The Lactose-Corn Steep Liquor Medium

Dr. Fleming had employed a nutrient broth as a culture medium, and Florey and his associates produced penicillin in a modified Czapek-Dox solution containing yeast extract and glucose. At the Northern Regional Research Laboratory, the nutrient solution was altered in many ways by A. J. Moyer, microbiologist, and many substances, known to promote the growth of micro-organisms, were investigated for their ability to increase penicillin production. Of such substances, corn-steep liquor, commonly referred to as "steep liquor," was found to be outstanding. At levels below those that actually inhibited mold growth, yields of penicillin were found to increase with the addition of increased amounts of this product. It was recognized that the steep liquor constituted the principal source of nitrogen and apparently contributed other important nutrients necessary for the formation of penicillin.

At the same time various sources of carbon were investigated, including glucose, sucrose, lactose, corn dextrin, and corn starch. Of these different carbon sources, lactose, or milk sugar, was found to be slowly assimilable by the mold and most generally favorable for penicillin production.

The Northern Regional Research Laboratory therefore recommended for the production of penicillin a culture medium whose principal ingredients were corn-steep liquor and lactose. Through Dr. A. N. Richards, chairman of the Committee on Medical Research, Office of Scientific Research and Development, these discoveries were made available to all producers of penicillin in the United States and allied countries. With certain modifications in the proportions of the ingredients used, depending upon the particular mold culture employed and the method of production, this medium has remained in general use.

The pioneer work of Professor Fleming had been done with surface or still cultures, as had also the equally important work of Professor Florey and associates. It was natural, therefore, that the same method should have been followed in our early work, and it was from studies with surface cultures that the lactose-steep liquor medium was developed. Experience with other fermentations, however, indicated that penicillin could, in all probability, be produced at a much lower cost, if a satisfactory tank or submerged fermentation could be developed. Attention was early directed toward this goal. Penicillin-producing molds were inoculated into lactose-steep liquor medium and subjected to continuous and vigorous agitation for several days, during which time the broth was assayed daily for penicillin content. When thus agitated, the mold grows submerged, usually assuming the form of small, rounded pellets.

The original Fleming strain and all substrains derived from it were found to produce disappointingly low yields of penicillin when grown submerged. Different molds in our collection that belonged to the P. notatum-chrysogenum group were then investigated. Another culture of P. notatum, designated NRRL 832, was found to produce promising yields. The details of the work were communicated to other research laboratories and to the producers of penicillin. Strain NRRL 832 was made available to the penicillin industry and for several months in 1943 and 1944 it was responsible for a large proportion of the penicillin production in this country. More productive strains have since supplanted it.

Culture solutions whose principal ingredients are corn-steep liquor and lactose are most favorable for the production of penicillin in both surface and submerged culture. For submerged production, however, the concentration of these nutrients should be approximately one-half that employed for production in surface culture. When increased amounts of steep liquor are employed, the growth of the mold is excessively heavy and the yield of penicillin is markedly reduced. Standard solutions for surface and submerged production were recommended.

Some modifications in the composition of the culture solution for both surface and submerged cultures can be made without seriously affecting Penicillin yields, and such alterations are often desirable when new equipment is employed or a new penicillin-producing mold is investigated.

The two methods of penicillin production worked out in the laboratory have their direct counterparts in industry. Penicillin has been made successfully on a commercial scale by both methods.

In the surface-culture method, the mold is grown upon the surface of a quiescent nutrient solution which is dispensed in flasks, bottles, or trays usually to a depth of 1/2 to 3/4 inch. A common practice is to use bottles of approximately 2-quart capacity and to incubate them on their sides to obtain the greatest possible amount of culture surf ace. This method entails a great deal of hand labor, and manufacturing costs are high. In some industrial plants as many as 30,000 bottles were inoculated each day and the plants were operated on a 6- to 10-day cycle. Maximum penicillin is produced at a temperature of about 24 to 25 C., and large incubating rooms had to be built to accommodate the 200,000 to 300,000 growing cultures.

The surface-culture method has now been supplanted by the submerged process. It was the process first developed on an industrial scale, however, and all the penicillin used in the clinical trials that first established the curative properties of the drug was produced by it.

All penicillin now made in the United States is produced by the submerged, or tank process. Inoculation with the mold may be in the form of spores or growing culture, commonly referred to as preformed inoculum. Fermentation periods vary somewhat, depending upon the mold employed and the equipment used, but commonly range from 2 1/2 to 4 days. Large amounts of sterile air are required and the mold growth must be constantly stirred. The fermentation must be run at a favorable temperature of approximately 24 to 25 C., and measures must be taken to remove or dissipate the heat generated by the vigorously growing mold.

Other methods of producing penicillin have been recommended but have not succeeded in large-scale operations.