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Документ Ethapolan synthesis by Acinetobacter sp. IMV B-7005 on the mixture of С2-С6-substrates and waste sunflower oil(2021) Voronenko, Andryi; Pirog, TatianaThe cultivation conditions of Acinetobacter sp. IMV B-7005, which would provide maximum synthesis of the microbial exopolysaccharide (EPS) ethapolan on the mixture of C2-C6–substrates (molasses, acetate, or ethanol) and waste sunflower oil was studied.Документ Intensification of microbial exopolysaccharide ethapolan synthesis on the mixture of energy-excessive substrates(2021) Voronenko, Andryi; Pirog, TatianaIntroduction. The cultivation conditions of Acinetobacter sp. IMV B-7005, providing maximum synthesis of exopolysaccharide (EPS) ethapolan on the mixture of ethanol and sunflower oil were studied, as well as the possibility of replacing refined oil in the mixture with ethanol on a waste one was demonstrated. Materials and methods. Strain IMV B-7005 was grown in liquid mineral media, containing the mixture of ethanol and sunflower oil of various quality, as well as appropriate monosubstrates. The optimal molar ratio of the concentrations of substrates in the mixture was calculated theoretically according to Babel’s concept. The EPS concentration was determined gravimetrically after precipitation with isopropanol, the EPS-synthesizing ability – as the ratio of the EPS concentration to the concentration of biomass and expressed in g EPS/g biomass. Results and discussion. The highest rates of ethapolan synthesis were observed with the molar ratio of concentrations of ethanol and refined sunflower oil in the mixture of 1:0.056, as close as possible to the theoretically calculated (1:0.076), and the use of inoculum grown on ethanol. Further increasing of the concentrations of ethanol and oil led to a decrease in pH of the culture fluid to a suboptimal level for the EPS synthesis (4.5-4.8). To ensure the synthesis of ethapolan on a medium with high concentrations of ethanol (4%) and oil (1.2%) ammonium nitrate was replaced with an equimolar amount of nitrogen KNO3 (0.8 g/l), which is transported into cells by the symport with proton; fractional introduction of substrates in five equal portions during cultivation was carried out and was increased the concentration of Mg2+ cations, which are one of the activators of acetyl-CoA synthetase in Acinetobacter sp. IMV B-7005 affecting the enzymatic activity of systems responsible for the catabolism of fatty acids. Under such cultivation conditions, regardless of the type of used sunflower oil (refined or mixed waste) in the mixture with ethanol, the concentration of ethapolan reached 13.5-16.0 g/l, and EPS-synthesizing ability – 3.1-3.7 g EPS/g of biomass, which were respectively 3.2-3.8 and 1.6-1.9 times higher than before optimization. Conclusions. Based on determining the optimal molar ratio of monosubstrate concentrations in the mixture, modification of the medium composition (replacement of ammonium nitrate with potassium nitrate, increasing the content of magnesium cations, replacement of refined oil on a mixed waste one) and fractional addition of substrates the possibility of intensification of ethapolan synthesis on the mixture of energy-excessive substrates (ethanol and sunflower oil) was established.Документ Non-traditional producers of microbial exopolysaccharides(2018) Pirog, Tatiana; Voronenko, Andryi; Ivahniuk, MykolaData on exopolysaccharides synthesis by psychrophilic fungi and bacteriae, halo- and thermophilic archaea and bacteriae, including those isolated from deep-sea hydrothermal vents − sources − were provided. Physiologic significance, physico-chemical properties and possible practical applications of exopolysaccharides from unusual sources were analyzed. Most of them have immunomodulating, antiviral, anticoagulant, antitumor, antioxidant activities promising for medical and pharmaceutical applications. Meanwhile, based on the literature date, the conclusion follows about the urgent necessity to develop efficient technologies for synthesis of these exopolysaccharides by nontraditional producers, which currently lags far behind common techniques. Наведено дані про синтез екзополісахаридів психрофільними грибами та бактеріями, гало- та термофільними археями та бактеріями, у тому числі виділеними з глибоководних гідротерм – джерел. Було проаналізовано фізіологічне значення, фізико-хімічні властивості та можливі практичні застосування екзополісахаридів з незвичайних джерел. Більшість із них мають імуномодулюючу, противірусну, антикоагулянтну, протипухлинну, антиоксидантну активність, перспективну для застосування в медицині та фармацевтиці. Водночас, виходячи з літературних даних, випливає висновок про нагальну необхідність розробки ефективних технологій синтезу цих екзополісахаридів нетрадиційними продуцентами, які на сьогодні значно відстають від загальноприйнятих.Документ Exopolysaccharides synthesis on industrial waste(2016) Pirog, Tatiana; Ivahniuk, Mykola; Voronenko, AndryiData from the literature and our own studies on the synthesis of microbial exopolysaccharides on various industrial waste (food industry, agricultural sector, biodiesel production, etc.) are reviewed here. Utilization of industrial waste to obtain exopolysaccharides will solve not only the problem of secondary raw materials accumulation, but also will reduce the costs of the biosynthesis of practically valuable metabolites. In addition, some kinds of waste have a number of advantages compared to traditional carbohydrate substrates: aside from environmental health benefits, there are technological ones, like the presence of growth factors. There is also no need to use anti-foam substances and substrate sterilization in the latter case.Документ Effect of environmental factors on the synthesis and properties of Acinetobacter sp. exopolysaccharides(1998) Pirog, Tatiana; Grinberg, Tamara; Malashenko, YuriEffects of external factors on the synthesis and physicochemical properties of Acinetobacter sp. exopolysaccharides (EPSs), which determine the biological functions of this microorganism, were studied. The cultivation temperature, medium pH, and oxygen concentration in the medium (p02) affected the viscosity of EPS solutions in the presence of monovalent cations, in the H+-form, and in a Cu2+-glycine system. All the EPSs studied were precipitated with heavy metal ions (Cr3+, Cu2+, Pb2+, Cd2+, etc.). No changes in the EPS yield were observed under unfavorable environmental conditions. At high pO2 values (up to 80% of saturation), the maximum specific rates of bacterial growth and EPS synthesis increased. It was suggested that Acinetobacter sp. EPSs perform different biological functions under optimal and nonoptimal conditions.Документ Formation of the Exopolysaccharide ethapolan by acinetobacter sp. IMV B-7005 on a fumarate–glucose mixture(2007) Pirog, Tatiana; Vysyatetska, Nadezhda; Korzh, YuliyaOur studies enabled us to intensify the synthesis of the microbial exopolysaccharide (EPS) ethapolan produced by Acinetobacter sp. IMV B-7005 grown on a mixture of fumarate (an energy-excessive substrate) and glucose (an energy-deficient substrate). Supplementing glucose-containing medium with sodium (potassium) fumarate at a molar ratio of 4 : 1 resulted in a 1.3–2.2-fold increase of the EPS amount synthesized and in a 1.3–2-fold increase of the EPS yield relative to the biomass compared to cultivation on monosubstrates. The conversion of the carbon of both substrates to EPS was the highest if the carbon/nitrogen ratio in the cultivation medium was 70.5 and inoculum grown on glucose monosubstrate was used.Документ Exopolysaccharide Production and Peculiarities of C6-Metabolism in Acinetobacter sp. Grown on Carbohydrate Substrates(2002) Pirog, Tatiana; Kovalenko, M.; Kuzminska, Yu.An Acinetobacter sp. strain grown on carbohydrate substrates (mono- and disaccharides, molasses, starch) was shown to synthesize exopolysaccharides (EPS). Glucose catabolism proved to proceed via the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. Pyruvate entered the tricarboxylic acid cycle due to pyruvate dehydrogenase activity. Pyruvate carboxylation by pyruvate carboxylase was the anaplerotic reaction providing for the synthesis of intermediates for the constructive metabolism of Acinetobacter sp. grown on C6-substrates. The C6-metabolism in Acinetobacter sp. was limited by coenzyme A. Irrespective of the carbohydrate growth substrate (glucose, ethanol), the activities of the key enzymes of both C2- and C6-metabolism was high, except for the isocitrate lyase activity in glucose-grown bacteria. Isocitrate lyase activity was induced by C2-compounds (ethanol or acetate). After their addition to glucose-containing medium, both substrates were utilized simultaneously, and an increase was observed in the EPS synthesis, as well as in the EPS yield relative to biomass. The mechanisms responsible for enhancing the EPS synthesis in Acinetobacter sp. grown on a mixture of C2- and C6-substrates are discussed.Документ Physicochemical Properties of the Microbial Exopolysaccharide Ethapolan Synthesized on a Mixture of Growth Substrates(2004) Pirog, Tatiana; Kovalenko, M.; Kuzminska, Yu.; Votselko, S.Some physicochemical properties of the microbial exopolysaccharide (EPS) ethapolan synthesized by Acinetobacter sp. 12S depended on whether the producer was grown on a mixture of ethanol and glucose or on a single substrate. Irrespective of the carbon source in the nutrient medium, the contents of carbohydrates, pyruvic acid, uronic acids, and mineral components in the EPS remained unchanged. The EPS were also identical in their monosaccharide composition: the molar ratio of glucose, mannose, galactose, and rhamnose was 3 : 2 : 1 : 1. EPS with a higher content of fatty acids was synthesized during growth on the mixture of ethanol and glucose. The average molecular mass and the content of high-molecular (M > 2 MDa) fractions were greater in ethapolan produced on the substrate mixture. In the presence of 0.1 M KCl, after transformation into the H+ form, and in the Cu2+–glycine system, solutions of these EPS showed higher viscosity than solutions of EPS synthesized on single substrates. The reasons for the improved rheological properties of the EPS produced on the substrate mixture are discussed.Документ Intensification of exopolysaccharide synthesis by Acinetobacter sp. on an ethanol–glucose mixture: aspects related to biochemistry and bioenergetics(2003) Pirog, Tatiana; Kuzminska, Yu.; Kovalenko, M.The possibility of intensifying the synthesis of microbial exopolysaccharides (EPS) by a strain of Acinetobacter sp. grown on a mixture of two substrates nonequivalent in terms of bioenergetics (ethanol + glucose) was shown. Based on theoretical calculations of the energy requirements for biomass and EPS synthesis from the energy-deficient substrate (glucose), the supplementary concentration of the energy-excessive substrate (ethanol) was determined that prevents the loss of glucose carbon that occurs when glucose is oxidized to CO2 to obtain energy for the processes of constructive metabolism. This made it possible to increase the efficiency of conversion of the substrate carbon to EPS. The introduction of ethanol into glucose-containing medium at a molar ratio of 3.1 : 1 allowed the amount of the EPS synthesized to be increased 1.8- to 1.9-fold; their yield relative to biomass increased 1.4- to 1.7-fold, and the yield of EPS relative to the substrate consumed increased 1.5- to 2-fold as compared to growth of the producer on single substrates. These results form the basis for the development of new technologies for obtaining secondary metabolites of practical value with the use of mixed growth substrates. Показана можливість інтенсифікації синтезу мікробних екзополісахаридів (ЕПС) при вирощуванні штаму Acinetobacter sp. на суміші двох енергетично-нерівноцінних субстратів (етанол + глюкоза). На основі теоретичних розрахунків енергетичних потреб синтезу біомаси та ЕПС на енергетично-дефіцитному субстраті (глюкоза) визначена "доповнююча" концентрація енергетично-надлишкового субстрату (етанол), що дозволяє заповнити втрати вуглецю глюкози при окисленні її до СО2 з метою отримання енергії для процесів конструктивного метаболізму, і підвищити ефективність конверсії вуглецю використовуваних субстратів в ЕПС. Введення етанолу в середу з глюкозою в молярному співвідношенні 3,1:1 дозволило збільшити кількість синтезованих ЕПС в 1,8-1,9 рази, їх вихід по відношенню до біомаси - в 1,4 - 1,7 рази, вихід ЕПС від субстрату - в 1,5 - 2 рази в порівнянні з вирощуванням продуцента на моносубстратах. Отримані результати є основою для створення нових технологій одержання практично цінних вторинних метаболітів при використанні змішаних енергетично-нерівноцінних ростових субстратів.Документ Isolation and characterization of Acinetobacter sp. mutants defective in exopolysaccharide biosynthesis(2000) Pirog, Tatiana; Stolyar, S.; Malashenko, YuriNitrosoguanidine-induced mutants ofAcinetobacter sp. defective in exopolysaccharide biosynthesis did not differ from the parent strain in distinguishing physiological and biochemical properties, such as requirements for growth factors, utilization of mono- and disaccharides, and resistance to antibiotics. The genetic relation of parent and mutant strains was shown by 16S rRNA PCR analysis. The comparative study of parent and mutant strains with respect to resistance to unfavorable environmental factors confirmed our hypothesis that Acinetobacter sp. exopolysaccharides perform protective functions. Hybridization experiments revealed the conjugal transfer of plasmid R68.45 from Pseudomonas putida BS228 (R68.45) to mutant but not to the parent Acinetobacter sp. strains. The role of the Acinetobacter sp. exopolysaccharides in providing the genetic stability of this bacterium is discussed. Методом нітрозогуанідінового мутагенезу отримані мутантні штами Acinetobacter sp., Що не утворюють Екзополісахариди (ЄПС). Вихідний і мутантні штами не відрізняються між собою по ряду характерних для цих бактерій фізіолого-біохімічних ознак (потреба в ростових факторах, асиміляція моно-і дисахаридів, стійкість до антибіотиків). Ідентичність вихідного і мутантних штамів встановлена також на основі аналізу їх 16S рРНК. Результати вивчення стійкості клітин вихідного та мутантних штамів до несприятливих факторів підтвердили захисні функції ЕПС Acinetobacter sp. по відношенню до клітин продуцента. При схрещуванні Acinetobacter sp. з Pseudomonas putida BS228 (R68.45) здійснено кон'югатівний перенесення плазміди R68.45 в клітини мутантних (але не початкового) штамів. Обговорюється роль ЕПС Acinetobacter sp. як одного з факторів, що забезпечують генетичну стабільність штаму-продуцента.