Diversity of Thermophilic Bacteria in Hot Springs and Desert Soil of Pakistan and Identification of Some Novel Species of Bacteria By By ARSHIA AMIN BUTT Department of Microbiology Quaid-i-Azam University Islamabad, Pakistan 2017 Diversity of Thermophilic Bacteria in Hot Springs and Desert Soil of Pakistan and Identification of Some Novel Species of Bacteria By ARSHIA AMIN BUTT Thesis Submitted to Department of Microbiology Quaid-i-Azam University, Islamabad In the partial fulfillment of the requirements for the degree of Doctor of Philosophy In Microbiology Department of Microbiology Quaid-i-Azam University Islamabad, Pakistan 2017 ii IN THE NAME OF ALLAH, THE MOST COMPASSIONATE, THE MOST MERCIFUL, “And in the earth are tracts and (Diverse though) neighboring, gardens of vines and fields sown with corn and palm trees growing out of single roots or otherwise: Watered with the same water. Yet some of them We make more excellent than others to eat. No doubt, in that are signs for wise people.” (Sura Al Ra’d, Ayat 4) iii Author’s Declaration I Arshia Amin Butt hereby state that my PhD thesis titled A “Diversity of Thermophilic Bacteria in Hot Springs and Deserts Soil of Pakistan and Identification of Some Novel Species of Bacteria” is my own work and has not been submitted previously by me for taking any degree from this University (Name of University) Quaid-e-Azam University Islamabad. Or anywhere else in the country/world. At any time if my statement is found to be incorrect even after my Graduate the university has the right to withdraw my PhD degree. Name of Student: Arshia Amin Butt Date: 27th March 2017. Certificate of Approval This is to certify that the research work presented in this thesis, entitled “Diversity of Thermophilic Bacteria in Hot Springs and Desert Soil of Pakistan and Identification of Some Novel Species of Bacteria” was conducted by Ms. Arshia Amin Butt under the supervision of Dr Iftikhar Ahmed No part of this thesis has been submitted anywhere else for any other degree. This thesis is submitted to the Department of Microbiology Quaid-e-Azam University Islamabad in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Field of Microbiology Department of Microbiology University of: Quaid-e-Azam University Islamabad Student Name: Arshia Amin Butt Signature: __________ Examination Committee: a) External Examiner 1: Name: Dr Ghazala Kaukab Signature:___________ (HOD- Department of Biochemistry, PMAS Arid Agriculture University Rawalpindi) b) External Examiner 2: Name Dr. Allah Nawaz Signature:___________ (Principal Clinical Bio Chemist Capital Hospital Islamabad) c) Internal Examiner: Name Signature:___________ Supervisor Name: Dr Iftikhar Ahmed Signature:____________ Name of Dean/HOD: Dr Fariha Hassan Signature:_____________ Plagiarism Undertaking I solemnly declare that research work presented in the thesis titled “Diversity of Thermophilic Bacteria in Hot Springs and Deserts Soil of Pakistan and Identification of Some Novel Species of Bacteria” is solely my research work with no significant contribution from any other person. Small contribution/help wherever taken has been duly acknowledged and that complete thesis has been written by me. I understand the zero tolerance policy of the HEC and University Quaid-e-Azam University Islamabad towards plagiarism. Therefore I as an Author of the above titled thesis declare that no portion of my thesis has been plagiarized and any material used as reference is properly referred/cited. I undertake that if I am found guilty of any formal plagiarism in the above titled thesis even after award of PhD degree, the University reserves the rights to withdraw/revoke my PhD degree and that HEC and the University has the right to publish my name on the HEC/University Website on which names of students are placed who submitted plagiarized thesis. Student /Author Signature: Name: Arshia Amin Butt Declaration I hereby declare that work presented in this thesis is my own effort except where others acknowledged and that the thesis is my own composition. No part of this thesis has previously been presented for any other degree. Arshia Amin Butt iv DEDICATED To My dearest Husband Malik JAWAD Saboor For his endless support, encouragment and making me believe in myself, and to my Lifeline, my son, Aali Jawad. My parents and parent in laws There is no doubt in my mind that without their continued support and counseling I could not have completed this process. v CERTIFICATE This thesis, titled “Diversity of Thermophilic Bacteria in Hot Springs and Desert Soil of Pakistan and Identification of Some Novel Species of Bacteria.” submitted by Ms. Arshia Amin Butt to the Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan, is accepted in its present form as satisfying the thesis requirement for the Degree of Doctor of Philosophy in Microbiology. SUPERVISOR: Dr. Iftikhar Ahmed Principal Scientific Officer / Director (IMCCP, NARC) CO-SUPERVISOR: Dr. Javed Iqbal Dasti Assistant Professor EXTERNAL EXAMINER: EXTERNAL EXAMINER: CHAIRPERSON: Dr. Fariha Hassan Associate Professor Dated: vi Acknowledgments First of all, all praises to ALLAH SUBHANAWATALLA, the Almighty, the creator and sustainer of all life, the origin of all power, knowledge and wisdom. His will is final. It was He who made me capable enough o understand the work, He who gave the command and the initiative to conclude this study. All praises to our beloved Prophet Hazrat Muhammad (SAWW) who got us out of the blinding ignorance and showed us the true path of life. I would like to convey my earnest gratitude to my husband, parents and parent in laws. They were always there to help, support, and share, and guide me every step of the way. To other family members Miss Aabaa, Zartashia Amin, Aisha Amin, Tayyaba Amin, Mehroze Amin, Tahir Butt, Fateh, Taymoor, Mansoor, Amlash, Shamil, Rehmat, Rafeh, who were always there to share my burdens. My family taught me about life and gave so many sacrifices for me just so I can be where I am today. May Allah shower His blessings upon them always Amen! I further express my deepest appreciation to my supervisor Dr Iftikhar Ahmed. His presence itself gave me hope and his open optimism towards the work gave me courage. He allowed me a free hand to pursue what I planned for the work and guided me accordingly. I believe it’s his pain staking efforts and devotion towards this field is what made me carry out my PhD in the first place. I feel honoured to be his doctoral student. My appreciation is also due to my co supervisor, Dr. Javed Iqbal Dasti. He guided me about everything related to him so I can easily carry out my work. I don’t want to Miss Dr Nauman Khalid who was very helpful whenever I asked him for guidance and discussed with me every bit of ambiguity. My special thanks to Dr Abdul Hameed, Dr Safia Ahmed, Dr Fariha Hassan, Dr Muhammad Imran, Dr Aamer Ali Shah who supported my work in every way possible and most importantly provided me with a platform for the study. Their advice and guidance was most candid and frank helping me ease my work whenever any hurdle came. I am extremely grateful to my supervisor in China, Prof. Dr. Wen Jun Li, at Sun Yat-Sen University, Guangzhou, who was Director of Yunnan institute of vii Microbiology during my entire stay in China, I was guided by him at each step. He understood my problems and went out way to support me. Consequently, his treatment of me was more as of a friend than a student which helped me enormously. My experience of travelling abroad and working in his well-equipped lab has been most fruitful due to his support. He guided me about the taxonomy as well as offered pyrosequencing study otherwise this work wouldn’t have reached a conclusion. I also appreciate him for introducing me with Dr Byung-Yong Kim and Dr Dharmesh Singh from Chun Lab, Seoul National University and Dr Xiao-Yang Zhi from YIM institute of Microbiology who gave me lot of time for guiding about pyrosequencing studies. I am greatly thankful to Mrs Xiao Min whose attitude was always extremely friendly and I am very grateful for all the help she provided me. Thanks, are also due to all my friends and research colleagues Miss Nadia, Saira, Safina, Yin Yrui, Liu Lan, Jhao Jhang yu, Xiao Mei, Ming Hong, Bing Bing, Wen, Jhan Jhao, Zhuging, Xian Wen Dong for all the general help and support with the lab work. My special gratitude goes off to Neeli Habib for taking time off in working with me, attending to my work and providing me the support whenever I needed it. She is one of those persons whose company gave me courage to complete my experiments. Great thanks to Firasat Hussain, Ghani Jan Usman and Inam who were a big moral support to stay in a new environment. I am very thankful to all funding agencies which were supporting us. Arshia Amin Butt viii Table of Contents Acknowledgments ...................................................................................................... vii CHAPTER 1 ................................................................................................................. 1 Executive Summary ..................................................................................................... 1 1.1 Actinobacterial and Bacterial diversity in hot water springs soil and desert soil 1 1.2. Novel taxa from hot water spring soil ................................................................. 2 1.2.1. Nocardioides pakistanensis sp. nov.............................................................