REDUCTION OF Cr(VI) COMPOUNDS BY THE IMMOBILIZED CELLS OF SULFATE-REDUCING BACTERIA DESULFOMICROBIUM SP. CrR3

Immobilization of microorganisms is an effective method of intensification of waste water purification form chromate-containing compounds. This paper presents a method of sulfate-reducing bacteria immobilization in agar developed in order to determine the effectiveness of water purification from toxic hexavalent chromium compounds. Depen dence of the influence of different factors on the reduction of hexavalent chromium compounds by immobilized cells of sulfate-reducing bacteria Desulfomicrobium sp. C rR3 has been studied. Both free and agar-immobilized Desulfomicrobium sp. C rR3 cells almost completely reduced Cr(VI) at the initial concentration of 1 mM. Immobilized cells reduced over 90 % of Cr(VI) in 4 days, and non-immobilized ones – in 6 days. Cr(III) content increased with the decrease of hexavalent chromium concentration. A possibility of multiple usage of agar-immobilized Desulfomicrobium sp. C rR3 cells for the purification of the model solution from Cr(VI) at the concentration of 0.5 mM and 1 mM during 60 h was confirmed. After three-time usage of the immobilized cells (1g/L), effectiveness of Cr(VI) reduction is 68 % at their initial concentration of 0.5 mM and 50 % – at 1 mM; at cells concentration 3 g/L – 87 % and 77 %; 5 g/L – 94 % and 92 %, and at cells concentration 8 g/L – 98 % and 96 %, respectively. As a result of the regres sion analysis of the influence of different factors on the purification of the model solution from Cr(VI) by agar-immobilized Desulfomicrobium sp. CrR3 cells a reliable dependence equation of the dependence of change of hexavalent chromium concentration per unit of time on the three analysed factors has been derived. Immobilization of Desulfomicrobium sp. C rR 3 cells is the promising way of water purification from hexavalent chro mium compounds. Effectiveness and duration of the process of water purification from Cr(VI) by the immobilized Desulfomicrobium sp. C rR3 bacteria depend on the initial hexavalent chromium content and cell concentration.

Immobilization of microorganisms is an effective method of intensification of wastewater purification form chromate-containing compounds. This paper presents a method of sulfate-reducing bacteria immobilization in agar developed in order to determine the effectiveness of water purification from toxic hexavalent chromium compounds. Dependence of the influence of different factors on the reduction of hexavalent chromium compounds by immobilized cells of sulfate-reducing bacteria Desulfomicrobium sp. CrR3 has been studied. Both free and agar-immobilized Desulfomicrobium sp. CrR3 cells almost completely reduced Cr(VI) at the initial concentration of 1 mM. Immobilized cells reduced over 90 % of Cr(VI) in 4 days, and non-immobilized ones -in 6 days. Cr(III) content increased with the decrease of hexavalent chromium concentration. A possibility of multiple usage of agar-immobilized Desulfomicrobium sp. CrR3 cells for the purification of the model solution from Cr(VI) at the concentration of 0.5 mM and 1 mM during 60 h was confirmed. After three-time usage of the immobilized cells (1g/L), effectiveness of Cr(VI) reduction is 68 % at their initial concentration of 0.5 mM and 50 % -at 1 mM; at cells concentration 3 g/L -87 % and 77 %; 5 g/L -94 % and 92 %, and at cells concentration 8 g/L -98 % and 96 %, respectively. As a result of the regression analysis of the influence of different factors on the purification of the model solution from Cr(VI) by agar-immobilized Desulfomicrobium sp. CrR3 cells a reliable dependence of the studied parameter change on time, initial content of hexavalent chromium and cells concentration was found. Determination indices have been calculated and the
Compared to the traditional methods of biological purification of wastewater in aera ted lagoons, immobilized microbiota has a number of advantages. First, such an "immobilized catalyzer" of the purification process is easy to remove from the reaction medium, which enables termination of the process at the right moment, while removal of active sludge from an aerated lagoon involves a long process of sedimentation and centrifugation. Besides, the carrier is suitable for multiple usage; purified water is not contaminated by microbial cells, so a secondary settler is not needed. Secondly, using the immobilized active sludge makes it possible to purify wastewater continuously and regulate the purification process by changing the flow velocity. Thirdly, immobilization of microbial biomass can increase the catalytic activity of enzymes [16].
Sulfate-reducing bacteria reduce sulfur oxoanions and oxidize organic compounds in anaerobic conditions in the process of sulfate respiration [5,10]. Previous studies show that as electron acceptors they can also utilize nitrate ions, hexavalent chromium compounds and some heavy metal ions with variable valency [7,13,15].
In this study, we made an attempt to immobilize sulfate-reducing bacteria in order to use them for water purification from toxic compounds of hexavalent chromium.

MATERIALS AND METHODS
Chromate-resistant sulfate-reducing bacteria Desulfomicrobium sp. CrR3, isolated from the wastewater of Lviv purification system, were used for this research [11].
Bacteria were grown in Postgate C medium [8] with potassium dichromate (1 mM of chromium) at 30 °C in 25 mL tubes. The choice of such a concentration of chromium was determined by the results of our previous study [12], according to which further increase of chromium content in the medium leads to the inhibition of bacterial growth. Water solution of potassium dichromate was added after sterilization. To achieve anaerobic conditions tubes were fully filled with the medium and stoppered with rubber corks. To confirm the anaerobic conditions, AnaerIndicator (bioMeriux, France) -an indicator of anaerobic conditions was used. Biomass was measured turbidimetrically using the photoelectrocolorymeter KFK-3 (λ = 340 nm, cuvette 3 mm). Chromate content was measured spectrophotometrically (λ = 540 nm, cuvette 10 mm) by biphenyl carbaside method [6]. Chromasurol S (λ = 590 nm, cuvette 10 mm) was used to measure Cr(ІІI) content [4].
To immobilize cells of sulfate-reducing bacteria on the synthetic carrier "VIIA", we grew them in the tube with synthetic fibres. After 14 days, "VIIA" were added to the model solution. To obtain Desulfomicrobium sp. CrR3 cells immobilized in agar, the grown bacteria were centrifuged at 6000 g for 30 min at 4 °C. The cells were washed twice with the isotonic solution of sodium chloride and added to 2% agar solution cooled to 40...45 °С. The agarized solution with the cells was poured into Petri plates to form a uniform layer. After solidification, the agar was cut into 7-8×7-8×7-8 mm cubes and added to the mo del solution of potassium dichromate containing Cr(VI) with pH adjusted to 7.
Statistical analysis was performed in R medium (3.6.3) in RStudio envelope (1.2.5033) using ggplot2 library [9]. Method of multiple linear regression with different variable combinations in linear model was used to determine the significance of the dependence of chromium concentration on such parameters as initial chromium concentration, time of measuring after the addition of chromium and concentration of cells.

RESULTS AND DISCUSSION
This study was focuses on a possibility to immobilize sulfate-reducing bacteria Desulfomicrobium sp. CrR3 on the synthetic carrier "VIIA", which is considered to be promising and readily available [14], and in agar. Effective growth of sulfate-reducing bacteria Desulfomicrobium sp. CrR3 in the Cr(VI) containing medium without sulfates and other terminal electron acceptors was reported previously [12]. After 14 days of Desulfomicrobium sp. CrR3 bacteria cultivation in Postgate C medium with "VIIA" carriers, bacterial biomass accumulated, but no immobilization occured. Bacterial biomass was equal in both culture liquids, with and without the synthetic fibres. Sedimentation of FeS, which stained the carrier in black colour, was observed in Postgate C medium with ferrous sulfate.
The synthetic carrier was substituted for agar, as immobilization on the synthetic carrier "VIIA" was not successful. The pattern of chromate ions utilization by the sulfatereducing bacteria Desulfomicrobium sp. CrR3 immobilized in agar proved to be similar to the pattern of Cr(VI) utilization by non-immobilized cells. The cells were immobilized in agar and their ability to reduce chromate ions was stu died. We found that both immobilized and non-immobilized cells almost completely redu ced Cr(VI) at the initial concentration of 1 mM (Fig. 1). The immobilized Desulfomic robium sp. CrR3 cells reduced over 90% of Cr(VI) within 4 days, and the free ones -within 6 days. Cr(III) content increa sed with the decrease of chromate ions concentration. The concentration of chromate ions did not change in the control sample (without cells) (Fig. 1A).   (Fig. 2B).
The increase in concentration of the added cells to 5 g/L accelerates the process of Cr(VI) reduction. Cr(VI) concentration in the medium reached 0.11 mM after 17 h. We observed an insignificant deceleration of Cr(VI) reduction in the case of a repeated addition of 0.5 mM. Cr(VI) concentration was 0.19 mM after 34 h. With the next addition of 0.5 mM of Cr(VI), its utilization decelerated (Fig. 2C). 0.06 mM of Cr(VI) was found in the medium 17 h after the addition of 8 g/L of agar-immobilized cells into the model solution with Cr(VI) concentration of 0.5 mM. Its concentration was 0.16 mM in 17 h (on the 34th hour) after the repeated addition of 0.5 mM of Cr(VI) (Fig. 2D).
A two-fold increase in the initial Cr(VI) concentration in the model solution results in different extent of its reduction by the agar-immobilized Desulfomicrobium sp. CrR3 cells depending on their concentration (Fig. 3).  (Fig. 3A).
After the addition of 3 g/L of agar-immobilized cells into the model solution, which contained 1 mM of Cr(VI), the utilization of 0.23 mM of Cr(VI) after 3 h was observed. Cr(VI) concentration was 0.53 mM after 17 hours. A repeated addition of 1 mM of Cr(VI) resulted in the 1.6-fold deceleration of CrO 4 2utilization by cells. With the next addition of 1 mM of Cr(VI) bacteria utilized only 0.29 mM of Cr(VI) (Fig. 3B).
The increase of cell concentration to 5 g/L accelerates the process of Cr(VI) reduction. Cr(VI) concentration reached 0.36 mM after 17 h. Its utilization decelerated in the case of a repeated addition of 1 mM of Cr(VI). After 34 h, Cr(VI) concentration was 0.52 mM. With the next addition of 1 mM of Cr(VI), its utilization decelerated (Fig. 3C).
After the addition of 8 g/L of agar-immobilized cells into the model solution with 1 mM of Cr(VI) 0.28 mM of Cr(VI) was found in the medium 17 h. 17 h after the repeated addition of 1 mM of Cr(VI) (in 34 hours), its concentration was 0.42 mM. With the next addition of 1 mM, utilization of Cr(VI) decelerated (Fig. 3D).
Thus, it is not only the concentration of cells, but also Cr(VI) concentration that influences the reduction of chromate ions by the immobilized Desulfomicrobium sp. CrR3 cells. After a three-time use of the immobilized Desulfomicrobium sp. CrR3 cells (1g/L), the effectiveness of chromate ions reduction is 68% at their initial concentration of 0.5 mM and 50% -at 1 mM; at cells concentration of 3 g/L -87% and 77%; 5 g/L -94% and 92%, and at cells concentration of 8 g/L -98% and 96%, respectively (see Table).

Ефективність відновлення хромат-йонів іммобілізованими клітинами
Desulfomicrobium sp. CrR3 Chromate ions concentration (mM) Concentration of cells (g/L) The dependence of chromium concentration on time is significant (p<0.001), but the determination coefficients that indicate the part of variation of the dependent variable, which can be explained by the variation of predictros, are low in this model (R 2 = 0.3234, R 2 adj = 0.320). It can be seen from equation 1 that Cr(VI) concentration changes by 0.009 mmol per hour.
At the initial Cr(VI) concentration of 1 mM in the model solution, the dependence of hexavalent chromium concentration on time is described by the equation [Cr] = 0.971 -0.022 · t, (2) where [Cr] -concentration of hexavalent chromium, mM; t -time after the moment of Cr(VI) addition to the medium, hours.
The dependence of chromium concentration on time is significant (p<0.001) and the determination coefficients are high (R 2 = 0.6873, R 2 adj = 0.6857). Cr(VI) concentration decreases by 0.022 mmol per hour (equation 2).
According to the regression analysis, the changes in Cr(VI) concentration when using agar-immobilized Desulfomicrobium sp. CrR3 cells conform with the interpretation of the visualization presented at Fig. 4. The multiple linear regression, performed for the dependent index -chromium concentration, and the independent indeces (predictors) -time and initial chromium concentration, shows that the change of Cr(VI) concentration is described by the equation In this case, the p-level of significance of dependence of Cr(VI) concentration on time is low (p = 0.0544), and the coefficient of this predictor is positive, which indicates that only time in this model is not a significant parameter. However, the p-level of significance of dependence of hexavalent chromium concentration on the initial Cr(VI) concentration is notable (p<0.001) in the same manner as for the interaction/combination of these factors (the extent of expression of measuring time factor significantly depends on the gradation of the initial chromium concentration, p<0.001). Determination coefficients for such a model indicate that a significant part of the chromium concentration variability is described by the variability of these two parameters (R 2 = 0.829, R 2 adj = 0.827). Thus, the influence of the factor of time depends significantly on other parameters, particularly, the initial concentration of Cr(VI), which by itself is a significant predictor.
The multiple linear regression, performed for the dependent index of chromium concentration and for the independent indices of time, Desulfomicrobium sp. CrR3 cells concentration and the initial Cr(VI) concentration, shows that the change of hexavalent The most significant predictor is the initial Cr(VI) concentration (p<0.001) and an aggregate of all three factors (expression of time as a factor depends on the initial chromium concentration factor gradation and cells concentration, p ≈ 0.001). Time in relation to the gradation of initial chromium concentration (p = 0.003) and concentration of cells (p = 0.046) are significant factors as well. Determination coefficients of the model indicate its conformity with the data variation (R 2 = 0.883, R 2 adj = 0.881). Cchanges in Cr(VI) concentration when using agar-immobilized Desulfomicrobium sp. CrR3 cells taking into consideration the factors of time, initial chromium content and concentration of cells conform with the interpretation of the visualization presented in Fig. 5.

CONCLUSIONS
Immobilization of Desulfomicrobium sp. CrR3 cells is a promising way of water purification from hexavalent chromium compounds, which can be used to develop technologies for bioremediation of the environment from these pollutants. Effectiveness and duration of the process of water purification from Cr(VI) by the immobilized Desulfomic-

Conflict of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Animal Rights: This article does not contain any studies with animal subjects performed by the any of the authors.