September, 1930
I N D U S T R I A L A N D ENGINEERING CHEMISTRY
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Stimulation of Cane Molasses Fermentation b y Certain Metallic Salts‘ F. M. Hildebrandt and F. F. Boyce U. S. INDUSTRIALALCOHOL COMPANY, BALTIMORE, MD.
been done, particularly o n I t is well known that small amounts of certain N RECENT years there yeast fermentation. metallic salts have a stimulating effect on green plants has been considerable inand the fungi. It is posslble to produce a slight but terest in the function of Previous Work definite increase in the yield of alcohol from yeast fersmall amounts of various subA large amount of work on mentation of cane molasses by the use of these salts. stances in the food of the poisons and stimulants, inManganese and copper salts were used with success, organism. These substances cluding an extensive bibliogand cyanides of potassium and sodium were also have been considered by some raphy, is collected in the effective. The stimulating effect was found to be students as nutrients and by monograph by Brenchley (3). more certain and consistent if the stimulants were others as stimulants. The Especial attention is paid to added in the yeast stage preceding the final fermentaolder investigators of nutricopper, zinc, arsenic, boron, tion. This yeast, treated with a suitable concentration tion who opened up the field of the salts, when put into a second solution of moand manganese. Generally of study of materials needed speaking, compounds of these lasses to which no salts have been added, will give a for cell a c t i v i t y confined higher yield of aicohol than untreated yeast. substances were f o u n d t o themselves necessarily to inhave a stimulating action on The use of the stimulants in the seed stage only vestigating those required in the higher plants over a cermakes it possible to control their action and also large amounts. On the side tain c o n c e n t r a t i o n range. reduces the amount of salt necessary to a very low of the organic foods their atMention is a l s o m a d e of figure. This technic of utilizing the stimulating tentionwas focused on the fat, such an effect on the lower effect of these salts makes their employment possible protein, a n d carbohydrate in industrial fermentations of molasses. plants. Johnston and Dore required. On the side of the (7’) found that very minute inorganic they considered the quantity of potassium, calcium, magnesium, etc , necessary. amounts of boron had a marked effect on the growth bf tomato A considerable knowledge of the food requirements of living plants. The oligodynamic action of copper is mentioned by Messerschmidt (11) and by Matsunaga (10). These authors things was thus built up. It has, however, been found necessary to supplement this emphasize the germicidal effect of small amounts of the metal knowledge by a study of other materials which play an impor- on bacteria. Schweizer (1‘7) investigated the inhibiting eftant part when present in very small amounts. I n the organic fect of copper, but could find no evidence of stimulation due portion of this study the function of vitamins, for instance, to the metal. Maquenne and Demoussy ( 8 , 9 ) regard copper furnishes a well-known example of the manner in which the and zinc as probably essential to plant growth. Attention older knowledge of food requirements has been extended. On is called to the wide distribution of copper in organisms by the inorganic side there is a constantly increasing interest Fleurent and Levi ( d ) , who found it in many plants, and also by Rose and Bodansky (14). Rose and Meunier (15) state in the biological effect of traces of salts of metals. This study of the effects of the metals includes both animals that care is necessary to avoid poisoning of yeast in copper and plants. The principle involved is apparently quite fermentation vessels. The role of manganese in the higher plants is discussed in general and may be expected to apply even to the simplest forms. It is the purpose of the present paper to describe ex- a paper by Bertrand and Rosenblatt (1). The authors show periments on the alcoholic fermentation of cane molasses by that practically all vegetable tissues contain manganese, and living yeast in which the course of the fermentation was state that it is most abundant in those tissues showing the altered by the use of small amounts of manganese and copper. greatest chemical activity. Harpuder (5) deals specifically Data are also included on sodium cyanide, although it is the with the effect of manganese and iron on alcoholic fermentacyanide and not the metal that is active in this case. These tion, stating that the former has a stimulating action a t certain substances produce a small but definite increase in the amount concentrations. Rosenblatt and March, referring to yeast of alcohol formed from a given amount of sugar in the mo- ( I @ , also state that manganese is without effect a t low conlasses. h method of applying the stimulating action has centrations, becomes stimulating as the amount is raised, and been developed that renders it controllable and thus gives finally exerts an inhibiting effect. The stimulating effect of other metals is mentioned by variit industrial possibilities in large-scale molasses fermentations. Although only three salts have been studied, it is probable ous authors. Richtet and Braumann ( I S ) found the lactic that others may be dsed in the same way. Indeed, if we con- acid fermentation to respond to extraordinarily minute sider this sort of action as a stimulation, it may he said that amounts of lanthanum, stimulation occurring a t concentragrams per liter. The action of silver most substances, toxic! in large quantities] have a stimulating tions of lo-* and effect in a certain concentration range. Branham (W), in on yeast was studied by Zerner and Hamburger (18). They a recent paper on the effect of antiseptics on yeast fermenta- were interested in the lethal dose and do not mention a stimution, found this to be the case. The literature on this sub- lating range. Branham ( 2 ) , dealing with the toxic and stimuject is too extensive to be covered here. Certain references lating effects of various substances on fermentation by yeast, are given, however, to indicate the scope of the work that has states that silver does not show stimulation in any concentration tried. 1 Received April 19, 1930. Presented under the title “Effect of Metallic The influence of selenium on mold spores is reported by Salts as Stimulants in Alcoholic Fermentation of Molasses” before the Ngmec and K&s(12). Very minute amounts of sodium selenate Division of Sugar Chemistry at the 79th Meeting of the American Chemical Society, Atlanta, Ga., April 7 l o 11, 1930. had a definite stimulating effect on the spore development.
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Development of Method
While no rigid generalization can be made on this subject, it may be said that certain metals, toxic in larger amounts, show stimulation within a certain concentration range. With this principle in mind an attempt was made to utilize these stimulating effects to increase the yield of alcohol produced by the fermentation of cane molasses with distiller's yeast. The literature quoted contains references to manganese, and it is stated specifically that salts of this metal, when added to a fermenting mash, produce an increased yield of alcohol as compared with yields from the same mash without the addition of salts of the metal.
CONCENTRAT/ON Figure I-Effect
of MnSOa o n Alcohol Yield-First
Run
Experimentation on a small scale with manganous sulfate did, in fact, show stimulation of the yield of alcohol in cane molasses, but the results were too erratic to be of practical value. The concentration a t which the maximum alcohol yield was obtained varied, and it was not always possible to obtain a smooth curve showing increase in yield as the concentration was raised to an optimum and then a fall in the yield as the dose was increased beyond this point. If use is to be made of such salts in factory operation, the optimum concentration must be rather definite and an increase in the concentration from a point below the optimum to a point above it must show a smooth rise and fall in yield. Otherwise, there would be no assurance that putting the salts in the mash would be a safe procedure from the operating point of view. A method was finally worked out by means of which the desired smooth stimulation curve could be obtained. Also, the point of maximum stimulation was stabilized sufficiently t o make the use of these salts a practical possibility. It was found that if the salts here reported are used in a solution of cane molasses containing living yeast and a small portion of this solution is used to seed a second similar solution from which the salts have been omitted, the stimulating effect of the salt is carried over into this second stage. I n terms of distillery practice this means that a stimulant may be used in the yeast stage and that this stimulated yeast will carry over into the principal fermentation the ability to produce slightly more alcohol than untreated yeast. Since the volume of the seed yeast is small compared with the volume of the main mash (4 to 6 per cent), small quantities of the metallic salts are sufficient to,accomplish the desired result. I n addition to this economy of material, when the metal salts are used as described, the stimulation curve smoothes out and the optimum point is stabilized rather definitely a t a certain salt concentration. The principle involved here has been found to apply with all of the salts used. It should be pointed out that the increases in alcohol obtained in this manner, although definite, are small. In order
Vol. 22, No, 9
to demonstrate these relatively small increases in alcohol produced from molasses, an experimental technic capable of a high degree of precision is necessary. This was accomplished by simplifying the method, eliminating volume measurements with attendant necessity of temperature control, and basing all results on weight determinations. The two critical measurements were (1) weight of molasses used in the run and (2) pycnometer weight of the alcoholic distillate obtained from this molasses after its fermentation by yeast. These two weights can be determined to a high degree of precision. Experimental Method
A large sample of the molasses was thoroughly mixed and its sugar content determined accurately, the sucrose by the Clerget method and the invert by the reduction of Fehling's solution. The sucrose was multiplied by 1.0528 to reduce it to the invert basis, and this figure added to the invert determined directly to get the total as invert. A 125-gram portion of this molasses was then weighed directly into a 1-liter Erlenmeyer flask in which the seed fermentation was to be carried out. To this molasses sufficient water was added to give a Brix of approximately 20". A record was kept of the amount of water added. The necessary quantity of a water solution of the stimulant and concentrated sulfuric acid to give the desired hydrogen-ion concentration were then added. The amount of acid was regulated in accordance with the method given in a previous paper ( 6 ) . The total volume was noted and the solution heated to boiling. It was then cooled and inoculated with pure culture yeast from a previously prepared batch of yeast grown in malt wort. This seed yeast was allowed to ferment until the carbon dioxide
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CONtENTRAT/ON
Figure 2-Effect
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of Manganese Sulfate on Alcohol YieldSecond R u n
evolved resulted in its losing 20 grams of weight. It was then about two-thirds fermented out, as the total weight loss from 125 grams of molasses of 50 per cent sugar fermented by yeast is approximately 30 grams. At this time it was used to seed a second flask. This flask was made up in all respects like the first, using 125 grams of molasses and the same amount of water and acid. T h e stimulating salt was omitted, however, and the diluted molasses was not heated to boiling. Twenty cubic centimeters of the treated yeast were then transferred to this second flask and fermentation was allowed to proceed to completion. Since the total volume of the seed yeast is known, the amount of molasses equivalent to this 20 cc. transferred can be calculated and added to the amount weighed into the second flask. Upon completion of fermentation, determined by the fact that the flask showed a weight loss of less than 0.5 gram in 24 hours, the entire content was washed into a distillation flask and half of the volume distilled into a graduated 250-cc. volumetric flask. The dis-
INDUSTRIAL AND ENGINEERING CHEMISTRY
September, 1930
tillate was made to volume a t 20' C., a portion weighed in a vacuum-jacketed pycnometer, and the total alcohol produced determined. The weight of molasses used and its sugar content being known, the total grams of sugar could be calculated. Each gram of invert sugar will produce theoretically 0.61 cc. of alcohol. The fermentation efficiency, or percentage of theoretical yield, was then calculated by dividing the total alcohol obtained by the 1,otalto be theoretically expected from the sugar present.
I n order to give an idea of the numerical magnitudes involved in these experiments, the data used in the graphs of Figure 1 are given in Tables I and 11. In these tables the quantities of alcohol determined by pycnometer in each of the duplicate runs are shown separately. From these determinations calculations are made to obtain the figures in the last column, which are plotted in Figure 1. The results for the other experiments were secured in the same way.
2
of Manganous Sulfate Used i n Main Fermentation EFFICIEUCY IN TERMSOF CONCN OF ALCOHOL COVTROL MnSO1 PRODUCED EFFICIENCY A 5 100 cc P e r cent Per cent Control 35 60 86 8 100 35 80 Av 35.70 1 t o 100 35 125 85 63 9s 63
3
1 t o 200
Table I-Effect
FLASK 1 la
2a
a5 an .~ .~
Av. 3a
4 4a
5
5a
1 t o 500
Av.
Av. 1 t o 1000 Av
6 6a
CONC~NTRAT/ON Figure 3-Effect
of Sodium Cyanide on Alcohol Yield
Each concentration of stimulant was run in duplicate and duplicate controls were run, the seed for which contained no stimulant. &o a series of concentrations were run together in order to secure data for plotting a stimulation curve. The results are given in the graphs. Results
In presenting the results as graphs the following scheme has been adopted. The efficiency figures obtained as previously noted were expressed in terms of the control taken as 100, and therefore give percentage increase or dwrease in alcohol yield produced by the treated yeast as compared with the untreated. These percentage yields are the ordinates of the graphs. The abscissas are the concentrations of the stimulant in the seed yeast, expressed as grams of molasses containing 1 gram of the substance under investigntion. It was also found that there was a logarithmic relation between the effect on fermentation and the concentration of the stimulant in the seed yeast. The abscissas are accordingly laid off in proportion to the logarithm of the concentration, so that the portion of the curves of alcohol yields involving the stimulating effect become symmetrical around the point of maximum stimulation. In several cases comparative curves are shown where the effect of using the stimulant directly instead of in the seed yeast map be compared with seed treatment by the stimulating salt. Figures 1 and 2 show the effect of using manganous sulfate in the seed yeast and in the main mash. In the case of Figure 1 the salt apparently had a depressing action in the main mash. However, seed yeast treated with the salt and then used in a second fermentation to which none of the salt was added, showed a smooth stimulation curve with a maximum a t about 1 gram of the salt per 5000 grams of molasses. Figure 2 shows a similar run on another lot of molasses. The salt in the main mash shows a stimulation curve, but the effect is slight and the maximum comes a t a concentration of 1 in 1000. Here again when manganrse-treated seed yeast was used a smooth stimulation is produced with a maximum a t about 1 to 5000 concentration.
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1 t o 5000
35.21 35.25 35.275 35.26 35.10 35.2'0 35.15 35.725 2.5 5.i _. _.
35.64 35.775 35 675 35 725
85.75
98.77
85.45
98.46
86.67
99.83
86 88
100 07
Av. Total sugar a s invert in molasses (Cuban). . . . . . . . . , . . , .5l.86% Theoretical yield of alcohol.. . . , . . . . . . . . . . . . . . . . 4 1 . 1 2 cc. 60' BC. sulfuric acid per 125 grams molasses t o adjust pH of fermentation solution t o 5.0.. . , . . . . . . . . , , , . . , . O . 5 cc.
. . .. .
.
Table 11-Effect of Treating the Seed Yeast with Manganous Sulfate EFFICIENCY IN CONCN. OF TERMS OF MnS04 I N ALCOHOL CONTROL FLASK SEEDYEAST PRODUCED EFFICIENCY AS 100 CC. Per cent Per cent 1 Control 35.75 85.32 100 la 35,5625 AV. 35.656 2 1 t o 100 84.94 99.55 35.45 2a 35.54 Av. 35,495 3 1 t o 200 85.50 100.21 35.835 3a 35.625 Av. 35.73 4 85.84 1 t o 500 100.61 35.95 4a 35.80 Av. 35.875 5 1 t o 1000 86.26 101.10 35.975 5a 36.125 Av. 36.05 6 101.59 86.68 1 t o 5000 36.175 63 36.275 Av. 36.225 100.80 7 86.00 1 t o 10,000 35.975 7a 35.90 Av. 35,9375 8 1 t o 30,000 S5.61 100.34 35,SO 8a 35.75 Av . 35.775 9 1 t o 50,000 85.31 99.98 35.725 9a 35.575 Av. 35.66 Total sugar a s invert in molasses (Cuban blackstrap). . . .52,70% Theoretical yield of alcohol. . . . , . . . . . . . . . . . . . . . . . . . .41.79cc. 60" BC.sulfuric acid per 125 grams molasses t o adjust p H of fermentation solution t o 5.0 . . . . . . . . . . . . . . . . . . . . O .55 cc.
.
Figure 3 shows the results of two series of experiments where sodium cyanide was added to the seed (curves 1 and 2) and one where it was added to the main fermentation (curve 3). There is no evidence of stimulation when the substance is used directly, but where it is used to treat the seed a definite increase in alcohol yield is obtained a t concentrations of around 1 to 11,000. This substance is more active than the manganese and the curve is therefore steeper, showing a sharp maximum. It is interestimg to note that when conditions throw the maximum point toward the higher concentrat'ions (curve 2 ) the total rise in the curve is not so great as where a more dilute solution gives the maximum stimulation (curve 1). Figure 4 shows three experiments using copper sulfate (CuS0g5H20). A marked effect is produced with maxima in two cases around 1 in 2000, and a t about 1 in 1500 in the third case. The salt was used in the seed yeast. As with the cyanide, one set of runs shows a maximum toward the
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1014
side of greater concentration, and in this case the highest alcohol yield is lower than when the maximum comes in the more dilute range.
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Here again there is no evidence of stimulation. It may thus be concluded that the effect of the salts is temporary and is lost after one growing in a molasses solution to which no salt has been added. Industrial Application
The effect of these salts when used to treat yeast which is subsequently employed for fermenting a second eolution is quite definite. The increases produced are not great in the absolute sense, but it must be remembered that a molasses solution seeded wit,h pure yeast will give a relatively high yield with no additions other than dilution water. By considering the margin between this and the theoretically obtainable, these increases take on more meaning. From the industrial point of view the increase in yield is well worth while, since it corresponds to a large quantity of alcohol over a period of t>ime. The quantities of salt necessary are very small and
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Figure 4-Effect
0
CONCENTRAT/ON of Copper Sulfate o n Alcohol Yield
Figure 5 shows a comparison of characteristics of each kind of salt by placing the three typical curves of stimulation on the same chart. It will be seen that cyanide and copper produce a greater effect than manganese, but the curves are steeper and the maximum point quite sharply defined. For industrial use a non-toxic salt such as manganese is preferable to the more active salts. It has a wider range of stimulation and an overdose will not poison the yeast. I n the case of the copper and especially in the case of cyanide a slight overdose would become toxic. Experiments with Successive Growings of Treated Seed
Since the stimulating effect of these salts is carried from a seed-yeast stage to a second-fermentation stage where the salts are omitted, it became of interest to know whether it would carry further. Three generations were run, therefore, to answer this question, and the graphs of Figure 6 give the
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coNcENrRar/oN
Figure 6-Successive
Growings of Manganese SulfateTreated Seed
the cost is correspondingly slight. It is not known how generally this principle applies It may be that many other substances will produce the effects shown in the case of the three here reported. Since the effect persists through one growing following the treatment] some semi-permanent change has evidently been made in the yeast by the salts. It would be of interest to know whether other microorganisms of the higher forms would show a similar behavior. Conclusion
Yeast treated with suitable concentrations of copper sulfate, manganous sulfate, or sodium cyanide in a solution of cane molasses and then used as seed in a second solution to which no salts have been added shows an increased alcohol production as compared with yeast not treated with these salts. Literature Cited
Figure 5-Comparative
CONCE’NTRAT/ON Effects, Copper, Manganese, and Cyanide
results. Treated seed was prepared in the usual way and used to seed the runs shown in the graph labeled “1st gen.” Here the usual stimulation was obtained with a maximum at 1 in 5000. The effect is not so great as is usually found, but the salt shows a smooth curve. These runs were used to seed a second generation (“2nd gen.”) and it will be seen that the stimulating effect has disappeared. Using the second series to seed a third series gave the results plotted as “3rd gen.”
Bertrand and Rosenblatt, Compt. rend., 173, 1118 (1921). Branham, J . Bact., 18, 247 (1929). Brenchley, “Inorganic Plant Poisons and Stimulants,” Cambridqe University Press, 1914. Fleurent and Levi, Bull. soc. chim., 27, 440 (1920). Harpuder, Biochem.Z.,183, 68 (1927). Hildebrandt, IND.ESG.CHEM.,21, 779 (1929). Johnston and Dore, Science, 67, 324 (1928). Maquenne and Demoussy, Bull.soc. chim., 27,266 (1920). Maquenne and Demoussy, Compl. rend., 170,87 (1920). Matsunaga, Cenlr. Bnkl. Parasilenk., I Abt. Orig., 82, 311 (1918). Messerschmidt, Zentr. Biorhem. Biophys., 19, 219 (1917). NGmec and K b s , Biochem. Z.,114, 12 (1921). Richtet and Rraumann, Compt. rend., 188, 1198 (1929). Rose and Bodansky, J . Bioi. Chem., 44, 99 (1920). Rose and Meunier, Bull. assocn. bldves inst. sup. fnmentations Gand, 28, 239 (1927). Rosenblatt and March, Biochem. Z., 170, 344 (1928). Schweizer, Mitt. Lebensm. I l y p . , 10, 261 (1919). Zerner and Hamburger. Biochcm. 2..222. 315 (1921).
