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STRUCTURAL AND METABOLIC STUDIES OF
CARBOHYDRATES IN ALGAE
A thesis presented to the Faculty
of Science of the University of London
in candidature for the degree of Doctor
December, 1971* Chemistry Department, Royal Holloway College, University of London, Englefield Green, Surrey.
ProQuest Number: 10096786
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I wish to thank Dr.E.E.Percival for her
supervision, interest and help in carrying out this work,
I would also like to thank Professor E.J,
Bourne for his interest and for providing laboratory
I am grateful to Professor R.G.S.Bidw'ell,
Queens University, Kingston, Ontario, Canada, for providing
facilities to carry out a part of this work in his laboratory.
Finally, I am indebted to the following
institutions for financial support:
Institute of Pharmacy, Oslo, I969 and 1970.A.C. Houeiis legat, Oslo, I969,Norsk Parmaceutiskr Selskap, Oslo, I969.Professor S.A,Sexes legat, University of Oslo,1970,Norsk Medisinaldepot, Oslo, 1970.International Federation of University Women, Summer 1970 for work in Canada.
The British Council, Oct.1970 - Dec.1971.
Investigation of the Carbohydrates synthesised by the marine
greed alga Acetabularia crenulata
By means of sequential extractions with different
solvents the following carbohydrates were isolated and
1, Prom an alcoholic extract ^glucose, B-fructose, allulose,
myo-inositol and an alcohol tentatively identified as
allo~-cfuercitol. This is only the second time allulose has
been found in Nature and the first time in any alga. An
homologous series of fructose-containing oligosaccharides
were also separated and characterised as 2,1linked units
terminated by a molecule of sucrose at the potential reducing
2. Aqueous extraction gave a mixture of a fructan (major) and
a sulphated heteropolysaocharide.
a) These two polysaccharides were separated on a column of
DEAE~oellulose, Using the classical techniques the fructan
was characterised as an inulin type polysaccharide,
b) (i) The sulphated polysaccharide contained D-glucuronic acid,
^galactose, ^rhamnose and small proportions of xylose and
4-0-methylgalactose, Each of the sugars were separated and
characterised. The presence of the last sugar has not been
reported.as a constituent of any green algal polysaccharide before*
a.H.C.' TP TP.l
(ii) By extraction of stalks and caps' separately with cold
and hot water and elution from the BEAE-cellulose with 0.5
and 1.0 M potassium chloride similar sulphated polysaccharidebpAO-
with variable proportions of the different sugars and portions
of sulphate were separated,
(iii) Structural studies by partial desulphation, mthylation,
periodate oxidation and partial hydrolysis established the'
essential similarity of these fractions and that the main
structural features are: highly branched molecules containing
1 ,3-lihked D-galaotose, ^-sulphate (rnajor) and 6-sulphate,
1 ,2-linked L-rhamnose^and glucuronic acid, galactose and
rhamnose all present as end groups. Glucuronic acid is linked
to both rhamnose and galactose and galactose units are
mutually linked in the macromolecule.
3. A j3-(l -^4)linked maiman was extracted with alkali. Mthylation,
periodate oxidation and gel filtration studies proved this
structure and indicated some degree of branching and a higher
molecular weight than those of previously reported for mannans
from green algae,
Photosynthetic studies on 1, Acetabularia mediterranea, 2,Fucus
vesicuXosus and 3* Ulva lactuca^14-1. Pulse labelling experiments with CO^ on A .mediterranea
followed by ethanolic extraction led to the separation of
labelled sucrose, glucose, fructose and the first three
oligosaccharides characterised in Part I (l), A possible
biosynthetic interconver&ion of these carbohydrates is
described from the results of these experiments*
142. a) Pulse labelling experiments with COg on Fucus vesiouloaus
a marine bcrown alga, show that of the low molecular weight
carbohydrates formed by photosynthesis, mannitol is formed first
The possible conversion of mannitol' into laminaran via mono-and
di- glucosylmannitol is discussed*
b) The polysaccharides, laminaran, xylogalactofucogluouronan (A),
xylogluQur-onogalactofacan (b ), fucoidan (C) and alginio acid
were extracted and separated by various fractionation techniques.
The radioactivity in each was measured as was the radioactivity
in the constituent sugars of the fucose-containing polysaccharides
(all of which are sulphated). From the changes in the radioactivity
of these polysaccharides in different samples it is postulated
that (a ) is synthesised first and transformed into (C) via (E).
Low molecular weight carbohydrates present in the acid extract
are suggested as precursors for the acid polysaccharides. The
residual material after acid and alkali extraction was hydrolysed
and the radioactivity of the sugars in the hydrolysate was
measured. Glucose was the major radioactive sugar.
3. Ulva lactuca, a marine green alga. Similar experiments on
U.laotuca were carried out.
a) Examincation of the 80^ ethanol extracts showed that sucrose
is the first sugar to be synthesised and this is followed by
glucose and fructose. Xylose, ribose(?) and myo-inositol also
b) Starch and a sulphated glucuronoxylorhamnan were extracted
and their radioactivities were measured. The former appears to be
synthesised most rapidly and to be an active metabolite and the
sulphated polysaccharide is laid down as a long terra storage
product or as part of the skeletal structure of the alga.
c) The residual material after ethanolic and aqueous extractions
was examined in the same way. Glucose is again the major sugar
and the results indicate that the carbohydrate is laid down in
the cell wall after other constituents.
PART I. Structural Studies of Carbohydrates in Algae
Introduction Page 1Experimental 26General methods 26
Carbohydrates of Acetabularia crenulata
Introduction 34Experimental and extraction
of the alga 39
Low Molecular Weight Fraction
Experimental 42Discussion 49
Introduction 51Experimental 53Discussion 5^
Experimental 61Discussion , 84
Introduction 102Experimental 103Discussion 113
CONTENTS . continued
14PART II. Photbsynthetic studies with CO^ Qct carbohydrates in Algae
1 Acetabularia mediterranea
Introduction Page 113Experimental 119Discussion ' 121
2. Fucus vesiculosus --- f
Introduction 132Experimental 139Discussion 154
3* Ulva lactuca
Introduction 165Experimental 169Discussion 176
Culture of A.mediterranea I8l
P A R T I
STRUCTURAL STUDIES OP CARBOHYDRATES IN ALGAE
Most of the plants growing in the sea belong to the
class known as algae. They are one of the most primitive groups
in the plant kingdom and evolved early in the earthfel history,
and morphologically they.differ very little from those found as
fossils. Although there are some freshwater species, they are
mainly found in marine waters. They vary in form from unicellular
plants, free-floating in the sea, to species which are several
metres long and are fastened to rock with a root-system called a
rhizoid. The algae are not differentiated into root, stem and
leaves as are the land plants. Instead they have a thallus which
varies in form from species to species, some looking almost like
flowers with a stem and "leaves, and others looking like lettuce,
for example, the sea lettuce, Ulva lactuca.
The algae are classified mainly according to their colour,
as the difference in pigments generally coincides with important
morphological distinctions. They are divided into the following
groups; Brown (Phaeophyceae), red (Rhodophyceae), green (Chloro-
phyceae), and blue-green (Cyanophyceae), They all possess chloro
phyll and photosynthesis as do the land plants. Generally the
green and brown seaweeds grow nearest to the surface of the sea,
while the red ones are found further down, but examples of all
types can be found at all levels from the surface.
Brown weeds were early collected for production of iodine,