Acidithiobacillus thiooxidans and application thereof

Abstract

The invention provides acidophilic thiobacillus thiooxidans and application thereof, and belongs to the technical field of acidophilic thiobacillus thiooxidans. The acidophilic thiobacillus thiooxidans is named as HS-PLS23-At.t, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.34267. The acidophilic thiobacillus thiooxidans is named as HS-PLS23-At.t. Research finds that the acidophilic thiobacillus thiooxidans can grow well when the initial pH is 1-6, and has strong acid resistance. Meanwhile, the acidophilic thiobacillus thiooxidans can generate sulfuric acid from elemental sulfur, so that the acidophilic thiobacillus thiooxidans can efficiently produce sulfuric acid and can also promote leaching of uranium and iron in uranium ores.

Description

Technical Field

[0001] This invention belongs to the technical field of acidophilic thiobacillus oxidizing, and particularly relates to an acidophilic thiobacillus oxidizing and its applications. Background Technology

[0002] Currently, the main leaching method used in uranium mining is conventional tank leaching. However, conventional tank leaching is more suitable for high-grade, small-particle-size uranium ore. It also has drawbacks such as long construction periods, high investment, high production costs, and environmental threats, which restrict mining operations. Compared with traditional processes, low-grade uranium ore leaching is better suited for biological heap leaching. Uranium ore bacterial heap leaching is a technology that integrates bacterial leaching with uranium ore heap leaching. It is less expensive, more environmentally friendly, and achieves higher mineral leaching rates. However, most bacterial strains (such as *Thiobacillus acidophilus*) require strictly controlled acidic environments. When the pH increases due to carbonate reactions during ore leaching, the activity of the strains decreases significantly, and they may even die. Therefore, finding an acidophilic bacterial strain for leaching uranium-bearing ore is crucial. Summary of the Invention

[0003] Therefore, the purpose of this invention is to provide an acidophilic thiobacillus and its applications.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides an acidophilic thiobacillus, named HS-PLS23-At.t, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo.34267.

[0006] Preferably, the initial pH for culturing the acidophilic thiobacillus is 1 to 6.

[0007] Preferably, the temperature for culturing the acidophilic thiobacillus is 20–40°C.

[0008] The present invention provides a sulfuric acid-producing microbial agent, wherein the microbial agent comprises the above-mentioned acidophilic thiobacillus.

[0009] This invention provides an application of the above-mentioned acidophilic thiobacillus in the preparation of sulfuric acid.

[0010] The present invention provides a method for preparing sulfuric acid, comprising the following steps: mixing and culturing the above-mentioned acidophilic thiobacillus with a culture medium containing a sulfur source.

[0011] Preferably, the sulfur source includes one or more of elemental sulfur, Na2S2O3, and K2S4O6;

[0012] The final concentration of sulfur source in the sulfur-containing culture medium is 1–20 g / L; the culture temperature is 20–40 °C; the pH of the sulfur-containing culture medium is 1–6; and the initial bacterial density of the acidophilic thiobacillus oxidizing is 1–5 × 10⁻⁶. 4 The inoculum size of the *Thiobacillus acidophilus* is 5%–15%;

[0013] The sulfur-containing culture medium comprises the following components in the following concentrations: final concentration of 0.1–0.3 g / L (NH4)2SO4, final concentration of 2–4 g / L K2HPO4, final concentration of 0.4–0.6 g / L MgSO4·7H2O, final concentration of 0.1–0.13 g / L CaCl2, and final concentration of 10–20 g / L sulfur source.

[0014] Preferably, the sulfur source is elemental sulfur, and the final concentration of the elemental sulfur is 10-20 g / L; the culture temperature is 25-35°C, and the pH of the culture medium containing the sulfur source is 2-5.

[0015] This invention provides an application of the above-mentioned acidophilic thiobacillus thiooxidans in the preparation of uranium ore leaching.

[0016] Preferably, the acidophilic thiobacillus promotes the leaching of uranium and iron from uranium ore.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides an acidophilic thiobacillus and its applications. The research found that this acidophilic thiobacillus can grow well at an initial pH of 1-6 and exhibits strong acid resistance. Furthermore, this acidophilic thiobacillus can convert sulfur sources into sulfuric acid; therefore, it can efficiently produce sulfuric acid and promote the leaching of uranium and iron from uranium ore.

[0019] Biological Preservation Information:

[0020] The acidophilic thiooxidizing bacillus of the present invention is named HS-PLS23-At.t, classified as Acidithiobacillus thiooxidans, and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34267, on April 18, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0021] Figure 1 The image shown is a scanning electron microscope image of the strain HS-PLS23-At.t obtained in Example 1.

[0022] Figure 2 pH and SO4 levels were measured for strain HS-PLS23-At.t under different initial pH conditions. 2- The results of SO4 concentration changes at different number of days: a) shows the pH change of HS-PLS23-At.t grown under different initial pH conditions as the culture time increases; b) shows the SO4 concentration change of HS-PLS23-At.t grown under different initial pH conditions as the culture time increases. 2- Results of concentration changes.

[0023] Figure 3 pH and SO4 levels at different temperatures were used to measure the growth of strain HS-PLS23-At.t. 2- The results of pH value changes at different days are shown in Figure a. a represents the pH value change of HS-PLS23-At.t grown under different temperature conditions with increasing culture time. b represents the SO4 value change of HS-PLS23-At.t grown under different temperature conditions with increasing culture time. 2- Results of concentration changes.

[0024] Figure 4 The pH and SO4 levels of strain HS-PLS23-At.t under different initial sulfur concentrations were compared. 2- The results of pH changes at different number of days are shown in Figure a. a represents the pH change of HS-PLS23-At.t grown under different initial sulfur concentrations as the culture time increases. b represents the SO4 concentration change of HS-PLS23-At.t grown under different initial sulfur concentrations as the culture time increases. 2- Results of concentration changes.

[0025] Figure 5 pH and SO4 levels at different inoculum sizes were used to measure the growth of strain HS-PLS23-At.t. 2- The results of pH changes at different inoculum sizes over different days are shown in Figure a. Figure a shows the pH changes of HS-PLS23-At.t grown at different inoculum sizes with increasing culture time. Figure b shows the SO4 concentration changes of HS-PLS23-At.t grown at different inoculum sizes with increasing culture time. 2- Results of concentration changes.

[0026] Figure 6 pH and SO4 content of strain HS-PLS23-At.t in different sulfur source media 2-The results of concentration changes at different days: a) shows the pH change of HS-PLS23-At.t with extended culture time in media with different sulfur sources; b) shows the SO4 concentration change of HS-PLS23-At.t with extended culture time in media with different sulfur sources. 2- Results of concentration changes.

[0027] Figure 7 The results of leaching uranium ore using strain HS-PLS23-At.t or sulfuric acid at different times are shown. a) pH changes with increasing culture time after treatment with HS-PLS23-At.t or sulfuric acid; b) leaching of uranium ions with increasing culture time after treatment with HS-PLS23-At.t or sulfuric acid; c) leaching of iron ions with increasing culture time after treatment with HS-PLS23-At.t or sulfuric acid. Detailed Implementation

[0028] This invention provides an acidophilic thiobacillus, named HS-PLS23-At.t, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo.34267.

[0029] In this invention, the initial pH for culturing the acidophilic thiocyanate bacillus is preferably 1-6, more preferably 2-5, and even more preferably 3-4, such as 3, 3.5, or 4. The acidophilic thiocyanate bacillus grows well at the above-mentioned initial pH. The temperature for culturing the acidophilic thiocyanate bacillus is preferably 20-40°C, more preferably 25°C-35°C, such as 30, 31, 32, 33, 34, or 35°C. Within the temperature range of 20-40°C, the acidophilic thiocyanate bacillus can generate sulfuric acid in a culture medium containing a sulfur source, while within the temperature range of 25°C-35°C, the generation of sulfuric acid is rapid and abundant, demonstrating a strong metabolic acid-producing capacity.

[0030] The present invention provides a sulfuric acid-producing microbial agent, wherein the microbial agent comprises the above-mentioned acidophilic thiobacillus.

[0031] This invention provides an application of the above-mentioned acidophilic thiobacillus in the preparation of sulfuric acid.

[0032] The present invention provides a method for preparing sulfuric acid, comprising the following steps: mixing and culturing the above-mentioned acidophilic thiobacillus with a culture medium containing a sulfur source.

[0033] In this invention, the *Thiobacillus acidophilus* is preferably in the logarithmic growth phase. The inoculum size of the *Thiobacillus acidophilus* is preferably 5%–15%, more preferably 10%–15%, such as 10%, 11%, 12%, 13%, 14%, or 15%. The inoculum size refers to the proportion of the volume of the *Thiobacillus acidophilus* seed culture to the volume of the culture medium after inoculation. This invention utilizes inoculum sizes within the above range to promote higher sulfate concentrations in the *Thiobacillus acidophilus*. The initial bacterial density of the *Thiobacillus acidophilus* is preferably 1–5 × 10⁻⁶. 4 The concentration of cells / mL is more preferably 2 to 4 × 10⁻⁶. 4 cells / mL, such as 3×10 4 The sulfur source includes one or more of elemental sulfur, Na2S2O3, and K2S4O6; the final concentration of the sulfur source in the culture medium containing the sulfur source is 1-20 g / L, more preferably 10-20 g / L, and even more preferably 10, 15, or 20 g / L.

[0034] In a preferred embodiment, the sulfur-containing culture medium comprises the following components in the following amounts: final concentration of 0.1–0.3 g / L (NH₄)₂SO₄, final concentration of 2–4 g / L K₂HPO₄, final concentration of 0.4–0.6 g / L MgSO₄·7H₂O, final concentration of 0.1–0.13 g / L CaCl₂, and final concentration of 10–20 g / L sulfur source. More preferably, the sulfur-containing culture medium comprises the following components in the following amounts: final concentration of 0.2 g / L (NH₄)₂SO₄, final concentration of 3 g / L K₂HPO₄, final concentration of 0.5 g / L MgSO₄·7H₂O, final concentration of 0.126 g / L CaCl₂, and final concentration of 10 g / L elemental sulfur, wherein the solvent of the culture medium is water.

[0035] This invention provides an application of the above-mentioned acidophilic thiobacillus thiooxidans in the preparation of uranium ore leaching.

[0036] The acidophilic thiobacillus of the present invention has a strong ability to oxidize low-valent sulfur to produce acid, which promotes the leaching of uranium and iron from uranium-containing ores.

[0037] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] 1. Isolation and screening of strains

[0041] The acid leaching solution from the Namibian Husab uranium mine was added to 9K+S medium and enriched at 30℃ and 150 rpm. The bacteria were filtered through a 0.22 μm filter and inoculated onto Waksman medium, then cultured with shaking at 30℃ and 150 rpm to screen for sulfur-oxidizing bacteria in the enriched solution. Further separation was performed using the extreme dilution method. In a clean bench, 8.6 mL of Waksman medium and 0.4 mL of thymol blue indicator were added to 30 test tubes, which were then shaken thoroughly. Each test tube was labeled 10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 and 10 -10 For each gradient, three parallel samples are made; towards 10 -1 Add 1 mL of bacterial sample to test tube #1, shake, and then randomly take 1 mL of the sample and add it to three vials of 10. -2 In test tube number 10; shake three tubes of 10 -2 After selecting the test tubes, randomly take one tube and add 1 mL of bacterial sample to each of the three tubes containing 10 mL of bacterial sample. -3 In test tube number 10, and so on, until 10... -10 Test tube number 1. Place in a 30℃ incubator and incubate statically. After 30 days, take the culture at the highest dilution with sulfur oxidation activity (changing from yellow to red) for further culture to obtain the selected acidophilic thiooxidizing bacteria HS-PLS23-At.t.

[0042] 9K+S medium: Solution A consists of (NH4)2SO4 3 g / L, KCl 0.1 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, and Ca(NO3)2 0.01 g / L; Solution B consists of FeSO4·7H2O 25 g / L. The pH is adjusted to 1.8 with dilute sulfuric acid. Solution A is autoclaved, and Solution B is sterilized by filtration through a 0.22 μm microporous membrane. When using, Solutions A and B are mixed, and then 10 g / L of elemental sulfur sterilized by UV irradiation and soaking in 70% ethanol is added. The solvent for the 9K+S medium is water.

[0043] Waksman medium: (NH4)2SO4 0.2 g / L, K2HPO4 3 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 0.126 g / L, pH adjusted to 4 with dilute sulfuric acid, autoclaved at high temperature and pressure to obtain solution C; elemental sulfur 10 g / L, sterilized by soaking in 70% alcohol and irradiating with ultraviolet light; when using, solution C and elemental sulfur are mixed, and the solvent of the Waksman medium is water.

[0044] Figure 1 The results showed that strain HS-PLS23-At.t was a short rod with rounded ends, 1.0–1.5 μm long and 0.3–0.5 μm wide.

[0045] 2. Identification of *Thiobacillus acidophilus* HS-PLS23-At.t

[0046] The isolated bacteria were identified by bacterial 16S rDNA sequencing, including genomic DNA extraction, 16S rDNA-specific primer PCR amplification, amplification product purification, DNA sequencing, and sequence alignment. Genomic DNA extraction was performed using the Ezup column-based bacterial genomic DNA extraction kit. The forward primer used for PCR amplification was 27F (AGAGTTTGATCMTGGCTCAG, SEQ ID NO.1), and the reverse primer was 1492R (GGTTACCTTGTTACGACTT, SEQ ID NO.2). The PCR reaction system consisted of 12.5 μL of 10×PCR Buffer, TaqPlus DNA Polymerase (5 U / μL), dNTPs (10 mmol / L), and MgSO4 (50 mmol / L), 1 μL each of 27F and 1492R primers (10 μmol / L), 1 μL of DNA, and 9.5 μL of ddH2O. The PCR reaction conditions were: 95℃ for 5 min; 94℃ for 30 s, 57℃ for 30 s, 72℃ for 90 s, for 30 cycles; and 72℃ for 10 min. DNA sequencing was performed using an ABI 3730-XL sequencer.

[0047] 16S rDNA sequence of the strain:

[0048] GGCTCAGATTGAACGCTGGCGGCATGCCTAACACATGCAAGTCGAACGGT

[0049] AACAGGTCTTCGGATGCTGACGAGTGGCGGACGGGTGAGTAATGCGTAGG

[0050] AATCTGTCTTTGAGTGGGGGACAACCCAGGGAAACTTGGGCTAATACCGC

[0051] ATAAGCCCTGAGGGGGAAAGCGGGGGATCTTCGGACCTCGCGCTGGAAG

[0052] AGGAGCCTACGTCTGATTAGCTAGTTGGTAGGGTAAAGGCCTACCAAGGC

[0053] GACGATCGGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAG

[0054] ACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTTCGCAATG

[0055] GGGGCAACCCTGACGAAGCAATGCCGCGTGAATGAAGAAGGCCTTCGGG

[0056] TTGTAAAGTTCTTTCGTGGAGGACGAAAAGGTGGGTGCTAATATCGCCTGC

[0057] TGTTGACGTGAATCCAAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCC

[0058] GCGGTAATACGGGGGGTGCAAGCGTTAATCGGAATCACTGGGCGTAAAGG

[0059] GTGCGTAGGCGGTGCATTAGGTCTGTCGTGAAATCCCCGGGCTCAACCTGG

[0060] GAATGGCGGTGGAAACCGGTGTACTAGAGTATGGGAGAGGGTGGTGGAAT

[0061] TCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAACATCAGTGGCG

[0062] AAGGCGGCCACCTGGCCCAATACTGACGCTGAGGCACGAAAGCGTGGGG

[0063] AGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATACT

[0064] AGATGTTTGGTGCCAAGCGTACTGAGTGTCGTAGCTAACGCGATAAGTATT

[0065] CCGCCTGGGAAGTACGGCCGCAAGGTTAAAACTCAAAGGAATTGACGGG

[0066] GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGA

[0067] ACCTTACCTGGGCTTGACATGTCTGGAATCCTGCAGAGATGCGGGAGTGCC

[0068] CTTCGGGGAATCAGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTC

[0069] GTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTG

[0070] CCAGCGGTTCGGCCGGGCACTCTAGGGAGACTGCCGGTGACAAACCGGA

[0071] GGAAGGTGGGGATGACGTCAAGTCCTCATGGCCTTTATGTCCAGGGCTACA

[0072] CACGTGCTACAATGGCGCGTACAGAGGGAAGCCAAGCCGCGAGGTGGAG

[0073] CAGAACCCCAGAAAGCGCGTCGTAGTTCGGATTGCAGTCTGCAACTCGACT

[0074] GCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATA

[0075] CGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGATTGTA

[0076] CCAGAAGCCGTTAGCCTAACCTTCGGGAGGGCGATGACCACGGTATGGTTCATGACTGGGGTGAAGTCGT (SEQ ID NO. 3).

[0077] Its sequence similarity with multiple strains of Acidithiobacillusthiooxidans in the genus Acidithiobacillus reached over 99%. The identification results showed that this strain is an acidophilic thiooxidizing bacterium and was named HS-PLS23-At.t.

[0078] The acidophilic thiobacillus HS-PLS23-At.t is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34267, deposited on April 18, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0079] Example 2

[0080] Optimal growth conditions of *Thiobacillus acidophilus* HS-PLS23-At.t prepared in Example 1

[0081] A single-factor shake-flask experiment was conducted in 100 mL Waksman medium to investigate the effects of different initial pH, temperature, S concentration, inoculum size, and sulfur source conditions on the growth of strain HS-PLS23-At.t, and to study the optimal growth conditions for the strain.

[0082] 1. Effect of initial pH on bacterial growth

[0083] Waksman liquid culture media with pH values ​​of 1, 2, 3, 4, 5, and 6 were prepared respectively. HS-PLS23-At.t bacterial suspensions in the logarithmic growth phase were inoculated at a 10% inoculum rate, with an initial bacterial density of approximately 3 × 10⁶. 4 The culture medium was collected at 30℃ and shaken at 150 rpm for 0, 2, 4, 6, 8, 10, 12, 14, or 16 days. The pH and SO4 content of the culture solution were measured after each of these 16 days. 2- concentration.

[0084] Preparation of the Waksman liquid medium: Add water to a final concentration of 0.2 g / L (NH4)2SO4, a final concentration of 3 g / L K2HPO4, a final concentration of 0.5 g / L MgSO4·7H2O, and a final concentration of 0.126 g / L CaCl2. Adjust the pH to 1, 2, 3, 4, 5, or 6 with dilute sulfuric acid. Sterilize under high temperature and high pressure to obtain solution C. Elemental sulfur with a final concentration of 10 g / L, wherein the elemental sulfur is elemental sulfur sterilized by soaking in 70% ethanol under ultraviolet irradiation. When needed, mix solution C and elemental sulfur to obtain the Waksman liquid medium.

[0085] like Figure 2 As shown, HS-PLS23-At.t oxidizes elemental sulfur (S 0 ) generates sulfuric acid (H2SO4), SO4 2- As the concentration increases, H in the environment +As the concentration increased, the pH decreased. Under initial pH conditions ranging from 1 to 6, the pH could be reduced to around 0.5, indicating a very strong ability of the bacteria to produce acid. This strain could grow well under initial pH conditions from 1 to 6 without significant inhibition, demonstrating its strong pH adaptability and suitability for bioleaching. The highest sulfate concentration (24.86 g / L) was achieved at an initial pH of 4; therefore, pH 4 was chosen as the optimal culture condition for this strain.

[0086] 2. Effects of temperature on strain growth

[0087] Prepare Waksman liquid medium with pH 3, and inoculate with HS-PLS23-At.t bacterial suspension in logarithmic growth phase prepared in Example 1 at a 10% inoculum rate, with an initial bacterial density of approximately 3 × 10⁻⁶. 4 The bacterial culture solution was prepared at 20℃, 25℃, 30℃, 35℃, and 40℃ and cultured at 150 rpm for 0, 3, 6, 9, 12, 15, or 18 days. The pH and SO4 levels of the culture solution were then measured at different incubation periods. 2- concentration.

[0088] Preparation of the Waksman liquid medium: Add water to a final concentration of 0.2 g / L (NH4)2SO4, a final concentration of 3 g / L K2HPO4, a final concentration of 0.5 g / L MgSO4·7H2O, and a final concentration of 0.126 g / L CaCl2. Adjust the pH to 3 with dilute sulfuric acid. Sterilize under high temperature and high pressure to obtain solution C. Add elemental sulfur with a final concentration of 10 g / L, wherein the elemental sulfur is elemental sulfur sterilized by soaking in 70% ethanol under ultraviolet irradiation. When needed, mix solution C and elemental sulfur to obtain the Waksman liquid medium.

[0089] like Figure 3 As shown, SO4 2- The concentration changed most rapidly and produced the most at 35℃, followed by rapid changes at 30℃ and 25℃, and slower changes at 20℃ and 40℃. The pH decreased rapidly between 25℃ and 35℃, indicating strong metabolic acid production at this temperature. The slow pH decrease at 20℃ and 40℃ indicated a low metabolic rate, low acid production, and incomplete sulfur oxidation, possibly due to low temperatures inhibiting enzyme activity, or high temperatures potentially damaging the cell structure or denaturing key enzymes, thus reducing metabolic activity. Therefore, 35℃ was chosen as the culture condition for the strain.

[0090] 3. Effect of initial sulfur concentration on strain growth

[0091] Prepare Waksman liquid medium 1 with pH 3, and inoculate with HS-PLS23-At.t bacterial suspension in logarithmic growth phase prepared in Example 1 at a 10% inoculum rate, with an initial bacterial density of approximately 3 × 10⁻⁶. 4 Bacterial culture medium was prepared by adding 0.1, 0.5, 1, 1.5, and 2 g of sterilized elemental sulfur to 100 mL of Waksman liquid medium 1, respectively, to achieve sulfur concentrations of 1, 5, 10, 15, or 20 g / L. The medium was then incubated at 150 rpm in a 30°C shaker for 0, 3, 6, 9, 12, or 15 days to obtain bacterial solutions after 0, 3, 6, 9, 12, or 15 days of incubation. The pH and SO4 precipitates of the bacterial solutions after different incubation periods were then measured. 2- concentration.

[0092] Preparation of Waksman liquid medium 1: Add 0.2 g / L (NH4)2SO4, 3 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, and 0.126 g / L CaCl2 to water. Adjust the pH to 3 with dilute sulfuric acid. Sterilize under high temperature and high pressure to obtain solution C, which is Waksman liquid medium 1.

[0093] like Figure 4 As shown, the higher the initial sulfur concentration, the lower the pH value and the higher the sulfate concentration. When the initial sulfur concentration added is 10–20 g / L, the pH decreases rapidly. At low sulfur concentrations (1–5 g / L), the pH decreases slowly because insufficient sulfur source leads to low acid production and SO4 levels. 2- The low concentration and slow change indicate that insufficient sulfur source limits cell growth and restricts metabolic activity. Therefore, an initial sulfur concentration of 15 g / L was selected.

[0094] 4. Effect of inoculum size on bacterial growth

[0095] Waksman liquid medium with a pH of 3 was prepared, and logarithmically growing HS-PLS23-At.t bacterial suspensions were inoculated at inoculum rates of 1%, 5%, 10%, 15%, or 20%, respectively. The suspensions were then incubated at 150 rpm in a 30°C constant-temperature shaker for 0, 3, 6, 9, 12, or 15 days to obtain the bacterial suspensions after 0, 3, 6, 9, 12, or 15 days of incubation. The pH and SO4 ions of the bacterial suspensions after incubation at different durations were measured. 2- concentration.

[0096] Preparation of the Waksman liquid medium: Add water to a final concentration of 0.2 g / L (NH4)2SO4, a final concentration of 3 g / L K2HPO4, a final concentration of 0.5 g / L MgSO4·7H2O, and a final concentration of 0.126 g / L CaCl2. Adjust the pH to 3 with dilute sulfuric acid. Sterilize under high temperature and pressure to obtain solution C. Elemental sulfur with a final concentration of 10 g / L is obtained; the elemental sulfur is elemental sulfur sterilized by soaking in 70% ethanol under ultraviolet irradiation. When needed, solution C and elemental sulfur are mixed to obtain the Waksman liquid medium.

[0097] like Figure 5 As shown, the pH decreased rapidly at inoculum sizes of 5%–15%, while it decreased more slowly at 20% inoculum. This may be due to excessive bacterial count, with dead cells releasing intracellular alkaline substances or metabolic byproducts accumulating and interfering with the acidification process. At 1% inoculum, the pH decreased slowly in the early stages and rapidly in the later stages. SO4 2- The growth was slow in the early stages and rapid in the later stages, possibly due to insufficient cell size in the early stages, which delayed growth. A 15% inoculum concentration achieved a higher sulfate concentration, indicating better growth; therefore, a 15% inoculum concentration was chosen.

[0098] 5. Effects of different sulfur sources on the growth of bacterial strains

[0099] Waksman liquid medium 1 with a pH of 3.2 was prepared, and 1 g / L of elemental sulfur, Na₂S₂O₃, or K₂S₄O₆ was added respectively. Elemental sulfur was sterilized by immersion in 70% ethanol and UV irradiation, while Na₂S₂O₃ and K₂S₄O₆ were sterilized by filtration through a 0.22 μm bacterial filter membrane. The logarithmic growth phase HS-PLS23-At.t bacterial suspension prepared in Example 1 was inoculated at a 10% inoculum, with an initial bacterial density of approximately 3 × 10⁻⁶. 4 The bacterial culture was incubated at 30℃ with shaking at 150 rpm for 0, 2, 4, 6, 8, 10, and 12 days. The pH and SO4 content of the culture were measured. 2- concentration.

[0100] Preparation of Waksman liquid medium 1: Add 0.2 g / L (NH4)2SO4, 3 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, and 0.126 g / L CaCl2 to water. Adjust the pH to 3 with dilute sulfuric acid. Sterilize under high temperature and high pressure to obtain solution C, which is Waksman liquid medium 1.

[0101] like Figure 6As shown, the pH of Waksman liquid medium 1 containing 1 g / L of elemental S, Na2S2O3, or K2S4O6 all showed a decreasing trend, while the sulfate concentration showed an increasing trend. This indicates that the strain can utilize not only elemental S, but also sulfur compounds Na2S2O3 or K2S4O6, and can oxidize sulfur in different low valence states to sulfate, demonstrating a strong sulfur oxidation metabolic capacity.

[0102] Example 3

[0103] The leaching capacity of *Thiobacillus acidophilus* HS-PLS23-At.t prepared in Example 1 for low-grade uranium ore

[0104] Prepare a Waksman liquid medium with a pH of 3, add 10g of uranium ore, and inoculate the logarithmic growth phase HS-PLS23-At.t bacterial culture prepared in Example 1 at an inoculation rate of 15%. Place it in a constant temperature shaker at 30℃ and shake at 150r / min for 12 days to carry out the uranium ore acclimatization of this strain, and obtain the acclimatized HS-PLS23-At.t bacterial culture.

[0105] The experiment was divided into a microbial leaching group and a sulfuric acid leaching group. For the microbial leaching group, 20g of uranium ore was added to a Waksman liquid medium with a pH of 3, and the domesticated HS-PLS23-At.t bacterial solution was inoculated at a 15% inoculum rate. For the sulfuric acid leaching group, 20g of uranium ore was added to the Waksman liquid medium, and sulfuric acid was added to adjust the pH to 1.2, simulating the pH conditions of heap leaching. Both the microbial leaching group and the sulfuric acid leaching group were placed in a 30℃ constant-temperature shaker and shaken at 150 rpm. The pH of the medium was measured after leaching for 0, 3, 6, 11, and 17 days, and the uranium ion concentration and iron ion concentration in the medium were measured using ICP-OES after leaching for 0, 3, 6, 11, and 17 days.

[0106] Preparation of the Waksman liquid medium: Add water to a final concentration of 0.2 g / L (NH4)2SO4, a final concentration of 3 g / L K2HPO4, a final concentration of 0.5 g / L MgSO4·7H2O, and a final concentration of 0.126 g / L CaCl2. Adjust the pH to 3 with dilute sulfuric acid. Sterilize under high temperature and pressure to obtain solution C. Elemental sulfur with a final concentration of 10 g / L is obtained; the elemental sulfur is elemental sulfur sterilized by soaking in 70% ethanol under ultraviolet irradiation. When needed, solution C and elemental sulfur are mixed to obtain the Waksman liquid medium.

[0107] like Figure 7As shown, during sulfuric acid leaching of uranium ore, due to the low initial pH, the highest concentration of leached uranium ions was 39.5 mg / L, and the highest concentration of leached iron ions was 246 mg / L. However, without the addition of sulfuric acid, the pH of the solution continuously increased, while the uranium concentration in the solution decreased. This indicates that the increase in pH may lead to uranium precipitation in the solution, which is not conducive to uranium leaching. In contrast, when using HS-PLS23-At.t for bioleaching of uranium ore, the highest concentration of leached uranium ions reached 41.33 mg / L, and the highest concentration of leached iron ions reached 1597 mg / L. The highest concentration of leached iron ions using HS-PLS23-At.t is 6.5 times that of sulfuric acid leaching. This strain can utilize sulfur to generate sulfuric acid, lowering the pH to around 1 without the need for additional sulfuric acid. Furthermore, this strain can grow well under acidic conditions, maintaining the pH at 1-2 for extended periods, providing an acidic environment for leaching low-grade uranium ore, promoting uranium leaching, and also exhibiting excellent leaching effects on iron in uranium ore.

[0108] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of acidophilic thiooxidizing thiobacillus, characterized in that, The acidophilic thiobacillus thiooxidans was named HS-PLS23-At.t and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo.34267.

2. The acidophilic thiobacillus thiooxidans according to claim 1, characterized in that, The initial pH for culturing the acidophilic thiobacillus was 1–6.

3. The acidophilic thiobacillus thiooxidans according to claim 1, characterized in that, The temperature for culturing the acidophilic thiobacillus is 20–40°C.

4. A sulfuric acid-producing microbial agent, characterized in that, The microbial agent includes *Thiobacillus acidophilus* as described in any one of claims 1 to 3.

5. The use of the acidophilic thiobacillus thiooxidans according to any one of claims 1 to 3 in the preparation of sulfuric acid.

6. A method for preparing sulfuric acid, characterized in that, The method includes the following steps: mixing and culturing the acidophilic thiobacillus thiooxidans according to any one of claims 1 to 3 with a culture medium containing a sulfur source.

7. The preparation method according to claim 6, characterized in that, The sulfur source includes one or more of elemental sulfur, Na2S2O3, and K2S4O6; The final concentration of sulfur source in the sulfur-containing culture medium is 1–20 g / L; the culture temperature is 20–40 °C; the pH of the sulfur-containing culture medium is 1–6; and the initial bacterial density of the acidophilic thiobacillus oxidizing is 1–5 × 10⁻⁶. 4 The inoculum size of the *Thiobacillus acidophilus* is 5%–15%; The sulfur-containing culture medium comprises the following components in the following concentrations: final concentration of 0.1–0.3 g / L (NH4)2SO4, final concentration of 2–4 g / L K2HPO4, final concentration of 0.4–0.6 g / L MgSO4·7H2O, final concentration of 0.1–0.13 g / L CaCl2, and final concentration of 10–20 g / L sulfur source.

8. The preparation method according to claim 7, characterized in that, The sulfur source is elemental sulfur, and the final concentration of the elemental sulfur is 10–20 g / L; the culture temperature is 25–35 °C, and the pH of the culture medium containing the sulfur source is 2–5.

9. The use of the acidophilic thiobacillus thiooxidans according to any one of claims 1 to 3 in the preparation of uranium ore leaching.

10. The application according to claim 9, characterized in that, The acidophilic thiobacillus promotes the leaching of uranium and iron from uranium ore.

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