Doctoraatsthesis Kris De Gussem

UNIVERSITEIT GENT FACULTEIT WETENSCHAPPEN Vakgroep Analytische Scheikunde Optimisation of Raman spectroscopy for the analysis of Basidiomycota: spores, latex and mycelium Optimalisatie van Ramanspectroscopie voor de analyse van Basidiomycota: sporen, latex en mycelium Kris De Gussem Academiejaar 2006 - 2007 Proefschrift neergelegd tot het verkrijgen van de graad van Doctor in de Wetenschappen, Scheikunde Promotor: Prof. Dr. Luc Moens Co-promotor: Prof. Dr. Annemieke Verbeken Co-promotor: Dr. Peter Vandenabeele The front page shows a picture of a specimen of Lepista nuda . It was photographed by Martine Decoussemaeker in De Panne (Westhoekreservaat). Lactarius deterrimus (Picture by Ruben Walleyn) Lactarius deterrimus as observed by a mycologist, microscopic view of a lamella (in 10% KOH) (Picture by Jorinde Nuytinck) Intensity (Arbitrary units) 400 600 800 1000 1200 1400 1600 1800 Raman shift (cm -1) Lactarius deterrimus as observed by a Raman spectroscopist, Raman spectrum of a single spore 1 cttggtcatt tagaggaagt aaaagtcgta acaaggtttc cgtaggtgaa cctgcggaag 61 gatcattatc gtacaaacat gtgnntgagn ngcgtgcgag ggctgtcgct gactttttga 121 ngacacaaaa gtcgtgcacg ccggagtgcg tcctctnnnn cacantaaaa ntccatctca 181 cccttttgtg caccaccgcg tgggtgccct tcngggatcg aatcgatcca ggagggggnc 241 ttgcgttttc acacaaaccc cttttaaaaa ngtgtagaat gaccccactt tgcgatnnna 301 cgcaatcaat acaactttca acaacggatc tcttggctct cgcatcgatg aagaacgcag 361 cgaaatgcga tacgtaatgt gaattgcaga attcagtgaa tcatcgaatc tttgaacgca 421 ccttgcgccc cttggtattc cgaggggcac acccgtttga gtgtcgtgaa aatctcaacc 481 ttctcggttt tcttctggac accgaaggag gcttggactt tggaggcctt tgctggcgtc 541 tctctcttct tntngagagc cnagctcctc ttaaacgaat tagcggggtc ctctttgccg 601 atcctcgaca tgtgataaga tgtttccatg acttggtttc tggctctgtt gcctttgggn 661 nnnnacctgc ttctaaccgt ctcgacgaga cgacgtttgg gcgcgtctcc cttctcggga 721 gactctctca accccnacga acccttgacc tcaaatcggg tgagactacc cgctgaactt 781 aagcatatca ataagcggag gaaaaganaa ctaacaagga ttcccctagt aactgcgagt 841 gaangcggga aaagctcaaa tttaaaatct gacggtcttt ggccgtccga gttgtaattt 901 agagaagcgt cttccg Lactarius deterrimus as observed by a microbiologist, 18S ribosomal RNA gene, partial sequence, Genbank: DQ922535 CONTENT WOORD VOORAF ....................................... ................. IX OUTLINE ............................................ .................... 5 Abbreviations and acronyms ......................... ......................... 9 PART I: INTRODUCTION AND THEORY . ................ 11 CHAPTER I: Raman spectroscopy and mycology . ................. 13 1 Introduction..................................... ................. 13 2 Raman spectroscopy ............................... ................ 15 2.1 The Raman effect ............................... ............ 16 2.2 Properties of Raman spectroscopy . ............... 17 a) Advantages of Raman spectroscopy ................. ..... 17 b) Disadvantages of Raman spectroscopy . ...... 18 2.3 Comparison of Raman spectroscopy with other techniques........... 18 3 Calibration and the use of chemometrics . ..................... 20 3.1 Accurate calibration of a Raman spectrometer . ................. 20 3.2 Pre-treatment of data .......................... .............. 21 3.3 Data analysis.................................. ............. 22 4 Applications of Raman spectroscopy in mycology . ..................... 22 4.1 Clinical applications .......................... ............... 23 4.2 Investigation of lichens ....................... ............... 23 4.3 Raman spectroscopic analysis of higher fungi . .................. 24 4.4 Other applications ............................. ............. 24 5 Future prospects of Raman spectroscopy in mycology..................... 25 6 Concluding remarks ............................... ................ 26 7 Notes on this publication ........................ ................... 26 CHAPTER II: Introduction to Basidiomycota ...................... ................... 27 1 Introduction..................................... ................. 27 2 General characteristics of fungi . ..................... 28 3 Life cycle of the Basidiomycota . .................... 28 4 Morphology of the basidiocarp . ................... 30 5 Systematics and taxonomy of Basidiomycota . ................... 31 CHAPTER III: Raman spectroscopy as a spectroscopic tool for the analysis of biomolecules . 35 1 Introduction..................................... ................. 35 2 Experimental..................................... ................ 37 2.1 Raman instrumentation .......................... ............ 37 2.2 Reference products ............................. ............. 38 3 Results and discussion ........................... .................. 40 3.1 Fatty acids and fats ........................... ............... 40 3.2 Saccharides.................................... ............ 44 4 Conclusions...................................... ................ 47 PART II: RAMAN SPECTROSCOPIC STUDY OF MACROFUNGI . ............ 49 CHAPTER I: Materials and methods .............................. ................. 51 1 Introduction..................................... ................. 51 2 Samples.......................................... ............... 52 2.1 The genus Lactarius ......................................... 52 2.2 The genus Russula .......................................... 54 2.3 The genus Laccaria ......................................... 54 2.4 The genus Collybia .......................................... 55 2.5 The genus Mycena .......................................... 56 3 Raman spectra collected from tissues of Lactarius species ................. 56 4 Collection of spore prints ....................... .................... 60 5 Chemometric analysis ............................. ................. 61 5.1 EMSC preprocessing ............................. ........... 63 a) Mathematical treatment of spectra ................ ....... 63 b) Mathematical modelling of spectra ................ ....... 64 c) The EMSC equation ................................ 65 5.2 2D correlation analysis ........................ ............... 66 CHAPTER II: Raman-spectroscopic study of Lactarius spores (Russulales, Fungi) . 73 1 Introduction..................................... ................. 74 2 Experimental..................................... ................ 75 2.1 Instrumentation................................ ............. 75 2.2 Samples ........................................ .......... 76 2.3 Reference materials ............................ ............. 76 2.4 Data processing ................................ ............ 76 3 Results and discussion ........................... .................. 77 3.1 Raman spectra of Lactarius spores ............................. 77 3.2 Raman spectra of reference materials . ................. 79 3.3 Interpretation of the Raman spectrum of L. controversus spores ....... 86 4 Conclusion ....................................... ............... 89 5 Notes on this publication ........................ ................... 89 CHAPTER III: Chemotaxonomical identification of spores of macrofungi .................. 91 1 Introduction..................................... ................. 92 2 Experimental..................................... ................ 93 2.1 Instrumentation................................ ............. 93 2.2 Instrument calibration......................... ............... 94 2.3 Samples and spectral dataset . ............... 94 2.4 Software ....................................... ........... 96 3 Results and discussion ........................... .................. 96 3.1 Chemotaxonomic identification of the spores . ................. 98 3.2 Spectral differences ........................... ............. 101 4 Conclusion ....................................... .............. 103 CHAPTER IV: Raman spectroscopic monitoring of Lactarius latex ....................... 105 1 Introduction..................................... ................ 106 2 Experimental..................................... ............... 108 2.1 Instrumentation................................ ............ 108 2.2 Samples and spectral acquisition . ............... 108 2.3 Instrument calibration......................... .............. 109 2.4 Data preprocessing ............................. ............ 109 2.5 2D correlation spectroscopy . .............. 109 3 Results and discussion ........................... ................. 109 3.1 Chemical reaction monitoring . ............. 110 3.2 2D correlation analysis of Lactarius lacunarum latex .............. 111 3.3 Explorative inter-species comparison of Lactarius latex Raman spectra ................................................... 116 3.4 Method evaluation .............................. ........... 118 4 Conclusion ....................................... .............. 119 5 Notes on this publication ........................ .................. 119 PART III: RAMAN SPECTROSCOPIC STUDY OF WOOD AND WOOD FUNGI . ......... 121 CHAPTER I: Raman spectroscopic study of wood inhabiting fungi . ..................... 123 1 Introduction..................................... ................ 123 2 Materials and methods ............................ ................ 124 3 Results and discussion ........................... ................. 125 3.1 Study of the Raman spectra of mycelium cultures ................. 125 a) Poria monticola mycelium ............................ 126 b) Gloeophyllum trabeum mycelium ....................... 127 c) Coniophora puteana mycelium ........................

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