Mikrobiolohichnyi Zhurnal
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The aim of this work was the isolation, purification and some properties investigation of two regulators of antibiotic biosynthesis of streptomycetes. Methods includes extraction of regulators from agar cultures and their concentration by vacuum rotary evaporator, thin layer chromatography and spectrophotometry. Results. Two strains of streptomycetes AN26 and B35 isolated from soils of different regions of Ukraine produce the regulators restoring the landomycin E biosynthesis and sporulation in mutant strain Streptomyces globispoprus 1912-B2. Both regulators were purified by thin layer chromatography and have the same Rf 0.69. Absorption curves of regulators were established by means of spectrophotometry. Maxima of absorption of regulators were 232.5 nm. The next study of the isolated regulators by means of NMR will give the possibility to elucidate their molecular structures. Conclusions. It is shown that two strains of streptomycetes isolated from the soils of Askania Nova and Brovary produce transcriptional regulators such as signaling molecules, which, like A-factor, restore the biosynthesis of antibiotics landomycin E and streptomycin in test strains S. globisporus 1912-B2 and S. griseis 1439, respectively. In terms of absorption maxima, they are similar and differ from similar indicators of known regulators of streptomycetes. It is possible that these compounds belong to new, not yet described signaling molecules, and the answer to this question will give future studies of their molecular structure by NMR spectroscopy.

Peculiarities of high-quality silage production are the use of biological products based on lactic acid bacteria. The composition of such starters varies greatly according to the use of bacterial cultures, so among the starters available on the market, the range of their effectiveness is also different. It is very common to use a one-sided approach to the choice of bacterial components, which in combination with imperfect production technology have low preservative activity. The study of combined preparations, which combine homo- and heterofermentative types of lactic acid fermentation, allows to stabilize the preservative properties throughout the ensiling time, and increase the aerobic stability of the silage after access of oxygen. Aim. Development of biotechnology of bacterial preparation for corn ensiling, optimization of cultivation conditions of newly created bacterial composition, and selection of cryoprotectants for its lyophilization. Methods. The combined preparation was created on the basis of heterofermentative strain Lactobacillus buchneri 3806 combining it in two- and three-strain compositions with other representatives of lactic acid bacteria, which are characterized by obligate homofermentative and facultative heterofermentative types of metabolism. Optimization of the environment and technological parameters was carried out using a central-compositional plan, further statistical analysis of the obtained data and determination of optimal values of input parameters according to the created mathematical model of optical density response. The effectiveness of the selected protective media was tested for the survival of bacteria after lyophilization. Results. The most effective bacterial composition was found during experiments: L. buchneri 3806, Enterococcus faecium C-8-12, L. plantarum 3216. The effectiveness of the obtained composition was tested by laboratory silage of corn. Tests of the drug based on the selected bacterial composition showed an improvement in the chemical composition of the silage compared to the untreated control and treated only with monoculture L. buchneri 3806, namely: there was a decrease in dry matter loss by 2.21% and 2.04%, 22 due to the increase of lactic acid content, and increase of aerobic stability of silage – 341 h against 57 h of the control sample, and 313 h in case of using monoculture. For the obtained bacterial composition, the culture medium of the following composition was optimized: base (hydrolyzed milk with the addition of the following components: monosubstituted potassium phosphate – 2 g/L; 5-aqueous manganese sulfate – 0.05 g/L; 7-aqueous magnesium sulfate – 0.2 g/L; twin-80 – 1.0 g/L); glucose – 19.7 g/L; yeast extract – 7.8 g/L; corn extract – 23.6 g/L; peptone – 9.1 g/L; sodium citrate – 6.6 g/L; sodium acetate – 3,4 g/L. Cultivation of the bacterial composition on an optimized medium made it possible to obtain the maximum biomass yield, at which the optical density was 2.01 units, which is almost twice as much as the value obtained by culturing the same composition in MRS medium. The optimal technological parameters of culturing the bacterial composition were established, namely the best growth was observed at a temperature of 36.4±0.4°C with constant maintenance of the pH value in the culture medium at the level of 6.5±0.1 units. In addition, the optimal composition of the protective medium containing sodium citrate, sucrose and agar was selected, and ensures the survival rate of lactic acid bacteria 98.4% after lyophilization. Conclusions. The newly formed bacterial composition can be used for the production of preparations for corn silage, and tested on other raw materials, in particular on some perennial legumes (alfalfa, clover), and the conditions of its production can be used to scale the technology.

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.

Every year the volume of production of poultry products all over the world is growing steadily. This contributes to a constant increase in the amount of by-products of poultry processing in the form of down and feather waste, which are dangerous for the environment due to the hard-to-degrade keratin protein and a large number of microbial pathogens. Therefore, the use of environmentally friendly methods for the destruction of keratin substrates due to keratinases of microorganisms is an urgent area of research. The aim of this work was to select the optimal cultivation conditions for the Bacillus megaterium strain UCM B-5710 to increase the activity of the keratinase synthesized by it. Methods. The culture was grown at 28°C, 201 rpm for 7 days on a basic nutrient medium containing defatted chicken feathers as the only source of carbon and nitrogen. The selection of optimal cultivation conditions was carried out according to the following parameters: temperature (21°C, 28°C, 42°C), stirring speed (201 rpm, 212 rpm), amount of inoculum (5%, 10%, 15% , 20%, 25%), the initial pH value of the nutrient medium (4.0–11.0), concentration of keratin-containing substrate (0.1%, 0.2%, 0.5%, 1.0%, 1.5%, 2.0%), additional carbon source (glucose, galactose, lactose, maltose, sucrose, mannitol, potato and corn starch, soluble starch, soybean meal) and nitrogen (NH4Cl, NH4NO3, (NH4)2SO4, NaNO3, urea, peptone, tryptone, yeast extract and soybean meal) at a concentration of 1%. Keratinase activity was assessed by the UV absorption at 280 nm of the hydrolysis products of keratin-containing raw materials. Protein was determined by the Lowry method. Results. The dynamics of the enzyme synthesis showed that the culture of B. megaterium UCM B-5710 exhibited the highest keratinase activity on the 3rd day, and complete splitting of feathers was observed on the 4–5th days. The selection of the concentration of the keratin-containing substrate showed that 0.5% is the optimal concentration. The study of the influence of the initial pH value of the nutrient medium indicates that the culture grew well at pH 6.0–7.0 and pH 9.0–11.0, but at pH 8.0 its growth was very weak. The culture exhibited the maximum keratinase activity at pH 10.0. In addition, at this pH value, complete splitting of feathers was visually observed. The influence of such a key factor as temperature on the growth and synthesis of the enzyme by B. megaterium UCM B-5710 culture demonstrated complete splitting of feathers already on the 2nd day of cultivation at 42°C, at 21°C the culture split feathers very poorly. The introduction of the inoculum into the composition of the nutrient medium in an amount of 15% of the volume of the medium and the mixing intensity of 212 rpm turned out to be optimal. Besides, it was shown that the introduction of an additional source of carbon or nitrogen had an ambiguous effect on the level of keratinase activity of B. megaterium UCM B-5710. Complete inhibition of enzyme synthesis was observed when ammonium sulfate was added to the nutrient medium, and partial inhibition was observed in the case of glucose, lactose, and maltose. Potato, corn, and soluble starch stimulated keratinase synthesis. The majority of inorganic nitrogen sources (ammonium chloride and nitrate) did not affect the synthesis of B. megaterium UCM B-5710 keratinase, while organic sources (urea, peptone, tryptone, yeast extract) increased the level of keratinase activity by 20–50%. However, the most effective result was obtained using soybean meal, the addition of which to the nutrient medium increased the keratinase activity by 2.5 times. Conclusions. As a result of the studies, the optimal conditions for cultivation of the B. megaterium UCM B-5710 strain were selected: the optimum temperature for the growth and development of the culture is 42°C, the initial pH value is 10.0, the stirring speed is 212 rpm and the amount of inoculum introduced is 15%, an additional source of carbon and nitrogen in the form of soybean meal at a concentration of 0.5%. This made it possible to increase the activity of keratinase by 4 times.

Bacteria of the genus Bacillus are known for their ability to mineralize organic phosphorus compounds. Phytates constitute up to 60–80% of the total plant phosphorus and almost 50% of soil organic phosphorus. Phytates phosphorus is unavailable for plants. Bacillus can synthesize phosphatases both wide spectrum of action, and highly specific phytases that catalyze the hydrolysis of phytates. Therefore, the aim of this work was to study the growth and phytase activity of Bacillus subtilis IMV B-7023, which is the component of the ”Azogran” complex bacterial preparation for plant growing. Methods. The growth activity of bacteria was studied by cultivation methods, the phytase activity – by measuring the amount of phosphate released from sodium phytate during the enzymatic reaction. Results. It was shown that B. subtilis IMV B-7023 assimilated phytate as the source of phosphorus nutrition during cultivation in media with 0.5, 1.0 and 2.0 g/L of sodium phytate. The highest growth activity of these bacteria was observed after two days of cultivation in medium with 1.0 g/L of phytate. The number of bacteria was (3.91±0.32)×109 CFU/mL under these conditions. At the same time, B. subtilis IMV B-7023 demonstrated a low level of phytate assimilation as a source of carbon nutrition. Thus, after two days of cultivation the number of bacteria increased from (4.12±0.09)×106 CFU/mL to (1.07±0.07–3.11±0.51)×107 CFU/mL in the presence of 0.5–2.0 g/L phytate in the medium and the absence of another carbon source. It was determined that strain B. subtilis IMV B-7023 had phytase activity, the highest activity (221.85±0.12 U/g) was on the first day of their cultivation in medium with inorganic phosphates. It should be noted that B. subtilis IMV B-7023 phytase activity was lower during cultivating in medium with sodium phytate as a source of phosphorus nutrition, than in medium with inorganic phosphates. The obtained fact may be due to phytate hydrolysis by widespecific phosphatases. Higher rates of phytase activity obtained on the first and third days compared to the second and fourth days of bacterial cultivation may indicate the expression of phosphatases genes only in the period required for maximum bacterial development, in the absence of these proteins in the media. At the same time, the phytase activity of B. subtilis IMV B-7023 after 2 days cultivation in a media with 0.5 and 1.0 g/L of sodium phytate (194.80±0.15 U/g and 160.90±0.13 U/g, respectively) as the source of carbon and phosphorus was higher compared to the activity of bacteria on medium with inorganic phosphates (137.79±0.10 U/g). This may be caused by the synthesis of a larger number of highly specific phosphatases (phytases) in bacterial cells at the presence of only phytate in the medium as a substrate. Conclusions. B. subtilis IMV B-7023 strain is characterized by growth on nutrient medium with sodium phytate and phytase activity. Because they are soil microorganisms used as the component of the ”Azogran” complex bacterial preparation for plant growing, the ability to hydrolyze and assimilate phytate is important for functioning of this strain in the rhizosphere. The obtained results extend the understanding of B. subtilis IMV B-7023 influence on phosphorus nutrition and development of plants.

Alveolar osteitis (AO) is one of the most common infectious complications after dental extractions. The data on the species composition of AO pathogens and their susceptibility to antimicrobial drugs can be the basis for their empirical use in case of inflammatory process aggravation. Objective. To determine the species composition and susceptibility to the antimicrobial agents of microorganisms, which were detected in patients with AO, who sought medical help in the oral surgery department of the dental medical center of Bogomolets National Medical University. Methods. Throughout 2018–2021, microbiological examination of tooth sockets from 30 patients with AO and 20 patients without AO was performed. The studied biological material was plated on appropriate nutrient media for isolation of aerobic, facultative and obligate-anaerobic microorganisms. Anaerobic conditions were achieved in GENbox 7.0 L and GENbox 2.5 L aerostats using GENbox anaerobic packages (“Biomerieux”, France). The genus and species identity of the bacteria were determined according to Bergey. Antibiotic susceptibility of the isolated strains was determined by disk diffusion method. Results. It was found that most commonly microorganisms from tooth sockets in case of AO are: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus spp., Escherichia coli, Bacteroides spp., Clostridium spp., and Candida species, as well as their mixed cultures of 3–5 species of microorganisms. These aerobic and facultative anaerobic bacteria were susceptible to amoxicillin, ceftriaxone and ciprofloxacin in 92.6–100% of cases. The growth of anaerobic bacteria in 100% of cases was inhibited by colistin and meropenem. Conclusions. AO developing is caused by pathological colonization of socket of the extracted tooth by representatives of endogenous microbiota, namely Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus spp., Escherichia coli, which are present mainly in the mixed cultures with Candida albicans. For empirical antibiotic therapy of complicated forms of AO, amoxicillin or ceftriaxone or ciprofloxacin in complex with colistin or meropenem should be used, since these drugs suppress the growth of 92.6–100% of strains of aerobic, facultative and obligate anaerobic microorganisms, which are potential pathogens of the purulent forms of AO.

Cerium dioxide nanoparticles (CeO2) have a wide range of biological properties, including antiviral activity. Preparations based on cerium oxide nanoparticles can be effective against animal RNA viruses, which have the greatest epizootic significance for Ukraine and potentially against human viruses, including SARS-CoV-2. In our studies, we determined the effect of cerium dioxide nanoparticles on bovine viral diarrhea virus (BVDV), a pestivirus that is the etiological agent of bovine viral diarrhea. To determine the antiviral efficacy of CeO2, a cytopathogenic strain of BVDV “BK-1” was used as an etiological agent of bovine viral diarrhea, which induces a cytopathic effect on cow embryo lungs (CEL) cell culture. When evaluating the antiviral efficacy of CeO2, it was determined that the MPC for CEL cell culture is 0.057 μg/cm3, and the CC50 is 0.077 μg/cm3. The antiviral activity of CeO2 was evaluated by the IC50 index, which was 0.036 μg/cm3 and the selectivity index, which after co-administration of CeO2 with BVDV was 2.14. There was a decrease in the titer of infectious activity of the virus during treatment with CeO2 by 2.09 lg TCD50/cm3.

Weeds as reservoirs for destructive plant pathogens have a significant impact on the viral epidemiology, ecology and, as a result, on local economy, and are therefore being investigated in many parts of the world. Thus, the aim of this study was to investigate virus occurrence in red dead-nettle plants (Lamium purpureum L.) widespread in urban and field conditions throughout the in the Kyiv region of Ukraine. Methods. Field crop observations, visual diagnosis, biological testing of the virus, immunoassay (ELISA), polymerase chain reaction with reverse transcription (RT-PCR), sanger sequencing of partial genome sequences of PVX, PVY, PVS, PVM. Results. The results obtained in the study indicate that Lamium plants could be alternative weed hosts of number important viral diseases including potatoes and other vegetables. Serological and molecular test results evidence plants were infected by Potato virus X, Potato virus Y, Potato virus M, Potato virus S and therefore Lamium L. species can serve as a potential source of inoculum for wide range of vegetables and ornamentals. This study is the first report of Lamium plants being naturally infected with Potato virus M and Potato virus S in central Europe. Conclusions. These plants are alternative host of mixed infection with viruses belonging to different families: Alphaflexiviridae, Betaflexiviridae and Potyviridae.

Monoaromatic compounds are related to widespread pollutants of soil and groundwater. Among them phenol is one of the most toxic and carcinogenic compounds. Therefore biodestruction of phenol is of much importance for environment protection. The use of metabolic potential of microorganisms for depolluting environment is a safe and economical alternative to widely used physicochemical methods. Aim. To assess efficacy of phenol detoxification with strain Rhodococcus aetherivorans UСM Ac-602 using the phytotesting method. Methods. Bacteria were cultivated in liquid mineral medium with initial concentration of phenol 500, 750 and 1000 mg/L as a single source of carbon and energy. Cultivation time was 24 h, 48 h and 72 h respectively. Phytotoxicity was determined in express-test with use of seeds of spring wheat variety “Pecheryanka” (Triticum aestivum L.). Plant seeds were incubated by temperature 20±2°C during 7 days in Petri dishes with filter paper treated with respective phenol aqueous solutions or post-fermentative cultural fluids (PFCFs). PFCFs were obtained after cultivation of strain in growth medium with same concentration of phenol. Morphometric parameters of wheat were assessed against control plants cultivated on distilled water. Comparative analysis of samples toxicity and toxicity class determination was performed according to Kabirov method by calculation of index of test factor toxicity (ITF). Results. Phenol aqueous solutions and PFCFs were much different in effect on wheat. Phenol solutions 500 and 700 mg/L have shown significant inhibitory effect on all initial growth parameters of test plants. The weakest growth inhibition was induced by phenol concentration of 500 mg/L which caused decrease in number of germinated seeds by 59.6%, shoot length – by 59.7%, root length – by 84.5%, sprout dry weight – by 35.0%. In the presence of phenol concentration of 750 mg/L these indicators increased by 7−30%; roots of test plants were the most sensitive to effect of phenol. Phenol concentration of 1000 mg/L caused total seed mortality. Unlike phenol aqueous solutions PFCFs have shown insignificant effect on all morphometric indicators of plants compared to control. Similar effects on plants were observed in the presence of PFCFs obtained from cultivation of strain R. aetherivorans UСM Ac-602 in the growth medium with initial concentrations of phenol of 500 and 750 mg/L. Under the influence of these PFCFs, the number of germinated seeds decreased on average by 15.8%, root length decreased by 19.8%, at the same time shoot length and their dry weight increased by 17.8% and 7.2% respectively. More negative effect on wheat was shown by PFCF obtained after strain cultivation on medium with phenol concentration 1000 mg/L. It caused reduction in number of germinated seeds by 18.0 %, shoot length – by 25.3%, root length – by 29.0%, sprout dry weight – by 7.2%. For phenol aqueous solutions ITFs had much lower values 0–0.40 than for PFCFs (0.71–1.0). Conclusions. Based on data obtained in this research it was concluded that strain R. aetherivorans UCM Ac-602 performs active detoxification of high-concentrated phenol-containing media. Analysis of calculation results for ITF medium values (ITFm) had shown that under the influence of studied strain there was a decrease in toxicity of phenol solutions (500, 750 and 1000 mg/L). According to Kabirov toxicity scale it was assessed that toxicity of phenol solutions with initial values of classes II (high) and I (very high) was decreased to IV (low) and V (normal level). Our results demonstrate ecological safety of the end products of phenol destruction with strain R. aetherivorans UCM AC-602 and prospects of its use in biotechnologies for environment detoxification from phenol pollutions.

Currently, a lot of researcher’s attention is devoted to the problem of microbiologically influenced corrosion (MIC), since it causes huge damages to the economy, initiating the destruction of oil and gas pipelines and other underground constructions. To protect industrial materials from MIC effects an organic chemical inhibitors are massively used. However, the problem of their use is associated with toxicity, dangerous for the environment that caused the need for development the alternative methods of MIC repression. At the review, the data about different types of inhibitors-biocides usage has provided. The chemical inhibitors features are given and the mechanisms of their protective action are considered. The screening results and use of alternative and eco-friendly methods for managing the effect of corrosion caused by sulfate-reducing bacteria (SRB) are highlighted. Methods of joint application of chemical inhibitors and enhancers, such as chelators, biosurfactants, which contribute to reducing the concentration of chemical inhibitors, are discussed. The possibility of disruption of the quorum sensing interaction in the bacterial community to prevent the biofilm formation is considered. The information about the use of natural plant extracts, food waste, as well as by-products of agro-industrial production to combat MIC is provided. The development of biological corrosion control methods (to combat MIC) is of great importance for creating the best alternative and eco-friendly approaches to managing the effect of corrosion caused by SRB. The analysis of the literature data indicates the need to find the best alternatives and environmentally friendly solutions.