Low-temperature saline-alkaline tolerant streptomyces microflavus and application thereof

Abstract

The invention discloses low-temperature salt-tolerant and alkali-tolerant streptomyces microflavus and application thereof, relates to the technical field of microorganisms, and is characterized in that the preservation number of the streptomyces microflavus is CCTCC (China Center for Type Culture Collection) M2023664. The streptomyces microflavus provided by the invention can well grow at the temperature of 10-30 DEG C. In a Congo red decoloration test, the ratio of the diameter (D) of a transparent circle to the diameter (d) of a bacterial colony is 3.6. The strain is applied to degradation of cellulose, has the advantages of degradation of cellulose at low temperature, high cellulase activity, salt resistance, alkali resistance and the like, and can be particularly used for cellulose degradation strain resources in low-temperature saline-alkali areas.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, more particularly, it relates to a low-temperature salt-tolerant Streptomyces felleus and application thereof. BACKGROUND

[0002] Cellulose is the main component of agricultural waste. Northeast China is an important grain production base, and a large amount of cellulose agricultural waste is produced every year. Cellulose is a high molecular compound connected by glucose through β-1, 4-glucosidic bond, which is composed of crystalline region and amorphous region. The molecules in the amorphous region are in amorphous state and arranged loosely, and most of the hydroxyl groups of the glucose ring are in free state, which is easy to be degraded by cellulase, and the product can also be fully utilized. The molecules in the crystalline region are arranged in order, and the hydroxyl groups in the cellulose are combined to form hydrogen bonds, and the intramolecular hydrogen bond attraction makes the molecular structure more solid. These hydrogen bonds form a huge hydrogen bond grid, forming a dense crystal structure, which seriously hinders the effective contact of chemical reagents or biological enzymes with the surface of cellulose and catalysis. This is also the important reason why natural cellulose is difficult to be hydrolyzed.

[0003] The disposal of cellulose agricultural waste by piling up occupies a large amount of space resources, and the piling up method of straw rotting is slow, causing serious resource waste. Biological treatment method is currently the most advocated method of agricultural waste resource utilization, which helps to accelerate the decomposition of agricultural waste, prevent waste accumulation, and has the advantages of safety, environmental protection, economy saving and the like, and is the main means for treating cellulose agricultural waste. Microorganisms can effectively degrade cellulose by secreting cellulase, and promote the resource utilization of cellulose agricultural waste. Among the microorganisms producing cellulase, actinomycetes can produce a large amount of mycelium and spores due to their unique morphological and cellular characteristics, and are widely distributed in different survival environments, especially in low temperature, high salt and high alkali environments, and can still produce spores, showing strong stress resistance. Among them, Streptomyces is the first large genus of actinomycetes, which dominates the culturable actinomycetes resources in the saline-alkali soil in northeast China.

[0004] In recent years, due to long-term high-intensity and overloading use of black soil in the Northeast region, serious black soil loss, soil impoverishment and land salinization have been caused. The salt-alkali soil is widely distributed in the Northeast region, and the total area of the salinized soil is 2986900 mu. Soil salinization leads to poor soil permeability, which further affects the growth of plants and microorganisms. From 1989 to 2019, the average annual temperature in the Northeast region was 4-10 DEG C, the temperature in spring and autumn was about 9 DEG C, and the temperature in winter was about-10 DEG C. The cellulose degradation ability of microorganisms is higher under the condition of medium-high temperature, and low temperature can seriously inhibit the cellulose degradation activity of microorganisms. The degree of soil salinization can also lead to the decrease of the cellulose degradation ability of microorganisms. The salt-tolerant cellulose-degrading bacteria have stronger environmental adaptability and stability, and are more suitable for the biodegradation of cellulose in the salt-alkali environment. Guichunyan screened and bred a salt-tolerant cellulase-producing bacillus YRD-19-35 from the soil sample of the salt-alkali land, and the CMCase activity of the bacillus YRD-19-35 was 229.6 U.mL-1 under the condition of 37 DEG C, initial pH 8.0 and fermentation for 48 h, which indicated that the bacillus YRD-19-35 had good low-temperature tolerance and salt-tolerance. Wangdan screened a Streptomyces DS-22, and the CMCase activity of the Streptomyces DS-22 was 6.08*10-2 U.mL-1 under the condition of 30 DEG C and pH 8.5. Although some strains capable of degrading cellulose under the condition of low temperature or salt-alkali have been found, the research on the cellulose-degrading strains under the coexistence condition of low temperature and salt-alkali is still less, and it is urgent to screen the cellulose-degrading strains with low-temperature tolerance and salt-tolerance to solve the problem that the cellulose agricultural waste in the Northeast region is slowly biodegraded under the condition of low temperature and salt-alkali. SUMMARY

[0005] The purpose of the present application is to solve the above problems, and provide a low-temperature salt-tolerant Streptomyces microflavus, which can grow well under the condition of temperature 10-30 DEG C and salinity 0-3.0 %. In the Congo red decolorization test, the ratio of the transparent circle diameter (D) to the colony diameter (d) is 3.6. When the low-temperature salt-tolerant Streptomyces microflavus is applied to the degradation of cellulose, the low-temperature salt-tolerant Streptomyces microflavus has the advantages of degrading cellulose under the condition of low temperature, high cellulase activity, salt tolerance and alkali tolerance, and can be used for the cellulose-degrading strain resources in the low-temperature salt-alkali region.

[0006] The above technical purpose of the present application is realized by the following technical scheme: a low-temperature salt-tolerant Streptomyces microflavus, the preservation number of the Streptomyces microflavus is CCTCC M2023664.

[0007] The present application is further provided as follows: the 16S rRNA sequence of the Streptomyces microflavus comprises the nucleotide sequence shown in SEQ ID NO. 1.

[0008]

[0009]

[0010] The application is further provided that the Streptomyces microflavus has a positive Congo red decolorization reaction.

[0011] The application is further provided that the Streptomyces microflavus has a culture temperature of 5-40℃, a salt tolerance of 0-3.0%, and an alkali tolerance of pH 8-9.

[0012] The application further discloses a microbial agent comprising the Streptomyces microflavus and / or a fermentation liquor of the Streptomyces microflavus.

[0013] The application further discloses a fermentation culture method of the Streptomyces microflavus, comprising the following steps: inoculating the Streptomyces microflavus into a culture medium and performing fermentation culture at 5-40℃.

[0014] The application further discloses application of the Streptomyces microflavus in cellulose degradation.

[0015] The application further discloses application of the microbial agent in cellulose degradation.

[0016] The application further discloses a method for degrading cellulose by the Streptomyces microflavus, comprising the following steps: inoculating the Streptomyces microflavus into a culture medium containing cellulose and performing fermentation culture.

[0017] The application further discloses a method for degrading cellulose by the microbial agent, characterized by comprising the following steps: inoculating the microbial agent into a culture medium containing cellulose and performing fermentation culture.

[0018] The cellulose-degrading Streptomyces provided in the application belongs to Streptomyces microflavus, and is named Streptomyces microflavus CLSD-1 (Streptomyces microflavus CLSD-1) when preserved. In the examples of the application, it is simply referred to as CLSD-1, and was sent to the China Center for Type Culture Collection (CCTCC) on April 25, 2023, and was preserved in the China Center for Type Culture Collection (CCTCC) on April 28, 2023. The address of the preservation center is Luo Jia Mountain, Baoyi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC M2023664. The strain is a low-temperature, salt-tolerant and alkali-tolerant cellulose-degrading actinomycete. In the process of growth and reproduction under low-temperature, salt and alkali conditions, the strain decomposes cellulose and other materials, has the advantages of degrading cellulose under low-temperature conditions, high cellulase activity, salt tolerance and alkali tolerance, etc.

[0019] The growth condition of the strain CLSD-1 can be obviously observed after culturing at room temperature for 24 hours, the colony size is 2mm, the early colony is round and convex, the milk yellow is translucent, the edge is neat, the surface is smooth, then the colony develops into white velvet, and the differentiation of intracellular hyphae and aerial hyphae is formed. Gram staining and microscope observation show that it is gram-positive filamentous structure, and the spore chain is sinuous. The 16S rRNA sequence of the Streptomyces microflavus includes the nucleotide sequence shown in SEQ ID NO. 1, and the 16S rRNA sequence phylogenetic tree shows that the strain is homologous to Streptomyces microflavus. The strain can grow well at a temperature of 10-30 DEG C and a salinity of 0-3.0%. In the Congo red decolorization test, the ratio of the transparent circle diameter (D) to the colony diameter (d) is 3.6. It can be used for cellulose degradation in low-temperature and saline-alkaline areas.

[0020] The microbial agent provided by the application can only contain the above-mentioned Streptomyces microflavus and / or the fermentation liquor of the above-mentioned Streptomyces microflavus, or can further contain other components in addition to the above-mentioned Streptomyces microflavus and / or the fermentation liquor of the above-mentioned Streptomyces microflavus, for example, auxiliary materials added to meet the needs of process, storage and the like, and other strains added without reducing the effect of the strain.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] 1. The Streptomyces microflavus provided by the application can grow well at a temperature of 10-30 DEG C and a salinity of 0-3.0%. In the Congo red decolorization test, the ratio of the transparent circle diameter (D) to the colony diameter (d) is 3.6. When it is applied to degrade cellulose, it has the advantages of degrading cellulose under low-temperature conditions, high cellulase activity, salt tolerance and alkali tolerance, and can be used for cellulose degradation in low-temperature and saline-alkaline areas.

[0023] 2. The fermentation culture method of the Streptomyces microflavus provided by the application is beneficial to the growth of the strain, and has the advantages of low-temperature tolerance, salt tolerance and the like.

[0024] 3. The method for degrading cellulose by using the Streptomyces microflavus provided by the application has the advantages of rapidly degrading cellulose in a short time and high cellulose degradation rate.

[0025] 4. The method for degrading cellulose by using the microbial agent provided by the application has the advantages of rapidly degrading cellulose in a short time, high cellulose degradation rate, low-temperature tolerance, salt tolerance and the like. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1is a colony morphology diagram of the strain CLSD-1 in the embodiment of the present application;

[0027] Figure 2 is a Congo red decolorization diagram of the strain CLSD-1 in the embodiment of the present application;

[0028] Figure 3 is a gram staining diagram (x40) of the strain CLSD-1 in the embodiment of the present application;

[0029] Figure 4 is a SEM diagram of the strain CLSD-1 in the embodiment of the present application;

[0030] Figure 5 is a phylogenetic tree of the strain CLSD-1 and related strains constructed in the embodiment of the present application;

[0031] Figure 6 is a filter paper weight loss rate of the strain CLSD-1 in different culture media in the embodiment of the present application. DETAILED DESCRIPTION

[0032] In order for those skilled in the art to better understand the present application, the technical solutions of the present application will be further described in detail below in combination with the embodiments of the present application and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

[0033] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below in combination with the embodiments.

[0034] The present application provides a Streptomyces, the strain name is Streptomyces microflavus CLSD-1 (CCTCC M2023664). In the embodiments of the present application, the strain is named CLSD-1.

[0035] The present application provides a cellulose-degrading Streptomyces, which belongs to Streptomyces microflavus, and is named Streptomyces microflavus CLSD-1 (CCTCC M2023664) when preserved. In the embodiments of the present application, it is simply referred to as CLSD-1. CLSD-1 is screened from 15℃ enrichment conditions, has the ability to degrade cellulose with salt and alkali tolerance, and has good Congo red decolorization effect.

[0036] The cellulolytic bacteria CLSD-1 of the present application is an actinomycete cultured in a modified inorganic salt medium at 15℃, Figure 1 The colony morphology observation is presented. The modified inorganic salt medium (g·L-1) comprises the following components: NaNO3 1.25, KH2PO4 0.5, MgSO4·7H2O 0.25, CaCl2 0.05, NaCl 30, CMC-Na 10, pH 9.0, and distilled water 1000.0 mL.

[0037] The identified physiological and biochemical characteristics of the strain CLSD-1 are as follows: the growth condition of the strain CLSD-1 can be observed obviously after 24h of culture at room temperature, the colony size is 2mm, the early-stage colony is round and convex, milky yellow and translucent, the edge is neat, the surface is smooth, then it develops into white villi, and differentiation of intramural hyphae and aerial hyphae occurs. Gram staining and microscopic observation show that it is a gram-positive filamentous structure, and the spore chain is sinuous. The hyphal length under electron microscope is greater than 10μm, the width is about 0.28μm-0.4μm, the intramural hyphae are mostly branched, and the two ends are blunt. The results of physiological and biochemical tests are shown in Table 1.

[0038] Table 1

[0039]

[0040] Note: "-" represents negative, "+" represents positive

[0041] The molecular biology identification results of the strain CLSD-1: through 16S rRNA sequence comparison, the strain CLSD-1 has the closest genetic relationship with Streptomyces microflavus NBRC 13062, and the similarity is 100%. The 16S rRNA sequence phylogenetic tree of the strain CLSD-1 shows that the strain CLSD-1 and Streptomyces microflavus are homologous.

[0042] The strain CLSD-1 provided by the present application can be cultured at 5-40℃.

[0043] The strain CLSD-1 can effectively degrade filter paper under low temperature and non-optimized culture conditions, and the degradation rate can reach 20.21% or more after 15d.

[0044] The separation and culture method of the cellulolytic actinomycete of the present application first enriches the soil sample, then re-enriches the cellulolytic bacteria in the soil by using a restrictive medium, then dilutes and spreads it on a modified inorganic salt medium for culture, screens the strain capable of using cellulose as the only carbon source, and finally analyzes the cellulolytic ability of the strain.

[0045] Example 1: Separation and culture of soil microorganism CLSD-1

[0046] 1. Sample collection: Soil samples were collected in Daqing City, Heilongjiang Province, China. The sampling point was located at (E124.992949°, N46.475373°). The soil samples were collected from the surface layer (0-10 cm) using a sampling shovel and immediately placed in sterile polyethylene bags for low-temperature transportation. The samples were divided into two parts, one for microbial isolation and the other for storage in 50 mL centrifuge tubes with PBS buffer at -80°C.

[0047] 2. Soil enrichment and isolation

[0048] 5 g of soil sample was weighed into a 100 mL conical flask, 50 mL of sterile water was added, and the mixture was shaken at 15°C and 150 rpm for 20 min. After 2 min of standing, the mixture was used as the original solution for plate dilution and plating. The soil original solution was diluted to 10-3, and 100 uL of 10-1, 10-2, and 10-3 dilutions were taken and plated on modified inorganic salt solid medium. The plates were incubated at 15°C, and the growth of colonies was observed regularly. The low-temperature and salt-tolerant cellulose-degrading bacteria were isolated and purified using plate streaking method, and the single colonies were obtained after multiple passages. The single colonies were transferred to liquid LB medium for cultivation and preservation by glycerol method.

[0049] 3. Strain screening

[0050] The purified strain was inoculated in the center of the modified inorganic salt solid medium and incubated at 15°C for 15 days. The strains producing Congo red decolorization rings were screened using Congo red staining method. The colony diameter and cellulose hydrolysis ring diameter were measured, and the size of the cellulose hydrolysis ring was calculated according to the formula. The strain with Congo red decolorization ability was obtained and transferred to liquid LB medium for cultivation and preservation by glycerol method.

[0051] Example 2 16S rRNA sequence analysis of CLSD-1

[0052] Strain CLSD-1 was sent to Shanghai Pisennol Bio-Technology Co., Ltd. for 16S rRNA sequencing analysis for strain identification.

[0053] Strain identification results: The sequence of the strain obtained by sequencing was compared with the sequences on NCBI, and the comparison results showed that the sequence of strain CLSD-1 had 100% similarity with Streptomyces microflavus sequence. Therefore, it was determined that strain CLSD-1 belonged to Streptomyces microflavus in molecular phylogenetic taxonomy.

[0054] Figure 5 The phylogenetic tree of strain CLSD-1 based on 16S rRNA sequence is shown in Figure 1.Figure 4 It can be seen that the strain most closely related to CLSD-1 is Streptomyces microflavus NBRC 13062, indicating that CLSD-1 belongs to Streptomyces microflavus in the molecular phylogenetic taxonomy.

[0055] Example 3 Filter paper degradation ability of cellulose-degrading bacterium CLSD-1.

[0056] The filter paper degradation effect in different culture media was determined by weight loss method. The filter paper was a 1 cm x 1 cm paper sheet and was added at 4 g L-1 to 100 mL / 250 mL of inorganic salt medium and liquid fermentation medium. The obtained strain was inoculated into the inorganic salt medium and liquid fermentation medium at an inoculation amount of 5%, and the control group was inoculated with 5% sterile water, and was cultured at 15°C with constant temperature oscillation at 150 rpm for 15 d. The filter paper degradation effect was determined by weight loss method.

[0057] Liquid fermentation medium (g L-1): KH2PO4 2.0, urea 0.3, MgSO4 7H2O 0.3, (NH4)2SO4 1.4, peptone 0.25, yeast extract 0.1, Tween 80 0.1 mL L-1, CaCl2 H2O 0.3, FeSO4 7H2O 0.005, MnSO4 H2O 0.0016, ZnSO4 7H2O 0.0014, CoCl2 6H2O 0.002, filter paper 4.0, NaCl 30, pH 9.0, distilled water 1000.0 mL.

[0058] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, and the modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A low-temperature, salt-alkali-tolerant Streptomyces tenuifolius strain, characterized by: The deposit number of the Streptomyces tenuifolius is CCTCCM2023664.

2. The low-temperature, salt-alkali-tolerant Streptomyces tenuifolius strain according to claim 1, characterized in that: The 16SrRNA sequence of the Streptomyces tenuifolius includes the nucleotide sequence shown in SEQ ID NO.

1.

3. The low-temperature, salt-alkali-tolerant Streptomyces tenuifolius strain according to claim 1, characterized in that: The Congo red decolorization reaction of the Streptomyces tenuifolius is positive.

4. The Streptomyces tenuifolius according to any one of claims 1 to 3, characterized in that: The culture temperature of the Streptomyces tenuifolius is 5-40° C., the salt tolerance is 0-3.0%, and the alkalinity tolerance is pH 8-9.

5. A bacterial agent characterized by: The method comprises the Streptomyces tenuifolius according to any one of claims 1 to 4 and / or the fermentation broth of the Streptomyces tenuifolius.

6. A fermentation culture method for Streptomyces tenuifolius according to any one of claims 1 to 4, characterized in that: The following steps are involved: Inoculate Streptomyces tenuifolius into the culture medium and ferment at 5°C-40°C.

7. Use of the Streptomyces tenuifolius according to any one of claims 1 to 4 in cellulose degradation.

8. Use of the bacterial agent according to claim 5 in cellulose degradation.

9. A method for degrading cellulose using the Streptomyces tenuifolius according to any one of claims 1 to 4, characterized in that: The following steps are involved: The Streptomyces tenuifolius is inoculated into a culture medium containing cellulose and fermented.

10. A method for degrading cellulose using the bacterial agent according to claim 5, characterized in that: The following steps are involved: The bacterial agent is inoculated into a culture medium containing cellulose and fermented.

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