Effect of Growth Conditions on Yield and Vitamin Bl of Yeast P. L. PAVCEK, W. H. PETERSON, AND C. A. ELVEHJEM University of Wisconsin, Madison, Wis.
R
EPORTS in the literature disagree as to the amount of the B vitamins in yeast
An apparatus is described for growing in batches of 1 to 4 kg. A study
a t a pH of 3.5 (sulfuric acid was used to adjust the acidity). After cooling, a sterilized soluwas made of various factors that affect and the factors responsible for tion of potassium pyrophostheir presence. Although the the growth and vitamin B1 content of phate and ferric pyrophosphate a commercially used strain of bakers' was added, and any slight preweight of evidence favors the belief that b r e w e r s ' yeast is yeast. Three types of media were used. cipitate was dissolved by adding richer than bakers' yeast in the Based on reducing sugar the of dry sterilized 40 per cent sulfuric B1 vitamin, the reports are far acid. The sterilized urea and yeast was about 30 per cent for the glufrom c o n s i s t e n t . Many of ammonium chloride solution was these discrepancies are probably cose-salts medium' 35 for the molassesthen added, and the pR adjusted salts, and 40 for the grain wort. Other to4.4 with sterilized 40 per cent related to differences in species factors affecting yield were investigated. s o d i u m hydroxide. By this of Yeast, conditions of growth, The vitamin B~ contents of yeasts procedure caramelization of the and method of biological assay. Because of the multiple characglucose was avoided and pre@'Own in the media as determined by ter of what was called "vitamin cipitation of the salts was reB," it is impossible to decide chick assay, Were: d u c o s e ~ 3 1. Per duced to a minimum. The ratio of carbon to nitrogen in the whether the B1 component or gram of dry yeast, molasses 5, and grain 10. Destruction of vitamin B1 in the above medium was 25 to 1. By some other dietary factor was being measured in many of the grain wort did not decrease either the increasing the amount of urea early experiments. and ammonium chloride, media yield Or B1potency Of the yeast' OmitI n this study the chick was were p r e p a r e d in which the used as the test animal, and ting aeration of the Wain medium reratios of carbon to nitrogen - were duced the yield of yeast nine-tenths but 18 to 1 and 14 to 1. the method is that r e c e ' n t l y mmnmended by Elvehjem (4) increased the Br potency threefold. The A molasses-salts medium of as giving satisfactory results. effect of several other factors on the B~ the following composition was In growing the yeast an effort used: crude b e e t m o l a s s e s , 'Ontent Of yeast was 'Om150 grams; calcium phosphate, was made to follow such procedures as have been generally mercial Yeasts ranged from 5 1. Per 1 gram; concentrated sulfuric gram of dry yeast (bakers') to 40 I. U. acid, 2 cc.; urea, 2 grams; adopted in commercial prac(brewers'). a m m o n i u m monohydrogen tice. As shown by the multiplicity of patents, commercial phosphate, 1 gram; tap water to make 1 liter. The m o l a s s e s , p r a c t i c e undoubtedly varies greatly, but there seems to be rather general agreement mlcium phosphate, and sulfuric acid were dissolved together and autoclaved in 5-gallon Pyrex bottles and, after the mixregarding the desirability of using a large inoculum, an acid reaction, abundant aeration, and a low concentration of ture was cool, the sterilized urea and ammonium dihydrogen phosphate solution was added. The pH of the medium sugar in the medium. The results indicate that under similar conditions of growth the experimental yeasts had about was adjusted to 4.4. the same vitamin B1 content as the commercial product. The grain wort medium was made from a mixture of corn, Variations from the regular procedure have been introduced malt, and sprouts in the ratio of 4 to 3 to 3. Two kilograms of finely ground whole corn were cooked at 15 pounds per with a view to obtaining information regarding the factors square inch pressure with about 8 liters of water for one-half that are responsible for the BI content of yeast. hour. At the same time 1.5 kg. of malt and 1.5 kg. of sprouts were mixed with 20 liters of water containing 5 cc. of conMedia for Growth of Yeast centrated hydrochloric acid and allowed to soak for one-half hour a t 25" C. About half of the autoclaved corn was then Three types of media were used-glucose-salts, molassesadded, the temperature of the mash was raised to 50" C. salts, and grain wort. and held there for one-half hour, and then the remainder of The composition of the glucose-salts was as follows: the corn and 5 cc. of concentrated hydrochloric acid were commercial glucose (cerelose), 70 grams; sodium chloride, added. The temperature was further increased to 62" C. 2 grams; magnesium sulfate, 2 grams; calcium chloride, 0.4 and held a t this point until the starch-iodine test was negagram; potassium pyrophosphate, 2 grams; ferric pyrophostive. The mash was filtered through burlap bags, the residue phate, 0.1 gram; copper sulfate, trace; urea (commercial), was washed once with warm water, and the filtrate was 2.4 grams; ammonium chloride, 0.15 gram; tap water to diluted to 30 liters and autoclaved in 5-gallon Pyrex bottles make 1 liter. Solutions of glucose, sodium chloride, maga t 15 pounds pressure for 45 minutes. The final wort had nesium sulfate, calcium chloride, and copper sulfate were made up in &liter batches in 5-gallon Pyrex bottles and a sugar concentration of 4.5 to 5.0 per cent reducing sugar calculated as glucose and a pH of 4.4. autoclaved for 1 hour a t 15 pounds per square inch pressure
u-
u-
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Apparatus for Growth of Yeast The fermentation apparatus is shown in Figure 1: Fermenter A consists of a 100-liter metal can with a closely fitting cover through which the medium, E , can be introduced aseptically. Aerator C is a Carborundum cylinder by means of which a fine stream of air can be passed through the fermentin solution. The fermenter is surrounded by an electrically heate3 water thermostat which is maintained at a temperature of 33" C . (the inside of the fermenter is thus kept at a temperature of 30" C.). Sterile water, F , is added from time to time to replace that lost by aeration. To start a fermentation, 200 grams of fresh pressed stock yeast (Saccharomyces cerevisiae grown in grain medium) were suspended in about 4 liters of sterile water and were introduced into the fermenter. The medium was then allowed to siphon into the yeast suspension at such a rate that never more than 0.5 per cent sugar (calculated as glucose) remained unfermented. T,he growing yeast was strongly aerated by means of compressed air. The amount of air passed through the medium was approximately 100 liters per minute during the day, but at night, t o avoid the possibility of loss by foaming, the amount of air was reduced to about half this figure. Periodic samplings were made in order to regulate the inflow of medium and, if necessary, to adjust the pH of the medium during the run. Microscopic examinations were made at frequent intervals, and, when budding of the yeast was well underway, the rate of addition of the nutrients, as well as the volume of air, was increased to a maximum. The temperature in all runs was maintained at 30" C. unless otherwise indicated. If the medium became contaminated with foreign organisms during the fermentation, the run was discarded. A t the end of the fermentation, the fermented medium was transferred to a cold room (4" C.) to allow settling of the yeast. After 20 to 30 hours, the supernatant liquid was siphoned off and the yeast pressed in canvas bags or centrifu ed off in a Sharples centiifuge. The yeast was dried at 60" for 20 hours and ground to pass a 30-40 mesh sieve.
culty of obtaining a sample that is representative of the whole volume. Another possibility is the accumulation of ethyl alcohol in one period and subsequent utilization of this alcohol for production of yeast in another period. Balls and Brown ( I ) showed that alcohol was utilized for the growth of yeast cells and the patent literature also reports increased yields of yeast from addition of alcohol to the fermenting medium.
Yeast Growth in Molasses-Salts and Grain Wort The rate of yeast formation and sugar utilization is given in Figure 3. The fermentation in the molasses medium was considerably slower than that in the synthetic medium, since for the utilization of 2300 grams of sugar, 92 hours were required, whereas in the glucose-salts medium 3000 grams were utilized in approximately 75 hours. The slow rate of yeast formation was probably due to growth-retarding substances in the crude molasses. The lag phase was considerably increased over that in the glucose-salts run. Sugar utilization and yeast formation increased together but did not bear a constant ratio to one another. With grain wort as the nutrient solution the lag phase was absent and the growth of yeast closely followed the sugar utilization (Figure 3). The time of fermentation is comparable to that of the glucose run since it required 45 hours for the utilization of 1400 grams of glucose.
Yields of Yeast under Various Growth Conditions
8
Analysis Sugar was determined by the macrosugar method of Stiles, Peterson, and Fred (9). The concentration of sugar in the medium during a run was followed roughly with a Balling saccharimeter. Yeast growth was determined by centrifuging 50-CC. samples at 2500 r. p. m. for 10 minutes, decanting the clear liquid, and drying the yeast deposit for 8 hours at 100" C. Nitrogen determinations were made on the medium, yeast, and beer by the Kjeldahl-Gunning method with copper selenide as the catalyst. Hydrogenion values were obtained colorimetrically on the glucose-salts runs and electrometrically (quinhydrone electrode) on the molasses and grain media.
Yeast Growth in Glucose-Salts In Figure 2 are given curves showing the rate of' yeast growth and the sugar utilization for a typical run (202) which was inoculated in the regular manner with 200 grams of fresh stock yeast and for another run (132) which was inoculated with 200 grams of the pressed yeast that had been grown on glucose-salts medium for one generation. Comparison of runs 132 and 202 shows that the lag phase was somewhat longer when the fresh stock yeast was used for seeding. Apparently the yeast requires some time to become accustomed to growing in the glucose-salts medium, but after this period reinoculations into fresh media are followed by a shorter lag phase. Although there is a general correspondence between sugar consumption and yeast production, the ratio between the two shows great variations at different times during the fermentation. The percentage of sugar converted into yeast varied from under 20 to over 40 for different periods. Part of t h i s v a r i a t i o n may be only apparent, because of the diffi-
537
Approximately forty batches of yeast have been grown in the glucose-salts medium , five in the molasses-salts medium, and four in the grain medium. Data for eleven of the glucose runs, two of the molasses runs, and all of the grain runs are recorded in Table I. The growth of yeast on the glucosesalts medium has been studied more extensively than any of the others because the medium was easily prepared, was comDosed of definite chemical compounds, and was not complicated by o r g a n i c s u b s t a n c e s of unknown character such as are found in the molasses and grain media. Several of the factors investigated are as follows:
EFFECTOF SIZE OF INOCULUM. Consideration of runs 107 and 1%) in Table I shows that use of a small inoculum increased the time of fermentation 100 per cent and
c
0
FIGURE 1. APPARATUSFOR GROWINQ YEAST
I
I
II
I I
A. B.
100-Iiter fermenter Thermostat
water)
INDUSTRIAL AND ENGINEERING CHEMISTRY
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ratio, 16 to 1) the nitrogen in the beer was 12 per cent of the total introduced, Yeast Inorease Xitroand, since the percentage i n this yeast Calod. gen was normal (6.40), it was assumed that Dry on in weight - -alucose Yeast sufficient nitrogen was present for good Grams % % cell development. In all later work a carbon-nitrogen ratio of 16 to 1 was 274 18.2 6.00 350 22.3 4.04 used. 350 22.0 4.23 EFFECTOF "GROWTH"FAc T O R S . 710 23.5 6.68 Bios. Nielsen (6), in small-scale experi962 30.6 4.40 1010 33.0 4.86 , ments and with small inoculum, found 888 27.1 7.00 that improved yields of yeast could be 1095 34.4 7.23 obtained when the synthetic media con1050 34.3 6 40 737 22.7 6:85 ' tained 1 per cent of wort. In run 121, 600 19.7 6.00 3 per cent of the medium was replaced by a grain wort. Comparison of runs 1300 35.0 7.07 120 and 121 indicates that the addition 769 33.6 7.88 of grain wort had no appreciable effect on the yield of yeast. Apparently 402 46.0 6.28 there was enough bios present in the 476 37.2 5.56 437 38.6 4.74 200 grams of inoculum to ensure normal 58 3.5 6.98 growth. As pointed out earlier, similar growth curves (132 and 202, Figure 2) were obtained when stock yeast and yeast grown on synthetic medium were used as inocula, indicating that bios was not a limiting factor for growth in these experiments. Vitamin B1. The work of Williams (11) seems to indicate that vitamin B1 is required by some strains of yeast for complete nutrition. From the present experiments it would appear either that there is enough B1 present in the inoculum or that the strain of yeast employed can synthesize enough B1 for its needs. In run 133, 1800 international units (I. U.) of B1 (Ohdake) were introduced into a synthetic medium, and 200 grams of a yeast which had been grown for one generation on a glucosesalts medium were used for the inoculum. The final yield of yeast was somewhat higher, 33.0 as compared with 30.6 per cent for the control, but it is doubtful whether this difference is significant. Awtoclaved Brewers' Yeast. Five hundred grams of dried brewers' yeast were dispersed in water and autoclaved 3 hours before being added to the medium. The same inoculum was used as in run 133. After deducting the added brewers'
TABLEI. YIELDSOF YEASTUNDER VARIOUSCONDITIONS OF GROWTH Run No.
DescriDtion of Run
Volume Liters Series I, 107 Regular 40 g. inoculum'" 28 120 Regular: 200 g. inoculumQ 27 121 Regular plus 3% wort 28.5 129 on0 P 43 132 30' C.. seast from 131 &a in47 133 50 134 46 135 52 54 202 206 56 207 51
-.
101 218 113 115 116 201 Q
. '
Regular Regular
Time Ratio of C/N Fermenin tation DH Medium Hours Glucose-Salts Medium 73 4 . 2 , 24/1 26/1 35 4.2 39 4 . 2 25/1 28/1 110 4.1
Sugar Utilized Grams 1510 1572 1592 3024
82 74 77
4.3 4.3 4.3
25/1 25/1 14/1
3145 3065 3294
77 74 75 79
4.1 4.0 4.3 6.2
18/1 l6/1 18/1 18/1
3185 3075 3255 3035
Series 11, Molasses-Salts Medium 59 143 4.7 3717 2296 56 92 4.4
"8;
Series 111, Grain Wort Medium Autoclaved medium 28 55 4.5 l5/l 4.1 20/1 50 Regular 28 71 4.3 17/1 Autoclaved medium 3.8 l6/l No aeration 41 66 Equivalent to 10 and 50 grams, reapectively, of dry yeast.
872 1270 1170 1546
decreased the yield of yeast by approximately 20 per cent. All runs listed except 107 were started with the larger inoculum (200 grams fresh yeast equivalent to about 50 grams on the dry basis). In the earlier runs where 1500 to 1600 grams of glucose were fermented, the maximum conversion of glucose to yeast was 22.3 per cent. In all the later work the fermentations were continued until approximately 3000 grams of glucose had been introduced; as a result the final conversion of glucose to yeast was as high as 34.3 per cent (run 202). EFFECT OF AQE OF INOCULUM. Run 202 was inoculated with 200 grams of fresh stock yeast whereas in run 206 the inoculum was taken from the same batch of stock yeast after it had been stored a t 4" C. for one month. The old inoculum gave about one-third less yeast than the fresh inoculum. Because of this marked decrease in yield, inoculating yeast older than 2 weeks was not employed in later fermentations. EFFECT OF TEMPERATURE. Run 129 was made at 20' C., and comparison with a 30" C. run (132 or 202) shows that the lower temperature increased the time of fermentation and also decreased the yield of yeast. EFFECT OF PH. It has been generally accepted that a p H of 4.0 to 4.3 is the optimum for growth of yeast. Taxner (10) states that the optimum initial pH is 3.4 to 3.9 and that the pH later attained by the yeast is not as important as this initial pH. These figures are for a brewers' yeast grown on a synthetic medium containing asparagine. Schwartz and Kautzmann (8) found that the yield of yeast on a malt wort was independent of the pH within the range 3.5 to 6.5. I n run 207 the pH of the fermenting medium was maintained a t 6.2 by the addition of sterile sodium hydroxide solution. The fermentation time was normal but the yield of yeast was considerably diminished. EFFECT OF CARBON-NITROGEN RATIO. I n the early experiments the ratio of carbon to nitrogen in the medium was 25 to 1; with this ratio the concentration of nitrogen appeared to be a limiting factor since in some cases (run 132) only 5 per cent of the total nitrogen introduced remained in the beer. The low percentage of nitrogen in the yeast in this case also indicated nitrogen insufficiency. When the nitrogen concentration was increased by making the carbonnitrogen ratio 14 to 1, the nitrogen in the yeast was greatly increased but neither the yield nor the vitamin B1 content of the yeast was improved. In run 202 (carbon-nitrogen
I
TIME
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HOURS
OF YEAST AND SUGARUTILIZATION IN FIGURE2. GROWTH GLUCOSE-SALTS MEDIUM
INDUSTRIAL AND ENGINEERING CHEMISTRY
MAY, 1937
539
procedure described later. It is evident that the lack of B1 had no appreciable effect on the final yield of yeast. Nonaeration of the medium during growth of the yeast resulted in a very marked decrease in yield; only 3.5 per cent of the sugar consumed was converted into new yeast cells (run 201).
Assay of Yeast Table I1 gives the data for the assays for vitamin B1 of the batches of yeast grown as described and also of a few samples of commercial bakers' yeast. The effect of various factors on the vitamin B1 content will be considered in the following paragraphs. TIME
0
- HOURS
Effect of Medium
OF YEASTAND SUQAR UTILIZATION IN MOLASSESFIGURE 3. GROWTH SALTSAND GRAINWORTMEDIA
yeast, thehet yleld was increased to 34.4per cent as compared with 30.6 for control run 132. The presence of some growthpromoting substance in the brewers' yeast may be responsible, but more experiments will be necessary before a definite conclusion can be drawn. Of
r"
I
x
Yeast
On
Molasses-Salts Medium
Batches of yeast that were produced under identical conditions except for differences in the composition of the medium are designated as regular runs. A comparison of these runs shows that yeast grown in grain wort (e. g., run 115) contained more vitamin B1 than that grown in molasses-salts (101); the latter, in turn, was richer in the vitamin than yeast grown in synthetic medium (120). The amount of yeast in the ration required to protect the chick against polyneuritis was roughly 2 per cent of grainwort yeast, 4 per cent of molasses-salts yeast, and 6 per cent of glucose-salts yeast. If the potencies of the three types of yeast are expressed in international units, the
The data for two typical runs are given in Table I. As mentioned before, the time of fermentation was considerably lengthened over that for the glucose-salts runs, The yields of ;east calculated on glucose fermented e are higher than the average for the glucose-salts media, but this is to be exON VARIOUS MEDIA TABLE 11. ASSAYOF YEASTGROWN pected. Claassen ( 2 ) pointed out that No. of No. of Chicks in Chicks molasses contains 12 to 15 per cent Group .Suf- Av. Weight per nonsugar carbon which can be used D e v e l o p vivine. Chick after: Proing after- c for yeast growth. On a factory scale tective Level Poly5 4 5 Yeast Description of Run or Level Fed neuritis Weeks weeks weeks Yeast NO. Claassen obtained yields of yeast as % Grams Grams % high as 39.5 per cent based on the Series I, Glucose-Salts Medium sugar content of the molasses. Balls 107 40 g. inoculum 6 4 None >6 and Brown (1) obtained yields of yeast 120 Regular 5 2 2 io5 iio 6 None 3 98 110 6 approximating 32 per cent of the glucose 121 Regular run plus 3% wort 4 3 . . . . . . None 5 None 40 98 5 in the molasses. The high nitrogen iii 129 Temp., 20° C. 4 2 2 90 in the final yeast as c o m p a r e d w i t h 6 None 2b 118 161 4 to 5 132 Regular run 6 None 4 135 174 6 nitrogen in yeast grown in other media, 5 4 None 133 1800 I. U. of B1 added None .. .. .. .. .. .. 134 Double nitrogen 6 3 is explained by the low ratio of carbon 8 8 None 4 96 134 to nitrogen in the m o l a s s e s - s a l t s 135 500 g. brewers' yeast added 2 1 3 126 172 4 None 3 140 201 2 medium. None 207 pH, 6.2 4 4 ......
Bi per Gram D r y Yeast I . u. n g s u g a r , , expressed as glucose, were obtained with grain media. The presence of considerable nonsugar carbon is probably responsible for this apparently high yield when expressed on a sugar basis. In connection with some experiments to be discussed later, the effect on yeast growth of the d e s t r u c t i o n of vitamin B1 in the grain medium should be observed. Runs 115 and 116 were made on identical media except that in 115 the wort contained the usual amount of B1 extractable from grain, whereas, in 116 the B1 was destroyed before inoculation by an autoclaving
101 218
Regular Regular
15
Regular
115
Regular
116
Autoclaved medium
201 6
118
No aeration Regular
Series 11, Molasses-Salts Medium 4 None 4 95 4 4 None 6 None 4 149 4 1 3 172 6 None 4 190
103 . . . . 205 ..
Series 111, Grain Wort Medium 1 4 None 2 None 4O 133 None 1 4 2 None 4a 150 None '/z 4 1 None 4 163 1 4 None 2 None 4 128
Brewers', A
222A Brewers', B 222B Bakers', A 208C Bakers', B 208D Bakers' Vienna 219 a
b
Bakers', C Test terminated after 4 weeks.
Only two chicks in group.
230 250
. . . . ... ..
. . . . ... .. . . . . 233 .. ......
Series IV. Commercial Yeasta 3 125 None ;/a 4 137 None 1 1 3 iii 4 None 1'/2 1 3 130 2 4 None 137 21 / 4 2 2 l'/n 91 4 2 None 2 2 'h 142 1 4 None 115 3 3 2 100 4 4 None
... ...
162
150 158
...
129 187 180
...
123
...
200 141 128
5
4 to5 4 to5
4 to5 4 to 5
2
10
2
10
'/1
27
2
10
I/a
40
1 1/2
13
2
10
l'/r to 2 10 t o 13 1 3 to 4
20 6 to 7
540
INDUSTRIAL AND ENGINEERING CHEMISTRY
figures are 10, 5, and 3, respectively. These values have been obtained repeatedly on batches of yeast grown a t different times and also assayed a t different times (compare, for example, runs 120 and 132, runs 15, 101, and 218, or runs 6 and 115). The 4 per cent level in run 101 is probably a borderline case, since the low weight of the chicks a t the end of the 5-week period is indicative of poor growth. In run 218 no level between 4 and 6 per cent was fed, but the good growth (230 grams) a t the end of 5 weeks seems to indicate that 5 per cent of this yeast would have been sufficient. The high vitamin B1 content of grain wort yeast indicates that, when Bf was available, the yeast obtained its vitamin by abstraction from the medium rather than by synthesis. Although there is definite evidence of B1 synthesis ifi the case of the glucose-salts batches, the yeast apparently preferred absorption to synthesis of the compound. Scheunert and Schieblich (7) found that very little of the B1 in the wort was left in the beer and suggested that yeast abstracts the vitamin almost quantitatively from the medium. If the vitamin B1 of the medium was destroyed by heating, the yeast proved its ability to regenerate the vitamin. Runs 115 and 116 were grown in identical media except that in the case of run 116 the B1 of the wort was destroyed by autoclaving for 3 hours a t pH 6.0. The assay showed that both batches of yeast prevented polyneuritis a t the same minimum level (2 per cent) and hence had the same B1 content. These results are in agreement with the work of Eijkman, van Hoogenhuijze, and Derks (3) who obtained qualitative data showing the regeneration of vitamin B1 by bakers' yeast in rice polish extract medium and by brewers' yeast in a beer wort. That the vitamin of the wort used here was actually destroyed was established by assaying it. The autoclaved wort was concentrated in a vacuum a t 28" C., evaporated on part of the ration, and fed a t a level equivalent to 4 per cent of yeast (about 250 cc. of wort per 100 grams of ration). Although this amount was twice the protective level required in case of the yeast, all of the chicks developed polyneuritis. Since molasses contains vitamin B1 (5), the yeast probably absorbs the vitamin from the medium as it does from grain wort and hence shows a higher vitamin B1 content than when grown in the synthetic medium. Synthesis of vitamin B1is shown in the case of the glucosesalts yeast. The 50 grams (dry weight) of stock yeast in. troduced as the inoculum accounted for 50 x 10 = 500 I. U, of B1. In a typical run, 1000 grams of dry yeast were obtained which assayed 3.3 I. U. B1per gram or 3.3 X 1000 = 3300 I. U. of B1 in the final yeast. This sixfold increase in vitamin B1 is due apparently to the ability of the yeast cell to synthesize this factor. In a grain medium, when considerable B1 is present, this synthesis is masked by collection (absorption) of the vitamin from the medium. In series IV, Table 11, are grouped assays of six commercial yeasts (from three different companies) grown on various media according to information furnished by these companies. Sample 118 represents a brewers' yeast grown on a beer wort medium a t 8" to 10" C. The high content of B1 is explained by the richness of the original medium in the vitamin and by the fact that the small yeast growth concentrated the vitamin from the medium in a small crop of yeast. Sample 222A is a beer yeast grown on a cereal extract medium, probably with aeration since the B1 content is much lower than that of regular brewers' yeast. Sample 222B is a bakers' yeast grown in a cereal extract plus molasses medium. This medium was apparently quite rich in vitamin B1, and the yeast grown upon it was about as potent as yeast 208C. The latter was grown in grain wort made from corn, malt, and sprouts and had about the same potency as the labora-
VOL. 29, NO. 5
tory yeasts produced in the same type of medium. The bakers' Vienna yeast was grown without aeration on a rich grain medium and, from the experiments (run 201) on nonaeration of grain wort, a high BI content was to be expected. Sample 219 was grown in a cereal extract molasses medium and was rather low in potency as compared with the other commercial bakers' yeasts.
Effect of Other Factors Other factors affecting the B1 content of the yeast were studied in the case of the glucose-salts medium because, as mentioned previously, it could be made easily and contained only compounds of known composition. The effects of several of the factors investigated on the B1 content of the final yeast are discussed below. Run 107, Table 11, was produced from a small inoculum40 grams of wet yeast equivalent to 10 grams of dry yeast. Comparison of this run with later runs (120 and 132) in which 200 grams of wet yeast were used, shows that the larger inoculum yields a final yeast somewhat more potent in vitamin B1. The difference in the amount of vitamin in the yeast is probably due to improved growth of the yeast rather than to the introduction of vitamin B1 in the inoculum, since in run 132 the inoculating yeast had been grown for one generation on a glucose-salts medium, thus "diluting" the vitamin originally present. In run 121 the regular glucose-salts medium was supplemented by 3 per cent grain wort. The data (Table 11) show that the yeast was slightly more potent in the B1 factor than a control with no grain wort supplement (run 120). The addition of 1800 I. U. of vitamin B1 (Ohdake) to the medium (run 133) was sufficient to decrease the protective level from the usual 6 to a 4 per cent if the added vitamin had been taken up completely by the growing yeast. The fact that a 5 per cent level of this yeast was not enough to prevent polyneuritis indicates that very little, if any, of the introduced vitamin was assimilated. The effect of adding autoclaved brewers' yeast was determined in run 135. Since the B1 of the brewers' yeast was destroyed by the autoclaving process, the increase in B1 in the yeast may be attributed to the ability of the growing yeast to resynthesize the vitamin from its decomposition products. This ability of the yeast to regenerate the vitamin was referred to previously in connection with experiments on autoclaved grain wort. The brewers' yeast contained originally 500 X 40 = 20,000 I. U. of B1 and the final yeast assayed 10 I. U. per gram. Since 1145 grams were obtained, 1145 X 10 = 11,450 I. U. of B1 were present in the yeast crop or about 57 per cent of that contained in the brewers' yeast. Apparently the B1 content of the yeast was not determined by the amount of B1 precursor in the medium. In the several runs (e. g., 129) which were made a t 20" instead of the usual 30" C., a slight increase in Bl content of the yeast was observed. In run 129 the 4 per cent level may be considered as a borderline case because only two of the four chicks survived the 5-week period. I n none of the numerous regular (30" C.) runs did 4 per cent of the yeast afford even slight protection. The indication is that a t the lower temperature synthesis of B1 by the yeast cell was favored. An increase of the pH to 6.2 (run 207) did not improve the synthesis of B1 since 4 per cent of yeast in the ration did not prevent the development of polyneuritis in the usual length of time. Higher levels of this yeast were not fed.
Acknowledgment The authors are indebted to H. R. Bird and R. L. Hutchinson for assistance in the early part of the work and to
MAY, 1937
INDUSTRIAL AND ENGINEERING CHEMISTRY
P. Hubanks for his part in the assay of the samples. C . H. ~~i~~~~of the Red Star yeastand products company gave many suggestions with a view to producing the yeast under conditions approaching those of commercial practice. The work was supported in part by a grant from the funds of the Graduate School, University of Wisconsin.
Literature Cited Balls, A. K., and Brown, 3. B., J . Biol. Chem., 62,789(1924). Claassen, H., 2.Ver.deut. Zucker.-lnd.,84,713(1934). Eijkman, C., van Hoogenhuijze, C. J. C., and Derks. T. J. G., J . Biol. Chem., 50, 311 (1922).
54 1
(4) Elvehjem, C. A,, J . Asaoc. 0.Uicial Agr. Chem., 18,354 (1935). (5) Nelson, v. E.,Heller, v. G.,and Fulmer, E. I., IND.ENQ. CHEM., 17, 199 (1925). (6) Nielsen, N., Compt. Tend. trav. lab. CaThheTg, 20, No. 1 (1934). (7) Scheunert, A,, and Schieblich, M., Z . Vitaminforach., 4 , 294 (1935). (8) Schwartz, W., and Kautzmann, R., Arch. Mikrohiol., 2, 537 (1931). (9) Stiles, H. R . , Peterson, W. H., and Fred, E. B., J . Bact., 12, 427 (1926). (10) Taxner,'C., $.Inat. Brewing, 41, 27 (1935). (11) Williams, R. J., and Saunders, D. H., Biochem. J., 28, 1887 (1934). RECEIVED October 9, 1936.
NEW CERAMIC TABLE TOPS
I
N 1928 when plans for
The commonly used materials are lacking in one or more properties
furnishing the new buildthat the ideal laboratory table top should possess. A recapitulation ing of Mellon Institute of the required properties is presented. A program of research resultwere being made, the selection ing in the formulation and manufacture of a new ceramic material of material for the laboratory table tops was discussed with is described. I n addition to the properties usually possessed by ceramic manufacturers of laboratory bodies, it is important that this material should have a low coefficient furniture. Since no material of expansion which imparts to it high resistance to thermal shock. appeared to be completely The body is made impervious to liquids by impregnation with bitumisatisfactory, it was suggested nous materials and subsequent special heat treatment by means of that through research a new product might be devised which the original pore spaces are filled with carbon in the form of coke. khat would possess all the adThe properties of the new material are described and other applicavantages of the commonly tions are considered. used materials and none of their shortcomings. After careful c o n s i d e r a t i o n the study of the problem was STUART M. PHELPS AND EDWARD E. MARBAKER begun in the institute as a Mellon Institute of Industrial Research, Pittsburgh, Pa. fellowship project under the s u p e r v i s i o n of the senior author. Research and deordinary wood top, it is often chosen with the idea that, if velopmont carried out during the ensuing five years finally anything serious should happen, it can be easily and economiresulted in the small-scale production stage a t the institute. cally replaced. If such replacement is not made when needed, During the past two years developmental work has been the utility of the top is diminished and its appearance is continued and actual commercial production has recently rendered less attractive. been attained Wood tops of the highest grade, made from maple or birch, Discussion of Table-Top Materials finished to resist acids, alkalies, and solvents, and constructed in thickness from l b / s to l3/4 inches, are almost as expensive The most extensively used material for laboratory table as tops made from soapstone or specially manufactured tops is wood (8),usually maple or birch, qrotected by fillers, materials. varnishes, lacquers ( 6 ) , rubber composltions, or special It is not unusual to install a wood top of ordinary construcmastic coatings, or made acid-resistant by chemical treatment. tion and then to cover it with some material to provide The principal advantages of wood are its relatively low cost protection against conditions peculiar to its use. For and its ease of procurement and application. Manufacturers example, in laboratories where special work involving the use of laboratory furniture use improved methods of construction of concentrated sulfuric acid is carried on, sheet lead is used. to prevent warping, but these procedures add to the cost. Such a protected top is acidproof but is not completely Wood is not fireproof, and many a table top has been ruined fireproof because of the heat conductivity and low melting by charring when a flame has been burned too long under a point (327" C., or 620.6' F.) of the lead. Ordinarily 6- or hot plate placed upon it, or when a lighted burner has been 8-pound sheet lead is used, and the joints are burned so that overturned. Resistance to the absorption. of liquids and to the cost is relatively high. Moreover it is never perfectly the attack of acids and alkalies, in which respects wood itself smooth and so does not present an attractive appearance. leaves much to be desired, depends mostly upon the properNickel ( 3 ) and stainless steel are being satisfactorily used as ties of the protective coating. Wood tops require considercoverings for wood tops; although both metals add much to able care if they are to be kept in good condition, and the the appearance of a laboratory, they are expensive. finish must be replaced from time to time so that this mainWood tops are sometimes protected by asbestos in the tenance expense must be added to the cost, Because of the form of sheet or board to decrease the fire hazard, but this low initial investment involved in the installation of an
