Observations on Photosynthesis and Related Systems. I. Influence of Anaerobiosis on Photosynthetic Rates During Continuous Irradiation* F. L. Allen From the Research Institutes
(Fels Fund), Received
April
University of Chicago, Chicago, Illinois 27, 1954
INTRODUCTION
The question whether molecular oxygen is a necessary ingredient of photosynthesis has been considered since the time of Boussingault (I), who in 1865 was the first to observe and record that plants lost their ability to “decompose” carbon dioxide after a temporary stay in an atmosphere devoid of oxygen. This observation was confirmed by Pringsheim (2), and the essentiality of oxygen was given considerable impetus by the detailed observations of Willstatter and Stoll (3). Willstatter supposed that a loosely bound complex between some acceptor molecule and oxygen was necessary before photosynthesis could proceed. Under anaerobiosis this complex would dissociate with the resultant inactivation of the photosynthetic machinery. Kautsky (4, 5), on the other hand, went further and made the somewhat generalized suggestion that all dye-sensitized reactions, of which photosynthesis is perhaps the most common, were mediated by oxygen, the mechanism presumably involving the transfer of energy from the dye to the oxygen molecule. In this manner he attempted to provide a reason for the observed inhibition of photosynthesis under anaerobiosis. 1 The present paper represents a portion of a doctoral dissertation submitted to the Committee on Instruction in Biophysics, Institute of Radiobiology and Biophysics, University of Chicago, in partial fulfillment of the requirements for the Ph.D. degree. The remainder of the dissertation will appear in a subsequent publication. The work was made possible through financial assistance from the Fels Fund (University of Chicago); the Office of Naval Research, Department of the Navy (contract No. 119-272); and from predoctoral fellowships from the U. S. Atomic Energy Commission and the National Science Foundation. 38
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Kautsky based his particular interpretation on his observation of florespence intensity in leaves exposed to different concentrations of oxygen. il variety of other considerations (6), however, made Kautsky’s proposal untenable, and it therefore did not meet with general approval. Nevertheless, t’he view remained that molecular oxygen in some manner or other played an essential role in the mechanism of photosynthesis. First serious doubt as to the validity of this assumption arose when the observations themselves were questioned. Gaffron, after noting t,he similarity between photosynthesis in green plants and photoreduction in purple bacateria (7), proceeded (8) to challenge this assumption. Studying photosynthesis in the algae ChZoreZla and Scenedesmus, Gaffron observed that the induction period in nitrogen after many hours of darkness was considerably longer than in air. In Chore& for example, it could last’ upwards of 20 min. However, even in this case, the final steady-state rate of oxygen production was the same in both nitrogen and in air. A variety of observations such as these, particularly on the shape and duration of the induction period, led Gaffron t’o point out that serious errors could be made if the effect of anaerobiosis on the induction period was not differentiated from the effect on the steadystate ratfe. The indications were that oxygen was not required, either in the free or the bound form, and that any theories in which reactions between chlorophyll and molecular oxygen played an essential role were unsound. The work of Xoack, Pirson, and Michels (9) and of Michels (IO) confirmed and extended Gaffron’s observations that the inhibition introduced by anaerobic incubation occurred only in an acid medium, and that this inhibition could be removed either by simply adding bicarbonate to the manometer vessel in order to raise the pH or by admitting oxygen to permit respiration. It was thus made clear that indeed some doubt existed as to the necessity of oxygen. That anaerobiosis had an inhibitory effect on photosynthesis was beyond denial, except, under some special conditions (10) by virtue of which the directness of the effect, was questioned. Following Gaffron’s suggestion, it was repeatedly pointed out that the effect could be purely indirect, perhaps caused by fermentat,ion products. It is interesting to note that the very early experiments of Reijerinck ( 11) using luminous organisms, and later those of Harvey (12)) although perhaps crude but at any rate elegant, which should have cast consider-
40
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ALLEN
able doubt on the phenomena at an earlier date, were not taken seriously or remained unnoticed. The most radical mechanism for the role of molecular oxygen in photosynthesis is, of course, the recent proposal of Burk and Warburg (13-16). These authors suggest that during the course of photosynthesis a fast back-oxidation of some substrate, presumably carbohydrate, occurs with molecular oxygen. The rate of this back-reaction is such that during the steady-state of photosynthesis the net oxygen production observed corresponds to about one-third of a molecule of oxygen per photon absorbed, although the primary reduction reaction is presumed to occur with the evolution of one oxygen molecule per photon absorbed. In short, approximately two-thirds of the oxygen initially produced is burned back. The energy thus made available presumably joins hands with a photon and drives photosynthesis forward, in the fashion of a one-quantum process, which clearly would otherwise be energetically impossible. The implication is that molecular oxygen per se is intimately necessary for photosynthesis. This mechanism is, of course, attrative and is based on observations which the authors interpret aa supporting evidence. On the other hand, there are at least three fairly recent observations which indicate that this fast back-reaction with molecular oxygen either does not occur or is not necessary. The most conclusive is that of Brown (17), who, using a mass spectrometer, has shown that molecular oxygen is not taken up from the gas phase by photosynthesizing algae at any rate comparable to that required for the operation of the mechanism proposed by Burk and Warburg. Weigl, Warrington, and Calvin (18) followed the content of Cl402 and CYO2in the gas phase, for barley leaves photosynthesizing in a closed system, and observed that during illumination the respiratory COPoriginates primarily from endogenous material and not from freshly assimilated carbon. Since they did not find a greatly enhanced dilution of the C402 in the gas phase by the CYO2 in the organism in the light, this was interpreted by Calvin et al. (19) as evidence against the theory of Burk and Warburg. Earlier, Franck, Pringsheim, and Lad (20) had demonstrated the evolution of oxygen by one single intense flash of light lasting about one-fiftieth of a second from algae previously in the dark and under strict anaerobiosis. Hence, there is some precedent for doubt as to the validity of the mechanism suggested by Burk and Warburg. More recently Franck (21) has shown that the observations of Burk and Warburg admit of another interpretation, which in addition is consistent with the bulk of experimental evidence in the field.
PHOTOSYNTHESIS.
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Inasmuch as the non-essentiality of molecular oxygen has again been questioned (22, 23), it seemed not only interesting but imperative to reinvestigate the effects of severe anaerobiosis on green algae. MATERIALS
AND METHODS
The algae, Chlorella pyrenoidosa strain Emerson and Scenedesmus obliquus strain DS , were grown in test tubes immersed in a constant-temperature bath at 24”C., in batches of about 30 ml. Agitation was provided by constantly bubbling 2% carbon dioxide in air through the tubes. Tw-o 20-w. fluorescent lamps illuminated the temperature bath from the aide. Essentially this method of growing algae has been described in detail by Myers (24). Both species of algae were grown in culture media of the following composition: 5.0 g. MgS04.7HzO 2.0 NHaN03 I .6 K,HPOI-~HZO 3.0 KHCOI 0.3 KzCOa NaCl 0.1 0.2 Ca(NOd 2 Distilled water 10.0 1. To each liter of this solution was added 1 ml. of improved Arnon solution (24) and 2 ml. of iron solution containing the following: 1.4 g. FeS04.7H20 2.4 FeNHI(S04) 2.12H20 DisGlled w-ater 100 ml. Roughly half the suspension was discarded every day, and the remaining was brought to the original volume with fresh nutrient. In this manner an activel! growing culture was always available. Since microscopic examination did not reveal any significant bacterial contamination, it was not considered necessary to provide sterile conditions. Normally the cultures were maintained growing at cell densities considerably higher than those used in most of the experimentIs de scribed. Dilutions were therefore made into fresh nutrient2 to the proper cell densities without centrifuging. Algae thus received a minimum of handling before actual use. Cell volume, as an index of concentration, was measured by centrifuging an aliquot of the culture suspension before dilution, in a cytocrit t,ube foi 20 min. at high speed (2000 X g). 2 While it is true that nitrate ion was present in the medium in which t,he algae were suspended, apparently its concentration was sufficiently low and that of carbon dioxide sufficiently high, since reduction of this ion did not appear to occur to a significant extent (26-28). No systematic differences were observed if, for an experiment, the algae were suspended in distilled water in place of the full nutrient. Further, careful search for nitrite ion by color reactions after long periods of anaerobic photosynthesis proved fruitless. The oxygen evolution observed in the present experiments cannot, therefore, be attributed to the reduction of NO,-, nor can the inhibitions observed be attributed to the possible concomitant accumulation of NO,-.
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Measurement of the oxygen production was made by the method described by Pollack, Pringsheim, and Terwoord (25). This method makes use of the quenching of the phosphorescence of the dye acriflavine adsorbed on silica gel. A carrier gas, in the present experiments nitrogen, enriched with 2% carbon dioxide to provide a carbon source for the plant. material, is made oxygen-free by passage through a column of pyrophoric copper kept at 25O”C., so that upon leaving the column the partial pressure of the oxygen in the carrier gas is less than 10-r mm. Hg. This carrier gas, which is allowed to bubble through water to humidify it (thus preventing the small suspensions of algae from drying out), then flows over the sample where it flushes away the oxygen produced during photosynthesis. After passing through a liquid nitrogen trap which serves to remove moisture, the gas flows over the adsorbed dye. Alternately, and in rapid succession, a light illuminates the dye and a photomultiplier tube measures the phosphorescence (Becquerel phosphoroscope), the degree of the quenching of this phosphorescence being a measure of the oxygen content of the gas. A Brown recorder continuously registers the output from the photomultiplier tube. By means of a calibration curve, obtained previously by exposing the adsorbed dye to known pressures of dry air, the trace from the Brown recorder can be converted to partial pressures of oxygen in the carrier gas. Since the flow rate of the carrier gas can be measured, the rate of oxygen production is given by : R=&F where R is the rate of oxygen production in cc./min., P is the partial pressure in mm. Hg, and F is the flow rate of the carrier gas in cc./min. The readable range of the present instrument is from 5 X 10-r to approximately 5 X 1OV mm. Hg of oxygen. A pressure of oxygen of 5 X 10-r mm. Hg is, for our purposes, entirely negligible. It is therefore the reference baseline of the instrument. The highest sensitivity obtains at the lowest pressures and decreases roughly exponentially with increasing oxygen pressure over this range. Flow rates of the carrier gas ranged from 10 cc./min. to 50 cc./min. CRITIQUE
OF THE METHOD
The difficult problem in performing experiments under anaerobic conditions with photosynthesizing algae, one that has been realized by workers using manometric techniques, is that of maintaining as closely as possible a state of anaerobiosis in spite of the rapid evolution of oxygen by the very process one is studying. The gas exchange between the relatively large volume of liquid in a manometer vessel and the oxygenabsorbing substance, as in the arm of the vessel, is necessarily a slow process3 Therefore, one can rightly suspect that very good states of an3 Gaffron has recently improved considerably such manometric measurements by the use of hydrogen atmospheres and a special palladium catalyst (30). He again found that the photosynthetic rates were the same under these anaerobic conditions as in air.
PHOTOSYNTHESIS.
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aerobiosis are very difficult, if not impossible, to maintain for even short periods of time in a manometer vessel in which the photosynthesizing organisms are evolving oxygen. The basic difficulty is, of course, connected with the low sensitivity of the manometric method. On the other hand, because of the extreme and selective sensitivity for oxygen of one part in log, the method of phosphorescence quenching is ideally suited to the measurement of oxygen evolution under conditions where a high degree of anaerobiosis is desired. Such a high sensitivity means that one is able to use very small and very dilute suspensions of algae. Whereas the usual manometric volume is a few cubic centimeters of a few tenths of a per cent suspension, the volumes used in the present experiments ranged from 0.01 to 0.125 cc. at concentrations varying from 0.0001 to 0.1%. Apart from using such small and dilute samples, in order to further decreasethe concentration of oxygen in the liquid phase itself during the time the algae are performing photosynthesis, the sample is rapidly stirred by means of a rotating magnet and iron ball sealed in glass, shown in Fig. 1. The result of rapid rotation is that the samples are spread over the lower inner surface of the sample holder, whose volume is but 0.3 cc. New layers of solution are continuously brought into contact with the
FIG. 1. Schematic diagram of the apparatus. stirred at a minimum of 200 r.p.m. The column with copper adsorbed on Fuller’s earth.
The glass-covered iron bead was for purifying the nitrogen is filled
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F. L. ALLEN
oxygen-free carrier gas nitrogen. In this manner the effective diffusion path of oxygen in the liquid is considerably reduced, and its flushing out is extremely efficient, as will be shown later. The volume and concentration of the algal suspensions were always chosen so that at the flow rates used of the carrier gas, the pressure of oxygen in the carrier gas never exceeded 25 X 1O-6 mm. Hg (except under special conditions) after passing over the sample during photosynthesis at saturation. In the decisive experiments this pressure never exceeded 0.4 X W6 mm. Hg. Under these favorable conditions, namely, such small sample volumes of very dilute algal suspensions, coupled with rapid stirring, we have been able to lower the concentration of oxygen in the liquid phase (expressedin terms of the pressure of oxygen which would be in equilibrium with this concentration) to a pressure smaller than 0.005 mm. Hg. In place of the usual 21% this represents a concentration of oxygen of only 0XK1066~~or a factor of at least 30,000 below normal aerobiosis-an even greater factor if one considers not normal aerobiosis but the concentration which would exist in the liquid phase during photosynthesis in a manometric vessel where the gas phase is ordinary air. Such a sustained degree of anaerobiosis is altogether unattainable with conventional manometric methods as well as with other techniques which permit accumulation of thd oxygen evolved. EXPERIMENTAL
RESULTS
Table I presents, for sample experiments, the values of the yield of oxygen at saturation expressedin volumes of oxygen per volume of algae TABLE Yield
I
of Oxygen at Saturation (1)
in Volume of Oxygen per Hour Algae During Anaerobiosis (T = 86°C.) (3) 6) (2) (4)
Experiment
Algae
pH of suspension
1-14-52
Chlorella Scenedesmus Chlorella Scenedesmus Chlorella Scenedesmus Scenedesmus
Hz0 5.5 6.2 8.2 8.2 8.2 8.2
10-8-52 2-12-52 7-22-52 5-28-52 10-8-53 10-g-53
Time min.
Volume of suspension cc. x lo-’
18
10
30 20 20
60 60 25 60 60 60
10 20 20
per Volume of Wet
(6)
(7)
Concentration %
Yield
0.064 0.01
20 35
0.01
18
0.02 0.05 0.00025
37 27 40 40
O.oool
PHOTOSYNTHESIS. I
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4.5
I I
I 1
FIG. 2. Brown recorder trace illustrating the effect of rapid stirring by the iron bead. The sample had been photosynthesizing but the light was turned off and the oxygen, indicated by the declining trace, was being slowly flushed out of the liquid, because the stirrer had been off during this time. When it was suddenly turned on, oxygen, which was “trapped” in the liquid, was released in a burst. The Brown recorder trace is not directly proportional to oxygen pressures but must be converted by means of the calibration curve. In this particular figure, 80 corresponds to a pressure of about 1.2 X 1F mm. Hg while 60 corresponds to a pressure of about 5 X 1OP mm. Hg. The baseline is at 94.5 and represents a pressure of at most 10eB mm. Hg. Abscissa: each division is 1 min.
obtained under the conditions of anaerobiosis discussed previously. These rates were measured after the samples had been subjected jointly to anaerobiosis and to complete darkness for the time indicated in col. (4). The pressure over the same at the start of illumination was at least below 1OV mm. Hg. This figure is known since our instrument can readily measure this pressure of oxygen. It will be noted that the rates observed compare very well with the usual 20-30 vol. oxygen/hr./volume algae observed by other workers (6) under ordinary aerobic conditions. In fact, the rates may be expected to be higher, and indeed they are, inasmuch as t’he well-known oxygen
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F. L. ALLEN
FIG. 3. When the light is turned off, the oxygen dissolved in the liquid sample will be flushed out as rapidly as the various constants of the system will permit. The shaded area is a measurement of this amount. For details, see text. Abscissa: each accented division is 20 seconds.
inhibition of photosynthesis (29, 30) either does not occur or is minimized at these very low partial pressures of oxygen, The yields as shown in Table I appear to be lower for Chlorellu than for Scenedesmus. The reason for this is found when one observes the value of the saturation rate with increasing duration of anaerobiosis, as shown in Fig. 4. Scenedesmus suspended in alkaline pH does not show any, or at most very slight, inhibition of the saturation rate for many hours of anaerobiosis. Even in acid pH, the inhibition appears slowly, while for Chlorella, regardless of whether the medium is acid or alkaline, the inhibition increases sharply during the first hour or two and for a given time of anaerobiosis is much more pronounced* than for Scenedes4 This is only an apparent contradiction to the observations of Gaffron mentioned earlier in the Introduction, i.e., that for Chlorella, although the induction period could be very long, the final steady-state rate at saturation in nitrogen was the same as in air. However, there is no disagreement because Gaffron started his experiments under anaerobic conditions, but permitted the oxygen evolved to accumulate, thus allowing respiration which in turn removed the inhibition.
PHOTOSYNTHESIS.
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mus. In other words, the three curves shown in Fig. 4 extrapolate to approximately the same yield at zero time. Concomitant with the decrease in the yield at saturation is an increase in the length of the induction period for oxygen. This is illustrated for Chlorella in Fig. 5. The longer the anaerobic incubation, the more extended t’he so-called induction period becomes. This lengthening is always much more pronounced for Chlorella than for Scenedesmus. In fact, for Scenedesmus suspended in alkaline media where we observed no inhibition for many hours, the induction period lasts only a couple of minutes, practically the same as the usual length under aerobic conditions. Inhibition of the saturation rate and lengthening of the induction period appear to run parallel. Figure 6 shows a typical set of saturation curves (rate vs. intensity) for Chlorella measured after various times under our conditions of anaerobiosis. The maximum rate of oxygen evolution is, of course, lower than under aerobic conditions, inasmuch as inhibition appears rapidly and is very pronouned in Chlorella. The values of the light intensity required to approximately reach saturation do not change with increasing anaerobic incubation, nor do they differ greatly from the values of the 40-
-SCENEDESMUS
0 TOTAL HOURS
5 ANAEAOBICITY
FIG. 4. Inhibition of the photosynthetic incubation. The yield is given in volumes algae.
IO PRECEDING
D3 PH 8 2
I5 ILLUMINATION
rate as a function of the total anaerobic of oxygen per hour per volume of wet
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FIG. 5. Lengthening of the induction period in Chlorella with increasing anaerobic incubation. Total anaerobic time is indicated above each curve. The ordinate is directly proportional to the photosynthetic rate. The yield at saturation after 18 min. of anaerobiosis is approximately 18 vol. oxygen/hr./vol. wet algae, calculated from the following data: no2 = 14 X 10~~ mm., algal volume = 6.4 X KP cc., flow rate = 10 cc./min. After the saturation rate is reached, the light has been turned off, as indicated by the falling oxygen pressure. intensity necessary to attain maximum oxygen evolution from the same algae under ordinary aerobic conditions (7). The per cent inhibition ob-
served is equal at all light intensities; that is, it does not disappear as the light intensity is decreased. At low light values, however, the saturation curve acquires a sigmoid character, particularly after prolonged periods of anaerobiosis.6 All this is not new, having been reported previously by Franck et al. (20). In the above experiments it is of particular interest to know as accurately as possible the concentration of oxygen in the sample of algae 6 Clearer evidence of this sigmoid character will be presented in a subsequent publication.
PHOTOSYNTHESIS.
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FIG. 6. Saturation curves for Chlorella, measured with white light. Total anaerobic time is indicated in hours above each curve. The sigmoid shape at very low light intensities is barely discernible in this figure. Ordinate is given in mm. Hg and is directly proportional to the photosynthetic rate. Temperature, 25°C.
which is performing photosynthesis under conditions of anaerobiosis. It is not sufficient to know the pressure of oxygen in the gas stream after it passes over the sample; one must know the concentration of oxygen in the liquid phase which may be, and indeed is, considerably higher. During the steady-state condition the algae will produce as much oxygen as is carried away by the gas stream flowing over the sample. However, the concentration of oxygen in the liquid phase is expected to be appreciably higher than that which would obtain if equilibrium existed between the liquid and the gas phases, a consequence of the existence of a diffusion barrier across the liquid-gas interface. The procedure followed to determine this concentration was simple and straightforward. If the light is turned off, the pressure in the gas stream as registered by the Brown recorder will drop (Fig. 3), eventually reaching the limit of detection of our instrument. This occurs within a minute or two, depending on the size and concentration of the sample, rate of photosynthesis, rate of stirring, etc. By integrating graphically under this curve, after transforming it to rate of oxygen evolution, one obtains the amount of oxygen which was dissolved or trapped in the liquid at the time the light was turned off. The oxygen contained in the finite gas space above the sample is small, some 10 % of the total amount, and is neglected. Since both the volume of the sample and Henry’s law constant for oxygen in water are known, one can calculate the pressure of oxygen in a
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F. L. ALLEN
gas atmosphere above the algal suspension which would be in equilibrium with this concentration. For entry 10-9 in Table I, this pressure was 0.004 mm. Hg of oxygen. For the more concentrated or larger samples, this pressure is naturally higher; it can reach values of 0.1 mm. Hg (or much higher for that matter) if the cell density is excessive, stirring inadequate, etc. DISCUSSION
Our results show that even for the conditions in which the partial pressure of oxygen is maintained at the very low value of 0.004 mm. Hg, the rate of photosynthesis is not impaired; rather, the yield is often better than obtains under aerobic conditions.6 This observation, then, that photosynthesis can proceed at rates equal to or higher than those observed in ordinary aerobic atmospheres when the partial pressure is reduced by a factor of some 40,000 is indicative that molecular oxygen is not required for the operation of the machinery of photosynthesis in the manner suggested by Burk and Warburg (14). It appears extremely improbable that a mechanism such as they suggest, which requires rather large amounts of oxygen to function, could proceed at the same rate at 160 mm. Hg and 0.004 mm. Hg of oxygen. It must also be remembered that under the conditions of our experiments, photosynthesis begins in a pressure of oxygen of less than KP mm. Hg. Neither are we in agreement with the results of Hill and Whittingham (22) who concluded “that the presence of a certain minimum pressure of oxygen (of the order of 2 mm.) is required for the full development of photosynthesis both in Chbrella and other green plants.” Our experiments indicate that if such a limit exists, it must lie below 0.004 mm. Hg. These results, rather, confirm and extend the earlier ones of Gaffron (S), Michels (lo), and Franck et al. (20),’ which indicate that oxygen 6 A possible exception to this is the value of 66 vol. oxygen/vol. wet algae/hr. reported by Warburg (36). 7 The dependence of the oxygen yield on the density of the algal suspension reported by Franck, Pringsheim, and Lad (20), and which apparently caused their low yields, has not been found under the conditions of the present experiments. Apparently this effect depends, as yet in an undetermined way, on the previous history of the algae, culture methods, age, or some other factors which have not been duplicated. Nevertheless, for the low concentrations of algae and for comparable total anaerobic times (at least 3 hr. in their case), the results are not too different. There is a misunderstanding by Hill and Whittingham (22) on this particuof lar result of Franck et al. They state: “In agreement with our interpretation
PHOTOSYNTHESIS.
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per se is not necessary for photosynthesis. As they did, we prefer to interpret the observed inhibition of the photosynthetic rate as t’he result of the appearance of an anaerobic metabolism. Algae, in the absence of oxygen, ferment, producing carbon dioxide and a variety of acids and other products of high molecular weight, which may, as assumed by Gaffron, act on the photosynthetic machinery as poisonous metabolites. The fact that the rates measured are as high as aerobic rates soon after the anaerobic state is established and then decrease wit’h time is suggestive of the appearance of such a fermentative process. Likewise, the fact that the simple artifice of suspending Scenedeswms in alkaline solution serves to prevent inhibition by anaerobiosis (10) for many hours is reminiscent of the well-known change of pathway of fermenting yeast with pH, the so-called Neuberg’s forms of fermentation. In one form, a change in the pH of the suspending medium from acid to alkaline changes one of the end products of fermentation from alcohol to acetic acid. Some bacteria present another example of organisms which produce predominantly acids in alkaline media and amines in acid media. This is not meant to imply a definite mechanism. It is merely meant to indicate a certain similarity which is again suggestive that the observed anaerobic inhibition is due to metabolic products of fermentation. If the enzyme concerned with the liberation of oxygen is inactivated in the dark by products of fermentation, illumination will reactivate it only partially, by oxidation via the peroxides, as long as not enough oxygen is produced or accumulated so that fermentation stops. Thus a balance will exist between two reactions: the fermentation causing the inactivation of this enzyme, and the photochemical production of photoperoxides causing reactivation. In the present rase where the oxygen concentration remains very low, the percentage of active enzyme will be proportional to the rate of photoperoxide production, i.e., photosynthesis. Hence t,he observation of equal per cent inhibition at all light intensities is not incompatible with an enzyme limitation. ITnfortunately, inactivation of an enzyme by anaerobic metabolism and its reactivation by the photoperoxides is not the only process which these experiments it was found by Franck et aZ. that the higher t.he cell concent,raCon, i.e., the greater the oxygen concentration developed in the cells, the higher the rate of photosynthesis finally attained.” On the contrary, Franck el al. found that the higher the cell concentration, the lower the final rat,e attained, i.e., thr lower the yield.
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F. L. ALLEN
has to be taken into account. Certain anomalies of the chlorophyll fluorescence and also some phenomena to be presented in a later publication give strong indications that oxidative reactions caused by the photoperoxides can result under certain conditions in the production of a new inhibitory influence. Franck (33-35) has suggested that this process may consist in the formation of substances which possess“narcotic” properties as soon as certain substrates are oxidized by photoperoxides. This question will be discussed in a subsequent publication from this laboratory. ACKNOWLEDGMENTS Special thanks are due to all members of the Fels Group, especially to Dr. J. Franck, who suggested the research and gave freely of his time, energy, and encouragement, and to Drs. Gaffron, Fager, and Rosenberg for many valuable discussions. SUMMARY
The extremely sensitive method of oxygen determination by the quenching of phosphorescenceand the technique for the study of anaerobic photosynthesis used previously by Franck et al. has been modified and improved to the point where it has been possible to establish a known anaerobic state of lO+ mm. Hg of oxygen within 10 min. During photosynthesis at saturation, the concentration of oxygen in the sample (expressed in terms of the pressure of oxygen in an atmosphere above the sample which would be in equilibrium with this concentration) has been maintained at a measured value of 0.004 mm. Hg. Under this severe oxygen restriction, the saturation rates observed are as good as, and often better than ordinary aerobic rates. The suggestion that molecular oxygen per se enters into the mechanism of photosynthesis by way of a fast back-reaction is, therefore, rendered untenable. These results, coupled with the studies of Brown et al. with the mass spectrometer and isotopically labeled oxygen and with the work of Calvin et al. on the dilution of a Cl*02 enriched gas phase with Cl202 from endogenous carbon, tend to discredit almost to the point of certainty the mechanism suggested by Burk and Warburg. REFERENCES 1. BOUSSINGAULT, J. B., Compt. rend. 61,493,605,657 (1865). 2. PRINGSHEIM, N., Sitzber. preuss. Akad. Wiss., Physik.-math. (1887).
Klasse
1887, 763
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R., AND STOLL, A., “Untersuchungen tiber die Assimilation der Kohlens&ure.” Julius Springer, Berlin, 1918. 4. KAUTSKY, H., Naturwissenschajten 19, 964 (1931). 5. KAUTSKY, H., AND EBERLEIN, R., B&hem. Z. 302,137 (1939). 6. RABINOWITCH, E. I., “Photosynthesis and Related Processes,” Interscience Publishers, New York, 1945, 1951. 7. GAFFRON, H., Biochem. Z. 260, 1 (1933). 8. GAFFRON, H., B&hem. Z. 280, 337 (1935). 9. NOACK, K., PIRSON, A., AND MICHELS, H., -~~at~cr~uisscnschujten 27,645 (1939). 10. MICHELA, H., Z. Botan. 36, 241 (1940). 11. BEIJERXCK, M. W., Proc. Roy. Acad. Amsterdam 4, -I5 (1902). 12. HARVEY, E. N., Plant Physiol. 3, 85 (1928). 13. BURK, D., AND WARBURG, O., Maturwissenschajten 37,560 (1950). 14. BURK, D., AND WARBURG, O., Z. Naturjorsch. 6b, 12 (1951). 15. BURK, I)., CORNFIELD, J., AND SCHWARTZ, M., Sci. Monthly 73, 213 (1951). 16. DAMAsCHKE, Ii., T~DT, F., BURK, D., ASD WARBI’RC, O., Biochim. et Hiophys. dcta 12, 347 (1953). 1’7. BROWS, A. I-I., Am. J. Botany 40, 719 (1953). 18. WEIGL, J. W., WARRINGTON, P. M., .~ND Ca~.v~x, >\I., .I. ,im. (‘hem. Sot. 73, 5038 (1951). I!). CALVIS, RI., BASSHAM, J. A., BENSON, A. A., ASD ,LIASSISI, I’., ;ltlr. Xev. Phys. (‘hem. 3, 215 (1952). 20. FRANVK, J., PRINGSHEIIII, P. ASD LAD, D. T., ;lrch. Biochem. 7, 103 (1945). 21. FRAN(!K, J., Arch. Biochem. and Bioph.ys. 46, 190 (1953). 22. HILL, R., AND WHITTINGHAM, C. P., Xew Phytologist 52, 133 (1953). 23. KRAI.I., A., Meeting of American Society of Plant Physiologists, University of Wisconsin, Lfadison, Wisconsin, September 7, 1953. 24. I\lYERS, J., AND Mo~cax, I,. O., Final rrport to OXR, (‘ontract X8onr 78000, Vnivcrsitp of Texas 1948-1950. 25. Por,mc~, 31., PRISGSHEIX, I’., ASU TER~OORI), II., -1. (‘hem. PItya. 12, 295 (1!)44). 26. \‘as SINI., C. B., AI,LEN, M. B., .4~i1)WRIGHT, B. I<:., Hiochi,tr. rt Biophys. Acttr 12, 6i (1953). 27. CR.~~~ER, RI., ASD ~IYERK, J., J. Gen. Phl/siol. 32, 92 (19-18). 28. DAVIS, I<. A., Pluxt Physiol. 28, 539 (1953). 3. WILLST~TTER,
29. Wi\RHI.RG, 30. G.I~ROS, Society 31. I’IR.IL)I.EY, 32. 33. 36. 35. 36.
O., Biochem. z. 103, 188 (1920).
II., Symposium on Autotrophic hlicroorganisms, I,ondon illeeting, for General Microbiology. Cambridge Univ. Press, April, 1954. D. F., ASD CALVIS, AI., C. S. i2t,omic l