Determination method for redox potential of soluble black carbon
By combining the reaction of quinone-reducing bacteria with dissolved black carbon under anaerobic conditions with the determination of total Fe(II) in ferrohydrate, the accuracy problem of determining the redox potential of dissolved black carbon in existing technologies has been solved, enabling measurement and evaluation that more closely resembles the real environment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG AGRI & ENG UNIV
- Filing Date
- 2026-02-13
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies cannot accurately determine the redox potential of dissolved black carbon in anaerobic environments such as soil and sediments. Commonly used electrochemical methods overestimate its electron transfer capacity and cannot truly reflect microbial-driven redox processes.
Quinone-reducing bacteria were mixed with dissolved black carbon under anaerobic conditions, and then ferrohydrate was added to carry out the reaction. The redox potential was calculated by measuring the total amount of Fe(II) in the liquid and solid phases and using AQDS as a reference, thus simulating the redox process of microorganisms under anaerobic conditions.
This method allows for more accurate measurement of the redox potential of dissolved black carbon under anaerobic conditions. It is simple to operate and provides stable and reliable results, enabling the evaluation of the environmental behavior of dissolved black carbon from different sources.
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Figure CN122017114A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of environmental analysis technology and relates to a method for determining the redox potential of dissolved black carbon. Background Technology
[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.
[0003] Dissolved black carbon contains abundant quinone and phenolic functional groups, possessing electron shuttle capabilities. It participates in microbial metabolism and pollutant transformation processes in anaerobic environments such as soil and sediments. For example, some studies have disclosed the effects of dissolved black carbon on… Geobacter sulfurreducens The effect of PCA on Fe(III) reduction has been studied. Currently, electrochemical methods are commonly used to determine their redox potential. For example, one study disclosed a quantitative prediction method for the electron transfer capacity of dissolved black carbon, using electrochemical methods to detect its electron transfer ability. However, this method usually overestimates the electron transfer capacity of dissolved black carbon in the actual environment and cannot truly reflect the microbial-driven redox process.
[0004] In particular, for the determination of the redox potential of DBC in anaerobic environments such as soil and sediment, there is an urgent need to develop a method that can simulate the above-mentioned anaerobic environment and accurately quantify the redox potential of dissolved black carbon. Summary of the Invention
[0005] To address the aforementioned issues, this invention provides an accurate and environmentally simulating method for determining the redox potential of dissolved black carbon (DBC), which can more accurately measure the redox potential of DBC in anaerobic environments such as soil and sediments.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a method for determining the redox potential of soluble black carbon, comprising: Under anaerobic conditions, soluble black carbon and quinone-reducing bacteria are mixed evenly in a culture medium and reacted. After the reaction is completed, the mixture is sterilized to obtain a mixed system. Add ferrohydrate to the mixture and react. After the reaction is complete, determine the total amount of Fe(II) in the liquid and solid phases. The redox potential of dissolved black carbon is calculated using AQDS as a reference.
[0007] The study found that in anaerobic environments such as soil and sediment, the main active group in DBC involved in the redox process in the natural environment is the quinone group. Therefore, quinone-reducing bacteria were selected to reduce DBC to more accurately simulate the redox process of DBC in the above-mentioned anaerobic environment and to more accurately measure the redox potential of DBC under the above conditions.
[0008] A second aspect of the present invention provides a system for determining the redox potential of soluble black carbon, comprising: quinone-reducing bacteria, culture medium, ferrohydrate, container, and ultraviolet-visible spectrophotometer.
[0009] Beneficial effects of the present invention (1) This invention uses microbial simulation of DBC to participate in the natural environment redox process in anaerobic environments such as soil and sediment, which is closer to the real environment; it can more accurately measure the redox potential of DBC in anaerobic environments such as soil and sediment, and can be used to evaluate the environmental behavior of dissolved black carbon from different sources.
[0010] (2) The present invention is simple to operate and the results are stable and reliable. Attached Figure Description
[0011] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. Exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.
[0012] Figure 1 This is a schematic diagram of the electron transfer efficiency of 300 DBC in Example 1; Figure 2 This is a schematic diagram of the electron transfer efficiency at 500 DBC in Example 2; Figure 3 This is a schematic diagram of the electron transfer efficiency of 700 DBC in Example 3. Detailed Implementation
[0013] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. The reagents and raw materials used in this invention are readily available through conventional means, and unless otherwise specified, they are used in accordance with conventional methods in the art or product instructions. Similarly, unless otherwise specified, the test methods of this invention are performed in accordance with conventional methods in the art or industry-standard methods or practices. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.
[0015] This invention mainly proposes a method for determining the redox potential of soluble black carbon, including: Under anaerobic conditions, soluble black carbon and quinone-reducing bacteria are mixed evenly in a culture medium and reacted. After the reaction is completed, the mixture is sterilized to obtain a mixed system. Add ferrohydrate to the mixture and react. After the reaction is complete, determine the total amount of Fe(II) in the liquid and solid phases. The redox potential of dissolved black carbon is calculated using AQDS as a reference.
[0016] The present invention does not impose any particular limitation on the preparation method of soluble black carbon. Preferably, the preparation method of soluble black carbon includes: mixing biochar with water, shaking, sonicating, filtering, and then desalting with resin to obtain a soluble black carbon solution, so as to improve the preparation efficiency.
[0017] The amount of water used affects the amount of dissolved black carbon released. Therefore, this invention studies the mass ratio of biochar to water. Preferably, the mass ratio of biochar to water is 1:100-120 to obtain more dissolved black carbon.
[0018] Dissolved black carbon in the solid phase can be released into the solution through oscillation and ultrasonic treatment. Therefore, the present invention has studied the oscillation time and ultrasonic time. Preferably, the oscillation time is 24-32 h; preferably, the ultrasonic time is 30-40 min, so as to improve the release efficiency of dissolved black carbon.
[0019] Filtration can remove impurities from water and improve the purity of dissolved black carbon. Therefore, this invention studies the filtration conditions. Preferably, the pore size of the filter membrane is 0.45 μm to improve filtration efficiency.
[0020] The amount of quinone-reducing bacteria used affects the reduction effect of soluble black carbon. Therefore, this invention studied the mixing ratio of soluble black carbon and quinone-reducing bacteria. Preferably, the mixing ratio of soluble black carbon and quinone-reducing bacteria is 100-120 mgC: 1.6 × 10⁻⁶. 9 -2×10 9cells, to achieve better reduction effect.
[0021] Temperature and stirring speed also affect the reduction effect of soluble black carbon. Therefore, this invention studies the reaction conditions of soluble black carbon and quinone-reducing bacteria. Preferably, the reaction conditions of soluble black carbon and quinone-reducing bacteria are anaerobic reaction at 35-37℃ and 150-160 r / min for 30-32 days to improve the efficiency of the reduction reaction.
[0022] The quinone-reducing bacteria of the present invention can be commercially available strains or can be cultured through domestication. Preferably, quinone-reducing bacteria are obtained by culturing and domesticating a microbial community with quinone-reducing function. The quinone-reducing bacteria were cultured to the logarithmic growth phase and centrifuged to obtain a bacterial suspension for better reduction.
[0023] To improve the reducing ability of quinone-reducing bacteria, this invention studied the acclimatization conditions. Preferably, the acclimatization medium is a mineral salt medium, the acclimatization temperature is 35-37℃, and the acclimatization period is 2-3 weeks, in order to obtain better reducing ability.
[0024] Inoculation concentration and temperature can affect the growth, metabolism, and products of bacterial strains. Therefore, this invention investigated the concentration of the quinone-reducing microbial inoculation solution, the culture temperature, and the culture time. Preferably, the concentration of the quinone-reducing microbial inoculation solution is 5 × 10⁻⁶. 7 -1×10 8 The cells / mL scale-up culture time is 28-35 days, and the scale-up culture temperature is 35-37℃ to improve culture efficiency.
[0025] To improve the accuracy of detection, this invention studies the concentration of ferrohydrate added. Preferably, the concentration of ferrohydrate added is 2-3 mM, and the reaction time is 72-76 h, so as to effectively detect the redox potential.
[0026] This invention studies a method for calculating the redox potential of soluble black carbon. Preferably, the formula for calculating redox potential is as follows: To more accurately assess the redox potential of dissolved black carbon.
[0027] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.
[0028] Unless otherwise specified, the reagents and instruments used in this invention are all commercially available products in the field or can be prepared by conventional methods.
[0029] In the following examples, the quinone-reducing bacteria were mixed bacteria, and the anaerobic sludge came from the anaerobic bioreactor of Beijing Tongzhou District Jingcheng Huitong Environmental Protection Co., Ltd.
[0030] Example 1: Redox potential of dissolved black carbon (300DBC) derived from pyrolyzed straw biochar at 300℃ A method for determining the redox potential of soluble black carbon, the specific steps of which are as follows: (1) Extraction of dissolved black carbon (DBC) solution: Weigh 5.00 g of biochar sample prepared from straw pyrolysis at 300℃ and place it in a 1 L clean glass bottle.
[0031] Add 500 mL of deionized water (resistivity ≥18.2 MΩ·cm) to make the solid-liquid ratio 1:100 (g / mL).
[0032] The glass bottle was placed in a constant temperature shaker and shaken at 25℃ and 150 r / min for 24 h.
[0033] The suspension was then sonicated for 30 min (300 W power, 40 kHz frequency).
[0034] The ultrasonically purified mixture was vacuum filtered through a pre-cleaned 0.45 μm glass fiber membrane, and the filtrate was collected.
[0035] The filtrate was desalted and concentrated by passing it through an activated PPL solid-phase extraction column at a flow rate of 10 mL / min. The eluent was eluted with methanol and collected. The eluent was then purged with nitrogen at 40 °C until nearly dry. The solution was reconstituted with deionized water and brought to a final volume of 100 mL to obtain a 300DBC stock solution with a concentration of 1000 mg C / L. The stock solution was stored at 4 °C in the dark.
[0036] (2) Cultivation, domestication, and collection of quinone-reducing bacteria: Mineral salt medium (MSM) was prepared with the following composition: NaCl 1000 mg / L, NH4Cl 800 mg / L, KH2PO4 500 mg / L, K2HPO4 600 mg / L, MgCl2 200 mg / L, CaCl2·2H2O 50 mg / L, yeast extract 10 mg / L, AQDS 1 mM, glucose 10 mM, pH=7.2. All glassware and the medium were sterilized at 121℃ for 30 min.
[0037] In an anaerobic glove box, the culture medium was purged with N2 for 10 min and the headspace was displaced for 5 min.
[0038] Establishment of the acclimatization system: Add 300 mL of anaerobic sludge inoculum to a 500 mL anaerobic serum bottle in MSM medium. After anaerobic incubation at 35℃ and 150 r / min in the dark for 2 weeks (OD600=0.8), the bacterial suspension is transferred to fresh MSM medium. After another 2 weeks of incubation, the previous generation bacterial suspension is transferred to fresh MSM medium again, for a total of four generations of acclimatization.
[0039] Take the acclimatized quinone-reducing bacteria, wash twice with sterile physiological saline, resuspend in physiological saline, and adjust the concentration to 8 × 10⁻⁶. 7 cells / mL, used as inoculum for quinone-reducing microorganisms.
[0040] (3) Synthesis of ferrohydrate: Prepare a 0.2 mol / L FeCl3 solution and a 1.0 mol / L NaOH solution.
[0041] While continuously and vigorously stirring, NaOH solution was added dropwise to FeCl3 solution, and the dropping rate was controlled so that the pH of the reaction system slowly rose to 7.0.
[0042] After stirring for another 30 minutes, the orange suspension was allowed to stand at 25°C for 2 hours to age.
[0043] Transfer the precipitate to a centrifuge tube, centrifuge with deionized water at 10000 r / min for 10 min, wash until the supernatant shows no white precipitate when tested with AgNO3 (Cl). - (Eliminated).
[0044] The washed ferrous ore precipitate was freeze-dried, ground, passed through a 100-mesh sieve, sealed, and stored at 4°C.
[0045] (4) Construction of anaerobic reaction system and microbial reduction: An anaerobic culture medium was prepared with the following composition: 0.14 g / L KH₂PO₄, 0.2 g / L NaCl, 0.3 g / L NH₄Cl, 0.5 g / L MgSO₄·7H₂O, 0.1 g / L CaCl₂, 20 mM CH₃COONa, and 22 mM NaHCO₃. High-purity nitrogen (N₂) was bubbled for 30 min to remove dissolved oxygen, and the pH was adjusted to 7.0.
[0046] Add the following to a 250 mL serum bottle in sequence: 80 mL of the above anaerobic culture medium, 20 mL of the quinone-reducing bacteria inoculation solution prepared in step (2), and 100 mL of the 300 DBC stock solution prepared in step (1) (the final concentration after mixing is 500 mg C / L).
[0047] Seal the bottle opening with a butyl rubber stopper and an aluminum cap, and continuously purge the headspace with N2 for 5 minutes to maintain an anaerobic environment.
[0048] The serum bottle was placed in a constant temperature shaker and reacted in the dark at 35℃ and 150 r / min for 30 days to allow the DBC to be fully reduced by microorganisms.
[0049] (5) Sterilization and chemical oxidation-reduction reaction: After the reaction was completed, the serum bottle was autoclaved at 121°C for 30 min to terminate the microbial activity.
[0050] After sterilization and cooling to room temperature, solid ferrohydrate was added to the bottle in an anaerobic glove box (N2 atmosphere, H2 < 1 ppm, O2 < 1 ppm) to achieve a final concentration of 2 mM in the system.
[0051] After being sealed again, the mixture was subjected to anaerobic and light-protected reaction at 35℃ and 150 r / min for 72 h, so that the reduced DBC could reduce Fe(III) in the ferrohydrate to Fe(II).
[0052] (6) Determination of total Fe(II): Liquid phase Fe(II) determination: After the reaction was complete, a portion of the reaction solution was filtered through a 0.45 μm filter membrane. 5.0 mL of the filtrate was taken, and 20 mL of acetate-sodium acetate buffer (pH 4.7) and 2 mL of o-phenanthroline reagent (10 g / L) were added. The volume was adjusted to 50 mL, mixed well, and allowed to stand in the dark for 10 min. The absorbance was measured at 510 nm using a UV-Vis spectrophotometer, and the Fe(II) concentration in the solution was calculated and recorded as Fe(II). AQ .
[0053] Extraction of Fe(II) by solid-phase adsorption: The filtered membrane and the retained solid were transferred together to a 50 mL centrifuge tube. The ferrohydrate solid was washed three times with deionized water, and 20 mL of 1 M HCl solution was added. Extraction was carried out by shaking at 25 °C and 150 r / min for 24 h. After filtering the extract through a 0.45 μm filter membrane, the Fe(II) concentration was determined using the same o-phenanthroline method. The concentration of Fe(II) adsorbed on the solid phase was calculated and denoted as Fe(II). Sorbed .
[0054] Calculation of total Fe(II) content: The total amount of Fe(II) produced in the reaction (unit: μmol) is calculated according to the following formula:
[0055] Where V is the total volume of the reaction system (0.2 L).
[0056] (7) Calculation of redox potential: Anthraquinone-2,6-disulfonate (AQDS) was used as a standard electron shuttle for control experiments. An electron equivalent (previously determined to be 265 μmol e) of the 300 DBC sample was weighed. - The reduced state of AQDS (g C) was reacted with ferrihydrite under the same conditions, and the total amount of Fe(II) produced was determined to be 257 μmol.
[0057] The redox potential of 300 DBC is characterized by electron transfer efficiency, calculated using the following formula:
[0058] The calculated electron transfer efficiency of 300 DBC in this embodiment is (77.9 μmol / 257 μmol) ×100% = 29.4%.
[0059] The results indicate that dissolved black carbon derived from straw pyrolysis at 300℃ has a reduction efficiency of approximately 29.4% for ferrohydrate under microbial-driven conditions, which can be used to assess its actual electron shuttle capability in a similar anaerobic environment.
[0060] Example 2: Dissolved black carbon (500DBC) derived from straw biochar pyrolysis at 500℃ The steps in this embodiment are basically the same as those in Embodiment 1, with the only difference being: In step (1), 500 DBC was extracted from biochar prepared by pyrolysis of straw at 500℃.
[0061] Step (6) determined that the concentration of Fe(II) in the liquid phase was 1×10⁻⁶. 4 μM; the total amount of Fe(II) adsorbed in the solid phase was 76 μmol. The total amount of Fe(II) generated was 180 μmol.
[0062] In step (7), the total amount of Fe(II) produced by the reduced state AQDS with equielectron equivalents is 316 μmol.
[0063] The calculated electron transfer efficiency of 500 DBC is (180 μmol / 316 μmol) × 100% ≈ 55.8%.
[0064] The results showed that when the pyrolysis temperature was increased to 500℃, the redox activity of soluble black carbon was significantly enhanced.
[0065] Example 3: Dissolved black carbon (700DBC) derived from pyrolyzed straw biochar at 700℃ The steps in this embodiment are basically the same as those in Embodiment 1, with the only difference being: In step (1), 700 DBC was extracted from biochar prepared by pyrolysis of straw at 700℃.
[0066] Step (6) determined that the concentration of Fe(II) in the liquid phase was 123 μM; the total amount of Fe(II) adsorbed in the solid phase was 78 μmol; and the total amount of Fe(II) generated was 201 μmol.
[0067] In step (7), the total amount of Fe(II) produced by the reduced state AQDS with equielectron equivalents is 313 μmol.
[0068] The calculated electron transfer efficiency of 700 DBC is (201 μmol / 313 μmol) × 100% ≈ 61.3%.
[0069] The results showed that soluble black carbon derived from straw pyrolysis at 700℃ had a higher redox potential.
[0070] Figures 1-3 The figures shown are schematic diagrams illustrating the electron transfer efficiency results for Examples 1-3, respectively. These examples clearly demonstrate that the method of the present invention can effectively determine the redox potential of dissolved black carbon from different sources.
[0071] Pyrolysis temperature is an important factor affecting the electron shuttle ability of soluble black carbon. Under the experimental conditions, the electron transfer efficiency increases with increasing pyrolysis temperature (29.4% → 55.8% → 61.3%), which is consistent with the results of existing studies (DOI: 10.1021 / acssuschemeng.1c01251, DOI: 10.1021 / es500906d), further confirming the feasibility of the method of the present invention.
[0072] This invention provides a more realistic redox potential assessment method by simulating the microbial reduction process, which can provide key parameters for studying the role of dissolved black carbon in soil carbon cycling, pollutant migration and transformation, and environmental remediation.
[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for determining the redox potential of soluble black carbon, characterized in that, include: Under anaerobic conditions, soluble black carbon and quinone-reducing bacteria are mixed evenly in a culture medium and reacted. After the reaction is completed, the mixture is sterilized to obtain a mixed system. Add ferrohydrate to the mixture and react. After the reaction is complete, determine the total amount of Fe(II) in the liquid and solid phases. The redox potential of dissolved black carbon is calculated using AQDS as a reference.
2. The method for determining the redox potential of soluble black carbon as described in claim 1, characterized in that, The method for preparing the soluble black carbon includes: mixing biochar with water, shaking, sonicating, filtering, and then desalting with resin to obtain a soluble black carbon solution; Alternatively, the mass ratio of biochar to water is 1:100-120; Alternatively, the oscillation time is 24-32 h; Alternatively, the ultrasound duration is 30-40 minutes; Alternatively, the pore size of the filter membrane is 0.45 μm.
3. The method for determining the redox potential of soluble black carbon as described in claim 1, characterized in that, The mixing ratio of the dissolved black carbon and quinone-reducing bacteria is 100-120 mg C: 1.6 × 10⁻⁶ 9 -2×10 9 cells.
4. The method for determining the redox potential of soluble black carbon as described in claim 1, characterized in that, The reaction conditions for the dissolved black carbon and quinone-reducing bacteria are anaerobic reaction at 35-37℃ and 150-160 r / min for 30-32 days in the dark.
5. The method for determining the redox potential of soluble black carbon as described in claim 1, characterized in that, Quinone-reducing bacteria were obtained by culturing and domesticating microbial communities with quinone-reducing functions. Quinone-reducing bacteria were cultured to the logarithmic growth phase and centrifuged to obtain a bacterial suspension.
6. The method for determining the redox potential of soluble black carbon as described in claim 5, characterized in that, The acclimatization medium is a mineral salt medium, the acclimatization temperature is 35-37℃, and the acclimatization period is 2-3 weeks.
7. The method for determining the redox potential of soluble black carbon as described in claim 5, characterized in that, The concentration of quinone-reducing microbial inoculum was 5 × 10⁻⁶. 7 -1×10 8 The cells / mL scale-up culture time is 28-35 days, and the scale-up culture temperature is 35-37℃.
8. The method for determining the redox potential of soluble black carbon as described in claim 1, characterized in that, The concentration of the added ferrohydrate was 2-3 mM, and the reaction time was 72-76 h.
9. The method for determining the redox potential of soluble black carbon as described in claim 1, characterized in that, The formula for calculating redox potential is: 。 10. A system for determining the redox potential of soluble black carbon, characterized in that, include: Quinone-reducing bacteria, culture medium, ferrohydrate, container, UV-Vis spectrophotometer.