Effect of Surface-Active Agents on
Penicillin Yields MILLICENT C. GOLDSCHMIDT AND HENRY KOFFLER Purdue University, Lqfayette, I d . Certain natural oils and commerdal sdace-active substances markedly inm+mee~penicillin yields obtained during the submerged growth of Penicillium chrysogenum 4-176 and other strains. These stimulatory decte can be reprodud by the unsaturated fatty adds which these substances contain. The mode of action by which t h i s increase is brought about has been studied in greater detail with lard oil and oleic add, but the only positive information available is that these compounds inmold growth as well as penicillin fields. This cannot be explained entirely by the ability of antifoam agents to aut foam, thereby i n c m i n g aeration. The Stimulation by oleic add apparently is not due to chemical contaminant. nor to the role whiah oleic acid may play as a precursor for types of penicillin with long side chains.
T
HE mediums used in the industrial manufacture of penicillin
produce such excessive amounts of foam when aerated that antifoam agents must be added during the fermentation to prevent the medium from overflowing or becoming contaminated. An ideal antifoam agent should break foam readily, without lowering penicillin yields. Using Penicium chqmgenum X-1612 in s h a k + W fermentations, Stefaniak, Gailey, Jarvis, and Johnson (96)noted that certain fatty materials auch as lard oil, lard, Nopco defoamer, and corn oil more than fulfilled these prerequisites, aa they actually increased penicillin yields. Thii phenomenon waa studied further with PeniciUium Chla(80. genum 9176 and other strains first by Gaby and Sawmiller (10) and later by Koffler and Goldschmidt ( I S ) , who stated that the increase in yields could be reproduced by the addition of the unsaturated acids which these fatty materials contained. The work reported here develops t h m observationsmore completely. MECEIODS
possible after samplimg. In several instances nitrogen analyses to determine mycelial growth were made on broth filtrates by the Kjeldahl method. Mediums. The fermentation medium contained 60 grams of corn-steep liquor, 30 grams of lactose (U.S.P.), and 10 grams of calcium carbonate in loo0 ml. of distilled water. Sodium phenylacetate was added as indicated above. The spores were obtained from culturea grown on a medium containing the following constituents in lo00 ml. of distilled water: Grams
Brer Rabbit gold-label molaasea Glyaerol Peptone NECl
lN@.."O
Agar
7.5 7.6 5.0 4.0 0.05 0.06 25.0
Cultures. For almost all these experiments, Penicillium chrysogenum Wisconsin 9176 was used. One experiment involved also the following strains of the Penicillium chrysogenum-
notatum group: NRRL 832, NRRL 1951-B25,NRRL 1984-NZ2, Carnegie Institution X-1612,Wisconsin 47-1380,and Wisconsin
47-911, INCREASE OF PENICILLIN YIELDS BY UNSATURATED FATTY ACIDS
The addition of several natural oils results in increased penic i l l i yields obtained during the submerged growth of strain &-176, as shown in Table I. The oils were added in levels that varied from 0.15 to 3.0 ml. per 100 ml. of medium. The concentrations which gave the highest yields are indicated in the table, as are the pH values of the samples on the day of the maximum yields. In almost all the experiments, maximum yields were obtained on the seventh or eighth day; in general, the addition of antifoam agents did not change the time to reach maximum yields.
In general the procedure was to add, at the time of inoculation, varying amounts of heat-sterilized or filtered surface-active test agents to 100-ml. portions of a sterile medium contained in TABLE I. EFFECFOF NATWBAL011sON PENICILLIN YIELDS 5OO-ml. Erlenmeyer flasks. The flasks were then inoculated with Max.Yield, pE on Day of Unita/MI. Max. Yield 1 ml. of a spore suspension of penicillin-producing organisms 0 t Conon oil Oil Oil oil and placed, a t 25' C., on a reciprocating shaker, which had a Agent &.j100~i: preeent ahent present absent 4 b c h stroke and moved at a rate of 85 strokes per minute. Corn oil 0.80 756 489 8.3 8.3 0.30 817 164 7.9 7.9 Lard oil* The spore suspension waa prepared from cultures which had been Olive oil0 0.16 808 290 8.2 8.2 stored in the refrigerator for no longer than 4 weeks. Sodium * Summary data from two sepsnrta rum. phenylacetate, a precursor of penicillin G, waa added to the shake flasks during the fermentation at a concentration of 0.176 gram per 100 ml. after 12,24, 48,60,72, 84,96,and 108 hours (9.4). The average control yield obtained with strain Q-176in the exOn the fifth, sixth, seventh, and eighth days, samples from periments described in this aper waa 280 units er ml. Althou h duplicate or triplicate flasks were withdrawn, pooled, and then data from experiments in wkch control yields Zeviated marke3y from this average should be used with caution they are included sssayed for penicillin by the cylinder-plat8 method with M i m e coccus pyogerzRp uar. aurewl FDA 209P, as test organism (899). in the tables to mdicate the maximum yields that can be reached in the presence of certain agents. They robably should not be A preparation of penicillin from the Food and Drug Adminisused to evaluate the relative stimulatory a h t y of various agents. tration, Washington, D. C., was used aa a reference standard. Because of the impurities that they may contain, natural oils To determine whether the antifoam agents had any inhibitory do not lend themselves readily to precise studies. Therefore, effects on the teat organism, assays were run on the uninooulated attempts were made to determine which components of these fermentation medium containing the highest level of the agent fats cawed the stimulation of yields. Table V shows that these used. In no caae waa any inhibition observed. The pH of oils contain large amounts of unsaturated fatty acids, and it liquor plus cells waa determined with a glass electrode as soon as 1819
,
1820
INDUSTRIAL A N D ENGINEERING CHEMISTRY
seemed likely that these acids were responsible for the increase in yields. The data in Table 11, obtained with chemicals of C.P. or U.S.P. grade whenever possible, strengthened this assump$ion. Oleic, linoleic, and linolenic acids stimulated penicillin yields strikingly. It is not surprising that esters of fatty acids did so, too, because Penicillium chrysogenum Q-176 contains lipases which can hydrolyze these esters and liberate the unsaturated fatty acids (If ). Glycerol, on the other hand, which would also be liberated from natural fats in this manner, stimulated yields much less markedly. Similarly, the saturated fatty acids were less stimulatory than the unsaturated fatty acids, and even inhibitory when they were added in comparable concentrations. UNSATURATED FATTY ACIDS, TABLE 11. EFFECTOF GLYCEROL, A N D ESTERS ON PENICILLIN YIELDS D Hon~Day of Max. Yieldb. Units/Ml. Max. Yidd No. Agent Agent Agent Agent Agenta Expts. present absent present absent 2 401 144 8.2 7.8 Glycerol 8.1 1142 10 259 8.1 Oleic acid (commercial) 8.2 1 1138 218 8.1 Methyl oleateC(99.8% pure) 8.1 2 1250 22 1 8.1 Methyl oleated (98% pure) 224 8.1 8.1 1 1280 Ethyl oleate 296 3 1335 8.0 7.9 Linoleic acid 296 1437 7.9 8.0 3 Linolenic acid 0 Concentration for all agents but glycerol was 0.002 M ; for glycerol i t was 0.004 M . b Average figures obtained from number of experimenta indicated in second column. c Obtained from Hormel Institute, Austin, Minn. d Obtained through the courtesy of F. W. Quackenbush, Purdue University '
To make sure that the increase in yields was not due to some chemical contaminants contained in the samples used, the experiments were repeated with highly purified methyl oleate in which the impurities amounted to less than 1%. The pure methyl oleate stimulated yields just as markedly as the less pure batches. Although oleic acid purchased from different sources was used in these experiments, the maximum yields obtained ranged only from 1000 to 1200 units per ml. The effects of oleic acid on penicillin yields are not restricted to strain Q-176. Almost all other strains of the Penicillium chrysogenum-nofutum group tested also responded to the addition of oleic acid, as shown in Table 111.
TABLE 111. EFFECTOF OLEIC ACID ON PENICILLIN YIELDS (During submerged growth of various strains of Penicillium notatumohrysooenum group) pH on Day of Max. Yield, Units/Ml. -___-Max. Yield Oleic acid0 Oleic acid Oleio acida Oleic acid Strain present absent present absent 14 7.6 31 832 8.1 i.'l 23 30 1951-B25 8.1 8.1 74 148 1984-N22 7.9 7.9 210 406 X-1612 8.1 8 . 0 392 1080 Q-176 8.0 7.8 294 940 47-1380 8.2 8 . 2 164 665 47-9 11 = 0.002 M .
Many commercially available surface-active compounds, some of which are being used as antifoam agents, are able to increase penicillin yields, as is shown in Table IV. Information on the chemical nature of some of the materials used is given in Table V; this information was either made available through the courtesy of the various manufacturers or taken from Fieser and Fieser (8). In order to obtain maximum penicillin yields it is important to control the concentration at which these agents are added. The optimum concentration for each compound is indicated in Table IV. Certain other compounds, such as Alkaterge C, Alkaterge 0, Alkaterge OX, Syntergent K, tributyl citrate, and tributyl phosphate, decreased penicillin yields or mold growth, or
Vol. 42, No. 9
both, even a t the lowest concentrations (usually 0.05 ml. per 100 ml.). However, batches of these commercially available compounds may differ and the conclusions given in Table IV, and mentioned here, need not always be true. With the exception of mineral oil, glycerol, and the silicone antifoam A, all compounds found to be stimulstory contained unsaturated fatty acids. Increases obtained with nearly all the glyceride antifoam agents were higher than those obtained with mineral oil and glycerol. Silicone antifoam A in two experiments proved as stimulatory as some of the other antifoam agents. EXPLANATIONS FOR INCREASED YIELDS
Stefaniak, Gailey, Jarvis, and Johnson (W), in trying to explain the increase in yields obtained on the addition of certain compounds, suggested that it might be associated with a more abundant supply of oxygen to the mold after the removal of foam by these substances. This suggeation sounds rather plausible. For instance, it has been shown that in shake flasks, foaming may seriously interfere with the diffusion of oxygen (g5). Moreover, the fermentation liquid in flasks to Fhich no antifoam agent had been added did froth intermittently in the experiments reported here. Nevertheless, this explanation does not seem to account fully for the dramatic increases in yields described. If the increase in aeration actually was the sole cause of these increases, the addition of any nontoxic agent capable of breaking foam should result in similar yields. However, this does not seem to be the case, as is indicated in Figure 1. Mineral oil is a paraffin hydrocarbon, and probably physiologically inert; up to approximately 10 ml. could be added per 100 ml. of medium without any decrease in penicillin production. Therefore, the increase in yields that could be accounted for by an increase in aeration through the removal of foam should not exceed that obtained with mineral oil. The ability of various substances to break foam can be compared in the following manner: A given amount of fermentation liquor lus cells is placed in a graduate cvlinder and is aerated throug: some suitable aerating device. The air flow is adjusted so that the foam that develops rises slowly in the cylinder. The amount of antifoam agent to reduce the volume of foam to a given level is then determined. By simulating fermentation conditions-that is, age of cells and li uor and type aerating and stirring devices-as closely as posshe and by keeping conditions throu hout various comparisons constant, a fair idea can be obtainecf of how effective a given antifoam agent will be in actual fermentations. Although the authors have such information on almost all of the agents used in this study, it is not pertinent because all the compounds that stimulated yields at least as much as did mineral oil prevented foaming in shake flasks when used in concentrations that allowed optimum increases in yields. Additional evidence for the belief that increases in yields beyond that brought about by mineral oil may be caused by other means has been furnished by Holtman (fa),who noted an in-
TABLEIv. EFFECT OF COMMERCIAL SURFACE-ACTIVE COMPOUNDS ON PENICILLIN YIELDS Opt. Concn., M1./100 MI. 0.15 0.15 3.00 0.05 0.05 1.50
pHa on Day of Max. Yieldn, Units/Ml. Max. Yield Agent Agent Agent Agent present absent present absent 290 8.3 8.2 972 7.8 7.9 164 790 8.0 8.0 261 585 715 293 7.9 8.0 8.0 7.9 293 850 236 7.6 8.0 69 1
No. Expts. Agent Defoamer 5 Defoamer 51 Mineral oil Monosulph Nopalcol 1-0 Nopco Glyceryl Oleate 212 7.8 1.50 2 466 h'opco GPI 486 8.3 0.15 1 1080 Penicillin defoamer 282 8.0 2 783 Silicone antifoam A 0.15 428 8.2 0.05 1 492 Tween 80 8.1 1 855 214 0.05 Vedfat Y a Average figures obtained from number of experimonts indicated in column.
8.0 8.3 8.1 8.2 8.0 third
INDUSTRIAL AND ENGINEERING CHEMISTRY
September 1950
1821
pH ON DAY OF MAX.
PENICILLIN YIELD
A - LARD OIL 0
- MINERAL OIL
MOLES OF OLElC ACID
0 - MINERAL O!L
