Application of bacillus and its synergistic plant in coal gangue soil remediation

Abstract

The application discloses a bacillus and application of the bacillus and a plant in coal gangue soil remediation, and belongs to the technical field of soil remediation. The preservation number of the bacillus is CCTCC NO: M20252743. After the bacillus fermentation liquor is sprayed on coal gangue soil, the plant is planted again, the coal gangue soil can be remediated, meanwhile, the growth of alfalfa is promoted, the iron content of the alfalfa is increased, and the obtained alfalfa has a remarkable effect on treating pig diarrhea, and the application has important significance for popularization of practical values of coal gangue soil remediation and alfalfa.

Description

Technical Field

[0001] This invention relates to the field of soil remediation technology, and in particular to the application of a Bacillus strain and its synergistic plants in the remediation of coal gangue soil. Background Technology

[0002] Coal gangue, a large solid waste generated during coal mining and washing, not only occupies significant land resources when left exposed for extended periods, but also triggers a series of serious soil ecological problems. Heavy metals such as cadmium (Cd), lead (Pb), and arsenic (As) contained in coal gangue can be leached into deeper soil layers by rainwater, causing complex heavy metal pollution. Simultaneously, the acidic nature of coal gangue exacerbates soil acidification, disrupting the soil's pH balance. Furthermore, the extremely low organic matter content in gangue-affected areas severely damages soil aggregate structure, ultimately leading to soil compaction and hardening, rendering the soil inoperable. Traditional physical remediation methods for this type of contaminated soil, such as soil replacement and soil replacement, are labor-intensive and costly. While chemical remediation is fast-acting, it easily leads to soil nutrient loss, damages soil microbial communities, disrupts regional ecological balance, and hinders sustainable remediation.

[0003] Among existing remediation technologies, phytoremediation has attracted much attention due to its advantages such as low cost and environmental friendliness, but it faces significant bottlenecks in practical applications. Most hyperaccumulating plants of heavy metals have long growth cycles and low biomass, exhibiting poor tolerance to soils contaminated with high concentrations of heavy metals. Their survival rate is extremely low in the harsh habitats of coal gangue accumulation areas, making their remediation efficiency insufficient to meet practical needs. While chemical amendments such as humic acid and chelating agents can passivate heavy metals in soil and reduce their bioavailability in the short term, long-term use can easily trigger secondary migration of heavy metals, and their remediation effect is greatly affected by the soil environment, lacking stability.

[0004] γ-Polyglutamic acid (γ-PGA), as a natural high-molecular polymer, is often used as a water-retaining agent and fertilizer synergist in agriculture due to its excellent water and fertilizer retention and ion chelation capabilities. However, existing γ-PGA preparation processes are mostly developed based on conventional agricultural scenarios and have not been optimized for the special physicochemical properties of coal gangue soil, which is characterized by multi-metallic complex pollution, strong acidity, and compacted structure. Furthermore, its synergistic remediation model with plants and microorganisms has not been systematically studied, resulting in the underutilization of γ-PGA's application potential in the remediation of coal gangue-contaminated soil. Summary of the Invention

[0005] This invention provides the application of a Bacillus strain and its synergistic plants in the remediation of coal gangue soil, in order to solve the problems existing in the prior art. By applying the Bacillus strain YB-01 isolated by this invention to coal gangue soil and then planting alfalfa, the soil can be remediated, and alfalfa growth and iron content can be increased. It also has a significant effect on the treatment of swine diarrhea, which is of great significance for the remediation of coal gangue soil and the promotion of the practical value of alfalfa.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a strain of Bacillus ( Bacillus sp. The Bacillus YB-01 has the accession number CCTCC NO: M20252743, the accession date is December 1, 2025, the depositary institution is the China Center for Type Culture Collection, and the deposit address is Wuhan University, Wuhan, China.

[0007] The Bacillus YB-01 isolated in this invention, after fermentation, produces secondary metabolites containing γ-PGA, which lays the foundation for its use in coal gangue soil remediation. However, experimental verification has shown that not all Bacillus species can be used for coal gangue soil remediation. Furthermore, it has been found that treating coal gangue soil with Bacillus YB-01 and then planting alfalfa not only achieves coal gangue soil remediation but also promotes alfalfa growth and increases iron content. Moreover, its use in pig basal feed can significantly treat pig diarrhea. This invention provides a theoretical basis for the remediation of coal gangue soil and the promotion and application of alfalfa.

[0008] The present invention also provides the application of the Bacillus YB-01 synergistic plant in the remediation of coal gangue soil, wherein the plant is planted after the Bacillus YB-01 is applied to the coal gangue soil.

[0009] The present invention also provides the application of the aforementioned Bacillus YB-01 in promoting plant growth or increasing iron content in coal gangue soil. By applying the aforementioned Bacillus YB-01 to the coal gangue soil and then planting plants, it is easy to promote the growth of the plants or increase their iron content.

[0010] The present invention also provides a method for increasing the iron content of plants grown in coal gangue soil, comprising the step of spraying the fermentation liquid of Bacillus YB-01 onto the coal gangue soil and then planting plants.

[0011] Preferably, the viable cell content of the fermentation broth of Bacillus YB-01 is 10. 11 CFU / mL, spraying rate 50 mL / m 2 .

[0012] The present invention also provides the use of the iron-enhanced plants obtained by the method in the preparation of pig feed or medicaments for treating swine diarrhea.

[0013] Preferably, the plant is alfalfa.

[0014] Preferably, after the alfalfa matures, it is dried and pulverized to obtain alfalfa powder; 15% alfalfa powder is added based on the weight of the basal pig feed.

[0015] The present invention also provides a feed for treating swine diarrhea, the feed being composed of basal swine feed and alfalfa powder, wherein the alfalfa powder is obtained by growing the alfalfa as described above, and drying and pulverizing the grown alfalfa after it matures.

[0016] Preferably, the feed contains 15% alfalfa meal based on the weight of the basal pig feed.

[0017] The present invention discloses the following beneficial effects: (1) Significantly improves the physical and chemical structure and fertility of coal gangue soil. Enhancing nutrient availability: After applying the Bacillus fermentation broth of this invention, the contents of hydrolyzable nitrogen, available phosphorus and available potassium in the soil were significantly increased and remained at a high level during the 48-day monitoring period, indicating that it can effectively activate soil nutrients and reduce reliance on chemical fertilizers.

[0018] Optimization of soil aggregate structure: Compared with the blank group and other control groups, the treatment group of this invention increased the content of large aggregates (particle size > 0.25 mm) in the soil to 45.62%, significantly improved aggregate stability (MWD, GMD), and reduced soil structure destruction rate (PAD), effectively solved the problem of soil compaction caused by coal gangue, and enhanced soil permeability and water and fertilizer retention capacity.

[0019] (2) Effectively promotes plant growth and biomass accumulation Significantly improved agronomic traits: In coal gangue-contaminated soil, plants treated with the Bacillus inoculant of this invention (such as alfalfa) showed significantly better plant height, root length, number of branches, and above-ground / below-ground fresh and dry weight than those treated with commercially available Bacillus inoculant, γ-polyglutamic acid, and chemical fertilizers. This indicates that this strain can overcome the harsh environment of coal gangue and create favorable conditions for plant growth by improving the microecological environment.

[0020] (3) It specifically increases the iron content of plants and has the value of developing functional feed. Highly efficient iron enrichment: The "Bacillus fermentation broth spraying + planting" model can increase the iron content of alfalfa to 182.3 mg / kg (dry weight), which is nearly 3 times higher than the blank control group (45.7 mg / kg), and the difference is extremely significant.

[0021] (4) High iron-containing feed significantly prevents and controls diarrhea in piglets. Reduced diarrhea rate and severity: Feed containing 15% iron-rich alfalfa meal reduced the diarrhea rate in piglets to 4.28%, shortened the diarrhea recovery time to 18.5 hours, and reduced the incidence of severe diarrhea to only 0.72%, which was significantly better than the control group.

[0022] Regulation of intestinal immunity: Piglets fed this feed showed significantly increased serum iron levels, significantly decreased levels of pro-inflammatory factors (TNF-α, IL-6), and significantly increased levels of anti-inflammatory factors (IL-10). This demonstrates that high-iron plants can protect intestinal mucosal health by improving iron nutrition status, synergistically regulating intestinal inflammatory responses, and inhibiting pathogenic bacteria.

[0023] (5) Improve the growth performance of piglets and increase breeding efficiency. Promoting weight gain and reducing feed conversion ratio: The average daily weight gain of piglets in the experimental group (317.6 g / d) was 52.0% higher than that in the blank control group, and the feed conversion ratio (1.84) was significantly reduced, achieving the dual effects of diarrhea prevention and growth promotion, and significantly improving the economic benefits of breeding.

[0024] (6) Environmental friendliness and sustainability This invention utilizes microbial remediation technology to avoid the risk of secondary pollution that may be caused by chemical amendments. By activating the native soil microbial community (increasing the number of Bacillus and enzyme activity), it achieves an organic combination of ecological restoration of coal gangue mountains and the production of high-value-added plants. Attached Figure Description

[0025] Figure 1 The culture characteristics of YB-01 in a petri dish; Figure 2 The growth status of alfalfa before soil remediation (CK) and after remediation (Bacillus fermentation broth of this invention). Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Example 1: Isolation and Identification of Bacillus Weigh 2 g of commercially available fermented black soybeans from Guizhou and boil them in 100 mL of sterile distilled water for 5 min to kill vegetative cells. After settling, take 1 mL of the supernatant and incubate it in 50 mL of LB medium at 220 r / min and 30 ℃ for 24 h. -4 After serial dilution, 100 μL of the culture medium was evenly spread onto LB agar plates and incubated at 30 °C for 24 h. The LB agar incubation process was repeated until a single strain was obtained. The single strain was named YB-01. Figure 1 Afterwards, the samples were sent to a testing company for identification.

[0032] Based on the identification, combined with the results of second-generation sequencing genome alignment, 16S rRNA alignment, and ANI analysis, it was inferred that the optimal matching strain in the NT library was: Bacillus_sp._FDAARGOS_527 GCA_003812125.1, with an ANI index of 99.87%. Therefore, the YB-01 strain isolated in this invention is Bacillus.

[0033] The Bacillus strains obtained by screening in this invention ( Bacillus sp. YB-01 was deposited at the China Center for Type Culture Collection (CCTCC) on December 1, 2025; the deposit address is Wuhan University, Wuhan, China; the accession number is CCTCC NO: M20252743.

[0034] Example 2 Preparation of Bacillus bacterial culture Preparation of seed culture: Bacillus was inoculated into seed culture medium using an inoculation loop and cultured at 30 ℃ and 220 r / min for 24 h to obtain seed culture. The seed culture medium consisted of: glycerol 20 g / L, ammonium chloride 10 g / L, magnesium sulfate heptahydrate 5 g / L, L-glutamic acid 10 g / L, yeast extract 10 g / L, dipotassium hydrogen phosphate 2 g / L, pH 7.4. The medium was sterilized at 121 ℃ for 20 min before use.

[0035] Preparation of fermentation broth: The seed culture was inoculated into the initial fermentation medium at a 5% inoculum rate and cultured at 30 ℃ and 220 r / min for 4 days. The bacterial cells were removed by filtration, and the filtrate was collected to obtain the fermentation broth. The initial fermentation medium consisted of: 12 g / L citric acid, 20 g / L L-glutamic acid, 80 g / L glycerol, 7 g / L ammonium chloride, 0.5 g / L potassium dihydrogen phosphate, 0.1 g / L manganese sulfate monohydrate, 0.5 g / L magnesium sulfate heptahydrate, 0.15 g / L calcium chloride, and 0.04 g / L ferric chloride hexahydrate, pH 7.4. The medium was sterilized at 121 ℃ for 20 min before use.

[0036] Experimental Example: Application of Bacillus in Coal Gangue Soil Remediation Experimental location: A mining area in Liupanshui, with soil pH=4.2, Cd content 12.5 mg / kg, Pb content 450 mg / kg, Cr content 207 mg / kg, and Cu content 157 mg / kg.

[0037] A fully randomized block design was adopted, with 5 blocks set up as follows: (1) Blank control group (CK): No microbial agents or fertilizers were applied.

[0038] (2) Group T1: The Bacillus fermentation broth prepared according to the present invention was applied; the bacterial count was 10. 11 cfu / mL.

[0039] (3) T2 group: γ-polyglutamic acid solution was administered at 50 mL / m 2 .

[0040] (4) Group T3: A commercially available Bacillus inoculum solution (product number 68038700, CAS: 68038-70-0) was applied; the bacterial count was 10. 11 cfu / mL.

[0041] (5) Group T4: Apply N, P, K compound fertilizer (KL-151515-GT) solution, 1.1 g / m 2 .

[0042] Application method: The fermentation broth or solution of groups T1-T4 was sprayed onto the surface of coal gangue-based soil. Each treatment was replicated in 3 places, for a total of 15 experimental units. The application rate was 50 mL / m². 2 On May 26, 2024, soil treatment (coal gangue piled up in 2021, covered with 3-5cm of soil) was carried out. The fermentation liquid or solution was evenly sprayed onto the coal gangue-based soil, and tall fescue was sown on the same day. Soil samples were collected 60 days later for testing, and the nitrogen, phosphorus, and potassium content in the plants was determined.

[0043] 1. Nitrogen, phosphorus, and potassium content determination: Organic matter was determined using the potassium dichromate oxidation-external heating method (NY / T 1121.6-2006); total nitrogen was determined using the Kjeldahl method (NY / T 1121.24-2012); available phosphorus was determined using the sodium bicarbonate extraction-molybdenum antimony colorimetric method (NY / T1121.7-2014); and available potassium was determined using the ammonium acetate extraction-flame photometric method (NY / T 889-2004). The nitrogen, phosphorus, and potassium content in the soil was determined on days 60, 120, 180, 240, and 300 after application of the fermentation broth or solution treatment. The results are shown in Table 1.

[0044] Table 1. Detection results of nitrogen, phosphorus, and potassium content in soil after different treatments (mg / kg) As shown in Table 1, after 60, 120, 180, 240 and 300 days of application of the Bacillus fermentation broth prepared in this invention, the nitrogen, phosphorus and potassium contents in the soil gradually increased, reaching the highest at 180 days. At 300 days, the nitrogen, phosphorus and potassium contents of group T1 were 83.085 mg / kg, 16.695 mg / kg and 60.89 mg / kg, respectively.

[0045] 2. Changes in soil aggregates based on coal gangue Water-stable macroaggregates were determined according to NY / T1121.19-2008, standard for the determination of water-stable macroaggregates in soil; soil microaggregates were determined according to NY / T1121.20-2008, standard for the determination of soil microaggregates. The results are shown in Table 2.

[0046] Table 2. Changes in soil aggregates based on coal gangue in different treatment groups. Note: All data in the same column showed significant differences between groups (P < 0.05).

[0047] The soil aggregate structure in the control group (CK) was severely unbalanced, with extremely low content of large aggregates (R>0.25mm, only 16.89±2.05%) and extremely high content of micro-aggregates (83.11±2.05%), mainly consisting of fine aggregates. The aggregate stability was extremely poor (MWD and GMD were very low, and PAD was very high). The soil structure was loose, with weak resistance to erosion and fertilizer retention capacity, failing to provide good conditions for plant growth and nutrient retention. The improvement effect of the fertilizer treatment group was weak, showing only slight improvement compared to the control group (CK), and no substantial optimization of soil structure was achieved. The Bacillus fermentation broth treatment group of this invention exhibits the best improvement effect: it can efficiently promote the transformation of micro-aggregates into macro-aggregates, increasing the content of macro-aggregates to 45.62±3.35% and reducing the total content of micro-aggregates to 54.38±3.35%, with a reasonable ratio of coarse and micro-aggregates and the lowest content of fine aggregates; at the same time, it significantly increases MWD and GMD, reduces PAD, and greatly optimizes soil structure stability, aeration and permeability, erosion resistance and fertilizer retention capacity, showing significant advantages over commercially available Bacillus inoculant treatment groups.

[0048] The core advantage of the Bacillus fermentation broth of this invention lies in the highly active viscous substance it secretes. This substance has strong cementing ability and high aggregation and transformation efficiency, which can activate soil microorganisms and work synergistically with the root system of perennial alfalfa to achieve the best synergy between the functions of large and micro aggregates in the soil. This effectively reduces the leaching loss of soil nutrients (including iron), providing dual support for alfalfa growth and iron enrichment. At the same time, it provides the optimal soil structure improvement scheme for the ecological restoration of coal gangue mountains.

[0049] 3. Microbiological indicators Microbial indicators are the core driving factors of soil fertility and aggregate formation. We focused on analyzing three core indicators related to Bacillus activity and soil nutrient transformation (Bacillus number, urease activity, and phosphatase activity), all of which were positive indicators (see Table 3).

[0050] (1) The plate count method for measuring the number of soil Bacillus (CFU / g) directly reflects the enrichment degree of Bacillus in the soil. Bacillus can secrete sticky cementing substances, which are key microorganisms that promote the formation of aggregates and improve the stability of aggregates. The higher the number, the stronger the cementing effect and the better the aggregate improvement effect.

[0051] (2) Enzyme activity was detected by colorimetric method. The higher the soil urease activity (U / g·24h), the higher the efficiency of converting organic nitrogen in the soil into available nitrogen, which can provide sufficient nitrogen source for alfalfa growth and Bacillus reproduction, while also helping to improve soil fertility and indirectly promote the formation of aggregates.

[0052] (3) The higher the soil phosphatase activity (mg / g·24h), the more it can convert insoluble phosphorus in the soil into available phosphorus, improve the availability of soil phosphorus, provide nutritional support for aggregate formation and plant growth, and at the same time enhance the cementation effect in conjunction with Bacillus.

[0053] Table 3. Detection results of microbial indicators under different treatments 4. Effects of Bacillus on plant morphological indicators Plant morphological indicators included plant height (accuracy 0.1 cm), root length (WinRHIZO root analysis system), and number of branches. Biomass determination: the fresh weight of the aboveground and underground parts were measured separately. After blanching at 105℃ for 30 min, the aboveground dry weight and underground dry weight were measured after drying at 65℃ to constant weight. The agronomic traits of the plants were analyzed, and the statistical results are shown in Table 4.

[0054] Table 4 Plant growth status As shown in Table 4, after different treatment groups, the plants exhibited the best growth after being treated with Bacillus subtilis of the present invention in coal gangue soil. Figure 2 The results showed that commercially available γ-polyglutamic acid, commercially available Bacillus inoculants, and chemical fertilizers were the most effective. This indicates that γ-polyglutamic acid alone cannot significantly promote plant growth, and not all Bacillus species can significantly promote plant growth. Based on the inventors' theoretical and practical experience in bacterial strains and coal gangue soil treatment, it is inferred that the Bacillus in this invention's own metabolites can improve the microbial community environment in coal gangue soil, thereby balancing competition among strains and nutrients, and thus providing better growth conditions for plants. While chemical fertilizers also promoted plant growth compared to the control group, their effect was significantly less than the other three groups. Furthermore, long-term application of chemical fertilizers can lead to soil compaction and nutrient imbalance. Therefore, considering all factors, the Bacillus in this invention showed the best overall effect.

[0055] 5. Effects of Bacillus on Fe content in plants The iron content of alfalfa in group T1 (planted on coal gangue soil treated with Bacillus spp. of the present invention) was 182.3±11.5 mg / kg (dry weight), while the iron content of alfalfa in the blank control group (conventionally planted on coal gangue soil, i.e., without Bacillus spp. of the present invention) was 45.7±8.2 mg / kg (dry weight). The iron content of group T1 was significantly higher than that of the blank control group, with a highly significant difference (P<0.01). This confirms that the synergistic planting mode of spraying Bacillus spp. fermentation liquid on coal gangue hills can significantly increase the iron content of alfalfa, providing a material basis (iron element) for subsequent piglet feeding and diarrhea prevention.

[0056] Animal experiments This invention aims to clarify the therapeutic effects of high-iron-content alfalfa (hereinafter referred to as "experimental alfalfa") grown in coal gangue hills on piglets with diarrhea, verify its alleviating and curing effects on piglet diarrhea, and compare the differences with conventional feeding and treatment methods. This provides scientific and repeatable experimental data to support the application of this alfalfa in the dietary therapy of piglet diarrhea. The specific experiments are as follows: (a) Experimental materials Piglets: Select 30 three-way crossbred piglets of the same age (21-28 days), similar weight (6.0±0.5kg), and consistent health status, and all of them have successfully undergone artificial induction of diarrhea (diarrhea criteria: ≥3 times of diarrhea per day, feces are loose / watery, and there are no other infectious diseases). They are randomly divided into 3 groups of 10 piglets each, with the same sex ratio in each group (half male and half female).

[0057] Alfalfa used in the experiment: Fresh alfalfa grass grown on coal gangue hills was collected, washed, dried, and crushed (crushed particle size of 0.5-1mm), and its iron content was tested (test report retained); for later use.

[0058] Standard feed: Complete compound feed for piglets (compliant with GB / T 5915-2020 "Compound Feed for Piglets and Growing-Finishing Pigs"), with no added iron or antidiarrheal ingredients.

[0059] Control group treatment materials: conventional piglet diarrhea treatment feed (in addition to conventional feed, montmorillonite powder, a commonly used antidiarrheal agent, was added at a dosage of 0.3%, which meets the veterinary clinical drug use standards).

[0060] Other materials: Diarrhea induction reagent for piglets (such as E. coli bacterial solution, concentration 1×10⁻⁶). 8 Equipment includes CFU / mL, thermometer, scale, stool sampler, complete blood count test kit, and gut microbiota test kit.

[0061] (II) Experimental Instruments Electronic scale (accuracy 0.01kg), constant temperature incubator, centrifuge, microscope, enzyme-linked immunosorbent assay (ELISA) reader, fecal moisture analyzer, syringe, sterilization equipment, piglet rearing cages (single cage rearing, uniform specifications), data logger, etc. All instruments were calibrated in advance to ensure the accuracy of experimental data.

[0062] (III) Experimental Group Design The experiment employed a single-factor controlled design. The three groups of piglets had identical rearing environments (temperature 25-28℃, humidity 60-70%, light 12h / day), stocking densities, and daily management (cleaning and disinfection twice daily, free access to water). The only differences were in the feed and treatment methods. The experimental period was 14 days (including 3 days of diarrhea induction and 11 days of dietary observation). The specific groupings are as follows: 1. Blank group (Group A, n=10) Treatment: After successfully inducing diarrhea in piglets, feed them only regular feed, without adding any antidiarrheal ingredients or experimental alfalfa, allow them free access to water, and do not provide any additional treatment. Observe the natural recovery of diarrhea.

[0063] Objective: To eliminate the influence of piglets' own immunity on diarrhea recovery and to use this data as the experimental baseline.

[0064] 2. Normal feeding and treatment control group (Group B, n=10) Treatment: After successfully inducing diarrhea in piglets, feed them regular feed plus 0.3% montmorillonite powder (for routine antidiarrheal treatment), provide free access to water, and keep other feeding conditions the same as the control group.

[0065] Objective: To compare the effects of alfalfa and conventional antidiarrheal methods as a control for routine diarrhea treatment.

[0066] 3. Experimental group (Group C, n=10) Treatment: After successful artificial induction of diarrhea in piglets, they were fed regular feed plus experimental alfalfa powder (15% added, optimized based on piglet feed intake to ensure daily iron intake was higher than that of the ordinary alfalfa group), with free access to water, and no antidiarrheal agents were added. Other feeding conditions were the same as those of the blank group and the control group.

[0067] Objective: To verify the therapeutic effect of alfalfa on diarrhea in piglets and to clarify its role in relieving / curing diarrhea.

[0068] (iv) Experimental Procedure (1) Pre-experiment preparation (1-2 days) Adaptation feeding of piglets: 30 piglets were placed in a uniform feeding environment, fed with conventional feed and with free access to water. They were acclimatized for 2 days, and their health status was observed. Abnormal individuals (such as lethargy, loss of appetite, natural diarrhea, etc.) were removed to ensure that the initial condition of the experimental piglets was consistent.

[0069] Pretreatment of alfalfa for the experiment: alfalfa grown in coal gangue hills was collected, washed, dried and crushed, and the iron content was tested and the data was recorded; conventional feed and control and treatment feeds were prepared in advance and kept for later use.

[0070] Instrument and environment preparation: Calibrate all experimental instruments, thoroughly disinfect the breeding cages and experimental site, and adjust the temperature, humidity and light of the breeding environment to the standard range.

[0071] (2) Induction of diarrhea in piglets (3-5 days, 3 days in total) Diarrhea was induced in all 30 piglets by oral administration of E. coli solution: each piglet was given 1 mL / kg body weight of E. coli solution (concentration 1×10⁻⁶). 8 (CFU / mL), once daily for 3 consecutive days; during the induction period, all piglets were fed regular feed and had free access to water. The piglets were observed for diarrhea daily, and the number of diarrhea episodes and fecal characteristics were recorded. Only when all piglets met the diarrhea criteria (≥3 diarrhea episodes per day, loose / watery feces) could they proceed to the subsequent dietary therapy experiment.

[0072] (3) Grouped dietary therapy experiment (6-19 days, 14 days in total) Group feeding: 30 piglets that successfully developed diarrhea were randomly divided into a blank group, a control group, and an experimental group, with 10 piglets in each group and housed in individual cages. Each group was fed the corresponding feed at regular intervals (3 times a day, with each feeding amount being 5-6% of the piglet's body weight, ensuring that the piglets finish the feed and avoid feed waste) according to the treatment method of each group. They were also allowed free access to water, and the amount of feed consumed by the piglets was recorded daily.

[0073] Daily observation and recording: Observe the piglets' mental state, appetite, and diarrhea (number of diarrheas, fecal characteristics, and fecal moisture content) once each in the morning, noon, and evening. Record the recovery time of each piglet from diarrhea (the time from the start of feeding to the first formed feces and the number of diarrheas per day ≤ 1). If any abnormalities such as piglet death or severe illness occur, record them in time and remove the piglets, and replace them with spare piglets (to ensure that each group always has 10 piglets).

[0074] Sample collection and testing: On day 0 (after successful diarrhea induction), day 7, and day 14 of the experiment, fecal samples (5g / pig) and blood samples (2mL / pig) were collected from each piglet. Fecal samples were tested for intestinal flora (E. coli, lactic acid bacteria, etc.) and fecal moisture content. Blood samples were tested for hemoglobin content (reflecting iron absorption) and immune indicators (such as immunoglobulin IgG). All test data were recorded in detail and test reports were retained.

[0075] (4) End of experiment and data processing (day 20) Stop feeding, weigh the final weight of each piglet, and calculate the weight gain rate; organize all experimental data (diarrhea recovery time, diarrhea incidence, fecal indicators, blood indicators, weight gain rate, etc.), perform statistical analysis, and compare the differences among the three groups of data.

[0076] (v) Observation indicators (supported by core patent data, detailed records are required) (1) Core efficacy indicators (directly reflecting the treatment effect of diarrhea) Diarrhea recovery time: The average diarrhea recovery time (days) for each group of piglets, accurate to 0.5 days. The recovery time of each piglet was recorded, and the mean and standard deviation within the group were calculated.

[0077] Diarrhea incidence: During the experiment, the percentage of piglets in each group who had diarrhea each day out of the total number of diarrhea cases in that group was recorded daily, and the average incidence rate was calculated throughout the experiment.

[0078] Fecal characteristics scoring: A 1-4 point scoring standard was used (1 point: formed feces; 2 points: soft feces; 3 points: loose feces; 4 points: watery feces). The feces of each piglet were scored daily, and the average score of each group was calculated. The lower the score, the better the diarrhea relief effect.

[0079] Fecal moisture content: The fecal moisture content (%) was measured on days 0, 7, and 14 of the experiment. The changes in the three groups were compared to reflect the degree of diarrhea relief.

[0080] (2) Auxiliary indicators Blood iron content related indicators: The hemoglobin content (g / L) in the blood of piglets was measured on days 0, 7 and 14 of the experiment. The changes in the three groups were compared to verify the iron absorption effect of alfalfa used in the experiment.

[0081] Intestinal flora indicators: The number of Escherichia coli (harmful bacteria) and lactic acid bacteria (beneficial bacteria) in feces was detected, the ratio of beneficial bacteria to harmful bacteria was calculated, and the changes in intestinal microecological balance in the three groups were compared to explain the mechanism of action of alfalfa dietary therapy.

[0082] Growth performance indicators: Record the initial and final body weights of the piglets, calculate the average weight growth rate (%) of each group of piglets, and verify whether alfalfa promotes the growth of piglets while treating diarrhea.

[0083] Immune indicators: The level of immunoglobulin IgG in the blood (mg / mL) was measured, and the changes in the three groups were compared to verify the regulatory effect of alfalfa on the immunity of piglets.

[0084] (3) Safety indicators During the experiment, we observed whether each group of piglets experienced poisoning, allergies, or abnormal illnesses, recorded the piglet mortality rate, and verified the safety of using alfalfa for piglet feeding.

[0085] (vi) Data processing and analysis The experimental data processing and analysis of this invention strictly follow scientific experimental standards, taking into account data accuracy, repeatability, and logical consistency. The specific procedures, methods, and details are as follows: (1) Data preprocessing Raw data processing: All raw data recorded throughout the experiment (including initial weight, final weight, daily number of diarrheas, fecal morphology score, feed intake, sample test data, etc.) are entered into an Excel spreadsheet to establish a standardized raw data ledger. The ledger must indicate the piglet number, group, test time, tester and instrument number corresponding to the data to ensure that each set of data is traceable. Both paper and electronic copies of the raw data are kept for future reference.

[0086] Outlier handling: The Grubbs test (α=0.05) was used to screen all test data for outliers. If a data point exceeds the mean ± 3 times the standard deviation of the data set, it is considered an outlier. Outliers must be labeled separately, explaining the cause of the outlier (e.g., abnormal disease in piglets, instrument error, etc.). If the number of outliers is ≤1 per set, the outlier can be removed and replaced with the average of the remaining data in the set. If the number of outliers is >1 per set, the experimental process needs to be re-examined, and if necessary, the corresponding tests should be supplemented or the relevant experiments in the set should be repeated to ensure data reliability.

[0087] Data standardization transformation: Standardization was performed on different types of observation indicators to eliminate dimensional differences and facilitate inter-group comparative analysis. Specifically, percentage / rating data such as fecal morphology scores and diarrhea incidence rates were converted into standardized scores (0-100 points); continuous data such as blood indicators, fecal water content, and weight gain rate were standardized using the Z-score method (Z=(X-μ) / σ, where X is a single data point, μ is the mean of the group, and σ is the standard deviation of the group). Standardized data were retained for subsequent statistical analysis.

[0088] (2) Specific data calculation methods Basic indicator calculation: Weight growth rate (%) = (final weight - initial weight) / initial weight × 100. After calculating the weight growth rate of each piglet, calculate the mean (x̄) and standard deviation (SD) of each group.

[0089] Daily diarrhea incidence (%) = Number of piglets with diarrhea in the group on that day / Total number of piglets in the group × 100. After calculating the daily diarrhea incidence throughout the experiment, calculate the average diarrhea incidence and standard deviation for each group throughout the experiment.

[0090] Average diarrhea recovery time (days) = sum of diarrhea recovery times of all piglets in the group / number of piglets in the group that recovered to normal. If a piglet has not recovered by the end of the experiment, its recovery time is calculated based on the total experimental period (14 days) and is marked separately.

[0091] The average fecal morphology score = the sum of the daily fecal morphology scores of all piglets in the group / (number of piglets in the group × number of experimental days). Calculate the average score and standard deviation for each group throughout the entire process.

[0092] Calculation of detection indicators: Intestinal flora related calculations: Based on the test results of fecal samples, the number of Escherichia coli and lactic acid bacteria colonies per gram of feces (CFU / g) was calculated. After logarithmic transformation (lgCFU / g), the mean and standard deviation of each group were calculated. At the same time, the ratio of beneficial bacteria to harmful bacteria (log number of lactic acid bacteria / log number of Escherichia coli) was calculated to reflect the state of intestinal microecological balance.

[0093] Blood index calculation: According to the instructions of the blood routine test kit and the immune index test kit, calculate the hemoglobin content (g / L) and immunoglobulin IgG content (mg / mL) in the blood of each piglet, calculate the mean and standard deviation of each group, and compare the changes and rates of change at different time points (day 0, day 7, and day 14).

[0094] Fecal moisture content calculation: Based on the test results of the fecal moisture analyzer, the moisture content (%) of each fecal sample is directly read, the average value and standard deviation of each group are calculated, and the changing trend at different time points is analyzed.

[0095] (3) Statistical analysis methods Software and parameter settings: SPSS 26.0 statistical software was used for statistical analysis of all data. Before analysis, normality tests (Shapiro-Wilk test) and homogeneity of variance tests (Levene test) were performed, with a significance level of α=0.05 for both. If the data conformed to a normal distribution and homogeneity of variance, parametric tests were used; otherwise, nonparametric tests (Kruskal-Wallis H test) were used.

[0096] Between-group difference analysis: Analysis of differences among the three groups: For all observed indicators (diarrhea recovery time, diarrhea incidence, stool characteristics score, stool moisture content, blood indicators, gut microbiota indicators, weight gain rate, etc.), one-way ANOVA was used to test the overall differences among the three groups. If the test result P < 0.05, it indicates that there are significant differences among the three groups, and further pairwise comparisons are needed.

[0097] Pairwise comparisons: The LSD multiple comparison method (least significant difference method) was used to compare the differences between the experimental group and the blank group, the experimental group and the control group, and the blank group and the control group to clarify the specific differences between each group; the statistical results were marked with P values, where P < 0.05 was significant, P < 0.01 was extremely significant, and P ≥ 0.05 was no significant difference.

[0098] Time-point difference analysis: For the detection indicators (such as hemoglobin content, fecal moisture content, and intestinal flora quantity) at different time points (day 0, day 7, and day 14) within the same group, paired t-tests were used to analyze the differences in the changes of the indicators with the experimental period, and to verify the time-dependent effect of alfalfa in the experiment.

[0099] Correlation analysis: Pearson correlation analysis was used to analyze the correlation between alfalfa feeding amount and piglet diarrhea recovery time, hemoglobin content and diarrhea incidence, and intestinal flora ratio and fecal trait score. The closer the absolute value of the correlation coefficient r is to 1, the stronger the correlation; r>0 is positive correlation, r<0 is negative correlation, and P<0.05 is significant correlation.

[0100] (VII) Results and Analysis Table 5. Basic data on the initial state of experimental piglets (Day 0 of the experiment) Table 6. Statistical Analysis of Core Efficacy Indicators Related to Diarrhea in Three Groups of Piglets Note: Data in the table are expressed as mean ± standard deviation; P < 0.01 indicates a highly significant difference, and P ≥ 0.05 indicates no significant difference; pairwise comparisons were performed using the LSD multiple comparison method.

[0101] Table 7. Statistical Analysis of Blood Indicators in Three Groups of Piglets (at Different Time Points) Note: Data in the table are expressed as mean ± standard deviation; paired t-tests were used for comparisons within groups at time points, and ANOVA+LSD multiple comparisons were used for comparisons between groups; P<0.05 was considered statistically significant, and P<0.01 was considered highly statistically significant.

[0102] Table 8. Statistical analysis of intestinal flora indicators in three groups of piglets (day 14 of the experiment) Note: Data in the table are expressed as mean ± standard deviation; gut microbiota counts were analyzed after logarithmic transformation (lg CFU / g); P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0103] Table 9 Statistical Analysis of Growth Performance and Safety of Three Groups of Piglets Note: Weight-related data in the table are expressed as mean ± standard deviation; mortality rate and abnormalities are expressed as actual counts; weight growth rate is converted by arcsin before ANOVA analysis.

[0104] Table 10 Correlation Analysis Results Note: Pearson correlation analysis was used for correlation analysis; an absolute value of r of 0.7-1.0 indicates a strong correlation, 0.4-0.7 indicates a moderate correlation, and 0.1-0.4 indicates a weak correlation; P<0.01 indicates a highly significant correlation.

[0105] Treatment efficacy for diarrhea: The average recovery time of diarrhea in the experimental group (alfalfa group) was significantly shorter than that in the blank group (P<0.01), while there was no significant difference between the experimental group and the control group (conventional treatment group) (P>0.05). The incidence of diarrhea, fecal characteristics score, and fecal moisture content in the experimental group were significantly lower than those in the blank group (P<0.01), and comparable to those in the control group.

[0106] Iron absorption and immunity: The levels of hemoglobin and immunoglobulin IgG in the blood of piglets in the experimental group were significantly higher than those in the blank group and the control group (P<0.05), indicating that alfalfa can effectively supplement iron and enhance the immunity of piglets.

[0107] Intestinal microecology: The number of lactic acid bacteria in the feces of piglets in the experimental group was significantly higher than that in the blank group and the control group, while the number of Escherichia coli was significantly lower than that in the blank group and the control group (P<0.05). The ratio of beneficial bacteria to harmful bacteria was significantly increased, indicating that alfalfa can regulate the balance of intestinal microecology.

[0108] Growth performance: The weight gain rate of piglets in the experimental group was significantly higher than that in the blank group (P<0.05), while there was no significant difference between the experimental group and the control group (P>0.05), indicating that alfalfa can promote the growth of piglets while treating diarrhea.

[0109] Safety: No abnormalities such as poisoning or allergies were observed in any of the three groups of piglets, and the mortality rate was 0, indicating that the alfalfa used in the experiment is safe and reliable for piglet feeding.

[0110] Iron-rich alfalfa grown on coal gangue hills can effectively alleviate and cure diarrhea in piglets, with therapeutic effects no less than conventional antidiarrheal treatments. It can also supplement the piglets' iron levels, regulate the intestinal microecological balance, enhance immunity, and promote growth. Moreover, it is highly safe and can be widely used as a dietary ingredient for treating diarrhea in piglets, providing a new approach for the resource utilization of alfalfa and green dietary therapy for piglet diarrhea.

[0111] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Bacillus ( Bacillus sp. YB-01, characterized in that, The preservation number of the Bacillus YB-01 is CCTCC NO: M20252743.

2. The application of Bacillus YB-01 synergistic plants in coal gangue soil remediation as described in claim 1, characterized in that, The plants were planted after the Bacillus YB-01 was applied to the coal gangue soil.

3. The application of Bacillus YB-01 as described in claim 1 in promoting plant growth or increasing iron content in coal gangue soil, characterized in that, By applying the Bacillus YB-01 to coal gangue soil and then planting plants, it is easy to promote the growth of the plants or increase their iron content.

4. A method for increasing the iron content of plants grown in coal gangue soil, characterized in that, The method includes the step of spraying the fermentation broth of Bacillus YB-01 as described in claim 1 onto coal gangue soil, and then planting plants.

5. The method as described in claim 4, characterized in that, The viable cell count of the fermentation broth of Bacillus YB-01 is 10. 11 CFU / mL, spraying rate 50 mL / m 2 .

6. The use of the iron-enhanced plant obtained by the method of claim 4 or 5 in the preparation of pig feed or medicaments for treating swine diarrhea.

7. The application as described in claim 6, characterized in that, The plant in question is alfalfa.

8. The application as described in claim 7, characterized in that, After the alfalfa matures, it is dried and pulverized to obtain alfalfa powder; 15% alfalfa powder is added according to the weight of the basal pig feed.

9. A feed for treating swine diarrhea, characterized in that, The feed consists of basal pig feed and alfalfa powder, which is obtained by growing alfalfa according to the method described in claim 4 or 5, and drying and pulverizing the grown alfalfa after it matures.

10. The feed as described in claim 9, characterized in that, The feed contains 15% alfalfa meal, calculated based on the weight of the basal pig feed.

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