Microbial active filler for degrading soil micro-plastics as well as preparation method and application of microbial active filler

Abstract

The invention relates to the technical field of microbial active fillers. The invention provides a microbial active filler for degrading soil micro-plastics as well as a preparation method and application of the microbial active filler. The microbial active filler comprises aspergillus fumigatus and a filler, the Latin name of the aspergillus fumigatus is Aspergillus fumigatus, the preservation name of the aspergillus fumigatus is aspergillus fumigatus F, the preservation place is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is September 18, 2024, and the preservation number is CGMCC NO.41513. The invention further discloses a preparation method of the aspergillus fumigatus. According to the filler disclosed by the invention, bacteria can be uniformly attached to the surface of the filler and can enter the filler, so that the bacterial cell loading amount of the microbial active filler is high, the microbial activity is improved, and the starting period is shortened. The components of the filler can adsorb the micro-plastics in the polluted soil, so that bacterial cells can be in contact with the micro-plastics in the soil, and migration and diffusion of the micro-plastics in the soil can be prevented and controlled.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial active fillers, and particularly relates to a microbial active filler for degrading soil microplastics, a preparation method thereof, and an application thereof. Background Art

[0002] As a new pollutant, microplastics (MPs) have rapidly become a hot issue in the environmental field. MPs generally refer to different types of polymers such as fibers, fragments, and films with a size upper limit of 5 mm. Widespread MPs have been detected in the ocean, soil, aquatic organisms and plants, and even in human blood. Early studies on MPs mainly focused on the source tracing, abundance, environmental behavior and effects of MPs in the aquatic environment. However, with the expansion of the research field, the problem of MPs pollution in the terrestrial environment has been gradually taken seriously. MPs in soil have multiple sources, wide distribution and great influence. MPs enter the soil environment through various channels such as agricultural film residues, wastewater irrigation, and fertilizer application. Due to the characteristics of strong migration, combined pollution, low recyclability, and weak degradability of MPs, MPs existing in soil for a long time affect the growth of animals and plants, change the soil structure and properties. At the same time, more and more studies have found that MPs can be absorbed into plants and then enter the human body through the food chain, threatening human health.

[0003] Due to its impact on the ecological environment and human health, Chinese and foreign scholars currently pay special attention to how to reduce microplastics. Currently, the main degradation methods include physical methods, chemical methods, and biological methods. The biological method has the advantages of low investment, high benefits, long duration, wide treatment scope, great development potential, and environmental friendliness, and is the main development direction for future environmental pollution treatment. However, the traditional bioremediation method of directly adding effective microbial agents to the polluted environment has problems such as the death of microorganisms due to their poor adaptation to the environment, and the instability of the floating of strains during pollution treatment and being easily taken away, so that the repair benefits of microorganisms cannot be maximally exerted. Therefore, the research and development of efficient, economic, environmentally friendly and long-term stable immobilized microbial composites is one of the research focuses of future bioremediation technologies, and is of great significance for realizing the bioremediation of soil microplastics. The immobilization technology has a strong ability to retain strains and a high concentration of strains, so it has a strong impact resistance and tolerance to toxic substances, and has strong pertinence in removing target pollutants, greatly improving or enhancing the treatment efficiency of the system, and becoming one of the effective ways to strengthen the treatment of toxic and refractory pollutants. Therefore, it is urgent to carry out research on the treatment of microplastic-polluted soil by immobilized microbial technology. Summary of the Invention

[0004] The object of the present invention is to provide a microbial active filler for degrading soil microplastics, its preparation method and application, which can enable bacteria to adhere evenly to the surface of the filler and can enter the pores inside the filler, so that the microbial active filler has a high bacterial cell loading amount, improves microbial activity, and shortens the startup period.

[0005] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0006] The present invention provides a microbial active filler for degrading soil microplastics, and the microbial active filler includes Aspergillus fumigatus and a filler;

[0007] The Latin name of the Aspergillus fumigatus is Aspergillus fumigatus, and the deposit name is: Aspergillus fumigatus F, the deposit location: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit date: September 18, 2024, the deposit number: CGMCC NO. 41513.

[0008] Preferably, the effective viable bacteria number of Aspergillus fumigatus in the microbial active filler is not less than 10 7 CFU / g.

[0009] Preferably, the type of the filler is one or more of corn straw biochar particles, rice straw biochar particles, activated carbon particles, steel slag particles, zeolite particles and wood chips.

[0010] Preferably, the density of the filler is 0.2 - 3.5 g / cm 3 , the porosity is 30 - 95%, and the particle size is 2 - 8 mm.

[0011] The present invention also provides a preparation method of the microbial active filler, including the following steps:

[0012] (1) Mix the Aspergillus fumigatus suspension and the filler, and after adsorption, freezing and drying, obtain the Aspergillus fumigatus immobilized filler;

[0013] (2) Mix the Aspergillus fumigatus immobilized filler and the carboxymethyl cellulose - sodium alginate mixed solution, add calcium chloride solution, and after crosslinking, obtain the microbial active filler.

[0014] Preferably, in step (1), the mass ratio of the Aspergillus fumigatus suspension to the filler is 10 - 20:1, and the preparation method of the Aspergillus fumigatus suspension is: ferment and culture Aspergillus fumigatus step by step according to an inoculation amount of 8 - 20%, and separate to obtain an Aspergillus fumigatus suspension with a cell concentration of 0.8 - 1.2 g / L;

[0015] The culture medium used for the fermentation and culture of Aspergillus fumigatus includes the following raw materials in parts by weight: KH 2 PO4 0.60 - 0.80 parts, K 2 HPO 4 0.60 - 0.80 parts, MgSO 4 ·7H 2 O 0.60 - 0.80 parts, NH 4 NO 3 0.90 - 1.10 parts, NaCl 0.004 - 0.006 parts, FeSO 4 ·7H 2 O 0.001 - 0.003 parts, ZnSO 4 ·7H 2 O 0.001 - 0.003 parts, MnSO 4 ·H 2 O 0.001 - 0.002 parts, micro - plastics 1 - 3 parts, water 950 - 1100 parts;

[0016] The medium is adjusted to a pH value of 7.0 - 7.2 before use and sterilized at 115 - 121 °C for 15 - 20 min.

[0017] Preferably, in the carboxymethyl cellulose - sodium alginate mixed solution in step (2), the mass fraction of carboxymethyl cellulose is 1 - 3%, the mass fraction of sodium alginate is 1 - 3%, the mass ratio of the Aspergillus fumigatus immobilized filler to the carboxymethyl cellulose - sodium alginate mixed solution is 1:4 - 1:10, the mass fraction of calcium chloride in the calcium chloride solution is 1 - 4%, and the dosage of the calcium chloride solution is 280 mL - 700 mL.

[0018] The present invention also provides the application of the microbial active filler in the degradation of micro - plastics.

[0019] Preferably, the types of the micro - plastics include polyethylene micro - plastics and / or polyethylene terephthalate.

[0020] The present invention also provides a method for degrading micro - plastics using the microbial active filler, comprising the following steps:

[0021] A. Place the microbial active filler in a liquid inorganic salt medium with micro - plastics as the sole carbon source at a mass ratio of 5 - 7%, and activate the microbial active filler for 1 - 10 days at 28 - 30 °C and 120 - 150 rpm to obtain the activated microbial active filler;

[0022] B. Uniformly add the activated microbial active filler to the micro - plastic - contaminated soil at a mass ratio of 7% - 9% and treat for 50 - 100 days.

[0023] The present invention provides a microbial active filler for degrading soil microplastics, a preparation method thereof, and an application thereof. The microbial active filler comprises Aspergillus fumigatus and a filler. The Latin name of Aspergillus fumigatus is Aspergillus fumigatus, and the deposit name is: Aspergillus fumigatus F. The deposit location is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is September 18, 2024, and the deposit number is CGMCC NO. 41513. The filler of the present invention can make the bacteria evenly adhere to the surface of the filler and can enter the pores inside the filler, so that the microbial active filler has a high bacterial cell loading amount, improves the microbial activity, and shortens the startup period. The components of the filler can adsorb microplastics in the polluted soil, facilitate the contact between bacterial cells and microplastics in the soil, and can control the migration and diffusion of microplastics in the soil.

[0024] Beneficial effects:

[0025] 1. The present invention uses biochar as a carrier to prepare a biochar-based microbial immobilized filler from Aspergillus fumigatus F with strong microplastic degradation ability screened from soil containing microplastics, and applies it to the degradation of soil microplastics, which can significantly reduce the content of microplastics.

[0026] 2. Aspergillus fumigatus F is fixed to biochar by adsorption, and then through the action of embedding crosslinking, sedimentation, adhesion, centrifugation and other mechanics, a biochar-based microbial immobilized filler is obtained. The bacterial cells can not only evenly adhere to the surface of the filler, but also enter the pores inside the filler, so that the microbial active filler has a high bacterial cell loading amount, improves the microbial activity, and shortens the startup period.

[0027] 3. Both biochar and cellulose components can adsorb microplastics in the polluted soil, facilitate the contact between bacterial cells and microplastics in the soil, and can control the migration and diffusion of microplastics in the soil and maintain a long-term stable soil microplastic remediation effect.

[0028] Deposit description

[0029] Aspergillus fumigatus, with the Latin name Aspergillus fumigatus, the deposit name is: Aspergillus fumigatus F. The deposit location is: China General Microbiological Culture Collection Center, the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 18, 2024, and the deposit number is CGMCC NO. 41513. Specific embodiments

[0030] The present invention provides a microbial active filler for degrading soil microplastics, and the microbial active filler includes Aspergillus fumigatus and a filler.

[0031] The Latin name of the Aspergillus fumigatus is Aspergillus fumigatus, and the preservation name is: Aspergillus fumigatus F. The preservation location is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The preservation date is: September 18, 2024, and the preservation number is: CGMCC NO. 41513.

[0032] In the present invention, the effective viable count of Aspergillus fumigatus in the microbial active filler is preferably not less than 10 7 CFU / g, and further preferably 10 7 ~10 8 CFU / g.

[0033] In the present invention, the type of the filler is preferably one or more of corn straw biochar particles, rice straw biochar particles, activated carbon particles, steel slag particles, zeolite particles, and wood chips.

[0034] In the present invention, the density of the filler is preferably 0.2~3.5 g / cm 3 and further preferably 1~2 g / cm 3 , the porosity is preferably 30~95%, further preferably 60~90%, and the particle size is preferably 2~8 mm, further preferably 2.5~6 mm, and still further preferably 3~5 mm.

[0035] The present invention also provides a preparation method of the microbial active filler, including the following steps:

[0036] (1) Mix the Aspergillus fumigatus suspension and the filler, and after adsorption, freezing, and drying, obtain the Aspergillus fumigatus immobilized filler;

[0037] (2) Mix the Aspergillus fumigatus immobilized filler and the carboxymethyl cellulose-sodium alginate mixed solution, add calcium chloride solution, and after crosslinking, obtain the microbial active filler.

[0038] In the present invention, in step (1), the mass ratio of the Aspergillus fumigatus suspension to the filler is preferably 10~20:1, and further preferably 15:1. The preparation method of the Aspergillus fumigatus suspension is preferably: ferment and culture Aspergillus fumigatus step by step according to an inoculation amount of 8~20%, and separate to obtain an Aspergillus fumigatus suspension with a cell concentration of 0.8~1.2 g / L. Further preferably: ferment and culture Aspergillus fumigatus step by step according to an inoculation amount of 15%, and separate to obtain an Aspergillus fumigatus suspension with a cell concentration of 1 g / L;

[0039] When fermenting and culturing the Aspergillus fumigatus, the medium preferably used includes the following raw materials in parts by weight: KH2 PO 4 0.60 - 0.80 parts, K 2 HPO 4 0.60 - 0.80 parts, MgSO 4 ·7H 2 O 0.60 - 0.80 parts, NH 4 NO 3 0.90 - 1.10 parts, NaCl 0.004 - 0.006 parts, FeSO 4 ·7H 2 O 0.001 - 0.003 parts, ZnSO 4 ·7H 2 O 0.001 - 0.003 parts, MnSO 4 ·H 2 O 0.001 - 0.002 parts, micro - plastics 1 - 3 parts, water 950 - 1100 parts. Further preferably: KH 2 PO 4 0.70 parts, K 2 HPO 4 0.70 parts, MgSO 4 ·7H 2 O 0.70 parts, NH 4 NO 3 1.0 part, NaCl 0.005 part, FeSO 4 ·7H 2 O 0.002 parts, ZnSO 4 ·7H 2 O 0.002 parts, MnSO 4 ·H 2 O 0.0015 parts, micro - plastics 2 parts, water 1000 parts;

[0040] Before use, the pH value of the culture medium is preferably adjusted to 7.0 - 7.2, and sterilized at 115 - 121 °C for 15 - 20 min. Further preferably: 7.1, sterilized at 118 °C for 17 - 18 min.

[0041] In the present invention, the types of the micro - plastics preferably include polyethylene micro - plastics and / or polyethylene terephthalate.

[0042] In the present invention, in step (2), the mass fraction of methylcellulose in the carboxymethylcellulose-sodium alginate mixed solution is preferably 1-3%, more preferably 2%, the mass fraction of sodium alginate is preferably 1-3%, more preferably 2%, the mass ratio of the Aspergillus fumigatus immobilized filler to the carboxymethylcellulose-sodium alginate mixed solution is preferably 1:4-1:10, more preferably 1:6-1:8, the mass fraction of calcium chloride in the calcium chloride solution is preferably 1-4%, more preferably 2-3%, and the dosage of the calcium chloride solution is preferably 280 mL-700 mL, more preferably 350-450 mL.

[0043] The present invention also provides the application of the microbial active filler in the degradation of microplastics.

[0044] In the present invention, the types of the microplastics include polyethylene microplastics and / or polyethylene terephthalate, and the particle size of the microplastics is less than 5 mm.

[0045] The present invention also provides a method for degrading microplastics by using the microbial active filler, comprising the following steps:

[0046] A. Placing the microbial active filler in a liquid inorganic salt medium with microplastics as the sole carbon source according to a mass ratio of 5-7%, and activating the microbial active filler for 1-10 days at 28-30°C and 120-150 rpm to obtain the activated microbial active filler;

[0047] B. Uniformly adding the activated microbial active filler to the microplastic-polluted soil according to a mass ratio of 7%-9% and treating for 50-100 days.

[0048] In the present invention, the ratio in step A is 6%.

[0049] In the present invention, the activation conditions in step A are preferably 29°C and 135 rpm.

[0050] In the present invention, the activation time in step A is preferably 2-5 days.

[0051] In the present invention, the ratio in step B is 8%.

[0052] In the present invention, the activation time in step B is preferably 75 days.

[0053] In the present invention, for the microplastic-polluted soil containing 100-120 microplastics per kg, after being treated with the above filler for 50-100 days, the degradation rate of the microplastics reaches 5-20%.

[0054] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] Example 1

[0056] A microbial active filler, the filler is loaded with Aspergillus fumigatus F, the filler is wheat straw biochar, the filler density is 0.8 kg / m 3 , the porosity is 89%, and the particle size is 3 mm; Aspergillus fumigatus F is loaded on the filler in the form of a bacterial suspension, and the mass ratio of the bacterial suspension to the filler is 10:1. The cell concentration of Aspergillus fumigatus F in the prepared microbial active filler is 10 7 CFU / g;

[0057] The preparation steps of the microbial active filler are as follows:

[0058] (1) Preparation of the bacterial suspension: Aspergillus fumigatus F is fermented and cultured step by step at an inoculation amount of 8%, and a bacterial suspension of Aspergillus fumigatus F with a cell concentration of 0.8 g / L is obtained by separation;

[0059] The preparation method of the Aspergillus fumigatus F bacterial suspension is as follows;

[0060] Aspergillus fumigatus F is fermented and cultured step by step at an inoculation amount of 8%, which requires 3 generations of rejuvenation process. The culture temperature at each level is 28 °C, the rotation speed is 150 rpm, the culture time is 48 h, and after fermentation, it is centrifuged and concentrated at 8000 rpm for 5 min. The supernatant is discarded and resuspended with a liquid inorganic salt medium to obtain a bacterial suspension of Aspergillus fumigatus F with a cell concentration of 0.8 g / L;

[0061] The composition of the medium used for fermentation culture (liquid inorganic salt medium) is: 0.70 g KH 2 PO 4 , 0.70 g K 2 HPO 4 , 0.70 g MgSO 4 ·7H 2 O, 1.00 g NH 4 NO 3 , 0.005 g NaCl, 0.002 g FeSO4·7H 2 O, 0.002 g ZnSO 4 ·7H 2 O and 0.001 g MnSO4·H 2 O, 1000 ml of water, pH value 7.0, sterilized at 121 °C for 20 min;

[0062] (2) Preparation of the microbial active filler:

[0063] Mix the Aspergillus fumigatus F bacterial suspension prepared in (1) with the filler at a mass ratio of the bacterial suspension to the filler of 10:1, and perform mixing adsorption at 30 °C and 150 r / min for 8 h. Then, freeze, dry, and grind to obtain the microbial immobilized biochar (particle size 3 mm). Add the microbial immobilized biochar (1%, w / w) to a mixed solution containing 1.5% (w / w) carboxymethyl cellulose and 1.5% (w / w) sodium alginate, stir evenly, and slowly drop it into a 2% calcium chloride solution for crosslinking for 16 h to obtain the immobilized filler. After rinsing the above immobilized filler 3 times with deionized water, dry it at low temperature to obtain the biochar-based microbial immobilized filler, and store it at 4 °C for later use;

[0064] Repeat step 3 times to completely spray the bacterial suspension with a mass ratio of 10:1 to the filler, so that the material surface and micropores are both loaded with bacterial cells, and obtain the Aspergillus fumigatus F cell concentration of 10 7 CFU / g microbial active filler.

[0065] Example 2 Preparation of microbial active filler containing Aspergillus fumigatus F

[0066] A microbial active filler, the filler is loaded with Aspergillus fumigatus F, the filler is wheat straw biochar, the filler density is 0.7 kg / m 3 , the porosity is 30%, and the particle size is 2 mm; Aspergillus fumigatus F is loaded on the filler in the form of a bacterial suspension, and the mass ratio of the bacterial suspension to the filler is 15:1. The Aspergillus fumigatus F cell concentration in the prepared microbial active filler is 10 8 CFU / g;

[0067] The preparation steps of the microbial active filler are as follows:

[0068] (1) Preparation of the bacterial suspension: Ferment and culture Aspergillus fumigatus F step by step at an inoculation amount of 10%, and separate to obtain an Aspergillus fumigatus F bacterial suspension with a cell concentration of 1 g / L;

[0069] The preparation method of the Aspergillus fumigatus F bacterial suspension is as follows;

[0070] Aspergillus fumigatus F is fermented and cultured step by step at an inoculation amount of 10%, which requires 3 generations of rejuvenation processes. The culture temperature at each level is 28 °C, the rotation speed is 150 rpm, the culture time is 48 h, and after fermentation, the culture is centrifuged and concentrated at 8000 rpm for 5 min. Discard the supernatant, and resuspend it with a liquid inorganic salt medium to obtain an Aspergillus fumigatus F bacterial suspension with a cell concentration of 1 g / L;

[0071] The composition of the medium used for fermentation and culture is (liquid inorganic salt medium): 0.70 g KH 2 PO 4 , 0.70 g K 2 HPO 4, 0.70 g MgSO 4 ·7H 2 O, 1.00 g NH 4 NO 3 , 0.005 g NaCl, 0.002 g FeSO4·7H 2 O, 0.002 g ZnSO 4 ·7H 2 O and 0.001 g MnSO4·H 2 O, 1000 ml of water, pH value 7.0, sterilized at 121 °C for 20 min;

[0072] (2) Preparation of microbial active filler:

[0073] Mix the Aspergillus fumigatus F bacterial suspension prepared in (1) according to the mass ratio of the bacterial suspension to the filler of 15:1, mix and adsorb at 30 °C and 150 r / min for 8 h, then freeze, dry, and grind to obtain immobilized microbial biochar (particle size 3 mm). Add the immobilized microbial biochar (1%, w / w) to a mixed solution containing 1.5% (w / w) carboxymethyl cellulose and 1.5% (w / w) sodium alginate, stir evenly, and slowly drop it into 2% calcium chloride solution for crosslinking for 16 h to obtain the immobilized filler. After rinsing the above immobilized filler 3 times with deionized water, dry it at low temperature to obtain the biochar-based microbial immobilized filler, and store it at 4 °C for later use;

[0074] Repeat step 3 times to spray all the bacterial suspension with a mass ratio of 15:1 to the filler, so that the surface and micropores of the material are both loaded with bacterial cells, and obtain an Aspergillus fumigatus F cell concentration of 10 8 CFU / g of microbial active filler.

[0075] Preparation of microbial active filler containing Aspergillus fumigatus F in Example 3

[0076] A microbial active filler, the filler is loaded with Aspergillus fumigatus F, the filler is wheat straw biochar, the filler density is 0.7 kg / m 3 , the porosity is 60%, and the particle size is 8 mm; Aspergillus fumigatus F is loaded on the filler in the form of a bacterial suspension, and the mass ratio of the bacterial suspension to the filler is 20:1. The cell concentration of Aspergillus fumigatus F in the prepared microbial active filler is 10 9 CFU / g;

[0077] The preparation steps of the microbial active filler are as follows:

[0078] (1) Preparation of bacterial suspension: Ferment and culture Aspergillus fumigatus F step by step according to an inoculation amount of 12%, and separate to obtain an Aspergillus fumigatus F bacterial suspension with a cell concentration of 1.2 g / L;

[0079] The preparation method of Aspergillus fumigatus F bacterial suspension is as follows;

[0080] Aspergillus fumigatus F is fermented and cultured step by step with an inoculation amount of 12%, and a rejuvenation process of 3 generations is required. The culture temperature at each level is 28°C, the rotation speed is 150 rpm, and the culture time is 48 h. After fermentation, the culture is centrifuged and concentrated at 8000 rpm for 5 min, and the supernatant is discarded. The bacterial cells are resuspended with a liquid inorganic salt medium to obtain an Aspergillus fumigatus F bacterial suspension with a cell concentration of 1.2 g / L;

[0081] The composition of the medium used for fermentation culture (liquid inorganic salt medium) is: 0.70 g KH 2 PO 4 ,0.70 g K 2 HPO 4 ,0.70 g MgSO 4 ·7H 2 O, 1.00 g NH 4 NO 3 , 0.005 g NaCl, 0.002 g FeSO4·7H 2 O, 0.002 g ZnSO 4 ·7H 2 O and 0.001 g MnSO4·H 2 O, 1000 ml of water, pH value 7.0, sterilized at 121°C for 20 min;

[0082] (2) Preparation of microbial active filler:

[0083] The Aspergillus fumigatus F bacterial suspension prepared in (1) is mixed at a mass ratio of the bacterial suspension to the filler of 20:1, and mixed and adsorbed at 30°C and 150 r / min for 8 h, and then frozen, dried, and ground to obtain microbial immobilized biomass charcoal (particle size 3 mm). The microbial immobilized biomass charcoal (1%, w / w) is added to a mixed solution containing 1.5% (w / w) carboxymethyl cellulose and 1.5% (w / w) sodium alginate, stirred evenly, and slowly dropped into 2% calcium chloride solution for crosslinking for 16 h to obtain the immobilized filler. After the above immobilized filler is rinsed 3 times with deionized water, it is dried at low temperature to obtain a biomass charcoal-based microbial immobilized filler, which is stored at 4°C for later use;

[0084] Repeat step 3 times to spray all the bacterial suspension with a mass ratio of 20:1 to the filler, so that the surface and micropores of the material are all loaded with bacterial cells, and an Aspergillus fumigatus F cell concentration of 10 9 CFU / g microbial active filler is obtained.

[0085] Example 4 Method for degrading soil microplastics using microbial active filler

[0086] The microbial active filler prepared in Example 1 was used for the biodegradation of soil microplastics. The specific soil conditions are as follows: The soil for this experiment was collected from the 0-100 cm underground of the soil where a PE plastic film with a length×width×thickness of 500 cm×45 cm×0.008 mm had been covered and sown all year round in the Hetao Irrigation Area of Inner Mongolia Autonomous Region as a sample. The soil type is mainly saline-alkali soil, with an average salt storage of about 1342 g / m 3 , with a pH of 8, an organic matter content of 10.9 g / kg, and a water content of 26.14%. Before the experiment, large impurities and stones were removed from the collected soil samples. After natural air drying, they were crushed and ground using an agate mortar, and then sieved through a 2 mm sieve. At the same time, PE and PET particles with a diameter of 0.75 μm dyed with Sudan red were added and thoroughly mixed to make the PE and PET particle contents reach 100 per kg respectively.

[0087] The specific degradation steps are as follows:

[0088] (1) Place the microbial active filler loaded with Aspergillus fumigatus F in a liquid inorganic salt medium with a PE film with a diameter less than 5 mm and PET particles with a diameter of 0.75 μm as the carbon source, and activate the microbial active filler at 28 °C and 150 rpm for 3 days. The concentrations of the PE film and PET particles are both 1 g / L;

[0089] The composition of the liquid inorganic salt medium with a PE film with a diameter less than 0.5 mm and PET particles with a diameter of 0.75 μm as the carbon source is: 0.70 g KH 2 PO 4 , 0.70 g K 2 HPO 4 , 0.70 g MgSO 4 ·7H 2 O, 1.00 g NH 4 NO 3 , 0.005 g NaCl, 0.002 g FeSO 4 ·7H 2 O, 0.002 g ZnSO 4 ·7H 2 O and 0.001 g MnSO 4 ·H 2 O, with 1 g each of the PE film and PET particles, 1000 ml of water, a pH value of 7.0, and sterilized at 121 °C for 20 min.

[0090] (2) After the activated microbial active filler is taken out of the activation medium, it is air-dried and evenly spread on the microplastic-contaminated soil at 7% (w / w, added according to the weight before activation), and degradation is carried out under natural conditions. Deionized water is added to keep the field water holding capacity at 30%. Samples are taken every 7 days, and the dyed PE and PET particles degraded by Streptomyces sudanensis are recovered and washed by sieving method, and the microplastic weight loss rate is calculated.

[0091] Use effect experiment: 50 days after the filler is used for the treatment of microplastics, the dyed PE and PET particles degraded by Streptomyces sudanensis are recovered and washed by sieving method, the mass of PE and PET particles in each kilogram of soil is measured, and it can be calculated by formula 1 that the degradation rates of PE film and PET particles reach 8.15% and 8.26% respectively.

[0092] Formula 1:

[0093] Microplastic weight loss rate (%) = [(original mass of microplastics - mass of microplastics after degradation) / original mass of microplastics] × 100%.

[0094] Method for improving physical and chemical properties of soil during microplastic degradation by microbial active filler in Example 5

[0095] The microbial active filler prepared in Example 1 is used for the soil microplastic biodegradation test. The specific land conditions are as follows: The soil samples used in this experiment are from the Hetao Irrigation Area in Inner Mongolia Autonomous Region, taken from a depth of 0 to 100 cm underground. In this area, plastic films have been used for covering cultivation all year round. After 5 years, 10 years, and 20 years of film mulching, the average abundance values of microplastics in the soil are 2,526 pieces / kg, 4,352.8 pieces / kg, and 6,070 pieces / kg respectively, mainly in the form of PE films and PET fibers with a particle size <1 mm. In addition, the soil in this area is mainly of the saline-alkali soil type, with an average salt storage of about 1,342 g / m 2 , the pH value is 8, the organic matter content is 10.9 g / kg, and the water content is 26.14%. Take 5 kg of air-dried and ground soil, add PE and PET particles with a particle size of 0.75 mm dyed with Sudan red, so that the contents of PE and PET particles reach 100 pieces / kg respectively, and evenly spread the microbial active filler prepared in Example 1 on the microplastic-contaminated soil at 7% (w / w, added according to the weight before activation). Take 5 g of soil samples every 7 days, and detect the contents of pH, salinity, total phosphorus, total nitrogen, organic matter, etc. in the soil before and after the experiment according to the methods specified in the national standards;

[0096] The specific methods for detecting the contents of pH, salinity, total phosphorus, total nitrogen, organic matter, etc. in the soil are as follows:

[0097] (1) pH: Mix the soil and water in a ratio of 1:5, stir evenly and let it stand still to fully dissolve the ions in the soil. Then, use a calibrated pH meter to measure the pH value of the supernatant, and repeat the measurement 3 times to obtain the pH value.

[0098] (2) Salinity: Indirectly reflect the soil salt content by measuring the electrical conductivity (EC) of the soil solution. During the measurement, usually mix the soil and water in a mass ratio of 1:5, oscillate and filter to obtain the leaching solution, and then use a conductivity meter to measure its EC value.

[0099] (3) Total phosphorus: Use the alkali fusion - molybdenum antimony anti - spectrophotometric method. This method first uses sodium hydroxide to melt the soil to convert the phosphorus in the soil into soluble orthophosphate. Then, dissolve the melt with dilute sulfuric acid and distilled water, and react with the molybdenum antimony anti - color reagent to generate phosphomolybdenum blue. Finally, measure the absorbance at a wavelength of 700 nm with a spectrophotometer, and find out the phosphorus content from the calibration curve.

[0100] (4) Total nitrogen: Generally use the alkaline potassium persulfate digestion - ultraviolet spectrophotometric method. This method is to use an alkaline potassium persulfate solution to convert the nitrogen in the nitrogen - containing compounds in the sample into nitrate at 122 °C, and then measure the absorbance at wavelengths of 220 nm and 275 nm respectively on an ultraviolet spectrophotometer, and calculate the corrected absorbance according to the formula to obtain the total nitrogen content in the soil.

[0101] (5) Organic matter: Generally use the potassium dichromate volumetric method. This method is to oxidize the organic matter in the soil with a potassium dichromate solution under heating conditions, and titrate the remaining potassium dichromate with a ferrous sulfate solution to calculate the organic matter content in the soil.

[0102] The results are shown in Table 1.

[0103] Table 1 Determination of soil physical and chemical properties before and after the action of microbial active filler

[0104]

[0105] Conclusion: As can be seen from Table 1, the preparation of the microbial active filler in this application can have various effects on the soil physical and chemical properties during the process of degrading soil microplastics, including adjusting the acidity and alkalinity, reducing the salt content, increasing the organic matter content, and redistributing or transforming phosphorus and nitrogen elements, etc. These effects help to improve the soil quality, maintain soil health and ecological balance. The specific effects are as follows: (1) Adjustment of acidity and alkalinity: The pH value of the soil gradually decreases from 8.46 to 8.21. This indicates that the microbial active filler has the ability to adjust the soil acidity and alkalinity, which helps the soil to maintain a suitable acid - base environment.

[0106] (2) Reduction in salt content: The salinity percentage shows a downward trend, decreasing from 0.428% to 0.408%. This indicates that the microbial active filler may help reduce the salt content in the soil, thereby improving the soil salinization condition;

[0107] (3) Increase in organic matter content: The percentage of organic matter increases significantly, from 4.73% to 6.77%. This shows that the microbial active filler plays a positive role in promoting the cycle of soil organic matter and increasing soil fertility;

[0108] (4) Redistribution or transformation of phosphorus and nitrogen elements: The contents of total phosphorus and total nitrogen show a trend of first increasing and then decreasing under the action of the microbial active filler. This may mean that during the process of degrading soil microplastics by the microbial active filler, the phosphorus and nitrogen elements in the soil are redistributed or transformed. Although the final content decreases, this change process may have an impact on the balance of the soil ecosystem.

[0109] As can be seen from the above embodiments, the present invention provides a microbial active filler for degrading soil microplastics, its preparation method and application. The microbial active filler includes Aspergillus fumigatus and a filler; the Latin name of Aspergillus fumigatus is Aspergillus fumigatus, and the deposit name is: Aspergillus fumigatus F, the deposit location: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit date: September 18, 2024, the deposit number: CGMCC NO. 41513. The filler of the present invention can make the bacteria evenly adhere to the surface of the filler and can enter the pores inside the filler, so that the microbial active filler has a high bacterial cell loading, improves microbial activity, and shortens the start-up period. The components of the filler can adsorb microplastics in the contaminated soil, which is beneficial for the contact between bacterial cells and microplastics in the soil and can control the migration and diffusion of microplastics in the soil.

[0110] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A microbial active filler for degrading soil microplastics, characterized in that: The microbial active filler comprises Aspergillus fumigatus and a filler; The Latin name of the Aspergillus fumigatus is (Aspergillus fumigatus), the preservation name is: Aspergillus fumigatus F, the preservation location: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the preservation date: September 18, 2024, and the preservation number: CGMCC NO.41513.

2. The microbial active filler according to claim 1, characterized in that: The effective viable bacteria count of Aspergillus fumigatus in the microbial active filler is not less than 10 7 CFU / g.

3. The microbial active filler according to claim 1, characterized in that: The filler is one or more of corn straw biochar particles, rice straw biochar particles, activated carbon particles, steel slag particles, zeolite particles and sawdust.

4. The microbial active filler according to claim 1, characterized in that: The density of the filler is 0.2-3.5 g / cm 3 The porosity is 30-95% and the particle size is 2-8 mm.

5. The method for preparing the microbial active filler according to any one of claims 1 to 4, characterized in that: The steps include: (1) mixing an Aspergillus fumigatus suspension and a filler, adsorbing, freezing, and drying to obtain an Aspergillus fumigatus immobilized filler; (2) The Aspergillus fumigatus immobilized filler and the carboxymethyl cellulose-sodium alginate mixed solution are mixed, and a calcium chloride solution is added, and the microbial active filler is obtained after cross-linking.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the Aspergillus fumigatus suspension to the filler in step (1) is 10-20:

1. The preparation method of the Aspergillus fumigatus suspension is as follows: fermenting and culturing the Aspergillus fumigatus step by step at an inoculation rate of 8-20%, and separating and obtaining an Aspergillus fumigatus suspension with a bacterial concentration of 0.8-1.2 g / L; The culture medium used in the fermentation culture of Aspergillus fumigatus includes the following raw materials in parts by weight: 0.60-0.80 parts of KH2PO4, 0.60-0.80 parts of K2HPO4, 0.60-0.80 parts of MgSO4·7H2O, 0.90-1.10 parts of NH4NO3, 0.004-0.006 parts of NaCl, 0.001-0.003 parts of FeSO4·7H2O, 0.001-0.003 parts of ZnSO4·7H2O, 0.001-0.002 parts of MnSO4·H2O, 1-3 parts of microplastics, and 950-1100 parts of water; The pH value of the culture medium is adjusted to 7.0-7.2 before use, and sterilized at 115-121° C. for 15-20 min.

7. The preparation method according to claim 5, characterized in that: In step (2), the mass fraction of methyl cellulose in the carboxymethyl cellulose-sodium alginate mixed solution is 1-3%, the mass fraction of sodium alginate is 1-3%, the mass ratio of the Aspergillus fumigatus immobilized filler to the carboxymethyl cellulose-sodium alginate mixed solution is 1:4-1:10, the mass fraction of calcium chloride in the calcium chloride solution is 1-4%, and the amount of the calcium chloride solution is 280 mL-700 mL.

8. Use of the microbial active filler according to any one of claims 1 to 4 in the degradation of microplastics.

9. The use according to claim 8, characterized in that: The types of microplastics include polyethylene microplastics and / or polyethylene terephthalate.

10. A method for degrading microplastics using the microbial active filler according to any one of claims 1 to 4, characterized in that: The steps include: A. placing the microbial active filler according to any one of claims 1 to 4 in a liquid inorganic salt culture medium with microplastics as the sole carbon source at a mass ratio of 5 to 7%, activating the microbial active filler at 28 to 30° C. and 120 to 150 rpm for 1 to 10 days to obtain an activated microbial active filler; B. Add the activated microbial active filler evenly into the microplastic contaminated soil at a mass ratio of 7% to 9% and treat for 50 to 100 days.

Images