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Diversity of Halophilic Bacteria in Saline Soil at Nong Bo Reservoir Was Determined Using the Ultimate Results of Bacterial Characterization

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Diversity of Halophilic Bacteria in Saline Soil at Nong Bo Reservoir Was Determined Using the Ultimate Results of Bacterial Characterization DIVERSITY OF HALOPHILIC BACTERIA IN SALINE SOIL AT NONG BO RESERVOIR, MAHASARAKHAM PROVINCE, THAILAND Mrs. Kannika Chookietwattana A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Environmental Biology Suranaree University of Technology Academic Year 2003 ISBN 974-283-006-1 ความหลากหลายของแบคทีเรียชอบเจริญในที่เค็มในดินเค็มบริเวณ อางเก็บน้ําหนองบอ จังหวัดมหาสารคาม ประเทศไทย นางกรรณิการ ชูเกียรติวัฒนา วิทยานิพนธนี้เปนสวนหนึ่งของการศึกษาตามหลักสูตรปริญญาวิทยาศาสตรดุษฎีบัณฑิต สาขาวิชาชีววิทยาสิ่งแวดลอม มหาวิทยาลัยเทคโนโลยีสุรนารี ปการศึกษา 2546 ISBN 974-283-006-1 Acknowledgement I would like to express the deepest gratefulness to my thesis advisor, Asst. Prof. Dr. Sureelak Rodtong, for kindly accepting me as her graduate student to discover the halophiles’ world in saline soil. She kindly provided me the laboratory facilities, chemicals, and microbiological media. All her advice is very valuable for my life and future career. My deepest gratitude also to my two co-advisors, Dr. Joseph A. Odumeru and Dr. Shu Chen, for giving me the opportunity to work in the Laboratory Services Division, University of Guelph, Canada, and for their continuous support, encouragement, and valuable suggestions from which I have greatly profited. My warmest thanks to all Thais in Guelph and all the staff of Guelph Molecular Supercentre and Food Microbiology, the Laboratory Services Division, for their readiness to help and warmth support for me throughout my stayed in Canada. All the experience I gained from the Laboratory Services Division is a great value. I would like to thank Dr. Usa Klinhom for encouraging me to study microbial diversity in saline soil and the kindly cooperative staff of Walairukhavej Botanical Research Institute, Mahasarakham University, in the soil samples collection. My special thanks to Mahasarakham University for providing funds through the University Development Scholarship to pursue my Ph.D. graduate work at Suranaree University of Technology (SUT) and the Mahasarakham University Staff Development Fund for training at the Laboratory Services Division. I also thank the SUT for partial funding for this work with the thesis grant. My thanks and great appreciation to the IV following people in the SUT: Areeya, Jitraporn, Kunthika, Nuttika, Phetchara, Wantanee, and Woottiput. Lastly, I would like to express my deepest gratitude to my mother, my father, and my husband for their greatest love, encouragement, help and support for me to overcome all the challenges and difficulties in this work. Kannika Chookietwattana Contents Page Abstract in Thai …………………………………………………………………. I Abstract in English …………………………………………………………..….. II Acknowledgement …………………………………………………………..…... III Contents ………………………………………………………………………… V List of Tables …………………………………………………………………… XII List of Figures …………………………………………………………. XIV List of Abbreviations ………………………………………………..…. XVI Chapters I Introduction ………..…………………………………………………... 1 1.1 Overview of saline soil problems and significance of halophilic bacteria study ………………………………………………………. 1 1.2 Research objectives …………………………………………………. 3 1.3 Expected results .……………………………………………………. 3 II Literature review .…..…………………………………………………... 4 2.1 Saline soil .……..…………………………………………………..... 4 2.1.1 Characteristics of saline soil ……………………………..… 4 2.1.2 Cause of soil salanisation ……………………………..……. 6 VI Contents (Continued) Page 2.1.3 Effects of soil salinity .………………………………..…… 6 2.1.3.1 Effects on soil structure properties .……….…....…. 7 2.1.3.2 Effects on plant growth .…………………….....….. 7 2.1.3.3 Effects on soil microorganisms .…………..…...….. 8 2.1.4 Saline soil in the northeast Thailand …………….……..….. 8 2.2 Bacterial diversity in soil …….…………………………….……… 12 2.2.1 Species concept in the study of bacterial diversity ……….. 12 2.2.2 Culturable and nonculturable bacteria in soil ……………... 13 2.2.3 Methods used in the study of soil bacterial diversity ……… 14 2.2.4 Bacterial systematics: the basic information for bacterial diversity ……………………………………….… 19 2.3 Halophilic microorganisms ………………………………………... 20 2.3.1 Systematics of halophilic bacteria …………………….…… 21 2.3.1.1 Extremely halophilic bacteria …………………….. 24 2.3.1.2 Moderately halophilic bacteria ……………….…… 24 1) Moderately halophilic eubacteria ……………... 25 2) Moderately halophilic archaebacteria ………… 25 2.3.2 Habitats of halophilic bacteria …………………………….. 26 2.3.2.1 Saline water ………………………………..….…… 26 VII Contents (Continued) Page 1) The Dead Sea ( at the border of Israel and Jordan) ……………………………………….. 27 2) The Great Salt Lake, Utah, U.S.A. …………… 27 3) The Solar Lake, Sinai, Egypt …………..…….. 28 4) Lakes at the Wadi Natrun ……………..……… 29 5) Inland saltern of La Mala, Granada, Spain …… 30 2.3.2.2 Saline soil ………………………………………… 30 2.3.2.3 Salted food ………………………………………... 31 2.3.3 Adaptation of halophilic bacteria …………………………. 32 2.3.3.1 Osmosensor and osmoregulation by bacteria …….. 33 2.3.3.2 Accumulation of compatible solutes by halophilic bacteria …………………………..……. 34 2.3.3.3 Stabilization of macromolecules …………….……. 36 2.3.4 Applications of halophilic bacteria ………………….……. 37 III Materials and methods ……………………………………………… 41 3.1 Chemicals and Reagents ……………………………………….….. 41 3.1.1 Chemicals and reagents for microbiological analysis ….… 41 3.1.2 Reagents for fatty acid extraction ………………………… 41 VIII Contents (Continued) Page 3.1.3 Reagents for nucleic acid analysis ………………………. 42 3.2 Instrumentation ………………………………………………….. 42 3.3 Site description ……………………………………………….….. 43 3.4 Saline soil sample collection …………………………………..…. 46 3.5 Physical and chemical analysis of saline soil samples …………..... 46 3.5.1 Determination of air and soil temperature ….……….…...... 47 3.5.2 Determination of soil moisture content ………….……….... 47 3.5.3 Determination of soil pH …………………….……..……… 48 3.5.4 Determination of soil salinity …………………..…….……. 48 3.6 Microbiological analysis of saline soil samples ………………….. 49 3.7 Characterization of bacterium isolates …………..………………. 50 3.7.1 Phenotypic characterization ………..………………….… 50 3.7.1.1 Morphological characteristics …………………... 50 3.7.1.2 Cultural characteristics ………………………..… 50 3.7.1.3 Physiological characteristics …………………..… 51 1) pH-, temperature-, and salt tolerance tests ………………………………..……….. 51 2) Biochemical tests ……………………..…….. 51 IX Contents (Continued) Page A. Nitrate reduction test ……………..……… 51 B. Methyl red test ……………………..……. 52 C. Voges-Proskauer test ……………..……… 52 D. Indole formation ……………………..…... ..53 E. Acid from carbohydrate utilization ….…...53 F. Starch hydrolysis ……………………...…. 54 G. Gelatin hydrolysis ……………….………. ... 54 H. Hydrogen sulfide production ………..……55 3) Enzymatic tests ……………………………... .. 55 A. Catalase test ……………………………... 55 B. Oxidase test …………………….….…….. 55 3.7.1.4 Antimicrobial sensitivity test ………….….….….. 55 3.7.2 Chemical analysis …………………………….……..……. 56 3.7.3 Genotypic characterization …………………………..….... 60 3.7.3.1 Genomic DNA extraction ……………….….….. 60 3.7.3.2 PCR amplification of 16S rDNA of bacterium isolates ………………………………….…..…… 61 3.7.3.3 Sequencing of PCR-amplicons ………..…..…… 63 3.7.3.4 16S rDNA sequence analysis ……………...…….. 63 X Contents (Continued) Page 3.8 Maintenance of selected bacterial isolates for future application ………………………………………………….……. 64 3.9 Data analysis ……………………………………………….…..… 64 IV Results and discussion …………………………………………....… 66 4.1 Physical and chemical properties of saline soil samples ….…... 66 4.2 Density of halophilic bacteria in saline soil ………..………..... 74 4.3 Diversity of halophilic bacteria in saline soil ……….……...…. 80 4.3.1 Phenotypic characteristics of halophilic bacterium isolates ……………………………………………..….. 83 4.3.2 Cellular fatty acid profiles of halophilic bacterium isolates ……………………………………………….. 92 4.3.3 Genotypic characteristics of halophilic bacterium isolates …………………………………..………….…. 97 V Conclusion and future perspective ……………………………..….. 109 References…………………………..………………………..………….… 115 XI Contents (Continued) Page Appendices ……………………………………………………………… 145 Appendix A Culture media and reagent preparations…….………… 146 Appendix B Examples of fatty acid composition report and fatty acid chromatogram …………………………….…. 159 Appendix C Physical and chemical properties, and halophilic bacterial density of saline soil samples …………….….. 165 Appendix D Results of statistical analysis …………….….…….…... 176 Appendix E Results of phenotypic characteristics tested and halophilic bacterial characterization ………………….… 180 Appendix F Examples of gel electrophoresis and electrophenogram ………………………..………..….. 200 Appendix G Preliminary study of DNA extraction from saline soil …………………………………………………….. 203 Curriculum Vitae …………………………………………………..…… 213 XII List of Tables Page Table 1 Interpretation of ECe and total salt content ……...……………….….... 5 2 Details of 5 sampling plots at Nong Bo Reservoir ……..………….… 43 3 Oligonucleotide primers used for PCR amplification and sequencing of 16S rDNA ……………………………..…………….… 62 4 Results of particle size analysis of soil samples collected at Nong Bo Reservoir From June 2001 to May 2002 ………………..... 67 5 Physical and chemical properties of saline soil samples collected at Nong Bo Reservoir from June 2001 to May 2002 ………….……… 73 6 Summarization of the number of halophilic bacterium isolates selected from this study ……………………………………….………. 81 7 Phenotypic features of 20 bacterial isolates in different species that were isolated from the media containing 3 % NaCl ………….…… 87 8 Phenotypic features of 11 bacterial isolates in different species that were isolated from the media
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