Temperature Profiles of Yeasts

Temperature Profiles of Yeasts N. VAN UDEN Laboratory of Microbiology, Gulbenkian Institute of Science, 2781 Oeiras Codex, Portugal

I. Introduction . . . . . . . 11. The elements of temperature profiles . . . A. Cardinal temperatures . . . . . B. Specific rates of growth and thermal death . C. Activation parameters . . . . . . . . 111. Types of temperature profiles A. Associative profiles. . . . . . B. Dissociative profiles . . . . . IV. Effects of drugs on the temperature profiles of yeasts A. Ethanol and other alkanols . . . . B. Other drugs . . . . . . . . . V. Targets of temperature effects . A. Basic aspects . . . . . . . B. Thermodynamiccompensation . . . C. Membranes and mitochondria . References . . . . . . . .

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195 196 196 199 202 206

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I. Introduction When an Arrhenius plot is prepared of the specific growth rates of a yeast for temperatures that range from suboptimal to supermaximal values and an Arrhenius plot of the specific rates of thermal death is superimposed on the Arrhenius plot of growth, one obtains by definition the temperature profile of this yeast. Temperature profiles of the specific rates of growth and thermal death may be enriched with curves displaying the temperature dependence of other significant parameters, such as growth yield, specific rates of production of extracellular enzymes and excretion of metabolic products. Temperature profiles of growth and death provide at least three types of information. (1) The position of the cardinal temperatures of the strain within the range of biological temperatures. (2) The dependence of the specific rates ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 25 ISBN 0-12-027725-4

Copyright 01984 by Academic Press, London All rights of reproduction in any form reserved.

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of growth and death on the temperature, i.e. their activation parameters. (3) The presence or the absence of linkage between growth and thermal death in the superoptimal temperature range. From an analysis of the temperature profiles, general predictions may be made regarding the relation between the target sites connected with the upper temperature limit for growth (Tma, sites) and the target sites connected with thermal death (thermal death sites). Although temperature profiles are an expression of the temperature dependence of growth and death in batch culture, once established they permit predictions regarding the temperature dependence of growth and death in open systems of industrial or ecological interest. Metabolites that accumulate in the medium and cells and added drugs of industrial, medical or general scientific interest may profoundly change the temperature profiles of yeasts. Analysis of such modified profiles may throw light on the nature and localization of the receptor sites and, depending on the case, allow predictions with respect to the temperature dependence of yeast performance in industrial fermentations, the temperature relations of the effects of preservatives on yeasts in food, wine and other beverages, and the effects of fever or deliberate hyperthermia on drug action in yeast infections of man and animals (Roberts, 1979). Finally the study of temperature profiles of yeasts, taken as models of eukaryotic cells, may give useful hints on possible effects of temperature, alone or combined with drug action or radiation, on growth and death of human cells and cell systems including malignant ones (Streffer, 1978).

II. The Elements of Temperature Profiles A. CARDINAL TEMPERATURES

A temperature profile covers, by definition, a temperature range that includes the maximum temperature for growth (T,,,,,), the optimum temperature for growth (Top)and, ideally, also the minimum temperature for growth (Tmi"). While the temperature range of microbial growth in general extends from several degrees below the freezing point of water to a few degrees below its boiling point at normal pressure (Precht et al., 1973; Ingraham, 1973; Ingraham and Stokes, 1959; Stokes, 1962; Farrell and Rose 1967a,b; Brock, 1967; Larkin and Stokes, 1968; Brock and Freeze, 1969; Bott and Brock, 1969; Babel et al., 1972), the temperature ranges of individual cell strains do not normally comprise more than 40 to 50°C and are often much narrower. This is also the case with the yeasts (Fig. 1). Depending on whether the T,,, value is well above 50°C, between about 25°C and 50"C, or below 25"C, micro-organisms are conventionally and roughly subdivided into three temperature groups. These are referred to as

197

TEMPERATURE PROFILES OF YEASTS

S d i izo.urrc.cliriro t ti !jce.v oct 0s)J o rll ,v Picli iti t t I e t t i / I rti t i ( i efucic.t Klrr ! l w r otti!lces.frtrgili.s

I

0

I

10

I

20

1

30

-1

40

SO

FIG. 1. Examples of temperature ranges for growth of several species of yeasts. From Phaff et al. (1978).

thermophilic, mesophilic and psychrophilic micro-organisms, respectively. Nearly all known yeasts are mesophilic, a few are psychrophilic while thermophilic yeasts, as defined above, have so far not been detected. Yeasts such as Cyniclomyces guttulatus, Saccharomyces telluster (Candida bovina) , Candida sloofii and Torulopsispintolopesii, which are able to grow only within a narrow range of temperatures with 20-30°C as the lower limit and 4245°C as the upper limit, are sometimes referred to as “thermophilic” yeasts (Watson et al., 1980) but are more appropriately called “psychrophobic” yeasts (do Carmo-Sousa, 1969). Widely scattered references indicate that many yeasts are able to grow at 37°C and some at 45°C (Guilliermond, 1920; Wickerham, 1951; van Uden, 1963; Bridge-Cooke, 1965; Lodder, 1970; Phaff et a/., 1978). Stokes (1 97 I), surveying the literature, compiled cardinal temperatures of 40 yeast strains belonging to 31 species. Interest in the biology of the Antarctic and other cold habitats, which reached a peak in the 1960s, led to the description of a number of psychrophilic yeasts with Tmax values around 20°C (Sinclair and Stokes, 1965; di Menna, 1966; Fell et al., 1969). At the other extreme, the highest T,,, value for a yeast so far reported is 49-50°C for a strain of Hansenula polymorpha (van Uden et al., 1968). Wickerham (1951) in his monograph on the genus Hansenula introduced the ability to grow at 37°C as a test in yeast identification. This test was adopted in the taxonomic treatment of a number of yeast genera (Lodder, 1970). Van Uden and Farinha (1958) and Van Uden and do Carmo-Sousa (1959) found that the T,,, value was fixed within narrow limits on the species level in a number of yeast species. The value for Tmaxas a character in yeast identification was later adopted by several authors. As a consequence, the T,,, ranges of a large number of yeast species belonging to the following genera

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N. VAN UDEN

have become available: Lipomyces (Slooff, 1970a); Metschnikowiu (Miller and van Uden, 1970); Nematospora (do Carmo-Sousa, 1970a); Schizossucharomyces (Slooff, 1970b); Candida (van Uden and Buckley, 1970); Oosporidium (do Carmo-Sousa, 1970b); Torulopsis (van Uden and Vidal-Leiria, 1970); Trichosporon (do Carmo-Sousa, 1970c) and Trigonopsis (Slooff, 1970c). Vidal-Leiria et al. (1979) determined the T,,, values of 594 yeast strains belonging to 1 12 species of the genera Candida, Torulopsis, Hansenula, Pichia, Metschnikowia and Leucosporidium. Less than 2% of the strains were psychrophilic having T,,, values below 24°C. More than 98% consisted of mesophilic strains with T,,, values ranging from 26 to 48"C, with the highest frequency in the 34-38°C range. No thermophilic strains were encountered (Fig. 2). The intraspecific variation of the T,,, value in 41 species of which more than five strains were studied did not exceed a range of 5°C in 78% of the species, the highest frequency pertaining to the 3°C range (Fig. 3). When the T,,, values of a collection of yeast strains supposedly belonging to the same species cover a wide range of temperatures, with subgroups of strains clustering around distinct T,,, values, these subgroups may represent distinct species. Thus Meyer et al. (1975) retained the name Candida sake' only for those of the strains included in this species by van Uden and Buckley (1970) that had their T,,, values around 30"C, and they excluded strain C . maltosa from C .sake' restoring it to species level based on its Tmax value around 40°C as well as on other characters. Similarly, Walsh and Martin (1977)

48

12

22 24

.rl! 26

28 30

32 :

LI I6 48

T,,, value P C )

FIG. 2. Distribution of the maximum temperature for growth (TmaX) among 594 yeast strains. From Vidal-Leiria et al. (1979).

21

TEMPERATURE PROFILES OF YEASTS 12

n E

3

4

6

7

Ronge of T,,,

199

8

volues ("C)

FIG. 3. Distribution of the range ofvariation in the maximum temperature for growth (Tmax)among 41 species of yeast of which six or more strains were studied. From Vidal-Leiria et al. (1979).

re-introduced the name Saccharomyces carlsbergensis for strains classified in Sacch. uvarum, as defined by van der Walt (1970), that had their T,,,,, values around 3 3 T , retaining the latter name only for strains with T,,, values around 39°C. B. SPECIFIC RATES OF GROWTH AND THERMAL DEATH

1. Growth When a yeast population is growing in a stirred liquid medium of suitable composition with aeration (if needed) at a constant suitable temperature and when a linear measure of the population density (turbidity, cell numbers or viable counts) is plotted on semilogarithmic paper against time, a number of sequential growth phases, characteristic for micro-organisms in general (Monod, 1942), become evident. Normally more than 90% of growth is represented in such a plot by a single straight line (above the Topvalue in some cases, as we shall see, two sequential straight lines with different slopes may characterize this phase). This linear relationship may be expressed as follows: In X , = In Xo+k,t

(1) where X , and XO are population densities at time t and time zero (on the straight line), respectively, and the constant k, is the so-called specific growth

200

N. VAN UDEN

rate. By taking antilogs on both sides of equation (l), it is seen why this growth phase is correctly called “exponential” (rather than “logarithmic”): X , = X , ekgr Differentiation of equation (2) leads to:

dX dt

-=

kgX

(2)

(3)

which, on re-arrangement, gives the definition of the specific growth rate:

k g-

dX 1 dt X

(4)

Exponential growth is “balanced” (Campbell, 1957; Painter and Marr, 1968) when all of the constituents of the biomass increase with the same specific rate:

where XI,X 2 . . . X,, are concentrations in the culture of biomass and biomass constituents (e.g. cells, protein or nucleic acids). When different methods for measuring biomass (e.g. turbidity or viable counts) lead to markedly different estimates of the specific growth rate, growth is likely to be unbalanced. The distinction between balanced and unbalanced growth is critical in determinating the T,,, value, which by definition is the temperature above which sustained and balanced exponential growth is impossible. At a given constant temperature at which sustained, balanced exponential growth is possible (i.e. between Tminand T,,,), the specific growth rate depends on a number of environmental factors such as the chemical composition of the growth medium (particularly the nature and concentration of the carbon source), pH value, water activity, oxygen tension, the presence of growth inhibitors (e.g. residual detergent on the glassware) and concentration of excreted metabolites (e.g. ethanol) in addition to other factors. If one wishes to study the temperature dependence of the specific growth rate of a given yeast with the objective of establishing its temperature profile, these variables should be under control. Even so, there is some evidence that unknown factors outside the control of the operator, possibly connected with meteorological conditions and solar activity (Bortels, 195l), may cause considerable variation of the specific growth rate of yeasts under otherwise controlled conditions (Stanley, 1964; Martinez-Peinado and van Uden, 1977). 2. Thermal Death When a suspension of micro-organisms is exposed to a high enough

TEMPERATURE PROFILES OF YEASTS

20 1

temperature, thermal death will occur. The semilogarithmic survival plot may be entirely linear or the linear part may be preceded by a shoulder, indicating a number of target sites greater than unity or the operation of repair mechanisms. When the population contains a subpopulation with greater heat resistance, the plot may display a "tail". Formal treatments of such plots were presented by Johnson et al. (1 954), Wood (1 956) and Moats (1971). The type of semilogarithmic survival plot commonly encountered in yeasts is shown in Fig. 4. The linear part of the semilogarithmic survival plot may be expressed by the following equation: In N1 = In No-kdt

(6)

where N1and NOare the numbers of viable cells after time t and time zero (on the straight line), respectively, and the constant kd is the so-called specific death rate: kd=

dN 1 dt N

(7)

By taking antilogarithms on both sides ofequation (6), it becomes evident that

50C 30C 20c

--ae

1oc

P ._ C 3

W

5

0 .> r

0 L

W

5 z

1c

e

c Time (min)

FIG. 4. Semilogarithmic survival plots of Saccharomyces cereuisiae exposed to various temperatures (A, 52°C; 0, 50°C; Q48"C). From van Uden et a/. (1968).

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N. VAN UDEN

the linear part of the semilogarithmic survival plot represents exponential death: Nt = NOe-kd' (8) The specific death rate for a given strain at a given temperature is a constant only under defined and controlled conditions. It may vary depending on the composition, pH value (also the chemical nature of the buffer; Cerny, 1980), the water activity of the suspending medium, the presence of death-enhancing drugs (including residual detergents on the glassware) and, most importantly, the physiological state of the cell population. If one wishes to study the influence of a single independent variable (such as the temperature or the concentration of a death-enhancing drug) on the kinetics of thermal death of a yeast strain, preparation of the cell populations should be carefully standardized. The composition and pH value of the growth medium, the method of stirring or shaking, the supply of oxygen (where needed) and the temperature of incubation should be the same from one experiment to the next. Furthermore, the cells to be used as inoculum in death experiments should be harvested at a defined point on the growth curve, for example in the mid-exponential phase of growth. The heat resistance of exponentially growing populations of Sacch. cereuisiue increased as the growth temperature was increased (Fintan Walton and Pringle, 1980). One should keep in mind that changes in growth temperature are accompanied by changes in specific growth rate. The separate effects of these two variables can be studied independently by using the chemostat at constant temperature and varying the dilution rate, or at constant dilution rate and varying the temperature (Hunter and Rose, 1972). Stationary-phase yeast cells are more heat resistant than exponentially growing cells (Schenberg-Frascino and Moustacchi, 1972; Parry et al., 1976). Use has been made of this fact for developing a procedure for enrichment of yeast mutants (Fintan Walton et ul., 1979). C. ACTIVATION PARAMETERS

When specific thermal death rates of a yeast are determined for a number of temperatures with the use of cell populations grown under carefully controlled conditions, typically, a plot of the logarithms of the specific rates against the reciprocals of the corresponding absolute temperatures is linear. Within temperature limits, the same is true for many other biological rates (Johnson et al., 1954). Thus, the specific thermal death rate behaves with respect to the temperature in the same way as a chemical rate constant as expressed by the Arrhenius equation; for detailed treatments of this subject, see textbooks on physical chemistry. Consequently we may write: kd = A e(-E/RT) (9)

TEMPERATURE PROFILES OF YEASTS

203

and E l In kd = ln A - - RT

where E is the “energy of activation”, R the gas constant and A an empirical parameter. A totally explicit form of equation (9) is based on the theory of absolute reaction rates (Eyring, 1935; Stearn, 1949; Johnson et al., 1954):

where k g is Boltzmann’s constant, h Planck’s constant, AS# the entropy of activation and AH’ the enthalpy of activation. Its logarithmic form is, for all practical purposes of experimental biology (i.e. over the temperature ranges normally used in biological experiments), a linear equation. However, for calculation of AS# and AH‘ from experimental data the following truly linear form should be used: kd kg AS’ In - = In -+--T h R

AH’ R

1 T

-

I shall refer to plots of In kd/T against 1/T as “modified Arrhenius plots”. In the following paragraphs an attempt will be made, for the benefit of the biological reader, to discuss the significance of equation (1 1) and the limits of its applicability to biological rates. Consider a simple chemical reaction: AeB

(13)

The first basic idea of the theory of absolute reaction rates is that the transformation of A into B passes through an activated form A # which, in most cases, has a greater free energy of formation than either A or B . The second basic idea is that A and A # are virtually at equilibrium with each other, so that: A‘ A

-=

Kf,

and that classical equilibrium thermodynamics may be applied to KG, the equilibrium constant of activation:

+-

Keg

( - A c + / R ) (IIT)

(15)

and consequently: #

Keg

-

( A S f / R )e ( - A H # / R )

(16)

where AG’, AS’ and AH‘ are the free energy, the entropy and the enthalpy of

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N. VAN UDEN

activation respectively (for a highly readable introduction into chemical thermodynamics, see Bent, 1965). The third basic idea of the theory is that the decay of activated form A + into product B is governed by a universal rate constant: dB dt

dA+ - kBT dr - h [A+]

According to classical chemical kinetics we also have:

-dB _ - k [A1 dt where k is the rate constant of the forward reaction. From equations (17) and (18) it follows that: kBT A + k =--h A

By substituting equations (14) and (16) in equation (19), equation (11) is readily obtained. The term A H + , the enthalpy of activation, is the difference (under standard conditions) in heat content between one mole of A + and one mole of A . Similarly A S + , the entropy of activation, is the difference in entropy content (a measure of disorder and randomness) between one mole of A + and one mole of A . When estimates of A H + and AS+ of thermal death are obtained from modified Arrhenius plots, the question arises as to what physical significance one may attribute to these values. If it is assumed that the specific thermal death rate is equal to the specific thermal inactivation rate of a thermosensitive target, A H + and A S + , if taken literally, would represent the difference in heat and entropy content between one mole of the activation target (a membrane protein for example) and one mole of the non-activated form (under standard conditions). Reservations must be made, even for the case of this most simplest of model, with respect to A S + . This thermodynamic quantity is contained in the value of the vertical intercept of the modified Arrhenius plot and may be calculated by the use of equation (1 2): AS#

=

kg R (Intercept - In -) h

(20)

However, since the universal rate constant kBT/h may need corrections from case to case by use of the appropriate “transmission coefficient” (Eyring, 1935; Stearn, 1949; Johnson et al., 1954), values for A S + calculated by the use of equation (20) may be only rough approximations. In most other cases of biological rates, it is not legitimate to estimate A S # values from modified Arrhenius plots. Consider, as a pertinent example, a yeast cell population in balanced exponential growth. The growth rate is

TEMPERATURE PROFILES OF YEASTS

205

related to the consumption of nutrient S through the appropriate yield coefficient y (Monod, 1942): dX dt

- --

Y

dS (-5)

By dividing both sides of the equation by the instant population density Xand applying equation (4), we obtain: k, = y ks

(22)

where ks, the specific rate of transfer into the biomass of nutrient S, is defined as:

The specific transfer rate ks is dependent on the capacity and affinity of transport systems and enzymes, and the concentrations of substrates, products, effectors and inhibitors of a complex multistep reaction system that leads from the extracellular nutrient to the final biomass and other end-products. Each of the enzymes and transport proteins involved in the system has a certain weight with respect to the value of ks as expressed by the so-called sensitivity coefficient (Kacser and Burns, 1968). For the present purpose, it is sufficient to consider the extremely simplified case in which the first transport step wholly controls the overall rate, i.e. its sensitivity coefficient is unity (van Uden, 1971) and behaves as a true “master reaction”. Under this condition and assuming that nutrient S is used at concentrations that saturate the transport step across the cytoplasmic membrane, equation (22) transforms into: (24) where ET is the total concentration per unit biomass of the carrier protein that transports the nutrient, and kT is the rate constant of transport by the loaded carrier. The form of equation (12) applicable to this extremely simplified case would be: k, ke AS+ AH+ 1 In - = In y + In ET+ In -+--h R R T T where the activation parameters refer to the transport step. Since estimates of ET are normally not available, a value for AS# cannot be estimated in the simple case presented, much less in realistic cases. What is usually done in these cases (Johnson et al., 1954) is to lump the known and unknown constants together in an empirical constant and write:

k,

=

y k-r ET

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N. VAN UDEN

When a straight line is obtained, AH# may be calculated from the slope and referred to as the enthalpy of activation. It may be seen that equation (26) is similar to equation (lo), the classical Arrhenius equation. The relation between the “energy of activation” calculated from a classical Arrhenius plot as expressed by equation (10) with the enthalpy of activation calculated from the modified Arrhenius plot is obtained by differentiating equation (1 1) which reveals that: E

=

AH#+RT

giving a difference of about 2.5 kJ (600 calories) at biological temperatures. In the author’s experience the use of activation thermodynamics in the analysis of Arrhenius plots of thermal death rates and of other biological rates is sometimes not well received by biologists, either because the equations are felt to be too formidable or because it is thought that the application of thermodynamics to living systems is being overdone. The basic experimental facts which are contained in a modified Arrhenius plot are the value of the slope and the value of the vertical intercept. Rejecting activation thermodynamics as a legitimate tool for processing these data should not lead to rejection of the data themselves, and thus of the information they may provide. Indeed, if so wished, the slopes and the intercepts may be used directly in the appropriate equation rather than the thermodynamic quantities calculated from them. For example, equation (39) given in Section IV (p. 229), which states that the entropy of activation of thermal death of Succh. cereuisiue in the presence of an alkanol is a linear function of alkanol concentration, might be rewritten to express directly the experimental results. This would reveal that the vertical intercept of the modified Arrhenius plot of thermal death of Succh. cereuisiue in the presence of an alkanol is a linear function of the concentration of the alkanol. This primary treatment would still lead to the verifiable prediction that, under isothermic conditions, the specific death rate is an exponential function of the alkanol concentration. However, it would be at a loss to provide a plausible explanation of why the slope of the linear relations between intercepts and alkanol concentrations are correlated with the lipid-buffer partition coefficients of the various alkanols. Though it should be kept in mind that the theory of absolute reaction rates contains speculative elements and that its applicability to biosystems is an open question, its use in the analysis of biological Arrhenius plots is potentially fruitful as it may lead to fresh theories open to experimental verification. 111. Types of Temperature Profiles

Up to the optimum temperature for growth, the specific growth rate of yeasts and other micro-organisms is an approximate Arrhenius function of the

207

TEMPERATURE PROFILES OF YEASTS

temperature. Ratkowsky et af. (1982) contested this, and reported to have observed in many bacteria and some yeasts a linear relationship between the square root of the specific growth rate and the growth temperature in the suboptimal temperature range. Though we have been unable to confirm their findings when applying their equation to our own yeast data, the matter warrants further study. At temperatures above To,,, in most instances the value for k, declines sharply till T,,, is reached (Fig. 5). Establishment of an optimum temperature for growth and the sharp decline of k, values in the superoptimal temperature range require the simultaneous occurrence of at least two opposite processes, namely a constructive one (i.e. biosynthesis leading to growth) with a relatively low enthalpy of activation and a destructive one with a relatively high enthalpy of activation. Growth-rate equations have been proposed on the basis of different concepts of the destructive process. Hinshelwood (1946) derived an equation based on the assumption that the destructive process is irreversible, whereas an equation proposed by Johnson et al. (1954) implies that the specific growth rate in the superoptimal temperature range is limited by the concentration of the native (i.e. catalytically active) form of a key

/

7

.;”

._

w

0

: c

40-

0

I

30 20:

c

E’

0

c

t ._

z

d

4-

I I

3-

I I

I I

’‘47 I m 6 4 1 1

310

1

1

1

I

I

315

I

I

I

I

I

‘3:’ :5 I

I

1

I

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25

30 Temperature (‘C) l

I

I

1

I

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I

1

1

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I I I I

320 325 330 335 340 105.Reciprocal of the absolute temperoture

I 15

1 1 345

FIG 5 . Relative rate of multiplication (G) of Escherichiu coli growing in a simple medium as a function of temperature. The maximum rate is arbitrarily taken as 100. The points are data from experiments, and the solid line is the curve calculated in accordance with the following equation and constants. G = c Tec-AHo#/Rv + 1+ e(-AHo/Rn where c = 0.3612e24.w, A@ = 150,000, A H + = 15,000 and AS‘ = 476.46. From Johnson et ul. (1954).

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N.

VAN UDEN

protein which is in thermodynamic equilibrium with its reversibly denatured form. Under the assumption that the sensitivity coefficient (Kacser and Burns, 1968) is sufficiently near to unity to provide it with dominant weight in the overall growth reaction, I shall refer to this protein as the T,,, site. At equilibrium we have: ED EN

-=

Keq

where EN and EDare the concentrations at equilibrium of the native and the reversibly denatured form of the T,,, site. From equation (28) it follows that:

where E is the sum of EN and ED.Following Johnson et al. (1954) the specific growth rate may now be expressed as: E k' k, = C'1 +Keq where k' is the rate constant of the step catalysed by the T,,, site (an enzymic reaction or a transport step) and C' is a proportionality coefficient. When k' and Keq are written as their respective temperature function and some of the constants are lumped together with C' of equation (30), one ends up with the equation originally proposed by Johnson et al. (1954): C T e(-AH#/R) (I/T) (31) kg = 1 + e (ASoIR) e ( - A H ~ / R T ) In this equation, AS" and AH0 are the standard entropy and the standard enthalpy of reaction (28), whereas A H Z is the enthalpy of activation of growth. For reasons explained on p. 205, ASz of growth cannot be determined and is therefore best lumped together with the other known and unknown constants in coefficient C. The equation fits some experimental results rather well (Fig. 5 ) , both in the suboptimal and the superoptimal range of temperatures. At relatively low temperatures, the slope of the Arrhenius plot of growth and thus the activation enthalpy are often found to increase. Readers interested in this aspect of temperature profiles may consult Ingraham (1973). Arrhenius plots of growth of yeasts may present peculiarities which depend on the relations with the respective Arrhenius plots of thermal death. The Arrhenius plots of thermal death of a number of mesophilic yeasts studied by van Uden et al. (1968) and by van Uden and Vidal-Leiria (1976) formed a positional sequence which followed the same order as the numerical sequence of the respective values of the maximum temperatures for growth. As can be seen in Fig. 6, the sequence related to T,,, value was no longer maintained by

c

209

TEMPERATURE PROFILES OF YEASTS

100

a?

c

e

:200

" ._ 5

la? Q n

.

10-

6-

0

z1 2-

1

I

300 I

60

305 I

50

I

56

I

1

I

31 0

lo5 * Reciprocal

54

I

52

I

50

I 325

I

31 5

320

of absolute temperature I

40

I

1

46

44

Temperature

I

42

I

40

I

30

l

36

l

34

(OC)

FIG 6. Arrhenius plots of the specific rates of thermal death of eight mesophilic and two psychrophilic yeasts. The mesophilic yeasts were A, Hansenula polymorpha (T,,, 4849°C); B, Candida albicans (T,,, 4546°C); C, Saccharomyces cerevisiae (T,,, 4142°C); D, Candida utilis (Tmax 4142°C); E, Torulopsis candida (T,,, 37-38°C); F, Torulopsis haemulonii (T- 37-38°C); G, Candida marina (T,,, 32-33°C); H , Torulopsisfujisanensis (T,,, 27-28°C). The psychrophilic yeasts were I, Candida nivalis (Tmax 22-23°C); J, Candida frigida (T,, 22-23°C). From van Uden et al. (1968).

sections of the plots extrapolated to much higher or much lower temperatures. The positioning of the plots was a consequence of an interplay between AH# and A S # of death in such a way that the plots, when extrapolated to the respective T,,, values, indicated values for the specific thermal death rate high enough to be measurable should they exist. This behaviour of the plots was formally expressed as follows (van Uden et al., 1968):

cY -

AHZ Tmax

+n

AS*

where Cyis a constant shared by the yeast strains and n is the number of degrees above the respective T,, value at which the constant applied. In the case of mesophilic yeasts, n was a small number between 1 and 4°C the constant having a value of from 301 to 330 J (72 to 79 cal) mol-l degree-'. Values of n for two psychrophilic yeasts (Fig. 6) were much higher (15-1 7"C), an expression of the fact the extrapolated Arrhenius plots indicated theoretical values for the specific death rate at Tma, values that were far below

210

N. VAN UDEN

measurable values. The results obtained by van Uden et al. (1968) suggested that there might be at least two distinct types of temperature profiles in yeasts, namely profiles in which there is some form of biologically significant association between the Arrhenius plots of growth and thermal death and other profiles in which growth and thermal death are dissociated. A. ASSOCIATIVE PROFILES

I . Batch Culture Wild and industrial strains of Sacch. cerevisiae (van Uden and MadeiraLopes, 1970; van Uden and Duarte, 1981; Loureiro and van Uden, 1982), but not necessarily genetic strains (Madeira-Lopes and van Uden, 1979), display typical associative profiles. The principal characteristics of such profiles are summarized in Table 1 and in Fig. 7. When a population of Sacch. cerevisiae growing at a suboptimal growth temperature is transferred to a liquid stirred medium incubated at a temperature between Topand T,,, and, when the logarithms of absorbance and viable cell counts are plotted against time, curves are obtained of the types depicted in Figs 8 and 9. In both cases, after a lag phase of longer or shorter duration (during which unbalanced growth may take place; Shaw, 1967) an initial period of exponential growth occurs during which the specific rate of mass growth (measured as absorbance) is equal to the specific rate of increase of the viable population (measured as viable cell counts). The duration of this first period and the respective specific growth rate decrease with increasing temperature. The first period of exponential growth is followed by a second period of exponential change during which exponential growth concurs with exponential thermal death. The true specific growth rate of the viable population during the second period is equal to k,, the specific growth rate of the first period. The apparent or net specific growth rate of the viable population during the second period is equal to the difference between k, of the first period and kd, the specific thermal death rate of the second period. the so-called final maximum temperature for growth, Between Topand Tmaxp k, is greater than kd and net exponential increase of the viable population takes place. The duration of the second exponential period between To, and Tmaxr is therefore unlimited as long as the culture medium is appropriate. The specific thermal death rate increases with the temperature whereas k, decreases. At Tmaxf k, equals kd and no net change of the viable population takes place during the second period (Krouwel and Braber, 1979). Between Tmaxf and T m q , the so-called initial maximum temperature for growth, the true specific growth rate of the viable population is smaller than its specific thermal death rate. Consequently, net exponential death prevails, which must lead to the eventual extinction of the viable population. Above Tmaxi, a first period

TEMPERATURE PROFILES OF YEASTS

TABLE 1. Characteristics of exponential growth and death as a function of temperature in yeasts with associative temperature profiles Process temperaturea

Characteristics of exponential periodsh

T < Top One exponential period (Fig. 7 curve A) Duration endless as long as the medium is appropriate:

dN 1 - k, dt N

--

Value for k, increases with increasing temperature. Top< T < Tmaxf Two exponential periods (Fig. 7, curve B) First period: duration decreases with increasing temperature:

dN 1 _ - - k, dt N Value for k g decreases with increasing temperature. Second period: duration endless as long as the medium is appropriate:

dN 1 dt N

-_

=

k,-kd

kg > kd Value fork, decreases while that for kd increases with increasing temperature.

Tmaxr< T < T,,,, Two exponential periods (Fig. 7, curve C) First period: duration decreases with increasing temperature: dN 1 - k, dt N Value for k , decreases with increasing temperature. Second period: population eventually becomes extinct: dN 1 - kg-kd dt N

kg < kd Value for k , decreases while that for kd increases with increasing temperature.

21 1

212

N. VAN UDEN

TABLE 1. (continued) Process temperaturea

Characteristics of exponential periodsh

Tmaxi
dN 1 -0 dt N Second period: population eventually becomes extinct: dN 1 _ _ dt N

=

-kd

Value for kd increases with increasing temperature. Abbreviations: T, process temperature; Top,optimum temperature for growth; TmXp final maximum temperature for growth; Tmaxi,initial maximum temperature for growth. Abbreviations: N , number of viable cells per unit volume; k,, specific growth rate; kd, specific death rate. (I

during which neither growth nor death occurs is followed by a second period of exponential thermal death without growth. As can be seen in Figs 8 and 9 the curve of the variation of biomass during the second period has a shape which is quite distinct from that of the curve of viable cells. This is a consequence of the simultaneous occurrence, during the second period, of exponential growth and death. For a detailed mathematical treatment of these relations van Uden and Madeira-Lopes (1970) should be consulted. When the specific growth rates of the first period and the apparent specific growth rates of the second period are plotted on semilogarithmic paper against the reciprocal of the absolute temperature and, when an Arrhenius plot of the specific thermal death rates, measured at supramaximal temperatures, is superimposed, one obtains the typical associative temperature profile of Sacch. cereuisiue (Fig. 10). The Arrhenius plot of growth in the supraoptimal temperature range displays two branches, one corresponding to the first exponential period of is its maximum temperature for growth, the other corresponding which Tmaxi to the second exponential period of which the maximum temperature is TmaXr The extrapolated Arrhenius plot of thermal death crosses the outer branch at Tmaxy, indicating that, at that temperature, k, of the first exponential period is

.

Time

FIG. 7. Schematic representation of exponential growth and death of various yeasts with associative profiles. Curve A, exponential growth below the optimum temperature for growth (Top)( T < Top);B, exponential growth and death between Topand the final maximum temperature for growth (T,,,,) (Top< T < T,,,,); C, exponential growth and death between Tmaxr and the initial maximum temperature for growth (Tmxi)(Tmaxr < T c T,,,,); D, exponential death above Tmaxi (Tmaxi< T). For further details see Table 1. From van Uden and Duarte (198 1).

1.0

1

FIG. 8. Growth in stirred liquid medium of Succhuromyces cereuisiue after transfer from a suboptimal to a supraoptimal (393°C) temperature below Tmxr Symbols: 0, viable counts; A, absorbance at 640 nm; -, hand fitted; - - - - theoretical curves. From van Uden and Madeira-Lopes (1970).

214

N. VAN UDEN

t

'.Or

If

>

I 5

I

8

I

10

1

15

Time ( h )

20

25

30

FIG. 9. Growth in stirred liquid medium of Saccharomyces cerevisiae after transfer from a suboptimal to a supraoptimal (40.5"C) temperature above Tmaxp Symbols: 0 , viable counts; A, absorbance at 640 nm; -, hand fitted; - - - -, theoretical curve. From van Uden and Madeira-Lopes (1970).

Reciprocal of the absolute temperature

FIG. 10. Diagram of an associative temperature profile as displayed by Saccharomyces cerevisiae, Candida albicans and other mesophilic yeasts. equal to k d of the second exponential period. Finally it should be stressed that the second periods discussed above are truly exponential growth phases (complicated by exponential death) quite distinct from the late stationary phase of microbial growth during which cell death may take place.

215

TEMPERATURE PROFILES OF YEASTS

So far, associative temperature profiles have been found in Sacch. cerevisiae (van Uden and Madeira-Lopes, 1970; Krouwel and Braber, 1979; van Uden and Duarte, 1981; Loureiro and van Uden, 1982), in Candida albicans (Lemos-Carolino et al., 1982; Fig. 11) and in a number of mesophilic yeasts studied by Oliveira-Baptista and van Uden (1971) including Torulopsis candida, Candida lusitaniae, C . kefyr and C . rnacedoniensis.The latter authors only determined specific growth rates based on absorbance which is not sufficient to exclude unbalanced growth, and their conclusions regarding the temperature profiles of these yeasts are open to question. Simdes-Mendes and Madeira-Lopes (1983), using a simplified methodology, found associative temperature profiles in strains of the following yeast species: Sacch. cerevisiae, Sacch. chevalieri, Sacch. carlsbergensis, Sacch. italicus, Torulopsis holrnii, T. glabrata and Pichia pastori. The temperature profile of the yield on glucose of a respiration-deficient strain of Sacch. cerevisiae was studied by Spencer-Martins and van Uden (1982). As shown in Fig. 12, the yield decreased in the supraoptimal temperature range from a maximum around To, to zero at Tmaxy. A similar change in yield had been observed earlier by Haukeli and Lie (1971) in strains of Sacch. cerevisiae and Sacch. carlsbergensis. As we shall see later (p. 220) this behaviour of the yield is due to dissipation of glucose by the non-viable

$ 1 e

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0

-

6

6

46 4442 4030 36 34 32 30 20 26 24 22 20 Temperature ("C) I 1

I

I

I

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I

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312 315 320 325 330 335 340 lo5 * Reciprocal of absolute temperature

FIG. 1 1 . Temperature profile of a strain of Candida uibicuns. Symbols: 0,specific death rates; A , specific growth rates of the first exponential period; 0,specific growth rates of the second exponential period. From Lemos-Carolino et ai. (1982).

216

N. VAN UDEN

1

27

I

30

35

40

1

FIG. 12. Temperature dependence of growth yields of (a) Lipomyces kononenkoae IGC 4052 and (b) Succhuromyces cereuisiue IGC 3507 in mineral medium with 1% (w/v) glucose. Symbols: 0,population density after 1 week expressed as absorbance at 640 nm; A, concentration of residual glucose. From Spencer-Martins and van Uden (1982).

fraction of the population. It remains to be seen whether this behaviour of the yield is characteristic for yeasts with associative profiles, in general. The fact that it was also found in Candida albicans (Lemos-Carolino et al., 1982) but not in several yeast species with dissociative profiles (see p. 223) seems to indicate this. If indeed this is so, associative yeasts cannot be used profitably for production of single-cell protein at temperatures above Top,a problem of practical importance in the bioconversion of paraffins with high molecular weights (Pozmogova, 1978; Kvasnikov and Isakova, 1978).

217

TEMPERATURE PROFILES OF YEASTS

2. Chemostat Cultures Let us consider a yeast with an associative temperature profile growing in a chemostat under carbon limitation at supraoptimal temperatures. When the population is in the steady state the kinetics of the second exponential period should apply, i.e. exponential death concurs with exponential growth and:

D

(33)

= kg-kd

where D is the dilution rate. For each supraoptimal temperature we may draw isotherms of the specific growth rate and the specific death rate as functions of the concentration of the limiting nutrient. Figure 13 shows semitheoretical isotherms for a strain of Sacch. cereuisiae growing under glucose limitation (van Uden and Madeira-Lopes, 1975). The principal assumption was that the relation between kg and glucose concentration is hyperbolic as expressed by

' / 38.50 39.40

50

0.00 0 0

.

100

150 0

200

250 2

Glucose concentration (mg 1-') I I I

37.5?38.Q0 38.5' 18.8 24.8 29.0

I

I

39.4O 39.6'

74.8 90.4

I

39.8O 140.8

I 39.90 210.0

Critical concentrations and respective temperatures

("C)

FIG. 13. Semitheoretical isotherms of specific growth rates (-) and of specific death rates (- - - -) of Succhuromyces cerevisiae IGC 3507 as a function of glucose

concentration at superoptimal temperatures. The specific death rates were determined in batch culture and were supposed to be independent of the glucose concentration. The isotherms of the specific rates are based on equation (12). The values of k,,, and K, were determined in batch culture and in the chemostat, respectively. From van Uden and Madeira-Lopes (1975).

~

218

N. VAN UDEN

the Monod (1942) equation:

c

(where S is the glucose concentration and K,the substrate constant) and that the specific death rate is independent of the concentration of the limiting nutrient and the dilution rate. The points of intersection between the hyperbolic k , isotherms and the linear kd isotherms indicate critical S values at which k , equals kd. In other words, the temperature of the isotherm is equal to Tmaxf for growth at the corresponding critical S value. A secondary plot of the Tmaxr values against the corresponding critical S values suggests that Tmaxf approaches Topwhen S approaches zero (Fig. 14). Measurements in glucose-limited chemostat cultures of Succh. cereuisiue at supraoptimal temperatures led to similar results (van Uden and MadeiraLopes, 1975), though the k , isotherms were sigmoid rather than hyperbolic (Fig. 15). The secondary plot (Fig. 16) shows that Tmaxf approaches Topwith decreasing concentration of the limiting nutrient. One conclusion was that Top becomes the effective maximum temperature for growth when microbial populations with associative temperature profiles are growing under nutrient limitation in natural ecosystems. In the supraoptimal temperature range k,,,, the maximum specific growth rate, and thus D, (the critical dilution rate), decrease steeply with increasing temperature. The value for D, may be expressed by the following equation (Herbert, 1958):

40.0 39.8

c

E

2

38.5

E

p

._

38.0

LL

1

37.51 I 0 - 50

I

100

I

150

I

200

I

250

I

300

I

350

I

400

I 450

Glucose concentration (rng 1.’)

FIG. 14. Secondary plot of Fig. 13. The temperatures of the isotherms were plotted against the S values that correspond to the points of intersection between the k , isotherms and the k d isotherms. At these critical S values the temperatures of the isotherms represent the final maximum temperatures for growth. From van Uden and Madeira-Lopes (1975).

219

TEMPERATURE PROFILES OF YEASTS

where Sr is the concentration of the limiting nutrient in the fresh medium. With Sacch. cerevisiae, and associative yeasts in general, we have in the supraoptimal range that D equals k,-kd. Since kd increases with the temperature, Dc decreases with increasing temperature even more steeply than kmax:

0 .12 0.12r

? I

P

- 0.10

It

c

c c

0.08

u

p0

0.061

_ _ _ _ _ _

".t

grn 0.04

._ r

'40.02a

v)

0.00 0

I

10

I

20

I

I

I

30

50

40

Glucose Concentration (mg

(-'I

I

60

-1 I 70

FIG. 15. Isotherms of specific growth rates (-) and of specific death rates (- - - A) of Saccharomyces cerevisiae IGC 3507 as a function of glucose concentrations, based on experimental data obtained at three superoptimal temperatures (0,38"C; 0,39"C; V, 40°C) in a glucose-limited culture in the MGV-medium described by van Uden (1967). From van Uden and Madeira-Lopes (1975).

37

L0

I

10

I

20

I

30

1

40

I

50

I 60

Critical glucose concentration (mg 1.')

FIG. 16. Secondary plot of Fig. 15, showing the dependence of the final maximum temperature for growth of Saccharomyces cerevisiae IGC 3507 on the glucose concentration. From van Uden and Madeira-Lopes (1975).

220

TEMPERATURE PROFILES OF YEASTS

The low specific growth rate that may be maintained in the chemostat at supraoptimal temperatures implies that maintenance requirements have a significantly depressing effect on y , the yield on carbon source, when the carbon and energy source is the limiting nutrient. The equation of Pirt (1975): 1- 1 - -+km Y Ymax -

1 kg

-

(37)

when applied to the case under consideration becomes:

This yield-depressing effect of maintenance at supraoptimal temperatures may be further aggravated when the value for the maintenance coefficient k m increases with the temperature (Topiwala and Sinclair, 1971). Van Uden and Madeira-Lopes (1976) obtained with Succh. cereuisiue a value of 0.14 h-’ for k , at 25°C as compared with 0.3 h-I at 39°C. The fraction of energy substrate which is diverted in the chemostat to maintenance normally increases with decreasing dilution rate. However, when death concurs with growth, this fraction decreases at very low dilution rates due to the increasing preponderance of the non-viable population (Fig. 17). However, the non-viable population may dissipate energy substrate as was observed with a strain of Succh. cereuisiue grown in the chemostat at 39°C under glucose limitation (Fig. 17; van Uden and Madeira-Lopes, 1976). For a formal treatment of the effect of death, maintenance and dissipation of yield in the chemostat of associative yeasts, these authors should be consulted.

-

0 0.00 0.02 0.04 0.06 0.08 0.10 Dilution rate (h-’)

FIG. 17. Glucose-limited growth of Saccharomycescereuisiae in a chemostat at 39°C showing relative rates of glucose utilization for growth (-) and maintenance(. . . .) of the viable cells and dissipation (- - - -) by the non-viable cells as a function of the dilution rate. From van Uden and Madeira-Lopes (1976).

TEMPERATURE PROFILES OF YEASTS

22 1

B. DISSOCIATIVE PROFILES

Yeasts in which the extrapolated Arrhenius plot of thermal death does not intersect with the extrapolated Arrhenius plot of growth at a biologically significant rate value are, by definition, dissociative with respect to their temperature relations. Thus, the Arrhenius plot of growth of such yeasts displays only one branch between the optimum and the maximum temperature for growth, and sustained exponential death does not occur over this temperature range. So far, dissociative profiles in yeasts have been reported for three species, namely, the psychrophilic yeast Candida curiosa (VidalLeiria and van Uden, 1980), the starch-converting yeast Lipomyces kononenkoae (Spencer-Martins and van Uden, 1982) and the pathogenic yeast Cryptococcus neoformans (Madeira-Lopes and van Uden, 1982). In addition, Simijes-Mendes and Madeira-Lopes (1983), using a simplified methodology, concluded that strains of the following species had dissociative temperature profiles: Sacch. bayanus, Sacch. kloeckerianus, Torulopsis colliculosa, T. dattila and Nematospora coryli. Above the T,,, value of such yeasts, defined as the highest temperature at which sustained balanced growth is possible, transient unbalanced growth may take place for quite some time after transfer of cells from a suboptimal to a supramaximal temperature. When only absorbance data are used for calculating specific growth rates, the phenomenon may not be detected, T,,, estimates will be too high and a pseudo-associative profile may emerge. With Candida curiosa, Tm,,, as defined above, was 17°C (Fig. 18). Above T,,,, up to about 26"C, transient growth in cell numbers occurred (see Fig. 21a, p. 224) while transient mass growth was detectable till about 33°C (see Fig. 2 1b, p. 224). With Lipomyces kononenkoae, the value of T,,, for sustained balanced growth was about 35°C (Fig. 19). From 35°C to about 4WC, transient growth occurred during periods of time that decreased from about 16 hours at 36°C to less than 1 hour at 40°C. This transient growth was linear rather than exponential and led, before the medium was exhausted, to a stationary phase of variable duration after which cell death set in. The behaviour of Cryptococcus neoformans (Madeira-Lopes and van Uden, 1982) around its T,,, value was different from the behaviour of C. curiosa and L. kononenkoae. At temperatures below 38.5"C, cells of Cr. neoformans transferred from cultures growing exponentially at 25°C had no difficulty in starting balanced exponential growth with the specific rate characteristic for the new temperature. Between 38.5"C and T,,, (near 40"C), initial growth was unbalanced, the specific rate of variation of biomass being positive and that of numbers of viable cells negative. However, within 24 hours, the population had adapted to the superoptimal temperature, and sustained balanced exponential growth was established. Above the T,,, value up to a temperature limit that was not determined, an initial period of transient mass growth

222

N. VAN UDEN

l--.2?Y

10-540 35

30

25

20

Temperature 1

1

1

1

lo3- Reciprocal of

1

15

10

("C) 1

1

1

3.20 3.25 3.30335 3.403.45 3.503.60

absolute temperature

FIG. 18. Arrhenius plots of growth and thermal death of Candida curiosa. A indicates specific rates of sustained exponential growth, 0 specific rates of death in buffer (of cells grown at 25"C), and 0 specific rates of death in growth medium containing glucose. From Vidal-Leiria and van Uden (1980).

I

315

I

lo5

-320

I

322

I 330

1

335

I

340

Reciprocal of absolute temperature

FIG. 19. Arrhenius plots of growth and thermal death of Lipomyces kononenkoae. 0 indicates specific rates of sustained exponential growth, and A specific rates of exponential thermal death (of cells grown at 25°C). From Spencer-Martins and van Uden (1982).

N.

VAN UDEN

223

occurred together with exponential decrease of the viable population from which there was no recovery. The difference in thermal behaviour between C . curiosa and L. kononenkoae, on the one hand, and Cr. neoformans on the other, is also apparent in the Arrhenius plots of thermal death. With the former pair, Arrhenius plots of growth and death were clearly dissociated, and this dissociation was maintained regardless of whether the cells for the death experiments had been grown at a suboptimal temperature or at a temperature near T,,, (see Figs 18 and 19, p. 222). In the latter species, Arrhenius plots of thermal death obtained from death experiments with cells grown at suboptimal temperatures intersected the Arrhenius plots of growth at biologically significant values, suggesting (as indeed was the case) that unadapted cells suffered transient thermal death below the T,,, value. Arrhenius plots of thermal death of cells adapted to supraoptimal temperatures no longer intersected the Arrhenius plot of growth at a significant temperature, but nevertheless remained closely similar (Fig. 20). Candida curiosa and L. kononenkoae displayed temperature profiles in which growth and death were clearly dissociated. This may indicate that the death sites and the T,, sites of these yeasts are quite distinct in nature. In yeasts such as Cr. neoformans, the dissociation is less clear. Possibly there is linkage between them, or identity of the death sites and the T,,, sites as is supposed to be so in truly associative yeasts. However, although sustained exponential growth and death concur between To, and T,,, values in associative yeasts such as Sacch. cerevisiae, sustained exponential growth and sustained exponential thermal death are mutually exclusive in yeasts that behave like Cr. neoformans. More research is needed to clarify these relations. The temperature profile with respect to the yield was similar in L. kononenkoae and the other three dissociative yeasts studied (see Fig. 12, p. 216). The yield on glucose in batch culture was constant up to the maximum temperature for growth, indicating the absence of non-viable cells dissipating energy. Yeasts with such a yield at supraoptimal temperatures have been called “thermotolerant” yeasts (Pozmogova, 1978; Kvasnikov and Isakova, 1978).

IV. Effects of Drugs on the Temperature Profiles of Yeasts A. ETHANOL AND OTHER ALKANOLS

During alcoholic fermentation in batch culture by Saccharomyces cereuisiae and other yeasts, the increasing concentration of ethanol adversely affects the yeast population including its specific growth rate, its specific rate of fermentation and its viability (Gray, 1941; Holzberg et al., 1967; Aiba et al.,

224

N. VAN UDEN

1 0-

-

10-

111 111

e

c

f

em

\

0

f 0

\

\ \

0

10-

\

U 0 ._ "._

"

\

0)

m m

\

\ \

\

1 0-

\

\

I_ \

1 0-

0

I

I 30

I

I

45

40

35

Temperature ('C)

1

3.10

I

3.15

I

I

3.20

3.25

lo3.Reciprocol of absolute temperature

I 3.30

FIG. 20. Arrhenius plots of growth and thermal death of Cryptococcus neoformans. A indicates specific rates of sustained exponential growth and 0 specific rates of exponential thermal death (of cells grown at 39°C). From Madeira-Lopes and van Uden (1982).

$ -4

0.8 0.4

0.2 0.0 0 5 10 15 2025 3 0 3 5 404550 Time (h)

0

2

4

Time (h)

6

8

FIG. 21. Transient growth of Cundidu curiosa at 23°C (a) and death and transient growth at 28°C (b). 0 indicates absorbance, A microscopic counts, dry weight and 0 plate counts. From Vidal-Leiria and van Uden (1980).

225

TEMPERATURE PROFILES OF YEASTS

1968; Day et al., 1975; Bazua and Wilke, 1977; Strehaiano et al., 1978; Thomas et al., 1978; Navarro and Durand, 1978; Thomas and Rose, 1979; Ghose and Tyagi, 1979; Beavan et al., 1982). With increasing temperature, some of these effects may become more severe (Gray, 1941; Nagodawithana and Steinkraus, 1976; Navarro and Durand, 1978; Brown and Oliver, 1982). The underlying mechanisms are many, and include irreversible denaturation (Llorante and Sols, 1969) and hyperbolic non-competitive inhibition (Nagodawithana et al., 1977) of glycolytic enzymes, exponential non-competitive inhibition of glucose transport (LeHo and van Uden, 1982b),depression of the optimum and the maximum temperature for growth (van Uden and Duarte, 1981; Loureiro and van Uden, 1982) and enhancement of thermal death (LeHo and van Uden, 1982a)and of petite mutation (CabeCa-Silva et al., 1982). Within the context of the present review, it is of interest to discuss the mechanisms that contribute to the effects of alkanols on temperature profiles. 1 . Eflects on the Maximum and the Optimum Temperaturesfor Growth

As shown in Fig. 22, ethanol added to a liquid batch culture had a pronounced depressing effect on the T,,, of Sacch. cerevisiae at concentrations above approximately 3% (w/v) (van Uden and Duarte, 1981). Under the experimental conditions used, estimates of T,,, values were intermediate between TmaXi and TmaxpThe plateau up to about 3% (w/v) of ethanol indicates a type of ethanol resistance that may be characteristic for Sacch. cerevisiae. As shown in Fig. 22 this plateau was not displayed by a strain of the inulin fermenter

50

20

r

I

0

I

1

I

2

I

3

I

4

I

5

I

6

I

7

1

8

I

9

I

10

Ethanol concentration (%, w / v )

FIG. 22. Effects of ethanol on the maximum temperatures for growth of Saccharogrowing in liquid mineral rnyces cerevisiae (- - - -) and Kluyverornycesfragilis (-) medium containing vitamins and glucose. From Sa-Correia and van Uden (1982).

226

N. VAN UDEN

Kluyveromyces fragilis (Sh Correia and van Uden, 1982) which has been proposed for industrial production of ethanol from tubers of the Jerusalem Artichoke (Guiraud et al., 1979, 1981a,b, 1982; Duvnjak et al., 1981; Margaritis et al., 1981). Since Sacch. cerevisiae has an associative temperature profile, the question arose whether the effect of ethanol on the T,,, value was only due to changes in the parameters of growth or also to changes in the parameters of thermal death. To answer this question, Arrhenius plots were prepared of growth and death in the presence and absence of 6% (w/v) ethanol (van Uden and Duarte, 1981; Fig. 23). Ethanol at the concentration used depressed the Topvalue by about 12°C from about 37°C to about 25"C, Tmaxi from 44°C to 36°C and Tmaxf from 40°C to 33°C. Consequently, the Arrhenius plot of growth in the new

'0

I

I

I

16

37

44

Temperature

I

3.1

I

3.2

lo3 .Reciprocal

("C) I

3.3

I

3.4

I

3.5

of absolute temperature

FIG. 23. Temperature profiles of Succhuromyces cerevisiue grown in liquid mineral medium containing vitamins and glucose. To the left are the profiles in the absence of added ethanol. indicates specific death rates, A specific growth rates of the first exponential period, and A net specific growth rates of the second exponential period. To the right are the profiles with added ethanol (6%, w/v). 0 indicates specific death rates, 0 specific growth rates of the first exponential period, and 0 net specific growth rates of the second exponential period. From van Uden and Duarte (1981).

TEMPERATURE PROFILES OF YEASTS

227

supraoptimal temperature range again displayed two branches. Furthermore, the Arrhenius plot of thermal death suffered a shift to lower temperatures in such a way that it intersected with the outer branch of the Arrhenius plot of In other words, ethanol at the growth at a temperature value very near TmaxF‘ concentration used shifted the temperature profile of Sacch. cerevisiae to lower temperatures without disrupting it. The results suggested that the following temperature relations govern yeast growth and thus fermentation performance in an alcoholic batch fermentation. Initially, while the ethanol concentration is sufficiently low, the yeast population displays its normal cardinal temperatures (Top,T m q and Tmq). During fermentation, while the ethanol concentration increases, the three cardinal temperatures decrease. At a critical ethanol concentration, which depends on the strain and the process temperature, the Topvalue will become value. Any further increase in ethanol concentration identical with the Tmaxi due to continuing fermentation will lead the population into the second exponential period of the supraoptimal temperature range during which death value does not decrease concurs with exponential growth. As long as the Tmaxr to the value for the process temperature, the specific growth rate will be greater than the specific death rate. At ethanol concentrations that depress the Tmaxf value below the process temperature, death will proceed at higher specific rates than growth, leading to the extinction of the viable population. This sequence of events may take place wholly, partially or not at all depending on the alcohol tolerance of the strain, the final ethanol concentration and the process temperature. At high process temperatures, such as may occur in fermentations of red wine and in the industrial production of fermentation ethanol in tropical countries, so-called “heat-sticking’’ (Ough and Amerine, 1960), due to the events already described, may stop the fermentation prematurely. 2. Effects on Thermal Death Lei50 and van Uden (1982b) prepared modified Arrhenius plots of thermal death of Sacch. cerevisiae grown in the presence or absence of various concentrations of added ethanol, propan-2-01, propanol and butanol. The plots constituted families of straight lines which were statistically parallel (Fig. 24). These results implied that the alkanols affected only the vertical intercepts of the plots, that is they affected A S # , the entropy of activation of thermal death but did not significantly change A H Z , the enthalpy of activation of thermal death. To calculate AS# values for each alkanol concentration, the average value of AH# (385 kJ mol-’; 92.1 kcal mol-I) was used and substituted in equation (12) together with a kd value of 2 . s-I and the respective experimental T value. Figures 25 and 26 show how the AS# value of thermal death depended on

10 I

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I

I

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7

, 6

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46

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I

313

lo5 *

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I I I 38 34 30 Temperature ("C) 1

I

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I

317 321 325 329 333 Reciprocal of absolute temperatures

I 22

I

337

FIG. 24. Temperature dependence of the specific rates of thermal death (kd) of Saccharomyces cerevisiae in the presence or absence of added alkanols. Modified Arrhenius plots according to equation (12). Numbers beside each plot indicate alkanol concentrations (%, w/v). From LeSio and van Uden (1982a).

229

TEMPERATURE PROFILES OF YEASTS 240r

*

v,

a210

I

00.2

I

1

I I I I I I I I I 1.0 1.4 1.8 2.2 2.6 Ethanol concentration (mol 1-’1

0.6

1

I

I 3.0

FIG. 25. Dependence on ethanol concentration of A S z , the entropy of activation of thermal death in Succhuromyces cerevisiue. From Le5o and van Uden (1982a).

00.2

0.6 1.0 1.4 1.8 Alkanol concentration (mot 1.’)

FIG. 26. Dependence on alkanol concentration of ASz , the entropy of activation of thermal death in Succhuromyces cerevisiue. 0 indicates propan-2-01, propanol and A butanol. From Lelo and van Uden (1982a).

the concentration of the alkanol. The relationship appeared to be linear with propan-2-01, propanol and butanol, and linear over the range of higher concentrations (above f0.3 M) in the case of ethanol. The following equation expresses these linear relations: AS$ = AS:+

CtX

(39)

where AS; and AS: represent the entropies of activation of thermal death at X and zero concentrations, respectively of the alkanol. The constant Ct,the “entropy coefficient for the aqueous phase”, was defined as the increase in entropy of activation of thermal death per unit concentration of alkanol in the culture medium. Values for Ct obtained for the four alkanols are listed in Table 2. They increased with the lipid solubility of the alkanols. Division by the respective lipid-buffer partition coefficients led to very similar values, i.e. CF, the entropy coefficient for the lipid phase defined as the increase in AS+ per unit concentration of alkanol in the membrane had nearly the same value for the four alkanols (Table 2).

230

N. VAN UDEN

TABLE 2. Increase in the entropy of activation of thermal death of Saccharomyes cerevisiae per mole of alkanol in a litre of the aqueous phase (C;)or in a kilogram of membrane (CF). From Lelo and van Uden (1982b) Entropy coefficientsa

Alkanol Ethanol Propan-2-01 Propanol Butanol

Lipid-buffer partition coefficient

From eqn. (3)

0.14 0.276 0.45

Ct

CF

c;

CF

5.1

36.4 37.0 38.7 38.9

3.6 9.6 15.3 60.0

25.7 34.8 34.0 40.0

10.2 17.4

58.3

1.5

From eqn. (6)

" C t is entropy units mol-l 1-' (aqueous phase); or CF is entropy units mol-I kg-' (membrane).

The parallelism of the Arrhenius plots implied that the specific death rates under isothermic conditions should be exponential functions of the alkanol concentrations (for a derivation, see Lelo and van Uden, 1982b): Ink:

=

In kj+-

AAS R

where k: and k j are the specific death rates at X and zero concentrations respectively of the alkanol. From equation (39), it follows that:

AASZ = Ct X

(41)

and substitution of equation (41) in (40) leads to: CA

Ink: = In kO,+J X R and: Lelo and van Uden (1982a) tested equation (43) experimentally and found that, under isothermic conditions, kd was indeed an exponential function of the alkanol concentrations (Figs 27 and 28). The slopes of the semilogarithmic plots increased with the lipid solubility of the alkanol and, as predicted by equation (43), were independent of the temperature. Estimates of Ct calculated from the slope using equation ( 5 ) were similar in value to estimates obtained from the AS# plots (Table 2).

23 1

TEMPERATURE PROFILES OF YEASTS

100

60

12

621

1

I

00.40.6

I

I

1

I

I

I

I

I

I

1.0 1.4 1.8 2.2 0 0.2 Alkanol concentration (rnol 1.’)

0.6

1.0

FIG. 27. Dependence at constant temperature (indicated on the figure) of the specific rate of thermal death in Sacchuromyces cerevisiue on the concentrations of ethanol (a) and propan-2-01 (b). From LeSlo and van Uden (1982a). 100

- .OF p “

0

10

6

0 0.2

c

0.6 1.0 0 0.2 0.6 Alkanol Concentration (rnol 1-’1

1.0

FIG. 28. Dependence at constant temperature (indicated on the figure) of the specific rate of thermal death in Sacchuromyces cerevisiue on the concentrations of propanol (a) and butanol (b). From Le5o and van Uden (1982a).

The results indicated that alkanols enhanced thermal death by acting in a non-specific way on membrane lipids (i.e. only dependent on the lipid solubility of the compound and, if at all, its molecular size but not its chemical structure). The results also suggested that the thermal death sites in Succh. cerevisiue, the rate of inactivation of which governs the kinetics of thermal death, are located in a cell membrane. The expression “enhancement of thermal death” implies that the alkanols rendered the thermal death sites more heat sensitive rather than acting on a death target of their own. The following evidence suggested that temperature-dependent death of Sacch. cerevisiue in the presence of alkanols has a molecular mechanism which is similar to or identical with that of thermal death. Firstly, ethanol enhanced death and depressed the maximum and the optimum temperatures for growth of Sacch. cerevisiue without disrupting its associative temperature profile.

232

N. VAN UDEN

Secondly, the alkanols did not significantly affect the A H z value of death or, using the terminology of students of disinfection (Bean, 1967), the temperature coefficient of death was the same in the presence or absence of alkanols. It was concluded that alkanols change the lipid vicinity of the thermal death sites in the membrane in such a way that the same amount of heat ( A H f ) leads to a greater amount of disorder ( A S z ) in the activated target molecules, the amount increasing linearly with the alkanol concentration. This determines a decrease in the free energy of activation (A@) at a given temperature, an increase in the steady-state concentration of the activated target molecules and, consequently, an increase in the rate of thermal denaturation of the targets at this same temperature and thus of thermal death.

3. A Model The effects of alkanols on the temperature profile of Sacch. cerevisiae (van Uden and Duarte, 1981) and on the activation parameters of thermal death (LeHo and van Uden, 1982a) are displayed in Fig. 29. On the basis of these relations, Loureiro and van Uden (1982) described and tested a model that

4

- _ _ TLx,

T&nx,

To

TX

Reciprocal of the absolute temperature

FIG. 29. Diagram showing the relation between alkanol-enhanced thermal death and the maximum temperature for growth in Succharomyces cerevisiae. Straight lines represent modified Arrhenius plots of thermal death at 0 and X concentrations of alkanol, 1/To and 1/Tx indicate intersections of these lines with the horizontal zero axis, curved lines show modified Arrhenius plots of growth at 0 and Xconcentrations of alkanol, Tiaxrare the final maximum temperatures for growth (in degrees K) at 0 and X concentrations of alkanol, k is the specific rate of growth or death, and T the absolute temperature. From Loureiro and van Uden (1982).

233

TEMPERATURE PROFILES OF YEASTS

relates the maximum temperature for growth of Succh. cereuisiae in the presence of an alkanol to the parameters of thermal death and the effects of the alkanol on the latter. Applying equation (12) to the relations depicted in Fig. 29, it can easily be shown (see Loureiro and van Uden, 1982) that:

where T:axr and TzaXfarethe final maximum temperatures for growth at X and zero concentrations respectively of the alkanol, AH# is the enthalpy of activation of thermal death and C; the entropy coefficient for the respective alkanol. The model was tested on an industrial wine yeast and a good fit was obtained (Fig. 30). The use of equation (44) for predicting the effect of ethanol on the maximum temperature for growth in an industrial batch fermentation may require the introduction of additional coefficients, the main difficulty being that the response of the yeast population to the extracellular concentration of the ethanol produced in the course of a batch fermentation changes in a more complex way that the observed responses to added ethanol in laboratory experiments (Navarro, 1980; Rose and Beavan, 1981).

-

-

0

P

-28

2

0

-

-

0

I 2

I

4

I 6

I

8 Ethanol concentration (%, v / v )

I

10

FIG. 30. Dependence of the Tmaxf value, the final maximum temperature for growth, of an industrial strain of Saccharomyces cereuisiae on the concentration of added ethanol. The solid line represents a theoretical curve calculated from equation (44) using a TLaXr value of 313 K, a AH# value of 456 kJ mol-' (108,860 cal mol-I, and a Ck value of 5.6 EU mol-' I-'. From Loureiro and van Uden (1982).

234

N. VAN UDEN

B. OTHER DRUGS 1. Sulphur dioxide

Sulphite, at concentrations used for the preservation of food and beverages and comparable to those found in polluted air, induced death in Sacch. cereuisiae (Schimz and Holzer, 1979; Schimz, 1980). The death effect was pH-dependent and correlated with the concentration of molecular sulphur dioxide in the suspending medium. Molecular sulphur dioxide, though transported selectively into the yeast cell (Macris, 1972), is virtually non-existent at pH 6, the approximate intracellular pH value of yeast (Uhl, 1960). The numerous intracellular effects of sulphite (see Schimz and Holzer, 1979) may therefore be ascribed to the action of the sulphite or the bisulphite ion or both, rather than to molecular sulphur dioxide. On the other hand, the death effect, if indeed primarily due to the action of molecular sulphur dioxide, should involve receptor sites on the cell surface to which sulphur dioxide has access at the low pH values that sustain significant concentrations of this chemical species. A membrane-bound adenosine triphosphatase (ATPase) was proposed as such a receptor site. Its activation by sulphur dioxide would cause the rapid depletion of intracellular ATP which in turn would cause sulphite death of Sacch. cereuisiae (Schimz and Holzer, 1979; Schimz, 1980). Death of Sacch. cerevisiae induced by sulphur dioxide was enhanced by increasing the temperature (Schimz and Holzer, 1979; Schimz, 1980). Anacleto and van Uden (1982) studied the temperature relations of sulphur dioxide-induced death in Sacch. cerevisiae. Modified Arrhenius plots of the specific death rates in the presence of potassium metabisulphite concentrations ranging from 5 to 150 p.p.m. constituted a family of straight lines which statistically were parallel (Fig. 31). The average A H # value for sulphiteinduced death was 151 kJ mol-' (36 kcal mol-I) which was less than one-half of the A H # value for thermal death. This suggested that sulphur dioxide acted on a death target of its own rather than enhancing the thermosensitivity of the thermal death sites. The A S # values of sulphur dioxide-induced death fell on a hyperbolic curve (Fig. 32):

AS: = ASt+AAS&,

X

-

K+X

(45)

where X is the potassium metabisulphite concentration, K the dissociation constant of a complex formed by sulphur dioxide and a receptor on the cell surface (expressed here as concentration of potassium metabisulphite), AS: the entropy of activation of sulphur dioxide-induced death at zero concentration (for an elucidation of this apparent contradiction the original paper of Anacleto and van Uden, 1982 should be consulted), AS: the entropy of

235

TEMPERATURE PROFILES OF YEASTS

r2

2

6ol

5

20

01 50

I

309

I

48 I

312

lo5

I

45

I I I 42 39 36 Temperature ("C)

I 33

I I I I I 321 324 327 315 318 Reciprocal of the absolute temperature

I

30 I

330

FIG. 3 1. Temperature dependence of sulphur dioxide-induced death in Saccharomyces cerevisiae at pH 3.4. Arrhenius plots were modified according to equation (12). Numbers beside each plot indicate concentrations of potassium metabisulphite in p.p.m. From Anacleto and van Uden (1982).

- 5 3

010 30 60 100 150 5 20 Concentration of potassium metabisulphite (p.p.m.)

FIG. 32. Dependence on potassium metabisulphite concentration of the ASz value, the entropy of activation of sulphur dioxide-induced death of Saccharomyces cereoisiae at pH 3.4. From Anacleto and van Uden (1982). activation a t concentration X , and AAS&x the difference between AS: and AS: a t saturating concentrations of the drug. A double reciprocal plot produced a straight line for concentrations below 60 p.p.m. potassium metabisulphite. At higher concentrations, the behaviour of the direct and the reciprocal plots was consistent with substrate inhibition, i.e. sulphur dioxide interfered with the efficient binding of sulphur dioxide. The observed

236

N. VAN UDEN

temperature relations led to the prediction that, under isothermic conditions, the specific rate of sulphur dioxide-induced death should be a hyperbolic function of the sulphur dioxide concentration. This prediction was confirmed experimentally (Fig. 33). Anacleto and van Uden (1982) proposed a model involving two types of receptor sites for sulphur dioxide on the cell surface; these were sulphur dioxide death (SDD) sites with a very high affinity for the drug and entropy modulating (EM) sites with a somewhat lower (but still very high) affinity. Since the chemical nature of sulphur dioxide makes strong interaction with membrane phospholipids an unlikely proposition, the SDD and the EM sites are likely to be membrane proteins and might well be different regions of the same protein. According to the proposal of Schimz and Holzer (1979), this protein would be a membrane-bound ATPase, but it cannot be excluded that the sites are located on another type of protein, denaturation of which by the drug would lead to leakage of intracellular components and cell death.

2. Sorbic Acid Potassium sorbate in concentrations up to 500 p.p.m. did not induce death in Sacch. cerevisiae and had no significant effect on thermal death in this yeast (J. Anacleto and N. van Uden, unpublished observations). However, the Arrhenius plots of growth were shifted to lower temperatures, the extent of the shift being dependent on the concentration of the drug (Fig. 34). As a consequence, sorbic acid transformed the associative temperature profile of Succh. cerevisiae into a dissociative one. Furthermore, sorbic acid decreased the yield on glucose, the decrease being an exponential function of sorbate

C -

-12-131

0

I

I I I I 20 30 40 50 Concentration of potassium metabisulphite (p.p.m.) I

I

57.510

1 60

FIG. 33. Dependence on potassium metabisulphite concentration of the specific rate of sulphur dioxide-induced death in Saccharomyces cerevisiae at pH 3.4 under isothermic condition. From Anacleto and van Uden (1982).

237

TEMPERATURE PROFILES OF YEASTS

concentration. The specific growth rate at permissive temperatures was also depressed exponentially by the drug with the same exponential inhibition constant as the effect on the yield. In the range of concentrations tested, there was no effect on the rates of glucose transport and glucose consumption. The results suggested that the target sites of sorbic acid in Sacch. cerevisiae are distinct from the ones that govern thermal death and T,,, value in this yeast. 3. Cycloheximide and Chloramphenicol

Similar results were obtained with cycloheximide (Madeira-Lopes and van Uden, 1983), a drug that inhibits protein synthesis on cytoplasmic ribosomes (Siege1 and Sisler, 1963). Cycloheximide strongly depressed the maximum temperature for growth of Succh. cerevisiae (Fig. 35) but, in the range of concentrations tested, had no effect on thermal death. As a consequence, Arrhenius plots of growth were shifted to lower temperatures and the temperature profile became dissociative (Fig. 36).

A

10-2

c

I \

Temperature I

310

I

315

lo5 * Reciprocal

I

320

I

("C)

325

I

330

I

335

of the absolute temperalure

W')

FIG. 34. Effects of potassium sorbate on the temperature profile of Saccharomyces cerevisiae. A , A indicates absence of potassium sorbate, 0 presence of 100 p.p.m. potassium sorbate, and 0 250 p.p.m. potassium sorbate. From J. Anacleto and N. van Uden (unpublished observations).

238

N. VAN UDEN

Cycloheximide concentration ( p g mC')

FIG. 35. Effects of cycloheximide on the maximum temperature for growth of Succhurornyces cereuisiue. From Madeira-Lopes and van Uden (1983).

1

45 I

3.1

I 40

I

35

I

30

Temperature ("C)

I

3.2

lo3*Reciprocal

I

I

25

I

3.3

20 J

3.4

of the absolute temperature

FIG. 36. Effects of cycloheximide on the temperature profile of Succhurornyces cerevisiue. 0 , A, indicate growth and death responses in the absence of cycloheximide, and 0,A in the presence of 1 pg cycloheximide ml-'. From Madeira-Lopes and van Uden (1983).

Chloramphenicol at saturating concentrations (about 4 mg ml- I ) lowered value of Succh. cereuisiue from about 40°C to slightly over 37°C the Tmaxf (Madeira-Lopes and van Uden, 1981). The Arrhenius plot of thermal death was shifted to lower temperatures, the Arrhenius plot of growth correspondingly shortened and an associative temperature profile was maintained (Fig. 37). The effect was probably due to the partitioning of the drug into a membrane system, presumably the inner mitochondria1 membrane (see p. 247).

239

TEMPERATURE PROFILES OF YEASTS f ’0 0

V .-

’c u

R

3i

10-2

r

I

10-61

I

50 46 I

3.10

1

-

3.15

lo3

I

42

38

Temperature I

1

1

34 30 (“C) 1

3.20 3.25

I 3.30

I 26 1

3.35

Reciprocal of the absolute temperature

FIG. 37. Temperature profiles of a petite mutant of Saccharomyces cereuisiae in the presence of 4 mg of chloramphenicol mi-’ (0, 0, A) and in its absence (B, A, 0 ) .0, describe specific rates of thermal death, A, A specific rates of growth during the first exponential period and 0,0 specific rates of growth during the second exponential period. From Madeira-Lopes and van Uden (1981).

V. Targets of Temperature Effects A. BASIC ASPECTS

Correlations between the heat-sensitivity of a microbial strain and the heat sensitivity of certain of its macromolecules or cell organelles (e.g. ribosomes; Pace and Campbell, 1967) do not warrant the conclusion that irreversible thermal denaturation of such macromolecules or organelles constitutes the “cause” of thermal death or that their reversible thermal denaturation is the determinant for the Tmax value of growth. When cells are subjected to supraoptimal or supermaximal temperatures many macromolecular cell constituents will suffer alterations. Furthermore, one may expect that these constituents will in general be more heat resistant the higher the T,,, value for growth and the greater the thermotolerance of the strain. The questions one should ask must be more precise. In the case of the T,,, value for growth they include: which of the reversible heat-induced denaturation processes of cell constituents essential for growth has the highest equilibrium constant at the T,,, value and may therefore be its principal determinant (Johnson et al., 1954)? Also, in the case of thermal death, of all irreversible denaturation processes, each of which by itself might lead to cell death, which is the one that has the highest specific rate at a given temperature and is therefore the principal determinant of the specific rate of thermal cell death at this same temperature? A great number of heat-induced alterations and disfunctions have been

240

N. VAN UDEN

detected in a wide variety of yeast species and have sometimes been proposed as the mechanisms that primarily underly thermal death or determine the position of T,,, on the temperature scale (Table 3). Though to the author's mind in no case has final proof been established with regard to their decisive weight in determining the temperature profile of the yeast in question, the observations in themselves are of importance for future quests for detervalues and the specific rates of thermal death in yeasts. minants of Tmax B. THERMODYNAMIC COMPENSATION

Families of chemical reactions which involve similar reactants or differ in external conditions may display linear thermodynamic compensation. An extensive review of the subject, with special attention to proteins, was given by Lumry and Rajender (1970). The first to report on the apparent occurrence of linear thermodynamic compensation in a microbial system were Barnes et al. (1969). They determined thermal inactivation rates in a strain of Sindbis virus suspended in media of different ionic strength. Arrhenius plots of the specific death rates varied in slope and vertical intercept in such a way that they intersected at or near a single point. Thus, at the temperature corresponding to this point, the so-called isokinetic temperature, death rates had the same value for all experimental conditions used. Values for A H + and AS' of thermal death calculated from the Arrhenius plots were linearly related and the slope of the A H + / A S + plot had the same value as the isokinetic temperature. When a family of Arrhenius plots has a single point in common corresponding to the isokinetic temperature Tc, the following form of equation (12) may be written:

where kdc is the specific death rate at the isokinetic temperature Tc. Making use of the relation:

AG' = AH'-TAS' equation (46) may rewritten as follows:

(47)

where ACZ is the free energy of activation of thermal death at the isokinetic temperature. Combining equations (46) and (48) leads to the following linear equation which expresses what is usually meant by linear thermodynamic compensation of rates: AH+ = AG,Z+TcASf (49)

TEMPERATURE PROFILES OF YEASTS

24 1

A less explicit form of equation (49), which is favoured by many authors, is the following: AS+

=

aAH+ -+ b

(50)

As can easily be seen T, = l/a and AGC = -b/a. Rosenberg et al. (1971) compiled activation parameters of the thermal death of yeasts (van Uden et al. 1968), bacteria, virus and Drosophila spp. The AH+ values of thermal death in this heterogeneous group of organisms were linearly related with the corresponding AS+ values and a value for T, of 325 K was obtained from the slope of the AH# versus AS+ plot. Similarly Elizondo and Labuza (1974) prepared a A H Z versus AS+ plot using their own data on thermal death of Sacch. cerevisiae in aqueous systems and in spray drying. They also included the yeast data of van Uden et al. (1968) and again a linear relationship was obtained. As was pointed out by Barnes et al. (1969), the occurrence of an isokinetic temperature in a family of biological rate processes suggests that an identical molecular mechanism is the rate-limiting common denominator. Using this reasoning Rosenberg et al. (1 971), having calculated almost identical isokinetic temperatures for thermal denaturation of proteins and for thermal death of yeasts and bacteria, suggested that protein denaturation is the cause of thermal death in unicellular organisms. Evans and Bowler (1 973) obtained a lower value for the isokinetic temperature of thermal death in multicellular animals and a protozoan, also calculated from a AH+ versus ASz plot, and suggested that, at least in these organisms, similar mechanisms do not underly thermal death and thermal protein denaturation. However, the linear AH+ versus AS+ plots which Rosenberg et al. (1971) and Elizondo and Labuza (1974) obtained using the activation parameters of thermal death of yeasts reported by van Uden et al. (1968) are not consistent with the experimental results of the latter authors. As is shown in Fig. 6 (p. 209), the Arrhenius plots of thermal death of these yeast strains did not intersect at a single common point. Thus, there was no isokinetic temperature, and the A H Z versus AS+ plot should not be linear. An analysis by Banks et al. (1972) of the mathematical procedures used for calculating AH+ and AS+ values led them to conclude that the A H Z versus AS+ plot is not sensitive enough for detecting non-linearity over the restricted range of rates and temperatures that are used in thermal death studies. The behaviour of Arrhenius plots of thermal death appears to be more relevant with respect to the occurrence, if at all, of thermodynamic compensation. Using this more direct approach, linear thermodynamic compensation was observed in certain cases when the same strain of a micro-organism suffered thermal death under different external conditions. Thus, stationary-phase populations of mesophilic yeast strains in the presence of various concentrations of NaCl (van Uden and Vidal-Leiria, 1976) as well

242

N. VAN UDEN

TABLE 3. Effects of supraoptimal and supramaximal temperatures on yeasts. Data are presented in chronological order.

Yeast Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Cryptococcus sp. (psychrophilic) Saccharomy ces cerevisiae Saccharomyces pastorianus Saccharomyces logos Saccharomyces bayanus Saccharomyces carlsbergensis Saccharomyces chevalieri Saccharomyces italicus Saccharomyces steineri Saccharomyces heterogenicus Saccharomyces fermentati Saccharomyces rosei Saccharomyces veronae Saccharomyces jlorentinus Kloeckera africana Candida sp. (psychrophilic) Candida nivalis (psychrophilic) Saccharomyces chevalieri

Temperature-sensitive cell constituent or process; conditions Petite mutation

Temperature range Supraoptimal

Haploid cells more Supramaximal thermosensitive than diploid cells Petite mutation Supraoptimal

References Ycas (1956) Wood (1956) Sherman (1959)

Inability to synthesize a-oxoglutarate

Shift from 16 to 30°C (supramaximal)

Hagen and Rose (1962)

Petite mutation

Supraoptimal

Bulder ( 1964)

Inactivation of pyruvate dehydrogenase Plasma-membrane leakage Plasma-membrane change: decrease in L-proline transport

Shift from 10 to

Evison and Rose (1965)

(supramaximal) Supramaximal

Nash and Sinclair (1 968)

25°C

Supramaximal

Schwencke and Magada-Schwencke (1971)

243

TEMPERATURE PROFILES OF YEASTS

TABLE 3 (continued)

Yeast Saccharomyces cerevisiae Saccharomyces carlsbergensis Kluyveromyces fragilis Leucosporidium stokesii Cryptococcus difluens

Temperature-sensitive cell constituent or process; conditions

Glucose-enhanced plasma membrane leakage

Unbalanced growth followed by death; aberrant cell wall synthesis Saccharomyces Mutations; mitotic cerevisiae gene conversion; mitotic chromosomal non-disjunction Schizosaccharomyces Synchronous cultures: most heat pombe sensitive in early G2 phase Candida tropicalis Continuous culture: leakage of amino acids; decrease in protein content Leucosporidium Depression of DNA stokesii synthesis Candida utilis Glycerol-limited chemostat culture: respiration less efficient Cryptococcus albidus Restriction of RNA synthesis Candida tropicalis Activation of Kluyveromyces anaerobic pathways fragilis and of cyanide-resistant respiration Saccharomyces Correlation kinetics cerevisiae of massive petite mutation and thermal death Saccharomyces Correlation kinetics cerevisiae ethanol-enhanced petite mutation and ethanol-enhanced death Saccharomyces Correlation cerevisiae lipid-buffer partition coefficients of alkanols and alkanol-enhanced death

Temperature range

Sub- and supramaximal

References

Hagler and Lewis (1974)

Dabbagh et al. (1974) Shift from 27 to 37°C (T,,, = 35°C) Shift from 39 to 52°C

Evans and Parry (1975)

Bullock and Coakley (1976) Submaximal

Kvasnikov et al. ( 1976)

Supramaximal

Silver et al. (1977)

Submaximal

Andreeva et al. (1977)

Shift from 23 to 37°C Submaximal

Stetler et al. (1978) Pozmogova ( 1978)

Supraoptimal

Simbes-Mendes et al. (1 978)

Supraoptimal

CabeCa-Silva et al. (1982)

Supramaximal

Leiio and van Uden (1982a)

244

N. VAN UDEN

as a strain of Sindbis virus (Barnes et al., 1969) displayed at least a tendency towards linear thermodynamic compensation, although exponential populations of the same yeast strains used by van Uden and Vidal-Leiria (1976) as well as the thermal death rates of spores of a Bacillus species in the presence of various concentrations of ethylene glycol, 1,2-propylene glycol and 2,3-butylene glycol clearly lacked an isokinetic temperature (Cerf et al., 1975). The significance of this erratic behaviour is open to question. The behaviour of Arrhenius plots of thermal death of heterogeneous groups of mesophilic yeasts studied by van Uden et al. (1968) and by van Uden and Vidal-Leiria (1976) indicated a type of non-linear thermodynamic compensation, the biological significance of which is more obvious. As is expressed in equation (32) the compensation between A H Z and ASZ of thermal death in the mesophilic yeasts tested was such that, regardless of the absolute values of these parameters, the Arrhenius plots of thermal death had values near the T,,, value for growth which were of the same order of magnitude as the specific growth rates which occurred in the respective superoptimal temperature ranges. Non-linear thermodynamic compensation of thermal death, correlated with the T,,, value for growth, as observed in many mesophilic yeasts, suggested that the thermal death sites and the T,,, sites in such yeasts were identical or had at least a thermosensitive component in common. C. MEMBRANES AND MITOCHONDRIA

A great amount of literature has accumulated pointing to biomembranes as

the primary target of temperature-dependent heat effects in prokaryotes which would determine the value of T,,, and the susceptibility of the cell population to thermal death. The basic idea with respect to bacteria is the following. With increasing growth temperature, the lipid composition of the membranes changes in an effort by the cell to counterbalance temperaturedependent changes in the physical state of the lipid bilayer (homoeoviscous adaptation). Eventually, temperatures are reached at which the adaptive responses of the cell are no longer adequate, and growth becomes increasingly difficult and stops at the T,,, value. Further increases in temperature render the membranes leaky, and cell death follows. In both processes, the interaction between membrane-bound proteins and their lipid vicinity is thought to play a key role (Overath et al., 1970; Esser and Souza, 1976; Welker, 1976). Fintan Walton and Pringle (1980) also explained the increased heat resistance of Sacch. cereuisiae, when the growth temperature was raised, in terms of a homoeoviscous adaptation implicitly suggesting that, in this yeast, membranes would constitute the primary target of thermal death. However, Hunter and Rose (1972) found little change in either the fatty-acyl composition or degree of unsaturation of the residues in batch-

TEMPERATURE PROFILES OF YEASTS

245

grown or chemostat-grown cells of Sacch. cerevisiae following a change in incubation temperature. In Candida utilis and in a psychrophilic yeast, on the other hand, McMurrough and Rose (1971, 1973) observed changes in lipid composition and degree of saturation depending on the growth temperature and other variables. Little influence of growth temperature on the fatty-acyl composition of Sacch. carlsbergensis was noted by Daum et al. (1977), whereas Nishi et al. (1 973) found a higher proportion of polyunsaturated fatty acids in Candida albicans grown at 35°C compared with 37°C. Although reported observations of temperature effects on the composition and degree of saturation of total yeast lipids are conflicting, a clearer picture emerged when attention was focused on mitochondrial membranes of yeasts. The degrees of unsaturation (d.u.) in the fatty-acyl residues of mitochondrial membranes expressed as [percentage of monoene + 2 (percentage of diene) + 3 (percentage of triene)] 100-I was correlated with the T,,, value in several yeasts studied by Arthur and Watson (1976): Leucosporidium frigidum (d.u. 2.3; T,,, 20"C), Candida lipolytica (d.u. 1.1; T,,, 35°C); C . parapsilosis (d.u. 0.9; T,,, 42°C); Saccharomyces telluris (d.u. 0.9; T,,, 42°C); Torulopsis bovina (d.u. 0.7; T,,, 45°C); C . sloofii (d.u. 0.6; T,,, 45°C). Some evidence has accumulated in addition to the findings of Arthur and Watson (1976) suggesting that, at least in Sacch. cerevisiae, the T,,, sites and the thermal death sites are located in the inner mitochondrial membrane. Kinetic (van Uden and Madeira-Lopes, 1970) and genetic (Madeira-Lopes, 1974) results indicated that the number of thermal death sites in Sacch. cerevisiae is of the same order of magnitude as that of mitochondria and that, as with mitochondria, they cannot be resynthesized by the cell once all of them have been inactivated. Marmiroli et al. (1976) found a Topvalue for growth of 30°C in a strain of Sacch. cerevisiae when growing on non-fermentable substrates, although the value for growth on fermentable sugar was 36°C. Furthermore, the optima for mitochondrial and cytoplasmic protein synthesis were also at 30 and 36"C, respectively. Elevated growth temperatures (at which exponential death concurs with exponential growth, as was shown later by van Uden and Madeira-Lopes (1970)), dramatically increased the spontaneous mutation rate to respiratory deficiency (petite mutation) in Sacch. cerevisiae and other yeasts (Ycas, 1956; Sherman, 1959; Bulder, 1964). Sherman (1959) was the first author to suggest that the thermal death sites of Sacch. cerevisiae are located in mitochondria. This hypothesis implied that the deficient residual mitochondria of viable petites are still functioning with respect to activities essential for cell multiplication. This appears to be true. h b i k et al. (1972) showed that mitochondria of stains of Sacch. cerevisiae viable petite retain non-identified functions vital for the yeast cell which require the availability of ATP in the mitochondrial matrix. It has not been shown yet, however, that these findings are applicable also to the residual repressed mitochondria of Sacch. cerevisiae

246

N. VAN UDEN

growing anaerobically and/or in the presence of high concentrations of glucose. As shown in Figs 38a and 39a, the kinetics of petite mutation in Sacch. cereuisiae followed closely the kinetics of thermal death in the supraoptimal temperature range (Simbes-Mendes et al., 1978). During the first period of exponential growth neither death nor net petite mutation occurred. As soon as the second period started, exponential growth concurred with exponential death and massive exponential petite mutation (Fig. 38a). Furthermore, massive petite mutation only occurred in the supraoptimal temperature range and the limits of its temperature profile coincided with those of exponential death concurrent with exponential growth (Fig. 39a). Ethanol, which had been found to enhance thermal death through effects on membranes (LeHo and van Uden, 1982b), to shift the temperature profile of Sacch. cereuisiae to

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247

TEMPERATURE PROFILES OF YEASTS

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FIG. 39. Temperature profiles of specific growth rates and specific petite mutation rates in a strain of Saccharomyces cereuisiae grown (a) without ethanol (reproduced with permission from Simdes-Mendes et al., 1978) and (b) with 5% (v/v) ethanol. From CabeGa-Silva et al. (1982). Abbreviations: ps, specific growth rate during the first exponential period (0); pg-pd, net specific growth rate during the second exponential period ( 0 ) ;pd, specific death rate; pm. specific petite mutation rate (0).

lower temperatures without disrupting it (van Uden and Duarte, 1981; Loureiro and van Uden, 1982) and to enhance petite mutation (Zakharov and Bandas, 1979; Bandas and Zakharov, 1980), had a temperature profile of mutagenic action which coincided with the supraoptimal range of temperature corresponding to the ethanol concentration used (Figs 38b and 39b; Cabeqa-Silva et al., 1982). The correlations observed suggest that, in Sacch. cereuisiae, the ethanoland temperature-sensitive petite mutation sites are located in the same membrane as the ethanol and temperature-sensitive T, sites and thermal death sites, namely the inner mitochondria1 membrane (Cabeqa-Silva et al., 1982). Should this hypothesis be confirmed, one should of course not exclude

248

N. VAN UDEN

a role for the nuclear genome of Sacch. cerevisiae in determining the temperature profile of this yeast and its sensitivity to ethanol and other drugs, since a large part of the mitochondria1 composition is under nuclear control. Furthermore, the mechanisms that underly the temperature profile of Sacch. cerevisiae and their sensitivity to drugs are not necessarily the same in all other yeasts or in eukaryotic cells in general.

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