882 EXTERNAL AND INTERNAL FACTORS CHAP. 26 



fig. 26.1 IB, is sufficient for saturation). Figure 26.12 shows the dedine 

 of the rate with advancing age; after 30 days, the rate was only one third 

 the original — in fair agreement with Wassink and Katz's results in figure 

 26.10. Respiration also declined during the same period, but a httle less 

 than photosynthesis (to about one half the initial rate). The concentra- 

 tion of chlorophyll and the size of the cells showed no marked change. 



The influence of the inhibitor produced by aged cultures can be recog- 

 nized even in the "second generation". The cultures prepared by inocula- 

 tion with a 3 day old culture showed, after 5 days' growth, a 10% higher 

 rate of photosynthesis than a similar culture prepared by inoculation with 

 material from a 23 day old culture. During the 5 days' growth, the num- 

 ber of cells has increased by a factor of 20, but the effect of the inhibitor 

 was noticeable even after this dilution. 



Pratt, Oneto and Pratt (1945) found that in Chlorclla cultures grown 

 in continuous light in inorganic medium, the inhibiting agent ("chlorel- 

 Hn") accumulated in the medium rapidly in the first 3-4 days; its con- 

 centration decreased sharply in the next 2-4 days, and then grew again, 

 reaching a constant level in about 2 weeks. The decline in chlorellin con- 

 centration occurs at the period when the increase in the number of cells 

 per unit volume is most rapid. Possibly, rapidly dividing cells use up all 

 the available chlorellin for their own metabolism (assuming it is a useful 

 product) ; alternatively (if chlorellin is merely a poison, not only for bac- 

 teria but also for the algae themselves), these cells may produce an anti- 

 dote to this poison. 



"Chlorellin" production was also studied, in co-operation with Pratt 

 and his group, by Spoehr and co-workers (1944, 1945, 1946). They worked 

 on improvement of the methods of cultivation of algae and extraction of 

 the antibiotic, first using cell-free culture medium, and later, the cells 

 themselves. The quantity of antibiotic extractable from dried cells was 

 found to increase by allowing the latter to undergo oxidation (by grind- 

 ing and exposing to light in air). Brown, partly crystalline material, ex- 

 tracted from dried cells with 80% methanol containing 2% KOH, and 

 transferred into petroleum ether after acidification, is nonantibiotic; it 

 becomes colorless and antibiotically active by oxidation in light. It thus 

 seems that living cells contain no antibiotic material ; the latter is formed 

 by oxidation, both in the medium and in dried and ground cells (it remains 

 to be proved that the two antibiotic products are identical). The cell- 

 derived antibiotic is a lipoid — probably a mixture of unsaturated fatty 

 acids. Pure acids of this type (linoleic, elaidic, etc.) also show no anti- 

 biotic activity before exposure to oxygen and light, but acquire antibiotic 

 properties after such exposure. Autoxidation of unsaturated compounds 

 is known to produce peroxides whose bactericidal effect is well established. 



