Effects of citric acid as an important component of the responses to saline and alkaline stress in the halophyte <Emphasis Type="Italic">Leymus chinensis</Emphasis> (Trin.)

Some plants accumulate some compatible solutes and exude various organic acids when exposed to environmental stress. These compatible solutes including proline have been suggested to be involved in stress tolerance by maintaining sufficient cell turgor for growth, thereby improving plant growth, protecting enzymes, and membranes. However, less evidence exists regarding the protective roles of organic acids under stress conditions. Here, we investigate the effects of citric acid as a component of the response to stress on plant growth and antioxidant enzyme activities in two genotypes of halophyte Leymus chinensis (Trin.) genotypes, LcWT07 and LcJS0107. Data showed that both saline stress (200 mM NaCl) and alkaline stress (100 mM Na₂CO₃) reduced plant growth on the relative growth rate and CO₂ assimilation rate, but increased the citric acid concentrations in 6-week-old plants over the 72 h experimental period. When 50 mg l⁻¹ citric acid was exogenously applied under stress conditions, it significantly improved the plant growth and internal citric acid concentration, and also induced defense mechanisms by increasing the activities of antioxidant enzymes. To compare with the mitigative effects of exogenous citric acid on stress, exogenous application of proline was also performed under same conditions, and similar effects on the improvement of growth were observed. Based on these results, we suggested that citric acid is an important component of the stress response in L. chinensis, and exogenous application of 50 mg l⁻¹ citric acid might play a positive role on stress tolerance.     

The kinetic basis of the role of Ca++ ions for higher yield of citric acid in a repeated-batch cultivation system

The present investigation deals with role of Ca⁺⁺ ions in increasing the yield of citric acid in a repeated-batch cultivation system (working volume 9-1) and its kinetic basis. Five different hyper-producing strains of Aspergillus niger were evaluated for citric acid production using clarified cane-molasses as basal substrate. Among the cultures, NG^GCB101 (developed by u.v./chemical mutation in our labs) gave maximum production of citric acid i.e., 87.98 g/1, 6 days after mycelial inoculation. The addition of CaCl₂ to the culture medium promoted the formation of small rounded fluffy pellets (1.55 mm, diameter), which were desirable for citric acid productivity. CaCl₂ at a level of 2.0 M, added during inoculation time, was optimized for commercial exploitation of molasses. During repeated-batch culturing, a yield of citric acid monohydrate of 128.68 g/1 was obtained when the sampling vs. substrate feeding was maintained at 4-1 (44.50% working volume). The incubation period was reduced from 6 to only 2 days. The values of kinetic parameters such as substrate consumption and product formation rates revealed the hyperproducibility of citric acid by the selected Aspergillus niger NG^GCB101 (LSD = 0.456a, HS). Case studies are highly economical because of higher yield of product, lower energy consumption and the use of raw substrate without any additional supplementation.     

Buffer-stimulated citrate efflux in Penicillium simplicissimum: an alternative charge balancing ion flow in case of reduced proton backflow?

Organic acids excreted by filamentous fungi may be used to win metals from industrial secondary raw materials. For a future commercial use a high production rate of organic acids is necessary. The conditions under which the commercially used fungus Aspergillus niger excretes high amounts of citric acid can not be maintained in metal leaching processes. However, Penicillium simplicissimum showed an enhanced citric acid efflux in the presence of an industrial filter dust containing 50% zinc oxide. Because Good buffers of high molarity were able to mimic the effect of zinc oxide, the high buffering capacity of zinc oxide and not an effect of the zinc ions was held responsible for the enhanced citric acid efflux. The presence of ammonium and trace elements reduced this buffer-stimulated citric acid efflux, whereas the plant hormone auxine canceled this reduction. This citric acid efflux was influenced by a depolarization of the membrane: the freely permeable compound tetraphenylphosphoniumbromide decreased the citric acid efflux, without decreasing intracellular citric acid or consumption of glucose and oxygen. Vanadate, an inhibitor of the plasma membrane H⁺-ATPase also reduced the buffer-stimulated citric acid efflux. The role of the efflux of citrate anions as an alternative charge balancing ion flow in case of impaired backflow of extruded protons because of a high extracellular buffering capacity is discussed.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - delta electrochemical potential gradient - DES diethylstilbestrol - DMSO dimethyl sulfoxide - TAPS N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid - TEA triethanolamine - TFP trifluoperazine - TPP tetraphenylphosphonium bromide     

Metabolic Pathway of alpha-Ketoglutarate in Citrus Leaves as Affected by Phosphorus Nutrition

The uptake and metabolism of α-[5-¹⁴C]ketoglutarate by phosphorus-deficient and full nutrient (control) lemon (Citrus limon) leaves were studied over various time intervals. After 45 minutes in P-deficient leaves, the bulk of incorporated ¹⁴C appeared in organic acids and much less in amino acids, while in the control leaves, the ¹⁴C contents of organic and amino acids were equal. In P-deficient leaves, after longer incubation times the ¹⁴C content of organic acids and amino acids increased, while that of CO₂ and residue fractions remained low. In full nutrient leaves the ¹⁴C content of amino acids and organic acids decreased after longer incubation time and increased in the insoluble residue and CO₂. In full nutrient leaves the organic and amino acid metabolism were closely related and accompanied by protein synthesis and CO₂ release, while in P-deficient leaves an accelerating accumulation of arginine and citric acid was linked together with inhibition of protein synthesis and CO₂ liberation.     

Physicochemical Aspects of Chitosan Dispersibility in Acidic Aqueous Media: Effects of the Food Acid Counter-Anion

Differences in formation of colloidal dispersions of chitosan in aqueous solutions of citric acid or lactic acid (25, 50 or 100 mM) were quantitatively studied. Protonation enthalpies, electrical conductivity and ζ-potential measurements were additionally undertaken, aiming at better understanding these differences at a molecular level. In dispersion kinetics assays, experimental data were well fitted (R²?>?0.9; MAPE?<?4 %) by a first-order kinetics model with two terms - one accounting for the fast, direct dispersion of biopolymers chains and another accounting for the slow dispersion of chains from lumps. In all cases, maximal dispersibility was reached after about 20?30 min of stirring. For both acids, the higher the acid concentration in the medium, the higher was the chitosan dispersibility. At a given acid concentration, chitosan showed higher dispersibility in lactic acid than in citric acid solutions. Protonation of chitosan -NH₂ groups was strongly exothermic, with ΔH values three times higher for citric acid (triprotic) than lactic acid (monoprotic) (ΔH?=??120 kJ?mol^{- 1} and ΔH?=??40 kJ?mol^{- 1}, respectively), indicating that chitosan -NH₂ protonation itself was not dependent on the type of acid. However, the electrical conductivity of suspensions of powdered chitosan in water evolved differently as these systems were titrated with citric or acid lactic. With citric acid, electrical conductivity remained virtually constant for acid concentration?<?of 15 mM, and then increased linearly as the acid concentration increased until 75 mM. Instead, with lactic acid, electrical conductivity progressively increased with increasing of acid concentration from 0 to 75 mM. The ζ-potential of chitosan dispersed particles was +28.5 mV and +52.1 mV in dispersions containing 10 mM of citric and lactic acids, respectively. The conjoint analysis of data from physicochemical analyses suggested that, contrarily to lactate anions, citrate anions bind more strongly on the electrical double layer of protonated, positively charged chains of chitosan, diminishing the inter-chains electrostatic repulsion, thus leading to a lower dispersibility of this polysaccharide in aqueous solutions of citric acid, compared to equimolar solutions of lactic acid.     

Ammonium ion & citric acid supplementation in batch cultures ofAspergillus niger B 60

Summary The effect of a single pulse of ammonium sulphate or of citrate upon the progress and final outcome of a batch citric acid fermentation was studied. It was found that the optimum addition time for the supplemental N was in the range of 40 to 75 h. Final citric acid concentration achieved was significantly increased when the concentration of N source added was between 0.25 and 0.5 kg m^–3. The mechanism of the observed stimulation seemed to be an indirect one. Addition of exogenous citric acid to the broth, led to an increase in citrate production by the culture. The optimum time for citric acid addition was around 90 h.Nomenclature Y_p/s Yield of citric acid produced (kg) on sucrose consumed (kg) - P/t Overall citric acid productivity (kg m^–3 h^–1)     

Cadmium — citric acid — xylem cell wall interactions in tomato plants

Abstract. Mutual interactions between cadmium ions, citric acid and xylem cell walls were examined. Cadmium and citric acid were measured as ¹¹⁵Cd and [1,5-¹⁴C] citric acid, respectively. Xylem cell walls were obtained by bacterial degradation of tomato stem sections (Lycopersicon esculentum Mill, cv. Tiny Tim), and applied as ion-exchange columns. The xylem column material carried 2·4 dm³ H₂O kg^?1 dry weight, and was temporarily capable of buffering perfusates at pH 5·7. Sorbed cadmium and citric acid were determined from H₂O and HCl rinses after perfusion periods. In all experiments, total cadmium and/or citric acid recoveries were better than 98%, indicating both the effectiveness of the rinses applied and the possibility of full regeneration of the xylem column. The results indicate that the presence of 2·45 mol m^?3 citric acid causes an approximately 50% reduction of adsorbed cadmium levels, irrespective of the applied total cadmium concentrations (0·04–0·4 mol m^?3 Cd(NO₃)₂.4H₂O). This reduction is probably related to a corresponding reduction to approximately 2% of the control applied free Cd²⁺ concentration, the latter also independent of the total cadmium concentrations. Furthermore, without inducing positively charged citrate complexes in the applied solution, the presence of cadmium resulted in increased levels of citric acid absorbed in the xylem column. The Donnan Free Space accumulation of citric acid in the presence of Cd(NO₃)₂.4H₂O, observed in the experiments described, could be expressed by its distribution coefficient, as approximately 15 times the control accumulation. These data indicate that the xylem column may operate as a ligand exchanger, suggesting the importance of metal ions for the longitudinal and lateral movement of organic complexing compounds in the xylem.     

Production of citric acid using its extraction wastewater treated by anaerobic digestion and ion exchange in an integrated citric acid-methane fermentation process

In order to solve the problem of extraction wastewater pollution in citric acid industry, an integrated citric acid-methane fermentation process is proposed in this study. Extraction wastewater was treated by mesophilic anaerobic digestion and then used to make mash for the next batch of citric acid fermentation. The recycling process was done for seven batches. Citric acid production (82.4 g/L on average) decreased by 34.1?% in the recycling batches (2nd–7th) compared with the first batch. And the residual reducing sugar exceeded 40 g/L on average in the recycling batches. Pigment substances, acetic acid, ammonium, and metal ions in anaerobic digestion effluent (ADE) were considered to be the inhibitors, and their effects on the fermentation were studied. Results indicated that ammonium, Na⁺ and K⁺ in the ADE significantly inhibited citric acid fermentation. Therefore, the ADE was treated by acidic cation exchange resin prior to reuse to make mash for citric acid fermentation. The recycling process was performed for ten batches, and citric acid productions in the recycling batches were 126.6 g/L on average, increasing by 1.7 % compared with the first batch. This process could eliminate extraction wastewater discharge and reduce water resource consumption.     

Ram horn peptone as a source of citric acid production by <Emphasis Type="Italic">Aspergillus niger</Emphasis>, with a process

The present study deals with the production of citric acid from a ram horn peptone (RHP) by Aspergillus niger NRRL 330. A medium from RHP and a control medium (CM) were compared for citric acid production using A. niger in a batch culture. For this purpose, first, RHP was produced. Ram horns were hydrolyzed by treatment with acids (6 N H₂SO₄, 6 N HCl) and neutralizing solutions. The amounts of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of the RHP were determined. RHP was compared with peptones with a bacto-tryptone from casein and other peptones. The results from RHP were similar to those of standard peptones. The optimal concentration of RHP for the production of citric acid was found to be 4% (w/w). A medium prepared from 4% RHP was termed ram horn peptone medium (RHPM). In comparison with CM, the content of citric acid in RHPM broth (84 g/l) over 6 days was 35% higher than that in CM broth (62 g/l). These results show that citric acid can be produced efficiently by A. niger from ram horn.     

Differential effects of drought and light levels on accumulation of citric and malic acids during CAM in Clusia

Night-time citrate accumulation has been proposed as a response to stress in CAM plants. To address this hypothesis, gas exchange patterns and nocturnal acid accumulation in three species of Clusia were investigated under controlled conditions with regard to water stress and responses to low and high photosynthetic photon flux density (PPFD). Under high PPFD, leaves of Clusia nocturnally accumulated large amounts of both malic and citric acids. Under low PPFD and well-watered conditions, substantial night-time citrate accumulation persisted, whereas malate accumulation was close to zero. Malate accumulation and night-time CO₂ uptake from the atmosphere declined in all three species during prolonged drought periods, whereas citrate accumulation remained similar or increased. Recycling of respiratory CO₂ was substantial for both well-watered and water-stressed plants. The suggestion that citrate accumulation is energetically more favourable than malate accumulation is not supported if the source of CO₂ for the formation of malate is respiratory CO₂. However, the breakdown of citric acid to pyruvate in the light period releases three molecules of CO₂, while the breakdown of malic acid releases only one CO₂ per pyruvate formed. Thus, citric acid should be more effective than malic acid as a mechanism to increase CO₂ concentration in the mesophyll and may help to prevent photoinhibition. Organic acid accumulation also affected the vacuolar pH, which reached values of 2·6–3·0 at dawn. At these pH values, the transport of 2H⁺/ATP is still feasible, suggesting that it is the divalent form of citrate which is being transported in the vacuoles. Since citrate is a well-known buffer, and Clusia spp. show the largest day-night changes in organic acid levels measured in any CAM plant, it is possible that citrate increases the buffer capacity of the vacuoles. Indeed, malate and titratable acidity levels are positively related to citrate levels. Moreover, Clusia species that show the highest nocturnal accumulation of organic acids are also the ones that show the greatest changes in citric acid levels.     

Influences of intracellular pyridine nucleotide redox states on fatty acid synthesis in isolated rat hepatocytes.

The regulation of fatty acid synthesis, measured by ³H₂O incorporation into fatty acids, was studied in hepatocytes from rats meal-fed a high carbohydrate diet. Ca²⁺ increased fatty acid synthesis, which became maximal at physiological concentrations of Ca²⁺. Ethanol markedly inhibited fatty acid synthesis. Maximum inhibition was reached at 4 mm ethanol. However, ethanol did not decrease lipogenesis in the presence of pyruvate. dl-3-Hydroxybutyrate increased fatty acid synthesis. Acetoacetate decreased lipogenesis when used alone and reversed the effect of dl-3-hydroxybutyrate when both were added. dl-3-Hydroxybutyrate moderately decreased flux through the pyruvate dehydrogenase system and markedly inhibited citric acid cycle flux. By measurement of glycolytic intermediates, two ethanol-induced crossover points were observed: one between fructose 6-phosphate and fructose 1,6-diphosphate and the other between glyceraldehyde 3-phosphate and 1,3-diphosphoglycerate. The concentrations of pyruvate and citrate were decreased by ethanol and increased by dl-3-hydroxybutyrate. Aminooxyacetate and l-cycloserine inhibited fatty acid synthesis and these effects were overcome by dl-3-hydroxybutyrate. Results indicate that in hepatocytes in a metabolic state favoring a high rate of lipogenesis, production of reducing equivalents in the cytosol via ethanol metabolism inhibits fatty acid synthesis from glucose by inhibition of both phosphofructokinase and glyceraldehyde 3-phosphate dehydrogenase and by promoting reduction of pyruvate to lactate. Production of reducing equivalents in the mitochondria via dl-3-hydroxybutyrate enhances fatty acid synthesis in liver cells by altering the partition of citrate between oxidation in the citric acid cycle and conversion to fatty acids in favor of the latter pathway. These interactions indicate the importance of the intracellular pyridine nucleotide redox states in the rate control of hepatic fatty acid synthesis.     

Purification, molecular weight, amino acid, and subunit composition of arylsulfatase A from human liver.

In isolated hepatic mitochondria, the oxidation of NAD⁺-dependent substrates was decreased after chronic consumption of ethanol or by the addition of acetaldehyde in vitro. Reversed electron transport from succinate to NAD^?, which requires transfer of electrons through the NADH dehydrogenase complex and energy transduction through coupling site 1, was depressed by ethanol feeding and by acetaldehyde in vitro, whereas NADH formation from glutamate, which is mediated directly by substrate oxidation and is not energy-dependent, was slightly increased. By contrast, reactions involving the terminal portion of the phosphorylation chain, e.g., ATP-³²P exchange or dinitrophenolstimulated ATPase activity, were not affected. Adenine nucleotide translocase activity was not altered by chronic consumption of ethanol or the addition of acetaldehyde in vitro. These data suggest that the NADH-ubiquinone oxidoreductase complex of the respiratory chain, a segment which contains several iron-sulfur centers which participate in electron transport and energy transduction, may be impaired by chronic consumption of ethanol and is especially sensitive to inhibition by acetaldehyde in vitro. Neither energy coupling sites II or III, nor the terminal reactions of oxidative phosphorylation share this sensitivity. CO₂ production from various labeled intermediates of the citric acid cycle was depressed after chronic consumption of ethanol and after the addition of acetaldehyde. Acetate had no effect on these reactions, indicating that the inhibition by acetaldehyde is not mediated via acetate. Impairment of the activities of the respiratory chain and the citric acid cycle, or both, may explain the decreases in oxygen uptake and CO₂ production from citric acid cycle intermediates and fatty acids, as well as the increase in ketone body production, found in mitochondria from ethanolfed rats.     

Untersuchungen zum Säurestoffwechsel von Aspergillus niger

Zusammenfassung Entwickelt sich das Mycel von Aspergillus niger ausgehend von Conidien bei 43, 30 bzw. 10° C auf einer Nährlösung mit Glucose (5%) als C-Quelle und KNO₃ (1%) als N-Quelle, so kann durch Zusatz von Glucon- bzw. Citronensäure vor und nach dem Zeitpunkt des fast völligen Glucoseverbrauches eine Mehranhäufung von Oxalsäure erzielt werden. Die auf die Kultur und auf den Verbrauch an Citronensäure bezogene Ausbeute ist von der Temperatur und dem Zeitpunkt des Zusatzes abhängig. Bei 43° C wurde für die Zeit nach dem fast völligen Glucoseverbrauch ein Einbau von C¹⁴ aus Gluconsäure-C¹⁴ (U) und Citronensäure-1,5-C¹⁴ in die Oxalsäure nachgewiesen. Unter diesen Bedingungen können also beide Säuren als C-Quelle für die Oxalsäureanhäufung dienen. Es wird vermutet, daß die Mehranhäufung von Oxalsäure durchweg mit einem Umsatz von Glucon- und Citronensäure u. a. in Oxalsäure verknüpft ist.

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Summary Aspergillus niger has been grown from conidia at 43, 30 or 10° C on a medium containing glucose (5%) as a source of carbon and KNO₃ (1%) as a source of nitrogen. Gluconic and citric acid have been added before and after glucose in the medium has been used up almost completely. By addition of each of the acids yield of oxalic acid related to flask could be increased. Yield of oxalic acid related to flask and to citric acid consumed depends on temperature and on time of addition. Incorporation of C¹⁴ from gluconate-C¹⁴ (U) and citric acid-1,5-C¹⁴ into oxalic acid has been demonstrated at 43° C for cultures having used up glucose almost completely. By this it is proofed that under these conditions gluconic and citric acid can be used as a source of carbon for accumulation of oxalic acid. It is suggested that increase of oxalic acid yield caused by addition of gluconic or citric acid is due to conversion of the two acids into oxalic acid (among other products) under all conditions investigated.

     

Transformation of Citric Acid to Acetic Acid,Acetoin and Diacetyl by Wine Making Lactic Acid Bacteria

A decrease in citric acid and increases in acetic acid, acetoin and diacetyl were found in the test red wine after inoculation of intact cells of Leuconostoc mesenteroides subsp. lactosum ATCC 27307. a malo-lactic bacterium, grown on the malate plus citrate-medium. Citric acid in the buffer solution was transformed to acetic acid, acetoin and diacetyl in the pH range of 2 to 6 after inoculation with intact cells of this bacterial species. It was concluded that citric acid in wine making involving malolactic fermentation, at first, was converted by citrate lyase to acetic and oxaloacetic acids, and the latter was successively transformed by decarboxylation to pyruvic acid which was subsequently converted to acetoin, diacetyl and acetic acid.

Both the activities of citrate lyase and acetoin formation from pyruvic acid in the dialyzed cell-free extract were optimal at pH 6.0. Divalent cations such as Mn²⁺, Mg²⁺, Co²⁺ and Zn²⁺ activated the citrate lyase. The citrate lyase was completely inhibited by EDTA, Hg²⁺ and Ag²⁺ . The acetoin formation from pyruvic acid was significantly stimulated by thiamine pyrophosphate and CoCl₂, and inhibited by oxaloacetic acid. Specific activities of the citrate lyase and acetoin formation were considerably variable among the six strains of malo-lactic bacteria examined. Some activities of irreversible reduction of diacetyl to acetoin were found in the cell-free extracts of four of the malo-lactic bacteria strains and the optimal pH was 6.0 for this activity of Leu. mesenteroides.     

Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin

Release of large amounts of citric acid from specialized root clusters (proteoid roots) of phosphorus (P)-deficient white lupin (Lupinus albus L.) is an efficient strategy for chemical mobilization of sparingly available P sources in the rhizosphere. The present study demonstrates that increased accumulation and exudation of citric acid and a concomitant release of protons were predominantly restricted to mature root clusters in the later stages of P deficiency. Inhibition of citrate exudation by exogenous application of anion-channel blockers such as ethacrynic- and anthracene-9-carboxylic acids may indicate involvement of an anion channel. Phosphorus-deficiency-induced accumulation and subsequent exudation of citric acid seem to be a consequence of both increased biosynthesis and reduced metabolization of citric acid in the proteoid root tissue, indicated by increased in-vitro activity and enzyme protein levels of phosphoenolpyruvate carboxylase (EC 4.1.1.31), and reduced activity of aconitase (EC 4.2.1.3) and root respiration. Similar to citric acid, acid phosphatase, which is secreted by roots and involved in the mobilization of the organic soil P fraction, was released predominantly from proteoid roots of P-deficient plants. Also ³³Pi uptake per unit root fresh-weight was increased by approximately 50% in juvenile and mature proteoid root clusters compared to apical segments of non-proteoid roots. Kinetic studies revealed a K _m of 30.7 μM for Pi uptake of non-proteoid root apices in P-sufficient plants, versus K _m values of 8.5–8.6 μM for non-proteoid and juvenile proteoid roots under P-deficient conditions, suggesting the induction of a high-affinity Pi-uptake system. Obviously, P-deficiency-induced adaptations of white lupin, involved in P acquisition and mobilization of sparingly available P sources, are predominantly confined to proteoid roots, and moreover to distinct stages during proteoid root development. Received: 10 September 1998 / Accepted: 22 December 1998     

Effects of several organic acids on the feeding behavior of Tilapia nilotica

Automatic recording of the frequency of feeding ‘bites’ was used to evaluate the effects of several organic acids (citric, metacectonic, lactic, acetic, and oxalic) on the stimulatory feeding behavior of Tilapia nilotica. Some of these acids are added to food stocks to retard spoilage. The results showed that citric acid at a concentration of 10^?2 to 10^?6 m , metacetonic acid at 10^?4 to 10^?6 m , and lactic acid at 10^?2 to 10^?5 m stimulated feeding. Fish tended to avoid metacetonic acid at 10^?3 m and acetic acid at 10^?3 m . Acetic acid at 10^?5 m and oxalic acid at 10^?6 m had no significant effects on fish feeding.     

The Effect of CO2-Concentration on the Occurrence of a Number of Acids from the Citric Acid Cycle in Tomato Leaves

Examination of the effect of CO₂-concentration and time of day on the content of malic acid, citric acid, aconitic acid, isocitric acid, succinic acid and fumaric acid in tomato leaves, revealed that the total content of these acids will rise with the CO₂-concentration up to 0.10 vol% CO₂. In the morning up to 0.22 vol% CO₂ was needed for optimal effect. Samples of leaves picked at 1 a.m. showed the lowest content of these acids. At 9 a.m. the content had increased, and at 4 p.m. the increase was considerable. The content of malic and citric acid constituted 36 and 34% of the total acid content. In the afternoon and the night the aconitic acid represented 14% and in the morning 20% of the total acid content. Isocitric acid, fumaric acid and succinic acid occurred only in relatively small concentrations.     

Citric acid production by recycling its wastewater treated with anaerobic digestion and nanofiltration

We have developed an integrated citric acid-methane fermentation process to solve the problem of wastewater pollution in the citric acid industry. Citric acid wastewater was initially treated by anaerobic digestion. After subsequent ultrafiltration and nanofiltration, the anaerobic digestion effluent (ADE) could be recycled as process water for the next fermentation, maintaining excellent citric acid production efficiency while eliminating wastewater discharge and reducing water consumption. Untreated ADE was not suitable for direct recycling. The effects on citric acid fermentation of components in the ADE were investigated. Production was inhibited when Na⁺ and Mg²⁺ concentrations in recycled ADE were >200 mg/L and >40 mg/L, respectively. This problem was resolved by treating the ADE using ultrafiltration and nanofiltration to reduce Na⁺ and Mg²⁺ concentrations to acceptable levels. Our results prove the technical feasibility of cleaner production in the citric acid industry and provide a strategy for management of wastewater in other submerged fermentation industries.     

Purification and structure of the host-specific toxin from Helminthosporium carbonum race 1

The host-specific toxin from Helminthosporiumcarbonum race 1 was purified from culture filtrates by solvent extraction, gel filtration, and high pressure liquid chromatography. High resolution mass spectrometry of the purified toxin gave a MW of 436.2318 and an elemental composition C₂₁H₃₂N₄O₆. Amino acid analysis and proton and¹³C-NMR indicated a peptide containing four amino acids. Their sequence was determined by gas chromatography mass spectrometry. Finally, digestion of the amino acids with D- and L-amino acid oxidases gave the complete structure cyclo[(L-2-amino-9, 10-epoxy-8-oxodecanoyl)-D-prolyl-L-alanyl-L-alanyl].     

C-NMR spectroscopic evaluation of the citric acid cycle flux in conditions of high aspartate transaminase activity in glucose-perfused rat hearts

A new mathematical model, based on the observation of ¹³C-NMR spectra of two principal metabolites (glutamate and aspartate), was constructed to determine the citric acid cycle flux in the case of high aspartate transaminase activity leading to the formation of large amounts of labeled aspartate and glutamate. In this model, the labeling of glutamate and aspartate carbons by chemical and isotopic exchange with the citric acid cycle are considered to be interdependent. With [U-¹³C]Glc or [1,2-¹³C]acetate as a substrate, all glutamate and aspartate carbons can be labeled. The isotopic transformations of 32 glutamate isotopomers into 16 aspartate isotopomers or vice versa were studied using matrix operations; the results were compiled in two matrices. We showed how the flux constants of the citric acid cycle and the ¹³C-enrichment of acetyl-CoA can be deduced from ¹³C-NMR spectra of glutamate and/or aspartate. The citric acid cycle flux in beating Wistar rat hearts, aerobically perfused with [U-¹³C]glucose in the absence of insulin, was investigated by ¹³C-NMR spectroscopy. Surprisingly, aspartate instead of glutamate was found to be the most abundantly-labeled metabolite, indicating that aspartate transaminase (which catalyses the reversible reaction: (glutamate + oxaloacetate ↔ 2-oxoglutarate + aspartate) is highly active in the absence of insulin. The amount of aspartate was about two times larger than glutamate. The quantities of glutamate (G_o) or aspartate (A_O) were approximately the same for all hearts and remained constant during perfusion: G₀ = (0.74 ±0.03) μmol/g; A₀ = (1.49±0.05) μmol/g. The flux constants, i.e., the fraction of glutamate and aspartate in exchange with the citric acid cycle, were about 1.45 min⁻¹ and 0.72 min⁻¹, respectively; the flux of this cycle is about (1.07±0.02) μmol min^-1 g^-1. Excellent agreement between the computed and experimental data was obtained, showing that: i) in the absence of insulin, only 41% of acetyl-CoA is formed from glucose while the rest is derived from endogenous substrates; and ii) the exchange between aspartate and oxaloacetate or between glutamate and 2-oxoglutarate is fast in comparison with the biological transformation of intermediate compounds by the citric acid cycle.