STUDIES I N PHOTOSYNTHESIS I N TROPICAL SUNLIGHT. PrlRT I BY G . GOPALA RAO AND N. R . DHAR
Formaldehyde Synthesis Under the action of sunlight, the green plants absorb carbon dioxide from the air and transform it with the intervention of water into carbohydrates. This phenomenon has been technically called photosynthesis. The reaction nCOz nHzO = (CH20)n n o z is endothermic and the quantity of energy absorbed is approximately 110,000gram calories for each gram molecule of carbon dioxide. Thus in the process of photosynthesis, nature has worked out a method of utilising the enormous floods of solar energy pouring on the earth. According to Warburg, no other endothermal photochemical reaction shows such a high utilisation factor. The green plant is thus an efficient converter of the inexhaustible supply of solar energy into potential energy in the shape of food materials, fuel, etc.; coal and petroleum are likewise the product of photosynthetic activity produced ages ago. Thus it becomes evident that all life on this planet and our gigantic industrial developments of the modern era depend on this phenomenon of photosynthesis, which in the ultimate analysis is the prime mover of civilisation. Because of its fundamental importance carbon assimilation early attracted the attention of scientific workers. Since the time of Stephen Hales ( I 7 0 7 ) , this has been the subject of numerous researches by such brilliant workers as Priestley, Ingenhousz, Senebier and de Saussure. In spite of countless suggestions the mechanism of the reaction is still obscure. The theory which has held the attention for over half a century and is still the pivot of the modern investigations, is the formaldehyde hypothesis of Baeyer given out in 1864. The general consensus of opinion is in favour of the view that formaldehyde is photochemically synthesised from carbon dioxide and water, and that the formaldehyde is then polymerised to form carbohydrate. The mechanism of the formation of formaldehyde from carbon dioxide and water will be discussed in Part 11. Evidence for the substantiation of Baeyer’s theory has been sought from more than one direction. These are: ( I ) Attempts to find in the plant the intermediate product, formaldehyde. Evidence on this head would not be of much value as the presence of formaldehyde in the leaves is not a sure indication of its being an intermediate product, especially in view of the work of Spoehr: and Moore and Webster? who showed that many substances of vegetable origin yield formaldehyde on exposure to ultraviolet light or sunlight. However, recent experiments of Klein and Wrernel3 seem to indicate that the existence of formaldehyde in
+
+
Biochem. Z . , 57, 95 (1913).
* Proc. Roy. Soc., 90, I68 3
(1918)
Biochem. Z., 169, 361 (1926).
PHOTOSYNTHESIS I N TROPICAL SUNLIGHT
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leaves is not due to the photolysis of certain substances existing in plants as has been suggested by the above-mentioned workers, but is really an intermediate product of assimilation. ( 2 ) Another direction in which evidence is sought for the above theory is the feeding of plants with formaldehyde. According to Grafe’ and Miss Baker2 plants can utilise small amounts of formaldehyde in light, though large amounts prove toxic. Sabalitschka and Riesenberg3 have reported that certain plants assimilate formaldehyde in the dark with the formation of sugar and starch. However, from the fact that a substance can serve as food for plants, it need not be concluded that it should be produced in the assimilation process. A more fruitful piece of evidence is furnished by the synthesis of formaldehyde “in vitro” under conditions simulating those obtaining in the plant. This phase of the problem has received new stimulus by the use of the quartz mercury vapour lamp and of fluorescent substances, which are capable of absorbing light of one wavelength and giving out light of another wavelength. Usher and Priestley4 obtained positive tests for formaldehyde by exposing quartz tubes containing water and carbon dioxide to ultraviolet light. Baly, Heilbron and Barker5 claim to have obtained direct synthesis of formaldehyde from carbon dioxide. These experiments have been contradicted by Spoehr,6 Baur and Rebman,’ and Porter and Ramsperger.8 Still recently Baly and coworkers9 seem to contradict their earlier results. They say that carbohydrate and not formaldehyde is the primary product of the action of ultraviolet light on a mixture of COZand water, and that the formaldehyde results from secondary photochemical action, the photolysis of the carbohydrate. On the other hand Mezzadroli and Gardano’O have obtained formaldehyde and small quantities of sugar by exposing solutions of bicarbonates of different metals. This work is confirmed by the later experiments of Mezzadroli and T’aretonlL and Mezzadroli and Babes.I2 There is unfortunately little agreement in the results of the various workers on the reduction of carbon dioxide in ultraviolet light. The question may well be asked of what significance for the natural process of photosynthesis are experiments on the action of ultraviolet light on carbon dioxide and water. That the ultraviolet part of the solar spectrum plays a very insignificant r6le in photosynthesis is almost certain. So atBer. bot. Gee., 27, 431 (1909); 29, 19 (1911). * A n n . Bot., 27, 411 (1913). Biochem. Z., 144, 545 (1924). Proc. Roy. SOC.,84B, 1 0 1 (1911). J. Chem. Soc., 119, 1025 (1921). J. Am. Chem. Soc., 45, 1 1 8 4 (1923). Helv. Chim. Acta., 5 , 928 (1922). 8 J. Am. Chem. Soc., 47, 79 (1925). 9 Proc. Roy. SOC., 116A, 2 1 2 (1927). 1OAtti Accad. Lincei, 6, 160 (1927). 11Zymologica, 3, 165 (1928). l*Gazz., 59, 305 (1929).
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0.GOPALA RbO A N D N. R. DHAR
tempts have not been wanting to obtain formaldehyde and carbohydrates in visible light in the presence of coloured organic and inorganic substances. Moore and Wehater' claimed to have obtained formaldehyde by exposing solutions of colloidal uranic hydroxide through which carbon dioxide was passed. Similar results axe obtained by Haly and collaborators.* On the other hand, entirely negative results were obtained by Baur and Rebman, and by Spoehr. Dhar and SangalYhave made an extensive investigation in t,his line. They find the frirniation of formaldehyde by passing carbon dioxide through suspensions of methyl orange, methylene blue, chlorophyll, ferric chloride, uranyl salt, chromium salt etc. These experiments of Sanyal and Dhar were recently contradicted by Burk.i The object of the present investigation is to see how far valid arc the objections raised by Burk. As will be discussed in the following pages, some of the experimental conditions of Burk differ from those of Dhar and Sanyal and are such as would not be conducive to photosynthesis. So we are constrained to modify one or two of his experimental conditions. Otherwise the procedure is just the same, and it. may be mentioned here that in most' cases positive results were obtained. Thus a comparison of the present investigation with that of Burk will clearly bring into prominence bhe importance of experimental conditions, and shows how a change in one or more of the conditions produces altogether contrary results. The present investigation consists of: (a) the reduction of carbon dioxide to formaldehyde in the presence of organic and inorganic photocatalysts; (b) the reduction of carbon dioxide to carbon monoxide in the presence of light and chlorophyll. Experimental In nearly all the work on photosynthesis repeated previously, with a few exceptions, the experimental conditions have not been described with the detailed precision which the subject demands. In the present investigation, the experimental methods and conditions have been carefully laid down with studied care, and the points of adverse criticism have been met with.
I . Reduction of carbon dioxide to formaldehyde. Carbon dioxide prepared from marble and dilute hydrochloric acid and purified by passage through two or three wash-bottles containing distilled water was passed through a two-way tube into pyrex beakers, one containing conductivity water and the other containing conductivity water to which the photosensitiser has been added. Both the beakers were covered over and exposed to the bright sun for three hours. The object of keeping a blank was to avoid the question of formaldehyde being attributed to impurities. l
Proc. Roy. SOC.,87B,163 ( 1 9 1 3 ) ;91B, 196 (1920). LOC.cit. J. Phys. Chem., 29, 926 (1925). J. Phys. Chem., 31, 1338 (1927).
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Porter and Ramsperger (loc. cit.) in their criticism of the work of Baly and collaborators attribute the origin of formaldehyde found to the impurities present such as stopcock grease,rubber tubing, sealing wax, etc. I n view of this criticism, we had to cut down the use of rubber tubing to the level of unavoidable requirements and, to prevent the action of light on rubber, it was coated over with black paper. Not content with this, we had resort to a very efficient method of proving the non-interference of impurities and it is that of putting a blank. The gas was passed through a two-way t.ube simultaneously into two beakers, one containing the reaction mixture and the other pure conductivity water. After three hours’ exposure to bright sunlight, both the solutions were tested for formaldehyde. In every case formaldehyde was detected in the reaction mixture and never in the conductivity water kept as a blank. If any formaldehyde were to arise from stopcock grease or rubber, it should have been found in both the beakers. The entire failure to detect formaldehyde in the blank solution effectively removes the question of the impurities and clearly brings about the prominence of the photocatalysts. The organic photocatalysts employed were: ( I ) methylene blue; ( 2 ) malachite green; (3) methyl orange; and (4) chlorophyll. Baur and Rebman’ and Baur and BUCK’ showed that formaldehyde is formed when various dyestuffs such as malachite green, eosin, rhodamine and phosphine are exposed to ultraviolet light. That aldehyde is also produced from chlorophyll and other aniline dyes in sunlight has also been shown by Osterhout.3 I n view of this work, in the experiments in which chlorophyll and other aniline dyes were used as photocatalysts we always exposed a blank solution of the dye under exactly identical conditions but no carbon dioxide was passed through it. The blank solution as well as the solution through which carbon dioxide was passed, were distilled and distillates tested for formaldehyde with Schryver’s or a modified Schiff’s reagent described by D 6 n i g e ~ . ~Comparative experiments showed that in every case in which carbon dioxide was passed considerably larger quantities of formaldehyde were produced than in the absence of carbon dioxide. These experiments repeated several times leave no doubt that carbon dioxide is reduced to formaldehyde in the presence of light and such dyestuffs. All the tests employed for the detection of formaldehyde were first critically studied starting with dilute solutions of the substance itself. The modified Schiff’s reagent was prepared as follows: One gram of rosaniline hydrochloride was dissolved in 500 C.C.of water and to this solution 2 5 grms of sodium sulphite and 1 5 C.C.of HC1 (sp. gr. 1 . 1 2 4 ) are added and the whole diluted to one litre. The solution is slowly decolorised and can be used after a few hours. I n pure solution formaldehyde can be detected in a concentration of I:~OO,OOO. Tests are carried out by Helv. Chim. Acta, 5, 1828 (1922). Helv. Chim. Acta, 6, 959 (1923). 3 Am. J. Bot., 5, 5x1 (1918). ‘ Compt. rend., 150,529 (1910).
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G . GOPALA RAO AND N. R. DHAR
adding I to z C.C.of hydrochloric acid (sp. gr. 1.124) to I O C.C. of the solution to be tested and then I C.C.of the reagent. Formaldehyde produces a blue to red violet coloration. With dilute solutions, several hours are required to develop the colour, which sometimes does not attain maximum intensity until after 20 hours. Other aldehydes give only a temporary coloration. The inorganic catalysts employed were colloidal ferric hydroxide , uranyl nitrate, and chromium sulphate and copper sulphate. In these cases also the solutions were distilled and distillates tested for formaldehyde. I n every one of these cases formaldehyde was detected in appreciable amounts.
II. Reduction
of carbon dioxide to monoxide. The decomposition of carbon dioxide in the extreme ultraviolet light was studied by Herchefinkel’ and later by Coehn and collaborators.2 Berthelot and Gaudechon3 state that, the reaction
COZ
* co +
(0)
is facilitated by the presence of reducing substances. We have now shown that such a decomposition takes place in visible light from a 500-watt gasfilled tungsten filament lamp by virtue of the reducing power of methylene blue or chlorophyll. The test employed for the detection of carbon monoxide is the well-known iodine pentoxide method of C. de La Harpe and Reverdine. Great precautions had to be taken in employing this test; for iodine pentoxide decomposes in light. So the U-tube containing iodine pentoxide was protected from all light both during and after preparation. All the joints were of glass, as iodine pentoxide is a notorious oxidising agent acting on cork, rubber and the usual lubricants. Blank experiments were always done. Traces of CO were always found in the carbon dioxide issuing out of a chlorophyll suspension exposed to light, The important bearing of this fact on the photosynthetic mechanism wi!l be discussed in another paper. From the experimental results recorded in Section I, ample confirmation is afforded for the work of Dhar and Sanyal, and the other workers in the field. We can now say, with a great degree of assurance that the experimental conditions of Burk were not conducive to successful photosynthesis; and hence the negative results. The distinctive feature of Bulk’s experimental technique was the use of condensed light. By a curious process of reasoning, Burk arrives a t the conclusion that the use of lenses was found indispensable. Many of the reasons assigned are hypothetical and the arguments unconvincing. To use his own words, “the plant leaf is a veritable nest of lenses; there is no question in my mind that these natural lenses do actually increase considerably the intensity of light inside the plant.” Therefore Burk used such lenses as would enable the intensity of sunlight to be increased to a maximum of five thousand fold. As against this, let us con-
’ Compt. rend.,
149, 395 (1909). Chem., 91, 347 (1916). Compt. rend., 150, 1690 (19x0).
* Z.physik.
PHOTOSYNTHESIS I N TROPICAL SUNLIGHT
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sider the common experience in photosynthesis in vivo, as well as in vitro. Falling off of photosynthesis was observed by Reinke in “Elodea” when the intensity of illumination surpassed sixty times that of sunlight and by Pantanelli when the light intensity was greater than that of sunlight. As regards experiments in vifro, Baly and coworkers1 report that with increasing intensity of light (using visible light from Ioo-watt lamps and using coloured substances like nickel and cobalt carbonates as photosensitisers) the quantity of carbohydrate synthesised per unit quantity of light decreases. The same writers report that P. F. R. Venables, in his experiments on the quantities of organic materials, soluble in alcohol, synthesised in presence of aluminum powder and light from COz and water, found that the amount synthesised decreases as the light intensity increases. I n the light of such extensive and unequivocal evidence, we have 110 doubt in asserting that the high intensities used in Burk’s experiments were harmful to photosynthesis.
Summary I. Formaldehyde has been obtained from carbon dioxide and water in the presence of the following photosensitisers and sunlight: (I) Chlorophyll, ( 2 ) methylene blue, (3) malachite green, (4) methyl orange, ( 5 ) ferric hydroxide sol, (6) uranyl nitrate, (7) chromium sulphate and (8) copper sulphate. Great precautions were taken in view of the adverse criticism. 11. Small quantities of carbon monoxide were obtained from the photochemical reduction of carbon dioxide by chlorophyll. Chemical Laboratories, University o j Allahabad, Allahabad, hTouember 18, 1930. 1
Proc. Roy. Soc., 116A, 197,
212-219,
(1927).
