THE PRODUCTION OF GLYCOLLATE DURING PHOTOSYOTHESIS 

 C. P. Vfliittingham, R. G. Hiller, and M. Bermingham 



A direct investigation of the compounds first fonned during photosynthesis 

 became possible only with the exploitation by M. Calvin of the radioactive 

 isotope of carbon, carbon 14, coupled with the technique of paper chroma- 

 tography and led to formulation of the photosynthetic carbon reduction cycled -'•), 



Most of the investigations by Calvin and his colleagues were of photosyn- 

 thesis at concentrations of carbon dioxide far higher than that in nonnal sir. 

 In our laboratory we have investigated the products of photosynthesis in 

 Ch lore 11a over a range of concentrations of COo above and below that in air. 

 We have confirmed (2) previous observations(3>47 that at low carbon dioxide 

 concentrations a large fraction of the carbon dioxide fixed in photosynthesis 

 is converted to glycolic acid, and to the amino acids glycine and serine. As 

 the carbon dioxide concentration is increased production of glycollic acid and 

 glycine decreases and sucrose becanes the major product. Other compounds, 

 e.g., alanine, malic and aspartic acids, are not markedly affected by changing 

 the carbon dioxide concentration within the range studied by us and represent 

 a relatively small fraction of the total carbon fixed. 



EXPERir-TENTAL [METHODS 



In all the experiraents three- to four-day-old cultures of Chlorella 

 pyrenoidosa (Bnerson Strain) grown on H% carbon dioxide in air, at constant 

 light intensity and temperature (22° C) , were used. Cells were harvested by 

 centrifugation, washed once with distilled water, and resuspended in potassium 

 dihydrogen phosphate solution (10~^ M; pH 4.5-5.0). A cell density of 1650 ul 

 wet packed volume of cells per 100 ml of solution was used. 



Ihe cell suspension was illuminated in a perspex chamber 5 mn thick and 

 19 cm in diameter, the incoming gas system aei^ting the suspension through a 

 finely perforated polythene tube. Carbon dioxide uptake during photosynthesis 

 was measured by passing the outflowing gas through an infrared CO2 analyser. 

 Glycollic acid in the medium was estimated by the colorimetric method of 

 Calkins(5)j after centrifuging the cells (3OOO x g for 5 min) and concentrating 

 the supernatant in vacuo at 30° C. A slight brownish color was given by 

 samples taken after the cells had been aerated in the dark for 30 min, and this 

 reading was subtracted from that of the bright purple obtained after 30 min 

 subsequent photosynthesis. Isoniazid gave no colour with the glycollic acid 

 reagent . 



The products of photosynthesis when Chlorella .was fed ^^C02 were determined 

 using the techniques developed by Calvin, et al^^-^ . The suspension was first 

 aerated in the dark for 30 min and then illuminated at 20,000 lux for a further 

 30 min, after which the non radioactive CO2 was replaced with -'■^002 at the same 

 flow rate and total CO2 concentration. Samples were taken directly into 

 boiling ethanol. After centrifligatlon, the cell residues were reextracted with 



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