Immobilization method of acidithiobacillus ferrooxidans

Abstract

The invention belongs to the field of microorganism immobilization, and relates to an immobilization method of acidophilic thiobacillus ferrooxidans. The method comprises the following steps: in a liquid culture medium containing Fe < 2 + >, carrying out semi-continuous culture on acidophilic thiobacillus ferrooxidans by taking superfine fibers as an immobilization material, and then carrying out continuous culture on the acidophilic thiobacillus ferrooxidans; wherein in the semi-continuous culture process, when Fe < 2 + > in a liquid culture medium is oxidized to 95% or above, 60-80% of a fresh liquid culture medium containing Fe < 2 + > is replaced for continuous culture; in the continuous culture process, a fresh liquid culture medium containing Fe < 2 + > is continuously introduced, meanwhile, the liquid culture medium is continuously discharged, and on the premise that the conversion rate of Fe < 2 + > is not lower than 95%, the flow rate of the liquid culture medium cannot be increased, so that immobilization is completed. The immobilization method provided by the invention has a better immobilization effect, and the Fe < 2 + > oxidation rate of A.ferrooxidans in unit time can be improved.

Description

Technical Field

[0001] The invention belongs to the field of microbial immobilization and relates to an immobilization method of acidithiobacillus ferrooxidans. Background Art

[0002] Bioleaching technology mainly relies on acid-producing chemotrophic microorganisms, among which Acidithiobacillus ferrooxidans with good oxidative activity is the dominant species. According to research, the direct effect of this species or the indirect effect of its metabolites are widely used in fields such as leaching of low-priced ore metals, recycling of copper and other metals in waste circuit boards, and hydrogen sulfide emissions from refineries.

[0003] Taking the recycling of copper and other metals from discarded circuit boards as an example, the principle is: Indirect effect: Fe 2+ Natural oxidation to Fe 3+ It is extremely slow and difficult, but in the presence of a large number of Acidithiobacillus ferrooxidans, this oxidation step can be completed quickly and easily. 2+ Oxidized to Fe at a very fast rate 3+ , Fe 3+ Oxidize copper and other metals in the circuit board to dissolve them, Fe 3+ Reduced to Fe 2+ , Fe 2+ Then it is rapidly oxidized by bacteria into Fe 3+ , and continue to act on the oxidation of metals such as copper. Direct action means that there is a layer of extracellular polymers on the surface of Acidithiobacillus ferrooxidans cells, which is conducive to the adsorption of Fe 3+ Attached to the surface of metals such as copper, making the Fe 2+ and Fe 3+ The concentration increases, forming an effective and strong corrosion to copper, accelerating the leaching of copper. Through indirect and direct action, copper and other metal ions can be leached in a shorter time. By using the immobilization method, a bioreaction film rich in acidophilic ferrothiobacillus is formed, and the oxidation reaction continues, that is, Fe 2+ Oxidized to Fe at a very fast rate 3+ , and keep the number of strains in the solution constant. Therefore, the oxidation efficiency of the immobilized Acidithiobacillus ferrooxidans determines its specific application effect. However, the inventors have found that the oxidation efficiency of the immobilized Acidithiobacillus ferrooxidans is currently low and needs to be further improved. Summary of the invention

[0004] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a method for immobilizing acidophilic ferrooxidans. The immobilization method provided by the present invention has a better immobilization effect and can increase the Fe content of A. ferrooxidans per unit time. 2+ Oxidation rate.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] On the one hand, a method for immobilizing acidithiobacillus ferrooxidans in a medium containing Fe 2+ In a liquid culture medium, using ultrafine fibers as immobilization materials, Acidithiobacillus ferrooxidans was first cultured semi-continuously and then continuously.

[0007] During the semi-continuous culture process, when Fe 2+ When the oxidation reaches 95% or more, replace 60-80% of fresh Fe 2+ During the continuous culture, fresh liquid medium containing Fe 2+ of liquid culture medium, while continuously discharging the liquid culture medium, in Fe 2+ Under the premise that the conversion rate is not less than 95%, the flow rate of the liquid culture medium cannot be increased any further, indicating that the immobilization is completed.

[0008] The immobilization materials that can be used in the present invention include reticulated polyurethane foam, cotton thread and ultrafine fiber (the ultrafine fiber in the present invention refers to the fiber with a monofilament fineness of 0.1 to 1 dtex). Studies have shown that the use of ultrafine fiber is more conducive to the fixation of acidophilic ferrothiobacillus, thereby increasing the number of immobilized bacteria, and then through semi-continuous culture and continuous culture, it is beneficial to further increase the number of bacteria in the immobilized material, thereby facilitating the increase of Fe 2+ Oxidation efficiency.

[0009] In some embodiments, the acidophilic ferrooxidans is A. ferrooxidans 14 strain. Studies have shown that after immobilization of A. ferrooxidans 14 strain, Fe 2+ Higher oxidation efficiency.

[0010] In some embodiments, in the semi-continuous culture, the liquid culture medium is replaced no less than 6 times. Studies have shown that when the liquid culture medium is replaced 6 to 7 times, the number of bacteria in the unit volume of the immobilized material increases faster.

[0011] During the semi-continuous culture and continuous culture, the air flow rate is 20 to 225 L / h. In some embodiments, during the semi-continuous culture and continuous culture, the air flow rate is 120 to 225 L / h. Studies have shown that under this condition, especially when the air flow rate is 170 to 190 L / h, the number of bacteria per unit volume of the immobilized material is greater, and Fe 2+ Higher oxidation efficiency.

[0012] In some embodiments, the liquid culture medium is a 9K liquid culture medium. 2HPO 4 0.4~0.6g / L,(NH 4 ) 2 SO 4 2~4g / L,KCl 0.05~0.15g / L,MgSO 4 7H 2 O0.4~0.6g / L,Ca(NO 3 ) 2 0.005~0.015g / L, pH is 1.5~2.5.

[0013] In one or more embodiments, the Fe 2+ The preparation process of the liquid culture medium is to prepare FeSO with a pH of 1.5 to 2.5. 4 solution, add it into 9K liquid culture medium and mix well.

[0014] In one or more embodiments, the Fe 2+ In the liquid culture medium, there is Fe 2+ The concentration is 14-16g / L.

[0015] In some embodiments, the temperature during semi-continuous culture or continuous culture is 25-35°C.

[0016] In some embodiments, the porosity of the reactor used in semi-continuous culture or continuous culture is 96-97%.

[0017] In another aspect, a strain immobilized material is obtained by the above-mentioned immobilization method.

[0018] In a third aspect, a strain immobilization device comprises an immobilization tank, wherein the immobilization tank is used to implement the above-mentioned immobilization method.

[0019] In a fourth aspect, a bacterial strain immobilization material or a bacterial strain immobilization device is used to oxidize Fe 2+ Application in.

[0020] The beneficial effects of the present invention are:

[0021] The present invention selects ultrafine fibers as immobilized materials, and can significantly increase the number of strains in the immobilized materials by first semi-continuous culture and then continuous culture; at the same time, ultrafine fibers as immobilized materials are beneficial to the distribution of acidithiobacillus ferrooxidans on the surface of the immobilized materials, thereby facilitating the increase of Fe 2+ Average oxidation rate.

[0022] The present invention can further improve the Fe content by using ultrafine fibers as immobilization materials to carry out semi-continuous culture and continuous culture in sequence, by selecting acidithiobacillus ferrooxidans, optimizing the number of semi-continuous culture replacement rounds, optimizing the ventilation volume, etc. 2+ Average oxidation rate, Fe in the optimized immobilization tank 2+ The maximum oxidation capacity can reach 18.22g / h, Fe 2+ The oxidation rate can reach 5.78 g / L / h, which is higher than the currently known immobilized Fe 2+ The oxidation rate is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 The Fe immobilized in each round of the three immobilized materials in the embodiment of the present invention is 2+ Average oxidation rate (A) and bacterial count per unit material (B);

[0025] Figure 2 The Fe immobilized in each round under different ventilation rates in the embodiment of the present invention is 2+ Average oxidation rate (A) and number of bacteria per unit volume of material (B);

[0026] Figure 3 The Fe content of the two immobilization tanks after each round of semi-continuous culture in the embodiment of the present invention is 2+ Average oxidation rate;

[0027] Figure 4 The medium flow rate and Fe in the continuous culture process in the embodiment of the present invention are 2+ The relationship between conversion rate;

[0028] Figure 5 It is the number of bacteria fixed on a unit volume of material during the entire immobilization process in the reaction tank in the embodiment of the present invention. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0030] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0031] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0032] Example

[0033] The liquid culture medium used is 9K-Fe liquid culture medium, and its preparation method is as follows: take FeSO 4 7H 2 O 39g, add appropriate amount of distilled water (H 2 SO 4 Adjust pH to 2.0) to 100 mL, filter and sterilize, and add 38.2 mL to 161.8 mL / 500 mL Erlenmeyer flask 9K liquid culture medium to make the final Fe 2+ The concentration is 15g / L.

[0034] 9K liquid culture medium is: K 2 HPO 4 0.5 g / L, (NH 4 ) 2 SO 4 3 g / L, KCl 0.1 g / L, MgSO 4 7H 2 O0.5g / L, Ca(NO 3 ) 2 0.01g / L, using H 2 SO 4 Adjust the pH to 2.0, sterilize by high pressure steam at 121°C for 20 min after packaging, take out and cool for later use.

[0035] The immobilized reaction tank used has an inner diameter of 9.5 cm, a height of 62 cm, and an effective volume of 3150 mL. The reaction liquid in the reaction tank is discharged by the outlet below the column, and the gas distributor for ventilation is placed at the bottom of the column. The outer diameter is 15 cm, and a sandwich is formed between the inner and outer diameters. The water inlet pipe at the lower left and the water outlet pipe at the upper right are connected to a constant temperature water bath device to maintain the temperature of the reaction tank in the column.

[0036] A.ferrooxidans 14 with high ferrous oxidation activity screened in our laboratory was used as the immobilized strain. The method of immobilizing A.ferrooxidans was adopted to increase the number of bacteria in unit volume of culture medium, so that the bacterial cells were enriched on the immobilized material so as to more effectively exert the oxidation effect. First, it was immobilized by semi-continuous culture (inoculation amount was 30% of the effective volume of the immobilization tank), and the parameters of the immobilization process were optimized with the number of bacteria fixed on unit material and ferrous oxidation ability as indicators, and then A.ferrooxidans 14 was immobilized with the optimized parameters.

[0037] Three materials, namely, reticulated polyurethane foam, cotton thread and soft microfiber, were selected as immobilization materials for the study. The number of bacteria fixed on each unit material and the Fe 2+ The average oxidation rate was used as an indicator to compare the immobilization effects of A. ferrooxidans on the three materials.

[0038] Using an immobilized fermentation tank, three materials, namely, reticular polyurethane foam, cotton thread and soft ultrafine fiber, were used as immobilized materials. Semi-continuous culture was adopted, that is, the initial inoculation amount was 30% of the effective volume of the immobilized reaction tank. The Fe 2+ ion concentration until the oxidation of ferrous ions is completed, and then 70% of the fresh culture medium is replaced for the next round of cultivation. 2+ The average oxidation rate is Figure 1 As shown in (A), at the end of each round, the number of bacteria fixed on a unit material is Figure 1 (B) shown.

[0039] Depend on Figure 1 (A) It can be seen that among the three immobilization materials, the Fe 2+ The average oxidation rate was greater than that of Fe when using reticulated polyurethane foam or cotton thread as the immobilization material. 2+ Average oxidation rate. 2+ In terms of oxidation efficiency, soft microfiber is superior to reticulated polyurethane foam and cotton thread.

[0040] Depend on Figure 1 (B) It can be seen that with each round of fixation, the number of bacteria per unit material of the three materials continued to increase. Among them, the number of bacteria per unit volume of soft microfiber was greater than that of reticulated polyurethane foam and cotton thread. Especially after 6-7 rounds, the number of bacteria per unit volume of material increased significantly. 2+ The average oxidation rate is also high, indicating that the more bacteria there are on the material, the higher the Fe2+ The average oxidation rate is also correspondingly higher. Therefore, soft microfibers have the best immobilization effect on A. ferrooxidans among the three immobilization materials.

[0041] Taking ventilation volume as a variable, Fe in solution was detected 2+ ion concentration until each round of ferrous ion oxidation is complete. Similarly, the concentration of Fe 2+ The average oxidation rate and the number of bacteria per unit volume of material were used as evaluation indicators to explore the effect of different ventilation volumes on the immobilization effect of A. ferrooxidans 14. The results are shown in Figure 3 shown.

[0042] Depend on Figure 2 (A) It can be seen that with the increase of ventilation, the oxidation rate of ferrous iron in the jar also increases, but when the ventilation volume increases to a certain amount, the oxidation rate no longer increases. As shown in the figure, when the ventilation volume is 180 and 225 L / h, the Fe 2+ The average oxidation rates are basically the same.

[0043] Depend on Figure 2 (B) It can be seen that when the ventilation rate is increased to 180L / h, the number of bacteria per unit volume of material after each round of immobilization reaches the maximum. When the ventilation rate is 225L / h, although Fe 2+ The average oxidation rate is not much different from the ventilation volume of 180L / h, but the number of fixed bacteria decreases a lot. Considering these two factors comprehensively, 180L / h should be selected as the ventilation volume parameter.

[0044] The immobilization method of A.ferrooxidans 14 was to use the optimized immobilization parameters of A.ferrooxidans 14 and the growth characteristics of A.ferrooxidans 14 to set the conditions for the culture and immobilization of the bacteria, namely: 30°C, initial pH 2.0, initial Fe 2+ The concentration was 15 g / L, the soft ultrafine fiber was used as the immobilization material, the porosity of the reaction tank was 96.64%, and the ventilation volume was 180 L / h. According to the above conditions, two reaction tanks (tank 1 and tank 2) were selected and semi-continuous culture was carried out for 10 days (each time the Fe 2+ When the oxidation reaches above 95%, replace with 70% fresh culture medium and continue to culture); then continue to culture to form biofilm (i.e. according to Fe 2+ Average oxidation, continuous supply containing Fe 2+ The culture medium is used to regularly take unit volume materials and determine the number of bacteria on the unit volume materials until the number of bacteria on the unit volume materials reaches a stable level.

[0045] The detection results during the immobilization of A.ferrooxidans 14 are as follows Figure 4 -5. Figure 4 It is the Fe after each round of immobilization in semi-continuous culture. 2+ The average oxidation rate was about 10 days in semi-continuous culture, and 7 rounds of immobilization were performed. The results showed that the Fe 2+ The average oxidation rate did not differ much. With each round of immobilization culture, Fe 2 + The average oxidation rate gradually increased and reached about 2000 mg / L / h after the 7th round. After semi-continuous culture, the immobilization process of continuous culture was entered. The Fe 2+ Concentration, calculate Fe 2+ The conversion rate is 95% of Fe 2+ The conversion rate is the index, and the flow rate is adjusted by the flow meter to control the flow of Fe 2+ The amount of Fe 2+ Under the premise of conversion rate, continuous cultivation until the liquid flow rate can no longer be increased indicates that the bacteria fixed on the material have reached saturation and the immobilization process is completed. At this time, the maximum Fe 2+ Oxidation rate, that is, the maximum Fe 2+ Oxidation capacity.

[0046] After the two immobilization tanks reached stability, Fe 2+ The inflow velocity and Fe 2+ The relationship between conversion rate and results Figure 5 As shown in the figure, as Fe 2+ With the increase of flow rate, the Fe 2+ The conversion rate showed a downward trend, with 95% Fe 2+ When the conversion rate is used as an indicator, the maximum flow rate is about 850 mL / h. 2+ The maximum oxidation capacity is 18.22g / h, Fe 2+ The oxidation rate was 5.78 g / L / h.

[0047] During the whole immobilization process, the number of bacteria on the unit immobilization material was detected at different times. The results are as follows: Figure 5 As shown in the figure, in the first 10 days of semi-continuous culture, the immobilization of bacteria was slow; after entering the continuous culture stage, the immobilization of bacteria was relatively fast, and it basically reached a balance state in about 20 days, indicating that the immobilization of bacteria was basically completed and a biofilm was formed. Finally, the difference in the number of bacteria per unit volume of material in the two immobilization tanks was not large, and the average could reach 2.05×10 10 cm -3The number of bacteria on the immobilized material in the reaction tank and the measured Fe 2+ The oxidation capacity corresponds to that when the bacteria fixed on the material reaches saturation, the maximum Fe 2+ Oxidation capacity.

[0048] The optimized culture and immobilization conditions of A.ferrooxidans 14 were as follows: 30°C, initial pH 2.0, initial Fe 2+ The concentration was 15 g / L, soft ultrafine fibers were used as immobilization materials, the porosity of the reaction tank was 96.64%, and the ventilation volume was 180 L / h. (2) Using the optimized immobilization condition parameters, A. ferrooxidans 14 was immobilized by semi-continuous culture and continuous culture to form a biofilm. The maximum number of bacteria immobilized per unit volume of material could reach 2.05 × 10 10 cm -3 The fixed reactor can reach a higher Fe content of 18.22 g / h 2+ Oxidation capacity and 5.78 g / L / h of Fe 2+ Oxidation rate. This oxidation rate is at a relatively high level reported in China.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for immobilizing Acidithiobacillus ferrooxidans, characterized in that: In the presence of Fe 2+ In a liquid culture medium, using ultrafine fibers as immobilization materials, Acidithiobacillus ferrooxidans was first cultured semi-continuously and then continuously. During the semi-continuous culture process, when Fe 2+ When the oxidation reaches 95% or more, replace 60-80% of fresh Fe 2+ During the continuous culture, fresh liquid medium containing Fe 2+ of liquid culture medium, while continuously discharging the liquid culture medium, in Fe 2+ Under the premise that the conversion rate is not less than 95%, the flow rate of the liquid culture medium cannot be increased any further, indicating that the immobilization is completed.

2. The immobilization method of Acidithiobacillus ferrooxidans according to claim 1, characterized in that: The acidithiobacillus ferrooxidans is A.ferrooxidans 14 strain.

3. The immobilization method of Acidithiobacillus ferrooxidans according to claim 1, characterized in that: In semi-continuous culture, the liquid culture medium is replaced no less than 6 times.

4. The immobilization method of Acidithiobacillus ferrooxidans according to claim 1, characterized in that: During semi-continuous culture and continuous culture, the air intake rate is 120-225 L / h.

5. The immobilization method of Acidithiobacillus ferrooxidans according to claim 1, characterized in that: The liquid culture medium is a 9K liquid culture medium. In the 9K liquid culture medium, K2HPO4 0.4-0.6 g / L, (NH4)2SO4 2-4 g / L, KCl 0.05-0.15 g / L, MgSO4·7H2O 0.4-0.6 g / L, Ca(NO3)2 0.005~0.015g / L, pH is 1.5~2.

5.

6. The method for immobilizing Acidithiobacillus ferrooxidans according to claim 5, characterized in that: Contains Fe 2+ The preparation process of the liquid culture medium is to prepare a FeSO4 solution with a pH of 1.5 to 2.5, add it to the 9K liquid culture medium and mix it evenly; Or, containing Fe 2+ In the liquid culture medium, there is Fe 2+ The concentration is 14-16g / L.

7. The method for immobilizing Acidithiobacillus ferrooxidans according to claim 1, characterized in that: The temperature during semi-continuous and continuous culture is 25-35°C; Alternatively, the porosity of the reactor used in the semi-continuous culture or continuous culture is 96 to 97%.

8. A bacterial strain immobilization material, characterized in that: Obtained by the immobilization method according to any one of claims 1 to 7.

9. A bacterial strain immobilization device, characterized in that: It comprises an immobilization tank, and the immobilization tank is used to implement the immobilization method according to any one of claims 1 to 7.

10. A bacterial strain immobilization material according to claim 8 or a bacterial strain immobilization device according to claim 9 in the oxidation of Fe 2+ Application in.

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