Citrate Fermentation by Lactococcus and Leuconostoc spp

Citrate and lactose fermentation are subject to the same metabolic regulation. In both processes, pyruvate is the key intermediate. Lactococcus lactis subsp. lactis biovar diacetylactis homofermentatively converted pyruvate to lactate at high dilution (growth) rates, low pH, and high lactose concentrations. Mixed-acid fermentation with formate, ethanol, and acetate as products was observed under conditions of lactose limitation in continuous culture at pH values above 6.0. An acetoin/butanediol fermentation with alpha-acetolactate as an intermediate was found upon mild aeration in continuous culture and under conditions of excess pyruvate production from citrate. Leuconostoc spp. showed a limited metabolic flexibility. A typical heterofermentative conversion of lactose was observed under all conditions in both continuous and batch cultures. The pyruvate produced from either lactose or citrate was converted to d-lactate. Citrate utilization was pH dependent in both L. lactis and Leuconostoc spp., with maximum rates observed between pH 5.5 and 6.0. The maximum specific growth rate was slightly stimulated by citrate, in L. lactis and greatly stimulated by citrate in Leuconostoc spp., and the conversion of citrate resulted in increased growth yields on lactose for both L. lactis and Leuconostoc spp. This indicates that energy is conserved during the metabolism of citrate.     

C Nuclear Magnetic Resonance Studies of Citrate and Glucose Cometabolism by Lactococcus lactis

C nuclear magnetic resonance (C-NMR) was used to investigate the metabolism of citrate plus glucose and pyruvate plus glucose by nongrowing cells of Lactococcus lactis subsp. lactis 19B under anaerobic conditions. The metabolism of citrate plus glucose during growth was also monitored directly by in vivo NMR. Although pyruvate is a common intermediate metabolite in the metabolic pathways of both citrate and glucose, the origin of the carbon atoms in the fermentation products was determined by using selectively labeled substrates, e.g., [2,4-C]citrate, [3-C]pyruvate, and [2-C]glucose. The presence of an additional substrate caused a considerable stimulation in the rates of substrate utilization, and the pattern of end products was changed. Acetate plus acetoin and butanediol represented more than 80% (molar basis) of the end products of the metabolism of citrate (or pyruvate) alone, but when glucose was also added, 80% of the citrate (or pyruvate) was converted to lactate. This result can be explained by the activation of lactate dehydrogenase by fructose 1,6-bisphosphate, an intermediate in glucose metabolism. The effect of different concentrations of glucose on the metabolism of citrate by dilute cell suspensions was also probed by using analytical methods other than NMR. Pyruvate dehydrogenase (but not pyruvate formate-lyase) was active in the conversion of pyruvate to acetyl coenzyme A. alpha-Acetolactate was detected as an intermediate metabolite of citrate or pyruvate metabolism, and the labeling pattern of the end products agrees with the alpha-acetolactate pathway. It was demonstrated that the contribution of the acetyl coenzyme A pathway for the synthesis of diacetyl, should it exist, is lower than 10%. Evidence for the presence of internal carbon reserves in L. lactis is presented.     

Redirection of pyruvate catabolism in Lactococcus lactis by selection of mutants with additional growth requirements

Based on requirements for acetate or lipoic acid for aerobic (but not anaerobic) growth, Lactococcus lactis subsp. lactis mutants with impaired pyruvate catabolism were isolated following classical mutagenesis. Strains with defects in one or two of the enzymes, pyruvate formate-lyase (PFL), lactate dehydrogenase (LDH) and the pyruvate dehydrogenase complex (PDHC) were obtained. Growth and product formation of these strains were characterized. A PFL-defective strain (requiring acetate for anaerobic growth) displayed a two-fold increase in specific lactate production compared with the corresponding wild-type strain when grown anaerobically. LDH defective strains directed 91-96% of the pyruvate towards alpha-acetolactate, acetoin and diacetyl production when grown aerobically in the presence of acetate and absence of lipoic acid (a similar characteristic was observed in an LDH and PDHC defective strain in the presence of both acetate and lipoic acid) and more than 65% towards formate, acetate and ethanol production under anaerobic conditions. Another strain with defective PFL and LDH was strictly aerobic. However, a variant with strongly enhanced diacetyl reductase activities (NADH/NAD+ dependent diacetyl reductase, acetoin reductase and butanediol dehydrogenase activities) was selected from this strain under anaerobic conditions by supplementing the medium with acetoin. This strain is strictly aerobic, unless supplied with acetoin.     

Effect of aeration and sodium on the metabolism of citrate by Klebsiella aerogenes.

Anaerobic growth of Klebsiella aerogenes NCDO 711 (NCTC 418) on citrate was dependent on the presence of Na+ in the medium, and fermentation of citrate was mediated via the fermentation pathway enzymes, citrate lyase and a Na+-dependent oxalacetate decarboxylase. This confirms the previous findings on strain NCTC 418. Growth under aerobic conditions was independent of Na+. The mean generation time for cells grown aerobically on either Na+ or K+ citrate medium was about 60 min, with a molar growth yield of about 40 g (dry weight) of cells per mol of citrate utilized. Citrate was apparently metabolized aerobically in both the Na+ and K+ citrate cells via the citric acid cycle, since cell extracts contained alpha-ketoglutarate dehydrogenase but not the citrate fermentation enzymes. The presence of theother enzymes of the citric acid cycle in K. aerogenes was shown in earlier studies. Under aerated conditions (no detectable oxygen tension in the culture), growth was faster on the Na+ citrate medium (mean generation time, 85 min) than on the K+ citrate medium (mean generation time, 120 min). Both cultures grew slower than under aerobic conditions, presumably because of oxygen limitation. Despite the faster growth rate, the molar growth yield of the aerated Na+ citrate culture was one-half that observed for the aerated K+ citrate culture. Citrate was metabolized via the citric acid cycle in cells grown in the K+ citrate medium under aerated conditions since alpha-ketoglutarate dehydrogenase, but not the fermentation enzymes, was detected in extracts prepared from these cells. Metabolism of citrate in the Na+ citrate medium under aerated conditions occurred via both the fermentation pathway (approximately 75 percent) and the citric acid cycle (about 25 percent), as evidenced by (i) the presence of the fermentation enzymes and alpha-ketoglutarate dehydrogenase in extracts of cells grown under these conditions, (ii) a molar growth yield which was intermediate between that obtained for anaerobic and aerated K+ citrate cultures, and (iii) the excretion of acetate, which also occurred in anaerobic cultures but not in aerated K+ citrate or aerobic cultures.     

Regulation of product formation during glucose or lactose limitation in nongrowing cells of Streptococcus lactis.

Nongrowing cells of Streptococcus lactis in a pH-stat were dosed with sugar to allow fermentation at the maximum rate or were fed a continuous supply of sugar at rates less than the maximum. Under anaerobic conditions, rapid fermentation of either glucose or lactose was essentially homolactic. However, with strain ML3, limiting the fermentation rate diverted approximately half of the pyruvate to formate, acetate, and ethanol. At limiting glucose fermentation rates, cells contained lower concentrations of lactate dehydrogenase activator (fructose 1,6-diphosphate) and pyruvate formate-lyase inhibitors (triose phosphates). As a result, pyruvate formate-lyase and pyruvate dehydrogenase play a greater role in pyruvate metabolism. In contrast to strain ML3, strain ML8 did not give the same diversion of products under anaerobic conditions, and cells retained higher concentrations of the above effector compounds. Lactose metabolism under aerobic conditions resulted in pyruvate excretion by both S. lactis ML3 and ML8. At 7% of the maximum utilization rate, pyruvate accounted for 69 and 35% of the lactose metabolized by ML3 and ML8, respectively. Acetate was also a major product, especially with ML8. The data suggest that NADH oxidase is involved in coenzyme recycling in the presence of oxygen and that pyruvate formate-lyase is inactivated, but the pyruvate dehydrogenase complex still functions.     

Regulation of product formation during glucose or lactose limitation in nongrowing cells of Streptococcus lactis

Nongrowing cells of Streptococcus lactis in a pH-stat were dosed with sugar to allow fermentation at the maximum rate or were fed a continuous supply of sugar at rates less than the maximum. Under anaerobic conditions, rapid fermentation of either glucose or lactose was essentially homolactic. However, with strain ML3, limiting the fermentation rate diverted approximately half of the pyruvate to formate, acetate, and ethanol. At limiting glucose fermentation rates, cells contained lower concentrations of lactate dehydrogenase activator (fructose 1,6-diphosphate) and pyruvate formate-lyase inhibitors (triose phosphates). As a result, pyruvate formate-lyase and pyruvate dehydrogenase play a greater role in pyruvate metabolism. In contrast to strain ML3, strain ML8 did not give the same diversion of products under anaerobic conditions, and cells retained higher concentrations of the above effector compounds. Lactose metabolism under aerobic conditions resulted in pyruvate excretion by both S. lactis ML3 and ML8. At 7% of the maximum utilization rate, pyruvate accounted for 69 and 35% of the lactose metabolized by ML3 and ML8, respectively. Acetate was also a major product, especially with ML8. The data suggest that NADH oxidase is involved in coenzyme recycling in the presence of oxygen and that pyruvate formate-lyase is inactivated, but the pyruvate dehydrogenase complex still functions.     

Glucose/citrate cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with impaired alpha-acetolactate decarboxylase

The pyruvate metabolism of a Lactococcus lactis subsp. lactis biovar diacetylactis mutant deficient in alpha-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations. A chemically defined medium was used containing glucose as carbon- and energy-source. The alpha-acetolactate decarboxylase deficiency had no effect on the specific growth rate. Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions. The product formation was monitored throughout the cultivations. The carbon- and redox-balances were within the accuracy of the experimental data. When citrate was added, alpha-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites. By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on alpha-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions. The strain with impaired alpha-acetolactate decarboxylase activity accumulated alpha-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.     

Study of the Citrate Metabolism of Lactococcus lactis subsp. lactis Biovar Diacetylactis by Means of C Nuclear Magnetic Resonance

The metabolic fate of citrate and pyruvate in four strains of Lactococcus lactis subsp. lactis biovar diacetylactis has been studied by means of C nuclear magnetic resonance, using as a substrate either [3-C]pyruvic acid or custom-synthesized citric acid that is C labeled either at carbons 2 and 4 or at carbon 3. The fermentations were carried out batchwise in modified M17 broth. For the actual conversions of the C-labeled substrates, cells at the end of their logarithmic growth phase were used to minimize the conversion to lactic acid. A mass balance of the main citric acid metabolites was obtained; the four strains produced from 50 to 70% (on a molar basis) lactic acid from either citrate or pyruvate. The remaining 50 to 30% was converted mainly to either alpha-acetolactic acid (for one strain) or acetoin (for the other three strains). One of the strains produced an exceptionally high concentration of the diacetyl precursor alpha-acetolactic acid. Another strain (SDC6) also produced alpha-acetolactic acid, but this was decarboxylated to acetoin at a high rate. The C nuclear magnetic resonance method confirmed that the biosynthesis of alpha-acetolactic acid occurs via condensation of pyruvate and "active" acetaldehyde. Diacetyl was not found as a direct metabolite of citrate or pyruvate metabolism.     

Metabolic Analysis of Wild-type Escherichia coli and a Pyruvate Dehydrogenase Complex (PDHC)-deficient Derivative Reveals the Role of PDHC in the Fermentative Metabolism of Glucose

Pyruvate is located at a metabolic junction of assimilatory and dissimilatory pathways and represents a switch point between respiratory and fermentative metabolism. In Escherichia coli, the pyruvate dehydrogenase complex (PDHC) and pyruvate formate-lyase are considered the primary routes of pyruvate conversion to acetyl-CoA for aerobic respiration and anaerobic fermentation, respectively. During glucose fermentation, the in vivo activity of PDHC has been reported as either very low or undetectable, and the role of this enzyme remains unknown. In this study, a comprehensive characterization of wild-type E. coli MG1655 and a PDHC-deficient derivative (Pdh) led to the identification of the role of PDHC in the anaerobic fermentation of glucose. The metabolism of these strains was investigated by using a mixture of ¹³C-labeled and -unlabeled glucose followed by the analysis of the labeling pattern in protein-bound amino acids via two-dimensional ¹³C,¹H NMR spectroscopy. Metabolite balancing, biosynthetic ¹³C labeling of proteinogenic amino acids, and isotopomer balancing all indicated a large increase in the flux of the oxidative branch of the pentose phosphate pathway (ox-PPP) in response to the PDHC deficiency. Because both ox-PPP and PDHC generate CO₂ and the calculated CO₂ evolution rate was significantly reduced in Pdh, it was hypothesized that the role of PDHC is to provide CO₂ for cell growth. The similarly negative impact of either PDHC or ox-PPP deficiencies, and an even more pronounced impairment of cell growth in a strain lacking both ox-PPP and PDHC, provided further support for this hypothesis. The three strains exhibited similar phenotypes in the presence of an external source of CO₂, thus confirming the role of PDHC. Activation of formate hydrogen-lyase (which converts formate to CO₂ and H₂) rendered the PDHC deficiency silent, but its negative impact reappeared in a strain lacking both PDHC and formate hydrogen-lyase. A stoichiometric analysis of CO₂ generation via PDHC and ox-PPP revealed that the PDHC route is more carbon- and energy-efficient, in agreement with its beneficial role in cell growth.     

Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio.

During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.     

Mechanism of citrate metabolism in Lactococcus lactis: resistance against lactate toxicity at low pH

Measurement of the flux through the citrate fermentation pathway in resting cells of Lactococcus lactis CRL264 grown in a pH-controlled fermentor at different pH values showed that the pathway was constitutively expressed, but its activity was significantly enhanced at low pH. The flux through the citrate-degrading pathway correlated with the magnitude of the membrane potential and pH gradient that were generated when citrate was added to the cells. The citrate degradation rate and proton motive force were significantly higher when glucose was metabolized at the same time, a phenomenon that could be mimicked by the addition of lactate, the end product of glucose metabolism. The results clearly demonstrate that citrate metabolism in L. lactis is a secondary proton motive force-generating pathway. Although the proton motive force generated by citrate in cells grown at low pH was of the same magnitude as that generated by glucose fermentation, citrate metabolism did not affect the growth rate of L. lactis in rich media. However, inhibition of growth by lactate was relieved when citrate also was present in the growth medium. Citrate did not relieve the inhibition by other weak acids, suggesting a specific role of the citrate transporter CitP in the relief of inhibition. The mechanism of citrate metabolism presented here provides an explanation for the resistance to lactate toxicity. It is suggested that the citrate metabolic pathway is induced under the acidic conditions of the late exponential growth phase to make the cells (more) resistant to the inhibitory effects of the fermentation product, lactate, that accumulates under these conditions.     

Regulation of lactose fermentation in group N streptococci

Group N streptococci, which have the lactose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) and phospho-beta-d-galactosidase (beta-Pgal), grew rapidly on lactose and converted more than 90% of the sugar to l-lactate. In contrast, Streptococcus lactis 7962, which does not have a beta-Pgal, grew slowly on lactose and converted only 15% of the sugar to l-lactate. With glucose and galactose, this strain had growth rates and fermentation patterns similar to those of other S. lactis strains, suggesting that the rapid and homolactic fermentation of lactose that is characteristic of group N streptococci is dependent upon a functional PEP-dependent PTS and the presence of beta-Pgal. Seventeen strains of group N streptococci were examined for the activator specificities of pyruvate kinase and lactate dehydrogenase. The properties of each enzyme from all the strains, including S. lactis 7962, were similar. Pyruvate kinase had a broad activator specificity, whereas activation of lactate dehydrogenase was specific for ketohexose diphosphate. All intermediates of lactose metabolism from the hexose phosphates to the triose phosphates activated pyruvate kinase. No activation was obtained with adenosine 5'-monophosphate. K and Mg were required for pyruvate kinase activity but could be replaced by NH(4) and Mn, respectively. Lactate dehydrogenase was activated equally by fructose-1,6-diphosphate and tagatose-1,6-diphosphate, the activation characteristics being pH dependent. The roles of pyruvate kinase and lactate dehydrogenase in the regulation of lactose fermentation by group N streptococci are discussed.     

Role of sodium in determining alternate pathways of aerobic citrate catabolism in Aerobacter aerogenes

In contrast to the absolute Na(+) requirement for anaerobic growth of Aerobacter aerogenes on citrate as sole carbon source, aerobic growth of this microorganism did not require the presence of Na(+). However, Na(+) (optimal concentration, 10 mm) did increase the maximal amount of aerobic growth by 60%, even though it did not change the rate of growth. This increase in growth was specifically affected by Na(+), which could not be replaced by K(+), NH(4) (+), Li(+), Rb(+), or Cs(+). Enzyme profiles were determined in A. aerogenes cells grown aerobically on citrate in media of varying cationic composition. Cells grown in Na(+)-free medium possessed all the enzymes of the citric acid cycle including alpha-ketoglutarate dehydrogenase, which is repressed by anaerobic conditions of growth. The enzymes of the anaerobic citrate fermentation pathway, citritase and oxalacetate decarboxylase, were also present in these cells, but this pathway of citrate catabolism was effectively blocked by the absence of Na(+), which is essential for the activation of the oxalacetate decarboxylase step. Thus, in Na(+)-free medium, aerobic citrate catabolism proceeded solely via the citric acid cycle. Addition of 10 mm Na(+) to the aerobic citrate medium resulted in the activation of oxalacetate decarboxylase and the repression of alpha-ketoglutarate dehydrogenase, thereby diverting citrate catabolism from the (aerobic) citric acid cycle mechanism to the fermentation mechanism characteristic of anaerobic growth. The further addition of 2% potassium acetate to the medium caused repression of citritase and derepression of alpha-ketoglutarate dehydrogenase, switching citrate catabolism back into the citric acid cycle.     

Citrate Metabolism in Aerobacter cloacae

Growth of Aerobacter cloacae on citrate either anaerobically or aerobically did not require and was not stimulated by the presence of Na(+) in the medium. Citrate was metabolized anaerobically via the fermentation pathway as evidenced by the (i) presence of oxalacetate decarboxylase, (ii) induction of citrate lyase, and (iii) repression of alpha-ketoglutarate dehydrogenase under anaerobic conditions. Thus, although all the other enzymes of the citric acid cycle were present in anaerobic cells, this pathway was not available for the metabolism of citrate. Citrate was metabolized aerobically via the citric acid cycle, since (i) citrate lyase but not oxalacetate decarboxylase was repressed and (ii) alpha-ketoglutarate dehydrogenase was induced under these conditions. The presence of Na(+) in the medium did not lead to a repression of alpha-ketoglutarate dehydrogenase as in the case of Aerobacter aerogenes. The oxalacetate decarboxylase was a soluble, constitutive enzyme, not activated by Na(+) nor inhibited by avidin. It was slightly inhibited by ethylenediaminetetraacetate but was not stimulated by Mg(2+) or Mn(2+). Thus, this enzyme differed markedly in its properties from the same enzyme found in citrate-grown A. aerogenes.     

Glucose metabolism and regulation of glycolysis in Lactococcus lactis strains with decreased lactate dehydrogenase activity.

The distribution of carbon flux at the pyruvate node was investigated in Lactococcus lactis under anaerobic conditions with mutant strains having decreased lactate dehydrogenase activity. Strains previously selected by random mutagenesis by H. Boumerdassi, C. Monnet, M. Desmazeaud, and G. Corrieu (Appl. Environ. Microbiol. 63, 2293-2299, 1997) were found to have single punctual mutations in the ldh gene and presented a high degree of instability. The strain L. lactis JIM 5711 in which lactate dehydrogenase activity was diminished to less than 30% of the wild type maintained homolactic metabolism. This was due to an increase in the intracellular pyruvate concentration, which ensures the maintained flux through the lactate dehydrogenase. Pyruvate metabolism was linked to the flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase, as previously postulated for the parent strain (C. Garrigues, P. Loubière, N. D. Lindley, and M. Cocaign-Bousquet (1997) J. Bacteriol. 179, 5282-5287, 1997). However, a strain (L. lactis JIM 5954) in which the ldh gene was interrupted reoriented pyruvate metabolism toward mixed metabolism (production of formate, acetate, and ethanol), though the glycolytic flux was not strongly diminished. Only limited production of acetoin occurred despite significant overflow of pyruvate. Intracellular metabolite profiles indicated that the in vivo glyceraldehyde-3-phosphate dehydrogenase activity was no longer flux limiting in the Deltaldh strain. The shift toward mixed acid fermentation was correlated with the lower intracellular trioses phosphate concentration and diminished allosteric inhibition of pyruvate formate lyase.     

13C Nuclear Magnetic Resonance Studies of Citrate and Glucose Cometabolism by Lactococcus lactis

¹³C nuclear magnetic resonance (¹³C-NMR) was used to investigate the metabolism of citrate plus glucose and pyruvate plus glucose by nongrowing cells of Lactococcus lactis subsp. lactis 19B under anaerobic conditions. The metabolism of citrate plus glucose during growth was also monitored directly by in vivo NMR. Although pyruvate is a common intermediate metabolite in the metabolic pathways of both citrate and glucose, the origin of the carbon atoms in the fermentation products was determined by using selectively labeled substrates, e.g., [2,4-¹³C]citrate, [3-¹³C]pyruvate, and [2-¹³C]glucose. The presence of an additional substrate caused a considerable stimulation in the rates of substrate utilization, and the pattern of end products was changed. Acetate plus acetoin and butanediol represented more than 80% (molar basis) of the end products of the metabolism of citrate (or pyruvate) alone, but when glucose was also added, 80% of the citrate (or pyruvate) was converted to lactate. This result can be explained by the activation of lactate dehydrogenase by fructose 1,6-bisphosphate, an intermediate in glucose metabolism. The effect of different concentrations of glucose on the metabolism of citrate by dilute cell suspensions was also probed by using analytical methods other than NMR. Pyruvate dehydrogenase (but not pyruvate formate-lyase) was active in the conversion of pyruvate to acetyl coenzyme A. α-Acetolactate was detected as an intermediate metabolite of citrate or pyruvate metabolism, and the labeling pattern of the end products agrees with the α-acetolactate pathway. It was demonstrated that the contribution of the acetyl coenzyme A pathway for the synthesis of diacetyl, should it exist, is lower than 10%. Evidence for the presence of internal carbon reserves in L. lactis is presented.     

13C-NMR analysis of glucose metabolism during citric acid production by Aspergillus niger

The effect of glucose concentration on glycolytic metabolism under conditions of citric acid accumulation by Aspergillus niger was studied with 13C-labelled glucose. The results show that during cultivation at high glucose (14%, w/v), most of the label in citric acid is in C-2/C-4, and is thus due to the pyruvate carboxylase reaction. However, a significant portion is also present in C-1/C-5, whose origin is less clear but most likely due to reconsumption of glycerol and erythritol. Formation of trehalose and mannitol is high during the early phase of fermentation and declines thereafter. The early fermentation phase is further characterized by a high rate of anaplerosis from oxaloacetate to pyruvate, which also decreases with time. At low glucose concentrations (2%, w/v), which lead to a significantly reduced citric acid yield and formation rate, labelling of citrate in C-2/C-4 is decreased and C-l/C-5 labelling increased. Growth on 2% glucose is also characterized by an appreciable scrambling of mannitol and considerable backflux from mannitol to trehalose (indicating tight glycolytic control at the fructose-6-phosphate step) and an increased anaplerotic formation of pyruvate from oxaloacetate. These data indicate that cultivation on high sugar concentrations shifts control of glycolysis from fructose-6-phosphate to the glyceraldehyde-3-phosphate dehydrogenase step.     

Influence of dissolved oxygen concentration on intracellular pH for regulation of Aspergillus niger growth rate during citric acid fermentation in a stirred tank bioreactor

In this paper we report the regulation of Aspergillus niger growth rate during citric acid fermentation in a stirred tank bioreactor. For this, the influence of dissolved oxygen concentration in a medium on intracellular pH values and consequently on overall microbial metabolism was emphasized. Intracellular pH of mycelium grown under different concentrations of dissolved oxygen in the medium was determined. Sensitivity of proteins toward proton concentration is well recognized, therefore pH influences on the activities of key regulatory enzymes of Aspergillus niger were determined at pH values similar to those detected in the cells grown under lower dissolved oxygen concentrations. The results have shown significantly reduced specific activities of hexokinase, 6-phosphofructokinase and glucose-6-phosphate dehydrogenase in more acidic environment, while pyruvate kinase was found to be relatively insensitive towards higher proton concentration. As expected, due to the reduced specific activities of regulatory enzymes under more acidic conditions, overall metabolism should be hindered in the medium with lower dissolved oxygen concentration which was confirmed by detecting the reduced specific growth rates. From the studies, we conclude that dissolved oxygen concentration affects the intracellular pH and thus growth rate of Aspergillus niger during the fermentation process.     

Metabolism of pyruvate and malate by isolated fat-cell mitochondria

1. Metabolism of pyruvate and malate by isolated fat-cell mitochondria incubated in the presence of ADP and phosphate has been studied by measuring rates of pyruvate uptake, malate utilization or production, citrate production and oxygen consumption. From these measurements calculations of the flow rates through pyruvate carboxylase, pyruvate dehydrogenase and citrate cycle have been made under various conditions. 2. In the presence of bicarbonate, pyruvate was largely converted into citrate and malate and only about 10% was oxidized by the citrate cycle; citrate and malate outputs were linear after lag periods of 6-9min and 3min respectively, and no other end products of pyruvate metabolism were detected. On the further addition of malate or hydroxymalonate, the lag in the rate of citrate output was less marked but no net malate disappearance was detected. If, however, bicarbonate was omitted then net malate uptake was observed. Addition of butyl malonate was found to greatly inhibit the metabolism of pyruvate to citrate and malate in the presence of bicarbonate. 3. These results are in agreement with earlier conclusions that in adipose tissue acetyl units for fatty acid synthesis are transferred to the cytoplasm as citrate and that this transfer requires malate presumably for counter transport. They also support the view that oxaloacetate for citrate synthesis is preferentially formed from pyruvate through pyruvate carboxylase rather than malate through malate dehydrogenase and that the mitochondrial metabolism of citrate in fat-cells is restricted. The possible consequences of these conclusions are discussed. 4. Studies on the effects of additions of adenine nucleotides to pyruvate metabolism by isolated fat-cell mitochondria are consistent with inhibition of pyruvate carboxylase in the presence of ADP and pyruvate dehydrogenase in the presence of ATP.