E. Pelosi, B. Pushparaj

Transcription

1 FISHERIES AND FiARINEI SERVICE Translation Series No The influence of culture conditions on the fatty acids composition of Scenedesmus SPP. II. The influence of the temperature by C. Paoletti, E. Pelosi, B. Pushparaj Original title: Influenza delle condizioni colturâli sulla composïzione in acidi grassi di Scenedesmus SPP. II. Influenza della temperatura From. La rivista italiana delle sostanze grasse (The Italian Review of Fatty Substances), 51(Feb.) , 1974 Translated by the Translation Bureau(MMNZ) Multilingual Services Division Department of the Secretary of.state of Canada Department of thè Environment Fisheries and Marine Service Halifax Laboratory Halifax, N.S pages typescript

2 DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION CANADA TRANSLATED FROM -. TRADUCTION DE INTO EN Italian SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES English freq e/9# AUTHOR AUTEUR C. PAOLETTI et al. TITLE IN ENGLISH TITRE ANGLAIS. The influence of culture conditions on the fatty acids composition of Scenedesmus SPP. II. The influence of the temperature. MTLEIN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACThES ROMAINS) Influenza delle condizioni colturali sulla cbmposizione in acidi grassi di - Scenedesmus SPP. II. Influenza della temperatura. REFERENCE IN FOREIGN LANGUAGE (RAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE,FOREIGN CHARACTERS. REFÉRENCE EN LANGUE ÉTRANGÉRE (NOM DU LIVRE OU PUBLICATION). AU COMPLET, TRANSCRIRE EN CARACM:RES ROMAINS. La rivista italiana delle sostanze grasse REFERENCE IN ENGLISH RÉFÉRENCE EN ANGLAIS The Italian Review of Fatty Substances. PUBLISHER-. ÉDITEUR Stazione sperimentale per. l'industria degli oli e dei grassi PLACE OF PUBLICATION LIEU DE PUBLICATION Milan, Italy. YEAR ANNÉE 1974 DATE OF PUBLICATION DATE DE PUBLICATION VOLUME LI ISSUE NO. NUMÉRO Feb. PAGE-flUMBERS IN ORIGINAL Numénos DES PAGES DANS Vommea HUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIÉES 10 rra REQUESTING DEPARTMENT MINISTRE-CLIENT Environment TRANSLATION BUREAU NO, NOTRE DOSSIER No BRANCH on DIVISION DIRECTION OU DIVISION Fisheries Service TRANSLATOR (INITIALS) TRADUCTEUR (INITIALES) MM PERSON REQUES71NG DEMAND'E PAR YOUR NUMBER VOTRE DOSSIER NO Dr. R.G. Ackman, Halifax Laboratory. DATE OF REQUEST 18th July, DATE DE LA DEMANDE UNI.:1-YiTi:-..;) TRANSLATION rz:r only NON s ;-ient SEP etnEY. 2/Ge) :.'"vets'o...e.rt"itre de«f-prcrreevyreterreey.rnr.,..,; MMPrenIN-lem'e!!ieertF, ',"IM'Ir`rld7e.`?i`..r r

3 DEPARTMENT OF THE SECRETARY OF STATE ' TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES CLIENT'S NO. DEPARTMENT DIVISION/BRANCH CITY NO DU CLIENT MINISTERE DIVISION/DIRECTION VILLE Environment Fisheries Service / Office of the Editor BUREAU NO. LANGUAGE TRANSLATOR(INITIALS) NO DU BUREAU LANGUE TRADUCTEUR (INITIALES) Ottawa Italian MMM 16th Sept The influence of culture conditions on the fatty acids composition of Scenedesmus SPP. II. The influence of the temperature. -62-* by C. Paoletti, E. Pelosi, B. Pushparaj National Research Council Autotroph Microorganisms Study Centre at the Institute of Technical and Agrarian Microbiology of the University of Florence. Director: Prof. Gino Florenzano. The Influence of Culture Conditions on the Fatty Acids Composition of Scenedesmus SPP. II: The Influence of the Temperature. The composition in fatty acids of three species of Scenedesmus (S. obliquus, S. acutus and S. obtusiusculus) grown at different temperatures and under identical lighting conditions was studied.. Although the three species had different optimum temperatures for growth, as regards the metabolism of the fatty acids, they show that they reflect the effect of this factor in the same way. The composition in fatty acids is-strongly influenced by.growth temperature. With the increase of this, an obvious diminution in the degree of unsaturation is obtained and an increase in the C16 acids compared with the C18 acids. Among the acids whose biosynthesis appears to be strongly influenced by temperature, there is a-linolenic acid, which, while it represents 33-36% of the fattg acids in the cells grown at 20 C, represents 10-13% in those grown at 35 C. THE INFLUENCE OF CULTURE CONDITIONS ON THE FATTY ACIDS COMPOSI- TION OF SCEt JEDESAiUS SPP. If. THE INFLUENCE OF THE TEMPERATURE The fatty acid pattern of threa Scenadesmus species ( S. obliqvus, S. acutus and S. obtusiusculus) grown in the same light conditions, but at different temperatures, has been Investigated. Although the three strains have different optimum temperatures, this factor Influence in the same manner the metaoclism of fatty acids. The fatty acid pattern vary widelq according to the incubation temoerature. Increasing the growth temperature causes a lowar degree of unsaturation and an higher C, acids content. The amount of a-ifnolenie acid changes from 33-36;ô of total fatty acids in cells grown at 20'C, to 10-13M. in cells grown at 35 C. *Number of page of original.,t^rir:!.' 11 :^ t^ -3T,?^D_ T^ { ^ijvl^^î:oi`^ only MON '>,EviSEE sc u!ernent SOS

4 2 Among the factors influencing liposynthesis, temperature plays an important role. In general, on the diminution of the temperature at which an organism is grown, there is an increase in the degree of unsaturation of the fatty acids produced. This behaviour was found in warm-blooded animals (1), in fish (2, 3), in arthropods (4, 5), in plants (6, 7), and in mircoorganisms (8-12). As regards the latter, the data in our possession indicate that the variations in the fatty acids' composition are more pronounced in bacteria than in fungi. The fact that the microorganisms studied up to now produce more unsaturated lipids at low temperatures has led various authors (9, 10, 13) to advance the hypothesis that the increase of unsaturated acids with the diminution of growth temperature is necessary to keep the physical state of the cellular membranes lipids, as an essential.condition for the preservation of the functional soundness of these cellular structures. As regards microalgae, the influence of the temperature on the synthesis of fatty acids has been only slightly investigated and the information presently available does not allow us to go beyond making some general comments (14-18). On pursuing the investigations intended to define the influences exercised by the culture conditions on the fatty acids content of the algal biomasses (19-21), we thought it necessary to study the influence exercised by temperature on the biosynthesis of the fatty acids in the microalgae species most suitable for culture in the open. In this note, the results relating to some species of the genus Scenedesmus are referred to. METHODS Organisms_and Culture Conditions Green algae of the genus Scenedesmils. (SC. acutus strain 8 M, Sc.

5 3 obliquus strain 170 V, and Sc. obtusiusculus strain 33M) belonging to the collection of the Centre were examined. The algae were grown under sterile conditions in the apparatus already used in previous research work (20), at a light intensity of 12,000 lux. The cultures inocculated with approximntely 50 mg of dry weight/liter and incubated at 20, 30 and 35 C in the culture medium referred to in one of our previous works (21), were collected at the same cellular concentration. Analysis Methods The cells gathered from the culture medium through centrifugation, were dried in a vacuum kiln at a temperature of C. The extraction of the lipids, the methylation of the fatty acids and the determination of the individual components were carried out according to the procedures set forth in a previous work (20). EXPERIMENTAL RESULTS Fig. 1 shows the growth trend of Sc. acutus strain 8 M and Sc. obliquus strain 170 V at the temperatures tested. Sc. obtusiusculus strain 33 M behaved in the same way as Sc. acutus. Ab the growth rate of the cultures was considerably influenced by the temperature, the time necessary to reach the preestablished concentration limit was different from time to time (thus for -63- Sc. acutus the incubation periods of the cultures at 20 0, 30 0 and 35 o C were respectively 120, 87 and 72 hours). By collecting the cultures at the same cellular concentration, it was possible to prevent extraneous influences, such as the one resulting from a different degree of lighting, superimposing themselves on the one caused by the temperature. Shown in Table I, in analytic form, are the data on the fatty acids

6 4 composition of the thtee microalgae species grown at different temperatures;. in Table II the same data are shown in a more summarized form to facilitate comparisons. Examination of Table II allows for the easy evaluation of the importance and nature of the influences exercised by growth temperature on the synthesis of fatty acids. With the increase in temperature there is a considerable increase in the acids with 16 carbon atoms compared with those with 18 carbon atoms and a marked diminution of the degree of unsaturation. Scen. acutus Scan. obliquus 1,5 35 C / /30 00 "20 C 20 C bo A ' 120 Houts Hours Fig. 1 Growth of "Seen. acutus" strain 8 M and of "Seen. obliguus" strain 170 V-at 20, 30 and 35 C. - TABLE. I Fatty Acids' Composition of "Scen. acutus" 8M, of "Scen. obtusiusculus" and of "Seen. oblicpus" 170 V grown at 20, 30 and 35 C. 33 M. 0 0 " e (Du el e e e n.. Organsms V; V; - 0 '-'.; g' c9. 5. 't?' a,.0 :7* ii..-1. a g a: a z _ ,. t) c.) x c..) 0 U >.' 0 0 U U U C.) U >.' U Sc. acutus... 1,6 1,4 tr 155 0,9 2,7 9, ,6 5, ,1 7, Sc. obtusiusculus. 12 1,4 tr 15,2 1,4 25 8,1 1,6 19,4 tr 6,0 2,6 36,0 25 0,9 1,1 Sc. obliquus ,2 oy 15,0 1, ,4 15,9 tr 6, ,3 3,1 1,3 1,2. 30 Sc. acutus..., 0,8 tr tr 215 1,5 tr 0,8 4,8. tly oy 8,0 15,1 28,6 1,7 1,4 0,5 Sc. obtusiusculus. 2,1 tr tr 20,7 2,0 1,1 25 3, ,3 sy 17,7 24,1 15 1,3 05 Sc. obliquus :.. Y '05 tr 05 21,9 2,5 0,9 0,5 3,4 13,9 tr 7,8 16,9 26,2 1,6 1, ,. Sc. acutus.. '. 1,9 ' tr tr 29,7 3,0 tr ,3 1,0 9,0 23,1 13,6 2,1 1,9 05 Sc. obtusiusculus. 22 tr tr 30, ,5 tr 7,0 7,0 0, ,1 12,9 OA 1,5 0,6 Sc. obliquus.. 12 tr tr. 32,9 4,3 oy tr tr MJ 22,6 10,3 0,4 tr 02.

7 (*) Xl= unsaturated acid with 14 carbon atoms; X2= unsaturated acid (probably n-heptadecenoic); X3= unsaturated acid with 20 carbon atoms. 5 TABLE II Summary description of the influences exercised by growth temperature on the fatty acids' composition of "Scenedesmus" spp cells. Organisms Unsaturated Acids/ Saturated Acids 20"C! 30"C 35"C Sc. acutus r 3,26 Sc. obtusiusculus 4,28 I 3,23 Sc. obliquus 4,28 r 3,12 2,02 2,01 1,91 C11,5/C13 e Unsaturated/, 16 Palmitic DrC 30-C I i 3D"C - 20`C i 30"C 35"C _ t 0,75 0,78 0,96 1,35 t 0,98 " 0,57 0,85 0,79 1,01 t 1,64 1, ,75 0,81 0,99 1;41 0,94! 0,49 Just as much in the C16 acids as in the CI8 acids, the variation of -64- the growth temperature causes profound changes in the relative abundance of the various fatty acids. These can be divided into three distinct groups according to the effect that the temperatute exercises'on their biosynthesis: Group_ 1 Comprises palmitic, palmitolinoleic, linoleic and, to a limited extent in Sc. obliquus, oleic acids, which increase considerably with increase in temperature. Group 2 Forming part of this group are a-linolenic, n-hexadecatetraenoic acids and as yet unidentified component (indicated as X2), 1 which undergo a strong diminution with increase in temperature. Group 3 Includes the secondary components which, although sometimes undergoing considerable variations, do not reach the point of affecting the overall ' composition to any great extent. Shown in Fig. 2 are the most-significant variations caused by temperature

8 6 on the percentage content of the most important fatty acids. As is seen, these variations are perfectly comparable in the three species of Scenedesmus examined. Therefore, the differences in the effects that temperature exercises on the metabolism of the fatty acids does not correspond to the differences respecting the optimal growth temperature existing between the strains under consideration. 30 Sc; acutus Sc. obtusiusculus Sc. abliquus Palmitic Acid n-hexadecatetraenoic Acid X2 15 Linoleic Acid Linolenic Acid Temperature r--,i hl eo 30 C Fire:335 oc Fig. 2. Table of the most important modifications caused by temperature on the fatty acids composition of the lipids of three species of Scenedesmus.

9 The most accentuated variations concern, in the C16 acids, palmitic and 7 n-hexadecatetraenoic acids. The former increases with the increase of temperature (from % at 20 C it passes to % at 35 C) while the latter behaves in the opposite way, since from % at 20 C its content drops to % at 30 C. As regards the C18 acids, linoleic and a-linolenic acids were shown to be very sensitive to temperature. The former increases with temperature increase (from % at 20 C it passes to % at 35 o C). Vice versa the percentage of a-linolenic acid decreases sharply with an increase in temperature; in fact at 20 C it is the main component among the fatty acids (22-36%), but at 35 C its content decreases by at least 15%. It is known that present-day knowledge on the synthesis of fatty acids in plants, claims that palmitic acid is the direct precursor of stearic acid through the elongation of two carbon atoms in the chain, and admits that the polyunsaturated acids derive from the saturated acid of the same number of carbon atoms through subsequent dehydrogenations. Therefore it is possible to attribute the modifications in the fatty acids' composition of cellular lipids, induced by different growth temperatures, to differing behaviour, in the light of this factor, of the enzymes which play a part in the metabolism of fatty acids. In this regard it is sufficient to admit that the enzymes which control the transformation of palmitic acid into stearic acid and the dehydrogenasis which act on palmitolinolenic and linoleic acids (changing them respectively into n-hexadecatetraenoic and a-linolenic acids) are, with respect to the other metabolism enzymes of the fatty acids, relatively less active at high temperatures in order to explain the main effects of temperature on the biosynthesis of fatty acids (increase, with increase in temperature, of the C16

10 8 acids and decrease of n-hexadecatetraenoic and ot-linolenic acids). In a report on the fatty acids of blue-green algae, Holton and Blecker (22) have thrown light on the existence of a certain relationship between composition in fatty acids and phylogenetic position, since in the simplest, and, therefore, most primitive, unicellular Myxophyceae often only monounsaturated acids are present, whilst the C16 acids clearly prevail over the C18 acids. Therefore the enzymes which give rise to polyunsaturated acids originated at a later time. This is in keeping with the hypothesis that these enzymes act better at relatively low temperatures, since it is admitted that adaptation to high temperatures is a property of primitive life. In this regard it is obvious that the composition in fatty acids of the strains of Scenedesmus grown at 35 C is much closer to that of the primitive M2cophyceae than that of the same strains grown at 20 C.

11 9 BIBLIOGR.APHY - I) DEAN H. K., 1Iann-m-1 T. P., The body fats of the pig. III.. The influence of body temperature on the composition of depot fats. Biochem. J., 27, 1950 (1933). 2) KNIPPIZATTI W. G., MEAD J. F., Influence of temperature on the fatty acid pattern of mosquitofislz (Garnbusia affinis) and guppies (Lebistes reticulatus). Lipids, 1, 113 (1966). 3) KNIPPR.ATH W. G., MEAD I. F., The effect of the environmental temperature on the fat!)' acid composition and on the in vivo incorporation of I-"C acetate in goldfish (Carassius auratus L.). Lipids, 3, 121 (1968). 4) FARKas T., HERODEK S., The effect of the:environmental temperature on the fatty acid composition of crustacean plankton. J. Lipid Res., 5, 369 (1964). 5) FRAENKEL G., HOPE H. S., Temperature adaptation and degree of saturation of phospaticies. Biochem. J., 34, 1085 (1940). 6) HARRIS P.,.IAmEs A. T., The effect of low temperatures on fatty acid biosynthesis in plants. Biochem. J.,.112, 325 (1969). - 7) HOWELL R., COLI.INS F. L, Factors affecting linolenic and linoleic acid content of soybean oil. Agron. J., 49, 593 (1957). 8) FARREL J., ROSE A. H., Low teinperature microbiology. Adv..Appl. Microbiol., 7, 335 (1965). 9) FAR/IEL J., ROSE A. H., Temperature effects on micro-organisms. Ann. Rev. Microbiol., 21, 101 (1967). 10) ROSE A. H., Physiology of micro-organisms at low temperatures. J. appl. Bact. 31, 1 (1968): 11) LONG S. K., WILLIM AS ' O. B., Lipids of Bacillus stearother- mophilus. J. Bacteriol., 79, 629 (1960). 12) MARR A. G., INGRAHAM J. L., Effect of temperature on the composition of fatty acids in Escherichia coll. J. Bacteriol., 84, 1260 (1962). 13) GAUGHRAti E. R., The saturation of bacterial lipids as a function of temperature. J. Bacteriol., 53, 506 (1947). 14) HOLTON R. W., BLECKER H. H., 0>».-3 M., Effect of growth' temperature on the fatty acid composition of a blue-green alga. Phytochemistry, 3, 595 (1964). 15) PArrERsoN G. W., Effect of culture temperature on fatty acid composition of ChIorella sorokiniana. Lipids, 5, 597 (1970). 16) KLEINSCHMIDT M. G., MCMAHON V. A., Effect of growth teinperature on the lipid composition of Cyanidium caldarium, I. Class separation of lipids. Plant Physiol., 46, 286 (1970). 17) KLEINSCHMIDT M. G., McMAtioN V.A., Effect of growth tem-. perature on the lipid composition of Cyanidium caldarium. IL Glycolipid and phospholipid compoezents. Plant Physiol., 46, 290 (1970). 18) IwAmom H., ho K., TSUKADA M., Effects of light, tempera. rature and glucose on the fat accumulation in Chlorellà. J. Fent. (Japan), 30 (1), 35 (1972). 19) BoDin V., PAotErri C., MATERASSI R., Indagini sulfa frazione lipidica di Spirulina platensis e di altre microalghe coltivate. Ann. Micr., 20, 65 (1970). 20) PAOLETTI C., /viaterassr R., PELOSI E., Variazione della cornposizione lipidica di alcuni ceppi mutanti di Spirulina platensis. Ann. /vlicr., 21, 65 (1971). 21) PAotr.rn C., PELost E., MATERAssr R., Influenza delle condizioni colturali sulla corn posizione in acidi grossi di Scenedesmus spp. I. Influenza della lizce. Riv. It. Sost. Grasse, 49, 428 (1972). 22) HOLTON R. W., BUCKER H. IL, Fatty acids of blue-green algae. In Zajic J. E. edit.: «Properties and products of algae». New York and London, Plenum Press, (1970). _

12 10 ITALIAN BIBLIOGRAPHIC ITEMS 19) Research on the lipid fraction of Spirulina platensis and other cultivated microalgae. 20) Variation in the lipid composition of some mutant strains of Spirulina Platensis. 21) Influence of growing conditions on the fatty açids' composition of Scenedesmus Spp. I. Influence of Light.