450 J. A. BASSHAM, M. KIRK 



the water is circulated in a thermostated bath. The temperature of this bath is set so 

 that during steady state photosynthesis the temperature indicated by the thermo- 

 meter in the algae suspension reads 25°. 



The algae chamber is connected to a side loop through which the algae suspension 

 is made to circulate by the flow of gas into the chamber. A beam from a small lamp 

 passes through a window in the side loop to a photovoltaic cell which measures the 

 light absorption and hence the density of the algae. Electrodes in the side loop 

 measure pH, which is recorded on a multipoint recorder. The pH meter output is also 

 connected to a control relay which, through the activation of a solenoid-operated 

 valve, can cause acid or base from a reservoir to be added in small volumes to the 

 algal suspension. Another reservoir within the closed system contains distilled water 

 or nutrient solution, which can be added to the algal suspension to dilute it to the 

 selected concentration as the algae grow. 



A solenoid-operated sampling valve at the bottom of the chamber permits one 

 to take I -ml samples rapidly (every 2 sec if desired). The inside of the algal chamber is 

 maintained at slightly above atmospheric pressure to force the algal sample out of 

 the chamber. When samples of algae are taken, they are run into 4 ml of methanol 

 at room temperature. This gives a mixture which contains about 80 % methanol 

 by volume. No significant difference in the resulting labehng pattern is seen whether 

 the algae are killed this way, in boiling ethanol, or in ethanol kept at — 40°. 



After the gas in the closed system bubbles through the algae, it passes through 

 instruments which measure COg, ^*C, and Og, and each measurement is automatically 

 recorded. From the known sensitivities of these instruments and the volume of the 

 system, one can calculate rates of exchange of these quantities and specific radio- 

 activity. A large reservoir and small reservoirs may be connected or disconnected 

 from the closed system to obtain closed systems of various sizes. The volume of 

 the largest system is 6400 ml, while the volume of the smallest system is 435 ml. The 

 system can be open during the pre-labeling period by means of a stopcock. 



Nutrient solution 



For steady state experiments it is necessary to supply the algae with all the in- 

 organic compounds required for them to photosynthesize and grow at a normal rate. 

 Unfortunately, the nutrient solution in which they are usually grown in the laboratory 

 contains quantities of salts which make impossible an adequate separation of labeled 

 compounds by two-dimensional paper chromatography. Therefore, the algae are 

 suspended in much more dilute nutrient solutions of which that in Table I is typical. 



TABLE I 



STARTING NUTRIENT SOLUTION FOR STEADY STATE EXPTS. l8 AND 28 



(NH^JijHPO^ 40 mg/1 



MgSOi-yHjO 2omg/l 



NH4CI 20 mg/1 



KNO3 20 mg/1 



Arnon's A-4 solution of trace elements plus 



CoClj-6HjO (40 mg/1) and M0O3 (15 mg/I)i2 i ml/1 



Fe'''+-versenol solution to give 90 xaM Fe++ i ml/1 



NH.VOj (23 mg/1) I ml/I 



106 



