The fate of "4C-labeled 2,4,6-trinitrotoluene (TNT) in an activated-sludge system was investigated. No [14C]TNT could be detected in the contents of an aerated reactor after 3 to 5 days of incubation. No significant 14C02 was formed, and the radioactivity was about equally divided between the floc and the supernatant. The radioactive carbon present in the microflora was mainly associated with the lipid and protein components, but the characteristic constituents of these compounds (e.g., fatty acids and amino acids) were not radioactive. The

Enterotoxin B, nuclease, and total exoprotein production by Staphylococcus aureus strain S-6 was studied in a 0.5-liter fermentor system. While these extracellular products were elaborated over a wide range of aeration rates, maximal production occurred within the very narrow range of 125 to 150 cm3 of air per min. The levels attained at the optimal aeration rate were not increased by maintaining a constant pH, although yield of enterotoxin:cell mass was highest at a constant pH of 7.0. During the growth cycle of the cultures, when aeration rate alone or aeration rate and pH were held constant, the dissolved oxygen (DO) levels, initially set at 100% of saturation, decreased to 5 to 10% 4 to 5 h after inoculation. The oxygen demand of the culture then maintained this level for an additional 4 to 6 h. This interval of low DO was characterized by maximal growth and exoprotein production. When the DO was controlled at a constant value throughout growth (by increasing or decreasing the airflow rate as appropriate), the culture demonstrated different optima for maximal growth and exoprotein production. A constant DO of 100% stimulated growth to extremely high densities, but the accumulation of toxin and nuclease was not observed. On the other hand, maintaining constant DO levels at 50 or 10% raised exoprotein levels higher than those achieved in a culture grown at the optimal aeration rate. Compared to the optimal aeration rate culture, the 10% DO culture yielded 20% more nuclease, 25% more toxin, and 40 to 50% more total exoprotein. These results indicate that it is the DO and not the aeration rate, per se, that is influential in controlling growth, toxin, nuclease, and total exoprotein production.

The production of enterotoxin A and nuclease by Staphylococcus aureus strain 100 was studied in a 1.0-liter fermentor. The effects of the gas flow rate, pH, and dissolved oxygen were evaluated. Toxin and nuclease secretion occurred under all conditions which permitted growth of the organism. Final yields of toxin and nuclease in cultures grown at constant air flow rates, ranging from 50 to 500 cm3 per min, were higher at successively higher flow rates. An optimum flow rate for either toxin or nuclease production was not observed. When the aeration rate alone or aeration rate and pH were held constant, the dissolved oxygen levels in the culture decreased from the initial 100% level to 0 to 5% 3 to 4 h after inoculation. The O2 demand of the culture then maintained this level for an additional 4 to 5 h. This low dissolved oxygen interval was characterized by rapid growth and extracellular protein production. Controlling the dissolved oxygen at a constant level throughout growth did not increase the final levels of toxin and nuclease above those achieved at the respective constant pH values. Growth under the influence of a constant aeration rate of 500 cm3 per min and a constant pH of 6.5 and 7.0 yielded the highest titers of nuclease (1,550 units/ml) and toxin (10.5 mug/ml) obtained in any of the fermentations conducted in this study. Sparging fermentor cultures with pure oxygen at a rate of 100 cm3 per min yielded growth and extracellular protein levels similar to those achieved at the sparge rate of 500 cm3 of air per min. Controlling the dissolved oxygen at 100% of pure oxygen saturation appeared to inhibit the culture, as the final cultural turbidity as well as the levels of toxin and nuclease were reduced. These data indicate that enterotoxin and nuclease secretions are closely associated with the growth of strain 100. Analyses of the production rates of these components indicated that early log phase was the most efficient production interval in the growth cycle and that this efficiency was increased by pH control at 6.7 to 6.8 and dissolved oxygen control at 10% of air saturation.

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