Citric Acid Synthesis Research Articles

Lipid composition of cAMP-dependent protein kinase mutants of Aspergillus niger

Lipid composition of cAMP-dependent protein kinase (PKA) Aspergillus niger mutants with overexpressed or deleted genes for either regulatory and/or the catalytic subunit of PKA was analyzed. Disruption of the gene encoding the PKA regulatory subunit resulted in 20% less total lipids, 30% less neutral lipids, four times more glycolipids and two-fold higher triacylglycerol lipase activity compared to the control strain. Concomitantly a five-fold decrease in phosphatidylcholine, accompanied with 1.5-, 1.8- and 2.8-fold increases in phosphatidylethanolamine, lysophosphatidylethanolamine and phosphatidylinositol, was determined, respectively. The lack of PKA activity, due to the disruption of a gene encoding the PKA catalytic subunit, resulted in a 1.6-times increase in total lipids with two times more neutral lipids associated with lower triacylglycerol lipase activity and a decrease in phospholipids. The mutants with unrestricted PKA activity synthesized twice as much citric acid as the control strain and three times more than strains lacking PKA activity. The results indicate the involvement of cAMP-mediated PKA activity in regulation of lipid biosynthesis as well as citric acid synthesis.

Oxygen requirements for growth and citric acid production of Yarrowia lipolytica.

During continuous cultivation of Yarrowia lipolytica N 1, oxygen requirements for growth and citric acid synthesis were found to depend on the iron concentration in the medium. A coupled effect of oxygen and iron concentrations on the functioning of the mitochondrial electron transport chain in Y. lipolytica N 1 was established. Based on the results obtained in continuous culture, conditions for citric acid production in a batch culture of Y. lipolytica N 1 were proposed. At relatively low pO(2) value and a high iron concentration, citric acid accumulation was as high as 120 g l(-1); the specific rate of citric acid synthesis reached 120 mg citric acid (g cells h)(-1). The mass yield coefficient was 0.87 and the energy yield coefficient was 0.31.

Biochemistry during the Life and Times of Hans Krebs and Fritz Lipmann

Biochemistry during the Life and Times of Hans Krebs and Fritz Lipmann

Geochemical roots of autotrophic carbon fixation: hydrothermal experiments in the system citric acid, H2O-(±FeS)−(±NiS)

Recent theories have proposed that life arose from primitive hydrothermal environments employing chemical reactions analogous to the reductive citrate cycle (RCC) as the primary pathway for carbon fixation. This chemistry is presumed to have developed as a natural consequence of the intrinsic geochemistry of the young, prebiotic, Earth. There has been no experimental evidence, however, demonstrating that there exists a natural pathway into such a cycle. Toward this end, the results of hydrothermal experiments involving citric acid are used as a method of deducing such a pathway. Homocatalytic reactions observed in the citric acid-H2O experiments encompass many of the reactions found in modern metabolic systems, i.e., hydration-dehydration, retro-Aldol, decarboxylation, hydrogenation, and isomerization reactions. Three principal decomposition pathways operate to degrade citric acid under thermal and aquathermal conditions. It is concluded that the acid catalyzed βγ decarboxylation pathway, leading ultimately to propene and CO2, may provide the most promise for reaction network reversal under natural hydrothermal conditions. Increased pressure is shown to accelerate the principal decarboxylation reactions under strictly hydrothermal conditions. The effect of forcing the pH via the addition of NaOH reveals that the βγ decarboxylation pathway operates even up to intermediate pH levels. The potential for network reversal (the conversion of propene and CO2 up to a tricarboxylic acid) is demonstrated via the Koch (hydrocarboxylation) reaction promoted heterocatalytically with NiS in the presence of a source of CO. Specifically, an olefin (1-nonene) is converted to a monocarboxylic acid; methacrylic acid is converted to the dicarboxylic acid, methylsuccinic acid; and the dicarboxylic acid, itaconic acid, is converted into the tricarboxylic acid, hydroaconitic acid. A number of interesting sulfur-containing products are also formed that may provide for additional reaction. The intrinsic catalytic qualities of FeS and NiS are also explored in the absence of CO. It was shown that the addition of NiS has a minimal effect in the product distribution, whereas the addition of FeS leads to the formation of hydrogenated and sulfur-containing products (thioethers). These results point to a simple hydrothermal redox pathway for citric acid synthesis that may have provided a geochemical ignition point for the reductive citrate cycle.

Optimization of the citric acid production by Aspergillus niger through a metabolic flux balance model

Idiophase, the citric acid producing stage of Aspergillus niger was mathematically modeled to identify required genetic manipulations to optimize citric acid production rate. For this reason, a consistent picture of cell functioning had to be achieved. The transient idiophase nature was established by stoichiometric analysis. The main intracellular fluxes were computed by application of material and physiological constraints (ATP, reduction equivalents, proton motive force) at culture time 120 hours. The HMP pathway accounts for 16% of the glucose input (carbon basis), the Krebs cycle for 13% and the citric acid synthesis for the remaining 71%. This profile implies an operative glycerol-P shuttle. It recycles 93% of the cytosolic glycerol-P to cytosolic DHAP thus coupling the transformation of cytosolic NADH to mitochondrial FADH. A cellular maintenance energy of 3.7 mmol ATP/g·h was determined. It would be spent in fueling cytoplasmatic (1.4 mmol H + /g·h) and mitochondrial (1.8 mmol H + /g·h) H + -ATPase pumps with efficiencies of 0.65 and 1.2 mmol H + /mmol ATP respectively. The role and extent of the alternative respiration system activity and polyol excretion is accounted by the model as well. In addition, the significance of GABA shunt and futile NH 4 + /NH 3 cycle were rejected. According to the developed model, the specific citric productivity would be increased in 45% by an unique change if glucose influx were duplicated. Differences with predictions from other model that required many manipulations are also discussed.

Solid-state fermentation for the synthesis of citric acid by Aspergillus niger

Solid-state fermentation was carried out to evaluate three different agro-industrial wastes, sugar cane bagasse, coffee husk and cassava bagasse for their efficiency in production of citric acid by a culture of Aspergillus niger. Cassava bagasse best supported the mould's growth, giving the highest yield of citric acid among the tested substrates. Results showed the fungal strain had good adaptation to the substrate (cassava bagasse) and increased the protein content (23 g/kg) in the fermented matter. Citric acid production reached a maximum (88-g/kg dry matter) when fermentation was carried out with cassava bagasse having initial moisture of 62% at 26°C for 120 h.

Effect of phosphorus supply on the formation and function of proteoid roots of white lupin (Lupinus albus L.)

We investigated (1) the effect of constant and altered inorganic phosphate (Pi) supply (1–100 mmol m–3) on proteoid root production by white lupin (Lupinus albus L.); and (2) the variation in citrate efflux, enzyme activity and phosphate uptake along the proteoid root axis in solution culture. Proteoid root formation was greatest at Pi solution concentrations of 1–10 mmol m–3 and was suppressed at 25 mmol m–3 Pi and higher. Except at 1 mmol m–3 Pi, the formation of proteoid roots did not affect plant dry matter yields or shoot to root dry matter ratios, indicating that proteoid roots can form under conditions of adequate P supply and not at the expense of dry matter production. Plants with over 50% of the root system as proteoid roots had tissue P concentrations considered adequate for maximum growth, providing additional evidence that proteoid roots can form on P‐sufficient plants. There was an inverse relationship between the Pi concentration in the youngest mature leaf and proteoid root formation. Citrate efflux and the activities of enzymes associated with citric acid synthesis (phosphoenolpyruvate carboxylase and malate dehydrogenase) varied along the proteoid root axis, being greatest in young proteoid rootlets of the 1–3 cm region from the root tip. Citrate release from the 0–1 and 5–9 cm regions of the proteoid root was only 7% (per unit root length) of that from the 1–3 cm segment. Electrical potential and 32Pi uptake measurements showed that Pi uptake was more uniform along the proteoid root than citrate efflux.

Synthetic study of nanocrystalline complex oxides LaFe1–yCoyO3

A precursor to LaFe1–yCoyO3 has been prepared using the polyethylene glycol-gel method. After calcination of the precursor at 450–550 °C for 1–2 h, nanocrystalline complex oxides LaFe1–yCoyO3(y= 0.5, 0.6, 0.8) with the perovskite structure and grain diameter < ca. 10 nm were obtained; the materials were single phase and of good crystallinity. The citric acid and self-propagating synthesis methods also gave nanocrystalline materials. The results indicate that the distribution of the various components in the precursor has an important effect on the subsequent solid-state reaction.

Patterns of gas exchange and organic acid oscillations in tropical trees of the genus Clusia.

Gas exchange patterns and nocturnal acid accumulation were examined in four species of Clusia under simulated field conditions in the laboratory. Clusia alata and C. major had midday stomatal closure, substantial net CO2 exchange ([Formula: see text]) during the night, and the highest water use efficiency (WUE). C. venosa showed a pattern similar to a C3 plant, with nighttime stomatal closure, while C. minor maintained positive [Formula: see text] continuously throughout a 24-h period. However, large changes in titratable acidity, which closely matched changes in citrate and malate levels, indicated that Crassulacean acid metabolism (CAM) is active in all four species. C. venosa showed dawn-dusk oscillations in titratable acidity that were higher than the values reported for other C3-CAM intermediates, while the nighttime acid accumulation of 998 mol m-3 observed in C. major is unsurpassed by any other CAM plant. Moreover, the dawn-dusk changes in citrate levels of over 65 mol m-3 in C. alata and C. minor, and over 120 mol m-3 in C. major, are 3-6 times higher than values reported for other CAM plants. Although these oscillations in citrate levels were quite large, and the nighttime dark respiration rates were high, the O2 budget analysis suggestes that only part of the reducing power generated by the synthesis of citric acid enters the respiratory chain. Dawn-dusk changes in malate levels were just over 50 mol m-3 for C. venosa but over 300 mol m-3 for C. major. Between 28% (C. major) and 89% (C. venosa) of the malate accumulated during the night was derived from recycled respiratory CO2. These daily changes in malate and citrate levels also contributed significantly to changes in leaf sap osmolality. This variability in CO2 uptake patterns, the recycling of nighttime respiratory CO2, and the high WUE may have contributed to the successful invasion of Clusia into a wide range of habitats in the tropics.

Enzyme-catalyzed synthesis of citric acid using acetyl-coenzyme a recycling in a two-phase system

This paper reports the first demonstration of a two-phase aqueous-organic system combining enzymatic catalysts and phase transfer catalysis. Various phase transfer catalysts were employed to catalyze the acylation of coenzyme A (CoA) with acetic anhydride. The acetyl-CoA was utilized by citrate synthase to form 964 eq of citric acid for each coenzyme A equivalent added to the reaction. This method offers the potential for preparing many different acyl-CoA derivatives of potential use as substrates for enzyme-catalyzed synthesis. The method opens up the possibility for synthesizing derivatives of natural products from simple acid anhydrides.

ChemInform Abstract: Oxidation of 4‐Methylenetetrahydropyran and 4‐Methyl‐5,6‐dihydro‐2H‐pyran by Nitric Acid. Synthesis of Citric and Oxalic Acid.

AbstractOxidation of 4‐methylenetetrahydropyran (I) with HNO3 in the presence of equimolar amounts of N2O3 or N2O4 yields citric acid (II) and oxalic acid (III).

Independent control of citrate production and ornithine decarboxylase by prolactin in the lateral lobe of the rat prostate

Prolactin stimulated the citric acid content of the lateral lobe of the prostate of androgenized-hypophysectomized rats in a time-dependent manner. This stimulation of citric acid levels was not blocked by pretreatment of the animals with the ornithine decarboxylase (ODC) inhibitor, alpha-difluoromethyl ornithine (DFMO), suggesting that the prolactin induction of citric acid in this organ is not mediated through activation of the ODC. The efficacy of the dose of inhibitor used was monitored by analysis of the diamine product of ODC, putrescine. Further evidence of an independent control of citric acid and polyamine synthesis in the lateral lobe was provided by their differing age distributions in intact animals. ODC activity decreased sharply with age, whereas the tissue concentrations of citric acid remained relatively constant. Both studies suggest that although citric acid and ODC are modulated by prolactin, their synthesis or activation are controlled independently of each other.

Function of Rhizodermal Transfer Cells in the Fe Stress Response Mechanism of Capsicum annuum L

A variety of red pepper (Capsicum annuum L., cv Yaglik) responds to Fe deficiency stress with simultaneously enhanced H(+) extrusion, reduction of ferric ions and synthesis of malic and citric acid in a swollen subapical root zone densely covered with root hairs. It is demonstrated that these stress responses temporally coincide with the development of rhizodermal and hypodermal transfer cells in this root zone. During stress response the transfer cells show a marked autofluorescence which could arise from endogenous iron chelators of the phenolic acid type. The presence of organelle-rich cytoplasm which often exhibits rotational cytoplasmic streaming points to high physiological activity and makes these cells, with their increased plasmalemma surface, particularly well suited for the entire stress response mechanism. Since Fe stress-induced acidification is diminished by vanadate and erythrosin B, both specific inhibitors of plasmalemma ATPases, it seems reasonable to suppose that H(+) pumping from transfer cells is activated by an ATPase located in their plasmamembrane. H(+) extrusion is also shown to be inhibited by abscisic acid. Raised phosphoenolpyruvate carboxylase activity and simultaneous accumulation of malate in the swollen root zone point to the action of a pH stat preventing a detrimental rise in cytoplasmic pH of transfer cells during enhanced H(+) extrusion. The simultaneous increase in citric acid concentration favors chelation of iron at the site of its uptake and thus ensures long distance transport to the areas of metabolic demand. A direct link between citrate accumulation and ferric ion reduction as proposed in recent literature further supports the crucial role of transfer cells in the response to Fe deficiency stress.

Enzymic synthesis of L-acetylcarnitine and citric acid using acetyl coenzyme A recycling

Enzymic synthesis of L-acetylcarnitine and citric acid using acetyl coenzyme A recycling

Use of cell recycle in the aerobic fermentative production of citric acid by yeast

An aerobic continuous stirred tank bioreactor with cell recycle was used to produce citric acid from glucose with a yeastSaccharomycopsis lipolytica NRRL Y7576. Specific rate of total acid production was 0.045h−1, yield on glucose was 0.86 g/g and volumetric productivity was 1.16 g acid/Lh; all higher than or similar to batch values. Effluent acid concentration was 75g/L. In batch, under nitrogen limited. conditions, stability of citric acid synthesis and excretion was constant over a period of 700 hours. Under conditions of cell recycle, cell concentration and rate of acid production were constant over 200 hours of operation.

Effect of iron on the activity of aconitate hydratase and synthesis of citric acid by Aspergillus niger

Two strains of A. niger were cultivated on beet molasses medium by the surface method, and the effects of iron on the mycelium formation, the rate of sugar utilization, the activity of aconitate hydratase, and synthesis of citric acid were determined. Among various sources of di- and trivalent iron, ferrous sulphate was chosen for studies, which most effectively limited the synthesis of organic iron, ferrous sulphate was chosen for studies, which most effectively limited the synthesis of organic acids. The molasses solutions were purified with potassium ferrocyanide and FeSO4 at a concentration of 20 mg/100 ml (in terms of Fe2+) was added. It was found in both strains that such an amount of Fe2+, additionally introduced into the molasses medium, only slightly effected the synthesis of the mycelium biomass and the rate and extent of sugar utilization, when at the same time a high increase in the activity of aconitate hydratase occurred. Citric acid production, however, was strongly inhibited by this Fe2+-concentration in case of A. niger 23 (about 94%), whereas A. niger Lcz showed a relatively small decrease (about 20%).

The Potential of Immobilized Biocatalysts for Production of Industrial Chemicals

The successful translation from conception to practice of processes based on immobilized biocatalyst technology has been slower than anticipated. There are severe barriers, both technical and economic, limiting the introduction of immobilized biocatalyst technology to replace conventional processing procedures and processes for the production of chemicals by synthetic or fermentative routes. A small number of immobilized enzyme processes are in operation commercially, the most noteworthy being in food-related processes and in the pharmaceutical industry, where they are used for carbohydrate conversions and antibiotic transformations, respectively. There does not, as yet, appear to be any large-scale industrial application of immobilized cell technology. Examples from our laboratory—immobilized yeast for ethanol production andAspergillus niger for citric acid synthesis—illustrate the problems that have to be overcome.

Joint utilization of glucose and n-alkanes in citric acid synthesis by Saccharomycopsis lipolytica

Fermentations for the overproduction of citrate and isocitrate with S. lipolytica in media containing both glucose and n-alkanes as mixed C-source have been performed. Biomass and product yields strongly depend on the C-source of the inoculation culture. If the inoculation culture had been taken from media containing glucose as sole C-source both glucose and n-alkanes were utilized for cell growth in the main culture whereas only glucose was utilized if the inoculation medium contained only n-alkanes. For idiophasic citrate and isocitrate production both glucose and n-alkanes were consumed independently of the C-source of the inoculum but that C-source was preferentially utilized which has been the C-source of the inoculation culture. These findings are reflected by the activities of the isocitrate lyase and the pyruvate carboxylase, respectively. In S. lipolytica both anaplerotic pathways are coexisting but the C-source of the inoculation culture determines the level of the specific activities even if the ratio of the cell-mass of the inoculum to the cell mass of the main culture at the end of the growth phase is about 1:35.

Attempts at improving citric fermentation on molasses solutions

The addition of sodium dithionite, potassium pyrosulphite or acid sodium bisulphite at concentrations of 0.005 to 0.015% to solutions of molasses of different origin, purified by potassium ferrocyanide, caused considerable increases in the yield of citric acid synthesis in five different strains of Aspergillus niger. This ranged from 13.5 to 86.6% with molasses mash not treated with stimulators. In the case of fermentations conducted by using A. niger Lcz strain in beakers containing molasses solutions having a total depth of 8 em, citric acid yield also increased considerably under the influence of the stimulators added and it ranged from 18.6 to 67.0%.

Biosynthesis of citric acid in relation to the activity of selected enzymes of the Krebs cycle in Aspergillus niger mycelium

The activities of ACH1, NAD-ICDH, NADP-ICDH and CS were determined in cell extracts of high and low citric acid-producing strains of A. niger, cultivated on molasses medium by the surface or submerged method. A high differentiation in the activities of the enzymes studied was found to occur at various accumulation stages of citric acid and during its decomposition by moulds. During intensive citric acid synthesis, the activity of ACH and that of both dehydrogenases decreased significantly (though they did not disappear completely) compared to their initial activities during the growth period of the mycelium. CS activity, however, was maintained at almost the same level over the whole fermentation period or increased slightly, particularly in the case of low citric acid-producing strains. The relationships between the activities of these enzymes were reversed during citric acid decomposition by moulds.