THE COMPLEX CARBOHYDRATES AND FORMS OF SUL- PHUR IN MARINE ALGAE OF THE PACIFIC COAST. BY D. R. IIOAGLAND AND L. L. LIEB. (From the Division of Agricultural Chemistry, Agricultural Experiment Station, Berkeley, California.) (Received for publication, August 18, 1915.) The giant kelps of the Pacific Coast have in recent years re- ceived considerable attention because of their high content of potash,l possibly of commercial value.2 The economic aspects of the subject are discussed elsewhere.3 Of far greater interest to the plant chemist and physiologist is the study of the chemical composition and metabolism of these remarkable plants. Their selective action on certain elements contained in sea water is very striking. Iodine is absorbed in comparatively large quantities from a solution containing only the smallest trace of this ele- ment. To a lesser degree there is a marked selective power for potassium. In a previous article the discussion of these points has received further elaboration.4 It is the purpose of the present paper to present the results of an investigation designed to de- termine the chemical nature of certain very characteristic organic constituents of several important species of algae growing along the Pacific Coast. The following species are now reported on: Macrocystis pyrifera, a brown sea weed belonging to the family ’ Balch, D. M., On the Chemistry of Certain Algae of the Pacific Coast, Jour. Incl. and Engin. Chem., 1909, i, 777-787. ? Cameron, F. Ii., and Moore, R. B., A Preliminary Report on the Fer- tilizer Resources of the United States, U. S. 62nd Congress, Senate Document 190, 1912, 290 p., 19 plates, maps. 3 Burd, J. S., The Economic Value of Pacific, Coast Kelps, California Bgricultural Experiment Station, Bull. 248, 183-21.5, 3 figs. 4 Hoagland, D. R., Organic Constituents of Pacific Coast Kelps, Jour. Agr. Resenrch, 1915, xv, 39958, 7 tables. 287 by guest on May 25, 2018 http://www.jbc.org/ Downloaded from
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THE COMPLEX CARBOHYDRATES AND FORMS OF SUL- PHUR IN MARINE ALGAE OF THE PACIFIC COAST.
BY D. R. IIOAGLAND AND L. L. LIEB.
(From the Division of Agricultural Chemistry, Agricultural Experiment Station, Berkeley, California.)
(Received for publication, August 18, 1915.)
The giant kelps of the Pacific Coast have in recent years re- ceived considerable attention because of their high content of potash,l possibly of commercial value.2 The economic aspects of the subject are discussed elsewhere.3 Of far greater interest to the plant chemist and physiologist is the study of the chemical composition and metabolism of these remarkable plants. Their selective action on certain elements contained in sea water is very striking. Iodine is absorbed in comparatively large quantities from a solution containing only the smallest trace of this ele- ment. To a lesser degree there is a marked selective power for potassium. In a previous article the discussion of these points has received further elaboration.4 It is the purpose of the present paper to present the results of an investigation designed to de- termine the chemical nature of certain very characteristic organic constituents of several important species of algae growing along the Pacific Coast. The following species are now reported on: Macrocystis pyrifera, a brown sea weed belonging to the family
’ Balch, D. M., On the Chemistry of Certain Algae of the Pacific Coast, Jour. Incl. and Engin. Chem., 1909, i, 777-787.
? Cameron, F. Ii., and Moore, R. B., A Preliminary Report on the Fer- tilizer Resources of the United States, U. S. 62nd Congress, Senate Document 190, 1912, 290 p., 19 plates, maps.
3 Burd, J. S., The Economic Value of Pacific, Coast Kelps, California Bgricultural Experiment Station, Bull. 248, 183-21.5, 3 figs.
4 Hoagland, D. R., Organic Constituents of Pacific Coast Kelps, Jour. Agr. Resenrch, 1915, xv, 39958, 7 tables.
of Laminariaceac;” Iridaea laminarioides; and Ulva fasciata. The two latter are so called rock weeds and do not attain great size. Macrocystis pyrifera is noteworthy because of its huge size and is said to have the widest distribution of any known plant.6
Studies of the Complex Carbohydrates.
Unlike most land plants marine algae do not, except perhaps in rare instances, contain simple carbohydrates or easily hydroly- zable polysaccharides. Starch is not present, and cellulose only in minor quantities. The most frequently occurring carbohydrates include pentosans, galactans, levulans, and methyl pentosans.
In “Nori” (Porphyra Zaciniata) Kintaro’identifiedas hydrolytic products galactose and mannose. From another species (Fucus) Gtinthersobtained fucose. Baucr’ states that he isolated from one of the Laminariaceae a sugar having the properties of dextrose. Kylin’o also found in Luminaria digifata a carbohydrate yielding dextrose on hydrolysis. He described several other carbohydrates obtained from Laminuria saccharina, FUCUS vesiculosus, and Ascophyllum nodosum. Muthe+ obtained mannitol from Fucus and Luminaria digit&a. According to Eule+’ Carragheen moss (Chondrus crispus) yielded galactose, fructose, and a methyl pentose on
j Setchell, W. A., The Kelps of the United States and Alaska, U.S. Gdnd Congress, Senate Document 190, Appendix K, 1912, 130-178.
6 McFarland, F. M., The Kelps of the Central Californian Coast, ibid., Appendix M, 1912, 194-208.
’ Kintaro, O., and Tollens, B., Ueber das Nori aus Japan, Ber. d. deutsch. them. Gesellsch., 1901, xxxiv, pt. ii, 1422-1424.
8 Giinthcr, A., and Tollens, B., Ueber die Fucose, einen der Rhamnose isomeren Zucker aus Scetang (Fucus-Arten), ibid., 1890, xxiii, pt. ii, 2585-2586.
D Bauer, It. W., Ucbcr einer aus Laminariaschleim entstehende Zucker- art, ibid., 1889, xxii, pt. i, 618.
lo Kylin, H., Zur Biochemie der Meeresalgen, Ztschr. f. physiol. Chem., 1913, lxxxiii, 171-197.
I1 Miither, A., and Tollens, B., Ueber die Producte der Hydrolyse von Seetang (Fucus), Laminaria und Carragheen-Moos, Ber. d. deutsch. them. Gesellsch., 1904, xxxvii, pt. i, 298-305.
I2 Euler-Chelpin, H. K. A. S. v., Grundlagen und Ergebnisse der Pflan- zenchemie, Braunschweig, 1908, pt. i, 238 p.
hydrolysis. Takahashi,l3 Saiki,‘4 Greenish,ls and Payen’ have identified in various species (Porphyra laciniata, Fucus amylaceus, Fucus evanescens)
pentosans, methyl pentosans, galactans, and mannans.
In all of the studies just referred to the procedure has consisted chiefly in hydrolyzing the sea weed and in establishing the general identity of the sugars thus formed.’ It would seem to be also of interest to study the properties of the original complexes themselves. This would lead to a better understanding of the chemical composition of the algae. In the present investigation data have been secured regarding the physical and chemical properties of certain well defined complexes in addition to the study of their hydrolytic products.
There are two main fractions of carbohydrates in the algae under consideration, one precipitated from an alkaline extract by acid (so called “algin”) and one precipitated by alcohol from aqueous solution. The discussion is accordingly divided into two parts.
Carbohydrates Precipitated by Acid.
The earliest study of this complex, as isolated from sea weeds of the Scottish Coast, was made by Stanford.“-20 Hc obtained a jelly-like sub- stance which he called “algin” or “alginic acid.” This he characterized
as a nitrogenous organic acid, having the structure, C&H76022 <
NH*. NH*
I3 Takahashi, E., iibcr die I~~rstandt~cilc von Fucus evanescens, Jour. College of AgKcuZkure, Sappiro, Japan, 1914, vi, pt. v, 109-116.
I4 Saiki, T., The Digestibility and Titilization of Some Polysaccharide Carbohydrates Derived from Lichens and Marine Algae, Jour. Biol. Chem., 1906-07, ii, 251-265.
l5 Greenish, H., Untersuchung von Fucus amylaceus, Ber. d. deutsch. them. Gescllsch., 1881, xiv, pt. i, 2253.
I6 Payen, M., Sur le &lose et lcs nids de salangane, Compt. rend. Acad. d. SC., i850, xlix, 521-530.
I7 Stanford, E. C. C., On Algin: A Ncm Substance Obtained from Some of the Commoner Species of Marine Algae, Chem. News, 1883, xlvii, 254- 257,267-269.
1s Stanford, On Algin, Jour. Sot. Cizem. In&s., 1SS4, iii, 297-301; dis- cussion, 301-303.
19 Stanford, .4 New &Iethod of Treating Seaweed, ibid., 1885, iv, 519- 520.
20 St,anford, On Alginic Acid and Its Compounds, ibid., 1886, v, 218-221.
He was unable to wash out the contaminating salts, and evidently worked with a sample only slightly purified.
Krefting, 2*s 22 experimenting on the sea weeds of Norway produced a “tang acid” similar to the alginic acid of Stanford, but claimed that his preparation was nitrogen free. Villon*3 and Kylinl” later described similar substances obtained from other sea weeds, indicating a wide distribution in plants from different localities. An entirely analogous complex is found in the kelps of the Pacific Coast,4 approximately 16-18 per cent of the crude substance in the case of Macrocystis pyrijera.
Preparation of #ample.-The preparation of a purified algin free of ash and organic impurities was difficult. The colloidal jelly absorbs a large quantity of dissolved organic and inorganic contaminating substances. The purest alginic acid prepared by StanfordzO and used in determining the molecular formula contained 2.3 per cent ash and 2.03 per cent nitrogen.
The following procedure was adopted in preparing the sample used in the present work. 1 kg. of crushed Macrocystis is covered with a 2 per cent Na2C03 solution for twenty-four hours. The mixture becomes thick and sticky. It is finally warmed and filtered through linen by suction. The addition of a slight ex- cess of HCl produces a white spongy precipitate, floating in the liquid. The color presently darkens to a deep brown. The precipitate is filtered off, redissolved in 2 per cent Na2C03, and the precipitation twice repeated. The final precipitation is made from an alkaline solution by addition of alcohol. Sodium alginate comes down as a stringy, non-gelatinous mass, and will keep indefinitely, preserved in alcohol. 1 kg. of dried Macrocystis yielded 160 grams of crude alginate, containing 33 per cent ash.
For final purification the sodium alginate, prepared as described above, is dissolved in water and placed in a parchment bag im- mersed in running water. After three days the solution is acidi- fied with HCl, which precipitates the alginic acid. The dialysis is continued for a few more days in tap water and finally in dis-
21 Krefting, A., An Improved Method of Treating Seaa-eed to Obtain Valuable Products (Alginic Acid, “Tang Acid”) Therefrom, Eng. Pat., 1896, 11,553, abstract in Jour. Sot. Chem. In&us., 1896, xv, 720.
22 Krefting, An Improved System or Apparatus for Treating Seaweed (Alginic Acid) for the Manufacture of Products Therefrom, Eng. Pat., 1898, 12,416, abstract in Jour. Sot. Chem. In&us., 1898, xvii, 846.
23 Villon, A. M., On “Algine,” Chem. News, 1893, Ixviii, 311.
tilled water until no test for chlorine is given. The alginic acid is then filtered off, dried at 100” C., and finally ground and dried to a constant weight. Samples thus prepared were free of more than traces of ash and nitrogen.
Properties.-Alginic acid is capable of absorbing 200 to 300 times its weight of water. When moist it is readily soluble in dilute alkali, but dried it becomes hard and horny and very resistant to solvents. It is readily precipitated from solution by alcohol and ether. As a colloid alginic acid may be considered an irre- versible gel. It is capable of absorbing salts to the extent of 60 per cent of its own weight, but has no selective action for potassium. Its optical activity is high. [&’ = - 169.2’. The index refraction is low; [N]?,O = 1.3373, for 1 gram of sodium alginate in 100 cc. of solution.
Metallic Derivatives.-A large number of metallic alginates may be formed as described elsewhere.4 Twenty insoluble and five soluble alginates were prepared by the addition of metallic salts to solutions of sodium alginate, slightly acidified with acetic acid.
Acidity.-Samples of the dialyzed alginic acid were titrated with 0.01 N NaJZ03; 325 grams were neutralized by 1 liter of normal alkali. The neutralization equivalent is therefore 325. This result indicates the weak acidity of the substance.
Decomposition of Algin.-Stanford” stated that algin was de- composed after several days’ standing in dilute alkaline solution. Preliminary work suggested a loss from chemical, bacterial, or enzymic action. Experiments proved that after some time considerable decomposition might take place as a result of bac- terial action.
Analytical Data.-Samples of ash- and nitrogen-free alginic acid gave the following data.
Molecular Formula.-Combustions indicated the following ulti- mate composition: C = 42.0, H = 4.5, 0 = 53.5 per cent. The simplest corresponding empirical formula is CZ1H2,0Z0 with molec- ular weight of 599. Analyses were made of compounds of al- ginic acid wit,h uni- and divalent metals. The results, using the formula given above, indicate two replaceable H atoms, Hz GJMM .
Percentages of Metals in Metallic Alginates.
FOUMl. per cent
Na in sodium alginate Na, (C21H250?0). 7.0 K in potassium alginate K?(CkH25020). . . 11.2 Ca in calcium alginate Ca(CslHzsOna). 6.3 Fe in ferrous alginate Fe (C21H2&0?0). . . . 8.7
Calcul:m?d. PC)’ cent
7.1 Il.5
6.3 8.5
The formula here advanced is obviously only an empirical one. No data are obtainable which would throw light on the manner in which the sugars are linked together. It may be said, however, that alginic acid, if not strictly speaking a definite chemical compound, is at least a homogeneous complex, which shows characteristic reactions.
Identification of Xugars.-Freshly precipitated alginic acid, hydrolyzed with 2 per cent HCl for four hours at 100°C. gave a strong reducing action. Considerable carbonization took place. To avoid this digestions were made for twenty-four hours at 80” C. The undissolved residue was filtered off and the filtrate neutralized with NaOH. The solution was then heated with a mixture of two parts phenylhydrazine hydrochloride and three parts sodium acetate, according to the method of Fischer.:5 Two osazones separated out, a yellow osazone crystallizing readily, and in lesser quantity a red amorphous form. After repeated crystalliza- tions from 50 per cent alcohol, the separation and purification of the yellow crystalline osazone was accomplished. Under the microscope fibrous needles, characteristically arborescent, were observed. The melting point of the cryst.als was 154-155°C. By comparison I-arabinose phenylosazone”F has a melting point
25 Fischer, IS., Verbindungen des Phenylhydrazins mit den Zuckerurten, Ber. d. deutsch. them. Gesellsch., 1884, xvii, pt. i, 579-584.
of 160°C. and I-xylose phenylosazone27 15%155°C. Optical activity was determined according to the method of Neuberg.28 0.2 gram of the pure osazone was dissolved in 4 cc. pyridine and 6 cc. absolute alcohol, then polarized in a 100 mm. tube, [cl]; = - 0” 10’. Under the same conditions for l-arabinose phenylosazone [a]: = + 1” 10’ and for I-xylose phenylosazone [a]; = - 0” 15”.
Solubilities.-The yellow osazone from the alginic acid is solu- ble in cold and hot water, benzene, ligroin, alcohol, ether, acetone, chloroform, and pyridine. I-Arabinose phenylosazone is insolu- ble in ether. I-Xylosc phenylosazone has solubilities similar to those of the osazone prepared from algin. The latter has a crystal- line structure distinctly different from that of arabinosazone. While the pentosazone here described is similar in many proper- t,ies to I-xylose phenylosazone, their identity cannot be positively asserted from data available. Very few pentoses have been isolated from plant,s and only three so completely described as to make identification certain.2’127
Carbohydrates Precipitated by Alcohol.
The alcohol-precipitable fraction of the carbohydrates of marine algae has not received attention from previous investigators. In Macrocystis pyrifera there is present approximately 11 per cent of alcohol-precipitable matter in the stems, and 6 per cent in the leaves. Iridaea sp. contains about 13 per cent.
The method of preparing the samples was the following. A kilogram of the crushed sea weed is first extracted cold with 2 per cent HCl. The liquid is then pressed from the sea weed and filtered. Strong alcohol is added to the filtrate and causes a light flaky mass to precipitate out and settle to the bottom, as a compact cream-colored layer. After a few days the super- natant liquid is drawn off, the precipitate washed with alcohol, and freed of liquid by suction. This precipitation is repeated twice. The final precipitate is preserved under alcohol.
17 Abderhalden, lot. cit., 297. 28 Neuberg, C., Ueber die Reinigung der Osazone und zur Bestimmung
ihrer opt&hen Drehungsrichtung, I3e~. d. deutsch. them. Gesellsch., 1899, xxxii, pt. iii, 3384-3388.
Properties.--When dried the alcohol-insoluble matter darkens and becomes sticky in the presence of moisture. The dried ma- terial is resistant to solution and to the action of dilute acids and alkalis.
The alcohol-insoluble matter from Macrocystis pyrifera is pre- cipitated partially by salts of Co, Zn, Sr, Sn, Cd, Ni, Al, Cr, and Cu. Complete precipitation is effected by ferric chloride, lead acetate, and lead subacetate. The alcohol-insoluble fraction from Iridaea is pure white, gelatinous, and gives no precipitate with metals except ferric chloride, lead acetate, and lead sub- acetate. The evidence in these cases points to complex mixtures of several compounds.
Sulphur Content.-An attempt was made to remove the large amount of inorganic elements found in the substance precipitated by alcohol. A clear water solution was made and dialyzed in parchment for six weeks. At the end of that period there still remained 35 per cent ash (CaSOJ in the preparation from Macro- cystis and 24 per cent in that from Iridaea. No precipitate, however, could be obtained by adding BaClz to the aqueous solutions. This would point to the absence of the SO4 ion. It might be assumed that the Ca and SO, are held in organic com- bination or else in some colloidal complex. After hydrolysis SO4 could be precipitated directly, as well as the Ca.
Carefully dialyzed samples were dried in vacua and the total sulphur was determined after peroxide fusion.2g Other samples were burned and the ash was analyzed. The following dat,a were obtained.
Allihn’s modification of Fehling’s solution and also estimations of the pentoses and methyl pentoses according to the method of Tollens and Ellett.30 The results indicated that nearly all the reducing action in the case of Macrocystis was due to a methyl pentose, while in the case of Iridaea no tests for pentose or methyl pentose could be obtained from the alcohol precipitate.
The hydrolyzed solution from Macrocystis was treated with phenylhydrazine hydrochloride and sodium acetate. Yellow osazones precipitated out on cooling. Three different crystalline forms were distinguishable under the microscope, including two sheaf-like forms to a comparatively small extent. Almost the entire mass was made up of an osazone resembling in general structure arabinose phenylosaxone prepared from gum arabic, although the grouping of the crystals was somewhat different. Repeated recrystallization from 50 per cent alcohol ‘gave pure crystals showing no variation in melting point on further crystalli- zation. The melting point is 17%173°C. The melting point of fucose phenylosazone”’ is 177” C. [a]: of the crystals by Neu- berg’s method was 0” 0’. They are insoluble in cold or hot toluene and alcohol. The melting point and solubilities closely resemble the corresponding properties of fucose phenylosazone. Fucose is the only methyl pentose so far found to occur in marine algae.3* It was prepared from Lam&aria digitata,8 Mucus vesiculosus,30 and Nori (Porphyra Zaciniata)’ by Tollens and his colleagues.
The hydrolysis of the preparation from Iridaea was accom- plished as previously described, and phenylosazones were pre- pared. The crystals obtained were identical with galactose phenylosazone crystals prepared at the same time.
“C.
M.P. purecrystals..................................... 187-188 M.P. galactosazone.................................... 188 [a];P of pure crystals.. . . . +O” 46’ [(Y]: of galactosazone. . . . . . . . . . +O” 48’
30 Ellett, W. B., and Tollens, B., Ueber die Bestimmung der Methyl- Pentosane neben den Pentosanen, Ber. d. deutsch. chew Gesellsch., 1905, xxxviii, pt. i, 492499.
The solubilities correspond to those of d-galactosazone.32 Mucic acid crystals were readily obtained and the hydrolyzed sugar solution gave Tollens’ reaction for galactose.33 There is little doubt that the sugar in question is d-galactose.
Forms of Xulphur in Algae.
Sulphur is a common and ,important constituent of marine algae.” The analyses of Peterson3’ made on land plants indicate much less total and organically combined sulphur than is found in the algae investigated. This condition is not astonishing when it is recalled that the sea water nutrient solution contains very large quantities of soluble sulphates, ‘as compared with the soil solution. Especially noteworthy for their sulphur content are the forms Iridaea and Ulva fasciata. The former has 8.2 per cent total sulphur, the latter 4.4 per cent. A detailed study of the forms of sulphur present was made for Ulva fasciata.
Total sulphur (a) was determined by fusion with sodium perox- ide, and inorganic sulphur (b)’ by leaching the sample with water until no further test for SO1 was given. Precipitations were made in each case by Folin’s2g method. Total sulphur was also deter- mined in the leached residue and in the ash. The difference be- twecn these two percentages represents the sulphur lost on burn- ing. Volatile sulphur was determined by steam distillation in the presence of 3 per cent HCl. The distillate was passed into bromine water, which was later evaporated to 2-3 cc., fused with sodium peroxide, and sulphur estimated in the usual way. The other fractions were obtained as indicated in the following table showing the distribution of sulphur.
DistributiorL oJ Sulphur in Ulva jasciata, Per Cent of Dried Material.
a. Total sulphur.. . . . . 4.44 b. Soluble sulphates, calculated to sulphur.. 2.85 c. Soluble organic sulphur (not precipitated by Bach) .36 d. Total soluble sulphur.. . . . . . 3.21
e. Insoluble sulphur, not volatilized on burning.. . . . 0.69 f. Insoluble sulphur, volatilized on burning.. . . . . . 0.47 g. Total insoluble sulphur.................................. 1.16 h. Sulphur, volatile with steam., . . . 0.11
Sulphur was observed to be lost on drying t,he sample at 105°C. This is relatively a small loss and is probably the same fraction which is volatilized by steam distillation. Determinations were made of total sulphur before and after drying. Closely agreeing duplicates yielded the following results.
Sulphur Volatilized at 105°C. (24 Hours).
(Calculated on air dried samples.)
Total S. Before drying. After drying. Loss of s.
per ceut per cent per cent &facroc!ysl’is pyrifera. 1.20 1.08 0.12 Iridaen Laminarioides.. . 8.97 8.76 0.21 Ulva .fasciata. 4.49 4.36 0.13
Steam distillations were made on the three plants named above and in each case sulphur compounds were fixed in the distillates by bromine water. When the residues from the distillates were being fused with sodium peroxide an odor like that of mustard oil was noted.
SUMMARY.
1. The carbohydrates of Macrocystis pyrifera and Iridaea
laminarioides were investigated and several complex polysac- charides described in detail with reference to their physical and chemical properties.
2. From the acid-precipitable complex known as “algin” a pentosazone, closely resembling I-xylosazone, was prepared in pure form and its properties were determined.
3. In the alcohol-insoluble carbohydrate fraction of Macrocys- tis pyrifera a methyl pentose, having the properties of fucose, was described. Iridaea laminarioides from a similar fraction yielded only galactose.
4. The high content of sulphur in marine algae, as typified by Ulva fasciata, was studied, and estimations were made of the sulphur held in various forms.
