UTILIZATION OF INDUSTRIAL WASTE (CHEESE WHEY) FOR THE BIOSYNTHESIS OF β- GALACTOSIDASE SYEDA UM-E-KALSOOM NAQVI Ph.D SCHOLAR DEPARTMENT OF ENVIRONMENTAL SCIENCE LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE 2017 SYEDA UM-E-KALSOOM NAQVI PhD 2017 THESIS UTILIZATION OF INDUSTRIAL WASTE (CHEESE WHEY) FOR THE BIOSYNTHESIS OF β– GALACTOSIDASE A THESIS SUBMITTED TO LAHORE COLLEGE FOR WOMEN UNIVERSITY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN ENVIRONMENTAL BIOTECHNOLOGY By SYEDA UM-E-KALSOOM NAQVI DEPARTMENT OF ENVIRONMENTAL SCIENCE LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE 2017 CERTIFICATE This is to certify that the research work described in this thesis submitted by Ms. Syeda Um-e-Kalsoom Naqvi to Department of Environmental Science, Lahore College for Women University has been carried out under my direct supervision. I have personally gone through the raw data and certify the correctness and authenticity of all results reported herein. I further certify that thesis data have not been used in part or full, in a manuscript already submitted or in the process of submission in partial fulfillment of the award of any other degree from any other institution or home or abroad. I also certify that the enclosed manuscript has been prepared under my supervision and I endorse its evaluation for the award of PhD degree through the official procedure of University. ________________ Name Supervisor Date: Verified By ________________ Name Chairperson Department of _______ Stamp _________________ Controller of Examination Stamp Date: ___________ DEDICATION This thesis is dedicated to my grandfather (Syed Zahur-ul-Hassan Naqvi), most beloved mother (Kaneez Sughra) and my very supportive and loving father (Syed Zain-ul-Aba Naqvi) for their unconditional love and prayers that motivates me to set highest targets in my life. I also dedicated my thesis to my husband (Syed Faisal Abbas) and my sons (Hussain Abbas and Nalain Abbas) for their love and moral support in my hard times. Also, this thesis is dedicated to my respected teachers for their unlimited kindness, scholarly guidance and moral support. ACKNOWLEDGMENTS Up and above anything else, all gratitude and praises are due to ALMIGHTY ALLAH alone, the most Gracious, Merciful and Compassionate, the Creator of the universe, who enabled us to complete this work successfully. We offer our humble and sincerest words of praise to the Holy Prophet HAZRAT MUHAMMAD (S.A.W) and FAMILY OF HOLY PROPHET (S.A.W) who forever is a torch of knowledge and guidance for humanity. I am greatly indebted to worthy Vice Chancellor Prof. Dr. Uzma Qureshi, Lahore College for Women University, Lahore for providing me a bright chance to carry out this research work. I offer my profound thanks to Prof. Dr. Bushra Khan, Dean Faculty of Natural Sciences and Head of Chemistry Department, Lahore College for Women University, for providing me this opportunity to avail the research work. It is a great honor and pleasure for me to express my deep feelings of gratitude to my respected Supervisor Prof. Dr. Arifa Tahir, Head of Environmental Science Department, Lahore College for Women University, for her encouragement, advice and criticism throughout the course of this study. My thanks are due to Prof. Christopher, Department of Plant and Environmental Sciences University of Copenhagen Frederiksberg, Denmark, for the invitation and guidance to continue my research work in the University of Copenhagen, Denmark. I am extremely obliged to my foreign supervisor Associate Prof. Peter Stougaard, Department of Plant and Environmental Sciences University of Copenhagen Frederiksberg, Denmark, for his supervision throughout the experimental work and for his help and guidance in the interpretation of results. Furthermore, I would like to thank, in particular Ph.D Student Yanan Qin, Department of Plant and Environmental Sciences University of Copenhagen Frederiksberg, Denmark, for her help to design the experimental plan of work and for intriguing scientific discussions related to my project. I want to express my heartiest gratitude to Ulla, Dorte and Susanne for technical assistance and also thankful to Ph.D students, Mikkel, Morten, Benhosh and Raju, Department of Plant and Environmental Sciences University of Copenhagen Frederiksberg, Denmark, for helping me in scientific calculations and protocols. Highly obliged to Associate Prof. Mikkel Andreas Glaring, Department of Plant and Environmental Sciences University of Copenhagen Frederiksberg, Denmark, for providing me the Antarctic samples and Witold for helping me in the interpretation of whole genome sequencing data. My greatest appreciation to Helle J. Martens, University of Copenhagen, Denmark for performing the transmission electron micrographs of strains NAQVI-58 and NAQVI-59T and Kjeld Pyrdol Nielsen, Process Technologist/ Process with responsibility for dairy and brewery, Institute of Food Science, IFV, University of Copenhagen, Denmark for the provision of Cheese Whey. I am extremely thankful to my father (Syed Zain-ul-Aba Naqvi) for his prayers, motivation, counseling and help to look after my kids during the course of my study, without his help it is almost impossible for me to complete my work. I would like to thank my husband (Syed Faisal Abbas) for his love, support, encouragement and motivation to complete my project at any cost. Extremely obliged to Uncle Riaz Shah and his family in Denmark for their help, support and loyalty towards my family which really helped me a lot to complete my project successfully. KALSOOM NAQVI CONTENTS Title Page No. List of Table i List of Figures ii List of Abbreviations x Abstract xi Chapter 1 : Introduction 1 Chapter 2: Review of Literature 10 Chapter 3: Materials and Methods 44 Chapter 4: Results 55 Chapter 5: Discussion 150 References 160 Annexures xiii Plagiarism Report ci List of Publications ciii i LIST OF TABLES Table No Description/Title Page No 1. Showing the highest sequence similarity with other bacterial 109 strains. 2. Whole genome sequencing results showing the sequence 111 similarity with other bacterial strains. 3. Description of new Pararhizobium sp. 111 4. Morphological characteristics of selected bacterial isolates 123 (NAQVI-58 and NAQVI-59). 5. Distinctive features of strains NAQVI-58 and NAQVI-59 are 127 represented in the table. 6. Biochemical characteristics of strains NAQVI-58,NAQVI-59 136 and three reference strains. 7. Cellular fatty acid (%) composition of strains NAQVI-58 and 139 NAQVI-59. ii LIST OF FIGURES Figure Description/Title Page No. No. 1 β-galactosidase producing bacterial strains from Antarctica on 56 R2 agar medium with X-gal. 2 Number of β-galactosidase producing bacterial colonies (turned 57 blue on X-gal) from Antarctica (Mc Murdo, USA) and Ikka columns (Greenland). 3 Isolation of β-galactosidase producing bacterial strains from 58 Antarctica on Marine agar without X-gal from Antarctic samples (1A, 2A, 3A, 4A and 5A). 4 Isolation of β- Isolation of β-galactosidase producing bacterial 59 strains on Marine agar without X-gal from Ikka column samples (1K, 2K, 3K, 4K, 5K, 6K, 7K, 9K and 10K). 5 Bacterial strains on Marine agar medium without X-gal 60 supplemented with cheese whey. 6 Bacterial strains on Marine agar medium supplemented with 62 cheese whey and X-gal (Plate 1A). 7 Bacterial strains on Marine agar medium supplemented with 63 cheese whey and X-gal (Plate 2A and 3A). 8 Bacterial strains on Marine agar medium supplemented with 64 cheese whey and X-gal (Plate 4A and 5A). 9 Number of β-galactosidase producing bacterial colonies from 65 Antarctica (Mc Murdo, USA) (Plate 1A). 10 Number of β-galactosidase producing bacterial colonies from 66 Antarctica (Mc Murdo, USA) (Plate 2A). 11 Number of β-galactosidase producing bacterial colonies from 67 Antarctica (Mc Murdo, USA) (Plate 3A). 12 Number of β-galactosidase producing bacterial colonies from 68 Antarctica (Mc Murdo, USA) (Plate 4A). iii 13 Number of β-galactosidase producing bacterial colonies from 69 Antarctica (Mc Murdo, USA) (Plate 5A). 14 β-galactosidase producing bacterial strains on Marine agar 70 medium with X-gal. 15 β-galactosidase producing bacterial strains from Antarctica 71 were categorized on the basis of their colour with X-gal on Marine agar medium. 16 Isolation of β-galactosidase producing bacterial strains from 73 Antarctic samples A1, A2, A3, A4 and A5 on R2 agar plates supplemented with cheese whey and X-gal. 17 β-galactosidase producing bacterial strains from Antarctica 74 were categorized on the basis of their size with X-gal on R2 agar medium. 18 Isolation of β-galactosidase producing bacterial strains from 75 Antarctic samples A1, A2, A3, A4 and A5 on R2 agar plates supplemented with cheese whey and X-gal. 19 Isolation of β-galactosidase producing bacterial strains from 76 Antarctic samples A1, A2, A3, A4 and A5 on Marine agar plates supplemented with cheese whey and X-gal. 20 β-galactosidase producing bacterial strains from Antarctica 77 were categorized on the basis of their size with X-gal on Marine agar medium. 21 Isolation of β-galactosidase producing bacterial strains on R2 80 agar medium with X-gal from Ikka samples I(1), I(2), I(3), I(4), I(5), I(6), I(7), I(8), I(9) and I(10). 22 Isolated colonies were purified again on new R2 agar medium 81 plates with X-gal. 23 β-galactosidase producing bacterial strains from Antarctica 82 were screened again on new R2 agar medium plates with X-gal. iv 24 Isolated colonies are purified again on new R2 agar medium 84 plates with X-gal. 25 β-galactosidase producing bacterial strains from Antarctica are 85 purified again on new R2 agar medium plates with X-gal. 26 β-galactosidase producing bacterial strains from Antarctica 86 were categorized on the basis of their color on Marine agar medium with X-gal. 27 Isolated colonies are purified again on new R2 agar medium 87 plates with X-gal.