Bacterium for degrading lignin under low-temperature alkaline condition and application thereof

Abstract

The invention discloses a bacterium for degrading lignin under a low-temperature alkaline condition and application of the bacterium, and relates to a bacterium for degrading lignin and application of the bacterium, the bacterium for degrading lignin under the low-temperature alkaline condition is Pseudomonas reidholzensis PR-6, the bacterium is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC 65470. The invention also discloses application of the catalyst in degradation of lignin under low-temperature alkaline conditions. According to the present invention, the Pseudomonas reidholzensis strain PR-6 is screened from the lignin-rich soil so as to obtain the PR-6 strain with the efficient degradation ability, and the PR-6 can secrete the enzymes related to the lignin degradation; the method disclosed by the invention has a beneficial effect of degrading lignin by the PR-6 strain under the low-temperature alkaline condition.

Description

Technical Field

[0001] The invention relates to bacteria for degrading lignin and application thereof. Background Art

[0002] Lignocellulosic biomass is the most abundant natural resource on earth, of which lignin is one of the main components and an important resource of aromatic polymers. Lignin has abundant reserves and is renewable. The total amount of crop straw in the world alone exceeds 2.2 billion tons. As the main component of most terrestrial plants, lignin accounts for more than 90% of the dry plant body. At the same time, lignin, as an important renewable carbon source, usually comes from agricultural and industrial waste such as straw and wood, and its reserves in nature are extremely large. 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.

[0003] Low temperature and alkaline resistant lignin degradation is an important research field, especially in northern my country, due to the low temperature in autumn and winter, the high pH of soda saline soil, and the low efficiency of traditional lignin degradation methods. Therefore, screening microorganisms that can efficiently degrade lignin under low temperature and alkaline conditions has important application value.

[0004] Bacteria have shown great potential in lignin degradation due to their wide presence and short culture time, and have become one of the research topics. Bacteria indirectly degrade lignin through their internal components, causing it to be modified. Studies have found that bacteria that degrade lignin exist widely. Although various bacteria have shown certain potential in the degradation and conversion of lignin, the conversion efficiency of bacteria is much lower than that of fungi. Therefore, obtaining bacteria that can efficiently degrade lignin has become a research hotspot. Summary of the invention

[0005] The invention discloses bacteria capable of degrading lignin under low-temperature alkaline conditions and application thereof.

[0006] The bacterium for degrading lignin under low temperature alkaline conditions is Pseudomonas reidholzensis PR-6, which is preserved in Guangdong Microbiological Culture Collection Center with a preservation number of GDMCC 65470.

[0007] The invention discloses an application of Pseudomonas reidholzensis PR-6 in degrading lignin under low-temperature alkaline conditions.

[0008] The present invention measures the lignin degradation performance of Pseudomonas reidholzensis PR-6, specifically including the measurement of lignin degradation enzyme activity and lignin degradation rate. The measurement results show that the bacterium PR-6 has a definite degradation ability for lignin, and has the effect of degrading lignin in the range of pH 7.0-11.0 and temperature 4-45 degrees Celsius.

[0009] Furthermore, the Pseudomonas reidholzensis PR-6 of the present invention secretes enzymes related to lignin degradation. Specifically, the enzymes related to lignin degradation include manganese-dependent peroxidase (MnP), peroxidase (LiP) and laccase (Lac). Single factor degradation experiments show that the optimal conditions for Pseudomonas reidholzensis PR-6 to degrade lignin are pH = 8.0, temperature 15.0 ° C, and inoculation amount 4%.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The Pseudomonas reidholzensis PR-6 strain of the present invention is screened from soil rich in lignin to obtain a PR-6 strain with high degradation ability. The Pseudomonas reidholzensis PR-6 can secrete enzymes related to lignin degradation. According to measurement, the activity of peroxidase (LiP) is 134U, the activity of manganese catalase (MnP) is 338U, and the activity of laccase (Lac) is 258U. After the culture conditions of the Pseudomonas reidholzensis PR-6 are optimized by response surface design, it is found that when the pH is 8.9, the temperature is 17.0°C, and the inoculation amount is 5.8%, the lignin degradation rate of the Pseudomonas reidholzensis PR-6 can be increased to 35.77%. The Pseudomonas reidholzensis PR-6 of the present invention produces a beneficial effect in degrading lignin under low temperature alkaline conditions.

[0012] The bacterium Pseudomonas reidholzensis PR-6 capable of degrading lignin under low-temperature alkaline conditions of the present invention is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC 65470. The deposit address is: 5th Floor, Building 59, Institute of Microbiology, Guangdong Academy of Sciences, No. 100, Xianlie Middle Road, Guangzhou, and the deposit date is November 12, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The aniline blue fading comparison results of the PR-6 strain;

[0014] Figure 2 is the phylogenetic tree;

[0015] Figure 3 The results of PR-6 lignin degradation rate at different temperatures;

[0016] Figure 4 The results of PR-6 lignin degradation rate at different pH values;

[0017] Figure 5 Results of determination of lignin degradation rate of PR-6 with different inoculation amounts;

[0018] Figure 6 are the contour lines and 3D response surface diagrams of initial pH and temperature;

[0019] Figure 7 Contour lines and 3D response surface plots of initial pH and inoculum size;

[0020] Figure 8 It is the contour line and 3D response surface diagram of temperature and inoculum size;

[0021] Fig. 9 It is the main product of lignin degradation by PR-6 bacteria. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0024] Specific implementation method 1: In this implementation method, the bacteria that degrade lignin under low temperature alkaline conditions are

[0025] Pseudomonas reidholzensis PR-6, which is deposited in Guangdong Microbiological Culture Collection Center with the deposit number of GDMCC 65470.

[0026] The Pseudomonas reidholzensis PR-6 isolation and screening method described in this embodiment is:

[0027] The following experimental methods, unless otherwise specified, are conventional methods, and the experimental materials used, unless otherwise specified, are purchased from commercial channels.

[0028] Soil source: Jingyuetan National Forest Park, Jingyue Avenue, Nanguan District, Changchun City, Jilin Province, January 2024

[0029] Culture medium:

[0030] 1. Enrichment medium: alkaline lignin 10g, (NH4)2SO4 2.0g, Na2HPO4 1.0g, KH2PO4 1.0g, NaCl1.0g, CaCl2 0.5g, distilled water to 1L, natural pH;

[0031] 2. Preliminary screening of solid culture medium: alkaline lignin 2g, (NH4)2SO4 2.0g, Na2HPO4 1.0g, KH2PO4 1.0g, NaCl 1.0g, CaCl2 0.5g, distilled water to 1L, natural pH;

[0032] 3. Liquid LB medium: 5.0 g yeast extract, 10.0 g NaCl, 10.0 g tryptone, distilled water to 1 L;

[0033] 4. Solid LB culture: 5.0 g yeast extract, 10.0 g NaCl, 10.0 g tryptone, 15 g agar, distilled water to 1 L;

[0034] 5. Aniline blue identification medium: Add 0.4 g / L aniline blue to solid LB medium;

[0035] 6. Lignin degradation medium: Add 1 g of alkaline lignin to liquid LB medium;

[0036] 7. Lignin enzyme production medium: alkaline lignin 2.0g, (NH4)2SO4 2.0g, KH2PO4 1.0g, Na2HPO40.2g, MgSO4 0.5g, anhydrous glucose 1.0g.

[0037] Screening and identification of Pseudomonas reidholzensis PR-6 strain

[0038] 1. Enrichment and initial screening

[0039] Take 10g soil, 100mL enrichment medium and glass beads and mix them in a 250ml conical flask. Cultivate at 15℃, 180r / min, and subculture after one week. After three generations, dilute the bacterial solution to 10 -1 ~10 -7 . Absorb 10 -3, 10 -5 and 10 -7 Four concentrations of suspension were coated on the solid culture medium for primary screening, and each concentration was repeated 3 times. The culture was cultured in a constant temperature incubator at 15°C for 72 hours, and bacteria with different colony morphologies were selected for separation and purification. Twenty strains of bacteria were obtained in the primary screening.

[0040] 2. Rescreening

[0041] The 20 selected bacterial strains were rescreened using the aniline blue decolorization method. The strains with strong decolorization ability generally have the function of lignin degradation. Figure 1 The aniline blue fading comparison results of strain PR-6.

[0042] 3. Strain identification

[0043] The selected cold-resistant strains were subjected to molecular biological identification. The extracted strain genomic DNA was used as a template, and PCR amplification was performed with universal primers 27F (5'-AGAGTTT-GATCCTGGCTCAG-3') and 1492R (5'-TACG-GTTACCTTGTTACGACTT-3') as primers, and then 16S rRNA sequencing was performed by Shanghai Paisono Biotechnology Co., Ltd. The sequencing results are shown in SEQ ID NO.1. The gene sequences obtained by sequencing were compared with homology in the NCBI database to find the strains with the closest relationship to the target strains, and then the strain phylogenetic tree was constructed using MEGA11 software.

[0044] The sequencing data set of strain PR-6 has been submitted, and its BioProject ID in the NCBI sequence read archive is PRJNA1176012. BLAST alignment was performed on NCBI, and the sequences with high similarity all belonged to Pseudomonas, with a consistency of more than 99%. It was preliminarily determined that PR-6 was Pseudomonas sp. The software MEGA11 was used to construct a Neighbor-Joining phylogenetic tree of strain PR-6 and other strains ( Figure 2 for phylogenetic tree).

[0045] 4. Optimization of culture conditions of Pseudomonas reidholzensis PR-6

[0046] 4.1. Preparation of bacterial suspension

[0047] Pseudomonas reidholzensis PR-6 was cultured in LB liquid medium at 15°C for 72 h, and the bacterial suspension was diluted to OD 600 =1.

[0048] 4.2 Determination of lignin degradation rate

[0049] By preparing alkaline lignin solutions of different concentrations, the OD 280 Value, draw its standard absorbance curve, get Y = 0.364X + 0.0021, R 2 =0.9938 linear relationship.

[0050] The bacterial suspension of Pseudomonas reidholzensis PR-6 was added to 50 mL of lignin degradation medium, cultured at 15°C, 180 r / min, sampled every 24 hours, centrifuged at 12000 r / min for 10 min, diluted 10 times with degradation medium without lignin components (control), and the OD value was measured at a wavelength of 280 nm. Repeat 3 times and input the absorbance value into the regression equation to calculate the lignin degradation rate.

[0051] 4.3 Single Factor Experiment

[0052] Under the conditions of initial medium pH 7 and 2% inoculation amount, the temperature was set at 4, 15, 25, 35, and 45 (unit: °C), 3 replicates were set for each treatment, and the shaking speed was 180 r / min for 7 days to detect the lignin degradation rate. Figure 3 The results of PR-6 lignin degradation rate at different temperatures are shown in Table 1. Figure 3 It can be seen that the degradation rate of lignin treated with PR-6 varies greatly in the range of 4 to 45 °C. The degradation rate of lignin is between 10 and 20 °C, especially at 15 °C, reaching the maximum degradation rate.

[0053] Under the conditions of a culture temperature of 15°C and an inoculation amount of 2%, the pH values ​​were set to 7.0, 8.0, 9.0, 10.0, and 11.0, and each treatment was repeated 3 times, and the subsequent treatments were the same as above. Figure 4 The results of PR-6 lignin degradation rate at different pH values ​​show that strains SC-2 and PR-6 can grow under pH values ​​of 7.0-11.0, and have a certain degradation effect on lignin. The degradation rate of lignin is highest in the range of 7.0-9.0, especially at 8.0, when the maximum degradation rate is reached.

[0054] At 15°C, the pH value of the culture medium was 7, and the inoculation amount in the culture medium was set to 2%, 4%, 6%, 8%, and 10%. Each treatment was repeated 3 times, and the subsequent treatments were the same as above. Figure 5 The results of the determination of lignin degradation rate of PR-6 with different inoculation amounts showed that the degradation rate of lignin was the highest when it was 4%.

[0055] 4.4. Optimization of Box-Behnken response surface

[0056] According to the Box-Behnken central composite design principle, the main factors affecting lignin degradation: pH (A), temperature (B), and inoculation amount (C) were used as independent variables, and the lignin degradation rate was used as the response value. A response surface analysis experiment with three factors and three levels was designed. The specific results are shown in Table 1.

[0057] Table 1 Response surface methodology test results

[0058]

[0059] Design Expert 13 software was used to perform multiple regression fitting analysis on the lignin degradation rates of and PR-6(Y), and the regression model was obtained:

[0060] Y=36.04-0.3462A+6.89B+1.83C-0.3025AB-0.14AC-0.83BC-1.51A 2 -12B 2 +

[0061] 0.8033C 2

[0062] The model was subjected to variance analysis, and the results are shown in Table 2. From the data in the table, it can be seen that the P value of the regression model of the bacteria is 0.0008, indicating that the model is extremely significant, the lack of fit term is greater than 0.05, and the model determination coefficient (R2) is greater than 0.9, indicating that the model fits the experimental situation well and can be used to analyze and predict the optimal lignin degradation conditions.

[0063] Table 2 Regression model analysis of lignin degradation rate of PR-6

[0064]

[0065] The response surface diagram of the experimental data was drawn, and the interaction relationship between it and each factor was analyzed intuitively. Figure 6-8 Contour lines and 3D response surface plots including response values ​​and test factors ( Figure 6 is the contour line and 3D response surface diagram of initial pH and temperature, Figure 7 Initial pH, inoculum size contours and 3D response surface plots, Figure 8 are the contour lines of temperature and inoculum size and the 3D response surface diagram).

[0066] through Figure 6 From the analysis in Table 2, it can be concluded that under the condition of 0% inoculation amount, the interaction effect of pH value and temperature has no obvious effect on the lignin degradation rate; when the pH value remains unchanged, with the increase of temperature, the degradation rate increases first and then decreases, and the degradation rate is the highest at 15-20℃.

[0067] through Figure 7 From the analysis in Table 2, it can be concluded that at 0°C, the interaction effect between pH value and inoculation size is not obvious; when the inoculation size is fixed, with the increase of pH, the lignin degradation rate increases first and then decreases, and the degradation rate is the highest when the pH value is 8.5-9.0.

[0068] through Figure 8 From the analysis in Table 2, it can be concluded that under the condition of pH 0, the interaction effect between temperature and inoculum size is not obvious; when the temperature is fixed, with the increase of inoculum size, the degradation rate shows a trend of first increasing and then decreasing. When the inoculum dose is 5.0%-6.0%, the degradation rate is the highest.

[0069] According to the results of response surface analysis, the influence of these three factors on the lignin degradation rate of the bacteria is: temperature > inoculation amount > pH, which may be related to the wide adaptability of the strain.

[0070] 4.5. Optimization and verification results of fermentation conditions When pH was 8.9, temperature was 17.0℃, and inoculation amount was 5.8%, the optimal analysis of the constructed mathematical model was performed using Design-Expert 13.0 software, and it was found that when the inoculation ratio was 5.8%, the temperature was 17.0℃, and the pH value was 8.9, the degradation rate of lignin by PR-6 could reach 35.77%. Considering the actual situation, the conditions were corrected to: pH 9.0, temperature 17℃, inoculation amount 5.5%. On this basis, the experimental verification degradation rate reached 36.82%. The error was small, reflecting that the lignin degradation conditions optimized by the response surface method are real and have certain practical value.

[0071] Specific implementation method 2: Application of the lignin-degrading bacteria described in the first implementation method to degrade lignin under low-temperature alkaline conditions.

[0072] Using the Pseudomonas reidholzensis PR-6 bacteria of this embodiment to degrade lignin:

[0073] Using LB liquid medium as the base medium, add an appropriate amount of lignin to be degraded, adjust the initial pH of the medium with KOH solution, and sterilize at 120°C for 20 minutes when the initial pH is 9.0. Inoculate PR-6 bacteria revived in LB liquid medium into the medium containing lignin at an inoculum of 5.5%, and culture at 17°C and 180rpm.

[0074] Samples were taken on the 3rd, 6th and 10th days of degradation, and the degradation products of lignin were determined by LC-MS. Fig. 9 It is the main product of lignin degradation in Pseudomonas reidholzensis PR-6. Fig. 9 It can be seen that the Pseudomonas reidholzensis PR-6 of the present invention achieves the degradation of lignin under low temperature alkaline conditions.

Claims

1. A bacterium that degrades lignin under low temperature alkaline conditions, characterized in that The bacterium that degrades lignin under low temperature alkaline conditions is (Pseudomonas reidholzensis) PR-6, which is preserved in Guangdong Microbiological Culture Collection Center with the preservation number of GDMCC 65470.

2. Use of the lignin-degrading bacteria according to claim 1 to degrade lignin under low-temperature alkaline conditions.

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