A type of Klebsiella variegata and its applications

Abstract

This invention belongs to the field of environmental microbiology, specifically a type of Klebsiella variicola and its applications. Klebsiella variicola, strain IFPBD3, is deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 25, 2023, with accession number CGMCC No. 27461. The rice straw degrading bacteria produced by the technical solution of this invention can effectively degrade rice straw, significantly increase straw fertility, and improve straw utilization rate.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology, specifically a type of Klebsiella variegata and its applications. Background Technology

[0002] my country is a major agricultural country with a rich variety of crops and huge yields. The production of straw has also increased accordingly, exceeding 900 million tons in 2020 and continuing to rise. Corn straw, rice straw, and wheat straw rank among the top three. However, the irrational disposal of straw resources in my country has caused serious resource waste and environmental pollution. Straw is an agricultural waste rich in lignocellulose produced during agricultural production. It retains various inorganic elements such as nitrogen, phosphorus, potassium, magnesium, calcium, and sulfur absorbed from the environment during crop growth, as well as assimilated carbon. It is an important renewable resource with extremely high resource value and excellent application prospects due to its huge yield. The ever-increasing straw production and the irrational utilization of straw resources in my country urgently require the exploration of more efficient and cleaner resource utilization methods.

[0003] Currently, the comprehensive utilization of straw can be summarized into five aspects: "fertilizer, feed, fuel, substrate, and raw material" (Huo Lili et al., 2022). Among them, fertilization is the most important method. In 2021, the amount of rice straw returned to the field was 113 million tons, accounting for 66.5% of the collectable rice straw. The application of straw as fertilizer generally involves crop harvesting, crushing, and decomposition. The operation is simple and easy to implement, but the efficiency of straw return to the field is reduced to some extent due to factors such as low level of crushing equipment, high purchase cost, and great influence from terrain. At the same time, straw return to the field cannot effectively eliminate pathogenic microorganisms, insect eggs, and other factors that harm crop growth, and the natural decomposition cycle is long, which may sometimes affect the sowing of the next crop and affect production and living efficiency. Therefore, it is necessary to further strengthen technological innovation and use various methods to improve the efficiency of straw application (Wang Hongmei et al., 2017).

[0004] Cellulose, hemicellulose, and lignin are the main components of straw. Cellulose accounts for 35%-50% of straw, making it a crucial component. Hemicellulose is also an important component, typically comprising 14%-32% of the total straw weight. Lignin, a complex phenolic polymer, usually accounts for 7%-25% of straw, and its molecules are linked by multiple covalent bonds, making it difficult to degrade. Enzymes capable of degrading lignin mainly include laccase, lignin peroxidase, and multifunctional peroxidases (Ambatkar et al., 2021).

[0005] Chemical treatment of straw can destroy its fiber structure and effectively improve its comprehensive utilization efficiency. However, the addition of acids and alkalis during the treatment process can cause the pH value of the straw to be too low or too high. Before comprehensive utilization, a large amount of reagents is needed to further adjust the pH to a suitable value. Furthermore, excessive acidity or alkali can corrode equipment and easily cause secondary environmental pollution. Biological treatment methods mainly use enzymes or microorganisms to degrade straw. Various lignocellulases act on the covalent and secondary bonds between components, destroying the tightly bound layer of lignocellulose, thereby improving degradation efficiency. This method is energy-efficient and produces no secondary pollution. Studies have shown that research on the microbial degradation of straw has been increasing year by year since the 21st century (Zhu Dan et al., 2020). However, current biological degradation methods have the drawback of being time-consuming, and there is an urgent need to explore and develop new biological degradation methods to improve degradation efficiency. Summary of the Invention

[0006] The purpose of this invention is to provide a Klebsiella variegata bacterium and its applications.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A strain of Klebsiella variicola, strain IFPBD3, is deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 25, 2023, with accession number CGMCC No. 27461.

[0009] An application of the aforementioned Klebsiella variegata in the degradation of rice straw.

[0010] The application of the described Klebsiella variegata in degrading rice straw under natural conditions.

[0011] A degrading microbial agent containing the aforementioned Klebsiella variicola.

[0012] Specifically, activated colonies were inoculated into LB liquid medium and cultured with shaking at 28°C. Based on the growth curve of the bacteria, and taking into account both high biomass and high division rate, the experimental inoculum was selected after 3 hours of inoculation.

[0013] An application of the aforementioned degrading microbial agent, wherein the microbial agent is used in rice straw.

[0014] Specifically: Add Mandels nutrient solution to straw and sterilize it at high temperature, then add the bacterial agent and cultivate it at 28-30℃ to achieve straw degradation.

[0015] The Mandels nutrient solution contained 2 g / L glucose, 8 g / L urea, 3 g / L (NH4)2SO4, 1 g KH2PO4, 0.3 g / L CaCl2, 0.3 g / L MgSO4·7H2O, 0.5 mg / L FeSO4·7H2O, 1.6 mg / L MnSO4·H2O, 1.4 mg / L ZnSO4·7H2O, and 2 mg / L CoCl2.

[0016] The mass-to-volume ratio (g / ml) of the straw to the inoculant is 3:1; the mass-to-volume ratio (g / ml) of the Mandels nutrient solution to the straw is 2:1.

[0017] A compound microbial agent, wherein the compound microbial agent is obtained by combining the strain with Schizophyllum commune; wherein the strain and Schizophyllum commune are added separately in a 1:1 (v / v) ratio.

[0018] The compound microbial agent is used in rice straw.

[0019] The advantages of this invention are:

[0020] The present invention relates to a highly viable strain of *Klebsiella variegata* obtained through large-scale isolation and screening from rice straw in wild farmland, which has the following advantages:

[0021] 1. Its cultivation method is simple, its growth rate is fast, and it is not prone to mutation;

[0022] 2. It has a strong ability to degrade rice straw. The strain produces a variety of enzymes during its growth process that can efficiently degrade rice straw. It has a high lignin degradation rate. After 42 days of degradation, the total weight of the straw was reduced by 14.23%, of which the lignin degradation rate was as high as 44.87%, and the hemicellulose and cellulose degradation rates were 50.94% and 16.60%, respectively.

[0023] 3. The rice straw substrate eluent after degradation by this bacterium showed an increase of 19.89% in soluble phosphorus and 55.47% in soluble sugar compared to the control, thus increasing the fertility of the rice straw.

[0024] 4. This bacterium has good affinity and no antagonistic effect with a variety of fungi, so it can be used to prepare compound bacterial agents.

[0025] This invention can improve the degradation efficiency of rice straw and the fertility of the biodegraded straw, thus protecting the ecological environment. Attached Figure Description

[0026] Figure 1The growth curves of strain IFPBD3 at different time periods provided in the embodiments of the present invention. Detailed Implementation

[0027] The present application will be further explained below with reference to the embodiments.

[0028] The bacteria of this invention can be propagated in solid or liquid culture, and then inoculated into sterilized rice straw for growth and degradation. It can efficiently degrade lignin in rice straw, with a lignin degradation rate as high as 44.87%, destroying the main structure of rice straw. The soluble phosphorus in the rice straw matrix eluent increased by 19.89% and the soluble sugar increased by 55.47% compared with the control, thus improving the fertility of rice straw.

[0029] Example 1

[0030] Isolation and identification of Klebsiella variicola:

[0031] 1. Isolation of the bacterial strain: The strain was isolated from rice straw piled up in the experimental field of Shenyang Agricultural Station, Chinese Academy of Sciences. 5-10g of severely decomposed rice straw was taken, rinsed with running water for 1-2 hours, then soaked in 75% (v / v) ethanol for 60 seconds, and treated with 2% (v / v) sodium hypochlorite for 15-20 minutes for surface sterilization. The straw was then rinsed four times with sterile water. Using the spread plating method, the final sterile water wash solution was spread onto LB agar and incubated at 30℃ for 1-2 days. Sterilization was considered successful when sterile growth was observed on LB agar. The completely sterilized material was placed in a sterilized mortar, chopped with sterile scissors, and then ground with an appropriate amount of PBS buffer. 0.1mL of the liquid was spread onto LB agar plates and incubated upside down at 28-30℃ for 2-3 days, observing colony growth. Colonies of different morphologies were picked out, purified, and cultured separately, then stored on new culture media.

[0032] 2. Identification of the strain:

[0033] 2.1 Morphological identification

[0034] The colonies are relatively large, pale yellow, raised, and semi-liquid, with regular edges and a smooth surface. Under a microscope, the BD3 strain is Gram-negative, relatively thick, short rod-shaped, measuring (0.5-0.8) μm × (1-2) μm, arranged singly, in pairs, or in short chains; it has no spores, but possesses pili and a capsule, the capsule being relatively thick but lacking flagella and non-motile.

[0035] 2.2 Homology Analysis

[0036] The 16S ribosomal RNA sequence of this strain is as follows:

[0037] CTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGCACAG

[0038] AGAGCTTGCTTCCGGGTGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTG

[0039] CCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAA

[0040] GACCAAAGTGGGGGACCTTCGGGCCTCATGCCATCAGATGTGCCCAGATGGGATT

[0041] AGCTGGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGGAGA

[0042] GGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAG

[0043] CAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGT

[0044] GAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGTGAGGT

[0045] TAATAACCTCATCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATA

[0046] Comparison with the NCBI database revealed that strain BD3 is most closely related to *Klebsiella variicola*, with a similarity exceeding 99%. Based on the morphological characteristics, physiological and biochemical features, and l6S rDNA sequence analysis results, strain BD3 was identified as *Klebsiella variicola*, specifically strain IFPBD3.

[0047] 3. Antagonism test of strains:

[0048] Klebsiella variegata has excellent colony inclusiveness and can be compounded with 16 fungi and 3 bacteria without producing antagonistic effects (Table 1, Table 2), making it one of the preferred strains for preparing compound bacterial agents.

[0049] Table 1. Results of antagonistic experiments between Klebsiella variegata and 16 fungi and 3 bacteria.

[0050]

[0051] Note: + indicates that the two bacteria can fuse and grow together without antagonism.

[0052] Table 2. Names and abbreviations of fungi and bacteria

[0053]

[0054]

[0055] The final purified strain obtained above is deposited at the China General Microbiological Culture Collection Center (CGMCC) (strain IFPBD3, located at Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), date of deposit: May 25, 2023, accession number: CGMCC No. 27461.

[0056] Example 2: Obtaining the bacterial agent

[0057] 1. Culture medium preparation:

[0058] (1) Solid LB medium: 3g beef extract, 10g peptone, 5g NaCl, 20g agar powder, 1000mL distilled water, natural pH value, sterilized at 121℃ for 30min and then cooled for later use.

[0059] (2) Liquid LB medium: 3g beef extract, 10g peptone, 5g NaCl, 1000mL distilled water, natural pH, sterilized at 121℃ for 30min and then cooled for later use.

[0060] All the above culture media were sterilized in an autoclave at 121°C for 20-30 minutes.

[0061] 2. Preparation of microbial agents:

[0062] Strain activation: Select the Klebsiella variegata strain described in the above examples and inoculate it onto a solid LB medium plate for activation. Incubate in a 28°C incubator in the dark until the colonies grow and are ready for use.

[0063] Preparation of bacterial inoculum: Two loopfuls of colonies were inoculated from the activated bacterial culture plate and added to 100 mL of LB liquid medium. The culture was incubated in a shaker at 140 rpm / min and 28 °C. Samples were taken every 2 hours (for a total of 24 hours) to measure the OD600 value of the bacterial strain to characterize its growth. Appropriate dilutions were performed to maintain an OD600 value less than 1.0. A growth curve of the bacterial strain was plotted with growth time on the x-axis and OD600 on the y-axis. Figure 1 Taking into account both the high biomass and high division rate of the bacterial strain, the experimental inoculum was selected after 3 hours of inoculation.

[0064] Example 3: Degradation of Straw

[0065] 1) Preparation of mushroom bags

[0066] Solid-state fermentation medium: The above-obtained inoculum and about 15g of dried rice straw segments with a length of 1-2cm were placed in a 12*27 bag. Mandels nutrient solution was added, and the bag was inverted several times until the nutrient solution completely wetted the straw sample (the amount of Mandels nutrient solution added should be enough to completely wet the straw; in this example, it is approximately 30mL). The bag was tied with a rubber band and sterilized at 121℃ for 30 minutes, then cooled for later use.

[0067] Mandels nutrient solution: glucose 2g / L, urea 8g / L, (NH4)2SO4 3g / L, KH2PO4 1g, CaCl2 0.3g / L, MgSO4·7H2O 0.3g / L, FeSO4·7H2O 0.5mg / L, MnSO4·H2O 1.6mg / L, ZnSO4·7H2O 1.4mg / L, CoCl2 2mg / L.

[0068] Add 5 ml of the culture collected in the above-mentioned preparation of the inoculum to the above-mentioned inoculum bag.

[0069] Meanwhile, straw mushroom bags with added Mandels nutrient solution and sterilized (without added inoculant) were used as a control.

[0070] 2) The three major components in straw were determined using the modified paradigm method.

[0071] The three major components of the rice straw matrix after 42 days of degradation were determined according to the method of Van Soest (1991), with modifications made by using cellulose filter bags during pretreatment.

[0072] 3) Determination of total nitrogen in straw

[0073] The total nitrogen content of the sample was determined using an elemental analyzer.

[0074] 4) Methods for determining nutrient elements in straw sample extracts

[0075] (1) Preparation of extract

[0076] Distilled water was added to the straw sample tube after degradation and drying at a ratio of 1:20 (sample mass g: distilled water volume mL). The tube was then fixed on a shaker and extracted at 160 rpm / min for 30 min. After filtration through four layers of gauze, the sample was centrifuged at 4200 rpm / min for 10 min and the supernatant was collected to obtain the extract to be tested.

[0077] (2) Determination of total phosphorus content in extract

[0078] The method for determining the total phosphorus content in the extract is based on the method for determining total phosphorus in water quality - ammonium molybdate spectrophotometry (GB11893-89). In this study, the potassium persulfate digestion method in the standard was selected for the digestion of the eluent.

[0079] (3) Determination of soluble sugar content in extract

[0080] The soluble sugar content was determined by the phenol-sulfuric acid method, which hydrolyzes polysaccharides into monosaccharides under the action of strong acid, and then dehydrates them to form a series of sugar aldehyde derivatives. These derivatives then react with phenol to produce a colored substance (orange-yellow) (Song Qi et al., 2022). The polysaccharide content was determined by photometric measurement.

[0081] 5) Research Results

[0082] After 42 days of degradation, the contents of important components in rice straw and the contents of soluble phosphorus and soluble sugars in the residue extract are shown in Table 3. The lignin degradation rate reached 44.87%, the total nitrogen content increased from 0.94% to 1.03%, the solubility of soluble phosphorus increased by 19.89%, and the release rate of soluble sugars increased by 54.49%, which greatly improved the fertility of rice straw.

[0083] Table 3. Nutrient content of residues and eluents from rice straw degradation by Klebsiella variegata.

[0084]

[0085] Example 4

[0086] Degradation effect of compound bacterial agents

[0087] The *Klebsiella variegata* obtained in the above examples was combined with the fungus *Schizophyllum commune* (which was commercially available and preserved in our laboratory) to prepare a compound inoculum for the degradation of rice straw. Specifically, 30 ml of Mandels nutrient solution was added to each 15 g bag of dry rice straw powder and sterilized at high temperature. Then, 2.5 ml of the *Klebsiella variegata* inoculum prepared in the above examples and 2.5 ml of the *Schizophyllum commune* inoculum (prepared according to the inoculum preparation method in Example 2, the difference being that *Schizophyllum commune* is a fungus and requires a longer cultivation time. In this example, the cultivation conditions were 28℃, 140 r / min constant temperature shaker for 7 days, and the mycelial balls were broken up with a sterilized homogenizer to prepare a uniform mycelial suspension, which is the fungal seed liquid) and placed in an incubator at 28-30℃ for 60 days.

[0088] The results showed that the degradation rate of straw lignin by the compound microbial agent, as well as the content of PK element and soluble sugar in the eluent of straw residue after degradation, were significantly higher than those of single microorganisms.

[0089] Table 4. Nutrient content of residues and eluent after degradation of rice straw by the compound microbial strain.

[0090]

Claims

1. A type of Klebsiella variegata, characterized in that: Klebsiella variegata ( Klebsiella variicola The strain IFPBD3 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 25, 2023, with accession number CGMCC No. 27461.

2. An application of Klebsiella variegata as described in claim 1, characterized in that: The application of Klebsiella variegata in the degradation of rice straw.

3. The application of Klebsiella variegata according to claim 2, characterized in that: The application of the described Klebsiella variegata in degrading rice straw under natural conditions.

4. A biodegrading agent, characterized in that: The bacterial agent contains the *Klebsiella variegata* as described in claim 1. Klebsiella variicola ).

5. The application of the degrading microbial agent according to claim 4, characterized in that: The application of the microbial agent in the degradation of rice straw; Mandels nutrient solution was added to the straw and sterilized at high temperature. Then, the bacterial agent was added and the straw was cultured at 28-30℃ to achieve straw degradation.

6. The application of the degrading microbial agent according to claim 5, characterized in that: The mass-to-volume ratio of straw to inoculant is 3:1; the mass-to-volume ratio of Mandels nutrient solution to straw is 2:

1.

7. A compound microbial agent, characterized in that: The compound microbial agent is the strain described in claim 1 and Schizophyllum commune (… Schizophyllum commune The strain described in claim 1 is obtained by combining with *Schizophyllum commune* (…). Schizophyllum commune Add them separately in a 1:1 ratio.

8. The application of the compound microbial agent according to claim 7, characterized in that: The application of the compound microbial agent in the degradation of rice straw.

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