MD4123C1 - Process for production of Fe2Se3O9·6H2O iron selenite and process for cultivation of Spirulina platensis cyanobacterium with the use thereof - Google Patents

Abstract

The present invention relates to a process for the production of iron selenite and for its use in the cultivation of Spirulina platensis cyanobacterium, with an increased content of selenium and iron, and can be used in medicine and food industry.It is proposed a simple process for production in mild conditions of hexahydrate iron(III) selenite - Fe2Se3O9·6H2O, providing for the interaction at 55…65°C of aqueous solutions of iron(II) and H2SeO3. The resulting product is a microcrystalline form of natural mandarinoit mineral.It is also claimed a process for cultivation of Spirulina platensis cyanobacterium, providing for the preparation of a nutrient medium, containing, g/L: NaHCO3 - 2.0, K2HPO4 - 0.5, NaNO3 - 2.5, K2SO4 - 0.5, NaCl - 1.0, MgSO4·7H2O - 0.2, CaCl2 - 0.04, FeSO4 - 0.01, EDTA - 0.08, as well as microelements, introduced as a separate solution - 1 mL, containing in its turn (g/L): H3BO3 - 2.86, MnCl2·4H2O - 1.81, ZnSO4·7H2O - 0.22, CuSO4·5H2O - 0.08 and MoO3 - 0.015. Further said cyanobacterium is inoculated in a concentration of 0.40…0.45 mg/L, it is carried out its cultivation during 6 days at the temperature of 30±2°C, light of 3000 lx and pH 9.5…10.5, and then is separated and dried the resulting biomass. At the same time, in the nutrient medium in the first three days of cultivation as a source of selenium and iron is added in portions in the form of suspension the said iron(III) selenite calculated for 0.015…0.030 g/L.The obtained technical result is to increase the content of selenium and iron in the biomass of cyanobacterium 2.0…2.9 and 1.4…2.9 times.

Description

The invention refers to a process for obtaining iron selenite and its use in the cultivation of the cyanobacterium Spirulina platensis, with an increased content of iron and selenium, and can be applied in medicine and the food industry.

Although selenium and inorganic iron in high concentrations are toxic, in low concentrations they are essential elements for the human body [1]. Selenium activates the process of tissue respiration, is a component of glutathione peroxidase and selenoproteins that are involved in the annihilation of oxygen radicals, produced as a result of oxidative stress in cells, has an immunostimulatory effect, influences the metabolism of proteins, especially those that include amino acids that contain sulphur. Iron is part of hemoglobin and has the function of enriching erythrocytes with oxygen. Iron deficiency causes anemia. The forms of selenium and organic iron are better assimilated by the body. Therefore, obtaining some nutritional additives, which would simultaneously contain iron and selenium in organic form, is of particular interest.

As a source of organic selenium, spirulina cyanobacteria biomass with an increased content of selenium, which in addition to selenium also contains cobalt, can serve. The process of cultivating spirulina with a high content of selenium is known. The process includes the steps: inoculation of spirulina in Gromov No. 16 nutrient medium, in which the concentration of NaHCO3 is 2 g/L. Cobalt(II) hydrogen selenite Co(HSeO3)2, in the amount of 15 mg/L, is additionally added to the medium. Cultivation is carried out under permanent lighting of 12...15 thousand erg/cm2·s in the first days and 18...21 thousand erg/cm2·s in the following days, at a temperature of 35±2°C. The pH of the environment is maintained within the limits of 8.4...8.6 by bubbling the suspension with CO2. On the 5th day of cultivation, the biomass is separated by filtration, washed with 1.5% ammonium acetate solution and then dried. The content of selenium in biomass is 93.0...98.6 mg%/100 g BAU [ 2].

The disadvantages of this process consist in the insufficient accumulation of selenium and cobalt in the biomass, as well as the low productivity of spirulina which decreases with the increase in the concentration of Co(HSeO3)2, because cobalt is a toxic element for spirulina. The iron content in biomass from Gromov reagent No. 16 constitutes 120 mg%/100 g BAU.

Spirulina biomass grown in the presence of inorganic compounds, which together contain selenium and iron, could serve as sources of these two elements, due to the ability of the cyanobacterium Spirulina platensis to bioassimilate these elements in organic form.

Iron selenite Fe2(SeO3)3 (Fe2Se3O9), with or without water molecules, could serve as a compound that simultaneously contains iron and selenium.

Iron selenite is known in the form of the natural mineral mandarinoite, which is a hexahydrate. It is widespread in the Pacajake mines near Hiaco, Colquechaca, Bolivia, together with other minerals, such as native selenium, siderite, penroseite, as well as their transformation (alteration) products, such as ahlfeldite ((Ni,Co)SeO3·2H2O ), cobaltomenite (CoSeO3·2H2O), chalcomenite (CuSeO3·2H2O) and molybdomenite (MoS2). The first data regarding the study of the chemical composition and crystalline structure of this mineral were presented in the work [3]. The authors named this mineral in honor of Dr. Joseph A. Mandarino of the Rozal Ontario Museum for his outstanding contribution to the development of both mineralogy in general and the study of selenium minerals in particular.

It was later determined that the crystal structure of mandarinite represents a three-dimensional coordination polymer (compound 1). Thus, the formula of compound 1 is more correctly represented as {[Fe2(SeO3)3(H2O)3]·3H2O}n [4].

Another compound with the same composition Fe2Se3O9·6H2O (2) is also known, which was obtained synthetically and disclosed in the paper [5]. The method of obtaining the compound consists of the following. Two aqueous solutions with a volume of 20 mL, containing respectively 0.3 mol Na2SeO3 and 0.2 mol FeCl3, were mixed in a Teflon-sealed vessel, after which they were kept at a temperature of 90°C for 40 days. At the initial stage of the experiment, a brown amorphous product was formed, which later turns into a green crystalline product. After 40 days it was separated by filtration, washed with water and dried at room temperature.

Following the analysis, it was found that the chemical composition of compound 2 is identical to that of the mandarinoite mineral and corresponds to the formula Fe2Se3O9·6H2O. On the other hand, the results of the X-ray study of compound 2 demonstrated that this compound 2 [5] and compound 1 [3,4] represent polymorphic forms having the same chemical composition (Fe2Se3O9·6H2O), but different crystal structure.

The disadvantage of the proposed synthetic process consists in carrying out the synthesis at high temperatures (90ºC) and in a long period of 40 days.

Although the important role of iron and selenium as bioelements is known, iron selenites have not been used as iron and selenium supplements in different biomass cultivation processes, except for the Spirulina platensis biomass cultivation process, whose extract was used in the process of obtaining cryoresistant bull semen [6]. The natural form of iron selenite (compound 1) was used for biomass cultivation, which has disadvantages caused by increased crystallinity and, as a result, very low solubility. Thus, the bioassimilation of the elements is reduced and unstable (variable content), despite the fact that additional iron is introduced into the growing medium in the form of a complex with ethylenediaminetetraacetic acid.

The problem that the present invention solves consists in developing a simple process for chemically obtaining Fe(III) selenite hexahydrate - Fe2Se3O9·6H2O and developing an effective process for cultivating the cyanobacterium Spirulina platensis with an increased content of selenium and iron in organic form with the use of this compound as a source of selenium and iron with increased and stable bioassimilability.

The essence of the invention is the development of a simple process for obtaining iron selenite under mild conditions. The applicants established that under certain conditions the synthesized iron(III) selenite Fe2Se3O9·6H2O can be used as a source of iron and selenium to obtain the biomass of the cyanobacterium Spirulina platensis enriched with these two bioelements.

The procedure for obtaining iron selenite provides for the interaction of aqueous solutions of FeSO4·(NH4)2SO4·6H2O and H2SeO3, heated to 55...65ºC, taken in the respective molar ratio of 2:1. The obtained mixture is maintained at a temperature of 50...60°C for 1 hour, after which it is filtered, washed and dried. The chemical composition and structure of the compound Fe2Se3O9·6H2O was investigated using several physical methods, such as the X-ray analysis method, the thermogravimetric method and Mossbauer spectroscopy. The analysis data proved that the obtained selenite represents a microcrystalline form of mandarinite, which has the structure {[Fe2(SeO3)3(H2O)3]·3H2O}n.

The process of cultivating the cyanobacterium Spirulina platensis includes the preparation of the nutrient medium, which contains, g/L: NaHCO3 - 2.0, K2HPO4 - 0.5, NaNO3 - 2.5, K2SO4 - 0.5, NaCl -1.0, MgSO4 ·7H2O - 0.2, CaCl2 - 0.04, FeSO4 - 0.01, EDTA - 0.08, as well as trace elements, introduced in the form of a separate solution - 1 mL, which in turn contains (g/L) : H3BO3 - 2.86, MnCl2·4H2O - 1.81, ZnSO4·7H2O - 0.22, CuSO4·5H2O - 0.08, MoO3 - 0.015, spirulina inoculation in a concentration of 0.40...0.45 mg/L and cultivation within 6 days at a temperature of 30±2°C, with an illumination of 3000 lx and the pH of the medium 9.5...10.5, after which the obtained biomass is separated and dried, at the same time in the nutrient medium in the first three days of cultivation as a source of selenium and iron, Fe(III) selenite hexahydrate - Fe2Se3O9·6H2O, obtained according to the claimed process, from the total calculation of 0.015...0.030 g/L, is added in portions in suspension.

The technical result obtained consists in the stable increase in the biomass of the selenium content by 2.0...2.9 times and the iron content by 1.4...2.9 times, compared to the known process.

The result obtained is due to the gradual dissociation of Fe2Se3O9·6H2O and the intracellular inclusion of selenium instead of sulfur in the composition of the thioamino acids cysteine and methionine, in polysaccharides, lipids, etc., as well as the intracellular binding of iron with organic compounds (proteins, amino acids, peptides, carbohydrates, etc. .) from the composition of the spirulina biomass, which ensures the synergistic effect of this compound, compared to the mixture consisting of 2 salts of these elements (FeCl3·6H2O and Na2SeO3), which contain similar concentrations of iron and selenium.

The invention is explained by fig. 1-4, which represent:

- fig. 1, diffractogram of the polycrystalline sample of the compound Fe2Se3O9·6H2O (3);

- fig. 2, thermogram Fe2Se3O9·6H2O (3);

- fig. 3, the Mossbauer spectrum of the compound Fe2Se3O9·6H2O (3) at T = 80K;

- fig. 4, the Mossbauer spectrum of the compound Fe2Se3O9·6H2O (3) at T = 300K.

Example of realization of the synthesis process of iron selenite hexahydrate

To the hot solution (t = 60°C) containing 7.84 g of FeSO4·(NH4)2SO4·6H2O (0.02 mol) dissolved in approximately 75 mL of water, the solution containing 1.29 g of H2SeO3 ( 0.01 mol) dissolved in about 25 mL of hot water. A red-brick colored crystalline sediment is obtained. The obtained mixture is maintained at a temperature of 50...60°C for one hour, then it is filtered. The obtained precipitate is washed several times with water, then with a little ethyl alcohol. Dry at room temperature. The product thus obtained is a brick-red microcrystalline substance, it is slightly soluble in water and organic solvents. The chemical composition and structure of the compound Fe2Se3O9·6H2O (3) was investigated using several analysis methods, such as the X-ray analysis method, the thermogravimetric method and Mossbauer spectroscopy.

The X-ray analysis of compound 3 obtained above was carried out using a ДРОН-3.0 linear diffractometer (FeKα radiation, λ = 1.93604 Å, θ/2θ scanning method), the samples being prepared according to the standard procedure. The X-ray experiment (fig. 1) demonstrated that the analyzed samples represent single-phase systems within the resolution limits of the method. Diffraction lines were indexed based on ICDD data [ICDD 1997 JCPDS-International Center for Diffraction Data. All right reserved PCPDFWIN v.1.30, 29-0719] and works [3, 4] within the space group P21/c of the monoclinic singion having the elementary cell parameters: a=16.810(4), b=7.880(2), c= 10.019(2) Å, β=98.26(2)°. The Miller indices, the sizes of the 2θ angles, as well as the sizes of the interplanar distances of the diffraction lines are presented in table 1.

Table 1

No. hkl Fe2Se3O9·6H2O (3) Fe2Se3O9·6H2O (1) [3,4] Fe2Se3O9·6H2O (2) [5] 2θ, ° d, Å d, Å hkl d, Å 1 200 13.40 8.30 8, 25 -101 7.101 2 110 15.70 7.09 7.10 101 6.426 3 011 18.14 6.14 6.14 011 6.314 4 002 22.60 4.94 4.94 210 4.681 5 -202 24.50 4 ... 32.90 3.42 3.43 022 3.166 10 411 34.26 3.29 3.31 311 3.033 11 -511 37.26 3.03 3.04 -302 2.934 12 -213 38.10 2.97 2, 97 013 2.739 13 222 40.60 2.79 2.80 103 2.701 14 322 43.82 2.59 2.59 302 2.623 15 123 46.60 2.45 2.45 023 2.443 16 204 50.6 0 2.27 2.27 132 2,418 17 -613 52.70 2.19 2.12 -412 2.341 18 304 53.80 2.14 331 2,236 19 124 56.68 2.04 2.04 141 2.195 20 -424 59 .18 1.96 1.96 004 2.149

Comparative analysis of the data presented in tab. 1 certainly leads to the conclusion that compound 3 obtained synthetically (according to the claimed process) and compound 1 previously studied [3,4] represent one and the same substance, the composition and structure of which corresponds to the mineral mandarinoite {[Fe2(SeO3)3( H2O)3]·3H2O}n. At the same time, these data demonstrate that compound 2 represents a polymorphic form having the same composition Fe2Se3O9·6H2O.

The thermal analysis of the compound Fe2Se3O9·6H2O (3) was performed using the Paulik-Paulik Erday System derivatograph in the temperature range 20...1000 °C at a speed of 5°/min in an air atmosphere (corundum crucible, Al2O3 standard). As can be seen from the derivative diagram shown in fig. 2, the thermolysis of the compound proceeds in three endothermic stages.

The first peak on the DTG curve is observed in the temperature range 40…120°C (with a maximum at 90°C) accompanied by a mass decrease equal to 9.2%, which corresponds to the elimination of three molecules of water of crystallization (calculated for three H2O molecules - 9.0%, resulting from composition 3). In the temperature range 220...330°C on the derivative diagram, another endothermic effect is observed with a maximum at 310°C. The mass decrease in this temperature range corresponds to the removal of three more H2O molecules from the iron coordination sphere (determined 9.5%; calculated for 3H2O 9.0%). The given unfinished effect overlaps with a set of superimposed endothermic effects in the range 330...620°C, the most intensive maximum being at 450°C. The resulting residue constitutes 25% of the initial mass of the compound (calculated 26.6%), which corresponds to the formation of Fe2O3 oxide.

The Mossbauer spectra of compound 3 (fig. 3, 4) were recorded at the Center "Physical Chemistry and Nanocomposites" within the Institute of Chemistry of AŞM, using a Mossbauer spectrometer type MS4 WRC (WEB Research, Edina, MN) equipped with a cyclic cooling system (4.5...300K) and a W106 temperature control system. A source of 57Co (3.7 GBq) in Rh matrix was used. Metallic α-Fe at 298K temperature was used as standard. The simulation of the experimental data was performed using the WMOSS software package (WEB Research, Edina, MN).

The Mossbauer spectra of the compound Fe2Se3O9·6H2O (3) at the temperature of 80 and 300K (fig. 3,4) were approximated by two doublets, the parameters of which (tab. 2) correspond to the Fe+3 ion in the high spin state S= 5/2.

Table 2

The parameters of the Mossbauer spectra of the compound Fe2Se3O9·6H2O (3)

T, K mm/s Srel mm/s Srel DIFe ±0.02 SC ±0.02 ΓL=Г R ±0.02 DIFe ±0.02 SC ±0.02 ΓL=Г R ±0.02 Doublet I Doublet II 300 0.43 0.71 0.43 0.63 0.43 0.37 0.33 0.63 80 0.52 0.72 0.38 0.54 0.52 0.34 0.37 0, 54

Mossbauer spectra data for the compound Fe2Se3O9·6H2O (3) show a perfect agreement with the chemical composition and crystal structure of mandarinite (1) [3]. First of all, the Mossbauer spectra demonstrated that the iron ions in compound 3 show the +3 oxidation state, although in the synthesis a bivalent iron sulfate was used as the initial compound. No species containing iron ions with oxidation state +2 were detected. At the same time, the presence of two doublets in the Mossbauer spectra is explained by the fact that the iron ions in the crystal structure of mandarinite occupy two crystallographically independent positions, having different octahedral surroundings.

In conclusion, the data presented above demonstrate that the compound Fe2Se3O9·6H2O (3), for which a new production process is proposed, is identical in chemical composition and structure to the mineral mandarinoite {[Fe2(SeO3)3(H2O)3]· 3H2O}n (1).

The ability of compound 3 to regulate the simultaneous content of iron and selenium in the biomass of Spirulina platensis was demonstrated in the examples below.

Example 1

In the experiments, the action of the selenite compound of Fe(III) hexahydrate - Fe2Se3O9·6H2O (3) on the accumulation process of selenium and iron in the biomass of Spirulina platensis was investigated.

Prepare the nutrient medium with the following composition (g/L): NaHCO3 - 2.0, K2HPO4 - 0.5, NaNO3 - 2.5, K2SO4 - 0.5, NaCl - 1.0, MgSO4·7H2O - 0.2 , CaCl2 - 0.04, FeSO4 - 0.01, EDTA - 0.08, a solution of microelements 1 mL/L is added, which in turn contains (g/L): H3BO3 - 2.86, MnCl2· 4H2O - 1.81, ZnSO4·7H2O - 0.22, CuSO4·5H2O - 0.08, MoO3 - 0.015. The spirulina suspension in a concentration of 0.45 mg/L is added to the prepared medium and it is cultivated within 6 days at a temperature of 30°C, with an illumination of 3000 lx and a pH of the medium of 9.5. Additionally, 0.005 g/L Fe(III) selenite hexahydrate - Fe2Se3O9·6H2O (final concentration 0.015 g/L) is added in portions to the nutrient medium during the first three days of cultivation. On the 6th day, the biomass was separated from the culture liquid by filtration and washed with 1.5% ammonium acetate solution. The biomass was dried and the content of selenium and iron was determined.

Example 2

Prepare the nutrient medium according to example 1. To the prepared medium, add the spirulina suspension in a concentration of 0.45 mg/L and cultivate within 6 days at a temperature of 28°C, with an illumination of 3000 lx and the pH environment 9.5. Additionally, 0.00667 g/L Fe(III) selenite hexahydrate - Fe2Se3O9·6H2O (final concentration 0.020 g/L) is added in portions to the nutrient medium during the first three days of cultivation. On the 6th day, the biomass was separated from the culture liquid by filtration and washed with 1.5% ammonium acetate solution. The biomass was dried and the content of selenium and iron was determined.

Example 3

Prepare the nutrient medium according to example 1. To the prepared medium, add the spirulina suspension in a concentration of 0.45 mg/L and cultivate within 6 days at a temperature of 28°C, with an illumination of 3000 lx and the pH environment 10.0. Additionally, 0.010 g/L Fe2Se3O9·6H2O (final concentration 0.030 g/L) is added in portions to the nutrient medium during the first three days of cultivation. On the 6th day, the biomass was separated from the culture liquid by filtration and washed with 1.5% ammonium acetate solution. The biomass was dried and the content of selenium and iron was determined.

The results regarding the accumulation of selenium and iron in the biomass of Spirulina platensis obtained according to the proposed invention and according to the closest solution are presented in tab. 3.

Table 3

Selenium and iron accumulation in spirulina biomass during cultivation in the presence of chemical regulators (Co(II) hydrogen selenite dihydrate, Fe(III) selenite hexahydrate)

Process used Compound Concentration Selenium content in biomass, mg% Iron content in biomass, mg% Productivity on the 6th day, g/L g/L mM Fe Se According to [2] Co(HSeO3)2·2H2O 0.015 0.020 0.030 - - - 0.085 0.114 0.171 94.0 102.0 - 120 120 - 0.90 0.68 0 (lethal dose) According to the invention Fe2Se3O9·6H2O 0.015 0.020 0.030 0.050 0.067 0 .100 0.075 0.100 0.150 187.0 216.0 296.0 165 220 330 0.96 0.94 0.92 Standard nutrient medium supplemented with Fe(III) and Se(IV) Control: FeCl3·6H2O and Na2SeO3 (added together in situ) 0.027 0.026 0.100 0.150 51, 0 205 0.90

Your data. 3 demonstrates that when Fe(III) selenite hexahydrate is used in concentrations of 0.015...0.030 g/L, the selenium content in the biomass is stable and increased compared to the solution [2] by 2.0...2.9 times, and the iron content by 1.4…2.9 times. The content of Fe(III) and Se(IV) in the spirulina biomass is considerably lower (205 and 51 mg%, respectively) when supplementing the mixture consisting of 0.027 g/L (0.100 mM) FeCl3·6H2O and 0.026 g/L ( 0.150 mM) Na2SeO3 taken together, compared to that accumulated in the case of administration in the medium of 0.030 g/L Fe2Se3O9·6H2O, with a similar content of iron and selenium. This demonstrates the advantage of using the compound proposed in the present invention.

1. Wolfgang Kaim, Brigitte Schwederski "Bioinorganic Chemistry: Inorganic Elements in the Chemistry of Life." John Wiley and Sons, 1994

2. Rudic V. New aspects of modern biotechnology. Chisinau, Science, 1993, p. 72-74

3. Pete J. Dunn, Donald R. Peacor, B. Darko Sturman // Canadian Mineralogist. Vol.16, pp. 605-609 (1978)

4. Frank C. Hawthorne // Canadian Mineralogist. Vol. 22, pp. 475-480 (1984)

5. Dhanpat Rai, Shas V. Mattigod and Dean A. Moore // Mat. Res. Bull., Vol. 23, pp. 1621-1628, 1988

6. MD 221 Y (2010.06.30)

Claims (2)

1. Process for obtaining Fe(III) selenite Fe2(SeO3)3·6H2O which provides for the addition with stirring to a solution with a temperature of 55...65°C, which contains 7.84 g of FeSO4·(NH4)2SO4·6H2O ( 0.02 mol) dissolved in 75 mL of water of a solution containing 1.29 g of H2SeO3 (0.01 mol) dissolved in 25 mL of hot water in the respective molar ratio of 2:1, the resulting mixture is further maintained at the temperature of 50...60°C for one hour, the obtained precipitate is filtered, washed with water, then with ethyl alcohol and dried. 2. Process for cultivating the cyanobacterium Spirulina platensis, which includes the preparation of a nutrient medium, which contains, g/L: NaHCO3 - 2.0, K2HPO4 - 0.5, NaNO3 - 2.5, K2SO4 - 0.5, NaCl - 1.0, MgSO4·7H2O - 0.2, CaCl2 - 0.04, FeSO4 - 0.01, EDTA - 0.08 and trace elements introduced in the form of a separate solution - 1 mL, which in turn contains (g /L): H3BO3 - 2.86, MnCl2·4H2O - 1.81, ZnSO4·7H2O - 0.22, CuSO4·5H2O - 0.08, MoO3 - 0.015; the inoculation of Spirulina platensis in a concentration of 0.40...0.45 mg/L and its cultivation within 6 days at a temperature of 30±2°C, illumination of 3000 lx and pH of the environment 9.5...10.5; separation and drying of the resulting biomass, at the same time, Fe(III) selenite hexahydrate (Fe2Se3O9·6H2O), obtained according to claim 1, from the total calculation of 0.015...0.030 g/ IT.