The paper analyzes the advanced technologies of wastewater treatment with a high content of organic pollutants and ammonium ions. Two different bi-stage scenarios for the treatment of such effluents are proposed. The first scenario includes the pretreatment in aerated lagoons and the final stage at the wastewater treatment plants after appropriate dilution with municipal sewages. The second scenario also includes the first stage in aerated lagoons with a tertiary treatment at the plant for cyanobacteria cultivation with the use of obtained biomass for biofuels production. The effects of the aeration periodicity on the leachate treatment efficiency and also on the composition of microbiocenosis in the aerated lagoons were investigated. The leachates of the Lviv landfill of municipal solid waste (MSW) were used in experimental investigations. The Lviv landfill of MSW was used for domestic and industrial wastes deposition for almost six decades, since the 1960s. It was found that the highest effect of ammoniacal nitrogen removal was obtained in the mode of periodic aeration, with the cycle duration of two hours including the one-hour aeration. It was found that the microorganisms extracted from the leachates of the Lviv MSW landfill are prospective for the new biotechnologies of treatment of the highly concentrated wastewaters, since this microbiocenosis is resistant to the widespread pollutants, in particular to heavy metal ions.

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The toxicity of metal ions to microorganisms, in particular at high concentrations, is one of the main impediments to their usage in remediation technologies. The purpose of this work is to analyze the possibility of usage by bacteria of the Desulfuromonas genus, isolated by us from Yavorivske Lake, of ferrum (ІІІ) and manganese (IV) ions at concentrations in the medium of 1,74–10,41 mM as electron acceptors of anaerobic respiration to assesss resistance of sulphur reducing bacteria strains to heavy metal compounds. Cells of Desulfuromonas acetoxidans ІМV V-7384, Desulfuromonas sp. Yavor-5 and Desulfuromonas sp. Yavor-7 were cultivated for 10 days at 30 °C under anaerobic conditions in Kravtsov-Sorokin’s medium without sulphate ions, sulphur, with cysteine as the sulphur source (0.2 g/l) and sodium lactate or citrate as the electron donor (17.86 g/l), in which were added sterile 1 M solutions of C6H5O7Fe and C4H4O4 (control) and also weights of MnO2 to their terminal concentrations 1.74, 3.47, 5.21, 6.94, 10.41 mM. Biomass was determined by the turbidimetric method. In the culture liquid the presence of Fe3+ and Mn4+ were qualitatively determined, and the content of Fe2+ in reaction with о-phenanthroline was determined quantitatively. It was established that sulphur reducing bacteria used with different intensity ferrum (ІІІ) and manganese (IV) ions as electron acceptors during the process of anaerobic respiration at concentrations of 1.74–10.41 mM C6H5O7Fe and MnO2 in the medium, which demonstrated the important role of the investigated microorganisms in reductive detoxication of natural and technogenic media from oxidized forms of transitional heavy metals. An insignificant difference in biomass accumulation during usage of 5.21–10.41 mM ferrum (ІІІ) ions and fumarate is caused by toxicity of the metal ions to cells since the high redox potential of the Fe(III)/Fe(ІІ) pair with increase in concentrations of electron acceptors in the medium did not lead to increase in the biomass accumulation level. The greatest biomass of the bacteria accumulated on the 8–10th days in the medium with the lowest concentration of C6H5O7Fe – 1.74 mM (up to 2.77 g/l), and the lowest biomass – with highest concentration – 10.41 mM (up to 2.41 g/l). After 10 days of cultivation the bacteria of all strains had fully used the ferrum (ІІІ) ions present in the medium. A biomass yield almost twice as low was revealed after manganese (IV) oxide was used by bacteria compared with its use of ferrum (ІІІ) citrate and fumarate at all studied concentrations of electron acceptors in the medium. The highest biomass of bacteria accumulated in the medium with the lowest MnO2 content – 1.74 mM (up to 1.35 g/l), and the lowest biomass in the medium with the highest content – 10.41 mM (up to 1.15 g/l). After 10 days of cultivation bacteria of all strains had not fully restored the manganese (IV) ions present in the medium. The greatest biomass compared with other strains after growth in medium with different C6H5O7Fe and MnO2 contents was accumulated by the strain Desulfuromonas sp. Yavor-7. Since sulphur reducing bacteria strains proved to be resistant to Fe3+ and Mn4+ high concentrations (up to10.41 mM) they can be successfully used in technologies of environmenal remediation from sulphur and heavy metal compounds.

Moroz, O. M., Hnatush, S. O., Tarabas, O. V., Bohoslavets, C. I., Yavorska, G. V., & Borsukevych, B. M. (2018). Sulfidogenic activity of sulfate and sulfur reducing bacteria under the influence of metal compounds. Biosystems Diversity, 26(1), 3-10.Due to their high content in natural environments, heavy metals exhibit toxic effects on living organisms, which leads to a decrease in the biological diversity and productivity of ecosystems. In niches with low oxidation reducing potential, sulfate and sulfur reducing bacteria carry out the reducing transformation of oxidized sulfur compounds with the formation of significant amounts of hydrogen sulfide. H 2 S produced by bacteria interacts with metal ions, precipitating them in the form of sulfides. The aim of this work was to investigate the influence of lead, cuprum (II), iron (II) and manganese (II) salts on the production of hydrogen sulfide by bacteria of the Desulfovibrio and Desulfuromonas genera, isolated from Yavorivske Lake, and to evaluate the efficiency of their use for purifying media, enriched with organic compounds, from hydrogen sulfide and heavy metals. The content of heavy metal ions in the water of Yavorivske Lake was determined by the spectrophotometric method. The bacteria were grown for 10 days at 30 °C in the Kravtsov-Sorokin medium under anaerobic conditions. To study the influence of metal ions on bacteria growth and their H 2 S production, cells were incubated with metal salts (0.5-4.0 mM), washed and grown in media with SO 4 2or S 0 . To determine the level of metal ions binding by H 2 S, produced by bacteria, cells were grown in media with metal compounds (0.5-4.0 mM), SO 4 2or S 0 . Biomass was determined by turbidimetric method. In the cultural liquid the content of H 2 S was determined quantitatively by spectrophotometric method, and qualitatively by the presence of metal cations. The content of metal sulfides in the growth medium was determined by weight method. Sulfate and sulfur-reducing bacteria were resistant to 2.0 mM Pb(NO 3 ) 2 , 2.5 mM CuCl 2 , 2.5 mM FeCl 2 × 4H 2 O and 2.0 mM MnCl 2 × 4H 2 O, therefore they are promising for the development of biotechnologies for the purification of water resources contaminated by sulfur and metal compounds. When present in a medium with sulfates or sulfur of 1.0-1.5 mM lead, cuprum (II), iron (II) or manganese (II) ions, they almost completely bind with the H 2 S produced by bacteria in the form of insoluble sulfides, which confirms the negative results of qualitative reactions to their presence in the cultural liquid.

Technogenic reservoirs mainly contain several possible electron acceptors of anaerobic respiration, many of which are dangerous to the environment. The succession of their reduction (and thus detoxification) by sulfur reducing bacteria is not yet sufficiently studied. We investigated the influence of ferrum (III) citrate, present in the cultivation medium, on the reduction of sulfur, nitrate and nitrite ions by sulfur reducing bacteria Desulfuromonas acetoxidans IMV B-7384, Desulfuromonas sp. Yavor-5 and Desulfuromonas sp. Yavor-7, isolated from Yavorivske Lake. It was established that ferrum (III) citrate inhibits the biomass accumulation and hydrogen sulfide production by bacteria of Desulfuromonas sp. after simultaneous addition to the medium of 3.47 mM S0 and 1.74–10.41 mM ferrum (III) citrate, as compared with growth and hydrogen sulfide production by bacteria in the medium with only sulfur. In the medium with the same initial content (3.47 mM) S0 and ferrum (III) citrate bacteria produced ferrum (II) ions at concentrations 3.5–3.9 times higher than that of hydrogen sulfide. Ferrum (III) citrate inhibits the biomass accumulation, the nitrate or nitrite ions reduction and the ammonium ions production by bacteria of Desulfuromonas sp. after simultaneous addition to the medium of 3.47 mM NaNO3 or NaNO2 and 1.74–10.41 mM ferrum (III) citrate. In the medium with the same initial content (3.47 mM) NaNO3 and ferrum (III) citrate, bacteria produced ammonium ions at concentrations in 1.1 times higher than that of ferrum (II) ions. In the medium with the same initial content (3.47 mM) NaNO2 and ferrum (III) citrate, bacteria reduced 1.5–1.6 times more ferrum (III) than nitrite ions with production of ferrum (II) ions at concentrations 1.7 times higher than that of ammonium ions. The process of nitrate reduction carried out by bacteria of Desulfuromonas genus was less sensitive to the negative influence of ferrum (III) citrate, compared to the process of nitrite ions reduction. When the reduction of nitrate ions by bacteria in the presence of 1.74–10.41 mM ferrum (III) citrate decreased by 1.4–2.2 times, then the reduction of nitrite ions decreased by 1.8–3.2 times compared to their reduction in media with only NaNO3 or NaNO2, respectively. Although the reduction of ferrum (III) by cells in media with 3.47 mM S0, NaNO3 or NaNO2 and 1.74–10.41 mM ferrum (III) citrate decreased by 1.6–2.7, 1.6–2.7 and 1.1–2.2 times, respectively, compared to the reduction in medium with only ferrum (III) citrate, the investigated strains of bacteria were resistant to high concentrations of trivalent ferrum compounds and can therefore can be used in technologies of complex purification of environments polluted by heavy metal and nitrogen compounds.

The effect of stressors, including heavy metal ions such as Cu2+, promotes activation of free radical processes in the cells of microorganisms, which causes changes in their physiological and biochemical properties and the structure of bacterial membranes. The aim of this work was to assess the influence of copper (II) sulphate on intensity of lipid peroxidation (LPO) of Chlorobium limicola IMV K-8 by measuring the content of primary (conjugated dienes and lipid hydroperoxides) and secondary lipid peroxidation products (TBA-reactive products). Microorganisms were cultivated at a temperature of 28 °C in GSB cultivation medium with exposure to light of wavelength 700–800 nm and intensity 40 lux. A suspension of C. limicola ІМV К-8 cells in the phase of exponential growth was treated for one hour with metal salt solution in concentrations 0.05–0.50 mM for investigation of the influence of copper (II) sulphate on its physiological and biochemical properties. The control samples did not contain any copper (II) sulphate. Biomass was determined by turbidity of diluted cell suspension by application of photoelectric colorimeter KFK-3. A mixture of n-heptane and isopropyl alcohol was added into cell-free extract for conjugated dienes determination. The samples were incubated at room temperature and centrifuged. Water was added into the supernatant and the samples were stirred. Ethanol was added to the heptanes phase and adsorption was measured at 233 nm. The content of lipid hydroperoxides was determined by a method based on protein precipitation by trichloroacetic acid followed by addition of ammonium thiocyanate. The concentration of TBA-reactive products in the cell-free extracts was determined by color reaction with malondialdehyde and thiobarbituric acid exposed to high temperature and acidity of the medium, which causes formation of trimetinic adduct with maximal absorption at 532 nm. It was shown that when CuSO4 was added to the incubation medium the content of conjugated dienes and lipid hydroperoxides increased with the enhancement of salt concentration. However, its value decreased by the seventh and eighth days of cultivation. The content of TBA-reactive products under the influence of copper (II) sulphate varied depending on the duration of cultivation and concentration of the metal. Its highest quantity was observed on the eighth day of cultivation. Thus it was determined that under the influence of CuSO4 the content of conjugated dienes, lipid hydroperoxides and TBA-reactive products increases. This indicates the increased activity of lipid peroxidation processes and the free radical chain reaction damage mechanism to lipids under these conditions.

To investigate the changes of fatty acid composition of green sulphur bacteria Chlorobium limicola ІМV К-8 cells at the influence of copper (II) sulfate. Methods. Microbiological, biochemical, biometrical. Results. The increase of content of long chain saturated fatty acids of C. limicola ІМV К-8 cells, in particular pentadecanoic, hexadecanoic, heptadecanoic and octadecanoic acids was observed under the influence of copper (II) sulfate in concentrations which caused decrease of biomass accumulation up to 70 %. Among the first reactions of adaptation of C. limicola ІМV К-8 cells under these conditions are cis/trans isomerisation of monounsaturated fatty acids and synthesis of cyclopropane fatty acids. Maintenance of appropriate level of membrane fluidity is provided by branched chain fatty acids. Conclusions. Under the influence of copper (II) sulfate on C. limicola ІМV К-8 cells fatty acids composition of membranes is changed, which causes the increase of membranes fluidity, and, probably, is contributed to more efficient efflux of Cu 2+ ions.

Photolithotrophic sulfur bacteria are involved in biota functioning and have a biotechnological potential for bioremediation of contaminated environment, but the mechanisms of xenobiotics, in particular of heavy metal ions damaging action and the pathways of photolithotrophic bacteria adaptation under these conditions have not been established. In this work, the biochemical indicators of green photosynthetic bacteria Chlorobium limicola response to Cu ions were studied. C. limicola cells were incubated during one hour in buffer containing copper (II) sulfate in 0.05-0.5 mM concentrations and grown for 8 days in GSB medium. The content of Cu 2+ in cells was estimated by atomic absorption spectroscopy. The activity of enzymes of antioxidant defense, photosynthetic pigments and glutathione content, indexes of lipids unsaturation and membrane viscosity as markers of membrane fluidity were estimated. It was shown that the response of green photosynthetic bacteria C. limicola to Cu 2+ action varied depending on cations concentration. Under the influence of metal salt at 0.05 mM concentration, the activity of antioxidant enzymes, GSH/GSSG ratio, the content of photosynthetic pigments and membrane fluidity indexes were higher as compared with control. Under the increase of copper (II) sulfate concentration to 0.25 mM, the activity of antioxidant enzymes was lower compared to the response of the cells under the influence of 0.05 mM copper (II) and the GSSG content was increased. Under the influence of 0.5 mM copper (II) the indexes of membrane fluidity did not differ from the control, but superoxide dismutase and peroxidase activity inhibition and the further decrease of GSH/ GSSG ratio were observed followed by the highest Cu 2+ cations accumulation in cells and significant decrease of bacteria biomass growth. K e y w o r d s: green bacteria, Cu cations, antioxidant protection, membrane fluidity, adaptation.