Production of Tyrothricin in Cultures of Bacillus brevis J
J. C. LEWIS, KEENE P. DIMICK, AND I. c. FEUSTEL Western Regional Research Laboratory,
U . S . Department of Agriculture, Albany, Calif.
for each treatment so that duplicates could be removed for assay at intervals. Before assays were made, evaporation losses were corrected by addition of water, so that all yield data are in terms of original volume of medium. Two types of media were used in these investigations: media in which asparagus juice was the sole or principal component, and media based on Bacto tryptone or other nitrogenous substances, plus glucose or other fermentable carbon compounds, and inorganic ingredients. Asparagus media were sterilized a t about pH 5 (the unaltered p H of the asparagus juice or diluted asparagus juice concentrate) by autoclaving for 20 minutes under 10 pounds of steam pressure. After the flasks had cooled, the medium was adjusted aseptically to p H 7.5 by the addition of predetermined aliquots of sterile 1 N sodium hydroxide (generally 0.5 to 3 ml. were required). Tryptone media were prepared in the same way, although no difference in results was obtained when they were sterilized at pH 7.5. Growth was estimated turbidimetrically. Suitable dilutions of the whole culture suspension were measured in a Klett-Summerson photoelectric colorimeter with a wide-band filter, with maximum transmission a t 660 millimicrons. For convenience the turbidity reading was multiplied b the dilution factor to give “Klett units” of bacterial growth. Ol&r cultures with heavy or wrinkled pellicles could not be dispersed completely by shaking; such cultures were dispersed in a homogenizer consisting of a squarebottom glass tube and a closely fitting, mechanically driven rubber cylinder. Blanks on the pH-adjusted uninoculated medium were subtracted. Tyrothricin was determined by a rapid method (I), based on the hemolytic action of tyrothricin on rat erythrocytes. The decrease in turbidity on hemolysis was measured photometrically. All results in this paper are expressed in terms of the standard described.by Dimick (I), Turbidity and tyrothricin determinations checked closely for replicate cultures. Calculations based on routine assays selected a t random showed that the over-all variation in culturing and assaying gave the following standard deviation for individual cultures: for turbidity, 50 Klett units or 3y0 of the actual mean values; for tyrothricin production, 0.07 gram per liter or 7% of the actual mean values. The hemolytic assay was checked by the isolation o f t rothricin in certain ex eriments. The procedure of Dubos and Sotchkiss (2) was con&cted on a scale that would permit the isolation of tyrothricin from 40- t o 100-ml. samples of culture suspension. The steps of their procedure were unchanged except that the solutions were heated to 50° C . on the steam bath after tyrothricin was precipitated with 1yosaline; this modification induced rapid flocculation and aided atration. The precipitate obtained wae dried to constant weight over phosphorus pentoxide in vacuo. The hemolytic activity of this material was determined after solution in alcohol. Yields of tyrothricin for a given treatment were fairly constant from one experiment to another. Thus a control medium containing 1.5% tryptone, 3% glucose, and inorganic salts was tested in connection with each of eight experiments carried out to obtain the comparisons given in Figures 7 and 8. The cultural work extended over a period of 9 weeks. The mean yields and standard deviations of the individual yields in grams per liter follow: 4 days of incubation, 1.23 =t0.09; 10 days, 2.14 * 0.11; 16 days, 2.32 A 0.19. The turbidities developed were equally regular; the corresponding values in Klett units were: 4 days of incubation, 1440 90; 10 days, 2590 f 90; 16 days, 3110 * 130. Nevertheless, each experiment was designed, as far as possible, to be complete within itself so that the interpretation of results would not depend on questionable differences in absolute v a l u ~ sfrom one experiment t o another.
Yields of tyrothricin in excess of 2 grams per liter of medium were obtained through the growth of Bacillus brevis (B.G.) in shallow layers of medium. Maximum yields were found after 10 or 16 days of incubation at about 35” C. with the medium disposed in 11-mm. layers. Complex sources of nitrogen, such as Bacto tryptone, acid hydrolyzate of casein, corn-steep liquor, tryptic digest of soybean meal, and press juice concentrates from waste asparagus butts, proved most suitable; relatively simple substances, such as glutamic acid, asparagine, or ammonium sulfate plus citric or malic acid, proved moderately effective in the presence of 0.2% Bacto tryptone. About 3 to 5 % of a fermentable carbon compound, auch as glucose, mannitol, or glycerol, was necessary for best yields; fructose, sucrose, lactose, and maltose proved much less effective. Requirements for calcium, magnesium, and manganese were demonstrated. Unlike the other nitrogen sources mentioned, properly processed asparagus concentrates yielded nutritionally complete media without addition of sugar or inorganic elements.
T”
ROTHRICIN, an antibiotic material formed during the growth of Bacillus brevis, is now produced commercially for medical and veterinary uses. I n the course of investigations of thb potential usefulness of press juices from vegetable wastes in industrial microbiological processes (6,6), shallow-layer cultures on asparagus-butt-juice media gave tyrothricin yields approaching 2 grams per liter (5). The yields reported elsewhere ( d , S , 9) are much lower for both shallow-layer and submerged cultures. This paper deals with the cultural and nutritional factors promoting high yields of tyrothricin in shallow-layer cultures and the use of juice concentrates from waste asparagus butts as culture media. Almost all of the published work on tyrothricin production has been done with a strain of Bacillus brevis isolated and designated “B.G.”by Dubos and Hotchkiss (2). The substock of this strain used principally in this study is identical with that discussed previously (6). Stock cultures were maintained on Bacto nutrient agar slants. The slants were incubated at 35” C . for 24 hours and then stored in the refrigerator. Inoculum cultures were prepared by suspending the surface growth from a freshly incubated nutrient agar slant culture i n sterile water, and transferring this t o 50 ml. of asparagus juice medium (concentrate E diluted toO.1570 nitrogen was enerally used, Figure 7). After incubation for 24 hours a t 35O the inoculum cultures were pooled and used a t the rate of 1 ml. per 50 ml. of experimental medium. The experimental cultures were usually grown on 50-ml. portions of medium i n 250-ml. Pyrex Erlenmeyer flasks. This method gave an 11-mm. depth of medium, which represented a compromise between the most rapid production and the need for suitable volumes of culture liquid. The cultures were incubated at 35’ C. in an incubator room maintained at about 50% relative humidity to diminish evaporation. Enough flasks were set up
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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
October, 1945
Nitrogen fractions, reducing sugars, and inorganic constituents were determined by .reoogniSed methods. Asparagus. re98 juces were found to oontan no a preciable amounts of aci8-hydrolyzable nonreducing su #onamino ot anic acids were approximated by titrating pH 7.8 to 2.8 10) and correcting for the butrering effect of the muno acids and hosphate present. An arbitrary e uivalent weight of 85 waa &for converting the d a b to a weig%t basis. Citric acid was determined by a colorimetric pentabromoacgtone method (7).
b,
EXPERIMENTAL RESULTS
A general parallelism was observed between tyrothricin production, turbidity and cellular-matter producbon, and nitrogen utilization. These and certain other variables are plotted in Figure 1, in relation to the period of incubation for two different media which are capable of producing high yields of tyrothricin. The asparagus medium conaisted of asparagus prees juice concentrate 101 (Figure 7),diluted fo give 0.18% nitrogen and, after sterilization, adjusted t o pH 7.6 by adding 1.76% of 1 N sodium hydroxide. The other medium contained 1.6% tryptone, 3% glucose, and inorganic salts (footnote a, Table 11). This medium also contained 0.18% nitrogen. The values for cellular matter were obtained by centrifuging aliquots of the cultures without pH adjustment, and drying and weighing the precipitate. The residual turbidity in the supernatant liquors and the additional weight of material centrifug-
In N y ,
2
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AS PAR AG US
M E DtlU M
I 0
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I 1.1
0
1
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able after a preliminary acidification, as in the isolation of tyrothricin, indicate that the values for cellular matter as determined are low by about 25 and IS%, respectively, for the asparagus and tryptone media. At each harvest period pooled cultures were centrifuged without adjustment of pH, and the liquors were clarified by filtration with the aid of a small amount of analytical-grade Celite. The culture atrates were then analyzed for total (Kjeldahl), amide, amino, and ammonia nitrogen, reducing sugar (as glucose), pH, and n o d n o organic acids. Approximately half of the total nitrogen disappeared from the culture filtrates after 14 or 18 days of incubation. Of the nitrogen taken up, about one third was converted into tyrothricin. Amino nitrogen disappeared almost completely during the 6rst 6 days of incubation; its disappearance was paralleled by a corresponding rise in ammonia nitrogen. Amide nitrogen remained approximately unchanged a t about 6% of the original total nitrogen on the tryptone medium; on the asparagus medium it dropped parallel with the drop in amino nitrogen from 12 to about 3% of the original total nitrogen. After 6 days of incubation the ammonia nitrogen began to disappear; this loss paralleled a drop in the nonamino organic acids, which began at about this time. About 0.025 milliequivalent of ammonia per ml. disap peared in either case; the decreases in organic acids amounted t o 0.024 milliequivalent per ml. of asparagus medium and 0.018 milliequivalent per ml. of tryptone medium. Only about 30% of the orjginal organic acid 0 content disappeared in either cme. These data are of interest in connection with experiments discussed later, which show that the presence of organic acids permits the K utilization of ammonia nitrogen for growth w and tyrothricin production. aka, -i Sugar was utilized grduaily over the K whole incubation period. On the tryptone w n. medium it was utilized almost completely after 18 days of incubation. The pellicles became increasingly heavy throughout the incubation period in proportion to theincrease in turbidity. Growth IIwas poor on the asparagus medium the W d first day, and diffuse arem in the pellicles Y did not disappear until the fourth day. Much precipitate was formed in the t r y p tone cultures but not in the asparagus cultures. The pellicles of the tryptone cultures t became creased with many fine intersecting P c m wrinkles between the seventh and tenth a d dm days of incubation; this was true to a lesser 3 W c Idegree of the pellicles on the asparagus t4 medium. Both media permitted the deP velopment of very heavy pellicles.
I
I
I
*
EFFECT OF CULTURAL CONDITIONS
o
0
w
DAYS O F I N C U B ~ T I O N TYROTHRICIN CELLULAR MATTER TURBIDITY REDUCING SUGAR
X
o 0
0
KJELDAHL NITROGEN AMMONIA NITROGEN AMINO NITROGEN PH
A
* v
+
Figure 1. Growth, Tyrothricin Production, Nitrogen a n d Sugar Utilization, a n d pH a f t e r Various Periods of Incubation Amparagus medium: Asparagus ooaeentrate 101 diluted to 0.18% nitrogen. Tryptons m c dium: 1.5% tryptone. 3% &looae, and inorganic salt. (0.18% nitrogen).
Growth and tyrothricin production were measured for seven incubation temperatures in the range 25.5' to 41.3' C., simultaneously with both asparagus-juice medium and tryptone medium (Figure 2); 35O C. proved to be a generally useful incubation temperature. Growth and tyrothricin production were measured for five depths of culture medium in the range 8 to 18 mm. simultaneously with both asparagus juice and tryptone (Figure 3). Growth and tyrothricin production increased with time of incubation, the shallowest cultures
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
998 TABLE I. EFFECTO F
Medium 1.5% tryptone. 3% glucose, &sparagus-concen%?ash (asHCIextract), 0.18% N Asparagus concentrate E diluted to 0.16% N
Vol. 37, No. 10
A few exploratory experiGROWTH AND TYROTHRICIN ments demonstrated that this PRODUCTION* stock of B. brevis, unlike that Tyrothricin Production, Growth Turbidity), in studied by Stokes and WoodComposition of in Grams/Liter, after Klett tinits, after Atmosphere, % Incubation for: Incubation for: ward (9),is able t o produce 01 NY 3 days 6 days 10 days 16 daya 3 days 6 days 10 days 16 days amounts of tyro-
OXYGEN
29-30 21 12-14
CONTENT
70-71 79
OF
1.34 1.32 0.92
AThlOSPHERE
1.36 1.22 1.12
1.70 1.67 1.46
ON
1.74 1.70 1.60
1890 1800 1350
2450 2360 1870
3200 3130 2530
3170 3200 3050
thricin in submerged Or shake cultures with complex sources of nitrogen. A yield of 1.4 grams 29-30 70-71 0.89 0.99 1.15 1.12 840 1360 1960 1830 21 79 0.90 0.90 1.12 1.26 770 1310 1780 is60 of tyrothricin per liter was ob12-14 86-88 0.51 0.81 1.05 1.23 550 850. 1410 1910 tained after 48 hours of sera0 The Basks were incubated in closed wooden boxes through which the gaB mixtures were passed continuoualy. tion on a medium containing Relative humidity was about 85%. Oxygen contents were checked by periodia gas analyses. 1.5% tryptone, 3% glucose, and inorganic salts. Fifty-milliliter portions of the same medium in 250-ml. Erlenmeyer flasks were also shaken in a reciprocating reaching a maximum at 5 to 10 days, while the deepest required shaker with a 1.5-inch stroke a t the rate of 140 cycles per minute. 16 days. A yield of 0.9 gram per liter was obtained after 4 days of shaking, Several comparisons of 50-ml. cultures in 250-ml. Erlenmeyer whereas the turbidity had reached a maximum within 24 hours flasks and 280-ml. cultures in Fernbach flasks (11 mm. depth of of incubation. Finally, on certain asparagus juices tyrothricin medium in either case) failed to disclose any significant differences production in aerated cultures lagged behind growth by one or in rate or extent of growth or tyrothricin production for pellicles more days. One asparagus juice, which gave excellent tyrothricin of differing mew. production in shallow-layer cultures, failed to permit significant Table I shows the effect of variation in the oxygen content of tyrothricin production in submerged cultures, although as good a the atmosphere. Experiments were conducted simultaneously level of growth was obtained. Our results, like those of Stokes with asparagus and tryptone media. A reduction of oxygen conand Woodward, indicate that some factor or factors in addition tent to 12-14% gave a small depressive effect for short incubato those that control growth are important for production of tion periods. An atmosphere with the oxygen content raised to tyrothricin in submerged fermentations. 29-30oJo was not better than normal air. No definite differences were obtained when cultures were incubated at 15 and at 85% relative humidity. Evaporation losses were greater in the former NUTRITIONAL REQUIREMENTS case, but such losses have been corrected in the experiments r+ A basal medium containing tryptone, glucose, and asparegua ported in this paper. concentrate ash was found to permit a high level of growth and Although the pellicle formed by B . &revis may be quite heavy, tyrothricin production. This medium, unlike asparagus juice, its cohesive strength is not high. A slight jarring usually suffices proved adaptable for investigations of the specific nutritional reto break up the pellicle so that it sinks to the bottom of the flask. On certain relatively poor media this happens spontaneously. Cultures in which the pellicle h t s been broken up by shaking daily or less frequently give depressed levels of growth and tyrothricin production.
ASPARAGUS
86-88
MEDIUM
U 1600
= 800 I-
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5
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I NCUB AT1 ON
O
TEMPERATURE
Figure 2. Effect of Incubation Temperature on Growth and Tyrothricin Production Asparagum padiumr Asparagus concentrate E diluted to 0.167 rpptone mediamt 1 % try tone, J 9% ~lucome, 0.4% nitrogen. asparagus concentrate ash (&nitrogen %I%
.
Figure 3. Effect of Depth of Culture Medium on Growth and Tyrothricin Production Asparagusmedium: Aspara us wncentrate E diluted toO.l6%nitrosen. Tryptone mediums l.S%tryptene. 3% slucose, 0.28% aspnragau wncentrata ash (0.18% nitrogen). For corn arinon, re ions 00spondtng to aertain arbitrary yields are indcated by dotted lime-.
October, 1945
INDUSTRIAL AND ENGINEERING CHEMISTRY
quirements of B. brm‘a,since the components could be eliminated or altered one at a time. The development of a synthetic medium was not attempted, however. Figure 4 combines the results of several experiments which demonstrated that both the glucose and the aah were essential components of the medium. Moderate variations of the concentration of glucose or of ash were without significant effect. The maximum yield was related to the amount of tryptone supplied. The ash was added directly to the medium in these preliminary trials. Subsequent work showed that it could be replaced by a hydrochloric acid solution of the ash, though not by a hot water extract. It could also be replaced by the mixture of inorganic salts described in footnote a, Table 11. Some effects of variations in the inorganic salts of the tryptoneglucose-salts medium are presented in Table 11. Of the elements supplied by the salt mixture, calcium, magnesium, and manganese were necessary for the highest yields; the others were either nonessential or were adequately supplied in the tryptone. Omission of calcium gave sharp reductions in tyrothricin yields, especially after 10 and 16 days of incubation. Lowering the calcium concentration to 50 p.p.m. or raising it to 200 p.p.m. have no marked effect. Asparagus aah supplied about 80 p.p.m. of calcium when it was used a t the 0.4% level. After incubation about half of the calcium supplied remained in solution; the other half was removed on centrifugation of the bacterial cells. The controls without added calcium contained about 10 p.p.m. of calcium. Calcium deficiency affected growth as markedly as it did tyrothricin production, so that calcium appears to be essential for the growth of B. brevis, at least for maximum levels. Within the authors’ knowledge this is the first time that calcium has been found to be essential for growth of a microorganism, although it has been recognized as important for other functions such as proteinase production or nitrogen fixation. The magnitude of the re-
999
quirement is noteworthy. W Definite depresk -I sive effects, al2.0 though less marked 5 P than with calcium, were noted when V) 1.5 m a g n e s i u m waa omitted from the medium. When I both calcium and 5 ‘*O m a g n e s i u m were 0 omitted, growth E and tyrothricin prod u c t i o n w e r e al0.5 most as poor as 2 when no salts were added to the basal 4 8 12 16 medium, and much DAYS OF INCUBATION poorer than when e i t h e r calcium or Figure 4, Tyrothricin Production magnesium was on Tryptone Media omitted. Omission of manganese resulted in marked reduction of the yields of tyrothricin and bacterial cell material. Figure 5 gives the results of a study of the magnitude of manganese requirement. The tryptone supplied 0.02 p.p.m. of manganese as shown by spectrographic analysis; the addition of 0.5 to 2 p.p.m. of manganese was necessary for best results. The data suggest that, if the medium is incubated as long as 16 days, relatively low amounts of manganese suffice for tyrothricin production, though not for growth. Figure 6 presents the influence of glucose concentration on growth and tyrothricin production. The need for sugar waa most evident for TABLE11. INORGANIC ELEMENT REQUIREMENTS OF BaciUw, the 10- and 16-day incubation periods. The brevis FOR TYROTHRICIN PRODUCTION sugar utilization in a similar medium (Figure 1) Tyrothricin Production, Relative Growth amounted to 0.4, 1.5, and 2.4% after 4, 10, and Addition to Basal Medium Grama Liter, af!er (Turbidit ) Klett Units, (1.5 Tryptone, Inoudation for. after Andubation for: 16 days of incubation; these concentrations me 3 2 Gluoose) Expt. 4 days 10 days 17 days 4 days 10 days 17 days similar to the limiting concentrations for maxi0.24 0.11 390 .4 None mum growth found i n this experiment. The 1.15 2.05 1760 ... As aragus ash (0.4oJo) 2.09 organism grew and produced a small amount 1.55 2180 ... H& ext. of asparagus ash 2.26 1.21 1790 ... Salt mixt., complete“ .., of tyrothricin after a 4-day lag in the presence 1.02 0.96 1890 Ca omitted 1.44 1.95 1900 Ca content halved ... 2070 of 14% glucose. 1.90 i.40 Ca content doubled 1.78 1.26 Mg omitted ... 1630 Glucose was the only satisfactory sugar tested; 770 1.04 0.48 M n omitted fructose, sucrose, lactose, and maltose were un2.05 1.29 ... 1940 Fe omitted ... 2.35 1.24 1890 Po4 omitted able to support maximum tyrothricin production 2.15 1.37 2080 804 omitted ... 2.30 1.33 2020 C1 replaaed by NOI ... (Table 111). Each was tested a t 1, 3, and 5% 2.15 1.29 2140 K replaced by Na ... concentration; the ratios of effectiveness in 0 . 1 8 0.18 0.12 160 260 660 B None comparison with glucose did not vary signi1.10 1.94 2.02 1620 3160 3110 Salt mixt complete“ 0.61 0.76 0.79 C s omi’tbd 980 1430 1480 ficantly for these concentrations. The ineffecMg omitted 0.92 1.51 1.05 1370 2200 2640 0.38 0.25 360 800 800 Ca and M g omitted 0.20 tiveness of fructose and lactose was not due to 740 1380 1680 1.05 1.76 Mn omitted 0.43 inhibitive action since combinations of glucose C None 0.10 0.10 ,. 180 460 with these sugars gave good results. Ca (100 p.p.m.) 0.68 1.11 .. 1490 1890 ,. Mn (2 p.p.m.) 0.13 0.35 ... 370 720 .. . The polyhydroxy alcohols, glycerol and manCa (100 p.p.m.) + Mn (2 P m) 0.94 1.61 1730 2620 nitol, were as effective as glucose. The pellicles C a ’ r i o d p p m ) + Mn 2 appeared distinctly different from those obtained p.m.) 4 Mg (SO p.p.mS 1.06 1.95 ,. 1980 3170 SaPi mixt., completes 0.98 2.24 1580 2760 . with glucose and had a fluffier texture. It canS i l t mixt. + Cu (0.1 p m.), M O (0.1 p.p.m.), %n*(O.6 not be stated to what extent glucose, glycerol, 0.86 2.18 1840 2950 Sari’St!. (Fe omitted) 1.02 1.83 , , 1940 2980 . and mannitol serve by removing ammonia and Salt mixt. (10 p.p.m. Fe preventing high cultural pH values. However, added) 1.00 2.40 1730 2810 the pH in cultures without such compounds or a . KHrPO4 0.070%, KC1 0.035%. Mg804.7HaO 0.060%, CaClt 0.025%. Fa (ad sulfate) 2.0 p.p.m Mn LE sulfate) 2.0 p.p.m. This salt mixture wan used whenever “inorganic with non-effective sugars (fructose, sucrose, lacnalta” areherre6 to in the reparation of a particular medium The approximate amounts of ions BU plied to the medPum by 1.6 0 tryptone were: K 66 No 400 Ca 10 M g 5, Po4 tose, and maltose) quickly rises and stays as 400 SO‘ f5 C1 50 Fe 1 and Mn O O r p . .m. In addition, dpproxim;tely 260 p.p:m. of Na’waa added duiing h adjuatmdnt 8onae uentl the totai amounts of ions in the high &B 8.8; in cultures with optimal levels of medium were: IT 400, &a 600, Ca 100; Mg 50, %O4 8 0, 804 200, C1400, Fe 3, and Mn 2 glucose, glycerol, or mannitol the pH rarely p.p.m. has risen above 8.2 and generally begins to fall
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INDUSTRIAL AND ENGINEERING CHEMISTRY
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I MANGANESE CONCENTRATION
- REM.
Figure 5. Manganese Effect on Growth and Tyrothricin Production with a Basal Medium of 1.501, Tryptone, 3% Glucose, and Inorganic Salts (except Manganese)
Vol. 37, No. 10
with the nitrogen content, which is dependent on the method of processing. The highest nitrogen contents (3.4to 4.3%,dry basis) were obtained by disintegrating the fresh butts and permitting the raw, cloudy press juice to stand for 4 hours a t 50' C. and pH 5.0. Four batches of concentrate totaling 6000 pounds were prepared by this process in cooperation with a commercial canning plant. The tyrothricin yields shown for KO. 101 in Figure 7 are representative for concentrates prepared by this process. Other methods of processing were based on a preliminary blanching of the raw butts as a n aid in obtaining clear juices. Concentrates of such juices had lower nitrogen contents (1.2to 2.7y0,dry basis), and proved less effective for tyrothricin production (concentrates C, D, E, and G of Figure 7). The juice obtained from blanched asparagus tips proved relatively ineffective (Figure 7), although it contained 4.5% total nitrogen, dry basis. Concentration of the juices to thick sirups (up to 75% solids) was carried out in vacuo without detrimental effects on color or usefulness in media for B. brevis. The concentrates contained 14 to 39% of the total nitrogen as free amino (formol) nitrogen, 10 to 24% as amide nitrogen (more than 90% is asparagine nitrogen), and 4 to 13% as ammonia nitrogen. The reducing sugar (as glu. cose) generally amounted to about 65% of the solids; the totel
between the fourth and tenth days of incubation, as is shown in Figure 1. Attempts to study the effect of culture pH by the addition of various buffers and by variation of the initial pH between 6.5 and 8.0 indicated that tyrothricin production was influenced more by t h t nature of the buffer substances added than by pH. For 0) TABLE 111. SPECIFICITY O F CARBOHYDR.4TE REQUIREMENT example, the addition to a medium containing 1.5% tryptone, Bacillus brevis FOR TYROTHRICIN PRODUCTION 3% glucose, and inorganic salts of 0.4% veronal (sodium diethyl Tyrothricin Production, barbiturate) (initial pH 8.0) or of 0.4% glycine (initial pH 7.4) in 01, of Controlso. gave good results, while the addition of 0.4% veronal plus 0.16% a f t e i h c u b a t i o n foi: Addition to Basal Medium Expt. (1.5% Tryptone, Inorganic Salts) 4 daya 10 days 16 day@ sodium acetate (initial pH 8.1)gave inferior results. The over100 100 A 100 all variation in pH was from 7.6to 8.2 in all three cases. On the 94 57 57 other hand, almost as good results were obtained in the addition of 89 59 70 36 81 51 0.4% veronal (initial pH 6.3) or 0.75% potassium dihydrogen 59 63 88 phosphate (,initial pH 6.4) in which cases the pH varied between 100 102 107 70 43 40 5.8 and 7.4 during incubation. The choice of pH 7.5 for adjustE Mannitol 91 106 107 ment of various media appeared to be satisfactory. Fructose (3 87 64 77 Table IV presents results of experiments dealing with nitrogen Fructose ( 2 % ] + glucose (2%) 96 113 111 Lnctoae (3 o) 59 52 41 requirements. Difco Casamino Acids was relatively ineffective 96 123 98 Lactose (2%) + glucose (2%) None 63 50 34 when tested alone; the addition of 0.2% tryptone made it as efa Except where otherwise indicated, each compound and glucose were fective as 1.5% tryptone. Fair yields were obtained with glutamic compared a t 1 3 and 5% concentration. the data are averagea of ratios acid alone; asparagine was ineffective. Both of these nitrogen calculated for 'ea& concentration. T h e 'actual assays for t Vothricin on media containing 3% glucose were approximately 0.9, 1.6, a d 2 grama per sources were improved when a small amount of liter in both experirnenta. tryptone was present. Ammonium sulfate was ineffectiveunless small amounts of both tryptone and TABLEIV. NITROQENREQUIREMENT OF Bacillus brevis FOR citric or malic acid were added. The pH of the amTYROTHRICIN PRODUCTION monium sulfate cultures dropped to about 5.0in the Tyrothricin Production, absence of the organic acids and to about 5.5 in Grams Liter after Addition to Basal Medium Content % N Incudat io; for: their presence. Sodium nitrate was completely Expt. (3% Gluooae, Inorganic Salts) of Medium 4 days 10 daya 16 dam ineffective. 1.62 0.186 0.95 1.97 Tryptone (1.5 These experiments indicate that relatively small 0.91 0.67 0.160 0.43 Casamino Acig)(1.55%) amounts of some particular nitrogen compounds or 1.92 1.98 0.185 0.96 Caaamino Acids + tryptone (0.2%) 0.63 1.26 0.36 0.160 Glutamic acid (1.67%) vitamins present in tryptone are required; in adG l u t p i a acid' (1.67%) + tryptone 0.84 1.13 1.46 0.185 (U.ir70~ dition, the main portion of the nitrogen probably 0:o 0.07 0.0 0.160 Asparagine hydrate (0.867) must be supplied in organic form or as a combinaAsparagine hydrate (0.86%) + ,tryp0.54 1.20 1.87 0.185 tone (0.2%) tion of ammonium nitrogen and organic acids. The 0.08 0.10 0.08 0.160 (NHdnSO4 (0 86 ) 0 .16 0.15 0.185 0 . 1 6 (NHr)BOr (0.'86& + tryptone (0.2%) results are similar to those reported by Kline, (NHdnSO4 (0.86%) + citric acid hyMacDonnell, and Lineweaver (6) for Bacillus 0.0 0.0 0.0 0.160 drate 0 5 % ) ( N H I ) I S (0.86%) ~ + citric acid hy- 0.185 subtilis smber. 0.71 0.68 0.29 drate 0 5%) + tryptone (0.2%) 0.16 0.16 0.0 0.160 (NH&Sb;(O.l6% + malic acid (05 7 ' ) Veronal was found to stimulate tyrothricin pro(NHr)oSO, (0.86%{ + malic acid (0:5%', duction markedly after long-continued incubation ; 0.88 1.25 0.31 0.185 + tryptone (0.2%) 0.09 0.10 0.0 0.160 NaNOa (0 9 7 7 ) yields above 3 grams per liter were observed (Table 0.12 0.12 0.13 0.185 NaNOa (0:97d) + tryptone (0.2%) IV). It was wholly ineffective when used as the 2.26 0.97 1.60 0.186 3.20 2.06 0.95 0.216 principal nitrogen source. The pH changes were 3.41 1.16 1.66 0.247 similar to those shown in Figure 1. 2.05 1.24 1.52 0.292 NITROGENOUS MEDIA
The suitability of concentrates of asparagus-butt press juices for tyrothricin production is correlated
Verona1
0.338 0.061
0.61
0.080
0.0 0.0
0.106
0.0 0.0
0.131
0.82 0.0 0.0 0.0 0.0
0.89 0.0 0.0
0.oj 0.0
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October, 1945
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Figure 6. Influence of Glucose Concentration on Growth and Tyrothricin Production with a Basal Medium of 1.5% Tryptone and Inorganic Salts
Figure 8 gives the tyrothricin-producing capacities of tryptone, corn-steep liquor, and soybean-meal hydrolyzate. The corn-steep liquor was a spray-dried commercial product containing 7.6% nitrogen, of which only 13% was present as free amino, amide, and ammonia nitrogen. The soybean-meal hydrolyzate was prepared by digestion of a solvent-extracted meal with 1% Fairchild's trypsin; the insoluble residue and heat-ooagulable materials were removed. It contained 10.2% nitrogen, with 25% present as free amino, amide, and ammonia nitrogen. The tryptone, corn-steep liquor, and soybean-meal hydrolyzate were employed in varying amounts in a b a d medium containing 3% of added glucose and inorganic salts in contrast t o the asparagus concentrates of Figure 7, which were simply diluted to the desired nitrogen levels. I n all casea tyrothricin production was related more or leas proportionably to the nitrogen supplied up t o levels varying, from below 0.1% for asparagus concentrate C to 0.2 or 0.3% for. the best asparagus preparations, tryptone, corn-steep liquor, and soybean-meal hydrolyzate. I n this region the tyrothricin yields varied from about 1 gram per gram of asparagus-tipjuice nitrogen to about 1.5 gram per gram of soybean-meal-hydrolyeate nitrogen. Maximum yields were obtained only after 10 or even 16 days of incubation. The ranges of nitrogen concbntrationsat which maximum yields were obtained were relatively narrow. With low concentrations the amounts of nitrogen compounds appeared t o be limiting; with high concentmtions, accompanying inhibitory
nonamino organic acids, from 4 to 13%; citric acid, from 0.2 t o 1.2%; and ash, from 8 to 12%. Before the processing of asparagus wastes' had been developed to the stage where high-nitrogen juices apparently could be obtained at will, the relatively ineffective juices C and D (Figure 7) were supplemented with nitrogenous materials. Among the Bacto products, tryptone was most effective, Caeamino Acids was less effective, and peptone, neopeptone, proteose-peptone, and protone were actually inhibitive at 0.4% concentration. The culture pH never rose above 8.2 with sny of these s u p plements. Glycine, asparaghe, and glutamic acid were only slightly stimulatory. 1 A detailed preaentation of anparagus-butt processing and analytical data will be made later. Mimeographed circular AIC-TO, describing equipment layout and giving cost estimatea for the production of anparagus oonoentrate by the disintegration and digeation proaeas, is avstlable on request t o this laborstow.
Figure 7. Relation of Tyrothricin Production to Incubation Period and to Concentration of Nitrogen for Several Asparagus Juicer, T h e concsntratioru of nitro ea coinaidant with 4 and8 % snsar and wit%0.9 % oreado acids in the uninoculatd medium are ind i e a t d to faailitate the discnssion of this Bgura.
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tained with tryptone, corn-steep liquor, and soybean-meal hydrolyzate tended to run somewhat higher but did not appear to be detrimental. Thus the soybean-meal hydrolyzate medium with 0.25% nitrogen was at pH 8.5 after 10 days of incubation, a period during which the rate of tyrothricin production was very good. Higher levels of nitrogen gave markedly lower yields, but the course of the pH was almost identical. Asparagus press juices have not been found deficient in any nutrient required for tyrothricin production other than nitrogen compounds. The predominant sugar is glucose; essentially all of the sugar is reducing sugar. The juices are adequately supplied with vitamixu and minerals. EFFECT OF ORGANIC ACIDS. Table V shows the effects on typothricin production of a number of organic acids which were added to the tryptone-glucose-salts medium. Citric acid was most inhibitive of the acids tested; the tyrothricin production was reduced by 50% in the presence of about 0.6% of anhydrous citric acid. All of the acids appeared t o be slightly stimulatory at the lower concentrations, The inhibitive concentrations of the acids listed in Table V are not sufficiently low t o account for inhibitory effects of the higher concentrations of asparagus juices and other materials shown in Figures 7 and 8. I n no case did citric acid amount to more than 20% of the total nonamino organic acids.
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PER C E N T NITROGEN Figure 8. Relation of Tyrothricin Production to Incubation Period and to Concentration of Nitrogen for Soybean-Meal Hydrolyzate, Corn-Steep Liquor, and Tryptone The nitro en sourcms were usmd i n e basal mmdium contaidnn 3 4 added glueass and inorganic salts. The COPcentrations of nitrogen colnaident with 0.5% organic acids in the uninoculated rnmdium ere indicatmd to facilitats the & c u d o n of this flgure.
substances appeared to affect the yields adversely. Figures 7 and 8 show that, with one exception, the peak of production was found a t levels of nitrogen coincident with about 4% of sugar or 0.6% of organic acids, whichever was reached first. Thus with asparagus concentrate C the peak of production was found at 4 to 5% sugar, while 8% was quite inhibitive; this is about the same resdt found when the glucose concentration was varied in a t r y p tone medium (Figure 6). Corn-steep liquor was the exception; in this case the peak of production appeared at about 0.8% of organic acid (sugar concentration did not exceed 4% at any level of nitrogen). The principal nonamino organic acid of corn-steep liquor is lactic acid, which proved to be one of the least inhibitive acids for tyrothricin production on tryptone medium (Table V). The depressive actions accompanying the higher concentrations of nitrogen (Figures 7 and 8 ) were not accountable by the pH of the cultures. The asparagus media gave pH trends similar to those shown in Figure 1; pH 8.2 was rarely exceeded for either high or optimal nitrogen levels. The maximum pH values at-
There was a question as to whether the high yields obtained with our stock of B. brevis were due solely to the use of better media, or whether the strain had varied spontaneously in this was laboratory. Accordingly, a second stock of B . brevis (B.G.) obtained and designated as B. brevis (X); the supplier found that it gave yields ranging from 0.15 to 0.5 (average 0.35)gram of isolatable tyrothricin per liter of medium containing 1% tryptone and 0.5y0sodium chloride in tap water a t pH 7.0. Preliminary tests in this laboratory showed i t to be inferior to our stock, designated as B. brevis (WRRL) ; ita tyrothricin-producing capacity appeared to be only 25 to 60% of that of the WRRL strain. The tyrothricin-producing capacity of B . brevis (X) was also tested after i t had been carried in slant cultures in this laboratory for periods of 2 and 6 months, respectively. For each of the WRRL and X stocks, six independent lines had been established -three on nutrient agar slants and three on 1%tryptone4).5% sodium chloride agar slants. Table VI gives typical growth and tyrothricin-production data for these lines of B. brauis. After 2 months of stock culturing (involving a total of seven serial transfers at approximately weekly intervals), three X lines (1, 4, and 6) showed significant improvement. After 4 more months of stock culturing (involving twelve more serial transfers), X line 6 was equivalent to the WRRL lines; X line 4, which had shown improvement at 2 months, was now very poor. At this time WRRL line 10 also gave inferior results. The reversal, after
TABLE V. EFFECT OF ORGANIC ACIDSON TYROTHRICIN PRODUCTION BY
Expt. A B
Acid Added Citric (monohydrate) Malonic Succinic Maleio 2-Malic d-Tartaric Lactic (8540%)
Bacillus brevis
Tyrothricin, a8 % of Control', at Acid Concentration of: 0.2% 0.4% 0.7% 1.0% 109 98 38 19 103 102 80 28 108 107 104 75 111 124 110 107
1120 ' 113 106
110 109 109 104
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94 99 96
0 The yielda on 1 .6 0 tryptone, 3% lucoae, inorganic salts medium without added organic aci% were designate3 as 100%. and the yielda with added orKanic acids oompared to it. Figures are average8 of ratios for tyrothrioin yields determined after 4, 10,.and 16 days of incubation. For these respeotive periods the control. yields were approximately 1.2, 2.4, and 2.4 grams per liter in both experiments.
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glucose-ash medium (Figure 2). qkewise, isolation data (experi-
TABLE VI. EFFECT OF STOCK CULTURING ON Two SUBSTOCKS ment C) confirm the strain variatiom described in Table VI. OF B. brevis The X strain, substocks 2 and 3,gave much lower yields than the Relative Tyrothricin Relative Growth WRRL strain, both by hemolytic assay and by isolation. ConProduction* in kTurbidi.ty)*, in arams/Liter. 'after lett Unitr after Stock Culturing for: Stock Culturikg for: 2mo. ( A ) 6mo.( B ) 2mo. ( A ) 6 m o . (8) 0.82 0.42 1470 1230 agar 0.20
