Thermoactinomycetes MM-1 and application thereof

Abstract

The invention discloses a thermoactinomycete MM-1 and application thereof, and belongs to the technical field of microorganisms. The invention discloses a thermoactinomycete MM-1, and the preservation number of the thermoactinomycete MM-1 is CCTCC (China Center for Type Culture Collection) NO: M20242858 The solid fungicide disclosed by the invention contains thermoactinomycetes MM-1, can be applied to corn straw returning and composting treatment, and promotes sustainable development of agricultural ecology.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more specifically to a thermophilic actinomycete MM-1 and application thereof. Background Art

[0002] With the improvement of global environmental protection awareness, the demand for efficient, environmentally friendly and sustainable agricultural waste treatment technologies is also growing. Corn straw, as a common agricultural waste, has a huge output worldwide. my country's annual corn straw output can reach about 200 million tons. Most of these straws are burned on site or piled up randomly, which not only wastes precious resources, but also may cause environmental problems and pose safety hazards. Therefore, how to improve its resource utilization rate has become an urgent problem to be solved. However, due to the complex structure of lignocellulose in corn straw, traditional acid and alkali treatment methods have problems such as low utilization rate and environmental pollution.

[0003] In recent years, microbial fermentation technology has received extensive attention in the field of biodegradation, and the degradation of corn stalks by microbial agents has become a research hotspot. For example, Zhang Sainan et al. developed a composite agent (CFF), which not only changed the structure of the indigenous microbial community, but also significantly improved the degradation rate of corn stalks. Ma Lan et al. successfully developed a composite agent that can efficiently degrade straw, and the agent has a certain growth-promoting effect on the growth and development of Chinese cabbage. Therefore, composite agents not only open up new ways for the resource utilization of agricultural waste, but also provide new methods for promoting the growth of crops.

[0004] Compared with bacteria and fungi, actinomycetes can survive in the form of spores under adverse conditions, and show advantages such as high temperature resistance and acid and alkali resistance. Actinomycetes are the dominant bacteria for decomposing cellulose during the high temperature period of composting. Nocardia, Streptomyces and Thermoactinomycetes are dominant thermophilic actinomycetes in composting, which play a role in degrading cellulose not only during the high temperature period of composting, but also during the cooling and maturity period. Therefore, thermoactinomycetes, with their unique biodegradation ability, can effectively decompose complex organic matter such as lignocellulose in corn stalks and convert them into valuable biofertilizers or biofuels. In this process, not only the accumulation of agricultural waste and environmental pollution are reduced, but also the recycling of resources is realized, providing strong support for the sustainable development of agriculture. Since enzymes are easily inactivated under high temperature conditions, the scarcity of such research has not only limited the application of cellulases in high temperature environments, but also hindered the in-depth understanding of the survival mechanism of microorganisms in extreme habitats.

[0005] Therefore, providing a thermophilic actinomycete MM-1 and its application is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a thermophilic actinomycete MM-1 and application thereof.

[0007] The preparation method and fermentation conditions of solid inoculant of thermophilic actinomycetes from corn stalk were studied to provide a theoretical basis for its practical application.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A thermostable actinomycete MM-1 with cellulose degradation ability was screened out from the thermostable compost sample and prepared into a solid bacterial agent.

[0010] A thermophilic actinomycete MM-1, with a deposit number of CCTCC NO: M 20242858, has been deposited in the China Center for Type Culture Collection, referred to as CCTCC, address: Wuhan University, Wuhan, China, with a deposit date of December 18, 2024, and is classified and named Streptomyces lusitanus MM-1.

[0011] Furthermore, the thermophilic actinomycete MM-1 is used in producing cellulase.

[0012] Furthermore, the thermophilic actinomycete MM-1 is used to produce cellulase under high temperature conditions.

[0013] Furthermore, the thermophilic actinomycete MM-1 is used in the degradation of corn stalks.

[0014] Furthermore, the thermophilic actinomycete MM-1 is used to degrade corn stalks under high temperature conditions.

[0015] Furthermore, a solid bacterial agent contains the thermophilic actinomycete MM-1.

[0016] Furthermore, the solid bacterial agent is used in degrading corn stalks.

[0017] Furthermore, the solid bacterial agent is used to degrade corn stalks under high temperature conditions.

[0018] It can be known from the above technical scheme that compared with the prior art, the present invention discloses a strain of thermophilic actinomycetes MM-1 and its application, and determines the ability of the strain to degrade cellulose by measuring the activity of cellulose-degrading enzymes. The enzyme activity of strain MM-1 reached the highest on the 13th day. In the corn straw degradation experiment, the degradation rate of the BM group with the addition of solid bacterial agents reached 30.83% and was significantly higher than that of the other two groups. The degradation rate was increased by 6.26 percentage points compared with the treatment group without the addition of bacterial agents. The present invention provides a theoretical basis for the screening of cellulose-degrading thermophilic actinomycetes, the preparation of bacterial agents and the fermentation conditions of solid bacterial agents. The obtained solid bacterial agents can be used in the return of corn straw to the field and composting treatment, promoting the sustainable development of agricultural ecology. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 The attached figure shows the transparent circle of the strain MM-1 of the present invention on Congo red-cellulose medium;

[0021] Figure 2 The accompanying drawing shows the colony morphology of the strain MM-1 of the present invention;

[0022] Figure 3 The accompanying drawing is a phylogenetic tree of the strain MM-1 of the present invention;

[0023] Figure 4 The accompanying figure shows the filter paper enzyme activity of the strain MM-1 of the present invention;

[0024] Figure 5 The accompanying figure shows the cellulase activity of the strain MM-1 of the present invention;

[0025] Figure 6 The accompanying drawings show the dynamic changes in the degradation rate of corn stalks in each treatment group of the present invention within 35 days. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Sample source: The bacterial source samples are cow dung and corn straw aerobic composting samples during the high temperature period.

[0028] (1) Primary screening culture medium:

[0029] Gao's medium No. 1 (g / L): agar 20.0, soluble starch 20.0, MgSO 4 7H 2 O 0.5, FeSO 4 7H 2 O 0.01, K 2 HPO 4 ·3H 2 O 0.5, KNO 3 1.0, NaCl 0.5, distilled water to 1L, pH 7.2~7.4.

[0030] (2) Rescreening culture medium:

[0031] Congo red-cellulose medium (g / L): CMC-Na 1, KH 2 PO 4 0.5,MgSO 4 7H 2 O 0.125, (NH 4 ) 2 SO 4 0.5, Congo red 0.05, agar 20, distilled water to 1L, pH 7.2-7.4.

[0032] (3) Actinomycete enzyme production medium (g / L): CMC-Na 5, K 2 HPO 4 ·3H 2 O 1, yeast extract 15, MgSO 4 7H 2 O1, NaCl 5, distilled water to 1L, pH 7.0~7.2.

[0033] Example 1 Screening and identification of strains

[0034] (1) Screening of strains

[0035] The samples came from the high temperature period of aerobic composting of cow dung and corn straw. Gao's No. 1 medium was used to screen the thermotolerant actinomycete strains at 45°C. After three purifications, the obtained pure colonies were inoculated on Congo red-cellulose medium and cultured at 45°C for 5 days. The ratio of the transparent zone diameter (D) to the colony diameter (d) was measured.

[0036] A thermostable actinomycete was screened and named MM-1, which produced a transparent zone on Congo red-cellulose medium ( Figure 1), indicating that this actinomycete has the ability to degrade cellulose. The D / d values ​​are shown in Table 1.

[0037] Table 1 Ratio of transparent zone diameter to colony diameter on Congo red-cellulose medium

[0038]

[0039] (2) Strain identification

[0040] ①Morphological identification: refer to "Classification and Identification of Actinomycetes" to conduct morphological identification of single colonies on Gao's No. 1 medium.

[0041] The strain MM-1 was streaked on Gao's medium No. 1 and cultured at 45°C for 5 days. Figure 2 The aerial hyphae of strain MM-1 are gray, do not produce pigments, are dry and easy to pick. According to the "Microbial Taxonomy", strain MM-1 was preliminarily identified as an actinomycete.

[0042] ② Molecular identification: 16S rDNA was sequenced using universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3'; SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3'; SEQ ID NO.2) (Beijing Liuhe BGI Genomics Co., Ltd.). The sequencing results were compared for homology using the National Center for Bioinformation (NCBI), and a phylogenetic tree was constructed using MEGA 5 software.

[0043] The strain 16S rDNA sequence obtained after sequencing (as shown in SEQ ID NO.3) was compared with the NCBI database and the sequences with higher homology were downloaded to construct a phylogenetic tree. The results are shown in Figure 3 The strain MM-1 was identified as Streptomyces lusitanus, which belongs to the genus Streptomyces.

[0044] 16S rDNA sequence:

[0045]

[0046] The deposit number of the thermophilic actinomycete MM-1 is CCTCC NO: M 20242858. It has been deposited in the China Center for Type Culture Collection, referred to as CCTCC, address: Wuhan University, Wuhan, China. The deposit date is December 18, 2024, and the classification name is Streptomyces lusitanus MM-1.

[0047] Example 2 Determination of cellulase activity

[0048] The strain MM-1 was placed in Gao's medium No. 1 and cultured at 45°C for 7 days. The spores were scraped off with a cell scraper and added to sterile water. After filtering through 8 layers of sterile gauze, the spores were diluted to a concentration of 1×10 6 CFU / mL, and obtain spore suspension. The spore suspension was inoculated into actinomycete enzyme production medium at a 2% inoculation rate, placed in a shaking incubator (150r / min) at 45°C, and sampled every 48h. The activity of filter paper enzyme (FPase) and cellulase (CMC) was determined by DNS method.

[0049] The filter paper enzyme activity of strain MM-1 is shown in Figure 4 , cellulase activity Figure 5 The activities of filter paper enzyme and cellulase reached their peaks on the 13th day, which were 2.5U / mL and 0.43U / mL, respectively. This showed that strain MM-1 had good cellulase production and cellulose degradation capabilities.

[0050] Example 3 Preparation of solid bacterial agent

[0051] Sawdust, starch and peptone were mixed in a mass ratio of 100:6:3 to obtain a solid culture matrix. The solid culture matrix was sterilized at 121°C for 30 min. After the temperature dropped to room temperature, 0.3 mL of spore suspension was inoculated per gram of solid culture matrix at a concentration of 1×10 6 CFU / mL, and then add sterile water to adjust the water content to 32%, and place it at 45℃ for static culture for 9 days to obtain a solid bacterial agent. The number of viable bacteria in the solid bacterial agent can reach 1.69×10 8 CFU / g.

[0052] Example 4 Corn straw degradation experiment

[0053] The solid bacterial agent prepared in Example 3 was used to conduct a straw degradation experiment to verify the degradation effect of the bacterial agent. Three treatment groups were set up for the corn straw degradation experiment: (1) BM group added 6g of unsterilized straw and inoculated with 10% solid bacterial agent; (2) MG group added 6g of sterilized straw and inoculated with 10% solid bacterial agent; (3) JG group added 6g of unsterilized straw. All three treatment groups were cultured at 45°C. The degradation rate of corn straw was determined by the weight loss method. The degraded straw residue was filtered with dry gauze, rinsed with distilled water, placed in a 75°C oven, dried to constant weight, and the degradation rate was calculated. Three parallels were set up for each group, and the test was conducted every seven days.

[0054] Straw degradation rate (%) = (W0-W1) / W0×100%, where: W0 represents the weight of the original straw added (g); W1 represents the weight of the remaining straw after drying after the cultivation is completed (g).

[0055] Figure 6 The dynamic changes of corn straw degradation rate showed that the corn straw degradation rate of each group increased the most during the 7th to 21st day of cultivation, and the degradation rate increased slowly after the 21st day and tended to be stable. On the 35th day of cultivation, the straw degradation rates of the BM, MG, and JG groups reached 30.83%, 17.28%, and 24.57%, respectively. Among them, the degradation rate of the BM group was significantly higher than that of the other two groups, and the degradation rate was increased by 6.26 percentage points compared with the treatment group without the addition of bacterial agents.

[0056] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A thermophilic actinomycete MM-1, characterized in that: Its deposit number is CCTCC NO: M20242858.

2. Use of the thermophilic actinomycete MM-1 according to claim 1 in producing cellulase.

3. Use of the thermophilic actinomycete MM-1 according to claim 1 in producing cellulase under high temperature conditions.

4. Use of the thermophilic actinomycete MM-1 according to claim 1 in degrading corn stalks.

5. Use of the thermophilic actinomycete MM-1 described in claim 1 in degrading corn stalks under high temperature conditions.

6. A solid bacterial agent, characterized in that: Contains the thermophilic actinomycete MM-1 according to claim 1.

7. Use of the solid bacterial agent according to claim 6 in degrading corn stalks.

8. Use of the solid bacterial agent according to claim 6 in degrading corn stalks under high temperature conditions.

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