Directional enrichment culture method of iron-oxidizing microorganisms
Through the directional enrichment culture method of reverse ferrite concentration gradient tube and improved culture medium, the problem of time-consuming and poor functional performance of iron oxidized microorganisms is solved, and efficient enrichment and separation of iron oxidized microorganisms is achieved, providing technical support for its research and application in environmental biogeochemical cycles.
Patent Information
- Application Number
- CN202510308802.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-17
- Publication Date
- 2025-05-06
AI Technical Summary
The prior art has the problem that large-scale cultivation of iron oxidized microorganisms under laboratory conditions has the problem of long time, difficulty in obtaining diverse microbial resources in nature, and pure cultured microorganisms have poor performance as well as mixed microbial communities.
Using a directional enrichment culture method designed by reverse ferrite concentration gradient tube and light culture combined with improved 9K liquid mineral culture medium, iron oxidation microorganisms were isolated by continuous passage and gradient dilution to form a circular iron oxidation band to identify their pure cultures.
It realizes efficient enrichment and separation of iron oxidized microorganisms, provides an efficient and controllable culture system, and promotes the research and industrial application of iron oxidized microorganisms in environmental biogeochemical cycles.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and in particular to a directed enrichment and cultivation method for iron-oxidizing microorganisms. Background Art
[0002] Iron (Fe) is the second most abundant metal element in the Earth's crust and is one of the essential nutrients for organisms. Iron plays a vital role in many key biochemical pathways, such as respiration, photosynthesis, denitrification, and nitrogen fixation. Especially under anaerobic conditions, Fe(II) and Fe(III) can be used as electron sources and terminal electron acceptors by iron-oxidizing and iron-reducing microorganisms, respectively. The redox cycle of Fe is not only an important part of geochemical processes, but also plays a key role in the formation of the electron transport network in the Earth's crust, affecting the migration and transformation of carbon, nitrogen, sulfur and other metal elements in the soil.
[0003] In the natural environment, Fe mainly completes redox reactions with the participation of microorganisms. Iron-oxidizing microorganisms generate Fe(III) by oxidizing Fe(II), and this process is essential for the iron cycle in soil. Iron-oxidizing microorganisms can be divided into microaerobic and anaerobic types according to the characteristics of their oxidation reactions. Anaerobic iron-oxidizing microorganisms are further subdivided into anoxygenic photosynthetic and nitrate (or sulfate) reducing types. Under anaerobic conditions, photoautotrophic iron-oxidizing microorganisms oxidize Fe(II) and use light energy to fix CO2 into organic matter by using c-type cytochromes. Nitrate (or sulfate) reduction-dependent Fe(II) oxidizing microorganisms couple Fe(II) oxidation with the reduction of nitrate and sulfate. Microaerobic iron-oxidizing microorganisms can oxidize Fe(II) through an oxygen (O2)-dependent mechanism under low oxygen content and near-neutral conditions, and produce a large amount of amorphous Fe(III) hydroxide. These microorganisms are widely present in iron-rich weakly acidic or near-neutral environments, such as groundwater and soil, and play an important role in iron oxidation, with a profound impact on the biogeochemical cycle of iron and other biogenic elements.
[0004] Although a large number of studies have revealed the mechanism of Fe oxidation driven by microorganisms, there are still many problems in the large-scale cultivation of iron-oxidizing microorganisms under laboratory conditions. On the one hand, the traditional microbial isolation method is mainly based on plate separation, and functional microorganisms need to be repeatedly purified to obtain pure culture, but this process is time-consuming and it is difficult to obtain diverse microbial resources in nature; on the other hand, the functional performance of pure cultured microorganisms in complex biological systems is usually not as good as that of mixed microbial communities, and the scope of utilization of complex substrates is limited, which restricts the actual application effect, causing problems such as slow growth and low biomass, and limiting the progress of its genetic research. Therefore, the research and development of effective targeted enrichment methods has important scientific significance and application value for a deep understanding of the metabolic pathways of Fe(II) oxidation and its microbial functions. Summary of the invention
[0005] The present invention provides a method for the targeted enrichment and cultivation of iron-oxidizing microorganisms, aiming to effectively enrich and separate microbial communities with iron oxidation ability from soil samples, thereby providing an efficient and controllable cultivation system for related research and promoting the research and industrial application of iron-oxidizing microorganisms in environmental biogeochemical cycles.
[0006] Another object of the present invention is to provide a method for identifying the above-mentioned iron-oxidizing microorganisms.
[0007] Another object of the present invention is to provide a method for determining the iron oxidation capacity of iron-oxidizing microorganisms
[0008] Another object of the present invention is to provide the use of the above-mentioned iron-oxidizing microorganisms in an iron oxidation process.
[0009] In order to achieve the above purpose, the technical solutions provided are as follows:
[0010] In a first aspect, the present invention provides a method for targeted enrichment and cultivation of iron-oxidizing microorganisms, characterized in that it comprises the following steps:
[0011] Step 1, preparing inoculation liquid: mixing fresh biological soil crust samples at different development stages with physiological saline at a ratio of 1:1 to form inoculation liquid;
[0012] Step 2: Prepare a reverse iron-oxygen concentration gradient tube: First, prepare 10.0 mL of the modified 9K liquid mineral culture medium and add it to a 15 mL culture tube; then, add 1.25 mL of freshly prepared iron sulfide (FeS) suspension to the culture medium. FeS will form a FeS layer at the bottom of the culture tube as an iron source, providing reducing ability while maintaining the redox gradient; finally, add NaHCO3 with a final concentration of 5 mmol / L, and after standing for 24 hours, a reverse iron-oxygen concentration gradient is formed in the tube;
[0013] Step 3: Screening culture: Add 0.5 mL of inoculum into the gradient tube and culture at 30°C for 15 days under light conditions; iron-oxidizing microorganisms preferentially grow at the redox interface, forming a distinct biofilm or iron oxidation zone; after the culture, a reddish-brown iron oxidation zone can be observed on the interface, indicating that active iron oxidation occurs;
[0014] Step 4: Continuous subculture: The iron-oxidizing microorganisms were isolated by continuous subculture and gradient dilution. The iron-oxidizing cell band in the enrichment culture was subcultured for 3 generations and then the gradient dilution method (dilution range of 10 -3 Up to 10 -8 ) were inoculated into new gradient tubes;
[0015] Step 5. Isolation of microorganisms: The cells grown in the highest dilution are further isolated and the operation is repeated until a pure culture is obtained to obtain iron-oxidizing microorganisms characterized by the formation of an annular iron-oxidizing zone at a specific depth in the tube.
[0016] Preferably, in step 2, the improved 9K liquid mineral culture medium comprises the following components: 3.0 g / L (NH4)2SO4, 0.1 g / L NaNO4, 0.1 g / L K2HPO4, 0.5 g / L KCl, 1 μL·mL -1 Vitamin C, 0.5g / L MgSO4·7H2O and 0.01g / L Ca(NO3)2·2H2O.
[0017] Preferably, in step 2, the pH value of the improved 9K liquid mineral culture medium is 7.00.
[0018] In a second aspect, the present invention provides a method for identifying iron-oxidizing microorganisms, characterized in that it comprises the following steps:
[0019] Step 1, extracting microbial DNA from iron-oxidizing microorganisms;
[0020] Step 2: amplify the 16S rDNA sequence using a bacterial 16S rDNA universal primer pair;
[0021] Step 3: Analyze community structure and phylogenetic information through amplicon sequencing.
[0022] Preferably, in step one, the whole genome DNA of the iron oxidizing microorganisms is extracted using an EZNA soil DNA extraction kit.
[0023] Preferably, in step 2, the bacterial 16S rDNA universal primer pair includes upstream primer 515F: 5'
[0024] -GTGCCAGCMGCCGCGGTAA-3', downstream primer 806R: 5'-GGACTACHVGGGTWTCTAAT-3'.
[0025] Preferably, in step three, the DNA quality is detected using a NanoDrop 2000c spectrophotometer to ensure that samples with A260 / A280>1.7 and A260 / A230>1.8 are used for subsequent experiments.
[0026] In a third aspect, the present invention also provides a method for determining the iron oxidation capacity of enriched microorganisms, characterized in that it comprises the following steps:
[0027] Step 1. Sample preparation: Take the iron oxidation zone (about 1-2 mL) formed in the iron sulfide gradient tube, centrifuge at 8000 rpm for 5 minutes, discard the supernatant, and dilute the precipitate to 1 mL with deionized water;
[0028] Step 2, detection of Fe(II) oxidation products: the sample treated in step 1 was quickly added to 10 mL of 0.5 mol / L HCl solution and extracted on a shaker at 150 rpm for 1.5 hours; then, the total iron concentration of the resulting solution was determined using the o-phenanthroline-spectrophotometer method, and each sample was measured three times to ensure the accuracy of the results;
[0029] Step 3: Data Analysis
[0030] The Fe(II) oxidation capacity of the enriched microorganisms was evaluated by calculating the concentration of iron oxidation products.
[0031] In a fourth aspect, the iron-oxidizing microorganisms obtained by the present invention are used for the research and industrial application of iron oxidation processes in biogeochemical cycles.
[0032] The beneficial effects of the present invention are:
[0033] (1) Efficient enrichment: Through reverse iron-oxygen concentration gradient, light culture and optimized culture medium design, the present invention can effectively enrich a variety of iron-oxidizing microorganisms, especially microaerobic iron-oxidizing microorganisms.
[0034] (2) Simple operation: The enrichment culture method is simple and easy to operate, suitable for laboratory-scale operation, and can obtain highly efficient enrichment of iron-oxidizing microorganisms in a relatively short time.
[0035] (3) Phylogenetic analysis: Through amplicon sequencing and community structure analysis, the present invention can provide detailed phylogenetic information on the functions and ecological effects of iron-oxidizing microbial populations.
[0036] (4) Strong repeatability: The culture method and determination technology of the present invention can be repeated many times and is applicable to soil samples from different sources, with good versatility and application prospects.
[0037] The method for directional enrichment and cultivation of iron-oxidizing microorganisms of the present invention provides strong technical support for further studying the ecological functions, metabolic pathways and roles of iron-oxidizing microorganisms in environmental biogeochemical cycles, and provides important technical guarantees for the development and application of microbial resources. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1It is a phylum-level composition diagram of the amplicon sequencing results of the iron-oxidizing microbial community enriched in Example 1 of the present invention, 61Y, 53Y, 44Y, 30Y and 17Y respectively indicate that the biological soil crust samples used for inoculation have undergone a development process of 61 years, 53 years, 44 years, 30 years and 17 years; the control indicates that the inoculation solution is sterilized physiological saline, the same below;
[0039] Figure 2 It is a composition diagram at the genus level of the amplicon sequencing results of the iron-oxidizing microbial community enriched in Example 1 of the present invention;
[0040] Figure 3 This is a diagram showing the effect of FeS oxidation by the enriched iron-oxidizing microorganisms in Example 2 of the present invention. The gradient tubes from left to right are inoculated with inoculation solutions made from biological soil crust samples that have undergone 17 years, 30 years, 44 years, 53 years, and 61 years of development, respectively;
[0041] Figure 4 This is the result of the iron-oxidizing microorganisms enriched in Example 2 of the present invention oxidizing the FeS-free liquid to produce Fe(III). DETAILED DESCRIPTION
[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0043] Example 1
[0044] Enrichment culture and amplicon sequencing of iron-oxidizing microorganisms
[0045] (1) Preparation of culture medium and formation of gradient tubes
[0046] Add 1.0 mL of freshly prepared iron sulfide suspension to 10.0 mL of modified 9K liquid mineral medium. The composition of 9K liquid mineral medium is (unit: g / L): (NH4)2SO43.0, NaNO40.1, K2HPO40.1, KCl0.5, vitamin C (1 μL·mL -1 ), MgSO4·7H2O 0.5 and Ca(NO3)2·2H2O 0.01, and the pH value was adjusted to 7.00. A 1.25mL FeS layer was added to the bottom of the culture tube as an iron source to provide reducing power while maintaining the redox gradient. A final concentration of 5mmol·L -1 NaHCO3, after standing for 24 hours, a reverse iron-oxygen concentration gradient is formed in the tube.
[0047] (2) Inoculation and culture conditions
[0048] Fresh biological soil crust samples at different developmental stages were mixed with physiological saline at a ratio of 1:1 as inoculum. 0.5 mL of inoculum was added to the gradient tube and cultured at 30 °C for 15 days under light conditions. Iron-oxidizing microorganisms preferentially grow at the redox interface, forming a distinct biofilm or iron oxidation zone. After the incubation, a reddish-brown iron oxidation zone can be observed on the interface, indicating that active iron oxidation occurs.
[0049] (3) Isolation and subculture of microorganisms
[0050] Iron-oxidizing microorganisms were isolated by continuous subculture and gradient dilution. After the iron-oxidizing cell band in the enrichment culture was subcultured for three generations, the gradient dilution method (dilution range of 10 -3 Up to 10 -8 ) were inoculated into new gradient tubes. Cells grown in the highest dilution were further isolated and the procedure repeated until a pure culture was obtained, characterized by the formation of a ring-like iron oxidation zone at a specific depth in the tube.
[0051] (4) DNA extraction and sequencing: The whole genome DNA of the enriched microorganisms was extracted using the EZNA soil DNA extraction kit;
[0052] Furthermore, the DNA quality was tested using a NanoDrop 2000c spectrophotometer to ensure that samples with A260 / A280>1.7 and A260 / A230>1.8 were used for subsequent experiments;
[0053] Furthermore, PCR amplification and purification were performed using bacterial 16S rDNA universal primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′);
[0054] Furthermore, the purified PCR products were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd., and the amplicon sequencing was performed using the Illumina Miseq PE300 platform. The sequencing data was returned via fastq files, and the phylogenetics and community structure of the enriched iron-oxidizing microorganisms were visualized and analyzed. The original sequencing data was visualized by clustering the taxonomic units OTU using RStudio software. The main analysis steps included quality control operations, redundancy removal operations, clustering operations, leveling operations, and species annotation.
[0055] Figure 1The composition characteristics of the iron-oxidizing microbial community at the phylum level in the enrichment culture system are shown as follows: the iron-oxidizing microbial community at the phylum level of the enrichment culture is composed of Firmicutes, Proteobacteria, Actinobacteria and Desulfobacterota. It should be noted that the iron-reducing microbial community obtained by enrichment culture using biological soil crusts developed for 17, 30, 44 and 53 years has a low relative abundance; the relative abundance of the iron-reducing microbial community obtained by enrichment culture using biological soil crusts developed for 61 years is the highest.
[0056] Figure 2 The composition of microorganisms at the genus level was further revealed: Enterococcus, Burkholderia, Gallionella and Pseudomonas were the dominant genera. The dominant iron-reducing microorganisms obtained by enrichment culture using biological soil crusts developed for 17, 30, 44 and 53 years all had low relative abundances; the dominant iron-reducing microorganisms obtained by enrichment culture using biological soil crusts developed for 61 years had the highest relative abundance, showing a significant time-dependent enrichment effect.
[0057] Example 2
[0058] Determination of Fe(II) Oxidation Capacity of Enriched Iron-Oxidizing Microorganisms
[0059] (1) Sample preparation
[0060] Take the iron oxidation zone (about 1-2 mL) formed in the iron sulfide gradient tube, centrifuge at 8000 rpm for 5 minutes, discard the supernatant, and dilute the precipitate to 1 mL with deionized water.
[0061] (2) Detection of Fe(II) oxidation products
[0062] The sample after the above treatment is rapidly added to 10mL 0.5mol / L HCl solution and leached for 1.5 hours on a 150rpm shaking table. Subsequently, the total iron concentration of the resulting solution is determined using o-phenanthroline-spectrophotometer method. Each sample is repeated 3 times to ensure the accuracy of the result.
[0063] (3) Data analysis
[0064] The Fe(II) oxidation capacity of the enriched microorganisms was evaluated by calculating the concentration of iron oxidation products. The experimental results showed that active iron-oxidizing microorganisms can significantly improve the efficiency of iron oxidation, further verifying their functional characteristics and biological effects.
[0065] Depend on Figure 4 It can be seen that after continuous enrichment culture, the iron-oxidizing enrichment culture method, the iron-reducing microorganisms showed significant iron-reducing ability, and the Fe(III) ion concentration in the culture solution gradually increased to 1.66-2.67 mmol / L. Among them, the iron-oxidizing microorganisms enriched and cultured using biological soil crust that had developed for 61 years had the strongest iron-reducing ability.
[0066] Figure 3 Describing the morphological differentiation of iron-oxidizing microorganisms during enrichment culture: When pH, redox potential, O2 and Fe in the gradient tube 2+ When the contents of and reached the appropriate range, Gallionella-type iron-oxidizing microorganisms that produced spiral stalks loosely attached to the tube wall. Their colonies were initially white flocculent and grew radially (<1 mm), then gradually turned rust red and eventually fell off the tube wall; the colonies of unicellular iron-oxidizing microorganisms that did not produce special extracellular structures were small and usually scattered on the tube wall. From the enrichment culture of iron-oxidizing species of biological soil crusts at different developmental stages, the 17Y and 30Y samples had fewer colonies attached to the tube wall, but there was rust-red colony precipitation at the bottom. The 61Y, 53Y and 44Y samples had more rust-red colonies attached to the tube wall, but less rust-red colony precipitation at the bottom.
[0067] Figure 4 Quantitative characterization of iron oxidation metabolic efficiency: In the FeS substrate system, the amount of Fe(Ⅲ) generated directly reflects the iron oxidation activity of microorganisms. Experimental data showed that the peak Fe(Ⅲ) concentration of the 61Y sample reached 2.67±0.12mmol / L (n=3), which was significantly higher than that of the 53Y (2.39±0.09mmol / L), 44Y (2.20±0.11mmol / L), 17Y (1.95±0.08mmol / L) and 30Y (1.66±0.10mmol / L) samples (P<0.05, ANOVA-Tukey test), confirming that the enriched bacterial community in the late stage of BSC development (53-61 years) has stronger iron oxidation metabolic capacity. The Fe(Ⅲ) concentration in the sterilized control group was only 0.19±0.03mmol / L (n=3), which was 88.3%-92.9% lower than that in the experimental group (P<0.01), confirming that the microbial-mediated iron oxidation process under neutral microaerobic conditions was dominant.
[0068] It can be seen from the above examples that by adopting the method for enriching iron-oxidizing microorganisms provided by the present invention, an enriched bacterial population with strong iron-oxidizing ability can be obtained in a relatively short time.
[0069] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.
Claims
1. A method for directional enrichment and cultivation of iron-oxidizing microorganisms, characterized in that: The steps include: Step 1, preparing inoculation liquid: mixing fresh biological soil crust samples at different development stages with physiological saline at a ratio of 1:1 to form inoculation liquid; Step 2: Prepare a reverse iron-oxygen concentration gradient tube: First, prepare 10.0 mL of the modified 9K liquid mineral culture medium and add it to a 15 mL culture tube; then, add 1.25 mL of freshly prepared FeS suspension to the culture medium. FeS will form a FeS layer at the bottom of the culture tube as an iron source, providing reducing ability while maintaining the redox gradient; finally, add NaHCO3 with a final concentration of 5 mmol / L, and after standing for 24 hours, a reverse iron-oxygen concentration gradient will be formed in the tube; Step 3: Screening culture: Add 0.5 mL of inoculum into the gradient tube and culture at 30°C for 15 days under light conditions; iron-oxidizing microorganisms preferentially grow at the redox interface, forming a distinct biofilm or iron oxidation zone; after the culture, a reddish-brown iron oxidation zone can be observed on the interface, indicating that active iron oxidation occurs; Step 4. Continuous subculture and separation of microorganisms: Use continuous subculture and gradient dilution method to separate iron-oxidizing microorganisms. After the iron-oxidizing cell band in the enrichment culture is subcultured for 3 generations, it is inoculated into a new gradient tube by gradient dilution method. The cells grown in the highest dilution are further separated and the operation is repeated until a pure culture is obtained to obtain iron-oxidizing microorganisms.
2. The method for directional enrichment and cultivation of iron-oxidizing microorganisms according to claim 1, characterized in that: In step one, the development age of the biological soil crust sample is 17 to 61 years.
3. The method for directional enrichment and cultivation of iron-oxidizing microorganisms according to claim 2, characterized in that: In step one, the development age of the biological soil crust sample is 61 years.
4. The method for directional enrichment and cultivation of iron-oxidizing microorganisms according to claim 1, characterized in that: In step 2, the modified 9K liquid mineral medium includes the following components: 3.0 g / L (NH4)2SO4, 0.1 g / L NaNO4, 0.1 g / L K2HPO4, 0.5 g / L KCl, 1 μL·mL -1 Vitamin C, 0.5g / L MgSO4·7H2O and 0.01g / L Ca(NO3)2·2H2O.
5. The method for directional enrichment and cultivation of iron-oxidizing microorganisms according to claim 1, characterized in that: In step 2, the pH value of the improved 9K liquid mineral culture medium is 7.
00.
6. The method for directional enrichment and cultivation of iron-oxidizing microorganisms according to claim 1, characterized in that: In step 5, the dilution range of the gradient dilution method is 10 -3 Up to 10 -8 .
7. A method for identifying iron-oxidizing microorganisms, characterized in that: The following steps are involved: Microbial DNA was extracted from iron-oxidizing microorganisms; 16S rDNA sequences were amplified using bacterial 16S rDNA universal primer pairs; community structure and phylogenetic information were analyzed by amplicon sequencing.
8. A method for identifying iron-oxidizing microorganisms according to claim 6, characterized in that: The whole genomic DNA of iron-oxidizing microorganisms was extracted using the EZNA soil DNA extraction kit; the bacterial 16S rDNA universal primer pair included upstream primer 515F: 5'-GTGCCAGCMGCCGCGGTAA-3', downstream primer 806R: 5'-GGACTACHVGGGTWTCTAAT-3'; the DNA quality was detected using a NanoDrop 2000c spectrophotometer to ensure that samples with A260 / A280>1.7 and A260 / A230>1.8 were used for subsequent experiments.
9. A method for determining the iron oxidation capacity of enriched microorganisms, characterized in that: The steps include: Step 1. Sample preparation: Take the iron oxidation zone (about 1-2 mL) formed in the iron sulfide gradient tube, centrifuge at 8000 rpm for 5 minutes, discard the supernatant, and dilute the precipitate to 1 mL with deionized water; Step 2, detection of Fe(II) oxidation products: the sample treated in step 1 was quickly added to 10 mL of 0.5 mol / L HCl solution and extracted on a shaker at 150 rpm for 1.5 hours; then, the total iron concentration of the resulting solution was determined using the o-phenanthroline-spectrophotometer method, and each sample was measured three times to ensure the accuracy of the results; Step 3. Data analysis: Evaluate the iron oxidation capacity of iron-oxidizing microorganisms by calculating the concentration of iron oxidation products.
10. An application of iron-oxidizing microorganisms enriched by the method according to any one of claims 1 to 6, wherein the iron-oxidizing microorganisms are used for the study of iron oxidation processes in biogeochemical cycles and for industrial applications.