Перегляд за Автор "Shevchuk, Tetiana"

Зараз показуємо 1 - 20 з 25

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.
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.
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.
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 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.
Glycerol metabolism in producers of surface-active substances rhodococcus erythropolis імv ас-5017, acinetobacter calcoaceticus імv в-7241 and nocardia vaccinіi к-8
(2012) Mashenko, Oksana; Shulyakova, Mariya; Pirog, Tatiana; Shevchuk, Tetiana
It was established that the strains Rhodococcus erythropolis ІМВ Ас-5017, Acinetobacter calcoaceticus ІМВ В-7241 and Nocardia vaccinіi К-8 metabolize glycerol to dihydroxyacetonephosphate via two known pathways (glycerol-3-phosphate pathway and dihydroxyacetone pathway). The data obtained could be the basis for theoretical calculations of the energy needs for surfactants and biomass synthesis on this substrate to determine the optimal molar ratio of energy unequivalent substrates in order to increase the synthesis of surfactants of R. erythropolis ІМV Ас-5017, A. calcoaceticus IMV B-7241 and N. vaccinii K-8 on the mixture of substrates.
Induction of auxins synthesis by Rhodococcus erythropolis IMV Ac-5017 with the addition of tryptophan to the cultivation medium
(2020) Pirog, Tatiana; Leonova, Natalia; Piatetska, Daria; Klymenko, Natalia; Zhdanyuk, Valentina; Shevchuk, Tetiana
The ability of surfactant producers to synthesize phytohormones expands the scope of their practical application and provides prospects for the development of microbial preparations with growth-stimulating properties. The possibility to intensify the phytohormone-stimulants synthesis by bacterial strains increases the efficiency of such preparations. Aim. The aim is to research the possibility of extracellular auxin synthesis induction in the presence of tryptophan in the cultivation medium of surfactant producer Rhodococcus erythropolis IMV Ac-5017 and establish the optimal concentration of tryptophan and time of introduction into the medium to ensure maximum synthesis of auxins. Methods. Biochemical, microbiological, biotechnological. Cultivation was performed in the liquid mineral medium using ethanol and waste sunflower oil as substrates. Tryptophan was added to the medium as a 1 % solution in an amount of 200 or 300 mg/l at the beginning of the cultivation process or at the end of the exponential growth phase. Phytohormones were isolated by triple extraction with organic solvents from the culture broth supernatant after surfactant extraction. Preliminary purification and concentration of phytohormones was performed by thin layer chromatography. Qualitative and quantitative determination of auxins was performed using high performance liquid chromatography. Results. It was found that regardless of the concentration and time of tryptophan introduction to the culture medium of R. erythropolis IMV Ac-5017 with both substrates, a significant increase (by two to three orders of magnitude) was observed in the amount of synthesized auxins compared to tryptophan-free medium. The highest concentration of auxins (5552–5634 μg/l) was achieved by adding 300 mg/l of tryptophan into the culture medium of R. erythropolis IMV Ac-5017 with ethanol, while without the precursor their amount was only 143 μg/l. In contrast to the cultivation of the strain on culture medium with ethanol, where the synthesis of auxins did not depend on the time of tryptophan introduction, R. erythropolis IMV Ac-5017 formed the maximum amount of auxins when 300 mg/l tryptophan was added to the culture medium with waste oil at the end of the exponential growth phase (2398 μg/l compared to 9.8 μg/l on the medium without tryptophan). As auxin compounds were identified: indole-3-acetic acid, indole-3-carboxylic acid and indole-3-butyric acid. However, the highest amount of indole-3-acetic acid was synthesized, the precursor of which is tryptophan. The synthesis of this auxin (the most common plant auxin) in the presence of 300 mg/l of tryptophan increased more than 40 times on ethanol medium and more than 700 times on medium with waste oil. Induction of auxin synthesis by strain R. erythropolis IMV Ac-5017 correlated with the activity of tryptophan transaminase: when cultured on ethanol without tryptophan, it was 138 nmol•min-1•mg-1 of protein, while cultured in the presence of precursor it was increased by 5.2 times (up to 714 nmol•min-1•mg-1 of protein). The obtained results suggest that indole-3-acetic acid biosynthesis by the strain IMV Ac-5017 occurs due to the formation of indole-3-pyruvate. Conclusions. Thus, it was established the possibility of increasing by two or three orders the amount of synthesized auxins in the case of low concentrations of tryptophan introducing to the culture medium of R. erythropolis IMV Ac-5017 not only with ethanol but also with industrial waste (waste oil). The obtained results can be considered as promising for use of exometabolites of R. erythropolis IMV Ac-5017 with growth-stimulating properties in crop production.
Influence of tryptophan on auxin-synthesizing ability of surfactant producer Acinetobacter calcoaceticus IMV B-7241
(2020) Pirog, Tatiana; Leonova, Natalia; Piatetska, Daria; Klymenko, Natalia; Shevchuk, Tetiana
Introduction. The aim of this work is to determine the optimal tryptophan concentration and the time of its addition into the culture medium of the surfactant producer Acinetobacter calcoaceticus IMV B-7241 to achieve maximum auxin synthesis. Materials and methods. Cultivation was carried out on a liquid nutrient mineral medium using as a substrate of ethanol and waste of biodiesel production (crude glycerol). Tryptophan was added into the medium as a 1% solution in an amount of 100, 200 or 300 mg/L at the beginning of the process or at the end of the exponential growth phase (48 h of cultivation). Phytohormones were isolated by three times extraction with organic solvents from the supernatant of the culture liquid after extraction of surfactants. The qualitative and quantitative determination of gibberellins was carried out by high performance liquid chromatography. Results and discussion. The results show that regardless of the time of addition of tryptophan in the culture medium of the strain IMV B-7241 with crude glycerol a significant increase in the synthesis of auxins compared with those on the medium without this precursor was observed. The highest concentration of auxins was achieved by adding 300 mg/L of tryptophan into the medium with both substrates. Thus, A. calcoaceticus IMV B-7241 synthesized 1404.73, 1295.04 and 4850.98 μg/L of auxins on the crude glycerol medium with 100, 200 and 300 mg/L tryptophan added at the end of exponential phase respectively (and without precursor the concentration of auxins was 175,4 μg/L). Increased synthesis of auxins by strain IMV B-7241 correlated with the activity of tryptophan transaminase (key enzyme of biosynthesis): under cultivation on crude glycerol without precursor it was 163 nmol•min-1•mg-1 of protein, while in the presence of 300 mg/L of tryptophan added at the end of the exponential growth phase the activity was in 3.2 times higher – 526 nmol•min-1•mg-1 of protein. The highest concentration of auxins under cultivation on ethanol was achieved when 300 mg/L of tryptophan were added at the beginning of cultivation – 2261.66 μg/L. Conclusion. The result of the work established the possibility of increasing by one or two orders of magnitude of synthesized auxins in the case of addition into the culture medium of A. calcoaceticus IMV B-7241 low concentrations of the precursor of their biosynthesis.
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).
Intensification of biosurfactant synthesis by Nocardia vaccinii ІМV В-7405 on a glucose–glycerol mixture
(2015) Pirog, Tatiana; Shevchuk, Tetiana; Beregova (Pokora), Khrystyna; Kudrya, Nadiya
The purpose of this study was to establish optimal molar glycerol to glucose ratio for enhanced biosurfactant synthesis by the cultivation of Nocardia vacсinii ІMV В-7405 on a mixture of energetically nonequivalent substrates (glucose and glycerol). Based on theoretical calculations of the energy requirements for biomass production and the synthesis of surface-active trehalose monomycolates on the energy-deficient substrate (glycerol), the concentration of the energy-excessive substrate (glucose), which increased the efficiency of the substrate carbon conversion to biosurfactant, was determined. It was found that the synthesis of extracellular biosurfactant on a mixture of glucose and glycerol at molar ratio of 1.0:2.5 increased 2.0–2.3-fold compared to that on corresponding monosubstrates. The increased level of biosurfactant on the mixed substrate is determined by intensification of surface-active trehalose monomycolate synthesis, confirmed by 1.2–5.7-fold increase in activity of the enzymes involved in their biosynthesis (trehalose phosphate synthase, phosphoenolpyruvatecarboxykinase, phosphoenolpyruvate-synthetase) compared to cultivation on monosubstrates glucose and glycerol. The results indicate the feasibility of using a mixture of energetically nonequivalent substrates for enhancing the synthesis of secondary metabolites, as well as possibility of achieving the high efficiency of these mixed substrates if they are properly chosen and the molar ratio of their concentrations are correctly determined. Метою роботи було визначення оптимального молярного співвідношення глюкози та гліцеролу для інтенсифікації синтезу поверхнево-активних речовин N. vaccinii ІМВ В-7405 на суміші енергетично нерівноцінних субстратів (глюкози і гліцеролу). На основі теоретичних розрахунків енергетичних потреб синтезу поверхнево-активных трегалозомономіколатів та біомаси на енергетично дефіцитному субстраті (гліцерол) встановлено концентрацію енергетично надлишкової глюкози, що дає змогу підвищити ефективність конверсії вуглецю використовуваних субстратів у поверхнево-активні речовини. За молярного співвідношення концентрацій глюкози та гліцеролу 1,0:2,5 кількість синтезованих позаклітинних поверхнево-активних речовин була у 2,0–2,3 раза більша, ніж на відповідних моносубстратах. Підвищення концентрації поверхнево-активних речовин на змішаному субстраті зумовлено збільшенням синтезу поверхнево-активних трегалозоміколатів, про що свідчило зростання в 1,2–5,7 раза активності ензимів їх біосинтезу (трегалозофосфатсинтази, фосфоенолпіруват-карбоксикінази, фосфоенолпіруватсинтетази) порівняно з культивуванням на моносубстратах глюкозі та гліцеролі. Отримані результати свідчать про доцільність використання суміші енергетично нерівноцінних ростових субстратів для підвищення синтезу вторинних метаболітів, а також про те, що високої ефективності таких змішаних субстратів може бути досягнено як за правильного вибору субстратів, так і коректного визначення молярного співвідношення їх концентрацій.
Intensification of surface-active substances synthesis of Rhodococcus Erythropolis IMV Ac-5017 and Acinetobacter Calcoaceticus IMV B-7241 on glycerol
(2012) Pirog, Tatiana; Shevchuk, Tetiana; Shulyakova, Mariya; Mashenko, Oksana; Konon, Anastasia
Proposed approaches such as use of glycerol as a cheap substrate, adding of exogenous precursors of biosynthesis, using a mixture of growth substrates, may be the basis for the development of economically beneficial industrial technologies of producing of surfactants of Acinetobacter calcoaceticus IMV B-7241 and Rhodococcus erythropolis IMV Ac-5017 and help to solve the problem of disposal of waste of biodiesel production. Запропоновані підходи, а саме використання гліцерину як дешевого субстрату, внесення екзогенних попередників біосинтезу, використання суміші ростових субстратів, можуть стати основою для розробки економічно вигідної промислової технології отримання поверхнево-активних речовин Acinetobacter calcoaceticus ІМВ В-7241 та Rhodococcus erythropolis ІМВ Ас-5017, а також вирішити проблему утилізації відходів виробництва біодизелю.
Intensification of Surfactant Synthesis in Rhodococcus erythropolis EK-1 Cultivated on Hexadecane
(2010) Pirog, Tatiana; Klimenko, Julia; Shevchuk, Tetiana
Activity of key enzymes of n-alkane metabolism was determined in cells of Rhodococcus erythropolis EK-1, a surfactant producer grown on n hexadecane. Potassium cations were found to inhibit alkane hydroxylase and NADP+-dependent aldehyde dehydrogenase, while sodium cations were found to activate these enzymes. Decreased potassium concentration (to 1 mM), increased sodium concentration (to 35 mM), and addition of 36 μmol/l Fe(II), required for alkane hydroxylase activity, resulted in increased activity of the enzymes of n-hexadecane metabolism and in a fourfold increase of surfactant synthesis. A 1.5–1.7-fold increase in surfactant concentration after addition of 0.2% fumarate (gluconeogenesis precursor) and 0.1% citrate (lipid synthesis regulator) to the medium with hexadecane results from enhanced synthesis of trehalose mycolates, as evidenced by a 3–5- fold increase in phosphoenolpyruvate synthetase and trehalose phosphate synthase, respectively.В клетках штамма-продуцента поверхностно-активных веществ (ПАВ) Rhodococcus erythropolis ЭК-1, выращенного на н-гексадекане, определена активность ключевых ферментов метаболизма н-алканов.
Intensification of surfactant synthesis of acinetobacter calcoaceticus IMV B-7241 on ethanol in presence of citrate and fumarate
(2012) Konon, Anastasia; Chebotaryova, Ksenia; Shevchuk, Tetiana; Pirog, Tatiana
The influence of the fumarate (C4-dicarboxylic acids precursors of the gluconeogenesis) and citrate (the regulator of lipids synthesizes), optimal pH of biosurfactant synthesis during Acineto-bacter calcoaceticus IMV В-7241 cultivation on ethanol was studied. It was determined that the neutralization of the medium by the KOH solution in the process of the IMV B-7241 strain cultivation with the subsequent adding of the fumarate (0.01 %) and ci-trate (0.01 %) at the end of the exponential growth phase was accompanied by the increasing sur-factant synthesis by 1.2-fold comparing to the same indexes without neutralization and by 3.5-fold comparing to the cultivation of bacteria on ethanol without organic acids and pH regulation. The increasing of the surfactant synthesis in the presence of the fumarate ant citrate was caused by the 1.7–7-fold increased activity of the biosynthesis enzymes of glycolipids (phosphoe-nolpyruvate (PEP)-synthetase and thregalozophosphate-syntase) and aminolipids (NADP+- depen-dent glutamatedehydrohenaze) as well as the simultaneous functioning of two anaplerotic ways (glyoxylate cycle and phosphoenolpyruvatecarboxylate reaction). Вивчено вплив фумарату (С4-дикарбонових кислот, попередник глюконеогенезу) і цитрату (регулятор синтезу ліпідів), а також рН на утворення поверхнево-активних речовин (ПАР) при культивуванні Acinetobacter calcoaceticus ІМВ В-7241 на етанолі. Встановлено, що нейтралізація середовища розчином КОН в процесі культивування штаму ІМВ В-7241 з подальшим внесенням в кінці експоненційної фази фумарату (0.01%) і цитрату (0.01%) супроводжувалося підвищенням кількості синтезованих ПАР в 1,2 рази порівнянно з показниками аналогічного процесу без нейтралізації і в 3,5 рази порівняно з культивуванням бактерій на етанолі без органічних кислот і регуляції рН. Підвищення синтезу ПАР у присутності фумарату і цитрату обумовлено збільшенням в 1,7-7 разів активності ферментів біосинтезу гліколіпідів (фосфоенолпіруватсінтетази і трегалозофосфатсінтази) і аміноліпідов (НАДФ +-залежної глутаматдегідрогенази), а також одночасним функціонуванням двох анаплеротіческіх шляхів (гліоксилатного циклу і фосфоенолпіруваткарбоксилазної реакції).
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, Sergey
Актуальною 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.
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 %.
Practically valuable metabolites of marine microorganisms
(2020) Pirog, Tatiana; Martyniuk, Anna; Skrotska, Oksana; Shevchuk, Tetiana
The review considers the modern literature data on the synthesis by fungi, actinobacteria, and bacteria isolated from marine ecosystems (seawater, bottom sediments, flora and fauna, mangrove biomes, glaciers), practically valuable metabolites. Marine microorganisms synthesize a wide range of practically valuable enzymes (cold-active galactosidase, agarase, alginate lyase, fucoidase, chitinase, etc.), surface-active glyco- and lipopeptides with emulsifying, antimicrobial and antiadhesive activity, as well as secondary metabolites with diverse biological activity (antimicrobial, antitumor, cytotoxic). However, the use of marine producers in biotechnological processes is constrained by their low synthesizing capacity and high costs of biosynthesis (complex nutrient media and expensive carbohydrate substrates). In biotechnology, marine microorganisms can be used as sources of genes encoding the synthesis of new biologically active substances with unique properties, including antimicrobial and antitumor.
Practically valuable properties of the surfactant synthesized by Rhodococcus genus actinobacteria
(2020) Pirog, Tatiana; Petrenko, Natalia; Skrotska, Oksana; Paliichuk, Olesia; Shevchuk, Tetiana; Iutynska, Galyna
Currently, microbial surfactants are the objects of intense research because of their surface-active and emulsifying properties, high antimicrobial, anti-adhesive activity, and ability to destroy biofilms. The review provides current literature data on the properties of surfactants synthesized by Rhodococcus genus actinobacteria, determining their practical significance. The researchers’ interest in the surfactants of Rhodococcus bacteria is primarily due to their key role in the destruction of xenobiotics (aliphatic, heterocyclic and polycyclic aromatic hydrocarbons). Information on the antimicrobial and anti-adhesive activity of surfactants of Rhodococcus genus bacteria remains scarce at present, while the immunomodulatory properties of these products of microbial synthesis are studied more actively than for other microbial surfactants known in the world. The data of our experimental studies on the practically valuable properties of surfactants synthesized by Rhodococcus erythropolis IMV Ac-5017 are presented. Unlike surfactants of other representatives of Rhodococcus genus bacteria, surfactants of IMV As-5017 strain are multifunctional preparations. Because in addition to the high efficiency of the destruction of oil pollution, including complex with heavy metals, surfactants are characterized by high antimicrobial and anti-adhesive activity, including the ability to destroy biofilms.
Production of Surfactants by Rhodococcus erythropolis Strain EK-1, Grown on Hydrophilic and Hydrophobic Substrates
(2004) Pirog, Tatiana; Shevchuk, Tetiana; Voloshina, Iryna; Karpenko, Elena
The ability of Rhodococcus erythropolis strain EK-1 to produce surfactants when grown on hydrophilic (ethanol and glucose) and hydrophobic (liquid paraffins and hexadecane) substrates was studied. The strain was found to produce surfactants with emulsifying and surface-active properties. The production of surfactants depended on the composition of the nutritive medium, nature and concentration of the sources of carbon and nitrogen, and duration of cultivation. Chemically, surfactants produced by Rhodococcus erythropolis EK-1 grown on ethanol are a complex of lipids with polysaccharide–proteinaceous substances. The lipids include glycolipids (trehalose mono- and dicorynomycolates) and common lipids (cetyl alcohol, palmitic acid, methyl n-pentadecanoate, triglycerides, and mycolic acids).
Synergism of antimicrobial and anti-adhesive activity of Nocardia vaccinii IMV B-7405 surfactants in a mixture with essential oils
(2020) Pirog, Tatiana; Kliuchka (Nykytyuk), Lilia; Kliuchka, Igor; Shevchuk, Tetiana; Iutynska, Galyna
An increase in the antibiotic resistance of pathogenic microorganisms has stimulated the search for alternatives to antibiotics substances of natural origin, which are essential oils (EO) and non-toxic biodegradable microbial surfactants. Aim. To investigate the antimicrobial and anti-adhesive activity of a mixture of EO and surfactants of Nocardia vaccinii IMV B-7405 synthesized on various oil-containing media. Methods. N. vaccinii IMV B-7405 was grown in medium containing as carbon source 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. The antimicrobial action of tea tree, cinnamon and lemongrass EO, surfactants and their mixtures was determined by index of the minimum inhibitory concentration (MIC). Synergistic effect of surfactants and EO was evaluated by indicator of fractional inhibitory concentration. The degree of bacteria and fungi biofilms destruction under the action of surfactants, EO and their mixtures was determined by spectrophotometric method. Results. It was found that N. vaccinii IMV B-7405 surfactants synthesized on all oil-containing substrates showed a synergistic antimicrobial and anti-adhesive activity with the investigated EO. MIC of a surfactants and EO mixture against bacteria (Bacillus subtilis BT-2 (spores), Escherichia coli IEM-1, Staphylococcus aureus BMS-1) and yeast (Candida albicans D-6, Candida utilis BVS-65 and Candida tropicalis RE-2) were 2–20 μg/ml and were significantly lower than each compound separately (156–625 and 8–80 μg/ml for EO and surfactants, respectively). The destruction of bacterial and yeast biofilms under the action of a mixture of surfactants (20–40 μg/ml) and EO (20–40 μg/ml) was 1.3–2.9 times higher compared with using of each component separately at similar concentrations. Conclusions. The data presented the possibility of using a mixture of EO and surfactants not only to reduce their MIC, but also as effective antimicrobial and anti-adhesive agents.
Synergistic effect of surfactants of Nocardia vaccinii IMV B-7405 and essential oils on Candida genus yeast
(2022) Pirog, Tatiana; Kliuchka (Nykytyuk), Lilia; Shevchuk, Tetiana; Muchnyk, Faina
The increase in the number of resistant strains of Candida genus representatives, capable of forming biofi lms on various surfaces, stimulates the search for new, alternative methods of combating them, one of which is the use of compounds of natural origin, such as essential oils. However, at the same time, their concentration should be minimal, which is due to the ability of essential oils to cause severe damage of the human’s central nervous system and aspiration pneumonia. This leads to the necessity of searching for new methods to reduce the concentration of essential oils and at the same time to preserve their properties, in particular, by their use in a mixture with other antimicrobial agents, which can be microbial surfactants. Previously, it was found that the degree of yeast biofi lm destruction under the action of Nocardia vaccinii IMV B-7405 surfactants dependson the nature of the growth substrate and is the highest in the presence of preparations synthesized on purified glycerol. Aim. To study the synergism of antifungal activity and the role in the destruction of biofilms of a mixture of Nocardia vaccinii IMV B-7405 surfactants synthesized on glycerol of different quality and essential oils. Methods. N. vaccinii IMV B-7405 was grown in a medium containing purified glycerol or waste from biodiesel production at a concentration of 2% (v/v) as carbon sources. The surfactants were extracted from the supernatant of cultural liquid by a modified Folch mixture. The antimicrobial activity of essential oils, surfactants, and their mixtures was determined by the index of the minimum inhibitory concentration. To assess the synergistic effect of a mixture of surfactants with essential oils, the fractional inhibitory concentration index was used. The degree of biofi lm destruction (%) was determined as the difference between the cell adhesion in untreated and treated with surfactants, essential oil, or their mixture wells of the polystyrene microplates. Results. It was found that the surfactants synthesized by N. vaccinii IMV B-7405 on both purifi ed glycerol and waste from biodiesel production showed synergistic antifungal activity in mixtures with cinnamon and lemongrass essential oils. Thus, the minimum inhibitory concentrations against Candida albicans D-6, Candida utilis BVS-65, and Candida tropicalis RE-2 of a mixture of surfactants synthesized on purified glycerol with cinnamon and lemongrass essential oils were 1.8—7.5 and 3.7— 15 μg/mL, respectively, and were lower than in the case of using surfactants (30-60 μg/mL), cinnamon or lemongrass essential oil (156—312 μg/mL) alone. The use of a mixture of surfactants obtained on waste from biodiesel production and cinnamon or lemongrass essential oils made it possible to reduce the minimum inhibitory concentrations of the latter against studied yeast test cultures by 14—56 times. At the same time, the index of fractional inhibitory concentration did not exceed 0.5, which indicates the synergism of the antifungal activity of the mixture of these compounds. The destruction of Candida yeast biofi lms under the action of surfactants synthesized on both purified glycerol and waste from biodiesel production in a mixture with cinnamon or lemongrass essential oils reached 60—67 and 67—77%, respectively, which is an average of 25—35% higher compared to the use of each monopreparation separately. Conclusions. The results presented in this paper confirm the previously obtained data that N. vaccinii IMV B-7405 surfactants, synthesized on both traditional substrates and toxic industrial wastes, have antimicrobial and antiadhesive synergistic action with essential oils, which allows us to consider them as potential components of the so-called «antifungal locks» in the fight against of Candida genus representatives.