Acidithiobacillus ferrooxidans immobilized material and application thereof in cell immobilization and desulfurization

Abstract

The invention belongs to the technical field of cell immobilization, and particularly relates to an acidophilic thiobacillus ferrooxidans immobilization material and application thereof in cell immobilization and desulfurization. According to the acidithiobacillus ferrooxidans immobilized material, Fe3O4 nano-clusters are used as raw materials, a PDA shell layer with a mesoporous structure is formed on the surfaces of the Fe3O4 nano-clusters by means of self-polymerization reaction of dopamine, a nano composite material Fe3O4 (at) mPDA is constructed, meanwhile, biotinylated chitosan derivatives Bio-CS are synthesized by using biotin as raw materials, and the acidithiobacillus ferrooxidans immobilized material is prepared. And combining the Fe3O4 (at) mPDA with a biotinylated chitosan derivative Bio-CS by virtue of an emulsification method, thereby obtaining the Fe3O4 (at) mPDA. The acidithiobacillus ferrooxidans immobilized material provided by the invention can be used for immobilizing acidithiobacillus ferrooxidans. The material provided by the invention not only can effectively maintain the biological activity of the acidithiobacillus ferrooxidans BY3, but also has the advantages of mild reaction conditions, no secondary pollution and capability of maintaining the cyclic utilization of Fe < 3 + >, so that the method meets the manufacturing requirements of a green biotechnology.

Description

Technical Field

[0001] This invention belongs to the field of cell immobilization technology, specifically relating to the immobilization material of *Thiobacillus acidophilus* and its application in cell immobilization and desulfurization. Background Technology

[0002] Acidithiobacillus ferrooxidan (A. ferrooxidan), a highly oxidative autotrophic leaching microorganism widely found in acidic mine wastewater, has been isolated and discovered in wastewater, soil, sulfur springs, and other environments since its discovery in 1947. In 1958, Kennecott Corporation in the United States first successfully applied A. ferrooxidan to copper leaching. Subsequently, A. ferrooxidan became one of the most extensively studied microbial strains in microbial hydrometallurgy and coal desulfurization. A. ferrooxidan primarily leaches Fe... 2+ Oxidized to Fe 3+ To obtain the energy required for life processes, its iron oxidation system mainly includes outer membrane carrier proteins, copper-containing proteins, cytochrome a, cytochrome c, and iron oxidases. It can also oxidize elemental sulfur, thiosulfates, and sulfide minerals. Simultaneously, studies have confirmed that temperature, pH, inoculum size, and Fe... 2+ Concentration, growth factors, and organic matter are among the many factors that influence the physiological activity of A. ferrooxidan.

[0003] H2S gas is a flammable and highly toxic hazardous chemical, widely found in biogas, natural gas, oil and gas, and other gases. It is one of the major air pollutants, and if not treated promptly, it can seriously threaten human health. Inhalation of small amounts can cause coma and respiratory failure, while large amounts can lead to multiple organ failure and myocardial infarction. Furthermore, in related industrial production, H2S causes severe catalyst inactivation and corrosion of pipelines and equipment, increasing safety risks and operating costs. Traditional desulfurization technologies are complex, have low purification levels, high energy consumption, and are prone to secondary pollution. In contrast, biological desulfurization technology, designed using selected microbial strains, offers advantages such as high desulfurization efficiency and simple operation and maintenance, and has become one of the main research directions in desulfurization technology in recent years, with broad application prospects.

[0004] Currently, existing biological desulfurization technologies utilizing microbial strains fall into three main categories: biological desulfurization processes in series with hydrodesulfurization, biological desulfurization processes that replace hydrodesulfurization, and biological desulfurization processes for removing sulfur from high-sulfur cracking feedstocks. However, existing biological desulfurization technologies utilizing microbial strains generally suffer from limited desulfurization efficiency, high by-product levels, unstable genetic performance of the strains, insufficient iron recycling, and low efficiency of related bioreactors, severely hindering the development of biological desulfurization technology in industrial applications. Summary of the Invention

[0005] To address the problems of limited desulfurization efficiency, high by-product levels, insufficient iron recycling, and low efficiency of related bioreactors in existing technologies, this invention adopts the following technical solution to achieve the above objectives:

[0006] This invention provides an immobilized material for *Thiobacillus acidophilus*. Using Fe3O4 nanoclusters as raw material, a mesoporous PDA shell is formed on the surface of the Fe3O4 nanoclusters via a dopamine self-polymerization reaction, constructing a nanocomposite material Fe3O4@mPDA. Simultaneously, a biotinylated chitosan derivative, Bio-CS, is synthesized using biotin, N-hydroxysuccinimide, N,N-dicyclohexylcarboimide, and N,N-dimethylformamide as raw materials. Then, Fe3O4@mPDA is combined with the biotinylated chitosan derivative Bio-CS using an emulsification method to obtain the *Thiobacillus acidophilus* immobilized material.

[0007] Preferably, the preparation of the *Thiobacillus ferrooxidans* immobilized material includes the following steps:

[0008] Preparation of Fe3O4@mPDA: Fe3O4 nanoclusters were prepared by solvothermal method using ferric chloride hexahydrate as raw material; Fe3O4@PDA core-shell structure was prepared using Fe3O4 nanoclusters, tris(hydroxymethyl)aminomethane and dihydroxyphenylalanine as raw materials; Fe3O4@mPDA was prepared using Fe3O4@PDA core-shell structure, F127, 1,3,5-trimethylbenzene and tris(hydroxymethyl)aminomethane as raw materials.

[0009] Preparation of chitosan derivative Bio-CS: Biotin, N-hydroxysuccinimide, and N,N-dicyclohexylcarboimide were dissolved in N,N-dimethylformamide, heated and stirred for 18–22 h, followed by ice bath for 0.5–1.5 h, and filtered to obtain crude active biotin ester precipitate; the crude active biotin ester precipitate was purified and dried to obtain Bio-NHS; Bio-NHS was dissolved in DMF solution, and then added to citrate synthase solution, stirred and reacted for 22–26 h, impurities were removed by dialyzing, and then freeze-dried to obtain Bio-CS, which was then dried and stored.

[0010] Preparation of Fe3O4@mPDA-CS nanoparticles: Fe3O4@mPDA was dispersed in water, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added for activation for 2-6 h. Bio-CS was added and the mixture was stirred for 22-26 h. The precipitate was collected by centrifugation and then freeze-dried to obtain Fe3O4@mPDA-CS nanoparticles, which are immobilized materials of Acidithiobacillus ferrooxidans.

[0011] Preferably, the preparation method of Fe3O4 nanoclusters includes the following steps: weighing FeCl3·6H2O and dissolving it in ethylene glycol to obtain a mixed solution; weighing NaAc and Na3Cit·2H2O and adding them to the mixed solution, stirring at 35-39℃ for 28-32 min; after complete dissolution, transferring the reaction solution and reacting at 195-205℃ for 8-12 h; after the reaction is completed, washing out the black particles with anhydrous ethanol, and the obtained black particles are Fe3O4 nanoclusters.

[0012] Preferably, the preparation method of Fe3O4@PDA core-shell structure includes the following steps: Fe3O4 nanoclusters are added to a mixture of ethanol and water and stirred for 0.5 to 1.5 min; tris(hydroxymethyl)aminomethane is dissolved in water and added to the mixture and mixed well; dihydroxyphenylalanine is dissolved in water and added to the mixture and stirred at room temperature for 22 to 26 h; the above solution is magnetically separated, the supernatant is discarded, and the lower precipitate is collected to obtain Fe3O4@PDA core-shell structure.

[0013] Preferably, the preparation method of Fe3O4@mPDA includes the following steps: F127 and 1,3,5-trimethylbenzene are simultaneously added to a mixture of ethanol and water, stirred for 28-32 min, and then the Fe3O4@PDA core-shell structure is added and stirred to obtain a mixture of TMB and F127.

[0014] Dissolve tris(hydroxymethyl)aminomethane in water and add it to a mixture of TMB and F127, then mix well. Dissolve dihydroxyphenylalanine in water and add it to the above solution. Stir at room temperature for 22–26 h. After the reaction is complete, centrifuge, discard the supernatant, and disperse the lower layer particles in a mixture of acetone and ethanol. Centrifuge again, discard the supernatant, and reflux the lower layer particles in the same mixture for 1.5–2.5 h. Centrifuge again, discard the supernatant, and disperse the lower layer particles in distilled water. The lower layer particles after centrifugation are Fe3O4@mPDA.

[0015] Preferably, the volume ratio of acetone to ethanol in the mixed solution of acetone and ethanol is 1:1.5 to 2.

[0016] Preferably, the purification steps for the crude active biotin ester precipitation are as follows: after dissolving the crude active biotin ester precipitate in hot isopropanol, it is placed in an ice bath for 3-5 hours to crystallize. The crystals are collected and dissolved again with DMF, followed by the addition of diethyl ether. After ice bath for 1.0-2.0 hours, the precipitate is filtered, collected, and vacuum dried to obtain Bio-NHS.

[0017] The present invention also provides the application of the aforementioned *Thiobacillus ferrooxidans* immobilization material in cell immobilization.

[0018] Preferably, the *Acidithiobacillus ferrooxidans* immobilization material serves as a magnetic composite carrier, with carboxyl, amino, and hydroxyl active functional groups on its surface, which are used to bind with the surface groups of *Acidithiobacillus ferrooxidans* BY3, thereby obtaining immobilized cells of *Acidithiobacillus ferrooxidans* BY3.

[0019] The present invention also provides the application of the aforementioned *Acidithiobacillus ferrooxidans* immobilization material in the desulfurization treatment of industrial hydrogen sulfide waste gas. The *Acidithiobacillus ferrooxidans* immobilization material serves as a magnetic composite carrier, and its surface has active functional groups of carboxyl, amino, and hydroxyl groups, which are used to bind with the surface groups of *Acidithiobacillus ferrooxidans* BY3, thereby obtaining immobilized cells of *Acidithiobacillus ferrooxidans* BY3.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. This invention provides an immobilization material for *Thiobacillus ferrooxidans* and its application in cell immobilization and desulfurization. The material provided by this invention is a novel Fe3O4 magnetic nanocarrier. This carrier is constructed by forming a mesoporous PDA shell on the surface of Fe3O4 nanoclusters through a dopamine self-polymerization reaction, thus creating a nanocomposite material Fe3O4@mPDA. Simultaneously, a biotinylated chitosan derivative, Bio-CS, is chemically synthesized. Then, Fe3O4@mPDA and the biotinylated chitosan derivative Bio-CS are combined using an emulsification method to form Fe3O4@mPDA-CS nanoparticles. These Fe3O4@mPDA-CS nanoparticles are the *Thiobacillus ferrooxidans* immobilization material (Fe3O4 magnetic nanocarrier) prepared in this invention.

[0022] 2. The *Acidithiobacillus ferrooxidans* immobilization material (Fe3O4 magnetic nanocarrier) provided by this invention can be used for the immobilization of *Acidithiobacillus ferrooxidans*. In this invention, the nanoparticles are sterilized by high temperature and ultraviolet light and then filled into a reactor. 9K medium is added to the reactor, and *Acidithiobacillus ferrooxidans* BY3 bacterial solution is inoculated at a 15% inoculum. The mixture is cultured at 30°C. The surface groups of *Acidithiobacillus ferrooxidans* BY3 bind to the synthesized Fe3O4@mPDA-CS. After the reaction continues for 72 hours, the 9K medium is removed, yielding immobilized *Acidithiobacillus ferrooxidans* BY3 nanoparticles. The immobilized *Acidithiobacillus ferrooxidans* BY3 nanoparticles prepared by this invention can be used for the desulfurization treatment of industrial hydrogen sulfide (H2S) waste gas. The *Acidithiobacillus ferrooxidans* immobilization material (Fe3O4 magnetic nanocarrier) provided by this invention not only effectively maintains the biological activity of *Acidithiobacillus ferrooxidans* BY3, but also features mild reaction conditions, no secondary pollution, and the ability to maintain Fe3O4 activity. 3+ The method involves recycling, thus meeting the requirements of green biotechnology manufacturing. Attached Figure Description

[0023] Figure 1 The synthetic route for the Fe3O4@mPDA-CS nanoparticles prepared in this invention is shown.

[0024] Figure 2 The image shows the SEM characterization of Fe3O4 nanoclusters and Fe3O4@mPDA-CS nanoparticles in this invention, where a represents Fe3O4 nanoclusters and b represents Fe3O4@mPDA-CS nanoparticles.

[0025] Figure 3 This is a particle size analysis of Fe3O4 nanoclusters and Fe3O4@mPDA-CS nanoparticles in this invention, where a is Fe3O4 nanoclusters and b is Fe3O4@mPDA-CS nanoparticles.

[0026] Figure 4 The image shows the cell fixation effect of Fe3O4@mPDA-CS nanoparticles detected by laser confocal microscopy in this invention. In this case, A represents co-culturing *Acidithiobacillus ferrooxidans* BY3 with Fe3O4@mPDA-CS nanoparticles for 24 h; B represents co-culturing *Acidithiobacillus ferrooxidans* BY3 with Fe3O4@mPDA-CS nanoparticles for 48 h; and C represents co-culturing *Acidithiobacillus ferrooxidans* BY3 with Fe3O4@mPDA-CS nanoparticles for 72 h.

[0027] Figure 5 Fe for immobilized cells and free cells in this invention 2+ Oxidation rate analysis.

[0028] Figure 6 This is a diagram of the experimental apparatus for H2S removal and iron recycling in this invention.

[0029] Figure 7 The removal rate of H2S gas at different concentrations in this invention is shown. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0031] The materials and reagents used in the following examples are as follows:

[0032] (1) Main reagents

[0033] Ferric chloride hexahydrate (FeCl3·6H2O), dopamine hydrochloride (DOPA), Pranic F127 (F127), biotin: Shanghai Maclean Biochemical Technology Co., Ltd.; Sodium acetate (NaAc), 1,3,5-trimethylbenzene (TMB), sodium citrate dihydrate (Na3Cit·2H2O): Tianjin Zhiyuan Chemical Reagent Co., Ltd.; Chitosan: Sigma-Aldrich, USA; N,N-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC): Sinopharm Chemical Reagent Co., Ltd.; Fluorescent staining kit SYTO 9: USA Invitrogen; Tris(hydroxymethyl)aminomethane (Tris), N,N-dimethylformamide (DMF): Chengdu Huaxia Reagent Co., Ltd.; Citrate synthase (CS): Beijing Solarbio Co., Ltd.; 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC): Zhengzhou Jacks Chemical Co., Ltd.; o-phenanthroline indicator: Shanghai Aladdin Reagent Co., Ltd.

[0034] (2) Main instruments

[0035] SU-8010 Scanning Electron Microscope: Hitachi, Japan; Tracer-100 Fourier Transform Near Infrared Spectrometer: Shimadzu, Japan; 7404 Vibrating Sample Magnetometer: Lake Shore, USA; Rigaku D / max-2400 X-ray Diffraction Energy Dispersive Spectrometer: Ningbo Xinzhi Biotechnology Co., Ltd.; Nano-ZS Dynamic Laser Particle Size Analyzer: Malvern, UK; FV1000MPE Two-Photon Laser Confocal Microscope (Upright): Olympus, Japan.

[0036] (3) Strains and culture media

[0037] Acidithiobacillus ferrooxidans BY3 was preserved and provided by the Laboratory of Synthetic Biology, School of Life Sciences, Xinxiang Medical University. See Qiyu Gao, Deping Tang, Peng Song, et al. Bio-adsorption and Bio-transformation of Arsenic by Acidithiobacillus ferrooxidans BY3, International microbiology, 2018, 21(4):207-214.

[0038] The culture medium uses 9K medium as the basal medium. The components of 9K medium include the following two parts: solution A and solution B:

[0039] A (basic salt solution): contains 3.00g of (NH4)2SO4, 0.50g of K2HPO4, 0.50g of MgSO4·7H2O, 0.10g of KCl, and 0.01g of Ca(NO3)2. To prepare the culture medium, dissolve each component of A in 700mL of distilled water to obtain solution A. Autoclave the solution at 15Psi and 121℃ for 20 minutes before use.

[0040] B (Energy component): Contains 44.78g FeSO4·7H2O; when preparing the culture medium, FeSO4·7H2O is dissolved in 300mL of distilled water, and after filtration and sterilization, solution B is obtained.

[0041] Solution B was mixed with solution A and the pH was adjusted to 1.8 with concentrated sulfuric acid to obtain 9K culture medium.

[0042] Example 1

[0043] A method for preparing an immobilized material of *Thiobacillus acidophilus* includes the following steps:

[0044] 1. The preparation steps of Fe3O4@mPDA are as follows:

[0045] (1) Synthesis of Fe3O4 nanoclusters by solvothermal method

[0046] Weigh 0.594 g of FeCl3·6H2O and dissolve it in 20 mL of ethylene glycol. Weigh 1.2 g of NaAc and 0.2 g of Na3Cit·2H2O, dissolving Na3Cit·2H2O in 0.25 mL of distilled water, and add them to the above solution. Stir at 37 °C for 30 min. After complete dissolution, transfer the reaction solution to a high-pressure reactor and place it in an electrically heated drying oven at 200 °C for 10 h. After the reaction is complete, wash the black particles with anhydrous ethanol, perform magnetic separation, wash three times with 200 mL of distilled water, and disperse in distilled water. The obtained black particles are Fe3O4 nanoclusters.

[0047] (2) Preparation of Fe3O4@PDA core-shell structure

[0048] Measure 65 mL of distilled water and 60 mL of ethanol and pour them into a 250 mL single-necked flask. Add 20 mg of Fe3O4 nanoclusters to the mixture of ethanol and water and stir mechanically for 1 min. Accurately weigh 0.0921 g of tris(hydroxymethyl)aminomethane (Tris), dissolve it in 1 mL of distilled water using a pipette, add it to the single-necked flask, and stir to mix. Weigh 0.03 g of dihydroxyphenylalanine (DOPA), dissolve it in 1 mL of distilled water, add it to the above mixed solution, and stir at room temperature for 24 h. Magnetically separate the above solution for 5 min, discard the supernatant, and collect the lower precipitate to obtain the Fe3O4@PDA core-shell structure for later use.

[0049] (3) Preparation of Fe3O4@mPDA

[0050] Measure 65 mL of distilled water and 60 mL of ethanol, pour them into a beaker and mix well. Accurately weigh 0.3621 g of F127 and transfer 625 μL of 1,3,5-trimethylbenzene (TMB). Add F127 and 1,3,5-trimethylbenzene (TMB) to the beaker simultaneously, cover with plastic wrap, and stir with a magnetic stirrer for 30 min. Then transfer to a 250 mL single-necked flask, add Fe3O4@PDA core-shell structure, and stir mechanically to obtain a mixture of TMB and F127.

[0051] Accurately weigh 0.0921 g of tris(hydroxymethyl)aminomethane (Tris), dissolve it in 1 mL of distilled water, and add it to the mixture of TMB and F127. Stir for 1 min. Weigh 0.0638 g of dihydroxyphenylalanine (DOPA), dissolve it in 1 mL of distilled water, and add it to the above solution. Stir at room temperature for 24 h. After the reaction is complete, centrifuge at 11000 rpm for 10 min. After centrifugation, discard the supernatant and disperse the lower layer particles in a mixed solution of acetone and ethanol (V:V = 1 / 2). Sonicate for 40 min, and centrifuge again at 11000 rpm for 10 min. After the second centrifugation, discard the supernatant and disperse the lower layer particles in a mixed solution of acetone and ethanol (V:V = 1 / 2) under reflux for 2 h. Centrifuge again at 11000 rpm for 10 min, discard the supernatant, and disperse the lower layer particles in distilled water. The lower layer particles after centrifugation are Fe3O4@mPDA.

[0052] 2. Preparation of chitosan derivative Bio-CS:

[0053] 10 mmol of biotin, 10 mmol of N-hydroxysuccinimide (NHS), and 12 mmol of N,N-dicyclohexylcarboimide (DCC) were dissolved in 200 mL of N,N-dimethylformamide (DMF). The mixture was magnetically stirred in a water bath at 50 °C for 20 h, and then filtered to obtain dicyclohexylurea (DCU). The filtrate was collected, and diethyl ether was added until the precipitation stopped increasing. The mixture was then transferred to an ice bath and reacted for 1 h. The precipitate of crude biotin ester (Bio-NHS) was obtained by filtration. The crude Bio-NHS precipitate was dissolved in hot isopropanol and then placed in an ice bath for 4 h to crystallize. The crystals were collected, dissolved again in DMF, and then diethyl ether was added. The mixture was then placed in an ice bath for 1.5 h, filtered, collected, and vacuum dried to obtain Bio-NHS.

[0054] Take 100 mL of 2% (w / w) citrate synthase (CS) solution, dissolve a certain amount of Bio-NHS in DMF solution, add it dropwise to the citrate synthase (CS) solution under magnetic stirring, stir and react for 24 h, remove impurities using an MD34-500-01 dialysis bag, freeze dry at -60℃ to obtain Bio-CS, and store dry.

[0055] 3. Preparation of Fe3O4@mPDA-CS nanoparticles:

[0056] 1.0 g of Fe3O4@mPDA was dispersed in deionized water and activated with 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) for 4 h. Then, 100 mg of Bio-CS was added, and the mixture was magnetically stirred for 24 h. The mixture was collected by centrifugation at 18000 r / min and freeze-dried at -60 °C to obtain Fe3O4@mPDA-CS nanoparticles, which are the immobilized materials of Acidithiobacillus ferrooxidans.

[0057] In Example 1 of this invention, an immobilized material of *Thiobacillus ferrooxidans* (Fe3O4@mPDA-CS nanoparticles) was prepared, and the synthesis route is as follows: Figure 1 As shown. To illustrate its performance, this invention uses the *Acidithiobacillus ferrooxidans* immobilized material (Fe3O4@mPDA-CS nanoparticles) prepared in Example 1 as an example, and performs SEM characterization and particle size analysis on it and the Fe3O4 nanoclusters prepared in Example 1, respectively. The specific research is as follows:

[0058] One g of Fe3O4@mPDA-CS nanoparticles prepared in Example 1 was washed three times with 10 mL of PBS for 10 min each time, then fixed with 1% ethyl tetroxide for 4 h, and subjected to gradient dehydration with 30%, 50%, 70%, 85%, 95%, and 100% ethanol for 10 min each time, including two dehydration treatments with 100% ethanol. Then, pure tert-butanol was used for three replacements, each for 15 min. After overnight storage at 4 °C, the sample was freeze-dried. The dried bacterial powder was adhered with carbon conductive tape and then sputter-coated with gold. It was then transferred to a particle size cup with dimensions of 500 mm x 410 mm x 420 mm, and the average particle size of the Fe3O4@mPDA-CS nanoparticles was measured using a Malvern nanoparticle size analyzer.

[0059] The average particle size of the Fe3O4 nanoclusters prepared in Example 1 was measured using the same procedure. The results are shown below. Figure 2 .

[0060] Depend on Figure 2 It can be seen that the Fe3O4 nanoparticle clusters exhibit a uniformly dispersed spherical morphology with a size of approximately 100 nm. However, the Fe3O4@mPDA-CS nanoparticles synthesized in Example 1 of this invention have a larger particle size, approximately 200-350 nm. The particle size analysis results are shown in […]. Figure 3 .

[0061] Depend on Figure 3 It can be seen that the particle size of Fe3O4 nanoclusters is 120nm (PDI: 0.15), and the particle size of Fe3O4@mPDA-CS nanoparticles is 320nm, thus successfully completing the construction of a nanocomposite material.

[0062] This invention provides an immobilized material for *Thiobacillus ferrooxidans* (Fe3O4@mPDA-CS nanoparticles). The experimental results demonstrate that the *Thiobacillus ferrooxidans* immobilized material (Fe3O4@mPDA-CS nanoparticles) provided by this invention can carry a large number of active functional groups such as carboxyl, amino, and hydroxyl groups. To illustrate its effectiveness in immobilizing *Thiobacillus ferrooxidans* and in desulfurizing industrial hydrogen sulfide waste gas, the *Thiobacillus ferrooxidans* immobilized material (Fe3O4@mPDA-CS nanoparticles) prepared in Example 1 was used as an example for the following study:

[0063] 1. Preparation of immobilized *Thiobacillus ferrooxidans* BY3:

[0064] 9K medium was added to the reactor, and *Acidithiobacillus ferrooxidans* BY3 bacterial suspension was inoculated at a 15% inoculum. 500g of Fe3O4@mPDA-CS nanoparticles prepared in Example 1 were added, and co-cultured at 30℃ and 150r / min. Fresh 9K medium was added by measuring the ferrous ion concentration in the 9K medium to maintain the ferrous ion concentration in the 9K medium at the steady-state concentration of the cells. This process was repeated three times. At this point, the numerous active functional groups such as carboxyl, amino, and hydroxyl groups carried by the Fe3O4@mPDA-CS nanoparticles combined with the surface groups of *Acidithiobacillus ferrooxidans* BY3. After the reaction continued for 72 hours, the 9K medium was removed, yielding immobilized cells of *Acidithiobacillus ferrooxidans* BY3, i.e., immobilized *Acidithiobacillus ferrooxidans* BY3 nanoparticles.

[0065] 2. Immobilized F. ferrooxidans BY3 nanoparticles and Fe from free cells 2+ Oxidizing capacity determination:

[0066] 4.5 mg of immobilized *Acidithiobacillus ferrooxidans* BY3 nanoparticles were added to 100 mL of 9K medium. Simultaneously, *Acidithiobacillus ferrooxidans* BY3 bacterial suspension was inoculated into 100 mL of 9K medium at a 15% inoculum. The process was carried out at 30℃ and 150 rpm, with samples taken every 4 hours to measure Fe. 2+ The oxidation rate was determined by the potassium dichromate method (Formula (1)). 2+ The changes.

[0067] In the potassium dichromate method, 1 mL of the reaction solution is mixed with 5 mL of distilled water, then 3 mL of a sulfuric acid-phosphoric acid mixed solution is added. After shaking well, 2-3 drops of o-phenanthroline indicator are added to the mixture. Titration is then performed with potassium dichromate solution. The titration is stopped when the test solution changes from reddish-yellow to purple. Fe is calculated using formula 1. 2+ The changes are shown in the experimental results. Figure 5 .

[0068] 6Fe 2+ +Cr₂O₇⁻ + 14H⁺ + =6Fe 3+ +2Cr 3+ +7H2O (1)

[0069] Depend on Figure 5 It can be seen that, after immobilization, *Acidithiobacillus ferrooxidans* BY3, compared with free cells, showed that its Fe content per unit time was significantly higher. 2+ Its oxidizing ability remains highly active.

[0070] 3. Laser confocal microscopy analysis of the binding effect between immobilized *Acidithiobacillus ferrooxidans* BY3 nanoparticles and *Acidithiobacillus ferrooxidans* BY3 cells:

[0071] Sterile circular glass slides (Ф6 mm) were placed in the wells of a 6-well cell culture plate. 100 mg of immobilized *Thiobacillus ferrooxidans* BY3 nanoparticles were added to each well. The plates were incubated statically at 30°C, with sterile water added every 48 hours. After 168 hours of incubation, the plates were stained using a bacterial staining kit (SYTO 9 fluorescent staining kit). Before staining, the fluorescent dyes SYTO 9 and PI were dissolved in sterile water, then diluted four times by volume. The stock solution was stored at -20°C in the dark. The immobilization effect was observed by laser confocal microscopy after mounting with glycerol. The experimental results are shown below. Figure 4 .

[0072] Depend on Figure 4 It can be seen that after co-culturing *Thiobacillus ferrooxidans* BY3 with Fe3O4@mPDA-CS nanoparticles for 24h, 48h, and 72h, free cells can be effectively fixed, but the cell immobilization effect is more significant after 72h.

[0073] 4. Desulfurization effect detection of immobilized *Thiobacillus ferrooxidans* BY3 nanoparticles:

[0074] H2S generated during the reaction process was removed using a chemical method. This gas is produced by the reaction of sodium sulfide and dilute sulfuric acid. Purified air was used to sequentially dilute the H2S to concentrations of 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, and 8 mg / L, and then... Figure 6 The diagram shows an experimental setup for H2S removal and iron recycling. The air, purified and diluted by an air purifier, first passes through a reaction apparatus containing sodium sulfide and dilute sulfuric acid. The reacted gas then enters a chemical reactor for further reaction. In the chemical reactor, the H2S gas is converted to Fe through a chemical reaction. 3+ The solution is oxidized to elemental S, while Fe... 3+ Reduced to Fe 2+ Subsequently containing Fe 2+ After sedimentation in a settling tank, the solution is pumped into a bioreactor where it reacts with immobilized *Thiobacillus ferrooxidans* BY3 nanoparticles. Fe... 2+ Oxidized Fe 3+ Fe produced by oxidation 3+ The gas is pumped back into the chemical reactor, thus completing the removal of H2S and realizing the recycling of iron. The chemical reactor is equipped with a gas outlet and a tail gas collector. The removal rate of H2S by the entire reactor was calculated by collecting and detecting the gas after H2S removal at the outlet. The iodometric method (GB / T 11060.1-1998) was used to detect the H2S removal rate of the reactor at different concentrations. The detection results of the removal rate of H2S gas at different concentrations are shown below. Figure 7 .

[0075] Immobilized thiobacillus ferrooxidans BY3 nanoparticles were added to the bioreactor.

[0076] Depend on Figure 7 It can be seen that the reaction device can effectively remove H2S gas. After 1 hour of reaction, the removal rates of H2S at concentrations of 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, and 8 mg / L are 89%, 88%, 86%, 86%, and 85%, respectively. After 2 hours, the effective rates are 93%, 91%, 90%, 89%, and 87%, respectively. After 3 hours of reaction, the removal rates of H2S at concentrations of 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, and 8 mg / L are 92%, 92.4%, 80%, 86%, and 85%, respectively. After 4 hours, the effective rates were 93.5%, 92.6%, 91%, 90%, and 89.5%, respectively. After 5 hours of reaction, the H2S removal rates for 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, and 8 mg / L were 92.8%, 92.1%, 91.2%, 90.2%, and 89.6%, respectively. After 6 hours of reaction, the effective rates were 93.3%, 92.4%, 90.8%, 90.1%, and 89.3%, respectively.

[0077] In the above H2S according to Figure 6 In the experimental setup shown, during the H2S removal reaction via a chemical reactor and a bioreactor, the H2S gas is reacted with Fe in the chemical reactor. 3+ The solution is oxidized to elemental S, while Fe... 3+ Reduced to Fe 2+ Subsequently containing Fe 2+ After precipitation, the solution is pumped into a bioreactor to react with immobilized *Thiobacillus ferrooxidans* BY3 nanoparticles in the bioreactor. Fe 2+ Oxidized Fe 3+ Fe produced by oxidation 3+ It is pumped back into the chemical reactor, thus completing the removal of H2S and realizing the recycling of iron.

[0078] This is achieved through reaction equations (2) and (3):

[0079]

[0080]

[0081] In summary, the *Acidithiobacillus ferrooxidans* immobilization material prepared in Example 1 of this invention specifically binds to *Acidithiobacillus ferrooxidans*, thereby forming immobilized cells, namely immobilized *Acidithiobacillus ferrooxidans* BY3. These immobilized cells are also referred to as immobilized *Acidithiobacillus ferrooxidans* BY3 nanoparticles. These immobilized cells (immobilized *Acidithiobacillus ferrooxidans* BY3 nanoparticles) can maintain the high Fe content of the original cells. 2+ It exhibits strong oxidizing power and stable genetic properties, and is easily separated from the reaction system, allowing for reusability. Using these immobilized cells (immobilized *Acidithiobacillus ferrooxidans* BY3 nanoparticles) for desulfurization can effectively remove H2S gas, achieving good desulfurization results; and it can maintain Fe2+ during the desulfurization process. 3+ The recycling process is efficient, with low byproducts and full recycling of iron. Furthermore, during desulfurization, the activity of *Acidithiobacillus ferrooxidans* BY3 can be maintained for a prolonged period, thus ensuring the stability of its genetic properties. Simultaneously, in accordance with... Figure 6 The experimental setup diagram for H2S removal and iron circulation shown indicates that when removing H2S, the bioreactor containing immobilized ferrooxidizobacterium ferrooxidans BY3 nanoparticles can maintain high reaction efficiency during the desulfurization process.

[0082] Existing biological desulfurization technologies utilizing microbial strains involve adding a culture medium containing appropriate nutrients (mainly ammonium sulfate and dipotassium hydrogen phosphate) to a bioreactor and inoculating it with appropriately cultured *Thiobacillus ferrooxidans* or other inorganic sulfur-removing microorganisms. This method of directly adding culture medium to the bioreactor suffers from limited desulfurization efficiency, high byproduct levels, unstable genetic performance of the strains, insufficient iron recycling, and low bioreactor efficiency. Compared to existing biological desulfurization technologies using microbial strains, the immobilized cells (immobilized *Thiobacillus ferrooxidans* BY3 nanoparticles) provided in this invention, applied to bioreactors for desulfurization, effectively solve the common problems of limited desulfurization efficiency, high byproduct levels, unstable genetic performance of the strains, insufficient iron recycling, and low bioreactor efficiency. The *Thiobacillus ferrooxidans* immobilized material provided in this invention can further promote the development of biological desulfurization technology in industrial applications.

[0083] This invention provides an immobilized material of *Thiobacillus ferrooxidans* and its preparation method, as well as the application of the immobilized material in cell immobilization and desulfurization. The material provided by this invention is a novel Fe3O4 magnetic nanocarrier. This carrier is constructed by forming a mesoporous PDA shell on the surface of Fe3O4 nanoclusters via a dopamine self-polymerization reaction, creating a nanocomposite material Fe3O4@mPDA. Simultaneously, a biotinylated chitosan derivative, Bio-CS, is chemically synthesized. Then, Fe3O4@mPDA and Bio-CS are combined using an emulsification method to form Fe3O4@mPDA-CS nanoparticles. These Fe3O4@mPDA-CS nanoparticles are the *Thiobacillus ferrooxidans* immobilized material (Fe3O4 magnetic nanocarrier) prepared according to this invention.

[0084] This invention provides an immobilized material for *Acidithiobacillus ferrooxidans* (Fe3O4 magnetic nanocarrier), which can be used for the immobilization of *Acidithiobacillus ferrooxidans*. The nanoparticles are sterilized by high temperature and ultraviolet light and then filled into a reactor. 9K medium is added to the reactor, and *Acidithiobacillus ferrooxidans* BY3 bacterial culture is inoculated at a 15% inoculum. The culture is carried out at 30°C. The surface groups of *Acidithiobacillus ferrooxidans* BY3 covalently bind to the synthesized Fe3O4@mPDA-CS. After the reaction continues for 72 hours, the 9K medium is removed, yielding immobilized *Acidithiobacillus ferrooxidans* BY3 nanoparticles. The immobilized *Acidithiobacillus ferrooxidans* BY3 nanoparticles prepared by this invention can be used for the desulfurization treatment of industrial hydrogen sulfide (H2S) waste gas. The *Acidithiobacillus ferrooxidans* immobilized material (Fe3O4 magnetic nanocarrier) provided by this invention not only effectively maintains the biological activity of *Acidithiobacillus ferrooxidans* BY3, but also features mild reaction conditions, no secondary pollution, and the ability to maintain Fe3O4. 3+ The method involves recycling, thus meeting the requirements of green biotechnology manufacturing.

[0085] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0086] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0087] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An immobilized material of *Acidithiobacillus ferrooxidans*, characterized in that, Using Fe3O4 nanoclusters as raw material, a PDA shell with a mesoporous structure was formed on the surface of Fe3O4 nanoclusters by means of the self-polymerization reaction of dopamine, thus constructing the nanocomposite material Fe3O4@mPDA. At the same time, biotinylated chitosan derivative Bio-CS was synthesized using biotin, N-hydroxysuccinimide, N,N-dicyclohexylcarboimide, and N,N-dimethylformamide as raw materials. Then, Fe3O4@mPDA and biotinylated chitosan derivative Bio-CS were combined by emulsification to obtain the immobilized material of *Thiobacillus ferrooxidans*.

2. The *Thiobacillus ferrooxidans* immobilization material according to claim 1, characterized in that, The preparation of the *Thiobacillus ferrooxidans* immobilized material includes the following steps: Preparation of Fe3O4@mPDA: Fe3O4 nanoclusters were prepared by solvothermal method using ferric chloride hexahydrate as raw material; Fe3O4@PDA core-shell structure was prepared using Fe3O4 nanoclusters, tris(hydroxymethyl)aminomethane and dihydroxyphenylalanine as raw materials; Fe3O4@mPDA was prepared using Fe3O4@PDA core-shell structure, F127, 1,3,5-trimethylbenzene and tris(hydroxymethyl)aminomethane as raw materials. Preparation of chitosan derivative Bio-CS: Biotin, N-hydroxysuccinimide and N,N-dicyclohexylcarboimide were dissolved in N,N-dimethylformamide, heated and stirred for 18-22 h, then placed in an ice bath for 0.5-1.5 h. The mixture was filtered to obtain crude biotin ester precipitate. The crude biotin ester precipitate was purified and dried to obtain Bio-NHS. Bio-NHS was dissolved in DMF solution, then added to citrate synthase solution, and stirred for 22-26 hours. After dialysis to remove impurities, it was freeze-dried to obtain Bio-CS, which was then dried and stored. Preparation of Fe3O4@mPDA-CS nanoparticles: Fe3O4@mPDA was dispersed in water, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added for activation for 2-6 h. Bio-CS was added and the mixture was stirred for 22-26 h. The precipitate was collected by centrifugation and then freeze-dried to obtain Fe3O4@mPDA-CS nanoparticles, which are immobilized materials of Acidithiobacillus ferrooxidans.

3. The *Acidithiobacillus ferrooxidans* immobilization material according to claim 2, characterized in that, The preparation method of Fe3O4 nanoclusters includes the following steps: weigh FeCl3·6H2O and dissolve it in ethylene glycol to obtain a mixed solution; weigh NaAc and Na3Cit·2H2O and add them to the mixed solution, stir at 35-39℃ for 28-32 min; after complete dissolution, transfer the reaction solution and react at 195-205℃ for 8-12 h; after the reaction is completed, wash out the black particles with anhydrous ethanol, and the obtained black particles are Fe3O4 nanoclusters.

4. The *Acidithiobacillus ferrooxidans* immobilization material according to claim 2, characterized in that, The preparation method of Fe3O4@PDA core-shell structure includes the following steps: Fe3O4 nanoclusters are added to a mixture of ethanol and water and stirred for 0.5 to 1.5 min; tris(hydroxymethyl)aminomethane is dissolved in water and added to the mixture and mixed well; dihydroxyphenylalanine is dissolved in water and added to the mixture and stirred at room temperature for 22 to 26 h; the above solution is magnetically separated, the supernatant is discarded, and the lower precipitate is collected to obtain the Fe3O4@PDA core-shell structure.

5. The *Acidithiobacillus ferrooxidans* immobilization material according to claim 2, characterized in that, The preparation method of Fe3O4@mPDA includes the following steps: F127 and 1,3,5-trimethylbenzene are added to a mixture of ethanol and water, stirred for 28-32 min, and then Fe3O4@PDA core-shell structure is added and stirred to obtain a mixture of TMB and F127. Dissolve tris(hydroxymethyl)aminomethane in water and add it to a mixture of TMB and F127, then mix well. Dissolve dihydroxyphenylalanine in water and add it to the above solution. Stir at room temperature for 22–26 h. After the reaction is complete, centrifuge, discard the supernatant, and disperse the lower layer particles in a mixture of acetone and ethanol. Centrifuge again, discard the supernatant, and reflux the lower layer particles in the same mixture for 1.5–2.5 h. Centrifuge again, discard the supernatant, and disperse the lower layer particles in distilled water. The lower layer particles after centrifugation are Fe3O4@mPDA.

6. The *Thiobacillus ferrooxidans* immobilization material according to claim 5, characterized in that, In a mixed solution of acetone and ethanol, the volume ratio of acetone to ethanol is 1:1.5 to 2.

7. The *Acidithiobacillus ferrooxidans* immobilization material according to claim 2, characterized in that, The steps for precipitating and purifying crude biotin ester are as follows: after dissolving the crude biotin ester precipitate in hot isopropanol, it is placed in an ice bath for 3-5 hours to crystallize. The crystals are collected and dissolved again with DMF, followed by the addition of diethyl ether. After ice bath for 1.0-2.0 hours, the precipitate is collected by filtration and vacuum drying to obtain Bio-NHS.

8. The application of the *Acidithiobacillus ferrooxidans* immobilization material according to claim 1 in cell immobilization.

9. The application according to claim 8, characterized in that, The aforementioned Acidithiobacillus ferrooxidans immobilization material serves as a magnetic composite carrier. Its surface is covered with active functional groups such as carboxyl, amino, and hydroxyl groups, which are used to bind with the surface groups of Acidithiobacillus ferrooxidans BY3, thereby obtaining immobilized cells of Acidithiobacillus ferrooxidans BY3.

10. The application of the *Acidithiobacillus ferrooxidans* immobilized material according to claim 1 in the desulfurization treatment of industrial hydrogen sulfide waste gas, characterized in that, The aforementioned Acidithiobacillus ferrooxidans immobilization material serves as a magnetic composite carrier. Its surface is covered with active functional groups such as carboxyl, amino, and hydroxyl groups, which are used to bind with the surface groups of Acidithiobacillus ferrooxidans BY3, thereby obtaining immobilized cells of Acidithiobacillus ferrooxidans BY3.

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