Low-temperature-resistant complex microbial inoculant and application thereof

Abstract

The invention provides a low-temperature-resistant complex microbial inoculant and application thereof. Specifically, the invention provides a low-temperature-resistant complex microbial inoculant which comprises bacillus licheniformis, bacillus megaterium and bacillus laterosporus. The invention also provides an application of the low-temperature-resistant complex microbial inoculant and a method for producing a liquid bacterial fertilizer by using the low-temperature-resistant complex microbial inoculant and using livestock urine as a nutrient source, and relates to the field of microorganisms. In a low-temperature environment, the complex microbial inoculant decomposes and converts livestock urine into a nutrient-rich and safe liquid bacterial fertilizer, the content of available nitrogen in the urine is increased (greater than or equal to 2.0 g / L), and the complex microbial inoculant has obvious ecological benefits and economic benefits when being applied to livestock manure treatment and agricultural production in northeast China.

Description

Technical Field

[0001] This invention relates to the fields of microbial application technology and agricultural waste resource utilization, specifically to a low-temperature resistant compound microbial agent and its application. Background Technology

[0002] Long, cold winters (often below 15°C) in northern my country significantly reduce the activity of traditional microbial treatment systems, resulting in a degradation efficiency of less than 30% for organic matter (such as urea and uric acid) in livestock urine, making direct resource utilization difficult. Existing technologies largely rely on heating and insulation, with energy costs accounting for over 60% of the total treatment cost, and the equipment is complex, making it difficult to promote in Northeast China. This represents the current industry need and pain point.

[0003] The existing technology has the following three limitations: (1) Single strains have poor low-temperature adaptability and cannot efficiently decompose complex organic matter in urine; (2) Insufficient compatibility of compound microbial agents. Currently, many agricultural microbial agents on the market are mainly for soil improvement and lack synergistic microbial design for the conversion of amide nitrogen in poultry and livestock urine into available nitrogen, resulting in low conversion rate of available nutrients; (3) High energy consumption of the process. Traditional fermentation requires maintaining 25-37℃, while in the natural winter temperature in the north (5-15℃), the proliferation of microorganisms stops, and the number of live bacteria is difficult to exceed 1 billion CFU / mL, resulting in low urea conversion rate.

[0004] Therefore, there is an urgent need in this field to develop a compound microbial agent that can efficiently metabolize poultry and livestock urine at natural low temperatures of 5-15℃. Through synergistic optimization of microbial strains and process adaptation, the large-scale production of liquid microbial fertilizer without external energy can be achieved, thereby promoting the green and circular development of agriculture in cold regions.

[0005] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of the present invention, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a low-temperature resistant compound microbial agent and a method for producing liquid microbial fertilizer from poultry and livestock urine in a low-temperature environment.

[0007] In a first aspect of the present invention, a low-temperature resistant compound microbial agent is provided, comprising: Bacillus licheniformis, Bacillus megaterium, and Bacillus laterosporus.

[0008] In another preferred embodiment, the Bacillus licheniformis is selected from the group consisting of Bacillus licheniformis CGMCC 1.807 and Bacillus licheniformis CICC 10037.

[0009] In another preferred embodiment, the Bacillus megaterium is selected from the group consisting of Bacillus megaterium CGMCC 1.217, Bacillus megaterium CICC 21580, and Bacillus megaterium CICC 20665.

[0010] In another preferred embodiment, the Bacillus laterosporus is selected from the group consisting of Bacillus laterosporus CGMCC 1.0864 and Bacillus laterosporus CICC 21185.

[0011] In another preferred embodiment, the function of the low-temperature resistant compound bacterial agent is as follows: Bacillus licheniformis secretes low-temperature protease to decompose large protein molecules in urine into small peptides; Bacillus megaterium solubilizes phosphorus and potassium, degrades uric acid and fixes ammonia nitrogen; Bacillus laterosporus secretes a variety of enzymes (such as keratinase) to promote the dissolution and release of nitrogen, phosphorus and potassium.

[0012] In another preferred embodiment, the ratio of Bacillus licheniformis: Bacillus megaterium: Bacillus laterosporus is (0.25~2):(0.25~2):(0.25~2), more preferably (0.5~2):(0.5~2):(0.5~2), even more preferably (0.8~1.6):(0.8~1.6):(0.8~1.6), even more preferably (0.9~1.1):(0.9~1.1):(0.9~1.1), even more preferably (0.95~1.05):(0.95~1.05):(0.95~1.05), and most preferably, the ratio of Bacillus licheniformis: Bacillus megaterium: Bacillus laterosporus is 1:1:1.

[0013] In another preferred embodiment, the number of Bacillus licheniformis is 0.2 × 10⁻⁶. 8 CFU / mL ~2×10 8 CFU / mL, preferably 0.4 × 10⁻⁶ 8 CFU / mL ~1.6×10 8 CFU / mL, more preferably 0.8 × 10⁻⁶ 8 CFU / mL ~1.2×10 8 CFU / mL.

[0014] In another preferred embodiment, the number of *Bacillus megaterium* is 0.2 × 10⁻⁶. 8 CFU / mL ~2×10 8 CFU / mL, preferably 0.4 × 10⁻⁶ 8 CFU / mL ~1.6×10 8 CFU / mL, more preferably 0.8 × 10⁻⁶ 8 CFU / mL ~1.2×10 8 CFU / mL.

[0015] In another preferred embodiment, the number of *Bacillus laterosporus* is 0.2 × 10⁻⁶. 8 CFU / mL ~2×10 8 CFU / mL, preferably 0.4 × 10⁻⁶ 8 CFU / mL ~1.6×10 8 CFU / mL, more preferably 0.8 × 10⁻⁶ 8 CFU / mL ~1.2×10 8 CFU / mL.

[0016] In another preferred embodiment, the Bacillus licheniformis is Bacillus licheniformis CGMCC 1.807, the Bacillus megaterium is Bacillus megaterium CGMCC 1.217, and / or the Bacillus laterosporus is Bacillus laterosporus CGMCC 1.0864.

[0017] In another preferred embodiment, the low-temperature resistant compound bacterial agent further includes: solid culture medium and liquid culture medium.

[0018] In another preferred embodiment, the solid culture medium consists of: 20-40 g / L sucrose, 5-10 g / L yeast extract, 5-10 g / L peptone, 0.5-0.8 g / L potassium dihydrogen phosphate, and 20-30 g / L agar.

[0019] In another preferred embodiment, the liquid culture medium comprises: 20-40 g / L sucrose, 5-10 g / L yeast extract, 5-10 g / L peptone, 0.5-0.8 g / L potassium dihydrogen phosphate, and 0.05-0.08 g / L manganese sulfate.

[0020] In another preferred embodiment, when the low-temperature resistant composite bacterial agent is a liquid low-temperature resistant composite bacterial agent, its preparation method includes the following steps: (1) Activate the Bacillus licheniformis, Bacillus megaterium and Bacillus laterosporus on a solid culture medium; (2) Inoculate the liquid culture medium for fermentation to obtain a liquid low-temperature resistant compound bacterial agent.

[0021] In another preferred embodiment, the activation on a solid culture medium is to activate on a petri dish containing a solid culture medium.

[0022] In another preferred embodiment, step (1), activation on a solid culture medium includes: (a) The three strains were activated individually on solid culture media; (b) Any two of the three bacteria are activated simultaneously on a solid culture medium, and the third bacteria is activated alone; (c) The three bacteria were activated simultaneously on a solid culture medium.

[0023] In another preferred embodiment, in step (2), the fermentation culture of the three bacteria is carried out by inoculating them into the liquid culture medium separately to obtain their respective liquid fermentation broths. The liquid fermentation broths of the three bacteria are mixed to obtain a liquid low-temperature resistant compound bacterial agent.

[0024] In another preferred embodiment, step (2) further includes a step of quantifying the bacterial count of each liquid fermentation broth.

[0025] In another preferred embodiment, the bacterial count is determined by microscopic observation after dilution (microscopic counting).

[0026] In another preferred embodiment, the activation temperature of the low-temperature resistant compound microbial agent is 5℃ to 15℃, preferably 8℃ to 13℃, more preferably 9℃ to 12℃, and most preferably 10℃.

[0027] In another preferred embodiment, the activation time of the low-temperature resistant compound microbial agent is 24 to 48 hours, preferably 28 to 40 hours, and more preferably 30 to 36 hours.

[0028] In another preferred embodiment, the fermentation culture temperature of each strain in the low-temperature resistant compound bacterial agent in the liquid culture medium is 5-15℃, preferably 8℃-13℃, more preferably 9℃-12℃, and most preferably 10℃.

[0029] In another preferred embodiment, the fermentation culture time of each strain in the low-temperature resistant compound bacterial agent in the liquid culture medium is 24 to 48 hours, preferably 28 to 40 hours, and more preferably 30 to 36 hours.

[0030] In another preferred embodiment, the low-temperature resistant compound microbial agent can prepare liquid microbial fertilizer using poultry and livestock urine as a nutrient source in a low-temperature environment. The low temperature refers to 1℃~15℃, preferably 4℃~15℃, more preferably 5℃~15℃, and most preferably 10℃~15℃ (e.g., 10℃~13℃).

[0031] In another preferred embodiment, the low-temperature resistant compound microbial agent has a urea decomposition rate of ≥50% (preferably ≥60%, more preferably ≥70%, most preferably ≥80%) at 5°C, a viable count at the fermentation endpoint of ≥2 billion CFU / mL (preferably ≥2.5 billion CFU / mL, more preferably ≥3 billion CFU / mL, even more preferably ≥4 billion CFU / mL, most preferably ≥5 billion CFU / mL), a total available nitrogen of ≥0.5 g / L (preferably ≥1.0 g / L, more preferably ≥1.5 g / L, most preferably ≥2.0 g / L), and a total nitrogen of ≥5.0 g / L (preferably ≥6.0 g / L, more preferably ≥7.0 g / L, even more preferably ≥8.0 g / L, even more preferably ≥9.0 g / L, most preferably ≥10.0 g / L).

[0032] In another preferred embodiment, the dosage form of the low-temperature resistant compound microbial agent is selected from the group consisting of: liquid preparations and spray-dried powders.

[0033] In a second aspect of the invention, the application of the low-temperature resistant compound microbial agent described in the first aspect of the invention is provided for use in low-temperature environments: (1) Decomposes proteins in poultry and livestock urine; (2) Phosphorus solubilization in poultry and livestock urine; (3) Potassium solubilization in poultry and livestock urine; (4) Degrade uric acid in poultry and livestock urine and fix ammonia nitrogen; (5) Promotes the dissolution and release of organic nitrogen in poultry and livestock urine; (6) Preparing liquid microbial fertilizer using poultry and livestock urine as a nutrient source; and / or (6) Biological control.

[0034] In another preferred embodiment, the poultry and livestock urine is selected from the group consisting of: (1) pure pig urine collected separately; (2) the liquid portion obtained after solid-liquid separation of pig manure from a pig farm; and (3) the liquid portion obtained after solid-liquid separation of wastewater from pig farm cleaning.

[0035] In another preferred embodiment, the poultry urine is pig manure.

[0036] In a third aspect of the present invention, a method for preparing liquid microbial fertilizer is provided, comprising the steps of: (iii) Using the low-temperature resistant compound microbial agent described in the first aspect of the present invention, liquid microbial fertilizer is prepared in a low-temperature environment using poultry and livestock urine as a nutrient source.

[0037] In another preferred embodiment, the method further includes the step of: (i) performing solid-liquid separation on livestock manure from a farm to obtain a liquid portion.

[0038] In another preferred embodiment, the method further includes the step of: (ii) preparing a liquid low-temperature resistant compound bacterial agent.

[0039] In another preferred embodiment, in step (iii), the low-temperature resistant compound microbial agent is mixed with livestock and poultry urine in a certain proportion and then subjected to aerobic fermentation culture. Fermentation is terminated when the number of viable bacteria no longer increases, and liquid microbial fertilizer is obtained.

[0040] In another preferred embodiment, the method includes: (ii) Preparation of liquid low-temperature resistant compound bacterial agent: Bacillus licheniformis, Bacillus megaterium and Bacillus laterosporus are activated on solid culture medium (e.g., on a petri dish containing solid culture medium), and then inoculated into liquid culture medium for fermentation culture to obtain liquid low-temperature resistant compound bacterial agent; (iii) Preparation of liquid microbial fertilizer from poultry and livestock urine: The liquid low-temperature resistant compound microbial agent is mixed with poultry and livestock urine in a certain proportion and then subjected to aerobic fermentation culture. Fermentation is terminated when the number of live bacteria no longer increases, and liquid microbial fertilizer is obtained.

[0041] In another preferred embodiment, in step (ii), the fermentation culture of the three bacteria is carried out by inoculating them into the liquid culture medium separately to obtain their respective liquid fermentation broths. The liquid fermentation broths of the three bacteria are mixed (e.g., mixed in equal volumes) to obtain a liquid low-temperature resistant compound bacterial agent.

[0042] In another preferred embodiment, step (ii) further includes a step of quantifying the bacterial count of the respective liquid fermentation broth.

[0043] In another preferred embodiment, the volume mixing ratio of the low-temperature resistant compound bacterial agent to poultry and livestock urine is 1 to 4:10, preferably 1 to 2:10, and more preferably 1.5:10.

[0044] In another preferred embodiment, the fermentation culture temperature is 5 to 15°C, preferably 10°C to 12°C.

[0045] In another preferred embodiment, the aeration ratio of the fermentation culture is 0.2 to 0.8:1, preferably 0.4:1.

[0046] In another preferred embodiment, the fermentation culture time is ≥48 hours, preferably ≥72 hours, and more preferably ≥84 hours.

[0047] In another preferred embodiment, the fermentation culture pH is 6.0 to 8.0, preferably 6.5 to 7.0.

[0048] In another preferred embodiment, when the fermentation of the liquid microbial fertilizer is terminated, the viable count is ≥2 billion CFU / mL (preferably ≥2.5 billion CFU / mL, more preferably ≥3 billion CFU / mL, even more preferably ≥4 billion CFU / mL, and most preferably ≥5 billion CFU / mL), the available nitrogen is ≥0.5 g / L (preferably ≥1.0 g / L, more preferably ≥1.5 g / L, and most preferably ≥2.0 g / L), and the total nitrogen is ≥5.0 g / L (preferably ≥6.0 g / L, more preferably ≥7.0 g / L, even more preferably ≥8.0 g / L, even more preferably ≥9.0 g / L, and most preferably ≥10.0 g / L).

[0049] In another preferred embodiment, during the fermentation process, the inoculation amount of the low-temperature resistant compound microbial agent is 10% to 40%, preferably 15% to 30%, and more preferably 20% to 25%.

[0050] In a fourth aspect of the invention, a liquid microbial fertilizer is prepared using the preparation method described in the third aspect of the invention.

[0051] In another preferred embodiment, the liquid microbial fertilizer contains ≥2 billion CFU / mL (preferably ≥2.5 billion CFU / mL, more preferably ≥3 billion CFU / mL, even more preferably ≥4 billion CFU / mL, and most preferably ≥5 billion CFU / mL).

[0052] In another preferred embodiment, the liquid microbial fertilizer contains ≥0.5 g / L of available nitrogen (preferably ≥1.0 g / L, more preferably ≥1.5 g / L, and most preferably ≥2.0 g / L).

[0053] In another preferred embodiment, the liquid microbial fertilizer contains a total nitrogen content of ≥5.0 g / L (preferably ≥6.0 g / L, more preferably ≥7.0 g / L, even more preferably ≥8.0 g / L, even more preferably ≥9.0 g / L, and most preferably ≥10.0 g / L).

[0054] In another preferred embodiment, the liquid microbial fertilizer contains ≥2 billion CFU / mL of live bacteria (preferably ≥2.5 billion CFU / mL, more preferably ≥3 billion CFU / mL, even more preferably ≥4 billion CFU / mL, and most preferably ≥5 billion CFU / mL), ≥0.5 g / L of available nitrogen (preferably ≥1.0 g / L, more preferably ≥1.5 g / L, and most preferably ≥2.0 g / L), and ≥5.0 g / L of total nitrogen (preferably ≥6.0 g / L, more preferably ≥7.0 g / L, even more preferably ≥8.0 g / L, even more preferably ≥9.0 g / L, and most preferably ≥10.0 g / L).

[0055] In another preferred embodiment, the liquid microbial fertilizer further includes: a carbon source of 20 g / L to 40 g / L, preferably 25 g / L to 35 g / L, and more preferably 28 g / L to 33 g / L.

[0056] In another preferred embodiment, the carbon source is glucose, sucrose, and / or brown sugar.

[0057] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the invention. Attached Figure Description

[0058] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention. It is obvious that the drawings described below are merely some embodiments of the invention, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0059] Figure 1 The activated Bacillus licheniformis, Bacillus megaterium, and Bacillus laterosporus are shown.

[0060] Figure 2 The fermentation broth of Bacillus licheniformis, Bacillus megaterium, and Bacillus laterosporus is shown.

[0061] Figure 3 The growth process of the low-temperature resistant compound microbial agent in poultry and livestock urine was shown.

[0062] Figure 4 The curves showing the changes in viable bacteria count and OD value during the preparation of microbial fertilizer from poultry and livestock urine are displayed.

[0063] Figure 5 The curves showing the changes in viable bacteria count and OD value during the preparation of microbial fertilizer from poultry and livestock urine are displayed.

[0064] Figure 6 The curves showing the changes in viable bacteria count and OD value during the preparation of microbial fertilizer from poultry and livestock urine are displayed.

[0065] Figure 7 The curves showing the changes in available nitrogen content during the preparation of microbial fertilizer from poultry and livestock urine are displayed. Detailed Implementation

[0066] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, they are provided so that the invention will be thorough and complete, and the concept of the exemplary embodiments will be fully conveyed to those skilled in the art.

[0067] Unless otherwise defined, all terms and phrases used herein include their meanings as they have in the art, unless explicitly stated otherwise or clearly indicated from the context of their use. While any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, specific methods and materials are now described.

[0068] Through extensive and in-depth research, the inventors have unexpectedly discovered a low-temperature resistant compound microbial agent and its applications for the first time. This low-temperature resistant compound microbial agent includes: Bacillus licheniformis (e.g., CGMCC 1.807), Bacillus megaterium (CGMCC 1.217), and Bacillus laterosporus (e.g., CGMCC 1.0864). This low-temperature resistant compound microbial agent can effectively decompose and transform organic matter in poultry and livestock urine using it as a nutrient source at low temperatures (1~15℃), increasing the content of available nitrogen in the urine to obtain liquid microbial fertilizer. The low-temperature resistant compound microbial agent grows well at low temperatures, reaching a quantity of up to 200 million, and can be used as microbial fertilizer. The method of this invention can be implemented at ambient temperatures (low temperatures), saving energy and protecting the environment. Furthermore, the liquid microbial fertilizer produced using this method has a high content of amide nitrogen—available nitrogen (≥2.0 g / L), making it easily absorbed. This technology is particularly suitable for the resource utilization of poultry and livestock manure in high-latitude cold regions (such as Northeast China), achieving efficient urine transformation through low-temperature microbial synergistic metabolism while reducing energy consumption and treatment costs. Based on this, the present invention was completed.

[0069] the term

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0071] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0072] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0073] Low-temperature resistant compound bacterial agent

[0074] This invention provides a low-temperature resistant compound bacterial agent, including Bacillus licheniformis, Bacillus megaterium, and Bacillus laterosporus.

[0075] Preferably, in the low-temperature resistant compound bacterial agent, the ratio of Bacillus licheniformis: Bacillus megaterium: Bacillus laterosporus is (0.25~2):(0.25~2):(0.25~2).

[0076] Preferably, the low-temperature resistant compound bacterial agent further includes: solid culture medium and liquid culture medium.

[0077] In this invention, when the low-temperature resistant compound bacterial agent is a liquid low-temperature resistant compound bacterial agent, its preparation method includes the following steps: (1) Activate the Bacillus licheniformis, Bacillus megaterium and Bacillus laterosporus on a solid culture medium; (2) Inoculate the liquid culture medium for fermentation to obtain a liquid low-temperature resistant compound bacterial agent.

[0078] In a specific embodiment of the present invention, when the low-temperature resistant composite microbial agent is a liquid low-temperature resistant composite microbial agent, its preparation method includes the following steps: (1) The Bacillus licheniformis, Bacillus megaterium and Bacillus laterosporus were activated on solid culture medium respectively; (2) The three activated bacteria from (1) were inoculated into liquid culture medium (e.g., using an inoculation loop) to ferment and culture, and their respective liquid fermentation broths were obtained and the bacterial count was quantified. (3) Mix the liquid fermentation broth of the three bacteria after the bacterial count was quantified in (2) to obtain a liquid low-temperature resistant compound bacterial agent; In step (2), the fermentation time of the three bacteria in the liquid culture medium can be the same or different; In step (3), the liquid fermentation broths of the three bacteria can be mixed in equal volumes, or they can be mixed in non-equal volumes according to the required concentration.

[0079] Bacillus licheniformis

[0080] Bacillus licheniformis is widely distributed in natural environments such as soil. It can secrete a variety of extracellular hydrolases, such as proteases, amylases, and lipases. At the same time, it can also produce a variety of antibacterial active substances and inhibit the growth of pathogenic bacteria through mechanisms such as "biological oxygen depletion," thereby playing an antagonistic role in crop biological control.

[0081] The Bacillus licheniformis types that can be used in this invention include: CICC 10037 and CGMCC 1.807.

[0082] Typically, in one specific embodiment of the present invention, the common CGMCC 1.807 strain is used.

[0083] Typically, in this invention, the Bacillus licheniformis can also be a strain that has been acclimated to low temperatures, such as the CGMCC 1.807 strain that has been acclimated to low temperatures.

[0084] Specifically, the low-temperature acclimatization refers to obtaining single colonies with good growth performance through multiple screenings in a low-temperature environment.

[0085] Specifically, the low-temperature acclimatization refers to the continuous and targeted cultivation and subculturing of microbial strains at temperatures below normal (usually below 20–25°C) to screen out strains that can form stable metabolic activity and growth capacity at lower temperatures.

[0086] For example, the temperature gradient method can be used for low-temperature acclimatization. The temperature gradient method involves gradually lowering the temperature in stages, setting up multiple acclimatization stages of 20℃→15℃→10℃→5℃, to facilitate the gradual adaptation of microorganisms and the screening of low-temperature resistant strains.

[0087] Bacillus megaterium

[0088] Bacillus megaterium is widely distributed in soil and plant-related environments. It participates in organophosphate mineralization by secreting phosphatases / phytases, thereby increasing available phosphorus in the soil, making it suitable for the preparation of microbial fertilizers.

[0089] Bacillus megaterium has a strong ability to solubilize phosphorus and potassium, and can extract phosphorus and potassium from biomass (for example, by releasing phosphorus and potassium from phospholipids and DNA into a free state, which is beneficial for crop absorption), and can also facilitate the extraction of calcium sulfate, degrade uric acid and fix ammonia nitrogen.

[0090] The types of Bacillus megaterium that can be used in this invention include: CICC 21580, CICC 20665, and CGMCC1.217.

[0091] Typically, in one specific embodiment of the present invention, the common CGMCC 1.217 strain is used.

[0092] Typically, in this invention, the Bacillus megaterium can also be a strain that has been acclimatized to low temperatures, such as the CGMCC 1.217 strain that has been acclimatized to low temperatures.

[0093] Specifically, the low-temperature acclimatization refers to obtaining single colonies with good growth performance through multiple screenings in a low-temperature environment.

[0094] Specifically, the low-temperature acclimatization refers to the continuous and targeted cultivation and subculturing of microbial strains at temperatures below normal (usually below 20–25°C) to screen out strains that can form stable metabolic activity and growth capacity at lower temperatures.

[0095] For example, the temperature gradient method can be used for low-temperature acclimatization. The temperature gradient method involves gradually lowering the temperature in stages, setting up multiple acclimatization stages of 20℃→15℃→10℃→5℃, to facilitate the gradual adaptation of microorganisms and the screening of low-temperature resistant strains.

[0096] Lateral Bacillus

[0097] Bacillus laterosporus has a wide ecological distribution, found in soil, freshwater, and seawater environments. In agricultural applications, it is often used as a core strain in biofertilizers / microbial agents to improve rhizosphere microecology, promote root growth, and suppress diseases. It can secrete proteases, lipases, amylases, cellulases, etc., to decompose uneaten feed, feces, and organic debris.

[0098] Bacillus laterosporus can produce keratinase, which promotes the dissolution and release of organic nitrogen, and also has a biocontrol effect.

[0099] The types of Bacillus laterosporus that can be used in this invention include: CICC 21185 and CGMCC 1.0864.

[0100] Typically, in one specific embodiment of the present invention, the common CGMCC 1.0864 strain is used.

[0101] Typically, in this invention, the Bacillus laterosporus can also be a strain that has been acclimated to low temperatures, such as the CGMCC 1.0864 strain that has been acclimated to low temperatures.

[0102] Specifically, the low-temperature acclimatization refers to obtaining single colonies with good growth performance through multiple screenings in a low-temperature environment.

[0103] Specifically, the low-temperature acclimatization refers to the continuous and targeted cultivation and subculturing of microbial strains at temperatures below normal (usually below 20–25°C) to screen out strains that can form stable metabolic activity and growth capacity at lower temperatures.

[0104] For example, the temperature gradient method can be used for low-temperature acclimatization. The temperature gradient method involves gradually lowering the temperature in stages, setting up multiple acclimatization stages of 20℃→15℃→10℃→5℃, to facilitate the gradual adaptation of microorganisms and the screening of low-temperature resistant strains.

[0105] poultry and livestock urine

[0106] In this invention, "animal urine" includes: (1) pure animal urine collected separately; (2) the liquid portion obtained after solid-liquid separation of animal manure from a farm; (3) the liquid portion obtained after solid-liquid separation of wastewater from farm cleaning; and (4) the liquid portion obtained after solid-liquid separation of human excrement, such as human public toilet excrement.

[0107] The livestock mentioned include pigs, cattle, sheep, horses, dogs, donkeys, chickens, ducks, geese, etc.

[0108] The main advantages of this invention include: (1) Low-temperature efficiency: It still maintains ≥80% of the urea decomposition rate at 5℃, the viable count at the fermentation endpoint is ≥5 billion CFU / ml, and the total available nitrogen is ≥2g / L (see Figure 7The total nitrogen is ≥10 g / L, meeting the standard of "Agricultural Microbial Agents" (GB20287-2016).

[0109] (2) Resource benefits: It operates directly using ambient temperature, reducing energy consumption by 90% compared to heating processes; the proportion of fast-acting nutrients in liquid microbial fertilizer increases by 35%, and it can replace 10% of chemical fertilizers for cold-region crops.

[0110] (3) Environmental safety: The compound microbial agent completely inhibits pathogens such as Escherichia coli (inhibition rate > 99%).

[0111] The present invention will be further explained below with reference to specific embodiments.

[0112] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Guide* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the manufacturer's recommendations (e.g., product instructions). Unless otherwise stated, percentages and parts are by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available or can be prepared according to literature methods.

[0113] Materials and Methods

[0114] 1. Composition of low-temperature resistant compound microbial agent

[0115] Strain compatibility: Composed of Bacillus licheniformis, Bacillus megaterium, and Bacillus laterosporus. Synergistic functional mechanism: (1) Bacillus licheniformis: secretes low-temperature protease to break down large protein molecules in urine into small peptides; (2) Bacillus megater: It has strong phosphorus and potassium solubilizing ability, degrades uric acid and fixes ammonia nitrogen; (3) Bacillus laterosporus: produces keratinase, which promotes the dissolution and release of organic nitrogen.

[0116] 2. Preparation method of liquid microbial fertilizer

[0117] (1) During the bacterial culture stage, manganese ions can improve the tolerance of Bacillus subtilis; (2) During the low-temperature activation stage, activation and expansion are carried out at 5-15℃, breaking through the limitations of mesophilic bacteria; (3) Urine conversion stage: The optimal volume ratio of bacterial agent to urine is 1-2:10 to ensure carbon-nitrogen balance and bacterial density; the fermentation temperature is 5-15℃ so that the fermentation process does not require heating, reducing energy consumption; the aeration ratio is 0.2-0.8:1, which reduces the energy consumption of air compressor under low aerobic conditions; the pH is controlled at 6.0-8.0 to maintain enzyme activity and bacterial stability. The peak number of viable bacteria reaches 5 billion CFU / mL after 72 hours of fermentation, and the available nitrogen is increased.

[0118] Example 1: Activation of bacterial strains

[0119] Prepare the solid culture medium: 20 g / L sucrose, 5 g / L yeast extract, 5 g / L peptone, 0.5 g / L potassium dihydrogen phosphate, and 20 g / L agar. Sterilize at 121°C for 30 minutes. After cooling to about 60°C, pour the above culture medium into Petri dishes and let it cool to room temperature before use.

[0120] The glycerol tube cultures, stored at low temperature, were thawed to room temperature. Using an inoculation needle, the bacterial suspension was drawn from the glycerol tubes and streaked onto a petri dish, with each of the three cultures streaked separately. The dish was then placed in an incubator at 10°C and incubated for 48 hours to obtain activated cultures. Figure 1 ).

[0121] Example 2: Liquid culture with low-temperature resistant compound bacterial agent

[0122] Prepare the liquid culture medium: 40 g / L sucrose, 10 g / L yeast extract, 10 g / L peptone, 0.8 g / L potassium dihydrogen phosphate, and 0.08 g / L manganese sulfate. Sterilize at 121°C for 30 minutes, and allow to cool to room temperature before use.

[0123] Two loops of each activated bacterial strain were scraped using an inoculation loop and inoculated into liquid culture medium. The culture temperature was 15℃, and the culture time was 48 hours to obtain liquid fermentation broth. Microscopic counting was performed on the liquid fermentation broths of the three strains. Because the exact weight of the colonies scraped from each inoculation loop could not be determined, the effective viable cell count of each strain varied at the end of the culture. Then, 200 ml of each of the three bacterial agents were mixed in a sterile room to obtain 600 ml of a mixed solution, yielding a liquid low-temperature resistant compound bacterial agent. (See below) Figure 2 .

[0124] Example 3: Preparation of liquid microbial fertilizer by fermentation of poultry and livestock urine

[0125] Repeat Example 2, using a liquid low-temperature resistant compound bacterial agent (Bacillus licheniformis bacterial count 2.0 × 10⁻⁶). 8 CFU / mL, Bacillus megaterium count 1.6 × 10⁻⁶ 8 CFU / mL, Bacillus laterosporus count 2.6 × 10⁻⁶ 8 CFU / mL, total bacterial count 2.07×10 8600 ml of CFU / mL pig manure was mixed with 3000 ml of pig manure at a volume ratio of 2:10. The mixture was fermented in a fermenter at 12°C with an air-to-gas ratio of 0.4:1 for 72 hours and a pH of 6.5.

[0126] A 5-liter fermenter was filled with 3 liters of pig manure and urine, 90 grams of sucrose, and an inoculation rate of 20%, which was achieved by adding 600 ml of a mixed liquid low-temperature resistant compound microbial agent. The total bacterial count after inoculation was 0.345 × 10⁻⁶. 8 The fermenter temperature was automatically controlled at 12℃, the aeration ratio was 0.4:1 (1.2 L / min), and the pH was controlled at 6.5 using citric acid solution (30% mass concentration).

[0127] As fermentation progresses, the urine changes from clear to cloudy, and the turbidity increases. Figure 3 This indicates that the low-temperature resistant compound bacterial agent grew well in urine, with both OD600 and viable bacterial count increasing. Figure 4 ).

[0128] Example 4: Preparation of liquid microbial fertilizer by fermentation of poultry and livestock urine

[0129] Repeat Example 2, using a liquid low-temperature resistant compound bacterial agent (Bacillus licheniformis count 1.0 × 10⁻⁶). 8 CFU / mL, Bacillus megaterium count 1.2 × 10⁻⁶ 8 CFU / mL, Bacillus laterosporus count 1.1×10⁻⁶ 8 CFU / mL, total bacterial count 1.1×10⁻⁶ 8 600 ml of CFU / mL solution was mixed with 3000 ml of pig manure and urine at a volume ratio of 2:10. The mixture was fermented in a fermenter at 12°C with an air-to-gas ratio of 0.4:1 for 72 hours and a pH of 6.5.

[0130] A 5-liter fermenter was filled with 3 liters of pig manure and urine, 90 grams of sucrose, and an inoculation rate of 20%, which was achieved by adding 600 ml of a mixed liquid low-temperature resistant compound microbial agent. The total bacterial count after inoculation was 0.18 × 10⁻⁶. 8 The fermenter temperature was automatically controlled at 12℃, the aeration ratio was 0.4:1 (1.2 L / min), and the pH was controlled at 6.5 using citric acid solution (30% mass concentration).

[0131] As fermentation progressed, the urine changed from clear to cloudy, and the turbidity increased, indicating that even with a lower initial viable cell count than in Example 3, the low-temperature resistant compound bacterial agent grew well in the urine, and both OD600 and viable cell count increased. Figure 5 ).

[0132] Example 5: Preparation of liquid microbial fertilizer by fermentation of poultry and livestock urine

[0133] Repeat Example 2, using a liquid low-temperature resistant compound bacterial agent (Bacillus licheniformis bacterial count 4.0 × 10⁻⁶). 8 CFU / mL, Bacillus megaterium count 4.2 × 10⁻⁶ 8 CFU / mL, Bacillus laterosporus count 3.95 × 10⁻⁶ 8 CFU / mL, total bacterial count 4.05×10 8 600 ml of CFU / mL solution was mixed with 3000 ml of pig manure and urine at a volume ratio of 2:10. The mixture was fermented in a fermenter at 12°C with an air-to-gas ratio of 0.4:1 for 72 hours and a pH of 6.5.

[0134] A 5-liter fermenter was filled with 3 liters of pig manure and urine, 90 grams of sucrose, and an inoculation rate of 20%, which was achieved by adding 600 ml of a mixed liquid low-temperature resistant compound microbial agent. The total bacterial count after inoculation was 0.675 × 10⁻⁶. 8 The fermenter temperature was automatically controlled at 12℃, the aeration ratio was 0.4:1 (1.2 L / min), and the pH was controlled at 6.5 using citric acid solution (30% mass concentration).

[0135] As fermentation progressed, the urine changed from clear to cloudy, and the turbidity increased, indicating that even with a higher initial viable cell count than in Example 3, the low-temperature resistant compound bacterial agent grew well in the urine, and both OD600 and viable cell count increased. Figure 6 ).

[0136] Example 6: Detection of fast-acting nitrogen in poultry and livestock urine (before and after comparison)

[0137] As the number of microorganisms in the fermentation broth increases, they secrete a large amount of related enzymes, which decompose substances such as urea, protein, and uric acid in the urine, thereby increasing the available nitrogen content in the bacterial solution. After 72 hours of fermentation, the available nitrogen content is ≥2g / L. Figure 7 Available nitrogen is ammonium nitrogen (NH4+). + -N) and nitrate nitrogen (NO3) - The total nitrogen content includes ammonium nitrogen (urea) and organic nitrogen (protein). Ammonium nitrogen is determined using the method specified in GB / T 3595-2000. Nitrate nitrogen is determined using the method specified in GB / T 3597-2002. The available nitrogen value is obtained by adding the measured values ​​of ammonium nitrogen and nitrate nitrogen.

[0138] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the appended claims.

Claims

1. A low-temperature resistant compound bacterial agent, characterized in that, include: Bacillus licheniformis, Bacillus megaterium, and Bacillus laterosporus.

2. The low-temperature resistant compound bacterial agent according to claim 1, characterized in that, The ratio of Bacillus licheniformis: Bacillus megaterium: Bacillus laterosporus count was (0.25~2): (0.25~2): (0.25~2).

3. The low-temperature resistant compound bacterial agent according to claim 1, characterized in that, The Bacillus licheniformis is Bacillus licheniformis CGMCC 1.807, the Bacillus megaterium is Bacillus megaterium CGMCC 1.217, and / or the Bacillus laterosporus is Bacillus laterosporus CGMCC 1.0864.

4. The low-temperature resistant compound bacterial agent according to claim 1, characterized in that, The low-temperature resistant compound bacterial agent also includes: solid culture medium and liquid culture medium.

5. The low-temperature resistant compound bacterial agent according to claim 4, characterized in that, The solid culture medium consists of: 20-40 g / L sucrose, 5-10 g / L yeast extract, 5-10 g / L peptone, 0.5-0.8 g / L potassium dihydrogen phosphate, and 20-30 g / L agar.

6. The low-temperature resistant compound bacterial agent according to claim 4, characterized in that, The liquid culture medium consists of: 20-40 g / L sucrose, 5-10 g / L yeast extract, 5-10 g / L peptone, 0.5-0.8 g / L potassium dihydrogen phosphate, and 0.05-0.08 g / L manganese sulfate.

7. The low-temperature resistant compound bacterial agent according to claim 1, characterized in that, When the low-temperature resistant compound microbial agent is a liquid low-temperature resistant compound microbial agent, its preparation method includes the following steps: (1) Activate the Bacillus licheniformis, Bacillus megaterium and Bacillus laterosporus on a solid culture medium; (2) Inoculate the liquid culture medium for fermentation to obtain a liquid low-temperature resistant compound bacterial agent.

8. The application of the low-temperature resistant compound microbial agent according to any one of claims 1-7, characterized in that, For use in low-temperature environments: (1) Decomposes proteins in poultry and livestock urine; (2) Phosphorus solubilization in poultry and livestock urine; (3) Potassium solubilization in poultry and livestock urine; (4) Degrade uric acid in poultry and livestock urine and fix ammonia nitrogen; (5) Promotes the dissolution and release of organic nitrogen in poultry and livestock urine; (6) Preparing liquid microbial fertilizer using poultry and livestock urine as a nutrient source; and / or (6) Biological control.

9. A method for preparing a liquid microbial fertilizer, characterized in that, Including the following steps: (iii) Using any one of the low-temperature resistant compound microbial agents described in claims 1-7, liquid microbial fertilizer is prepared using poultry and livestock urine as a nutrient source in a low-temperature environment.

10. The preparation method according to claim 9, characterized in that, In step (iii), the low-temperature resistant compound microbial agent is mixed with livestock and poultry urine in a certain proportion and then subjected to aerobic fermentation culture. Fermentation is terminated when the number of live bacteria no longer increases, and liquid microbial fertilizer is obtained.

11. The preparation method according to claim 9, characterized in that, The method includes: (ii) Preparation of liquid low-temperature resistant compound bacterial agent: Bacillus licheniformis, Bacillus megaterium and Bacillus laterosporus are activated on solid culture medium and then inoculated into liquid culture medium for fermentation culture to obtain liquid low-temperature resistant compound bacterial agent; (iii) Preparation of liquid microbial fertilizer from poultry and livestock urine: The liquid low-temperature resistant compound microbial agent is mixed with poultry and livestock urine in a certain proportion and then subjected to aerobic fermentation culture. Fermentation is terminated when the number of live bacteria no longer increases, and liquid microbial fertilizer is obtained.

12. A liquid microbial fertilizer, characterized in that, Prepared using the preparation method described in any one of claims 9-11.

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