Extreme acidophilus flora and application thereof in bioleaching

Abstract

The invention provides an extreme acidophilic bacterium flora. The extreme acidophilic bacterium flora comprises acidophilic thiobacillus with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.14839 and iron-sulfur oxidizing bacteria with the preservation number of CGMCC No.26454. According to the invention, two strains of extreme acidophilic bacteria obtained by screening are compounded to construct a double-bacteria mineral leaching system. The compounded flora can realize metabolism cooperation of autotrophic bacteria and abnormal bacteria, can realize coupling of iron-sulfur oxidation, can be applied to efficient leaching of pyrite, and has a more efficient ore leaching effect compared with a single strain. And the artificial mixed flora has definite microbial composition, so that the controllability in the bioleaching process is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of industrial microbial application, and in particular relates to an extreme acidophilic bacteria group and application thereof in biological leaching. Background Art

[0002] Mineral resources are an important material basis for human society and national economic construction. my country is a country with large reserves of nonferrous metal mineral resources and a large consumer. After more than half a century of production and consumption, there are only a few mines that are easy to mine and beneficiate, making the status of low-grade complex mines, which are the main source of mineral resources to be mined in my country, increasingly important. The existing conventional physical and chemical beneficiation methods are no longer suitable for the sustainable development of social economy due to problems such as low recovery rate, large resource loss, high production cost and serious environmental pollution. Under this circumstance, the role of microorganisms in mineral separation has gradually attracted people's attention. It can be used for in-situ leaching of minerals, as well as for factory mineral treatment and wastewater and waste residue treatment. In addition, microbial leaching has the advantages of low production cost, low investment, short process flow, simple equipment, environmental friendliness, and the ability to treat low-grade complex polymetallic minerals. Therefore, the widespread application of bacterial leaching will cause major changes in the traditional mineral processing industry and open up broad prospects for the sustainable development of mankind, resources and the environment.

[0003] Bioleaching refers to the method of dissolving valuable metals from ores using solvents containing microorganisms. When using microorganisms to leach minerals, bacterial communities often show higher leaching efficiency than single strains. This is because compared to single bacteria, bacterial communities have a wider spectrum of substrate utilization and higher leaching efficiency. However, when natural bacterial communities are applied to mineral leaching, there are problems such as complex microbial composition, difficult mechanism analysis, and low operability. Summary of the invention

[0004] The purpose of the present invention is to provide an extreme acidophilic bacteria community and the application of the same in bioleaching, so as to more efficiently carry out bioleaching treatment on low-grade ores such as pyrite.

[0005] The invention firstly provides an extreme acidophilic bacteria group, which comprises an acidithiobacillus Ameehan AC strain with a preservation number of CGMCC No.14839 and an iron-sulfur oxidizing bacteria (Sulfobacillus thermotolerants) hq2 strain with a preservation number of CGMCC No.26454.

[0006] The Acidithiobacillus Ameehan AC strain (hereinafter referred to as AMEEHan strain) used in the present invention has a deposit number of CGMCC No. 14839, which was deposited in the General Microbiological Culture Collection Center of China General Microbiological Culture Collection Committee (referred to as CGMCC) on October 27, 2017. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The other strain is the Sulfobacillus thermotolerants hq2 strain (abbreviated as hq2 strain), whose preservation number is CGMCC No.26454. It was deposited in the China General Microbiological Culture Collection Center (CGMCC for short) on January 12, 2023. The address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The AMEEHan strain is prepared by fermentation in a 9K medium supplemented with 10 g / L of elemental sulfur;

[0009] The hq2 strain was grown in a medium supplemented with 44.2 g / L FeSO 4 .7H 2 O was prepared by fermentation in 9K medium.

[0010] The specific formula of 9K culture medium is as follows: (NH 4 ) 2 SO 4 3g, KCI 0.1g, MgSO 4 .7H 2 O 0.5g, K 2 HPO 4 0.5 g, Ca(NO 3 ) 2 0.01g, adjust pH to 2.0, make up to 1L, and sterilize at 121℃ for 20min.

[0011] In yet another aspect, the present invention provides an application of the bacterial flora, which is application in oxidizing ferrous ions.

[0012] The present invention also provides another use of the bacterial community, which is use in biological leaching.

[0013] The application of the biological leaching method is the application in the biological leaching of pyrite.

[0014] The present invention compounds two strains of extreme acidophilic bacteria obtained by screening to construct a dual-bacteria leaching system. The compounded bacterial community can realize the metabolic cooperation between autotrophic bacteria and heterotrophic bacteria, and realize the coupling of iron-sulfur oxidation, which can be applied to the efficient leaching of pyrite, and has a more efficient leaching effect than a single strain. Moreover, the artificial mixed bacterial community has a clear microbial composition, which improves the controllability of the biological leaching process. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 :Single colony culture characteristics of strains Aciditiobacillus sp.AMEEHan (left) and Sulfobacillus sp.hq2 (right);

[0016] Figure 2 :Fe in co-culture experiments 2+ Change trend chart, where CK is the blank control without bacteria; AC and ST are AMEEHan and hq2 single culture groups respectively; AC+ST group is the mixed culture group of the two.

[0017] Figure 3 : ORP change trend diagram in the co-culture experiment, where CK is the blank control without bacteria; AC and ST are AMEEHan and hq2 single culture groups respectively; AC+ST group is the mixed culture group of the two.

[0018] Figure 4 : CFU change trend diagram in the co-culture experiment, where CK is the blank control without bacteria; AC and ST are AMEEHan and hq2 single culture groups respectively; AC+ST group is the mixed culture group of the two.

[0019] Figure 5 :The effect diagram of the metabolites of strain Aciditiobacillus sp.AMEEHan on the ferrous oxidation capacity of Sulfobacillus sp.hq2;

[0020] Figure 6 : The effect diagram of single and mixed bacterial community coupling leaching of pyrolusite, where AC and ST are AMEEHan and hq2 single culture groups respectively; AS group is the mixed group. Specific implementation plan

[0021] The present invention is described in detail below in conjunction with embodiments and drawings.

[0022] Example 1: Strain compounding experiment

[0023] 1. Information about strains

[0024] The Acidithiobacillus Ameehan (AC) strain (hereinafter referred to as AMEEHan strain) used in the present invention has a deposit number of CGMCC No. 14839, which was deposited in the General Microbiological Culture Collection Center of China General Microbiological Culture Collection Committee (referred to as CGMCC) on October 27, 2017. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0025] The other strain is the Sulfobacillus thermotolerants hq2 strain (abbreviated as hq2 strain), whose preservation number is CGMCC No.26454. It was deposited in the China General Microbiological Culture Collection Center (CGMCC for short) on January 12, 2023. The address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0026] 2. Culture conditions

[0027] AMEEHan strain was cultured in 9K medium with 10 g / L sulfur added, and cultured in a constant temperature shaker at 180 rpm and 37°C. After weighing, the sulfur powder was wrapped in tin foil and sterilized by dry heat at 105°C for 24 hours.

[0028] hq2 strain was grown on 9K medium with 44.2 g / L FeSO added 4 .7H 2 O.FeSO 4 .7H 2 O is prepared into 10 times stock solution with dilute sulfuric acid solution, the final pH of the stock solution is 1.2, and sterilized by filtering with a membrane.

[0029] The specific formula of 9K culture medium is as follows: (NH 4 ) 2 SO 4 3g, KCI 0.1g, MgSO 4 .7H 2 O 0.5g, K 2 HPO 4 0.5 g, Ca(NO 3 ) 2 0.01g, adjust pH to 2.0, make up to 1L, and sterilize at 121℃ for 20min.

[0030] Aciditiobacillus sp.AMEEHan is a chemoautotrophic acidithiobacillus, a Gram-negative bacterium with an optimal growth temperature of 37°C and 45°C and an optimal growth pH of 2.0. 2 O 6 S 4It can grow chemoautotrophically on the surface, but not on the surface of Na 2 S 2 O 3 , FeS 2 、FeSO 4 7H 2 Sulfobacillus sp.hq2 is a mixed-trophic iron-oxidizing bacterium, a Gram-positive bacterium with an optimal growth temperature of 37°C and 42°C and an optimal growth pH of 2.0. It can oxidize Fe in the presence of 0.02% yeast. 2+ , S 0 , K 2 S 4 0 6 Growth cannot utilize Na 2 S 2 O 3 To grow. Figure 1 These are photos of the morphology of single colonies of the two strains.

[0031] 3. Compounding experiment

[0032] The AMEEHan strain and the hq2 strain were cultured to the exponential phase using the above-mentioned culture method. AMEEHan was cultured in a 9K medium containing elemental sulfur at 37°C for about 7-8 days, and hq2 was cultured in a medium containing ferrous sulfate heptahydrate at 37°C for about 5-6 days to obtain the seed liquid of the two strains used in the experiment.

[0033] The mixed culture experiment set up three experimental groups: AMEEHan and hq2 bacteria were cultured alone, and AMEEHan and hq2 were mixed. The CK group without bacteria was used as a blank control, and three parallels were made for each group. The experiment used 9K inorganic salt culture medium, the addition of sulfur was 10g / L, and the addition of ferrous sulfate heptahydrate was 44.2g / L. In the experiment, AMEEHan and hq2 were inoculated in the mixed group at a ratio of 1:1.

[0034] The inoculation volume of hq2 was 30%, and the bacterial solution was centrifuged at 10,000 rpm for 15 minutes, the supernatant was discarded, and the bacteria were washed three times with 9K basic inorganic salt medium, and then resuspended in 9K medium. After counting under a microscope, they were used as inoculated bacteria. AMEEHan obtained inoculated bacteria in the same way, and the inoculation volume was kept the same as hq2.

[0035] 4. AMEEHan metabolite culture hq2 experiment

[0036] AMEEHan strain was cultured, and bacterial liquid was collected on days 0-7. The bacterial cells were removed by centrifugation at 10,000 rpm for 15 min. The supernatant was sterilized by filtration with a 0.22 μm filter membrane, and then 44.2 g / L of ferrous sulfate heptahydrate was added for the culture of hq2 strain. Fresh bacterial liquid of hq2 strain was collected, and bacterial cells were obtained by centrifugation according to 30% inoculation volume. The bacterial cells were washed three times with fresh 9K and inoculated into the prepared AMEEHan metabolites. Samples were taken every day to detect changes in various indicators.

[0037] During the experiment, samples were taken every two days to measure pH, ORP, and Fe. 2+ , CFU and SO 4 2- pH was measured by a pH meter (METTLERTOLEDO, Shanghai, China). ORP was measured by an ORP probe (METTLER TOLEDO, Shanghai, China). 2+ The national standard o-phenanthroline spectrophotometric method was used for detection, and CFU was directly counted under a microscope. 4 2- Using hashing Sulfate reagent kit (HACH 2106769-CN, Shanghai, China) was used for detection.

[0038] 5. Experimental results

[0039] A compound experiment was conducted on AMEEHan and hq2, that is, the two target strains were cultured separately and mixed in an iron-sulfur medium without any organic matter. The uninoculated culture medium group was used as a blank control, and samples were taken every two days to detect changes in various indicators.

[0040] 1) The two strains have a synergistic effect in ferrous ion oxidation

[0041] The change trend of ferrous ions is as follows Figure 2 As shown in the figure, when AMEEHan and hq2 were cultured alone, they did not show the ability to oxidize ferrous ions. However, when they were mixed and cultured, they could completely oxidize the ferrous ions in 44.2 g / L ferrous sulfate heptahydrate within 14 days. Figure 3 As shown, the mixed culture group also showed the highest redox potential (ORP) compared with the single culture.

[0042] 2) The two strains have a synergistic effect in biomass accumulation and ferrous ion oxidation

[0043] The number of bacteria in each experimental group during single culture and mixed culture was detected by microscopic counting. The experimental results are as follows Figure 4 As shown. Figure 4It can be seen that hq2 cannot grow alone under experimental conditions, and its cell number gradually decreases during the counting process. Compared with the AMEEHan single culture group, the mixed culture group showed the highest biomass accumulation. This shows that due to the presence of AMEEHan bacteria, the hq2 strain can grow in an environment where it cannot grow originally, and exert its ferrous iron oxidation function. This shows that the two have a strong synergistic effect in ferrous iron oxidation.

[0044] 3) The promoting effect of metabolites on iron oxidation function

[0045] The metabolites of Aciditiobacillus sp.AMEEHan at different stages were used to culture the Sulfobacillus sp.hq2 strain. The experimental results are as follows Figure 5 The ORP index showed that the metabolites of AMEEHan on days 4-7 could promote the oxidation of ferrous ions by the hq2 strain without adding any organic matter.

[0046] Example 2: Leaching experiment

[0047] In order to further clarify the effects of AMEEHan and hq2 on mineral leaching together and alone, coupled leaching experiments of pyrolusite and pyrite were carried out.

[0048] The pyrolusite used in the experiment was collected in Hunan Province, and the manganese content was 7%. The remaining elements were Fe5.64%, Mg 0.25%, S 0.06%, Si 58%, Ca 0.37%, and Al 3.91%.

[0049] The liquid-to-solid ratio of the microbial leaching system used in the experiment was 1:2.5, of which 1g of pyrolusite was added, 0.18g of pyrite was added, 0.6g of sulfuric acid was added, 4mL of pre-leaching solution (the leaching solution of pyrite bioleaching by AMEEHan and hq2), and 8.62mL of sterile water. AMEEHan and hq2, as well as a 1:1 mixture of the two, were inoculated in the above system. At the same time, the leaching reaction without adding microorganisms was used as a control, and the pre-leaching solution was partially replaced by water. The reaction was set at 30°C and 90°C, and the sampling time was 4h and 16h, respectively. The potassium periodate method was used to detect the soluble Mn in the leaching solution after the reaction. 2+ concentration.

[0050] The specific steps of the potassium periodate method for determining the concentration of soluble manganese ions are as follows:

[0051] Determination of manganese leaching rate: Potassium periodate spectrophotometry was used in the test. Take a certain amount of leaching solution, centrifuge it, take the supernatant and transfer it to a 50mL volumetric flask, add 10mL potassium pyrophosphate-sodium acetate buffer and 3mL potassium periodate solution, dilute to the mark, mix well, let stand for 10 minutes, and use pure water as a reference. Use a 50mm colorimetric dish to measure the absorbance at 525nm. The calculation formula is as follows:

[0052] C=m / V

[0053] Where m is the manganese content obtained from the calibration curve, μg; V is the sample volume, mL.

[0054] The experimental results are as follows Figure 6 As shown, the results show that at 30℃, the effect of mixed bacteria is significantly better than that of single bacteria. The leaching concentration of manganese by mixed bacteria for 4h is 4.23g / L, and the leaching effect decreases slightly with the extension of reaction time. The leaching effect at 90℃ is significantly better than that at 30℃, and the leaching effect of mixed bacteria is also significantly better than that of single bacteria. The leaching concentration of Mn2+ by mixed bacteria for 4h is 12.18g / L.

[0055] From the above results, it can be seen that the leaching effect of mixed bacteria under different conditions is significantly better than that of single bacteria, which means that the synergistic effect of the two can promote the leaching of minerals.

Claims

1. An extreme acidophilic bacteria group, characterized in that: The extremely acidophilic bacteria group includes an acidithiobacillus strain with a preservation number of CGMCC No.14839 and an iron-sulfur oxidizing bacteria strain with a preservation number of CGMCC No.26454.

2. The extreme acidophilic bacteria group according to claim 1, characterized in that The acidithiobacillus strain is prepared by fermentation in a 9K medium added with elemental sulfur.

3. The extreme acidophilic bacteria group according to claim 1, characterized in that The iron-sulfur oxidizing bacteria are prepared by fermentation in a 9K culture medium added with FeSO4.7H2O.

4. The extreme acidophilic bacteria group according to claim 2 or 3, characterized in that The formula of the 9K culture medium is as follows: (NH4)2SO4 3g, KCI 0.1g, MgSO4.7H2O 0.5g, K2HPO4 0.5g, Ca(NO3)2 0.01g, adjust the pH to 2.0, make the volume to 1L, and sterilize at 121°C for 20min.

5. Use of the extreme acidophilic bacteria group described in claim 1 in oxidizing ferrous ions.

6. Use of the extreme acidophilic bacteria group described in claim 1 in biological leaching of minerals.

7. A method for bioleaching, characterized in that: The method is to use the extreme acidophilic bacteria group described in claim 1 to carry out bioleaching of ore.

8. The method according to claim 7, characterized in that The ore is pyrite ore.

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