Effect of L-malic and citric acids metabolism on the essential amino acid requirements for Oenococcus oeni growth

AIMS: The purpose of this work was to study the effect of L-malic and/or citric acids on Oenococcus oeni m growth in deficient nutritional conditions, and their roles as possible biosynthetic precursors of the essential amino acids. METHODS AND RESULTS: Bacterial cultures were performed in synthetic media. Bacterial growth rate was reduced or annulled when one amino acid was omitted from basal medium, especially for members of aspartate family, except lysine. The organic acids increased or restored the growth rates to the respective reference values. In each medium deficient in one essential amino acid, the L-malic acid utilization was accompanied by an increase of L-lactic acid concentration and accounted for approximately 100%l-malic acid consumed. D-lactic acid formation from glucose decreased in the medium without cysteine. Except for tyrosine, the recovery of glucose-citrate as D-lactic acid was lower than in the complete medium when asparagine, isoleucine or cysteine were excluded. The ethanol and acetate production was not modified. CONCLUSIONS: L-malic and citric acids favoured Oenococcus oeni m growth in nutritional stress conditions. Specifically citric acid was involved in the biosynthesis of the aspartate-derived essential amino acids and glucose in the cysteine biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Such beneficial effect of l-malic and citric acids on amino acids requirements of Oenococcus oeni m have great significance considering the low amino acids concentration in wine.     

Citric acid production by Yarrowia lipolytica cultivated on olive-mill wastewater-based media

Yarrowia lipolytica ACA-DC 50109 cultivated on olive-mill wastewater (O.M.W.)-based media, enriched with commercial-industrial glucose, presented an efficient cell growth. Parameters of growth were unaffected by the presence of O.M.Ws in the growth medium. In diluted O.M.Ws enriched with high glucose amounts (initial sugar concentration, 65 g l(-1)), a notable quantity of total citric acid was produced (28.9 g l(-1)). O.M.W.-based media had a noteworthy stimulating effect on the production of citric acid, since both final citric acid concentration and conversion yield of citric acid produced per unit of sugar consumed were higher when compared with the respective parameters obtained from trials without added O.M.W. Adaptation of the strain in O.M.W.-based media favoured the biosynthesis of cellular unsaturated fatty acids (principally of oleic and palmitoleic acids). Additionally, a non-negligible decrease of the phenolic compounds in the growth medium [up to 15% (wt/wt)], a slight decrease of the phyto-toxicity, and a remarkable decolourisation of the O.M.W. were observed. All these results suggest the potentiality of O.M.Ws utilisation in the fermentation process of citric acid production.     

Optimization of L-malic acid production by Aspergillus flavus in a stirred fermentor

Effects of various nutritional and environmental factors on the accumulation of organic acids (mainly L-malic acid) by the filamentous fungus Aspergillus flavus were studied in a 16-L stirred fermentor. Improvement of the molar yield (moles acid produced per moles glucose consumed) of L-malic acid was obtained mainly by increasing the agitation rate (to 350 rpm) and the Fe(z+) ion concentration (to 12 mg/L) and by lowering the nitrogen (to 271 mg/L) and phosphate concentrations (to 1.5 mM) in the medium. These changes resulted in molar yields for L-malic acid and total C(4) acids (L-malic, succinic, and fumaric acids) of 128 and 155%, respectively. The high molar yields obtained (above 100%) are additional evidence for the operation of part of the reductive branch of the tricarboxylic acid cycle in L-malic acid accumulation by A. flavus. The fermentation conditions developed using the above mentioned factors and 9% CaCO(3) in the medium resulted in a high concentration (113 g/L L-malic acid from 120 g/L glucose utilized) and a high overall productivity (0.59 g/L h) of L-malic acid. These changes in acid accumulation coincide with increases in the activities of NAD(+)-malate dehydrogenase, fumarase, and citrate synthase.     

Histidine decarboxylase production by Lactobacillus hilgardii: Effect of organic acids

Histidine decarboxylase production from Lactobacillus hilgardii 5w, isolated from wine, was inhibited by the presence of l-malic acid in the basal culture medium. The inhibition was related to l-malic acid concentration. The maximal production of the enzyme at 12 h of culture incubated at 30°C was inhibited 71% by 2 g/L l-malic acid and 47% by 0.5 g/L. In these conditions l-malic acid consumption was 16% and 20% respectively. The addition of 300 mg/L citric acid to the basal medium stimulated the enzyme production from 9 to 45 nmoles/min/mg dry weight, and the increase was correlated with citric acid concentration. When different concentrations of l-malic acid were added to the basal medium plus 200 mg/L citric acid, reversion of stimulation was observed, achieving the maximum at a concentration of 2 g/L. In this case, citric acid comsumption was not modified, whereas L-malic acid utilization was higher.     

Lactobacillus casei metabolic potential to utilize citrate as an energy source in ripening cheese: a bioinformatics approach

AIMS: To identify potential pathways for citrate catabolism by Lactobacillus casei under conditions similar to ripening cheese. METHODS AND RESULTS: A putative citric acid cycle (PCAC) for Lact. casei was generated utilizing the genome sequence, and metabolic flux analyses. Although it was possible to construct a unique PCAC for Lact. casei, its full functionality was unknown. Therefore, the Lact. casei PCAC was evaluated utilizing end-product analyses of citric acid catabolism during growth in modified chemically defined media (mCDM), and Cheddar cheese extract (CCE). Results suggest that under energy source excess and limitation in mCDM this micro-organism produces mainly L-lactic acid and acetic acid, respectively. Both organic acids were produced in CCE. Additional end products include D-lactic acid, acetoin, formic acid, ethanol, and diacetyl. Production of succinic acid, malic acid, and butanendiol was not observed. CONCLUSIONS: Under conditions similar to those present in ripening cheese, citric acid is converted to acetic acid, L/D-lactic acid, acetoin, diacetyl, ethanol, and formic acid. The PCAC suggests that conversion of the citric acid-derived pyruvic acid into acetic acid, instead of lactic acid, may yield two ATPs per molecule of citric acid. Functionality of the PCAC reductive route was not observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This research describes a unique PCAC for Lact. casei. Additionally, it describes the citric acid catabolism end product by this nonstarter lactic acid bacteria during growth, and under conditions similar to those present in ripening cheese. It provides insights on pathways preferably utilized to derive energy in the presence of limiting carbohydrates by this micro-organism.     

Influence of Glucose and Saturated Free-Fatty Acid Mixtures on Citric Acid and Lipid Production by Yarrowia lipolytica

In the present report, the effect of glucose and stearin (substrate composed by saturated free-fatty acids) on the production of biomass, reserve lipid, and citric acid by Yarrowia lipolytica ACA-DC 50109 was investigated in nitrogen-limited cultures. Numerical models that were used in order to quantify the kinetic behavior of the above Yarrowia lipolytica strain showed successful simulation, while the optimized parameter values were similar to those experimentally measured and the predictive ability of the models was satisfactory. In nitrogen-limited cultures in which glucose was used as the sole substrate, satisfactory growth and no glucose inhibition occurred, although in some cases the initial concentration of glucose was significantly high (150 g/l). Citric acid production was observed in all trials, which was in some cases notable (final concentration 42.9 g/l, yield 0.56 g per g of sugar consumed). The concentration of unsaturated cellular fatty acids was slightly lower when the quantity of sugar in the medium was elevated. In the cases in which stearin and glucose were used as co-substrates, in spite of the fact that the quantity of cellular lipid inside the yeast cells varied remarkably (from 0.3 to 2.0 g/l – 4 to 20% wt/wt), de novo fatty acid biosynthesis was observed. This activity increased when the yeast cells assimilated higher sugar quantities. The citric acid produced was mainly derived from the catabolism of sugar. Nevertheless, citric acid yield on sugar consumed and citrate specific production rate, as evaluated by the numerical model, presented substantially higher values in the fermentation in which no fat was used as glucose co-substrate compared with the cultures with stearin used as co-substrate.     

Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala

DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol.s-1.mg (dry weight)-1; Km, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, alpha-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis-Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0.     

Sugar and organic acid metabolism in mixed cultures of Pediococcus pentosaceus and Leuconostoc oenos isolated from wine

Pediococcus pentosaceus 12p and Leuconostoc oenos X₂L isolated from Argentinian wine were examined for growth and changes in the concentrations of glucose, fructose, sucrose and mannitol and malic, citric, acetic and lactic acids in pure and mixed cultures. In mixed cultures a mutualistic growth response and a change in the balance of end-products of sugar and organic acid metabolism were observed. The production of mannitol and acetic acid was lower while D(-) and L(+) lactic acids were detected in higher levels than in pure cultures. Malic and citric acids were metabolized simultaneously, but the amount of citric acid consumed was lower than in pure culture of Leuc. oenos.     

Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala.

DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol.s-1.mg (dry weight)-1; Km, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, alpha-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis-Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0.     

Isolation, characterization, and fermentative pattern of a novel thermotolerant Prototheca zopfii var. hydrocarbonea strain producing ethanol and CO2 from glucose at 40 °C

A novel thermotolerant strain of the achlorophyllous micro-alga Prototheca was isolated from a hot spring. The isolate was found to produce an appreciable amount of ethanol and CO2 from glucose under anoxic conditions at both 25 and 40 degrees C; this type of alcohol fermentation has not yet been reported in the genus Prototheca. Moreover, it also evolved gas from sucrose after a time lag at 40 degrees C. Its taxonomic characteristics coincided with those of Prototheca zopfii var. hydrocarbonea, and phylogenetic analysis, based on a small-subunit (SSU) rDNA sequence, also revealed a close relationship between the two strains. D-lactic acid, ethanol, CO2 and a trace of acetic acid were produced from glucose, but L-lactic acid, formic acid, and H2 were not. At 25 degrees C, D-lactic acid and ethanol were produced in approximately equimolar amounts under N2/H2/CO2, whereas ethanol production was predominant under N2. More ethanol was produced at 40 degrees C than at 25 degrees C irrespective of the gas composition in the atmosphere. This is the first report on gas production from glucose and on the changes in the fermentative patterns as a function of temperature for the genus Prototheca.     

Characterization of Schizosaccharomyces pombe malate permease by expression in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, L-malic acid transport is not carrier mediated and is limited to slow, simple diffusion of the undissociated acid. Expression in S. cerevisiae of the MAE1 gene, encoding Schizosaccharomyces pombe malate permease, markedly increased L-malic acid uptake in this yeast. In this strain, at pH 3.5 (encountered in industrial processes), L-malic acid uptake involves Mae1p-mediated transport of the monoanionic form of the acid (apparent kinetic parameters: Vmax = 8.7 nmol/mg/min; Km = 1.6 mM) and some simple diffusion of the undissociated L-malic acid (Kd = 0.057 min(-1)). As total L-malic acid transport involved only low levels of diffusion, the Mae1p permease was further characterized in the recombinant strain. L-Malic acid transport was reversible and accumulative and depended on both the transmembrane gradient of the monoanionic acid form and the DeltapH component of the proton motive force. Dicarboxylic acids with stearic occupation closely related to L-malic acid, such as maleic, oxaloacetic, malonic, succinic and fumaric acids, inhibited L-malic acid uptake, suggesting that these compounds use the same carrier. We found that increasing external pH directly inhibited malate uptake, resulting in a lower initial rate of uptake and a lower level of substrate accumulation. In S. pombe, proton movements, as shown by internal acidification, accompanied malate uptake, consistent with the proton/dicarboxylate mechanism previously proposed. Surprisingly, no proton fluxes were observed during Mae1p-mediated L-malic acid import in S. cerevisiae, and intracellular pH remained constant. This suggests that, in S. cerevisiae, either there is a proton counterflow or the Mae1p permease functions differently from a proton/dicarboxylate symport.     

Effect of sugars, hydrogen ion concentration and ammonium nitrate on the formation of citric acid by Aspergillus niger.

Aspergillus niger Mulder strain when grown on a synthetic medium containing urea as the sole source of nitrogen at pH 5.2, formed a mixture of citric and gluconic acids. On growing the organism at pH 2.0 the gluconic acid content was reduced but citric acid yield remained low. Addition of NH4NO3 to the medium lowered the gluconic acid yields to undetectable levels with a simultaneous increase in the citric acid content. Of the sugars used for the production of citric acid, sucrose in an unautoclaved medium was found to be the best carbon source. Sucrose medium if autoclaved at pH 2.0, or a mixture of glucose and fructose instead of sucrose gave lower yields of citric acid. Under optimum conditions only citric acid was produced and the yield was 66-68 per litre after a growth period of about 10 days.     

Effect of phenolic compounds on the co-metabolism of citric acid and sugars by Oenococcus oeni from wine

AIMS: The goal of this study was to examine the growth of Oenococcus oeni in the presence of phenolic compounds under wine conditions and to see how these compounds affect bacterial metabolism. METHODS AND RESULTS: Phenolic compounds have been added to a basal medium that simulates the composition of wine. Fifty milligrams per litre or more of phenolic compounds stimulated bacterial growth. Oenococcus oeni seemed to use citric acid and trehalose, if they were present, before glucose and fructose. Citrate was completely exhausted in three days and the yield of acetate was higher when phenolic compounds were present. CONCLUSIONS: Phenolic compounds reduced the rate of sugar consumption and enhanced citric acid consumption, increasing the yield of acetic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: This study allows a better knowledge of co-metabolism of citric acid and sugars by O. oeni in the presence of phenolic compounds of wine.     

Channelling of glucose by methanol for citric acid production from Aspergillus niger

Citric acid produced by Aspergillus niger was increased from 4.6g l^-1 to 7.8gl^-1 by supplementing basal medium with methanol (30mll^-1). While stimulating citric acid production, methanol did not improve membrane permeability of the fungus for citric acid. Methanol inhibited the germination of Aspergillus spores. An increase in glucose concentration from 50gl^-1 to 100gl^-1 in the presence of methanol (30mll^-1) improved citric acid production (1.6-fold) while at higher levels of glucose concentration methanol had no effect on citic acid production.     

温特曲霉转富马酸为L-苹果酸的初步研究*

从大量霉菌中选育到一株具有较高富马酸酶活性的温特曲霉(Aspergillus wentii) A5-61。在摇瓶培养条件下,32℃ 96小时,产L-苹果酸达10.49g/100ml,对富马酸的转化率达90.80%。利用菌体细胞,进行酶转化试验,结果表明：1.6g湿菌体接入25ml含富马酸10.0%（用NaOH中和至pH7.0）的转化液中,35℃16～24小时,连续转化三次,分别产生L—苹果酸9.61g/100ml、9.73g/100ml、6.93g/100ml。对菌体整体细胞酶学性质的研究表明,其最适反应温度35℃,最适反应pH7.0,Cu2＋对该酶有明显的抑制作用,该酶的Km＝0.154mol/L,Vmax＝0.0571mol/L·h。     

Influence of wine-like conditions on arginine utilization by lactic acid bacteria

Wine can contain trace amounts of ethyl carbamate (EC), a carcinogen formed when ethanol reacts with carbamyl compounds such as citrulline. EC is produced from arginine by lactic acid bacteria (LAB), e.g., Lactobacillus and Pediococcus. Although the amounts of EC in wine are usually negligible, over the last few years there has been a slight but steady increase, as climate change has increased temperatures and alcohol levels have become proportionately higher, both of which favor EC formation. In this study, resting cells of LAB were used to evaluate the effects of ethanol, glucose, malic acid, and low pH on the ability of non-oenococcal strains of these bacteria to degrade arginine and excrete citrulline. Malic acid was found to clearly inhibit arginine consumption in all strains. The relation between citrulline produced and arginine consumed was clearly higher in the presence of ethanol (10-12%) and at low pH (3.0), which is consistent with both the decreased amount of ornithine produced from arginine and the reduction in arginine degradation. In L. brevis and L. buchneri strains isolated from wine and beer, respectively, the synthesis of citrulline from arginine was highest.     

Growth and end product formation of two psychrotrophic Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T at different pH values and temperatures

Lactobacillus viridescens, Lactobacillus sp. strain 173 (homofermentative), and Brochothrix thermosphacta ATCC 11509T were studied at different pH values and temperatures in aerobic and anaerobic batch cultures. The growth rates were higher in aerobic than in anaerobic cultures. L. viridescens grew faster at pH 5.8 than at pH 6.3, whereas the opposite was true for B. thermosphacta. Lactobacillus sp. strain 173 was inhibited in air or at 8 degrees C in anaerobic culture. B. thermosphacta did not grow in anaerobic culture at pH 5.3. The following variations in growth yields were found in the different environments studied: Lactobacillus sp. strain 173, 23 to 25 g (dry weight) per mol of glucose consumed; L. viridescens, 11 to 23 g/mol; B. thermosphacta, 16 to 38 g/mol. In air, L. viridescens produced D-lactic acid, ethanol, and acetic acid, whereas no acetic acid was produced anaerobically. Acetic acid and ethanol together constituted 41 to 48% of the total product yield irrespective of pH and temperature. Lactobacillus sp. strain 173 produced a racemic mixture of D- and L-lactic acid at pH 6.3, whereas the proportion of L-lactic acid was higher than that of D-lactic acid at pH 5.3. In air, product formation of B. thermosphacta varied from a domination of L-lactic acid to increasing yields of acetoin, acetic acid, 2,3-butanediol and isovaleric acid. The effect of pH and temperature on product formation was difficult to separate from the effect of O2 availability in aerobic cultures. However, it was indicated that more 2,3-butanediol and less acetoin were produced with a decreasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth and end product formation of two psychrotrophic Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T at different pH values and temperatures.

Lactobacillus viridescens, Lactobacillus sp. strain 173 (homofermentative), and Brochothrix thermosphacta ATCC 11509T were studied at different pH values and temperatures in aerobic and anaerobic batch cultures. The growth rates were higher in aerobic than in anaerobic cultures. L. viridescens grew faster at pH 5.8 than at pH 6.3, whereas the opposite was true for B. thermosphacta. Lactobacillus sp. strain 173 was inhibited in air or at 8 degrees C in anaerobic culture. B. thermosphacta did not grow in anaerobic culture at pH 5.3. The following variations in growth yields were found in the different environments studied: Lactobacillus sp. strain 173, 23 to 25 g (dry weight) per mol of glucose consumed; L. viridescens, 11 to 23 g/mol; B. thermosphacta, 16 to 38 g/mol. In air, L. viridescens produced D-lactic acid, ethanol, and acetic acid, whereas no acetic acid was produced anaerobically. Acetic acid and ethanol together constituted 41 to 48% of the total product yield irrespective of pH and temperature. Lactobacillus sp. strain 173 produced a racemic mixture of D- and L-lactic acid at pH 6.3, whereas the proportion of L-lactic acid was higher than that of D-lactic acid at pH 5.3. In air, product formation of B. thermosphacta varied from a domination of L-lactic acid to increasing yields of acetoin, acetic acid, 2,3-butanediol and isovaleric acid. The effect of pH and temperature on product formation was difficult to separate from the effect of O2 availability in aerobic cultures. However, it was indicated that more 2,3-butanediol and less acetoin were produced with a decreasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of L- AND D-lactate by Pediococcus pentosaceus in batch and steady-state continuous culture

The fast growth and acid production of a strain of Pediococcus pentosaceus , used as a starter culture in the production of dry sausages, was dependent on the presence of acetate. In a batch culture on a mixture of glucose and sucrose both sugars were consumed simultaneously. Similar growth rates and product yields were obtained on glucose and sucrose, d - AND l -lactate were produced via a D- and L-lactate dehydrogenase (LDH), respectively, and no racemase was present. In batch cultures about 15% of the lactic acid produced was the D-isomer, whereas in a sucrose-limited, continuous culture the fraction of D-lactic acid increased with decreasing dilution rate. The results are discussed in relation to the two LDH activities.     

Stimulation of lactic acid production in chick embryo fibroblasts by serum and high pH in the absence of external glucose

Lactic acid production by chick embryo fibroblasts occurs in the absence of exogenous glucose. Fifteen to 50-fold less lactic acid is formed in the absence of glucose than in its presence. Nevertheless, serum and pH stimulation enhances this residual lactic acid production to the same relative extent as when glucose is present. The amount of lactic acid formed cannot be accounted for by the catabolism of residual glucose in the medium since its concentration is less than one-tenth that of the lactic acid eventually produced. Moreover, the residual glucose concentration remains constant or increases during the course of the experiment. To a large extent lactic acid accumulation in the absence of external glucose is dependent on the presence of amino acids in the medium, but amino acid transport is not affected by the stimulatory agents used in this study. The results suggest that treatments which stimulate cell multiplication also activate those enzymatic pathways which convert amino acids to pyruvic and thence to lactic acid.