Bacterium capable of efficiently degrading cellulose under low-temperature condition and application thereof

Abstract

The invention discloses a bacterium capable of efficiently degrading cellulose under a low-temperature condition and application of the bacterium. The low-temperature-resistant cellulose degrading bacterium Paenibacillus sp.JN-1 provided by the invention is preserved in the China Center for Type Culture Collection on July 22, 2024, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M20241624. The strain can well grow and secrete filter paper enzyme and carboxymethyl cellulose under the conditions of 30 DEG C and 15 DEG C. After the JN-1 strain is cultured for one month under the conditions of 30 DEG C and 15 DEG C, the actual degradation rates of the JN-1 strain to corn straw respectively reach 34.04% and 34.23%, and the strain resource can be provided for straw decomposition efficiency in a low-temperature environment.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and specifically relates to a bacillus strain capable of efficiently degrading cellulose under low temperature conditions and application thereof, in order to provide strain resources for straw resource utilization in low temperature areas. Background Art

[0002] Straw is a byproduct of crop stems and leaves, containing various nutrients required for plant growth. Returning straw to the field is beneficial to improving straw utilization, providing crops with the necessary nutrients, and improving soil fertility. However, if straw is returned directly to the field, it may not be completely degraded before the next crop is sown, which is not conducive to the growth of the next crop and will aggravate the occurrence of diseases and insect pests. It is also affected by various factors such as temperature and soil texture. Therefore, reasonable straw treatment is particularly important. There are many ways to process straw resources, among which microbial degradation methods are widely used due to their high safety and low energy consumption.

[0003] Corn harvesting in northern my country is usually in late autumn, and currently the suitable temperature for cellulose-degrading bacteria is mostly 20℃-50℃, which is difficult to adapt to in cold environments. Therefore, research on low-temperature and efficient cellulose-degrading bacteria and exploring bacterial resources suitable for local climatic conditions are of great practical significance for promoting straw decomposition, improving straw utilization and improving soil fertility. Summary of the invention

[0004] The purpose of the present invention is to provide a bacterium that can efficiently degrade cellulose under low temperature conditions. During the growth and reproduction process under low temperature conditions, the strain can effectively produce cellulase and degrade corn stalks.

[0005] The low-temperature-resistant cellulose-degrading bacteria Paenibacillus sp. JN-1 provided by the present invention was deposited in the China Center for Type Culture Collection on July 22, 2024, with a deposit number of CCTCC NO: M20241624.

[0006] The low-temperature resistant cellulose degrading bacteria JN-1 provided by the present invention is a Gram-negative bacterium that can grow normally in a sodium carboxymethyl cellulose culture medium. The colony morphology on the solid culture medium is round, white and opaque, with a rough waxy surface and a certain degree of extensibility at the edge, such as Figure 1 As shown. The JN-1 strain was Figure 2 Shown as a rod.

[0007] The 16SrDNA sequence of the strain JN-1 provided by the present invention is shown in SEQ ID NO.1.

[0008] The low-temperature resistant cellulose degrading bacteria JN-1 provided by the present invention has a D (diameter of transparent zone) / d (colony diameter) of 3.0-3.83 and 2.65-3.28 at 30°C and 15°C respectively after being stained and decolorized by Congo red staining on a solid culture medium.

[0009] The invention discloses an application of the low-temperature resistant cellulose degrading bacteria Paenibacillus sp. JN-1 in the preparation of low-temperature cellulase or filter paper enzyme.

[0010] The invention discloses an application of the low-temperature resistant cellulose degrading bacteria Paenibacillus sp. JN-1 in low-temperature straw degradation.

[0011] As a preferred embodiment of the present invention, the low temperature is 15-30°C.

[0012] The low-temperature resistant cellulose degrading bacteria JN-1 provided by the present invention can secrete filter paper enzyme and carboxymethyl cellulase when cultured at 30°C and 15°C. After continuous measurement every 24 hours for 15 days, the maximum values ​​of the two cellulase activities at the following different temperatures are:

[0013] At 30°C: filter paper enzyme activity 2.19U / mL, carboxymethyl cellulase activity 3.48U / mL;

[0014] At 15°C: filter paper enzyme activity 1.93U / mL, carboxymethyl cellulase activity 4.11U / mL;

[0015] After the low-temperature resistant cellulose degrading bacteria JN-1 provided by the present invention are added into the straw fermentation medium, the degradation rates of corn straw at 30° C. and 15° C. after 30 days are 34.04% and 34.23% respectively.

[0016] A low-temperature cellulase, wherein a crude enzyme solution of the low-temperature cellulase is prepared by the following method: low-temperature resistant cellulose degrading bacteria Paenibacillus sp. JN-1 is cultured in a sodium carboxymethyl cellulose liquid enzyme production medium at low temperature and 120r / min constant temperature shaking for 15 days, the fermentation liquid is centrifuged to obtain the supernatant as the crude enzyme solution, and the low temperature is 15-30°C.

[0017] As a preferred embodiment of the present invention, the formula of the sodium carboxymethyl cellulose liquid enzyme production culture medium is: peptone 10.0g, beef extract 10.0g, CMC-Na 10.0g, NaCl 1.5g, KH2PO41.0g, MgSO4·7H2O 0.3g, distilled water 1000mL, PH7.0-7.2.

[0018] A low-temperature filter paper enzyme. The crude enzyme liquid of the low-temperature filter paper enzyme is prepared by the following method: the low-temperature resistant cellulose degrading bacteria Paenibacillus sp. JN-1 is cultured in a filter paper liquid enzyme production culture medium at a low temperature and a constant temperature shaker of 120r / min for 15 days, the fermentation liquid is centrifuged and the supernatant is taken as the crude enzyme liquid, and the low temperature is 15-30°C.

[0019] As a preferred embodiment of the present invention, the filter paper liquid enzyme production culture medium is formulated as peptone 10.0 g, beef extract 10.0 g, filter paper strip 50.0 mg, NaCl 1.5 g, KH2PO4 1.0 g, MgSO4·7H2O 0.3 g, distilled water 1000 mL, PH 7.0-7.2.

[0020] A method for low-temperature straw degradation comprises adding straw into straw fermentation medium, inoculating 2% culture solution of low-temperature cellulose-degrading bacteria Paenibacillus sp. JN-1 into the straw fermentation medium, and culturing under shaking conditions of 30°C to 15°C and 120 r / min for 20 to 35 days to degrade the straw.

[0021] As a preferred embodiment of the present invention, the formula of the straw fermentation medium is: 1.0 g NH4NO3, 0.5 g MgSO4·7H2O, 1.0 g KH2PO4, 1.0 g peptone, and 1000 mL distilled water.

[0022] Beneficial effects:

[0023] After strain JN-1 was screened from the soil, it was subcultured and enriched at a low temperature of 15°C for many times, and corn straw was used as the only carbon source for limiting carbon source screening. This method retains the natural property of strain JN-1 to degrade straw stably under low temperature conditions to a greater extent. Even at 15°C and under unoptimized culture conditions, strain JN-1 can still effectively degrade corn straw. This provides a potential excellent microbial resource for solving the problem of returning corn straw to the field in situ in Northeast China, and provides a material basis and technical support for the subsequent development of microbial agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the growth of strain JN-1 on solid medium;

[0025] Figure 2 This is an optical microscope photo of the bacterial morphology of strain JN-1;

[0026] Figure 3 This is the electrophoresis diagram of 16SrDNA PCR products of strain JN-1;

[0027] Figure 4This is the appearance of strain JN-1 after staining and decolorization by Congo red clearing zone staining method;

[0028] Figure 5 The changes of enzyme activity of strain JN-1 after continuous cultivation at 30℃ and 15℃ for 15 days;

[0029] Figure 6 is the actual degradation rate of corn straw by strain JN-1 at 30℃ and 15℃.

[0030] Biomaterial Deposit Information

[0031] Paenibacillus sp.JN-1 was deposited in the China Center for Type Culture Collection on July 22, 2024, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M20241624. DETAILED DESCRIPTION

[0032] The present invention provides a low-temperature cellulose-degrading bacterium, the specific contents of which are as follows:

[0033] Medium used:

[0034] Enrichment medium: NaNO3 0.5 g, KCl 0.5 g, KH2PO4 1.0 g, Fe2(SO4)3·7H2O (prepare into 1% mother solution first, then take 1 mL), MgSO4·7H2O 0.5 g, corn stalk powder (crush the corn stalk and pass it through a 60-mesh sieve) 10 g, add distilled water to 1000 ml;

[0035] Isolation medium (NA medium): 3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, 18.0 g agar, 1000 mL distilled water, pH 7.0-7.2;

[0036] Identification medium (sodium carboxymethyl cellulose medium): CMC-Na 20.0 g, (NH4)2SO4 2.0 g, MgSO4·7H2O 0.5 g, KH2PO4 1 g, NaCl 0.5 g, distilled water 1000 mL, agar 20 g, pH 7.0-7.2;

[0037] Straw fermentation medium: NH4NO3 1.0 g, MgSO4·7H2O 0.5 g, KH2PO4 1.0 g, peptone 1.0 g, distilled water 1000 mL;

[0038] LB medium: 10 g tryptone, 5 g sodium chloride, 5 g yeast powder, 1000 mL distilled water, pH 7.0-7.2;

[0039] Sodium carboxymethyl cellulose liquid enzyme production medium: peptone 10.0g, beef extract 10.0g, CMC-Na 10.0g, NaCl 1.5g, KH2PO4 1.0 g, MgSO4·7H2O 0.3g, distilled water 1000mL, pH 7.0-7.2;

[0040] Filter paper liquid enzyme production medium: formula is peptone 10.0g, beef extract 10.0g, filter paper strip 50.0mg, NaCl 1.5g, KH2PO4 1.0 g, MgSO4·7H2O 0.3g, distilled water 1000mL, pH 7.0-7.2;

[0041] Reagents used:

[0042] 0.1% Congo red stain;

[0043] 1mol / LNaCl;

[0044] DNS reagent;

[0045] Citric acid-sodium citrate buffer: weigh 21.01 g of citric acid, add distilled water, stir evenly, and dilute to 1000 mL to prepare a 0.1 mol / L citric acid solution; weigh 29.41 g of sodium citrate, add distilled water, stir evenly, and dilute to 1000 mL to prepare a 0.1 mol / L sodium citrate solution; mix 9.2 mL of 0.1 mol / L citric acid solution and 10.8 mL of 0.1 mol / L sodium citrate buffer to obtain a citric acid-sodium citrate buffer with a pH value of 4.8;

[0046] 1% CMC-Na solution: Weigh 10g CMC-Na and add 990mL citric acid-sodium citrate buffer. After it is completely dissolved, it becomes 1% CMC-Na solution.

[0047] 1mg / L glucose standard solution: Accurately weigh 0.1g of glucose dried to constant weight at 105℃, dissolve it in a small amount of distilled water, transfer it into a 100ml volumetric flask, make up to volume, and shake well.

[0048] 1. Isolation of Cellulolytic Bacteria

[0049] Weigh 10g of soil sample collected in Lishu County, Siping City, Jilin Province, and put it into 90mL of sterile water containing glass beads, shake at 150r / min for 1h, and then let it stand for 5min. Take 2mL of supernatant in the sterilized and cooled enrichment medium, shake and culture at 20℃ and 180r / min for 3d. The enriched culture solution was diluted stepwise, and 10 -6 , 10 -7 , 10 -80.1 mL was spread on NA medium for separation, and 3 groups of parallels were set up for each gradient and inverted cultured at 30°C for 1 to 2 days.

[0050] 2. Purification of cellulose-degrading bacteria

[0051] After plate culture, select representative colonies based on the number, morphology, color and other characteristics of the colonies. Use a sterile inoculation loop to separate the colonies on NA medium until a purified single colony is obtained. Then, store the single colony in LB medium and store it at 4°C for future use after it has fully grown.

[0052] 3. Screening of cellulose-degrading bacteria

[0053] like Figure 1 As shown, the purified strains were inoculated on CMC-Na culture plates with an inoculation loop, and cultured at 30°C and 15°C, respectively. The strains that could grow on the plates were selected and stained with Congo red transparent circle staining method, that is, the identification culture plates were stained with 0.1% Congo red dye for 20 minutes, and then decolorized with 1mol / L NaCl solution for 30 minutes, and the excess solution on the plates was poured out. Finally, the cellulose-dominant decomposing bacteria were screened out according to the ratio of the transparent circle diameter (D) on the culture plate to the colony diameter (d). The D / d values ​​were calculated, and the D / d of the JN-1 strain under the culture conditions of 30°C and 15°C were 3.0-3.83 and 2.65-3.28, respectively.

[0054] 4. Identification of strain JN-1

[0055] The morphological characteristics and physiological and biochemical properties of strain JN-1 were identified: JN-1 can grow normally in sodium carboxymethyl cellulose medium, and the colony morphology on solid culture medium is round, white and opaque, with a rough waxy surface and a certain degree of extensibility at the edge, such as Figure 1 The bacteria were subjected to Gram staining test and the morphology of the bacteria was observed under a 16×100 microscope. Figure 2 It is rod-shaped as shown, and further observation of the bacterial color shows red, so JN-1 is a Gram-negative bacterium.

[0056] The strain JN-1 was sequenced and identified, and its 16SrDNA sequence was shown in SEQ ID NO.1, and was identified as Paenibacillus sp.

[0057] 5. Preparation of crude enzyme solution

[0058] The test strains were inoculated into sodium carboxymethyl cellulose liquid enzyme production medium and filter paper liquid enzyme production medium, with each strain repeated 3 times, and cultured in a constant temperature shaker at 30℃ and 15℃, 120r / min for 15 days, and samples were taken at a fixed time every day. The samples were centrifuged at 10000r / min for 3min, and the supernatant was taken as the crude enzyme solution.

[0059] 6. Determination of carboxymethyl cellulase activity

[0060] Determination of carboxymethyl cellulase activity: Add 200 μL crude enzyme solution to 200 μL prepared 1% CMC-Na solution (add 200 μL crude enzyme solution inactivated in boiling water bath to the control group), mix well and place in a 50°C water bath for 30 min, add 600 μL DNS reagent, shake well and place in a boiling water bath for 10 min, cool rapidly to room temperature in an ice water bath, and dilute to 5 mL with distilled water. Use a spectrophotometer to measure the absorbance at 540 nm. According to the glucose standard curve (y=1.0569x+0.0109, R 2 =0.9941) to calculate the change of carboxymethyl cellulose activity, such as Figure 5 As shown, the maximum activity of carboxymethyl cellulase at 30°C was 3.48 U / mL, and the maximum activity of carboxymethyl cellulase at 15°C was 4.11 U / mL.

[0061] 7. Determination of filter paper enzyme activity

[0062] Add 7.5 mg of filter paper as substrate into the test tube, add 250 μL of crude enzyme solution (the control group adds 250 μL of crude enzyme solution inactivated in a boiling water bath), mix well and place in a 50°C water bath for 30 minutes, add 1000 μL of DNS reagent, shake well and place in a boiling water bath for 10 minutes, cool rapidly to room temperature in an ice water bath, and dilute to 5 mL with distilled water. Use a spectrophotometer to measure the absorbance at 540 nm, and according to the glucose standard curve (y=1.0569x+0.0109, R 2 =0.9941) to calculate the change of filter paper enzyme activity, such as Figure 5 As shown, the highest enzyme activity of the filter paper at 30°C was 2.19 U / mL, and the highest enzyme activity of the filter paper at 15°C was 1.93 U / mL.

[0063] Enzyme activity is defined as: 1 mL of enzyme solution hydrolyzes the substrate to produce 1 μmol of reducing sugar in 1 minute, which is 1 enzyme unit (U / mL). The enzyme activity calculation method is as follows:

[0064]

[0065] X: enzyme activity to be measured; W: amount of reducing sugar; V1: volume of crude enzyme solution after dilution; V2: volume of enzyme solution to be measured added to the reaction; V: volume of crude enzyme solution taken

[0066] 8. Actual straw degradation capacity of cellulose-degrading bacteria

[0067] Cut the sterilized corn stalks into 2 cm long segments and dry them to constant weight for later use. Take 200 ml of natural straw liquid fermentation liquid and 20 g of treated straw segments and divide them into 500 ml conical flasks to make natural straw fermentation culture. The obtained strain JN-1 was inoculated into the straw fermentation medium with a culture liquid inoculation amount of 2%, and oscillated and cultured at 30°C and 15°C, 120r / min for 30 days. The degradation rate of the straw in each group was measured to study the degradation effect of JN-1 on the straw. The mass of dry matter after fermentation was weighed and recorded as Ml (g), and the mass of straw before fermentation was recorded as M0 (g). The weight loss rate (D) of the straw was calculated by the formula. It was measured that after one month of cultivation at 30°C and 15°C, the actual degradation rate of the JN-1 strain for corn straw reached 34.04% and 34.23%, respectively. Figure 6 shown.

[0068]

Claims

1. A cold-resistant cellulose-degrading bacterium Paenibacillus sp. JN-1, characterized in that: It was deposited in the China Center for Type Culture Collection on July 22, 2024, with the deposit number being CCTCC NO: M20241624.

2. Use of the low-temperature resistant cellulose degrading bacterium Paenibacillus sp. JN-1 according to claim 1 in the preparation of low-temperature cellulase or filter paper enzyme.

3. Use of the low-temperature resistant cellulose degrading bacteria Paenibacillus sp. JN-1 according to claim 1 in low-temperature degradation of straw.

4. The use according to claim 3, characterized in that The low temperature is 15-30°C.

5. A low-temperature cellulase, characterized in that The crude enzyme solution of the low-temperature cellulase is prepared by the following method: the low-temperature resistant cellulose degrading bacteria Paenibacillus sp. JN-1 described in claim 1 is cultured in a sodium carboxymethyl cellulose liquid enzyme production medium at low temperature and a constant temperature shaker at 120 r / min for 15 days, the fermentation liquid is centrifuged and the supernatant is taken as the crude enzyme solution, and the low temperature is 15-30°C.

6. The low-temperature cellulase according to claim 5, characterized in that The formula of the sodium carboxymethyl cellulose liquid enzyme production culture medium is: 10.0g of peptone, 10.0g of beef extract, 10.0g of CMC-Na, 1.5g of NaCl, 1.0g of KH2PO4, 0.3g of MgSO4·7H2O, 1000mL of distilled water, and PH7.0-7.

2.

7. A low temperature filter paper enzyme, characterized in that The crude enzyme liquid of the low-temperature filter paper enzyme is prepared by the following method: the low-temperature resistant cellulose degrading bacteria Paenibacillus sp. JN-1 described in claim 1 is cultured in a filter paper liquid enzyme production medium at a low temperature and a constant temperature shaker of 120r / min for 15 days, the fermentation liquid is centrifuged and the supernatant is taken as the crude enzyme liquid, and the low temperature is 15-30°C.

8. The low-temperature filter paper enzyme according to claim 7, characterized in that The formula of the filter paper liquid enzyme production culture medium is 10.0g of peptone, 10.0g of beef extract, 50.0mg of filter paper strip, 1.5g of NaCl, 1.0g of KH2PO4, 0.3g of MgSO4·7H2O, 1000mL of distilled water, and the pH value is 7.0-7.

2.

9. A method for low temperature straw degradation, characterized in that The straw is added into the straw fermentation medium, the low-temperature resistant cellulose degrading bacteria Paenibacillus sp. JN-1 described in claim 1 is inoculated into the straw fermentation medium at a culture solution inoculation rate of 2%, and the medium is shaken and cultured at 30°C to 15°C and 120r / min for 20 to 35 days to degrade the straw.

10. The method according to claim 9, characterized in that The formula of straw fermentation medium is: NH4NO3 1.0 g, MgSO4·7H2O 0.5 g, KH2PO4 1.0 g, peptone 1.0 g, and distilled water 1000 mL.

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