Bacillus JX-7 and application thereof

Abstract

The invention relates to the technical field of biology. The invention discloses a strain bacillus sp. JX-7, and the preservation number of the strain bacillus sp. JX-7 is CGMCC (China General Microbiological Culture Collection Center) No.27978. The preservation date of the strain is July 24, 2023, the preservation unit is China General Microbiological Culture Collection Center, the China General Microbiological Culture Collection Center is called CGMCC for short, and the address of the China General Microbiological Culture Collection Center is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The bacillus sp. JX-7 provided by the invention can be used for effectively increasing the content of organic carbon in soil and has efficient melanin synthesis capability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. Background Technology

[0002] Melanin is widely found in plants, animals, and microorganisms. It is a class of complex polyaromatic compounds formed by the oxidation and polymerization of polyhydroxyphenols or indoles. Melanin has relatively diverse structures, with surface morphology consisting of small, spherical or irregularly shaped particles and a porous structure. Its structure, elemental composition, and physicochemical properties are very similar to humus, and it is also known as a humus-like substance. Melanin-like aromatic carbons have strong antioxidant capabilities, are not easily oxidized, can be deposited in the soil, and possess eco-friendly and biocompatible characteristics, thus contributing to the long-term storage of carbon in the soil.

[0003] Furthermore, melanin possesses excellent photothermal stability, absorbing all visible light, with maximum absorption in the ultraviolet spectrum (200-400 nm). The presence of its complex conjugated molecules is the reason for this ultraviolet absorption. Melanin is also an organic semiconductor, exhibiting electrical conductivity. Additionally, melanin has strong redox capabilities, readily accepting or losing an electron, thus possessing free radical scavenging functions. It also exhibits antibacterial and tumor-inhibiting activities, leading to its widespread application in food, cosmetics, and biomedicine. It can also be used to neutralize toxic phenolic compounds in the environment.

[0004] Currently, melanin can be produced through chemical extraction and oxidative synthesis, but these processes are complex and inefficient. Microbial melanin production, on the other hand, has a shorter production cycle, is not limited by environmental or geographical conditions, and is lower in cost, showing great application potential. Therefore, the discovery and selection of melanin-synthesizing microbial strains have significant application prospects for melanin biosynthesis, soil organic matter transformation, and improving stress resistance.

[0005] Microbial melanin synthesis mainly includes two types: eumelanin and 1,8-dihydroxynaphthalene (DHN) melanin. Their biosynthesis typically occurs via either the DOPA pathway (conversion of tyrosine) or the propionyl-CoA pathway (DHN pathway). In the DOPA pathway, the melanin precursor tyrosine is converted to L-DOPA (3,4-dihydroxyphenylalanine), which is then converted to dopaquinone by tyrosinase and laccase. Dopaquinone undergoes spontaneous oxidative polymerization to ultimately form melanin. In the DHN pathway, under enzymatic catalysis, five molecules of propionyl-CoA undergo decarboxylation condensation to produce 1,3,6,8-tetrahydroxynaphthalene (THN), which then undergoes reduction and dehydration reactions to generate 1,8-dihydroxynaphthalene (DHN), which polymerizes to form melanin. Both pathways exist in bacteria and fungi, with most bacteria and basidiomycetes synthesizing melanin via the DOPA pathway, such as *Streptomyces*, *Bacillus*, *Rhizobium*, *Vibrio tyrosinans*, pink thermophilic microbes, and some marine bacteria. Ascomycetes produce melanin via the DHN pathway. Melanin produced through the DHN pathway is endogenously generated and adheres tightly to the inner side of the cell wall, making extraction difficult. In contrast, melanin synthesized by exogenous tyrosine or tyrosine derivative substrates is produced extracellularly, which facilitates extraction. Summary of the Invention

[0006] In view of the above problems, this invention provides a strain of Bacillus sp. JX-7, with the accession number CGMCC No. 27978. The accession date of this strain is July 24, 2023, and the depository institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The Bacillus sp. JX-7 provided by this invention can effectively increase the organic carbon content in soil and has a high efficiency in melanin synthesis. Attached Figure Description

[0008] Figure 1 This is a growth diagram of Bacillus JX-7 on a screening plate.

[0009] Figure 2 This is a diagram of melanin production in Bacillus JX-7.

[0010] Figure 3 This is a diagram of melanin production by Bacillus JX-7 fermentation. Detailed Implementation

[0011] The technical solution of the present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the raw materials used in the present invention are all conventional commercially available products; unless otherwise specified, the methods used in the present invention are all conventional methods in the art.

[0012] Example 1.

[0013] Discovery and identification of melanin-producing strains

[0014] On August 21, 2022, soil samples were collected from the forest area of ​​Jixi City, Heilongjiang Province. The collected samples were thoroughly mixed with sterile water, heated in an 80°C water bath for 15 minutes, and serially diluted 10-1. -1 ~10 -6 Take the diluted sample solution and spread it onto the casein selection medium. Incubate it upside down at room temperature for 3 to 7 days. After the colony grows, select colonies that are visible to the naked eye and have black or brown color, as well as colonies on the surrounding medium that are black or brown. This will give you a preliminary sample of melanin-producing strains.

[0015] The initially screened colonies were repeatedly streaked and purified to obtain single-strain culture plates. The obtained initially screened strains were then inoculated onto casein selection medium for secondary screening. Figure 1 As shown, the growth and melanin secretion of the strain were observed, and the strain was passaged and preserved, named JX-7. 16S rDNA sequencing was performed on the pure culture (base sequence shown as SEQ ID NO.1 in the sequence listing), and after comparison with the NCBI database Blast, it was identified as Bacillus sp., a member of the genus Bacillus.

[0016] The casein solid culture medium consists of 10g peptone, 1g glucose, 5g NaCl, 0.1g CaCl2, 5g casein, 2g L-tyrosine, and 15g agar powder, diluted to 1L with distilled water, pH 7.2, and autoclaved at 121℃ for 15min.

[0017] Bacillus sp. JX-7 was deposited on July 24, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNo.27978, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0018] Example 2

[0019] Bacillus JX-7 fermentation to produce melanin

[0020] A standard curve was prepared using melanin solutions of different concentrations. The standard curve was calculated as: y = 0.0101x + 0.0005(R²). 2 =0.99).

[0021] Table 1. Determination of melanin standard curve

[0022]

[0023] Single colonies of Bacillus JX-7 were selected and cultured in LB liquid medium until the logarithmic growth phase to prepare a seed culture. This seed culture was then inoculated into LB-Tyr medium with an initial OD value of 0.2, and cultured on a shaker at 30°C and 150 rpm. Every 24 hours, 1 mL of cell culture medium was collected, centrifuged at 9000 rpm for 10 min, and the supernatant was diluted 5-fold and the OD value was measured using a microplate reader. 400 (OD 400 (The absorption peak of melanin is shown), with LB-Tyr medium used as a control for zeroing. The OD value of the supernatant was compared with the standard curve to calculate the melanin content in the sample, such as... Figure 3 As shown.

[0024] The LB-Tyr medium consisted of 10g tryptone, 5g yeast extract, 10g NaCl, and 2g tyrosine, diluted to 1L with distilled water, pH 7.2, and autoclaved at 121℃ for 15min.

[0025] Experimental results showed that during LB-Tyr liquid fermentation, the melanin production of strain JX-7 significantly increased to 208.91 mg / L on days 0-1, reaching a peak of 256.08 mg / L on day 4. These results demonstrate that JX-7 exhibits highly efficient melanin synthesis during LB-Tyr liquid fermentation.

[0026] Example 3

[0027] Bacillus JX-7 effectively increases the organic carbon content in soil.

[0028] The melanin-producing Bacillus strain JX-7 was inoculated into 10 mL of LB liquid medium and cultured overnight at 37°C. Seed culture was inoculated at a 1% inoculum into 500 mL of LB liquid medium and cultured on a shaker at 37°C and 200 rpm for 12 hours. Garden soil was sieved through a 4-mesh sieve to remove stones and other impurities. 10 g of corn stalks were weighed, added to 50 mL of the prepared bacterial solution, mixed thoroughly, and then mixed with 1.8 kg of garden soil. The mixture was then placed in boxes measuring 11.8 cm * 12.6 cm * 15 cm and placed at room temperature (25–30°C), with regular watering to maintain soil moisture at no less than 40%. The control group (CK) was mixed with 50 mL of sterile water and chopped straw. After 30 days of incubation, 10 g of soil samples were taken from the boxes using a five-point sampling method, dried, and sieved through a 100-mesh sieve. The organic carbon content was analyzed using a total organic carbon analyzer.

[0029] The experimental results showed that after 30 days of incubation at room temperature, the soil organic carbon content was significantly increased compared with the CK control group, with an increase of more than 70%. This can promote the accumulation of organic matter and improve soil structure and properties.

[0030] Table 2. Changes in organic carbon content in the box test.

[0031]

[0032] Example 4

[0033] Neurospora crassa N7, with accession number CGMCC No. 40738, was deposited on July 24, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0034] 1. Antagonistic effects between microorganisms in compound microbial agents

[0035] The presence of antagonistic effects between Neurospora crassa N7 and Bacillus sp. JX-7 was tested using the plate confrontation culture method.

[0036] First, activate Neurospora crassa N7 on a PDA plate and incubate it at room temperature for one week.

[0037] After the fungus culture is complete, activate the Bacillus sp. JX-7 monoclonal strain one day in advance using liquid LB medium.

[0038] Take more than 10 μL of activated Bacillus sp. JX-7 bacterial suspension and spot it on both sides of WA plate. Inoculate the center of the plate with Neurospora crassa N7 fungal hyphae. At the same time, set up a plate without bacterial suspension as a control. Incubate at room temperature and observe whether the diameter of fungal colonies is affected by bacterial growth compared with the control.

[0039] If the diameter of fungal colonies remains unchanged regardless of the presence or absence of bacteria, it indicates that there is no antagonistic effect between Neurospora crassa N7 and Bacillus sp. JX-7; otherwise, it indicates that there is an antagonistic effect between the two.

[0040] WA medium (Waksman's agar): peptone 15 g / L, glucose 10 g / L, beef extract 3 g / L, NaCl 5 g / L, agar 15 g / L.

[0041] The results of confrontation culture on WA plates showed that, compared with the control group which was inoculated with a single fungal hyphae of the test fungus in the middle of the plate, the hyphae of Neurospora crassa N7 spread to the bacterial colony and did not show any antagonistic reaction with the bacterial Bacillus sp. JX-7, which can be used for the development of compound bacterial agents.

[0042] 2. Low-temperature decomposition ability of microbial agents in straw-inorganic salt culture medium

[0043] Neurospora crassa N7 strain was activated using Vogel's agar plates and cultured at room temperature for 7 days. Spores were collected by rinsing the plates with sterile water to prepare a spore suspension. After dilution, the spore concentration was counted using a hemocytometer, and the final concentration was recorded as 1×10⁻⁶. 5 Inoculate with straw-based inorganic salt medium at a concentration of 1 × 10⁶ spores / mL. Additionally, inoculate Bacillus JX-7 into 10 mL of LB liquid medium and incubate overnight at 37°C. Take 1% of the overnight culture and inoculate with straw-based inorganic salt medium. Inoculate with the above-mentioned Neurospora crassa N7 strain (containing at least 1 × 10⁶ spores). 7 ( / mL) and Bacillus sp. JX-7 strain (containing a viable count of not less than 1×10⁻⁶). 9 The culture medium (cfu / mL) was cultured at 25℃ and 15℃ with shaking at 150 rpm for 21 days. After 21 days of low-temperature culture, 2 mL of the culture medium was taken into a sterile centrifuge tube and centrifuged at 12000 rpm for 5 min. The supernatant was the crude enzyme solution, which was used to determine the activity of laccase.

[0044] The method for preparing the straw inorganic salt culture medium is as follows: first, cut the corn straw into straw segments about 2cm long, then cut each straw segment into four pieces, weigh 2g of the cut straw pieces, add 10mL of 5×M9 solution and 38mL of distilled water into a 150mL shake flask, and autoclave at 115℃ for 20min. After cooling, add the following filtered and sterilized solution: 500 μL NaNO3 (2.5 g / 10 mL), 500 μL CaCl2 (0.11 g / 10 mL), 100 μL MgSO4·7H2O (2.465 g / 10 mL), 50 μL FeCl3·6H2O (0.162 g / 10 mL), 50 μL CuSO4·5H2O (2.496 g / 10 mL), 50 μL ZnSO4·7H2O (0.288 g / 10 mL), and 50 μL MnSO4·H2O (0.34 g / 10 mL).

[0045] 5×M9 solution: 64g Na2HPO4·7H2O, 15g KH2PO4, 2.5g NaCl, 5g NH4Cl, dissolve in water and bring to a final volume of 1000mL, then adjust the pH to 7.2.

[0046] Laccase Activity Assay

[0047] Add 25 μL of crude enzyme solution to 160 μL of 0.2 mol / L sodium acetate buffer (pH = 4.5), and initiate the reaction with 15 μL of 20 mM ABTS. Measure the absorbance at 420 nm and record the change in absorbance over 3 minutes. Enzyme activity unit definition: The amount of enzyme required to oxidize 1 μmol of substrate ABTS per minute per milliliter of crude enzyme solution is defined as one enzyme activity unit.

[0048] At 25℃ and 15℃, there was no significant difference in the laccase activity and decomposition rate of the inoculant, indicating that the utilization efficiency of the applied microbial agent on straw was not affected by low temperature. At 15℃, the straw decomposition rate of the microbial agent after 21 days of shake-flask culture could reach more than 40%.

[0049] 3. Soil pot experiment on in-situ decomposition of straw

[0050] Spores of Neurospora crassa N7 were inoculated into a 250 mL shake flask containing 50 mL of Vogel's liquid medium and incubated on a shaker at 25 °C and 150 rpm for 48 h. At least 15 mL of the bacterial culture was then inoculated into 500 mL of Vogel's liquid medium and incubated on a shaker at 25 °C and 150 rpm for 48 h.

[0051] Bacillus sp. JX-7 was inoculated into 10 mL of LB liquid medium and cultured overnight at 37°C. Seed culture was transferred to 500 mL of LB liquid medium at a 1% inoculation rate and cultured on a shaker at 37°C and 200 rpm for 12 h.

[0052] The study found that the bacterial agent contained 1 × 10⁻⁶ N7 of Neurospora crassa. 7 1 spore / cell, corresponding to an inoculum size of 1 × 10⁶ Bacillus sp. JX-7. 8 The two strains of bacterial sludge were mixed evenly according to different treatment groups, and distilled water was added to make up to 50 mL to obtain the bacterial solution to be applied.

[0053] Garden soil was sieved through a 4-mesh sieve to remove stones and other debris. 10g of straw was weighed, mixed thoroughly with 50mL of prepared bacterial solution, and then combined with 1.8kg of garden soil. The mixture was then placed in a box measuring 11.8cm x 12.6cm x 15cm. The box was placed in an indoor environment (4-8℃) or at room temperature, and watered regularly to maintain soil moisture at no less than 40%. The control group (CK) used 50mL of sterile water to mix with the straw. After 30 days of incubation at room temperature, the soil in the box was sieved through a 4-mesh sieve to remove undecomposed straw. The removed straw was rinsed with 80mL of distilled water, dried in a 60℃ oven, and the straw decomposition rate was measured. A 10g sample of soil from the box was taken using a five-point sampling method, sieved through a 100-mesh sieve, and analyzed for organic matter content using a potassium dichromate oxidation-spectrophotometric method.

[0054] The method for determining the straw decomposition rate is as follows: After washing the straw using the above treatment method, drying it at 60℃, and weighing the remaining straw, the decomposition rate is calculated using the following formula:

[0055]

[0056] Wherein, W0: original dry weight of straw, and W1: dry weight of straw after decomposition.

[0057] The method for determining the organic matter content is as follows: After air-drying the soil sample, pass it through a 0.25 mm sieve. Weigh a certain amount of soil sample into a test tube, add 10 mL of 0.4 mol / L potassium dichromate-sulfuric acid solution and shake well. Place a glass funnel at the mouth of the test tube, insert it into an iron wire furnace, and place it in oil at 170-180℃. Let the solution in the test tube boil for 5 minutes. After cooling, transfer the liquid and soil sample in the test tube to an Erlenmeyer flask. Also transfer the washing solution used to clean the test tube and funnel to the Erlenmeyer flask, bringing the final volume to 50-60 mL. Add 3 drops of o-phenanthroline indicator and titrate the remaining potassium dichromate with ferrous sulfate solution. Take about 0.2 g of ignited pumice powder or soil as a blank test. The formula for calculating soil organic matter is as follows:

[0058]

[0059] In the formula, SOM is the mass fraction of soil organic carbon (g / kg); c is the concentration of ferrous sulfate standard solution (mol / L); V0 is the volume of ferrous sulfate standard solution consumed in the blank test (mL); V is the volume of ferrous sulfate standard solution consumed in the sample test (mL); 0.003 is the millimolecular mass of 1 / 4 carbon atom (g); 1.10 is the oxidation correction coefficient; m is the mass of the dried sample (g); 1.724 is the conversion factor of organic matter to organic carbon; and 1000 is the coefficient for converting the result to content per kilogram.

[0060] Test results in a soil pot experiment under low-temperature conditions (4–8℃) showed that the in-situ decomposition rate of straw in the control group without inoculant application was 10.33±1.96%, while the decomposition rate in the experimental group with inoculant application reached 42.76±3.13%, a fourfold increase. After 30 days of in-situ decomposition of straw, the total organic matter (SOM) content in the control group was 1.08±0.09 g / kg, while that in the experimental group was 1.85±0.34 g / kg, representing a 71% increase in soil organic matter content. Soil pot experiments verified that the straw decomposition carbon-collecting inoculant of this invention can effectively improve straw decomposition efficiency and soil organic carbon content, and promote the accumulation of organic matter in the soil under low-temperature conditions (4–8℃).

[0061] Table 3 Evaluation of the effect of microbial agents on in-situ decomposition and quality improvement of straw under low temperature (4-8℃) conditions in soil.

[0062] control group experimental group Decomposition rate (%) 10.33±1.96 42.76±3.13 Organic matter content (g / Kg) 1.08±0.09 1.85±0.34

[0063] Test results in a soil pot experiment under laboratory temperature conditions showed that the in-situ decomposition rate of straw in the control group without inoculant application was 58.53±6.37%, while the decomposition rate in the experimental group with inoculant application reached 88.41±4.62%, an increase of 51%. After 30 days of in-situ decomposition of straw, the total organic matter content of the control group was 1.32±0.2 g / kg, while that of the experimental group was 1.79±0.3 g / kg, an increase of 36% in organic carbon content. Soil pot experiments verified that the straw decomposition and carbon-collecting inoculant of this invention can effectively improve straw decomposition efficiency and soil organic carbon content, promoting the accumulation of organic matter in the soil.

[0064] Table 4 Evaluation of the effect of microbial agents on in-situ decomposition and quality improvement of straw in soil under room temperature conditions.

[0065] control group experimental group Decomposition rate (%) 58.53±6.37 88.41±4.62 Organic matter content (g / Kg) 1.32±0.2 1.79±0.3

[0066] 4. In-situ decomposition of straw in cornfields by microbial agents

[0067] To accurately determine the in-situ decomposition performance of corn stalks after returning them to the field, this invention employs a nylon mesh bag method to measure the in-situ decomposition of corn stalks. Experimental and control groups were placed in cornfields during the spring planting season. A 40×60cm mesh bag was used. 40g of dry corn stalks were mixed with 200mL of experimental microbial agent and soaked for 30 minutes. This mixture was then stirred with a certain amount of soil sample, placed in the mesh bag, and positioned in the cornfield. The experimental microbial agent contained *Neurospora crassa* strain N7 (containing at least 1×10⁻⁶ spores). 7 ( / mL) and Bacillus sp. JX-7 strain (containing a viable count of not less than 1×10⁻⁶). 9A composite microbial culture medium (cfu / mL) was prepared. The control group used 200 mL of sterile water mixed with straw. The experimental results were consistent with those described in Example 3.

[0068] In cornfields in Northeast China, after two months of experiments in May and June, the decomposition rate of total corn stalks in nylon mesh bags was measured. The decomposition rate of the control group without microbial agent application was 62.4±6.7%, while the decomposition rate of the experimental group with microbial agent application was 73.4±5.8%, an increase of 17.6%. Soil samples were taken from the nylon mesh bags using the five-point sampling method. The organic matter content of the control group was 6.8±0.8%. After the straw was decomposed in situ with the microbial agent, organic matter accumulated, reaching 8.2±0.6%, an increase of 20.1%.

[0069] 5. The in-situ decomposition of straw in paddy fields using the invented microbial agent.

[0070] To accurately determine the in-situ decomposition performance of rice straw, this invention employs a PVC pipe method. A perforated PVC pipe with a diameter of 12-16 cm, a length of 23-25 ​​cm, and a thickness of 3-5 mm is selected. 34 g of rice straw is weighed and mixed thoroughly with 170 mL of microbial agent, then mixed with a certain amount of soil sample. The mixture is placed inside the PVC pipe, covered with a mesh bag, and positioned in the field. The experimental microbial agent contains *Neurospora crassa* strain N7 (containing at least 1 × 10⁻⁶ spores). 7 / mL) and Bacillus sp. JX-7 (containing a viable count of not less than 1×10⁻⁶) 9 The experimental group used a compound microbial culture medium (cfu / mL). The control group used 170 mL of sterile water mixed with straw. Both the experimental and control groups were introduced into the paddy field during the spring plowing season. Nine parallel groups were designed for each group. Sampling was carried out regularly to determine the straw decomposition rate and soil organic matter content, and to evaluate the in-situ decomposition effect of the microbial agent on rice straw and the soil improvement effect.

[0071] In rice paddies in Northeast China, after two months of experiments in May and June, the decomposition rate of total rice straw in PVC pipes was measured. The decomposition rate of the control group without microbial agents was 47.9±5.0%, while the decomposition rate of the experimental group with microbial agents was 63.6±4.3%, an increase of 32.8%. Soil samples were taken from the PVC pipes using the five-point sampling method. The organic matter content of the control group was 8.5±1.6%. After the straw was decomposed in situ with microbial agents, organic matter accumulated, reaching 9.2±0.3%, an increase of 8.2%.

[0072] In summary, the invented microbial agent promoted straw decomposition and increased soil organic matter during field experiments of in-situ return of corn and rice straw, demonstrating soil improvement effects. Furthermore, comparative analysis of microbial diversity showed that the microorganisms in the agent exhibited good colonization ability in both corn and rice fields, further indicating that the increased decomposition efficiency and organic matter content in the experimental groups are directly related to the functional microorganisms in the agent.

Claims

1. Bacillus sp. JX-7, with accession number CGMCC No.27978.

2. A microbial inoculant containing Bacillus sp. JX-7 as described in claim 1.

3. A composition comprising Bacillus sp. JX-7 as described in claim 1.

4. The application of Bacillus sp. JX-7 according to claim 1 in melanin production.

5. The application of Bacillus sp. JX-7 according to claim 1 in increasing the organic carbon content in soil.

6. A compound microbial agent, characterized in that, Contains Bacillus sp. JX-7 and Neurospora crassa N7 as described in claim 1; The preservation number of Neurospora crassa N7 is CGMCC No. 40738.

7. The compound microbial agent according to claim 6, characterized in that, The number of spores of Neurospora crassa N7 is not less than 1 × 10⁻⁶. 7 / mL.

8. The compound microbial agent according to claim 6, characterized in that, The viable count of the Bacillus sp. JX-7 is not less than 1×10⁻⁶. 9 cfu / mL.

9. The application of the compound microbial agent according to any one of claims 6 to 8 in the preparation of microbial agents for in-situ decomposition of straw and / or enhancement of soil organic matter content.

10. The application of the compound microbial agent according to any one of claims 6 to 8 in the preparation of microbial agents for in-situ decomposition of straw and / or enhancement of soil organic matter content, characterized in that, The straw is either corn straw or rice straw.

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