Riboflavin Production by Candida Yeasts J
HAROLD LEVINE, JULIAN E. OYAAS, LEONARD WASSERMAN, J. C. HOOGERHEIDE, AND ROBERT M. STERN Pabst Brewing Company, Milwaukee, Wis.
T h e yeast species, Candida guilliermondia and Candida jZareri, produce high yields of riboflavin on a simple synthetic medium of low- cost. With the former species, either ammonium sulfate or urea can serve as the only source of nitrogen, while with the latter, urea is the nitrogen source. Asparagine or glycine is not necessary in unsteriIized mediums for either organism; however, in sterile mediums, they are necessary for high yields with Candida jzareri. The high riboflavin yields are obtainable only when the total iron content of the medium is held to a minimum 40 to 607 per liter. With Candida guilliermondia, riboflavin yields as high as 1757 per ml. were se-
cured, while with Cundidajlareri, yields as high as 5677 per ml. were obtainable. Molasses, treated to remove iron, can replace pure biotin in the medium. Various metallic salts (manganese, copper, zinc, tin, nickel, and aluminum) added to the medium do not inhibit riboflavin synthesis. Preparations having a riboflavin potency as high as 97,0007 per gram of solids were prepared from liquid cuIture mediums fermented by Candida jlareri. Pilot plant fermentations in 24-liter batches gave riboflavin yields of 1187 per ml. with Candida guilliermondia and yields of 3257 per ml. with Candida jlareri using nonsterilized culture mediums.
T
ml.) containing 25 ml. of inoculated medium were shaken on a Cenco-Meinzer mechanical shaker for a period of 4 t o 5 days in a 30" C. room. At the end of this period, the fermentation suspension was centrifuged in order t o separate the yeast crop from the fermentation liquor. The yeast crop was determined by drying overnight at 103O C. and the soluble solids by drying overnight a t SO" C. The p H of the liquor was determined electrometrically. The riboflavin content of the liquor was determined fluorometrically, frequent checks being made by the microbiological assay.
HE biosynthesis of riboflavin in synthetic mediums con-
taining asparagine has been reported by Burkholder (1, 3 ) and by Tanner and co-workers ( 5 ) . Employing the yeast Candida guilliermondia, Burkholder (1 ) reported riboflavin potencies in shaken cultures as high as 757 per ml. in a synthetic medium in which asparagine was used to supplement ammonium sulfate as a source of nitrogen, and iron was added to the extent of 507 per liter, With glycine in place of asparagine, Burkholder (1) reported yields as high as 607 per ml. Tanner and co-workers ( 5 )confirmed Burkholder's findings but reported that riboflavin formation by Candida guilliermondia was extremely variable, ranging from 2 to 807 per ml. under seemingly identical conditions. Upon further investigation, they showed t h a t riboflavin formation was related t o the iron content of their medium xhich contained both urea and asparagine as nitrogen sources. When the iron content was reduced markedly below the level of 507 per liter used by Burkholder, riboflavin values as high as 1577 per ml. were obtained. Tanner and co-workers (6) also reported that Candida $ a r m produced as high as 2 1 6 ~of riboflavin per ml. provided the iron content of the medium was maintained at a low level. I n the authors' work with the two above-mentioned yeast strains, the findings of Tanner et al. ( 5 )relative to the importance of the level of iron in the medium for maximum riboflavin yields have been conflrmed. I n the mediums developed by the above workers ( 1 , 5 ) expensive asparagine or glycine was used to supplement either amnlonium sulfate or urea. I n the authors' laboratory it was found that if iron is not added to a medium similar to that used by the above workers, i t was possible to secure as high yields without asparagine or glycine in the medium as with these expensive ingredients. By this modification, the cost of the medium becomes relatively inexpensive. I n the work reported here with the two above-mentioned yeast strains, the development of the authors>medium and the results secured when either ammonium sulfate or urea serve as the sole source of nitrogen in the absence of added iron will be described. LABORATORY STUDIES
RESULTB WITH Candida guilliermondia.
The technique of cultivating and carrying out the shake flask fermentations was essentially that of Burkholder (1). Erlenmeyer flasks (125
I n the authors' preliminary work, the findings of Burkholder and Tanner et al. were confirmed. Burkholder's medium containing either ammonium sulfate alone or supplemented with urea, asparagine, or glycine in the presence of 507 of added iron per liter was used. When the medium contained ammonium sulfate alone or with supplementary urea, low riboflavin values (average, 137 and 177 per ml., respectively) resulted. Somewhat higher values (average, 437 and 347 per ml., respectively) were obtained when either asparagine or glycine supplemented ammonium sulfate in the medium. As will be shown later in the authors' work, much higher values are secured with these sources of nitrogen when the added iron is omitted. I n one experiment, the results of Tanner et al. relative to the influence of the iron level were confirmed by studying nine levels of added iron ranging from 0 to 50507 per liter of Burkholder's medium containing glycine. The inhibitory effect of iron on riboflavin production was clearly demonstrated. After these preliminary observations, the mediums used by the above workers were simplified in order t o make the production of riboflavin less expensive. This simplification involved the elimination of expensive asparagine or glycine and the omission of added iron a5 used by Burkholder. It was found t h a t with these changes, i t was possible t o secure as high riboflavin potencies when either ammonium sulfate or urea was the only source of nitrogen as when these nitrogenous compounds were supplemented with either asparagine or glycine. Table I shows the composition of mediums 4A and 4B, the former containing 2 % sugar and the latter 4%. Medium 4B, except for the omission of asparagine and reduction in magnesium sulfate concentration, is otherwise quite similar to the medium of Tanner et al. ( 5 ) . AMedium4B was employed after the authors' initial findings with medium 4A showed that asparagine was not necessary. Except for potassium dihydrogen phosphate, i t was 1665
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found possible to dispense with C.P. chemicals. Urea WRS employed as the sole nitrogen source in this medium. In line with the findings of Tanner et al., it was possible to dispense with calcium chloride and potassium iodide, ingredients present in Burkholder's medium. It was also found that 0.2 gram of magnesium sulfat,e (AIgS0~.7H20)per liter gave as good results as a level of 0.5 gram per liter. A commercial grade of this ingredient was as effective as the C.P. grade. An inexpensive source of urea ( D u Pont) gave as good results as the Eastman Kodal: compound. Pure biotin (1Ierck & Company, Inc.) could be replaced by deionized molasses or by cane molasses after treatment to remove iron by the method used by Elvehjein (3) and by Pappenheirner arid Shaslian (4).
TULE I. COMPOSITIOS OF MEDIUMS 4A
Ingredient Water, distilled or tapb
1000 ml.
0.50 g.
KHiPOi
3igSOA.7 H z 0 (NH4)zSOr or Urea ( D u P o n t ) Sucrose o r glucose, C.P. Biotin (Merck 8: Co., Inc.) C T r a c e elements B (as HaBOd) h f n (as hInSOa) Zn (as ZnSOa) Cu (as CuSOd >Io [As(NHi)alIorOzr.4HzO] p H with distilled water p H with t a p water
0 . 2 0 g. 2 . 0 0 g. or 0.92 g. 20 g. 1.oy
4Ba 1000 ml. 0.50 6. 0 . 2 0 g.
....
1 . 8 4 g.
40 g.
TABLE 111. DATA SHOWING DAILI CHASGES~ DURIKG FERMEIVTATION
The results secured with mediums 4.k and 4B with supplements to these inediuins are set forth in Table 11. The authors found it unnecessary to st'erilize the mediums in order to obtain good riboflavin yields with Candida gztilliermondia. Lsing medium 4 8 , the results show that ( a ) distilled and tap water gave the same results because Milwaukee t a p water coming from Lake Michigan is low in iron content, ( b ) added iron is inhibitory, ( c ) glycine or asparagine superimposed on the medium does not give increased vitamin yields, ( d ) the level of ammonium sulfate does not influence the yield, and ( e ) urea and ammonium sulfate at the same level of nitrogen gave the same results. A commercial grade of ammonium sulfate treated t o remove iron by precipitation as ferric hydroxide was found to be as satisfactory
TABLE 11. IKFLUEKCE O F IXGREDIENTS A S D SrJPPLEXESTS O F VARIOUSMEDICXSON THE YIELDOF RIBOFLAVIN CSISG Candida guilliermondia
Range in Average Riboflavin Riboflavin Potencs, Potency, y/bIl. y/hll. 63- 81 (12jU 76 61- 79 (12) 69 63- 84 (12) 79 57- 89 (12) 78 66 45- 84 (101) 64 48- 87 (113)
2 . 0 g , (KH
