Klebsiella, compound microbial agent and application of Klebsiella

Abstract

The invention relates to a microbial composite bacterial agent, which is prepared from Klebsiella sp. CS11, Trichoderma longibrachiatum and Azotobacter chroococcum according to a mass ratio of 1: 1: 1, the classification name of the bacterial strain is Klebsiella sp. CS11, and the preservation number of the bacterial strain is CCTCC M 20242943. When the Klebsiella CS11 strain is applied to aerobic composting of corn straw and chicken manure, H2S gas emission can be reduced, and the sulfur metabolism efficiency in the composting process can be improved. On the basis, the composite microbial agent further accelerates decomposition and conversion of organic matters, reduces H2S gas emission and improves sulfur fixation efficiency, meanwhile, the content of amino acid, humic acid and the like of compost is adjusted, high-quality organic fertilizer is produced, the soil environment is improved, cyclic utilization of resources is achieved, and agricultural sustainable development is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a Klebsiella strain, a composite microbial agent and applications thereof. Background Art

[0002] Composting is an important means of recycling organic waste, but it also produces 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, the control of odorous gas emissions during composting has always been an important topic in scientific research and engineering practice. Hydrogen sulfide is one of the main malodorous gases in the composting process, mainly derived from the anaerobic decomposition of sulfur-containing organic matter in animal manure. Hydrogen sulfide is a toxic gas. Long-term exposure can cause damage to the human respiratory tract, endocrine system, and nervous system, and may also induce diseases such as cancer.

[0003] Furthermore, if the sulfur content is too high during the composting process, under anaerobic conditions, sulfur can be easily reduced to pungent gases such as hydrogen sulfide. This not only pollutes the air at the composting site and surrounding environment, affecting air quality, but can also attract pests and pathogens, adversely affecting the quality and usability of the compost. However, sulfur is a key component of many enzymes and coenzymes in microbial metabolism. Excessively low sulfur content can limit microbial activity, slowing the decomposition of organic matter during composting and prolonging compost maturity, impacting compost production efficiency and quality. Ultimately, compost with too low a sulfur content cannot meet the sulfur needs of plants, leading to sulfur deficiency symptoms such as leaf chlorosis and yellowing, and stunted and weak plants. This can affect photosynthesis and other physiological functions, reducing crop yield and quality.

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

[0005] The present invention aims to provide a microbial agent, which includes a strain of Klebsiella, which can inhibit the generation of H2S and synthesize sulfate, regulate the amino acid content in compost, and improve the quality of compost.

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

[0007] The object of the present invention is achieved through the following technical solutions: A strain of Klebsiella, characterized in that: the strain is classified as Klebsiella ( Klebsiellasp. )CS11, deposited in China Center for Type Culture Collection, the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC NO. M 20242943, and the deposit date is December 30, 2024.

[0008] Furthermore, Klebsiella CS11 can metabolize sulfide in the environment and convert it into organic sulfur and inorganic sulfur.

[0009] Furthermore, Klebsiella CS11 can synthesize inorganic sulfur such as sulfate by regulating its own metabolism in a sulfur-free or sulfur-deficient environment.

[0010] Furthermore, Klebsiella CS11 inhibited the growth of H2S-producing bacteria by changing the microbial community structure during composting, thereby reducing H2S production.

[0011] The application of the above-mentioned Klebsiella CS11 in compost fermentation.

[0012] A microbial composite agent, characterized in that: it is based on Klebsiella ( Klebsiellasp. )CS11, long-branch mold ( Trichoderma longibrachiatum ) and Azotobacter chrysosphaeroides ( Azotobacter chroococcum ) are composed in a mass ratio of 1:1:1.

[0013] The strain is classified as Klebsiella Klebsiellasp. ) CS11, deposited in China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with the deposit number CCTCC NO. M20242943 and the deposit date December 30, 2024; The long branch mold ( Trichoderma longibrachiatum ) is deposited with GDMCC No.: 3.139, and the brown ball nitrogen-fixing bacteria ( Azotobacter chroococcum )GDMCC No.:1.272.

[0014] Application of the above composite microbial agent in fermentation composting.

[0015] Furthermore, the fermentation compost is made of corn straw and chicken manure as raw materials, and is inoculated with a composite microbial agent for compost fermentation. The composite microbial agent is composed of Klebsiella CS11, Trichoderma longifolia and Nitrogen-fixing Bacterium brown in a mass ratio of 1:1:1, and the inoculation amount of the composite microbial agent in the compost raw material is 5%.

[0016] During the composting process, CS11 metabolism changes the amino acid content in the compost, thereby improving the quality of the fertilizer.

[0017] The initial C:N of corn stalks is high, and even when compounded with chicken manure with a high N content, it is still high and not suitable for the growth of sulfur-fixing bacteria, resulting in unsatisfactory sulfur-fixing effect of the compost. In the present invention, a composite bacterial agent is used to participate in compost fermentation, wherein the long-branched Trichoderma decomposes complex organic matter such as lignocellulose in corn stalks, releasing small molecular carbon sources and sulfur-containing organic matter, providing energy and substrates for nitrogen-fixing bacteria and sulfur-fixing bacteria. Nitrogen-fixing bacteria synthesize ammonium nitrogen and organic nitrogen through growth metabolism, directly supplement the compost nitrogen source, and adjust the compost carbon-nitrogen ratio to a more suitable carbon-nitrogen ratio range. The combination of the composite bacterial agent in this process reduces the anaerobic fermentation of sulfur-containing substances, inhibits the production of H2S, and improves the sulfur-fixing effect of CS11. At the same time, it shortens the composting cycle, changes the amino acid content, humus content and electrical conductivity in the compost, and improves the quality of the compost.

[0018] Application of the above-mentioned composite microbial agent in soil improvement.

[0019] The above-mentioned composite microbial agent can be inoculated into nitrogen-deficient and sulfur-deficient soils to increase the nitrogen and sulfur content in the soil. The sulfur-fixing bacteria CS11 can synthesize organic sulfur and inorganic sulfur (sulfate, sulfite, thiosulfate, etc.) from sulfide in the soil. Sulfate is a form of sulfur that can be absorbed and utilized by plants. The activity of CS11 increases the sulfate content in the soil and promotes plant growth. In addition, the production of sulfate during the sulfur oxidation process releases hydrogen ions (H + ), it has a certain regulating effect on soil pH and can improve alkaline soil.

[0020] The present invention has the following technical effects: Klebsiella CS11 The strain, when applied to aerobic composting of corn stalks and chicken manure, can reduce H2S emissions and improve sulfur metabolism efficiency during the composting process. Furthermore, the composite microbial agent of the present invention further accelerates the decomposition and conversion of organic matter, reduces H2S emissions, and improves sulfur fixation efficiency. It also regulates the amino acid and humic acid content of the compost, producing high-quality organic fertilizer, improving the soil environment, enabling resource recycling, and promoting sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Changes in the expression levels of sulfur metabolism-related genes in bacterial strains at different time points, a: inorganic sulfur metabolism-related genes; b: organic sulfur metabolism-related genes.

[0022] Figure 2 :Changes in cysteine ​​and methionine contents 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.

[0023] Figure 3 :Determination of inorganic sulfur content in strain CS11, a: SO4 2- ; b:HS - ;c:SO3 2- ;d:S2O3 2- .

[0024] Figure 4 :Contents 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- .

[0025] Figure 5 :Changes in amino acid content of strain CS11 during composting, a: total amino acids, b: free amino acids.

[0026] 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.

[0027] Figure 7 : Changes in carbon-nitrogen ratio during composting in different treatment groups with composting time.

[0028] Figure 8 : Changes in electrical conductivity during composting in different treatment groups along with composting time.

[0029] Figure 9 : Changes of pH value during composting in different treatment groups along with composting time.

[0030] Figure 10 : Changes in moisture content during composting in different treatment groups along with composting time. DETAILED DESCRIPTION

[0031] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to 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-mentioned contents of the present invention.

[0032] The long branch mold used in the present invention ( Trichoderma longibrachiatum ) is deposited with GDMCC No.: 3.139; Brown ball nitrogen-fixing bacteria ( Azotobacter chroococcum) GDMCC No.: 1.272 was purchased from Guangdong Microbial Culture Collection Center (GDMCC).

[0033] The corn straw used in the present invention was taken from the experimental field of Forestry 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 Biology.

[0034] The culture medium used in the present invention is as follows: LB medium (g / L): yeast extract 5.0, NaCl 10.0, tryptone 10.0; 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.) Starky -Na2S2O3 medium (g / L): (NH4)2SO40.3, KH2PO43.5, MgSO40.5, CaCl2·2H2O0.33, FeSO4·7H2O 0.018, Na2S2O310.0; 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. (Sulfide-free medium lacks sulfide; all other components are the same.)

[0035] Example 1 Isolation, screening and identification of strain CS11 (1) Separation: Fresh chicken manure samples were collected from the Gou Gou Poultry Farm in Liaoyuan City in the late egg-laying period. Stones, feathers and other debris were removed, the samples were sealed in ziplock bags and stored at -20 °C for later use.

[0036] (2) Screening: Weigh 10 g of chicken manure sample, dissolve it in 90 mL of sterile water, add 3 mm small glass beads, and incubate at 30°C and 180 r / min for 30 min; let it stand for 5 min, take 10 mL of supernatant and inoculate it into 100 mL of liquid enrichment medium, and incubate it at 30°C and 180 r / min for 2 days; take 5 mL of enrichment liquid and inoculate it into selective medium and incubate it for 3 days. After continuous transfer 3 times, perform gradient dilution, and the dilution multiples are 10-5, 10-6, 10-7, 10-8, 10-9, 10-10, 10-11, and 10-12 respectively. Take 200 μL of bacterial liquid and spread it on solid selective medium, repeat 3 times, and incubate it at 30°C for 24 h. Pick single colonies with different morphologies, perform multiple streaking culture, and obtain monoclonal strains by purification. It was transferred to selective culture medium and cultured at 30°C and 180 r / min for 24 h. The SO 42- concentration in the culture 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 the higher conversion rate was selected and numbered CS11.

[0037] (3) Identification: Strain Morphological Identification: Colonies of strain CS11 are round, with a smooth, raised surface and regular margins. They are opaque, milky white, easy to pick, and relatively moist. Gram staining is negative. The bacteria are short rod-shaped and small. Strain CS11 is a medium-sized rod-shaped bacterium, 0.7-1.0 μm wide and 1.0-2.2 μm long, with blunt ends and dispersed distribution.

[0038] Physiological and biochemical characterization: This bacterium can utilize glucose, maltose, mannitol, and sucrose, but not lactose. It can degrade cellulose and hydrolyze starch, but does not produce H2S. The VP test is positive and the MR test is negative, and it does not produce phenylalanine deaminase.

[0039] After sequencing the PCR-derived sequence using BLAST, the strain was found to be highly homologous to Klebsiella spp., with a similarity of 99.7%. The closest relative of strain CS11 was Klebsiella spp. Based on morphological characteristics and physiological and biochemical identification results, strain CS11 was identified as Klebsiella spp. Klebsiella sp. ).

[0040] The strain is biologically preserved, and the strain is specifically classified as Klebsiella ( Klebsiellasp.)CS11, deposited in China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with the deposit number CCTCC M 20242943 and the deposit date of December 30, 2024.

[0041] Example 2 Sulfur metabolism of Klebsiella CS11: 5% CS11 cells were inoculated into sulfide culture medium and the expression and content of sulfur metabolism-related genes were measured at different times. 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 (Fusheng Industrial).

[0042] 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 were measured at different times.

[0043] (1) Changes in gene expression and substance content of Klebsiella CS11: The expression levels of sulfur metabolism-related genes in bacterial strains at different time points are shown in Figure 2. Figure 1 As shown, Figure 1 a is a gene related to inorganic sulfur metabolism, sbp 、 cysD 、 cyh 、 CysI 、 SiR The gene content was the highest at 36 hours. The reason for the increase in the content of these genes may be due to the metabolic needs of the strain: these genes are involved in sulfur metabolism in the organism ( ‌cysD 、 ‌cysH 、 ‌CysI 、 SiR )‌ or transcriptional regulation (‌SBP)‌,‌ when the organism is in a specific physiological state or environmental conditions,‌ the demand for corresponding metabolites increases,‌ thereby driving the increased expression of these genes.‌ cysN 、 cysC The gene expression peaked at 48 hours, likely because in CS11, these genes are involved in the assimilation and reduction of sulfate, providing the organism with an essential sulfur source. When sulfur is deficient in the environment or when the organism is in a specific physiological state, the expression of these genes increases to meet the organism's sulfur needs. Figure 1 b is related to organic sulfur metabolism. Compared with inorganic metabolism, the expression levels of genes in the organic metabolism process are mostly increased compared with those in the sulfur-free environment. CTH It is an enzyme that promotes the decomposition of cysteine, but metBThe gene encodes the protein homocysteine ​​synthetase, which is involved in the methionine biosynthesis pathway. metB The gene expression level was always lower than CTH This is because CTH The enzyme encoded by the gene is involved in another key step in cysteine ​​metabolism, the synthesis of cysteine ​​from cysteine, so in the process of cysteine ​​metabolism, metB Gene expression levels are usually lower than CTH Gene. At 24h metH The highest content is related to the oxidative stress in the process of cysteine ​​metabolism. Methamphetamine can affect the metabolism of intracellular cysteine ​​by inducing oxidative stress, thereby leading to the methylation of genes ( metH ) expression level increased. cysK The gene promotes the metabolism of sulfide to cysteine. CysK The larger increase at 48h was related to the presence of more cysteine ​​in the supernatant at this time. metH and metA The reason for this is that, at 48 hours, most of the conversion occurs to cysteine, with a small amount converted to methionine. Furthermore, inorganic content decreases overall after 48 hours, prompting more inorganic sulfur to be converted to organic sulfur. This suggests that CS11 can utilize inorganic sulfur and convert it into organic sulfur through metabolism.

[0044] (2) Changes in cysteine ​​and methionine content in strain CS11: Figure 2 As shown in a, in the sulfur-free medium, the initial cysteine ​​content was 0, and then increased. The cysteine ​​content reached a maximum of 0.0438 μmol / mL at 36 h, and then continued to decrease, as shown in Figure 2As shown in Figure b, the initial cysteine ​​content in strain CS11 was 0.1042 μmol / mL. In sulfur-free medium, the cysteine ​​content in the cells gradually decreased, reaching a minimum of 0.0200 μmol / mL at 48 hours. Cysteine ​​content showed a downward trend before 12 hours, as intracellular cysteine ​​exists primarily as glutathione or cysteine ​​residues, which participate in cellular antioxidant and reactive oxygen species scavenging processes. In sulfur-containing medium, the cysteine ​​content reached a maximum of 0.1289 μmol / mL at 24 hours and a minimum of 0.0441 μmol / mL at 36 hours. When cells are exposed to oxidative stress, these sulfur-containing amino acids are released extracellularly, leading to an increase in cysteine ​​content in the supernatant. Alternatively, the strain may secrete cysteine-containing proteins or enzymes, which increase the cysteine ​​content in the supernatant. Simultaneously, the release of these secretions can lead to a decrease in intracellular cysteine. The substances that contribute to the content are all present in the form of sulfur-containing compounds. The increase in content at 24 hours may be due to the metabolism of sulfur-containing compounds by the strain, from sulfide to cysteine. The subsequent decrease in content and subsequent increase may be due to the metabolism of methionine during the conversion to cysteine. In sulfur-free medium, the significant increase in content at 36 hours may be due to the body's own growth and metabolism, the increase in cysteine ​​content caused by other metabolic processes, and the long growth period of the strain, which leads to a decrease in the number of viable cells and the release of some cysteine.

[0045] Figure 2 The overall change trend of methionine content in the sulfur-free medium was small, and the methionine content in the sulfur-containing medium at 36 h was the highest in the bacterial and external bacteria treatment groups. Figure 2 As shown in Figure d, the initial methionine content in the strain was 125.1551 pg / mL. In the sulfur-free medium, the strain showed an overall upward trend and reached 352.4492 pg / mL at 48 h. However, in the sulfur-containing medium, the strain showed an overall downward 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 showed an overall upward trend, which is consistent with the gene expression levels in the first 24 h that we saw previously. CTH, metC The methionine content was the highest at 36 h, which indicates that the synthesis or metabolism of methionine may be at its peak at this time point. The cysteine ​​content was the lowest, which may mean that the consumption or conversion of cysteine ​​was relatively active at this time point. CTH The gene expression level was the highest at 36h. CTHThe gene encodes cysteine ​​hydrolase, which is responsible for catalyzing the decomposition of cysteine. In the supernatant, the methionine content in the sulfur-containing culture medium increased significantly at 36 hours, and various metabolites were secreted during the culture process, including amino acids, organic acids, etc. As the culture time increases, these metabolites will gradually accumulate in the supernatant, resulting in an increase in the methionine content. At the same time, the strain may increase the synthesis or release of methionine by adjusting its own metabolic process, thereby increasing the methionine content in the supernatant. However, as the culture time increases, the bacteria may gradually use these methionines as a nutrient source, resulting in a decrease in the methionine content in the supernatant at 48 hours.

[0046] (3) Effects of Klebsiella CS11 on sulfur metabolism: Figure 3 a is SO4 generated by CS11 in sulfate culture medium 2- Concentration, the highest SO4 2- The concentration is 29.239 mg / L in 20h. Figure 3 b shows the change in sulfide concentration of CS11 metabolized in sulfide medium. The initial sulfide concentration was 200 μg / mL. As the strain metabolized, the sulfide concentration dropped to 0 at 12 h and then remained at 0.

[0047] 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. Sulfite concentration peaked at 120 μg / mL at 24 h and then decreased continuously as the strain metabolized the sulfite. The initial thiosulfate concentration was 112 μg / mL, reaching a peak of 168 μg / mL at 24 h before decreasing. 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 for sulfite and sulfite were similar, indicating that CS11 can utilize thiosulfate and sulfite as nutrients to provide energy for subsequent metabolic processes. Therefore, Klebsiella CS11 is able to metabolize sulfide, thiosulfate, and sulfite, providing a reference for subsequent studies of its sulfide-producing mechanism. Furthermore, in the absence of external sulfur, CS11 can also synthesize inorganic sulfur by regulating its metabolic processes.

[0048] (4) Content of sulfur metabolism-related substances in bacterial strains at different time points At different time points, the content of sulfur metabolism-related substances in the culture medium changes as follows Figure 4 As shown. Figure 4As shown in a, the overall sulfur content showed an upward trend, and the sulfate content reached the highest at 12h, which may be due to sbp 、 cysD、cysN Genes convert external sulfur-containing substances into sulfate-containing substances in cells. Sulfide changes such as Figure 4 As shown in b, the initial sulfide content in the sulfur-containing medium was 7.49 mg / mL, which was 0 at the 12th hour and continued until 48 hours. The sulfide content in the sulfur-free medium was always 0. This means that strain CS11 can metabolize and convert sulfide, thereby reducing the sulfide content. Figure 4 As shown in Figure c, the sulfate content reached its highest at 12 h, reaching 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 time may be due to the fact that the strain was inoculated in a sulfur-free environment. In the absence of sulfur, it can use related nutrients to convert them into sulfate and other substances to maintain its growth and development. Figure 4 As shown in Figure d, the sulfite content showed an overall downward trend in the sulfur-free medium, but increased to 11.60 μg / mL at 24 h in the sulfur-containing medium. Figure 4 As shown in Figure e, thiosulfate levels showed an overall downward trend in sulfur-free medium, reaching a peak of 16.80 μg / mL at 24 h in sulfur-containing medium. In sulfur-free medium, the strain can metabolize thiosulfate into sulfite through its own metabolism. Sulfite also undergoes further metabolic processes within the strain, converting it into other substances, such as organic sulfur compounds like cysteine.

[0049] Example 3 Composting strain CS11: Three groups of composts mixed with chicken manure and corn straw were set up, with the mass ratio of straw to chicken manure being 2.5:1. They were the CK group, the CS11 group (chicken manure mixed with corn straw and added with 5% CS11 microbial agent), and the CABA group (chicken manure mixed with corn straw and added with a commercially available microbial agent).

[0050] Changes in amino acid content during composting: Depend on Figure 5It is known that during the composting process, 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 the composting process progresses, resulting in 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. At the same time, during the composting process, the decomposition of organic matter and the evaporation of water cause the total mass of the compost material to decrease, resulting in a relative decrease in the total organic sulfur content. At the end of the composting period, the cystine content in the CS11 group was the highest 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. The results indicate that the addition of strain CS11 can slightly increase the content of cysteine ​​and methionine, thereby promoting their conversion to cystine.

[0051] Example 4 Composting the compound bacterial agent: Three compost piles were set up with chicken manure and corn straw mixed at a 2.5:1 straw-to-chicken manure mass ratio. A 5 wt.% composite microbial agent was added. The composite agents were F1 (CS11, Trichoderma longibrachiatum, and Aspergillus niger combined at a mass ratio of 1:1:1), F2 (CS11, Aspergillus niger, and Azotobacter chrysogenum combined at a mass ratio of 1:1:1), F3 (CS11, Trichoderma longibrachiatum, and Azotobacter chrysogenum combined at a mass ratio of 1:1:1), and F4 (CS11, Trichoderma longibrachiatum, Aspergillus niger, and Azotobacter chrysogenum combined at a mass ratio of 1:1:1). (These composting tests were conducted in winter.)

[0052] On days 1 to 15 and 21 and 35 after the start of composting in Examples 3 and 4, gas samples were measured using the static chamber method. On days 0, 3, 7, 14, 21, 27, 35, and 45, solid samples were collected from the upper, middle, and lower layers of the compost using a five-point sampling method and mixed evenly for the determination of total sulfur, sulfate, and sulfide indicators. SO4 was determined according to the method of Mussa SAB et al. (2009). 2－ Sulfide content. Sulfide determination refers to HJ883-2017. Sulfite determination refers to GB5009.34-2003 (CK group without any bacterial agent added).

[0053] Hydrogen sulfide gas determination: At 2:30 PM every day, a self-made static box was placed above the pile until 8:30 AM the next day. The H2S concentration was measured using a pump-type portable H2S meter, and the H2S gas emission rate and cumulative emission formula were calculated as follows: (1) Emission rate:

[0054] F : Gas emission rate (mg / kg / h); h : Height of the gas extraction space above the pile (m); t : average temperature of the gas collection space (°C); M : molar mass of the gas (g / mol); V m : molar volume of gas under standard conditions (L / mol); dC / dt : gas concentration change rate; A : Game surface area (m 2 ); m : Total mass of the pile (kg).

[0055] (2) Calculation formula for cumulative gas emissions:

[0056] f : Cumulative gas emissions during the entire composting process (mg / kg); F i 、F i+1 : gas emission rates of the i-th and i+1-th composting respectively; t i+1 -t i : The time interval between two consecutive samplings, in d.

[0057] The test results are as follows Figure 6 As shown in (a), the initial total sulfur contents of the treatment groups were 2.44%, 2.48%, 2.36% / 2.33%, 2.68%, and 2.53%, respectively. The overall trend was first decreasing and then increasing. On the 45th day of composting, the total sulfur contents of the treatment groups were 13.43%, 15.56% / 12.36%, 11.99%, 16.77%, and 14.72%, respectively. Figure 6 (b) Initial SO4 of each treatment group 2- The contents were 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 had a maximum of 5.68 g / kg on the 14th day, and on the 21st day, the CS11, F1, F3 and F4 groups all reached their maximum values, which were 12.69 g / kg, 6.09 g / kg, 9.77 g / kg and 6.83 g / kg respectively. On the 45th day of composting, the SO4 2-The contents of SO4 in group F3 were reduced 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 the first day. 2- The content increased by 0.11g / kg compared with the 0th day. Figure 6 In (c), the CK group reached a maximum sulfite content of 3.56 g / kg on day 7, while the other treatments (CS11, F1, F2, F3, and F4) reached their maximum values ​​on day 27, with sulfite contents of 4.85 g / kg, 3.51 g / kg, 3.33 g / kg, 5.17 g / kg, and 4.35 g / kg, respectively. On day 45, sulfite contents in 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 Sulfide levels in all treatments in (d) showed an overall trend of first decreasing, then increasing, and then decreasing again. Sulfide levels in the CK and F1 groups reached their maximum values ​​of 24.16 mg / kg and 23.85 mg / kg on day 21. On day 27, sulfide levels in the CS11, F2, F3, and F4 groups reached their maximum values ​​of 26.96 mg / kg, 28.70 mg / kg, 31.13 mg / kg, and 29.55 mg / kg, respectively. On day 45 of composting, sulfide levels 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. However, sulfide levels in the CS11, F3, and F4 groups increased by 0.62 mg / kg, 1.88 mg / kg, and 1.12 mg / kg, respectively. The total amount of inorganic sulfur in the final compost of the F3 group was higher than that of the CS11 group, which showed that the synergistic effect of the F3 composite microbial agent improved the sulfur fixation capacity of CS11. Figure 6 The cumulative emissions in (e) show the control of H2S by each treatment. The cumulative H2S emissions of 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 indicates that Klebsiella CS11 significantly reduced the total H2S emissions. On this basis, the composite bacterial agents in the F3 and F4 groups further reduced H2S emissions. Figure 6(f) On day 3, H2S emission rates 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 for the CK, CS11, F1, F2, and F3 groups, respectively. However, the F4 group reached its maximum value of 0.25 mg / kg / h on day 2. This is because readily decomposable organic matter rapidly decomposes during the warming and high-temperature periods of the composting process, leading to microbial consumption of large amounts of O2 and localized hypoxia, resulting in concentrated H2S emissions during this period. H2S gas emissions were zero for the CK, CS11, and F1 groups on day 14, while those for the F2, F3, and F4 groups were zero by day 9.

[0058] After composting, we tested the compost for quality indicators. A conductivity meter measured the electrical conductivity of the extract, a pH meter tested the acidity and alkalinity of the extract, and potassium dichromate oxidation was used to determine the total amount of amino acids (ninhydrin colorimetry) and the proportion of essential amino acids (high-performance liquid chromatography) after composting. The results are shown in Table 1. All indicators in the table represent the corresponding test indicators for each group of compost when it was fully composted.

[0059] Table 1:

[0060] As shown in the table, the addition of microbial agents during the composting process shortened the compost maturity period to varying degrees, significantly affecting the carbon-nitrogen ratio. The F3 compost shortened the compost maturity period by 17 days, achieving a carbon-nitrogen ratio of 10.56:1 compared to the CK and CS11 composts. A low carbon-nitrogen ratio can negatively impact microbial activity, nitrogen utilization, soil ecology, and crop growth. However, the addition of microbial agents suppressed this low carbon-nitrogen ratio. Furthermore, the F3 compost had higher amino acid and essential amino acid ratios than the CK compost. Essential amino acids are directly absorbed by plant roots and are more readily available than inorganic nitrogen (such as ammonium). This is particularly true under low temperatures or when root activity is low, such as during the seedling stage. Furthermore, the compost had the highest humus content and an electrical conductivity of 3.25 mS / cm, significantly contributing to soil improvement. The carbon-nitrogen ratio, conductivity, pH and total amino acid content of the CK, CS11, F1, F2, F3 and F4 groups changed with the extension of composting time. Figure 7-10 shown.

Claims

1. Application of a strain of Klebsiella CS11 in compost fermentation, characterized in that: The strain is classified as Klebsiella Klebsiellasp. )CS11, deposited in China Center for Type Culture Collection, the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC NO.M 20242943, and the deposit date is December 30, 2024.

2. A microbial composite agent, characterized in that: Klebsiella ( Klebsiellasp. )CS11, long-branch mold ( Trichoderma longibrachiatum ) and Azotobacter chrysosphaeroides ( Azotobacter chroococcum ) are composed in a mass ratio of 1:1:

1.

3. The microbial composite agent according to claim 2, wherein: The strain is classified as Klebsiella Klebsiellasp. ) CS11, deposited in China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with the deposit number CCTCC NO. M 20242943 and the deposit date December 30, 2024; The long branch mold ( Trichoderma longibrachiatum ) is deposited with GDMCC No.: 3.139, and the brown ball nitrogen-fixing bacteria ( Azotobacter chroococcum )GDMCC No.:1.

272.

4. Use of the composite microbial agent as claimed in claim 2 or 3 in fermentation composting.

5. The use according to claim 4, characterized in that: The fermentation compost is prepared by using corn stalks and chicken manure as raw materials and inoculating a composite microbial agent for compost fermentation. The composite microbial agent is composed of Klebsiella CS11, Trichoderma longifolia and Azotobacter chlorosphaer in a mass ratio of 1:1:1, and the inoculation amount of the composite microbial agent in the compost raw materials is 5%.

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