STUDIES ON CHLORELLA VULGARIS. XL RELATION BETWEEN SURFACE TENSION AND ACCUMULATION OF CHLORELLIN

A SERIES of investigations on Chlorella vulgaris reported in this journal between 1940 and 1943 (Pratt et al.) showed that thjere is produced in cultures of this organism a substance or complex of substances capable of inhibiting the growth of Chlorella and respiration and photosynthesis in Chlorella and other algae. It was shown to possess antibiotic activity in vitro against several Gram-positive and Gram-negative pathogenic bacteria, and for convenience of reference it was designated by the name chlorellin (Pratt et al., 1944). Later it was shown that chlorellin accumulates in the culture solution when growth in population is relatively slow and that during the period of most rapid increase in density of population it disappears from the external solution (Pratt et al., 1945). Spoehr et al. (1945) observed that chlorellin accumulates in the nutrient solution under conditions that previously had been shown to favor production of more highly reduced carbon compounds, such as fats and hydrocarbons, in Chlorella cells. Extracts prepared from the cells, and assumed to be similar to those originally obtained from the culture filtrates, were shown to be lipoidal. They concluded that the antibiotic activity of chlorellin (in this instance isolated from cultures of Chlorella pyrenoidosa, but presumably similar to, if not identical with, the material from Chlorella vulgaris) is due to a mixture of unsaturated fatty acids, or at least is associated with such compounds. Since the presence of such substances in a culture medium might be expected to lower the surface tension, it seemed of interest to determine whether a correlation exists between changes in surface tension in cultures of Chlorella and antibiotic activity. This is the subject of the present report. MATERIALS AND METHODS.-The organism used in these studies was the same strain of Chlorella vulgaris that was used previouslv in this laboratory (loc cit.). The cells were cultured in 500 ml. Florence flasks, each of which contained 250 ml. of an essentially inorganic nutrient solution of the following composition: KNO3, 0.025 M; MgSO4 7H20, 0.02 M; KH2PO4, 0.018 M; FeSO47H20, 5X10-5 M; and potassium citrate, 5X10-5 M. The initial density of population in each culture was 100 cells per mm3. The inocula for differentexperiments were always taken from a vigorously growing 4day old culture, one of a long series of 4-day old cultures, each of which had been inoculated from a 4-day old culture. Continuous illumination was provided by two 40-watt white fluorescent lamps in a reflector. The light intensity at the surface of the cultures was 200 f. c. Sterile humidified air was bubbled through the cultures at the rate of approximatelv 5-6 volumes of air per volume of culture I Received for publication May 5, 1948. liquid per minute. This served the dual purpose of aerating the cultures and of continuously agitating them so that the cells were uniformly suspended at all times. No settling occurred. It should be noted that the illumination and aeration differed in these experiments from the conditions previouslv employed. These differences, especially the lack of CO2-enriched air, account for the reduced amount and rate of growth in the present experiments. Due to temporary local conditions it was not practicable to use the customary mixture of 5 per cent CO2 in air. The temperature varied from 220-250C. Surface tension was measured at 220C. by means of a deNouy precision tensiometer and by the capillary rise method. Results obtained by the two methods were in close agreement. EXPERIMENTS AND RESULTS.-Forty flasks were inoculated and then cell counts and surface tension measurements were made daily using samples that were removed from the cultures by means of a pipette. In sampling cultures in which foaming occurred (2 weeks or more in age) the pipette was inserted down into the body of culture so that foam was not included in the sample. In none of the cultures was the foaming of sufficient magnitude to cause any loss of material from the flasks. At appropriate intervals pH determinations were made and cell-free filtrates from the cultures were extracted and the extracts were tested for antibiotic activity as described below. Antibiotic tests were made on extracts from each of three flasks at each time. Subsequent determinations of cell count, surface tension, etc., were made on all remaining cultures. Since the cultures were sacrificed in making extracts, the number of flasks employed in making later observations was reduced by three each time. Thus 40 flasks were used the first day, 37 flasks the second to eighth days, 34 the ninth to eleventh days, etc. The points plotted in fig. 1 are average values of all cultures examined on the particular day. Growth, surface tension and pH.-The points plotted in fig. 1 show the population and accompanying changes in surface tension and in pH values in the cultures at different times. The growth curve (solid circles) is of the characteristic sigmoid shape, although the final height attained and the slope during the logarithmic phase are less than obtained in earlier experiments under different conditions of illumination and aeration (loc. cit.). The irregularities in the later portion of the curve (approximately day 30 to day 50 or 55) may merit special notice in passing. It can be seen that the variations in cell count appear not to vary in a random manner but seem to indicate periods of decrease in cell number alternating with periods of increase at approximately 3-5 day intervals. The