Method for immobilizing thiobacillus ferrooxidans by using basalt fiber as carrier and basalt fiber carrier

Abstract

The invention relates to the crossing field of a microorganism utilization technology and an environmental protection technology, and provides a method for immobilizing thiobacillus ferrooxidans by using basalt fiber as a carrier and the basalt fiber carrier. The method comprises the following steps: 1, mixing a thiobacillus ferrooxidans active bacterium solution with a 9k liquid culture medium to prepare an immobilized culture solution; 2, carrying out acid etching modification on the basalt fiber carrier; and 3, filling the prepared immobilized culture solution and the basalt fiber carrier subjected to acid etching modification into an immobilization reactor, and circulating the immobilized culture solution to immobilize the thiobacillus ferrooxidans. The application limitation caused by slow growth of the thiobacillus ferrooxidans under conventional conditions is overcome, and the biomass and the biological oxidation efficiency of the thiobacillus ferrooxidans are greatly improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the cross field of microbial application technology and environmental protection technology, and specifically relates to a method for immobilizing Acidithiobacillus ferrooxidans by using basalt fiber as a carrier and a basalt fiber carrier with a symbiotic system of biofilm and secondary mineral on the surface obtained by using the method. BACKGROUND

[0002] Acidithiobacillus ferrooxidans (A. ferrooxidans for short) is an important microorganism widely used in the fields of mineral bioleaching and environmental remediation. Its main characteristics include being able to survive in extremely acidic environments (pH 1.5-3.0) and using ferrous iron and sulfide as energy sources to promote the dissolution and transformation of metal minerals. This strain has important application value in microbial leaching, acid mine drainage treatment, and secondary mineral recovery. However, in actual application processes, due to its slow growth in free state, susceptibility to environmental factors, difficulty in recovery and reuse, its promotion and efficient use in the industrial field are limited.

[0003] Microbial immobilization technology is a technology that immobilizes microorganisms on specific carriers, allowing them to survive stably in specific environments and continuously exert their functions. Immobilization not only improves the biological catalytic capacity of microorganisms, but also enhances their resistance to environmental changes, prolongs their service life, and facilitates the recovery and reuse of microorganisms. Currently, common immobilization carriers include natural mineral materials (such as zeolite, bentonite), synthetic polymers (such as sodium alginate, polyvinyl alcohol), and carbon-based materials (such as activated carbon, graphene). However, these carriers have problems such as insufficient mechanical strength, unsatisfactory biocompatibility, short service life, or high preparation cost, making it difficult to meet the needs of actual industrial applications.

[0004] Therefore, it is necessary to provide a method to improve the shortcomings of existing Acidithiobacillus ferrooxidans immobilization methods and promote the further development of microbial metallurgy and environmental remediation technology.

[0005] It should be noted that the information disclosed in the above background section is only used to enhance the understanding of the background of the present application, and therefore it may contain information that does not constitute prior art known to those skilled in the art. SUMMARY

[0006] In order to solve one or more of the above problems in the prior art, the present application aims to provide a method for immobilizing Acidithiobacillus ferrooxidans using basalt fiber as a carrier and a basalt fiber carrier.

[0007] According to an aspect of the present application, there is provided a method for immobilizing Thiobacillus ferroxidans using basalt fiber as a carrier, the method comprising the following steps: Step 1: mixing Thiobacillus ferroxidans active bacteria solution with 9k liquid medium to prepare an immobilized culture solution; Step 2: acid-etching and modifying the basalt fiber carrier with sulfuric acid; Step 3: loading the acid-etched and modified basalt fiber carrier and the prepared immobilized culture solution into an immobilized reactor, and circulating the immobilized culture solution to immobilize Thiobacillus ferroxidans. According to an embodiment of the present application, before the acid-etched and modified basalt fiber carrier is loaded into the immobilized reactor, the acid-etched and modified basalt fiber carrier is filled into a flow-off ball to prepare a plurality of unit carriers.

[0008] According to an embodiment of the present application, the plurality of unit carriers are arranged in a vertical direction in the immobilized reactor, and in the immobilized reactor, the immobilized culture solution flows from a lower end to an upper end and then flows into the reactor from the lower end of the reactor again, and the circulation is repeated.

[0009] According to an embodiment of the present application, the diameter of the basalt fiber is 17 μm to 18 μm, and in Step 2, the basalt fiber is braided into a pigtailing filler form.

[0010] According to an embodiment of the present application, the diameter of the flow-off ball is 50 mm to 60 mm, and the filling ratio of the basalt fiber in the flow-off ball is 50% to 70%.

[0011] According to an embodiment of the present application, the bacterial concentration of the Thiobacillus ferroxidans active bacteria solution is in the range of 6.8 × 10 7 CFU / mL to 8.2 × 10 8 CFU / mL. The Thiobacillus ferroxidans active bacteria solution can be a bacteria solution in the middle and later stages of the logarithmic growth phase obtained by shake flask culture.

[0012] According to an embodiment of the present application, the mixing ratio of the Thiobacillus ferroxidans active bacteria solution to the 9k liquid medium is 0.5:1 to 1.5:1.

[0013] According to an embodiment of the present application, in Step 4, the aeration amount of the immobilized reactor is 0.7 L / min to 0.9 L / min, the heating temperature is 28°C to 32°C, and the flow rate of the immobilized culture solution is 0.5 L / h to 1.0 L / h.

[0014] According to an embodiment of the present application, when the oxidation rate of the Thiobacillus ferroxidans to ferrous iron in the culture solution reaches 90% to 100%, the immobilized culture solution is replaced.

[0015] The basalt fiber carrier is modified by acid etching using 1:1 sulfuric acid, the 1:1 sulfuric acid refers to the ratio of sulfuric acid and distilled water being 1:1, the 1:1 sulfuric acid can be prepared by slowly adding 500 mL of concentrated sulfuric acid into 500 mL of distilled water under an ice water bath, and the acid etching time using the 1:1 sulfuric acid is 24 h-48 h.

[0016] According to the embodiment of the present application, the components of the 9k liquid medium include (NH4)2SO4, K2HPO4, KCl, MgSO4·7H2O, Ca(NO3)2 and FeSO4·7H2O, wherein the concentration of (NH4)2SO4 is 3.0 g / L, the concentration of K2HPO4 is 0.5 g / L, the concentration of KCl is 0.1 g / L, the concentration of MgSO4·7H2O is 0.5 g / L, the concentration of Ca(NO3)2 is 0.01 g / L, and the concentration of FeSO4·7H2O is 44.2 g / L.

[0017] According to the embodiment of the present application, the pH range of the active bacteria solution of the Thiobacillus ferroxidans is 1.7-2.0, the pH range of the 9k liquid medium is 1.9-2.1, the pH of the immobilized culture solution is 1.8-2.0, and the initial pH of the 9k liquid medium is adjusted using 1:1 sulfuric acid.

[0018] According to the embodiment of the present application, the bacteria of the immobilized culture solution replaced for the first four to six batches of the immobilized reactor can be collected by centrifugation, and the collected bacteria are re-added into the immobilized reactor for rapid accumulation of the early biomass.

[0019] According to the embodiment of the present application, the bacteria of the immobilized culture solution replaced for the first four to six batches can be collected by the following steps: removing the pyrite type precipitate in the culture solution to obtain supernatant by centrifugation at 4℃, 2000 r / min for 5 min, and then centrifuging the supernatant at 4℃, 10000 r / min for 8 min to obtain bacteria precipitate.

[0020] According to another aspect of the present application, a basalt fiber carrier is provided, the surface of the basalt fiber carrier has a biofilm and a secondary mineral symbiotic system, and is obtained by the method as described above.

[0021] Beneficial effects:

[0022] The method of the present application can be widely applied to the treatment of acid mine drainage and acid industrial wastewater and the recycling of secondary minerals, can shorten the growth time of Thiobacillus ferroxidans, improve the oxidation leaching performance of the bacteria, improve the treatment efficiency of the wastewater, and reduce the limitation of the application scene of the secondary minerals on the Thiobacillus ferroxidans through the attachment recovery. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this specification or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the embodiments of this specification. For those skilled in the art, other drawings can be obtained based on these drawings.

[0024] Figure 1 This is a schematic diagram of the reactor body and related components used in the implementation of this invention;

[0025] Figure 2 Line graph showing the change of ferrous concentration over time in the immobilization culture medium of *Thiobacillus ferrooxidans* in the middle and late batches (batch 8) of Group A (Examples 4, 5, Comparative Examples 2 and 4) and Group G (Comparative Examples 1 and 2).

[0026] Figure 3 The adsorption amount of secondary minerals on the carrier after immobilization is shown for Examples 4 and 5, Comparative Examples 2 and 4 in Group A, and Comparative Examples 1 and 2 in Group G. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. It should be noted that, unless otherwise specified, the implementation methods and features in the implementation methods of the present invention can be combined, separated, interchanged, and / or rearranged. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Also, the terminology used in the present description is for the purpose of describing particular embodiments and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "a" or "an," or any variation thereof, are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising" as an assisting legal principle applied to this patent application. It is also noted that the terms "substantially," "approximately," and other similar terms, as used herein, are used as terms of approximation and not as terms of degree, unless otherwise indicated, so that, as would be recognized by those of ordinary skill in the art, such terms encompass amounts that would be deemed reasonably close to a stated value by those of ordinary skill in the art performing similar measurements or calculations.

[0029] In the field of microbial utilization of Acidithiobacillus ferrooxidans, its biological oxidation performance is a key factor affecting the treatment effect. With the development of environmental remediation and resource utilization technology, biological oxidation method has attracted widespread attention due to its green and efficient characteristics. Acidithiobacillus ferrooxidans can catalyze the oxidation reaction of ferrous ions and sulfide under acidic conditions to generate secondary minerals such as jarosite and shiveite. These minerals not only have high added value, but also can be used as catalysts or adsorbents in subsequent processes.

[0030] In traditional processes, the bacterial cells are often in a free state, which is easily affected by environmental factors, resulting in reduced activity and loss of bacterial cells. At the same time, traditional carriers have deficiencies in corrosion resistance, mechanical strength, and biocompatibility for the culture environment of Acidithiobacillus ferrooxidans. In addition, the overall reaction efficiency may be affected by the shedding of bacterial cells or the blocking of carriers during the operation of the immobilized reactor.

[0031] Basalt fiber (BF) is a new type of inorganic non-metallic material made of natural basalt by high-temperature melting and drawing. It has excellent mechanical properties, chemical corrosion resistance, and good biocompatibility. Its surface can be modified to improve the adhesion of microorganisms, making it an ideal immobilized carrier for microorganisms. In addition, the high specific surface area of basalt fiber carrier helps to increase the loading capacity of microorganisms and provides a stable growth environment. During the process of biomineralization, the oxidation of ferrous iron and sulfide by microorganisms can promote the formation of secondary minerals such as jarosite and shiveite. If Acidithiobacillus ferrooxidans can be effectively immobilized and the deposition and recovery of secondary minerals can be controlled, it will provide a new technical approach for environmental pollution control and mineral resource recovery. Therefore, the present application selects basalt fiber with excellent corrosion resistance, high mechanical strength, and biological inertness as an immobilized carrier, and provides a method for immobilizing Acidithiobacillus ferrooxidans.

[0032] According to an embodiment of the present application, the method for immobilizing Thiobacillus ferroxidans by using basalt fiber as a carrier comprises the following steps (the operation sequence of steps one and two is not distinguished in advance): Step one: mixing the active bacteria solution of Thiobacillus ferroxidans with 9k liquid medium to prepare an immobilized culture solution; Step two: using sulfuric acid to acid-etch modify the basalt fiber carrier; Step three: loading the acid-etch modified basalt fiber carrier and the prepared immobilized culture solution into an immobilized reactor, and circulating the immobilized culture solution to immobilize Thiobacillus ferroxidans. By using the acid-etch modified basalt fiber carrier to immobilize Thiobacillus ferroxidans, a biofilm of Thiobacillus ferroxidans can be formed, thereby improving the biomass and bio-oxidation efficiency of Thiobacillus ferroxidans. In the above process, the biofilm-secondary mineral symbiotic system formed by the bacteria on the surface of the basalt fiber not only effectively enriches the bacteria, but also makes the reactants fully contact with the microorganisms, thereby greatly improving the oxidation reaction rate and the attachment efficiency of the secondary minerals on the carrier. Through periodic or continuous operation, the generated secondary minerals can be adsorbed and separated in the reactor, realizing the resource utilization and environment-friendly treatment process.

[0033] The acid-etch modification of the basalt fiber carrier can use 1:1 sulfuric acid, wherein the 1:1 sulfuric acid refers to the ratio of sulfuric acid to distilled water being 1:1, and the 1:1 sulfuric acid can be prepared by slowly adding 500 mL of concentrated sulfuric acid into 500 mL of distilled water under an ice water bath.

[0034] In addition, according to an embodiment of the present application, the components of the 9k liquid medium include (NH4)2SO4, K2HPO4, KCl, MgSO4·7H2O, Ca(NO3)2 and FeSO4·7H2O. Among them, the concentration of (NH4)2SO4 is 3.0 g / L, the concentration of K2HPO4 is 0.5 g / L, the concentration of KCl is 0.1 g / L, the concentration of MgSO4·7H2O is 0.5 g / L, the concentration of Ca(NO3)2 is 0.01 g / L, and the concentration of FeSO4·7H2O is 44.2 g / L. The preparation of the 9k medium can include the following steps: adding 3.0 g of (NH4)2SO4, 0.5 g of K2HPO4, 0.1 g of KCl, 0.5 g of MgSO4·7H2O and 0.01 g of Ca(NO3)2 into 600 mL of distilled water to prepare a 9k basic salt medium, adjusting the pH with 1:1 sulfuric acid and then placing it in a high-pressure sterilization pot for sterilization at 121°C for 20 min; dissolving 44.2 g of FeSO4·7H2O in 400 mL of distilled water, filtering and sterilizing through a 0.22 μm filter membrane. Mixing the 9k basic salt medium and the FeSO4·7H2O solution to obtain the 9k liquid medium.

[0035] According to embodiments of the present invention, before loading the acid-etched modified basalt fiber carrier into the immobilization reactor, the acid-etched modified basalt fiber carrier can be filled into a free-floating sphere to prepare multiple unit carriers. Here, a unit carrier includes a free-floating sphere and the basalt fiber carrier filled therein. By filling the acid-etched modified basalt fiber carrier into the free-floating sphere, it is convenient to load the basalt fiber carrier into the immobilization reactor and it is beneficial to improve the formation of the biofilm of *Thiobacillus ferrooxidans*. In addition, it is convenient to recover the basalt fiber carrier with the biofilm and secondary mineral symbiotic system on its surface after the reaction is completed.

[0036] The acid etching modification of basalt fiber carriers specifically includes the following steps: after cleaning and drying the basalt fibers, an acid etching modification process is performed to enhance their surface activity and hydrophilicity, giving them good bacterial adhesion performance.

[0037] Figure 1 An immobilization reactor for immobilized *Thiobacillus ferrooxidans* according to an embodiment of the present invention is shown. Figure 1 As shown, the immobilized reactor includes: a reactor body 9, a free-floating ball 1 (i.e., a unit carrier) filled with basalt fiber carrier, and an immobilized culture medium; a flow-adjustable peristaltic pump 5 for driving the flow of the immobilized culture medium; and a culture medium storage tank 6, located outside the reactor body 9 and connected to the upper outlet and lower inlet of the reactor body 9 via pipelines. Furthermore, according to an embodiment of the invention, the immobilized reactor may also include: an overflow weir 7 for solid-liquid separation of the culture medium before it flows out of the reactor 9, preventing potentially detached secondary minerals from clogging the culture medium circulation channel; an air pump 2 that provides aeration to the reactor body 9 via a gas flow meter 3 into an aeration stone 4; and a temperature-adjustable heating device 8 surrounding the reactor body 9 for temperature control of the reactor.

[0038] According to an embodiment of the present invention, multiple unit carriers 1 can be arranged vertically in sequence in the immobilized reactor (i.e., in the reactor body 9 of the immobilized reactor). In the immobilized reactor, the culture medium enters from the bottom and flows upwards, then flows back into the reactor body 9 from the bottom, repeating this cycle. This upward circulating flow improves mass transfer efficiency, optimizes microbial distribution, and prevents secondary mineral blockage of the reactor. Furthermore, the upward circulating flow reduces fluctuations in the culture medium, minimizing its impact on the attachment of *Thiobacillus ferrooxidans* and biofilm formation.

[0039] According to the embodiment of the present application, the diameter of the basalt fiber can be 17-18 μm, and the basalt fiber can be braided into a braided filler form before being filled into the flow-off ball, and the time for acid etching using 1:1 sulfuric acid can be 24-48 h. The fine diameter of the basalt fiber is such that the braided filler formed therefrom can provide high specific surface area of the attachment sites for the Thiobacillus ferroxidans, while resisting the acidic environment in which the Thiobacillus ferroxidans grows and research has shown that the braided filler has an advantage of promoting the formation of a biofilm. The surface of the basalt fiber carrier can be modified by acid etching, further improving the bioaffinity thereof.

[0040] According to the embodiment of the present application, the diameter of the flow-off ball is 50-60 mm, and the filling ratio of the basalt fiber in the flow-off ball is 50-70%, i.e., the volume of the basalt fiber accounts for 50-70% of the total volume inside the flow-off ball. The flow-off ball used in the present application is usually made of high-density polyethylene (HDPE), polypropylene (PP) or modified plastic, and has the characteristics of corrosion resistance, impact resistance and long service life.

[0041] The flow-off ball is provided with a porous structure, so that the adhesion capacity of the microorganism can be enhanced by affecting the flow of the culture solution. The filling ratio of the basalt fiber in the flow-off ball is set to 50-70%, so that sufficient adhesion sites of the microorganism can be provided while ensuring uniform dispersion of the culture solution, reducing the position difference caused by uneven flow rate of the culture solution.

[0042] According to the embodiment of the present application, the bacterial concentration of the active bacteria solution of the Thiobacillus ferroxidans ranges from 6.8 x 10 7 CFU / mL to 8.2 x 10 8 CFU / mL. The active bacteria solution of the Thiobacillus ferroxidans can be a bacteria solution in the middle and late logarithmic growth phase of the shake flask culture, at which time the energy substances in the bacteria solution are almost consumed and the bacterial metabolism is at the peak, and each metabolic product is at the peak. The bacteria solution in this stage can quickly enter the state of forming a biofilm in the immobilized reactor.

[0043] According to the embodiment of the present application, the mixing ratio of the active bacteria solution of the Thiobacillus ferroxidans to the 9k liquid medium is 0.5:1-1.5:1. When the ratio of the two is 0.5:1-1.5:1, the bacteria can be provided with sufficient energy substances while maintaining a certain level of abundance, so that the immobilized culture solution can play a role in a short time after being put into the immobilized reactor.

[0044] According to the embodiment of the present application, the aeration amount of the immobilized reactor is 0.7 L / min to 0.9 L / min, the heating temperature is 28℃ to 32℃, and the flow rate of the immobilized culture solution is 0.5 L / h to 1.0 L / h. That is, the aeration amount of the aeration stone 4 is set to 0.7 L / min to 0.9 L / min, the heating temperature of the heating device is set to 28℃ to 32℃, and the flow rate of the flow-adjustable peristaltic pump 5 is set to 0.5 L / h to 1.0 L / h. The above conditions are obtained by orthogonal response surface experiments considering the suitable conditions of the Acidithiobacillus ferrooxidans and are within the optimal condition range of the immobilized reactor. By suitable setting, the conditions conducive to the formation of biofilm are provided for the Acidithiobacillus ferrooxidans. The regulation range of the aeration amount and the flow rate of the culture solution makes the physical conditions in the immobilized reactor fluctuate, which is conducive to the attachment of microorganisms and the formation of biofilm.

[0045] According to the embodiment of the present application, when the oxidation rate of ferrous iron in the culture solution by the Acidithiobacillus ferrooxidans reaches 90% to 100%, the immobilized culture solution is replaced. Ferrous iron in the culture solution is an energy source for the Acidithiobacillus ferrooxidans, and after most of it is consumed, the growth and metabolism of the bacteria will begin to weaken, and the secretion of extracellular polymers and other secretory products related to biofilm will also stop. Therefore, when the oxidation rate of ferrous iron reaches 90% to 100%, the culture solution is replaced in time to obtain an environment conducive to the immobilization of the Acidithiobacillus ferrooxidans in the reactor, which helps to maintain a high-activity bacterial population in the immobilized reactor for the construction of biofilm.

[0046] In addition, the bacteria in the immobilized culture solution replaced in the first four to six batches of the immobilized reactor can be collected by centrifugation, and the collected bacteria can be readded to the immobilized reactor for rapid accumulation of biomass in the early stage. In addition, the bacteria in the immobilized culture solution replaced in the first four to six batches can be collected by the following steps: removing the jarosite-type precipitate in the culture solution by centrifugation at 4℃ and 2000 r / min for 5 min to obtain a supernatant, and then centrifuging the supernatant at 4℃ and 10000 r / min for 8 min to obtain a bacteria precipitate.

[0047] According to another embodiment of the present application, a basalt fiber carrier is provided, which is obtained by the above method and has a biofilm and secondary mineral symbiotic system on the surface. The basalt fiber bio-carrier obtained by the present application combines the growth characteristics of Acidithiobacillus ferrooxidans. Since Acidithiobacillus ferrooxidans produces jarosite-type secondary minerals during growth, by selecting basalt fibers and designing the immobilized reactor, a basalt fiber carrier with a biofilm and secondary mineral symbiotic system can be obtained. The carrier has the characteristics of acid corrosion resistance and high mechanical strength, making its application scenarios more diverse.

[0048] In summary, the application uses basalt fiber as a carrier, through pretreatment of the carrier and combination of the carrier with the fluidized ball, and simultaneously optimizes the design of the immobilized reactor culture conditions, improves the immobilization efficiency of the Thiobacillus ferroxidans, and ensures sufficient contact between the reactants and the biofilm, thereby improving the adsorption rate of the secondary minerals. The method not only effectively reduces the risk of loss of bacterial bodies and reactor blockage, but also realizes a continuous and stable biological oxidation process, has high economic benefits and environmental benefits. The adsorbed secondary minerals have high added value and can play a catalytic, adsorbing and environmental remediation role in subsequent processes, thereby providing a solid theoretical basis and technical support for practical application.

[0049] The application will be further described in detail below in combination with specific examples, but the application is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not mentioned are conventional conditions in the industry.

[0050] I. Experimental steps

[0051] The Thiobacillus ferroxidans strain used in the application is selected from activated sludge of a sewage treatment plant in Qingdao City by the laboratory. The basalt fiber is purchased from Jiangsu Nongchaoer Composite Material Co., Ltd.

[0052] The inventors have completed the following seven groups of experiments, wherein the examples and comparative examples in the seven groups of experiments include the following implementation steps (the operation sequence of step one and step two is not distinguished first or second):

[0053] Step one: adding the Thiobacillus ferroxidans active bacteria liquid to the freshly prepared 9k culture medium;

[0054] Step two: soaking the basalt fiber carrier prepared into a plaited material in 1:1 sulfuric acid for etching;

[0055] Step three: adding the fluidized ball loaded with the basalt carrier to the immobilized reactor, and starting the immobilized reactor.

[0056] Each time the ferrous oxidation rate reaches 95% is a batch, and the immobilization process is set to 10 batches. After immobilization, the basalt fiber carrier with a symbiotic system of biofilm and secondary minerals on the surface is obtained.

[0057] In addition, in the following seven groups of experiments, the 48h ferrous oxidation rate in the culture liquid in the late batch (8th batch) of immobilization of each group is obtained by changing the concentration of the bacteria liquid in the immobilized culture liquid, the composition ratio of the culture liquid, the filling ratio of the basalt fiber carrier, the aeration flow, the heating temperature, and the flow of the immobilized culture liquid in the reactor body.

[0058] Note that in the following experiments, only part of the experimental data is listed in each table as an example. For example, in Table 1, the broth concentration of the active Thiobacillus ferroxidans broth is listed as a variable for Examples 1-9 and Comparative Examples 1-4, while the broth and culture medium composition ratio, the basalt fiber carrier packing ratio, the aeration flow rate, the heating temperature, and the culture medium flow rate are only selected as values near the middle of the corresponding ranges as examples.

[0059] However, in fact, the inventors have also selected various broth and culture medium composition ratios (i.e., any value within the range of 0.5:1 to 1.5:1), various basalt fiber carrier packing ratios (any value within the range of 50% to 70%), various aeration flow rates (any value within the range of 0.7 L / min to 0.9 L / min), various heating temperatures (any value within the range of 28°C to 32°C), and various culture medium flow rates (any value within the range of 0.5 L / h to 1.0 L / h) as fixed amounts, and only the broth concentration as a variable for experiments. The results of these experimental data are similar to those listed in Table 1, and thus, since the experimental data and results of Table 1 clearly express the effect of the broth concentration change on the experimental results, only these values are selected as examples in Table 1. However, those skilled in the art will understand that other experimental results within the above ranges also conform to the change trend of the experimental results of the data in Table 1, i.e., the description of the experimental results of Table 1 also applies to other data within the above ranges.

[0060] Tables 2 to 6 are similar to Table 1, and thus a detailed description thereof is omitted.

[0061] 1. Broth concentration difference group (Group A)

[0062] Table 1

[0063]

[0064]

[0065] 2. Culture medium composition ratio difference group (Group B) Table 2

[0066]

[0067]

[0068] 3. Basalt fiber carrier packing ratio difference group (Group C) Table 3

[0069]

[0070]

[0071] 4. Aeration flow rate difference group (Group D) Table 4

[0072]

[0073]

[0074] 5. Temperature difference group (Group E) Table 5

[0075]

[0076] 6. Culture solution flow rate difference group (Group F)

[0077] Table 6

[0078]

[0079] 7. Other comparative example group (Group G)

[0080] Comparative Example 1: Iron-oxidizing Thiobacillus was immobilized using zeolite instead of basalt fiber as the carrier, and other conditions were the same as Example 5 of the first group of experiments.

[0081] Comparative Example 2: The flow-off balls filled with basalt fiber carriers were placed in a large conical flask to simulate shake flask culture, and the conditions before being placed in the large conical flask were the same as Example 5 of the first group of experiments.

[0082] As can be seen from the examples and comparative examples in Table 1, when the concentration of the bacterial solution was 6.8 x 10 7 CFU / mL to 8.2 x 10 8 CFU / mL (Group A Examples 1-9), the ferrous content rapidly decreased with the culture solution circulation time. As the concentration of the bacterial solution was gradually increased from 6.8 x 10 7 CFU / mL to 3.0 x 10 8 CFU / mL, the 48-hour ferrous oxidation rate steadily increased to 95.61%, which was the highest value. Subsequently, when the concentration of the bacterial solution was increased to 8.2 x 10 8 CFU / mL, the oxidation rate remained at a relatively high level of 91.85%. However, when the concentration of the bacterial solution was further increased, the oxidation rate began to decrease. This indicates that there is an optimal range for the concentration of the bacterial solution, and too high a concentration can lead to a decrease in metabolic efficiency due to increased oxygen transport resistance, competition among bacteria, and group sensing regulation, etc.

[0083] From the examples and comparative examples of Table 2, it can be seen that in the group B of the ratio difference of the culture solution, the ratio of the bacterial solution to the culture solution is changed from 0.3:1 to 1.8:1. The experiment shows that when the ratio is 0.5:1 or 1.5:1, a higher oxidation efficiency can be maintained, and the ferrous oxidation rate is higher than 89% in 48 hours. The oxidation rate is reduced when the ratio is too high or too low, especially when the ratio is 0.3:1, the oxidation rate is only 69.72%, which is significantly lower than that of other groups, indicating that a low ratio of bacterial solution is not conducive to the full fixation of bacterial cells, and a high ratio may affect the balance of the components of the culture solution.

[0084] In the group C of the filling ratio difference of the basalt fiber carrier, the filling ratio is set to 40% to 80%. The results show that when the filling ratio is 50% to 70%, the oxidation rate is above 88%. Among them, when the filling ratio is 60%, the oxidation rate is the highest (95.61%). A filling ratio lower than 50% results in insufficient effective attachment area, and a ratio higher than 50% causes a decrease in liquid flowability and mass transfer resistance in the reactor, which further reduces the oxidation effect.

[0085] In the group D of the difference in aeration flow, the oxygen supply is most coordinated with bacterial metabolism by controlling the air flow to be 0.5 L / min to 0.9 L / min, and the oxidation rate is as high as 91.82% to 93.61%. Too low or too high air flow will cause a decrease in efficiency.

[0086] In the group E of the difference in heating temperature, the temperature interval is set to 25°C to 35°C. The results show that when the temperature is 28°C to 32°C, the metabolic activity and enzyme activity of the bacterial cells are relatively strong, and the oxidation rate is between 90.95% and 95.61%. When the temperature is lower than 28°C or higher than 32°C, the physiological state of the bacterial cells is disordered or the protein structure is unstable, indicating that 28°C to 32°C is the ideal temperature control interval.

[0087] In the group F of the difference in the flow of the culture solution, the liquid flow rate is set to 0.5 to 1.0 L / h, the bacterial cells can be stably adsorbed, the system residence time is moderate, and the oxidation rate is maintained at 88.68% to 95.61%. Higher than 1.0 L / h will increase the shear force and reduce the fixation time, at which point the oxidation rate of ferrous iron depends on the number of cycles. Lower than 0.5 L / h will result in insufficient liquid supply and slow reaction, leading to excessive mineral deposition and breaking the balance between biofilm formation and mineral adsorption.

[0088] Figure 2 The figures of the change of the ferrous content with time in the culture solution in the late batch (8th batch) of the immobilization of the group A of example 4, example 5, comparative example 2 and comparative example 4, and comparative example 1 and comparative example 2 in the group G are given, and the concentration of the bacterial solution directly affects the speed of biofilm formation and the effect of immobilization. The level of decrease of the ferrous concentration can reflect the effect of immobilization on the one hand, and also represents the biological oxidation ability of the bacteria on the other hand.

[0089] As can be seen from Figure 2 , compared with the zeolite carrier and the shake flask culture system, the ferrous iron oxidation rate and oxidation capacity of the optimal embodiment show obvious advantages in the circulating immobilized culture liquid immobilization system, and the ferrous iron oxidation ratio can reach 90%-98.9%. Meanwhile, it can be obtained from group A embodiment 1 to embodiment 9 that the immobilization effect of the strain is directly affected by the strain concentration of the immobilized culture liquid. In the circulating immobilized culture liquid system with basalt fiber as the carrier, the ferrous iron content can be reduced to 95% within 36h, which indicates that the biomass in the immobilization system is improved and the biological oxidation capacity of the strain is increased. Compared with group G comparative example 1 and comparative example 2, the immobilized Leptospirillum ferrooxidans effect is better in the circulating immobilized culture liquid system with basalt fiber as the carrier.

[0090] Group A embodiment 4, embodiment 5, comparative example 2 and comparative example 4 and group G comparative example 1 and comparative example 2 are compared in terms of ferrous iron concentration changes, and the experimental results are directly presented in the form of a line graph, so that the dynamic changes of key indicators under different conditions can be more clearly compared. The difference group of culture liquid composition ratio, the difference group of filling ratio of basalt fiber carrier, the difference group of aeration flow, the difference group of temperature and the difference group of culture liquid flow are described in detail in words, and the influence of each parameter on the immobilization of Leptospirillum ferrooxidans and the biological oxidation capacity is displayed. The parameter settings of the immobilized reactor directly affect the biological membrane formation effect and the biological oxidation capacity.

[0091] Figure 3 The amount of secondary minerals adsorbed by the carrier after immobilization in group A embodiment 4, embodiment 5, comparative example 2 and comparative example 4 and group G comparative example 1 and comparative example 2 is given. As can be seen from Figure 3 , the adsorption amount of secondary minerals in the circulating immobilized culture liquid system with basalt fiber as the carrier is obviously better than that of the zeolite carrier and the non-circulating immobilized culture liquid system, which indicates that the selection of basalt fiber as the carrier and the regulation of the immobilization system have obvious advantages in the immobilization of Leptospirillum ferrooxidans and the adsorption amount of secondary minerals in the process.

[0092] In conclusion, the optimal effect can be obtained when the immobilization conditions are controlled as follows: the strain liquid concentration of the Leptospirillum ferrooxidans active bacteria liquid is 6.8x10 7 CFU / mL-8.2x10 8 CFU / mL, the composition ratio of the bacteria liquid and the culture liquid is 1:1, the filling ratio of the basalt fiber carrier is 50%-70%, the aeration amount of the reactor is 0.7L / min-0.9L / min, the heating temperature is 28℃-32℃, and the flow rate of the immobilized culture liquid is 0.5L / h-1.0L / h.

[0093] The method for fixing Thiobacillus ferroxidans by using basalt fiber as a carrier according to the application can provide good fixing effect, and the ferrous ion oxidation efficiency of the Thiobacillus ferroxidans is increased by 30%-50% compared with ordinary inoculation. The treated basalt fiber is a new type of inorganic non-metallic material, has excellent mechanical properties, chemical corrosion resistance and good biocompatibility, and can be used as a carrier with excellent physical and chemical properties under the application conditions of the Thiobacillus ferroxidans. Due to the particularity of the basalt fiber carrier, the biomass and biological oxidation performance of the Thiobacillus ferroxidans are improved through the circulation of the fixed culture solution system, and the secondary minerals formed in the adsorption process are also adsorbed, further stabilizing the fixing effect of the basalt fiber carrier. In addition, the basalt fiber carrier provided by the application can be reused after simple treatment after use or effect reduction, and in this process, the resource recycling of the adsorbed secondary minerals on the carrier can be realized by combining other technologies.

[0094] The method for fixing Thiobacillus ferroxidans by using basalt fiber as a carrier according to the application can provide good fixing effect, and the ferrous ion oxidation efficiency of the Thiobacillus ferroxidans is increased by 30%-50% compared with ordinary inoculation. The treated basalt fiber is a new type of inorganic non-metallic material, has excellent mechanical properties, chemical corrosion resistance and good biocompatibility, and can be used as a carrier with excellent physical and chemical properties under the application conditions of the Thiobacillus ferroxidans. Due to the particularity of the basalt fiber carrier, the biomass and biological oxidation performance of the Thiobacillus ferroxidans are improved through the circulation of the fixed culture solution system, and the secondary minerals formed in the adsorption process are also adsorbed, further stabilizing the fixing effect of the basalt fiber carrier. In addition, the basalt fiber carrier provided by the application can be reused after simple treatment after use or effect reduction, and in this process, the resource recycling of the adsorbed secondary minerals on the carrier can be realized by combining other technologies.

[0095] The above specific embodiments further illustrate the purpose, technical solutions and advantages of the application, and it should be understood that the above description is only a specific embodiment of the application and is not used to limit the protection scope of the application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the application should be included in the protection scope of the application.

Claims

1. A method for immobilizing *Thiobacillus ferrooxidans* using basalt fiber as a carrier, characterized in that, The method includes preparing an acid-etched modified basalt fiber carrier and an immobilization culture medium, loading the acid-etched modified basalt fiber carrier and the immobilization culture medium into an immobilization reactor, and then circulating the immobilization culture medium to immobilize *Thiobacillus ferrooxidans*. The immobilized culture medium is prepared by mixing active bacterial solution of *Thiobacillus ferrooxidans* with 9K liquid culture medium, and the acid-etched modified basalt fiber carrier is obtained by acid etching modification of the basalt fiber carrier with sulfuric acid.

2. The method according to claim 1, characterized in that, Before the acid-etched modified basalt fiber carrier is loaded into the immobilization reactor, the acid-etched modified basalt fiber carrier is filled into free-floating spheres to prepare multiple unit carriers.

3. The method according to claim 2, characterized in that, The multiple unit carriers are arranged vertically in sequence in the immobilization reactor. In the immobilization reactor, the immobilized culture medium enters from the bottom and flows from bottom to top, and then flows back into the reactor from the bottom again outside the reactor, thus circulating in this way.

4. The method according to claim 1, characterized in that, The basalt fibers have a diameter of 17μm to 18μm, and in step two, the basalt fibers are braided into a braided filler shape.

5. The method according to claim 2, characterized in that, The diameter of the free-floating sphere is 50mm to 60mm, and the filling ratio of the basalt fiber carrier in the free-floating sphere is 50% to 70%.

6. The method according to claim 1, characterized in that, The bacterial concentration range of the *Acidithiobacillus ferrooxidans* active bacterial solution is 6.8 × 10⁻⁶. 7 CFU / mL ~8.2×10 8 CFU / mL.

7. The method according to claim 6, characterized in that, The mixing ratio of the active bacterial solution of *Thiobacillus ferrooxidans* to the 9k liquid culture medium is 0.5:1 to 1.5:

1.

8. The method according to any one of claims 1-7, characterized in that, In the immobilization step of *Thiobacillus ferrooxidans*, the aeration rate of the immobilization reactor is 0.7 L / min to 0.9 L / min, the heating temperature is 28℃ to 32℃, and the flow rate of the immobilization culture medium is 0.5 L / h to 1.0 L / h.

9. The method according to claim 1, characterized in that, When the oxidation rate of ferrous iron in the culture medium by *Thiobacillus ferrooxidans* reaches 90%–100%, the immobilized culture medium is replaced, and / or the bacteria in the first 4–6 batches of the replaced immobilized culture medium in the immobilized reactor are collected by centrifugation, and the collected bacteria are added back into the immobilized reactor.

10. The method according to claim 1, characterized in that, The 9k liquid culture medium comprises (NH4)2SO4, K2HPO4, KCl, MgSO4·7H2O, Ca(NO3)2, and FeSO4·7H2O, wherein the concentration of (NH4)2SO4 is 3.0 g / L, the concentration of K2HPO4 is 0.5 g / L, the concentration of KCl is 0.1 g / L, the concentration of MgSO4·7H2O is 0.5 g / L, the concentration of Ca(NO3)2 is 0.01 g / L, and the concentration of FeSO4·7H2O is 44.2 g / L.

11. A basalt fiber carrier, characterized in that, The surface of the basalt fiber carrier has a symbiotic system of biofilm and secondary minerals, and is obtained by the method described in any one of claims 1 to 10.

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