Penicillin Production by d
Pigment-Free Molds R. F. ANDERSON, L. 31. WHITMORE, JR., W. E. BROWN, W. H. PETERSON, B. W. CHURCHILL. F. R . ROEGNER, T. H. CAMPBELL, M. P. BACKUS, AND J. F. STAUFFER Llniversity of Wisconsin, Madison 6, Wis.
0
XE of the disadvantages of the R-idely used penicillin
culture, P. chrysogenum Q176, is its secretion of yellow pigments. A pigmentless mutant of Q176 has been obtained by treating the spores n-ith ultraviolet radiation. From this ancestral stock, a family of several thousand isolates free of pigment has been secured by a process of selection and further mutagenic treatment. Six strains from t,his family, selected mainly on the basis of superior production of the antibiotic in screening tests, have been investigated in regard to their fermentation behavior. The best culture, 49-133, gave nearly double the penicillin yield of Q176 in flask fermentations, and in pilot plant runs gave approximately equal yields. About 99% of the product was penicillin G. Metabolism studies of the highest yielding ferment’ation showed good mycelial growth, a steady p H condition, good utilization of lactose, efficient use of precursor, and a high aeration and agitation requirement. Another mutant, 49-2103, although derived from the pignientless strain 49-133, produces a brox-n pigment. In preliminary tests its production of penicillin has been slightly better than 49-133, but optimal conditions for its performance have not yet been worked out. Cultures differ in metabolic characteristics; hence every strain must be carefully studied before its full capabilities can be learned. Molds produce over 35 ell-identified pigments, plus many more Khich have not been characterized. -4bout one third of these pigments are produced by various species of the ubiquitous penicillia. The pigments encountered in penicillin production are those produced by the P. chrysogenumnotatum group. Three pigments from this group have been isolated and well characterized. In 1932, Clutterbuck, Lovell, and Raistrick ( 4 ) isolated from several strains of P. chrysogenum a strongly levorotatory, yellow pigment which they called chrysogenin. The empirical formula is C18H220B. Stodola et al. (19) working with a strain of P. notatum obtained a yellow, nitrogen-containing pigment which they named penitrinic acid. This compound contained one carboxyl group and appeared to be a phenol. Cram and Tishler (6) isolated a compound from clinical penicillin which had the same ultraviolet absorption spectrum as penitrinic acid. They also obtained 8-penitrin, a degradation product of penitrinic acid. Another pigment from clinical penicillin n-as studied by Cram ( 5 ) . This compound was well characterized and designated as ~orbicillin. These compounds probably do not represent all of the pigments produced by the penicillia which have been used in the production of penicillin. The presence of these pigments in the ferrnentation broth is troublesome in that it necessitates additional treatment in processing in order to obtain a white product. For this reason considerable effort has been made to secure strains which do not secrete pigments into the broth but do give good yields of penicillin. The first report in the literature that has been noted is that of de Somer ( I ? ) . H e treated the parent culture with x-rays and from 95 selected colonies obtained 7 strains that produced no pigment and gave yields (170 units per ml.) equal or superior to the parent. H e pointed out the importance of these cultures in respect to the purity of the commercial product. A patent on
the use of a pigmentless mutant of the long-used culture, P . chrysogenum Q176, was granted t o Woodruff and Larsen (21) in 1950. This culture was obtained by irradiating spores of Q176 nith ultraviolet and selecting survivors which secreted no pigment and gave high yields of penicillin. Yields of 1720 units of penicillin per ml. of culture in comparison to 605 for Q176 were reported. The percentage of benzylpenicillin for the mutant is given as 82 as compared to about 60 for the parent. The figures for Q176 are much lower than those reported by others. For example, Backus et al. ( 2 )reported 920 units per ml. in 1943 a t the time Q176 wa8 discovered, Higuchi et al. ( 8 ) obtained 1045 units per ml. in 1946, Peterson ( 1 2 ) reported 1550 units per ml. in 1947, and BroR-n and Peterson ( 3 )obtained about 2000 units per ml. in 1950. The percentages of benzylpenicillin givrn in these papers ranged from 77 to 98%. It is probable that still higher yields from Q176 can be obtained by more favorable conditions of fermentation. This conclusion does not imply, of course, that better cultures than Ql76 cannot be or have not been obtained; but in setting up a comparison, the best known performance of each of the cultures should form the basis of the comparison. In a recent paper Arima (I) described the isolation, morphology, and industrial use of an ultraviolet pigmentless mutant of Q176. In tank fermentations yields averaged only 700 to 800 units per nil. on a lactose-corn steep medium with phenylacetic acid as precursor. The percentage of penicillin G was low, amounting to only 50 to 70% of the total. This culture is said to be used in most of the penicillin plants of Japan ($0). The work on pigmentless cultures reported in the present paper was begun in July 1947 and since that time hundreds of mutants have been tested for penicillin-producing ability. Seven of the best cultures obtained in the course of the studies have been tested in both flasks and pilot plant fermentors, with the results given in this paper. Reports on the performance of these cultures have been sent to nearly all of the penicillinproducing companies in this country, and transfers of the cultures have been sent to penicillin producers in England, France, Sweden, Holland, Denmark, Australia, Brazil, Japan, and many other foreign countries, and to any individual asking for them. Many favorable reports have been received and it is probable that several of the cultures have been tried in large scale fermentations. ORIGIN AND RELATIONSHIP O F CULTURES
The strains of Penicillium chrysogenuin involved in the fermentation studies reported here are all descendants of the wellknown strain Wis. Q176 ( I S ) . This culture, which gives high yields of penicillin but also produces yellow pigment, was made available for industrial use late in 1945. Early in the summer of 1947 there was obtained from Ql76 a notable strain, designated as 14%. BL3-Dl0, which produced no yellow pigment in the medium. This strain was secured following subjection of conidia of Q176 t o a very mild treatment with ultraviolet radiation of 2750 A. It was selected as a breeding stock because, although it showed penicillin yields inferior to those of its parent, its production of the antibiotic was of
768
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
April 1953
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I
greater magnitude than that from any other pigmentless strain previously encountered by the authors. Through a selection program extending over more than two years and involving study of several thousand isolates stemming from culture BL3-D10, a number of improved strains were secured, among them the seven involved in the tests described in this paper. Five of the seven strains were obtained by selection of spontaneously occurring variants in a series of three generations. Two strains were obtained as survivors following treatment of spores with nitrogen mustard [methyl-bis(@-chloroethy1)amine] in the later portion of the breeding program. The method of treating the conidia with the nitrogen-mustard compound has been described by Stahmann and Stauffer (18). T h e interrelationships of the various strains are indicated in the genealogical chart (Figure 1). WIS. 4176 WIS. BL3-Dl0
S
1
I
WIS. 47-650 IS WIS. 47-1380
WIS. 47-638
1
WIS. 47-911
IS
WIS. .47-1584
Is
WIS. 48-701
WE. 48-749
WIS. 49-133
TABLEI. YIELDS
IN SHAKEN FLASKS (Rotary shaker, 3 flasks in each run)
Precursor Levela,
Time t o Max. Yield Hour; 91 91 115 138 114 140 114 140 115 140 140 140
Mean Yield, Units/MI. 0 268 f 13b 0.05 368 8 0.10 386 24 0.20 347 f 8 47-1380 0 260 f 21 0.05 416 f 9 0.10 405 f 15 0.20 393 24 47-1564 0 317 =t 5 0.05 508 f 14 30 0.10 535 464 & 52 0.20 48-701 , 0 372 i 1 1 0.05 589 f 12 0.10 558 f 12 0.20 487 f 22 48-749 0 254 f 9 0.05 407 f 22 0.10 373 f 18 0.20 384 f 16 49-133 0 648 i 55 0.05 1037 i 14 0.10 994 i 58 0.20 667 i 28 49-2105 0 460 f 0 0 05 1030 i 18 0 10 963 i 28 0 20 755 f 86 a Amount added every 12 hours, nine additions in hours. b Standard deviation of mean. Strain 47-9 11
%
** *
*
88
113 113 113 91 115 91 115 139 139 114 139 114 114 139 114 all,
Av. p H Values a t Time
of M a x .
Yield 7 3 7.4 7.5 7.6 7.4 7 5 7.5 7 6 7.4 7.6 7.6 7.6 7.4 7.5 7.6 7.6 7.3 7.5 7.4 7.5 7.6 7.9 7.6 7.7 7 5 7 6 7 9 7 4 beEinning at 24
FERMENTATION EQUIPMENT AND METHODS
of the Department of Biochemistry. A reciprocating shaker was employed t o provide aeration of flask cultures in the botany tests; in the biochemistry fermentations a rotary shaker was used All the larger scale work was done in t h e latter department. FLASKFERMENTATIONS ON RECIPROCATING SHAKER. Spore inoculum obtained from 7- t o 10-day agar tube cultures (6% honey, 1% Difco Bacto-Peptone, 2% agar) was employed in the slow shaker trials. One milliliter of spore suspension was used t o inoculate each flask. The shaker ran a t about 92 cycles per minute with a 4-inch stroke, while the temperature in the fermentation room was kept at 24-25' C. The fermentation medium contained the following basal ingredients: distilled water, corn steep solids 2.0%, lactose 4.0%, sodium nitrate 0.3%, otassium dihydrogen phosphate 0.05%, magnesium sulfate eptahydrate 0.025%, calcium carbonate 0.3 t o 0.4%. Onehundred-milliliter quantities of this mixture were placed in 500ml. Erlenmeyer flasks, t o each of which 3 drops of Dow Corning Antifoam A (silicone) emulsion and a quantity of P-phenylethylamine (as acetate) were added separately. The amount of precursor was varied; concentrations as low as 0.1% were used in some of the earlier experiments, but levels of 0.2 and 0.25% have been employed almost exclusively in recent tests, including those from which antibiotic yield data are tabulated here. The medium was sterilized for 15 minutes a t 15 pounds steam pressure. FLASK FERMENTATIONS ON ROTARY SHAKER. Six-ounce bottles containing 25 ml. of the agar sporulation medium of Gailey et al. ( 7 ) , distributed on the flat side, were seeded with spores from a soil stock. Five-hundred-milliliter conical flasks containing 100 ml. of a 6% dextrin-2% corn steep solids medium reviously autoclaved a t 15 pounds for 30 minutes were inocuated with 1 ml. of a n aqueous suspension of spores from the 6ounce bottle. The flasks were then incubated for 46 t o 50 hours on a reciprocating shaker (4-inch stroke, 90 strokes per minute) a t 25" C . Four milliliters of vegetative growth from these flasks were then introduced into the flasks containing the fermentation medium composed of 2.5% lactose, 1yo commercial glucose, 2% corn steep solids, and 0.4% calcium carbonate. Three dro s of lard oil containing 6% Allcaterge C (Commercial Solvents Eorp., Terre Haute, Ind.) were added t o each flask every 24 hours t o prevent accumulation of foam. A penicillin precursor, phenylacetic acid, was added as the sodium salt in the levels indicated in Table I. The flasks were incubated a t 25" C. on a rotary shaker which describes a 2-inch circle a t a speed of 220 t o 240 r.p.m.
Fermentations were carried out in 500-ml. Erlenmeyer flasks and in 30-liter fermentors. Flask tests were conducted both in the microbiological laboratories of t h e Botany Department, where the strains were developed, and in the fermentation laboratories
LARGER SCALE FERMENTAT1ONS* The 30-1iter fermentors used in this work have been described by Rivett et al. ( 1 4 ) and Brown and Peterson (3). The automatic antifoam addition
N I,
WIS. 49-2105
Figure 1. Derivation of Strains UV.
s.
NM.
..
769
Selection following ultraviolet irradiation Selection without treatment Selection following nitrogen-mustard treatment
Six of the strains are characterized by the fact that their mycelia give off either no yellow pigment or so little that it cannot be readily detected. This lack of pigment production, contrasting sharply with the situation encountered not only among all wild forms of the species but also with t h a t in all the early improved cultures, was the outstanding feature of the ancestral form BL3D10; the character proved t o be a very durable one, for it has been transmitted t o thousands of descendants over a considerable number of generations, both in the face of further mutagenic treatments and when subsequent treatment was withheld. Indeed, no descendant of BL3-D10, in these laboratories a t least, has ever shown reversion t o production of yellow pigment. However, one interesting nitrogen-mustard derivative, Wis. 49-2105, was found to secrete a new pigment of reddishbrown color into the fermentation medium. Since this strain belongs to the same family stock as the Wisconsin pigmentless cultures and, in addition, has a very good capacity to produce penicillin, it has been included along with the pigmentless strains in the present studies. The various strains included in this investigation differ from one another in cultural characteristics and especially in their population patterns. These details and further information concerning the development of the Wisconsin family of cultures from which these strains have been selected will be published elsewhere.
R
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INDUSTRIAL AND ENGINEERING CHEMISTRY
110
system was modified to obtain more positive and reliable foam control. I n the system used, antifoam is forced from the reservoir by filtered air which maintains a constant pressure of about 10 pounds gage on the reservoir. The passage of antifoam from the reservoir t o the fermentor is regulated by a metering valve shown in Figure 2. This valve is a modification of a model designed by the National Research Council of Canada ( 1 1 ) . I t is placed on the antifoam line after the fermentors and reservoirs have been sterilized. As indicated in Figure 2, t h e various parts of the valve are mounted in a shallow cylinder, A , with one end open. The flow of antifoam through the rubber tubing, B, is controlled by two cam-operated rocker clamps, C, t h a t open and shut alternately; each clamp is closed completely before the other one opens. The amount of antifoam added during each revolution of cam D is contained in the rubber tubing between the two clamps. With the cam in the position shown in Figure 2, antifoam is flowing into the fermentor. The amount of antifoam delivered can be regulated by the pressure maintained on t h e reservoir, the size of tubing used in the antifoam line, and the position of the flat spring, E. The valve is set to deliver about 1 ml. of antifoam per cycle; this slow rate of addition gives the antifoam time to act between additions and thus prevents the addition of excess antifoam. Antifoam from reservoir
Vol. 45, No. 4
by Brown and Petwson (S) and Gailey et al. ( 7 ) . The antifoam was lard oil containing 670 Blkaterge C. Mediums and agitation and aeration rates are given in Table 111. In all cases, the fermentations were incubated a t 24-25 'C . Penicillin precursor, sodium phenylacetate, was added in equal portions a t 12-hour intervals in the amounts indicated in Table 111. The fermentors were sterilized with the antifoam and precursor reservoirs in place by autoclaving for 75 minutes a t 15 pounds steam pressure. I n experiments where the pH mas controlled by manual addition of 2 aV sulfuric acid, an acid reservoir was also attached t o the fermentor previous t o sterilization.
YIELDS IN SHBKEN FLASKS~ (Reciprocating shaker) Penicillin, Units/MI. s o . of 6 Days 7 Days Flasks .4v. Range AV. Range
T-4BLE
Strain 47-9 11 47-1380 47-1564 48-701 48-749 49-133 49-2105
8 8 8 7 8 8 8
11.
419 401 494 510 420 857 1013
(345-448) (360-475) (415-552) (325-560) (440-560) (790-990) (840-1170)
2;:
505 517 520 809 1019
380-475) 350-540) (420-602) (465-625) (446-630) (698-995) (900-1160)
{
a With 0.20% P-phenylethylamine, added as precursor.
5 ANALYTICAL METHODS
Inches
AntifoLm to fermentor
Figure 2. A. B. C. D.
E. F. G. H. I.
Antifoam Metering Valve
Aluminum housing, depth S/r-inch Rubber tubing, '/a*-iuch i.d., '/winch wall Rocker clamps Eccentric cam Flat spring Coil spring Tubing guide Adjustable screws Recess for rocker clamp
The eccentric cam, D , is driven b y a 1r.p.m. motor which starts automatically when foam rises enough to contact an electrode extending 3 inches below the top of t h e fermentor. The electrode consists of an insulated No. 14 copper wire with the lower end bare, which is inserted into the fermentor through a rubber stopper. Accumulation of mycelium along the electrode may cause short circuits between the electrode and the grounded fermentor, resulting in an addition of excess antifoam. To prevent this the metering valve is actuated by an electronic relay so adjusted t h a t addition of antifoam is initiated only when the electrode resistance reaches a value below 1000 ohms. Antifoam addition ceases when the resistance increases t o a value of about 1200 ohms. This range is variable, so that the system may be adapted t o fermentations having different foam characteristics. Control of agitation, aeration, and temperature was accomplished as described by Brown and Peterson (3)for the standard type of fermentors. As mentioned in t h a t paper, two of the four agitator blades were set in a vertical position and the other two blades at 30 O t o the horizontal. Fourteen liters of medium were used in the fermentors. The vegetative inoculum was raised and used in the manner described
Samples of the broth were withdrawn from the fermentors a t desired intervals by means of air pressure or, in some instances, by use of a water aspirator. After the pH had been determined with a glass electrode and an aliquot withdrawn for penicillin assay, the samples were stored for chemical analysis according t o the methods of Gailey et al. ( 7 ) . In the case of flask cultures, small amounts of broth were removed a t apDropriate periods -_ with sterile pipets. The total penicillin titer mas determined by a modification of the cvlinder-date method of Schmidt and Mover 116). The penicfilin G content of the broth was determine; b y the paper chromatographic procedure of Karnovsky and Johnson (10). For sugar determination the filtered broth was hydrolyzed by heating at 120' C. for 30 minutes in 0.75 S hydrochloric acid and analyzed by the Shaffer and Somogyi method (16) (reagent 50 with 5 grams of potassium iodide was used). A standard reference curve was obtained with hydrolyzed U.S.P. lactose. Soluble Kjeldahl nitrogen was determined at the time of inoculation and at intervals throughout the fermentation by the method described by Johnson (9). Mycelial nitrogen content was salculated by subtracting the soluble Kjeldahl nitrogen of the filtered broth from the total nitrogen a t the time of inoculation. Ammonia nitrogen was determined on the undiluted, filtered broth. A sample of suitable size (1 t o 2 ml.) was made alkaline with l ml. of saturated sodium carbonate and the ammonia was steam-distilled into an aliquot of standard sulfuric acid. RESULTS AND DISCUSSION SHAKEX
FLASKS.I n fermentation tests on the reciprocating
shaker, culture 47-1564 has almost alviays given slightly better penicillin yields than other selected strains of the 47-series, and cultures 48-701 and 48-749 in turn commonly give yields 25 to 50Y0 above those obtained with their parent 47-1564. The highest yields, hoviever, have consistently come from the nitrogenmustard derivative strains 49-133 and 49-2105. Yields obtained in a representative test are listed in Table 11. It is perhaps significant that the fermentation conditions employed for the tests have closely approximated those employed in the screening work done in selecting the races in question from among the large number of variants developed in the breeding program. I n fact, for all the Botany Department fermentations a relatively uniform set of conditions has been maintained for a long time. Essentially the only factor intentionally varied has been the concentration of precursor; and within recent months even that has been shifted through only a narrow range. X o s t of the pigmentless strains considered in this report were selected in screening fermentations in which the precursor level
April 1953
INDUSTRIAL AND ENGINEERING CHEMISTRY
OF MUTANTS OF P. chrysogenum Q176 TABLE 111. COMPARISON
IN
30-LITER FERMENTORS
Av. PrecurTime sor No. of to AgitaL./L,'/ tion. Level, FermenMean Yield, Max., Strain Min. R.P.M. % tors Units/Ml. Hours 9 914 zt 1 ~ 7 5 ~ 88 540 0.05 47-9 11 0.64 90 1005 6 5 540 0.05 0.64 47-1380 1015 zt 136 91 3 0.05 540 48-701 0.64 1044 zt 31 98 2 0.10 540 0.64 48-70 1 640 1070 f 32 70 0.05 48-749 0.64 AX-7A0 1040 f. 65 0.10 86 . 0.64 40 91 990 0.05 47-1564 0.98 1040 zt 80 89 0.10 47-1564 0.98 1105 zt 35 90 0.05 49-133 0.56 17 94 1165 0.10 0.56 49-133 70 105 1300 0.05 0.56 49-2105 98 1470 65 0.10 0.56 49-2105 a Standard deviation of mean. All fermentations contained 1% Cerelose, 0.5% CaCOa, O.>% MEDIUIM. Na2SO4, and slightly varying amounts of lactose and corn steep solids. Twenty-five grams per liter of lactose and 40 grams per liter of corn steep solids were used with first four cultures and 30 grams per liter of both lactose and corn steep solids with last three cultures. Precursor was added every 12 hours in amounts given in column 4. Seven additions were made beginning at 24 hours. Aeration
*
__ __
* **
*
.
.
was 0.2y0 and all the precursor was supplied in a single addition just prior t o sterilization of the medium. When a given group of isolates has been tested repeatedly even under supposedly identical fermentation conditions and using the same soil stocks as initial inoculum, yields have varied considerably from run t o run; and no satisfactory explanation for this situation can be offered at the present time. However, the various strains have usually maintained approximately the same relative positions when ranked according to their performance in the individual runs. It seems certain that under the conditions of low aeration, rapidly falling precursor level, etc., which characterize the Botany Department fermentation setup, the strains rarely, if ever, display their full potencies as penicillin producers. Nevertheless, the general success of the strain development program (IS) suggests t h a t this type of test gives a rather good indication of the worth of a culture. Results obtained in flask fermentations carried out on the rotary shaker are shown in Table I. Strains 49-2105 and 49-133 were definitely superior to the other cultures tested, yielding 1030 units per ml. Strains 48-701 and 47-1564 gave from 500 t o 600 units per ml. The remaining strains, 47-911, 47-1380, and 48-749, were relatively inferior, giving yields of about 400 units per ml. I n flasks nine additions of 0.0570 phenylacetic acid (PAA) at 12-hour intervals gave approximately the same yields as did higher amounts of precursor. When 0.10% phenylacetic acid was added at 12-hour intervals lower yields were frequently 5 ,
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obtained, and 0,20y0 was definitely inhibitory in all cases. It is possible t h a t for at least some of the cultures the optimum precursor rate may be less than 0.05% per 12 hours. The media, conditions of fermentaIN~O-LITER FERMENTORS. tion, and yields are given in Table 111. The strain giving the highest yields was 49-2105 (1470 units per ml.). The average yield for 49-133, 1165 units per ml., is not significantly better than the yield from 48-701 and 48-749. The remainder of the cultures appear t o be about equal under the conditions used and gave yields ranging from 915 to 1045 units per ml. The conditions given in Table I11 are probably not those most suitable for each of the cultures. Evidence for this conclusion has been obtained in the case of 49-133. With increased aeration and agitation, yields of 1850 units per ml. have been obtained as compared with 1165 units per ml. given in Table 111. Detailed discussion of this fermentation is given in connection with Figures 5 and 7. None of the pigmentless cultures to date have surpassed the best yields (2000 units per ml.) obtained previously in this equipment with Q176 ( 3 ) . However, it is probable t h a t when these cultures have been studied as intensively as Q176 has been studied, equal or higher yields will be obtained. Under conditions of higher aeration and agitation 49-133 has practically equaled the highest yields obtained with Q176. Strain 49-133 appears to be more uniform in its performance and has the important advantage of producing no pigment. CHEMICAL CHANGES.The chemical changes for some typical fermentations are shown in. Figures 3 to 7. The course of a fermentation by 47-1564 or 47-911 is similar t o t h a t by 47-1380 (Figure 3). Although the chemical changes produced in a fermentation by mutants of Q176 vary somewhat, certain general conclusions apply to all these cultures. Early rapid growth of mycelium is desirable in a penicillin fermentation. For this reason glucose, which is utilized rapidly by the mold, is included in the medium. I n the fermentation by 48-701 (Figure 4 ) mycelium production was slow a t the start and was not complete until 50 hours, while with the other strains mycelium development was essentially complete a t 30 hours. Unfortunately, this run was terminated before maximum yields of penicillin were reached. However, the actual amount of mycelium does not seem t o determine penicillin yields. Both 49-133 and 49-2105 (Figures 5 and 6 ) produced only about 50% as much mycelium as the other cultures but gave higher penicillin titers than did the dther strains. This indicates t h a t these cultures are more efficient producers of penicillinLe., penicillin produced per unit of mycelium is greater. Mycelial growth depeleted the ammonia nitrogen of the medium, as shown in Figure 5. This ammonia nitrogen curve is characteristic for all the cultures tested. As mycelial growth is com-
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Figure 3. *2
40
60 80 TIME, HOURS
100
110
Chemical Changes in 47-1380 Fermentation
N HzSOn added. 100 ml. at 37 hours, 200 ml. at 49 hours
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Figure 4.
40
60 80 TIME, HOURS
100
120
Chemical Changes in 48-701 Fermentation
5
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
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PENICILLIN
-celium production was double that in the low aeration fermentation. The ammonia nitrogen curve x a s not changed to any large extent. The yield of penicillin, 1850 units per nil., is the highest obtained with any of the piymentless cultures to date, and is believed to be a reflection of the vigorous metabolism of 49133. -4n outstanding feature of this culture is its efficient use of penicillin precursor. Only about 0.35y0of phenylacetic acid has been used with it and still over 99% of the penicillin in the broth has been G. Probably less than 0.35% would be sufficient. for optimal production of benzylpenicillin. ACKNOWLEDGMENT
The authors wish t o acknowledge their indebtedness to Margaret Larson and Mary Hershberger for the penicillin assays and to Sanford Anderson for the construction of the metering valve. The research was aided by grants from Eli Lilly and Co. and Cutter Laboratories. LITERATURE CITED
(1) Arima, K., J . Antibiotics (Japan), 4, 289 (1951). (2) Rackus, hI. P., Stauffer, J. F., and Johnson, M. J., J-. Am. Chem. SOC.,68, 152 (1946).
INDUSTRIAL AND ENGINEERING CHEMISTRY
April 1953 (3)
-
Brown, W. E., and Peterson, W. H., IND. ENG.CHEM.,42, 1823 (1950).
(4)
Clutterbuck, P. W., Lovell, R., and Raistrick, H., Biochem. J . ,
(12) (13)
(14)
2 6 , 1907 (1932).
. .
Cram, D. J., J . Am. Chem. Soc., 70, 4240 (1948). Cram, D. J., and Tishler, M., Ibid., 70, 4238 (1948). (7) Gailey, F. B., Stefaniak, J. J., Olson, B. H., and Johnson, M. J.,
(5) (6)
J . Bact., 52, 129 (1946).
Higuchi, K., Jarvis, F. G., Peterson, W. H., and Johnson, M. J., J . Am. Chena. SOC.,6 8 , 1669 (1946). (9) Johnson, &I. J., J . Biol. Chem., 137, 575 (1941). (IO) Karnovsky, M. L., and Johnson, M. J., A n a l . Chena., 21, 1125 (8)
(1949). (1:)
National Research Council, Canada, Radio and Electrical Engineering Division, Rept. E.R.A.-166 (February 1949).
(15) (16) (17) (18) (19)
773
Peterson, W, H., Harvey Lectures, Ser. XLII, 276 (1946-47). Raper, K. B., Mycologia, 44, 1 (1952). Rivett, R. W., Johnson, M. J., and Peterson, W. H., IND.ENG. CHEM.,42, 188 (1950). Schmidt, W. H., and Moyer, A. J., J . Bact., 47, 199 (1944). Shaffer, P. A., and Somogyi, -M., J . Biol. Chem., 100, 695 (1933). Somer, P. de, Bull. SOC. chim. biol., 29, 364 (1947). Stahmann, M. A , , and Stauffer, J. F., Science, 1 0 6 , 3 5 (1947). Stodola, F. H., Wachtel, J. L., Moyer, A. J., and Coghill, R. D., J . Biol. Chem., 159, 67 (1945).
Taira, T . , Yamatodani, S.,Fujii, S., Komatsu, H., and Takamoto, I., J . Antibiotics ( J a p a n ) , 4 , 103 (1951). (21) Woodruff, H. B., and Larsen, A. H., U. S. Patent 2,532,980 (Dec. 5 , 1950). (20)
RECEIVED for review June 16, lS52.
ACCEPTED
January 2, 1953.
NYLON-COATED LEATHER FRED LEONARD, T. B. BLEVINS, W. S. WRIGHT, AND M. G. DEFRIES Army Prosthetics Research Laboratory, Walter Reed Army Medical Center, Washington, D . C .
L
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E A T H E R is used extensively in fabricating prostheses. The leather is usually worn in direct contact with the skin during use of the prosthesis and is subject to acid and alkaline sweat and bacterial skin flora. Experience has indicated that uncoated leather causes clothing stains, retains odors, and undergoes cracking, discoloration, and degradation. These effects are especially noted during summer months, and in certain instances the useful life of samples of chrome-tanned horsehide harnesses has been as short as 4 weeks. Any coating t o be considered as a protective coating for leather in this application should possess the following attributes: Act as a n osmotic membrane, allowing diffusion of water vapor, b u t be an effective barrier t o bulk sweat, larger nitrogenous molecules, and proteolytic bacteria Have sufficient strength and flexibility to withstand repeated flexings without cracking from the leather Be resistant t o sweat of varying p H Show qood adhesion Be easily cleansible after wear Be easily applied Show good abrasion and scuffing resistance
Of the commercially available plastic materials which were considered for this application, it appeared that a n alcoholsoluble type nylon designated FLM-6501(I), manufactured by D u Pont, showed most promise. Bull et al. (8) have described the use of a n alcohol-soluble type nylon in occlusive dressings for wounds and burns, and their data indicate t h a t this type film was water vapor permeable and acted as an effective barrier against microorganisms. This paper describes the use and properties of FM-6501 S y l o n ( I ) as a protective coating for leather in contact with the skin. FM-6501 Nylon has been used to coat leather worn next t o the skin of amputees. The results indicate t h a t the coated leather is far superior t o uncoated leather in this application. EXPERIMENTAL
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The nylon solution is prepared PREPARATION OF SOLUTION. by allowing 20 grams of Nylon FM-6501 t o dissolve with stirring in 200 cc. of 85% 2-propanol-water solution heated t o 170' F. Once dissolved, the nylon remains in solution for several days, after which the solution gels. T o redissolve the nylon, it is merely necessary to reheat the solution t o 170' F. COATINGTHE LEATHER. The leather in either the fabricated or "as received" form is first wiped clean with alcohol and dried in a circulating-air oven a t 60' C. for 5 minutes. It is then carefully brush-coated with five coats of nylon solution, allowing approximately 5 minutes to elapse between each application.
Following this technique, a transparent coating of approximately 0.008 inch is obtained. ilfter the final coat the sample is allowed to dry in a circulating-air oven a t 60" C. for 15 minutes. ABRASIONRESISTANCE.The abrasion resistance was measured with a Taber Abrasor, Research Model, Taber Instrument Co., North Tonawanda, New York, using a CS-8 wheel and a 250-gram u-eight. The samples were allowed to run for 3000 cycles. FLEXERE.The nylon-coated leather samples were flexed by fixing one end of the sample t o a rigid support and the other end of the sample t o a horizontally reciprocating rod. At each pull stroke the sample received a slight stretch; on the return stroke, the sample was flexed. The machine was operated at a speed of 65 cycles per minute for 40,000 cycles. SIIIULTANEOUS FLEXING AND ABR4DING I N SYNTHETIC SWEAT MEDIUM. A strip of nylon-coated leather was placed on a glass tray in a Gardner washability machine No. 105 (Henry 8. Gardner Laboratories, Inc., Bethesda, Md.). The trough of the machine was filled with a synthetic sweat medium until the nyloncoated sample was immersed. The brush was allowed t o move over the surface of the sample and arranged so t h a t the action of the brush caused the leather to flex while being abraded. The synthetic sweat ( 3 ) was composed of the following ingredients: Sodium chloride, 57 millimoles Lactic acid, 3280 mg. Urea, 390 mg. Creatinine, 10 mg. Uric acid, 15 mg. Water, qs. t o make 1 liter RESISTAKCE TO SYNTHETIC SWEAT. A weighed sample of the nylon pellets was placed in 250 cc. of the synthetic sweat composition at 40' C. for 7 days. After immersion, the sample was washed with water, filtered, dried, and reweighed. STRESS-STRAIN PROPERTIES. Tensile strength and ultimate elongation were measured with a Scott L6 rubber tester in accordance with A.S.T.M. Method D-142, using die C. WATERVAPORPERMEABILITY. Ointment jars, 2-ounce capaoity, with metal screw tops were used. The inside diameter of the tops was 1.85 inches. Circular specimens of the leather samples were cut t o this diameter. Iloles 34 mm. in diameter were cut out in the center of each lid. Thus, when the circular specimens were inserted inside the tops and the caps screwed in place, an square meters of the specimen surface was area of 9.07 X exposed. The diffusion jars were filled within inch of the top with Drierite. The diffusion jars were placed in a desiccator containing acid sweat at 40" C. At regular intervals a jar was removed, weighed, and replaced in the desiccator. The gain
