Station, Pacific Grove, California.

STUDIES ON SOME GROWTH FACTORS OF YEASTS1 PAUL R. BURKHOLDER, ILDA McVEIGH, AND DOROTHY MOYER Osborn Botanical Laboratory, Yale University, New Haven, Connecticut Received for publication April 19, 1944 A knowledge of the factors concerned in the growth of yeasts is of value for theoretical plant science and biochemistry, and provides information for practical application in the field of microbiological assays, the production of yeast for fodder and food, and the development of fermentative industrial processes. In this paper some data are presented on the vitamin requirements of 163 strains of yeasts belonging in 110 named species and varieties. Dosage response curves of a yeast which is deficient for 6 B vitamins and an illustration of the -application of such information to the assay of vitamins are given. No attempt is made here to review the literature in this field; some valuable references may be found in the citations given in our earlier papers (Burkholder, 1943; Burkholder and Moyer, 1943). Grateful acknowledgment is made to the following persons who supplied cultures for this work: L. J. Wickerham, Northern Regional Research Laboratory, U. S. Department of Agriculture, Peoria, Ill.; E. M. Mrak, Fruit Products Laboratory, University of California, Berkeley, Calif.; V. E. Graham, Department of Dairy Husbandry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; A. G. Lochhead, Division of Bacteriology and Dairy Research, Central Experimental Farm, Ottawa, Canada; and C. B. Van Niel, Hopkins Marine Station, Pacific Grove, California. Vitamin deficiencies. The methods employed in this part of the work were essentially the same as those which were reported earlier (Burkholder, 1943). Stock cultures of yeasts were carried on Difco malt agar. The synthetic basal medium employed in determining the vitamin requirements of the different strains contained the following compounds in each liter of solution: cp glucose, 20 g; recrystallized asparagine, 2.0 g; KH2PO4, 1.5 g; MgSO4c7H20, 0.5 g; CaCl2-2H20, 0.33 g; (NH4)2SO4, 2.0 g; and KI, 0.1 mg. Trace elements were added to this medium in ppm. as follows: B, 0.1; Mn, 0.01; Zn, 0.07; Cu, 0.01; Mo, 0.01; and Fe, 0.05. Vitamin supplements were added in micrograms per liter of medium as follows: thiamin hydrochloride, 200; pyridoxine hydrochloride, 200; nicotinic acid, 200; biotin, 2.0; calcium pantothenate, 200; and inositol, 10,000. In some of our work with certain yeasts, especially osmophilic types, the amount of glucose was increased to 200 g per liter. Vitamin auxanograms in nutrient agar also were employed, but this method requires purified agar, very clean glassware, and is more difficult to evaluate than the culture tube method. Nine kinds of media were prepared for testing the vitamin requirement of each yeast as follows: no vitamins, 6 vitamins, 6 vitamins plus 0.5 g per liter of This work was supported by a grant from the Research Corporation, New York City. 385

386 P. R. BURKHOLDER, I. MCVEIGH, AND D. MOYER Wilson's liver concentrate, and single omissions of each of the 6 vitamins. The media were adjusted to about ph 5.0 and dispensed with an automatic burette to provide 5 ml per culture tube. The tubes were plugged and autoclaved at 15 pounds' pressure for 15 minutes. Yeast inoculum was prepared by transferring a small amount of stock culture into 5 ml of sterile medium, free of all vitamins. After thorough mixing to obtain uniform suspension of the cells, a loopful was used to inoculate each culture tube. Duplicate sets of media were employed for each kind of yeast, and when disagreement occurred in duplicate tubes, an entire new series was set up for further observation. Growth was allowed to proceed in still cultures for 72 hours at approximately 25 C. The amount of growth was measured turbidimetrically with a Klett photoelectric colorimeter, and average values were computed for duplicate tubes showing satisfactory agreement. Growth responses of 100 strains of yeasts not reported in our earlier papers were determined, and a considerable number of these yeasts exhibited no outstanding vitamin deficiencies under the cultural conditions employed. Examples of yeasts showing no outstanding vitamin deficiencies are the following: Candida krusei, C. pelliculosa, Debaryomyces dekkeri, Hansenula anomala, H. anomala v. longa, H. anomala v. heteromorpha, H. anomala v. sphaerica, H. ciferri, H. lambica, H. saturnus, Mycoderma cerevisiae v. pulverulenta, M. decolorans, Pichia chodati v. fermentans, P. membranaefaciens, Rhodotorula longissima, Saccharomyces aceris-sacchari "R", S. exiguus, Schizoblastosporion starkeyi-henricii, Torulopsis utilis, T. utilis v. major, T. utilis v. thermophila, Trichospora sp., Zygopichia chevalieri. Some species of yeasts were found to be deficient for as many as 6 vitamins, i.e., Hanseniospora melligeri, Kloeckera africana, K. brevis, and K. linderi. Between these extremes, many single and multiple deficiencies occur. Our new determinations include the following biotin-deficient yeasts: Candida chalmersi, C. deformans, C. guilliermondi, C. interm.edia, C. suaveolens, Debaryomyces fabrii, D. guilliermondi, D. hudeloi, D. matruchoti v. subglobosus, D. membranaefaciens v. hollandicus, Hansenula suaveolens, Mycoderma valida, Saccharomyces italicus, S. tubiformis, Schwanniomyces occidentalis, Torulaspora delbruckii, T. fermentati, Torulopsis molischiana, Zygosaccharomyces bisporus, and Z. pini. The list of yeasts deficient for biotin and thiamin includes Candida brumpti and Zygosaccharomyces bisporus. Yeasts requiring biotin and pantothenic acid are Saccharomyces logos, Zygosaccharomyces barkeri, Z. felsineus, Z. japonicus v. soja, Z. mandshuricus, Z. nadsonii, Z. rugosus, Z. variabilis. Biotin, pantothenic acid, and niacin are required by Candida pseudotropicalis, Torulopsis sphaerica, Zygosaccharomyces marxianus. Biotin and niacin are required by Zygosaccharomyces lactis. Saccharomyces macedoniensis is deficient for biotin, pantothenic acid, niacin, and thiamin. Zygosaccharomyces japonicus requires biotin, pantothenic acid, inositol, and thiamin. Biotin, pyridoxine, and pantothenic acid are required by Saccharomyces chodati. The yeast Zygosaccharomyces priorianus requires biotin, pantothenic acid, and inositol. Torulopsis dattila and T. uvae require biotin,

STUDIES ON SOME GROWTH FACTORS OF YEASTS pyridoxine, and thiamin. Yeasts which require only thiamin are Hansenula subpelliculosa, Pichia dombrowskii, Rhodotorula mucilaginosa, and R. rubra. Brettanomyces bruxellansis requires pyridoxine. Biotin and pyridoxine are required by Pichia kluyveri. Torulopsis stellata requires biotin, inositol, and thiamin. Schizosaccharomyces pombe requires biotin, pantothenic acid, niacin, and inositol. The addition of liver concentrate to the basal medium containing 6 B vitamins resulted in greater growth of many of these yeasts, indicating the possible utilization of other compounds, such as amino acids, nitrogenous bases, etc. Also, some yeasts grew more abundantly in media containing higher amounts of sugar (200 g/liter), but the results do not suggest that variation in the amount of sugar obviates the requirements for vitamins. These data are offered as a preliminary survey of vitamin requirements of yeasts grown under special conditions, and no claims are made concerning yeasts cultivated under other circumstances. A summary of the essential data embodied in our two previous reports and in the present paper is presented in table 1. No attempt has been made to calculate what-proportion of described yeasts may be deficient for vitamins; the results presented herewith are concerned with only 110 named species and varieties studied in our laboratory. It appears to be of some significance that nodeficiency for riboflavin has been observed thus far in yeasts, although some groups of bacteria are notably deficient in this vitamin. In the list of 110 species and varieties, 24 appear to be autotrophic with respect to the B vitamins. Among the remaining 86 vitamin-heterotrophic yeasts, 78 require biotin, 33 benefit by an external supply of thiamin, and 30 must have pantothenic acid. Deficiencies for pyridoxine, niacin, and inositol are 13, 13, and 15, respectively. Among a total of 163 laboratory strains studied, 130 were found to be deficient for one or more B vtamit s. Stating the occurrence of vitamin deficiencies in terms of percentage of these heterotrophic strains, we find that biotin deficiency occurred in 87 per cent, thiamin and pantothenic acid deficiency each in about 35 per cent, and pyridoxine, niacin, and inositol each in approximately 12 per cent of the cases. Response to varied dosages of vitamins. For the purpose of studying growth in response to varied dosage levels of vitamins the yeast Kloeckera brevis was selected because it was found to require 6 B vitamins, i.e., biotin, thiamin, pantothenic acid, nicotinic acid, inositol, and pyridoxine. The culture, obtained originally from cider, was supplied by L. J. Wickerham, who identified the species. The basal medium supplemented with these 6 vitamins was considered adequate for this experiment. Vitamins were supplied in the following amounts expressed as micrograms per liter of medium: biotin, 1.0; thiamin hydrochloride, 50; calcium pantothenate, 50; niacin, 200; inositol, 5,000; and pyridoxine hydrochloride, 50. In testing the dosage response for any one vitamin, an appropriate medium was made containing the other five vitamins, and increments of the particular vitamin under investigation were set up so as to cover satisfactorily the range of growth response. Four tubes were used for each concen- 387

388 P. R. BURKHOLDER, I. McVEIGH, AND D. MOYER tration. The methods employed were essentially like those described earlier in this paper except that growth was allowed to proceed for about 24 hours at approximately 30 C. The results obtained are presented graphically in figure 1. Growth of the yeast in response to each of the 6 vitamins, in a medium containing other required growth factors in excess, provides a set of curves typicalof this kind of experiment. It seems probable that any of the 3 species of kloeckera investigated might be employed for microbiological assay of all 6 vitamins, provided an adequate basal TABLE 1 Vitamin requirements of 110 species and varieties of yeast grown for 72 hours at 25 C in a medium comprised of salts, glucose, asparagine, and vitamins (+ = required; - = not required) NUM'BER OF PY11- PANTO- INOSZ- ORDONETEI TO REPRESENTATIVE SPECIES SPECIES BIOTIN THENC NAIN OL THIAM VARIETIES Hansenula lambica... 24 Candida chalmersi... 29 + - - Saccharomyces globosus... 11 + _- + Saccharomyces logos... 10 + - + -_ Mycoderma lipolytica... 7 + Kloeckera brevis... 5 + + + + + + Candida pseudotropicalis... 3 + - + + - Saccharomyces chodati... 3 + + + -_ Zygosaccharomyces lactis... 2 + - - + _ Saccharomyces fragilis... 2 + - + + + Zygosaccharomyces japonicus... 2 + + + + Zygosaccharomyces priorianus... 2 + + + Torulopsis dattila... 2 + + -_ + Saccharomyces cerevisiae v. ellipsoideus.. 2 + - + + + Schizosaccharomyces pombe... 1 + - + + + Torulopsis stellata... 1 + - - - + + Pichia belgica... 1 + + - - + + Pichia kluyveri... 1 + + - - _ Brettanomyces bruxellansis... 1 - + - - _ Saccharomyces uvarum... 1 + - + - Totals, 20 types... 110 78+ 13+ 30+ 13+ 15+ 33+ medium is used. Our studies on the influence of various organic substances added to the basal medium indicate that a supply of amino acids is essential in order to obtain maximum growth. Acid-hydrolyzed casein, supplied in amounts of the order of 0.2 to 2.0 g per liter of medium, stimulated growth appreciably. Additions of cystine, tryptophane, adenine, guanine, and uracil apparently do not promote growth of Kloeckera brevis above the amount obtained in the basal medium supplemented with the 6 vitamins and casein hydrolysate. Addition of peptone to the synthetic medium at the rate of 5 g per liter results

STUDIES ON SOME GROWTH FACTORS OF YEASTS in somewhat greater growth than has been obtained thus far with any synthetic medium. Whether this further stimulation is brought about by unknown factors or by more favorable proportions of known nutrients is not yet apparent. 389 z i a- 1001 X) 75i- I- z NIACIN.... I 0 0.5 MICROGRAM PYRIDOXINE THIAMIN I, I.. 1.0 0 Q5 1.0 0 OF VITAMIN IN 5 ML OF MEDIUM I 0.5 1.0. Downloaded from http://jb.asm.org/ >- 50 - ct 25!PANTOTHENIC AC. BIOTIN INOSITOL. l -.- I --. I... l -I s I I I a. * I I. I* 0 QO 02 0.3 0 Ql Q2 Q3 0 0.1 Q2 03 MICROGRAM OF VITAMIN IN 5 ML OF MEDIUM FIG. 1. DOSAGE REsPONSE CURVES of Kloeckera brevis CULTIVATED IN SYNTHETIC MEDIA CONTAINING GLUCOSE, SALTS, ASPARAGINE, AND APPROPRIATE SUPPLIES OF VITAMINS Values for thiamin should be multiplied by 10-1, values for pyridoxine by 10-, values for biotin by 10-3, and values for inositol by 10 in order to make the abscissae read correctly. on March 11, 2019 by guest Yeast assay for vitamins. The use of yeasts in microbiological determination of vitamins in samples of food, clinical materials, etc., has received considerable attention recently (Kogl and T6nnis, 1936; Snell, Eakin, and Williams, 1941; Atkin, Schultz, Williams, and Frey, 1943; Hertz, 1943; etc.) and is worthy of

390 P. R. BURKHOLDER, I. MCVEIGH, AND D. MOYER further consideration. Some of the yeasts which we, have studied appear to have potential usefulness in this connection. A brief description of the method used for assaying inositol with the yeast Kloeckera brevis will be given herewith to indicate the procedure which may be followed in making such tests. This method appears to offer considerable advantage over some other yeast methods which have been proposed for assaying inositol (Williams et al., 1941). Calculated values for different dosage levels of plant tissue extracts show excellent agreement, and results obtained in recovery experiments with vegetable extracts and inositol are also satisfactory when the dosage levels occur in the middle region of the dosage response curve (see figure 1). Routine tests for the 5 other vitamins have not been conducted with this yeast, but preliminary studies indicate that satisfactory assays probably can be made for them in the usual way. It should be recognized, however, that the growth response of Kloeckera to "vitamers" and "provitamers" has not yet been determined. The procedure used in assaying inositol is similar to the meth'od which has been described by Snell and co-workers (1941) for the determination of biotin with a yeast method (Burkholder and Moyer, 1943). A satisfactory culture medium contains the following constituents per liter: KH2P04... 2.0 g. Hydrolyzed casein (Borden's MgSO4-7H20... 0. 5 g. vitamin-free)... 0.0 g. CaCl2.2H20... 0.33 g. Biotin... 1,ug (NH4)2S04... 2.0 g. Niacin... 600 pg Asparagine... 2.0 g. Thiamin HCI... 100 pg Glucose... 20.0 g. Pyridoxine HOC... 100,ug KI... 0.1 mg. Ca pantothenate...250 pg A small volume of stock solution of trace elements is added to supply the following amounts in ppm: B, 0.01; Mn, 0.01; Zn, 0.07; Cu, 0.01; Mo, 0.01; and Fe, 0.05. Appropriate small volumes of a standard solution of inositol are pipetted into clean culture tubes and the total volume adjusted with water to equal 1 ml in each tube. These tubes provide a standard curve for reference. In a similar way aqueous extracts of samples are pipetted into tubes to provide suitable levels for assay. The open tubes, containing the standard solution and samples, and the medium in a flask are steamed at 100 C for 10 minutes. When cool, the medium is inoculated with yeast from an agar slant so as to provide 2 mg of moist yeast per liter of medium. Five milliliters of inoculated medium are then transferred with a sterile automatic pipette into each culture tube. The tubes are shaken in order to mix the 6 ml of liquid, and the cultures are allowed to incubate for 24 hours at 30 C. Incubation on a shaking machine results in more rapid and more uniform growth and diminishes by several hours the time required for making the test. Growth is determined turbidimetrically, and smooth curves having a satisfactory dosage response range are obtained. Appropriate corrections for light absorption by deeply colored or turbid samples may be made with suitable control tubes containinng known volumes of extract.

STUDIES ON SOME GROWTH FACTORS OF YEASTS 391 SUMMARY The B vitamin requirements of 163 strains of yeasts belonging in 110 named species and varieties have been determined. Among the 86 named kinds found to be heterotrophic for one or more vitamins, deficiencies occurred as follows: biotin, 78; thiamin, 33; pantothenic acid, 30; inositol, 15; nicotinic acid, 13; pyridoxine, 13. No deficiency for riboflavin has been observed thus far in yeasts. Growth curves of Kloeckera brevis in response to increasing doses of 6 B vitamins are presented, and the use of this yeast in assays for inositol is described. REFERENCES ATKIN, L., SCHULTZ, A. S., WILLIAMS, W. L., AND FREY, C. N. 1943 Yeast microbiological methods for determination of vitamins. Pyridoxine. Ind. Eng. Chem., Anal. Ed., 15, 141-144. BURKHOLDER, P. R. 1943 Vitamin deficiencies in yeasts. Am. J. Botany, 30, 206-211. BURKHOLDER, P. R., AND MOYER, DOROTHY. 1943 Vitamin deficiencies of fifty yeasts and molds. Bull. Torrey Botan. Club, 70, 372-377. HERTZ, R. 1943 Modification of the yeast-growth assay method for biotin. Proc. Soc. Exptl. Biol. Med., 52, 15-17. KbGL, F., AND T6NNIS, B. 1936 Uber das Bios-Problem. Darstellung von krystallisiertem Biotin aus Eigelb. Z. Physiol. Chem., 242, 43-73. SNELL, E. E., EAXIN, R. E., AND WILLIAMS, R. J. 1941 Assay method for biotin. Univ. of Texas Pub., No. 4137, 18-21. WILLIAMS, R. J., STOUT, ANNE K., MITCHELL, H. K., AND MCMAHON, J. R. 1941 Assay method for inositol. Univ. of Texas Pub., No. 4137, 27-30. Downloaded from http://jb.asm.org/ on March 11, 2019 by guest