106 The Maximum Efficiency of Photosynthesis 



two-vessel method under favorable conditions. But the irregulär behavior of the 

 cells employed, especially their long latent periods, seemed to us so unsatisfactory 

 that we decided to reinvestigate from the beginning the problem of the efficiency 

 of photosynthesis. 



While our new investigation was proceeding in Bethesda in early 1949, a series 

 of papers on the efficiency problem appeared in book form in "Photosynthesis in 

 Plants" 8 . The results reported in this volume seemed to be conclusive and final: 

 with three different and independent methods — manometric, Polarographie, and 

 calorimetric — minimum quantum requirements of 10 — 12/molecule of O2 pro- 

 duced were obtained. 



New Experiments 



1. Cültivation of Chlorella 



A strain of Chlorella pyrenoidosa, isolated and identified many years ago by Dr. Florence Meier 

 of the Smithsonian Institution, was eultivated in Drechsel gas washing bottles containing 200 ml. 

 of the following sah Solution: 5 g. MgS0 4 ■ 7 H-jO, 2.5 g. KNO3, 2.5 g. KHTO4, 2 g. NaCl, and 

 5 mg. FeSOi • 7 H^O containing Zn impurity, in 1 1. of filtered unsterilized well water (pH of the 

 Solution 4.5 — 5.0). The eultures were maintained at room temperatures of 25 — 30 C. and were 

 aerated with 5% CO2 in air at a rate of about 500 cc. gas/min., rapid enough to prevent cell Sedi- 

 mentation, and were constantly illuminated with a 100-watt incandescent lamp at a distance of 

 about 30 cm. The inoculum was 100 cu. mm., sometimes even 200 cu. mm., of cells per flask. 

 When after a few days the cells had multiplied to 600 — -800 cu. mm., they were washed in fresh 

 eulture medium in a No. 2 International Centrifuge at the lowest speed giving nearly complete 

 settling in about 10 min., and were then either reinoculated or used for efficiency determinations. 

 Bacterial growth was found to be negligible, due to the aeid reaction, the lack of added organic 

 matter — in the salt Solution, and possibly antibiotics produced by the Chlorella. 



Cultivated by this method, used for many years by one of us (D. B.), the Chlorella had a relatively 

 stable respiration and gave high photosynthetic efficiencies. The main drawback of the old eulture 

 method 1 was very likely the cell Sedimentation, which unfortunately was praised as an advantage 

 in 1922 and was therefore favored by later investigators. In the course of the years the tall Erlen- 

 meyer flasks originally employed became broader, and the aeration became so slow that the greater 

 part of the cells sedimented and suffered in the Sediments from lack of oxygen*, carbon dioxide, 

 and light. 



There still remains the question as to the seeming superiority of well water over 

 distilled water. Thus far, high photochemical efficiencies have been reported only 

 for Chlorella cultivated with tap, well, or lake water, the active substances of 

 which, if any, must be widely distributed, since equally efficient yields have been 

 obtained with waters from Berlin, Liebenberg, Bethesda, and Woods Hole. It is 

 probably safer, for the present, and in the absence of specific investigation, to 

 continue with the use of well or natural waters for the eultures. 



2. Intermittency of Illumination 



When Chlorella in optically thin suspensions is illuminated with increasing light 

 intensities, the rate of O2 produetion at first increases proportionally to the light 

 intensity, then increases more slowly, and finally, at "saturation intensity", be- 



*In a recent paper" on the eulture of Chlorella, carried out by the methods of 1922, damage 

 from lack of oxygen was cited as being due to nitrite poisoning. This is not true generally, but 

 only when the salt medium contains free nitric aeid 1 ( p- 385 )- 



