Bacillus subtilis and application thereof
By using Bacillus subtilis FM-63 to degrade lignin under slightly soluble oxygen conditions, the problem of low bacterial degradation efficiency under aerobic conditions in the prior art was solved, and a high lignin degradation rate and enzymatic efficiency were achieved.
Patent Information
- Application Number
- CN202510063987.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art When bacteria degrade lignin under aerobic conditions, NADH consumption leads to high fermentation energy consumption and low degradation efficiency.
Bacillus subtilis FM-63 is used to reduce lignin under slightly soluble oxygen conditions and improve the enzymatic lysis efficiency.
Under slightly soluble oxygen conditions, the lignin degradation rate of corn stalks reached 17.69%, and the enzymatic efficiency of pretreated residues increased by 20.36% compared with the raw materials.
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Figure CN119979377A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of lignocellulose pretreatment technology, specifically relating to Bacillus subtilis and its applications. Background Technology
[0002] Lignin is an important component of lignocellulose biomass, and its complex structure makes cellulose and hemicellulose difficult to degrade. Pretreatment to increase enzyme accessibility to cellulose is key to achieving high-value utilization in lignocellulose biorefining. Biological pretreatment typically utilizes microorganisms or their products to decompose lignocellulose, which is more environmentally friendly than chemical methods (such as acid or alkali treatment) and does not generate large amounts of chemical waste or require higher energy consumption.
[0003] Bacteria, too, can secrete the enzyme system required for lignin degradation, metabolizing poplar dioxane lignin and breaking down the biphenyl structure of lignin, making them important participants in the lignin degradation process. Furthermore, compared to fungi, bacteria reproduce rapidly, are highly adaptable to their environment, and are easier to apply on a large scale; their smaller genomes also facilitate gene manipulation and large-scale recombination expression of key enzymes. Therefore, bacteria are potential candidates for improving the enzymatic efficiency of lignin biodegradation in the future.
[0004] Current research on bacterial degradation of lignin mainly focuses on aerobic degradation. Under aerobic conditions, most of the NADH produced in Bacillus cells enters the respiratory chain and is consumed. Aerobic fermentation has high energy consumption, resulting in low lignin degradation efficiency. Summary of the Invention
[0005] One technical problem solved by this invention is to provide a Bacillus subtilis FM-63. Another technical problem to be solved by this invention is to provide the application of Bacillus subtilis FM-63 in lignin degradation and in enhancing the enzymatic hydrolysis of lignocellulose pretreatment. Under micro-dissolved oxygen conditions, the lignin degradation rate of corn straw raw material is 17.69%, and the enzymatic hydrolysis efficiency of the pretreatment residue is increased by 20.36% compared with the raw material, thus solving the problems existing in aerobic degradation.
[0006] Technical Solution: To solve the above-mentioned technical problems, the present invention adopts the following technical solution:
[0007] A Bacillus subtilis strain FM-63, classified as Bacillus subtilis, was deposited on May 10, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30579. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0008] Bacillus subtilis FM-63 was obtained by screening rotten wood from the back hill of Nanjing Forestry University.
[0009] Biological characteristics of Bacillus subtilis FM-63: When cultured in LB slant agar, the colonies of this strain are white, opaque, and have a rough surface. Gram staining confirms that it is a Gram-positive bacterium.
[0010] The 16S rRNA identification results of Bacillus subtilis strain FM-63 are shown in SEQ ID NO. 1. A phylogenetic tree was constructed by BLAST comparison in NCBI. The phylogenetic tree showed that strain FM-63 belongs to Bacillus subtilis. Based on the morphological identification and 16S rRNA identification results, this strain was identified as Bacillus subtilis.
[0011] The application of Bacillus subtilis FM-63 in lignin degradation involves inoculating Bacillus subtilis FM-63 into a liquid culture medium containing only carbon source for lignocellulose raw materials, and degrading lignin under micro-aerobic conditions.
[0012] The application of Bacillus subtilis FM-63 in lignin degradation involves treatment at 30-40℃ for 8-12 days; dissolved oxygen content of 2%-5%; and inoculum size of 2%-4%; preferably, dissolved oxygen content of 2%. The inoculum size is 2%, and the pretreatment conditions are treatment at 37℃ for 6 days.
[0013] The application of Bacillus subtilis FM-63 in enhancing the enzymatic hydrolysis effect of lignocellulose pretreatment involves inoculating Bacillus subtilis FM-63 into a liquid culture medium containing only carbon source for lignocellulose raw materials, pretreating the lignocellulose raw materials under micro-oxygen conditions, and then performing enzymatic hydrolysis after pretreatment.
[0014] The application of Bacillus subtilis FM-63 in the enzymatic hydrolysis enhancement of lignocellulose pretreatment involves pretreatment at 30-40℃ for 8-12 days; dissolved oxygen content of 2%-7%; and inoculum size of 2%-4%; preferably, dissolved oxygen content of 5%; inoculum size of 4%; and pretreatment at 35℃ for 10 days.
[0015] The application of Bacillus subtilis FM-63 in the enzymatic hydrolysis enhancement of lignocellulose pretreatment, wherein the lignocellulose raw material is one or more of rice straw, wheat straw, corn stalks, poplar, eucalyptus, pine, and bamboo.
[0016] The application of Bacillus subtilis FM-63 in enhancing the enzymatic hydrolysis of lignocellulose pretreatment includes the following steps:
[0017] (1) Dry the wood fiber raw material that has been crushed through a 40-80 mesh sieve to a constant weight; preferably, the raw material is crushed to 60 mesh and dried at 60°C.
[0018] (2) Inoculate the Bacillus subtilis FM-63 bacterial culture that has been cultured to the logarithmic growth phase into the liquid fermentation medium;
[0019] (3) Wash and dry the fermented lignocellulose raw material, add 0.1 mol / L citrate buffer, adjust the pH value, preheat in a water bath, then add cellulase and β-glucosidase, and hydrolyze in a constant temperature water bath.
[0020] (4) After hydrolysis, stop the enzyme hydrolysis with ice and filter to obtain the enzyme hydrolysate.
[0021] The application of Bacillus subtilis FM-63 in the enzymatic hydrolysis enhancement of lignocellulosic pretreatment, the formula of the liquid fermentation medium is as follows: (NH4)2SO4 1g / L, MgSO4∙7H2O 0.01g / L, KH2PO4 1.0g / L, FeSO4-7H2O 0.05g / L, ZnSO4-7H2O 0.001g / L, CaCl2-2H2O 0.1g / L, K2HPO4 1.0g / L, MnSO4-H2O 0.01g / L, CuSO4·5H2O 0.001g / L, lignocellulosic raw material 1.0g / L, pH 6.5.
[0022] The application of Bacillus subtilis FM-63 in the enzymatic hydrolysis enhancement of lignocellulose pretreatment involves adjusting the pH to 4.5-5.0 with citrate buffer; preheating in a water bath at 45-55℃ for 15-25 min; then adding cellulase and β-glucosidase; and hydrolyzing in a shaking incubator at 45-55℃ at a shaking speed of 100-200 r / min for 48-96 h. Preferably, the pH is adjusted to 4.8 with citrate buffer; preheating in a water bath at 50℃ for 20 min; then adding cellulase and β-glucosidase; and hydrolyzing in a shaking incubator at 50℃ at a shaking speed of 150 r / min for 72 h.
[0023] Beneficial effects: Compared with the prior art, the present invention has the following advantages:
[0024] The Bacillus subtilis FM-63 of this invention can degrade straw lignin at a rate of 17.69% under micro-dissolved oxygen conditions, and the enzymatic hydrolysis efficiency of the pretreated residue is 20.36% higher than that of the raw material. Attached Figure Description
[0025] Figure 1 Phylogenetic tree diagram of FM-63;
[0026] Figure 2 This is a graph showing the growth rate and lignin degradation rate under FM-63 micro-dissolved oxygen conditions in Example 2. OD 600For growth, OD 280 This refers to the lignin concentration.
[0027] Figure 3 The images show the infrared spectra of corn stalks before and after pretreatment in Example 4.
[0028] Figure 4 The image shows the degradation effect of the three major elements in poplar wood chips under FM-63 micro-dissolved oxygen conditions in Example 5. Detailed Implementation
[0029] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.
[0030] The lignin-degrading bacterium used in this invention is Bacillus subtilis FM-63, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30579 and address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.
[0031] Example 1
[0032] The Bacillus subtilis FM-63 strain of this invention was screened from decaying wood collected from the back hill of Nanjing Forestry University. The screening method included the following steps:
[0033] 1) Add 15 mL of LB medium to 4 g of rotten wood and incubate at 33°C for about 36 h to enrich the microbial strain;
[0034] 2) Dilute the enriched bacterial strain 8 times and heat it in an 80℃ water bath for 15 minutes;
[0035] 3) Pour an appropriate amount of lignin-based solid culture medium as the sole carbon source into a sterile petri dish. The formula is: (NH4)2SO4 1g / L, MgSO4-7H2O 0.01g / L, KH2PO4 1.0g / L, FeSO4-7H2O 0.05g / L, ZnSO4-7H2O 0.001g / L, CaCl2-2H2O 0.1g / L, K2HPO4 1.0g / L, MnSO4-H2O 0.01g / L, CuSO4·5H2O 0.001g / L, alkali lignin 1.0g / L, agar 15g / L, pH 6.5. Take 0.2mL and serially dilute it 10... -4 Spread the bacterial solution at a ratio of 1:1, then pour in solid culture medium, cool it, and then pour in molten solid paraffin to create a micro-aerobic environment. Invert the container and incubate at 35°C.
[0036] 4) Selected target colonies and purified them three times by streak plating until the colony characteristics were stable. The colonies were then transferred to LB slant agar and screened to obtain a strain FM-63. Its colonies were white, opaque, and had a rough surface. Gram staining confirmed it as a Gram-positive bacterium. 16S rRNA identification was performed, and a phylogenetic tree was constructed using BLAST alignment in NCBI. The results are shown below. Figure 1 This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30579, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0037] The nucleotide sequence of this strain is as follows:
[0038]
[0039] Example 2
[0040] Prepare alkaline lignin aqueous solutions of different concentrations and determine OD. 280 The value was used to construct a standard absorption curve for lignin, and the regression equation was obtained as Y = 203.15X + 0.5751, R² = 0.999, where X is the absorbance and Y is the lignin concentration.
[0041] Lignin degradation rate % = (C 原 -C 降 ) / C 原 ;
[0042] Among them, C 原 The concentration of undegraded lignin, C 降 The concentration of lignin after degradation.
[0043] Scrape the FM-63 slant culture and inoculate it into 30 mL of LB medium. Incubate overnight at 37°C with 180 rpm. Then, inoculate 2% of the culture into an alkaline lignin-only carbon source medium with the following composition: (NH4)2SO4 1 g / L, MgSO4-7H2O 0.01 g / L, KH2PO4 1.0 g / L, FeSO4-7H2O 0.05 g / L, ZnSO4-7H2O 0.001 g / L, CaCl2-2H2O 0.1 g / L, K2HPO4 1.0 g / L, MnSO4-H2O 0.01 g / L, CuSO4·5H2O 0.001 g / L, alkali lignin 1.0 g / L, pH 6.5. Set the dissolved oxygen content of the fermentation broth to 5% and incubate at 37°C for 6 days.
[0044] Every 24 hours, 2 mL of culture medium was taken into a centrifuge tube, centrifuged at 12000g for 5 min, and the absorbance of the sample at 600 nm was measured using a spectrophotometer to determine the growth of the strain.
[0045] Take 2 mL of culture medium into a centrifuge tube, centrifuge at 12000 g for 5 min, then boil in 100℃ boiling water for 15 min, and centrifuge again. Measure the absorbance of the sample at 280 nm using a spectrophotometer. Determine the lignin concentration according to the lignin standard absorption curve, and calculate the lignin degradation rate. The results are shown below. Figure 2 After 6 days of degradation, the lignin degradation rate reached 22.5%.
[0046] Example 3
[0047] Scrape the FM-63 slant culture and inoculate it into 30 mL of LB medium. Incubate overnight at 37°C with 180 rpm. Then, inoculate 2% of the culture into an alkaline lignin-only carbon source medium with the following composition: (NH4)2SO4 1 g / L, MgSO4∙7H2O 0.01 g / L, KH2PO4 1.0 g / L, FeSO4·7H2O 0.05 g / L, ZnSO4·7H2O 0.001 g / L, CaCl2·2H2O 0.1 g / L, K2HPO4 1.0 g / L, MnSO4·H2O 0.01 g / L, CuSO4·5H2O 0.001 g / L, alkali lignin 1.0 g / L, pH 6.5. Set the dissolved oxygen content of the fermentation broth to 2% and incubate at 37°C for 6 days.
[0048] Every 24 hours, 2 mL of culture medium was taken into a centrifuge tube, centrifuged at 12000g for 5 min, and the absorbance of the sample at 600 nm was measured using a spectrophotometer to determine the growth of the strain.
[0049] Take 2 mL of culture medium into a centrifuge tube, centrifuge at 12000 g for 5 min, then boil in boiling water at 100℃ for 15 min and centrifuge again. Use a spectrophotometer to measure the absorbance of the sample at 280 nm, determine the lignin concentration according to the lignin standard absorption curve, and calculate the lignin degradation rate. After 6 days of degradation, the lignin degradation rate reached 25.34%.
[0050] Example 4
[0051] The method of pretreating corn straw using Bacillus subtilis FM-63 under micro-dissolved oxygen conditions is as follows:
[0052] (1) Dry the corn stalks that have been crushed through a 60-mesh sieve at 60°C until they reach a constant weight;
[0053] (2) The bacterial culture that has been cultured to the logarithmic growth phase was inoculated into liquid fermentation medium at an inoculation rate of 4%. The formula was (NH4)2SO4 1g / L, MgSO4-7H2O 0.01g / L, KH2PO4 1.0g / L, FeSO4-7H2O 0.05g / L, ZnSO4-7H2O 0.001g / L, CaCl2-2H2O 0.1g / L, K2HPO4 1.0g / L, MnSO4-H2O 0.01g / L, CuSO4.5H2O 0.001g / L, corn stalks 1.0g / L, pH 6.5; dissolved oxygen content was 5%, and the culture was carried out at 35℃ for 10 days. The control group was not inoculated with bacterial culture.
[0054] (3) After the cultivation is completed, the straw solid loss rate and the composition of the three major elements are determined.
[0055] Solid loss rate % = (W 原 -W 降 ) / W 原 ,
[0056] Among them, W 原 For the quality of undegraded straw, W 降 The quality of the straw after degradation;
[0057] Cellulose degradation rate % = (W 原 -W 降 ) / W 原 ,
[0058] Among them, W 原 For the quality of undegraded cellulose, W 降 The weight of the fiber after degradation;
[0059] Hemicellulose degradation rate % = (W 原 -W 降 ) / W 原 ,
[0060] Among them, W 原 For the mass of undegraded hemicellulose, W 降 The mass of the hemifiber after degradation;
[0061] Lignin degradation rate % = (W 原 -W 降 ) / W 原 ,
[0062] Among them, W 原 For the quality of undegraded lignin, W 降 The quality of lignin after degradation.
[0063] After fermentation under microaerobic conditions, the solid loss rate of corn stalks was 10.36%, the cellulose degradation rate was 6.75%, the hemicellulose degradation rate was 9.36%, and the lignin degradation rate was 17.69%.
[0064] After 10 days of fermentation, the straw was washed and dried. 1 g of sample was added to 50 mL of 0.1 mol / L citrate buffer, and the pH was adjusted to 4.8. The mixture was preheated in a 50℃ water bath for 20 min, then cellulase (enzyme activity 251.6 FPU / g) and β-glucosidase (enzyme activity 9.3 CBU / mg) were added at amounts of 20 FPU / 10 CBU / g substrate, respectively. The mixture was then hydrolyzed for 72 h in a 50℃ constant-temperature water bath with shaking at 150 rpm. After hydrolysis, ice was used to stop the enzymatic hydrolysis, and the mixture was filtered. The sample was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. After appropriate dilution, the content of various sugars was determined by HPLC. The enzymatic hydrolysis efficiency was calculated using the following formula:
[0065] ;
[0066] Wherein, M0 represents the dry weight of the raw material used in the enzymatic hydrolysis, in g; C glu —Glucose content after hydrolysis, g / L; 0.9 —Conversion coefficient between cellulose and glucose; C pret —The cellulose content of the raw material after pretreatment,%.
[0067] The enzymatic hydrolysis efficiency of straw cellulose was increased by 20.36% after microaerobic pretreatment with the strain.
[0068] Infrared spectral analysis of straw before and after pretreatment is shown in the figure. Figure 3 During the pretreatment process, no new chemical structures were formed in the raw materials; only the peak intensities changed. (2900, 1420, and 1330 cm⁻¹) -1 The characteristic peak of cellulose is 1732 cm⁻¹. -1 The absorption peak at 1510 cm⁻¹ represents either the C=O bending vibration of the acetyl groups in hemicellulose and xylan, or the ester bond between lignin and carbohydrates. After pretreatment, this peak changed to varying degrees, indicating that hemicellulose underwent deacetylation during fermentation, and the bond between lignin and hemicellulose was disrupted. -1 and 1604cm -1 The vibration of the lignin benzene ring skeleton is located at 1460 cm⁻¹. -1 The relative absorption intensity of these absorption peaks is also weakened due to the CH vibration of the methoxy group in lignin, which proves that the molecular structure of lignin has also been damaged.
[0069] Example 5
[0070] The method for pretreating poplar sawdust using Bacillus subtilis FM-63 under micro-dissolved oxygen conditions is as follows:
[0071] (1) Dry the poplar wood chips that have been crushed through a 60-mesh sieve at 60°C to a constant weight;
[0072] (2) The bacterial culture that has been cultured to the logarithmic growth phase was inoculated into liquid fermentation medium at an inoculation rate of 4%. The culture medium formula was (NH4)2SO4 1 g / L, MgSO4-7H2O 0.01 g / L, KH2PO4 1.0 g / L, FeSO4-7H2O 0.05 g / L, ZnSO4-7H2O 0.001 g / L, CaCl2∙2H2O 0.1 g / L, K2HPO4 1.0 g / L, MnSO4-H2O 0.01 g / L, CuSO4·5H2O 0.001 g / L, poplar sawdust 1.0 g / L, pH 6.5, 35℃, dissolved oxygen 5%, and cultured for 10 days. The control group was not inoculated with bacterial culture.
[0073] (3) After cultivation, the straw solid loss rate and the composition of the three major elements were measured, and the results are as follows: Figure 4 As shown.
[0074] After fermentation under microaerobic conditions, the solid loss rate of poplar wood chips was 7.25%, indicating that some components of the poplar wood chips were degraded during the pretreatment process.
[0075] Further analysis of the components before and after fermentation showed that the degradation rate of cellulose in the straw was 4.68%, the degradation rate of hemicellulose was 6.01%, and the degradation rate of lignin was 15.38% after fermentation by the strain.
[0076] Poplar sawdust fermented for 10 days was washed, dried, and 1g of sample was added to 50mL of 0.1mol / L citrate buffer to adjust the pH to 4.8. The mixture was preheated in a 50℃ water bath for 20min, then cellulase (activity 251.6 FPU / g) and β-glucosidase (activity 9.3 CBU / mg) were added at 15FPU / 15CBU / g substrate, respectively. The mixture was then hydrolyzed for 72h in a 50℃ constant-temperature shaking incubator at 150r / min. After hydrolysis, ice was used to stop the enzymatic hydrolysis, and the sample was filtered. The sample was then centrifuged at 8000r / min for 10min, and the supernatant was collected. After appropriate dilution, the content of various sugars was determined by HPLC. The enzymatic hydrolysis efficiency was calculated; the enzymatic hydrolysis efficiency of poplar sawdust cellulose after microaerobic pretreatment was 16.45% higher than that of untreated raw material.
[0077] By investigating the microaerobic degradation of natural lignocellulosic raw materials by Bacillus subtilis FM-63, we can provide high-quality microbial strains and more sufficient scientific basis for constructing an efficient biological lignocellulosic raw material pretreatment strategy.
[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A Bacillus subtilis FM-63, which is classified and named as Bacillus subtilis FM-63, has been deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.30579, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.
2. The use of Bacillus subtilis FM-63 according to claim 1 in degrading lignin, characterized in that: Bacillus subtilis FM-63 was inoculated into a liquid culture medium with lignin as the only carbon source, and lignin was degraded under slightly dissolved oxygen conditions.
3. The use according to claim 2, characterized in that: The degradation is carried out at 30~40℃ for 8~12 days; 2%~5%, and the inoculation amount is 2%~4%.
4. Use of the Bacillus subtilis FM-63 according to claim 1 in enzymatic hydrolysis enhancement of wood fiber pretreatment.
5. The use according to claim 4, characterized in that: Bacillus subtilis FM-63 was inoculated into the liquid culture medium of the sole carbon source of the lignocellulosic raw material, the lignocellulosic raw material was pretreated under slightly dissolved oxygen conditions, and then enzymatic hydrolysis was performed after the pretreatment.
6. The use according to claim 4, characterized in that: The pretreatment conditions are 30~40℃ for 8~12 days; the dissolved oxygen content is 2%~7%, and the inoculation amount is 2%~4%.
7. The use according to claim 4, characterized in that: The wood fiber raw material is one or more of rice straw, wheat straw, corn straw, poplar, eucalyptus, pine and bamboo.
8. The use according to claim 4, characterized in that: The following steps are involved: (1) Dry the crushed wood fiber raw material through a 40-80 mesh sieve to a constant weight; (2) inoculating the bacterial liquid of Bacillus subtilis FM-63 cultured to the logarithmic growth phase into the liquid fermentation medium; (3) Wash and dry the fermented wood fiber raw material, add 0.1 mol / L citric acid buffer, adjust the pH value, and preheat in a water bath, then add cellulase and β-glucosidase, and hydrolyze in a constant temperature water bath; (4) After the hydrolysis is completed, use ice cubes to terminate the enzymatic hydrolysis and filter to obtain the enzymatic hydrolyzate.
9. The use according to claim 8, characterized in that: The formula of liquid fermentation medium is: (NH4)2SO4 1g / L, MgSO4-7H2O 0.01g / L, KH2PO4 1.0g / L, FeSO4-7H2O 0.05g / L, ZnSO4-7H2O 0.001g / L, CaCl2-2H2O 0.1g / L, K2HPO4 1.0g / L, MnSO4-H2O 0.01g / L, CuSO4.5H2O 0.001g / L, wood fiber raw material 1.0g / L, pH 6.
5.
10. The use according to claim 8, characterized in that: Add citric acid buffer to adjust the pH to 4.5-5.0; preheat in a 45-55°C water bath for 15-25 min, then add cellulase and β-glucosidase, and shake in a 45-55°C constant temperature water bath at a speed of 100-200 r / min for hydrolysis for 48-96 h.