High-temperature-resistant bacillus aryabhattai and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to high-temperature-resistant bacillus aryabhattai and application thereof, the bacillus aryabhattai is named as LYGW-K2, and the preservation number of the bacillus aryabhattai is CCTCC (China Center for Type Culture Collection) NO: M2024963. Soil is collected in a field test field, lignocellulose degrading bacteria are obtained through enrichment culture, Congo red dyeing and aniline blue fading preliminary screening, then the degrading bacteria are sequenced by adopting 16srDNA through determination of the activity of cellulase, hemicellulase and ligninase of the degrading bacteria, a filter paper strip disintegration test and straw degradation rate research, and the content of the lignocellulose is determined. And finally, a strain of high-temperature-resistant bacillus aryabhattai LYGW-K2 with a relatively high degradation rate on lignin and cellulose is selected out. The bacillus aryabhattai is used for straw decomposition, can still keep excellent biological activity at a high composting temperature, generates cellulose degrading enzyme and lignin degrading enzyme, accelerates the straw decomposition process, and is good in high temperature resistance and obvious in straw degradation effect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a high temperature resistant Bacillus agni and an application thereof. Background Art

[0002] According to statistics, the current annual output of crop straw exceeds 800 million tons, and the overall output shows a slowly increasing trend; straw is rich in organic substances such as cellulose, hemicellulose, lignin, protein, as well as a large amount of nutrients such as nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, silicon, etc., and is a high-quality agricultural resource.

[0003] However, due to the tight and complex structure, high degree of polymerization and strong resistance to decomposition of lignin, cellulose and hemicellulose in straw, a large amount of straw is not fully utilized. In the process of using microorganisms to degrade straw, there are problems such as slow degradation of lignocellulose components, long fermentation cycle, incomplete use of materials, and low degree of maturity. Due to the high temperature during composting, most microorganisms are not suitable for survival.

[0004] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a high temperature resistant Bacillus aeruginosa and its application, aiming to solve the problems of high temperature during straw decomposition, which makes it difficult for microorganisms to survive and leads to slow degradation of lignocellulose components.

[0006] The technical solution of the present invention is as follows:

[0007] The invention discloses a high temperature resistant Bacillus arginii, wherein the Bacillus arginii is named as LYGW-K2 and has a preservation number of CCTCC NO: M2024963.

[0008] A bacterial agent containing thermostable Bacillus aggradi.

[0009] The bacterial agent, wherein the bacterial agent also includes auxiliary materials and / or carriers.

[0010] A method for preparing a bacterial agent containing thermostable Bacillus argyi, comprising the steps of:

[0011] activating the Bacillus arginii to obtain activated Bacillus arginii;

[0012] The activated Bacillus arnoldii is inoculated into a PDA liquid culture medium to obtain a bacterial agent.

[0013] The method for preparing the bacterial agent, wherein the activation treatment step comprises:

[0014] The Bacillus arnoldii was inoculated on a PDA solid culture medium by a spread plate inoculation method, and cultured at 29° C.-31° C. for 23-25 ​​hours to complete the activation treatment.

[0015] In the method for preparing the bacterial agent, the activated Bacillus aggregatii is inoculated into the PDA liquid culture medium in an inoculation amount of 1%-5% by volume.

[0016] The preparation method of the bacterial agent, wherein the number of viable bacteria in the bacterial agent is 5.0×10 -9 cfu / ml-6.0×10 -9 cfu / ml.

[0017] An application of the heat-resistant Bacillus arnoldii in straw composting.

[0018] A use of the thermostable Bacillus arnoldii in lignin degradation and / or cellulose degradation.

[0019] Beneficial effect: The present invention provides a high temperature resistant Bacillus arginii and its application, the Bacillus arginii is named LYGW-K2, and the deposit number is CCTCC NO: M2024963. The present invention collects soil in a field test field, obtains lignocellulose degrading bacteria through enrichment culture and Congo red staining and aniline blue fading initial screening, and then determines the cellulose, hemicellulose and lignin enzyme activities of the degrading bacteria, filter paper strip disintegration test and straw degradation rate research, and uses 16s rDNA to sequence the degradation strain, and finally selects a high temperature resistant Bacillus arginii LYGW-K2 with a high degradation rate of lignin and cellulose, which is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M2024963. The Bacillus arginii is used in straw decomposition, can still maintain excellent biological activity at a higher composting temperature, produce cellulose degrading enzymes and lignin degrading enzymes, accelerate the decomposition process of the straw, and the strain has good high temperature resistance and obvious straw degradation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a biofilm of the bacterial plaque obtained in Example 1;

[0021] Figure 2 The phylogenetic tree constructed based on 16s rDNA sequence in Example 1;

[0022] Figure 3 This is a rendering of the filter paper strip degradation experiment in Example 3;

[0023] Figure 4 is the activity data diagram of β-D glucosidase;

[0024] Figure 5is the activity data graph of hemicellulase;

[0025] Figure 6 is the activity data graph of laccase;

[0026] Figure 7 This is the data chart of straw degradation rate. DETAILED DESCRIPTION

[0027] The present invention provides a thermostable Bacillus agglutinosa and its application. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as generally understood by those skilled in the art in the art to which the present invention belongs. It should also be understood that terms such as those defined in general dictionaries should be understood to have meanings consistent with the meanings in the context of the prior art, and will not be interpreted with idealized or overly formal meanings unless specifically defined as herein.

[0029] The present invention provides a high-temperature resistant Bacillus aggrad, the Bacillus aggrad is named LYGW-K2, and the strain has been deposited in the China Center for Type Culture Collection (CCTCC for short, address: Wuhan University, Wuhan, China) on May 23, 2024, with a preservation number of CCTCC NO: M2024963.

[0030] In this embodiment, soil is collected in a field test field, and then lignocellulose degrading bacteria are obtained by enrichment culture, Congo red staining, and aniline blue fading. Then, the degradation bacteria are sequenced by 16s rDNA through the determination of cellulose, hemicellulose and lignin enzyme activities, filter paper strip disintegration test, and straw degradation rate determination. Finally, a high-temperature resistant Bacillus agni LYGW-K2 with a high degradation rate of lignin and cellulose is selected and preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M2024963. The Bacillus agni is used in straw decomposition, and can still maintain excellent biological activity at a higher composting temperature, produce cellulose degrading enzymes and lignin degrading enzymes, accelerate the decomposition process of straw, and the strain has good high temperature resistance and obvious straw degradation effect.

[0031] In some embodiments, the 16s rDNA sequence of the thermostable Bacillus arnoldii is as shown in SEQ ID NO.1.

[0032] In some embodiments, the colony morphology of the thermostable Bacillus arnoldii is characterized by: the colony morphology on the enriched medium is round, the positive and reverse colors are both yellow, opaque and with regular edges, the surface is convex, smooth and glossy, and relatively moist.

[0033] In addition, the present invention also provides a bacterial agent containing the thermostable Bacillus arnoldii.

[0034] In this embodiment, when the bacterial agent containing the heat-resistant Bacillus arnoldii is used in straw decomposition, it can still maintain excellent biological activity at a higher composting temperature, produce cellulose degrading enzymes and lignin degrading enzymes, accelerate the decomposition process of the straw, and the strain has good high temperature resistance and a significant effect on straw degradation.

[0035] In some embodiments, the bacterial agent further comprises auxiliary materials and / or carriers, which can provide survival conditions for the Bacillus argyi and ensure its activity.

[0036] In addition, the present invention also provides a method for preparing a bacterial agent containing thermostable Bacillus arnoldii, comprising the steps of:

[0037] Step S10: activating the Bacillus arginense to obtain activated Bacillus arginense;

[0038] Step S20: inoculating the activated Bacillus aeruginosa into a PDA liquid culture medium to obtain a bacterial agent.

[0039] In this embodiment, the heat-resistant Bacillus arnoldii can be made into a bacterial agent through the preparation method, and the bacterial agent can be used in straw decomposition. It can still maintain excellent biological activity at a higher composting temperature, produce cellulose degrading enzymes and lignin degrading enzymes, and accelerate the decomposition process of the straw. The strain has good high temperature resistance and a significant effect on straw degradation.

[0040] In some embodiments, the activation treatment step includes: inoculating the Bacillus aeruginosa on a PDA solid culture medium by a spread plate inoculation method, and culturing at 29° C.-31° C. for 23-25 ​​hours to complete the activation treatment.

[0041] Specifically, the Bacillus aggregati strain is taken out of the ultra-low temperature refrigerator for activation: inoculated on a PDA solid culture medium by a spread plate inoculation method, and cultured at 29° C.-31° C. for 23-25 ​​hours; after the activation, a sterilized inoculation loop is used to gently scrape a portion of the bacterial film, and inoculated into a PDA liquid culture medium (without agar) to prepare a liquid bacterial agent.

[0042] In some embodiments, after the liquid bacterial agent is prepared, the liquid bacterial agent can be added to the constructed carrier to prepare a bacterial agent co-body.

[0043] In some embodiments, the carrier includes but is not limited to bentonite, diatomaceous earth, sodium alginate-gelatin, and the like.

[0044] In some embodiments, the preparation step of the PDA solid culture medium includes: weighing 200g of peeled potato pieces, boiling for 30min, collecting the filtrate, adding ultrapure water to 1.0L, then adding 20.0g of glucose and 20.0g of agar, adjusting the pH value to 6.9-7.1, and sterilizing in a high-pressure steam sterilizer to obtain a PDA solid culture medium. Compared with the PDA solid culture medium, the PDA liquid culture medium does not contain agar.

[0045] In some embodiments, the activated Bacillus aeruginosa is inoculated into the PDA liquid culture medium in an inoculation volume ratio of 1%-5%.

[0046] In a preferred embodiment, the activated Bacillus aeruginosa is inoculated into the PDA liquid culture medium in an inoculation amount of 1% by volume.

[0047] In some embodiments, the number of viable bacteria in the bacterial agent is 5.0×10 -9 cfu / ml-6.0×10 -9 cfu / ml.

[0048] In addition, the present invention also provides an application of high temperature resistant Bacillus arnoldii in straw composting.

[0049] In addition, the present invention also provides an application of the high temperature resistant Bacillus arnoldii in lignin degradation and / or cellulose degradation.

[0050] In this embodiment, the Bacillus arnoldii is used in straw decomposition and can maintain excellent biological activity at higher composting temperatures, produce cellulose degrading enzymes and lignin degrading enzymes, accelerate the decomposition process of the straw, and the strain has good high temperature resistance and a significant effect on straw degradation.

[0051] The following examples are further given to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention.

[0052] The culture medium required for the following examples includes:

[0053] Alkali lignin solid culture medium: alkaline lignin 5g, (NH4)2SO4 2g, K2HPO4 1g, KH2PO4 1g, MgSO4·7H2O 0.2g, NaCl 1.5g, H2O 1L, agar powder 10g, pH=7.5, sterilization at 121℃ for 20min.

[0054] Sodium carboxymethyl cellulose (CMC-Na) solid culture medium: CMC-Na 5.0 g, (NH4)2SO4 2.0 g, K2HPO4 1.0 g, NaNO3 3.0 g, KCl 0.5 g, MgSO4 0.5 g, agar 10.0 g, H2O 1 L, humidity 50%, high pressure sterilization at 121 ° C for 20 min.

[0055] Enrichment culture medium: alkaline lignin liquid culture medium, sodium carboxymethyl cellulose liquid culture medium; compared with solid culture medium, liquid culture medium does not contain agar.

[0056] Aniline blue rescreening culture medium: yeast powder 15g, glucose 15g, K2HPO4 0.5g, KNO3 1.0g, MgSO4 0.5g, aniline blue 0.1g, H2O 1L, agar powder 10g, pH=7.5.

[0057] PDA medium: 1.0 L potato boiled liquid, 20.0 g glucose, 20.0 g agar (not included in liquid medium). Potato boiled liquid: weigh 200 g peeled potato pieces, boil for 30 min, collect the filtrate and add water to 1.0 L.

[0058] Straw powder culture medium: straw powder 20g, K2HPO4 1g, NaCl 0.1g, MgSO4 0.3g, (NH4)2SO4 0.5g, FeCl3 0.01g, CaCl2 0.1g, H2O 1L, pH=7.2.

[0059] Filter paper strip degradation medium: filter paper strip 0.2 g, K2HPO4 1 g, NaCl 0.1 g, MgSO4 0.3 g, (NH4)2SO4 0.5 g, FeCl3 0.01 g, CaCl2 0.1 g, H2O 1 L, pH = 7.2.

[0060] Example 1

[0061] This embodiment performs the initial screening of strains, including the following steps:

[0062] Weigh 5 g of soil sample into a conical flask containing glass beads, add 45 mL of enrichment medium, and shake at 180 r min in an oscillator. -1 After shaking for 72 hours, an enriched soil suspension was obtained.

[0063] Use sterile water to dilute the soil suspension in a gradient manner. -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 100 μL of each soil suspension was evenly spread on PDA medium, and 3 parallels were made for each concentration. The medium was inverted and cultured at 50°C for 3 days.

[0064] When colonies grow in the culture medium, pick out a single colony and enrich it in a liquid culture medium. Purify the obtained colony isolate on the corresponding plate culture medium using the three-zone line method until a single colony is obtained. Record the morphology, color, size, colony edge, gloss, transparency, surface, degree of protrusion, etc. of the single colony. Number the obtained single colonies and store them in a -80°C refrigerator for later use. The morphological characteristics of the colonies are shown in Table 1, and the actual picture of the colonies is shown in Figure 1 shown.

[0065] Table 1 Morphological characteristics of colonies

[0066] shape Positive Color Invert Color size edge smooth transparent luster Wet and dry surface round yellow yellow middle rule √ × √ Wet Convex

[0067] 1) Hydrolysis zone determination test of the thermostable Bacillus argyi LYGW-K2 strain obtained by screening

[0068] The strains were screened and cultured on sodium carboxymethyl cellulose solid medium for 3 days. -1 After staining with Congo red for 15 min, the staining solution was discarded and 1 mol L -1 The cells were eluted with NaCl solution, and the ratio of the transparent zone diameter to the colony diameter was measured with a ruler; the cells were cultured with aniline blue rescreening medium for 3 days, and the ratio of the transparent zone diameter to the colony diameter was measured with a ruler. Specifically, the ratio of the transparent zone diameter to the colony diameter is shown in Table 2.

[0069] Table 2 Ratio of transparent circle diameter to colony diameter

[0070] Experiment Name Transparent ring diameter (mm) Colony diameter (mm) Transparent circle diameter / colony diameter Congo red staining 10.0 8.0 1.250 Aniline blue fading 9.5 8.9 1.067

[0071] 2) Identification of thermotolerant Bacillus aggregati LYGW-K2 strain

[0072] The 16s rDNA was used to sequence the thermostable Bacillus arginii LYGW-K2 strain to determine the genus of the thermostable Bacillus arginii LYGW-K2 strain. The DNA of the strain was extracted by sampling column method and magnetic bead method. The forward primer was 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; the reverse primer was 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'. PCR reaction system: PCR Mix 21μL, Primer F (5p) and Primer R (5p) 1μL each, template DNA template amount (2ngμL -1 )2μL, the total system volume was 25μL. The amplification program was: 96℃ preheating for 5min; 96℃ 20s, 62℃ 30s, 72℃ 30s, 35 cycles; 72℃ final extension for 10min, and then maintained at 4℃. The PCR product was purified by gel excision and gene sequencing was completed. The sequencing results were compared with NCBI-BLAST and analyzed by MEGA11 software. The neighbor-joining method was used to construct a phylogenetic tree; the phylogenetic tree is shown in Figure 2 shown.

[0073] The obtained 16s rDNA sequence of Bacillus agni is shown as SEQ ID NO.1.

[0074] Example 2

[0075] The determination of the enzyme activity secreted by the Bacillus argyi LYGW-K2 strain in this example is as follows:

[0076] The enzyme activity of the screened Bacillus lyglucoside LYGW-K2 was determined, and the β-D-glucosidase activity and hemicellulase activity were determined by the 3,5-dinitrosalicylic acid method; the laccase activity was determined by the ABTS oxidation method. The enzyme activity data table is shown in Table 3.

[0077] Table 3 Enzyme activity data

[0078] type Enzyme activity (U / ml) β-D-glucosidase 468 Hemicellulase 344 Laccase 2.074

[0079] Example 3

[0080] This example mainly studies the degradation effect of Bacillus argyi LYGW-K2 (represented by K2), including the following:

[0081] 1) Filter paper strip degradation experiment

[0082] The bacterial liquid was inoculated into 50 mL of filter paper strip degradation medium at a 1% inoculation volume and cultured for 5 days. The degree of degradation of the filter paper strip was recorded and observed. The degradation effect was judged according to the degree of damage to the filter paper. The disintegration of the filter paper strip is shown in Table 4; "+" indicates that the edge of the filter paper has expanded; "++" indicates that the filter paper has expanded as a whole and has bent downward; "+++" indicates that the filter paper is in an indeterminate shape; "++++" indicates that the filter paper is in a paste state. The effect diagram of the filter paper strip degradation experiment is shown in Table 4. Figure 3 shown.

[0083] Table 4 Disintegration of filter paper strips

[0084] 1d 2d 3d 5d + +++ ++++ ++++

[0085] 2) Straw degradation rate test

[0086] Weigh 2g of straw powder into a nylon bag and place it in a 250ml triangular flask. Inoculate the single strain obtained by screening into 50mL of sterilized straw powder medium at a 1% inoculum. Repeat each treatment 3 times. Meanwhile, a control (CK) without any bacterial strain inoculation was used. The culture was incubated at 30℃ and 150r min. -1 The straw powder was fully rinsed with sterile water, dried at 80℃ to constant weight, and weighed. The degradation rate of the straw powder was calculated by weight loss method. After 15 days, the degradation rate was as high as 28.24%.

[0087] Example 4

[0088] In this example, the high temperature resistance of Bacillus argyi LYGW-K2 strain was measured, as follows:

[0089] High temperature resistance experimental data: Bacillus arginense LYGW-K2 strain was screened out under 50℃ culture conditions. The high temperature resistance of Bacillus arginense LYGW-K2 strain was tested to explore its ultimate tolerance temperature. +: indicates the number of bacteria, "++" indicates the number of colonies>50; "+" indicates the number of colonies<50; "-" indicates that the number of colonies is 0. The results are shown in Table 5. It can be concluded that LYGW-K2 is inactivated at 80℃ and stops working. It still has good activity at 70℃ and can play a role.

[0090] Table 5 High temperature resistance test data

[0091] Temperature ℃ LYGW-K2 60 ++ 70 + 80 -

[0092] Comparative Example 1:

[0093] For comparison, two strains K3 and K6 were isolated from farmland soil and identified using conventional methods. The results showed that K3 was a Bacillus subtilis strain and K6 was a Bacillus sp.

[0094] Taking K3 and K6 as comparative examples, they were also subjected to enzyme activity determination test, filter paper strip degradation test, straw degradation rate test, and high temperature resistance test. The enzyme activity data comparison table is shown in Table 6, and the activity data of β-D glucosidase is shown in Figure 4 As shown, the activity data of hemicellulase is shown in Figure 5 The activity data of laccase are shown in Figure 6 The filter paper strip disintegration table is shown in Table 7; the straw degradation table is shown in Table 8, and the straw degradation rate data is shown in Table 9. Figure 7 The high temperature resistance performance comparison is shown in Table 9.

[0095] Table 6 Enzyme activity data comparison table

[0096] serial number β-D-glucosidase (U / ml) Hemicellulase (U / ml) Laccase (U / ml) K3 286 321 1.556 K6 307.8 211 1.728 LYGW-K2 468 344 2.074

[0097] Table 7 Disintegration of filter paper strips

[0098] serial number 1d 2d 3d 5d K3 + +++ +++ +++ K6 No change + ++ ++ LYGW-K2 + +++ ++++ ++++

[0099] Table 8 Straw degradation status

[0100] serial number Degradation rate % K3 23.22 K6 24.31 LYGW-K2 28.32

[0101] Table 9 High temperature resistance performance comparison table

[0102] Temperature ℃ K3 LYGW-K2 K6 60 ++ ++ ++ 70 + + - 80 - - -

[0103] In summary, the present invention provides a thermostable Bacillus arginii and its application, the Bacillus arginii is named LYGW-K2, and the deposit number is CCTCC NO: M2024963. The present invention collects soil in a field test field, obtains lignocellulose degrading bacteria by enrichment culture and Congo red staining and aniline blue fading initial screening, and then determines the cellulose, hemicellulose and lignin enzyme activities of the degrading bacteria, filter paper strip disintegration test and straw degradation rate research, and uses 16s rDNA to sequence the degradation strain, and finally selects a thermostable Bacillus arginii LYGW-K2 with a high degradation rate of lignin and cellulose, which is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M2024963. The Bacillus arginii is used in straw decomposition, and can still maintain excellent biological activity at a higher composting temperature, produce cellulose degrading enzymes and lignin degrading enzymes, accelerate the decomposition process of the straw, and the strain has good high temperature resistance and obvious straw degradation effect.

[0104] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A thermostable Bacillus agglutinosa, It is characterized in that The Bacillus arnoldii is named as LYGW-K2, and its deposit number is CCTCC NO: M2024963.

2. A bacterial agent containing the thermostable Bacillus aeruginosa as claimed in claim 1.

3. The bacterial agent according to claim 1, It is characterized in that The bacterial agent also includes auxiliary materials and / or carriers.

4. A method for preparing a bacterial agent containing thermostable Bacillus arguta. It is characterized in that Includes steps: activating the Bacillus arginii as claimed in claim 1 to obtain activated Bacillus arginii; The activated Bacillus arnoldii is inoculated into a PDA liquid culture medium to obtain a bacterial agent.

5. The method for preparing the bacterial agent according to claim 4, It is characterized in that The activation treatment step comprises: The Bacillus arnoldii was inoculated on a PDA solid culture medium by a spread plate inoculation method, and cultured at 29° C.-31° C. for 23-25 ​​hours to complete the activation treatment.

6. The method for preparing the bacterial agent according to claim 4, It is characterized in that The activated Bacillus aggregatii is inoculated into the PDA liquid culture medium in an inoculation amount of 1%-5% by volume.

7. The method for preparing the bacterial agent according to claim 4, It is characterized in that The number of viable bacteria in the bacterial agent is 5.0×10 -9 cfu / ml-6.0×10 -9 cfu / ml.

8. Use of the thermostable Bacillus aeruginosa as claimed in claim 1 in straw composting.

9. Use of the thermostable Bacillus aeruginosa as claimed in claim 1 in lignin degradation and / or cellulose degradation.

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