proton production and consumption pathways in yeast metabolism. a chemostat culture analysis

YEAST VOL. 11: 1353-1365 (1995)

Proton Production and Consumption Pathways in Yeast Metabolism. A Chemostat Culture Analysis J. I. CASTRILLO, I. DE MIGUEL AND U. 0. UGALDE*

Unit of' Biochemistry, Drpurtment of Applied Chemistry, Fuculty of Chemistry, University of the Basque Country. PO B ~ . Y 1072, 20080-Sun Sebustikn, Spain

Received 20 April 1995; accepted 28 June 1995

In this investigation, a method for the accurate quantitative determination of net proton production or consumption in biological cultures has been devised. Cells are cultured under constant pH conditions. The specific rate of proton production or consumption by the culture (qH', mmol h - ' per g biomass) is proportional to the mmol of base or acid required to maintain constant pH per unit time, and this equivalence is independent of the buffering capacity of the culture medium.

The above method has been applied to chemostat cultures of Cundidu utilis growing on glucose or glycerol as carbon source, and different nitrogen sources. The results indicate that the nitrogen assimilation pathway alone determines the value of qH', and a fixed stoichiometric relationship between nitrogen uptake rate qN (meq h ' per g biomass) and qH' has been found for each nitrogen source employed. Thus, qH'/qN values of + 1, 0 and - 1 were found for ammonium ions, urea and nitrate respectively. Under oxidative metabolism, the contribution of carbon catabolism to the value of qH' was undetectable.

Since qN may be related to growth and production of type 1 compounds in fermentation processes, the parameter qH' was incorporated into a model of growth and energy metabolism in chemostat culture (Castrillo and Ugalde, Yt'usf 10, 185-1 97, 1994), resulting in adequate simulations of experimentally observed culture performance. Thus, it is suggested that qH' may be employed as a simple and effective control parameter for biotechnological processes involving biomass-related products.

KEY WORDS ~ yeast; nitrogen pathway; chemostat culture; proton production; pH; metabolic model; control

INTRODUCTION

Cellular activity entails control of internal conditions, among which proton concentration (pH) is a central parameter. Cell-medium proton exchange processes precisely regulate intracellular p H by acting at different sites of the cell (Vallejo and Serrano, 1989; Sigler and Hofer, 1991) and also maintaining localized transmembrane pH gradients which drive the active transport of solutes and metabolites (Serrano, 1985, 1988).

Extracellular medium acidification by cell cultures has been reported in yeast (Kotyk, 1989; Sigler and Hofer, 1991), fungi (Roos and Luckner, 1984) as well as plant cells (Barr et al., 1990). The capability of cells for pumping protons out of the cytosol has been demonstrated by means of spec- trophometric methods using acid/base indicators

*Corresponding author

CCC (~749~503>095/141353~13 ( 1995 b> John Wiley & Sons Ltd

and fluorophores in vesicles and whole cells (Bashford and Smith, 1979; Pascual and Kotyk, 1982; Palmgren, 1990, 1991). Glucose-induced medium acidification has been specially docu- mented in yeast cultures (Sigler et al., 1981a; Kotyk, 1989; Ramos et ul., 1989), although it is not clear to what extent this phenomenon is associated with net metabolic generation of protons, and/or the activation of a proton trans- location mechanism (Haworth et ul., 1991; Sigler and Hofer, 1991). In some cases, on-line measurement of the volume of acid/base consumed has been applied (Votruba et al., 1986; Huth et ul., 1990a,b,c). However, in many cases factors such as the contribution of the acidlbase dissociation of other molecules to proton concentration, o r the buffering capacity of the medium have not been accurately considered, either being ignored or underestimated.

1354 J. I. CASTRILLO ET AL.

Pump B+-7 I SUBSTRATES

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Y - B + H+ ---> BH (3) - R, pH ---> p- + H+

(2) S + H+ --a SH PRODUCTS (PH, S)

Figure 1. A visual representation of the factors influencing proton concentration ([H']) in biological cultures. Protons generated or consumed (qH+) as a result of cellular processes (1). The dissociation equilibria of substrates and products (2) and the buffering capacity (R,) of the medium (3) exert a modulating effect on the final detected concentration of protons.

In this paper, a general method for the accurate determination of net proton production or consumption (qH+) in biological cultures is presented. The method can be applied to different types of culture (batch, fed-batch and chemostat) for any organism and a wide range of culture conditions. The method has been applied to chemostat cultures of Candida utilis growing on different carbon and nitrogen sources. The results point to the nitrogen assimilation pathway as the main con- tributing route of net metabolic protons. The parameter qH+ can be incorporated into a previous model of growth and energy metabolism which allows for the simulation of experimentally observed culture performance.

MATERIALS AND METHODS Determination of the speclJic rate of proton exchange (qH+): general description

The cell-medium proton exchange responsible for the observed variations of pH in biological

cultures is a multicomponent process in which different mechanisms are involved (Figure 1). On the one hand, metabolic activity generates or con- sumes protons, depending on the prevailing metabolic pattern of the organism at any one time. Secondly, the dissociation equilibria of added substrates, or products can contribute to the appear- ance or disappearance of protons, and third, other medium components provide a buffering capacity 'Rn', which varies according to the medium and culture conditions.

The contribution of the dissociation equilibria of substrates can be eliminated if they are added at same pH as that of the culture. Furthermore, in many cases (i.e. cultures on sugars under aerobic conditions) the contribution of the external production of acid or basic metabolites can be considered negligible.

In these cases, under constant pH conditions, any changes in proton concentration are compen- sated for by the addition of acid/base to the medium. Since the compensating acid or base is also subject to the buffering capacity of the

PROTON PRODUCTION AND CONSUMPTION PATHWAYS

GAS PHASE

1355

- PH electrode

- -

Figure 2. Schematic description of the rationale used for the calculation of qH' in pH-stat cultures. Protons produced (F') are modulated by the buffering capacity (R,) resulting in F net protons. The corresponding pH changes are counterbalanced by addition of base (N). However this is subject to the same buffering capacity, since R,,+= R,,, - (Day and Underwood, 1986; Castrillo, 1992). Thus, the equivalents of OH- titrated (N') give a direct measurement of the net metabolic proton production (F').

medium, this element is cancelled out (Figure 2) and the equivalents of added acid or base can be used as a direct measurement of the net metabolic H ' production/consumption by the culture. Thus, the design of a simple method for the determination of the specific rate of cell-medium proton exchange (qH+) becomes possible under constant pH conditions.

The method comprises two sequential steps:

(1 ) determination of the flux of acid/base ( N l , eq

(2) application of mass balance for protons in the h - ') required to maintain constant pH;

reactor medium.

The equivalents of acid/base ( N , ) required to maintain constant pH can be determined as Ni=Q[w, where Q is the flux of acid/base consumed by the culture (1 h- ' ) and [w is the nor- mality of the incoming acid/base solution. Q can be determined by continuous titration of the volume of acid/base consumed versus time, and

subsequent determination of the slope by linear regression (preferably > I5 points).

After determination of Mi, application of the mass balance for H + in the reactor allows for the determination of the specific rate of cell-medium proton exchange qH+ (meq H + g ' per h).

General mass balance for H+ in the reactor volume:

[Accumulation] = [Input-Output] + [Appeared/disappeared in the medium due to cells] - [Appeared/ disappeared in the medium due to external addition of acid or base]

Vd[H']/dt=FL([H']i[H'l,)+F- N , [=] eq h - ' [l]

where Y is the volume of the culture (1); FL is the liquid flux (1 h - ') for continuous and semi- continuous cultures; [H+], is the concentration of protons in the input (i) and output (0) medium (eq 1 - '); F are the protons appeared/disappeared in the medium coming from the net metabolic

1356 J . 1. CASTRILLO ET AL.

activity of the cells, and N , are the equivalents of H+/OH appeared in the medium due to external addition of acid/base (eq h - ') (same nomenclature as in Figure 2).

During the determination, pH is controlled and constant (d[H+]/dt=O), the input medium can be fixed at the same pH as that of the culture ([H+],=[H+],), R,=N'/N=F'/F, and F', net proton production by the cells (eq h - I ) can be expressed as F'=(1/1000) qH+ * X - V, where X is the biomass concentration (g 1- I ) . Thus, for the case of external acidification, qH' can be formulated:

qH'=(1000/V) * (N'&)/X [=](meq g- ' per h) [2]

where N , is the flux of base (eq h I ) required to be added externally to maintain constant pH.

Similarly, for the case of net consumption of protons:

qH+= - (lO0OlV) * (N',+)/X [=](meq g- ' per h ) [3]

The above expressions can be applied to different culture configurations, batch, fed-batch and chemostat culture.

Determination of qHt in chemostat cultures of C. utilis Organism and culture conditions Candida utilis CBS 621 was maintained on agar slopes in YM medium (Difco; 2% w/v) at 4°C. Five-day-old solid cultures grown at 30°C were used to inoculate 250-ml Erlenmeyer flasks containing 100 ml of synthetic medium (concentration of glucose 5 g 1-'; Fiechter et al., 1987). Incubation took place with constant shaking at 60 cycles per min at the same temperature for 10 h and the contents of the flask were then transferred into a sterile glass bioreactor containing 2 1 of the synthetic medium (concentration of glucose 10 g 1 - I ) . The culture was aerated (2vvm) and incubated for 14 h at 30°C. The contents were finally transferred into a sterile 14-1 capacity Chemap bioreactor (internal diameter 22 cm) with control of agitation (double flat-bladed stirrer, diameter 7.5 cm), temperature (30°C) and pH 3-5 (Chemap, Volketswil, Switzerland). Fresh medium was administered at a fixed rate of 10 ml min- ' with a Watson Marlow flow inducer (Watson Marlow, Falmouth, U.K.) until a working volume of 5 1 was reached.

Chemostat culture For the study of the influence of the nitrogen source, a series of glucose-limited chemostat cultures with different nitrogen sources

(sodium nitrate, urea and ammonium sulphate) were used for a program of increasing dilution rates (between 0.05 and 0.35 h I ) . The concentration of the nitrogen (expressed in gram-atoms of nitrogen) was the same in all cases.

The synthetic medium, adapted from Fiechter and coworkers (1987), consisted of glucose (30g 1 I ) supplemented with (supplementation per gram-atom of carbon): KH,PO,, 3.0 g; gram-atom of nitrogen, 0.120 (either in the form of NaNO,, 10.2 g; urea, 3.6 g; or (NH,),SO,, 7.9 8); MgSO, 7H20, 0.45 g; CaCl,, 0.23 g; NaCI, 0.7 g; FeCl, 6H20, 15 mg; MnSO,H,O, 16 mg; ZnSO, 7H,O, 9mg; CuSO, 5H20, 2.41ng; Na,MoO, 2H20, 4 mg; CoCl, 6H,O, 0.3 mg; meso-inositol, 60 mg; calcium pantothenate, 30 mg; nicotinic acid, 6 mg; thiamine hydrochloride, 6 mg; pyridoxine hydrochloride, 1.5 mg; d-biotin, 30 pg. The pH of the medium was aseptically adjusted to 3.5 immediately before use. Urea and vitamins were sterilized by filtration through 0.45-pm pore sterile filters (Millipore, U.S.A.), and the rest sterilized by autoclaving ( 1 20°C, 1.2 bar, for 40 min).

A constant volume (5 1) was maintained through an automatic weight-control device which acti- vated the output of fermentation medium at the same rate as the input flow. The pH was maintained at 3.5 ( & 0.01) by addition of 3 N-NaOH or H,SO, through Preciflow peristaltic pumps (Lambda, Naters, Switzerland). The temperature was kept at 30 ( * 0.1)"C. Aeration was fixed in 2.8 vvm providing oxygen concentrations above 5E mmol 1 - (>20"/0 of air saturation) at all times. The system evolved until a steady state was reached, as determined by the attainment of a constant value for the concentration of biomass, dissolved oxygen, oxygen (h,) and carbon dioxide (hCJ gas fractions, for a minimum period of three residence times (Stafford, 1986).

In the studies of the influence of the carbon assimilation pathway, glycerol-limited chemostat cultures at intermediate dilution rates (0.1- 0.15 h - I ) were performed with the same medium and culture conditions. In these cultures sudden pulses of glucose (2 g 1 - ') were implemented by means of a direct injection of a sterile concentrated solution of the sugar.

Determinution of qHt The equivalents of H + (OH-) per unit of time required to maintain constant pH for each dilution rate in steady state were measured by titration with H,SO$NaOH 0.2 N during a minimum of 30 min.

PROTON PRODUCTION AND CONSUMPTION PATHWAYS 1357

Applictrtion of mass balance f o r protons For a steady-state chemostat culture with [H+],=[H+],, equations [2] and [3] were used (see above).

Other. uizaljtical metlzods Biomass and dissolved oxygen concentration were determined as in Castrillo and Ugalde (1993). For off-line analysis. samples were drawn, rapidly placed on ice, centri- fuged and the supernatant was immediately frozen in liquid nitrogen, and stored at - 20°C. Glucose concentration and determination of the absence of other products of fermentation (ethanol, acetate, acetaldehyde and ethyl acetate) were performed as described in Castrillo and Ugalde (1993). Nitrate, urea and ammonia were determined enzymatically with Boehringer test-kits 905658 and 542946. In order to certify the negligible contribution of other possible interfering processes (i.e. urea hydrolysis or external production of ammonia by the cells) in those chemostat series in which nitrate and urea were used, determination of ammonia was also included.

Mod~~l l ing The metabolic model presented elsewhere

(Castrillo and Ugalde, 1994) was extended to include the participation of the metabolic compo- nent of the nitrogen assimilation pathway. For that purpose a direct relationship between the flux gram-atom of carbon I - ' hK' (QG) and the gram-atom of nitrogen assimilated 1- ' h (QN) is included (see Results). Application of the adequate stoichiometric relationships allows for the theoretical prediction of the specific rate of proton exchange qH+.

Duia toialysis and statistical treatment Specific rates different from qHt were calcu-

lated from the material balance for each compo- nent in steady state (Fiechter et ul., 1987). All data are the average of at least three measurements. Standard error limits have been omitted for clarity but never exceeded more than 6% of the presented values, being in the majority of cases lower than 3'%,.

gen sources (ammonium sulphate, urea and sodium nitrate). Cultures on ammonium sulphate were characterized by a progressive decrease of pH (final pH=3.0), whereas cultures on nitrate re- sulted in the opposite behaviour, with progressively increasing pH (final pH above 7.0). In cultures on urea, pH remained unchanged.

Chemostat cultures of C. utilis: influence of the nitroaen source

In order to certify the influence of the nitrogen assimilation pathway in net proton production, a series of glucose-limited chemostat cultures with different nitrogen sources (ammonium sulphate, urea and sodium nitrate) were implemented. For all series, the obtained metabolic pattern was exclusively oxidative (respiratory), which was certified by the absence of production of ethanol, acetate and other metabolites, and a respiratory quotient RQ=QC0,/Q02 equal to unity. In series with urea and nitrate, also no nitrogen compounds apart from urea or nitrate were detected, certifying the absence of possible interfering mechanisms (i.e. urea hydrolysis, or ammonia extrusion by the cells).

The results are presented in Figure 3. In a series of chemostat cultures with ammonium as the nitrogen source, when increasing dilution rates were progressively implemented, net increasing rates of proton production (qH+) were obtained). On the other hand, in the case of chemostat cultures in which nitrate was used as nitrogen source, the behaviour was characterized by a progressively increasing net proton consumption.

These results, which point to a possible participation of the nitrogen assimilation pathway in net proton exchange, were related with the specific rates of nitrogen source assimilation. When the obtained specific rates of proton production qH+ (meq g ' per h) were related to the specific rates of nitrogen assimilation qN (meq g K ' per h), a mainly constant relationship was obtained for each case, corresponding to + 1, 0 and - 1 protons interchanged per gram-atom of nitrogen assimilated respectively in the form of ammonium, urea and nitrate (Table 1).

RESULTS Preliminary studies were performed using flask cultures of C. utilis (5 g 1 glucose, synthetic medium and culture conditions as described in Materials and Methods) on three different nitro-

Cltemostat culturrs of C. utilis: influence of the

In order to study the influence of the route of assimilation of the carbon substrate (glycolysis) on the total proton interchange, glycerol-limited

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Relationships between specific rates of proton production (qH') and specific rates of glucose and nitrogen

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chemostat cultures (dilution rates 0=0.10- 0.15 h ~ ') with urea as nitrogen source were performed. The existence of exclusively oxidative (respiratory) metabolism was again certified as above. The specific rates of proton production were below 0.01 meq g ' per h in all cases, resembling those obtained on glucose chemostat cultures.

When pulses of glucose (2 g 1 - ') were suddenly imposed on this system, no changes in pH were detected (results not shown). These results point to a null or negligible contribution of the glycolytic pathway on the cell-medium net proton exchange. As a general conclusion, the presented results point to the nitrogen assimilation pathway as the main pathway responsible for the net proton productionlexchange.

Modelling The main characteristics of the implemented

model have been presented elsewhere (Castrillo and Ugalde, 1994). In order to predict the specific rate of proton production by the cells as a function of the nitrogen assimilation pathway, the model was adapted to incorporate fixed relationships between proton production rates and nitrogen assimilation. The following assumptions were made.

(a) Assimilation of each nitrogen source occurs through its distinct metabolic pathway (Huth and Liebs, 1 988; Price and Stevens, 1989; Webb, 1992). Ammonium ions are assimilated via the reaction catalysed by glutamate dehyrdrogenase:

NH,' +a-ketoglutarate "-)+NAD(P)H+ H++glutamate ( ' -)+NAD(P)++H,O

This reaction does not necessarily entail the net generation of a net proton. However, a proton may arise from a previous step, leading to the

generation of the ketoacid (see Discussion). Thus, a ratio of one proton produced per ammonium ion assimilated is postulated.

Urea assimilation, in the case of urease-negative yeasts (Succharomyces cerevisiue, C. utilis, Kluy- veromyces murxiurzus), occurs along the pathway catalysed by urea carboxylase and allophanate hydrolase (Phaff et al., 1978).

Urea+ 2 a-ketoglutarate'2 - )+

2(NAD(P)H+HC)+ATP+ + > 2 glutamate" )+

2NAD(P)+ +CO,+H,O+ADP+P,

Irrespective of the assimilation route employed for this compound, urea can be viewed as equivalent to (C02+2NH,) upon hydrolysis. On conversion into NH,+, every N atom incorporates one proton from the medium, therefore cancelling out the proton derived from subsequent ammonium assimilation (above). Thus, the net balance is 0.

For nitrate ions, the pathway includes the participation of nitrate reductase, nitrite reductase and glutamate dehydrogenase, in that order (Huth and Liebs, 1988; Large, 1986). The conversion into ammonium has the following stoichiometry (NO, +2H'+[8H]-t>NE-I,++3H20). As ammonium assimilation results in production of 1 H + , the net balance is the consumption of ]Hi per assimilated N atom.

NO, - + H + +a-ketoglutarate (' - )+

S(NAD(P)H+H+)-+glutamate" - )+ SNAD(P)+ +4H,O

These metabolic pathways result in the following stoichiometric relationships:

For NH,': 1 net H' produced; 1 NAD(P)H+H+ needed.

1360 J . I . CASTRI1,L.O FT A I .

For urea: 0 net H' produced; 2(NAD(P)H+H+) needed.

For nitrate: 1 net H + consumed; S(NAD(P)H+H+) needed.

(b) The NAD(P)H required in these reactions is obtained from part of the carbon flux (glucose) which is directed to the pentose phosphate pathway (Stryer, 1995):

3 glucose-6-P+6NADP+ + SNAD' + 5Pi+8ADP+5 pyruvate+6NADPH+SNADH +

8ATP+8H++3C02+2H,0

Thus, three molecules of glucose-6-P yield 6NADPH + 5NADH +8ATP, or expressed in the manner of reducing power: 3 glucose+ - 13.7 NAD(P)H (considering a relation of 3ATP per mol of NAD(P)H).

(c) Under balanced growth, the total carbon flux can be considered as the sum of two components: the carbon flux channelled through the pentose phosphate pathway and the glycolytic flux, associated with the production of energy and biomass, of a fixed elemental composition (CH,,O,N,.) (Roels, 1980).

The model, which was originally able to predict the pattern of metabolic behavioup for different types of yeasts under different culture conditions, after incorporation of the above-mentioned assumptions and relationships is able to predict, under balanced growth, the specific rates of nitrogen consumption (qN, meq g - ' per h). Further- more, when the stoichiometric relationships + 1, 0 and - 1 meqH' (meqN)-' (relations confirmed experimentally) are applied, this results in a model which is able to predict the specific rates of net proton production (qH+).

The model was tested in order to explain the experimental behaviour of chemostat cultures of C. utilis. The model projections of the specific rates of proton production qH+ for glucose- limited chemostat cultures of C. utilis on different nitrogen sources are presented in Figure 4. The model is able to reproduce qualitatively and quantitatively the observed experimental ten- dencies and patterns of behaviour. The model focuses on the nitrogen assimilation pathway as the main source and sink for net proton production. In this respect, changes in carbon substrate concentration (glucose or glycerol) were confirmed not to influence the value of q H + in the culture.

DISCUSSION

The changes in proton concentration observed i n the extracellular medium during cell culture rc- spond to a combination of processes, including metabolic activity, redistribution between cellular compartments and the extracellular environment. and finally the dissociation of solutes. The siniulta- neous estimation of all the elements involved in this complex system seems impossible in simple batch culture, where conditions are changing constantly. Most studies have been conducted in these conditions, and are necessarily qualitative, or should be taken with extreme caution. In this study, we have implemented a pH-stat analysis in chemostat cultures, in order to eliminate interference by two of the above-mentioned elements (transitory inter- compartmental proton exchanges and dissociation of solutes), thus isolating the source of metabolic protons in aerobic culture. This issue alone is therefore the subject of this investigation.

Many studies have been carried out dealing with changes in medium pH associated with growth processes, and metabolic activity has been recog- nized as the principal cause (Kotyk, 1989; Sigler and Hofer, 1991). However, most investigations have been centered on a transitory glucose-induced acidification phenomenon (Sigler et ( I / . , 198 1 a,b; Sigler and Hofer, 1991) requiring increases i n sugar concentrations which do not reflect on the majority of culture conditions found in microbial cultures. Thus, they provide an interesting insight into the reactions operating in glycolysis, but m a y not be extrapolated to the steady growth-related pH change trends in yeast cultures. Other efforts to relate medium pH changes. to the production of CO, and organic acids have shown that their contribution is not enough to account for the high extracellular acidification observed (Kotyk, 1989). In some studies, constant pH has been maintained with on-line measurement of the acid/base consumed (James and Lummry, 1983; Huth ct 01.. 1990a,b,c). These have pointed to nitrogen assimilation pathways as the principal producers and/or consumers of extracellular protons. However, these studies have been performed in batch and fed-batch cultures, and aspects such as the contribution of cellular metabolites on proton concentration, or the buffering capacity of the medium have not been accurately considered. The results can only be given a limited value, and do not exclude the possibility of participation of other pathways or mechanisms.

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1362 J . I . CASTRILLO ET AL.

For the chemostat series performed in this investigation, a strict control over proton production and consumption points to the nitrogen assimilation pathway as the main source and sink of protons, and practically rules out any relevant contribution by other pathways under steady-state conditions, and oxidative metabolism.

Assimilation of inorganic nitrogen sources into organic forms entails a tight coordination with carbon and energy metabolism (Cooper, 1982; Large, 1986). The first enzyme which links both types of metabolism is glutamate dehydrogenase (GDH) in a reaction through the conversion of ammonia (NH,) to the central nitrogen intermediate glutamate (Mazon, 1978; Large, 1986; Huth and Liebs, 1988). Two GDH activities have been reported in yeast. The NADP-dependent GDH (EC 1.4.1.4), which catalyses the direct reaction towards the production of glutamate, and NAD- dependent GDH (EC 1.4.1.2), which catalyses the inverse reaction (Smith et al., 1975; Large, 1986). The activities of both enzymes are regulated by glucose and different nitrogen sources (Hemmings, 1978; Bogonez et al., 1985; Large, 1986). Thus, in the presence of ammonia or nitrogen sources which result in intracellular ammonia generation (i.e. ammonium salts, nitrate, nitrite and urea), very low or null activities of the NAD-dependent enzyme have been reported in yeast (Burn et al., 1974; Hemmings and Sims, 1977). In the presence of these nitrogen sources, GDH drives the reaction towards the incorporation of nitrogen into the cell in the form of glutamate. The fact that the enzyme is induced by glucose, being rapidly inactivated in the absence of the sugar (Hemmings, 1978; Mazon, 1978) can reflect the crucial role of the enzyme in the coordination between carbon and nitrogen metabolism. On the basis of its regulatory properties, glutamate dehydrogenase has been claimed to play a crucial role as an anabolic route (Polakis and Bartley, 1965).

Assimilation of ammonium ions through this central route entails the consumption of one pair equivalent (NADPH+H+) and the liberation of a net acid proton H+ to the cytosol. The tests carried out in this investigation preclude the mechanism and step at which the proton is generated. The net proton could be liberated from the ammonium ion (pKa=4.8) as presented by some authors (Huth and Liebs, 1988). However, it seems more likely that it arises from the previous generation of the ketoacid destined for amination (a-ketoglutarate,

pKa, =2.5; pKa,=4.7). Further investigations will be required to clarify this point.

With respect to the assimilation of nitrate, two previous enzymatic steps, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.6.6.4) are required to converge in the central GDH reaction. The net balance from nitrate results in the requirement of 5NADPH+H+, and a global consunip- tion of one net acid proton from the cytosol (Huth and Liebs, 1988). In the case of urea, yeasts which do not exhibit urease activity (urease-negative yeasts, i.e. S. cerevisiiw, C. utilis, K. r i i r i r . r i r r r i i r s )

(Phaff ef ul., 1978; Large, 1986) incorporate the nitrogen by means of the urea carboxylase- allophanate hydrolase pathway (Roon and Levenberg, 1972). In this case the net balance results in the requirement of 2NADPH+H' t o yield glutamate, without net production/ consumption of protons in the cytosol. The net acid protons H' participating in the above processes (produced or consumed) have to be mobi-

pathway for urea should not be decisive with respect to proton production and consumption balances, and all assimilation pathways should be neutral in this respect.

The essential redox equivalents needed for the incorporation of the different nitrogen sources (i.e. the case of nitrate, 5 NADPH+H+) are provided by the channelling of part of the carbon f lux through the pentose phosphate pathway (Stryer. 1995). In the case of yeast growing 011 glucose with ammonium as nitrogen source, the pentose phosphate pathway was found to account for 2.5% of the total metabolism of glucose (Gancedo and Lagunas, 1973). We have .calculated that, in the case of nitrate assimilation, the reducing power required would be provided by an increase i n fux of the pentose phosphate pathway of more than 20'%1. This is also reflected by the relative increase in qglucosc/qN observed in Table I , markedly higher in the case of nitrate. The nature of the regulatory pathways responsible for coordinating glycolysis and the pentose phosphate pathways may not be ascertained in this study. Recent studies haw suggested the possibility of the existence o f :I

mechanism for coordinated regulation of glycolysis and the pentose phosphate pathway mediated by xylulose 5-P (Nishimura ef ( I / . , 1994).

The stoichiometry of one net proton produced per molecule of ammonium sulphate consumed has been reported by other authors with ditferent systems and organisms (Roos and Luckner, 19x4;

lized in the cytosol. The actual assiniil, 'I t ' loll


Huth et al., 1990~). Our results are also in good agreement with data obtained in studies report- ing a stoichiometry of one proton equivalent consumed per equivalent of nitrate or nitrite consumed (Eddy and Hopkins, 1985).

The study of strategies based on pH changes of the medium for the control of a fermentation process has been the subject of continuous investigation (Veres et al., 1981; San and Stephanopoulus, 1984; lshizaki et al., 1994; Iversen et al., 1994). In the majority of cases the wide variety of processes and conditions have restricted the studies to estab- lish empirical relations, which are only valid for the specific process under investigation. On the other hand, very limited knowledge of the metabolic real- ity of the system has been put to use in these at tempts.

The presented model is a simple metabolic model. which establishes a formal stoichiometric relationship between the metabolic pathways involving nitrogen assimilation and proton production or consumption. In turn, nitrogen metabolism is related to energy metabolism and growth through previously established relationships (Castrillo and Ugalde, 1994).

Models based on formal relationships, such as this one, present the advantage of being applicable to all organisms presenting similar metabolic characteristics. It is therefore predictable that the relationships described in this system will be applicable to other yeasts, which assimilate nitrogen sources in a similar way. The model does not, however, apply to processes in which a sugar is not the carbon and energy source, or cases in which respiro-fermentative metabolism, with production of organic acids and alcohols is observed. These are presently under investigation.

Despite the limited array of processes in which qHf may be used as a control parameter under the described conditions, further detailed investigations should provide valuable guidelines as to the adequacy of using this parameter for control purposes. A priori the advantages envisaged include the following.

(1) I t allows for simple monitoring of the state of the culture, which is a direct reflection of the metabolic activity. (2) It enables a fast response action over the system, compared to other conven- tional systems of control characterized by a slower response (i.e. control systems based on control of the RQ, or a substrate or product concentration). (3) It is cheap, durable, easily applicable without incorporation of new costs to the industrial plant

and with a comparatively low cost of maintenance compared to other systems (i.e. specific sensors).

ACKNOWLEDGEMENTS

We thank the Departamento de Educacion, Universidades e Investigacion of the Basque Gov- ernment (Project PGV9241) and the Diputacion Foral de Guipuzcoa for financial support.

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