Bacillus velezensis X014-2H and application thereof

Abstract

The invention discloses bacillus velezensis X014-2H and application thereof, and belongs to the technical field of microorganisms and agriculture. The bacillus velezensis X014-2H disclosed by the invention is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the bacillus velezensis X014-2H is CCTCC M 2024413. The invention discloses a microbial inoculum comprising the strain. The invention discloses an application of the bacillus velezensis X014-2H or a microbial inoculum of the bacillus velezensis X014-2H in decomposition of cellulose, and further discloses an application of the bacillus velezensis X014-2H or the microbial inoculum in decomposition of cellulose. The bacillus velezensis X014-2H disclosed by the invention has the advantages of low nutritional requirements, mild conditions, high temperature resistance and stronger enzyme production capacity.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and agriculture, and particularly relates to a strain of Bacillus velez X014-2H and application thereof. Background Art

[0002] Crop straw is an important biological resource in the agricultural production system. According to incomplete estimates, the world produces nearly 2 billion tons of straw every year. China is a large agricultural country and one of the countries with the richest straw resources; and with the increase in crop yields, straw production will also increase accordingly. In the traditional agricultural stage, straw resources are mainly used directly as fertilizer, fuel and feed without any treatment. With the development of society, the problem of excess straw resources in major agricultural production areas has become increasingly prominent; burning straw will lead to resource waste and environmental pollution.

[0003] The use of biological methods to degrade straw has the advantages of low cost, environmental protection and safety. In the prior art, microorganisms with the function of decomposing cellulose are generally used to degrade straw. However, the prior art has the problems of low cellulase activity and unstable fiber decomposition function in degrading cellulose.

[0004] Therefore, providing a strain that has good and stable function of decomposing plant fiber has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] One of the purposes of the present invention is to provide a strain of Bacillus velezensis X014-2H, which can decompose cellulose, has low nutritional requirements, mild conditions, can withstand high temperatures, and has strong enzyme production ability.

[0006] The second object of the present invention is to provide the application of Bacillus velezensis X014-2H.

[0007] The third object of the present invention is to provide a bacterial agent containing Bacillus velezensis X014-2H.

[0008] A fourth object of the present invention is to provide an application of a bacterial agent containing Bacillus velezensis X014-2H.

[0009] To realize the above technical solution, the technical solution adopted by the present invention is as follows:

[0010] The invention discloses a strain of Bacillus velezensis X014-2H, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2024413; the deposit time is March 7, 2024, and the deposit location is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0011] The invention discloses a bacterial agent, which comprises the bacterial agent of Bacillus velezensis X014-2H.

[0012] In some embodiments of the present invention, the bacterial agent further comprises a carrier acceptable in fungal preparations.

[0013] The invention discloses application of the Bacillus velezensis X014-2H or bacterial agent in decomposing cellulose.

[0014] The invention discloses application of the Bacillus velezensis X014-2H or bacterial agent in decomposing crop straw.

[0015] The invention discloses application of the Bacillus velezensis X014-2H or bacterial agent in composting and decomposing crop straw.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The Velez Bacillus X014-2H of the invention has the characteristics of low nutritional requirements, mild conditions, high temperature resistance and strong enzyme production ability.

[0018] The Velez Bacillus X014-2H of the present invention is screened out by constant temperature culture in CMC-Na medium at 45°C. Carboxymethyl cellulose is a directly utilized carbon source, and no additional nutrients need to be added during the culture process. The Velez Bacillus X014-2H of the present invention has low nutritional requirements. The culture temperature of 45°C is often used as a temperature condition for screening medium- and high-temperature bacteria, and the strain of the present invention is resistant to high temperatures. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Attached Figure 1 is the colony morphology of strain X014-2H;

[0020] Attached Figure 2 is the microscopic morphology of strain X014-2H;

[0021] Attached Figure 3 is the developmental tree diagram of strain X014-2H;

[0022] Attached Figure 4 A heat map was generated for the strain X014-2H using the strain-wide genome calculated using the OAT software;

[0023] Attached Figure 5 This is a standard curve diagram of Example 5 of the present invention;

[0024] Attached Figure 6 This is a graph showing the test results using the API ZYM test kit. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific embodiments, which are explanations of the present invention rather than limitations. In the embodiments, if specific conditions are not specified, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0026] Example 1 discloses the isolation and screening method of strain X014-2H, and Examples 2-4 disclose the 16S rRNA gene sequence analysis results, physiological and biochemical characteristics detection results and whole gene sequencing results of the strain by China Center for Type Culture Collection, respectively.

[0027] Example 1

[0028] The strain X014-2H of the present invention was isolated from compost in Hongyuan area of ​​Aba Prefecture, Sichuan Province. This embodiment discloses the separation and screening method of the strain X014-2H of the present invention. The details are as follows:

[0029] Use a sterile sampling shovel to collect compost from the pastures around Hongyuan area as samples. Weigh 0.5g of sample, add 4.5ml of fecal diluent, vortex to mix, let stand and stratify. Take 100μl of the supernatant after stratification in 4.9ml of CMC-Na liquid culture medium, mix well and culture at 185rpm and 45℃ for 3 days.

[0030] The CMC-Na liquid medium after culture was heated to 10 -4 , 10 -5 , 10 -6 After gradient dilution, the cells were spread on CMC-Na Congo red medium and then cultured at 45°C.

[0031] The strains with better growth and obvious transparent zones were selected for plate streak purification, and a strain was obtained and named X014-2H.

[0032] The colony morphology of X014-2H on the plate is shown in the attached Figure 1 As shown, the micrograph (1000X) is shown in the attached Figure 2 As shown,

[0033] Example 2

[0034] The strain of the present invention was sent to China Center for Type Culture Collection for identification, and the results of 16S rRNA gene sequence analysis were as follows:

[0035] 1. The 16S rRNA gene sequence of strain X014-2H of the present invention is as follows:

[0036] GTTAATACCGGAGGCTGGATTGACCGGCATGTTTCAATCATAAAAGGTGG

[0037] CTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGA

[0038] GGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGAT

[0039] CGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCA

[0040] GTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTG

[0041] AGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAAG

[0042] TACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACG

[0043] GCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTC

[0044] CGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTTCTTAAGTCTGATGT

[0045] GAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTG

[0046] AGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAG

[0047] AGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGA

[0048] CGCTGAGGCGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTA

[0049] GTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTT

[0050] AGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAA

[0051] GACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCAT

[0052] GTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCT

[0053] CTGACAACCCTAGAGATAGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTG

[0054] GTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCA

[0055] ACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAA

[0056] GGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCA

[0057] TCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAA

[0058] AGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGT

[0059] TCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAAT

[0060] CGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCG

[0061] CCCGTCACAACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTT

[0062] TTGGAGCC

[0063] 2.2) BLAST results of 16S rRNA gene sequencing of strain X014-2H

[0064] Table 1 BLAST results of X014-2H 16S rRNA gene sequencing

[0065]

[0066]

[0067] 3. Evolutionary tree

[0068] The Neighbor-Joining phylogenetic tree of strain X014-2H was constructed based on the 16S rRNA gene sequence alignment results with Brevibacillus brevis DSM 30 (AB101593) as the outer branch as shown in the attached figure. Figure 3 shown.

[0069] Example 3

[0070] The strain of the present invention was sent to China Center for Type Culture Collection for identification. The enzyme activity and carbon source assimilation of the strain of the present invention are shown in Table 2.

[0071] Table 2 Physiological and biochemical characteristics of strain X014-2H - enzyme activity, carbon source assimilation

[0072] Test items Test results NO3 Nitrate reduction to nitrite + TRP Indole - GLU acidified glucose - ADH arginine dihydratase - URE + ESC hydrolyzes esculin (β-glucosidase) + GEL Hydrolyzed Gelatin (Protease) + PNPG β-galactosidase - GLU assimilation of glucose + ARA assimilation of arabinose + MNE assimilates mannose + MAN assimilates mannitol + NAG assimilation of N-acetyl-glucosamine + MAL Assimilated Maltose + GNT Assimilation Gluconate + CAP assimilates decanoic acid - ADI assimilated adipic acid - MLT Anabolic Malate + CIT assimilation citric acid + PAC assimilates phenylacetic acid -

[0073] +: positive reaction; -: negative reaction.

[0074] The carbon source acid production of the strain of the present invention is shown in Table 3.

[0075] Table 3 Physiological and biochemical characteristics of strain X014-2H - carbon source acid production

[0076] Serial number Reagent strips corresponding to tubes / substrates Test results Serial number Reagent strips corresponding to tubes / substrates Test results 0 Comparison - 25 Aesculin + 1 glycerin + 26 Salicylate - 2 Erythrocarb - 27 Cellobiose + 3 D-arabinose - 28 maltose + 4 L-arabinose + 29 lactose + 5 Ribose + 30 Melibiose - 6 D-Xylose - 31 sucrose + 7 L-Xylose - 32 Trehalose + 8 Adonol - 33 Inulin - 9 β-Methyl-D-xyloside - 34 Melezitose - 10 Galactose - 35 Raffinose + 11 glucose + 36 starch - 12 fructose + 37 Glycogen - 13 Mannose + 38 Xylitol - 14 sorbose - 39 Nougat - 15 Rhamnose - 40 D-turanose - 16 Dacron - 41 D-Lyxose - 17 Inositol + 42 D-Tagatose - 18 Mannitol + 43 D-Rock Sugar - 19 Sorbitol + 44 L-Rock Sugar - 20 α-Methyl-D-mannoside - 45 D-arabinitol - 21 α-Methyl-D-glucoside - 46 L-arabinitol - 22 N-acetyl-glucosamine - 47 Gluconate - 23 Amygdalin - 48 2-Keto-gluconate - 24 Arbutin - 49 5-Keto-gluconate -

[0077] +: positive reaction; -: negative reaction.

[0078] As can be seen from Table 3, the strain X014-2H of the present invention can metabolize cellobiose.

[0079] Example 4

[0080] The strain of the present invention was sent to the China Center for Type Culture Collection for identification. The whole genome sequencing results of the microbial strain X014-2H of the present invention showed that the ANI value of the strain X014-2H of the present invention was compared with the whole genome data of the model strain of Bacillus velezensis (NRRL-B-41580) was: 97.94%, which was greater than the threshold value of 96% for ANI same-species judgment.

[0081] The heat map of the strain X014-2H genome calculated by OAT software is shown in the attached figure. Figure 4 shown.

[0082] The content of X014-2H (G+C) mol% was determined to be 46.66%.

[0083] The strain X014-2H of the present invention is identified as: Bacillus velezensis.

[0084] Example 5

[0085] This example discloses the evaluation of the cellulose degradation ability of the strain of the present invention, and the cellulose degradation ability of the Bacillus is evaluated by measuring the cellulose filter paper enzyme activity by the dinitrosalicylic acid (DNS) method. The specific method is as follows:

[0086] Preparation of crude enzyme solution: The single strain to be evaluated was cultured in a cellulose filter paper + LB liquid medium at 37°C constant temperature shaker for 24 hours, inoculated into the fermentation enzyme production medium at a 2% inoculation amount and cultured for 48 hours, and then centrifuged at 5000r / min for 15 minutes to collect the supernatant as the crude enzyme solution for cellulase activity determination. The crude enzyme solution after 100°C water bath for 15 minutes was used as the control group, and 3 experimental groups and 3 control groups were set up for each strain.

[0087] Drawing of glucose standard curve: Use 1mg / ml glucose standard solution and UP water to prepare glucose standard series with concentrations of 0, 0.1, 0.2, 0.2, 0.4, 0.6, 0.8mg / ml, use 0mg / ml glucose solution as blank control, add 2ml DNS reagent and 2ml UP water to glucose solutions of each concentration, mix the solution evenly, place it in a boiling water bath for 5min, cool it to room temperature, and measure the absorbance at a wavelength of 540nm. Use absorbance as the horizontal axis and the corresponding milligrams of sugar in the standard glucose solution as the vertical axis to list the linear regression equation.

[0088] Cellulase activity determination: 1×2cm cellulose filter paper + citric acid buffer was used as the substrate for the enzyme activity reaction. 0.5ml of crude enzyme solution and 1×2cm cellulose filter paper were added to 1ml of citric acid buffer, and after 30min of 50℃ water bath, 2ml of DNS reagent was immediately added to stop the reaction. After 10min of boiling water bath for the reaction mixture, it was cooled to room temperature, UP water was added to make up to 5ml, and the absorbance value was measured at a wavelength of 540nm.

[0089] Calculation formula of cellulase activity:

[0090]

[0091] Table 4 Glucose standard curve determination results

[0092] Glucose concentration (mg / mL) Absorbance A 0.1 0.003 0.2 0.235 0.4 0.745 0.6 1.244 0.8 1.699

[0093] Perform linear regression on the data in Table 4 and draw a standard curve, as shown in the attached figure. Figure 5 It shows that glucose has a good linear relationship in the range of 0.1-0.8 mg / mL.

[0094] The original data measured by the DNS experiment of X014-2H is:

[0095] Table 5. DNS experimental results of strain X014-2H

[0096] Absorbance A Parallel one Parallel 2 Parallel Three Experimental Group 0.164 0.171 0.186 Control group 0.114 0.117 0.134

[0097] The enzyme activity of X014-2H cellulose filter paper was calculated to be 24.05±1.04U / ml.

[0098] Experimental Example 6

[0099] In this example, the API ZYM kit was used to test the enzyme activity of strain X014-2H, and the specific operation was carried out according to the instructions. The results are shown in the attached Figure 6 As shown in the following table:

[0100] Table 6 X014-2H enzyme activity table

[0101] Enzymes Enzyme activity Enzymes Enzyme activity Alkaline phosphatase 4 Naphthol-AS-BI-phosphohydrolase 2 Esterase (C4) 4 α-Galactosidase 2 Lipid esterase (C8) 4 β-Galactosidase 0 Lipase (C14) 0 β-Uronidase 0 Leucine arylaminease 0 α-Glucosidase 0 Valine arylaminease 0 β-Glucosidase 1 Cysteine ​​arylaminease 0 N-acetyl-glucosaminidase 0 Trypsin 0 α-Mannosidase 0 Trypsin 0 α-Fucosidase 5 Acid phosphatase 0 / /

[0102] The results showed that the activity of α-fucosidase of strain X014-2H was stronger than that of α-galactosidase and β-glucosidase tested by API ZYM.

[0103] Finally, it should be noted that the above embodiments are only preferred embodiments of the present invention to illustrate the technical solution of the present invention, but not to limit it, and certainly not to limit the patent scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention without any substantial meaning, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention; in addition, the direct or indirect application of the technical solution of the present invention in other related technical fields is also included in the patent protection scope of the present invention.

Claims

1. A strain of Bacillus velezensis X014-2H, characterized in that: It is deposited in China Center for Type Culture Collection with the accession number CCTCC NO: M 2024413.

2. A bacterial agent, characterized in that A bacterial agent comprising the Bacillus velezensis X014-2H according to claim 1.

3. The bacterial agent according to claim 2, characterized in that Also included are acceptable carriers in fungus preparations.

4. Use of the Bacillus velezensis X014-2H according to claim 1 or the bacterial agent according to any one of claims 2 to 3 in decomposing cellulose.

5. Use of the Bacillus velezensis X014-2H according to claim 1 or the bacterial agent according to any one of claims 2 to 3 in decomposing crop straw.

6. Use of the Bacillus velezensis X014-2H according to claim 1 or the bacterial agent according to any one of claims 2 to 3 in composting and decomposing crop straw.

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