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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 Improvement of the technology for surfactant synthesis by Аcinetobacter calcoaceticus ІМV В-7241(2015) Pirog, Tatiana; Konon, AnastasiaThe aim of the work was to intensify the synthesis of surfactants by Acinetobacter calcoaceticus IMV-7241 cultivated on ethanol and other carbon substrates. A. calcoaceticus IMV-7241 was grown in the medium with mono- (ethanol, glycerol, liquid paraffin, n-hexadecane, glucose) and mixed substrates in the presence of organic acids or heavy metal cations (0.1–2.0 mM Cu2+, Cd2+, Zn2+, Pb2+). The synthesis of surfactants was evaluated by emulsification index of cultural liquid, conditional concentration and concentrations of extracellular surfactants, which were determined gravimetrically after their extraction from supernatant with the mixture of methanol and chloroform. It was shown that addition of citrate and fumarate (0.01%) at the end of exponential growth phase of A. calcoaceticus ІМV В-7241 in the medium with ethanol (2%) and the maintenance of neutral pH increased the surfactants’ concentration in 3.5 times (up to 6.0 g/l). The quantity of extracellular surfactants synthesized by the strain ІМV В-7241 in the medium containing mixture of n-hexadecane and glycerol (molar ratio 1:7) and C/N 30 was increased in 2.6–3.5 times in comparison with cultivation on corresponding monosubstrates. Addition of 2.0 mM Cu2+ at the stationary growth phase of A. calcoaceticus ІМV В-7241 in medium with liquid paraffin and n-hexadecane led to the increase of surfactants’ synthesis in 2.3–2.5 times compared with those in the medium without Cu2+. Approaches to intensification of surfactants’ synthesis by A. calcoaceticus ІМV В-7241 (including addition of biosynthesis precursors and cultivation on the mixture of substrates) can be used to increase the efficiency of microbial technologies.Документ Synthesis of exopolysaccharide ethapolan during сultivation of auxotroph Acinetobacter sp. IMV B-7005 in the medium with sunflower oil(2014) Ivahniuk, Mykola; Pirog, Tatiana; Penchuk, YuriThe possibility to use the multivitamin complex «Complevit» as a source of calcium pantothenate by auxotrophic strain Acinetobacter sp. IMV B-7005 – the producer of microbial exopolysaccharide ethapolan was shown. The cultivation conditions for the producer were established to provide the synthesis of 6.6-6.7 g/l of the desired product in the medium with high (4-5 %) content of sunflower oil – cheap and available substrate for microbial technologies.Документ Intensification of surfactants’ synthesis by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii K-8 on fried oil and glycerol containing medium(2013) Pirog, Tatiana; Sofіlkanich (Morozova), Anna; Konon, Anastasia; Shevchuk, Tetiana; Ivanov, SergeyАктуальною проблемою сьогодення є пошук шляхів для переробки відходів. Біотехнологія є одним з найбільш екологічно привабливих методів, що дають змогу вирішити проблему утилізації відходів і одержання цінних мікробних продуктів, наприклад, поверхнево-активних речовин (ПАР). Встановлено, пересмажена (відпрацьована) соняшникова олія, олієвмісні відходи (фузи) і гліцерин можуть бути використані для синтезу ПАР Rhodococcus erythropolis IMВ Ac-5017, Acinetobacter calcoaceticus ІМВ В-7241 і Nocardia vaccinii К-8. Внесення глюкози (0,1%) у середовище з відпрацьованою олією (2% об.) супроводжувалося збільшенням у 4 рази кінцевої концентрації поверхнево-активних речовин (6,8 г/л). У разі одночасного добавлення фумарату і цитрату (0,01-0,2%) у середовище культивування штамів IMВ B-7241 і К-8 спостерігали підвищення кількості позаклітинних ПАР у 2-2,5 рази порівняно з вирощуванням без органічних кислот. Збільшення концентрації ПАР штаму IMВ B-7241 зумовлене одночасним функціонуванням двох анаплеротичних шляхів, а також підвищенням у 3-5 разів активності ферментів біосинтезу поверхнево-активних речовин. Культивування на суміші гліцерину і н-гексадекану (0,5-1,0 об.%) супроводжувалося підвищенням у 1,5-3 рази синтезу ПАР. Препарати ПАР штамів IMВ B-7241 (0,15-0,22 мг/мл) і IMВ Ac-5017 (0.61-2.1 мг/мл) проявляли антимікробну активність щодо Escherichia coli IEM-1 (загибель 67% клітин), а також вегетативних клітин (загибель 45-100%) і спор (загибель 75%) Bacillus subtilis БТ-2. Searching for the ways to process waste has become very topical today. Biotechnology is one of the most environmentally attractive methods, which has the ability to solve the problem of waste utilization and to produce the valuable microbial products, for example biosurfactants. We concluded that fried sunflower oil, oil-containing wastes (soapstock) and glycerol can be used for biosurfactant production by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii K-8. Glucose addition (0.1%) into the medium with fried oil (2 vol.%) led to a 4-fold increase of final surfactant concentration (6.8 g/L). The simultaneous addition of fumarate and citrate (0.01–0.2%) into the IMV B-7241 and K-8 strains’ cultivating medium was accompanied by an increase of the exocellular biosurfactant quantity by 2–2.5-fold compared to the cultivation without organic acids. An increase in surfactant concentration of IMV B-7241 strain was the result of the simultaneous functioning of two anaplerotic pathways, also resulting in a 3–5-fold increase in activity of biosynthesis enzymes. Cultivating on a mixture of glycerol and n-hexadecane (0.5–1.0 vol.%) led to a 1.5–3-fold increasing surfactant synthesis. Biosurfactant preparations of IMV B-7241 (0.15–0.22 mg/mL) and IMV Ac-5017 (0.61–2.1 mg/mL) strains were effective against Escherichia coli IEM-1 (67% of cell loss), and vegetative (45–100% of cell loss) and spore (75% of spore loss) cells of Bacillus subtilis BT-2.Документ Biosurfactants of Rhodococcus erythropolis IMV Ас-5017: synthesis intensification and practical application(2013) Pirog, Tatiana; Sofіlkanich (Morozova), Anna; Shulyakova, Mariya; Shevchuk, TetianaIntensification of the surfactant synthesis by Rhodococcus erythropolis IMV Ac-5017 on different substrates, including industrial waste, as well as the use of surfactant preparations for oil degradation were studied. It was established that the addition of fumarate (0.2 %) and citrate (0.1 %) into the medium with ethanol, n-hexadecane, or glycerol (1–2 %) was accompanied by an increase of conditional surfactant concentration by 1.5–1.7 times compared to the indexes in the medium without organic acids. The intensification of surfactant synthesis in the presence of fumarate and citrate is caused by the increased activity of isocitrate lyase (by 1.2–15-fold) and enzymes of the surfactant biosynthesis (by 2–4.8-fold) compared to their activity in the medium without precursors. The possibility of surfactant synthesis intensification (by 3–4-fold) while cultivating of R. erythropolis IMV Ac-5017 in the medium with oil containing substrates (2 %) and glucose (0.1 %) was shown. The introduction of 0.01 mM Cu2+ in the exponential growth phase of strain IMV Ac-5017 in the medium with ethanol accompanied by the increasing conditional surfactant concentration by 1.9 times. The highly efficient remediation (92–95 %) of oil (2–2.6 g/L) and Cu2+ polluted water after treatment with surfactant preparations (native cultural liquid) at low concentrations (5 %) was determined.Документ Intensification of biosurfactant synthesis by Acinetobacter calcoaceticus IMV B-7241 on a hexadecane–glycerol mixture(2012) Pirog, Tatiana; Konon, Anastasia; Shevchuk, Tetiana; Bilets, I.The possibility of enhanced biosurfactant (BS) synthesis by the cultivation of Acinetobacter calcoaceticus IMV B-7241 on a mixture of energetically nonequivalent substrates (hexadecane and glycerol) was shown. Based on theoretical calculations of the energy requirements for biomass production and the synthesis of surface-active trehalose monomycolate from the energy-deficient substrate (glycerol), the concentration of the energy-excessive substrate (hexadecane), which increased the efficiency of the substrate carbon conversion to BS, was determined. The synthesis of extracellular BS on a mixture of hexadecane and glycerol in a molar ratio of 1 : 7 at C/N ratio of 30 increased 2.6–3.5-fold compared to that on single_substrate media. Increased BS synthesis by Acinetobacter calcoaceticus IMV B-7241 grown on a hexadecane–glycerol mixture was accompanied by a 1.3–2.4-fold increase in activities of the enzymes involved in their biosynthesis, as well as by simultaneous functioning of two anaplerotic pathways (the glyoxylate cycle and the phosphoenolpyruvate carboxylase reaction). Досліджено можливість підвищення синтезу поверхнево-активних речовин (ПАР) за умов росту Acinetobacter calcoaceticus ІМВ В-7241 на суміші енергетично нерівноцінних субстратів (гексадекану і гліцерину). На основі теоретичних розрахунків енергетичних потреб синтезу поверхнево-активних трегалозомономіколатів і біомаси на енергетично дефіцитному субстраті (гліцерин) визначена концентрація енергетично надлишкового гексадекану, що дає змогу підвищити ефективність конверсії вуглецю використовуваних субстратів у ПАР. За молярного співвідношення концентрацій гексадекану і гліцерину 1:7 та співвідношенні С/N, що дорівнює 30, кількість синтезованих позаклітинних ПАР збільшувалася у 2.6−3.5 рази порівняно з такою на моносубстратах. Підвищення синтезу ПАР A. calcoaceticus ІМВ В-7241 на суміші гексадекану і гліцерину зумовлене збільшенням в 1.3−2.4 рази активності ферментів їх біосинтезу, а також одночасним функціонуванням двох анаплеротичних шляхів (гліоксилатного циклу і фосфоенолпіруват-карбоксилазної реакції).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 Peculiarities of C2-Metabolism and Intensification of the Synthesis of Surface-Active Substances in Rhodococcus erythropolis EK-1 Grown in Ethanol(2008) Pirog, Tatiana; Korzh, Yuliya; Shevchuk, Tetiana; Tarasenko, D.Oxidation of ethanol, acetaldehyde, and acetate in Rhodococcus erythropolis EK-1, producer of surface-active substances (SAS), is catalyzed by N,N-dimethyl-4-nitrosoaniline (DMNA)-dependent alcohol dehydrogenase, NAD+/NADP+-dependent dehydrogenases (optimum pH 9.5), and acetate kinase/acetyl-CoAsynthetase, respectively. The glyoxylate cycle and complete tricarboxylic acid cycle function in the cells of R. erythropolis EK-1 growing on ethanol; the synthesis of phosphoenolpyruvate (PEP) is provided by the two key enzymes of gluconeogenesis, PEP carboxykinase and PEP synthetase. Introduction of citrate (0.1%) and fumarate (0.2%) into the cultivation medium of R. erythropolis EK-1 containing 2% ethanol resulted in the 1.5- and 3.5-fold increase in the activities of isocitrate lyase and PEP synthetase (the key enzymes of the glyoxylate cycle and gluconeogenesis branch of metabolism, respectively) and of lipid synthesis, as evidenced by the 1.5-fold decrease of isocitrate dehydrogenase activity. In the presence of fumarate and citrate, the indices of SAS synthesis by strain R. erythropolis EK-1 grown on ethanol increased by 40–100%. Окисление этанола у штамма Rhodococcus erythropolis ЭК-1 – продуцента поверхностно-активных веществ (ПАВ), осуществляется 4-нитрозо-N,N-диметиланилин (НДМА)-зависимой алкогольдегидрогеназой, окисление ацетальдегида – НАД+- и НАДФ+-зависимыми дегидрогеназами с оптимумом рН 9.5, окисление ацетата – ацетаткиназой и ацетил-КоА-синтетазой. При росте на этаноле в клетках R. erythropolis ЭК-1 функционирует как глиоксилатный цикл, так и полный цикл трикарбоновых кислот, синтез фосфоенолпирувата (ФЕП) обеспечивается двумя ключевыми ферментами глюконеогенеза – ФЕП-карбоксикиназой и ФЕП-синтетазой. Внесение в среду культивирования R. erythropolis ЭК-1, содержащую 2 % этанола, цитрата (0.1 %) и фумарата (0.2 %) сопровождалось усилением глюконеогенеза, что подтверждается повышением в 1.5 и 3.5 раза активности изоцитратлиазы и ФЕП-синтетазы (ключевых ферментов глиоксилатного цикла и глюконеогенетической ветви обмена веществ соответственно), а также синтеза липидов, о чем может свидетельствовать снижение в 1.5 раза активности изоцитратдегидрогеназы. В присутствии фумарата и цитрата показатели синтеза ПАВ штаммом R. erythropolis ЭК-1 на этаноле повышались на 40–100 %.Документ Specific features of the synthesis of the exopolysaccharide ethapolan on a mixture of energy-deficient growth substrates(2007) Pirog, Tatiana; Vysyatetska, Nadezhda; Korzh, YuliyaIntensification of the synthesis of the microbial exopolysaccharide ethapolan by Acinetobacter sp. B-7005 was shown to occur on a mixture of energy-deficient growth substrates (acetate + glucose). When the bacterium grew on the substrate mixture, both substrates were utilized simultaneously; acetate was taken up by means of active transport at the expense of the energy of the proton-motive force. When acetate was present in the form of a sodium salt, the activities of acetyl-CoA synthetase and phosphoenolpyruvate synthetase (the key enzyme of gluconeogenesis) were tenfold higher than in the presence of potassium acetate, and the indexes of ethapolan synthesis were two times higher. The positive effect of Na+ on ethapolan synthesis is supposed to consist in the creation of ion gradients on the membrane, necessary for the generation of the proton-motive force. Simultaneous functioning of the glyoxylate cycle and pyruvate carboxylase reaction, as well as an increase in the activity of isocitrate lyase, malate synthase, and phosphoenolpyruvate synthetase, provide evidence of increased gluconeogenesis in the presence of the acetate + glucose mixture (as compared to gluconeogenesis on the corresponding monosubstrates).