A method for culturing thiobacillus ferroxidans

Abstract

The application provides a culture system and culture method of Thiobacillus ferroxidans, wherein the system comprises a nutrient solution tank, a culture tank, a communication device, an overflow separator, a clear liquid tank, a sedimentation tank and a reflux device, the overflow separator is provided with a solid discharge control valve at the bottom, the precipitate is discharged from the solid discharge control valve, and the overflow liquid returns to the culture tank, so that the precipitate and the Thiobacillus ferroxidans are separated; the culture method realizes the efficient culture of the Thiobacillus ferroxidans through the steps of preparing a nutrient solution, inoculation, aeration culture, circulating flow addition, precipitation separation and continuous culture, solves the problem that the precipitate such as iron alum affects the growth of microorganisms in the culture process, guarantees the fresh culture liquid supply, promotes the continuous propagation and growth of the microorganisms, and thus improves the output efficiency and the living quality of the Thiobacillus ferroxidans. The obtained microbial suspension can be prepared into a microbial agent product and can also be directly used in the fields of bio-metallurgy, environmental remediation, bio-electrochemistry and bio-fuel cells.

Description

[0001] This invention is a divisional application based on the application filed on March 20, 2023, with application number 202310272191.6 and entitled "A culture system and culture method for ferrooxidase". Technical Field

[0002] This invention relates to a method for culturing *Thiobacillus ferrooxidans*, and relates to microorganisms, particularly those used in biometallurgy, environmental remediation, bioelectrochemistry, and biofuel cells. Background Technology

[0003] Biometallurgy utilizes microorganisms or their metabolic products to leach valuable metals from ores. It features simple equipment, short process flow, low construction and operating costs, environmental friendliness, and the ability to utilize low-grade, complex, and difficult-to-process ores. Biometallurgical technology can significantly improve the recovery rate of metals such as copper, gold, and uranium, reduce energy consumption and emissions, and promote resource conservation and recycling.

[0004] Currently, microbial metallurgy technology has been successfully applied in many countries and regions at home and abroad, mainly involving the recovery of valuable metals such as copper, uranium, and gold. Here are some specific examples: (1) Kennecott Copper Company in the United States pioneered heap leaching of copper ore in 1958, and was the pioneer of modern microbial metallurgy industrial application; (2) Canada used microbial leaching technology to treat low-grade pyrite-type gold ore, which increased the gold recovery rate from 30% to more than 90%; (3) China has established a number of microbial leaching plants in Inner Mongolia, Yunnan, Jiangxi and other places to treat low-grade copper sulfide ore and copper oxide ore, and achieved a significant improvement in resource utilization.

[0005] Acidithiobacillus ferrooxidans (A. ferrooxidans) is one of the most commercially valuable leaching bacteria in the metallurgical industry and one of the most studied species. It is a Gram-negative bacterium that uses the oxidation of ferrous iron, elemental sulfur, or partially oxidized sulfides as an energy source. This microorganism can oxidize sulfides in minerals to sulfuric acid, thereby increasing the acidity and oxidizing properties of the solution, which is beneficial for metal leaching. It has many other industrial applications, including biometallurgy, industrial desulfurization, environmental protection, and waste electronic product processing.

[0006] These acidophilic microorganisms are characterized by their ability to thrive in low pH and high Fe environments. 2+It grows in an environment that utilizes ferrous oxide ions as an energy source. Cultivating *Thiobacillus ferrooxidans* requires a special culture medium, the main components of which are ferrous sulfate heptahydrate, ammonium sulfate, anhydrous magnesium sulfate, potassium chloride, calcium nitrate, and dipotassium hydrogen phosphate. When inoculating *Thiobacillus ferrooxidans*, it is necessary to select strains that have already been domesticated to improve their tolerance to extreme environments.

[0007] Industrial cultivation methods typically utilize fermenters for liquid culture. During cultivation, the dissolved oxygen rate is increased by increasing the contact area between the liquid and oxygen or raising the oxygen partial pressure, thereby promoting microbial growth. Industrial cultivation of *Thiobacillus ferrooxidans* requires selecting a suitable culture medium formulation and carefully controlling conditions such as pH, temperature, and inoculum size to ensure the stability and activity of the strain. Specific cultivation methods may vary depending on different production objectives and scales, requiring optimization and adjustment based on actual circumstances.

[0008] However, some precipitates are produced during the cultivation of *Thiobacillus ferrooxidans*, mainly jaundice or sodium jaundice. These precipitates affect the growth of the bacteria and the oxidation of Fe. 2+ It has no direct impact, but it will increase the turbidity and viscosity of the culture medium, affecting culture efficiency and cost. The effect of precipitates on microorganisms depends on the nature and quantity of the precipitate and the type of microorganism. Generally, precipitates reduce the activity and growth rate of microorganisms, but some microorganisms can utilize precipitates for detoxification or adsorption.

[0009] The precipitate produced during the cultivation of *Thiobacillus ferrooxidans* is mainly due to the reaction between phosphate and iron ions in the culture medium. Some researchers have proposed methods to reduce precipitate formation, such as adjusting pH and phosphate concentration, or replacing nitrogen and phosphorus sources. However, these operations alter reactor conditions such as temperature, pH, aeration rate, and nutrient levels, making it impossible to guarantee optimal microbial growth. Summary of the Invention

[0010] This invention discloses a culture system and method for *Thiobacillus ferrooxidans*, which solves the problem of precipitates such as iron alum affecting microbial growth during the culture process, and enables continuous culture of *Thiobacillus ferrooxidans*.

[0011] A culture system for *Thiobacillus ferrooxidans* includes: a nutrient solution storage tank, a culture tank, a communicating vessel, an overflow separator, a clear liquid tank, a settling tank, and a reflux device. The culture tank and the overflow separator are connected through the communicating vessel. An air aerator is installed at the bottom of the culture tank and is connected to an air input pipe. The overflow separator is characterized by having a solid discharge control valve at its bottom, through which precipitates are discharged into the settling tank, thereby separating the precipitates from *Thiobacillus ferrooxidans*.

[0012] Furthermore, the overflow separator is equipped with an inner tube connected to the communicating vessel in the middle, a baffle sleeve in the upper middle part of the overflow separator, an overflow weir and an overflow separator outlet in the upper part of the overflow separator. The liquid flows sequentially into the inner tube of the upper overflow separator, the baffle sleeve, the overflow weir and the overflow separator outlet, and flows out of the overflow separator outlet, returning to the clear liquid tank. The reflux device sends the liquid in the clear liquid tank back to the culture tank through the reflux pipe, realizing the circulation of the solution in the culture tank and the overflow separator, and realizing the cyclic culture of ferrooxidizobacillus.

[0013] Furthermore, a nutrient solution tank output control is installed in the lower part of the nutrient solution tank. The nutrient solution tank output control is connected to the nutrient solution tank output pipe. The solution in the nutrient solution tank flows into the culture tank through the nutrient solution tank output control and the nutrient solution tank output pipe, thereby realizing the continuous culture of ferrooxidizum.

[0014] Furthermore, the culture system is applicable to strains or mixed bacterial communities with ferrous oxide capabilities, specifically a mixed bacterial community composed of *Acidithiobacillus ferrooxidans* and *Acidithiobacillus thiooxidans*, *Leptospirillum ferrooxidans*, *Sulfobacillus thermosulfidooxidans*, *Acidithiobacillus caldus*, *Ferroplasma acidiphilum*, *Acidithiobacillus ferrivorans*, *Acidithiobacillus albertensis*, and *Acidithiobacillus ferridurans*.

[0015] A method for culturing *Thiobacillus ferrooxidans*, characterized by comprising the following steps: S1. Preparation of nutrient solution: Pour clean water into the culture tank, turn on the reflux device, and let the solution circulate in the reactor body and overflow separator. Calculate the amount of basic nutrient salt to be added according to the volume of the solution in the reactor. Then take out a certain amount of solution from the main reactor, add the basic nutrient salt, dissolve it completely, and pour it back into the reactor body to reach the basic nutrient salt concentration. Repeat the same process to dissolve the corresponding amount of ferrous sulfate. Mix the solution in the culture system evenly to reach the ferrous ion concentration. Adjust the pH value and control the solution temperature. S2. Microbial inoculation: Replace a certain amount of solution in the system with a cultured solution of Thiobacillus ferrooxidans, and allow the bacterial solution to circulate and mix evenly in the system to confirm the cell concentration of Thiobacillus ferrooxidans in the liquid. S3. Aeration culture: Turn on the air aerator of the culture tank to blow in fresh air, adjust the aeration rate to the specified value, and control the dissolved oxygen value in the culture tank. S4. Circulation culture: After the solution fills the entire reactor system, turn on the reflux device to allow the solution to circulate in the main reactor and overflow chamber, and control the temperature and pH value within the specified range to allow ferrooxidizing thiobacillus to grow. S5. Sedimentation Separation: The precipitate, ferrous sulfate, is separated from the circulating solution in an overflow separator. The system culture cycle is controlled to be 7–14 days. When the concentration of *Thiobacillus ferrooxidans* in the clear liquid tank stabilizes at 3 × 10⁻⁶... 8 A concentration of 10⁶ cells / mL or higher was obtained to obtain *Thiobacillus ferrooxidans* of the present invention. Timely separation of iron alum reduced microbial adsorption and increased cell concentration, compared to the traditional 3 × 10⁶ cells / mL. 6 The concentration / mL has now been increased to 3 × 10⁻⁶. 8 The cell yield per mL was increased by two orders of magnitude. The separation of iron alum facilitated pH stability, ensured more uniform temperature conditions, prevented aeration head clogging due to the absence of particulate matter, and facilitated timely solid separation, extending the continuous production cycle, reducing cleaning frequency, and guaranteeing long-term stable culture output. The cell yield was improved, and the cells in the precipitated iron alum could be processed separately, washed out with fresh nutrient solution, and recycled for the next batch of the system. S6. Continuous culture: The nutrient solution with a temperature of 25℃~35℃ and a pH value of 1.5~2.5 is added to the culture tank, and the cultured ferrous thiobacillus is discharged from the clear liquid tank. The flow rate of the solution is controlled to form a continuous culture.

[0016] Furthermore, in step S1, the pH value is adjusted to 1.5-2.5 using 98% concentrated sulfuric acid, and the solution temperature is controlled at 25℃-35℃.

[0017] Further, in step S1, the concentrations of the basic nutrients are: (NH4)2SO4 1.0–3.0 g / L, K2HPO4 0.1–0.5 g / L, KCl 0.05–0.1 g / L, MgSO4·7H2O 0.1–0.5 g / L, Ca(NO3)2 0.005–0.01 g / L, and the concentration of ferrous sulfate 5–10 g / L.

[0018] Further, in step S2, the amount of solution replaced by the *Thiobacillus ferrooxidans* solution in the system is 8%–12% of the total solution, preferably 10%, and the cell concentration of *Thiobacillus ferrooxidans* in the control solution is ≥1×10⁻⁶.8 per mL.

[0019] Furthermore, in step S3, the aeration rate is adjusted to a specified value of 0.005–0.05 Nm per minute. 3 / m 3 The dissolved oxygen value in the controlled culture tank is ≥5 mg / L.

[0020] Furthermore, in steps S4 and S6, the controlled temperature is 25°C to 35°C, and the pH value is adjusted to 1.5 to 2.5 using 98% concentrated sulfuric acid.

[0021] The ferrooxidans is one of A. ferrooxidans ATCC23270, A. ferrooxidans ATCC19859, A. ferrooxidans ATCC53993, A. ferrooxidans ATCC33020, etc., with A. ferrooxidans ATCC23270 being preferred.

[0022] A method for culturing *Thiobacillus ferrooxidans* is implemented in the above-mentioned culture system.

[0023] The present invention has the following advantages: (1) It solved the problem of iron alum affecting the growth of microorganisms during the cultivation process.

[0024] (2) Timely separation of iron alum reduces microbial adsorption and increases cell concentration, from the traditional 3×10 6 The number / mL was increased to 3 × 10 8 This represents an increase of two orders of magnitude in terms of cells / mL.

[0025] (3) The separation of iron alum is conducive to the stability of pH value and more uniform temperature conditions.

[0026] (4) No particulate matter interference, avoiding clogging of aeration heads.

[0027] (5) Timely separation of solids extends the continuous production cycle, reduces the number of cleaning operations, and ensures long-term stable cultivation output.

[0028] (6) Because the entrainment of ferrous ions is reduced, the overall ferrous energy utilization efficiency is improved, and the addition of salt is reduced.

[0029] (7) While increasing the cell yield, the cells in the precipitated iron alum can also be processed separately and washed out with fresh nutrient solution for the next batch of system recycling. Attached Figure Description

[0030] Figure 1This is a diagram of a culture system for a type of ferrooxidobacterium.

[0031] Numbered in the diagram: 1. Nutrient solution storage tank; 11. Nutrient solution storage tank output control; 12. Nutrient solution storage tank output pipe; 2. Culture tank; 21. Culture tank air input pipe; 22. Culture tank air aerator; 3. Connecting device; 4. Overflow separator; 41. Overflow separator inner pipe; 42. Baffle sleeve; 43. Overflow weir; 44. Overflow separator outlet; 45. Solid discharge control valve; 5. Clear liquid tank; 6. Settling tank; 7. Return device; 71. Return pipe. Detailed Implementation

[0032] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0033] Example 1 (1) Nutrient solution preparation: Inject a dilute sulfuric acid solution with a pH of 2.0 into the culture tank, turn on the reflux device, and the solution will circulate in the reactor body and overflow separator due to the liquid level difference. Calculate the amount of basic nutrients to be added based on a solution volume of 10,000 L in the culture system. Weigh out 30,000 g of (NH4)2SO4, 5,000 g of K2HPO4, 1,000 g of KCl, 5,000 g of MgSO4·7H2O, and 100 g of Ca(NO3)2 for later use. Take 100 L of acidic solution from the main reactor, add the prepared basic nutrients, dissolve them completely, and pour them back into the main reactor. Dissolve the corresponding amount of ferrous sulfate in the same way. Continue to turn on the reflux device to allow the solution to circulate, ensuring the solution in the entire system is evenly mixed. The ferrous sulfate concentration is 5 g / L. Add 98% concentrated sulfuric acid to adjust the pH of the solution in the system to 1.5. Turn on the temperature control device and maintain the temperature at 35℃.

[0034] (2) Microbial inoculation: Adjust the system temperature to the specified temperature, confirm pH stability, replace 8% of the solution in the system with a cultured *Thiobacillus ferrooxidans* solution, and continue to operate the reflux device to allow the bacterial solution to circulate within the system. The cell concentration of the microorganisms in the inoculum used must not be less than 1 × 10⁻⁶. 8 per mL.

[0035] (3) Aeration culture: Turn on the air aerator in the culture tank to blow in fresh air, and adjust the aeration rate to the specified value of 0.01 Nm. 3 / m 3•min, control the dissolved oxygen value in the culture tank to above 5mg / L, and you can see obvious bubbles churning in the main reactor.

[0036] (4) Circulation culture: After the solution fills the entire reactor system, turn on the reflux device to allow the solution to circulate in the main reactor and overflow chamber, and control the temperature at 35°C and the pH value at 1.5 within the specified range so that the microorganisms can grow under the best culture conditions.

[0037] (5) Precipitation separation: *Thiobacillus ferrooxidans* utilizes Fe... 2+ As an energy source, it is oxidized to Fe. 3+ Subsequently, it reacts with OH in the solution. - A precipitate forms and separates from the circulating solution in the overflow separator. The initial culture period is 7 days. The culture period continues until the microbial concentration in the clear liquid tank stabilizes at 3 × 10⁻⁶. 8 A concentration of more than 100 cells / mL indicates that the solution is ready for leaching or to be used as a seed solution.

[0038] (6) Continuous culture: Fresh culture medium at 35°C and pH 1.5 is added from the nutrient solution storage tank port, and the cultured *Thiobacillus ferrooxidans* is discharged from the clear liquid tank. The same rate is maintained to form a continuous culture. During the continuous culture process, the sediment at the bottom of the overflow separator can be gradually discharged into the settling tank in batches through the valve according to the accumulation of sediment. The bottom of the settling tank can be filtered by a filter press to obtain iron ferric sulfate residue, and the clear liquid is returned to the culture system for circulation.

[0039] In this case, the ferrooxidans bacillus was A. ferrooxidans ATCC23270.

[0040] Example 2 (1) Nutrient solution preparation: Inject a dilute sulfuric acid solution with a pH of 2.0 into the culture tank, turn on the reflux device, and the solution will circulate in the reactor body and overflow separator due to the liquid level difference. Calculate the amount of basic nutrients to be added based on a solution volume of 10,000L in the culture system. Weigh out 10,000g of (NH4)2SO4, 1,000g of K2HPO4, 500g of KCl, 1,000g of MgSO4·7H2O, and 50g of Ca(NO3)2, and set aside. Take 100L of acidic solution from the main reactor, add the prepared basic nutrients, dissolve thoroughly, and pour back into the main reactor. Dissolve the corresponding amount of ferrous sulfate in the same way. Continue to turn on the reflux device to circulate the solution, ensuring the solution in the entire system is evenly mixed. The ferrous sulfate concentration is 8g / L. Add 98% concentrated sulfuric acid to adjust the pH of the solution in the system to 2.0, turn on the temperature control device, and maintain the temperature at 30℃.

[0041] (2) Microbial inoculation: Adjust the system temperature to the specified temperature, confirm that the pH value is stable, replace 10% of the solution in the system with a cultured *Thiobacillus ferrooxidans* solution, and continue to turn on the reflux device to allow the bacterial solution to circulate within the system. The cell concentration of the microorganisms in the inoculum used must not be less than 1 × 10⁻⁶. 8 per mL.

[0042] (3) Aeration culture: Turn on the air aerator in the culture tank to blow in fresh air, and adjust the aeration rate to the specified value of 0.035 Nm. 3 / m 3 •min, control the dissolved oxygen value in the culture tank to above 5mg / L, and you can see obvious bubbles churning in the main reactor.

[0043] (4) Circulation culture: After the solution fills the entire reactor system, turn on the reflux device to allow the solution to circulate in the main reactor and overflow chamber, control the temperature at 30℃ and the pH value at 2.0 within the specified range, so that the microorganisms can grow under the best culture conditions.

[0044] (5) Precipitation separation: *Thiobacillus ferrooxidans* utilizes Fe... 2+ As an energy source, it is oxidized to Fe. 3+ Subsequently, it reacts with OH in the solution. - A precipitate forms and separates from the circulating solution in the overflow separator. The initial culture period is 11 days. The culture period continues until the microbial concentration in the clear liquid tank stabilizes at 3 × 10⁻⁶. 8 A concentration of more than 100 cells / mL indicates that the solution is ready for leaching or to be used as a seed solution.

[0045] (6) Continuous culture: Fresh culture medium at 30°C and pH 2.0 is added from the nutrient solution storage tank port, and the cultured *Thiobacillus ferrooxidans* is discharged from the clear liquid tank. The same rate is maintained to form a continuous culture. During the continuous culture process, the sediment at the bottom of the overflow separator can be gradually discharged into the settling tank in batches through the valve according to the accumulation of sediment. The bottom of the settling tank can be filtered by a filter press to obtain iron ferric sulfate residue, and the clear liquid is returned to the culture system for circulation.

[0046] In this case, the ferrooxidans bacillus is A. ferrooxidans ATCC19859.

[0047] Example 3 (1) Nutrient solution preparation: Inject a dilute sulfuric acid solution with a pH of 2.0 into the culture tank, turn on the reflux device, and the solution will circulate in the reactor body and overflow separator due to the liquid level difference. Calculate the amount of basic nutrients to be added based on a solution volume of 10,000 L in the culture system. Weigh out 20,000 g of (NH4)2SO4, 3,000 g of K2HPO4, 800 g of KCl, 4,000 g of MgSO4·7H2O, and 90 g of Ca(NO3)2, and set aside. Take 100 L of acidic solution from the main reactor, add the prepared basic nutrients, dissolve thoroughly, and pour back into the main reactor. Dissolve the corresponding amount of ferrous sulfate in the same way. Continue to turn on the reflux device to allow the solution to circulate, ensuring the solution in the entire system is evenly mixed. The ferrous sulfate concentration is 10 g / L. Add 98% concentrated sulfuric acid to adjust the pH of the solution in the system to 2.5. Turn on the temperature control device and maintain the temperature at 25℃.

[0048] (2) Microbial inoculation: Adjust the system temperature to the specified temperature, confirm that the pH value is stable, replace 12% of the solution in the system with a cultured *Thiobacillus ferrooxidans* solution, and continue to turn on the reflux device to allow the bacterial solution to circulate within the system. The cell concentration of the microorganisms in the inoculum used must not be less than 1 × 10⁻⁶. 8 per mL.

[0049] (3) Aeration culture: Turn on the air aerator in the culture tank to blow in fresh air, and adjust the aeration rate to the specified value of 0.005 Nm. 3 / m 3 •min, control the dissolved oxygen value in the culture tank to above 5mg / L, and you can see obvious bubbles churning in the main reactor.

[0050] (4) Circulation culture: After the solution fills the entire reactor system, turn on the reflux device to allow the solution to circulate in the main reactor and overflow chamber. Control the temperature at 25°C and the pH value at 1.9 within the specified range so that the microorganisms can grow under the best culture conditions.

[0051] (5) Precipitation separation: *Thiobacillus ferrooxidans* utilizes Fe... 2+ As an energy source, it is oxidized to Fe. 3+ Subsequently, it reacts with OH in the solution. - A precipitate forms and separates from the circulating solution in the overflow separator. The initial culture period is 14 days. The culture continues until the microbial concentration in the clear liquid tank stabilizes at 3 × 10⁻⁶. 8 A concentration of more than 100 cells / mL indicates that the solution is ready for leaching or to be used as a seed solution.

[0052] (6) Continuous culture: Fresh culture medium at 25°C and pH 1.9 is added from the nutrient solution storage tank port, and the cultured *Thiobacillus ferrooxidans* is discharged from the clear liquid tank. The same rate is maintained to form a continuous culture. During the continuous culture process, the sediment at the bottom of the overflow separator can be gradually discharged into the settling tank in batches through the valve according to the accumulation of sediment. The bottom of the settling tank can be filtered by a filter press to obtain iron ferric sulfate residue, and the clear liquid is returned to the culture system for circulation.

[0053] In this case, the ferrooxidans bacillus was A. ferrooxidans ATCC33020.

[0054] Example 4 (1) Nutrient solution preparation: Inject a dilute sulfuric acid solution with a pH of 2.0 into the culture tank, turn on the reflux device, and the solution will circulate in the reactor body and overflow separator due to the liquid level difference. Calculate the amount of basic nutrients to be added based on a solution volume of 10,000 L in the culture system. Weigh out 15,000 g of (NH4)2SO4, 3,000 g of K2HPO4, 700 g of KCl, 3,000 g of MgSO4·7H2O, and 100 g of Ca(NO3)2, and set aside. Take 100 L of acidic solution from the main reactor, add the prepared basic nutrients, dissolve thoroughly, and pour back into the main reactor. Dissolve the corresponding amount of ferrous sulfate in the same way. Continue to turn on the reflux device to circulate the solution, ensuring the solution in the entire system is evenly mixed. The ferrous sulfate concentration is 9 g / L. Add 98% concentrated sulfuric acid to adjust the pH of the solution in the system to 1.6. Turn on the temperature control device to maintain the temperature at around 28°C.

[0055] (2) Microbial inoculation: Adjust the system temperature to the specified temperature, confirm pH stability, replace 11% of the solution in the system with a cultured *Thiobacillus ferrooxidans* solution, and continue to operate the reflux device to allow the bacterial solution to circulate within the system. The cell concentration of the microorganisms in the inoculum used must not be less than 1 × 10⁻⁶. 8 per mL.

[0056] (3) Aeration culture: Turn on the air aerator in the culture tank to blow in fresh air, and adjust the aeration rate to the specified value of 0.04 Nm. 3 / m 3 •min, control the dissolved oxygen value in the culture tank to above 5mg / L, and you can see obvious bubbles churning in the main reactor.

[0057] (4) Circulation culture: After the solution fills the entire reactor system, turn on the reflux device to allow the solution to circulate in the main reactor and overflow chamber. Control the temperature at 28°C and the pH value at 1.6 within the specified range so that the microorganisms can grow under the best culture conditions.

[0058] (5) Precipitation separation: *Thiobacillus ferrooxidans* utilizes Fe... 2+ As an energy source, it is oxidized to Fe. 3+ Subsequently, it reacts with OH in the solution. - A precipitate forms, which is then separated from the circulating solution in an overflow separator. The initial culture period is 10 days. When the microbial concentration in the clear liquid tank stabilizes at 3×10 8 A concentration of more than 100 cells / mL indicates that the solution is ready for leaching or to be used as a seed solution.

[0059] (6) Continuous culture: Fresh culture medium at 28°C and pH 1.6 is added from the nutrient solution storage tank port, and the cultured *Thiobacillus ferrooxidans* is discharged from the clear liquid tank. The same rate is controlled to form a continuous culture. During the continuous culture process, the sediment at the bottom of the overflow separator can be gradually discharged into the settling tank in batches through the valve according to the accumulation of sediment. The bottom of the settling tank can be filtered by a filter press to obtain iron ferric sulfate residue, and the clear liquid is returned to the culture system for circulation.

[0060] The microorganisms in this case are a mixed community of A. ferrooxidans ATCC23270 and Leptospirillum ferrooxidans ATCC29047.

[0061] As attached Figure 1 As shown, a culture system for *Thiobacillus ferrooxidans* includes: a nutrient solution storage tank 1, a culture tank 2, an overflow separator 4, and a connector 3. The culture tank 2 and the overflow separator 4 are connected via the connector 3. An air aerator 22 is installed at the bottom of the culture tank 2 and is connected to an air inlet pipe 21. The overflow separator 4 is equipped with a solid discharge control valve 45 at its bottom. The precipitate, ferrous sulfate hematite, is discharged from the solid discharge control valve into a settling tank 6, thereby separating the precipitate from *Thiobacillus ferrooxidans*.

[0062] The overflow separator 4 is equipped with an inner overflow separator tube 41 connected to the connector 3 in the middle. The upper middle part of the overflow separator 4 is equipped with a baffle tube sleeve 42. The upper part of the overflow separator 4 is equipped with an overflow weir 43 and an overflow separator outlet 44. The liquid flows into the upper overflow separator inner tube 41, the baffle tube sleeve 42, the overflow weir 43 and the overflow separator outlet 44 in sequence, and flows out of the overflow separator outlet 44 and returns to the clear liquid tank 5. The return device 7 sends the liquid in the clear liquid tank 5 back to the culture tank 2 through the return pipe 71, so as to realize the circulation of the solution in the culture tank 2 and the overflow separator 4, and realize the cyclic culture of ferrooxidizobacillus.

[0063] The lower part of the nutrient solution storage tank 1 is equipped with a nutrient solution storage tank output control 11, which is connected to the nutrient solution storage tank output pipe 12. The solution in the nutrient solution storage tank 1 flows into the culture tank 2 through the nutrient solution storage tank output control 11 and the nutrient solution storage tank output pipe 12, thereby realizing the continuous culture of ferrooxidizum.

[0064] Examples 1, 2, 3, and 4 describe a method for culturing *Thiobacillus ferrooxidans* as described in the attached... Figure 1 The diagram shows a culture system for culturing *Thiobacillus ferrooxidans*.

[0065] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for culturing *Thiobacillus ferrooxidans*, characterized in that, Includes the following steps: S1. Preparation of nutrient solution: Pour clean water into the culture tank, turn on the reflux device, and use the liquid level difference to make the solution circulate in the culture tank and overflow separator. Calculate the amount of basic nutrient salt to be added according to the solution volume in the culture system. Then take a certain amount of solution from the culture tank to dissolve the basic nutrient salt. After it is fully dissolved, pour it back into the culture tank to reach the basic nutrient salt concentration. Repeat the same method to dissolve the corresponding amount of ferrous sulfate. Mix it evenly in the culture system, adjust the pH of the solution to 1.5 to 2.5, and control the solution temperature to 25℃ to 35℃. S2. Microbial inoculation: Replace 8%–12% of the solution in the system with a suspension of *Thiobacillus ferrooxidans* to obtain a cell concentration of ≥1×10⁻⁶ cells of *Thiobacillus ferrooxidans*. 8 A control solution of [number] cells / mL; S3. Aeration culture: Turn on the air aerator in the culture tank and control the dissolved oxygen value in the culture tank to ≥5mg / L; S4. Circulating culture: After the solution fills the entire culture system, turn on the reflux device, control the temperature at 25-35℃ and the pH at 1.5-2.5 to allow *Thiobacillus ferrooxidans* to grow. S5. Sedimentation Separation: The precipitate, ferrous sulfate, is separated from the circulating solution in an overflow separator. The system culture cycle is controlled to be 7–14 days. When the concentration of *Thiobacillus ferrooxidans* in the clear liquid tank stabilizes at 3 × 10⁻⁶... 8 A number of bacteria per mL or higher was obtained to obtain well-cultured *Thiobacillus ferrooxidans*. The cultivation system includes a nutrient solution storage tank, a cultivation tank, a communicating vessel, an overflow separator, a clear liquid tank, a settling tank, and a reflux device. The cultivation tank and the overflow separator are connected through the communicating vessel. An air aerator is installed at the bottom of the cultivation tank and is connected to the air input pipe of the cultivation tank. The bottom of the overflow separator is equipped with a solid discharge control valve. The precipitate, potassium ferric sulfate, is discharged from the solid discharge control valve into the settling tank, thereby separating the precipitate from ferrous thiobacillus.

2. The method for culturing *Thiobacillus ferrooxidans* according to claim 1, characterized in that, The overflow separator is equipped with an inner overflow separator tube connected to the communicating vessel in the middle. The upper middle part of the overflow separator is equipped with a baffle sleeve, and the upper part is equipped with an overflow weir and an overflow separator outlet. The liquid flows into the inner overflow separator tube, the baffle sleeve, the overflow weir and the overflow separator outlet in sequence, and flows out through the overflow separator outlet back to the clear liquid tank. The reflux device sends the liquid in the clear liquid tank back to the culture tank through the reflux pipe, realizing the circulation of the solution in the culture tank and the overflow separator, and realizing the cyclic culture of ferrooxidizobacillus.

3. The method for culturing *Thiobacillus ferrooxidans* according to claim 1, characterized in that, The nutrient solution storage tank is equipped with a heating and temperature control function, and a nutrient solution storage tank output control is installed in the middle and lower part. The nutrient solution storage tank output control is connected to the nutrient solution storage tank output pipe. The solution in the nutrient solution storage tank flows into the culture tank after passing through the nutrient solution storage tank output control and the nutrient solution storage tank output pipe, thereby realizing the continuous culture of ferrooxidizum.

4. The method for culturing *Thiobacillus ferrooxidans* according to claim 1, characterized in that, In step S1, the concentrations of the basic nutrients are: (NH4)2SO4 1.0–3.0 g / L, K2HPO4 0.1–0.5 g / L, KCl 0.05–0.1 g / L, MgSO4·7H2O 0.1–0.5 g / L, and Ca(NO3)2 0.005–0.01 g / L; the concentration of ferrous sulfate is 5–10 g / L.

5. The method for culturing *Thiobacillus ferrooxidans* according to claim 1, characterized in that, In step S2, 10% of the system solution is replaced with a suspension of *Thiobacillus ferrooxidans*. The ferrooxidizing thiobacillus is a mixed flora consisting of Acidithiobacillus ferrooxidans or Acidithiobacillus ferrooxidans and strains with ferrous oxidizing ability; the strains with ferrous oxidizing ability are at least one of Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans, Sulfobacillus thermosulfidooxidans, Acidithiobacillus caldus, Ferroplasma acidiphilum, Acidithiobacillus ferrivorans, Acidithiobacillus albertensis, and Acidithiobacillus ferridurans.

6. The method for culturing *Thiobacillus ferrooxidans* according to claim 5, characterized in that, The ferrooxidans is one of Acidithiobacillus ferrooxidans ATCC 23270, Acidithiobacillus ferrooxidans ATCC 19859, Acidithiobacillus ferrooxidans ATCC 53993, and Acidithiobacillus ferrooxidans ATCC 33020.

7. The method for culturing *Thiobacillus ferrooxidans* according to claim 1, characterized in that, In step S3, the aeration rate of the air aerator in the culture tank is 0.005–0.05 Nm³ / min. 3 / m 3 .

8. The method for culturing *Thiobacillus ferrooxidans* according to claim 1, characterized in that, In steps S1 and S4, 98% concentrated sulfuric acid is used to adjust the pH value.

9. The method for culturing *Thiobacillus ferrooxidans* according to any one of claims 1-8, characterized in that, It also includes the following steps: Nutrient solution with adjusted pH and temperature is added to the nutrient solution storage tank, and the cultured *Thiobacillus ferrooxidans* is discharged from the clear solution tank. The flow rate of the solution is controlled to form a continuous culture.

10. The method for culturing *Thiobacillus ferrooxidans* according to claim 9, characterized in that, Adjust the pH of the nutrient solution to 1.5–2.5 and the temperature to 25°C–35°C; The pH value was adjusted using 98% concentrated sulfuric acid.

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