t h e U n i v e r s it y o f G l a s g o w Effects of culture conditions on the lipid composition of Acinetobacter sp. NCIB 8250 being a thesis submitted for the degree of Doctor of Philosophy at the University of Glasgow by Martin J. McAvoy @Martin J. McAvoy:September, 1993 ProQuest Number: 13834102 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13834102 Published by ProQuest LLC(2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 To my may ske live in peace and my Fa+ke**, may ke rest in peace Summary 1 This thesis describes the effects of a wide variety of growth conditions on the wax ester content and composition of Acinetobacter sp. NCIB 8250. It also describes the effects of these different growth conditions on the fatty acid composition of both the wax esters and the phospholipids found in this bacterium. 2 When Acinetobacter sp. NCIB 8250 was grown in continuous culture at 30 °C under ammonium limitation the wax ester content was significantly greater at low specific growth rates [approximately 4 0 -4 5 mg (g dry weight)"1] than at specific growth rates approaching the maximum specific growth rate [ 1 - 2 mg (g dry weight)'1]. This trend in the wax ester content was true for all ammonium- limited cultures at all growth temperatures investigated. 3 Wax esters were not accumulated to any great extent when Acinetobacter sp. NCIB 8250 was grown in succinate-limited continuous culture. 4 Using continuous culture it was shown that both the specific growth rate and the growth temperature affected the wax ester composition. When the specific growth rate was increased, there was a decrease in the overall chain length of the wax esters but an increase in their degree of unsaturation. As the growth temperature was increased there was no discernible change in the overall chain length but the wax esters became more saturated at the elevated growth temperatures. 5 In continuous culture only the growth temperature had a significant effect on the fatty acid composition of the wax esters, the affect being an increase in the degree of unsaturation as the growth temperature was lowered. However, the increase in the degree of unsaturation in the fatty acid composition was insufficient to account for the increased degree of unsaturation in the wax ester composition, indicating that there was also a change in the degree of unsaturation in the fatty alcohol composition. 6 When Acinetobacter sp. NCIB 8250 was grown at 30 °C in continuous culture the fatty acid composition of the phospholipids was shown to be dependent on the growth temperature, becoming more unsaturated as the growth temperature decreased. However, the growth rate and nutrient limitation did not have a significant affect on the fatty acid composition of the phospholipids. 7 When Acinetobacter sp. NCIB 8250 was grown at 30 °C in batch culture using a medium with a low concentration of (NH^SCU the wax ester content during stationary phase was greater [10 - 15 mg (g dry weight)'1] than during the exponential phase [0-5 - 1 mg (g dry weight)'1]. Also, in batch culture exponential and stationary phase bacteria, grown on the medium with a low concentration of succinic acid there was only 0-2 - 0-5 mg wax esters (g dry weight) ' 1 accumulated. 8 The wax ester composition of Acinetobacter sp. NCIB 8250 grown in batch culture was dependent on the growth temperature and on the phase from which the culture was harvested. At low growth temperatures the wax esters were significantly more unsaturated than when the bacterium was grown nearer its optimum growth temperature. Also, when cultures were harvested from stationary phase the wax ester composition became more saturated and consisted predominantly of shorter chain length wax esters when compared with the wax ester compositions of bacteria harvested during the exponential phase. 9 The fatty acid composition of the wax esters of Acinetobacter sp. NCIB 8250 harvested from stationary phase was predominantly C i 6:o whereas the fatty acids of the wax esters in the bacteria harvested from exponential phase were more unsaturated and the predominant fatty acid was Ci&o- 10 Unambiguous evidence is shown for the presence of diglycerides in the lipids extracted from Acinetobacter sp. NCIB 8250. However, it is not clear whether these compounds originated from the phospholipids or whether they are present in the bacterium as diglycerides. Acknowledgements I am indebted to Professor Charles Fewson and Dr. Les Fixter for their support and guidance during the course of my research and during the writing of this thesis. Also, I am grateful to the late Professor Martin Smellie and Professor Miles Housley for the use of the facilities in their department. I would like to thank Dr. Bob Anderson (Department of Forensic Medicine, University of Glasgow) and Bioflux Ltd (Institute of Biochemistry, University of Glasgow) for the use of the glc-ms and total carbon analyser, respectively. I am also indebted to Mr. T.C. Aitchison and Mr. D. Watt (Department of Statistics, University of Glasgow) and to Mr. D. Buchanan (Paisley College, Paisley) for the statistical analysis of the data. The collection, harvesting, disruption of cell pellets and assaying of NADP- dependent alcohol dehydrogenase activity in samples of Acinetobacter sp. NCIB 8250 was carried out by M. Wales. I am forever indebted to Alan Scott, James Jardine and Patrick Ferry for sharing their superb technical expertise with me and to Dr. El-Mansi M.T. El-Mansi, for sharing! Special thanks go to my diving friends at L.E.S.A.C. - for their sympathetic ears - and to Mags Rozycna - for introducing me to the secrets of juggling, the practice of which helped keep me sane during the latter stages in the writing of this thesis!! I produced this thesis on my Apple Macintosh™ using Word™ and CricketGraph™ Contents page Summary i Acknow ledgements iii Contents iv List of Tables X List of Figures xi Abbreviations xiii Chapter One Introduction 1 1.1 The genus Acinetobacter 2 1.2 Wax esters 5 1.2.1 Structure and properties 5 1.2.2 Distribution and importance 8 1.2.3 Biosynthesis of fatty acids, fatty alcohols and wax esters 10 1.2.3.1 Biosynthesis of fatty acids 10 1.2.3.2 Biosynthesis of fatty alcohols 17 1.2.3.3 Esterification of fatty acids and alcohols to form wax esters 22 1.3 Phospholipids 24 1.3.1 Biosynthesis of phospholipids 24 1.3.2 Regulation of membrane fluidity 28 1.4 Aims of this project 31 Chapter Two Methods 33 2.1 Materials 34 2.2 Culture Storage 34 2.3 Growth media .35 2.3.1 Complex media 35 2.3.1.1 Nutrient broth 35 2 3 .1 2 Nutrient agar 35 2.3.2 Minimal media 35 2.3.2.1 Standard salts medium 35 2.3.2.2 Medium containing a low concentration of succinic acid 35 23.2.3 Medium containing a low concentration of (NH 4 )2 SC>4 36 23.2.4 Mandelate/salts agar 36 2.3.3 Trace metal supplement 36 iv 2.4 Sterilization 37 2.4.1 Steam 37 2.4.2 Dry heat 37 2.4.3 Ethylene oxide 37 2.5 Culture growth conditions 37 2.5.1 Preparation of the inocula 37 2.5.2 Batch culture 38 2.5.3 Continuous culture 38 2.6 Optical density measurement 40 2.7 Harvesting of cultures 41 2.8 Extraction of bacterial lipids for total wax ester content and fatty acid and wax ester composition analysis 41 2.9 Separation and localization of the different classes of lipids by thin layer chromatography 42 2.9.1 Preparation of the thin layer chromatography plates 42 2.9.2 Separation and localization of the different classes of lipids 42 2.10 Estimation of wax ester content and fatty acid and wax ester compositions 43 2.10.1 Chemical synthesis and purification of the internal standard 43 2.10.2 Gas liquid chromatographic analysis of wax esters 43 2.10.3 Gas liquid chromatographic analysis of the fatty acid methyl esters 45 2.10.3.1 Preparation of the fatty acid methyl esters 45 2.10.3.2 Analysis of the FAME by gas liquid chromatography 45 2.10.4 Gas liquid-mass spectrometry 46 2.11 Estimation of organic carbon and identification of the organic acids in culture supernatants 46 2.11.1 Estimation of the organic carbon concentration 46 2.11.2 Identification of the organic acids and estimation of the concentrations 47 2.12 Estimation of the ammonia concentration 47 2.13 Collection of Acinetobacter sp. NCIB 8250 from continuous culture, the preparation of cell free extracts and assaying for NADP-dependent alcohol dehydrogenase activity 48 2.13.1 Collection and preparation of the cell free extracts used in the NADP-dependent alcohol dehydrogenase assay 48 2.13.2 NADP-dependent alcohol dehydrogenase assay 48 2.14 Estimation of the protein concentration 49 2.15 Oxidase test 49 2.16 Statistical analysis of data 49 v 2.17 Cleaning of glassware 50 2.17.1 General purpose glassware 50 2.17.2 Growth flasks 50 2.17.3 Glassware used for the extraction of lipids 50 2.17.4 Pipettes 50 2.18 Distilled Water 50 2.19 Safety 50 Chapter Three Validation of Experimental Methods 52 3.1 Introduction 53 3.2 Results and Discussion 53 3.2.1 Calibration curves of optical density against dry weight and against the carbon content of cells 53 3.2.2 Determination of the nutrient limitation in culture supernatants 56 3.2.3 Extraction and analysis of the lipids in Acinetobacter sp.