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.

The article covers the patterns of oxidation of sulfide and thiosulfate ions by bacteria Rhodopseudomonas yavorovii Ya-2016 under different cultivation conditions. In the environments with 1.4-5.6 мМ Na 2 S 2 O 3 , R. yavorovii Ya-2016 bacteria accumulated biomass of 1.4-1.6 g/l, which was higher than biomass (1.2-0.6 g/l) accumulated by the bacteria with the same concentrations of Na 2 S × 9H 2 O. The efficiency of oxidation of 1.4, 2.8, 5.6 мМ sulfide-and thiosulfateions as donors of electrons by the bacteria equaled 97.4, 42.6, 18.7 and 68.8, 28.0, 3.7%, relatively. As a result bacteri' oxidation of 1.4 мМ hydrogen sulfide and sodium thiosulphate in the environment caused accumulation of 0.13-1.30 мМ sulfate-ions, and the element sulfur became an intermediate metabolite in the environment with Na 2 S × 9H 2 O. R. yavorovii Ya-2016 bacteria are capable of using sulfate-ions as a single source of sulfate at increase in photptrophs. In the environment with 2.5 мМ sulfate-ions concentration the bacteria biomass was 1.4 g/l, the bacteria assimilated 17.7% of sulfates. Because purple non-sulfur bacteria R. yavorovii Ya-2016 are capable of using sulfide-ions as donors of electrons of anoxygenic photosynthesis and using sulfate-ions as a single source of sulfate, they could be successfully used in the technologies of remediating the environment from compounds of sulfur.

Виділено чисту культуру пурпурових несіркових бактерій із води озера Яворівське (Львівська область, Україна), яке утворилося в результаті затоплення території сіркового кар'єру. Суспензія виділеного штаму Yа-2016 має рожево-червоне забарвлення. Вібріоїдні клітини є рухомими, грамнегативними, не утворюють спор. Розміри клітин 1,5-1,8 х 0,4-0,46 мкм. У клітинах бактерій виявлені внутрішні мембрани везикулярного типу. Ріст штаму пригнічується за внесення NaCl (1 г/л). Як субстрати для фототрофного росту бактерії використовують сульфід, тіосульфат і низку органічних сполук. Для росту потребують вітамін В 12. Нуклеотидна послідовність консервативної ділянки гена 16S рРНК виявляє високу подібність (99 % ідентичних залишків у попарному вирівнюванні методом BLASTN) до 16S рРНК бактерій роду Rhodopseudomonas. На основі дослідження морфофізіологічних характеристик і аналізу нуклеотидних послідовностей гена 16S рРНК пурпурові фотосинтезувальні несіркові бактерії штаму Yа-2016, виділені з води озера Яворівське, ідентифіковані як Rhodopseudomonas sp. Yа-2016. Ключові слова: фототрофні бактерії, пурпурові несіркові бактерії, Rhodopseudomonas sp.

In this article, we characterized the regularities of oxidation of nitrite ions by phototropic purple non-sulfur bacteria Rhodopseudomonas yavorovii IMV B-7620, which were isolated from the water of Yavorivske Lake (Lviv Region, Ukraine). The bacteria were cultivated anaerobically at the light intensity of 200 lux and aerobically without illumination for 13 days in the modified ATCC No. 1449 medium. The concentration of nitrite ions was determined turbidimetrically by the turbidity of the solution by method of diazotization of sulfanilic acid by the nitrite ions and the interaction of the formed salt with n-(l-naphtyl)ethylenediamine dihydrochloride. The concentration of nitrate ions was determined turbidimetrically by the turbidity of the solution by method of diazotization. Zinc powder was used as a reducing agent. Efficiency of oxidation of 0.7–5.6 mM nitrite ions as electron donors by these bacteria was 100–7%, on the 10-th day of cultivation. It was established that nitrate ions were accumulated in the medium as a result of oxidation of nitrite ions by bacteria. The largest biomass (1.6 g/L) bacteria accumulated on the thirteenth day of growth in a medium with 2.8 mM NO2–. We found that R. yavorovii can use nitrate ions and urea as the only source of nitrogen for phototrophic growth. At a concentration of 1.9 mM ammonium chloride, sodium nitrite and urea in the cultivation medium, the biomass of bacteria was 1.2, 0.8, 1.0 g/L, respectively. The ability of the studied microorganisms to oxidize nitrite ions and to use nitrate ions indicates the significant impact of purple non-sulfur bacteria on the redistribution of streams of nitrogen compounds in ecosystems and the essential role of these microorganisms in the nitrogen biogeochemical cycle.

Життя сьогодні існує завдяки процесу фотосинтезу. Перетворення енер-гії світла в енергію хімічних зв'язків здійснюють рослини та фотосинтезу-вальні прокаріоти [10]. На відміну від рослин, деякі фотосинтезувальні бак-терії не використовують воду як донор електронів у процесі фотосинтезу і не виділяють кисень. Аноксигенними фотосинтетиками є пурпурові несіркові бактерії (ПНСБ), пурпурові сіркові бактерії (ПСБ), зелені сіркові та несіркові бактерії. ПНСБ є метаболічно, таксономічно та морфологічно універсальною групою мікроорганізмів. Основними пігментами фотосинтезу цих бактерій є бактеріохлорофіл a або b і каротиноїди [1].Штами ПНСБ були використані для очищення стічних вод, акваріумних вод, сільськогосподарських стоків. Також вони можуть продукувати водень, індол-3-оцтову кислоту і 5-амінолевулінову кислоту [14].ПНСБ можуть використовувати у процесі життєдіяльності різні ор-ганічні речовини, що забезпечує їм відносно високу швидкість росту. ПНСБ очищують водойми від сірководню і, будучи компонентами трофічних ланцю-

Hydrogen production by microorganisms is studied by using different sources of carbon for their cultivation. Purple non-sulfur bacteria are capable of producing molecular hydrogen phototrophically with the simultaneous accumulation of biomass on organic substrates that may be waste from various industries. That fact makes the study of this group of microorganisms very much promising. The aim. The determination of the ability of purple non-sulfur bacteria Rhodopseudomonas yavorovii IMV B-7620 to produce hydrogen consuming different organic substrates and their effects on the main metabolic indicators of culture growth. Methods. Bacteria were grown in 100 mL glass jars in liquid modified ATCC No. 1449 medium for 14 days at temperature +27...+30ºC and at constant light (200 lux). Biomass accumulation and hydrogen production in the cultivation medium were determined using sodium acetate (12 and 36 mM), malate (12 and 36 mM), succinate (36 mM), glucose (36 mM), starch (36 mM), sodium citrate (36, 60, 90 mM). Biomass was determined turbidimetrically, the composition of the gas phase was determined using a gas chromatograph LHM-8-MD, redox potential and pH were estimated potentiometrically. The volume of gas synthesized was measured on a syringe scale. Determination of the content of organic acids in the culture liquid was analyzed by high performance liquid chromatography. Results. The utilization of organic compounds (malate, glucose, starch, sodium citrate) by R. yavorovii IMV B-7620 is accompanied by hydrogen synthesis. Under the growth with sodium acetate, bacteria produce small amounts of succinate. The malate metabolism results in the production of small amounts of fumarate on the 7th day of cultivation and isocitrate on the 10th day of cultivation. On the 14th day of cultivation, the cultural liquid contains a small amount of succinate. On the 14th day of cultivation, R. yavorovii IMV B-7620 produces 7.64±0.04% of hydrogen in the medium with malate (36 mM). However, the maximum concentration of hydrogen in the gas phase (21.26±0.08%) was gained on the 14th day of cultivation in the medium with sodium citrate. The maximum concentration of H2 in the gas phase during the growth in the medium with sodium citrate (60 mM) and NH4+ was 27.83±5.46% on the 7th day of cultivation and 35.69±0.40% with increasing concentration of sodium citrate up to 90 mM on the 10th day of cultivation. The total volume of hydrogen was 25.54±0.49 mL of H2 during the growth of R. yavorovii IMV B-7620 in the medium with 90 mM sodium citrate and NH4+. That is 1.5 times more than the amount of H2 produced during the growth of bacteria in the medium with 60 mM sodium citrate with the addition of NH4+. Conclusion. Purple non-sulfur bacteria R. yavorovii IMV B-7620 synthesize hydrogen during photofermentation of organic compounds. Bacteria were isolated from the water of Yavoriv Lake (Lviv region, Ukraine) formed as a result of flooding of the sulfur quarry. Bacteria consume sodium citrate, malate, glucose, starch and emit hydrogen. The total volume of hydrogen during the growth of R. yavorovii IMV B-7620 in the medium with 90 mM sodium citrate and NH4+ is 25.54±0.49 mL H2.

This article presents the regularities of reduction of sulfur, nitrate and nitrite ions by sulfur reducing bacteria Desulfuromonas sp., which were isolated from the water of the man-made Yavorivske Lake (Lviv Region, Ukraine), under the influence of potassium dichromate. This bacteria in the process of anaerobic respiration can use and reduce different electron acceptors, such as sulfur, nitrates, nitrites, oxidized forms of heavy metals, in particular, hexavalent chromium. Technogenically altered ecotopes are characterized by complex pollution, so several electron acceptors are available to bacteria at the same time. Strains of microorganisms isolated from such ecotopes are adapted to unfavourable conditions and therefore have high biotechnological potential. The purpose of this work was to investigate the regularities of elemental sulfur, nitrate or nitrite ion usage by sulfidogenic bacteria of Desulfuromonas genus in conditions of simultaneous presence in the medium of another electron acceptor – Cr(VI), to establish the succession of reduction of electron acceptors by strains of these bacteria and to evaluate the efficiency of their possible application in technologies of complex purification of the environment from metal compounds and other inorganic toxicants. Bacteria were grown under anaerobic conditions in Kravtsov-Sorokin medium without SO42– and without Mohr’s salt for 10 days. To study the efficiency of sulfur, nitrate or nitrite ions’ reduction at simultaneous presence in the medium of Cr(VI) bacteria were sown in media with elemental sulfur, NaNO3, NaNO2 or K2Cr2O7 to final S0, NO3–, NO2–or Cr(VI) concentration in the medium of 3.47 (concentration of SO42– in medium of standard composition) or 1.74, 3.47, 5.21, 6.94 and 10.41 mM. Biomass was determined by the turbidimetric method, and the concentrations of nitrate, nitrite, ammonium ions, hydrogen sulfide, Cr(VI), Cr(ІІІ) in cultural liquid were determined spectrophotometrically. It has been established that Cr(VI) inhibits the biomass accumulation and hydrogen sulfide production by bacteria of Desulfuromonas sp. after simultaneous addition into the medium of 3.47 mM S0 and 1.74–10.41 mM Cr(VI). In the medium with the same initial content (3.47 mM) of S0 and Cr(VI) bacteria produced Cr(III) at concentrations 3.3–3.4 times higher than that of hydrogen sulfide. It has been shown that K2Cr2O7 inhibits biomass accumulation, nitrate ions’ reduction and ammonium ions’ production by bacteria after simultaneous addition into the medium of 3.47 mM NO3– and 1.74–10.41 mM Cr(VI) or 1.74–10.41 mM NO3– and 3.47 mM Cr(VI). In the medium with the same initial content (3.47 mM) of NO3– and Cr(VI) bacteria reduced up to 1.2 times more nitrate ions than Cr(VI) with the production of ammonium ions at concentrations the same times higher than those of Cr(III). It has been established that K2Cr2O7 inhibits biomass accumulation, nitrite ions’ reduction and ammonium ions’ production by bacteria after simultaneous addition into the medium of 3.47 mM NO2– and 1.74–10.41 mM Cr(VI) or 1.74–10.41 mM NO2– and 3.47 mM Cr(VI). In the medium with the same initial content of (3.47 mM) NO2– and Cr(VI) the reduction of Cr(VI) by bacteria was only slightly, up to 1.1 times, lower than the reduction of nitrite ions, almost the same concentrations of trivalent chromium and ammonium ions were detected in the cultural liquid. The processes of nitrate and nitride reduction carried out by bacteria of Desulfuromonas genus were revealed to be less sensitive to the negative influence of sodium dichromate, as compared with the process of sulfur reduction, because in the media with the same initial content (3.47 mM) of NO3– or NO2– and Cr(VI) bacteria produced 1.1–1.2 times more NH4+ than Cr(III), but in the medium with the same initial content (3.47 mM) of S0 and Cr(VI) ) bacteria produced over than three times more Cr(III) than hydrogen sulfide. Our data allow us to conclude that bacteria of Desulfuromonas genus, the investigated strains of which are adapted to high concentrations (up to 10.41 mM) of inorganic toxicants, play an important role in the geochemical cycles of sulfur, nitrogen and chromium in aquatic environments that have been under anthropogenic influence.

Phototrophic purple non-sulfur bacteria (PNSB) mineralize organic waste. In media with different organic substances they are metabolized from simple aliphatic organic acids to complex polysaccharides. These bacteria can produce H2, indole-3-acetic acid and 5-aminolevulinic acid. PNSB purify waters from hydrogen sulfide and, as components of ecosystem trophic chains, participate in process of sulfur cycle. The ability of these bacteria to denitrification is of particular interest to specialists because nitrates concentrations in industrial effluents are often much higher than the maximum permissible concentrations. The use of industrial, agricultural and household wastes to produce PNSB biomass is economically profitable. Therefore, the aim of our work was to investigate the influence of wastewater of the yeast plant and the water of the settler № 3 filtrate of the Lviv Solid Household Waste Landfill (LSHWL) on the biomass accumulation by PNSB Rhodopseudomonas yavorovii IMV B-7620 and the changes in the composition of these waters in the process of cultivation of the investigated bacteria. Bacteria were grown for 21 days anaerobically at an illumination intensity of 200 lux in 500 ml flasks at +29 ... +30 °C. The wastewater of the yeast plant and the water of the settler № 3 filtrate of the LSHWL were used as the cultivation media. Water was diluted at 10; 30; 50; 100 times. In water, diluted at 10 times, the pH, total ferrum content, Ca2+, Mg2+, Cl-, NO3-, NO2-, SO42-, HS- and chemical oxygen demand (COD) were determined. The biomass of bacteria was measured turbidimetrically at λ = 660 nm. The concentration of HS- was determined spectrophotometrically (λ = 665 nm) by the formation of methylene blue. The content of sulfate ions was determined turbidimetrically (λ = 520 nm) after their precipitation with barium chloride. The concentrations of Ca2+ and Mg2+ were determined titrometrically by reaction with trilon B; Na+ and K+ were analyzed by flame emission spectrometry method (lK = 768 nm, lNa = 589 nm). The pH of the water was measured potentiometrically. The concentration of Cl- was determined titrometrically by reaction with AgNO3. The concentration of nitrite ions was determined spectrophotometrically by the method of sulfanilic acid diazotization by nitrite ions and the interaction of the formed salt with n-(naphthyl)ethylenediamine dihydrochloride (λ = 540 nm, l = 10 mm). The concentration of nitrate ions was determined spectrophotometrically by the diazotization method. Zinc powder was used as the reducing agent. COD was measured with using of KMnO4. It is characterised the patterns of biomass accumulation by R. yavorovii IMV B-7620 bacteria in the wastewater of the yeast plant and the water of the settler № 3 filtrate of the LSHWL, diluted at 10–100 times. The optimal dilution (at 10 times) of water for bacteria cultivation was selected. Bacteria R. yavorovii IMV B-7620 accumulate biomass of 2.2 g/l during growth in a yeast plant wastewater that is twice as large as the biomass, accumulated by bacteria in the diluted water of the settler № 3 filtrate. The organic compounds oxidation efficiency of the yeast plant wastewater and of the settler № 3 filtrate of LSHWL, determined by chemical oxygen demand, was 42.3 and 65.6 %, respectively, on the 21st day of cultivation. The established possibility of the influence of R. yavorovii IMV B-7620 on the decreasing of the content of total Fe, Ca2+, Mg2+, NO3-, NO2-, SO42-, Cl-, HS- in wastewater has complemented and broadened the understanding of the role of purple non-sulfur bacteria in ecosystems, which can significantly influence on the biogeochemical cycles of these compounds in nature. The results obtained may form the basis for the development of effective biotechnologies for wastewater treatment from hydrogen sulfide, chlorine, ferrum, nitrogen, sulfur compounds with the participation of these microorganisms.