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Автор: search.filters.author.Shevchuk, Tetiana

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  • Ескіз
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    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.
  • Ескіз
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    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.
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    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.
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    Synthesis and biological activity of Аcinetobacter calcoaceticus IMV B-7241 surfactants depending on monovalent cations content in cultivation medium
    (2021) Pirog, Tatiana; Lutsay, Dariya; Shevchuk, Tetiana; Iutynska, Galyna
    Microbial surfactants (biosurfactants) are multifunctional preparations due to a combination of physicochemical (reduction of surface and interfacial tension, emulsifying activity) and biological (antimicrobial and antiadhesive activity, the ability to destroy biofilms) properties. However, the disadvantage of biosurfactants synthesized as a complex of compounds is the possibility of changing the biological activity depending on the conditions of producer cultivation. Aim. To study the effect of potassium and sodium cations on the NADP+-dependent glutamate dehydrogenase activity of cell-free extract of Acinetobacter calcoaceticus ІМV B-7241 with subsequent appropriate modification of the nutrient medium composition and determination of antimicrobial and anti-adhesive activity of surfactant synthesized. Methods. A. calcoaceticus ІМV B-7241 strain was grown in media containing 2% of sunflower oil waste as a carbon source, as well as various concentrations of potassium and sodium chloride (basal – 1.0 g/l NaCl, medium # 1 that did not contains NaCl, medium # 2 in which the concentration of NaCl was 2.0 g/l, medium # 3 in which the concentration of NaCl and KCl was 1.0 g/l each). The surfactants were extracted from the supernatant liquid culture with a modified Folch mixture. Anti-adhesive activity and the degree of biofilms degradation were determined by spectrophotometric method, antimicrobial activity − by the indicator of the minimum inhibitory concentration (MIC). Activity of enzymes of surface-active aminolipids biosynthesis (NADP+-dependent glutamate dehydrogenase) and glycolipids (phosphoenolpyruvate (PEP) carboxylase, PEP-synthetase, PEP-carboxykinase, trehalose phosphate synthase) were analyzed in cell-free extracts obtained after the destruction of cells by ultrasound. Results. It was found that potassium and sodium cations in concentrations of 50 and 100 mM are inhibitors of NADP+-dependent glutamate dehydrogenase, and in lower concentrations (5–20 mM) – activators of this enzyme, as well as PEP-carboxykinase and PEP-synthetase. The increase in the biosurfactant concentration to 6.1−7.7 g/l during cultivation of A. calcoaceticus ІМV B-7241 in medium # 1 and # 3 was due to the predominant synthesis of glycolipids under such conditions, which was evidenced by the increase in 1.8−6.5 times in the activity of PEP-carboxylase, PEP-carboxykinase, PEP-synthetase and trehalose phosphate synthetase compared to the indicators on the basal medium. The concentration of surfactants synthesized in the basal medium was 3.6 g/l, but such surfactants were characterized by the highest antimicrobial and anti-adhesive activity. Their MIC against the test-cultures of studied bacteria (Pseudomonas sp. MI-2, Bacillus subtilis BT-2, Escherichia coli IEM-1, Staphylococcus aureus BMS-1, Enterobacter cloaceae C-8) and fungi (Candida albicans D-6, Rhizopus nigricans P1, Aspergillus niger P-3, Fusarium culmorum T-7) were 0.88−56 μg/ml and were by 2−3 orders of magnitude lower compared to established for surfactants synthesized in modified media # 1–3. In the case of treatment of abiotic materials with surfactant solutions obtained on the basal medium, the adhesion of bacteria and fungi was on average 10–20 % lower than after surface treatment by the surfactant synthesized in modified media. In the presence of 148−296 μg/ml of surfactants obtained in the basal medium, destruction of S. aureus BMS-1 and B. subtilis BT-2 biofilms was 45−66 %, and C. albicans D-6 yeast – 39−44 %. Under the action of similar concentrations of surfactants synthesized in modified media, the destruction of bacterial and yeast biofilms was lower: 6-52 and 20–46 %, respectively. Conclusions. The obtained results are consistent with the data of our previous studies on the possibility of regulating the antimicrobial and antiadhesive activity of surfactants in the process of producer cultivation by changing the content of cations in the medium, which are inhibitors/activators of enzymes responsible for the synthesis of components of the surfactants complex, which have certain biological properties.
  • Ескіз
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    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.
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    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.
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    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.
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    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.
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    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.
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    Synthesis of gibberellins by surfactant producers Nocardia vaccinii IMV В-7405, Acinetobacter calcoaceticus IMV В-7241 and Rhodococcus erythropolis IMV Ас-5017
    (2020) Leonova, Natalia; Pirog, Tatiana; Piatetska, Daria; Shevchuk, Tetiana; Kharkhota, Maxym; Iutynska, Galyna
    In this study, we established that the producers of surfactants Nocardia vaccinii IMV В-7405, Acinetobacter calcoaceticus IMV B-7241 and Rhodococcus erythropolis IMV Аc-5017 were able to synthesize phytohormones of gibberellic nature during cultivation on the traditional substrates (ethanol, hexadecane) and on the industrial waste sunflower oil and biodiesel production waste). The results of specific biotesting showed that the treatment of cucumber seedlings with phytohormonal extracts of all studied strains at 1:500 and 1:600 dilutions stimulated the extension of the cucumber hypocotyls as compared to the water control in values close to the treatment with gibberellic acid. The results of high-performance liquid chromatography showed N. vaccinii IMV B-7405, A. calcoaceticus IMV B-7241 and R. erythropolis IMV Аc-5017 produce highly active forms of gibberellins GA3 and GA4. The level of its synthesis was nearly the same (6.0-10.0 μg∙L-1) under cultivation of strains on every substrate. The exception was strain N. vaccinii IMV В-7405 which synthesized almost 47.0 μg∙L-1 GA3 and GA4 while is growing on the waste oil from meat frying. The obtained results are the groundwork for the development of an economically profitable technology for the recycling of toxic wastes using N. vaccinii IMV B-7405, A.calcoaceticus IMV B-7241 and R. erythropolis IMV Аc-5017. Such technology will allow us to develop complex microbial preparations with various biological properties in a single process. У цьому дослідженні встановлено, що продуценти ПАР Nocardia vaccinii ІМВ В-7405, Acinetobacter calcoaceticus ІМВ В-7241 та Rhodococcus erythropolis ІМВ Ас-5017 здатні синтезувати фітогормони гіберелової природи при культивуванні на традиційних субстратах (етанол, гексадекан) а також на промислових відходах соняшникової олії та відходах виробництва біодизеля). Результати специфічного біотестування показали, що обробка проростків огірка фітогормональними екстрактами всіх досліджуваних штамів у розведенні 1:500 та 1:600 ​​стимулювала подовження гіпокотилів огірка порівняно з контролем у воді в значеннях, близьких до обробки гібереліновою кислотою. . Результати високоефективної рідинної хроматографії показали, що N. vaccinii IMV B-7405, A. calcoaceticus IMV B-7241 і R. erythropolis IMV Аc-5017 продукують високоактивні форми гіберелінів GA3 і GA4. Рівень його синтезу був приблизно однаковим (6,0-10,0 мкг∙л-1) за культивування штамів на кожному субстраті. Виняток становив штам N. vaccinii ІМВ В-7405, який синтезував майже 47,0 мкг∙л-1 ГА3 та ГА4 під час вирощування на відпрацьованому маслі від смаження м’яса. Отримані результати є основою для розробки економічно вигідної технології переробки токсичних відходів з використанням N. vaccinii ІМВ В-7405, A.calcoaceticus ІМВ В-7241 та R. erythropolis ІМВ Ас-5017. Така технологія дозволить розробляти комплексні мікробні препарати з різними біологічними властивостями в одному процесі.