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. Antiadhesive 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.
Until recently, there were rare scientific reports on the biotechnological potential of non-pathogenic bacteria of the Acinetobacter genus. Although the first reports about the practically valuable properties of these bacteria date back to the 70s and 80s of the twentieth century and concerned the synthesis of the emulsan bioemulsifier. In the last decade, interest in representatives of the Acinetobacter genus as objects of biotechnology has significantly increased. The review presents current literature data on the synthesis by bacteria of this genus of high-molecular emulsifiers, low-molecular biosurfactants of glyco- and aminolipid nature, enzymes (lipase, agarase, chondroitinase), phytohormones, as well as their ability to solubilize phosphates and decompose various xenobiotics (aliphatic and aromatic hydrocarbons, pesticides, insecticides). Prospects for practical application of Acinetobacter bacteria and the metabolites synthesized by them in environmental technologies, agriculture, various industries and medicine are discussed. The data of own experimental studies on the synthesis and biological activity (antimicrobial, anti-adhesive, ability to destroy biofilms) of biosurfactants synthesized by A. calcoaceticus IMV B-7241 strain and their role in the degradation of oil pollutants, including complex ones with heavy metals, are presented. The ability of A. calcoaceticus IMV B-7241 to the simultaneous synthesis of phytohormones (auxins, cytokinins, gibberellins) and biosurfactants with antimicrobial activity against phytopathogenic bacteria allows us to consider this strain as promising for practical use in crop production to increase crop yields.
Microbial surfactants are multifunctional products because they cannot only reduce the surface tension at the interface and emulsify various substrates, but also display antimicrobial and anti-adhesion activity (including the ability to destroy biofilms). However, under various conditions of producer’s cultivation the surfactant composition and their properties can vary. One of the approaches to increasing antimicrobial and anti-adhesion activity of the surfactant can be an increase in medium of producer cultivation content of activators of key enzymes biosynthesis of aminolipids − the most effective antimicrobial agents. Activators of NADP+-dependent glutamate dehydrogenase in Acinetobacter calcoaceticus IMV B-7241 are cations of calcium, magnesium and zinc, Rhodococcus erythropolis IMV Ac-5017 and Nocardia vaccinii IMV B-7405 – calcium.Surfactants were extracted from supernatant of cultural liquid by mixture of chloroform and methanol (2:1). Antimicrobial activity of surfactants was determined by index of minimum inhibitory concentration (MIC), antiadhesive − by spectrophotometry. The degree of biofilm destruction was determined as difference between the number of adhered cells in untreated and treated with surfactant holes of polystyrene immunological plate containing pre-formed biofilm of test cultures and was expressed as a percentage. It was found that addition of CaCl2 (0.1 g/l) into medium cultivation of R. erythropolis IMV Ac-5017, increasing concentration of this salt to 0.4 g/l in medium for N. vaccinii IMV B-7405 growth, introduction of CaCl2(0.1 g l) and increasing MgSO4·7H2O content to 0.2 g/l, or adding Zn2+(38 μM) into medium cultivation of A. calcoaceticus IMV B-7241 was accompanied by synthesis of surfactants MICs of which against test cultures were 1.2–13 times lower, their adhesion on abiotic surfaces treated with such surfactants was on average 10−40 % lower, and the degree of biofilms destruction was 7−20 % higher than indicators established for surfactants obtained on the base medium. The obtained data indicate the possibility of regulating antimicrobial and anti-adhesion activity of microbial surfactants under producer cultivation.
здатність поверхнево-активних речовин (ПАР), синтезованих за умов росту Acinetobacter calcoaceticus IMB B-7241 на різних субстратах (етанол, гліцерин, н-гексадекан), руйнувати біоплівки деяких бактерій і дріжджів. Методи. ПАР екстрагували з супернатанту культуральної рідини сумішшю хлороформу і метанолу (2:1). Ступінь руйнування біоплівки визначали як різницю між кількістю адгезованих клітин у необроблених і оброблених ПАР лунках полістиролового планшету з попередньо сформованою біоплівкою тест-культури і виражали у відсотках. Кількість адгезованих клітин визначали спектрофотометричним методом. Результати. Встановлено залежність ступеня руйнування біоплівок під впливом ПАР A. calcoaceticus IMB B-7241 від природи джерела вуглецю у середовищі культивування продуцента, концентрації поверхнево-активних речовин у препаратах і типу тест
Порівняння біологічних властивостей (антимікробної та антиадгезивної активності, у тому числі й роль у руйнуванні біоплівки) поверхнево-активних речовин Acinetobacter calcoaceticus ІМВ В-7241, синтезованих на очищеному та технічному гліцерині (відходи виробництва біодизелю). Методи. Поверхнево-активні речовини екстрагували з супернатанту культуральної рідини сумішшю хлороформу і метанолу (2:1). Кількість адгезованих клітин і ступінь руйнування біоплівки визначали спектрофотометричним методом, антимікробні властивості − за показником мінімальної інгібуючої концентрації. Результати. Антимікробна активність поверхнево-активних речовин A. calcoaceticus ІМВ В-7241, синтезованих на технічному гліцерині, була на 1−2 порядки нижчою, адгезія тест-культур на абіотичних матеріалах, оброблених такими препаратами -в 1,2−4 рази нижчою, а ступінь руйнування бактеріальних біоплівок -в 1,3−4 рази меншою порівняно з показниками, встановленими для поверхнево-активних речовин, одержаних на середовищі з еквімолярною за вуглецем концентрацією очищеного гліцерину. Зниження антимікробної та антиадгезивної активності поверхнево-активних речовин може бути зумовлене інгібуючим впливом надлишкової концентрації К + та Na + (складових технічного гліцерину) на активність НАДФ + -залежної глутаматдегідрогенази: за наявності в реакційній суміші 50-100 мМ катіонів калію та натрію ферментативна активність знижувалась в 1,2-1,8 рази. Висновки. Незважаючи на нижчу антимікробну та антиадгезивну активність поверхнево-активних речовин, синтезованих A. calcoaceticus ІМВ В-7241 на відходах виробництва біодизелю, ці показники (мінімальні інгібуючі концентрації 0,96-15,2 мкг/мл, руйнування 50 % біоплівки при концентрації 124 мкг/мл) є співставимими із визначеними для відомих мікробних поверхнево-активних аміноліпідів.
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