Bacillus amyloliquefaciens C-50-11 with high H2O2 yield and application of bacillus amyloliquefaciens C-50-11

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus amyloliquefaciens with high H2O2 yield and application of the bacillus amyloliquefaciens. According to the bacillus amyloliquefaciens C-50-11, H2O2 can be continuously generated in the high-temperature period of compost, H2O2 free radicals can oxidize and decompose complex organic matter to promote humus condensation in a mediated mode, the content of humus in the compost in the high-temperature period is further effectively increased, and compared with the content of humus in uninoculated compost, the content of the humus after inoculation is increased by 20% or above. According to the method disclosed by the invention, chemical degradation is realized by combining an endogenous H2O2 strengthening effect of thermophilic bacteria and utilizing an oxidizing agent generated by organisms, so that the condition of a high-temperature period of compost is fully utilized, and a new method way is provided for increasing the content of humus in the compost.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a thermophilic amyloliquefaciens C-50-11 strain with high H2O2 production and application thereof. Background Art

[0002] Traditional aerobic composting, operating at high temperatures of 50°C to 65°C, suffers from long maturation times, high odor emissions, and the rebound of residual antibiotic resistance genes (ARGs). Existing microbial enhancement methods, such as SBI agents, shorten maturation times but fail to systematically utilize hydrogen peroxide, which is active at high temperatures, to promote organic matter mineralization and humus formation.

[0003] The efficiency of humus formation during composting currently needs to be improved, and some composting technologies may not fully utilize the high-temperature conditions of the composting period to promote effective humus synthesis. Existing technologies primarily rely on physical manipulation, such as ultra-high-temperature fermentation or the addition of external inhibitors. This suggests that existing technologies focus solely on physical adsorption or the addition of conditioning agents, failing to utilize biologically produced oxidants for chemical degradation. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a thermophilic amyloliquefaciens C-50-11 with high H2O2 production and its application.

[0005] The technical solutions of the present invention are as follows.

[0006] The first aspect of the present invention provides a high-yield H2O2 accumulation thermophilic Bacillus C-50-11, the thermophilic Bacillus ( Caldibacillus thermoamylovorans ) The deposit number of C-50-11 is CGMCC No.33544, and the deposit date is February 17, 2025.

[0007] The present invention uses canteen kitchen waste and sawdust from the China Agricultural University Organic Recycling Research Institute (Suzhou) in a 3:1 mass ratio and a 25:1 carbon-nitrogen ratio. Water is then added to a moisture content of 65%, and 0.25 wt% permonosulfate is added to provide an active oxygen environment. Composting is then carried out. When the compost reaches a temperature of 50°C or higher and enters the high-temperature phase, a high-H₂O₂-producing bacterial strain, designated C-50-11, is isolated and identified as Thermomylolytic Bacillus amylolyticus. The Thermomylolytic Bacillus amylolyticus C-50-11 can continuously produce H₂O₂ at temperatures between 50°C and 65°C. H₂O₂ free radicals can oxidatively decompose complex organic matter, mediating the condensation of humus, thereby effectively increasing the humus content in the high-temperature compost. This fully utilizes the high-temperature phase of the compost, utilizing the H₂O₂ produced by Thermomylolytic Bacillus amylolyticus C-50-11 as an oxidant to achieve chemical degradation and accelerate the rate of decomposition.

[0008] The second aspect of the present invention provides the use of the thermophilic amyloliquefaciens C-50-11 in promoting the generation of H2O2 in composting.

[0009] In another preferred embodiment, the thermophilic Bacillus amyloliquefaciens C-50-11 is used to generate H2O2 during the high temperature period of composting.

[0010] In another preferred embodiment, the high temperature period of composting refers to composting at a temperature of 50°C to 65°C.

[0011] The third aspect of the present invention provides the use of the thermophilic amyloliquefaciens C-50-11 in increasing the humus content in compost.

[0012] In another preferred embodiment, said increasing humus in compost refers to promoting the formation of humus during the high temperature period of compost.

[0013] In another preferred embodiment, the high temperature period of composting refers to composting when the temperature reaches 50°C to 65°C.

[0014] A fourth aspect of the present invention provides a method for increasing the humus content in compost, comprising the following steps: When the compost temperature reaches 50°C to 65°C, inoculating the thermophilic Bacillus amyloliquefaciens C-50-11; The inoculation amount of the thermophilic amyloliquefaciens C-50-11 was 1×10 8 CFU / g~ 1×10 9 CFU / g.

[0015] In another preferred embodiment, the time for inoculating Thermomylobacillus amyloliquefaciens C-50-11 for compost fermentation is 4 to 12 days.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention involves mixing kitchen waste and sawdust from the canteen of the China Agricultural University Organic Recycling Research Institute (Suzhou) in a 3:1 mass ratio and a 25:1 carbon-nitrogen ratio. Water is then added to a moisture content of 65%, and 0.25 wt% permonosulfate is added to provide an active oxygen environment. Composting is then carried out. When the compost reaches a temperature of 50°C or higher and enters the high-temperature phase, a bacterial strain, designated C-50-11, is isolated and identified as Thermomylolytic Bacillus. The H2O2 produced by Thermomylolytic Bacillus C-50-1 can oxidatively decompose complex organic matter and promote humic condensation through free radicals. By inoculating Thermomylolytic Bacillus C-50-11 during the high-temperature phase of composting, the invention effectively increases the humus content in the compost, achieving a 20% increase compared to uninoculated compost.

[0017] The thermophilic Bacillus amyloliquefaciens C-50-11 of the present invention can improve the quality of compost products, making them more conducive to soil improvement and plant growth. In addition, the method has the advantages of simple operation and low cost, and is easy to promote and apply in actual compost production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a colony morphology diagram of the thermomyloliquefaciens C-50-11 of the present invention.

[0019] Figure 2 This is a growth curve of the thermomyloliquefaciens C-50-11 grown for 48 hours.

[0020] Figure 3 is the OD value of the thermomyloliquefaciens C-50-11 grown for 48 h. 600 Value result graph.

[0021] Figure 4 This is a graph showing the H2O2 production results of the thermomyloliquefaciens C-50-11 grown for 48 hours in the present invention.

[0022] Figure 5 This figure shows the results of the 48-hour growth of Thermomylobacillus amyloliquefaciens C-50-11 for compost yield; wherein HA represents humic acid, FA represents fulvic acid, and DP represents the degree of polymerization, which characterizes the stability of the humic acid structure; OH represents the C-50-11 inoculation treatment group, and CK represents the control group. DETAILED DESCRIPTION

[0023] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0024] The efficiency of humus formation during current composting processes needs to be improved, and some composting technologies may not fully utilize the high-temperature conditions of the composting period to promote effective humus synthesis. Existing technologies primarily rely on physical manipulation, such as ultra-high-temperature fermentation or the addition of external inhibitors, without incorporating the endogenous H₂O₂-enhancing effects of thermophilic bacteria. This suggests that existing technologies focus solely on physical adsorption or the addition of conditioning agents, failing to utilize biologically produced oxidants for chemical degradation.

[0025] In the prior art, thermophilic amyloliquefaciens is commonly used in anaerobic fermentation of organic solid waste to effectively increase methane production and organic matter conversion rate, or has protein hydrolysis function. This invention screened 68 bacterial strains from high-temperature compost and identified their extracellular hydrogen peroxide production ability. One of the bacteria was found to have the ability to produce high levels of H2O2 at 60°C. Molecular biological identification was performed on this strain, which was identified as thermophilic amyloliquefaciens ( Caldibacillus thermoamylovorans ), named C-50-11, and deposited in the General Microbiology Center of China Culture Collection of Microorganisms, with the deposit number: CGMCC No.33544, the deposit date: February 17, 2025, and the deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0026] The Thermomylolytic Bacillus C-50-11 in the present invention can continuously generate H₂O₂ during the high-temperature period of composting. H₂O₂ free radicals can oxidatively decompose complex organic matter, mediating the promotion of humus condensation, thereby effectively increasing the humus content in compost during the high-temperature period. Compared to the humus content in uninoculated compost, the humus content after inoculation increased by more than 20%. By combining the endogenous H₂O₂ enhancing effect of thermophilic bacteria and utilizing biologically produced oxidants to achieve chemical degradation, the present invention fully utilizes the conditions of the high-temperature period of composting, providing a new method and approach for increasing the humus content in compost.

[0027] According to the conventional wisdom regarding the invention patent application CN108339828B, entitled "Restaurant Waste Treatment System," restaurant waste, commonly known as swill, is waste generated by restaurants (including households) during the catering and cooking process. This waste can be in three forms: gas, liquid, and solid. These include solid forms such as table scraps and kitchen scraps; liquid forms such as oily wastewater like pot and dishwashing water; and gaseous forms such as kitchen and restaurant fumes. The restaurant waste in the embodiments of the present invention is in the solid form.

[0028] Example 1 Isolation, Culture and Identification of Thermomylobacillus C-50-11 (1) Isolation and culture of thermophilic amyloliquefaciens C-50-11 Ten grams of a 60°C thermophilic compost sample was extracted in 100 mL of sterilized 0.85 wt% NaCl. The extract was then diluted serially to identify 68 bacterial strains resistant to 60°C. The extracellular hydrogen peroxide production capacity of these 68 strains was determined, and one strain, designated C-50-11, was found to be capable of producing hydrogen peroxide at 60°C. The thermophilic compost sample was prepared by mixing canteen waste from the China Agricultural University (Suzhou) Institute of Organic Recycling with commercially available sawdust in a 3:1 mass ratio and a 25:1 carbon-nitrogen ratio. Water was then added to a moisture content of 65%, and 0.25 wt% permonosulfate was added to provide an active oxygen environment. The compost was then fermented and collected after the compost had heated to above 50°C.

[0029] The C-50-11 isolated above was cultured in the following manner: Culture medium formula: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 1000 mL distilled water, pH adjusted to 7.0, autoclaved at 121°C for 20 min.

[0030] A single colony was taken and cultured in a culture medium at 55°C, a shaking speed of 160 r / min, and a culture time of 16 h.

[0031] (2) Molecular biological identification of strains The C-50-11 strain was collected for whole-genome sequencing. Average nucleotide identity (ANI) is one of the most powerful metrics for identifying the closeness of bacterial genome relationships. It reflects the evolutionary distance between genomes by comparing the average of all orthologous protein sequences within the genome. An ANI > 95% indicates that two genomes belong to the same species. The ANI analysis results for the genome and the reference genome are shown in Table 1.

[0032] Table 1 ANI analysis results Note: ANI represents average nucleotide identity, Mapped_fragment represents mapped fragment, Query_fragment represents query fragment, and Taxon represents taxonomic group.

[0033] Example 2: Growth characteristic detection of thermophilic Bacillus amyloliquefaciens C-50-11 The thermomyloliquefaciens strain C-50-11 selected in Example 1 was activated in LB liquid medium and cultured at 55°C and 150 pm until the logarithmic growth phase. 600 About 0.5, where the growth curve and OD 600 like Figure 1 and Figure 2 shown.

[0034] The production of H2O2 was measured during the cultivation process, and the results were as follows: Figure 3 As shown, from Figure 3 It can be seen that the amount of H2O2 increases with time, and reaches the maximum when cultured for 40 hours.

[0035] Example 3: Promotion of Humic Substance Synthesis by Thermomylolytic Bacillus C-50-11 The isolated thermomyloliquefaciens C-50-11 was inoculated into the culture medium in Example 1, and cultured at 55° C. with a shaking speed of 160 r / min for 16 h for later use.

[0036] Kitchen waste and sawdust were mixed as compost raw materials, with the weight of kitchen waste accounting for 75% of the weight of the compost raw materials and the weight of sawdust accounting for 25% of the weight of the compost raw materials. When the moisture content of the compost raw materials was adjusted to 65%, they were placed in a 500mL micro-intelligent composting reactor for aerobic fermentation. When the compost temperature reached above 50℃, indicating that the compost had entered the high temperature period, the thermophilic amyloliquefaciens C-50-11 was added at a rate of 10 8 The inoculation rate of CFU / g was inoculated into the high temperature compost, which was recorded as the OH group, and the control group without inoculation of thermophilic amylobacillus C-50-11 was recorded as the CK group. The humus content in the compost was regularly tested. The results are as follows. Figure 5 shown.

[0037] from Figure 5 As can be seen, the humic acid content in the compost of the OH group increased overall over time compared to the CK group. Humic acid content in the OH group was significantly higher than in the CK group between days 4 and 8 of composting. This indicates that the thermomyloliquefaciens C-50-11 strain promoted the formation of humic acid. Humic acid, a major component of humic acid, improves soil structure and increases soil fertility. Inoculation with the thermomyloliquefaciens C-50-11 strain helped increase the humic acid content in the compost, thereby improving the quality of the compost.

[0038] The fulvic acid content in the OH group was also higher than that in the CK group at most time points. Between days 4 and 8 of composting, the fulvic acid content in the OH group was significantly higher than that in the CK group. Fulvic acid also plays an important role in soil quality and plant growth, such as regulating soil pH and promoting plant absorption of nutrients. Therefore, inoculation with Thermomyloliquefaciens C-50-11 helped increase fulvic acid content, indicating that Thermomyloliquefaciens C-50-11 can promote the transformation of organic matter during the composting process.

[0039] The trend of humic acid's degree of polymerization (DP) reflects the stability of the humic acid structure. As shown in the figure, the DP value of the OH group was higher than that of the CK group for most of the time, particularly between days 4 and 12 (the high-temperature period of composting), when the DP value of the OH group was significantly higher than that of the CK group. This indicates that inoculation with Thermomylobacillus amyloliquefaciens C-50-11 helps increase the degree of polymerization of humic acid, making the humic acid structure more stable and promoting its long-term preservation and function in the soil.

[0040] In addition, from Figure 5 The results show that when composting lasts longer than 12 days, the humic and fulvic acid contents decrease significantly, as does the humic acid degree of polymerization. This is primarily due to the rapid proliferation and metabolism of Thermomylocybin C-50-11 between days 4 and 12, which decomposes a large amount of organic matter, including humic acid and fulvic acid. By day 10, metabolism likely shifts from a rapid decomposition of organic matter to a phase where intermediates are utilized for other metabolic activities. This significant consumption of readily decomposable organic matter reduces the availability of precursors for humic and fulvic acid formation, leading to a decrease in humic and fulvic acid content. Furthermore, the decrease in humic acid degree of polymerization may also be due to changes in the activity of enzymes or related substances involved in humic acid polymerization. Therefore, after the high-temperature period of composting, the humic content in the compost reaches its highest level after 10 days of inoculation with Thermomylocybin C-50-11, increasing by 20% compared to the control compost without Thermomylocybin inoculation.

[0041] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0042] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A high-yielding H2O2 thermophilic amyloliquefaciens strain C-50-11, characterized in that: The thermophilic amyloliquefaciens ( Caldibacillus thermoamylovorans ) The deposit number of C-50-11 is CGMCC No.33544, and the deposit date is February 17, 2025.

2. Use of the thermomyloliquefaciens C-50-11 according to claim 1 in promoting H2O2 production in composting.

3. The use according to claim 2, characterized in that The thermophilic amyloliquefaciens C-50-11 is used for the generation of H2O2 during the high temperature period of composting.

4. The use according to claim 3, characterized in that The high temperature period of composting refers to the composting temperature of 50°C to 65°C.

5. Use of the thermomyloliquefaciens C-50-11 according to claim 1 in increasing the humus content in compost.

6. The use according to claim 5, characterized in that The increasing of humus in compost refers to promoting the formation of humus during the high temperature period of compost.

7. The use according to claim 5, characterized in that The high temperature period of composting refers to the composting period when the temperature reaches 50°C to 65°C.

8. A method for increasing the humus content in compost, characterized in that: The following steps are involved: When the compost temperature reaches 50°C to 65°C, inoculating the thermophilic Bacillus amyloliquefaciens C-50-11 according to claim 1 for compost fermentation; The inoculation amount of the thermophilic amyloliquefaciens C-50-11 is 10 8 CFU / g~ 10 9 CFU / g.

9. The method for increasing the humus content in compost according to claim 8, characterized in that: The time for compost fermentation by inoculating Thermomylobacillus amyloliquefaciens C-50-11 is 4 to 12 days.

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