Lignin degrading bacterium, separation method, microbial inoculum and application of lignin degrading bacterium in promoting biomass degradation

Abstract

The invention belongs to the technical field of microbial metabolism engineering, and particularly relates to a lignin degrading bacterium, a separation method, a microbial agent and application of the lignin degrading bacterium in promoting biomass degradation. The strain is named as Trabulsiella odontobacteria L1, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.35692. The invention further discloses a preparation method of the strain. The invention provides a lignin degrading bacterium L1 which can degrade lignin under both an aerobic condition and an anaerobic condition, so that the degradation and conversion of biomass are promoted, and the yield is increased. Therefore, compared with the prior art, the lignin degrading bacterium L1 disclosed by the invention is unique in function, low in oxygen demand and beneficial to lignin degradation demands in different scenes.

Description

Technical Field

[0001] This invention belongs to the field of microbial metabolic engineering technology, and particularly relates to a lignin-degrading bacterium and its isolation method, bacterial agent, and its application in promoting biomass degradation. Background Technology

[0002] Lignocellulose is the most abundant renewable biomass resource on Earth, with an annual production exceeding 20 billion tons. It can be degraded into a range of chemicals. Lignocellulose is generally composed of cellulose, hemicellulose, and lignin, with lignin being a key anti-degradation barrier. Lignin and hemicellulose are intertwined through a complex and diverse network of chemical bonds, forming a tight network structure. This structure tightly surrounds cellulose, forming a robust barrier that not only ensures the integrity and mechanical strength of plant cells but also provides them with sufficient rigidity, effectively resisting the erosion of various chemicals in nature, as well as the degradation by exogenous enzymes and microorganisms.

[0003] In related technologies, most microorganisms that degrade lignin are aerobic, primarily using oxidases and peroxidases to attack and degrade lignin under aerobic conditions. However, a small number of microorganisms can substantially degrade lignin under anaerobic conditions. Anaerobic degradation eliminates the need for oxygen supply and associated costs, undoubtedly possessing unique application potential and value in industrial applications, and is expected to inject new momentum into the biomass conversion industry.

[0004] Therefore, there is an urgent need for a lignin-degrading bacterium to solve the technical problem that few strains can decompose lignin-based biomass under anaerobic conditions.

[0005] It should be noted that the information disclosed in this background section is only for understanding the background technology of this application concept, and therefore may include information that does not constitute prior art. Summary of the Invention

[0006] This disclosure provides at least one lignin-degrading bacterium and its isolation method, a bacterial agent, and its application in promoting biomass degradation.

[0007] In a first aspect, embodiments of this disclosure provide a lignin-degrading bacterium, wherein the lignin-degrading bacterium is bacterium L1. Trabulsiella odontotermitis L1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35692.

[0008] In one optional embodiment, the lignin-degrading bacteria L1 is stored in 20% glycerol at -80°C.

[0009] Secondly, this disclosure also provides a method for isolating lignin-degrading bacteria as described above, including the following steps: Step S1, take black-winged subterranean termite worker ants, wash and crush them to obtain a homogenate; Step S2: Take 1 mL of homogenate supernatant and inject it into 15 mL of anaerobic enrichment medium mMRS using a syringe. Incubate anaerobically at 35°C for 48 h. Step S3: The enriched bacterial culture was serially diluted 10-fold with phosphate buffer. After thorough shaking, 50 μL of the diluted solution was taken and spread onto solid culture plates. The culture was anaerobic at 35°C for 48 h. The strain was further purified by streak plating until a pure culture was obtained, yielding lignin-degrading bacteria L1.

[0010] In one optional implementation, the mMRS culture medium is prepared as follows: Step R1: Take 1g of yeast powder, 5g of NH4Cl, 1g of alkaline lignin, 1.5g of KH2PO4, 2.9g of K2HPO4×3H2O, 0.2g of MgCl2×6H2O, 75mg of CaCl2×2H2O, 0.05g of MnSO4, 2g of CaCO3, and 5g of KHCO3. Step R2: Add 1L of distilled water and adjust the pH to neutral; Step R3: After boiling the culture medium, purge it with nitrogen for 10 minutes, then continue to add cysteine ​​salt under nitrogen atmosphere to a final concentration of 0.5 g / L and dispense it into anaerobic tubes, then sterilize at 121°C for 20 minutes.

[0011] Thirdly, this disclosure also provides a microbial inoculant, including the lignin-degrading bacteria L1 as described above.

[0012] In an optional embodiment, the microbial agent further includes lignin-degrading bacteria. Klebsiella aerogenes TL3 , CGMCC 1.61912.

[0013] In an optional embodiment, the microbial agent further includes cellulose-degrading bacteria. Ruminiclostridium cellulolyticum DSM 5812, Lactobacillus pentosaccharide Lactobacillus pentosus ATCC8041, Lactococcus Lactococus sp .CGMCC 24341.

[0014] Fourthly, the embodiments of this disclosure also provide the application of the lignin-degrading bacterium L1 described in the first aspect above in lignin degradation and the conversion of lignocellulose into organic acids.

[0015] Fifthly, the embodiments of this disclosure also provide the application of the microbial agents described in the third aspect above in lignin degradation and the conversion of lignocellulose to produce organic acids.

[0016] The beneficial effects of this invention are that it provides a lignin-degrading bacterium L1, which can degrade lignin under both aerobic and anaerobic conditions, thereby promoting biomass degradation and conversion and increasing yield. Therefore, compared with the prior art, the lignin-degrading bacterium L1 described in this invention has unique functions, low oxygen requirements, and is beneficial for lignin degradation needs in different scenarios.

[0017] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention are realized and obtained in the structures particularly pointed out in the description, claims and drawings.

[0018] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 This is a colony morphology diagram of a lignin-degrading bacterium L1 on a plate, provided in an embodiment of this disclosure. Figure 2 A flowchart illustrating the isolation process of a lignin-degrading bacterium L1 provided in this embodiment of the present disclosure; Figure 3 A line graph showing the growth and lignin degradation of a lignin-degrading bacterium L1 using lignin as a substrate, provided in an embodiment of this disclosure; Figure 4 A bar chart illustrating the synergistic degradation of lignin by TL3, a lignin-degrading bacterium, under anaerobic conditions, as provided in this embodiment of the present disclosure. Figure 5 This is a stacked bar chart illustrating how a lignin-degrading bacterium L1 promotes the transformation of corn cobs into organic acids, as provided in an embodiment of this disclosure. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Research has revealed that most microorganisms involved in lignin degradation are aerobic, primarily using oxidases and peroxidases to attack and degrade lignin under aerobic conditions. However, a small number of microorganisms can also substantially degrade lignin under anaerobic conditions. Anaerobic degradation eliminates the need for oxygen supply and associated costs, undoubtedly possessing unique application potential and value in industrial applications and promising to inject new momentum into the biomass conversion industry.

[0023] Therefore, there is an urgent need for a lignin-degrading bacterium to solve the technical problem that few strains can decompose lignin-based biomass under anaerobic conditions.

[0024] The shortcomings of the above solutions are the result of the inventor's practical experience and careful research. Therefore, the discovery process of the above problems and the solutions proposed in this disclosure below should be considered as the inventor's contribution to this disclosure.

[0025] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the embodiments and features described below can be combined with each other. Furthermore, in the accompanying drawings, the thickness of components may be exaggerated or reduced for the purpose of effectively describing the technical content.

[0026] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0027] Based on the above research, this disclosure provides a lignin-degrading bacterium, wherein the lignin-degrading bacterium is bacterium L1. Trabulsiella odontotermitis L1, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35692, is a lignin-degrading bacterium. L1 is stored in 20% glycerol at -80°C.

[0028] In addition, refer to Figure 1 This disclosure also provides a method for isolating the above-mentioned lignin-degrading bacteria, which is obtained by screening from the intestines of black-winged subterranean termites. The original biological material was collected from the suburbs of Zhenjiang City, Jiangsu Province, and the method specifically includes the following steps: Step S1: Take black-winged subterranean termite worker ants, wash them, and crush them to obtain a homogenate. Step S2: Take 1 mL of homogenate supernatant and inject it into 15 mL of anaerobic enrichment medium mMRS using a syringe. Incubate anaerobically at 35°C for 48 h. Step S3: The enriched bacterial culture was serially diluted 10-fold with phosphate buffer. After thorough mixing, 50 μL of the diluted solution was taken and spread onto solid culture plates. The plates were then anaerobically cultured at 35°C for 48 h. Strains with larger colonies were further purified by streaking until a pure culture was obtained, thus obtaining the lignin-degrading bacterium L1.

[0029] The aforementioned lignin-degrading bacteria L1 can also be simply referred to as bacteria L1.

[0030] In at least one embodiment, the method for preparing the mMRS culture medium described above is as follows: Step R1: Take 1g of yeast powder, 5g of NH4Cl, 1g of alkaline lignin, 1.5g of KH2PO4, 2.9g of K2HPO4×3H2O, 0.2g of MgCl2×6H2O, 75mg of CaCl2×2H2O, 0.05g of MnSO4, 2g of CaCO3, and 5g of KHCO3. Step R2: Add 1L of distilled water and adjust the pH to neutral; Step R3: After boiling the culture medium, purge it with nitrogen for 10 minutes, then continue to add cysteine ​​salt under nitrogen atmosphere to a final concentration of 0.5 g / L and dispense it into anaerobic tubes, then sterilize at 121°C for 20 minutes.

[0031] The final identification of bacterial species L1 using molecular biological methods is as follows: Morphological identification, refer to Figure 1 The L1 bacterial colonies are round, slightly transparent, and consist of short rods; they do not form spores.

[0032] Identification of genetic characteristics (strain 16S rDNA sequence): The L1 bacteria selected by the above method were amplified using universal bacterial primers, namely the forward primer Eubac27F (SEQ ID NO:1) and the reverse primer Eubac27F (SEQ ID NO:2), as follows: Forward primer: Eubac27F: 5-AGAGTTTGATC-CTGGCTCAG-3 (SEQ ID NO:1); Reverse primer: Eubac1492R: 5-GGTTACCTTGTTACGACTT-3 (SEQ ID NO:2); PCR amplification was performed by mixing 0.5 μL each of the forward and reverse primers from 23 μL of 1×sPfu Master Mix primers with 1 μL of bacterial culture, followed by sequencing to obtain the 16S rDNA gene sequence of bacterial L1 (SEQ ID NO:3). The 16S rDNA sequence of this strain and its close relatives were compared and analyzed in the NCBI database using the BLAST method. The comparison analysis showed that this strain was similar to... Trabulsiella odontotermitis It has the highest species similarity, at 99.0%, and is therefore named... T. odontotermitis L1 was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on August 20, 2025, with accession number CGMCC No. 35629.

[0033] SEQ ID NO:3:

[0034] Furthermore, this disclosure also provides the application of the lignin-degrading bacterium L1 described above in degrading lignin under both aerobic and anaerobic conditions. The specific operation method is as follows: Bacterial L1 is inoculated into MSMG medium, and the OD is detected... 600 and OD 310 Bacterial growth and lignin content were monitored separately. The MSMG medium formula (g / L) is as follows: K2HPO4 1.0, KH2PO4 1.0, (NH4+) 4） 2.0 g of MgSO4, 0.2 g of MgSO4, 0.1 g of CaCl2, 0.01 g of FeSO4, 0.01 g of MnSO4, 1.0 g of alkali lignin, and 1.0 g of glucose were added. The pH was adjusted to neutral and the mixture was sterilized at 115°C for 30 min. Anaerobic culture medium required nitrogen purging and the addition of cysteine ​​hydrochloride reducing agent to a final concentration of 0.5 g / L, as described in Example 1. Aerobic culture medium required no additional treatment.

[0035] Results reference Figure 3 As shown, L1 grows rapidly, with OD at 37℃ under aerobic conditions. 600 It reaches its peak at 48 h, at approximately 1.9. OD under anaerobic conditions. 600 It also reached a peak of approximately 0.8 at 48 h. Bacterial L1 exhibited similar lignin degradation effects under both aerobic and anaerobic conditions. Lignin degradation was faster under aerobic conditions, but stalled after 24 h, while degradation continued under anaerobic conditions. Ultimately, the lignin content under anaerobic conditions (measured by OD) was [data missing]. 310 The content (indicating) is slightly lower than that under aerobic conditions, indicating that the lignin degradation rate is higher under anaerobic conditions, reaching 18.6%.

[0036] On the other hand, besides lignin, bacteria L1 can also metabolize some common substrates. Tests showed L1's ability to metabolize lignocellulosic substrates and their monomers in mMRS medium under anaerobic or aerobic conditions. Therefore, the lignin in the original medium formulation was replaced with a series of test substrates (10 g / L): microcrystalline cellulose, glucose, xylose, corn cob, and basic lignin. Fermentation conditions were 180 rpm at 37°C for three days. Product detection was performed using liquid chromatography with 4 mM sulfuric acid as the mobile phase, an Aminex HPX-87H sugar column, and a parallax detector.

[0037] Table 1. T.odontotermitis L1 metabolism acid production from five substrates Note: ND, Not Detected.

[0038] The results are shown in Table 1. Bacterium L1 can utilize the five substrates mentioned above, and the main small-molecule organic compounds under both aerobic and anaerobic conditions are lactic acid and acetic acid, although the differences are significant. Under anaerobic conditions, L1's ability to accumulate organic acids from different carbon sources is higher than that under aerobic conditions. Anaerobic metabolism using glucose and xylose as substrates yielded the highest lactic acid and acetic acid production, approximately 1.64 g / L and 1.63 g / L, respectively. Under anaerobic conditions, except for the degradation of glucose where lactic acid production was higher than acetic acid production, the acetic acid production from other carbon sources was higher than that from lactic acid, especially xylose, where the acetic acid production was more than twice that of lactic acid. Under aerobic conditions, except for xylose and glucose, no lactic acid or acetic acid accumulation was observed with the other three substrates, but significant cell growth was observed.

[0039] Furthermore, this disclosure also provides a microbial inoculant, including the lignin-degrading bacterium L1 as described above, and adding other strains to form a novel microbial inoculant, which has better degradation efficiency than a single bacterium L1.

[0040] In at least one embodiment, bacteria L1 was combined with another lignin-degrading bacterium also derived from black-winged subterranean termites. Klebsiella aerogenes TL3 These microbial agents, used synergistically, contribute to the degradation of lignin under anaerobic conditions. The culture medium used remains the anaerobic MSMG medium.

[0041] Results reference Figure 4 Under anaerobic conditions, lignin degradation approached its limit after 72 hours. L1 showed slightly better lignin degradation ability than TL3, with degradation rates of 18.6% and 15.9%, respectively. The bacterial community composed of L1 and TL3 could increase the lignin degradation rate to 29.6%.

[0042] The dense three-dimensional structure of lignin affects the accessibility of cellulose substrates to microorganisms and cellulases. Overcoming the degradation barrier of lignin plays an important role in improving biomass conversion.

[0043] To further investigate the effect of L1 on promoting organic acid production in microbial communities, in at least one embodiment, cellulose-degrading bacteria were used. Ruminiclostridium cellulolyticum DSM 5812, Lactobacillus pentosaccharide Lactobacillus pentosus ATCC 8041, Lactococcus Lactococus A microbial inoculum composed of sp. CGMCC 24341 was used for the anaerobic degradation and transformation of corn cob, a lignocellulose substrate, and the effect of L1 addition on product accumulation was compared. The culture medium used was mMRS medium, and the fermentation conditions were 180 rpm, 35°C, for three days. The corn cob used as the carbon source was only pre-treated by grinding to 60 mesh.

[0044] The results are as follows Figure 5As shown, both microbial agents mainly produced acetic acid and lactic acid, while common metabolites such as ethanol, propionic acid, and butyric acid did not accumulate significantly. The basic agent without L1 produced 1.24 g / L of lactic acid and 1.22 g / L of acetic acid, while the agent with added L1 produced 0.83 g / L of lactic acid and 2.13 g / L of acetic acid. The significant increase in acetic acid production and the 20% increase in total organic acid production indicate that L1 can promote the degradation of corn stalks.

[0045] In summary, T.odontotermitis L1 can degrade lignin and various carbon sources under both aerobic and anaerobic conditions. T.odontotermitis L1 can also be with K.aerogenes TL3 and other bacteria can be combined to enhance lignin degradation under anaerobic conditions, and can also be combined with cellulose-degrading bacteria and lactic acid bacteria to improve substrate transformation and organic acid accumulation. These anaerobic degradation processes have certain application value in the field of biomass conversion under anoxic conditions.

[0046] In the several embodiments provided in this application, it should be understood that the disclosed methods can be implemented in other ways. The apparatus embodiments described above are merely illustrative. Based on the above-described ideal embodiments according to the present invention, and through the above description, those skilled in the art can make various changes and modifications without departing from the technical spirit of the disclosed embodiments. The technical scope of the embodiments of this disclosure is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A lignin-degrading bacterium, characterized in that, The lignin-degrading bacteria is bacterium L1. Trabulsiella odontotermitis L1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35692.

2. The lignin-degrading bacteria as described in claim 1, characterized in that, The lignin-degrading bacteria L1 was stored in an environment of 20% glycerol and -80°C.

3. A method for isolating lignin-degrading bacteria as described in claim 1, characterized in that, Includes the following steps: Step S1: Take black-winged subterranean termite worker ants, wash them, and crush them to obtain a homogenate. Step S2: Take 1 mL of homogenate supernatant and inject it into 15 mL of anaerobic enrichment medium mMRS using a syringe. Incubate anaerobically at 35°C for 48 h. Step S3: The enriched bacterial culture was serially diluted 10-fold with phosphate buffer. After thorough shaking, 50 μL of the diluted solution was taken and spread onto solid culture plates. The culture was anaerobic at 35°C for 48 h. The strain was further purified by streak plating until a pure culture was obtained, yielding lignin-degrading bacteria L1.

4. The method for isolating lignin-degrading bacteria as described in claim 3, characterized in that, The preparation method of the mMRS culture medium is as follows: Step R1: Take 1g of yeast powder, 5g of NH4Cl, 1g of alkaline lignin, 1.5g of KH2PO4, 2.9g of K2HPO4×3H2O, 0.2g of MgCl2×6H2O, 75mg of CaCl2×2H2O, 0.05g of MnSO4, 2g of CaCO3, and 5g of KHCO3. Step R2: Add 1L of distilled water and adjust the pH to neutral; Step R3: After boiling the culture medium, purge it with nitrogen for 10 minutes, then continue to add cysteine ​​salt under nitrogen atmosphere to a final concentration of 0.5 g / L and dispense it into anaerobic tubes, then sterilize at 121°C for 20 minutes.

5. A microbial inoculant, characterized in that, Includes the lignin-degrading bacteria L1 as described in any one of claims 1-2.

6. The microbial agent as described in claim 5, characterized in that, The microbial agent also includes lignin-degrading bacteria. Klebsiella aerogenes TL3 , CGMCC 1.61912.

7. The microbial agent as described in claim 5, characterized in that, The microbial agent also includes cellulose-degrading bacteria. Rumiclostridium cellulolyticum DSM5812, Lactobacillus pentosaccharide Lactobacillus pentosus ATCC 8041, Lactococcus Lactococus sp CGMCC24341.

8. The application of the lignin-degrading bacterium L1 as described in claim 1 or 2 in lignin degradation and the conversion of lignocellulose to produce organic acids.

9. The application of the microbial agent according to claim 5 in lignin degradation and the conversion of lignocellulose to produce organic acids.

10. The application of the microbial agent according to claim 6 or 7 in lignin degradation and the conversion of lignocellulose to produce organic acids.

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