PHOTOSYNTHESIS OF CARBON COMPOUNDS 451 



This medium was adequate to maintain nearly a constant rate of photos3aithesis 

 in experiment steady state No. i8. In other experiments, such as steady state 28, the 

 algae growing under steady state conditions would in time exhaust the supply of 

 ammonium ion contained in this medium. However, it has been observed that as the 

 algae take up ammonium ion, the pH of the medium tends to decrease, presumably 

 due to the exchange into the medium of hydrogen ions for ammonium ions. Therefore, 

 dilute NH4OH was added to the algae suspension automatically by the pH control 

 system, thereby maintaining constant pH. At the same time ammonium ion concen- 

 tration was maintained approximately constant. The nutrient solution for pH 

 control was diluted by trial and error until its addition kept the algae density constant. 

 To it were added other inorganic ions in a ratio to the ammonium ion which was 

 estimated to provide the algae with an adequate level of these ions for growth for a 

 limited period. The resulting pH control medium used in steady state experiment 28 

 is shown in Table II. 



TABLE II 



CONTROL MEDIUM USED IN STEADY STATE EXPERIMENT 28 



(NH^jHPO^ 6.6 mg/1 



(NHi)jS04 6.6 mg/1 



NH4OH 0.55 mg/1 



FeClj-6H20 50 mg/l 



KCl 8.0 mg/1 



Trace elements as in starting medium 



Administration of^*C 



During the first part of the experiment the algae are kept photosynthesizing in 

 the light with a constant supply of 1.5 to 2 % unlabeled CO^ in air for 0.5-1 h. Constant 

 pH, temperature, and light intensity are maintained during this time, and during 

 the subsequent exposure to "COg. In the experiments reported here the pH was kept 

 at 6. Rate measurements of CO2 uptake and O2 evolution are made by making the 

 closed system small, 435 ml for a few minutes, and observing the rate of change of 

 CO2 and O2 tensions as indicated on the recorder. The closed gas system is made 

 large again, and at zero time, ^^COg is added to the system by turning a stopcock. 

 At the same instant a solution of NaH'^COj is injected directly into the algal suspen- 

 sion. The amount and specific radioactivity of the injected bicarbonate solution is so 

 calculated that it will immediately bring the specific radioactivity of the dissolved 

 COg and bicarbonate already present in the algal suspension to its final value. This 

 is the specific radioactivity which will obtain for all the CO 2 and bicarbonate in the 

 gas and liquid phases of the closed system after complete equilibration has occurred. 

 An example of this calculation is given in Table III. Samples of the algae suspension 

 of uniform size are taken every 5 or 10 sec for the first few minutes, and then less 

 frequently for periods up to i h. Each sample is taken directly into 4.0 ml of methanol 

 (room temperature) in a centrifuge tube (preweighed) . Sample tubes are reweighed 

 to give the sample size (± i %). After an hour at room temperature, the samples are 

 centrifuged and the 80 % methanol extract removed, i ml of methanol is added to 

 the residue and stirred a few minutes, then 4 ml water is added and the mixture 



107 



