Frateuria L17, bacterial inoculant and application

Abstract

The present application relates to a frangibacterium L17, bacterial agent and application, including frangibacterium L17, the preservation number is CCTCC M20221102;Frangibacterium L17 and bacterial agent in aniline blue decolorization, alkali lignin degradation and / or lignin degradation, wheat straw degradation application.Its advantages are that frangibacterium L17 has obvious decolorization effect on aniline blue, has strong oxidation capacity;Frangibacterium L17 of the present application can produce lignin-degrading enzyme, can effectively degrade lignin, and has good degradation effect on alkali lignin and lignin in wheat straw, can be used for the development and utilization of biomass resources, and has great application value.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, and in particular to a Francoid L17 bacterial agent and its application. Background Technology

[0002] Agricultural waste contains abundant lignocellulose resources. Bio-fermentation of lignocellulose can generate clean energy, thereby realizing the reuse of biomass resources.

[0003] However, lignin, as a physical barrier, with its inherent heterogeneity and stubbornness, hinders the effective refining and utilization of cellulose, severely reducing the utilization rate of cellulose.

[0004] Depolymerization and removal of lignin structure facilitates the effective release of cellulose and hemicellulose, which can improve energy production efficiency and alleviate the current situation of high dependence on fossil fuels. At the same time, after converting lignin macromolecules into aromatic compound monomers, their application in chemical, food, pharmaceutical engineering and other industrial production can improve the economic value of lignin and agricultural by-products.

[0005] Microbial degradation of lignin is an effective means of breaking down the physical barrier of lignin. This invention aims to propose a Francoid L17, a microbial agent, and its application. Francoid L17 and microbial agents containing Francoid L17 can effectively degrade lignin. Summary of the Invention

[0006] The purpose of this invention is to provide Francoidobacterium L17, its inoculum agent, and its application. The Francoidobacterium L17 and its inoculum agent of this invention can effectively degrade lignin.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a Francoid L17 strain, with accession number CCTCC M 20221102.

[0009] In some of these embodiments, the Francoid L17 contains a gene sequence as shown in sequence number (ID):1.

[0010] In some of these embodiments, the Francoid L17 produces laccase and lignin peroxidase.

[0011] In a second aspect, the present invention provides a microbial agent comprising Francoid L17 as described in the first aspect.

[0012] In some embodiments, the microbial agent also comprises fermentation broth or metabolites of Francoid bacteria L17 as described in the first aspect.

[0013] Thirdly, the present invention provides a method for preparing the microbial agent as described in the second aspect, comprising the following steps:

[0014] Francoid L17, as described in the first aspect, was inoculated into LB liquid medium and cultured until OD. 600 The concentration was 0.8–1.2, and the resulting activated culture medium contained Francoid bacteria L17.

[0015] In some of these embodiments, the inoculum amount of Francoid L17 was 0.8–1.2% of LB liquid medium, by concentration percentage.

[0016] The incubation temperature is 35–40℃;

[0017] The incubation rotation speed is 140–160 r / min.

[0018] In some of these embodiments, the inoculum size of Francoid L17 was 1% of LB liquid medium, by concentration percentage.

[0019] The incubation temperature is 37℃;

[0020] The incubation speed was 150 r / min.

[0021] Fourthly, the present invention provides the application of Francoid L17 as described in the first aspect or the bacterial agent as described in the second aspect in aniline blue decolorization.

[0022] Fifthly, the present invention provides the use of Francoid L17 as described in the first aspect or the bacterial agent as described in the second aspect in the degradation of alkali lignin and / or lignin.

[0023] In a sixth aspect, the present invention provides the application of Francoid L17 as described in the first aspect or the microbial agent as described in the second aspect in the degradation of wheat straw.

[0024] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0025] This invention relates to Francoidobacterium L17, its inoculum agent, and its applications. Francoidobacterium L17 exhibits a significant decolorization effect on aniline blue and possesses strong oxidizing ability. Furthermore, Francoidobacterium L17 can produce lignin-degrading enzymes, effectively degrading lignin, and showing good degradation effects on alkali lignin and lignin in wheat straw. It can be used for the development and utilization of biomass resources and has significant application value. Attached Figure Description

[0026] Figure 1 This is a microscopic image of the colony of Franconibacter sp. L17 according to an embodiment of the present invention;

[0027] Figure 2 This is a phylogenetic tree of Franconibacter sp. L17 according to an embodiment of the present invention;

[0028] Figure 3 This is a diagram illustrating the decolorization effect of Franconibacter sp. L17 on aniline blue according to an embodiment of the present invention.

[0029] [Preservation Information] Franconibacter sp. L17, with accession number CCTCC M20221102, deposited on July 13, 2022, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0031] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0033] Example 1

[0034] This embodiment relates to Franconibacter sp. L17 of the present invention and its application.

[0035] A Franconibacter sp. L17 strain, with accession number CCTCC M 20221102.

[0036] The aforementioned Franconibacter sp. L17 was deposited at the China Center for Type Culture Collection (CCTCC) on July 13, 2022. The address of the depository is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, 430072, China.

[0037] The Franconibacter sp. L17 with accession number CCTCC M 20221102 is a Gram-positive, short rod-shaped bacterium with a smooth, opaque, milky-white colony surface.

[0038] In some of these embodiments, Francoid L17 contains a gene sequence as shown in sequence number (ID):1.

[0039] In some of these embodiments, Francoid L17 produces laccase and lignin peroxidase.

[0040] An application of the above-mentioned Franconibacter sp. L17 in aniline blue decolorization.

[0041] Specifically, Franconibacter sp. L17 can secrete lignin-degrading enzymes, especially lignin peroxidase, which has a strong oxidizing ability and can oxidize aniline blue dye and cause it to fade.

[0042] An application of the above-mentioned Franconibacter sp. L17 in alkali lignin degradation and / or lignin degradation.

[0043] Franconibacter sp. L17 contains laccase and lignin peroxidase. According to literature reports, laccase and lignin peroxidase can catalyze the cleavage of phenolic hydroxyl groups or β-O-4 on the benzene ring in alkali lignin, thereby depolymerizing the network structure of alkali lignin.

[0044] Franconibacter sp. L17 produces lignin-degrading enzymes, the most important of which are laccase (Lac) and lignin peroxidase (LiP), which are used to biodegrade lignin.

[0045] An application of the aforementioned Franconibacter sp. L17 in the degradation of wheat straw.

[0046] Example 2

[0047] This embodiment relates to the bacterial agent of the present invention, the preparation method of the bacterial agent, and the application of the bacterial agent.

[0048] An agent containing Franconibacter sp. L17 as described in Example 1.

[0049] In some of these embodiments, the inoculum also includes fermentation broth or metabolites of Franconibacter p. L17, such as in Example 1.

[0050] A method for preparing the above-mentioned microbial agent includes the following steps:

[0051] Franconibacter sp. L17, as described in Example 1, was inoculated into LB liquid medium and cultured until OD. 600 The concentration was 0.8–1.2, and the resulting activated culture medium contained Franconibacter sp. L17.

[0052] In some of these embodiments, the inoculum of Franconibacter sp. L17 was 0.8–1.2% of LB liquid medium, by concentration percentage.

[0053] The incubation temperature is 35–40℃;

[0054] The incubation rotation speed is 140–160 r / min.

[0055] Furthermore, the inoculum size of Franconibacter sp. L17 was 1% of LB liquid medium, expressed as a percentage of concentration.

[0056] The incubation temperature is 37℃;

[0057] The incubation speed was 150 r / min.

[0058] Application of the above-mentioned bacterial agent in aniline blue decolorization.

[0059] Franconibacter sp. L17 produces laccase as the active ingredient in the bacterial agent. Laccase is a copper-containing polyphenol oxidase that breaks the polyphenol bonds of aniline blue, thereby decolorizing the aniline blue.

[0060] The application of the above-mentioned microbial agent in alkali lignin degradation and / or lignin degradation.

[0061] Franconibacter sp. L17, as the active ingredient in the inoculant, produces lignin-degrading enzymes. The most important of these enzymes are laccase (Lac) and lignin peroxidase (LiP), which biodegrade lignin by producing lignin-degrading enzymes.

[0062] Application of the above-mentioned microbial agent in the degradation of wheat straw.

[0063] Example 3

[0064] This embodiment is a modified embodiment of the preparation method of the bacterial agent in Example 2.

[0065] A method for preparing the bacterial agent of Example 2 includes the following steps:

[0066] Franconibacter sp. L17, as described in Example 1, was inoculated into LB liquid medium and cultured until OD. 600 The value was 1.0, and the resulting activated culture medium was an inoculum containing Franconibacter sp. L17.

[0067] The inoculum size of Franconibacter sp. L17 was 0.8% of LB liquid medium, expressed as a percentage of concentration.

[0068] The incubation temperature is 35℃;

[0069] The incubation speed was 140 r / min.

[0070] Example 4

[0071] This embodiment is a modified embodiment of the preparation method of the bacterial agent in Example 2.

[0072] A method for preparing the bacterial agent of Example 2 includes the following steps:

[0073] Franconibacter sp. L17, as described in Example 1, was inoculated into LB liquid medium and cultured until OD. 600 The value was 1.0, and the resulting activated culture medium was an inoculum containing Franconibacter sp. L17.

[0074] The inoculum size of Franconibacter sp. L17 was 1.2% of LB liquid medium, based on the concentration percentage.

[0075] The incubation temperature is 40℃;

[0076] The incubation speed was 160 r / min.

[0077] Example 5

[0078] This embodiment describes the isolation, screening, and identification of Francobacter sp. L17 of the present invention; verification of the decolorization effect of Francobacter sp. L17 on aniline blue; determination of the lignin-degrading enzyme activity of Francobacter sp. L17; verification of the degradation effect of Francobacter sp. L17 on alkali lignin; and verification of the degradation effect of Francobacter sp. L17 on wheat straw.

[0079] (I) Isolation, screening and identification of strain L17

[0080] 1. Isolation and screening of strain L17

[0081] (1) Cow dung and wheat straw were composted. 1g of the sample was added to 100mL of alkali lignin screening medium and cultured at 37℃ and 150r / min in a shaker for 7 days. The screening medium formula (in g / L) was: NaNO3, 2.5; MgSO4,

[0082] 1.0; NH4Cl, 1.0; NaCl, 1.0; KH2PO4, 1.0; CaCl2, 0.1; Alkali lignin, 1.0; Sterile water; The pH of the screening medium was 7.0-7.5.

[0083] (2) Take 10g of the bacterial suspension obtained in step (1) -1 -10 -7 After gradient dilution, the culture was spread on LB solid medium plates, and single colonies were selected for streak isolation to obtain strain L17 single colonies.

[0084] 2. Identification of strain L17

[0085] (1) Gram staining and microscopic examination of strain L17

[0086] like Figure 1 As shown, strain L17 is a Gram-positive bacterium, short rod-shaped, with a smooth, opaque, milky-white colony surface.

[0087] (2) 16S rDNA identification

[0088] Main culture medium: LB liquid medium formula (unit: g / L): NaCl, 10; tryptone, 10; yeast extract, 5; sterile water.

[0089] Specific experimental methods:

[0090] Add 1% of the frozen pure culture strain L17 to LB liquid medium and incubate in a shaker at 37°C and 150 rpm until OD. 600 The concentration was 0.8–1.2, and an activated culture medium for strain L17 was obtained.

[0091] Genomic DNA was extracted from strain L17 according to the instructions of the Tiangen Bacterial Genomic DNA Extraction Kit. PCR amplification of the DNA was performed using primers 27F and 1492R. PCR reaction mixture (in μL): sterile water, 1 g; Mix, 25 g; 27F, 2 g; 1492R, 2 g; DNA, 2 g; PCR conditions: 95℃, 3 min; 95℃, 30 s; 60℃, 30 s; 72℃, 90 s; 35 cycles; 72℃, 10 min.

[0092] The amplification products were detected by 1.5% agarose gel electrophoresis. The PCR products were sent to Sangon Biotech for 16S rDNA sequencing. The sequences were aligned to the NCBI database using BLAST, and a phylogenetic tree was constructed using MEGA 5.05 to classify and identify the species of strain L17.

[0093] Experimental results:

[0094] The 16S rDNA sequence of strain L17 obtained after sequencing is shown in the nucleotide and amino acid sequence listing in the instruction manual. Based on the sequencing results, strain L17 has a 98.68% similarity to *Franconibacter daqui* strain YBB12.

[0095] (3) Constructing a developmental tree

[0096] Phylogenetic trees were constructed using MEGA 5.05, as follows: Figure 2 As shown, based on the position of strain L17 in the phylogenetic tree, this bacterium was identified as belonging to the genus *Franconibacter*. The sequence of *Franconibacter sp.* L17 was uploaded to GenBank, obtaining accession number OK509193.

[0097] (II) Verification of the decolorization effect of Franconibacter sp. L17 on aniline blue

[0098] Table 1. Formulation of Aniline Blue Decolorizing Medium

[0099]

[0100] According to Table 1 above, prepare aniline blue medium. Add 60 μL of Franconibacter sp. L17 bacterial suspension to the wells of the aniline blue medium. The method for preparing the bacterial suspension is the same as the method for preparing the activation culture medium for the 16S rDNA identification strain L17 mentioned above. Incubate the aniline blue decolorizing medium in a 37°C biochemical incubator and observe and record the decolorization effect of Franconibacter sp. L17.

[0101] Franconibacter sp. L17, after growing on aniline blue plates for 12 hours, oxidizes aniline blue dye, causing it to fade, and a distinct decolorization zone appears around the wells. The results are as follows: Figure 3 As shown.

[0102] (III) Determination of lignin-degrading enzyme activity by Franconibacter sp. L17

[0103] Microbial biodegradation of lignin is mainly accomplished by the production of a series of lignin-degrading enzymes. The activity of these enzymes determines the strength of the microbial degradation ability of lignin and is an important indicator for measuring the lignin degradation ability of a strain. Among these enzymes, the most important are laccase (Lac) and lignin peroxidase (LiP).

[0104] Francoidacter sp. L17 was inoculated into LB liquid medium and cultured for 48 h. After centrifugation at 12000×g for 5 min, the supernatant of the Francoidacter sp. L17 culture broth was used as crude enzyme solution for enzyme activity assay. The preparation method of LB liquid medium was the same as that used in the 16S rDNA identification above.

[0105] Using an ultraviolet spectrophotometer, the results were obtained at 420 nm (ε) 420 =36000L·mol -1 ·cm -1 ), 310nm (ε 310 =9300 L·mol -1 ·cm -1 ), 468nm (ε 468 = 49600 L·mol -1 ·cm -1At a wavelength of [wavelength missing], using 2,2'-adiazono-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and resveratrol as substrates, the absorbance changes of the reaction system were monitored over 5 minutes, and the enzyme activities of two lignin-degrading enzymes, Lac and LiP, were calculated. Enzyme activity was defined as the amount of enzyme required to convert 1 μM of substrate into product per minute, which is one international enzyme activity unit (U). All experiments were repeated three times, and the results were averaged.

[0106] The LiP enzyme activity of Francobacter sp. L17 was higher than that of Lac, reaching 0.717 U / L, while that of Lac was (0.046±0.024) U / L. This indicates that in the presence of lignin stimulation, Francobacter sp. L17 secretes laccase and lignin peroxidase, with lignin peroxidase being the predominant activity.

[0107] (iv) Verification of the degradation effect of Franconibacter sp. L17 on alkali lignin

[0108] Franconibacter sp. L17 was inoculated into a culture medium with alkali lignin as the sole carbon source, and the degradation effect of Franconibacter sp. L17 on alkali lignin was determined.

[0109] Table 2 Alkali lignin degradation culture medium

[0110]

[0111] According to Table 2 above, the alkali lignin degradation medium was prepared. Franconibacter sp. L17 was activated in LB medium and then added to the alkali lignin degradation medium at a dosage of 10%. The culture was incubated in shake flasks at 37°C and 150 r / min in an air bath for 7 days. The culture solution was then centrifuged at 5000 rpm for 10 min to obtain the supernatant.

[0112] The absorbance of the supernatant at 280 nm was measured and recorded using a UV spectrophotometer. The alkali lignin content was determined after treatment with Franconibacter sp. L17 for 7 days, and the degradation ability of Franconibacter sp. L17 on alkali lignin was quantitatively determined.

[0113] The experiment found that, compared with the blank lignin degradation medium without inoculation, the lignin content in the lignin degradation medium treated with Francoidacter sp. L17 was reduced, and the lignin degradation rate was (22.06±0.66)%, indicating that Francoidacter sp. L17 can effectively degrade lignin.

[0114] (V) Verification of the degradation effect of Franconibacter sp. L17 on wheat straw

[0115] The formulation of the wheat straw degradation medium is as follows (unit: g / L): NaNO3, 2.5; MgSO4, 1.0; NH4Cl, 1.0; NaCl, 1.0; KH2PO4, 1.0; CaCl2, 0.1; wheat straw, 3.0.

[0116] Specific experimental methods:

[0117] Prepare the wheat straw degradation medium according to the above formula. Add 10% of the activated culture solution of Francobacter sp. L17 to the wheat straw degradation medium. Incubate in an air bath at 150 rpm and 37℃ for 7 days. After filtration, obtain the degraded wheat straw and dry it in a 105℃ oven until constant weight. The difference in mass of wheat straw before and after degradation by Francobacter sp. L17 is the weight loss rate.

[0118] The content of acid-soluble and acid-insoluble lignin in wheat straw was determined according to the method of the National Renewable Energy Laboratory (NREL) in the United States. 0.3 g of absolutely dried wheat straw was weighed and added to 3.00 mL of 72% concentrated sulfuric acid, and stirred until the raw materials were thoroughly mixed. The mixture was then placed in a water bath at 30°C and kept warm for 60 min, stirring every 10 min to ensure uniform mixing. After removal, 84 mL of deionized water was added for dilution and mixing, and the mixture was sterilized at 121°C for 1 h. The residue and filtrate were obtained by vacuum filtration using a G3 sintered glass funnel. The absorbance of the filtrate was measured at 250 nm using a UV spectrophotometer, and the content of acid-soluble lignin in the wheat straw after degradation by Franconibacter sp. L17 was recorded. After rinsing the filtered residue with hot deionized water, the residue was dried in an oven at 105°C to constant weight to calculate the acid-insoluble lignin content of wheat straw after degradation by Franconibacter sp. L17. Wheat straw not inoculated with Franconibacter sp. L17 was used as a control group.

[0119] Table 3. Effects on wheat straw degradation

[0120]

[0121] The degradation results of wheat straw by *Franconibacter* sp. L17 are shown in Table 3. During the degradation process, bacteria damage the cell walls of wheat straw, leading to the loss of internal proteins and waxes, resulting in weight loss. Therefore, the weight loss rate of wheat straw can be used as an indicator of the degradation effect of *Franconibacter* sp. L17 on wheat straw. The weight loss rate of untreated wheat straw after 7 days of culture was (9.43±0.43)%. This may be because the structure of the crushed wheat straw was damaged after high-pressure steam sterilization, and some proteins and sugars dissolved in the culture medium during the 7-day shake-flask culture, causing weight loss. The weight loss rate of wheat straw treated with *Franconibacter* sp. L17 for 7 days was (22.23±0.26)%, an increase of 12.8% compared to the control group. Meanwhile, L17 also showed significant degradation effects on acid-soluble and acid-insoluble lignin in wheat straw, with degradation rates of (14.69±3.91)% and (5.33±2.78)%, respectively. This indicates that Franconibacter sp. L17 can effectively degrade wheat straw.

[0122] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Francoid bacteria ( Franconibacter sp.)L17, characterized in that, Its accession number is CCTCC:M20221102.

2. A microbial agent, characterized in that, The bacterial agent comprises Francoid L17 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The inoculum also includes the fermentation broth of Francoid L17 as described in claim 1.

4. A method for preparing the microbial agent as described in claim 2 or 3, characterized in that, Includes the following steps: Francoid L17 as described in claim 1 was inoculated into LB liquid medium and cultured until OD. 600 The concentration was 0.8~1.2, and the resulting activated culture medium was a bacterial agent containing Francoid bacteria L17.

5. The method according to claim 4, characterized in that, The inoculum size for Francoid bacteria L17, expressed as a percentage of concentration, is 0.8–1.2% of LB liquid medium. The incubation temperature is 35~40℃; The incubation rotation speed is 140~160 r / min.

6. The use of Francoid L17 as described in claim 1 or the bacterial agent as described in claim 2 or 3 in aniline blue decolorization.

7. The use of Francoid L17 as described in claim 1 or the inoculum as described in claim 2 or 3 in the degradation of alkali lignin and / or lignin.

8. The application of Francoid L17 as described in claim 1 or the inoculant as described in claim 2 or 3 in the degradation of wheat straw.

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