Перегляд за Автор "Pirog, Tatiana"

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A method for determining the mass-molecular composition of microbial exopolysaccharides
(1993) Votselko, S.; Pirog, Tatiana; Malashenko, Yuri; Grinberg, Tamara
A method of determining the molecular mass composition of microbial exopolysaccharides (EPS) by centrifuging them in a combined density gradient created by NaCl and CsCl solutions and using the molecular mass of dextranes as standards is developed. The process of determining the molecular mass distribution pattern of EPS is simplified and made considerably less time-consuming. This method allows the analysis of native EPS with molecular masses ranging from 13 700 to 2 000 000.
A two-stage cultivation technique for producing microbial exopolysaccaride ethapolan with improved rheological properties
(2001) Pirog, Tatiana; Malashenko, Yuri; Votselko, S.
A two-stage technique was proposed for cultivating producers of microbial exopolysaccharide ethapolan. The practical value of ethapolan is determined by its rheological properties. The use of a formaldehyde-supplemented medium at the second stage of cultivation improved the rheological properties of ethapolan without reducing its yield. This effect of formaldehyde was due to its binding to the exopolysaccharide, which altered the molecular-weight characteristics of the latter and protected cells against the toxic action of formaldehyde. At all stages of its purification, ethapolan had improved rheological properties, suggesting that it was tightly bound to formaldehyde.
Antimicrobial activity of a mixture of surfactants produced by Acinetobacter calcoaceticus IMV B-7241 with antifungal drugs and essential oils
(2022) Pirog, Tatiana; Kliuchka, Igor; Kliuchka (Nykytyuk), Lilia
Introduction. The aim of the work was to study the effect of a mixture of surfactants synthesized by Acinetobacter calcoaceticus IMV B-7241 under various cultivation conditions with antifungal drugs (clotrimazole and fluconazole) and essential oils (cinnamon and lemongrass) on yeast of genus Candida. Material and methods. The cultivation of A. calcoaceticus IMV B-7241 was carried out in a basic medium that did not contain NaCl (medium 1), contained NaCl, 2.0 g/l (medium 2), contained NaCl, 2.0 g/l, and KCl, 1.0 g/l (medium 3). The surfactants were extracted from supernatant of cultural liquid by modified Folch mixture. Antimicrobial properties of the surfactants, antifungal drugs and essential oils were determined by index of the minimum inhibitory concentration (MIC). To assess the synergistic effect of a mixture of surfactants with antifungal drugs or essential oils the fractional inhibitory concentration index was used. Results and discussion. Surfactants synthesized by A. calcoaceticus IMV B-7241 on the basic medium were the most effective antimicrobial agents against the yeasts strains Candida albicans D-6, C. tropicalis RE-2 and C. utilis BVS-65 with MIC 22.5–45 μg/ml that were 2.6–17 times lower than the values determined for surfactants synthesized on modified media. At the same time, regardless of the strain cultivation in different media, all surfactants showed synergism of antifungal activity with clotrimazole, fluconazole, cinnamon or lemongrass essential oils. Thus, in the presence of surfactants synthesized on basic and modified media in a mixture with antifungal drugs, MIC of clotrimazole and fluconazole against the studied yeast test cultures decreased by 4–32 times. The use of a mixture of essential oils with surfactants synthesized by A. calcoaceticus IMV B-7241 growing in different media made it possible to reduce MIC of cinnamon and lemongrass oils against yeasts of Candida genus 4–18 and 8–32 times, respectively. At the same time, the index of fractional inhibitory concentration did not exceed 0.5, which indicates the synergism of anifungal activity between the studied compounds. Conclusion. The results confirm the possibility to reduce the minimum inhibitory concentrations of antifungal drugs or essential oils against members of genus Candida by their mixture with microbial surfactants.
Antimicrobial activity of exocellular metabolites of Acinetobacter Calcoaceticus IMB B-7241, Rhodococcus Erythropolis IMB Ac-5017, Nocardia
(2012) Konon, Anastasia; Sofіlkanich (Morozova), Anna; Pirog, Tatiana; Beregova (Pokora), Khrystyna; Chebotaryova, Ksenia
It was showed that exocellular metabolites of strains IMV B-7241, IMV Ac-5017 and K-8 inherent antimicrobial properties against some phytopathogenic microorganisms. Therefore, these preparations can be used as environmentally safe antimicrobial products, which exhibit high efficiency against a number of pathogenic bacteria resistant to existing traditional preparations.
Antimicrobial activity of surfactances of bacteria Nocardia, Rhodococcus and Acinetobacter genera
(2021) Pirog, Tatiana; Kliuchka, Igor; Lutsay, Dariya; Kliuchka (Nykytyuk), Lilia; Skrotska, Oksana
It was found that the minimum inhibitory concentrations against bacteria and yeast of Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Ac-5017 and Nocardia vaccinii IMV B-7405 surfactants, synthesized on traditional substrates, were 9–120 μg/ml and were within the limits defined for the surfactants known in the world. It was for the first time established that surfactants synthesized by the study strains on wastes of biodiesel production and fried sunflower oil were characterized by high antimicrobial activity against bacteria and yeast (minimum inhibitory concentrations 0.45–120 and 1.9–142 μg/ml respectively). It was found that the added of both live and inactivated Escherichia coli ІEM-1 and Bacillus subtilis BT-2 cells in R. erythropolis IMV Ac-5017 and N. vaccinii IMV B-7405 medium cultivation was accompanied by synthesis of surfactants, minimum inhibitory concentrations of which were several times lower than those showed for surfactants synthesized without competitive microorganisms. The obtained results indicate the possibility of using the studied surfactants as effective antimicrobial agents.
Antimicrobial activity of surfactants of microbial origin
(2019) Pirog, Tatiana; Lutsay, Dariya; Kliuchka (Nykytyuk), Lilia; Beregova (Pokora), Khrystyna
The recent literature data about the antibacterial and antifungal activity of microbial surfactants (lipopeptides synthesized by representatives of genera Bacillus, Paenibacillus, Pseudomonas, Brevibacillus, rhamnolipids of bacteria Pseudomonas, Burkholderia, Lysinibacillus sp., sophorolipids of yeasts Candida (Starmerella) and Rhodotorula), and our own experiments data concerning antimicrobial activity of surfactants synthesized by Acinetobacter calcoaceticus IMB B-7241, Rhodococcus erythropolis IMB Aс-5017 and Nocardia vaccini IMV B-7405 were presented. The analysis showed that lipopeptides were more effective antimicrobial agents compared to glycolipids. Thus, the minimum inhibitory concentrations (MIC) of lipopeptides, ramnolipids and sophorolipids are on average (μg/ml): 1–32, 50–500, and 10–200, respectively. The MIC of surfactants synthesized by the IMV B-7241, IMV Ac-5017 and IMV B-7405 strains are comparable to those of the known microbial lipopeptides and glycolipids. The advantages of glycolipids as antimicrobial agents compared with lipopeptides were the possibility of their synthesis on industrial waste and the high concentration of synthesized surfactants. The literature data and our own results indicate the need to study the influence of microbes’ cultivation conditions on the antimicrobial activity of the final product. Останні літературні дані про антибактеріальну та протигрибкову активність мікробних поверхнево-активних речовин (ліпопептидів, синтезованих представниками родів Bacillus, Paenibacillus, Pseudomonas, Brevibacillus, рамноліпідів бактерій Pseudomonas, Burkholderia, Lysinibacillus sp., софороліпідів дріжджів Candida (Starmerella) і Rhodotorula), та наведено дані власних експериментів щодо антимікробної активності поверхнево-активних речовин, синтезованих Acinetobacter calcoaceticus IMB B-7241, Rhodococcus erythropolis IMB Aс-5017 та Nocardia vaccini IMV B-7405. Аналіз показав, що ліпопептиди є більш ефективними протимікробними агентами порівняно з гліколіпідами. Так, мінімальні інгібуючі концентрації (МІК) ліпопептидів, рамноліпідів і софороліпідів становлять у середньому (мкг/мл): 1–32, 50–500 і 10–200 відповідно. МІК ПАР, синтезованих штамами ІМВ В-7241, ІМВ Ас-5017 та ІМВ В-7405, можна порівняти з показниками відомих мікробних ліпопептидів і гліколіпідів. Перевагами гліколіпідів як протимікробних засобів порівняно з ліпопептидами є можливість їх синтезу на промислових відходах і висока концентрація синтезованих ПАР. Дані літератури та власні результати свідчать про необхідність вивчення впливу умов культивування мікробів на антимікробну активність кінцевого продукту.
Application of surface-active substances produced by Nocardia vaccinii ІМВ В-7405 for the treatment of vegetables
(2019) Pirog, Tatiana; Beregova (Pokora), Khrystyna; Geichenko, Bogdana; Stabnikov, Victor
Application of surface-active substances (SAS) produced by Nocardia vaccinii ІМV В-7405 for shelf live extension of vegetables was studied.Materials and methods. Organic vegetables such as tomato, cucumbers, and squashes were washed with the solution of SAS produced by N. vaccinii ІМV В-7405 with concentrations of 0.25 or 0.5 g/L. Microbiological analysis was done before the beginning of the vegetables storage. Evaluation of vegetable quality was conducted by viewing during time of the storage. Results and discussions. The results of our research showed the efficiency of the application of biosurfactant produced by Nocardia vaccinii ІМV В-7405 using industrial wastes for shelf life extension of vegetables. Results of visible observations as well as microbiological analysis showed that the treatment of vegetables with SAS solutions at the concentrations of 0.25 and 0.5 g/L was more effective than washing them with tap water. The total number of heterotrophic bacteria and fungi in the samples decreased after treatment of vegetables with SAS of N. vaccinii ІMV B-7405 by 16–34 and 3–14 times, respectively, meanwhile the washing of vegetables with tap water decreased total microbial number only by 2–2.5 times. It was shown that vegetables washed with water spoiled faster than those treated with SAS solution. The advantages of application of this biosurfactant for vegetables post-harvest treatment are that (1) it can be used at the lower by 2–6 times concentration in comparison with other reported in literature microbial SAS, and (2) it can be produced using industrial wastes that will reduce the cost of its production.Conclusion. Biosurfactant produced by Nocardia vaccinii ІМV В-7405 can be used for the treatment of vegetables to extent their shelf life. Досліджено застосування поверхнево-активних речовин (ПАР) Nocardia vaccinii ІМV В-7405 для подовження терміну зберігання овочів. Матеріали і методи. Органічні овочі, такі як помідори, огірки, кабачки, мили розчином ПАР виробництва N. vaccinii ІМВ В-7405 з концентрацією 0,25 або 0,5 г/л. Мікробіологічне дослідження проводили перед початком зберігання овочів. Оцінку якості овочів проводили шляхом огляду протягом часу зберігання. Результати та обговорення. Результати наших досліджень показали ефективність застосування біосурфактанту виробництва Nocardia vaccinii ІМВ В-7405 з використанням промислових відходів для подовження терміну зберігання овочів. Результати наочних спостережень, а також мікробіологічного аналізу показали, що обробка овочів розчинами ПАР у концентрації 0,25 і 0,5 г/л була більш ефективною, ніж миття їх водопровідною водою. Загальна кількість гетеротрофних бактерій та грибів у зразках зменшувалася після обробки овочів ПАР N. vaccinii ІМВ В-7405 у 16–34 та 3–14 разів відповідно, тоді як миття овочів водопровідною водою зменшувало загальну мікробну кількість. лише в 2–2,5 рази. Показано, що вимиті водою овочі псувалися швидше, ніж оброблені розчином ПАР. Переваги застосування цього біосурфактанту для післязбиральної обробки овочів полягають у тому, що (1) його можна використовувати в меншій у 2–6 разів концентрації порівняно з іншими мікробними ПАР, зареєстрованими в літературі, та (2) його можна отримувати з використанням промислових відходів, що здешевить його виробництво. Висновок. Біосурфактант Nocardia vaccinii ІМV В-7405 можна використовувати для обробки овочів з метою збільшення терміну їх зберігання.
Application of surface-active substances produced by Rhodococcus erythropolis IMB Aс-5017 for post-harvest treatment of sweet cherry
(2022) Pirog, Tatiana; Stabnikov, Victor; Antoniuk, Svitlana
Introduction. The aim of the present study was testing of the supernatant of Rhodococcus erythropolis ІМВ Ас-5017 with different concentration of surface-active substances (SAS) for treatment of sweet cherry for shelf-life extension. Materials and methods. R. erythropolis ІМВ Ас-5017 were grown in the medium with ethanol. Supernatant with concentration of SAS from 0.1 to 0.5 g/L was used for the treatment of sweet cherry fruit. Concentration of SAS in supernatant was determined by weight method. The total number of heterotrophic bacteria and fungi were determined by the plate dilution method. Results and discussion. The treatment of sweet cherries with a supernatant containing 0.5 g/L SAS diminished the numbers of bacteria and fungi on the fruit’s surface by 10 and 5 times, respectively, in comparison with cherries washed with water. The treatment of sweet cherries with supernatant containing 0.2 g/L SAS diminished the numbers of bacteria and fungi on the fruit’s surface by 5 and 3 times, respectively; treatment with supernatant containing 0.1 g/L diminished the numbers of bacteria and fungi by 2 times in comparison with cherries washed with water. The treatment with supernatant with concentration SAS 0.5 g/L was most effective. Treated with supernatant sweet cherries fruits did not show signs of decay even on 7th day of storage, while untreated or washed with water fruits lost moisture, fruit’s skin became wrinkled, cracks and decayed areas appeared on it. Content of fungal cells on the surface of sweet cherry pretreated with supernatant with concentration of SAS from 0.1 to 0.5 g/L and after that contaminated with spore’s suspension of Aspergillus niger Р-3 were by 2 – 11 times lower than on the surface of fruits washed with water after 5 days of incubation. The possibility of multiple usage of supernatant was shown. Application of supernatant with concentration of 0.5 g/L resulted in decrease of bacterial concentration after first usage by 10 times, after second usage it was diminished by 5 times and after third usage it was diminished by 3 times, meanwhile concentration of fungi decreased by 9, 5 and 4 times after I, II, and III usage of supernatant. Conclusion. Surface-active substances synthesized by Rhodococcus erythropolis IMB Ac-5017 could be used for treatment of sweet cherry to extend their shelf life.
Biological activity of Acinetobacter calcoaceticus IMV B-7241 surfactants synthesized in the presence of competitive bacteria Bacillus subtilis BT-2
(2023) Pirog, Tatiana; Ivanov, Mykyta; Shevchuk, Tetiana
Currently, the effectiveness of technologies of microbial surfactant, which are characterized by a complex of practically valuable physicochemical and biological properties, is lower than that of synthetic analogues. To reduce the cost of these products of microbial synthesis, industrial waste is used as substrates for their biosynthesis. In previous studies, it was established that surfactants synthesized by Acinetobacter calcoaceticus IMV B-7241 on crude glycerol had lower antimicrobial activity compared to those obtained on purified glycerol. The main approaches to the regulation of the biological activity of microbial surfactants are their post-fermentation chemical modification, as well as the improvement of producer strains by methods of metabolic and genetic engineering. In recent years, the great amount of studies have appeared on the co-cultivation of producers of antimicrobial compounds with competitive microorganisms (biological inductors), in response to the presence of which the antimicrobial activity of the final product increases. Aim. To study the effect of live and inactivated cells of Bacillus subtilis BT-2, as well as the corresponding supernatant, on the antimicrobial, anti-adhesive activity and the ability to destroy biofilms of A. calcoaceticus IМV B-7241 surfactants, synthesized in a medium with glycerol of different degrees of purification. Methods. The IMV B-7241 strain was grown in the liquid mineral medium with purified and crude glycerol, into which live and inactivated B. subtilis BT-2 cells, as well as the supernatant after growing the BT-2 strain (2.5−10%, v/v) were added. Surfactants were extracted from the supernatant of the culture liquid with Folch's mixture. Anti-adhesive activity and the degree of destruction of biofilms were determined by the spectrophotometric method, antimicrobial activity − by the indicator of the minimum inhibitory concentration. The activity of enzymes of surface-active aminolipids biosynthesis (NADP+-dependent glutamate dehydrogenase) and glycolipids (phosphoenolpyruvate (PEP)-carboxylase, PEP-synthetase, PEP-carboxykinase, trehalose-phosphate synthase) was analyzed in cell-free extracts obtained after сells sonication. Results. It was established that the introduction of inactivated B. subtilis BT-2 cells and supernatant into the medium with both substrates did not affect the indicators of the surfactants synthesis, while in the presence of live cells of the BT-2 strain in the medium with purified glycerol, a decrease in the concentration of the final product by 1.5 times, and in the culture medium with crude glycerol - an increase of 1.4 times were observed compared to the indicators without the inductor. The study of the antimicrobial activity of surfactants showed that the most effective of the used inductors (live, inactivated cells, supernatant) were live cells of B. subtilis BT-2. The introduction of BT-2 strain live cells into the culture medium with both substrates was accompanied by the formation of surfactants, the minimum inhibitory concentrations of which in relation to bacterial (Bacillus subtilis BT-2, Staphylococcus aureus BMS-1, Proteus vulgaris PA-12, Enterobacter cloacae С-8 ) and yeast (Candida albicans D-6, Candida tropicalis PE-2) test-cultures were 3-23 times lower than established for those synthesized on the medium without this inductor. Anti-adhesive activity of surfactants obtained on purified and crude glycerol in the presence of all types of inductors was higher than those synthesized in the culture medium without inductors (cells adhesion of bacterial and yeast test-cultures on polyvinyl chloride was 13−70 and 33−96%, respectively). Introduction into A. calcoaceticus IMV B-7241 medium cultivation of both live and inactivated B. subtilis BT-2 cells, as well as the supernatant, was accompanied by the synthesis of surfactants in the presence of which the disruption of bacterial biofilms was on average 10-20 % higher compared to using surfactants synthesized without an inductor. In the presence of B. subtilis BT-2 in the medium, in the cells of the IMV B-7241 strain the activity of NADP+-dependent glutamate dehydrogenase (a key enzyme of aminolipids biosynthesis) increased by 1.5-2 times, while the activity of glycolipids enzymes biosynthesis remained practically at the same level as without an inductor. Such data indicated that the higher biological activity of surfactants obtained by A. calcoaceticus IMV B-7241 in the presence of biological inductors might be due to an increase in the content of aminolipids in their composition. Conclusions. As a result of research, it was established the possibility of regulating the antimicrobial and anti-adhesive activity, as well as the ability to disrupt biofilms of A. calcoaceticus IМV B-7241 surfactants by introducing into the culture medium of competitive bacteria B. subtilis BT-2. It is important that under such cultivation conditions the antimicrobial activity of surfactants synthesized on toxic crude glycerol significantly increased.
Biosurfactant of acinetobacter calcoaceticus імv в–7241: influence of Cu2+ on synthesis and use in bioremediation processes
(2012) Andrushchenko, Yaroslav; Konon, Anastasia; Antoniuk, Svitlana; Pirog, Tatiana; Parfenyuk, Sergey
The influence of Cu2+ on surfactant synthesis by Acinetobacter calcoaceticus IMV B-7241 and possibility of their using for the degradation of complex oil pollution of soil and water was studied
Biosurfactants of Rhodococcus erythropolis IMV Ас-5017: synthesis intensification and practical application
(2013) Pirog, Tatiana; Sofіlkanich (Morozova), Anna; Shulyakova, Mariya; Shevchuk, Tetiana
Intensification 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.
Biotechnological potential of the Acinetobacter genus bacteria
(2021) Pirog, Tatiana; Lutsay, Dariya; Muchnyk, Faina
Until recently, there were rare scientific reports on the biotechnological potential of non-pathogenic bacteria of the Acinetobacter genus. Although the first reports about the practically valuable properties of these bacteria date back to the 70s and 80s of the twentieth century and concerned the synthesis of the emulsan bioemulsifier. In the last decade, interest in representatives of the Acinetobacter genus as objects of biotechnology has significantly increased. The review presents current literature data on the synthesis by bacteria of this genus of high-molecular emulsifiers, low-molecular biosurfactants of glyco- and aminolipid nature, enzymes (lipase, agarase, chondroitinase), phytohormones, as well as their ability to solubilize phosphates and decompose various xenobiotics (aliphatic and aromatic hydrocarbons, pesticides, insecticides). Prospects for practical application of Acinetobacter bacteria and the metabolites synthesized by them in environmental technologies, agriculture, various industries and medicine are discussed. The data of own experimental studies on the synthesis and biological activity (antimicrobial, anti-adhesive, ability to destroy biofilms) of biosurfactants synthesized by A. calcoaceticus IMV B-7241 strain and their role in the degradation of oil pollutants, including complex ones with heavy metals, are presented. The ability of A. calcoaceticus IMV B-7241 to the simultaneous synthesis of phytohormones (auxins, cytokinins, gibberellins) and biosurfactants with antimicrobial activity against phytopathogenic bacteria allows us to consider this strain as promising for practical use in crop production to increase crop yields.
Calcium and magnesium cations influence on antimicrobial and antiadhesive activity of Acinetobacter сalcoaceticus ІMV B-7241 surfactants
(2016) Pirog, Tatiana; Sidor, Inga; Lutsay, Dariya
The aim of the work was to study the effect of calcium and magnesium cations on NADP+-dependent glutamate dehydrogenase activity (key enzyme of biosynthesis of Acinetobacter calcoaceticus ІMV B-7241 surface-active aminolipids) followed by modification of medium composition and determining antimicrobial and antiadhesive activity of synthesized surfactants. The strain IMV B-7241 was grown in medium with ethanol. NADP+-dependent glutamate dehydrogenase activity of the cell-free extract was analyzed using the formation of glutamate in the oxidation of NADPH. Surfactants were extracted from supernatant of cultural liquid by mixture of chloroform and methanol (2:1). Antimicrobial against bacteria properties of the surfactants were determined by index of the minimal inhibitory concentration. The number of attached cells and the degree of biofilm destruction were analyzed spectrophotometrically. It was established that in the presence of 10 mM Cа2+ and Mg2+ NADP+-dependent glutamate dehydrogenase activity in the cell-free extract increased to 1.5 times in comparison with that without cations. Increasing concentration of magnesium sulfate to 0.2 g/l, or adding CaCl2 (0.1 g/l) into cultivation medium of IMV B-7241 strain was accompanied by rise of NADP+-dependent glutamate dehydrogenase activity in 2.4 and 3.0 times respectively, as well as increasing antimicrobial and antiadhesive activity of synthesized surfactants. Minimal inhibitory concentration of surfactants synthesized in modified media against some bacteria was in 1.3–3.5 times, adhesion on abiotic surfaces treated with such surfactants in an average of 5–17% lower, and the degree of biofilm destruction in 7–13% higher as compared to indicators for the surfactant produced in the base medium. The obtained results indicate the possibility of regulating antimicrobial and anti-adhesive activity of surfactants under producer cultivation.
Comparative characteristics of ethapolan and xanthan exopolysaccharides as agents for the increasing secondary oil extraction
(2018) Ivahniuk, Mykola; Pirog, Tatiana
The purpose of research – to compare the cost of nutrient mediums for obtaining culture fluids of Acinetobacter sp. IMB B-7005 and Xanthomonas campestris strains (xanthan gum producers) for its using in the secondary oil extraction. Materials and methods. Calculation of the culture fluid amount and the cost of nutrients for the ethapolan (producer – Acinetobacter sp. IMB B-7005) and xanthan (producers of Xanthomonas campestris strains) exopolysaccharides production as oil extraction agents for 262 oil wells of Oil and Gas Production Administration “Okhtyrkanaftogas” of JSC “Ukrnafta” was carried out taking into account the following parameters:4 times treatment per year of wells with the 15 m3 of EPS solution with its concentration 0,05%). Results and discussions. On the basis of the data on the concentration of synthesized polysaccharides by strainsproducer, the annual need for a culture fluid (545–1849 m3) was calculated for the treatment of wells of the OGPA “Okhtyrkanaftogas” and the amount of product per fermentation cycle according to selected technologies. Theoretical calculations have shown that costs for the nutrient medium preparing for Acinetobacter sp. IMB B-7005 culture fluid obtaining on waste sunflower oil after frying meat, necessary for increasing secondary oil extraction at “Okhtyrkanaftogas” wells in 1.8–5.4 times less than in the case of ethapolan obtaining on C2-C6 substrates, molasses or their mixtures. Comparison of nutrient medium cost for the preparation of ethapolan and the most famous polysaccharide xanthan obtained on molasses on technical glycerine was made. The cost of polysaccharide xanthan obtaining (in amount that would provide oil extraction in equivalent quantity like in case of ethapolan using) in 12.7–16.7 times higher compared to the ethapolan obtaining. Conclusion. Obtained results confirm the economic feasibility of the ethapolan synthesis on waste oil for the polysaccharide using in secondary oil production in comparison with the technologies for xanthan obtaining. Мета дослідження – порівняти вартість поживних середовищ для отримання культуральних рідин Acinetobacter sp. IMB B-7005 і штами Xanthomonas campestris (продуценти ксантанової камеді) для використання у вторинній екстракції олії. Матеріали та методи. Розрахунок кількості культуральної рідини та вартості поживних речовин для виробництва екзополісахаридів етаполану (продуцент – Acinetobacter sp. IMB B-7005) та ксантану (продуценти штамів Xanthomonas campestris) як нафтовидобувних агентів для 262 нафтових свердловин Нафтогазовидобувного управління. «Охтирканафтогаз» ВАТ «Укрнафта» проводилась з урахуванням наступних параметрів: 4-разова обробка на рік свердловин 15 м3 розчину ЕПС з концентрацією 0,05%). Результати та обговорення. На підставі даних про концентрацію синтезованих полісахаридів за штамами-продуцентами розраховано річну потребу культуральної рідини (545–1849 м3) для обробки свердловин НГПА «Охтирканафтогаз» та кількість продукту за цикл бродіння за до обраних технологій. Теоретичні розрахунки показали, що витрати на приготування живильного середовища для Acinetobacter sp. Отримання культуральної рідини ІМБ В-7005 на відпрацьованій соняшниковій олії після смаження м’яса, необхідної для збільшення вторинного видобутку нафти на свердловинах «Охтирканафтогаз» в 1,8–5,4 рази менше, ніж при отриманні етаполану на субстратах С2-С6, мелясі або їх сумішах. Проведено порівняння вартості живильного середовища для приготування етаполану та найвідомішого полісахариду ксантану, отриманого на мелясі на технічному гліцерині. Вартість одержання полісахариду ксантану (у кількості, яка б забезпечила екстрагування нафти в еквівалентній кількості, як при використанні етаполану) у 12,7–16,7 разів вища порівняно з одержанням етаполану. Висновок. Отримані результати підтверджують економічну доцільність синтезу етаполану на відпрацьованих оліях для використання полісахариду у вторинному виробництві олії порівняно з технологіями отримання ксантану.
Destruction of biofilms on silicone tubes under the action of a mixture of Nocardia vaccinii IMV B-7405 surfactants with other biocides
(2021) Pirog, Tatiana; Kliuchka (Nykytyuk), Lilia; Shevchuk, Tetiana; Iutynska, Galyna
The formation of pathogenic microorganisms biofilms on the central venous catheter is the cause of catheter-associated infections. An alternative method of combating biofilms is the use of “antibacterial” and “antifungal” locks, which are solutions of antibiotics or antifungal drugs in a mixture with other natural compounds, which can be microbial surface-active substances (surfactants) or essential oils. Aim. To investigate the role of Nocardia vaccinii IMV B-7405 surfactants mixture with other antimicrobial compounds in the destruction of biofilm on silicone tubes. Methods. N. vaccinii IMV B-7405 was grown in medium containing as carbon source purified glycerol and waste from biodiesel production, refined sunflower oil, oil after frying French-fried potatoes, Potato wedges and meat. The surfactants were extracted from supernatant of cultural liquid by modified Folch mixture. 2 mL of surfactant solutions, antifungal drugs (nystatin, fluconazole) or tea tree essential oil of the same concentration (5–640 μg/mL) were added to test tubes with silicone tubes (with pre-formed biofilm from test culture). To study the synergistic effect of the biofilms destruction, a mixture of surfactant solutions and antifungal substances (or essential oil) of the same concentration in a ratio of 1:1 (1 mL of each solution) was added to the test tubes. Sterile tap water (2 mL) was added to control test tubes instead of surfactants preparations, antifungal substances or essential oil. The degree of biofilm destruction (%) was determined as the difference between the adhesion of cells on the inner side of silicone tubes, untreated and treated with surfactants, antifungal drugs, essential oil, or their mixture. Results. It was found that surfactants synthesized by N. vaccinii IMV B-7405 on all substrates showed synergism of yeast and bacterial biofilms destruction on silicone tubes in a mixture with nystatin, fluconazole and tea essential oil in the whole range of investigated concentrations (5–640 μg/mL), but the highest effect was achieved at a concentration of 20–40 μg/mL. Thus, the degree of Candida albicans D-6, Candida utilis BVS-65 and Candida tropicalis PE-2 biofilms destruction under the action of a mixture of surfactants synthesized on waste from the biodiesel production and waste oil, with antifungal drugs was 45.8–71.8 % and was higher than with only surfactants (21.2–41.6 %), nystatin (22.4–24.1 %) or fluconazole (28.1–31.3 %). The destruction of Candida genus yeast biofilms under the action of surfactants synthesized on oil-containing substrates in a mixture with both nystatin and fluconazole reached 50.1–71.2 %, which is 10–30 % higher compared to the use of surfactants alone or only antifungal agents. The degree of Pseudomonas sp. MI-2, Escherichia coli IEM-1, Staphylococcus aureus BMS-1, Bacillus subtilis BT-2 (spores) biofilms destruction on silicone tubes treated with a mixture of tea tree essential oil and surfactants synthesized on all oil-containing substrates was 10–29 % higher than in the case of using only solutions of surfactants (11.5–45.4 %) or essential oil (21.4–34.5 %) for the tubes treatment. Conclusions. The data obtained make it possible to consider surfactants synthesized by N. vaccinii IMV B-7405 on a wide range of cheap and accessible substrates as promising components of “antibacterial” and “antifungal” locks in combination with essential oils and antifungal agents.
Destruction of biofilms under the influence of Acinetobacter calcoaceticus IMV B-7241 surfactants, synthesized in the presence of competitive microorganisms
(2022) Pirog, Tatiana; Ivanov, Mykyta
Introduction. The aim of this study was to investigate the role of surfactants synthesized by Acinetobacter calcoaceticus IMV B-7241 in media with glycerol in the presence of biological inductors in destruction of biofilms. Materials and methods. Cultivation of A. calcoaceticus IMV B-7241 was carried out in a mineral medium using refined glycerol or crude glycerol, the waste of biodiesel production, as carbon sources. Biological inductors were introduced as live or inactivated cells of Bacillus subtilis BT-2, as well as the supernatant after strain BT-2 cultivation. Surfactants were extracted from the supernatant of the culture liquid with a modified mixture of Folch (chloroform and methanol, 2:1). The degree of biofilm destruction in the presence of surfactants was determined by spectrophotometric method. Results and discussion. Regardless of the substrate used, the introduction of both live and inactivated cells of B. subtilis BT-2 into medium used for cultivation of A. calcoaceticus IMV B-7241 was accompanied by the synthesis of surfactants, the degree of biofilm destruction of which was higher than those obtained in the medium without an inductor. The degree of destruction of bacterial and yeast biofilms achieved by the action of A. calcoaceticus IMV B-7241 surfactants obtained on refined glycerol in the presence of inductor cells was 36.5–85% and was 1.5-3 times higher compared to using surfactants synthesized in medium without inductors. Note that, surfactants synthesized in the presence of biological inductors destroyed biofilms of the test cultures at fairly low (7.5–960 μg/ml) concentrations. Similar results were observed for the usage of surfactants obtained on the waste of biodiesel production. Therefore, introduction of live cells of B. subtilis BT-2 into the medium with the crude glycerol was accompanied by synthesis of surfactants, which at concentration 1.8-960 μg/ml caused destruction of B. subtilis BT-2, Proteus vulgaris PA-12 and Enterobacter cloacae C-8 biofilms at 30.1–80.7% and was higher than using similar surfactant concentrations obtained during cultivation without inductors (24.1–75%). The destruction of biofilms of Staphylococcus aureus BMS-1, Candida albicans D-6 and Candida tropicalis PE-2 under the action of surfactants (1.8-960 μg/ml) synthesized on crude glycerol in the presence of both live or inactivated cells of B. subtilis BT-2 was 1.5–8 times higher than surfactants synthesized in medium without inductor. Conclusion. The possibility to regulate the ability to destroy bacterial and yeast biofilms of surfactants synthesized by A. calcoaceticus IMV B-7241 by introducing into the medium competitive bacteria B. subtilis BT-2 was found.
Effect of cations on the activity of NADP+-dependent glutamate dehydrogenase in Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Ac-5017 and Nocardia vaccinii IMV B-7405 grown on industrial waste
(2021) Pirog, Tatiana; Paliichuk, Olesia; Lutsay, Dariya; Kliuchka (Nykytyuk), Lilia; Shevchuk, Tetiana
Introduction. It is studied the activity of NADP+-dependent glutamate dehydrogenase in the presence of mono- and divalent cations (potential activators of this key enzyme of surface-active aminolipids biosynthesis) in A. calcoaceticus IMV B-7241, R. erythropolis IMV Ac-5017 and N. vaccinii IMV B-7405 during cultivation on waste of biodiesel production and sunflower oil waste. Materials and methods. Cultivation of strains was performed in liquid mineral media using as substrates: refined and waste (after frying potato) sunflower oil, refined glycerol and waste of biodiesel production. NADP+-dependent (EC 1.4.1.4) glutamate dehydrogenase activity in cell-free extracts was analyzed for glutamate formation during oxidation of NADPH at 340 nm. Monovalent (Na+, K+) and divalent (Mg2+, Ca2+, Zn2+) cations in the form of salts of NaCl, KCl, MgSO4 × 7H2O, CaCl2 and ZnSO4 × 7H2O were added to the reaction mixture, as well as into the medium for strains cultivation. Results and discussion. Calcium cations were found to be activators of NADP+-dependent glutamate dehydrogenase activity in R. erythropolis IMV Aс-5017 and N. vaccinii IMV B-7405 grown on refined and waste sunflower oil: in the presence of 1–5 mmol Ca2+ in the mixture, the activity of the enzyme increased 1.3–2 times compared with that without these cations. The increase in the concentration of CaCl2 to 0.2−0.4 g/l in oil-containing medium of strains IMV Ac-5017 and IMV B-7405 cultivation was accompanied by an increase in NADP+-dependent glutamate dehydrogenase activity by 1.3–1.5 times compared with that on basic medium. When additional quantity of CaCl2 (0.1−0.2 g/l) was introduced into the medium with purified glycerol for the cultivation of A. calcoaceticus IMV B-7241, an increase in NADP+-dependent glutamate dehydrogenase activity was observed by almost 2.5−3 times compared with those for strain IMV B-7241 on the basic medium. There was no impact of activating cations magnesium, zinc, potassium and sodium on NADP+-dependent glutamate dehydrogenase activity of all strains grown on oil-containing substrates and glycerol of different degrees of purification. Conclusion. The results demonstrate the possibility to increase activity of key enzymes of the biosynthesis of the desired product: the composition of the medium should be modified by changing the content of enzymes’ activators.
Effect of environmental factors on the synthesis and properties of Acinetobacter sp. exopolysaccharides
(1998) Pirog, Tatiana; Grinberg, Tamara; Malashenko, Yuri
Effects 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.
Effect of tryptophane on synthesis of certain exometabolites by bacteria of genus Acinetobacter, Nocardia, and Rhodococcus and their properties
(2022) Pirog, Tatiana; Piatetska, Daria; Zhdanyuk, Valentina; Leonova, Natalia; Shevchuk, Tetiana
The efficiency of integrated microbial biotechnologies for obtaining several practically valuable metabolites in one technological process is determined both by the maximum concentration of these substances and their properties. This is especially true for secondary metabolites, the composition and properties of which vary depending on the cultivation conditions of the producer. Aim. To research the eff ect of tryptophan (a precursor of auxin biosynthesis) in the culture media on the synthesis of certain exometabolites by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241, and Nocardia vaccinii IMV B-7405 as well as their properties. Methods. R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-724, and N. vaccinii IMV B-7405 were cultivated in a medium containing refi ned and waste sunflower oil, biodiesel waste, or ethanol as a carbon source. The concentration of tryptophan in the medium was 300 mg/L. Surfactants were extracted from the supernatant of the cultural liquid with a modified Folch mixture. Phytohormones were isolated from the supernatant by sequential extraction with organic solvents after surfactant extraction. Thin-layer chromatography was used for preliminary purification and concentration of phytohormones. Qualitative and quantitative determination of auxins was performed using high-performance liquid chromatography. The antimicrobial activity of surfactants was analysed by the minimum inhibitory concentration. The activity of enzymes of surface-active glycoand aminolipids biosynthesis (phosphoenolpyruvate synthetase, phosphoenolcarboxykinase, and NADP+-dependent glutamate dehydrogenase) was determined spectrophotometrically during the oxidation of NADH or NADP. Results. It was found that the presence of tryptophan in the culture medium of the strains under study did not affect the number of synthesized surfactants, which was 1.80−1.90, 1.55−1.75, and 1.50−1.65 g/L, respectively. At the same time, cultivation of R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-724, and N. vaccinii IMV B-7405 in the media with tryptophan increased the number of phytohormones: it was higher than the amount of phytohormones synthesized during cultivation without a precursor. The introduction of tryptophan into the culture medium of the strains was accompanied by the formation of surfactants. These compounds showed 2−4 times higher antimicrobial activity against the phytopathogenic bacteria (Agrobacterium tumefaciens UCM B-1000, Pseudomonas syringae UCM B-1027T, Xanthomonas vesicatoria UCM B-1106, Pectobacterium carotovorum UCM B-1075T, Clavibacter michiganensis IMV B-102 and Pseudomonas syringae pv. tomato IMV B-9167) than compounds synthesized on a medium without a precursor. The antimicrobial activity of surfactants synthesized by A. calcoaceticus IMV B-7241 in the presence of tryptophan either did not change compared to that for surfactants obtained without tryptophan, or increased slightly. Data on the activity of surfactant biosynthesis enzymes correlated with the indicators of their antimicrobial activity. In the presence of tryptophan in the culture medium of N. vaccinii IMV B-7405 and R. erythropolis IMV Ac-5017, NADP+-dependent glutamate dehydrogenase activity in the cells of these strains (a key enzyme for biosynthesis of aminolipids responsible for antimicrobial activity) increased almost by 1.4 times compared to that on a tryptophan-free medium. Conclusions. As a result of this work, it was found that the presence of tryptophan in the culture medium of researched strains did not affect the number of surfactants. The antimicrobial activity of surfactants against phytopathogenic bacteria either increased or remained unchanged compared to that established for surfactants synthesized without a precursor of auxin biosynthesis. The obtained data testify to the high efficiency of the potential use of surfactants complex preparations and phytohormones in crop production to stimulate the growth of plants and biocontrol of phytopathogenic bacteria.
Effect on phytopathogenic microorganisms of surfactants of microbial origin
(2021) Pirog, Tatiana; Piatetska, Daria; Yarova, Hanna; Iutynska, Galyna
Biodegradable non-toxic surfactants of microbial origin are multifunctional preparations, which due to antimicrobial activity are promising for use in crop production to control phytopathogenic microorganisms. Studies on the prospects of using microbial surfactants to control the number of phytopathogenic microorganisms are conducted in three directions: laboratory studies of antimicrobial activity of surfactants in vitro, determination of the effect of surfactants on phytopathogens in vegetative experiments in the process of plants growing in a laboratory or greenhouse, post-harvest treatment of fruits and vegetables with solutions of microbial surfactants to extend their shelf life. The review presents literature data on antimicrobial activity of surfactants against phytopathogenic bacteria and fungi in vitro. Antimicrobial activity of surfactants is evaluated by three main parameters: minimum inhibitory concentration, zones of growth retardation of test cultures on agar media and inhibition of growth of test cultures on agar or liquid media. The vast majority of available publications relate to the antifungal activity of surfactant lipopeptides and rhamnolipids, while data on the effect of these microbial surfactants on phytopathogenic bacteria (representatives of the genera Ralstonia, Xanthomonas, Pseudomonas, Agrobacterium, Pectobacterium) are few. The researchers determined the antimicrobial activity of either total lipopeptides extracted with organic solvents from the culture broth supernatant, or individual lipopeptides (iturin, surfactin, fengycin, etc.) isolated from a complex of surfactants, or culture broth supernatant. Lipopeptides synthesized by members of the genus Bacillus exhibit antimicrobial activity on phytopathogenic fungi of the genera Alternaria, Verticillium, Aspergillus, Aureobasidium, Botrytis, Rhizoctonia, Fusarium, Penicillium, Phytophora, Sclerotinia, Curvularia, Colletotrichum, etc. in sufficiently high concentrations. Thus, the minimum inhibitory concentrations of lipopeptides against phytopathogenic fungi are orders of magnitude higher (in average 0.04–8.0 mg/mL, or 40–8000 μg/mL) than against phytopathogenic bacteria (3–75 μg/mL). However, the antifungal activity of lipopeptidecontaining supernatants is not inferior by the efficiency to the activity of lipopeptides isolated from them, and therefore, to control the number of phytopathogenic fungi in crop production, the use of lipopeptidecontaining supernatants is more appropriate. Rhamnolipids synthesized by bacteria of the genus Pseudomonas are more effective antimicrobial agents comparing to lipopeptides: the minimum inhibitory concentrations of rhamnolipids against phytopathogenic fungi are 4–276 μg/mL, which is an order of magnitude lower than lipopeptides. In contrast to the data on the antifungal activity of rhamnolipids against phytopathogens, there are only a few reports in the literature on the effect of these surfactants on phytopathogenic bacteria, whilst the minimal inhibitory concentrations are quite high (up to 5000 μg/mL). The advantage of rhamnolipids as antimicrobial agents compared to lipopeptides is the high level of synthesis on cheap and available in large quantities industrial waste. Currently in the literature there is little information about the effect of surface-active sophorolipids of microbial origin on phytopathogenic fungi, and all these works are mainly about the antifungal activity of sophorolipids. We note that in contrast to surfactant lipopeptides and rhamnolipids, the effective concentration of most sophorolipids, which provides the highest antimicrobial activity against phytopathogens, is higher and reaches 10,000 μg/mL.