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.

The capacity of sulfur-reducing bacteria Desulfuromonas acetoxidans Geobacter sp. CB35 and Desulfuromusa sp. CB30 and green photosynthesizing bacteria Chlorobium limicola their growth in wastewater of industrial and municipal origin. The strains of exoelectrogens, which are characterized by resistance to heavy metal ions, were isolated from the man-made Yavorivske lake located in the Lviv Oblast in Ukraine (D. acetoxidans Ch. limicola Geobacter sp. CB 30 and Desulfuromusa sp. CB 35). Bacteria D. acetoxidans fuel cell (MFC) with the application of D. acetoxidans W/m 2 and the reduction of chemical oxygen demand of wastewater was 99%. The new approach to improving the MFC performance was investigated. It includes a combination of phototrophic microorganisms Ch. limicola and heterotrophic microorganisms, which reduce the content of nitrates, nitrites, ammonia, sulfates, sulfites, hydrogen sulfide, while simultaneously generating electric current.

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.

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.

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.

Desulfuromonas acetoxidans IMV B-7384 is exoelectrogenic obligate anaerobic sulfur-reducing bacterium. Its one of the first described electrogenic bacterium that performs complete oxidation of an organic substrate with electron transfer directly to the electrode in microbial fuel cell (MFC). This bacterium is very promising for MFC development because of inexpensive cultivation medium, high survival rate and selective resistance to various heavy metal ions. The size of D. acetoxidans IMV B-7384 cells is comparatively small (0.4-0.8×1-2 μm) that is highly beneficial while application of porous anode material because of complete bacterial cover of an electrode area with further significant improvement of the effectiveness of its usage. The interconnection between functioning of reductive stage of tricarboxylic acid (TCA) cycle under anaerobic conditions, and MFC performance was established. Malic, pyruvic, fumaric and succinic acids in concentration 42 mM were separately added into the anode chamber of MFC as the redox agents. Application of malic acid caused the most stabile and the highest power generation in comparison with other investigated organic acids. Its maximum equaled 10.07±0.17mW/m 2 on 136 hour of bacterial cultivation. Under addition of pyruvic, succinic and fumaric acids into the anode chamber of MFC the maximal power values equaled 5.80±0.25 mW/m 2 ; 3.2±0.11 mW/m 2 , and 2.14±0.19 mW/m 2 respectively on 40, 56 and 32 hour of bacterial cultivation. Hence the malic acid conversion via reductive stage of TCA cycle is shown to be the most efficient process in terms of electricity generation by D. acetoxidans IMV B-7384 in MFC under anaerobic conditions.

Бактерії Desulfuromonas acetoxidans ІМВ В-7384 є перспективними для роз-роблення біотехнології очищення стічних вод від іонів металів зі змінною валент-ністю. Дослідження механізмів захисту клітин D. acetoxidans ІМВ В-7384 за стресо-вих умов, зокрема, від зростаючих концентрацій іонів важких металів, є важливим для розуміння способів регулювання метаболізму бактерій у процесі очищення стічних вод. Зміни жирнокислотного складу ліпідів розглядають як один із можли-вих способів захисту бактерійної клітини за умов стресу. Внесення ферум цитрату в середовище для вирощування бактерій D. acetoxidans ІМВ В-7384 зумовлювало зміни жирнокислотного складу, порівняно з контролем. За умови внесення різних концентрацій ферум цитрату спостерігали зниження вмісту насичених жирних ки-слот із непарною кількістю атомів карбону. За впливу солі металу в клітинах бакте-рій зафіксовано зростання вмісту насичених жирних кислот із парною кількістю атомів карбону, жирних кислот із циклопропановим кільцем і розгалуженим карбо-новим ланцюгом. За цих умов знижується індекс ненасиченості жирних кислот. Ступінь цис-транс ізомеризації ненасичених жирних кислот клітин D. acetoxidans ІМВ В-7384 за впливу ферум цитрату не зростає. Ключові слова:Desulfuromonas acetoxidans, ферум цитрат, жирні кислоти. ВСТУПМікроорганізми у природі не завжди є в оптимальних умовах, особливо за ан-тропогенного забруднення довкілля важкими металами. Одним із основних напря-мів очищення навколишнього середовища від забруднення є застосування біотех-нологій, які ґрунтуються на ефективних біологічних механізмах детоксикації небез-печних речовин мікроорганізмами. Бактерії здатні ензиматично відновлювати ме-тали у метаболічних шляхах, які безпосередньо не пов'язані з асиміляцією цих металів. Представники класу Deltaproteobacteria, зокрема бактерії Desulfuromonas acetoxidans, здатні використовувати S 0 , Fe (III) і Mn (IV) як акцептори електронів у разі окиснення органічного карбону, що забезпечує їхню особливу адаптацію до змін довкілля. Ці бактерії є одними із перших мікроорганізмів, у яких встановлена здатність отримувати енергію для їхнього росту поєднанням повного окиснення Biol.

Desulfuromonas acetoxidans obtains energy for growth by the anaerobic oxidation of organic compounds with the carbon dioxide formation. It was found that ferrum and manganese are used as terminal electron acceptors in the processes of anaerobic respiration, such as dissimilative Fe3+- and Mn4+-reduction, carried out by these bacteria (Lovely, 1991). D. acetoxidans ІМV B-7384 can be used as anode biocatalyst in microbial fuel cell with high electron recovery through acetate oxidation to the electric current as a result of electron transfer to the anode or 3d-type transition metals, such as ferrum and manganese, in the process of their reduction. Investigation of biochemical changes of D. acetoxidans ІМV B-7384 under the influence of Fe (III) compounds is important for optimization of the process of bacterial electricity generation. ATP-hydrolase is located in cytoplasmic membrane, and its subunits are exposed to both the cytoplasm and the external environment. Therefore, the changes of that enzyme activity can be used as an indicator of various stress exposure. Presence of ferric iron ions in the bacterial growth medium could catalyze generation of organic reactive oxygen species, such as peroxyl (ROO-) and alkoxyl (RO-) radicals. Lipid peroxidation is one of the main reasons of cell damage and it’s following death under the influence of reactive oxygen metabolites. It is known that lipid peroxidation and membrane transport processes are somehow interrelated, but mechanisms of such interaction are still unidentified. In our previous researche we have shown the influence of ferric (III) citrate on the intensity of lipid peroxidation of D. аcetoxidans ІМV В-7384. Significant increase of the content of lipid peroxidation products (lipid hydroperoxides, conjugated dienes and malondialdehyde) in bacterial cells has been observed under the addition of ferric (III) citrate into the cultural medium. The increase of the concentration of lipid peroxidation products in bacterial cells confirms free radical mechanism of oxidation of polyunsaturated fatty acids. Thus, for fulfiling complete analyses of cell response against oxidative stress it was reasonable to investigate the influence of ferric (III) citrate on specific ATP-hydrolase activity, Na+, K+-ATP-hydrolase activity and Mg2+-ATP-hydrolase activity of D. acetoxidans ІМV В-7384. Bacteria were cultivated in the modified Postgaite C medium during four days under the anaerobic conditions and temperature +27°С with addition from 10 to 20 mM of ferric (III) citrate into the growth medium. Control samples didn’t contain investigated metal salt. Chosen concentrations of metal salt caused inhibition of bacterial growth by 20–50%. Activities of ATP-hydrolases were investigated as described. It was shown, that specific ATP-hydrolase activity of D. acetoxidans ІМV В-7384 is changing in dependance on duration of ferric (III) citrate exposure and concentration of the metal salt. Addition of the ferric (III) citrate in relatively low concentrations (10–12 mM) causes increasing of specific ATP-hydrolase activity of D. acetoxidans IMV B-7384 in comparison with control. Activity of investigated enzymes was inhibited under the increasing of metal salt concentration in bacterial growth medium. Increase of duration of D. acetoxidans IMV B-7384 cultivation causes decrease of ATP-hydrolase activity. Addition of ferric (III) citrate causes simultaneous increasing of Na+, K+-ATP-hydrolase activity and inhibition of Mg2+-ATP-hydrolase activity during four days of bacterial cultivation.