CARBOHYDRATES in ALGAE Berit Smestad a Thesis Presented to The

STRUCTURAL AND METABOLIC STUDIES OF CARBOHYDRATES IN ALGAE Berit Smestad A thesis presented to the Faculty of Science of the University of London in candidature for the degree of Doctor of Philosophy, December, 1971* Chemistry Department, Royal Holloway College, University of London, Englefield Green, Surrey. ProQuest Number: 10096786 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10096786 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ACKNOWLEDGEMENTS 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 facilities, I am grateful to Professor R.G.S.Bidw'ell, Queen’s 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. ABSTRACT PART I 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 characterised, 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 2,1—linked units terminated by a molecule of sucrose at the potential reducing end. 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 11 (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 polysaccharideb pAO- with variable proportions of the different sugars and portions of sulphate were separated, (iii) Structural studies by partial desulphation, méthylation, 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. Méthylation, 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, PART II Photosynthetic studies on 1, Acetabularia mediterranea, 2,Fucus vesicuXosus and 3 * Ulva lactuca^ 14- 1. Pulse labelling experiments with CO^ on A.mediterranea Ill 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* 14 2. 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 IV 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 incorporated radioactivity. 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. CONTENTS PART I. Structural Studies of Carbohydrates in Algae Introduction Page 1 Experimental 26 General methods 26 Carbohydrates of Acetabularia crenulata Introduction 34 Experimental and extraction of the alga • 39 Low Molecular Weight Fraction Experimental 42 Discussion 49 The Fructan Introduction 51 Experimental 53 Discussion 5^ Acidic Polysaccharide Experimental 61 Discussion , 84 The Mannan Introduction 102 Experimental 103 Discussion 113 CONTENTS «.••• continued 14 PART II. Photbsynthetic studies with CO^ Qct carbohydrates in Algae 1• Acetabularia mediterranea Introduction Page 113 Experimental 119 Discussion ' 121 2. Fucus vesiculosus — --- f Introduction 132 Experimental 139 Discussion 154 3* Ulva lactuca Introduction 165 Experimental 169 Discussion 176 Appendix Culture of A.mediterranea I8l References 183 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, soda and potash, but now there are other cheaper ways of getting these products. Brown and red weeds have been used as food both for human beings and for animals, particularly in Asian countries. Industries using algae for production of gelling materials such as alginate and agar can be found in several parts of the world. The carbohydrates of the red and brown weeds have received the most attention because of their economic importance. It is only during the last few decades that interest has arisen in the carbo hydrates of the green algae. Pew chemical studies have been carried out on those of the blue-green algae. The carbohydrates present in algae can be roughly divided into three groups according to their solubility in various solvents, I Low molecular weight carbohydrates,

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