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Документ Ways of auxin biosynthesis in microorganisms(2022) Pirog, Tatiana; Piatetska, Daria; Klymenko, Natalia; Iutynska, GalynaAmong plant hormones, auxins, in particular indole-3-acetic acid (IAA), are the most studied and researched. Almost all groups of soil microorganisms, both plant-associated and non-plant-associated bacteria, fungi, and phytopathogenic microorganisms are capable of producing auxins. Th e development of preparations for crop production is directly related to the production of bacterial strains with high auxin-synthesizing potential, which is possible only with a full understanding of the ways of regulation and synthesis of auxins in bacteria. Th e synthesis of auxins in microorganisms can take place in two ways: by the gradual conversion of tryptophan to IAA (tryptophan-dependent pathway) or by the use of other intermediates (tryptophan-independent pathway). Th e latter is poorly clarifi ed, and in the literature available today, there is only a small amount of information on the functioning of this pathway in microorganisms. Th e review presents literature data on the ways of auxin biosynthesis in diff erent groups of microorganisms, as well as approaches to the intensifi cation of indole-3-acetic acid synthesis. Th e formation of IAA from tryptophan can be carried out in the following ways: through indole-3-pyruvate, through indole-3-acetamide, and through indole-3-acetonitrile. Th e vast majority of available publications are related to the assimilation of tryptophan through the formation of indole-3-pyruvate as this pathway is the most common among microorganisms. Th us, it functions in rhizospheric, symbiotic, endophytic, and free-living bacteria. Th e concentration of synthesized IAA among natural strains is in the range from 260 to 1130 μg/mL. Microorganisms in which the indole-3-acetamide pathway functions are characterized by lower auxin-synthesizing ability compared to those that assimilate tryptophan through indole-3-pyruvate. Th ese include bacteria of the genera Streptomyces, Pseudomonas, and Bradyrhizobium and fungi of the genus Fusarium. The level of synthesis of IAA in such microorganisms is from 1.17•10−4 to 255.6 μg/mL. To date, only two strains that assimilate tryptophan via the indole-3-acetonitrile pathway and form up to 31.5 μg/mL IAA have been described in the available literature. To intensify the synthesis of indole-3-acetic acid, researchers use two main approaches: the fi rst consists in introducing into the culture medium of exogenous precursors of biosynthesis (usually tryptophan, less oft en indole-3-pyruvate, indole-3-acetamide, and indole-3-acetonitrile); the second — in increasing the expression of the corresponding genes and creating recombinant strains-supersynthetics of IAA. Th e largest number of publications is devoted to increasing the synthesis of IAA in the presence of biosynthesis precursors. Depending on the type of bacteria, the composition of the nutrient medium, and the amount of exogenously introduced precursor, the synthesis of the fi nal product was increased by 1.2—27 times compared to that before the intensifi cation. Th us, in the presence of 11 g/L tryptophan, Enterobacter sp. DMKU-RP206 synthesized 5.56 g/L, while in a medium without the precursor, it yielded only 0.45 g/L IAA. Recombinant strains Corynebacterium glutamicum ATCC 13032 and Escherichia coli MG165 formed 7.1 and 7.3 g/L IAA, respectively, when tryptophan (10 g/L) was added to the culture medium. Th e level of auxin synthesis in microorganisms may be increased under stress conditions (temperature, pH, biotic and abiotic stress factors), but in this case, the IAA concentration does not exceed 100 mg/L, and therefore this method of intensifi cation cannot compete with the others above.Документ Effect on phytopathogenic microorganisms of surfactants of microbial origin(2021) Pirog, Tatiana; Piatetska, Daria; Yarova, Hanna; Iutynska, GalynaBiodegradable 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.Документ 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, GalynaThe 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.Документ 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, GalynaMicrobial 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.Документ 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, GalynaAn 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.Документ 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, GalynaIn 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.Документ Microbial synthesis of phytohormones(2018) Pirog, Tatiana; Iutynska, Galyna; Leonova, Natalia; Beregova (Pokora), Khrystyna; Schevchuk TetyanaThe aim of the review was to analyze current literature data and the results of own studies on the synthesis of auxins, cytokinins, and gibberellins by plant-associated microorganisms (living in rhizosphere, endophytic, nitrogen-fixing, and phytopathogenic), and by those not involved in symbiotic interactions. Many microorganisms can generate phytohormones, and microbial synthesis of indole-3-acetic acid can be enhanced which can be used in producing it instead of extracting it from plants or by chemical synthesis. Recent progress in intensifying the synthesis of gibberellic acid in deep and solid-phase producer cultivation allows substantially reducing the prime cost of biotechnological production of that phytohormone. The ability of microorganisms to simultaneously synthesize phytohormones and other biologically active compounds with antimicrobial, nematocidal, and other various effects enables creating complex polyfunctional microbial preparations with various biological properties for use in crop production to stimulate plant growth and pest control. Метою огляду було проаналізувати сучасні дані літератури і результати власних досліджень синтезу ауксинів, цитокінінів,гіберелінів як асоційованими з рослинами мікроорганізмами (ризосферними, ендофітними, азотфіксувальними, фітопатогенними), так і тими, які не беруть участі у такій взаємодії. Виявлена у широкого кола мікроорганізмів здатність до утворення фітогормонів, а також успіхи у підвищенні ефективності мікробного синтезу індоліл-3-оцтової кислоти свідчать про можливість такого способу її одержання замість екстракції з рослин або хімічного синтезу. Досягнення останнього десятиліття щодо інтенсифікації синтезу гіберелінової кислоти за умов глибинного і твердофазного культивування продуцентів дають змогу суттєво знизити собівартість цього фітогормону, одержуваного біотехнологічним способом. Здатність мікроорганізмів до одночасного синтезу фітогормонів та інших біологічно активних сполук з антимікробною, нематоцидною та ін. активністю підтверджує можливість створення комплексних поліфункціональних мікробних препаратів з різноманітними біологічними властивостями з метою використання у рослинництві для стимуляції росту рослин і контролю чисельності шкідників.Документ Practically valuable properties of the surfactant synthesized by Rhodococcus genus actinobacteria(2020) Pirog, Tatiana; Petrenko, Natalia; Skrotska, Oksana; Paliichuk, Olesia; Shevchuk, Tetiana; Iutynska, GalynaCurrently, 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.