Biological material for efficiently removing organic pollutants in frozen and thawed soil as well as preparation method and application of biological material
By using biomaterials immobilized with a mixture of biochar and alpine spore fungi, the problem of efficient degradation of polycyclic aromatic hydrocarbon pollutants in freeze-thawed soils was solved, achieving significant soil remediation results.
Patent Information
- Application Number
- CN202511671164.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-14
- Publication Date
- 2026-02-10
AI Technical Summary
Existing technologies are insufficient for efficiently removing polycyclic aromatic hydrocarbons (PAHs) from freeze-thawed soils, especially in cold northern regions, where the remediation process is lengthy and the survival rate of microorganisms is low.
Using mixed biochar as a carrier, Pseudomonas sp. and Mortierella alpine inoculants were immobilized to prepare biomaterials. The mixed inoculants were then immobilized by adsorption and used to degrade organic pollutants in freeze-thawed soil.
It achieved efficient degradation of polycyclic aromatic hydrocarbons in freeze-thawed soil, with remediation efficiencies of 72.47% ± 2.31% and 69.52%, which is of great significance for purifying soil contaminated with organic pollutants.
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Figure CN121495918A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biodegradation of organic pollutants, and particularly relates to a biomaterial for efficiently removing organic pollutants from freeze-thawed soil, its preparation method, and its application. Background Technology
[0002] Polycyclic aromatic hydrocarbons (PAHs) are persistent organic compounds that are difficult to degrade and are among the most challenging organic pollutants to manage. They can accumulate in the food chain through biomagnification, harming the health of organisms. PAHs are highly toxic, carcinogenic, teratogenic, and can persist in the environment for extended periods, posing a serious threat to the ecological environment and human health. Currently, 16 PAHs are listed as priority pollutants by the U.S. Environmental Protection Agency. The main harms of PAHs to humans include increased cancer risk, cardiovascular disease, and impaired neurodevelopment. Low-ring-number PAHs are associated with impaired lung function.
[0003] Microbial remediation technology has limitations such as long remediation cycles and low microbial survival rates. However, the combined use of biochar and bioremediation technologies can improve bioavailability, promote the degradation of organic pollutants, and enhance the remediation effect of PAH-contaminated soils. In cold northern regions, soils are affected by temperature, and most PAH-contaminated soils are at low to medium levels. The combined use of immobilized, cold-resistant fungi and bacteria has shown significant remediation effects on PAH-contaminated soil sites in these areas.
[0004] Biochar, with its unique physical structure including varying specific surface areas and porosity, provides an excellent living environment for microorganisms, making it a superior microbial carrier material. Corn cobs and wheat straw are natural biodegradable materials that can be reused to avoid resource waste. Summary of the Invention
[0005] The purpose of this invention is to provide a biomaterial for efficiently removing organic pollutants from freeze-thawed soil, its preparation method, and its application. A mixed microbial agent is immobilized using corn cobs and wheat straw as carriers for degrading organic pollutants in soil.
[0006] The technical solution adopted in this invention is: a biomaterial for efficiently removing organic pollutants from freeze-thawed soil. The biomaterial is prepared by immobilizing a mixed microbial agent using mixed biochar as an immobilization carrier and employing an adsorption method. The mixed biochar is made by mixing carbonized corn cob biochar and carbonized wheat straw biochar. The mixed microbial agent consists of Pseudomonas sp. SDR4 (CGMCC NO.14048) and Mortierella alpine JDR7 (CGMCC NO.15183).
[0007] Furthermore, by mass ratio, corn cob biochar : wheat straw biochar = 1 : (1-3).
[0008] Furthermore, by volume ratio, Pseudomonas sp. SDR4 : Mortierella alpine JDR7 = 1 : 1.
[0009] A method for preparing a biomaterial that efficiently removes organic pollutants from freeze-thawed soil includes the following steps:
[0010] 1) Preparation of mixed biochar: Corn cobs were washed, air-dried for 2-3 days, dried at 70℃-80℃, and then carbonized under anaerobic conditions to obtain corn cob biochar; wheat straw was washed, air-dried for 2-3 days, dried at 70℃-80℃, and then carbonized under anaerobic conditions to obtain wheat straw biochar; corn cob biochar and wheat straw biochar were mixed and sterilized to obtain mixed biochar;
[0011] 2) Preparation of mixed bacterial agent: Pseudomonas sp. SDR4 (CGMCC NO.14048) and Mortierella alpine JDR7 (CGMCC NO.15183) were inoculated into complete culture medium and cultured for 2-3 days. Then, they were inoculated into liquid culture medium and cultured continuously for 72-74 hours at 120 rpm in a constant temperature shaking incubator at 15℃ to obtain mixed bacterial agent.
[0012] 3) Mix the mixed biochar with the proliferation culture medium, and after thorough soaking, inoculate the mixed bacterial agent at a rate of 10% per day for 3-4 days. Finally, place the culture system in a constant temperature environment of 15℃ for cultivation to obtain biomaterials that can efficiently remove organic pollutants from freeze-thawed soil.
[0013] Further, in step 1), the corn cob biochar is obtained by carbonization at 450℃-550℃ for 4h-5h under anaerobic conditions; the wheat straw biochar is obtained by carbonization at 250℃-350℃ for 4h-5h under anaerobic conditions; the sterilization treatment is carried out in an autoclave at 120℃-122℃ for 60min-70min.
[0014] Further, in step 2), the complete culture medium consists of: 9g-11g peptone, 4g-6g yeast extract, 4g-6g NaCl, 18g-22g agar, distilled water added to 1000mL, pH 7.1-7.2, sterilized at 121℃ for 20min-30min; the liquid culture medium consists of: 7g-9g sucrose, 5g-7g yeast extract, 0.8g-1.2g KH2PO4, 3g-5g (NH4)2HPO4, 0.4g-0.6g MgSO4·H2O, pH 6.0-6.5, sterilized at 121℃ for 20min-30min.
[0015] Further, in step 3), the biochar and culture medium are mixed according to the material-to-liquid ratio: 1g:(1mL-2mL); the culture medium is composed of: 75g-85g sucrose, 55g-65g yeast extract, 8g-12g KH2PO4, 35g-45g (NH4)2HPO4, 4g-6g MgSO4·H2O, pH 6.0-6.5, sterilized at 121℃ for 20min-30min, and stored at 4℃.
[0016] The application of the biomaterials provided by this invention in the efficient removal of organic pollutants from freeze-thawed soil.
[0017] Further, the method is as follows: In freeze-thawed soil, the biological material is mixed in situ with the soil from the surface to a depth of 10cm-30cm.
[0018] Furthermore, the freeze-thawed soil refers to soil used for growing soybeans or corn in Northeast China; the organic pollutant is polycyclic aromatic hydrocarbons.
[0019] The beneficial effects of this invention are:
[0020] The biomaterials provided by this invention have advantages such as low cost, non-toxicity, and biodegradability, and possess a large adsorption capacity for microorganisms. The biomaterials provided by this invention exhibit high degradation efficiency for polycyclic aromatic hydrocarbons (PAHs) in freeze-thawed soil. In this invention, *Pseudomonas* and *Mortierella alpina* can utilize PAHs as a carbon source to degrade organic pollutants. After a one-year remediation cycle, the remediation efficiencies in soils planted with soybeans and corn were 72.47% ± 2.31% and 69.52% ± 2.01%, respectively, which is of great significance for purifying soils contaminated with organic pollutants. Attached Figure Description
[0021] Figure 1 This is a degradation rate diagram of PAHs in soybean and corn soil based on Example 1 of the present invention. Detailed Implementation
[0022] The following examples are for illustrative purposes only and do not limit the scope of application of the invention.
[0023] Example 1: A biomaterial for efficiently removing organic pollutants from freeze-thawed soil and its application.
[0024] (I) A method for preparing biomaterials for efficiently removing organic pollutants from freeze-thawed soil, comprising the following steps:
[0025] 1. Preparation of mixed biochar:
[0026] 1.1) Preparation of corn cob biochar
[0027] Rinse the corn cobs three times with tap water, then rinse them twice with distilled water. Let them air dry naturally for two days, and then dry them in a 75℃ oven.
[0028] The dried corn cobs were packed into an iron can (10 cm in diameter and 10 cm in length), the biomass was compacted, and nitrogen gas was introduced until there was no oxygen inside. The iron can was then placed in a muffle furnace and heated to 500°C at a rate of 10°C / min. It was then pyrolyzed at 500°C for 4 hours, cooled to room temperature, and removed to obtain corn cob biochar that had undergone anaerobic carbonization.
[0029] 1.2) Preparation of wheat straw biochar
[0030] After rinsing the wheat straw three times with tap water, rinse it twice with distilled water, let it air dry naturally for two days, and then put it into a 75℃ oven to dry.
[0031] The dried wheat straw was packed into an iron can (10 cm in diameter and 10 cm in length), the biomass was compacted, and nitrogen gas was introduced until there was no oxygen inside. The iron can was then placed in a muffle furnace and heated to 300°C at a rate of 10°C / min. It was then pyrolyzed at 300°C for 4 hours, cooled to room temperature, and removed to obtain wheat straw biochar that had been carbonized under anaerobic conditions.
[0032] 1.3) Mixing
[0033] The corn cob biochar and wheat straw biochar were mixed in a mass ratio of 1:2, and then placed in an autoclave at 121℃ for 60 minutes to complete the sterilization process, obtaining the mixed biochar. 2. Preparation of the mixed inoculant:
[0034] 2.1) Culture of Pseudomonas sp. SDR4
[0035] LB bacterial culture medium: 0.5g yeast extract, 1g peptone, 1g NaCl, 1.5g-2g agar powder, pH 7.0-7.2, sterilized at 121℃ for 30min.
[0036] Take Pseudomonas sp. SDR4, preserved on an agar slant with accession number CGMCC NO.14048, and inoculate it into LB bacterial medium at a 5% inoculum. Incubate at 15°C and 120 rpm for 30-40 hours on a shaker until the bacterial density reaches 6 × 10⁻⁶. 8 CFU·mL -1 A suspension of Pseudomonas sp. SDR4 bacteria was obtained.
[0037] 2.2) Cultivation of Mortierella alpine JDR7
[0038] PDA solid medium: 20g potato, 2g glucose, 1.5g-2g agar, add distilled water to 100mL, pH 5.6-6.0, sterilize at 115℃ for 20min.
[0039] Mortierella alpine JDR7, preserved on an agar slant medium with accession number CGMCC NO.15183, was inoculated at a rate of 5% into PDA solid medium and cultured at 15°C and 120 rpm for 30-40 hours until the bacterial density reached 6 × 10⁻⁶. 8 CFU·mL -1 A suspension of Mortierella alpine JDR7 fungus was obtained.
[0040] 2.3) Mixing
[0041] The complete culture medium consists of: 10g peptone, 5g yeast extract, 5g NaCl, 20g agar, distilled water to 1000mL, pH 7.1-7.2, sterilized at 121℃ for 20min.
[0042] The liquid culture medium consisted of: 8g sucrose, 6g yeast extract, 1.0g KH2PO4, 4g (NH4)2HPO4, 0.5g MgSO4·H2O, pH 6.0-6.5, and was sterilized at 121℃ for 20min.
[0043] At a volume ratio of 1:1, a bacterial suspension of Pseudomonas sp. SDR4 (CGMCC NO.14048) and a fungal suspension of Mortierella alpine (CGMCC NO.15183) were inoculated into complete culture medium at a 10% inoculation rate. After culturing for 2 days, the mixture was then inoculated into liquid culture medium and cultured continuously at 120 rpm for 72 hours in a constant temperature shaking incubator at 15°C to obtain a mixed inoculum.
[0044] 3. Preparation of biomaterials
[0045] The proliferation culture medium consisted of: 80g sucrose, 60g yeast extract, 10g KH2PO4, 40g (NH4)2HPO4, 5g MgSO4·H2O, pH 6.0-6.5, sterilized at 121℃ for 20-30 minutes, and stored at 4℃.
[0046] The ratio of mixed biochar to proliferation culture medium is 1g:1mL. Mix the mixed biochar with the proliferation culture medium, and after thorough soaking, inoculate the mixed bacterial agent at a rate of 10% per day for 3 days. Finally, place the culture system in a constant temperature environment of 15℃ for cultivation to obtain a biological material that can efficiently remove organic pollutants from freeze-thawed soil.
[0047] (II) Application of biomaterials for efficient removal of organic pollutants from freeze-thawed soils
[0048] 1. In-situ remediation experiments of PAHs in soybean and corn farmland
[0049] The remediation experiment used soil from Shenyang, Northeast China. The soil freeze-thaw period was from early November to April of the following year, with an average annual temperature of 4℃-16℃. The lowest temperature was in January (average -14℃), and the highest temperature was in July (average 25℃). The experimental soil had the following characteristics: pH 6.31, organic matter 5.36%, and PAHs content 72.71 μg·kg⁻¹. -1 .
[0050] The in-situ soil PAHs biodegradation test method is as follows:
[0051] 1.1) The experimental site was set up as a control area and an experimental area, separated by a bundle edge pool. The bundle edge pool was square (3m×3m). The control area was a blank control area without any treatment, and the experimental area was an experimental area with added biological materials.
[0052] 1.2) In freeze-thawed soil, the prepared biological material was evenly spread on the soil surface of the experimental area using a fertilizer spreader, and then mixed into the soil by tilling and tillage. The biological material was mixed in situ with the soil at a depth of about 20 cm. The biological material was mixed in situ with the soil at a mass percentage of 2%.
[0053] 1.3) The experiment was launched in mid-May 2023 and ended in mid-April 2024. Topsoil samples from the 0cm-20cm depth were collected in the middle of each month. Equal amounts of soil samples were taken from each sampling point and mixed to form composite samples, which were sealed, preserved, and the sampling information was recorded. The samples were then transported back to the laboratory.
[0054] 2. PAHs Extraction and Detection Methods
[0055] 2.1) PAHs Extraction
[0056] After the soil samples were transported back to the laboratory and purified, they were placed in a well-ventilated, cool environment to dry naturally. Once the samples were completely dry, they were sieved using a standard test sieve (2mm aperture) for testing.
[0057] Add 1.5g of diatomaceous earth, 1.5g of the soil sample to be tested, and another 1.5g of diatomaceous earth to the extraction column. Add a filter membrane, tighten, and place in the extraction chamber. Use a rapid extractor at 100℃ and 100MPa, with a mixed solvent of hexane and acetone (volume ratio 1:1) as the extractant, and extract for 30 min. Store the extract in a concentration bottle. In a fume hood, use a parallel evaporator at 40℃, 184mbar, and 90r / min to concentrate the extract. Evaporate to dryness, then add 5mL of hexane for further concentration. Repeat this extraction process three times to ensure complete extraction of the target substance from the sample. Then, add 4mL of acetone as the extraction solvent to the sample and treat with an ultrasonic cleaner for 60s. Use a sterile syringe to filter the remaining solution through a 0.22μm organic phase filter membrane to remove particulate impurities. Transfer the filtrate to a pre-labeled liquid chromatography sample vial, seal, and store for subsequent instrumental analysis.
[0058] 2.2) PAHs detection
[0059] The PAH content was determined by gas chromatography-mass spectrometry (GC-MS). The instrument parameters for GC-MS detection were as follows: injection volume: 1 μL; injection port temperature: 280℃; injection mode: splitless injection; ion source: electron impact source; ionization energy: 70 eV; ion source temperature: 230℃; detector temperature: 280℃.
[0060] Scanning mode: Select ion mode (SIM). Furnace temperature ramp-up program: initial 80°C, hold for 1 min; then initially at 15°C / min. -1 The temperature is rapidly increased to 160°C; then increased at a rate of 5°C per minute. -1 Increase the temperature slowly to 300°C and hold for 10 minutes.
[0061] 2.3) Experimental Results:
[0062] The effect of adding biological materials to actual farmland on the degradation of PAHs in the soil is as follows: Figure 1 .
[0063] like Figure 1 As shown, in soybean-grown soil, a one-year soil remediation experiment revealed that, without human intervention, the natural degradation rate of PAHs in farmland soil was only 5.87% ± 1.22%, indicating relatively slow remediation. The experimental group with added biological materials exhibited a strong remediation effect on PAHs throughout the entire remediation cycle, achieving a remediation efficiency of 72.47% ± 2.31% after one year.
[0064] In soils planted with corn, the remediation effect was similar to that in soils planted with soybeans. The soil's self-remediation capacity was 6.17% ± 1%. The experimental group with added biological materials achieved a PAH removal rate of 69.52% ± 2.01%.
Claims
1. A biomaterial for efficiently removing organic pollutants from freeze-thawed soil, characterized in that, The biomaterial is prepared by immobilizing a mixed microbial agent using a mixed biochar as an immobilization carrier and employing an adsorption method. The mixed biochar is made by mixing carbonized corn cob biochar and carbonized wheat straw biochar. The mixed microbial agent consists of Pseudomonas sp. SDR4 (CGMCC NO.14048) and Mortierella alpine JDR7 (CGMCC NO.15183).
2. The biomaterial for efficiently removing organic pollutants from freeze-thawed soil according to claim 1, characterized in that, By mass ratio, corn cob biochar : wheat straw biochar = 1 : (1-3).
3. The biomaterial for efficiently removing organic pollutants from freeze-thawed soil according to claim 1, characterized in that, By volume ratio, Pseudomonas sp. SDR4 : Mortierella alpine JDR7 = 1 :
1.
4. A method for preparing a biomaterial for efficiently removing organic pollutants from freeze-thawed soil as described in claim 1, 2, or 3, characterized in that, The preparation method includes the following steps: 1) Preparation of mixed biochar: Corn cobs were washed, air-dried for 2-3 days, dried at 70℃-80℃, and then carbonized under anaerobic conditions to obtain corn cob biochar; wheat straw was washed, air-dried for 2-3 days, dried at 70℃-80℃, and then carbonized under anaerobic conditions to obtain wheat straw biochar; corn cob biochar and wheat straw biochar were mixed and sterilized to obtain mixed biochar; 2) Preparation of mixed bacterial agent: Pseudomonas sp. SDR4 (CGMCC NO.14048) and Mortierella alpine JDR7 (CGMCC NO.15183) were inoculated into complete culture medium and cultured for 2-3 days. Then, they were inoculated into liquid culture medium and cultured continuously for 72-74 hours at 120 rpm in a constant temperature shaking incubator at 15℃ to obtain mixed bacterial agent. 3) Mix the mixed biochar with the proliferation culture medium, and after thorough soaking, inoculate the mixed bacterial agent at a rate of 10% per day for 3-4 days. Finally, place the culture system in a constant temperature environment of 15℃ for cultivation to obtain biomaterials that can efficiently remove organic pollutants from freeze-thawed soil.
5. The method for preparing a biomaterial for efficiently removing organic pollutants from freeze-thawed soil according to claim 4, characterized in that, In step 1), the corn cob biochar is obtained by carbonization at 450℃-550℃ for 4-5 hours under anaerobic conditions; wheat straw biochar is obtained by carbonization at 250℃-350℃ for 4-5 hours under anaerobic conditions; the sterilization process is carried out in an autoclave at 120℃-122℃ for 60-70 minutes.
6. The method for preparing a biomaterial for efficiently removing organic pollutants from freeze-thawed soil according to claim 4, characterized in that, In step 2), the complete culture medium consists of: 9g-11g peptone, 4g-6g yeast extract, 4g-6g NaCl, 18g-22g agar, distilled water added to 1000mL, pH 7.1-7.2, sterilized at 121℃ for 20-30min; the liquid culture medium consists of: 7g-9g sucrose, 5g-7g yeast extract, 0.8g-1.2g KH2PO4, 3g-5g (NH4)2HPO4, 0.4g-0.6g MgSO4·H2O, pH 6.0-6.5, sterilized at 121℃ for 20-30min.
7. The method for preparing a biomaterial for efficiently removing organic pollutants from freeze-thawed soil according to claim 4, characterized in that, In step 3), the biochar and culture medium are mixed according to the material-to-liquid ratio: 1g:(1mL-2mL); the culture medium is composed of: 75g-85g sucrose, 55g-65g yeast extract, 8g-12g KH2PO4, 35g-45g (NH4)2HPO4, 4g-6g MgSO4·H2O, pH 6.0-6.5, sterilized at 121℃ for 20min-30min, and stored at 4℃.
8. The application of the biomaterials described in claim 1, 2 or 3 in the efficient removal of organic pollutants from freeze-thawed soil.
9. The application according to claim 8, characterized in that, The method is as follows: In freeze-thawed soil, the biological material described in claim 1, 2 or 3 is mixed in situ with the soil from the surface to a depth of 10cm-30cm.
10. The application according to claim 8, characterized in that, The freeze-thawed soil refers to soil in Northeast China used for growing soybeans or corn; the organic pollutant is polycyclic aromatic hydrocarbons.