Studies of the extracellular polysaccharide of the blue-green alga Anabaena spiroides

182 Pages·2016·2.92 MB·English

Iowa State University Capstones, Theses and
Retrospective Theses and Dissertations
Dissertations
1979
Studies of the extracellular polysaccharide of the
blue-green alga Anabaena spiroides
George Edwin Rottinghaus
Iowa State University
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Rottinghaus, George Edwin, "Studies of the extracellular polysaccharide of the blue-green alga Anabaena spiroides " (1979).
Retrospective Theses and Dissertations. 6670.
https://lib.dr.iastate.edu/rtd/6670
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UniversiW
MicrxSilms
Intemationcil
300 N. ZEEB ROAD, ANN ARBOR, Ml 48106
18 BEDFORD ROW, LONDON WC1 R 4EJ, ENGLAND
7924268
IROTTXNOHAUSr GEORGE EDWIN
STUDIES or THE EXTRACELLULAR POLYSACCHARIDE
OF' THE BLUE-GREEN ALGA ANABAENA SPIROIDES.
IOWA STATE UNIVERSITY, PH.D.# 1979
Univers%
Micrailms
International 300 N. ZEES ROAD. ANN ARBOR, M) 48106
Studies of the extracellular polysaccharide of
the blue-green alga Anabaena splroldes
by
George Edwin Rottlnghaus
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of
The Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Department: Biochemistry and Biophysics
Major: Biochemistry
Approved :
Signature was redacted for privacy.
In Charge of Major Work
Signature was redacted for privacy.
Po] bment
Signature was redacted for privacy.
Iowa State University
Ame s. Iowa
1979
11
TABLE OP CONTENTS
Page
ABBREVIATIONS x
DEDICATION xi
I. INTRODUCTION 1
II. LITERATURE REVIEW 3
III.. MATERIALS AND METHODS l8
A. Materials 18
B. Methods l8
1. Collection of lake samples l8
2. Microscopic counts of algae 19
3. Isolation of dissolved carbohydrate 20
4. Acid hydrolysis of samples 20
5. Paper chromatography 21
6. Thin-layer chromatography 22
7. Gas chromatography of aldltol acetates 23
8. Total carbohydrate analysis 25
9. Culture medium 26
10. Monitoring of cell growth and
extracellular polysaccharide
. production 28
11. Isolation of Anabaena spiroides poly­
saccharides 30
12. Culturing of bacteria associated with
Anabaena spiroides 33
13. Purification of algal extracellular
polysaccharide 34
14. Fractionation techniques employed for
the determination of homogeneity 36
15. Methylation analysis 37
16. Perlodate oxidation (128,129) 4l
17. Partial acid hydrolysis 43
18. Fractionation of oligosaccharides 43
19. Analytical and physical measurements 44
20. Viscosity measurements 47
21. ^^C labeled extracellular polysaccharide 48
22. Bacterial uptake of labeled extra­
cellular polysaccharide 49
Ill
IV. RESULTS AND DISCUSSION 51
A. Naturally Occurring Dissolved Carbohydrates 51
B. Polysaccharides of Anabaena splroldes 64
1 ii
C. C Tracer Studies of the Symbiotic
Relationship of Anabaena splroldes and
Its Associated Bacteria l46
V. CONCLUSIONS 153
VI. BIBLIOGRAPHY 155
VII. ACKNOWLEDGEMENTS l68
Iv
LIST OF TABLES
Page
Table 1. Monosaccharide composition of the
mucilaginous sheath of Cyanophyceae
reported in the literature 7
Table 2. Monosaccharide composition of extracellular
polysaccharides from Chlorophyceae and
Cyanophyceae reported in the literature 9
Table 3. Bloom species of planktonic blue-green algae
collected from Iowa lakes, 1972 and 1974,
and their maximum density 52
Table 4, R- values of standard sugars for solvent
systems A and B 54
Table 5. R_ values and color reactions of fucose
and xylose standards 55
Table 6. Retention times of reduced and acetylated
monosaccharide standards relative to
inositol hexaac'etate 56
Table 7. Monosaccharide components of dissolved
carbohydrate isolated from Iowa lake
water during blue-green algal blooms 57
Table 8. Molar ratios of the monosaccharides present
in the crude extracellular poly­
saccharides isolated from axenic and
mixed algal-bacterial cultures of
Anabaena spiroides 72
Table 9. Monosaccharide constituents of micro­
organisms associated with Anabaena
spiroides 82
Table 10, Fractionation by solvent extraction of
cell wall material from Anabaena
spiroides 83
Table 11. Cetavlon fractionation of the crude
Anabaena spiroides extracellular
polysaccharide and gas chromatographic
analysis of the monosaccharide
composition of each fraction 84
V
Table 12. Composition and molar ratios of the
purified intracellular, sheath, and
extracellular polysaccharides of
Anabaena spiroldes as determined by
gas chromatography 95
Table 13. Relative retention times and molar ratios
of partially methylated alditol
acetates derived from the hydrolyzed
permethylated extracellular poly­
saccharides of Anabaena spiroldes 123
Table l4. Thin-layer chromatography of the partially
methylated monosaccharide components of
the methylated extracellular poly­
saccharide of Anabaena spiroldes 125
Table 15. Comparison of the mole per cent composition
of the purified extracellular poly­
saccharide and its methylated derivative 127
Table l6. Prominent peaks (m/e) in the mass spectra
of acetates of methylated alditols 129
vl
LIST OP FIGURES
Page
Figure 1. Extracellular polysaccharide production by
Anabaena spiroides in culture and
increase in chlorophyll a content as a
function of time 65
Figure 2. Gas chromatogram of the crude extracellular
polysaccharide of Anabaena spiroides
after hydrolysis, reduction and
acetylation C3% ECNSS-M) 68
Figure 3. Gas chromatogram of the crude extra­
cellular. polysaccharide of axenic
Anabaena spiroides (UTEX-1552) after
hydrolysis, reduction and acetylation
(3% ECNSS-M) 71
Figure 4. Gas chromatogram of the crude sheath
polysaccharide of Anabaena spiroides
after hydrolysis, reduction and
acetylation i3% ECNSS-M) 74
Figure 5. Gas chromatogram of the crude intracellular
polysaccharide from Anabaena spiroides
after hydrolysis, reduction and
acetylation (3% ECNSS-M) 76
Figure 6, Gas chromatogram of the crude cell wall
material of Anabaena spiroides after
hydrolysis, reduction and acetylation
CO.75% HiEFF-lBP, 0,25% EGSS-X and
0,1% 144-B) 78
Figure 7. A comparison of the gas chromatograms of
the crude extracellular polysaccharide
C ) and crude sheath polysaccharide
(. ) of Anabaena spiroides after
hydrolysis, reduction and acetylation
C3% ECNSS-M) 81
Figure 8. Gas chromatogram of the hot water extract
from the cell wall material of Anabaena
spiroides after hydrolysis, reduction
and acetylation i3% ECNSS-M) 87
vil
Figure 9. Gas chromatograra, after hydrolysis,
reduction and acetylatlon, of the
pH 8, pH 12 fractions and the
polysaccharide remaining in solution
on Cetavlon fractionation of the crude
extracellular polysaccharide of
Anabaena spiroldes (0.75% HIEFF-IBP,
0.25% EGSS-X, and 0.1% 144-B) 89
Figure 10. Gas chromatogram, after hydrolysis,
reduction and acetylatlon, of the
pH 10 fraction resulting from
Cetavlon fractionation of the crude
extracellular polysaccharide of
Anabaena spiroldes (3% ECNSS-M) 91
Figure 11. Gas chromatogram, after hydrolysis,
reduction and acetylatlon, of the
purified extracellular polysaccharide
of Anabaena spiroldes eluted from
DEAE-cellulose column chromatography
(3% ECNSS-M) 94
Figure 12. Profile of the extracellular poly­
saccharide of Anabaena spiroldes on
elution from a DEAE-cellulose column (B) 98
Figure 13. Infrared spectrum of the free acid form of
the purified extracellular poly­
saccharide of Anabaena spiroldes 101
Figure l4. Infrared spectrum of the sodium salt form
of the purified extracellular poly­
saccharide of Anabaena spiroldes 102
Figure 15. Periodate uptake (moles perlodate consumed
per anhydrosugar residue) by the
purified extracellular polysaccharide
of Anabaena spiroldes 104
Figure l6. Viscosity vs^ concentration of extra­
cellular polysaccharide of
Anabaena spiroldes 107
Figure 17. Viscosity vs pH of dispersions of the
extracellular polysaccharide of
Anabaena spiroldes (0.2% w/v) IO8