Bacillus subtilis LU5, lignocellulose degrading flora and culture method and application of lignocellulose degrading flora

Abstract

The invention provides a bacillus subtilis LU5 and a lignocellulose degrading flora as well as a culture method and application thereof, and belongs to the technical field of microorganisms. According to the invention, a strain is screened from surface soil of composted straws and identified as bacillus subtilis, the strain is combined with bacillus velezensis SS6 and trichoderma longibrachiatum according to a specific proportion, co-culture is carried out in a specific culture medium, a flora capable of degrading lignocellulose is obtained, all microorganisms of the flora are in synergistic symbiosis, and the effect of degrading lignocellulose is achieved. The produced various cellulases have a synergistic effect, so that the aim of degrading lignocellulose is fulfilled. And by adopting the flora provided by the invention, the efficiency and the stability of the lignocellulose degradation process are remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Bacillus subtilis LU5 and a lignocellulose degrading bacterial community, and a culture method and application thereof. Background Art

[0002] Crop straw is a kind of plant fiber waste with abundant production and wide distribution. Crop straw was once widely used as a fuel resource and is an important energy resource. However, with the changes in economic structure and people's lifestyles, crop straw has been discarded or burned in large quantities, resulting in huge waste of resources and serious environmental pollution, which in turn affects human health. How to develop efficient, clean, green and friendly straw processing technology, release the energy contained in straw, and improve the efficiency of straw resource utilization is an important measure to promote the comprehensive utilization of crop straw.

[0003] Straw is mainly composed of rich cellulose substances (such as lignocellulose), a small amount of minerals, water, crude protein and crude fat. Lignocellulose, as its main component, is mainly composed of cellulose (about 40%-50%), hemicellulose (about 20%-30%) and lignin (about 10%-25%) (Gao Z, Alshehri K, Li Y, et al. Advances in biological techniques for sustainable lignocellulosic waste utilization in biogas production [J]. Renewable and Sustainable Energy Reviews, 2022, 170: 112995.). The presence of three phenylpropane compounds in the structure of lignocellulose is the main reason for the stability of the structure of lignocellulose and its high resistance to degradation; cellulose molecules are tightly wrapped by hemicellulose and lignin, which together form the skeleton of the plant cell wall, enhance the rigidity of the plant and form a natural barrier. These three components are tightly combined to form a complex and stable supramolecular structure in the plant body, making cellulose degradation difficult and limiting the process of straw cellulose conversion and utilization (Zhang Shuang. Screening of low-temperature cellulose-degrading bacteria and research on their degradation effect on corn straw [D]. Northeast Agricultural University, 2019.). In the cellulose structure, glucose molecular chains aggregate and combine into fibrils. Because they contain high-density and high-strength crystalline regions, it is difficult for degrading enzymes to penetrate and play a role, thereby reducing the degradation efficiency of the cellulose structure. In practical applications, how to effectively improve the degradation efficiency of lignocellulose is an important issue that needs to be solved in the fields of bioenergy development and ecological environment protection.

[0004] The main ways to degrade lignocellulose include physical, chemical and biological methods. Compared with the other two treatment methods, the biological method is more efficient, environmentally friendly and economical. Using microorganisms to degrade lignocellulose is an important way to efficiently utilize straw, but the rate of microbial degradation is greatly affected by environmental factors (Liu Yu. Screening of a cold-resistant bacteria and its decomposition effect on corn straw [D]. Northeast Agricultural University, 2023.). For example, in cold and dry areas, microorganisms are more active in summer. Affected by the climate characteristics of spring and winter, the abundance and activity of microorganisms are low, and the rate of straw cellulose degradation is slow. Screening and using microbial strains that efficiently produce cellulose-degrading enzymes for straw cellulose degradation has become a research hotspot to solve the current large amount of straw resources, low biodegradation efficiency, promote the degradation rate of lignocellulose and improve straw utilization. The development of composite bacterial agents suitable for local climate characteristics is of great significance to solving the problem of regional straw cellulose degradation difficulties. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a strain of Bacillus subtilis LU5 and a lignocellulose degrading bacterial community and a culture method and application thereof. The present invention utilizes the synergistic symbiotic mechanism between Bacillus subtilis LU5 and other microorganisms to mix and culture a variety of cellulose degrading bacteria, and achieves the goal of degrading lignocellulose through the synergistic action of a variety of cellulases produced by the bacterial community. The bacterial community in the present invention significantly improves the efficiency and stability of the lignocellulose degradation process.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a Bacillus subtilis LU5, wherein the Bacillus subtilis LU5 was deposited in the China Center for Type Culture Collection on June 15, 2023, with a deposit number of CCTCC No: M 20231029.

[0008] Preferably, the sequence of 16S rRNA of the Bacillus subtilis LU5 strain is as shown in SEQ ID NO.1.

[0009] The present invention provides a lignocellulose degrading bacterial community, which comprises the following components in a mass ratio: Bacillus velezensis SSF6: Bacillus subtilis LU5: Trichoderma longibrachiatum = 1:1.5-3:1.5-3; the number of live bacteria in the bacterial community is 1×10 8 ~1×10 10CFU / mL; the Bacillus subtilis LU5 is the strain.

[0010] Preferably, the activity of the Bacillus velezensis SSF6 endo-glucosidase is greater than 54 U / mL;

[0011] The Bacillus subtilis LU5 endo-glucosidase activity is greater than 75 U / mL;

[0012] The endo-glucosidase activity of the Trichoderma longibrachiatum is greater than 141 U / mL.

[0013] Preferably, the Bacillus velezensis SSF6 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 14, 2022, with the deposit number CGMCC No.25298.

[0014] The invention provides a method for culturing a lignocellulose-degrading bacterial flora. Bacillus Velezii SSF6, Bacillus subtilis LU5 and Trichoderma longifolia are mixed and placed in a fermentation medium for co-fermentation and culture to obtain a lignocellulose-degrading bacterial flora.

[0015] Preferably, the fermentation medium uses water as solvent and includes components in the following concentrations: starch 10-20 g / L, soybean meal 0.5-1.5 g / L, bran 25-75 g / L, peptone 1-5 g / L, NaCl 3-7 g / L, (NH4)2SO4 0.5-2 g / L, KH2PO4 0.5-2 g / L, and MgSO4 0.1-0.5 g / L.

[0016] Preferably, the conditions for the co-fermentation culture are: culture time of 55-60 h, culture temperature of 35-45° C., addition amount of bacterial flora of 1%-5% of the volume of the fermentation medium, and initial pH of 6-10.

[0017] The invention also provides the use of the lignocellulose degrading bacterial community in degrading straw.

[0018] Preferably, the lignocellulose degrading bacteria are used as a fermentation agent to degrade the straw.

[0019] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a strain of Bacillus subtilis LU5 and a lignocellulose degrading bacterial community and a cultivation method and application thereof. The present invention obtains a strain by screening in the surface soil of mature straw, identifies it as Bacillus subtilis, combines the strain with Bacillus Velezii SSF6 and Trichoderma longibrachiatum in a specific ratio, and then, in a mixture of 10-20 g / L starch, 0.5-1.5 g / L soybean meal, 25-75 g / L bran, 1-5 g / L peptone, and NaCl In a culture medium composed of 3-7g / L, (NH4)2SO40.5-2g / L, KH2PO40.5-2g / L, and MgSO40.1-0.5g / L, the culture time is 55-60h, the culture temperature is 35-45°C, the addition amount is 1.5%-2.3%, and the initial pH value is 6-10. The bacterial community that can degrade cellulose is obtained. The present invention utilizes the synergistic symbiotic mechanism between microorganisms to mix and culture a variety of cellulose-degrading bacteria, and achieves the purpose of degrading cellulose through the synergistic action of a variety of cellulases produced by the bacterial community. The bacterial community in the present invention significantly improves the efficiency and stability of the cellulose degradation process, which is of great significance to improving the enzyme production efficiency of the composite bacterial community, and has important practical significance for promoting the cellulose degradation process and solving the problem of difficult cellulose degradation and low utilization rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the glucose standard curve;

[0021] Figure 2 These are pictures of molecular biological identification of Bacillus subtilis LU5, where A is the gel electrophoresis of Bacillus subtilis LU5, and B is the phylogenetic tree of Bacillus subtilis LU5;

[0022] Figure 3 The effect of culture time on the enzyme activity of composite bacterial consortium;

[0023] Figure 4 The effect of culture temperature on the enzyme activity of composite bacterial consortium;

[0024] Figure 5 The effect of inoculation ratio on the enzyme activity of composite bacterial consortium;

[0025] Figure 6 The effect of medium pH on the enzyme activity of composite bacterial flora;

[0026] Figure 7 The effect of the interaction between culture time and culture temperature on the enzyme activity of composite bacterial consortium;

[0027] Figure 8The effect of the interaction between the culture time and the amount of composite bacterial consortium added on the enzyme activity of the composite bacterial consortium;

[0028] Fig. 9 The effect of the interaction between culture time and initial pH value on the enzyme activity of composite bacterial consortium;

[0029] Fig.10 The effect of the interaction between culture temperature and the amount of composite bacterial consortium added on the enzyme activity of composite bacterial consortium;

[0030] Fig.11 The effect of the interaction between culture temperature and initial pH value on the enzyme activity of composite bacterial consortium;

[0031] Fig.12 The effect of the interaction between the addition amount of composite bacteria and the initial pH value on the enzyme activity of composite bacteria;

[0032] Fig.13 The change of the degradation rate of corn straw by composite bacteria;

[0033] Fig.14 Figure 3 shows the changes in the decomposition degree of corn straw at different fermentation times, where A is the effect diagram of the decomposition degree of corn straw fermentation on the 0th day, B is the effect diagram of the decomposition degree of corn straw fermentation on the 6th day, C is the effect diagram of the decomposition degree of corn straw fermentation on the 12th day, D is the effect diagram of the decomposition degree of corn straw fermentation on the 18th day, E is the effect diagram of the decomposition degree of corn straw fermentation on the 24th day, and F is the effect diagram of the decomposition degree of corn straw fermentation on the 30th day.

[0034] Biological Deposit Description

[0035] Bacillus subtilis LU5, Latin for Bacillus subtilis, is deposited in China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: June 15, 2023, deposit number: CCTCC No: M 20231029.

[0036] Bacillus velezensis SSF6, Latin name Bacillus velezensis, is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, address: No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit date: July 14, 2022, deposit number: CGMCC No.25298. DETAILED DESCRIPTION

[0037] The present invention provides a Bacillus subtilis LU5, wherein the Bacillus subtilis LU5 was deposited in the China Center for Type Culture Collection on June 15, 2023, with a deposit number of CCTCC No: M 20231029.

[0038] In the present invention, the sequence of 16S rRNA of the Bacillus subtilis LU5 strain is shown in SEQ ID NO.1, which is as follows:

[0039]

[0040] The present invention provides a lignocellulose degrading bacterial community, which comprises the following components in a mass ratio: Bacillus velezensis SSF6: Bacillus subtilis LU5: Trichoderma longibrachiatum = 1:1.5-3:1.5-3; preferably 1:2:2, and the number of live bacteria in the bacterial community is 1×10 8 ~1×10 10 CFU / mL; the Bacillus subtilis LU5 is the strain.

[0041] In the present invention, the enzyme activity of the Bacillus velezensis SSF6 endo-glucosidase is greater than 54 U / mL; the enzyme activity of the Bacillus subtilis LU5 endo-glucosidase is greater than 75 U / mL; and the enzyme activity of the Trichoderma longibrachiatum endo-glucosidase is greater than 141 U / mL.

[0042] In the present invention, the Bacillus subtilis LU5 was deposited in the China Center for Type Culture Collection on June 15, 2023, with a deposit number of CCTCC No: M 20231029; the long-branched Trichoderma was purchased from the China Industrial Microbiological Culture Collection Administration Center, with a deposit number of CICC 40136; the Bacillus velezensis SSF6 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on July 14, 2022, with a deposit number of CGMCC No.25298.

[0043] The invention provides a method for culturing a lignocellulose-degrading bacterial flora. Bacillus Velezii SSF6, Bacillus subtilis LU5 and Trichoderma longifolia are mixed and placed in a fermentation medium for co-fermentation and culture to obtain a lignocellulose-degrading bacterial flora.

[0044] In the present invention, the fermentation medium uses water as a solvent, and the water is preferably distilled water; the components of the fermentation medium are: starch 10-20 g / L, preferably 12-18 g / L, and more preferably 15 g / L;

[0045] Soybean meal 0.5-1.5 g / L, preferably 0.8-1.2 g / L, more preferably 1.0 g / L;

[0046] Bran 25-75 g / L, preferably 45-65 g / L, more preferably 50 g / L;

[0047] Peptone 1-5 g / L, preferably 2-4 g / L, more preferably 3 g / L;

[0048] NaCl 3-7 g / L, preferably 4-6 g / L, more preferably 5 g / L;

[0049] (NH4)2SO4 0.5-2 g / L, preferably 0.8-1.5 g / L, more preferably 1 g / L;

[0050] KH2PO4 0.5-2 g / L, preferably 0.7-1.5 g / L, more preferably 1 g / L;

[0051] MgSO4 0.1~0.5g / L, preferably 0.2~0.4g / L, more preferably 0.3g / L.

[0052] In the present invention, the conditions for the co-fermentation culture are: the culture time is 55 to 60 hours, preferably 56 to 59 hours, and more preferably 58 hours; the culture temperature is 35 to 45°C, preferably 37 to 42°C, and more preferably 40°C; the amount of bacterial flora added is 1% to 5% of the volume of the fermentation medium, preferably 1.5% to 3%, and more preferably 1.9%; the initial pH value is 6 to 10, preferably 7 to 9, and more preferably 8.

[0053] The invention also provides the use of the lignocellulose degrading bacterial community in degrading straw.

[0054] In the present invention, the lignocellulose degrading bacteria are used as a fermentation agent to degrade straw, and the straw is one or more of corn straw, wheat straw, rice straw, rape straw, and cotton straw.

[0055] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0056] Fermentation medium: 15 g starch, 1.0 g soybean meal, 50 g bran, 3 g peptone, 5 g NaCl, 1 g (NH4)2SO4, 1 g KH2PO4, and 0.3 g MgSO4 were dissolved in 1000 mL of distilled water and sterilized at 121°C for 20 min.

[0057] LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, adjust pH to 7.0 with 1% NaOH solution, and sterilize by high pressure at 121°C for 20 min.

[0058] PDA medium: weigh 200g of potato, add water and boil until soft, filter with gauze, add 15g / L glucose and 15g / L agar, stir evenly, cool slightly after the agar is completely dissolved, make up to 1000mL, divide into portions, and sterilize under high pressure at 115℃ for 20min.

[0059] The culture medium used in the subsequent examples was prepared according to the above method.

[0060] Example 1 Isolation of Bacillus subtilis LU5 strain

[0061] 1. Sample Collection

[0062] Straw tillage and return to the field was carried out in Qingshuihe County, Hohhot City, Inner Mongolia (111°0.68′E, 39°0.92′N). Corn was planted in the field in 2019 and 2020. Straw tillage and return to the field were carried out after the autumn harvest, and soil samples were collected before sowing in the spring of 2021. Farmland soil with a straw degradation degree greater than or equal to 50% was selected as the sample for this study. The “five-point sampling method” was used for sampling. The surface soil was lightly scraped to collect the decomposed straw surface soil 5 to 10 cm from the surface, and the soil was mixed and stored in a sterile bag at 4°C for later use.

[0063] 2. Screening of cellulose-degrading bacteria

[0064] 1) Mix 5 g of the collected soil sample in a conical flask containing 45 mL of enrichment culture medium and culture at 30°C and 170 rpm for 2 days.

[0065] 2) Take 1 mL of enriched cultured bacterial solution in a 9 mL saline tube and -5 ~10 -9 Five dilution gradients were diluted and thoroughly shaken to mix. 200 μL of each gradient suspension was spread on sodium carboxymethyl cellulose (CMC-Na), microcrystalline cellulose (microcrystalline cellulose) and starch solid culture medium, repeated 3 times, and cultured at 30°C for 24h to 48h until colonies appeared on the culture medium.

[0066] 3) Use Congo red dye to stain sodium carboxymethyl cellulose and microcrystalline cellulose culture media for 30 minutes, wash with 1% NaCl solution for 30 minutes, pour out the solution, observe and measure the colony diameter (d) and its transparent circle diameter (D), use dilute iodine solution to stain starch culture medium for 30 minutes, wash with 1% NaCl solution for 1 hour, observe and measure the colony diameter (d) and its transparent circle diameter (D), and repeatedly purify, isolate and culture the strain.

[0067] The hydrolysis ratio (HCR) of the strain can directly reflect the effect of microorganisms in degrading lignocellulose. The hydrolysis ratio is calculated by the diameter of the colony degradation circle (D) and the colony diameter (d). The larger the corresponding ratio, the better the degradation effect. It is generally believed that a hydrolysis ratio (HCR) ≥ 2 has a better cellulose degradation effect. The hydrolysis rate of Bacillus subtilis LU5 on CMC medium is 3.47±0.13%, the hydrolysis rate on microcrystalline cellulose medium is 3.00±0.01%, and the hydrolysis rate on starch medium is 2.56±0.11%. Based on the hydrolysis performance on CMC-Na, microcrystalline cellulose and starch medium, Bacillus subtilis LU5 has a high potential for lignocellulose degradation.

[0068] 3. Determination of cellulase activity of isolated strains

[0069] 1) Drawing of glucose standard curve

[0070] Take 8 20mL test tubes and add 0.0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, 1.2mL, and 1.4mL of 1mg / mL glucose standard solution respectively to make the volume 2.0mL. Add 2mL DNS solution to each test tube, shake well, and boil in water for 10min, take out and cool immediately, and make the volume to 20mL with distilled water.

[0071] At 540nm absorbance, use the solution without glucose as the blank control to adjust the zero point, measure the OD value of the solutions in other tubes, and draw a glucose standard curve with the glucose content as the horizontal axis and the absorbance value at 540nm as the vertical axis. Figure 1 .

[0072] The glucose standard curve was drawn with the glucose content as the horizontal axis and the absorbance at 540 nm as the vertical axis. The regression equation of the standard curve obtained by linear fitting was: y = 1.6267x + 0.0071, R 2 =0.9982, the correlation coefficient is greater than 0.995, the standard curve is well fitted and can better reflect the relationship between the absorbance value and the glucose content, which can be used as the standard curve for the determination of cellulase activity and then calculate the cellulase activity.

[0073] 2) Determination of cellulose-degrading enzyme activity

[0074] The purified strain was inoculated into LB liquid culture medium for activation. The bacterial solution was inoculated into a conical flask containing 100 mL of LB culture medium and fermented at 37°C and 170 rpm for 1 day. After the fermentation, the bacterial solution was aspirated and centrifuged at 12000 rpm and 4°C for 10 min. The supernatant was aspirated as the crude enzyme solution.

[0075] Determination of total cellulase activity: The enzyme activity was determined by filter paper enzyme activity (FPA). 0.1 g of filter paper strips were added to a stoppered colorimetric tube, 2 mL of citric acid-sodium hydrogen phosphate buffer with pH = 4.8 was added to soak the filter paper strips, 1 mL of crude enzyme solution was added to shake and mix (the blank group was 1 mL of sterilized crude enzyme solution), and the mixture was placed in a water bath at 50°C for 30 min, 3 mL of DNS colorimetric agent was added, and the mixture was boiled in a boiling water bath for 10 min, and the volume was adjusted to 20 mL after cooling to room temperature, and the absorbance was measured at 540 nm after mixing, and each tube was repeated 3 times.

[0076] Determination of endo-β-1,4-glucosidase activity: Carboxymethyl cellulose (CMC) enzyme activity determination method was used. Add 2 mL of 1% CMC-Na solution (citric acid-disodium hydrogen phosphate buffer at pH = 4.8) to a stoppered colorimetric tube, add 1 mL of crude enzyme solution and shake to mix (blank group is 1 mL of sterilized crude enzyme solution), water bath at 50°C for 30 minutes, add 3 mL of DNS reagent color developer, mix well and boil in a boiling water bath for 10 minutes, cool to room temperature and dilute to 20 mL, mix well and measure the absorbance at 540 nm, repeat 3 times for each tube.

[0077] Determination of exo-β-1,4-glucosidase activity: Microcrystalline cellulose (MCC) enzyme activity determination method was used. Add 2 mL of 1% microcrystalline cellulose solution (citric acid-disodium hydrogen phosphate buffer at pH = 4.8) to a stoppered colorimetric tube, add 1 mL of crude enzyme solution and shake to mix (blank group is 1 mL of sterilized crude enzyme solution), place in a 50°C water bath for 30 minutes, add 3 mL of DNS reagent color developer, mix well and boil in a boiling water bath for 10 minutes, cool to room temperature and dilute to 20 mL, mix well and measure the absorbance at 540 nm, and repeat 3 times for each tube.

[0078] β-1,4-glucosidase activity determination: Salicinase activity determination method was used. Add 2 mL of 1% salicin solution (citric acid-disodium hydrogen phosphate buffer solution with pH = 4.8) to a stoppered colorimetric tube, add 1 mL of crude enzyme solution and shake to mix (blank group is 1 mL of sterilized crude enzyme solution), water bath at 50°C for 30 minutes, add 3 mL of DNS reagent colorimetric agent, mix well and boil in a boiling water bath for 10 minutes, cool to room temperature and dilute to 20 mL, mix well and measure the absorbance at 540 nm, repeat 3 times for each tube.

[0079] Calculation of cellulase activity: One unit (U) of enzyme activity is defined as the amount of enzyme required to release 1 μmol of reducing sugar per milliliter per minute. The formula for calculating cellulase activity is as follows:

[0080]

[0081] Endoglucosidase, exoglucosidase and β-glucosidase are the key enzyme systems for cellulose degradation, and filter paper enzyme can reflect the total enzyme activity of these three cellulase complex enzyme systems to a certain extent. The cellulase activity produced by the isolated strains was analyzed using filter paper enzyme, endo-glucosidase, exoglucosidase and β-glucosidase. The activity of filter paper enzyme produced by Bacillus subtilis LU5 was 56.13±0.23U / mL, the activity of endo-glucosidase was 52.75±0.07U / mL, the activity of exoglucosidase was 52.36±0.34U / mL, and the activity of β-glucosidase was 36.60±0.07U / mL. It can be seen that Bacillus subtilis LU5 has a high potential for cellulose degradation.

[0082] Example 2 Molecular Biological Identification of Bacillus subtilis LU5 Strain

[0083] 1. Extraction of strain DNA

[0084] The strain obtained in Example 1 was inoculated into LB liquid culture medium, and cultured at 37°C, 170 rpm, and shaking for 24 h. After the culture was completed, the bacterial sludge was collected by centrifugation at 12000 rpm and 4°C for 10 min. Genomic DNA was extracted using a bacterial genomic DNA purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP302).

[0085] 2. PCR Amplification

[0086] Universal primer amplification: Using the extracted DNA as a template, select forward primer 27F: (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.2) and reverse primer 1429R: (5'-TACGGCTACCTTGTTACGACTT-3', SEQ ID NO.3) to perform 16S rRNA gene PCR. The PCR amplification reaction system is 50 μL, including: forward primer (1 μL), reverse primer (1 μL), Taq DNA polymerase (25 μL), DNA template (1 μL), and ddH2O (22 μL).

[0087] The PCR amplification reaction program was as follows: 95°C, 5 min pre-denaturation; 95°C, 1 min denaturation; 55°C, 1 min re-annealing; 72°C, 1 min extension, 30 cycles, and finally keeping at 72°C for 10 min.

[0088] After the PCR reaction, the quality of the amplified product was checked by electrophoresis on a 1% agarose gel at 200V for 15 min, using the maker as a control. The gel system was used for observation and photography.

[0089] 3.16S rRNA sequence determination

[0090]

[0091] The strains were identified based on 16S rRNA. Figure 2 shown.

[0092] Depend on Figure 2 It can be seen that PCR amplification and electrophoresis detection of the strain DNA using 16S rRNA gene universal primers obtained a brighter target band of about 1500 bp, and the amplified product was bidirectionally sequenced, and the 16S rRNA gene sequence was compared with the GenBank nucleotide database sequence. The results showed that the strain LU5 obtained by the present invention was identified as Bacillus subtilis.

[0093] Example 3

[0094] Bacillus Velez SSF6, Bacillus subtilis LU5 and Trichoderma longifolia were mixed in a ratio of 1:2:2 to obtain a bacterial community, and the bacterial community was added to a fermentation medium at a temperature of 40°C and a pH of 8.0 at 1.9% of the volume of the fermentation medium, and cultured for 58 hours to obtain a lignocellulose-degrading bacterial community. The endo-glucosidase activity of the bacterial community was 330.86 U / mL.

[0095] Example 4

[0096] Bacillus Velez SSF6, Bacillus subtilis LU5 and Trichoderma longifolia were mixed in a ratio of 1:1.5:1.5 to obtain a bacterial community, and the bacterial community was added to a fermentation medium at a temperature of 40°C and a pH of 8.0 at 1.9% of the volume of the fermentation medium, and cultured for 58 hours to obtain a lignocellulose-degrading bacterial community. The endo-glucosidase activity of the bacterial community was 322.38 U / mL.

[0097] Example 5

[0098] Bacillus Velez SSF6, Bacillus subtilis LU5 and Trichoderma longifolia were mixed in a ratio of 1:3:2 to obtain a bacterial community, and the bacterial community was added to a fermentation medium at a temperature of 40°C and a pH of 8.0 at 1.9% of the volume of the fermentation medium, and cultured for 58 hours to obtain a lignocellulose-degrading bacterial community. The endo-glucosidase activity of the bacterial community was 267.88 U / mL.

[0099] Example 6

[0100] Bacillus Velez SSF6, Bacillus subtilis LU5 and Trichoderma longifolia were mixed in a ratio of 1:2:3 to obtain a bacterial community, and the bacterial community was added to a fermentation medium at a temperature of 40°C and a pH of 8.0 at 1.9% of the volume of the fermentation medium, and cultured for 58 hours to obtain a lignocellulose-degrading bacterial community. The endo-glucosidase activity of the bacterial community was 221.28 U / mL.

[0101] Comparative Example 1: Study on strain compatibility

[0102] Bacillus velzensis SSF6 (B. velzensis SSF6) strain, Bacillus subtilis LU5 (B. subtilis LU5), Bacillus siamensis 20751 (B. siamensis 20751, purchased from China Industrial Microbiology Center CICC, strain collection number CICC 20751), Aspergillus niger (A. niger, purchased from China Industrial Microbiology Center CICC, strain collection number CICC 40273), and Trichoderma longifolia (T. longifolia) strains were selected for the construction of a composite bacterial community.

[0103] The test strains were transferred to liquid LB medium for recovery and cultured at 37°C with shaking at 170 rpm until 1×10 8 CFU / mL, fungi were cultured on PDA solid medium in an incubator at 28°C.

[0104] Cross-cross method: The cross-cross method is used for the antagonism experiment between bacteria. The inoculation loop is dipped into the bacterial solution and the cross-scratch method is used to draw lines on the culture medium and make them intersect. Three groups of parallel experiments are set up and cultured inverted for 1 day. Observe whether antagonism occurs during the growth of the strains.

[0105] Plate confrontation method: The antagonism experiment between bacteria and fungi was tested by the plate confrontation method. The bacterial solution was dipped into an inoculation loop and a line was drawn in the center of the plate. The fungal spores were picked up with a sterile toothpick and spotted on the plate. The plate was wrapped with a sealing film and placed in a 28°C incubator for culture. At the same time, the spores were inoculated onto the plate using a sterile toothpick, and a blank control was set up to monitor the growth and interaction of the strains. If both adjacent strains grow well and merge at the junction, it indicates compatibility; if the growth of one colony slows down and there is a gap at the junction, it indicates partial compatibility; if one colony obviously inhibits the other and the junction is clear, it indicates incompatibility. Strains with obvious antagonism were not used for composite flora construction. The results are shown in Table 1.

[0106] Table 1 Antagonistic effect of cellulose degrading strains

[0107]

[0108] Note: “+” indicates that the strains can be co-cultured without antagonism; “-” indicates that antagonism exists or may exist between the strains.

[0109] As shown in Table 1, there was no growth inhibition between B.subtilis LU5 and B.velzensis SSF6, B.siamensis 20751, T.longifolia and Aspergillus niger, and all of them could grow well; there was no antagonism between B.velzensisSSF6 and B.subtilis LU5 and B.siamensis 20751, and there was slight incompatibility between B.velzensisSSF6 and T.longifolia and A.niger; there was slight antagonism between B.siamensis 20751 and T.longifolia and A.niger; there was antagonism between T.longifolia and A.niger.

[0110] Comparative Example 2

[0111] B. velzensis SSF6, B. subtilis LU5, and T. longifolia strains were inoculated into LB medium or PDA liquid medium for culture, respectively. The test strains were combined in equal proportions for a bacterial flora compound test. The constructed bacterial flora was inoculated into the fermentation medium at an inoculum size of 3% of the volume of the fermentation medium, and cultured at 30°C and 170 rpm for 48 h. The supernatant was taken to determine the endo-glucosidase activity (CMCase) of the constructed bacterial flora. The results are shown in Table 2.

[0112] Table 2 Construction of composite bacterial community and its cellulase activity

[0113]

[0114] Note: Different letters indicate that the cellulase activities of different strains were significantly different (P<0.05).

[0115] As shown in Table 2, 4 different strain composition ratios were designed according to equal ratio addition. The combination D of mixed fermentation of B.velzensis SSF6, B.subtilis LU5 and T.longifolia showed higher cellulase activity than other combinations. The enzyme activity of combination D (B.velzensis SSF6+B.subtilis LU5+T.longifolia) was the highest at 230.03U / mL, followed by combination C (B.velzensis SSF6+T.longifolia), with an enzyme activity of 202.87U / mL. This shows that the symbiosis and nutritional complementarity between B.velzensis SSF6+B.subtilis LU5+T.longifolia can effectively overcome the influence of adverse factors produced in fermentation, thereby enhancing the fermentation enzyme production effect.

[0116] Comparative Example 3

[0117] Based on the combination D (SSF6:LU5:T.longifolia) obtained in Comparative Example 2, 4 different addition ratios were designed to optimize the composition ratio of the composite bacterial community D. The other conditions were the same as in Comparative Example 2. The results are shown in Table 3.

[0118] Table 3 Composite flora D and its flora ratio

[0119]

[0120] Note: Different letters indicate that there are significant differences in the cellulase activities produced by different combinations (P<0.05).

[0121] As shown in Table 3, the composition ratio of different composite bacterial communities D and the bacterial community enzyme activity are arranged from large to small as D3>D1>D2>D4, and the CMCase enzyme activity of bacterial community D3 is the highest at 242.23U / mL. The efficient composite bacterial community constructed by the present invention is B.velzensis SSF6: B.subtilis LU5: T.longifolia=1:2:2. Different strains can coexist with each other through synergistic effects, and this synergistic effect provides a basis for each strain to jointly exert biodegradation to degrade lignocellulose.

[0122] Comparative Example 4

[0123] 1. Effect of fermentation time on enzyme activity of composite bacterial flora

[0124] The composite bacterial consortium was inoculated into the fermentation enzyme production medium at an addition rate of 3% of the fermentation medium volume, and 8 fermentation time gradients were set: 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, and cultured in a constant temperature shaker at 30℃ and 170rpm. The CMCase enzyme activity of the composite bacterial consortium at different fermentation times was measured, and the changes in the enzyme activity of the composite bacterial consortium were analyzed to determine the optimal fermentation time. The results are shown in Figure 3 shown.

[0125] Depend on Figure 3 It can be seen that the CMCase activity first increased and then decreased with the increase of fermentation time, and reached the maximum enzyme activity of 157.04U / mL at 48h of fermentation time.

[0126] 2. Effect of fermentation temperature on enzyme activity of composite bacterial flora

[0127] The composite bacterial consortium was inoculated into the fermentation enzyme production medium at an addition rate of 3% of the fermentation medium volume, and seven fermentation temperature gradients were set: 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, and 45°C, and the culture was shaken at 170rpm until the optimal fermentation time. The CMCase enzyme activity of the composite bacterial consortium at different fermentation temperatures was measured, and the changes in the enzyme activity of the composite bacterial consortium were analyzed to determine the optimal fermentation temperature. The results are shown in Figure 4 shown.

[0128] Depend on Figure 4 It can be seen that the CMCase enzyme activity is the highest at 412.36U / mL when the culture temperature is 40℃, indicating that it has certain heat-resistant characteristics and is conducive to the growth and metabolism of the composite flora under certain temperature conditions. Its enzyme production activity is relatively high, and deviation from the optimal temperature range is not conducive to the metabolic activities of the composite flora.

[0129] 3. Effect of inoculation amount on enzyme activity of composite bacterial community

[0130] The composite bacterial consortium was inoculated into the fermentation enzyme production medium, and 7 inoculation gradients were set: 1%, 2%, 3%, 4%, 5%, 6%, and 7%. The culture was shaken at 170 rpm at the optimal fermentation temperature until the optimal fermentation time. The CMCase enzyme activity produced by the composite bacterial consortium was measured, and the changes in the enzyme activity of the composite bacterial consortium were analyzed to determine the optimal inoculation amount. The results are shown in Figure 5 shown.

[0131] Depend on Figure 5 It can be seen that the CMCase enzyme activity has two peaks at inoculation amounts of 2% and 7%, with enzyme activities of 310.80U / mL and 255.55U / mL respectively. Analysis shows that the reason for this may be due to the nutritional competition of a single bacterium in the composite bacterial agent.

[0132] 4. Effect of initial pH value on enzyme activity of composite bacterial community

[0133] The composite bacterial consortium was inoculated into fermentation enzyme production medium with pH values ​​of 3, 4, 5, 6, 7, 8, 9, and 10 at an optimized inoculation amount. The culture was shaken at 170 rpm at the optimal fermentation temperature until the optimal fermentation time. Samples were taken to measure the CMCase activity of the composite bacterial consortium, and the changes in the enzyme activity of the composite bacterial consortium were analyzed to determine the optimal initial pH value. The results are shown in Figure 6 shown.

[0134] Depend on Figure 6 It can be seen that the change in pH value changes the degree of dissociation between the enzyme active center group and the substrate, which in turn affects the binding of cellulase to the substrate. The enzyme activity of the composite bacterial community gradually increases with the increase in pH, showing a certain tolerance to high pH environment. The highest enzyme production is 279.13U / mL at a pH of 8.

[0135] It can be seen that the composite bacterial community can tolerate high temperature and strong alkaline environment. This characteristic is of great significance for the development of microbial agents suitable for northern saline-alkali soils.

[0136] 5. Optimization of fermentation conditions

[0137] The Box-Benhnken model of Design Expert 8.0.6 was used to design a four-factor three-level experiment to further optimize the four influencing factors of fermentation time, fermentation temperature, inoculation amount and initial pH. Based on the results of the single-factor experiment, the optimal fermentation conditions were selected as the center point, and the CMCase activity produced by the composite bacterial community was used as the response value. The experimental design factors and levels are shown in Table 4.

[0138] Table 4 Response surface test factors and levels

[0139]

[0140] Taking CMCase activity as the response value, the Box-Benhnken model was used to design and optimize the fermentation time (A), fermentation temperature (B), addition amount (C) and initial pH value (D) and their levels. The experimental results are shown in Table 5.

[0141] Table 5 Response surface test results

[0142]

[0143] The quadratic regression equation prediction model obtained based on the test data is as follows:

[0144] Y(CMCase)=298.04-29.64×A+10.93×B-13.44×C-17.58×D+25.22×AB-12.64×AC-1 6.08×AD+8.93×BC-9.04×BD+8.13×CD-68.83×A2-79.33×B2-48.64×C2-68.40×D2.

[0145] In order to test whether the prediction model is established accurately, the obtained regression equation is subjected to variance analysis and significance test, and the results are shown in Table 6. As shown in Table 6, the model F value is 64.6, the P value is less than 0.0001, the regression model is extremely significant, the model lack of fit term P = 0.0809> 0.05, and the regression model lack of fit term is not significant, indicating that the prediction model is reliable and can better predict and analyze the optimal conditions for the fermentation of composite bacterial community D3 enzyme production. The influence on the production of cellulase activity of composite bacterial community D3 is ranked from high to low as (B) fermentation temperature> (D) initial pH value> (C) addition amount> (A) fermentation time.

[0146] Table 6 Analysis of variance of regression model

[0147]

[0148] Note: ** represents extremely significant difference (P<0.01); * represents significant difference (P<0.05).

[0149] The response surface curve diagram can intuitively reflect the impact of the interaction of any two variables on the response value. According to Table 6, the response surface curve diagram ( Figure 7 to Figure 12 ),Depend on Figure 7 to Figure 12 It can be seen that when the temperature remains unchanged, the CMCase activity increases with the increase of culture time, and with the increase of inoculation amount and initial pH value, it shows a trend of first increasing and then decreasing. When other factors remain unchanged, with the increase of temperature, the cellulase activity first increases and then decreases. The cellulase activity of the composite microbiota is significantly affected by the interaction of culture temperature and time, while the interaction between fermentation temperature, inoculation amount, and initial pH value has no significant effect on the enzyme activity of the composite microbiota.

[0150] With the increase of inoculation amount, the cellulase activity of the composite bacterial community first increased and then slowly decreased, and increased with the increase of temperature. When the initial pH value of the culture medium remained unchanged, the cellulase activity first increased and then decreased with the increase of inoculation amount, and increased slightly and then slowly decreased with the extension of fermentation time.

[0151] The BBD model was used to analyze and optimize the fermentation conditions of the composite bacterial community D3, and the optimal fermentation conditions of the composite bacterial agent D3 were obtained as follows: culture time 57.8h, culture temperature 40.19℃, addition amount 1.88%, and initial pH value 7.88. Based on the actual experimental conditions, the optimal fermentation conditions were adjusted to: culture time 58h, culture temperature 40℃, addition amount 1.9%, and initial pH value 8.0. In order to verify the accuracy and reliability of the enzyme activity response model of the composite bacterial community D3, fermentation enzyme production verification was carried out under the optimal enzyme production conditions, and the actual value of CMC enzyme activity was measured to be 330.86U / mL. Compared with before optimization, the enzyme activity of the composite bacterial community D3 was significantly increased by 1.3 times, indicating that the model can accurately predict the results of the composite bacterial community fermentation optimization test.

[0152] Comparative Example 5

[0153] 1. Choice of carbon source nutrients and addition amount

[0154] The composite bacterial community was inoculated at an inoculation rate of 1.9% of the culture medium volume into fermentation medium containing corn straw powder, wheat straw powder, microcrystalline cellulose, sucrose and starch as carbon source nutrients, respectively. The added amount of carbon source was 5g / L, 7.5g / L, 10g / L, 12.5g / L and 15g / L. The culture was carried out at 40°C for 58h. The CMCase activity value of the composite bacterial community was used as an indicator to determine the effects of different carbon sources and their addition amounts on the CMCase activity value of the composite bacterial community. The results are shown in Table 7.

[0155] Table 7 Effects of different carbon sources and their addition amounts on the cellulase activity of composite bacterial flora

[0156]

[0157] Note: Different letters indicate that the cellulase activity produced by the composite flora at different addition amounts was significantly different (P<0.05).

[0158] As shown in Table 7, the type of carbon source and its addition amount have a great influence on the enzyme activity of the composite flora. When starch is used as the carbon source, the CMC enzyme activity is higher, and the highest enzyme activity is about 435.39U / mL when the starch addition amount is 15g / L; when microcrystalline cellulose is used as the carbon source, the CMC enzyme activity is lower, and the enzyme activity is the lowest when the addition amount is 5g / L, about 151.77U / mL. According to the test results, the addition of starch plays a positive role in the fermentation of cellulase by composite flora, which is of great significance to promote the industrialization of cellulase production.

[0159] 2. Selection of nitrogen source nutrients and addition amount

[0160] The composite bacterial community was inoculated at an inoculation rate of 1.9% of the culture medium volume into fermentation medium containing ammonium sulfate, lysine, glutamic acid, soybean meal and urea as nitrogen source nutrients, respectively. The amount of nitrogen source added was 1g / L, 2g / L, 3g / L, 4g / L and 5g / L. The culture was carried out at 40°C for 58h. The CMCase activity value of the composite bacterial community was used as an indicator to determine the effects of different nitrogen sources and their addition amounts on the CMCase activity value of the composite bacterial community. The results are shown in Table 8.

[0161] Table 8 Effects of different nitrogen sources and their addition amounts on the cellulase activity of composite bacterial flora

[0162]

[0163] Note: Different letters indicate that the cellulase activity produced by the composite flora at different addition amounts was significantly different (P<0.05).

[0164] It can be seen from Table 8 that when soybean meal is used as nitrogen source nutrition, the CMC enzyme activity of the bacterial community is the highest, and the CMC enzyme activity is the highest when the addition amount is 1g / L, which is 230.29U / mL; using ammonium sulfate as nitrogen source nutrition has no obvious effect on the enzyme production of the composite bacterial community fermentation, and the enzyme production of the bacterial community decreases with the addition of ammonium sulfate. When the addition amount is 2g / L, the enzyme activity is the lowest, which is 192.96U / mL.

[0165] 3. Optimization of culture medium composition

[0166] The composite bacterial consortium was inoculated into the fermentation enzyme production medium under different factor levels at an inoculation rate of 1.9% of the culture medium volume, and cultured at 40°C for 58 h. Three replicates were set for each group of experiments. Based on the results of Tables 7 and 8, a 4-factor 4-level orthogonal experiment L16 (4 5 )(Table 9), the optimal fermentation and enzyme production medium of the composite bacterial community was finally determined according to the CMCase activity value.

[0167] Table 9 Levels of orthogonal factors of carbon and nitrogen sources

[0168]

[0169] Table 10 Orthogonal test table

[0170]

[0171]

[0172] Table 11 Results of one-way ANOVA

[0173]

[0174] Note: R 2 =0.955 (adjusted R2 =0.776).

[0175] It can be seen from Table 10 that the effects on the activity of cellulase produced by composite bacterial agent fermentation are B nitrogen source > A carbon source > D nitrogen source addition > C carbon source addition, and the optimal culture medium component combination is A2B3C4D1, i.e. starch 15.0 g / L, soybean meal 1.0 g / L, bran 50.0 g / L, peptone 3.0 g / L, NaCl 5.0 g / L, (NH4)2SO4 1.0 g / L, KH2PO4 1.0 g / L, MgSO4 0.3 g / L, and distilled water 1000 mL.

[0176] As shown in Table 11, the type of nitrogen source has a significant effect on the activity of cellulase produced by the composite bacterial community. The type of carbon source, the amount of carbon source added and the amount of nitrogen source added have an important influence on the fermentation and enzyme production process of the composite bacterial community, indicating that the composition of nitrogen source nutrients plays a decisive role in the fermentation and cellulase production process of the composite bacterial community D3.

[0177] The optimal composite bacterial community fermentation conditions obtained by the present invention are: culture time 58h, culture temperature 40 DEG C, addition amount 1.9%, initial pH value 8.0, and the CMC enzyme activity value measured after optimization is 330.86U / mL, and the enzyme activity of the bacterial community is significantly increased by 1.3 times compared with that before optimization; a group of culture medium formulas for optimal composite bacterial community D3 fermentation enzyme production are obtained, namely: starch 15.0g / L, soybean meal 1.0g / L, bran 50.0g / L, peptone 3.0g / L, NaCl 5.0g / L, (NH4)2SO4 1.0 g / L, KH2PO4 1.0g / L, MgSO4 0.3 g / L, and distilled water 1000mL. The CMC enzyme activity value of the bacterial community is measured after optimization to be 450.49U / mL, and the enzyme activity of the bacterial community is significantly increased by 1.85 times compared with that before optimization.

[0178] Effect example

[0179] Weigh 100g of washed corn stalks, cut them into pieces of about 5cm, apply the composite bacterial agent obtained in Example 3 at a dosage of 3%, adjust the carbon-nitrogen ratio to 30%, add sterile water to adjust the humidity to 60%, ferment at 25℃ for 30 days, and take samples every 6 days to measure the stalk degradation rate. Fig.13 and Fig.14 .

[0180] Depend on Fig.13 and Fig.14It can be seen that with the increase of fermentation time, the color of the straw gradually darkened, the texture became softer, and it emitted a decomposition smell, and the straw degradation rate gradually increased. On the 6th, 12th, 18th, 24th, and 30th days of fermentation, the straw degradation rates were 8.96%, 10.58%, 15.66%, 21.02%, and 40.33%, respectively. The straw degradation rate reached a maximum of 40.33% on the 30th day of straw fermentation, indicating that the composite bacterial agent has a high cellulose degradation ability, and the addition of the composite bacterial agent effectively accelerated the straw degradation process.

[0181] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Bacillus subtilis LU5, characterized in that: The Bacillus subtilis LU5 was deposited in the China Center for Type Culture Collection on June 15, 2023, with the deposit number being CCTCC No: M20231029.

2. The strain according to claim 1, characterized in that The sequence of 16S rRNA of the Bacillus subtilis LU5 strain is shown in SEQ ID NO.

1.

3. A lignocellulose-degrading bacterial community, characterized in that: The bacterial community includes the following components in a mass ratio: Bacillus velezensis SSF6: Bacillus subtilis LU5: Trichoderma longibrachiatum = 1:1.5-3:1.5-3; The number of viable bacteria in the bacterial colony is 1×10 8 ~1×10 10 CFU / mL; The Bacillus subtilis LU5 is the strain according to claim 1.

4. The lignocellulose degrading bacterial community according to claim 3, characterized in that The activity of the Bacillus velezensis SSF6 endo-glucosidase is greater than 54 U / mL; The Bacillus subtilis LU5 endo-glucosidase activity is greater than 75 U / mL; The endo-glucosidase activity of the Trichoderma longibrachiatum is greater than 141 U / mL.

5. The lignocellulose degrading bacterial community according to claim 3 or 4, characterized in that: The Bacillus velezensis SSF6 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 14, 2022, with the deposit number CGMCC No.25298.

6. The method for culturing lignocellulose-degrading bacteria according to claim 3, characterized in that: Bacillus Velez SSF6, Bacillus subtilis LU5 and Trichoderma longibrachiatum were mixed and placed in a fermentation medium for co-fermentation and culture to obtain a lignocellulose-degrading bacterial community.

7. The culture method according to claim 6, characterized in that: The fermentation medium uses water as a solvent and includes components with the following concentrations: 10-20 g / L starch, 0.5-1.5 g / L soybean meal, 25-75 g / L bran, 1-5 g / L peptone, 3-7 g / L NaCl, 0.5-2 g / L (NH4)2SO4, 0.5-2 g / L KH2PO4, and 0.1-0.5 g / L MgSO4.

8. The culture method according to claim 6, characterized in that: The conditions for the co-fermentation culture are: a culture time of 55 to 60 hours, a culture temperature of 35 to 45° C., an added amount of the bacterial flora of 1% to 5% of the volume of the fermentation medium, and an initial pH value of 6 to 10.

9. Use of the strain according to claim 1 or 2 or the lignocellulose degrading bacterial community according to any one of claims 3 to 5 in degrading straw.

10. The use according to claim 9, characterized in that: The lignocellulose degrading bacteria group acts as a fermentation agent to degrade the straw.

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