Bacillus thuringiensis ZL-5, composting biological agent, and preparation method and application of bacillus thuringiensis ZL-5

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus thuringiensis ZL-5, a compost biological agent as well as a preparation method and application of the compost biological agent. The preservation number of the bacillus thuringiensis ZL-5 is CCTCC (China Center for Type Culture Collection) NO: M 20241070. According to the method, the leafy vegetable waste is used as a main material, the straw is used as an auxiliary material, the provided composting biological agent is added to compost the leafy vegetable waste, the temperature of a compost body can be increased to 60 DEG C or above in the 2th day and can be kept at the high-temperature period of 6-8 days, and the leafy vegetable waste is completely decomposed about the 20th day. In a compost body added with the biological agent, the percentage value of Hu-rich and the percentage value of humic acid are 9.0%-12% and 20%-30% respectively, after composting, the leaf vegetable organic fertilizer remarkably improves the germination index of lettuce seeds, and biochemical humic acid remarkably promotes the dry quality, plant height, chlorophyll content and leaf area of the overground part, the dry weight of the underground part and the total root tip number of the lettuce.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Bacillus thuringiensis ZL-5, a composting biological bacterial agent, and a preparation method and application thereof. Background Art

[0002] Vegetables are the second largest industry in my country's planting industry after grain. However, the waste ratio of various types of vegetables ranges from 16.5% to 63.5%, with an average of 33.6%. The total amount of vegetable waste in my country is about 2.52×10 8 t. Most of these vegetable wastes are abandoned in vegetable production areas, distribution points, etc., with low utilization rates and high water content. If not handled in time, they will quickly rot and produce a large amount of leachate, which will cause non-point source pollution to soil, rivers, lakes and groundwater. At present, most vegetable growing areas in my country often treat vegetable waste by deep burial, dumping, etc., and some areas also use natural composting, biogas production and other methods for treatment. The utilization rate of solid waste is low and generally cannot meet the requirements of harmless treatment. Therefore, it is necessary to adopt more effective resource treatment methods based on the characteristics of vegetable waste. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a strain of Bacillus thuringiensis ZL-5, a composting biological agent and a preparation method and application thereof. The present invention adds Bacillus thuringiensis ZL-5 to a pile of leafy vegetable waste, significantly improves the heating rate of the pile, prolongs the duration of the high temperature period, improves the compost quality, and promotes the rapid humification of the leafy vegetable waste.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a Bacillus thuringiensis ZL-5, wherein the Bacillus thuringiensis ZL-5 was deposited in China Center for Type Culture Collection on May 27, 2024, with a deposit number of CCTCC NO: M20241070.

[0006] The present invention also provides a composting biological bacterial agent for humifying leafy vegetable waste, which contains the Bacillus thuringiensis ZL-5 described in the above technical solution.

[0007] Preferably, the bacterial content of Bacillus thuringiensis ZL-5 in the compost biological agent is 10-15%.

[0008] The present invention also provides a method for preparing the compost biological agent described in the above technical solution, comprising the following steps:

[0009] 1) performing seed culture on the Bacillus thuringiensis ZL-5 to obtain a seed solution;

[0010] 2) mixing the seed solution obtained in step 1) with a fermentation medium and a nutrient solution and fermenting the mixture to obtain a fermentation product;

[0011] 3) Drying the fermented product obtained in step 3) to obtain a composting biological agent.

[0012] Preferably, the step 1) seed culture comprises:

[0013] The culture medium used included LB medium, and the culture was carried out at 30°C and 150 rpm until the logarithmic growth phase.

[0014] Preferably, in step 2), the volume ratio of the seed solution to the mass of the fermentation medium and the nutrient solution is 20 mL: 1 kg: 20 mL.

[0015] Preferably, the fermentation medium comprises the following components in parts by weight: 600 parts of soybean meal, 200 parts of furfural residue, 150 parts of sawdust and 50 g of diatomaceous earth;

[0016] The nutrient solution uses water as solvent and includes the following components: 5 g / L glucose, 1 g / L ammonium nitrate, KH 2 PO 4 0.5g / L, Na 2 HPO 4 1.5g / L, NaCl 1g / L and MgSO 4 0.5 g / L, natural pH.

[0017] Preferably, the drying temperature in step 3) is 30°C.

[0018] The present invention also provides the use of the composting biological bacterial agent described in the above technical solution in composting leafy vegetable waste.

[0019] Preferably, the application includes: mixing the composting biological agent with leafy vegetable waste and straw for composting;

[0020] The mass ratio of the composting biological agent to the total mass of the leafy vegetable waste and the straw is 3%;

[0021] The mass ratio of the leafy vegetable waste to the straw is 10-20:1.

[0022] Beneficial effects of the present invention:

[0023] The Bacillus thuringiensis ZL-5 provided by the present invention ferments leafy vegetable waste, has a simple preparation method, is easy to operate, can increase the temperature of the compost body, prolong the duration of the high temperature period, improve the quality of the compost, and promote the rapid humification of the leafy vegetable waste. The price is low, and no other waste is generated during the preparation process. It is a green and environmentally friendly method with strong social benefits.

[0024] The present invention uses leafy vegetable waste as the main material and straw as the auxiliary material, and adds the provided composting biological bacterial agent to compost the leafy vegetable waste. The pile can be heated to above 60°C on the second day, and can maintain a high temperature period (≥50°C) for 6-8 days. Around the 20th day, the leafy vegetable waste reaches complete decomposition. The humic acid ratio and humic acid percentage values ​​in the pile with the biological bacterial agent are respectively increased by 9.0%-12% and 20%-30%. After composting, the leafy vegetable organic fertilizer significantly improves the germination index of lettuce seeds, and the biochemical humic acid significantly promotes the aboveground dry weight, plant height, chlorophyll content and leaf area of ​​lettuce, underground dry weight and total root tip number. This study shows that the ZL-5 exogenous microbial agent can increase the temperature of the pile, prolong the duration of the high temperature period, improve the quality of the compost, and promote the rapid humification of leafy vegetable waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0026] Figure 1 This is the colony morphology of strain ZL-5;

[0027] Figure 2 This is a scanning electron micrograph of strain ZL-5;

[0028] Figure 3 Gram stain for strain ZL-5;

[0029] Figure 4 is the phylogenetic tree of strain ZL-5;

[0030] Figure 5 Coarse screening for Congo red staining of strain ZL-5;

[0031] Figure 6 The four enzyme activities of strain ZL-5 are endo-nuclease (CMC), cellulose holoenzyme (FPA), exo-nuclease (C1) and β-glucosidase (β-Gase);

[0032] Figure 7 The effect of biological agents on the changes of humic acid in organic fertilizers for leafy vegetables;

[0033] Figure 8 The influence of the change of Hufu ratio of biological microbial agent leafy vegetable organic fertilizer;

[0034] Fig. 9 The influence of humic acid changes in leafy vegetable organic fertilizers using biological microbial agents;

[0035] Fig.10 The influence of temperature change on bio-microbial leafy vegetable compost;

[0036] Fig.11Indicates the compost quality index during the composting process of leafy vegetable waste;

[0037] Fig.12 This study is the effect of leafy organic fertilizer on the physiological indicators of lettuce.

[0038] Biological Deposit Description

[0039] Bacillus thuringiensis ZL-5, Latin name Bacillus thuringiensis, was deposited in the China Center for Type Culture Collection on May 27, 2024, with the address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M20241070. DETAILED DESCRIPTION

[0040] The present invention provides a strain of Bacillus thuringiensis ZL-5, which was deposited in the China Center for Type Culture Collection on May 27, 2024, with a deposit number of CCTCC NO: M20241070. In the present invention, the 16S rDNA sequence of the Bacillus thuringiensis ZL-5 is shown in SEQ ID No. 1, which is as follows:

[0041]

[0042] The present invention also provides a composting biological agent for humifying leafy vegetable waste, which contains the Bacillus thuringiensis ZL-5 described in the above technical solution. In the present invention, the bacterial content of Bacillus thuringiensis ZL-5 in the composting biological agent is preferably 10%-15%.

[0043] The present invention also provides a method for preparing the compost biological agent described in the above technical solution, comprising the following steps:

[0044] 1) performing seed culture on the Bacillus thuringiensis ZL-5 to obtain a seed solution;

[0045] 2) mixing the seed solution obtained in step 1) with a fermentation medium and a nutrient solution and fermenting the mixture to obtain a fermentation product;

[0046] 3) Drying the fermented product obtained in step 3) to obtain a composting biological agent.

[0047] In the present invention, the Bacillus thuringiensis ZL-5 is subjected to seed culture to obtain seed liquid. In the present invention, the seed culture preferably comprises: the culture medium used comprises LB culture medium, and the culture is carried out at 30° C. and 150 rpm to the logarithmic growth phase.

[0048] The present invention mixes the obtained seed liquid with a fermentation medium and a nutrient solution and then ferments to obtain a fermented product. In the present invention, the volume ratio of the seed liquid to the mass of the fermentation medium and the nutrient solution is preferably 20mL:1kg:20mL. In the present invention, the fermentation medium preferably includes the following components in parts by weight: 600 parts of soybean meal, 200 parts of furfural residue, 150 parts of sawdust and 50g of diatomaceous earth. In the present invention, the nutrient solution preferably uses water as a solvent and includes the following components: 5g / L glucose, 1g / L ammonium nitrate, KH 2 PO 4 0.5g / L, Na 2 HPO 4 1.5g / L, NaCl 1g / L and MgSO 4 0.5 g / L, natural pH. In the present invention, the fermentation conditions preferably include: fermentation at a temperature of 28° C. and under aerobic conditions for 48 hours.

[0049] The present invention dries the obtained fermented product to obtain a compost biological agent. In the present invention, the drying temperature is preferably 30°C, and the moisture content of the fermented product after drying is ≤35%. In the present invention, the drying is preferably carried out under ventilation. The present invention preferably pulverizes after drying to obtain a compost biological agent.

[0050] The present invention also provides the use of the composting biological bacterial agent described in the above technical solution in composting leafy vegetable waste.

[0051] In the present invention, the application preferably includes: mixing the composting biological agent with leafy vegetable waste and straw for composting. In the present invention, the mass ratio of the total mass of the leafy vegetable waste and straw to the composting biological agent is preferably 3%. In the present invention, the mass ratio of the leafy vegetable waste to the straw is preferably 10 to 20:1. In the present invention, the leafy vegetable waste is preferably cut into 2 to 4, and the straw is preferably crushed to 1 to 2 cm before fermentation. In the present invention, the straw is preferably corn straw.

[0052] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1

[0054] Isolation and identification of Bacillus thuringiensis ZL-5:

[0055] (1) Weigh 5 g of soil sample and add it into a flask containing 95 mL of sterile water. Incubate at 35 °C and 150 rpm / min. -1 The culture was shaken on a shaker for 72 hours. After standing for 5 hours to allow the solution to separate, 2 mL of the supernatant was inoculated into the liquid enrichment medium and cultured at 35°C and 150 rpm·min-1 for 7 days. During this period, the scale line on the triangular flask was marked and the water level was replenished every 12 hours. Dilute it to 10 according to the gradient dilution method. -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Concentration. Pipette 100 μL of each dilution onto a solid enrichment medium and incubate inverted at 35°C in the dark for 24 h. Use an inoculation loop to pick strains with different colony morphology, streak on the solid enrichment medium, separate and purify, and store by number.

[0056] (2) The purified strains were inoculated into the screening medium, with each strain being repeated three times. The colonies were grown in the dark at 35°C and stained with 1 g / L Congo red colorimetric solution for 20 min. The Congo red colorimetric solution was discarded. The plate surface was rinsed 2-3 times with 1 mol / L NaCl solution. The production of transparent circles was observed and the diameter of the hydrolysis zone (D) and colony diameter (D / d) on the plate were accurately measured with a vernier caliper. The ratio of the hydrolysis zone diameter to the colony diameter was calculated. The cellulase activity of the isolated strain was preliminarily determined based on the ratio. Figure 1This is the sodium carboxymethylcellulose-Congo red staining image of ZL-5. It can be seen that the ratio of the transparent circle (D) to the colony (d) of ZL-5 is greater than 1.

[0057] (3) Preliminary isolation of strains The strains were subjected to conventional identification and molecular biological identification. Conventional identification includes morphological characteristics and physiological and biochemical characteristics. Morphological characteristics include microscopic morphology and culture characteristics; physical and chemical characteristics include nutritional type, carbon and nitrogen source utilization ability, various metabolic reactions, enzyme reactions, etc. Molecular biological identification mainly involves extracting the bacterial genome of the preliminary screening strains, amplifying the 16s rDNA of the preliminary screening strains using 16s universal primers, and identifying the preliminary screening bacteria as Bacillus thuringiensis by constructing a developmental tree, and naming it ZL-5.

[0058] (4) Use the Gram stain kit to observe the smear of Bacillus thuringiensis ZL-5. Take the bacterial solution and drop it on the slide smear for fixation. First, add crystal violet staining solution to the fixed smear for 1 minute, and use distilled water to absorb the residual dye; then add Gram iodine solution, and wash the residual dye with distilled water after 2 minutes; then add decolorizing solution to decolorize for 5 minutes, and wash with distilled water; finally, add safranin counterstaining solution for 3 minutes, wash the residual dye with distilled water, and examine under a microscope after drying. Gram staining of Bacillus thuringiensis ZL-5 is blue-purple, and it is identified as Gram-positive bacteria.

[0059] (4) Determine the logarithmic growth phase of Bacillus thuringiensis ZL-5 in LB liquid medium by measuring the growth curve of Bacillus thuringiensis ZL-5 in LB liquid medium. Use an inoculation loop to take Bacillus thuringiensis ZL-5 into LB liquid medium, place it in a shaking incubator at 35°C and 150 rpm for 40 h, and measure the OD every 4 h using a UV spectrophotometer. 600 The absorbance value at 100 ℃ was used to determine the logarithmic growth period of Bacillus thuringiensis ZL-5 to be 8-12 h. Figure 6The strain ZL-5 had four enzyme activities, namely endo-nuclease (CMC), cellulose holoenzyme (FPA), exo-nuclease (C1) and β-glucosidase (β-Gase). The enzyme activity of endo-nuclease (CMC) was 1.75U / mL, the enzyme activity of cellulose holoenzyme (FPA) was 4.2U / mL, the enzyme activity of exo-nuclease (C1) was 5.45U / mL, and the enzyme activity of β-glucosidase (β-Gase) was 2.25U / mL. That is, ZL-5 had higher enzyme activities of cellulose holoenzyme (FPA) and exo-nuclease (C1), and lower enzyme activities of endo-nuclease (CMC) and β-glucosidase (β-Gase).

[0060] Example 2

[0061] Preparation of biological agents

[0062] (1) Bacillus thuringiensis ZL-5 was activated on a culture medium, and the cultured bacteria were inoculated into a seed bottle at a volume ratio of 10%, and cultured at 30° C. and 150 rpm with shaking until the logarithmic growth phase to obtain a seed solution;

[0063] (2) 20 mL (OD 600 =1) adding 1000 g of fermentation medium, and at the same time adding 20 mL of nutrient solution to 1000 g of fermentation medium, carrying out aerobic conditions at 28° C. for 48 hours, and drying the fermented product under ventilation at 30° C. until the moisture content is ≤35%, and then crushing and packaging to obtain a bacterial agent of ZL-5 strain for strong humification of leafy vegetable waste compost, wherein the bacterial content is controlled at 10%-15%;

[0064] The composition of each 1000 g fermentation medium by weight percentage is: 600 g soybean meal, 200 g furfural residue, 150 g sawdust, and 50 g diatomaceous earth as a stabilizer;

[0065] The composition of each 1000mL nutrient solution by weight percentage is: glucose 5g / L, ammonium nitrate 1g / L, KH 2 PO 4 0.5g / L, Na 2 HPO 4 1.5g / L, NaCl 1g / L, MgSO 4 0.5 g / L, the balance is water, pH natural.

[0066] Example 3

[0067] Effects of adding biological agents on humification of leafy vegetable waste compost

[0068] Detect physiological and biochemical indicators such as humic acid, fulvic acid, and humic acid-fulvic acid ratio to reflect the composting process and judge the maturity of the compost. Use pH, electrical conductivity (EC), organic matter (TOC), total nitrogen, total phosphorus, total potassium, and seed germination index to evaluate the maturity and quality of aerobic composting.

[0069] (1) The prepared rapid humification leafy vegetable waste composting biological agent was unsealed at room temperature, and the biological agent was mixed with the dried and cut waste cabbage leaves at a mass ratio of 3%, and corn stalks were added at the same time. The mass ratio of the waste cabbage leaves to the stalks was 10 to 20:1. After 36 days of composting and humification reaction, the moisture content and temperature of the pile were measured every day, and samples were taken every 4 days and stored at -20°C.

[0070] (2) Humic acid content was determined according to NY / T 525-2021, using Na 4 P 2 O 7 -NaOH mixed solution extraction, pH was adjusted to separate fulvic acid (FA) and humic acid (HA), and the content was determined by potassium dichromate volumetric method.

[0071] (3) Weigh 1g of sample, add it to a centrifuge tube, add 25mL of 0.5mol / L NaOH solution, seal the centrifuge tube, and use an oscillator to oscillate the centrifuge tube for 1.5h (30min for liquid samples). After oscillation, rinse the bottle cap with 1% NaOH solution. Centrifuge at 2000rpm for 20min. (All centrifugation in this test is based on this standard). Transfer the supernatant to a second batch of centrifuge tubes of known weight. Add 5mL of 1% NaOH solution to the original centrifuge tube, oscillate thoroughly and centrifuge. Similarly, collect the supernatant into a second batch of centrifuge tubes of known weight. Add 10ml of concentrated hydrochloric acid to the supernatant, oscillate thoroughly and centrifuge. Discard the supernatant, add 12.5mL (distilled water with pH <1) to the precipitate, oscillate thoroughly to mix, centrifuge, discard the supernatant, and repeat twice. Place the centrifuge tube in an oven and dry it at 60℃ to constant weight. Cool it in a desiccator for 2-3h and weigh it.

[0072] (4) Determination of humic acid and fulvic acid content: Weigh 4g of soil sample that has passed through a 60-mesh sieve and place it in a 50ml centrifuge tube, add 40ml of sodium hydroxide-sodium pyrophosphate mixed extract, and shake at 25℃ for 30min. After standing at 25℃ for 24h, mix well, filter or centrifuge, and the filtrate is the test solution. Take 5-20ml of the filtrate (determined by the color depth) and place it in a digestion tube, and neutralize it with sulfuric acid to pH = 7. Then evaporate it in a water bath, and determine the humic acid + fulvic acid content according to the determination method of organic carbon.

[0073] (5) Total organic carbon (TOC): Determined by potassium dichromate heating method in accordance with the Chinese national standard (NY 525-2021).

[0074] (6) Total nitrogen (TN), total phosphorus (TP), and total potassium (TK): Accurately weigh about 0.1000 g of compost sample and place it in a digestion tube. Add 5 mL of concentrated sulfuric acid and hydrogen peroxide. Digest at 350 °C until the solution becomes milky white. Adjust the volume and take part of the solution to measure total nitrogen using a Kjeldahl nitrogen analyzer. Measure total phosphorus using the molybdenum antimony colorimetric method-spectrophotometer according to NY-T299-1995. Measure total potassium using a flame spectrophotometer according to GB-T34203-2017.

[0075] Figure 7-9 . indicates the humification of leafy vegetable waste compost. Pha is the percentage of humic acid in humic acid, and HA / FA is the ratio of humic acid to fulvic acid, indicating the polymerization rate of humus. In the pile with 3% ZL-5 bacterial agent added, the HA content of each treatment increased rapidly in the first 12 days of composting. The degradation and humification rate of organic matter during composting was the highest, and reached a maximum value of 3-3.5% at the end of composting. The humic acid ratio and humic acid percentage values ​​in the pile with biological bacterial agent were 9.0%-12% and 20%-30% higher, respectively. The temperature of the pile rose rapidly and reached the high temperature period (>60℃) on the third day. The high temperature period of each group of piles lasted until the eighth day, and the highest temperature of the treatment group was significantly higher than that of the control group.

[0076] Fig.11 .Indicates the compost quality index during the composting process of leafy vegetable waste. In the first 7 days of composting, as the temperature rose, the pH value of the pile treated with ZL-5 inoculant increased rapidly. On the 12th day, the pH value of each group decreased briefly, decreasing to 8.89-9.07. On the 21st day, the pH value of each group rose to a maximum value of 9.35. On the 35th day, the pH value of each treatment stabilized at around 8.35. In the early stage of composting, the pile treated with ZL-5 inoculant increased with the increase of composting time compared with the control group. On the 3rd day, the EC of the pile reached a maximum value of 1357.33±21.36μS / cm, and continued to decline after the 14th day to the 35th day. At the end of composting, the conductivity of each group stabilized between 1022.67 and 1123.00μS / cm. In the early stage of composting, the TOC content decreased slowly. The TOC content of each group of compost decreased fastest from the 7th to the 14th day. On the 12th day, the TOC content of each group of compost decreased to 38.02%. In the early stage of composting, the TN content of the compost decreased rapidly and reached the lowest value on the 8th day.

[0077] Comparative Example 1

[0078] Effect of adding biological inoculants on composting humification of leafy vegetable waste In step 1 of Example 3, no ZL-5 inoculant was added. The remaining parameters were exactly the same as those in Example 3, and the process was also the same.

[0079] In the early stage of composting, the EC of the pile without adding microbial agent reached the highest value of 1384.33±17.47μS / cm. The TOC content decreased slowly in the early stage of composting, and the TOC content of each group decreased fastest from the 8th to the 12th day. On the 12th day, the TOC content of the pile dropped to 41.72%. At the end of the composting on the 36th day, the TOC content dropped to 31.98±0.49.

[0080] Application Example 1

[0081] Effects of organic fertilizer from leafy vegetable waste on crops

[0082] (1) Weigh 10 g of the compost sample obtained in Example 3 and mix them at a solid-liquid ratio of 1:10 (g mL -1 ) add 100 mL of deionized water, oscillate at 200 r·min-1 at 25 ℃ for 30 min, filter the mixed solution and set aside, take 10 mL of supernatant into a 9 cm culture dish padded with 2 qualitative filter papers, evenly put 10 lettuce seeds with full grains on it, and culture in a light-proof incubator at (25±2) ℃ for 48 h. Count the germination rate and measure the taproot length. At the same time, use distilled water as a blank test.

[0083] The seed germination index (GI) was calculated according to the following formula.

[0084] GI = (seed germination rate of organic fertilizer extract group * seed root length) / (seed germination rate of control group × seed root length) × 100%

[0085] At the same time, physiological and biochemical indicators such as chlorophyll, proline, and active oxygen in lettuce were determined.

[0086] (2) Extraction of proline

[0087] Grind the sample material with liquid nitrogen, weigh an appropriate amount of sample powder (0.1-0.2 g) into a 2 mL centrifuge tube, then add 1.5 mL of 3% sulfosalicylic acid solution to each tube, shake and mix. Centrifuge at 12000 g for 10 min, and transfer 1 mL of the supernatant to a new 10 mL centrifuge tube.

[0088] (3) Proline determination

[0089] Add 1mL of glacial acetic acid and 1mL of 2.5% acidic ninhydrin reagent, heat in a boiling water bath for 60min, the solution will turn red. After cooling, add 2mL of toluene, shake for 30s, and gently pipette the upper layer of proline red toluene solution into a colorimetric cup. Use the toluene solution as a blank control and measure the absorbance (A) value at a wavelength of 520mm.

[0090] Find the proline content in the sample solution from the standard curve and calculate the proline content in the sample according to the following formula:

[0091]

[0092] The formula for the fresh weight of sample x (g) is the amount of extract used (mL) during determination: C---the proline content (g) obtained from the standard curve; dilution factor; total amount of extract---1.5mL, the amount of extract used during determination---the amount absorbed after extraction with 1.5mL sulfosalicylic acid, generally 1mL.

[0093] Fig.12 The effect of organic fertilizer on the physiological indexes of lettuce is shown in Figure 2. Chlorophyll, as a pigment in the chloroplasts of green plants, plays a vital role in photosynthesis. It is an important indicator to measure the strength of plant stress resistance and is usually related to the nutrients, water and light during the growth of the plant. Fig.12 As shown in the figure, on the 21st day, the development of spinach leaves was close to complete, and the chlorophyll content reached its peak at this time; reactive oxygen species (ROS) are important signal molecules in the normal growth and development of plants, mainly including superoxide anion free radicals (O 2 - ), hydroxide ion (OH - ), hydroxyl radicals (·OH), hydrogen peroxide (H2O2), etc. ROS-related redox signals often play a vital role in plant growth and development and adaptation to the environment. In plants, each cell has a redox network pathway regulated by reactive oxygen and controls almost all physiological processes of the cell, such as gene expression and translation, metabolism, aging and death. As a protein oxidant, reactive oxygen not only changes protein function, but is also a signal molecule released by lipid molecules, playing an important physiological function in plant defense response and programmed cell death, stomatal movement, plant regeneration and other pathways. On the 36th day, the content of reactive oxygen was the highest, reaching 85; proline is one of the more abundant amino acids in plants and is widely present in various parts of the plant. In plants, proline is involved in a variety of physiological processes, such as maintaining the plant immune system, regulating plant metabolism, protecting plant cell membranes from oxidative damage, etc., including the drought adaptation process of plants. Its content reached a peak of 84 on the 36th day.

[0094] Compared with the comparative example and the blank group, the present invention has a higher improvement in the temperature of the pile, the extension of the duration of the high temperature period, and the improvement of the compost quality. It can significantly promote the rapid humification of leafy vegetable waste, and within a certain proportion range, it improves with the increase of ZL-5 concentration.

[0095] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus thuringiensis ZL-5, characterized in that: The Bacillus thuringiensis ZL-5 was deposited in the China Center for Type Culture Collection on May 27, 2024, with the deposit number being CCTCC NO: M20241070.

2. A composting bio-microbial agent for humifying leafy vegetable waste, characterized in that: Contains the Bacillus thuringiensis ZL-5 according to claim 1.

3. The composting biological agent according to claim 2, characterized in that: The bacterial content of Bacillus thuringiensis ZL-5 in the compost biological bacterial agent is 10-15%.

4. A method for preparing a composting biological agent according to claim 2 or 3, characterized in that: The following steps are involved: 1) performing seed culture on the Bacillus thuringiensis ZL-5 to obtain a seed solution; 2) mixing the seed solution obtained in step 1) with a fermentation medium and a nutrient solution and fermenting the mixture to obtain a fermentation product; 3) Drying the fermented product obtained in step 3) to obtain a composting biological agent.

5. The preparation method according to claim 4, characterized in that: The step 1) seed cultivation comprises: The culture medium used included LB medium, and the culture was carried out at 30°C and 150 rpm until the logarithmic growth phase.

6. The preparation method according to claim 5, characterized in that: In the step 2), the volume ratio of the seed solution to the mass of the fermentation medium and the nutrient solution is 20 mL: 1 kg: 20 mL.

7. The preparation method according to claim 5 or 6, characterized in that: The fermentation medium comprises the following components in parts by weight: 600 parts of soybean meal, 200 parts of furfural residue, 150 parts of sawdust and 50 g of diatomaceous earth; The nutrient solution uses water as a solvent and includes the following components: Glucose 5g / L, ammonium nitrate 1g / L, KH2PO4 0.5g / L, Na2HPO4 1.5g / L, NaCl 1g / L and MgSO4 0.5 g / L, natural pH.

8. The preparation method according to claim 5, characterized in that: The drying temperature in step 3) is 30°C.

9. Use of the composting biological agent according to claim 2 or 3 in composting leafy vegetable waste.

10. The use according to claim 9, characterized in that: The application includes: mixing the composting biological agent with leafy vegetable waste and straw for composting; The mass ratio of the total mass of the leafy vegetable waste and the straw to the compost biological agent is 3%-5% (100:3-100:5); The mass ratio of the leafy vegetable waste to the straw is 10-20:1.

Images