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Molecular Identification and Physiological Characterization of Halophilic and Alkaliphilic Bacteria Belonging to the Genus Halomonas

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Molecular Identification and Physiological Characterization of Halophilic and Alkaliphilic Bacteria Belonging to the Genus Halomonas Molecular Identification and Physiological Characterization of Halophilic and Alkaliphilic Bacteria Belonging to the Genus Halomonas By Abdolkader Abosamaha Mohammed (BSc, Agricultural Sciences, Sebha University, Libya) (MSc, Environmental Engineering, University of Newcastle Upon Tyne, Uk) A Thesis Submitted for Degree of Doctor of Philosophy Department of Molecular Biology and Biotechnology The University of Sheffield 2013 Abstract Alkaline saline lakes are unusual extreme environments formed in closed drainage basins. Qabar - oun and Um - Alma lakes are alkaline saline lakes in the Libyan Sahara. There were only a few reports (Ajali et al., 1984) on their microbial diversity before the current work was undertaken. Five Gram-negative bacterial strains, belonging to the family of Halomonadaceae, were isolated from the lakes by subjecting the isolates to high salinity medium, and identified using 16S rRNA gene sequencing as Halomonas pacifica, Halomonas sp, Halomonas salifodinae, Halomonas elongata and Halomonas campisalis. Two of the Halomonas species isolated (H. pacifica and H. campisalis) were chosen for further study on the basis of novelty (H. pacifica) and on dual stress tolerance (high pH and high salinity) shown by H. campisalis. Both species showed optimum growth at 0.5 M NaCl, but H. campisalis alone was able to grow in the absence of NaCl. H. pacifica grew better than H. campisalis at high salinities in excess of 1 M NaCl and was clearly a moderately halophile. H. pacifica showed optimum growth at pH 7 to 8, but in contrast H. campisalis could grow well at pH values up to 10. 13C - NMR spectroscopy was used to determine and identify the compatible solutes accumulated by H. pacifica and H. campisalis grown in rich and minimal media at different concentrations of NaCl. H. pacifica and H. campisalis accumulated betaine in rich (LB) medium with ectoine only appearing at the highest salinity tested (2.5 M NaCl). In contrast, in M9 minimal medium, no betaine was detected and ectoine and hydroxyectoine were accumulated at high salinities. H. campisalis was able to grow well with urea or nitrate as the sole source of nitrogen and was shown to be capable of efficiently removing nitrate from the medium under aerobic assimilatory conditions, where it is incorporated into biomass. i Acknowledgements First and foremost, I thank Allah for His guidance to make this work possible and to give me the ability and patience to complete this research. It is a pleasure for me to acknowledge the precious help and support provided to me by my research supervisor, Dr James Gilmour during a period of my PhD study. His guidance, advice and productive criticism have been invaluable in the shaping and synthesis of this research work. In addition, I also would like to thank my two advisors professor Milton Williamson and Dave Kelly for their guidance and support during each stage of my research. I am very grateful to Professor Mike Williamson, for his help to use NMR spectroscopy. I must thank my parents and bothers for their love and fully devotion towards my studies. I do have especial thanks to my wife ‘Imkultoum’ and my kids Buthiena, Mohammed and Fatima who shared the life with me in the Uk and makes the success and happiness during my study is possible. Last but not least, I would like to thank all my colleagues in the Department of Molecular Biology and Biotechnology in the University of Sheffield. ii Contents CHAPTER 1 General Introduction ...................................................................... 1 1 General Introduction .................................................................................................... 2 1.1 Hypersaline Environments ...................................................................................... 2 1.1.1 The Dead Sea .......................................................................................................... 4 1.1.2 The Great Salt Lake ................................................................................................ 6 1.1.3 Soda lakes ............................................................................................................... 8 1.1.3.1 Wadi Natrun Lakes ............................................................................................... 10 1.1.3.2 Lake Magadi ......................................................................................................... 12 1.2 Biodiversity of Halophilic and Halotolerant Microorganisms .............................. 15 1.2.1 Halophilic and Halotolerant Microorganisms ....................................................... 16 1.2.2 Haloalkaliphilic Microorganisms .......................................................................... 18 1.3 Diversity of Compatible Solute ............................................................................. 21 1.3.1 Glycine Betaine ..................................................................................................... 25 1.3.2 Ectoine and Hydroxyectoine ................................................................................. 27 1.3.3 Osmoregulation of compatible solutes in prokaryotic cells .................................. 29 1.4 Molecular Method Used to Identfiy Microorganisms ........................................... 31 1.5 Biotechnological Applications of Halotolerant and Halophilic Microorganims .. 34 1.5.1 Compatible solutes ................................................................................................ 34 1.5.2 Enzymes ................................................................................................................ 35 1.5.3 Bioremediation ...................................................................................................... 35 1.6 Aims of the project ................................................................................................ 34 CHAPTER 2 Materials and Methods ............................................................... 39 2 Materials and Methods............................................................................................... 40 2.1 Study Sites ............................................................................................................. 40 2.2 Sample Collection ................................................................................................. 40 2.3 Isolation of Microorganisms ................................................................................. 43 2.3.1 Growth media and culture conditions ................................................................... 43 2.3.1.1 Luria-Bertani (LB) medium .................................................................................. 43 2.3.1.2 M9 minimal salt medium ...................................................................................... 43 iii 2.3.2 Initial isolation of microorganisms ....................................................................... 46 2.3.3 Maintenance of the halophilic microorganisms .................................................... 46 2.4 Initial Identification of Microorganisms ............................................................... 46 2.4.1 Cell morphology ................................................................................................... 46 2.4.2 Motility .................................................................................................................. 47 2.5 Molecular Identification of Environmental Isolates ............................................. 47 2.5.1 Genomic DNA extraction ..................................................................................... 47 2.5.2 Agarose gel electrophoresis .................................................................................. 49 2.5.3 Polymerase chain reaction (PCR) amplification of 16S rRNA gene .................... 49 2.5.4 Purification of PCR products ................................................................................ 50 2.5.5 DNA sequencing and phylogenetic analysis ......................................................... 51 2.6 Electron Microscopy ............................................................................................. 51 2.6.1 Preparation of cells for scanning electron microscopy (SEM) ............................. 51 2.6.2 Transmission electron microscopy (TEM) ........................................................... 52 2.7 Growth experiments .............................................................................................. 54 2.7.1 Anaerobic growth .................................................................................................. 54 2.7.2 Determination of optimum salinity ....................................................................... 54 2.7.3 Determination of pH range for growth.................................................................. 54 2.7.4 Growth at optimum temperature ........................................................................... 55 2.7.5 Determination of growth curve ............................................................................. 55 2.7.6 Carbon source utilization ...................................................................................... 56 2.8 Measurement of Oxygen Uptake in the Bacterial Cells ........................................ 57 2.8.1 Preparation and calibration of oxygen electrode ................................................... 57 2.8.2 Oxygen (O2) uptake calculation ..........................................................................
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