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  Spectral fluorescence of chlorophyll and phycobilins as an in-situ tool of phytoplankton analysis - models, algorithms and instruments

Beutler, M. (2003). Spectral fluorescence of chlorophyll and phycobilins as an in-situ tool of phytoplankton analysis - models, algorithms and instruments. PhD Thesis, Christian-Albrechts-Universität, Kiel.

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Beutler, Martin1, Author           
Hansen, U.-P., Advisor
Wiltshire, K. H.1, Referee           
Affiliations:
1Department Ecophysiology, Max Planck Institute for Limnology, Max Planck Institute for Evolutionary Biology, Max Planck Society, ou_976547              

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 Abstract: Fingerprints of excitation and emission spectra of chlorophyll and phycobilin fluorescence can be used to differentiate 'spectral groups' of microalgae in vivo and in situ , e. g. vertical profiles can be taken within a few minutes. The investigated spectral groups of algae (green group - chlorophyta; blue - blue cyanobacteria; brown - heterokontophyta, haptophyta, dinophyta; red - red cyanobacteria and mixed - cryptophyta) are each characterised by a specific composition of photosynthetic antennae pigments and, consequently, by a specific excitation and emission spectrum of the chlorophyll and phycobilin fluorescence. Particularly relevant are chlorophyll a, chlorophyll c, phycocyanin, phycoerythrin, fucoxanthin and peridinin. In a first approach, a laboratory based instrument and a submersible instrument were constructed containing light-emitting diodes to excite chlorophyll fluorescence in five distinct wavelength ranges to facilitate the differentiation of four spectral algal groups (green, blue, brown, mixed). They were measured under a fixed emission wavelength. Norm spectra were determined for the four spectral algal groups (several species per group). Using these norm spectra and the actual five-point excitation spectrum of a water sample, an estimate of the group-specific chlorophyll concentration is rapidly obtained for each algal group. This was accomplished by the development of a fast mathematical fit procedure. In vivo and in situ measurements based on calibration experiments were compared with results obtained by high performance liquid chromatography and biovolume estimations from the light microscope. Depth profiles of the distribution of spectral algal groups taken over a time period of few minutes were shown. The described method for algal differentiation opens new research areas and monitoring and supervision facilities related to photosynthetic primary production in aquatic environments. Yellow substances (coloured dissolved organic matter) may interfere with the measurement because of an overlap of the excitation spectra with those of phytoplankton. The use of an ultra-violet excitation source (370 nm light-emitting diode) enabled differentiation between algal fluorescence and fluorescence by yellow substances. The resulting six-point excitation spectra were deconvoluted on the basis of norm spectra. A mean norm spectrum for yellow substances was obtained from natural samples. In a new submersible instrument the correction of chlorophyll fluorescence measurements for the influence of yellow substances was tested in in vivo and in situ experiments. Specific problems associated with the principle of a free-falling depth profiler for algae discrimination were also considered. When F0, F and Fm are determined sequentially with one measuring cell, then phytoplankton inside the cell experiences a different light history according to different locations in the cell. This leads to a superposition of different induction curves of chlorophyll fluorescence. Mathematical algorithms were developed that enable theevaluation of the integral fluorescence signal (averaged for 1s) for different velocities of the falling probe. This yields a correction factor which allows the usage of calibration factors obtained from stationary suspensions for the determination of algal concentrations in flowing suspensions. The predictions of the model were compared with measurements in flowing suspensions containing chlorophyta, cyanobacteria, cryptophyta and diatoms. The comparison showed the reliability of the algorithms. The requirement of corrections by the algorithm was high for dark-adapted cells and less important for light-adapted cells. Fluorometric determination of the chlorophyll content of cyanobacteria is impeded by the unique structure of their photosynthetic apparatus, that is, the phycobilisomes in the lightharvesting antennae. The problems are caused by the variations in the ratio of the pigment phycocyanin to chlorophyll a resulting from adaptation to varying environmental conditions. In order to improve fluorometric analysis of algae a simplified energy distribution model describing energy pathways in the cyanobacterial photosynthetic apparatus was conceptualised. Two sets of mathematical equations were derived from this model and tested. Fluorescence of cyanobacteria was measured with a new fluorometer at seven excitation wavelength ranges and at three detection channels (650 nm, 685 nm and 720 nm) in vivo. By employing a new fit procedure it became possible to correct for variations in the cyanobacterial fluorescence excitation spectra and to account for other phytoplankton signals. The effect of energy state transitions on the phycocyanin fluorescence emission of phycobilisomes were documented. The additional use of the phycocyanin fluorescence signal at 650 nm in combination with the previously developed mathematical approach for phytoplankton analysis based on chlorophyll fluorescence spectroscopy allows a more detailed study of cyanobacteria and other phytoplankton in vivo and in situ. The detection of red cyanobacteria with these newly developed methods is not possible because of adaptation processes of the cyanobacterial photosynthetic apparatus and spectral interferences with cryptophyta. To overcome these problems a simplified energy distribution model accounting for energy pathways in the red cyanobacterial photosynthetic apparatus and the apparatus of cryptophyta was designed. Mathematical equations were derived that enabled the calculation of the pigment content in both organisms: cryptophyta and red cyanobacteria. This resulted in the extension of a fluorometer previously developed for phytoplankton, with seven excitation wavelengths and four detection channels (600 nm, 620 nm, 650 nm and 685 nm). An extension of the fit procedure allowed corrections for variations in the fluorescence excitation spectra of red cyanobacteria and cryptophyta in the presence of other phytoplankton signals. The new approach provided correct fluorometric pigment estimation also in the presence of energy state transitions. The combination of fluorescence emission excitation matrices, the fluorescence models and the enhanced fit algorithm provides valuable information for phytoplankton analysis. Finally, the set-up was tested successfully with natural samples. It enabled the determination of chlorophyll in five spectral groups of phytoplankton and of the phycobilins in three spectral types. A correction for yellow substances was included using mean fluorescence spectra of filtered natural samples. The pigment estimation of this method was compared to reference estimates obtained by high performance liquid chromatography and wet chemicalanalysis of natural freshwater samples. Reference and fluorometric methods showed similar results. This measuring principle was installed as a submersible instrument which makes possible the measurement of fluorescence depth-profiles via pigment estimation in situ in a time scale of a few minutes. Profiles obtained with the instrument were compared to those obtained with the stationary instrument. The correlation between both methods was very high. This demonstrates the success and the large step forward in phytoplankton analysis achieved by this method.

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Language(s): eng - English
 Dates: 2003-04-28
 Publication Status: Accepted / In Press
 Pages: 170 p.
 Publishing info: Kiel : Christian-Albrechts-Universität
 Table of Contents: 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
2
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 ................................................................................................................ 48
4.3 Materials and methods................................................................................................49
4.3.1 Algal growth .......................................................................................................... 49
4.3.2 Chlorophyll determination..................................................................................... 49
4.3.3 Fluorometer set-up................................................................................................. 49
4.3.4 Mathematical evaluation........................................................................................ 50
4.3.5 Model for the depth profiler .................................................................................. 53
4.4 Results ........................................................................................................................ 58
4.4.1 Fluorometer tests ................................................................................................... 58
4.4.2 Fluorescence induction curves............................................................................... 59
4.4.3 Tests with flowing suspensions ............................................................................. 60
4.5 Discussion................................................................................................................... 63
Chapter 5. A reduced model of the fluorescence from the cyanobacterial
photosynthetic apparatus designed for the in situ detection of
cyanobacteria ............................................................................................................. 65
5.1 Abstract....................................................................................................................... 65
5.2 Introduction ................................................................................................................ 66
5.3 Materials and methods................................................................................................69
5.3.1 Growth experiments............................................................................................... 69
5.3.2 Fluorescence measurements .................................................................................. 70
5.3.3 Determination of chlorophyll concentrations ........................................................ 72
5.3.4 Phycobilin determination....................................................................................... 72
5.3.5 Number of photosystem II reaction centres........................................................... 72
5.4 Results ........................................................................................................................ 74
5.4.1 Effect of pigment variability on two different evaluation problems ..................... 74
5.4.2 Cyanobacteria with different pigmentation for the investigation for
variability of spectra and for testing the model..................................................... 74
3
5.4.3 A simplified model of energy transfer in the antennae of cyanobacteria ..............75
5.4.4 Test of the first assumption with cyanobacteria of different
pigmentation types (Equation (B8) in the Appendix A) ........................................76
5.4.5 Estimation of chlorophyll and photosystem II by fluorescence
in cyanobacteria.....................................................................................................77
5.4.6 Variation of cyanobacterial fluorescence excitation spectra..................................79
5.4.7 Norm spectra of other spectral algal groups ..........................................................81
5.4.8 Tests using dilution experiments............................................................................82
5.4.9 Aging cultures ........................................................................................................83
5.4.10 Sensitivity to changed light intensity ...................................................................84
5.5 Discussion...................................................................................................................86
5.5.1 Variation of cyanobacterial pigments ....................................................................86
5.5.2 Applicability of the model in Figure 5.5 for the estimation
of cyanobacterial pigments....................................................................................86
5.5.3 Variation of cyanobacterial pigments ....................................................................87
Chapter 6. An algorithm and practical fluorescence models from red cyanobacteria
and cryptophyta designed for the in situ detection of phytoplankton ........................90
6.1 Summary.....................................................................................................................90
6.2 Introduction.................................................................................................................91
6.3 Materials and methods ................................................................................................93
6.3.1 Growth experiments...............................................................................................93
6.3.2 Fluorescence measurements...................................................................................94
6.3.3 Pigment determination ...........................................................................................95
6.4 Results........................................................................................................................96
6.4.1 Red cyanobacteria grown with different pigmentation..........................................96
6.4.2 Growth of cryptophyta under different light intensities and
growth of different species ....................................................................................97
6.4.3 Simplified models of energy transfer in the antennae of
red cyanobacteria and cryptophyta ........................................................................98
6.4.4 Validation of fluorescence model of the red cyanobacterial apparatus ...............100
6.4.5 Comparison of the calculated and measured phycoerythrin,
chlorophyll and photosystem II centre concentrations in cryptophyta................101
6.4.6 Identification in the presence of other phytoplankton..........................................104
6.4.7 Aging of red cyanobacterial cultures ...................................................................105
6.4.8 Sensitivity to state transitions (coupling of phycobilisome to photosystem II) ...106
6.5 Discussion.................................................................................................................108
6.5.1 Variation of cyanobacterial pigments and cryptophytal pigments.......................108
6.5.2 Reliability of prediction of pigment contents in red cyanobacteria
by the model in Appendix D ................................................................................108
6.5.3 Prediction of pigment contents in cryptophyta by the model in Appendix E.......109
Chapter 7. In situ analysis of phytoplankton by deconvolution of fluorescence
excitation and emission matrices..............................................................................111
7.1 Abstract.....................................................................................................................111
4
7.2 Introduction .............................................................................................................. 112
7.3 Materials and methods.............................................................................................. 114
7.3.1 Fluorescence measurements ................................................................................ 114
7.3.2 Fluorescence profiler ........................................................................................... 115
7.3.3 Pigment determination......................................................................................... 116
7.4 Results ...................................................................................................................... 117
7.5 Discussion................................................................................................................. 126
Conclusion ............................................................................................................................... 128
Abstract ................................................................................................................................. 131
Zusammenfassung.................................................................................................................... 134
Appendix ................................................................................................................................. 137
Appendix A. Fitting by norm spectra. ...................................................................... 137
Appendix B. Energy distribution model (blue cyanobacteria)................................. 139
Appendix C. Mathematical fit for several excitations wavelengths and detection
wavelengths with variable cyanobacterial norm spectra .......................................... 143
Appendix D. Fluorescence model of phycoerythrin-containing cyanobacteria ....... 145
Appendix E. Energy distribution model of cryptophytes......................................... 151
Appendix F. Mathematical fit for cyanobacterial spectra with several excitations
wavelengths and detection wavelengths using variable cyanobacterial
norm spectra .............................................................................................................154
References................................................................................................................................ 158
Danksagung.............................................................................................................................. 168
Curriculum vitae ....................................................................................................................... 169
 Rev. Type: -
 Identifiers: eDoc: 111901
URI: http://e-diss.uni-kiel.de/diss_717/
 Degree: PhD

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