Klebsiella bacteria, complex microbial inoculant and application thereof

Abstract

A microbial compound inoculant, based on Klebsiella pneumoniae (Klebsiella pneumoniae) Klebsiella sp. CS11, long-branch mold ( Trichoderma longibrachiatum ) and Azotobacter brownii ( Azotobacter chroococcum The strain, Klebsiella pneumoniae CS11, was composed of bacteria in a mass ratio of 1:1:1 and its preservation number is CCTCC M 20242943. CS11 When applied to aerobic composting of corn stalks and chicken manure, the strain can reduce H2S emissions and improve sulfur metabolism efficiency during composting. Furthermore, the compound microbial agent in this invention further accelerates the decomposition and transformation of organic matter, reduces H2S emissions, and improves sulfur fixation efficiency. Simultaneously, it regulates the content of amino acids and humic acid in the compost, producing high-quality organic fertilizer, improving the soil environment, achieving resource recycling, and promoting sustainable agricultural development.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Klebsiella pneumoniae, a compound microbial agent, and their applications. Background Technology

[0002] Composting is an important means of resource utilization of organic waste, but it generates various odorous gases, such as ammonia (NH3), hydrogen sulfide (H2S), and volatile organic compounds (VOCs). The emission of these gases not only affects the surrounding environment but also poses a threat to public health and safety. Therefore, controlling odorous gas emissions during composting has always been an important issue in scientific research and engineering practice. Hydrogen sulfide is one of the main odorous gases in composting, primarily originating from the anaerobic decomposition of sulfur-containing organic matter in animal manure. Hydrogen sulfide is a toxic gas; long-term exposure can damage the human respiratory, endocrine, and nervous systems, and may even induce diseases such as cancer.

[0003] Furthermore, during composting, if the sulfur content is too high, under anaerobic conditions, sulfur is easily reduced to hydrogen sulfide and other gases with pungent odors. This not only pollutes the air at the composting site and in the surrounding environment, affecting air quality, but may also attract pests and pathogens, adversely impacting the quality and use of the compost. However, sulfur is an important component of many enzymes and coenzymes in microbial metabolism. Too low a sulfur content limits microbial activity, slowing the decomposition of organic matter during composting, prolonging the composting time, and affecting the production efficiency and quality of the compost. Moreover, if the final compost has too low a sulfur content, it cannot meet the sulfur requirements of plants, leading to sulfur deficiency symptoms such as chlorosis and yellowing of leaves, stunted growth, and weakness, affecting photosynthesis and other physiological functions, and reducing crop yield and quality.

[0004] Klebsiella pneumoniae ( Klebsiella Klebsiella pneumoniae is a type of Gram-negative bacterium that is widely distributed in nature and is often used in composting. Different Klebsiella pneumoniae produce different effects. For example, the Klebsiella pneumoniae disclosed in CN113913348A can produce gases such as hydrogen sulfide and methanethiol, while the Klebsiella pneumoniae disclosed in CN119120301A can degrade and remove NH3 and H2S. Summary of the Invention

[0005] The purpose of this invention is to provide a microbial agent comprising a strain of Klebsiella pneumoniae that can inhibit the generation of H2S and synthesize sulfate, thereby regulating the amino acid content in compost and improving compost quality.

[0006] Another object of the present invention is to provide the application of the above-mentioned microbial agents.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A strain of Klebsiella pneumoniae, characterized in that: the strain is classified as Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiellasp. CS11, deposited at the China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with accession number CCTCC NO. M 20242943, and deposited on December 30, 2024.

[0009] Furthermore, Klebsiella CS11 can metabolize sulfides in the environment into organic and inorganic sulfur.

[0010] Furthermore, Klebsiella CS11 can synthesize inorganic sulfur such as sulfate in sulfur-free or sulfur-deficient environments through its own metabolic regulation.

[0011] Furthermore, Klebsiella CS11 reduces H2S production by altering the microbial community structure during composting and inhibiting the growth of H2S-producing bacteria.

[0012] The application of Klebsiella CS11 in composting fermentation.

[0013] A microbial compound inoculant, characterized in that: it is composed of Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiellasp. CS11, long-branch mold ( Trichoderma longibrachiatum ) and Azotobacter brownii ( Azotobacter chroococcum It is composed of ingredients in a mass ratio of 1:1:1.

[0014] The strain is classified as Klebsiella pneumoniae (Klebsiella pneumoniae) Klebsiellasp. CS11, deposited at the China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with accession number CCTCC NO. M20242943, and deposit date December 30, 2024;

[0015] The long-branch mold ( Trichoderma longibrachiatum The accession number is GDMCC No.: 3.139, and the described *Azotobacter brownis* ( Azotobacter chroococcum GDMCC No.: 1.272.

[0016] The application of the above-mentioned compound microbial agents in fermentation composting.

[0017] Furthermore, the fermented compost is made from corn stalks and chicken manure as raw materials, and inoculated with a compound microbial agent for composting fermentation. The compound microbial agent is composed of Klebsiella CS11, Trichoderma longifolia and Azotobacter chrysophagus in a mass ratio of 1:1:1, and the inoculation amount of the compound microbial agent in the compost raw materials is 5%.

[0018] During the composting fermentation process, CS11 metabolism alters the amino acid content in the compost, thus improving the quality of the fertilizer.

[0019] The initial C:N ratio of corn stalks is high, even when combined with chicken manure, which has a high N content, making it unsuitable for the growth of sulfur-fixing bacteria and resulting in unsatisfactory sulfur fixation in composting. This invention utilizes a compound microbial agent in composting fermentation. Trichoderma longicornis decomposes complex organic matter such as lignocellulose in corn stalks, releasing small-molecule carbon sources and sulfur-containing organic matter, providing energy and substrate for nitrogen-fixing and sulfur-fixing bacteria. Nitrogen-fixing bacteria synthesize ammonium nitrogen and organic nitrogen through growth and metabolism, directly supplementing the nitrogen source in the compost and adjusting the carbon-nitrogen ratio to a more suitable range. The combined use of the compound microbial agent reduces anaerobic fermentation of sulfur-containing substances, inhibits H2S production, and improves the sulfur fixation effect of CS11. It also shortens the composting cycle and alters the amino acid content, humus content, and electrical conductivity of the compost, thus improving compost quality.

[0020] The application of the above-mentioned compound microbial agents in soil improvement.

[0021] The aforementioned compound microbial inoculant can increase the nitrogen and sulfur content of nitrogen- and sulfur-deficient soils by inoculating them. The sulfur-fixing bacterium CS11 can synthesize organic and inorganic sulfur (sulfates, sulfites, thiosulfates, etc.) from sulfides in the soil, with sulfates being a form of sulfur that plants can absorb and utilize. The activity of CS11 increases the sulfate content in the soil, promoting plant growth. Furthermore, the production of sulfates during sulfur oxidation releases hydrogen ions (H+). + It has a certain effect on regulating soil pH and can improve alkaline soil.

[0022] The present invention has the following technical effects:

[0023] Klebsiella CS11 The strains, when applied to aerobic composting of corn stalks and chicken manure, can reduce H2S emissions and improve sulfur metabolism efficiency during composting. Furthermore, the compound microbial agent in this invention further accelerates the decomposition and transformation of organic matter, reduces H2S emissions, and improves sulfur fixation efficiency. Simultaneously, it regulates the content of amino acids and humic acid in the compost, producing high-quality organic fertilizer, improving the soil environment, achieving resource recycling, and promoting sustainable agricultural development. Attached Figure Description

[0024] Figure 1 Changes in the expression levels of sulfur metabolism-related genes in bacterial strains at different time points: a: genes related to inorganic sulfur metabolism; b: genes related to organic sulfur metabolism.

[0025] Figure 2Changes in cysteine ​​and methionine content during the metabolism of strain CS11: a: cysteine ​​in the supernatant, b: cysteine ​​in bacterial cells, c: methionine in the supernatant, d: methionine in bacterial cells.

[0026] Figure 3 Determination of inorganic sulfur content in strain CS11, a:SO4 2- b:HS - c:SO3 2- ;d:S2O3 2- .

[0027] Figure 4 The content of sulfur metabolism-related substances in bacterial strains at different time points: a: TOS; b: SO4 2- ;c:HS - ;d:SO3 2- e:S2O3 2- .

[0028] Figure 5 The changes in amino acid content during composting were observed in strain CS11. a: total amino acids, b: free amino acids.

[0029] Figure 6 Changes in sulfur content during composting in different treatment groups; a: Total sulfur; b: SO4 2- c:SO3 2- ;d:H2S - e: Cumulative hydrogen sulfide emissions; f: H2S emission rate.

[0030] Figure 7 Changes in the carbon-nitrogen ratio over time during composting in different treatment groups.

[0031] Figure 8 Changes in conductivity over time during composting in different treatment groups.

[0032] Figure 9 pH changes over time during composting in different treatment groups.

[0033] Figure 10 The change in moisture content of different treatment groups during composting over time. Detailed Implementation

[0034] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0035] The long-branch mold used in this invention ( Trichoderma longibrachiatum The accession number is GDMCC No.: 3.139; *Azotobacter brownii* ( Azotobacter chroococcum Purchased from Guangdong Provincial Microbial Culture Collection Center (GDMCC) with GDMCC No. 1.272.

[0036] The corn stalks used in this invention were taken from the experimental field of the Forestry and Agriculture University, the chicken manure was provided by Dapeng Animal Husbandry Co., Ltd., and the commercially available microbial agent, named Organic Material Composting Agent, was purchased from Yihao Biotechnology.

[0037] The culture medium used in this invention is as follows:

[0038] LB medium (g / L): yeast extract 5.0, NaCl 10.0, tryptone 10.0;

[0039] Sulfate medium (g / L): NH4Cl 0.4, glucose 0.4, K2HPO4 1.2, MgCl2·6H2O 0.2, FeC6H5O7 0.01, KH2PO4 1.2, Na2S·9H2O 0.04~0.08; (Wang HX, Jiang LY, Wu XW, Chen JM.)

[0040] Starky-Na2S2O3 medium (g / L): (NH4)2SO4 0.3, KH2PO4 3.5, MgSO4 0.5, CaCl2·2H2O 0.33, FeSO4·7H2O 0.018, Na2S2O3 10.0;

[0041] Sulfide medium (g / L): KH2PO4 2.0, NH4Cl 0.5, Na2CO3 0.5, MgCl2·6H2O 0.2, Na2S·9H2O 0.2, pH adjusted to 7.0–7.2. (Sulfur-free medium is the same as sulfide medium except that it lacks sulfides).

[0042] Example 1

[0043] Isolation, screening and identification of strain CS11

[0044] (1) Separation: Fresh chicken manure samples were collected from poultry farms in Liaoyuan City during the later stages of egg production. Stones, feathers and other debris were removed, and the samples were sealed in self-sealing bags and stored at -20 ℃ for later use.

[0045] (2) Screening: Weigh 10 g of chicken manure sample, dissolve it in 90 mL of sterile water, add 3 mm glass beads, and incubate at 30 ℃ and 180 r / min for 30 min; let stand for 5 min, take 10 mL of supernatant and inoculate it into 100 mL of liquid enrichment medium, and incubate at 30 ℃ and 180 r / min for 2 days; take 5 mL of enrichment solution, inoculate it into selective medium and incubate for 3 days, and perform serial dilution after 3 consecutive transfers, with dilution factors of 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, and 10⁻¹². Take 200 μL of bacterial suspension and spread it on solid selective medium, 3 replicates, and incubate at 30 ℃ for 24 h. Pick single colonies of different morphologies and perform multiple streak cultures to obtain monoclonal strains through purification. The strain was transferred to a selective medium and cultured at 30 °C and 180 r / min for 24 h. The concentration of SO₄²⁻ in the medium was determined according to the method of Sun Haofen et al., and the conversion rate was calculated. The higher the conversion rate, the better the sulfur oxidation performance of the strain. The strain with a high conversion rate was selected and numbered CS11.

[0046] (3) Identification:

[0047] Morphological identification of the strain: Colonies of strain CS11 are round, with a smooth and raised surface, relatively regular edges, opaque, milky white, easy to pick up, and relatively moist. Gram staining is negative. The bacterial cells are short rods, relatively small. Strain CS11 is a short rod, 0.7–1.0 μm wide and 1.0–2.2 μm long, belonging to medium-sized rod-shaped bacteria. The ends of the bacterial cells are blunt and rounded, and they are scattered.

[0048] Physiological and biochemical identification: This bacterium can utilize glucose, maltose, mannitol, and sucrose, but not lactose. It can degrade cellulose and hydrolyze starch, but does not produce H₂S. It is positive for VP test and negative for MR test, and cannot produce phenylalanine deaminase.

[0049] BLAST analysis of the PCR-obtained sequence revealed a high degree of homology (99.7%) with Klebsiella spp. The closest phylogenetic relationship of strain CS11 is to Klebsiella spp. Based on morphological characteristics and physiological and biochemical identification results, strain CS11 was identified as Klebsiella. Klebsiella sp. ).

[0050] The strain was biopreserved, specifically the strain classified as Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiellasp.CS11, deposited at the China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with accession number CCTCC M 20242943, and deposited on December 30, 2024.

[0051] Example 2

[0052] Sulfur metabolism in Klebsiella CS11:

[0053] 5% CS11 was inoculated into sulfide medium, and the changes in the expression and content of sulfur metabolism-related genes at different time points were measured. Cysteine ​​content was determined using a cysteine ​​assay kit (Nanjing Jiancheng Bioengineering Institute). Methionine content was determined using a methionine enzyme-linked immunosorbent assay (ELISA) kit from Fusheng Industrial Co., Ltd.

[0054] 5% of CS11 strain was inoculated into sulfate medium, Starky-Na2S2O3 medium and sulfide medium respectively, and the changes in sulfate, sulfide, sulfite and thiosulfate contents at different time points were measured.

[0055] (1) Changes in gene expression and substance content of Klebsiella CS11:

[0056] The expression levels of sulfur metabolism-related genes in bacterial strains at different time points are as follows: Figure 1 As shown, Figure 1 a is a gene related to inorganic sulfur metabolism. sbp , cysD , cysH , CysI , SiR The gene content was highest at 36 hours, and the increase in these gene contents may be due to the metabolic needs of the strain: these genes are involved in sulfur metabolism in the organism. cysD , cysH , CysI , SiR This can be attributed to transcriptional regulation (SBP), or specific physiological or environmental conditions. When an organism is in a particular physiological state or environment, the increased demand for corresponding metabolites drives increased expression of these genes. cysN , cysC The gene content is highest at 48 hours, possibly because these genes are involved in the assimilation and reduction of sulfate in CS11, providing the organism with the necessary sulfur source. When sulfur is scarce in the environment or the organism is in a specific physiological state, the expression of these genes increases to meet the organism's need for sulfur. Figure 1 Genes b are related to organic sulfur metabolism. Compared to inorganic metabolism, the expression levels of most genes involved in organic metabolism are increased in the sulfur-free environment. CTH It is an enzyme that promotes the breakdown of cysteine, but metB The gene encodes a protein called homocysteine ​​synthase, which participates in the methionine biosynthesis pathway. metB The gene expression level was consistently lower than CTH This is because CTH Gene-encoded enzymes participate in another key step of cysteine ​​metabolism, synthesizing cysteine ​​from cysteine. Therefore, in the process of cysteine ​​metabolism, metB Gene expression levels are typically lower than CTH Genes. At 24 hours metH The highest content is associated with oxidative stress in cysteine ​​metabolism. Methamphetamine can influence intracellular cysteine ​​metabolism by inducing oxidative stress, thereby leading to methylation of genes (…). metH Increase in expression levels. cysK Genes promote the metabolism of sulfides into cysteine. CysK The significant increase at 48 hours is related to the higher concentration of cysteine ​​in the supernatant at this time. Besides... metH and metA It's not that all the components except for the highest content were at their highest levels. This is because, at 48 hours, most of the conversions were to cysteine, with a small amount to methionine. Simultaneously, the overall inorganic content showed a downward trend over 48 hours, promoting the conversion of more inorganic sulfur into organic sulfur. This indicates that CS11 can utilize inorganic sulfur and convert it into organic sulfur through metabolism.

[0057] (2) Changes in cysteine ​​and methionine content in strain CS11:

[0058] Figure 2 As shown in Figure a, in sulfur-free medium, the initial cysteine ​​content was 0, which then increased, reaching a maximum of 0.0438 μmol / mL at 36 h, and subsequently decreased. Figure 2As shown in b, the initial cysteine ​​content in the CS11 bacterial strain was 0.1042 μmol / mL. In sulfur-free medium, the intracellular cysteine ​​content gradually decreased, reaching a minimum of 0.0200 μmol / mL at 48 h. The cysteine ​​content showed a decreasing trend before 12 h because intracellular cysteine ​​mainly exists in the form of glutathione or cysteine ​​residues, participating in the cell's antioxidant and reactive oxygen species scavenging processes. In sulfur-containing medium, the cysteine ​​content was highest at 24 h (0.1289 μmol / mL) and lowest at 36 h (0.0441 μmol / mL). When cells are subjected to oxidative stress, these sulfur-containing amino acids are released extracellularly, leading to an increase in cysteine ​​content in the supernatant. This increase may also be due to the strain secreting cysteine-containing proteins or enzymes, resulting in an increase in cysteine ​​content in the supernatant. Simultaneously, the release of these secretions also leads to a decrease in intracellular cysteine. The substances present in the culture medium are all sulfur-containing compounds. The increase in content after 24 hours may be due to the metabolism of sulfur-containing compounds by the strain, converting sulfides into cysteine. The subsequent decrease followed by an increase may be due to the metabolism of methionine during its conversion to cysteine. In sulfur-free culture medium, the significant increase in content after 36 hours may be due to the organism's own growth metabolism and other metabolic processes leading to an increase in cysteine ​​content. Additionally, the longer growth time of the strain may result in a decrease in viable cell count, thus releasing some cysteine.

[0059] Figure 2 The methionine content in medium c showed a relatively small overall trend in the sulfur-free medium, but at 36 h in the sulfur-containing medium, the methionine content was the highest among both the bacterial cells and the external bacteria in this treatment group. Figure 2 As shown in Figure d, the initial methionine content in the strain was 125.1551 pg / mL. In sulfur-free medium, the strain showed an overall increasing trend, reaching 352.4492 pg / mL at 48 h. However, in sulfur-containing medium, the content showed an overall decreasing trend in the first 24 h, reaching a minimum of 95.6898 pg / mL at 24 h, and a maximum of 188.6310 pg / mL at 36 h. During the period of decreasing methionine content, the cysteine ​​content generally showed an increasing trend, which is consistent with the gene expression levels observed previously in the first 24 h. CTH, metC The increase in content is related to the fact that methionine content is highest at 36 hours, indicating that methionine synthesis or metabolism may be at its peak at this time. Cysteine ​​content is lowest at this time, which may mean that cysteine ​​consumption or conversion is relatively vigorous at this point. However... CTH The gene expression level was highest at 36 hours. CTHThe gene encodes a cysteine ​​hydrolase, responsible for catalyzing the breakdown of cysteine. In the supernatant, the methionine content in the sulfur-containing medium significantly increased after 36 hours. During cultivation, various metabolites, including amino acids and organic acids, are secreted. With prolonged cultivation, these metabolites gradually accumulate in the supernatant, leading to an increase in methionine content. Simultaneously, the strain may increase methionine synthesis or release by regulating its own metabolic processes, further increasing the methionine content in the supernatant. However, with extended cultivation time, the bacteria may gradually utilize these methionines as a nutrient source, causing the methionine content in the supernatant to decrease after 48 hours.

[0060] (3) The effect of Klebsiella CS11 metabolism on sulfur:

[0061] Figure 3 a represents SO4 generated by CS11 in sulfate medium. 2- Concentration, highest SO4 2- The concentration was 29.239 mg / L over 20 hours. Figure 3 b represents the change in sulfide concentration during the metabolism of CS11 in sulfide medium. The initial sulfide concentration was 200 μg / mL. As the strain metabolized, the sulfide concentration decreased to 0 at 12 h and remained at 0 thereafter.

[0062] The concentrations of sulfite and thiosulfate generated by CS11 in Starky-Na2S2O3 medium are as follows: Figure 3 c and Figure 3 As shown in Figure d, the initial sulfite concentration was 80 μg / mL. With the strain's metabolism, the sulfite concentration reached its highest point at 24 h, at 120 μg / mL, and then continuously decreased. The initial thiosulfate content was 112 μg / mL, reaching its highest point at 24 h at 168 μg / mL, and then decreased. The overall thiosulfate content was greater than the sulfite content. At 48 h, the sulfite and thiosulfate contents were 60 μg / mL and 84 μg / mL, respectively. The overall trends of sulfite and sulfite were basically consistent, indicating that CS11 can utilize thiosulfate and sulfite as nutrients to provide energy for subsequent metabolic processes. Therefore, Klebsiella CS11 can utilize sulfides, thiosulfates, and sulfites for metabolism, providing a reference for further research on its sulfide mechanism. Furthermore, even in the absence of external sulfur, CS11 can synthesize inorganic sulfur by regulating its own metabolic processes.

[0063] (4) Content of sulfur metabolism-related substances in bacterial strains at different time points

[0064] The changes in the content of sulfur metabolism-related substances in the culture medium at different time points are as follows: Figure 4 As shown. Figure 4 As shown in figure a, the overall sulfur content shows an upward trend, with the sulfate content reaching its highest level at 12 hours, which may be due to... sbp , cysD、cysN Genes convert sulfur-containing substances from the external environment into sulfate-containing substances within the cell; the changes in sulfides are as follows: Figure 4 As shown in b, the initial sulfide content in the sulfur-containing medium was 7.49 mg / mL, which decreased to 0 at 12 h and remained at 0 until 48 h. The sulfide content in the sulfur-free medium remained at 0 throughout. This indicates that strain CS11 can metabolize and transform sulfides, thereby reducing the sulfide content. Figure 4 As shown in Figure c, the sulfate content reached its highest level at 12 h, with 14.85 μg / mL in the sulfur-free medium and 23.3 μg / mL in the sulfur-containing medium. The increase in sulfate content at this point is likely due to the strain being inoculated in a sulfur-free environment. Under sulfur-free conditions, the strain can utilize relevant nutrients to convert them into sulfate and other substances to maintain its growth and development. Figure 4 As shown in d, the sulfite content generally decreased in the sulfur-free medium, but increased to 11.60 μg / mL in the sulfur-containing medium after 24 h. Figure 4 As shown in Figure e, the thiosulfate concentration generally decreased in the sulfur-free medium, reaching its highest level of 16.80 μg / mL in the sulfur-containing medium after 24 hours. In the sulfur-free medium, the strain can convert its thiosulfate into sulfite through its own metabolism. Sulfite undergoes further metabolic processes within the strain, converting into other substances such as organic sulfur compounds like cysteine.

[0065] Example 3

[0066] Compost strain CS11:

[0067] Three groups of compost were prepared, consisting of a mixture of chicken manure and corn stalks, with a mass ratio of 2.5:1 between the straw and chicken manure. These groups were designated as CK, CS11 (chicken manure and corn stalks mixed with 5% CS11 microbial agent), and CABA (chicken manure and corn stalks mixed with commercially available microbial agent).

[0068] Changes in amino acid content during composting:

[0069] Depend on Figure 5It is known that during composting, microorganisms decompose organic sulfur compounds into volatile sulfur compounds, such as dimethyl sulfide (Me2S). These volatile sulfur compounds are gradually released into the atmosphere as composting progresses, leading to a decrease in the total organic sulfur content. During aerobic composting, sulfur-oxidizing bacteria oxidize organic sulfur compounds and elemental sulfur into sulfates, thereby reducing the total organic sulfur content in the compost material. Simultaneously, during composting, the decomposition of organic matter and evaporation of moisture reduce the total mass of the compost material, resulting in a relative decrease in the total organic sulfur content. At the end of composting, the CS11 group showed the highest cysteine ​​content at 0.487 mg / g, an increase of 0.007 mg / g compared to the CK group and 0.031 mg / g compared to the CABA group. These results indicate that the addition of strain CS11 can slightly increase the content of cysteine ​​and methionine, thereby promoting their conversion to cysteine.

[0070] Example 4

[0071] Composting with compound microbial agents:

[0072] Three groups of compost were prepared, consisting of a mixture of chicken manure and corn stalks at a mass ratio of 2.5:1, with 5 wt.% of a compound microbial agent added. The compound microbial agents were: F1 (CS11, *Trichoderma longifolia*, and *Aspergillus niger* in a mass ratio of 1:1:1), F2 (CS11, *Aspergillus niger*, and *Azotobacter chrysogenum* in a mass ratio of 1:1:1), F3 (CS11, *Trichoderma longifolia*, and *Azotobacter chrysogenum* in a mass ratio of 1:1:1), and F4 (CS11, *Trichoderma longifolia*, *Aspergillus niger*, and *Azotobacter chrysogenum* in a mass ratio of 1:1:1) (the compost tests were conducted in winter).

[0073] In Examples 3 and 4, gas samples were measured using the static chamber method on days 1-15 and days 21 and 35 of the composting process. Solid samples were collected from the top, middle, and bottom layers of the compost pile at days 0, 3, 7, 14, 21, 27, 35, and 45 using a five-point sampling method. These samples were thoroughly mixed and used to determine total sulfur, sulfate, and sulfide levels. SO4 was determined according to the method of Mussa SAB et al. (2009). 2－ Content. Sulfide determination refers to HJ883-2017. Sulfite determination refers to GB5009.34-2003 (no bacterial agent was added to the CK group).

[0074] Hydrogen sulfide gas determination:

[0075] Every afternoon at 2:30 PM, a self-made static box was placed on top of the stack until 8:30 AM the following morning. H2S concentration was measured using a pump-type portable H2S analyzer, and the H2S emission rate and cumulative emission formula were calculated, as follows:

[0076] (1) Emission rate:

[0077]

[0078] F : Gas emission rate (mg / kg / h); h : Height of the gas extraction space above the reactor body (m); t : Average temperature of the gas extraction space (°C); M : Molar mass of the gas (g / mol); V m Molar volume of a gas under standard conditions (L / mol); dC / dt : Rate of change of gas concentration; A : Surface area of ​​the game (m 2 ); m Total mass of the stack (kg).

[0079] (2) Formula for calculating cumulative gas emissions:

[0080]

[0081] f Cumulative gas emissions (mg / kg) throughout the composting process. F i 、F i+1 : represent the gas emission rates of the i-th and (i+1)-th composting operations, respectively; t i+1 -t i : The time interval between two consecutive samples, in days.

[0082] Test results are as follows Figure 6 (a) It can be seen that the initial total sulfur content of each treatment group was 2.44%, 2.48%, 2.36% / 2.33%, 2.68%, and 2.53%, respectively. The overall trend was first decreasing and then increasing. On day 45 of composting, the total sulfur content of each treatment group was 13.43%, 15.56% / 12.36%, 11.99%, 16.77%, and 14.72%, respectively. Figure 6 (b) Initial SO4 in each treatment group 2-The SO42 content was 4.27 g / kg, 5.38 g / kg, 4.95 g / kg, 4.83 g / kg, 5.43 g / kg, and 4.99 g / kg, respectively. The CK group reached its maximum value of 5.68 g / kg on day 14. On day 21, the CS11, F1, F3, and F4 groups all reached their maximum values, at 12.69 g / kg, 6.09 g / kg, 9.77 g / kg, and 6.83 g / kg, respectively. On day 45 of composting, the SO42 content in the CK, CS11, F1, F2, and F4 groups was [not specified]. 2- The SO4 content decreased by 1.22 g / kg, 0.73 g / kg, 1.10 g / kg, 0.80 g / kg and 0.34 g / kg respectively compared with day 0, while the SO4 content in group F3 was lower. 2- The content increased by 0.11 g / kg compared to day 0. Figure 6 In (c), the CK group reached its maximum sulfite content of 3.56 g / kg on day 7, while the other treatment groups (CS11, F1, F2, F3, and F4) reached their maximum values ​​on day 27, at 4.85 g / kg, 3.51 g / kg, 3.33 g / kg, 5.17 g / kg, and 4.35 g / kg, respectively. At day 45, the sulfite content of each treatment group increased by 0.45 g / kg, 1.792 g / kg, 1.05 g / kg, 1.61 g / kg, 2.97 g / kg, and 2.34 g / kg, respectively. Figure 6 (d) The sulfide content in each treatment group showed an overall trend of first decreasing, then increasing, and then decreasing again. The sulfide content in the CK group and F1 group reached its maximum on day 21, at 24.16 mg / kg and 23.85 mg / kg, respectively. On day 27, the CS11, F2, F3, and F4 groups reached their maximum values, at 26.96 mg / kg, 28.70 mg / kg, 31.13 mg / kg, and 29.55 mg / kg, respectively. On day 45 of composting, the sulfide content in the CK, F1, and F2 groups decreased by 3.18 mg / kg, 1.36 mg / kg, and 0.10 mg / kg, respectively, compared to day 0, while the CS11, F3, and F4 groups increased by 0.62 mg / kg, 1.88 mg / kg, and 1.12 mg / kg, respectively. Furthermore, the total inorganic sulfur content in the final compost of group F3 was higher than that of group CS11, indicating that the F3 compound microbial agent produced a synergistic effect that improved the sulfur fixation capacity of CS11. Figure 6(e) shows the cumulative emissions, indicating the control of H2S by each treatment. The cumulative H2S emissions for the CK, CS11, F1, F2, F3, and F4 groups were 39.336 mg / kg, 31.356 mg / kg, 36.984 mg / kg, 28.712 mg / kg, 23.373 mg / kg, and 20.125 mg / kg, respectively. This demonstrates that Klebsiella CS11 significantly reduced the total H2S emissions. Furthermore, the combined bacterial agents in the F3 and F4 groups further reduced H2S emissions. Figure 6 (f) On day 3, the H2S emission rates of groups CK, CS11, F1, F2, and F3 reached their maximum values ​​of 0.40 mg / kg / h, 0.31 mg / kg / h, 0.34 mg / kg / h, 0.3 mg / kg / h, and 0.27 mg / kg / h, respectively. Group F4 reached its maximum value of 0.25 mg / kg / h on day 2. This is because easily decomposable organic matter decomposes rapidly during the warming and high-temperature periods of composting, leading to the consumption of large amounts of O2 by microorganisms, resulting in localized hypoxia and concentrated H2S emissions during this period. On day 14, H2S emissions from groups CK, CS11, and F1 were zero, while H2S emissions from groups F2, F3, and F4 were zero on day 9.

[0083] After composting, quality-related indicators of the compost were tested. A conductivity meter was used to measure the conductivity of the leachate, a pH meter was used to measure the acidity / alkalinity of the compost leachate, and the total amino acid content (ninhydrin colorimetric method) and the percentage of essential amino acids (high performance liquid chromatography) were determined after humus content was matured using the potassium dichromate oxidation method. The results are shown in Table 1, where all indicators represent the test indicators corresponding to the complete composting of each group.

[0084] Table 1:

[0085]

[0086] As shown in the table, the addition of microbial inoculants shortened the composting cycle to varying degrees during the composting process and significantly affected the carbon-nitrogen ratio. Specifically, F3 shortened the composting time by 17 days, and its carbon-nitrogen ratio was adjusted to 10.56:1 compared to the CK and CS11 groups. An excessively low carbon-nitrogen ratio can have a chain of negative effects on microbial activity, nitrogen utilization, soil ecology, and crop growth. The use of microbial inoculants effectively suppressed this low carbon-nitrogen ratio. Furthermore, the F3 group had a higher proportion of amino acids and essential amino acids than the CK group. Essential amino acids can be directly absorbed by plant roots and are more readily utilized than inorganic nitrogen (such as ammonium nitrogen), especially under low temperatures or when root activity is weak (such as in the seedling stage). In addition, the F3 group had the highest humus content and its electrical conductivity was adjusted to 3.25 mS / cm, demonstrating a significant effect on soil improvement. The changes in carbon-to-nitrogen ratio, electrical conductivity, pH, and total amino acid content in groups CK, CS11, F1, F2, F3, and F4 during composting with increasing composting time are as follows: Figures 7-10 As shown.

Claims

1. A microbial compound inoculant, characterized in that: Klebsiella pneumoniae ( Klebsiellasp. CS11, long-branch mold ( Trichoderma longibrachiatum ) and Azotobacter brownii ( Azotobacter chroococcum The bacteria were composed of bacteria in a mass ratio of 1:1:1, and the strain was classified as Klebsiella pneumoniae. Klebsiellasp. CS11, deposited at the China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with accession number CCTCC M20242943, and deposited on December 30, 2024.

2. The microbial compound inoculant as described in claim 1, characterized in that: The long-branch mold ( Trichoderma longibrachiatum The accession number is GDMCC No.: 3.139, and the described *Azotobacter brownis* ( Azotobacter chroococcum GDMCC No.: 1.

272.

3. The application of the compound microbial agent as described in claim 1 or 2 in fermentation composting.

4. The application as described in claim 3, characterized in that: The fermented compost is made from corn stalks and chicken manure as raw materials, and inoculated with a compound microbial agent for composting fermentation. The compound microbial agent is composed of Klebsiella CS11, Trichoderma longifolia and Azotobacter chrysophagus in a mass ratio of 1:1:1, and the inoculation amount of the compound microbial agent in the compost raw materials is 5%.

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