Penicillin Production by High-Yielding Strains ofPenicillium chrysogenum'

ERIK G. M. TORNQVIST2 AND WILLIAM H. PETERSON

Department of Biochemistry, University of Wisconsin, Madison, Wisconsin

Received for publication May 17, 1956

The development and study of strains of Penicilliumchrysogenum that give high yields of penicillin has beena major project at the University of Wisconsin duringthe past 12 years. Many thousands of cultures havebeen screened for their penicillin-producing ability inthe Botany Department of the University. The methodsused for the production and screening of mutants, thegenealogy of the outstanding strains and their culturalcharacteristics have been described recently by Backusand Stauffer (1955). In recent years, interest hascentered on cultures that produce no pigment but givehigh yields of penicillin. Many of the superior cultureshave been studied intensively both in shaken flasks andpilot plant fermentations in the Biochemistry Depart-ment (Anderson et al., 1953, 1956; Davey and Johnson,1953; Soltero and Johnson, 1954; and Owen and John-son, 1955). In the present paper we shall report onfactors affecting penicillin production by some of therecent high-yielding strains. These are numberedW50-935, W50-1583, W51-20, W51-616, W51-20F3, andW51-20F3-64.

MATERIALS AND METHODS

The fermentations were run in 500-ml Erlenmeyerflasks containing 100 ml of medium. The flasks wereaerated by shaking on a rotary shaker describing a 2-incircle at 250 rpm. The fermentation temperature was24 to 25 C. As a rule, 3 replicate flasks were used in eachexperiment.The media had the composition given in table 1.

Medium I was used in the first series of fermentationsand medium III, a richer medium, was designed on thebasis of the data obtained with the first medium. Theother media, most of which are more concentrated thanIII, were' used in a few experiments to show whatcombinations of corn steep and lactose would be bestfor the new cultures under our experimental conditions.The pH of the media was adjusted with strong potas-

sium hydroxide to 5.2 to 5.6 before sterilization.Lard oil, containing 3 per cent octadecanol, was used

1 This investigation was supported in part by researchgrants from Bristol Laboratories, Inc. and Cutter Laboratories.Presented in part before the Fermentation Subdivision of theAgricultural and Food Chemistry Division at the 126th meetingof the American Chemical Society, New York, 1954.

2 Fellow of the Sweden-America Foundation.

as an antifoam agent. It was added as described in theindividual experiments. Phenylacetic acid (PAA, asolution of the potassium salt containing 5 per centPAA) was added as a precursor, usually in incrementsof 0.05 per cent beginning at 24 hours and at intervalsof 24 hours thereafter until near the end of the fermen-tation. From 4 to 6 additions were made giving a totalof from 0.2 to 0.3 per cent. In a few experiments, a 5per cent solution of #3-phenylethylamine (PEA) neutral-ized to pH 6.0 with H2SO4 was used as precursor. Whenextra lactose was added to the fermentations, a 15 percent W/V solution was used. Ammonium acetate wasadded as a 20 per cent solution.The flasks were inoculated with 4 ml of a mycelium

suspension grown in medium containing 6 per centdextrin and 2 per cent corn steep solids. One hundredml of the medium in a 500-ml Erlenmeyer flask wasinoculated with 5 ml of a spore suspension and the flaskwas shaken for 40 to 48 hours. The spore inoculum wasprepared in the following way: Flat 6-ounce bottles con-taining about 35 ml of medium (6 per cent honey, 1 percent Difco Bacto-peptone, 2 per cent agar) were seededwith spores from a soil stock. The bottles were incubatedon the flat side at 25 C for 7 to 10 days for growth andsporulation and were stored at 4 C until needed. Thespore suspension was made by adding 25 ml of sterilewater to a bottle and loosening the spores from theagar surface with an inoculation loop.

Analytical methods. Samples of the broth were takenfrom the fermentations at desired intervals and the pHwas 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

MediumComponent*Component*

~ I I II I III IV V VI

g per 100 ml

Corn steep solids (C. S. S.) ...... 2.0 2.0 2.5 3.0 3.0 3.5Lactose ........................ 2.5 4.0 4.0 4.0 5.0 5.0Sodium sulfate or sodium thio-

sulfate pentahydrate........... 0.1 0.1 0.1 0.1 0.1 0.1Ratio C. S. S. per lactose........ 0.8 0.5 0.630.750.6 0.7

* In addition, the medium contained 0.4 per cent CaCO3unless otherwise stated.

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ERIK G. M. TORNQVIST AND WILLIAM H. PETERSON

(1944) with Micrococcus pyogenes var. aureus H as thetest organism. Residual sugar in the filtered broth was

determined (after hydrolysis at 120 C for 30 minuteswith 1 N HCl and neutralization) according to the micromethod of Shaffer and Somogyi (1933). Soluble nitrogenin the broth was determined by the micro method ofJohnson (1941), nitrogen in the washed and driedmycelium by the method of Hiller, Plazin, and van

Slyke (1948), and ammonia in the filtered broth by a

modification of the method of Umbreit and Bond(1936). One-half ml of the broth was made alkaline withsaturated sodium carbonate solution, the ammonia was

removed by aeration, collected in standard dilutesulfuric acid, and nesslerized according to Johnson(1941).

Residual fat in the broth was extracted by etherfrom a slightly acidified sample (50 to 100 ml) in a

250-ml separatory funnel. The ether extract was trans-ferred to a weighed 100-ml Erlenmeyer flask, the etherwas evaporated, and the fat dried to constant weightat 105 C.

Fat in the mycelium was obtained by washing withether to remove adhering oil, drying, grinding, digestingwith boiling methanol, and extracting with ether. Theether was removed from the extract and the residualsolids were dried and weighed.The iodine numbers of residual and mycelial fat were

determined according to Roseumund and Kuhnhenn(1923).When mycelial dry substance was determined on

samples to which no oil had been added, the broth was

acidified first to dissolve CaCO3 and other insolublesalts formed during fermentation. The mycelium was

filtered off on a Buchner funnel, washed with water,transferred to a weighed Petri dish, and dried to con-

stant weight. Nitrogen in the mycelium was determinedby the Kjeldahl method.

Preliminary tests on penicillin production by differentstrains. The most promising new pigmentless strains,W50-935, W50-1583, W51-20, and W51-616, were testedsimultaneously on medium I with PAA as precur-sor and lard oil as defoamer. Average yields of 1110(units) per ml were obtained with W50-935 and 1270u per ml with W51-20. The yields of the other cultureswere only about half of these values, quite in agreementwith the values obtained in the Botany Department. Assimilar results also have been reported by Soltero andJohnson (1954) in a synthetic medium, only the twohigher-yielding strains were given further study. One ofthe best of the older cultures, W49-133, was includedfor comparison.

Since these cultures had been selected with PEA as

precursor, some preliminary tests were made comparingit with PAA. Culture W49-133 was used in these tests.PAA proved to be as good a precursor as PEA, or

possibly slightly better. It gave the best results when

added in small amounts beginning at approximately 24hours after inoculation and continuing at 24-hourintervals during fermentation. A total of 0.3 per cent ofPAA had to be added as compared to 0.15 per cent forPEA. Under such conditions PAA gave 1280 u per mlas compared to 1180 u per ml for PEA. These observa-tions are in agreement with results obtained earlier bySingh and Johnson (1948) with culture Q176. No im-provement in penicillin production was obtained whencombinations of PEA and PAA were used.PEA proved to be toxic in many cases when added at

the beginning of the fermentation. Thus, it almost com-

pletely inhibited growth and gave penicillin yields ofless than 500 u per ml when 0.25 per cent was added at0 hour or at 24 hours. An inhibitory level of PEA alsocould be built up by adding slightly more than 0.05 per

cent of PEA at 0 hour and every following 24 hours. Themaximum yield dropped to about 900 u per ml. Thetoxicity was not due to the presence of impurities, as

freshly distilled colorless PEA was as toxic as the yellow-

colored technical product. By adding PEA beforesterilization, the toxic effect was eliminated partly. Thisindicates that, during sterilization, PEA is either com-

bined with some compound in the medium to form a

less toxic substance, or it is changed into a less toxicbut still active precursor.

After these preliminary tests, a series of runs were

made with three of the cultures. The results are sum-

marized in table 2. All of these cultures gave similarpenicillin yields. W51-20 seemed, however, to be slightlybetter than the others. Although promising, W51-20was not used in further tests because of its somewhatirregular behavior and poor sporulation. While thiswork was in progress, Backus and Stauffer (1955) iso-lated several promising substrains of W51-20. Two ofthese, W51-20F3 and W51-20F3-64, did not possess thetroublesome qualities mentioned above and were used,therefore, instead of their parent.

Importance of amount of inoculum. Results from a

great number of experiments indicated that some of thevariations in penicillin yield depended upon variations

TABLE 2. Penicillin production by strainsChrysogenum (Medium I)

of Penicillium

No. of Penicillin Time to pH During LardCulture Ns Maximum Maximum Penicillin OilRunsMaximum ~Production Added*

u/mli hr ml

W49-133 3 1480-1500 114-119 7.3-8.1 0.75avg. 1490

W50-935 4 1300-1700 114-119 7.3-8.0 0.75avg. 1515

W51-20 3 1340-1770 114-119 7.3-8.0 0.75avg. 1630

* 0.1 ml oil was added at 24 hr and usually 0.2 ml every 24hr thereafter.

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PENICILLIN PRODUCTION BY PENICILLIUM CHRYSOGENUiM2

1i 5W51-20F3'1U500_XO

/ >s W49-133

3 1000

0e500

25 50 75 100 125

Inoculum, mg. dry mycelium/100 ml.

FIG. 1. Influence of size of inoculum on penicillin production

in the amount of inoculum. Figure 1 shows the relationbetween inoculum size and penicillin yield. A 48-hourinoculum of both W49-133 and W51-20F3 was used.The different points on the graphs correspond to 1, 2, 4,and 8 ml of inoculum, containing 15 mg of dry myceliumper ml. Maximum penicillin yields were obtained withthe two highest amounts of inoculum. With a smallerinoculum, a slightly higher yield was obtained at 110hours than at 86 hours.

It is evident that the usual amount of inoculum, 4 per

cent by volume, was too low for maximum yield. Inorder to obtain the best results with this amount, theinoculum should contain about 15 mg of dry myceliumper ml. An inoculum of 5 to 6 per cent by volume was

indicated, especially for W51-20F3 which appears torequire a larger inoculum for maximum yield than doesW49-133. Therefore, 5 ml of inoculum was used in laterexperiments. Culture W51-20F3 was a distinctly betterpenicillin producer at all levels than W49-133.A thick inoculum can be obtained by inoculation with

a small number of spores and allowing a long growthtime, or by inoculation with a large number of sporesand allowing a shorter growth time. In shaken flaskfermentations, inocula developed in these two ways willbe very different because of the varying number ofmycelial units. An inoculum from a small number ofspores has a tendency to form big pellets in the finalfermentation and to give low yields. In pilot plantfermentations this may not be of so great importance as

the mycelium is partly broken up by the agitator in thetank.

Effect of supplements on penicillin yields with medium

I. Since analytical data of previous runs indicated thatboth ammonia and lactose were exhausted before theend of the fermentation, additions of these compoundsand also of oil were made.3 The results from the variousadditions are given in table 3. Lactose had no appreci-able effect but oil increased penicillin production 300 uper ml or about 25 per cent (Run 28). In Run 30 theincrease was nearly 45 per cent. Without oil, the penicil-lin maximum was reached in 111 hours after which thepenicillin yield declined to 760 u per ml at 135 hours.With oil, the penicillin content reached 1148 u per ml in111 hours, rose to 1330 u at 135 hours, and then fell to1210 u at 159 hours. Oil gave not only faster penicillinproduction but it also prolonged the period of pro-duction.

Addition of ammonium acetate at the lower level (100mg) had no appreciable effect; however, at 250 mg perflask an increase of nearly 20 per cent over the oilfigure was obtained. The higher level of ammoniumacetate is equivalent to about twice the maximumamount of ammonia nitrogen present in the usual cornsteep medium (figures 2 and 3). Perhaps, under morecarefully controlled conditions, a mycelium of highernitrogen content and presumably greater activity couldbe obtained.

Oil utilization and composition of mycelium. When theresidual oil was separated from the broth in fermenta-tions with culture W49-133 (table 2) about 250 mg offat per flask was recovered. Therefore, about 75 percent of the oil was utilized since 1.1 ml or 1.0 g had beenadded to each flask. The recovered fat was crystallinein nature and had almost the same acid number as thefatty acids from alkali-hydrolyzed oil. This indicatedthat it was composed almost exclusively of free fattyacids. The iodine number was nearly constant for thethree samples, varying from 58.6 to 59.9. This is lowerthan the iodine number of the added oil, 65.8, whichshows some preferential utilization of the unsaturatedpart. Oil recovered from other flask experiments gavesimilar results. As a rule, the best utilization of the oiloccurred when it was added in small portions every 24hours. When 0.5 ml of oil was added at one time, forma-tion of pellets of calcium soap was very marked, andthis necessarily affected the utilization of the fattyacids.The effect of oil and lactose additions on the amount

of mycelial dry substance formed is given in table 3.The lowest mycelial dry weight was obtained when noadditions were made to the medium. When oil or lactosewas added, nearly 50 per cent more mycelium wasformed. The per cent of nitrogen in the mycelium was

3An increased production of penicillin from the addition ofoil has been reported by a number of investigators. We havereviewed the literature on the subject recently (Anderson,Tornqvist and Peterson, 1956) and will not repeat a discussionof it here.

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ERIK G. M. TORNQVIST AND WILLIAM H. PETERSON[

TABLE 3. Effect of supplements on penicillin yields and mycelium composition (Culture WV49-133, Medium I)

pH of Penicillin ~~~~Mycelium NSupplements Maximum Penicillin pH of Penicillin Mycelium Dry wt NDirect Calculated

umi. hr g/lOO ml % mIg/100 ml mg/100 ml

Run 28None .............................. 1140 131 7.2-8.2 1.31 5.2 68.1 89.2Lactose* .............................. 1210 131 7.2-7.4 1.97 4.8 94.3 103.7Lard oilt .............................. 1440 131 7.2-8.0 1.92 5.4 102.7 96.5

Run 30None ................................. 920 111 7.2-7.7 Not determinedLard oil .............................. 1330 135 7.2-7.6 Not determinedLard oil and NH4C2H302t

(a) .............................. 1420 135 7.2-8.1 Not determined(b). .-1560 159 7.2-8.1 Not determined

* Lactose: 0.3 g at 48 hr, and so forth; total 1.2 g/100 ml.t Lard oil: 0.2 ml at 24 hr and every 24 hr thereafter; total, 1.1 ml/100 ml of medium.t NH4C2H302: (a) 25 mg at 48 hr and so forth; total 100 mg/100 ml.

(b) 50 mg at 48 hr. and so forth; total 250 mg/100 ml.

0 30 60 90 120 150 0 30 60 90 120 150 0 30 60 90 120 150Time, hrs.

FIG. 2. Chemical changes in penicillin fermentations (Medium III)

2000

1500

S

1000 z

500

0

highest with added lard oil and lowest when lactose wasadded.The amount of nitrogen in the mycelium can be ob-

tained in two ways, either from the dry weight of themycelium and its nitrogen content or by the differencebetween the total nitrogen and the nitrogen not takenup from the medium. Results from such calculations aregiven in table 3. The two methods check fairly wellexcept for the fermentation on the basal medium.Values calculated from the nitrogen uptake should beapproximately 5 to 10 per cent higher than those ob-

tained from the weight and nitrogen content of themycelium because of the dilution that resulted fromadditions of precursor and lactose.

Fermentation of a richer medium. The results obtainedwith medium I indicated that this medium was toodilute to give maximum penicillin production. There-fore, the richer medium III was used for some furtherstudies. The superiority of the more concentratedmedium was obvious from the first experiments. Yieldsfrom 1400 to 1870 u per ml were obtained with culture.W49-133 and from 2000 to 2800 u per ml with culture

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PENICILLIN PRODUCTION BY PENICILLIUM CHRYSOGENUM 28

I I .I I I I

60 90 120 150 0 30 60 90Time,

FIG. 3. Chemical changes in gradec

W51-20F3. The average for the two cultures was about1550 u per ml and 2400 u per ml, respectively. Althoughthe variation between different runs amounted to as

much as 20 per cent, the variation between individualflasks in a particular run seldom amounted to 10 percent.

Chemical changes in typical fermentations withcultures W49-133 and W51-20F3 in this medium are

shown in figure 2. The more rapid and higher penicillinproduction of W51-20F3 is especially noticeable. Themetabolic rate of this culture was lower than that ofW49-133 as indicated by its slower utilization of lactoseand ammonia. The more even pH plateau of W51-20F3on the two media seems also to be characteristic of thisculture.Of interest also are the changes in the nitrogen con-

tent of themycelium. Up toabout 30 hours, therewas notonly a rapid increase in weight of mycelium, but thenitrogen content of the mycelium rose to a maximum ofaround 10 per cent. It then decreased to about 6 percent at the end of the fermentation. The decrease in

nitrogen content was not the result of a loss of nitrogenfrom the mycelium as is shown by the constant level ofnitrogen present in the filtrate. Other data show thatthe drop in per cent of nitrogen is balanced by a cor-

responding increase in weight of mycelium. While itmay be that nitrogen compounds are excreted and thenreabsorbed to make the new mycelium, the over-allphenomenon is one of translocation of nitrogen withinthe mycelium.

Ijg "c, / 8.0

0. . . . . . . 7.0

/.6.5

conten 1s penicillin 6.0

Medium / Mediuxn4wVi'5Mediumn 9 _ Medium V

120 150 0 30 60 90 1 20 150hrs.

I media (Culture W51-20F3-64)

The data in figure 2 show that the shift in nitrogencontent is associated with penicillin formation. Penicil-lin appears in the medium when the nitrogen content ofthe mycelium reaches a maximum, and continues toform as the nitrogen content falls, and ceases when thelatter approaches a low, constant level. Perhaps at thistime the forms of nitrogen in the mycelium are pre-dominantly of an inert chitinous type. A systematic anddetailed study of the forms of nitrogen in the myceliumat the two stages should be highly informative.

Effect of oil and calcium carbonate additions to mediumIII. Because of the great stimulation of penicillin pro-duction which was obtained by adding oil to medium I,similar experiments were made with medium III. Aspreliminary tests indicated that oil depressed penicillinproduction in medium III when only 0.1 per cent CaCO3was present, fermentations were run with varying con-centrations of this compound. Table 4 shows some datafrom two of these experiments. For the sake of compari-son, medium I was also used with culture W49-133. Theresults with that culture will be discussed further.No great differences in penicillin production resulted

from variation in CaCO3 level when no oil was added.Real stimulation from the addition of oil was obtainedon medium I when 0.4 per cent CaCO3 was added tothe medium. With less than 0.2 per cent CaCO3, ad-dition of oil had a depressing effect. As the oil is hy-drolyzed rapidly by the mold with formation of freefatty acids and glycerol, oil additions lowered the pHmarkedly, for example, 6.2 at 51 hours, when no CaCO3

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TABLE 4. Effect of oil and calcium carbonate on penicillinproduction

Max Penicillin Hr to Max pH RangeMedium Yield, u/mi Yield p ag

Culture MediuN CaCO3

Nio oil* o Oil No oil Oil*oil oil

W49-133 I 0 965 318 98 98 7.0-8.1 7.0-6.90.1 1030 710 98 121 7.1-8.2 7.1-7.90.2 1020 1040 98 98 7.2-8.1 7.2-7.70.4 1100 1480 98 98 7.3-8.0 7.3-7.8

III 0.1 1680 730 121 121 7.1-8.0 7.0-6.70.4 1620 1590 121 121 7.2-8.1 7.2-7.7

W51-20F3 III 0 1890 470 115 115 6.8-7.5 6.8-6.60.1 1850 1260 115 115 7.1-7.6 7.0-7.40.4 1990 1935 115 136 7.2-7.6 7.3-7.50.6 1950 2000 136 115 7.2-8.0 7.3-7.2

* 0.2 ml lard oil was added at 24 hr and every 24 hr there-after.

was added. On medium III, no stimulation by oil wasobserved even when 0.4 per cent CaCO3 was includedin the medium. The depressing effect of oil on the peni-cillin production, however, was even greater with only0.1 per cent CaCO3 in this medium than it was withmedium I.The results with culture W51-20F3 on medium III

were quite similar to those for W49-133. Penicillin pro-duction did not vary much with different amounts ofCaCO3 when no oil was used, but the tendency towardlower yields in the oil-low CaCO3 fennentations wasmarked still more than for culture W49-133. Thepenicillin yields obtained when oil and adequateCaCO3 were used were in no cases definitely higherthan when no oil was added.The lower pH values that resulted from oil additions

are of particular interest and are given in detail infigure 4. The pH dropped markedly with decreasingconcentrations of CaCO3 and reached a minimumaround 65 hours. The pH drop was small and of aboutthe same magnitude with 0.4 and 0.6 per cent CaCO3present in the medium. This would be expected as 0.4per cent CaCO3 is about the amount needed for neu-tralization of the acids liberated from the oil, andadditional CaCO3 would be of no benefit. With noCaCO3 the pH never reached the level for good peni-cillin production, that is, pH 7 to 7.5. The same un-favorable but less marked pH picture was noted with0.1 per cent CaCO3 in the medium. The variation inpenicillin production was as striking as that for pH.The more even the pH values the better was the yield,a result that emphasized the long-recognized importanceof proper pH control.

Further data on strength and balance of media. Theimprovement in yields obtained with medium III sug-gested that even this medium might be too weak formaximum penicillin production by the pigmentless

20 40 60 0 100 120Time, brs.

FIG. 4. Effect of calcium carbonate on pH and penicillinproduction in medium containing oil (Culture W51-20F3,Medium III, Table 4).

cultures. Therefore, some experiments were made inwhich all six media listed in table 1 were used.

Figure 3 shows the results from one of these experi-ments, in which culture W51-20F3-64 was used. Thisculture is one of a hundred single spore cultures selectedfrom W51-20F3, and gave about the same yields as theparent culture in screening tests. As was the case withthe other cultures, the penicillin yields increased withincreasing concentration of nutrients up to medium Vwhere the yield was 1980 u per ml. Medium VI whichwas slightly richer than medium V gave somewhatlower yields, probably because some other condition,such as air supply, had become the limiting factor.With better aeration as in pilot plant fermentors, itshould be possible to use such a heavy medium withformation of more nitrogen-rich mycelium and higherpenicillin yields.An interesting result from these fermentations is ob-

served when data within each pair of media are com-pared. In each pair, the highest yields were obtainedfrom the medium with the lowest corn steep-lactoseratio. From this, it appears that, in addition to amedium of optimal concentration, a proper balancemust exist between corn steep solids and lactose inorder to obtain maximum yields. The best value forthis ratio seems to be about 0.6.The nitrogen content of the mycelium,4 in general,

increased with increasing concentration of corn steepsolids. In general, the mycelium with the highest nitro-

4The determination of mycelial nitrogen on samples fromshaken flasks was somewhat difficult. During the early stages inthe fermentation, the amount of mycelium was small. Duringlater stages it was easy to obtain the actively growing myceliumin the flask mixed with old mycelium which had been stickingto the wall of the flask. Great care was taken to avoid this, butit was not possible to avoid such mixing in every case. Thismight explain some unexpected irregularities in the curve formycelial nitrogen.

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gen content was also the best penicillin producer. Thenitrogen content reached a maximum around 45 to 50hours and then fell steadily for about 50 hours, afterwhich it remained relatively unchanged. The fall innitrogen and a steady or slowly rising pH are the out-standing features of the penicillin phase. It is probablethat maximal penicillin yields will be obtained if andwhen these two factors can be controlled. The cultureis, of course, the prime factor, but maintenance of avigorous physiologic state is also of great importance.

SUMMARY

Some new pigmentless strains of Penicillium chrys-ogenum have been tested for penicillin-producingability on corn steep-lactose media in shaken flasks.The use of a suitable precursor as well as the impor-tance of using a proper inoculum was investigated.

Yields up to 1150 u per ml were obtained on astandard medium [2 per cent corn steep solids (C. S. S.)and 2.5 per cent lactose] with the best culture. Additionof oil, preferably in small portions every 24 hoursduring fermentation, stimulated penicillin production,giving yields of about 1800 u per ml. More myceliumand higher yields (up to 2800 u per ml) were obtainedin a richer medium (2.5 per cent C. S. S., 4 per centlactose). Oil did not stimulate penicillin production inthis medium, indicating that the oil acted primarily asan extra source of energy. The effect of oil in bothmedia depended upon the amount of calcium carbonatepresent. If less than 0.4 per cent calcium carbonate waspresent, oil depressed the yield because of the low pHthat followed the rapid hydrolysis of the oil by themold lipase. Experiments in graded media indicatedthat a C. S. S. concentration of about 3 per cent and aratio between C. S. S. and lactose of 0.6 gave optimalresults.

Penicillin production was associated in all cases witha decrease in the nitrogen content of the myceliumfrom approximately 9 per cent to approximately 6 per

cent. This decrease was not the result of a net excretionof nitrogen, which indicates that more mycelium oflower nitrogen content was formed. A mycelium of highnitrogen content and a suitable pH are two factorsfavorable to good penicillin production.

REFERENCES

ANDERSON, R. F., WHITMORE, L. AM., JR., BROWN, W. E.,PETERSON, W. H., CHURCHILL, W. W., ROEGNER, R. F.,CAMPBELL, T. H., BACKUS, M. P., AND STAUFFER, J. F.1953 Penicillin production by pigment-free molds. Ind.Eng. Chem., 45, 871-874.

ANDERSON, R. F., TORNQVIST, E. G. M., AND PETERSON, W. H.1956 Penicillin production: Effect of oil on pilot plantfermentations. Agri. and Food Chem., 4, 556-559.

BACKUS, M. P., AND STAUFFER, J. F. 1955 The productionand selection of a family of strains in Penicillium chryso-genum. Mycologia, 47, 429-463.

DAVEY, V. F., AND JOHNSON, M. J. 1953 Penicillin pro-duction in corn steep media with continuous carbohydrateaddition. Appl. Microbiol., 1, 208-211.

HILLER, A., PLAZIN, J., AND VAN SLYKE, D. D. 1948 A studyof conditions for Kjeldahl determination of nitrogen inproteins. J. Biol. Chem., 176, 1401-1420.

JOHNSON, M. J. 1941 Isolation and properties of a pureyeast polypeptidase. J. Biol. Chem., 137, 575-586.

OWEN, S. P., AND JOHNSON, M. J. 1955 The effect of tempera-ture changes on the production of penicillin by Penicilliumchrysogenu?n W 49-133. Appl. Microbiol., 3, 375-379.

ROSENMUND, K. W., AND KUHNHENN, W. 1923 A new methodfor the determination of the iodine number in fats and oilsby the use of pyridine sulfate dibromide. Z. Nahr.Genussm., 46, 154-159.

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

SHAFFER, P. A., AND SOMOGYI, Ml. 1933 Copper-iodometricreagents for suigar determination. J. Biol. Chem., 100,695-713.

SINGH, K., AND JOHNSON, MI. J. 1948 Evaluation of precur-sors for penicillin G. J. Bacteriol., 56, 339-355.

SOLTERO, F. V., AND JOHNSON, M. J. 1954 Continuous ad-dition of glucose for evaluation of penicillin-producingcultures. Appl. MIicrobiol., 2, 41-44.

UMBREIT, W. W., AND BOND, V. S. 1936 Analyses of planttissue: Application of a semi-micro Kjeldahl method.Ind. Eng. Chem. Anal. Ed., 8, 276-278.

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