Sphingomonas fluorescens and application thereof in lignin degradation

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to sphingomonas fluorescens and application thereof in lignin degradation. The invention discloses a sphingomonas fluorescens H39, the preservation number of the sphingomonas fluorescens H39 is CCTCC (China Center for Type Culture Collection) No: M 2025936, and the sphingomonas fluorescens H39 is preserved in the China Center for Type Culture Collection on April 29, 2025. The strain not only has remarkable peroxidase activity (85.34 U / mL), but also can stably grow under the condition of multiple nitrogen sources (such as ammonium sulfate, peptone, yeast extract and the like), the culture period is short (3-5 days), and the lignin degradation rate of agricultural wastes such as corn straw and the like is remarkably improved. The invention finds that a new microbial resource is provided for biodegradation of lignin, and resource utilization of agricultural wastes and development of environmental protection industry are expected to be promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a Novosphingobium fluoreni and application thereof in lignin degradation. BACKGROUND

[0002] Lignin, as one of the main components of plant cell walls, is an aromatic polymer with the second highest content in nature after cellulose, and widely exists in agricultural and forestry wastes such as straw and wood. The complex three-dimensional network structure and stable chemical bonds between phenolic monomers make lignin extremely difficult to degrade, which becomes a key bottleneck restricting the efficient utilization of biomass resources. At present, the treatment methods of lignin mainly include physical method, chemical method and biological method. Although the physical method (such as crushing and pyrolysis) and the chemical method (such as acid and alkali treatment and oxidative degradation) can partially destroy the structure of lignin, they have problems such as high energy consumption, serious equipment corrosion and secondary pollution, which are difficult to meet the demand of green and sustainable development.

[0003] The biological degradation method has become a research hotspot in recent years due to its advantages such as mild reaction conditions and environmental friendliness. Existing researches show that fungi (such as white rot fungi) are the main microbial groups for lignin degradation, but they have defects such as long culture period (usually 7-14 days), complex enzyme system, low yield and sensitivity to environmental conditions, which limit the industrial application. In contrast, bacteria have characteristics such as fast growth rate, strong metabolic capacity and wide environmental adaptability, and show great potential in the field of lignin degradation. However, the reported bacterial strains (such as Bacillus and Acinetobacter) generally have problems such as insufficient lignin degradation enzyme activity, strong substrate specificity and limited ability to utilize complex nitrogen sources, which make it difficult to meet the industrialization requirements in actual degradation efficiency.

[0004] In view of the above technical bottlenecks, it is necessary to screen bacterial strains with high-efficiency lignin degradation ability, stable enzyme activity and simple culture conditions, which is the key to break through the high-value utilization of biomass resources. SUMMARY

[0005] The application isolates a Novosphingobium fluoreni H39 from potato and rice bran fermentation, which not only has significant peroxidase activity (85.34 U / mL), but also can stably grow in various nitrogen sources (such as ammonium sulfate, peptone and yeast extract), has a short culture period (3-5 days) and significantly improves the lignin degradation rate of agricultural wastes such as corn straw. The discovery provides a new microbial resource for the biological degradation of lignin, and is expected to promote the resource utilization of agricultural wastes and the development of environmental protection industry.

[0006] The application provides a strain of Novosphingobium fluoreni H39, characterized in that the preservation number of the Novosphingobium fluoreni H39 is CCTCC No: M 2025936, and the Novosphingobium fluoreni H39 has been preserved in the China Center for Type Culture Collection on April 29, 2025.

[0007] The application provides an application of the strain of Novosphingobium fluoreni H39 in lignin degradation.

[0008] The application provides an application of the strain of Novosphingobium fluoreni H39 in preparation of a lignin degradation product.

[0009] Further, the 16S rDNA sequence of the Novosphingobium fluoreni H39 is shown in SEQ ID NO: 1.

[0010] The application provides a microbial inoculant, which comprises the Novosphingobium fluoreni H39.

[0011] Compared with the prior art, the application has the following beneficial effects:

[0012] The application separates a strain of Novosphingobium fluoreni H39 from potato and rice bran fermentation products, the strain has significant peroxidase activity (85.34 U / mL), can stably grow under the condition of various nitrogen sources (such as ammonium sulfate, peptone, yeast extract, etc.), has a short culture period (3-5 days), and can significantly improve the lignin degradation rate of agricultural waste such as corn straw; with the lapse of time, the degradation degree of the straw increases. The straw weight loss rate of the sodium nitrite group and the peptone group is significantly higher than that of the blank group at 7 days and 14 days, which indicates that the two treatments may promote the degradation of the straw. The loss rate of the peptone group at 14 days is slightly higher than that of the sodium nitrite group, which indicates that the peptone may have a stronger promoting effect on the degradation of the straw in the later period, and these results provide a reference for further research on the promotion method of straw degradation.

[0013] Biological preservation instructions:

[0014] Preservation agency: China Center for Type Culture Collection;

[0015] Preservation number: CCTCC No: M 2025936;

[0016] Preservation date: April 29, 2025;

[0017] Preservation address: Wuhan University, Wuhan, China;

[0018] Taxonomic designation: Novosphingobium fluoreni H39. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure 1 is a phylogenetic tree based on 16S rRNA for Example 1.

[0020] Figure 2 Figure 2 is a schematic of the morphology of H39 on LB plates for Example 1.

[0021] Figure 3 Figure 3 is a plot of the size of transparent zones / colony diameters on Congo Red for Example 1.

[0022] Figure 4 Figure 4 is a bar graph of the effect of different nitrogen sources on lignin degradation by H39 for Example 2.

[0023] Figure 5 Figure 5 is a plot of the rate of straw degradation at different times for Example 3. DETAILED DESCRIPTION

[0024] 1. Sample Source

[0025] Fermentation of potato with rice bran

[0026] 2. Culture Medium

[0027] PDA liquid medium: Potato 200.0 g, glucose 20.0 g, distilled water 1 L.

[0028] Lignin primary screening liquid medium: Alkali lignin 2.0 g, ammonium sulfate 1.0 g, magnesium sulfate heptahydrate 0.5 g, potassium dihydrogen phosphate 1.0 g, sodium hydrogen phosphate 0.2 g, distilled water 1 L.

[0029] Aniline blue coloration medium: Yeast extract powder 5.0 g, sodium chloride 10.0 g, protein peptone 10.0 g, agar 20.0 g, aniline blue 0.25 g, distilled water 1 L.

[0030] Congo red cellulose plate medium: Congo red 0.2 g, microcrystalline cellulose 1.88 g, magnesium sulfate heptahydrate 0.25 g, potassium hydrogen phosphate 0.50 g, agar 20.0 g, distilled water 1 L.

[0031] Carboxymethylcellulose sodium (CMC-Na) medium: Ammonium sulfate 1.0 g, calcium chloride 0.02 g, magnesium sulfate heptahydrate 0.20 g, carboxymethylcellulose sodium (CMC-Na) 10.0 g, ferric chloride hexahydrate 0.05 g, potassium hydrogen phosphate 1.0 g, agar 20.0 g, distilled water 1 L.

[0032] Lignin enzyme production medium: Alkaline lignin 3.0 g, dipotassium hydrogen phosphate 1.0 g, magnesium sulfate heptahydrate 0.10 g, calcium chloride 0.08 g, ferrous sulfate heptahydrate 0.05 g, manganese chloride 0.02 g, potassium dihydrogen phosphate 1.0 g, proteose peptone 2.0 g, distilled water 1 L.

[0033] Cellulose enzyme production fermentation medium: Sodium carboxymethyl cellulose 10.0 g, proteose peptone 5.0 g, ammonium sulfate 2.0 g, potassium dihydrogen phosphate 2.0 g, yeast powder 1.0 g, magnesium sulfate heptahydrate 0.5 g, calcium chloride 0.1 g, sodium chloride 0.5 g, ferrous sulfate heptahydrate 0.05 g, manganese sulfate 0.02 g, for cellulase strain fermentation.

[0034] The above medium, unless otherwise specified, is sterilized at 121°C for 15 min before use.

[0035] Example 1

[0036] Isolation and screening of strains

[0037] (1) Strain enrichment and primary screening: 20 g of potato and rice bran fermentation product was weighed and poured into a conical flask containing 200 mL of PDA liquid medium, and cultured at 28°C and 120 r / min for 3 days to obtain a fermentation suspension. 5 mL of the fermentation suspension was added to the sodium carboxymethyl cellulose primary screening enrichment medium and cultured at 28°C for 3 days. The enrichment culture was gradient diluted with normal saline, and different concentrations of dilutions (10^ 3 -10^ 5 ) were coated on Congo red cellulose plate medium, and each concentration was repeated three times. The plates were placed in an incubator at 28°C and cultured for 3-5 days.

[0038] (2) After the colonies grew on the Congo red cellulose plate medium, single colonies with relatively obvious transparent circles were purified on the Congo red cellulose plate medium by three-zone streaking method until single colonies were obtained. The single colonies were transferred to LB liquid medium for enrichment culture for 3 days. Gradient dilution was performed using normal saline, and 10^3-10^5 dilutions were plated on CMC-Na plates for plate streaking purification. After 3-4 times of separation and purification, the pure strain was transferred to LB liquid medium, numbered, and stored in a -80°C refrigerator with glycerol.

[0039] (3) After the bacteria were cultured, DNA group was extracted, and 16s identification was performed by Shanghai Shengong.

[0040] The results of the second-generation sequencing genome alignment, 16S rRNA alignment information, such as Figure 2ANI analysis etc. to infer: it retrieves the best matching strain in the NT database as:

[0041] Novosphingobium fluoreni DSM 27568 GCA_014196615.1,

[0042] ANI index is 97.19%, which is more than 95%, identified as species level.

[0043] Taxonomic name is:

[0044] Bacteria | Pseudomonadota | Alphaproteobacteria | Sphingomonadales | Sphingomonadaceae | Novosphingobium | Novosphingobium fluoreni.

[0045] 16s rRNA sequence is SEQ ID NO: 1

[0046] > H39

[0047]

[0048] As Figure 3 shown in Fig. 1 and Table 1, the comparison result shows that the strain is Sphingomonas flava, named Sphingomonas flava H39.

[0049] Table 1

[0050]

[0051] (4) Strain re-screening: the strains obtained in step (2) were respectively inoculated into LB liquid medium at an inoculation amount of 1% and cultured for 3 d to obtain seed liquid (2.08×10^7 CFU / mL), and the seed liquid (at an inoculation amount of 1%) was respectively inoculated into lignin enzyme production medium and cellulose enzyme production fermentation medium, and cultured at 28 ℃ and 120 r / min constant temperature shaker for 5 d, and the fermentation liquid was centrifuged at 4 ℃ and 8000 r / min for 10 min to obtain the supernatant, which was the crude enzyme liquid, and the supernatant was used to determine the activities of laccase, lignin peroxidase, manganese peroxidase and cellulase, as shown in Table 2 and Table 3.

[0052] Table 2 Enzyme activity determination

[0053]

[0054] Table 3

[0055]

[0056] Among them, the enzyme activity is defined as the amount of enzyme required to catalyze 1 μmol of substrate to product per minute, which is an enzyme activity unit (U).

[0057] Enzyme activity (U / mL) = (▲OD) / (▲T)×10^6 / ε×Vtotal / Venzyme

[0058] In the formula:

[0059] Vtotal: total reaction volume (mL) when measured;

[0060] Venzyme: enzyme liquid volume (mL) when measured;

[0061] ▲T: reaction time (min);

[0062] ▲OD: measured absorbance value;

[0063] ε: molar extinction coefficient (M-1.cm-1), wherein the molar extinction coefficient of veratryl alcohol is 9300 M-1.cm-1, the molar extinction coefficient of ABTS is 36000 M-1.cm-1, and the molar extinction coefficient of Mn2+ is 49600 M-1.cm-1

[0064] 106 - Coefficient to convert mol to pmol.

[0065] Cellulase activity determination: The total reaction volume was 5 mL: 200 μL of 1% CMC-Na in citric acid buffer solution and 200 μL of crude enzyme solution were added, mixed well, and then placed in a 50 ℃ water bath for 30 min. Then, 600 μL of DNS reagent was added, shaken well, and placed in a boiling water bath for 10 min. After ice water cooling, the volume was adjusted to 5 mL with distilled water. 200 μL of the solution was taken and added to an enzyme-labeled plate, and the absorbance was measured at 540 nm. The activity of carboxymethyl cellulase was calculated according to the glucose standard curve that had been drawn.

[0066] Enzyme activity (U / mL) = (W x Δf x 1000) / (180.16 x ΔT)

[0067] Wherein: W is the amount of glucose produced by enzymatic hydrolysis reaction, which can be obtained according to the glucose standard curve; Δf represents the dilution, i.e. the ratio of the total volume of the test tube to the volume of the enzyme solution added; 1000 represents unit conversion; 180.16 represents the molecular weight of glucose; ΔT is the reaction time of the enzyme and the substrate (30 min).

[0068] Total cellulase activity (FPAase) determination: The experimental group was added with dry 1 cm x 6 cm filter paper strips, and 0.5 mL of crude enzyme solution was added. The blank group was added with 0.5 mL of water. After mixing with a pipette gun, the test tubes were placed in a 50 ℃ water bath for reaction for 60 min. After taking out, 1.5 mL of DNS reagent was added, and boiling water bath was performed for 10 min. After cooling to room temperature, the volume was adjusted to 10 mL with distilled water, and vortex shaking was performed for mixing. The absorbance value was measured at 540 nm, and the reducing sugar content was determined according to the glucose standard curve to calculate the enzyme activity.

[0069] Exocellulase (C1) activity determination: 1.5 mL of citric acid buffer was added to each test tube, 0.5 g of absorbent cotton was added, and then 0.5 mL of crude enzyme solution was added. The blank group was added with 0.5 mL of water. Water bath was performed at 50 ℃ for 1 h. After water bath, 1.5 mL of DNS reagent was added, 100 ℃ boiling water bath was performed for 10 min, and then cooled with tap water. The volume was adjusted to 10 mL. The absorbance was measured at a wavelength of 540 nm.

[0070] Enzyme activity unit (U / mL) definition: 1 mL of enzyme solution catalyzes the hydrolysis of 1 μg of glucose per minute, and the amount of cellulase required is one unit of enzyme activity.

[0071] (5) Viable cell count and transparent circle measurement on Congo red medium

[0072] Strain activation and counting: 200 μL of glycerol bacteria was taken from the -80 ℃ refrigerator into LB liquid medium, and the strain was cultured at 28 ℃ for 24 h, then 1 % of the bacterial liquid was taken into new LB liquid medium, and after being cultured at 28 ℃ for 24 h, gradient dilution counting was performed, and the rest was stored in a 4 ℃ refrigerator for standby use.

[0073] Preparation of standard: 10 U / mL cellulase standard solution was prepared and sterilized by 0.22 um filtration. It was stored at 4 ℃ for standby use.

[0074] Oxford cup method for measuring transparent circle: under sterile conditions, 4 Oxford cups with a diameter of about 7.8 mm were placed on the Congo red cellulose medium in turn, then 100 μL of culture was injected into the Oxford cup,

[0075] Among them: positive control: 100 μL of filtered cellulase standard solution;

[0076] Experimental group: 100 μL of bacterial liquid; negative control: 100 μL of filtered physiological saline;

[0077] After incubation in a 4 ℃ refrigerator for 2 h, it was placed in a constant temperature incubator for 1 d, and the transparent circle diameter and colony diameter were measured. Each experiment was repeated three times, and the average value was taken.

[0078] The results are shown in Figure 3 and Table 4, viable cell count, transparent circle / colony diameter on Congo red;

[0079] The viable cell count of H39 on LB plate was 2.08×10^7 CFU / mL, wherein H4-2 was a strain isolated at the same time as H39 in the laboratory.

[0080] The above results show that the strain H39 has high lignin peroxidase enzyme activity and also has certain fiber degradation capacity.

[0081] Table 4

[0082]

[0083] Example 2

[0084] Effect of H39 on lignin degradation under different nitrogen sources

[0085] Nitrogen source in lignin enzyme-producing medium: ammonium sulfate, urea, peptone, yeast extract, ammonia water, diammonium hydrogen phosphate, and sodium nitrite; each medium was divided into 3 tubes of 20 mL (50 mL centrifuge tube) and 1 tube of blank, inoculated with 3 % seed liquid obtained in step (4) of Example 1, and cultured at 30 ℃ for 3 days. After the crude enzyme solution was filtered through a 0.22 μm membrane, the OD value was measured at 280 nm.

[0086] Degradation rate = (A0 - An) / A0;

[0087] A0: the concentration of lignin in the culture medium before inoculation;

[0088] An: the concentration of lignin in the culture medium after inoculation for n hours;

[0089] The results are shown in Figure 4 Strain H39 was almost not affected by the nitrogen source. This suggests that H39 can utilize nitrogen sources from multiple sources, including inorganic nitrogen and organic nitrogen.

[0090] Example 3

[0091] In order to verify the ability of strain H39 to degrade lignin, indoor liquid fermentation culture method was used to conduct degradation test with corn stalk as the research object.

[0092] The corn stalks were washed under flowing tap water, dried in an oven at 80°C, and cut into small pieces about 1 cm long.

[0093] Inoculate the strain in LB liquid medium (at a 1% inoculation amount), and cultivate in a shaking incubator at 30°C for 3 days to prepare the bacterial solution.

[0094] Test group: add 3% of the bacterial solution to the conical flask containing 100 mL of 5 g of straw fermentation culture medium, and place it in a constant temperature incubator at 30°C for static culture. Set up an equal amount of sterile water added to the straw fermentation liquid as a blank control (CK), and cultivate for 7-14 days. After washing with distilled water, place it in a drying oven to dry to constant weight, weigh the dry weight of the straw, and measure the degradation rate of the straw at 7 days and 14 days of cultivation, respectively;

[0095] The formula for calculating the weight loss rate of the straw (η) is:

[0096] × 100%;

[0097] M is the dry weight of the degraded straw, M0 is the dry weight of the initial straw, and η is the weight loss rate of the straw after cultivation (%).

[0098] The results are shown in Figure 5 All treatment groups had a higher straw weight loss rate at 14 days than at 7 days, indicating that the degree of degradation of the straw increased over time.

[0099] The straw weight loss rates of the sodium nitrite group and the protein peptone group were significantly higher than that of the blank group at 7 days and 14 days, indicating that these two treatments may promote the degradation of the straw.

[0100] The loss rate of the protein peptone group at 14 days was slightly higher than that of the sodium nitrite group, indicating that protein peptone may have a stronger promoting effect on the degradation of the straw in the later stage.

[0101] These results provide a reference for further study of the promotion method of straw degradation.

[0102] For example, as shown in Table 5:

[0103] Table 5

[0104]

[0105] Among them, Curvularia clavata J1 is recorded in Institute of Industrial Microbiology, Chinese Academy of Sciences. Lignin-degrading bacteria and applications thereof: 202411022370.5 [P]. 2024-10-11.

[0106] Myrothecium wuxin is recorded in Institute of Industrial Microbiology, Chinese Academy of Sciences. A Myrothecium sp. capable of degrading lignin and its application: 202411022558.X [P]. 2024-08-27.

[0107] Aspergillus kawakamii F25, Geotrichum candidum K10, Acinetobacter F16 are recorded in Beijing Agricultural University, Beijing Hanlin Mufeng Agricultural Technology Co., Ltd. A composite microbial agent capable of degrading lignin and cellulose at low temperature and its application: 202410404376.2 [P]. 2025-02-11.

[0108] Bacillus pumilus ZB1 is recorded in Jiangsu Tobacco Industry Co., Ltd. A Bacillus pumilus capable of degrading lignin and its application: 202410927281.9 [P]. 2024-09-27.

[0109] Bacillus sp. HR-1 is recorded in Northwest Agriculture and Forestry University. A lignin-degrading bacteria and its application: 202310475440.1 [P]. 2023-10-03.

[0110] Bacillus hainanensis XZB-5 is recorded in Institute of Tropical Bioscience and Technology, Chinese Academy of Tropical Agricultural Sciences, and Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences. A Bacillus hainanensis with lignocellulose-degrading activity and its application: 202410616095.3 [P]. 2024-11-15.

[0111] The above-described embodiments are only descriptions of the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A strain of Novosphingobium fluoreni H39, characterized in that, The preservation number of the fluorescent Novosphingobium H39 is CCTCC No: M 2025936, which has been preserved in China Center for Type Culture Collection on April 29, 2025.

2. Application of the fluorescent Novosphingobium H39 in lignin degradation according to claim 1.

3. Application of the fluorescent Novosphingobium H39 in preparing lignin degradation product according to claim 1.

4. The fluorescent Novosphingobium fluoreni H39 of claim 1, characterized in that, The 16S rDNA sequence of the fluorescent Novosphingobium H39 is shown as SEQ ID NO:

1.

5. A microbial inoculant, characterized in that, The fluorescent Novosphingobium (Novosphingobium fluoreni) H39 according to claim 1.

Images