PHOTOSYNTHESIS WITH AMMONIA BY DEAN BURK
The present paper1 reports photochemical experiments attempting ( I ) to induce ammonia to build up complex biochemical nitrogen compounds with carbonic acid, formaldehyde, glucose, and other carbonaceous substances; ( 2 ) to reduce carbonic acid, bicarbonates, and carbonates; and (3) to oxidize ammonia to hydroxylamine, nitrites, and nitrates. Sunlight in combination with colored catalysts was used, and the experimental conditions, particularly of temperature and illumination, were made to correspond as closely as possible to the ordinary conditions of plant growth. The work is a natural sequence to the researches of Baudisch,* B a l ~ and , ~ S p ~ e h r their , ~ coworkers, and others, who have worked along similar lines, using chiefly ultra violet light, and who have obtained suggestive results as to the mechanism of photosynthesis in plants. The one serious criticism to be made of the important work of these investigators is that the ultra violet light region in the spectrum of sunlight is neither wide nor intense, nor does it extend t o the very short wave lengths, thus limiting any direct application of their results to plant phenomena Furthermore it is known that plants may grow normally either in ( I ) sunlight which has passed through glass, which removes all but about five hundred Angstrom units of the sun's ultra violet light; or ( 2 ) artificial light which contains no ultra violet light, In all of the writer's experiments exposures were made behind glass vessels, the thickness of the walls of which varied from five to ten hundredths of a millimeter. The question of the effect of long wave ultra violet light ( 3 3 0 p p to 3 9 0 p p ) upon plant growth as a whole is still unsettled,$as is indeed also, the questicn 1 d brief rhumb of a dissertation submitted by the writer in partial satisfaction of the requirements for the Degree of Doctor of Philosophy at the I-niversity of California, Slay 192;. 111 nearly all the work on photosynthesis reported previously, the experimental conditions have not, been descrihed with the detailed precision which the subject demands, and in addition, it has not been apparent always that such detaded data were available. The present discussion is based upon a mass of detail which would be impracticable to puhlish, but which may he found in its entirety in the dissertation. 2 Naturwissenschaften, 1914, Heft 9, I O , and many other papers. 3 J. Chem. Foe., 119, 1025 (1921); 121, 1078 (1922); 123, 185 (1923!; J . Ind. Eng. Chem., 16, io16 fI924). 4 Biochem. Z., 57, I I O (1913);plant Korld, 19, I , ~ 1 9 1 6 )J. ; Am. Chem. doc., 45, 1184 (1923). ' It would he well t o make mention of a common misconception among plant physiologists. Some believe that by passing sunlight through window glaes. ns might he done in a greenhouse, that all the ultra violet light is removed. Such is not the case. Ordinary glass transmits wave lengths as short as 330 to 340 p p (and sometimes even shorter). and since the visible rays end at 380 to 390 p p , there are thus some 40 to 50 pil (400 to j o 1 Kngstrijm units) of ultra violet light which may he transmitted. i n ternyerate zones tk.e ultra violet region does not eutend much beyond 300 p p (290 p p in Indial and ordinarily not beyond 310 to 320 p p . Furthermore, the region 310 t o 330 p p ie very much lrss intense (one half to one tenth) than the region 370 t o 380 p p , so tha? i t may he stated safely that glass transmits nor only 60 to so(!; of the ultra violet wave length region, but also that t t e actual amount of energy transmitted as ultra violrt light is even greater than these values. It is true that these figures (which err, if a: all, on the side of consrrvntism: tippend somewhat upon the int.ensity of the light, and the thickness, age, and cleanliness of the glass, hlat in general, variations in these factors can make little diterenre in the conclusions. On t h e other hand, it is well to hear in mind that passing bright sunlight through glass ma!' remove the r a m 300 to 330 p p and that these particular rays may happen to he those ultra violet rays RT'hiLh aff'ect organic substantes i i i Farticular reattiors, n hile the m y ? 330 t o 390 p p may he relatively ineffective.
PHOTOSYNTHESIS WITH AMMONIA
I339
of the effect of infra red light. Upon the basis of numerous, but from the modern point of view, insufficiently critical experiments, Daubeny‘ and Draper2 were the first to deny the essentialness of either of these kinds of invisible light.3 Furthermore, these workers were able to establish the incorrectness of the prevailing view of the previous sixty years, which derived initial support from the experiments of Senebier,4namely, that the violet and chemical rays mere the most powerful rays of sunlight for the growth of plants. But so far as the writer knows, the first and only experiments concerning the essentialness of invisible light, for plant growth to be carried out in a manner long hoped for by plant physiologists, have been performed within the last year or two at the Boyce Thompson Institute for Plant Research by P ~ p p . ~ For the first time, presumably, many species of higher green plants have been grown somewhat extensively over their entire growth period in differently colored sunlights, both the intensities and wave length compositions of which were simultaneously and accurately known and controlled. From all the criteria used Popp could find no significant’ changes resulting from growing plants in light from which wave lengths shorter than 389 p p has been excluded. Sumerous changes, however, were observed when parts of the visible spectrum &e. wave lengths less than 529 p~ or even 427 pp) were excluded. In 1779 Sir Humphry Davy6 made the first attempt to reduce carbonic acid artificially without, the aid of plants, by means of sunlight, and similar attempts have been made ever since, often in the presence of both inorganic and organic catalysts. Vithin the last few years success with inorganic catalysts and sunlight has been claimed by Moore7 and Dhar and SanyaLY Moore claimed that in the essential presence of ferric chloride, carbon dioxide and water could be reduced to formaldehyde. Dhar and Sanyal, who used tropical sunlight, now dispute Davy’s results and claim that no catalyst at all is necessary, adding that “we are of the opinion that the intensity and prolonged exposure have much to do with this photosynthesis of formaldehyde in sunlight.” They make no mention of the fact that their results contradict seemingly the Grotthuss-Draper First Law of Photochemistry that “only those rays that are absorbed produce chemical change.” According to h s c h k i n a s ~ some , ~ one hundred and fifty feet of water are required to reduce by absorption either the blue, green, or yellow, rays to .37 their initial intensity. Porter and Rarnsperger’O have recently pointed out the need for the complete
’ Phil. Trans., 126, 149 (1836). Phil. Hag.. 23, 161 (1843). Am. J. Bot., 13, ;oh (1926). 1Ii.moires Physico-Chimiques, Geneva, 1762. The infra red ravs, h e it recalled, were discovered first bv Fir \Yilliam Herschel in 1600 (Phil. Trans., 1800,p. 2 5 5 ) , and the ultraviolet rays first h v k i t t e r ((:ilb. .Ann. Phrsik., 12,406. 1603) and TVollaston (Sirholson‘s Journal, 8,293, (1864))simultaneously in i 8 0 1 . Beddors: “Contributions to Science,” 161. Biochemistry, (1921). J . Phys. Chem., 29, 926 (1925). TYied. Ann., 5 5 , 401 (1895). l0J.Am. Chem. Sor., 47, ;9 (1925). 2
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absence of organic matter in the experimental vessels. I t is not apparent that Dhar and Sanyal took this precaution, and the view that the formaldehyde they obtained was derived from organic matter is strengthened inferentially by the fact that whereas one hour’s exposure in open vessels was sufficient to allow detection of traces of formaldehyde, ninety hour’s exposure were required in sealed tubes, in which no atmospheric dust could accumulate.
Experimental Methods of Exposure. The distinctive feature of the writer’s technique, which should be kept in mind when comparing it with that of others, was the use of condensed light, The employment of lenses was found to be desirable, indeed indispensable. The lenses were large, spherical, colorless, common-glass flasks twelve to sixteen inches in diameter filled with clear water, and mounted on stationary iron tripods; at times, small four and one half inch plano-convex glass lenses were used also. Calculations indicated that the intensity of sunlight could be increased to a maximum of approximately five thousand fold; for noon sunlight this would be about five million times the intensity of an ordinary forty watt house lamp. The increase in total intensity was considered not to be so advantageous or to be desired particularly, as was the increased intensity of particular lines, perhaps rather weak in normal sunlight, which might be instrumental in causing certain photosynthetic reactions. It was estimated that exposure behind one of these lenses on a clear day was equal, as regards the amount or total calorific value of the light, to exposure to ordinary light for a month of clear days; experiments sustained this assumption. It may be mentioned that the ordinary ultra violet mercury lamp has a total energy yield about equal to good sunshine. And so, since these experiments were conducted with intensities of light which have been used seldom before, the hope was entertained that either of two effects might be observed; ( I ) that the rate of reaction would be increased more than linearly with a linear increase in intensity, as has been observed to a small extent in the hydrogen chlorine reaction, where the rate of reaction divided by the intensity is slightly greater a t greater intensities; or ( 2 ) that new photochemical reactions would take place only above certain critical intensities or photochemical threshholds. I t may be stated here that neither of these hopes was realized; although it is possible still, that the intensities used were not high enough. There are other advantages to the use of these lenses, and practically no disadvantages. For instance, it might be imagined that their effect would cause difficulties, but a simple water bath can keep the temperature of the exposed solution below 3oOC. I t is true that over a small localized area, such as a few square microns, the temperature might become slightly higher than presumed, but this could be true of ordinary sunlight also. Dhar and Sanyal stated that results obtained at one time of year were difficult to repeat at other times The writer has had no such experience, presumably because of his continuous use of condensed light. I t cannot be objected that the great intensities used could be harmful, or shield results, because the beam of focused light never covered the whole
PHOTOSYNTHESIS WITH AMMOSIA
1341
surface possible, but generally one tenth only or less. The products formed could be removed to relatively unilluminated parts of the exposed tube, where they would not be decomposed further by the intense light. This condition has the same good effect as that which Baly obtains when he shakes the chambers he is exposing to ultra violet light, thus carrying the primary products of the reaction to the back of the chamber out of danger of further decompx&ion. The use of lenses is by no means without example in plant processes. I t may be stated, with considerable assurance, that the active concentration or intensity of photosynthesizing light inside any particular plant under observation can never be known; the plant is a veritable nest of lenses. First, the external cells of the leaves of many plants have been observed to function exactly like convex lenses, the light entering them being forced to a focus before being used; this is well illustrated in the case of cells of Schzstostega osmundacea which grow in weak cave light, the chloroplasts forming an aggregate at the back of the cell where the light intensity is the highest. Second, the chloroplasts themselves are almost perfect double convex lenses. Finally, every fat globule or other similar heterogeneous phase in the protoplasm is found quite generally to have the approximate shape of a lens. These instances are mentioned not because the writer believes that these natural lenses bring about some unobserved or unaccounted for effect in plants (indeed, one might fear harm, especially in brilliant sunlight), but to show plainly that in using lenses he was not making use of some advantage not possessed by plants. There is no question in his mind that these natural lenses do actually increase considerably the intensity of the light inside the plant. The exposed solutions were made up with water twice distilled which had a conductivity not greater than 5 X I O + reciprocal ohms. Baker's C. P. chemicals without further purification were nearly always employed. Most of the solutions were exposed in glass bulbs blown on the end of common soft-glass tubes about a foot in length and twelve millimeters in diameter. The bulbs were quite spherical, had a capacity of fifteen to sixty cubic centimeters, were just as thin as safety would allow (from five to ten hundredths of a millimeter), and were very transparent. In most of the experiments the tubes were sealed, often under vacuum. In some of the experiments they were not sealed, in order to observe what effects the constituents of the atmosphere might have. The unsealed tubes were protected by hoods of loose tin foil bent over in such a manner as to allow free entrance of air but to exclude foreign matter. Exposures were made on top of a roof, several blocks away from any laboratory which might give off undesirable fumes. The tubes were always cleaned with chromic acid at 100' C, followed by hot alcoholic potash, hot concentrated nitric acid, many rinsings of distilled water (which effectively removed all nitrates), and then were dried at 100' C and put away until used. By means of suitable supports an exposed tube was held dipped in a liter beaker of water in such a manner that the bulb part of it was about an inch under water and in the direct path of the focused beam of light. The focal point, at which the highest intensity of light would be
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obtained, was never used ordinarily. The beam was usually one quarter of an inch or more in width at the point where it entered the bulb. The position of the bulb required changing every twenty minutes. KO heliostat was employed. The tubes were exposed to lenses for four to six hours a day, but they also received the ordinary sunlight, direct and diffuse, throughout the entire day. h battery of twenty lenses and tubes was generally kept in operation. Exposures were continued from one day to two weeks, depending upon the nature of the experiment and the speed with which tubes could be prepared for exposure. and analyzed subsequently. Many miscellaneous photochemical reactions were observed and in all cases three days’ exposure to lenses, equal to three months’ exposure to ordinary light, sufficed to approach closely the equilibrium or stationary state. The tubes were prepared most often in duplicate. One was exposed, while the other, as a control, was wrapped heavily in tin foil, so as to exclude light entirely, and placed alongside the first in the same thermostat. This system of maintaining controls was ideal; any difference between the two tubes appearing upon examination could be considered immediately as being the result of either primary or secondary effects of radiant energy.
Methods of Analysts. The methods used to analyze the different exposed solutions and their respective controls varied widely, and it would be impossible to give here more than a list of most of the tests used quite generally, and to add that on the average each exposed solution was examined by about twenty five different tests. Most of the tests were performed as described in Mulliken,’ but changes and improvements were sometimes necessary. The sensitiveness of each test, as carried out either in distilled water or in the experimental solutions, was always known. The sensitiveness of the majority of tests ranged from one to fifty parts per million by weight, although several, notably Schryver’s, Griess, and Trommsdorff were considerably more sensitive. I t should be understood that when negative results were obtained with unknown solutions, traces of the substance being examined for were added to the unknown solution and the test repeated, in order to insure correct interpretation. The pH was often adjusted to neutrality before testing. Where the solutions contained inorganic catalysts they were either distilled first, cr examined directly, with or without filtering. In the following list of tests attention needs to be called to the fact that each reagent is usually a test for many other substances besides those specifically mentioned, and hence trustworthy conclusions should obtain when a whole series of tests is performed on a solution, with negative results: reduction of permanganate, mercuric oxide, silver nitrate, and Tollens’ reagent for formic acid, hydroxylamine, etc. ; Schryver’s, resorcin, and gallic acid tests for formaldehyde; Schiff’s reagent for aldehydes in general; Molisch test for carbohydrates ; Prussian Blue test for nitrogen, cyanide; Fehling’s test for reducing sugars
’
“A Method for the Identification of Pure Oiganic Compounds.” 3 vols. (1904, 1911, 1916).
PHOTOSYNTHESIS WITH AMMONIA
I343
and formaldehyde; Benedict’s tests (second modification) for reducing sugars, but not formaldehyde ; Pierart’s, Barfoed’s, dilute iodine solution, saccharimeter, and aniline acetate paper pentosan tests for various carbohydrates; the various reagents for proteins and alkaloids (Millon’s, xanthoproteic, Biuret, mercuric chloride, picric acid, phosphotungstic acid, etc.,) Rimini, Simon, potassium ferrocyanide, carbylamine, Nessler, concentrated iodine in potassium iodide, formaldehyde and concentrated iodine in potassium iodide (Thatcher’s test), sublimation of solids (darkening, melting, odor, burning of sublimate, residue) tests for the various amines and nitrogen bases; diphenylamine, brucine, Griess, powdered zinc and Griess, Trommsdorff tests for oxygen nitrogen compounds and oxidizing agents (Le. hydrogen peroxide, chlorates, chlorine, ferric ion, etc.,) ; potassium ferricyanide, potassium ferrocyanide, and potassium thiocyanate for ferrous and ferric ions; organic solvent extractable and precipitable fractions. The pH, titration values to various pH’s, titration values with N/zo and I\J/zoo permanganate, color and odor were usually noted. Various forms of nitrogen were measured quantitatively by the modified Gunning-Kjeldahl method, Devarda method, Van Slyke method, distillation in alkali at 100’ C, vacuum distillation in dilute alkali at 40 to 45’ C, vacuum distillation in dilute acid at 40 to 45’ C.
Statement and Discussion of Results.
A good portion of the work was concerned with hexamethylenetetramine, CaHI2N4,a weak mono-acid base which hydrolyses in water according to a second order reaction and a t a rate depending upon the concentration, pH, and temperature, to give six molecules or formaldehyde and four molecules of ammonia, This substance can be obtained easily in a highly purified state and by adjusting properly the pH with sodium hydroxide and sulfuric acid any desired equilibrium concentration of formaldehyde may be obtained. A highly buffered range exists between pH 7.4 and 4.0. The whole range of concentrations was used, with and without catalysts. I n one of the earliest experiments without catalysts, in which the technique of exposure differed from that described already, 300 cc. of .8 M hexamethylenetetramine (or, expressed in terms of the hydrolyzable constituents 4.8 M formaldehyde and 3.2 M ammonia) were exposed in a sealed Jena glass flask over a period of 54 days to cloudless sunlight for 2 I O hours, and to 68 hours of sunlight condensed by simultaneous reflection from a maximum of thirty silver mirrors through glass lenses four and one half inches in diameter. The most exhaustive analysis could detect no difference between either this or similarly exposed tubes and the respective controls. Owing to the amount and duration of radiant energy supplied it would be difficult to make a more effective demonstration of the Grotthuss-Draper law. A significant fraction of the infra red was absorbed, no doubt, When catalysts were used (and the methods of exposure described initially, employed) the usual range of concentration was from .I to I%, but much smaller and larger amounts were often employed. Ferric tartrate,
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sodium citrate, potassium permanganate, copper sulfate,' magnesium ribbon and ferrous sulfate, yellow mercuric oxide, and a compounded mixture of equal parts by weight of the sulfates of ferric, ferrous, magnesium, manganese, and copper and also ferric oxide were tried. Entirely negative results were obtained, with the following minor exceptions. The ferric tartrate and potassium permanganate decomposed photochemically after about a day's exposure to lenses to give ferrous ion and manganese dioxide, and also presumably carbonate, since no formic acid was found. Traces of nitrates ( I p.p.m.) were found often when ferric ion was present. When free acid existed along with hexamethylenetetramine, Le., when the pH was greater than 4, small amounts of formic acid and a primary amine (methylamine presumably) were produced thermochemically after a week's standing a t a maximum temperature of 30' C. The amounts obtained were neither increased nor decreased by the presence of either catalysts or light, and the reaction observed was no doubt that described by Werner,' who worked at somewhat higher temperatures (50 to 110' C), but for much shorter periods of time. At 50 to 60' C relatively large amounts were obtained by the writer after 2 4 hours. Dhar and Sanyal stated that when the system XHdOH, CO,, HCOH, was exposed for twenty hours, methylamine was produced. I n the absence of experimental details, particularly in regard to controls, it would be difficult to criticize this observation, but suffice to say that the writer believes that if enough carbon dioxide had been present to yield a pH of less than 7 , and if the temperature was not kept below about 40' C, or even less the reaction observed was thermochemical, probably. With mercuric oxide, which was used up in the reaction, formates, carbonates, nitrates, and nitrites were produced photochemically but not thermochemically. The amount of nitrites and nitrates formed depended greatly upon the ammonium ion concentration, and mas related closely to the amount of surface rather than bulk of mercuric oxide used. The relative amount of nitrate to nitrite apparently increased with length of exposure, although no special experiments were made to determine whether mercuric oxide would oxidize photochemically riitrites to nitrates. Experiments with identical results were made with ammonium salts, as well as with hexamethylenetetramine. Moore claimed that all substances of biological origin yield formaldehyde upon exposure to sunlight. Plausible theoretical schemes for the building up of plant protein compounds directly from glucose and ammonia, without going through nitrate, nitrite, or formhydroxamic acid steps have been sug1 Two miscellaneous reaction mav he mentioned. Very pure metallic copper may be prepared by the photochemical reduction of copper sulfate organic acid solution, pr0vidin.g alkali is absent. When alkali is present, as in Benedict's or Fehling's solutions, copper IS not produced in quantity until the reduction (to cuprous oxide) has been about corn leted. Sodium nitrite and methyl alcohol, in either the presence or absence of copper sulfate a t the time of exposure, were observed to yield rapidly a nitrogeneous organic acid, the copper salt of which was insoluble in water. Formhydroxamic acid or an isomer had been produced no doubt, according t o Baudisch, but time did not allow further study and adequate identification. J. Chem. SOC.,111: 2, 844 ( 1 9 1 7 ) .
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gested.1 Experiments of the writer do not lend support to either of these views. Vacuum sealed solutions of widely varying concentrat,ions (600 fold) of the purest glucose obtainable, with and without ammonium chloride, ferric chloride, and ferrous sulfate present also, at varying hydrogen ion concentrations (pH 3 to 7 . 5 ) , were exposed to condensed light’ for 7 0 hours over a period of two weeks. Saccharimeter readings were made when the concentrations of glucose permitted. All of the numerous tests mentioned previously were employed to detect photochemical change, but no indication of the formation of any new organic substances was obtained. Most of the ferric ion was reduced to ferrous ion, after a time, of course. I t is a criticism of these experimenh, perhaps, that oxygen gas was excluded. The hypotheses of Stewart and the, statement of Moore were formulated, however, without reference to oxygen: Dhar and Sanyal reported that they obtained reducing sugars from formaldehyde when exposed to tropical sunlight in the essential presence of ferric chloride or methyl orange. This result was not confirmed by the writer. Solutions of vacuum redistilled, colorless formaldehyde at three different concentrations (15, .6, and .024YG)were exposed in the presence of ferric chloride at three different concentrations ( 2 , .oz and o x ) . z and 0% ferrous sulfate were also tried. The solutions were made up with redistilled water, sealed under vacuum, and exposed to 3 5 hours of condensed sunlight over a period of two weeks. The pH was varied from about z to 7 . j . The ferric ion was reduced photochemically to ferrous ion, the rate depending a great deal upon the pH. No reducing sugars, pentoses, other carbohydrates, or odorous substances were produced.
Carbon Dioxide Reduction. Ammonium carbonate is what might be termed the ideal plant nutrient; not so much because it supplies the four main essential elements, but because these four main elements are in those two radicles, carbonaceous and nitrogenous, which are most rapidly absorbed and efficiently used by plants. While nitrate enters rapidly, the general concensus of opinion and experimental findings is that ammonium ion enters even more rapidly. That ammonium ion is used more efficiently than nitrate is not surprising, since it is the form in which nitrogen appears in the plant. Nitrate would have to be reduced, at an expense of energy. The ammonium carbonate used was Baker’s (NH )2C03NH,COlXH2. Ammonium formate was used also. Formic acid is the first reduction product of carbonic acid (Le., dihydroxyformic acid), and results might perhaps be obtained with formate rather than carbonate, although it is to be remembered that much more energy is needed to reduce formic acid to formaldehyde than carbonic acid to formic acid2 (about five times). Spoehr3 has stated that he Stewart: “Recent Advances in Organic Chemistry,” 258 (1918). the relationsexisting if hydro en gas were involved, in a paper by the writer “The Free Energy of Nitrogen Fixstion by j i v i n g Forms,” J. Gen. Phvsiol., 10, 566 (1927). 3 Riochem. Z., 57, 95 ( 1 9 1 3 ) .
* See in this connection
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tried for years in many different ways to reduce formic acid to formaldehyde by means of radiant energy, but without success. The catalysts used were uranyl sulfate, mercuric oxide, copper sulfate, ferric and ferrous chloride, a compound catalyst consisting of equal parts by weight of the sulfates of ferrous ion, ferric ion, magnesium, manganous ion, and copper, and also without any catalyst at all. The results mere entirely negative, except that traces of nitrites and nitrates were formed in the respective cases of mercuric oxide and ferric ion, and that uranyl formate decomposed to give uranium oxide and carbon dioxide, a reaction noticed previously by Schiller.’ The ammonium ion was not oxidized by the uranyl sulfate. The reaction went rapidly to completion, . I gm being decomposed by a I 2-inch water lens in a few minutes. To illustrate the power of the condensed light used, it may be mentioned that a freshly prepared solution of ammonium carbonate and uranyl sulfate can be exposed to very bright sunlight for 1 5 minutes with scarcely perceptible change in color, but upon placing the same tube in the path of a beam of condensed light, a marked reaction is noticed in no more than a second, and in less than a minute the whole solution is entirely black or dark gray, filled with precipitated uranium oxide. During the first five seconds, the formation of intermediate oxides, indicated by a purple pink color, can be noticed. The active concentration of carbon dioxide in the plant during the photoreduction process is, like the active concentratiou of light, not known, within fairly wide limits even, nor is its form (i.e. CO?, HCO3‘, COS”, etc.). For the present, it would not be unreasonable to suppose that the concentration, calculated as a gas might be, say, I O atmospheres, which, expressed as carbonate would be about . 5 hl. Experiments were designed therefore, to obtain very much higher concentrations than have been used hitherto in similar experiments. This was accomplished by adding to ainnioniuin carbonate various amounts of concentrated C.P. 3 5 . 0 N sulfuric acid. The acid was contained in a side arm sealed into the neck of the tube, and was not mixed with the rest of the contents in the bulb part until after vacuum sealing and until just a few minutes previous to exposure. .18 cc. of the acid neutralized .joo gms. of ammonium carbonate to full methyl orange, pH 3.0. The amounts of acid used varied from none at all to complete neutralization where as high as I O atmospheres of gaseous carbonic acid were obtained. This arrangernent allowed each form COZ, HC03’!and COZ” to predominate separately. The following catalysts were used: ferrous sulfate; ferric chloride; ferrous sulfate and ferric chloride; a mixture of equal parts by weight of inanganous sulfate, nickel sulfate, copper sulfate, cobalt sulfate, chromic sulfate, ferrous sulfate, and ferric chloride; nickel sulfate; chromic and cobaltous sulfate; white zinc oxide; manganous chloride; inanganous sulfate; malachite green ( j o p.p.m.) ; uranyl sulfate. Each exposed solution was examined by soine thirty different tests. Not the slightest trace of detectable organic matter, nitrogenous or otherwise, 1
Z. physik. Chem., 80,641
(1912).
PHOTOSYSTHESIS WITH AMMONIA
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was formed photochemically from the ammonium carbonate. Special attention may be called to the ineffectiveness of uranyl sulfate as a catalyst. There is reason to believe that if any carbonaceous substance had been formed, its transient existence would have been indicated by an irreversible photochemical reduction of the uranyl compound. The experiments of Usher and Priestley in which formic acid was formed in some such system as was used here seem to be quite contradicted by these experiments, as well as by those of Baur and Rebmann.? The only photochemical reaction observed was the oxidation of ammonia to nitrite or nitrate in the presence of ferric ion, the mixed catalyst and zinc oxide. The amounts found in the cases of the first two catalysts were the same and hence it may be assumed that the other substances in the catalyst neither promoted nor prevented the formation of nitrates especially since no nitrates were formed when each of the other substances was tried separately by itself. Zinc oxide was the most vigorous photocatalytic oxidizer of ammonia discovered some hundreds of parts per million of nitrate being produced upon a day’s exposure in the presence of zyc ammonium chloride or carbonate. Presumably the active wave lengths are 3 5 0 to 400 pp, which are absorbed by the white zinc oxide. Solid zinc oxide phosphoresces in the ultra violet and if exposed beforehand to sunlight will cause the oxidation when added in the dark to aqueous ammonium salts. Exposed solid zinc oxide when added in the dark to the Griess reagent will give the nitrite test but not when similarly added to the Trommsdorff reagent, the reason being that the former contains nitrogen-hydrogen compounds while the latter contains no nitrogen compounds at all. The reaction was observed with either carbonate, chloride, or sulfate of ammonia. The rate depended directly upon the ammonium ion concentration and the surface of zinc oxide exposed, and did not seem to be affected by accumulation of end-products. There was no dark reaction over a considerable period of time if the zinc oxide had been kept away from the light previously. Neither clear saturated solutions of zinc oxide nor solutions of zinc salts, the chloride or the sulfate, possessed any catalytic activity, providing that all particles of solid zinc oxide were excluded. The experiments with malachite green were extended considerably, and the results are detailed el~ewhere.~Suffice to say that solutions of malachite green in the absence of any other substance were observed to decompose in sunlight to yield formaldehyde, amines, and under aerobic alkaline conditions, nitrites, in no case in concentrations greater than I O p.p.m, Since the concentration of nitrite was only I p.p.m., there was no way of determining if nitrates were produced also. The nitrites were formed presumably from the nitrogenous decomposition products of malachite green, amines as well as ammonia. Proc. Roy. SOC., 78B,318 (1906).
* Chim. Acta. Helv., 5, 828 3
(1922).
J. Am. Chem. SOC.,in press.
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Fenton's observation' that metallic magnesium may react with carbonates to give formaldehyde was confirmed. It was found also, that the amounts formed were not affected by the presence of either sunlight or ferric chloride. Although the experiments of Moore and Dhar and Sanyal on synthesis are unconvincing, they have attracted wide attention nevertheless. The extensive experiments of the writer, in which more detailed analyses were made, are directly contrary to the results of these workers, and throw doubt upon the sufficiency of their technique. I t is true that since ammonium sulfate, in addition to carbonic acid, was present in the writer's experiments, the experimental conditions were not exactly comparable; but so far as can be anticipated, this substance should have no critically harmful influence; quite the contrary, if formaldehyde and oxygen gas were the photochemical end-products of the reaction, the ammonia might assist in removing both of them, by forming hexamethylenetetramine (or other bases) and nitrites and nitrates. The negative character of the writer's experiments are upheld further by the fact that the C. P. chemicals used must have contained traces of many catalytic substances not introduced purposely.
The Oxidation of Ammonia by Ferric Chloride. By way of positive results of interest particularly for the understanding of plant processes is the photochemical oxidation of ammonia by ferric chloride, one nitrate and eight ferrous ions being formed stoichiometrically, X'itrites can never be detected even after sunlight has been excluded from the exposed chamber for some time. I n examining exposed solutions, the iron was removed by filtration after bringing the pH to 7 . The following results with the filtered solution prove the formation of nitrates. The diphenylamine reaction was given, and only by those solutions exposed previously, but not by the controls. The latter fact alone precludes the possibility of traces of ferric ion being the causal agent, even though only I O p.p.m. would be sufficient. A t pH 7 an infinitesimal amount of iron can exist in solutions free from organic matter, and furthermore no tests with the three iron reagents previously mentioned were given. The fact that the Griess and Trommsdorff reactions were negative precludes, to a concentration of . I ppm., nitrites, chlorine, chlorates, and hydrogen peroxide. Hydroxylamine has no effect on the diphenylamine reagent, and furthermore there was no reduction of N / zoo permanganate, even though hydroxylamine reduces permanganate instantly in the cold. The fact that the reaction is given only when ammonia has been present, together with the observed stoichiometric relation (the ferrous ion was determined quantitatively with N,'zoo permanganate and qualitatively with ferricyanide) is good inferential evidence. The conclusive proof, however, is given by the fact that when powdered zinc is added, either when testing reagents are present at the same time.or afterward, nitrites are formed in less than a minute in the cold, as shown by the Griess reaction, which is entirely specific for nitrites, and also by the Trommsdorff reaction, which, while not entirely specific for nitrites, is not given by nitrates. The zinc does
* J. Chem. Soc.,
91, 687 (1907).
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not affect the Griess reagent, even upon long standing, and while it affects slowly the Trommsdorff reagent, advantage may be taken of the relative rates. Incidentally, this excellent but little known met'hod of proving by reduction with zinc dust in the cold the specific presence of nitrates, in either the total or relative absence of nit'rites, should prove welcome to analysts. The writer has seen no mention of it in the literature or in the standard analytical works. The extensive proof above is given to avoid the usual criticism which can be made so justly of inany physiological papers in which a single line of evidence for the existence of nitrates (usually the diphenylamine or phenoldisulphonic acid reaction) is considered sufficient, The amounts of nitrate formed may reach as high as one hundred parts per million, and depend upon the concentration, pH, and intensity of light (as shown by using simultaneously different sized lenses and also by comparing with ordinary sunlight), but there is no direct proportionality with any of these factors. h stationary state, the cause of which is not yet understood, is reached, under any given set of conditions, after about a day's exposure. The half way point to this state is reached in less than an hour of bright sunlight. The reaction is unaffected one x a y or the other by the presence of oxygen gas. The back reaction of oxidation of ferrous ion in the dark, in either the presence or absence of air, is very slow, not greater, relatively, than one hundredth or more, nitrites being produced. Bonazzil believes that hydrogen peroxide is probably involved in the mechanism of nitrification by bacteria, where it is known that iron is essential for the process. No hydrogen peroxide (Le,, less than . I p.p.m.) mas observed in the writer's experiments. The question of whether nitrates or nitrites are produced by any of the observed catalysts is interesting from a theoretical viewpoint, but probably the relations would be changed if the systems involved were not so homogeneous, and thus from the plant viewpoint, it is probably satisfying to know only that either is produced. In practically every photochemical experiment of the writer's where ammonium ion and ferric ion existed together, nitrates (but not nitrites) were found, indicating that the reaction may be of very general occurrence. The oxidation of ammonia to nitrite or nitrate a t ordinary temperatures in the dark is accomplished with difficulty by even the strongest oxidizing agents, such as concentrated permanganate, chromate, etc., and hence the importance of the photochemical reaction is established. According to Baudisch and Baly the photochemical elaboration of nitrogen compounds may proceed from the nitrite stage and go through the formhydroxamic acid stage. The end-products of synthesis in plants are protein like bodies in which the nitrogen is in an ammonia-like form (Le.) NH, XH?, NH,). The question would arise then, when plants are nourished with ammonium salts, must these be oxidized to nitrites, then be reduced to formhydroxamic acid, and then proceed to an ammonia form? There is no reason J. Bact., 8, 343 (1923).
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why a plant should make use of what seems to be the most direct path, and the writer believes that his work, in combination with that of Baudisch and Baly, shows that such an indirect path in plants is possible, and furthermore, may proceed without loss of efficiency, which could occur, for instance, by the formation and loss of nitrogen gas, as an intermediate product. I t is true that Baly has another suggestion1 for taking care of the synthesis from ammonia, by means of the activated formaldehyde postulation. Since this discussion applies chiefly to what may occur in plants, it’is not difficult to imagine other quite possible paths; and furthermore it is well known that the elaboration of proteins in plants can take place in the dark. However, if elaboration is photochemical, the probability of the indirect path suggested above is strengthened furt’her by the unsuccessful attempts of the writer in trying to accomplish synthesis by the direct path. In speculative vein, it may be suggested that nitrification in soils may be accomplished on the surface by means of sunlight and photocatalysts. There are reasons to believe, that in addition to the catalysts observed, many oxides, particularly those which absorb the sun’s ultra violet light, could function also as catalysts, becoming reoxidized by the air. Conceivably the “nitre spots” in Colorado, reported by Headden and Sackett2 in a series of papers since 1910, are formed for the reason, in part, that the soil contains some very efficient photochemical nitrifying catalyst which is able to oxidize quickly the ammonia fixed by the nitrogen fixing bacteria. I t should be remembered that the formation of nitre spots always requires fairly moist conditions, just as the photochemical reaction presumably would. The severe criticism to be made of the biological explanation as being entirely sufficient for the accumulation of nitrates, is that’ the concentrations of nitrate formed (as great as jYG of the bulk of the soil) should preclude the action of organisms in the later stages. The suggested photochemical mechanism of nitrification would not be open to this objection, however, if the writer’s experiments with zinc oxide may be used as an indication. The accumulated nitrate had no noticeable effect on the amount formed; nor could it be expected to have, a priori, in the sense that the reaction, being photochemical, is hardly subject, according to present views, to the mass law.
Summary Some five hundred photosynthesis experiments with systems involving ammonia and various carbonaceous substances, including carbon dioxicic, formic acid, formaldehyde, and glucose, were performed. Sunlight condensed through twelve-inch lenses, in combination with colored inorganic catalysts, was used, the exposures being made in very thin glass vessels. Only one type of photochemical change with ammonia was observed ; in. the presence of ferric chloride, ammonia was oxidized to nitrates; in the presence of zinc oxide and mercuric oxide to nitrites and nitrates. Hydroxylamine was never produced. ‘Rice Inst. Pamphlet, 12, S o . I , p. 8s. * Col. Exp. Sta. Bull., 155, 178, 179, 186, 193, 239, 258, 299, 277, 291.
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KO photochemical reduction of carbonic acid was observed. No complex biochemical nitrogen compounds were produced from ammonia and carbonaceous substances. I t is felt that owing to the enormous range of concentrations of sunlight used, number of experiments performed, variety of conditions maintained, carefulness of technique employed, involving the necessary precautions to avoid contamination by organic matter, and the extensiveness of the methods of analysis, considerable doubt has been thrown upon the positive photosynthesis results of Moore, Dhar and Sanyal, and others, Jvho have employed sunlight and inorganic catalysts also. The writer wishes to express appreciation of the advice and assistance given by Professor Dennis R. Hoagland and Professor Charles ITr. Porter throughout the course of this investigation. Davasaon of Plant Sutrztaon and Department o j Chemastry, rntaerszty o j Calzfornaa, Berkeley.
