LIGHT INTENSITY INFLUENCES ON ALGAL PIGMENTS, PROTEINS AND CARBOHYDRATES ...

326 Pages·2011·3.17 MB·English

LIGHT INTENSITY INFLUENCES ON ALGAL PIGMENTS, PROTEINS AND
CARBOHYDRATES: IMPLICATIONS FOR PIGMENT-BASED
CHEMOTAXONOMY
by
Cidya Grant

A Dissertation Submitted to the Faculty of
The Charles E. Schmidt College of Science
in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy

Florida Atlantic University
Boca Raton, FL
December 2011

ACKNOWLEDGEMENTS

Special thanks to my research advisor Dr. J. W. Louda, for his guidance and
support during this dissertation research. To the members of my dissertation committee:
Drs. J. E. Haky, C. Parkanyi and S. Hagerthey, for answering pertinent questions and
steering me on the right path to fulfilling the objectives and goals of this research.
To the FAU-Harbor Branch Oceanographic Institute for NMR sample analyses:
special thanks to Dr. Amy Wright for granting permission for instrument use and to her
post- doctoral associate Dr. P. Winder for her assistance with experiment set-up.
To the West natural products research group at FAU, particularly Dr. L. West, his
post-doctoral associate Dr. P. Gupta and graduate student T. Vansach: thank you for the
technical assistance with LC-MS analyses and NMR interpretation.
To my teaching supervisors and mentors at FAU: Drs. D. Chamely-Wiik and E.
Rezler, thank you for always challenging me to reach the highest academic standards, in
research and teaching. The encouragement and assistance were all greatly appreciated.
Funding for this material is based in part upon work supported by the National
Science Foundation under Grant no. DGE: 0638662. Any opinions, findings and
conclusions or recommendations expressed in this material are those of the author and do
not reflect the views of the National Science Foundation.

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ABSTRACT
Author: Cidya Grant
Title: Light Intensity Influences on Algal Pigments, Proteins and
Carbohydrates: Implications for Pigment-Based Chemotaxonomy
Institution: Florida Atlantic University
Dissertation Advisor: Dr. J. W. Louda
Degree: Doctor of Philosophy
Year: 2011

Phytoplankton Chlorophyll a (CHLa), total protein, colloidal carbohydrates,
storage carbohydrates and taxonomic pigment relationships were studied in two
cyanophytes (Microcystis aeruginosa and Synnechococcus elongatus), two chlorophytes
(Dunaliella tertiolecta and Scenedesmus quadricauda), one cryptophyte (Rhodomonas
salina), two diatoms (Cyclotella meneghiniana and Thalassiosira weissflogii) and one
dinophyte (Amphidinium carterae) to assess if algal biomass could be expressed in other
indices than just chlorophyll a alone. Protein and carbohydrates are more useful
currencies for expressing algal biomass, with respect to energy flow amongst trophic
levels. These phytoplankton were grown at low light (LL = 37 µmol photons m-2 s-1),
medium light (ML = 70-75 µmol photons m-2 s-1), and high light (HL= 200 µmol photons
m-2 s-1). Even though pigment per cell increased with increasing light intensity,
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statistically light had very little effect on the CHL a: taxonomic marker pigment ratios, as
they covaried in the same way. Protein, colloidal carbohydrates and storage
carbohydrates per cell all increased with increasing light intensity, but they did not co-
vary with CHLa. Statistical data showed that light intensity had a more noticeable effect
on protein: CHL a, colloidal carbohydrate: CHLa, storage CHO: CHLa, therefore a
general mathematical expression for these relationships cannot be generated. This study
showed that light intensity does have an influence on these biomass indices, therefore,
seasonal and latitudinal formulas may be required for meaningful algal biomass
estimation. However, more studies are needed if that goal is to be realized.
While studying the effects of light intensity on algal pigment content and
concentration, a new pigment was isolated from a cyanophyte (Scytonema hofmanii)
growing between 300-1800 µmol photons·m-2·s-1 and from samples collected in areas of
the Florida Everglades. This pigment was characterized and structurally determined to
possess indolic and phenolic subunits that are characteristic of scytonemin and its
derivatives. In addition, the pigment has a ketamine functionality which gives it its
unique polarity and spectral properties. Based on the ultra violet/visible absorbance data,
this pigment was postulated to be protecting the chlorophyll a and cytochrome Soret
bands as well as α and β bands of the cytochromes (e.g. cyt-c ) in the photosynthetic
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unit.

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LIGHT INTENSITY INFLUENCES ON ALGAL PIGMENTS, PROTEINS AND
CARBOHYDRATES: IMPLICATIONS ON PIGMENT-BASED
CHEMOTAXONOMY

LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES ............................................................................................................
X
I. INTRODUCTION ........................................................................................................... 1
The working hypothesis .................................................................................................. 4
BACKGROUND ............................................................................................................ 4
Methods for estimating algal biomass ........................................................................ 4
Converting CHLa to biomass.................................................................................... 10
Select algal metabolites which may serve as biomass indices .................................. 17
Photosynthesis overview ........................................................................................... 23
Novel sunscreen pigment .............................................................................................. 30
Overall goals of this study ............................................................................................ 33
II. MATERIALS AND METHODS ................................................................................. 34
Experimental organisms................................................................................................ 34
Algal culturing .............................................................................................................. 36
Culture conditions ..................................................................................................... 37
Cell counting. ................................................................................................................ 38
Chemical Analyses........................................................................................................ 39
Algal protein extraction ................................................................................................ 39
Algal protein measurement ....................................................................................... 39
Algal colloidal and storage carbohydrate extraction .................................................... 40
Algal colloidal and storage carbohydrate measurement ........................................... 40
Algal total organic carbon (TOC) extraction ................................................................ 41
Colorimetric determination of extracted TOC samples ............................................ 42
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Nutrient analyses ........................................................................................................... 42
Pigment Analyses.......................................................................................................... 43
Ultra Violet - Visible (UV/Vis) Analyses of Extracts .............................................. 45
High Performance Liquid Chromatography (HPLC) ................................................... 46
HPLC Data Calculations ........................................................................................... 47
Statistical analyses ........................................................................................................ 49
Isolation and characterization of a new pigment. ............................................................. 50
IR analysis ................................................................................................................. 52
Mass Spectrometry .................................................................................................... 52
NMR analyses ........................................................................................................... 54
Acetylation reactions ................................................................................................ 54
Deuterium exchange reactions .................................................................................. 55
III. RESULTS - STATISTICAL ANALYSES ................................................................. 56
Significance of the algal species used in this study .................................................. 56
Analyses overview .................................................................................................... 59
Synechococcus elongatus .............................................................................................. 60
Microcystis aeruginosa ................................................................................................. 70
Dunaliella tertiolecta .................................................................................................... 78
Scenedesmus quadricauda ............................................................................................ 87
Rhodomonas salina ....................................................................................................... 95
Cyclotella meneghiniana ............................................................................................ 103
Thalassiosira Weissflogii ............................................................................................ 111
Amphidinium carterae ................................................................................................ 119
IV. DISCUSSION ........................................................................................................... 127
Growth patterns ........................................................................................................... 127
Phytoplankton protein as a biomass indicator ............................................................ 128
Phytoplankton colloidal carbohydrate (CHO) as a biomass indicator ........................ 137
Phytoplankton storage carbohydrate (CHO) as a biomass indicator .......................... 139
Marker pigments as indicators of algal biomass ......................................................... 140
Phytoplankton chlorophyll a, protein and carbohydrate relationships to biovolume . 145
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V. CONCLUSION: IMPLICATIONS FOR CHEMOTAXONOMY ............................ 147
VI. CHARACTERIZATION OF NOVEL PIGMENT .................................................. 149
The ‘scytoneman’ skeleton ......................................................................................... 149
New pigment – putative structure elucidation ............................................................ 155
Mass interpretation ...................................................................................................... 159
IR analysis ................................................................................................................... 166
Ecological significance of the new pigment ............................................................... 167
VII. APPENDICES ......................................................................................................... 170
I- Pigment calculation and data handling .................................................................... 171
II. Select photoprotectorant and accessory pigments .................................................. 179
III- Spectroradiometric output .................................................................................... 181
IV- Calibration curves and equations ......................................................................... 185
V- Retention times and UV-Vis maximas .................................................................. 187
VI-ANOVA tables ...................................................................................................... 190
VII- Cellular concentration of CHLa and photosynthates .......................................... 264
VIII- Typical chromatograms of species studied ........................................................ 267
IX- Specific growth rate (µ) curves ............................................................................ 271
X – NMR SPECTRA .................................................................................................. 275
XI – Mass Spectra ....................................................................................................... 284
VIII. REFERENCES....................................................................................................... 295

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LIST OF TABLES

Table 1: Methods for estimating algal biomass .................................................................. 5
Table 2: Marker pigments having stoichiometric relationships with CHLa in biomass
estimations .......................................................................................................... 12
Table 3: Colloidal and storage carbohydrate composition of the taxonomic groups
studied. ............................................................................................................... 20
Table 4: Gradient program used in FAU OGG laboratory ............................................... 47
Table 5: Gradient program used for LC-MS runs ............................................................. 53
Table 6: Cellular concentration of chlorophyll a and products of photosynthesis ......... 130
Table 7: Protein:CHLa (log ) ratios of the species as influenced by irradiance ........... 134
10
Table 8: Colloidal CHO/CHLa (log ) ratios as a function of irradiance ...................... 138
10
Table 9: Storage CHO/CHLa (log ) ratios as a function of irradiance ........................ 140
10
Table 10:Three new pigments isolated form Scytonema sp. ........................................... 152
Table 11: Scytonemin- a comparison of literature and observed values ........................ 153
Table 12: 1H and 13C NMR data for putative structure of pigment ............................... 157

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LIST OF FIGURES

Figure 1: Structure of Chlorophyll a ................................................................................... 2 
Figure 2:. Xanthophyll cycling in (a) Chrysophytes and (b) Chlorophytes. .................... 26 
Figure 3: Structure and UV/Vis spectra of Scytonemin ................................................... 31 
Figure 4: New pigments isolated form Scytonema sp.. ..................................................... 32 
Figure 5: Flow Chart of Analytical Scheme. .................................................................... 35 
Figure 6: Schematic of inoculation procedure. ................................................................. 36 
Figure 7: Synechococcus elongatus Marker pigment/CHLa ........................................... 62 
Figure 8: Synechococcus elongatus Protein/CHLa relationships ..................................... 64 
Figure 9: Synechococcus elongatus Colloidal CHO/CHLa .............................................. 66 
Figure 10: Synechococcus elongatus Storage CHO/CHLa .............................................. 69 
Figure 11: Microcystis aeruginosa Markerpigment/CHLa .............................................. 71 
Figure 12: Microcystis aeruginosa Protein/CHLa relationships ...................................... 73 
Figure 13: Microcystis aeruginosa Colloidal CHO/CHLa ............................................... 75 
Figure 14: Microcystis aeruginosa Storage CHO/CHLa.................................................. 77 
Figure 15: Dunaliella tertiolecta Marker pigment/CHLa ................................................ 79 
Figure 16: Dunaliella tertiolecta Protein/CHLa relationships ......................................... 81 
Figure 17: Dunaliella tertiolecta Colloial CHO/CHLa .................................................... 84 
Figure 18: Dunaliella tertiolecta Storage CHO/CHLa ..................................................... 86 
Figure 19: Scenedesmus quadricauda Marker pigment/CHLa ........................................ 88 
Figure 20: Scenedesmus quadricauda Protein/CHLa relationships ................................. 89 
Figure 21: Scenedesmus quadricauda Colloidal CHO/CHLa .......................................... 92 
Figure 22: Scenedesmus quadricauda Storage CHO/CHLa ............................................. 94 
Figure 23: Rhodomonas salina Marker pigment/CHLa ................................................... 96 
Figure 24: Rhodomonas salina Protein/CHLa .................................................................. 98 
Figure 25: Rhodomonas salina Colloidal CHO/CHLa ................................................... 100 
x