Rhodopseudomonas palustris sx2, applications thereof, iron oxidation methods, compositions, and cultures

Abstract

The application provides a Rhodopseudomonas palustris SX2 and an application thereof, an iron oxidation method, a composition and a culture, and belongs to the technical field of environmental microorganisms. The strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 38191. The strain can efficiently oxidize free state and organic combined state Fe(II) under anaerobic light conditions, especially has stronger oxidation capacity for the organic combined state Fe(II), and the oxidation process is coupled with cell growth and inorganic carbon fixation; the photo-iron oxidation product is goethite short-range ordered nanocrystalline state iron mineral, and the iron minerals formed under different iron forms are different in spatial distribution and particle size. The Rhodopseudomonas palustris SX2 provided by the application provides a microbial material with excellent performance for environmental remediation, iron resource, sewage treatment and divalent iron pollution treatment.

Description

Technical Field

[0001] This application relates to the field of environmental microbiology technology, and in particular to a species of Rhodopseudomonas palustris SX2 and its applications, iron oxidation methods, compositions and cultures. Background Technology

[0002] Phototrophic iron oxidation is an important microbially driven process in anaerobic environments and a key link in the biogeochemical iron cycle. This process is mainly mediated by anaerobic photosynthetic bacteria (such as Rhodopseudomonas palustris), which can utilize light energy to oxidize soluble Fe(II) into insoluble Fe(III) minerals. It is often accompanied by metabolic activities such as bacterial growth and inorganic carbon fixation, thereby realizing energy conversion and material cycling in the ecosystem.

[0003] In naturally anoxic environments, Fe(II) exists primarily in two chemical forms: free and organically bound, with organically bound Fe(II) often being the dominant form. The chemical properties of different iron forms vary significantly, directly affecting the photo-iron oxidation efficiency, metabolic response, and product characteristics of microorganisms. However, existing research largely focuses on evaluating the photo-iron oxidation performance under single iron forms (especially free Fe(II)), lacking a systematic comparison of the oxidation efficiency of different iron forms within the same strain. Furthermore, it lacks comprehensive characterization of the microstructure, spatial distribution, and crystal form characteristics of iron oxidation products.

[0004] Therefore, a highly efficient phototrophic iron-mineralizing bacterium is needed. Summary of the Invention

[0005] In view of the above, in order to at least partially solve at least one of the aforementioned technical problems, this application provides a Rhodopseudomonas palustris SX2, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 38191.

[0006] According to an embodiment of one aspect of this application, an application of Rhodopseudomonas palustris SX2 is provided, comprising at least one of the following: (1) oxidizing free Fe(II); (2) oxidizing organically bound Fe(II); (3) oxidizing soluble Fe(II) into insoluble iron minerals; (4) performing phototrophic iron oxidation under anaerobic light conditions; (5) coupling cell growth during iron oxidation; (6) fixing inorganic carbon during iron oxidation; (7) producing ordered nanocrystalline iron minerals; (8) generating iron minerals in the periplasmic space and / or on the cell membrane surface; and (9) generating intracellular nanoparticle iron minerals.

[0007] According to an embodiment of another aspect of this application, an application of Rhodopseudomonas palustris SX2 is provided, including at least one of the following: (1) for environmental remediation; (2) for pollution control; (3) for soluble Fe(II) pollution control; (4) for driving the biogeochemical cycle of iron; (5) for wastewater treatment; and (6) for iron resource utilization.

[0008] According to an embodiment of another aspect of this application, an application of Rhodopseudomonas palustris SX2 is provided, wherein at least one of the following is used: (1) for the study of the biogeochemical cycle of iron; (2) as a phototrophic iron mineralization microbial resource; (3) for the study of the microbial oxidation efficiency of various iron forms; (4) for the study of the formation mechanism of microbial-mediated iron mineralization products.

[0009] According to another embodiment of this application, a method for iron oxidation is provided, comprising a culture step of inoculating Rhodopseudomonas palustris SX2 into soluble Fe(II).

[0010] According to another embodiment of this application, a composition is provided containing Rhodopseudomonas palustris SX2.

[0011] According to another embodiment of this application, a culture is provided comprising Rhodopseudomonas palustris SX2, a mineral salt culture medium, and sodium lactate.

[0012] According to the implementation scheme of this application, the *Rhodopseudomonas palustris* SX2 can simultaneously oxidize both free and organically bound Fe(II) under anaerobic light conditions. In particular, its oxidation capacity for organically bound Fe(II) (oxidation efficiency reaching 85%) is superior to that for free Fe(II) (oxidation efficiency reaching 50%). This photo-oxidation process achieves coupled metabolism of bacterial growth and inorganic carbon fixation, with the oxidation product being goethite-like short-range ordered nanocrystalline iron minerals. This enhances the efficiency and product controllability of phototrophic iron oxidation, providing a high-performance microbial material for environmental remediation, iron resource utilization, wastewater treatment, and the treatment of ferrous iron pollution. Attached Figure Description

[0013] The above and other objects, features and advantages of this application will become clearer from the following description of embodiments with reference to the accompanying drawings, in which:

[0014] Figure 1 The following is a graph showing the screening and identification results of Rhodopseudomonas palustris SX2 in Example 1 of this application: (a) Neighbor-joining phylogenetic tree based on 16S rRNA gene; (b) ANI value heatmap of SX2 and closely related strains; (c) Fe(II) concentration change graph of SX2 under different culture conditions.

[0015] Figure 2 The following is a graph showing the results of photo-iron oxidation of different iron forms by *Rhodopseudomonas palustris* SX2 in Example 2 of this application: (a) Dynamic curves of the concentration changes of free Fe(II) and organically bound Fe(II); (b) OD values ​​of the bacterial cells in the three experimental groups. 600 Growth curve; (c) End-stage δ cell count 13 Test result image;

[0016] Figure 3 The images show the HR-TEM and EDS characterization of the iron oxidation products of Rhodopseudomonas palustris SX2 in Example 3 of this application. (a) and (f) are TEM images of the cells in the free and organically bound Fe(II) groups, respectively; (b) and (g) are high-resolution lattice fringe patterns of the iron minerals; (c)~(e) and (h)~(j) are elemental distribution patterns of Fe, S, and P in the iron minerals; (k) is a TEM image of the cells in the blank control group; (l) is a high-resolution lattice fringe pattern of the iron minerals in the blank control group; and (m)~(o) are elemental distribution patterns of Fe, S, and P in the iron minerals in the blank control group.

[0017] Figure 4 This is the XRD pattern of the photoiron oxidation product of Rhodopseudomonas sphygmoides SX2 in Example 3 of this application. Detailed Implementation

[0018] The embodiments of this application will now be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of this application. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of this application for ease of explanation. However, it will be apparent that one or more embodiments may be implemented without these specific details. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this application.

[0019] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0020] When using expressions such as "at least one of A, B, and C," they should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art.

[0021] In realizing the concept of this application, it was found that current research on phototrophic iron-mineralizing bacteria is mostly focused on the oxidation capacity of a single iron form, lacking comparative studies on the oxidation efficiency of strains under different iron forms, and the characterization of iron oxidation products is not systematic enough.

[0022] Based on this, according to one aspect of the present application, a bacterium SX2, *Rhodopseudomonas palustris*, is provided.

[0023] Preservation Instructions

[0024] Bacterial species name: Rhodopseudomonas palustris

[0025] Strain number: SX2

[0026] Preservation period: March 30, 2026

[0027] Preservation Center: China General Microbiological Culture Collection Center (CGMCC)

[0028] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0029] CGMCC Registration Number: CGMCC No. 38191

[0030] According to the implementation scheme of this application, the *Rhodopseudomonas palustris* SX2 can simultaneously oxidize both free and organically bound Fe(II) under anaerobic light conditions. In particular, its oxidation capacity for organically bound Fe(II) (oxidation efficiency reaching 85%) is superior to that for free Fe(II) (oxidation efficiency reaching 50%). This photo-oxidation process achieves coupled metabolism of bacterial growth and inorganic carbon fixation, with the oxidation product being goethite-like short-range ordered nanocrystalline iron minerals. This enhances the efficiency and product controllability of phototrophic iron oxidation, providing a high-performance microbial material for environmental remediation, iron resource utilization, wastewater treatment, and the treatment of ferrous iron pollution.

[0031] According to an embodiment of one aspect of this application, an application of Rhodopseudomonas palustris SX2 is provided, comprising at least one of the following: (1) oxidizing free Fe(II); (2) oxidizing organically bound Fe(II); (3) oxidizing soluble Fe(II) into insoluble iron minerals; (4) performing phototrophic iron oxidation under anaerobic light conditions; (5) coupling cell growth during iron oxidation; (6) fixing inorganic carbon during iron oxidation; (7) producing ordered nanocrystalline iron minerals; (8) generating iron minerals in the periplasmic space and / or on the cell membrane surface; and (9) generating intracellular nanoparticle iron minerals.

[0032] According to the implementation scheme of this application, phototrophic iron oxidation is carried out using Rhodopseudomonas palustris SX2 under anaerobic light conditions. It can simultaneously oxidize free and organically bound Fe(II). The oxidation efficiency for free Fe(II) is 50%, while the oxidation efficiency for organically bound Fe(II) is 85%. Soluble Fe(II) is converted into insoluble iron minerals. During the oxidation process, coupled metabolism of cell growth and inorganic carbon fixation is achieved. The inorganic carbon fixation value of free Fe(II) is 4.3‰, while that of organically bound Fe(II) is 12.5‰. The generated iron minerals are goethite-type short-range ordered nanocrystalline states, and can form nanoparticles in the periplasmic space, cell membrane surface, or intracellular space of the cells, thereby improving the iron oxidation efficiency, enhancing the stability and controllability of the products, and providing technical means and material basis for the treatment of environmental iron pollution.

[0033] According to an embodiment of another aspect of this application, an application of Rhodopseudomonas palustris SX2 is provided, including at least one of the following: (1) for environmental remediation; (2) for pollution control; (3) for soluble Fe(II) pollution control; (4) for driving the biogeochemical cycle of iron; (5) for wastewater treatment; and (6) for iron resource utilization.

[0034] According to the implementation scheme of this application, by using Rhodopseudomonas palustris SX2 in environmental remediation, pollution control, and wastewater treatment, soluble Fe(II) pollution in water bodies or sediments can be effectively removed. Its oxidation efficiency for free Fe(II) is 50%, while its oxidation efficiency for organically bound Fe(II) is 85%, converting it into stable insoluble iron minerals, thereby reducing the bioavailability and migration risk of iron. At the same time, it can drive the biogeochemical cycle of iron, promote the transformation of iron forms in the ecosystem, and convert dispersed and harmful iron ions (usually Fe²⁺) into solid iron minerals with specific structures, compositions, and functions. The converted iron minerals can be used as environmental functional materials (such as adsorbents or catalysts), industrial raw materials (such as magnetite as a precursor for magnetic materials), and biogeoengineering materials (such as cementing loose particles to improve the strength and stability of soil or sediments). It can also reduce iron pollution and improve water quality in the process of wastewater treatment, combining the functions of environmental governance and element cycle regulation, and providing an effective microbial technology solution for the remediation and sustainable management of iron-polluted environments.

[0035] According to another embodiment of this application, an application of Rhodopseudomonas palustris SX2 is provided, wherein at least one of the following is used: (1) for the study of the biogeochemical cycle of iron; (2) as a phototrophic iron mineralization microbial resource; (3) for the study of the microbial oxidation efficiency of various iron forms; (4) for the study of the formation mechanism of microbial-mediated iron mineralization products.

[0036] According to the implementation scheme of this application, by using Rhodopseudomonas palustris SX2 to conduct research on the biogeochemical cycle of iron, it can serve as a phototrophic iron mineralization microbial resource, providing experimental materials for analyzing the microbial oxidation efficiency of different iron forms (free and organically bound), and can be used to explore the formation mechanism of microbial-mediated iron mineralization products. This lays a solid theoretical and material foundation for further revealing the mechanism of phototrophic iron oxidation, improving the iron cycle model, and developing new microbial mineralization technologies.

[0037] According to another embodiment of this application, a method for iron oxidation is provided, comprising a culture step of inoculating Rhodopseudomonas palustris SX2 into soluble Fe(II).

[0038] According to the implementation scheme of this application, under anaerobic light conditions, Rhodopseudomonas palustris SX2 is inoculated into a culture system containing soluble Fe(II). By utilizing the phototrophic iron oxidation capacity of the strain, free or organically bound Fe(II) is converted into insoluble goethite-like short-range ordered nanocrystalline iron minerals, and the coupled metabolism of cell growth and inorganic carbon fixation is achieved during the oxidation process.

[0039] According to the implementation scheme of this application, an organic carbon source is added during the cultivation process, preferably sodium lactate.

[0040] According to the implementation scheme of this application, *Rhodopseudomonas palustris* SX2 is inoculated into a culture system containing soluble Fe(II). An organic carbon source (preferably sodium lactate) is then added during the culture process to provide the strain with a metabolizable carbon source, promoting photo-iron oxidation under anaerobic light conditions and enhancing the synergistic effect between bacterial growth and inorganic carbon fixation. This improves the oxidation efficiency of both free and organically bound Fe(II), allowing soluble Fe(II) to be more fully converted into goethite-like short-range ordered nanocrystalline iron minerals. The iron-rich nanoparticles of free Fe(II) are mainly distributed in the periplasmic space and on the cell membrane surface, while the iron minerals formed by organically bound Fe(II) are intracellular nanoparticles, and the particle size of the organically bound Fe(II) iron minerals is larger than that of the free Fe(II) group.

[0041] According to the implementation scheme of this application, the cultivation environment is an anaerobic light environment.

[0042] According to the implementation scheme of this application, Rhodopseudomonas palustris SX2 is inoculated into a culture system containing soluble Fe(II), and an organic carbon source (such as sodium lactate) may be selectively added. The culture environment is controlled under anaerobic light conditions so that the strain can fully exert its phototrophic iron oxidation capacity.

[0043] According to another embodiment of this application, a composition is provided containing Rhodopseudomonas palustris SX2.

[0044] According to the embodiments of this application, the composition contains *Rhodopseudomonas palustris* SX2, which can oxidize soluble Fe(II) into goethite-like short-range ordered nanocrystalline iron minerals under anaerobic light conditions, and achieve coupled metabolism of bacterial growth and inorganic carbon fixation. The products under different iron forms exhibit characteristic differences in particle size and spatial distribution. Free Fe(II) iron-rich nanoparticles are mainly distributed in the periplasmic space and cell membrane surface, while the iron minerals formed by organically bound Fe(II) are intracellular nanoparticles, and the particle size of the organically bound Fe(II) iron minerals is significantly larger than that of the free Fe(II) group. Therefore, it plays an important role in environmental remediation, wastewater treatment, and the treatment of ferrous iron pollution.

[0045] According to another embodiment of this application, the composition is a microbial agent.

[0046] According to the implementation scheme of this application, the bacterial agent contains Rhodopseudomonas palustris SX2, which can efficiently oxidize soluble Fe(II) into goethite-like short-range ordered nanocrystalline iron minerals under anaerobic light conditions, and simultaneously achieve bacterial growth and inorganic carbon fixation.

[0047] According to another embodiment of this application, a culture is provided comprising Rhodopseudomonas palustris SX2, a mineral salt culture medium, and sodium lactate.

[0048] According to the implementation scheme of this application, the culture contains Rhodopseudomonas palustris SX2, mineral salt culture medium and sodium lactate, which can achieve efficient oxidation of free and organically bound Fe(II) under anaerobic light conditions, and simultaneously promote cell growth and inorganic carbon fixation, converting soluble Fe(II) into goethite-like short-range ordered nanocrystalline iron minerals.

[0049] To make the objectives, technical solutions and advantages of this application clearer, the following detailed description is provided in conjunction with specific embodiments and the accompanying drawings. Unless otherwise specified, all reagents used are commercially available reagents commonly used in the art.

[0050] Example 1: Screening and identification of Rhodopseudomonas palustris SX2

[0051] 1. Enrichment and isolation of bacterial strains

[0052] 1.1 Strain enrichment

[0053] Iron-rich sediment samples were collected, and 1 g of fresh sample was inoculated into a sterile serum bottle containing 50 mL of liquid mineral salt medium (MSM, pH 7.0±0.2) containing 1 mM sodium lactate. After thorough mixing, high-purity N2 was continuously introduced into the bottle to replace the air for more than 10 min to create a strictly anaerobic environment. The bottle was sealed with a butyl rubber stopper and placed in a 28℃ constant temperature anaerobic culture device. A 40 W incandescent light source was turned on and the vertical distance between the light source and the culture system was fixed at 20 cm. The culture was continuously irradiated and cultured at constant temperature for 8 days. The state of the culture medium was observed daily during the period. Finally, an enriched culture medium containing phototrophic iron mineralization functional microorganisms was obtained for subsequent strain purification and isolation.

[0054] MSM medium components (1 L): potassium dihydrogen phosphate (KH2PO4) 0.5 g, ammonium chloride (NH4Cl) 0.3 g, sulfuric acid heptahydrate (MgSO4·7H2O) 0.5 g, calcium chloride dihydrate (CaCl2·2H2O) 0.1 g, vitamin solution 10 mL, SL10 trace element solution 1 mL, pH 7.0-7.2.

[0055] 1.2 Culture medium preparation

[0056] First, prepare liquid mineral salt medium (MSM). Add basic inorganic salts, trace elements, and vitamin solutions according to the formula. After thorough dissolution, adjust the pH of the medium to 7.0±0.2. Dispense into sterile containers and autoclave at 121℃ for 20 min. After sterilization, cool to room temperature in a sterile environment. After the medium cools, add filtered sterilized sodium lactate to the liquid MSM in a sterile anaerobic workbench to a final sodium lactate concentration of 1 mM. Mix well and seal for later use in the enrichment, purification, and culture of bacterial strains. Simultaneously, prepare solid MSM agar plates by adding 1.5% (w / v) agar powder to the liquid MSM formula. After thorough dissolution, adjust the pH to 7.0±0.2. After autoclaving under the same conditions, pour the plates while hot in a sterile anaerobic workbench. After the agar has completely solidified and cooled, seal and store at 4℃ for later use in the streak purification and single colony isolation of bacterial strains.

[0057] 1.3 Strain purification

[0058] The above-mentioned enriched culture medium was subjected to 10 [units of treatment] in a sterile anaerobic operating table. -1 Up to 10 -6A series of serial dilutions were performed, and 100 μL of each dilution of bacterial culture was evenly spread on solid MSM agar plates. The plates were then placed under the same anaerobic light conditions as the bacterial enrichment (28℃, 40 W incandescent light, light source 20 cm away from the culture system) and incubated. After single colonies grew on the plates, uniform and well-grown single colonies were picked with a sterile inoculation loop and subjected to multiple streak purification operations on fresh solid MSM agar plates to finally obtain pure strain SX2.

[0059] 2. Strain identification

[0060] 2.1 Sequencing of the 16S rRNA gene of strain SX2

[0061] The purified SX2 bacterial culture was used to extract genomic DNA using a bacterial genomic DNA extraction kit. Using the extracted DNA as a template, PCR amplification was performed using universal primers for the bacterial 16S rRNA gene. After verification by agarose gel electrophoresis, the qualified amplification products were sent for sequencing to obtain the 16S rRNA gene sequence of the strain. The sequence was compared for homology with known bacterial 16S rRNA gene sequences in the GenBank database. A neighbor-joining phylogenetic tree was constructed using bioinformatics software. The results are as follows: Figure 1 As shown in (a) of the diagram.

[0062] Figure 1 (a) is a neighbor-joining phylogenetic tree diagram of Rhodopseudomonas palustris SX2 based on the 16S rRNA gene in an embodiment of this application.

[0063] according to Figure 1 As can be seen from (a) in the figure, based on the comparison results and the clustering relationship of the phylogenetic tree, the taxonomic position of this strain is preliminarily determined to be a strain belonging to the genus Rhodopseudomonas.

[0064] 2.2 Whole genome sequencing

[0065] After culturing the purified Rhodopseudomonas palustris SX2 strain to obtain sufficient bacterial cells, high-quality genomic DNA was extracted using a genomic DNA extraction kit. After the DNA concentration, purity, and integrity were tested and found to be qualified, a genome sequencing library was constructed. The qualified library was sent to the Illumina HiSeq sequencing platform for whole-genome sequencing. After sequencing, the raw data was filtered, assembled, and annotated using bioinformatics analysis to obtain the whole-genome sequence information of the strain. Subsequently, the whole-genome sequencing data was submitted to the NCBI database to complete the molecular information registration of this bacterial species.

[0066] 2.3 Average Nucleotide Identity (ANI) Analysis

[0067] The whole genome sequencing assembly sequence of the purified strain in this experiment was obtained. Simultaneously, the whole genome reference sequences of closely related strains of *Rhodopseudomonas palustris* were downloaded from the NCBI public database. Using analysis software, with the whole genome sequences as the analysis object, the software's analysis algorithm was used to perform whole genome mean nucleotide identity (ANI) comparison analysis between the target strain and the closely related *Rhodopseudomonas palustris* strain, calculating the ANI value between them. The results showed that the ANI value of this strain and the *Rhodopseudomonas palustris* strain TIE-1 was 97.49%. Figure 1 As shown in (b) of the diagram.

[0068] Figure 1 (b) is a heatmap of ANI values ​​for Rhodopseudomonas palustris SX2 and closely related strains in the embodiments of this application.

[0069] according to Figure 1 As can be seen from (b) in the figure, based on the ANI value determination criteria for microbial species identification, this strain is determined to be a strain that conforms to the species Rhodopseudomonas palustris.

[0070] Based on a comprehensive integration of homology alignment from 16S rRNA gene sequencing, neighbor-joint phylogenetic tree clustering results, and genome-wide ANI analysis, a multi-dimensional species determination was performed. According to a series of results—16S rRNA gene sequence similarity >97% with *Rhodopseudomonas palustris* strains, clustering with *Rhodopseudomonas palustris* in the phylogenetic tree, and a genome-wide ANI value of 97.49–97.53% with closely related strains of *Rhodopseudomonas palustris*, meeting the standard threshold for microbial species identification—this strain was identified as *Rhodopseudomonas palustris* and formally named *Rhodopseudomonas palustris* SX2. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 38191.

[0071] 3. Study on photo-iron oxidation function

[0072] This experiment set up four treatment groups: Fe(II) + light group, Fe(II) dark control group, Fe(II) + sodium lactate + light group, and Fe(II) + sodium lactate dark control group, with three biological replicates for each group. The logarithmic growth phase of the SX2 strain was inoculated into a mineral salt medium containing 1 mM free Fe(II) (with 1 mM sodium lactate added as needed), and placed in a strictly anaerobic environment at 28℃. The light group was continuously illuminated by a 40 W incandescent lamp (20 cm from the culture system), while the dark group was kept in complete darkness. The culture was carried out continuously for 8 days, with regular sampling. The concentration change of soluble Fe(II) in the system was determined using the phenanthridine method under anaerobic conditions to analyze the photo-iron oxidation efficiency of the strain and the effects of light and sodium lactate on its photo-iron mineralization ability. The results are as follows: Figure 1 As shown in (c) in the figure.

[0073] Figure 1 (c) in the figure is a graph showing the change of Fe(II) concentration of Rhodopseudomonas palustris SX2 under different culture conditions in the embodiments of this application;

[0074] according to Figure 1 As can be seen from (c), the Fe(II) oxidation (photo-iron mineralization) process of Rhodopseudomonas palustris SX2 is strictly dependent on light. Sodium lactate, an organic carbon source, can significantly improve the photo-iron oxidation efficiency of the strain and is a key growth factor for the strain to exert its photo-iron mineralization function.

[0075] Example 2: Study on the photo-iron oxidation efficiency of Rhodopseudomonas palustris SX2 for different iron forms

[0076] 1. Preparation of experimental materials

[0077] 1.1 Preparation of bacterial culture

[0078] A single colony of purified *Rhodopseudomonas palustris* SX2 was picked using a sterile inoculation loop and inoculated into a 100 mL sterile serum bottle containing 50 mL of 1 mM sodium lactate solution (MSM). High-purity N2 was bubbled into the bottle for at least 10 min to replace the air. The bottle was then sealed and placed in a 28°C incubator under anaerobic conditions with shaking under 40 W incandescent light (20 cm from the system). The OD of the bacterial culture was periodically measured using a visible spectrophotometer. 600 Value, wait until the bacterial culture grows to the logarithmic phase (OD). 600 When the nutrient content reaches 0.6~0.8, the culture is stopped, and the experimental seed solution is prepared. It is then anaerobically stored at 4℃ for a short period of time and inoculated within 24 hours.

[0079] 1.2 Iron Morphology Configuration

[0080] Throughout the process, two iron-form stock solutions were prepared using anaerobic water in an anaerobic operating room to prevent Fe(II) oxidation. The free Fe(II) stock solution was a 100 mM FeCl2 solution prepared by accurately weighing anhydrous FeCl2 powder and dissolving it in anaerobic water to a final volume. The organically bound Fe(II) stock solution was a 100 mM Fe(II)-NTA complex solution prepared by weighing anhydrous FeCl2 and NTA powder at a 1:1.2 molar ratio, dissolving them in anaerobic water, and thoroughly stirring to form a complex. Both stock solutions were sterilized by filtration through a 0.22 μm sterile membrane and then anaerobically sealed for later use. Before use, they were diluted to the final experimental concentrations of 1 mM Fe(II) and 1 mM Fe(II) and 1.2 mM NTA, respectively. Simultaneously, a 1 M solution was prepared... 13 C-labeled NaHCO3 stock solution was filtered and sterilized, then anaerobic for later use. Also prepared sterile liquid MSM, 1 mM sodium lactate solution, 100 mL sterile serum bottle, pipette, sterile pipette tips, high-purity N2, centrifuge tubes and other necessary consumables and reagents for the experiment.

[0081] 2. Experimental group design

[0082] The entire reaction system was constructed in a sterile anaerobic operating room. Three experimental groups were set up: a blank control group (CK), a free Fe(II) group, and an organically bound Fe(II) group, with three biological replicates in each group. The total volume of the reaction system for each experimental group was 50 mL. The blank control group consisted of 49 mL of liquid MSM containing 1 mM sodium lactate, 1 mL of logarithmic seed culture, and a final concentration of 10 mM sodium lactate added to a 100 mL sterile serum bottle. 13 C-labeled NaHCO3 was used without adding iron. The free Fe(II) group was prepared by adding free Fe(II) stock solution to the blank control group to make the final FeCl2 concentration 1 mM. The organically bound Fe(II) group was prepared by adding Fe(II)-NTA stock solution to the blank control group to make the final Fe(II) concentration 1 mM and the final NTA concentration 1.2 mM. After adding samples to each system, they were thoroughly mixed. High-purity N2 was continuously purged into each serum bottle for no less than 10 min to completely replace the air in the bottle and ensure that the oxygen concentration of the system was below 0.1%. Then the bottle mouth was sealed with butyl rubber stopper and pressed tightly with aluminum cap to complete the construction of all experimental systems.

[0083] 3. Cultivation conditions

[0084] All sealed serum bottles were placed in a 28°C constant temperature incubator for uniform incubation. A 40W incandescent light source was turned on and fixed at a vertical distance of 20 cm from the serum bottles to provide uniform and continuous light for all experimental groups. The culture environment was kept sealed throughout the entire incubation process to prevent the serum bottles from failing to seal and to ensure that the system maintained a strictly anaerobic state. The incubation was carried out under constant temperature and light for 8 days. During the incubation period, the serum bottles were not shaken or opened to avoid the entry of external oxygen and affecting the experimental results.

[0085] 4. Detection Indicators and Methods

[0086] 4.1 Fe(II) concentration and oxidation efficiency

[0087] In this experiment, Fe(II) concentration, photooxidation efficiency, and bacterial growth were simultaneously measured on an anaerobic operating table at days 0, 2, 4, 6, and 8 of culture. One mL of bacterial culture from each replicate was placed in a sterile anaerobic centrifuge tube. Fe(II) concentration was determined using the phenanthridine method. Anaerobic 1M HCl was added to the sample to dissociate the Fe(II)-organic complex. After reaction with phenanthridine chromogenic reagent, absorbance was measured at 562 nm using a visible spectrophotometer. The concentration was calculated according to the Fe(II) standard curve. Photooxidation efficiency was calculated using the formula: (initial Fe(II) concentration - remaining Fe(II) concentration after culture) / initial Fe(II) concentration × 100%. No calculations were performed for the blank control group. Results... Figure 2 As shown in (a) of the diagram.

[0088] Figure 2 (a) is a graph showing the dynamic changes in the concentrations of free Fe(II) and organically bound Fe(II) when Rhodopseudomonas palustris SX2 oxidizes iron in different forms according to the embodiments of this application.

[0089] according to Figure 2 As can be seen in (a), after 8 days of culture, no iron oxidation was detected in the blank control group. The Fe(II) concentration in the free Fe(II) group decreased from the initial 1.0 mM to about 0.5 mM, with an oxidation efficiency of about 50%. The Fe(II) concentration in the organically bound Fe(II) group decreased from the initial 1.0 mM to below 0.15 mM, with an oxidation efficiency of over 85%.

[0090] 4.2 Cell growth

[0091] The bacterial growth was measured by an optical density (OD) of the bacterial suspension at a wavelength of 600 nm using a visible spectrophotometer. 600 Using liquid MSM containing 1 mM sodium lactate as a blank reference to eliminate background interference, OD was recorded at each time point. 600 Value representation, results as follows Figure 2 As shown in (b) of the diagram.

[0092] Figure 2 (b) shows the bacterial OD values ​​of Rhodopseudomonas palustris SX2 in three sets of experiments according to the embodiments of this application. 600 Growth curve.

[0093] according to Figure 2 As can be seen in (b), the OD of the blank control group 600 The OD of the free Fe(II) group is approximately 0.55. 600 Approximately 0.50, OD of organically bound Fe(II) group 600 The growth rate was approximately 0.72, and its growth was significantly higher than that of the control group and the free Fe(II) group.

[0094] 4.3 Inorganic carbon fixation capacity

[0095] Inorganic carbon fixation capacity was determined at the end of the 8-day culture experiment. The serum bottle was opened on an anaerobic workbench, and the bacterial culture was transferred to a sterile centrifuge tube. The tube was centrifuged at 4°C and 8000 r / min for 10 min, and the supernatant was discarded. The bacterial pellet was resuspended in sterile phosphate buffer (pH 7.2) and washed twice to remove surface adsorbates. The washed bacterial pellet was freeze-dried to constant weight, ground into powder, and passed through a 100-mesh sieve. An appropriate amount of powder was used to determine the δ¹⁸O values ​​using an isotope ratio mass spectrometer (IRMS, MAT253Plus). 13 The C value, with Vienna Pinisi stone (VPDB) as the standard reference, is measured with an accuracy of ±0.15‰, based on δ 13 The C-value characterizes the inorganic carbon fixation capacity of the strain, and the results are as follows: Figure 2 As shown in (c) in the figure.

[0096] Figure 2 (c) in the text represents the δ-cell count of *Rhodopseudomonas palustris* SX2 culture endpoint in the embodiments of this application. 13 C test results.

[0097] according to Figure 2 As can be seen in (c), the bacterial cell δ of the blank control group... 13 C is approximately 1‰, with almost no inorganic carbon fixation, and the free Fe(II) group of bacteria has a high δ-carbon content. 13 C is approximately 4.3‰, and the δ of organically bound Fe(II) group bacteria is... 13 The carbon content was approximately 12.5‰. Both groups achieved significant inorganic carbon fixation, with the latter showing significantly stronger carbon fixation capacity (p < 0.001).

[0098] Key findings: Rhodopseudomonas palustris SX2 exhibits significantly better photooxidation efficiency for organically bound Fe(II) than for free Fe(II), and the photooxidation efficiency is positively correlated with cell growth and inorganic carbon fixation capacity, indicating that the photooxidation process of this strain is highly coupled with cell metabolism and carbon fixation.

[0099] Example 3 Characterization of photoiron oxidation products of Rhodopseudomonas palustris SX2

[0100] 1. Sample preparation

[0101] After the culture reached the experimental endpoint, the bacterial cells and iron mineral mixture of each experimental group were collected under anaerobic conditions. The mixture was placed in a centrifuge tube, and the precipitate was collected by low-temperature centrifugation. The precipitate was then washed multiple times with sterile deionized water to remove unreacted culture medium and soluble impurities. The washed sample was then freeze-dried to obtain a solid sample that could be used for subsequent characterization and analysis. At the same time, the blank control group was prepared using the same centrifugation, washing, and freeze-drying process.

[0102] 2. Characterization methods

[0103] 2.1 High-resolution transmission electron microscopy (HR-TEM)

[0104] The prepared samples were first pre-fixed with glutaraldehyde, then fixed a second time with osmium tetroxide and uranium acetate, and then dehydrated stepwise with graded ethanol. After that, the samples were embedded in resin and made into ultrathin sections. The spatial distribution, micromorphology and particle size of iron minerals were observed using a JEOL-2100F high-resolution transmission electron microscope. At the same time, high-resolution lattice fringe imaging was performed on the target area to determine and analyze the lattice spacing of iron minerals.

[0105] 2.2 Energy-dispersive X-ray spectroscopy (EDS)

[0106] Using an EDS detector paired with HR-TEM, the target area of ​​iron-bearing minerals was selected under a transmission electron microscope. Elemental surface distribution scanning and qualitative and quantitative analysis of elemental composition were performed on the sample to obtain images of the types, relative contents, and spatial distribution of the main elements in the sample. The co-location characteristics of iron with other related elements were clarified, thereby determining the elemental composition and occurrence state of the iron minerals.

[0107] 2.3 X-ray diffraction (XRD)

[0108] After freeze-drying, the iron mineral sample was thoroughly ground into a uniform powder. An appropriate amount of sample was spread evenly on an XRD sample holder and compacted to ensure a smooth and impurity-free sample surface. A Bruker D8 Advance XRD instrument was used with Cu Kα as the radiation source (λ=1.5406Å). Appropriate test parameters were set (e.g., tube voltage 40 kV, tube current 40 mA, scanning range 2θ=10°~80°, scanning step size 0.02°, scanning speed 5° / min) to perform X-ray diffraction tests on the sample and obtain diffraction patterns. By comparing the measured diffraction peak positions and intensities with JCPDS standard cards, the crystal structure characteristics, phase composition, and crystallinity of the iron mineral were analyzed to determine the specific phase type of the iron mineral.

[0109] 3. Characterization results of photoferric oxidation products

[0110] Figure 3 This is the HR-TEM and EDS characterization diagrams of the photoferric oxidation products of Rhodopseudomonas palustris SX2 in Example 3 of this application. (a) and (f) are the TEM diagrams of the bacterial cells in the free and organically bound Fe(II) groups respectively; (b) and (g) are the high-resolution lattice fringe diagrams of iron minerals; (c)-(e) and (h)-(j) are the Fe, S, and P elemental surface distribution diagrams of iron minerals; (k) is the TEM diagram of the bacterial cells in the blank control group; (l) is the high-resolution lattice fringe diagram of iron minerals in the blank control group; (m)-(o) are the Fe, S, and P elemental surface distribution diagrams of iron minerals in the blank control group;

[0111] 3.1 Spatial distribution characteristics

[0112] Combined with the observations of HR-TEM and the results of EDS elemental analysis, the iron-related particles in the samples of each experimental group were located and morphologically characterized, and the results are as shown in (a) and (f) in Figure 3 as follows.

[0113] Figure 3 In (a) and (f) in the following, they are the TEM diagrams of the bacterial cells in the free and organically bound Fe(II) groups of the photoferric oxidation products of Rhodopseudomonas palustris SX2 in Example 3 of this application respectively.

[0114] According to Figure 3 in (a) and (f) as follows, it can be seen that in the free Fe(II) group, the iron-rich aggregate nanoparticles are mainly distributed in the periplasmic space and on the cell membrane surface through HR-TEM imaging. In the organically bound Fe(II) group, it is determined that the formed iron minerals are intracellular nanoparticles, and through image particle size statistical analysis, it is obtained that the particle size of the iron minerals in this group is significantly larger than that in the free Fe(II) group; the blank control group was detected by the same characterization process, and no iron-containing particles were found, so as to clarify the spatial distribution differences and particle size characteristics of the iron minerals formed by R. palustris SX2 under different iron forms.

[0115] 3.2 Elemental composition characteristics

[0116] With the help of the EDS detector supporting HR-TEM, elemental surface distribution scanning and composition analysis were carried out on the two groups of iron mineral samples, and the results are as shown in (c)-(e) and (h)-(j) in Figure 3 as follows.

[0117] Figure 3 In (c)-(e) and (h)-(j) in the following, they are the Fe, S, and P elemental surface distribution diagrams of the iron minerals of the photoferric oxidation products of Rhodopseudomonas palustris SX2 in Example 3 of this application.

[0118] according to Figure 3 As shown in (c)~(e) and (h)~(j), Fe, P, and S, the three core elements, were clearly detected in both the free Fe(II) group and the organically bound Fe(II) group of iron minerals. Furthermore, the elemental surface distribution images revealed a significant spatial co-localization characteristic among these three elements. Combined with biochemical analysis, it was found that P mainly originates from the phospholipid components of the bacterial cell membrane, while S primarily comes from sulfur-containing amino acids in proteins. This co-localization phenomenon indicates a close binding relationship between the formed iron minerals and biomolecules such as phospholipids and proteins in the bacterial cells, thus confirming that biomolecules participate in the formation process of iron minerals and their subsequent stability maintenance.

[0119] 3.3 Crystal Structure and Phase Characteristics

[0120] High-resolution lattice fringes imaging of two groups of iron mineral samples was performed using HR-TEM, and the results are as follows: Figure 3 As shown in (b) and (g) in the figure.

[0121] Figure 3 (b) and (g) are high-resolution lattice fringes of iron minerals produced by the photoiron oxidation of Rhodopseudomonas swampus SX2 in the embodiments of this application.

[0122] according to Figure 3 As can be seen from (b) and (g) in the data, the lattice spacing of the iron minerals in both groups of samples is approximately 0.338 nm, which is highly consistent with the characteristic lattice spacing of goethite.

[0123] Simultaneously, a Bruker D8 Advance XRD instrument (Cu Kα radiation) was used to perform phase analysis on the sample, and the results are as follows: Figure 4 As shown.

[0124] according to Figure 4 It can be seen that the obtained diffraction pattern exhibits broad and weak diffraction peaks, and does not show the sharp reflection peaks corresponding to well-crystallized iron minerals (such as goethite and lepidocrocite).

[0125] Based on the above characterization results, it is clear that the iron minerals produced by the photo-oxidation of iron by this strain are goethite-type short-range ordered nanocrystalline minerals. It is further inferred that the phospholipids, proteins and other biomolecules secreted by the bacteria play a key role in the formation of iron minerals. By tightly binding with the iron minerals, they inhibit their long-range crystal growth and phase transformation processes, ultimately forming a short-range ordered nanocrystalline structure.

[0126] Key findings: The photooxidation products of different iron forms by Rhodopseudomonas palustris SX2 are all short-range ordered nanocrystalline minerals of the goethite type, accompanied by co-localization of P and S elements; different iron forms only affect the spatial distribution and particle size of iron minerals, without changing the core phase and crystal structure characteristics of iron minerals.

[0127] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of this application. It should be understood that the above descriptions are merely specific embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A species of Rhodopseudomonas palustris SX2, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38191.

2. An application of Rhodopseudomonas palustris SX2, wherein, It is at least one of the following: (1) Oxidized free Fe(II); (2) Oxidation of organically bound Fe(II); (3) Oxidize soluble Fe(II) into insoluble iron minerals; (4) Phototrophic iron oxidation under anaerobic light conditions; (5) Coupled bacterial growth during iron oxidation; (6) Fixing inorganic carbon during iron oxidation; (7) Production of ordered nanocrystalline iron minerals; (8) Iron minerals are generated in the periplasmic space and / or on the cell membrane surface of the bacteria; (9) Intracellular nanoparticle iron minerals are generated.

3. An application of Rhodopseudomonas palustris SX2, wherein, It is at least one of the following: (1) Used for environmental remediation; (2) Used for pollution control; (3) Used for the control of soluble Fe(II) pollution; (4) Used to drive the biogeochemical cycle of iron; (5) Used for sewage treatment; (6) Used for iron resource utilization.

4. An application of Rhodopseudomonas palustris SX2, wherein, It is at least one of the following: (1) Used for research on the biogeochemical cycle of iron; (2) As a phototrophic iron mineralization microbial resource; (3) Used to study the microbial oxidation efficiency of various iron forms; (4) Used to study the formation mechanism of microbial-mediated iron mineralization products.

5. A method for iron oxidation, wherein, This includes the culture step of inoculating Rhodopseudomonas palustris SX2 into soluble Fe(II).

6. The method according to claim 5, wherein, It also includes adding an organic carbon source during the cultivation process, preferably sodium lactate.

7. The method according to claim 5 or 6, wherein, The culture environment is an anaerobic light environment.

8. A composition, characterized in that, The composition contains Rhodopseudomonas palustris SX2.

9. The composition according to claim 8, wherein, The composition is a microbial agent.

10. A culture wherein, Includes Rhodopseudomonas palustris SX2, mineral salt culture medium, and sodium lactate.

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