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The fermentations were run in 500-ml Erlenmeyer flasks containing 100 ml of medium.Presented in part before the Fermentation Subdivision of the Agricultural and Food Chemistry Division atthe 126th meeting of the American Chemical Society, New York, 1954.The spore inoculum was prepared in the following way: Flat 6-ounce bottles containing about 35 ml of medium (6 per cent honey, 1 per cent Difco Bacto-peptone, 2 per cent agar) were seeded with spores from a soil stock.The flasks were inoculated with 4 ml of a mycelium suspension grown in medium containing 6 per cent dextrin and 2 per cent corn steep solids.2.5 4.0 4.0 4.0 5.0 5.0 Sodium sulfate or sodium thiosulfate pentahydrate...........Analytical methods.2.0 2.0 2.5 3.0 3.0 3.5 Lactose........................2

The fermentations were run in 500-ml Erlenmeyer flasks containing 100 ml of medium. The flasks were aerated by shaking on a rotary shaker describing a 2-in circle at 250 rpm. The fermentation temperature was 24 to 25 C. As a rule, 3 replicate flasks were used in each experiment. The media had the composition given in table 1. Medium I was used in the first series of fermentations and medium III, a richer medium, was designed on the basis of the data obtained with the first medium. The other media, most of which are more concentrated than III, were' used in a few experiments to show what combinations of corn steep and lactose would be best for the new cultures under our experimental conditions. The pH of the media was adjusted with strong potassium hydroxide to 5.2 to 5.6 before sterilization. Lard oil, containing 3 per cent octadecanol, was used as an antifoam agent. It was added as described in the individual experiments. Phenylacetic acid (PAA, a solution of the potassium salt containing 5 per cent PAA) was added as a precursor, usually in increments of 0.05 per cent beginning at 24 hours and at intervals of 24 hours thereafter until near the end of the fermentation. From 4 to 6 additions were made giving a total of from 0.2 to 0.3 per cent. In a few experiments, a 5 per cent solution of #3-phenylethylamine (PEA) neutralized to pH 6.0 with H2SO4 was used as precursor. When extra lactose was added to the fermentations, a 15 per cent W/V solution was used. Ammonium acetate was added as a 20 per cent solution. The flasks were inoculated with 4 ml of a mycelium suspension grown in medium containing 6 per cent dextrin and 2 per cent corn steep solids. One hundred ml of the medium in a 500-ml Erlenmeyer flask was inoculated with 5 ml of a spore suspension and the flask was shaken for 40 to 48 hours. The spore inoculum was prepared in the following way: Flat 6-ounce bottles containing about 35 ml of medium (6 per cent honey, 1 per cent Difco Bacto-peptone, 2 per cent agar) were seeded with spores from a soil stock. The bottles were incubated on the flat side at 25 C for 7 to 10 days for growth and sporulation and were stored at 4 C until needed. The spore suspension was made by adding 25 ml of sterile water to a bottle and loosening the spores from the agar surface with an inoculation loop. Analytical methods. Samples of the broth were taken from the fermentations at desired intervals and the pH was measured with a glass electrode. After filtration, certain other analyses were made on the broth. The penicillin titer was determined by a modification of the cylinder-plate method of Schmidt and Moyer TABLE 1. Composition of media used for penicillin production Component* Component* Medium ~ I I II I III IV V VI g per 100 ml 1 This investigation was supported in part by research grants from Bristol Laboratories, Inc. and Cutter Laboratories. Presented in part before the Fermentation Subdivision of the Agricultural and Food Chemistry Division atthe 126th meeting of the American Chemical Society, New York, 1954. 2 Fellow of the Sweden-America Foundation. Corn steep solids (C. S. S.) ...... 2.0 2.0 2.5 3.0 3.0 3.5 Lactose........................ 2.5 4.0 4.0 4.0 5.0 5.0 Sodium sulfate or sodium thiosulfate pentahydrate........... 0.1 0.1 0.1 0.1 0.1 0.1 Ratio C. S. S. perlactose........ 0.8 0.5 0.630.750.6 0.7 * In addition, the medium contained 0.4 per cent CaCO3 unless otherwise stated. Downloaded from https://journals.asm.org/journal/am on 28 November 2024 by 41.201.97.205. 2