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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.
Effect on phytopathogenic microorganisms of surfactants of microbial origin
(2021) Pirog, Tatiana; Piatetska, Daria; Yarova, Hanna; Iutynska, Galyna
Biodegradable non-toxic surfactants of microbial origin are multifunctional preparations, which due to antimicrobial activity are promising for use in crop production to control phytopathogenic microorganisms. Studies on the prospects of using microbial surfactants to control the number of phytopathogenic microorganisms are conducted in three directions: laboratory studies of antimicrobial activity of surfactants in vitro, determination of the effect of surfactants on phytopathogens in vegetative experiments in the process of plants growing in a laboratory or greenhouse, post-harvest treatment of fruits and vegetables with solutions of microbial surfactants to extend their shelf life. The review presents literature data on antimicrobial activity of surfactants against phytopathogenic bacteria and fungi in vitro. Antimicrobial activity of surfactants is evaluated by three main parameters: minimum inhibitory concentration, zones of growth retardation of test cultures on agar media and inhibition of growth of test cultures on agar or liquid media. The vast majority of available publications relate to the antifungal activity of surfactant lipopeptides and rhamnolipids, while data on the effect of these microbial surfactants on phytopathogenic bacteria (representatives of the genera Ralstonia, Xanthomonas, Pseudomonas, Agrobacterium, Pectobacterium) are few. The researchers determined the antimicrobial activity of either total lipopeptides extracted with organic solvents from the culture broth supernatant, or individual lipopeptides (iturin, surfactin, fengycin, etc.) isolated from a complex of surfactants, or culture broth supernatant. Lipopeptides synthesized by members of the genus Bacillus exhibit antimicrobial activity on phytopathogenic fungi of the genera Alternaria, Verticillium, Aspergillus, Aureobasidium, Botrytis, Rhizoctonia, Fusarium, Penicillium, Phytophora, Sclerotinia, Curvularia, Colletotrichum, etc. in sufficiently high concentrations. Thus, the minimum inhibitory concentrations of lipopeptides against phytopathogenic fungi are orders of magnitude higher (in average 0.04–8.0 mg/mL, or 40–8000 μg/mL) than against phytopathogenic bacteria (3–75 μg/mL). However, the antifungal activity of lipopeptidecontaining supernatants is not inferior by the efficiency to the activity of lipopeptides isolated from them, and therefore, to control the number of phytopathogenic fungi in crop production, the use of lipopeptidecontaining supernatants is more appropriate. Rhamnolipids synthesized by bacteria of the genus Pseudomonas are more effective antimicrobial agents comparing to lipopeptides: the minimum inhibitory concentrations of rhamnolipids against phytopathogenic fungi are 4–276 μg/mL, which is an order of magnitude lower than lipopeptides. In contrast to the data on the antifungal activity of rhamnolipids against phytopathogens, there are only a few reports in the literature on the effect of these surfactants on phytopathogenic bacteria, whilst the minimal inhibitory concentrations are quite high (up to 5000 μg/mL). The advantage of rhamnolipids as antimicrobial agents compared to lipopeptides is the high level of synthesis on cheap and available in large quantities industrial waste. Currently in the literature there is little information about the effect of surface-active sophorolipids of microbial origin on phytopathogenic fungi, and all these works are mainly about the antifungal activity of sophorolipids. We note that in contrast to surfactant lipopeptides and rhamnolipids, the effective concentration of most sophorolipids, which provides the highest antimicrobial activity against phytopathogens, is higher and reaches 10,000 μg/mL.
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.
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.
Ways of auxin biosynthesis in microorganisms
(2022) Pirog, Tatiana; Piatetska, Daria; Klymenko, Natalia; Iutynska, Galyna
Among 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.