STERN: THE NUTRITION OF YEAST. PART 111. 943 C.-Yhe Nutrition of ITeast. Pm*t IIL By ARTHUR L. STERN, D.Sc. THE work described in the two preceding parts (Trans., 1899, 75, 202 ; J. Peed. Inst. Brewing, 1899, 399) mas continued with the object of obtaining a knowledge of the effect which conditions other than those already discussed had on the growth of yeast, its composition, and the relations of these t o the progress of the fermentation. The experimental method has already been described in Part I ; briefly, i t is as follows. Into a flask holding rather more than a litre are introduced 500 C.C. of a solution of dextrose (&glucose) of definite strength, 1.5 grams of crgstallised asparagine, 0.5 gram of potassium phosphate, 0.2 gram of magnesium suIphate, and 0.02 gram of calcium sulphate.The neck of the flask is closed with a cotton wool plug, and the contents are boiled for 10 minutes. When cool, the yeast is added.944 STZRN: THE NUTRITION OF YEAST. PART This was obtained in the manner described in Part I from the same culture of Burton yeast then prepared. The sugar solution when thus seeded was kept at a constant tem- perature until the conclusion of the experiment. The yeast was then removed by filtration, dried a t 100' until constant, and the nitrogen contained in it estimated. The optical activity of the fermented solu- tion indicated the amount of sugar remaining unfermented. No difficulty mas experienced in filtering 08 the yeast, except when the fermentation was still proceeding. In these experiments, the addi- tion of 0.5 gram of salicylic acid dissolved in 10 C.C.of alcohol t o the 500 C.C. of fermenting solution a t once stopped the fermentation, and the yeast could be easily removed. (1) The InJEuence of Concentrution.-J. Archleb (Zeit. Spiriturrind., 11, 243), using maltose solutions varying in concentration between 1 and 25 per cent., found t h a t as the Concentration of the sugar was in- creased, (a) from 1 to 5 per cent., the weight of yeast obtained increased; ( b ) from 5 t o 10 per cent,, the weight of yeast crop in- creased but slightly; ( c ) from 10 to 14 per cent., the weight of yeast crop increased rapidly; (d) from 14 t5 19 per cent., the weight of the yeast crop diminished; (e) from 19 to 25 per cent., the weight of the yeast crop again increased.These experiments indicate that the weight of the yeast crop varies irregularly as the concentration of the sugar is increased. Experiments were made according to the method described above t o obtain further information on this point. They were performed at 2 5 O , and the amount of seeding was 1.5 cells per 1/4000 cub. mm. The results are given in Tables l a and l b (p. 945). The only differ- ence in the conditions of the two series of experiments is that those of Table l b were performed with 0.75 gram of asparagine per 500 C.C. of solution instead of 1.5 grams used in all the other experiments. As the standard time of fermentation (7 days) allowed in the earlier experiments was found to be insufficient when fermenting solutions containing more than 15 per cent.of dextrose, experiments with these solutions mere repeated, allowing a longer time for fermentation. Curve 1 (p. 946) was drawn by joining the points obtained by taking as abscissae the weights of nitrogen contained in the yeast, and as ordin- ates the concentration of the sugar at the commencement of the fer- mentation ; it clearly shows that as the concentration rises from zero the amount of nitrogen assimilated increases rapidly at first, then more slowly, until with concentrations of more than 15 per cent. the in- crease appears almost to cease ; there is, however, much difficulty in com- pleting the fermentation with concentratious exceeding 20 per cent. The percentage of nitrogen in the yeast does not greatly vary, but is highest with the weakest and strongest solutions here employed.STERN: THE NUTRITION OF YEAST, PART IIJ.985 Duration of fermen ta- tion. Duration of fermenta- tion. Percentage of dextrose before fermen ta- tion. 0 1 3 5 7.5 10 12.5 15 20 25 30 12.5 15.0 17'5 20.0 22.5 TABLE l a . Weight of nitrogeii in yeast crop. Gram per 100 C.C. 0 0028 0*0081 0.0129 0.0178 0.0200 0.0216 0'0246 0.0275 0.0167 0.0148 0.0122 0.0230 0.0238 0.0232 0*0252 0.0253 Percentage of nitrogen in yeast crop. 7.0 8'6 7'8 7'9 7 -1 6.9 7.0 7-2 6.7 7 .O 7 -7 7'4 7.3 7.6 7.8 7 'ti TABLE 16. Percentage of dextrose before fermenta- tion. -~ _____~ 0 1 3 5 7.5 10 1 2 5 15 20 Weight of nitrogen in yeast. Gram per 100 c.c. Percentage of nitrogen in yeast. 0.0028 0-0076 0.0122 0'0165 0'0194 0.0212 0'0236 0.0199 0'0190 0'0160 0'0129 7.0 9-0 7 *4 7'4 6'6 6'6 6-6 6.8 6.5 7 - 1 7'4 Percentage of dextrose inferm en ted, - 0 1 *5 2 '8 3.1 4.2 6.0 6.6 37'4 51.5 65'2 - 5.3 5 '1 5.4 6'6 10.5 Weight of yeast crop.Gram per 100 C.C. 0.040 0'094 0.166 0.226 0.280 0.314 0-352 0.382 0.24E' 0.211 0'160 0.311 0.326 0.306 0-322 0.320 Percentage of sugar nnfermented. - 4 *7 1-8 1.9 2.2 5-8 5'0 12'6 34'5 50.4 62 0 Weight of yeast. Gram per 1co C.C. 0-040 0.085 0.165 0'221 0'293 0'323 0.361 0-291 0293 0.224 0-175 No yeast growth was obtained when no sugar was present. (2.) In,uence of Temperature.-R. Pedersen (Carlsberg Lab. Reports, 1878, 22) found that the yeast cells increase in number most rapidly at 2S0, but that the same total number was produced at 13.5' as a t 23", and that no growth took place at 38O.The experiments summarised in Table 2 were all carried out with946 STERN: THE NUTRITION OF YEAST. PART 111. 10 per cent. dextrose solutions, and the inorganic and nitrogenous nutriment stated above at the temperatures noted. The amount of seed yeast was the same as before, I n order that the fermentations I'crcentage of s u p r before femm&tion. could be completed, the time of fermentation had to be considerably longer at the lower temperatures. These results indicate that at temperatures between 12' and 35" there is little difference in the weight of yeast, and the amount ofSTERN: THE NUTRITION OF YEAST. PART 111. 947 Temperature. nitrogen contained by it, but that a t 30° and above there is a decrease of these factors, and at 37" i t is evident that the functions of the yeast are seriously weakened.Duration of ferments- tion. TABLE 2. 21 25 30 37 9 > Y 7 I Y 5 2 ) 3 Y Y 12" 13 days 15.5 1 16 ), 18 1'2 Y Y Weiglit of nitrogen in yeast crop. Gram pcr 100 C.C. 0.0194 0'0198 0.0221 0'0224 0.0212 0'0173 0.0085 Percentage of nitrogen in yeast crop. 7.7 6.8 6% 6.6 6 -9 7 . 2 7.8 Percent age of sugar unfermented. 14-7 3'6 4.7 9'2 4'4 5 . 6 40.1% Weiglit of yeast crop. Gram per 100 C.C. 0.252 0.291 0-337 0-339 0.310 0.243 0.110 * Six days' fermentation produced but a slight increase in the amount of sugar fermented 2nd a decrease i n the weight of yeast. (3.) The Efect of Vuwjing the Amount of Seed Yeast.-A. J. Brown (Trans., 1892,61, 369) has shown that for any one fermentable solution there is a constant number of yeast cells up to which the yeast will increase if the solution be seeded with a smaller number, and that if it be seeded with a larger number no increase in the number of cells will take place. The experiments summarised in Table 3 were made more particularly t o ascertain the relationship between the weight of nitrogen contained in the seed yeast and that contained in the final yeast crop at the conclusion of the fermentation. They were carried out a t 2 5 O with the quantities of nitrogenous and inorganic nutriment given above, but in view of the great importance of the results three series of experi- ments were made with 5, 10, and 15 per cent.dextrose solutions. On comparing the figures obtained by subtracting the weight of nitrogen added in the seed yeast from that contained in the yeast crop, it is found (except where the seeding is less than 5 per cent.of the crop) that within the limits of experimental error the figures are the same for any one solution, and that, as might be expected from the previous experiments on the effect of concentration, the average for the 5 per cent. solutions is the least, and that for the 15 per cent. solution the greatest. Jn the experiments where the seeding was less than 5 per cent. of thc crop, these differences are below the average. As the percentage of nitrogen contained in the yeast does not vary very materially, these results may be taken as also applying to the94s STERN: THE NUTRITION O F YEAST. PART 111. Weight of nitrogen in tlto I TABLE 3. Difference between the values in the first two columns.Gram per Gram per 100 C.C. ( a ) Five per cent. sugar solutions : 0.0121 0.0144 0'0151 0.0167 0.0176 0-0179 0.0236 0 -0005 0-0006 0'0014 0'0019 0'0040 0.0056 0'0112 Mean., , , , , . ( b ) Ten per cent. sugar solutions : 0.0143 0.0155 0'0161 0-0196 0'0204 0'0218 0.0217 0'0247 0'0269 0.0308 0.0352 0.0002 0'0006 0*0008 0'0017 0 -0023 0-0045 0 0050 0-0066 0'0096 0'0125 0-0172 Mean ........ (c) Fifteen ~ e r cent. solutions : 0.0173 0'0201 0'021 4 0'0248 0'0228 0.0318 0.0009 0*0013 0.0027 0*0040 0.0072 0.0135 I Mean.. , .... 0-0116 0.0138 0.0137 0.0148 0'0136 0.0123 0'0124 0.0132 0-0141 0'0149 0'0153 0.0179 0 0181 0.01 73 0*0167 0-0181 0-0173 0.0183 0.0180 0.0177" 0.0164 0'0188 0.0187 0.0208 0.0156 0'0183 0'0181 Percentage of nitrogen in the yeast.6 .t3 7-1 6.7 7'4 7'3 7'3 7.7 6-9 7.0 6.8 7 '1 6'6 6 -5 5.1 6 -5 7 '0 6.6 7 '1 7 - 1 6-3 7.0 6 '1 6 -7 6 '6 * Mean of the last eight values. Percentage of sugar unfermented. 2.8 1'9 1'1 1 '9 2 *8 2'8 3'4 3 '0 2'0 11 *3 5.1 6'2 2.8 3'0 3 '3 3'0 2.3 3'0 5 .O 9.5 3 9 6'3 4'6 3.9 weight of the yeast, and except when the seeding is very small, a general statement of the result may be made as follows. If t o equalSTERN: THE NUTRITION O F PEAST. PART 111. 949 volumes of any one fermentable solution quantities of yeast u, b, c be added, the resultant crop of yeast a t the end of the fermentation will be a + p , b + p , c +p, where p is a constant quantity dependent on the composition of the solution. If the seeding is very large, this statement must be modified, as yeast always contains some dead or weak cells, and the decomposition of these would introduce more yeast nutriment into the solution. (4) Efect of Time.-It is generally stated that most of the yeast is formed during the early stages of the fermentation and that in the later stages there is little, if any, increase of yeast (Mohr, Wochen- schr.B~azc., 1S96, 3, 210; F. Schonfeld, ibid., 421 ; Delbruck, Bied. Centy., 1880, 217 ; Boulanger, Ann. Inst. Pastew, 1896, 10, 598). To determine the relation of the weight and nitrogen contents of the yeast to the amount of sugar fermented at successive time inter- vals, experiments were performed a t 25' with 10 per cent. dextrose solutions, and the amounts of nitrogenous and inorganic nutriment already noted; the results are given in Table 4 (p.950). The amount of seed yeast in the four series of experiments a, b, c, and d was in the proportions of 1, 3, 9, and 14. Curve 2 (p. 951) was drawn by joining the points obtained by taking as abscissze the weight of nitrogen in the yeast, and as ordinates the corresponding amount of sugar fermented. I t clearly shows that the increase of the yeast, and of the weight of nitrogen contained in it, goes on to the end of the fermentation. After a certain time, which is least in series c, the curve becomes a straight line; that is, the ratio of the amount of sugar fermented t o the weight of nitrogen remains constant during the remainder of the fermentation. The curves show that before constancy is reached, the nitrogen increases more rapidly than the amount of sugar fermented, when the seeding is less than in c (compare cb and 6), and less rapidly when it is greater than in c (compare d).As the percentage of nitrogen in the yeast does not vary much throughout the fermentation, these statements are practically also true for the ratio of the weight of yeast to the weight of sugar fermented. The experiments described in this investigation were primarily made with the object of obtaining a knowledge of the effect of different conditions on the growth of the yeast and its composition, and the relation of these to the progress of the fermentation. As it was necessary to work under conditions which could at any time be exactly reproduced, the choice of yeast foods was restricted to such as could be obtained pure in reasonable quantity.It is known that when employing such yeast foods as are contained in malt worts, yeast water, &c., larger and more nitrogenous yeast crops are obtained. It is thus possible that the results here obtained may be in some VOL. LXXIX. 3 T950 STERN: THE NUTRITION OF YEAST, PART 111. respects abnormal. the future, The author hopes to investigate this question in TABLE 4. Time. Series a : 0 2 days 4 9 ) 6 Y Y lo ) I Series b : 0 1 day 2 Y Y 3 ? t 4 3 3 5 Y Y 7 3 9 Series c : 0 1 day 2 9 9 3 $ 3 5 > 9 Series d ; 0 14 hours 38 9 Y 86 9 s ~~ Weight of nitrogen in yeast. Gram per 100 C.O. 0~0009 0'0045 0'0095 0'0116 0.0138 0-0026 0.0120 0*0150 0*0180 0-0191 0'0205 0-0212 0.0078 0.0123 0.0178 0'0220 0.0249 0.0125 0.0157 0.0259 0-0340 Percentagc of nitrogen in yeast.7.5 7'2 6.5 6 * 0 6.2 7.0 8.0 7 '0 7-0 6.9 6.9 6.9 7.1 6.6 6 *1 6 '1 6.1 6'1 6 '8 6'2 6'2 Percentage of sngar un- fermented. 100.0 90.9 66.0 38.9 6 '0 100.0 75.6 50.8 30.8 18.6 8-4 4-4 100.0 77 *6 49 '3 25 -1 5-4 100*0 75'6 32.0 3.5 Weight of yeast. Gram per 100 C.C. 0.012 0.065 0.147 0.193 0.223 0,037 0,161 0.217 0.260 0'279 0,296 0.310 0.110 0.185 0.290 0.364 0-406 0.206 0.229 0'419 0.548 The results here obtained may be summarised as follows : 1. Any increase of nitrogenous or inorganic nutriment beyond a definite limit will not increase either the amount of nitrogen assimi- lated by the yeast or the weight of the yeast. This limit is but little greater than the largest amount which the yeast is able to assimilate under the conditions of the experiment (Part I, Zoc.cit.). 2. An increase of the sugar is accompanied by an increase in the weight of nitrogen assimilated and in the weight of the yeast, This increase continues up to the strongest concentrations which can be completely fermented. The rate of increase is greatest at the lowest cnncentrations and falls off gradually as the concentration rises.STERN: THE NUTRITION OF YEAST. PART 111. 951 j 20 CURVE 2 (TABLE 4). I- / 2’ 40 60 so Percentayc of sicgas* fermented. 7 r“ 100 3. Temperatares between 1 2 O and 25’ have lout little influence on the weight of nitrogen assimilated and the weight of the yeast crop. A t higher temperatures, reproduction is weakened. 4. The total weight and nitrogen content of the yeast crop obtained at the completion of fermentation are dependent solely on the weight 3 ~ 2952 STERN: THE NUTRITION OF YEAST, PART 111.and nitrogen content of the yeast added to produce fermentation and on the composition of the fermentable solution. 5. During a portion of the fermentation the growth of yeast is pro- portional t o the amount of sugar fermented, and proceeds as long as any sugar remains unfermented. Although the chief object of this work was to obtain these results, yet it was thought that when obtained they would throw additional light on the mechanism of fermentation and yeast growth ; that they do so will be seen from the following. Statements 1 and 2 show that there must be a great difference between the functions of the nitrogenous and inorganic nutriment, on the one hand, and of the eugar on the other; whilst the function of the first two is to supply the necessary material to form the yeast, that of the sugar is twofold-to supply the yeast with material and with, energ y.The chemical products of the decomposition of the sugar (alcohol and carbon dioxide) are useless t o the yeast, and when present in sufficient quantity even deleterious t o i t ; this decomposition of the sugar, however, sets free an amount of energy which has been cal- culated as from 33 to 67 K ; the heat developed has been measured, and amounts to 23.5 R according to Bouffard (Compt. ?*end, 1895, 121, 357), and to 21.4 K according to A. J. Brown (J. Fed. Inst. Brewing, It has been previously suggested by A.J. Brown (Trans,, 1894, 59, 911), by Reynolds Green (Ir’ewuentation, p. 334), and others that the need for this energy is the reason for the fermentation of the sugar, part of i t being employed to carry on the vital actions of the yeast, and part to raise the temperature of the solution, a temperature slightly above the ordinary being most favourable to the yeast. It is a much debated question whether the fermentation of the sugar is the work of an enzyme or of the vital functions of the yeast. Buchner (Ber., 1897, 30, 117, 1110, 2668; 1898, 31, 209, 568, 1084, 1090, 1531 ; 1899, 32, 127), Abeles (Ber., 1898, 31, 2261), and others state that they have isolated this ferment from yeast ; this explanation of their experiments has, however, been combated by Mscfadyen, Morris, and Rowland (Proc.Rog. Soc., 1900, 6’7, 250) and Wrdblewski (Centy. Phyiol., 1899, 22). The great difficulty attending the investigation of the fermentative function of yeast has been to eliminate disturbances due to change in the fermentative power of the plant-that is, to change in the amount of fermentative enzyme, supposing such to be present. Dumas (Ann. Chint. Phys., 1874, [v], 3, S l ) and A. J. Brown (Trans., 1892,61, 369) employed such an excess of yeast as was shown by the latter to result in fermentation without any numerical increase of yeast, and found 1901, 7, 93).STERN: THE NUTRITION OF YEAST. PART 111. 953 that under these conditions equal quantities of sugar were fermented in equal times. If no change had taken place in the fermentative power of the yeast during the experiment, this result would negative the enzyme theory.I t is, however, quite possible that the fermenta- tive power of the yeast has changed in the course of the experiments, and for the following reasons. It is shown in Table 4 that fermentation is closely connected with yeast growth, and if yeast growth has taken place, the fermentative power of yeast as a whole has probably changed. Now, in two of A. J. Brown’s experiments (Zoc. cit., 377), in which a large excess of yeast was employed, and no numerical increase took place, the weight of the yeast had appreciably increased during the ferment a t ion. Moreover, J. O’Sullivan (J. Fed. Inst. B~ewing, 1899, 5, 101) showed that whilst the invertive function of living yeast could be and was usually exercised without yeast growth taking place, this was not the case with the fermentative function.Other evidence in- dicating the intimate connection between yeast growth and fermenta- tion is to be found in the experiments described above (statements 4 and 5) and in the stimulating effect of nutriment in increasing the rapidity of fermentation of a non-multiplying yeast (J. O’Sullivan, J. SOC. Chem. Ind., 1898, 17, 559). Even supposing that, by using a large excess of yeast, i t mere possible to obtain fermentation, without any apparent increase either in the weight of the yeast or in the number of yeast cells, yet it would be quite possible for yeast growth to take place a t the expense of the decomposition products of dead cells which are always present, decom- posed yeast being an excellent yeast food, If yeast growth has taken place, there will probably be some change in the fermentative power OF the yeast as a whole. The experiments of Dumas and Brown do not then disprove the enzyme theory. There is thus no proof that the fermentation of sugar by yeast is not caused by an enzyme, and whilst the converse has not yet been definitely proved, the evidence at present available points in this direction. The enzyme (if existent) is exceedingly un- stable; i t diminishes in amount, even in living yeast cells which have remained quiescent for a short time,* but when required by growing yeast i t is abundantly secreted. This view derives some support from the fact that the change in the appearance of the yeast cells when sown in a fermentable soIution is similar to that which takes place in the columnar cells in the germinating barley embryo when secreting diastase. * Buchner and Rapp (Bey., 1897, 30, 2668) state that it can only be prepared from fresh yeast.