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M. Calvin and P. Massini: The Path of Carbon in Photosynthesis 



[ExperieniiaVol.VIII/12] 



biological material, of the compound in question. 

 Furthermore, having once achieved a relatively uni- 

 form label in these photosynthetic intermediates, it 

 becomes possible to follow the behavior of the reservoir 

 size as a function of change in external variables, for 

 example, light intensity. We have chosen to include in 

 this review a more or less detailed description of just 

 this determination of the effect of light intensity upon 

 reservoir sizes as a means of describing the general ex- 

 perimental technique which is involved. 



Steady state and reservoir sizes— Methods and results 



The apparatus used for these experiments was con- 

 structed to permit the algal suspension to be left under 

 controlled external conditions (illumination intensity, 

 temperature, carbon dioxide and oxygen concentration) 

 while photosynthesizing for at least one hour. Further- 

 more, it was required that the change, natural to radio- 

 active carbon dioxide, which was to be circulated in a 

 closed system, and the withdrawal of several samples 

 at given time intervals be accomplished with a mini- 

 mum of change in these conditions. 



The apparatus consisted of : 



(a) A square illumination vessel A (Fig. 7) made out 

 of Lucite (polyacrylic plastic), 49 cm high, 11 cm wide 

 and 0-7 cm thick (inside dimensions). The bottom was 

 provided with a gas inlet tube with five small holes to 

 allow good contact between gas and liquid and a drain 

 tube closed with a screw clamp. The top of the vessel 

 was provided with a gas outlet tube. A water-alcohol 

 mixture from a constant temperature bath was allowed 

 to flow over the outer surfaces of the vessel in order to 

 control the temperature of the suspension. 



Fig. 7.— Diagram of the assembly for steady state photosynthesis. 

 (For explanation of the letters, see text.) 



[b) Two illumination banks (represented by B), each 

 with four fluorescent tubes (General Electric, quality 

 white, 20 W each), providing an almost uniform illumi- 

 nation over the whole surface of the vessel, of 7 x 10* 

 ergs. /cm ^ (roughly 700 footcandles). 



Kig. S.— .\sscnibly for steady state photosynthesis. (For explanation 

 of the letters, sec text.) 



(c) An ionization chamber C, connected to a record- 

 ing vibrating reed electrometer, to record the activity 

 of the gas leaving the vessel continually during the run. 



{d) Three gas traps D, to permit the addition of a 

 known amount of radioactive carbon dioxide to the 

 system, and trap the remaining radioactivity after 

 the run. 



(e) A flask E, of 5 1 volume, containing a mixture of 

 1% radioactive carbon dioxide in air. The reservoir 

 contained so much carbon dioxide that the algae assi- 

 milated no more than 20% of it during a run. 



(/) A gas circulating pump F of the rubber tubing 

 type, and a flow meter G. 



(g) A system of four-way stopcocks H, which per- 

 mitted the vessel to be flushed with a mixture of 1 % 

 ordinary carbon dioxide in air, from the cylinder I. The 

 assembly is shown in Figure 8. 



In a typical experiment, 2 cm' (wet packed) of one- 

 day old Scenedesmus, washed and resuspended in 200 

 cm' of deionized water, were placed in the vessel and 

 aerated with the ordinary gas mixture for at least one- 

 half hour, while the mixture of radioactive carbon 

 dioxide circulated in the gas system for thorough 

 mixing, without passing through the vessel. The sus- 

 pension was kept at 24 °C. After this time, during which 

 a steady state of photosynthesis had been reached, the 

 radioactive mixture was passed through the vessel in 

 place of the ordinary gas mixture, by a manipulation 



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