THE PRODUCTION OF PENICILLIN X IN SUBMERGED CULTURE

KENNETH B. RAPER AND DOROTHY I. FENNELL

Fermentation Division, Northern Regional Research Laboratory, Peoria, Illinoise

Received for publication March 11, 1946

It is now generally known that members of the Penicillium notatum-chrysoge-num group are able to produce at least four different penicillins, known as F, G,X, and K,2 when grown under the usual conditions of laboratory or commercialculture. It is likewise recognized that two or more of these penicillins may occurin the same culture broth, the proportions being influenced to a considerabledegree by the particular strain used and the specific cultural conditions employed.Since these penicillins differ in chemical characteristics and in their inhibitoryeffect upon susceptible bacteria, it has long seemed reasonable that they mightpossess different possibilities in clinical application. The first clinical evidencein support of this possibility was provided by Welch, Putnam, Randall, andHerwick (1944). They reported that penicillin X in vitro was more effective thancommercial penicillin (primarily penicillin G) against a strain of Klebsiella pneu-moniae type A and Bacillus cereus, and that it was three to five times as effectiveas commercial penicillin in protecting mice against 10,000 lethal doses of pneumo-coccus type I. The same investigators found penicillin X to offer particularpromise in the treatment of gonorrhea: bacteriological cures were obtained in 64of 68 cases (94 per cent), mostly sulfonamide-resistant, which were treated withsingle 25,000-unit intramuscular injections; whereas only 37 cures were obtainedin 58 comparable cases (64 per cent) treated with single 25,000-unit intramuscularinjections of commercial penicillin. They further observed that patients givenpenicillin X maintained a consistently higher blood concentration, and that thispenicillin was excreted into the urine more slowly than commercial penicillin. Amore recent report by Ory, Meads, and Finland (1945) confirms the earlier workof Welch et al. (1944). Group A hemolytic streptococcus, gonococcus, andmeningococcus were found to be from two to eight times more sensitive to prepa-rations containing 65 or more per cent of penicillin X than to commercial peni-cillin (primarily penicillin G), whereas most strains of pneumococcus andStreptococcus viridans were twice as sensitive to penicillin X. As reported byWelch et al. (1944), staphylococci were found to be equally sensitive to the twokinds of penicillin. Levels of penicillin activity in the serum were significantlyhigher and were sustained longer after intramuscular injections of penicillin Xthan after injections of the same number of units of commercial penicillin. Sub-sequent to that work, Libby and Holmberg (1945) found penicillin X to be moreeffective than penicillin G against a number of different bacteria including various

1 This is one of four regional laboratories operated by the Bureau of Agricultural andIndustrial Chemistry, Agricultural Research Administration, U. S. Department ofAgriculture.

2 These penicillins are known as I, II, III, and IV (?), respectively, in the United King-dom (Coghill and Koch, 1945).

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7KENNETH B. RAPER AND DOROTHY I. FENNELL

streptococci and pneumococci, and Flippin and associates (1945) reported thesuccessful use of penicillin X in the treatment of a case of bacterial endocarditis(Streptococcus viridans) which had failed to respond to repeated administra-tions of commercial penicillin.

Realizing that the different penicillins might have special applications, mem-bers of the Fermentation Division had given some attention to the developmentof culture conditions favoring the production of particular penicillins before theappearance of the paper by Welch et at. (1944), but no concerted attempt hadbeen made to select, isolate, or develop an organism characterized by the pro-duction of increased amounts of a particular penicillin as opposed to an increasein total yield. Because of the possible-important clinical role of penicillin X,and because of its interesting chemical possibilities, such a course now seemedwarranted.The presence of penicillin X was observed first in surface cultures of NRRL

1249.B21, a derivative of the Fleming strain (Raper, Alexander, and Coghill,1944; Raper and Alexander, 1945; and Moyer and Coghill, 1946a); and thematerials employed by Welch et al. (1944) and Ory and associates (1945) wereundoubtedly of this origin. Surface culture trials made at this laboratory showedthis strain to be the best penicillin X producer among ten selected cultures withmaximum total yields of 155 to 160 units per ml, of which 50 to 57 units per ml,or about 30 to 35 per cent, represented penicillin X. Although this ratio wasvery promising, it was realized that if penicillin X were to be made in quantity,some strain capable of producing substantial amounts in submerged culture wouldbe required, and no derivative of the Fleming culture had ever been found satis-factory for this method of production. Attention was thus directed toward good"submerged" strains. Differential assays (Schmidt, Ward, and Coghill, 1945;and Schmidt, 1946) were made upon samples taken from rotary drum and vat fer-mentations then being conducted by Dr. G. E. Ward and associates (unpublisheddata). No detectable amount of penicillin X was found in cultures of Peniciltiumchrysogenum NRRL 1951.B25, or either of three substrains derived from it,including X-1612, the highest known producer of penicillin in submerged cul-ture. However, yields of this penicillin amounting to somewhat more than 15per cent of the total were produced by P. chrysogenum NRRL 1984.A. Withthis background, the writers set about in March, 1945, to secure, if possible, asubstrain of 1984.A capable of producing substantially increased ratios of peni-cillin X. By the end of August such a strain had been developed and its per-formance repeatedly verified. The present report is concerned with this de-velopment.

MATERIALS AND METHODS

Molds included in study. A considerable number of different cultures wereinvestigated during the present study. To individuals generally familiar withthe whole penicillin development in this country, the origins and numericaldesignations of these strains will be commonplace. In the belief that some in-formation regarding the source and known potentialities of these cultures will

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prove of interest and value to other readers, a somewhat detailed list is presented.NRRL 1249.B21 Penicillium notatum, derived from the Fleming culture

(Raper and Alexander, 1945), a substrain developed at the Northern RegionalResearch Laboratory which produced high yields of penicillin in surface culture(Raper, Alexander, and Coghill, 1944, and Moyer and Coghill, 1946a). Thisstrain has been used in industry more than any other for the surface productionof penicillin in this country and abroad.NRRIL 832.A2 P'enicillium notatum, a substrain of NRRL 832 which normally

produces somewhat higher yields of penicillin than the parent (Raper and Alexan-der, 1945). The latter culture was the first mold found to produce good yieldsof penicillin in submerged culture (Raper, Alexander, and Coghill, 1944; Moyerand Coghill, 1946b), and was generally employed in industry for this type of pro-d(uction during the period prior to the summer of 1944. Maximum yields ofabouit 100 units per ml have been obtained.

NRRIL 1951.1B25 Penicillium chrysogenum, a substrain developedl at theNorthern Regional Research Laboratory from NRRL 1951 (isolated from acantaloupe collected in Peoria, Illinois), which produced substantially greateramounts of penicillin in submerged culture than NRRL 832 and substrainsderived therefrom (Raper and Alexander,1945). Yields in excess of 200 units perml have been obtained in large-scale laboratory apparatus. This cultture waswidely used in industry for submerged production during the latter half of 1944and the early months of 1945. It found limited use for surface production, beingless sensitive to temperatures in excess of 24 C than NRRL 1249.B21. It wNasselected as a promising strain for X-ray and ultraviolet irradiation, and anumberof noteworthy substrains were developed from it (see below).

Stanford 25099 Penicillium chrysogenum, a substrain of NRRL 19511.B25 re-

sulting from conidia exposed to ultraviolet radiation, distributed by ProfessorG. WV. Beadle and associates as a superior submerged culture and studied inmany laboratories.

Stanford 35217 Penicillium chrysogenutm, a substrain of NRRL 19511.B25 re-stulting from conidia exposed to ultraviolet radiation, distributed by ProfessorlBeadle and associates as a superior submerged culture. As studied in manylaboratories, this substrain generally produced somewhat better yields than theparent cullture, and it has found limited use in industry.X-l612 Penicillium chrysogenum, an X-ray-induced mutation of NRRLI,

19511.B25, produced by Dr. M. Demerec and associates at the C(arnegie Instittuteof Washington, Cold Spring Harbor, New York, tested by Professor C. M.Christensen and associates at the University of Minnesota, and forwarded toProfessors W. H. Peterson and M. J. Johnson of the University of Wisconsin as a

superior strain. When tested in 80-gallon fermenters by the latter investigators,this mutant was found to be an exceptionally good penicillin producer, and yieldsin excess of 500 units per ml were obtained (unpublished data). The strain wassubsequently tested at the Northern Regional Research Laboratory (with yieldsup to 450 units per ml) and other laboratories, and its superior qualities were

confirmed.

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764 KENNETH B. RAPER AND DOROTHY I. FENNELL

NRRI, 1984 IPenicillium chrysogenum, received as Minn. R-13 from ProfessorC. M. Christensen of the University of Minnesota where it was isolated fromMinnesota soil. The culture was recommended for submerged production andrepresents the parent of commercially important strains.

,r

FIG. 1. APIPARATIUS AND TECHNIQUE EmPLOYED FOR THE IRRADIATION OF PENICILLIUIMSPORES

Minn. 9SS251 Penicilliuim chrysogenum, a substrain of Minn. R-13, of mono-spore origin, selected at the University of Minnesota and distributed as a high-yielding submerged culture.NRRI, 1984.A Penicilliuim chrysogenum, a substrain of NRRI, 1984 (Minn.

R-13) selected at the Northern Regional Research Laboratory and found inrepeated trials to produce somewhat greater yields than the parent. Thisstrain has been intensively studied at the NRRL and is successfully used inindustry. Yields in shaken flasks up to 150 to 160 units per ml are not uncom-

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PRODUCTION OF PENICILLIN X IN SUBMERGED CULTURE

mon, and yields up to 260 units per ml have been obtained in rotary drumfermenters.NRRL 1984.N22 Penicillium chrysogenum, a substrain of NRRL 1984.A

resulting from conidia exposed to ultraviolet radiation. This strain producesgood submerged yields, of which approximately half the total units present, asassayed with Staphylococcus aureus, represent penicillin X. The present paperis largely concerned with the development of this strain.NRRL 1975.B Penicillium chrysogenum, a substrain selected at the Northern

Regional Research Laboratory from a culture isolated from Nebraska soil at theUniversity of Minnesota And sent to us by Professor C. M. Christensen as Minn.R-123. Yields up to 170 to 175 units per ml have been obtained in shaken flasksand approximately 250 units per ml in rotary fermenters, but performance is notso consistent as NRRL 1984.A.

Irradiation technique. A 4-watt General Electric germicidal lamp, of bent-tube construction, emitting 500 milliwatts of 2,537 A (95 per cent of the totalradiation), was employed as a source of ultraviolet light (figure 1). The lampwas supported on a ring stand in a horizontal position at either 2 or 4 inchesabove the base. The spores (conidia) to be irradiated were thoroughly dis-persed in sterile water containing sodium lauryl sulfonate in a concentration of1 to 10,000 as a wetting agent. During the irradiation the suspension was con-tained, to a depth of approximately 2 to 3 mm, in a flat-bottomed, uncoveredpetri dish placed on the base of the ring stand. The suspension was continuouslystirred by means of a bent glass rod throughout the whole period of irradiation toinsure as uniform exposure as possible. One-ml samples were removed from thesuspension at 0, 1, 2, 4, 6, 8-; 12, and 16 minutes, the greater portion of the totalsuspension being removed in the eight samples. Appropriate dilutions weremade and the samples plated in Czapek's solution agar. Viability counts weremade at 4 or 5 days, and random isolations were made from developing coloniesfrom selected exposures. The cultures thus obtained were then tested in shaken-flask culture for their capacity to produce penicillin X when grown submerged.

Culture conditions. The following standardized culture solution, essentiallylike that previously employed for testing new isolates or variants (Moyer andCoghill, 1946b; Raper, Alexander, and Coghill, 1944; and Raper and Alexander,1945) for their capacity to produce penicillin in submerged cultures, was em-ployed:

Lactose .............................................................. 20.0 gMgSO4.7H20 ...................................... 9.25 gKH2P04...................................... 0.5 gNaNO,3...................................... 3.0 gConcentrated corn steeping liquor ............. ............. 40.0 mlCerelose (commercial glucose) .......................... 1.2 gDistilled water.......................... 1.0 L

Cultures were grown in cotton-stoppered, 1-liter Erlenmeyer flasks containing200-ml quantities of culture solution, to each of which was added 1.6 g of sterileCaCO8 and two drops of sterile lard oil before inoculation. Cultures were seeded

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76ENETH B. RAPER AND DOROTHY I. PENNELL

with spore suspensions washed from 5- to 6-day-old cultures, and a quantitysufficient to contain approximately 20 to 40 million spores was added to eachflak. Cultures were tested in duplicate or triplicate flasks depending upon theasay facilities available. Incubation was at 24 to 25 C. Continuous agitationwas effected by incubation on a rocker-type shaker making 100three-inchstrokes per minute.

Chloroform extraction. Penicillin X was isolated in pmire form in one large-scalefermentation conducted at the end of this investigation. In all other experi-ments, the presence of this penicillin has been presumed when (1) an appreciablefraction of the total penicillin was not extracted by chloroform at pH 2.0 to2.5, and (2) the unextracted penicillin gave a high assay ratio (see below) whentested against Bacillus subtilis (rough) and Staphylococcus aureus. The extrac-tion technique' was as follows: Add 5.0 ml of broth to 5.1 ml of purified chloro-form, cool in an ice bath, and add 1:1 phosphoric acid to pH 2.0 to 2.5. Two tofive drops are usually required. The quantity is determined on a separate por-tion. Shake vigorously and centrifuge about 2 minutes in a chilled centrifugehead. Replace in the ice bath and at once pipette 0.5 ml of the supernatantaqueous layer into 9.5 ml of buffer at pH 7.0. This may be used for asay, ordiluted further with pH 6.0 buffer. The chloroform is washed, before use, withNaHCO3, followed by two washings with water. The buffer, pH 7.0, consists ofK,HP04, 22.2 g, and KH2PO4, 7.8 g, dissolved and made up to one liter.

Assays. Assays 6f total penicillin were usually made daily on the thirdthrough the seventh days, but differential assays of chloroform-insoluble peni-cillin were usually made on the fourth, fifth, and sixth days only. Assays wereperformed by William H. Schmidt and assistants, and were made by the cylinder-plate method as reported by Schmidt and Moyer (1944), and as later improved bySchmidt (1946). Total penicillin was determined by assaying the untreatedculture broth against Staphylococcus aureus, NRRL B-313 (F.D.A. no. 209P).The amount of chloroform-insoluble penicillin, presumably penicillin X, wasdetermined by assaying an extracted broth against both Staphylococcus aureus,B-313, and the rough phase of Bacillus subtilis, NRRL B-558. As reported bySchmidt, Ward, and Coghill (1945), and by Schmidt (1946), the differentialassay is based upon the varied responses of these organisms to penicillinX. Purepenicillin X asays approximately 950 units per mg against S. aureus, B-313,when pure penicillin G at 1,667 units per mg is used as a standard; against thesame standard, its assay value varies from 1,200 to 1,900 units per mg againstBacillu 8ubtili8, B-558(R), depending upon the phase of the test organism. Thepenicillin present in an extracted broth is presumed to be primarily penicillin Xif this broth shows a differential assay ratio4 which corresponds closely to theassy ratio for the pure penicillin X standard for the same day. Amsay ratios

' Chloroform extractions were performed by Z. Louise Smith and G. E. N. Nelson underthe supervision of Drs. G. E. Ward and R. G. Benedict.

4The asay ratio for a given sample is defined as the assay value shown by that sampletoward Bacillus subtilis NRRL B-5(R), divided by the asay value shown by the samesample toward Staphylococcus aureus NRRL B-313. Pure penicillin G is used as thestandard in both cases.

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PRODUCTION OF PENICILLIN X IN SUBMERGED CULTURE

fluctuate from day to day (table 1), increasing with an increase in the degree ofroughness of B. subtilis, B-558. Ratios between 1.3 and 2.0 may be regarded assatisfactory, although the upper half of this range is preferred.

Total yields of penicillin and yields of penicillin X as shown in the figuresand as cited in the text refer in all cases to values obtained with S. aureus, B-313.The assay values obtained with B-558(R), when interpreted in relation to thepure penicillin X assay ratio for the same day, are useful primarily as a checkon the identity of the penicillin remaining in the extracted broth. For example,if the assay ratio is approximately 2.0 for a particular day, and the B-313 assayvalues in penicillin units are approximately one-half the units obtained withB-558(R) for a particular sample, then it can be assumed that the extraction hasbeen satisfactory, and that the penicillin being measured is primarily penicillin X.If, however, the assay ratio is approximately 2.0, but the B-313 and the B-558(R)assays are approximately equal, then the active material present cannot be re-garded as containing an appreciable amount of penicillin X, and one can usuallyassume that the chloroform extraction was poor. It is not believed that anyuseful purpose would be served by listing the B-558(R) assays generally, or inattempting to show them in the graphs. In table 1, where comparisons aremade between penicillin X production by NRRL 1984.A and NRRL 1984.N22,the ultraviolet-induced mutation produced from it, the general relationshipsbetween B-313 assays of total yields, and B-313 and B-558(R) assays on chloro-form extracts for a selected group of samples are shown. The agreement thatcan be expected between assay ratios for chloroform extracts and for pure peni-cillin X, on the same days, is also indicated.

EXPERIMENTAL

Preliminary trials. Although the highest yielding "submerged" cultures hadbeen included in Ward's tests, the writers felt that, in seeking better penicillin-X-producing strains, some preliminary trials should be made which would includea greater variety of known good submerged cultures. The results of such testsare graphically presented in figure 2. The yields shown, in all cases, representthe average maxima for triplicate shaken-flask cultures. Stock cultures wereemployed for these tests, but in the case of NRRL 1984.A a reisolation from anearlier shaken-flask culture was also included. The total yields obtained in thisexperiment with some strains, notably NRRL 1951.B25 and Stanford 25099 and35217, were below the levels usually produced by these strains when grown undersimilar culture conditions. This, however, was not believed to invalidate theusefulness of the results as a measure of the relative capacities of the differentcultures to produce chloroform-insoluble penicillin.

Confirming the results obtained by Ward and associates in larger laboratoryapparatus, best yields of chloroform-insoluble penicillin were obtained fromNRRL 1984.A. Total yields from the parent culture, NRRL 1984, were ap-preciably lower, but the proportion of penicillin X was essentially the same as inthe more productive substrain. The same general relationship applied to Minn.9SS251, a substrain of Minn. R-13 (= NRRL 1984) of monospore origin dis-tributed by Professor Christensen. Substantially higher total yields were

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KENNETH B. RAPER AND DOROTHY I. FENNELL

obtained with X-1612 than with any other culture, but yields of chloroform-insoluble penicillin were very low. Low yields were likewise obtained withNRRL 1951.B25, the parent of X-1612, and Stanford strains 25099 and 34217,likewise derived from NRRL 1951.B25. Yields of penicillin X from strains

170

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FIG. 2. COMPARATIVE YIELDS OF TOTAL PENICILLIN AND OF CHLOROFORM-INSOLUBLE PENI-CILLIN, OR PENICILLIN X, PRODUCED BY NINE SELECTED "SUBMERGED" CULTURES

Tested in shaken flasks.

NRRL 832.A2 and 1975.B were intermediate between those obtained from1984.A and the much lower yields of 1951.B25 and derivative strains.Upon the basis of these tests, it appeared that cultures of common ancestry

tended to show similarities in their ability to produce penicillin X. Furtherefforts, therefore, were directed toward the development of improved substrainsfrom the most promising culture available, NRRL 1984.A.

768

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PRODUCTION OF PENICILLIN X IN SUBMERGED CULTURE

Selection of natural variants of NRRL 1984.A. Applying the general methodspreviously employed for the isolation of such superior penicillin-producingstrains as NRRL 1249.B21 and 1951.B25 (Raper and Alexander, 1945), thewriters attempted to develop naturally occurring variants characterized by theproduction of substantially increased amounts of penicillin X. Streak plateswere made from high-yielding shaken-flask cultures and observed for the de-velopment of obvious cultural variants differing from normal in such char-acteristics as color, colony texture, rate of growth, and the amount of sporulation.Altogether about 50 substrains were selected and subsequently tested in shaken-flask culture. The majority of these substrains produced yields approximatelyequal to the parent strain; others, including most of the more striking variants,produced consistently lower yields either of total penicillin, of penicillin X, or ofboth. A few substrains appeared to be better producers when first tested butfailed to show consistently higher yields upon repeated cultivation. Thus theprimary objective of these experiments was not realized. They were significant,however, since they demonstrated that NRRL 1984.A, like NRRL 1249.B21,NRRL 1951.B25, and other good penicillin-producing strains, is characterized bygreat cultural variability.

Ultraviolet irradiations. Our attention was next directed toward the use ofultraviolet radiation as a possible means of developing substrains of 1984.Acapable of producing increased amounts of penicillin X. The technique had beenused successfully in the development of better itaconic-acid-producing strains ofAspergillus terreus (Hollaender, Raper, and Coghill, 1945; Raper, Coghill, andHollaender, 1945; Lockwood, Raper, Moyer, and Coghill, 1945), and at StanfordUniversity Professor G. W. Beadle and associates had employed it with somesuccess in developing molds characterized by increased penicillin production.Working with other fungi, primarily species of Neurospora, and employing bothX-ray and ultraviolet radiation, the Stanford group (Beadle and Tatum, 1941;Bonner, Tatum, and Beadle, 1943; Horowitz and Beadle, 1943; Tatum andBeadle, 1942a, 1942b) had developed a whole series of mutations possessingaltered biochemical properties. By means of X-ray radiation, Dr. M. Demerecand associates had produced a mutant, X-1612, characterized by greatly in-creased penicillin production. The technique of radiation as a suitable means ofartificially producing mold mutations was thus well established, and it seemedreasonable to believe that mutations might be produced in which the ratio ofdifferent penicillins would be altered sufficiently to produce a substantiallygreater proportion of penicillin X.

First irradiation of NRRL 1984.A. Conidia from a week-old tube culture(25 by 150 mm) of 1984.A growing upon sporulation media (Moyer and Coghill,1946a) were thoroughly suspended in sterile water and irradiated in the manneralready described. The suspension contained approximately 250,000,000conidia per ml, and, in this case, the lamp was supported at a distance of 2 inchesabove the liquid surface. Samples were removed and plated as noted above,and the resulting plates were incubated at room temperature. The plates wereexamined and population counts made after 4 days. The intensity of the irradi-

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7KENNEDTH B. RAPER AND DOROTHY I. FENNELL

ation wa misjudged, with the result that the killing curve was unusually steep.Less than 1 per cent of the conidia remained viable after 2 minutes' exposure,and only occasional colonies developed in the 4-minute-exposure plates. Isola-tions upon agar slant tubes were made at random from the 2-minute plates, atotal of 50 colonies being picked off in this initial series. These were subse-quently replanted upon sporulation media to insure the development of adequate

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FIG. 3. COMPARATIVE; YIELDS OF TOTAL PENICILLIN AND OF CHLOROFORM-INSOLUBLE PENI-CILLIN, OR PENICILLIN X, PRODUCED BY NRRL 1984.A (CONTROL) AND THIRTY SUB-

STRAINS OF 1984.A RESULTING FRiOM CONIDIA IRRADIATED WITH ULTRAVIOLETTested in shaken flasks.

conidia for seeding the test production flasks. Limitations in the number ofassays that could be assigned to this work necessitated the testing of these cul-tures during two different weeks, but the results obtained in the two experimentsare believed to be wholly comparable. Average total yields and average yieldsof chloroform-insoluble penicillin for a block of 30 consecutively numbered cul-tures that are representative of the whole series are presented in figure 3. Thecontrol represents the average of yields produced by four unirradiated stocks.

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PRODUCTION OF PENICILLIN X IN SUBMERGED CULTURE

The cultures resulting from irradiated conidia showed wide variation both intotal penicillin production and in the formation of chloroform-insoluble penicillin,or penicillin X. Although they could not be so classified in all cases, these cul-tures tended to fall into four more or less tangible groups: (1) cultures producinglow total yields but with penicillin X in approximately normal proportion tothe whole; (2) cultures producing high total yields but relatively small amounts ofpenicillin X; (3) cultures producing approximately normal yields of both totalpenicillin and of penicillin X (these were believed to represent, in the main, cul-tures resulting from conidia only slightly or not at all affected by the radiation);and (4) a single culture, no. 22, producing a normal total yield and an unusuallyhigh ratio of penicillin X. The latter culture was of immediate and particularinterest, and in the next succeeding experiment was retested with no. 18 (hightotal but low penicillin X) and no. 25 (high total and intermediate penicillin X)to determine whether the observed ratios were reproducible. The results werewholly confirmatory; no. 18 showed total yields averaging 115 units per ml, ofwhich penicillinX represented 20 units per ml; no. 25 showed total yields averag-ing 124 units per ml, of which penicillin X represented 30 units per ml; whereasno. 22 showed total yields averaging 155 units per ml, of which penicillinX repre-sented 54 units per ml.Two other cultures, not included in figure 3, were of special interest: no. 11

was characterized by tan-colored rather than blue-green conidia, but producednormal amounts of total penicillin and of penicillin X; no. 16 was characterizedby restricted, light sporulating colonies and when tested produced no detectablequantity of penicillin.

Second irradiation of NRRL 1984.A. In the hope of obtaining an even betterpenicillin X producer than no. 22 of the first series, conidia ofNRRL 1984.A wereagain irradiated. In this case, the lamp was placed at a distance of 4 inchesabove the surface of the suspension, and the killing rate was much less rapid.Fifty random isolations were made from the 4-, 6-, and 8-minute-exposure plates,recultivated upon sporulation media, and tested for their capacity to producepenicillin. In general, the results paralleled those obtained from the first seriesof irradiations, but no new culture was found which equaled no. 22 of the firstseries in the proportion of chloroform-insoluble penicillin produced.

Irradiation of NRRL 1984.N22. For the third and last series, it was decidedto irradiate conidia of culture no. 22, or NRRL 1984.N22 as it was now desig-nated. The procedure employed was the same as that used for the secondirradiation. The killing rate was regarded as satisfactory, and random isolationswere made from the 1-, 4-, 6-, and 8-minute-exposure plates. Altogether 80cultures resulting from "twice" irradiated conidia were surveyed for penicillinproduction in three successive experiments. The results obtained from 30consecutively numbered cultures, all from conidia exposed for 4 minutes, aregraphically presented in figure 4. The general groupings noted for the firstseries as shown in figure 3 could be applied here, although only one culture, no.30, showed a reasonably high total yield coupled with a low penicillin X contentand no strain showed an outstanding increase in the proportion of penicillin X

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772 KENNETH B. RAPER AND DOROTHY I. FENNELL

produced. In the initial test, cultures no. 14 and no. 29 appeared mostpromising, both from the standpoint of total production and penicillin X forma-tion. But when retested, no. 14 produced average total yields of 125 units perml, of which 68 units per ml represented penicillin X; and no. 29 produced average

170

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FIG. 4. COMPARATIVE YIELDS OF TOTAL PENICILL1N AND OF CHLOROFORM-INSOLUBLEPENICILLIN, OR PENICILLIN X, PRODUCED BY NRRL 1984.N22 (CONTROL) ANp TmHwR

SUBSTRAINS OF 1984.N22 RESULTING FROM CONIDIA IRRADIATED WITH ULTRAVIOLT3j,Tested in shaken flasks.

total yields of 124, of which 61 units per ml represented penicillin X; whereas inthe same experiment the parent culture 1984.N22 produced an average total yieldof 115 units per ml, of which 65 units per ml represented penicillin X. Amongthe cultures of this irradiation series which are not included in figure 4, there weretwo others which gave high yields of total penicillin and of penicillin X; but like

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PRODUCTION OP PEMCILLIN X IN SUBMERGXD CULTURE

nos. 14 and 29, these failed to show any apparent improvement in the amountof either total penicillin or penicillin X production when retested. In none ofthese cases were the observed differences regarded as significant.The 80 cultures resulting from "twice" irradiated conidia included a number

of striking cultural mutations which were of considerable interest from a myco-logical point of view. To some degree, the yields of penicillin produced by thesecould be correlated with particular cultural types. Most abundant among thecultures were forms producing colonies essentially normal in appearance, andthese generally produced good total yields of penicillin, of which approximatelyhalf represented penicillin X. It can be assumed that many of these developedfrom conidia not affected by the radiation. Two cultures were characterized bywhite to light buff conidia, and from one of these fair yields were obtained, fromthe other good yields. A few cultures produced restrictedly growing but heavilysporulating colonies, and these gave uniformly good yields. Another group ofcultures characterized by restrictedly growing, but close-textured and lightlysporulating, colonies produced poor yields. A single culture that, grew verysparingly upon Czapek's solution agar and that appeared to possess some nu-tritional deficiency grew well in shaken-flask cultures and produced high totalyields of penicillin and of penicillin X.

It is to be regretted that the writers did not have sufficient time and facilitiesat their disposal, during the course of this investigation, to make a more thoroughmycological study of the interesting cultural mutations which were produced,or to survey a sufficiently large number of irradiated substrains to permit somestatistical analyses.

In view of the limited number of cultures studied, the question arises as towhether NRRL 1984.N22 represents an induced mutation, or whether it repre-sents merely a natural variant, presumably of monospore origin, which wasfortuitously isolated from the dilution plates. No proof can be presented onthis matter, but we do know that among approximately 50 substrains, repre-senting obvious variants and other selected isolates of natural origin, none ap-peared appreciably better than the parent 1984.A. Furthermore, in two otherirradiation series no substrain appeared which showed consistently higher yieldsthan the parent stock under investigation. It is, therefore, presumed that thehigh penicillin-X-producing strain, NRRL 1984.N22, represents a true mutationof NRRL 1984.A, artificially induced by ultraviolet radiation. The perform-ances of both parent and mutant substrain are consistent and would seem tosupport this view. The latter regularly produced yields of penicillin X amount-ing to about 50 per cent of the total, as measured by differential assay, which isabout 65 to 70 per cent of the total yields upon a weight basis.

Comparison ofNRRL 1984.A and 1984.N22. It should be borne in mind thata considerable degree of variation in yields of penicillin can always be expectedin shaken-flask tests from one experiment to another (Raper and Alexander,1945), and that it is often necessary to test a culture repeatedly to be sure of itsimproved performance. Such evidence has been provided in ample measure for

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7KENNETH B. RAPER AND DOROTHY I. FENNELL

NRRL 1984.N22. In at least a dozen experiments in which this strain hasbeen grown in shaken-flask culture as the principal object of investigation, or asthe control for additional irradiated cultures, it has produced total yields ofpenicillin ranging from 100 to 130 units per ml, of which amount an average ofapproximately one-half of the activity represented chloroform-insoluble peni-cillin, or penicillin X.The relative productivity of the two strains is clearly demonstrated in table 1

in which are presented representative yields from a series of different experiments.The assay values presented are averages for duplicate or triplicate flasks andrepresent maximum yields, usually occurring on the fifth day. Assay ratios for

TABLE 1Penicillin production by Penicillium chrysogenum NRRL 1984.A and NRRL 1984.NU,

an ultraviolet-induced mutation characterized by substantially increasedpenicillin X production, in shaken-flask cultures

NRRL 1984.A NRRL 1984N22

Differential assay | Differential assay

Expt.Whole Chloroform- Assay ratiosEnpt. broth extracted b Asrt Expt.

no. _no.

Whole Chloroform- Asamtobroth extracted broth Assay ratios

On ex- OnpueOcx-OnprB-313 B-558(R) B-313 tracted peOn.p B-313 B-558(R) B-313 tracted Openpebroth pe.Xbroth pn

u/mi u/mi u/ml u/mi u/mi u/mi155 127 49 28 1.7 1.5 160 128 96 49 1.9 1.7156 110 58 27 2.1 1.8 161 97 77 54 1.4 1.4157 128 47 32 1.5 1.6 164 117 115 54 2.1 1.9157 118 44 31 1.4 1.6 165 97 106 60 1.8 1.8159 111 52 31 1.7 2.0 165 96 116 55 2.1 1.8165 96 46 28 1.6 1.8166 107 40 25 1.6 1.7 170 115 81 64 1.3 1.4167 119 61 29 2.1 2.0 171 113 108 61 1.8 1.8170 121 32 26 1.3 1.3 172 115 103 65 1.6 1.7

the chloroform extracts have been calculated and are presented parallel withasay ratios for pure penicillin X as determined on the same days.

Culturally the mutant NRRL 1984.N22 cannot be distinguished from theparent NRRL 1984.A. Both are characterized by heavily sporulating, es-sentially velvety colonies which fit reasonably well the species concept for Peni-cillium chrysogenum Thom.

Vat fermentations and the recovery of peniciuin X. The capacity of NRRL1984.N22 to produce penicillin X in the proportions indicated above was verifiedby conducting 600-liter fermentations in a pilot plant vat fermenter, and bysubsequently recovering the penicillin from the fermented culture broth. Twoof these fermentations were made by R. G. Benedict and D. H. Traufler. Inthe second of these runs the operational details and the yields obtained were as-follows:

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PRODUCTION OF PENICILLIN X IN SUBMERGED CULTURE

Medium: Lactose 2.5 per cent, corn steep liquor 6 per cent (by vol.), calciumcarbonate 0.5 per cent, and tap water to make 600 liters.

Conditions: Temperature-24 to 25CAeration-C0 liters per minuteAgitation-250 rpm

Inoculum: 12 liters of germinated spores of P. chrysogenum NRRL 1984.N22,age 2 days.

Samples were removed at appropriate intervals throughout the period of thefermentation and assayed for total penicillin and penicillin X. At 90 hours, ap-proximately 50 per cent of the total penicillin assayed as chloroform-insolublepenicillin, or penicillin X. By weight this represented approximately 67 percent of the total penicillin.

Assay values at 90 hours (units/ml)

WHOLE BROTH BROTH AFTER CHLOROFORM EXrRACTION ASSAY RATIO ONPURE PEN. X

S. aureus, B. subtilis, S. aurcus, B. subtilis,NRRL B-313 NRRL B-558(R) NRRL B-313 NRRL B-558(R)

u/mi u/ml S/mW u/ml

192 273 102 136 1.4

The fermentation was terminated at 90 hours, and the recovery operationswere taken over by J. L. Wachtel. From 200 liters of ifitered culture liquor,more than 2 grams of crystalline sodium penicillin X were isolated in two cropsof 1,820 and 390 mg, respectively.' Both crops gave X-ray diffraction patternscharacteristic of penicillin X. The first crop assayed 920 units per mg againstS. aureus, B-313, and its assay value against B. subtilis, B-558(R), was 1.6 timesgreater. The ultraviolet absorption was correct for sodium penicillin X, andcarbon and hydrogen values on the first crop were in good agreement with theory.In concluding his report of this work, Dr. Wachtel states: "This isolation experi-ment shows without doubt that the chloroform-insoluble penicillin produced byP. chrysogenum NRRL 1984.N22 is penicillin X, approximately 50 per cent ofthe activity being due to this analogue."

SUMARY

Of several good penicillin-producing strains investigated, Penicillium chryso-genum NRRL 1984.A, a substrain derived from Minn. R-13, was found to be the

In industrial and laboratory practice a recovery of penicillin of "commercial grade"representing 50 per cent of the potency in the original broth is considered satisfactory. Theproduct thus obtained usually contains 50 to 75 per cent penicillin by weight, with the re-mainder representing impurities. Additional losses are encountered in the successive stepsleading to the production of a crystalline salt from this material. The isolation of 2.21 gof pure sodium penicillin X from a potential of roughly 20 g in the whole broth (as measuredby differential assay) is considered satisfactory.

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KENNETH B. R&APER AND DOROTHY I. FENNELL

best producer of chloroform-insoluble penicillin, or penicillin X, when grownin submerged culture.By ultraviolet irradiation, a substrain of 1984.A, designated NRRL 1984.N22,

was developed, which produced substantially higher yields of penicillin X, gen-erally amounting to approximately 50 per cent of the total yield as measured bydifferential assays, and representing approximately 65 to 70 per cent of the totalyield upon a weight basis.The superior performance of NRRL 1984.N22 as a producer of penicillin X

was repeatedly observed in shaken-flask cultures, and was subsequently demon-strated in 600-liter vat fermentations. From one of these fermentations purepenicillin X was isolated in a quantity which verified the approximate correctnessof the survey data based upon differential assays only.The technique of irradiation, the method of performing chloroform extractions,

and the theory and practice of the differential assay used in demonstrating thepresence and determining the amount of penicillin X are presented.

REFERENCESBEADLE, G. W., AND TATUM, E. L. 1941 Genetic control of biochemical reactions in

Neurospora. Proc. Natl. Acad. Sci. U. S., 27, 499-506.BONNER, D., TATUM, E. L., AND BEADLE, G. W. 1943 The genetic control of biochemical

reactions in Neuro8pora: A mutant strain requiring isoleucine and valine. Arch.Biochem., 3, 71-91.

COGfILL, ROBERT D., AND KOCH, Roy S. 1945 Penicillin-a wartime accomplishment.Chem. Eng. News, 23, No. 21, December 10.

FLIPPIN, H. F., MAYOCK, R. L., MURPHY, F. D., AND WOLFERTH, C. C. 1945 Penicillin inthe treatment of subacute bacterial endocarditis. Preliminary report on twenty casestreated over one year ago. J. Am. Med. Assoc., 129, 841-43.

HOLLAENDER, A., RAPER, K. B.,AND COGHILL, R.D. 1945 The production and characteri-zation of ultraviolet-induced mutations in Aepergillus terreus. I. Production of themutations. Am. J. Botany, 32, 160-165.

HOROWITZ, N. H., AND BEADLE, G. W. 1943 A microbiological method for the determina-tion of choline by use of a mutant of Neuro8pora. J. Biol. Chem., 150, 325-333.

IABBY, R. L., AND HOLMBERG, N. L. 1945 The activities of penicillins G and X in vitro.Science, 102, 303-304.

LOcKwOOD, L. B., RAPER, K. B., MOYER, A. J., AND COGHILL, R. D. 1945 The productionand characterization of ultraviolet-induced mutations in Aepergillus terreus. III. Bio-chemical characteristics of the mutations. Am. J. Botany, 32, 214-217.

MOYER, A. J., AND COGHILL, R. D. 1946a Penicillin. VIII. Production of penicillin insurface cultures. J. Bact., 51, 57-78.

MOYER, A. J., AND COGHILL, R. D. 1946b Penicillin. IX. The laboratory scale pro-duction of penicillin in submerged culture by Penicillium notatum Westling (NRRL832). J. Bact., 51, 79-93.

ORY, E. M., MEADS, M., AND FINLAND, M. 1945 Penicillin X: Comparison with penicillinG with respect to sensitivity of pathogenic organisms and serum levels. J. Am. Med.'Assoc., 129, 257-261.

RAPER, K. B., ALEXANDER, D. F., AND COQHLL, R. D. 1944 Penicillin. II. Naturalvariation and penicillin production by Penicillium notatum and allied species. J. Bact.,48, 639-659.

RAPER, K. B., AND ALEXANDER, D. F. 1945 Penicillin. V. Mycological aspects of peni-cillin production. J. Elisha Mitchell Sci. Soc., 61, 74-113.

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RAPER, K. B., COGHILL, R. D., AND HOLLAENDER, A. 1945 The production and characteri-zation of ultraviolet-induced mutations in Aspergillus terreus. II. Cultural and mor-phological characteristics of the mutations. Am. J. Botany, 32, 165-176.

SCHMIDT, W. H., AND MOYER, A. J. 1944 Penicillin. I. Method of assay. J. Bact., 47,199-208.

SCHMIDT, W. H., WARD, G. E., AND COGHILL, R. D. 1945 Penicillin. VI. Effect ofdissociation phases of Bacillus subtilis on penicillin assay. J. Bact., 49, 411-412.

SCHMIDT, W. H. 1946 A modification of the cylinder plate method for the assay of peni-cillin. The League of Nations, Bulletin of the Health Organization. In press.

TATUM, E. L., AND BEADLE, G. W. 1942a The relation of genetics to growth factors andhormones. Growth, 6, 27-35.

TATUM, E. L., AND BEADLE, G. W. 1942b Genetic control of biochemical reactions inNeurospora: An "aminobenzoicless" mutant. Proc. Natl. Acad. Sci. U. S., 28, 234-243.

WEIWCH, H., PUTNAM, L. E., RANDALL, W. A., AND HERWICK, R. P. 1944 Penicillin X:Successful treatment of gonorrhea with a single intramuscular injection. J. Am. Med.Assoc., 126, 1024.

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