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Rajiv Kumar M.Phil PHYLOGENETIC ANALYSIS OF BACTERIAL DIVERSITY OF HEAVY METAL AFFECTED SOIL OF BATHINDA REGION (SOUTH WEST PUNJAB) A Dissertation submitted to the Central University of Punjab For the award of Master of Philosophy In Biosciences BY Rajiv Kumar Supervisor Dr. Sanjeev K. Thakur Centre for Biosciences School of Basic and Applied Sciences Central University of Punjab, Bathinda August, 2012 1 CERTIFICATE I declare that the dissertation entitled “PHYLOGENETIC ANALYSIS OF BACTERIAL DIVERSITY OF HEAVY METAL AFFECTED SOIL OF BATHINDA REGION (SOUTH WEST PUNJAB)” has been prepared by me under the guidance of Dr. Sanjeev K. Thakur, Assistant Professor, Centre for Biosciences, School of Basic and Applied Sciences, Central University of Punjab. No part of this dissertation has formed the basis for the award of any degree or fellowship previously. Rajiv Kumar Reg. No.:- CUP/M.Phil.-Ph.D/SBAS/BIO/2010-11/05 Centre for Biosciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda - 151001 DATE: i CERTIFICATE I certify that Rajiv Kumar has prepared his dissertation entitled “PHYLOGENETIC ANALYSIS OF BACTERIAL DIVERSITY OF HEAVY METAL AFFECTED SOIL OF BATHINDA REGION (SOUTH WEST PUNJAB)”, for the award of M.Phil. degree of the Central University of Punjab, under my guidance. He has carried out this work at the Centre for Biosciences, School of Basic and Applied Sciences, Central University of Punjab. Dr. Sanjeev K. Thakur Assistant Professor Centre for Biosciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda - 151001 DATE: ii ABSTRACT Phylogenetic Analysis of Bacterial Diversity of Heavy Metal Affected Soil of Bathinda Region (South West Punjab) Name of student: Rajiv Kumar Registration Number: CUP/M.Phil.-Ph.D./SBAS/BIO/2010-11/05 Degree for which submitted: Master of Philosophy Name of supervisor: Dr. Sanjeev K. Thakur Centre: Centre for Biosciences School of Studies: School of Basic and Applied Sciences Key words (Five minimum): Heavy metals, 16S rRNA gene, bacterial diversity, Phylogenetics. The soil of Bathinda region is affected by heavy metals due to anthropogenic activity, industrial effluent and fly ash. Three soil samples in triplicate were collected from three different places of Bathinda region, abandoned soil, agricultural soil, thermal power plant fly ash soil. Based on 16S rRNA gene cloning and sequence analysis, phylogentic analysis of bacterial diversity was performed. 366 clones were picked and through colony PCR 128 positive clones were chosen. Out of 128 clones, 126 clones were sequenced in three fragments and all three fragments aligned using SeqMan Lasergene ver.10.0 (DNA STAR) software. BLAST analysis of sequenced and aligned samples was carried out at NCBI to find out the homology with different groups of bacteria. All 126 clones aligned for phylogenetic analysis by MEGA 5 (Neighbor Joining method). 32 genus belong to 8 phyla were observed in all three samples. Most dominant phylum is Proteobacteria followed by Bacteriodates. Pseudomonas spp. is dominant in agricultural and thermal power plant soil. Abandoned soil showed maximum diversity followed by thermal power plant soil. In agricultural soil minimum diversity was observed. So with this investigation it can be concluded that lands without anthropogenic activities are rich in bacterial diversity as compared to more anthropogenic active areas. (Rajiv Kumar) (Dr. Sanjeev K. Thakur) iii ACKNOWLEDGEMENT Only one name appeared on the cover of this M.Phil dissertation, but actually there are many hidden names that helped me to make contents in between the covers. I take this opportunity to express my gratitude to all of them. I did not find words that could express my thanks to a comparable extent for the help and support that he provided me, but still trying to thank my supervisor Dr. Sanjeev K. Thakur, who guided my M.Phil. dissertation and provided all facilities. I have no words to express my gratitude for him. He has motivated me very much and this M.Phil. work would not have been completed without his help. I thank you sir, for all the help and advice, you have given me at right time and in right direction. My special thank to Dr. Pankaj Bhardwaj who gave me a chance to work in his lab and provided me the opportunity to work on an aspect that altogether changed my attitude toward science especially microbiology. I thank you sir, for your prosperous guidance during practical and thesis writing. I would like to thank Dr. Felix Bast and Dr. Sandeep Singh for their guidance and help during my experimental work. I am thankful to Dr. Dhanwinder Singh, Sr. Soil Chemist, Department of Soil Sciences, Punjab Agricultural University, Ludhiana, for testing soil samples. I would also like to thankfully acknowledge Dr. R.G. Saini, Coordinator of Centre, Centre for Biosciences and Dr. P. Ramarao, Dean, Academic Affairs for their valuable guidance. I am very thankful to Prof. (Dr.) Jai Rup Singh, Honorable Vice Chancellor, Central University of Punjab, Bathinda for creating and providing world class research facilities. I am extremely thankful to all my friends in CUP, Bathinda, Devendra Gahlot, Bibekananda Sarkar, Ravi Cholia, Md. Zahid, Abhimanyu Kumar, Shweta Thakur and Seema S. for their cooperation, guidance, support and help during M.Phil and to qualify CSIR-JRF (Dec.2011). I am also thankful to all seniors and juniors for their support and help during M.Phil. degree. I am thankful to Ashwani Kumar, Gopal Soni, Sona and Pritpal Singh to help and assist in lab during experimental and computer work. It wouldn’t have been possible for me to complete my work without the love, care, understanding and support of my parents, sisters and all other family members who iv were always in front of me to extend their support at times of desperation and shaping my dissertation. Finally I would like to thankfully acknowledge UGC and Central University of Punjab, Bathinda, for providing me the Rajiv Gandhi Fellowship and financial support during my stay in CUP Bathinda. Rajiv Kumar v TABLE OF CONTENTS S. No. Contents Page no. 1. Introduction 1-4 2. Review of Literature 5-18 3. Materials and Methods 19-23 4. Results 24-39 5. Discussion 40-47 6. Conclusion 48-49 7. References 50-65 vi LIST OF TABLES Table no. Tables description Page no. 2.1 Heavy metal and metalloid reducing and tolerant bacterial 10 species 2.2 Heavy metals and metalloids with their effects on human 13 physiology 2.3 Different method used to study microbial diversity 18 3.1 Primers of 16S rRNA 21 4.1 Physiochemical analysis of soil samples 26 4.2 Heavy metal analysis of soil samples 26 4.3 Result of BLASTN (www.blast.ncbi.nlm.nih.gov) analyses of 35 the bacterial clone sequences of all three soil samples in groups vii LIST OF FIGURES Figure no. Figure description Page no. 3.1 Map of Bathinda showing site of sample collection 20 3.2 Primers of 16S rRNA gene with its internal fragments 21 3.3 Genome walking to align sequenced fragments of 16S 23 rRNA gene 4.1 Extracted DNA of three different soils in triplicate 27 4.2 Amplified 16S rRNA gene (~1500bp) of bacteria from all 27 three soil samples 4.3 Amplified fragments of 16S rRNA gene 27 4.4 Amplified fragments of 16S rRNA gene 28 4.5 Colony PCR showing positive and negative clones 28 amplification 4.6 Microbial profile of Thermal Power Plant, Abandoned and 29 Agricultural soil 4.7 Evolutionary relationships of taxa of abandoned soil 32 4.8 Evolutionary relationships of taxa of agricultural soil 33 4.9 Evolutionary relationships of taxa of thermal power plant 34 soil viii LIST OF ABBREVIATIONS S. No. Abbreviation Full Form 1. US Unused/Abandoned Soil 2. AS Agricultural Soil 3. TS Thermal Power Plant Soil 4. rRNA Ribosomal Ribonucleic acid 5. S Sedimentation Coefficient 6. bp Base pairs 7. G+C Guanine + Cytosine 8. HCl Hydrochloric acid 9. HNO3 Nitric acid 10. ICAP-AES Inductively Coupled Argon Plasma- Atomic Emission Spectrophotometry 11. DNA Deoxyribonucleic acid 12. CTAB Cetyltrimethyl ammonium bromide 13. M Molar 14. mg/Kg Milligram/kilogram 15. 0C Degree Celsius 16. ml Milliliter 17. ng Nanogram 18. µl Micro liter 19. F Forward 20. R Reverse 21. PCR Polymerase Chain Reaction 22. U Unit 23. BLAST Basic Local Alignment Sequence Tool 24. pH Hydrogen ion concentration 25. S Siemens 26. EC Electrical Conductivity 27. OC Organic Carbon 28. MilliQ H2O Distilled water 29. dNTP Deoxynucleoside triphosphates 30. ~ Approximately 31. NCBI National Centre for Biotechnology Information ix Chapter I Introduction Microorganisms are the unseen majority on earth and their diversity is enormous. They are the most important component of biodiversity, and their activities are essential for survival of other forms of life. Soil microbes play pivotal role of biogeochemical cycles on earth, and are responsible for cycling of organic matter. These soil microbes influence soil fertility, soil structure and of above ground ecosystem by contributing plants nutrition. Microbes also help in removing toxins, soil formation, to suppress soil borne diseases of plants, in promoting growth factors and changes in vegetation of particular site (Garbeva et al., 2004; Kirk et al., 2004). They also help in biotransformation, metals and minerals transformation, decomposition, bioweathering and soil formation in biosphere. Both prokaryotes and eukaryotes and their association involve in biogeochemical cycling (Gadd, 2010). Environmental factors like pH, oxygen concentration, particle size of soil, organic carbon content, nutrition availability and water content influence the microbial diversity. Microbial diversity and community vary by physiological and metabolic function of microbes along with the soil profile and location (Hansel et al., 2008). Some of the heavy metals are essential and required by the organisms as micronutrients (trace elements) (Co, Cr, Ni, Fe, Mn and Zn).
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