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PA STEUR EFFECT IN YEAST

PROEFSCHRIFT

TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE TECHNISCHE WETENSCHAP AAN DE TECH-NISCHE HOGESCHOOL TE DELFT OP GEZAG VAN DE RECTOR MAGNlFICUS Ir. H. J. de WIJS. HOOG-LERAAR IN DE AFDELING DER MIJNBOUWKUNDE. VOOR EEN COMMISSIE UIT DE SENAAT TE VER-DEDIGEN WOENSDAG 17 OKTOBER 1962 DES

NAMIDDAGS TE 2 UUR DOOR

EDMUND STEFAN IDZIAK

GEBOREN TE MONTREAL ~:,~

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ti

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MET STEUN VAN

HET AGRICULTURAL INSTITUTE OF CANADA (OVERSEAS SCHOLARSHIP),

DE NEDERLANDSE REGERING

(NETHERLANDS GOVERNMENT SCHOLARSHIP 1957/1958),

EN

HE T DELFTS HOGESCHOOLFONDS.

De leden van de Wetenschappelijke en Technische staf van het Laboratorium voor Microbiologie moge ik oprecht en hartelijk danken voor hun toegewijde hulp bij mijn werk.

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p. 102; line 1:

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p. 134, line 40:

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Schwartz

CBS 81

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1956)

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(FEINGOLD et al.

1956)

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H.J.W.Kreger-van Rij

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Rivière, J.W.M. la.

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INTRODUCTION 9

A Brief Review of the Subject 9

The Aim of the Present Investigation 11

CHAPTER I 12

Materials and Methods 12

1. Materials 12

2. Organisms 13

3. Media Used for Growing the Yeasts 14

4. Preparation ofSuspensions of Non-Proliferating

(Resting) CeUs 16

5. Preparation of CeU-Free Extracts 16

6. Techniques for Measuring Fermentation 18

7. Chromatographic Procedures 23

CHAPTER II 26

Experimental Results with Whole CeUs 26

1. Preliminary Fermentation Experiments 26

a. Screening for the Negative Pasteur

Ef-fed 26

b. Effect of Aeration during Growth on

Subsequent Fermentation by Resting

CeUs under Various Conditions 31

c. Fermentation by Resting CeUs

Sus-pended in Different Solutions 35

d. Effect of Varying the Pretreatment on

Subsequent Fermentation by Resting

CeUs 36

2. Detailed Study of the Negative Pa steur Effect

in

Saccharomyces uvarum Beijerinck, CBS 426

39

a. Fermentation by Resting CeUs "after

Increased Flushing Times 39

b. Fermentation by Resting eeus from

Cultures of Different Ages 47

c. Fermentation by RestingCeUs Suspended in Buffers of Different Molarity and pH 53

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. d. Localization of the Inhibition of Anaerobic Fermentation in the Pathway of Sugar

Metabolism 59

e. Interaction ofthe Components of the

Buf-fer Systems with the Resting Cells 65

f. Prevention and Reversal of the Negative

Pasteur Effect 84

CHAPTER lil 95

Experimental Results with Cell-Free Extracts 95

1. Crude Extracts 96

a. Definition 96

b. Effect of Dilution 96

c. Influence of Various Additions on the Anaerobic and Aerobic Fermentation of

Glucose by Crude Extracts 97

d. Aerobic Fermentation of Glucose by Various Fractions of the Crude Extract 101

2. Purified Extracts 104

a. Definition 104

b. Some Properties ofthe Purified Extracts 104 c. Influence of Various Additions on the

Anaerobic and Aerobic Fermentation of

Glucose by Purified Extracts 107

d. Reversal of Succinic Acid-Sodium Suc-cinate Inhibition of Fermentation by Different Intermediates of the Glycolytic

Pathway 112

GENERAL DISCUSSION 122

SUMMARY 127

REFERENCES 130

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A Brief Review of the Subject

In the course of extensive studies involving yeasts as re-gards their fermentation and oxybiontic metabolism ofvarious carbohydrates in the absence of known inhibitors, other than oxygen, a general phenomenon, referred to as the Pasteur effect or reaction, was observed. This term was first coined

by WARBURG (1926) but he, unfortunately, immediately

placed restrictions on its use. According to WARBURG

(1926), the term should be applied to situations where th~ fermentation is inhibited by respiration. In 1939, BURK disputed the validity of this restriction on the ..basis that PASTEUR (1876) made the general statement that oxygen gas inhibited the fermentation, without specifying whether this was as a re sult of respiration or some other mechanism(s), direct or indirect. He therefore suggested that the Pas-teur effect should have a general connotation and denoce simply the inhibition of fermentation processes by gaseous oxygen.

Many hypotheses (cf. BURK, 1939) have been proposed to explain the phenomenon of the Pasteur effect. By way

of example, DIXON and HOLMES (1935) have suggested

that oxygen affects the permeability of· the cells as to set a limit to the rate at which glucose can reach the cell enzymes. Under anaerobic conditions the cell becomes more permeable and the substrate becomes more accessible to the intracellular enzymes. WU (1959) has, however, shown that under anaerobic conditions the uptake of' glucose is of the same magnitude as that under aerobic conditions. In 1937, GEMMILL and HELLERMANshowed that iodine inhibi-tion of glycolysis under aerobic condiinhibi-tions could be rever-sed by the addition of cysteine or glutathione. A study of the results of the latter authors and those of his own experiments enabled LIPMANN (1933, 1934, 1942) to propose that the oxygen reduced the rate of glycolysis. by inactivating one or more of the glycolytic enzymes containing sulfhydryl groups, the latter being reversibly oxidized. Many other authors (JOHNSON, 1941; LYNEN, 1941, 1958; LYNEN and KOENlGS-BERGER, 1951; LYNEN et al., 1959; WU and RACKER, 1959 a, b; GATT and RACKER, 1959 a, b; CHANCE, 1961; CHANCE andHESS, 1956, 1959; CHANCE et al., 1955; HOLZER, 1959 a, b; HOLZER and HOLZER, 1953; HOLZER and GRUNICKE, 1961; KORFF, 1959; KOTYK, 1961, 1962) have presented evidence in favour of the assumption of a competition, under aerobic conditions, between the respiratory. and glycolytic

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processes for the co-factors inorganic phosphate and ade-nosine diphosphate.

In 1940, CUSTERS showed that conditions which should produce a normal Pasteur effect in yeasts in reality did not always do so. He demonstrated that oxygen increasedthefer-mentation rate of cells of

Bretlanomyces claussenii

Custers as compared to that under anaerobic conditions. In the ensuing years' no further work had been done on this topic. Then in 1961 it became a subject of intensive study and the phenomenon was found, to be of a general occurrence in yeasts of this genus (WIKEN et al., 1961; cf. SCHEFFERS, 1961). CUSTERS (1940) proposed the use of the term 'nega-tive Pasteur effect' when referring to a situation where there is an inhibition of alcoholic fermentation under strictly an-aerobic conditions and a stimulation in the presence of mol-ecu lar oxygen. Whereas LIPMANN (1933,1934,1942) has pro-posed an inactivation of an essential enzyme of the glycolytic chain by reversible oxidation of its sulfhydryl groups in explaining the Pasteur effect, CUSTERS (1940), in turn, has proposed a reversible reductive inactivation of one or more enzymes of the glycolytic chain under strictly anaero-bic conditians in explaining the negative Pasteur effect.

SCHEFFERS (1961) has shown that acetaldehyde, acetone, pyruvic acid, and other carbonyl compounds such as formal-dehyde', dihydroxyacetone, acetoin, etc., were active in eliminating the negative Pasteur effect. Oxidized coenzyme 1 (DPN) enhanced the anaerobic fermentation to an extent dependent on its concentration. He suggested that the car-bonyl compounds act as hydrogen acceptors in enzymatic dehydrogenations, thus enabling the oxidation of the re-duced DPN to take place, and in ·this manner restore the anaerobic fermentatiol1.

In the genus

Saccha1'omyces,

a negative Pasteur effect has also been found although its occurrepce, for the pre-sent, ,appears to be m9re restricted. WIKEN and coworkers

(WIKEN, 1961; WIKEN and PFENNIG, 1957, 1958, 1959;

WIKÉN and RICHARD, 1953 a, b, 1954 a, b, 1955 a, b) and PFENNIG and WIKÉN, (1960 a, b) have demonstrated, quite readily, a negative Pasteur effect in resting cells from young cultures of yeasts of the genus

Saccharom)'ces

in succinic acid-sodium or potassium succinate at pH 4.9.

WIKÉN and PFENNIG (1957) have examined the rate of

alcoholic fermentation in succinic acid-succinate buffers of various concentrations within the range of 0.04 M to 0.3 M. They found that at low' concentrations , viz., 0.04 M to 0.08 M, no normal Pasteur effect at all or only a small one was observed. A further increase in the concentration

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of the buffer brought about a marked decrease in the anaerobic fermentation and a subsequent increase in the magnitude of the negative Pasteur effect. Resting cells from young cultures suspended in distilled water or in solutions of primary po-tassium phosphate or in buffers of organic acids other than

succinic acid, of the same pH and in the same concentration range, did not demonstrate this phenomenon. Further cha-racteristics of the negative Pasteur effect as observed and studied by the above-mentioned authors, in a strain of

Sac-cha1'omyces earlsbergensis ,

shall be cited when we discuss

our own findings on this effect in a strain of

Saccharomyces

uvarum

Beijerinck.

From the work thus far presented in the literature. no definite conclusions as to the actual mechanism of the inhibi-tion of fermentainhibi-tion under anaerobic condiinhibi-tions, as demon-strated in yeasts of the genus

Saccharomyces,

can be drawn. A hypothesis on the mechanism of the negative Pasteur effect

in yeasts of the genus

Brettanomyces

can be formulated

from the results presented by SCHEFFERS (1961) and WIKÉN et al.. (1962). Further, two main differences in the negative Pasteur effect in yeasts of the two genera have thus far been observed. Firstly, in yeasts of the genus

Saeeharomyces,

the effect is specific for succinic acid-succinate at physiological concentrations, whereas, in yeasts of the genus

Brettanom-yees,

no specificity is involved. Secondly, in yeasts of the

latter genus, there is an interdependence of the magnitude of the negative Pasteur effect on the external potassiuml sodium ion ratio, whereas. in yeasts of the former genus, there appears to be no such dependence. No other differences in the effects, as found in yeasts of these two genera have, as yet, been shown.

The Aim of the Present Investigation

The primary objective of this study is to evolve and elucidate the mechanism of the negative Pasteur effect of the type demonstrated in yeasts of the genus

Saccharomyces.

In pursuing this aim we hope, in addition, to be able to observe new differences and/ or similarities between the ef-fect as observed in yeasts of the genus

Saecharomyces

and that found in yeasts of the genus

Bretlanomyces.

Such in-formation should be useful in ascertaining whether the two effects are related or not.

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MATERlALS AND METHODS

1.

Materials

The compounds used in the preparation of the culture media were obtained from the following firms: (+)-biotin puriss. and pyridoxal hydrochloride puriss. froIl} Fluka AG, Buchs SG, Switzerland; d-pantothenic acid, calcium salt, from Merck & Co. !nc., New Jersey; citric acid p. a. from E Merck AG; Darmstadt; inositol from Eastman Organic Chemicals, New York; {3-alanine and nicotinic acid U. S. P. from S. A. F . Hoffman-La Roche & Co. Ltd., Basel;riboflavin U. S. P. and thiamine hydrochloride U. S. P. from General Biochemicals Inc. , Ohio;and casein hydrolyzate, vitamin free, from Difco Laboratories, Michigan, The glucose was of a technical grade. All other compounds used in the preparation of the culture media were of analytical quality.

The compounds used in the fermentation experiments were obtained from the following sources: glucose, pure vitamin free, S. A. F. Hoffman-La Roche & Co. Ltd., Basel; 2-de-oxyglucose (2-DG), A grade glucose free, from the Cali-fornia Corporation for Biochemical Research, Los Angeles; glucose-6-phosphate (G-6-P) as the sodium salt, fructose-phosphate (F-P) as the barium salt, and fructose-I, 6-diphosphate (F-l, 6-P) as the disodium salt from Boehringer '

& Soehne, GmbH, Mannheim; phosphoglyceric acid (PGA)

as the barium salt. from Schwartz Laboratories !nc., New York; dihydroxyacetone (DHA) cryst. , adenosine triphosphate (ATP) as the disodium salt, fumaric acid puriss. , and maleic acid puriss. from Fluka AG, Buchs SG, Switzerland; ade-nosine diphosphate (ADP) as the sodium salt from Nutritional Biochemical Corp., Ohio; diphosphopyridine nucleotide oxid-ized (DPN) and reduced .(DPNH) from the Koninklijke Neder-landsche Gist- en Spiritus Fabriek, Delft; succinic acid p. a. from the Amsterdamsche Chininefabriek, Amsterdam; L-malic acid from L. Light and Co. Ltd., Colnbrook; and citric acid p. a. from E Merck AG. Darmstadt. The barium salts of F-6-P and PGA were converted to their sodium salts before use. All other compounds utilized in the fermentation ex-periments were of analytical quality. The solvents employed in chromatography were redistilled prior to use.

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2. Organisms

We have chosen from the yeast culture collection of the Centraalbureau voor Schimmelcultures in Delft several Saccharomyces species, two Candida species, and one Hanse-nula species for this study. A large number of Saccharomyces species were selected in view of th"e fact that a negative Pasteur effect had already been observed in strains of at least three species of this genus (see WIKÉN, 1961). The Candida and Ha1lsenula species were inc luded for

compa-rative purposes in the early stages of this work and were later, with all but one of the Saccharomyces species, eli-minated from further study. We have purposely not included any Breltanomyces species because the negative Pasteur effect in these yeasts is already the subject of a detailed investigation (SCHEFFERS, 1961; WIKÉN et al., 1961).

The yeasts chosen wère the following:

Candida pulcherrima (Lindner) Windisch CBS 2240 Strain received from Porchet (Switzerland) in 1938,

isolated from cherries

Candida pulcherrima (Lindner) Windisch Strain received from Mrak (USA) in 1940, isolated from grapes

Hansenula ([l1omala (Hans en) H et P Sydow Strain received from Fabian (USA) in 1934, isolated fr om m2.ple syrup

SaccharoJl1yces ca1'lsbergensis Hansen

Hansen's authentic strain received from Helm (Denmark) in 1947

CBS 2245

CBS 442

CBS 1513

SacclzaroJl1yces cere1.:isiae Hansen var ellipsoideus CBS 1311 (Hans en) Dekker

Strain received from Atwood (USA) in 1936, used in manufacture of whiskey / .

Sacclzaromyces"cerevisiae Hansen var elliPsoideus CBS 1396 (Hansen) Dekker

Strain 'Champagne cramant' receivecj from Schoen (France) in 1926 /,1.,

Sacclzaro17l\'ces clzet'alieri Guilliermond Strain (Sac'clzaromyces lil1dl1eri) sent by GuilliE;!rmond (France) in 1925

CBS 405

Sacc1zaro17lyces ot'iformis Oster\\"alder CBS 1250 Strain received from S::::handerl (Germó.ny) in 1938

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Sacclwrom)'ces rosei (Guilliermond) Lodder et CBS 81 van Rij

(Torlllospora rosei Guilliermond) original strain l'cceived from Guilliermond (Franée) in 1927

Sacclwrom)'ces ltl'aYUIIl Beijerinck CBS 377

Strain obtained from Osterwalder (Switzerland) in 1934

Sacc!wromyccs ut'aYWIl Beijerinck CBS 426

Strain l'eceived from Verona (Italy) in 1947, isolated from honey

The nomenclature is in agreement with that of LODDER and KREGER-VAN RIJ (1952).

3. Media Used for GYoll'illg the Yeasts

The media employed for the cultivation of the organisms were those used by WIKÉN and RICHARD (1951).

MediulIl-A Glucose 20.0 g Malt extract 5.0 g NH-lCl 0.5 g KH2P 0-l 0.5 g MgS0-l.7H;:O 0.5 g

FeCl3 solution, 1 mg FeC1 3 /ml 1.0 mI

Distilled water to 1000 mI

The mixture was heated for 20 min at lOOoC, cooled, fil-tered till c lear through the same filter paper and made up to volume with distilled water. The pH was adjusted to 4.8-5.0 with sodium hydroxide.

Medium B Glucose 50.0 g (NH-!)2 S0-l 6.0 g KH2P 0-l 2.0 g MgS04 · 7H2O 0.25g CaCl2 0.25g H3 B 03 solution, 1 mg H3BÜ:J /ml 1.0 mI ZnS04 solution, 1 mg ZnS04/ml 1.0 mI

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MnCl z solution, 1 mg MnCl z /ml 1. 0 mI Tl z S04 solution, 1 mg Tl zS04 /ml 1.0 mI FeCl3 solution, 0.5 mg FeC13/ml 1.0 mI CuS0 4 solution, 0.1 mg CuS04 /ml 1. 0 mI :<1 solution, 0.1 mg KI/mI 1. 0 mI

Distilled water to 1000 mI

The glucose was dissolved separately in 250 mI of distillea water; the other ingredients in 700 mI of distilled water.

The two solutions were autoclaved at 120°C for 20 min after which they were cooled, filtered, and combined. Thc pH of the solution was adjusted to 4. 8 with potassium hydroxide and the volume made up to 1000 mI with distillcd water. Medium C Glucose 50.0 g KHzP04 0.55 g KCI 0.425 g CaCl z ·2Hz O 0.125 g MgS04· 7HzO 0.125 g FeCLj.6H z O 2.5 mg MnS04·4HzO 2.5 mg Citric acid 4.5 g

Casein hydrolyzate, vitamin free,

10% w/v solution 40.0 mI

Distilled water to 1000 mI

The ingredients were dissolved in 700 mI of distilled water and the pH adjusted to 4. 5 with potassium hydroxide. The solution was then autoclaved at 120°C for 20 min after which it was cooled a!1d filtered twice through the same filter paper. The pH was then adjusted to 4.8. Finally, the volume was made up to 1000 mI with distilied water.

Throughout this work four growth factors were mainly used,

\'iz. , biotin, calcium pantothenate, meso-inositol, and {3-alanine at concentrations of 5 ~g/ 2.5 mg/l, 25 mg/l, and 2.5 mg/l, respectively. They ~\'ere add€d immediately

prior to making up the volume of the medium to 1000 mI. Final sterilization of all media was accomplished with

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three consecutive steamings for 20 min at 100°C, 24 hr apart.

4. Pre/Jaratiol1 of Sllspel1siol1s of Non-Proliferalil1g (Resting) Cells

Our reference stock cultures were maintained on malt agar s lopes at 25°C and were transferred every two months. In the preliminary experiments with the various yeasts, working stock cultures were maintained in Medium A. These were transferred every 96 hr. In the particular experiments with SacclwroJ/l)'cC's UVanl1l1 Beijerinck, CBS 426, the stock cultures were grown in Medium B, with and without the addition of the four above-mentioned growth factors, as weIl as in Medium C without any addition of growth factors.

The cells examined for fermentative capacity and respi-ratory ability (uptake of gaseous oxygen) were grown in cultures set up with 125 mI of medium in 250 mI Florence flasks. These were inoculated with four mI of a ceU suspen-sion from a stock culture. The flasks were then shaken at 400 rev/rnin on a rotary shaker or left standing, with occasional shaking, at 25°C. Af ter various times of incubation, the ce Us were harvested by centrifugation at 3000 rev

I

min for 15 min at room temperature, washed twice with distiUed water, once in the proper buffer, and resuspended in the same buffer. The particular experiments with SacclzaroJ/lyce s

lll'arlllll Beijerinck, were performed mostly with cells from shaken cultures grown in Medium B supplemented with biotin, calcium pantothenate, meso inositol, and j3-alanine.

In the manometric determination of the fermentative and respiratory abilities of yeast cells, a final concentration of approximately 1. 7 mg of dry ceU substance per mI of sus-pension was generally used; in the volumetrie measurements it was 17.0 mg of dry ceU substance per mI of suspension.

5. Preparatioll of Cell-Free Extracts

We are in this study mainly interested in the alcohol fermentation of yeast. For a comparison of the activity of ceU-free extracts with that of intact ceUs, the extracts must contain aU enzymes essential for the glycolytic process mentioned in a highly active state and in the proper propor-tions . In later sections it shall be shown that the fermentation of glucose with whole ceUs and with ceU-free extracts of the particular yeast examined, viz., Saccharomyces llVarUnl

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proceeding according to the Parnas-Embden-Meyerhof scheme and giving ethyl alcohol and carbon dioxide as end products. We have used various methods of mechanical disintegration of cells in trying to obtain an extract with the above-men-tioned characteristics. The glycolytic activity of the extracts was determined by adding 2% w

Iv

of glucose, final concen-tration. to the extract and measuring subsequent carbon dio-xide production. Acetone-dried cells were ground with alumina in achilIed mortar at 4°C (McILWAIN, 1948). After five min grinding and addition of

o. OlM pho sphate so lution, pH 7. 0,

we obtained a suspension with only a few ruptured cells.

In an attempt to increase the percentage of disintegrated cells, we subjected, for various intervals of time, a thick cell suspension in phosphate solution to vibrations at a frequency of approximately 10 kilocycles per sec in an atmosphere of air or nitrogen. The source of the vibrations was the Raytheon sonic oscillator (CAMPBELL and SCHOEN-LEBER, 1949; REPASKE and WILSON, 1953). The results, however, proved to be indifferent. At one time, the percentage of cells ruptured approached 50% and the extract was active, 5 t-ll of carbon dioxide being formed per hr per mg of protein; at other times, under identical experimental conditions,· very little rupturing of the ce lls occurred and the extract was completely inactive. The Mickle shaker (MICKLE,1948), with ballotini beads, also proved ineffective due to the fa ct that a very low percentage of the cells were disintegrated.

Similarly, cell suspensions, with and without added car-borundum, were mostly left intact af ter passage through the Hughes press (HUGHES, 1951). Both of the latter meth-ods gave extracts with very low activity, viz., 0.05 to O. 1 t-ll of carbon dioxide per hr per mg of protein.

Due to one or more reasons the above-mentioned methods proved to be unsatisfactory for our particular nee ds. Final-ly we tried the method of HOFSTEN and TJEDER (1961). They described a dry ice homogenization technique for obtaining active extracts from yeast. This method, with a slight modification, gave excellent reproducible results and was used throughout this work to obtain cell-free extracts Four mI of a stock culture in Medium B, supplied with the four growth factors, were used to inoculate each of twenty 500 mI Florence flasks containing 250 mI of fresh medium. The cells were harvested from the shaken cultures after 24 hr incubation at 25°C and washed twice with dis-tilled water. The cells were then resuspended in a small volume of distilled water and filtered. with suction, till dry. The resultant yeast cake was frozen in dry ice.

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homoge-nizer, type Braun Multimix. The frozen yeast cake was

broken up into small lumps and. added to the powdered ice in

the homogenizer and the mixture disrupted. Because the contents tended to adhere to the walls of the container, five one min intervals were used for homqgenization with tiine in between to jar loose the contents from the walls.

The finely powdered mass was transfer red to a beaker which was then placed in streaming cold water. The re-sulting melted paste was transferred to a centrifuge tube and the beaker rinsed with a little distilled water. The washing was added to the paste and the mixture centrifuged at 0 °c, in a refrigerated centrifuge, for 20 min at 8000 rev /min (8170 x g); the resulting supernatant solution was decanted and stored at 4° C. The residue was frozen in dry ice, homogenized, and defrosted as above. The resulting paste, with the small amoum of wash water, was added to the supernatant and the mixture centrifuged at 16000 rev /min (32700 x g) for 100 min, at the end of which time the super-natant was coUected and stored, till needed, at -15°C. Fractional centrifugation of the extract will be described in another section.

The slightly modified biuret me,thod of WEICHSELBAUM (1946), as described by LA RIVIERE (1958), was used to determine the protein content of the extracts •

6.

Techniques lor Measuring Fermentation

The standard manometric techniques developed byWarburg (cf. UMBREIT et al., 1957) and the volumetric techniques

according to von Euler, Myrbäck, Nilsson, and Alm (cf.

NILSSON, 1941) were used in measuring the fermentative rates, while only the former method was used in deter-mining the oxygen uptake in respiration. Anaerobic conditions were obtained by flushing the cell suspensions with oxygen-free nitragen; aerobic conqitions, by shaking the cell sus-pensions in air. The fermentation flasks were shaken at a rate of 100 complete oscillation per min at 25°C.

Unless otherwise stated, glucose at a concentration of 2% w/v was used as the carbahydrate to be metabolized.

In· the preliminary experiments the nitrogen was freed

from the last vestiges af oxygen by passag.e of the gas

through .an alkal~e pyrogallol solution (KÛSTER, 1921;

METZGER and MULLER, 1959) and an alkaline hyposulfite

solution containing sodium anthraquinone-ê-sulfonate as

catalyst (FIESER, 1924; METZGER and MULLER, 1959). The effluent gas from the vessels was tested for the presence

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of oxygen with Fildes' alkaline glucose-methylene blue solution (FILDES, 1931>.

In the later experiments, a palladium catalyst was used

for the removal of traces of oxygen from the cylinder rutrogen gas (ZELINSKY and BORISOFF, 1924; WINKLER and BRUNCK, 1927). The catalyst was prepared as follows. Twenty grams of palladium chloride were dissolved, with

slight warmi.lg, ln a minimum of water. Sixty mI of 40%

formaline were then mixed with the dark brown solution. Sub s equently , asbestos was added till the solution was com-pletely absorbed and a jelly-like mass obtained. Seventy to 80 mI of 40% sodium hydroxide solution were then cautiously added, with stirring, till the mass no longer darkened. At this point all of the palladium had precipitated out on the asbestos. The mixture was placed on a boiling water bath for one hr and then left overnight at room temperature. The following day the mixture was filtered and washed with 10% acetic acid solution and finally with water. The residue was

dried at 100-110oC and broken up into small slivers.

The catalyst receptacle, with a glass wool layer at the bottom, was filled with the catalyst to a depth of about 8 cm. The catalyst was loosely packed to ensure an undisturbed and even gas flow.

Before use the catalyst was saturated with hydrogen. Prior to this, the catalyst was flushed with nitrogen gas to remove most of the oxygen in the receptacle. This was do ne to prevent excessive overheating of the catalyst with possible formation of large cinders and subsequent decrease in effectiveness.

Passage of the purified nitrogen gas from the catalyst tube to the fermentation vessels was through glass tubing joined

together, where unavoidable, w~th 1. 5 mm

thickpolyvinyliden-echloride polymer tubing (trade name: Saran). The nitrogen

gas .was blown through the vesseis at a rate of 100 mI per

mjn. A Warburg apparatus with attachments for flushing the vessels with nitrogen can be seen in Figures la and lb.

Figure la: Warburg apparatus with attachments for flushing vessels with nitrogen. The cylinder nitrogen is purified once by passage through a common catalyst and once again by passage through separate catalysts, one per manometer.

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Warburg"" A..-.---..._

ma_t.rv ... T~

.1

Figure lb: Detail of the separate catalysts.

glass wool

t~:

separate

.:{' ealalyst

:-:.

to vess.l

With the galvanic method of HERSCH (1956) for the meas-urement of gaseous oxygen we determined the purity of the nitrogen af ter passage through the catalyst as weU as the oxygen content of the atmosphere in the fermentation vessels

af ter flushing them with the purüied nitrogen for various intervals of time.

The 'dry' galvanic ceU used cortsisted of a lead foil as anode and silver gauze as cathode with a 5 N potassium hydroxide solution as electrolyte. The reaction taking place in the galvanic cell when oxygen is passed through may be represented as foUows:

Silver electrode:

t

02 + H 20 + 2e - 2

OH-Lead electrode: Pb + 3 OH- - Pb0 2H- + H 20 + 2e A detailed diagram of the galvanic ceU can be seen in

Figure 2d. When aU components of the element were.

fastened in place with silver wire, the element was placed ih a tubE: and flooded with a 5 N potassium hydroxide solu-tion. Vacuum was then applied tiU cessation of bubbling in the liquid. At this time the element was completely saturated with the base. The element was then quickly placed in its container and the lead and silver electrodes short-circuited, with platinum wire, over a 100 ohm resistance. The incoming

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gas was bubbled through a solution of 5 N potassium hy-droxide to prevent drying out of the cello

At oxygen concentrations between 10-5 and 10-3 percent by volume (vol %), the element produces a current directly proportional to the oxygen content. This current, in turn, produces a propordonal drop in the voltage over the 100 ohm resistance. This voltage drop was then autoroatically recorded on a Philips mV recorder, PR 4069,M/04. Diagrams and photographs of the apparatus 1) can be seen in Figures

2a, b, c, and d. 145 V oe'} Jl-ll-+----' + vol tage stabilizer resistances A: 3.83 ohms B: 15.5 ohms . C:l00 ohms "or more 5 !'IN recorder switches choice betwHn I : resistancn A and B n :cirC'IJit with electrolyt ic ceU

and that wilh measuring element

Figure 2a: Electrical circuit of the oxygen measurement apparatus.

The standard mixtures of nitrogen and oxygen used in

calibratin~ the galvanic cell were obtained in an electrolytic cell according to the method of HERSCH (1956). A 5 N solution of potassium hydroxide was electrolyzed, under standard conditions, between platinum electrodes (Figure 2c). The apparatus must, of necessity, be free from oxygen when calibration curves are being prepared. This was accomplished by incorporation of a palladium catalyst into the apparatus (Figure 2b). The nitrogen gas · entering the system would then be completely free of oxygen. When, however I the oxygen content of a gas mixture was to be

1) My thanks are due to Messrs. J.C.A. Knetemann and J. Doodewaard. Chemical Engineers, for their help in these experiments. A detailed repon on. the use of the pafladiuJI1 catalyst and the oxygen measurement apparatus will soon be published by WIKf:N et al. (1963).

I also wish to tltank Professor Dr. P. Karsten for advice and help in the design and construction of the particular apparatus used in this investigation.

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Figure 2b. Oxygen measuremen. apparatus. 1: Catalyst; 2: by-pass: 3: electrolytic eeU: 4: galvanic eell: 5: flowrator.

I & Ir: plat inum eled rodes :m:5 N KOH solutlon

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N KOH solution

., N . . ,...,.t fllt., ~ Whatrnan Hr 50

::;~l:' C':r'1Vin,l chloride tublng.

Figure 2d: Detail of galvanic cello

deterrnined, the mixture was, of course, allowed to by-pass the catalyst and enter the system directly. The rate of flow of the gases through the apparatus was set at 100 mI/per min.

When we wished to measure the oxygen content in the vessels during the time of flus hing with oxygen-free nitrogen by passing the effluent gas fr om one of the vessels through the measuring apparatus, we discovered that the gas would not flow through the system due to the resistance of several centimeters of potassium hydroxide solution. Ta overcome this difficulty, a vacuum pump was attached to the outlet of the apparatus and suction, sufficient to overcome this resistance, applied.

7.

Chromatographic Procedures

Two methods of chromatographic separationof the various compounds were tested, the one being column chromatography, the other paper chromatography. With column chromatography silica gel (ELSDEN, 1941; ISHERWOOD, 1941; MARVEL and·

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RANDS, 1950; BULEN et al., 1952; DAJAN and ORTEN, 1958) as weU as ion exchange resins (PALMER, 1955) were used as the adsorbing solids with different solvents as the eluents. Although these methods were quite suitable for separation of certain compounds, they were discarded due to mechanical difficulties with our fraction collector. With paper chromatography, the descending technique was used. Whatman no 1 chromatographic paper, 58 x 68 cm, without further pretreatment was employed throughout. Samples were brought unto the paper with a standard platinum loop and the number oi applications to one spot varied from 15 to 25.

Various developing solvents were tried to obtain both proficiency in the manipulations and a suitable solvent For separation of the acids of the Krebs citric acid cyclE., the developing solventfs tested contained n-butyl alcohol with chloroform (DONALDSON et al., 1952, or glacial acetic

acid (WOIWOD, 1949) and ethyl alcohol with ammonia

(BROWN, 1950; ELLIOTT, 1954) or ammonia, mesityl oxide and formic acid (JAMES and ELLIOTT, 1955). A combina-tion of mesityl oxide and formic acid without ethyl alcohol was also used (BRYANT and OVERELL, 1953). In addition, n-propyl alcohol with ammonia (PANEK, 1959) and n-amyl alcohol with formic acid (BUCH et al., 1952) were tried. Although the above-mentioned methods were all quite suitable for the separation of the di- and tricarboxylic acids, I chose, because of the slightly better separation and ease of ma-nipulation, the method of BUCH et al. (1952) for the routine separation of organic acids. The method was as follows. The solvent, n-amyl alcohol - 5 M aqueous formic acid in a ratio of 1 : 1 v/v, was prepared at least three hr before use. The aqueous phase was employed for equilibration; the organic phase, for development. Thj<; was complete in 24 to 36 hr. The chromatograms were then removea from the developing tank and allowed to dry. at room temperature for 24 hr. The organic acids were localized by spraying the dried chromatograms with a solution which was 0.05 N as regards sil ver nitrate and ammonium hydroxide (BUCH et al., 1952) and leaving the chromatograms in the dark for four hr. The acids appeared on a brown background as differently coloured spots or, where the concentration was too great, as bands.

With radioactive material, the development was the same as above. Af ter the localization and identification of the spots, the chromatograms were, in the dark, ·laid. flat and in close contact with X-ray film, Ilford Nuclear· Research Emulsion, G.5, for 14 days. The X-ray film was removed and developed. The spots containing radioactive material were detected as exposed areas .on the film.

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Similarly in the chromatographic separation of substances of a carbohydrate nature many different solvents and sprays were tested. The solvents included water nbutyl alcohol

-pyridine (JEANES 'et al., 1951), water nbutyl alcohol

-glacial acetic acid (PARTRIDGE, 1949), and iso-propyl

al-cohol - glacial acetic acid - water (FEINGOLD, 1956).

A greater variety of spraying reagents wer€ tried and these

included solutions of periodate - permanganate (LEMIEUX

and BAUER, 1954), meta periodate - benzldine (CIFONELLI and SMITH, 1954), benzidine alone (BACON and EDELMAN, 1951), phloroglucinol (BORENFREUND and DISCHE, 1957)", anthrone (JOHANSON, 1953) and p-anisidine hydrochloride (HOUGH et al., 1950). With the exception of the first two spraying solutions, which often did not clearly localize the spots due to reaction with the developing solvent and the paper, all the sprays were quite suitable. For the routine separation, however, I chose the method of FEINGOLD et al. (1956) for developing the chromatograms and the solution of HOUGH et al. (1950) for detecting the acids.

The developing solvent was iso-propyl alcohol - glacial acetic acid - water in a ratio of 7 : 1 : 2 v/v. Develop-ment time was 24 to 36 hr at room temperature. Mter development the chromatogram was dried at room tempe-rature . The chromatogram was then sprayed with a four

% w/v solution of p-anisidine hydrochloride in n-butyl

al-cohol. Subsequently, the chromatogram was placed in a thermostat at 100°C for five min. The various carbohydrate substances appeared as differently coloured spots on a

light-brown background. With radioactive material, the

procedure was the same as that mentioned for organic acids. For the separaLon of the amina acids the developing

sol-vent used was phenol - water in a ratio of 4 : 1

wl

v

(BLOCK et al., 1952). A beaker containing 100 mg of

sodium cyanide in four to six mI of water was placed in the developing chamber to prevent too rapid a decomposition of the phenol (BLOCK et al., 1952). Development time was

24 to 36 hr. Mter development, the chromatograms were

dried at 40°C in a current of warm air and then sprayed with a 0.1% w/v solution of ninhydrin in n-butyl alcohol

(CONSDEN et al., 1944; BERRY. and CAIN, 1949). The

chromatograms 'were then placed in a thermostat at 110°C

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EXPERIMENTAL RESULTS WITH WHOLE CELLS

1.

Preliminary Fermentation Experiments

a. Screening for the Negative Pasteur Effect

As previously mentioned, CUSTERS (1940) was able to

demonstrate a negative Pasteur effect in cells of

Brettanom-yees claussenii

Custers suspended in phosphate solutions. A

screening of the different yeast strains, listed on p. 13, for the presence of a negative Pasteur effect was, therefore, performed with suspensions of these organisms in 0.16 M primary potassium phosphate solution at pH 4.5.

Samples of one mI were removed from the stock cultures of the various yeasts in Medium A, streaked on malt agar plates and incubated at 25°C. Af ter various times of incuba-tion the cells were harvested and further treated as described on p. 16.- The final suspension had a density of approximately 15 mg of wet yeast per 1.8 mI of phosphate solution. Fer-mentation was measured manometrically with 30 min flushing of the resting cell suspension with oxygen-free nitrogen or shaking in air prior to the addition of 2% w/v of glucose. The results of this experiment can be seen in Table 1.

From the results in Table 1 we see that in 0.16 M primary.

potassiumphosphate solution, pH 4.5, a normal Pasteur effect was recorded with resting cells of all yeast species irrespec-tive of their age. Thus a negativè Pasteur effect of the type observed i;l yeasts of the

Brettanomyees

species (CUSTERS, 1940; WIKEN et al., 1961) does not appear to be of a general occurrence in yeasts. WIKÉN and RICHARD (1953 a, b) were also unable to observe a negative Pasteur effect with resting cells of the Salenegg strain of

Saeeharomyees uvarum

Beije-rinck, CBS 2986, the D~zaley strain of

Saeeharomyees

eere-visiae

Hansen, CBS 2987, and a strain of

Saeeharomyees

earlsbergensis

Hansen, CBS 2834, isolated from a Fendant

stf'rter, when these cells were suspended in primary potas-sium phosphate solution but did, however, observe an inhibition of the anaerobic fermentatiC'n in resting cells of the same yeasts suspended in 0.16 M succinic acid-sodium succinate buffer, pH 4.8-4.9. In view of this, we decided to examine

whether our

Saeeharomyees

species would produce similar

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ages and suspended in 0.16 M solution of primary potassium phosphate, pH 4.5.

Species Strain Fermentation Fermentation

CBS no conditions in plof C02 per mg of yeast dry substance per 150 min age of cultures in hr

12 15 18 24 36 42 48 60 62 72 120

Cantida 2240 Anaerobic 97 212 292 182 37

EUlc errima Aerobic 48 38 40 18 8

Candida 2245 Anaerobic 252 155 187 105 90 Eulcherrima Aerobic 58 38 45 22 29 Hansenula 442 Anaerobic 55 100 160 ~ Aerobic 22 13 20 SacchaI2ll)y~ 1513 Anaerobic 65 74 242 80 120 ~g~ Aerobic 15 42 38 20 48 Saccharomy~ 1311 Anaerobic 639 318 200 118 I cerevisiae Aerobic 218 130 45 20 N -::J 1396 Anaerobic 130 210 287 95 I Saccharomy~ cer~visiae Aerobic 102 152 80 50 Saccharomy~ 405 Anaerobic 167 405 212 135 212 chevalieri Aerobic 80 80 32 53 ~il~chIHl2my~ 1250 Anaerobic 68 28 332 92 85 oVltOrmis Aerobic 60 30 260 15 40 Saccharomyces 817 Anaerobic 160 247 300 120 89 ~ Aerobic 100 40 85 22 20 Saccharomyces 377 Anaerobic 266 55 172 238 172 !!r!!!!!ll Aerobic 210 30 110 105 58 Saccharomy'ces 426 Anaerobic 197 85 569 220 147 ~ Aerobic 103 54 230 50 56

The cells were harvested, at the times indicated in the Table, from malt agar plates incubated at 25OC. The resting cell suspensions were flushed _ with oxygen-free nitrogen (Anaerobic) 0<5 shaken in air (Aerobic) for 30 min prior to the addition of 'l'/o w/v of glucose. Fermentation was measured at 25 C with the Warburg te<;}mique. The

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one set of conditions, we preferred to test one strain under

a great variety of conditions. S.

uvarum,

CBS 426, was

ar-bitrarily chosen for this purpose. The cells which were harvested at various times from malt agar plates were sus-pended in succinic acid-sodium succinate buffer and primary potassium phosphate solution, for comparative purposes, of different molarities at pH 4.5. The anaerobic and aerobic fermentation rates were measured after 30 min flushing with oxygen-free nitrogen or shaking in air in the absence of glucose. The results of these experiments can be seen in Table 2.

TABLE 2

Influence of an increase in the molarities of succinic acid-sodium succinate buffer and primary potassium phosphate solution. pH 4.5, on the anaerobic and aerobic

fermen-tation of glucose by resting cells of ~~. CBS 426. Age of cultures from which resting cells were obtained hr 18 24 36 48 96 18 24 36 48 96 Fermentation

in plof CO2 per mg of yeast dry substance per 150 min

resting cells suspended in

solution of ~rimary potassium succinic acid-sodium succinate

osphate buffer M M 0.02 0.16 0.34 0.50 0.02 0.16 0.34 0.50 Anaerobic 339 354 623 628 347 3 16 0 660 971 790 487 866 734 11 8 448 511 396 368 632 212 3 2 912 151 658 327 215 170 29 23 6 2 3 4 4 3 8 3 Aerobic 309 333 587 519 380 53 14 0 858 903 550 366 702 717 61 23 535 474 564 294 709 194 62 4 '752 95 557 267 92 104 57 12 5 4 1 1 9 10 4 5

The cells were harvested, at the times indicated in the tabie. from malt agar plates incubated at 25OC. The resting cell suspensions were flushed with oxygen-free nitrogen (Anaerobic) or shaken in air (Aerobic) for 30 min prior to the addition of '2f1/o w Iv of

glucose. Fermentation was measured at 250C with the Warburg technique. The results were not corrected for oxygen uDtake. There was practically no endogenous fermentation.

As can be seen from the results in Table 2, varying thc concentration of the primary potassium phosphate solution dlu not, in general eliminate the normal Pasteur effect in

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however, vary with the age of the cultures as wen as with the concentration of the phosphate solution. The cens which were harvested after 24 hr growth on malt agar were the most active under both anaerobic and aerobic conditions. In

general, the activity decreased with further increase in the age of the cultures till cens harvested from 96 hr cultures exhibited practically no fermentation activity. Irregularities in the results are probably due to the fact that cens harvested from malt agar plates have not all reached the S&flle stage in development. Whereas the cells in the b0ttom layer of the colonies have an abundant supply of nutrients but only a limited supplyof air, the cells in the uppermost 1ayer have the oppo-site, viz., an abundant supply of air but only a limitedsuPPly of nutrients. Between these two layers there are innumerable different growth conditions all of which contribute to physio-logical variations in the one culture.

Further from the results in Table 2 we see that the an-aerobic and an-aerobic fermentation of glucose by resting cells suspended in succinic ::l.cid-sodium succinate buffer of vary-ing concentration is greatly altered from that by restvary-ing cells suspended, under identical conditions, in phosphate solutions. Resting cells fr om 18 hr cultures produced more carbon dioxide in 0.16 M succinic acid-sodium succinate buffer under aerobic than under anaerobic conditions, 53 and 3.u1 per 150 min, respectively. Similar results were noted with resting cells from 24 hr cultures suspended in 0.34 M and O. 5 M succinic acid -sodium succinate buffers where the aerobic and anaerobic fermentation rates per 150 min were ,61 and 11 .uI and 62 and 3.u1, respectively. An exception was the fermentation in 0.02 M succinic acid-sodium succinate buffer. The anaerobic fermentation of resting cens from 24 and 36 hr cultures was greater in 0.02 M succinic acid-sodium succinate buffer than in 0.02 M primary potassium phosphate solution. Aerobically, resting cells from 18 and 36 hr cul-tures produced more carbon dioxide in the 0.02 M succinate buffer than in the corresponding phosphate solution.

Although we have throughout this work measured the oxygen uptake by the resting cells suspendedin the various buffers, we have not included the results obtained in the proper tables because we considered them to be of little i~portance in the elUcidation of the problem dealt with. WIKEN et al.

(1961) have considered the corrections that are involved in determining the aerobic glycolysis in yeasts that have prac-tically no or a low to high respiration. The result was th at the introduction of these corrections ultimately led to a higher value for the aerobic glycolysis than tha,t directly measured. This in turn led to, in some instances, a

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sub-800

stantial increase in the magnitude of the negativE' Pasteur effect. We we re fortunate, in these preHminary experiments and more so in the later experiments, to observe a large negative Pasteur effect without recourse to these corrections. I must, however, add th at a correction for the respiration

should be applied in instances where we wish to obtain the absolute value of the negative Pasteur effect.

In these preliminary experiments, fermentation has been recorded as tll of carbon dioxide produced per mg of yeast

dry substance. This method of stating results is very com-mono It may, however, under certain conditions lead to erroneous conclusions. A cell mass consists of living and dead cells. Should the activity of the living cells be averaged out with the complete inactivity of the dead cells? From the results in Figure 3 onE' can see tne different conclusions which may be drawn when the data are recorded in these two ways. The viabie cell content of the resting cell sus-pt::nsions was determined by direct count with methylene blue solution (FINK and WEINFURTNER, 1930) and by diluting and plating a sample in malt agar. The results obtained from these two methods agreed to within seven

%.

A

Figure 3:

72 96

9

24 48 72 96 AGE OF CELlS (HOURS)

C o

ul C02 per mg VI.bl. y .. st ul C02 per mg vlabl. r.ut 10000

8000

AGE OF CELLS

Anaerobic fermentatioo of glucose by resting cells of S. uvarum, CBS 426. suspended in 0.16 M (Curve 1), 0.34 M (Curve 2), O-:-s-M(Cutve 3) ór'

0.68 M (Curve 4) succinic acid-sodium succinate buffer, pH 4.5. The cells were harvested from shaken (A and C) or stationary cultures (B and D) af ter 24 to 96 hr of growth (x axis) at 250C in Medium B with added biotin. calcium pantothenate. meso-inositol, and p-alanine at concentrations of 5 lig/i, 2.5 mg/l, 25 mg/l, and 2.5 mg/l. respectively. The resting cell suspensions were flushed with oxygen-free nittogen for 30 min prior to the additioo of 2 "10 w /v of glucose. Fermentation was measured at 250C with the Warburg technique. There was practicaUy no endogenous fermentauOll.

The data in Figure 3 show that the rate of anaerobic fermentation by resting cells harvested from a 24 hr shaken

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culture and suspended in 0.16 M SUCClnlC acid-sodium succinate buffer, as recorded per mg of viabIe cells (C curve 1), is approximately three times that recorded per mg of yeast dry substance (A curve 1); withrestingcells from 36 hr cultures it is 12.5 times greater. The activity of rest-ing cells obtained from a 24 hr shaken culture and suspended in 0.16 M succinic acid-sodium succinate buffer is about one

fourth th at of cells from a 36 hr shaken culture. as recorded

per mg of viabIe cells (C curve 1); whereas the activity is the same when recorded per mg of yeast dry substance (A

curve 1). Sinlilar statements can be made on comparing

ac-tivities of the resting cells from cultures of different ages. Thus when studying the differences in the activities of resting cells from cultures of different ages, the above

re-sults should be kept in minde If, however, a comparison is

made between the activities vf cells within one age limit, as is the case throughout most of the work in this thesis, then the results need not be converted from J.ll of carbon dioxide per mg of yeast dry sub stance to J.ll of carbon dioxide per mg

of viabIe cells. The difference in the activity will remain

re-latively the same because the same factor would be used in all the conversions. The discrepancy arises in the comparison of activities of resting cells from cultures of various ages where different conversion factors have to be applied. b. Effect of Aeration during Growth on Subsequent

Fermen-tation by Resting Cells under Various Conditions

We have shown that cells harvested from malt agar plates and suspended in succinic acid-sodium succinate buffer pro-duce, under certain conditions, a negative Pasteur effect. Further fermentation studies in succinic acid-sodium succinate and succinic acid-potassium succinate buffers were performed with cells harvested fromshaken and stationary cultures grown in Medium B with added biotin, calcium pantothenate, meso-inositol, and t3-alanine at concentrations of 5 f.lg/l,

2.5 mg/l, 25 mg/l, and 2.5 mg/l, respectively. We used

fluid medium and shaking conditions to eliminate as much as possible heterogeneous growth conditions by allowing equal

exposure of the yeast cells to the nutrients and oxygen· (see

also p. 29). Buffer systems containing potassium salts' and

sodium salts were used to see whether the sodium ions or

potassium ions would in any way influence the fermentation

of glucose by the resting cells. WIKÉN et al. (1961) have

shown th at the rate of fermentation with resting cells of

Brettanomyces claussenii,

in buffers containing mixtures of potassium and sodium salts of the acid concerned, increased

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with increasing potassium ion concentration and decreased with increasing sodium ion concentration at constant total salt concentrations. The results of a typical fermentation expe-riment with resting eells harvested from cultures of different ages and suspended in either succinic acid- sodium succinate or succinic acid-potassium suceinate buffer can be seen in Tables 3 and 4.

The data in Table 3 show th at cultivation of cells with increased aeration, or shaking, lowered the age limit under which the resting cells from these cultures were still ca-pable of producing a negative Pasteur effect in 0.34 M suc-cinic acid-sodium succinate and sucsuc-cinic acid-potassium succinate buffers, pH 4.5. The greatest negative Pasteur effect was noted with resting cells from an 18 hr shaken culture in 0.34 M succinic acid-sodium succinate and suc-cinic acid-potassium succinate buffers where the aerobic

and anaerobic fermentation rates were 100.8 and 8. 4 ~l and

86.6 and 5. 8 ~l, respectively. The aerobic and anaerobic

fer-mentation rates were nearly the same in resting cells har-vested from a 36 hr shaken culture and suspended in O. 34 M succinate buffer; whereas cells from a 72 hr stationary culture still exhibited a noticeable negative Pasteur effect. The sodium and potassium ions can be used interchangeably without causing too great a variation in the rates of fermentation. These latter results are in agreement with those obtained by WIKÉN and PFENNIG (1957). A normal or near normal Pasteur ef-fect was demonstrated with resting cells from shaken and stationary cultures of all ages suspended in 0.17 M succinic acid-sodium succinate and succinic acid-potassium succinate buffers, pH 4.5 (Tabie 4).

The rates of growth of the cultures under shaking and stationary conditions cannot be used here to explain differences in the anaerobic and aerobic fermentation rates because from the results of several of our experiments, not recorded here, we have seen that the growth rates are approximately the same in both instances. Microscopical examination of the growing cultures has shown that the cells begin to rupture slightly after 36 hr shaking. This latter phenomenon may aid in explaining the great decrease in anaerobic and aerobic fermentation rates (see Table 3) of resting cells in 0.34 M succinic acid-sodium succinate and succinic acid-potassium succinate buffers, pH 4.5, from 36 hr and older shaken cultures as compared with those of resting cells from sta-tionary cultures of the same age. The rupturing would per-mit the increased absorption of not only glucose but also of succinic acid-sodium succinate or succinic acid-potassium succinate. The inhibition may thus be due to an inactivation

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Anaerobic and aerobic fermentation of glucose by resting cens harvested from shaken and stationary cultures of ~. ~. CBS 426. and suspended in 0.34 M succinic acid-sodium succinate and

succinic acid-potassium succinate buffers at pH 4.5. Age of cultures from which resting cens were obtained hr 18 24 36 48 72 96 18 24 36 48 72 96 Fp.rmentation

in plof C02 per mg of yeast dry substance per 150 min resting cells originated from shaken cultures and

we re suspended in

stationary cultures and were suspended in SUCClfllC acid-sodium succinate 11.5 92.6 25.0 30.8 1.4 .5 125.5 170.5 21. 6 17.5 1.6 succinic acid-potassium succinate succinic acid-sodium succinate Anaerobic 8.4 6.6 79.4 74.1 12.5 133.9 19.0 128.1 2.0 69.8 9 6.8 Aerobic 100.8 94.7 189.5 221.1 13.5 177.8 9.8 165.4 2.9 89.1 succinic acid-potassium succinate 5.8 41.5 116.9 118.7 44.1 1.8 86.6 216.2 171. 3 184.9 58.8

The cens were harvested at the times indicated in the Table from shaken or stationary cultures grown at 250C in Medium B with added biotin. calcium pantothenate. meso-inositol. and p-alanine at concentrations of 5 pg/l. 2.5 mg/l. 25 mg/l. and 2.5 mg/l, respectively. The resting cell suspensions were flushed with oxygen-free nitrogen (Anaèrobic) or shaken in air (Aerobic) for 30 min prior to the addition of 'lfI/o w /v of glucose. Fermentation was measured at 250C with the Warburg technique. The results were not corrected for oxygen uptake. There was practically no endogenous fermentation.

I C..:l C..:l

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I

"'"

Cf.) I

Anaerobic and aerobic fermentation of glucose by resting cells harvested from shaken and stationary cultures of.2.. uvarum, CBS 426, and suspended in 0.17 M succinic acid-sodium succinate and

succinic acid-potassium succinate buffers at pH 4.5. Age of cultures from which resting cells were obtained hr 18 24 36 48 72 96 18 24 36 48 72 96 Felmentation

in lil of CO2 per mg of yeast dry substance per 150 min

resting cells originated from shaken cultures and

were suspended in

stationary cultures and were suspended in SUCClnlC acid-sodium succinate 234.5 230.9 212.7 164.0 75.7 .6 222.3 218.2 225.5 158.2 84.4 .7 succinic acid-potassium succinate SUCCilllC acid-sodium succinate Anaerobic 289.3 248.1 199.1 260.6 186.7 216.3 183.9 203.8 86.1 103.8 1.5 19.6 Aerobic 272.4 235.6 224.4 246.7 201.2 185.5 195.2 188.5 83.9 114.7 1.6 8.1 succinic acid-potassium succinate 178.9 184.7 234.1 256.6 101.6 21.7 178.5 190.3 183.7 239.3 112.6 16.9

The cells were harve~ted at the times indicated in the Table from shaken or stationary cultures

grown at 250C in Medium B with added biotin, calcium pantothenate, meso-inositol. and )J,-alanine

at concentrations of 5 IIgll, 2.5 mg/l. 25 mg/l, and 2.5 mg/l. respectively. The resting cell suspensions were flushed with oxygen-free nitrogen (Anaerobic) or shaken in air (Aerobic) for 30

min prior to the addition of '2P/o w Iv of glucose. Fermentation was measured at 250C with the

Warburg technique. The results wele not corrected for oxygen uptake. There was practically no endogenous fermentation.

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of one or more enzyme systems brought about by a decrease in the cellular pH or by the snccinic acid-succinate buffer itself.

c. Fermentation by Resting Cells Suspended in Different Solutions

In the previous sections we have shown that resting cells of S.

uvarum,

CBS 426, suspended in succinic acid-sodium succinate buffer, pH 4.5, demonstrated, under certain con-ditions, a negative Pasteur effect. Under otherwise identical conditions, however, this effect was not observed in resting cells suspended in primary potassium phosphate solution. The next series of experiments involves suspensions of resting cells of S.

uvarum,

CBS 426, in other organic acid buffer systems under conditions th at produced a negative Pasteur effect with succinic acid-sodium succinate buffer. The pur-pose of this was to determine whether the negative Pasteur effect with S.

uvarum,

CBS 426, is a characteristic exc1usive to resting cells suspended in succinic acid-sodium succinate buffer or a more general characteristic of resting cells sus-pended in any organic acid buffer system. The anaerobic and aerobic fermentation of glucose by resting cells of S.

uvarum,

CBS 426, after such treatments is recorded in Table 5. The results in Table 5 indicate that the negative Pasteur effect is restricted to resting cells suspended in succinic acid buffer systems. The anaerobic rate of fermentation by resting cells suspended in the other organic acid buffer sys-tems was either approximately the same or slightly higher than the corresponding aerobic rates. These results are in agreement with those of WIKÉN and PFENNIG (1957). Further from the results in Table 5 we noted that the anaerobic and aerobic rates of fermentation by resting cells from a 24 hr shaken culture of S.

uvarum,

CBS 426, were greater in fumaric acid-sodium fumarate, L-malic acid-sodium malate, and in citric acid- sodium citrate than in succinic acid- sodium succinate buffer. These results appear to refute the postula-tion of RUNNSTRÖM andSPERBER (1930) and BRANDT (1942) th at succinic acid-sodium succinate is an indifferent system not poisoning yeast; The former authors suspénded their yeast in 0.2 M succinate buffer at pH 5.0 or above. As will be shown later, at such a pH and concentration the succinic acid- sodium succinate buffer IS an indifferent system not poisoning yeast. It is only at. a lower pH and higher con-centration that the succinic acid- sodium succinate affects the fermentation of the resting cell suspensions. Thus, whereas the above mentioned authors were justified in making

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TABLE 5.

Anaerobic and aerobic fermentation of glucose by resting cells of..§.. !!YMl!!!!. CBS 426. suspended in various 0.34 Mand 0,17 M buffers at pH 4.5.

I\cid component of the buffer Fumaric Maleic Succinic L-Malic Citric Fumaric Maleic Succinic L-Malic Citric Fermentation

in plof C02 per mg of yeast dry substance per 60 min resting cells suspended in buffer comprising the acid

component and

its sodium salt its potassium salt

molarity of buffer molarity of buffer

0.34 0.17 0.34 0.17 Anaerobic 280.8 331. 4 263.3 275.2 273.6 345.1 222.2 316.1 92.8 230.9 79.2 199.1 210.7 334.8 201.4 270.9 307.2 325.1 225.4 253.2 Aerobic 260.8 280.6 235.6 288.9 222.3 299.7 216.3 287.8 170.6 218.3 189.5 224.4 224.3 299.2 207.8 240.6 294.9 287.2 228.2 242.1

The cells were harvested from a 24 hr shaken culture grown at 250C in Medium B with

added biotin. calcium pantothenate, meso-inisitol, and P-alanine at concentrations of 5 IJg/l. 2.5 mg/l. 25 mg/l. and 2.5 mg/l. respectively. The buffer consisted of the acid component and its corresponding sodium or potassium salt. The resting cell suspensions were flushed with oxygen-free nitrogen (Anaerobic) or shaken in air (Aerobic) for 30 min prior to the addition of 'i!'/o w/v of glucose, Fermentatioo was measured at 250C with the Warburg technique. The results were not corrected for oxygen uptake. There was practically no endogenous fermentation.

such a statement for a fixed set of conditions, they were not justified in making it a generality without further experimen-tal evidence.

d. Effect of Varying the Pretreatment on Subsequent Fer-mentation by Resting Cells

Till now the resting cell suspensions, prior to the meas-urement of fermentation, had been treated in a standard way; viz., harvested, washed twice in distilled water, once in an appropriate buffer, and then resuspended in the same buffer. After flushing with oxygen-free nitrogen or shaking in air, glucose, dissolved in the same buffer as present in the cell suspension, was added. A variation in the washing, suspending, and glucose addition procedures, as indicated in Table 6,

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