THE INSTITUTE OF BREWING RESEARCH SCHEME

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1 November, 1939] 533 THE INSTITUTE OF BREWING RESEARCH SCHEME THE BIOS REQUIREMENTS OF VARIOUS STRAINS OF SACCHAROMYCES CEREVISIAE By C. Rainbow, B.Sc, Ph.D. (Received by the Secretary: 7/5/39) A study of the numerous papers on bios reveals a number of conflicting statements on the subject. This creates considerable confusion, and the present work was undertaken with a view to find out the reason for this state of affairs and, in doing so, to correlate the author's bios work with the large volume of work from other sources. The present investigation is a stud/ of seven different strains of Saccharotnyces cerevisiae, from the point of view of their individual bios requirements. These /easts varied in their requirements under the same conditions of growth-test and have been classified as Class A, B and C /easts according to their reactions to the bios fractions. Furthermore, for the same yeast the bios requirements varied according to: (a) the source of nitrogen (ammonium phosphate or aspartic acid) in the synthetic medium; and (6) the conditions of sub-culture of the yeast. Consideration of these factors should account largely for the different observa tions of various workers on bios. The importance of avoiding generalisations, even with regard to a single strain of Saccharomyces cerevisiae, is evident. Historical In order to give an idea of some of the points brought out by researches on the biosproblem, the following brief summary is appended: E. Wildiers (la Cellule, 1901, 18, 313) gave the name "bios" to a substance present in extracts such as yeast water, which had to be supplied, in addition to sugar, salts and nitrogen (the known nutrients) for the nutrition of yeast cells. W. L. Miller and his co-workers found that for good growth their Toronto yeast re quired three fractions of biosbios I, bios IIA and bios KB. E. V. Eastcott (J. Phys. Chem., 1928, 32, 1094) identified bios I as meso-inositol and bios IIA has been identi fied as /3-alanine supplemented by the action of Meucine (Miller, Trans. Boy. Soc. Canada, 1936, in, 30, 99). F. Kogl and B. Tonnis (Zt.it. Phys. Chem., 1936,242,43) have described the preparation of crystalline "biotin" from dried egg yolks. this substance being intensely active in the presence of inositol. /3-Alanine was found to have some effect, but Meucine was inactive. For good growth, only biotin and inositol were necessary. Kogl (Chem. and Ind., 1938, 57, 49) reported on the stimulatory effect of vitamin B\ (aneurin) on bis yeast (Race M) and distinguished between the action of aneurin and biotin (a hormone-like potency) and that of inositol and /3-alanine (specific nutritional - factors), the former definitely constituting part of an enzyme system. For the purpose of the present paper, the work of R. J. Williams and his co-workers can be summarised in five points: (1) Different yeasts have different bios requirements (R. J. Williams, J. L. Wilson, and F. H. Von der Ahe, J. Amer. Chem. Soc., 1927, 49, 227). (2) Inositol was a detectable factor with all the yeasts tested (R. J. Williams and D. H. Saundera, Biochem. J., 1934, 28, 1887).

2 534 baikbow: bios requirements of stbatns of s. cbrbvisiae [November, 1939 (3) Of prime importance among bios factors was a substance called by the authors "pantothenio acid" (B. J. Williams, C. M. Lyman, G. H. Goodyear, J. H. Truesdail and D. Holaday, J. Amer. Chem. Soc., 1933, 55, 2912). (4) Some form of assimilable organic nitrogen was necessary for good yeast growth, hence asparagine was incor porated into the test medium (B. J. Williams and Saunders, loc. tit.). (5) B. J. Williams and E. Bohnnan (J. Amer. Chem. 8oc., 1936, 58, 695) discovered the activity of /J-alanine, the effect of which was only brought out when asparagine was absent from the culture medium. From the foregoing brief account it is apparent that differences occur between the results of one worker and another. Varia tions occur also in the media used, in the technique of carrying out the test reactions and in the criteria of growth adopted. The writer wishes particularly to point out the latter. In these laboratories, the ultimate aim has been the production of "wort growth" (L. B. Bishop and C. Bainbow, this Journ., 1939, 33); (C. Eainbow and L. B. Bishop, this Journ., in a paper to be pub lished later). The present writer believes that some workers, at least, have concerned themselves with much smaller stimulations, and in so doing may have overlooked factors which are essential for the comparatively large "wort growth," but not so essential for smaller stimulations. The importance of this is discussed later in the present communication. Technique.The synthetic culture medium (medium I) used for the tests described in Sections (1) and (2) of this investigation had the following composition, per litre: 100 grms. glucose, 2-0 grms. potassium dihydrogen phosphate (KHjPO4), 1-0 grm. magnesium sulphate (MgSO4-7H2O), 0-2 grm. calcium sulphate (CaSO4-2H2O), 001 grm. ferrous sulphate (FeSO4-7HaO), 4-0 grms. diammonium hydrogen phosphate 12 ml. potassium lactate (50 per cent, wt/wt.), and syrupy lactic acid to pu 4-9 (about 3-6 ml.). For Section (3) a modified medium (medium II), containing aspartic acid was used. This medium is described under that Section. The salts and lactic acid were of "Analyti cal Reagent" quality, whilst the glucose was of a high degree of purity ("Dextrosol" from Messrs. Corn Products, Ltd.) and free from bios. The presence of the lactate-lactic acid mixture in the medium gave it additional buffering power so that the fall in pb as the growth tests proceeded was like that in malt wort. The growth tests were carried out in plugged 100 ml. conical flasks containing 50 ml. synthetic medium, together with such additions of test materials as were necessary. Sterilisation was effected by bringing to the boil and allowing to simmer so that steam just continued to issue from the plug. After allowing to cool, each test was seeded with 1 ml. ofa suspension of the pure culture yeast, introduced by means of a sterile, plugged 1 ml. pipette. Throughout the investigation the seeding rate ranged from 2 million to 4 million cells per ml. or 22-5 to 45 mgrms. moist yeast per test flaska reading of 8 to 16 per cent, on the drum of the nephelometer (see below). For the growth period, the inoculated test flasks were placed on a cradle erected in an incubator at C. This cradle was main tained in rotation at constant speed in an horizontal plane, so that the yeast in the test flasks was kept just in suspension. Growth readings were taken by gently shaking by hand the test flask until an homogeneous yeast suspension was obtained. One ml. of this suspension was withdrawn by pipette, diluted to 50 ml. with distilled water and shaken. This diluted suspension was placed in a beaker of parallel sides (36 mm. in ternal diameter) and read in the Zeiss-Pulfrich nephelometer, using the green filter LII and the disc of brightness 4. The Zeiss standard turbidity prism indicated 96 to 98 per cent, on the drum of the nephelometer throughout this investigation. All the readings given in this paper are percentages on the drum of the nephelometer and were obtained in this standard manner. For the convenience of those accustomed to expressing yeast crops as grms. of moist or dry yeast, the following information will be of

3 November, 1939] rainbow : bios requirements of strains of a. cerevisiae 535 assistance in translating the growth figures given in this paper into those terms: The equation D = T, where D = grms. dry yeast per 100 ml., and T nephelometer reading (as stated in this paper), enables the dry weight to be obtained from the nephelometer readings as given in this paper. Simple multiplication of D by 4-5 will then convert the weight of dry yeast into moist yeast per 100 ml. Moist yeast can be further reduced to cells per ml. using the approximation that 25 grms. moist yeast per litre is equivalent to 100 million cells per ml. For example, taking a growth reading of 100, the weight of dry yeast per 100 ml. is X 100, or 0135 grm. dry yeast, or x 4-5 = 0-61 grm. moist yeast per 100 ml. = ^ x 100,000,000 cells per ml. = about 25,000,000 cells per ml. Fvtt Wort Growth.*It has been pointed out in a previous paper (Bishop and Rainbow, this Journ., 1939, 33) that, whereas workers have been using comparatively small stimula tions as growth criteria for bios work, in this laboratory the ultimate aim of bios prepara tions has been to produce "full wort growth"; that is, to produce an equal rate and extent of yeast growth in a synthetic medium as in a malt wort of the same specific gravity. Using the present technique for a particular malt wort of sp. gr. 1,040, prepared from a commercial malt extract, the growth of the Institute of Brewing English brewery top yeast is such as to give a reading on the drum of the nephelometer of 300 to 500 after 48 hours' growth period. As will be shown below, "full wort growth" varies according to the yeast in question. Towards the end of this paper, the author has appended a few further remarks on attainment of "full wort growth" with bios preparations. The Yeasts.The yeasts examined are listed below, the reference number on the left being used subsequently to designate the yeast for the sake of brevity. Each of these yeasts is a strain of Saccharomyces cerevisiae. * In this investigation unhopped wort was used. Ref. No. Yeast. 1. Institute of Brewing yeast (English Brewery top fermentation yeast). 2. 2,160 (National Collection of Type Cul tures) (Prof. J. C. Drummond's and Narayanan's yeast). 3. Race M (Kogl's yeast). 4. Wildier's yeast. 5. Gebruder Mayer (baker's yeastused by Williams and his co-workers and by Eddy and his co-workers). 6. Frohberg yeast (Institute of Brewing; bottom yeast). 7. Frohberg yeast (76) (Institute of Brew ing; bottom yeast). Yeasts 2, 3, 4 and 5 were kindly Bent by Miss A. Britten of Prof. J. C. Drummond's laboratories. These yeasts arrived on agar slopes and were maintained in pure culture for the subsequent experiments by sub-culture in sterile malt wort by Dr. R. S. W. Thome, of these laboratories. Sub-culture took place once a week. The Test Materials.In all the following experiments, unless otherwise stated, the following fixed quantities of materials were used for tests in 50 ml. synthetic medium: Bios.0003 ml. of a solution con taining 0-05 grm. solid matter in 25 ml. This preparation was obtained from egg yolk by the rapid method described in a paper to be published later. Bios HA ml. of a solution con taining 0-23 grm. solid matter in 25 ml. This was prepared as for. Inositol1 mg. (Messrs. B.D.H., Ltd.). j3-alanine grm. (sourcesynthesised by the author by Holm's modifica tion {Arch. Pharm., 1904, 242, 590) of Hoogewerff and van Dorp's method (Bee. Trav. Chitn., 10, 4),».e., by the action of bromine and potash on succinimide. Vitamin B1.O grm. crystalline aneurin(messrs. B.D.H., Ltd.). Lactoflavin grm. (Messrs. B.D.H., Ltd.). Nicotinio acid amid grm. (by kindness of Dr. B. C. J. G. Knight). Vitamin B gnn. (crystalline from Messrs. Merck & Co., Darmstadt).

4 536 rainbow: bios requirements of strains of s. cerevisiab [November, 1939 "Thiazole" (4-methyl-5-/3-hydroxyethyl thiazole) gnn. (by kindness of Dr. F. Bergel). " Aminopyrimidine " (2-methyl-4-amino 5- thioformido-methyl pyrimidine).0*00001 gnn. (by kindness of Dr. F. Bergel). i-leucine gnn. (Messrs. Hoff man la Roche, Ltd.). "Hydrolysed" bios IIA and "hydrolysed" bios (see p. 538).Quantities equivalent to those of bios IIA and bios above. Growth in Wort.The growth of yeasts 1 to 7 in this medium of sp. gr. 1,040 are tabulated below. Yeast. 1. (I. of B.) 2. ( 2160) 3. (Race M) 4. (Wildiera').. 5. (Gob. Mayer).. 0. (Frohberg).. 7. (Frohberg 76) Table 45 hours I Growths. 70 hours hours )7 893 From these figures it will be seen that the yeasts vary considerably in the extents to which they grow in wort. Wildiers' yeast gives comparatively small crops, whilst the two Frohberg yeasts (both "bottom" yeasts), Race M and Gebriider Mayer give large crops. The Institute's yeast ("top" yeast) and Professor Drummond's yeast were inter mediate. Section I. Reactions of the Yeasts to the Author's Preparations Effect oflnoaitol, Bios IIA and Bios.- In another communication from this labora tory (C. Rainbow and L. R. Bishop, loc. dt.), the recognition of three constituents of bios is discussed. The effects of concentrates containing these factorsbios I (inositol), bios IIA and bios on the yeasts under test in this investigation were determined by means of growth tests, of which details are given in Table II. From a study of the figures recorded in Table II it is possible to divide the yeasts into three classes: Class A Yeasts (yeasts 1 and 2), which are stimulated slightly by bios IIA but not by inositol or bios. Inositol plus IIA gives a higher stimulation than IIA alone, but as a supplement to IIA gave no greater yeast crop than that obtained by the addition of IIA only. Strong stimulation was only obtained when all three fractions were together. Thus, yeast 2 resembles the Institute of Brewing yeast in that it requires the same complement of bios fractions for good growth. Class B Yeasts (yeasts 3, 4 and 5) are slightly stimulated by inositol alone, IIA plus inositol increasing the stimulation. alone stimulates to about the same extent as inositol, but together with inositol the crop is considerably increased in all cases. High growths are obtained only when all three factors are present. Table II Effect of Inositol, Bios IIA and Bios. Growth Figures after 46 Hours. Seeding Rate 12. Yeast. 50 ml. synthetic medium containing. 1 (I. of B.). 2 ( 2160). (Race M). (Wildiers'). 5 (Gob. Mayer). 6 (Frohberg). 7 (Froh berg 76). Inositol IIA.. Inositol + IIA Inositol + IIA + Inositol + IIA

5 November, 1930] rainbow: bios requirements of strains of s. cerevisiab 531 Table III Effect of /J-Ai.anine GnowTHS apteb 46 Hours' Incubation Seeding Rate 8. Yeast. Medium containing. I (I. of B.). 2 ( 2160). 3 (Raco M). 4 (Wildiere1). 5 (Gob. Mayor). 6 (Frohberg). 7 (Frohberg 76). Inositol Inositol + /J-alanino t Inositol+IIA Inositol+IIA + /3-olanino Inositol+ Inositol + + /3-nlnnino Inositol+ IIA+.. Inositol+IIA+11B + /3-alanino Class C Yeasts (yeasts 6 and 7) grow slightly in the medium without bios addi tions. Very little effect is produced by adding any one of the factors, although inositol may have a slight effect on both yeasts. Inositol plus IIA shows a slight increase over the inositol figure. High growths are obtained where inositol and are present together. IIA causes compara tively little increase over the inositol plus figure so that, for these yeasts, bios IIA docs not appear to be a necessary constituent. It should be noticed, in addition, that inositol and bios are essential factors for good growth of all the yeasts. The main points arising from this classifica tion consist in the following. Class A yeasts and class B yeasts resemble each other in requiring a complement of three bios factors, but, whereas inositol plus bios partly stimulates class B yeast, class A yeasts are unaffected by these factors in the absence of bios IIA. Class C yeasts require inositol plus bios, bios HA having little effect. Thus there is considerable evidence that the yeasts differ in their response to the constituents of the bios complex. These differences bear out Williams' observations in his experiments with various yeasts. In order to pursue these points of difference further, various substances of known con stitution, which have been reported to be growth factors both for yeasts and for other organisms, were tested. These substances include several of the factors of the vitamin B complex, /9-alanine, etc. Effect of P-Alanine.j3-Alanine (/3-aminopropionic acid) CH^NH^CHjCOOH has been reported to have bios activity by Williams and Rohrman and also by Miller and his co-workers. Table III gives the growth figures obtained from a series of tests designed to show the effect of /?-alanine on the yeasts under investigation. It will be seen that /?-alanine adequately replaces bios IIA for yeasts 3,4 and 5 (Class B yeast) but not for yeasts 1 and 2 (Class A yeasts). With yeasts 6 and 7 (Class C yeasts), which do not require bios IIA, j8-alanine has no effect. /}-Alanine, therefore, functions as bios IIA for some yeasts but not for others; so that disagreement among workers over the activity of js-alanine can be regarded as due to differing reactions of their yeasts. Effect of Vitamin Bl (Aneurin).Crystal line vitamin Bx had no effect on the yeasts with the exception of Nos. 4 and 5. With yeast 5, a slight inhibition was noticed, but the effect, although duplicablo, was usually very small. With yeast 4, a considerable

6 538 rainbow: bios requirements of strains of 5. cbrbvisiae [November, 1930 Table IV Effect of "Aminopybimidinb" Obowth Fiouhks after 47 Houits Seedino Base 13. Yeasts. Medium containing inositol plus " Aminopyrimidino " IIA* : IIA*+" aminopyrimidine " " aminopyrimidine" IIA*+ IIA*+ + "aminopyrimidine " * For yoaste 3, 4 and 5, /3-alonino was used as bios IIA. stimulation was produced (from 30 to 50 per cent, on the drum of the nephelometer after 48 hours' growth period). The stimulatory effect was observed by comparison of media containing (a) inositol, /J-alanine (as IIA) and, and (6) inositol, /J-alanine, and vitamin Bj. Effect of Lactoflavin and Nicolinic Acid Amide.Neither lactoflavin nor nicotinic acid amide showed significant effect on any of the yeasts 1 to 7. Effect of Crystalline Vitamin BQ.Vitamin Bs had no effect except on yeast 4. With this yeast the increase was about 40 per cent, on the drum of the nephelometer in the 48 hours' yeast crop in the presence of inositol, /J-alanine (as IIA) and. Effect of "Thiazole" and "Aminopyrimi dine."the vitamin Bj molecule consists of a combination of the molecules of "thiazole" (4 methyl-5-hydroxyethyl thiazole) and " aminopyrimidino " (2-methyl-4-amino-5- thioformidomethyl pyrimidino). A. S. Schultz, L. Atkin and C. N. Frey (J. Amer. Chem. Soc., 1937,59,2457; 1938,60,490) have described a fermentation method of assay of vitamin Bj and also described the effect of "thiazole" and "aminopyrimidine" as bios factors. They divide yeasts into two classes: Type A yeastsstimulated by vitamin Bj and a new factor (bios VI); "thiazole" and "amino pyrimidine" partly stimulated these yeasts, Mos HA plus plus inositol having little effect. " Thiazole " and " aminopyrimi dine" together gave "complete" stimulation and, with bios VI, still higher growths were recorded. Type B yeasts were inhibited by vitamin Bi, slightly inhibited by "aminopyrimidine," but not affected by "thiazole." These yeasts were stimulated by inositol plus IIA plus. On the seven yeasts tested by the author, "thiazole" had no effect. "Aminopyrimi dine" stimulated yeasts 1 and 2 to a small extent in the presence of inositol, IIA and, whilst yeast 4 was stimulated to about the same extent as by vitamin Bj. The two substances together stimulated yeast 4 to the same extent as "aminopyrimidine" by itself, whilst yeast 2 was slightly inhibited by these substances used together. No effect from both substances used together was detectable with yeasts 1,3,6 and 7. Table IV gives a set of growth figures obtained during the tests on "aminopyrimidine." Effect of ULeucine.This showed no effect on any of the yeasts, either alone or together with /j-alanine. Effect of "Hydrolysed" Bios UA and "Hydrolysed" Bios 115.The significance of " 'hydrolysed' bios IIA" lies in the fact that it has been found that bios IIA con centrates heated with dilute acids or alkalis are inactivated with respect to the Institute of Brewing yeast (1). (Rainbow and Bishop, loc. cit.). Since /J-alanine is unaffected by such

7 November, 1939] rainbow: bios requirements of strains of s. 539 treatment, those yeasts for which /J-alanine can replace bios IIA [(3), (4) and (5)] should respond also to "hydrolysed" bios IIA, whilst the other yeasts [(1) and (2)], (which do not respond to /3-alanino as bios IIA) should not respond to the "hydrolysed" concentrate. Experiment has shown this to be correct. Bios was found to be more stable under the conditions outlined above (Rainbow and Bishop, loc. tit.). By heating for three hours at 100" C. with 25 per cent, sulphuric acid, decomposition was affected. This "hydrolysed" bios concentrate proved inactive as bios for all the yeasts tested. On the whole, the results of the tests on the pure substances were disappointing. None of the substances tested, other than /3-alanine, was found to be able to replace either IIA or. Apparently all of the yeasts tested fall into the "type B yeasts" of Schultz, Atkin and Frey. The small stimula tions shown in certain cases with vitamin Blt vitamin B6 and "aminopyrimidine" were only given when inositol, IIA and were present together: omission of either the IIA or fraction eliminated the stimulative effect. Consequently, although differences between certain of the yeasts were indicated by this method, the effects were not striking enough to add materially to the evidence obtained by testing j9-alanine. The author considers that the stimulations due to vitamin B6, vitamin Bx and "aminopyrimidine" cannot be taken as indication that those substances form an essential part of the bios complex, required by the yeasts examined. Section 2. Effect of Sub-Culture on the reactions of the yeasts It has already been stated that yeasts 2, 3, 4 and 5 arrived for investigation on agar slopes and that sub-culture of each of these was made in sterile malt wort (sp. gr. 1,030 ). Sub-culture was maintained in this liquid medium throughout this investigation. The author noticed, however, that after about two months of sub-culture, perceptible differences in the reaction of certain of the yeasts towards bios factors had taken place. After three months of sub-culture (subculturing taking place roughly once a week), these differences were strongly emphasised. Reference to Tables II and III, the figures of which have already been discussed, gives the reactions of the yeasts after one or two sub-culturings in wort from the original agar slopes. Yeasts 1, 6 and 7 being yeasts from this laboratory, had been maintained in sub-culture in malt wort for a much longer period and did not show any variation in reaction during this investigation. Thus the differences were apparent only in those yeasts which were originally on agar slopes and were subsequently cultured in malt wort. Yeast 2 was exceptional in that its reactions remained consistent. Table V (see p. 540) sets out, side by side, typical results obtained (a) shortly after the first sub-culture from the agar slopes (lefthand column), and (b) after three months' sub-culture in malt wort (right-hand column). If we neglect for the moment differences in the magnitude of growth, it will be noticed that, whereas the earlier experiments showed the necessity for js-alanine (as bios IIA) for yeasts 3, 4 and 5since the inositol plus /3-alanine plus growth figures are higher than those due to inositol plus in the later experiments the effect of /J-alanine is either insignificant (yeast 3) or comparatively small (yeasts 4 and 5). It seems, therefore, that after a period of sub-culture in the liquid medium, the effect of /3-alanine, and thus the need for bios IIA, becomes less significant for these three yeasts. Section 3. Effect of the Source of- Nitrogen Aspartic Acid as Nitrogen Source.Aspartic acid has been used by workers as the source of nitrogen in the culture fluid used for yeast growth investigations. Williams has sug gested that differences in bios investigations may be due, in addition to other causes, to the composition of the medium. He has obtained particularly interesting results with aspartic acid and asparagine in his media. It was therefore deemed important that some investigation of the effect of varying the source of assimilable nitrogen should be conducted. For this purpose, a series of growth tests in a medium containing aspartic acid instead of the usual ammonium phosphate was per formed. This modified medium contained 8-0 grms. aspartic acid per litre and the usual addition of potassium lactate, but no lactic acid, adjustment to pn 4-9 being made by potassium hydroxide (A.R.): in all other res pects the media were identical.

8 540 rainbow: bios requirements of strains of 5. CEREVisiAE [November, 1939 Table V Comparison of Reactions of Yeasts 2, 3, 4 and 5, after Initial Sub-culture and After 3 Months of Sub-culture Seeding Bate 11. After initial sub culture. After 3 months of sub culture. Medium containing inositol. Growth (46 hours). Growth (46 hours). Yeast ) IIA IIA+ +/3-alnniiio Yeast 3 (Raco M) /3-alanino (as IIA). /9-alanino Yeast 4 (Wildieis) fi-alanine (as IIA) 0-alanine YeastS (Gebriider Mayer) /9-alanino (as IIA) 0-alanino GO The tests carried out with this medium took the form of those which are recorded in Table II. The growth figures are given in Table VI. On the basis of the results of the similar series of tests using the medium containing ammonium phosphate (see Table II), the yeasts were classified into Class A, Class B and Class C yeasts. The results set out in Table VI will be briefly summarised, utilising Table this classification. It should be observed that this experiment was performed with the yeasts which had been sub-cultured for 3 months. Class A Yeasts react in the same way to the aspartic acid medium (medium II) as they do to the medium containing ammonium phosphate (medium I), with the exception that the growth due to inositol plus IIA is not very much less than that due to inositol VI Effect of Inositol (I), Bios IIA* and Bios in Asfartic Acid Medium Growth Figures after 48 Hours Seeding Rati: 8. Medium containing. 1 (I. of B.). 2 ( 2160). 3 (Raco M). 4 (Wildiors1). 5 (Gob. Mayer). 6 (Frohberg). 7 (Frohberg 76). I IIA I+IIA I+ IIA+ I+nA+EtB.. I+nB + /3-alanine * /3-olanine was used as bios IIA for yeasts 3, 4 and 5.

9 November, 1939] rainbow:bios requirements of strains of s. cehevisiak 541 plus IIA plua ; in other words, the effect of bios is small, and this factor does not appear to be essential for high growths. Class B Yeasts grow somewhat in medium II without bios additions. Inositol alone stimulates yeast 3 and, to a less degree, yeast 4. Bios IIA (/3-alanine) alone has no effect on yeast 5 and slightly inhibits yeasts 3 and 4. Bios slightly increases the crop of yeasts 3 and 5 and slightly inhibits yeast 4. As a supplement to inositol, js-alanino has a small stimulatory effect on yeasts 3 and 4, and a larger effect on yeast 5; bios plus inositol strongly stimulates yeasts 3 and 5, but has no greater effect on yeast 4 than inositol alone. js-alanine plus inhibits all three yeasts somewhat. The growths due to inositol plus /3-alanine plus bios are very high in every case, the effect of /3-alanine in the presence of other factors being most striking. Class C Yeasts in medium II behave very much in the same manner as in medium I, with the following exceptions. First, yeast 6 is inhibited somewhat by alone and IIA plus and perhaps also by IIA alone. Second, yeast 7 seems to require both bios HA and bios in the presence of inositol. One other point of interest arises from Table VI, namely, the small activity of /3-alanine as bios IIA for yeast 2 in the aspartic acid medium. Most of the con firmatory tests showed this small activity, although on at least one occasion the result was negative, so that the effect was not wholly consistent. The author considers that this effect of /3-alanine provides some indication that bios IIA for yeast 2 (and perhaps even for yeast 1) may consist of some acid and alkali labile derivative of /3-alanine. Discussion (1) With Respect to Medium I.In Section I of the experimental part of this paper it was shown that the yeasts under investiga tion had different bios requirements. For instance, five of the yeasts (1, 2, 3, 4 and 5) required, for good growth, three fractions of biosinositol (bios I), bios IIA and bios. For three of these yeasts (3, 4 and 5), jbalanine adequately replaced the bios IIA, but failed to do so for yeasts 1 and 2. Yeasts 6 and 7 differed from the others in that good growth was obtained without the addition of bios IIA as concentrate or as /3-alanine. Bios and inositol were essential factors for all the yeasts. From this it is apparent that the various strains of Saccharomyces cerevisiae have different bios requirements, and on these grounds alone, knowing that various authors have been working with different strains, it is to be expected that discrepancies in the findings of bios workers will occur. Since it is unlikely that the yeasts investigated during the present research are completely repre sentative of all the strains of S. cerevisiae, the writer does, not contend that the results out lined in this section will assist in interpreting the results of every worker, but they should assist somewhat in this connection. For instance, W. L. Miller (Trans. Roy. Soc. Canada, 1934, 28, 175) using ammonium nitrate as a source of nitrogen, finds that his Toronto yeast requires bios I (inositol), bios IIA and bios, and later established that bios IIA can be adequately re placed by js-alanine plus Meucine. Now, until the present investigation was carried out, it was perplexing for the author to find that his yeast (yeast 1), although requiring three apparently similar bios fractions, did not respond to /3-alanine plus Meucine when these substances were substituted for the bios IIA concentrate. The matter is now much clearer, since the /3-alanine has been found to be active as bios IIA for three yeasts. The activity of Meucine has not been confirmed, but Miller's result need occasion no surprise, especially since Miller's Toronto yeast has not been examined by the author; and further, Miller's results seem to show that the effect of Meucine is subsidiary, that of /3-alanine being of primary importance. The relevance of /3-alanine to the results of Kogl and of Williams is discussed later. (2) With Respect to the Effect of Sub-culture of the Yeasts.Changes of the reaction of three of the yeasts towards the bios fractions after some months of sub-culture in malt wort from the original agar slopes have been described. These changes manifest them selves mainly in the lessening of the effect of /3-alanine (as bios IIA) with yeasts 3, 4 and 5 after prolonged sub-culture. As a result of the effects described under this heading, the writer suggests that the reactions of the yeasts after 3 months sub culture are in reality more comparable with his own conditions of test than the earlier work described in Section (1) since the

10 642 rainbow: bios requirements of strains of s. ckrevisiae [November, 1039 writer's yeast (yeast 1) had been maintained in sub-culture for a long period. Thus it is necessary for workers who wish to examine other yeasts to sub-culture the yeasts under their own conditions for some time before bios tests are carried out. If this condition is accounted for, variations such as have been observed under the present heading will be eliminated, and the true reactions of the yeasts under the particular set of conditions employed by an author will be under test. (3) With Respect of Media containing Aspartic Acid (Medium II).In his later work, with which the author is concerned here, Williams used media containing, in addition to ammonium salts, an organic source of nitrogen such as aspartic acid or asparagine. The beneficial effect of the latter in the culture medium had been noted previously and the importance of aspartic acid as. a yeast nutrient was later emphasised. In fact, Williams regarded some sort of organic nitrogen as a general nutrient, essential for good yeast growth; so that, the results obtained with the aspartic acid medium II in the present investigation are more suitable for comparison with Williams' results. To facilitate comparison, the following table has been drawn up: ibid,, 1930, 61, 454) help to throw light on this matter. Before the publication of these papers the author bad favoured the view that this bios and pantothenlc acid corresponded with each other or might even be identical. How ever, the properties of pantothenic acid described in the above papersparticularly with respect to the instability to acids and alkalis, the non-precipitability as the mer cury compounds and the suggestion that pantothenic acid may consist of j3-alanine linked to an hydroxy acidpoint to the fact that pantothenic acid very closely resembles, and may even be identical with, bios IIA. Reference to the table above shows that for yeast 2160 (2) bios IIA does indeed correspond to pantothenic acid. With Wildiers' yeast (4) and Gebriider Mayer (5) a direct comparison cannot be made (since /3-alanine was used during the present work to replace bios IIA for these yeasts) unless /}-alanine be assumed to be equivalent to pantothenic acid for these yeasts. Under the writer's conditions, where bios is necessary in addition to /3-alanine, this may indeed be true, but Williams and Bohrman ibid., 1936, 58, 695) found that for "complete growth" pantothenic acid was necessary in addition to f$-alanine. Although it Yeast. Gobrudor Mayer (Yeast 5). Wildiers' (Yeast 4). Williams' results. Inositol alonenegativo. Pantothenic acidstrikingly active Inositolnegative. Pantothenic acid negative. Pantothonic acid + inosi tolstrikingly active Author's results (Medium II). Iiuwitol alonenegative. Bios aloneslightly active Bios finositol-activo. /3-alanino (as IIA)- negative /3-alanine+inositolvery active. Tnoaitolslightly active. Bios slightly inhibitive. Bios + inosi tolno greater effect than inositol alone. 0-alanine (as IIA)-slightly inhibitory. fl-alanine + inositol considerable activity (Yeast 2). Inositolvery slightly active Panto- Inositolnegative. Bios nogativo. thenic aciddefinite increase. Bios -f inositolnogative. Pantothonic acid+inositolstrikingly Bios IIAslightly inactive. Bios IIA active + inositolvery active. It is interesting at this juncture to attempt to fit Williams' "pantothenic acid" into the scheme of things suggested in this paper. Two recent papers, appearing since this work was concluded (Williams, Truesdail, Weinstock, Bohrman, Lyman and McBurney, J. Amer. Chem. Soc., 1938,60,2719; Williams, Weinstock, Bohrman, Truesdail and Meyer, may have been that the increased effect produced by addition of j3-alanine to the pantothenic acid was, in reality, no more than increasing the dose of the latter (if /3-alanine can replace pantothenic acid for the particular yeast in question, as has just been suggested), it seems perhaps more likely that, with Williams' medium and under his

11 November, 1939] rainbow: bios requirements of strains of s. cerevisiae 543 conditions, these yeasts (4) and (5) require for high growths the presence of bios HA (= pantothenic acid?) and that /3-alanine will not replace bios HA under these condi tions, but only supplement its action. It is noteworthy that Williams makes no mention of a factor which might correspond to the writer's bios. It is probable that here, again, the composition of the medium is an all-important factorwilliams' medium may eliminate the necessity for a further bios () factor, just as the author found that for certain yeasts (1) and (2) bios is not necessary in aspartic acid medium. Another possible explanation is dealt with under section (4) of the discussion. Reference has been made above to the view advanced by Williams and his co-workers, that pantothenic acid may consist of /3-alanine linked to an hydroxyacid possibly through the amide linkage. This supposition fits the properties of the authors' bios HA (Rainbow and Bishop, toe. dt.) and also establishes some connection between bios HA and /3-alanine. This connection is necessary since it has been shown that /J-alanine can adequately replace bios HA for certain yeasts. The author's bios seems to have its counterpart in Kogl's "biotin" (Kogl and Tonnis, Zeit. Phys. Chem., 1936, 242, 43), which was also prepared from dried egg yolks. In its properties, "biotin" strongly resembles bios and, further, the reactions of Kogl's yeast, Race M (3) to bios nb in medium I as recorded in this investigation [Section (2)], confirm Kogl's results with biotin. Kogl employed a synthetic medium similar to medium I described in this paper. Before leaving Williams' work it is worth noticing that he pointed out the relatively large amount of inositol necessary to give good growth (Williams and Saunders, Biochem. J., 1934, 28, 1887). This has been amply confirmed in this laboratory and is reported in another paper (Rainbow and Bishop, toe. tit.). Kogl's conclusions concerning the activity of /3-alanine are clear to the writer when the results of experiments with medium II. are taken into account. Kogl and Tonnis found that /3-alanine was inactive by their 5 hours' test, but possessed a certain amount of acti vity in the presence of inositol and a#partic acid. Like Kogl, the writer found that /8-alanine had very little effect on his (Kogl's) yeast (Race M) [see under Section (2) of the present paper] unless aspartic acid was present in the culture fluid, in which case the effeot of /3-alanine was brought out [Section (3)]. (4) Other Considerations.Under this head ing it is proposed- to discuss variations in technique and criteria of growth which may be factors responsible for varying results in bios research. These points have not been investigated in the experiments reported in this paper but are set forth as being sugges tive. Here, again, to facilitate comparison, the following Table has been drawn up, contain ing some details of the technique adopted by some of the workers who are mentioned in this disctassion. Author. Technique, etc. Williams. Growths at 30 C. Readings after 18 hours. Test flasks not shaken daring growth period. Soedings0-12 mg. moist yeast per culture of 12 ml., i.e., 40,000 cells per ml. Miller. Growths at 25 C. Readings after 24 (and 48) hours. Tests shaken (rock ing tubes). Soedings 250,000 to 500,000 cells per ml. Cultures 10 ml. Kogl. Growths at 30 C. Readings after 5 hours. Tests shaken. Seedings3-25 million cells por culture of 2 ml. Culture diluted to 22 ml. before determining yeast crop. Yeast "ac climatised" for 45 minutes in culture fluid before uso for inoculation. Rainbow. Growtlis at C. Readings after 48 hours. Tests shaken. Seedings 100 to 0 million cells por culture of 50 ml. Before proceeding with the disoussion, the writer wishes to point out that in the matters of seeding ("pitching") rate, temperature of incubation and the ultimate magnitude of growth, his conditions have been chosen so as to approximate to brewery fermentations. From the data above, it will be readily appreciated that the workers vary a great deal in their conditions of bios testing (temperature and time of incubation, agita tion and seeding rates). These factors each have a bearing on the rate and extent of yeast growth. Thus cultures incubated at 30 C. and shaken during the growth period will tend to give'higher crops than unshaken cultures incubated at C. and, again, the longer the growth period, the higher will be the yeast crop, and so on. This brings us on to the question of "growth criteria." K3

12 544 rainbow: bios requirements of strains of a. OERBViaiAB [November, 1939 What degree of stimulation should be taken as a positive reaction in a bios test?. The author believes that many workers on bios take comparatively small stimulations as growth criteria. This will particularly apply when small seedings and short growth periods are used in testing. The conditions during the present series of experiments utilised comparatively large growths and compara tively long growth periods, with a view to obtaining a " wort growth " of yeast. Under these conditions, it is likely that the bios requirements are considerably more stringent than those investigated in connection with small growths. For instance, inositol plus bios IIA will give a small stimulation (yeasts 1 and 2, medium I) (i.e., "small" by the author's criteria), but those additions alone never give growths approaching " wort growth"; only when, in addition, bios is added is the "wort growth" figure ap proached. Therefore, it is possible that, using a criterion of growth which is much lower by comparison, the effect of such a factor as bios in the present instance might be overlooked. Some Theoretical Considerations.It has been suggested that bios may be of a coenzyme nature. For instance, aneurin (vita min B^ combines with pyrophosphoric acid to form co-carboxylase. This conception may help to throw light on certain bios phe nomena and also supply some theoretical grounds for explaining differences hi bios results. Thus, a change in the source of nitrogen available to the yeast for assimila tion would probably necessitate the bringing into action of a different enzyme or set of enzymes within the yeast cell hi order to bring about the assimilation. This change would involve the appropriate change of coenzyme, or, hi other words, the yeast would show a difference hi bios requirements. The change would not necessarily entail the need for more bios factors; on the contrary, a change to a more readily assimilable form of nitrogen might obviate the necessity of a particular enzyme (and consequently its coenzyme) and thus eliminate a bios factor. The differences hi the results obtained with media I and II may be explained on such a theory and similarly, those recorded by Williams using aspartic acid and asparagine may be explained. These ideas may not only apply to the source of nitrogen but also to the particular sugar employed hi the culture fluid. If the same reasoning applies, it is quite likely that workers using cane sugar with then* media will get bios results different hi some respects from those workers using glucose or maltose. If, hi addition to this, we accept the con ception of "training" or "acclimatising" yeast to substrates, this hypothesis can be extended to explain the difference hi the results obtained early on hi this investigation and those obtained after considerable sub culture of the yeasts, described hi Section (2) of this paper. It is well established that many micro-organisms, including yeast, can be "trained" to utilise certain nutrients out side then* usual range. The "training" of yeast to ferment galactose is an example. This process must involve the production (or activation) of enzymes within the cell in order that the cell can utilise the new nutrient. Thus a "trained" yeast may have different co-enzyme (bios) requirements from the "untrained" yeast. Here then is an extension of the hypothesis which may explain the results described under Section (2) of this paper, the "training" effect being introduced by the prolonged sub culture hi liquid medium from the original culture on agar slopes. These considerations raise a further point which must be taken into account in bios researches, namely, the conditions of culture of the yeast used for seeding ("pitching") the bios tests. Note on "Wort Orowth."Mention has already been made that the criterion of "wort growth" has been adopted in this laboratory. Before the bios problem has been satisfactorily solved, the writer con siders that the rate and extent of yeast growth in synthetic solutions containing bios must be equal to that of yeast hi its natural medium, which, for the author's yeast and other strains of S. cerevisiae em ployed hi brewing, is malt wort. From the figures given in Table VII it will be seen that the author's bios prepara tions are not wholly satisfactory from the point of view of "wort growth." It will be seen that hi wort the yeast growth is more rapid and almost reaches the maximum ex tent of growth after 70 hours. In contrast to this, the growth of yeast hi the synthetic solution, whilst almost equalling the extent

13 November, 1939] bainbow: bios requirements of strains of s. oerbvisiab 546 Table VII Comparison of Growth of Yeast 1 in Wobt and in Synthetic Medium containing Bios. Growth figures. Medium. 45 Hours. 70 Hours. 94 Hours. 118 Hours. Malt wort (1040 ) Synthetic medium containing inositol, bios IIA and bios of growth in wort after a period of 5 days, does not grow so rapidly during the initial stages. Thus, the rate of growth in the bioscontaining synthetic solution is lower than that in wort, although the extent of growth, which attains the maximum after 5 days, is approximately the same in both cases. This could be remedied by addition of a larger dose of bios, but the increased amount necessary to bring about the desired rate of growth for "wort growth" was considerably greater than was expected, even if a law of decreasing returns was taken into considera tion. Thus the author was led to consider that there are other factors which influence the rate of growth and these may include the effect of substances like " aminopyrimidine." Such substances may be, at least partially, responsible for the effects discussed above, whilst they are not essential for the produc tion of high growths for the yeasts examined herein, being required in order to attain the high rate of yeast growth met with in wort cultures. For certain yeasts, however, vita min 3$! and " aminopyrimidine " appear to be essential (Williams and Saunders, loo. cit.; Schultz, Atkin and Frey, loc. cit.) Summary.As a result of the experiments described in this paper the author considers that the apparent differences in the results obtained by various workers on the bios problem are due- to, and may to a large extent, be eliminated by consideration of the following factors: (1) The type of yeast used; (2) The composition of the synthetic solu tion ; (3) The methods employed for the sub culture of the seeding yeast; (4) The general techniqueseeding rate, length of growth period, temperature of incubation, agitation conditions and criteria of growth. Acknowledgment.The author wishes to express his gratitude to Mr. J. N. Scott of this laboratory for bis assistance in the pre paration of the bios concentrates used in this investigation. Institute of Brewing Laboratories, University of Birmingham.