Separation and analysis method for soil boron isotope released by sulfur-oxidizing microbial functional flora

Abstract

The invention provides a separation and analysis method for soil boron isotope released by sulfur-oxidizing microbial functional flora, and belongs to the technical field of isotope analysis. According to the method, a stable sulfur oxidation functional flora is obtained through enrichment and passage domestication, boron in soil is efficiently activated through the metabolism effect of the stable sulfur oxidation functional flora with elemental sulfur as energy, a culture solution with complex components is further deeply purified through ion exchange resin, impurities are effectively removed, and high recovery of boron is achieved; and finally, the accurate determination of the boron isotope composition (delta < 11 > B) is realized by combining a high-precision multi-receiving inductively coupled plasma mass spectrometry technology.

Description

Technical Field

[0001] This invention belongs to the field of isotope analysis technology, specifically relating to a method for separating and analyzing soil boron isotopes released by functional microbial communities of sulfur-oxidizing microorganisms. Background Technology

[0002] Boron isotopes ( 10 B and 11 B) Due to their significant relative mass difference, boron undergoes substantial fractionation in physical, chemical, and biological processes, making them a key tool for tracing biogeochemical processes in surface systems. In soil, microorganisms are the core engine driving boron migration, transformation, and cycling. They can influence the speciation and spatial distribution of boron through a series of processes, including bioadsorption, extracellular complexation, and transmembrane transport, and trigger significant boron isotope fractionation. However, existing research largely focuses on non-biologically driven physicochemical processes, lacking precise quantitative assessment of the contribution and mechanisms of fractionation caused by microbial activity, constituting a significant gap in this field. Summary of the Invention

[0003] The purpose of this invention is to address the current lack of precise quantification and systematic analysis of microbial-driven soil boron isotope fractionation processes in existing technologies, and to provide a systematic analysis and quantitative assessment scheme for these processes. To this end, this invention provides a method for the separation and analysis of soil boron isotopes released by functional microbial communities of sulfur-oxidizing microorganisms.

[0004] This invention provides a method for separating and analyzing soil boron isotopes released by functional microbial communities that oxidize sulfur, comprising the following steps: ① Enrich the functional microbial community of sulfur-oxidizing microorganisms from plant rhizosphere soil; ② The sulfur-oxidizing microbial functional group was inoculated into a liquid culture medium containing the soil to be tested, and shaken culture was carried out to activate and release boron in the soil to be tested, and the boron-containing culture solution was collected. ③ The boron in the culture medium is separated and purified using an ion exchange resin to obtain a purified boron-containing solution; ④ The purified boron-containing solution was analyzed for boron isotope composition using multi-receiver inductively coupled plasma mass spectrometry.

[0005] Preferably, step ① involves enriching sulfur-oxidizing microbial functional communities from plant rhizosphere soil using a liquid culture medium, which includes: inoculating plant rhizosphere soil into a culture medium, performing shaking culture and / or subculturing, enriching sulfur-oxidizing microbial functional communities during the shaking culture and / or subculturing process, and obtaining a culture solution; the liquid culture medium is an inorganic salt culture medium with elemental sulfur as the sole energy source. The inorganic salt culture medium contains the following components: 0.3 g / L (NH4)2SO4, 0.01 g / L KCl, 0.05 g / L K2HPO4, 0.05 g / L MgSO4·7H2O, 0.001 g / L Ca(NO3)2 and 1 g / L elemental sulfur.

[0006] Preferably, the temperature for the shaking culture and / or subculture is 28~32℃; and the rotation speed for the shaking culture and / or subculture is 170~180 rpm. The subculture includes the following steps: when the cell density of the culture medium reaches (1~4)×10⁻⁶... 8 When the cells / mL is reached, transfer to fresh culture medium at an inoculum volume of 10%; The number of subcultures is 15 to 20.

[0007] Preferably, the liquid culture medium in step ② is an inorganic salt culture medium with elemental sulfur as the sole energy source; The inorganic salt culture medium contains the following components: 0.3 g / L (NH4)2SO4, 0.01 g / L KCl, 0.05 g / L K2HPO4, 0.05 g / L MgSO4·7H2O, 0.001 g / L Ca(NO3)2 and 1 g / L elemental sulfur.

[0008] Preferably, the inoculation amount of the sulfur-oxidizing microbial functional flora in step ② is 5-15% of the volume of the liquid culture medium; In step ②, the amount of soil to be tested added to the liquid culture medium is 1~10 g of soil to be tested per 100 mL of culture medium.

[0009] Preferably, the temperature of the shaking culture in step ② is 28~32℃; the rotation speed of the shaking culture is 170~180 rpm; and the shaking culture time is 9 days.

[0010] Preferably, the culture medium is pretreated before the separation and purification operation is performed; The pretreatment includes: centrifuging the culture medium collected in step ② at a centrifugal force of 1000~3000×g for 5~10min, collecting the supernatant, filtering the supernatant using a filter membrane with a pore size of 0.4~0.5 μm, collecting the filtrate, and adjusting the pH of the filtrate to 6~7 using a 0.1 M sodium hydroxide aqueous solution.

[0011] Preferably, the separation and purification includes separating and purifying boron in the culture medium using a three-step tandem method with ion exchange resins; The three-step tandem method of ion exchange resins includes: boron-specific adsorption resin for impurity removal, nitric acid elution, and anion-cation exchange resin for impurity removal.

[0012] Preferably, the plant rhizosphere soil includes the rhizosphere soil of one or more of the following plants: pepper, cucumber, tomato, wheat, and rapeseed.

[0013] The present invention also provides the application of the separation and analysis method described above in the separation and / or analysis of boron isotopes in soil, wherein the soil includes one or more of black soil, sandy loam, saline-alkali soil, brown soil and red soil.

[0014] Beneficial effects: This invention provides a method for separating and analyzing soil boron isotopes released by sulfur-oxidizing microbial functional communities, comprising the following steps: ① enriching sulfur-oxidizing microbial functional communities from plant rhizosphere soil; ② inoculating the sulfur-oxidizing microbial functional communities into a liquid culture medium containing the soil to be tested, performing shaking culture to activate and release boron from the soil to be tested, and collecting the boron-containing culture solution; ③ separating and purifying the boron in the culture solution using ion exchange resin to obtain a purified boron-containing solution; ④ performing boron isotope composition analysis on the purified boron-containing solution using multi-receiver inductively coupled plasma mass spectrometry.

[0015] This invention obtains a stable sulfur-oxidizing functional bacterial community through enrichment and subculturing. Utilizing its metabolic activity using elemental sulfur as an energy source, it efficiently activates boron in the soil. Furthermore, it employs ion exchange resin to deeply purify the complex culture medium, effectively removing impurities and achieving high boron recovery. Finally, combined with high-precision multi-collector inductively coupled plasma mass spectrometry, it achieves accurate determination of boron isotope composition (δ¹²). 11 B) Accurate determination. As verified by examples, this invention successfully established a microbial-mediated soil boron release system, accurately elucidating the microbial-driven process of soil boron isotope fractionation, filling a gap in microbial research on boron isotope fractionation. The method established in this invention provides a novel research paradigm for systematically tracking and quantitatively assessing the geochemical behavior and environmental effects of boron in the soil-microbe-solution system, and also provides reliable technical support for accurately quantifying the microbial-driven boron isotope fractionation effect and deeply analyzing the behavior of boron in biogeochemical cycles. Detailed Implementation

[0016] This invention provides a method for separating and analyzing soil boron isotopes released by functional microbial communities that oxidize sulfur, comprising the following steps: ① Enrich the functional microbial community of sulfur-oxidizing microorganisms from plant rhizosphere soil; ② The sulfur-oxidizing microbial functional group was inoculated into a liquid culture medium containing the soil to be tested, and shaken culture was carried out to activate and release boron in the soil to be tested, and the boron-containing culture solution was collected. ③ The boron in the culture medium is separated and purified using an ion exchange resin to obtain a purified boron-containing solution; ④ The purified boron-containing solution was analyzed for boron isotope composition using multi-receiver inductively coupled plasma mass spectrometry.

[0017] This invention enriches sulfur-oxidizing microbial functional communities from plant rhizosphere soil. As one embodiment, step ① of this invention, using a liquid culture medium to enrich sulfur-oxidizing microbial functional communities from plant rhizosphere soil, includes: inoculating plant rhizosphere soil into a culture medium, performing shaking culture and / or subculturing, enriching the sulfur-oxidizing microbial functional communities during the shaking culture and / or subculturing process, and obtaining a culture solution; the liquid culture medium is an inorganic salt culture medium using elemental sulfur as the sole energy source; the inorganic salt culture medium contains the following components: 0.3 g / L (NH4)2SO4, 0.01 g / L KCl, 0.05 g / L K2HPO4, 0.05 g / L MgSO4·7H2O, 0.001 g / L Ca(NO3)2, and 1 g / L elemental sulfur. In one embodiment, the temperature for the shaking culture and / or subculture of the present invention is 28~32℃; the rotation speed for the shaking culture and / or subculture is 170~180 rpm; the subculture includes the following steps: when the cell density of the culture medium reaches (1~4)×10 8 When the cells / mL concentration is 10%, the culture is transferred to fresh culture medium at an inoculum concentration of 10% by volume; the number of subcultures is 15-20 times. As one embodiment, the temperature for the shaking culture and / or subculture is 30°C; the rotation speed for the shaking culture and / or subculture is 175 rpm.

[0018] This invention involves inoculating the sulfur-oxidizing microbial functional flora into a liquid culture medium containing the soil to be tested, followed by shaking culture to activate and release boron from the soil, and collecting the boron-containing culture solution. As one embodiment, the liquid culture medium in step ② is an inorganic salt culture medium using elemental sulfur as the sole energy source; the inorganic salt culture medium contains the following components: 0.3 g / L (NH4)2SO4, 0.01 g / L KCl, 0.05 g / L K2HPO4, 0.05 g / L MgSO4·7H2O, 0.001 g / L Ca(NO3)2, and 1 g / L elemental sulfur. As one embodiment, the inoculation amount of the sulfur-oxidizing microbial functional flora in step ② is 5-15% of the volume of the liquid culture medium; the amount of soil to be tested added to the liquid culture medium in step ② is 1-10 g of soil to be tested per 100 mL of culture medium. In one embodiment, the shaking culture temperature in step ② of the present invention is 28~32℃; the shaking rotation speed is 170~180 rpm; and the shaking culture time is 9 days. In another embodiment, the shaking culture temperature is 30℃; and the shaking rotation speed is 175 rpm. In yet another embodiment, the inoculum size of the sulfur-oxidizing microbial functional flora is 10% of the liquid culture medium volume.

[0019] This invention uses ion exchange resin to separate and purify boron in the culture medium, obtaining a purified boron-containing solution. As one embodiment, before the separation and purification operation, the invention further includes pretreatment of the culture medium; the pretreatment includes: centrifuging the culture medium collected in step ② at 1000~3000×g for 5~10 min, collecting the supernatant, filtering the supernatant using a filter membrane with a pore size of 0.4~0.5 μm, collecting the filtrate, and adjusting the pH of the filtrate to 6~7 using a 0.1 M sodium hydroxide aqueous solution. As one embodiment, the separation and purification of this invention includes using a three-step tandem ion exchange resin method to separate and purify boron in the culture medium; the three-step ion exchange resin method includes: boron-specific adsorption resin for impurity removal, nitric acid elution, and anion and cation exchange resin for impurity removal. As one embodiment, in the pretreatment operation, the culture medium collected in step ② is centrifuged at 4000 rpm for 5 min. As one embodiment, the pore size of the filter membrane in this invention is 0.45 μm.

[0020] This invention employs multi-receiver inductively coupled plasma mass spectrometry to analyze the boron isotope composition of the purified boron-containing solution.

[0021] As one embodiment, the plant rhizosphere soil of the present invention includes the rhizosphere soil of one or more of the following plants: pepper, cucumber, tomato, wheat, and rapeseed.

[0022] This invention also provides the application of the separation and analysis method described above in the separation and / or analysis of boron isotopes in soil, wherein the soil includes one or more of black soil, sandy loam, saline-alkali soil, brown soil, and red soil. As one embodiment, this invention obtains stable sulfur-oxidizing functional bacterial communities through enrichment and subculturing, utilizes their metabolic activity using elemental sulfur as an energy source to efficiently activate boron in the soil, and further employs ion exchange resin to deeply purify the complex culture medium, effectively removing impurities and achieving high boron recovery. Finally, combined with high-precision multi-receiver inductively coupled plasma mass spectrometry, it achieves the analysis of boron isotope composition (δ¹² + δ¹²)... 11 B) Accurate determination. As one implementation method, this invention successfully established a microbial-mediated soil boron release system, accurately analyzed the microbial-driven soil boron isotope fractionation process, filled the gap in microbial research on boron isotope fractionation, provided a new research paradigm for systematically tracking and quantitatively evaluating the geochemical behavior and environmental effects of boron in the soil-microbe-solution system, and also provided reliable technical support for accurately quantifying the microbial-driven boron isotope fractionation effect and deeply analyzing the behavior of boron in biogeochemical cycles.

[0023] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a method for separating and analyzing soil boron isotopes released by functional microbial communities of sulfur-oxidizing microorganisms provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1 Separation and analysis methods for boron isotopes released from soil by microorganisms 1. Microbial community enrichment and domestication Pepper rhizosphere soil samples were inoculated at a ratio of 5% (w / v) into fresh culture medium powered by elemental sulfur and cultured with shaking at 28–32°C and 170–180 rpm. When the bacterial cell density in the culture reached 1.0–4.0 × 10⁻⁶, the inoculum was cultured. 8 When the number of cells / mL is 10%, transfer the inoculum to fresh culture medium and subculture for 15-20 times to obtain a stable bacterial population.

[0025] 2. Biological activation and release of boron in soil The stabilized sulfur-oxidizing microbial functional community was inoculated into fresh culture medium at an inoculation rate of 5-15% (v / v), and 1-2% (w / v) of the soil to be tested was added. The medium was then cultured under the same shaking conditions as in step 1 until the cell density reached 1.0-4.0 × 10⁻⁶ again. 8 The boron concentration was increased by 100 cells / mL to complete the activation and release of boron, resulting in a boron-containing culture medium.

[0026] 3. Leachate Pretreatment Centrifuge the above boron-containing culture medium at 1000~3000×g for 5~10 min, take the supernatant and filter it through a 0.40~0.50μm filter membrane, and adjust the pH of the filtrate to 6~7 with 0.1M sodium hydroxide solution to obtain a neutral solution.

[0027] 4. Isolation, purification, and isotope determination of boron Boron in the leachate was separated and purified using a three-step tandem ion exchange resin method (sequential adsorption with boron-specific resin, desorption and concentration with nitric acid, and deep purification with a mixed anion and cation exchange resin) to obtain a high-purity boric acid solution. The boron isotope composition was then determined using multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). The specific steps are as follows: ① Boron-specific resin was used to adsorb borate ions from the leachate; ② The borate-loaded resin column was desorbed with 0.1M nitric acid, and the desorbed solution was collected and concentrated by evaporation at 50-60°C in a forced-air drying oven to obtain a concentrated boron solution; ③ This concentrated solution was diluted with pure water and then deeply purified using a mixed anion and cation exchange resin column to collect a high-purity boric acid solution; ④ The obtained high-purity boric acid solution was evaporated at 50-60°C until a volume of 2-3 mL was reached, and then transferred to a centrifuge tube for analysis; ⑤ The boron isotope composition (δ¹²) of the solution was determined using MC-ICP-MS. 11 (B value).

[0028] Example 2 Effects of different treatment methods on soil boron release and isotope fractionation 1. Experimental setup Based on a 50 mL liquid system (placed in a 100 mL polypropylene conical flask), 0.5 g of the same soil sample was added to each treatment (i.e., the soil addition amount was 1% w / v), and six different treatments were set up: ①Group CK, the liquid system is deionized water; ②CM group, the liquid system is a basic culture medium with an initial pH of 7; ③CM2.0 group, the liquid system is a basic culture medium adjusted to pH 2.0 with concentrated sulfuric acid; ④Group IB: The liquid system is a basic culture medium with an initial pH of 7, inoculated with 10% (v / v) sulfur-oxidizing microbial functional groups, and 1 g / L of elemental sulfur is added. ⑤ BS group, the liquid system is a suspension of sulfur-oxidizing microbial functional flora (without fresh culture medium). ⑥FBS group, the liquid system is the filtrate obtained by filtering a suspension of sulfur-oxidizing microbial functional groups through a 0.22 μm filter membrane.

[0029] All treatments were placed in a shaking incubator at 30℃ and 175 rpm for 7 days for constant temperature incubation.

[0030] 2. Sample processing and analysis After cultivation, each treated liquid was centrifuged at 4000 rpm for 5 min, and the supernatant was filtered through a 0.45 μm filter membrane. After adjusting the pH of the filtrate to 7.0, boron was separated and purified using the "three-step tandem method of ion exchange resin" described in step 4 of Example 1. Subsequently, its isotopic composition was analyzed using multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). 11 B / 10 B) Perform high-precision measurements and calculate the δ of the sample using Equation I. 11 B value: , Formula I; Where Sample is the sample 11 B / 10 The measured value of the B ratio, NBS 951 is the result of two standards adjacent to the sample. 11 B / 10 The average measured value of the B ratio.

[0031] 3. Test Results The results of soil boron leaching efficiency and boron isotope composition for different treatment groups are shown in Table 1. According to Table 1, the treatment with inoculation of functional microorganisms and supply of energy sulfur (IB) achieved the highest boron leaching efficiency (54.94%) and the most significant negative fractionation of boron isotopes (δ¹²⁴⁰). 11 B = -47.83‰), significantly different from other control treatments, demonstrating that microbial metabolic activity is the core factor driving efficient boron release and isotope fractionation.

[0032] Table 1. Soil boron leaching efficiency and boron isotope composition

[0033] Example 3 Verification of the effect of microbial boron release in different soil types 1. Soil sample The soil samples used in the experiment were collected from the following locations from south to north: paddy field red soil in Yonghe Town, Liuyang City, Hunan Province (28°17′N, 113°74′E); brown soil in Mengyin County, Linyi City, Shandong Province (35°42′N, 117°56′E); farmland sandy loam in Tancheng County, Linyi City, Shandong Province (34°37′N, 118°21′E); saline-alkali soil in Kenli District, Dongying City, Shandong Province (37°59′N, 118°55′E); and cornfield black soil in Gongzhuling City, Jilin Province (43°30′N, 124°48′E).

[0034] 2. Experimental Setup Two treatments were set up for each soil type: a control (CK, containing only basal medium) and a microbial treatment (SOB, containing basal medium and inoculated with 10% by volume sulfur-oxidizing microbial functional groups and 1 g / L elemental sulfur). For each treatment, 50 mL of medium was added to a 100 mL Erlenmeyer flask, and 5 g of the corresponding soil was added (i.e., the soil addition was 10% w / v). All treatments were incubated at 30℃ and 175 rpm with shaking for 9 days.

[0035] 3. Sample processing and analysis After cultivation, the leachate was pretreated, boron was separated and purified, and isotope analysis was performed according to steps 3 and 4 of Example 1. The δ of the sample was determined. 11 The calculation of the B value is the same as in Example 2.

[0036] 4. Test Results The results of soil boron leaching efficiency and boron isotope composition are shown in Table 2. As shown in Table 2, for all tested soil types, the boron leaching efficiency of the SOB microbial treatment was significantly higher than that of the corresponding CK control. Simultaneously, microbial activity led to significant boron isotope fractionation, and the direction and magnitude of the fractionation (δ) were also significant. 11 The variation in B-values ​​varies depending on soil type. This fully demonstrates that the method of this invention can be effectively applied to a variety of soil systems to study boron release behavior driven by microorganisms and its specific isotope fractionation effect.

[0037] Table 2 Soil boron leaching efficiency and boron isotope composition

[0038] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for separating and analyzing soil boron isotopes released by functional microbial communities that oxidize sulfur, characterized in that, Includes the following steps: ① Enrich the functional microbial community of sulfur-oxidizing microorganisms from plant rhizosphere soil; ② The sulfur-oxidizing microbial functional group was inoculated into a liquid culture medium containing the soil to be tested, and shaken culture was carried out to activate and release boron in the soil to be tested, and the boron-containing culture solution was collected. ③ The boron in the culture medium is separated and purified using an ion exchange resin to obtain a purified boron-containing solution; ④ The purified boron-containing solution was analyzed for boron isotope composition using multi-receiver inductively coupled plasma mass spectrometry.

2. The separation and analysis method according to claim 1, characterized in that, Step ①, which involves enriching sulfur-oxidizing microbial functional communities from plant rhizosphere soil using liquid culture medium, includes: inoculating plant rhizosphere soil into a culture medium, performing shaking culture and / or subculturing, enriching sulfur-oxidizing microbial functional communities during the shaking culture and / or subculturing process, and obtaining a culture solution; the liquid culture medium is an inorganic salt culture medium with elemental sulfur as the sole energy source. The inorganic salt culture medium contains the following components: 0.3 g / L (NH4)2SO4, 0.01 g / L KCl, 0.05 g / L K2HPO4, 0.05 g / L MgSO4·7H2O, 0.001 g / L Ca(NO3)2 and 1 g / L elemental sulfur.

3. The separation and analysis method according to claim 2, characterized in that, The temperature for the shaking culture and / or subculture is 28~32℃; the rotation speed for the shaking culture and / or subculture is 170~180 rpm; The subculture includes the following steps: when the cell density of the culture medium reaches (1~4)×10⁻⁶... 8 When the cells / mL is reached, transfer to fresh culture medium at an inoculum volume of 10%; The number of subcultures is 15 to 20.

4. The separation and analysis method according to claim 1, characterized in that, The liquid culture medium mentioned in step ② is an inorganic salt culture medium with elemental sulfur as the sole energy source; The inorganic salt culture medium contains the following components: 0.3 g / L (NH4)2SO4, 0.01 g / L KCl, 0.05 g / L K2HPO4, 0.05 g / L MgSO4·7H2O, 0.001 g / L Ca(NO3)2 and 1 g / L elemental sulfur.

5. The separation and analysis method according to claim 1, characterized in that, The inoculation amount of the sulfur-oxidizing microbial functional flora mentioned in step ② is 5-15% of the volume of the liquid culture medium; In step ②, the amount of soil to be tested added to the liquid culture medium is 1~10 g of soil to be tested per 100 mL of culture medium.

6. The separation and analysis method according to claim 1, characterized in that, The temperature of the shaking culture in step ② is 28~32℃; the shaking rotation speed is 170~180 rpm; and the shaking culture time is 9 days.

7. The separation and analysis method according to claim 1, characterized in that, Before performing the separation and purification operation, the culture medium is also pretreated. The pretreatment includes: centrifuging the culture medium collected in step ② at a centrifugal force of 1000~3000×g for 5~10min, collecting the supernatant, filtering the supernatant using a filter membrane with a pore size of 0.4~0.5 μm, collecting the filtrate, and adjusting the pH of the filtrate to 6~7 using a 0.1 M sodium hydroxide aqueous solution.

8. The separation and analysis method according to claim 1, characterized in that, The separation and purification process includes the separation and purification of boron in the culture medium using a three-step tandem method with ion exchange resins. The three-step tandem method of ion exchange resins includes: boron-specific adsorption resin for impurity removal, nitric acid elution, and anion-cation exchange resin for impurity removal.

9. The separation and analysis method according to claim 1, characterized in that, The plant rhizosphere soil includes the rhizosphere soil of one or more of the following plants: pepper, cucumber, tomato, wheat, and rapeseed.

10. The application of the separation and analysis method according to any one of claims 1 to 9 in the separation and / or analysis of soil boron isotopes, characterized in that, The soil includes one or more of the following: black soil, sandy loam, saline-alkali soil, brown soil, and red soil.

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