Strains with the ability to degrade pepper straw and culture method and application thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a group of strains with the ability of degrading pepper stalks, and a culture method and application thereof. The present application finds that three strains isolated from pepper stalks have the degradation effect on agricultural wastes, and can be used for the reuse of agricultural wastes. The fermentation liquor of the strains can effectively degrade pepper stalk waste. The strains of the present application are simple to make and low in cost, and are non-toxic, harmless and non-polluting to the environment, which is conducive to the safe and effective degradation of pepper stalk waste, and is also conducive to environmental protection.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a group of strains capable of degrading chili straw, their cultivation methods, and applications. Background Technology

[0002] Chili peppers are a highly nutritious fresh vegetable, rich in vitamins C and E, carotenoids, and other nutrients, and are widely cultivated in my country. According to statistics from the National Major Vegetable Industry Technology System, the annual planting area of ​​chili peppers in my country has remained stable at 2.1 × 10⁻⁶ in recent years. 6 hm 2 The total output reached 6.4 × 10⁴. 7 (Zou Xuexiao, Ma Yanqing, Dai Xiongze, et al. Spread and industrial development of chili pepper in China [J]. Journal of Horticulture, 2020).

[0003] As the planting area of ​​chili peppers in my country continues to expand, a large amount of chili pepper straw is also generated during the large-scale production of chili peppers. Since the allelochemicals produced during the decomposition of chili pepper straw have a certain inhibitory effect on crop growth and development, it is not suitable to directly return chili pepper straw to the field (Hou Yongxia, Zhou Baoli, Wu Xiaoling, et al. Study on allelochemicals of chili pepper straw decomposition [J]. Chinese Journal of Applied Ecology, 2006). In most areas, the main method for disposing of chili pepper straw is direct disposal or on-site burning. This method not only damages soil structure but also causes a series of environmental pollution problems such as smog.

[0004] Chili pepper straw contains nitrogen, phosphorus, potassium, and various trace elements necessary for plant growth, making it an important source of organic fertilizer. Cellulose, hemicellulose, and lignin, three organic compounds, account for over 80% of the total solids in straw (LI Y, ZHANG R, LIU G, et al. Comparison of methane production potential, biodegradability, and kinetics of different organic substrates[J]. Bioresource Technology, 2013, 149:565-569.). However, the development and utilization of chili pepper straw in my country is still in a stage of high pollution, high consumption, and low output (Shen Yuli, Niu Xinlu, Jia Ruoyun, et al. Research status of efficient utilization of chili pepper straw[J]. Modern Horticulture, 2021). Straw has a complex high-energy hydrogen-bonded crystal structure, making it difficult to degrade naturally, which has become a technical bottleneck for its resource utilization. Achieving efficient straw degradation using biotechnology has become a research focus in recent years. Currently, there are many methods for treating the main components of straw, mainly physical, chemical, and biodegradation. Biodegradation involves fermenting and enzymatically hydrolyzing straw using microorganisms. Compared to physicochemical treatments (such as acid or alkali treatments), microbial treatment is safer, produces no secondary pollution, and is energy-efficient, environmentally friendly, and economical.

[0005] Therefore, finding efficient chili straw degrading bacteria has become an important way to solve this problem. Summary of the Invention

[0006] This invention discovered three strains isolated from chili pepper plants that have a degradation effect on agricultural waste, and based on this, the invention was completed.

[0007] In a first aspect, the present invention provides a group of strains with degradation capabilities, wherein the strains are selected from one or more of XNCD-3, XNCD-4 and / or XNCD-7; wherein the XNCD-3 has accession number CGMCC NO. 34327, accession name XNCD-3, and suggested classification name is Pseudomonas, and the DNA sequence of the strain is shown in SEQ ID NO.1; wherein the XNCD-4 has accession number CGMCC NO. 34328, accession name XNCD-4, and suggested classification name is Microbacteria, and the DNA sequence of the strain is shown in SEQ ID NO.2; wherein the XNCD-7 has accession number CGMCC NO. 34329, accession name XNCD-7, and suggested classification name is Klebsiella, and the DNA sequence of the strain is shown in SEQ ID NO.3.

[0008] Furthermore, the straw in question is chili straw.

[0009] In a second aspect, the present invention provides a method for culturing the strain described in the first aspect, the method comprising the following steps: S1. The strain was inoculated into R2A liquid medium and cultured to obtain fermentation broth; S2. Centrifuge the fermentation broth obtained in step S1, discard the supernatant, and the precipitate is the bacterial block of this strain; S3. Store the mycelium blocks in glycerol.

[0010] Furthermore, in step S1, the culture temperature is selected from 25-38℃, preferably 30℃.

[0011] Furthermore, in step S1, the culture is carried out in a shaker, the rotation speed of which is selected from 120-360 r / min, preferably 200 r / min.

[0012] Furthermore, in step S1, the culture time is selected from 2-5 days, preferably 3 days.

[0013] Furthermore, in step S2, the centrifugation temperature is selected from 0-10℃, preferably 4℃.

[0014] Furthermore, in step S2, centrifugation is carried out by a centrifuge, the centrifuge speed of which is selected from 2200-3600 rpm, preferably 3000 rpm.

[0015] Furthermore, in step S3, the concentration of the glycerol is selected from 25-38%; preferably 33%.

[0016] Furthermore, the strains are selected from one or more of XNCD-3, XNCD-4, and / or XNCD-7; the XNCD-3 has the accession number CGMCC NO. 34327, accession name XNCD-3, and suggested classification name *Pseudomonas*, and its DNA sequence is shown in SEQ ID NO. 1; the XNCD-4 has the accession number CGMCC NO. 34328, accession name XNCD-4, and suggested classification name *Microbacteria*, and its DNA sequence is shown in SEQ ID NO. 2; the XNCD-7 has the accession number CGMCC NO. 34329, accession name XNCD-7, and suggested classification name *Klebsiella*, and its DNA sequence is shown in SEQ ID NO. 3.

[0017] Furthermore, the straw in question is chili straw.

[0018] Thirdly, the present invention provides a microbial agent for degrading straw, wherein the strain is selected from one or more of XNCD-3, XNCD-4, and / or XNCD-7; the XNCD-3 has the accession number CGMCC NO. 34327, accession name XNCD-3, and suggested classification name *Pseudomonas*, and the DNA sequence of the strain is shown in SEQ ID NO. 1; the XNCD-4 has the accession number CGMCC NO. 34328, accession name XNCD-4, and suggested classification name *Microbacterium*, and the DNA sequence of the strain is shown in SEQ ID NO. 2; the XNCD-7 has the accession number CGMCC NO. 34329, accession name XNCD-7, and suggested classification name *Klebsiella*, and the DNA sequence of the strain is shown in SEQ ID NO. 3.

[0019] Furthermore, the straw in question is chili straw.

[0020] Furthermore, the bacterial agent is available in liquid and solid formulations.

[0021] Furthermore, the microbial agent includes nutrients and auxiliary components.

[0022] Furthermore, the nutrients include one or more of the following: carbon source, nitrogen source, phosphorus source, trace elements, and / or water.

[0023] Furthermore, the auxiliary ingredients include one or more of surfactants, surfactants, pH adjusters, humectants, stabilizers, penetrants, and / or antioxidants.

[0024] Fourthly, the present invention provides the application of the strain described in the first aspect of the present invention in the preparation of a microbial agent for degrading straw, said strain being able to reduce the cellulose content of straw.

[0025] Furthermore, the strains are selected from one or more of XNCD-3, XNCD-4, and / or XNCD-7; the XNCD-3 has the accession number CGMCC NO. 34327, accession name XNCD-3, and suggested classification name *Pseudomonas*, and its DNA sequence is shown in SEQ ID NO. 1; the XNCD-4 has the accession number CGMCC NO. 34328, accession name XNCD-4, and suggested classification name *Microbacteria*, and its DNA sequence is shown in SEQ ID NO. 2; the XNCD-7 has the accession number CGMCC NO. 34329, accession name XNCD-7, and suggested classification name *Klebsiella*, and its DNA sequence is shown in SEQ ID NO. 3.

[0026] Furthermore, the straw in question is chili straw.

[0027] Furthermore, the bacterial agent is available in liquid and solid formulations.

[0028] Furthermore, the microbial agent includes nutrients and auxiliary components.

[0029] Furthermore, the nutrients include one or more of the following: carbon source, nitrogen source, phosphorus source, trace elements, and / or water.

[0030] Furthermore, the auxiliary ingredients include one or more of surfactants, surfactants, pH adjusters, humectants, stabilizers, penetrants, and / or antioxidants.

[0031] Beneficial effects The three bacterial strains provided by this invention have the ability to degrade chili straw waste and can be used for the reuse of agricultural waste. The fermentation broth of these strains can effectively degrade chili straw waste.

[0032] When the three strains were used to treat straw individually, the average straw degradation rate reached 52.18%; the average hydrolysis value reached 2.96; and the average cellulose degradation rate reached 30.5%, all of which were significantly higher than the water-treated control group. The weight loss rate of the chili straw from the composite microbial community was 50.39%, an increase of 88.02% compared to the control group. The strains of this invention are simple and low-cost to prepare, non-toxic, harmless, and environmentally friendly, which is beneficial for the safe and effective degradation of chili straw waste and also contributes to environmental protection. Attached Figure Description

[0033] Figure 1 This is a phylogenetic tree of 16S rDNA gene sequences constructed based on three straw-degrading strains.

[0034] Figure 2 This image shows the effect of a single strain on straw degradation.

[0035] Note: The control group consisted of water.

[0036] Figure 3 This image shows the effect of the compound microbial community in degrading straw.

[0037] Note: The control group was water; CC was a complex bacterial group. Detailed Implementation

[0038] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0039] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0040] Example 1 Screening of chili straw degrading bacteria Preprocessing The straw was cut into small pieces and placed in a liquid specialized carbon-deficient medium containing 4% (by volume) of inoculum. It was then cultured under restricted conditions at 30°C and 200 r / min.

[0041] Using the turbid bacterial culture from 10 days as the inoculum, 5 mL was inoculated into a new liquid specialized carbon-deficient medium. All other materials and procedures remained unchanged, and the culture was continued with shaking. This restricted subculturing was repeated for at least 5 generations to obtain a specialized, highly efficient, and synergistic degradative bacterial community capable of stably degrading chili straw.

[0042] The bacterial culture was serially diluted 10-fold, and the serially diluted solutions were spread on LB medium for incubation. Different morphological bacteria were picked and streaked repeatedly to obtain single strains, which were then coded and stored in LB slant culture.

[0043] Initial screening Initial screening was performed using Congo red staining. The isolated single strains were inoculated onto screening medium and cultured. After staining with 1 mg / mL Congo red solution, the strains were destained with 1 mol / L NaCl solution. The diameter of the clear zone and the colony diameter were measured, and the hydrolysis value was calculated as: hydrolysis value = diameter of clear zone / colony diameter.

[0044] Secondary screening Strains with high hydrolysis values ​​were selected for secondary screening using the filter paper strip disintegration method. Filter paper strips and bacterial culture were added to Hutchison Inorganic Salt Medium and cultured, with an equal volume of sterile water added as a blank control. The disintegration of the filter paper was observed. Simultaneously, the aniline blue decolorization method was used to detect whether the screened strains possessed lignin degradation ability.

[0045] Example 2 Molecular biological identification of three chili straw degrading strains 16S rDNA gene sequencing and phylogenetic tree construction. Bacterial DNA was extracted using a DNA extraction kit, and specific PCR amplification was performed using the universal bacterial primer 27F (TACGGYTACCTTGTTACGACTT)-1492R (AGAGTTTGATCMTGGCTCAG). The PCR products were sequenced, and the 16S rDNA nucleotide sequence lengths of each strain were determined to be 1332 bp, 1389 bp, and 1247 bp, respectively. Similarity analysis was performed between these sequences and relevant data in GenBank. Phylogenetic trees were constructed by comparing the 16S rDNA sequences with those of strains in NCBI.

[0046] Based on the comprehensive analysis of the culture characteristics, morphological and physiological biochemical characteristics of the strains and the results of 16S rDNA sequence analysis, XNCD-3, XNCD-4 and XNCD-7 were finally identified as Actinomycetota Microbacterium, Gammaproteobacteria Stenotrophomonas and proteobacteria Klebsiella, respectively.

[0047] Example 3: Validation of the Degradation Function of a Single Straw Variety in Chili Pepper Stalks Test methods Hydrolysis value The isolated single strains were inoculated onto screening medium and cultured. After incubation, they were stained with 1 mg / mL Congo red solution and destained with 1 mol / L NaCl solution. The diameter of the transparent zone and the diameter of the colony were measured, and the hydrolysis value was calculated as follows: hydrolysis value = diameter of transparent zone / diameter of colony.

[0048] Straw weight loss rate Single-source bacterial suspensions of XNCD-3, XNCD-4, and XNCD-7 were cultured separately. 5 g of straw was cut into small pieces and placed in 125 mL of liquid carbon-deficient medium containing 4% (v / v) of the single-source bacterial strain. Restrictive culture was performed at 30℃ and 200 r / min. After 10 days, the straw was removed from the medium, blanched, and dried to constant weight. The straw weight loss rate was calculated by measuring the weight after drying: Straw weight loss rate = (straw weight before treatment - straw weight after degradation) / straw weight before treatment × 100%.

[0049] Cellulose degradation Add filter paper strips and bacterial culture to Hutchison Inorganic Salt Medium and culture. Add an equal amount of sterile water as a blank control. Observe the disintegration of filter paper and calculate the cellulose degradation rate. Cellulose degradation rate = (mass of filter paper - mass after degradation) × 100%.

[0050] Test results Hydrolysis value Hydrolysis zone experiments showed that the hydrolysis value of strain XNCD-3 was 3.04; that of strain XNCD-4 was 2.96; and that of strain XNCD-7 was 2.88; with an overall average of 2.96. This indicates that each individual strain possesses a strong ability to secrete cellulase, suggesting that these strains have potential application value as cellulose-degrading bacteria.

[0051] Straw weight loss rate Through straw degradation experiments, phenotypic observation revealed that the chili straw treated with various degrading strains had many pores on its surface and was loose like cotton, indicating that the degrading bacteria rapidly and deeply decomposed the chili straw. In contrast, the straw treated with CK (water) softened but retained its shape, indicating a poor degradation effect.

[0052] As shown in Table 1, after water treatment, the straw weight loss rate of the CK group was 31.81%; the straw weight loss rate of strain XNCD-3 was 48.8%; the straw weight loss rate of strain XNCD-4 was 56.66%; and the straw weight loss rate of strain XNCD-7 was 51.09%; the overall average reached 52.18%. The straw weight loss rate under each strain treatment increased by 53.41%, 78.12%, and 60.61% compared with the CK treatment, respectively, indicating that each degrading strain had a significant promoting effect on straw degradation, with XNCD-4 showing the most significant degradation effect.

[0053] Table 1. Validation of the degradation function of chili straw by a single strain Note: Different lowercase letters in the table indicate different meanings. P The difference was significant at the <0.05 level.

[0054] Cellulose degradation Cellulose degradation characteristic tests showed that different strains had different abilities to degrade cellulose.

[0055] The cellulose degradation rate of strain XNCD-3 was 35.94%; that of strain XNCD-4 was 26.31%; and that of strain XNCD-7 was 29.25%; the overall average was 30.5%. XNCD-3 exhibited the strongest degradation ability, demonstrating that each individual strain had a high cellulose degradation rate.

[0056] Example 4: Verification of the degradation function of chili straw by compound microbial community Test methods Straw weight loss rate Five g of straw was cut into small pieces and placed in 125 mL of a specialized carbon-deficient liquid culture medium containing 4% (v / v) XNCD-3, XNCD-4, and XNCD-7 in a 1:1:1 v / v ratio of inoculum. The medium was then used for restrictive culture at 30℃ and 200 r / min. After 10 days, the straw was removed from the culture medium, blanched, and dried in an oven to constant weight. The weight loss rate of the straw was calculated by measuring its weight after drying: Straw weight loss rate = (straw weight before treatment - straw weight after degradation) / straw weight before treatment × 100%.

[0057] Test results The straw degradation test showed that when XNCD-3, XNCD-4 and XNCD-7 were mixed in a volume ratio of 1:1:1, the weight loss rate of the chili straw by the compound microbial community was 50.39%, which was 88.02% higher than that of the CK treatment. This indicates that the compound microbial community can enhance the decomposition rate of straw.

Claims

1. A group of bacterial strains capable of degrading straw, wherein the strains are selected from one or more of XNCD-3, XNCD-4 and / or XNCD-7; wherein XNCD-3 has accession number CGMCC NO. 34327, accession name XNCD-3, and suggested classification name is *Pseudomonas*, and the DNA sequence of the strain is shown in SEQ ID NO. 1; wherein XNCD-4 has accession number CGMCC NO. 34328, accession name XNCD-4, and suggested classification name is *Microbacterium*, and the DNA sequence of the strain is shown in SEQ ID NO. 2; wherein XNCD-7 has accession number CGMCC NO. 34329, accession name XNCD-7, and suggested classification name is *Klebsiella*, and the DNA sequence of the strain is shown in SEQ ID NO.

3.

2. The strain with straw degradation ability as described in claim 1, wherein the straw is chili straw.

3. The method for culturing the strain according to claim 1, wherein the method comprises the following steps: S1. The strain was inoculated into R2A liquid medium and cultured to obtain fermentation broth; S2. Centrifuge the fermentation broth obtained in step S1, discard the supernatant, and the precipitate is the bacterial block of this strain; S3. Store the mycelium blocks in glycerol.

4. The method of claim 3, wherein the strain is selected from one or more of XNCD-3, XNCD-4, and / or XNCD-7; the XNCD-3 has accession number CGMCC NO. 34327, accession name XNCD-3, and suggested classification name *Pseudomonas*, and the DNA sequence of the strain is shown in SEQ ID NO. 1; the XNCD-4 has accession number CGMCC NO. 34328, accession name XNCD-4, and suggested classification name *Microbacteria*, and the DNA sequence of the strain is shown in SEQ ID NO. 2; the XNCD-7 has accession number CGMCC NO. 34329, accession name XNCD-7, and suggested classification name *Klebsiella*, and the DNA sequence of the strain is shown in SEQ ID NO.

3.

5. The method as described in claim 3, wherein the straw is chili straw.

6. A microbial agent for degrading straw, said microbial agent containing the strain as described in claim 1.

7. The bacterial agent according to claim 6, wherein the bacterial strain is selected from one or more of XNCD-3, XNCD-4 and / or XNCD-7; wherein the XNCD-3 has accession number CGMCC NO. 34327, accession name XNCD-3, and suggested classification name is *Pseudomonas*, and the DNA sequence of the strain is shown in SEQ ID NO. 1; wherein the XNCD-4 has accession number CGMCC NO. 34328, accession name XNCD-4, and suggested classification name is *Microbacterium*, and the DNA sequence of the strain is shown in SEQ ID NO. 2; wherein the XNCD-7 has accession number CGMCC NO. 34329, accession name XNCD-7, and suggested classification name is *Klebsiella*, and the DNA sequence of the strain is shown in SEQ ID NO.

3.

8. The microbial agent as described in claim 6, wherein the straw is chili straw; the microbial agent comprises nutrients and auxiliary components; the nutrients comprise one or more of carbon sources, nitrogen sources, phosphorus sources, trace elements and / or water; the auxiliary components comprise one or more of surfactants, surface surfactants, pH adjusters, humectants, stabilizers, penetrants and / or antioxidants.

9. The application of the strain described in claim 1 in the preparation of a microbial agent for degrading straw, wherein the strain is able to reduce the cellulose content of straw.

10. The application as described in claim 9, wherein the strain is selected from one or more of XNCD-3, XNCD-4, and / or XNCD-7; the XNCD-3 has accession number CGMCC NO. 34327, accession name XNCD-3, and suggested classification name *Pseudomonas*, and the DNA sequence of the strain is shown in SEQ ID NO. 1; the XNCD-4 has accession number CGMCC NO. 34328, accession name XNCD-4, and suggested classification name *Microbacterium*, and the DNA sequence of the strain is shown in SEQ ID NO. 2; the XNCD-7 has accession number CGMCC NO. 34329, accession name XNCD-7, and suggested classification name *Klebsiella*, and the DNA sequence of the strain is shown in SEQ ID NO. 3; and the straw is chili straw.

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