RU2099412C1 - Method of culturing thiobacillus ferrooxidans and a method of extraction at least one metal from ore difficult for concentrating - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: culture Thiobacillus ferrooxidans is cultured on mineral medium containing formate at amount 100 mcM, not above. Ore difficult for concentrating is treated with culture T. ferrooxidans grown on mineral medium containing formate. EFFECT: improved method of culturing. 15 cl, 3 tbl, 4 dwg

Description

 The invention relates to a method for cultivating Thiobacillus ferrooxidans bacteria cultures to a very high density by feeding bacterial growth-limiting formate as the only energy source. Such bacteria in large quantities are required for more effective bioleaching of sulfide-containing metal ores, and are also used for other purposes.

 This method allows you to grow "Thiobacillus ferrooxidans" ("Th.ferrooxidans") to such high concentrations that cannot be achieved by previously known methods. This significantly improves the quality of bioleaching of sulfide-containing metal ores associated with the use of "Th.ferrooxidans". Any interruption in the supply of these bacteria can overlap with the quality of industrial operations where these bacteria are used.

 The term "ore" in this description can mean both tails and raw ore. Tails are ores that have already been processed or graded to separate or recover more easily recoverable metals or metal fractions. Raw ore, which must be oxidized before leaching, is a common ore, the oxidation and leaching of which is carried out by conventional methods that ensure its economical purification, for example, by cyanidation. Oxidation before leaching is usually done in the processing of sulfide-containing and carbonate-containing ores, which are very difficult to process ores.

 Leaching in the art is also known as hydrometallurgical recovery.

 It is known that the leaching method can be improved by oxidizing ores containing sulfide components using chemical and biological agents, conducting such oxidation before further purification by cyanidation. For example, a noble metal ore, which is difficult to concentrate due to the presence of a carbonate-containing component in it, can be treated with gaseous chlorine, and then cyanidation using carbon in alkali to release carbon from the oxidation process. This method has many advantages, but it also has disadvantages. Chlorine is expensive and dangerous to handle. With an increase in the carbon content in sulfide ores, production and environmental costs increase. Ore, which is difficult to concentrate due to the presence of sulfides and / or carbonate-containing components in it, can also be pretreated with oxygen under pressure or through other chemical oxidation processes.

 Another traditional processing method is roasting at high temperatures, designed to oxidize sulfide and / or carbon-containing components of ore or other material to be processed. Such processes require significant costs for production and environmental protection, which increase with increasing sulfide content in the processed ores. Ores that are difficult to process because they contain both carbonate and sulfide components are very expensive to oxidize by conventional chemical methods, so it is best to pre-treat them by bioleaching followed by extraction with thiourea (materials from the 1985 AIME conference in San Diego in November 1985 g.).

There is also known a method of adding "Th.ferrooxidans" in amounts exceeding those that occur in nature. Bacteria are added to tails in the presence of additional nutrient media (for example, (NH ₄ ) ₂ SO ₄ ) that promote bacterial growth. The resulting population of "Th.ferrooxidans" increases the oxidation rate of sulfide-containing ores compared with the oxidation rate that is observed in the tailings dump under natural conditions. (PB Marchant, Fundamental and Applied Biohydrometallurqy, New York, NYpp. 53-57. 1986.)
Bioleaching has many advantages over traditional methods of oxidizing sulfide-containing ores in order to increase the recovery of useful components during leaching. Bioleaching allows avoiding the use of process components such as gaseous chlorine, the use of expensive high-pressure oxygen injection equipment, and also avoids the problems associated with the release of sulfur dioxide and volatile arsenic compounds into the atmosphere during the high-temperature firing process. (Hutchins SR Davidson MS and etal. Ann. Rev. Mierobiol. 40: 311-336. 1986). On the other hand, during bio-leaching, arsenic and other toxic materials remain in the leach, which can be disposed of using methods acceptable to the environment. Another advantage of leaching is more efficient metal recovery compared to other methods. (PBMarchant, Fundamental and Applied Biohydromentallurqy, Elsevier New York, NYpp. 53-57. 1986).

 It is known to use the Th.ferrooxidans bacteria when leaching gold, which is manifested in the absorption mechanisms of pyrite or other sulfide minerals that clog up useful components, due to which the ore particles are destroyed and the clogged metal is released, followed by an increase in its extraction efficiency through cyanidation (US Patent N 4727882).

 Thus, this method is the closest analogue to the method of extracting metal from refractory ore and is characterized by the cultivation of Th.ferrooxidans; treatment of refractory ore with a Th.ferrooxidans culture; the extraction of metals from the processed ore, and the culture of Th.ferrooxidans is grown on a solution of mineral salts. However, there has been a very significant increase in the recovery of gold from leached tailings using Th.ferrooxidans followed by cyanidation, compared to the recovery of gold from tailings subjected to chemical oxidation followed by cyanidation. (B. Marchant, Fundamental and Applied Biohydrometallurqy Elsevier, pp. 53-77. 1986). However, the use of such a process for biooxidation of pyrite will require inoculation of reactors or heaps with concentrated Th.ferrooxidans culture in large quantities.

 The only known method of growing "Th.ferrooxidans" in which compounds containing ferrous iron or reduced sulfur is used as a culture substrate (Inqledew W.J. Biochim.Biophys.Acta. 683 89-117. 1962). As a culture medium, these inorganic substrates have the following disadvantages. Growing crops on them requires their high concentration in order to try to compensate for the low density of biomass. A high concentration of substrates affects the accumulation of mineral salts and / or precipitates of ferric iron in such a culture, which sharply limits the density of biomass obtained using such substrates. Growing a culture on minerals causes an increase in costs and reduces the amount of "Th.ferrooxidans" available for leaching.

 It is well known in the art that formate can serve as an energy source for growing heterotrophic and autotrophic bacteria.

 Their toxicity to acidophilic microorganisms is also known. (Inqledew WJ Biochim. Biophys.Acta. 683, 89-117 (1982); Alexander B. and etal, J. Gen. Microbiol. 133, 1171-1179. 1987.) It was a complete surprise to discover that "Th. Ferrooxidans" can use formate as a carbon source. In the technical and scientific literature there are neither materials nor even suggestions that Th.ferrooxidans can oxidize formate or use it as a substrate for growth.

 This should be an important discovery in the art and provide an alternative culture medium for Th.ferrooxidans, which would allow the growth of high density biomass and from which Th.ferrooxidans would be able to grow on sulfur-containing minerals. For the area under consideration, it would also be important to provide a culture medium that allows the cultivation of “Th.ferrooxidans” with a high biomass density to ensure the number of bacteria that is optimal for bioleaching metal-containing ores, desulphurization of coal, desulphurization of wastewater and for other purposes at minimal cost.

 An object of the present invention is to provide a method for culturing high concentration Th. Ferrooxidans bacteria cultures, as well as a method for extracting metal from refractory ores, which can significantly improve the quality of bioleaching of sulfide-containing metal ores. The task is achieved by this method of cultivation of "Th.ferrooxidans", which consists in the cultivation of "Th.ferrooxidans" on a medium containing mineral salts, and the medium additionally contains formate with a concentration of less than 100 mm.

 Preferably, when culturing in the initial stage, a seed culture is periodically introduced, and upon reaching the desired biomass density, a continuous process for growing biomass is carried out. It is also preferable that during the cultivation process the biomass is recycled from the reactor. And also after growing "Th.ferrooxidans" on a formate-limited culture, the formate concentration in the medium is increased at a rate sufficient to avoid the accumulation of formate in the culture to a level not exceeding 100 mM.

 Preferably, a formated culture is fed periodically or continuously.

 As mineral salts, salts containing ferrous iron and reduced sulfur compounds are used.

 It is also preferred that in the cultivation method the strain "Th.ferrooxidans" is characterized by the strain designated LMD 81.69 (ATCC 21834). The problem is also achieved by this method of extracting at least one metal from refractory ore, including growing Th.ferrooxidans on a solution of mineral salts, treating refractory ores with a Th. Ferrooxidans culture and extracting at least one metal from ore, moreover, a solution of mineral salts contains formate.

 Preferably, in the method, the metal is selected from the group consisting of gold, silver, copper, lead, bismuth, zinc, uranium. A refractory ore is selected from the group consisting of sulfide gold ore, sulfide silver ore, sulfide copper ore, sulfide lead ore, sulfide bismuth ore, sulfide zinc ore, sulfide uranium ore and sulfur-containing coal. Or refractory ore is a combination of carbonate-containing ore and sulfide materials.

 Preferably, in the method the strain "Th.ferrooxidans" has the characteristic of a strain designated LMD 81.69 (ATCC 21834).

 The invention further relates to a method for treating mineral-containing effluents, comprising contacting the effluents with "Th.ferrooxidans" under conditions conducive to treating the minerals "Th.ferrooxidans" contained in the effluents grown to high density using a culture with a limited formate concentration. In particular, the mineral contained in the waste may be a ferrous compound or a reducible sulfur compound.

In FIG. 1 shows a linear graph of the growth of "Th.ferrooxidans" on an evaporated formate (pH 2.0, 30 ^° C.) as a function of time; figure 2 a linear graph of the growth rate of "Th. ferrooxidans" on the evaporated formate when changing the concentration of formate in the tank; figure 3 is a graph of the dependence of the rate of oxygen consumption on the concentration of the substrate; figure 4 two graphs of the effect of pH on the oxidation of formate cells "Th.ferrooxidans" and cell-free extracts (o).

 Figure 1 1 optical concentration 420; 2 time, day.

 In FIG. 2 1 growth rate, mg dry weight on the day of inoculation; 2 concentration of formic acid, by volume.

 In Fig.3 1 the rate of oxygen consumption, 2 the concentration of formate, μm.

In FIG. 4 1 oxygen consumption rate, nmol • min ^-1 mg ^-1 ; 2 reduction reaction of dichlorophenolindophenol, nmol • min ^-1 • mg of protein.

 Thus, it was found that the well-known effect of formate toxicity on "Th. Ferrooxidans" in batch culture can be avoided by using conditions under which substrate concentrations during bacterial growth will be limited, for example, a chemostatic culture or fed uterine culture, when growing "Th. ferrooxidans "to achieve high biomass densities in the culture. In order to identify the ability to oxidize formate when its concentration is low (100 μM), many acidophilic bacteria, both heterotrophs and facultative autotrophs, were studied. The oxidation parameters of formate by acidophilic bacteria were within wide limits (table 1).

The highest oxidation rate of formate was shown by the optional autotrophic acidophilic microorganism "Thiobacillus acidophilus" grown autotrophically on formate (table 1). Significant oxidation rates of formate ((30 + 63) nmol O ₂ • min ^-1 • (mg mass in dry state) ^-1 ) were obtained in all cases of studies of acidophilic bacteria. Typically, the oxidation rates shown by obligate autotrophic bacteria were lower (Table 1). Thiobacillus thiooxidans and Thooacillus concretivorus cells grown on elemental sulfur did not oxidize formate at all (Table 1). The oxidation rates of formate by Th.ferrooxidans cells grown on ferrous iron compounds varied depending on the strain used (Table 1). Among the Th.ferrooxidans strains, the highest oxygen uptake rates depending on the formate concentration were shown by Th.ferrooxidans (ATCC 21834). These data correlate well with the ability of Th.ferrooxidans (ATCC 21834) to use low concentration formate as the only energy source under growth conditions with limited formate concentration.

This is a completely unexpected result, unforeseen in the art. Attempts to grow Th.ferrooxidans on low concentration formate were not previously known. Moreover, it was found that the cultivation of "Th. Ferrooxidans" using the process of this invention retains the ability of such bacteria to grow on and oxidize minerals containing ferrous sulfides. It is not known reliably in the field of science and technology whether bacteria that have adapted to the second substrate can retain the ability to instantly use a previously applied substrate (Hazeu W. Bijleveld W. Grotenhuis JTC Kakes E. u Kuenen JGAntonie van Leeuwenhoek J. Microbiol. 52, 507- 518. 1986.)
The published reports on the toxicity of formate for Th.ferrooxidans were confirmed by the failure that accompanied the attempt to grow Th.ferrooxidans (ATCC 21834) in a uterine culture with formate at a concentration of 20 mM as the only energy source. To avoid the poisoning of "Th.ferrooxidans" with formate, several different methods of delivery of formate to the culture, ensuring its low concentrations, were used. One method was to grow Th.ferrooxidans in a uterine-fed type culture by inoculating Th.ferrooxidans in shaken narrow-necked flasks equipped with a central reservoir of formic acid, from which the substrate could enter the growth medium only by evaporation of formic acid from the central reservoir and carried out its dissolution in the growth medium (cells of those cultures that were grown as periodic on divalent iron were inoculated as duplicate cultures). With this method, growth rates varied linearly over time, when the concentration of formic acid in the reservoir was 15% (in volume ratio) (Fig. 1). Growth rate in cultures (independent third cultures were inoculated in the form of cells of chemostatic cultures with a limited concentration of formate (D = 0.01 h ^-1 ), pH 2.0, T = 30 ^o C, dry masses were evaluated by the optical density of the cultures using calibration curves constructed in the analysis of chemostatic cultures with a limited concentration of formate) was linearly dependent on the concentration of formic acid in the reservoir (Fig. 2). Under such conditions, growth rates increased with an increase in the concentration of formic acid in the reservoir to 35% (by volume). With a further increase in concentration, growth did not occur.

 When "Th.ferrooxidans" grows on a mineral medium, growth on formate does not affect the accumulation of mineral salts. When formic acid (HCOOH) is used as a substrate, it is usually completely oxidized to form carbon dioxide and water. Thus, the accumulation of salts does not set a limit on the maximum concentration of formic acid in the medium in the tank.

Such a system for feeding crops through the evaporation of formic acid has been found to be useful for demonstrating the ability of amidophilic microorganisms to use formate as an energy source. Nevertheless, a chemostatic culture was found to be more useful, into which a liquid phoripate-containing medium was directly introduced. Using a culture into which formate was continuously introduced and which was chemostatic, concentrations of Th. Ferrooxidans biomass were obtained in excess of 0.7 g · l ^-1 while maintaining its ability to oxidize ferrous iron at a high rate.

 According to the invention, advantageous growth rates of "Th.ferrooxidans" on formate in a chemostatic culture are achieved. One way to significantly reduce the time required to achieve high biomass densities is to start cultivating the culture as a fed uterine culture (Pirt SJ "Batch, Fed Batch, and Chemostat Cultivation of Micro-orqanisms" Principles of Microbe and Cell Cultivation, Blackwell Scientific Publications, Oxford, UK. Chapter 21,211-218). Another way to significantly reduce the time required to achieve high biomass densities is to use a biomass recycling system in which part of the Th.ferrooxidans biomass is returned to the culture. In particular, part of the medium is removed from the bioreactor, the cultured microorganism contained in it is isolated, and then placed back into the bioreactor to ensure continuous growth (Van Verseveld H.W. et. Al. 52, 325-342. 1986).

Example 1. The conditions of existence of cultures and organisms
Pure cultures obtained from the collection of cultures Laboratory of Microbioloqy and Enzymoloqy, Delft were used in this and the following examples. A preferred strain of "Th.ferrooxidans" is LMD 81.69 (American Type Culture Collection, Bethesda, MD) (ATCC N 21834). Methods of uterine cultivation and growth media consistent with those described by Kuenen et. el. (Kuenen JG and Tuovinen OH The Prokaryotes pp. 1023-1036 (1981).

Growing on formate in uterine culture
To grow "Th.ferrooxidans" on evaporated formate in fed uterine cultures, improved 500 ml Erlenmeyer flasks were used. Glass tubes (5 cm long, 1 cm in diameter) were attached to the bottom of the flasks. The flasks were filled with mineral medium (pH 2) in an amount of 100 ml, in which there was no carbon source, and inoculated with a suspension of Th. Ferrooxidans cells (either cells of the mother crop grown on ferrous iron or a suspension of cells of the crop of chemostat cultures grown in medium with a limited concentration of formate) until a density of approximately 15 mg of dry weight per liter is reached. After that, 2 ml of concentrated (5-50)% by volume of formic acid, which could enter the nutrient medium only by evaporation, was added to the central tube of the tank. The cultures were incubated at 30 ^o C on a rotary shaker, the rotation speed of which was 200 rpm

Sourdough Chemostatic Crops
Chemostatic cultures were inoculated by uterine cultivation in a mineral medium (Mackintosh MEJGen. Microbiol 105, 215-218 (1978) at pH = 1.6 in 9 K medium (Silverman MN and Lundqren DGGBacteriol. 77, 642-648 (1959)) and additions 180 mM FeSO ₄ • 7H ₂ O. The mineral medium used in the continuous cultivation of Th.ferrooxidans per liter of demineralized water contained: (NH ₄ ) ₂ SO ₄ 3.0 g; K ₂ HPO ₄ 1.0 g; MgSO ₄ • 7H ₂ O 0.5 g; CaCl ₂ • 2H ₂ O
0.05 g; Na ₂ SO ₄ 1.0 g; FeSO ₄ • 7H ₂ O 5 mg; ZnSO ₄ • 7H ₂ O 1 mg; CuSO ₄ • 5H ₂ O 2 mg; MnSO ₄ 1 mg; NaMoO ₄ • 2H ₂ O 0.5 mg; CoCl ₂ • 6H ₂ O 0.5 mg; Na ₂ SeO ₄ • 10H ₂ O 1 mg; NiCl ₂ • 6H ₂ O - 1 mg.

Adjustment of the pH to a value of 1.8 was carried out by adding concentrated sulfuric acid and autoclaving at a temperature of 120 ^o C. Solutions of formic acid were sterilized by autoclaving at 110 ^o C.

Experimental conditions
Experiments with chemostatic cultures were carried out at a temperature of 30 ^o C in laboratory bioreactors with a capacity of 20 l with a working volume of substances of 18 l. The cultures were aerated with air that was saturated with water at a flow rate of 2.5 L / min and mixed at a rotation speed of 500 rpm. During uterine ferrous growth, the pH of the culture was 1.6; this was achieved by automatic titration of unipolar H ₂ SO ₄ . Continuous growth on formic acid does not require titration of crops whose pH is maintained at 1.8.

Chemical substances
Ribulose 1,5-biophosphate was obtained from Siqma Chemical Co. ¹⁴ c-NaHCO ₃ (2.11 TW • mol ^-1 from Amersham International PLC. Formic acid (“bhd”) was obtained from Merck, Darmstadt, Germany. All other chemicals corresponded to the degree of purity required for chemical reagents, and were obtained from various commercial sources.

Culture purity control
The purity of chemostatic cultures grown on formate was regularly checked using immunofluorescence microscopy using special antisera to detect pure cultures of a specific organism (Muyzer G. et. El. Appl. Environm. 53, 660-664.1987).

 Example 2

Analytical methods
Measurement of oxygen consumption rate as a function of formate concentration
Batch culture cells were harvested either by centrifugation (15,000 g, 10 min) or by filtration through filters with a polycarbonate membrane (0.2 μm pore diameter, manufactured by Nuclepore, Pleasantor, CA, USA). Cells were washed and resuspended in mineral medium (for Th.ferrooxidans pH 1.8; for other acidophilic bacteria pH 3.0). Oxygen consumption was measured at 30 ^° C. using a Clark type electrode element (Yellow Sprinqs Instruments Inc. Yellow Sprinqs, OH). Oxygen consumption rates were calculated by assuming that the oxygen concentration in the air-saturated water was 236 μM adjusted for endogenous respiration.

 Cells of chemostatic cultures with limited concentrations of formate were tested directly in liquid culture or after it was diluted with a mineral medium. The endogenous respiration rates of cells grown on chemostatic cultures were negative.

Analysis of carbon, formate and protein
To determine the carbon content in the cultures in general and in the supernatant of the cultures, an Beckman 915B organic carbon analyzer was used. The carbon content in bacteria was calculated by difference.

 The mass of bacteria in the dry state was determined using the method developed by Pronk et.al. Kuenen J.G. Arch Microbiol 153, 392-398. 1990).

 Formate concentrations in the tank material were measured using the method developed by Lanq and Lanq, Z.anal. Chem. 260, 8-10. 1972).

 Protein concentration in cell-free extracts was determined using the method developed by Bradford Anal. Bio-Chem 72, 248-254 (1976)); bovine serum albumin was used as a reference.

 Example 3

Oxidation of formate by acidophilus bacteria
A number of acidophilic bacteria, both heterotrophs and (optional) autotrophs, were investigated to determine their ability to consume oxygen and oxidize formate at its low concentration (100 μM).

Judging by the results of experiments, the ability to oxidize formate was found to be widespread among acidophilus bacteria and varied widely (table 1). The oxygen consumption rates depending on the formate concentration were measured at 30 ^° C in the presence of cell suspensions taken directly from a chemostatic culture with a limited formate concentration (D = 0.01 h ^-1 ), pH 1.8, T = 30 ^° C). The activity of 100% corresponds to the oxygen consumption rate of 55 nmol O • min ^-1 mg ^-1 . In all cases, the stoichiometry of formate oxidation was as expected (approximately 50 μM of oxygen was consumed during the oxidation of formate at a concentration of 100 μM).

The highest oxidation rate of formate was shown by the optional autotroph "Thiobacillum acidophilus". This organism showed a pronounced ability to oxidize formate in a number of experiments under various growing conditions. Significant oxidation rates of formate (30-63) nmol O ₂ • min ^-1 (mg mass in a dry state) ^-1 were also shown by all studied acidophilic heterotrophs. The oxidation rates of formate, which are shown by obligate autotrophs, were usually lower. Thiobacillus berrooxidans cells LMD 81.55 (American Type Culture Collection, Bethesda, MD) (ATCC N 8085) and Thiobacillus concretivorus LMD 81, 54 (American Type Culture Collection, Bethesda MD) (ACTT N 19703) grown on elemental sulfur , did not oxidize formate at all. The oxidation rates exhibited by Th. Ferrooxidans cells varied greatly with the strain used. The highest oxygen uptake rates were shown by Th.ferrooxidans LMD 81.69 (American Type Culture Collection, Bethesda, MD) (ATCC N 21834).

 Example 4

Growing "Th.ferrooxidans" on formate
"Th. Ferrooxidans" were pre-grown as a uterine culture on divalent iron with a concentration of 180 mM in a 1 L bioreactor. At the end of the cultivation of the uterine culture, its development was switched to continuous cultivation at a dilution rate of 0.01 h ^-1 and a pH that was adjusted to 1.6, suppressing the precipitation of ferric iron, and then increased to 1.8. The reservoir medium initially had formic acid at a concentration of 20 mM, which is the only source of energy. It was this concentration that was chosen in order to ensure that the ability of the culture to oxidize formate would be sufficient to suppress the accumulation of formate in it. The concentration of formic acid in the tank medium was increased in stages. After increasing the formate concentration for every 20%, it was maintained at the same level until the increase in the biomass concentration was leveled (usually after 2-4 days). An increase in the concentration of formic acid introduced by more than 30% at a time led to leaching of the culture.

 The resulting chemostatic cultures with limited formate concentrations were monitored for purity using special antisera to detect "Th. Ferrooxidans" (Muyzer Appl. Environ Microbiol 53, 660-664.1987). All of the cells present in the cultures gave a positive immunofluorescence reaction to the fluorespinisothiocyanate (F1TC) antisera intended for the detection of Th.ferrooxidans, but did not react with the antisera designed to detect the heterotrophic acidophiles Th.acidophilus and Acidiphilium crypt.

The oxidation of formate by the introduced Th.ferrooxidans cells was inhibited sharply at substrate concentrations above 100 μM (Fig. 3). This observation explains the inability of Th.ferrooxidans strains to grow on formate in uterine cultures. The optimum pH for the oxidation of formate was approximately 3.0 (FIG. 4). Typical pH range is 1.3-3.5; a preferred pH range is 1.8-3.0. However, to reduce the likelihood of contamination, the pH of the culture was maintained at 1.8. The oxidation rate at this pH of -53 nmol O ₂ • min ^-1 • mg ^-1 was essentially the same as the rate observed in cells of uterine cultures grown on ferrous iron (table 3). With the observed biomass yields (see below), the required formate oxidation rate for growing in a medium with limited formate concentrations at a dilution rate of 0.01 h ^-1 is 60 nmol O ₂ • min ^-1 • mg ^-1 . This rate is consistent with the oxidation rates observed during the experiments. Further increases in the rate of growth and consumption of formate are possible by optimizing conditions such as temperature, aeration, and composition of the medium.

The molar yield of “Th.ferrooxidans” cultivation on the formate shown in the experiments, with its concentration limited by a dilution rate of 0.01 h ^-1, was 1.36 g of the dry mass (mol of formate) or 0.68 g of the dry mass • ( mole of electrons) ^-1 . This yield is higher than that shown by the body when grown on ferrous iron 0.23 g dry weight • (mol F ^2t ) (Hazen W. et. El. Proc.Forth Europ.Conqr.Biotechnol 3,497-499. 1987). The yield on formate is less than the maximum yield for chemostatic cultures with limited concentrations of tetrathionate previously reported for this organism (0.92 g dry weight (mole of electrons) ^-1 (Hazeu W. et. El. Proc. Forth Europ.Conqr.Biotechnol. 497-499. 1987). The observed Th.ferrooxidans cultivation yield was lower than that shown by a number of other bacteria autotrophically grown on formate (Table 2). The carbon and protein content of those grown on the Th. Ferrooxidans formate "was 48%, respectively. The requirements for maintaining a culture can substantially iyat to harvest the cells at low dilution rates, which have been used in experiments with hemostatichnymi cultures.

To calculate the theoretically maximum cell yield (Y _max ) of Th.ferrooxidans, it is necessary to take into account the culture maintenance coefficient ( ^M s) for the case of growing it on a formate of limited concentration. Assuming that the maintenance coefficient ( ^M s) of this culture is of the same order of magnitude as the maintenance coefficient "Pseudomonas oxalacticus" (Dijkhuizen L. Wiersma M. and Harder W. "Enerqy production and qrowth of" Pseudomonas oxalacticus "OXI on oxylate and formate ". Arch. Microbiol. 115, 229-236. 1977)), then Y _max " Th. ferrooxidans "should be approximately 2.5 g • mol ^-1 . This value is similar to the yield of cells grown on formate, shown by other autotrophic bacteria (table 2).

 Surprisingly, cells of chemostatic cultures grown on limited concentration formate retain the ability to oxidize ferrous iron at high rates (Table 3). The specified rates of iron oxidation remained unchanged after long-term cultivation (over three months) in chemostatic cultures with a limited concentration of formate and did not depend on the density of the culture. This contradicts previous observations of the cultivation of “Th. Ferrooxidans” on reducible sulfur compounds when the ability to oxidize ferrous iron was lost (Hazen W. et.el. J. Microbiol 52, 507-518. 1986). "Th.ferrooxidans" grown with limited formate concentration also showed oxidation of sulfides and elemental sulfur at noticeable rates.

The effect of pH on oxygen consumption rates, depending on the formate concentration, was measured using cell suspension of a chemostatic culture with a limited formate concentration (D = 0.01 h ^-1 ), pH 1.8, T = 30 ^o C). The suspension was adjusted to an appropriate pH by adding dilute H ₂ SO ₄ or KOH. Oxidation of formate with cell-free extracts was tested as dichlorophenolindophenol reduction (DCP1P) dependent on the formate concentration. The oxidation parameters of formate were measured with cell suspensions (o) and extracts without cells (o) of “Th.ferrooxidans" (Fig. 4). The observed oxygen consumption rates by cells grown on formate and ferrous iron, in the presence of ferrous iron, sulfide, or elemental sulfur as substrates, were compared. In the presence of thiosulfate, tetrathionate, methanol, formaldehyde and molecular hydrogen, oxygen uptake was not observed.

Cell-free extracts were prepared in chemostatic cultures grown on formate containing the RuBPCase enzyme, whose activity was 10 nmol • min ^-1 (mg protein) ^-1 . This activity is identical to the carbon assimilation rate calculated from the observed cell yields and cell composition, which indicates the use of formate by Th.ferrooxidans cells via the Calvin cycle.

 Example 5

Oxidation of formate with acellular extracts "Th.ferrooxidans"
Preparation of acellular extracts
The products of chemostatic cultures with a limited concentration of formate were collected at a temperature of 4 ^o C. Cells were removed by centrifugation (15000 g; 15 min) and washed in freshly prepared buffer (pH = 7.0) containing 50 mM MOPS (2- (N-morpholino) -hydropropanesulfonic acid), 2 mM-cysteine and 0.2 mM Fe (NH ₄ ) ₂ (SO ₄ ) ₂ (FOXB. G.et.el. J. Biol. Chem. 264, 10023-10033.1988). Cells were resuspended in the same buffer to a final concentration of about 10 mg of dry weight per ml and crushed by sonication at 0 ^° C using a 150 W MSE ultrasound transducer (10 times for 30 seconds with intermediate cooling) . All cells and residues were removed by centrifugation at a centrifugal force of 45,000 g for 20 minutes. Pure supernatant residues containing (2-5) mg of protein per milliliter were used as acellular extracts.

Enzyme analysis
The stained formate dehydrogenase was tested by spectrophotometry at 30 ^o C, selecting for the experiment a mixture in an amount of 1 ml containing potassium phosphate concentration of 100 mm, MgSO ₄ • 7H ₂ O concentration of 10 mm, 0.2 millimolar 2,6-dichlorophenolindophenol ("DCPIP ") and cell-free extract. The reaction was started by adding potassium formate to a final concentration of 40 mM. The enzyme activity was calculated from the decrease in absorbance at a wavelength of 522 nm using an optical absorption coefficient for DCP1P, equal to 8.6 mM ^-1 cm ^-1 (Armstronq J.McD.Biochim.Biophys.Acta 86, 194-197. 1964). Enzyme activities were proportional to the amount of enzyme added.

 Formate dehydrogenase bound by nicotinamide adenine dinucleotide phosphate (N "NAD (P)") was tested in the same sample system, but replacing DCPIP with 1 mmol nicodinamide adenine dinucleotide ("NAD") or with NADP. The recovery of NAD and NADP was monitored by spectrophotometry at a wavelength of 340 nm.

 The carboxylase of ribulose 1,5-bisphosphate (RuBPase EC 4.1.1.39) EC 4.1.1.39) was tested according to the method of Beudeker et. el. Arch. Microbol 124, 185-189. 1980).

Thus, cell-free extracts of the Th.ferrooxidans culture-grown formate LMD 81.69 (ATCC N 21834) catalyzed the oxidation of DCPIP formate as an artificial electron acceptor. The optimum pH of the formate dehydrogenase was approximately 6 (Fig. 4), confirming the localization of the cytoplasm. At this pH, the Michaelis constant for formate was approximately 0.1 mM ("K _m ") at a maximum speed ("V _max ") of 80 nmol DCPIP • min ^-1 • (mg protein) ^-1 . The recovery rates of DCPIP depending on the formate concentration did not increase with the addition of 0.2 mmol phenazine methosulfate ("PM S"). In contrast to the observations made on intact cells (Fig. 1), inhibition of the substrate by formate was not observed until a concentration of 80 mM was reached. Formate dehydrogenase activity in cell-free extracts was unstable at 4 ^° C, and the half-life was approximately 4 hours. In the presence of NAD and NADP acceptors, activity was not observed.

Claims (15)

 1. The method of cultivation of Thiobacillus ferrooxidans on a medium containing mineral salts, characterized in that the medium further comprises formate with a concentration of less than 100 mmol.  2. The method according to claim 1, characterized in that it includes at the initial stage the introduction of a seed culture as a periodically fed culture, and upon reaching the desired biomass density, cultivation is carried out.  3. The method according to claim 2, characterized in that in the process of cultivation, the biomass is removed with recycling from the reactor.  4. The method according to claim 2, characterized in that the formated culture is fed periodically.  5. The method according to claim 2, characterized in that the form-limited culture is constantly fed.  6. The method according to claim 1, characterized in that after growing Thiobacillus ferrooxidans on a formate-limited culture, an increase in the concentration of formate in the medium is carried out at a rate sufficient to avoid the accumulation of formate in the culture to a level exceeding 100 mmol.  7. The method according to p. 1, characterized in that the salts of ferrous iron and reduced forms of sulfur are used as mineral salts.  8. The method according to PP.1 to 7, characterized in that use the strain of Thiobacillus ferrooxidans LMD 81.69 (ATCC 21834).  9. A method for extracting at least one metal from refractory ore, comprising growing a Thiobacillus ferrooxidans culture in a solution of mineral salts, treating refractory ore with a Thiobacillus ferrooxidans culture and recovering at least one metal from the ore, characterized in that the mineral salt solution further comprises formate.  10. The method according to claim 9, characterized in that the metal is selected from the group consisting of gold, silver, copper, lead, bismuth, zinc, uranium.  11. The method according to claim 9, characterized in that the refractory ore is selected from the group comprising sulfide gold ore, sulfide silver ore, sulfide copper ore, sulfide lead ore, sulfide bismuth ore, sulfide zinc ore, sulfide uranium ore and sulfur-containing coal.  12. The method according to claim 9, characterized in that the refractory ore is a combination of carbonate-containing ore and sulfide materials.  13. The method according to claim 9, characterized in that when processing mineral-containing waste, the waste is brought into contact with Thiobacillus ferrooxidans under conditions conducive to the processing of waste minerals, and the Thiobacillus ferrooxidans culture is grown to a high biomass density by formatively limited cultivation.  14. The method according to claim 9, characterized in that the minerals are selected from the group consisting of ferrous iron and reducible sulfur compounds.  15. The method according to PP.9 to 14, characterized in that use the strain of Thiobacillus ferrooxidans LMD 81.69 (ATCC 21834).

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