298 I~HlLlPS TECHNICAL REVIEW VOL. 1<1-, Ne. 10

Summary. Starting from the supposition that' the voltageproduced by an oscillator is approximately sinusoidal and thatthe amplitude-controlling mechanism has no effect upon thephase of the oscillator voltage, the phase shift of that voltagedue to an interfering pulse has been calculated.i'I'his is followedby an elementary explanation of the synchronization of anoscillator by a periodic disturbance differing in the durationof its cycle from that of the oscillator. By solving a differential

. equation the phase and frequency variations under the in-

fluence of a weak sinusoidal interference are calculated. Asthe strength of the interference increases, or the frequencydifference decreases, the solution passes continuously into thestate of synchronization. Further, the non-linearity of thephenomena is discussed, i.e, the dependency of the effect ofan interference upon the presence of other interferences. Thisis followed by an explanation how the method followed canbe applied for calculating the influence of noise voltages in the.oscillator upon the frequency constancy .of the oscillator.

PHOTOSYNTHESIS

by R. van der Veen. 581.132.1

In the final analysis, al1life on this earth depends on the transformation of sunlight intochemical energy. This process, called photosynth;sis, takes place exclusively in green plantsand even there the output is small. If in this respect mankind could succeed in making itselfindependent of tbe vegetable world, the prospects for the energy provision of the whole worldwould be very favourably influenced. If we really intend to reach this goàl it is important toobtain a fuller insight into the complicated process of photosynthesis; at present only the[undamental principles are known. It has been found that we can contribute to our knowledgeby studying theJlrtorescence ofliving elements oJthe vegetable world.

The energy radiated from the sun to the earthamounts to about 1018 kWh a year. The energyconsumption in a country as highly developed asthe USA amounts only to about 1013 kWh a year,while the figure for the whole world is only 3 timesas large as that for the USA. By far the largest partof the energy used by mankind has been derivedsince time immemorial from the coal and oildeposits found in the earth's crust, formed long agoby solar energy. These deposits however are notinexhaustible, and assuming that present-day tech-nical development proceeds at the same speed, ithas been calculated that after some centuries thesedeposits' will have been completely consumed. (Ifwe tried to replace the solar energy supplied to usevery day by the energy stored in -the oil and coaldeposits we would consume the whole stock with-in 3 days!)

As might be expected science is already carryingout experiments to .compensate the effects of thisdepletion. A modest step in this direction has beenmade by using atomic energy released from thoriumand uranium but the available quantities of theseelements are limited. If we could succeed in trans-muting hydrogen into helium in a technically work-able process, we- certainly would postponc the dayof exhaustion for many hundreds of thousands of

years, but to achieve this we have still a long wayto go.lt is obviously desirable to exploit the possibility

of making direct use of solar energy. A small per-centage of the energy in the world is generated inthis way, viz. by making use of wind and waterpower, but it is improbable that the amount ofenergy so obtained can be considerably increased.Now, it is known that part of the total solar energysupplied is transformed by green plants into chemi-cal energy. This 'process - photosynthesis - is thebasis of all life and consists of the formation ofcarbohydrates from water and the carbonic acidof the atmosphere by the influence of sunlight. Thisphotosynthesis results in the accumulation ofenergy to an amount of 1015 to 1016 kWh a year.However, this energy is lost again for the greaterpart without being used to an appreciable 'extent bymankind, by decomposition of the organic sub-stances through bacteria, etc.If we look at these figures it is evident that all

our worries about the exhaustion of our energydeposits would disappear at once if we could onlysucceed in making a more efficient use of thisphotosynthesis. The first step in this direction is toobtain an insight into the very complicated systemof reactions which constitute photosynthesis.

APRIL 1953 PHOTOSYNTHESIS 299

Photosynthesis

Photosynthesis is the most important chemicalreaction taking place on earth. All life is dependen ton the chemical energy stored in its own organicsubstance. The processes which govern life are keptgoing by the release of this energy by respiration, i.e.by the combustion of organic substance. Conse-quently a primary condition for the conservationof life is the formation of organic matter in one wayor another. This formation takes place mainly by asingle method, viz ., by the influence of sunlight ona certain substance present in green plants -chlorophyll. (Certain organisms capable of usinglight for building organic substances are not greenbut close examination has shown that they do con-tain chorophyll, the green colour being suppressedby other colours.)It is remarkable that the higher developed beings

such as the animals, are unable to make direct use ofphotosynthesis. Not even in the laboratory has thisproved possible. This means that all the higherdeveloped creatures including mankind have to liveparasitieally on the green plants.

In general the ehlorophyll present in the leavcsof a plant is in the form of granules or ch l 0 r 0 -p l a st s, moving about in the plasma of the cell.In jig . .1 an electron micrograph of some cbloro-plasts is shown. It is clearly to be seen that each

•ft

Fig. 1. Electron micrograph of chlorophyll granules (chloro-plasts ), The distance between the black squares in the right-hand top corner = 1 fL. It can he seen that the granules arebuilt up from small corpusculae, the grana, embedded in thecolourless stroma.

chloroplast consists of a number of lenticularbodies, the gr a n a, embedded in a colourless gel,the stroma. Fig. 2 shows a fragment of a chloro-plast at a higher magnification.

The photosynthesis process as such takes placeinside the chloroplasts, Schematically, photosyn-

thesis can be represented by the reaction:

Though this formula looks very simple, the processthat really happens is extremely complicated and

Fig. 2. Electron micrograph of a fragment of a cborophyllgranule. Four grana are visible, embedded in a patch ofstroma. The distance between the white squares in the rigbt.hand bottom corner = 1 [J ••

••

up till now it has been only partly unravelled. Thcfinal product, represented by (CH20), and whichshould be read as a collection of higher carbohydrates(sugars) is the organic substance which suppliesthe energy for the conservation of life.

Thus in fact photosynthesis consists of ther edu ct ion of CO2 to cabohydrates, whereas thcrespiration process corresporids to the mversereaction, i.e. the oxidation of carbohydrates toCO2•

Mechanism of the reaction.

It has been found that the chloroplasts alonecannot effect a photosynthesis of carbohydratesfrom CO2 and water. After having been exposed tolight, the chloroplasts do possess a certain reducingpower, as can be concluded from the rednetion ofeasily reducible snbstances as e.g. ferric salts toferrous salts, during which process oxygen is liber-ated. In orderto reduce carbon dioxide, however,a preliminary treatment of this substance is neces-sary. This preliminary treatment takes place in theprotoplasm, independent of the light; for thisreason it is called a dark-reaction. The lightreaction proper, coupled with a number of dark-reactions, takes place in the chloroplasts.From the foregoing it is already evident that the

photosynthesis process as a whole is infinitely morecomplicated than is suggested by the simple equat-

300 PHILIPS TECHNICAL REVIEW VOL. 14, No. 10

ion givèn above..To clarify the picture - without have succeeded in gaining a, better insight into'undue complication - jig. 3 illustrates how the the various reactions which take place ..reaction system looks according to present-day' The first author has shown that under specialknowledge. . circumstances it is' possible to effect the CO2 con-

In many [largely unknown] intermediate stages - version by the chloroplasts, by adding diphos-the, CO

2fr0lf- the atmosphere is 'converted into a p h op yr i.d ine n u cl eot ide (ElPN) to a suspension

Light

76046

Fig. 3. Schematic reproduetion of the photosynthesis process. The dark-reactions are onthe left. On' the right is the reduction of H202 .to H20 and O2 by the enzyme catalase. Theactuallight reaction in the chloroplast narrows down to the decomposition of water into an(H)-component and an (OH)-component. All the oxygen which finally escapes originatesfrom the water, not from absorbed CO2,

complexity of unknown chemical compounds, indi-cated in the scheme of fig. 3 as [cg]. At the sametime water is decomposed by the chlorophyll-protein complex of the chloroplasts under the influ-ence of light into a reducing component, indicatedby the symbol (H) and an oxidizing componentindicated by (OH). It has not yet been agreed as towhat is meant exactly by these compounds; it ispossible that we have to deal with H-atoms andOH-radicals.

The (H)-compound serves to reduce the [cg]- complex, formed in the dark-reaction, to carbo-hydrates and water. The (OH)-components pro-bably combine to H202-molecules, which, under theinfluence of the enzyme catalase (a substance,which has been isolated and is present in a relativelyhigh concentration in every chloroplast) decomposeimmediately m water and oxygen. The oxygenescapes.

According to this scheme, the oxygen whichfinally escap!!soriginates entirely from the wat e I'in the plant. From the symbolic equation atthe beginning of this paper one might expect thathalf the liberatèd oxygen - or even more -originates from the CO2, but experiments carriedout with the oxygen isotope 018 have completelydisproved this simple conception.

Much research work has been carried out toclarify those details of the photosynthesis which are.still obscure. Among others, Oc h o a ") and Oalv iu ê)

1) S. Ochoa, Symposia Soc. Exp. BioI. 5, 29, 1951.2) M. Calvill, J. Chem. Educ. 26, 639, 1949.

of chloroplasts. DPN is a complicated, phosphorus-containing organic substance, which seems to bepresent in all live cells and which is reducible.However, when other reducible substances andcertain enzymes are present, it is easy to re-oxidizethe reduced DPN. Ochoa showed that in thepresence of reduced DPN and certain enzymes,CO2_can combine with pyruvic acid to form malicacid. During this reaction the reduced DPN isoxidized, but it is immediately reduced again by thechloroplast which has been exposed to light. Thus,in a sense, the DPN acts as a catalyst. By the com-bined action of chloroplast, light energy, DPN andenzymes, the reaction always proceeds in the samedirection, i.e. in the direction of the reduction ofCO2 to carbohydrates.

By making use of radio-active carbon, Cal vin hasproved, however, that the CO2,fixed during thephotosynthesis is first found, not in malic acid, butin the form of phosphoglyceric acid. After sometime the radio-active C-atoms spread over a largenumber of other compounds. It may well be thatthese results are not opposed to those of 0 cho a:in the sense that both 'results are based on thereduction of DPN by the chloroplasts. It is inter-esting to note that organic phosphates are necessaryto effect photosynthesis, a fact which has beenestablished by Wassink, Winte'rmans andTjia3).

3) E.C. Wassink, J.F.G.M. Winter mans and J. E. Tjia,Proc. Kon. Akad. Wet. Amst., Series C, 54, 4.96-502,1951(No. 5).

APRIL 1953· • PHOTOSyNTHESIS 301

The question àrises - why is the course of thephotosynthesis reaction so extremely complicated?To . answer this question it should be realizedthat in nature only those processes which lead tothe, final product in an efficient manner have achance to 'succeed. In order to effect so radical areaction as the synthesis of carbohydrates by meansof the relatively small energy (light and heat)available, nature has to rely on subdividing thereaction into a very large number of intermediatereactions, the direction (sense) of which can .beeasily influenced by the circumstances. Now thepresence of light and certain enzymes results in aslight shift of the equilibria in the plant cellstowards. the reducing direction. This requires onlya very small energy-absorption per process. More-over, it should be taken into account that thecompounds formed not only play a part in thereactions considered, but also have very definitefunctions in the organisms, which results in theincrease of the "efficiency" of the entire process.

Liglu. saturation

The speed of photosynthesis is determined by theslowest reaction in the chain of the light and dark-reactions. The rate of progress of the light-reactionincreases with the increase of the light intensity.In the temperature region where physiologicalprocesses can normally take place, i.e. betweenabout 5-30 °C, this speed is almost independentof the temperature. The dark-reactions, on thecontrary, are pure chemical reactions, of which the

73818

-I

Fig. 4. Speed v of photosynthesis as a function of the illu-mination intensity I and the temperature. The light-reactiondetermines the speed in the sloping part of the curve, whereasthe speedof the reaction is completely limited by the dark-reactions in the horizontal part, where light saturation sets in.The rate of progress of these dark-reactions increases as thetemperature becomes higher. The light-reaction is almostindependent of the temperature, the result being that lightsaturation at a higher temperature sets in only at a higherlight intensity.

rate of progress rapidly increases with the tempera-ture. At a given temperature an increase of thelight intensity will result in an increase of photo-synthesis, but only as long as the light-reaction isthe limiting factor. When one of the dark-reactionsbecomes limiting, an increase of the light intensitycan no longer cause a change in the rate of photo- .synthesis. light saturation sets in. The higherthe temperature, the greater the rate of progressof the dark-reactions, and light saturation will set .in at higher intensities. This has been represented'schematically in jig. 4.

Analysis of the light-reaction hy means of fluor-escence

As already demonstrated, only the fundamentalprinciples of the process of photosynthesis areunderstood at present. The transitions which takeplace in the dark-reactions are still unknown andso is the light-reaction. But there is a possibilityof becoming better acquainted with the latter.

Only part of the absorbed light energy is trans-formed in chemical energy during the light-reac-tion. In nature the output is only a few percent,whereas it has been possible to obtain an output ofof about 30%or more in the laboratory when wor-king with low light intensities. The remainder ofthelight energy is transformed into heat and for asmall part into fluorescent radiation. Thelatter fact is most fortunate, for by studying thefluorescence, it is possible to follow the course of thelight-reaction to a certain extent. Itmay be assumedthat the relation between the energy given off asheat and the energy given off as fluorescenceis always constant, whatever the percentage ofthe absorbed radiation energy used for thechemical transformation. Consequently the intensityof the fluorescence is a yardstick for the quantityof light energy bound chemically, and so for theoutput of the photosynthesis: the intensity ofthe fluorescence will be smaller according as theoutput is higher.It is evident' that the measurement of the inten-

sity of the fluorescence can teach us only about theprogress of the light-reaction. As far as the progress'of the dark-reactions (including those following thelight reaction) are concerned, this method will bringus no further. It may well be that part of thechemically bound energy is liberated in a l~ter stageof the process as heat. This is'the case e.g. if a leaf is'exposed to light in the absence of CO2, In that casethere is no material that can be reduced and nooxygen is set free. However, the fluorescence ishardly any higher than it would be if CO2 were

VOL. 14., No. 10302 PHILIPS TECHNICAL REVIEW •

present. Thè fundamental light-reaction, i.é. thesplitting-up of water into (H)- and (OH)-compounds, obviously takes place at the same rate,the presence or absence of CO2 being immaterial.However, CO2 being absent, these compounds com-bine again to water, during which process heat isgenerated a,nd from a chémical point of view, it. appears that nothing has happened.

The Philips Laberatory has carried out extensiveresearches on the phenomena which occur at the"begin n in g of the illumination in the processof photosynthesis. In particular the CO2-absorptionand the fluoresence were studied 4).

c

I13819

7min_t

F

i

Fig. 5. a) Progress of the CO2-absorption C per unit of time.b) Intensity of the Iluorescence F, as function of the time tafter applying the illumination. With the exception of thevery early part, the two curves are nearly reflected images.

The general course of the CO2-absorption isillustrated in fig. Sa. 'During the first moments(about 10 seconds) rather a large quantity of CO2 isabsorbed, but then the rate of absorption decreases. quickly. About a minute after illumination isstarted, there is a gradual increase of thephotosynthesis (adaptation period), until analmost constant level of the CO2-absorption isreached, the height of the level being dependent on, the intensity of illumination and the plant in ques-tion. The fluorescence shows, apart from the earlybeginning period, closely corresponding variations

4) See, for example, R. van der Vee n, Physiologia Plant a-rum 4, 486-494 1951 (No. 3).

, (fig: Sb): the CO2 absorption at the bèginning isreflected in a short-period decrease, and the adap-tation period as a continued gradual decrease.Fluctuations in the photosynthesis which may set

la r ,

F

1

\\ 25°C\.._.,."",------------

'181J _t

Fig. 6. Intensity of fluorescenceF as function of the time tafter applying the illumination, at two temperaturcs, 13°Cand 25 °C.Though the ultimate level is the same in both cases,it is attained much earlier at the higher tcmperature. Themeasurements have been carried out on a dahlia-leaf.

in later are completely reproduced in the intensityof the. fluorescence. In the very early part, however,the fluorescence shows a sharp peak, the relation ofwhich to the photosynthesis is still obscure.

The influence of the temperature, the light inten-sity and the duration of the dark period precedingthe illumination has been investigated. In theearly stages the rate of photosynthesis is muchlower at low temperatures (say, 13 CO)than at high

100

~F 11

i 'I "...1 / <,,( / ",'1/ -,: ti -,

-,<,,-----~----

73821 -t

Fig. 7. Intensity of fluorescenceF as function of the timc tafter applying the illumination at two intensities, differingbya factor 3. Curve 1 appertains to the low intensity, curve 2 tothe high one.Measurements have been carried out on a tomatoleaf.

·APRIL ·1953 PHOTOSY~THESIS 303

temperatures (25 °C). This is reflected in the highvalue of the fluorescence at 13°C as compared withthat at 25 °C, during the first minutes (jig. 6J.It can be seen, however, that the ultimate level ofthe fluorescence is not influenced by the tempera-ture. This confirms the assertion that the lightreaction depends little on the temperature.

The intensity of the fluorescence increases whenthe light intensity is increased, as would be expected.It is remarkable, however, that the increase is muchmore. marked when the illumination is first begunthan in later periods (fig. 7J.

100

73822-t

Fig. 8. The influence of the length of the preceding darkperiod on the fluorescence curve. The figures against eachcurve represent the length of the period in minutes. Themeasurements were carried out on a tomato leaf.

The influence of the preceding dark period isrepresented infig. 8. The longer theperiod, the higherare the various maxima. This agrees completely withwhat has been found by other, more direct means.It seems that the plant remembers the previousillumination, provided' this took place not longerthan 20 minutes previously.It is impossible as yet to have a clear picture of

the physical and chemical background of all thesephenomena, but it is certain that the study offluorescence is of material aid to investigatiomsinto photosynthesis.

I

Summary. Photosynthesis, the basis of alllife on earth, takesplace only in green plant parts. The substance called chloro-phyll is an essential link in the transformation process oflight into chemical energy. The process of photosynthesis,cssentially consistingof the reduction of CO2 to carbohydratesand accompanied by the liberation ofoxygen- and as suchto beconsidered as the inverse of the respiration process - is verycomplicated. The actual light-reaction takes place in thechloroplasts of the plant, but it is preceded by a large seriesofdark-reactions, occurring partly in the protoplasm, whicheffect a preliminary transition of the CO2 into an easilyreducible substance. The light-reaction can be considered asthe splitting up of water, under the influence of light, into areducing and an oxidizing compound; the former effectingthereduction of CO2 to carbohydrates and water, and the latterultimately liberating oxygen. It has been found that the pre-sence of organic phosphates in the dark-reactions is veryimportant. The output of the photosynthesis in nature is onlya small percentage, whereas in the laboratory, at low lightintensities, an output of 30% and higher has been attained.A large part of the absorbed light energy is lost as heat, and asmall part as fluorescent radiation. By studying the fluores-cence of the green parts of plants, science has succeeded ininvestigating the early part of the light-reaction. The resultsof the fluorescence research indicate that the intensity offluorescence is often a measure of the process of photosyn-thesis."