A salt-tolerant Bacillus strain and its application

Abstract

This invention relates to the field of biotechnology, and particularly to a salt-tolerant Bacillus strain and its applications. The invention applies a salt-tolerant Bacillus strain to the composting of high-salt organic waste, and provides an industrially feasible process and key parameter window. The salt-tolerant Bacillus strain is a microorganism that can maintain activity and promote rapid hydrolysis of starch and protein under high-salt / high-osmotic / low-water-activity conditions, along with a matching composting process, to achieve rapid, stable composting and low-odor treatment of tobacco waste, providing technical support for circular agriculture in tobacco-growing areas.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a salt-tolerant Bacillus strain and its applications. Background Technology

[0002] Composting is an important way to utilize organic solid waste as a resource. Through the hydrolysis, oxidation, and humification of organic matter by aerobic microorganisms, food waste, agricultural byproducts, and livestock manure can be transformed into products that can be used for soil improvement and fertilization. However, high-salt organic waste generally has characteristics such as high osmotic pressure, high ionic strength, and low water activity. Conventional composting microbial communities are easily inhibited during the start-up and high-temperature stages, resulting in slow temperature rise of the compost pile, insufficient effective high-temperature period, and incomplete degradation of easily decomposable components such as protein and starch. This leads to significant ammonia and putrid odors, prolonged composting period, and decreased product stability and safety. Microorganisms with protease and amylase activity can rapidly initiate composting and control odors in starch / protein-containing organic waste.

[0003] Existing composting enhancement technologies mostly employ mixed microbial agents or thermophilic agents to improve the composting temperature rise rate and organic matter degradation efficiency. For example, some publicly available methods combine various thermophilic bacteria (including some Bacillus halotolerans strains) with other microorganisms for composting fermentation to improve initial microbial activity and shorten primary fermentation time; others provide specific salt-tolerant Bacillus halotolerans strains and their preparations, primarily targeting salt-tolerant applications. While these technologies can improve general composting efficiency or obtain salt-tolerant strains, they do not provide a complete and rapid composting method specifically for organic waste scenarios characterized by "high salt / low water activity + starch / protein," including reproducible salinity / water activity constraints, inoculum size, ventilation and turning windows, and composting criteria.

[0004] The existing technology has the following main drawbacks in the high-salt organic waste composting scenario: (1) Salt stress inhibition leads to slow temperature rise, making it difficult to quickly enter and maintain the high-temperature composting stage of ≥55℃; (2) Insufficient protein hydrolysis leads to prominent ammonia and putrid odors, affecting the working environment and causing nitrogen loss; (3) The composting cycle is long and the batches fluctuate greatly, the degree of humification is insufficient, and the risk of plant toxicity in the finished product is high. The fundamental reason is that conventional composting microorganisms are not adaptable to high-salt / high-osmotic environments and lack the synergistic design of C / N, moisture content and ventilation windows for high-salt materials.

[0005] Tobacco, as an important economic crop in my country, generates a large amount of organic waste during its production. Specifically, during the field management stage, topping and pruning produce a large amount of tender tissues such as buds and branches. These wastes are rich in protein (15%–25%) and starch, making them typical high-protein / high-starch organic waste. Simultaneously, during the harvesting and initial curing stages, the removed lower leaves, diseased leaves, damaged leaves, and green leaves resulting from improper curing also contain high levels of starch and sugars. Furthermore, the long-term use of chemical fertilizers and pesticides during tobacco cultivation has led to soil salinization in some tobacco fields, subjecting these wastes to a combined stress of high salt and high osmotic pressure during composting. Currently, the disposal of these wastes in tobacco-growing areas often involves indiscriminate dumping or simple landfilling, which not only wastes resources but also produces large amounts of ammonia due to incomplete protein degradation, causing foul odors and posing risks of environmental pollution and disease transmission due to the difficulty in degrading nicotine residues.

[0006] In the field of tobacco production waste treatment, existing technologies mainly suffer from the following problems: First, for lignocellulosic waste such as tobacco stalks, conventional straw composting agents or simple composting are mostly used, lacking specialized treatment technologies for high-protein / high-starch waste such as tobacco buds and tobacco forks; Second, for tobacco buds and forks produced by topping and pruning, tobacco-growing areas generally use random field dumping or simple landfilling, relying on natural decomposition, which has a long treatment cycle (usually 3-6 months), insufficient protein degradation leading to severe odor, and nicotine residues are difficult to effectively degrade; Third, a few studies have attempted to mix tobacco waste with other livestock and poultry manure for composting, but no specialized microbial agents and supporting processes have been developed for the high salt (partially saline tobacco fields), high protein, and high starch characteristics of tobacco waste, and the problems of slow composting start-up and unstable composting quality remain prominent.

[0007] Therefore, to achieve high-value composting utilization of tobacco production waste (especially tobacco buds and branches produced by topping and pruning, as well as low-grade tobacco leaves produced during the harvesting stage), it is urgent to develop a microorganism and supporting composting process that can maintain activity and promote rapid hydrolysis of starch and protein under high salinity / high osmotic pressure / low water activity conditions. This would enable rapid, stable decomposition and low-odor treatment of tobacco waste, providing technical support for circular agriculture in tobacco-growing areas. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a salt-tolerant Bacillus strain and its application in the composting of high-salt organic waste, specifically including providing an industrially feasible process and key parameter window. The described salt-tolerant Bacillus strain is a microorganism capable of maintaining activity and promoting rapid hydrolysis of starch and protein under high-salt / high-osmotic / low-water-activity conditions, along with a corresponding composting process, to achieve rapid, stable composting and low-odor treatment of tobacco waste, providing technical support for circular agriculture in tobacco-growing areas.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a salt-tolerant Bacillus halotolerans, with accession number CGMCC No. 36428.

[0011] The present invention also provides a microbial agent comprising the above-mentioned salt-tolerant Bacillus.

[0012] In some specific embodiments of the present invention, the above-mentioned microbial agent is a liquid microbial agent; or a powder or granule obtained by carrier adsorption and / or low-temperature drying.

[0013] The present invention also provides any of the following applications in organic waste composting:

[0014] (i) the above-mentioned salt-tolerant Bacillus species;

[0015] (ii) The above-mentioned microbial agents.

[0016] In some specific embodiments of the present invention, the organic waste used in the above applications includes high-salt organic waste or high-osmotic organic waste.

[0017] In some specific embodiments of the present invention, the organic waste described above has at least one of the following properties:

[0018] (i) The mass fraction of NaCl is 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5% or 6%;

[0019] (ii) The electrical conductivity EC is 4 mS / cm, 6 mS / cm, 8 mS / cm, 10 mS / cm, 12 mS / cm, 14 mS / cm, 16 mS / cm, 18 mS / cm or 20 mS / cm;

[0020] (iii) The water activity a_w is 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97 or 0.98.

[0021] The present invention also provides a composting method comprising composting based on any of the following:

[0022] (i) the above-mentioned salt-tolerant Bacillus species;

[0023] (ii) The above-mentioned microbial agents.

[0024] In some specific embodiments of the present invention, the above composting method includes the following steps:

[0025] The composting raw materials are mixed with acceptable adjuvants or additives, the C / N ratio is adjusted to 20-35, the moisture content is adjusted to 50%-65%, and the pH is adjusted to 6.5-9.0 to obtain compost material. The adjuvants or additives include a leavening agent.

[0026] The salt-tolerant Bacillus or the inoculant is inoculated into the compost material to achieve a viable bacterial count of 10. 5 ~10 9 CFU / g, after decomposition and post-fermentation, compost products are obtained.

[0027] In some specific embodiments of the present invention, the above composting method includes the following steps:

[0028] (1) Select high-salt organic waste or high-osmotic organic waste as composting raw materials;

[0029] (2) Add a leavening agent and adjust the C / N ratio to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35; adjust the moisture content to 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65%; adjust the pH to 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0;

[0030] (3) Culture halophilic Bacillus to obtain bacterial culture;

[0031] (4) Inoculate the compost material with the microbial agent to achieve a final viable count of 10 in the compost pile. 5 CFU / g, 10 6 CFU / g, 10 7 CFU / g, 10 8 CFU / g or 10 9 CFU / g;

[0032] (5) Ferment under aerobic conditions by turning or aeration, maintaining an effective high temperature period until the compost is fully decomposed;

[0033] (6) After the composting is complete, it is further fermented and screened to obtain compost products.

[0034] In some specific embodiments of the present invention, the above composting method is as follows:

[0035] The composting raw materials are high-salt organic waste or high-osmotic organic waste;

[0036] The high-salt organic waste or high-osmotic organic waste includes high-salt kitchen waste and / or tobacco waste;

[0037] The acceptable excipients or additives also include at least one of zeolite, bentonite, and humic acid.

[0038] The leavening agent includes at least one of straw, rice husk, sawdust, and biochar;

[0039] The composting process includes fermentation and composting using methods such as turning over the pile or aeration.

[0040] In some specific embodiments of the present invention, the above composting method is as follows:

[0041] The amount of the leavening agent added is 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the wet basis mass of the composting raw material;

[0042] The inoculation includes inoculating 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.9%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5% or 6% of the wet substrate weight of the salt-tolerant Bacillus or the inoculum.

[0043] The frequency of turning over the pile is once every 2 days, every 3 days, every 4 days, or every 5 days;

[0044] The aeration rate is 0.1 L / (min·kg wet material), 0.2 L / (min·kg wet material), 0.3 L / (min·kg wet material), 0.4 L / (min·kg wet material) or 0.5 L / (min·kg wet material);

[0045] The composting temperature is 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃ or 70℃, and the time is 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days or 25 days.

[0046] In some specific embodiments of the present invention, the conditions for completion of composting in the above-described composting method are at least two of the following:

[0047] (a) GI ≥ 80%;

[0048] (b) The reactor temperature is close to the ambient temperature and remains stable for more than 48 hours;

[0049] (c) The ratio of ammonium nitrogen to nitrate nitrogen tends to stabilize;

[0050] (d) Respiratory activity decreased significantly.

[0051] The present invention has the following beneficial effects:

[0052] Compared to conventional composting, this invention utilizes the salt tolerance and hydrolytic metabolism of halophilic Bacillus, which can maintain high activity under high salt / high osmotic conditions. As a result, the compost pile starts up faster, heats up more rapidly, and forms a more stable effective high-temperature period. Under the rapid hydrolysis and mineralization of easily perishable substrates such as protein and starch, ammonia and putrid odors can be reduced, nitrogen loss can be reduced, and the humification process can be accelerated, so that maturity indicators such as GI can reach the target earlier. This shortens the maturity cycle and improves the stability and safety of compost products.

[0053] Biological Preservation Instructions

[0054] Biological material: BHA1, taxonomically named Bacillus halotolerans, was deposited on October 30, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number: CGMCC No. 36428. Attached Figure Description

[0055] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below.

[0056] Figure 1 A flowchart illustrating a method for rapid composting and maturation of high-salt organic waste. Detailed Implementation

[0057] This invention discloses a salt-tolerant Bacillus strain and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0058] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0059] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0060] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0061] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0062] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0063] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0064] The technical terms and explanations involved are as follows:

[0065] High-salt organic waste: Organic solid waste with a high content of soluble salts, usually characterized by a NaCl mass fraction ≥ 0.5% or an electrical conductivity (EC) ≥ 4 mS / cm;

[0066] High osmotic pressure / low water activity: refers to an environment with high osmotic pressure and low water activity a_w (e.g., a_w 0.85~0.98), which will inhibit the metabolism and reproduction of most conventional composting microorganisms;

[0067] C / N: Carbon-nitrogen ratio, the mass ratio of organic carbon to total nitrogen in composting raw materials, with a suitable range of 20-35;

[0068] Leavening agents: used to improve the porosity and air permeability of structural materials, such as straw, sawdust, rice husks, biochar, etc.

[0069] Composting: The process by which compost transforms degradable organic matter into stable humus and achieves safe utilization by plants. It is often evaluated using criteria such as GI, ammonia nitrogen, and respiration activity.

[0070] Post-fermentation: After the high-temperature period of composting ends, the slow degradation and humification process takes place under normal or medium temperature conditions. Easily degradable organic matter is further decomposed, the structure of humus tends to stabilize, the microbial community evolves into a mature microbial community, the adaptability of compost plants continues to improve, and finally reaches a state of composting that can be safely applied.

[0071] GI: Germination Index, used to characterize the toxicity of compost plants and the degree of decomposition. A GI ≥ 80% is often used as a reference threshold for decomposition.

[0072] EC: Electrical Conductivity, which reflects the soluble salt content and ionic strength, and is used to characterize salt changes in high-salt environments and during the composting process;

[0073] L / (min·kg wet material): The aeration rate per minute per kilogram of wet material, expressed in liters.

[0074] An industrially feasible process and key parameter window of the present invention is shown in the form of a black and white block diagram, see [link / reference]. Figure 1 .

[0075] In practice, a strain of halophilic Bacillus is used in the composting of high-salt organic waste to promote rapid decomposition, including the following steps:

[0076] (1) Raw material selection: High-salt / high-osmotic organic waste is selected as compost raw material;

[0077] (2) Ingredient control: Add leavening agent and adjust C / N ratio to 20~35, moisture content to 50%~65%, and pH to 6.5~9.0;

[0078] (3) Preparation of bacterial culture: Obtain bacterial culture by culturing salt-tolerant Bacillus;

[0079] (4) Inoculation and composting: Inoculate the composting material with the microbial agent so that the final viable count of the compost pile reaches 10^5~10^8 CFU / g;

[0080] (5) Aerobic fermentation: Fermentation is carried out under aerobic conditions by turning or aeration, maintaining an effective high temperature period until the compost is fully decomposed;

[0081] (6) Post-fermentation and finished product: After fermentation, post-fermentation is carried out and the compost is screened to obtain compost products.

[0082] In one example, the high-salt organic waste meets at least one of the following criteria:

[0083] The mass fraction of NaCl is 0.5%~6%;

[0084] The electrical conductivity EC is 4~20 mS / cm;

[0085] The water activity a_w is 0.85~0.98.

[0086] In one example, the leavening agent in step (2) is selected from straw, rice husk, sawdust, biochar or a combination thereof, and the amount of leavening agent added is 5% to 40% of the wet basis mass of the compost raw material.

[0087] In one example, salt acclimatization in step (3) involves culturing the strain in a medium containing 0.5% to 4% NaCl for 12 to 36 hours to improve its adaptability to high-salt composting environments.

[0088] In one example, the inoculum amount in step (4) is 0.1% to 5% (w / w, on a wet basis) or the final viable count of the pile reaches 10. 5 ~10 9 CFU / g.

[0089] In one example, the turning frequency in step (5) is once every 2 to 5 days, or the aeration rate is 0.1 to 0.5 L / (min·kg wet material).

[0090] In one example, in step (5), the pile temperature reaches 55~70℃ and is maintained for 3~7 days as an effective high-temperature period, with a total fermentation time of 10~25 days.

[0091] In one example, zeolite, bentonite, humic acid or a combination thereof may be added in step (2) to reduce ammonia odor and improve composting stability.

[0092] In one example, the maturity determination in step (6) includes meeting at least two of the following: GI ≥ 80%; pile temperature close to ambient temperature and stable for more than 48 hours; NH4 + / NO3 - The condition tends to stabilize; respiratory activity decreases significantly.

[0093] In one example, the above method ensures rapid entry into and maintenance of an effective high-temperature composting stage through coordinated control of "ingredient window (C / N, moisture content, leavening agent) + inoculum amount + ventilation and turning / aeration".

[0094] In one example, the detailed steps include:

[0095] S1 Raw Material Selection and Judgment: High-salt / high-osmotic organic waste is selected as the composting material. The waste can be high-salt kitchen waste, pickling and processing byproducts, seafood processing waste, salt-containing starch residue / protein residue, etc. Preferably, it meets at least one of the following criteria: NaCl mass fraction 0.5%~6%; or EC 4~20 mS / cm; or water activity a_w 0.85~0.98.

[0096] S2 Pretreatment and Batching: The raw materials are crushed / mixed, and a leavening agent is added to improve porosity and air permeability; the C / N ratio is adjusted to 20-35, the moisture content to 50%-65%, and the pH to 6.5-9.0. Leavening agents can be straw, rice husks, sawdust, biochar, or combinations thereof; zeolite or other adsorbent materials can be added to reduce ammonia odor and buffer salt impact.

[0097] S3 Inoculum Preparation: Using tobacco leaf microorganisms as the material for isolation experiments, a salt-tolerant Bacillus strain was screened using protease-producing plates. The salt-tolerant Bacillus was then subjected to salt acclimation in tryptic soybean broth (TSB) medium with sequential additions of 0.5%, 0.75%, 1%, 1.5%, 2%, and 4% NaCl to improve its initial adaptation in high-salt composting. The obtained salt-tolerant Bacillus was deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 36428. After activation with TSB medium, a seed culture was prepared from the salt-tolerant Bacillus, and then expanded to obtain a highly active bacterial solution or spore suspension. The inoculum can be prepared as a liquid inoculum or as a powder / granule through carrier adsorption / low-temperature drying. The formulation for the protease-producing plate: 10 g / L tryptone, 3 g / L beef extract, 5 g / L sodium chloride, 15 g / L skim milk powder, deionized water, and 18 g / L agar powder. The TSB medium formula is: 17 g casein peptone, 3 g soybean peptone, 5 g sodium chloride, 2.5 g glucose and 2.5 g dipotassium hydrogen phosphate. The pH is adjusted to 7.3±0.2 before sterilization.

[0098] S4 Inoculation and Pile Building: Inoculate the material with microbial agents at the pile building or turning point to achieve a final viable count of 10⁻⁶ in the pile. 5 ~10 9 CFU / g (on a wet basis). After inoculation, mix thoroughly, with a pile height of 0.8~1.8 m and a width of 1.5~3.5 m, ensuring continuous ventilation channels.

[0099] S5 Aerobic composting fermentation control: Maintain an aerobic state by turning the compost pile or by forced aeration. The preferred frequency of turning the compost pile is once every 2-5 days, or the aeration rate is 0.1-0.5 L / (min·kg wet material). Maintain the moisture content at 50%-65%, and ideally, maintain an effective high-temperature period of 55-70℃ for 3-7 days; the total fermentation time is 10-25 days or until the maturity criteria are met.

[0100] S6 Maturity Assessment and Post-fermentation: During fermentation, temperature, moisture content, EC, pH, ammonia (NH3), and changes in ammonium nitrogen / nitrate nitrogen are monitored. The degree of maturity is comprehensively assessed using GI and respiration activity (CO2 release rate). After reaching maturity, post-fermentation is carried out for 7-20 days, and the compost product is obtained by screening.

[0101] In practice, salt-tolerant Bacillus is selected as the core functional bacteria. After domestication, it is made to still have the ability to produce protease and to start and maintain metabolism in a high-salt / high-osmotic / low-water-activity composting environment.

[0102] In practice, a reproducible decomposition endpoint is achieved by using a combination of criteria such as GI, ammonia nitrogen, and respiratory activity.

[0103] In one example, the composting raw material is organic waste mainly containing starch and / or protein, selected from kitchen waste, soybean residue / distillers' grains, by-products of salt-containing grain processing, by-products of salt-containing aquatic product processing, tobacco waste from saline tobacco fields, or combinations thereof.

[0104] In one example, the Bacillus halotolerans produces proteases to facilitate the rapid hydrolysis of proteins and starches in compost.

[0105] In one example, the bacterial agent is a liquid bacterial agent or a powder / granule obtained by adsorption on a carrier and low-temperature drying, which can be rehydrated before inoculation.

[0106] In one example, the liquid bacterial agent can be replaced with a carrier-adsorbed powder / granule or microcapsule formulation.

[0107] In one example, the drying method may be low-temperature vacuum drying, fluidized bed drying, etc., and the seeding should be rehydrated before use.

[0108] In one example, the turning method can be replaced by static forced aeration, intermittent aeration, or a combination of turning and aeration.

[0109] In one example, the aeration rate and turning frequency can be adjusted within the aforementioned window.

[0110] In one example, the leavening agent can be replaced or combined among straw / rice husk / wood chips / biochar; zeolite, bentonite and other adsorbent materials can be added to further reduce ammonia, buffer salt content and improve composting stability.

[0111] In one example, a phased inoculation method was used in the early stages of pile construction to enhance colonization and initiation in high-salt feedstock.

[0112] The present invention will be further illustrated below with reference to the embodiments.

[0113] Example 1: Preparation of microbial agents and composting of high-salt kitchen waste

[0114] (1) Raw material preparation and pretreatment

[0115] 1) Raw material composition and pretreatment: High-salt kitchen waste and soybean residue are used as the main substrates. The kitchen waste is first mechanically cleaned (removing foreign objects such as plastic, bones, and metal), and large pieces are crushed to a particle size ≤20 mm to improve mixing uniformity and mass transfer efficiency. Chopped straw is used as a leavening agent / conditioner, and the straw is chopped to a length of 3~5 cm to improve porosity and aeration.

[0116] 2) Salinity / Conductivity Measurement and Target Range: Take a representative sample of the mixed raw materials, extract by shaking at a ratio of "sample:deionized water = 1:5 (w / v)" for 30 min, let stand for 10 min, and then take the supernatant to measure EC; in this example, the EC of the raw materials is approximately 8~12 mS / cm. If the EC is too high, it can be controlled within the preset range by adding low-salt adjuvants (straw, sawdust, etc.) or an appropriate amount of water in stages.

[0117] 3) C / N and Moisture Content Control: Adjust the C / N ratio to approximately 25-30: preferably by adjusting the proportion of soybean residue and straw; if still insufficient, a small amount of nitrogen-containing raw materials (such as a small amount of manure or soybean meal) can be added to avoid ammonia volatilization caused by directly adding large amounts of available nitrogen. Adjust the moisture content to approximately 58%: use "forming a ball when squeezed, with water visible between fingers but not dripping" as a quick on-site judgment standard, and verify using the drying method (drying at 105℃ to constant weight). If the moisture content is too high, increase the straw / dry material; if it is too low, spray clean water in several batches and mix thoroughly until uniform. Moisture adjustment should be done by adding water in several batches, and mixing for at least 3-5 minutes after each addition to avoid localized over-wetting and the formation of anaerobic patches.

[0118] (2) Preparation of bacterial culture

[0119] Bacillus halotolerans, a halophilic spore-forming bacterium deposited at the China General Microbiological Culture Collection Center (CGMCC) No. 36428, was revived from streaks on culture slant / glycerol tubes and cultured on TSB medium to obtain single colonies. Typical single colonies were picked and inoculated into TSB liquid seed medium and cultured on a shaker until the end-log / stationary phase to obtain the seed culture. After scaling up the culture to obtain the bacterial suspension, the viable cell concentration was corrected to 1 × 10⁻⁶ using plate counts. 9 CFU / mL. The TSB medium was formulated with 17 g casein peptone, 3 g soybean peptone, 5 g sodium chloride, 2.5 g glucose and 2.5 g dipotassium hydrogen phosphate, and the pH was adjusted to 7.3±0.2 before sterilization.

[0120] (3) Inoculation, composting, ventilation and turning and process control

[0121] 1) Trial grouping and replication: Set up an inoculated group (BH group) and an uninoculated control group (CK group); it is recommended to have at least 3 parallel stacks to facilitate statistical comparison.

[0122] 2) Inoculation and mixing: Add bacterial solution at 0.5%~1.0% (w / w) of wet substrate (e.g., add 0.5~1.0 kg of bacterial solution per 100 kg of wet substrate), spraying and mixing simultaneously. Mixing standard: Randomly select 3 samples and measure the moisture content difference ≤2%, ensuring no obvious "wet clumps / dry clumps".

[0123] 3) Stacking specifications: Use windrows or static aeration stacks. For windrow stacks, the recommended stack height is about 1.2 m and the stack width is about 1.8~2.5 m (adjusted according to site conditions). If forced aeration is used, perforated aeration pipes (hole diameter 2~4 mm, hole spacing 10~20 cm) should be laid at the bottom, and an air-permeable layer should be added to prevent clogging.

[0124] 4) Aeration and Turning System: Forced aeration rate of 0.2~0.3 L / (min·kg wet material) (estimated and recorded using a blower flow meter or valve calibration). Turn the pile every 3 days; the turning method is "outer turning to inner, top turning to bottom" to ensure that the edges and center of the pile are evenly exposed to high temperatures. Maintain the moisture content at 50%~65%: if the moisture content decreases during turning, water can be added in a mist; if leachate or stickiness occurs, increase the drying and loosening agent and enhance ventilation.

[0125] 5) Temperature monitoring: Temperature probes or thermometers are inserted in the center of the reactor body and in the upper, middle and lower layers, and recorded at least twice a day; and the time when the temperature reaches 55°C, the highest temperature and the duration of the high temperature are recorded.

[0126] (4) Sampling and testing methods and maturity assessment

[0127] 1) Sampling method: Sampling was carried out on days 0, 3, 6, 9, 12, 15 and 18. Samples were taken from ≥5 points in the center, edge and upper, middle and lower parts of the pile. The samples were mixed to form a mixed sample. Representative samples were taken using the quartering method for testing.

[0128] 2) Detection indicators and methods: Moisture content was determined by drying at 105℃ to constant weight; pH / EC was determined using an extract with a sample:water ratio of 1:5 (w / v); ammonia nitrogen (NH4+) was determined by colorimetric / ionic method after KCl extraction. + -N) and nitrate nitrogen (NO3) - -N); odor / ammonia smell can be assessed using a semi-quantitative scoring system (0-5 points) or a portable ammonia detector (ppm); GI (germination index) is determined using seeds such as radishes, based on compost extract (1:10 w / v); maturity is determined by GI ≥ 80%, NH4+ + -N decreased significantly and NH4 + / NO3 - The temperature drops and approaches ambient temperature, and the odor weakens significantly.

[0129] (5) Experimental results

[0130] Table 1. Comparison of process performance (inoculation group vs. control group)

[0131]

[0132] Table 2 Key physicochemical and composting indicators (days 0 and 18)

[0133]

[0134] Results show that, compared with the uninoculated control, the mounds inoculated with halophilic Bacillus exhibited a faster heating rate and a more stable high-temperature period under high-salt conditions (EC approximately 8–12 mS / cm), and lower ammonia odor intensity and ammonia nitrogen accumulation levels; the germination index (GI) reached or exceeded 80% earlier at maturity, and NH4+ was also lower. + / NO3 - It reduces the composting time to the typical range of mature compost more quickly, thereby shortening the composting cycle and improving product stability under high salinity / high osmotic conditions.

[0135] Example 2: Preparation of microbial agents and composting of tobacco waste from saline tobacco fields

[0136] (1) Raw material preparation and pretreatment

[0137] 1) Raw material composition and pretreatment: Tobacco waste from saline tobacco fields is used as the main substrate. The tobacco waste is crushed to a particle size ≤30 mm to improve mixing uniformity and mass transfer efficiency.

[0138] 2) Salinity / Conductivity Measurement and Target Range: A representative sample of the mixed raw materials was taken and extracted by shaking for 30 min at a ratio of "sample:deionized water = 1:5 (w / v)". After standing for 10 min, the supernatant was taken to measure EC. In this example, the EC of the raw materials was approximately 10~15 mS / cm (derived from the background of saline tobacco field soil and the salt accumulation of the tobacco leaves themselves). If the EC is too high (>12 mS / cm), it can be diluted by increasing the proportion of tobacco straw fragments or adding a small amount of low-salt additives such as straw powder and sawdust, and controlled within the range of 8~12 mS / cm.

[0139] 3) C / N and Moisture Content Control: Tobacco waste itself has a low C / N ratio (approximately 15-20:1). By adding tobacco stalk fragments (C / N approximately 60:1), the overall C / N ratio can be adjusted to approximately 25-30:1. If necessary, a small amount of urea (0.5%-1.0%) can be added to optimize the nitrogen source for microbial metabolism, avoiding direct large-scale addition that could cause ammonia volatilization. The moisture content should be adjusted to approximately 60%-65%: the standard for rapid on-site judgment is "the mixture should be able to form a ball, with water visible between the fingers but not dripping," and verified using a drying method (drying at 105℃ to constant weight). If the moisture content is too high, increase the proportion of tobacco stalk fragments; if it is too low, spray water in several stages and mix thoroughly until uniform. Moisture adjustment should be done by adding water in stages, mixing for at least 3-5 minutes after each addition to avoid localized over-wetting and the formation of anaerobic patches.

[0140] 4) Nicotine and protein content records: The initial nicotine content and crude protein content were measured as baseline data for evaluating the degradation effect during the composting process.

[0141] (2) Preparation of bacterial culture

[0142] Using Bacillus halotolerans (CGMCC No. 36428), a salt-tolerant spore-forming bacterium preserved at the China General Microbiological Culture Collection Center, the bacterium was revived from streaks in slant / glycerol tubes and cultured on TSB medium to obtain single colonies. Typical single colonies were picked and inoculated into TSB liquid seed medium and cultured at 30–37°C and 180 rpm for 12–16 h until the end-logarithmic / stationary phase to obtain the seed culture. The culture was expanded at an inoculum of 2% (v / v), and the viable cell concentration was corrected to 1 × 10⁻⁶ by plate counting. 9 CFU / mL.

[0143] (3) Inoculation, composting, ventilation and turning and process control

[0144] 1) Experimental grouping and replication: An inoculated group (BH group) and an uninoculated control group (CK group) were set up, with 3 parallel piles in each group and a pile volume of approximately 1.5 m³.

[0145] 2) Inoculation and mixing: Add bacterial solution at 0.8%~1.2% (w / w) of wet substrate (e.g., add 0.8~1.2 kg of bacterial solution per 100 kg of wet substrate), spraying and mixing simultaneously. Mixing standard: Randomly select 3 samples and measure the moisture content difference ≤2%, ensuring no obvious "wet clumps / dry clumps".

[0146] 3) Composting specifications: Use windrow composting, with a height of approximately 1.0~1.2 m, a width of approximately 1.5~2.0 m, and a length adjusted according to the site. Lay a 10 cm thick layer of straw powder at the bottom as a breathable isolation layer.

[0147] 4) Aeration and Turning System: Natural ventilation combined with manual turning, turning the pile every 2-3 days; the turning method is "outside turning inside, top turning bottom" to ensure that the edges and center of the pile are evenly exposed to high temperatures. Moisture content is maintained at 55%~65%: if the moisture content drops to <55% during turning, add water in a mist; if leachate or stickiness occurs (moisture content >70%), increase the amount of tobacco straw fragments and enhance ventilation.

[0148] 5) Temperature monitoring: Temperature probes were inserted into the center, surface and upper, middle and lower layers of the stack, and recorded three times a day, morning, noon and evening. The time when the temperature reached 55℃, the highest temperature, the duration of ≥55℃ and the temperature stability during the critical period of nicotine degradation (the first 7 days) were recorded.

[0149] (4) Sampling and testing methods and maturity assessment

[0150] 1) Sampling method: Sampling was conducted on days 0, 3, 6, 9, 12, 15, and 18. Samples were taken from at least 5 points in the pile, including the center, edge, and upper, middle, and lower parts. The samples were mixed thoroughly to form a composite sample, and a representative sample was taken using the quartering method for testing. Nicotine-specific test samples were added on days 6 and 12.

[0151] 2) Detection Indicators and Methods: Basic Indicators: Moisture content was determined by drying at 105℃ to constant weight; pH / EC was determined using an extract with a sample:water ratio of 1:5 (w / v); Nitrogen Forms: Ammonia nitrogen (NH4) was determined by colorimetric / ionic method after KCl extraction. + -N) and nitrate nitrogen (NO3) - -N); Protein degradation: Total nitrogen was determined by the Kjeldahl method, and changes in soluble protein and small peptides were determined by the TCA precipitation method; Odor / ammonia odor: Semi-quantitative scoring (0~5 points) combined with a portable ammonia detector (ppm); Maturity: GI (germination index) was determined using cabbage seeds in compost extract (1:10 w / v); Maturity criteria: GI≥80%, NH4+ + -N<400 mg / kg, NH4 + / NO3 - <0.5, nicotine degradation rate ≥60%, temperature drops and tends to ambient temperature, odor is significantly reduced.

[0152] (5) Experimental results

[0153] Table 3. Comparison of process performance (inoculation group vs. control group)

[0154]

[0155] Table 4 Key physicochemical and composting indicators (days 0 and 18)

[0156]

[0157] Results show that, compared with the uninoculated control, tobacco waste piles inoculated with halophilic Bacillus exhibited significant advantages under high-salt conditions (EC approximately 12-15 mS / cm): the heating rate was twice as fast, the high-temperature period was nearly twice as long, and the nicotine degradation half-life was shortened by 50%; the germination index (GI) at maturity reached ≥80% 7-10 days earlier, and NH4+ was reduced. + / NO3 - The degradation rate decreased more rapidly to the safe range for mature composting (<1.0), the crude protein degradation rate increased by approximately 30 percentage points, and the peak ammonia odor was significantly reduced. This indicates that the salt-tolerant strain can effectively adapt to the high-salt / high-osmotic environment of tobacco field waste, simultaneously achieving rapid protein hydrolysis, efficient nicotine degradation, and a shortened composting cycle, providing effective technical support for the resource utilization of organic waste and soil health management in tobacco-growing areas.

[0158] Comparative Example 1: Composting Experiment of Bacillus sp. Bs-2 in Tobacco Waste

[0159] (1) Source and background of strains

[0160] Strain Bs-2 was isolated from ordinary farmland soil and screened in tobacco field soil in the same batch as Bacillus halotolerans CGMCC No. 36428 of this invention. In simulated composting experiments of tobacco waste, it exhibited problems such as slow heating, short high-temperature period, and incomplete decomposition, serving as a representative strain with poor performance in the screening experiment.

[0161] (2) Preparation of bacterial culture

[0162] Bacillus sp. Bs-2 was revived by streaking from preservation slant / glycerol tubes and cultured on TSB medium to obtain single colonies. Typical single colonies were picked and inoculated into TSB liquid seed medium and cultured at 30–37°C and 180 rpm for 12–16 h until the end-log / stationary phase to obtain the seed culture. The culture was scaled up at a 2% (v / v) inoculum, and the viable cell concentration was corrected to 1 × 10⁻⁶ by plate counting. 9 CFU / mL.

[0163] (3) Raw material preparation and pretreatment

[0164] 1) Raw material composition and pretreatment: Tobacco waste from saline tobacco fields is used as the main substrate. The tobacco waste is crushed to a particle size ≤30 mm to improve mixing uniformity and mass transfer efficiency.

[0165] 2) Salinity / Conductivity Measurement and Target Range: Take a representative sample of the mixed raw materials, extract by shaking at a ratio of "sample:deionized water = 1:5 (w / v)" for 30 min, let stand for 10 min, and then take the supernatant to measure EC. The EC of the comparative raw materials is approximately 12~15 mS / cm. If the EC is too high (>12 mS / cm), it can be diluted by increasing the proportion of tobacco straw fragments or adding a small amount of low-salt auxiliary materials such as straw powder or sawdust, and controlled within the range of 8~12 mS / cm.

[0166] 3) C / N and Moisture Content Control: Tobacco waste itself has a low C / N ratio (approximately 15-20:1). By adding tobacco stalk fragments (C / N approximately 60:1), the overall C / N ratio can be adjusted to approximately 25-30:1. If necessary, a small amount of urea (0.5%-1.0%) can be added. The moisture content should be adjusted to approximately 60%-65% on-site. The standard for quick judgment is "can be formed into a ball, and water can be seen between the fingers but not dripping," and this should be verified using the drying method (drying at 105℃ to constant weight). Moisture adjustment should be done by adding water in stages, and stirring for at least 3-5 minutes after each addition to avoid localized over-wetting and the formation of anaerobic patches.

[0167] 4) Nicotine and protein content records: The initial nicotine content and crude protein content were measured as baseline data for evaluating the degradation effect during the composting process.

[0168] (4) Inoculation, composting, ventilation and turning and process control

[0169] 1) Experimental Grouping and Replication: Three treatment groups were set up: ① no bacterial control (CK); ② Bacillus sp. Bs-2 (Bs-2 group), which showed poor efficacy; ③ Bacillus halotolerans CGMCC No. 36428 (BH group), the strain of this invention. Each group consisted of 3 parallel piles, with a pile volume of approximately 1.5 m³. 3 .

[0170] 2) Inoculation and mixing: Add bacterial solution at 0.8%~1.2% (w / w) of wet substrate (e.g., add 0.8~1.2 kg of bacterial solution per 100 kg of wet substrate), spraying and mixing simultaneously. Mixing standard: Randomly select 3 samples and measure the moisture content difference ≤2%, ensuring no obvious "wet clumps / dry clumps".

[0171] 3) Composting specifications: Use windrow composting, with a height of approximately 1.0~1.2 m, a width of approximately 1.5~2.0 m, and a length adjusted according to the site. Lay a 10 cm thick layer of straw powder at the bottom as a breathable isolation layer.

[0172] 4) Aeration and Turning System: Natural ventilation combined with manual turning, turning the pile every 2-3 days; the turning method is "outside turning inside, top turning bottom" to ensure that the edges and center of the pile are evenly exposed to high temperatures. Moisture content is maintained at 55%~65%: if the moisture content drops to <55% during turning, add water in a mist; if leachate or stickiness occurs (moisture content >70%), increase the amount of tobacco straw fragments and enhance ventilation.

[0173] 5) Temperature monitoring: Temperature probes were inserted into the center, surface and upper, middle and lower layers of the stack, and recorded three times a day, morning, noon and evening. The time when the temperature reached 55℃, the highest temperature, the duration of ≥55℃ and the temperature stability during the critical period of nicotine degradation (the first 7 days) were recorded.

[0174] (5) Sampling and testing methods and maturity determination

[0175] 1) Sampling method: Sampling was conducted on days 0, 3, 6, 9, 12, 15, and 18. Samples were taken from at least 5 points in the pile, including the center, edge, and upper, middle, and lower parts. The samples were mixed thoroughly to form a composite sample, and a representative sample was taken using the quartering method for testing. Nicotine-specific test samples were added on days 6 and 12.

[0176] 2) Detection Indicators and Methods: Basic Indicators: Moisture content was determined by drying at 105℃ to constant weight; pH / EC was determined using an extract with a sample:water ratio of 1:5 (w / v); Nitrogen Forms: Ammonia nitrogen (NH4) was determined by colorimetric / ionic method after KCl extraction. + -N) and nitrate nitrogen (NO3) - -N); Protein degradation: Total nitrogen was determined by the Kjeldahl method, and changes in soluble protein and small peptides were determined by the TCA precipitation method; Odor / ammonia odor: Semi-quantitative scoring (0~5 points) combined with a portable ammonia detector (ppm); Maturity: GI (germination index) was determined using cabbage seeds in compost extract (1:10 w / v); Maturity criteria: GI≥80%, NH4+ + -N<400 mg / kg, NH4 + / NO3 - <0.5, nicotine degradation rate ≥60%, temperature drops and tends to ambient temperature, odor is significantly reduced.

[0177] (6) Experimental results

[0178] Table 5 Comparison of the composting effects of different Bacillus strains on tobacco waste

[0179]

[0180] Results show that compared with the ineffective strain Bs-2, the strain Bacillus halotolerans CGMCC No. 36428 of this invention exhibits significant advantages in the composting of high-salt tobacco waste. Although Bs-2 has some activity (slightly faster heating and slightly longer high-temperature period than the control), its nicotine degradation rate (42%~50%) and crude protein degradation rate (32%~40%) are much lower than those of the BH group (68%~75%, 55%~65%, respectively), and its ammonia odor control effect is limited (score 3~4 points). The composting cycle is only shortened by 2~4 days compared to the control. In contrast, the salt and high-temperature tolerance characteristics obtained by the BH group through systematic domestication achieved unexpected technical effects such as a 43%~50% increase in heating rate, a 75%~80% extension of the high-temperature period, and a 50%~79% increase in nicotine degradation rate, providing an effective solution for the rapid composting of high-salt organic waste.

[0181] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A halophilic Bacillus halotolerans, characterized by, The accession number is CGMCCNo.36428.

2. A microbial agent, characterized in that, Includes the salt-tolerant Bacillus as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, for: (i) Liquid bacterial agent; or (ii) Powders or granules obtained by carrier adsorption and / or low-temperature drying.

4. Any of the following applications in organic waste composting: (i) The salt-tolerant Bacillus as described in claim 1; (ii) The microbial agent according to claim 2 or 3; The organic waste includes high-salt organic waste or high-osmotic organic waste.

5. The application as described in claim 4, characterized in that, The organic waste has at least one of the following properties: (i) The mass fraction of NaCl is 0.5%~6%; (ii) The electrical conductivity EC is 4~20 mS / cm; (iii) The water activity a_w is 0.85~0.

98.

6. A composting method, characterized in that, Including composting based on any of the following: (i) The salt-tolerant Bacillus as described in claim 1; (ii) The microbial agent according to claim 2 or 3.

7. The composting method as described in claim 6, characterized in that, include: The composting raw materials are mixed with acceptable adjuvants or additives, the C / N ratio is adjusted to 20-35, the moisture content is adjusted to 50%-65%, and the pH is adjusted to 6.5-9.0 to obtain compost material. The adjuvants or additives include a leavening agent. The salt-tolerant Bacillus or the inoculant is inoculated into the compost material to achieve a viable bacterial count of 10. 5 ~10 9 CFU / g, after decomposition and post-fermentation, compost products are obtained.

8. The composting method as described in claim 7, characterized in that, include: The composting raw materials are high-salt organic waste or high-osmotic organic waste; The high-salt organic waste or high-osmotic organic waste includes high-salt kitchen waste and / or tobacco waste; The acceptable excipients or additives also include at least one of zeolite, bentonite, and humic acid. The leavening agent includes at least one of straw, rice husk, sawdust, and biochar; The composting process includes fermentation and composting using methods such as turning over the pile or aeration.

9. The composting method as described in claim 8, characterized in that, include: The amount of the leavening agent added is 5% to 40% of the wet basis mass of the compost raw material; The inoculation includes inoculating 0.1% to 5% of the wet weight of the salt-resistant Bacillus or the bacterial agent; The frequency of turning the pile is once every 2 to 5 days; The aeration rate is 0.1~0.5 L / (min·kg wet material); The composting temperature is 55~70℃, and the time is 10~25 days.

10. The composting method as described in claim 7, characterized in that, The conditions for complete fermentation are at least two of the following: (a) GI ≥ 80%; (b) The reactor temperature is close to the ambient temperature and remains stable for more than 48 hours; (c) The ratio of ammonium nitrogen to nitrate nitrogen tends to stabilize; (d) Respiratory activity decreased significantly.

Images