Spectral fluorescence of chlorophyll and phycobilins as an in-situ tool of phytoplankton analysis – models, algorithms and instruments Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von Martin Beutler Kiel, März 2003 Referent: Prof. Dr. U.-P. Hansen........................................... Korreferent: ........................................................................... Tag der mündlichen Prüfung:................................................ Zum Druck genehmigt: Kiel, den.......................................... ....................................................... Der Dekan Vorbemerkung Teilergebnisse dieser Dissertation sind bereits wie folgt vorab veröffentlicht bzw. eingereicht worden: Veröffentlicht: Beutler, M., K. H. Wiltshire, B. Meyer, C. Moldaenke & H. Dau (1998): Rapid depth-profiling of the distribution of spectral groups of microalgae in lakes, rivers and the sea. In. Photosynthesis: Mechanisms and effects V (ed. G. Garab). pp. 4301-4304. Kluwer Academic Publishers, Dordrecht. Beutler, M., Wiltshire, K. H., Meyer, B., Moldaenke, C. & Dau, H. (2001). In situ profiles of phytoplankton: algal composition and biomass determined fluorometrically. In Proceedings of the Ninth International Conference on Harmful Algal Blooms, Hobart, Australia, 7-11 February 2000 (ed. G. M. Hallegraeff, S. I. Blackburn, C. J. Bolch and R. J. Lewis), pp. 202-205. Intergovernmental Oceanographic Commission of UNESCO, Paris. Beutler, M., Wiltshire, K. H., Lüring, C., Moldaenke, C. & Lohse, D. (2002). Fluorometric depth-profiling of chlorophyll corrected for yellow substances. In Aquaculture environment and marine phytoplankton, vol. 34 (ed. G. Arzul), ISSN 0761-3962. pp. 231- 238. Ifremer, Brest. Beutler, M., Wiltshire, K. H., Meyer, B., Moldaenke, C., Lüring, C., Meyerhöfer, M., Hansen, U.-P. & Dau, H. (2002). A fluorometric method for the differentation of algal populations in vivo and in situ. Photosynthesis Research 72, 39-53. Beutler, M., Wiltshire, K. H., Arp, M., Kruse, J., Reineke, C., Moldaenke, C. & Hansen, U.-P.. A reduced model of the fluorescence from the cyanobacterial photosynthetic apparatus designed for the in situ detection of cyanobacteria. (In Druck) Biochimica et Biophysica Acta-Bioenergetics. Eingereicht: Schimanski, J., Moldaenke, C., Hansen U.-P. & Beutler, M. A model for correcting the fluorescence signal from a free-falling depth profiler. (Eingereicht) Photosynthesis Research. Beutler, M., Wiltshire, K. H., Reineke, C. & Hansen U.-P. An algorithm and practical fluorescence models from red cyanobacteria and cryptophyta designed for the in situ detection of phytoplankton. (Eingereicht) Biochimica et Biophysica Acta-Bioenergetics. In Vorbereitung: Beutler, M., Wiltshire, K. H., Reineke, C., Ruser, A. and Hansen, U.-P. In situ analysis of phytoplankton by deconvolution of fluorescence excitation and emission matrices. (Manuskript fertig zum Einreichen). Diese Arbeit wurde finanziert vom BMBF (Projektnummer 03F0287A). 1 Content Chapter ................................................................................................................................ Page Abbreviations ................................................................................................................................5 Introduction ...................................................................................................................................7 Chapter 1. Biophysical background ..............................................................................................9 1.1 Photosynthesis ..............................................................................................................9 1.1.1 Photosystems and spectral algal groups.................................................................11 1.1.2 Light harvesting in cyanobacteria ..........................................................................12 1.1.3 Electron transport chain and linear electron transport ...........................................12 1.1.4 Photosystem II chlorophyll fluorescence...............................................................13 1.1.5 Origins of chlorophyll fluorescence.......................................................................14 1.1.6 Quenching mechanisms .........................................................................................14 1.1.7 Photosystem II chlorophyll fluorescence transients...............................................15 Chapter 2. A fluorometric method for the differentiation of algal populations in vivo and in situ. ..................................................................................................................18 2.1 Abstract.......................................................................................................................18 2.2 Introduction.................................................................................................................19 2.3 Materials and methods................................................................................................21 2.3.1 Fluorescence measurements...................................................................................21 2.3.2 Algal cultures .........................................................................................................23 2.3.3 Growth of cultures and sampling ...........................................................................24 2.3.4 Determination of chlorophyll concentrations.........................................................24 2.3.5 Determination of biovolume ..................................................................................26 2.3.6 Mathematical evaluation........................................................................................26 2.3.7 Norm spectra ..........................................................................................................28 2.4 Results.........................................................................................................................29 2.4.1 Measurement of norm spectra...............................................................................29 2.4.2 Linear independence of the norm spectra ..............................................................30 2.4.3 Laboratory tests: common dilution factor for all classes. ......................................30 2.4.4 Influence of pre-illumination on the norm curves..................................................32 2.4.5 Influence of scattering on the norm curves............................................................33 2.4.6 In situ test: comparison with HPLC determinations ..............................................34 2.4.7 In situ test: depth profiles.......................................................................................35 2.4.8 In situ tests: depth profiles and biovolume ............................................................36 2.5 Discussion...................................................................................................................38 2.5.1 Reliability...............................................................................................................38 2.5.2 Detection limit........................................................................................................39 2.5.3 Relation of fluorescence to chlorophyll content and productivity.........................39 2.5.4 Relation of fluorescence to biovolume concentrations ..........................................39 2.5.5 Future developments to overcome present limitations...........................................39 1 Chapter 3. Fluorometric depth-profiling of chlorophyll corrected for yellow substances................................................................................................................... 41 3.1 Abstract....................................................................................................................... 41 3.2 Introduction ................................................................................................................ 42 3.3 Materials and methods................................................................................................ 43 3.3.1 Set-up of the submersible instrument .................................................................... 43 3.3.2 Determination of chlorophyll concentrations and algal cultures........................... 43 3.3.3 Mathematical evaluation........................................................................................ 43 3.3.4 Fluorescence-offsets of natural water-samples...................................................... 43 3.4 Results and discussion................................................................................................ 44 3.4.1 Norm spectra of phytoplankton compared with spectra of yellow substances...... 44 3.4.2 Variability of natural fluorescence offsets............................................................. 44 3.4.3 Dilution experiment and in situ profile.................................................................. 45 Chapter 4. A model for correcting the fluorescence signal from a free-falling depth profiler........................................................................................................................ 47 4.1 Abstract....................................................................................................................... 47 4.2 Introduction