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Contents of Phd Thesis 博士論文 Isolation and Identification of Novel Species of Heavy-metals Tolerant Bacteria from Pakistan for Their Potential Usage in Bioremediation and in Agriculture （パキスタン由来の重金属耐性を示す新種細菌の 単離•同定及びそのバイオレメディエーションや 農業への応用の可能性についての研究） By SAIRA ABBAS サイラ アバス DEPARTMENT OF APPLIED BIOLOGICAL CHEMISTRY GRADUATE SCHOOL OF AGRICULTURE AND LIFE SCIENCES THE UNIVERSITY OF TOKYO TOKYO-JAPAN 2016 博士論文 Isolation and Identification of Novel Species of Heavy- metals Tolerant Bacteria from Pakistan for Their Potential Usage in Bioremediation and in Agriculture （パキスタン由来の重金属耐性を示す新種細菌の 単離•同定及びそのバイオレメディエーションや 農業への応用の可能性についての研究） By SAIRA ABBAS サイラ アバス DEPARTMENT OF APPLIED BIOLOGICAL CHEMISTRY GRADUATE SCHOOL OF AGRICULTURE AND LIFE SCIENCES THE UNIVERSITY OF TOKYO TOKYO-JAPAN 2016 （ パ of Heavy of Species Novel Identification Isolation and キ ス タ ン 由 来 の 重 金 属 耐 農 性 業 を へ 示 の す 応 新 - 用 種 in from for and Usage Bioremediation Their Potential Pakistan Bacteria Tolerant metals の 細 可 菌 能 の 性 単 に 離 つ • い 同 て 定 の 及 研 び 究 そ ） の バ イ オ レ メ デ ィ エ ー シ ョ ン や サ Agriculture in SAIRA ABBAS イ ラ ア バ ス Isolation and Identification of Novel Species of Heavy- metals Tolerant Bacteria from Pakistan for Their Potential Usage in Bioremediation and in Agriculture （パキスタン由来の重金属耐性を示す新種細菌の 単離•同定及びそのバイオレメディエーションや 農業への応用の可能性についての研究） By SAIRA ABBAS サイラ アバス A thesis submitted as a requirement for the degree of DOCTORATE OF PHILOSOPHY IN ENVIRONMENTAL MICROBIOLOGY AND BIOTECHNOLOGY DEPARTMENT OF APPLIED BIOLOGICAL CHEMISTRY GRADUATE SCHOOL OF AGRICULTURE AND LIFE SCIENCES THE UNIVERSITY OF TOKYO TOKYO-JAPAN 2016 Dedicated to My loving Mother and Father Who always pray To see the bud of Their wishes Bloom into a Flower CERTIFICATE ''It is certified that the contents and form of the thesis entitled “Isolation and Identification of Novel Species of Heavy-metals Tolerant Bacteria from Pakistan for Their Potential Usage in Bioremediation and in Agriculture ” submitted by Ms. SAIRA ABBAS, has been found satisfactory for the award of degree of the Doctorate of Philosophy under JSPS Ronpaku Program.'' (Sign and Official seal) The Dean, Faculty of Agricultural and Life Sciences The University of Tokyo Tokyo, Japan Committee: CHAIRMAN: Professor Dr. Toru Fujiwara Research Supervisor from Japan Laboratory of Plant Nutrition and Fertilizer Department of Applied Biological Chemistry The University of Tokyo, Japan MEMBER: Dr. Iftikhar Ahmed Research Supervisor from Pakistan Principal Scientific Officer / Director (IMCCP), NARC, Islamabad, Pakistan MEMBER: Dr. Moriya Ohkuma Head (Microbes Division), JCM, Riken BioResource Centre, Tsukuba, Japan MEMBER: Professor Dr Masaharu Ishii Laboratory of Applied Microbiology, Department of Biotechnology The University of Tokyo, Japan MEMBER: Professor Dr. Keishi Senoo Soil Science Laboratory Department of Applied Biological Chemistry The University of Tokyo, Japan ACKNOWLEDGEMENT I would like to begin the acknowledgement by thanking the “ALMIGHTY ALLAH” for the countless blessings He has bestowed upon mankind in the form of “life on earth”. I offer my humble thanks to the “HOLY PROPHET (PEACE BE UPON HIM)” who is forever a torch of guidance and knowledge for entire humanity. I express my sincere gratitude to Professor Dr. Toru Fujiwara sansei for his continuous guidance, encouragement and facilitation in research work during my stay in Japan. Special thanks to Dr. Moriya Ohkuma sansei and all other colleagues, especially Dr. Takuji Kudo san, Dr. Takashi Itoh and Dr. Toshiya Iida san from Japan Culture of Microorganisms (JCM), Riken, Tsukuba, Japan for providing me a chance to work in their labs and facilitating my research work by all means. I also appreciate for all the assistance of Ms. Sachiko Yamashita and Ms. Kayo Kusaoke, who spared a lot of time to facilitate me in many ways. As a matter of fact I have no appropriate words that fully express the immense indebtedness and deep gratitude I owe to my worthy learned research Supervisor Dr. Iftikhar Ahmed, Principal Scientific Officer / Director (IMCCP), NARC, Islamabad, Pakistan for his keen interest, admirable guidance, constructive suggestion, affectionate supervision, inspiring behavior and valuable knowledge, which he contributed to this work. Whenever I needed, he was there for me and always swayed me out of the disaster. His personality has always been like a beacon of light in the darkness of ignoranc e. May Allah bless him with all the successes in his life. Special thanks to Dr. Nauman Khalid for his technical help, scientific advices and discussions, particularly helping me in writing the dissertation and for critically reading/editing the manuscripts and his valuable comments. I appreciate Dr. Muhammad Iqbal Senior Scientific Officer NIGAB NARC, Islamabad, Pakistan for his valuable suggestion, support and interest in my research work. I also express my sincere and deep regard to respected Prof. Dr. Muhammad Subhan and Prof. Dr. Sultan Mahmood, University of Science & Technology, Bannu, for their support and cooperation during the course of my research endeavor. Many thanks to all the members of my Lab; Ms. Neeli Habib, Ms. Asia Bangash, Mrs. Asia Sakina, Mr. Muhammad Ehsan, Mr. Nazir Ahmad, for providing me friendly working environment and the splendid assistance in the laboratory. I appreciate the encouragement of my great friends Ms. Nadia, Ms. Romana Malik and Ms. Aysha Farooq, for their help, encouragement and cooperation during the whole tenure of my studies. Their valuable advice s can never be expressed by words. I gratefully acknowledge financial support of Higher Education Commission (HEC) of Pakistan in the form of Indigenous Ph.D. Scholarship in Pakistan and the IRSIP fellowship to facilitating my stay in Japan for conducting research project in technologically advanced country. I am also thankful to Professor Dr. Toru Fujiwara for providing me the financial support in the extended period of my stay in Japan. Special thanks to Japanese Society for Promotion of Science (JSPS), JAPAN for financial support under Ronpaku Program to present and defend my research project in the University of Tokyo for my PhD degree. In the last but not least, I sincerely acknowledge my family members for their devotion, sacrifices, patience and countless prayers. I have no words to pay my heartiest thanks to my parents and my grandmother for their encouragement, full support and prayers to pursue my studies peacefully. No expression, verbal or written, can express my heart feeling for my family for their unbound love, support and inspiration that built up my confidence to complete this tedious task. I am really thankful to my sisters, Suman Abbas and Arzamia Abbas for providing me more care and love. (SAIRA ABBAS) LIST OF CONTENTS CHAPTER # TITLE PAGE # ACKNOWLEDGEMENT LIST OF CONTENTS LIST OF TABLES LIST OF FIGURES I EXECUTIVE SUMMARY 1 II INTRODUCTION 6 III REVIEW OF LITERATURE 10 IV Isolation and molecular characterization of heavy metal 31 tolerant bacteria for their potential usage in bioremediation and in agriculture V Heavy metal-tolerant and psychrotolerant bacterium 58 Acinetobacter pakistanensis sp. nov. isolated from a textile dyeing wastewater treatment pond VI A heavy metal tolerant novel bacterium, Alcaligenes 83 pakistanensis sp. nov., isolated from industrial effluent in Pakistan VII A heavy metal tolerant novel bacterium, Bacillus malikii sp. 108 nov., isolated from tannery effluent wastewater VIII GENERAL CONCLUSION 128 REFERENCES 131 LIST OF TABLES TABLE # TITLE PAGE # 3.1. Densities of some heavy metals and other substances 10 3.2. Examples of PGPR ability to tolerate a variety of heavy 23 metals in plants 3.3. Heavy metal removal from different agricultural waste 30 biosorbents. 4.1 Set of primers used in present study. 38 4.2 Physicochemical analysis of effluent samples 39 4.3 Morphological characteristics of isolated heavy metal 40 tolerant strains 4.4 Identification of isolated Heavy metal tolerant strains 47 based on 16S rRNA gene sequence and their accession numbers published in DNA database 4.5 Results of nifH and acdS genes amplification with 51 different sets of primers 5.1. Amplification and sequencing primers for gyrB, rpoB, 62 atpD and infB genes. 5.2. Differentiating phenotypic and biochemical 65 characteristics of strain NCCP-644T and the type strains of closely related Acinetobacter species. 5.3 Analysis of housekeeping genes Acinetobacter 70 pakistanensis NCCP-644T in comparison with the reference species and their accession numbers published in DNA database 5.4 DNA-DNA relatedness between strain (NCCP-644T) of 77 Acinetobacter pakistanensis sp. nov. with the type strains of their most closely related species. 5.5 Cellular fatty acid profiles (%) of strain NCCP-644T 79 compared to the reference type strains of the genus Acinetobacter. 6.1. Amplification and sequencing primers for phenol 87 hydroxylase and nitrite reductase (nirK) genes 6.2. Characteristics that differentiate novel strain NCCP-650T 91 from the type strains of closely related species of the genus Alcaligenes 6.3. Sequence similarity index (%) of gyrB gene for NCCP- 97 650T and NCCP-667 with closely related reference strains and their accession numbers for gyrB gene published in DNA database 6.4. Sequence similarity index (%) of nirK gene for NCCP- 98 650T and NCCP-667 with closely related reference strains and their accession numbers for gyrB gene published in DNA database 6.5. DNA-DNA relatedness (%) between strain NCCP-650T 99 of Alcaligenes pakistanensis sp. nov. with the type strains of their most closely related species. 6.6. Cellular fatty acid profiles (%) of strains NCCP-650T and 101 type strains of reference species of the genus Alcaligenes 6.7. Comparison of polar lipids of type strains of (a) 105 Alcaligenes pakistanensis NCCP-650T, (b) A. aquatilis LMG 22996T, (c) A. faecalis subsp. parafaecalis DSM 13975T, (d) A. faecalis subsp. faecalis JCM 20522T, (e) A. faecalis subsp. phenolicus DSM 16503T 7.1. Characteristics that differentiate novel strain NCCP-662T 115 from the type strains of closely related species of the genus Bacillus 7.2.
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