Bacillus tropicus and application thereof in degrading plastic and repairing plastic-polluted soil

Abstract

The application provides a Bacillus tropicus SH-XP1, which is preserved in the China General Microbiological Culture Collection Center (CGMCC) and has a preservation number of CGMCC No. 37380. The application also provides a straw immobilized bacterial agent and application of the straw immobilized bacterial agent in degradation of plastics and remediation of plastic contaminated soil. The application uses straw as a carrier to provide physical attachment points and protective barriers for plastic degrading bacteria, so that the plastic degrading bacteria are protected from adverse environmental factors in the soil, and intermediate products generated in the degradation process of the straw itself can act as co-metabolic substrates to stimulate microbial growth, thereby synergistically promoting the degradation of plastics. The application significantly improves the survival rate, colonization ability and degradation activity of the bacterial agent in the soil, and solves the problems of easy loss and short activity duration of the free bacterial agent. The method of the application can significantly shorten the remediation period and improve the removal rate of plastics in the soil.

Description

Technical Field

[0001] This invention relates to a strain of tropical Bacillus and its application in degrading plastics and remediating plastic-contaminated soil. Background Technology

[0002] Plastic pollution, particularly "white pollution" caused by agricultural mulch film residue, has become a global soil environmental problem. Traditional plastics such as polyethylene (PE) are difficult to degrade in the natural environment, while biodegradable plastics such as polybutylene adipate / terephthalate (PBAT) also degrade slowly in soils lacking specific microorganisms and suitable conditions, potentially leading to the accumulation of microplastics. Existing physical and chemical remediation methods suffer from high costs and the risk of secondary pollution. Microbial remediation technology shows great potential due to its environmental friendliness and lower cost. However, directly applying free microbial agents to the soil is susceptible to environmental factors, resulting in low survival rates, poor colonization, and unstable remediation efficiency. Summary of the Invention

[0003] To address the problems existing in the prior art, this invention provides a strain of Bacillus tropicalis and its application in degrading plastics and remediating plastic-contaminated soil. This invention screens a strain of Bacillus tropicalis capable of degrading plastics and uses agricultural waste straw as a carrier to immobilize the aforementioned Bacillus tropicalis, preparing a straw-immobilized bacterial agent with good stability and degradation performance. This agent is used to remediate plastic-contaminated soil, significantly improving the degradation efficiency of plastics while simultaneously achieving the recycling of straw resources.

[0004] The technical solution adopted in this invention is as follows:

[0005] In a first aspect, the present invention provides a strain of Bacillus tropicus, wherein the Bacillus tropicus is Bacillus tropicus SH-XP1, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37380.

[0006] In a second aspect, the present invention provides a Bacillus tropicus bacterial suspension, characterized in that it is obtained by expanding the culture of the Bacillus tropicus CGMCC No.37380 strain described in the first aspect;

[0007] Preferably, the expanded culture involves inoculating the Bacillus tropicus CGMCC No. 37380 strain described in the first aspect into LB liquid medium, and culturing at a temperature of 28–32°C, a shaking speed of 160–200 rpm, and a culture time of 10–14 h to obtain a bacterial solution.

[0008] In a third aspect, the present invention provides a method for preparing a straw immobilized microbial agent, wherein activated Bacillus tropicus of the first aspect or Bacillus tropicus of the second aspect is mixed with pretreated crop straw, and the mixture is subjected to oscillation adsorption and immobilization under sterile conditions, followed by solid-liquid separation, and finally the crop straw immobilized with the microbial agent is sterilely dried to obtain the straw immobilized microbial agent;

[0009] As a preferred method, use 10 mL OD per gram of crop straw. 600 Bacterial solution with a value of 1.0.

[0010] Preferably, the pretreatment includes the step of pulverizing crop straw and then soaking it in an alkaline solution;

[0011] Preferably, the alkaline solution is a 1.5% to 2.5% NaOH solution;

[0012] Preferably, the soaking conditions are: soaking at 60 °C for 1.5–2.5 h;

[0013] Preferably, the process also includes rinsing with deionized water after soaking until the filtrate is neutral;

[0014] Preferably, the process also includes drying and sterilization after rinsing.

[0015] Preferably, the oscillation adsorption is performed at 28–32 °C;

[0016] Preferably, the frequency of the oscillation adsorption fixation is 130–170 rpm;

[0017] Preferably, the oscillation adsorption fixation time is 36–60 h.

[0018] Preferably, the crop straw includes at least one of corn straw, wheat straw, rice straw, and rapeseed straw; preferably, the crop straw is corn straw.

[0019] In a fourth aspect, the present invention provides a straw immobilization microbial agent, which is prepared by the method described in the third aspect.

[0020] In a fifth aspect, the present invention provides the application of Bacillus tropicus as described in the first aspect, Bacillus tropicus bacterial solution as described in the second aspect, or straw immobilized bacterial agent as described in the fourth aspect in degrading plastics and remediating plastic-contaminated soil.

[0021] Preferably, the plastic includes at least one of PE, PBAT, and PBAT black film.

[0022] Preferably, when remediating plastic-contaminated soil, the dosage of the straw immobilization microbial agent is 1.5–2.5 g / kg soil, more preferably 1.8–2.2 g / kg soil.

[0023] As a preferred method, when remediating plastic-contaminated soil, the soil moisture content can be adjusted, for example, to 58% to 62% of the field capacity.

[0024] Crop straw is a common agricultural waste, mainly composed of cellulose, hemicellulose, and lignin. Its rich porous structure and large specific surface area make it an excellent natural biomass carrier. Immobilizing functional bacterial strains using crop straw can provide physical protection and nutritional sources, improving their survival rate and activity in polluted environments, thus achieving efficient and long-lasting bioremediation. Currently, there are no reports on using crop straw as a carrier to immobilize bacterial strains and synergistically apply it to the remediation of plastic-contaminated soil. Therefore, screening core microbial degradation strains and applying them through crop straw immobilization is of great significance for degrading plastic-contaminated soil.

[0025] The beneficial effects of this invention are as follows:

[0026] (1) The present invention uses crop straw as a carrier, which not only provides a physical attachment point and protective barrier for tropical Bacillus, protecting it from adverse environmental factors in the soil, but also the intermediate products generated by the crop straw itself during the degradation process may serve as co-metabolite substrates to stimulate microbial growth, thereby synergistically promoting the degradation of plastics.

[0027] (2) The immobilization method of the present invention significantly improves the survival rate, colonization ability and degradation activity of the microbial agent in the soil, and solves the problems of easy loss and short-lasting activity of free microbial agents.

[0028] (3) This invention realizes the high-value-added resource utilization of agricultural waste straw, turning waste into treasure and reducing remediation costs. The entire remediation process does not introduce chemical reagents, is environmentally friendly, and has no secondary pollution.

[0029] (4) The method of the present invention can significantly shorten the remediation cycle and improve the removal rate of plastics in the soil.

[0030] (5) The addition of straw immobilization microbial agents can improve the physical and chemical properties of soil and has a certain soil improvement effect. Attached Figure Description

[0031] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0032] Figure 1 This is a process flow diagram of the method of the present invention.

[0033] Figure 2 A phylogenetic tree for Bacillus tropicus SH-XP1 based on the 16S rRNA gene sequence.

[0034] Figure 3 The graph shows the changes in the weight loss rates of PE, PBAT, and PBATB films in the soil in Example 3.

[0035] Figure 4 This is a scanning electron microscope (SEM) image of the surface morphology of PE, PBAT, and PBATB films in the soil in Example 3.

[0036] Figure 5 The growth indicators of plants under different treatments are: (a) plant growth phenotype; (b) root length and (c) plant height; (d) leaf area; (e) fresh weight and (f) dry weight; and (g) chlorophyll content.

[0037] Figure 6 Soil physicochemical properties under different treatments: (a) pH value and (b) electrical conductivity (EC); (c) available phosphorus (AP) and (d) available potassium (AK); (e) NH4. + -N; (f) Total organic carbon (TOC). Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to embodiments and comparative examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods. Unless otherwise specified, the raw materials or test materials used in the following examples are typical commercially available products. The quantitative experiments in the following examples and comparative examples were all performed in triplicate, and the results were averaged.

[0040] The technical solution of the present invention is briefly described as follows:

[0041] (1) A strain of Bacillus tropicus SH-XP1, which has a high degradation capacity for both polyethylene (PE) and polybutylene adipate / terephthalate (PBAT), was isolated, screened and identified from the long-term plastic pollution environment. Its 16S rRNA gene sequence has been submitted to GenBank (accession number PX599112). The bacterium has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No.37380. The bacterium was further cultured and prepared into a bacterial solution.

[0042] (2) Agricultural waste straw is used as a natural biomass carrier. After pretreatment and sterilization with alkali, it is used to immobilize the degrading bacterium Bacillus tropicus SH-XP1, providing physical protection, attachment sites and nutrient sources, and improving the survival rate and activity of the bacterial agent in the soil.

[0043] (3) The bacterial solution is combined with the pretreated straw by oscillation adsorption fixation and then air-dried to prepare straw immobilized bacterial agent (XP1). The process is simple, low-cost and easy to scale up.

[0044] (4) The immobilized microbial agent was uniformly mixed into the plastic-contaminated soil at an application rate of 1.5–2.5 g / kg soil, preferably 1.8–2.2 g / kg, and the soil moisture content was controlled at 58%–62% of field capacity. The remediation period was 45 days, achieving a synergistic remediation effect of plastic degradation, soil improvement, and plant growth promotion. The plastic included at least one of PE, PBAT, and PBAT black film.

[0045] Figure 1 This is a process flow diagram of the method of the present invention.

[0046] Specifically, the preparation method of the straw immobilization microbial agent of the present invention includes the following steps:

[0047] (1) Pre-treat crop straw:

[0048] (2) Mix the bacterial solution of Bacillus tropicus SH-XP1 with the pretreated crop straw, and then perform oscillation adsorption fixation under sterile conditions. Then, separate the solid and liquid, and finally sterilely dry the crop straw with the bacterial agent to obtain the crop straw immobilized bacterial agent.

[0049] In a preferred embodiment of the present invention, step (1) includes the step of pulverizing crop straw and then soaking it in an alkaline solution.

[0050] As a preferred embodiment, the alkaline solution is a NaOH solution with a mass fraction of 1.5% to 2.5%.

[0051] As a preferred embodiment, the soaking conditions are: soaking at 60 ℃ for 1.5 to 2.5 h.

[0052] As a preferred embodiment, the pretreatment further includes rinsing with deionized water after soaking until the filtrate is neutral.

[0053] As a preferred embodiment, the pretreatment further includes the steps of drying and sterilizing after rinsing.

[0054] In a preferred embodiment of the present invention, in step (2), the bacterial solution is prepared by inoculating the above-mentioned Bacillus tropicus CGMCC No.37380 strain into LB liquid medium, and culturing at a temperature of 28-32°C, a shaking speed of 160-200 rpm, and a culturing time of 10-14 h to obtain the bacterial solution.

[0055] As a preferred method, use 8–12 mL OD per gram of crop straw. 600 Bacterial solution with a value of 1.0.

[0056] In a preferred embodiment of the present invention, in step (2), the oscillation adsorption fixation is carried out at 28-32 °C.

[0057] As a preferred embodiment, the rotation speed of the oscillation adsorption fixation is 130–170 rpm.

[0058] As a preferred embodiment, the oscillation adsorption fixation time is 36–60 h.

[0059] In a preferred embodiment of the present invention, the crop straw includes at least one of corn straw, wheat straw, rice straw and rapeseed straw; preferably, the crop straw is corn straw.

[0060] Corn stalks are mainly composed of cellulose, hemicellulose, and lignin. Their surface has a dense waxy layer and a lignin barrier, resulting in closed natural pores and a small specific surface area, which is unfavorable for microbial adsorption and colonization. Alkali pretreatment has four aspects:

[0061] 1. Breaking down surface wax and lignin: NaOH can hydrolyze the surface wax of straw, degrade some lignin, open up closed pores, and greatly increase the specific surface area of ​​straw, providing more attachment sites for bacterial strains.

[0062] 2. Loosening the fiber structure: It breaks down the cross-linking structure between cellulose and hemicellulose, making the straw fiber fluffy and enhancing its adsorption capacity.

[0063] 3. Remove soluble impurities: Dissolve free tannins, phenols and other antibacterial substances in straw, eliminating their inhibitory effect on the target strain.

[0064] 4. Appropriate modification of the carrier: After alkali treatment, the functional groups on the surface of straw change, which enhances the binding force with bacteria and increases the amount of immobilized bacteria and the retention rate of bacteria.

[0065] The 1.5%–2.5% NaOH, 60 °C, and soaking time of 1.5–2.5 h used in this invention is the optimal pretreatment process after single-factor optimization, requiring no further optimization. The entire alkali pretreatment process is simple to operate, low in cost, and produces no secondary pollution, making it suitable for large-scale production.

[0066] In this invention, there is no fermentation reaction between crop straw and the bacterial strain, and the crop straw will not undergo fermentation and decomposition.

[0067] Although crop straw contains cellulose, the bacterial solution in the entire oscillation and adsorption system is a nutrient-rich LB culture medium. LB contains readily available carbon and nitrogen sources such as yeast powder, peptone, and glucose. Tropical Bacillus will preferentially utilize the readily available nutrients of LB for proliferation and will not decompose straw fiber as a carbon source, thus lacking the metabolic driving force for straw fermentation.

[0068] After pretreatment with 2% NaOH at 60℃, the soluble sugars, hemicellulose, and easily fermentable small molecules in the crop straw were dissolved by the alkali solution and then completely removed by subsequent washing with water until neutral. The remaining straw mainly consists of stable lignin and crystalline cellulose. Under short-term shaking conditions of 2 days, the strain cannot degrade the straw fiber in large quantities, and there is no phenomenon of straw fermentation producing acid or gas.

[0069] The design logic of this step is only physical adsorption biofilm formation: relying on the porous structure of straw to adsorb bacteria, allowing live bacteria to be fixed inside the pores of the carrier; the process is matched with a short cycle of 2 days and a sterile environment, without setting the long-term anaerobic / facultative anaerobic fermentation conditions required for straw fermentation, and the system has no fermentation substrate, no fermentation environment, and no fermentation design intent.

[0070] Adsorption ≠ fermentation:

[0071] Adsorption process: The bacteria attach to the pores on the surface of the straw by electrostatic and hydrophobic forces. The straw only serves as an inert carrier, and its structure and components do not decompose significantly.

[0072] Fermentation process: Microorganisms decompose organic substrates (straw sugars / fibers), producing organic acids, gases, and alcohols, while straw continues to degrade and be lost.

[0073] In this step, only the microbial cells are adsorbed and colonized; the straw carrier is hardly decomposed, and there is no fermentation reaction.

[0074] The finished microbial agent is then dried to a moisture content of <8% and stored. If the straw ferments during this step, the straw will become soft and rotten, miscellaneous bacteria will proliferate, and a large number of live bacteria will be inactivated. This contradicts the experimental results of the high bacterial load and high survival rate of the microbial agent in this invention, and conversely proves that there is no straw fermentation in this process.

[0075] The bacterial load of the bacterial agent prepared using the method of this invention is 6.5 × 10⁻⁶. 8 CFU / g ~ 8.2 × 10⁻⁶ 8 CFU / g, survival rate was above 83.5% for all samples.

[0076] Example 1: Screening and Identification of Bacillus tropicus

[0077] Five g of soil samples from farmland plastic film residue areas in Lanzhou, Gansu Province, were inoculated into 100 mL of inorganic salt medium with 1 g / 100 mL PE (or PBAT) powder as the sole carbon source. The samples were cultured at 30 °C and 180 rpm for 7 days with shaking to enrich microorganisms. One mL of the soil supernatant was added to 9 mL of LB medium and cultured for 12 h, followed by serial dilutions to 10⁻⁶. -3 The diluted solution was spread onto an inorganic salt solid medium with PE (or PBAT) film as the sole carbon source to screen for potential degrading strains. An inorganic salt solid medium without PE or PBAT film was used as a negative control to eliminate autotrophic microorganisms. The medium was then incubated upside down at 30°C for 3–5 days. Finally, colonies growing at the edge of the plastic film were picked and transferred to fresh nutrient agar medium. This process was repeated until single colonies were obtained.

[0078] The inorganic salt solid culture medium consists of: 1.0g KH2PO4, 1.5g Na2HPO4, 2.0g NH4Cl, 0.1g CaCl2·2H2O, 0.2g KCl, and 0.2g MgSO4·7H2O, diluted to 1L, and pH adjusted to ≈7 (20g agar was added to the solid culture medium). The medium was then autoclaved at 120℃ for 20min.

[0079] A strain exhibiting high degradation rates for both PE and PBAT was screened. Genomic DNA was extracted from the strain, its 16S rRNA gene sequence was amplified and sequenced, and the 16S rRNA gene sequence was submitted to GenBank (accession number PX599112). A phylogenetic tree was constructed (see...). Figure 2The strain was confirmed to belong to Bacillus tropicus and named Bacillus tropicus SH-XP1. It was deposited on January 14, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 37380.

[0080] Figure 2 A phylogenetic tree for Bacillus tropicus SH-XP1 based on the 16S rRNA gene sequence.

[0081] The physicochemical properties of the above-mentioned strain Bacillus tropicus SH-XP1 were analyzed, and the results are as follows:

[0082] The phylogenetic tree results of the 16S rRNA gene of this strain show that strain NR XP1 closely clusters with the standard tropical Bacillus reference sequence NR 157736.1 in the same evolutionary branch, confirming its taxonomic position as Bacillustropicus; however, the two form an independent subbranch, and strain SH-XP1 has characteristic gene base variation sites, which distinguishes it from all published tropical Bacillus reference strains, making it a completely new and independent strain.

[0083] Although this strain possesses the common characteristics of tropical Bacillus, such as Gram-positive, endospore-producing, and mesophilic aerobic growth, it has several core advantages that distinguish it from existing tropical Bacillus strains: (1) Polymer plastic degradation function: Existing tropical Bacillus strains can only utilize natural sugars and cellulose, while this strain can use PE powder and PBAT film as the sole carbon source to complete proliferation, secrete specific degradation enzymes to destroy the microstructure of plastics, and achieve efficient degradation of PE and PBAT in the soil system within 45 days. It is currently the only tropical Bacillus strain that has both polyethylene and PBAT polyester degradation activity; (2) Soil stress tolerance and crop growth promotion function: The strain is tolerant to the toxic low molecular weight substances released by PE and PBAT plastics, and at the same time secretes IAA and phosphate-solubilizing potassium metabolites, which can improve crop biomass while repairing plastic-polluted soil. There are no reports of existing tropical Bacillus strains achieving plastic repair and plant growth promotion simultaneously; (3) Growth and environmental tolerance advantages: The strain can reach OD after 10-14 h of shaking culture at 30℃. 600 =1.0, with a growth rate 15%~20% higher than existing tropical Bacillus strains; it can proliferate stably in the pH range of 5.5~8.5, has stronger acid resistance, and is suitable for acidified farmland soil environments.

[0084] Meanwhile, the topological parameters of the soil microbial co-occurrence network confirmed that this strain can enhance the richness of the microbial community in plastic-polluted soil, strengthen the symbiotic cooperation among species, open up the metabolic pathways of plastic degradation in the microbial community, and construct a stable microbial network with degradation functions; while the existing publicly available Bacillus tropicalis can only colonize the soil for a short period of time, cannot reshape the soil microbial co-occurrence structure, and does not have this ecological regulation function.

[0085] Based on comprehensive verification from three dimensions—molecular evolution, physiological and biochemical analysis, and soil microecology—the Bacillus tropicus SH-XP1 strain of this invention differs from all existing publicly available Bacillus tropicus strains in that it possesses multiple unique and innovative characteristics, including the ability to degrade PE / PBAT plastics, regulate soil microbial communities, mitigate plastic toxicity, and promote crop growth.

[0086] Preparation of Bacillus tropicus SH-XP1 bacterial culture:

[0087] The selected Bacillus tropicus SH-XP1 strain was inoculated into LB liquid medium and cultured at 28–32°C with shaking at 160–200 rpm for 10–14 h until the bacterial culture reached OD500. 600 When the value reaches 1.0, the bacterial solution is obtained.

[0088] Example 2 Preparation of straw immobilized microbial agent

[0089] Crush corn stalks to a particle size of 2 mm, weigh 20 g of corn stalk powder, add 2% (w / w) NaOH solution, and soak at 60℃ for 2 hours. Filter and rinse repeatedly with deionized water until the filtrate is neutral. Dry in a 60℃ oven and sterilize at 121℃ for 20 min, then cool to room temperature under aseptic conditions for later use.

[0090] The Bacillus tropicus SH-XP1 strain selected in Example 1 was inoculated into LB liquid medium and cultured at 30°C with shaking at 180 rpm for 12 hours until the bacterial culture reached OD. 600 The value reaches 1.0.

[0091] Weigh 10g of pretreated straw powder and place it in a sterile Erlenmeyer flask. Add 100mL of the above bacterial solution. Place the flask in a shaker at 30℃ and shake at 150 rpm for 2 days for adsorption and fixation. After adsorption and fixation, centrifuge at 5000 rpm for 5 min and discard the supernatant.

[0092] The obtained mixture was filtered through sterile gauze to remove excess liquid, spread evenly on a sterile tray, and dried in a 30°C ventilated drying oven until the moisture content was below 8%, thus obtaining the corn straw immobilized Bacillus tropicalis inoculant, named XP1.

[0093] The bacterial load of the bacterial agent XP1 was 8.2 × 10⁻⁶. 8 CFU / g, survival rate 91.2%.

[0094] Example 3: Remediation of PE, PBAT, and PBAT black film contaminated soil using straw immobilized microbial agents

[0095] Typical slightly alkaline farmland soil from Northwest China, with a pH of 8.0 and free of plastic, heavy metals, and pesticide residues, was air-dried and passed through a 2 mm sieve to remove impurities such as stones and plant debris. The soil retained its original indigenous microbial community and had uniform fertility. Polyethylene (PE), polybutylene adipate / terephthalate (PBAT), and PBAT black film (PBATB) were cut into 3×3 cm fragments and added to the soil, with each fragment having a concentration of 1 g / kg. The mixture was then thoroughly mixed to prepare simulated contaminated soil.

[0096] Prepare the corn stalk immobilized Bacillus tropicalis agent XP1 prepared in Example 2. Apply it evenly to plastic-contaminated soil and till it appropriately to mix it evenly with the soil. The dosage is 2g / kg soil.

[0097] Seven treatment groups were set up: CK (no plastic soil), PE (PE plastic pollution only), PE+XP1 (PE plastic pollution + XP1 microbial agent), PBATB (PBAT black film pollution only), PBATB+XP1 (PBAT black film pollution + XP1 microbial agent), PBAT (PBAT plastic pollution only), and PBAT+XP1 (PBAT plastic pollution + XP1 microbial agent). Each group had three replicates. Each treatment group was placed in flowerpots, with 500g of soil in each pot. The soil moisture content was adjusted to 60% of field capacity. After sowing Chinese cabbage seeds, each pot was incubated at room temperature for 45 days. Changes in soil plastic content and physicochemical properties were monitored regularly.

[0098] After cultivation, the plastic films from each treatment group were removed, rinsed three times with ultrapure water, and then immersed in a 2% sodium dodecyl sulfate (SDS) solution for ultrasonic treatment for 30 minutes. Subsequently, the films were immersed in a 20% alcohol solution for 5 minutes. Finally, they were repeatedly rinsed with deionized water and dried. The dried films were weighed using an analytical balance, and the weight loss rate of the degraded plastic films was calculated; the weight loss was the degradation rate. Furthermore, scanning electron microscopy (SEM) was used to observe the microscopic features of the microplastic particles before and after degradation, examining for features such as pits and wrinkles on the microplastic surface. Results are as follows: Figure 3 and Figure 4 As shown.

[0099] Figure 3 The graph shows the changes in the weight loss rates of PE, PBAT, and PBATB films in the soil in Example 3.

[0100] Figure 4 This is a scanning electron microscope (SEM) image of the surface morphology of PE, PBAT, and PBATB films in the soil in Example 3.

[0101] Figure 5 The growth indicators of plants under different treatments are: (a) plant growth phenotype; (b) root length and (c) plant height; (d) leaf area; (e) fresh weight and (f) dry weight; and (g) chlorophyll content.

[0102] Figure 6 Soil physicochemical properties under different treatments: (a) pH value and (b) electrical conductivity (EC); (c) available phosphorus (AP) and (d) available potassium (AK); (e) NH4+. + -N; (f) Total organic carbon (TOC).

[0103] like Figure 3 As shown, the weight loss rate of the film during the cultivation process was calculated using the following formula. After cultivation under the same conditions, the degradation rate of the film inoculated with XP1 was significantly higher than that of the corresponding film not inoculated with XP1.

[0104] Formula 1:

[0105] like Figure 4 As shown, the film surface before degradation is relatively flat and smooth. After degradation, especially after inoculation with XP1, the film surface becomes rougher, with local unevenness, and more obvious grooves, pits, and wrinkles appear, resulting in damage.

[0106] After cultivation, plant and soil samples were collected for subsequent testing. Plant height, root length, fresh weight, and dry weight were measured. Leaf area was measured using a leaf area meter (LAM-B), and chlorophyll content (SPAD value) was determined using a plant nutrient analyzer (SY-S02A, Shijiazhuang Fansheng Technology Co., Ltd.). The pH and electrical conductivity (EC) values ​​of the soil leachate (soil to water ratio of 1:10) were analyzed using a pH meter (PHS-3C) and an electrical conductivity meter (POS-307A, Shanghai Instrument & Electronics Scientific Instruments Co., Ltd.), respectively. Soil ammonium nitrogen (NH4) was also measured. + Available nitrogen (N), available phosphorus (AP), and available potassium (AK) were determined using a rapid soil nutrient analyzer (YT-TRB, Shandong Yuntang Intelligent Technology Co., Ltd.). Total organic carbon (TOC) was determined using a total organic carbon analyzer (Heitai Automation Technology Co., Ltd.). Results are as follows: Figure 5 and Figure 6 As shown.

[0107] like Figure 5 As shown, compared with the control group, the addition of XP1 significantly increased root length and plant height, especially in the PBATB+XP1 treatment group, where these increased by 17.2% and 11.7%, respectively. Under the same plastic pollution conditions, the fresh weight and dry weight of the XP1-added treatment groups were both increased. Furthermore, the leaf area of ​​the XP1-added treatment groups was significantly expanded, and chlorophyll content was effectively restored. The addition of XP1 significantly alleviated the toxicity of plastic pollution to plants, which is attributed to XP1 promoting plastic degradation, soil nutrient absorption, and soil microbial activity, thereby promoting plant growth and development.

[0108] like Figure 6 As shown, under the same plastic pollution conditions, the pH and EC values ​​increased with the addition of XP1 compared to the group without XP1. AP increased by 3.6%–10.9% compared to the group without XP1, AK increased by 4.5%–13.1%, and NH4 increased... + -N increased by 2.2%–7.6% compared to the untreated group. TOC also significantly increased after the addition of XP1. The application of XP1 improved soil quality and promoted plant growth. This resulted in pH stability, increased EC, and enhanced AP, AK, and NH4+. + XP1 enhances the availability of nutrients such as nitrogen (N). Furthermore, XP1 effectively promotes carbon sequestration by facilitating the conversion of recalcitrant organic matter into stable soil carbon.

[0109] This embodiment fully demonstrates that the straw immobilization microbial agent XP1 provided by the present invention can not only effectively degrade PE, PBAT, and PBAT black film, but more importantly, it can significantly reduce the stress of plastics on plants and promote their growth and development by improving soil physicochemical properties and regulating microbial communities, thereby creating a healthier rhizosphere microenvironment. This technology achieves a leap from "pollution removal" to "ecological function restoration" and has extremely high agricultural and environmental application value.

[0110] Example 4: Soil pH range suitable for the microbial agent of the present invention

[0111] To clarify the suitable soil pH range for the microbial agent of this invention, farmland soil from Lanzhou with a baseline pH of 8.0 was used as the material, and the soil pH was adjusted to eight gradients: 6.0, 6.5, 7.0, 7.5, 8.0, 8.4, 8.5, and 9.0. The straw immobilization microbial agent prepared in Example 2, PE, and PBAT were added to each group of soil. The application rate of the microbial agent was 2 g / kg of soil, and the addition rates of PE and PBAT were both 1 g / kg. Each group had three replicates. The soil moisture content was uniformly adjusted to 60% of field capacity, and the soil was continuously incubated at room temperature for 45 days. The experimental results are shown in Table 1.

[0112] Table 1 Comparison of the effects of microbial agents on soil remediation under different pH conditions

[0113]

[0114] Experimental results show that the general tolerance range of this straw immobilized microbial agent to soil pH is 6.0–8.5; the optimal application pH range is 7.5–8.4, consistent with the original isolation environment of the strain and the soil pH of the simulated experiment in this patent. Within this range, the survival rate of viable bacteria, plastic degradation efficiency, and crop growth promotion effect are the best. When the soil pH is below 6.0, the activity of the strain is significantly reduced, and the plastic degradation ability is greatly weakened; when the soil pH is above 8.5, the strongly saline-alkaline environment will cause a large number of bacteria to be inactivated, and the microbial agent will lose its repair function, making it unsuitable for this technology. The microbial agent of this invention is particularly suitable for the weakly alkaline farmland in Northwest my country, and can also be promoted and applied in neutral farmland in the north.

[0115] Example 5: Comparative Experiment of Straw Immobilized Microbial Agent and Free Bacillus Tropicalis Incense

[0116] To verify the effect of straw immobilization on the remediation performance of bacterial strains, a control experiment was set up with equal amounts of free Bacillus tropicalis bacterial solution (prepared in Example 1) and straw immobilized bacterial agent (prepared in Example 2). (The straw immobilized bacterial agent was applied at a rate of 2 g / kg soil; based on the preparation ratio of 1 g carrier adsorbing 10 mL of bacterial solution with OD600=1.0, 20 mL of Bacillus tropicalis bacterial solution with OD600=1.0 was directly added per kilogram of soil in the free bacterial treatment group). The experiment used uniformly simulated plastic-contaminated soil, with polyethylene (PE), polybutylene adipate / terephthalate (PBAT), and PBAT black film (abbreviated as "PBATB") added respectively, at a plastic addition rate of 1 g / kg soil. The experiment included a blank control group, a single plastic pollution group, a plastic + straw immobilized microbial agent group, and a plastic + equal volume of free microbial solution group, with three replicates per group. Each pot contained 500 g of soil, and the soil moisture content was adjusted to 60% of field capacity. The soil was incubated at room temperature for 60 days. The number of viable bacteria in the soil and the weight loss rate of the plastic film were measured on days 15, 30, 45, and 60 of incubation. The experimental results are shown in Table 2.

[0117] On the day the soil, plastic, and microbial agent mixture was filled into flowerpots, 12 plump and uniformly sized Chinese cabbage seeds were sown in each pot. The soil moisture content was maintained at 60% field capacity throughout the process, with no additional fertilization, and the plants were cultivated under natural light and at room temperature. After 45 days of cultivation, all Chinese cabbage plants were harvested at once, and rhizosphere soil samples were collected from each pot simultaneously. A complete set of growth indicators for the Chinese cabbage and the full range of soil physicochemical properties were then analyzed. The experimental results are shown in Table 3.

[0118] Table 2 Summary of Soil Viable Bacteria Count and Plastic Degradation Rate at Different Culture Cycles

[0119]

[0120] Table 3 Summary of measured values ​​of crop growth promotion and soil physicochemical nutrients after 45 days of cultivation.

[0121]

[0122] The results showed that free bacteria had poor survival ability after being applied to the soil, with a significant decrease in the number of viable bacteria after 30 days of cultivation, and almost no active bacteria were detectable after 45 days. In contrast, the strains in the straw-immobilized microbial agent could stably colonize the soil for a long period. Within the standard 45-day remediation cycle, the degradation rate of PE, PBAT, and PBAT black film by the straw-immobilized microbial agent was higher than that of free bacteria at the same dosage. Free bacteria only showed a certain degradation effect in the first 30 days after application; after 30 days, the degradation process essentially stopped, and extending the cultivation time did not significantly improve the plastic degradation rate. Furthermore, the improvement and crop growth promotion effects of free bacteria on plastic-contaminated soil were weak and short-lived, while the straw-immobilized microbial agent could exert its effects for a long time.

[0123] In summary, direct application of free degrading bacteria has drawbacks such as easy loss of bacterial cells, short survival time, low degradation efficiency, and short action cycle. Using straw immobilization can significantly improve the survival rate, colonization ability, and long-term degradation activity of the strains in the soil, and is a necessary technical means to ensure the remediation effect of soil plastic pollution.

[0124] The following is a partial optimization experiment of the method of the present invention:

[0125] Optimization Experiment 1: Screening Experiment for Different Carrier Materials

[0126] Various carriers were prepared into immobilized bacterial agents through adsorption and aseptic drying according to the method in Example 2. Simulated plastic-contaminated soil was prepared by mixing air-dried farmland soil as a matrix with 2 g of immobilized bacterial agent and 1 g of PE plastic per kilogram of air-dried soil. The dosage of carriers in each group was completely uniform. This experiment used carrier material as the only variable to compare the comprehensive performance of different carriers under the same dosage. Due to differences in carrier porosity and surface adsorption characteristics, the bacterial load per unit mass varied among groups, and the total viable bacteria input in the soil naturally varied with carrier performance. The bacterial load, 7-day survival rate at room temperature, and 45-day PE plastic degradation capacity were uniformly measured.

[0127] Experimental carrier setup: crop straw (corn straw, 2 mm); inorganic carrier group: diatomaceous earth (0.1–0.2 mm), zeolite (0.2–0.5 mm); organic synthetic carrier: polyvinyl alcohol (PVA) microspheres (1–2 mm in diameter), calcium alginate gel beads (1–2 mm in diameter); other agricultural and forestry waste: sawdust (2 mm). Experimental results are shown in Table 4.

[0128] Table 4. Screening Test Results of Immobilization Effects of Different Carrier Materials

[0129]

[0130] The experimental results showed that the bacterial load, cell survival rate, and plastic degradation effect of inorganic carrier group, organic synthetic carrier, and sawdust were significantly lower than those of crop straw (corn straw). After screening multiple types of carriers, this invention finally determined that crop straw is the optimal immobilization carrier suitable for this degrading strain.

[0131] Optimization Experiment 2: Screening Experiment of Different Crop Straws

[0132] Experimental procedure:

[0133] I. Experimental Materials and Grouping

[0134] 1. Test strain: Bacillus tropicus SH-XP1 (CGMCC No. 37380), cultured to OD. 600 =1.0.

[0135] 2. Test straws: wheat straw, rice straw, rapeseed straw, and corn straw. All straws were uniformly crushed to a particle size of 2 mm.

[0136] 3. Test soil and plastics: Uncontaminated farmland soil (air-dried and passed through a 2 mm sieve); three types of plastics, polyethylene (PE), polybutylene adipate / terephthalate (PBAT) film and PBAT black film, were mixed into the soil and cut into 3 cm × 3 cm fragments. The amount of each type of plastic added to the soil was uniformly 1 g / kg.

[0137] 4. Experimental groups (3 replicates per group):

[0138] CK group: blank soil (no plastic, no immobilized bacterial agent);

[0139] Experimental group 1: Wheat straw immobilized microbial agent;

[0140] Experimental Group 2: Rice straw immobilized microbial agent;

[0141] Experimental Group 3: Rapeseed straw immobilized microbial agent;

[0142] Experimental group 4: Corn straw immobilized microbial agent;

[0143] Pure plastic pollution control group: soil only + the above-mentioned plastics, without adding bacterial agents, as a reference for the effect.

[0144] Experimental groups 1-4 above were prepared using the method described in Example 2 of this invention to prepare straw immobilization microbial agents. II. Preparation of microbial agents and remediation of PE, PBAT, and PBAT black film contaminated soil.

[0145] Various straw microbial agents were prepared according to the methods in Examples 2 and 3, and used for the remediation of soil contaminated with PE, PBAT, and PBAT black film.

[0146] III. Detection Indicators and Detection Methods

[0147] 1. After the basic performance of the carrier is immobilized, the bacterial load (CFU / g) and cell survival rate are measured to evaluate the adsorption and protection ability of straw on the bacterial strain.

[0148] 2. The degradation effect of plastics was determined according to the method in Example 3. The film was cleaned, ultrasonicated with SDS, soaked in alcohol, dried and weighed. The weight loss rate (degradation rate) of PE and PBAT films was calculated. The microstructure (pits, wrinkles and damage) of the film surface was observed by scanning electron microscopy (SEM).

[0149] 3. After the soil physicochemical remediation was completed, the soil pH, electrical conductivity (EC), and ammonium nitrogen (NH4+) were tested according to the method in Example 3. + -N), available phosphorus (AP), available potassium (AK), and total organic carbon (TOC).

[0150] 4. Crop growth indicators were uniformly determined by sowing Chinese cabbage. Root length, plant height, leaf area, fresh weight, dry weight, and chlorophyll SPAD value were measured to evaluate the ability of the microbial agent to mitigate plastic toxicity and promote growth. IV. Experimental Results

[0151] Table 5. Summary of Measured Values ​​of Comprehensive Performance of Immobilized Microbial Agents from Four Crop Straws

[0152]

[0153] Wheat straw: Its physicochemical structure, bacterial carrying capacity, and degradation effect are very similar to those of corn straw. It is widely planted throughout the country and can replace corn straw. It is compatible with all the process parameters in this invention and is the optimal alternative carrier.

[0154] Rice straw: Raw materials are abundant in rice-producing areas in the south. Although the bacterial load and degradation effect are slightly reduced, local sourcing can significantly reduce transportation and remediation costs, making it suitable for regional large-scale application. Due to its high silicon content, it is not recommended as a universal main carrier nationwide.

[0155] Rapeseed straw: It has a high organic matter content and outstanding effects on soil improvement and carbon sequestration, making it suitable for use in oilseed crop producing areas.

[0156] V. Conclusion

[0157] Besides corn stalks, wheat stalks, rice stalks, and rapeseed stalks can all be used as immobilization carriers for Bacillus tropicus CGMCC No. 37380 in this invention, for preparing straw immobilized bacterial agents and remediating plastic-contaminated soil. Among them, wheat stalks have similar comprehensive performance to corn stalks and are the preferred alternative carriers. Rice stalks and rapeseed stalks can be used as regional supplementary carriers, and all can stably achieve the core effects of plastic degradation and soil improvement.

[0158] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of tropical Bacillus tropicus, characterized by: The tropical Bacillus is Bacillus tropicus SH-XP1, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37380.

2. A bacterial suspension of Bacillus tropicus, characterized in that: It was obtained by expanding the culture of Bacillus tropicus CGMCC No.37380 strain as described in claim 1; Preferably, the expanded culture involves inoculating the Bacillus tropicus CGMCC No. 37380 strain according to claim 1 into LB liquid medium, and culturing at a temperature of 28–32°C, a shaking speed of 160–200 rpm, and a culture time of 10–14 h to obtain a bacterial solution.

3. A method for preparing a straw immobilized microbial agent, characterized in that: The activated Bacillus tropicus of claim 1 or Bacillus tropicus of claim 2 was mixed with pretreated crop straw, and the mixture was shaken and adsorbed under sterile conditions. Then, the solid and liquid were separated, and the crop straw immobilized with the bacterial agent was aseptically dried to obtain the straw immobilized bacterial agent. As a preferred method, use 8–12 mL OD per gram of crop straw. 600 Bacterial solution with a value of 1.

0.

4. The method for preparing a straw immobilized microbial agent according to claim 3, characterized in that: The pretreatment includes the step of pulverizing crop straw and then soaking it in an alkaline solution; Preferably, the alkaline solution is a NaOH solution with a mass fraction of 1.5% to 2.5%; Preferably, the soaking conditions are: soaking at 60 °C for 1.5–2.5 h; Preferably, the process also includes rinsing with deionized water after soaking until the filtrate is neutral; Preferably, the process also includes drying and sterilization after rinsing.

5. The method for preparing a straw immobilized microbial agent according to claim 3, wherein the oscillation adsorption and immobilization is carried out at 28–32 °C; Preferably, the rotational speed of the oscillation adsorption fixation is 130–170 rpm; Preferably, the oscillation adsorption fixation time is 36–60 h.

6. A method for preparing a straw immobilized microbial agent according to any one of claims 3 to 5, characterized in that: The crop straw includes at least one of corn straw, wheat straw, rice straw, and rapeseed straw; preferably, the crop straw is corn straw.

7. A straw immobilization microbial agent, prepared by the method described in any one of claims 3 to 6.

8. The application of Bacillus tropicus as described in claim 1, Bacillus tropicus bacterial solution as described in claim 2, or straw immobilized bacterial agent as described in claim 7 in degrading plastics and remediating plastic-contaminated soil; Preferably, the plastic includes at least one of PE, PBAT, and PBAT black film.

9. The application according to claim 8, characterized in that: When remediating plastic-contaminated soil, the dosage of the straw immobilization microbial agent is 1.5–2.5 g / kg soil, preferably 1.8–2.2 g / kg soil.

10. The application according to claim 8, characterized in that: When remediating plastic-contaminated soil, adjust the soil moisture content to 58%–62% of field capacity.

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