Nitrate Decompsn

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THE MECHANISM OF REDUCTION OF NITRATES ANDNITRITES IN PROCESSES OF ASSIMILATION.

BY OSKAR BAUDISCH.(From the Department of Chemistry, Yale University, New Haven.)

(Received for publication, July 30, 1921.)The mechanism of reduction of nitrates of the alkali metalsto ammonia and the formation of amino nitrogen in biochemicalsyntheses from inorganic nitrogen compounds have not beenexplained satisfactorily. Some investigators explain the trans-formation by assuming a direct reduction of the nitrates to am-monia by action of nascent hydrogen, while others assume anintermediate reduction of the nitrate to nitrite, from which, as thereduction proceeds, ammonia is produced. The exact chemical

procedure by which bacteria or molds are able to produce ammoniaor nitrites from alkali nitrates also has not been satisfactorilyexplained.Schimper,l the botanist, has been able to demonstrate experi-mentally that the reduction of nitrates in green leaves is connectedintimately in some manner, not only with the influence of light,but also with the action of iron compounds in the leaves. Thisobservation was so interesting to the writer that it led him to aninvestigation of the question whether iron actually takes partin the reduction of nitrates by means of bacteria. This workhas proved very productive and, as a matter of fact, experimentshave shown conclusively that the cholera bacillus, which possessesextraordinary reducing power for nitrates, has the ability toaccumulate iron2 in its organism, and its reducing power maypossibly be a function, not only of its ability to absorb oxygenby respiration, but also of its iron content.The data revealed through this biological research, which wasinterrupted by the War and political disturbances in Europe,

1 Schimper,A. F. W., Bot. Z., 1890, xlv i, 73.2 Unpublished data. 489

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490 Nitrates and Nitrites in Assimilationwarranted a purely chemical investigation of the reduction ofinorganic nitrates by means of iron salts. The opportunity tocontinue this work has now been offered to me, and, as a result,the study of this interesting problem has been taken UP.~

An interesting paper by Menaul which recently appeared in thisJournal,4 brings up for discussion a very important biochemicalchange. This investigator describes the action of formaldehydeon saltpeter in aqueous solution when exposed to sunlight, and theobservation is made by him that in such solutions small quantitiesof hydrocyanic acid can be detected easily. This quite remarkableaction of sunlight on nitrates was observed by the writer severalyears ago5 and this preliminary paper is now contributed to bringthe results of this work before the American reader, and at thesame time to present a summary of the principal results of hisearlier investigations on the photochemical reduction of nitratesand the reduction of nitrates and nitrites with iron salts, whichhave appeared in various scientific publications during the last10 years6 This summary will be presented in three parts asfollows: (1) Reduction of nitrates of alkali metals by means oflight and also iron salts; (2) Reduction of nitrites of alkali metalsby means of light and also iron salts; and (3) Synthesis of organiccompounds containing nitrogen from inorganic compounds ofnitrogen.

The Reduction of Nitrates.In the course of researches on the photochemical decomposi-

tion and synthesis of nitrates and nitrites, the observation wasmade, for the first time, that one oxygen atom in nitrates must bebound in the molecule in a manner quite different from that ofthe oxygen atoms in nitrites. The writer has used the specificterm nitrate oxygen atom? to designate that oxygen atom which

a Through cooperation with Prof. Treat B. Johnson, who has suppliedrare research material, it has been possible to extend the field of investi-gation into the pyrimidine and purine series. The results of these re-searches, which are of immediate biochemical interest, will be publishedat a later date.4 Menaul, P., J. Biol. @hem., 1921, xlv i, 297.5 Baudisch, O., Ber. them. Gee., 1916, xlix, 1151.BBaudisch, O., Ber them. Gee., 1911, xliv, 1009.r Baudisch, O., Ber. them. Ges., 1912, XIV, 2879; 1916, xlix, 1176.

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0. Baudisch 491ia easily split off from such salts either under the influence of lightor of metallic iron. That we are dealing here with such a labileoxygen linking in nitrates is demonstrated to us by nature, in thatthe various nitrifying and denitrifying bacteria have the powerto differentiate between nitrate and nitrite oxygen. The questionnaturally arises, under what influence or by means of what poweran atom of oxygen can be split out of potassium nitrate, for ex-ample, with the formation of a nitrite. This problem was firstattacked from a purely chemical standpoint, but it is now proposedto continue the study also from a biological point of view.

In explanation of the photochemical reduction of nitrates inaqueous solution, we may assume, according to Werners theory8of reaction, an activation of the residual valence of an oxygenatom of the nitrate and of the oxygen atom of the water, resultingin the attraction of molecules of water into the inner sphere ofthe nitrate molecule. There follows a dissociation of the nitratemolecule with the formation of oxygen as is expressed by thefollowing equation:

In other words, this reduction process takes place under the influ-ence of light even in an atmosphere of oxygen, and stops at thenitrite stage. The reverse process can also take place and nitriteis readily formed from ammonia by photochemical oxidationwithout, however, any production of nitrate.

It is apparent from these facts that light can readily split off anatom of oxygen from nitrates of the alkali metals, without thepresence or influence of either nascent or molecular hydrogen.This dissociation of nitrate into oxygen and nitrite can also bebrought about by means of metallic iron as well as under theinfluence of the energy of light. If a neutral oxygen-free solutionof potassium nitrate be shaken in A vacuum with active iron pre-pared by reduction with hydrogen, the supernatant liquor obtained

8 Werner, A., Neuere Auschauungen auf dem Gebiete der anorganischeChemie, Brunswick, 4th edition, 1920.

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492 Nitrates and Nitrites in Assimilationafter the iron powder has been allowed to settle will give everyreaction applicable for the detection of nitrous acid.s In otherwords, metallic iron will easily reduce potassium nitrate to potas-sium nitrite in the cold in the absence of every trace of oxvgen,qnd under conditions such that neither the action on the iron bywater nor the effect of nascent hydrogen can possibly come intoconsideration. These results lead to the assumption, therefore,that iron readily splits off an oxygen atom of the nitrate afterhaving first entered into a loose combination with it. Thischange may be expressed by the following equation:

+ Fe __) K N . 0 ._.___....] -+KNO*+FeO.

From these examples it is seen hat the photochemical reductionof nitrates to nitrites and their reduction by means of metalliciron are similar in nature, and in both cases there occurs eitheran activation or mobilization of the valence energy leading tothe formation of an unstable addition product, which finallybreaks down into the final products of reaction. Although ithas been possible to show a relation between the photochemicalreduction of nitrates to nitrites and the corresponding biologicalreduction in green leaves, it has not yet been possible to connectknown chemical reduction processeswith the biological reductionsoccurring naturally in bacteria or in molds.It formerly appeared scarcely possible to attach any biologicalimportance to ferrous hydroxide in these reduction processesbecause, as was commonly believed, the reaction proceededstoichiometrically and ferric hydroxide was formed at the expenseof the oxygen split off from the nitrate. In biological processes,however, one cannot speak of stoichiometrical reactions in connec-tion with metals, because, as s well known, they are present only intraces. Their action must be explained, on the contrary, by aparticular energy inherent in tlieir molecules, and consequently thewriter has been accustomed to consider the metals as function-ing in biological processeseither as mobilizers or catalysts,

0 Baud&h, O., Ber. them. Ges., 1921, liv , 406.

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0. Baud&hhaving the power to bring into play the latent energy of cer-tain organic molecules.

From the point of view of Werners coordination theory, it ispossible to conceive of a relation between the mobil izing powerof such a catalytic agent, and the peculiar power expressed byresidual valency, which, as is well known, has the ability to drawnew atoms, molecules, and radicals into the sphere of action ofthe internal nucleus of the metal, resulting in the most variedtypes of reactions. In fact, it is well known that extremely finelydivided metals, such as platinum, palladium, or iron, possess thesevalence powers to a large degree, and their specific action hasoften been placed in parallel with purely biological processes.

The reduction of nitrates with ferrous hydroxide assumed anew interest when it was discovered that this reagent alone doesnot split off nitrate oxygen as had been ari;sumed, but reducesnitrates only under the influence of oxygen.lO As it is well knownthat white ferrous hydroxide is converted instantaneously intogreen ferrous hydroxide peroxide by. the oxygen of the air, itis reasonable to say, therefore, that this polymolecular combina-tion or peroxide is the active reagent which brings about thistransformation of nitrates into nitrites.

02

[ 1e (OH) a(OH3o(Coordination formula for ferrous hydroxide peroxide.)The mechanism of this reduction has not yet been explained,but the attempt will be made here to show that the free energyof ferrous hydroxide is increased enormously by a loose combina-tion with an oxygen molecule, and that this increase in energymakes itself apparent both physically and chemically. The strik-ing effect on the color of white ferrous hydroxide, which is causedby the smallest trace of oxygen, shows that the oxygen entersint*o the inner sphere of the iron nucleus. Schiiferll has provedspectroscopically that such extraordinary alterations in color,either in the visible or in the invisible part of the spectrum, canonly take place simultaneously with changes in the inner spherelo Baudisch, O., Ber. them. Ges., 1921, liv , 410.*l Schiifer, K., 2. anorg. Chem., 1918, lxxxvi, 221.

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494 Nitrates and Nitrites in Assimilationof the molecule. It has, however, not yet been shown how manyoxygen molecules are present in such a molecular combination,It may be possible that the observation of Meyer,12 who dis-covered that strongly magnetic substances were rich in absorp-tion bands, whereas diamagnetic substances were poor in absorp-tion bands, has some connection with these facts. This coincides.completely with the action of the above mentioned iron compound,because, while the white ferrous hydroxide has practically no mag-netic properties, the green to black ferrous hydroxide peroxide pos-sesses, magnetic properties which are almost equal to that of me-tallic iron. According to Hilpert,la when a stream of air or oxy-gen is led through a precipitate of ferrous hydroxide, the magneticproperties of the precipitate increase rapidly. QuartarolP has alsoshown that oxidation with air converts the ordinary non-magneticferrous hydroxide into mixed ferro-ferri oxides, which possess a sus-,ceptibility almost a hundred times greater than ferric salts. Infact the magnetic susceptibility of Fea04 approaches that of themetal iron itself. It may be concluded, therefore, from our presentknowledge, that there is a very closerelationship between the phys-ical properties of metallic iron and those of ferrous hydroxideperoxide, and it will be of the greatest interest and importanceto determine whether there is also any direct relationship betweenthe peculiar chemical properties of this peroxide and those of finelydivided metals. The simplest explanation, therefore, for the reduc-tion of nitrates is the assumption that freshly precipitated, colloi-dal ferrous hydroxide peroxide acts catalytically as a fi;lely dividedmetal.It seems very probable that the peculiar properties resultingfrom the colloidal nature of ferrous hydroxide peroxide and theproperties of the metal resulting from its position as the centralatom of a complex system actually coalesce. Furthermore, thesecharacteristic properties apply only to the peroxide and not toferrous hydroxide, because the latter compound is not only unableto bring about a reduction of nitrates, but also will not react toform polynuclear compounds. Not until brought under theinfluence of oxygen does the central iron nucleus of ferrous hy-

19Meyer, S., Pied. Ann., 1899, Ixviii , 325.l* Hilpert, S., Ber. &em. Gee., 1909, xlii, 2248.I4 Quartaroli, A., Chem. Zentr., 1917, i, 729.

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0. Baudisch 495droxide or other ferrous salts acquire the property of attractingnew molecules of ferrous hydroxide into its inner sphere. Thiscoordination combination exists, according to Werner, not betweeniron and iron, but between the active hydroxyl oxygen atomsof the ferrous hydroxide, which are attracted to the central ironnucleus of the peroxide by its residual valency. It is also possiblefor the iron atoms of ferrous hydroxide to be held in combinationthrough the peroxide oxygen atoms, as has been demonstratedin the case of cobalt compounds by the classical researches ofWerner. The mechanism of the autooxidation of ferroushydroxide and the formation of strongly magnetic Fe304.zH20 maybe expressed by the following formulas:[ e(OH,)e 1OH)z + 0, -+ Fe[ 1OH,)2 (OH), + 2Fe (OHIs6

(OH)2

The structure of this polynuclear combination may be expressedgraphically as follows :

HO,y. . /p,,

HO

. ..\...,,,b;Fe . ..\.\.

OHaIH

From these graphic representations it is seen that ferroushydroxide is converted by the absorption of oxygen into a per-oxide of greater potential energy, whose iron nucleus, as experiencehas shown, possesses the property of intensifying the activity ofand of entering into loose combination with the residual valenceof oxygen atoms in other molecules of ferrous hydroxide present.

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496 Nitrates and Nitrites in AssimilationWith the coordination theory of Werner as a basis, it becomes ap-parent from the foregoing that ferrous hydroxide may be trans-formed by the absorption of oxygen into a complex salt whoseiron nucleus, just as finely divided metals, may enter into a widerange of reactions. A nitrate oxygen atom may be split off aswell by means of light as by means of metallic iron or by ferroushydroxide peroxide. All three of these processesof reduction maybe considered to depend upon the same principle; namely, themobilization or activation of the energy in the residual valenceof the reacting materials Ferrous hydroxide peroxide reactsmost probably in a very similar manner to finely divided ironor platinum.

The Reduction of Nitrites.Aqueous solutions of potassium nitrite containing easily oxidi-zable substances, such as alcohols, aldehydes, sugars, starches, etc.,

suffer a comparatively rapid reduction and decomposition underthe influence of diffused daylight, and the change may be expressedvery simply as follows:KNO s=KNO+O.

The presence of potassium nitrosyl in solution may be detectedby means of its condensation reaction with aldehydes (Angelisaldehyde reaction) .15 Hydroxamic acids are formed as productsof reaction, and as is well known, these acids give characteristiccomplex salts with iron which are colored a deep reddish violet.This reduction of potassium nitrite to potassium nitrosyl can alsobe accomplished by means of complex iron salts. The smoothreduction of potassium nitrite via potassium nitrosyl to ammoniaby means of glucose, in the presence of very small quantitiesof iron, possessesparticular biological interest. The system,glucose + iron + alkali, which is a fundamentally new reducingcombination, does not attack in the least the alkali salts of nitricacid.16 It is therefore possible to make a quantitative separation

Is Angeli, A., Samml. Chem. u. them. Techn. Vortr., 1908, xiii.16A method, ba.sed on this observation, has been developed for thequantitative determination of nitrites and nitrates in the presence of othernitrogen compounds in soil extracts. PfeZer, T., and Simmerbacher, W.,Land. Versuchsstat., 1916, xciii, 65; J. Sot. Chem. Ind., 1919, xxxviii, 507.

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0. Baudischbetween nitrate and nitrite by means of a grape sugar-iron-alkalisolution. Grape sugar, which is absolutely free from iron, does notcause the slightest reduction, even on heating, of nitrites; and alsochrysarobin (1 ,%dioxy-3-methyl-anthranol), which occurs quitewidely in the vegetable kingdom, has not the ability of reducingthe salts of nitrous acid. On the other hand, the addition of tracesof any iron salt to an alkaline solution of either of thesesubstancesenables them to reduce immediately the nitrites by way of nitrosylto free ammonia. The particular part that iron plays in thesereactions remained a mystery for a long time, but seems now tohave been explained quite satisfactorily.

To explain this reduction process andits application for the quan-titative separation of nitrates and nitrites, we may assume thatthe unsaturated, trivalent nitrogen atom of the nitrite moleculeenters, through its activated residual valency, into a loose com-bination with the central iron nucleus of whatever complex saltis present, and then dissociates through the intermediate forma-tion of nitrosyl into NO and K. The residual valency of thenitrate oxygen atom is not suhiciently active or powerful to dis-place the molecules or radicals already present in the inner sphereof the iron nucleus, and therefore these compounds are not at-tacked. That these reductions may all be considered as complexsalt reactions, or in other words nuclear exchange or displace-ment reactions may be shown by the following example: Asolution of 1 gm. of KJ?e (CN),, 1 gm. of NaN02, and 5 gm. ofsodium carbonate in 200 cc. of water, is distilled in a stream ofoxygen. After interrupting the stream of oxygen, it is possibleto detect nitrous acid in the distillate. The oxygen, under theinfluence of heat, has displaced the cyanogen group from the innersphere, and in its place a molecule of nitrite has entered. Thenitrite, however, decomposesand its scission product, NO, whichat first takes its place in the inner sphere of the iron nucleus, isin turn displaced by the oxygen, and finally passesover into thedistillate where it is easily detected as nitrous acid.The fact that this remarkable reaction may not only be in-fluenced by daylight, but in some caseswill not take place exceptunder the influence of daylight, is of particular chemical andbiological interest. For example, if a freshly prepared solutionof potassium ferrocyanide be treated with an aqueous alcohol

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498 Nitrates and Nitrites in Assimilationsolution of nitrosobenzene and placed under the influence of dif-fused daylight, the solution which in the beginning possesses aweak, greenish yellow color, changes in a few minutes to a deepreddish violet. The mechanism of the reaction may be expressedas follows:1 1e(CN)a Kq + CaHbNO + ~e~~b~o]K8+KCN.

On treating the aqueous solution of.this reddish violet compoundwith an excess of potassium nitrite and again placing the solutionunder the influence of diffused daylight the following decomposi-tions take place:KNOa + KNO+OKNO ------f K+NO

HzOfK - KOH+H

The deep violet color of the solution disappears very rapidly andthe reddish yellow or potassium nitroprusside takes its place.By means of this reaction, the reduction of sodium nitrite by meansof a complex iron salt and light is demonstrated.These processesof reduction of nitrites by way of nitrosyl toammonia, may be drawn into intimate relation with biologicalreductions of nitrite, particularly as sugar, or its products ofdecomposition, and iron constantly accompany the nitrates inplants or in bacteria. Kostyschew and Tswekowal state thatthe reduction of nitrate to nitrous acid takes place without thepresence of any sugar, but that the further conversion of thenitrous acid, at least n the caseof Mucor racemosus, s accomplishedonly in the presence of sugar. It seems ikely, from investiga-tions with cholera bacteria, that nitrates are reduced to nitritesby way of nitrosyl, because t was possible to detect the alkalinedecomposition products of nitrosyl, for example, NO and NH8(the latter as a reduction product of NO), in the volatile portionsof alkaline cholera peptone cultures.l*

17 Kostyschew, S., and Tswekowa, E., 2. physiol. Chem., 1920, cxv, 171.18 Baud&h, O., Ber. &em. Ges., 1916, xlix, 1148.

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0. Baudisch 499The assumption that nitrite is converted into nitrosyl finds a

further support in the qualitative and quantitative compositionof the gases which are produced during the photochemical reduc-tion of nitrites in the presence of formaldehyde and during thebiological reduction. For example, those bacteria which havethe property of decomposing nitrates, produce a fermentationgas which consists of about 65 to 72 per cent Nz0.6 In additionto the nitrous oxide, there is also always formed a little nitrogen,traces of NO, andalso of prussic acid. A formaldehyde-potassiumnitrite solution produces a gas under the influence of diffuseddaylight, which contains 64 per cent NzO, as well as very smallquantities of NO and HCN. The latter two gases were detectedqualitatively by sensitive reactions. Clawson and Younglg havedetected prussic acid in cultures of Bacillus pyocyaneus and ofother bacteria.

To summarize, the photochemical reduction of the alkalinenitrites proceeds by way of the reactive intermediate product,potassium nitrosyl, which may be detected by means of aldehydes,as well in the case of the photochemical reduction of nitrites as-in the case of a reduction with the system, grape sugar + iron+ alkali. Certain complex iron salts possess the property ofreducing nitrites, whereas under these conditions nitrates remainunchanged. The reduction of the alkali nitrites by means ofcomplex iron salts depends most likely upon the residual valencyof the central iron nucleus and in all these changes light as wellas heat exercises a very fundamental influence.Synthesis of Organic Compounds Contai@ng Nitrogen from Inorganic

Compounds of Nitrogen.Nitrosyl, which is formed in the reduction of alkali nitrates,

interacts readily with formaldehyde with formation of formhy-droxamic acid (Angelis aldehyde reaction). This reactionproceeds, as was shown by the writer and Coert,20 through theintermediate formation of nitroso methyl alcohol. Formhydrox-amic acid is then formed from this by molecular rearrangement,and may be detected easily by means of its characteristic ironand copper salts, which are highly colored.

19 Clawson, B. J., and Young, C. C., J. Sol. Chem., 1913, xv , 419.*OBaudisch, O., and Coert, J. H., Ber. them. Ges., 1912, XIV, 1775.

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500 Nitrates and Nitrites in AssimilationIF\ /No

OH/H*CHO+RN0 - C - HC

/\ \H OH NOHFormhydroxamic acid, under the influence of light, loses anatom of oxygen and is converted into formaldoxime, which isalso characterized by its great reactivity. The stable form of

this compound is altered under the influence of light and alsoalkali, and is transformed into an extremely labile modification.This transformation may be expressed as follows:21\ 1

0 . H~=NC-JHf$ H\ IC=N 1OH* I/ I I

\C = NOH + KOH0 _..........

\ IC=N/ ---+ / IH 1 H H _

1OHAs is well known, formaldoxime exhibits a strong tendency topolymerize with the formation of three-carbon chain compounds.Its labile form is also capable of reacting further with aldehydeswith the formation of three-carbon compounds, and furthermore

under the influence of light combines with formaldehyde to formcyclic combinations containing both nitrogen and carbon. Underthe influence of light formaldoxime undergoes, in part, a Beck-mann rearrangement with-formation of formamide, and, in part,a complete dissociation into prussic acid, water, and ammonia,These changes are expressed below:0-m HIH&3 = NOH -- 1 CHs=N 11 OH + H\C-NH+HOI /- aH 1 OH

H\ ~ H * CONHoC=NHHO / HCN+HrO

$1 Baudisch, O., Ber. &em. Ges., 1916, xlix, 1159.

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0. Baudisch 501The small quantities of prussic acid that are always foundaccompanying the treatment of formaldehyde in nitrate solutionswith light, or by the reduction of nitrates with bacteria, mayhave been formed in accordance with the above reaction from thealdoximes. The photochemical formation of nitroso methylalcohol (or hydroxamic acid) from formaldehyde, methyl alco-hol, and nitrosyl may be the chemical counterpart of a possiblebiochemical formation of carbon-nitrogen-containing organicsubstances from inorganic nitrogen. The next step to aminonitrogen is simpler and may either take place by reduction or,as in the case of aldoxime, by simple rearrangement. It seemsprobable that the proof of the biological importance of nitrosomethyl alcohol or formhydroxamic acid is found in its markedreactivity and in the pronounced tendency which it has to rear-rangement, to polymerization, and to the formation of complexsalts, particularly of iron. It seems extremely possible to intro-

duce amino nitrogen into the higher alcohols, sugars, starches,etc., by means of nitrosyl which is formed photochemicallyfrom the nitrites and which is capable of entering into such widelydifferent reactions.

KNOe + Hz0 + CHsOHL--y-Fe(OH), and air/r( A4reduction oxsdataonKNO. HCHO.

synthesisNOH//HC

\ OHand

H,C = NOHRecent worklo has revealed the fact that light may be replacedin certain cases by means of ferrous hydroxide and oxygen. Fer-

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Nitrate Decompsn

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