the photosynthesis of naturally occurring...

12. Since rigid tests established the freedom of the carbon dioxide and all the materials from all organic impurity, and since over 200 control experiments, periodically carried out, invariably gave entirely negative results, it would seem impossible that the photosynthesised carbohydrates arise from organic impurity.

We acknowledge our indebtedness to Messrs. Brunner, Mond & Co. for their generous financial assistance, which has enabled this work to be carried out.

Photosynthesis o f Naturally Occurring Compounds. 219

The Photosynthesis of Naturally Occurring Compounds.—III. Photosynthesis in vivo and in vitro.

By E. C. C. B aly , F.R.S., and J. B. D a v ies , Liverpool University.

(Received July 28, 1927.)

The evidence adduced in the two preceding communications leads to the belief that the direct photosynthesis of complex carbohydrates in a single operation from carbonic acid has now been achieved in the laboratory. There still remains, however, the question as to how far the results take us in the explanation of the natural process as it occurs in the living leaf. I t must be admitted that the natural process has ever presented many difficulties, and in view of the foregoing results the problem of its explanation is one of peculiar interest.

In the first place, we may refer to the difficulty arising from the complete absence of ordinary formaldehyde in the living leaf. The elegant work of Willstatter, proving that the molecular ratio of the carbon dioxide assimilated and the oxygen transpired is unity, offers a very definite proof that the first product in the photosynthesis is formaldehyde, and, in consequence, the fact of its entire absence from the leaf during photoassimilation of carbon dioxide was very difficult to understand. This difficulty has been completely eliminated by our results. Theoretical considerations based on the formation of activated carbonic acid as the initial stage in the process lead to the view that activated formaldehyde is then produced, which at once undergoes polymerisation to give the hexoses. De-activated or ordinary formaldehyde should not, therefore, take part in the reaction and, in consequence, should not be found at any

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stage. These theoretical deductions have been proved to be correct, since in the photosynthetic production of carbohydrates vitro the complete absence of ordinary formaldehyde has been proved. So far as this fact is concerned there is agreement between the laboratory and living processes.

The second point of interest lies in the fact that the photosynthesis vitro has been achieved by the use of a surface, and the question at once arises as to whether the natural process is or is not a photochemical surface reaction. There exists a considerable amount of evidence that a limiting surface not only exists in the chloroplast but is necessary for the normal photosynthesis to take place. I t is generally agreed that in the living plant the photosynthesis takes place in the chloroplasts, these being heterogeneous systems consisting of water,, proteins, lipoids, and the plant pigments. Price,* * * § as the result of ultra- microscopic observations, states that the chloroplast appears as a slightly opaque and heterogeneous body with a motionless gel structure. Sternf drew attention to the importance of the surface between the two phases of the chloroplast, namely, the lipoid and the aqueous phases. The photosynthetic process only takes place normally when this surface is intact. Then, again, Warburg J investigated the influence of certain surface-acting substances, such as phenylurethane and methylurethane and its homologues, and he found that they retarded natural photosynthesis, his conclusion being that their action depended on changes in a limiting surface. The relation between the rate of photosynthesis and the concentration of the narcotic is expressed by a curve which is similar to the well-known adsorption isotherm of Freundlich. These results indicate strongly that natural photosynthesis is a heterogeneous reaction, and it thus would appear that a second point of similarity has been established between the process in vivo and in vitro.

Perhaps the greatest difficulty in the way of understanding the natural photosynthetic process has been the utilisation of visible light by the leaf. This is well shown by the following values of the photosynthetic efficiency at different wave-lengths observed with Chlorella by Warburg and Negelein§ :—

X = 660ji.fi, 578fj,[i, 546p.fi. 436fi.fi,

Efficiency 59 53-5 44-4 33-8 per cent.

* ‘ Ann. Botany,’ vol. 28, p. 601 (1919).t ‘ Z. f. Bot.,’ vol. 13, p. 193 (1921).t ‘ Biochem. Z.,’ vol. 100, p. 230 (1919); vol. 103. p. 188 (1920); and vol. 146, p. 486-

(1924).§ ‘ Z. Phys. Chern.,’ vol. 106, p. 191 (1923).

E. C. C. Baly and J. B. Davies.



The difficulty can at once be realised from a consideration of the equation, derived from thermochemical data—

6H2C03 — C6H120 6 -f- 602 — 673,800 calories.

It is clear from this that the minimum quantity of energy required to activate a single molecule of carbonic acid is 112,300 calories. If the activation were achieved by photochemical means only, then we would have—

hvN = 112,300 calories = 4-6941 X 1012 ergs,

where h is the Planck constant, 6-547 X 10-27, v is the frequency of the light, and N is the Avogadro constant 6-1 X 1023. The value of v is found to be 1-1754 X 1015, which corresponds to a wTave-length of 255-2 pp.

This calculation is based on the minimum quantity of energy and, as can be ; seen, the wave-length lies far beyond the region of the spectrum which promotes photosynthesis in the leaf. As a matter of fact, carbonic acid has no power of absorbing light of the wave-length 255-2 pp, and in the experiments described in the first communication, which proved the existence of a photo-stationary state when carbonic acid is exposed to ultra-violet light in the absence of a surface, it was found that light of a wave-length X = 210 pp is necessary. It would follow from this that the light required to achieve photosynthesis of carbohydrates from carbonic acid by purely photochemical means must have this wave-length. This accentuates the remarkable nature of the natural process if this is considered as a purely photochemical reaction.

It has, however, been shown in these communications that in all probability the photosynthesis is a photochemical reaction on a surface, and this enables us to offer a suggestion which appears to eliminate the difficulty. We believe that the adsorbed layer of carbonic acid is partially activated—that is to say, the molecules have a higher energy content than they have in aqueous solution. In order to complete the activation to that stage required to give the activated formaldehyde, less energy will be needed than when the carbonic acid is in

, solution. Although we cannot at present define the increment of energy gained by the adsorbed layer of carbonic acid, it seems certain that the completion of the activation necessary for the photo-synthesis to take place is a photochemical reaction which can be achieved by means of visible light. In other words, the total quantity of energy necessary for the photosynthesis to take place is supplied in two separate amounts, one quantity being given when the adsorption on the surface takes place, and the second quantity being given by light. The first amount is sufficiently large to enable the second stage to be achieved by means of visible light. Our results justify us in offering this

Photosynthesis of Naturally Occurring Compounds. 221



222 E. C. C. Baly and J. B. Davies

explanation, and if it is correct we would point out that the essential difficulty in explaining photosynthesis in vivo has been solved.

The question might well be asked as to the peculiar merit of a visibly coloured surface for adsorbing the carbonic acid. I t is difficult to postulate that carbonic acid adsorbed on a coloured surface contains more energy than when adsorbed on a white surface, but this would seem at first sight to follow from our results. On the other hand, if it be true that the energy gained by the adsorbed carbonic acid is derived from the surface, then that surface must be re-activated by the supply of energy before it can give up energy to a fresh layer of carbonic acid, after the first layer has been converted to carbohydrates. Such re-activation of the surface would probably be secured by the absorption of light, and for this purpose a white powder would require ultra-violet light, whilst a visibly coloured powder would be re-activated by visible light. We venture to suggest this as a possible explanation of the peculiar efficacy of a coloured surface. I t is also possible that the decrease in efficacy after exposure which Zenghelis observed with his paper surfaces and which was mentioned in the preceding communication is due to the want of re-activation of the surface.

In offering these suggestions, we recognise their speculative nature, which necessarily must be the case when we are dealing with a somewhat novel phenomenon, namely, a photochemical heterogeneous reaction.

In the preceding papers the poisoning of the surface by the oxygen set free in the photosynthetic reaction has been referred to, and evidence has been given which shows that the poisoned surface slowly recovers itself, the yield of carbohydrates per unit quantity of light energy absorbed being greater with smaller light intensity. In other words, the de-poisoning of the surface is a slow reaction compared with the photosynthetic reaction. I t is of some interest to consider the function of the plant pigments from this standpoint.

The theory advanced by Willstatter and Stoll states that each molecule of oxygen evolved converts a molecule of chlorophyll A into chlorophyll B according to the equation

f^B^OsN^Mg + 02 = C55H70O6N4Mg, H2O,

and that there must be some mechanism for reversing the reaction, since the ratio of chlorophyll A to chlorophyll B remains constant during photoassimilation of C02. It is possible that this equilibrium is maintained by the carotin, this pigment being oxidised to xanthophyll in accordance with the equation

C40H56 + 0 2 = C40H06O2.

If this be correct, it will follow that the tendency will be for the xanthophyll/



carotin ratio to increase during photoassimilation of C02, a change which was observed by Willstatter and Stoll. In support of this view’, it may be stated that Dr. Stead, working in these laboratories, has succeeded in proving that it is possible to oxidise carotin to xanthophyll in two ways. In the first place, carotin in chloroform solution is oxidised by atmospheric oxygen, the first product being xanthophyll, and, in the second place, xanthophyll is formed wrhen a dilute solution of ferric chloride in acetone is cautiously added to a solution of carotin in acetone. The details of this investigation wi 11 form the subject of a further communication.

It is obvious that if the molecular ratio of oxygen transpired to carbon dioxide absorbed be unity, the relative quantities of all four pigments will be constant. The fact that Willstatter and Stoll observed an increase in the xanthophyll/carotin ratio, suggests at once that one of the pigment reactions is slow compared with the photosynthetic reaction, and, indeed, that a further similarity between photosynthesis in vivo and in vitro exists. Since thechlorophyll ratio remains constant whilst the carotinoid ratio changes, it follows that the slow reaction must be the one in which the xanthophyll is reduced again to carotin, a reaction winch includes the transference of the oxygen from the chloroplasts to the stomata. I t also follows that for the maximum efficiency to be secured the photosynthetic reaction must proceed at a rate wrhich is not greater than that of this slow reaction. In the laboratory we find, as already stated in the second paper, that if the photosynthesis proceeds at a greater rate than the de-poisoning of the surface, the carbohydrate yield is materially reduced, and, further, that the use of strong light for long periods causes oxidation of the carbohydrates.

These considerations offer an explanation of the fatigue effect which is well known in connection with the photosynthetic activity of the living leaf. This was demonstrated by Ursprung,* who showed that the yield of starch decreased after exposure to strong light. The phenomenon has also been studied by Ewart,f who found that prolonged exposure to intense light caused the destruction of the chlorophyll.

There is yet another phenomenon shown by the living leaf which finds a possible explanation from our results. During the day the leaves are exposed to light of varying intensities, including that of direct sunlight on a clear day. In the photosynthesis in vitro it is a simple matter so to adjust the light intensity that the photosynthetic rate does not greatly exceed the rate of

* ‘ Ber. bot. Ges.,’ vol. 35, p. 57 (1917).t ‘ Ann. Botany,’ vol. 11, p. 439 (1897); and vol. 12, p. 379 (1898).

Photosynthesis o f N aturally Occurring Compounds. 223



224 E. C. C. Baly and J. B. Davies.

de-poisoning of the surface. Since the plant cannot do this, it is not unreasonable to ask whether there is not present in the leaf some internal mechanism whereby the rate of photosynthesis is controlled, so that it does not at any time materially exceed the rate of the slow reaction which may be assumed to be a constant for any one leaf. We venture to suggest that the well-known variation of the orientation of the chloroplasts with respect to the light rays is one of the details of such an internal mechanism. By change in the orientation of the chloroplasts the surface exposed to the light is decreased when the light intensity is increased. The result will be that, although the quantity of carbohydrate formed on unit area of surface is increased, the total quantity formed per unit time does not exceed the limit set by the velocity of the slow reaction. In connection with this the results recorded by Puriewitsch* are of considerable interest. He determined the increase in the heat of combustion obtained with excised leaves of four species of plants after exposure to sunlight, the total intensity of the light being determined by means of a recording bolometer. He found that there is an inverse ratio between the amount of energy used by the leaves and the total amount of energy which fell on them, which is the result to be expected from the above considerations.

The quantitative data given by Puriewitsch enable us to make a comparison between the quantities of carbohydrates synthesised in our experiments and in the living leaf. Puriewitsch gives the following average values of the increase in heat of combustion in calories per sq. cm. per hour :—

Acer plantanoides .. .. 0-34, 0-27, 1-09, 0*53.Polygonum sacchalinense .. 4 • 1, 1 • 7, 1 • 4, 2 • 0, 0 • 3, 0 • 9.Helianthus annuus .. .. 1-3.Saxifragia cordifolia.. .. 1-5.

As stated in our previous communication, the maximum yield of carbohydrate that we have succeeded in obtaining under the best conditions yet secured is 0-075 gram in two hours. Assuming for the present purpose that the product is glucose, this corresponds to a gain in calorific value of 674,000 X 0-075/180 = 280-8 calories, or 140-4 calories per hour.'}' Since the surface exposed was 294 sq. cm., the gain in calorific value was 0-48 calorie per sq. cm. per hour. I t would seem, therefore, that the yield of carbohydrates obtained in the laboratory is not seriously at variance with that observed in nature.

Although the results described and discussed in this and the two preceding* ‘ Jahrb. wiss. Bot.,’ vol. 53, p. 229 (1914).t Similar results would be given if any other simple carbohydrate were assumed, since

the heat of combustion is, to a first approximation, proportional to the molecular weight.



papers would seem to show that a definite advance has been made towards the solution of the problem of photosynthesis, it must be remembered that the actual nature of the carbohydrates synthesised in the laboratory has still to be determined. If it be true that these are the same as those which are photosynthesised by the action of ultra-violet light on ordinary formaldehyde, then valuable information will be secured by the complete study of the latter compounds. As has already been noted, Irvine and Francis have proved that glucose is one of the products. A systematic investigation of these products in these laboratories is now approaching completion, and it is hoped soon to communicate the results of this work. Sufficient evidence has already been obtained to justify the statement that the similarity between photosynthesis in vivo and in vitro is greater than is implied by the fact that glucose is formed in each case.

Then, again, it must be remembered that the compounds obtained in the laboratory are optically inactive, a fact which marks a sharp differentiation between the natural and the laboratory processes. Although the explanation of the asymmetric synthesis in the leaf still remains to be found, yet it may be suggested that the presence of a suitably oriented surface may be the determining factor. It is at any rate easier to envisage the possibility of asymmetric photosynthesis in a heterogeneous than in a homogeneous system.

Conclusions.1. A marked similarity exists between photosynthesis in vivo and that now

recorded as having been achieved in vitro. This is established by the following features which appear to be common to both.

2. Ordinary formaldehyde does not take part in the reaction in either case.3. The laboratory process has been realised by the action of light on carbonic

acid adsorbed on a surface. There seems little doubt that a limiting surface exists in the chloroplast and is necessary for the photosynthesis to take place.

4. A visibly coloured surface and visible light function in each of the two processes.

5. A marked fatigue effect is observed when the living leaf is exposed to too long and intense illumination, and very intense illumination destroys the chlorophyll. In the laboratory a similar fatigue effect, due to poisoning of the surface by oxygen, is observed. Intense and prolonged illumination destroys the carbohydrate, there being present nothing which is analogous to the chlorophyll.

6. In both processes there is a slow recovery reaction, and it appears that inQ

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VOL. CXVI.— A.



226 Photosynthesis o f Naturally Occurring Compounds.

both the photosynthesis must not proceed at a more rapid rate than that recovery reaction.

In addition to the foregoing, the following conclusions have been reached :—7. The total yield of organic products photosynthesised vitro per hour

per sq. cm. of surface is not seriously at variance with that produced in the leaves of four plant species.

8. I t is possible that the constant ratio of chlorophyll A to chlorophyll B, observed by Willstatter and Stoll in the living leaf, is maintained by the carotin, which becomes oxidised to xanthophyll. Since the ratio of xanthophyll to carotin tends to increase during photosynthesis, it may be suggested that the slow recovery process present in the leaf is that in which the xanthophyll is again reduced to carotin.

9. I t follows from 6 that it is to be expected that there must be present in the living leaf some internal mechanism which controls the rate of photosynthesis so that it does not exceed that of the slow-recovery reaction. I t is suggested that the orientation of the chloroplasts with respect to the direction of the light rays is one of the details of this mechanism.

We again express our thanks to Messrs. Brunner, Mond & Co. for their valuable assistance. The experimental work which has led to the arguments advanced in this paper was alone rendered possible by the generous grants which this firm has from time to time made to these laboratories.



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