Cover picture: Vibrio cholerae , magnification approximately 10.000 × Reprinted permission from Macmillan Publishers Ltd: Nature 406, 469-470. M.K. Waldor and D. RayChaudhuri. Bacterial genomics: Treasure trove for cholera research. Copyright 2000 Vakgroep Analytische Chemie Vakgroep Biochemie, Fysiologie en Microbiologie INW Laboratorium voor Microbiologie (Lm-Ugent) Proeftuinstraat 86 K.L. Ledeganckstraat 35 9000 Gent 9000 Gent Raman spectroscopy as a tool for studying bacterial cell compounds Joke De Gelder Academic year 2007-2008 Dissertation submitted in fulfillment of the requirements for the degree of Doctor (Ph.D.) in Sciences, Chemistry Promotor: Prof. Dr. Luc Moens Co-promotor: Prof. Dr. Peter Vandenabeele Co-promotor: Prof. Dr. Paul De Vos Content Abbreviations and acronyms Chapter 1: Introduction and aim ......................................................................... 1 Chapter 2: Microbiological aspects ..................................................................... 7 2.1 Bacteria in the pool of living organisms ..................................................... 9 2.2 The bacterial cell ................................................................................... 9 2.2.1 General bacterial cell constitution ............................................... 9 2.2.2 Nutrition and metabolic systems ................................................ 11 2.2.3 Sporulation............................................................................. 15 2.2.4 PHB production ...................................................................... 17 2.3 Bacterial growth .................................................................................. 19 2.4 Taxonomy........................................................................................... 20 2.4.1 Definition and techniques ......................................................... 20 2.4.2 Taxonomic ranks and Bergey’s manual....................................... 22 2.4.3 Taxonomic position of species studied in this work ....................... 24 Cupriavidus necator .......................................................... 24 Cupriavidus metallidurans .................................................. 25 Bacilus species................................................................. 26 Paenibacillus lactis ........................................................... 27 Enterococcus faecalis ........................................................ 27 Chapter 3: Raman spectroscopy ....................................................................... 35 3.1 Introduction ........................................................................................ 37 3.2 The Raman effect: quantum theory ......................................................... 37 3.3 The Raman effect: classical theory and selection rules ............................... 38 3.4 Raman shift and intensity ...................................................................... 39 3.5 Fluorescence....................................................................................... 41 3.6 Advantages and disadvantages ............................................................... 41 Chapter 4: Data processing ............................................................................. 45 4.1 Introduction ........................................................................................ 47 4.2 EMSC ................................................................................................ 48 4.3 Modified polynomial fit.......................................................................... 50 4.4 PCA .................................................................................................. 50 4.5 Calculation of dot products ................................................................... 53 Chapter 5: Reference database of Raman spectra of biological molecules ................ 57 5.1 Introduction ........................................................................................ 59 5.2 Experimental ....................................................................................... 60 5.2.1 Raman instrumentation............................................................. 60 5.2.2 Reference products.................................................................. 61 5.3 Results and discussion .......................................................................... 63 5.3.1 DNA and RNA bases................................................................ 64 5.3.2 Amino acids............................................................................ 70 5.3.3 Fatty acids and fats ................................................................. 72 5.3.4 Saccharides ............................................................................ 75 5.3.5 Primary metabolites................................................................. 78 5.3.6 Others................................................................................... 80 5.3.7 Examples of Raman spectra of biological materials........................ 81 5.4 Conclusions ........................................................................................ 82 Chapter 6: Raman spectroscopic study of bacterial endospores ............................. 87 6.1 Introduction ........................................................................................ 89 6.2 Experimental ....................................................................................... 90 6.2.1 Strains and culturing conditions................................................. 90 6.2.2 Sample preparation.................................................................. 92 6.2.3 Products ................................................................................ 92 6.2.4 Raman spectroscopy ................................................................ 92 6.3 Results and discussion .......................................................................... 93 6.3.1 Monitoring sporulation of Bacillus licheniformis LMG 7634 ............ 93 6.3.2 Discrimination between endospores from different species, strains or cultivation conditions......................................................... 98 6.4 Conclusions .......................................................................................104 Chapter 7: Methods for extracting biochemical information from bacterial Raman spectra: an explorative study on Cupriavidus metallidurans LMG 1195..................111 7.1 Introduction .......................................................................................113 7.2 Experimental ......................................................................................115 7.2.1 Strain and medium..................................................................115 7.2.2 Sample preparation.................................................................115 7.2.3 Raman spectroscopy ...............................................................116 7.2.4 Data processing .....................................................................117 7.3 Results and discussion .........................................................................118 7.4 Conclusions .......................................................................................127 Chapter 8: Methods for extracting biochemical information from bacterial Raman spectra: focus on a group of structurally related molecules - fatty acids ................133 8.1 Introduction .......................................................................................135 8.2 Experimental ......................................................................................136 8.2.1 Strains and culturing conditions................................................136 8.2.2 Raman spectroscopy ...............................................................137 8.2.3 Gas chromatographic fatty acid methyl ester analysis (FAME).......138 8.3 Results and discussion .........................................................................138 8.3.1 FAME analysis.......................................................................138 8.3.2 Discussion of the Raman spectra of fatty acids............................140 8.3.3 Discussion of Raman spectra B. coagulans and E. faecalis .............142 8.3.4 Chemometric approach for the extraction of fatty acid information from bacterial Raman spectra .................................................147 8.4 Conclusions .......................................................................................152 Chapter 9: Monitoring poly-3-hydroxybutyrate in Cupriavidus necator DSM 428 with Raman spectroscopy ...............................................................................157 9.1 Introduction .......................................................................................159 9.2 Experimental ......................................................................................160 9.2.1 Strains, culturing conditions and sampling..................................160 9.2.2 Products ...............................................................................162 9.2.3 Raman spectroscopy ...............................................................162 Instrument 1...................................................................162 Instrument 2...................................................................163 9.2.4 Data analysis .........................................................................163 9.2.5 HPLC analysis .......................................................................163