Different molds are employed for different methods of production. The strain isolated by Fleming was employed for all of the early studies, and in early tests made at this laboratory it was found to produce higher yields than any other unimproved strain when grown in surface culture. The Fleming strain was observed to be quite unstable in laboratory culture, and substrains possessing different cultural characteristics could be separated from it. Although most of the latter failed to equal the productivity of the parent strain, one of them, designated NRRL 1249.1321, produced yields approximately double those of the parent culture and raised the titre, or penicillin content, from about 75 to 100 units per milliliter to 150 to 200 units per milliliter. It was made available to producers and was thereafter generally employed for the production of penicillin by the surface-culture method.

More spectacular success has been achieved in developing improved cultures for the production of penicillin in submerged culture. As it became apparent that the submerged fermentation was industrially feasible, the need for developing higher-yielding submerged cultures was recognized. Strain NRRL 832, the culture employed for this type of production, was studied intensively. Efforts to obtain from it a natural variant characterized by substantially increased production were unsuccessful. Attention was then directed toward the isolation of new strains from nature. Previous work had shown that almost all members of the P. notatum-chrysogenum group produced some penicillin. It seemed probable, therefore, that new strains possessing greater productive capacity than NRRL 832 might be obtained if a large number of isolates were examined. Such a search was undertaken early in 1943.

New cultures were obtained from moldy food products, fruits and vegetables in early stages of spoilage, and from fertile soil collected from various stations in the United States and from many foreign countries.

The most important culture discovered, however, was isolated from a moldy cantaloupe in Peoria. The culture represented a strain of Penicillium chrysogenum Thom, a species closely allied to P. notatum, and was designated NRRL 1951 in our collection of cultures. When first studied, it produced penicillin in slightly greater yields than NRRL 832, but within a few months a natural variant, which more than doubled the amount, was developed from it. This substrain, designated NRRL 1951.1125, was studied intensively here, and was at the same time made available to the penicillin industry in 1944. It was soon generally adopted for submerged production.

Faced with the demand by the armed forces for ever-increasing amounts of penicillin, the Office of Production Research and Development of the War Production Board early in 1944 set up projects at the University of Wisconsin, Stanford University, and the Carnegie Institution of Washington to discover or develop more productive cultures. Largely because of the work already done at the Northern Regional Research Laboratory, it then seemed probable that cultures capable of producing greatly increased yields of penicillin might be obtained by one or more of the following means: The isolation of new strains from nature; the selection of natural variants from such new stocks; and the production of induced mutations from known good producing strains by X-ray and ultraviolet radiation, or by other artificial means.

At the Carnegie Institution a mutation was produced that possessed Outstanding merit. This culture, designated X-1612, was produced by X-ray radiation of spores of NRRL 1951.B25. It was first tested at the University of Minnesota, but its real potentialities were established at the University of Wisconsin in small vat fermenters. The superiority of the strain was subsequently verified at this laboratory. Yields more than twice those produced by NRRL 1951.1125 were obtained from X-1612 and it soon supplanted the parent culture as the principal strain for commercial production. Another great step forward was made by exposing spores of X-1612 to ultraviolet. In this way, the Wisconsin group succeeded in producing a mutation, designated Q-176, which doubled the yield produced by strain X-1612. The development of this outstanding culture for submerged production can be summarized as follows:

Table 5

The importance of the foregoing developments to present penicillin production cannot be overemphasized, because current yields of 750 to 900 units per milliliter are obtained in nutrient solutions of approximately the same composition as those used to produce maximum yields of 75 to 100 units per milliliter with NRRL 832 just a short time ago.

Some difficulties were encountered when these high-yielding strains were first adopted for commercial production, for they were found to produce primarily penicillin K, a type that is rapidly destroyed in the animal body and hence is much less useful clinically. However, if phenyl-acetic acid or phenylacetamide is added to the production medium, these strains can be made to produce primarily the more useful penicillin G. This procedure has been adopted by industry.