REDUCTION OF SULFATE, NITRATE AND NITRITE IONS BY DESULFOVIBRIO SP. UNDER THE INFLUENCE OF FERRUM (III) CITRATE

The influence of ferrum (III) citrate added to the cultivation medium, on with Na 2 SO 4 × 10 H 2 O, NaNO 3 or NaNO 2 decreased 1.1−2.1, 1.6−2.7 and 1.1−2.5 times, respectively, compared with its reduction in the medium with only FeC 6 H 5 O 7 . T he investigated strains of bacteria were resistant to high concentrations of ferrum (III) citrate and, therefore, can be applied in the technologies of complex environment purification from pollution with ferrum, sulfur, and nitrogen compounds.

The influence of ferrum (III) citrate added to the cultivation medium, on the reduction of sulfate, nitrate, and nitrite ions by sulfate-reducing bacteria Desulfovibrio desulfuricans IMV K-6, Desulfovibrio sp. Yav-6, and Desulfovibrio sp. Yav-8 isolated from Yavorivske Lake was studied. It was established that ferrum (III) citrate inhibits the biomass accumulation, SO 4 2reduction, and H 2 S production by the bacteria after addition of 1.74-3.47 mM Na 2 SO 4 ×10 H 2 O and 1.74-10.41 mM FeC 6 H 5 O 7 to the medium, in comparison with the growth and level of the reduction of sulfate ions by bacteria in the medium supplemented with only Na 2 SO 4 ×10 H 2 O. At conditions of the bacteria cultivation in the presence of an equimolar amount (3.47 mM) of Na 2 SO 4 ×10 H 2 O and FeC 6 H 5 O 7 , they reduced 2.5−2.7 times more Fe(ІІІ) than SO 4 2with Fe 2+ production at a concentration 2.4−2.7 times greater than H 2 S. FeC 6 H 5 O 7 inhibited growth, NO 3 -or NO 2 reduction and NH 4 + production by the bacteria in the presence of 1.74−3.47 mM NaNO 3 or NaNO 2 and 1.74−10.41 mM FeC 6 H 5 O 7 in the medium, compared to the growth and level of nitrate or nitrite ions reduction in the medium with only NaNO 3 or NaNO 2 . In the medium with the same initial content of 3.47 mM NaNO 3 and 3.47 mM FeC 6 H 5 O 7 , the bacteria reduced 1.4 times more NO 3 than Fe(ІІІ), with NH 4 + production at concentration 1.1 times higher than that of Fe 2+ . In the medium with 3.47 mM NaNO 2 and 3.47 mM FeC 6 H 5 O 7 , the cells reduced 1.4−1.6 times more Fe(ІІІ) than NO 2 -, with Fe 2+ production at concentration 1.5−1.6 times higher than NH 4 + . Ferrum (III) citrate had more inhibitory effect on the dissimilatory reduction of sulfate by the bacteria than of nitrate and nitrite ions, since the SO 4 2reduction by the bacteria at its presence in the medium decreased 2.0−4.7 times. The reduction of NO 3 and NO 2 decreased only 1.3−1.9 and 1.7−3.1 times, respectively, as compared with their reduction in the media with only Na 2 SO 4 ×10 H 2 O, NaNO 3 or NaNO 2 . Despite the fact that the reduction by cells of 1.74−10.41 mM Fe(III) in the media

INTRODUCTION
In process of the anaerobic respiration, sulfidogenic bacteria of Desulfovibrio, Desulfotomaculum, Desulfobacterium, Desulfobacter, Geobacter etc. genera oxidize organic compounds using different electron acceptors [1,2,3,16,31,37]. Sulfate-reducing bacteria produce H 2 S, as a result of the dissimilatory reduction of sulfate ions that occurs in their cytoplasm with a formation of the adenosine-5′-phospho sulfate (APS), as an intermediate product. The stages of sulfate reduction are catalyzed by ATP sulfurylase, APS reductase, and a number of sulfite reductases [16]. These bacteria play an important role in regulating the level of not only compounds of sulfur and carbon, but also of nitrogen and metals in the environment [2,23,33,[38][39][40]. Pollution of media by heavy metals influences on the physiological and biochemical processes which are carried out by bacteria [10,22,24,25,33].
Facultative anaerobic bacteria carry out a dissimilatory reduction of nitrates with the formation of NO 2 -, NO, N 2 О, and N 2 or nitrite ions with the participation of NAD(P)H or reduced menaquinone can be directly reduced to NН 4 + [16,39]. Oxidized compounds of nitrogen are reduced by the microorganisms that synthesize nitrate and nitrite reductases [16]. Nitrate reductase NarGHI is an enzymatic complex that includes multi-heme b-type cytochrome, proteins with Fe-S clusters, and Mo-containing cofactor [13,21]. Nitrate reduction with the formation of nitrite and its further reduction by complex of periplasmic dissimilatory nitrite reductases to NH 4 + was descri bed in Desulfovibrio desulfuricans, Desulfotomaculum sp., Desulfobacter sp., and Wolinella succinogenes [4,11,20,25,29,39].
Sulfidogenic and metal-reducing bacteria occupy close ecological niches, providing various links in a cycle of chemical elements in nature [2,40]. The structure and properties of the components of electron transport chain and enzymes, involved in the process of dissimilatory reduction of oxidized metal forms, have been intensively studied in recent years in connection with the ability of metal-reducing bacteria (Desulfovibrio, Geobacter, Desulfuromusa, Desulfuromonas, Desulfotomaculum, Shewanella, Wolinella, Pseudomonas etc.) in process of the anaerobic respiration release electrons into the medium [5,7,31,32,34], due to which these bacteria are considered as anode biocatalysts in the microbial fuel cells [6,10,34].
In natural conditions most often there are several possible electron acceptors of anaerobic respiration, and bacteria, first of all, reduce acceptors with higher standard oxidation-reduction potential. Although a succession of reduction of electron acceptors by the microorganisms is determined by electrochemical laws, it is not sufficiently studied. In different microorganisms, the succession of the elements with a variable valence reduction is determined genetically and controlled by profound regulatory mechanisms [13,16].
Ferrum enters the aquatic and soil environments with industrial, agricultural and household effluents, as well as due to natural processes of chemical weathering and dissolution of rocks [14]. In the bacteria cells, ferrum is an essential trace element that participates in the processes of photosynthesis, N 2 fixation, methanogenesis, H 2 synthesis, respiration, regulation of gene expression and DNA biosynthesis [19]. The toxic effect of ferrum, as well as other metals, on the bacterial cell is its binding with the surface structures of the cell wall, the change in the electrophysiological properties of the cytoplasmic membrane, the blocking of transport systems, the replacement of the nece ssary ions from active centers of the enzymes, binding with functional groups of cell metabolites [27,28]. Because the Fe(ІІІ)/Fe(ІІ) pair at pH 7.0 has a very high oxidation-reduction potential (E 0 ′ = +0.77 V), which, however, is highly dependent on the acidity of the medium [7,8,16], at high concentrations in the cytoplasm the Fe(ІІІ) is the catalyst of Fenton and Haber-Weiss reactions that result a formation of toxic forms of oxygen [10,19,27]. Therefore, an important mechanism for bacteria protecting from the toxic influence of heavy metals is their ability to extracellular metal reduction by a system of membranebound metal reductases (multi-heme c-type cytochromes) [5,6,31,32,34]. Another way of eliminating heavy metals by sulfidogenic bacteria from the natural cycle, is their immobilization in the form of sulfides formed as a result of interaction with H 2 S [12-14, 17, 24].
A selection of resistant to pollutions strains of sulfate-reducing bacteria isolated from technogenically altered ecotopes, capable to reductive transformation of various nature pollutants, is especially actual task for the creation of biotechnologies for purification [3,13,14,18,37]. Previously we have shown that the bacteria of Desulfovibrio genus in addition to oxidized forms of sulfur or nitrogen can reduce oxidized forms of heavy metals, in particular, ferrum (III), transforming them into compounds less toxic for the living organisms [23,25]. The purpose of this work was to investigate the regularities of sulfate, nitrate or nitrite ions usage by these bacteria at conditions of simultaneous presence in the medium of ferrum (III) citrate to establish a succession of electron acceptors reduction by strains of sulfidogenic bacteria of Desulfovibrio genus, isolated by us from the Yavorivske Lake, and to evaluate an efficiency of their application in technologies of complex purification of environment pollution by ferrum, sulfur and nitrogen compounds.

MATERIALS AND METHODS
Sulfate-reducing bacteria Desulfovibrio desulfuricans IMV K-6, Desulfovibrio sp. Yav-6, Desulfovibrio sp. Yav-8 isolated from the Yavorivske Lake, were identified at the Department of Microbiology of Ivan Franko National University of Lviv. They are stored at the Depository of D. K. Zabolotny Institute of Microbiology and Virology of the NAS of Ukraine and/or at the collection of the Department of Microbiology [26,30].
Bacteria were grown for 10 days in Kravtsov-Sorokin medium [9] without SO 4 2and without Mohr's salt of such composition (g/L): , at a temperature of 30 o C in test tubes, volume 25 ml, completely topped up by the medium. Before bacteria seeding 0.05 ml of Na 2 S×9 H 2 O (1%) sterile solution was added to the medium. pH of the medium was 7.2. Bacteria were sown in medium to initial concentration of cells of 0.1 mg/ml. Solutions of sodium fumarate (C 4 H 3 NaO 4 ), Na 2 SO 4 ×10 H 2 O, NaNO 3 , NaNO 2 , FeC 6 H 5 O 7 were sterilized separately and placed into the medium before seeding of the cells. To media with C 4 H 3 NaO 4 , FeC 6 H 5 O 7 and/or NaNO 3 or NaNO 2 cysteine (0.017 mM) was introduced to meet the assimilation needs of bacteria in sulfur [16]. To media supplemented with NaNO 3 , NaNO 2 and FeC 6 H 5 O 7 or without it NH 4 Cl was not added.
To study the unfluence of ferrum (III) citrate on the kinetics of sulfate or nitrate ions usage by bacteria, the cells, previously grown in medium with sodium fumarate (3.47 mM), as an electron acceptor, and sodium lactate (17.86 mM), as an electron donor, to middle of exponential growth phase, were sown in medium with sodium citrate (Na 3 C 6 H 5 O 7 ) as an electron donor (17.86 mM) to which sterile 1 M solutions of Na 2 SO 4 ×10 H 2 O or NaNO 3 and FeC 6 H 5 O 7 were added at concentrations of 1.74 mM to obtain the total content of electron acceptors in medium of 3.47 mM (SO 4 2concentration in the medium of standard composition). The cells were also sown in media with only 3.47 mM Na 2 SO 4 ×10 H 2 O, NaNO 3 or FeC 6 H 5 O 7 to verify their growth in media with SO 4 2-, NO 3 or Fe(ІІІ) as the sole electron acceptor (control). As control the spontaneous reduction of sulfate, nitrate ions and ferrum (III) in media without cells with Na 2 SO 4 ×10 H 2 O, NaNO 3 and/or FeC 6 H 5 O 7 at concentrations of 1.74 or 3.47 mM was investigated. The biomass, and the concentrations of sulfate or nitrate, ferrum (II) ions, ferrum (III), hydrogen sulfide or ammonium ions in cultural liquid were determined on 2, 4, 6, 8 and 10 days of growth.
To determine the efficiency of reduction of sulfate, nitrate, or nitrite ions at simultaneous presence in the medium of ferrum (ІІІ) citrate, cells were previously cultivated in medium with sodium fumarate (3.47 mM) and sodium lactate (17.86 mM) to the middle of the exponential growth phase. They were sown in the medium with sodium citrate (17. 4 2-, NO 3 -, NO 2 or Fe(ІІІ) (control). The biomass, the concentrations of sulfate, nitrate or nitrite, ferrum (II) ions, ferrum (III), hydrogen sulfide or ammonium ions in cultural liquid were determined on 10 day of growth. According to difference between the initial and residual content of electron acceptors in the medium, the efficiency (%) of their reduction by bacteria, based on the ratio of molar concentrations of reduced by bacteria sulfate, nitrate, nitrite ions or ferrum (III) in the process of anaerobic respiration and their concentrations at the beginning of cultivation taken as 100 % was calculated.
To determine the concentrations of ferrum (II) ions and hydrogen sulfide, the precipitate of FeS formed after bacteria cultivation in medium with Na 2 SO 4 ×10 H 2 O and FeC 6 H 5 O 7 was dissolved after interaction with HCl according to the equation: FeS + 2HCl ⇄ FeCl 2 + H 2 S (the HCl concentration exceeded twice the concentration of SO 4 2in the medium and was 3.5 mM or 7.0 mM). The biomass was determined by the turbidimetric method using optical density of the cell suspension measured at 340 nm wavelength in a cuvette with an optical way of 3 mm and calculated using the formula: C, g/L = (E 340 × n) / K, where E 340 − extinction (λ = 340 nm); n − dilution factor; K -coefficient of recalculation, obtained from the calibration curve of the dependence of extinction upon the mass of dry cells, determined by the weight method, and equal 0.19 [9]. In a cultural liquid separated from the cells by centrifugation (6,000 rpm, 15 min), the concentrations of sulfate ions by turbidimetric method for the formation of barium sulfate were determined after sulfates precipitation by barium chloride. Concentrations of nitrate ions were measured after their reduction to nitrites in the presence of Zn:MnSO 4 (1:100) powder, as a reducing agent, while concentrations of nitrite ions were measured by spectrophotometric method in the reaction with Griss reagent (n-(1-naphthyl)ethylenediamine dihydrochloride, sulfanil and acetic acid). Concentrations of ferrum (III) were determined after its reduction to ferrum (II) ions in the acidic medium by interaction with hydroxylamine, ferrum (II) ions -by spectrophotometric method in reaction with o-phenanthroline, hydrogen sulfide -by spectrophotometric method of the formation of the methylene blue, and the ammonium ions -by the colorimetric method of the formation of the indophenol [9].
Experiments were repeated three times with three parallel staging for each variant of the experimental and control conditions. The obtained data were processed using the Microsoft Excel 2010 program. To evaluate the certainty of the difference between the statistical characteristics of the two alternative sets of data, Student's coefficient t was counted. The difference was considered to be significant at P<0.05 [15].

RESULTS AND DISCUSSION
The intensity of the anaerobic respiration of microorganisms in the contaminated ecotopes is determined by the level of their adaptation to unfavorable environmental conditions, in particular, increased content of metal compounds [18,34,35,37,38]. In the technogenic reservoir that arose on the place of Yavoriv sulfur deposit open pit, high concentrations of toxic to living organisms compounds of sulfur, nitrogen and heavy metals were detected [24,36]. The concentrations of Fe(III) were significantly higher than maximum permissible concentrations (MPC) (0.1−0.3 mg/L [14]) at a depths of 50−70 m and reached to 0.28−0.34 mg/L, while the content of Fe(II) at no depth exceeded the MPC [24]. The influence of Fe(III) at concentrations significantly higher, than in the reservoir, on sulfate-, nitrate-and nitrite-reducing activity of Desulfovibrio genus bacteria, isolated from Yavorivske Lake, was studied. Earlier we have shown that these bacteria in the process of the anaerobic destruction of the organic compounds, except of sulfate, nitrate or nitrite ions, can use oxidized forms of heavy metals with a variable valence, in particular, Fe(III) as electron acceptors [23,25].
To study the influence of ferrum (III) citrate on the sulfate ions reduction by sulfatereducing bacteria, they were sown in a medium with sodium citrate as an electron donor, to which 1.74 mM Na 2 SO 4 ×10 H 2 O and 1.74 mM FeC 6 H 5 O 7 were added to obtain total content of electron acceptors in the medium of 3.47 mM. The cultures were also sown in the medium with sodium citrate and 3.47 mM Na 2 SO 4 ×10 H 2 O or 3.47 mM FeC 6 H 5 O 7 (Fig. 1, Table 1). The biomass of bacteria in medium with Na 2 SO 4 ×10 H 2 O did not differ from the biomass accumulated by cells in medium with FeC 6 H 5 O 7 . After a simultaneous introduction of Na 2 SO 4 ×10 H 2 O and FeC 6 H 5 O 7 to the cultivation medium, a slight inhibition of biomass accumulation by bacteria, compared with their growth in the media with one electron acceptor was observed. In the medium with Na 2 SO 4 ×10 H 2 O and   (Table 1). It was established that in medium with the same initial content (1.74 mM) Na 2 SO 4 ×10 H 2 O and FeC 6 H 5 O 7 bacteria reduced 2.9−3.9 times more Fe(III) than sulfate ions with ferrum (II) ions production at concentration 2.2−3.6 times higher than that of the hydrogen sulfide.  The efficiency of the biological methods for purifying the environment from the pollutants depends not only upon the metabolic activity of the selected strains of bacteria, but primarily upon their resistance to metal compounds [2,7,10,14,40]. Therefore, we studied the ability of these bacteria to reduce in the process of the anaerobic respiration of sulfate, nitrate or nitrite ions at a simultaneous presence in the medium of 1 with increasing of ferrum (III) citrate concentrations, a gradual (from 2.0 to 4.7 fold) decrease in the efficiency of sulfate ions reduction by bacteria as compared with their reduction in the medium with only Na 2 SO 4 ×10 H 2 O (95−98 %) was detected (Fig. 2, A). In that medium, the cells produced 0.65−1.32 mM of hydrogen sulfide (control: 2.54−2.76 mM) ( Table 2). The efficiency of Fe(ІІІ) reduction by the bacteria in media with Na 2 SO 4 ×10 H 2 O and 1.74−3.47 mM FeC 6 H 5 O 7 practically did not differ from their reduction in the medium with ferrrum (III) citrate (92−94 %). It was found to be 1.1−2.1 times lower at FeC 6 H 5 O 7 concentrations in the medium of 5.21−10.41 mM (Fig. 2, B). In the media with Na-    Although at simultaneous presence SO 4 2and oxidized form of ferrum in medium the bacteria were used to a greater extent FeC 6 H 5 O 7 , at all investigated concentrations its effect on the microorganisms was more or less toxic. That was confirmed by inhibition of dissimilatory reduction of the sulfate ions. Despite the fact that the reduction of metal oxidants by membrane-bound metal reductases is mainly carried out outside the cell [7,32,34], with an increase in the concentration of soluble FeC 6 H 5 O 7 in the medium increase in the degree of Fe(ІІІ) penetration through the cytoplasmic membrane of bacteria into the cytoplasm. Here its interaction with intracellular metabolites occured, oxygen radicals are formed, and ferrum (II) ions accumulated as a reduced end product, that caused inhibition of bacteria growth and their metabolic activity [32,38].
To study the influence of ferrum (III) citrate on the nitrate ions usage by bacteria, they were seeded in the medium without NH 4 Cl with sodium citrate to which 1.74 mM NaNO 3 and 1.74 mM FeC 6 H 5 O 7 were added to obtain the total content of electron acceptors in the medium of 3.47 mM. The bacteria were also seeded in a medium without NH 4 Cl with sodium citrate to which 3.47 mM NaNO 3 or 3.47 mM FeC 6 H 5 O 7 was added (Fig. 3, Table 3). After simultaneous addition of NaNO 3 and FeC 6 H 5 O 7 into the cultivation medium, the bacteria were accumulated the biomass 1.2 times higher than in the medium with NaNO 3 . That appeared 1.2 times lower than it was in the medium with  (Table 3). It was shown that in the medium with an equimolar initial content (1.74 mM) of NaNO 3  The bacteria were cultivated in the medium without NH 4 Cl with sodium citrate to which 3.47 mM NaNO 3 and FeC 6 H 5 O 7 at different concentrations were added. The bacteria were also sown in the medium with sodium citrate and 3.47 mM NaNO 3 or 3.47 mM FeC 6 H 5 O 7 (Table 4). After 10 days of growth, the biomass of bacteria in the medium with NaNO 3 revealed the same one as in the medium with FeC 6 H 5 O 7 . After simultaneous introduction into the medium of NaNO 3 and FeC 6 H 5 O 7 with increasing of ferrum (III) citrate concentrations a gradual repression of bacteria growth was observed, compared with growth in the medium with NaNO 3 or FeC 6 H 5 O 7 . In the medium with NaNO 3 and 10.41 mM FeC 6 H 5 O 7 , the growth of bacteria decreased 1.7−1.9 times, compared with the growth in the media only with NaNO 3 or FeC 6 H 5 O 7 . In the media with NaNO 3 and FeC 6 H 5 O 7 with increasing of ferrum (III) citrate concentrations, there was also a gradual (1.3−1.9 times) decrease in the efficiency of nitrate ions reduction by cells, compared with their reduction in the medium only with NaNO 3 (96−97 %) (Fig. 4, A). In the media  with NaNO 3 and FeC 6 H 5 O 7 , the bacteria produced 0.78−1.91 mM of ammonium ions (control: 2.00−2.10 mM) ( Table 4). The efficiency of Fe(ІІІ) reduction by cells with increasing its concentrations in the media with NaNO 3 and FeC 6 H 5 O 7 was revealed from 1.6 to 2.7 times lower than its reduction in the medium with only FeC 6 H 5 O 7 (89−92 %) (Fig. 4, B). In the media with NaNO 3 and FeC 6 H 5 O 7 , the bacteria were produced 0.92−3.61 mM of ferrum (II) ions (control: 3.05−3.18 mM) ( Table 4). In a medium with NaNO 3 and FeC 6 H 5 O 7 without bacteria, the efficiency of NO 3 and Fe(ІІІ) reduction did not exceed 4.3 and 4.6 %, respectively (Fig. 4). It was shown that ferrum (III) citrate inhibits the biomass accumulation, the nitrate ions reduction and the ammonium ions production by the bacteria of Desulfovibrio sp. after simultaneous addition into medium of NaNO 3 and FeC 6 H 5 O 7 (1.74−10.41 mM). In medium with the same initial content (3.47 mM) of NaNO 3 and FeC 6 H 5 O 7 , the bacteria reduced 1.4 times more nitrate ions than Fe(ІІІ) with the production of ammonium ions at concentration 1.1 times higher than that of ferrum (II) ions.   The bacteria were grown in the medium without NH 4 Cl with sodium citrate to which 3.47 mM NaNO 2 and FeC 6 H 5 O 7 at different concentrations were added. The bacteria were also sown in the medium with sodium citrate and 3.47 mM NaNO 2 or 3.47 mM FeC 6 H 5 O 7 ( Table 5). The biomass of bacteria in the medium with NaNO 2 was revealed 1.2 times lower than that in the medium with FeC 6 H 5 O 7 . After simultaneous addition into the medium of NaNO 2 and FeC 6 H 5 O 7 with increasing concentrations of the ferrum (III) citrate there was a decreasing in the bacteria growth, compared with growth in a medium with NaNO 2 or FeC 6 H 5 O 7 . In the medium with NaNO 2 and 10.41 mM FeC 6 H 5 O 7 , the growth of bacteria was decreased 2.6−3.1 times, compared with growth in media only with NaNO 2 or FeC 6 H 5 O 7 . In the media with NaNO 2 and FeC 6 H 5 O 7 with increasing concentrations of the ferrum (III) citrate, there was a gradual (1.7−3.1 times) decrease in the efficiency of nitrite ions reduction by the bacteria, as compared with their reduction in the medium with NaNO 2 (96−97 %) (Fig. 5, A). In the media containing NaNO 2 and FeC 6 H 5 O 7 , the cells produced 0.52−1.77 mM of ammonium ions (control: 1.82−1.88 mM) ( Table 5). The efficiency of Fe(ІІІ) reduction by the bacteria with increasing its concentration in the media with NaNO 2 and FeC 6 H 5 O 7 was revealed from 1.1 to 2.5 times lower than its reduction in the medium with FeC 6 H 5 O 7 (92−93 %) (Fig. 5, B). In the media with NaNO 2 and FeC 6 H 5 O 7 , cells produced 1.30−4.16 mM of the ferrum (II) ions (control: 2.88−2.97 mM) ( Table 5). In the media with NaNO 2 and FeC 6 H 5 O 7 without bacteria, the reduction of NO 2 and Fe(ІІІ) did not exceed 4.0 and 3.5 %, respectively (Fig. 5). Thus, it was established that ferrum (III) citrate inhibits the biomass accumulation, the nitrite ions reduction, and the ammonium ions production by the bacteria of Desulfovibrio sp. after simultaneous addition into the medium of NaNO 2 and FeC 6 H 5 O 7 (1.74−10.41 mM). In the medium with the same initial content (3.47 mM) NaNO 2 and FeC 6 H 5 O 7 , the bacteria reduced 1.4−1.6 times more Fe(ІІІ) than the nitrite ions with production of ferrum (II) ions at concentration 1.5−1.6 times higher than that of the ammonium ions.    In the medium with the same initial content (3.47 mM) of NaNO 3 and FeC 6 H 5 O 7 , the bacteria reduced 1.4 times more nitrate ions than of Fe(ІІІ), and in medium with the same content (3.47 mM) of NaNO 2 and FeC 6 H 5 O 7 strains reduced 1.4−1.6 times more Fe(ІІІ) than the nitrite ions. Nevertheless, FeC 6 H 5 O 7 at all concentrations in the medium showed an inhibitory action on nitrate and nitrite reduction, that was carried out investigated strains of the bacteria. Negative influence of Fe(ІІІ) on the activity of molybdenum-containing membrane-bound respiratory and/or dissimilatory nitrate reductase [21], as well as periplasmic nitrite reductase, containing siro heme as a prosthetic group [16,20], in the bacteria of Desulfovibrio genus can be due to a damage of the cytoplasmic membrane structure or modification of active conformation and denaturation of protein molecule, as a result of the replacement of the necessary metal ion by the ferrum in the active center of the enzyme. Although at pH 7.0 the standard oxidation-reduction potential of the Fe(III)/Fe(II) pair (E 0 ′= +0.77V) is lower than that of the oxidation-reduction NO 3 -/NO 2 pair (E 0 ′ = +0.78V), but higher than that of NO 2 -/NH 4 + pair (E 0 ′ = +0.34V) [16,32], the efficiency of electron acceptor reduction by the microorganisms is primarily determined by the difference between the donor and electron acceptor potentials, that depend on the pH of the medium and change during cultivation of the bacteria [8]. Sulfate-reducing bacteria of the Desulfovibrio genus oxidize organic substrates only to acetic acid. Among them are species able to ferment, except fumarate, lactate, pyruvate or other organic acids [16]. Therefore, energy supply of cells in the process of the anaerobic respiration depends not only on the oxidation-reduction potential of present in the medium electron acceptor, but also on the ways of ATP synthesis in process of electron donor oxidation -by substrate or electron transport phosphorylation.
The processes of nitrate and nitride reduction carried out by the bacteria of the Desulfovibrio genus was less sensitive to negative influence of ferrum (III) citrate, as compared with the process of sulfate ions reduction. When sulfate ions reduction by the bacteria in the presence of 1.74−10.41 mM FeC 6 H 5 O 7 decreased 2.0−4.7 times, the nitrate ions reduction − 1.3−1.9 times, nitrite ions − 1.7−3.1 times, in comparison with their reduction in the media with only Na 2 SO 4 ×10 H 2 O, NaNO 3 or NaNO 2 respectively. It is possible that at growth in the medium with FeC 6 H 5 O 7 and sulfate, nitrate or nitrite ions, nitrate and nitrite reductases of the investigated strains are less sensitive to negative influence of ferrum (III) citrate than the cytoplasmic enzymes involved in sulfate respiration of these bacteria -ATP sulfurylase, pyrophosphatase, APS reductase, sulfite reductase, as described [16,21,27]. This can be explained by the fact that at high concentrations in the medium Fe(III) can interact not only with functional groups of a number of bacteria cellular metabolites, but also cause an oxidative stress [10,28]. Despite the fact that the reduction of 1.74−10.41 mM Fe(ІІІ) by cells in the media with Na 2 SO 4 ×10 H 2 O, NaNO 3 or NaNO 2 decreased by 1.1−2.1, 1.6−2.7 and 1.1−2.5 times, respectively, compared with its reduction in the medium with only FeC 6 H 5 O 7 , the obtained results suggest that the investigated strains of bacteria are adapted to high concentrations of trivalent ferrum compounds (up to 10.41 mM) and, therefore, can survive in environments contaminated by heavy metals. The isolated strains are perspective for application in the technologies of complex purification of the environment from heavy metals, sulfur and nitrogen compounds, since they are capable of active reductive transformation of these pollutants.

CONCLUSIONS
Sulfate-reducing bacteria, oxidizing organic compounds, beside sulfates, can use other electron acceptors in process of the anaerobic respiration. These are oxidized metal forms, in particular, ferrum, nitrates or nitrites that are dangerous for biological organisms. In the media with Na 2 SO 4 ×10 H 2 O or NaNO 2 and FeC 6 H 5 O 7 at all tested concentrations, the bacteria reduced more Fe(III) than SO 4 2-or NO 2 -. In the media with NaNO 3 and FeC 6 H 5 O 7 at all concentrations, the bacteria reduced more NO 3 -, than of Fe(III). Nevertheless, at all concentrations in the medium FeC 6 H 5 O 7 showed a toxic effect on dissimilatory sulfate-, nitrate-and nitrite reduction, carried out by the bacteria. Due to the exoelectrogenic properties, the investigated strains of Desulfovibrio sp., demonstrated high metal-reducing activity even in the media with two various electron acceptors, can be applied as the anode biocatalysts in the microbial fuel cells for the formation of electric current during the oxidation of the organic matter. A resistance of strains of Desulfovibrio genus, isolated from Yavorivske Lake, to different pollutants can be a basis for their application in different biotechnologies, aimed at bioremediation of the polluted soils and waters.