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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 Рroduction of exopolysaccharide ethapolan under Acinetobacter sp. IMV B-7005 cultivation on the mixture of acetate and sunflower oil(2020) Pirog, Tatiana; Voronenko, Andryi; Yarosh, MarynaThe aim of the work was to establish the cultivation conditions of Acinetobacter sp. IMV B-7005 for providing the maximum indicators of the exopolysaccharide ethapolan synthesis on the mixture of acetate and sunflower oil, as well as to study the possibility of replacing the refined oil in the mixture with acetate on a waste one. The optimal molar ratio of concentrations for refined sunflower oil and acetate in the mixture was calculated theoretically according to Babel’s concept of “auxiliary substrate”. The EPS concentration was determined gravimetrically after precipitation with isopropanol. The EPS-synthesizing ability was calculated as the ratio of the EPS concentration to the concentration of biomass and expressed in g EPS/g biomass. Based on theoretical calculations of energy requirements for EPS synthesis and biomass of Acinetobacter sp. B-7005 on energy-deficient substrate (acetate) it was found that molar ratio for the concentrations of sodium acetate and oil in the mixture, at which the maximum EPS synthesis was achieved, should be 1:0.13. It was experimentally confirmed that at this ratio of monosubstrate concentrations and the use of the inoculum grown on refined oil, the synthesis rates of ethapolan were higher than at the other ratios of acetate and oil concentrations in the mixture. However, the assimilation of sodium acetate through the symport with proton led to an increase in pH of the culture liquid to 9.0−9.3, which is not optimal for EPS synthesis. Decrease of the medium alkaline component and fractional introduction of substrates enabled not only to stabilize pH at the level of 7.8−7.9, but to increase the amount of synthesized ethapolan to 16−17 g/l, which was achieved regardless of the type of used oil (refined or mixed waste) in the mixture with acetate. Метою роботи було встановити умови культивування Acinetobacter sp. ІМВ В-7005, які б забезпечували максимальні показники синтезу мікробного екзополісахариду (ЕПС) етаполану на суміші ацетату та соняшникової олії, а також дослідити можливість заміни рафінованої олії в суміші з ацетатом на відпрацьовану. Оптимальне молярне співвідношення концентрацій рафінованої соняшникової олії та ацетату в суміші розраховували теоретично згідно з концепцією «допоміжного субстрату» Бабеля. Концентрацію ЕПС визначали ваговим методом після осадження ізопропанолом, ЕПС-синтезувальну здатність — як відношення концентрації ЕПС до біомаси та виражали у г ЕПС/г біомаси. На основі теоретичних розрахунків енергетичних потреб синтезу ЕПС і біомаси Acinetobacter sp. ІМВ В-7005 на енергетично дефіцитному субстраті (ацетат) встановлено, що молярне співвідношення концентрацій ацетату натрію та олії в суміші, за якого досягається максимальний синтез ЕПС, має становити 1:0,13. Експериментально підтверджено, що за даного співвідношення концентрацій моносубстратів та з використанням інокуляту, вирощеного на рафінованій олії, показники синтезу етаполану були вищими, ніж за інших співвідношень концентрацій ацетату та олії в суміші. Проте асиміляція ацетату натрію симпортом з протоном призводила до підвищення рН культуральної рідини до 9,09,3, що є неоптимальним для синтезу ЕПС. Зниження вмісту лужної складової середовища та дробне внесення субстратів дало змогу не лише стабілізувати рН на рівні 7,87,9, а й підвищити кількість синтезованого етаполану до рівня 1617 г/л, якого було досягнено незалежно від типу використаної олії (рафінованої або змішаної відпрацьованої) в суміші з ацетатом.Документ Production of exopolysaccharide ethapolan by Acinetobacter sp. IMV B-7005 on fried oil and oil-containing mixed substrates(2020) Voronenko, Andryi; Ivahniuk, Mykola; Pirog, TatianaNowadays, the majority of microbial EPS are not produced on an industrial scale because of high cost and low yield of the target product. In this study, the cultivation conditions of Acinetobacter sp. IMV B–7005 for the efficient process of exopolysaccharide ethapolan synthesis on refined or cheaper waste oil and oil–containing mixed substrates were established. The production efficiency on the selected substrates was evaluated by the amount of synthesized ethapolan (g/l) and EPS–synthesizing ability (g EPS/g biomass). Regardless of the quality (sunflower, corn, olive, rapeseed) and type (after frying meat or potatoes) of waste oil in the biosynthesis medium, it was found that the polysaccharide synthesis and its rheological properties were at the level obtained using the refined substrate. Use of waste oil, especially mixed one, in mixture with molasses or acetate allows increasing the amount of synthesized EPS to 14–16 g/l. The obtained results show the possibility of developing a universal technology of ethapolan production on the oil–containing substrates and their mixtures, which is independent of the type and quality of the waste oil, as well as its supplier.