Straw degrading bacterium and application thereof

Abstract

The invention relates to a straw degrading bacterium and application thereof, and belongs to the technical field of biology. The straw degrading bacterium is bacillus megaterium XLL1, the preservation number of the straw degrading bacterium in the China Center for Type Culture Collection is CCTCC NO: M20242410, the preservation date is October 31, 2024, and the preservation address is Wuhan University, Wuhan University, No. 299 on eight road, Wuchang District, Wuhan City, Hubei Province. The strain belongs to one of bacilli, shows good straw degradation capability, provides a new microorganism for microbial degradation of straw, and has great practical significance for promoting straw decomposition. The strain can also be used as a feed raw material, so that the feed cost can be reduced, the development of circular economy is promoted, the emission of straw converted carbon is reduced, and the purpose of carbon neutralization is achieved. The straw degradation amount of the straw degrading bacterium provided by the invention within 7 days is 0.2820 g, and compared with the degradation amount of some found straw degrading bacteria within 7 days, the straw degrading bacterium provided by the invention has a more obvious degradation effect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to straw-degrading bacteria and applications thereof. Background Art

[0002] As a major agricultural country, my country produces a large amount of straw every year, mainly from crops such as rice, wheat, and corn. Straw resources are distributed throughout the country, especially in major agricultural production areas such as North China, Northeast China, and the Yangtze River Basin. Every year, about 30% of straw is not fully utilized and is directly discarded or burned on the spot, resulting in waste of resources and adverse effects on the ecosystem. For example, traditional methods such as burning, returning to the field, and feed production have problems such as environmental pollution, waste of resources, and low efficiency. Straw burning causes air pollution, and improper returning to the field may cause pests and diseases and soil problems. The current technical needs for straw degradation problems often lie in converting straw into high-value-added products such as bioenergy and organic fertilizer, improving resource utilization, reducing pollution caused by burning, improving air quality, and promoting sustainable agricultural development.

[0003] At present, the main methods of straw treatment include biodegradation: using microorganisms or enzymes to decompose cellulose, hemicellulose and lignin in straw to generate organic fertilizer or biogas; chemical degradation: treating straw with acid, alkali or oxidant to convert it into sugars or other chemicals; physical degradation: using mechanical crushing, high temperature and high pressure to change the structure of straw for subsequent use. However, the above three methods have advantages and disadvantages. Biodegradation: There are many studies and the technology is relatively mature, but the efficiency and cost still need to be optimized. Chemical degradation: High efficiency, but there are environmental pollution and cost issues. Physical degradation: Simple operation, but the effect is limited when used alone, and it is often combined with other technologies.

[0004] Biodegradation avoids air pollution caused by burning straw and reduces greenhouse gas emissions. At the same time, no harmful substances are produced during the degradation process, and there is no pollution to the soil and water, which is conducive to ecological protection. The result of biodegradation is the efficient utilization of resources, which converts straw into high-value-added products such as organic fertilizer, biogas, biofuel, etc., improves resource utilization efficiency, promotes the recycling of agricultural waste, and reduces resource waste. In addition, the products of biodegradation can increase the organic matter content of the soil and improve the soil structure. Organic fertilizer can also provide nutrients needed by plants, reduce the use of chemical fertilizers, and reduce agricultural costs. Biodegradation has low costs, and degradation products such as organic fertilizers and biogas can also be sold to increase farmers' income.

[0005] In biological straw degradation, aerobic composting or anaerobic digestion are usually used. During aerobic composting, microorganisms first decompose the easily degradable organic matter in the straw, and then gradually decompose complex organic matter such as cellulose, hemicellulose and lignin. The residual organic matter that is not completely degraded will be converted into humus. In anaerobic digestion, microorganisms hydrolyze, acidify, produce acetic acid and produce methane in sequence under anaerobic conditions, and some substances are eventually converted into humus-like components. Humus can increase the soil's ability to retain water, heat, air and fertilizer, and is conducive to promoting the formation of soil aggregate structure. Mixing it with chemical fertilizers can make up for the defects of chemical fertilizers that cause soil compaction and reduce water and fertilizer retention.

[0006] At present, straw-degrading bacteria under natural conditions cannot promote the rapid decomposition of straw, resulting in an extremely slow conversion process of straw into humus after returning to the field, which is difficult to meet the immediate demand for improving soil fertility in agricultural production. The microbial flora in the soil used to degrade straw is easily affected by chemical agriculture. The large-scale use of chemical fertilizers, herbicides, fungicides, etc. will cause a sharp decrease in soil microbial flora, affecting straw degradation and humus formation. Summary of the invention

[0007] The invention provides a straw degrading bacteria and application thereof, so as to solve the problem that the existing straw degrading bacteria cannot promote the rapid decomposition of straw, resulting in waste of straw resources and environmental pollution.

[0008] The technical solution adopted by the present invention is: A straw degrading bacterium Bacillus megaterium XLL1, its deposit number in the China Center for Type Culture Collection is CCTCC NO:M20242410, the deposit date is October 31, 2024, and the address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University.

[0009] The straw degrading bacteria of the present invention Bacillus megaterium The nucleotide sequence of XLL1 is shown in SEQ ID No.1.

[0010] The straw degrading bacteria of the present invention Bacillus megaterium Application of XLL1 in straw degradation.

[0011] The straw degrading bacteria of the present invention Bacillus megaterium Application of XLL1 in feed.

[0012] The straw degrading bacteria of the present invention Bacillus megaterium The cultivation method of XLL1 is to inoculate the straw-degrading bacteria into the culture medium, and then put it into the SHZ-82A air bath constant temperature oscillator, set the temperature to 28°C, the oscillation speed to 120rpm, and continuously observe. The cultivation time is 7 days.

[0013] The culture medium of the present invention comprises 2-3 g of tryptone, 4-6 g of straw and 2-3 g of NaCl, the pH value of which is adjusted to 7.0 with a 1 mol / L NaOH solution, the volume is fixed to 250 ml with distilled water, the culture medium is stirred evenly with an HJ-6 magnetic heating stirrer, and the culture medium is sterilized at 121° C. and 10.4 kPa for 20 min.

[0014] The invention has the advantages of developing a new microbial strain, achieving efficient degradation of straw, accelerating the decomposition speed of straw, and producing humus. The invention is a dominant strain in composting, and its extract has high cellulase activity in the process of cellulose decomposition, effectively promoting the degradation of cellulose. It can secrete a small amount of cellulose extracellular enzymes and has a strong ability to degrade lignocellulose in corn straw. The present invention belongs to Bacillus megaterium, has rich protein storage capacity, can be used as feed for secondary utilization, and greatly increases economic benefits. Its own protein storage capacity has a high utilization value. Bacillus megaterium contains a large amount of water, protein, fat and carbohydrates. The strain is put into feed processing as a raw material, which can further improve the utilization rate of straw digestion process products. The present invention can be used as a microbial pretreatment bacterial system to improve the anaerobic digestion efficiency of cellulose biomass, thereby improving the utilization rate of corn stalks and increasing protein content. The present invention can improve the nutritional quality of poultry feed. By feeding Bacillus megaterium, the feed cost of broilers can be reduced and their weight can be increased. The spores of Bacillus megaterium can restore their activity in the animal intestine and stably colonize in the intestine, improve the intestinal flora composition of livestock and poultry, enhance the disease resistance of animals, provide enzymes required by various animals, and promote the growth and development of animals. Compared with feeding basic concentrate, the daily weight gain of beef cattle fed with Bacillus megaterium fermented feed is improved, and the crude protein digestibility and crude fiber digestibility are both improved. Through experiments, it was found that the present invention can degrade straw for 7 days and stably produce humus, thus solving the problem of slow humus formation cycle; the present invention can degrade straw for 15 days and obtain a degradation rate of 46.24%, which is higher than the efficiency of most straw-degrading bacteria; after the straw is degraded by the present invention, it can also be used as a raw material for feed processing, thereby improving the utilization rate of the strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the OD value graph in the culture medium before and after the proliferation of the straw-degrading strain; Figure 2 It is an identification chart of humus output; Figure 3 This is a graph showing the changes before and after straw degradation; Figure 4 This is a diagram of the application of straw-degrading bacteria as a protein source in feed. DETAILED DESCRIPTION

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the range are also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the range. Unless otherwise specified, all technical and scientific terms used herein have the same meanings commonly understood by those skilled in the art of the present invention. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and / or materials related to the documents. When conflicting with any incorporated documents, the content of this specification shall prevail.

[0018] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0019] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0020] The experimental materials involved in the following examples are as follows: 1. Main components of culture medium 2.5g tryptone, 5g straw, 2.5g NaCl. Adjust pH to 7.0 with 1mol / L NaOH solution, add distilled water to 250ml. Stir evenly with HJ-6 magnetic heating stirrer. Autoclave at 121℃ for 20min.

[0021] Experimental Example 1: Isolation and purification of straw-degrading bacteria Bacillusmegaterium XLL1 Isolation and purification of straw-degrading bacteria Bacillusmegaterium XLL1. The soil sample was petrochemical-contaminated soil, collected in Jilin City, Jilin Province. The isolation and purification steps are as follows: Add 5 grams of the petrochemical contaminated soil sample collected to a 250 ml Erlenmeyer flask containing 100 ml of enrichment medium and culture on a shaker at 30°C and 180 rpm for 5 to 7 days. Add 1.5% to 2.0% agar to the enrichment medium to make a solid medium. Use the dilution plate method for separation. Dilute the enrichment culture solution in a gradient manner, such as 10 -1 ~10 -6 , take 0.1 ml of the dilution and spread it on the separation medium plate, and spread 3 plates for each dilution. Invert the plate and culture it in a constant temperature incubator at 30°C for 3-5 days to observe the colony morphology. Pick single colonies with different morphologies, streak and purify them on new separation medium plates, and repeat 2-3 times until a pure culture is obtained. The purified strains were inoculated into liquid culture medium containing straw powder and cultured at 30°C and 180 rpm for 7 days. After the culture, the strains with the strongest degradation ability were preliminarily screened by measuring the reducing sugar content and straw weight loss rate in the culture medium. After identification, it belongs to Bacillus megaterium. The Bacillus megaterium XLL1 strain was deposited in the China Center for Type Culture Collection on October 31, 2024. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCCNO: M20242410. The gene sequence of Bacillus megaterium XLL1 strain is shown in SEQ ID NO.1.

[0022] Experimental Example 2 Changes in cell weight of straw-degrading bacteria Bacillus megaterium XLL1 strain during 7-day culture The XLL1 strain was inoculated into the culture medium provided by the present invention, and then placed in a SHZ-82A air bath constant temperature oscillator, the temperature was set to 28°C, the oscillation speed was set to 120 rpm, and continuous observation was performed. The culture was carried out for 7 days, and the cell weight of each 1.5 ml of culture solution was measured during the 7 days. The results are shown in Table 1.

[0023] Table 1 Changes in cell weight of XLL1 strain during 7-day culture Time / Day 1 2 3 4 5 6 7 Cell weight / mg 28.87 52.32 49.36 52.72 70.07 76.65 75.95 After seven days of culture, the cell proliferation was detected by using a microplate reader and CCK-8 assay. About 100ul of cell suspension was added to each well of a 96-well plate using a pipette, and a small amount of CCK-8 solution was added. The culture dish was gently shaken to allow the cell suspension to fully contact with the reagent. Figure 1 As shown, the OD value of the culture medium was measured to be 0.2997. After incubating the cells for 1 hour, the OD value of the culture medium was measured to be 0.4395.

[0024] Experimental Example 3 Identification of degradation products of straw-degrading bacteria Bacillusmegaterium XLL1 strain and calculation of degradation rate like Figure 2 After the strain was cultured for 7 days, the culture fluid was tested for three-dimensional fluorescence spectroscopy. The test found that the high expression part of the culture fluid was in the excitation wavelength of 320nm to 360nm and the emission wavelength of 400nm to 440nm. The analysis showed that this part was humus. Figure 3 shown.

[0025] The flocculation weight was tested by alcohol precipitation method. 3 ml of stock solution was taken, 3 ml of anhydrous ethanol was added, and finally a small amount of 1 mol / L sodium chloride solution was added. The flocculation weight was 0.2820 after centrifugation. That is, the straw degradation amount of XLL1 strain in 7 days was 0.2820 g. After a total of 15 days of culture, the final degradation amount was obtained, and the degradation rate was calculated.

[0026] Degradation rate = (degradation amount / 5g)*100% Compared with the straw-degrading bacteria data provided by some published patents, the straw-degrading bacteria provided by the present invention have better degradation effect.

[0027] Table 2 Degradation rates of four straw-degrading bacteria Bacteria Degradation rate Bacillus smegateriumXLL1 46.24% QZ-Z1&EF-Z1 strains 43.31% NJAU-X3b2 21.17% NJAU-M3a1 21.00% Experimental Example 4 Application of straw-degrading bacteria Bacillus megaterium XLL1 strain in feed After the strain completes the straw degradation process, the fermentation liquid is centrifuged at 4°C and 8000-10000rpm for 10-15 minutes, the supernatant is removed, and the bacteria are collected. Wash the bacteria 2-3 times with sterile saline or buffer to remove the culture medium residue. Use lysozyme to remove the residual cell wall. The washed bacteria are resuspended in spore induction medium, in which the carbon source and nitrogen source content can be appropriately reduced and the phosphate concentration can be increased. Continue to culture on a shaker at 37°C and 150 rpm for 24-48 hours to induce spore formation. After the culture is completed, the bacterial liquid is centrifuged again and the precipitate is collected. At this time, the precipitate is mainly spores. To further purify the spores, differential centrifugation or density gradient centrifugation can be used to remove bacteria and other impurities that have not formed spores. The collected spore suspension is placed in a freeze-drying bottle, pre-frozen to below -40°C, and then sublimated and dried under vacuum conditions to remove water directly from solid to gas to obtain dry Bacillus powder.

[0028] like Figure 4As shown in (a), the amount of earthworms required is calculated based on the protein content of dry earthworms. Beef cattle under 300kg need 1320~1600g of earthworms. Beef cattle from 300kg to 600kg need 2090~2400g of earthworms. If the XLL1 strain is used as the entire protein source for feeding beef cattle. Then beef cattle under 300kg need 1323.72~1720.84g of earthworms. Beef cattle from 300kg to 600kg need 2089.89~2716.86g of earthworms. If Chlorella is used as the entire protein source for feeding beef cattle. Then beef cattle under 300kg need 1244.82~1455.38g of earthworms. Beef cattle from 300kg to 600kg need 1965.32~2297.75g of earthworms.

[0029] like Figure 4 (b) If earthworms, Chlorella, and XLL1 strain are used to replace the protein in concentrated feed at a ratio of 1:1:1, the feed formula is shown in Table 3.

[0030] Table 3 Ratio of ingredients in feed prepared by bacterial strains

[0031] Experimental Example 5: The edibility of feed was tested by a mouse feeding experiment. The scheme and results are as follows: Experimental Materials Experimental animals: A number of healthy mice were divided into two weight range groups: under 30g and 30-60g, with 30 mice in each group.

[0032] Experimental feed: earthworms, Chlorella, and XLL1 strain mixed in a ratio of 1:1:1.

[0033] Experimental equipment: electronic scale, mouse cage, feed bowl, waterer, etc.

[0034] Experimental procedures Grouping and initial body weight measurement: Mice were grouped according to weight ranges and randomly divided into 3 groups, each with 10 mice. The initial body weight of each mouse was measured using an electronic scale and recorded.

[0035] Feeding: According to the feed usage ratio of beef cattle, the mixed feed usage of mice is converted. For mice under 30g, feed a mixed feed equivalent to the usage ratio of beef cattle under 300kg every day, which is about 416mg; for mice 30-60g, feed a mixed feed equivalent to the usage ratio of beef cattle 300-600kg every day, which is about 728mg. Feed regularly and quantitatively every day to ensure that each mouse can get enough feed, and provide sufficient clean drinking water.

[0036] Weight monitoring: Measure the weight of mice every 5 days using an electronic scale, record the data, and observe the growth and mental state of the mice.

[0037] Experimental results: (1) Weight changes of mice below 30g: Initial body weight: average body weight was (25.5±2.0) g; Body weight on the 5th day: The average body weight increased to (27.0±2.2) g, with an average increase of 1.5 g; Body weight on day 10: The average body weight was (29.5±2.5) g, an increase of 4.0 g compared to the initial body weight; Body weight on day 15: average body weight reached (32.0±2.8) g, an increase of 6.5 g; Body weight on day 20: average body weight was (35.0±3.0) g, an increase of 9.5 g; Body weight on day 25: average body weight reached (38.0±3.2) g, an increase of 12.5 g; Body weight on day 30: The average body weight was (41.0±3.5) g, an increase of 15.5 g compared to the initial body weight.

[0038] (2) Weight changes of 30-60g mice Initial body weight: average body weight was (45.0±3.0) g; Body weight on the 5th day: The average body weight increased to (47.5±3.2) g, with an average increase of 2.5 g; Body weight on day 10: average body weight was (50.5±3.5) g, an increase of 5.5 g compared to the initial body weight; Body weight on day 15: average body weight reached (54.0±3.8) g, an increase of 9.0 g; Body weight on day 20: average body weight was (58.0±4.0) g, an increase of 13.0 g; Body weight on day 25: average body weight reached (62.0±4.2) g, an increase of 17.0 g; Body weight on day 30: The average body weight was (66.0±4.5) g, an increase of 21.0 g compared to the initial body weight.

[0039] Experimental conclusion: The results of a 30-day feeding experiment showed that when mice were fed with a 1:1:1 mixture of earthworms, chlorella, and XLL1 strain, the weight of mice in different weight ranges showed a continuous growth trend, and the weight growth of mice in the 30-60g weight range was more obvious than that of mice below 30g. This shows that the mixed feed can meet the nutritional needs of mammals and promote growth, and can be put into use.

[0040] The above-mentioned experimental examples only express several embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A straw-degrading bacterium Bacillus megaterium XLL1, characterized by: Its preservation number at the China Center for Type Culture Collection is CCTCCNO:M20242410, the preservation date is October 31, 2024, and the address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University.

2. A straw degrading bacteria according to claim 1 Bacillus megaterium XLL1, characterized by: The straw degrading bacteria Bacillus megaterium The nucleotide sequence of XLL1 is shown in SEQ ID No.

1.

3. A straw degrading bacteria as claimed in claim 1 Bacillus megaterium Application of XLL1 in straw degradation.

4. A straw-degrading bacterium as claimed in claim 1 Bacillus megaterium Application of XLL1 in feed.

5. A straw-degrading bacterium as claimed in claim 1 Bacillus megaterium The method for cultivating XLL1 is characterized by: The straw-degrading bacteria were inoculated into the culture medium, and then placed in a SHZ-82A air bath constant temperature oscillator, the temperature was set to 28°C, the oscillation speed was set to 120 rpm, and continuous observation was performed. The culture time was 7 days.

6. A straw-degrading bacterium according to claim 5 Bacillus megaterium The method for cultivating XLL1 is characterized by: The culture medium comprises 2-3 g of tryptone, 4-6 g of straw and 2-3 g of NaCl. The pH value is adjusted to 7.0 with 1 mol / L NaOH solution, and the volume is fixed to 250 ml with distilled water. The culture medium is stirred evenly with a HJ-6 magnetic heating stirrer, and sterilized at 121° C. and 10.4 kPa for 20 min.

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