A study of the effects of caffeine upon the growth and metabolism of yeast

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A STUDY OF THE EFFECTS OF CAFFEINE UPON THE GROWTH AND METABOLISM OF YEAST

A THESIS Presented to The Faculty of the Graduate School University of Southern California

In Partial Fulfillment of the Requirements for the Degree Master of Science /

by Harold Pennington Vind

UMI Number: EP41312

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'so i/T ' / C f

This thesis, written by

'bf* i .Kamid_ _P jenriingtqel..Y.ln.d.........

JI

under the guidance of h ..ls.. Faculty Committee, and approved by a ll its members, has been presented to and accepted by the Council on Graduate Study and Research in p a rtia l f u l f i ll­ ment of the requirements fo r the degree of

..... Mas.ter....of...Scienc.e______________

1.950.....

Date

Faculty Committee

/// t

V ~

/*■ Chairman

IjS**

TABLE OF CONTENTS CHAPTER I. II.

PACE

AN INTRODUCTION TO THE P R O B L E M............

1

A REVIEW OF THE LITERATURE................ .

2

Effect of caffeine on yeast. . . . . . .

2

Related studies

........................

k

..........................

5

METHODS OF ANALYSIS U S E D ..................

7

Measurement of growth r a t e ..............

7

Measurement of sugar consumption ........

10

Measurement of alcohol production

....

12

Introduction ..........................

12

The p r o c e d u r e .......................

13

Experm e n t a l ..........................

1^

Summary III.

IV.

THE EFFECT OF CAFFEINE UPON THE GROWTH RATE OF YEAST

.

19

Preliminary preparations . ..............

20

Nutrient salt m i x t u r e ................

20

Bios Concentrate.....................

20

Ammonium phosphate solution

..........

20

Basal Medium A .........................

21

Basal Medium B .........................

21

E x p e r m e n t a l ............................

21

Procedures

..........................

21

iii CHAPTER

PAGE I n o c u l a .........................

2k

Effect of caffeine upon pH. . . . . .

.

R e s u l t s .........................

25 25

Deviations from averages .............. V.

40

THE EFFECT OF CAFFEINE UPON THEFERMENTATION RATE OF Y E A S T ..................... Preliminary preparations

k2

..............

k2

Nitrogen-free m e d i u m ............... . .

VI.

k2

Yeast suspension.................

kj

E x p e r m e n t a l .......................

kj

P r o c e d u r e .......................

kj

R e s u l t s .........................

kk

A COMPARISON OF THE EFFECT OFCAFFEINE UPON GLUCOSE CONSUMPTION BY YEAST IN AIR AND UNDER O I L ......................... Procedure

VII.

DISCUSSION

50 . .

50

........................

53

Growth experiments................... . .

53

Fermentation experiments ................

55

Comparison of caffeine-effeet upon growth and fermentation rates. . . . .

57

Comparison of the caffeine-effeet in air and under o i l ....................

60

iv CHAPTER

PAGE Comparison to the effects of caffeine

VIII.

upon frog m u s c l e ....................

62

S U M M A R Y ..................................

6k

B I B L I O G R A P H Y.........

66

A P P E N D I X ........................................

69

LIST OF TABLES

TABLE I.

PAGE Effect of Standing Time Upon the Titration in Bichromate Alcohol Determinations . . . .

II.

Milli-equivalents of Dichromate Reduced per Millileter of Standard Alcohol Solution

III.

IV.

16

. .

17

Respective Experiments ....................

23

Media and Sizes of Inocula Employed in the

Increase in Yeast Concentration with Time at Various Caffeine Concentrations, Experiment One

V.

...............

70

Increase in Yeast Concentration with Time at Various Caffeine Concentrations, Experiment Two

VI.

..........................

71

Increase in Yeast Concentration with Time at Various Caffeine Concentrations, Experiment F o u r ..........................

VII.

72

Increase in Yeast Concentration with Time at Various Caffeine Concentrations, Experiment F i v e ..........................

VIII.

7^

Rates of Growth of Yeast at Various Caffeine Concentrations as Estimated from the Times Required to Attain Half Maximum Growth, Experiment One

....................

26

vi TABLE IX.

PAGE Bates of Growth of Yeast at Various Caffeine Concentrations as Estimated from the Times Required to Attain Half Maximum Growth, Experiment T w o ...........

X.

27

Rates of Growth of Yeast at Various Caffeine Concentrations as Calculated "from the Growth Attained at Age 13 Hours, Experiment Three

XI.

. . . . .

. . . . .

28

Rates of Growth of Yeast at Various Caffeine Concentrations as Estimated from the Times Required to Attain Half Maximum Growth, Experiment F o u r ..........................

XII.

30

Rates of Growth of Yeast at Various Caffeine Concentrations as Estimated from the Times Required to Attain Half Maximum Growth, Experiment Five

XIII.

........................

The Effect of Caffeine Upon the Consumption of Glucose by Yeast,

XIV.

Experiment Six

....

k?

The Effect of Caffeine Upon the Consumption of Glucose by Yeast,

XV.

31

Experiment Seven

...

**8

A Comparison of Alcohol Produced to Glucose Consumed by Yeast in Media Containing Caffeine, Experiment Seven ................

^9

vil TABLE XVI.

PAGE A Comparison of the Effect of Caffeine Upon Glucose Consumption by Yeast in Air and Under Gil Aerobic Series ..........................

51

Anaerobic Series

52

......................

LIST OF FIGURES FIGURE

PAGE

1. Standard Curve for

the Conversion of Klett

Colorimeter Readings to Yeast Concentration 2. Standard Curve for

. .

9

the Conversion of ml. of

0.1 N Sodium Thiosulfate Solution to mg. of Glucose 3.

. . . . . . . . . . . . ..........

Yeast Concentration Versus Time, Caffeine Concentration Constant

k.

11

. . ..................

33

Yeast Concentration Versus Caffeine Concentration, Time Constant

.........

5.

Growth Rate Versus

6.

Fermentation Rate Versus Log Caffeine Concentration

Log Caffeine Concentration

35 . .

..............................

39

k6

CHAPTER I

AH INTRODUCTION TO THE PROBLEM Experiments designed to elucidate the mode of action of caffeine are of considerable practical importance since caffeine is used in medicine, and since caffeine-containing beverages are used almost universally. The literature dealing with the physiological effects of caffeine upon various cells, tissues, organs, and organ­ isms is very extensive.

In spite of the vast amount of work

that has been reported, there is as yet no specific enzyme or biochemical process which can be Identified as a locus of caffeine action.

It is the purpose of this thesis to pre­

sent data bearing on this question. It has seemed possible that caffeine might exert its effects by discoupling energy-yielding from energy-requiring processes.

This question has been approached by comparing

the relative effects of caffeine upon growth and metabolism of yeast cells.

CHAPTER II

A REVIEW GF THE LITERATURE Reviews on special aspects of caffeine action, par­ ticularly those pertaining to Its effect on mammalian nerve and muscle, have been undertaken.

However, a comprehensive

discussion of metabolic effects of caffeine has not appeared in recent literature.

The present discussion of the liter­

ature is largely limited to studies pertaining to the effect of caffeine upon yeast metabolism. Effect of caffeine on yeast.

Newberg and Sandberg

(1921) stated that living yeast cells are not stimulated by caffeine, but they presented no data to substantiate their statement.

They did, however, present data to show that

several other alloxuric compounds did stimulate the fermen­ tation of glucose by yeast.

The reported stimulations were

not great and would appear to be within the possible range of error or chance variation. Bellisia and Aanda (1929) reported that the carbon dioxide evolution of yeast suspension was stimulated by low concentrations of caffeine and was retarded by high concen­ trations of caffeine.

These workers made thirty-five deter­

minations of carbon dioxide evolution at each of five caffeine concentrations and thirty-five determinations

without caffeine*

3 Although each determination was conducted

upon identical weights of freshly supplied baiter' s yeast and all other conditions were identical, they were not all conducted on the same batch of yeast.

Because equipment

permitted only two determinations at a given time, they were forced to use different batches of yeast cells each day so that all of their work might be upon freshly pre­ pared yeast suspensions.

The very great deviations in

values obtained for the rate of carbon dioxide evolution at a given caffeine concentration and the subsequent overlap­ ping of data somewhat decrease the importance of their work. Amati and Sgarzi (1935) reported that caffeine at a concentration of 20 to JO mg. per ml. had a retarding effect upon the alcoholic fermentation of molasses by yeast, but that at a concentration of 5 mg. per ml. caffeine had a stimulating effect upon the fermentation of molasses. Enders and Wienlnger (193?) presented excellent data on the toxicity of alkaloids to yeast.

They concluded that

caffeine retarded the rate of fermentation to a greater extent than the rate of multiplication.

It is possible,

however, to interpret their data differently.

They cul­

tured a given strain of yeast in a series of flasks con­ taining glucose media And various concentrations of caffeine.

A small initial inoculum was used.

At various

times during the growth of the cultures, turbidity measure-

k ments and sugar determinations were made.

Extrapolating

from their analytical results, they calculated that a caffeine concentration of from 1.1 to 1.2 mg. per ml. in­ hibited glucose consumption by 25 per cent, and that a con­ centration of from 2.2 to 2.5 mg. per ml. inhibited growth by 25 per cent.

Had their data been calculated in terms of

glucose consumed per yeast cell instead of per flask, how­ ever, the inhibition of fermentation by a given concentra­ tion of caffeine would not have been greater than the inhi­ bition of growth. Related studies.

Certain other studies, while not

pertaining directly to yeast, have a bearing upon the investigation.

These studies suggested the hypotheses that

caffeine might exert its effect by discoupling energyyielding from energy-requiring processes. Saslow (1937) conducted extensive studies on the effect of caffeine upon the heat production of frog muscle. His observations strengthened the hypothesis of Meyerhoff

(1921) and Hartree (1920) that "The action of caffeine upon muscle is merely to release, slowly and continuously, the chemical processes, anaerobic or oxidative, normally induced suddenly and discontlnuously by stimulation." Some of Saslow*s experimental findings, which he ex­ plained on the basis of the above hypothesis, include the

5 following:

1*

Frog sartorii in Ringer solution containing

caffeine showed large increases in resting heat production hoth in an atmosphere of oxygen and in an atmosphere of nitrogen.

2.

On stimulation the poisoned muscle developed

greater twitch tension than the uneaffelnized muscle both in oxygen and in nitrogen.

3*

Caffeinized muscle produced

but little “delayed” heat after a contraction.

For con­

traction, normal muscle utilized the energy from the delayed (recovery) heat production.

In the case of the caf­

feinized muscle, utilization of the energy from the increased resting heat production replaced the delayed heat closer to the process of contraction,

k.

Caffeine-treated muscles

have an R. Q. of 1.00 for the resting heat production as compared to O.83 for the untreated muscle. Cheney (19^5) observed similarities between the ac­ tion of caffeine and sodium cyanide on the oxygen consump­ tion and cell division of fertilized sea urchin eggs.

Thus,

he suggested that caffeine may, like cyanide, act upon the cytochrome oxidase enzyme.

He found cell division to cease

when oxygen uptake is decreased by fifty per cent or more by either inhibitor. Summary.

In the following statements an attempt is

made to summarize the results of previous work upon the effects of caffeine on yeast.

6

In low concentrations caffeine has been stated to stimulate the evolution of carbon dioxide by suspensions of yeast, and to stimulate the alcoholic fermentation of molasses by beer yeast.

Other workers have challenged the

statement that yeast are stimulated by caffeine.

The data

bearing upon the stimulatory effects of caffeine upon yeast is not conclusive. On the other hand there is conclusive data regarding the inhibitory effects of high concentrations of caffeine upon the growth rate and metabolism of yeast.

However, the

data are primarily qualitative rather than quantitative. The general trend of Increasing inhibition with increasing concentration has been established, but a quantitative expression relating inhibition to concentration has not. A comparison of the effect of caffeine upon cell division of yeast to the effect of caffeine upon the carbo­ hydrate metabolism of yeast has been attempted.

In the

experiment, however, the two processes were not separated. Therefore, that which has been stated to be the effect of caffeine upon the fermentation rate is actually the com­ bined effect of caffeine upon both the fermentation rate and the growth rate.

CHAPTER III

METHODS OF ANALYSIS USED In these experiments it was essential to measure the growth rate of yeast, the rate of sugar consumption by yeast, and the rate of alcohol production* I*

MEASUREMENT OF GROWTH RATE

Thorne (19^6), Pulley and Greaves (19^6), and others have successfully employed turbidity to measure the growth rate of yeast.

Because of its simplicity, we chose the

turbidimetrle method.

The cells were grown in tubes which

would fit directly into a Klett colorimeter.

Consequently,

it was not necessary to transfer or sample the growing cultures. Gargille* sample tubes (screw cap vials of 10 ml* capacity) were found to fit nicely into the colorimeter. Although the tubes varied slightly in diameter, a prelimin­ ary check upon the readings obtained with a standard solu­ tion gave consistent enough results to disregard these variations.

Only a few of the tubes varied sufficiently in

diameter to be unusable.

To calibrate the tubes an arbl-

* R. P. Cargille, Sample Storage Sets, Model A-SC, 118 Liberty Street, New York 6, N. Y.

8 trary color standard consisting of 25 ml. of washable blue ink diluted to one liter with distilled water was used. The tubes were also calibrated with distilled water only. The average corrected reading was 282 Klett units. average diviation from 282 was

Klett units.

highest reading was 292; the lowest, 268.

The

The

Seventy-nine

per cent of the tubes gave readings of 282 plus or minus 5> ninety-four per cent, plus or minus 8.

The readings indi­

cated that, with the exception of six tubes, the tubes were of nearly constant diameter. A yeast calibration curve was prepared.

One gm. of

Flelschmann's yeast cake was suspended in a 5 per cent glu­ cose solution.

The suspension was centrifuged and the

supernatent decanted.

The yeast cells were resuspended in

sufficient 5 pe** cent glucose solution to give a total vol­ ume of 100 ml.

From this suspension various dilutions of

yeast in 5 P©** cent glucose solution were prepared.

The

turbidities of the various dilutions were determined with the Klett colorimeter.

A blue filter was used and the

readings were made in one of the sample tubes already de­ scribed.

Results of the determinations are represented

graphically in figure 1 which was used to convert colori­ meter readings to yeast concentrations in the experiments which follow.

100

^5a5-jt--t5ncei FIGURE 1 STANDARD CURVE FOR THE CONVERSION OF KLETT COLORIMETER READINGS TO YEAST CONCENTRATION

II. MEASUREMENT OF SUGAR CONSUMPTION

Sugar consumption was followed by a modification of the Shaffer-Hartman (1921) macro sugar method.

The modifi­

cation consisted simply in dividing the quantities of all reagents used by five, and conducting the determinations in 50 ml. erlenmeyer flasks rather than the 250 ml. flasks. A 10 ml. micro-burette was used instead of the 50 ml. burette, and a 25 minute heating period in a boiling water bath was substituted for the nine minutes heating period over a bunsen burner. A curve was prepared relating sugar content to vol­ ume of 0.100 N sodium thiosulfate solution.

The curve was

prepared from values obtained from determinations run upon known quantities of C. P. glucose. graphically in figure 2.

The results are shown

Actual calculations were based on

an enlargement of figure 2 in which the horizontal axis represented mg. of copper reduced rather than ml. of sodium thiosulfate solution. Caffeine had no effect upon sugar determinations. As much as 5 ml* of a one per cent caffeine solution did not alter the position of the end point in a blank deter­ mination.

-CO----

it—

FIGURE 2 STANDARD CURVE FOR THE CONVERSION OF ML. OF 0.1 N SODIUM THIOSULFATE SOLUTION TO MG. OF GLUCOSE

III.

MEASUREMENT OF ALCOHOL PRODUCTION

Considerable care was exercised in choosing a suit­ able method for determining alcohol content.

Several

investigations were carried out in relation to the analyt­ ical procedures. Introduction.

Where large quantities of sample are

available and the necessary equipment is at hand physical procedures are generally used to estimate alcohol concen­ trations.

Estimations of alcohol content from specific

gravity or refractive index are examples of such procedures. These methods are especially suitable for the estimations of alcohol concentration when the alcohol concentration is high.

It was decided that a chemical method would be more

suitable for the needs of the investigations to be under­ taken. In recent years several micro-methods for determin­ ing alcohol in tissue and body fluids have been described which are based upon the oxidation of ethyl alcohol to acetic acid by dichromate with the subsequent determination of the unused dichromate.

Such methods differ only in the

manner in which the alcohol is separated and in the manner in which the unused dichromate is determined. Conway (19^7) reviewed a micro-dichromate method introduced by Widmark (1922).

In Widmark’s alcohol method

13 the alcohol passed by diffusion from a small cup, which was suspended from the stopper, into a solution of dichromate in sulphuric acid in the bottom of a 50 ail. flask.

The

unused dichromate, left after the oxidation of the alcohol, was determined iodometrically. The principle of the micro method of Widmark ap­ peared to be suitable for the needs of the investigations to be undertaken.

For greater accuracy, however, the

method was modified into what might be described as a semi­ micro method. The procedure.

Ten ml. of 0.5 N potassium dichromate

in 1:1 sulphuric acid and 10 ml. of distilled water were placed in the bottom of an iodine flask. taining alcohol was placed in a test tube.

The sample con­ The test tube

was Inserted into the iodine flask and was supported by the bottom and sides of the flask.

The top of the test

tube extended well above the surface of the dichromate solution.

A strip of filter paper placed Inside of the

test tube facilitated more rapid evaporation of the sample. The iodine flask was then sealed with a well greased stop­ per.

When the sample had completely evaporated from the

test tube, the test tube was removed with a pair of tweezers, and the dichromate reagent adhering to the out­ side surface was rinsed back into the flask with distilled

water.

14 The dichromate not used in the oxidation of alcohol

to acetic acid was then determined by an iodometric titra­ tion. In carrying out the titration 50 ml. of water were first added to dilute the dichromate reagent. KI solution was then added.

Ten per cent

The unused dichromate liberat­

ed an equivalent amount of iodine.

A sufficient excess of

the KI solution was added to render the liberated iodine soluble and thus reduce the loss of iodine as vapor from the solution.

The liberated iodine was titrated at once

with standard 0.1 N sodium thiosulfate solution.

A one per

cent soluble starch solution was used as an indicator. When fermentation liquors containing alcohol were tested one ml. of 0.1 H NaOH was added to the aliquot of the sample in the inserted test tube.

This was to prevent

errors from volatile organic acids and to stop fermentation. Excellent agreement was obtained with duplicate analysis. Again it was necessary that the strip of filter paper be completely dry before the dichromate reagent was titrated. Experimental.

An experiment was performed to ascer­

tain the time required for complete oxidation of the alcohol by the dichromate reagent.

In a series of six determina­

tions equal volumes of an alcohol and water solution were added directly to the dichromate reagent.

At various

15 intervals the contents of the flasks were titrated lodoraetrleally in the manner already described. are indicated in table I.

The results

It was concluded that the ensu­

ing reaction is completed in 15 minutes or less after the alcohol reaches the dichromate reagent.

No further reac­

tion was indicated even in an additional Zk hours. An experiment was performed to determine the quanti­ ties of dichromate reduced by various quantities of alcohol. Various volumes of 0.132 M ethyl alcohol solution were added directly to the dichromate reagent in a series of 10 determinations.

After 15 minutes the contents of the flasks

were titrated iodometrically. table II.

The results are indicated in

From table II it is evident that a straight line

relationship exists between the quantity of alcohol in the aliquot for a determination and the milli-equivalents of dichromate reduced by the alcohol.

The balanced equation

for the oxidation of ethyl alcohol to acetic acid by dichromate would indicate that a one molar ethyl alcohol solution would have a normality of four.

On this basis

the normality of the alcohol standard was 0.528 which compares quite favorably with the value of 0 .520, the average value for the milli-equivalents of dichromate reduced per ml. of alcohol standard (omitting the value obtained on the one ml. sample).

Inasmuch as the so-

TABLE I EFFECT OF STANDING TIME UPON THE TITRATION IN DICHROMATE ALCOHOL DETERMINATIONS Standing time

Titer

B l a n k ............................... ^9*3 nil.

Titrated immediately..............31.5 11 Titrated after 10 m i n u t e s ....... 2k, 0 11 Titrated after 30 m i n u t e s ....... 23,8 " Titrated after 1 h o u r ........... 23.9 H Titrated after 2 h o u r s ......... 23.8 n Titrated after 2k h o u r s ......... 23.9 rt

17

TABLE II MILLI-EQTJIVALENTS OF DICHROMATE REDUCED PER MILLILITER OF STANDARD ALCOHOL SOLUTION Vol. of alcohol standard 0 ml.

Titer of 0.101 N

Na2®2^3 49.8 ml .

0

ti

49.7

1

ii

2

Blank minus titer

Milli-equiv. dichromate per ml. ale.

0.0

11

0.0

44.8

II

4.95

0.500

ii

39.6

II

10.15

0.515

3

ti

34.35

It

15.40

0.520

4

ti

29.3

II

20.45

0.518

24.0

II

25.75

0.520

18.7

H

31.05

0.523

13.7

It

36.05

0.520

5

ii

6

it

7

ti

8

n

8.5

II

41.25

0.521

9

N

3.^

If

46.35

0.520

18 called 95 per cent alcohol, which was used in preparing the standard, is usually slightly less than the stated 95 per cent, it is probable that the difference between 0,520 and 0,528 was experimental error.

It was concluded that the

reaction was stoichiometric rather than empirical, and that determinations could be calculated directly from the titra­ tions without the need of tables or graphs.

Four equiva­

lents of dichromate are reduced by one mole of alcohol* A number of trials were made to test the efficiency of the diffusion process.

If the titrations were not made

until the sample had completely evaporated from the insert­ ed test tube and the filter paper therein had become dry, the results were always identical to controls in which the same quantities of alcohol solution were added directly to the dichromate reagent. The time required for complete evaporation and diffusion depended entirely upon the shape of the Inserted test tube, size of the strip of filter paper, temperature, volume of sample used, and such considerations.

In some

cases the strip of filter paper did not beeome dry until after 72 hours.

One flask was prepared by suspending a

shortened test tube from the stopper to the flask by means of a glass rod.

In this flask evaporation of a one ml.

sample was complete in 12 hours at room temperature.

CHAPTER IV

THE EFFECT OF CAFFEINE UPON THE GROWTH RATE OF YEAST A series of five experiments were performed to determine the effect of caffeine, under various conditions, upon the rates of growth of yeast.

The caffeine concentra­

tions, the quantities of yeast in the inocula, and the compositions of the media were varied. The experiments were to determine what caffeine con­ centrations, if any, stimulated yeast growth.

The experi­

ments were also to provide a means of comparing the effects of caffeine upon yeast growth in a synthetic medium to its effects upon yeast growth in a medium containing yeast extract.

The experiments were further to provide a means

of comparing the effects of caffeine upon yeast growth when large quantities of inoculating yeast are used to its effects when small quantities are used.

Finally, the experi­

ments were to provide data which could later be used to compare the effects of caffeine upon growth rates to its effect upon the fermentation rates of yeast. The strain of yeast studied was a Saccharomyces cerevisiae obtained from the department of Bacteriology at the University of Southern California.

It was designated

as the G-. M. Strain to denote its origin.

It was isolated

20

at the General Mills Laboratories, Minneapolis, Minnesota, where it is used for vitamin assay. I.

It is a “bottom yeast.*

PRELIMINARY PREPARATIONS

Nutrient salt mixture.

A nutrient salt mixture was

prepared containing *K) gm. of KHgPO^, 20 gm. of MgSOj^. ?H20 , 0.2 gm. of FeSO/j,.?H20 , a few crystals of ZnSO^, a few crys­ tals of MnSO^, and sufficient water to give a final volume of 250 ml.

In the preparation of media 12.5 nil. of the

nutrient salt mixture were used per liter of media. Bios concentrate.

A bios concentrate was prepared

containing 0.*K)10 gm. of inositol, 0.0099 gm. of calcium pantothenate, 0.0033 gm. of thiamine hydrochloride, 0.0053 gm. of pyridoxine hydrochloride, one ml. of a saline solu­ tion containing 30 micrograms of biotin per liter, and suf­ ficient distilled water to give a final volume of 100 ml. The bios concentrate thus prepared was sterilized in an autoclave for 15 minutes at 15 lbs. gauge pressure.

Five

ml. of the bios concentrate were used per liter of media. Ammonium phosphate solution.

A solution of 15 gms.

of ammonium phosphate in 100 ml. of distilled water was pre­ pared. media.

Two ml. of this solution were used per liter of

21

Basal Medium A.

A synthetic medium, designated

Basal Medium A, was prepared containing 320 gms. of anhyd­ rous glucose, 20 ml. of sterile bios solution, *K) ml. of 85 per cent lactic acid, 375 ml* of 1 N KOH solution, 50 ml. of the nutrient salt mixture, 8 ml. of the ammonium phos­ phate solution, and sufficient distilled water to give a final volume of k liters. Basal Medium B.

Basal Medium B consisted of Basal

Medium A to which 2.0 gms. of Dlfco yeast extract were added per liter. II. Procedures.

EXPERIMENTAL

Aside from minor differences in the

manner in which the various caffeine concentrations were prepared, the same procedure was employed in all five ex­ periments.

Yeast were grown in media to which varying

quantities of the free caffeine base were added.

At inter­

vals during the growth of the yeast the concentration of yeast in each culture was estimated from the turbidity of the culture. Only one culture was grown at each caffeine concen­ tration in the first experiment.

The second and third ex­

periments were run in triplicate; experiments four and five, in duplicate.

A total of some 160 cultures were grown in

22

the five experiments. The culture tubes were left open during growth in an attempt to standardize the quantity of oxygen available to each tube.

For the same reason an attempt was made to

standardize the shaking of the tubes previous to reading the turbidity.

Aside from the mixing, which was necessary

for the turbidity measurements, the tubes were stationary during growth and were incubated at 27^ C. In each experiment the concentrations of caffeine were varied.

The composition of the basal medium and of

the quantities of inoculating yeast were constant through­ out a single experiment but were varied from experiment to experiment.

The medium employed and the relative quantity

of yeast in the inoculum employed in the respective experi­ ment is designated in table III.

The figures in the column

headed "Relative Size of Inoculum" represent the concentra­ tions of yeast in mg. per ml. existing in each of the cul­ ture tubes of the respective experiment immediately after Inoculation (i.e., at age zero hour). In experiment three the basal medium was filtered because a slight cloudiness developed when the nutrient salt mixture was added.

The growth of yeast in the

filtered medium was decidedly slower than that in the media used in the first two experiments.

This characteris­

tic of the medium did not interfere in the experiment as it

23

TABLE III MEDIA AND SIZES OF INOCULA EMPLOYED IN THE RESPECTIVE EXPERIMENTS Experiment number

Relative size of inoculum

1

.004

A

2

4

A

3

41

A

4

75

B

5

0,75

B

Basal medium employed

zk was a consistent factor in all of the cultures of experi­ ment three, Inocula.

The inocula were developed from a slant on

100 ml, portions of medium of the same basal composition as that which was used in the experiment. for approximately ^8 hours at 27^ C.

They were incubated Prior to use, if

dilutions were required, they were adjusted to the appro­ priate yeast concentration by diluting with medium of the same composition.

For example, the culture tubes in

experiment five were Inoculated at the same time as the culture tubes in experiment four with a 1:100 dilution of the same Inoculum as that used in experiment four. In the first experiment 0.1 ml. serological pipettes were used to measure the inocula.

In the other experiments,

for greater accuracy, non-sterile, 1 ml., volumetric pip­ ettes were used.

Previous to the time of inculation, how­

ever, all preparations were autoclaved and the Inocula were prepared using sterile techniques.

In none of the experi­

ments was gross contamination encountered. In the first experiment before inoculation each of the culture tubes was filled with 6 ml. of the caffeine containing medium.

Dilutions of the caffeine concentra­

tions by the 0.1 ml. portions of inoculum were considered to be negligible.

In the other experiments before inocu-

25 lation each of the culture tubes were filled with 5 ml* of the caffeine containing medium.

Dilution of the caffeine

concentration by the 1 ml. portions of inoculum in these experiments was considerable.

Hence, each of the caffeine

concentrations was multiplied by five sixths before being entered in the tables. Effect of caffeine upon pH.

A solution of 2 gms. of

the free caffeine base in 100 ml. of distilled water had a pH of about 5-6 which was not very much above that of the media.

When 2 gms. of caffeine were added to 100 ml. of

the basal synthetic medium, the pH was changed from 4.5 to 4.7.

When less caffeine than 2 gms. per 100 ml. were used

the effect upon the pH was negligible.

Only in experiment

one was there an attempt to adjust the pH to a uniform value at the various caffeine concentrations.

In the other

experiments the maximum caffeine concentration was 1 gm. per 100 ml. and, therefore, the differences in pH due to the caffeine were considered to be negligible. Results.

The results are reported in detail in

tables IV through VII of the appendix and are partially summarized in tables VIII through XII of this chapter.

The

data of these tables will primarily be of interest only to those requiring detailed information. the results are presented graphically.

For greater clarity

26

TABLE VIII RATES OF GROWTH OF YEAST AT VARIOUS CAFFEINE CONCENTRATIONS AS ESTIMATED FROM THE TIMES REQUIRED TO ATTAIN HALF MAXIMUM GROWTH EXPERIMENT ONE Caffeine concentration in mg. per ml*

Time to attain growth of 225 mg. per 100 ml.

Relative growth rate compared to control as 100

20

Infinite

0

10

576 hr s.

11

5

425

n

14

1

78

tt

77

0.5

73

n

82

i-t • o

68

tt

88

0.05

6l

tt

99

0.01

63

it

95

0.001

54

h

111

0.0

6l

r

99

0.0

60

tt

100

27

TABLE IX RATES OF GROWTH OF YEAST AT VARIOUS CAFFEINE CONCENTRATIONS AS ESTIMATED FROM THE TIMES REQUIRED TO ATTAIN HALF MAXIMUM GROWTH EXPERIMENT TWO Caffeine concentration in mg. per ml.

Time to attain growth of 158 mg. per mOO ml.

Relative growth rate compared to control as 100

8.33

46 hrs.

22

4. 17

32

"

31

o. 833

14

"

71

0.417

13

11

77

O.O833

11

"

91

—1 H

as

91

O.OO833

ioi

"

95

O.OOO833

O H

a

100

0.000000

10

«

100

1

0.0417

TABLE X RATES OF GROWTH OF YEAST AT VARIOUS CAFFEINE CONCENTRATIONS AS CALCULATED FROM THE GROWTH ATTAINED AT AGE 13 HOURS EXPERIMENT THREE Caffeine concentration in mg. per ml*

Growth attained at age 13 hoars

Relative growth rate compared to control as 100

8.33

47

11

6.25

51

15

4. 17

62

31

2.08

82

52

0.833

129

86

0.625

134

89

0.417

137

90

0.208

146

95

0.0833

148

96

0.0625

148

96

0.0417

153

99

0.0208

158

101

O.OO833

158

101

0.00417

160

102

O.OOO833

156

100

0.000417

158

101

29 TABLE X (continued) RATES OF GROWTH OF YEAST AT VARIOUS CAFFEINE * CONCENTRATIONS AS CALCULATED FROM THE GROWTH ATTAINED AT AGE 13 HOURS EXPERIMENT THRIVE Caffeine concentration in mg. per ml.

Growth attained at age 13 hours

Relative growth rate compared to control as 100

O.OOOO833

156

100

0.0000000

156

100

NOTE: At age zero hour the yeast concen­ tration was 41 mg. per 100 ml. The growth at­ tained at age 13 hours is expressed in terms of mg. per 100 ml.

TABLE XI RATES OF GROWTH OF YEAST AT VARIOUS CAFFEINE CONCENTRATIONS AS ESTIMATED FROM THE TIMES REQUIRED TO ATTAIN HALF MAXIMUM GROWTH EXPERIMENT FOUR Caffeine concentration in mg* per ml.

Time to attain growth of 600 mg. per 100 ml.

Relative growth rate compared to control ae 100

8.33

72 hours

2k

3.87

28

II

61

1.80

22

II

77

0.833

18*

n

92

0.387

17 3 A 11 H 17*

96

96

0.0387

17 3 A H II 16*

103

0.0180

17*

H

97

0.00833

17*

II

97

0.00387

16 3/4 H

101

0.00180

16*

If

103

O.OOO833

16*

II

103

0.000387

16 3/k II

101

0.000180

16

II

106

0.0000833

16*

H

105

0.0000000

17

H

100

0.180 0.0833

99

31 TABLE XXI RATES OF GROWTH OF YEAST AT VARIOUS CAFFEINE CONCENTRATIONS AS ESTIMATED FROM THE TIMES REQUIRED TO ATTAIN HALF MAXIMUM GROWTH EXPERIMENT FIVE Caffeine concentration in mg. per ml. 8.33

Time to attain growth of AOO mg. per 100 ml. 130 hr s.

Relative growth rate compared to control ae 100 19

3-8?

67



37

1.80

hi



60

0.833

31

"

79

0.387

26f *

93

0.180

25i *

97

O.0833

25

*

98

O .0387

25i *

97

0.0180

25

*

98

0.00833

25

*

98

0.00387

25

*

98

0.00180

25

»

98

O.OOO833

25



98

0.000387

25

*

98

0.000180

2h§- “

100

O.OOOO833

25

®

98

0,0000000

2h| 11

100

32 Three different methods of graphical representation were used.

In essence the three methods were to plot the

yeast concentration against time for a given caffeine con­ centration; to plot the yeast concentration against the caffeine concentration for a given time; and to plot a func­ tion of the caffeine concentration (logarithm of the caf­ feine concentration) against a function of time (relative growth rate) for a given yeast concentration.

Thus the

three variables— caffeine concentration, time, and yeast concentration— were each in turn made constant while the other two variables, or functions of the other two varia­ bles, were compared to one another on Cartesian coordinates. For the first method a plot of the concentration of yeast against age of the culture was made for each caffeine concentration in each of the five experiments. are usually called growth curves.

Such plots

A series of selected

growth curves obtained in experiment one are illustrated in figure 3«

The growth curves obtained in experiment one were

typical of those obtained in the other experiments.

In

general the growth curves of the cultures containing caffeine fell below the growth curves of the controls.

In

a few instances the growth curves of caffeine containing cultures rose slightly higher than the curves for the corresponding controls but these results were not consistent features in duplicate cultures containing the same

200 "W

-05

FIGURE 3 YEAST CONCENTRATION VERSUS TIME, CAFFEINE CONCENTRATION CONSTANT

3k concentration of caffeine. In the second method of graphical representation of the results the yeast concentrations attained at a given time were plotted against the caffeine concentrations of the media.

The yeast concentrations at the given time were

taken from the growth curves.

Comparable times for plotting

the comparisons were selected in all of the growth experi­ ments.

The times chosen were the approximate times that the

control cultures, containing no caffeine, attained half maximum growth, which, as pointed out by Buchannon and Fulmer (1928), are the periods of most rapid multiplication. The results obtained in experiment one were illustrated in figure k.

The plot indicated a rapid fall in yeast coneen-

tration'as the caffeine concentration was increased from zero to 5

P©** ®1*

With higher caffeine concentrations

there was only scanty growth of yeast.

The curve had its

maximum height at a caffeine concentration of zero mg. per ml.

These results were typical of the results obtained in

the other experiments as well. In the third method of graphical representation of the results, the relative growth rates were plotted against the logarithms of the caffeine concentrations of the media. Before the plots could be constructed, however, it was first necessary to calculate the relative growth rates. T h ome (19^6) claims that the growth curves of yeast

150

I)

13

fff-^ijiengpncentrirFidiiiiTltlnjg|. p
and the caffeine concentration is given in mg. per ml.

The curve indicates that there can not

be a negative inhibition (i.e. stimulation). Stated in another way, the straight line curve in­ dicates that caffeine concentrations of 0.316 mg. per ml. or less have little or no effect upon the rate of the multi­ plication of yeast; that caffeine concentrations of 15.9 mg. per ml. or greater prevent multiplication of yeast altogether; and that caffeine concentrations intermediate between 0.316 mg. per ml. to 15.9 nig. per ml. inhibit yeast growth.

This inhibition varies between zero and 100 per

cent with the per cent of inhibition being directly propor-

C5

¥?-

m JjtS.

i&a

iti

m

PP*

FIGURE 5 GROWTH RATE VERSUS LOG CAFFEINE CONCENTRATION

ho tlonal to the logarithm of the caffeine concentration except at very low per cent inhibition* In figure 5 there appears to be a consistent devia­ tion of the points from a straight line at caffeine concen­ trations of 0*63 mg. per ml. and less. inserted with a dotted line.

The correction is

Thus the stated 0.316 mg. per

ml. as a caffeine concentration exhibiting no inhibition upon the rate of growth of yeast is not correct.

It is a

concentration exhibiting only slight inhibition upon yeast growth.

The plot would indicate that there is a definite

and consistent Inhibition of yeast growth by caffeine concentration of 1.0 mg. per ml.

There Is definitely no

inhibition of yeast growth with caffeine concentrations of 0.01 mg. per ml. or less.

At some intermediate caffeine

concentration inhibition of yeast growth first becomes apparent. Deviations from averages.

In general, the agreement

of values obtained for the growths of duplicate and tri­ plicate cultures was very satisfactory.

Each yeast concen­

tration listed in tables IV through VII or in table X, represents the average or mean value for duplicate or triplicate cultures grown at the respective caffeine con­ centration.

The average deviation of the values from the

mean was calculated in each case.

As experiment one was conducted on single cultures, no average deviations could be calculated for this experi­ ment.

The best agreement was obtained in experiments two

and three which were cultured in triplicate. * In these ex­ periments average deviations from the mean ranged from zero to 21 in the extreme case.

The average of the average de­

viations from the mean was slightly greater than 3.

The

very close agreement of values in triplicate cultures is similar to that which would have been expected from tripli­ cate readings on single cultures.

For experiment four and

five the two values obtained for a given set of readings at one caffeine concentration were in general fairly close to­ gether but the values did not agree as well as the values for triplicate cultures in the preceding two experiments. The average deviation of single readings from the average value reported in the tables was 15-3 but the average de­ viation over the range of calculations was much smaller. The rather high deviations for later values was caused by the rapid settling of the very abundant yeast growth.

When

the concentration of yeast was greater than 500 mg. per 100 ml. it was difficult to obtain a reading since the settling of the cells interfered.

This problem was not as serious

in the experiments in which synthetic media were employed inasmuch as growth was much less luxurient in these media.

CHAPTER V

THE EFFECT OF CAFFEINE UPON THE FERMENTATION RATE OF YEAST Two experiments, six and seven, were performed to determine the effects of various concentrations of caffeine upon the fermentation rate of yeast.

The experiments were

to determine what caffeine concentrations, if any, stimu­ lated the fermentation rates of yeast.

The experiments

were also to provide a means of comparing the effects of caffeine upon the fermentation rates of a dilute yeast suspension to the effects of caffeine upon the fermentation rates of a concentrated yeast suspension.

In addition, the

experiments were to provide a means of comparing the effects of caffeine upon the consumption of glucose by a yeast suspension to its effects upon the production of alcohol.

Finally, the experiments were to provide data to

be used in comparing the effects of caffeine upon the growth rate of yeast to its effects upon the fermentation rate. 4

I.

PRELIMINARY PREPARATIONS

Nitrogen-free medium.

A medium, similar to the

Basal Medium A used in the growth rate experiments but lacking in a nitrogen source, was prepared for the fermen­ tation experiments.

One hundred and sixty gms. of anhyd-

rous glucose, 10 ml. of sterile bios solution, 5 ml. per cent lactic acid, 50 ml.

1 ® ^OH solution, and 25 ®1«

of the nutrient salt mixture were diluted to a volume of 2 liters with distilled water. Yeast suspension.

Yeast cells were developed on k

liters of glucose yeast-extract medium.

The medium had

the same composition as the yeast-extract media used in the growth experiments except for the addition of one extra gm. of yeast-extract per liter of medium.

After kQ hours most

of the supernatant liquor was decanted and the residues were spun down in a centrifuge.

The yeast cells thus obtained

had a wet weight of 22.2 gms.

They were suspended in 1^0

ml. of distilled water and placed in the freezing compart­ ment of the ice box.

The suspension was designated stock

yeast suspension and was kept frozen when not in use.

As

the stock yeast suspension was needed 25 ml. portions were spun down, the supernatant liquid decanted, and the cells resuspended in 100 ml. of the nitrogen-free medium. II. Procedure.

EXPERIMENTAL

Ten fermentation bottles, divided into

two sets of five, were each filled with 50 ml. portions of nitrogen-free media.

The media had been previously adjusted

to caffeine concentrations of 10, 1 , 0.1 , 0.01, and zero mg.

kk per ml. by means of a serial dilution technique.

To each

of the first set of five bottles, 8 ml. of sterile nitrogenfree medium and 2 ml. of yeast suspension were added.

To

each of the second set of five bottles, 10 ml. of the yeast suspension were added.

The fermentation bottles were then

incubated at 27° G. and samples were withdrawn at various intervals.

During fermentation the bottles were covered

with only loosely fitting cotton plugs.

Glucose determin­

ations were run upon each of the samples withdrawn.

In

addition, alcohol determinations were also run upon the last set of samples withdrawn from the second set of fermentation bottles. The first set of fermentations, which contained the lesser quantity of yeast suspension, constituted experiment six.

The second set of fermentations, which contained the

greater quantity of yeast suspension, constituted experiment seven.

The quantity of yeast suspension in each fermentation

bottle of experiment seven was five times greater than the quantity in each bottle of experiment six. Results.

The results of experiments six and seven

are given in detail in tables XIII through XV. are also presented graphically in figure 6 .

The results

In figure 6 the

results of experiments six and seven were plotted on a single graph.

The plot of a straight line was much less

certain than was in the case of figure 5*

Fewer points

were available and those that were available were much more scattered.

The curve may be represented mathematically by

the expression: per cent inhibition = k (log caffeine con. + 0 .5 ) where k is 1^*3 , where the caffeine concentration is ex­ pressed in mg. per ml., and where the per cent inhibition may not be less than zero.

_

_ _“



h3 1 1 L* 13< u

"1

""

t\ f

1 01)

pi

f

T

_Hw V)

\ © V

0

1

Jo p

i

c Vi

& r "Tf © .M l & I «n F S2 n [© - - -

>H,

- - -

-

-

i

r

5 w j K

~ r !

-S

i_

? 0;

- -

© Hfr

-

F

3.Ct s JS —> s..

“ fct- 8 0 - -

• bi

o p ©r [jCf LW C ~V' — & 1*y»■

c L -N n S, m L@J

- *

JS ~© Ei

s i

1

iv ■v)

h

J_ .

j_

•_ E G3m& k« > ra4 Xi9

..



i —

|F» m m “1 _jon SL -S H a m u _S - SC M ~j □ Ig J! © p. jf & _ i \ gr H !h P P it L u i _ —s© rr ! !

r"

_ 0 • ir

j w ree a N ^ r l _J

E3 t

i 4 o:Et J! jfl - 3%at3 '- 1 ©I* 33

r~

■w

i r 3 r r - - -- - n —-

FIGURE 6 FERMENTATION RATE VERSUS LOG CAFFEINE CONCENTRATION

4? TABLE XIII THE EFFECT OF CAFFEINE UPON THE CONSUMPTION OF G-LUCOSE BY YEAST EXPERIMENT SIX

Age of

Caffeine concentration in mg. per ml.

culture 8,3.3

O .833

0.0833

0.00833

0.0000

Concentration of glucose in gms. per 100 ml. Zero hour

7.92

7.92

7.92

7.92

7.92

17 hour

7.53

7.58

7.55

7.58

7.62

4l hour

6.94

6.96

,.6.97

6.80

6.80

72 hour

6.70

6.67

6.62

6.64

6.68

182-J- hour

4 .76

4.56

4.32

4.10

4.13

336 hour

2.90

2.50

2.1^

1.92

1.99

Gms. of glucose consumed per 100 ml. of media 182^ hour

3.16

3.36

3.60

3.82

3.79

336 hour

5.02

5.^2

5.78

6.00

5.93

Relative rate of glucose consumption compared to the control as 100 1824 hour

83.5

88.5

95.0

101

100

336 hour

84.7

91.5

97.5

101

100

TABLE XIV THE EFFECT OF CAFFEINE UPON THE CONSUMPTION OF GLUCOSE BY YEAST

EXPERIMENT SEVEN

Age of

Caffeine concentration in mg. per 100 ml.

culture 8.33

O.833

0.0833

0.00833

0.000

Concentration of glucose in gms. per 100 ml. Zero hour

7.92

7.92

7.92

7.92

7.92

13 hour

6.43

6.28

6.15

6.07

6.10

174 hour

6.22

5.70

5.48

5.45

5.46

40 hour

3.92

2.74

2.42

2.56 '

2.61

48 hour

3.29

1.87

1.49

1.67

1.74

60 hour

2.12

0.75

0.44

0.60

0.66

Sms. of glucose consumed per 100 ml. of media 48 hour

4.63

6.05

6.43

6.25

6.18

60 hour

5.80

7.17

7.48

7.32

7.26

Relative rate of glucose consumption compared to the control as 100 48 hour

75-0

98.0

104

101

100

60 hour

78.5

98.7

!03

101

100

49 TABLE XV A COMPARISON OF ALCOHOL PRODUCED TO GLUCOSE CONSUMED BY YEAST IN MEDIA CONTAINING CAFFEINE EXPERIMENT SEVEN

Caffeine concentration in mg. per ml. 8.33

0.833

0.0833

0.00833

0.000

Gins, glucose consumed

5.80

7.17

7.48

7.32

7.26

Gms. alcohol produced

2.59

3.20

3.24

3.21

3.19

0.447

0.446

0.434

0.439

0.439

Gms. alcohol produced per gm. glucose consumed

NOTE: All values are based upon the analytical results obtained on the 60 hour samples.

50 CHAPTER VI A COMPARISON OF THE EFFECT OF CAFFEINE UPON GLUCOSE CONSUMPTION BY YEAST IN AIR AND UNDER OIL

An experiment was performed to compare the effect of caffeine upon the consumption of glucose by yeast in air to the effect of caffeine upon the consumption of glucose by yeast in the absence of air. Procedure.

A serial dilution technique was employed

to prepare caffeine solutions of 10, 1, 01, 0.01, and zero mg. of caffeine per ml. in the nitrogen-free media.

Each

of the five solutions were divided into four 10 ml. portions. Two 10 ml. portions of each caffeine concentration were placed in 50 ml. Erlenmeyer flasks and the other two 10 ml. portions were placed in 15 ml. test tubes.

To each of the

20 aliquots thus prepared were added 2 ml. portions of the yeast suspension.

The series of 10 Erlenmeyer flasks were

incubated at 27° 0. uncovered.

The series of 10 test tubes,

however, were each covered with a 2 ml. layer of C. P. min­ eral oil and incubated at 27^ C.

The fermentations con­

ducted in the open Erlenmeyer flasks were designated the aerobic series and those in the test tubes, whose contents were covered with mineral oil, were designated the anaerobic series.

At the end of 48 hours the 20 fermentations were

harvested, and determinations of the residual glucose were made.

The analytical results are tabulated in table XVI.

51 TABLE XVI A COMPARISON OF THE EFFECT OF CAFFEINE UPON GLUCOSE CONSUMPTION BY YEAST IN AIR AND UNDER OIL

AEROBIC SERIES

Caffeine concentration in mg. per ml.

0 hour glucose in gm. per 100 ml.

48 hour glucose in gm. per 100 ml.

Gms. glucose used per 100 ml.

Per cent of max. rate of glucose utilization

8.33

7.92

4.11

3.81

73.4

8.33

7.92

3.80

4.12

79.3

0.833

7.92

4.20

3.72

71.5

0.833

7.92

3.95

3.97

76.2

O.O833

7.92

3.66

4.26

81.8

0.0833

7.92

3.48

4.44

85-3

0.00833

7.92

3.52

4.40

84.5

O.OO833

7.92

3.48

4.44

85.3

0.00000

7.92

3.02

4.92

94.4

0.00000

7.92

3.44

4.48

86.0

(NOTE: A consumption 5*21 gms. of glucose in the 48 hour period was taken as 100 and the other rates are expressed relative to this.)

52 TABLE XVI(continued) A COMPARISON OF THE EFFECT OF CAFFEINE UPON GLUCOSE CONSUMPTION BY YEAST IN AIR AND UNDER OIL

ANAEROBIC SERIES

0 hour glucose in gm. per 100 ml.

48 hour glucose in gm. per 100 ml.

Gms. glucose used per 100 ml.

Per cent of max. rate of glucose utilization

8-33

7.92

5.01

2.91

55.8

8.33

7-92

4.78

3.14

60.2

0.833

7.92

3.58

4.34

83.^

0.833

7.92

3.54

4. 38

84.0

0.0833

7.92

3.18

4.74

91.0

0.0833

7.92

3.02

4.90

95-0

O.OO833

7.92

2.96

4.96

95.2

O.OO833

7.92

2.79

5.13

98.5

0.00000

7.92

3.36

4.56

87.5

0.00000

7.92

2.71

5.21

100.0

Caffeine concentration in mg. per ml.

(NOTE: A consumption 5*21 gms. of glucose in the 48 hour period was taken as 100 and the other rates are expressed relative to this.)

CHAPTER VII

DISCUSSION Growth experiments.

The correlation of results in

figure 5 is rather remarkable when it is considered that the five growth rate experiments differed from each other in several respects.

Although the hydrogen ion concentration

and glucose concentration were constant throughout the five experiments, other components of the medium were varied. The sizes of the inocula varied widely. Caffeine concentrations of 15*9 mg. P®** ml. or greater prevented multiplication of yeast altogether.

However, a

microscopic examination of the yeast cultures grown in high concentrations of caffeine revealed normal yeast cells even after **8 hours.

The action of caffeine in preventing yeast

growth appeared to be fungistatic rather than fungicidal. Caffeine concentrations of O.316 mg. per ml. or less had little or no effect upon the growth rate of yeast. At no concentration was caffeine found to stimulate the growth rate of yeast.

In each experiment there were

occasional cultures which grew slightly more rapidly than the controls.

These results did not represent a reproducible

feature at a given concentration of caffeine.

The differ­

ences were not great and were merely chance variations rather than consistent features.

5^ • Caffeine concentrations intermediate between O .316 and 15.9 mg. per ml. inhibited the growth of yeast.

This

inhibition varied between zero and 100 per cent with the per cent of inhibition being directly proportional to the logarithm of the concentration of caffeine except at very low per cent inhibition.

At very low per cent inhibition,

the pattern of inhibition appeared to deviate slightly from a straight line. The extent of the inhibition of yeast growth by caffeine appeared to be Independent of the numbers of yeast cells being acted upon by the caffeine. upon the concentration of caffeine.

It depended only

In experiment one the

initial inoculum was so light that visible evidence of turbidity could not be observed for the first twenty-four hours of growth.

In experiment five the concentration of

yeast in the culture medium at the start of the experiment was O.75 mg. per 100 ml.

In experiment four the concentra­

tion of yeast in the culture medium at the start of the experiment was 75

per 100 ml.

Yet, in all of these

cases the pattern of inhibition by caffeine was the same. When the results of experiments one, two, and three were compared with the results of experiments four and five, it was found that the per cent of inhibition at a given caffeine concentration was the same regardless of whether the medium was strictly synthetic or whether it contained

55

yeast-extract.

It is thus improbable that caffeine exerted

its effect by competing with any substance present in yeast extract. A comparison of the results of experiment one with those of three or four, or a comparison of the results of experiment four with those of experiment five, might justify the conclusion that yeast either do not develop tolerance to caffeine or that they develop tolerance very slowly.

In

experiment one the yeast cells had been under the influence of caffeine for a great number of generations before a concentration of cells equal to the size of the initial inoculum in experiment three had been developed.

Likewise,

a similar relationship existed between experiments four and five.

Yet, the inhibition patterns were all alike. Had tolerance to caffeine been rapidly developed by

the yeast, figure five would not have been a single straight line.

The points from experiment one would have determined

a line whose slope was less steep than the line determined by the points from experiment three.

Similarily, the points

from experiment five would have determined a line whose slope was less steep than the line determined by the points from experiment four.

As evidenced by figure 5, all of the

points fell on one line. Fermentation experiments.

In general the results of

the fermentation experiments were less consistent and less conclusive than were the growth rate experiments.

This was

partly due to the fact that fewer and less extensive exper­ iments were performed* The effect of caffeine upon the consumption of glu­ cose and the production of alcohol by a yeast suspension was much less pronounced than was the effect of caffeine upon the rate of growth of yeast.

Even with caffeine

concentrations of 10 mg. per ml. the inhibition of fermen­ tation rate was not more than around 20 per cent. The inhibition that was observed appeared to be independent of the concentration of yeast.

In experiment

seven the suspension employed had a concentration of yeast cells five times greater than the concentration of yeast cells in experiment six.

In both experiments the fermenta­

tion rate was inhibited to about the same extent by a given concentration of caffeine. The production of alcohol by yeast was Inhibited by caffeine to the same extent as was the consumption of glucose.

This would indicate that caffeine did not block

the intermediary metabolism of glucose at any point between glucose and alcohol unless it blocked the Initial reaction involving glucose itself. No concentration of caffeine stimulated the utiliz­ ation of glucose or the production of alcohol by yeast

57 suspensions. Tolerance to caffeine was not developed by the yeast suspension or it developed very slowly. the fermentations were very slow.

In experiment six

The inhibition of fermen­

tation by caffeine was constant throughout the 336 hours that the fermentations were conducted.

This fact also

substantiated the statement, previously made, that the ac- ' tion of caffeine upon yeast is fungistatic rather than fungicidal.

Had the action of caffeine been fungicidal, the

per cent of inhibition of fermentation would have increased with time.

Had tolerance been rapidly developed to the

action of caffeine, the per cent of inhibition of fermen­ tation would have decreased with time. Comparisons of the caffeine-effects upon growth and fermentation rates.

At no concentration did caffeine stim­

ulate either the growth rate or the fermentation rate of yeast.

The lack of stimulatory effects is not in accord

with a widely held view that all agents toxic to a cell are stimulatory in sub-toxic concentrations.

Arndt and Schulz

(1888) proposed this view in the form of a statement which has come to be known as the Arndt-Schulz Law.

More recent

evidence favoring the acceptance of such a view was the report by Curran and Evans (19^7) that traces of penicillin or streptomycin stimulate the growth of some bacteria.

Rahn

58 (1932) discusses the dual role of poisons as stimulants and inhibitors.

He compares the action of chemical agents on

enzymes to the action of temperature and derives a mathemat­ ical expression for the treatment of data concerning the action of chemical agents on enzyme systems.

His precise

treatment does not predict that all chemical agents possess the ability to effect biological systems in a dual manner but does explain how some chemical agents might produce opposite effects at different concentrations. Inhibition of both the fermentation rate and the growth rate approximated a linear function of the logarithm of the caffeine concentration.

Compounds which produce a

biological effect proportional to the logarithm of the concentration of the compound are common among drug or metabolite antagonists.

A new scale for the measurement of

drug antagonism, which in some respects resembles the pH scale, has been introduced by Schild (19^7 ).

The scale is

based upon the logarithm of the concentration of the antag­ onist and has been applied to such antagonists as neoantergan, benadryl, pethidine, and atropine. The straight line relationship would exist if caf­ feine were directly involved in antagonizing a monomolecular reaction.

This follows from the general mass action law

for chemical reactions of the first order.

The straight

line relationship, however, does not prove that caffeine is

59 an antagonist.

As one of several alternatives caffeine

could itself be reacting in a chemical reaction which was monomolecular with respect to caffeine.

Rahn (19^5)

discussed the frequent occurrence ©f the logarithmic pattern in biological processes and warned of the limita­ tions involved in interpreting the mechanism of action from the mathematical pattern. The limiting caffeine concentration which had little or no effect upon either the fermentation rate or the growth rate was in both cases 0.316 mg. per ml.

A concen­

tration of 15.9 mg. of caffeine per ml. inhibited the fermentation rate by 25 per cent and the growth rate by 100 per cent. With many inhibiting agents the extent of inhibition depends not so much upon the concentration of the inhibiting agent as it does upon the quantity of inhibiting agent per yeast cell or per bacterial cell. with caffeine.

This was not the case

The extent of inhibition of both the growth

rate and the fermentation rate was independent of the numbers of yeast cells being acted upon but depended only upon the concentration of caffeine.

These results are in

some respects analagous to the process described by Lineweaver and Burke (193*0 as “noncompetitive inhibition.u Tolerance to caffeine did not appear to be developed by either growing yeast cells or by a mature yeast suspen-

6o slon. The effects of caffeine upon the processes of ferm­ entation and multiplication were thus qualitatively parallel in all respects but were quantitatively disproportionate. An equation was developed relating the per cent of inhibi­ tion to the concentration of caffeine.

The equation was as

follows: per cent inhibition = k (log caffeine con. + 0.5) The values of k were 1^.3 for the fermentation rate and 58.8 for the growth rate. Comparison of the caffeine-effect in air and under ■■■M M M M HM M NM M VM

oil.

M M M M M M M M M M m M M M M M MM M M M M M M M M H M M MM M M M M M M M M M M *

The effect of caffeine upon the glucose consumption

by yeast suspensions appeared to be more pronounced when the supply of oxygen was restricted than when free access to air was permitted.

This premise cannot be stated with certainty

as only a single experiment was performed upon which to base the conclusion. Certain criticisms of the methods chosen to obtain anaerobic and aerobic conditions are in order.

Were the

cultures designated as anaerobic actually anaerobic?

Were

those designated as aerobic actually aerobic? It must first be understood that the terms aerobic and anaerobic are not so much terms of opposite meaning as they are terms designating relative oxygen tensions.

In

61 experiment eight the attempt was made to maintain two sets of fermenting yeast suspensions in environments of relative ly different oxygen tensions. In the anaerobic experiments the fermentations were conducted in test tubes. tation liquor.

A layer of oil covered the fermen­

Although oxygen is somewhat soluble in oil,

the oxygen which diffused through the layer of oil had to also penetrate a relatively great distance of culture medium before reaching the yeast cells which were ,fbottom yeast.M In the aerobic series the fermentations were con­ ducted in Erlenmeyer flasks.

Thus a relatively large

surface area was exposed for the absorption of oxygen and a relatively short distance was required for the oxygen to diffuse through before reaching the yeast cells at the bottom of the flask. Yeast have the property of rapidly lowering the oxygen tension of

the medium in which they are suspended.

In spite of the precautions taken it may be that the oxygen tension was very low in both sets of fermentations and thus the differences in oxygen tension between the two sets may have been small. Comparison to the effect of caffeine on sea urchin eggs. Cheney (19^5) studied the effects of caffeine upon

fertilized sea urchin eggs.

He found cell division to be

completely inhibited if oxygen uptake is decreased by fifty per cent or more. With sea urchin eggs as with yeast cell division is inhibited to a greater extent by a given concentration of caffeine than are certain energy-producing metabolic proc­ esses.

We may state that the fermentative enzyme system of

a mature yeast cell or the oxidative enzyme system of a sea urchin egg is less sensitive to caffeine than are the energyrequiring reproductive processes of the respective cells. Or, we may state that cell division will cease when the energy-yielding systems are only partially inhibited.

The

two statements may be synonomous but it seems that they represent two different interpretations of the effect of caffeine.

The former implies that caffeine acts simultane­

ously but to different degrees upon the two systems.

The

later implies that the action of caffeine upon the energyrequiring processes is manifested through its effect upon the energy-liberating processes.

In either Interpretation

there appears to have been a discoupling of the energyyielding from energy-requiring processes. Comparison to the effect of caffeine upon frog muscle. Saslow (1936) studied the effect of caffeine upon frog muscle.

His observations strengthened the hypothesis of

63

Meyerhoff (1921) and Hartree (1920) that "the action of caffeine upon muscle is merely to release, slowly and continuously, the chemical processes, anaerobic or oxida­ tive, normally Induced suddenly and discontinuously by stimulation." A certain relationship was observed between the effect of caffeine upon frog muscle and the effect of caf­ feine upon yeast.

The relationship, however, was less

direct than in the case of sea urchin eggs.

In frog muscle

as with yeast the normal relationship of energy-yielding processes and energy-requiring processes was changed by caffeine.

CHAPTER VIII

SUMMARY The biochemical explanation of the many physiological effects of caffeine is not known.

It has seemed possible

that caffeine might exert its effect by discoupling energyyielding from energy-requiring processes.

This question has

been approached by comparing the relative effects of caf­ feine on growth and metabolism of yeast cells. The effects of caffeine upon the rate of growth of yeast and upon the rates of glucose-consumption and alcoholproduction by yeast suspensions have been studied.

A com­

parison of the effects of caffeine upon the consumption of glucose by yeast in air and under oil has also been made. At no concentration did caffeine stimulate either the growth rate or the fermentation rate of yeast.

Inhibition

of both rates approximated a linear function of the logarithm of the caffeine concentration.

Caffeine concentrations of

O.316 mg. per ml. or less had little or no effect upon either the fermentation rate or the growth rate.

A concentration

of 15.9 rag. of caffeine per ml. inhibited the fermentation rate by 25 per cent and the growth rate by 100 per cent.

An

equation was developed relating per cent Inhibition to the caffeine concentration.

The equation was as follows:

per cent inhibition =s k (log caffeine con. -f 0.5)

65 The values of k were 14.3 ^or the fermentation rate and 5S,3 for the growth rate. In these experiments, in which the pH and glucose concentration of the media were constant, the extent of in­ hibition of both the growth rate and the fermentation rate appeared to be Independent of the numbers of yeast cells being acted upon but depended only upon the concentration of caffeine.

The presence or absence of yeast-extract In

the medium did not alter the extent of inhibition of the growth rate.

The production of alcohol by yeast was in­

hibited by caffeine to the same extent as was the con­ sumption of glucose.

Tolerance to caffeine was not devel­

oped by yeast, or it developed very slowly.

The action of

caffeine was probably fungistatic rather than fungicidal. The effect of caffeine upon the glucose consumption by yeast suspensions appeared to be more pronounced when the supply of oxygen was restricted than when free access to air was permitted.

This premise cannot be stated with certainty

. as only a single experiment was performed upon which to base the conclusion. The results were in accord with but did not necessa­ rily prove the original contention that caffeine might exert Its influence by discoupling energy-yielding from energyrequiring processes.

BIBLIOGRAPHY

BIBLIOGRAPHY

Amati, A,, and Sgarzi, L. , "The Action of Some Alkaloids on the Alcoholic Fermentation of Molasses," Giornale di biologie Indus triale, agrarle ed allmentare. 5:5 2-5>2 * 1935. Abstracted in Chemical Abstracts, 30:6125, 1936* Arndt, and H. Schulz, Pflugers* Archlv fftr die gesamte Physiolog!e des Menschen und der tiere, 42:517. 1S88 and Biologlsche Studlen. Greifswald, 1&82. Reviewed and abstracted In Chemical Abstracts, 21:2289, 1921. Bellisai, Jole, "How the Activity of the Fermentation of Beer Varies in the Presence of Caffeine," Archives Internationales de pharmacodynamle et de therapie, 3 5 :**7 4-4 6 0 , 1929* Buchanan, R. E., and Ellis I. Fulmer, Physiology and Biochemistry of Bacteria. Volume I, Chapter 1, Baltimore Williams and Wilkins, 1928. Cheney, Ralph H., "Effects of Caffeine on Oxygen Consumption and Cell Division in Sea Urchin Eggs," Journal of General Physiology, 29:63-72, 1945. Conway, Edward J., Microdiffusion Analysis and Volumetric Error. London: Crosby Lockwood and Son, Ltd., 1947. Curran, Harold R., and Fred R. Evans, "Stimulation of Sporogenic and Nonsporogenic,Bacteria by Traces of Penicillin and Streptomycin," Proceedings of the Society for Experimental Biology and Medicine, 64:231-233. 1947. Enders, C., and F. M. Wienlnger, "The Effects of Alkaloids on the Fermentation and Multiplication of Yeast," Blochemische Zeitschrlft, 293:22-29, 1937. Hartree, W., and A. V. Hill, "The Heat Production of Muscles Treated with Caffeine or Subjected to Prolonged Discon­ tinuous Stimulation," Journal of Physiology, 58:441-454, 1920. Lineweaver, Hans and Dean Burk, "Determination of Enzyme Dissociation Constants," Journal of the American Chem­ ical Society. 56:658-606, 1934. Lockwood, A. G. , and G. B. Landerkin, "Nutrllite Requirements of Gsmophilic Yeasts," Journal of Bacteriology, 44:343351, 1942.

68 Meyerhoff, Otto, "Energy Transformation in Muscle,11 Pflugers1 Archlv fSr die gesamte Physiologle des Menschen und der fliere. 188:1^3, 1921* Neuberg, C., and Marta Sandberg, “The Stimulators of Alcoholic Sugar Decomposition," Biochemlsche Zeitschrlft. 125:202-219, 1921. Porter, John Roger, Bacterial Chemistry and Physiology. York: Wiley and Sons, Inc., 19 W>.

New

Pulley, H. C. and J. Dudley Greaves, “An Application of the Autocatalytic Growth Curve to Microbiological Metabo­ lism," Journal of Bacteriology. 2*Kl45-lo8, 1932. Saslow, George, "Delayed Heat Production of Caffeinized Frog Muscle," Journal of Cellular and Comparative Physiology. 8:89-99, 193&. Saslow, George, "Twitch Tension and Initial Heat in Caffeinized Frog Muscle," Journal of Cellular and Comparative Physiology. 8:387-401, 19 3&* Saslow, George, "Oxygen Consumption and Respiratory Quotient of Caffeinized Frog Muscles," Journal of Cellular and Comparative Physiology. 10:385-39^, 1937. Schlld, H. 0., "pA, A New Scale for the Measurement of Drug Antagonism," British Journal of Pharmacology and Chemo­ therapy, 2:187-206, 19^7. Schaffer, P. A. , and Hartman, “A Method for the Determina­ tion of Reducing Sugars in Blood and Urine," Journal of Biological Chemistry. ^5:377, 1921. Thorne, R. S. W., "The Nitrogen Nutrition of Yeast," Communications of the Wallerstein Laboratories, August 19^

-----------------------------------------------------

Rahn, Otto, Physiology of Bacteria. ton Co., 1932. Rahn, Otto, Microbes of Merit. Press, 19^5.

Philadelphia:

Lancaster:

Blakis-

Jaques Cattell

Widmark, Biochemlsche Zeitschrlft, 131:^73* 1922.

APPENDIX

TABLE IT INCREASE IN YEAST CONCENTRATION WITH TIMS AT VARIOUS CAFFEINE CONCENTRATIONS EXPERIMENT ONE

Caffeine concentration

0 Hour

20 Hour

40 Hour

60 Hour

80 Hour

100 Hour

140 Hour

180 Hour

0

0

0

0

0

0

0

1

10.0

"

0

0

0

0

1

7

24

56

5.0



«

0

0

0

2

12

30

78

152

1 .0

H

«

0

3

69

175

236

278

337

367

0 .5

w

«

0

7

117

191

242

274

320

358

0.1

»

it

0

8

134

209

238

258

291

316

0.05

*

H

0

7

120

220

276

325

405

456

0.01

«

11

0

4

105

213

272

306

351

378

0.001 «

H

0

7

141

258

316

358

424

486

0

10

122

220

280

314

376

419

0

7

129

224

289

325

371

405

a

mg. ml.

o o

20.0

It

0 .0



It

NOTE:

The fig u r es in the table represent mg. of yeast per 100 ml. of media.

TABLE V INCREASE IN YEAST CONCENTRATION WITH TIME AT VARIOUS CAFFEINE CONCENTRATIONS EXPERIMENT TWO Caffeine concentration in mg. per ml.

Hour

3 3 /4 Hour

5 1 /3 Hour

6* Hour

10 Hour

1 5i Hour

71 Hour

4

14

11

12

15

21

40

126

4.17

4

14

15

17

19

29

69

187

0.833

4

22

28

40

48

89

189

248

0.417

4

24

34

50

64

108

196

266

0.0833

4

26

36

55

71

133

198

272

0.0417

4

28

37

56

72

134

200

274

0.00833

4

29

37

59

76

145

207

283

0.000833

4

30

38

59

78

156

222

320

0.000000

4

25

35

55

71

158

213

314

0 Hour

8.S3

NOTE: The figures in the table represent mg. of yeast per 100 ml, of media. Eaeh figure is the average value for the three tubes at that concentration.

TABLE VI INCREASE IN YEAST CONCENTRATION WITH TIME AT VARIOUS CAFFEINE CONCENTRATIONS EXPERIMENT FOUR Caffeine concentration in mg. per ml.

6 3/4 Hour

8* Hour

18* Hour

25 Hour

33 Hour

44 Hour

93

104

113

170

234

332

450

75

108

133

150

273

481

723

907

1.80

75

132

175

207

468

730

974

1106

0.833

75

157

206

275

593

853

1009

1086

0.387

75

188

290

358

620

865

1019

1140

0.180

75

188

288

369

638

870

1042

1116

0.0833

75

195

288

378

608

825

927

1043

0.0387

75

201

302

391

661

942

1086

1173

0.0180

75

191

286

374

625

854

989

1124

i Hour

4& Hour

8.33

75

3.87

NOTE: The figures in the table represent mg. of yeast per ICO ml. of media* Each figure is the average value for the two tubes at that concentration.

S3

TABLE VI (continued) INCREASE IN YEAST CONCENTRATION WITH TIME AT VARIOUS CAFFEINE CONCENTRATIONS EXPERIMENT FOUR Caffeine concentration in mg. per ml.

Hour

0.00833

i

8i

Hour

6 3/4 Hour

Hour

18 i Hour

25 Hour

33 Hour

44 Hour

75

193

281

375

625

820

983

1110

0.00387

75

193

287

385

646

895

1045

1145

" 0.00180

75

199

298

401

648

908

1060

1160

0.000833

75

199

301

405

650

847

1007

1099

0.000387

75

193

297

424

630

842

1022

1089

0.000180

75

200

302

408

668

925

1091

1206

0.0000833

75

192

304

390

671

938

1069

1156

0.0000000

75

194

300

385

615

845

988

1095

NOTE: The figures in the table represent mg. of yeast per 100 ml. of media* Each figure is the average value for the tvo tubes at that concentration.

table vii

INCREASE IN YEAST CONCENTRATION WITH TIME AT VARIOUS CAFFEINE CONCENTRATIONS EXPERIMENT FIVE Caffeine concentration in mg. per ml.

0 Hour

9* Hour

18 Hour

24i Hour

274 Hour

33 Hour

45 Hour

8.33

0

4

9

4

9

20

25

49

3.87

0

4

14

12

22

38

65

186

1.80

0

4

15

22

50

89

195

514

0.833

0

5

40

60

158

281

463

682

0.387

0

3

64

133

317

435

547

718

0.180

0

5

82

163

347

463

579

724

0.0833

0

8

89

182

363

471

612

758

0.0387

0

7

79

164

345

457

605

738

0.0180

0

7

84

175

349

466

610

789

21 Hour

NOTE: The figures in the table represent mg. of yeast per 100 ml. of media. Eaeh figure is the average value for the two tubes at that concentration.

TABLE VII (continued) INCREASE IN YEAST CONCENTRATION ®ITH TIME AT VARIOUS CAFFEINE CONCENTRATIONS EXPERIMENT FIVE

University of Seuthern M ffe m fa

Caffeine concentration in mg. per ml.

0 Hour

9i Hour

18 Hour

21 Hour

24* Hour

27i Hour

33 Hour

45 Hour

0.00833

0

11

88

185

360

471

625

820

0.00387

0

10

94

199

371

465

616

738

0.00180

0

9

96

191

364

489

627

752

0.000833

0

11

96

209

364

470

627

750

0.000387

0

10

96

205

366

472

588

736

0.000180

0

12

97

215

387

488

638

798

0.0000833

0

12

96

208

377

491

628

865

0.0000000

0

10

96

204

386

492

635

834

NOTE: The figures in the table represent mg. of yeast per 100 ml. of media. Each figure is the average value for the two tubes at that concentration. *>3

Vn