Isocitric acid (ICA) refers to a group of promising regulators of energy metabolism which has antistress, antihypoxic, and antioxidant activities. In this paper, we reported a process of ICA production from rapeseed oil using yeast... more
Isocitric acid (ICA) refers to a group of promising regulators of energy metabolism which has antistress, antihypoxic, and antioxidant activities. In this paper, we reported a process of ICA production from rapeseed oil using yeast Yarrowia lipolytica VKM Y-2373 in a 500-L fermentor. The producer synthesized 64.1 g/L ICA with a product yield of 0.72 g/g and a productivity 0.54 g/L·h. We also developed an effective purification method, including a cell separation, clarification, concentration, acidification, and crystallization process, which resulted in the formation of the crystals of monopotassium salt of ICA with a purity of 99.0–99.9%. To the best of our knowledge, this is the first report on an ICA production process at an upscaled bioreactor level.
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Yarrowia lipolytica yeast is well known to be able to synthesize citric acid (CA) in large amounts. This study deals with CA biosynthesis, the production of biomass, as well as the accumulation and composition of proteins and lipids in Y.... more
Yarrowia lipolytica yeast is well known to be able to synthesize citric acid (CA) in large amounts. This study deals with CA biosynthesis, the production of biomass, as well as the accumulation and composition of proteins and lipids in Y. lipolytica VKM Y-2373 grown in media with glucose at different concentrations of ammonium sulfate (from 2 to 10 g/L). It was found that these concentrations of nitrogen source are limiting for the growth of Y. lipolytica and that nitrogen deficiency is the main cause of CA excretion. At the high concentration of (NH4)2SO4 (10 g/L), the accumulation of cell biomass, biomass yield (YX/S), and protein concentration was higher than in the medium with 2 g/L ammonium sulfate by 4.3 times, 143%, and 5.1 times, respectively. CA was accumulated in meaningful quantities only in media containing 3–10 g/L (NH4)2SO4 with the maximum concentration of CA (99.9 g/L) at 4 g/L ammonium sulfate. Also of interest is the technological mode with 6 g/L (NH4)2SO4, which i...
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Ester–aldehyde fraction (EAF) is a by-product of ethyl-alcohol-producing companies whose purification requires an expensive process. The results of this study illustrate the environmentally friendly and alternative possibility of using... more
Ester–aldehyde fraction (EAF) is a by-product of ethyl-alcohol-producing companies whose purification requires an expensive process. The results of this study illustrate the environmentally friendly and alternative possibility of using EAF to increase their value as substrate to produce α-ketoglutaric acid (KGA) using different yeasts. It was found that some species of the genera Babjeviella, Diutina, Moesziomyces, Pichia, Saturnispora, Sugiyamaella, Yarrowia and Zygoascus grown under thiamine deficiency accumulate KGA in the medium with an EAF as the sole carbon source. The strain Y. lipolytica VKM Y-2412 was selected as the producer. To reach the maximum production of KGA, the cultivation medium should contain 0.3 µg/L thiamine during cultivation in flasks and 2 µg/L in the fermentor; the concentration of (NH4)2SO4 should range from 3 to 6 g/L; and the optimal concentrations of Zn2+, Fe2+ and Cu2+ ions should be 1.2, 0.6 and 0.05 mg/L, respectively. EAF concentration should not ex...
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ABSTRACT The synthesis of arachidonic acid (15–20% of lipid) from biodiesel-derived waste by Mortierella alpina strains was demonstrated. An inverse correlation between lipid- and arachidonic acid (AA) production was revealed in media... more
ABSTRACT The synthesis of arachidonic acid (15–20% of lipid) from biodiesel-derived waste by Mortierella alpina strains was demonstrated. An inverse correlation between lipid- and arachidonic acid (AA) production was revealed in media with different concentrations of carbon substrate. A method of increasing the AA content of M. alpina lipid based on the additional incubation of harvested mycelium was developed. The effect of mycelium age, time of incubation, and temperature on the fatty acid composition of lipid was studied. Under optimal conditions (incubation of a harvested 7-day mycelium at room temperature for 4–7 days), the AA content of lipid in M. alpina strains NRRL-A-10995 and LPM-301 grown on biodiesel-derived waste and in the pure-glycerol grown strain LPM-301 increased by 162–199% of initial level (from 14.6 to 29.0%, from 19.8 to 36.5%, and from 35.0 to 56.8%, respectively).PRACTICAL APPLICATIONSArachidonic acid (AA) has found wide applications in medicine, pharmacology, diet, and infant nutrition as a precursor of several key eicosanoid hormones and pharmacologically active metabolites, as well as in agriculture as an elicitor of plant resistance to phytopathogens. Microbiological processes for the AA production usually used carbohydrate substrates. Results of this study indicate that AA (15–20% of lipid) can be produced by M ortierella alpina strains from biodiesel-derived waste, inexpensive and renewable carbon substrate. A method of increasing the AA content of M. alpina lipid based on additional incubation of harvested mycelium was developed. Under optimal conditions (incubation of a harvested 7-day mycelium at room temperature for 4–7 days), the AA content of lipid in M. alpina strains increased by 162–199%% of initial level.
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ABSTRACT Microbiological production of physiologically active AA usually used carbohydrates as substrates. Recently, glycerol attracted attention as a promising renewable substrate for biotechnological industry. The effect of pure... more
ABSTRACT Microbiological production of physiologically active AA usually used carbohydrates as substrates. Recently, glycerol attracted attention as a promising renewable substrate for biotechnological industry. The effect of pure glycerol on the growth, lipid synthesis, and AA production by earlier selected Mortierella alpina strains LPM-301 and NRRL-A-10995 was studied. It was shown that AA amount varied from 22–29 to 63–68% of lipid in dependence on the initial glycerol concentration in the medium. The transition from glycerol- to nitrogen limitation of the growth was accompanied by a reverse correlation between lipid content of biomass and AA level of lipid. Under selected optimal conditions (nitrogen limitation of fungal growth at glycerol concentrations of 75–81 g/L), AA production by 14-day cultures reached 40–43% of lipid and 11–13% of biomass indicating that glycerol can be successfully used as a carbon substrate for AA production. Practical applications: AA has found wide application in medicine, pharmacology, diet, and infant nutrition as a precursor of several key eicosanoid hormones and pharmacologically active metabolites. It can also be used in agriculture as an elicitor of plant resistance to phytopathogens. Microbiological processes for AA production usually used carbohydrate substrates. Results of this study indicate that AA can be produced from glycerol, which is known as a promising renewable carbon substrate. Under selected optimal conditions (nitrogen limitation of fungal growth at glycerol concentrations of 75–81 g/L), AA production by Mortierella alpina strains LPM-301 and NRRL-A-10995 reached 40–43% of lipid and 11–13% of biomass. These values are comparable with those obtained for carbohydrate-grown Mortierella fungi.
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The synthesis of succinic acid in ethanol-containing media has been tested in 32 yeasts of different genera (Debaryomyces, Candida, Pichia, Saccharomyces, Torulopsis). The capability of succinic acid synthesis was revealed in 29 strains,... more
The synthesis of succinic acid in ethanol-containing media has been tested in 32 yeasts of different genera (Debaryomyces, Candida, Pichia, Saccharomyces, Torulopsis). The capability of succinic acid synthesis was revealed in 29 strains, from which two most effective producers were selected. When grown in a fermentor under high aeration in mineral medium with pulsed addition of ethanol, the strain Candida catenulata VKM Y-5 produced succinic acid up to 5.2 g/L with mass yield of 32.6 % and energy yield of 14.8 %; the other strain, Candida zeylanoides VKM Y-2324, excreted 9.4 g/L of succinic acid with mass and energy yields of 39 and 17.8 %, respectively. It was indicated that succinic acid formation in the yeasts was accompanied by the synthesis of considerable amounts of malic acid, which was apparently due to a high activity of the glyoxylate cycle. Growth characteristics of both strains were studied in dependence on the concentrations of ethanol, zinc ions and nitrogen in the med...
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The possibility of using glycerol and glycerol-containing waste from biodiesel manufacture as a carbon and energy source for microbiological production of citric acid has been studied. Acid formation on the selective media had previously... more
The possibility of using glycerol and glycerol-containing waste from biodiesel manufacture as a carbon and energy source for microbiological production of citric acid has been studied. Acid formation on the selective media had previously been tested in 66 yeast strains of different genera (Candida, Pichia, Saccharomyces, Torulopsis and Yarrowia). Under growth limitation by nitrogen, 41 strains (belonging mainly to species Yarrowia lipolytica) produced acids; unlike 25 strains of the genera Debaryomyces, Candida, Pichia, Saccharomyces and Torulopsis. Among the 41 acid-producing strains, mutant strain Yarrowia lipolytica N15 was selected since it was able to produce citric acid presumably in high amounts. The citric acid production by the selected strain was studied in dependence on the medium pH, aeration and concentration of glycerol. Under optimal conditions, the mutant Y. lipolytica N15 produced up to 98 g/L of citric acid when grown in a fermentor with the medium containing pure ...
Research Interests: Microbiology, Chemistry, Food Science, Fermentation Technologies, Biotechnology, and 15 moreBiodiesel, Industrial Biotechnology, Optimization, Biofuels, Industrial microbiology, Food Technology and Biotechnology, Biofertilizers, Organic Fertilizers, Biodiesel Production, Fermentation, Glycerol, Fertilizers, Industrial and Fermentation Microbiology, Food Sciences, and Mutants
During the cultivation of a thiamine-auxotrophic yeast strain Yarrowia lipolytica VKM Y-2412 on ethanol, the growth limitation by thiamine leads to the production of α-ketoglutaric acid. The α-ketoglutaric acid synthesis has been studied... more
During the cultivation of a thiamine-auxotrophic yeast strain Yarrowia lipolytica VKM Y-2412 on ethanol, the growth limitation by thiamine leads to the production of α-ketoglutaric acid. The α-ketoglutaric acid synthesis has been studied in dependence on pH, oxygen supply and ethanol, zinc and iron concentrations. Under optimal conditions, Y. lipolytica produced 88.7 g/L of α-ketoglutaric acid. The culture broth containing α-ketoglutaric acid was subjected to chemical treatment with hydrogen peroxide, which led to the formation of succinic acid in significant quantities (71.7 g/L). Further direct esterification of succinic acid with excess absolute ethanol yielded diethyl succinate. Biomass of Y. lipolytica, a superproducer of α-ketoglutaric acid, was characterized by a high content of protein and essential amino acids, free amino acids, and unusually large amount of γ-aminobutyric acid. The unique amino acid composition of the producer makes it possible to use this biomass as a com...
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It was obtained that the yeast Torulaspora globosa VKPM Y-953 is suitable for the production of biodiesel fuel. Zinc plays an important regulatory role in the metabolism of the studied strain. The study of the growth parameters and the... more
It was obtained that the yeast Torulaspora globosa VKPM Y-953 is suitable for the production of biodiesel fuel. Zinc plays an important regulatory role in the metabolism of the studied strain. The study of the growth parameters and the fatty acid profile of the yeast T. globosa showed that the limitation of its growth by ethanol, at different concentrations of zinc in the medium, considerably influences the chemical composition and the energy content of yeast cells, but not their yield by weight (Yx/s). The increased concentrations of zinc in the medium, in combination with the yeast growth limitation by ethanol, elevated the content of lipids in the cells by 28% and diminished the content of proteins by 14%. At the same time, the limitation of yeast growth by zinc decreased Yx/s and energy (ηX/S) by 2.6 and 3.1 times, respectively. In this case, the content of lipids in cells fell by 72%, while that of proteins rose by 65%. The fatty acid profile of the T. globosa cells was used to...
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The basic tendency in the field of plant protection concerns with reducing the use of pesticides and their replacement by environmentally acceptable biological preparations. The most promising approach to plant protection is application... more
The basic tendency in the field of plant protection concerns with reducing the use of pesticides and their replacement by environmentally acceptable biological preparations. The most promising approach to plant protection is application of microbial metabolites. In the last years, bactericidal, fungicidal, and nematodocidal activities were revealed for citric, succinic, α-ketoglutaric, palmitoleic, and other organic acids. It was shown that application of carboxylic acids resulted in acceleration of plant development and the yield increase. Of special interest is the use of arachidonic acid in very low concentrations as an inductor (elicitor) of protective functions in plants. The bottleneck in practical applications of these simple, nontoxic, and moderately priced preparations is the absence of industrial production of the mentioned organic acids of required quality since even small contaminations of synthetic preparations decrease their quality and make them dangerous for ecology and toxic for plants, animals, and human. This review gives a general conception on the use of organic acids for plant protection against the most dangerous pathogens and pests, as well as focuses on microbiological processes for production of these microbial metabolites of high quality from available, inexpensive, and renewable substrates.
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The optimal cultivation conditions ensuring the maximal rate of citric acid (CA) biosynthesis by glycerol-grown mutant Yarrowia lipolytica NG40/UV7 were found to be as follows: growth limitation by inorganic nutrients (nitrogen,... more
The optimal cultivation conditions ensuring the maximal rate of citric acid (CA) biosynthesis by glycerol-grown mutant Yarrowia lipolytica NG40/UV7 were found to be as follows: growth limitation by inorganic nutrients (nitrogen, phosphorus, or sulfur), 28 °C, pH 5.0, dissolved oxygen concentration (pO₂) of 50 % (of air saturation), and pulsed addition of glycerol from 20 to 80 g L⁻¹ depending on the rate of medium titration. Under optimal conditions of fed-batch cultivation, in the medium with pure glycerol, strain Y. lipolytica NG40/UV7 produced 115 g L⁻¹ of CA with the mass yield coefficient of 0.64 g g⁻¹ and isocitric acid (ICA) amounted to 4.6 g L⁻¹; in the medium with raw glycerol, CA production was 112 g L⁻¹ with the mass yield coefficient of 0.90 g g⁻¹ and ICA amounted to 5.3 g L⁻¹. Based on the activities of enzymes involved in the initial stages of raw glycerol assimilation, the tricarboxylic acid cycle and the glyoxylate cycle, the mechanism of increased CA yield from glycerol-containing substrates in Y. lipolytica yeast was explained.
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Summary The synthesis of succinic acid in ethanol-containing media has been tested in 32 yeasts of different genera (Debaryomyces, Candida, Pichia, Saccharomyces, Torulopsis). The capability of succinic acid synthesis was revealed in 29... more
Summary The synthesis of succinic acid in ethanol-containing media has been tested in 32 yeasts of different genera (Debaryomyces, Candida, Pichia, Saccharomyces, Torulopsis). The capability of succinic acid synthesis was revealed in 29 strains, from which two most effective producers were selected. When grown in a fermentor under high aeration in mineral medium with pulsed addition of ethanol, the strain Candida catenulata VKM Y-5 produced succinic acid up to 5.2 g/L with mass yield of 32.6 % and energy yield of 14.8 %; the other strain, Candida zeylanoides VKM Y-2324, excreted 9.4 g/L of succinic acid with mass and energy yields of 39 and 17.8 %, respectively. It was indicated that succinic acid formation in the yeasts was accompanied by the synthesis of considerable amounts of malic acid, which was apparently due to a high activity of the glyoxylate cycle. Growth characteristics of both strains were studied in dependence on the concentrations of ethanol, zinc ions and nitrogen in...
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There is ever increasing evidence that isocitric acid can be used as a promising compound with powerful antioxidant activity to combat oxidative stress. This work demonstrates the possibility of using waste product from the alcohol... more
There is ever increasing evidence that isocitric acid can be used as a promising compound with powerful antioxidant activity to combat oxidative stress. This work demonstrates the possibility of using waste product from the alcohol industry (so-called ester-aldehyde fraction) for production of isocitric acid by yeasts. The potential producer of isocitric acid from this fraction, Yarrowia lipolytica VKM Y-2373, was selected by screening of various yeast cultures. The selected strain showed sufficient growth and good acid formation in media with growth-limiting concentrations of nitrogen, sulfur, phosphorus, and magnesium. A shortage of Fe2+ and Ca2+ ions suppressed both Y. lipolytica growth and formation of isocitric acid. The preferential synthesis of isocitric acid can be regulated by changing the nature and concentration of nitrogen source, pH of cultivation medium, and concentration of ester-aldehyde fraction. Experiments in this direction allowed us to obtain 65 g/L isocitric ac...
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Svetlana V. Kamzolova1*, Igor G. Morgunov1, Andreas Aurich2, Oksana A. Perevoznikova1, Nadezda V. Shishkanova1, Ulrich Stottmeister2 and Tatiana V. Finogenova1 G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms,... more
Svetlana V. Kamzolova1*, Igor G. Morgunov1, Andreas Aurich2, Oksana A. Perevoznikova1, Nadezda V. Shishkanova1, Ulrich Stottmeister2 and Tatiana V. Finogenova1 G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, p-t Nauki 5, Pushchino, Moscow region, 142290 Russia UFZ Centre for Environmental Research Leipzig-Halle, Department of Environmental Biotechnology, Permoserstrasse 15, D-04318 Leipzig, Germany
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Isocitric acid (ICA) has found wide application in medicine as a promising compound with powerful antioxidant activity to combat oxidative stress. In the known microbiological processes of ICA production by non-conventional yeast Yarrowia... more
Isocitric acid (ICA) has found wide application in medicine as a promising compound with powerful antioxidant activity to combat oxidative stress. In the known microbiological processes of ICA production by non-conventional yeast Yarrowia lipolytica, the pure carbon sources are commonly used. ICA can be also synthetized by Y. lipolytica from ester-aldehyde fraction (EAF)-waste of the ethanol production process. A highly effective method of ICA production from EAF based on regulation of key enzymes (aconitate hydratase and isocitrate lyase) by metabolic regulators (iron and itaconic acid) and aeration was developed. It is recommended to cultivate Y. lipolytica VKM Y-2373 under nitrogen deficiency conditions, a high aeration (60% of air saturation), an addition of 15 mM itaconic acid, and 2.4 mg/L iron. Under optimal conditions, Y. lipolytica VKM Y-2373 produced 83 g/L ICA with isocitrate to citrate ratio of 4.1:1 and mass yield of 1.1 g/g. The putative mechanism of ICA overproduction...
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There is ever increasing evidence that isocitric acid can be used as a promising compound with powerful antioxidant activity to combat oxidative stress. This work demonstrates the possibility of using waste product from the alcohol... more
There is ever increasing evidence that isocitric acid can be used as a promising compound with powerful antioxidant activity to combat oxidative stress. This work demonstrates the possibility of using waste product from the alcohol industry (so-called ester-aldehyde fraction) for production of isocitric acid by yeasts. The potential producer of isocitric acid from this fraction, Yarrowia lipolytica VKM Y-2373, was selected by screening of various yeast cultures. The selected strain showed sufficient growth and good acid formation in media with growth-limiting concentrations of nitrogen, sulfur, phosphorus, and magnesium. A shortage of Fe2+ and Ca2+ ions suppressed both Y. lipolytica growth and formation of isocitric acid. The preferential synthesis of isocitric acid can be regulated by changing the nature and concentration of nitrogen source, pH of cultivation medium, and concentration of ester-aldehyde fraction. Experiments in this direction allowed us to obtain 65 g/L isocitric ac...
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Summary The possibility of using glycerol and glycerol-containing waste from biodiesel manufacture as a carbon and energy source for microbiological production of citric acid has been studied. Acid formation on the selective media had... more
Summary The possibility of using glycerol and glycerol-containing waste from biodiesel manufacture as a carbon and energy source for microbiological production of citric acid has been studied. Acid formation on the selective media had previously been tested in 66 yeast strains of different genera (Candida, Pichia, Saccharomyces, Torulopsis and Yarrowia). Under growth limitation by nitrogen, 41 strains (belonging mainly to species Yarrowia lipolytica) produced acids; unlike 25 strains of the genera Debaryomyces, Candida, Pichia, Saccharomyces and Torulopsis. Among the 41 acid-producing strains, mutant strain Yarrowia lipolytica N15 was selected since it was able to produce citric acid presumably in high amounts. The citric acid production by the selected strain was studied in dependence on the medium pH, aeration and concentration of glycerol. Under optimal conditions, the mutant Y. lipolytica N15 produced up to 98 g/L of citric acid when grown in a fermentor with the medium containi...
Research Interests: Microbiology, Renewable Energy, Fermentation Technologies, Biotechnology, Biodiesel, and 14 moreIndustrial Biotechnology, Optimization, Biofuels, Industrial microbiology, Ph, Food Technology and Biotechnology, Biofertilizers, Organic Fertilizers, Fermentation, Fertilizers, Industrial and Fermentation Microbiology, Food Sciences, Mutants, and Strains
The NAD+-dependent isocitrate dehydrogenase of the organic acid-producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid... more
The NAD+-dependent isocitrate dehydrogenase of the organic acid-producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid precipitation, hydrophobic chromatography, and gel-filtration chromatography. The enzyme was purified 129-fold with a yield of 31% and had a specific activity of 22 U/mg protein. The molecular mass of the enzyme was found to be 412 kDa. The enzyme consists of eight identical subunits with a molecular mass of about 52 kDa. The Km for NAD+ is 136 microM, and that for isocitrate is 581 microM. The effect of some intermediates of the citric acid cycle and nucleotides on the enzyme activity was studied. The role of isocitrate dehydrogenase (NAD+) in the overproduction of citric and keto acids is discussed.
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Isocitric acid exists in the form of four stereoisomers, of which only the -Ds-form (ICA) is a natural active compound, an intermediate of Krebs cycle, and suitable for nutritional and pharmaceutical use. In this paper, we propose a... more
Isocitric acid exists in the form of four stereoisomers, of which only the -Ds-form (ICA) is a natural active compound, an intermediate of Krebs cycle, and suitable for nutritional and pharmaceutical use. In this paper, we propose a method for ICA production from ethanol by yeast . The effects of temperature, pH of the medium, and aeration on the growth of the producer VKM Y-2373 and synthesis of ICA were studied. An optimal fermentation regime, which ensures a good growth of the producer and directed synthesis of the target product, was determined. The producer is advised to carry out cultivation at 29°C and various pH of the medium and the oxygen concentration (pH 5 and pO 20-25% (of saturation) during the growth period and pH 6 and pO 50-55% (of saturation) during the acid formation) on a nutrient medium containing an increased content of zinc (0.6 mg/L), iron (1.2 mg/L), and 30 mM itaconic acid (inhibitor of isocitrate lyase-the key enzyme of ICA metabolism) should also be intro...
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The microbiological production of isocitric acid (ICA) is more preferable for its application in medicine and food, because the resulting product contains only the natural isomer—threo-DS. The aim of the present work was to study ICA... more
The microbiological production of isocitric acid (ICA) is more preferable for its application in medicine and food, because the resulting product contains only the natural isomer—threo-DS. The aim of the present work was to study ICA production by yeast using sunflower oil as carbon source. 30 taxonomically different yeast strains were assessed for their capability for ICA production, and Y. lipolytica VKM Y-2373 was selected as a promising producer. It was found that ICA production required: the limitation of Y. lipolytica growth by nitrogen, phosphorus, sulfur or magnesium, and an addition of iron, activating aconitate hydratase, a key enzyme of isocitrate synthesis. Another regulatory approach capable to shift acid formation to a predominant ICA synthesis is the use of inhibitors (itaconic and oxalic acids), which blocks the conversion of isocitrate at the level of isocitrate lyase. It is recommended to cultivate Y. lipolytica VKM Y-2373 under nitrogen deficiency conditions with ...
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Within this work, the microbial synthesis of (2R,3S)-isocitric acid (ICA), a metabolite of the nonconventional yeast Yarrowia lipolytica, from biodiesel waste, has been studied. The selected strain Y. lipolytica VKM Y-2373 synthesized ICA... more
Within this work, the microbial synthesis of (2R,3S)-isocitric acid (ICA), a metabolite of the nonconventional yeast Yarrowia lipolytica, from biodiesel waste, has been studied. The selected strain Y. lipolytica VKM Y-2373 synthesized ICA with citric acid (CA) as a byproduct. This process can be regulated by changing cultivation conditions. The maximal production of ICA with the minimal formation of the byproduct was provided by the use of a concentration of (NH4)2SO4 (6 g/L); the addition of biodiesel waste to cultivation medium in 20–60 g/L portions; maintaining the pH of the cultivation medium at 6, and degree of aeration between 25% and 60% of saturation. Itaconic acid at a concentration of 15 mM favorably influenced the production of ICA by the selected strain. The optimization of cultivation conditions allowed us to increase the concentration of ICA in the culture liquid from 58.32 to 90.2 g/L, the product yield (Y) by 40%, and the ICA/CA ratio from 1.1:1 to 3:1. Research on l...
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The effect of biologically active form (threo-Ds-) of isocitric acid on oxidative stress was studied using the infusorian Paramecium caudatum stressed by hydrogen peroxide and salts of some heavy metals (Cu, Pb, Zn, and Cd). Isocitric... more
The effect of biologically active form (threo-Ds-) of isocitric acid on oxidative stress was studied using the infusorian Paramecium caudatum stressed by hydrogen peroxide and salts of some heavy metals (Cu, Pb, Zn, and Cd). Isocitric acid at concentrations between 0.5 and 10 mM favorably influenced the infusorian cells with oxidative stress induced by the toxicants studied. The maximal antioxidant effect of isocitric acid was observed at its concentration 10 mM irrespective of the toxicant used (either H2O2 or heavy metal ions). Isocitric acid was found to be a more active antioxidant than ascorbic acid. Biologically active pharmaceutically pure threo-Ds-ICA was produced through cultivation of the yeast Yarrowia lipolytica and isolated from the culture liquid in the form of crystalline monopotassium salt with a purity of 99.9%.
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Comparative study of 43 natural yeast strains belonging to 20 species for their capability for overproduction of citric acid (CA) from glucose under nitrogen limitation of cell growth was carried out. As a result, natural strain Yarrowia... more
Comparative study of 43 natural yeast strains belonging to 20 species for their capability for overproduction of citric acid (CA) from glucose under nitrogen limitation of cell growth was carried out. As a result, natural strain Yarrowia lipolytica VKM Y-2373 was selected. The effect of growth limitation by biogenic macroelements (nitrogen, phosphorus, or sulfur) on the CA production by the selected strain was studied. It was shown that yeasts Y. lipolytica grown under deficiency of nitrogen, phosphorus, or sulfur were able to excrete CA in industrially sufficient amounts (80-85g/L with the product yield (YCA) of 0.70-0.75g/g and the process selectivity of 92.5-95.3%). Based on the obtained data on activities of enzymes involved in the initial stages of glucose oxidation, the cycle of tricarboxylic acids, and the glyoxylate cycle, the conception of the mechanism responsible for the CA overproduction from glucose in Y. lipolytica was formulated.
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The NAD+-dependent isocitrate dehydrogenase of the organic acid-producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid... more
The NAD+-dependent isocitrate dehydrogenase of the organic acid-producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid precipitation, hydrophobic chromatography, and gel-filtration chromatography. The enzyme was purified 129-fold with a yield of 31% and had a specific activity of 22 U/mg protein. The molecular mass of the enzyme was found to be 412 kDa. The enzyme consists of eight identical subunits with a molecular mass of about 52 kDa. The Km for NAD+ is 136 microM, and that for isocitrate is 581 microM. The effect of some intermediates of the citric acid cycle and nucleotides on the enzyme activity was studied. The role of isocitrate dehydrogenase (NAD+) in the overproduction of citric and keto acids is discussed.
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The growth of wild type strain Yarrowia lipolytica VKM Y-2373 and its mutant Yarrowia lipolytica N 15 as well the biosynthesis of citric and isocitric acids on sunflower oil were studied. It was indicated that cell growth was associated... more
The growth of wild type strain Yarrowia lipolytica VKM Y-2373 and its mutant Yarrowia lipolytica N 15 as well the biosynthesis of citric and isocitric acids on sunflower oil were studied. It was indicated that cell growth was associated with the simultaneous utilization of glycerol and free fatty acids produced during oil hydrolysis. The activities of enzymes of glycerol metabolism (glycerol kinase), fatty acid assimilation enzymes of glyoxylate cycle (isocitrate lyase and malate synthase) and citric acid cycle were comparatively assayed in Y. lipolytica grown on sunflower oil, glycerol and oleic acid. Glycerol kinase and enzymes of glyoxylate cycle were active during the whole period of cell cultivation on sunflower oil. Citric acid production and a ratio between citric and isocitric acids depended on both the strain used and the medium composition. It was revealed that wild type strain Y. lipolytica VKM Y-2373 produced almost equal amounts of citric and isocitric acids at pH=4.5 a...
In the present work, the possibility of using glycerol and glycerol-containing waste from biodiesel manufacture as a carbon and energy source for microbiological production of citric acid was studied. Acid-formation ability on selective... more
In the present work, the possibility of using glycerol and glycerol-containing waste from biodiesel manufacture as a carbon and energy source for microbiological production of citric acid was studied. Acid-formation ability on selective media was preliminary tested for 66 yeast strains of different genera (Candida, Pichia, Saccharomyces, Torulopsis, and Yarrowia). Under growth limitation by nitrogen source, 41 strains belonging mainly to the species Yarrowia lipolytica were found to excrete organic acids to the culture broth. In contrast, 25 strains of the genera Debaryomyces, Candida, Pichiа, Saccharomyces and Torulopsis did not show acid excretion. Among the 41 acid-producing strains, the wild strain Y. lipolytica VKM Y-2373 and the mutant strain Y. lipolytica N 15 exhibited the highest ability to accumulate citric acid in the culture broth. The production of citric acid by these two strains was studied as a function of the medium pH, degree of aeration and the concentration of gl...
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The production of technical-grade sodium citrate from the glycerol-containing biodiesel waste by Yarrowia lipolytica was studied. Batch experiments showed that citrate was actively produced within 144 h, then citrate formation decreased... more
The production of technical-grade sodium citrate from the glycerol-containing biodiesel waste by Yarrowia lipolytica was studied. Batch experiments showed that citrate was actively produced within 144 h, then citrate formation decreased presumably due to inhibition of enzymes involved in this process. In contrast, when the method of repeated batch cultivation was used, the formation of citrate continued for more than 500 h. In this case, the final concentration of citrate in the culture liquid reached 79-82 g/L. Trisodium citrate was isolated from the culture liquid filtrate by the addition of a small amount of NaOH, so that the pH of the filtrate increased to 7-8. This simple and economic isolation procedure gave the yield of crude preparation containing trisodium citrate 5.5-hydrate up to 82-86%.
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In this study, physiologo-biochemical characteristics of citrate-producing yeast Yarrowia lipolytica grown on glycerol-containing waste of biodiesel industry were studied by an investigation of growth dynamics, the consumption of... more
In this study, physiologo-biochemical characteristics of citrate-producing yeast Yarrowia lipolytica grown on glycerol-containing waste of biodiesel industry were studied by an investigation of growth dynamics, the consumption of glycerol, and the fatty acid fractions from waste as well as by measuring the activities of enzymes involved in the metabolism of waste. It was shown that Y. lipolytica realizes concurrent uptake of glycerol and the fatty acid fractions during conversion of glycerol-containing waste, although glycerol was utilized at a higher rate than fatty acids. Under optimal feeding of glycerol-containing waste by portions of 20 g l(-1), the citric acid production and the ratio between citric acid and isocitric acid depended on the strain used. It was revealed that wild strain Y. lipolytica VKM Y-2373 produced citrate and isocitrate with a ratio of 1.7:1, while the mutant strain Y. lipolytica NG40/UV7 synthesized presumably citric acid (122.2 g l(-1)) with a citrate-to-...
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The involvement of transamination in the respiration of liver mitochondria in ground squirrels during hibernation and arousal has been studied. It was shown by HPLC that, in the presence of glutamate and malate, the formation of... more
The involvement of transamination in the respiration of liver mitochondria in ground squirrels during hibernation and arousal has been studied. It was shown by HPLC that, in the presence of glutamate and malate, the formation of alpha-ketoglutarate (KGL), a transamination marker, and fumarate, a product of succinate oxidation, takes place. During arousal, the formation of KGL increased fourfold, and the respiration sensitive to the inhibitor of aspartate transaminase aminooxyacetate (AOA) increased threefold. The function of transamination upon arousal is related to the elimination of oxaloacetate, an endogenous inhibitor of succinate dehydrogenase. In addition, being more resistant to oxidative stress than oxidation, transamination is probably involved in the antioxidant defense required during the rapid rise of body temperature upon arousal. Our experiments showed an increase in the concentration of malonic dialdehyde (MDA), an end product of lipid peroxidation, in liver mitochond...
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The native strain Yarrowia lipolytica VKM Y-2373 grown in a complete medium exhibited the maximum lipase activity at the concentration of rapeseed oil of at least 5.0 g/l. In the course of yeast growth, no considerable changes were... more
The native strain Yarrowia lipolytica VKM Y-2373 grown in a complete medium exhibited the maximum lipase activity at the concentration of rapeseed oil of at least 5.0 g/l. In the course of yeast growth, no considerable changes were observed in the glycerol concentration, the proportions of the major free fatty acids formed via oil hydrolysis, or the fatty acid composition of oil. Under nitrogen limitation of cell growth, the accumulation of citric acids reached 77.1 g/l with predominance of isocitric acid at pH 6.0, whereas at pH 4.5, almost equal amounts of citric and isocitric acids were produced. Cultivation of the mutant strain Y. lipolytica N 1 at pH 4.5 resulted in the predominant accumulation of citric acid (66.6 g/l) with an insignificant amount of isocitric acid. In the period of intense acid synthesis, high production of lipase was observed.
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It is generally accepted that the glyoxylate cycle exists in microorganisms and higher plants but absent in higher animals. the hypodhesis of the glyoxylate cycle in the tissues of higher animals with a high level of physiological... more
It is generally accepted that the glyoxylate cycle exists in microorganisms and higher plants but absent in higher animals. the hypodhesis of the glyoxylate cycle in the tissues of higher animals with a high level of physiological activity was first proposed by Kondrashova and Rodionova in 1971. The goal of this work was yo verifv this in newborn rats, which possess a 2.5-fold hygher physiological activity and oxygen consumption rate than adult rats. Newborn (7-day-old) anradult 1 ats were used for this experiment. The activities of the key enzymes of the glyoxylate cocle (isecitrate lyse and nmalate synthase) were measured by HPLC and spectroscopic methods. The activities of isocitrate lyase and malate synthase were found in the liver homogenates prepared from newborn rats, but not from adult rats. The activities of the enzymes common to both the Krebs cycle and the glyoxylate cycle (citrate synthase, aconitase, and malate dehydrogenase) were 20-40% higher in newborn than in adult ...
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The decrease in metabolism is one of mechanisms for hibernating animals to resist hypoxia and oxidative stress. Assuming that the inhibition of mitochondria; respiration in torpor and its activation upon arousal are accompanied by changes... more
The decrease in metabolism is one of mechanisms for hibernating animals to resist hypoxia and oxidative stress. Assuming that the inhibition of mitochondria; respiration in torpor and its activation upon arousal are accompanied by changes in the content of mitochondrial substrates, we estimated the levels of endogenous metabolites of the tricarboxylic acid (TCA) cycle in the liver, brown adipose tissue, and the brain of the arctic ground squirrels as possible indicators of mitochondrial processes. The level of lactate in the same tissues and serum was determined as marker of hypoxia. It was found that the isocitrate (ISC) concentration in all tissues was one order of magnitude higher than that of alpha-ketoglutarate (KGL), while succinate was not detected in any of tissues, indicating the inhibition at the initial stages of the TCA cycle. During the torpor, the concentrations of ISC, KGL and lactate predominantly decreased in tissues. Serum lactate decreased five-fold in torpor and ...
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The production of α-ketoglutaric acid by yeast Yarrowia lipolytica VKMY-2412 from ethanol and its subsequent chemical conversion to succinic acid (SA) were investigated. A highly effective and environmentally friendly process of... more
The production of α-ketoglutaric acid by yeast Yarrowia lipolytica VKMY-2412 from ethanol and its subsequent chemical conversion to succinic acid (SA) were investigated. A highly effective and environmentally friendly process of α-ketoglutaric acid production was developed using a special pH-controlling strategy, in which the titration of the culture broth with KOH in the acid-formation phase was minimal, that allowed accumulation of only low amounts of inorganic wastes in the course of SA recovery. The culture broth filtrate containing α-ketoglutaric acid (88.7 g l(-1)) was directly employed for SA production; the amount of SA produced comprised 71.7 g l(-1) with the yield of 70% from ethanol consumed. SA was isolated from the culture broth filtrate in a crystalline form with the purity of 100%. The yield of isolated SA was as high as 72% of its amount in the culture broth filtrate. The antimicrobial and nematocidic effects of SA of microbial origin on pathogenic organisms that cau...
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The process of succinic acid (SA) production represents the combination of microbial synthesis of α-ketoglutaric acid from rapeseed oil by yeast Yarrowia lipolytica VKM Y-2412 and subsequent decarboxylation of α-ketoglutaric acid by... more
The process of succinic acid (SA) production represents the combination of microbial synthesis of α-ketoglutaric acid from rapeseed oil by yeast Yarrowia lipolytica VKM Y-2412 and subsequent decarboxylation of α-ketoglutaric acid by hydrogen peroxide to SA that leads to the production of 69.0 g l(-1) of SA and 1.36 g l(-1) of acetic acid. SA was isolated from the culture broth filtrate in a crystalline form. The SA recovery from the culture filtrate has certain difficulties due to the presence of residual triglycerides of rapeseed oil. The effect of different methods of the culture filtrate treatment and various sorption materials on the coagulation of triglycerides was studied, and as a result, the precipitation of residual triglycerides by acetone was chosen. The subsequent isolation procedures involved the decomposition of H2O2 in the filtrate followed by filtrate bleaching and acidification with a mineral acid, evaporation of filtrate, and SA extraction with ethanol from the residue. The purity of crystalline SA isolated from the culture broth filtrate achieved 97.6-100 %. The product yield varied from 62.6 to 71.6 % depending on the acidity of the supernatant.
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The ability of taxonomically different yeasts to synthesize pyruvic acid (PA) from glucose was studied. The study showed that many yeasts are able to produce PA from glucose under the condition of growth limitation by thiamine. This... more
The ability of taxonomically different yeasts to synthesize pyruvic acid (PA) from glucose was studied. The study showed that many yeasts are able to produce PA from glucose under the condition of growth limitation by thiamine. This ability was found in the yeast Blastobotrys adeninivorans for the first time. The production (oversynthesis) of PA in this yeast can be explained by disturbance in the function of thiamine-dependent pyruvate dehydrogenase. Namely, the partial inhibition of this enzyme brings about the excretion of PA from the yeast cells. Due to incomplete inhibition of pyruvate dehydrogenase, the formation of acetyl-CoA continues, although at a lower level, maintaining the synthesis of α-ketoglutaric acid (KGA) in the tricarboxylic acid (TCA) cycle. KGA is no longer oxidized in the TCA cycle, because thiamine limitation inhibits α-ketoglutarate dehydrogenase. As a result, KGA is excreted from the yeast cells as a byproduct of PA oversynthesis. Furthermore, the increased level of KGA in the yeast cells inhibits NAD-dependent isocitrate dehydrogenase in the TCA cycle and enhances the production and excretion of citric acid, another byproduct of PA oversynthesis. During cultivation in a fermentor, the strain Blastobotrys adeninivorans VKM Y-2677 produced 43.2 g l(-1) PA from glucose with a product yield (YPA) of 0.77 g PA/g glucose. The proportion of PA to byproducts was 18:1 for KGA and 8:1 for citric acid.