CARBON DIOXIDE REDUCTION WITH MOLECULAR HYDROGEN IN GREEN ALGAE

HYDROGEN HAS been used very often as a means to replace air in experiments with plant material when anaerobic conditions were desired. No effects of this gas on the metabolism of plants other than those observed in an atmosphere of nitrogen have been reported. Hydrogen has been considered, therefore, as an inert gas for the plant. (For references see Spoehr, 1926; Stiles, 1936.) This paper furnishes the experimental evidence of a new type of photosynthesis which can be induced artificially in some strains of algae belonging to the genera Scenedesmus and Rhaphidium. Under the proper conditions the photochemical reduction of carbon dioxide will proceed with the simultaneous absorption of twice the volume of molecular hydrogen. The reaction bears a striking resemblance to the photosynthesis with hydrogen found a few years ago in purple bacteria. (Roelofsen, 1934; Gaffron, 1935; French, 1937.) METHODS.-The plant used in most of the experiments was the alga Scenedesmus spec. D 3. Rhaphidium,2 which gave similar results, appeared to be more easily injured by the experimental procedure. The algae were grown in absolutely pure cultures. Culture methods and general properties of the alga have been described elsewhere (Gaffron, 1937, 1939b, 1940). The gas exchange was measured manometrically; 0.05 to 0.200 ml. of cells were suspended in 2 to 4 ml. of a bicarbonate or phosphate solution. The gas space of the manometer was filled either with air, N2, 02, H2, or with the same gases containing approximately 4 per cent C02, according to the special experimental conditions. The light sources used were a 1,000 W. incandescent lamp and a specially constructed neon lamp. The neon lamp was a frame, somewhat larger than the thermostat window, completely filled with parallel tubes of commercial neon sign tubing. Light of shorter wave length than 5,500 A' was filtered out by a sheet of red cellophane. The intensity of illumination could be changed in a measurable way with the aid of gray glass filters from Schott and Gen. Jena. The relative ratios of the different intensities given in figure 2 are accurate within a few per cent. (See Gaffron, 1937.) The absolute value of the intensities is only approximately correct. It was measured with an ordinary photronic 1 Received for publication December 18, 1939. The experiments reported were made at Hopkins Marine Station, Pacific Grove, California, where the author was the guest of Dr. C. B. van Niel to whom he feels greatly indebted. The manuscript has been kindly read by Dr. F. Rieke. A short communication has appeared in Nature, Vol. 143, 204, 1939. 2 Dr. Yamanouchi was kind enough to identify the alga as Rhaphidium polymorphium var. aciculare. cell as used in photography. No effort has been made to correct the values found in respect to the color sensitivity of the photo-electric cell. In the experiments reported only the relative intensities are of iinportance. The diameter of the neutral glass filters, however, was so small that only two vessels could be illuminated simultaneously. Later therefore the glass filters were exchanged for sheets of colored commercial cellophane large enough to cover the entire front window of the thermostat. The number of red filters was increased until the visible spectrum did not change in quality by the addition of two more filters. Under these conditions green cellophane filters could be used practically like neutral gray filters to changc the intensity of the light without altering its spectrum. For measurements of the dark metabolism, the manometer vessels were wrapped in tinfoil and the thermostat covered with a lid and black cloth. Under aerobic conditions the gas exchange in the dark is due to respiration; under strictly anaerobic conditions to fermentation. In computing the light metabolism, corrections have been applied accordingly. But it is sometimes difficult to decide which kind of dark reactions prevail when photosynthesis begins under anaerobic conditions and, by producing oxygen, changes these conditions gradually into aerobic ones. As a rule in the following experiments a correction for respiration has been made only where so much oxygen had been liberated by the illuminated cells that a true, measurable respiration resulted. Whether or not an oxygen partial pressure below one per cent of an atmosphere is sufficient to maintain normal respiration is still a matter of controversy. Generally the respiratory quotient begins to rise at very low oxygen pressures because additional carbon dioxide is formed due to the beginning of fermentation. From the point of view of the method of measuring the gas exchange, the terms liberation, formation, production, etc., of oxygen by photosynthesis should be used only if oxygen gas escapes from the cell and can be found outside of it. As long as we have no means of distinguishing between free and bound oxygen (photoperoxides) inside the cell, it is meaningless, for instance, to say that the plant produces molecular oxygen which is consumed instantly and completely by respiration in such a way that only the formation of carbon dioxide can be observed. This practical attitude appears to be particularly justified in the case of the microscopically small unicellular algae. Contrary to widespread belief, the oxygen partial pressure inside these cells depends less on their photochemical activity than on the oxygen concentration of the surrounding me-