Application of novel bacillus velezensis Y-6 in efficient degradation of lignin and biochemical treatment of fulvic acid

Abstract

The invention discloses application of novel bacillus velezensis Y-6 in efficient degradation of lignin and biochemical treatment of fulvic acid, and belongs to the technical field of microorganisms. The strain is preserved in the Guangdong Microbial Culture Collection Center on September 26, 2024, the preservation address is the 5th floor of the building 59, No.100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCCNO: 65214. The bacillus velezensis provided by the invention shows the activity of three lignin degrading enzymes, and can efficiently degrade lignin under the conditions of short time and low energy consumption. Meanwhile, lignin is used as a substrate, and the bacillus velezensis can degrade the lignin to generate fulvic acid in a fermentation manner.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to application of a novel strain of Bacillus velez Y-6 in efficient degradation of lignin and biochemical fulvic acid. Background Art

[0002] Cellulose has abundant reserves and is renewable. The total amount of crop straw alone exceeds 2.2 billion tons worldwide. As the main component of most terrestrial plants, cellulose accounts for more than 90% of dry plants. At the same time, cellulose, as an important renewable carbon source, usually comes from agricultural and industrial waste such as straw and wood, and has a huge reserve in nature. Among them, lignin content is second only to cellulose, and it is the type with the highest energy content among the three types of biomass. Lignin mainly plays a supporting role in plants, and also forms a physical barrier that makes plant cell walls difficult to degrade. The existing lignin high-value technologies include pyrolysis, hydrogenolysis / hydrocracking liquefaction, and alkali / acid catalysis. The reaction conditions are relatively harsh, and there are many disadvantages such as disordered depolymerization, complex cracking products, and difficult to control directional conversion.

[0003] The main reason why lignin is difficult to develop and utilize is closely related to its own chemical properties. First of all, lignin is a complex phenolic polymer, mainly composed of p-coumarol (H), coniferyl alcohol (G) and sinapyl alcohol (S). These three phenylpropanol units form a complex three-dimensional network structure through different connection methods. In different plants, the content and distribution of the above three phenylpropanol units are different. For example, in softwood lignin such as oak, the main phenylpropanol unit is S, accounting for more than 90%; in hardwood lignin such as birch, the content of S and G units each accounts for about 50%; and lignin of herbs such as straw contains all three phenylpropanol units. Due to the great differences in the chemical structures of various types of wood lignin, it is difficult to find a highly universal degradation and transformation plan. Secondly, the units between lignin are mainly connected by CO bonds and CC bonds, which are mainly divided into β-O-4, α-O-4, 4-O-5, 5-5, β-5 and β-β. Among them, β-O-4 bonds are the dominant chemical bonds in lignin bonds, accounting for more than half. At the same time, as a treasure trove of aromatic resources, lignin's degradation products can meet the pharmaceutical industry's demand for aromatic substances, and it is a breakthrough to get rid of dependence on non-renewable resources such as coal and oil. Summary of the invention

[0004] The purpose of the present invention is to provide a novel strain of Bacillus Velezii Y-6 for use in efficient lignin degradation and biochemical fulvic acid, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a strain of Bacillus velezensis Y-6, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 26, 2024. The storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the storage number is GDMCC No: 65214.

[0007] The second technical solution of the present invention is a microbial agent, including the Bacillus Velezii Y-6.

[0008] The third technical solution of the present invention is the use of the Bacillus Velez Y-6 or the microbial agent in degrading lignin.

[0009] A fourth technical solution of the present invention is the use of the Bacillus Velez Y-6 or the microbial agent in the production of fulvic acid.

[0010] A fifth technical solution of the present invention is a method for degrading lignin, using the Bacillus Velez Y-6 or the microbial agent to ferment and degrade lignin.

[0011] The sixth technical solution of the present invention is a method for producing fulvic acid, which uses the Bacillus Velez Y-6 or the microbial agent to ferment and degrade lignin to produce fulvic acid.

[0012] The seventh technical solution of the present invention is a product for producing fulvic acid by degrading lignin, comprising the Bacillus Velez Y-6 or the microbial agent.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] The Bacillus Velez provided by the present invention exhibits three lignin degrading enzyme activities, and can efficiently degrade lignin in a short time and with low energy consumption. At the same time, with lignin as a substrate, the Bacillus Velez can degrade lignin to produce fulvic acid by fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is the colony morphology of Y-6.

[0017] Figure 2 It is the morphological characteristics of the colonies under a microscope after Gram staining.

[0018] Figure 3 is the standard concentration curve of lignin.

[0019] Figure 4 The effect of temperature on the lignin degradation rate of strain Y-6.

[0020] Figure 5 The effect of initial pH on the lignin degradation rate of strain Y-6.

[0021] Figure 6 The effect of inoculation amount on the lignin degradation rate of strain Y-6.

[0022] Figure 7 The effect of different carbon sources on the lignin degradation rate of strain Y-6.

[0023] Figure 8 The effect of different carbon-nitrogen ratios on the lignin degradation rate of strain Y-6.

[0024] Fig. 9 is the degradation rate of lignin in compost by strain Y-6.

[0025] Fig.10 is the degradation rate of lignin residue by strain Y-6.

[0026] Fig.11 is the fulvic acid content in the lignin residue.

[0027] Fig.12 It is the fulvic acid content in biogas residue. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0034] An embodiment of the present invention provides a strain of Bacillus velezensis Y-6, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 26, 2024, with the deposit address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number being GDMCC No: 65214.

[0035] The embodiment of the present invention also provides a microbial agent, including the Bacillus Velezii Y-6.

[0036] The embodiment of the present invention also provides the use of the Bacillus Velez Y-6 or the microbial agent in degrading lignin.

[0037] The embodiment of the present invention also provides the use of the Bacillus Velez subtilis Y-6 or the microbial agent in the production of fulvic acid.

[0038] The embodiment of the present invention also provides a method for degrading lignin, using the Bacillus Velez Y-6 or the microbial agent to ferment and degrade lignin.

[0039] In some specific embodiments, the fermentation degradation conditions include: a degradation temperature of 37°C, an initial pH of 7, an inoculation amount of 3% to 16% (v:v), peptone as a nitrogen source in the degradation system, and a carbon-nitrogen ratio of 10:1.

[0040] The embodiment of the present invention also provides a method for producing fulvic acid, using the Bacillus Velez Y-6 or the microbial agent to ferment and degrade lignin to produce fulvic acid.

[0041] In some specific embodiments, the fermentation degradation conditions include: degradation temperature of 37°C, initial pH of 7, inoculation amount of 3% to 16% (v:v), peptone as nitrogen source in the degradation system, carbon-nitrogen ratio of 10:1, and degradation time of 1 to 10 days.

[0042] The embodiment of the present invention also provides a product for producing fulvic acid by degrading lignin, comprising the Bacillus Velez Y-6 or the microbial agent.

[0043] The present invention uses a two-round strain selection method of lignin enrichment culture, primary screening, and rescreening to screen a strain that efficiently degrades lignin. After 18s DNA comparison analysis, sequencing results show that strain Y-6 is highly similar to Bacillus velezensis (NR075005.2). Combined with morphological and sequence analysis, Y-6 was identified as Bacillus velezensis.

[0044] The present invention further provides a method for culturing Bacillus Velezii Y-6, which comprises culturing the culture medium at 28-37° C. in a solid plate using lignin as the sole carbon source, or culturing the culture medium in a liquid medium containing lignin.

[0045] Example 1

[0046] 1. Screening of strains

[0047] Culture medium:

[0048] Primary screening medium (g / L): lignin 5.0000 g, KH2PO4 1.0000 g, K2HPO4 1.0000 g, MgSO4 0.2000 g, FeSO4 0.0200 g, (NH4)SO4 2.0000 g, MnSO4 0.0500 g, CaCl2 0.0200 g, distilled water 1.0 L, pH natural;

[0049] LB solid medium: peptone 10.0000 g, yeast extract 5.0000 g, sodium chloride 10.0000 g, agar 18.0000 g, distilled water 1.0 L, natural pH;

[0050] LB liquid medium: peptone 10.0000 g, yeast extract 5.0000 g, sodium chloride 10.0000 g, distilled water 1.0 L, pH natural.

[0051] 1.1 Preliminary screening

[0052] The biogas residue samples from Fuyang were placed in sterilized test tubes, 100.0 mL of sterilized deionized water was added, and the samples were placed in a 37°C constant temperature incubator and shaken at 160 rpm for 12 hours. After sufficient shaking, 200 μL of the sample was pipetted and applied to the primary screening culture medium, which was placed in an inverted culture medium at 37°C for 1 day. About 10 strains that could grow on the plate were marked as Y1-Y10, and were preliminarily identified as strains that could degrade lignin. These primary screening strains were separated and purified.

[0053] 1.2 Rescreening

[0054] The Y-5, Y-6, and Y-7 strains obtained in the initial screening were inoculated into the rescreening medium (LB liquid medium), cultured at 37°C and an initial pH of 7 for 7 days, the lignin degradation rate was determined, and the strain with the highest degradation rate was screened out. The strains were inoculated into the application medium (inorganic salt medium: 2.0 g ammonium sulfate, 1.0 g dipotassium hydrogen phosphate, 1.0 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, 0.1 g calcium chloride, 0.05 g ferrous sulfate, 0.02 g manganese sulfate, 1 L distilled water. Sterilized in an autoclave for 20 min, and then 5 g lignin was added.), cultured at 37°C and an initial pH of 7 for 7 days, the absorbance was measured by fluorescence photometry to calculate the lignin degradation rate, and the humic acid content produced after lignin degradation was measured by titration, and the humic acid content was calculated as the lignin degradation rate.

[0055] The three strains screened initially were inoculated in the secondary screening medium, and all had good degradation effects on lignin. Among them, Y-6 had the most obvious effect, with a lignin degradation rate of 35.39%, and the degradation rate reached 30.56% on the 5th day, proving that this strain had the strongest lignin degradation ability among the tested strains and could be used as a subsequent test strain.

[0056] 1.3 Single Factor Experiment

[0057] Under the conditions of initial medium pH 7 and 20.0mL bacterial inoculation volume, the temperature was set at 28, 37, 46, 55, and 60 (unit: ℃), and each treatment was repeated 3 times. The culture was left to stand for 7 days and the lignin degradation rate was detected. The results showed that the degradation rate of lignin was between 25% and 35%, especially at 37℃, the maximum degradation rate was reached ( Figure 4 ).

[0058] Under the conditions of 37℃ culture temperature and 20.0mL inoculum, the pH values ​​were set to 3, 5, 7, 9, and 11, and each treatment was repeated 3 times, and the subsequent treatments were the same as above. The results showed that Y-6 could grow well under the conditions of pH values ​​of 3.0 to 11.0, and had a certain degradation effect on lignin; the degradation rate of lignin was the highest at pH 7.0, reaching 34.81% ( Figure 5 ).

[0059] At 37°C, the pH of the culture medium was 7 and the volume of the culture medium was 127.17 cm 3 The inoculation volume in the culture medium was set to 4.0mL, 8.0mL, 12.0mL, 16.0mL, and 20.0mL. Each treatment was repeated 3 times, and the subsequent treatments were the same as above. The results showed that the degradation rate of lignin was the highest at 20mL, reaching 34.43%. At the inoculation ratio of 20.0mL, the degradation rate of lignin was the highest ( Figure 6 ).

[0060] Under the conditions of initial medium pH 7 and 20.0mL bacterial inoculation volume, different carbon sources were set as peptone, ammonium nitrate, ammonium sulfate, and yeast extract. Each treatment was repeated 3 times, and the subsequent treatments were the same as above. The results showed that when peptone was used as the nitrogen source, its degradation ability was the greatest, and the maximum degradation rate of Y-6 reached 30.69% ( Figure 7 ).

[0061] Under the conditions of initial medium pH 7 and 20.0mL bacterial inoculation volume, different carbon-nitrogen ratios were set at 30:1, 20:1, 10:1, 1:10, and 1:20, and each treatment was repeated 3 times. The results showed that the degradation rate of lignin was the highest at 10:1, reaching 35.39%. At a carbon-nitrogen ratio of 10:1, the degradation rate of lignin was the highest ( Figure 8 ).

[0062] 2 Identification of strains

[0063] 2.1 Morphological observation and physiological and biochemical identification

[0064] Smear: First, take a clean glass slide and drop a drop of distilled water in the center of the slide. Next, carefully pick a small portion of the colony with an inoculation loop in the sterile area of ​​the alcohol lamp and mix it with the water drop. Then, place the smear in a safe place and let it dry naturally.

[0065] Fixation: With the smear facing up, pass it through a flame three times to fix the colonies on the smear.

[0066] Primary staining: Place a drop of crystal violet stain on the smear, wait for about one minute, and then gently rinse off the excess stain with water.

[0067] Mordanting: Add iodine solution to the coated area, wait for one minute, and then gently rinse with water.

[0068] Decolorization: Add 95% ethanol to the smear and gently shake the slide until the purple color will not fade further. After decolorization is completed, wash with water immediately.

[0069] Safranin counterstaining: Add safranin stain to the smear area for counterstaining, and the staining time is one minute.

[0070] Microscopic examination: oil immersion microscopy.

[0071] The results are shown in Table 2.

[0072] Table 2 Morphological observation and physiological and biochemical identification of strains

[0073]

[0074] 2.2 Molecular identification

[0075] The strain was sent to Beijing Ruibo Biotechnology Co., Ltd. for sequencing. The BLASTN software and EZbiocloud website were used to compare the partial sequence homology of the strain 16SrDNA. The MEGA5.0 software was used to establish a molecular evolutionary tree and analyze the relationship of the isolated bacteria. Strain Y-6 was highly similar to Bacillus velezensis (NR075005.2). Combining morphological and sequence analysis, Y-6 was identified as Bacillus velezensis.

[0076] The Bacillus velezensis Y-6 provided by the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 26, 2024, with the deposit address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number being GDMCC No: 65214.

[0077] Example 2

[0078] 1 The extracted bacteria are used to degrade lignin residue to produce fulvic acid

[0079] 1.1 Production of fulvic acid from biogas residue

[0080] (1) After drying the biogas residue, 1 g of the residue was taken out and added into 70 mL of 7% sodium hydroxide in a 100 mL reactor. The mixture was reacted at 160° C., 170° C., 180° C., 190° C. and 200° C. for 2 h, 4 h, 6 h, 8 h and 10 h, respectively. After natural cooling, the solid residue was filtered and washed thoroughly with water to retain the filtrate.

[0081] (2) The filtrate was adjusted to pH 1 with 1 M hydrochloric acid, and then allowed to stand for 2 h and centrifuged for 10 min. The solid obtained was fulvic acid, which could be further characterized after freeze drying.

[0082] 1.2 Determination of lignin degradation rate

[0083] By preparing alkaline lignin solutions of different concentrations, the OD 700 Value, draw its standard absorbance curve ( Figure 3), we get Y = 0.0457X-0.0321, R 2 =0.99478 linear relationship.

[0084] Drawing of the standard curve of lignin concentration: Take several test tubes and number them, take 1.5mL of lignin of different concentrations (10-40μg / mL) and add them to the test tubes, then add 100μL of 8mmol / LK3Fe(CN)6 and 100μL of 0.1mol / L FeCl3 to the test tubes in turn, mix well and react in a shaker at 30℃ for 5-10min. Measure the absorbance at a wavelength of 700nm, and use deionized water as a blank control. Draw the standard curve of lignin concentration with lignin concentration (μg / mL) as the horizontal axis and the corresponding OD value as the vertical axis, repeat 3 times, input the absorbance value into the regression equation, and calculate the lignin degradation rate.

[0085] Determination of lignin concentration and calculation of degradation rate: After the liquid alkaline lignin culture medium is diluted a certain multiple, its absorbance is determined according to the above method, and the OD value is substituted into the lignin concentration standard curve to calculate the lignin concentration and degradation rate.

[0086] The results show that: from the regression line and high correlation coefficient (R 2 =0.99478) It can be seen that the degree of fit between the data points and the regression line is very high, and almost all the data points are closely distributed on the regression line. There is a strong linear relationship between concentration and absorbance. As the concentration increases, the absorbance also increases accordingly. The slope (0.0457) in the regression equation y=0.0457x-0.0321 means that for every 1μg·mL-1 increase in concentration, the absorbance increases by an average of 0.0457OD value.

[0087] 1.3 Degradation rate of lignin residue

[0088] (1) After drying the corn cob, 5.0000 g of the corn cob was taken out and added to 50.0 mL of 8% sodium hydroxide and 50.0 mL of 12% urea in a 250.0 mL flask, and reacted at 60°C for 45 minutes. After natural cooling, the solid residue was filtered and washed thoroughly with water to retain the filtrate. The filtrate was adjusted to pH 1 with 1M hydrochloric acid, allowed to stand for 2 hours, and then centrifuged for 10 minutes (this operation was repeated three times). The solid product was placed in a freeze dryer and dried to obtain the lignin residue.

[0089] (2) Degradation of lignin residue by Y-6

[0090] The obtained lignin residue was added to the inorganic salt culture medium prepared in advance, and then 20.0 mL of bacterial solution amplified in the liquid culture medium for 12 h was added, and the mixture was placed in a shaker at 37°C for reaction for 1d, 3d, 5d, 7d, and 9d, respectively, and the degradation rate of the lignin residue was measured.

[0091] Results show that as time goes by, the degradation rate of lignin gradually increases. In the first day, the degradation rate is as low as 5.77%, but as time goes by, the degradation rate increases steadily, especially between 5 and 9 days, the degradation rate increases greatly from 20.68% to 30.42%.

[0092] 1.3 Determination of fulvic acid content in treated lignin residues and digestate

[0093] Take out 1 mL of the primary screening culture medium that has been shaken for 1-9 days, add silver sulfate, potassium dichromate and sulfuric acid in a large test tube, add at 160-170℃ for 5 minutes, then take out, cool and pour into a 250.0mL conical flask, add distilled water to 60.0-70.0mL, add 3-4 drops of o-phenanthroline indicator and titrate with ammonium ferrous sulfate. Observe the color of the solution change from orange→green→red, record the volume of ammonium ferrous sulfate solution consumed and calculate the fulvic acid content.

[0094] The results showed that the content of fulvic acid in lignin residue and biogas residue increased with the increase of oscillation time. The growth rate of fulvic acid content was faster in the initial 1-5 days, and slowed down after 7-9 days.

[0095] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A strain of Bacillus velezensis Y-6, characterized in that: The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 26, 2024. The collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the collection number is GDMCC No: 65214.

2. A microbial agent, characterized in that: The invention comprises the Bacillus Velez Y-6 as described in claim 1.

3. Use of the Bacillus Velez Y-6 according to claim 1 or the microbial agent according to claim 2 in degrading lignin.

4. Use of the Bacillus Velez Y-6 according to claim 1 or the microbial agent according to claim 2 in the production of fulvic acid.

5. A method for degrading lignin, characterized in that: The Bacillus Velez Y-6 described in claim 1 or the microbial agent described in claim 2 is used to ferment and degrade lignin.

6. The method according to claim 5, characterized in that The fermentation degradation conditions include: degradation temperature of 37°C, initial pH of 7, inoculation amount of 3% to 16% (v:v), peptone as nitrogen source in the degradation system, and a carbon-nitrogen ratio of 10:

1.

7. A method for producing fulvic acid, characterized in that: The Bacillus Velez Y-6 described in claim 1 or the microbial agent described in claim 2 is used to ferment and degrade lignin to produce fulvic acid.

8. The method according to claim 7, characterized in that The fermentation degradation conditions include: degradation temperature of 37°C, initial pH of 7, inoculation amount of 3% to 16% (v:v), peptone as nitrogen source in the degradation system, and a carbon-nitrogen ratio of 10:

1.

9. A product for producing fulvic acid by degrading lignin, characterized in that: It includes the Bacillus Velez Y-6 described in claim 1 or the microbial agent described in claim 2.

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