Oxygen Isotope Fractionation during Photosynthesis in a Blue-Green and a Green Alga

Abstract

Oxygen isotope fractionation (180/160) at the natural abundance level has been measured during photosynthesis of a blue-green and a green alga. When sufficient attention is paid to removal of contaminating air 02 before and during the experiments, then the photosynthetic 02 evolved, as compared to the water 02, had an average difference of-0.36%0 for a blue-green alga and-0.80%0 for a green alga. These experiments suggest that there is no reason to invoke an inverse isotope effect in photosynthesis as part of the explanation for the 180 enrichment in atmospheric 02 relative to 02 in oceanic waters. In addition, in an indirect way, the experiments also support the argument that the bulk of 02 evolved during photosynthesis comes fronm water. A 10%s contribution of 02 arising froni CO2 would have been detectable in the present work. Oxygen isotope fractionation during photosynthesis is of interest from two standpoints. First, our visualization of the reaction mechanisms of photosynthesis requires that the source of the evolved 02 iS from water. Through the years the observations of Ruben et al. (7), Holt and French (5), and Vinogradov et al. (10) support this hypothesis. However, the experimental assumptions necessary in the interpretation of each of these results has precluded rigorous proof of this hypothesis. A second interest in photosynthetic 02 isotope fractionation began with the observations of Dole (3) that 02 in the atmosphere is 23%, enriched in 180 relative to 02 in oceanic waters. The Dole effect obviously should be related to the two major biological transformations , photosynthesis and respiration. Moreover, measurements of 02 isotope fractionation at the natural abundance level during photosynthesis have led to the supposition that an inverse isotope effect is occurring, that is, the evolved 02 iS isotopically heavier than the water used. Such an anomaly is difficult to explain , and if true would perhaps allow some predictions to be made on the nature of the reactions leading to 02 evolution during photosynthesis. The work reported herein bears mostly upon the 180 enrichment anomaly extant in the literature. In addition, because of the manner in which the experiments were done, the data can also be used to support the idea that photosynthetically evolved 02 derives primarily from water. The accuracy of our measurements does not allow us to comment upon the bicarbonate stimulation of 02 evolution (see Stemler and Govindjee [8] for a recent discussion) and the possible implication that a small part of photosynthetically evolved 02 does indeed arise from CO2. MATERIALS AND METHODS The blue-green alga, Agmenellum quadruplicatum, strain PR-6, was grown in modified ASP-2 (9); the green alga, Chlorella pyrenoidosa, high temperature strain TX-71105, was grown in EDTA Knops (6). Axenic growth occurred at 35 C in test tube cultures, with continuous bubbling of 1 % CO2 in air. Illumination consisted of four (two on each side of the bath) F20T12/ CWX fluorescent lamps 11 cm from the growth tubes. Cells for isotopic experiments were purposely harvested at different stages of growth by centrifugation, then injected to give various final cell concentrations (see Table I) into a sealed 4-liter bottle containing 3 liters of fresh medium, magnetically stirred. The bottle was held at 35 C and illuminated on two sides by seven 100-w tungsten lamps, about 22 cm from the center of the bath. Oxygen content of the algal suspension during the degassing phase or during periods of photosynthesis or respiration was monitored with an 02 electrode (Gilson Medical Electronics Clark-Type electrode, OX700, connected to a Keithley 150A microvolt-ammeter and Brown recorder), see Figure 1. The 02 electrode was routinely calibrated using air-saturated medium (checked by the Winkler method) and an 02-free solution (0.16 M Na2SO3 in 10 mM Na2B407). The removal of atmospheric 02 from an algal suspension was attained by (a) evacuation, using a water aspirator, in conjunction with bubbling with 1 % or 2%o CO2 in N2 (G, in Table