STUDIES ON CHLORELLA VULGARIS II. FURTHER EVIDENCE THAT CHLORELLA CELLS FORM A GROWTH‐INHIBITING SUBSTANCE

PREVIOUS STUDY of the growth of the unicellular green alga Chlorella vulgaris showed that under the conditions employed the maximum density of population attained in different cultures is independent of the size of the inoculum, that the rate of multiplication throughout the growth period in different cultures varies inversely with the initial density of population, and that the rate of multiplication, as measured by the increase in number of cells per hour per cell in a given culture, decreases during nearly the entire period of growth (Pratt, 1940). It was pointed out that the data could be interpreted as affording evidence of the presence of a growth-inhibiting substance that was produced by the cells. If such an agent is liberated into the culture medium as the result of the metabolic activities of the cells, it should be possible to measure its effects by studying the increase in the population of cultures prepared with media in which some growth has already occurred. Further experiments and data bearing on this subject are presented herewith. MATERIALS AND METHODS. -General. -The experimental technique was essentially the same as that which was previously employed except that in the present work cell counts were made less frequently than in the earlier work and were obtained by averaging haemacytometer counts of two samples withdrawn from single cultures instead of four samples taken from triplicate cultures. The values obtained in the present work approximated reasonably closely those obtained for comparable cultures in the detailed earlier work. Unless otherwise stated, cells for all experiments were taken from four-day-old, rapidly growing stock suspensions and were washed in three changes of distilled water by repeated centrifugation and decantation. Preparation of nutrient media. -A number of cultures were prepared as previously described (Pratt, 1940) with an initial population of 100 cells/cu. mm. and were permitted to grow for different lengths of time. The culture solution was prepared with distilled water and contained KNO3, 0.025 M. ; MgSO4 7H21, 0.02 M. ; KH2PO4, 0.018 iLI. ; FeSO4 7H20, 0.00001 M. ; potassium citrate 0.00001 M. ; and Zn, 0.1 ppm. ; Mn, 0.1 ppm. ; B, 0.05 ppm. ; and Cu, 0.001 ppm. A gas mixture containing 5 per cent CO2 and 95 per cent air was bubbled continuously through the solutions, and the cultures were illuminated continuously by Mazda lamps yielding about 20,000 lux at the level of the flasks. The temperature at the level of the flasks was about 200C. 20. At suitable intervals several of the cultures were centrifuged and the supernatant solution was drawn 1 Received for publication March 20, 1940. through sintered Jena glass filters, number 1 G 4. The filtrate was then used, either diluted or undiluted according to the experiment, to prepare new nutrient solutions. Since it was not feasible to analyze the different solutions to ascertain the precise amount of each salt that had been removed during the growth period, uniform quantities of the salts employed were added to each solution and the pH was adjusted by the addition of H2SO4 to 4.45, the pH of similar media prepared with distilled watpr.2 The total salt concentrations were not unifor jm in all solutions, therefore. The minimum concentration was 0.063 M. (media prepared with distilled water). The maximum concentration could not have exceeded 0.12 M. (media prepared with 90 per cent filtrate and 10 per cent distilled water), and may have been considerably below that value. In no instance was there any evidence of growth over a period of two weeks in uninoculated media so prepared. It may be concluded, therefore, that the treatment effectively removed all cells. EXPERIMENTS AND RESULTS. -Influence of concentration of nutrient solution on the growth of Chlorella. -Since the concentrations of the salts in the different solutions were not identical, it seemed desirable to ascertain the influence of the concentration of the culture medium on growth. Figure 1 shows the results of this investigation. It is evident that the variations in concentration were not significant for the work reported below, since the total molar concentration of the nutrient medium exerted relatively little influence on the rate or amount of growth of Chlorella between 0.01 M. and 0.1 M. The inset shows that the final cell counts in solutions containing 0.01 mole and 0.1 mole per liter were 97 and 95 per cent, respectively, of those in the standard control solution which contained 0.063 mole per liter. The several salts and the micrometabolic elements were furnished in the same proportions in each solution. Only the total concentration was varied. In preliminary experiments it was found that fair growth occurred in filtrate diluted to twice its original volume with distilled water. This seems to afford ample evidence that depletion of nutrients is not a primary factor in determining the final level or gradually decreasing slopes of growth curves of Chlorella cultured under the experimental conditions employed. However, since it was found that the concentration of the standard solution could be doubled without seriously impairing growth, the filtered medium was fortified in the present work by an addi2 As growth proceeds in the standard solution employed in this work, the pH value rises from an initial level of 4.45 to approximately 6.5-7.0.