STUDIES IN YEAST METABOLISM. I. BY AK BALLS AND JB BROWN.

34 Pages·2003·1.39 MB·English

STUDIES IN YEAST METABOLISM. I.
BY A. K. BALLS AND J. B. BROWN.
(From the Henry Phipps Institute and the Department of Pharmacology,
University of Pennsylvania, Philadelphia.)
(Received for publication, July 18, 1924.)
I.
INTRODUCTION.
The experiments described in this paper were undertaken for
the purpose of investigating some of the more conspicuous changes
associated with the active and rapid growth of baker’s yeast
(Saccharomyces cerevisice). Such a growth is essentially aerobic,
and necessitates experimental conditions which fulfill this re-
quirement, and accordingly differ from the conditions suitable
for the study of alcoholic fermentation.
The methods employed permitted the construction of time-
concentration curves of the most important constituents of a
yeast mash which, as far as laboratory facilities would permit,
represented the conditions known empirically in the yeast industry
to produce the largest crops.
Lately there has been a great change in the methods of pro-
ducing commercial yeast. Sugar, nitrogen, and phosphoric acid,
formerly derived from grain, are now more cheaply obtained
from molasses, ammonium salts, and phosphates. By very
vigorous aeration of the liquid, far larger quantities of yeast are
produced per unit of raw material. These “ammonia-molasses”
processes have now been generally adopted by yeast manu-
facturers both in this country and Europe.1
1 A typical example of the modern ammonia-molasses process is shown
in United States Patent, No. 1, 449, 127, to Nilsson and Harrison. It em-
bodies the first technically successful method for obtaining these immense
yeast crops, the production of which in the last few years has practically
revolutionized the industry.
789
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Studies in Yeast Metabolism. I
II.
General Method.
The yeast was grown on a medium composed of:
gm. per 1.
Beetmolasses.............................................. 48
Ammonium dihydrogen phosphate.. . . . . . . . . . . . . . . . . . . 1.2
“ sulfate”........................................ 1.6
In preparation for an experiment the necessary weight of molasses to
furnish 30 liters of mash was taken, diluted fourfold with water, heated to
70-75°C. with an excess of powdered chalk, filtered on a Btichner funnel,
and washed. This solution, together with the salts, the yeast used for
seeding the culture, and sufficient water to make 30 liters, were placed in a
cylindrical stoneware crock which served as a “fermenter.” The crock was
equipped with heating, cooling, and aerating coils, the last sufficient to
produce a very vigorous aeration.
The molasses used in our several experiments was from the same stock
supply. It contained 50 per cent sucrose, only a trace of reducing sugar,
and 2 per cent nitrogen. This was present as betaines and partially hy-
,drolyzed proteins; none was precipitable by trichloroacetic acid.
The yeast used to seed the fermenter was treated as usual with dilute
sulfuric acid. As soon as the yeast was added, necessary manipulations
were made as rapidly as possible, and samples of the liquid withdrawn to
represent the zero hour in at most 2 minutes. The mashes were allowed to
grow with constant attention from 20 to 24 hours, and similar samples with-
drawn at stated intervals, usually hourly, during the run. These samples
were used in determinations of yeast content, sugars, ammonia, amino and
total nitrogen, alcohol, specific gravity, and total non-volatile solid matter.
Sugars and ammonia were determined at once, the rest in the course of 6
or 8 hours.
The specific gravity and pH of the whole mash were also determined,
and the appearance of the yeast was observed frequently under the micro-
scope. Should the presence of foreign yeasts or bacteria become promi-
nent, the run was naturally discarded. This happened once (in the case
of Mash B).
III.
Analytical Methods.
En general, two samples were removed, one for the deter-
mination of the various soluble constituents, the other for the
measurement of the yeast crop and pH. The first sample was
centrifuged at once, the clear liquid removed and cooled to 2-3”.
2 In the first experiment (Mash A) ammonia was used as a nitrogen
source.
A. K. Balls and J. B. Brown
Total solids were determined by evaporating 10 cc. of this liquid in a
shallow porcelain dish and drying to constant weight at 110”.
Reducing sugars were determined, using 10 or 25 cc. samples which, with-
out clarification, were treated with 50 cc. of Fehling’s solution according
to Munson and Walker (l), the copper being finally determined as directed
by Low (I). The results are expressed in grams of invert sugar per liter of
mash.
Sucrose was taken as the difference between the reducing sugar and the
total sugar determined in the same manner after inversion. The inversion
was performed by adding 5 cc. of 38 to39 per cent hydrochloric acid solution
to 50 cc. of the liquid freed from yeast, then setting aside for 24 hours at
20-25”. The liquid was then neutralized to phenolphthalein, diluted to
100 cc., and aliquots were taken equivalent to the sample used for reducing
sugars. Several determinations were tried in which the sugar was inverted
in the presence of the yeast. The results were not appreciably different.
The tables of Munson and Walker are used in the computation of the
sugars from the copper oxide, and the reducing sugar is computed as in the
absence of sucrose. Sucrose is also expressed as grams per liter of invert
sugar. Since the inversion is very rapid, the error appears to be small.
Beet molasses contains only traces of sugars other than sucrose, and no
great error has been introduced by not considering these. The probable
error in the determinations of both sugars is, in our opinion, less than 1
per cent.
Alcohol was determined by distilling the mash from a small quantity of
precipitated chalk until a few drops less than half of the volume had been
distilled over. This was then diluted to precisely half the original sample,
and the specific gravity determined with a 25 cc. gravity bottle at tempera-
tures within half a degree of 15.6”. A correction for this deviation from the
standard temperature was made by the customary formula
1 - D’
D = D’ * d 0.00014 + r)
where D and D’ are the apparent and the observed specific gravities, and
d = t - 15.6, where t is the temperature of the determination in degrees
Centigrade.
Ammonia and amino nitrogens were determined on the centrifuged mash
liquor. For the ammonia determination 10 cc. of the solution were placed
in a Folin aeration tube, made alkaline with potassium carbonate, and a
current of acid-washed air was passed through and into 10 cc. of 0.1 N acid.
The back titration was made with Congo red.
The total of amino and ammonia nitrogens was estimated by Sorenson’s
formaldehyde titration. 50 cc. of the liquid were diluted to about 150 cc.,
and neutralized by alkali until giving a distinct pink tinge with phenol-
phthalein. 50 cc. of approximately 10 per cent formaldehyde, also neutral-
ized to phenolphthalein, were added, and the solution was brought back by
standard alkali to the same end-point as before.
Studies in Yeast Metabolism. I
Nitrogen in yeast and total nitrogen of the mash were determined by the
ordinary Kjeldahl method, using copper sulfate as a catalyst. For the
former, dried yeast crop determinations served as samples; for the latter,
50 cc. portions of the mash, taken under precautions to prevent settling of
the yeast during measurement of the sample. The determination of nitro-
gen in the yeast crop samples was not very satisfactory, especially at the
beginning of the experiments, where the yeast crops were small. The total
mash nitrogen was found to be more consistent and so replaced the former
determination in our later runs.
The determination of pH on the whole mash we found could be made by
adopting the collodion membrane method of Dale and Evans (2), because
the mash liquid contains considerable buffer. A few cubic centimeters of
the mash were dialyzed against an equal volume of distilled water for 5 to
10 minutes. Equilibrium is obtained long before much of the coloring mat-
ter has diffused through. By using brom-thymol blue or methyl red, as the
case might require, the pH of the dialysate could be determined at once.
Readings were made only to tenths of the pH index, and there was no
attempt to prevent the escape of carbon dioxide, but the amount present
at any time in the thoroughly aerated liquid was found to be very small.
Method of Determining Yeast Crops.-It seemed to us that data based
upon the weight increase of the yeast would be best since the expression
of our other results must necessarily be in terms of weight. This method,
we are aware, has met with very little favor in the past. Pasteur, however,
used it, and Bokorny (3) in recent times has used it again. Counting the
cells was out of the question, since increase in the size of the individual cells
is seen under the microscope to be the most potent factor in the yeast in-
crease during the latter part of an aerated run. It is to be noted that the
weight probably increases as some simple function of the cube of the cell
diameter.
After many trials a procedure was adopted consisting of filtering a known
volume, usually 100 cc. of mash, through a Gooch crucible, the mat of which
must be composed of loose long fibred asbestos, preferably in a thick layer.
Very little suction is used, otherwise the filter will clog. The cake of yeast
so formed is then washed twice with 25 cc. of water and twice with 25 cc. of
alcohol. The alcohol washing is necessary to remove traces of the oil
originally used as a foam destroyer in the fermenter. The dead cells are
dried at 110”. After drying, the material is very hygroscopic, and the
crucible was placed before coolin g in a tared weighing bottle of just the
proper size to contain it.
Tests on the method as outlined, employing water suspensions
of known amounts of yeast, gave very satisfactory results, which
were practically independent of the degree of dilution of the
yeast. The probable error seems to be about f 2 per cent.
Since the liquids in which our yeast was grown were well buffered,
A. K. Balls and J. B. Brown 793
and no very great change in pH was allowed to take place, nothing
was done to regulate the acidity of the samples used for deter-
mining yeast crops. If the method were to find general use,
samples before filtering would naturally have to be adjusted to
nearly the same pH by appropriate buffering. The method has
been carefully checked by Professor A. W. Hixson at Columbia
University and found to give good results in the hands of his
students.
EXPERIMENTAL.
Seven consecutive experiments of the type outlined were run.
One was rejected because of infection. It was not possible to
make all the determinations desired each time, and particularly
after the first run, some modifications of the procedure were
made. The tabulated data should make this clear. It should
be noted, however, that the spent liquor (beer) from Run D was
used instead of water in diluting Run F. This experiment was
meant to show whether any growth-inhibiting substances had
been excreted into the medium by the yeast during its growth in
Run D, but no such influence was demonstrated.
On the other hand, Run E was interrupted at the 8th hour by
the addition of more molasses (and ammonium salts) in order
to show the effect of replenishing the food supply at the point
where the original sugar had disappeared. In this way a marked
increase was caused in the growth rate.
The measurement of carbon dioxide production was made on
Run H, only 6 liters of medium being used in a covered vessel,
the carbon dioxide being absorbed by potassium hydroxide from
the issuing air current. Due to the resistance of the apparatus,
only about half the usual aeration was obtained.
G z F
yeast. m yeast. anisms. ms.
of the of Kah n org organis
V. Data. TABLE I. Data on Mash A. gn. per 1. position : olasses...................................................... 48 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 - - Additions during Microscopic appearance experiment. Qm. Qrn. perl. perl. 1.09 0.14 1.01‘ 1.014 29.0 N added as 1.014 29.5 1.04 0.12 1.01‘ NHIOH. 30.0 0.03 Budding well; trace 1.05 0.14 1.01‘ 29.0 0.03 1.01~ Chains with no foreig1.011 29.0 0.07 1.01 29.0 l.Ol( 0.10 1.006 29.0 0.07 1.00: Clusters; no foreign 1.004 29.0 0.03 l.OO( l.OO! 1.003 28.8 No foreign organisms. 1.004 29.0 0.93 0.17 1.00t 1.004 28.7 l.OO( 1.004 28.8 0.80 0.13 1.oot 1.004 28.7 l.OO( I&SO+,
comgarmO4. Qm. perl. 0.15 0.18 0.43 0.49
u$
Original BeetsNH4H 2 .8 4 ~. p”2. &2. 0 0.32 0.59 0.73 1.13 0 1.47 1.6 2.141 6.0 3.721 7.0 4.66 7.0 5.04 6.0 5.27 5.64 5.0 5.84 6.02
i D 2 2 ‘B s 2 _- Qm. PW0 1.62 4.92 7.50 8.98 5.26 4.94 1.40 1.00 0.53 0.48 0.53
m a 2 0 8 8 8 8 7 6 5 6 8 9 1
6.6.5.5.5.5.5.5.5.5.5.5.6.
i E F - hrs. 0 1 2 3 4 5 6 7 8 9 10 11 12
ms. nisms.
ganis m. orga
ms. or Kah
Pairs; no foreign organis Fewer buds; no foreign Very few buds; trace of No foreign organisms. “ “ “ Cells discrete; no foreign No foreign organisms. “ “ ‘I “ “ “ I‘ ‘I ‘I
HzSO4 &SO4
28.8 28.7 28.6 28.5 28.7 29.0 29.0 28.5 28.5 28.5 28.7 28.6 28.6
h.
1.005 1.004 1.004 1.005 1.004 1.005 1.005 1.005 1.005 1.005 1.005 1.005 1.004 of mas
1.007 1.007 1.007 1.007 1.008 1.008 1.007 1.008 1.007 1.006 1.007 1.007 1.007 liter
0.51 0.80 0.12 0.47 0.69 0.10 0.55 0.69 0.07 0.70 0.06 0.58 0.69 0.07 0.54 0.68 0.04 0.65 0.05 0.58 0.63 0.05 in the yeast per hours.
6.09 5.0 6.23 6.28 6.68 6.76 1.0 6.97 7.38 7.44 7.61 8.32 8.12 7.90 8.14 as grams of N 4%. and 6.17
6.3 0.51 6.4 6.3 6.3 0.48 6.2 6.2 6.2 0.52 6.1 6.2 6.1 6.4 6.2 9.44 5.6 Expressed Respectively
13 14 15 16 17 18 19 20 21 22 23 24 25 - * t
m. “ ‘I
the yeast. no KahI‘ “ good.
C. gm. per 1. 48 s................. 1.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 Additions during Microscopic appearance of experiment “C. 29 NaHC03 Trace of Kahm in stock. “ “ “ “ “ 29 “ “ “ “ “ 28 27 1.5 Apparently pure culture; ‘I “ “ 28 “ “ ‘I 27 28 Few small cocci; otherwise 27 No change. “ “ 28 “ I‘ 29 “ “ 29
II. Mash k-2 .f: k. ,oI .-a g% * .01: .Ol! .01: .Ol( . oat . oat .oot . OO! . OO! OO! ,001
TABLE on Oli .OlE ,012 ,011 . OOE . 00: OOE . OOE ooi 00s ,008
Data Original composition: Molasses................................................... NH&PO d.................................... (NH4)2S04................................................................... - - - i i d s g . . t L B e i i 2 2 d 2 .': a % d ,a2 d ;: .s z % a g 82 g . 2 “0 -2 -2 2 8 “-2 ,” E$ .- E I $p .- s g Ee z z * z 4 Fr 4 4 2 -- - -- - gm. gm. sm. gm. gm. gm. gm. 9erz. ,er 1. per 1 perl. per1 $. 2Ci per 1 per I 1 ).O! 0 !t 5.3< 4 1.5s I( I.55 ..51 1.550.4; 1.0: )( 2.55 ( I.79 1.530.4t ..4( i( j.0: 3.8: L7E 11 !.510.14 1.500.4i 1.t .3$ )( ).OL 0.35 4.5i c i. 19 0.31 4.c I.41 1 .2c I( ,.;o: 1.14 7.43 :E i.530.34 6.: i 2: j.260.23 .oc I.04 i( 3.5E 5 E i. 35 0.53 5.: ). 19 0.15 1.8E ).OL i( 3.51 5.32 !E i.650.57 5.: 5 I! ). 16 0.11 1.8: j.01 I( 3.5: 7 ‘.210.72 3.c 1 1.130.OE 1.7s I.04 C 3.3 1.61 7 I. 42 0.72 12 ).120.OE I. 71 ;I( I. 01 3.54 i ‘. 89 0.65 ).100.09 1.75 !‘C 1.0: 3.54 L76 ie i.040.70 1.080.06 1.72 L !- -
$ 9 z .: s 2 2 -~ ie?i, 5.8 5.9 4.05 5.29.99 5.08 83 5.60.73 5.8’0.71 6.0 0.43 6.50.42 6.00.43 6.20.43 6.30.43
- E” F - hrs 0 2 4 6 8 10 12 14 16 18 20 -
ure. ‘I ‘I “ “ “ “ ‘I “ “ “ ‘I
ult
c
pure “ “ “ “ “ “ I‘ ‘I “ “ “
m. per 1. 48 1.2 1.6 Ipparently “ “ “ “ ‘I “ “ “ “ “ ‘<
g
...... ...... ...... C03 1.0 0.5 WI 0.2
TABLE III. Data on Mash D. Original composition: Molasses............................................................ NHdH2POa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (NH&S04.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - i ?a c :: sm. Qm. Qm. Qm. Qm. Qm. "C. per 1. per 1. per 2. perz. perl. pe71. 1 4.17 1.45 0.55 0.48 0.071.0141.014 28 NaH2.77 0.55 0.49 0.061.0141.014 29 8.74 1.27 0.49 0.43 0.061.0131.012 28 3.E 0. II 4.11 0.45 0.37 0.29 0.081.0111.008 28 1.12 0.21 8.21 8.C * 3.36 0.9E 0.31 0.26 0.051.0081.005 27 L5.50 4.9; 0.4: 0.89 1o.c 0.54 0.7i 0.25 0.17 0.081.0061.005 29 6.t 5.3( 0.4 0.55 0.8: 0.21 0.15 0.061.0061.004 30 &6.: 15.54 5.58 0.41 0.20 0.13 0.071.0051.004 29 0.5: 0.7: 4.c 5.9; 0.56 0.78 0.20 0.16 0.041.0061.005 30 14.02 6. li 0.5: 0.58 0.72 0.18 0.12 0.061.008 28 6.4: 0.5’ 0.50 0.7E 0.18 0.11 0.071.0081.006 29 14.05 6.7f 0.5: 0.17 0.11 0.061.0071.007 29 0.5‘ 0.51 0.7i 6.8( 0.14 0.09 0.05 30 13.43 7.4f 0.5‘ 0.61 0.48
Qm. per 1. 0.13 6.82 2.69 0.95 8.93 3.00’ 0.80 0.28 0.42 0.44 0.33 0.43 0.37 urs.
o
h
m a 6.0 5.7 5.3 5.0 4.2 5.0 5.0 6.4 6.6 6.0 6.2 6.2 6.4 6.4 6.2 6f
..- d Ei ‘G hrs. 0 2 4 5 6 7 8 9 10 12 13 14 16 18 20 22 24 *At
gm. per 1. 48 1.2 1.6 - Stock looks pure. I Little change. Many buds; no infection. Yeast looked healthy and grew well, but contained a trace of Kahm throughout. are made here on account
................ ................ ................ Additions during experiment. NaHC03 0.5 2.0 QWL. per 2. 36 0.65 0.89 NaHCOs 1.0 No corrections
TABLE IV. Data on Mash E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . $, .‘1 > c b0.c ii P- a’0 * “C. gm. cm. Qm. petl. per 1. PCTl. 0.44 1.01, 27 0.11 1.41 0.41 1.01, 0.10 29 0.38 1.01. 26 0.08 1.001 30 0.07 0.26 1.00, 0.04 1.37 0.21 29 w volume made 8 to 8; hrs. Molasses ............................ NHdHzPO., ... .( ..................... (NH&S04 .......................... - 0.13 28 27 1.012 29 0.18 29 30 0.08 served in the liquids analyzed.
Original composition: Molasses. ................. NHaHzPO , ................ (NH&S04 ................ !Tm. Qm. !Jm. Qm. w: !7m. lm. per 1. per 1. PWZ. pPr 1. per I. per 1. 0.55 0 5.6 0.9( 24.3( 42.21 0.54 1.39 0.51 2 6.0 9.6f 0.94 22.3( 0.46 4 5.6 12.0! 16.7( 32.3, 2.06 0.34 6 4.8 4.9: 4.08 6.1: 0.24 8 4.8 0.7: 2.3( 15.81 5.15 0.93 Additions calculated to necc. Old volume......................... 4,790 New “ . . . . . . . . . . . . . . . . . . . . . . . . . 5,360 ;$I 5.61 4.33’1 17.331 40.401 .63 1.251 1 10; 0.89 I I 7.32 11 6.8 12 6.5 12; 6.8 0.74 14; 6.8 16 6.5 0.67 1.36 * Concentrations from 8; hrs. on are those obof the change in volume.