ml) and nalidixic acid (20 μg/ ml).

6.3.1 Sample Collection and Processing

6.3.2 Isolation of Fish Gut-Associated Actinobacteria

l

l

l

Serially dilute the sample using sterile seawater blank up to 106 dilutions. Transfer 100 μl of aliquot from all the dilutions onto isolation agar plates, and spread using sterile L-rod. Incubate all the plates at 25–30
C for 1–3 months.

l

During incubation, recover morphologically different actinobacterial colonies, and purify using ISP2 or oatmeal agar plates.

l

Dereplicate morphologically similar isolates by cultural and micromorphological features, and select different actinobacterial cultures.

l

Preserve all the actinobacterial cultures that will be stored in ISP2 agar slants as well as in 15% glycerol stock.

Total number of actinobacterial population in the given sample (see Table 5) calculated using following formula:

Isolation of Actinobacteria from Insect Gut

17

Table 5 Dilution factor and total number of actinobacterial colonies Dilution factor 10

Number of actinobacterial colonies

1

102 103 104 105 106

CFU ¼

No: of colonies counted  Dilution f actor Volume of sample ðorÞ inoculum

The total actinobacterial population present in the given plant sample is estimated as ____ CFU/g of fish gut sample. 6.4

7 7.1

Notes

When working for isolation of fish gut-associated actinobacteria, perform all the sample processing, isolation, and recovery from the place doing all the actinobacterial work in order the avoid crosscontamination of isolation agar plates by other actinobacterial cultures.

Isolation of Actinobacteria from Insect Gut Introduction

Insect digestive tracts support communities of symbiotic and transient microorganisms that are increasingly the subjects of studies of microbial diversity and novel bioactive microbial products. In general, insect gut microbiota make significant contributions to the nutrition of the insect host, as demonstrated in well-studied examples such as termites, cockroaches, wood-feeding beetles, and aphids. With the advancement of new sequencing methods, gut microbial communities have been analyzed in an even wider range of insects. Associations with bacteria can play important roles in the ability of insects to adapt to novel environments and food sources. These roles include protection from pathogens, parasites, and predators. Many of these interactions involve Actinobacteria, a phylum known to produce a diversity of antimicrobial secondary metabolites. Numerous studies to date have investigated antimicrobial activity of Actinobacteria isolated from a variety of insects, including termites, for the purpose of discovering novel antimicrobials.

18

Sample Collection, Isolation, and Diversity of Actinobacteria

Actinobacteria are consistently identified in culture-independent and culture-dependent studies of both higher and lower termite lineages. Usually the insect samples were slightly frozen and surface sterilized using 70% ethanol to avoid surface contamination. The dissected gut content is homogenized and plated on suitable isolation agar media which are made up of complex substrates such as chitin pectin, cellulose, or starch [9]. 7.2

7.3

Materials

Methods

l

Insect samples.

l

Dissecting tools.

l

Starch casein agar, cellulose agar and chitin agar.

l

Water blank.

l

70% ethanol.

l

Sterile saline.

l

Collect the insect like termite, honeybee, or cockroach from different locations.

l

Within 12 h of capture, externally sterilize the insects with 70–100% alcohol, and dissect under sterile conditions.

l

Remove the digestive tract and pool from crop to the rectum.

l

Homogenize and suspend the whole digestive tract in saline.

l

Serially dilute the processed sample using sterile saline up to 106 dilutions.

l

Prepare chitin agar, starch casein agar, and cellulose agar supplementing with filter-sterilized cycloheximide (100 μg/ml) and nalidixic acid (20 μg/ml).

l

l

Transfer 100 μl of aliquot from each dilution onto all the isolation agar plates. Incubate all the plates at 28–30
C for 2–6 weeks.

l

After incubation, select the filamentous bacterial colonies that appeared powdery, fuzzy, or leathery and nonfilamentous pigmented colonies.

l

Purify the actinobacterial colonies using YEME agar or chitin agar medium, and preserve at 80
C using 20% (v/v) glycerol.

Total number of actinobacterial population in the given sample (see Table 6) calculated using following formula: CFU ¼

No: of colonies counted  Dilution f actor Volume of sample ðorÞ inoculum

Recovery and Purification of Actinobacteria

19

Table 6 Dilution factor and total number of actinobacterial colonies Dilution factor 10

Number of actinobacterial colonies

1

102 103 104 105 106

The total actinobacterial population present in the given insect gut sample is estimated as ____ CFU/g of insect gut sample. 7.4

8

Notes

Like fish gut-associated actinobacteria, when working for isolation of insect gut-associated actinobacteria, perform all the sample processing, isolation, and recovery from the place doing all the actinobacterial work in order the avoid cross-contamination of isolation agar plates by other actinobacterial cultures.

Recovery and Purification of Actinobacteria

8.1

Introduction

Recovery of pure actinobacterial colonies from the isolation agar plates containing mixture of bacterial and fungal colonies is a cumbersome process. Certain actinobacterial genera like Streptomyces which produce powdery colonies are easy to recover, whereas other genera which produce leathery colonies with submerged substrate mycelium are very difficult to recover. To avoid touching of adjacent colonies, usually an L-shaped needle is used for the recovery of actinobacterial colonies. While recovering the rough leathery colonies, the inoculation needle needs to be heated and inserted into the agar medium in order to melt the agar medium which facilitates the recovery of substrate mycelium from the agar medium [1]. Then the mycelium along with the agar portion was transferred and spread onto the agar plates. The recovery plates should be carefully observed during incubation in order to remove the contaminants or further subculture the uncontaminated portion of the actinobacterial colonies [2].

8.2

Materials

l

Starch casein agar plates with actinobacterial colonies.

l

Sterile L loop.

l

ISP2 agar or oatmeal agar.

l

Incubator—28
C.

20

8.3

Sample Collection, Isolation, and Diversity of Actinobacteria

Methods

l

Prepare ISP2 agar plates and allow to dry inside the laminar air flow cabinet.

l

Take starch casein agar plates with actinobacterial colonies.

l

Open the plate near the flame inside the cabinet.

l

Take an L-shaped inoculation needle and sterilize by direct heat using the flame.

l

Before recovery, mark the colonies with suspected actinobacterial morphology on the reverse side of the SCA plate using glass marker.

l

Gently take the spores of actinobacterial colonies from SCA plates using sterile L-shaped needle.

l

Take the ISP2 agar plate and open inside the cabinet near the flame.

l

Inoculate the actinobacterial spores on ISP2 agar plates, and spread well to form a mother inoculum.

l

Then sterilize the L-shaped needle, and spread the mother inoculum on one direction as a straight line or continuous lines.

l

Spread the culture on four sides by streaking as mentioned above.

l

8.4

9 9.1

Notes

Incubate all the plates at 28
C for 7 - 14 days.

l

Observe all the plates for actinobacteria growth without any mixed growth.

l

If needed (in case any mixed growth observed/suspected), again recover the actinobacterial cultures from primary isolation plate and subculture on fresh ISP2 agar plates by phase streaking as mentioned above (see Table 7).

l

Experienced eyes have greater value in the selection of actinobacterial colonies.

l

Care should be taken while taking the actinobacterial colonies from SCA plates to avoid the mixing of nearby colonies.

l

Perform the recovery in duplicate to avoid loss of colonies due to contamination and increase the recovery rate.

Metagenomic Analysis of Uncultured Actinobacterial Diversity Introduction

Metagenomics is a culture-independent technique that strives to collect and analyze the complete genomes contained in a given environmental sample. It’s defined as the direct genetic analysis of genomes contained with an environmental sample. Metagenomics is a rapidly growing field of research that has had a dramatic effect on the way we view and study the microbial world. This new field of

Metagenomic Analysis of Uncultured Actinobacterial Diversity

21

Table 7 Actinobacteria colonies from pure/mixed cultures S. no.

Actinobacterial colony no.

Pure/mixed culture

1 2

biology has proven to be rich and comprehensive and is making important contributions in many areas including ecology, biodiversity, bioremediation, bioprospection of natural products, and in medicine [10]. Metagenomics provides access to the functional gene composition of microbial communities and thus gives a much broader description than phylogenetic surveys, which are often based only on the diversity of one gene, for instance the 16S rRNA gene. On its own, metagenomics gives genetic information on potentially novel biocatalysts or enzymes, genomic linkages between function and phylogeny for uncultured organisms, and evolutionary profiles of community function and structure. The rapid and substantial cost reduction in next-generation sequencing has dramatically accelerated the development of sequence-based metagenomics. In the future, metagenomics will be used in the same manner as 16S rRNA gene fingerprinting methods to describe microbial community profiles. It will therefore become a standard tool for many laboratories and scientists working in the field of microbial ecology [10]. 9.2

9.3

Materials

Methods

l

Soil sample.

l

Skimmed milk solution (0.4% w/v).

l

SDS extraction buffer (0.3% SDS in 0.14 M NaCl, 50 mM sodium acetate, pH 5.1).

l

Tris saturated phenol.

l

Isopropanol.

l

70% Ethanol.

l

Primers.

l

Sample processing is the first and most crucial step in any metagenomics project.

l

Collect the samples from different extreme environments placed over sterile container.

l

Store the sample in a cooler box with ice packs, and transport aseptically to the laboratory for further processing.

9.3.1 Sample Collection

22

Sample Collection, Isolation, and Diversity of Actinobacteria

9.3.2 Soil DNA Isolation

l

l

To the supernatant, add 2 ml of SDS extraction buffer (0.3% SDS in 0.14 M NaCl, 50 mM sodium acetate, pH 5.1), and mix well by vortexing.

l

Add equal volume of Tris-saturated phenol and vortex again for 2 min.

l

l

l l

Centrifuge at 12,000  g for 10 min and collect the supernatant. Precipitate the DNA from the supernatant by adding equal volume of ice-cold isopropanol at 20
C for 1 h. Recover the DNA pellet by centrifugation at 12,000  g for 10 min. Wash the pellet twice with 70% ethanol and air-dry. Dissolve the DNA pellet in sterile water and store at 20
C deep freezer.

l

Amplify the actinobacterial 16S rRNA gene V3 and V4 regions using primers containing Illumina adapters following Illumina’s 16S metagenomics protocol.

l

Briefly, use Kapa Library Amplification Kit for PCR, and clean the products using Beckman Coulter Agencourt AMPure XP Beads according to the 16S Metagenomics protocol.

l

l

l

9.3.4 Bioinformatics and Statistical Analysis

Vortex the soil solution well and centrifuge at 12,000  g for 10 min.

l

l

9.3.3 Amplification and Sequencing

Ground 1 g of soil to fine powder under aseptic conditions, and suspend in 0.4% w/v solution of skimmed milk.

Use the actinobacterial-specific primer ACT283F 50 -GGGTAG CCGGCCUGAGAGGG-30 and ACT1360R 50 -CTGATCTG CGATTACTAGCGACTCC-30 to create a single amplicon of approximately 250 bp. Adjust the concentrations to 4 μM, and prepare for loading on the Illumina MiSeq according to Illumina’s 16S metagenomics protocol. Pool and denature the sample, and load on the Illumina MiSeq at 8pM, and sequence paired end (2  120 300) using a MiSeq® Reagent Kit v3. 1. Use QIIME 1.9.1 pipeline for the entire downstream analysis. 2. Perform the quality check using FastQC0.11.7, and PHRED score reads with >Q30 to consider for further analysis. 3. Adaptor trim the high-quality reads, and stitch the paired-end reads using FLASH 1.2.11 to make consensus FASTA sequences.

References

23

Table 8 Base pair length of sample

V3 –V4 region Sample name Numbers

Total length (bp)

Max. length (bp)

Min. length (bp)

4. Form the consensus reads with 0 mismatch having an average contig length of 350–450 bp and queried to UCHIME to remove all the chimeric sequence which was subsequently pooled and clustered into operational taxonomic units (OTUs) based on their sequence similarity using UCLUST. 5. Identify the representative sequence for each OTU against SILVA OTUs database using PyNAST. 6. Analyze the microbial diversity within the samples by calculating Shannon, Chao1, and observed species metrics. 7. The Chao1 metric estimates the species richness, while Shannon metric is the measure to estimate observed OTU abundances and accounts for both richness and evenness. 8. The species metric to be observed is the count of unique OTUs identified in the sample (see Table 8).

References 1. Balagurunathan R, Radhakrishnan M (2007) Actinomycetes diversity and their importance. In: Trivedi PC (ed) Microbiology–applications and current trends. Pointer Publishers, Jaipur, India, pp 297–329 2. Radhakrishnan M, Suganya S, Balagurunathan R, Kumar V (2010) Preliminary screening for antibacterial and antimycobacterial activity of actinomycetes from less explored ecosystems. World J Microbiol Biotechnol 26:561–566 3. Radhakrishnan M, Pazhanimurugan R, Gopikrishnan V et al (2014) Streptomyces sp D25 isolated from Thar Desert soil, Rajasthan producing pigmented antituberculosis compound only in solid culture. J Pure Appl Microbiol 8 (1):333–337

4. Radhakrishnan M, Balagurunathan R, Sakthivel M (2013) Antibacterial substance from actinobacterial strain TA4 isolated from Western Ghats, India. Indian J Appl Microbiol 16 (2):9–16 5. Radhakrishnan M, Deeparani K, Balagurunathan R (2006) Production of bioactive compounds by solid state fermentation from actinomycetes of Andaman sediments. Indian J Appl Microbiol 6(1):92–98 6. Balagurunathan R, Radhakrishnan M, Somasundaram ST (2010) L-glutaminase producing actinomycetes from marine sediments–selective isolation, semi quantitative assay and characterization of potential strain. Aust J Basic Appl Sci 4(5):698–705

24

Sample Collection, Isolation, and Diversity of Actinobacteria

7. Chen P, Zhang C, Ju X, Xiong Y, Xing K and Qin S (2019) Community composition and metabolic potential of endophytic actinobacteria from coastal salt marsh plants in Jiangsu, China. Front Microbiol 10:1063 8. Egerton S, Culloty S, Whooley J, Stanton C, Ross RP (2018) The gut microbiota of marine fish. Front Microbiol 48(8):1280–1285

9. Arango RA, Carlson CM, Currie CR, et al (2016) Antimicrobial activity of actinobacteria isolated from the guts of subterranean termites. Environ Entomol 45(6):1415–1423 10. Thomas T, Gilbert J, Meyer F (2014) Metagenomics–a guide from sampling to data analysis. Microb Inf Exp 2:3

Chapter 2 Dereplication and Ex Situ Conservation of Actinobacteria Abstract Actinobacteria are morphologically diverse which can be easily differentiated based on the micromorphological differences as well as their ability to produce morphologically different colonies. Notably several actinobacterial genera produce different colors of pigments either extracellular or intracellular in nature. However, the differentiation and selection of actinobacterial colonies based only on their microscopic and cultural features are inadequate for differentiating several actinobacterial genera which includes the non-sporulating, unicellular actinobacteria like Arthrobacter and Modestobacter and monosporulating species like Micromonospora species which produce similar micro- and colony morphology. In such situations, either chemotaxonomic like FT-IR-based whole cell fingerprinting and/or molecular-based methods like RFLP, RAPD, or 16s rRNA analysis will be used for dereplicating the actinobacterial colonies. Dereplication of actinobacterial colonies by any means will be highly needed for the further selection and preservation of distinct actinobacterial cultures for taxonomic and bioprospecting research. There are several methods in practice for the ex situ preservation of actinobacterial cultures. However, each method has its own merits and limitations. Lyophilization remains as the best method for long-term preservation of actinobacterial cultures. In this chapter, protocols for actinobacterial dereplication and preservation are discussed. Keywords Dereplication, Micromorphology, Cultural, FT-IR, RFLP , RAPD , Preservation, Lyophilization

1 Dereplication of Actinobacterial Cultures Based on Color Grouping and Micromorphology 1.1

Introduction

Members of the actinobacterial phylum are able to produce different pigments either intracellularly in the mycelium and spores or secrete extracellularly into its surrounding medium. For several decades, actinobacterial forms especially members of the genus Streptomyces are selected and differentiated based on their aerial mycelia color. When we are growing actinobacterial cultures on suitable medium for a specified incubation time, they produce colonies with different aerial mass color as well as different reverse side substrate mycelia with different reverse side color [1]. This will be enabling us to discard the similar isolates and select the different ones. Further, nutritional composition of the culture medium and incubation conditions will also influence the pigment production by the actinobacterial cultures. Yeast extract malt extract (ISP2)

Ramasamy Balagurunathan et al., Protocols in Actinobacterial Research, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0728-2_2, © Springer Science+Business Media, LLC, part of Springer Nature 2020

25

26

Dereplication and Ex Situ Conservation of Actinobacteria

medium is very commonly used to study the morphological features of actinobacteria in particular the Streptomyces. In addition, using other media like oatmeal agar, glycerol asparagine agar, etc. will enable us to get the different morphological or pigment-producing actinobacterial cultures. In addition to mycelial color, observation of structure and arrangement of mycelia and spores will also be useful for differentiating actinobacterial cultures. Usually the culture plates are directly observed under bright-field microscope by inverted position to see the mycelia structure and arrangements of actinobacterial colonies [2]. 1.2

1.3

Materials

Methods

l

Actinobacterial cultures.

l

ISP2 agar plates.

l

Bright-field microscope.

l

Prepare ISP2 agar plates as thin layer preferably using disposable plastic petri plates.

l

Inoculate the actinobacterial cultures on ISP2 agar plates using sterile loop under aseptic condition.

l

1.4

Notes

Incubate the plates at 28
C for 7–14 days.

l

Visually observe the cultures grown on ISP2 agar plates for every 24 h to visualize, and record the following morphological properties: growth, colony consistency, aerial mycelia color, reverse side pigment, soluble pigment production, and any other special features (see Table 1).

l

Observe all the cultures grown on ISP2 agar surfaces under bright-field microscope by keeping the plates under inverted position.

l

Record the presence of mycelium, length, arrangement, fragmentation, etc.

l

Tabulate all the cultural and microscopic morphology of the actinobacterial cultures (see Table 2).

l

Discard actinobacterial cultures showing similar morphological features, and select the different cultures for preservation.

l

Actinobacterial cultures showing similar colors will be grouped together.

l

The similarities and differences among the cultures present in each color group will be observed.

l

Actinobacterial cultures will be dereplicated by discarding similar cultures and selecting different ones from each color group.

Dereplication of Actinobacterial Cultures Based on FT-IR Analysis

27

Table 1 Cultural morphology

S. no

Strain no

Growth

Consistency

Aerial mass color

Reverse side pigment

Soluble pigment

Table 2 Micromorphology

S. no

2 2.1

Strain no

Aerial

Substrate

Spore chain

Mycelia

mycelium

mycelium

morphology

fragment

Any other structure

Dereplication of Actinobacterial Cultures Based on FT-IR Analysis Introduction

Nowadays, several physicochemical whole-organism fingerprinting techniques are in practice to provide rapid and highly reproducible discrimination of bacteria including actinobacteria. Recently, FT-IR has been used as a nondestructive spectroscopic approach, in combination with other methods, for whole-organism fingerprinting. This technique is based on the absorption of IR light directed onto a sample. The amount of light absorbed depends on the molecules found within the sample [3]. It measures dominantly vibrations of the functional groups and highly polar bonds. Therefore, it gives a lot of information about the total biochemical composition of a sample regarding the molecule composition, structure, and interactions. Established reflection-based sampling protocol has the major advantages that it is nondestructive, reproducible, and is very rapid technique both for single sample (1–10 s is typical) and the automated high throughput of samples in batches of 96 or 384. Thus, FT-IR can be used as a rapid dereplication technique to differentiate microbial strains [4].

28

2.2

2.3

Dereplication and Ex Situ Conservation of Actinobacteria

Materials

Methods

l

Actinobacterial cultures.

l

ISP2 agar plates.

l

FT IR spectroscopy.

l

Sterile nylon membrane.

l

0.9% NaCl solution.

l

Prepare ISP2 agar plates and aseptically place the sterile nylon membrane on the top.

l

Inoculate the actinobacterial cultures over the top of nylon membrane.

l l

l

Notes

Take the actinobacterial biomass containing nylon membrane, and put it in sterile 0.9% NaCl solution, and subsequently homogenize to disrupt the mycelia lump. Load 10 μL of each sample into the 96-well aluminum IR plate, and dry at 50
C for 30 min.

l

Run six biological replicates, i.e., each isolate cultivates six times, and three machine replicates, i.e., analyze each of the biological replicate for three times by FT IR.

l

Run the sample equipped with mercury–cadmium–telluride (MCT) detector cooled with liquid N2 to collect the FT-IR spectra.

l

Collect FT-IR spectra over the wave number ranging 4000–600 cm1.

l

2.4

Incubate the plates at 28
C for 7–14 days.

Acquire the spectra at a rate of 10 s1 with the spectral resolution of 4 cm1. Co-add 256 scans to improve the signal noise ratio, and take average.

l

Collect all the reflectance mode and display in terms of absorbance as calculated from the reflectance–absorbance spectra using OPU.

l

Compare the FT IR spectra of all the actinobacterial cultures, and select the cultures showing different FT IR spectra (see Table 3).

l

Ranges of wave numbers can be associated with special chemical bonds.

l

l

The wave numbers 3050 to 2800 cm1 is the so-called fatty acid region, where peaks show the vibration of CH2 and CH3 groups of fatty acids. The amide section, 1750 to 1500 cm1, is where protein and peptide bonds dominate.

Dereplication of Actinobacterial Cultures by RFLP Analysis

29

Table 3 Wave number and indication

Wave number

l

l l

3

Indication for

The range from 1500 to 1200 cm1 is a mixed region containing vibrations of fatty acids, proteins, and polysaccharide. Polysaccharide dominates the region from 1200 to 900 cm1. The fingerprint region range is from 900 to 700 cm1. This contains bonds which are most characteristic at the species level, but only a few peaks can be assigned to the vibrations of special substances.

Dereplication of Actinobacterial Cultures by RFLP Analysis

3.1

Introduction

A number of nucleic acid fingerprinting studies have been designed to separate closely related species and strains of the genus Streptomyces. Strains belonging to different Streptomyces species were subjected to restriction endonuclease digestion of genomic DNA to determine the relationships between them. Since the generation of complex fingerprints contained a large number of low-molecularweight fragments, it did not provide sufficient resolution for determining taxonomic relationships at species level. In previous studies, there are considerable variations in RFLP patterns of representatives of various Streptomyces species been observed which evidenced that RFLP analysis of ribosomal RNA genes appeared to be an accurate and rapid strain identification tool for establishing relationships between closely related Streptomyces species. This method was considered to be useful for discriminating between Streptomycetes at strain level [5].

3.2

Materials

l

Actinobacterial cultures.

l

DNA isolation kit.

l

PCR.

l

Restriction enzymes.

l

Agarose gel.

l

Gel electrophoresis.

l

Buffer.

30

3.3

Dereplication and Ex Situ Conservation of Actinobacteria

Methods

3.3.1 Cultivation and DNA Extraction

3.3.2 PCR Amplification and RFLP Analysis

l

Grow the actinobacterial cultures on non-sporulation agar plates for 3 days at 28
C.

l

Prepare the solutions to be used in this experiment from dilution of stock solutions of the main reagents.

l

Prepare the stock solutions according to standard procedures.

l

Prepare the genomic DNA from strains of Streptomyces according to the guanidine thiocyanate extraction method.

l

Study the PCR amplifications of the interspacer region of 16S– 23S rDNA in a Perkin Elmer DNA Thermal Cycler 480 using 0.5 ml PCR microfuge tubes.

l

Optain the Taq DNA polymerase, MgCl2 solution, Taq buffer and Deoxyribonucleotides (dNTPs) at a concentration of 100 mM.

l

Prepare the working stock solution of dNTPs by mixing in a master stock in equimolar ratio to produce the final concentration of individual dNTP at 25 mM.

l

For PCR amplification, use the forward primer GP1 (50 -GCGAT TGGGACGAAGTCG-30 ) and reverse primer GP2 (50 -TATCG TGGC CTCCCACGTCC-30 ).

l

Mix the reagents by vortexing, and collect at the bottom of the tubes by a short-pulse centrifugation (5 s).

l

Keep the tubes in ice until they are placed in the thermocycler.

l

After the addition of the DNA sample, place the reaction mixture in the thermocycler block, and heat at 96
C for 5 min in an initial denaturation, and then add 0.5 μl of Taq polymerase.

l

Perform the PCR amplification according to the following temperature profile: an initial denaturation at 95
C for 5 min, 35 cycles of denaturation (1 min at 95
C), annealing (1 min at 55
C), extension (1 min at 72
C), and final extension at 72
C for 10 min.

l

l

Keep the PCR products at 4
C, and then check by agarose electrophoresis (1%, w/v in 0.5TBE) on 0.5TBE running buffer, containing 0.5 μg/ml ethidium bromide. Mix approximately 5 μl of the PCR reaction with 1 μl of gel-loading buffer, and load into wells in the agarose gel slab.

l

Run the electrophoresis at 100 V for 1 h, and identify the size of amplified fragments (interspacer region of 16S and 23S rDNA) by comparison with 100 bp molecular-size marker at the position of 500-bp.

l

Separate the PCR-amplified 16S rDNA by preparative agarose electrophoresis.

l

After separation, elute it and purify using the agarose gel by a kit.

Dereplication of Actinobacterial Cultures by Random Amplified Polymorphic. . . 3.3.3 Digestion with the Restriction Enzyme

31

l

Digest the PCR products (8.5 ml) with three restriction endonucleases, Bsp143I, Hae III, and MnlI, at 37
C for 3 h.

l

Analyze the digested DNA by horizontal electrophoresis in 4% NuSieve 3:1 agarose gel.

l

Run the electrophoresis at 100 V for 220 min with a gel electrophoresis apparatus in TBE buffer.

l

Use the super ladder-low 20-bp ladder DNA marker. After electrophoresis, analyze the scanning images of the gel, and estimate the fragment size under UV light, and document on a BioRad illuminator connected to a computer program called the Gel Compare Program. Do not consider the restriction fragments shorter than 99 bp in the analysis.

l

Transfer the restriction patterns of each enzyme for whole test strains as TIF files for analyzing in the Molecular Analysts Program.

l

Score the informative bands derived from restriction enzyme digestion by their presence or absence and similarity, and calculate the divergence.

l

Construct a similarity percentage distance matrix and dendrogram. Use unweighed pair group method using arithmetic averages (UPGMA; 16) to construct the dendrogram.

4 Dereplication of Actinobacterial Cultures by Random Amplified Polymorphic DNA: Polymerase Chain Reaction (RAPD-PCR) Analysis 4.1

Introduction

A large number of actinobacterial strains isolated and selected empirically by taxonomists have been subjected to microbial screenings. For improving the efficiency of screenings, we need a method that could eliminate strains, particularly similar ones isolated from different samples. The random amplified polymorphic DNA (RAPD) method is a DNA polymorphism analysis system based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. These primers detect polymorphisms in the absence of specific nucleotide sequence information. Genomic fingerprinting assays using random amplified polymorphic DNA (RAPD) have already been shown to be useful for the differentiation of bacterial strains. This method is based on the amplification of distinct genomic DNA sequences under low-stringency conditions during annealing using an oligonucleotide of arbitrary sequence. The primer is not directed at any specific sequence within the template, making previous knowledge of the genome nonessential. The efficacy of the amplification procedure is primarily dependent on sufficient sequence similarity at the 30 end of the oligonucleotides to allow adequate priming. The resulting

32

Dereplication and Ex Situ Conservation of Actinobacteria

pattern of amplification products of varying size can be subsequently used as a genetic fingerprint of the organisms used in the analysis [6]. 4.2

4.3

Materials

Methods

4.3.1 DNA Isolation (CTAB Method)

l

Actinobacterial cultures.

l

Restriction enzymes.

l

PCR.

l

Reaction mixture.

l

Agarose gel electrophoresis.

l

Lysis buffer.

l

5 M NaCl.

l

10% SDS.

l

5 M Potassium acetate.

l

Isopropanol.

l

50 mM Tris

l

10 mM EDTA (pH 8).

l

3 M sodium acetate.

l

70% Ethanol.

l

TE buffer.

l

Grow the actinobacterial culture to the late exponential phase in TSB at 28
C.

l

Resuspend 0.5–1.0 g of cells in 5 ml lysis buffer (25 mM Tris; 25 mM EDTA (pH 8.0); 10–15 mg lysozyme; 50 pg/ml RNaseA), and incubate for 30–80 min at 37
C.

l

Following the addition of 1 ml of 5 M NaCl solution; agitate the suspension on a vortex mixer until the cell suspension becomes translucent.

l

Lyse the cells by the addition of 1.2 ml of 10% SDS and incubate for 15–30 min at 65
C.

l

Add 2.4 ml of 5 M potassium acetate, and mix the solution by vortexing, and keep it on ice for a minimum of 20 min.

l

Remove the precipitate by centrifugation for 30 min at 4000  g (or 10 min at 16,000  g), and adjust the volume of the supernatant to 8 ml.

l

Recover the DNA by precipitation with isopropanol. Dissolve the precipitate in 700 μl of 50 mM Tris/10 mM EDTA (pH 8.0).

l

Spin off any insoluble substances, and transfer the aqueous phase to a 1.5 ml microfuge tube.

Ex Situ Conservation of Actinobacteria l

l

RAPD PCR

5

l

33

Subsequently, add 75 μl 3 M sodium acetate and 500 μl isopropanol, and centrifuge the solution for 30 s to 2 min. Wash the precipitate with cold 70% ethanol; dry and dissolve in 100 μl TE (10 mM Tris/1 mM EDTA, pH 8.0) or distilled water. Perform the amplification reaction with the final volume of 25 μl with the following conditions: 94
C for 4 min as a primary denaturation, 40 cycles of 94
C for 50 s, 40
C for 50 s, 72
C for 1 min, and final extension for 10 min at 72
C.

l

Run the electrophoresis of PCR products on 1.5% agarose gel with molecular marker of 1 kb DNA ladder.

l

After RAPD-PCR, analyze the polymorphic DNA band patterns, and calculate the genetic relationships among the Streptomyces isolates using NTSYS-PCTM.

l

Calculate the dendrogram and distance matrix by a UPGMA (Unweight Pair-Group Method by Arithmetic Average) program in order to determine genetic relationships among the isolates.

Ex Situ Conservation of Actinobacteria

5.1

Introduction

Microbial consortia, especially those which are useful to produce high-value products or process, are generally considered knowledge hubs for the life sciences and underpin biotech industries. Exploring and conserving industrially important microbes like actinobacteria from unexplored ecosystems are worth pursuing which results in the isolation of novel taxa and metabolites. Ex situ conservation of useful microorganisms avoids the need for costly and timeconsuming re-isolation protocol. Good infrastructure and expertise in long-term preservation, characterization, and identification of diverse group of microorganisms are necessary for good culture collection. Microbial culture collections for their ex situ conservation are established in many countries around the world having a variety of purposes. These range from small specialized collections that support a small group of researchers to the large international public service collections that provide reference materials and service to the scientific community and bioindustries. The selection of actinobacterial preservation method depends on the type of genera, duration of preservation, and purpose of storage [7].

5.2

Materials

l

ISP2 agar slants.

l

Oatmeal agar.

l

Actinobacterial cultures.

34

5.3

Dereplication and Ex Situ Conservation of Actinobacteria

Methods

5.3.1 Preservation Using Slant Cultures

l

Soil.

l

Paraffin tape.

l

20% Glycerol.

l

Cryo vials.

l

Nutrient agar.

l

SDA.

l

Lyophilization medium [10% Skim milk (or) 10% sucrose].

l

Microbial freeze-drying medium.

l

Prepare ISP2 agar slants.

l

Inoculate the spores of each actinobacterial culture under aseptic condition onto three ISP2 agar slants.

l

5.3.2 Preservation Using Glycerol Stock

l

Seal the cotton plug of the ISP2 agar slants using paraffin tape or melted liquid paraffin.

l

Preserve the slants under refrigerated conditions.

l

Prepare oatmeal agar plates.

l

Inoculate the spores of actinobacterial culture on to oatmeal agar plates.

l l

l l

l

5.3.3 Preservation Using Soil Stock

Incubate all the slants at 28
C for 7–14 days.

Incubate the culture plates at 10–14 days at 28
C. Prepare 20% glycerol broth and aliquot each 2 ml in 5 ml cryo vial or screw-capped bottle. Sterilize the glycerol broth by autoclaving at 121
C for 15 min. Transfer the spores of each actinobacterial cultures from oatmeal agar plates into three to five bottles or vials containing 20% glycerol broth. Store the glycerol stock vials at 80
C in deep freezer.

l

Collect the soil sample from where the actinobacteria is to be isolated.

l

Keep the sample for 3–5 days at room temperature to completely dry.

l

Divide the sample into aliquots, and sterilize intermittently by autoclaving at 121
C for 30 min for 3 consecutive days.

l

Check the sterility by streaking the soil sample on nutrient agar, SDA, and ISP2 agar plates.

l

Aliquot 1–2 g of sterile soil sample on to 5 ml size presterilized screw-capped vials.

Ex Situ Conservation of Actinobacteria l

Transfer each 1 ml of actinobacterial culture on to each three vials of sterile soil, and mix well.

l

Incubate the soil samples at room temperature.

l

Periodically take 100 mg of soil sample vials, and serially dilute and spread on ISP2, SDA, and nutrient agar plates.

l

5.3.4 Preservation by Lyophilization

Incubate the plates at 28
C for 7–14 days to check for viability, purity, and contamination.

l

Preserve the soil stock vials at room temperature.

l

Prepare each 50 ml or required quantity of ISP2 broth, and sterilize by autoclaving at 121
C for 15 min.

l

Inoculate the spores of actinobacterial culture on the above medium aseptically.

l

Incubate all the flasks in rotary shaker with 95 rpm for 5 days at 28
C.

Culturing of Actinobacteria

l

Collect the cells by centrifugation at 2700  g for 10 min.

l

Suspend the pellet in equal volume of lyophilization medium.

l

For agar cultures, flood the plate/tube with 5–10 ml of lyophilization medium.

l

Using a sterile pipette, flush the medium over the colonies to dislodge the cells.

l

Transfer the cell suspension to a sterile tube.

l

Freeze-Drying Process: Shelf Lyophilizer

35

Aliquot 250–500 μl of the cell suspension into sterile vials or tubes.

l

Place split stoppers on the vials or loosely plug tubes with glass wool.

l

If a vial contains upward of a billion bacteria, one or two contaminating microbes become insignificant when that culture is rehydrated and streaked. If there is fear of contamination, then glass wool or cotton can be placed under the stopper to prevent contamination.

l

Turn on the lyophilizer and start the condenser. If there is an external condenser using a dry ice/ethanol mixture, then prepare this as well. The shelf can be set to 4
C.

l l

Set the shelf to 4
C and center the vials on the shelf. Using either manual or programmed controls, freeze the samples down to 40
C. This step should take approximately 30–60 min and is very dependent upon the instrument. If the rate of freezing can be controlled, then a drop of 1
C/min is a practical rate. Once the samples reach temperature, they should

36

Dereplication and Ex Situ Conservation of Actinobacteria

be visibly frozen (clear liquid turns opaque and skim milk appears solid). l

5.4

Notes

Allow the sample to sit at 40
C for 1 h to ensure complete freezing. Vials at the center of a cluster may freeze more slowly than those on the outside.

l

Turn on the vacuum pump. Within 10–20 min, the vacuum should be under 200 millitorr (mtorr).

l

Once the vacuum is below 200 mtorr, increase the temperature of the shelf for primary drying.

l

Primary drying is the longest phase of the freeze-drying process. The idea is to keep the sample colder than condenser (or ice trap) but still sufficiently warm so that water sublimes rapidly. The temperature of the shelf can be raised to above the melting temperature as long as the sublimation process removes the heat flowing into the sample sufficiently fast to prevent melting and sample collapse.

l

Samples still contain moisture following primary drying. The amount is debatable, but it is somewhere between 2 and 4%. This moisture level needs to be reduced, and that is done by pumping heat into the sample during the secondary drying phase. This phase is relatively short, lasting 1–2 h, but important for long-term viability. However, overdrying of the bacteria can be detrimental as well. Once again, based on the idiosyncrasies of your lyophilizer and samples, the ideal time for secondary drying needs to be determined experimentally. Generally, raise the shelf temperature to 20
C and dry for 2 h.

l

With the vacuum in place, stopper the vials using the stoppering plate/mechanism. Release the vacuum, remove the vials, and further secure the rubber bungs/stoppers with foil crimp seals. It is best to store the vials at 4
C in the dark. 1. Some authors used glycerol broth with 25–50% glycerol concentration. 2. Glycerol broth may be prepared in same media used for cultivation like YEME broth instead of using distilled water alone. 3. Depending on the instrument, pressure may be reported in mtorr, mbar, Pascal, or “inches Hg” on a vacuum gauge. For reference, 100 mtorr = 0.133 mbar = 13.3 Pascal = 29.9” Hg = 0.000132 atm = 99.99% vacuum. 4. The temperature of the shelf is dependent upon the lyophilization medium. For sucrose, keep the shelf temperature at 25
C. For Reagent 18 or Microbial Freeze-Drying Buffer, the shelf can be as high as 15
C. In any case, the greater the difference in temperature between the shelf and the

References

37

condenser/ice trap, more efficient the primary drying process will be. 5. The time for primary drying will also depend upon the volume of the sample. For bacteria, samples rarely need to be large and typically are 0.25–0.5 ml. A limited number of samples (10–20) in a shelf dryer can be completed in just a couple of hours. A fully loaded dryer with several hundred samples will take longer. Safely, a primary drying period which is overnight should work, but test this first before you attempt to freeze-dry large numbers of vials. As a standard guide, freeze-dry overnight. 6. Test the freeze-dried cells for viability as compared to the original culture. Additionally, monitor the stability/viability of the freeze-dried cultures by testing at 30, 90, 180, and 365 days. A good protocol will yield nearly 100% viable cells. Anything above 50% is considered acceptable by many labs. Skim milk will yield 10–20%. However, % viable after freezedrying is not as important as the number viable following storage.

References 1. Axenov-Gribanov DV, Voytsekhovskaya IV, Tokovenko BT et al (2016) Actinobacteria isolated from an underground lake and Moonmilk Speleothem from the biggest Conglomeratic Karstic Cave in Siberia as sources of novel biologically active compounds. PLos One 11(3): e0152957 2. Behie SW, Bonet B, Zacharia VM et al (2017) Molecules to ecosystems: actinomycete natural products in situ. Front Microbiol 7:2149 3. Zhao H, Parry RL, Ellis DI et al (2006) The rapid differentiation of Streptomyces isolates using Fourier transform infrared spectroscopy. Vib Spectrosc 40(2):213–218 4. Brandes Ammann A, Brandl H (2011) Detection and differentiation of bacterial spores in a

mineral matrix by Fourier transform infrared spectroscopy (FTIR) and chemometrical data treatment. BMC Biophys 4:14 5. Atalan E (2001) Restriction fragment length polymorphism analysis (RFLP) of some Streptomyces strains from soil. Turk J Biol 25:397–404 6. Rivas R, Vela´zquez E, Valverde A et al (2001) A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species. Electrophoresis 22(6):1086–1089 7. Filippova SN, Surgucheva NA, Galchenko VF (2012) Long-term storage of collection cultures of actinobacteria. Microbiology 81(5):630–637

Chapter 3 Characterization and Identification of Actinobacteria Abstract The characterization of a strain is a key element in prokaryote systematics. Actinobacteria are quite different from other eubacteria with respect to cellular morphology and cell wall chemistry. A number of different methods have been used to classify Actinobacteria. However, its taxonomy was traditionally based on morphological characteristics such as size, shape, and color of the colonies on specific media. Gram staining and acid fastness and pigment production are the other parameters used while classifying using morphology. Other morphological features that are taxonomically important include the color, colony morphology, and surface arrangement of conidiospores. Physiological attributes such as nutritional requirements, fermentation products, and growth conditions such as oxygen, temperature, and inhibitory products are also important when classifying Actinobacteria. The composition of cell walls varies greatly among different group of actinobacteria. The presence of diaminopimelic acid (DAP) isomers is one of the most important cell wall properties of the Gram-positive bacteria including the Actinobacteria. Chemotaxonomy also involves the analysis of other macromolecules such as the isoprenoid quinines (menaquinones and ubiquinones), lipids (lipopolysaccharides and fatty acids including mycolic acids), polysaccharides, and related polymers (methanochondruitin and wall sugars) and proteins. Although chemotaxonomy is still considered useful in actinobacterial taxonomy, it is not always reliable as several genera may exhibit similar chemical properties. In addition, the techniques are cumbersome and time-consuming. The comparison of the DNA nucleotide sequences of actinobacterial strains provides a rapid and accurate method of establishing relatedness. 16s rRNA is a major component of the small (30S) ribosomal subunit which is important for subunit association and translational accuracy. The 16S rRNA gene consisting of 1542 bases is highly conserved among the microorganisms and is therefore an excellent tool for studying phylogenetic relationships. The 16s rRNA genes of many phylogenetic groups have characteristic nucleotide sequences called oligonucleotide signatures. Analysis of the 16S rRNA gene offers a time-saving alternative to the classical methods of identification. 16s rRNA sequencing has been used to reclassify actinobacterial species that were incorrectly classified using classical identification methods. Different methods for phenotypic, chemotaxonomic, and molecular characterization of actinobacteria are discussed in this chapter. Keywords Characterization, Taxonomy, Identification, Phenotypic, Chemotaxonomy, Molecular

1 1.1

Micromorphological Characterization of Actinobacteria Introduction

Micromorphological characteristics of Actinobacteria are very much important for their identification and taxonomy. But, due to high variation, morphological features are not exclusively attributed a specific actinobacterial taxon. In general, actinobacterial genera exist in the forms of filaments, rods, cocci, or rod-cocci [1]. Mycelial genera like Streptomyces generally develop two types

Ramasamy Balagurunathan et al., Protocols in Actinobacterial Research, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0728-2_3, © Springer Science+Business Media, LLC, part of Springer Nature 2020

39

40

Characterization and Identification of Actinobacteria

of mycelia such as substrate or vegetative mycelia and aerial/reproductive mycelia. Microscopic observation of cell structure is primely important for the initial selection and recovery of actinobacterial cultures. Microscopic observation of special structures like sporangium and mycelia arrangement will also be useful for generic identification of Actinobacteria. 1.2

1.3

Materials

Methods

1.3.1 Agar-Embedded Technique

l

Actinobacterial cultures.

l

ISP2 agar medium.

l

Sterile petri plates.

l

Sterile cover slip.

l

Forceps.

l

Bright-field microscope. 1. Prepare ISP2 agar plates. 2. Make a trough on the center of the petri plate. 3. Place a sterile cover slip over the trough made on ISP2 agar. 4. Aseptically inoculate the spores of actinobacterial cultures on the four sides of the cover slip by touching the ISP2 agar medium. 5. Incubate the plates at 28
C for 7–14 days. 6. After incubation, take the cover slip which contains the actinobacterial spores and mycelium using sterile forceps. 7. Place the cover slip over the microscopic slide and fix it using cellophane tape. 8. Observe the slide under bright-field microscope at different magnifications. 9. Record the different micromorphological structures of given actinobacterial culture (see Table 1).

1.3.2 Slide Culture Method

1. Prepare each 2 ml of ISP2 agar medium in test tubes, and sterilize by autoclaving at 121
C for 15 min.

Table 1 Micromorphological structures and observation Micromorphological structures Aerial mycelium Substrate mycelium Spore chain morphology Mycelia fragmentation Any special structures like sporangium

Observation

Scanning Electron Microscopic Studies on Actinobacteria

41

2. Inoculate the spores of actinobacterial cultures onto sterile ISP2 agar medium. 3. Mix the agar medium and actinobacterial spores aseptically, and pour as a thin layer over the surface of sterile microscopic slides. 4. Keep the slides under sterile petri plates and incubate at 28
C for 10 days. 5. Keep the sterile cotton or blotting paper humidified with sterile water on both sides of the culture slide in the petri plate. 6. During incubation observe the slides under bright-field microscope at 40 magnification. The microscopic characteristics to be recorded include the presence of aerial mycelium, substrate mycelium, mycelial fragmentation, and spore chain morphology (see Table 1). 1.4

2 2.1

Notes

l

Direct observation of colonies grown on isolation agar plates under bright-field microscope at lower magnification will be helpful for the selection of actinobacterial colonies.

l

Observing the mycelia structure in undisturbed conditions is highly useful for identification of certain actinobacteria at generic level.

l

However, the direct observation of non-sporulating actinobacterial cultures is less useful for generic identification. Instead the non-sporulating cultures will be observed after Gram staining.

Scanning Electron Microscopic Studies on Actinobacteria Introduction

Usually, the basic morphology of hyphae and spores is observed by light microscopy. The scanning electron microscope is an easy and suitable tool to study the external features of microbial cells at high magnification and resolution. Since the initial work on actinomycetes, a large number of strains have been examined, and further information on the range of forms of the members of this group has been obtained. In addition, improved specimen preparation and instrument operation have resulted in better resolution at higher magnifications [1]. Streptomyces species were compared on the basis of their cultural characteristics and spore chain morphology and spore surface. Spores of Streptomyces were examined by transmission and scanning electron microscopy. The procedure described below is so simple and fast, and the samples can be prepared in any microbiological laboratory without any expertise; therefore, it can be used for routine identification of Streptomyces without disturbing their spore chain morphology in a short span of time [2].

42

2.2

2.3

Characterization and Identification of Actinobacteria

Materials

Methods

l

Yeast extract malt extract agar.

l

Actinobacterial cultures.

l

Circular aluminum stub.

l

Petri dish.

l

Sterilize the circular aluminum stubs, Petri dishes, and yeast extract malt extract agar media by autoclaving at 121
C for 15 min.

l

Cool the medium at 45
C before pouring into the Petri dishes to avoid excess of moisture formation.

l

Pour approximately 30 ml of media in each Petri dish, and allow solidifying at room temperature.

l

Insert the sterilized stubs with the help of sterile forceps at an angle of about 45o into yeast extract malt extract agar medium plates, until about half the stub was dipped in the medium.

l

Insert the sterile cover slips also at the same angle on the sample plate for the same culture as a control. Incubate the plates at 37
C for 24 h to check the bacterial contamination during the handling procedures. Discard the contaminated plates.

l

After 24 h, spread the inoculum of given actinobacterial culture along the line where the surface of the stubs met the medium, using inoculating loop.

l

Repeat this process with cover slips as well.

l

Incubate the plates at 28
C for 10 days.

l

Check the length of incubation period needed for each strain to produce mature sporing structures by cover slip method.

l

Carefully withdraw the stub from the medium which contains the line of actinobacterial growth.

l

Coat the upper surface of each stub, under vacuum, with a film of gold for 15–20 min.

l

Once coated with gold, the specimen is ready for examination under the scanning electron microscope.

l

View the gold-coated metal stubs on the SEM at an accelerating voltage of 20 kV, a probe diameter of 102 pA, to obtain secondary electron images.

l

Scan the field at low magnification until the line of growth was detected.

l

Select the areas with clear, intact sporing structures for examination at higher magnification. Photograph the suitable fields in the preparation.

Cultural Characterization of Actinobacteria

3

43

l

Similarly, also withdraw the cover slips, and mount on the glass slide having one drop of methylene blue (0.3 g in 10 ml distilled water).

l

Fix the cover slips with petroleum jelly and observe under light microscope.

Cultural Characterization of Actinobacteria

3.1

Introduction

Cultural characteristics of actinobacteria refer to the growth characteristics and morphology in various kinds of culture media. It is usually determined after incubation for 7-14 days at 28
C strictly according to methods used in the International Streptomyces Project (ISP). The colors of substrate and aerial mycelia and any soluble pigments produced were determined by comparison with chips from the ISCC-NBS color charts. The colors of the mature sporulating aerial mycelium are recorded in a simple way (white, gray, red, green, blue, and violet). The yeast extract malt extract agar and inorganic salt starch agar are commonly used to study cultural characteristics. The groupings are made on the production of melanoid pigments (i.e., greenish brown, brownish black, or distinct brown, pigment modified by other colors) on the media such as ISP1 and ISP7. The strains are grouped as melanoid pigment produced (+) and not produced () [1]. The strains are divided into two groups, according to their ability to produce characteristic pigments on the reverse side of the colony, namely, distinctive (+) and not distinctive or none (). In case a color with low chroma such as pale yellow, olive, or yellowish brown occurs, it is included in the latter group (). The strains are divided into two groups by their ability to produce soluble pigments other than melanin, namely, produced (+) and not produced (). The color is recorded (red, orange, green, yellow, blue, and violet) [3].

3.2

Materials

l

Different ISP2 agar plates.

l

Actinobacterial cultures.

l

Incubator.

l

Prepare each 50 ml of ISP2 broth in 250 ml conical flask, and sterilize by autoclaving at 121
C for 15 min.

l

Transfer actinobacterial spores aseptically onto ISP2 broth.

3.3

Methods

l

Incubate the flask in rotary shaker at 28
C with 120 rpm for 72 h.

l

Prepare different ISP agar medium [ISP1—ISP7 agar] plates.

l

Inoculate each 1 ml of actinobacterial culture onto all the ISP agar media plates as straight line.

44

Characterization and Identification of Actinobacteria

Table 2 Culture characteristics of actinobacterial strain Media

Growth

Consistency

Aerial mass color

Reverse side pigment

Soluble pigment

ISP 1 ISP 2 ISP 3 ISP 4 ISP 5 ISP 6 ISP 7 l

3.4

4 4.1

Notes

Incubate all the plates for 10 days at 28
C.

l

Record the different cultural characteristics such as the nature of growth, consistency, aerial mass color (AMC), presence of reverse side pigment (RSP), and the details of soluble pigment (SP) production, if any (see Table 2).

l

Care should be taken while preparing different ISP media.

l

Prepare the trace salt solution freshly and use it for different ISP media preparation.

Physiological Characterization of Actinobacteria Introduction

Different members of the phylum Actinobacteria tolerate different physiological conditions. There are many environmental factors having pronounced effects on the growth, differentiation, and metabolism of Actinobacteria. Carbon, nitrogen, and mineral contents of the environment or culture medium majorly affect the growth of Actinobacteria. Amino acids like methionine and tryptophan, humic and fulvic acids, and complex organic media are also the factors reported to be favorable for sporulation of actinobacteria [4]. Other than nutrient parameters, medium pH, incubation temperature, and medium consistency are also found to have great impact of actinobacterial growth and metabolism. Physiological characterization is very importantly used as a criteria for phenotypic characterization and identification of actinobacteria. Utilization of different sugars is used as a characterization and identification of Streptomyces species for more than six decades. Determination of effect of pH, temperature, and salinity is also useful to identify the different physiological groups of Actinobacteria like acidophiles, alkaliphiles, psychrophiles, thermophiles, and halophilic actinobacteria [5].

Physiological Characterization of Actinobacteria

4.2

4.3

Materials

Methods

4.3.1 Preparation of Basal Medium

l

Basal medium.

l

Sugars [glucose, sucrose, fructose, xylose, mannitol, starch, inositol, cellulose].

l

Amino acids [L-asparagine, L-glutamine].

l

Diethyl ether.

l

Actinobacterial cultures.

l

Measure required quantity of salts and dissolve in 1000 ml of distilled water.

l

Prepare trace salt solution and transfer 1 ml of it into basal medium.

l

Transfer each 100 ml of basal medium into each 250 ml conical flask.

l

4.3.2 Carbon and Nitrogen Utilization Test

4.3.3 Temperature Tolerance

Adjust pH of the medium to 7.0  0.2, add 1.5% agar, and then sterilize by autoclaving at 121
C for 15 min.

l

Weigh each 1 g of different sugars and amino acids as listed in the table, and transfer in to a beaker.

l

Add 10 ml of diethylether in to each beaker and mix well.

l

Allow the solvent portion to dry under fume hood.

l

Transfer each sugar and amino acids into each 100 ml of molten basal medium, and mix well.

l

Pour the medium into sterile petri plate and allow to solidify.

l

Inoculate the actinobacterial spores on all the sugar- and amino acid-containing plates in triplicate as a straight line.

l

pH Tolerance

45

Incubate all the plates at 28
C for 7–14 days.

l

Record the growth characteristics as the nature of growth, consistency, aerial mass color, presence of reverse side pigment, and the details of soluble pigment production, if any.

l

Prepare ISP2 agar plates with different pH values, viz., 5, 7, 9, and 11.

l

Inoculate the actinobacterial spores on ISP2 agar with different pH ranges in triplicate as straight line.

l

Incubate the plates at 28
C for 7–14 days.

l

Prepare ISP2 agar plates.

l

Inoculate actinobacterial spores onto ISP2 agar plates.

l

Incubate at different temperatures, viz., 20, 30, 40, and 50
C.

l

After 7–14 days, observe the plates for actinobacterial growth.

46

Characterization and Identification of Actinobacteria

Table 3 Cultural characteristic of actinobacterial strains Factors

Variables

Sugars

Glucose

Growth

Consistency

AMC

RSP

SP

Fructose Sucrose Rhamnose Raffinose Xylose Inositol Cellulose Amino acids

L-asparagine L-glutamine

pH

5 7 9 11

Temperature (
C)

20 30 40 50

NaCl (%)

0 1 5 10

4.3.4 NaCl Tolerance

l

Prepare ISP2 agar plates supplementing with different concentrations of sodium chloride, viz., 0, 1, 2.5, 5.0, 7.5, and 10%.

l

Inoculate actinobacterial spores onto ISP2 agar plates with different concentration of NaCl.

l

Incubate all the plates at 28
C for 7–14 days, and then observe the plates for actinobacterial growth (see Table 3).

Analysis of Actinobacterial Cell Wall for Amino Acids and Sugars

5

47

Analysis of Actinobacterial Cell Wall for Amino Acids and Sugars

5.1

Introduction

Actinobacteria could be separated into broad groups at the generic level on the basis of morphology and cell wall composition. The compositions of cell wall diaminopimelic acid isomers and wholecell sugars have become widely accepted as the taxonomic markers for the generic grouping of actinobacteria. A method for analysis of the diaminoacids of peptidoglycan from whole cells is rapid, simple, inexpensive and requires only small amount of biomass. Thin-layer chromatography, is quite suitable method for separation of cell wall diaminopimelicacids [6]. Like call wall amino acids, for the classification and identification of actinobacteria, the analysis of sugars from whole cells is needed. For discrimination of meso-diaminopimelic acid containing actinomycetes, five whole-cell sugar patterns have been recognized, based on the presence of distinct sugars (A, arabinose and galactose; B, madurose; C, no diagnostic sugars; D, arabinose and xylose; E, rhamnose). The combination of the characteristic diaminoacid and some amino acids used cell wall sugars to describe eight wall chemotypes to distinguish actinomycetes. The analysis of diaminoacids and sugars from whole-cell preparations is less timeconsuming and often allows an allocation to the correct wall chemotype (see Table 4) [7].

5.2

Materials

l

ISP2 broth.

l

Actinobacterial culture.

l

6 N HCl.

Table 4 Chemotypes of actinobacterial cell wall Cell wall chemotype

Characteristic cell wall component

I

L-Diaminopimelic

II

meso-Diaminopimelic acid, glycine

III

meso-Diaminopimelic acid

IV

meso-Diaminopimelic acid, arabinose, galactose

V

Lysine and ornithine

VI

Variable presence of aspartic acid and galactose

VII

Diaminobutyric acid, glycine

VIII

Ornithine

IX

meso-Diaminopimelic acid, various amino acids

X

meso-Diaminopimelic acid, L-Diaminopimelic acid

acid, glycine

48

Characterization and Identification of Actinobacteria l

Cellulose TLC sheet.

l

1% DL–diaminopimelic acid.

l

Methanol.

l

Distilled water.

l

Pyridine.

l

Ninhydrin.

l

Sugars (galactose, glucose, mannose, arabinose, xylose, and ribose).

l

N-butanol, toluene.

l

Acid aniline.

Methods

l

5.3.1 Cell Wall Amino Acid Analysis

Grow the actinobacterial culture in 5 ml ISP2 broth in rotary shaker with 95 rpm for 5 days at 28
C.

l

5.3

l

Transfer the cells to screw-capped tube and add 0.1 ml of 6 N HC1

l

Steam the content in an autoclave at 15 lb for 15 min.

l

After cooling, spot 1 μl hydrolysate on cellulose TLC sheet.

l

Spot also one microliter of 1% DL-DAP that contain both the LL- and meso-DAP on the same sheet as a standard.

l

Run the chromatogram using methanol-distilled water-6 N HC1-pyridine (80:26:4:10, v/v), as the solvent system for 4 h.

l

After drying, detect the amino acids in the chromatogram by spraying with acetonic-ninhydrin (0.1% w/v)

l

5.3.2 Cell Wall Sugar Analysis

Harvest the cells by centrifugation at 13,000  g for 15 min, and wash the cells twice with distilled water.

l

l

Heat the TLC sheet at 100
C for 2 min to reveal the spots. Grow the actinobacterial culture in 5 ml ISP2 broth in rotary shaker with 95 rpm for 5 days at 28
C. Harvest the cells by centrifugation at 13000  g for 15 min, and wash the cells twice with distilled water.

l

Transfer the cells to screw-capped tubes and add 0.1 ml of 0.25 N HCl.

l

Autoclave the content at 15 lb for 15 min.

l

l

After cooling, spot 1 μl of the hydrolysate on a cellulose TLC sheet in parallel with 1 μl of a standard solution, which contains galactose, glucose, mannose, arabinose, xylose, and ribose each at 1% concentration. Perform the ascending thin-layer chromatography with a solvent system of n-butanol-distilled water-pyridine-toluene (10:6:6:1, v/v) for 3 h.

Analysis of Actinobacterial Cell Wall for Polar Lipids l

6

49

After drying the sheet, detect the sugars by spraying with acid aniline and heating at 100
C for 4 min.

Analysis of Actinobacterial Cell Wall for Polar Lipids

6.1

Introduction

Polar lipids are important components of bacterial plasma membranes. Bacterial plasma membranes are composed of amphipathic polar lipids associated with specific membrane proteins. Amphipathic polar lipids consist of hydrophilic head groups usually linked to two hydrophobic fatty acid chains. Phospholipids are the most common polar lipids, including phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and other phosphatidyl glycolipids. In addition, glycolipids and acylated ornithine or lysine amides also fall into this category. For the description and differentiation of actinobacteria, five phospholipid types (PI–PV) have been recognized [6]. During the extraction process, the lower, mainly chloroform, layer contains the polar lipids, whereas non-lipid components remain in the upper aqueous phase. Extraction with hexane removes nonpolar components such as isoprenoid quinones; in this way menaquinones and polar lipids can be extracted from a single sample of biomass [7].

6.2

Materials

l

Actinobacterial cultures.

l

Teflon-lined screw-capped tubes.

l

0.85% Aqueous NaCl.

l

Methanol.

l

Petroleum ether.

l

Chloroform.

l

Acetic acid.

l

Water.

l

Silica gel TLC sheet.

6.3

Methods

6.3.1 Extraction of Polar Lipids

1. Transfer 100–200 mg of dried actinobacterial cell biomass into a tube with Teflon-lined screw cap (50 ml). 2. Add 2 ml of 0.85% aqueous NaCl, followed by 15 ml methanol in to the biomass and mix well. 3. Heat the tube at 100
C for 10 min in a boiling water bath. 4. After cooling at room temperature, add 10 ml chloroform and 6 ml 0.85% aqueous NaCl, then shake for 10 min.

50

Characterization and Identification of Actinobacteria

5. Centrifuge the treated actinobacterial biomass at 7000  g for 10 min and collect the lower layer. 6. Then collect the lower layer and evaporate to dryness under reduced pressure at 40o C on a rotary evaporator. 6.3.2 Separation of Polar Lipids by 2D-TLC

1. Dissolve the dried polar lipids in 100 μl of petroleum ether (boiling point, 70–90
C). 2. Spot 10 μl of sample to the bottom of a clean, silica gel coated TLC plate (10  10 cm size) using a glass capilliary tube. 3. Develop the chromatogram in the first dimension using chloroform–methanol–water (65:25:4, v/v) as solvent system. 4. After overnight drying in a fume cupboard, develop the chromatogram again with chloroform–acetic acid–methanol–water (80:18:12:5, v/v) in the second dimension. 5. Again dry the TLC plate in a fume cupboard.

6.3.3 Identification of Polar Lipid Component

Identify the various lipids by comparison of their Rf values in the plates and staining behavior with following procedure. 1. Dissolve ninhydrin (0.1%, w/v) in acetone. 2. Spray the TLC–chromatogram plate, and heat at 100
C for 5 min to reveal lipids which contain amino groups as pink spots. Mark the pink spots with a soft pencil to prevent them from fading on storage. 3. Use the same plate for the detection of lipid phosphorus using molybdenum reagent. Molybdophosphoric Acid for Total Lipids

1. Dissolve 10% (w/v) molybdophosphoric acid in 95% (v/v) ethanol. 2. Spray the TLC plate and heat at 150
C for at least 10 min. Lipids show as dark spots on a light-green background. α-Naphthol Reagent for Lipids Containing Sugar Groups

1. Dissolve 15 g α-naphthol in 100 ml 95% (v/v) ethanol. 2. Mix 10.5 ml of this solution with 6.5 ml H2SO4, 40.5 ml ethanol, and 4 ml water to make a working solution. 3. Spray the Dragendorff reagent over the plate lightly and heat at 100
C for 10 min. Glycolipids appear as purple-brown or brown spot.

Detection of Actinobacterial Menaquinones in Actinobacterial Cell Wall

51

Dragendorff Reagent for Lipids Containing Quaternary Nitrogen Groups

1. Add bismuth nitrate (1.7 g) to 100 ml of 20 % acetic acid (solution A). 2. Add potassium iodide (40 g) to 100 ml water (solution B). 3. Mix solution A (3.5 ml) and solution B (5 ml) with acetic acid (20 ml) and water (50 ml) to make a working solution. 4. Spray the plate lightly at room temperature; lipids containing quaternary nitrogen show as orange-red spots. Mark the orange-red spots with a soft pencil to prevent them from fading on storage. 5. The same plate can be used for the detection of lipid phosphorus using molybdenum reagent. Zinzadze Reagent for Phosphorus-Containing Lipids

1. Add molybdenum trioxide (40.11 g) to 1 L of 25 N H2SO4, and boil gently in a fume cupboard until all the residue dissolves (solution A). 2. Add powdered molybdenum (1.78 g) to 500 ml of solution A, and boil the mixture gently for 15 min and allow to cool (solution B). 3. Mix equal volumes of solutions A and B, and dilute with two volumes of distilled water to make a working solution. 4. Spray the plate with Zinzadze reagent very lightly at room temperature. 5. Lipids containing phosphorus show as blue spots.

7 7.1

Detection of Actinobacterial Menaquinones in Actinobacterial Cell Wall Introduction

Respiratory isoprenoid quinones are constituents of the bacterial cytoplasmic membrane as well as the mitochondrial membrane where they play an important role in the electron transport chain. The potential of analyzing the quinone system for the characterization of bacteria is based on the different types of quinones, the length of isoprenoid side chain, and the number of saturated isoprenoid units. To date, menaquinones are the only type of respiratory isoprenoid quinones found in actinobacteria, and the variations in the number of isoprene units and hydrogenated double bonds make these membrane constituents of considerable chemotaxonomic value [8]. Menaquinones are free lipids that can be readily extracted from freeze-dried cells with organic solvents or with their mixture such as acetone, chloroform, and hexane. However, they are susceptible to

52

Characterization and Identification of Actinobacteria

strong acid or alkaline and photooxidation in the presence of oxygen and strong light conditions. But it is not necessary to work in a nitrogen atmosphere or dim light. The menaquinones in these extracts are purified by preparative slilica gel thin-layer chromatography (TLC) with hexane-diethylether as developing solvent, and analysis is then performed by HPLC. Purified menaquinones are revealed by using UV light at 254 nm [9]. 7.2

7.3

Materials

Methods

7.3.1 Extraction of Menaquinones

l

Actinobacterial cultures.

l

Solvents.

l

Magnetic stirrer.

l

Filter papers.

l

Rotary evaporator.

l

TLC plates.

l

0.45 μm Filter membrane.

l

Menaquinones.

l

Silica gel F254 sheet. 1. Take approximately 100 of lyophilized cells, and mix with 40 ml of chloroform–methanol (2:1 v/v) for approximately 1 h or overnight using a magnetic stirrer to extract the menaquinones. 2. Pass the cell/solvent mixture through filter paper to remove cell debris. 3. Collect the eluate in a flask, and evaporate to dryness under reduced pressure at 40
C on a rotary evaporator.

Purification of Menaquinones by TLC

1. Dissolve the dried menaquinones in 800 μl of acetone. 2. Apply the diluted menaquinone sample as a uniform streak (5 cm long) to a silica gel F254 sheet. 3. Develop the plate in methylbenzene for time ~20 min. 4. Allow the plate to dry in a fume cupboard (~5 min); view menaquinones by brief irradiation with ultraviolet light at 254 nm. The menaquinones appear as dark-brown/purple bands on a green fluorescent background, Rf ~ 0.7. 5. Scrap gel containing menaquinones from the plate with spatula, dissolve scraped gel in 500 μl of methanol, and elute through syringe and 0.45 μm filter membrane for further HPLC analysis.

Detection of Mycolic Acid in Actinobacterial Cell Wall

8

53

Detection of Mycolic Acid in Actinobacterial Cell Wall

8.1

Introduction

Mycolic acids (MAs), 2-alkyl, 3-hydroxy long-chain fatty acids (FAs), are the hallmark of the cell envelope of Mycobacterium tuberculosis and related species. They are found either unbound, extractable with organic solvents such as esters of trehalose or glycerol, or esterifying the terminal pentaarabinofuranosyl units of arabinogalactan (AG), the polysaccharide that, together with peptidoglycan, forms the insoluble cell wall skeleton. Both forms presumably play a crucial role in the remarkable architecture and impermeability of the cell envelope, participating in the two leaflets of the mycobacterial outer membrane, also called the mycomembrane. Detection of mycolic acid has the taxonomic value in the detection for mycolic acid containing actinobacterial genera such as Corynebacterium, Dietzia, Gordonia, Mycobacterium, Nocardia, Rhodococcus, Turicella, and Tsukamurella. For the extraction and analysis of mycolic acids, different methods have been described based on TLC, GC, or HPLC [10].

8.2

Materials

l

Silica gel-coated TLC sheet.

l

Methanol.

l

Toluene.

l

Conc. H2SO4.

l

Petroleum ether.

l

Diethyl ether.

l

Ammonium hydrogen carbonate.

l

Glass column.

l

Rotary evaporator.

l

Acetone.

l

Molybdophosphoric solution.

8.3

Methods

8.3.1 Extraction of Mycolic Acids from Whole Cell

1. Add 50–100 mg freeze-drying cells into a clean, dry test tube. 2. Add 3 ml mixture solvent of methanol, toluene, and conc. sulfuric acid (30:15:1) into the test tube, and tightly seal the test tube. 3. Place the test tubes into a water bath at 75
C. 4. Cool the test tube down to room temperature, and add 2 ml petroleum ether (b.p. 60–80). 5. Centrifuge the mixture for 10 min at low speed (1000  g), and collect the upper solvent phase. 6. Prepare a small column of ammonium hydrogen carbonate, and prewash the small column with diethyl ether.

54

Characterization and Identification of Actinobacteria

7. Pipette the upper solvent phase into a small column (ca. 1 cm), and collect the eluent in a small centrifuge tube (5 ml), then wash the small column again with diethyl ether. 8. Combine the washed eluent, and evaporate to dryness under reduced pressure at 40
C on a rotary evaporator. 1. Dissolve the dried mycolic acids in 200 μl of petroleum ether.

Analysis of Mycolic Acids from Whole Cell

2. Spot 10 μl to the bottom of a 10  10 cm of thin-layer plate coated with silica gel. 3. Develop single dimentional chromatogram with petroleum ether, acetone (95:5, v/v) and dry the plates in a fume cupboard. Develop the same sheet of chromatogram in second direction by toluene, acetone (97:3) and dry the plates in a fume cupboard. 4. Stain, spray plate with molybdophosphoric acid, and heat at 150
C for 5 min to reveal mycolic acids.

9

Isolation of Chromosomal DNA from Actinobacteria

9.1

Introduction

The isolation of bacterial genomic DNA is one of the primary requirements in the areas of bacterial genetics, molecular biology, and biochemistry. Purified DNA is required in many applications such as studying structure and chemistry of DNA, examining DNA–protein interactions, carrying out DNA hybridizations, sequencing, PCR, and gene cloning. The isolation of DNA from actinobacteria is a relatively simple process [11]. The common step in all the procedures is that a cell is first broken, and then the DNA is separated from other intrusive compounds such as proteins, RNA, lipid, and carbohydrates. Purity of the DNA is essential as slight contaminants can inhibit further experiments like restriction digestion, polymerase chain reaction, sequencing, etc. [12].

9.2

Materials

l

Actinobacterial culture—Streptomyces.

l

Tryptic soy broth.

l

Shaking incubator.

l

Centrifuge.

l

Sodium chloride.

l

2 ml Micro-centrifuge tubes.

l

Tris–HCl.

l

EDTA.

l

Lysozyme.

l

RNase A.

l

Proteinase K.

Isolation of Chromosomal DNA from Actinobacteria

9.3

Methods

l

Sodium dodecyl sulfate.

l

Cetyltrimethylammonium bromide.

l

Water bath.

l

Phenol.

l

Chloroform.

l

Isopropanol.

l

Ethanol.

l

20
C Refrigerator.

l

Nanodrop UV spectrophotometer.

55

1. Grow the actinobacterial culture in 20 ml of TSB for 7 days at 30
C with 90 rpm shaking in incubator, and then centrifuge at 1700  g for 5 min. 2. Resuspend the mycelial pellet in 500 μl of 5 M NaCl and transfer to a 2 ml tube. 3. Centrifuge the cells at 10,500  g for 30 s. 4. Resuspend the pellet in 1 ml of 10 mM Tris–HCl–1 mM EDTA (pH 7.5) (TE) containing 20 mg of lysozyme/ml and 20 mg of RNase A/ml, and incubate at 37
C for 1 h. 5. Following incubation, add 250 μl of 0.5 M EDTA, 250 μl of TE containing 5 mg of proteinase K/ml, and 100 μl of 10% SDS, and incubate at 37
C for 1 h. 6. Mix the tubes by inversion after the addition of 250 μl of 5 M NaCl. 7. Immediately thereafter, add 200 μl of CTAB solution (10% CTAB plus 0.7 M NaCl), and incubate the tubes at 65
C water bath for 10 min. 8. Remove the cellular debris by centrifugation at 7000  g for 5 min, and transfer the supernatant solution to a new 2 ml centrifuge tube. 9. Remove the proteins and lipids by the addition of 0.3 volume of phenol-chloroform, and mix the phases by inversion, and centrifuge at 17000  g for 5 min. 10. Transfer the aqueous phase to a new tube, and precipitate the DNA with an equal volume of isopropanol. 11. Rinse the pellet with 70% ethanol to remove traces of salt, dry, and redissolve in 50 μl of TE for immediate use or storage at 20
C. 12. Analyze the quantity and quality of DNA using a “NanoDrop” UV spectrophotometer.

56

Characterization and Identification of Actinobacteria

Table 5 Absorbance at 260/280 (nm) and their OD value

Absorbance at 260/280 (nm)

OD value

13. Switch on machine. Start up the NanoDrop software and log in. Select the “nucleic acid” option. 14. Follow the onscreen instructions—place 1.5 μl of ultrapure water onto the sample pedestal, close the arm, and click on OK to initialize the instrument. 15. Once the instrument has initialized, lift the arm, and carefully wipe the sample pedestal and arm. Perform a blanking reaction using 1.5 μl of whatever your sample is dissolved in buffer, close down the arm, and click on the blank button. 16. Once the instrument has blanked (graph with zeroed baseline), lift the arm, and carefully wipe the sample pedestal and arm. Then place 1.5 μl of your sample onto the sample pedestal, name it in the sample ID window, close down the arm, and click on the measure button. 17. Record the concentration (ng/μl) and the OD 260/280 ratio. A ratio of ~1.8 is generally accepted as “pure” for DNA (see Table 5).

10

PCR Amplification of 16s rRNA Gene

10.1

Introduction

The polymerase chain reaction (PCR) involves the primer-mediated enzymatic amplification of DNA. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. Primer is needed because DNA polymerase can add a nucleotide only onto a preexisting 30 -OH group to add the first nucleotide. DNA polymerase then elongates its 30 end by adding more nucleotides to generate an extended region of double-stranded DNA [11, 13].

10.2

Materials

l

DNA template: The double stranded DNA (dsDNA).

PCR Amplification of 16s rRNA Gene

57

Table 6 PCR reaction

10.3

Methods

Components

Concentration

Volume (μl)

DNA

25–50 ng

2

Ex Taq PCR buffer

10

5

MgCl2

1.5 mM

5

dNTP

10 mM

2

Forward primer

50 pMol

2.5

Reverse primer

50 pMol

2.5

Taq polymerase

0.5 U

0.5

Water

–

30.5

l

DNA polymerase: Usually a thermostable Taq polymerase that does not rapidly denature at high temperatures (98
) and can function at a temperature optimum of about 70
C.

l

Oligonucleotide primers: Short pieces of single-stranded DNA (often 20–30 base pairs) which is complementary to the 30 ends of the sense and antisense strands of the target sequence.

l

Deoxynucleotide triphosphates: Single units of the bases A, T, G, and C (dATP, dTTP, dGTP, dCTP) which provide the energy for polymerization and the building blocks for DNA synthesis.

l

Buffer system: Includes magnesium and potassium to provide the optimal conditions for DNA denaturation and renaturation; it is also important for polymerase activity, stability, and fidelity.

l

Thin-walled PCR tubes.

l

Ice bucket.

l

Molecular grade water.

l

PCR machine. 1. Design primers. 2. Place thin-walled PCR tubes on ice. 3. Set up a 50 μL PCR reaction as mentioned below (keep all your reagents on ice) (see Table 6). 4. Place reaction tubes in PCR machine. 5. Set PCR condition as mentioned below. l

Initial Denaturation Increase the reaction temperature to 94
C for 5 min to ensure that all complex, double-stranded DNA (dsDNA) molecules are separate into single strands for amplification.

58

Characterization and Identification of Actinobacteria l

Denaturation This step involves heating the reaction mixture to 94
C for 45 s. During this, the double-stranded DNA is denatured to single strands due to breakage in weak hydrogen bonds.

l

Annealing Lower the reaction temperature rapidly to 55
C for 1 min. This allows the primers to bind (anneal) to their complementary sequence in the template DNA.

l

Elongation This step usually occurs at 72
C for 1 min 30 s. In this step, the polymerase enzyme sequentially adds bases to the 30 each primer, extending the DNA sequence in the 50 to 30 direction. Under optimal conditions, DNA polymerase is added about 1000 bp/min. During the first extension, the template will not be length limiting, and so templates will be synthesized that exceed the amplicon length. In subsequent extension steps, the amplicon length will be defined by the primer sequence at each end. Note: Repeat: Steps 2–4 are performed in a cyclical manner (34 cycles), resulting in exponential amplification of the amplicon.

l

Final Extension Carry out the final extension step at 72
C for 10 min, a final extension to fill-in any protruding ends of the newly synthesized strands.

6. Run 2 μl on an electrophoresis gel to check size and concentration of PCR product.

11

Separation of Actinobacterial DNA by Agarose Gel Electrophoresis

11.1

Introduction

Agarose gel electrophoresis is a method used to separate DNA fragments. DNA molecules will be separated according to their sizes and charges. The location of DNA fragments within the gel can be determined directly by staining with appropriate dyes and can be detected with naked eyes [11, 13].

11.2

Materials

l

Agarose powder.

l

10 TAE/TBE buffer.

l

Ethidium bromide.

l

Loading dye.

l

DNA ladder.

l

Deionized distilled water.

Separation of Actinobacterial DNA by Agarose Gel Electrophoresis l

11.3

Methods

59

Gel chamber with gel tray and comb. 1. Prepare a 10 TAE/TBE buffer solution by using distilled water. The prepared buffer solution can be stored under 4
C for several days. 2. Use agarose powder and prepared buffer solution to make a 1.5% agarose solution. l

Add agarose powder into a conical flask containing buffer solution.

l

Heat the mixture in microwave oven for about 2 min until the agarose powder is completely dissolved.

l

Observe the process of heating closely to avoid boiling up of the mixture.

l

A hot plate may be used instead of microwave oven.

3. Seal the lateral openings of the gel mold with cellotape properly. 4. Pour the agarose solution into the gel mold slowly. 5. Place the comb into the gel mold at one side. 6. Allow the agarose solution to solidify for about 30 min. 7. Remove the comb. A row of “wells” is formed in the gel. 8. Place the gel in the gel tank. The side with the wells should be placed at the cathode side of the tank. 9. Add buffer solution to immerse the gel completely. 10. Remove the sharp end of the syringe needle with a pair of scissors. 11. Use the syringe to load the marker solution into the well gently. 12. Rinse the syringe with buffer solution for several times before every loading. 13. Load the samples in the wells, respectively. 14. Cover the gel tank properly with the lid. 15. Connect the electrodes and turn on the power supply. A 120 V DC voltage is needed. 16. Migration of DNA fragments: l

Allow the DNA fragments to move in the gel.

l

The tracking dye in the samples indicates the progress of electrophoresis.

l

When the tracking dye reaches about one-third to half of the length of the gel, it is the time to collect.

17. In general, 30 min is long enough to finish the electrophoresis process. Switch off the power supply. Take out the gel carefully.

60

Characterization and Identification of Actinobacteria

18. Visualize the gel under UV light

12

Sequencing of Actinobacterial 16S rRNA Gene

12.1

Introduction

Sanger’s method of gene sequencing is also known as dideoxy chain termination method. It generates nested set of labeled fragments from a template strand of DNA to be sequenced by replicating that template strand and interrupting the replication process at one of the four bases. A DNA primer is attached by hybridization to the template strand, and deoxynucleosides triphosphates (dNTPs) are sequentially added to the primer strand by DNA polymerase. The primer is designed for the known sequences at 30 end of the template strand [13]. The reaction mixture also contains dideoxynucleoside triphosphate (ddNTPs) along with usual dNTPs. If during replication ddNTPs is incorporated instead of usual dNTPs in the growing DNA strand, then the replication stops at that nucleotide. The ddNTPs are analogue of dNTPs. The ddNTPs lack hydroxyl group (–OH) at c3 of ribose sugar, so it cannot make phosphodiester bond with nest nucleotide, thus terminating the nucleotide chain. A respective ddNTPs of dNTPs terminates chain at their respective site. Similarly, ddCTP, ddGTP, and ddTTP terminate at C, G, and T site, respectively [11].

12.2

Materials

l

Primers.

l

Sequencer.

l

Actinobacterial DNA.

l

PCR reaction mixture.

12.3 12.3.1

Methods

l

Purification

Add 50 μl of AM-Pure XP beads (Vortex AMPure XP beads before each use) to PCR products, and pipette to mix well.

l

Incubate at room temperature for 5 min.

l

Place the tubes on a magnetic stand and wait 2 min for liquid to get clear.

l

Remove and discard all supernatant from each well. Add 180 μl fresh 80% EtOH to wash each well. Then again incubate on the magnetic stand for 30 s.

l

Remove and discard all supernatant from each well.

l

Air-dry on the magnetic stand for 15 min. Remove from the magnetic stand.

l

Add 52.5 μl RSB to each well and pipette to mix.

l

Incubate at room temperature for 2 min.

l

Place on a magnetic stand and wait 2 min for liquid is to get clear.

Phylogenetic Tree Construction and GenBank Submission of Actinobacterial. . . l

61

Transfer 50 μl supernatant to a new tube.

12.3.2 QC Report for Purified PCR Products

Perform Qubit quantification on purified samples. Also select a few samples, and run an agarose gel to ensure there is a major product at approximately 1400 bp.

12.3.3

Sequencing

The tube should contain 6 μl of PCR product at 25 ng/μl concentration and 3 μl of the desired primer at 1.1 μM concentration. The sequencing facility suggests 10 ng of DNA per 100 bp. All samples should be prepared in 0.2 mL strip tubes and placed in the sequencer.

12.3.4

Interpret Results

Assemble the sequencing reads using assembly software either DNA Baser or Geneious. Ab1 files for all three amplicons can be dragged onto the DNA Baser v.4 used to produce a consensus contig. Take the highest quality portion of the full-length consensus sequence, and search for the best match using NCBI-BLAST.

13 Phylogenetic Tree Construction and GenBank Submission of Actinobacterial 16S rRNA Gene Sequence 13.1

Introduction

The Molecular Evolutionary Genetics Analysis (MEGA) is widely using software for the phylogenetic analysis of DNA and protein sequence to construct phylogenetic tree using various statistical methods such as maximum likelihood, neighbor joining (NJ), parsimony, and distance methods. By using the NJ method to infer phylogenetic tree in MEGA, researchers often discovered that the NJ method generated a tree quickly for datasets containing many sequences and that differences between NJ trees and those produced by other time-consuming methods were localized to parts of the trees that were usually statistically weakly supported. These firsthand experiences spurred a more widespread appreciation of statistical methods for phylogenetic analysis, which was clearly reflected in the fast-growing citations of the NJ method. The imprint of context-dependence principle is seen throughout MEGA. For example, the distribution of the computational functionalities and display properties into input data explorers and output result explorers is also a product of the context-dependence design imperative, as it enables the user to conduct simple downstream analyses easily using the results presented. To submit a sequence in NCBI, we need certain tools, which are easily found in the NCBI page itself. If we want to submit a single sequence and assume it in GenBank, then we will be requiring BankIt or Sequin, which are sequence submitting tools [14].

62

13.2

13.3

Characterization and Identification of Actinobacteria

Materials

Methods

13.3.1 Phylogenetic Tree Construction

l

Desktop system.

l

MEGA software.

l

Sequence sample. 1. Paste our aligned original sequence and reference sequence collected from NCBI-BLAST in Notepad, and save it. 2. Go to Clustal X 2, click File button, and then load sequence. After that browse Notepad saved file, and save here another file, and it will be saved as “aln” file automatically. 3. Then open MEGA software, and open saved “aln” file, and now on the same window, click on conversion sign, and save file without star as MEGA file, and now close this window 4. Open mega again and click on phylogeny and followed by neighbor-joining method. 5. Select to nucleotide sequence and standard, then software will progress on the same window to open phylogeny, and the tree will be constructed.

13.3.2 GenBank Submission Through BankIt

1. Register through the NCBI login. 2. Sequence data can be either cut and pasted as text or uploaded as file (multiple sequences must be in a FASTA format). 3. Add date for public release (immediate or at a specified future date). 4. Provide basic information (authors and a working title) for a corresponding reference paper, name(s) of the organism (s) from which the sequence data were isolated, and any other related descriptive data. 5. Give sequence features (e.g., CDS, gene, rRNA, tRNA, with nucleotide intervals and product names). 6. Add molecule type either genomic DNA or mRNA or genomic RNA or cRNA, etc. 7. Select topology either linear or circular (circular must be complete, such as a complete plasmid). 8. Include source modifiers contain strain, clone, isolate, specimen voucher, isolation source, country, location, etc. 9. Features of the sequence—upload files, or use input forms to add all applicable features (e.g., CDS, gene, rRNA, tRNA, microsatellite, exon, intron). 10. Once all the required information filled, click Submission button.

References Through Sequin

63

Sequin is a stand-alone software tool developed by the National Center for Biotechnology Information (NCBI) for submitting and updating sequences to the GenBank, EMBL, and DDBJ databases. Sequin has the capacity to handle long sequences and sets of sequences (segmented entries, as well as population, phylogenetic, and mutation studies). It also allows sequence editing and updating and provides complex annotation capabilities. In addition, Sequin contains a number of built-in validation functions for enhanced quality assurance. To submit sequence in Sequin, follows these steps: 1. Open Sequin Form. 2. Click to Start New Submission button. 3. Give the tentative title on the submission page. When the article is published, the sequence record will be updated with the new citation. 4. Fill the the name, phone number, and email address of the person responsible for making the submission on the contact page. 5. In the Authors page, enter the names of the people who should get scientific credit for the sequence presented in this record. 6. Fill the institutional affiliation of the primary author in the affiliation page. 7. Before you begin, prepare your sequence data files using a text editor, perhaps one associated with your laboratory sequence analysis software. 8. Select sequence type either Single Sequence or Segmented Sequence. 9. Choose Original Submission if directly sequenced the nucleotide sequence. 10. Choose Third Party Annotation if you have downloaded or assembled sequence from GenBank and modified it with your own annotations. 11. The Nucleotide page will have one of three appearances, based on whether you have chosen to import a single sequence, a set of sequences, or an alignment. 12. Once all the required information is filled, the page should be sent to [email protected].

References 1. Li Q, Chen X, Jiang Y, Jiang CL (2016). Morphological Identification of Actinobacteria. In: Actinobacteria - Basics and Biotechnological Applications, Dharumadurai Dhanasekaran

and Yi Jiang (Ed), 10.5772/61461.

IntechOpen,

DOI:

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2. Radhakrishnan M, Balaji S, Balagurunathan R (2007) Thermotolerant actinomycetes from Himalayan Mountain – antagonistic potential, characterization and identification of selected strains. Malays Appl Biol 36(1):59–65 3. Radhakrishnan M, Gopikrishnan V, Suresh A et al (2013) Characterization and phylogenetic analysis of antituberculous compound producing actinomycete strain D25 isolated from Thar desert soil, Rajasthan. Bioinformation 9 (1):18–22 4. Radhakrishnan M, Gopikrishnan V, Balaji S (2014) Bioprospecting of actinomycetes from certain less explored ecosystems active against Mycobacterium tuberculosis and other non-mycobacterial pathogens. Int Scholarly Res Notices 2014:812974 5. Balagurunathan, R and M. Radhakrishnan. 2007. Actinomycetes: Diversity and their importance. In: Microbiology – Applications and Current Trends. P.C. Trivedi (editor), Pointer publishers, Jaipur, India, Pp: 297–329. 6. Wang Y and Jiang Y (2016) Chemotaxonomy of Actinobacteria, Actinobacteria - Basics and Biotechnological Applications. Dharumadurai Dhanasekaran and Yi Jiang, IntechOpen, DOI: 10.5772/61482. 7. Barka EA, Vatsa P, Sanchez L et al (2015) Taxonomy, physiology, and natural products of Actinobacteria. Microbiol Mol Biol Rev 80 (1):1–43

8. Rahlwes KC, Sparks IL, Morita YS (2019) Cell walls and membranes of Actinobacteria. Subcell Biochem 92:417–469 9. Ser HL, Tan LT, Palanisamy UD et al (2016) Streptomyces antioxidans sp. nov., a novel mangrove soil Actinobacterium with antioxidative and neuroprotective potentials. Front Microbiol 7:899 10. Gago G, Diacovich L, Arabolaza A (2011) Fatty acid biosynthesis in actinomycetes. FEMS Microbiol Rev 35(3):475–497 11. Magarvey NA, Keller JM, Bernan V (2004) Isolation and characterization of novel marine-derived actinomycete taxa rich in bioactive metabolites. Appl Environ Microbiol 70 (12):7520–7529 12. Nikodinovic J, Barrow KD, Chuck JA (2003) High yield preparation of genomic DNA from Streptomyces. Biotechniques 35(5):932–936 13. Vetrovsky T, Baldrian P (2013) The variability of the 16S rRNA gene in bacterial genomes and its consequences for bacterial community analyses. PLoS One 8(2):e57923 14. Dewhirst FE, Shen Z, Scimeca MS et al (2005) Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: implications for phylogenetic inference and systematics. J Bacteriol 187(17):6106–6118

Chapter 4 Screening of Actinobacteria for Biological Activities Abstract Members of the phylum actinobacteria have the tremendous potential to produce various bioactive metabolites and also exhibit different biological activities. Screening is the first step in any microbial bioprospecting program. Screening may be defined as the use of highly selective procedures to allow the detection and isolation of only those microorganisms of interest from among a large microbial population. Thus, screening must in one or a few steps allow the discarding of many valueless microorganisms, while at the same time allowing the easy detection of the small percentage of useful microorganisms that are present in the population. The usual screening procedure includes two stages such as primary screening and secondary screening. Primary screening allows the detection and isolation of microorganisms that possess potentially interesting industrial applications. Primary screening is usually conducted on agar plates. Primary screening determines only which microorganisms are able to produce a compound but not providing much idea of the production or yield potential for the organisms. There are several primary screening methods described in this chapter to detect the various biological activities of actinobacterial strains. Keywords Primary screening, Agar plug, Disk diffusion, Well diffusion, LRP assay, Broth dilution, Anti-TB, Anticancer, Antiviral, Antioxidant, Enzymes, Pigments

1 1.1

Screening of Actinobacteria for Antimicrobial Activity by Agar Plug Method Introduction

Antagonism is a native property of certain microorganisms which can be exhibited by them through the production of secondary metabolites. Based on the determination of antagonistic activity, secondary metabolites from such microorganisms are further isolated, characterized, and explored for the development of antibiotics [1]. In general, antagonism through the production of secondary metabolites is the native property of filamentous organisms like actinobacteria, fungi, and cyanobacteria. Methods like crowded plate technique, cross-streak method, cross-spot method, broth dilution method, agar dilution method, and agar plug method are commonly used to determine the antimicrobial activity of actinobacteria. Agar plug method is a simple method used for the detection of antagonistic activity of actinobacteria. In this method, agar plug with 5 mm diameter is cut from the culture medium grown with the respective bacteria and placed over the

Ramasamy Balagurunathan et al., Protocols in Actinobacterial Research, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0728-2_4, © Springer Science+Business Media, LLC, part of Springer Nature 2020

65

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Screening of Actinobacteria for Biological Activities

nutrient agar/Mueller–Hinton agar medium seeded with test pathogens. Appearance of zone of inhibition around the agar plug after 24 h of incubation at 37
C indicates that the given actinobacteria secreted antagonistic molecules extracellularly into the agar medium and inhibited the test pathogen [2]. 1.2

1.3

Materials

Methods

l

Actinobacterial culture—Streptomyces.

l

Bacterial pathogens—E. coli and S. aureus.

l

ISP2 agar.

l

Nutrient broth.

l

Nutrient agar.

l

Inoculation loop.

l

Sterile cotton swab.

l

Well cutter.

l

Incubator—37
C.

l

Prepare ISP2 agar plates.

l

Inoculate actinobacterial cultures into ISP2 agar plates and incubate at 28
C for 5–7 days.

l

Prepare sterile nutrient broth each 2 ml in test tubes.

l

l

Prepare nutrient agar plates.

l

Spread the bacterial culture from nutrient broth into nutrient agar plates using sterile cotton swab.

l

Take ISP2 agar plates which contain the well-grown actinobacterial cultures.

l

Cut 5 mm agar plug of actinobacterial cultures from ISP2 agar using sterile well cutter.

l

Place the agar plug over the surface of nutrient agar plates inoculated with bacterial pathogens.

l

1.4

Notes

Inoculate bacterial pathogens into it and incubate at 37
C for 18 h.

Incubate all the plates at 37
C for 24 h.

l

Observe all the plates for zone of inhibition around the agar plug, and express in millimeter in diameter (see Table 1).

l

The presence of zone of inhibition around the agar plug indicates the antimicrobial activity of respective actinobacteria.

l

The absence of zone of inhibition around the agar plug indicates that there is no antimicrobial activity exhibited by the given actinobacterial culture.

Screening of Actinobacteria for Antimicrobial Activity by Agar Diffusion Method

67

Table 1 Antibacterial activity Test pathogens (zone of inhibition in mm in dm) S. no.

Actinobacterial strain no.

S. aureus

E. coli

1 2

2

Screening of Actinobacteria for Antimicrobial Activity by Agar Diffusion Method

2.1

Introduction

Selection of suitable screening method greatly influences the success of detecting the antimicrobial activity of microbes or their extracts/compounds. The agar diffusion test is used to assess the antimicrobial activity of microbes, their extracts, and compounds. Agar diffusion refers to the movement of molecules through the matrix that is formed by the gelling of agar. When performed under controlled conditions, the degree of the molecule’s movement can be related to the concentration of the molecule. This phenomenon forms the basis of the agar diffusion assay that is used to determine the susceptibility or resistance of a bacterial strain to an antimicrobial agent [1]. The agar diffusion assay allows bacteria to be screened in a routine, economical and easy way for the detection of resistance. There are two common methods used under this principle which includes disk diffusion method and well diffusion method. In disk diffusion method, the extract- or moleculeimpregnated filter paper disk is placed over the surface of screening agar plates previously seeded with test pathogens, whereas in agar well diffusion method, the cell-free supernatants, natural extract, or the purified compounds dissolved in suitable vehicle solvents are loaded in a well made on screening agar medium seeded with test pathogens. In both methods, the bioactive material loaded in the disk or in the well are diffused into the medium and produce zone of inhibition. Actinobacterial extracts and cell-free supernatants are well studied for antimicrobial activity by both methods [2].

2.2

Materials

l

Actinobacterial culture—Streptomyces.

l

Bacterial pathogens—E. coli and S. aureus.

l

ISP2 broth.

l

Nutrient broth.

l

Nutrient agar.

l

Inoculation loop.

l

Sterile cotton swab.

l

Well cutter.

l

Incubator—37
C.

68

2.3

Screening of Actinobacteria for Biological Activities

Methods

2.3.1 Agar Well Diffusion Method

l

Prepare each 50 ml of ISP2 broth and sterilize by autoclaving at 121
C for 15 min.

l

Inoculate actinobacterial cultures into ISP2 broth, and incubate in rotary shaker at 95 rpm speed 28
C for 5–7 days.

l

Prepare sterile nutrient broth each 2 ml in test tubes.

l

l

Prepare nutrient agar plates.

l

Spread the bacterial culture from nutrient broth into nutrient agar plates using sterile cotton swab.

l

Collect the cell-free supernatant (CFS) of actinobacterial cultures by centrifugation at 10800  g for 10 min.

l

Cut 6 mm well on nutrient agar plates previously inoculated with test bacterial pathogens.

l

2.3.2 Disk Diffusion Method

Observe all the plates for zone of inhibition around the well, and express in millimeter in diameter (see Table 2).

l

Prepare the stock concentration by dissolving the given actinobacterial extract in 10% DMSO in such a way to get 10 mg/ml concentration. Load 10 μl of stock extract to the sterile filter paper disk, and allow to dry under aseptic condition.

l

Prepare the inoculum of test bacterial pathogens using sterile nutrient broth, and adjust to 0.5 McFarland standard.

l

Prepare nutrient agar plates.

l

Spread the bacterial culture from nutrient broth into nutrient agar plates using sterile cotton swab.

l

Place the extract impregnated disc on nutrient agar plates previously inoculated with test pathogens.

l

Notes

Add 100 μl of CFS on to the well and incubate all the plates at 37
C for 24 h.

l

l

2.4

Inoculate the bacterial pathogens into it and incubate at 27
C for 18 h.

Incubate the plates at 37
C for 24 h.

l

Observe all the plates for zone of inhibition around the disk, and express in millimeter in diameter (see Table 2).

l

The presence of zone of inhibition around the well indicates the antimicrobial activity of respective actinobacteria

l

Absence of zone of inhibition around the well indicates that there is no antimicrobial activity exhibited by the given actinobacterial culture

Screening of Actinobacterial Extracts for Antimicrobial Activity by. . .

69

Table 2 Antimicrobial activity Test pathogens (zone of inhibition in mm in dm) S. no.

Actinobacterial strain no.

S. aureus

E. coli

1 2

3 Screening of Actinobacterial Extracts for Antimicrobial Activity by Microbroth Dilution Method and Determination of Minimal Inhibitory Concentration (MIC) 3.1

Introduction

Dilution susceptibility testing methods are used to determine the minimal concentration of antimicrobials to inhibit or kill the microorganism. This can be achieved by dilution of antimicrobial in either agar or broth media. Broth dilution method and agar dilution methods are used for testing MIC. The broth dilution method is a simple procedure for testing a small number of isolates, even single isolate. It has the added advantage that the same tubes can be taken for MBC tests also. Broth dilution is a technique in which a suspension of bacterium of appropriate concentration is tested against varying concentrations of an antimicrobial agent (usually serial twofold dilutions) in a liquid medium. The broth dilution method can be performed either in tubes containing a minimum volume of 2 ml (macrodilution) or in smaller volumes using microtitration plates (microdilution) [1]. Agar dilutions are most often prepared in petri dishes and have the advantage that it is possible to test several organisms on each plate. If only one organism is to be tested, e.g., M. tuberculosis, the dilutions can be prepared in agar slopes, but it will then be necessary to prepare a second identical set to be inoculated with the control organism. The dilutions are made in a small volume of water and added to test agar medium which has been melted and cooled to not more than 60
C. It would be convenient to use 90 mm diameter petri dishes and add 1 ml of desired drug dilutions to 19 ml of broth. Both the broth dilution and the agar dilution method may be labor-intensive if automated equipment is not used [2].

3.2

Materials

l

Actinobacterial extracts.

l

Bacterial pathogens—E. coli and S. aureus.

l

Nutrient broth.

l

Inoculation loop.

l

Sterile cotton swab.

70

3.3

Screening of Actinobacteria for Biological Activities

Methods

l

Sterile filter paper disc (5 mm diameter).

l

Incubator—37
C.

l

l

l

Take eight microfuge tubes of 2 ml capacity and mark from 1 to 8. Add about 900 μl of distilled water to the first tube and 500 μl of distilled water to the remaining tubes. Transfer about 100 μl of 10 mg/ml of actinobacterial extract to the first tube, and mix well. Then transfer 500 μl of the diluted sample from the first tube to the second tube, and continue the dilution till the eighth tube.

l

Suspend the freshly grown colonies of bacterial cultures in 2 ml of sterile nutrient broth, and adjust the turbidity to 0.5 McFarland standards.

l

Take ten sterile glass tubes and mark from T1 to T10.

l

l

l

l

Add each 800 μl of nutrient broth to each of the tubes T1 to T8 and 900 μl of nutrient broth to each of T9 and T10 tubes. Add 100 μl of 0.5 McFarland’s standard cells to the tubes T1 to T9, and subsequently add 100 μl of diluted actinobacterial extract from the working stock added to T1 to T8. Consider the tubes T9 and T10 as culture control and medium control, respectively. Incubate all the tubes at 37
C for 18–24 h, and then observe for growth inhibition by spectroscopically at 600 nm (see Table 3).

Table 3 Antimicrobial activity Inhibition of test pathogens S. no.

Actinobacterial extract/compound [μg/ml]

1

100

2

50

3

25

4

12.5

5

6.25

6

3.125

7

1.6

8

0.78

S. aureus

E. coli

Screening of Actinobacterial Cultures for Antimycobacterial Activity Using. . .

3.4

Notes

71

The lowest concentration of extract showing the inhibition against the test pathogens is considered as minimal inhibitory concentration (MIC).

4 Screening of Actinobacterial Cultures for Antimycobacterial Activity Using Mycobacterium smegmatis 4.1

Introduction

Agar plug diffusion method is often used to highlight the antagonism between microorganisms, and the procedure is similar to that used in the disk diffusion method. It involves making an agar culture of the strain of interest on its appropriate culture medium. During their growth, microbial cells secrete molecules which diffuse in the agar medium. After incubation, an agar plug or cylinder is cut aseptically with a sterile cork borer and deposited on the agar surface of another plate previously inoculated by the test microorganism [3]. The substances diffuse from the plug to the agar medium. Then, the antimicrobial activity of the microbial secreted molecules is detected by the appearance of the inhibition zone around the agar plug. Screening extracts against M. tuberculosis can be an aseptically rigorous procedure owing to its extremely infective nature and it requires a biological safety level 3 (BSL3) cabinet . M. tuberculosis also grows slowly, with a generation time of 16–24 h. The slow-growing and highly infectious nature of M. tuberculosis has slowed down the discovery of new anti-TB agents. In previous study, Vinita et al. (2007) used the noninfective M. smegmatis because of its fast-growing nature and basic similarities with M. tuberculosis. These results highlight the utility of M. smegmatis as surrogate host for primary screen to shortlist compounds for advanced screening against MDR M. tuberculosis [4].

4.2

Materials

l

Actinobacterial cultures.

l

ISP2 agar plates.

l

G7H9 broth.

l

G7H11 agar.

l

Albumin dextrose.

l

0.5% Glycerol.

l

Inoculate the given actinobacterial cultures on to ISP2 agar plates.

4.3

Methods

l l

Incubate the ISP2 agar plates at 28
C for 7–14 days. Inoculate a loopful of M. smegmatis culture into 0.3 ml of Middlebrook 7H9 broth in a sterile Bijou bottle added with glass beads.

72

Screening of Actinobacteria for Biological Activities l

l

Cut 5 mm plug of actinobacterial culture from ISP2 agar using sterile cork borer.

l

Place the actinobacterial plug over the surface of 7H11 agar plates previously inoculated with M. smegmatis culture.

l

Notes

Then add 200 μl of M. smegmatis suspension to 5 ml of molten 7H11 agar and pour immediately onto Middlebrook 7H9 agar plate.

l

l

4.4

Vortex the M. smegmatis suspension for 30 seconds and keep undisturbed for 5 minutes and then make up the suspension up to 5 ml using Middlebrook 7H9 broth.

Incubate the plate at 28
C for 48–72 h. Observe the plates for zone of inhibition around the actinobacterial plug.

The presence of inhibition zone around the actinobacterial plug indicates its inhibitory activity against M. smegmatis, whereas the absence of inhibition zone indicates the absence of inhibitory activity.

5 Screening of Actinobacterial Extracts/Compounds for Antimycobacterial Activity by Luciferase Reporter Phage (LRP) Assay 5.1

Introduction

With the increasing need for drugs to combat TB, there is an urgent need for rapid, low-cost, high-throughput assays for screening of new drug candidates. Due to the slow growth of M. tuberculosis, incubation times for drug susceptibility assays which rely on the development of colonies or turbidity are excessively long. Moreover, conventional method using LJ medium cannot be used for testing novel extracts and compounds as their heat stability is not known: it requires mixing these novel compounds with the egg medium and inspissating it [5]. There has been a number of mycobacterial drug susceptibility assays described over the period of time and also used for the screening of natural products for anti-TB activity. These include the classical disk diffusion method, agar dilution methods, and broth dilution assay, radiometric (BACTEC), dye-based, and fluorescent/luminescent reporter assays. Most of these assays, however, lack one or more of the attributes of a mass screening assay: rapidity, high throughput, and low cost of the supplies and equipment [6]. Assay strategies employing reporter phages are an attractive alternative to cumbersome conventional method. Drug susceptibility was assessed based on efficient production of photons by viable mycobacteria infected with specific reporter phages expressing firefly luciferase gene. In the presence of drugs, resistant organisms continue to produce light, while susceptible organisms do not, as

Screening of Actinobacterial Extracts/Compounds for Antimycobacterial. . .

73

they get killed [5]. The first LRP constructed was TM4-derived phage that is capable of infecting both fast and slow-growing mycobacteria. LRP assay is a rapid, less laborious, and less timeconsuming method for high-throughput screening of a large number of compounds for antimycobacterial activity [6]. Overall 480 extracts were tested, and about 99% agreement was observed between luciferase assay and microplate alamarBlue assay. Investigators also found that drug activities in LRP assay were parallel to MIC determined by conventional and BACTEC 460. Natural products from various sources and synthetic compounds are being screened for antimycobacterial activity by adopting LRP assay. Antimycobacterial activity of chalcone derivatives, novel 1,3,5-triphenyl 1-2-pyrazolines, quinoline-coupled 1,2,3triazoles, and novel 4-(morpholin-4-yl)-N0 -(arylidene) benzohydrazides were reported by using LRP assay. Radhakrishnan et al. (2010; 2011; 2014) screened the crude extracts prepared from terrestrial and marine actinomycetes against standard and clinical isolates of M. tuberculosis using LRP assay. Crude solvent extracts prepared from selected marine fungal isolates were also screened for M. tuberculosis H37Rv using LRP assay [7]. Assay strategies employing reporter phages are an attractive alternative to cumbersome conventional method. Drug susceptibility was assessed based on efficient production of photons by viable mycobacteria infected with specific reporter phages expressing firefly luciferase gene. In the presence of drugs, resistant organisms continue to produce light, while susceptible organisms do not, as they get killed [8]. 5.2

5.3

Materials

Methods

5.3.1 Preparation of Actinomycete Extracts

l

Extracts from actinobacterial strains.

l

Test organisms—standard strain M. tuberculosis H37Rv.

l

G7H9 broth.

l

Albumin dextrose.

l

0.5% Glycerol.

l

DMSO.

l

D-luciferin.

l

phAE129.

l

Luminometer.

l

Lowenstein–Jensen (LJ) medium.

l

Inoculate the actinobacterial culture into YEME agar plates (20 ml/plate), and incubate at 28
C for 7–14 days.

l

Cut the whole agar medium into pieces and extract using 50 ml of methanol.

74

Screening of Actinobacteria for Biological Activities

Phage Propagation and Calculation of Phage Titer

l

After 24 h, collect the methanol portion and concentrate using Eppendorf concentrator at 45
C.

l

Measure the quantity of extract using pre-weighed Eppendorf tubes.

l

Make the decimal dilutions of the phage phAE129/phAE TRC202 by diluting 50 μl of the phage lysate in 500 μl of MP buffer.

l

l

l

l l

l

l

Prepare the suspension of M. smegmatis mc2155 in Middlebrook 7H9 complete medium to a turbidity of 0.8 OD at 600 nm equivalent to 5  108 cfu/ml. Add 500 μl of the cell suspension to one in ten dilutions of the phage, and incubate the mixture for 30 min at 37
C. After incubation, remove 200 μl from the mixture, and mix with 5 ml of 7H9 top agar at 50
C, and pour on 7H9 base agar plates. Incubate the plates at 37
C overnight. Flood the plates showing a “lacey” pattern of plaques (approximately 300 plaques) with about 5 ml of MP buffer each, and allow to stand for 4 h at room temperature. Aspirate the lysate and filter by passing through 0.45 μm membrane filter. For calculating the titer, include higher dilutions of phage during propagation and set up in triplicates. Select plates showing approximately 30 plaques. Count actual number of plaques, and calculate the average. Calculate the titer using the following formula: No:of plaques  10  Dilution factor ¼ plaque forming units=ml:

Preparation of Extracts and Cell Suspensions

l

l

l

Prepare the stock solution of crude extracts (10 mg/ml) using 10% dimethyl sulfoxide (DMSO), and filter using 0.45 μm filters. Dilute about 100 μl of stock extract solution in 900 μl of sterile distilled water to get 1 mg/ml concentration of working solution. Prepare the cell suspension equivalent to #2 MacFarland units by inoculating the log phase culture of M. tuberculosis from LJ slope into G7H9 broth in Bijou bottles with few glass beads and vortex.

Screening of Actinobacterial Extracts/Compounds for Antimycobacterial. . . LRP Assay

l

l

75

Take about 350 μl of G7H9 broth supplemented with 10% albumin dextrose complex and 0.5% glycerol in 2 ml cryo vials. Add 50 μl of crude extract in order to get the final concentration of 100 μg/ml.

l

Add hundred microliters of M. tuberculosis H37Rv cell suspension to all the vials.

l

Follow the above procedure for all the three drug-sensitive and MDR M. tuberculosis isolates. Include DMSO (1%) also in the assay as solvent control.

l l

l l

l

l

Incubate all the vials at 37
C for 72 h. After incubation, add 50 μl of high-titer mycobacteriophage phAE129/phAE TRC202 and 40 μl of 0.1 M CaCl2 solution in to the test and control vials. Incubate all the vials at 37
C for 4 h. After incubation, transfer 100 μl of reaction mixture from each vial into luminometer cuvette. Add 100 μl of D-Luciferin, and measure the relative light unit (RLU) using luminometer (see Table 4). Calculate the % of inhibition using the following formula: Percentage RLU reduction ¼ Control RLU  Test RLU=Control RLU  100

5.4

Notes

Extracts showing RLU reduction by 50% or more when compared to control were considered as having antitubercular activity.

Table 4 Antimycobacterial activity

Extract no

% Reduction in RLU MTB H37Rv

MTB drug sensitive

MDR MTB

76

Screening of Actinobacteria for Biological Activities

6 Screening of Actinobacteria for Anti-TB Activity by Microplate AlamarBlue Assay (MABA) 6.1

Introduction

Dilution bioassays have one major advantage over diffusion bioassays, namely, the test compound concentration in the medium is defined. In addition, the dilution assays are regarded as the method of choice to study the MIC of given drugs. The advent of microtiter plates has led to significant reductions in test compound concentrations. However, test samples that are not fully soluble may interfere with turbidity readings, making interpretation difficult, emphasizing the need for a negative control or sterility control, i.e., extract dissolved in blank medium without microorganisms. An important alternative can be the use of oxidation/reduction indicator dye like alamarBlue with which the growth/inhibition can be read visually and the reduced form of these dyes can also be quantitated colorimetrically by measuring absorbance at 570 nm or fluorimetrically by exciting at 530 nm and detecting emission at 590 nm [9]. In general, dilution methods are appropriate for assaying polar and nonpolar extracts or compounds to determine MIC and MBC/MFC-values. The alamarBlue oxidation–reduction dye is a general indicator of cellular growth and/or viability; the blue, nonfluorescent, oxidized form becomes pink and fluorescent upon reduction. Growth can therefore be measured with a fluorometer or spectrophotometer or determined by a visual color change. Moreover, alamarBlue has been used as a nonradioactive assay used and compared with other anti-TB screening assays [10].

6.2

Materials

l

Middlebrook 7H9 broth with supplements.

l

Middlebrook 7H 11 broth.

l

M. tuberculosis H37Rv.

l

Actinobacterial extract.

l

DMSO.

l

AlamarBlue reagent.

Methods

l

6.3.1 M. tuberculosis Culture Preparation

Prepare 10 ml of Middlebrook 7H9 broth supplemented with 10% oleic acid–albumin–dextrose–catalase and 0.2% of glycerol.

l

Inoculate the 0.4–0.6 ml of freezer stock of M. tuberculosis H37Rv in a 50 ml conical tube.

6.3

l

l

Grow the culture to mid-log phase on the wheel at 37
C, until OD600 ¼ 0.4–0.8. Dilute the culture using 7H9 broth without Tween 80 to OD600 ¼ 0.001. This results in a culture with approximately 105 CFU/ml.

Screening of Actinobacteria for Anti-TB Activity by Agar Dilution Assay l

6.3.2 MABA

Dilute the working solutions of the test extracts in Middlebrook 7H9 broth supplemented with oleic acid–albumin–dextrose– catalase to obtain the final sample concentrations ranged from 250 μg/ml to 10 mg of sample to a clean NMR tube. 2. Dissolve the sample in 0.7 ml NMR-grade solvent (CDCl3). 3. Cap the NMR tube carefully and write the sample name on the cap. 4. Shake the sample gently to dissolve all the materials completely. 5. Clean the outside of the NMR tube and spinner with 2-propanol and lab tissues in order to remove fingerprints and dirt. Insert the NMR tube carefully into a spinner. 6. Place the spinner in a sample depth gauge to ensure that the bottom of the NMR tube is not inserted too far into the NMR probe. 7. Place the sample in the NMR spectrometer and run. 8. After completion of the NMR measurement, process the spectrum, and assign the peaks in the spectrum.

6.4

Notes

1. Process the spectrum with a suitable program. Also the operational steps may be vary one instrument to another, based on the make and model. 2. Correlate the different peaks to the NMR shifts in Table 2. The chemical shifts gives a hint of what type of environment the protons exist in. 3. Integrate the peaks to give the number of hydrogens corresponding to each peak. Integration of all peaks gives a relative number of total protons.

X-Ray Diffraction (XRD) Analysis of Actinobacterial Metabolites

161

Table 2 Common proton and carbon NMR chemical shifts Type of proton

Shift (δ, ppm)

Type of carbon

Shift (δ, ppm)

1. Alkyl, RCH3

0.8–1.2

1. Alkyl, RCH3

2. Alkyl, R2CH2R

1.2–1.5

2. Alkyl, R2CH2R

10–50

3. Alkyl, RCHR2

1.4–1.8

3. Alkyl, RCHR2

15–50

Allylic, R2C¼CRCH3

1.6–1.9

Alkene, C¼C

100–170

Ketone, RC(¼O)CH3

2.1–2.6

Aryl, C in aromatic ring

100–170

Ether, ROCH2R

3.3–3.9

Alcohol or ether, R3COR

Alcohol, HOCH2R

3.3–4.0

Carboxylic acid or ester, RC(¼O)OR

160–185

Vinylic, R2C¼CH2

4.6–5.0

Aldehyde or ketone, RC(¼O)R

182–215

Vinylic, R2C¼CRH

5.2–5.7

Aromatic, Ar H

6.0–8.5

Aldehyde RC(¼O)H

9.5–10.5

Alcohol hydroxyl, ROH

0.5–6.0

Carboxylic, RC(¼O)OH

10–13

0–40

50–90

4. Evaluate the splitting of the proton peaks, which indicate the number of neighbors. 5. Measure the J-coupling to see how the protons are connected to each other.

7 7.1

X-Ray Diffraction (XRD) Analysis of Actinobacterial Metabolites Introduction

X-ray diffraction is used extensively to determine the structure of crystalline and also of noncrystalline and amorphous materials. For crystalline materials, the directions (θ) and wavelength (λ) of the diffracted beams are given by Bragg’s law; this is 2d sin θ ¼ nλ where n is the order of diffraction from crystalline lattice planes of spacing d. The equation may be rewritten as 2dn sin θ ¼ λ where dn ¼ d/n and the equation now represents first order diffraction from planes d/n. Using Bragg’s law, a measurement of θ and λ for a diffracted beam enables the spacing dn to be determined. For cubic lattices:

162

Bioassay-Guided Isolation and Characterization of Metabolites from Actinobacteria

d ¼ a √h2 þ k2 þ l2 where a is the side length of the cubic lattice cell (the lattice constant) and h, k, and l are the Miller indices of the lattice planes [11, 12]. 7.2

Methods

l

Install the powdered crystalline target and repeat the beam– target–detector alignment.

l

Accumulate data and store the resulting spectrum.

l

Change the detector angle by a few degrees and repeat the accumulation.

l

The diffraction peaks shift position in the spectrum; the XRD peaks do not.

l

After this provisional classification into XRD and diffraction lines, keep the detector angle fixed, and accumulate until a “smooth” spectrum is obtained.

l

A “smooth” spectrum is a very qualitative term which simply means that the center of the peaks of interest may be determined by visual inspection to approximately 1 channel.

l

It is useful to keep an eye on the X-ray machine current and voltage readings during accumulation since occasionally the machine trips OFF; if this occurs, restart.

l

When the accumulation is completed, CLOSE the X-ray beam shutter, and turn the X-ray current and HV OFF.

l

Determine the energies of the XRD and diffraction peaks.

l

Using the characteristic X-ray energy tables, identify the elements in the powdered sample. Also the operational steps may be vary one instrument to another, based on the make and model.

7.2.1 Alignment Spectrum Accumulation

7.2.2 Data Analysis

References 1. Dehnad A, Hamedi J, Derakhshan-Khadivi F et al (2015) Green synthesis of gold nanoparticles by a metal resistant Arthrobacter nitroguajacolicus isolated from gold mine. IEEE Trans Nanobioscience 14(4):393–396 2. Gopikrishnan V, Radhakrishnan M, Shanmugasundaram T et al (2019) Isolation, characterization and antifouling activity of taxifolin from

Streptomyces sampsonii PM33 isolated from mangrove ecosystem, South India. Nat Sci Rep 9:12975 3. Choma IM, Grzelak EM (2011) Bioautography detection in thin-layer chromatography. J Chromatogr A 1218(19):2684–2691 4. Nunez-Montero K, Lamilla C, Abanto M et al (2019) Antarctic Streptomyces fildesensis So13.3

References strain as a promising source for antimicrobials discovery. Sci Rep 9(1):7488 5. Raghava Rao KV, Mani P, Satyanarayana B et al (2017) Purification and structural elucidation of three bioactive compounds isolated from Streptomyces coelicoflavus BC 01 and their biological activity. 3 Biotech 7(1):24 6. Uzair B, Menaa F, Khan BA et al (2018) Isolation, purification, structural elucidation and antimicrobial activities of kocumarin, a novel antibiotic isolated from actinobacterium Kocuria marina CMG S2 associated with the brown seaweed Pelvetia canaliculata. Microbiol Res 206:186–197 7. Atta HM (2015) Biochemical studies on antibiotic production from Streptomyces sp.: taxonomy, fermentation, isolation and biological properties. J Saudi Chem Soc 19(1):12–22

163

8. Shaik M, Girija Sankar G, Iswarya M et al (2017) Isolation and characterization of bioactive metabolites producing marine Streptomyces parvulus strain sankarensis-A10. J Genet Eng Biotechnol 15(1):87–94 9. Graham Solomons TW, Fryhle CB (2011) Organic chemistry, 10th edition, Wiley, p 387 10. Clayden J, Greeves N, Warren S, Wothers P (2001) Proton nuclear magnetic resonance. Organic chemistry, Chapter 11, Oxford University Press, 269 11. Lenz EM (2010) Nuclear magnetic resonance (NMR)-based drug metabolite profiling. Methods Mol Biol 708:299–319 12. Dass R, Grudzia ZK, Ishikawa T et al (2017) Fast 2D NMR spectroscopy for in vivo monitoring of bacterial metabolism in complex mixtures. Front Microbiol 8:1306

Chapter 9 Evaluation of Actinobacteria for Environmental Applications Abstract Environmental pollution due to toxic pollutants is the major problem around the world. The use of chemical and physical approach for environmental cleanup is detrimental to the environment as well as to the associated microorganisms. In recent years microorganisms are widely explored to fight against the environmental pollution. Actinobacterial members are widely distributed in various environments including the extreme ecosystems. This is mainly due to their ability to degrade and utilize a wide range of complex and recalcitrant substrates by means of producing several enzymes. Several actinobacterial genera like Streptomyces, Nocardia, and Rhodococcus are reported to resist as well as accumulate different heavy metals in the environment. There are several Streptomyces strains reported to degrade environmental pollutants such as pesticides, textile dyes, hydrocarbons, etc. These properties made them to explore as potential candidates to clean up to toxic pollutants from the environment. Recently several studies reported that actinobacterial extracts/compounds are capable of exhibiting anti-biofouling activity against macroand microfouling organisms. Interestingly, certain members of actinobacteria are reported to produce carbonic anhydrase—an important enzyme in CO2 sequestration. In this chapter, methods for screening actinobacteria for biodegradation properties, antibiofouling activity, as well as carbonic anhydrase production will be studied. Keywords Metal resistance, Dye degradation, Pesticide, Antifouling, Anticorrosion, Carbonic anhydrase

1 1.1

Screening of Actinobacteria for Heavy Metal Resistance/Accumulation Introduction

Microorganisms are also dominating the heavy metal-contaminated soil where they can easily convert heavy metals into nontoxic forms. Microorganisms mineralize the organic contaminants to end products such as carbon dioxide and water or to metabolic intermediates during the bioremediation process. Microorganisms can produce various enzymes for the target pollutants as well as they can resist high concentrations of heavy metals in the contaminated environments. Different mechanisms of bioremediation exhibited by microbes include biosorption, metal–microbe interactions, bioaccumulation, biomineralization, biotransformation, and bioleaching. They are capable of dissolving metals and reducing or oxidizing transition metals. Different methods by which microbes restore the environment are oxidizing, binding, immobilizing, volatizing, and transformation of heavy metals [1, 2].

Ramasamy Balagurunathan et al., Protocols in Actinobacterial Research, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0728-2_9, © Springer Science+Business Media, LLC, part of Springer Nature 2020

165

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Evaluation of Actinobacteria for Environmental Applications

Table 1 Heavy metal accumulation/resistance Heavy metal concentration (ppm) S. no.

100

300

500

700

900

1100

1300

1500

Arsenic Chromium Nickel

1.2

1.3

1.4

Materials

Methods

Notes

l

Actinobacterial cultures.

l

Lab-grade heavy metals—arsenic, chromium, and nickel.

l

Conical flask.

l

ISP2 agar.

l

ISP2 broth.

l

UV-visible spectrophotometer.

l

Prepare ISP2 agar medium with different concentration of heavy metal (100–1500 ppm), and sterilize at 121
C for 15 min.

l

After the sterilization pour the agar medium into sterile petri dish and allow to solidify.

l

Spot actinobacterial cultures on the agar medium and incubate at 28
C for 7-14 days. Then observe the growth of actinobacterial cultures.

l

On the other hand, prepare 95 ml of ISP2 broth with 3 ml of 100 ppm of metal solution, and sterilize the medium.

l

Then add 2 ml of given actinobacterial cultures, and incubate at 28
C for 120 rpm in shaking incubator for 7-14 days.

l

Measure the OD of the sample for every 24-h interval at 600 nm by using UV-visible spectrophotometer (see Table 1) and compare with the actinobacterial culture in ISP2 broth alone as control.

The strain which shows good growth in medium supplemented with maximum concentration of heavy metals would be considered as heavy metal resistant or tolerant strain.

Biodegradation of Pesticides by Actinobacteria

2

167

Biodegradation of Pesticides by Actinobacteria

2.1

Introduction

The complete biodegradation of the pesticide involves the oxidation of the parent compound resulting into carbon dioxide and water; this provides energy to microbes. In the soil where innate microbial population cannot be able to manage pesticides, the external addition of pesticide-degrading microflora is recommended. Degradation of pesticides by microbes not only depends on the enzyme system but also the conditions like temperature, pH, and nutrients. Some of the pesticides are easily degraded; however, some are recalcitrant because of the presence of anionic species in the compound. Enzymes are also involved in the degradation of pesticide compounds, both in the target organism, through intrinsic detoxification mechanisms and evolved metabolic resistance, and in the wider environment, via biodegradation by soil and water microorganisms [3, 4].

2.2

Materials

l

Actinobacterial strain.

l

Pesticide-organochlorine.

l

Conical flask.

l

Mineral salt agar (MSA) medium.

l

Mineral salt liquid medium.

l

UV-visible spectrophotometer.

l

Prepare the mineral salt agar medium with 100 mg/l of pesticide (organochlorine).

l

Then inoculate the given actinobacterial strains as spot on the mineral salt medium.

l

Prepare the MSA medium without pesticide and inoculate the actinobacterial strain to serve as control.

2.3

Methods

l

2.4

Notes

Incubate the plates at 28
C for 7-14 days.

l

Prepare the mineral salt liquid medium with 20, 40, 60, 80, 100, 300, 500 and 700 mg/l concentrations of organochlorine.

l

Add 2 ml of actinobacterial strain in mineral salt liquid medium, and incubate at 28
C for 7-14 days.

l

Measure the OD of mineral salt liquid samples with 1-, 3-, 5-, 7-day intervals using UV- visible spectrophotometer at 590 nm and compare with the actinobacterial culture in ISP2 broth alone as control (see Table 2).

l

The strain which shows good growth in the medium supplemented with maximum concentration of pesticide is considered as resistant or tolerant strain.

168

Evaluation of Actinobacteria for Environmental Applications

Table 2 Pesticide degradation Pesticide concentration (mg/l) S. no.

20

40

60

80

100

300

500

700

Strain 1 Strain 2 Strain 3 Strain 4 Strain 5

3

l

Care should be taken while handling the pesticides.

l

It is advisable to wear mask while handling the pesticides.

Isolation of Dye-Decolorizing Actinobacteria by Using Dilution Plate Method

3.1

Introduction

3.2

Materials

Microbial discoloration can occur via two principal mechanisms, biosorption and enzymatic degradation, or a combination of both have been used to remove dyes. In order to develop a practical bioprocess for treatment of dye wastewater, it’s necessary to isolate and investigate the microorganisms capable of degrading dyes. Microbial or enzymatic decolorization are eco-friendly and, costeffective when compared the physicochemical method [5]. Two mechanisms in bacteria for the decolorization of dyes under anaerobic conditions. The first one consists of direct electron transfer to dyes as terminal acceptors via enzymes during bacterial catabolism connected the ATP generation. The second one involves a free reduction of dyes by the end products of bacterial catabolism, not linked to ATP generation. During anaerobic degradation, a reduction of the bond in the molecules is observed. Then, aerobic conditions are required for the complete mineralization of the reactive dye molecule. The aromatic compounds produced by the initial reduction are degraded via hydroxylation, and opening in the process is necessary in which oxygen is introduced after the initial anaerobic reduction of the bond has taken place [5]. 1. Soil sample. 2. Synthetic dyes - malachite green. 3. Petri plates. 4. Starch casein agar medium. 5. Test tube. 6. Sterile distilled water.

Screening the Actinobacterial Compounds for Anticorrosion Activity

3.3

3.4

4 4.1

Methods

Notes

169

l

Prepare the dye solution at the ratio of 100 mg/l of water and sterilize.

l

Then prepare 100 ml of the starch casein agar (SCA) medium by supplementing 100 μl of malachite green dye solution.

l

Mix 1 g of soil with 100 ml of sterile distilled and serially dilute up to 106 using 9 ml distilled water blanks.

l

Take 0.1 ml of sample and spread over the agar plate and incubate at 28
C for 4–7 days

l

Recover and preserve the actinobacterial colonies showing zone of decolourization on SCA plates

l

Care should be taken while handing the dyes.

l

Wear protective cloths and gloves while handling the dyes.

Screening the Actinobacterial Compounds for Anticorrosion Activity Introduction

The factors that generally influence corrosion of reinforcement in concrete structures are pH value, carbonation of concrete, chlorides, moisture, oxygen, ambient temperature and relative humidity, severity of exposure, quality of construction materials, permeability of concrete, cover to reinforcement, initial curing conditions, formation of cracks, high carbon content in the reinforcement, high stress levels, inadequate grouting of prestressed tendons, rusted reinforcement prior to embedment, alkali aggregate reaction, potential difference associated with liquid, contact with other metals, stray currents, and absence of periodical maintenance. Inhibitors are substances or mixtures that in low concentration and in aggressive environment inhibit, prevent, or minimize the corrosion [6]. The following are some of the mechanisms by which the corrosion inhibitor works: l

Oxidation by passivation of the surface

l

Formation of barrier layers

l

Influencing the environment in contact with the metal

The most commonly used inorganic inhibitors include silicates, chromates, arsenates, carbonates, nitrites, and phosphates. Organic inhibitors can be extracted directly from natural sources like plants and of synthetically manufactured [7]. 4.2

Materials

l

Metal steel.

l

Actinobacteria.

l

Petri plates.

170

4.3

Evaluation of Actinobacteria for Environmental Applications

Methods

l

Starch casein agar medium.

l

Test tube.

l

Sterile distilled water.

l

The corrosion study is done for the steel specimen. This study is used to evaluate the inhibition efficiency of actinobacterial extracts.

l

Accelerated corrosion techniques are used to induce the corrosion in specimens within a reasonable time limit.

l

This method helps us to control the corrosion in steel rod.

l

Take a steel specimen and stainless steel of 12 mm diameter and immerse in 3.5% NaCl solution act as an anode and cathode.

l

The length of the steel specimen and stainless steel is 20 cm. Immerse 10 cm of specimen in solution.

l

Connect the positive terminal of the DC supply to anode (steel) and the negative terminal to cathode (stainless steel).

l

Pass the 1.5 V current to the specimens for the period of 48 h.

l

Directly pass the 1.5 V current to cathode then through the electrolytic solution (3.5% NaCl); it reaches anode and gets corroded.

l

After the corrosion test, clean all steel specimens per ASTM G1-03. Finally, to determine the % of weight loss, initial weight is to be noted before the initiation of test and after the test period of 48 h.

l

Note the final weight. %Weight Loss ¼ ððW 1  W 2Þ=W 1Þ∗100% where, W1 ¼ Initial Weight W2 ¼ Final Weight

l

4.4

Notes

From this formula, % weight loss can be determined for steel specimen. Follow the same methods for steel coated with actinobacterial extracts or compounds.

The inhibition efficiency of the extracts shows that % weight loss of the extract is lesser than other coatings as well as the control steel specimen. So that extract will be considered as promising anticorrosion agent.

Screening the Actinobacterial Compounds for Antifouling Activity

5

171

Screening the Actinobacterial Compounds for Antifouling Activity

5.1

Introduction

Antagonism is a native property of certain microorganisms which can be exhibited by them through the production of secondary metabolites. Based on the determination of antagonistic activity, secondary metabolites from such microorganisms are further isolated, characterized, and explored for the development of antibiotics. In general, antagonism through the production of secondary metabolites is the native property of filamentous organisms like actinobacteria, fungi, and cyanobacteria. The anti-biofilm effects of natural products are mainly relying on the following aspects, the inhibition of formation of polymer matrix, suppression of cell adhesion and attachment, interrupting extracellular matrix (ECM) generation, and decreasing virulence factors production, thereby blocking quorum sensing (QS) network and biofilm development [8, 9].

5.2

Materials

l

Biofouling bacteria, algae (Nostoc sp.,), and mollusk (Perna indica).

l

Nutrient agar.

l

ISP2 agar.

l

Petri plates.

l

Actinobacteria.

l

Forceps.

l

Chamber Neubauer.

l

PVC panel.

l

Boat wooden part.

l

Test the ethyl acetate extract of the actinobacterial strain for antimicrobial activity against biofilm forming bacteria by disk diffusion method.

l

Impregnate the sterile filter paper disks (5 mm diameter) with crude actinobacterial extracts, and allow it for drying.

l

Then place the disks on the surface of nutrient agar plates inoculated with overnight broth culture of the biofouling bacteria.

5.3

Methods

5.3.1 Activity of Extracts on Biofouling Bacteria

l

5.3.2 Algal Spore Assay

Incubate the plates at 28
C for 48 h. Measure the zone of inhibition after incubation.

l

Treat the algal spores with the extract at the concentrations of 1000, 100, and 10 μg/ml in 96-well microtiter plate.

l

Incubate plate in dark condition for 6 h.

172

Evaluation of Actinobacteria for Environmental Applications l

5.3.3 Mollusk Foot Adherence Assay

5.3.4 Field Assay

5.4

Notes

After the treatment period, incubate the plates at 18
C for 16 h: 8 h, light; dark cycle for 6 days.

l

Observe the spores under light microscope (400) (see Table 3) and compare with the algal spore counts of untreated control. Then, calculate the reduction of algal spores % using the formula: (No. of spores in control - No. of Spores in Test/ No. of Spores in Control) x 100.

l

Collect the specimens of Perna indica from the coastal area.

l

Select and place on a polyvinyl petri plate, and add 20 ml of seawater with various concentrations (200–1000 μg/ml) of extract.

l

Maintain the healthy mollusks in seawater without extract as control.

l

Perform the experiments in triplicate with mild aeration.

l

Not supplement the tests and controls with any additional feed during the assay period.

l

After 24 h, study the anti-adherence property of extract on mollusks (see Table 4).

l

Note EC50 and LC50 value.

l

Take the wooden parts of boats or PVC panels.

l

Mix the phytogel with actinobacterial extract.

l

Spot the extract containing phytogel on the wooden and PVC panels.

l

Keep the treated and untreated (control) panels for 1–2 months in marine environment and observe for biofouling inhibition and compare with control.

l

Presences of zone of inhibition around the disks indicate the antifouling activity.

the

desired

concentration

Table 3 Algal spore assay

Extract concentrations [µg/ml]

Reduction of algal spore (%)

of

Screening the Actinobacterial Compounds for CO2 Sequestration

173

Table 4 Mollusk foot adherence assay Extract concentrations

Mollusk (24–48h)

[µg/ml]

6

Active

Inactive

Dead

l

Reduction of algal spore germination indicates the antifouling activity.

l

Inhibition of adherence property of mollusk indicates antifouling activity.

l

No growth around the spot in the panels indicates antifouling activity

Screening the Actinobacterial Compounds for CO2 Sequestration

6.1

Introduction

Sequestration of carbon dioxide (CO2) is defined as the capture and long-term storage of this greenhouse gas such that it is removed from the atmosphere. Many microorganisms have the ability to sequester atmospheric CO2. Some microorganisms such as autotrophic bacteria can fix CO2 by non-photosynthetic pathway. In addition, microorganisms can be used to generate bioenergy. The advantages of using microorganisms for CO2 sequestration are (1) rapid production; (2) high photosynthetic conversion; (3) high capability for environmental bioremediation, such as CO2 biofixation from the atmosphere or flue gas; and (4) high capacity to produce a wide variety of additive products. Some previous studies reported that many actinobacteria contain putative carbonic anhydrase genes from more than one class and some even contain genes from all three known classes [10, 11].

6.2

Materials

l

Soil samples.

l

ISP2 agar supplemented with 3 mM para-nitro phenyl acetate (p-NPA).

l

Petri plates.

l

Actinobacteria.

6.3

Methods

Screen the carbon sequestration ability of actinobacteria by analyzing carbonic anhydrase production. Supplement the ISP2 agar medium with 3 mM para-nitro phenyl acetate (p-NPA).

174

Evaluation of Actinobacteria for Environmental Applications

Spot the actinobacterial culture on the above medium and incubate at 28
C for 5–7 days. 6.4

Notes

l

Appearance of yellow coloration around the actinobacterial growth indicates the production of carbonic anhydrase.

References 1. Dixit R Wasiullah, Malaviya D, Pandiyan K, Singh UB et al (2015) Bioremediation of heavy metals from soil and aquatic environment: an overview of principles and criteria of fundamental processes. Sustainability 7:2189–2212 2. El Baz S, Baz M, Barakate M et al (2015) Resistance to and accumulation of heavy metals by actinobacteria isolated from abandoned mining areas. Sci World J 2015:1–14 3. Schrijver AD, Mot RD (1999) Degradation of pesticides by actinomycetes. Crit Rev Microbiol 25(2):85–119 4. Briceno G, Fuentes MS, Saez JM et al (2018) Streptomyces genus as biotechnological tool for pesticide degradation in polluted systems. Crit Rev Environ Sci Technol 48(10–12):773–805 5. Lavanya C, Dhankar R, Chikara S, Sheoran S (2014) Degradation of toxic dyes: a review. Int J Curr Microbiol App Sci 3(6):189–199 6. Rosa JP, Tibu´rcio SR, Marques JM et al (2016) Streptomyces lunalinharesii 235 prevents the formation of a sulfate-reducing bacterial biofilm. Braz J Microbiol 47(3):603–609

7. Agarwal A, Mehra A, Karthik L et al (2014) Antibiofouling property of marine actinobacteria and its mediated nanoparticle. Int J Nanopart 7(3/4):294 8. Gopikrishnan V, Radhakrishnan M, Shanmugasundaram et al (2016) Antibiofouling potential of quercetin compound from marinederived actinobacterium, Streptomyces fradiae PE7 and its characterization. Environ Sci Pollut Res 23(14):13832–13842 9. Gopikrishnan V, Radhakrishnan M, Shanmugasundaram et al (2019) Isolation, characterization and antifouling activity of taxifolin from Streptomyces sampsonii PM33 isolated from mangrove ecosystem, South India. Nat Sci Rep 9:12975 10. Feng B, An H, Tan E (2007) Screening of CO2 adsorbing materials for zero emission power generation systems. Energy Fuel 21 (2):426–434 11. Ghelani AD, Bhagat CB, Dudhagara PR, Gondalia SP, Patel RK (2015) Biomimetic sequestration of CO2 using carbonic anhydrase from calcite encrust forming marine actinomycetes. Sci Int 3(2):48–57

Chapter 10 Evaluation of Actinobacteria for Agricultural Applications Abstract Microbes as bioinoculants and biopesticides are an alternative to chemical fertilizers to reduce environmental pollutions. Actinobacteria can be utilized as biofertilizers for sustainable agriculture as they can enhance plant growth and soil health though different plant growth-promoting attributes such as solubilization of phosphorus, potassium, and zinc; production of Fe-chelating compounds phytohormones hormones such indole acetic acids, cytokinin, and gibberellins; as well as by biological nitrogen fixation. The Actinobacteria also plays an important role in mitigation of different abiotic stress conditions in plants. The members of phylum Actinobacteria such as Actinomyces, Arthrobacter, Bifidobacterium, Cellulomonas, Clavibacter, Corynebacterium, Frankia, Microbacterium, Micrococcus, Mycobacterium, Nocardia, Propionibacterium, Pseudonocardia, Rhodococcus, Sanguibacter, and Streptomyces exhibited the multifarious plant growthpromoting attributes and could be used as biofertilizers for crops growing under natural as well as under the abiotic stress conditions. Methods for screening actinobacteria for plant pathogen control and plant growth promotion are described in this chapter. Keywords Agriculture, Plant pathogen control, Plant growth promotion, Biofertilizer

1 1.1

Screening of Actinobacterial Metabolites for Anti-phytopathogenic Activity Introduction

The most important constraint limiting crop yield in developing nations worldwide, and especially among resource-poor farmers, is soil infertility. Therefore, maintaining soil quality can reduce the problems of land degradation, decreasing soil fertility and rapidly declining production levels that occur in large parts of the world needing the basic principles of good farming practice. Minerals, organic components, and microorganisms are three major solid components of the soil. They profoundly affect the physical, chemical, and biological properties and processes of terrestrial systems. Biofertilizer are the products containing cell of different types of beneficial microorganisms. Thus, biofertilizers can be important components of integrated nutrient management. Organisms that are commonly used as biofertilizer component are nitrogen fixers (N-fixer), solubilizer (K-solubilizer), and phosphorus solubilizer (P-solubilizer) or with the combination of molds or fungi. These potential biological fertilizers would play a key role in productivity and sustainability of soil and also protect the environment as

Ramasamy Balagurunathan et al., Protocols in Actinobacterial Research, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0728-2_10, © Springer Science+Business Media, LLC, part of Springer Nature 2020

175

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Evaluation of Actinobacteria for Agricultural Applications

eco-friendly and cost-effective inputs for the farmers. With using the biological and organic fertilizers, a low-input system can be carried out, and it can help in achieving sustainability of farms. There are several studies reported the phytopathogenic and plant growth promoting properties of actinobacteria isolated from different sources like rhizosphere soil, marine sediments, and plants [1, 2]. 1.2

1. Laminar air flow.

Materials

2. Inoculation loop. 3. MHA and MHB. 4. Petri plate. 5. Actinobacterial cultures 6. Phytopathogens - Rolstonia solanacearum. 1.3

Methods

l

Prepare Pseudomonas solanacearum agar medium using distilled water.

l

Heat with frequent agitation and boil to dissolve the medium completely. Sterilize by autoclaving at 121  C for 15 min.

l

l

From a fresh culture of bacterial pathogen, take four or five colonies with a inoculation loop.

l

Transfer colonies to 2 ml of R. solanacearum broth.

l

1.4

Notes

Cool the agar medium to 40–50  C. Pour the agar into sterile glass plate or plastic petri plate, and allow to solidify.

Incubate the broth at 30  C until it achieves the turbidity of 0.5 McFarland standards.

l

Reduce turbidity by adding sterile saline or broth.

l

Dip a sterile cotton swab into the standardized bacterial suspension, if needed.

l

Remove excess inoculum by lightly pressing the swab against the tube wall at a level above that of the liquid.

l

Inoculate the agar by streaking with the swab containing the inoculum of R. solanacearum.

l

Take 5 mm agar plug of actinobacterial culture from ISP2 agar and place over the agar medium seeded with R. solanacearum.

l

Measure the inhibition zone around the agar plug after 24 h of incubation at 28 oC.

l

Handle the actinobacterial cultures in a place away from handling plant pathogens.

Screening of Actinobacterial Isolates for Its Plant Growth-Promoting Properties

2

177

Screening of Actinobacterial Isolates for Its Plant Growth-Promoting Properties

2.1

Introduction

Plant growth-promoting and extracellular enzymes producing rhizobacteria represent a wide variety of rhizosphere-inhabiting bacteria which colonize the root systems of plants and can stimulate plant growth by direct or indirect mechanisms. Direct mechanisms of plant growth promotion include biofertilization, stimulation of root growth, rhizoremediation, and plant stress control, while mechanisms of biological control include reducing the level of disease, antibiosis, induction of systemic resistance, and competition for nutrients and niches. In other words, the rhizobacteria can stimulate plant growth, increase yield, reduce pathogen infection, as well as reduce biotic or abiotic plant stress, without conferring pathogenicity. Indirect plant growth promotion is mediated by antibiotics or siderophores produced by PGPR that decrease or prevent the deleterious effects of plant pathogenic microorganisms. Direct promotion of growth by PGPR is including production of metabolites that enhance plant growth such as IAA, cytokinins, and gibberellins and also through the solubilization of minerals. Phosphate-solubilizing bacteria have been shown to enhance the solubilization of insoluble P compounds through the release of organic acids and phosphatases. Organic acid production is the main mechanism by which phosphate-solubilizing bacteria mobilizes phosphate from sparingly soluble phosphate. Microbes that assist in plant nutrient acquisition act through a variety of mechanisms including augmenting surface area accessed by plant roots, nitrogen fixation, P-solubilization, siderophore production, and HCN production. Therefore, manipulating microbial activity has great potential to provide crops with nutritional requirements [1, 2].

2.2

Materials

l

Actinobacteria cultures.

l

ISP2 broth.

l

Tryptophan.

l

Shaking incubator.

l

Salkowski reagent.

l

Succinate medium.

l

Chrome azurol S.

l

Fe3+.

l

Hexadecyl trimethyl ammonium bromide.

l

Jensen’s medium.

l

Peptone broth.

l

Chitin agar.

l

Mineral salt agar.

178

2.3

Evaluation of Actinobacteria for Agricultural Applications

Methods

2.3.1 Indole Acetic Acid (IAA) Production

l

Carboxy methyl cellulose.

l

Starch agar.

l

Skim milk agar.

Screen the antagonistic actinobacterial cultures for in vitro plant growth-promoting properties (indole acetic acid production and siderophore production) and soil fertility enhancement (N2 fixation, P and Zn solubilization, extracellular enzyme production). l

l

2.3.2 Siderophore Production

After incubation, separate the cell-free supernatant by centrifugation at 1000  g for 10 min.

l

Mix 2 ml of the supernatant with 1 ml of Salkowski reagent. The development of pink color is the indication for IAA production (see Table 1).

l

Inoculate the actinobacterial cultures on iron-free succinate medium amended with the tertiary complex chrome azural S (CAS)/ Fe3+/hexadecyl trimethyl ammonium bromide as an indicator. Incubate the CAS agar plates at 28  2  C for 96 h.

l

Appearance of orange color zone surrounding the actinobacterial growth indicates the production of siderophore (see Table 1).

l

To confirm nitrogen fixation, inoculate all the actinobacterial cultures inoculated into nitrogen-free medium, namely, Jensen’s medium (Sucrose - 20 g/l, K2HPO4 - 1 g/l, MgSO4 - 0.5 g/l, NaCl - 0.5 g/l, FeSO4 - 0.1 g/l, Na2MoO4.2H2O - 0.005 g/l, CaCO3 - 2 g/l, Agar - 15 g/l).

l

2.3.4 Phosphate Solubilization

Incubate the flask in rotary shaker for 7 days at 28  C.

l

l

2.3.3 Nitrogen Fixation

Inoculate the well-grown actinobacterial cultures into the ISP2 broth supplemented with tryptophan (2 mg/ml).

l

l l

Incubate at 28  C for 10 days for the growth of actinobacteria (see Table 1). Inoculate the actinobacterial cultures into Pikovskaya’s agar (Yeast extract - 0.5 g/l, Dextrose - 10 g/l, Ca3(PO4)2 - 5 g/l, (NH4)2SO4 - 0.5 g/l, KCl - 0.2 g/l, MgSO4 - 0.1 g/l, MnSO4. H2O - 0.0001 g/l, FeSO4 - 0.0001 g/l, Agar - 15 g/l) plates. Incubate the plates at 28  C for 7 days. The clear halo formation around the actinobacterial growth is the indication for phosphate solubilization (see Table 1).

Screening of Actinobacterial Isolates for Its Plant Growth-Promoting Properties

179

Table 1 PGP and enzyme activities PGP activities

Positive/negative

IAA Phosphate solubilization Siderophore production Nitrogen fixation Ammonia production Enzymatic activities Amylase Protease Chitinase Cellulose

2.3.5 Ammonia Production

l

l

2.3.6 Extracellular Enzyme Activity

Incubate the culture tubes at 28  C for 7 days in rotary shaker.

l

Development of pink color after the addition of 0.5 ml of Nessler’s reagent is the indication for ammonia production (see Table 1).

l

For chitinase activity inoculate the actinobacterial culture into chitin agar medium.

l

For cellulase activity inoculate the actinobacteria culture into mineral salt agar containing carboxy methyl cellulose (CMC) as carbon source.

l

For production of amylases and proteases, inoculate the actinobacterial culture into starch agar and skim milk agar plates, respectively.

l

2.3.7 Mass Cultivation and Formulation of PGP-Actinobacteria for Laboratory and In Planta Valuation

For ammonia production, inoculate the actinobacterial cultures into each 5 ml of peptone broth.

Incubate all the plates at 28  C for 7 days (see Table 1).

l

Inoculate the actinobacterial culture into 50 ml of soybean meal inoculation medium, and incubate in rotary shaker at 120 rpm for 48 h at 28  C.

l

Then, transfer 10% of inoculum into soybean meal production medium, and keep it in rotary shaker with 120 rpm for 120 h at 28  C.

l

After fermentation, mix the broth culture with talc-based formulation.

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Evaluation of Actinobacteria for Agricultural Applications

Table 2 In planta/field study

Actinobacteria no.

2.3.8 Field Evaluation and Tolerance Effects of PGP

Shoot length

Root length

Fresh weight

Dry weight

Yield

l

For 1 kg of talc-based formulation, mix 10 g of CMC in order to maintain the pH of the microbial formulation.

l

Mix 1 l of broth culture with talc-based formulation and dry.

l

Actinobacteria will be cultured in ISP2 broth at 28  C for 7 days.

l

Inoculate the three-week-old plant seedlings with 500 mL of the bacterial suspension (final density, 1.5  106 CFU/mL on a tray containing 300 seedlings) or the dry actinobacterial formulation.

l

After the inoculation, incubate the plant seedlings in a greenhouse for 5 days prior to transplantation to the experimental fields.

l

Evaluate the effects of the actinobacterial inoculation on plant growth by measuring the shoot length, root length, and tiller number of nine plants for each cultivar with a square pattern (three plants) inside the square pattern with or without inoculation at 37 and 58 days after transplanting (DAT).

l

Determine the statistical difference in plant length or tiller number between non-inoculation and inoculation by two-sided student’s t-test.

l

For in planta and field study, measure the plant height, weight, and yield (see Table 2).

References 1. Vurukonda SSKP, Giovanardi D, Stefani E (2018) Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. as Endophytes. Int J Mol Sci 19(4):952 2. Betancur LA, Naranjo-Gaybor SJ, VinchiraVillarraga DM et al (2017) Marine

Actinobacteria as a source of compounds for phytopathogen control: An integrative metabolic-profiling/bioactivity and taxonomical approach. PLoS One 12(2):e0170148

Chapter 11 Evaluation of Actinobacteria for Aquaculture Applications Abstract In response to the increased seafood demand from the ever-going human population, aquaculture has become the fastest-growing animal food-producing sector. However, the indiscriminate use of antibiotics as biological control agents for fish pathogens has led to the emergence of antibiotic-resistant bacteria. Probiotics are defined as living microbial supplements that exert beneficial effects on hosts as well as improvement of environmental parameters. Probiotics have been proven to be effective in improving the growth, survival, and health status of the aquatic livestock. Members of the phylum actinobacteria notably the genus Streptomyces are reported to exhibit probiotic properties to apply in aquaculture sector. Countable number of studies reported the antimicrobial activity against fish pathogens, inducing immune response in fishes, tolerance to gut conditions, growth-enhancing effect, and water quality amelioration effect of Streptomyces. In this chapter, we have described the methods to study the probiotic properties of Actinobacteria for aquaculture applications. Keywords Aquaculture, Probiotics, Pathogen control, Immune enhancement, Gut tolerance

1 Preexperimental Screening of Actinobacterial Cultures for Aquaculture Applications 1.1

Introduction

Aquaculture is the fastest food-producing sector in the global market. Microbial infections are the major limiting factor for aquaculture productivity. Applying probiotics is the promising approach to maintain the health and productivity of aquacultures animals. Bacterial species from fish gut is naturally prescreened for probiotic properties. Chemotherapeutic agents have been banned for disease management in aquaculture systems due to the emergence of antibiotic resistance gene and enduring residual effects in the environments. Instead, microbial interventions in sustainable aquaculture have been proposed, and among them, the most popular and practical approach is the use of probiotics. A range of microorganisms, especially the members of Lactic Acid Bacteria, have been used so far as probiotics. The results are satisfactory and promising; however, to combat the latest infectious diseases in aquaculture, the search for a new strain for probiotics is essential. The selection for probiotic candidate organisms was based on in vitro antagonism, as well as on the results of adhesion, colonization, and growth in

Ramasamy Balagurunathan et al., Protocols in Actinobacterial Research, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0728-2_11, © Springer Science+Business Media, LLC, part of Springer Nature 2020

181

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Evaluation of Actinobacteria for Aquaculture Applications

intestinal mucus. Marine-derived actinobacteria are the promising source for bioactive compounds. There are several studies that report the antagonistic activity of marine actinobacteria against bacterial and viral pathogens of fishes and prawn. Testing the antagonistic potential against aquaculture pathogens and their tolerance against physiological conditions is the prerequisite for the selection of actinobacteria as aquaculture probiotics [1, 2]. 1.2

1.3

Materials

Methods

1.3.1 Preexperimental Screening for Probiotic Properties Antagonistic Activity

l

Actinobacterial strain.

l

Fish pathogen -Aeromonas hydrophila.

l

ISP2 agar medium.

l

Nutrient broth.

l

Nutrient agar.

l

Incubator.

l

Test tubes.

l

Petri dishes.

l

Blood agar.

l

Sodium chloride.

l

Tryptic Soy Agar (TSA) plates .

l

Sodium chloride.

l

Congo red.

l

Phosphate-buffered saline (PBS).

l

Xylene and vortex.

l

Casein agar.

l

Test the antagonistic activity of actinobacterial strains against the fish pathogen Aeromonas hydrophila by agar plug method.

l

Prepare ISP2 agar plates.

l

Inoculate actinobacterial cultures into ISP2 agar plates and incubate at 28
C for 7-14 days.

l

Prepare sterile nutrient broth each 2 ml in test tubes.

l

Inoculate the cell suspension of A. hydrophila into it and incubate at 28
C for 18 h.

l

Prepare nutrient agar plates and inoculate the A. hydrophila cultures using sterile cotton swab.

l

Take ISP2 agar plates which contain the well-grown actinobacterial cultures.

l

Cut 5 mm agar plug of actinobacterial cultures from ISP2 agar using sterile well cutter.

l

Place the agar plug over the surface of nutrient agar plates inoculated with A. hydrophila.

Preexperimental Screening of Actinobacterial Cultures for Aquaculture. . . l

1.3.2 Tolerance Studies

l

Hemolytic Activity

l

1.3.3 Test for Hydrophobicity

l

l

183

Incubate all the plates at 28
C for 24 h and observe for zone of inhibition. Streak the actinobacterial cultures on blood agar plates containing 5% human blood and 2.5% sodium chloride (NaCl). Incubate for 7-14 days at 28
C and then observe for zone of haemolysis. Streak the actinobacterial cultures on TSA plates containing 1% sodium chloride and 0.03% Congo red Incubate at 28
C for 7-14 days.

Hydrophobicity of Actinobacterial Strains Using the Congo Red Method The Bacterial Adherence to Hydrocarbons (BATH) Test

1.3.4 Tolerance to Salt, pH, Bile, and Acidic Conditions Salt Tolerance

pH Tolerance

Bile Tolerance

l

Screen the actinobacterial cultures by BATH test for measuring the cellular affinity for organic solvents.

l

Inoculate the actinobacterial strain in tryptic soy broth (TSB) at 28
C for 7-14 days under shaking conditions.

l

Harvest the cells by centrifugation, and then wash three times with phosphate-buffered saline (PBS).

l

Measures the OD of the cells at 540 nm and adjust with 0.8 in PBS.

l

Subsequently, mix 3 ml of each cell suspension with 1 ml of xylene, and vortex for 30 s at room temperature.

l

After 30 min, measure the OD of the aqueous phase at 540 nm.

l

Prepare the ISP2 agar plates with different sodium chloride concentrations (0, 1, 2.5, 5, 7.5 and 10%).

l

Spot the actinobacterial strains in all the plates.

l

Incubate the plates at 28
C for 7–14 days and observe for growth.

l

Prepare the TSA plates with a pH of 3, 5, 7, 9 and 11.

l

Spot the actinobacterial strains.

l

Incubate the plates at 28
C for 7–14 days.

l

l

The same medium supplementing with different concentration of bile salt (0.1, 0.2, and 0.3%). Inoculate the culture and incubate the plates at 28
C for 7–14 days

184

1.4

Evaluation of Actinobacteria for Aquaculture Applications

Notes

l

Zone of inhibition around the agar plug indicates antagonistic activity.

l

Strains with a reddish color indicates positive for the hydrophobicity test.

l

Strains with a translucent to white color indicate negative.

l

>50%—strongly hydrophobic when the values.

l

20–50%—moderately hydrophobic.

l