Bacillus composti with high ammonia assimilation capacity and application thereof

Abstract

The application discloses a high-efficiency ammonia-assimilating Bacillus compostus and application thereof, and relates to the technical field of microorganisms. Bacillus stercoris The strain is QY6, which has been preserved in the China General Microbiological Culture Collection Center (No. 3, Beichen West Road, Chaoyang District, Beijing) on December 23, 2024, with a preservation number of CGMCC No. 33139. The strain or a biological bacterial agent prepared from the strain can be used for composting of organic solid waste, so that ammonia emission can be significantly reduced; the composting product can be applied to soil, so that the contents of soil available nitrogen, organic matter and humic acid can be significantly increased; and the product can be used for cultivation of Chinese cabbage, so that the fresh weight can be significantly increased and the content of soluble sugar can be increased. The application provides a high-efficiency nitrogen-preserving and decay-promoting microbial strain, which can be used in the fields of agricultural waste resource utilization, composting quality improvement and efficiency enhancement and agricultural planting.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Bacillus compostii strain QY6 with highly efficient ammonia assimilation capabilities and its applications. Background Technology

[0002] Composting is an important technology for reducing, rendering harmless, and recycling organic solid waste. However, the significant nitrogen loss (74%–90%) caused by NH3 volatilization during composting and the low efficiency of humic acid synthesis are core issues restricting the quality of compost products. Humic acid, a key indicator of compost maturity, depends on the coupling of carbon and nitrogen metabolism driven by microorganisms, while severe nitrogen loss significantly hinders the humification process. Currently, the main technical routes for reducing NH3 emissions from composting include nitrification, denitrification regulation, physical adsorption, and acidification adjustment. Among these, nitrifying bacteria can convert NH4+ into nitrogen. + The conversion of -N to NO3⁻-N is the most common microbial regulatory pathway. However, this type of technology only changes the inorganic form of nitrogen and does not convert it into a stable organic nitrogen pool. It still has problems such as nitrate nitrogen leaching, denitrification producing N2O greenhouse gas, and limited effect on promoting humification.

[0003] In contrast, NH4 is assimilated via microbial ammonia pathways. + -N is directly converted into organic nitrogen and further participates in humic formation, which is considered an ideal pathway that can simultaneously achieve the coupling of "nitrogen retention and decomposition promotion". Ammonia assimilation converts NH4+ into organic nitrogen through the glutamate dehydrogenase pathway or the glutamine synthase-glutamate synthase pathway. + The conversion of nitrogen (-N) into amino acids provides precursors for humic acid polymerization and is a crucial mechanism for nitrogen retention and decomposition in composting. Existing ammonia assimilation microbial agents suffer from limitations, including some strains exhibiting significant ammonification effects but limited nitrogen retention efficiency. Furthermore, there is insufficient research on the screening, functional verification, and systematic application in composting, soil improvement, and crop cultivation of highly efficient ammonia assimilation strains. Moreover, most existing composting microbial agents focus on nitrification or organic matter degradation, with extremely limited research on the screening and application of compost-specific microbial strains that utilize ammonia assimilation as the dominant nitrogen transformation pathway. Strains with engineering applicability and functional stability are also lacking.

[0004] By enhancing the ammonia assimilation process, promoting composting humification, and improving the functional activity of humic acid in compost, humic acid becomes more advantageous in heavy metal remediation and microbial application. On the one hand, humic acid can affect the form and mobility of metals through complexation adsorption and mediating redox reactions; on the other hand, as a carrier, humic acid can enhance the immobilization efficiency and environmental stability of microorganisms, thereby improving the activity retention and remediation effect of the microbial system.

[0005] Therefore, developing a functional microbial strain capable of nitrogen transformation driven by ammonia assimilation in a composting system is of great practical significance for overcoming the limitations of traditional nitrification-based ammonia reduction technology, achieving efficient nitrogen retention and synergistic humification during the composting process, addressing the pain points of nitrogen loss and insufficient humification in composting, improving the quality and functional characteristics of compost products, and promoting the efficient utilization of agricultural waste and the development of green agriculture.

[0006] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a composting Bacillus strain with ammonia assimilation capabilities, which can convert NH4+ into nitrogen during composting. + -N is converted into organic nitrogen, thereby inhibiting NH3 volatilization and promoting humification, achieving efficient biological fixation and stabilization of nitrogen in the composting system. This strain can significantly reduce ammonia emissions when used in composting, and when applied to soil, it can significantly increase crop fresh weight and soluble sugar content, effectively contributing to the resource utilization of agricultural waste.

[0008] To achieve the above objectives, this invention provides a composting Bacillus strain with highly efficient ammonia assimilation capabilities (…). Bacillus stercoris This strain, QY6, was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33139. This Bacillus composting strain QY6 can release NH4+ in a composting environment. + -N is converted into organic nitrogen via the glutamate dehydrogenase pathway or the glutamine synthase-glutamate synthase pathway, and further participates in the formation of humic substances, thus preferentially converting NH4 under conditions where the ammonia assimilation pathway is dominant. + -N represents organic nitrogen, achieving a coupling of nitrogen retention and decomposition promotion during the composting process, while inhibiting NH3 volatilization.

[0009] The present invention also provides a composting nitrogen-retaining biological agent that uses ammonia assimilation as the nitrogen conversion pathway, the biological agent comprising the aforementioned Bacillus compostii.

[0010] Preferably, the effective viable count of Bacillus compostingus QY6 in the above-mentioned biological agent is ≥ 1×10⁻⁶. 8 This biological agent, with a concentration of cfu / mL, can be used to reduce ammonia emissions and increase the total nitrogen and humic acid content of compost during the composting process.

[0011] This invention also provides a nitrogen retention method for composting based on the ammonia assimilation pathway, wherein the above-mentioned biological agent is inoculated into the composting raw materials during the composting process to retain NH4+. + -N is converted into organic nitrogen through microbial assimilation and participates in the humification process.

[0012] Preferably, the above-mentioned nitrogen retention method for composting further includes improving the efficiency of humic acid synthesis by promoting the formation of humic acid precursor substances, and by regulating microbial metabolic pathways and organic matter decomposition to form more precursors that can participate in humic acid condensation in the composting system, thereby promoting the generation and enrichment of humic acid.

[0013] Preferably, in the above-mentioned composting nitrogen retention method, the prepared compost-source humic acid can be used for heavy metal pollution remediation. The compost-source humic acid is directly added to the heavy metal pollution system, or used as a carrier to load heavy metal remediation microorganisms to form a composite remediation material, so as to achieve synergistic removal and stabilization of heavy metals. The heavy metal is hexavalent chromium Cr(VI), and the dosage of compost-source humic acid is 10–500 mg / L. Alternatively, Bacillus heavy metal remediation bacteria can be loaded onto the compost-source humic acid as a carrier to form a humic acid-microorganism composite material for the reduction and removal of Cr(VI) in water or soil.

[0014] Preferably, in the above-mentioned composting nitrogen retention method, the inoculation amount of biological agent is 1% of the mass of compost raw materials.

[0015] The present invention also provides a soil conditioner, which is a composting product of the aforementioned Bacillus compostii.

[0016] The present invention also provides a soil improvement method by applying the above-mentioned soil conditioner during crop cultivation.

[0017] The Bacillus spp. QY6 provided by this invention can be used in any of the following ways: promoting nitrogen retention in compost; promoting the formation of humic acid precursors; promoting humification of compost; soil improvement; promoting crop growth; for preparing or obtaining compost-derived humic acid; for remediating heavy metal pollution; and / or for preparing compost-derived humic acid as a carrier to immobilize microorganisms for remediating heavy metal pollution.

[0018] The present invention has the following advantages: Compared with existing composting ammonia reduction technologies that are mainly based on nitrification or physical adsorption, the Bacillus spp. QY6 for composting provided by this invention uses ammonia assimilation as the nitrogen transformation pathway, and can convert NH4+ into nitrogen. + -N is directly fixed into organic nitrogen, thereby significantly reducing NH3 volatilization loss and simultaneously promoting humic formation, increasing the total nitrogen and humic acid content in compost. This pathway avoids the risks of nitrate nitrogen accumulation and its leaching, as well as greenhouse gas emissions, achieving a synergistic enhancement of nitrogen stabilization and humification.

[0019] This invention provides a Bacillus composting strain QY6 with ammonia assimilation capabilities. This strain exhibits excellent nitrogen retention efficiency in composting by assimilating NH4+ through an ammonia assimilation pathway. + -N is converted into amino acids, which greatly increases the nitrogen content of compost.

[0020] This strain has a prominent humification-promoting effect, which can significantly increase humic acid content, accelerate the conversion of unstable organic matter into stable humic acid, shorten the composting cycle, and improve the compost maturity.

[0021] When this strain or its prepared biological agent is applied to the soil, it can improve the soil, significantly enhance soil fertility, significantly increase the total organic carbon content of the soil, enhance the soil activation capacity, and maintain the soil pH within a suitable range.

[0022] This strain can significantly improve crop growth indicators, promoting plant height, root length, and fresh biomass; it also significantly improves crop quality, increasing soluble sugar, soluble protein, and total nitrogen content. Applying this strain helps reduce the need for chemical fertilizers, mitigates environmental pollution caused by nitrogen loss from composting, and contributes to the agricultural ecological cycle. Attached Figure Description

[0023] Figure 1 NH4 of the 7 strains initially screened in this invention + -N conversion capability status.

[0024] Figure 2 The NH4+ of strain QY6 screened in this invention at different times + -N transformation capacity and growth of the strain on streak plates.

[0025] Figure 3 The results of measuring various nitrogen contents and humification indicators after composting were obtained from the inoculation of strain QY6 in a small-scale composting trial.

[0026] Figure 4 This report describes the determination of humic acid, fulvic acid, and HA / FA in the compost after inoculation with QY6 in a small-scale composting trial.

[0027] Figure 5 The results of TOC, amino acids, carboxyl groups, polyphenols, reducing sugars, and polysaccharides in compost after inoculation with QY6 in a small-scale composting trial. Figure 6 This study describes the determination of the content of various forms of nitrogen in compost after inoculation with QY6 in large-scale composting.

[0028] Figure 7 This report describes the determination of humic acid, fulvic acid, and HA / FA in the compost after inoculation with QY6 in large-scale composting.

[0029] Figure 8 The results show the determination of TOC, amino acids, carboxyl groups, polyphenols, reducing sugars, and polysaccharide in the compost after inoculation with QY6 in large-scale composting.

[0030] Figure 9 This study verifies the soil improvement effect of QYO, a compost product obtained after inoculation with QY6.

[0031] Figure 10 This study verifies the effect of applying QYO, a compost product obtained after inoculation with QY6, to potted Chinese cabbage.

[0032] Figure 11 The effect of compost-derived humic acid extracted from the compost product obtained by inoculating QY6 on Cr(VI) remediation, and the comparison of the effect of compost-derived humic acid as a carrier loaded with microorganisms on Cr(VI) remediation. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0035] Example 1: Isolation, Screening and Identification of Ammonia Assimilating Bacteria 1. Screening of strains Eutrophic lake bottom sludge was collected, and 20 bacterial strains were obtained through enrichment culture and screening. These strains were then inoculated onto ammonia assimilation solid medium, and 7 viable strains, designated QY1 to QY7, were screened from the plates. This indicates that the initially screened strains can utilize NH4+. + -N was used as a nitrogen source for ammonia assimilation. Single colonies were picked from the plates and inoculated into 50 mL of sterile ammonia assimilation medium. The ammonia assimilation medium was formulated as follows: 0.25 g ammonium chloride, 5 g glucose, 1 g dipotassium hydrogen phosphate, 3.2 g potassium dihydrogen phosphate, 0.1 g ferric ammonium citrate, 0.5 g sodium chloride, 0.1 g magnesium sulfate heptahydrate, and 1 L pure water.

[0036] The initially screened strains were cultured in a shaker at 170 rpm and 33℃ for 48 h. The initial culture medium and the bacterial suspension cultured for 48 h were centrifuged at 6000 rpm for 10 min to obtain supernatants. Then, 200 μL of the supernatant was added to a 20 mL test tube, followed by the addition of 5 mL of sodium phenolate and 5 mL of sodium hypochlorite. The volume was then adjusted to 20 mL with pure water, allowed to stand for 30 min for color development, and the OD was measured. 630 The value of nm was used. Furthermore, an absorbance-concentration standard curve was plotted using ammonium chloride to calculate the NH4 content in the sample. + -N content. Results are shown in... Figure 1 As shown, it can be seen that QY6's NH4 + -N conversion ability is the best, NH4 at 48 h + -N conversion capacity reached 86.53%, followed by QY3, and NH4 at 48 h. + -N conversion capability reaches 76.43%.

[0037] 2. Identification of strains The initially screened strain QY6 was activated. Glycerol culture, stored at -80℃, was removed and 500 μL was added to 49.5 mL of sterile ammonia assimilation medium. The culture was incubated in a shaker at 170 rpm and 33℃. Samples were taken every 12 h after inoculation, and the initial culture medium and bacterial suspensions at different incubation times were centrifuged at 6000 r / min for 10 min to obtain supernatants. 200 μL of the supernatant was then added to a 20 mL test tube, followed by the addition of 5 mL sodium phenolate and 5 mL sodium hypochlorite. The volume was brought to 20 mL with pure water, and the mixture was allowed to stand for 30 min for color development. The OD was then measured. 630 The absorbance-concentration standard curve was plotted using ammonium chloride to calculate the NH4 content in the sample. + -N content.

[0038] The results are as follows Figure 2 As shown, where, Figure 2 (a) in the figure represents NH4 at different incubation times. + -N conversion rate, Figure 2 (b) shows the streak plate culture of strain QY6. It can be seen that after 24 h of culture, the NH4+ of the ammonia-assimilating bacteria... + -N conversion rate reached 65.6%, and NH4+ was reduced after 72 h of cultivation. +The -N conversion rate reached 93.5%, and the culture was essentially stable. On LB medium, the colonies of strain QY6 were slightly white, rough, round, and convex in the center. Comparison with the standard classification characteristics in Bergey's Manual of Bacteriology showed that the morphological characteristics and physiological properties of QY6 conformed to the description of the genus *Bacillus*. 16S rRNA sequencing yielded a 1470 bp gene fragment. Blast analysis of the sequencing results with the NCBI database confirmed the strain as *Bacillus compostii*, with the taxonomic name [not specified]. Bacillus stercoris .

[0039] The strain QY6 was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33139.

[0040] Example 2: Application Verification of QY6 Biological Agent in Composting Small-Scale Test 1. Preparation of biological inoculant: QY6 was inoculated into LB medium and cultured in a shaker at 170 rpm and 33℃ until OD. 600 =0.80±0.10, its viable bacterial count is approximately 1×10 8 CFU / mL was used to obtain the seed culture.

[0041] 2. Composting Experiment Design Control group (CK): compost raw materials without inoculation with microbial agent; Treatment group (T1): inoculated with ammonia assimilation microbial agent (Bacillus compostii QY6).

[0042] The composting material consisted of a mixture of shiitake mushroom residue, oyster mushroom residue, and distiller's grains in a 20:3:5 ratio. The total organic carbon (TOC) of the mixture was 394.01 g / kg, the total nitrogen (TN) was 15.01 g / kg, and the carbon-to-nitrogen ratio (C / N) was 26.25. 30 kg of material was added to an aerobic composting reactor, along with 1% QY6 inoculum. An equal volume of LB liquid culture medium was added to the control group, and the moisture content was adjusted to 55%. The compost was turned on days 0, 2, 5, 9, 16, 23, 30, and 45. After composting, the nitrogen content and humification indicators were measured. The results are as follows: Figure 3 As shown, where, Figure 3 In the figure, a, b, c, d, and e represent the total nitrogen content, cumulative ammonia emissions, and NH4+ content in the system, respectively. + -N content, NO3 --N content and acid-hydrolyzed organic nitrogen status, where AAN, AN, ASN, and HUN represent amino acid nitrogen, ammonia nitrogen, amino sugar nitrogen, and acid-hydrolyzed unknown nitrogen, respectively. It can be seen that at the end of composting, the cumulative NH3 emission significantly decreased after adding the microbial agent, and the addition of QY6 microbial agent significantly increased the acid-hydrolyzed organic nitrogen content; the TN content in group T1 was 23.72% higher than that in group CK; and the cumulative NH3 emission was 33.76% lower than that in group CK. p <0.05); the NH4⁺-N content in group T1 was 37.50% lower than that in group CK; the content of acid-hydrolyzed organic nitrogen was 25.82% higher than that in group CK.

[0043] Further measurements were taken of humic acid (HA), fulvic acid (FA), and the HA / FA ratio in the compost of the two treatment groups. Specific results can be found in the table below. Figure 4 As shown in a, b, and c, the results indicate that adding QY6 inoculant can significantly increase the humic acid content in the composting system. The humic acid content in group T1 was 25.73% higher than that in group CK. p <0.05); the HA / FA ratio in group T1 increased by 30.55% compared to group CK. This indicates that QY6 effectively reduces nitrogen loss and promotes composting and humification through ammonia assimilation.

[0044] The changes in TOC and humic acid precursors in the two treatment groups during composting are as follows: Figure 5 As shown, where, Figure 5 a, b, c, d, e, and f in the table represent the measured results of TOC, amino acids, carboxyl groups, polyphenols, reducing sugars, and polysaccharides in the compost, respectively. TOC is total organic carbon. The results show that adding the microbial agent QY6 can effectively promote the production of humic acid precursors. The amino acid content in group T1 was 43.30% higher than that in group CK. p <0.05); the polyphenol content in group T1 increased by 39.13% compared to group CK (<0.05); p <0.05); The reducing sugar content in group T1 was 21.43% higher than that in group CK (<0.05); p <0.05). Meanwhile, the lower TOC content and higher humic acid content in group T1 indicate that the addition of QY6 can effectively promote the production of carbon precursors of humic acid. At the same time, the higher amino acid content in group T1 is also conducive to its condensation with carbon precursors, thereby accelerating humic acid polymerization and improving the humification process.

[0045] Example 3: Application Verification of QY6 Biological Agent in Large-Scale Factory Composting Group design: Control group (CK): uninoculated bacterial residue; Treatment group (T1): inoculated with the above-mentioned ammonia assimilation bacterial agent (Bacillus compostii QY6 seed liquid).

[0046] Mushroom residue was selected as the composting material (a mixture of shiitake mushroom residue, oyster mushroom residue, and distiller's grains in a 20:3:5 ratio). The TOC of the mixture was 430.83 g / kg, TN was 15.84 g / kg, and C / N ratio was 27.20. Each pile initially contained 30 t of material with a moisture content of 50%. QY6 was added at a 1% (wet weight) inoculum concentration, while the control group received the same volume of sterile LB liquid culture medium. The compost pile was turned every 2 days for the first 10 days, every 5 days from days 11 to 30, and every 10 days from days 30 to 62, for a total of 62 days of composting.

[0047] The measurement results are as follows Figure 6 As shown, where, Figure 6 In the figure, a, b, c, d, and e represent the total nitrogen content, cumulative ammonia emissions, and NH4+ content in the system, respectively. + -N content, NO3 - -N content and acid-hydrolyzed organic nitrogen status. It can be seen that at the end of composting, the TN content in group T1 was 27.12% higher than that in group CK; the cumulative NH3 emissions in group T1 were 25.66% lower than those in group CK. p <0.05); the content of acid-hydrolyzed organic nitrogen in group T1 increased by 10.62% compared with group CK.

[0048] Further measurements were taken of humic acid, fulvic acid, and HA / FA levels in the compost of the two treatment groups. Specific results can be found in the table below. Figure 7 As shown in a, b, and c, the results indicate that the addition of QY6 bacterial agent significantly increased the humic acid content, with the humic acid content in group T1 being 12.23% higher than that in group CK. p <0.05); the HA / FA ratio in group T1 increased by 18.40% compared to group CK. This indicates that QY6 has good functional stability in large-scale composting.

[0049] Changes in TOC and humic acid precursors in the two treatment groups during large-scale plant composting are as follows: Figure 8 As shown, Figure 8 a, b, c, d, e, and f in the table represent the measured results of TOC, amino acids, carboxyl groups, polyphenols, reducing sugars, and polysaccharides in the compost, respectively. TOC refers to total organic carbon. The results showed that the amino acid content in group T1 was 4.40% higher than that in group CK; the carboxyl content in group T1 was 29.27% ​​higher than that in group CK. p <0.05); the polyphenol content in group T1 was 13.95% lower than that in group CK (<0.05); p <0.05); the polysaccharide content in group T1 decreased by 24.14% compared to group CK ( p <0.05). Meanwhile, the lower TOC content and higher humic acid content in group T1 indicate that the addition of QY6 can effectively promote the production of humic acid precursors and accelerate precursor polymerization, thereby promoting humic acid synthesis, improving the humification process, and accelerating the compost stabilization cycle.

[0050] Example 4: Verification of the soil improvement effect of QY6 compost product QYO Preparation of QYO compost product: The compost product obtained in Example 2 was aged at room temperature for 15 days and then ground through a 5 mm sieve for later use.

[0051] Three treatment groups were set up: NAS (without compost product), CKS (with CKO), and QYS (with QYO). CKO was the final compost product obtained from the CK treatment group after composting, and its soil-improved group was designated CKS. QYO was the final compost product obtained from the T1 treatment group in Example 2 after composting, and the soil improved using QYO was designated QYS. The compost product was mixed with the soil at a ratio of 5%, and 1.5 kg of the mixed soil (soil + compost product) was filled into each pot. Water was added to adjust the moisture content to 60%. The soil was watered regularly daily to adjust the soil moisture holding capacity (watering once a day, 100 mL per pot each time). After 30 days of outdoor cultivation, soil samples were taken to determine conventional indicators and nutrient content.

[0052] See results Figure 9 As shown, where, Figure 9 In the equation, a, b, c, d, e, f, g, h, and i represent soil pH, soil electrical conductivity, soil organic matter ratio, total organic carbon content, available phosphorus content, available potassium content, available nitrogen content, total nitrogen content, and humic acid content, respectively. The results indicate that the application of QYO altered soil properties and significantly increased soil nutrient content (…). p <0.05). The application of compost products had no significant effect on soil pH, remaining around 7.40; however, the application of compost products significantly increased soil EC (…). p <0.05), the soil EC of the QYS group was 27.68% higher than that of the NAS group. The higher EC indicates that the application of QYO enhanced the soil activation capacity; the application of compost products significantly increased the soil organic matter and total organic carbon content. p <0.05), compared with NAS, the soil organic matter and total organic carbon content in the QYS group were 62.08% and 201.11% higher, respectively; compared with other treatments, the available phosphorus content in the QYS group was 200.04% and 33.46% higher, respectively, and the available potassium content was 369.50% and 13.94% higher, respectively. Application of QYO can increase the content of available nutrients in the soil, which is beneficial for crop absorption and utilization; compared with other treatments, the available nitrogen content in the QYS group was 139.35% and 59.98% higher, respectively. p <0.05), while the TN content in the soil of the QYS group was 166.33% and 22.45% higher, respectively ( p<0.05), indicating that the addition of QYO can promote soil nitrogen content, and the increase in its available nitrogen content is beneficial to plant nitrogen absorption; compared with the other treatments, the humic acid content in the soil of the QYS group was 156.64% and 21.34% higher, respectively. p <0.05), and its increased content can inhibit the leaching of ionic nutrients and increase the proportion of available nutrients in the soil. The above results indicate that adding QYO can effectively increase soil nutrient content and improve soil properties.

[0053] Example 5: Verification of the effect of QY6 compost product QYO on potted cabbage. The compost product obtained in Example 4 was used as a preparatory step. Three treatment groups were set up: NAP (no compost product added), CKP (CKO added), and QYP (QYO added). CKO was the final compost product obtained from the CK treatment group after composting, and its plant growth-promoting group was designated CKP. QYO was the final compost product obtained from the T1 treatment group in Example 2 after composting, and the group using QYP for growth-promoting verification was designated QYP. The compost product was mixed with soil at a ratio of 5%, and 1.5 kg of the mixed soil (soil + compost product) was filled into each pot. Water was added to adjust the moisture content to 60%. One week after emergence, seedlings of equal size were transplanted into pots, and the soil moisture content was adjusted by watering regularly every day (100 mL / pot once a day). After 30 days of outdoor cultivation, samples were taken to determine the agronomic traits and quality indicators of the Chinese cabbage.

[0054] See results Figure 10 As shown, where, Figure 10 In the figure, a, b, c, d, e, f, g, and h represent plant height, root length, fresh weight biomass, dry weight biomass, chlorophyll content, soluble sugar content, soluble protein content, and total nitrogen content, respectively. The results showed that plant height reflects the plant's growth status, and its growth and development are closely related to yield traits. Compared with other treatments, the QYP group of Chinese cabbage had plant heights that were 62.13% and 32.29% higher, root lengths that were 86.24% and 48.65% higher, biomass (fresh weight) that were 81.27% and 45.19% higher, and biomass (dry weight) that were 52.32% and 25.53% higher, respectively. p <0.05), indicating that QYO compost products have a significant promoting effect on the growth of Chinese cabbage. Compared with the other treatments, the chlorophyll content of the QYP group was 45.26% and 4.33% higher, respectively, indicating that the application of QYO can effectively promote plant photosynthetic capacity and increase chlorophyll content. The soluble sugar content of the QYP group was 312.90% and 89.16% higher than that of the other groups, respectively. p<0.05), applying QYO is beneficial for the synthesis of soluble sugars and avoids external stress on Chinese cabbage. Compared with other treatments, the soluble protein content in the QYP group was 30.74% and 8.81% higher, respectively, and the total nitrogen content in plants was 22.03% and 17.93% higher, respectively. This indicates that applying QYO has a strong nitrogen supply capacity for Chinese cabbage growth, which is conducive to the absorption of nitrogen in the soil and its conversion into plant nitrogen, thus reducing nitrogen loss and improving the quality of Chinese cabbage.

[0055] Example 6: Preparation of compost-derived humic acid by QY6 and its application in heavy metal remediation 1. Preparation of compost-source humic acid using QY6: Take 100 g of the air-dried sample of QY6 compost product obtained in Example 2 into a 3 L polyethylene bottle, add 2 L of sodium pyrophosphate-sodium hydroxide extract, shake at 135 rpm for 1 h at room temperature, and let stand overnight. After thoroughly shaking the extract, centrifuge at 4000 rpm for 10 min, collect the supernatant, adjust the pH to 1-1.5 with 1 M sulfuric acid solution under 80℃ water bath conditions until flocculent precipitate appears, keep warm for 30 min, let stand overnight, centrifuge at 4000 rpm for 10 min, wash the precipitate with 0.05 M sulfuric acid solution until the washing liquid is colorless, centrifuge at 4000 rpm for 10 min, and dry in a 100℃ oven to obtain compost-derived humic acid.

[0056] The sodium pyrophosphate-sodium hydroxide mixed extract was obtained by dissolving 44.6 g of sodium pyrophosphate and 4.0 g of sodium hydroxide in water and diluting to 1 L.

[0057] 2. Application of compost-derived humic acid in heavy metal remediation (1) Construction of Cr(VI) simulated pollution system A solution with an initial Cr(VI) concentration of 200 mg / L was prepared using potassium dichromate (K₂Cr₂O₇) as the Cr(VI) source. The initial pH was adjusted to a neutral or weakly acidic range (pH 6.0–7.0) using dilute sulfuric acid / sodium hydroxide. The solution was dispensed into 250 mL Erlenmeyer flasks, with 100 mL of the above Cr(VI) solution added to each flask. The reaction was carried out in a constant temperature shaking incubator (e.g., 28–30 °C, 135 rpm).

[0058] (2) Humic acid-borne bacteria Place the compostable humic acid or commercially available mineral-derived humic acid powder (purity ≥99%) obtained in Part 1 above into a sterile mixing container, and add an appropriate amount of sterile water according to the mass of humic acid to adjust the moisture content to 20%–40% (w / w) to form a plastic wet material; add 10 8A CFU / mL CRB-7 bacterial suspension was evenly sprayed onto the surface of the wet material using a misting method, while simultaneously stirring for 10 minutes to ensure uniform bacterial loading onto the humic acid matrix. After mixing, the mixture was allowed to stand at 20–30°C for 2 hours for adsorption, yielding the humic acid-loaded bacterial material. Without affecting bacterial viability, the material was further dried at ≤40°C using low-temperature air drying or vacuum drying to reduce the moisture content to 10%–20%. CRB-7 is a Bacillus subtilis (…). Bacillus The strain (sp.) is selected from the article 'Wu M, Wang Q, Wang C, Zeng Q, Li J, Wu H, Wu B, Xu H, Qiu Z. Strategy for enhancing Cr(VI)-contaminated soil remediation and safe utilization by microbial-humic acid-vermiculite-alginate immobilized biocomposite. Ecotoxicol Environ Saf. 2022Sep 15;243:113956'. This strain has heavy metal remediation capabilities and can be used for Cr(VI) pollution remediation.

[0059] (3) Processing settings and dosing methods Set up the following processing groups (3 parallel processes per group): ① M-HA: Add mineral-derived humic acid to the Cr(VI) solution; ② QY6-HA: Add compost-derived humic acid obtained from the compost product obtained by inoculating QY6 to the Cr(VI) solution; ③ CRB-7: Inoculate the heavy metal remediation bacteria CRB-7 into the Cr(VI) solution; ④ M-HA@CRB-7: Add the bacterial material loaded with CRB-7 using mineral-derived humic acid as a carrier to a Cr(VI) solution; ⑤ QY6-HA@CRB-7: Add the bacterial material loaded with CRB-7 using QY6-HA as a carrier to the Cr(VI) solution.

[0060] (4) Heavy metal remediation effect The results are as follows Figure 11As shown, there were significant differences in the Cr(VI) removal rates among the treatment groups at 24 h. Compared with M-HA, the compost-derived humic acid extracted from QY6 compost products (QY6-HA) showed higher Cr(VI) removal rates, at 27.11% and 20.15%, respectively, indicating that compost-derived humic acid has a superior removal effect when used to remediate Cr(VI) pollution. The removal rate of the remediation bacterium CRB-7 alone was 62.13%. When CRB-7 was immobilized with humic acid as a carrier, the remediation effect was further improved, with the Cr(VI) removal rate of QY6-HA@CRB-7 reaching 88.35%, significantly higher than that of M-HA@CRB-7 (76.0%). p The value <0.05 indicates that compost-derived humic acid, when used as a carrier to immobilize microorganisms for the remediation of Cr(VI) pollution, can achieve better synergistic removal effects.

[0061] After 48 h, the Cr(VI) removal rates of each treatment group further improved. The removal rate of QY6-HA@CRB-7 increased to 92.01%, still the highest among all treatments; M-HA@CRB-7 was 80.01%, and CRB-7 was 76.22%. Meanwhile, the removal rates of QY6-HA and M-HA were 33.0% and 23.32%, respectively, further indicating that the compost-derived humic acid contributed more to the removal of Cr(VI) than the mineral-derived humic acid under the same conditions.

[0062] The combined results from 24 h and 48 h show that the compost-sourced humic acid can be directly used for the remediation of heavy metal pollution and exhibits a superior removal effect. It can also be used as a carrier for immobilizing remediation microorganisms, thereby significantly improving the removal and stabilization effect of the microbial remediation system on Cr(VI).

[0063] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A composting Bacillus strain with highly efficient ammonia assimilation capabilities ( Bacillus stercoris ), characterized in that, The strain, QY6, was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33139. This Bacillus composting strain QY6 releases NH4+ into the composting system. + -N is preferentially converted into organic nitrogen, which can inhibit the volatilization of NH3.

2. A composting nitrogen-retaining biological agent that uses ammonia assimilation as the nitrogen conversion pathway, characterized in that, The biological agent comprises the composting Bacillus as described in claim 1.

3. The biological agent according to claim 2, characterized in that, The effective viable count of Bacillus compostingus QY6 in the biological agent is ≥ 1×10⁻⁶. 8 The biological agent, with a concentration of cfu / mL, can be used to reduce ammonia emissions and increase the total nitrogen and humic acid content of compost during the composting process.

4. A composting nitrogen retention method based on the ammonia assimilation pathway, characterized in that, During the composting process, the biological agent described in claim 2 or 3 is inoculated into the composting raw materials to allow NH4 to... + -N is converted into organic nitrogen through microbial assimilation and participates in the humification process.

5. The composting nitrogen retention method based on the ammonia assimilation pathway according to claim 4, characterized in that, The method further includes improving humic acid synthesis efficiency by promoting the formation of humic acid precursor substances, and by regulating microbial metabolic pathways and organic matter decomposition to form more precursors that can participate in humic acid condensation in the composting system, thereby promoting the generation and enrichment of humic acid.

6. The application of the composting nitrogen retention method as described in claim 4 or 5, characterized in that, The compost-derived humic acid prepared by the method is used for the remediation of heavy metal pollution. The compost-derived humic acid is directly added to the heavy metal pollution system, or used as a carrier to load heavy metal remediation microorganisms to form a composite remediation material, so as to achieve synergistic removal and stabilization of heavy metals. The heavy metal is hexavalent chromium Cr(VI). The dosage of the compost-derived humic acid is 10–500 mg / L, or the compost-derived humic acid is used as a carrier to load Bacillus heavy metal remediation bacteria to form a humic acid-microorganism composite material for the reduction and removal of Cr(VI) in water or soil.

7. The composting nitrogen retention method according to claim 4, characterized in that, The inoculation amount of the biological agent is 1% of the mass of the compost raw materials; wherein, the effective viable count of Bacillus compostii QY6 in the biological agent is ≥1×10⁻⁶. 8 cfu / mL.

8. A soil conditioner, characterized in that, The soil conditioner is the composting product of Bacillus compostingus as described in claim 1.

9. A method for soil improvement, characterized in that, Apply the soil conditioner as described in claim 8 during crop cultivation.

10. The use of the Bacillus composting strain of claim 1 in any of the following, comprising: Promotes nitrogen retention in compost; Promotes the formation of humic acid precursors; Promotes composting and humification; Soil improvement; Promote crop growth; Used to prepare or obtain compost-source humic acid; For the remediation of heavy metal pollution; and / or The compost-source humic acid is used as a carrier to immobilize microorganisms for the remediation of heavy metal pollution.

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