CI PHOTOSYNTHESIS BY H. A. SPOEHR LABORATORY FOR PLANT PHYSIOLOGY CARNEGIE INSTITUTION OF WASHINGTON American Chemical Society Monograph Series BOOK DEPJ RTMENT The CHEMICAL CATALOG COMPANY, Inc. 19 EAST 24th STREET, NEW YORK, U. S. A. 1926 Copyright, 1926, by The CHEMICAL CATALOG COMPANY, Inc. All rights reserved Printed in the United States of America by J. J. LITTLE AND IVES COMPANY, NEW YORK GENERAL INTRODUCTION American Chemical Society Series of Scientific and Technologic Monographs By arrangement with the Interallied Conference of Pure and Applied Chemistry, which met in London and Brussels in July, 1919, the American Chemical Society was to undertake the pro- duction and publication of Scientific and Technologic Mono- graphs on chemical subjects. At the same time it was agreed that the National Research Council, in cooperation with the American Chemical Society and the American Physical Society, should undertake the production and publication of Critical Tables of Chemical and Physical Constants. The American Chemical Society and the National Research Council mutually agreed to care for these two fields of chemical development. The American Chemical Society named as Trustees, to make the necessary arrangements for the publication of the mono- graphs, Charles L. Parsons, Secretary of the American Chemical Society, Washington, D. C; John E. Teeple, Treasurer of the American Chemical Society, New York City; and Professor Gellert Alleman of Swarthmore College. The Trustees have arranged for the publication of the American Chemical Society series of (a) Scientific and (b) Technologic Monographs by the Chemical Catalog Company of New York City. The Council, acting through the Committee on National Policy of the American Chemical Society, appointed the editors, named at the close of this introduction, to have charge of securing authors, and of considering critically the manuscripts prepared. The editors of each series will endeavor to select topics which are of current interest and authors who are recognized as author- ities in their respective fields. The list of monographs thus far secured appears in the publisher's own announcement elsewhere in this volume. 3 4 GENERAL INTRODUCTION The development of knowledge in all branches of science, and especially in chemistry, has been so rapid during the last fifty years and the fields covered by this development have been so varied that it is difficult for any individual to keep in touch with the progress in branches of science outside his own specialty. In spite of the facilities for the examination of the literature given by Chemical Abstracts and such compendia as Beilstein's Handbuch der Organischen Chemie, Richter's Lexikon, Ostwald's Lehrbuch der AUgemeinen Chemie, Abegg's and Gmelin-Kraut's Handbuch der Anorganischen Chemie and the English and French Dictionaries of Chemistry, it often takes a great deal of time to coordinate the knowledge available upon a single topic. Consequently when men who have spent years in the study of important subjects are willing to coordinate their knowledge and present it in concise, readable form, they perform a service of the highest value to their fellow chemists. It was with a clear recognition of the usefulness of reviews of this character that a Committee of the American Chemical Society recommended the publication of the two series of mono- graphs under the auspices of the Society. Two rather distinct purposes are to be served by these mono- graphs. The first purpose, whose fulfilment will probably render to chemists in general the most important service, is to present the knowledge available upon the chosen topic in a readable form, intelligible to those whose activities may be along a wholly different line. Many chemists fail to realize how closely their investigations may be connected with other work which on the surface appears far afield from their own. These monographs will enable such men to form closer contact with the work of chemists in other lines of research. The second purpose is to promote research in the branch of science covered by the mono- graph, by furnishing a well digested survey of the progress already made in that field and by pointing out directions in which investigation needs to be extended. To facilitate the attainment of this purpose, it is intended to include extended references to the literature, which will enable anyone interested to follow up the subject in more detail. If the literature is so voluminous that a complete bibliography is impracticable, a critical selection will be made of those papers which are most important. GENERAL INTRODUCTION 6 The publication of these books marks a distinct departure in the policy of the American Chemical Society inasmuch as it is a serious attempt to found an American chemical literature with- out primary regard to commercial considerations. The success of the venture will depend in large part upon the measure of cooperation which can be secured in the preparation of books dealing iidequately with topics of general interest; it is earnestly hoped, therefore, that every member of the various organizations in the chemical and allied industries will recognize the impor- tance of the enterprise and take sufficient interest to justify it. AMERICAN CHEMICAL SOCIETY BOARD OF EDITORS Scientific Series:— Technologic Series:— William A. Noyes, Editor, Harrison E. Howe, Editor, Gilbert N. Lewis, Walter A. Schmidt, Lafayette B. Mendel, F. A. Lidbury, Arthur A. Noyes, Arthur D. Little, Julius Stieglitz. Fred C. Zeisberg, John Johnston, R. E. Wilson, E. R. Weidlein, C. E. K. Uees, F. W. Willard. American Chemical Society MONOGRAPH SERIES PUBLISHED The Chemistry of Enzyme Actions (Revised Edition). By K. George Falk. Price $5.00. The Chemical Effects of Alpha Particles and Electrons. By Samuel C. Lind. Price $3.75- Organic Compounds of Mercury. By Frank C. Whitmore. Price $7.50. Industrial Hydrogen. By Hugh S. Taylor. Price $4.50. Zirconium and Its Compounds. By F. P. Venable. Price $4.00. The Vitamins. By H. C. Sherman and S. L. Smith. Price $5.50. The Properties of Electrically Conducting Systems. By Charles A. Kraus. Price $6.50. The Origin of Spectra. By Paul D. Foote and F. L. Mohler. Price $5.50. Carotinoids and Related Pigments. By Leroy S. Palmer. Price $6.00. The Analysis of Rubber. By John B. Tuttle. Price $3.50. Glue and Gelatin, By Jerome Alexander. Price $4.50. 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Application of Statistical Mechanics to Physical-Chemical Problems. By Richard C. Tolman. Hydrochloric Acid and Sodium Sulfate. By N. A. Laury. Vapor Phase Catalytic Oxidation of Organic Compounds and Ammonia. By J. M. Weiss, C. R. Downs and Dorothy A. Hahn. Absorption Spectra. By Victor Henri and Emma P. Carr. The Thermal Decomposition of Organic Compounds. By Charles D. Hurd. ( Preface The popularity which the general subject of the utilization of solar energy has enjoyed within recent years has led to much speculation re- garding means of accomplishing this. These speculations have con- cerned themselves not only with mechanical contrivances designed to utilize the energy of the sun, but in many cases have also endeavored to explain the manner in which the green plant plays the role of a converter of radiant energy. In this book only the latter subject is discussed. Un- fortunately much of the speculation regarding the manner in which the green plant utilizes solar energy has not been restrained by a knowledge of certain facts concerning the process. It is quite true that the subject with which this monograph deals is still in a condition of development ; our knowledge of many phases of the subject is fragmentary and incomplete. There are nevertheless certain well established facts which cannot be disregarded in any consideration of the problem. In the following pages the results of experimental investigations have been stressed rather than the conclusions which have been arrived at by observations in the field or by empirical methods. There can be little doubt that the greatest advances in our knowledge of the phenomenon of photosynthesis have been made through experimental study, that is, through analysis of the phenomenon by the exact control of the different factors which affect the process. These experimental studies have also served to emphasize the complexity of the subject and to demonstrate that great experimental skill is required in order to penetrate more deeply into the nature of the phenomenon. While the direct object of this book is to give the present status of the subject, it is hoped that it may serve to stimulate the interest of those familiar with allied branches of science and bring other and more refined methods to bear on the problems involved. The problem of photosynthesis borders on so many sciences that there have developed a number of avenues of approach. Each of these presents an aspect somewhat different from the other, each sees certain features in relief which from another approach are only vaguely discernible. It is very much like looking at a mountain from different sides. It is as yet impossible to determine which is the surest path of ascent. But of this we can be quite certain, that a single approach will not give us a complete view of our objective. Only from knowledge obtained by a study of the different aspects can we construct a true picture. The sciences of chemistry, plant physiology, physics, geology, oceanog- raphy and others have offered a view of the phenomenon of photosynthesis. To many viewing this process from one such restricted field it has ap- 9 10 PREFACE pearecl that they have obtained a perfect impression and they have en- deavored to construct a complete picture on the basis of their knowledge. Very few of these pictures, which have taken the form of theories of the mechanism of photosynthesis, have stood the test of time. They have served by pointing out the need of accumulating more precise knowledge and by emphasizing the necessity of applying a wider range of vision in formulating conceptions of the working of organic nature. Because of the fact that the phenomenon of photosynthesis has been considered from many different sides, publications on the subject are scattered through many different scientific journals. The literature of the subject is also very extensive. It has not been the object to achieve the inclusion of full references on all of the subjects discussed. Nor are all papers which have been published given discussion ; this would result in great prolixity. An endeavor has been made to discuss the salient features of the subject with some consideration of those theoretical as- pects which offer promise of fruitful development on the basis of experi- mental study. H. A. S. Carmel-by-the-Sea California April, 1926 TABLE OF CONTENTS PAGE Chapter 1. The Origin of Organic Matter and the Cosmical Function of Green Plants '^^ 1. Historical Introduction— 2. Sunlight, the Prime-Mover of Civilization— 3. The Green Plant as a Converter of Solar Energy. Chapter 2. The Nature of Photosynthesis as Determined by Observations of Gaseous Interchange and the Forma- tion OF Organic Matter o2 1. The Gaseous Interchange— a. The Path of Gaseous Ex- change— &. The Sources of Carbon Dioxide— c. The Evolution of Oxygen— (/. The Photosynthetic Quotient— 2. Factors which In- fluence the Rate of Photosynthesis- a. The Principle of Limit- ing Factors— &. The Influence of Light— c. Partial Pressure of Carbon Dioxide— (/. The Influence of Temperature— e. Chloro- phyll—/. Water Supply—^. The Time Factor—/?. Internal Fac- tors—/. Efifect of Various Substances, of Age, Electricity, etc.— 3. The Compensation Point. Chapter 3. The Products of Photosynthesis 182 1. The Liberation of Oxygen— 2. The Carbohydrates of the Leaf — a. Dioses and Trioses— &. Tetroses. — c. Pentoses.— d. Methods of Analysis of Pentoses— c. Quantitative Methods of Pentose Determination—/. Methylpentoses.— ^r. Hexoses — Asymmetric Synthesis — /?. Analysis of Hexoses — i. Cycloses — /. Heptoses — k. The Conjugated Carbohydrates— 3. The Transformation of Carbohydrates in the Leaf— a. Starch— ^. Disaccharides and Monosaccharides. The First Sugar Formed— 4. Are Other Substances Besides Carbohydrates Formed in Photosynthesis? Chapter 4. The Methods of Measuring Photosynthetic Ac- tivity L General Considerations— 2. The Liberation of Oxygen— a. The Use of Leucobases— /?. Luminous Bacteria — c. Motile Bac- teria Method — d. Optical Properties of Hemoglobin — e. Bubble Counting Method—/. Gas Analytical Methods— gr. The Deter- mination of Oxygen in Water — h. Apparatus for Gas Analysis — 3. The Absorption of Carbon Dioxide— 4. Formation of Organic Matter. 225 12 TABLE OF CONTENTS PAGE Chapter 5. The Chemistry of Photosynthesis 256 1. The Theories Regarding the Reduction of Carbon Dioxide and Water to Carbohydrates — a. Organic Acids — b. Formalde- hyde — The Baeyer Theory — c. The Condensation of Formalde- hyde by Means of Alkali — d. The Condensation of Formaldehyde with Weak Alkalies and under the Influence of Light — e into the atmosphere. The fundamentally important point is in relation to the energy changes. The energy expended by the man has been permanently lost to a large ex- tent. Similarly that obtained from fuel. The reconversion of the carbon dioxide into food or fuel material can be accomplished only by the use of a great deal of energy. The cycle is made possible only by the intro- duction of energy from without. This energy is derived from the sunlight which the plant, unlike the animal, is able to utilize and so convert the waste carbon dioxide again into food or fuel material. If our earth were an isolated system in which there were no imports and no exports, our state of affairs would be very different from that which now presents itself to us. According to our experience formulated in the laws of thermodynamics, in all naturally occurring transformations the tendency is to arrive at a condition of stable equilibrium. Thus the sub- stances on the earth are constantly tending to arrive at a condition of greatest entropy, meaning "rundownness." Most of the metals, for in- " As yet no term has been proposed for this process which is entirely satis- factory. This is due largely to the difficulty of expressing adequately in a suc- cinct term a process which appears to be highly complex and about which we have as yet incomplete information. Carbon assimilation used very generally by British writers, describes the process but very incompletely, especially as the word assimilation has been employed in so many different connotations. Further- more the light factor is in no way suggested, nor that of chlorophyll. The same criticism applies to the German Kohlensacureassimilation. On the other hand the French assimilation chlorophyllienne is cumbersome and not very much more ex- pressive. The term photosyntax suggested by Barnes, Bot. Gaz. 18, 403-411 (1893), has found little favor. While photosynthesis is not an altogether adequate expression, it is not too narrow and has come into very general use to mean the synthesis of complex carbon compounds out of carbon dioxide and water, in the presence of chlorophyll, through the action of light. The following have been sug- gested as more definite phrases : "Photosynthesis of carbon compounds," "Chloro- phyll-photosynthesis of carbohydrates," "Photosynthetic utilization of carbon dioxide," "Photochemical synthesis of carbo-hydrates," "Photosynthetic assimilation of carbon," "Photosynthetic appropriation of carbon." 24 PHOTOSYNTHESIS stance, are oxidised to their most stable oxides and converted into other compounds which under existing conditions are extremely stable. Our ores are those stable oxides or salts. Although this condition has not been uniformly attained in the earth, while there are still, for example, natural deposits of metallic copper and silver, yet, unquestionably that is the direc- tion in which the chemical changes are proceeding. Now most of these substances before they can be made use of require certain chemical changes which are a reversal of the naturally occurring ones. The ores, oxides or salts of the metals, must be reduced to the ele- mental metals. This, of course, is the reversal of the processes occurring in nature, and to accomplish such a reversal, work must be done, energy must be supplied. If then, the tendency is to attain the dead level, this state of equilibrium on our earth, what are the agencies or sources of energy which counteract this tendency and make possible the reverse reaction, the pumping of water up-hill, as it were? In searching for such possible sources of energy which might serve this purpose, we find that a little heat is probably given to the surface of the earth from the interior, another very small amount is the result of certain radio-active chemical changes, the action of the tides contributes some, and a further amount is received from radiation from the stars and moon. But these amounts are quite inadequate and insignificant when compared with the primal source of our energy, the sun. The radiations from the sun constitute our main source of energy. This is our main and most consequential import, the only potent factor which counteracts the tendency of complete running down. It is important not only in such reactions as the smelting of ores, but equally to the life on the planet. All living things on the earth demand for their maintenance and propa- gation a continuous supply of energy. In final analysis, plants are the fundamental source of energy of all animals as well as man. Just as the herbivorous land animals are the source of food of the carnivor?e, the diatoms are the fundamental source of food of the sea. It thus becomes evident that all life on the planet depends upon the energy derived from the sun through the intermediary of the plant, i.e., through the process of photosynthesis. Mankind lives entirely on the energy derived from the sun through the pursuits of agriculture. Ikit in addition to this we are squandering the principal of an enormous legacy of solar energy accumulated during the past ages. The plants, which alone are capable of utilizing the enormous floods of solar energy pouring upon the earth, have been at work for many ages prior to man's appearance upon the earth, and have, during time which would make the total s})an of human history appear as but a moment, built the foundations upon which all his present eminence rests. This fossil vegetation, pre- served as coal and oil. represents a very small fraction of the energy which has been falling upon the earth and which has been conserved for man. It is kindled, its energy liberated and used in a thousand ways, and the THE ORIGIN OF ORGANIC MATTER 25 rays of sunlight stored beneath the earth for millions of years give birth to a civilization such as the vv^orld has never known. Hut this great civilization of coal and steel is at the same time a most squandrous and profligate one ; it is using the principal of its legacy in numberless new ways. A year's consumption of coal at the present rate represents the accumulation of hundreds of years. The quest for further sources of energy in the form of coal and petroleum is being pushed with a feverish intensity revealing at times man's least attractive nature in personal and national greed. Our civilization is dependent upon the amount of available energy in the form of food and fuel. Fundamentally, our source of food and of fuel is the same. Photosynthesis supplies us with food directly. The accumulation of the products of photosynthesis during the past ages represents our present fuel supply. When these accumulations are exhausted or impracticable to utilize, our daily ration of solar energy will represent almost our entire means of livelihood. Our civilization now based upon the inanimate forces, must give way to one in which human physical efifort is again the driving force. It is one function of the scientist to care for the material welfare of man. His horizon should extend beyond the domain of the present, his view penetrate the future and by the exercise of his foresight guide us to ever increasing assurance of mastery over the world. And therefore the scientific world is realizing the necessity of considering our available sources of energy. Our ever increasing population and the development of civilization demand an ever increasing supply of energy in the form of food and fuel. National power is pre-eminently dependent upon such commodities. Liquid fuel, especially in the form of petroleum, is rapidly becoming the most prized possession. Our main source of energy is coal and, although it is less than one hundred years since it has been put to extensive use as fuel, the present annual consumption is stupendous, about 650,000,000 tons. Each decade has brought a very decided increase in the rate of consumption. Another very considerable source of energy is that developed from the water powers. Theoretically, this is virtually an inexhaustible supply and one of relatively high efficiency. The late Mr. Charles P. Steinmetz ^ has calculated on the basis of every raindrop which falls in the United States being collected and all the power it could produce on its way to the ocean being developed, that there would be possible about three hun- dred million horse-power. This enormous figure represents about the amount received from our present total consumption of coal. Thus this theoretical hydro-electric power would just about cover our present coal consumption, but leave nothing for future increased needs nor to cover other sources of energy now in use. Moreover, this figure for hydro- electric power is purely hypothetical, of which only a small fraction repre- sents that actually available, which, when united with other difficulties "Steinmetz, C. P., Survey Graphic, 1, 1035 (1922). 26 PHOTOSYNTHESIS such as cost of equipment and limitations of distribution, shows very clearly that all the water powers of the country cannot suffice. Much attention has been given to the production of liquid fuel other than petroleum. Thus far the investigations along these lines have almost universally led to the opinion that the substance best suited to these methods is alcohol. This is on the basis that alcohol can be produced from vegetable material and is the most direct route from solar energy. It is thus proposed to develop a photosynthetic industry on the basis of agri- culture, the products of which are to be converted into alcohol by means of fermentation. It is characteristic of most discussions of energy that they finally revert to a consideration of our primal source of energy, the sun. It is equally significant that when we arrive at this stage we realize that the green plant still remains the only large scale converter of solar energy through the process termed photosynthesis. In the process of photosynthesis nature has worked out a method of utilizing solar energy. In principle this method is probably the most effective imaginable. It is the only chemical reaction we know, induced by visible light, in which there is a great accumulation of energy. The products are easily stored, transported and capable of transformation in numberless ways. As we shall see, the natural process of photosynthesis is not very efficient ; but it can serve as a most valuable guide to the de- velopment of a method of the utilization of solar energy. The chemist need not be timid about competing with nature. He has many cases to his credit in which he has learned to surpass nature both in efficiency and reliability. 3. The Green Plant as a Converter of Solar Energy Photosynthesis is a complex process; for its successful operation a number of elements or factors are essential. These include light, carbon dioxide, water, oxygen, the minute corpuscles called chloroplasts which contain the chlorophyll, and a temperature at which the plant is able to exist. It must also be realized that the process of photosynthesis is inti- mately connected with the life processes of the plant. It is not merely a manufacturing of food which makes possible the life activities of the organism, but the operation of the photosynthetic process is apparently as much dei>endent upon the interplay of enzymatic reactions and the struc- ture of the organism as is respiration. When we consider the difficulties which have been encountered in unravelling the chemistry of the respira- tory process, as for instance the oxidation in the organism of glucose to carbon dioxide and water, it is not surprising that the synthetic reaction, starting with the relatively inert carbon dioxide and involving photo- chemical reactions about which until very recently we knew practically nothing, should appear as such a difficult task. Of all the factors which are essential to photosynthesis, light has prob- THE ORIGIN OF ORGANIC MATTER 27 ably received the most thorough study. This is the case not only on the part of the physicist in the laboratory but as well by the astronomer and meteorologist for solar radiation. The measurement of the total supply of solar radiation as well as the wave lengths and wave frequencies which constitute this radiation have been the subject of careful and exhaustive investigation. The pioneer work in this field was done by the American physicist, Langley, and has been greatly extended by the investigations of Abbot and his collaborators. In most of these investigations * the intensity of solar radiation has been measured by means of the heat pro- duced when the radiation is absorbed on a black surface at right angles to the rays, and has been converted into calories per square centimeter per minute. The values thus obtained depend, among other factors, upon ele- vation above sea-level, i.e. the thickness of atmosphere which the rays must penetrate, and the angle distance which the sun is from the zenith. Thus Abbot states ("The Sun," p. 284) "The maximum intensity of solar radia- tion as measured near sea level at Washington when the sun is not more than 45° from the zenith usually ranges from 1.15 to 1.45 calories per square centimeter per minute on cloudless days, depending on the clearness and dryness of the air. At IMount Wilson in California, over one mile above sea level, the values observed range from 1.45 to 1.62 calories, and on Mount Whitney in California, nearly three miles in altitude, the ob- served values reach 1.75 calories." Observations have been made over extended periods of time at many different points on the earth with a number of different instruments. The total annual insolation (for clear sky) varies with the latitude, is greatest at the equator and diminishes toward the poles. It should be emphasized that the values of total insolation have only an indirect bearing on possible photosynthetic activity, because the latter activity depends upon a number of factors besides insolation which greatly complicates the situation. In Figure 1 is shown the maximum radiation on normal and horizontal surfaces during the course of the year. The lower maxima at normal incidence during the summer are due to the dust and moisture in the atmosphere during this season.^ Solar radiation is greatly affected both in intensity and composition by its passage through the earth's atmosphere. This is due to the fact that the lower layers of the air contain dust particles and water vapor which absorb and refract the light rays. Because of the fact that the quantity and quality of the dust and the distribution of water vapor are changing from day to day it is not possible to predict accurately the in- * Annals of the Astrophvsical Observatory of the Smithsonian Institution, 2 (1908); 3 (1913); 4 (1922). Abbot. C. G., The Sun, Appletons, New York, 1911 Bigelow, F. H., Treatise on the Sun's Radiation and Other Solar Phe- nomena, New York, 1918. Kimball. IT. II., Bull. Mt. Weather Observatory, 1, parts 2 and 4; 2, part 2; 3. part 2 (1910). Pulling, H. E., Sunlight and Its Meas- urement. The Plant World, 22, 151-171; 187-209 (1919). Dorno, C, Studie uber Licht und Luft des Hochgebirges. Braunschweig, 1911. ° Kimball, Monthly Weather Rev., 43, 102 (1915). 28 PHOTOSYNTHESIS crease in total solar radiation with increasing elevation. Abbot ^ states that the loss of radiation in passing through the last mile of air is almost as great as the entire loss sustained above Mount Wilson (5,675 feet). In their passage through the earth's atmosphere some wave lengths are more absorbed than others. The absorption bands of oxygen, ozone, water vapor and carbon dioxide are chiefly in the infra-red and some in the extreme ultra-violet portions of the spectrum. The solar radiation received on the earth lies almost entirely within the wave lengths 0.29 ^ and 2.5 |-i. The spectral energy curve shows that the maximum intensity is at .47 \i, decreasing to almost zero in the ultra-violet and infra-red 0:tA„ Oecembcs 1 2 3 Januabt 1 2 3 TcBRUARr 1 2 3 March I 2 3 April 1 2 3 May I 2 3 JONC 1 2 3 July 1 2 3 August 1 2 3 StpicMetR 12 3 OcTOotr 1 1 2 31 Kov£l.:aER ! 1 2 3| December: 1 2 3 1 t 1.80 1.70 1.60 I.SO UC I.JO 1.20 110 1.00 .90 o S_ o / / o O ■&- o I 1. . -.A 7 o _•_ m_ r*- o \ \> JL, - -4- -f- — } t y ^ ■4= ^ ■*- + ^ c • -t- .4^ 'n! s -+- ^ -+- ^ / 'i / TS V, ^ ^ > o, / > '. •n >. CN \ o o / / / \ • \ \ V N o o - V _!_ — - -s- 7^ o 7 V > o "" _£, m • ■< K V, ;k • M • "^ d Fig. 1. — Maximum solar radiation per minute in gram calories per square centimeter at Washington, D. C. I. Solar radiation at normal incidence. II. Solar and sky radiation on a horizontal surface, with clouds near the sun, but not obscur- ing it. III. Solar radiation on a horizontal surface, with cloudless sky. (From Kimball.) regions beyond the limits mentioned and exhibiting a number of depres- sions at the Fraunhofer lines and in the regions where the earth's at- mosphere exercises selective absorption. Abbot ' has made extensive studies of the energy of the solar spectrum and by means of the spectro- bolometer has prepared energy curves, called holographs, which show the distribution of solar radiation and the transmission of the atmosphere at all parts of the spectrum. These investigations are of fundamental im- portance for every phase of the question of solar energy. He has also pre- pared a table of the transmission of the atmosphere at various wave lengths. This is reproduced in Table i. "The ^ values represent the frac- tion of intensity of the solar beam outside the atmosphere which would remain in a direct beam transmitted vertically to the earth's surface. Aver- age values for cloudless (but not necessarily hazeless) days are given, as •Abbot, The Sun. p. 293. ' .\bbot, Smithsonian Inst. Ann. Astrophysical Obser., 3, 21 (1913). " Abbot, Ibid., 197. THE ORIGIN OF ORGANIC MATTER 29 they have been found at Washington, Mount Wilson and Mount Whitney, for various wave lengths. To compute the transmission for other than zero zenith distances, the coefficients here given must be raised to a povv^er equal to the secant of the zenith distances. This does not hold closely for zenith distances above 75°." TABLE 1 Abbot's Table of Me^.'in Coefficients of Atmospheric Transmission. Washington Mount Wilson ]Mount Whitney Wave length 1902-1907 1909-1910 1909-1910 0.30 0.70? 0.325 .... (0.550) 0.635 0.35 0.612 0.715 0.375 0.662 0.776 0.39 .0'.445 0.694 0.800 0.42 0.586 0.764 0.831 0.43 0.600 0.778 0.844 0.45 0.640 0.800 0.875 0.47 0.671 0.827 0.902 0.50 0.705 0.858 0.919 0.55 0.739 0.876 0.930 0.60 0.760 0.89a 0.940 0.70 0.839 0.942 0.964 0.80 0.865 0.964 0.976 1.00 0.901 0.973 0.975 1.30 0.916 0.972 0.967 1.60 0.930 0.975 0.963 2.00 0.909 0.957 0.932 2.50 0.870 (0.900) 0.945 3.00 . . • • .... 0.916 The intensity and composition of solar radiation varies considerably v^^ith the altitude ; and also, at a given place, the composition of the light varies considerably, almost from minute to minute. This is especially true in the violet end of the spectrum. Probably of even greater significance than the gaseous constituents of the atmosphere for the absorption of solar radiation is the dust in the upper atmosphere. The origin of this dust is usually volcanic. Benjamin Franklin already suggested that the hard winters which had been recorded in history may be due to the decrease in solar radiation by the fine volcanic dust and that these abnormally cold periods may be synchronous with volcanic activity in dififerent parts of the world. That such is the case, at least to a considerable degree, now seems highly probable. Thus. Abbot ^ found, following the volcanic eruption of Mount Katmai. Alaska, June 6-7, 1912, that evidence of the dust appeared at Bassour, Algeria, on or before June 19, and at Mount Wilson, California, on or before June 21. The effect reached its maximum in August and reduced the total direct radiation of the sun by nearly 20 per cent. The complicated effects produced by such layers of volcanic dust on solar radiation have been studied also by Kimball '" and by Humphreys.'^ The latter has compiled "Abbot, Smithsonian Inst. Ann. Aslroplivsical Obscr., 3, 214 (1913). "Kimball, H. H., Monthh Weather Revietv, 46, 355 (1918). " Humphreys, W. J., Physics of the Air. Philadelphia, 1920, p. 569. 30 PHOTOSYNTHESIS very interesting data on the relation of volcanic disturbances to solar radia- tion and atmospheric temperatures. It appears that not all eruptions result in a decrease in surface temperatures ; usually only those in which a large quantity of fine dust is thrown into the high isothermal layer of the at- mosphere. The end results depend upon a number of factors, chiefly the absorption by the dust of the direct solar radiation and the effect of the dust on the earth's radiation into space. But there seems to be httle room for doubt that both volcanic dust and solar activity, the latter evi- denced by sun-spots, have decided effects both on the direct solar radiations and on atmospheric temperatures. The effect of such changes is dis- cussed by Humphreys : "For instance, during the summer, or growing season, a change of 0.5° C. produces a latitude shift by fully 80 miles. Hence, if there is but little or no volcanic dust to interfere, during sun- spot minima cereals and other crops may be successfully grown 50 to 150 miles farther north (or south in the southern hemisphere) than at times of sun-spot maxima. This alone is of great practical importance, espe- cially to those who live near the thermal limits of crop production. "In addition to changing the area over which crop production is pos- sible, a change of average temperature also affects, in some cases greatly, the time of plant development. Thus Walter ^- has shown that a change of only 0.7° C. may alter, and in Mauritius has been observed actually to alter by as much as an entire year, the time required for the maturing of sugar cane. Hence the temperature changes that normally accompany sun- spot variations, though small in absolute magnitude, are of great impor- tance, and, by availing ourselves of the reasonable foreknowledge we have of these changes, may easily be made of still greater importance." The astronomer is particularly interested in the amount of solar radia- tion received at the outside of the earth's atmosphere. This quantity he obtains by making determinations of the amount of radiation received at the surface of the earth and calculating the amount of loss sustained by passage through the atmosphere. The value thus obtained is called the "solar constant" of radiation. For this measure Abbot ^^ has given the value of 1.946 calories (15°) per square centimeter per minute for the epoch 1912 to 1920 determined by 1,244 observations. Bigelow ^* arrived at another value by means of different methods ; his results are summarized below, in values of gram calories per square centimeter per hour : 1. True Solar intensity of radiation 5.85 calories 2. Effective Solar intensity at the distance of the earth 3.98 " 3. Effective intensity by the bolometer 3.98 " 4. Effective intensity by thermodynamics 3.98 " 5. Extrapolated intensity by the pyreheliometer 1.95 " 6. Intensity at sea level by the pyreheliometer 1.50 " ""On the Influence of Forests on Rainfall and Probable Effect of 'Deboisement' on Agriculture in Mauritius" (1908), quoted from Humphreys. 1. c, p. 602. "Abbot, Smithsonian Inst. Ann. Astrophysical Obser., 4, 192 (1922). Bigelow, Treatise on the Sun's Radiation. New York, 1918, p. 211. 13 THE ORIGIN OF ORGANIC MATTER 31 It is important to bear in mind that the value for the solar constant (Abbot 1.95 and Bigelow 3.98) refers to the intensity at the outside of the atmosphere. While the establishment of the true value of this influx of energy is of very great importance, it has only indirect bearing on the problem of photosynthesis. In calculating the amount of solar energy available on the earth, the value of the solar constant has occasionally been used. That this is erroneous is evident. Fortunately there appears to be little dispute regarding the amount of solar energy received at the surface of the earth and it is this factor which is of more immediate importance to the problem of photosynthesis. Abbot has summarized the total solar radiation on normal incidence and horizontal surface assuming the sun to shine 261,000 minutes per year and has calculated the square feet required per horse-power on the basis of complete absorption and transformation. Table 2 is taken from Abbot's "The Sun," page 386. TABLE 2 Normal Incidence Horizonta 1 Surface ititude Sea level 6,000 feet Sea level 6,000 feet 20° 30° 38° 45° 292,000 287,000 271,000 270,000 362,000 355,000 342,000 340,000 185,000 170,000 152,000 137,000 226,000 203,000 185,000 169,000 Calories per sq. cm. per year. 20° 30° 38° 45° 10.5 10.7 11.3 11.4 8.5 8.8 9.0 9.1 16.6 18.1 20.2 22.4 13.6 15.1 16.6 18.2 Average sq. ft. per horse power. Abbot ^^ makes the following statement to give an idea of the total amount of energy received by the earth from the sun : "Expressed in another way, the measurements indicate that if the sun's rays could be completely employed to melt ice exposed continuously to them at right angles, they would suffice to melt a layer 426 feet thick in a year. Such a layer at the earth's mean distance, if it entirely surrounded the sun. would weigh 4 X 10^^ (4 followed by 25 ciphers) tons, and the complete melting of it each year would represent as many heat units as the burning of 4 X 10-^ tons of anthracite coal. This, then, is a measure of the sun's yearly output of radiation." It is a very interesting fact that the value of the solar constant is sub- ject to certain periodic fluctuations. These are, firstly, long period variations which indicate that the emission of radiation varies with solar activity as made evident by sun-spots and other phenomena. With increasing num- ber of sun-spots solar radiation increases, though the relation is not a di- rect numerical one. Thus Abbot reports that the increasing solar activity of 1914 resulted in a 3.5 per cent increase in the value of the solar constant over 1913. The solar constant is also subject to short-period variations. "Abbot, The Sun, p. 299. 32 PHOTOSYNTHESIS Thus from day to day there have been extreme fluctuations observed rang- ing over nearly 10 per cent. These short-period variations are apparently associated with changes in the opacity of the outer solar layers.^" The correlation between the variations in solar activity and climatic conditions on the earth may be of great significance for the photosynthetic activity of plants. It should be stated, however, that there is no direct correlation between variations of the solar constant and terrestrial temperatures and that the interrelations appear to be very complex. The foregoing is of necessity only a very brief consideration of the factor of solar radiation. Many phases have not been touched upon but the reader will find in the literature cited a wealth of material for this most fascinating study. We have, however, sufficient data to enable us to draw some general conclusions on the relation of solar energy to photo- synthetic activity ; a more detailed consideration of the light factor in photosynthesis will be found in Chapter 2. In order to form a conception of the amount of solar energy received at the surface of the earth and the proportion of this energy which is utilized by the plant and which thus represents the amount of energy now available for use by man, let us make the following calculations. Instead of 1.5 calories per square centimeter per minute as the amount of solar energy received at the surface of the earth we shall take 1.35 calories. Such a calculation should be based upon good agriculture conditions and should exclude, naturally, extreme conditions of solar intensity such as exist on the desert. In a six hour day at 1.35 calories per square centi- meter per minute there would be received 486 calories per square centi- meter. From Kimball's ^^ values, obtained with a Callender pyreheliom- eter, at Washington, D. C, and Mount Weather, Va., the mean daily normal for the 92 days of May, June and July, 1910-1914, is 522 gram calories per square centimeter of horizontal surface. This somewhat higher value is due to the fact that it represents the amount of radiation received during the entire day instead of only six hours, but it serves to show that we have not taken too high a quantity in the 1.35 gram calories. We shall take 90 days as the growing season, and convert the total amount of solar radiation received on an area of one U. S. acre into terms ex- pressed by the energy derived from the combustion of coal. Thus, taking the heat of combustion of anthracite coal equal to 8,000 kilogram calories per kilogram, we conclude that the solar energy received on an acre of land during a growing season of 90 days is equal to the energy contained in 243 tons of anthracite coal. For the purix)se of comparison, we can calculate on the same basis, the amount of this total energy which the plant fixes and which is found in the jx^tential energy of the substances which have been elaborated by the plant in the photosynthetic process. We shall take a yield of 25 bushels "Abbot, C. G., Smithsonian Inst. Ann. Astro physical Obser. 4 16 177 184 (1922). Humphreys, W. J., Astrophx. Jour.. 32, 97 (1910). "Kimball, Monthly Weather Reznezv, 43, 108 (1915). THE ORIGIN OF ORGANIC MATTER ZZ of corn (bushels of 56 pounds) per acre and consider the heat value of the grain only. De Baufre ^^ reports the heat value of fully cured corn, containing 10 per cent of water, as 6,700 B.t.u. per pound. The energy of the crop of corn from an acre of land is thus equivalent to 0.325 of a ton of anthracite coal. That is, by raising corn on a given area of land about .13 per cent of the total amount of solar energy received is "fixed" by conversion into potential energy of carbon compounds. If the yield of corn from one acre were fermented in order to obtain alcohol, the energy obtainable would be still further reduced. Thus, from 25 bushels of corn at 2.7 gallons per bushel there would be obtained 67.5 gallons of alcohol. The heat of combustion of this amount of ethyl alcohol corresponds to about 0.20 tons of anthracite coal or about 0.08 per cent of the total energy of solar radiation per acre for a period of 90 days. On the basis of a conversion factor of 10 per cent, the yield- of corn from one acre, when fed to steers, would produce meat corresponding to about 0.033 tons of coal per acre. A similar calculation can be made of the production of material syn- thesized by forest trees. ^^ We shall take one of the fastest growing trees, the redwood {sequoia scmpervirens) . The average annual growth of 20-60 year trees is about 300 cubic feet, or 75 cubic feet for a period of three months. This includes the stem and top but not the bark and limbs. The heat of combustion of one cubic foot of redwood is 159,000 B.t.u. This would yield an energy equivalent of 0.41 tons of anthracite from one acre of redwood in a period of three months. While this does not represent the entire yield of material synthesized by the trees, it is a liberal account- ing of the merchantable timber. Another very rapid growing tree is Eucalyptus globulus. This tree averages about 355 cubic feet of wood per year for 20 year trees, or 89 cubic feet for three months. The heat of combustion of one cubic foot of this wood is 268,000 B.t.u. On this basis an acre of eucalyptus trees would yield energy corresponding to 0.826 tons of coal in a period of three months. This is about double the quantity obtained from redwood ; the latter is notoriously poor fuel while eucalyptus though fast growing, is a hard wood of high heat of combustion. The foregoing figures have been given in order to present an approxi- mate idea of the plant as a converter of solar energy. They cannot be taken as a true index of the efficiency of the photosynthetic process ; this will be discussed in a later chapter. The values have a "practical" rather than a strictly scientific meaning. For instance, in the determination of the per cent of conversion in corn we did not include the stalks, which would probably double the total value. Yet the use of such material for fuel or for the production of alcohol presents many difficulties which make them of questionable value. One of the practical questions of the "De Baufre, Pozver, 56, 212 (1922). ^ Bruce, University of California, Agricultural Experiment Station. Bulletin No. 361, Metcalf, Ibid., 380. U. S. Dept. of Agriculture, Agriculture Bull., 753 (1919). 34 PHOTOSYNTHESIS photosynthesis problem is whether the production of industrial energy from solar radiation through the intermediary of the plant seems feasible. In Figure 2 is shown graphically the relation of the total energy of solar radiation to that portion which is fixed or transformed into usable form by the plant. The great difference in these quantities is very evident. We are now utilizing about 0.1 per cent of the solar energy falling on a given area of the earth's surface, and this only during a fraction of the year. For this utilization we are still wholly dependent upon the inter- 0.41 0.83 E F 0.33 0.20 0.033 BCD Fig. 2.— a, amount of sol^'r energy received on an area of one acre in a growing season of 90 days expressed in tons of anthracite coal. B, 25 bushels of corn from an acre of land, energy obtainable therefrom expressed in tons of coal. C, energy of alcohol obtained'by fermentation of 25 bushels of corn. D, energy from meat when corn obtained from an acre is fed to steers. E, energy m terms of coal from an acre of redwood in 3 months. F, energy in terms of coal from an acre of Eucalyptus. mediary of the chlorophyllous plant. The causes which account for this low order of efficiency are rather complicated. Moreover, dififerent species of plants vary greatly in the various factors which must be taken into account in analyzing the effectiveness of plants as storers of solar energy. We have prepared what may be termed a composite picture of the relation of plants to solar energy. In Figure 3 is shown the disposal by the leaf of the energy incident on it. All of this energy is not absorbed by the leaf ; approximately 70 per cent is absorbed, 30 per cent being transmitted or reflected. In land plants a very considerable portion of the energy absorbed by the leaf is dissipated through evaporation of water from the leaf. This has the effect of cooling the leaf. The quantity of energy thus lost naturally varies greatly with external conditions. We can place this amount at 50 per cent. When there is relatively little evaporation the absorption of solar THE ORIGIN OF ORGANIC MATTER 35 radiation raises the temperature of the leaf above that of its surroundings. The leaf thus also loses energy by reradiation and convection cooling. With a loss of energy by transpiration of 50 per cent, that lost by reradia- tion can be placed at 19 per cent. As directly determined the amount of energy used up in the endothermic process of photosynthesis is relatively very small. It also varies considerably with external conditions and in different plants, but a fair value for the present purpose is about 1 per cent. This value of 1 per cent of the total incident solar radiation used in photosynthesis represents the value for mature active leaves. In Figure 4 is shown the distribution of material which has been manu- factured in the photosynthetic process. The plant can, of course, produce carbohydrates only during the hours of illumination. For its life activities r PHOTO - SYNTHESIS l/C Fig. 3. — What happens to solar radiation incident on a chlorophyllous leaf. The values indicated give approximate disposal of the energy ; the ratio varies with changes in external conditions. a certain amount of the materials elaborated are used up by the process of respiration. This in a sense represents the operating cost of the manu- facture and amounts to approximately 20 per cent. The material of the plant can be divided into the crop, the leaves and stalks, and the roots. Here again there are great variations in different species of plant. For corn which has served as an example we can take the ratio as given in Figure 4. Thus we can account for a further reduction in the ratio of total incident energy to energy recovered in the plant crop. The 1 per cent of the incident solar energy used in photosynthesis is the ratio of the energy utilized in photosynthesis to the total radiation falling on the leaf. In our first analysis we concluded that about 0.13 per cent of the total incident energy was recovered in the crop over a period of 90 days. The 36 PHOTOSYNTHESIS 1 per cent used in photosynthesis represents a value determined for the absorbing leaves and not for an entire plant. Moreover, in a growing period of 90 days we include the plant from the seedling stage to the time of harvest. During this period the photosynthetic activity of the entire plant varies from virtually nothing to maximum and then decreases agam. Also the entire acre of land on which we based our calculation is not covered by leaf surface. These various factors, then, contribute to reduc- ing the net conversion by the plant to about 0.1 per cent of the total incident solar radiation. The foregoing brief discussion gives a general idea of the efficiency of the green plant as a converter of solar energy. Under natural conditions Pig 4_ — Approximate distributiuii ui material manufactured in the photosynthetic process. the rate of photosynthetic activity undergoes wide fluctuations according to the changes in external conditions to which it is subjected. Before entering upon an analytical study of the influence of each of these various factors we shall consider the function of green plants on our earth in its more general aspects. The composition of the atmosphere is, of course, of very great impor- tance in relation to the photosynthetic activity of plants. While there is still considerable discussion regarding the composition of the atmosphere to its outer limits, we are concerned primarily with the composition at the surface of the earth. Here the different gases are present in the following volume percentages : Nitrogen 78.03 Hydrogen 0.01 • Oxygen 20.99 Neon 0.0012 Argon 0.94 Helium 0.0004 Carbon dioxide 0.03 THE ORIGIN OF ORGANIC MATTER 37 In view of the fact that the amount of oxygen required for photo- synthetic activity is relatively very small we can confine our attention to the carbon dioxide. The calculated difference in the percentage distribu- tion of gases in the atmosphere with increasing altitude -'' are also of no immediate significance to our subject, as we must naturally confine our attention to land areas capable of supporting vegetative growth. There is still some debate regarding the exact value of the carbon dioxide content of the atmosphere, more i>articularly the constancy of the amount present with change in location and weather conditions. It should be stated immediately that it is highly probable that many of the dis- cordant results unquestionably are due to the fact that different methods of analysis were used and, simple as the problem may seem, thoroughly reliable methods of air analysis have been worked out only relatively re- cently. The tension of the water vapor in the atmosphere may cause a considerable variation and some of the analyses have apparently been cor- rected for moisture while others have not. Moreover, the place of taking the sample whether this is, for instance, close to the ground or not may be of considerable influence. Sources of carbon dioxide even when these are some distance away may influence results. Thus Reiset -^ took samples 8 kilometers from the city of Dieppe and found a mean value of 0.02917 volume per cent COo. The proximity of a drove of sheep raised this value to 0.03178. Benedict ^^ reports remarkably constant values for carbon dioxide and oxygen. He concludes : "The results of analyses of air taken near the laboratory showed no material fluctuations in oxygen percentage during a period extending from April 15, 1911, to January 30, 1912. This con- stancy was maintained in spite of all possible alteration in weather condi- tions, changes in barometer, thermometer, humidity, and wind direction and strength; furthermore, the experiments were made before, during, and after the vegetative season. The average result of 212 analyses showed 0.031 per cent of carbon dioxide and 20.938 per cent of oxygen. The analyses of air collected over the ocean, at two dift'erent times of the year, and on top of Pike's Peak, gave essentially similar results." The carbon dioxide-content in crowded city streets was found to be slightly higher than normal, while samples taken in New York and Boston subway stations contained about twice the normal amount. Among the older analyses reported one finds very considerable varia- tions in the amount of CO2. By some writers much importance has been attached to these variations. In fact Reinau ^=* has developed an elaborate theory on the basis of these observations, in which he stresses the signifi- cance of periodic variations in carbon dioxide content of the atmosphere. '"Humphreys, F. J., Physics of the Air. Philadelphia, 1920, p. 60. ^Reiset, J. A., Compt. rend., 88, 1007 (1879). c^ . ^ r '■" Benedict, F. G., The Composition of the Atmosphere with Special Reference to its Oxygen Content. Carnegie Inst, of Washington. Pub. No. 166 (1912), p. 114. "'Reinau, E., Kohlensaure und Pflanzen. Halle, 1920. 23 38 PHOTOSYNTHESIS Without entering here upon a discussion of this theory, it is nevertheless well to bear in mind that the older analyses were carried out by means of different methods and at a time when all the precautions essential for accurate air analyses were not yet realized. Lundegardh,'* working on the island of Hollands Vadero, 3.2 kilo- meters from the mainland of Sweden, reported the following analyses for carbon dioxide during the summer months : 1920 1921 1922 1923 612 0.5603 0.5267 0.5565 mg. per Liter 03295 0.03031 0.02843 0.0300 vol. per cent 19.97 17.3 16.48 15.39 mean temperature The mean variation during any one summer was ± 14 to ±16 per cent. That there is a variation in the COo-concentration during day and night has been reported by a number of investigators. Thus Reiset,^'' M^unz and Aubin,-'^ and Letts and Blake ^' conclude that the diurnal CO2 content is about 12 per cent lower than the nocturnal when the analyses were made at some distance from the sea. Such variations were not ob- served in analyses made at sea (Thorpe) ^^ or on a small island (Lunde- gardh).^^ Schulz ^° reports variations ranging in extremes from 2.7 to 3.2 m the air over the North Sea and Baltic. He considers these as due to in- accuracies in analyses rather than actual differences in the carbon dioxide content of the atmosphere and regards a mean value of 0.000293 at- mosphere as the partial pressure of carbon dioxide over these bodies of water. The entire question of the carbon dioxide content of the atmosphere is in need of a thorough investigation with uniform methods at different points on the earth and extending over a number of years. It is only in this way that a solution of such questions as convection, the complete mix- ing of the atmosphere and the influence of land and sea is to be hoped for. KendalL'^ concludes his discussion of the carbon dioxide content of the atmosphere with the following comment : "The fact that exceed- ingly large variations (as much as 700 per cent) have been recorded by numerous observers is probably due to the faulty methods of estimation that have been generally employed. The most careful and recent analyses of pure out-door air indicate relatively constant values. Unfortunately most of the observations on record have been obtained in connection with non-chemical investigations, and these frequently betray the fact that at "* Lundegardh H., Der Kreislauf der Kohlensaure in der Natur. Jena, 1924, p. 9. *»Reiset, J. a'., Compt. rend., 88, 1007 (1879). "Mirnz A., aAd Atibin, E.. Compt. rend., 92, 247, 1229 (1881), 93, 797 (1881), 94, 1651 (1882), 96, 1793 (1883). "Letts and Blake, Roy. Soc. Dublin Proc. N. S., 9, 107 (1899-1902). ''Thorpe, Ann. Chem., 145, 94 (1868). " Lundegardh, I.e., p. 16. »" Schulz, Arch, deutsch. Seewarte, 40, 16 (1922), 41, 6 (1923). "Kendall, J., Jour. Amer. Chem. Soc, 38, 1490 (1916). THE ORIGIN OF ORGANIC MATTER 39 least a little knowledge of chemistry would have been of great assistance to the exi:)erimenter, both in the choice of method and in the necessary manipulations." Many years ago Regnault ^^ inaugurated an extensive international investigation of the composition of the atmosphere. While the results from this elaborately planned cooperative investigation were considerably curtailed by the political disturbances existing in Europe at the time, they are sufficient to demonstrate the value of such an undertaking. With the advance in methods of analysis, of communication and of transportation a similar undertaking now ought to be capable of execution and would certainly justify itself. If photosynthesis actually has an appreciable influence on the carbon dioxide content of the atmosphere, this could only be detected because of imperfect and slow mixing of the gases of the air.^^ As nearly as can be calculated there are about 2.2 X 10^^ tons (about 2 X 10^^ kilograms) of carbon dioxide in the atmosphere. The total photosynthetic carbon dioxide consumption can be put at about 54 X 10^- kilograms per year on the basis of the calculations of Sachs given below. The annual consump- tion therefore would be roughly only one fiftieth of the total supply of atmospheric carbon dioxide. These figures are obviously very rough, but indicate that if perfect mixing took place, the difference in carbon dioxide concentration of the atmosphere, due to photosynthesis, would be so slight that it could not be detected by analyses. Day and night variations in carbon dioxide content, if they exist, are therefore probably due to a combination of local conditions. On the other hand, with no wind, when there is imperfect mixing it is highly probable that densely vegetated areas show a carbon dioxide content below the "normal." A larger reservoir of carbon dioxide than the atmosphere is the sea.^* Sea-water contains approximately 50 cc. of carbon dioxide per liter, but varies with the depth at which the water is drawn. Moreover, the oceans comprise fully 70 per cent of the surface of the earth, so that they play an important role in the carbon dioxide cycle. In absolute amount also the sea contains considerably more carbon dioxide than the atmosphere ; the latter contains approximately 2100 billion kilograms while the former contains about 16,000 billion kilograms, though these figures can be taken only as approximations. Different estimates give the oceans eighteen to twenty-seven times as much carbon dioxide as the atmosphere. Just how great are the potentialities of the sea for food it appears' difficult to determine. In limited areas the sea produces as much or slightly *' Regnault, Ann. Chim. ct Phys. Ill, 36, 385 (1852). ^ Clarke, Data of Geo-chemistry. Bui. U. S. Geological Survey No. 770, 1924, p. 50. ''Krogh, A., Compt. rend., 139, 896 (1904). Krummel, O., Handb. der Ozeanog- raphie. Vol. I, 312 (1923). Schulz, A., Naturwissenschaften, 12, 105-113, 126-133 (1924). Politzsch, S., Compt. rend, dcs travaux du Laboratoire de Carlsberg, 11, 199 (1916). Wells, R. C, U. S. Geol. Survey, Professional Paper IZQ-A, 1-16 (1918). 40 PHOTOSYNTHESIS more food than an equal area of land. East ^^ considers that most of the plants, which are as much the primary source of food of sea animals as of land animals, are found in waters not over 200 feet in depth, and that such areas of the sea would approximately equal 5 per cent of the land area of the globe. With the possible exception of certain rather restricted regions as in the North Sea and Baltic, where the economic importance of sea life has long been recognized, few careful surveys have been made of the production of the sea. In most cases the products of photosynthesis in the sea are harvested indirectly. That is, while some algae are used as a source of food in China and Japan, this is not very general and man confines himself largely to the consumption of animals which feed upon sea vegetation or other sea animals. In many cases this is several steps removed from the original plant source. The result is a tremendous waste of energy, so that man finally obtains an exceedingly small quantity of food from a very large amount of carbon dioxide reduced by the marine plants. The diatoms and holophytic peridines are the great marine con- verters of solar energy. Upon these and other sea plants we depend for our sea food ; the very intricate relationships between the various forms of marine life make the determination of the "food producing value" of an area of water an exceedingly complex problem. The chemistry of the sea and its relation to the gases of the atmosphere has been the subject of much experimental and theoretical study. ^" While at first these problems appeared to be relatively simple, when studied more minutely, particularly in relation to the activities of living organisms, many complex conditions became apparent. The solubility of the gases of the atmosphere, the influence of the salinity of the ocean and of temperature on this and similar conditions determine the fitness of the ocean for plant and animal life. While most of the constituents of the ocean are main- tained in relatively constant proportions the concentration of carbon dioxide and to a lesser degree also oxygen are found to undergo consider- able variation. These variations together with the intimately associated hydrogen ion concentration of the sea are of paramount importance to living organisms. Schulz ^' has made a very careful study of the aeration of the North Sea and Baltic Sea and his recent results are here given to illustrate the conditions existing for life in the sea. For more detailed information the literature cited must be consulted. Table 3, taken from Schulz's publica- tion shows the percentages of the different gases of the atmosphere in natural waters. '° East, Mankind at the Crossroads, p. 71. ''Murray, J., and Hjort, J., The Depths of the Ocean. London, 1912. For- chammer, Phil. Trans. Roy. Soc, 155, 203-262 (1865). Dittmar, Challenger Report, Physics and Chemistry, vol. 1, 1-251 (1884). Quinton, R., L'eau de Mer. Paris (1904). Clarke, Data of Geochemistry, Bull. U. S. Geol. Survey No. 770 (1924). Stieglitz, Carnegie Inst, of Washington Pub. 107 (1909). Schulz, Arch, deutsch Seezvarte, 40, 1 (1922), 41, 1 (1923). "Schulz, B., Die Naturmissenschaften, 12, 105-113, 126-133 (1924). THE ORIGIN OF ORGANIC MATTER 41 TABLE 3 Gases of the Atmosphere Contained in Fresh and Sea- water. (From Schulz.) Nitrogen -\- A, in the Atmosphere J J"^'^^^''^- ^ ^ In per cent In 1,000 cc. In per cent In 1,000 cc. In per cent In 1,000 cc. In per cent In 1,000 cc. ^ In per cent B, In Fresh water [At 0° \ [At 20° C, In Sea- water with Salt Con- tent of 3.5 fAt 0° Nitrogen + Argon 790 cc. 79 18.64 cc. 63 12.59 cc. 65 per cent \ L At 20° 14.42 63 10.42 65 cc. cc. Oxygen 210 cc. 21 10.29 cc. 35 6.57 cc. 34 8.04 cc. 35 5.36 cc. 34 Carbon Dioxide 0.3 cc. 0.03 0.51 cc. 1.7 0.26 cc. 1.3 0.44 cc. 1.9 0.23 cc. 1.4 Oxygen -|- Carbon Dioxide 29.4 cc. 100 19.4 cc. 100 22.9 cc. 100 16.0 cc. 100 Owing to differences in the solubility of the gases of the atmosphere the ratios of oxygen, nitrogen and carbon dioxide are different in water than in air. The amounts of the gases present in the water are, of course, greatly influenced by temperature, as well as by the salinity; both of these factors vary greatly in different parts of the world. It has been found that in the summer the oxygen content of sea-water at depths of about 20 meters is greater than that obtaining with sea-water and air in equilibrium. The explanation offered for this is that during the summer months photo- synthesis by marine plants is in excess of respiration and the oxygen lib- erated during photosynthesis accumulates in the water. At the surface of the sea where there is opportunity for diffusion into the atmosphere. TABLE 4 Increased Oxygen Content of Sea-water Due to Photosynthesis in Summer. (From Schulz.) "Poseidon." Station 13. 56° 40' July N, 2° 14' E. 19, 1921. Water Oxygen epth Temperature Salt content Relative m. °C. °/00 cc./L. Content 14.60 34.25 5.77 100 5 14.53 34.25 5.87 101 10 13.78 34.60 6.05 103 20 12.17 34.99 6.46 107 30 11.56 34.99 6.13 100 40 10.96 35.01 6.08 98 50 6.71 35.07 5.34 79 60 6.70 35.03 5.36 79 70 6.72 35.07 5.34 79 78 6.74 35.08 4.66 69 42 PHOTOSYNTHESIS the oxygen-content is equal to that of sea-water in equihbrium with air. In Tal)lc 4 are given the results of one of Schulz's analyses. By "relative oxygen-content" is meant the ratio in per cent of sea-water in equilibrium with air to the amounts actually found. In the winter the conditions are changed ; due to cooling, convection currents are set up which result in more thorough mixing of the water. The changes throughout the year are, however, greatly affected by local conditions.^^ . There is a slight increase in the amount of free carbonic acid in sea- water with depth though there is apparently no invert ratio between the oxygen-content and that of carbon dioxide. The quantitative relation of these two gases is of great importance to the life of plants and animals in the sea and the relative content in the sea is probably to a considerable extent also determined by the activity of plants and animals. TABLE 5=" Average Amount of Free Carbonic Acid in Sea-water at Various Depths in MG. PER Liter. c,,rfare 42.6 300 fathoms 44.0 25 fathom;-::::::::.: 33.7 4oo ;; 41.0 CO " 48.8 800 " 42.2 ^00 " ' ' 43.6 More than 800 fathoms 44.6 200 " :::::: 44.6 Bottom 47.4 One of the most striking properties of sea-water is the constancy of its hydrogen ion concentration. Thus Politzsch *» found that in samples of water taken from the Atlantic, the Mediterranean, the Black Sea and the Baltic the hydrogen ion concentration varied only from IQ-^'^^ to IQr^-^^. Atkins*' has made determinations of the hydrogen ion concentration of sea-water over several years and found that the changes in the pH values are associated with a variety of factors but that the variations are very ' small. The variations in pH are somewhat greater where there is abundant plant and animal life, as near the coast. Photosynthesis causes the water to become more alkaline, while the carbon dioxide liberated in respira- tion tends to decrease the alkalinity. Where large streams meet the sea the water may, of course, vary in composition. Also in some regions sulphurous acid is found in the lower depths of the sea which naturally increases the hydrogen ion concentration in that locality. McClendon *' and his collaborators have made a careful study of hydro- gen ion concentration and carbon dioxide-content of sea-water. They conclude that the pH of sea-water is determined "solely by the ratio of the concentration of bufifers, including carbon dioxide and other weak '" Schulz, Arch, deutsch. Seewarte, 41, 8 (1923). •'"From Clarke, Data of Geochemistry (1924), p. 146. '"Politzsch, S., Biochem. Zeit., 37, 116 (1911). "Atkins W. R. G.. Journ. Marine Biol. Ass., 12, 717-771 (1922), 13, 93-118 (1923), 13,' 437-446 (1924). "McClendon, Carnegie Inst, of Washington, Pub. No. 251, 23-69 (1917). THE ORIGIN OF ORGANIC MATTER 43 acids, to the concentration of bases combined with them (excess base over strong acid)." They have worked out methods of determining the pH of sea-water and therefrom the carbon dioxide-tension thereof. For many years the influence which the oceans may exert on the carbon dioxide-content of the atmosphere has been a subject of much discussion. If chemical reactions in the sea or the activity of hving organisms therein influence the carbon dioxide-content of the atmosphere, it is evident that such factors may be of great significance in the photosynthetic activity of land plants. Carbon dioxide is continually being added to and lost from the sea. Submarine volcanic springs and the action of marine ani- mals, the latter forming normal calcium carbonate from bicarbonates, both are among the factors which tend to increase the carbon dioxide-con- tent, while the development of the marine flora tends to remove carbon dioxide. According to a theory of Schlossing *^ the oceans serve as the great regulators of the carbon dioxide-content of the atmosphere. Ac- cording to this view, due to the presence of carbonates and bicarbonates of the alkaline metals and alkaline earths, sea-water has the capacity of absorbing or giving ofif carbon dioxide, depending upon the partial pres- sure of this gas in the atmosphere. If the carbon dioxide-content of the atmosphere increases, the excess will l)e taken up by the sea-water and if there is a decrease in atmospheric carbon dioxide, this gas will be liberated from the sea-water until equilibrium is reached again. In view of the fact that complete concordance has not been attained in regard to the carbon dioxide-content of the air over the sea and on land, there still exists some difficulty in establishing the validity of Schlossing's theory. Krogh ** reported the carbon dioxide-tension of the water of the North Atlantic corresponded to a carbon dioxide-content of the atmosphere of 0.023 per cent. This has been interpreted by Reinau to mean that the carbon dioxide equilibrium is shifting in favor of sea-water and that the oceans are actually absorbing carbon dioxide, although Schlossing stated that the sea does not continuously absorb the carbon dioxide and that the adjustment is a very slow one. Reinau *=^ concludes that there is little connection between the carbon dioxide reservoir of the atmosphere and that of the sea. In fact, he considers that the only regulator of carbon dioxide is the life activity of plants and animals, and that the variations in the carbon dioxide-content are to be ascribed entirely to the factor which regulates "assimilation'' and "dissimilation" of carbon, namely, light and heat, or, ultimately the sun. There appears to be little doubt as to the possibility of an interchange of carbon dioxide between the sea and the atmosphere. The conditions afifecting this equilibrium over long periods of time are, however, numerous and apparently impossible to evaluate and correlate in a quantitative sense. The possible significance of variations in carbon dioxide-content of the ''Schlossing, Cnmpt. rend.. 90. 1410 (1880). " Krogh. Compt. rend., 139, 896 (1904). *•' Reinau, F... Kohlcnslhirc nnd Pflanzcn, p. 140. 44 PHOTOSYNTHESIS atmosphere in relation to the great chmatic alternations as evidenced by glacial periods and epochs of luxuriant vegetative growth, is, after all, a matter of speculation upon which we cannot enter here. These are questions which involve thorough discussion from the geological view- l>oint **^ and upon which there still exist divergent opinions. The condi- tions of interchange of carbon dioxide between atmosphere and sea as they exist at present are sufficiently complex. The biochemistry of the sea is of itself a chemical constellation. The living organisms in the sea exert a decided influence on the composition of the water. The plants absorb large quantities of carbon dioxide and convert it into organic compounds. United with these are hydrogen, nitrogen, sulphur and phosphorus. These compounds are in turn appropriated by the animals of the sea, constituting a system fully as complex as the life of land plants and animals. Many of these animals liberate carbon dioxide from bicarbonates, precipitating calcium carbonate, and thus enormous calcareous deposits of tufa and travertine are formed. After death the bodies of both plants and animals decompose yielding ammonia, hydrogen sulphide and carbon dioxide. These compounds are again drawn into the metabolism of other organisms. The whole forms an enormously complex series of cycles involving fine adjustments and stupendous quantities of material, an evaluation of which has not yet been attained. The whole question of the carbon dioxide-content of the atmosphere in different parts of the world, under varying conditions and its relation to the sea and the life therein is in need of thorough study over a longer I^eriod of time. It is only within recent years that reliable methods of analysis have been perfected. Such a survey should include studies not only at marine stations near the coast but also at localities far distant from the mainland and with consideration of the effect of the great streams. McClendon *' has made some very valuable contributions to the meth- ods of analysis of sea-water. Some of his conclusions are here quoted. "It has been supposed that the amount of CO2 in sea-water regulates the growth of seaweed, but the reverse is probably more nearly correct. The respiratory quotient of marine organisms seems to be about 0.7 to 1.0 and the respiration of animals and plants reciprocal. Some marine bacteria take their oxygen from nitrates, but this effect must be minute, since the supply of nitrates is small. The atmosphere cannot be the chief regu- lator of the CO2 of the sea, since there is about 30 times as much CO2 in the sea as in the air. There is always a superabundance of COo in sea- water to supply the needs of green, red, or brown seaweed, but by using it the plants increase the pH of the water. It seems probable that the plants grow rapidly until the pH that is most favorable to them is ex- ^•Chamberlin, T. C, and Salisbury, R. D., GcoJoqv. New York, 1907. Vol. I, 640. Arrhenius, 5". Phil. Mag. (5), 41, 237 (1896). Abbot and Fowle, Ann. Astrophxs. Obs. Smithsonian Inst., 2, 172, 175 (1908). "McClendon, J. F., Carnegie Inst. Wash. Pub. No. 251, p. 37 (1917). THE ORIGIN OF ORGANIC MATTER 45 ceeded. This is in harmony with the fact that the pH of the great oceans to the depth penetrated by light is more constant than the CO2 tension, the pH varying from about 8.0 to 8.25 and the CO, tension from about 0.00015 to 0.0005 atmosphere. The sea may be compared to the body of one of the higher vertebrates. The mammal regulates the pH of the blood through the action of the respiratory center. The sea regulates the pH of its surface-water most probably through the action of seaweed. The limit in the supply of oxygen probably prevents animal life from getting the upper hand temporarily and thus endangering the communal life in the sea. "It seems probable that seaweeds regulate the CO2 of the atmosphere. The gaseous exchange between sea and air is necessarily at the surface and is comparatively slow. Bohr observed that the absorption of CO2 from an atmosphere of the pure gas by CO.; — free water that is stirred (probably more vigorously than the sea ever is) is about 0.1 cc. per square centimeter of surface per minute. Since the difference in COo tension between air and sea seems never to exceed 0.02 per cent of that in Bohr's experiment, except in the polar regions, the rate of diffusion would not exceed 0.00001 cc. per square centimeter per minute or 0.1 cc. per square meter per minute in a storm, and necessarily much less in calm weather on account of the lessened rate of stirring at the surface. When we consider the volume of the sea and air compared to the sea-air surface, the fact becomes intelligible that the CO2 in the air is relatively constant (3 per 10,000) in the different regions of the world where it has been accurately measured, whereas the CO2 tension of the sea-surface varies from 1.5 to 5 per 10,000. The air is stirred more rapidly than the sea, and the CO2 of the air seems to be determined by an equilibrium between gain in CO2 over some regions of the sea-surface and loss over others. The partial pressure of CO2 in the air is therefore the average CO2 tension of the sea-surface. The burning of billions of tons of coal per year is probably changing the CO2 content of the sea and of the rocks and not of the atmosphere." McClendon's analysis of the relation of the carbon dioxide in the atmosphere and in the sea is much more thorough than that of Reinau. McClendon points out that Krogh's results show that the carbon dioxide- tension of sea-water increases with depth and that the tension of the sea- surface is not in equilibrium with the air. This is also evident from the work of Schulz already cited. Moreover, the carbon dioxide-tension of sea-water varies; Krogh found it to vary from 1.5 to 3 per 10,000 in the North Atlantic and McClendon from 3.3 to 4.7 per 10,000 at Tortugas near the Gulf Stream. McClendon assumes that the water maintains a relatively constant carbon dioxide-content and that on its flow northward the carbon dioxide-tension decreases with the lower temperatures. In general it would appear that McClendon's investigations support the gen- eral theory of Schlossing. It has been assumed, on the basis of some analytical data, that there 46 PHOTOSYNTHESIS is a cap or belt of air of low carbon dioxide-content at the poles of the earth. The mixing of a portion of this mass of cold air is supposed to be intimately associated with formation of cyclones. It might be expected therefore that the passage of a "low"' would give some indication in the carbon dioxide-content of the air. Some evidence of this is claimed by Lundegardh,*® though a clear demonstration thereof is made difificult on account of the influence of other factors. The carbon dioxide-content of the sea and atmosphere represents a dynamic equilibrium. There are, on the one hand, sources which are producing carbon dioxide in prodigious quantities and, on the other hand, agents which are removing the carbon dioxide from air and sea. The nature of these sources and agents differs widely. The situation can be schematically represented about as follows : CO, PRODUCTION Springs Volcanoes Combustion of Coal, Wood and Petroleum \ Respiration of lower organisms, plants and animals -> Atmosphere and Sea CO2 CONSUMPTION Photosynthesis by Plants CaCOs-forming organisms - Chemosynthesis of certain bacteria The weathering of rocks It is not possible to discuss here exhaustively all the factors which effect this equilibrium. We are dealing with chemical changes taking place on an enormous scale and in many cases involving extensive periods of time. Nor are we probably familiar with all the factors which have a part in this great drama in which the life of man plays but an insignificant role. For this reason most calculations are but rough approximations. Let us consider first the sources of carbon dioxide. What may be termed the inorganic sources are perhaps of the greatest influence. Thus, there are ancient craters (e.g. Agnano near Naples) and gas wells which have been discharging carbon dioxide since time immemorial. The composition of the gases of volcanic eruptions varies greatly but carbon dioxide is a frequent component.*^ The observations of Lewy ^° on the island of Guadaloup, though not very accurate, indicate that after the erup- tion of the volcano the atmosphere contained fifty times the normal amount of carbon dioxide. No consistent efforts have been made to determine whether the great volcanic disturbances result in changes in the atmospheric carbon dioxide-content at distances from the place of eruption. Even less is known concerning the volcanic eruptions at the bottom of the sea. Min- eral springs constitute a very important source of carbon dioxide ; the waters of some of them are supersaturated with the gas as they issue *' Lundcgardh, Der Krcislauf der Kohlcnslhire. p. 38. "Allen, E. T. J., Franklin Inst., 193, 29 (1922) for bibliography and chemical analyses of volcanic gases. ''Lewy, M., Ann. Chim. Phys., 8, 425 (1843). THE ORIGIN OF ORGANIC MATTER A7 from the earth. However, of none of these sources can an approach at quantitative estimation be made. More accurate estimations can be made of carbon dioxide produced through the combustion of mineral fuels. A detailed compilation of the world's coal production for 1920 has been made by Sievers." According to these figures the annual production of coal amounted to a total of 1,317,000,000 metric tons (2204 lbs.) or 1,317 X 10' kilograms. This in- cludes anthracite, bituminous and lignite or corresponds at the most to about 70 per cent carbon which when burned would give about 338 X 10^° kilograms of carbon dioxide. Thus we have 338 X 10^° kilograms car- bon dioxide from the burning of coal compared to the 21 X 10^* kilo- grams of carbon dioxide in the atmosphere, or an annual production of 0.16 per cent of the existing carbon dioxide. At this rate, other condi- tions remaining the same, it would take about 650 years to double the concentration of carbon dioxide in the atmosphere. ^- If the amount of carbon dioxide produced annually by the burning of coal were distributed through the entire atmosphere it would exert but a very slight effect and could not be detected by our present methods of analysis. It is possible, however, that the mixing above about 100 meters is slow, so that it may be expected that the carbon dioxide-concentration near large industrial centers is higher. Coal has been used on a large scale for only about one hundred years. If the rate of increase in consumption continues it might be expected that this would result in an accumulation of atmospheric carbon dioxide. On the other hand such an increase in carbon dioxide-content of the atmosphere would result in augmented photosynthetic activity, which in turn would tend to reestablish the equiUbrium. To what extent such an increase in carbon dioxide-content would be noticeable in the vegetation of the earth it is difficult to calculate. Increase in the rate of photosynthesis is about directly proportional to increase in carbon dioxide-concentration under what may be termed natural conditions. That is, for instance, with bright sunlight, at 20-25°, and ample water supply a plant will about double its photosynthetic rate when the carbon dioxide concentration is raised from 0.03 to 0.06 per cent. However, the influence of external factors on the rate of photosynthesis is very complex, as will be shown later ; moreover, photosynthesis is but one factor affecting the development of a plant, so that it is very difficult to make predictions regarding the final effect of any single factor. More- over, an increase in the carbon dioxide-content of the atmosphere would in time be largely equalized by the influence of the sea. During the last few years there has been much discussion and specula- ■^Sievers, E. G., Gas Age Record, 51, 757-761 (1923). "'The annual coal consumption has been constantly increasmg so that various older estimations are at slight variance with this figure. Krogh, Meddelelscr om Groenland. 26, 419 (1904). Van Hise, Mon. U. S. Geol. Survey, 47, 964 (1904^ Dittmar, Challenger Report, Vol. 1, pt. 2, 954. Chamberlm, Jour. Geol., 7, 6b2 (1899). 48 PHOTOSYNTHESIS tion regarding- the influence of the carbon dioxide-content of the atmosphere on our economic plants. Many schemes have been advanced for increas- ing the atmospheric carbon dioxide. These have included the setting afire of the coal deposits of the polar regions. Nernst has suggested that this coal, which is inaccessible for mining purposes, be ignited and kept burning by means of shafts. It would thus become useful to man by increasing the carbon dioxide-content of the atmosphere, resulting in higher crop yields. It has also lieen suggested that powdered coal of low grade be spread over the cultivated land. Through auto-oxidation the coal would yield carbon dioxide which would be directly available for the plant. ^^ The respiration of plants including fungi and bacteria yields very con- siderable quantities of carbon dioxide. Fundamentally, of course, most of the material which is thus burned is of photosynthetic origin, so that these organisms are simply reconverting the products of photosynthesis into carbon dioxide and water. The rate of respiration of most of the lower organisms is relatively very high, so that even when the period of their activity is short the amount of work accomplished is great. The amount of carbon dioxide produced by micro-organisms per unit surface or weight is many times that of man. The number of substances which bacteria can convert into carbon dioxide is very great ; this assures the complete conversion of plants and animals after death to the simplest substances. The complete process of the degradation of carbon compounds by lower organisms presents in detail an exceedingly complex and intricate picture of the interrelation of these organisms. The chief product of photosynthesis is probably cellulose. This is converted by various organisms into soluble carbo- hydrates or in the absence of air into formic, acetic, butyric acids, carbon dioxide, hydrogen and methane. The latter two gases are further oxidized by special organisms to carbon dioxide and water, so that while there are enormous quantities of hydrogen and methane produced in this manner annually and have been for eons, only traces of these gases are found in our atmosphere. The soluble carbohydrates may be fermented to alcohols and fatty acids which in turn are converted by other bacteria and some fungi to carbon dioxide and water. The anaerobic cellulose-destroy- ing organisms ])lay an enormously important role in the carbon dioxide cycle, as much of the cellulose is finally buried under water or soil. Similarly other substances, such as proteins, which contain besides car- bon, hydrogen and oxygen also nitrogen, phosphorus and sulphur are com- pletely broken down to simple inorganic compounds. In these processes, many of which are analogous to step-reactions, there often exists a close interrelationship between aerobic and anaerobic organisms. Were it not for the lower organisms such as bacteria and fungi which rapidly convert the components of dead ])lants and animals to carbon dioxide and other simple compounds, it is conceivable that large quantities of carbon dioxide "Reinau, E., Kohlcnsdure und Pnanzen. Halle, 1920, p. 125. THE ORIGIN OF ORGANIC MATTER 49 would in time be withdrawn from the atmosphere, and that the rates of change in the carbon dioxide cycle would be greatly retarded. The effect of such a state of affairs on the life of our planet is impossible to imagine. An important factor in the production of carbon dioxide by decaying animal and vegetable matter is that it is produced under or on the surface of the ground where it soon becomes directly available again for photo- synthesis. Air analyses show that the carbon dioxide content near the ground is often many times that at several feet higher. The amount of carbon dioxide produced by lower organisms cannot be calculated with any degree of accuracy. Over a period of years it would, of course, not be in excess of the amount of carbon dioxide reduced by the chlorophyllous plants, though probably it is greater than the amount produced by higher plants and animals. The higher chlorophyll-bearing plants cannot be regarded as carbon dioxide producers, for in the total span of their life history they reduce carbon dioxide and convert it largely into carbohydrates. Of course all of the carbon dioxide reduced by a chlorophyllous plant is not perma- nently laid down as carbohydrate. The plant is a living organism and requires energy for its life processes ; this it obtains from the oxidation of carbon compounds previously produced. Thus a portion of the ma- terial produced in photosynthesis is oxidized by the plant and is in a sense the operating expense of the factory. This factor varies with different species, but in most cases 15 to 20 per cent of the material synthesized is consumed in this manner. The carbon dioxide production of germinating seeds is very high. In some cases as much as 25 per cent of the dry material is consumed within 24 hours. In this manner most of the stored material is depleted by the time the plant becomes a self-supporting organism. But taken as a whole and during the entire course of its life the chlorophyllous plant is on the minus side of the carbon diox- ide cycle. The carbon dioxide production of animals is in a sense a reversal of the photosynthetic process. The food of animals is directly or indirectly a product of photosynthesis. The amount of carbon dioxide which can be produced by the respiration of animals is naturally dependent upon the quantity of material produced by photosynthesis. The respiration of animals, therefore, tends to maintain the constancy of the atmospheric carbon dioxide. Not all of the carbon in the food of animals is exhaled as carbon dioxide. In mammals about 5 per cent of the carbon is excreted in the form of organic compounds. The latter are in turn broken down by micro-organisms. The rate of carbon dioxide production and the degree of oxidation of the organic food materials varies greatly with different animals. Some idea can be gained of this source of carbon dioxide from a calculation of the amount produced by man. While the amount varies greatly with weight and muscular work, we can take the value of 900 grams of CO.. produced per individual per day. The 1750 million in- 50 PHOTOSYNTHESIS habitants ®* of the earth would thus produce 1575 million kilograms of carbon dioxide or 575 X 10^ kilograms per year. Compared to the atmospheric supply of 2 X 10^^ kilograms this is a very insignificant amount, about 0.02 per cent; it nevertheless corresponds to 156 X 10^ kilograms of carbon or approximately 230,000,000 tons of coal. It is impossible to estimate the quantity of carbon dioxide produced by other animals. In general, however, it is probably safe to state that of the total of what chlorophyllous plants produce in the form of carbon compounds through photosynthesis, the greater portion is reconverted into carbon dioxide not by animals, but by lower plants and micro- organisms. Turning now to the other side of the carbon dioxide cycle, that of COa-consumption, photosynthesis by the chlorophyllous plants is the factor of foremost importance. While some attempts have been made to esti- mate the amount of photosynthesis on the surface of the entire earth, this involves so many variables that the results of such calculations must be regarded as subject to more or less drastic revision. Such a calculation, it has been claimed, would be of value in determining the total amount of food the earth is capable of producing and consequently contribute to the general probleni of world population. But different plants differ enormously in their behavior under like conditions which, together with differences between tropical and temperate regions, makes such a calcula- tion, with the available data, impossible. The amount of carbon dioxide which plants remove from the atmos- phere is very considerable. Thus Noll ^^ has calculated that a tree of 5000 kilograms dry weight contains about 2500 kilograms of carbon. In order to have obtained this amount of carbon the tree must have re- moved the carbon dioxide from about 12 million cubic meters of air. Similarly the old calculations of Sachs, ^^ while they cannot be taken as an exact measure of photosynthetic activity, give an idea of the order of magnitude of the function of plants. The leaves of an ordinary sun- flower plant have an area of about 1.5 square meters. Sachs found that such a plant absorbs 660 cc. or 1.3 grams of carbon dioxide per hour. In a ten hour day the plant would absorb about 400 grams of carbon dioxide per month. If the entire land area of the earth were covered with sunflowers so that on each square meter there was one plant or a million plants to each square kilometer, the plants covering the 135 million square kilometers of land would absorb 54 X 10^^ kilograms carbon dioxide in a month. At this rate, the 21 X 10" kilograms carbon dioxide of the atmosphere would last about 40 months. The limited significance of such calculations requires no further comment. The concentration of atmospheric carbon dioxide represents a condition of dynamic equilibrium in which photosynthesis is but one factor. East, E. M., Mankind at the Crossroads. N. Y., 1924, p. 111. Noll, Strasburger, Lehrbuch der Botanik. Jena, 1906. 8th Edition, p. 181. Sachs, J., Arbeiten aus dem bot. Inst. Wilrzburg, 3, 1-33 (1884). THE ORIGIN OF ORGANIC MATTER 51 The quantity of carbon dioxide consumed in the weathering of rocks can also not be given any precise valuation though undoubtedly considerable amounts are thus consumed. The carbon in the coal and petroleum de- posits and probably also that in the sedimentary rocks was drawn from the atmosphere ages ago. It has already been stated that photosynthesis constitutes the chief means of counteracting the "running down" in energy of our planet. Photosynthesis is essentially a process of reduction, in which a carbon compound containing the maximum amount of oxygen is reduced to com- pounds of carbon containing hydrogen and oxygen in the proportion in which the latter two elements are found in water, i.e. carbohydrates. This is an endothermal reaction and the energy for it is obtained from the radiations of the sun. Similarly, nitrates are reduced to amino com- pounds. Further reductions are carried on through the metabolic activity of the plant resulting in alcohols, fats and hydrocarbons. For the latter reactions, so far as we know, solar energy is not essential, the plant appar- ently being able to utilize the energy derived from the oxidation of a por- tion of its carbohydrate supply for these reductions. The chemical kinetics of the metabolic reduction reactions taking place in plants is still an unsolved problem. Largely by means of these reactions there are produced the enormous number of organic compounds found in plants and for which these have become valuable to man. The reducing power of the metabolic activity of plants is illustrated by the fact that a wide variety of compounds can be reduced, many of which never enter into the normal metabolism of plants. This is clearly demonstrated by experi- ments with the yeast plant.^' If this plant has an ample supply of sugar it is capable of reducing many different compounds including aliphatic, aromatic and cyclic aldehydes, ketones, nitro-compounds, sodium thio- sulfate and others. Thus the plant in its photosynthetic and metabolic activities may be considered as a reducing machine, providing materials which are capable of combining with oxygen and thus serve man as food and fuel. The object of agriculture is, of course, the production of food materials for man by means of the photosynthetic process. Our present chief sources of energy in the form of coal and petroleum are likewise the product of photosynthetic activity, produced ages ago. It is only comparatively recently that man is realizing that he is using energy at a greater rate than it is being placed at his disposal, that his industries depend upon the accumu- lation of centuries. Especially in the case of petroleum is it being realized that, while depletion of the supply may not be imminent, nevertheless this source of energy is not in the form of a continuous flow, and that the question of its exhaustion should be given timely consideration.^^ While opinions are still divided as to the extent of the world's petroleum reserves, "Neuberg and Ehrlich, Biocliem. Zcit., 101, 276 (1919-1920). ''White, D., Sihlcv J own. Eng., 1920, p. 156. Burrell, G. A., Oil Gas Journ., 1920, p. 84. Smith, G. O., Amer. Petroleum Institute, Bull. 132 (1920). 52 PHOTOSYNTHESIS the necessity of investigating other forms of fuel has been very generally recognized. Probably foremost among these "synthetic fuels" is alcohol, ihis has been used in limited amounts for motor fuel for many years. Owmg to a complex of economic reasons the development of the industry has had many difficulties to contend with. There are in general two methods of alcohol formation : in the first can be included those synthetic methods which start with ethylene or acetylene and in another the fermentation of various vegetable materials. Regarding the first of these Monier- Williams =^^ concludes: "On the general question as to whether, apart from cost of production, it is sound policy to look to synthetic alcohol as one of the motor fuels of the future, the following points may be considered. The source of the carbon in synthetic alcohol is coal, while in fermentation alcohol it is derived from atmospheric carbon dioxide. Although the world's reserves of coal are ample, and there is little danger of their being exhausted within a reasonable period, it is likely that the cost of raising the coal will gradually increase as the more easily worked seams are used up and the accessibility of the material diminishes. "Economy in the use of coal is a matter of national importance, and it is questionable whether alcohol is the most economic form in which the available carbon can be utilized as motor fuel. On the face of it, it would seem preferable to work in the direction of utilizing more directly the heat of combustion of acetylene, possibly by polymerizing it into hydro- carbons of higher boiling-point, rather than to add to it the elements of water which represent so much dead weight in the resulting fuel. Quite apart from this there is the question of the electrical energy required for the manufacture of calcium carbide from lime and coke. Where ample water power is available, the demands made upon coal are limited to that necessary for the actual formation of the carbide in the furnace. It is claimed, however, that under the most favorable conditions electrical power may be obtained almost as cheaply in the neighborhood of coal- fields as near waterfalls. Where coal is used as a source of power, it has been estimated that the amount consumed in the manufacture of synthetic alcohol is nine times as great as that required for the same quantity of fermentation alcohol. "Another point which has been raised has reference to the value as a fertilizer of calcium cyanamide, prepared from calcium carbide by the Frank-Caro process. One ton of carbide will yield approximately 110 gallons of alcohol. If the carbide is used for the production of calcium cyanamide, Ca.N.CN, the nitrogen thereby made available for crops amounts to 550 to 600 pounds. Applied to potato land under normal weather conditions this quantity of nitrogen should result in an increase of over 20 tons in the crop yield. Twenty tons of potatoes will yield at " Monier-Williams, C. W., Power Alcohol. Oxford Technical Publications. London, 1922, p. 184. THE ORIGIN OF ORGANIC MATTER 53 least 400 gallons of alcohol, or nearly four times as much as could be obtained direct from one ton of carbide." For our purpose we shall confine ourselves to a brief discussion of the production of alcohol through the fermentation of the products of photosynthesis. This in short depends upon the hydrolysis of various polysaccharides such as cellulose, starch or sucrose to monosaccharides and the conversion of the latter according to the general equation, CeHiaOe -> 2 C2H5OH -|- 2 CO2, by means of fermentation. From the standpoint of fuel the most important constituent is the carbon, of this about 66 per cent is converted into alcohol, the remainder passing into the useless CO2. The amount of fermentable material contained in different vegetable materials varies greatly. Moreover, the cost of hydrolysis varies also TABLE 6 Alcohol Obtainable from Various Vegetable Material. Total Average Fermentable Crop Yield 95 Per cent Yield 95 Per cent Carbo- Yield Alcohol per Ton Alcohol per Acre hydrates per Acre U. S. U.S. Material. Per cent in Tons Gallons Liters Gallons Liters CEREALS Wheat 65 0.5 99.6 375 50 189 Barley 60 0.65 90 341 59 223 Rye 64 0.4 96 i6i 38 144 Oats 55 0.6 84 318 50 189 Maize 67 0.7 103 390 72 273 Sorghum grain .... 67 0.7 103 390 72 273 Rice (cleaned) .... 76 0.43 102 386 44 167 TUBERS Potatoes 17.5 6.5 25 95 164 621 Cassava 30 9 44 167 396 1,499 Jerusalem artichoke. 17 7 23 87 160 602 Sweet potato 27 4 42 159 168 636 Yam 17 5 24 91 120 454 ROOTS Sugar beet 15 14 25 95 353 1,336 Mangolds 5 20 8 31 168 636 Sugar mangolds ... 8 20 13 49 264 999 Sugar cane 13 15 20 76 306 1,158 Sorghum cane 14 15 14 53 224 848 Molasses (cane) ..58 .. 88 ?,2,i Molasses (beet) ... 50 .. 72 273 TREES Mowra flowers (dried) 50 1.3 83 314 108 400 Nipa palm (sap)... 12 15 18 68 260 984 Zamia palm (pith).. 13 .. 22 83 Grass tree (Xanthorrhaea) .... .. 23 87 Horse chestnuts and acorns .. 43 163 Iceland and Reindeer moss ... 60 . . 76 288 Sea-weed (dry) .. 34 129 54 PHOTOSYNTHESIS with different raw materials. Table 6 is taken from the book of Monier- W'illiams ; the values have been converted into U. S. gallons and liters. There are, of course, a great many more plants than those given in Table 6 from which alcohol is produced, the plants used in different parts of the world depending upon a number of different agricultural and eco- nomic conditions. It is only the hexose sugars, d-glucose, d- fructose and d-mannose that can be converted readily into alcohol by the enzyme of yeast. Galactose is present in the products of hydrolysis of many plants, but this sugar is fermented with difficulty. The pentose sugars which comprise a considerable proportion of many plants are not fermented with pure yeast. Recently other organisms have been found which convert the pentose sugars into a variety of products, but this is not as yet carried out on a large scale. The production of alcohol on a large scale has usually been consid- ered from the economic viewpoint. Under present circumstances this is essential and must include the many agricultural conditions related to soil, climate, water supply, fertilizers, etc., as well as the factors of methods of production, labor, transportation, markets, by-products, etc. The problem therefore becomes an exceedingly complicated one. But here we are, for the moment, not concerned with alcohol as competitive or even supplementary to petroleum but rather with the perhaps somewhat specu- lative idea of whether alcohol can replace petroleum, i.e. whether it can serve as one of our principal sources of energy. In view of the fact that the raw materials from which alcohol can be produced are all plant products the question immediately arises what effect such a gigantic undertaking would have on the production of foodstuffs. There is no sound indication that man will be able to derive his food from any other source but the soil. So that if his means of obtaining industrial energy is to be the same as that for obtaining food- stuffs, the cjuestion is raised whether there will be a competition for arable land to be devoted to food production on the one hand or fuel production on the other. Boyd ®° has made an interesting analysis of the possibilities of using vegetation as a source of motor fuel. The fol- lowing figures give an idea of the situation : Average annual U. S. production of corn, 1913-1919, bushels. .2,740,000,000 Average annual Acreage in corn, 1913-1919 106,000,000 Alcohol from corn at 2.75 gallons per bushel 7,500,000,000 Concerning this Boyd states: "The heating value of this amount of alcohol is about equal to that of 5,000,000,000 gallons of gasoline. The production of gasoline in the U. S. during 1920 was very close to this amount, it having been about 4,900,000,000 gallons. The average acreage in corn as given above is equal to 166,000 square miles, which is more than four times the total area of Ohio. In view of the fact the possible alcohol production from corn represents close to 60 per cent of the total "Boyd, T. A., Jouni. Ind. and Bug. Chcin., 13, 836-841 (1921). THE ORIGIN OF ORGANIC MATTER 55 possible amount of alcohol that could be prepared from all the starch and sugar containing foodstuffs produced in the United States, and that such a large acreage is required for its production, the possibility of a sufficiently large increase in production of such materials to be diverted to the manufacture of motor fuel seems very unlikely. At any rate if large quantities of motor fuels are to be prepared from vegetation another material, if not instead of foodstuffs, at least in addition to foodstuffs, must be relied upon as a source." A similar analysis has been made by Lane and Bauer " v^ho discuss the various means of developing motor fuels. In considering alcohol as a possible motor fuel, they conclude: "For example, the corn that can be grown on an average acre of Oklahoma soil in a year will yield about 3 barrels of alcohol. Many of the oil fields of Oklahoma have an ultimate production of about 3,500 barrels of crude petroleum per acre, of which about 1000 barrels can be considered as gasoline. One would have to grow corn on an acre of Oklahoma soil land for about three hundred years to obtain a volume of alcohol equal in fuel value to a supply of gasoline produced in from five to ten years. ... Of course, the area of oil fields in the country is but a small part of the total area of the United States, but this simply emphasizes the fact that while we can grow corn over an area many times as large as the area of the producing oil lands, it will then have to be harvested and brought to the distilleries, a laborious and expensive task. Furthermore, edible grain of any kind is usually too valuable to be used in the manufacture of alcohol. And even if we had a surplus it would be a mere drop in the bucket. A surplus of a 1,000,000,000 bushels of corn would sound tremendous to the farmers of this country, but it would yield only 250,000,000 gallons of motor fuel, enough to run our cars and trucks about three weeks." There is no doubt that the growing of plants for the purpose of obtain- ing fuel would require such enormous areas of land that it would ma- terially affect the production of foodstuffs under present systems of agri- culture. It has happened that corn has been used directly as fuel. When prices for corn were exceedingly low (32 cents a bushel) it was an economy for the farmer to burn his corn rather than buy coal at 16 dollars a ton. Under such conditions it has happened that even large public utilities have been run with grain as fuel. But this represents extremely abnormal economic conditions and must be considered in the light of an emergency measure rather than as a contribution to the fuel problem. De Baufre *'- has determined the heat value of corn and concludes that with coal at $10.00 a ton, com on the cob must be less than 20 cents a bushel of 70 pounds to make the use of corn as fuel economical. At the same time in the production of food by means of agriculture there are tremendous quantities of wajte materials. These have high energ}^ content and many of them could be converted into fuel by alcoholic Lane, F. W., and Bauer, A. D., Ind. and Eng. Chen.. 15, 479 (1923). De Baufre, Power, 36, 212 (1922). 56 PHOTOSYNTHESIS fermentation and similar processes. From the waste molasses of the sugar industry alone there are some two hundred million gallons of alcohol produced annually. However, in the utilization of such surplus and waste materials for large scale production a number of difficulties are encountered. The supplies of such materials are often uncertain and irregular, so that operation of a plant is overtaxed at one time and idle at another. Storage of a great deal of such waste material is often im- possible or expensive, which has made the continuous operation of the distilleries impossible. One of the requisites for the successful operation of an industry of this kind seems to be that the supply of the product is continuous or synchronous with the demand, thus obviating the expense and hazard associated with the storage of enormous quantities of alcohol. With seasonal crops this has been exceedingly difficult to accomplish. Probably the most promising waste material of vegetable origin is the cellulose from the lumber industry. Such material answers many of the requirements of a source of fuel : it is easily produced and its supply can, with proper management, be continuous. The chemical methods for hydrolyzing the cellulose to fermentable sugars are, in principal at least, very simple and the waste products from the fermentation process can be returned to the soil to maintain its fertility.®^ Although this problem has been the subject of much intensive research there is still much to be done to assure good yields of alcohol. At present there are obtained from 15 to 25 gallons of 95 per cent alcohol from a ton of sawdust which is about 50 per cent cellulose or a yield of approximately 35 per cent of the theoretical amount. The increase in these yields is dependent upon the development of the chemistry of the processes involved. Whether there is sufficient waste cellulose available to supply the needs is very difficult to ascertain. It has been estimated that there are approxi- mately 5,000,000,000 cubic feet of waste in the woods and at the mills. .Assuming a cubic foot of wood to weigh 30 pounds this would be 75,- 000,000 tons which at 20 gallons of alcohol per ton would yield 1,500,- 000,000 gallons of alcohol. This quantity represents about one third of our present motor fuel requirements. In all probability the quantity of waste material could be increased and it is also to be exp>ected that the yield of alcohol can be increased. P)y making what appear to be rather generous allowances for increase in waste material by various means, Hibbert calculates that about 75 per cent of present requirements can be cared for. Calculations of this nature are. of course, of limited value, as they do not include the various economic factors of labor, transporta- tion, interest charges, etc., which usually are the determining ones. But such approximations as can be made, even in their most sanguine aspect, do not assure a continuous supply of fuel in quantities to meet present petroleum consumption. There is one other factor which may play a very important role in "Sherrand, E. C, Chem. Age, 29, 53 (1921). Hibbert, H., Journ. Ind. and Eng Chem., 13, 841 (1921). THE ORIGIN OF ORGANIC MATTER 57 supplying the needs for fuel through vegetable material/"* This factor is the productivity of the tropics. While agriculturalists are apparently not in agreement on the potentialities of the tropics, there is no question but that these regions are very favorably suited for the conversion of solar energy through photosynthesis. Ikit, on the whole, the conditions in the tropics are even less amenable to careful survey over a period of years than the temperate zones. The question of the kind of plants best suited for fuel production has received little attention, apparently even as to whether these should be sugar and starch or oil producing plants. In the production of alcohol fuel is one of the most important items ; every gallon of 95 per cent alcohol requires about 15 pounds of coal. In tropical countries refuse from crops must constitute the main fuel supply. In the cane sugar industry the residual fibre or "bagasse" constitutes about one fourth of the weight of the canes. This waste material is un- suitable for the preparation of ethyl alcohol, and as yet probably can be used only as fuel directly."^ If alcohol is used as fuel, starting with a 10 per cent solution of alcohol by weight and producing 91 per cent alcohol, about one third of the alcohol produced will be required to furnish the heat necessary for distil- lation."" While a solution of alcohol 91 per cent by weight would probably be satisfactory for fuel, considerably more heat would be required to produce 95 per cent alcohol. These and many other economic and technical problems such as transportation and water supply make the enterprise of fuel alcohol a very complicated one. But if in the future we must look to agriculture for our industrial energy, there is no doubt that the tropics will play a very important part. How such a state of affairs would affect the production of food it is impossible to say. The temperate portions of Europe and Asia with the exception of Russia are already overpopulated. Tropical Asia largely is in the same condition. It is doubtful whether these as well as the arable portions of the United States and Canada can be counted on for anything but the production of foodstuffs. There still remain huge agri- cultural areas in tropical South America and central Africa where the population is relatively sparse. Whether these areas are destined to offer relief to the increasing population of Europe and to over-populated Asia, or can be put to other uses than food production raises economic and political questions far beyond the domain of this work. But the relation of land areas to population is one of the most funda- mental economic conditions affecting the lives and destiny of a people. Hence any new factor introduced into these conditions is certain to have profound consequences. The utilization of land areas in the United States has been the subject of much careful study particularly by the «4 65 Whitford, H. N., Jotirn. Ind. and Eng. Chcm., 14, 151 (1922). Sherrand, E. C, and Blanco, C. W., Joiirn. Ind. and Eng. Chem., 12, 1160 (1920). ** From private communication of Dr. W. H. Rodebush. 58 PHOTOS] 'N THESIS Department of Agriculture.*'' We cannot here enter upon a discussion of the intricate problem of land requirements in relation to increasing population. Suffice it to state that from the surveys which have been made it appears exceedingly doubtful v^hether any considerable land areas in the United States could be spared from agricultural needs for the production of fuel. From the report of the U. S. Department of Agri- culture, 1923, we learn that 94 per cent of all the land available for crops, pasture and forest are now employed for these purposes, although of these, large areas are and always will be of low productiveness and other areas are under-used. Table 7 is taken from the above-mentioned report. TABLE 7 Crop and Pasture Land that Would Be Required for 150,000,000 People As- suming No Change in per Capita Consumption and Production PER Acre, Also No Exports of Agricultural Products AND No Change in per Capita Imports. Use of Land Area (Acres) Crop land 43L000,000 Woodland pasture 237,000,000 Other humid pasture 336,000,000* Semi-arid pasture 587,000,000 Total 1,59L000,000 —J * As a result of assuming the acreage of semi-arid pasture and woodland pasture to remain constant, the area of other humid pasture is increased in greater proportion than the increase in population. The report continues: "It has already been noted that if the present policy continued the area of land in forests, beginning with approximately 402,000,000 acres of standing timber, will rapidly diminish until the point of approximate exhaustion is reached. On the other hand, if we wish to provide enough forest land to grow our timber, a much larger quantity of land will be required ; at the present rate of growth and of waste and consumption per capita the enormous area of 1,465,000,000 acres would be needed for a population of 150,000,000 people. The impos- sibility of such an outlook is emphasized by combining this area with the 1,591,000,000 acres of crop and pasture land wliich, as shown above, would be required under similar assumptions. The total resulting require- ment would be 2,819,000,000 acres after allowing for duplications, or about 48 per cent more than the present land area of the continental United States. "The result suggests that if we are to maintain our present degree of self-sufficiency, for a population of 150,000,000 we must increase the average production per acre of our crop, pasture, and forest land, effect "Gray, L. C, Baker, O. E.. Marschncr, F. J., Weitz. B. O., ChapHne, W. R., Shepard, W., Zon, R., U. S. Department of Agriculture, Yearbook for 1923, 415-506. Baker, O. E., and Strong, H. M., U. S. Department of Agriculture, Yearbook for 1918, 433-441. East, E. M., "Mankind at the Crossroads," N. Y., 1924. THE ORIGIN OF ORGANIC MATTER 59 marked reductions in per capita consumption of farm and forest products, or make changes in both regards." It is at least highly probable that the relative increasing scarcity of land resources will in a measure be met by increased production per acre and readjustments of standards and habits of living. But if we accept the conclusions of the foregoing report, it certainly appears highly im- probable that any considerable area of land can be devoted to the produc- tion of industrial energy. There remains in this connection one factor the value of which it is as yet impossible to evaluate. This applies to the arid or desert regions. There are about 468.000.000 acres of arid grazing land most of which is of low productivity and about 67.000.000 acres of waste desert. It is calculated that there are about 30,000,000 acres capable of irrigation. But the greater portion of the arid lands will probably never be cultivated. Most of these regions are subjected to solar radiation of high intensity and have a large percentage of clear days. If science devises a means of directly utilizing solar energy, may not these be the regions of greatest value for such an undertaking? Recognizing the difficulties which beset the production of fuel through the intermediarv of crop plants, the suggestion has repeatedly been made that all that is necessary is that the botanist or horticulturalist create some kind of a "super-plant." That is. while it is realized that present crops are not adequate to supply the necessary amount of carbohydrate or oil. it ought not to be difficult for the biologist to produce a plant which instead of yielding 300 gallons of alcohol per acre would, for instance, yield 3,000 gallons. This is. of course, the same question which is constantly recurring in regard to the problem of food prodaction : why does not the agricultural explorer bring in new plants which yield two bushels in place of one ? The answer is the same for both cases, namely, that the food-plants which constitute our chief means of subsistence were already brought into cultivation by primitive man. While there is a large yariety of plants which are used as food in one form or another, man almost the world over, derives most of his food from the grass family, the grains. These together with the seeds of some legumens and oil-bearing nuts constitute the mainstay of man's existence. Undoubtedly our standards of living will in time undergo some readjustments and the yields per unit area be increased somewhat, but it is doubtful whether these will more than keep step with the increasing demand for food occasioned by a growing population. In advocating the production of material to replace our present sources of energy by means of agriculture not only is the question of area an im- portant one but a number of other factors immediately present themselves. For the production of energy on a large scale reliance could not be placed upon extant material. Recourse would have to be taken to very ex- tensive cultivation. \\''hile, as has been stated, there is still considerable arable land not yet under cultivation, it is very clear that any interfer- 60 PHOTOSYNTHESIS ence with the areas of land required for food production would lead to severe economic disturbances. Moreover, the fact is too readily forgotten that agriculture is a very sensitive and highly complex industry. Agriculture deals primarily with biological processes involving all the fine adjustments and balances of a growing organism. In the growth of a plant sunlight is but one of a number of determining factors. The fact that during critical periods in the development of a plant slight changes in climatic conditions during a short time may greatly reduce or entirely destroy a crop, serves to empha- size the hazard of obtaining energy through the intermediary of plants. In agriculture water supply and temperature are far more variable and determining factors than light intensity. On the proper coordination of these two factors, probably more than any other, depends the success of crop production. The multiplicity of pests and diseases which annually destroy a large per cent of our crops greatly increase the hazards of this industry. It is evident, therefore, that the plant at best is not only very inefficient in storing solar energy but is also rather unreliable. The plant not only produces organic compounds of great complexity from carbon dioxide and water by the use of solar energy, but also uses a con- siderable portion of this material for its own life activities. Most plants are, however, thrifty beings. Of the products of their labor they lay by a small portion. From these savings the succeeding generation gains its strength for growth until it is a self-supporting organism. Man lives largely by taking this surplus from the plant for the maintenance of his own life. Agriculture has been largely concerned with the study of con- ditions of soil, climate and cultivation for the production of this surplus of the plant. It has given little consideration to the vital question, the process by which the plant manufactures its products, i.e., photosynthesis. As has been stated, a clearer understanding of this process is of importance not only that we may understand the internal working of the plant, but now also as a guide to accomplish outside of the living organism what the plant is doing. *■■ i'W'jflJi'^f In conclusion, then, we may say that solar radiation is the greatest and an inexhaustible supply of energy for our earth. The chlorophyllous plant is a converter of this energy into potential energy ; it is from a chemical viewpoint a great reducing mechanism, producing compounds which can combine with oxygen. The transformatioi. of matter involved m this conversion of energy, that is, the chemistry of photosynthesis and metabolism present an exceedingly complex picture. The main reason for this ai>parent complexity is that photosynthesis is intimately connected with the vital process of the plant and hence subject to the manv fine adjustments characteristic of living protoplasm. No analysis of the process of photosynthesis in ])lants is reliable which does not give due regard to this fact. This need not mean, however, that a photosynthesis attaming the same or analogous results as the plant can never be achieved THE ORIGIN OF ORGANIC MATTER 61 without the action of living protoplasm. Only, up to the present time no chemical system has been devised which can approach the plant in efficiency or usefulness. An examination of the photosynthetic mechan- ism of the plant may, therefore, not be without value for discovering the fundamental principles of the transformation of matter by radiant energy. Chapter 2 The Nature of Photosynthesis as Determined by Observations of Gas Interchange and the Formation of Organic Matter 1. The Gaseous Interchange The phenomenon of photosynthesis was discovered as a result of investigations on the composition of the atmosphere and the influence of plants on the same. Thus the study of the problems of gaseous inter- change or the efifect of green plants on the composition of the air sur- rounding them has been so intimately associated with the development of our conceptions of the phenomenon of photosynthesis, that a consideration of this subject is of primary importance both on account of its direct bearing and because of its historical significance. In fact it can be said that historically considered the subject of plant physiology had its real inception as a recognized branch of experimental science with the study of the interchange of gases by de Saussure. a. The Path of Gaseous Exchange. In submerged aquatic plants the carbon dioxide reaches the interior of the plant by means of diffusion of the dissolved gas through the outer walls of the epidermal cells. This is probably also the case in many of the lower land plants. In the leaves of the higher land plants, on the other hand, there are special differentiated organs, the stomata, through which gaseous carbon dioxide passes to the chlorophyll-bearing cells and although the existence of stomata was known for a long time their func- tion in photosynthesis was a subject of great dispute for many years. It had to be determined what portion of the carbon dioxide used by the plant actually passed through the stomata and what portion diffused directly through the outer cuticle of the leaf. In brief, the stomata are minute mouth-like openings in the surface of the leaf. They are usually on the under side of the leaf though in some species the upper surface also has stomata. There exists consider- able variation in the structure of the stomata according to species and habitat of the plant.' The cross diameter of the stomata is exceedingly small, 0.0006-0.02 mm. Brown and Escombe ^ found the average area ' Haberlandt, G., Physioloqische FHanseiumatomie . Leipzig, 1904, p 395 "Brown and Escombe, Phil. Trans. Roy. Soc. B., 193, 275 (1900). 62 THE NATURE OE PHOTOSYNTHESIS 63 of the elliptical cross-section of a perfectly open sunflower stomatum to be 0.0000908 sq. mm. which is equal to the area of a circle 0.0107 mm. in diameter. Their number varies widely, 40 to 300 stomata to the square millimeter according to the species with as high as 700 in some cases. The number of openings in a single leaf thus runs to enormous numbers, a medium sunflower leaf containing about 13 million stomata. The morphology and details of the functioning of the stomata can- not be entered upon here.^ Suffice it to state that the cells bordering the openings, the guard-cells, are capable of controlling the size of the open- ing in response to certain external and internal conditions. It must be borne in mind that the stomata do not serve only for the ingress of carbon dioxide into the leaf, but as well for the egress thereof when the plant is in the dark as well as for the corresponding egress and ingress of oxygen and the escape of water vapor. Thus, for instance, when the stomata close under conditions of extreme heat the rapid loss of water is prevented, but at the same time the conditions are more unfavorable for the absorption of carbon dioxide. In experimental work on photosynthesis it is essential that changes in the size of the stomatal openings of this sort be constantly borne in mind in order to avoid epurious results. The factors effecting the opening and closing of the stomata are of an extremely complex nature. A very important role in these movements of the stomata is played by the starch grains in the guard-cells. The fluctuations in the starch-content of these cells do not run entirely parallel to that of the rest of the leaf, and the behavior of the chloroplasts of the guard-cells is in many respects quite different from that of the chloro- plasts of the other cells. The movements of the guard-cells appear to be intimately connected with variations in their osmotic pressure. This in turn is brought about by the hydrolysis and formation of the starch through enzyme action. The influence of various factors on the starch economy of guard-cells has been studied by Iljin * who ascribes great importance to certain kations and the hydrogen ion concentration in this phenomenon. Many external factors are of influence on the move- ments of the stomata. In general, light results in the opening of the stomata and darkness in their closing. This is, however, not a universal rule, for after prolonged darkness the stomata may be wide open. Lloyd ^ found that in blue light stoma^^a open but not so much as normally or as in red light. Considerable difference of opinion still exists regarding the effect of air enriched in CO2 on the condition of the stomata. Linsbauer ^ concludes that increasing the COo-content of air results in a closing of the stomata, ' Lloyd, F. E., The Physiologv of Stomata. Pub. No. 82. Carnegie Institution of Washington (1908). * Iljin, W. S.. Biochcm. Zcit., 132, 494, 511, 543 (1922). Wiggans, R. G., Am. J. Botany. 8, 30-40 (1921). ''Lloyd, I.e., 114. "Linsbauer, K., Flora, 109, 100 (1916). 64 PHOTOSYNTHESIS and the opposite effect is attained with COa-free air. Lloyd ^ states : "My conclusion is, therefore, that the presence or absence of COo has no direct influence on stomata, and that, physiologically, they are not at all dependent upon photosynthetic processes within the guard-cells. If this is true, the guard-cell is set off as distinct physiologically from the chlorenchyma- cell. . . . The mere fact that stomata open in the absence of COo shows conclusively that the movement is not directly connected with photosyn- thetic activity, even if the process takes place normally in the stomata. "The reduction and increase of starch in the guard-cells, in the absence of carbon dioxide, points rather clearly to the activity of an enzyme, presumably a sort of diastase, as a factor in the mechanism. . . ." Undoubtedly the causes underlying the opening and closing of the stomata are of a very intricate nature. In view of the fundamental importance of stomata to the photosynthetic process the subject is in great need of thorough investigation on the basis of experimental pro- cedure in which the various factors are subject to careful control. In this connection the findings of Molisch and others (discussed here in the chapter on carbohydrate transformations) of the conversion of starch into soluble sugars in wilting leaves may be of considerable significance. The old debate as to the path of gaseous exchange has been quite definitely settled. The idea that the COo is absorbed through the cuticle of the leaf, as maintained by Boussingault ^ and Barthelemy,^ has been replaced by the establishment of the stomata as the main path of gaseous exchange. By stopping-up the stomata, Mangin and Stahl ^° were able to demonstrate the retardation of gaseous exchange. Quantitative rela- tions were first established by Blackman ^^ who measured the quantity of carbon dioxide passing in and out of living leaves of which the distribu- tion of the stomata had been determined. The plugging of the stomata with vaseline or water does not completely prevent carbon dioxide diffusion, for Blackman found that more carbon dioxide passes through the surface in which the stomata had been stopped-up with vaseline than through the unvaselined upper surface containing no stomata. Under such artificial conditions there is a slight diffusion of carbon dioxide through the cuticle, but the results seem to indicate that under normal conditions the stomata are the chief path of gaseous exchange in the leaf. Blackman used spe- cially devised glass chambers which were sealed to both sides of the leaves and air was passed through these chambers. By determining the difference in the amount of carbon dioxide in the air before and after passing through the chambers the quantity of carbon dioxide given off by the leaf was 'Lloyd, I.e., 125. 'Boussingault, Aqronomie, 4, 359 (1868). 'Barthelemy, Ann. Set. Nat. Bot., V Ser., 9, 287 (1868) ; 19, 131 (1874) ; Comnt rend.. 84, 663 (1877); 85. 1055 (1877). "Mangin, Compt. rend., 105, 879 (1887). Stahl. Bot. Zeitg., 52, 117 (1894) Garreau, Ann. Sci. Mat. Bot., ITT Ser., 13, 321 (1850). Merget, Compt. rend 84 376, 957 (1877): 86. 1492 (1878). Wiesner and Molisch, Sit:;bcr. K Akad Whs Wicn.. 79, I, 368 (1879). "Blackman, PhU. Tran.';. Roy. Soc. London, B., 186, 503-562 (1895). THE NATURE OF PHOTOSYNTHESIS 65 established. These quantities were then correlated to the number of stomata on each surface. A similar procedure was followed in order to establish the path when carbon dioxide is taken up by the leaf in the light. It was concluded that in both cases the carbon dioxide passes primarily through the stomata. Jorgensen and Stiles ^- have summarized the result obtained by Black- man of the quantities of carbon dioxide emitted from the two surfaces of various leaves in proportion to the number of stomata on each surface. TABLE 8 Amounts of Carbon Dioxide Given Out from the Two Surfaces of Leaves. (From Jorgensen and Stiles.) Stomatic Ratio. CO2 Respired. Upper Surface Upper Surface Plant Peculiarity Lower Surface Lower Surface Neriuni oleander Very thick cuticle 3 6 100 100 100 Primus laurocerasus " " " 4 100 100 100 Hedera helix " " " _0_ J_ 100 100 Plantanus occidentalis Thin cuticle 3 Too 100 Ampelopsis hcderacea " " 3 loo 100 Polygonum sacclialinense . . " " 6 loo 100 135 120 ^r , A ■ , >r 100 100 Alisma plantago Aquatic plant. More J35 stomata on upper -rp^ surface ^ ^ 100 100 Iris germanica Isobilateral leaf 100 105 110 iOO 100" 100 Populus nigra Stomata on both sur- 100 100 faces, fewer on upper 575 375 Helianthus tubcrosus " 100 100 240 273 Tropceolum majus " 100 100 200 265 From Table 8 it can be seen that the quantities of carbon dioxide emitted by the various leaves in most cases is in direct proportion to the stomatic ratio, at least within the experimental error. Similar experi- ments were carried out by Blackman with illuminated leaves, in which "Jorgensen and Stiles, Carbon Assimilation. London, 1917, p. 53. 66 PHOTOSYNTHESIS he found that the absorption of carbon dioxide by the two leaf surfaces also followed the stomatic ratio. With some improvements in methods, Brown and Escombe '' repeated the work of Blackman and determined the amounts of carbon dioxide emitted and absorbed by the two sides of various leaves on which the distribution of the stomata was ascertained by actual counting under the microscope. The results obtained for carbon dioxide evolution are given in Table 9. TABLE 9 Emission of Carbon Dioxide from Upper and Lower Surface of Leatcs during Respiration. (From Brown and Escombe.) Stomatic Ratio. CO2 Emitted. Upper Surface Upper Surface Lower Surface Lower Surface Time in Plant Hours Canna indica 4.75 •' 5.00 4.23 Runicx alpinuin 5.50 Leaf Area in Sq. Cm. 28.27 28.27 28.27 59.44 100 246 100 246 100 246 100 269 100 246 100 322 100 210 100 286 Similarly the results obtained for the absorption of COo during photo- synthesis are given in Table 10. TABLE 10 Absorption of Carbon Dioxide on Upper and Lower Surface of Leaves during Photosynthesis. (From Brown and Escombe.) Stomatic Ratio. CO2 Fixed. Time in Hours Leaf Area in Sq. Cm. LJppt ;r .suriaLc Kj\)\ ^ci oui ieriments illuminated the upper surface of the leaves, it is not surprising that they should have found an apparent excess of photosynthesis on this surface. The one sided illumination in all probability brought about the partial oi>ening of the stomata on the illuminated surface and, moreover, the light active in photo- synthesis would be largely absorbed by the chloroplasts of the parenchyma into which the stomata of the illuminated surface open. This would result in a steeper diffusion gradient between the atmosphere and the 68 PHOTOSYNTHESIS intercellular spaces of the upper .surface, a condition which would favor a more rapid absorption of carbon dioxide by the upper surface. Brown and Escombe ^* then made a very thorough study of the purely physical process by which the carbon dioxide of the atmosphere enters the leaf, and presented the results of their laborious investigations in a masterful fashion. The difficulties in accepting simple diffusion as an explanation of the way in which carbon dioxide gets into the leaf are: (1) the relatively large amount of carbon dioxide absorbed by a leaf during active photosynthesis, about 0.1 cc. CO2 per sq. cm. of leaf sur- face per hour; (2) the low partial pressure of carbon dioxide in the atmosphere, 0.031 per cent by volume; (3) the very small portion of the surface of a leaf which represents stomatic openings, 1 to 3 per cent. Thus, Brown and Escombe state: "As a concrete example we will take the case of a leaf with which we have done a considerable amount of work, that of Catalpa bijnomoides, in which we have carefully determined both the number of stomata (which here occur only on the lower sur- face) and the area of the stomatal slits when fully opened. This leaf when placed under favorable conditions for assimilation, can abstract from ordinary air containing three parts COo per 10,000, about .07 cc. of carbon dioxide (measured at 0° and 760 milHms. bar.) per sq. cm. of leaf surface per hour. The stomatal slits when fully open have an area of .0000618 square millim., and since there are 145 of them on each square mm. of leaf, the area of the stomatal openings only represents .9 per cent of the total surface of the leaf on which they occur. It follows from this that if we regard the whole of the carbon dioxide as entering the leaf through these openings, diffusion must take place through 100 X .07 „ ^^ , ^T • them at the rate of = / .77 cc. per hour. Now it will be seen later on that the surface of a strong solution of caustic soda, when freely exposed to moderately still air containing the normal amount of carbon dioxide (three parts per lO.OCX) by volume), absorbs that gas at ordinary temperatures at the rate of only about .120 cc. per sq. cm. per hour, and when the rate of the air current passing over the surface is in- creased the maximum absorption is found to be .177 cc. per sq. cm. per hour. It follows, therefore, that the absorption of atmospheric carbon dioxide by the whole of the under surface of an assimilating leaf like that of the Catalpa, must proceed at about one-half the rate which the same absorptive surface of leaf would possess if it were covered with a constantly renewed film of a solution of caustic alkali; we may say, in fact, that the coefficient of absorption of the leaf surface under these conditions is about half that of the surface of the alkaline solution. If, however, we assume that the absorption of carbon dioxide in the leaf takes place only through the stomatal openings — which occupy at the "Brown and Escombe, Phil. Trans. Roy. Soc, B., 193, 223 (1900). Brown, H. T., Jour. Chem. Soc, 113, 559 (1918). THE NATURE OE PHOTOSYNTHESIS 69 outside not more than .9 per cent of this leaf area — we arrive at the somewhat remarkable conclusion that during assimilation the absorption [yer unit area of these openings must be from 43 to 64 times as fast as the absorption of a unit area of a freely exposed solution of caustic alkaH. In other words, under the natural conditions of assimilation the stomatal openings, supposing them alone to be operative, must take in carbon dioxide from the air about 50 times as fast as they would do supposing it were possible to fill them with a constantly renewed solution of caustic alkali." In order to explain these facts Brown and Escombe carried through an elaborate investigation of the diffusion of gases and liquids under a variety of conditions. They departed somewhat from the conventional method of studying interdiffusivity of gases in which these are at equal pressure so that one gas has to diffuse against an equal and opposite flow of the other, but rather set the conditions so as to study the rate of diffusive flow of carbon dioxide of very low initial pressure down a stationary column of air on its way to an absorbent surface. The absorbent was caustic soda and the surface thereof was 20 to 25 times the cross-section area of the column, thus simulating the conditions extant in the stomata. Their experiments show a variation in the diffusive flow which is inversely proportional to the length of the column and the re- sults may be stated by the following expression : k(p — pQA.t ^~ L in which O represents the amount of carbon dioxide flowing down the cylinder towards the absorptive surface, A the area of the cross-section, L the length of the column, t the time, p and pi the partial pressure of carbon dioxide in the outer air and at the surface of the absorbent re- spectively and k the diffusivity constant of carbon dioxide and air, i.e. the number of cc. of the gas, measured at the temperature of the experi- ment, which will pass across a section of 1 square cm. in area P — Pi when the fall of pressure is 1 atmosphere in 1 cm. — is the concentration gradient of the carbon dioxide and must be constant for unit thickness of any two adjacent layers at right angles to the axis of the cylinder. If the alkaline solution is a perfect absorbent, pi at the immediate surface of the absorbent is zero and the concentration gradient then becomes ~. If the absorption is not perfect pi will possess a sensible value and then the value for k will be lower than it should be. Thus Brown and Escombe give the following experimental results : "The diffusivity, K°, of the atmospheric carbon dioxide at 0° C, in C. G. S. units, is obtained from the equation : 70 PHOTOSYNTHESIS 3600 p p \T ) Qy = the number of cc. of CO2, measured at the mean temperature and pressure, which passes across a sq. cm. of the cross-section of the tube in one hour. p r= the volume of CO2 contained in unit volume of air. L = the length of the tube in centims. T° = the zero of absolute temperature. T = the mean temperature during the experiment, expressed on the abso^ lute scale, p =r the mean barometric pressure in millims. of mercury." TABLE 11 Results of Brown and Escombe on the Rate of Diffusion of Carbon Dioxide. CO2 Diffused per Sq. Cm. per Hour Measured Mean at Temp. COa Duration Total and Pres- Mean Content of Ex- Length CO2 sure of Tempera- of Air periment of Tube Diam. Diffused Experi- ture Mean in in Cm. Cc.atO°, ment Barom- Parts per Hours Corrected Cm. 760 Mm. Qvt. ° C. eter 10,000 K° 500.9 18.0 2.32 23.89 .01175 7.7 751.2 3.70 .151 501.0 75.0 2.32 6.33 .00311 7.7 751.2 3.70 .167 501.0 18.0 2.32 20.88 .01036 9.1 763.4 3.61 .133 384.0 20.1 2.28 14.91 .00989 13.3 766.3 3.39 .146 383.7 19.9 2.30 15.49 .01260 13.3 766.3 3.39 .185 384.8 36.6 2.23 8.81 .00609 13.3 766.3 3.39 .164 386.4 37.3 2.20 7.91 .00559 13.3 766.3 3.39 .154 Mean K° 157 The mean value for K° of all their experiments was 0.158. Below are given the values obtained by other investigators for the interdiffusivity of carbon dioxide and air with higher partial pressures of the former gas apd with both gases at the same pressure. K° Loschmidt " 142 von Obcrmayer '* 132 Waitz " 151-.158 Waitz, recalculated by von Obermayer for dry gases 131-137 Brown and Escombe calculated that their results are but very slightly afifected by the presence of water vapor in the air through which the carbon dioxide was passing. "Loschmidt, IVien. Akad. Ber., 61 (2), 62, 367, 468 (1870). "Von Obermayer, ibid., 85 (2), 147, 748; 87 (2), 188; 81, 1102 (1880). "Waitz, Ann. Phys. Chem., 17, 201 (1882). See also Meyer, O. E., Kinetic Theory of Gases, Eng. translation, p. 267 (1899). Stefan, IVieti. Akad. Ber. 83 (2), 613 (1881); 79 (2), 161; 78 (2), 161 (1877). THE NATURE OE PHOTOSYNTHESIS 71 Brown and Escombe having thus determined that, "when a condi- tion of static equiHbrium has been estabhshed in a diffusing column of gas, vapor, or sokite, as the case may be, the amount of diffusion, under Hke conditions, is proportional to the sectional area of the column, . . ," they proceeded to apply these findings to conditions which simulate those existing in the leaf. They discovered that when a septum with a circular aperture is interposed in the line of flow, diffusion is modified in a re- markable manner. It was found that when the aperture was reduced to a certain point, the carbon dioxide i>assing through unit area of aperture in a given time showed a marked increase, "which could not be satisfac- torily accounted for by the mere difference in the gradient of i)artial pres- sures of the gas inside and outside the covered disk." The experiments were carried out with great care and the apparatus consisted, in brief, of flasks containing sodium hydroxide to absorb the carbon dioxide. Over the mouths of the flasks were cemented septa with apertures of differ- ent diameters. After observing all precautions to maintain all experiments under precise conditions of carbon dioxide-content, temperature, freedom from convection currents, etc., the amount of carbon dioxide absorbed by the sodium hydroxide was determined. Thus it developed that as the size of the restriction decreased the flow per unit area of aperture rapidly increased, and as the aperture was diminished below a certain size rela- tive to the cross-section of the column, the amount of carbon dioxide which passed became i>roportional to the linear dimension of the apertures. The following table is taken from Brown and Escombe's pai>er and with the exceptions of Nos. 2 and 3 the results show that the rates of diffu- sion follow very closely the ratios of the diameters of the openings as well as the relative increase of carbon dioxide diffused, per area of the aperture per hour. In Nos. 2 and 3 the diameter of the aperture was more than one-half that of the unrestricted opening of the flask in which case other conditions prevail. TABLE 12 Rate of Diffusion of Carbon Dioxide through Apertures. (Brown and Escombe.) CO2 Diffused Ratio of Ratio of Diameter Time of Total CO2 per Sq. Ratio of Diam- CO. of Aper- Diffusion CO, Diffused Cm. of Areas of eters of Diffused ture, in Diffused, per Hour, Aperture Aper- Aper- in Unit Jo. mm. Hours cc. cc. per Hour tures tures Time 1 22.7 0.23800 .0588 1.00 1.00 1.00 2 12.06 476.5 44.22 0.09280 .0812 .28 .53 .39 3 12.06 477.1 48.57 0.10180 .0891 .28 .53 .42 4 5.86 478.8 26.61 0.05558 .2074 .066 .25 .23 5 6.03 643.1 40.21 0.06252 .2186 .07 . .26 .26 6 3.233 863.0 34.41 0.03988 .4855 .023 .14 .16 7 3.216 863.8 34.30 0.03971 .4852 .020 .14 .16 8 2.00 1007.8 24.16 0.02397 .7629 .007 .088 .10 9 2.117 1007.3 26.28 0.02608 .8253 .008 .093 .10 72 PHOTOSYNTHESIS It was also found that the rate of diffusion of water vapor is con- trolled by the linear dimensions of the apertures. This applies equally when the water is evaporating from a surface of water through an aper- ture or is absorbed by, e.g., sulfuric acid. In order to explain their experimental results on the rate of diffusion of carbon dioxide through apertures and the "diameter law" Brown and Escombe pictured "lines of creep" of the carbon dioxide as it passes through the air towards the disc to replace that absorbed. Thus, the simplest case would be a circular disc, capable of absorbing carbon dioxide, freely exposed to the air and the disc surrounded with a rim, in the same plane, three or four times the diameter of the disc. If the air is perfectly still there will be established a steady gradient density of car- bon dioxide surrounding the disc. If lines are drawn through all the points of the same carbon dioxide-density above the disc, curved surfaces are formed. These surfaces will be in the form of shells surrounding the disc. If the latter is a perfect absorbent of carbon dioxide each shell will represent a carbon dioxide-density varying from zero at the absorb- ing surface to the maximum density which will be that of the carbon dioxide in the air. This maximum is at a distance of 5 or 6 diameters from the disc. The gradient of density will thus be a line perpendicular to the shells of equal density. The problem is very similar to that of the lines of force of an electric field. Stefan and others studied the exact converse case, that of the evapora- tion from a circular surface. A mathematical analysis of this problem showed that the amount of evaporation is proportional to the linear dimen- sions of the liquid surface and not to the area. The shells in this case form an orthozonal system of ellipsoids having their foci in the edge of the disc. Larmor worked out the following formula for the absorption of atmospheric carbon dioxide by a perfectly absorbing circular disc : Q = 2kpD, in which Q is the amount absorbed in a given time, k the coefficient of diffusion of carbon dioxide in air, p density of atmospheric carbon dioxide, and D the diameter of the disc. By using discs of very small diameter and perfectly quiet air Brown and Escombe were able to obtain results which showed an absorption de- pending upon the linear dimensions of the surface according to the formula given above. In Figure 5 are shown Brown and Escombe's conception of diffusion shells. A represents the shells in the case of an absorbent disc with rim; the convergent hyperbolic lines of flow, representing the carbon dioxide gradient, terminate in the surface of the disc. In the case of perforations in a septum which divides two regions of dift'erent density the conditions are very different. In the former case, as the experiments show, the absorption, on the basis of linear dimen- sions, is exceedingly sensitive to any disturbances which affect the hypo- THE NATURE OF PHOTOSYNTHESIS 73 thetical shells above the disc. In tlie case of the i^erforated septum, as shown m C, Figure 5, the lines of flow are convergent as they approach the septum, bend around their foci, situated in the edges of the aperture, and form a divergent system on the other side. If the absorbing surface below the septum is a i)erfect one, there will be formed in the chamber between this surface and the septum (provided the chamber is sufficiently large) a system of shells exactly similar to those on the outside. In these inner shells the gradient of carbon dioxide-density will, of course, decrease away from the septum. In the latter case diffusion on the basis of linear dimensions of the aperture is not affected by movement of B Fig. 5. — Brown and Escombe's diffusion shells or gradients of CO2 density about a single opening above an absorbing medium. the outer air. The inner shells will be protected from the action of air movement (if the aperture is not large) and are quite indei>endent of the outer shells. They will be as effective, therefore, in regulating relative rates of inflow of carbon dioxide according to the linear dimensions of the aperture as the outer shells. The absolute rates of diffusion through an aperture are decidedly influenced by conditions permitting a single or double system of shells. The experimental observations on the rates of diffusion through small apertures are capable of explanation on the assumption that the con- verging and diverging lines of flow to and from the aperture "result in a system of shells of equal density, which locally alter the gradient in the immediate neighborhood of the septum." Brown and Escoml^e attempted to demonstrate the formation of ellipsoidal shells by allowing a weak solution of methylene blue in 5 per cent gelatine to diffuse into colorless gelatine through an aperture. The formation of a limiting surface of color, ellipsoidal in shape, is clearly shown. Similar arrangements for demonstrating the zones of density 74 PHOTOSYNTHESIS gradient in diffusion through apertures are possible by the use of inter- acting substances such as sulphates and chromates into gels of agar con- taining barium chloride. The Liesegang phenomena can also be used in such a manner that the reacting substances produce the rhythmical series of. zones which follow the contours of surfaces of equal density. Brown and Escombe's investigations thus established that the effect of interposing a diaphragm pierced by a single circular aperture on the diffusion of a gas was quite significant. The velocity of the flow through unit area of such an aperture varies inversely as the diameter. The original object of their study was to determine the mechanism of gas interchange through the minute apertures in the surface of a leaf and to develop experimental conditions which simulated such a gas interchange. The discovery of the "diameter law" warranted the anticipation that under proper circumstances a thin septum might be pierced by the cor- rect number, size and distribution of apertures so that the septum would cause but little obstruction to the diffusive flow of the gas while the aggregate area of the apertures would represent but a small percentage of the entire area of the septum. That a leaf l^ehaves as such a system had been established by their earlier studies. Experimental studies with multi-perforated septa showed that these conditions could be realized. Brown and Escombe used celluloid dia- phragms fixed over short glass tubes. The latter were provided with a side tube and stop-cock for running in the sodium hydroxide solution to any desired height, as well as another tube for drawing off the solution for analysis. In the multi-perforate septa each hole was 0.38 mm. in diameter and the septum itself was 0.1 mm. in thickness. The factor which was varied in the experiments was the distance the holes were apart, i.e., the number of holes per square centimeter. The experiments were carried out in still air, and the amounts of carbon dioxide which had diffused through the multi-perforate septa were compared with the amounts which would have diffused down the open tube if there had been no obstructing septum present. The latter amount was calculated on the basis of previous results of (1) the diffusion coefficient of carbon dioxide in air, (2) the dimensions of the tube and (3) the mean density of carbon dioxide in the air during the experiment. In the last column are given the calculated values of the relation of the carbon dioxide diffusion through the septum to the rate of diffusion without the septum. It becomes evident that the multi-perforate septum exerts but a slight effect on the rate of diffusion. The slight increase in No. 1 is due to the fact that in this experiment the air was not per- fectly still. In No. 2, for example, 43.2 per cent of the open tube diffusion has taken place although the aggregate area through which the gas could pass was only 2.82 per cent of the cross-section of the tube. In the second portion of the table it will be seen that when the distance between the septum and the absorbing surface is decreased, the actual amount of carbon dioxide absorbed is increased, but the septum diffusion is a smaller THE NATURE OF PHOTOSYNTHESIS 75 percentage of the oi>en tube diffusion. It is, moreover, evident that as the distance between the holes is increased, the efficiency of the holes, i.e., the area of perforations increases. For the optimum efficiency of each perforation, these should be about ten diameters apart. Under these circumstances the rate of diffusion is about forty times the amount it would be if the diffusion were proportional to the area of the cross-sec- tion of the tube. The results are summarized in the following table : TABLE 13 Diffusion of Carbon Dioxide Through Septa with Holes 0.380 mm. in Diam- eter. The Distance between the Septa and the Sodium Hydroxide Solu- tion IN THE Two Experiments was 1 cm. and 4 cm. (Brown and Escombe.) cc. CO2 Percent- Diffused cc. CO. age of Through Open Tube Percent- Septum No. of Septum Diffusion age .\rea Diffusion Area of Holes per Distance per Hour, per Hour, of Holes of Open Tube, Sq. Cm. .\part in 0°.760 0°,760 in Tube No. Sq. Cm. Septum Diameters (Length mm. f tube = 4 mm. cm. ) Septum Diffusion 1 9.348 100.00 2.63 0.361 0.346 11.34 104.3 2 9.186 25.00 5.26 0.148 0.342 2.82 43.2 3 9.456 11.11 7.80 0.131 0.352 1.25 37.2 4 9.511 6.25 10.52 0.110 0.353 0.70 31.1 5 9.456 2.77 15.70 (Length c 0.0683 f tube = 1 0.334 cm.) 0.31 20.4 6 9.347 100.00 2.63 0.433 0.771 11.34 56.1 7 9.186 25.00 5.26 0.401 0.775 2.82 51.7 8 9.456 11.11 7.80 0.312 0.768 1.25 40.6 9 9.511 6.25 10.52 0.241 0.767 0.70 31.4 10 9.186 4.00 13.10 0.156 0.744 0.45 20.9 11 9.347 2.77 15.70 0.106 0.740 0.31 14.0 Brown and Escombe picture this phenomenon as shown in Figure 6. This is simply a multiplication of the case shown in Figure 5. Assuming perfectly quiet air at some distance from the septum the lines of -flux in a diffusive column are approximately parallel. As the stream lines approach the aperture they converge and gradually assume parallelism again on the lower side of the septum. The ellipsoidal shells come closer together at the edges than in the center which would indicate that in these regions the gradient of density is greater and results in greater rate of flow per unit area. The lines of flow, which are at right angles to the gradients of density, thus also converge at the edges of the aper- ture, and after passing through the opening, again diverge. The lines of flow from adjacent apertures cannot cross each other, as there would then be shells of dift'erent density crossing each other, which is impos- sible. The lines of flow must, therefore, bend around the aperture and again become parallel, the velocity of the flow decreasing at the same time. As is shown in the tallies there is an acceleration of flow per 76 PHOTOSYNTHESIS unit area as the size of the aperture is diminished. This is caused in the main by the degree of convergence and divergence of the lines of flow. With a smaller aperture the convergence and divergence increase, result- ing in an increase in the gradient of density. When the apertures are ten diameters or more apart each perforation behaves independently without interfering vi^ith its neighbors and follows the "diameter law." On this basis Brown and Escombe explain the fact that they were able to block out nearly 90 per cent of the cross-section of a column of diffusing gas leaving 100 circular apertures, and still produce no sensible effect in obstructing the diffusion of carbon dioxide through air. They were able to obtain photographs of the zones of equal density produced by intermittent diffusion of two reacting substances through apertures. Fig. 6. — Brown and Escombe's conception of the lines of flux in a diffusive column through septa. Thus this behavior, as would be expected, is characteristic not only for gases but also for substances in solution. In applying these results to the gaseous exchange of leaves, Brown and Escombe studied an herbaceous plant which has been extensively used for photosynthesis investigations. The sunflower, HeliantJms an- nuiis, has an arrangement of stomata in the surface of the leaves corre- sponding to a multi-perforate diaphragm below which is a very large absorptive surface on the sides of the intercellular spaces. In these spaces is ample room for the formation of negative shells as described above. If the stomata are regarded as circular apertures they are about eight diameters apart, permitting nearly full efficiency of each opening. When they are partly closed, full efficiency of each is very probably attained. When the stomata are wide open they form straight-sided tubes about 0.014 mm. in length. This opening corresponds to a circle 0.0107 mm. in diameter and an area 0.0000908 sq. mm. Assuming the following most favorable conditions : wide-open stomata, constant partial pressure of carbon dioxide at the mouth of the stomata by a moving current of air and zero partial pressure of carbon dioxide in the inter- THE NATURE OF PHOTOSYNTHESIS 77 cellular spaces, by complete absorption, the amount of gas in cc. absorbed by one sq. cm. of leaf per hour will be represented by: kp . A . y . 3600 ^ L+x k r=: diffusion constant of CO2 = 0.145. p ^ density of COo in outer air in atmospheres = 0.0003. A = area of stomata = 10"" X 9.08 cm. y = number of stomata per sq. cm. == 33,000. L r^ length of stomatic tube = 0.0014 cm. X ^ resistance of the column of diffusive flow = y^Tt X diameter = 0.00042 cm. Q = 2.578 cc. CO2 per hour per sq. cm. If it is supposed that the air over the leaf is perfectly still Brown and Escombe develop the equation so that the denominator becomes L -|- 2x and Q = 2.095. A comparison with the actually observed rates of photosynthesis will show that these rates are very much lower than those theoretically possible on the basis of Brown and Escombe's calculations. Prob- ably the chief factor in this discrepancy is the fact that the walls of the intercellular spaces into which the stomata lead are not perfect absorbers of carbon dioxide. In 1850, Graham, ^^ in his Bakerian Lecture, pointed out that the "liquid diffusion of carbonic acid is a slow process compared with its gaseous diffusion, quite as much as days are to minutes." The gradient between the carbon dioxide on the outside and inside of the leaf is therefore much smaller than is assumed in the formula. A considera- tion of the absorptive capacity of the leaf material is taken up in Chapter 5. The outstanding contribution of Brown and Escombe's studies is the fact that the structure of a leaf with its minute stomata is admirably adapted to the work it has to perform. The surface of a leaf, with the physical properties of a multi-perforate septum, having only 1 to 3 per cent of open area, still permits free gaseous interchange. The stomata can. in fact, be closed to 5 per cent of their maximum and yet permit sufficient carbon dioxide to pass to account for the maximum photo- synthesis, provided the absorption is perfect. This aspect of the photosynthesis problem has received very little attention aside from the very careful studies of Brown and Escombe. It would be highly desirable to have their results verified and extended. Jeffreys ^^ has made a mathematical study of the laws of evaporation of water from circular surfaces and from cylinders. The laws of evapora- tion have a close analogy to those of absorption and from the results of Jeffrey's studies some of Brown and Escombe's conclusions are called " Graham, Chemical and Physical Researches, p. 446. ''Jeffreys, H., Phil. Mag., 35, 270 (1918). Thomas and Ferguson, ibid., 34, 308 (1917). 78 PHOTOSYNTHESIS in question. This applies more particularly to the conclusions of the latter authors as to the influence of the distance between openings. Brown and Escombe consider that when the small apertures are more than ten diameters apart they influence each other very little, while Jeffreys con- cludes that the stomata in a leaf must close to a diameter %o of that of their full aperture before they act independently. There exists thus such a wide divergence between the conclusions of Jeffreys and what has been held for a long time regarding the function of stomata that a reinves- tigation of the subject seems highly desirable. b. The Sources of Carbon Dioxide. The question has repeatedly arisen whether the atmosphere is the only source of carbon dioxide or is a sufficient supply of this gas for the photosynthetic activity of the plant. From the time of Senebier who maintained that plants absorb the carbon dioxide through the roots this question has been a topic of much controversy. The careful and numer- ous analyses of Benedict -° show a very constant per cent of carbon dioxide in the atmosphere, 0.031 per cent. Samples of air collected over the ocean, at different times of the year and on the top of Pike's Peak gave essentially the same results. An appreciable increase in the car- bon dioxide-content of the atmosphere is detectable only where there is not perfectly free movement of the air, in cities and industrial centers where the liberation of enormous quantities of carbon dioxide is taking place. The soil air may vary greatly from the normal, and under circum- stances, as for instance when the soil is dunged or in pasture land, the carbon dioxide in the soil may rise considerably. When the air supply is cut off the carbon dioxide-content of the soil may rise to very high percentages.-^ Thus, in India under monsoon conditions Leather found that the carbon dioxide rose to 16-20 per cent. Lundegardh '- has made an extensive study of the carbon dioxide in the soil with a specially devised apparatus. At a depth of 15-20 cm. the carbon dioxide-content of the soil atmosphere ranges from about 2.5 to 0.12 per cent depending upon the type of soil, kind of fertilizer, season, etc. Through the action of the bacteria in the soil large quanti- ties of carbon dioxide are constantly being formed in the soil. Lunde- gardh points out that the amount of carbon dioxide formed in the soil approaches that which is absorbed by i^lants in photosynthesis per unit area. As a consequence of the formation of carbon dioxide in the soil the concentration of this gas immediately above the ground is often con- siderably higher than that usually reported. Thus, for example, Lunde- gardh found in a well fertilized field of beets in October the carbon "Benedict, Carnegie Institution of Washington, Pub. No. 166 (1912). '^ Russell,^ E. J., Soil Conditions and Plant Grozi'fh, p. 229 (1921). ""Lundegardh, Der Kreislanf dcr Kohlcns'dnre (1924), p. 144. Literature cita- tions. THE NATURE OF PHOTOSYNTHESIS 79 dioxide-content at the surface 0.0534 to 0.284 volume per cent, above the leaves 0.0401 to 0.0674 and at a height of one meter 0.0375 to 0.0720. From analyses of this nature it has been concluded that the available carbon dioxide for many crop plants may be very much higher than that of the free atmosphere, and since the concentration of carbon dioxide, perhaps more than any other factor, determines the rate of photosynthesis, it is apparent that the production of carbon dioxide by the soil may exert a decided influence on the development of plants. Boussingault -' showed definitely that plants grown in an atmosphere lacking carbon dioxide did not increase in carbon content, while Moll '* was able to demonstrate that plants growing in a carbon dioxide rich humus soil formed no starch in leaves which were kept in an atmosphere free of carbon dioxide. The leaves growing in normal air produced starch as usual. It is conceivable, however, that conditions may exist in which the carbon dioxide absorbed by the roots may contribute to the total amount of carbon dioxide reduced by the plant.^^ Neverthe- less, the chief source of carbon dioxide for the plant must be recognized as being the atmosphere. In aquatic plants the conditions of carbon dioxide-absorption are in some respects different from those obtaining in land plants. The exten- sive anatomical investigations of aquatic plants have disclosed that in submerged leaves there are generally no stomata, though there appear to be some exceptions to this. In floating leaves the stomata are usually confined to the upper surface. In submerged aquatic plants the ingress of carbon dioxide into the leaf must therefore be accomplished by dififu- sion through the epidermal cells to the cells containing chloroplasts. In the mosses which are without stomata similar conditions must exist. The mechanism of this diffusion has been more closely studied by Devaux.^* Ordinarily the carbon dioxide-content of pure water is, of course, determined by the partial pressure of the carbon dioxide in the atmos- phere over the water. Under certain circumstances the carbon dioxide- and oxygen-content of water are greatly influenced by the presence of decaying organic matter on the one hand and by active photosynthesis of aquatic plants on the other. Of special importance to the carbon dioxide supply of natural waters is the presence of carbonates and bicarbonates. It has been known for a long time that aquatic plants form incrustations of calcium carbonate and that these deposits arise from the conversion of the calcium bicar- bonate into the more insoluble carbonate by the withdrawal of carbon dioxide through photosynthetic activity. Draper -' first showed that ^' Boussingault, Ann. Chim. et Phys., (5), 8, 433 (1876). ~Moll, Arbeit, hot. Inst. Wiirzburg, 2, 105 (1878). ^ .„ ^ ="Pollacci G., Bull. Soc. Bot. Ital. Genoa, 208 (1918). Cailletet, Compt. rend., 152 1215 (1911). Maquenne, ibid., 1811. Moillard, ibid., 154, 291 (1912). ^"Devaux, Ann. Set. Nat., (7) 9, 35 (1890). "Draper. Ann. chim. et phys., (3) 11, 223 (1844). 80 PHOTOSYNTHESIS plants were able to liberate oxygen in the light when grown in solutions of sodium bicarbonate and Hassak ^^ carried out quantitative experiments with potassium bicarbonate to demonstrate this fact. In this process the water naturally becomes alkaline. If a solution of sodium bicarbonate is allowed to stand in contact with air containing carbon dioxide, an equilibrium will be established between the hydrolyzed bicarbonate solution and the carbon dioxide in the air. This equilibrium will depend upon the partial pressure of the carbon dioxide in the air and will consist of a solution containing a high concentration of HCO3- and relatively small amounts of normal car- bonate. The concentration of HCO3- will be higher with increased partial pressure of COo in the air and will be decreased by the addition of normal sodium carbonate or alkali to the solution. These relations have been clearly expressed by Warburg.-^ From the equations of the electrolytic dissociation of carbonic acid : H^ X HCO3- _ /, ^,_ ,,:_„„;„,:._„ ..,„.,, ^/ HXO3 CO2 H^ X COs / . . HXOsN :l kj = the dissociation constant X ^^ — j = K HCO3- through division : (HC03-)^ =^'-K (1) CO3-XCO, k„ If in mixtures of NaHCOo and Na2C03 the values of HCO3- and CO3— are known we can calculate the COo concentration in each case if K is known. If NaHCOs and NaoCOg represent the total concentra- tion of bicarbonate and carbonate in moles per liter, a the dissociation of (T-TCO ~ \ ,, ,, ' I and j5 the dissociation of the carbonate NaHCOs / -^-—- I . we get from ( 1 ) NaoCOa/ aHNaHCOo)- pNaXOa X COo — K (NaHC03)^_^^P_^^, ^2) NaoCOs X CO3 a In solutions which are not too dilute, i.e. where the hydrolysis can be neglected this equation agrees with that of McCoy ^° and Seyler and * Hassak, Unters. hot. Inst. Tiihingni. 2, 465-477 (1888). Pringsheim, N., Jalirb. wiss. Bol., 19, 138 (1888). "Warburg, Riochcm. Zcit.. 100, 238 (1919). *'McCov, Amcr. Chem. Jour., 29. 437 (1903). THE NATURE OF PHOTOSYNTHESIS 81 Lloyd. ^^ K^ is constant when the total Na-content is kept constant. When the latter is varied — changes, and with it K'. If c designates the total Na-content in milli-equivalents per liter, War- burg gives the empirical formula K^rr: 8739— 1671 log c. (3) as holding when c = 100 to 1000 at 25°. Although there are no determinations of K^ for temperatures other than 25°, Warburg calculated the CO.-concentration at other tempera- tures. This was done by calculating the change of K^ with temperature from the heat of decomposition of the bicarbonate. Unfortunately the latter are not very accurate. In the dissociation of 2 moles of NaHCOa (aq.) in 1 mole CO, (aq.) and 1 mole NagCOs (aq.) 2028 calories are absorbed. On the further supposition that the degree of dissociation of the salts does not change ^markedly with temperature there is obtained for absolute temperature T log K^ ,,, ^ V2XT 2X29S;^ (4) 2.3 By means of equations (2) (3) and (4) Warburg has calculated the C02-concentration at three diiTerent temperatures of various mixtures of NaaCOs and NaHCO-.,. These must, of course, be regarded in the light of the assumptions made in the equations. In mixtures of this sort many acjuatic plants can carry on photosyn- thetic activity for longer or shorter periods depending upon conditions of light intensity, temj>erature and the nature of the plant itself. In general, the length of time during which plants can survive in the mix- tures depends upon the hydrogen-ion concentration, i.e. the time de- creases with increasing alkalinity. Great care must be exercised in the use of these solutions and preliminary experiments must be carried out to determine whether the plants can live in the solutions without injury.^^ The presence of a living plant in anyone of these carbonate mixtures disturbs the equilibrium either by the liberation of carbon dioxide from the plant in the dark or by the absorption of carbon dioxide from the solution in the light. In the latter case when carbon dioxide is removed from the solution the equilibrium is shifted, so that bicarbonate is de- composed into carbonate and carbon dioxide, and the concentration of all '^Seyler and Lloyd, Jour. Chcm. Soc. HI, 138 (1917). See also Kendall, /. Am. Chew. Soc, 38, 1480 (1916). .A.uerbach and Pick, Art. Reichsgcsundh.. 38, 274 (1911). For dilute solutions McCoy has shown that in the equation NaHCOs + HOH ^ NaOH -|- H2CO3 the amount of NaOH is about Mio of the amount calculated for a normal hydrolvsis, because of the secondary reaction : NaOH 4- NaHCOs ^ H2O + Na^COs. =' Harder, R., Jahrb. wiss. Bat., 60, 538 (1921). CM O ^r ooooooocDOOO ^3 xxxxxxxxxxx •^h roooooo<^<^'-i<^o O y lO O r^ M3 ro CO CM -^ OMO fO <5 o '— I »- 1 CM "^ a\ OOOOOOOOQOO r, ooooooooooo txj o O O O •-I ^^ CM CM fO <^ -^ ID IT) irj lo lo "^ OO^^CMCMfOfO"^ 'O fc o »-'t~^'— it^r^CMO OOr-^CM-^K '"'^ OOOOOoOoOoO ^ ooooOoooooo K/ 1- -rr Tf Tj- lo lo t^ f^ 00 00 0\ ►^ \0 \0 ^ VO*^ VO^ vo ^ \o *o 2; 8 o u I u a o Q o < t — . QJ HH o Ok B o u f- X ir;0"^OOo"^"^"^0"^ oooor^t^^io'^CM-^'— lO 5 ^ ^2 u-)OioOOO"^"^"^0"^ ■— iCMCM'0'^iD^t^OOO\C?s IJ-)0>-0000101010010 oooot^r^^"T~ocM'-i«-" O 3 T-HCsjro-^i-o'or^cooNO'— 1 83 THE NATURE OE PHOTOSYNTHESIS 83 components changes. However, if the quantity of carhon dioxide taken up by the plant is small in comparison to the total amount of the carbonate salts present, the changes of concentration are negligible and the carbonate mixtures play the role of buffers. Thus, for example, taking Warburg's figures for 25° for the carbonate mixture No. 9 CNaHCOa = 0.085 moles per liter. Ceo = 91 X 10-*' " " " K^ " = 5.3 X 10^ " " " If from 10 cc. of this mixture 0.2 cc. COo are removed, there will be taken 0.9 X lO"' moles CO, per liter. Thereby the same amount of NezCOs molecules will be formed and twice the number of NaHCOa molecules will disappear. So that from equation (2) r (0.085-1.8X10-^)^ -g-^xio-B ^°^- (0.015 + 0.9 X 10-=*) X 5.3 X 10^ i.e. Cec\, decreases from 91 X lO"' to 82 X 10"' or about 10 per cent. Nathanson ^^ has tried to show that it is not the accumulation of OH ions but only the reduction of free CO2 which is responsible for the de- crease in the rate of photosynthesis in carbonate mixtures as these be- come more alkaline. This is based upon the fact that certain aquatic plants which are capable of storing small quantities of carbon dioxide can utilize this for photosynthesis in relatively strongly alkaline solu- tions. In view of the complexity of such a system the evidence is not altogether convincing, however. Large bodies of water and particularly the sea are solutions of car- bonates which must be viewed in the light of the equilibrium between atmospheric carbon dioxide and carbonates. The water, being in con- stant contact with the atmosphere, an equilibrium has been established be- tween solution and gas so that the carbon dioxide pressure in the air is about equal to that in the water. The conditions under which plants carry on photosynthesis in the sea and in the atmosphere are therefore about the same as far as the carbon dioxide supply is concerned. Although the total carbon dioxide-content of sea- water, i.e. both free and com- bined CO2, is about 50 times greater than that of the atmosphere, the larger portion of this is not available to the plant. It appears that no other gas can take the place of carbon dioxide in the photosynthetic process. The effect of carbon monoxide in low concentra- tions is like other indifferent gases, e.g. nitrogen, and plants do not survive when confined in an atmosphere containing carbon monoxide but no carbon dioxide.^* Higher concentrations of carbon monoxide are "Nathanson, Stoffwechsel der Piianzen, p. 166. (Leipzig. 1910.) " Boussingault, Agronomic, 4, 300 (1868). Stutzer, Ber. chem. Ges., 9, 1570 (1876). 84 PHOTOSYNTHESIS toxic to phanerogams.^'' Bottomley and Jackson,^*' however, report that this gas can to some extent replace carbon monoxide in photosynthesis. Boussingault also tried hydrocarbons without success. c. The Evolution of Oxygen. As has already been pointed out, one of the first facts which was observed in connection with the phenomenon of photosynthesis was that in the light carbon dioxide was taken up by the plant and oxygen was emitted. Thus the emission of oxygen as well as the absorption of car- bon dioxide have been utilized as a means of studying the phenomenon. While a variety of qualitative methods, based upon the evolution of oxygen have been devised, relatively little work has been done on the laws governing this phase of the phenomenon. The methods which have been used in studying the emission of oxygen are described in Chapter 4. That gas is emitted in the process of photosynthesis can be observed most easily in submerged aquatic plants. These absorb the dissolved car- bon dioxide and emit the oxygen in form of minute bubbles. Leaves of land plants are not always suited to this demonstration, because the gas is not allowed to escape from the minute stomata on account of the capillary surface formed by the water. Leaves covered with a waxy sur- face are therefore more suitable. The demonstration of the evolution of oxygen in a striking manner is made possible by the use of the leuco compound of methylene blue or indigo carmine. An aquatic plant is allowed to remain over night in water the surface of which is covered with a layer of paraffin oil. When the oxygen has been removed from the water by the respiratory activity of the plant, a small quantity of the leuco dye is added to the water and the plant is illuminated. The evolution of oxygen from the illuminated plant is made evident by the formation of the bright colored dye close to the plant. It should be stated at once that the gas which is thus emitted is not pure oxygen, but contains from 25-85 per cent of this gas with ad- mixtures of nitrogen. Since it is impossible to obtain an exact analysis of the gas as it is emitted from the leaves of land plants, the results obtained with aquatic plants give more reliable data on this point. In land plants the path of oxygen escape is through the stomata, while in aquatic plants under normal conditions the oxygen escapes from the whole leaf surface by diffusion into the surrounding medium. The diffusion coefficient of oxygen is slightly higher than that of carbon dioxide. According to Carlson ^^ kco„ =1-378 and ko„ =1.607 (per sq. cm. per 24 hrs. at 16°) giving a ratio between the coefficients of oxygen and carbon dioxide of 1.166. "^ Richards and MacDougal, Bull. Torrey. Bot. Club, 31, 57 (1904). '"Bottomley and Jackson, Proc. Rov. See, 72, 130 (1903). Krascheninnikoff, Rev. Gen. Bot., 21, 177 (1909). "Carlson, /. Am. Chem. Soc, 33, 1027 (1911). THE NATURE OF PHOTOSYNTHESIS 85 A practical method of determining the rate of oxygen evolution is the counting of the huhhles which escape from the cut end of a submerged aquatic plant. In this case the gas passes through the gas-filled inter- cellular spaces of the stem thus taking the path of least resistance. The determination of rates of escape of the gases by counting the bubbles is the basis of a method of comparative measurement of the rate of photosynthesis which has been in use for a long time. The description and necessary precautions of this method are given in Chapter 4. The rate at which these bubbles escape varies with the factors which afifect photosynthesis, more particularly the light intensity. It is evident that a direct proportionality between the rate of the escaping bubbles and photosynthesis can exist only if the gas is pure oxygen or contains only negligible traces of other gases, or if the per cent of oxygen in the gas bubbles is constant and independent of their rate of escape. That these conditions ordinarily are not met has been demonstrated by Kniep.^^ He showed that the oxygen-<:ontent of the liberated gas varies as much as 30 per cent. Below are given Kniep's analyses of the gas escaping from an illuminated plant of Heleodea canadensis. The percentages of oxygen and nitrogen are calculated for the total volume of gas after removal of the carbon dioxide. The rate is figured on the basis of the time required for 20 bubbles to escape from the plant. TABLE IS Composition of Gas Escaping from an Aquatic Plant with Different Rates of Photosynthesis. (Percentages of O2 and N2 calculated for total volume after removal of CO2.) Rate : 20 Bubbles Per Cent Per Cent Per Cent in Seconds O2 COs N2 7.0 49.6 0.9 50.4 5.2 54.4 0.6 46.6 14.7 36.6 1.2 63.4 The oxygen-content of the emitted gas increases with increased rate of bubble formation and vke versa. From this it follows, that with changing light intensity the rate of gas escape increases or decreases more slowly than the true rate of photosynthesis (as determined by the oxygen-content of the gas). A certain amount of the oxygen formed will, of course, escape by diffusion into the surrounding water. This fact, however, hardly affects the foregoing conclusion, because the diffu- sion out will certainly not be less when the intercellular spaces have a high oxygen-content than when they have a low one. The explanation of this phenomenon is probably to be sought in the difference of the solubility of the gases rather than in the difference of their diffusion coefficients as is maintained by Kniep. The plant obtains "Kniep, Jahrb. miss. Bot., 56, 460 (1915). 86 PHOTOSYNTHESIS its carbon dioxide from the surrounding water in which the gas is dissolved. The water is in equilibrium with oxygen and nitrogen of the atmosphere, the solubility of these two gases in water being relatively low as compared with carbon dioxide. The carbon dioxide reaches the centers of photosynthetic activity, the chloroplasts, by diffusion. Here, through photosynthesis, the carbon dioxide is reduced, and we may assume with a fair degree of safety, an equal volume of oxygen is formed. This oxygen can migrate either an exceeding small distance to the intercellular spaces, or in the other direction, out of the plant into the surrounding water. What actually happens is that the oxygen escapes from the plant through the intercellular spaces. If it were simply a matter of dififerences in diffusion coefificients the oxygen would never appear as bubbles of the gas. Kniep erroneously assumes that carbon dioxide has a very much higher diffusion coefficient than oxygen. Actually the reverse is the case and, strictly speaking, the diffusion coefficient is independent of solubility. However, carbon dioxide is much more soluble in water than oxygen and the water is already saturated with the latter gas at ordinary pressures and temiDeratures. The oxygen which is formed in photosynthesis produces a slight increase in pressure, partially dissolves and diffuses out, but mainly escapes into the inter- cellular spaces. Assuming the water is under atmospheric pressure, the gas will escai^e from the intercellular spaces when the pressure therein has attained a value slightly higher than atmospheric pressure and suffi- cient to overcome the resistance of the capillary surfaces in the inter- cellular canals. As this pressure increases more oxygen is dissolved in the water bounding the intercellular sj^aces and escapes by diffusion. Therefore, all of the oxygen produced in photosynthesis does not escape as a gas, although on account of the low solubility of oxygen in water the quantity removed by diffusion is very small compared to the gaseous emission. The higher the rate of oxygen formation the faster the gas will escape from the intercellular spaces. But the water contains dissolved, not only oxygen, but also nitrogen and carbon dioxide. Therefore these gases are also present in the intercellular spaces. The bubbles which escape from these spaces contain these gases also. As has been stated, the higher the rate of oxygen formation, the faster the gas will escape, sweeping out with it the nitrogen and carbon dioxide. As the pressure of these latter gases is reduced in the intercellular spaces the equilibrium with the water bounding the spaces is disturbed and some nitrogen and carbon dioxide pass from the dissolved state into the gaseous form. When the rate of photosynthesis is high and the escaping gas stream correspondingly high, the oxygen sweeps out some of the nitrogen and carbon dioxide faster than it can be replaced. Thus, per unit time, with a high rate of oxygen formation, much nitrogen is carried out. This represents the condition in which the oxygen-content of the escaping gas THE NATURE OF PHOTOSYNTHESIS 87 is high during rapid photosynthesis. Conversely when the rate of photo- synthesis is low, oxygen formation is low, the escaping gas stream is slow, and the nitrogen and carbon dioxide which have been swept out are replaced in the intercellular spaces from the surrounding water. Under these conditions the gas which escapes from the plant contains a higher percentage of nitrogen and carbon dioxide than when the photo- synthetic rate is high. Kniep's results also show that when the rate of the escaping gas stream is high the absolute amount of nitrogen evolved in unit time is higher than during a low rate of gas emission. This is in spite of the fact that the percentage of nitrogen is lower during a high rate than during a low one. This would indicate that during high rate of gas emission the partial pressure of nitrogen in the intercellular spaces is sufficiently reduced to cause a relatively rapid escape of this gas from the surrounding water into the intercellular spaces. In all probability oxygen is the only gas which is emitted by the plant during photosynthesis. Pollacci ^^ reported the presence of small quantities of hydrogen and some hydrocarbon in the emitted gas, but this single observation has never been substantiated. Carbon monoxide ^^ has been found in plants as a product of respira- tory activity but has not been found in the gas emitted during photo- synthesis. Most of the analyses of gases emitted during photosynthesis are based upon relatively small samples of gas ; it might prove interesting if large quantities of the gas were analyzed for traces of other gases. 40 d. The Photosynthetic Quotient. In the study of the respiration of both animals and plants the deter- mination of the ratio of the volume of oxygen absorbed to that of carbon dioxide expired has been a most valuable means of determining the nature of the oxidation processes. Similarly in photosynthesis the ratio of the volume of carbon dioxide absorbed to that of oxygen emitted has been extensively studied in attempting to establish the nature of the reduction process. The much quoted early work of de Saussure, Boussingault and some others, while it established that this ratio was close to unity, is nevertheless, in the light of modern methods, not suffi- ciently accurate for our present needs. There are a number of factors in the determination of the ratio which exert a profound influence on the values obtained, but which are extremely difficult to regulate. Prob- ably the most significant of these is respiration. The oxidative processes comprising respiration, in which carbon dioxide is formed, it is generally assumed, are going on simultaneously with photosynthesis during the ^Pollacci, Atti deir R. hist. Bot. Pavia. 7, 97 (1902). "-Langdon, G. C, Science, 49, 573 (1919). ■^Boussingault, Agronomic, etc., 3, 271 (1864). 88 PHOTOSYNTHESIS periods of illumination. It has been an exceedingly difficult problem to separate these two reactions, proceeding, as it were, in opposite direc- tions. It needs no further analysis to realize that even slight variations in the respiratory ratio would affect profoundly the results of the photo- synthetic ratio. It must be borne in mind that respiration is an exceed- ingly complex process, the resultant of a series of reactions, some of which lead to the synthesis of material necessary for the life of the plant, others to end or waste products. Many of the latter are rich in oxygen and are broken down only under certain conditions. So that the absorp- tion of oxygen and liberation of carbon dioxide may be separated by many intermediate reactions.*^ Unfortunately there exists in the literature a great deal of confusion as to the manner of designating the photosynthetic and respiratory O Oo ,CO, ^ , quotients. The former has been written -zr:r-, 7^7;~» ^^'^'-'TS — ' these C02 . pages th e photosynthetic quotient will be designated by -— — , i.e. vol. COo absorbed , . . Oo ^, . : and similarly the respiratory quotient 7:77-. ihis vol. Oo emitted CO3 will necessitate that in some of the quoted works the symbols must be specially noted because they are given as they stand in the original pub- lications. Bonnier and Mangin ■*- endeavored to separate the photo- synthetic and respiratory processes and made a number of determinations of the photosynthetic quotient under special conditions. In their pub- lication there are a number of misprints and arithmetical errors, which, however, it is difficult to correct on account of the lack of original ex- perimental data. By means of the following four methods it was attempted to separate the gaseous exchange due to photosynthesis and respiration. These methods have been extensively used for this purpose, with little further critical examination. 1. — Comparison of the gas interchange in the dark and in light. The rate of respiration is determined in the dark, and in the light the gas interchange due to photosynthesis plus respiration is measured. From this data it is possible to obtain the values for the changes in the com- position of the gas surrounding the plant due to photosynthesis alone. Thus, if in a given time c is the volume of carbon dioxide emitted in the , o O2 dark, o is the volume of oxygen absorbed in the dark — = = r, the C V^vJ2 respiratory quotient, and if in the same time Ci is volume of carbon dioxide absorbed in the light, Oi is volume of oxygen emitted in the light, the total carbon dioxide absorbed in photosynthesis is c + Ci and the total oxygen produced, o + O], and the true photosynthetic quotient is "Spoehr, H. A., Carnegie Institution of Washington Pub. No. 287, 1-23 (1919). *^ Bonnier and Mangin, Ann. Sci. not. Bot.. (7) 3, 1-44 (1886). THE NATURE OF PHOTOSYNTHESIS 89 c + ci COo o + oi O, =: P. That is, the photosynthetic quotient is determined from the difference in the carbon dioxide and oxygen-content of the air surrounding the plant in the two determinations. In employing this method there are a number of corrections which must be applied. Great care must be exercised to avoid fluctuations in temperature in the two experiments. It is a question whether the rate of respiration is the same in the light and in the dark. Under the experimental conditions of Bonnier and Mangin *'■'' there were a number of disturbing influences which unquestionably affected their results. They also attempted to separate the two processes of respiration and photosynthesis by comparing the rate of respiration of plant parts that contained chlorophyll with other portions that contained no pigment. It is apparent now, however, that such procedure is not permissible. 2_Inhibition of Photosynthesis by means of narcotics. Bonnier and Mangin, based upon the old observations of Bernard ** with chloroform, found that photosynthetic activity is much more sensitive to the action of ether than respiration. It was attempted to determine the photo- synthetic quotient by determining the gas interchange in parallel experi- ments with and without ether. The method is not exact, because it is very difficult to establish the exact dosage of the narcotic which completely inhibits photosynthesis and does not affect respiration (see the effect of narcotics on photosynthesis discussed later). It is very doubtful whether the rate of respiration in an anesthetized plant is the same in the light and in the dark.*^ 3_Suppression of photosynthesis by removal of carbon dioxide. The method depends upon the comparison of the gas interchange of leaves in two similar vessels. One of these contains a solution of barium hydroxide in order' to absorb the carbon dioxide of the atmosphere and that liberated in respiration, the other contains an equal volume of water. From the difference between the oxygen-content and carlion dioxide-content of the two vessels at the end of the experiment the quantities of oxygen evolved and carbon dioxide absorbed during photosynthesis can be calculated. This method is open to serious error on account of the fact that the rate of carbon dioxide emission (respiration) in the two vessels is not the same. Spoehr and McGee *^ have shown that the rate of carbon dioxide emission of leaves is greatly influenced by the partial pressure of carbon dioxide in the surrounding atmosphere. They showed that when the carbon dioxide-content of the air surrounding a leaf is changed from a «Bonftier and Mangin, Compt. rend., 96, 1075 (1883), 99. 160 (1.884). Anyi. Set. mt. Bot., (6) 18, 293 (1884). . ■"Bernard, Legons sur les Phcnomcnes de la Vie, 1. 278. Pans (1878). « Usher and Priestley, Proc. Roy. Soc, B 77, 369 (1906), 78, 318 (1906). ^Spoehr and McGee, Amer. J. Bot., 11, 493 (1924). 90 PHOTOSYNTHESIS higher to a lower concentration, the leaf shows a primary increased rate of carbon dioxide emission. Conversely, when the carbon dioxide-content of the air changes from a lower to a higher concentration the leaf shows a reduced rate of carbon dioxide emission. In the method just described, the leaf in the vessel containing barium hydroxide would therefore emit carbon dioxide at a higher rate than the leaf in the vessel containing only water. This would result in an apparently higher rate of respiration than is actually the case in the vessel containing a higher concentration of carbon dioxide. There are a number of modifications of this method, but the principle and errors involved are the same. 4— Comparison of leaves with different chlorophyll-content. Hereby the gaseous interchange of leaves of the same plant which are unequally green is measured, on the assumption that the two varieties dififer only as to photosynthetic rates. One difficulty in this method is to obtain leaves of the same age and yet dififering in their chlorophyll-content. Willstatter and StoU *' have shown that there is no direct proportionality between chlorophyll-content and photosynthetic activity. Also Plester ** has shown that the light-green or aurea varieties, with low chlorophyll-content have a low respiratory activity as compared with the normal varieties. By means of these methods Bonnier and Mangin determined the photo- synthetic quotient of a large number of plants. Following their notation, '- the values obtained for the photosynthetic quotient were usually vol. CO2, greater than 1, and ranged between 1.1 and 1.3. In the 40 years since the publication of the work of Bonnier and Mangin a number of publications have appeared on the subject of the photosynthetic quotient notably by H. Jumelle ^^ and Th. Schloessing fils.^° With the exception of Willstatter and Stoll, whose work will be considered later, no contributions of note have been made to the problem of measuring separately the respiratory and photosynthetic activities. Maquenne and Demoussy,^^ employing essentially the first method of Bonnier and Mangin, made an extensive investigation of the quotients of gas interchange. Their results of the respiratory and photosynthetic quotients are given in Table 16. They used a closed chamber and the composition of the gas was determined after exposure to light and darkness. In 29 of the 34 plants studied the photosynthetic quotient was equal to or very slightly less than the respiratory quotient. After making cer- tain corrections Maquenne and Demoussy conclude that the volume of oxygen emitted is equal to the volume of carbon dioxide decomposed. "Willstatter and Stoll, Untcrsuchuiigcn ucber die Kohlensaeiireassilation, p. 86. Berlin (1918). ** Plester, Bcitragc zur Biol. d. PHanzcn. 11, 249 (1912). "Jumelle, Compt. rend., 112, 888 (1891); 113, 920 (1891); Rev. gen. hot., 4, 49 (1892). "Schloessing, Compt. rend., 115, 881, 1017 (1892) ; 117, 756, 813 (1893). " Maquenne and Demoussy, Echanges Gaseux des Plantes Vertes avec fAtmos- sphere. Paris, 1913. THE NATURE OE PHOTOSYNTHESIS 91 TABLE 16 Respiration and Photosvnthetic Quotient Determined by Maquenne and Demoussy. Respiratory Photosynthetic Quotient Quotient COa _0_ Species O COa Ailanthus 1.08 1.02 Aspidistra 0.94 1.00 Aneuba 1.11 1.10 Begonia 1.11 1.03 Cherry-laurel 1.03 0.97 Chrysanthemum 1.02 1.01 Dahlia 1.07 1.07 Geranium 1.02? 1.05? Grape-vine 1.01 0.99 Ivy 1.08 1.00 Kidney bean (young) 1.12 1.12 " (average) 1.07 1.07 Lilac 1.07 1.03 Lily(?) 1.07 1.00 Mahonia (autumn) 0.95 0.99 Pea 1.07 1.04 Pear 1.10 1.08 Poppy 1.09 1.09 Privet 1.03 1.02 Rhubarb 1.02 1.00 Ricinus 1.03 1.03 Rose 1.02 1.10 Rose Laurel 1.05 1.01 Spindle-tree 1.08 1.02 Sorrel 1.04 1.04 Tobacco 1.03 . 1.04 Turnip 1.11 1.06 Wheat 1.03 1.02 It is probably evident from the foregoing that the cliief difficulty in obtaining an exact determination of the photosynthetic quotient is occa- sioned by the fact that an error in the determination of the respiratory rate also appears in that of the photosynthetic quotient. The separation of the two processes is exceedingly difficult so that no matter v^hether the respira- tion is determined in the dark, with narcotics in the light, or with leaves poor in chlorophyll, every inexactness of the respiratory quotient affects the value of the photosynthetic quotient. Willstatter and Stoll " have endeavored to surmount this difficulty by determining the photosynthetic quotient under conditions in which the photosynthetic activity is very much greater than respiration, so that small errors in the determination of the latter would have but a very slight effect on the accuracy of the photo- synthetic quotient. They made another departure in that instead of using a closed system, a stream of air was passed over the leaves. This is the only method by which the leaves are exposed to constant conditions "Willstatter and Stoll, I.e., 325. 92 PHOTOSYNTHESIS of photosynthesis. Of course the differences in the composition of the gas to be analyzed are very much smaller than in the closed chamber method, but these differences can be determined with modern methods of gas analysis. For this purpose Willstatter and Stoll employed conditions of maximal photosynthetic activity, so that respiration was 1/20 to 1/30 of photosynthesis. Thus slight variations in respiration, as occasioned, for instance, by the differences in respiration in light and in the dark, had but an insignificant effect on the determination of the photosynthetic quotient. Carbon dioxide-concentrations of 5-6.5 volume per cent and light intensity of about 45,000 lux were used. Under these conditions the photosynthetic quotient —^ at 10 to 35° was found to be constant and exactly 1. This applied to a variety of plants including Ilex aquifolimn, Samhucus niger, Pelargonium sonale, and Aesculus hippocastanum. Even under conditions of photosynthetic inhibition through the accumulation of products (see below) the value of 1 was maintained. The importance which attaches to the value of the photosynthetic quotient can be readily seen from the following considerations. Just as from the value of the respiratory quotient definite information can be gained as to the nature of the material which is oxidized, the photosynthetic quotient indicates the nature of the material formed in the decomposition of carbon dioxide. It has been found that the respiratory quotient vol. COo emitted , . , ,. r i/^/^ r i /^ — of annuals on a diet of 100 per cent fat and per cent vol. O2 absorbed, carbohydrate is 0.707, and when on a diet of i>er cent fat and 100 per cent carbohydrate is 1.00. Quotients intermediary between 0.707 and 1.00 indicate that mixtures of the two materials are being used. This principle has become one of the most useful tools in the hands of the animal physiologist and has been elaborated to include a large variety of substances and conditions. TABLE . 17 Calculated Photosynthetic Quotients for Different Prim Carbon Dioxide Reduction. ARY Products of Compound Formic Oxalic Glyoxalic Acid Acid Acid Glvcollic Acid Malic Acid Glycol- Formal- aldehyde dehyde Photosynthetic quotient CO, 2.0 4.0 2.0 1.33 1.33 1.0 1.0 o Similarly the photosynthetic quotient may yield some information relative to the first product formed in photosynthesis. Between carbonic acid and carbohydrates there are a number of possible reduction products. A great deal of speculation has centered about the question as to which is the first substance formed in the process of photosynthesis. In the THE NATURE OF PHOTOSYNTHESIS 93 previous table are given a number of the possible reduction products together with the photosynthetic quotient which corresponds to their formation calculated from the carbon dioxide absorbed and oxygen split off. When the photosynthetic quotient is exactly 1 it is evident that the carbon dioxide has been reduced to carbon, CO2 ^ C + O2, or in the hydrated form carbonic acid has been reduced to a carbohydrate. Since the best evidence indicates that the photosynthetic quotient is 1, this would signify that carbonic acid is reduced to the formaldehyde stage. How- ever, the evidence of the value of the photosynthetic quotient does not throw any light on the question as to the exact compound which is formed. It may be formaldehyde, glycolaldehyde, or any other carbohydrate of the general formula CnH^-uOn, so that the results of the gaseous exchange during photosynthesis can contribute little toward settling this much de- bated question. The evidence of the photosynthetic quotient does con- tribute to determining whether the first products formed in photosynthesis are compounds other than those possessing an empirical formula CnHonOn. The old theory of Liebig ^^ that organic acids are the precursors of carbo- hydrates in photosynthesis still finds adherents who have modified and elaborated the original theory. These views will be discussed in another chapter. Suffice it to point out here, that it is now clearly established that the hydroxy-acids which play so important a role in the Liebig theory are products of a modified respiratory activity and that a photosynthetic quotient of 1 excludes them from being the first direct products of carbon dioxide reduction. Similarly the theory that fats are the first products of photosynthesis finds no support in the values of the photosynthetic CO2 absorbed quotient, for these substances would demand a quotient, -— -, ;; — ^ O2 emitted in the neighborhood of 0.7. One other point regarding the photosynthetic quotient deserves con- sideration. It has been known for a long time that the fleshy plants or succulents exhibit respiratory and photosynthetic quotients which differ greatly from the values obtained with thin leaf species. These plants ordinarily show a very low photosynthetic ratio. The structural arrangements of the succulents are such that the plant loses relatively little water through transpiration. The ratio of the sur- face to the volume is low and there are relatively few stomata. This results in inhibiting the gaseous exchange. A striking feature of the metabolism of these plants is the accumulation of organic acids at night or in the dark and the decomposition of these acids during periods of illumi- nation. This phenomenon has been investigated very thoroughly recently by Richards '^* who also discusses the older literature. The accumulation ■"Liebig, Die Chcviic in Hirer Anzvendtmg auf Agrikultur iind Physiologic, I, 52 (1862). *• Richards, Carnegie Inst. Washington, Pub. No. 209 (1915). Hempel, Compt. rend, du Laboratoire de Carlsberg, 13, 1-129 (1917). 94 PHOTOSYNTHESIS of organic acids results from the incomplete oxidation of sugars. In the light these acids disappear, partly on account of a greater oxygen- supply due to photosynthesis, and also due to the direct photolysis of the acids as described by Spoehr.''^ There are therefore several compli- cations which arise in determining the photosynthetic quotient. The organic acids (e.g. malic acid in the cacti) break down in the light with the liberation of carbon dioxide. Thus it happens, that, as Richards records, carbon dioxide is given off by the plant when exposed to diffuse light or direct sunlight. The oxygen also varies greatly under these circumstances ; in diffuse light there is an absorption of oxygen while in intense illumination oxygen is emitted. It must also be realized that in the interior of the plant the breaking down of the acids serves as a source of carbon dioxide, which in the light is used in photosynthesis. When this oxygen escapes from the plant it results in a greater vol- ume of oxygen being emitted than carbon dioxide is absorbed, i.e., CO2 absorbed , . , • • , , r , , , — — < 1 ; m some cases this ratio has been found to be less O2 emitted than 0.5. With long continued exposure to light the ratio increases and slowly approaches unity. These processes have the general significance that in plants which are protected against great water loss the gaseous exchange is slow and carbon dioxide is dealt with most economically. CO2 Kostytschew ^^ has brought out the very interesting fact that the — — — O2 ratio during illumination varies with time. The initial carbon dioxide content in his experiments was about 6 per cent, i.e. considerably above normal air. He found that during photosynthesis the leaves absorb initially a great deal more carbon dioxide than oxygen is given off ; about one third the absorbed carbon dioxide is fixed without oxygen emission. After a short time these conditions are reversed, more oxygen is given COo off than carbon dioxide is absorbed, and finally the — — ratio attains a constant value of 1. Kostytschew found that these relations maintain for aquatic plants as well as for leaves of land plants. These observations are found in accord with those of Spoehr and McGee ^^ on the absorptive capacity of leaf material for carbon dioxide. They are especially impor- tant in their bearing on the methods of measuring photosynthetic activity. If this behavior is universal it would follow that photosynthetic activity should be measured by the amount of carbon dioxide absorbed rather than by the amount of oxygen emitted. It is also of direct bearing on the theory of a primary chemical reaction of carbon dioxide with an absorbing substance as the first step in photosynthesis. "'Spoehr, Biochcm. Zeit., 57, 95 (1913). • '" Kostvtschew, Ber. hot. Gcs.. 39, 319 (1921). "Spoehr and McGee, Amer. Jour. Bot., 11, 493 (1924). THE NATURE OF PHOTOSYNTHESIS 95 2. Factors Which Influence the Rate of Photosynthesis a. The Principle of Limiting Factors. In endeavoring- to understand the photosynthetic process it is essen- tial to bear in mind that we are dealing with a complex system. It is complex because there are a number of factors involved, each one is necessary for the successful and continuous operation of the process. It is complex because each of these factors influences the process more or less independently. Add to this that we are dealing with a series of chemical reactions of different types including photochemical and catalytic reactions, and it must be evident that to express these relationships in quantitative terms becomes an exceedingly difficult task. And finally the fact must never be lost sight of that photosynthesis is a function of the living plant. Although a great deal of work has been done to determine the influence of the various factors involved in photosynthesis it is evident now that no one of these factors can be studied without at the same time taking into consideration all of the others. The factors which are primarily concerned in determining the rate of photosynthesis in any chlorophyllous organ are as follows : 1 — The partial pressure of carbon dioxide in the air or water sur- rounding the plant. 2 — The intensity and frequency of the light used. 3 — Temperature, more particularly that of the chloroplasts. A — The amount and composition of the chlorophyll. 5 — The amount of water available. 6 — Certain internal factors. It has been customary for physiologists to endeavor to determine three cardinal points in studying quantitatively the influence of various factors or conditions on any physiological phenomenon. These cardinal points are: the minimum, below which the phenomenon ceases, the optimum, at which the phenomenon takes place to the highest degree, and the maxi- mum, above which it ceases. Many attempts which were made to estab- lish the cardinal points of the various factors operative in photosynthesis led to contradictory results. It was F. F. Blackman ^* who in 1905 called attention to the fact that in a phenomenon such as photosynthesis where there are several factors at work simultaneously, the focusing of attention on a single factor with disregard to the influence of the others leads to erroneous results. A study of the "inter-relation of conditioning factors" led Blackman to formulate his principle of limiting factors which he stated in the following axiom : "When a process is conditioned as to its rapidity by a number of separate factors, the rate of the process is limited by the pace of tbe 'slowest' factor." Any of the factors mentioned above can ''Blackman, Ann. of Bot., 19. 281 (1905). 96 PHOTOSYNTHESIS act as the limiting one and thus determine the rate of photosynthetic activity. There has been some debate and misunderstanding regarding this theory. Below is given Blackman's original illustration. "Suppose a leaf in a glass chamber to have enough light falling upon it to give energy equal to decomposing 5 cc. of carbon dioxide per hour. Then, as one gradually increases the carbon dioxide in the air current through the cham- l)er from the amount (or pressure) that causes 1 cc. to diffuse into the leaf through its stomata up to five times that pressure, so steadily the assimilation will increase from 1 cc. to fivefold. After that, further in- crease of carbon dioxide will produce no augmentation of the assimilation. 30 2b 20 -O r, — 10 / / / / / n/ 7 ■ f -C S A % t^ i% i %% 5% i% Fig. 7. — Blackman's conception of the effect of limiting factors on the rate of photosynthesis. but will give continually an effect of 5 cc. of carbon dioxide — the light being now the limiting factor. The curve obtained will be of the form ABC. (Figure 7.) "Ultimately, if the supply of carbon dioxide in the air current be increased up to 30, 50, 70 per cent, the carbon dioxide will have a general depressing effect on the whole vitality, and before suspension of all function a diminution of assimilation undoubtedly occurs ; this is, however, quite a separate process. Now, secondly, suppose light falling on the leaf to be sufficient for the decomposition of 10 cc. of carbon dioxide per hour, then twice the external pressure of carbon dioxide will be required to reach the limit and the angle of the curve, which will now be A B D E. With still stronger light we should get A B D F G. Those who would be prepared to admit that a curve like ABC shows an optimum, only with a very long drawn-out top, would have to further admit that for each intensity of light falling on a leaf there is a different optimum amount of carbon dioxide. This is not to be entertained. THE NATURE OF PHOTOSYNTHESIS 97 "The light energy available fixes an upper limit to the carbon dioxide that can be decomposed, and when that amount is attained, which even for direct sunlight could be provided with a current of air containing less than 1 per cent, if the current were sufficiently fast, the limit of effect of carbon dioxide is reached : any more provided is wasted, and has no further effect till many times that concentration is reached and a general depressing effect comes in. . . ." Similarly, any of the other factors can act as the limiting one. It is evident that in all experimental work all of these factors must be taken into account and that it is essential to determine that no other factor beside the one under consideration is acting as the limiting one. The principle of limiting factors has its analog in the step-reactions of chemistry. Here if a reaction is made up of a number of steps, the rate of the total reaction will be determined by the rate of that step which is proceeding at the lowest rate. Also, as early as 1843, in his book, "Chemistry in its application to Agriculture and Physiology," Liebig had formulated his well-known Law of Minimum which stated : "by the deficiency or absence of one necessary constituent, all the others being present, the soil is rendered barren for all those crops to the life of which that one constituent is indispensable." At the time, of course, Liebig was not familiar with all the factors which affect the growth of plants and it required a great deal of experimental work by many investigators before this principle found an approximate mathematical expression. It is of interest here on account of the fact that the problem of crop yield is very similar to that of photosynthetic activity and has been dealt with in much the same way. Liebig's law of minimum has undergone many modifications and the facts which this law endeavored to express have finally been put into mathematical form though there still exists some controversy regarding it. It can be dealt with here but very briefly. ^^ There are five soil factors which influence the growth of plants : (1) Water supply, (2) Air supply, (3) Temperature, (4) Supply of min- eral nutrients, (5) Injurious factors. The effect of these factors may be determined by the total amount of dry matter formed by the plant under definite conditions. The effect of the fourth factor, the supply of mineral nutrients can be most easily determined. When then, the weights of dry matter formed under conditions in which but one essential factor is varied, are determined, there are obtained smooth curves which are amenable to mathematical expression. Mitscherlich has done this on the basis of many crop experiments. From these results it is evident that in the law of minimum we are dealing with a logarithmic function. The crop yields obtained with a single varying factor can be expressed in curves which are asymptotic to a maximum. This maximum would be attained if all condi- tions were ideal and there is a shortage in yield corresponding to the ™ Russell, E. J., Soil Conditions and Plant Grou'th. London, 1921. Pfeiffer, T.. Der Vegetationsversuch. Berlin, 1918. 98 PHOTOSYNTHESIS deficiency of any essential factor. The exact mathematical formula for these curves is still a matter of controversy. •'° The important fact is that they are logarithmic curves. In the problem of crop yield and the manner in which this is affected by various factors, there has been obtained, through a great mass of experimental data, a fairly accurate mathematical expression of the in- fluence of the deficiency of one of these factors. This expression is un- doubtedly an outgrov^rth of the original law^ of minimum, though in its course of development it has undergone considerable change. In the principle of limiting factors the photosynthetic activity, as first formulated by Blackman, vit have a state of affairs w^hich is in many re- spects analogous to that of crop yield. Blackman's theory has been one of the most helpful conceptions w^hich has been contributed to the study of the problem of photosynthesis. Within the last few years there has been some agitation regarding the general applicability of Blackman's theory.*'^ Some of the writers have gone so far as to maintain that the principle is entirely erroneous. It would seem, however, that these are rather developmental phases such as occur in the evolution of any funda- mental theory. In the more valid objections which have been raised, the conflict centers about the graphic or mathematical expression to be given to the theory rather than to the more general underlying principles. With more refined methods and extensive data it is not surprising that some modifications should come to light which may have escaped the origi- nal propounders with their rather limited experimental methods and data. Benecke "^ has given a very good elucidation of the principle of limiting factors. He points out that Blackman's theory is usually stated in such a manner as to predicate that only one of the various factors is limiting, while probably Jost's ^^ position is more tenable. The latter states that it may happen that all factors but one have so high an intensity or con- centration, that this single factor alone may determine the rate of photo- synthesis. Benecke shows that the curve representing the rate of photo- synthesis, when only one factor is varied, does not, when the intensity of this factor is augmented, exhibit a straight line increase and then a sharp turn when some other factor prevents the further increase in the photo- synthetic rate. But rather, the curve is a logarithmic one as, for example, is shown by Warburg's curve of the efifect of light intensity. Fig. 8. At first this is practically directly proportional to the light intensity, then it gradually bends to the horizontal. Here the photosynthetic rate is no longer directly proportional to the intensity of a single factor. This is the region where other factors interact and more than one factor limits. Finally, the curve is almost parallel to the abscissa, and here some other factor besides the one which was varied becomes the limiting one. As "Baule. Landtv. Jahrb., 54. 495 (1920). "Hooker, Science. 46, 197 (1917). Crocker, Bot. Gaz., 65, 287 (1918). "Benecke, Zeit. f. Bot, 13, 424 (1921). "Jost, Bot. Zeitg., 64, 72 (1906). THE NATURE OF PHOTOSYNTHESIS 99 will be shown under the discussion of the effect of light intensity accord- ing to Harder 's experiments, there is a portion of the curve where more than one factor is limiting. Benecke considers that any factor is in "absolute minimum" only when that factor is entirely absent, e.g., light is in "absolute minimum" when the plant is in darkness. If we start with very weak light and then grad- ually increase the intensity, the light factor is in "relative minimum" the moment of first illumination. Other factors can at the same time be in "relative minimum." The difficulty of obtaining strictly mathematical results lies in the experimental complexities involved, in the fact that it is almost impossible to alter only one factor without at the same time affecting some other factor. Even if corrections can be applied to such factors as temperature on bicarbonate dissociation or solubility of carbon dioxide, it is impossible to correct for the effect of external agencies on internal conditions, as e.g., the action of light on chlorophyll and certain stimulatory effects. The principles involved in the theories of limiting factors can be more clearly elucidated in the discussions of the influence of various individual factors in the following sections. b. The Influence of Light. Investigations on the influence of light on photosynthesis have been directed in two main courses. One of these has been chiefly concerned with the effect of different intensities of illumination on photosynthetic activity and the other with the influence of different frequencies or wave lengths. Some of the earlier work on both of these phases of the problem is contradictory and confusing. This is due, first of all, to a lack of realization on the part of the older workers of the interrelation of the various factors affecting photosynthesis, which was first clearly recognized in Blackman's theory of limiting factors, and secondly, to technical diffi- culties in the measurement of light intensity. With a clearer understand- ing of the influence of other factors involved in the photosynthetic process, that of light too was somewhat clarified. Also, the recent development in our conceptions of the nature of radiant energy, the quantum theory, the laws of photochemical action have contributed much to better experimental procedure. It may be expected that the mastery of these newer concep- tions of the nature and action of light by experimenters in this field will be of great service in gaining a better understanding of the role which light plays in photosynthesis. Already the two lines of investigation men- tioned, that of the effect of different intensities and that of the influence of different frequencies have been brought closer by the approach from the viewpoint of the quantum theory. In the dark a chlorophyllous plant absorbs oxygen and gives off car- bon dioxide in the process of respiration. Even illumination of very low intensity is apparently capable of inducing a reduction of carbon dioxide 100 PHO TOS YN THESIS by the chloroplasts. However, under these circumstances photosynthesis does not become evident by an evolution of oxygen, but only by an appar- ently lower rate of respiration, that is, the amounts of carbon dioxide given off and of oxygen absorbed are less. There is no evidence that feeble illumination affects the rate of respiration itself, so that it has been concluded that the lower rate of carbon dioxide evolution and of oxygen absorption are due to the fact that in the very low light intensity a por- tion of the carbon dioxide evolved in the process of respiration, is taken up by the chloroplasts and a small amount of oxygen is evolved which in turn is immediately utilized in respiration again. This results in a dimin- ished gas interchange of the plant with the atmosphere. With increasing illumination a point is finally reached where the rate of photosynthesis just balances that of respiration and there is no gas interchange whatso- ever. A discussion of this state, the so-called compensation-point, will be taken up later. Exact determinations of the minimum intensity of light which is necessary for carbon dioxide reduction have not yet been made.*** Most of the measurements consider the point of oxygen evolution, that is, where photosynthesis overbalances respiration. But this, of course, is not the lowest intensity of light necessary for photosynthesis, but merely an intensity where the rate of photosynthesis is greater than that of respiration. Early experiments showed that photosynthetic activity increases pro- portionately to the intensity of light and that there is a limit to the in- crease above which increase in the light intensity results in no augmenta- tion of the photosynthetic rate. Moreover, every plant has its own specific requirements and the capacity to use light as well as the efficiency varies widely in different species. The underlying principles for these phe- nomena have been brought to light by more recent investigations. Some reference to the influence of light on the rate of photosynthesis has already been made under the discussion of the effect of carbon dioxide. It has repeatedly been stated that none of the factors affecting the rate of photosynthesis can be studied alone or without regard to the influence of other factors. From a consideration of the principles which have been given expression in the theory of limiting factors, it is evident that changes of light intensity have an effect on the rate of photosynthesis only when light is the limiting factor or "in minimum." Considering first Blackman's conclusions in formulating this conception we can summarize, that where carbon dioxide supply and temperature are in excess the rate of photosynthesis is proportional to the intensity of illumination. This, of course, assumes that other factors affecting photosynthesis, as chlorophyll-content and water supply are constant. Blackman and Matthaei '•' used sunlight and perforated screens to reduce the light in- •" For Moonlight: Boussingault, J. B., Ann. d. sci. nat. (Ser. V), 10, 335 (1869). Ursprung, A., Ber. bot. Ges.. 35, 62 (1917). Midnight-sun: Curtel, G., Rev. gen. bot 2 7 (1902). Kostytschew, Ber. bot. Ges., 39, 334 (1921). ^Blackman and MaUhaei, Proc. Roy. Soc. London, B 76, 402 (1905). Black- man and Smith, ibid., B 83, 389 (1911). THE NATURE OF PHOTOSYNTHESIS 101 tensity. Exact values for the light intensities were not obtained. But they were able to determine what proportion of direct sunlight was required to give the maximum possible photosynthesis at a definite temperature. Thus the photosynthetic rates determined at 29.5° at mid-day of August 8 and 16 respectively were : Cherry-laurel: 0.28 sunlight gives 0.0116 g. CO2 per 50 sq. cm. per hour. Helianthus: 0.62 " " 0.0224 " " " " " " " These values then represent the minimum light intensity which is suffi- cient for a maximum rate of photosynthesis at that temperature and as- suming there is no other factor limiting. Helianthus is therefore capable of a much higher rate of photosynthesis than cherry-laurel at the same temperature, and neither of the plants is capable of utilizing for photo- synthesis the whole of the solar radiation. The highest photosynthetic rate observed by Blackman and Matthaei equaled about 2900 cc. CO2 per square meter per hour for Helianthus at 29.5°. According to the Black- man view, in order to determine whether photosynthesis as observed at any given time is Hmited by light intensity, temperature or some other factor, it is necessary only to alter the intensity of each factor separately. It has just been stated that the two ditTerent species of leaves, cherry- laurel and Helianthus, exhibit different maxima of photosynthetic activity at the same temperature. This raises the question whether different types of leaves have different specific photosynthetic characteristics. Blackman and Matthaei have also investigated this question using different plants as Helianthus, Tropaeolum, Bomarca and Aponogeton. They conclude that "leaves in general have the same coefficient of economy in the photo- synthetic process." While at 29.5° Helianthus can assimilate twice as much carbon dioxide as the cherry-laurel, at low temperatures, they have similar photosynthetic maxima. Blackman and Matthaei harmonize the latter statement with their assumption of "equal coefficients of economy" by pointing out that Helianthus requires twice as much light to attain the double assimilation at 29.5°. "The fundamental existing specific differ- ences would seem to lie in their different coefficients of acceleration of activity with increase in temperature." For a rise of 10° the increase with cherry-laurel is 2.1 and with Helianthus it is about 2.5. The question of the specific photosynthetic value of leaves requires further investigation before Blackman and Matthaei's dictum can be en- tirely accepted. There are two points which suggest themselves at once. The first of these is that the differences of acceleration due to temperature of photosynthetic activity are probably related to enzyme action or what we here include under internal factors, which, under the experimental conditions, were governing the rate of photosynthetic activity. Of this we know as yet very little. Secondly, it must be kept in mind that leaves differ greatly in their photosynthetic activity according to their chlorophyll- content, but that there is probably no direct relationship between the 102 PHOTOSYNTHESIS chlorophyll-content and photosynthetic activity. Willstatter has shown that the effect of the internal factor is increased by raising the temperature. However, an increase in the activity of the internal factor is of influ- ence on photosynthesis only in leaves with a sufficiently high chlorophyll- content. The latter question is discussed in another section of this book. It is a matter of common observation that some plants thrive in the shade, protected from the direct rays of the sun, while others do best under conditions of direct insolation. Undoubtedly the causes which underlie such an adaptation to environmental conditions are complicated. Yet under these different conditions of illumination the respective plants are capable of carrying on their photosynthetic activity with a total net gain to each in dry matter. Boysen-Jensen ^^ has made a study of the photosynthetic activity of some typical "shade" and "light" plants with special reference to their gain in dry matter, i.e. "the amount of dry matter produced in unit time, calculated in per cent of dry matter in the plant in question at the beginning of the experiment." As an example of a light plant Sinapis alba is taken, and as a shade plant Oxalis acetosella. In Boysen-Jensen's experiments the former were grown in "full daylight" and the latter in "a light intensity as small as possible." His own con- clusions are as follows : "In Sinapis the intensity of COo assimilation is very great, rising to at least 6 mg. CO2 per 50 cm.^ per hour at 20°. Also the respiration in the leaves is great, about 0.8 mg. CO2 per 50 cm.^ per hour at 20°. The point of equilibrium between CO2 assimilation and respiration lies at a light intensity of 1.0 (Bunsen units X 100). The de- velopment of a Sinapis plant is very quick. In four weeks the dry matter content rises from 0.5 g. to 38 g. per 100 plants. In favorable conditions the daily per cent production of dry matter can be estimated as about 15. "In Oxalis the maximal intensity of COo assimilation is very small, alx)ut 0.8 mg. CO, per 50 cm.- per hour at 20°. Also the respiration of the leaves is small, about 0.1-0.2 mg. COo per 50 cm.=^ per hour at 20°. The point of equilibrium between COo assimilation and respiration lies at a light intensity of 0.2. The daily per cent production of dry matter is 2.1." From these experiments it is apparent that, as nearly as two such plants can be compared, the one growing in the sunlight does more photo- synthetic work than the other, a conclusion which is not surprising. In both cases respiration took about 13 per cent of the material synthesized. Boysen-Jensen also calculated from .Combes' ''^ data the per cent produc- tion of dry matter of a number of rapidly growing plants. The results are given in Table 18. Of course, the foregoing results contribute very little to the question of the efficiency of different plants in utilizing light for photosynthetic work. This question is discussed in the section on energy relations. Some "Boysen-Jensen, Bot. Tidsskrifl, 36, 219 (1918). Johansson. Svcns K. Bot. Tidsskrift, 17, 215 (1923). •"Combes, Ann. d. Sci. nat., IX Ser. Bot., 11, 75 (1910). See also Weis, R, Compt. rend., 137, 801 (1903). Weber, Arb. Bot. Inst. Wiirzburg, 2, 346 (1879). THE NATURE OF PHOTOSYNTHESIS 103 TABLE 18 Per Cent Production of Dry AIatter of Different Plants. (Boysen-Jensen.) Number of Per Cent Production Plant Days of Dry Matter Triticum vulgare ; . . . . 35 6.5 77 5.9 Raphanus satious 19 16.8 Pisum sativum 17 5.9 Tropaeolum majus 11 11.2 30 13.4 Salsola kali 24 10.1 results of Warburg and Negelein *^* are of direct bearing on this question and of interest in this relation. They studied the photosynthetic activity of the unicellular alga Chlorella and observed great variations in the efficiency of these plants. They found that when the plants are raised under conditions of high light intensity, the amount of radiant energy utilized is low. That is, plants raised under these conditions convert but a small fraction of the absorbed radiant energy into chemical energy. While plants grown under low light intensity convert a larger portion of the absorbed energy into chemical energy. In other words, Warburg and Negelein consider that they can at will produce "light" and "shade" plants and that the photosynthetic efficiency of the two types of plants is different. It would be interesting to determine whether shade plants such as the Oxalis used by Boysen-Jensen, while they may in total produce a smaller amount of dry matter, do not utilize a greater proportion of the light ab- sorbed than plants growing in the direct sunlight. In a previous section some discussion has already been devoted to the interaction of the different factors which influence photosynthesis. Ac- cording to the original theory of Blackman on "limiting factors" "the rate of photosynthesis is determined by the intensity of the weakest factor." If the intensity of this latter factor is increased, the rate of photosynthesis increases until some other factor becomes relatively the weakest or limiting one. Further increase in the intensity of the first factor does not result in an augmented photosynthetic rate because this factor is no longer the limiting one. These conditions have been described graphically under the sections devoted to the "Principle of Limiting Fac- tors" and the "Partial Pressure of Carbon Dioxide," where the recent in- vestigations of Warburg and of Harder are also discussed. The latter has followed the rate of photosynthesis when one factor only is changed as well as when two factors (light intensity and COa-concentration) are altered. Harder's results appear to necessitate a modification of Blackman's original conception of the influence of various factors, in the sense that the effect of the factor which is varied is not the same throughout the ''Warburg and Negelein, Zdt. physik. Chem., 102, 246 (1922). 104 PHOTOSYNTHESIS course of the curve but depends upon the intensity of the other factors. When the other factors, carbon dioxide-concentration, temperature, etc., are relatively high and light intensity is low, the increase in the rate of photosynthesis is about proportional to the increase in light intensity. As the latter factor continues to be augmented the effect on the rate of photo- synthesis becomes less, so that the curve flattens, and the influence of other factors becomes noticeable. Such curves of the effect of light intensity have been obtained by Boysen-Jensen,«^ Warburg '' and Harder '^ and are reproduced in Figure 8. 24U 220 ^^ 200 180 ^^,^ 140 ^ 120 100 / (lU 60 40 20 / / 1 I 3 t 5 5 7 B » 1 11 1 2 13 1 4 15 1 6 17 Fig. 8. — The influence of light intensity on the rate of photosynthesis of Chlorella. The ordinate represents the rate of photosynthesis, the abscissa the illumination intensity. (From Warburg.) Harder measured the photosynthetic activity of Fontinalis at different light intensities and varying carbon dioxide-concentrations. The light intensity of the highest was 216 times that of the lowest intensity, and the carbon dioxide-concentration was varied from 0.01 to 0.32 per cent. The results are shown in Table 19 and represent true photosynthetic rates. '^ Boysen-Jensen, P., Bot. Tidsskrift, 36, 220 (1918). '"Warburg, O., Biochcm. Zcit., 100, 255 (1919). "Harder, R., Jahrb. wiss. Bot., 60, 531 (1921). THE NATURE OF PHOTOSYNTHESIS 105 TABLE 19 Rate of Photosynthesis of "Fontinalis" at Six Different Light Intensities AND Four Different Concentrations of Carbon Dioxide. (From Harder.) -Light Intensity (in Meter Candles) KHCO3 167 667 2000 6000 18,000 36,000 0.01 per cent... 0.12 0.42^^ *^-^^ ^'^^ ^'^^ ^'^^ 0.04 per cent... 0.26 88^'^^ ^'^^ ^'^^ ^'"^^ ~ 0.16 per cent... - i?f(l.lO 3.45 6.40 li;47 i 11.35 - 1-1^ > 10.75 J ^„ . 17^ IfioK^O 8.60 15.83 15.20 16.645 0.32 per cent... — 1.^3 4.oU> 15.70 J If the table is read from left to right it becomes evident that each increase in light intensity results in an increased photosynthetic rate ; also if the table is read down it can be seen that each increase in carbon dioxide concentration produces an increase in the rate of photosynthesis. Harder therefore maintains that the rate of photosynthesis under all circumstances is dependent upon the light intensity as well as upon the concentration of carbon dioxide. He takes the stand that Blackman's conception of the principle of limiting factors as well as Liebig's law of minimum have no application to the photosynthetic process. There is apparently little doubt that conditions exist in which the in- tensity of two or possibly more factors determines the rate of photo- synthesis. It is here that the difficulty with Blackman's theory arises. Blackman and Smith" state: "When several factors are possibly con- trolling a function, a small increase or decrease of the factor that is limiting, and that factor only, will bring about an alteration of the magnitude of the functional activity." Contrary to this dictum Harder's experiments show that changes in both carbon dioxide-concentration and light intensity are able to eilect changes in the photosynthetic rate. The intensity of one factor influences the effect produced by changes in intensity of some second factor. Or, what amounts to about the same, the effect produced by changes in intensity of this second factor is not the same for all in- tensities of the first factor. Thus, for example, the increase in rate of photosynthesis produced by augmented light intensity is dependent upon the concentration of carbon dioxide; the effect prodticed by augmented light intensity is greater the higher the concentration of carbon dioxide. When determining the influence of a single factor on the rate of photo- synthesis, all other factors being kept constant, the amount by which an increase in this single factor augments the photosynthetic rate depends, according to Harder, in every case on the concentration of the factors kept constant. The higher the concentration of these factors, the higher is "Blackman and Smith, Proc. Roy. Soc. London, B 83, 397 (1911). 106 PHOTOSYNTHESIS each point on the curve of the photosynthetic rate. Moreover, a change in that factor which in comparison to the others is in minimum concen- tration (or intensity) has the greatest efifect on the rate of photosynthesis. Thus with high Hght intensity and low carbon dioxide-concentration, an increase in the concentration of the latter produces the greatest change and vice versa. Theoretically it is inconceivable that only one factor can determine the photosynthetic rate, though Harder concedes that practically it may occur that a single factor is so greatly in minimum concentration compared to the others that it appears as though the concentration of this one alone is determining the rate of photosynthesis. Harder also points out (inconsidering primarily the influence of the two factors, carbon dioxide-concentration and light intensity) that in every photosynthetic rate curve there are two portions to be differentiated: the one in which light intensity is the determining factor, the other in which carbon dioxide-con- centration plays this role. Between these two portions of the curve there is a point at which the two factors are of equal influence. This point represents the most favorable relative combination of the two factors, and Harder concludes that the product of the two intensities at this point is lower than for any other combination of the two factors producing the same photosynthetic rate. The interaction of the various factors is undoubtedly complex. Harder's chief point is that the augmenting effect on the rate of photosynthesis of a single factor is not directly proportional to the intensity of this factor at all concentrations of the other factors. The higher the intensity of light the greater is the augmenting effect of an increase in carbon dioxide- concentration, and conversely the higher the carbon dioxide-concentration, the greater is the augmenting effect of an increase in light intensity. Finally, an important point in Harder's conclusions is this: that the augmenting effect in the photosynthetic rate just described pertains to absolute rates. Relatively the augmenting effect of an increase in carbon dioxide-concen- tration is greatest with low light intensity as is also the relative effect of light greatest with low carbon dioxide-concentration. Lundegardh " has formulated a theory which is essentially the same as that of" Harder. To this Lundegardh has given the name Relativity Law of Photosynthetic Factors. According to this theory the rate of photosynthesis is determined by both the light intensity and the carbon dioxide-concentration. The more either one of these factors is relatively in minimum concentration, the greater will be the resulting augmentation in the photosynthetic rate when this factor is increased. Under natural conditions sunlight is available in excess, while the relatively minimum factors are carbon dioxide and chlorophyll content. Recently Miss M. Henrici ^* obtained curves of the rate of photo- synthesis with increasing light intensity which show two maxima. At low illumination intensities the rate rises sharply, with increasing intensity "Lundegardh, Der Kreislaiif der Kohlensaure in der Natur. Jena (1924), p. 74. "Henrici, Verhand. Naturf. Ges. Basel, 32, 107 (1921). THE NATURE OF PHOTOSYNTHESIS 107 drops again and attains a second and principal maximum. Thereafter increased light intensity has no influence on the rate, so that the curve becomes almost parallel to the abscissa on which the light intensities are plotted. With the exception of the two maxima the curves bear a strong resemblance to the original curves of Blackman in that they are in no sense logarithmic. Miss Henrici's experiments, however, were appar- ently carried out with atmospheric air, so that the limiting influence of the carbon dioxide concentration is clearly evident. These investigations are also discussed under the influence of temperature on photosynthesis. Turning now to the effect on photosynthesis of light of different wave- lengths, this subject has for over a century engaged the attention of plant physiologists and has resulted in a most voluminous literature. The re- sults, however, are by no means commensurate with the efforts expended. The problem of the effect of different wave-lengths involves experimental difficulties which at first glance are not apparent and in many cases have entirely escaped the notice of the experimenter. These difficulties are concerned with obtaining a satisfactory source of light, the measure- ment of energy in the different portions of the spectrum, methods of ob- taining light of sufficient intensity of various wave-lengths either through filters or a prism, the relative absorption by the leaf of light of different frequency, temperature effects, as well as devising reliable methods of measuring photosynthetic rates. The most common error is the disregard of the intensity or more properly, the energy of the different wave-lengths employed. Colored glasses and light filters rarely yield monochromatic light ; reliable data on their transmission can be gained only from spectro- graphs. In using prisms consideration must be given to the degree of dispersion of the different spectral regions. A perusal of the many different investigations which have been under- taken to determine the effect on photosynthesis and plant growth in general of the various portions of the spectrum lead to the conclusion that the cause for the disappointing outcome of many of these elaborate and costly experiments lies in a disregard of some of the fundamental principles of the physics of light. There is a wealth of information originating in physical and chemical laboratories which has direct application to these problems and cannot be disregarded if intelligible results are to be hoped for. The question of the source of light used in such experiments is of paramount importance. \\'hile it seems desirable often to adhere to nat- ural conditions of illumination by using sunlight, for experimental work, in spite of the high intensity, this source of light is not very satisfactory. The cause is that sunlight, at the surface of the earth, varies both in inten- sity and composition from hour to hour and from day to day. The high intensity of the infra-red rays in sunlight also introduces experimental difficulties. Artificial sources of light have many advantages furthermore, from the viewpoint of regulating intensity, constancy and composition of the light. Only it is necessary that some study be devoted to the physical problems involved, so that the experimenter is thoroughly familiar with the 108 PHOTOSYNTHESIS tools he is using. The great industrial development in illumination during recent years and the wealth of exact physical information which has ac- companied it has made available tremendously valuable facilities for experimental work in this field. There are also a number of authentic compilations of data in book form relative to these matters. ^^ In Table 20 is given the relative distribution of energy in the visible spectra of a number of common sources of light with wave-length 0.59 [i equal to 100. It is evident from this that differences in photosynthesis are to be expected when different sources of light are employed. TABLE 20 Relative Distribution of Energy in the Visible Spectra of Different Sources OF Light. (From Luckiesh, "Color and Its Application." 1921.) Tungsten Tungsten Incandescent Incandescent Lamp Lamp Carbon (Vacuum) (Gas) Black Body Incandescent 1.25 0.5 Wave- at 5000° Lamp w.p.m.h.c. w.p.m.h.c. D.C. length Abs. Noon Blue 3.1 7.9 Lumens 22 Lumens Arc M- Sunlight Sky w.p.m.h.c. per Watt per Watt (Open) 0.41 72.0 177.0 4.0 — 16.5 — 0.43 79.0 185.0 7.0 — 22.5 21.8 0.45 84.3 187.0 12.0 16.7 30.0 29.0 0.47 91.0 180.0 18.0 23.5 38.0 37.0 0.49 92.5 162.0 25.5 32.7 47.0 45.5 0.51 96.0 146.0 34.5 42.6 56.5 55.0 0.53 98.0 132.0 47.0 54.9 67.0 65.5 0.55 99.0 120.0 62.0 68.6 78.0 76.0 0.57 100.0 108.0 79.0 83.4 88.0 88.0 0.59 100.0 100.0 100.0 100.0 100.0 100.0 0.61 100.0 93.0 123.0 117.0 111.0 113.5 0.63 98.5 87.0 148.0 136.0 121.5 127.0 0.65 97.1 82.0 176.0 157.0 131.0 142.0 0.67 95.5 77.0 204.0 179.0 140.0 156.0 0.69 93.5 72.5 234.0 202.0 147.5 170.0 The first to observe a difference in photosynthetic activity in light of different color was Senebier.'" This Swiss scientist devised the double walled bell jars, capable of being filled with solutions of different color, which have found extensive use in plant-physiology. Senebier found that plants growing under identical conditions under white, red, and blue light formed most oxygen in the white, next in the red, and least in the blue "Sheppard, S. E., Photochcniistry. Longmans, Green Co., 1914. Plotnikow, J., Photochcmische Vcrsuchstcchnik. Leipzig, 1912. Idem., Allgemeine Photochemie. Berlin and Leipzig, 1920. Luckiesh, M., Ultraviolet Radiation. Van Nostrand Co., 1922. Idem., Artificial Light. Its Influence Upon Ciznli:;ation, 1920. Idem., Color and Its Application, 1921. Ellis and Wells, The Chemical Action of Ultra- violet Rays. Chemical Catalog Co., 1925. Hiibl, A., Die Lichtfllter. Halle, 1921. " Senebier, Jean, "Memoires physico-chimiques sur I'influence de la lumiere solaire pour modifier les etres des trois regncs de la nature et surtout ccux du regne vegetale," Geneve, 1782. German translation by F. H. Jacobaer, Leipzig, 1785, Vol. I, p. 153. THE NATURE OF PHOTOSYNTHESIS 109 light. Draper," using a prismatic spectrum, concluded that the plant re- duced carbon dioxide most readily in yellow light. A number of in- vestigators endeavored to establish the region in the spectrum of maximum photosynthesis. Most of these concurred that maximum activity is in the red portion of the spectrum though they differed as to the precise frequency. Most of these investigations contributed some data of a quali- tative rather than of a precise quantitative nature, also a number of inter- esting demonstration experiments and much experience in the experimental difificulties entailed. Against the conclusion that photosynthetic activity exhibits a single maximum in the red-yellow portion of the spectrum were the results pub- lished by Timiriazeff, Englemann and later by Kohl. Timiriazeft' used the solar spectrum and determined the rate of photosynthesis by means of gas analyses. In practical agreement with these were the results of Engle- mann who employed his very sensitive bacteria method in a microspectrum. These investigators maintained that besides the photosynthetic maximum in the red there was a second maximum in the blue. We shall not enter into the lengthy discussions arising from these different results. The ex- perimental methods employed contained numerous errors and the variant results can in part be ascribed to errors due to dispersion and to the unequal absorption of the different wave-lengths in the screens and prisms. In most of the older investigations not sufficient attention was devoted to the distribution of energy in the various s^^ectral regions studied. The impetus for most of these researches was the desire to determine whether the regions in the spectrum of maximum or high photosynthetic activity correspond to the absorption bands of chlorophyll. While it is true that there can be no photochemical action without absorption of light, it does not follow that absorption of light results in photochemical action. Within recent time several very thorough investigations have ap- peared which elucidate most of the problems with which the older investi- gators struggled with only partial success. The earlier experiments there- fore have largely only an historical interest and we shall confine our dis- cussion to the more recent publications. A new attempt to study this subject was made by the investigations of Kniep and Minder.'^ These investigators started with the realization that it was essential to determine accurately the energy of each region of the spectrum they were using and the relation of the different wave-lengths to each other and to the source of light. When sunlight is used as a source of light it must be borne in mind that the intensity of different portions of the "Draper, J. W., "Scientific Memoirs," New York, 1878. p. 184. Phil. Mag.. 25, 169 (1844). Doubenv, C, Phil. Trans. Roy. Soc. London, 126, 149 (1836). Lommel, E., Pog. Ann., 143, 568 (1871). Sachs, J., Bot. Zeitg., 22, 353-358, 361-367, 369- 372 (1864). Pfeffer, W., Arbeit, bot. Inst. Wiirzburq, 1, 1-76 (1871). Timiriazeff, C, Ann. Chim. Ph\s., Ser. V, 12, 355 (1877). Reinke, J., Bot. Zeitg.. 42, 1, 16, 2>i, 49 (1884). Richter, A., Rev. gen. Bot.. 14, 151-169, 211-215 (1902). Engle- mann, T. W., Bot. Zeitg., 40, 419 (1882); 42, 81, 97 (1884). Kohl, F. G., Ber. Bot. Ges., 15, 111 (1897) ; 24, 39 (1906). "Kniep and Minder. Zeit. f. Bot., 1, 619' (1909). no PHOTOSYNTHESIS spectrum may vary independently of one another. Kniep and Minder, working in Naples, consider that the intensity of sunlight about the noon hours is relatively constant, and checked each determination with direct measurement of the energy falling on the leaf. For measuring the in- tensity of radiation they used a Rubens thermopile with 20 elements which permitted the determination of less than 0.0000001° and was calibrated to a scale of gram calories per unit time. As a source of light sunlight between 11 A.M. and 2.30 P.M. was used; the light was filtered through water to absorb the heat rays. Filters of colored glass were used to produce different spectral regions : (1) a red glass fiker passing wave-lengths in \i \i, with the coefficient of transmission D from infra-red to about 620 \i \i, and (2) a blue filter. 1. 2. ^^ 644 578 546 509 D 0.846 0.00056 0.000057 0.000 Uti 546 509 480 436 405 384 361 340 332 D 0.00 0.0109 0.177 0.455 0.395 0.267 0.078 0.010 0.000 Green light was produced by the use of a filter made by mixing a solution of potassium chromate and ammoniacal copper hydroxide. This solution allowed light of wave-lengths 512 to 524 \i \i to pass through, though no quantitative data are given. From the data on the coefficients of trans- mission and from the curve of distribution of energy in the sun's spectrum it was possible to construct curves of the distribution of energy in the various regions passing through the light filters. The plant employed was Elodea canadensis and for determining the rate of photosynthesis the bubble counting method was used (see chapter on Methods of Measuring Photosynthetic activity). Unfortunately this method is subject to many errors, as was later recognized by the authors who subsequently made a careful analysis of the sources of error involved. As a consequence they had to reduce the intensity of radiation by a series of screens which to a measure diminished the value of their careful deter- minations of radiation distribution. The conclusion is drawn that red and blue light of the same intensity produce about the same rate of photo- synthesis. The light intensity in these experiments was very low and probably was the factor of relatively minimum intensity, though little regard was apparently paid to the other factors. The real value of Kniep and Minder's investigations is the demonstration of the fact that there is no sense in considering the photosynthetic activity in different colored light without at the same time determining the energy relations of the light employed. The maximum photosynthesis in the red-yellow portion of the spectrum, observed by earlier investigators could thus be ascribed to the higher intensity of these wave-lengths in the spectra used. THE NATURE OF PHOTOSYNTHESIS 111 By throwing a si>ectrum on a leaf Timiriazeff "^ endeavored to demon- strate the capacity of different wave-lengths for photosynthetic work. The method has been used by several investigators and consists essen- tially in allowing the light to act for a certain length of time, extracting the chlorophyll, and treating the leaf with an iodine starch reagent. The starch formed in photosynthesis thus becomes blue to black and the depth of color serves as a rough measure of the rate of photosynthesis. By means of the iodine reagent the starch is thus "developed" producing an effect not entirely dissimilar to the development of a photographic plate. Ursprung ^° has used the method with prism and grating spectra and a variety of light sources. Timiriazeff had obtained hardly any photo- synthetic effect with blue and violet rays. Ursprung found the lower limit of starch formation at about 759 ^i ^. There was no starch formation in the infra-red. In the violet he found starch formation up to 330 [i [i. With the use of quartz apparatus no photosynthetic action could be de- tected in the ultra-violet. Considering the region of maximum activity as that in which starch formation is first detected, the region 687-656 |.i j.i would receive this value. This applies for light from the sun. a carbon arc or filament lamp with prism and grating spectra. There appears to be little doubt that all light between wave-lengths 760-330 ^i |i is capable of producing starch if sufficient time of illumination is allowed ; in the normal solar spectrum staixh formation in the blue requires longer time than in the red. The exact spectral limits of photosynthesis have not been determined and it is, in fact, probable that considerable variation exists in different plants in this regard. In general, the method of measuring the rate of photosynthesis by the appearance of starch in the leaf cannot be considered as being very accurate. The formation of starch is itself not dependent upon the presence of light, as is indicated by the fact that starch can be formed by the leaf from glucose, sucrose and other sub- stances in the dark. Between the actual reduction of carbon-dioxide by 'light and the appearance of starch in the leaf there are a number of steps all of which may be influenced by light and other factors. A somewhat different line of approach was followed by Wurmser.®^ This author has made a careful study of the effect of light on chlorophyll solutions and the decoloration of such solutions in different wave-lengths. The rate of decomposition of an acetone solution of chlorophyll by light of different wave-lengths was utilized by Wurmser as a measure of the incident light. He used the green alga Ulva lacfuca and the method of Osterhout and Haas for determining the rate of photosynthesis. The latter method, which is described in another chapter of this book, is based upon the fact that certain marine algae, when exposed to light convert some of the dissolved bicarbonates into carbonates and thus increase the hydroxyl ion concentration of the solution, the rate of increase of the "Timiriazeff, Proc. Rox. Soc. London, B. 72, 424 (1903). '"Ursprung, Ber. bot. Ges.. 35, 44 (1917). ^Wurmser, "Recherches sur rassimilation chlorophjilienne," Paris, 1921. 112 PHOTOSYNTHESIS latter serving as a measure of the rate of photosynthesis. For light filters Wurmser used solutions of potassium dichromate, transparent up to about 560 \i \i, cuprous chloride transmitting from 560 \i \i to 460 ^ \i and ammoniacal copper sulphate, transmitting from about 540 fi [x to 450 ^ ^. Wurmser's results, which are expressed in arbitrary values are summarized as follov^s : Red Light Green Light Violet Light Photosynthesis = v 100 24 80 Enegry absorbed = Pa 100 6 *^1 -,. Ratio = v:Pa 1.0 4.00 2.35 This is indeed a surprising conclusion, while the highest value for photo- synthesis was found to be in the red and next in the blue light, the highest value in proportion to the energy absorbed was in the green. From these observations Wurmser formulates a hypothesis of the mechanism of photo- synthesis. This assumes that the photochemical reaction takes place on the surface of the "pigment." A more exact statement would probably be that the photochemical reaction takes place on the surface of the chloroplasts, the chlorophyll acting as a photochemical sensitizer. ^ The photochemical reaction is but the first step in a series of reactions in the subsequent steps of which the activity of the protoplasm plays an impor- tant role. The rate of photosynthesis thus depends not only upon the energy absorbed but upon the activity of the protoplasm as well. Views quite analogous to this have recently been expressed by a number of in- ve.^tigators of the problem and will be discussed in greater detail in an- other chapter. Wurmser's results do not permit, however, of an accurate calculation of the photosynthetic efficiency in light of different wave- lengths especially because his method of measuring light intensity has no direct physical basis. Probably the most successful investigations of the efifect of different wave-lengths on photosynthetic activity have been carried out by Warburg and Negelein.^2 These are described in greater detail in the chapter on Energy Relations of Photosynthesis. Warburg and Negelein conclude that the efficiency of the photosynthetic process becomes less with decreas- ing wave-length. There is apparently no relation between photosynthetic efficiency and the absorption bands of chlorophyll. Thus the yield in the red, a region of high absorption is greater than in the green, a region of low absorption ; while the yield in the green is greater than in the blue, the region of highest absorption. For the reduction of one molecule of carbon dioxide with red (660 ^i i-i) or yellow (578 ^a h) light there are required about four quanta, with blue light (436 n \i) about five quanta. There are undoubtedly several factors which affect these results. Prob- ably the most important of these is the concentration of the chlorophyll and carotin in the plants. That these vary with the conditions of previous culture of the plants is a matter of common experience. The influence "'Warburg and Negelein, Zcit. physik. Cheni., 106, 191 (1923). )epth in Orange Meters Red Yellow 5 Z.7 2.5 50 0.0021 100 0.001 1000 1500 THE NATURE OF PHOTOSYNTHESIS 113 of light as a cultural condition has already been alluded to ; its effect on plants under natural conditions has been a subject of long investigation from which some important conclusions have already been drawn. Englemann ^^ first offered the hypothesis that the color of certain marine plants was complementary to the color of the light to which they were exposed. It is a familiar fact that water has a higher absorption for the longer wave-lengths of light than for the shorter wave-lengths. As a consequence the deeper the water, the greater is the relative proportion of blue light. Thus, if 1000 represents the light intensity at a depth of one meter the distribution of the different wave-lengths is about as follows : ^* Blue Green Blue Violet 230 450 866 0.0003 0.001 0.003 — — 0.0001 Shelford and Gail ®' found that the brown algal zone is at a depth of 5 to 20 meters where the shorter wave-lengths have an intensity of about 10 per cent of full sunlight and the longer, red wave-lengths about 1 per cent. The red algal zone lies between 10 and 30 meters in depth where the blue light is 2 to 10 per cent of full sunlight and the red light is 0.032 to 1 per cent. Gaidukow *" reported that he was able to alter the color of certain marine algae by growing them under colored light and thus obtained plants with a color complementary to the color of the light in which they were grown. These and other similar observations gave rise to the theory of complementary chromatic adaptation. The theory and some of the rami- fications and deductions which have been drawn therefrom have been at- tacked from different sides, a discussion of which would lead too far afield.^^ Some of the principles involved are, however, of direct bearing on the photosynthesis problem. In its broader significance, of course, the question resolves itself into the old one of why vegetation is green. This question has been subjected to an interesting theoretical analysis by Stahl. The theory is really an extension to land plants of Engle- mann's theory of the distribution of marine plants. Stahl ^^ points out "=• Englemann, Bot. Zeitg., 42, 81 (1884). '* Monaco, The Prince of, Sci. Monthly, 13, 177 (1921). ^ Shelford, V. E., and Gail, F. VV., Pub. Puget Sound Biological Station, 1920, 141. Gail, F. W., ibid., 177-193. Geitler, I., Int. Rev. d. gcs. Hydrobiol Hydro., 10, 681 (1922). Pascher, Bot. Arch.. 3, 311 (1923). ^Gaidukow, Bcr. bot. Ges., 21, 484, 517 (1903) ; 24, 1, 23 (1906). "Baresch, K., Jahrb. zmss. Bot., 52, 145 (1913) ; Bcr. hot. Gcs.. 37, 25 (1919) ; ibid., 39, 93 (1921); Zeit. f. Bot., 13, 65 (1921). Magnus, W., and Schindler, B., Ber. hot. Gc.^., 30, 314 (1912). Pringsheim, E., Cohn's Beitr. z. Biol. d. Pnanzen. 12, 49 (1914). Tobler, F., Die Naturimssenschaftcn. 1, 845 (1913). Savageau, C., Compt. rend. Soc. bioL, 64, 95 (1908). ^^ Stahl, "Laubfarbe und Ilimmelslicht," Jena, 1906. 114 PHOTOSYNTHESIS that the chlorophyll apparatus is adapted to the conditions of illumina- tion to which the plant is exposed. Thus a plant receives not only the direct rays of the sun, but also dififuse light, which in composition has been considerably altered by reflection and dispersion. While the direct rays of sunlight are altered by absorption of the blue-violet rays in passing through the atmosphere, in dififuse light the blue-violet rays are relatively in excess. In the direct rays of the siin there is therefore a relative pre- ponderance of red-yellow light while in diffuse light there is a preponder- ance of blue light. This is based upon the well-known theory of Lord Rayleigh. Stahl sees in the absorption of light by chlorophyll a direct adaptation to this condition. The pigments of the leaf are of two dififerent colored components capable of absorbing the different spectral components of the light incident on them. Stahl considers that in the chlorophyll, the blue-green pigment serves to absorb the red-yellow rays of direct sun- light and the yellow pigment in leaves absorbs the diffuse reflected blue rays. That the plant does not absorb the green and infra-red rays he explains on the ground that in diffuse light these rays are of such low in- tensity that the plant can make no use of them, while in direct sunlight these rays are of such high intensity that their absorption would result in an increase of temperature harmful to the leaf. Stahl considers that the plant absorbs and utilizes those rays which are the most constant com- ponents of the light to which it is exposed, at the same time avoiding the absorption of rays which have little value for photochemical reactions. These views are still largely speculative. It appears to be true that light over a wide range of frequency is capable of inducing photosynthesis. Interesting in this relation, it may parenthetically be stated, is the fact, observed by Stern,*'' that the chlorophyll in the cell fluoresces. Chloro- phyllous cells illuminated with yellow, green or blue light fluoresce red light, 630-650 \i \\. and 660-690 |.i ^i, that is, light which is in the red absorp- tion band of chlorophyll. In view of the fact that marine plants growing at some distance below the surface are subjected to illumination of much more restricted spectral regions than land plants, the theory of complementary adaptation has been studied primarily in relation to marine life. It is also in these plants that differences in color, due to pigments besides chlorophyll, are most striking. According to the Engelmann theory the production of these pigments is the reaction to a particular factor in the environment, the frequency of the light in which the plants are growing ; and these pigments play an im- portant role in the photosynthetic process of the plants. On the basis of experimental work Richter ^° comes to a very different conclusion. Work- ing with a variety of marine algae of different color and with light of vari- ous intensities and frequencies he concludes that it is not the wave-length of the light which determines the color of the algae but the intensity of the light. While Gaidukow. already cited, stressed particularly the de- ■^ Stem, Ber. bot. Ges., 38, 28 (1920). "Richter, Ber. bot. Ges., 30, 280 (1912). THE NATURE OF PHOTOSYNTHESIS 115 velopment of color in marine plants i^rown under light of different color, Richter emphasizes that only the photosynthetic activity of the variously colored plants under the different conditions of illumination is of real biological significance. Richter therefore takes the stand that the distribu- tion of the different colored algae is a matter of light intensity, quite analogous to "light" and "shade" land plants. The vertical zonal dis- tribution is a matter of light intensity or "Lichtgenuss" in the sense of Wiesner, and the other pigments in the plants play no essential role in photosynthesis. Thus Richter concludes that only the chlorophyll proper plays a role in photosynthesis. Regarding the phenomenon of zonal distribution of aquatic plants primarily from the viewpoint of photosynthesis, an aspect of much interest is whether the blue and red complementary pigments, phycocyanin and phycoerythrin play a role in the photosynthetic process. It is evident from the work of Richter, just cited, that consideration must be given the in- tensity of the light as well as the wave-length in interpreting the com- plementary color adaptation phenomenon. Also from what has already been said it is evident that attention must be paid to the intensity and wave length of the light not only during the course of the determinations of photosynthetic rates but as well for a period prior to making such de- terminations. Considerable information on these cjuestions has been gained through an investigation by Harder.^^ He used a blue-green alga, Phonnidium foveolarum which in red light becomes green and in blue light takes on a purple color. Boresch ^- has shown that the purple form contains phycoerythrin and that the green form contains the blue pigment phycocyanin. By means of light filters, Harder obtained blue and red Hght the intensity of which he measured with a Rubens thermopile. The rate of photosynthesis was determined from the oxygen content of the water by Winkler's method. Harder upholds the conclusions of Englemann of complementary adaptation; the purple varieties of the alga, containing the red pigment, phycoerythrin, have a higher rate of photosynthesis in blue light than in red, while the blue-green varieties containing the blue pigment, phycocyanin, show higher rate of photosynthesis in red light than in blue. These re- sults apply also when equal intensities of the different colored lights are used ; the rate of photosynthesis is relatively and absolutely higher in those wave-lengths which are complementary to the color of the plant. That the intensity of light in which plants have been growing, and to which they have in a sense become adjusted, is a very important factor in the rate of photosynthesis has been recognized for some time. This is, of course, the basis of the behavior of "hght" and "shade" plants and has already been touched upon. It is, in fact, possible to produce arti- ficially with the same species, as far as their light requirements for photo- " Harder, Zcit. f. Bof., 15, 305 (1923). *' Boresch, Arch. f. I'roslistcnkundc, 44, 1 (1921). 116 PHOTOSYNTHESIS synthesis are concerned, both "Hght" and "shade" varieties. ^^ By cultivat- ing the same alga under different intensities of light, Harder found that the plants grown under light of low intensity can utilize lower intensities of light for their photosynthetic work than plants grown under high light intensity, while plants grown in light of high intensity attain a higher rate of photosynthesis in light of high intensity than those grown in light of low intensity. The color of the plant, i.e., the presence of phycocyanin or phycoerythrin seems to be of little significance herein. This adaptation or accommodation to light of different intensity is not recognizable by any outward appearance of the plant, but is a more important factor than color. For this reason it is extremely important that it be taken into consideration in all determinations of the rate of photosynthesis. Just what the nature is of these accommodations to light of different intensities it is difficult to tell. Presumably it is associated with the chlorophyll-content and pos- sibly also with internal plasmatic factors. There exists, it is evident, a contradiction between the conclusions of Richter and those of Harder. The former regards that the color of the plant or the presence of pigments besides chlorophyll is of no consequence to photosynthesis. Harder considers that this is an extreme view. There is no doubt that the light to which the plants are accommodated is an im- portant factor. This is evident from the following extreme conditions : plants which have been raised in weak red light (blue-green "shade" plants) show relatively higher photosynthesis in weak blue light than in strong red light, and plants raised in strong blue light (red "light" plants) show relatively higher photosynthesis in strong red light than in blue light of low intensity. That is, the typical behavior of "light" and "shade" plants is maintained even when the light is not complementary to the color of the plants. This would be quite in agreement with Richter's claims. How- ever, Harder points out, that when the light intensity is duly taken into consideration, the factor of wave-length is always discernible, in the sense that the plants show relatively higher photosynthesis in those wave-lengths which are complementary to the color of the plant. This action is often covered by results of differences of intensity. Harder comes to this conclusion from the following facts. Photosynthesis of different colored plants in light of different wave-lengths, hut of the same intensity, is highest in those wave-lengths which are complementary to the color of the plant. Also, when plants are accommodated to light of different wave- lengths but of the same intensity, photosynthesis is highest in those wave- lengths which are complementary to the color of the plant, no matter what intensities of other wave-lengths are used. Harder's conclusion that "light" plants show greater photosynthesis in light of high intensity than "shade" plants and the latter greater photo- synthesis in light of low intensity than "light" plants, is in apparent con- tradiction to the conclusions of Warburg and Negelein, already referred "Lubimenko, W., Ann. sci. not. (9), 7, 321 (1908). Rose, ibid. (9), 17, 1 (1913). THE NATURE OF PHOTOSYNTHESIS 117 to. It will be recalled that these investigators found that the unicellular alga used by them, when cultivated in light of high intensity, converted but a small amount of the light absorbed in the process of photosynthesis, while the plants cultivated in light of low intensity converted a large portion of the absorbed energy in the photosynthetic process. It is essential to bear in mind that \\'arburg and Negelein worked with light of low inten- sity only, they did not expose their "light" plants to illumination of high intensity. Thus their results as far as they go are really in agreement with those of Harder. Finally, it should be pointed out that the results of Harder and those of Warburg and Negelein are not strictly comparable. The latter investigators were concerned with photosynthetic efficiency, namely, the proportion of radiant energy, absorbed which is converted into chemical energy. This is a strictly quantitative and physical method of measurement. Harder was satisfied with the relative amounts of oxygen liberated by the two types of plants under different conditions of illumina- tion ; there is no measure of the ratio of energy absorbed to that converted. Thus there is no information on the efficiency of the two types of plants under the different conditions. The investigations of Harder and those of Warburg and Negelein were carried out from different viewpoints and had not the same goal in view. Aquatic plants lend themselves better to experimentation of the nature just discussed than land plants. Particularly on account of errors due to temperature changes within the leaf and variations in the stomatal open- ings which give rise to alterations in the gaseous exchange and water loss ; thus, experimentations with land plants are associated with many diffi- culties. While the principles underlying the reactions involved in photo- synthesis are probably the same in aquatic and land plants, it is in the latter that greatest interest centers. Lubimenko ^* has worked with a number of land plants in relation to their behavior in light of different wave-lengths. His results are difficult to interpret on the basis of other findings. Thus, he concludes that while the reduction of carbon dioxide is greater in red light than in blue or violet the increase of dry substance is greater in blue light than in red. Concerning the role of ultraviolet rays in the photosynthesis process, it may be stated that it is highly improbable that these are of significance under natural conditions. The intensity of ultraviolet light at sea-level is exceedingly low. The solar spectrum ends very abruptly near wave- length 300 ji |i ; even at great heights above sea-level there is no appre- ciable energy of wave-lengths shorter than 290 j-ii-i.**^ Moreover photo- synthesis is carried on normally by plants under glass which is opaque to ultraviolet light. Whether it is possible for photosynthesis to take place in ultraviolet light is as yet uncertain. Bonnier and Mangin's "'' ex- ** Lubimenko, Zentr. Biochcm. Bioph\s., 10, 803 (1910); Rev. gen. hot., 23, 1 (1911); Compt. rend., 145, 1191 (1907): 177, 606 (1923). "'Luckiesh, "Ultraviolet Radiation," 1922, p. 24. • "* Bonnier and Mangin, Compt. rend., 102, 123 (1886). 118 PHOTOSYNTHESIS perinients would indicate that this was the case, yet there is considerable doubt as to whether their filters excluded all other light of lower fre- quency. The problem is made difficult on account of the harmful action of ultraviolet light on plants and the complications arising from the effect of these rays on enzymes, starch and other components of the plant. ^" Similarly, there is no evidence that the infra-red rays play a part in the reduction of carbon dioxide, although it appears that chlorophyll absorbs some infra-red rays. On the other hand active photosynthesis is caused in purple bacteria by the infra-red rays (800-900 \i \i).^^ The composition of the light is changed as it penetrates successive layers of chlorophyll so that the chloroplasts in the center of a leaf are exposed to light of different composition from those near the surface. This is one factor which may account for the varying behavior of different species of leaves. Thus the red rays (700-660 \i \i) cause very active photosyn- thesis, but are also largely absorbed by the outer layers of the leaf and can thus act upon fewer chloroplasts than the yellow rays which pene- trate more deeply. It is conceivable therefore that the maximum carbon dioxide reduction in relation to the amount of light of different wave- lengths absorbed will vary in relation to the thickness of the leaf, and in thick leaves maximum photosynthesis will be shifted toward the blue. The reflection as well as the absorption of light by a leaf are both of considerable importance in determinations of photosynthesis. Undoubt- edly some leaves in nature, due to their glossy surface, reflect a large proportion of the light incident on them. The absorption spectra of alcoholic solutions of chlorophyll show a slight displacement toward the blue compared with spectra of chloro- phyllous leaves. Although there has been considerable controversy as to whether a leaf is capable of carrying on photosynthesis in light fil- tered through a chlorophyll solution or through another leaf, there appears to be little doubt that this is possible if the intensity of the light is sufficiently high as was shown by Ursprung ^^ with direct sunlight. There is some evidence that there is a difference of effect on photo- synthesis between continuous and intermittent light. Warburg ^°'' has tested this by means of rotating sectors which in one revolution cut off one half the light so that the period of illumination was equal to that of darkness. Comparison between the effects of continuous and inter- mittent illumination were made on the basis of equal periods of illumina- "'Kluyver, A. J., Oestrcich. Boi. Zcit., 63, 49 (1913) ; Sitsher. Wicn. Akad., 120, I, 1137 (1911). Bovie, W. T., Bot. Gaz.. 59, 149 (1915) ; 61, 1 (1916). Maquenne and Demoussy, Compt. rend., 149, 756 (1909). Stoklasa, Centralbl. f. Bakt., 31, II, 477 (1912). Raybaud, L., Rev. gen. hot., 25, 38 (1913). Ursprung and Blum, Ber. bot. Ges., 35, 385 (1917). "» Englemann, Bot. Zeitg., 40, 419 (1882) ; 41, 1 (1883) ; 42, 81 (1884). Pfeffer, W., Arb. bot. Itist. IViirsburq., 1, 41 (1871). Ursprung, Ber. bot. Ges., 35, 55 (1917). Warburg and Negelein, Zcit. phvsik. Chcm., 106, 191 (1923). ""Ursprung, Ber. bot. Ges., 35, 64 (1917). *'" Warburg, Biochem. Zeit., 100, 260 (1919). THE NATURE OE PHOTOSYNTHESIS 119 tion and not simply on the basis of equal time. With high illumination intensity equal quantities of radiant energy reduced more carbon dioxide when the light was intermittent than when it was continuous. The ex- cess of photosynthesis with intermittent illumination was about 100 per cent when the alternations of the rotating sector were 8000 per minute, and only al)out 10 per cent when the alternations were 4 per minute. With low light intensity there is no difference between intermittent and continuous illumination. It is important that in a period of time which is long in comparison with the length of time occupied by a single flash of light in the intermittent illumination, the amount of carbon dioxide reduced is the same whether the light is continuous or intermittent. There are two possible explanations for the effect of intermittent illumination. Either photosynthesis continues at an undiminished rate during the periods of darkness, which would be in accord with an older conception of Tswett,^"^ or photosynthesis is interrupted during the periods of darkness and is doubled during the periods of illumination. It would seem that the latter is the more probable explanation. Thus, during the dark periods carbon dioxide would have an opportunity to enter the centers of photosynthetic activity and synthesized material move away, both of which would tend to increase the rate of photosynthesis. When the chloroplast is then again illuminated there are available higher concentrations of dissociable material. With light of low intensity, in which there is little difference between equal periods of illumination of the continuous and intermittent type, the reduction of the concentration of carbon dioxide in a given length of time of illumination is less ; the rate of replacement is therefore also less and the influence of the periods of darkness is less pronounced. This is virtually the same explanation that Willstatter and Stoll "^ give for the results of Brown and Escombe ^°^ with intermittent light. c. Partial Pressure of Carbon Dioxide. The early observations on the influence of various concentrations of carbon dioxide were naturally of a qualitative nature.^"* Thus it has been known for a long time that leaves which ordinarily produce only soluble carbohydrates but no starch can be induced to form the latter by exposing them to an atmosphere enriched in carbon dioxide. More- over Kreusler ^°^ and others clearly demonstrated that an increase in the partial pressure of the carbon dioxide resulted in increased photosynthetic activity. The object of these investigators was to establish the optimal 101 ■ 102 'Tswett, Zcit. phxsik. Chcm., 76, 413 (1911). 'Willstatter and Stoll, "Untersuchungen ii. die Ass. der Kohlens," Berlin, 1918, p. 240. '"'Brown and Escombe, Proc. Roy. Soc. London, 76 B, 86 (1905). •" Godlewski, Flora, 56, 378 (1873) ; 60, 215 (1877). Schimper, Bot. Zeitg., 43, 737 riS8S) "'Kreusler, Landiv. Jahrb.. 14, 951 (1885). 120 PHOTOSYNTHESIS percentage of carbon dioxide which the plant was capable of utiHzing. The results of different workers varied greatly ; in some cases 4 per cent carbon dioxide produced injurious effects, in others 10 per cent produced optimal activity. As is now evident the cause for these con- tradictions lay primarily in the fact that the other factors which influence photosynthetic activity were not taken into consideration, that is, the various investigators were using different light intensities, temperatures, plants with unknown chlorophyll-content, etc., and while focussing their attention on the influence of carbon dioxide-concentration, became en- tangled in complications resulting from the interaction of these other factors. Treboux,^"^ in his study of the effects of a variety of substances on photosynthesis, established a direct proportionality between this activ- ity and carbon dioxide-concentration and concluded that with low light intensity the carbon dioxide optimum shifts with the light intensity. It was Blackman who first broke away from the conception of a single optimum factor and developed the theory of the interaction of various factors. Blackman and Smith ^°^ studied the effect of varying concentrations of carbon dioxide on the photosynthetic activity of the aquatic plants, Fontinalis antipyretica and Elodca. They obtained curves which are of the nature of those shown in the diagram in Fig. 7. Of these they state : 'Tn the weaker solutions of CO, the curve shows steadily increasing assimilation proportional to the increase of COg-supply. Here the light and temperature are in excess, but, at a certain point, sharply defined, increase of CO, is no longer followed by further increase of assimilation but the value of the assimilation remains at a fixed level. . . . This part of the curve is due undoubtedly to the limiting action of either illumination or the temperature. . . . "Had a more intense light and higher temperature been fixed upon, then the ascending part of the curve would have been prolonged further and a fixed (but higher) level only attained with a greater concentra- tion of COg. With less light the limiting value would have been arrived at sooner." The outstanding feature of Blackman and Smith's conclusions on the relation of the rate of photosynthesis to the environmental factors, COo- supply, temperature and light intensity is that photosynthesis "in every combination of these factors is determined by one or the other of them acting as a limiting factor. "The identification of the particular limiting factor in any definite case is carried out by applying experimentally the following general principle. When the magnitude of a function is lifiiited by one of a set of possible factors, increase of that factor, and that alone, ivill be found to bring about an increase of the magnitude of the function." Blackman and Smith also give an interpretation of the results of ""•Treboux, Flora., 92, 63 (1903). ""Blackman and Smith, Proc. Roy. Soc, 83 B, 389 (1911). THE NATURE OF PHOTOSYNTHESIS 121 Treboux (cited above) and of Pantanelli ^"^ on the basis of limiting factors; these seem to lend considerable substantiation to the theory. It is necessary to bear in mind that Blackman and Smith's ^"'^ experiments were the first in which a conscious effort was made to test out this conception of the influence of environmental factors. The method used for determining photosynthetic activity was new and the results from which the final curves were plotted were taken from experiments ranging over three years. 280 240 _-— 200 / 1(iU / 120 j 80 - 40 ( I 1 2 : 10 ' to 50 ( SO ro iO \ w Fig. 9.— The rate of photosynthesis at different concentrations of carbon dioxide. The ordinate represents the rate of photosynthesis, the abscissa the concentra- tion of carbon dioxide. (Constructed from values given by Warburg.) Within recent years conceptions differing from those of Blackman have been advanced by a number of investigators. These modifications of Blackman's views are largely the result of careful experimentation with a variety of different plants and improved means of measuring photo- synthetic rates. In 1919 Warburg"" devised a method of determining the rate of *»» PantaneUi, Jahrb. f. n'iss. Bot., 39, 167 (1903). ^Blackman and Smith, Froc. Ro\. Sac, 83 B, 374 (1911). ""Warburg, Biochem. Zeit.. 100, 230 (1919). 122 PHOTOSYNTHESIS photosynthetk activity of the unicellular green alga ChlorcUa. This method makes use of the principle of the Haldane-Barcroft method of hlood gas analysis and i)ermits rapid and accurate determinations to he made. Ky using solutions of sodium carhonate and sodium bicarbonate the concentration of carbon dioxide in the water could be controlled with great accuracy. The source of light was a metal filament lamp of 1500 watts current consumption which at 15 cm. distance exceeded the light intensity of direct sunlight. The concentrations of carbon dioxide in solution ranged from that which is in equilibrium with air containing 34 o to 10 times the normal amount. The temperature was 25°. It is thus quite certain that carbon dioxide was at first the limiting factor. In Figure 9 is given the curve which Warburg obtained under these con- ditions. Warburg's curve indicates that at low concentrations of carbon dioxide the rate of photosynthesis is closely proportional to the carbon dioxide- concentration. Above a concentration of about 2 X 10"*' moles per liter progressive increase in the carbon dioxide-concentration results in a con- tinuously smaller increase in the rate of photosynthesis until finally the latter seems to be independent of carbon dioxide-concentration. War- burg interprets the form of the curve on the basis that the rate of photo- synthesis is proportional to the concentration of carbon dioxide and to the concentration of a second substance which reacts with carbon dioxide. Thus if A represents the total amount of this absorbing substance in a cell, x and A — x res]>ectively the amounts which are in the free and combined condition and Ceo., the concentration of carbon dioxide, then in any steady state ^^- — would be constant. A — X Warburg's conception of the photosynthetic process thus involves that of an absorbing substance for carbon dioxide, a conception the necessity of which has been demonstrated in a number of dififerent ways and of which there is further discussion in another part of this chapter. An im- portant assumption in the idea that carbon dioxide first goes into com- bination is that the rate of photosynthesis, even at the lowest carbon dioxide-concentrations is governed by the rate of a chemical reaction and not only by dififusion. It would seem that Warburg's experimental con- ditions have certain advantages over previous ones for determining photo- .synthetic rates, because the use of the unicellular organism reduces the element of diffusion to a minimum. In higher plants possessing an in- ternal atmosphere it is evident that these conditions are more complex. This was already found by Blackman, who showed that the Bryophyte, Fontinalis, has a photosynthetic rate about half that of the Phanerogam, Elodea. The fact that carbon dioxide diffuses more rapidly as a gas than in solution may account for this difference. In the higher plants, which are of a more complex structure, under conditions of low carbon dioxide supply, there would rarely be equilibrium between the carbon dioxide- THE NATURE OF PHOTOSYNTHESIS 123 concentration surrounding the plant and that of the centers of photo- synthetic activity. In the case of the higher plants there would thus be introduced the factor of diflfusion in determining the rate of photo- synthesis for any one condition of carbon dioxide-concentration and con- sequently be more complex than under the conditions studied by Warburg. There is no rational basis upon which the photosynthetic activity of two plants of widely different structure can be compared. Such meas- ures as area, fresh weight, chlorophyll-content and number of chloro- plasts, which have occasionally been used, are either not universally applicable or can be determined only for certain types of plants. A quanti- tative comparison of two such widely different plants as a unicellular alga and the leaf of a sunflower is impossible as yet. Harder ^^^ has made a rather extensive study of the principle of limiting factors. He was particularly concerned with the form of the curves of photosynthetic activity under varying external conditions and subjects Blackman's theory to rather severe criticism. In order to test this theory Harder sets the following conditions : 1. The course of the rate of photosynthesis must be followed when all factors but one are maintained constant. This single factor must be gradually increased. Under these conditions it will be disclosed whether the photosynthetic rate curve rises in a straight line and then abruptly becomes horizontal or whether the transition to the horizontal is gradual. 2. The course of the rate of photosynthesis must be followed when more than one factor is increased. Under these conditions it can be determined whether the rate of photosynthesis is actually affected only by the one factor which is the limiting one, or whether the other factors which are changed, also influence the rate. As experimental material Fontinalis antipyretica, CincUdotus aqiicatlles, and two species of Cladophora were employed ; the rates of photosynthesis were determined by analysis of the water for oxygen, and metal filament lamps used as the source of light. Harder found that with all of the plants used, when carbon dioxide- concentration or light intensity alone are altered, the photosynthetic rate curve does not exhibit an abrupt change, but changes gradually from the almost vertical to a line approaching the horizontal. The form of the curves varies with different plants ; an example is given in Figs. 10 and 11. So Harder concludes that when only one factor is changed the curve representing the rate of photosynthesis does not exhibit an abrupt turn ; it is rather a logarithmic curve. In other words the point at which one factor ceases to be the limiting one and some other factor becomes such is not a sharply defined one, but the transiticMi is a gradual one. Harder furthermore, attempted to apply the mathematical formula which Baule ^^^ "'Harder, R., Jahrh. u-tss. Bat., 60, 531 (1921). "'Baule, Landw. Jahrb., 51, 361 (1918). 124 PHOTOSYNTHESIS had developed for the effect of nutrients on crop yield, to the effect of carbon dioxide on the rate of photosynthesis. He found, however, that the calculated values are not in accord with the experimental results. In the second set of Harder "s experiments he endeavored to determine the effect on the rate of photosynthesis of altering two factors. He argues that according to Blackman's view, only that factor which is limiting should affect the photosynthetic rate. The experimental results do not 2,000 6.000 10,000 14,000 18,000 M.C. Fig. 10. — Effect of change in light intensity on the rate of photosynthesis of Fontinalis with different concentrations of carbon dioxide. (From Harder.) support this conclusion. A series of experiments with six different light intensities and four concentrations of potassium bicarbonate showed that an increase in either light intensity or carbon dioxide-concentration re- sulted in an augmented photosynthetic rate. The latter is therefore de- pendent upon both these factors under all circumstances. Harder's results are reproduced in Figures 10 and 11, From these curves it appears that with either factor held constant an increase in the other factor results in a rise in the rate of photosyn- thesis. The effect of the factor which is varied is not the same through- out the course of the curve but depends upon the intensity of the other THE NATURE OF PHOTOSYNTHESIS 125 factor. Harder therefore concludes that a variation in the intensity of that factor which is relatively to the greatest degree in minimum con- centration or intensity produces the greatest change in the photosynthetic rate. This is a rather awkward statement. It is Harder's conception that the relation of the photosynthetic rate to the two factors, carbon dioxide-<:oncentration and light intensity, is a complicated one and that these two factors are mutually interdependent. The augmenting effect 15 ^ 18.000 H.C. 10 ^ 6.000 M.C. / 5 / / ^^ , - 2.000 H.C. 3 2 / 667 M.C. F^^ I .01 .0 4 0.1 .1 6 2 0.3 0. 32 y- KHC( )3 Fig. 11. — Effect of change in concentration of carbon dioxide on the rate of photo- synthesis with different intensities of light. (From Harder.) of an increase of one of these factors is not the same at all concentra- tions or conditions of intensity of the other factor. In fact, the augment- ing effect of the first factor will be higher the greater the intensity of the second factor. In other words, the higher the light intensity, the greater will be the augmenting effect of an increase in carbon dioxide- concentration and the higher the existing carbon dioxide-concentration, the greater will be the rise in the photosynthetic rate caused by an in- crease of the light intensity. From what has already been said it is evident that in any endeavor to determine the effect of carbon dioxide-concentration on photosynthesis, in any quantitative sense whatsoever, it is essential that the other factors 126 PHOTOSYNTHESIS be accurately controlled. For this reason many of the older investigations, in which the influence of the other factors was not recognized or in which these are described in uncertain terms, can contribute but little. The determination of the influence of varying concentrations of car- bon dioxide on the photosynthetic activity of land plants is associated with many more difiiculties than is the case with aquatic plants. These difficulties are largely due to the more complex structure of land plants ; the opening and closing of the stomata are occasioned by water supply of the leaf, temperature, light and carbon dioxide-concentration and are of profound ini'uence on the gaseous exchange of the leaf, and hence, of course, also on the rate of photosynthesis. It is therefore not surpris- ing that some of the earlier work is difficult to interpret. Brown and Escombe ^^^ found a direct proportionality between car- bon dioxide-concentration and rate of photosynthesis. However, the num- ber of experiments was small and there was some indication of patho- logical effects. They also reported an increase in the chlorophyll-con- tent in plants intermittently exposed to higher concentrations of carbon dioxide. This has also been found to be the case by Lundegardh. An increase in chlorophyll-content would of itself tend to increase the photo- synthetic activity. Lundegardh, ^^'* working with leaves of land plants, has arrived at very similar conclusions to those of Warburg and Harder. Lundegardh's curves are very much like those of Harder. In Table 21 are given the results of experiments on the photosynthetic rate with varying carbon dioxide-concentrations. TABLE 21 Photosynthesis with Varying C02-Concentrations and Light Intensities ViQ, -5'L'O, AND 1/4 that OF DiRECT SuNLIGHT, IN TeRMS OF CO2 FiXED. (From Lundegardh.) Photos jTithe sis per SO Sq. cm. per Hour- CO. 2 J_ 1 Concentration 40 20 4 Plant Per Cent mg. mg. mg. Oxalis acctosclla 0.03 0.45 LI 2.3 0.06 0.9 2.3 2,.7 0.09 L3 2.5 4.9 0.12 1.6 2.7 6.3 0.24 1.6 — — Stellaria memorum 0.03 0.7 LO L5 0.06 0.9 L7 2.3 0.09 1.0 2.4 3.0 0.12 L3 4.0 3.9 0.24 1.9 — 5.3 ""Brown and Escombe, Proc. Rov. Soc. London. V, 70, 397 (1902). Kreusler, Lamhc. Jahrb., 16, 711 (1887); 17. 161 (1888); 19, 649 (1890). Giltay, E., Ann. Jard. Buitcnzorg., 15, 43 (1898). "♦Lundegardh, H., Svensk. bot. Tidsskrift.. 15, 59 (1921): Biol. Ccufralbl., 42, 337 (1922). "Der Kreislauf dcr Kohlensaeure in dcr Natur." Jena, 1924, p. 74. THE NATURE OF PHOTOSYNTHESIS 127 Lundegardh's results would indicate that with light intensity %o that of direct sunlight an increase in carbon dioxide-concentration above the normal (0.03 per cent) results in an increased photosynthetic rate. Also, with increasing carbon dioxide-concentration the augmentation of the photosynthetic rate is high at first and gradually becomes less. With higher light intensities, i.e., illumination approximating conditions exist- ing in nature, the photosynthetic rates are raised considerably by an in- crease in carbon dioxide-concentration. In absolute measure these latter increases are high, though relatively they are not as high as with low illumination. This point is taken up under the discussion of the effect of light intensity. From what has been said of the effect on photosynthetic activity of increasing the carbon dioxide concentration, it may be concluded that the normal carbon dioxide-concentration of 0.03 per cent in the atmosphere represents a condition in which this factor is ordinarily to be expressed as the "limiting one" in terms of the Blackman conception or as being "in minimum" in the terms of Harder and Lundegardh. From the prac- tical \newpoint of agriculture the question of photosynthetic rate is naturally a very important one and the problem of how to increase this rate has occupied plant physiologists for a long time, for on the rate of the photosynthetic process depends more or less directly the amount of the plant product, the crop. The question then arises, can an increase in crop yield be attained by raising the carbon dioxide-concentration in the atmosphere surrounding the plants? The question has received much attention especially during the past few years. A full discussion of all the aspects and developments of the problem would reach beyond the purpose of this monograph. The prob- lem has been widely discussed in Gennany where flue gases from the blast furnaces have been used as a source of carbon dioxide."^ Many technical problems have arisen which still require much study. It is becoming evident that increasing growth and dry material is not simply a matter of increasing the photosynthetic rate. There are questions of water relations, and migration of the material formed which play a very important role. Different species exhibit very different behavior toward air enriched in carbon dioxide. Finally, growth, the formation of new tissue, and the laying down of storage material, are in part only depend- ent upon the photosynthetic process. There are many other factors which also come into play. As a result of the neglect of due consideration of this interrelation of various factors in the development of plants, many contradictory observations have been made and erroneous conclusions drawn. This is probably inevitable when it is attempted to apply intricate "'Reinau, "Kohlensaure and Pflanzen," Halle, 1920. Bornemann, "Kohlensaure und Pflanzenwachstum." Berlin, 1923. Classen, H., Chem. Zeitq.. 44, 585 (1920). Cerighelli, R., Ann. Sci. Awon., 38, 68 (1921). Fischer, H., "Pflanzenbau und Kohlensaeure," Stuttgart, 1921. Wagner, H.. Die Kohlensaeure als Wachstums- faktor. Die Umschau., 27, 758 (1923). Lundegardh, "Der Kreislauf der Kohlen- saeure in der Natur.," Jena., 1924. Jess, Jour. agr. pract., N. S. 35, 250 (1921). 128 PHOTOSYNTHESIS scientific principles to a large scale industrial undertaking. In some cases the experiments were carried out in glasshouses during the winter with no measurements of the intensity and duration of the light. Under these conditions it is not surprising that an atmosphere enriched in carbon dioxide produced only a very slight increase or even deleterious efifects. Thus, for instance, Tjebbes and Uphof ''^^ who carried out ex- periments in greenhouses in December found that air enriched with car- bon dioxide produced an effect only by the aid of electric light. Similarly, when total growth or crop yield are taken as a criterion, careful atten- tion must be given the questions relating to soil, such as mineral nutrients, aeration, etc. Among the first to study the effect of enriching the atmosphere with carbon dioxide was Demoussy."^ He used glass chambers of about one cubic meter. The control plants were exposed to an atmosphere of nor- mal tarbon dioxide-content while others were in an atmosphere of 0.15- 0.18 per cent. After two months the plants in the enriched atmosphere were cut and weighed; they were 122-262 per cent or an average of 157.6 per cent in excess of the control plants. The increased yields of the plants grown in the enriched atmosphere were not, however, directly proportional to the carbon dioxide-concentration ; also different plants vary greatly in the increases shown. TABLE 22 Effect of Increased Carbon Dioxide-Concentration on Fresh-Weight Yield. (Results of Lundegardh.) CO2 — House Control House Excess Cucumbers— shoots 1.807 Kg. 0.890 Kg. 0.917 Kg. = 103% —fruits 25.879 " 15.187 " 10.692 " = 74 Tomatoes— shoots 2.700 " 1.310 " 1.390 " =124 —fruits 1.210 " 1.120 " 0.090 " = 8 Bean-fruits 7.080 " 3.343 " 2>.7Z7 " =112 Total 38.676 " 21.850 " 16.826 " = 77% Mean CO^content 0.065% 0.043% 0.22% =51% The recent results obtained by Lundegardh ^^^ in Sweden give a good idea of the effect of carbon dioxide fertilization. The experiments were carried out in glasshouses, the carbon dioxide was continuously injected and the plants grew for ten weeks. Carbon dioxide detenninations were made daily. In Table 22 are given some of Lundegardh's results in terms of the fresh weight of the different plants. It would have been of greater value if the results had also been calculated on the basis of dry weight. Similar experiments have been carried out on a larger scale in the open field.^^^ Here the carbon dioxide, taken from cylinders or blast- "• Tjebbes and Uphof, LandiiK Jahrb., 56, 313 (1921). "'Demoussy, Compt. rend.. 139, 883 (1904). "' Lundegardh, "Der Kreislauf der Kohlensaure in der Natur.," p. 127. "• Lundegardh, 1. c, p. 135. Bornemann, 1. c, p. 85. THE NATURE OF PHOTOSYNTHESIS 129 furnace exhausts, is piped in or on the ground. In general an increased yield has been obtained. But the results are naturally dependent upon cli- matic conditions and soil fertilization, and it is difficult to calculate any regular or reliable ratio between increased yield and the carbon dioxide factor. High concentrations of carbon dioxide exert a narcotic effect on photo- synthesis. In pure carbon dioxide photosynthesis quickly ceases. Dif- ferent species exhibit a wide variation in the amount of carbon dioxide they can withstand.^-" Mosses and the lower plants are especially re- sistant to the effects of asphyxiating gases. It is highly probable that the effect of high concentrations of carbon dioxide is due to a toxic influence on the protoplasm. Higher concentrations of this gas, 15-25 per cent, retard and finally completely inhibit growth.^-^ Darwin ^" has also found that in an atmosphere rich in carbon dioxide the stomata tend to close. The efifect of high concentrations of carbon dioxide on photo- synthesis is undoubtedly of a complex nature in which other functions of the plant play an important role. d. The Influence of Temperature. The wide range of temperature under which plants are found to thrive in nature indicates that the photosynthetic process can be carried on at these temperatures, or at least that the photosynthetic apparatus is not permanently injured at the extreme temperatures noted. Thus McGee ^-^ found that joints of a prickly pear (Opuntia) reach a tempera- ture of 55° in the open without injury. On the other hand the marine algae of the polar regions must be exposed to temperatures of about zero degrees for long periods ; while Jumelle ^-* reports that Picea excelsa still showed photosynthesis at — 35° and Junipcriis at — 30 to —40°, while Physcira cilaris and Cladonia rangifernia ceased at - — 25° and Primustri at — 37°. Miss Henrici ^^^ found the threshold temperature of certain alpine phanerogams at — 16° and for some lichens at — 20°. Ewart ^^^ has shown that recovery of the capacity to do photosynthetic work after exposure to extreme temperatures depends very much upon the length of time of exposure. Similar results were obtained by Wurmser ^^^ and Jacquot who found that marine algae, subjected to tem- peratures of 36 to 45° for from 1 to 15 minutes had a lower rate of photosynthesis when returned to the normal temperature of 16°, the de- "" Ewart, Journ. Linncan Soc. 31, 404 (1896). ""Chapin, Flora., 91, 348 (1902). Brown and Escombe, Proc. Roy. Soc, 70 B, 397 (1902). '=" Darwin, Phil. Trans. Roy. Soc, 190, 531 (1898). ^ McGee, Carnegie Inst, of Washington, Year Book, 20, 47 (1921). ""Tumelle, Compt. rend., 112, 1462 (1891). •^''Henrici, Verli. Natur. f. Ges. Basel, 32, 107 (1921). "'Ewart, Joxirn. Linncan Soc, 31, 368 (1896). "'Wurmser, Bull. Soc. chim. Biol, 5, 305 (1923). 130 PHOTOSYNTHESIS gree of depression depending upon the temperature and length of time the plants were exposed to the higher temperatures. The methods of studying photosynthesis in the field have not been perfected so that reliable results can be obtained from plants growing under natural conditions. As has been repeatedly emphasized the inter- action of so many factors makes it impractical to determine all of these simultaneously, and even if this were possible, to attain a rational inter- pretation of the results of photosynthesis determinations under constantly varying conditions leads only to spurious results and false conclusions. So that, highly desirable as it undoubtedly is to obtain determinations of the influence of various external conditions, as existing in the field, on the photosynthetic activity of different plants, we are, nevertheless, con- fined to laboratory experimentation in order to attain results approaching scientific accuracy. One of the most difficult problems in the determination of the influ- ence of temperature on photosynthesis is the ascertaining of the tempera- ture of the plant itself. In aquatic plants the problem is somewhat simplified because with plants of small volume their temperature during illumination probably does not dift'er greatly from that of the surrounding water. But with land plants the problem is much more difficult. An illuminated leaf absorbs radiant energy, only a small portion of which is converted into chemical energy ; the greater proportion of the absorbed energy is converted into heat which results in the evaporation of water from the leaf, in alterations in the size of the stomatal openings and in other more obscure internal changes. In working with land plants it is therefore essential to know the internal temperature of the leaf and to obtain this, several methods have been employed.^-* Brown and Escombe ^-^ recognized that a certain amount of the radiant energy absorbed by a leaf is dissipated through the evaporation of water and they endeavored to calculate this amount. The following physical and chemical changes taking place within the leaf are of influence on the thermal relations of the leaf and its environments : 1— the evaporation of water, 2— photosynthesis, 3— respiration. Of these 1 and 2 are endothermic while 3 is exothermic. The actual determination of each of these factors and the calculation of their energy relations is associated with considerable difficulty. It is, first of all. necessary to know the thermal emissivity of the leaf. This term includes the gain or loss of heat due to radiation, convection and conduction of a unit area of leaf in unit time with unit difference of temperature between the leaf and its surroundings. The simplest condition is that of a leaf in the dark in an atmosphere saturated with water-vapor. Owing to the oxidative processes taking place within the leaf there is a tendency of the leaf to rise in temperature. This thermal disturliance due to respiration is, "'Shreve, Edith, Plant World. 22, 100 (1919). Miller and Saunders, Jour. Agric. Res.. 24, 15 (1923). "•Brown and Escombe, Proc. Rov. Sac. London, B 76, 69 (1905). THE XATURE OF PHOTOSYNTHESIS 131 however, exceedingly small. Brown and Escombe have calculated that the heat of respiration of a Hcliunthus ainuius leaf is 0.000582 calory per square centimeter of leaf lamina per minute when the leaf is respiring 0.7 cc. CO. per sq. decimeter per hour. Brown and Wilson ^^° have de- termined the thermal emissivity of such a leaf per square centimeter for still air and for a temperature excess in the leaf of 1" C. as being 0.015 callory per sq. cm. per minute. This amount must be doubled for the two sides of the leaf so that the rate of cooling becomes 0.030 calory 0.000582 per sq. cm. per minute for 1" excess. Thus, - =0.019 will represent the maximal excess temperature which the leaf will attain above its surroundings under still-air conditions when there is no transpiration, and respiration remains constant at 0.7 cc. CO2 per sq. decimeter per hour. Such conditions are, of course, purely theoretical. Any rise of tem- perature within the leaf will increase the partial pressure of the water- vapor in the intercellular spaces of the leaf : this will diffuse from the leaf into the surrounding atmosphere and even this small theoretical ex- cess temperature of 0.019"" will not be reached. Moreover, in experi- mental work we can rarely deal with still-air conditions nor with fully saturated atmosphere. Also, the loss of heat due to transpiration is nor- mallv of a much greater magnitude than the energy liberated in respiration. If the slight exothermic disturbance due to respiration is neglected, Brown and Escombe consider that: "the amount of water, Q, lost by unit-area of leaf surface in unit-time is a measure of the energy flowing into the leaf from its surroundings, and if we know the tem^^erature difference between the leaf and its surroundings, i.e. the temperature gradient 6 — ©n we can determine the rate of interchange of energy be- tween the leaf and its surroundings in absolute units for a temperature difference of P C, that is to say, the coefficient of thermal emissivity." This method has been applied to leaves in still and moving air by Brown and Wilson. Brown and Escombe regard that the following data are required to determine the thermal relations of a leaf to its surroundings when it is exposed to direct solar radiation : 1. The total amount of radiant energy incident on the leaf per unit time and area. 2. The amount of this energy absorbed by the leaf. 3. A measure of the internal work due to (a) evaporation of water, (b) photosynthesis. 4. The influence of air currents on the thermal emissivity of the leaf. The first. 1. can be determined by direct measurement of the intensity of solar radiation ; 2 is the absorption coefficient of the leaf. A measure ""Brown and Wilson, Proc. Roy. Soc, B 76, 122 (1905). 132 PHOTOSYNTHESIS of the evaix)i-ation of water, 3, is the water lost by transpiration per unit area and unit time Q; 592.6 calories is the latent heat of vaporization of one gram of water at 20\ The internal work from evaporation of water thus becomes 592.6 Q calories. The energy of photosynthesis can be calculated from the quantity of carbon dioxide absorbed. The heat of combustion of a hexose carbohydrate is 3760 calories per gram, and one cc. of carbon dioxide corresponds to 0.001336 grams hexose N.T.P., so that the conversion of one cc. of carbon dioxide into hexose sugar corresponds to the absorption of 0.001336 X 3760 = 5.02 calories. With the volume of carbon dioxide absorbed in photosynthesis per square centimeter per minute designated by c, the energy absorbed in photo- synthesis is expressed by 5.02 c per square cm. per minute. Alterations in external conditions afifect mainly the thermal emissivity of the leaf. Thus, the emissivity of a leaf in still air is doubled by an air current of 44.2 meters per minute. This and other influences which affect the emissivity of the leaf make Brown and Escombe's approach to this prob- lem exceedingly complex and indicate how, under natural conditions, the thermal relations of a leaf with its surroundings must change from moment to moment. Brown and Escombe's method involves a number of determinations which are exceedingly difhcult to make so that it has found little application in further investigations. One example of Brown and Escombe's results will indicate the differ- ence between the energy used in photosynthesis and that of transpiration : Energy used for photosynthesis 0.66 " transpiration 48.39 Total energy expended in internal work 49.05 Solar energy transmitted by leaf "^r 'cc Energy loss by "thermal transmission" 19.55 100.00 External conditions such as temperature and humidity greatly influ- ence the results so that wide variations in the various items are obtained. We shall enter into these more full}- in the consideration of the Energy Relations of Photosynthesis. We wish here to emphasize that the tem- perature of the leaf and its surroundings greatly affect the performance of the leaf as to its photosynthetic function. In plants well adapted for transpiration the superfluous or excessive radiant energy absorbed by the leaf can be readily dissipated through the evaporation of water. Transpiration rates of 500 to 1000 cc. of water per sq. meter per hour are not unusual; this would correspond to the dissipation of about 0.5 to 1.0 calory per sq. cm. per minute. With light intensity of about 1 calory per sq. cm. per minute incident on the leaf it can readily be seen that the plant may be kept at the temperature of the surroundings or absorb heat therefrom. In plants specially adapted to resist transpira- tion as in the succulents, thermal emission of absorbed radiant energy THE NATURE OF PHOTOSYNTHESIS 133 becomes highly important. Under natural conditions the problem becomes much more complex with the introduction of such factors as the angle of incidence of the light, etc. It may here also be mentioned, that since the photosynthetic process is endothermic, it has been suggested that photosynthetic activity would reduce the transpiration from a leaf to a certain degree. A plant in an atmosphere freed of carbon dioxide and so incapable of photo- synthesis, would thus show a higher rate of transpiration than when the plant was photosynthetically active. Claims of having observed such a phenomenon have been affirmed and denied from several sources. ^^' In view of the fact that in land plants but a small proportion of the absorbed energy is converted into chemical energy, the "cooling effect" of photosynthesis would be relatively slight and it is possible that the phenomenon, when observed, was actually due to some other cause. In this connection the observation of Detlefsen "^ and of Puriewitsch '^^ are of interest. Both found that the amount of radiant energy absorbed was greater when leaves were in an atmosphere containing carbon dioxide than in one free of this gas. The photosynthetically active leaf absorbs 1.7 to 11.7 per cent more energy than the leaf in a carbon dioxide free atmosphere. Blackman and Matthaei ^^* have made careful determinations of the internal temperature of leaves exposed to bright illumination. They used small thermocouples of copper and constantan connected with a galvanome- ter. One junction was inserted in the mid-rib of the leaf, and the other junction was placed in a water bath. The internal temperatures of the leaves were measured by bringing the temperature of the water bath to a point where the galvanometer deflection was zero. The temperature of the two couples was then the same and the temperature of the water bath was taken as that of the leaf. The leaves used for the determina- tion of the photosynthetic rates were in a special frame submerged in a water bath. The temperature of the bath varied only slightly, while "the leaf temperature oscillated up and down with the varying fine shades of natural illumination through a range of 9°." Miss ]\Iatthaei has made a study of the influence of temperature on photosynthesis by determining the amount of carbon dioxide absorbed. The smoothed curve which she constructed from a number of separate determinations is shown in Figure 12. This gives the rates of photo- svnthesis from — 6° to 43° under optimal conditions of light and carlx)n dioxide supply. Miss ]\Iatthaei's work was carried out with the viewpoint of Black- "^^Deherain, Ann. sci. nat. (VI), 4, 177 (1876). Jumelle, Rev. gen. hot., 1, 2,7 (1889); 2, 417 (1890); 3, 241 (1891). Burgerstein, "Transpiration der Pflan- zen " 46. '"'Detlefsen, Arh. hot. Inst. Wiirzhurg., 3, 534 (1888). "'Puriewitsch, Jahr. iciss. Bot., 53, 210 (1914). "^ Blackman and Matthaei, Proc. Roy. Soc. London, B 76, 402 (1905); Phil. Trans. Roy. Soc. London, B 197, 75 (1904). 134 PHOTOS YNTHESIS man's theory of limiting factors. She found that increasing the tem- perature produces an increase in the rate of photosynthesis only up to a certain point. Here some other factor, either light intensity or carbon dioxide-concentration determines the rate and a further increase in tem- perature has very little effect on photosynthesis. However, when there 260 220 180 A A / ^ \ 140 \ eo 2t y -w 10* 20* SO* 40* 50* Fig. 12. — Influence of temperature on the rate of photosynthesis. (From Matthaei.) is an excess of light and carbon dioxide, it is ai)parent that the rate of photosynthesis increases rapidly with increasing temperature and it is con- cluded that "corresponding to each temperature there is a certain definite amount of assimilation which may be termed 'maximal assimilation' for that temperature. This cannot be exceeded and will not be reached unless both light and carbon dioxide supply are adequate." Most of the older work on the influence of temperature on photo- THE NATURE OF PHOTOSYNTHESIS 135 synthesis is of limited value either because the effect of other factors as light and carbon dioxide-concentration, were not fully recognized or on account of inadequate methods of measuring the temperature and the photosynthetic rate.^^^ A second, very important element of the effect of temperature on photosynthesis is that this is greatly influenced by time. At high tem- 260 A 1 \ 180 \ \ 1 . A / \ 140 / 1 \ //: .'■ 100 r ,*' \ \ '[ / / \ 3 / / I 60 / 4 20 X /' / y -1 0* ( )• 1 0' 2 ©• •i V 4 0° 5 0' 6 0* Fig. 13. — Influence of temperature and time on tlie rate of photosynthesis. (From Duclaux.) peratures a leaf can maintain a maximal rate of photosynthesis only for a short time. The higher the temperature the shorter is the time the maximal rate can be maintained and the steeper is the slope of its de- cline. As a consequence the rate of photosynthesis at higher tempera- tures is not the same during two successive hours and the maximum value "'Kreusler, U., Landw. Jahrb., 16, 711 (1887). 136 PHOTOSYNTHESIS observed will depend uixjii the time that has elapsed between the beginning of the experiment and the determination of the rate. Ihis effect is known as the "time factor" and is discussed in more detail in a later section of this book. In Figure 13 is shown schematically the manner in which the time factor afifects the rate of photosynthesis. At lower temperatures the rate of photosynthesis is fairly constant for successive periods of time. Above about 24° there is a constant falling off of the rate with time. The exact temperature at which this begins very probably varies with differ- ent leaves. In Figure 13 the broken lines 2, 3, 4, indicate rate of photo- synthesis after the second, third and fourth hours. The result thus is a shifting of the maximal rate to lower temperatures. As a result of theoretical considerations developed by lilackman ^^'^ and others the influence of time on the optimal rate of photosynthesis has been interpreted on the basis that there are two op|X)sed reactions in- volved. This principle of superposition of two curves has been em- ployed for a variety of reactions. ^^^ From Miss Matthaei's results it can be determined that the temperature coefficient, Qio, for the rate of photo- synthesis of leaves of the cherry laurel is 2.1 for temperatures between 5° and 25°. That is, between these temperatures the rate of photosynthe- sis follows closely what is frequently called the van't Hoff rule. Above and below these temperatures it does not follow this rule and, as has been observed in other reactions of living organisms, when the limits of temperatures are approached there is a great variation in the rates from that expected from the van't Hoff rule. The type of curves obtained from a study of the effect of tempera- ture on photosynthetic rate bears a strong similarity to the curves of the effect of temperature on the rate of catalysis of a number of different chemical reactions. The interpretation of these latter reactions have been used to explain the results obtained with photosynthesis. More particularly have the results of Tammann '^^ and of Duclaux ^^^ on the effect of temperature on fermentation been taken as types of reactions which find application to the photosynthesis problem. These workers have shown that the relation of temperature to the rate of a fermentation re- action depends upon two different factors, the temperature and the con- centration of the ferment. The latter are thermolabile substances, i.e., they are destroyed or inactivated at ordinary temperature and the rate of this destruction increases with temperature. As the temperature of the fermenting mixture is increased, the rate of the fermentation reaction is accelerated at the same time; however, the ferment is also being de- stroyed, as this cannot endure high temperatures for any length of time. The latter reaction reduces the concentration of the ferment which re- "* Blackmail, Ann. of Bot., 19, 282 (1905). Kanitz, "Temperatur und Leben- svorgange," Berlin, 1915, p. 16. Jost, Biol. Zcntralb., 26, 225 (1906). "'Bredig, G., Ergeh. Physiol., 1, I, 198 (1902). "'Tammann, Zeits. phvsiol. chem., 16, 317 (1892). "* Duclaux, "Traite de microbiologic," 2, 193, Pans, 1899. THE NATURE OF PHOTOSYNTHESIS 137 suits in the fermentation reaction attaining a maximal rate at a certain temperature but with a continuation of this temperature decreasing and finally falling to zero when all the ferment has been destroyed. Duclaux has pictured the phenomenon as shown in Figure 14. In this the curve OA represents the rate of reaction with increasing tem- A D / \ \ / iTx ^ / \ \ \ y ^ C O y^ 1( )• 2) )• 3 0* 4 D* 5( )• 6 »• 7( )* 80 Fig. 14. — Influence of temperature and time on the rate of photosynthesis. (Blackman.) perature, DB is the rate of destruction of the ferment with temperature and OAIC the observed rate of the fermentation reaction with increas- ing temperature. This general view has been applied to the observed maximal rates of photosynthesis at different temperatures. Thus OA would represent the rate of photosynthesis with increasing temperature, DB would be the inactivation of the chloroplasts due to higher tempera- 138 PHOTOSYNTHESIS ture and OMC the rate of photosynthesis at the different temperatures observed after longer periods of time. This interpretation of the influence of temperature and time on the rate of photosynthesis can probably not be taken to picture the kinetics of the reaction in every detail. As a matter of fact we know very little about the inactivation of the chloroplasts, either as to the rate thereof or of the causes which bring this about. As will be shown later the activity of the chloroplasts can be diminished not only by high tempera- tures but as well by. high light intensity and by the accumulation of the products of photosynthesis. The importance of this is emphasized by the fact that in order to obtain uniform results on the rate of photosyn- thesis, it is essential that the plants be subjected to the same treatment for some time previous to the experiment. Just what form the curve DB takes is therefore uncertain. The foregoing also illustrates how exceed- ingly difficult it is to obtain constants of biological reactions if such con- stants are to have even remotely the same meaning as our physical chemical values. The temperature coefficient of photosynthesis has been determnied by Warburg ^■*° over a range of temperatures. He used the unicellular alga Chlorella and his method had the advantage that it avoided the necessity of determining the internal temperature of a leaf, a procedure that is associated with considerable experimental difficulty and some uncertainty. In view of the fact that the temperature coefficient shows decided changes at different temperatures the method of indicating this for 10" C. is not strictly correct. For the sake of comixirison, however, Warburg cal- culated his results on this basis. He found that when the concentration of carbon dioxide and light intensity are high the temperature coefficient is not constant. Thus he found the following temperature coefficients, calculated for Qio as the indicated temperature differences : TABLE 23 Temperature Coefficients of Photosynthesis at Different Temperatures. (Warburg.) ^T^rlTl^^."'.''''':.... 5-10° 16-25° 5.4-10° 10-20° 20-30° 15-25° 25-32° Q^^ 4.7 2.0 4.3 2.1 1.6 1.06 1. Relative light intensity... 16 16 45 45 45 1.8 1. It is apparent from this that the temperature coefficient decreases with increasing temperature. When the light intensity is low the temperature coefficient is about unity. The latter is in agreement with Blackman's results. Osterhout and Haas ^" working with Ulva rigida found that the temperature coefficient of photosynthesis between 17- and 27° is 1.81. ^^'Wavhurg, Biochcm. Zcit., 100, 2SS {1919} ^^, ,,_._^ . . „. "*i Osterhout and Haas, Jour. Gen. Physiol., 1, 295 (1919). van Amstel, Rev. trav. hot. Neerlandais, 13, 1 (1917). THE NATURE OF PHOTOSYNTHESIS 139 The fact that the temi^eratiire coefficient varies with temperature has been employed to sul)Stantiate the view held by a number of investigators that the photosynthetic process involves two reactions ( Willstatter and Stoll, Warburg, Osterhout and Haas and others). These various hypothe- ses differ somewhat in detail, but the facts upon which they are based are about as follows. At high illumination intensity a rise of ten degrees, from 15'' to 25", causes the velocity of photosynthesis to double. Here a chemical reaction is evidently determining the rate of the process ; this reaction Warburg has designated as the "Blackman reaction." At low illumination intensity the rate of photosynthesis is' independent of the temperature between 15' and 25^. This is what could be expected if under these conditions a photochemical reaction, as, for instance, the photolysis of a chlorophyll-carbonic acid complex were determining the velocity. The assumption here is that all photochemical reactions have low temi^erature coefficients. The photosynthetic process is made up of two reactions, one an ordinary chemical reaction, which determines the rate of the process at high illumination intensities and has a high temperature coefficient, secondly a photochemical reaction which deter- mines the rate of the process at low illumination intensities and has a low temperature coefficient. These theories are taken up in greater de- tail under the discussion of the chemistry of the photosynthetic process. There is no satisfactory explanation of the high temperature coefficients at low temperatures. It should l3e stated, however, that high values of Qio at low temj^eratures are of very common occurrence in biological processes. ^*^ A great deal more experimental work is required in order to elucidate fully the relation between these two steps in the photosynthetic process, the "dark" and the "light" reaction. In fact it cannot be accepted as established that two such reactions actually exist or that their assumption will prove adequate to explain what still api)ears to be an exceedingly complex system. That the temperature coefficient varies with differ- ent temperatures and with other external conditions there is little doubt. Many of these facts have been brought to accord with the general prin- ciples first outlined by Blackman's theory of limiting factors. This theory and its modifications, however, tell us little of the kinetics of the photosynthetic process ; they are the first attempts at an accurate expres- sion of the relation of this process to the various factors which influence it. In a sense this mode of attack is analogous to the thermodynamic treatment of chemical reactions, though we are not in possession of the exact data which such a method requires. But the thermodynamic treat- ment is largely indej^endent of any molecular hypothesis we may have formed regarding any process. In photosynthesis we are constantly en- deavoring to determine the nature of the molecular changes involved and to this end data are frequently applied which have but very indirect *"Kanitz, A., "Temperatur uiid Lebensvorgange," Berlin, 1915, p. 27. 140 PHOTOSYNTHESIS bearing. As a consequence much controversy and confusion has arisen which could have been avoided if the points at issue had been kept more clearly in mind.^*^ Willstatter and StoU ^** observed that there was considerable differ- ence in the influence of temperature on the rate of photosynthesis between leaves of high and low chlorophyll-content. The leaves of low clilorophyll- content exhibit a lower acceleration with increasing temperature than do the leaves of high chlorophyll-content. Thus leaves of Ulnius with low chlorophyll-content showed a temperature coefificient of 1.34 and with high chlorophyll-content of 1.53 for 15° to 25°. Willstatter and Stoll call attention to the fact that in these experiments an increase in illumina- tion intensity was without effect on the photosynthesis of leaves rich in chlorophyll ; in fact the light intensity could be reduced by ^ without affecting the rate. They explain this on the ground that in these leaves the chlorophyll-content was relatively of much higher concentration than the enzyme which they consider plays an important role in the photosyn- thetic process. This enzyme is a protoplasmic (internal) factor and in- duces a "dark" reaction. On the other hand, in the leaves low in chloro- phyll, which have a low Qm, the enzyme ("dark reaction") is relatively in excess and chlorophyll is relatively in minimum concentration. The rate of the reaction is here determined by the chlorophyll-content and an increase or decrease in light intensity is of decided influence on the rate of photosynthesis. These observations are quite in harmony with the ideas expressed above, of the influence of those factors which are rela- tively in minimum concentration. Willstatter and StoU's experiments are particularly valuable, because of their careful control of the chlorophyll factor and their realization of the importance of the internal factor. The nature of this internal factor they regard as being enzymatic. While it must be admitted that virtually nothing is known of this "enzyme," nevertheless the internal factor is doubtless a protoplasmic activity and in so far as we largely depend upon the conception of enzyme activity to account for protoplasmic activity the term seems permissible. More- over, the effect of temperature on this factor corresponds to that which has been observed for enzymatic reactions. Thus Willstatter and Stoll point out that both types of leaves, those high and low in chlorophyll- content, are equally equipped with the enzymatic factor. Temperature exercises an accelerating influence on the activity of this internal factor. However, this accelerating influence will result in a higher photosynthetic rate only in those leaves which have a high chlorophyll-content, that is, in those leaves which can make use of high intensities of light. In the leaves low in chlorophyll-content the accelerating influence of increased temperatures on the internal factor will not result in a higher rate of "'Brown and Heise, Philippine Jour, of Sci., 12, 1, 85 (1917). Brown, ibid., 13, 345 (1918). Smith, Ann. Bot., 33, 517 (1919). '^^ Willstatter and Stoll, "Untersuchungen ii. d. Kohlensaureassimilation," Berlin, 1918, pp. 56, 112, 143. THE NATURE OF PHOTOSYNTHESIS 141 photosynthesis, because the rate here is determined by the chlorophyll- content, namely, the amount of radiant energy which the leaf is capable of utilizing. On this basis the difference in the temperature coefficients of the two types of leaves becomes intelligible. With the leaves low in chlorophyll- content, a light reaction was determining the rate, resulting in a low Qio- In the other case, the leaves of high chlorophyll-content, this factor was relatively less in minimum concentration, and the influence of tempera- ture on the enzymatic reaction was more clearly expressed. Willstatter and StoU also found that the temperature coefficient of photosynthesis was lower at higher temperatures. It is not surprising that the temperature coefficient of photosynthesis should vary from that of simple chemical reactions. The rate of photo- synthesis is influenced by a number of factors. As an example of this we may mention the lower solubility of carbon dioxide at higher tempera- tures which may result in a lessened supply of this gas in the chloroplasts. An interesting phenomenon discovered by Miss Henrici and described in the section of the Time Factor is related to the effect of starch forma- tion on the photosynthetic rate. The formation of starch in the leaf is greatly affected by temperature. The presence of starch in the chloro- plast also is of influence on the rate of photosynthesis. Temperature can, therefore, influence the rate of photosynthesis in this rather indirect manner of affecting the starch formation in the chloroplasts. The threshold of photosynthesis in alpine shade plants and lichens is con- siderably below 0°. This is probably due to their low freezing point arising from the high osmotic pressure of the cell sap which contains no starch but much soluble sugar. Even when ice crystals appear in the interior of the cells photosynthesis still occurs ; this is especially true of the lichens. e. Chlorophyll. That chlorophyll is of paramount importance in the photosynthetic process was concluded by the first investigators of the phenomenon. Only those portions of plants containing chlorophyll are capable of reducing carbon dioxide to carbohydrates. Frequently plants are colored red or brown by an admixture of other pigments, but these usually also con- tain chlorophyll. There are lower organisms capable of reducing carbon dioxide, certain bacteria which do not contain chlorophyll, but the re- actions here are very different from those taking place in the chlorophyl- lous plants. Some of these bacteria do not require light as a source of energy for the carbon dioxide reduction, but are apparently capable of accomplishing the reduction by means of chemical energy as, for ex- ample, the oxidation of hydrogen or ammonia. In those plants which use light as a source of energy for the photosynthetic reaction, chlorophyll is an essential component. There exists, however, some difference of 142 PHOTOSYNTHESIS opinion as to whether all plant parts containing chloroplasts exhibit the capacity for photosynthesis. In view of the fact that frequently the chloroplasts are rendered inactive by injurious external agencies, it is difficult to establish this point experimentally.^*^ While, then, it is generally accepted that chlorophyll is essential for photosynthesis, the quantitative determination of the role of this factor is exceedingly difficult, because it is impossible to vary experimentally the concentration of the chlorophyll in the plants under investigation. In considering chlorophyll alone as a factor in photosynthesis, it is well to bear in mind that this is done only for the sake of analysis; photo- synthesis can take place only when the chlorophyll is associated with the living plasma in the chloroplast. We shall discuss, then, the effect on photosynthesis of plants containing varying amounts of chlorophyll, bear- ing in mind always that chlorophyll is but one part of a complex mechanism to the successful functioning of which other parts are essential. On account of the impossibility of varying the concentration of chloro- phyll experimentally in any one plant, the method which has been fol- lowed most generally has been to compare leaves of the same species but of different chlorophyll-content, as for example, normal varieties, rich in chlorophyll, with the yellow varieties, poor in chlorophyll. While this is probably the best method of approach now available to the prob- lem of the influence of variation in chlorophyll-content on photosynthe- sis, it is not altogether satisfactory. Two varieties of the same species of a plant may differ in chlorophyll-content according to differences in certain environmental conditions, e.g., light intensity and soil, or this dift'erence in chlorophyll-content may arise from more deep-seated heredi- tary causes. The question arises whether associated with the differences in chlorophyll-content there may not be other differences which also affect photosynthesis. Chlorophyll is itself a product of the metabolism of the plant ; for its formation, in most plants, light is essential as is also oxygen. But the respiratory and metabolic activity of the plant is of con- siderable significance in the photosynthetic activity thereof. It is. there- fore, not entirely improbable that the factors which produce differences in chlorophyll-content affect other portions of the photosynthetic mecha- nism. Thus the differences in photosynthetic activity which in some cases have been ascribed to differences in chlorophyll-content may actually be due to a complex of factors. Lubimenko ^^^ found that the light requirements of "shade" plants for photosynthesis is considerably lower than that of "Hght" plants, i.e. the former type of plant can accomplish the same amount of photosynthetic work with a lower illumination intensity than the latter type. This fact "'Dehnecke, Dissertation, K51n (1880). Friedel, Comt>t. rend.. 142, 1092 (1906). F.wart, Journ. Linnean Soc, 31, 436 (1896). "*Lubimenko, Rev. gen. Bot.. 17, 381 (1905); 20, 162, 217, 253, 285 (1908); Ann. Sci. Nat. (9), 7, 321 (1908) ; Compt. rend., 145, 1347 (1907). Griffon, Ann. Sci. Nat. (8), 10, 1 (1899). THE NATURE OF PHOTOSYNTHESIS 143 Lubimenko endeavored to correlate with differences in chlorophyll-con- tent, for he found that "shade" plants contain more chlorophyll than the "light" plants. The "shade" plants show an optimal light intensity for photosynthesis; if the light intensity is increased above this optimal intensity the rate of photosynthesis decreases. This, it is assumed, is due to the fact that the "shade" plants have a high chlorophyll-content and consequently a high absorption coefficient. On the other hand, "light" plants, with a low chlorophyll-content show maximum photosynthesis at the highest light intensities. This can be interpreted, that in "light" plants the amount of light absorbed when illuminated by highest light intensities, is not sufficient to produce an inhibition in the activity of the chloroplasts. The optimum temperature and optimum light intensity for photosynthesis of a plant decreases with increasing chlorophyll-content according to this view. In the course of its development a plant can regulate the amount of light it absorbs by changes in its chlorophyll- content. It is, however, not only the light intensity which determines the chlorophyll-content, but also temperature. With increasing tempera- tures the intensity of light required for maximal chlorophyll formation decreases. Plester^*^ studied the relation of carbohydrate formation (by means of the half leaf method) to the chlorophyll-content of the light green or yellow and normal varieties of a number of plants. He found that the light green varieties have a lower chlorophyll-content than the normal varieties per equal area of leaf surface; the former ranged from about 30 to 50 per cent of the latter. From Plester's results it cannot be concluded that there is any direct ratio between the rate of photosyn- thesis and chlorophyll-content. The light green varieties showed a lower rate of photosynthesis than the normal varieties, ranging from 34.2 to 59.5 per cent. On the other hand in some cases the light green or yellow varieties showed a much higher rate of photosynthesis than would correspond to their chlorophyll-content. This points to the fact that other factors besides chlorophyll-content are playing a role. Also, the light green varieties had a lower rate of respiration than the normal plants, though there was no direct parallelism between respiration and chlorophyll- content. A closer relationship seems to exist between the rate of photo- synthesis and that of respiration. Hence, to a measure at least, the lower rate of photosynthesis in the light green varieties is compensated for by a lower rate of carbohydrate consumption. It is, however, still a question whether the lower rate of respiration is simply the result of a lower supply of carbohydrates, or whether the other conditions or fac- tors which produce a higher rate of respiration (protoplasmic factors, enzymes) do not also affect the rate of photosynthesis. Plester also found, contrary to Willstatter and Stoll (see below) that plants which contain besides chlorophyll admixtures of red pigments, had a lower rate of photosynthesis than the same species without these pig- "' Plester, Beitr. Biol. d. Pflansen, 11, 249 (1912). 144 PHOTOSYNTHESIS ments. But in this case also, those plants that had a lower rate of photo- synthesis also had a lower rate of respiration. Undoubtedly, however, in these cases questions of light intensity and absorption coefficient enter which make the interpretation of the results very difficult. The results of Henrici,'*^ who made a study of the effect of altitude on photosynthesis and chlorophyll-content of the various plants, are also of interest in this relation. The photosynthetic activity and chlorophyll- content was compared of Anthyllis vulneraria, Bcllis perenis, Primula farinosa, and Taraxacum officiuale, grown at different altitudes, the low land material at Basel at 450 m., the alpine material at 1400 to 2700 m. Differences in the structure of leaves, particularly of the chlorophyll bearing portions, with changes in altitude have been observed repeatedly.^*^ Henrici found that the plants grown at high altitudes contain less chloro- phyll than those of the same species from lower altitudes. This is pre- sumably due to the greater intensity of light at higher altitudes. How- ever, whether the lower chlorophyll-content of- plants grown under high illumination intensity can be directly ascribed to the destructive action of such light (especially the red-yellow rays) on chlorophyll, does not seem entirely established.^ '° There is no variation in the chlorophyll-con- tent during the course of the day nor do plants taken from one alti- tude to another change in this respect within two weeks. The photosyn- thetic relations of the plants grown at different altitudes is more complex than would be expected from simply a difference in the chlorophyll-con- tent and is a function of light and temperature. While photosynthesis in the alpine plants (low chlorophyll-content) commences at higher light intensity than the low altitude plants (high chlorophyll-content), the temperature minimum of the former is much lower than the latter. With high light intensity, photosynthesis of the alpine plants is higher than that of the low land plants at any temperature. With low light intensity the photosynthesis of the alpine plants is higher than that of the low land plants only at low temperatures. At higher temperatures, more light is required in order that the photosynthesis of the alpine plants equal that of the low land plants. These results are in agreement with the findings of Willstiitter and Stoll discussed below. The alpine plants can endure a wide range of temperature, and, with their low chlorophyll- content, also high illumination intensities. These plants are apparently well adapted to the conditions of high altitudes, though the conditions of such an adaptation lie not only in a single factor such as the chlorophyll- content, but as well in other factors as the temperature relations of the plants. Henrici's experiments show marked specific differences in the adaptation of the various species of plants used. Her work also serves to demonstrate the great difficulty, if not the impossibility, of obtaining ^^^ Henrici, Dissertation, Basel, 1918. ""Bonnier, G., Ann. Sci. nat. Bot. (7), 20, 247 (1895). ^'" Dangerard, P. H., Compt. rend., 151, 1386 (1910). Ewart, Jour. Liinu'an Soc. 31, 436 (1896). THE NATURE OF PHOTOSYXTHESIS 145 a quantitative analysis of the influence of the various factors afifecting photosynthesis under field conditions. A great advance in the knowledge of the chlorophyll factor in photo- synthesis is due to the researches of Willstatter and Stoll/^^ These workers, on the basis of their thorough investigations of the chemistry of chlorophyll and other plant pigments devised reliable quantitative methods for the determination of chlorophyll, a contribution of the first importance toward the solution of these problems. A description of these methods will be found in Chapter 7 of this book. According to Willstatter and Stoll chlorophyll is composed of two components designated as a and h : Chlorophyll a: [MgN4C3oH3oO ICO.CHs . CO0C20H39 Chlorophyll /' : [MgN^CaoH^sOojCOoCHs . CO0C00H39. Accompanying the chlorophyll pigments in the chloroplasts there are two yellow pigments, carotinoid c and x : Carotin : C40H56, Xanthophyll : C4oHg602. The chlorophyll-content of leaves ranges as follows : 0.15-0.35 gram per 100 grams fresh weight. 0.6-1.2 gram per 100 grams dry weight. 0.3-0.7 gram per 1 sq. m. leaf surface. The yellow pigments range from 0.07 to 0.20 per cent of the dry weight or about 0.0 to 0.07 gram per sq. m. of leaf surface. The ratio of c the two yellow components, O — , is fairly constant, equal on an average .X. to 0.60. The ratio of the chlorophvH components, O — , also varies but ~ b slightly and is equal to 2.9. In this work of Willstatter and Stoll there are two points of particular importance bearing on the question of the relation of chlorophyll-content to photosynthetic rate. These are: 1, that the chlorophyll-content does not change during the course of photosynthesis, and 2, that the ratio of chlorophyll a and b also remains nearly constant during photosynthesis. The second point is about as significant as the first, for in most of the subsequent analyses the two components are determined together. If during photosynthesis the ratio of the two components showed a great variation, it would not be permissible to compare mixtures composed of the two. Willstatter and Stoll studied the relation of photosynthesis to chlorophyll-content of leaves in different stages of development and in leaves which exhibit rather extreme conditions such as yellow varieties, "* Willstatter and Stoll, "Untersuchungen iiber die Assimilation der Kohlensaure," Berlin, 1918. 146 PHOTOSYNTHESIS etiolated and chlorotic plants. The conditions under which the experi- ments were carried out were such that neither temperature, carbon dioxide-concentration nor light intensity were determining the rate of photosynthesis. This is, of course, necessary in order to emphasize or demonstrate the influence of other factors, in the cases studied, more especially chlorophyll-content. In most of the experiments excised leaves were used. Willstatter and Stoll introduced as a measure of the ratio of chlorophyll- content to rate of photosynthesis the term "Assimilationszahl" or photo- synthetic number. This is a measure of maximal photosynthesis expressed in grams of CO2 absorbed per hour by a leaf mass containing 1 gram of chlorophyll under favorable conditions of temperature. Or briefly it is grams of carbon dioxide absorbed per hour i:>er gram of chlorophyll; this is the photosynthetic number which for the sake of brevity we shall designate by Pc- hourly photosynthesis, grams COo P.= grams chlorophyll. This term expresses the highest possible rate of photosynthesis under the particular conditions of temperature and carbon dioxide-concentra- TABLE 24 Photosynthetic Number, Pc, of Various Lux, Determined by VV Species Temp. Aesculus Hippocastanum 25 Acer Negundo ^^ Acer Pseudoplatanus j^ Ampelopsis quinquefolia 25 Hostia plantaginea 3U Hydrangia opulodes 30 Pelargonium zonale ^^ Primula ^^^ Prunus Laurocerasus ■^ Rubus ^^o Sambuscus nigra ^^^ Tilia cordata j^ Ulmus ^^ Cucurbita Pepo 25 Clerodendron trichotomum Thumb 25 Fragaria vesca j-^^ Helianthus annuus 25 '.'.'.'.'.'.'.'...■■ 25° Populus pyramidalis hort 25° Pelargonium peltatum (from green-house) ^^ Plants, 5 Per Cent CO2, About ILLSTATTER AND StOLL. From 10 g. Fresh Leaves : Dry Chloro- Weight phyll Grams Mg. 48,000 2.94 2.22 3.58 2.00 1.00 1.21 0.96 0.90 3.40 3.60 2.75 3.19 2.94 1.3 2.07 3.00 1.72 1.94 1.67 3.19 .60 Photosynthesis : gram COjper Hour, per 1 g. Dry Weight Pc 24.7 0.054 6.4 24.8 0.086 7.7 40.0 0.058 5.2 28.8 0.089 6.2 10.0 0.088 8.8 9.2 0.050 6.5 12.5 0.097 7.4 11.4 0.117 9.1 12.2 0.029 8.1 32.4 0.052 5.8 22.2 0.034 6.6 28.1 0.028 6.6 16.2 0.022 6.9 17.5 0.164 12.1 15.0 0.089 12.3 17.7 0.078 10.6 16.5 0.134 14.0 15.0 0.129 16.7 20.8 0.137 10.9 19.0 0.060 10.0 8.2 0.198 14.5 THE NATURE OF PHOTOSYNTHESIS 147 tion. Willstatter and Stoll in discussing the theoretical value of Pc emphasize the temperature factor; but it seems essential to define the car- bon dioxide-concentration, for different plants show specific differences in the concentration of carbon dioxide they can utilize. In Willstatter and Stoll's experiments some of the leaves which had a very low chlorophyll- content did not attain maximum photosynthesis tecause the light intensity was not sufficiently high. In Table 24 are given the values of Pc for normal leaves, that is, excluding those low in chlorophyll, young and old leaves. Approximately constant values of Pc for a given temperature would indicate that under these conditions the rate of photosynthesis was determined by the chlorophyll-content. The values of Pc in the first section of Table 24 shows a fair constancy, for 25° ranging from 5.2 to 7.7, for 30° from 6.5 to 9.1. In these cases there is a rough parallelism between the rate of photosynthesis and chlorophyll-content. In the lower section of Table 24 are given the values of Pc for plants which have a notably rapid growth and high photosynthetic rate. In these Pc is considerably higher. It is already evident from these values that there is no simple quantitative ratio between chlorophyll-content and rate of phv^tosyntl-c ■'. In this coni.L\:tior .t has been an interesting question as to what are the relations in very young leaves between the rate of photosynthesis, chloro- phyll formation and respiration. The latter activity is notably high and decreases with time and the development of the leaf.^^^ Under favor- able conditions of temperature and light the development of chlorophyll is quite rapid. Thus, for example, Willstatter and Stoll observed increases in chlorophyll-content, at 25° and 48,000 Lux, in 80 minutes as follows: Tilia cordata, 32 per cent of the original, Acer negundo, 29 per cent, Quercus Robur, 22 per cent, Popuhis pyramidaUs hort., 15 per cent, Acer Pseudoplatamis, 10 per cent. With this increase in chlorophyll-content there is also an increase in photosynthesis. There is, however, not a direct proportionality between chlorophyll-content and photosynthetic rate. The latter, 8 to 10 days after the first determination, made when the leaves were just unfolding, show about a constant value for equal areas but a decreased value on the basis of dry weight. On the basis of chlorophyll- content after about nine days there is a decrease in photosynthesis, that is, Pc decreases. These relations are given in Table 25 which show an in- crease in Pc after about nine days and thereafter again a decrease. The table is taken from the results of Willstatter and Stoll. A perusal of Table 25 will show an increase with time of the dry weight of the leaves ; on the basis of the dry weight there is a decrease in photo- synthesis. The leaves also show a consistent increase in chlorophyll content, but this is not associated with an increase in photosynthesis after the first nine days. This results in a considerable variation of the Pc. '"Willstatter and Stoll, 1. c, p. 87. 148 PHOTOSYNTHESIS These facts point to the existence of some other internal factor and give weight to the theory that the rate of photosynthesis, under constant and favorable external conditions is determined not only by the chlorophyll- content but also by another internal factor. The latter apparently is more rapid in development than chlorophyll, resulting in a high Pc during the first days of the life of the leaf. After full development of the leaf and TABLE 25 Rate of Photosynthesis, Chlorophyll-Content and Photosynthetic Number, AT 25°, 5 Per Cent CO2, About 48,000 Lux, Determined by WiLLSTATTER AND StOLL. Species From 10 g. Fresh Leaves : Photosynthesis : g. C02per Hour Dry Weight grams Chloro- phyll mg. Date Per 1 g. Dry Weight Perl sq. dm. Pc Apr. 29 May 7 June 3 Oct. 8 Aesculus Hippo- castanum 2.10 2.06 2.94 3.62 10.1 15.1 24.7 31.2 0.054 0.088 0.054 0.041 0.043 0.039 0.033 0.044 11.1 12.1 6.4 4.8 May 1 May 8 July 14 Sambuscus nigra 1.85 2.25 2.56 11.7 23.1 23.5 0.078 0.101 0.057 0.046 0.057 0.032 12.2 9.8 6.2 May 4 May 12 June 5 June 25 Tilia cordata 2.18 2.15 3.52 3.19 8.3 11.5 28.8 28.1 0.040 0.085 0.058 0.058 0.015 0.024 0.029 0.028 10.6 16.0 7.1 6.6 May 5 May 14 June 24 Acer Pseudo- platanus 2.60 2.55 3.58 10.7 18.7 40.0 0.035 0.078 0.058 0.017 0.022 0.026 8.6 10.7 5.2 May 10 May 19 June 8 Ampelopsis quinquefolia 1.84 1.98 2.00 7.4 15.4 28.8 0.042 0.105 0.089 0.018 0.038 0.028 10.5 13.5 6.2 May 11 May 20 June 9 June 20 Quercus Robur 2.76 2.64 4.14 4.50 6.6 8.6 21.6 25.0 0.026 0.051 0.047 0.044 0.013 0.024 0.038 0.041 10.9 15.8 9.0 7.8 increase in the chlorophyll-content this other factor is relatively less active than the chlorophyll factor. Very similar results vi^ere obtained by Willstatter and Stoll in a study of the photosynthetic activity of leaves of diiTerent ages. They compared the activity of a light-green leaf from the end of a branch with that of a dark-green leaf from the base of the same branch. Some examples of the differences between old and young (but almost full grown) leaves are given in Table 26. The results of Willstatter and Stoll of the photosynthetic activity of leaves in different stages of development show that although the chloro- THE NATURE OF PHOTOSYNTHESIS 149 phyll-content of the leaves increases with age and that the photosynthetic activity also increases, the two are by no means parallel. In autumn, with the change in color of the leaves, the conditions be- come very complex. In general there is a decrease in photosynthesis (g. CO2 per hour) when this is calculated on the basis of leaf area, dry or fresh weight. With the yellowing of the leaves there is also a decrease in TABLE 26 Photosynthesis of Leaves from the Same Plant but in Different Stages of Development, at 25°, 5 Per Cent CO2, About 48,000 Lux, from THE Determinations of Willstatter and Stoll. Date Description • of Leaves 10 g. Fresh Leaves : Photosynthesis: g. COzper Hour: Species Drv Weight grams Chloro- phyll mg. Per 1 g. Dry Weight Per 1 sq. dm. Leaf Area Pc Acer pseudo- platanus June 23 4th-6th leaf from end of branch Z.Z 8.3 0.030 0.016 11.8 " 24 From base of branch . . 3.58 40.0 0.058 0.026 5.2 TiUa cor- data June 25 " 26 Young, light- green Lower dark- 2.56 6.5 0.036 0.018 14.2 green from same branch 3.19 28.1 0.058 0.028 6.6 Laurus nobilis June 30 July 1 Light - green leaves Dark - green 3.10 12.7 0.024 0.019 5.9 leaves of previous year 4.95 21.2 0.016 0.023 Z.7 the chlorophyll-content. Under conditions of maximal photosynthesis (25°, high CO2 and light intensity) the activity of the leaves turning yel- low, on the basis of chlorophyll-content (i.e. the Pc) is not very different from the normal leaves. This is due to the fact that synchronous with the decrease in chlorophyll there is a decrease in the activity of the protoplasmic factor. As a consequence the Pc also decreases with the yellowing of the leaves. In some cases the two factors, chlorophyll-content and protoplasmic activity, do not decrease at the same rate so that it may occur that the Pc of autumnal leaves is higher than in midsummer. Willstatter and Stoll observed all possible variations in the Pc with the yellowing of the leaves, rising, constant and falling values. Such values, it must be recalled, repre- sent the photosynthetic activity on the basis of chlorophyll-content. In absolute terms, the amount of carbon dioxide reduced in leaves turning yellow is a tenth or less than that of normal leaves. We have already mentioned experiments in which the photosynthesis 150 PHOTOSYNTHESIS of normal and yellow varieties of various plants vi^as compared (Plester). These demonstrated that there was no direct proportionality between chlorophyll-content and rate of photosynthesis. Willstatter and Stoll made similar experiments using 5 per cent carbon dioxide and high light inten- sity. The yellow varieties of the plants used by them contained 3 to 15 per cent, and even less, of the chlorophyll of the normal varieties. It was found that on the basis of chlorophyll-content the yellow varieties show a much greater photosynthetic activity than the normal ones. The Pc of the former was in some cases almost 20 times that of the latter ranging from 6 to 12 for the normal varieties against 50 to 120 for the yellow varieties. TABLE 27 Photosynthesis of the Green and Yellow Varieties of Elm, 5 Per Cent CO2, About 24,000 Lux. (Willstatter and Stoll.) Photosynthesis : g. CO2 per Hour : A Variety Temp. Wt. of Leaves grams Dry Weight Leaf Surface sq. cm. Chloro- phyll mg. 8g. Fresh Leaves 1 1 sq. m. Surface Chlorophyll, poor . . 25° . 15° 8.0 Same leaves 2.00 2.00 321 321 0.95 0.95 0.075 0.056 2.3 1.7 Chlorophyll, rich . . 25° . 15° 8.0 Same leaves 2.35 2.35 421 421 "13.0 13.0 0.089 0.058 2.1 1.4 A most striking case of the disproportionality between chlorophyll- content and photosynthetic rate was found by Willstatter and Stoll in the elm. These results are reproduced in Table 27. In the case of the elm (Table 27) though there is a great difiference in the chlorophyll-content of the two varieties, the rate of photosynthesis shows but slight differences, and at 15° the one poor in chlorophyll has a higher rate of photosynthesis on the basis of equal surface. Experiments of this nature may be of considerable value in analyzing the photosynthetic mechanism. Temperature variations do not afifect the rate of photosynthesis of the yellow varieties as much as of the normal ones. Dififerences in light intensity have apparently a profound effect on the yellow varieties. Undoubtedly it will be essential to gain more knowl- edge of the nature of the internal protoplasmic factor before a clear under- standing can be had of the whole photosynthetic process. The time factor seems to become apparent in the yellow varieties more slowly than in the normal ones. Willstatter and Stoll point out that the relatively high photosynthetic rates of yellow varieties cannot be attributed to the carotinoids. The content of the latter pigment in the yellow varieties is in many cases con- siderably less than in the normal varieties. What is the function of the THE NATURE OF PHOTOSYNTHESIS 151 yellow pigments of the leaf is still unknown.^^^* It has been given a role in photosynthesis as well as in the respiratory process, though most of these ideas are largely hypothetical. The photosynthetic activity of etiolated plants, or more accurately, of plants in which the chlorophyll is just developing, demonstrates the dis- proportionality between photosynthesis and chlorophyll-content. Will- statter and Stoll investigated the photosynthesis of etiolated leaves under conditions of maximal activity, i.e. 25°, 5 per cent CO. and high light intensity, about 48,000 Lux, using cultures of Phaseolus vulgaris and Zea mays. The etiolated plants, as soon as the first traces of chlorophyll are formed in the light, are remarkably active. For example, Phaseolus, with a chlorophyll-content of 0.7 mg. per 10 grams fresh leaves, had a Pc of 133, while the control plants grown in light with 18.6 mg. chlorophyll on the same basis showed a Pc of 9.4. In general the Pc of etiolated leaves is much higher than that of young leaves which developed in the light. This high photosynthetic rate of etiolated leaves, or those which have only just become green, holds not only when calculated on the basis of chloro- phyll-content but as well as an absolute measure. Etiolated leaves which had been in the light used in the experiment 2 to 4 days, showed a higher rate of photosynthesis, on the basis of either, dry weight, fresh weight or area than the control plants raised in light. This greater activity of the etiolated plants also holds for increase in dry weight which was about twice that of the control plants, after the former had developed about 75 per cent of the normal chlorophyll-content. Willstatter and Stoll in- terpret these facts by assuming that, while chlorophyll formation is in- hibited in the dark, the development of the protoplasmic or enzymatic factor is not suppressed ; in fact the absence of light appears to favor the development of this factor. As a result of the accumulation or higher development of this enzymatic agent in etiolated leaves, these, after they develop a small quantity of chlorophyll are able to surpass leaves grown in the light. Miss Irving ^^* has also studied the photosynthetic activity of etiolated plants. She endeavored to determine whether etiolated plants were capable of utiHzing for photosynthesis the carbon dioxide produced by their respiration. Her results do not show a decrease in carbon dioxide evolu- tion when the plants were illuminated. This is in disagreement with the results of Willstatter and Stoll. It is possible that Miss Irving's results may be explained on the basis that the light intensity employed in her experiments was very low (light from a north window) though no intensities are given. Another condition of interest in relation to chlorophyll-content and photosynthesis is that of chlorotic plants. When plants are grown in such a manner that no iron salts become available to the leaves, they remain "^Palmer, L. S., "Carotinoids and Related Pigments," Chemical Catalog Co., 1922, p. 262. '"" Irving, Ann. Bot.. 24, 805 (1910). Ewart, Jour. Linnean Soc, 31, 554 (1897). 152 PHOTOSYNTHESIS very pale green or colorless with restricted development of the chloroplasts. This condition continues even under conditions of high illumination in- tensity. The condition of chlorosis can be caused by a number of circum- stances, but probably the most common is the absence of iron. Willstatter and Stoll cultivated plants w^ith nutrient solutions con- taining no iron. While other types of leaves also poor in chlorophyll such as the light green or yellow^ varieties, autumnal and etiolated leaves, showed high photosynthetic activity on the basis of their chlorophyll-content, the chlorotic leaves had a very low rate of photosynthesis. Thus chlorotic Heliantlius leaves with a chlorophyll-content for 10 grams fresh leaves of 1.9, 2.9, 3.8 mg. had a Pc respectively of 13, 21.7 and 19.5 compared to the normal leaves with 11.6 mg. and a Pc of 11.5. From this it is apparent that in chlorotic leaves the chlorophyll is only partially utilized and that in such leaves not only is the chlorophyll-content low but other essential parts of the photosynthetic mechanism are imperfectly developed. Chlorosis, then, means besides insufficient chlorophyll, inadequacy in other parts of the photosynthesis apparatus as well. Chlorophyll itself contains no iron and we have no adequate explanation of the nature of the condi- tion termed chlorosis nor of the role of iron in the activity of the chloro- plasts. Benjamin Moore ^^^ has reported the presence of iron in the color- less portion of the chloroplasts and considers iron essential for their for- mation. His conclusions of the role of iron, are, however, not entirely convincing and further investigation in this field seems necessary. In this connection the observations of Curtel ^^° are of interest who found that chlorotic plants have a lower rate of respiration and transpiration than normal plants. Willstatter and Stoll also showed that while the rate of photosynthesis in the extreme light-green and yellow varieties was low, the photosynthetic number was very high. On the other hand, in albino or variegated leaves, which do not contain the yellow pigments, the rate of photosynthesis is low as is also the Pc- The anthocyanin pigments which in some leaves accompany the chloro- phyll are apparently without direct influence on the rate of photosynthesis. The photosynthetic activity of fruit skins is very similar to that of leaves. An important fact brought out by the investigations of Willstatter and Stoll is that leaves of the light green or yellow variety, in their photo- synthetic activity, are affected more by differences in light intensity, while the leaves rich in chlorophyll are more affected by changes in temperature. This is one of the facts which has led to the assumption of the existence of an internal factor which Willstatter and Stoll consider to be of enzymatic nature. They interpret varying effects of light and temperature on leaves of different chlorophyll-content as follows. The excess or larger quantity of chlorophyll in normal leaves does not result in a photo- '" Moore, Proc. Roy. Soc, E 87, 556 (1914). "Biochemistry," Longmans, Green & Co., London, 1921, p. 53. '=* Curtel, Compt. rend., 130. 1074 (1900). THE NATURE OF PHOTOSYNTHESIS 153 synthetic activity much greater than that of the yellow varieties. Hence the latter are constituted as well in regard to the activity of the proto- plasmic factor as the former. Increase in temperature augments the activ- ity of this internal protoplasmic factor. But the activity of this internal factor is only one step in a series of reactions. An increase in its activity will result in an augmentation of the rate of the total reaction only if the other steps are proceeding at a higher rate, i.e. if the rate of the re- action of the internal factor is determining the total rate. Therefore, an increase in temperature will become effectual and result in an augmented photosynthetic rate only in those leaves which have enough chlorophyll to absorb the radiant energy necessary to maintain this increased rate. In leaves poor in chlorophyll an increase in temperature results in but a slight rise of photosynthetic rate, because here the reactions depending upon the absorption of radiant energy by the chlorophyll are determining the total reaction. In normal leaves which usually have more light available than they can utilize in photosynthesis, the internal factor is relatively in minimum concentration and is determining the rate of the process, at least under experimental conditions with ample carbon dioxide supplied. In the leaves poor in chlorophyll the amount of absorbed radiant energy is de- termining the rate of the process and the internal factor is relatively in excess. Further discussion of the role of the internal factor will be found in another section. The work of Willstatter and Stoll clearly establishes the dispropor- tionality between chlorophyll-content and photosynthetic rate. Tliese studies also demonstrate the complexity of the reactions constituting the photosynthetic process. They emphasize the importance of agents about which we know as yet practically nothing beyond the fact that they exist, namely the protoplasmic or enzymatic factors. These investigations, more- over, make evident the fruitfulness of the quantitative chemical treatment of physiological problems and the value of chemical and physical concep- tions in interpreting such complex phenomena. f. Water Supply. That water enters chemically into the photosynthetic reaction was clearly established by de Saussure. The eft'ect of the water-content of the plant and of the water vapor in the atmosphere on the stomatal oi>enings through which the carbon dioxide passes to the centers of photosynthetic activity has also been recognized for a long time. Thus water is an essen- tial compound in the chemical reactions comprising the photosynthetic process and through its action on the stomata indirectly influences the rate of this important function of the plant. The latter fact has been brought out by Kreusler's ^^' investigations. Moreover, the fact that the water- content of a leaf influences the carbohydrate ratio thereof, that is, the ^"'Kreusler, Landw. Jalirb., 14, 913 (1885). 154 PHOTOSYNTHESIS ratio of polysaccharides to .soluble monosaccharides, serves to emphasize the important role which water plays in photosynthesis, for it is well established that the accumulation of starch has an inhibitory influence on photosynthesis. Deherain and Maquenne ^^^ as well as lljin have shown how greatly the rate of photosynthesis of leaves is affected by their water-content. In the investigations of lljin the rate of photosynthesis was related to the degree of oj^ening of the stomata. The latter was measured by means of a Darwin porometer. The methods employed cannot be considered as yield- ing strictly quantitative results, but indicate that alterations in the size of the stomatal openings can have a profound influence on the rate of photosynthesis. lljin ^^° has also investigated the eft'ect of water loss in leaves on the disappearance of starch from the guard-cells of the stomata. It has been found that leaves which had become flaccid or wilted through loss of water and had again become turgid and of normal appearance when an adequate supply of water was given, did not under the latter circumstances attain their original photosynthetic activity. The cause for this lies in a disturb- ance of the stomatal function. The rate of photosynthesis depends to a large measure upon the rate at which carbon dioxide is supplied to the chloroplasts. The carbon dioxide reaches the chloroplasts through the stomatal openings. If these are not open or are partially closed, obviously the ingress of carbon dioxide into the leaf will be hampered and the rate of photosynthesis will be decreased. Now lljin found that leaves which had recovered after severe loss of water and appeared normal, still on careful examination revealed the fact that a large proportion of the stomata had been killed and were closed. Also, the opening and closing of the stomata under normal conditions is apparently accomplished by alterations in the osmotic pressure of the cell sap of the stomatal guard- cells. These changes in osmotic pressure are, in part at least, due to the transformation of starch into soluble carbohydrates and vice versa. Thus, in a humid atmosphere, the osmotic pressure of the guard-cells increases and the stomata open ; while in a dry atmosphere the soluble carbohydrates are converted into starch, which can be detected in the guard-cells, the osmotic pressure decreases and the stomata close. This mechanism is probably dependent upon the activity of an enzyme and lljin has been able to demonstrate that ample water supply favors soluble carbohydrate formation while desiccation favors the formation of starch. This is quite in harmony with other observations of the effect of water content on the carbohydrate ratio in plants. ^"^ However, if the desiccation is carried too far the activity of the enzyme is apparently impaired, consequently the regulatory action of the guard-cells is inhibited and the stomata do not ^"^ Deherain and Maquenne, Compt. rcitd.. 103, 167 (1886). lljin, Flora 116 306 (1923). Kreusler, Lamhv. Jahrb., 14, 951 (1885). "•lljin, Jahrb. zinss. Bot., 61. 670 (1922). "~Spoehr, H. A., Pub. No. 287, Carnegie Inst, of Wash., 1919, p. 57. THE NATURE OF PHOTOSYNTHESIS 155 react to changes in the water-content of the plant. No doubt there are some features of the mechanism of the movement of the stomata still to be explained, but Iljin's investigations have indicated one important influence of water on the rate of photosynthesis. In experimental work it is therefore highly important that due con- sideration be given the question of the humidity of the atmosphere to which the plant is subjected. In mosses and leaves possessing no stomata this factor is according to some authors ^"^ not of such importance, while others have found considerable variation in photosynthetic activity with changes of water-content. Lichens and some mosses can be dried to a powdery consistency, in which condition the power of photosynthesis is, of course, temporarily lost ; on the addition of water these plants regain almost immediately their photosynthetic activity. In the leaves of higher plants loss of water beyond a certain point results in death, and while such dried and powdered leaves still exhibit a post mortal respiration with the formation of carbon dioxide the power of photosynthesis is apparently permanently lost. It should be stated, however, that this question has not yet been definitely answered and that by the use of proper methods it may be possible to obtain photosynthesis with leaf material which has previously been dried. This has been shown to be possible by the investi- gations of Molisch ^"^ which are discussed in the section under Internal Factors. Dastur,^"^ from investigations in which the starch-iodine test was employed concludes that with advancing age there occurs a decay of photo- synthetic activity. This is at first noticeable in the mesophyll cells of the margin and intravascular regions of the leaves, supposedly the regions in the leaf farther removed from the vascular system supplying water. There is some question, however, whether the iodine test is reliable for deter- mining photosynthetic activity especially where small differences are concerned. For plants growing under natural conditions the humidity of the air and water supply of the soil are of great importance for the photosynthetic activity of the plant. These factors to a large measure determine the open- ing of the stomata and consequently the absorption of carbon dioxide by the leaf. In general it is the latter factor more than any other, namely, the amount of carbon dioxide which the plant takes up, that determines the rate of photosynthesis and hence the vegetative development of the plant. In xerophytic plants, such as the cacti, which exercise special economy of their water-content, the gaseous exchange is as a consequence also greatly affected. This applies naturally not only to the absorption of carbon dioxide for photosynthesis but as well to the absorption of oxygen for respiration. The result is that these plants exhibit certain modifica- '" Jumelle. Rev. (fcn. Bot., 4. 168. 318 (1892). Rastit, ibid.. 3, 521 (1891). T'-sson. Compt. mid.. 119. 440 (1894). Htnrici, Verli. Nattirf. Ccs. Ba.<;el., 32, 157 (1921). "'Molisch, H.. Zc-t. f. Dot.. 17. 577 (1925); Bot. Zcitg., 62, 1 (1904). ^'^Dastur, Ann. Bot., 38, 779 (1924). 156 PHOTOSYNTHESIS tions in their metabolic activities in which organic acids, such as malic acid, play an important role. While there is no reason for believing that in these plants, the chemical reactions constituting the photosynthetic process are different from other plants, owing to the modified gaseous exchange the study of photosynthesis in xerophytes offers specially complex conditions.^"* But water supply is of importance to the photosynthetic process not only on account of its indirect influence through affecting the stomatal openings but also in a very direct manner. This question is more ex- haustively discussed in the chapter on the Chemistry of Photosynthesis. Suffice it to recall that de Saussure demonstrated that water enters into the chemical reactions constituting photosynthesis. He showed that of the dry material synthesized by a plant less than half could be ascribed to the weight of the carbon in the carbon dioxide taken up by that plant. The rest he regarded as coming from the water. The reaction for photosynthesis is usually written in the following manner with the molecular weight relations as indicated : 6 CO, + 6 HoO -> CeHxoOe + 6 O2. 264 108 180 192 The basis for this is that the ratio of the volume of carbon dioxide ab- sorbed to that of oxygen emitted is very close to unity which would indi- cate the formation of a compound of the composition CnHanOn. The value of n is, of course, not established thereby, but from the widespread oc- currence of hexoses in leaves the assumption is frequently made that the equation represents empirically the course of the reaction. Similarly the relation of the weight of carbon dioxide absorbed to that of material formed in photosynthesis in many instances supports this view. From the equation it is apparent that the molecular ratio of water to carbon dioxide in photosynthesis is : H2O : CO, = 108 : 264. It is rather sur- prising that in much of the writing on photosynthesis the authors disregard the fact that we are dealing with carbonic acid and not simply with CO,, that the water actually enters into the chemical reaction and is not only a solvent. From theoretical as well as experimental considerations, therefore, we can conclude that water is essential for the photosynthetic process. Each 100 grams of material synthesized, calculated as CsHioOg requires 60 grams of water. Interesting in this connection are also the osmotic relations of the photosynthetically active cells. Treboux ^'^^ has studied the effect of dif- ferent concentrations of various substances which are considered as non- toxic to the photosynthetic activity of Elodea. A noticeable effect was first observed with a 0.1 per cent solution of KNO3. Solutions of KCl, *" Richards, H. M., Pub. No. 209, Carnegie Inst, of Wash., 1915. Spoehr, H. A., Biochem. Zeit, 57,95 (1913). „. ,0^ ,ionox "'Treboux, Flora., 92, 53 (1903). Jacobi, Flora, 86, 326 (1899). THE NATURE OF PHOTOSYNTHESIS 157 NaNO.i. sucrose and glycerine isotonic with 0.5 per cent KNO3 showed an equal inhibiting effect. This inhibiting effect increases with concentration. Concentrations which did not cause plasmolysis produced no permanent effects ; that is, the plant regained its original rate when the salt solution was replaced by water. If plasmolysis had occurred (2.5 per cent KNO3) the plant did not recover its original rate when placed in water. Some- what contrary to these results are those of Kny ^"''' who found that Spyrogyra cells which had been in a 40 per cent solution of sucrose for less than an hour, showed evolution of oxygen when placed in a 20 per cent solution and were illuminated. When the concentration of the sucrose solution was gradually increased from 10 to 40 per cent, some cells still showed photosynthesis after 24 hours. There appears to be no doubt, however, that plasmolysis greatly decreases the rate of photo- synthesis, though the activity may not be entirely prevented. Fromageot ^^~ concludes from his studies with Ulva lactuca in different concentrations of sea-water that there is a distinct optimal concentration for photosvnthesis and that this corresponds to that of sea-water (A = 1.94)'. g. The Time Factor. Investigations of the eft'ect of temperature on photosynthetic rates have shown that the maximum rate cannot be maintained for any length of time, but that with time this maximum rate shifts to a lower temperature. This is apparently due to the fact that there are two opposed reactions involved. This has been shown graphically in Figure 14. If the point M is taken as the optimum, this point is not a fixed one. Its position will be determined by all factors which affect the rates of the opposing reactions O A and D B, and these factors are manifold. In the case of photo- synthesis the curve of inactivation, D B, is a function not only of tem- perature but of other factors as well, such as light intensity and the accumulation of the products of photosynthesis. Similarly the curve of acceleration, O A, is a function not only of temperature but also of chloro- phyll-content and certain internal factors. As a consequence the position of the optimum point, M, is dependent upon the previous treatment of the plant material in regard to temperature and illumination intensity and the rate at which the temperature is raised to this optimum point. Blackman and Matthaei have shown that for cherry laurel the rate of photosynthesis remains fairly constant at temperatures below 25°. Above this temperature the initial rate cannot be maintained, but decreases with time. It is highly probable that different species of plants differ as to the point where this time' factor first becomes apparent. As has been stated, the inactivation of photosynthetic activity can be brought about not only by higher temperatures but also by long exposure »-Kny, Ber. hot. Ges., 15, 396 (1897). Klebs, Biol. Centralb., 7, 166 (1887). "'Fromageot, Compt. rend., 177, 779 (1923). 158 PHOTOSYNTHESIS to high illumination. This has been demonstrated by Ursprung ^^* with the formation of starch in the chloroplasts. Starting with a starch-free leaf, the formation of starch on illumination and the gradual accumulation thereof with continued illumination of the leaf can be clearly followed by the depth of color produced when the leaf is treated with iodine. If ex- posure to bright sunlight is continued the amount of starch in the leaf gradually decreases again. Thus a leaf of PJuiscolus after 5 hours of illumination showed very deep coloration of the starch-iodine, while after 8.5 hours of illumination the reaction was faint. This phenomenon can be produced by almost any source of light of sufficient intensity, namely, sunlight, electric arcs and filament lamps, and the time required is pro- portional to the intensity of the light. By using a spectrum the effect is first brought about in the red-orange portion, the region showing, under the circumstances, the highest photosynthetic rate. With higher intensity the shorter wave-lengths bring it about in correspondingly shorter time. It is therefore apparently proportional to the photosynthetic activity. Ursprung has given the name of solarization to this phenomenon on account of its analogy to the eft'ect produced in photographic plates under similar circumstances. ^^^ W^e have here another case of the inactivation of the chloroplasts. These organs after long exposure to intense light cease to function al- though they are not killed, and, after a period of darkness, again produce starch normally. It is possible that the phenomenon is in some manner associated with the oxygen produced in photosynthesis.^"*' It must be borne in mind that solarization as observed by Ursprung, has been confined to the presence of starch ; it would be highly desirable if simi- lar experiments would be carried out in which more complete analyses of the carbohydates in the leaf were made and consideration given to the influence of temperature. The inhibiting effect on photosynthesis of long exposure to light of high intensity has also been investigated by Ewart.^"^ From these results it would appear that the inhibiting effect is due to destruction of the chlorophyll. The question arises whether the inhibition of photosynthesis under conditions of continued exposure to high illumination intensity is due merely to the destruction of the chlorophyll or whether the chloroplast ])lasma is also injured. Pantanelli ^'- inclines to the opinion that both pigment and stroma are affected ; that the fatigue effects observed by him in bright light are possibly due to a combination of chlorophyll destruction and injury to the chloroplast plasma. It is evident that our limited knowl- edge of the relation of pigment to stroma in the chloroplasts and of the mechanism of these latter bodies prevents a more thorough understanding '"'Ursprung, Ber. hot. Ges., 35, 57 (1917). ""■' Plotnikow, "Handbuch d. Photochemie," Berlin-Leipzig, 1920, p. 645. ''" Pringsheim, Jahrb. unss. Bot., 12, 288 (1879). ^^'Ewart, Ann. of Bot., 11, 439 (1897) ; 12, 379 (1898). '^■'Pantanelli, Jahrb. miss. Bot., 39, 167 (1903). THE NATURE OF PHOTOSYNTHESIS 159 of the phenomenon of the inhibition of photosynthesis by high light intensity. It has been known for a long time that the photosynthetic rate de- creases with the accumulation of the products of photosynthesis. This is especially noticeable when the leaves which are being observed have been removed from the rest of the plant. Boussingault ^" first noticed the gradual decrease in the amount of carbon dioxide reduced by leaves which had been removed from a plant. This is undoubtedly associated with the fact that in excised leaves the capacity for translocation of synthesized carbohydrates does not exist. Under normal conditions the material formed in the leaves travels to other parts of the plant where it is con- sumed or stored. When the leaves are cut from the plant this is not possible, and accumulation readily occurs. SaposchnikofT ^'* has demonstrated the inhibitory power of an accumu- lation of carbohydrates and that these cannot increase beyond a certain point. When the leaves of Vitis vinifera contain 23 to 29 per cent carbo- hydrates of the dry weight there was a cessation of photosynthesis. Leaves which had accumulated a certain amount of carbohydrates showed a de- creased photosynthetic rate, while a decrease in this accumulation of carbohydrates resulted in an increased photosynthetic rate. It is evident that the rate of the dififerent steps comprising the photosynthetic process exercises a profound effect upon the total reaction and the movement of the products away from centers of activity is of importance for con- tinuous action. This applies not only to the accumulation of carbohydrates but apparently also to oxygen, for pressures of oxygen above %o at- mosphere tend to decrease the photosynthetic rate when high light inten- sity is used. These facts are of particular importance in experimental work, when excised leaves are used, though they must be taken into consideration under all circumstances. When the rate of photosynthesis is high as under conditions of high carbon dioxide concentration, strong illumination and elevated temperatures, this effect is especially to be watched for. Ewart ^" made similar observations with a variety of plants, but found that the plants which have thus been inactivated do not always regain their photosynthetic capacity by being kept in darkness. The case of Allium cepa is of interest because this plant does not form starch. When leaves of this plant are exposed to bright light for a long time, 14 days, or for a shorter period while being fed sugar, the evolution of oxygen finally ceases. This inactivation apparently does not injure the cells or chloro- plasts. After a few days in darkness the capacity for photosynthesis is regained. It is possible that this phenomenon is associated with the osmotic relations of the cell, for when the cell sap reaches a certain concentration photosynthesis ceases. '''Boussingault, "Agronomic Chimie et Agriculture," Vol. 4, pp. 286, 312 (1868). >'* Saposchnikoff, Ber. bot. Ges.. 11, 391 (1893) ; 9, 293 (1891) ; 8, 233 (1890). "'Ewart, Jour. Linncan Soc, 31, 429 (1896). 160 PHOTOSYNTHESIS There is as yet not sufficient information to enable a clear conception to be formed as to the manner in which the accumulation of starch in- hibits the photosynthetic activity of the chloroplasts. In fact, in view of the behavior of plants which form no starch and show an inhibition through high concentration of soluble carbohydrates, it is possible that the accumulation of starch is not directly associated with the cause of the inhibition, but is merely an accompanying phenomenon. At the same time, if we accept the theories of Wurmser, of Briggs, and of Warburg, that the primary photochemical action takes place on the surface of the chloroplast, it is conceivable that the accumulation of starch in the chloro- plast would materially afifect this action. In starch-laden leaves, the starch grains are frequently larger than the chloroplasts, the latter being partially covered by the starch. Under these conditions the surface of the chlorophyll solution is materially reduced. As a consequence as much carbon dioxide could not reach the chlorophyll as when the chloroplasts are starch-free, and the amount of carbon dioxide entering the photochemical reaction would be reduced with a resulting decrease in photosynthesis. Thus, any substance which displaces carbon dioxide from the surface would tend to inhibit the rate of photosynthesis. Miss Henrici ^~^ has described some interesting curves showing the indirect effect which temperature and light may exert on the photo- synthetic rate. These curves for alpine phanerogamic shade plants and lichens do not show the single maximum, either as a function of tempera- ture or illumination intensity when only one factor is changed. There are, in fact, two maxima. These plants form starch under conditions of higher light intensity or higher temperature than normally occur in the winter. For example, while at 0° in these alpine plants there is never any starch, when these are exposed to light of high intensity starch ap- pears. The threshold of temperature and illumination intensity for photo- synthesis is much lower than that of starch formation. As a result when either factor is increased the rate of photosynthesis rises to a maximum; during this time no starch is formed. With continued increase of either factor the rate decreases ; this decrease is synchronous with the appearance of starch in the chloroplasts. With further increase of either Hght or temperature the rate of photosynthesis passes a minimum value, then rises to a second maximum and finally drops rapidly again. Plants which habitually form no starch do not behave in this manner and, as has been stated, the feeding of soluble sugar and consequent starch formation, rapidly decreases the photosynthetic rate. These effects are apparently closely associated with those observed by Ursprung. Prolonged exposure to sunlight may cause temporary inhibition of the photosynthetic capacity of plants. The causes of this action are still obscure, though it appears that the plasma of the chloroplasts is more sensitive under certain conditions than the protoplasm of the cell.^"'' ""Henrici, Vcrh. Naturf. Ges. Basel, 32, 107 (1921). "' Pringsheim, Jahrh. zviss. Bot., 12, 326 (1882). THE NATURE OF PHOTOSYNTHESIS 161 h. Internal Factors. The external conditions which influence photosynthetic activity, namely, temperature, light intensity, and partial pressure of carbon dioxide can be easily determined and the intensity of each of these influences can also be controlled experimentally. As a consequence the efifect of these factors on the photosynthetic process can be studied experimentally, and although the analysis of the reactions involved has not proved to be a simple task nor is as yet complete, it has been possible to gain considerable informa- tion. The study of the influence of these external factors has served to emphasize the complexity of photosynthesis and to bring to light the fact that there are conditions or influences within the cell which are of equal or greater significance in determining the rate of photosynthesis. The most evident of these is probably the chlorophyll-content of the plant and the relation of this to photosynthetic activity has already been dis- cussed. The existence of these internal factors becomes evident from the fact that under circumstances the photosynthetic rate varies inde- pendently of the external factors such as temperature, light intensity and carbon dioxide-concentration. Moreover, with constant external condi- tions, the rate of photosynthesis does not run parallel with the chlorophyll- content. So that, besides the latter factor, there must be some other, internal factor which is determining the rate of the reactions. Conclusions regarding the existence of an internal factor have been arrived at through inference rather than by direct experimental demon- stration. Our knowledge regarding internal factors has not progressed very iar ; in fact, a good deal of the subject is still purely hypothetical. It is an exceedingly difficult experimental task ; external conditions can be altered at will, but to deal with material or conditions inside of the cell, material about which we know virtually nothing, presents unusual ob- stacles. Moreover, it is uncertain whether there are several internal factors, or whether different properties of the same thing have been described. Pantanelli ^^^ came to the conclusion that a very important role in the photosynthetic process is played by the protoplasmic activity of the color- less components of the chloroplasts. This was based upon the observa- tion that the photosynthetic activity becomes inhibited in time and after a period of darkness again attains its original rate. The inhibition in Pan- tanelli's experiments was probably not due to an accumulation of the products of photosynthesis, for the duration of illumination was short, nor was apparently the chlorophyll-content decreased though the illumination intensity was high. The photosynthetic activity shows the phenomenon of fatigue and recovery very similar to that of the animal muscle. With this inhibition of photosynthetic activity there occurs a retardation of the protoplasmic streaming. A disturbance in protoplasmic streaming con- "* Pantanelli, Jahrh. zi'iss. Bol., 39, 184 (1903). 162 PHOTOSYNTHESIS current with photosynthetic inactivity has been observed frequently. ^^^ This is associated vi^ith the aggregation of the chloroplasts into a central clump. In this condition the chlorophyll in the chloroplast is destroyed if illumination is continued. It is interesting that protoplasmic stream- ing, after retardation by light of high intensity, recommences sooner than does the recovery of the original rate of photosynthesis. While the aggre- gation of the chloroplasts probably plays an important role in the inhibi- tion of the photosynthetic activity, it cannot be said that this fact in itself explains the phenomenon. In the fatigue effects observed by Pantanelli the destruction of chlorophyll probably plays no part. But as to the real cause of the temporary inhibition of photosynthesis no definite statement can be made. It is very doubtful that in photosynthesis chlorophyll is successively decomposed and reformed, for Willstatter and Stoll could observe no alteration in the chlorophyll-content during photosynthesis. And when chlorophyll is decomposed, as occurs during very intense illumi- nation, the reforming thereof is a slow process. Therefore it seems at least plausible that in the fatigue effects the colorless portion of the chloro- plasts plays a part. It is doubtful whether we can make much advance in these problems until we know more about the relation of chlorophyll to the colorless portion of the chloroplast. There is, on the one hand, the theory that the chlorophyll and the plasmatic stroma are morphologically separate, and, on the other, that the two are chemically united. In view of the ease with which chlorophyll can be extracted from the plant it is doubtful whether the union of the pigment and the proteinaceous stroma can be of a more stable nature than exists in emulsions. An exceedingly important fact in this connection is that photosynthetic activity is apparently intimately associated with the vital activity of the plant. Any disturbance in the respiratory activity of the plant also affects the photosynthetic activity. This is particularly true of narcotics and poisonous substances. Whether the photosynthetic activity is affected by the action of these substances on the protoplasm or on certain enzymes elaborated by the protoplasm is not certain. From the results of Molisch who was able to detect the evolution of oxygen in leaves that had been dried, and the protoplasm consequently killed, it would appear that enzymes play an important role. It may be said that ascribing these functions to an enzyme about which we know virtually nothing, rather than to the protoplasm is just shifting the burden and constitutes no real advance in our knowledge ; it is nevertheless a step in the same direction in which Buchner's discovery of zymase aided in elucidating the phenomenon of fermentation. The absence of oxygen also has a profound effect on the rate of photo- synthesis. Boussingault ^^° showed that in an atmosphere of hydrogen, nitrogen or methane plants lose the power of photosynthesis and attributed "'Ewart, Ann. Bot., 12, 385 (1898)_; Journ. Linncan Soc, 31, 439 (1896). ™ Boussingault, "Agronomie, chimie agricole et physiologic," Paris, 1868, 4, p. 329. THE NATURE OF PHOTOSYNTHESIS 163 it to a disturbance of respiration. Later Pringsheim ^^^ studied the effects of lack of oxygen by subjecting the plants to a stream of hydrogen and carbon dioxide. Under these conditions protoplasmic streaming soon ceases, and if the cells are then illuminated no photosynthesis occurs. Cells in this condition of "inanition" can be revived by the supplying of oxygen. Even in continuous illumination in an atmosphere of hydrogen and carbon dioxide, i.e., starting with active photosynthesis, cells of Cliara gradually showed stoppage of photosynthesis and protoplasmic streaming. If this condition of "inanition" is not continued too long photosynthesis and streaming can be revived by addition of traces of oxygen. Pringsheim interpreted these facts on the basis that photosynthesis and respiration were intimately associated. He argued that with photosynthesis inde- pendent of respiration, the former process in an atmosphere containing carbon dioxide and with sufficient light intensity should continue to evolve oxygen, and respiration and protoplasmic streaming could continue. This. his experiments showed, is not the case. Ordinarily photosynthesis pro- duces more oxygen than is used in respiration. Pringsheim's ^®- next deduction does not seem as well founded. He concluded that in photosynthesis no oxygen is formed within the cell. But rather that in the decomposition of carbonic acid in the cell a sub- stance, possibly hydrogen peroxide, is formed which passes out by diosmosis and on the surface of the cell decomposes with the liberation of oxygen. He maintained that we have no experimental evidence of the formation of oxygen within the cell. That all our conclusions are based upon methods of gas analysis which can yield results only of the final end products. Of the chemical nature of the hypothetical oxygen compound Pringsheim did not venture a conjecture but emphasized that the carbon dioxide decomposition and oxygen evolution are two separate processes. This he sought to support by his observations of oxygen evolution from dying cells. There is as yet not sufficient experimental evidence to permit the formulation of a theory of either the kinetics of oxidation or of reduction in living cells. In connection with the fact, which seems quite well established, that a small amount of oxygen is essential for the reduction of carbon dioxide in the photosynthetic process, similar conditions which have been observed in the catalytic hydrogenation by means of platinum are of interest. Adams and others ^^-^ have called attention to the fact that oxygen is essential to the activity of platinum black as a hydrogena- tion catalyst, though the role of oxygen in these reactions is not yet definitely established. 2*^ Pringsheim, Sit::b. prcuss. Akad. Wiss., 1887, 763. Friedel, Compt. rend., 131, 477 (1900). ''== Pringsheim, Jahrb. wiss. Bot., 17, 178 (1886). ''"■ Carothers and Adams, Jour. Am. Clicin. Soc, 47, 1047 (1925). Waldschmidt- Leitz and Seitz, Ber. Chcm. Ges., 58 B, 563 (1925). 164 PHOTOSYNTHESIS It does not seem pertinent to follow Pringsheim's further deductions. Rut his experimental results indicate this fact. That a normal cell con- taining chlorophyll in an apparently unchanged condition and exposed to bright light and an atmosphere containing carbon dioxide is incapable of photosynthesis in an atmosphere free of oxygen. It appears, there- fore, that besides the conditions of light, chlorophyll, temperature, and carbon dioxide supply, other conditions are essential and these are inti- mately associated with the respiration of the protoplasm. The latter is an important internal factor affecting photosynthesis, in addition to the chlorophyll-content and the number of chloroplasts. The exact manner in which these two processes of respiration and photosynthesis may be linked is still an open problem. There have been observed some quantitative relations between photo- synthetic activity and respiration. The leaves of the light-green or yellow varieties of the plants investigated by Plester ^^^ showed a lower rate of photosynthesis than the varieties rich in chlorophyll. Similarly the former had a lower rate of respiration. Thus, while it is evident that there is no parallelism between chlorophyll-content and rate of photosynthesis, the quotient respiration — photosynthesis is more constant. This quotient for the light-green varieties was found to be as follows: Ptelea= 1.77. Catalpa—1.72, Mirabilis = 2.0, Ulmus = 2.0, Populus = 2.1, Atiplex =. 1.3. The latter plant exhibits rather erratic photosynthetic rates. A correlation between respiration and photosynthetic rates was also noticed by Henrici.^^* She found in a study of alpine and low land plants, that those plants which had a high photosynthetic rate also had a high rate of respiration and vice versa. Boysen-Jensen ^^^ found that plants which have a high rate of respiration also have a high rate of carbohydrate formation. Similar results have also been obtained by Spoehr and McGee."' Kny^®" has shown that photosynthesis soon ceases when the chloro- plasts are separated from the cytoplasm of the cell, although there appears to be no direct parallelism between injury to the cytoplasm and photo- synthetic inhibition. Disorganization of the cell nucleus is not detrimental to photosynthetic activity. Ewart^'*' has also made observations on photosynthesis as affected by an atmosphere of hydrogen and carbon dioxide. Even in mosses, which show this effect more slowly than other plants, stoppage of photo- synthesis finally follows exposure to such an atmosphere. When oxygen is made available the plants recover their photosynthetic ability if they have not been kept in the mixture of hydrogen and carbon dioxide too long. ^"Plester, Beit. Biol, dcr Pnanzcn., 11, 249 (1912) _ . • », tt- '**Henrici "Chlorophyllgehalt und Kohlensaure-Assimilation bei Alpen-hben- pflanzen," Inaug. Diss., Basel, 1918, P- 90. '^^Boysen-Jensen, 5ofam5^ r((fj.y" Kny, Ber. bot. Ges., 15, 388 (1897). ""Ewart, Linnean Soc, 31, 403 (1897). THE NATURE OF PHOTOSYNTHESIS 165 Willstatter and Stoll ^^^ found that various plants exhibit a wide varia- tion in their resistance to lack of oxygen. It is perhaps not without significance that the mosses which are resistant to the absence of oxygen have no stomata and hence the gaseous interchange with the atmosphere is more difficult. According to Willstatter and Stoll the partial pressure of oxygen can be reduced to one hundredth of that in air without disturb- ing photosynthesis if the rest of the atmosphere is nitrogen. After com- plete displacement of oxygen for two hours the leaves were no longer capable of photosynthesis on illumination. This is quite in agreement with the older observations. Under these conditions the leaves show no visible signs of injury. After exposures to an oxygen-free atmosphere of shorter duration (one hour) some plants as Cyclamen curopacuni, poly- trichum juniperinmn, the photosynthetic activity is decreased but not entirely inhibited. Of special interest are the observations that the plants just mentioned when kept in an oxygen-free atmosphere for 15-24 hours, on illumination, show no photosynthesis, but after about 30 minutes begin to evolve oxygen and increase in this activity to a high rate. The long continued exposure to an oxygen-free atmosphere is not without permanent injurious effect on the photosynthetic mechanism ; the plant does not again attain its original photosynthetic rate. The longer the exposure to an oxygen-free atmosphere, the lower is the subsequent rate of photo- synthesis and the more incomplete is the recovery. Lack of oxygen there- fore produces a permanent injury of the protoplasm; the degree of this injury depending upon the length of time the plant is deprived of oxygen and the individual constitution of the plant. Willstatter and Stoll conclude that oxygen is absolutely essential for photosynthesis, but that a very small quantity of oxygen suffices for sup- plying the photosynthetic apparatus. Moreover, they consider that free oxygen is not necessary but that oxygen held in an easily dissociable form can do the work. The conception of oxygen loosely bound in the plants is largely based upon those cases which were kept in an oxygen-free atmosphere and on illumination again recovered their photosynthetic activity. It is suggested that oxygen is removed from the plant in two stages: 1, the replacement of the free oxygen by an oxygen-free at- mosphere, and 2, the removal of loosely bound oxygen, through the dis- sociation of an oxygen compound when the oxygen tension of the atmos- phere becomes less than that of this hypothetical compound. The second stage is slower than the first. Hence in plants which have lost oxygen chiefly by the first stage, a recovery of photosynthesis is possible on illumi- nation. Only the more resistant plants can withstand the second stage. On the basis of their work on the relation of chlorophyll-content to photosynthetic rates, Willstatter and Stoll came to the conclusion that there is no direct ratio between these two. Besides chlorophyll as an internal factor influencing the rate of photosynthesis, there is, according "* Willstatter and Stoll, "Untersuchungen iiber die Assimilation," Berlin, 1918. p. 349. 166 PHOTOSYNTHESIS to this conception, also an enzymatic agent. This latter protoplasmic factor is contained in hoth the chlorophyll-rich and light-green or yellow varieties in about the same amount. Temperature affects primarily this factor. Now the temperature coefficient of photosynthesis is considerably higher in leaves rich in chlorophyll than in leaves poor in chlorophyll. This Willstatter and Stoll explain on the basis that an increase in tem- perature and consequent augmented activity of the protoplasmic factor, can result in a higher rate of photosynthesis only under conditions in which the other steps in the photosynthetic process are also raised, or at least are not lower than the ones determined by the enzymatic factor. That is, it must be possible for the plant to absorb enough light to supply the greater energy necessary to carry on these other steps at a rate which is equal to that of the enzymatic factor, now augmented by a rise in tem- perature. Therefore, higher temperature can result in a higher rate of photosynthesis only if there is sufficient chlorophyll present which can absorb this increased amount of energy required. These latter conditions are met only in leaves rich in chlorophyll. In leaves poor in chlorophyll the activity of the enzymatic factor may also be raised by higher tempera- tures but it will not result in a total increase in photosynthesis, because the leaf cannot absorb sufficient energy to increase the rate of the purely photochemical steps in the series of reactions comprising photosynthesis. Willstatter and Stoll found that etiolated leaves after they had de- veloped a fraction of the chlorophyll-content of normal leaves surpassed the latter in photosynthetic activity. They assume that this higher activity of etiolated leaves is due to a greater development of the internal enzymatic factor. It is possible, on this assumption, that light is not so favorable for the development of this enzymatic factor as darkness or at least in high intensity exerts an inhibiting effect. Although there is hardly suffi- cient evidence to permit of speculation, is it not possible that if light does exert an inhibiting effect on this internal factor, that the "time factor" of photosynthetic activity may be explained on this basis? Also it may be possible that the fatigue effects, described by Pantanelli and discussed above, are to be explained on the ground that light, while essential for the photochemical reactions, is injurious to the protoplasm in high illumina- tion intensities. The deleterious effects of light, particularly the blue- violet rays, on enzymes and proteins have been repeatedly demonstrated.^^" In fact Ewart^^^ has shown that "it is possible to produce a condition of permanent light rigor, i.e., death, in the chlorophyll grains over an exposed region of a cell of Chara without affecting the vitality of the cell, i.e. the plasma of the chlorophyll grain appears to be more sensitive than the general protoplasm of the cell." ^Reynolds-Green, Trans. Rov. Soc. London, 188, 167 (1897). Emmerling, Ber. chem Ges., 34, 3811 (1901). Chauchard, Conipt. rend., 158, 1575 (1914). Pin- cussen, Biochem. Zeit., 134, 457 (1922). Schanz, Arch. Ges. Physiol, 164, 445 (1916). ^^Ewart, /. c, 443. THE NATURE OF PHOTOSYNTHESIS 167 The high photosynthetic rates observed by Willstatter and StoU in leaves poor in chlorophyll make Pringsheim's protective theory very im- probable. Pringsheim/'-*- starting from the erroneous hypothesis that di- rect light accelerates respiration, developed the theory that chlorophyll acts as a protection against light. He supposed that light might induce photo- synthesis in colorless cells as well as those containing chlorophyll ; the pigment acting as a protection against light and permitting the reaction to become more prominent in the green cells than in the colorless ones. The theory is quite untenable, for the cells containing chlorophyll are, if anything, more sensitive to light than the colorless ones. From the fatigue effects of Pantanelli, the inhibition experiments of Ewart and others and the disproportionality betvi^een chlorophyll-content and photosynthesis as demonstrated by Willstatter and Stoll, it is apparent that the protoplasm of the cell plays an important role in the process. Now, of course, it must be realized that the nature of this protoplasmic factor or enzyme has not been established. Willstatter and Stoll consider it probable that it behaves as a dissociable oxygen compound (dissozierbare Sauerstoft'verbindung) . In relation to the question of the dependence of photosynthesis on protoplasmic activity mention should be made of the attempts which have been made to detect photosynthetic activity in dead leaves. The first reports regarding this subject were not clear and decidedly contradictory. Frie- deP^^ was the first to claim that photosynthesis was possible in dried leaf material to which water had again been added, but was himself unable to substantiate his first findings. Recently Molisch has again made a very careful study of this problem. For the detection of photosynthesis he used a very sensitive luminescent bacteria method to the technique of which he had previously made many valuable contributions. In the earlier ex- periments on the photosynthetic activity of dried leaves, the latter were ground in water and the mixture was then filtered. JMolisch altered the procedure so that the entire paste was used ; this proved to be an impor- tant point, for material prepared in this manner after being kept in the dark for a time and subsequently illuminated, even for a very brief period, showed the evolution of oxygen. The leaves were dried at 30 to 35° for 3 to 4 days and in some cases were kept in a desiccator over calcium chloride or sulphuric acid for weeks. Some leaves which had been heated to 84° for 5 hours still showed the capacity to form oxygen, but the leaves could not endure a temperature of 96° for one hour. When heated to higher temperatures in the moist condition the leaves lose the capacity to form oxygen in the light. Similarly, if the leaves are treated with ether and dried they no longer are able to form oxygen. Leaves which have been killed by freezing are also able to emit oxygen when illuminated. "' Pringsheim, Jahrb. iviss. Bot., 12, 288 (1882). ^'"Friedel Compt. rend.. 132. 1138 (1901). Harroy. ibid.. 133, 890 (1901). Herzog, Zeit. phvsiol. Chcm., 35. 459 (1902). Molisch, Bot. Zeitg., 62, 1 (1904); Zeit.f. Bot. 17, 577 (1925). 168 PHOTOSYNTHESIS Dried leaves can be kept for a long time, up to three months, without completely losing the capacity for oxygen formation in the light. That chlorophyll is essential for the formation of oxygen in the light by killed leaves is shown by the fact that etiolated leaves do not possess this prop- erty, although the amount of chlorophyll that is necessary is exceedingly small. Molisch demonstrated this phenomenon with a wide variety of plants. Some plants with a relatively high acid content on being dried and tested for oxygen evolution gave very slight or negative results. When the acids are neutralized during the preparation of the leaf material positive results are obtained, and it is not improbable that the negative results in the first case are to be attributed to the effect of the acid on the chlorophyll during the process of drying. Molisch regards that these experiments on the oxygen production of killed leaves in the light, demonstrate the existence of an enzyme which is essential for the photosynthetic process ; that the life of the plant is not necessarily essential for the photosynthetic activity any more than the living yeast plant is essential for the fermentation process. While the results of Molisch perhaps do not entirely remove the photosynthetic process from the living activity of the plant to the same degree that the isolation of zymase did for the process of alcoholic fermentation, they nevertheless are a distinct contribution toward establishing the enzymatic nature of photosynthesis, a trend which has already been fostered by the results of Willstatter and Stoll, Pringsheim and others. Returning to the influence on photosynthesis of lack of oxygen, it is apparent that this is largely one of degree; some plants are easily per- manently inhibited while others withstand absence of oxygen for long periods. All show some injury. In viewing these facts critically it is well to bear in mind that it is extremely difficult to free any plant from the last traces of a gas. Plants differ greatly in their rate of oxygen consumption as well as in the rate of gaseous interchange with the sur- rounding atmosphere. Also it is difficult to free a gas from the last traces of oxygen; this is apparently impossible by the use of glowing copper. Moreover, the injurious effects of lack of oxygen may well be due to injury of the protoplasm caused by the accumulation of toxic substances. It is well known that plants deprived of oxygen continue to consume carbohydrates, but that the course of metabolism is greatly altered, usually resulting in the formation of ethyl alcohol. Different plants vary in the rate of alcohol formation. So also does the protoplasm of different plants vary in resistance to toxic substances, such as alcohol, produced by the plant itself, or administered from without. Apparently photosynthesis is more sensitive to protoplasmic disturb- ances than is respiration. Thus Wurmser and Jacquot ^^* found that when certain marine algae were subjected to higher temperatures (36° to 45°) '^^ Wurmser and Jacquot, Bull. Soc. Chim. biol, 5, 305 (1923) ; 6, 169 (1924). THE NATURE OF PHOTOSYNTHESIS 169 for from 1 to 15 minutes, the rate of photosynthesis was always depressed when the plants were returned to the temperature of their previous environ- ment, 16°. The depression is greater the higher the temperature and the longer the exposure to the higher temperature. Similar effects were produced with glycerine, which are taken to show that the photosynthetic apparatus is more delicate than that of respiration. Warburg ^^^ has also shown that the photosynthetic rate is reduced by hydrocyanic acid and urethanes of extreme dilutions in which the respiratory activity is not affected or in certain cases is even stimulated. The effect of narcotics on photosynthesis is discussed in Section i. of this chapter. Warburg ^^*' found that, while respiration is not influenced by different partial pressures of oxygen (probably above a certain minimum) photo- synthesis is less at higher pressures. This decrease in the photosynthetic rate is noticeable only at high illumination intensities. A change in the partial pressure of oxygen from %o to 1 atmosphere reduces the photo- synthetic rate by about one-third. From this it would appear that the reaction nCOa + nHoO -^ (CH20)n + nOa, can be checked by a higher concentration of oxygen. This may be of considerable significance in determining the kinetics of the reaction and should be investigated more fully. In the discussion of the temperature coefficient of photosynthesis at- tention has already been called to the fact that the photosynthetic process is composed of two reactions. One of these is a photochemical reaction with a low Qio, the other an ordinary chemical reaction with a Qio of about 2, in approximate agreement with the van't Hoff rule. There is a variety of evidence which is in harmony with this conception. It is the ordinary chemical reaction which is associated with the protoplasmic ac- tivity of the plant. It is this reaction which is affected by changes in temperature and is sensitive to narcotics. The nature of this reaction is not known, but has been the subject of considerable speculation. War- burg ^^' has called this reaction the "Blackman Reaction" and advanced a theory that the reaction resulted in the formation of a photochemical acceptor, a compound of carbon dioxide sensitive to light. Willstatter and Stoll proposed the formation of a peroxide in the reduction of carbonic acid, and that the splitting off of oxygen from the peroxide constitutes the "Blackman Reaction." Later Warburg considered that his original theory was not tenable and on the basis of measurements of the inhibiting action of certain narcotics on the decomposition of hydrogen peroxide by Chlorella supported Willstatter and Stoll 's theory. This phase of the "= Warburg, Biochem. Zeit., 100, 264 (1919) : 103, 196 (1920). ^Warburg, /. c, 103, 193 (1920). "nVarburg, Biochem. Zeit., 100, 230 (1919) ; 103, 188 (1920) ; 146, 486 (1924). Willstatter and Stoll, "Untersuchungen iiber die Kohlensaure Assimilation," 1918, p. 395. 170 PHOTOSYNTHESIS photosynthesis problem requires still a great deal more investigation before any definite conclusion can be arrived at. Finally, as internal factors which affect the rate of photosynthesis there come into play the position of the chloroplasts, the accumulation and translocation of the material synthesized. These are discussed in the sections devoted to chloroplasts and to the time factor. i. Effect of Various Substances, of Age, Electricity, etc. It has long been a question vi^hether other substances could be substi- tuted for carbon dioxide in the photosynthetic process. There are a num- ber of substances which when fed to plants produce starch, but most of these are of practically an equal energy content as hexose sugars, so that radiant energy is not necessary and the starch formation is not a photo- synthesis. We are concerned first with those substances which require reduction, in which the reaction step to sugar is endothermic and hence would require the absorption of energy. The first to suggest itself in this relation is the lower oxide of carbon. Carbon monoxide: de Saussure found that the behavior of plants in this gas is the same as in an atmosphere of nitrogen. ^^^ There were no evidences of photosynthesis. Virtually the same conclusions were arrived at by Boussingault'^^^ and by Stutzer.^oo Richards and MacDougaP°^ found that larger quantities of carbon m.onoxide are toxic for phanerogams. Krascheninnikofif also found that it could not be used, while the results of Bottomley and Jackson -''- indicate that this gas can, to a measure at least replace carbon dioxide in photosynthesis. Recently Wehmer ^^^^ has reported that carbon monoxide up to a concentration of 50 per cent exerts no injurious effect on the development of seedlings. Formic Acid: Usher and Priestley 2°* report the formation of oxygen from Elodca in 0.02 per cent solution of this acid, on illumination. Spoehr ^o^ has also found that in an atmosphere containing small quantities of formic acid but no carbon dioxide, leaves of the sunflower show an appreciable gain in dry weight and form starch in bright sunlight. Plants cannot endure high concentrations of this acid, and in the dark the toxicity soon becomes evident. Ketene, CH2 : CO, and Carbon Suboxide C3O2, have as far as we know not been investigated and on account of their poisonous and reactive properties probably offer little of interest. Collie '°« suggested that deriva- "'Ostwald's "Klassiker d. Exakten Wissenschaften," No. 16, p. 27. ""' Boussingault, "Agronomic chimic agricole," etc., 4, 300 (1868). ''" Stutzer, Ber. chcm. Gcs., 9, 1570 (1876). Just, IValhiys Forsch. Agnk. pliysik., 5 79 (1882). Krascheninnikoff, Rev. gen. bot.. 21, 173 (1909). *"' Richards and MacDougal, Bull. Torrev Bot. Club, 31, 57 (1904). ="' Bottomley and Jackson, Proc. Roy. Soc, 72, 130 (1903). ='"' Wehmer, Ber. bot. Gcs., 43, 184 (1925). ==»' Usher and Priestley, Proc. Rov. Soc, 78 B, 322 (1906). '"•^Spoehr, Plant World, 19, 15 (1916). ^Collie, Jour. Chcm. Soc, 91, 1806 (1907). THE NATURE OF PHOTOSYNTHESIS 171 tives of these substances may play an important role in the formation of organic compounds found in plants. Formaldehyde: This substance has played a xtvy important role in the theoretical discussions of the photosynthetic process. The behavior of plants toward formaldehyde is considered in Chapter 5. Anesthetics: The inhibiting efifect of chloroform on the photosyn- thesis of aquatic plants was first described by Claude Bernard and has been repeatedly verified. If the anesthetization is not continued too long permanent injury to the chloroplasts can be avoided and they regain their activity. Bonnier and Mangin,-"' like Bernard,-"* showed that photosyn- thesis is more sensitive to the action of ether and chloroform than is respiration and that by carefully regulating the quantities of these sub- stances administered, a separation of the two processes can be attained. Thus it is possible to stop photosynthesis and allow the respiration to con- tinue. The separation is, however, not a very sharp one, for with the amounts of ether or chloroform required to stop photosynthesis, the rate of respiration is also afifected : either it is somewhat inhibited or with small quantities of the anesthetic it is accelerated. Dift'erent species of plants show a wide variation in their reaction to ether and chloroform. A thor- ough investigation of the effect of anesthetics has been made by Irving. From this it is apparent that small doses of chloroform affect the gaseous exchange so that light produces little effect thereon. Very small concen- trations of ether and chloroform exert a stimulating influence on respira- tion, but it is very doubtful whether any such acceleration occurs in photo- synthesis. From the investigations of Willstatter and Stoll it is apparent that quantities of chloroform which inhibit the rate of photosynthesis effect a decomposition of the chlorophyll in leaves of Prunus laurocerasus. With ether, while recovery of the photosynthetic activity is not complete, there is apparently no appreciable alteration in the chlorophyll and caroti- noid components. Narcotics: morphine, cocaine, and quinine, inhibit photosynthesis. Treboux reports a 0.005 per cent solution of quinine hydrochloride of very slight effect, while a 0.15 per cent solution of the same salt inhibits completely.-"^ Antipyrene is equally poisonous. Warburg has investi- gated the effect of urethanes. The inhibiting action of a homologous series depends apparently upon the absorption of these substances. Photo- synthesis is far more sensitive to these substances than respiration. The latter is stimulated by weak solutions, which inhibit photosynthesis. The concentrations affecting respiration are about twenty times as strong as those affecting photosynthesis. The following table from Warburg illus- *" Bonnier and Mangin, .hni. Sci. Xat. (7), 3, 14 (1886). Schwartz, Untcrs. a. d. Bot. Inst. Tiibingcn, 1, 102 (1881). Ewart, Jour. Limican Soc, 31, 408 (1896). Irving, Ann. Bot.. 25, 1077 (1911). Bose, "The Physiology of Photosynthesis," London, 1924, p. 60. *" Bernard, "Legons sur les phenomenes de la vie," 1878, p. 278. ^"Jacobi, Flora. 86, 323 (1899). Treboux, ibid.. 92, 56 (1903). Warburg, Bio- chetn. Zeit., 100, 264 (1919); 103, 196 (1920). 172 PHOTOSYNTHESIS trates this difference ; the experiments were carried out with high carbon dioxide-concentration, high hght intensity, and at 25 : Photosynthetic Respiration Inhibition of 50 Inhibition of 50 Per Cent by milli- Per Cent by milli- Narcotic moles per liter moles per liter Methyl-urethane 400 1200 Ethyl-urethane 220 . 780 Propyl-urethane 50 100 Butyl-urethane (iso) 17 43 Amyl-urethane (iso) 12 32 Phenyl-urethane 0.5 6 Inorganic Compounds: Some inorganic salts, even in great dilution, have a very decided effect on photosynthesis. The toxicity of solutions is not only a matter of the concentration but also of the total amount of the toxic salt available. For example, Treboux -" found that twigs of Elodea in 100 cc. of 0.000015 per cent CUSO4, 0.00016 per cent ZnSO^ and 0.00015 per cent C0SO4 were but very slightly affected; in 500 cc. of solutions one-tenth these concentrations, the plants were soon killed. The salts just mentioned, and those of mercury, are very toxic. The inhibiting effect on photosynthesis decreases gradually with concentration and appears to be about the same as the toxicity for the plant cells. Ferrous sulfate and ferric chloride, in not too dilute solutions, accelerate photosynthesis. The action may be due to the fact that these solutions have an acid re- action. Mercury vapor is very toxic. Briggs ^^^ studied the effect of a deficiency of various mineral nutrients on photosynthesis. In cultures lacking one of the essential elements, namely, potassium, magnesium, iron and phosphorous, photosynthetic activity of Phaseolus vulgaris was decidedly less than in leaves grown in solutions with all the essential nutrients present. The same depression was observed whether external conditions such as light or temperature were limiting factors. Briggs endeavored to explain this on the basis that a lack of any of the essential elements resulted in a reduction of the re- active surface of the chloroplasts ; the reactive surface affecting both the temperature and light reactions. Briggs also found that the photo- synthetic activity of a plant grown in what is usually considered a com- plete mineral nutrient solution to be less than that of a plant grown in soil. Very little is known regarding the role of mineral nutrients in photosynthesis. ^^^ Galwialo ^^^ reports that in leaves which have been cut into pieces and placed into water containing carbon dioxide, the rate of photosynthesis soon decreases and that photosynthesis can be revived by the addition to '•"Treboux, /. c, 56. Rumm, Bcr. hot. Gcs., 11, 79 (1893). Frank and Kruger, ibid., 12, 8 (1894). ^Briggs, Proc. Roy. Soc, 94 B, 20 (1922). "^ Stoklasa and 'Matousek, "Beitrage zur Kenntniss der Zuckerriibe," Jena, 1916. Andre, Cornpt. rend.. 162, 563 (1916). "' Galwialo, Biochnn. Zeit., 158, 65 (1925). n3 ; THE NATURE OF PHOTOSYNTHESIS 173 the water of salts extracted from roots of the plants. He concludes that there are two elements necessary for photosynthesis, electrolytes and a ferment, that in a mixture of these components from the plants (maize, pea, bean, potato) and tlie electrolytes in water solution a reduction of carbon dioxide in sunlight is attainable. In the absence of more exact experimental data it is, however, difficult to judge of the value of these results. Acids and Alkalies: \>ry dilute solutions of acids (0.0001 N.) exert a stimulating effect on photosynthesis. Treboux -^* found that this was the case with HCl, HXO3, H2SO4, CrOs, H3PO4, acetic, succinic, oxahc, tartaric and citric acids, as well as with KHSO4 and KH2PO4. Higher concentrations are inhibitory. Bose reports that one part of HNO3 in two billion parts of water caused an increase of nearly 200 per cent in the rate of photosynthesis. This author has also observed a stimulating effect of traces of iodine and of the extract of thyroid gland. The effect of alkalies on the photosynthetic process is complicated because this becomes involved in the carbon dioxide-concentration avail- able to the plant. Potassium Cyanide in very dilute solutions produces inhibition of photo- synthesis in aquatic plants. Lund and Holt ^^^ found complete but re- versible inhibition in 0.00008 molal solution with fronds of the Pacific Coast kelp Nereocystis. Higher concentrations (0.0006 molal) produce permanent injury in the light, but when the plants are kept in these solu- tions in the dark no permanent injury was observed. Warburg also ob- served that the photosynthetic activity is affected by much lower concen- trations of cyanide than is respiration. He found reversible inhibition of photosynthesis by Chlorella in a 0.0001 normal solution of prussic acid while respiration was not affected by a 0.01 normal solution. War- burg further reports that while his algae were very sensitive to low con- centrations of c}^nide in the absorption of carbon dioxide, the cyanide did not prevent the utilization of the carbon dioxide produced in respira- tion. This fact is brought out when different light intensities are used : with high light intensity the rate of photosynthesis normally greatly ex- ceeds that of respiration, and in a 0.05 normal HCN solution photosyn- thesis is greatly inhibited. As the light intensity is decreased, and the rate of photosynthesis consequently also decreases, an illumination intensity is reached at which photosynthesis equals respiration (compensation point). At this point all the carbon dioxide produced in respiration, but no more, is utilized in photosynthesis. A 0.05 normal solution of HCN does not affect this photosynthesis. In fact, according to Warburg, the degree of inhibition of cyanide solutions depends greatly upon the intensity of light. In a 0.0001 normal cyanide solution with high light intensity ="^ Treboux, /. c, 65. Wider and Hartlieb, Ber. hot. Ges., 18, 348 (1900). Bose, /. c, 64. "'Lund and Holt, Proc. Soc. Expt. Biol, and Med., 20, 232 (1923). Warburg, Bxochem. Zeit., 100, 267 (1919) ; 103, 199 (1920). 174 PHOTOSYNTHESIS (about 19,000 Lux) photosynthesis was inhibited 65 per cent. The same concentration of cyanide with weaker Hght (1800 Lux), which normally utilized slightly more than the carbon dioxide that was produced by respira- tion, did not inhibit photosynthesis. Warburg has made use of these observations in his theoretical deductions. Further investigation of the sub- ject with other methods and plants seems desirable. Age: For a long time it was thought that leaves showed active photo- synthesis only after they had attained a certain size and condition of de- velopment. While different species of plants vary greatly in their rate of development it is highly probable that the foregoing conclusion is erroneous. Undoubtedly the development of the capacity for photosyn- thesis depends very much upon the conditions of temperature and light under which the plants are growing, yet it appears from the resuhs of Willstatter and Stoll that photosynthesis is quite high in leaves which are just unfolding. Young leaves have an exceedingly high rate of res- piration which decreases to one-quarter of this rate when the leaf ma- tures. The results of photosynthesis measurements depend in many in- stances upon a proper determination of respiration and such results vary according as to whether they are calculated on the basis of area, fresh weight or dry weight. Willstatter and Stoll's experiments show that the rate of photosynthesis after about nine days of growth is almost constant for equal areas of leaf surface. Calculated on the basis of dry weight and in some cases on the basis of fresh weight, the rate of photo- synthesis decreases with age. On the basis of chlorophyll-content there is a decided decrease in photosynthetic activity. It is important, there- fore, to consider carefully on what basis photosynthesis measurements are determined and also the methods which are employed in making such measurements. Willstatter and Stoll also compared young leaves with those of the previous year of Laurus nobilis and of Taxus baccata and found that on the basis of fresh weight there was little difference, while on the basis of dry weight the older leaves had a lower photosynthetic rate. In the autumn, when the leaves are just beginning to change in color, on the basis of dry or fresh weight as well as on the basis of area, they show a decided decrease in photosynthesis. It is, however, hazardous to draw any very general conclusions regarding the effect of age and develop- ment on the photosynthetic activity of plants. The marked individual and specific peculiarities of different species together with their adaptive modifications resulting in differences of structure as well as composition of the leaves become evident in photosynthesis. In general, functional activity decreases with age, but owing to the complication of factors which is involved in producing this diminished activity, a quantitative expression of the causes underlying it is as yet impossible."^'^ ^'Ewart, ;. c, 452. Kreusler, Landii>. Jahrb., 14, 913 (1885). Willstatter and Stoll, /. c, 86. THE NATURE OF PHOTOSYNTHESIS 175 Electricity : For more than a century the question of electro culture and the influence of electric currents on the production of crops has, in the hands of different investigators, been the subject of almost continuous investigations. Few of these researches have been devoted to the more immediate causes of the reported acceleratory action of electric currents on the grovi^th of plants and the production of crops. There are a num- ber of general theories, such as that of Lemstrom, that the electric cur- rent "produces an augmentation of the energy with which the circula- tion of the juices is going on." Also the acceleratory action has been variously ascribed to the formation of nitric and nitrous acids, ammonia and ozone. Relatively little work has been done on the effect of electric currents on photosynthesis. Thouvenin has reported a decided acceleration of the oxygen evolution by some aquatic plants in the light. Several Daniel's cells in series were connected so that the two poles were at opposite ends of a branch of Elodea. A current of 0.0005 to 0.001 ampere increased the rate of oxygen emission taking place in the light. Plants in which the photosynthesis was inhibited by chloroform were not affected by the electric current. Pollacci reported an acceleration of starch formation in land plants under the influence of direct current. His conclusion that the electric current can replace radiant energy is of great interest, but probably needs further confirmation. The direction of flow of the current through the plant is apparently also of significance. The entire subject is in need of thorough working over with a view of coordinating and ex- tending the isolated observations and of establishing more accurately all the chemical and electrical conditions involved.-^' Henrici ^" has made the interesting observation that the rate of photosynthesis is influenced by the degree of ionization of the atmosphere. In her experiments the air containing carbon dioxide was either discharged or its conductivity increased by means of thorium oxide. The rate of photosynthesis is greatly accelerated in an atmosphere of high conduc- tivity. Under favorable conditions the rate of photosynthesis in ionized air is 1.5-4 times that in discharged air. Thus, in an atmosphere of high conductivity it is possible to obtain a rate of photosynthesis with a light intensity which in unionized air produces no photosynthesis. How- ever, no matter how great the conductivity, it is not possible to obtain photosynthesis without light. The influence of the atmospheric ioniza- tion is apparently closely associated with the carbon dioxide in the air. Individual plants and different species exhibit a wide variation in their reaction to ionized and unionized air and a great deal more experimental "' Lemstrom, "Electricity in Agriculture and Horticulture," London. Thouvenin, Rev i/cii bat.. 8, 432 (1896). Pollacci. Atti. tst. Bnt. Paria (2), 13 (1907); Bot. Cent.' 99, 544 (1905). Koltonski. Beih. Bot. Cent.. 23, 1, 204 (1908). Bo.se. "Physiology of Photosynthesis," p. 72. Knv, Ber. bot. Ges.. 15, 398 (1897). Waller, Attn. Bot., 39, 516 (1925). ^Henrici, Arch. Sci. Phy. Nat. (5). 3. 276 (1921). Spoehr, Bot. Gas., 59, 366 (1915). 176 PHOTOSYNTHESIS work is necessary in order to employ these observations for the formula- tion of a theory of the rate of atmospheric ionization in photosynthesis. 3. The Compensation Point The light intensity at which the respiratory and photosynthetic activities compensate each other, i.e., where there is an equilibrium, or steady state, CnHonOn + uO. ^ uCOo + uHoO, and the gaseous exchange is conse- quently zero, has been designated by Plaetzer ~'^^ as the compensation point. As this point is dependent upon the rate of respiration it varies greatly in dififerent plants. Plaetzer studied only acj[uatic plants and em- ployed the bubble counting method with the precautions prescribed by Kniep for plants with an intercellular system. (See Chapter 4.) In using plants without an intercellular system the titration method of Winkler was employed for determining the oxygen in the water. The efifect of temperature on the compensation point as determined by Plaetzer is of considerable interest. It appears that with decreasing tem- perature the compensation point is lowered. Thus, the compensation point, expressed in Hefner candles, changes with temperature as follows: Spirogxra. 174 at 20° ; 26.7 at 5°. Fontinalis, 150 at 20° ; 40 at 5°. Cladofhora, 253.3 " " ; 62.9 " " Cinclidotus. 400 " " ; 75 " " It would appear from the above that with a given light intensity photo- synthesis increases with decreasing temperature. In fact, Plaetzer was able to demonstrate that a light intensity which at 20° represented the compensation point of Cinclidotus, at 5^ produced a decided evolution of oxygen due to photosynthesis. That is, with the same light intensity which at 20' just balances the energy transfers, at 5° there is a gain for the plant. It should be noted, however, that the actual total rate of photosynthesis at 5° is lower than at 20", but in relation to the respira- tion it is higher. Paradoxical as this appears at first glance, it must be clearly borne in mind that we are dealing here with light of low intensity, i.e. at these temperatures the light is the limiting factor. It must be re- called again that in photosynthesis the three chief external factors, any one of which may, according to circumstances, be "limiting"' are carbon dioxide-concentration, light intensity and temperature. Now then, given an ample supply of carbon dioxide, if a plant at a definite temperature, is exposed to a light intensity just sufficient to surpass the compensa- tion point, oxygen will be emitted. If the light intensity is increased, oxygen emission (photosynthesis) will also increase; if the temperature is increased photosynthesis will not increase. That indicates that the intensity of illumination — not the temperature — determines the rate of photosynthesis, i.e. light is the limiting factor. If the light intensity is further increased, a point will be finally reached at which photosynthesis, even with the most intense light, will not increase. Here light is no '""Plaetzer, Vcrhandlungen Physik-Mcd. Gcs. IVurzhurg N. F., 45, 31 (1917). THE NATURE OF PHOTOSYNTHESIS \77 longer the determining or limiting factor. At this point (still provided there is ample carbon dioxide) temperature becomes the determining fac- tor and an increase in temperature increases the rate of photosynthesis. The results of Plaetzer on the compensation point must be regarded in the light of these facts. The observation that photosynthesis de- creases vi^ith increasing temperature can hold only for a limited range of light intensity. When this is increased so that it no longer is a limiting factor at the lower temperature, but rather the temi^erature constitutes the limiting factor, then photosynthesis will increase with increasing temperature. Perhaps a clearer insight of the phenomenon of apparent decreasing photosynthesis with increasing temperature when light is the limiting fac- tor can be obtained from Plaetzer's quantitative data. Let i represent the light intensity which at 5" produces compensation between respiration and photosynthesis. It will be recalled that at 20° i no longer is the com- pensation point and that with light intensity i at 20°, carbon dioxide is given ofif by the plant, i.e., oxygen is taken up and respiration overbalances photosynthesis. The question then arises, is the absolute amount of oxygen produced in photosynthesis with light intensity / at 5° the same as at 20°. The following simplified data answer the question: a = respiration (CO, emitted) at 20° in the dark. b = respiration (CO2 emitted) at 5° in the dark, i.e., the respiration which at the compensation point i is equal to photosynthesis, hence also b = photosynthesis at 5°. c = oxygen consumed at 20° under the influence of light intensity i. Then a = b + c if the light intensity is the limiting factor. If this equa- tion does not hold the action of / is different at 5° than at 20°, i.e., greater or less. Plaetzer's results are : a = 4.3 b = 1.38 c = 2.97 b + c = 4.35. In the equation a r= b + c the assumption is, of course, made that the rate of respiration is the same in the dark as in the light. This is still a debated question and no conclusive evidence is as yet available. If i has the same influence, x, on respiration at 5° and at 20°, then c would remain unchanged, for (a -f x) — (b + x) = c. The nature of x is still unknown, though it is possible that the difference between a == 4.3 and b + c = 4.35 indicates a slight stimulation of respiration. The main result shows quite definitely, however, that with light constant and the limiting factor, a change in temperature does not alter the absolute rate of photosynthesis, while respiration is directly influenced. 178 PHOTOSYNTHESIS Plaetzer furthermore showed with CladopJwra that with increasing temperature, the compensation point (i.e. light intensity required to main- tain respiration — photosynthesis at an equiUbrium) rises more rapidly than the rate of respiration, determined in the dark. Thus, with an increase in temperature from 5° to 25° the rate of respiration showed a proportionate rise of 1 : 4.8. The light intensity required to maintain the compensation point over this same range of temperatures showed a corresponding proportion of 1 : 6.69. The explanation of this phenomenon is in all probability not a simple one. Aside from the possibility (not yet established positively) that light has a stimulating action on respiration, it would appear that photo- synthesis, under these conditions, did not increase in proportion to the increase in light intensity. Such a direct proportionality could exist only under conditions where light was the limiting factor. Now it is highly probable that with the increasing light intensity a point was reached where light was no longer the limiting factor and temperature played the role. Since it has been found that with high illumination and high carbon dioxide-concentration the temperature coefficient of photosynthe- sis decreases with increasing temperature (4.3 at 5° to 1.6 at 32°) it would be expected that the rate of photosynthesis would not increase at the same rate as respiration with increasing temi>erature. From the re- cent work of Harder, ^^° it is apparent, moreover, that where one factor ceases to be a limiting factor and another factor commences to play this role is not a definite point, but rather that there is a gradual transition from one condition to another. This fact must also be taken into con- sideration in interpreting the phenomenon just described. Finally the relative decrease in the rate of photosynthesis as compared to that of respiration with increasing temperature must also be viewed in the light of the influence of the "time factor.'' The latter, as well as a discussion of limiting factors have been taken up in a previous section. The position of the compensation point of a plant in regard to tem- perature is naturally of great importance to the life of the plant and its relation to the environment. The fact that with decreasing tempera- ture respiration decreases more rapidly than photosynthesis has also been recorded by Harder. ^-^ His results indicate that CladopJwra, at low light intensity, has a higher photosynthetic activity at low temperatures than at higher temperatures. Harder points out that conditions must exist in nature where at higher temperatures the plant gains no material through photosynthesis on account of the excessive respiratory activity, while at lower temperatures with the same light intensity nutritive material is formed in the plant. These conditions would, of course, apply particularly to the seas of the polar zones where the light intensity is low. Harder gives the following ratio of : =*' Harder, Jahrb. zviss. Bot., 60. 531 (1921). ^'^'^ Harder, Jahrb. wiss. Bot., 56, 281 (1915). THE NATURE OF PHOTOSYNTHESIS 179 Photosynthesis . ,. „ — — 7 — -. for ditferent temperatures: Respiration 20 — 22° 0.5882 0.4227 0.4280 2 — 3.5° 1.603 0.9207 2.059 Plaetzer found, that while the different species vary greatly in the light intensity at which compensation is attained, the same species seems to possess a definite comi^ensation point. Harder,^^^ on the other hand, was able to demonstrate that the compensation point is a variable quantity, varying more than 100 per cent, for any one species depending upon the previous conditions of illumination of the plant. Thus, ivy leaves which had grown in the direct sunlight showed a compensation point of 2477 Lux, while similar leaves, grown in the shade, one of 1133 Lux. Harder was able, moreover, to demonstrate that with continued dark- ness the compensation point is reduced although the difference between the two sets of plants, sun and shade plants, is maintained. Thus, two sets of Fontinalis antipyrctica, the one taken from a sunny, the other from a shady habitat, possessed, after corresponding periods in darkness the following compensation points : Date ■ Sun Plants Shade Plants Sept. 20 152 Lux 95 Lux 26 118 79 30 Ill 74 Oct. 2 84 64 5 59 < 41 10 27 about 10 Harder also showed that by starting with the same culture of Clado- pliora and keeping one portion in diffuse light and another in direct sunlight great differences in the compensation point result within seven days. Similar results have been obtained by Boysen-Jensen."^ In Shiapis alba, a light plant, the compensation point lies at 1.0 (Bunsen units X 100) ; while in Oxalis acetosella, a shade plant, the compensation point lies at 0.2. It is evident, therefore, that the compensation point is by no means of constant value for any single species of plant and that it is essential that the previous condition of the plant as to illumination must be taken into consideration before attempting to draw conclusions from the value of the compensation point. Harder's results show that under circumstances in Fontinalis photosynthesis more than overbalances respiration at an illumination of 10 Lux, while under other circumstances 150 Lux is not sufficient to accomplish this. To show that the compensation point can Harder, Ber. hot. Ges.. 41, 194 (1923). Boysen-Jensen, Bot. Tidsskr., 36, 219 (1918). Staelfelt, "Meddel. fran Statens Skogsforsoksanst.," Stockholm, 18, No. 5 (1921). 322 223 180 PHOTOSYNTHESIS rise very high. Harder quotes Klehs who found that the leaves of the beech tree which had been kept under constant and high illumination still emitted carbon dioxide at 6250 Lux. The great importance of a considera- tion of such circumstances to plants in nature needs no special elaboration. The investigations of O. Warburg"* on the efficiency of the photo- synthetic process are pertinent to the subject of the nature of the com- pensation point. He found wide fluctuations in the quotient: absorbed radiant energy to chemical work performed, depending upon the previous treatment of the plants studied. It was established that when the plants are cultivated under conditions of high light intensity they are capable of utilizing only a small amount of the absorbed radiant energy. On the other hand, when the plants are cultivated under conditions of low light intensity they are capable of utilizing a relatively large proportion of radiant energy absorbed. By cultivation under conditions of either high or low light intensity, one type of plant can apparently be converted into the other within a few days, and its photosynthetic efficiency altered accordingly. It is doubtful on the basis of the work of Willstatter and Stoll, that this alteration can be entirely at least ascribed to alterations in chlorophyll-content, as they found no direct j)roportionality between this and photosynthetic rates. It is apparent, therefore, that merely increasing the light intensity or the period of illumination of a plant does not result in a higher photo- synthetic efficiency. Some very interesting observations of the effect of hydrocyanic acid on the compensation point have been made by Warburg"^ on the green alga Chlorella. A 0.0001 normal solution of hydrocyanic acid exerts a decidedly inhibiting effect on the photosynthetic activity; on removing the plant from the HNC solution normal photosynthesis is again attained. Respiration, on the other hand, is not affected in the same manner. A 0.01 N. solution of HNC slightly stimulates the oxygen consumption and carbon dioxide emission and only after several hours exerts a poisonous or inhibiting effect. By studying the influence of various concentrations of HNC on photosynthesis Warburg found that oxygen evolution is inhibited by very small amounts of HNC, but that there is a point be- yond which even relatively high concentrations of the poison have no effect. Warburg's results seem to indicate that in 0.05 normal HNC solution the plants are incapable of taking up and reducing carbon dioxide even in high light intensity. That is, plants with a compensation point of 500 Lux in 0.05 normal HNC solution produced no more oxygen at a light intensity of 19,000 Lux than with 500 Lux. Also the compensation point is not affected by the HNC, for the carbon dioxide produced by respi- ration is reduced by means of light of low intensity at the same rate in plants exposed to cyanide as in plants under normal conditions. Thus it appears that relatively high concentrations of HXC (0.05 normal) do "* Warburg, Z. physik. Chew.. 102, 246 (1922). =" Warburg, Biochem. Zeit.. 103, 199 (1920). THE NATURE OF PHOTOSYNTHESIS 181 not affect the mechanism of photochemical reaction, as is indicated by the maintaining of the compensation point in plants treated with HNC, but that this substance does affect the photochemical reactivity of the carbon dioxide which the plant normally takes up from the surrounding medium. An example of the effect of low concentration of cyanide on photo- synthesis, as found by Warburg, is given below : Approximate Light Intensity in Lux. Cyanide Cone, in moles per liter Length of Experiment in minutes Photo- synthesis 1,800 1,800 19,000 19,000 0.0001 0.0001 30 30 5 5 61 61 540 192 In these plants the comi>ensation point was about 500 Lux. It appears, therefore, that the light intensity of the compensation point does not determine the point where the inhibiting action of HNC commences; the critical light intensity is higher. In 0.0001 normal solution of HNC, carbon dioxide is absorbed and reduced, but the rate is considerably decreased as compared with plants not treated with cyanide. It seems highly probable that an interpretation of Warburg's results will finally be found in the effect of HNC on the absorptive capacity of the material in the plant which first takes up the carbon dioxide from the surrounding medium. Chapter 3 The Products of Photosynthesis The substances which are known to be produced in photosynthesis are oxygen and carbohydrates. It was, in fact, through the formation of oxygen that photosynthesis was discovered. Oxygen and carbohydrates must be regarded as the final products of what is apparently a series of reactions comprising the photosynthetic process. We have as yet no definite and experimentally satisfactory evidence regarding the intermedi- ate products in these reactions. The intermediate or first formed product never accumulates to any extent so that it has been extremely difficult to ascertain the precise manner in which carbonic acid is converted into carbohydrates and oxygen. For years it has been the object of chem- ists and physiologists to describe the chemical reactions involved, but probably the greatest obstacle has been a lack of reliable knowledge con- cerning the first formed product. In the absence of direct observational evidence many theories have been proj^osed to "boost" us over this obstruction. In this chapter we shall confine the discussion to the products which are known to be produced as a result of photosynthesis. In the chapter on the chemistry of photosynthesis the different theories which have been formulated to describe the various steps in the process are discussed. As has been stated, oxygen and carbohydrates are the two chief products of photosynthesis, and while the formation of each of these is chemically closely related, their production in photosynthesis has to a considerable extent been investigated separately. The quantitative relation between the liberation of oxygen and the formation of carbohydrates has received little attention. 1. The Liberation of Oxygen While it can be demonstrated very easily that the atmosphere sur- rounding a plant which is photosynthetically active becomes richer in oxygen and depleted of carbon dioxide, the composition of the gas which is emitted by the plant can be more accurately determined with aquatic than with land plants. The gas which is emitted from an aquatic plant during photosynthesis is not pure oxygen, but contains 25 to 85 per cent of this gas and varying amounts of nitrogen and carbon dioxide. The percentage of oxygen in the escaping gas increases with the rate of 182 THE PRODUCTS OF PHOTOSYNTHESIS 183 photosynthesis and vice versa; this jjhenomenon has already been dis- cussed in Chapter 2. The quantitative relations of the amount of carbon dioxide absorbed to that of oxygen emitted have been subjected to careful examination. These values, termed the photosynthetic quotient, have been found to be very close to unity and are also discussed in greater detail in Chapter 2. Oxygen emission commences immediately on illumination of the green parts and stops when the light exposure ceases. According to Kostyts- chew ^ the amount of oxygen emitted is considerably less than that of carbon dioxide absorbed during the first few minutes of illumination ; after CO2 a short period of illummation (15 to 30 minutes) the — =— ratio attams U2 unity. . 2, The Carbohydrates of the Leaf The early work on photosynthesis was to a great extent confined to investigations on the gaseous exchange of plants, and, owing largely to the undeveloped state of organic chemistry, little advance was made re- garding the substances which were formed from the decomposition of the carbon dioxide. Ingen-Housz, Senebier and de Saussure realized that the carbon dioxide absorbed during photosynthesis was converted into material which was appropriated by the plant for the formation of new tissue and was used to maintain respiration. But the chemistry of these substances was treated only very meagerly. The first decisive step in identifying carbohydrates with the photo- synthetic process was made by Sachs. Since the time of Ingen-Housz and Senebier it was realized that photosynthesis was dependent upon chlorophyll. Von Mohl, the discoverer of protoplasm in plants, in 1837, had made an intensive study of chlorophyll, had described the chloroplasts and recognized starch in them. A great deal of confusion existed re- garding the nature and function of the chloroplasts, but von Mohl de- veloped the conception that the starch which was associated with the chloroplasts was of the nature of reserve food material. These investi- gations together with those of other anatomists, notably Nageli, deter- mined the structure, and to some degree also the function, of the chloro- plasts. Gries " then demonstrated that the starch in the chloroplasts dis- appears when the leaves were kept in the dark. It was, however, not certain whether starch or chlorophyll was the first to appear in the illuminated plant so that the genetic relationship of these two substances was still uncertain. With these facts as a background Sachs^ undertook his classical re- searches on the function of chlorophyll. These established the fact that ' Kostytschew, Ber. hot. Ges., 39, 319 (1922). 'Gries, Ann. Sci. Nat. Bot., 8, 179 (1857). 'Sachs, Jahrb. wiss. Bot., 3, 184 (1863); Bot. Zeifg., 20, 365 (1862); 22, 289 (1864) ; Arbeiten aus dem bot. Inst. Wiirzburg, 3, 1 (1884). 184 PHOTOSYNTHESIS starch is a product of photosynthesis and is formed in the chloroplasts. These organs have the power of forming starch in the Hght and again dissolving it in the dark. Sachs developed the macrochemical method of determining the amount of starch in a leaf on the basis of the colora- tion produced with iodine. By means of this method he also demonstrated the necessity of light, chlorophyll and carbon dioxide for starch forma- tion. He showed that starch represents a reserve food material and his investigations indicate that starch formation is the result of an accumula- tion of the products of photosynthesis. That starch is deposited in the chloroplasts, even in the dark, when leaves are floated on solutions of soluble carbohydrates was demonstrated by Bohm.* This was an exceedingly important observation. Already at that time, as still, there existed an active controversy regarding the first product of photosynthesis. Sachs took the stand that starch was the first visible product. This method of producing starch in plants kept in the dark, from soluble carbohydrates has been extensively investigated.^ Starch is formed under these circumstances in the plastids, whether they contain chlorophyll or not. Starch formation in photosynthesis is there- fore probably a secondary reaction and cannot be regarded as a direct product of photosynthesis, but is formed when the concentration of the simple, soluble carbohydrates attains a sufficiently high concentration. Plants form starch in the dark, not only from the carbohydrates, glucose, fructose, galactose, mannose, sucrose and maltose, but also from other organic compounds. Thus alcohols such as mannitol, dulcitol, erythritol and glycerine also are capable of being used for starch forma- tion in the plant. The pentose, arabinose, is also capable of conversion into starch according to reports of Polonovski '^ and Morillez. The fact that such a variety of substances can be utilized by the plant for starch formation would indicate that it is fallacious to draw conclusions re- garding the first product of photosynthesis from the observation that any particular substance produces starch. Nor can it be concluded that all substances which form starch are normally the products of photo- synthesis. The independence of starch formation and photosynthesis is further illustrated by the fact that some plants, although they carry on active photo- synthesis, never produce starch. Bohm showed this to be the case and Meyer made a classification of the plants which form starch and those which do not. The plants which form no starch contain disaccharides and monosaccharides. There are also some plants which store their carbohydrate in the form of inulin instead of starch. Bohm also showed that some plants in which normally no starch is found, e.g. Galanthus, *Bohni, Bot. Zcitq., 41, iZ, 49 (1883). "Meyer, Bot. Zeitg., 44, 81, 105. 129, 145 (1886). Laurent, Bull. Sac. bot. Belgium, 26, 243 (1887). Klebs, Untcrs. bot. Inst. Tiibmgen., 2, 489 (1888). Bokorny, Biol. Centrabl., 17. 1 (1897