Vol. 175, No. 15JOURNAL OF BACTERIOLOGY, Aug. 1993, p. 4809-48160021-9193/93/154809-08$02.00/0Copyright © 1993, American Society for Microbiology

Growth and Metabolism of Saccharomyces cerevisiae inChemostat Cultures under Carbon-, Nitrogen-, or

Carbon- and Nitrogen-Limiting ConditionsCHRISTER LARSSON,t* URS VON STOCKAR, IAN MARISON, AND LENA GUSTAFSSONt

Department of Chemical Engineering, Swiss Federal Institute of Technology, CH-1O15 Lausanne, Switzerland

Received 21 December 1992/Accepted 24 May 1993

Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dualcarbon- and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammo-nium concentration was varied among different experiments and different dilution rates. It was found that bothglucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of mediumC/N ratios and dilution rates. To a small extent, this was due to a changing biomass composition, but muchmore important was the ability of uncoupling between anabolic biomass formation and catabolic energysubstrate consumption. When ammonium started to limit the amount of biomass formed and hence theanabolic flow of glucose, this was totally or at least partly compensated for by an increased catabolic glucoseconsumption. The primary response when glucose was present in excess of the minimum requirements forbiomass production was an increased rate of respiration. The calculated specific oxygen consumption rate, atD = 0.07 h-', was more than doubled when an additional nitrogen limitation was imposed on the cellscompared with that during single glucose limitation. However, the maximum respiratory capacity decreasedwith decreasing nitrogen concentration. The saturation level of the specific oxygen consumption rate decreasedfrom 5.5 to 6.0 mmol/g/h under single glucose limitation to about 4.0 mmol/g/h at the lowest nitrogenconcentration tested. The combined result of this was that the critical dilution rate, i.e., onset of fermentation,was as low as 0.10 h-1 during growth in a medium with a low nitrogen concentration compared with 0.20 h-obtained under single glucose limitation.

During growth of Saccharomyces cerevisiae in a chemo-stat with glucose as the sole limiting substrate, energy ratherthan carbon limits biomass formation (7, 22, 29), i.e., it canbe regarded as a catabolic limitation. If, on the other hand, a

nitrogen source such as ammonium is used as the limitingsubstrate, this can instead be viewed as an anabolic limita-tion. Several studies, concerning bacteria, have shown thatanabolism and catabolism do not seem to be tightly coupled(3, 5, 12, 16-20, 25). The catabolic activity is often higherthan what can be explained by the anabolic requirements forATP when the energy source is present in excess. Whenglucose-grown chemostat cultures of the bacterium Kleb-siella aerogenes were subjected to a limitation other than a

glucose limitation, it was found that the specific respirationrate increased compared with that of glucose-limited cells. Inaddition, when the energy source was in excess, the cellsresponded by excreting a number of products more oxidizedthan the carbon source, i.e., not a fermentative metabolism,a phenomenon named overflow metabolism (3, 12, 17-20,25). K aerogenes is the most extensively studied in thisrespect, but several other bacteria seem to respond in a

similar way (16, 20). In regards to the yeast S. cerevisiae,uncoupling between catabolism and anabolism has also beenreported. When glucose-limited chemostat cultures of S.cerevisiae were pulsed with excess glucose, the catabolicactivity increased more or less immediately, whereas therate of biomass production remained unchanged for the first30 min (26). However, these cells were adapted to an energylimitation when they were faced with a sudden energy excess

* Corresponding author.t Present address: Department of General and Marine Microbiol-

ogy, University of Goteborg, S-413 19 Goteborg, Sweden.

through glucose pulsing. In contrast, this work describesmetabolic uncoupling under steady-state conditions for cellsadapted to energy excess.The main aim of this study was to investigate metabolic

uncoupling of S. cerevisiae cultivated aerobically in chemo-stat cultures with glucose as a carbon and energy source.Uncoupling was induced by gradually decreasing the nitro-gen concentration of the feed medium and thereby imposingan increasing anabolic constraint on the cells. Furthermore,S. cerevisiae is capable of both respiratory and fermentativemetabolism, and the relative importance of these two path-ways for uncoupling was determined.The transition from one limitation to another is not very

sharp (1, 12). Consequently, a dual limitation, in which boththe carbon as well as the nitrogen substrate is limiting, mayoccur (3, 5, 6, 15). A limiting substrate is in this case definedby von Stockar and Auberson (31) as a substrate which isconsumed at the maximal possible rate, i.e., the feed rate,and this rate is not determined by the concentration or feedrate of any other substance in the medium. Although boththe carbon and nitrogen sources constitute limiting sub-strates if the dual limitation is induced by reducing thenitrogen concentration, the cells are facing an energy excess.Actually, glucose will be available-and also consumed-inexcess of the minimum requirements for the formation ofbiomass that is possible in nitrogen-limited situations.

Finally, the influence of growth limitation, i.e., by carbonand/or nitrogen, and dilution rate on biomass compositionwas studied. An influence of C/N ratio in the medium onbiomass composition has previously been reported for thebacteria Hyphomicrobium X (5) and HyphomicrobiumZV620 (9) as well as for the yeasts Hansenula polymorpha(6) and Candida valida (15). For S. cerevisiae, it seems as if

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Inc., Ann Arbor, Mich.) was water saturated by passing theB 50 1 __ l airstream through a bubble column with a temperature of

330C before it entered the chemostat. These conditions340 ensured a dissolved oxygen concentration above 60% of air

3saturation. The pH was kept constant at 4.5 by automatic

U 30 1 ,_O o |addition of 4 M NaOH with a pH controller (Bioengineering20

/o~ AG, Wald, Switzerland). The feed medium was thermo-.53 20 [ | F ~ | stated to 30'C before it entered the chemostat.

e0 c~The total activity (rate of heat production) of the culture6 10 *( t * * * * * * * * | was continuously monitored by the calorimeter. Before

inoculation of the medium, a baseline for the calorimetric0 signal was established by running the calorimeter (chemo-

0 0.1 0.2 03 0.4 stat) under conditions identical to those of the actual exper-Dilution rate (1/h) iment for at least 12 h. The heat transfer coefficient (W m-2

FIG. 1. Changes in the C/N ratio of the feed medium at different K-1) was then determined by measuring the response to thedilution rates for high (0)-, intermediate (0)-, and low (E)-nitrogen- activation of a 10-W in situ calibration heater for 20 min. Theconcentration media. principles of the calorimetric measurement procedure have

been described previously (32).After inoculation, substrate feeds for continuous opera-

the growth rate is an important parameter in determining the tion were initiated once the cells had reached the latecellular nitrogen concentration (27, 28). exponential or stationary phase of batch growth. The exper-

iments were always performed by starting at the lowest

MATERIALS AND METHODS dilution rate and subsequently increasing the dilution ratestep by step. Apart from continuous on-line measurement ofYeast strain and media. S. cerevisiae CBS 426 was main- the rate of heat production by the cells, samples were taken,

tained on YPD agar slants (1% [wt/vol] yeast extract, 2% once a steady state had been established, for determinationpeptone, 2% glucose, and 2% agar). Cells were cultured in a of biomass concentration and extracellular concentrations ofmedium having the following composition (per liter of solu- glucose, ethanol, acetate, glycerol, and ammonium and fortion): glucose, 20 g; KH2PO4, 2 g; MgSO4 . 7H20, 0.4 g; determination of the elemental composition of the biomass.CaCl2 2H20, 0.2 g; NaCl, 0.5 g; yeast extract, 1 g; H3B03, Test for steady-state conditions. A constant heat produc-1 mg; ZnSO4 - 7H20, 1 mg; MnCl2, 1 mg; FeCl3 3H20, 0.5 tion rate, as recorded by the calorimeter, was used as themg; CUS04 *5H20, 0.1 mg; Na2MoO4, 1 mg; KI, 0.1 mg; criterion for the establishment of steady-state conditions. Allmyo-inositol, 25 mg; 1-alanine, 2.5 mg; biotin, 7 pLg; folic reactions in a cell give rise to a heat change; therefore, theacid, 2 ,ug; p-aminobenzoic acid, 200 pg; nicotinic acid, 400 calorimetric technique can be used as a measure of the total,g; pyridoxine hydrochloride, 400 ,ug; thiamine hydrochlo- activity of the culture (2, 8, 11).ride, 400 ,ug; riboflavin, 200 ,ug; and Sigma Antifoam A, 0.2 Dry weight determinations. Samples (two, 4 ml each) wereml. centrifuged for 5 min at 3,500 x g, washed twice withThe concentration of (NH4)2SO4 was varied between the deionized water, dried for 24 h at 105°C, and stored in a

different experiments. The experiment in which the (NH4)2 desiccator before being weighed. The duplicates differed byS04 concentration in the feed was constant at 6 g/liter is ±0.6%.referred to in the text as the high or highest nitrogen Determinations of glucose, ethanol, acetate, glycerol, andconcentration in medium. In the remaining experiments, the ammonium concentrations. Samples (two, 4 ml each) werefeed concentration of (NH4)2SO4 was different for each centrifuged for 5 min at 3,500 x g. The supernatants weredilution rate. This was achieved by the use of two indepen- stored in the freezer (-20°C). Glucose, ethanol, acetate,dently controlled medium flow rates. The flow of the first glycerol, and ammonium concentrations were determinedmedium, containing (NH4)2SO4, was maintained at a con- with enzyme combination kits (Biochemica Test Combina-stant, while the flow of an identical medium, lacking tion; Boehringer Mannheim GmbH, Mannheim, Germany).(NH4)2S04, was varied in order to achieve the desired Gas analysis. The concentrations of CO2 and ethanol in thedilution rate. Thus, increasing the dilution rate resulted in a exhaust gas stream from the reactor were determined bydecrease in the feed concentration of (NH4)2SO4. Further- infrared spectroscopy (model PSA-401; Servomex, Crow-more, two different reservoir concentrations of (NH4)2SO4 borough, United Kingdom). The sample line from the reac-were employed. The first (13.4 g/liter) is referred to as the tor and the gas distribution system were heated in order tointermediate nitrogen concentration, while the second (4.6 prevent condensation of ethanol in the gas stream.g/liter) is referred to as low or lowest nitrogen concentration Determination of elemental composition and ash and resid-in medium. The resulting C/N ratios of the feed medium at ual water content of the biomass. The procedure used fordifferent dilution rates are shown in Fig. 1. determination of elemental composition and enthalpy ofGrowth conditions and calorimetry. The chemostat cul- combustion of biological material has been described previ-

tures were conducted in a 2-liter bench-scale calorimeter ously by Gurakan et al. (10). The elemental composition of(model RC-1; Mettler-Toledo AG, Greifensee, Switzerland) each sample of freeze-dried biomass samples was measuredmodified for biological work. The working volume was 1.5 four times, and the standard deviations were 0.11, 0.11, andliters, the temperature was 30°C, and the stirring rate was 0.06% (wt/wt) for carbon, hydrogen, and nitrogen, respec-700 rpm. Aeration was controlled at 1.5 volume of air per tively. The residual water and ash determinations werevolume of liquid per minute by a mass flow meter (5850 TR; performed in duplicate. The residual water content rangedBrookes Instrument, B.V., Veenendal, The Netherlands). from 5.13 to 8.15% (wt/wt) of the lyophilized sample weight,The sterile filtered air (pore size, 0.45 ,um; Gelman Sciences, and the duplicates differed by +0.01%. The ash content

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ranged from 6.76 to 10.71% (wt/wt), and the duplicatesdiffered by ±0.02%.

Calculation of carbon flow and oxygen consumption. Thefractions of the carbon substrate used for anabolism (Sa) andcatabolism (Se) were calculated for different dilution ratesand different C/N ratios in medium. In addition, the catabolicfraction was further divided into a respiratory (Scr) and afermentative (SCf) part.The anabolic fraction (Sa, C-mol per C-mol; 1 C-mol is the

amount of an organic compound containing 1 mol [e.g., 12 g,of carbon]) was determined by dividing the measured rate ofbiomass produced (dX/dt, C-mol per liter per hour) and theaccompanying carbon dioxide (dCO2,/dt, C-mol per liter perhour) by the measured rate of glucose consumed (dS/dt,C-mol per liter per hour):

Sa = [(dX/dt) + (dCO2a/dt)]/(dS/dt)The rate of carbon dioxide produced in anabolism wasdetermined by using the concept of degree of reduction (14,22). The biomass carbon content was determined by anelemental analyzer, and the results were calculated on anash- and water-free basis (see above).The catabolic fermentative part (SCP C-mol per C-mol)

was calculated by dividing the measured rates of formationof the fermentation products (dP/dt, C-mol per liter per hour)ethanol, acetate, glycerol, and the corresponding carbondioxide by the rate of glucose consumed (dSldt, C-mol perliter per hour):

Scf = (dP/dt)I(dS/dt)The carbon dioxide formed in the fermentative pathways(dCOQ/dt, C-mol per liter per hour) could be determined,since 1 C-mol of acetate or ethanol produced corresponds to0.5 C-mol of carbon dioxide formed.The catabolic respiratory part (Sc, C-mol per C-mol) was

calculated by taking the measured total rate of carbondioxide produced (dCO2,,0dt, C-mol per liter per hour)minus the carbon dioxide production rate which accompa-nied the formation of fermentation products (dCO2/dt,C-mol per liter per hour) and biomass (dCO2ajdt, C-mol perliter per hour) and dividing by the rate of glucose consumed(dS/dt, C-mol per liter per hour):

Scr = [(dC02tot/dt) - (dCO21dt) - (dCO2a/dt)]/(dS/dt)The specific oxygen consumption rate (dO2spldt, moles per

gram per hour) was obtained by taking the measured totalrate of carbon dioxide produced (dCO2,,Jdt, C-mol per literper hour) minus the carbon dioxide production which ac-companies the formation of ethanol (dCO2,tOhldt, C-mol perliter per hour) and biomass (dCO2jdt, C-mol per liter perhour) minus 0.5 times the rate of glycerol produced (dGlyc/dt, moles per liter per hour) divided by the actual biomassconcentration (X, grams per liter):

dO2wldt = {(dCO2W/dt) - (dCO2,toh/dt)- (dCO2aIdt) - [0.5 (dGlyc/dt)]}/X

Production of 1 mol of glycerol consumes 1 mol of NADH,and 0.5 mol less 02 will be consumed per mol of glycerolproduced. Carbon dioxide production accompanying acetateformation is followed by production of NADH, i.e., 1 CO2produced yields 2 NADH and hence consumption of 1 02-Consequently, the 02 consumption is already accounted forby the dCO2,tjdt term.

0.8

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Dilution rate (1/h)

FIG. 2. Changes in growth yield at different dilution rates duringgrowth of S. cerevisiae in high (0)-, intermediate (0)-, and low(-)-nitrogen-concentration media.

The results are shown in Fig. 4 for the different nitrogenconcentrations in medium. The heights of the columns alsoshow the carbon balance for each dilution rate; ideally, thisshould always equal 1. The average values of the carbonbalance for the different dilution rates were 0.99 ± 0.05(standard deviation; n = 10) during growth in high andintermediate nitrogen concentrations in medium and 0.99 +0.06 (standard deviation; n = 10) during growth in low-nitrogen medium. It was assumed in all the calculations thatthe carbon content of the yeast extract, measured to 32.05mmol of C per g, was consumed. Calculations on theassumption that none of the carbon content of the yeastextract was consumed yielded an average carbon balance of1.05, indicating that the former assumption is more correct.

In addition, calculations were also made to determine therespiratory yield (SrX, C-mol per C-mol). The respiratoryyield was calculated by dividing the rate of glucose respired(dScrgdt, C-mol per liter per hour) by the rate of biomassformation (dX/dt, C-mol per liter per hour) plus the accom-panying carbon dioxide production (dCO2,/dt, C-mol perliter per hour):

Srl = (dScr/dt)/[(dX/dt) + (dC02/dt)]The respiratory yield can be obtained from Fig. 4 by dividingthe size of the respiratory fraction (grey part) with the size ofthe anabolic fraction (black part) of the bars.

RESULTS

Chemostat cultures of S. cerevisiae were performed atdifferent dilution rates and at different C/N ratios of themedium.Growth characteristics. (i) High N concentration. When the

nitrogen feed concentration was high, the growth yieldremained approximately constant over the dilution raterange 0.06 to 0.17 h-1 (Fig. 2). Above 0.17 h-1, the growthyield fell with increasing D. In these experiments, glucosewas the sole limiting substrate, as shown by the low residualglucose concentration (Fig. 3A), while the residual ammo-nium concentration remained above 36 mM for all values ofD (Fig. 3A). The critical dilution rate, Dc, representing theonset of respirofermentative activity, was 0.20 h-, asshown by the appearance of ethanol in the medium. Atvalues of D < 0.20 h-1, the ethanol concentration was <1

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90

60

30

60

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30

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60

45

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15

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0 0.1 0.2 03 0.4Diion rate (1/h)

I

. . *b - To0 0.1 0.2 03 0.4

Diluton rate (1/h)

0 0.1 0.2 03 0.4

Dilution rate (1/h)FIG. 3. Changes in measured parameters at different dilution

rates during growth of S. cerevisiae in high (A)-, intermediate (B)-,and low (C)-nitrogen-concentration media. Residual glucose (0),ammonium (A), and extracellular ethanol (0) concentrations areshown.

mM, while the maximum concentration (123 mM) was ob-served at the highest dilution rate (0.35 h- ) tested (Fig. 3A).

(ii) Intermediate N concentration. Three distinct phasescould be distinguished. (i) D = 0.05 to 0.08 h-1. This wascharacterized by a high growth yield (Fig. 2), low residualglucose (<0.4 mM), and high residual ammonium concentra-tion (>12 mM) (Fig. 3B) and corresponds to glucose limita-tion of the culture. (ii) D = 0.11 to 0.26 h-1. This wascharacterized by a reduced growth yield, low residual glu-cose (<0.4 mM), and low residual ammonium concentration(<0.3 mM) and corresponds to dual carbon and nitrogenlimitation of the culture. (iii) D > 0.29 h-1, where the growthyield continued to fall and the residual glucose concentration

increased, whereas the ammonium concentration remainedlow (Fig. 2 and 3B). This phase corresponds to a nitrogen-limited culture. Furthermore, DC decreased (0.14 h-1) com-pared with the high-N-concentration experiment, and themaximum ethanol concentration of 123 mM was obtained atD = 0.29 h-1.

(iii) Low N concentration. In this case, the growth yield fellcontinuously with increasing dilution rate (Fig. 2). Theresidual ammonium concentration remained low (<0.3 mM)over the whole range of dilution rates studied while theresidual glucose concentration was <1 mM up to D = 0.16h-1 and increased at higher values of D (Fig. 3C). Thus, twodistinct phases could be distinguished: (i) D ' 0.16 h-1characterized by low residual glucose and ammonium con-centrations and corresponding to a dual carbon and nitrogenlimitation of the culture and (ii) D 2 0.19 h-1, characterizedby an increasing residual glucose and low residual ammo-nium concentrations (Fig. 3C), corresponding to nitrogenlimitation of the culture. Furthermore, DC decreased (0.10h-1) compared with both of the previous experiments, andthe maximum ethanol concentration of 111 mM was obtainedatD = 0.19 h-1.Carbon flow and oxygen consumption. (i) High N concen-

tration. The anabolic fraction remained more or less con-stant between D = 0.06 h-1 and D = 0.17 h-1 (Fig. 4A). Inthis interval, there was no fermentation; only respirationoccurred. Consequently, there was a steady increase inoxygen consumption with increasing dilution rate. The ratioof glucose respired per biomass formed, the so-called respi-ratory yield, attained its maximal value of 0.66 C-mol/C-molat D = 0.17 h-1. Fermentation was used in addition torespiration at D = 0.20 h-1. The oxygen consumption rate of5.4 mmol/g/h at this dilution rate seemed to be close to themaximum capacity during these conditions (Fig. 4A). Thefermentative part accounted for almost all of the increasedconsumption rate of glucose as the dilution rate was in-creased above 0.20 h-1 (Fig. 4A).

(ii) Intermediate N concentration. At D = 0.11 h-1, theanabolic fraction already started to decrease (Fig. 4B). Atthis point, the respiratory yield began to increase, and themaximum value of 0.79 C-molIC-mol was obtained at D =0.14 h-1. Fermentation in addition to respiration started toappear at a dilution rate of 0.14 h-1 (Fig. 4B). The specificoxygen consumption rate seemed to have reached its maxi-mum value of about 5.0 mmol/g/h at D = 0.17 h-1. Therespiratory rate remained more or less constant, whereas thefermentation rate increased when the dilution rate wasincreased above 0.17 h-1 (Fig. 4B).

(iii) Low N concentration. The anabolic fraction decreasedcontinuously with increasing dilution rate up to D = 0.22 h-1(Fig. 4C). Further increase of the dilution rate resulted in aconstant anabolic fraction of approximately 0.18 C-mol/C-mol. The respiratory yield was very high, with a maximumvalue of 1.09 C-molIC-mol at D = 0.07 h-1. In fact, thevalues for this ratio at the three different dilution rates 0.07,0.10, and 0.13 h-1 were all well above the maximum valueobtained during growth in the highest nitrogen concentrationin the feed medium (Fig. 4A and C). Consequently, theoxygen consumption rate increased rapidly with increasingD at low dilution rates. Fermentation in addition to respira-tion started at D = 0.10 h-1. The specific oxygen consump-tion rate at this point was 3.5 mmol/g/h (Fig. 4C). When thedilution rate was further increased, the fermentative rateincreased; there was also a small increase in the respiratoryrate as well. The maximum specific oxygen consumption

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1.2

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l 104 X A

3 t 5

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o o o

Dilution rate (1/h)

0.40 0.1 0.2 03

Diluton rate (1/h)

FIG. 5. Changes in the heat yield (heat produced per unit of0 biomass formed) at different dilution rates during growth of S.

cerevisiae in high (0)-, intermediate (0)-, and low (-)-nitrogen-concentration media.

1.2

jl 1

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a

__4_4

Dilution rate (1/h)FIG. 4. Changes in carbon flow and specific oxygen uptake rate

at different dilution rates during growth of S. cerevisiae in high (A)-,intermediate (B)-, and low (C)-nitrogen-concentration media. Thecalculated specific oxygen consumption rate (U) and fraction ofglucose used for biomass production (black bar), respiration (greybar), and fermentation (unfilled bar) are shown. The total heights ofthe columns also show the carbon balance at each dilution rate.

rate was about 4.0 mmol/g/h (except for the highest dilutionrate, at which 4.6 mmol/g/h was recorded).Heat production. The amount of heat produced per amount

of biomass formed, the so-called heat yield, was calculatedby dividing the steady-state rate of heat production (kilo-joules per liter per hour) by the rate of ash-free biomassformed (grams per liter per hour). During growth in thehigh-nitrogen-concentration medium, the heat yield in-creased with increasing dilution rate as long as fermentationwas absent (Fig. 5). The values increased from 9.4 kJ/g at D= 0.06 h-' to 12.5 kJ/g at D = 0.17 h-1, with a very lowvalue of 7.3 kJ/g at D = 0.09 h-1. Because of the small heatchange accompanying the fermentation of glucose to etha-nol, the heat yield decreased as the fermentative part ofcatabolism became increasingly important. In the intermedi-ate-nitrogen-concentration medium, the heat yield valuesobtained for the two lowest dilution rates were of the same

order of magnitude as in the high-nitrogen-concentrationmedium (Fig. 5). However, as nitrogen, in addition toglucose, became limiting, the heat yield values increased toa maximum of 15.9 kJ/g at D = 0.14 h-1 (Fig. 3B and 5).Because of the increased fermentative part of catabolism,the heat yield started to decrease at dilution rates above 0.14h-1. Furthermore, during growth in the low-nitrogen-con-centration medium, the heat yield value was comparable tothe others at the lowest dilution rate (Fig. 5). However, therewas a dramatic increase in the heat yield to 23.8 kJ/g as

nitrogen in addition to glucose became limiting at D = 0.07h-1. As observed before, with an increase in fermentationrate, at dilution rates above 0.07 h-1, the heat yield valuesstarted to decrease.Biomass composition. The carbon content was more or less

similar during growth at all dilution rates and nitrogenconcentrations in medium, with typical values of 50% (wt/wt). There was, however, a slight tendency to a lowercarbon content at the very lowest dilution rates in all threemedia, with values of about 48%. The hydrogen content wasalways about 7% irrespective of dilution rate and mediumcomposition. For the glucose-limited culture during growthin the high-nitrogen-concentration medium, there was a

steady increase in nitrogen content of the biomass from 7.5%at D = 0.06 h-1 to 9.6% at D = 0.35 h-1. Consequently, theC/N ratio of the biomass decreased from 6.6 to 5.1 g of C perg of N with increasing dilution rate (Fig. 6). When ammo-

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i8

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Dilution rate (1/h)FIG. 6. Changes in the biomass C/N ratio at different dilution

rates during growth of S. cerevisiae in high (0)-, intermediate (0)-,and low (E)-nitrogen-concentration media.

nium also became limiting during growth in intermediate-and low-nitrogen-concentration media at the dilution rates0.14 and 0.07 h-', respectively, the nitrogen content of thebiomass decreased. The result was that the C/N ratio of thebiomass made a shift in the otherwise steady decrease withincreasing dilution rate (Fig. 6). When the dilution rate wasfurther increased, above the point at which ammoniumstarted to become limiting, the C/N ratio of the biomassdecreased again continuously with increasing dilution rate(Fig. 6). This is despite the fact that the actual nitrogenconcentration in the feed medium decreased with increasingdilution rate (Fig. 1). The lowest nitrogen content of thebiomass was 5.2% obtained at D = 0.07 h-1 during growth inlow-nitrogen-content medium, resulting in a C/N ratio of thebiomass of 9.8 g of C per g of N.

DISCUSSION

When S. cerevisiae is grown aerobically in a chemostatunder glucose limitation, a strictly respiratory metabolism isobtained at low dilution rates. However, as the dilution rateis increased above a certain critical value, fermentation inaddition to respiration becomes apparent, a behavior whichhas been attributed to the limited respiratory capacity of S.cerevisiae (21, 24). This study showed that the value of thecritical dilution rate was very much dependent on thenitrogen concentration of the medium. During growth in thelowest-nitrogen-concentration medium, significant amountsof ethanol were produced at a dilution rate as low as 0.10 h- 1

(Fig. 3C). When a nonlimiting concentration of ammoniumwas supplied and only glucose was limiting, ethanol was notdetected until a dilution rate of 0.20 h-1 (Fig. 3A). Thereason for this behavior seems to be that the maximumrespiratory capacity, i.e., the maximum specific oxygenconsumption rate, decreased with decreasing nitrogen con-centrations in the medium (Fig. 4). The maximum valuedecreased from about 5.5 to 6.0 mmol/g/h under singleglucose-limiting conditions during growth in the highest-nitrogen-concentration medium, down to 4.0 mmol/g/h at thelowest nitrogen concentration tested. Similar to the obser-vations by Rieger et al. (21), the specific consumption rate ofoxygen remained more or less constant when the dilutionrate was increased above the critical value (Fig. 4). Thesmall decrease in nitrogen concentration in the medium withincreasing dilution rates, at dilution rates above the criticalvalue, did not seem to affect the oxygen consumption rate.

This is not surprising, since as shown in Fig. 1, this decreasewas very small compared with the differences in nitrogenconcentrations in the medium among the three differentexperiments. Both the critical dilution rate as well as themaximal specific oxygen consumption rate were somewhatlower compared with those reported by Rieger et al. (21),who used a different strain of S. cerevisiae. Increasing thedilution rate in small steps as suggested by Rieger et al. (21)did not change the situation. Ethanol always accumulated ata dilution rate of about 0.20 h-1 when glucose was the solelimiting substrate.However, not only the fermentation rate was affected by

the nitrogen concentration of the medium. In fact, theprimary response when ammonium started to limit theamount of biomass formed was an increased rate of respira-tion. The amount of glucose respired per amount of biomassformed, the so-called respiratory yield, increased from amaximum value of 0.66 C-mol/C-mol during single glucoselimitation to a maximum value of 1.09 C-mol/C-mol duringgrowth at the lowest nitrogen concentration (calculated fromFig. 4A and C). Consequently, the specific oxygen consump-tion rate was more than doubled during growth at D = 0.07h-1 under dual carbon and nitrogen limitation comparedwith single glucose limitation at similar dilution rates (Fig.4). The increased respiratory yield was not due to anincreased acetate concentration during growth in the low-nitrogen-concentration medium. The acetate concentrationswere between 0.5 and 1.0 mM irrespective of nitrogenconcentration in the medium (data not shown). When therespiratory capacity became saturated, an increased rate offermentation was observed. To summarize, as the C/N ratioof the feed medium increased and nitrogen started to limitthe anabolic flow of carbon, this was compensated for,totally or at least in part, by an increased catabolic carbonflow.An increased catabolic compared with anabolic flow might

result in an overproduction of ATP unless the cells are ableto adjust either the amount of ATP produced or the ATPyield. An increased degree of fermentation relative to respi-ration at conditions of low nitrogen concentration may be away of producing less ATP when the requirement is de-creased in the anabolic pathway because of the limitedsupply of nitrogen. Of course, the decrease in the maximumspecific oxygen consumption rate might also simply be areflection of a reduced synthesis of nonessential respiratoryenzymes during nitrogen-limiting conditions. However, theincreased respiratory yield when an additional nitrogenlimitation was imposed on the cells implies that the ATPyield and/or the P/O ratio must be able to change. Assuminga constant ATP yield and adopting the value of 16 g ofbiomass per mol of ATP reported for S. cerevisiae underoptimal growth conditions (28) yielded a P/O ratio of about 1to 1.5 during glucose limitation at low dilution rates andvalues of about 0.3 at high dilution rates under nitrogenlimitation. If instead a fixed P/O ratio of 1 is used (23, 29, 30),the corresponding ATP yield can be calculated to a maxi-mum of about 20 g per mol of ATP under glucose limitationand low dilution rates down to values below 10 g of biomassper mol of ATP under nitrogen limitation. In fact, even forthe culture in which only glucose was limiting, a decrease inthe ATP yield could be calculated with increasing dilutionrate. Assuming a P/O ratio of 1 gave an average ATP yield ofabout 18 g of biomass per mol of ATP at low dilution rateswhereas the value at high dilution rates was about 13 g ofbiomass per mol of ATP. This is in line with the resultsobtained for anaerobically grown glucose-limited cultures of

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DUAL LIMITATION CHEMOSTAT CULTURES OF S. CEREVISL4E 4815

S. cerevisiae, in which a decrease in the ATP yield was

obtained with increasing dilution rate (28). It has also beenargued that a constant YATp value for S. cerevisiae atdifferent dilution rates is highly unlikely, since the proteincontent increases significantly with increasing growth rate(27-29).The obtained heat yield values (Fig. 5) below the critical

dilution rate support the view that the ATP yield and/or theefficiency of energy generation, i.e., the P/O ratio, decreasedwith increasing dilution rate and when an additional nitrogenlimitation was introduced, i.e., the heat yield increased. Themaximum value was 23.8 kJ/g compared with 12.5 kJ/g ofbiomass formed during growth in low- and high-nitrogen-concentration media, respectively. At dilution rates abovethe critical value, the heat yield decreased (Fig. 5) because ofthe very low heat change accompanying fermentation ofglucose to ethanol compared with respiration of glucose.Respiration of glucose yields -468.7 kJ/C-mol, whereasfermentation only provides -16.7 kJ/C-mol (14). Neverthe-less, the heat yield values obtained at different dilution ratesabove the critical value were significantly higher duringgrowth in the lowest-nitrogen-concentration medium com-

pared with growth under single glucose limitation (Fig. 5).This was despite the fact that the fermentative part was even

more dominating during growth in the lowest-nitrogen-con-centration medium. Part of the increased heat yield and thesuggested decrease in ATP yield during growth in lownitrogen concentrations can most probably be explained byan increased uncoupling between ATP production and bio-mass formation during these conditions. However, it is alsoprobable that the decrease in ATP yield and increase in heatyield are in part due to the increased energy requirements forammonium transport during growth in the low nitrogenconcentration. The Km values of the two ammonium trans-port systems in S. cerevisiae have been reported to be 2 and0.25 mM (4).

It seemed as if the cells changed their C/N ratio of thebiomass according to the C/N ratio of the feed medium atlow dilution rates. However, this relationship between bio-mass and C/N ratio of the medium gradually disappeared as

the dilution rate increased. The C/N ratios of the biomass atthe highest dilution rates tested were 5.1, 4.8, and 5.1 g of Cper g of N in high-, intermediate-, and low-nitrogen-concen-tration media, respectively (Fig. 6). The increase in C/Nratio with increasing D is probably, at least in part, due to a

decreasing content of reserve carbohydrates at high dilutionrates (13). However, if this was the only reason for thedifference in C/N ratio it should also be reflected in a

changing carbon content of the cells. This was not the case,except for the very lowest dilution rates, at which a slightlylower carbon content of the biomass was obtained. Conse-quently, the main reason for the decreasing C/N ratio at highdilution rates is probably an increasing protein content underthese conditions (27, 28). This view is supported by the factthat the nitrogen content of the biomass did increase withincreasing dilution rates.To summarize, the primary response when nitrogen

started to limit biomass formation and the energy and carbonsource was present in excess was an increased respiratoryactivity. When the respiratory capacity became saturated,an increased rate of fermentation was observed. The satura-tion level of respiration decreased with decreasing nitrogenconcentration in medium. Consequently, the critical dilutionrate, i.e., onset of fermentation, decreased concomitantlywith decreasing nitrogen concentrations in the medium.Both the energy and carbon source (glucose) as well as the

nitrogen source (ammonium) were limiting over a range ofdilution rates and medium C/N ratios. This dual limitationwas to a small extent due to a changing biomass composi-tion, but much more important was the ability of uncouplingbetween anabolic biomass formation and catabolic energysubstrate consumption.

ACKNOWLEDGMENT

This work was supported by a grant from the Swiss NationalScience Foundation (FNRS).

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