Biotechnologia Acta

12-31

Authors: Klyuchko O. M.

The work was aimed to summarize the results describing the mechanisms of interaction of some aromatic hydrocarbons, isolated from Arthropodae, with biological membranes. Neurophysiological properties of the substances obtained from spider venoms, derivatives of phenol and indole with polyamine substituents, were studied by the method of transmembrane electrical currents registration in voltage clamp mode. Their influence was revealed as a result of chemo sensitive ionic currents characteristics changes due to the action of these substances on the membranes of rat hippocampal neurons. All of the studied substances were antagonists of glutamate channel-receptor complexes. Their blocking activity characteristics were examined as follows: caused reversible and irreversible blocking effects, the effects dependence on membrane holding potential, difference of their action on activated and inactivated receptors, kinetics of blocking actions, type of antagonism. Antagonists amphiphilic properties and the role of aromatic groups and polyamine substituents during interaction with the membranes were analyzed. The results of such analysis were accepted as a basis for development of physical model of artificial memory device. Principles of its work were explained as well. The results regarding the influence mechanisms of the studied substances on biological membranes were summarized. Some characteristics and functioning peculiarities of the suggested elements of storage devices were discussed. The properties of the proposed substances-analogues meet a number of requirements for the elements of such nano-devices.

38-47

Authors: D. R. Abdulina, G. O Iutynska, A. I. Aniskina, M. M. Nikitin2

The study of detection effectiveness of sulfate-reducing bacteria (SRB) in the samples from various ecotopes by microbiological (cultural by serial dilutions) and molecular biological (by real-time PCR) methods with designed test-systems lyophilized on the silicon microchip was performed. The developed DSRM and SRB2 test-systems for detection of the functional gene dsrA presence, encoding one of the key enzyme of dissimilatory sulphate-reduction pathway — dissimilatory sulfite reductase were used. It was found that the minimal determined SRB titres in water samples were 104 cells/ml and in soil samples they were 102–105 cells/g of absolutely dry soil. In natural and man-caused samples, the amount of SRB detected by the microbiological method was correlated with calculated values determined by the molecular biological method, Pearson’s indexes were r = 0.41–0.69 (k = 11, P ≤ 0.01–0.05). Thus, real-time PCR assay with designed test systems lyophilized on the silicon microchips is a high-quality and rapid method for the detection of SRB in various natural and man-caused ecotopes.

56-64

Authors: Relina L. I., Suprun O. H., Boguslavskyi R. L., Didenko S. Yu., Vecherska L. A., Golik O. V.

Although wheat has never been considered an oil crop, oil from wheat germs and bran is valuable because it contains important bioactive compounds. Most of studies in this area were conducted with traditional commercial wheat varieties. At the same time, the interest of breeders, producers and consumers is going back to ancient and underutilized wheats species. In this respect, we set the purpose to evaluate tetraploid wheat species (Triticum. dicoccoides var. pseudojordanicum, Triticum dicoccum, Triticum timofeevii, Triticum persicum var rubiginosum, Triticum durum var. falcatamelanopus, Triticum polonicum var. pseudocompactum and Triticum aethiopicum var. densimenelikii) for fatty acid composition. Grain was harvested in 2015, 2016, 2017, 2018 and 2019. Fatty acid methyl esters were prepared by the modified Peisker method. Fatty acid composition was analyzed by gas chromatography. Six major fatty acids were found in grain of tetraploid wheat species, with linoleic acid being the most abundant. The ratio of unsaturated acids to saturated ones in grain of wild emmer T. dicoccoides var. pseudojordanicum was slightly lower than in the domestic emmer varieties. T. timofeevii, emmer varieties Holikovska and Romanivska and radium wheat variety Spadschina had the most beneficial unsaturated/saturated ratios. As conclusion there was no evidence of deterioration in the grain quality in terms of unsaturated fatty acid levels, and we observed no patterns in variability of fatty acid contents across the species under investigation. Fatty acid composition was analyzed by gas chromatography. Six major fatty acids were found in grain of tetraploid wheat species, with linoleic acid being the most abundant. The ratio of unsaturated acids to saturated ones in grain of wild emmer T. dicoccoides var. pseudojordanicum was slightly lower than in the domestic emmer varieties. T. timofeevii, emmer varieties Holikovska and Romanivska and durum wheat variety Spadschina had the most beneficial unsaturated/saturated ratios. There was no evidence of deterioration in the grain quality in terms of unsaturated fatty acid levels. We observed no patterns in variability of fatty acid contents across the species under investigation.

15-29

Authors: О. V. Kyrychenko

The purpose of the work was to evaluate the symbiotic productivity of soybean‒rhizobium and wheat‒azotobacter phyto-bacterial systems under the action of N-acetyl-D-glucosamine (0.01 M; 0.1 M) in vitro on the cultures of nitrogen-fixing microorganisms Bradyrhizobium japonicum 634b and Azotobacter chroococcum Т79. We have used such indicators as plants biological and seed productivity, rhizobia nodulation ability and nitrogenase activity of soybean symbioses as well as wheat rhizosphere microbiota. It was shown that the biological activity of N-acetyl-D-glucosamine during incubation with soybean nodule bacteria had a higher level of realization of the rhizobia nodulation ability: plants were more actively infected (by 12%), a greater number of nodules were formed (1.2‒2.3 times) with their greater total mass per plant (1.4‒2.1 times) and the mass of each root nodule (1.2 times), as well as the nitrogen-fixing activity of the symbiosis (1.7 times) and the functional capacity of each morpho-structural symbiotic unit (1.4 times). It provided higher (by 14‒29%) seed productivity of this system as compared with the symbiosis formed by the rhizobia monoculture. Activation of basic physiological processes in wheat such as nitrogen fixation (the activity of the rhizosphere microbiota was increased by 1.1‒1.4 times) and photosynthesis (the content of chlorophylls in leaves was increased by 1.1‒1.2 times) with N-acetyl-D-glucosamine-modified azotobacter provided a higher level of realization of the productive potential of this system in comparison with both non-infected plants (by 15%) and the variant of seed inoculation with bacteria only (by 7%). While inoculants bacteria + glucosamine had positive effect on seed productivity of the symbiotic and associative systems, it was shown no significant change in the biological productivity of soybean and wheat plants. So, the use of N-acetyl-D-glucosamine as an additional agent of carbohydrate nature in inoculants with soybean nodule bacteria and soil diazotrophs of Azotobacter genus led to a more complete realization of the symbiotic and productive potential of phyto-bacterial symbiosis and association when compared to using a diazotrophs only. Our results indicated the possibility of practical use of acetylated glucose-containing aminosaccharide in the creation of complex inoculants based on nitrogen-fixing bacteria.

38-44

Authors: D. A. Ivanchenko

The work was aimed to study the synergy of the antimicrobial activity of the prodigiosin pigment with antibiotics against bacteria of the genera Bacillus, Staphylococcus and Streptococcus. The serial dilution method was used to evaluate antimicrobial compositions, which included inhibitors of cell wall synthesis: ampicillin, benzylpenicillin, vancomycin, cefazolin, and metronidazole (nitroimidazole derivatives) in combination with the pigment prodigiosin isolated from Serratia marcescens. Each combination was tested against the studied strains. The fractional inhibitory concentration index (FICI) for each combination was calculated to determine synergy, and the results were interpreted as follows: FICI ≤0.5 ‒ synergism; FICI > 4.0 ‒ antagonism; and FICI > 0.5-4 ‒ neutralism. It was shown that the ethanol extract of prodigiosin in combination with benzylpenicillin, vancomycin, cefazolin, and metronidazole interacted differently synergistically depending on the type of microorganism. The combinations of prodigiosin and metronidazole showed a synergistic effect against Bacillus subtilis, vancomycin and cefazolin against Staphylococcus aureus and benzylpenicillin against Streptococcus pyogenes. Other combinations of prodigiosin and antibiotics showed a neutral effect, and in the case of cefazolin against Str. pyogenes, even an antagonistic effect. Thus, the study showed the synergism of prodigiosin with antibiotics depending on the type of microorganism, contributed to a several-fold decrease in the minimum inhibitory and bactericidal concentrations of each component separately, and the results indicated that prodigiosin acted separately more efficiently against gram-positive non-spore-forming bacteria. This synergistic combination of antimicrobial agents had great potency to prevent bacterial resistance.

56-63

Authors: L. Sablii, M. Korenchuk

The aim of the work is to determine the rational parameters of the malt plant wastewater treatment from ferric compounds in a flow-through experimental bioreactor with the use of duckweed. The main tasks of the study were as follows: to determine the effect of the initial concentration of the ferrum, the amount of biomass introduced and the duration of the purification process to reduce the content of ferric compounds in the wastewater. The studies were carried out on existing sewage treatment plants. They used a semi-production unit incorporated into the technology before the decontamination step. The results show that the logarithmic nature of the biological process was detected in the concentration range from 0.2 to 1.3 mg/dm3. Under these conditions, the rational values of the duration of the purification process is found to be 3-8 h with the Lemna minor biomass value not exceeding 12 g/dm3. It has been first demonstrated that the effect of sewage treatment from ferric ions in the bioreactor with Lemna minor was up to 40% and depended on the initial concentration of the ferrum compounds in water at the existing wastewater treatment plants at a semi-production unit for biological treatment of wastewater from ferric compounds.

12-24

Authors: Öznur Özge ÖZCAN, Mesut KARAHAN

Recent research focused on finding new strategies in cancer therapy that did not have significant side effects and was more effective than traditional modules including the surgical intervention, radiation and chemotherapeutics. In this regard the nanoscale structures provide useful approaches for cancer treatment. So, the nanoparticle systems improve the efficiency of therapeutic drugs reducing their side effects. Although many studies reported the development of novel cancer cell therapies for future, the clinical success is lacking understand the effects of nanoparticle type, size and dose with their usage areas. Thus, this review was aimed to illustrate the usage of nanoparticles in cancer diagnostic, imaging and treatment.

35-43

Authors: Sakovich V. V., Stohnii Ye. M., Zhernosekov D. D., Rebriev A. V., Korolova D. S., Marunych R. Yu., Chernyshenko V. O.

The aim of this work was to identify and to study physical and chemical properties of the enzyme preparation which was obtained from the cultural liquid of Pleurotus ostreatus. The protease containing fraction was obtained from the cultural liquid by sodium chloride precipitation followed by dialysis and concentration procedures. Gelatinase and milk-clotting activity were defined by standard methods. The content of the protein component of the fraction was analysed by HPLC, Laemmli electrophoresis and MALDI-TOF analysis. Protease activity was proved by enzyme-electrophoresis. To identify the protease, mass-spectrometry was carried out using the MatrixScience database. To study the specificity of protease action, the series of chromogenic substrates were used: S2238, S236, S2251, S2765, Leu-pNa, Ala-pNa and S2302. The inhibitory analysis was carried out using EDTA, benzamidine, PMSF, PCMB. The obtained fraction possessed maximal protease activity at 45 °C. Meanwhile maximal milk-clotting activity was observed at 35 °C. The highest milk-clotting activity was shown at pH 5.0 and less than 3.0. The highest protease activity was shown at pH 6.0. Using HPLC method, it was found the main protein component and some minor proteins. According to the electrophoresis results, the main protein component of the fraction had molecular mass 45 kDa. Enzyme electrophoresis demonstrated that protease activity of the fraction was present in the zone corresponding to 45 kDa. When identifying trypsinolysis products, no homology was found with other known proteinases. It was shown that the protease hydrolyzed peptide bonds which were formed by carboxyl group of amino acids with hydrophobic side chains. The enzyme was inhibited by EDTA (ІС50 = 2.5 mМ). The maximal enzyme activity towards gelatin and Leu-pNa was shown in the presence of 5 mM calcium chloride. The new calcium-dependent metalloprotease with molecular weight 45 kDa was found in the cultural liquid of Pleurotus ostreatus. The enzyme had no homology with other known proteases and hydrolyzes peptide bonds formed by carboxyl groups of amino acids with hydrophobic side chains.

46=55

Authors: T. P. Pirog, B. S.Geichenko, A. O. Zvarych

The aim of the work was to study the possibility of Nocardia vaccinii IMV B-7405, Acinetobacter calcoaceticus IMV B-7241 and Rhodococcus erythropolis IMV Ac-5017 supernatants usage with various concentrations of surfactants for post-harvest processing of vegetables. N. vaccinii IMV B-7405, A. calcoaceticus IMV B-7241 and R. erythropolis IMV Ac-5017 were grown on used sunflower oil and ethanol. For vegetables treatment, supernatants of the culture fluid with surfactant concentration of 0.01‒0.5 g/l were used. The concentration of surfactants was determined by the gravimetric method after extraction with Folch mixture. The total number of heterotrophic bacteria and fungi on the surface of vegetables was determined by the Koch method on meat-peptone agar and wort agar, respectively. It was shown that treatment of broccoli, Brussels sprouts, sweet pepper and tomatoes with N. vaccinii IMV B-7405, A. calcoaceticus IMV B-7241 and R. erythropolis IMV Ac-5017 supernatants was accompanied by 6‒17 and 8–50 times decrease of bacteria and fungi number on their surface, respectively, compared with that on the surface of vegetables washed with tap water. The possibility of double use of the same supernatant for various batches of vegetables washing was established. Non-treated and water-washed vegetables spoiled faster than those treated with surfactant-containing supernatants did. N. vaccinii IMV B-7405, R. erythropolis IMV Ac-5017 and A. calcoaceticus IMV B-7241 exometabolites used for treating vegetables to extend their shelf life have the following advantages in comparison with known microbial surfactants: they exhibit high antimicrobial activity when the surfactant concentration was sometimes lower and in the form of supernatant, which lets you exclude the expensive stage of isolation and purification of the target product from the technological process. In addition, surfactant-containing supernatants are highly effective in their repeated use.

65-70

Authors: Malynovska I. M.

The aim of the work was to study the direction and intensity of mineralization and immobilization processes in different layers of gray forest soil at fallow fields, extensively and intensively used agrosoils. The research included laboratory analysis, microbiological studies, and statistical processing. For the fallow plots, the layers chosen for study were Hd – turf (010 cm), He – humus-eluvial (11–40 cm), Hi – humus-illuvial (4174 cm), Ih – illuvial-humus (75115 cm), Ip – transitional from the illuvial layer to bedrock (116156 cm), Pi – bedrock with occasional insertions of illuvial soil (157191 cm); for the agrosoils of the stationary experiments: He – humus-eluvial (0-10 and 11–40 cm), Hi – humus-illuvial (4174 cm), Ih – illuvial-humus (75115 cm), Ip – transitional from the illuvial layer to bedrock (11656 cm). We found that humus mineralization differed in some layers of the gray forest soil under these management regimes. At fallows, the intensity of humus mineralization tended to decrease with depth, and it was interrupted in the Ih and Ip layers. In the intensively used agrosoil, humus mineralization was more active in Hi- and Ih- layers. Comparatively, the activity of humus mineralization smoothly decreased in the profile of the extensively used agrosoil from the uppermost layer to the lower by 97.2%. The mineralization coefficient of Nitrogen compounds gradually decreased in the fallow ground and extensively used agrosoil, unlike the intensively managed agrosoil, in which the intensity of mineralization-immobilization of nitrogen compounds increased in the He and Hi layers. It was shown that the fallow ground had the more efficient system to transfer substrates and mineral ions down the profile to the lower layers. The difference in biologic activity between the upper and lower layers was maximum in the fallow ground, intermediate in the intensively used agrosoil and minimum in the extensive agrosoil.

29-41

Authors: O. Tigunova, N. Beyko, G. Andriiash, S. Shulga

Literary data on the organization of the genome, the connection of genes with the production and regulation of solvents, and the metabolic engineering of solventogenic clostridia are presented. The transition from the phase of the formation of acids to the phase of the formation of alcohols and the relationship of the latter with sporogenesis are analyzed. The main key genes (ak, pta, buk, ptb, thl, crt, bcd, BdhAB, ctfAB, adc, rub, Spo0A, adhE, hdb) that affect their course have been identified. The possibility of improving strains by genetic manipulations (inactivation of genes, entering of genes of other microorganisms, etc.) is shown. The effect of gene inactivation on solvent production is shown. An analysis of methods for increasing the accumulation of butanol showed the need to create effective recombinant producer strains for butanol supra synthesis using renewable raw materials.

53-71

Authors: S. V. Gorobets , L. A. Yevzhyk, I. A. Kovalchuk, O. V. Kovalev

The aim of the study was to produce magnetically controlled biosorbent based on fungi of champignon and shiitake, to determine the proportion of the magnetically controlled phase of the biomass of fungi when the magnetic fluid (MF) was added to the substrate and to explore the efficiency of extraction Fe3+ ions by shredded biomass of the fungus. The object of the study was mushrooms champignon Agaricus bisporus and shiitake Lentinula edodes grown in the laboratory. An effective and cheap way to remove waste biosorbent from the working environment is a high-gradient magnetic separation (HGMS), which takes place in high-speed mode. The separation of the magnetically controlled phase of fungi biomass A. bisporus and L. edodes was carried out by HGMS methods. It was investigated that when using the biomass of champignon grown on MF, the properties of the sorbent were significantly improved, the full saturation was 6 times faster in comparison with the biosorbent based on the biomass of the fungus grown without MF.

82-88

Authors: L. Sablii, M. Korenchuk, M. Kozar

The aim of this work was to establish the possibility of Lemna minor usage to improve the efficiency of wastewater treatment from nitrogen and phosphorus compounds. Due to their pollution resistanse, Lemna minor is often used for wastewater remediation. It’s capability of absorption and transformation of various compounds, promotes the deposition of suspended matter, saturates water with oxygen, intensifies the purification processes. Due to the rapid growth rate, duckweed absorbs a large amount of pollutants, thereby purifying water from them. However, there is insufficient data and information on the efficiency of nitrogen and phosphorous compounds extraction by Lemna minor wastewater. That is why the research direction was to determine the efficiency of wastewater treatment from biogenic nitrogen and phosphorus compounds by higher aquatic plants of Lemna minor. The necessary conditions for cultivation of higher aquatic plants of Lemna minor in autumn and winter were established. The efficiency of using duckweed of different mass for purification of waste water from biogenic compounds of nitrogen and phosphorus was investigated. The usage of duckweed together with other higher aquatic plants to investigate wastewater from biogenic nitrogen and phosphorus compounds was investigated. It was found that the efficiency of sewage treatment in the autumn-winter period should adjust the mode of illumination and temperature regime.

19-36

Authors: Gudzenko O. V., Varbanets L. D., Seifullina I. I., Martsinko E. E., Pirozhok O. V., Chebanenko E. A.

The aim of the work was to determine the influence of a number of coordination compounds of germanium, in particular cation-anionic type, on the activity of α-L-rhamnosidases of three producers: Cryptococcus albidus, Eupenicillium erubescens and Penicillium tardum ІМВ F-100074. Activity of α-L-rhamnosidases was determined according to Davis method, using naringin as substrate. The specific α-L-rhamnosidase activity of preparations was 12 units/mg of protein for C. аlbidus, 120 units/mg for E. еrubescens and 27 units/mg for P. tardum. Protein concentration in preparations was 0.01 mg/ml. Gernanium compounds were synthesized in alcohol-water solution according to standardized methods. In all compounds, the same bis (citrato) germanate anion is realized; however, the composition of the cation changes: the protonated form of phenanthroline and bipyridine or the complex cations of d-metals. It was established that none of the complex compounds exhibited an inhibitory effect on the activity of the enzymes under study. Maximum activating effect was observed with compound tris(bi-pyridine)nickel(II) bis(citrate)germanate monohydrate in concentration of 0.1% on α-L-rhamnosidases of C. albidus (10-fold), E. еrubescens (2.5-fold) and P. tardum (5-fold). Notably, enzymatic activity increased by 45, 47 and 60% respectively in presences of compound bi-pyridine bis(citrate)germanate dihydrate in same concentration. Hence, it was shown that these two compounds can be used as effectors of studied enzymes.

34-41

Authors: Poyedinok N. L., Tugay T. I., Tugay A. V., Mykchaylova O. B., Sergiichuk N. N., Negriyko A. M.

The aim of the work was to study the effect of nitrogen concentrations on photo-induction of growth, enzymatic activity and synthesis of melanin by the medicinal fungus Inonotus obliquus (Ach.: Pers.) Pilát from the Collection of cultures of cap mushrooms of Kholodny Institute of Botany of the National Academy of Sciences of Ukraine. Irradiated by light of low intensity, different coherence and in different wavelength ranges, mycelium was cultivated in a dynamic mode on a glucose-peptone medium with different concentrations of total nitrogen. The concentration of the nitrogen source was not shown to significantly affect the photo-induced stimulation of the growth of I. obliquus. The increase in biomass accumulation of mycelium photoactivated in different modes was almost the same in all variants of the experiment, compared with the biomass of not-irradiated mycelium. A reliable dependence of the photo-stimulation of melanin synthesis on the concentration of nitrogen in the medium was established. Reduced nitrogen concentration more than twice increased the stimulating effect of low-intensity laser radiation with a wavelength of 488 nm. Using substrate with a reduced content of the nitrogen source is advisable to increase the photo-induced stimulating effect in the production of extracellular catalase, tyrosinase and polyphenol oxidase, intracellular peroxidase. Thus, the cultivation parameters of I. obliquus and the light treatment regimes of the inoculum should be adjusted according to the target bioactive components.

50-56

Authors: Sablii L., Korenchuk M., Kozar M.

The aim of the research was to determine the degree of influence of nitrate wastewater concentration on the process of removal of phosphorus compounds by sequential water treatment in anoxic and aerobic conditions in an activated sludge system. Model wastewater solutions were used for research with following parameter: biochemical oxygen demand for 20 days –200 mgО2/l; phosphate concentrations – 11.87‒12.38 mg/l; nitrate concentrations – 21.0; 36.0 and 48.0 mg/l. Аctivated sludge was added to them with content in solutions 2.2 mgО2/l to provide biological processes. For simulation of the biological process of dephosphotation in wastewater with usage of activated sludge in sequentially formed anoxic and aerobic conditions, a model sequential reactor ‒ SBR reactor ‒ was used. As the results show, with the increase in the concentration of nitrates at the inlet from 21.0 to 48.0 mg/l, the phosphate concentration in the treated solutions at the outlet from the bioreactor increases by 7.3%. Thus, from the work presented here, it can be concluded that for successful and effective implementation of the dephosphotation process the elimination of the nitrate present in wastewater is required. It is reasonable to separate processes of denitrification and dephosphotation in separate structures with the provision of minimal nitrate influence on the phosphorus removal from wastewater.