Continuous citric acid secretion by a high specific pH dependent active transport system in yeast Candida oleophila ATCC 20177
Keywords: active citrate export, citric acid fermentation, energy consuming citric acid secretion, specific active transport system.
in Biotechnology, Co., Vat. #: 108851559. Avgi/Sohos, 57002
The pH influence on continuous citric acid secretion was investigated in Candida oleophila ATCC 20177 (var.) under NH4+ limiting state steady conditions, using glucose. Highest citric acid concentration of 57.8 g/l, citrate/isocitrate ratio of 15.6, space-time yield of 0.96 g/(l x hr) and biomass specific productivity of 0.041 g/(g x hr) were obtained at pH 5 and 60 hrs residence time. Only 22.8 g/l (39.4%) and a ratio of 9.9 were achieved at pH 6 pH and 12.4 g/l (21.5%) and a ratio of 3.7 at pH 3. Under non producing conditions, in excess of nitrogen, biomass concentration increased at raising pH. An iron concentration of 200 ppm was determined in biomass of C. oleophila at pH 5, compared with only 26 ppm found at pH 3 (factor 7.7). Intra- and extracellular concentrations of citrates and glucose confirmed the existence of a high specific, pH dependent active transport system for citrate secretion, while isocitrate isn't a high-affine substrate, displaying a strong correlation with ATP/ADP ratio. Differences between extra- and intracellular concentration of citrate higher than 1 and up to about 60 were determined. The active transport systemfor citrate excretion appears to be the main speed-determining factor in citrate overproduction by yeasts.
citric acid production using mutant strains of A.
Overproduction of citric acid in moulds and yeast has been reported to be triggered out by limitations of certain elements, like N, P, Mn, Fe or Zn, essential for citrate accumulation in A. niger (Shu and Johnson, 1948a; Shu and Johnson, 1948b; Noguchi and Johnson, 1961; Kisser et al. 1980; Kubicek and Röhr, 1980; Kapoor et al. 1982; Kristiansen et al. 1982; Crueger and Crueger, 1989; Dawson and Maddox, 1989; Grewal and Kalra, 1995), as well as N, P, S and Mg in yeasts Yarrowia lipolytica and Candida oleophila (Lozinov et al. 1974; Behrens et al. 1987; Stottmeister and Hoppe, 1991; Anastassiadis, 1994; Anastassiadis et al. 2001; Anastassiadis et al. 2002; Anastassiadis et al. 2004). The yeasts can use various carbon sources for the formation of citric acid (Ikeno et al. 1975; Stottmeister and Hoppe, 1991; Grewal and Kalra, 1995; Mansfeld et al. 1995; Crolla and Kennedy, 2001; Crolla and Kennedy, 2004; Venter et al. 2004) or lipid production (Papanikolaou and Aggelis, 2002). Intracellular nitrogen limitation and low intracellular nitrogen content (Briffaud and Engasser, 1979; Moresi, 1994; Anastassiadis et al. 2002; Anastassiadis et al. 2004), occurring after extracellular nitrogen exhaustion and entering a transition phase, and the increase in intracellular NH4+ concentration, possibly caused by proteolysis, are the most important factors influencing and triggering out citric acid formation and secretion in yeasts (Anastassiadis et al. 1993; Anastassiadis 1994; Anastassiadis et al. 1994; Anastassiadis et al. 2001; Anastassiadis et al. 2002; Anastassiadis et al. 2004). Intracellular accumulation of NH4+ found in cytoplasm of A. niger (Röhr and Kubicek, 1981; Habison et al. 1983) and in Candida oleophila (Anastassiadis et al. 2002), possibly caused by a disturbances in protein or nucleic acid turn over, uncouples citrate feed back inhibitory effect on phosphofructokinase, enabling an unlimited flow through glycolysis. A further increase of glycolysis flow is obtained by the stimulation of pyruvate kinase through fructose bi-phosphate (Habison et al. 1979; Kubicek and Röhr, 1980; Habison et al. 1983; Kubicek et al. 1984; Milson and Meers, 1985). A negative effect on pyruvate kinase isn't known, so there is no need for any kind of control at this point (Meixner-Monori et al. 1984).
Utilizing glucose as carbon source, the basic principle, extensively investigated in A. niger, of oxaloacetate formation by anaplerotic sequences, e.g. by the key enzyme pyruvate carboxylase for citric acid production, (Kapoor et al. 1982; Milson, 1987; Peksel et al. 2002) and phosphoenol carboxykinase (Crueger and Crueger, 1989), the flux delay and inhibition of TCA cycle (α-oxoglutarate dehydrogenase inhibition) and high activity of citrate synthase, is also valid for yeast strains. Glyoxylate cycle is involved in citric acid synthesis in case that acetate, other C2 sources or aliphatic compounds are used as carbon sources. A positive control of phosphofructokinase by ammonium ions, enabling supply of citrate synthase by acetyl-Co A and oxaloacetate, even a possible repression of α-ketoglutarate dehydrogenase through high glucose and ammonium concentration (Kubicek and Röhr, 1978; Röhr et al. 1983), or inhibition of its activity by oxaloacetate (Meixner-Monori et al. 1985) doesn't explain completely intracellular accumulation and secretion of citric acid. Cis-aconitate has also been assumed to inactivate the only irreversible reaction of tricarboxylic acid cycle (Kubicek and Röhr 1986). The increasing concentration of α-ketoglutarate caused by oxaloacetate inhibition, inhibits isocitrate dehydrogenase and thus a further α-ketoglutarate formation. As a result, increasing citrate concentration inhibits isocitrate dehydrogenase and reaching a critical level it stops its further metabolism (Agrawal et al. 1983; Meixner-Monori et al. 1985; Grewal and Kalra, 1995), causing a complete block of TCA cycle. An additional block of citrate cycle occurs at succinate dehydrogenase level by oxaloacetate.
Significant cytological, morphological and physiological changes (e.g. cell wall composition, cell compartmentalization, pellet formation, vacuolization and formation of storage compounds and polyols) take place in both microbial systems and clear variations are occurring in terms of electron transport and energy coupling (Kisser et al. 1980; Honecker et al. 1989; Papagianni et al. 1999; Paul et al. 1999; Pera and Callieri, 1999; Anastassiadis et al. 2002; Haq et al. 2002). Alternative respiration chains with a higher oxygen demand, functioning without yielding of ATP has also been reported to be involved in citrate accumulation (Kubicek et al. 1980; Zehentgruber et al. 1980; Röhr et al. 1983; Wallrath et al. 1991), leading to higher glucolysis rate and substrate phosphorylation (Wallrath et al. 1991). Byproducts (e.g. polyols) are produced in late fermentation phases that can be reconsumed forming citric acid.
overall success of citric acid production depends to a large extent
on the regulation of the TCA cycle. However, the excretion mechanism
central aspect of present work was to investigate the influence of
pH on continuous citric acid secretion by a specific active transport
system, as well as on the elemental biomass composition in free growing
chemostat cultures of Candida oleophila ATCC
oleophila ATCC 20177 var. (obtained from Dr. Siebert, Jungbunzlauer
Co. and later H and R, Bayer,
influence of pH on continuous citric acid fermentation and secretion
was investigated in chemostat experiments carried out in 1 litter
magnetically stirred double glass fermenter (Research
Centre Jülich, RCJ, Germany) with a working volume of about 460
basic fermentation medium of following composition was used in a series
of experiments for the preliminary orientation's investigation of
pH influence on iron uptake and citrate formation (BM): 3 g/l NH4Cl,
120 g/l glucose, 0.7 g/l KH2PO4, 0.35 g/l MgSO4
x 7H2O, 0.11 g/l (
basic medium, however with 6 g/l NH4Cl, 120 g/l glucose
a second series of experiments, a production medium with 4.5 g/l NH4Cl,
analogously increased concentrations of residual compounds (at factor
1.5 higher than in medium with 3 g/l NH4Cl), however with
g/l NH4Cl, 250 g/l glucose, 1.05 g/l KH2PO4,
0.525 g/l MgSO4 x 7H2O, 0.2475 g/l (
oil or polypropylene glycol was used as antifoaming agent. The 20
litter medium was sterilized in autoclave for 30-60 min at
density (OD) was measured at 660 nm using a spectrophotometer (CPS-240,
biomass was measured using the filter method. 10 ml of fermentation
broth were quickly filtered through a
acids, glucose, ammonia nitrogen and intracellular concentrations
were analysed as described in Anastassiadis, 1993;
Anastassiadis et al. 1993; Anastassiadis,
1994; Anastassiadis et al. 2001 and Anastassiadis
Intracellular concentrations of citric, isocitric acid and glucose were precisely evaluated using the above HPLC methods after their extraction following next procedure.
1. 10 ml of fresh fermentation broth of known biomass concentration (tubes were hold in ice) were immediately filtered using a vacuum pump and washed 3-4 times using a cold 0.9% NaOH solution.
2. Intracellular acids and glucose were extracted by placing the filter with biomass in boiling ethanol for 5 min. Thereafter, filter and solids were dried using a vacuum rotator and resolved in 5 ml distilled water.
3. The sample was filtered using a filter with 0.2 μm pores and analysed. No destruction of metabolites was found in the controls submitted to the same treatment method.
4. Intracellular concentrations of various metabolites were determined and presented in g per gram dried biomass or in mM (under the consideration that cell volume corresponds to an average value of about 2.2 µl/mg dry weight that was determined) based on the report of Marchal et al. (1980) for cell volume of Saccharomycopsis lipolytica. The authors give an average value of 0.43 ml x (g wet weight)-1. The above value of 2.2 µl/(mg dry weight) was calculated according to the ratio between wet and dried weight of about 5 determined for cells of C. oleophila (used in present work).
Similar values were determined for cell volume of C. oleophila as well, using a method involving a radioactive polymer, which can not diffuse into the internal volume of yeast cells (D. Brücher, RCJ). In generally, cell volume of microorganisms arranges between 2 and 4 µl/(mg dry weight) (Knowles, 1977). Höfer et al. 1985 determined a value of 2 µl/(mg dry weight) for the cell volume of Rhodotorula gracilis (glutinis).
and ADP ratio was determined at the Institute of Biotechnology 1 of
Centre Jülich (
Ammonium nitrogen was analysed as has been described in Anastassiadis et al. (2002).
The influence of pH on the growth and elemental biomass composition of C. oleophila and citric acid secretion was investigated in different series of chemostat experiments.
The influence of pH on growth of C. oleophila and continuous citric acid formation was investigated in a series of preliminary orientation's experiments, carried out in chemostat cultures at a residence time of about 40 hrs (D = 0.025 h-1) using the basic fermentation medium (BM) with 3 g/l NH4Cl and 120 g/l glucose. Figure 1 illustrates the course of biomass, glucose, citrate and isocitrate concentration as well as the ratio between citrate and isocitrate as a function of pH. Citric acid was continuously produced at a pH range between 2 and 6. Only 4.22 g/l of citric acid were measured at pH 2 and 4.02 g/l at pH 3. Increasing the pH from 3 to 4, citric acid concentration increased continuously by a factor of about 6 reaching a steady state concentration of 24.1 g/l after several days. Isocitric acid reached in generally a stationary steady state concentration within shorter times compared with citrate. Highest citrate concentration of 37.6 g/l was achieved at pH 5, compared with 24.1 g/l and only 10.2 g/l reached at pH 4 and pH 6, respectively. Isocitric acid reached concentrations between 0.5 and 3.36 g/l. A ratio between citrate and isocitrate of around 12 was determined at most of pH values. Residual glucose concentrations between 27 g/l (pH 5) and 70.2 g/l (pH 6) or 69.7 g/l (pH 2) were measured under steady state conditions, corresponding to conversions (conversion means the consumed glucose/feeding medium glucose, scale 0-1, which corresponds to 0-100%; 100% conversion corresponds to total conversion of glucose) between 40% and about 76% (Figure 2). The decreasing of pH, resulted to continuous increase of biomass concentration, starting with about 11 g/l biomass at pH 6 and reaching 15.3 g/l at pH 2 (factor 1.4) (Figure 1). A correlation factor between 0.27 (pH 5.5) and 0.34 (pH 2) has been determined between biomass and OD (Biomass/OD).
As Figure 2 shows, product concentration, space-time yield (STY, volumetric productivity that means g product/[litter x hr]), biomass specific productivity (BSP, it means g product/[g dry biomass x hr]) and additional specific parameters were very poor at lower pH values (e.g. pH 2 and 3) in opposite of higher biomass concentrations. Highest STY of 0.94 g/(l x hr) was achieved at pH 5 and BSP of 0.067 g/(g x hr) at pH 4.8 compared with 0.11 g/(l x hr) and 0.0069 g/(g x hr) found at pH 3. Highest selectivity (selectivity means g of product/g of converted glucose; scale 0-1 or in %) of 44.3%, yield (yield means g of product/g of feeding glucose; scale 0-1 or in %) of 33.6% and conversion of 75.9% were determined at pH 5 as well. About 7.7% selectivity, 3.5% yield and 45% conversion were determined at pH 3 instead (Figure 2). Similar results were observed in further experiments with 3 g/l NH4Cl, 120 g/l glucose and without any iron supply (data not shown here), indicating that traces of iron are present in other media components.
series of chemostat experiments was carried out at pH between 2 and
7 for the investigation of pH influence on continuous growth of C.
oleophila, independent from citric acid production. The basic
medium as has been described in the material and methods was used,
which contained an excessive NH4Cl concentration of 6 g/l
elemental biomass composition in biomass of C. oleophila, obtained
from chemostat cultures grown at pH 3 and pH 5, was analysed by the
Analytical Chemistry Department of RCJ (Jülich,
a new series of chemostat experiments, the pH influence on intracellular
concentrations of citrates and glucose and on citric acid secretion
was investigated at a residence time of about 60 hrs (corresponds
to a dilution rate of about D=~0.017 h-1), using an optimised
production medium with 4.5 g/l NH4Cl, 250 g/l glucose,
6 shows space-time yield (STY), biomass-specific productivity
(BSP), the ratio between ATP and ADP and the specific parameters product
selectivity, conversion and product yield as a function of pH. Highest
STY of 0.96 g/(l x hr) was obtained at optimum pH 5, compared with
only 0.21 g/(l x hr) or 0.38 g/(l x hr) reached at pH 3 (21.5%) and
pH 6 (39.5%). Highest BSP of 0.041 g/(g x hr) was also found at pH
The existence of an active transport system for citric acid secretion in C. oleophila became obvious, based on citric acid transport over the cell membrane against a very high concentration gradient between intra- and extracellular citrate concentration. The active transport system was influenced by various parameters such as air saturation, temperature, medium composition and growth state of chemostat cells (residence time, growth or dilution rate). It showed a very high specificity for citrate over isocitrate (specificity factor of 33). Furthermore, the highest intracellular concentrations of citrate, isocitrate and glucose and simultaneously the lowest extracellular citric acid concentrations were determined under none or low producing conditions. In contrary, maximum extracellular citric acid concentrations were reached under conditions, where the lowest intracellular concentrations of citrate and isocitrate appeared. Intracellular isocitric acid concentration exceeded citric acid concentration significantly. Under producing conditions, isocitrate was drawn out from aconitase equilibrium towards citrate, resulting to a higher glycolysis rate and to lowering of intracellular concentration of glucose and isocitric acid (Anastassiadis et al. 1993; Anastassiadis, 1994; Anastassiadis et al. 1994; Anastassiadis et al. 2001). Based on intracellular measurements of glucose, citrate, isocitrate as well as of ATP and ADP (ATP/ADP ratio) has been investigated, whether there would be a pH dependency for citric acid secretion by active transport system.
Figure 7 shows, the highest intracellular
glucose concentration of 372 mg/g dried biomass, citrate concentration
of 57.6 mg/g (~136,3 mM, under the consideration that the cell volume
of C. oleophila corresponds to 2.2 µl/mg dry weight, calculated
from the cell volume for wet weight; Marchal et al.
1980, under the condition that
reports exist related to the influence of initial or operating pH
and other fermentation parameters on citric acid fermentation. Basically,
most investigations were carried out in batch experiments running
in stirrer fermenter or shake flasks (Nubel et al. 1971;
Briffaud and Engasser, 1979; Kozlova
et al. 1981; Lozinov and Finogenowa, 1982; Enzminger
and Asenjo, 1986; Behrens et al. 1987; Rane
and Sims, 1993; Antonucci et al. 2001; Crolla
and Kennedy, 2001). However, no information was found about the
influence of pH on the continuous citric acid secretion and a little
is known regarding the real pH effect on citrate formation. Kinetic
data obtained in chemostat cultures give essential information for
sophisticated process design, process development and scale up. This
type of information for continuous citric acid fermentation is rather
rare in literature. For instance, looking in the internet about 15,200
results was found as compared with 89 for chemostat (Anastassiadis
et al. 2004). Tisnadjaja et al. (1996) reported
about a higher productivity of citric acid using C. guilliermondii
in continuous culture compared with the batch process. About a four
stage process for continuous production of citric acid using A.
active citric acid export found for the first time in yeasts has been
shown in present work to be a strongly pH dependent process. The pH
had a remarkable effect on the growth, the elemental biomass composition
and the secretion of citric acid in C. oleophila, displaying
a production optimum and higher biomass iron content at pH 5. Already
very low iron (cofactor of aconitase) concentrations (<20 µM) affect
dramatically citric acid formation and ratio between citrate and isocitrate.
On the other side, Crolla and Kennedy (2001) reached
highest citrate production in C. lipolytica using 10 mg/l ferric
nitrate. Iron is essential for yeast growth, however a little is known
about the mechanism of its assimilation. Shavloskii
et al. (1988) identified an active iron uptake system in Pichia
quilliermondii with an optimum at pH 5.3 and
of published works regarding citric acid production by yeasts were
performed at pH between 4.5 and 5.5 (Briffaud and Engasser,
1979: pH 4.7; Enzminger and Asenjo, 1986: pH
5.0; Klasson et al. 1989: pH 5.5; Grewal
and Kalra, 1995: pH >5.5; Anastassiadis et al.
2004). Nubel et al. (1971) reported on the other
side about citric acid production in Candida (Yarrowia)
lipolytica ATCC 20228, occurring even at lower pH. The yeast
process differs from Aspergillus
lower citric acid production occurring at lower pH in yeasts could
be theoretically attributed to reduced intracellular citrate formation
or/and inhibition of citrate transport over the mitochondrial or cytoplasm
membrane. Furthermore, citric acid secretion in yeasts has been reported
to occur only as a result of passive diffusion of the undissociated
acid through plasma membrane (Marchal et al. 1980;
Netik et al. 1997). However, present results showed
clearly that citrate secretion by the specific pH dependent active
transport systemand not intracellular citrate accumulation alone is
the crucial speed determining factor for overproduction in yeasts
(Figure 8). The highest extra-/intracellular
ratio of 6.8 for citrate and of 0.24 for isocitrate that have been
determined at optimum pH 5 revealed the high specificity of transport
system for citrate over isocitrate. The difference between extra-
and intracellular isocitrate concentration is lower than 1, indicating
that it doesn't seem to be an affine substrate for the active transport
system and a diffusive excretion of isocitrate into medium may also
take place, forced by gradient imbalances. Netik et
al. (1997) reported about an active secretion of citric acid in
Intracellular accumulation and secretion of citrates are obviously two totally different phenomena, influencing each other, every time in a different way based on varying environmental conditions. Highest extracellular citrate concentrations were identified under optimised conditions (e.g. optimum air saturation, optimum temperature, optimum pH, optimum concentrations of important trace elements), where under also the lowest intracellular concentrations of citrates were identified (Anastassiadis et al. 1993; Anastassiadis, 1994). Thus, the active transport system seems to be induced by other factors, either than by the intracellular accumulation of citric acid. However, a certain critical intracellular level of citrate (~20 mM), determined at optimum air saturation of 20%, was found to be necessary for functioning of active transport system (Anastassiadis et al. 1993; Anastassiadis, 1994; Anastassiadis et al. 1994; Anastassiadis et al. 2001). Higher intracellular concentrations of citrates were also found in chemostat cultures of C. oleophila under non producing conditions at lower residence times, compared with higher producing residence times, confirming once again that citrate secretion by the specific energy consuming active transport system is the main speed determining event in citric acid overproduction (Anastassiadis et al. 1993; Anastassiadis, 1994), rather than intracellular citrate accumulation alone, as has been broadly thought before in international scientific community. Such transport systems are generally carrier proteins (Krämer and Sprenger, 1993).
acid is taken out from aconitase equilibrium towards citric acid,
followed up by citrate's secretion, resulting under optimum fermentation
conditions to lowest intracellular concentrations of citric and isocitric
acid. The lower biomass iron content found at pH 3 would mean a lower
aconitase activity. However, lower aconitase activity caused by iron
deficiency wouldn't explain the 3-fold intracellular isocitrate concentration
compared with citrate at pH
The mechanism of citrate accumulation in yeasts has been studied for several years (Marchal et al. 1980; Gutierrez and Maddox, 1993; Anastassiadis, 1994; Netik et al. 1997; Anastassiadis et al. 2002). While transport of mitochondrial citrate and isocitrate into cytoplasm is well known, a little was known about excretion of citrate into culture medium. Vacuolisation of cytoplasm has also been discussed in connection with citrate production in C. lipolytica (Kozlova et al. 1981; Behrens et al. 1987). However, in case that citrate excretion would be mediated by vacuoles, specificity between excretion of citrate and isocitrate would be attributed to aconitase equilibrium. An argument against this hypothesis is the lower intracellular iron concentration that indicates lower aconitase activity and in the mean time the higher intracellular isocitrate concentration found in present work at pH 3.
Marchal et al. (1980) and McKay et al. (cited in: Gutierrez and Maddox, 1993) proposed a passive diffusion of citrate and isocitrate over yeast plasma membrane into fermentation medium, in a ratio reflecting their intracellular equilibrium, and the existence of a selective transport of mitochondrial isocitrate to cytoplasm, based on intra- and extracellular concentrations of citrate found in experiments using C. lipolytica and paraffins as sole C-source. The higher intracellular isocitrate concentrations indicate a high aconitase activity and in accordance to Marchal et al. (1980) a selective accumulation of isocitrate in cytoplasm. Citric acid production by yeasts seems to be a paradox, because cell accumulation of citric acid occurs under high ratio between ATP and ADP (Anastassiadis, 1994), although the process is considered to be non-growth related, triggered out by nitrogen limitation. The strong correlation found between ATP/ADP ratio and citric acid accumulation varying the pH can be considered as a consequence of a high glycolytic flow under nitrogen limitation and intracellular NH4+ accumulation. A relatively high intracellular NH4+ concentration of about 1.2 mg/g biomass (~37.4 mM) was found in C. oleophila during the production phase (Anastassiadis, 1994; Anastassiadis et al. 2002). The pH dependent specific active transport is providing the explanation for citrate overproduction in yeasts. Active transport seems to be a way for regenerating reduction equivalents and converting excessive ATP, gained by intensive glycolysis under growth limiting conditions.
are divided in two categories, namely the lipogenous and non-lipogenous
citric acid accumulating strains, which can also include strains of
the same genera. Under nitrogen limitation, yeasts belonging to the
first category predominantly form fatty acids from intracellular citric
acid by ATP:citrate lyase (located in cytosol), whereas yeasts belonging
to the latter category produce citric acid (Evans and
Ratledge, 1985). Thus, fatty acid synthesis and citric acid secretion
by active transport system can be considered as a means to cut down
energy overload and surplus amount of NAD(P)H2. It seems
also useful to consider polyol formation under the aspect of regulation,
because polyol formation is discussed in relation to regeneration
of reduced pyridine nucleotides in yeasts (Lozinov and
Finogenova, 1982) as well as in Aspergillus
overproduction of precursors for di- and polysaccharide formation
(e.g. trehalose, glycogen and pullulan) under various nutrient
limitations by certain yeasts and moulds may be regulated by feedback
control of an elevated cytosolic pool of citrate (Evans
and Ratledge, 1985; Anastassiadis, 1994). The
existence of active transport system for citrate secretion and the
strong correlation between ATP/ADP ratio and citrate overproduction,
found in C. oleophila, goes well together with reports of Lozinov
and Finogenova (1982) about a non phosphorylating alternative
oxidase, identified in yeasts, that completes electron flow without
ATP regeneration, competing citrate production. Active citric acid
producing strains showed lower alternative oxidase activity instead.
Whether the energy charge is the driving force of citrate excretion
Citric acid production is obviously a very complicated process, whereby numerous events such as growth limitations, enzyme activities, energy gain and energy state, intracellular acid accumulation, as well as uptake and transport systems display different optima and regulation mechanisms, which are however somehow interconnected and interrelated in a synergistic mode. Essentially higher intracellular isocitrate concentrations found in producing cells of C. oleophila in comparison to citrate indicate a high aconitase activity. However, isocitrate doesn't seem to be a high-affine substrate for active transport system. Thus, the specific active transport of citrate is resulting to decreasing intracellular whole acid and cytoplasmatic isocitrate concentration under optimum fermentation conditions. This phenomenon is a further evidence for the existence of a specific active transport system for citrate secretion in yeasts, well explaining the overproduction of citric acid against a very strong concentration gradient. Figure 8 resumes most crucial evens influencing citric acid overproduction in yeasts.
Professor Dr. U. Stottmeister (UFZ Ctr. Envtl. Res.
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The experiments of the
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