Complex microbial inoculant for promoting degradation of lignocellulose as well as preparation method and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant for promoting lignocellulose degradation and a preparation method and application thereof, and relates to the technical field of microorganisms, the complex microbial inoculant comprises brevundimonas sp. AFBI (Brevundimonas sp.), alcaligenes faecalis AFBII (Alcaligenes faecalis), Achromobacter AFBIII (Achromobacter sp.), Staphylococcus aureus GBSI (Staphylococcus sp.), Glutamic Bacter GBS II (Glutamic Bacter sp.) and Bacillus (Bacillus sp.). According to the composite microbial agent provided by the invention, all strains have no antagonistic action and are mutually cooperated, and lignocellulose can be efficiently and stably degraded.

Description

[0001] This application claims the priority of a Chinese application titled "A Composite Bacterial Agent for Promoting the Degradation of Lignocellulose, Its Preparation Method and Application", with the application number 202410464148.4, which was filed with the Chinese Patent Office on April 17, 2024. The entire content of this application is incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of microbial technology, and particularly to a composite bacterial agent for promoting the degradation of lignocellulose, its preparation method and application. Background Art

[0003] With the continuous development of agricultural production, the total amount of agricultural solid waste, such as forestry waste, agricultural and livestock waste, and domestic waste, is also increasing continuously. Due to problems such as a large base number and insufficient attention, the recovery rate of agricultural waste is low, resulting in resource waste. At the same time, solid waste will occupy a large amount of land area, and secondary pollution will occur after long-term stacking. In severe cases, it will damage the surrounding atmosphere and pollute the soil. Reasonable utilization of agricultural solid waste has important environmental significance.

[0004] Agricultural solid waste is rich in a large amount of lignocellulose. Lignocellulosic biomass is mainly composed of three intertwined polymer components: cellulose, hemicellulose, and lignin, and exists as a natural resistant biological complex. Due to the heterogeneous and crystalline structural characteristics of lignocellulosic biomass, its resource utilization is difficult and the utilization rate is low. Hemicellulose is the most easily degradable matrix in lignocellulose, lignin is the most difficult to degrade, and cellulose is the second. The technology for the conversion and utilization of lignocellulose has gradually become a research hotspot in this field. These conversion technologies mainly include physical methods, chemical methods, physicochemical methods, and biological methods.

[0005] In recent years, biodegradation technology has shown great potential in the field of lignocellulose conversion. Among them, the method of using enzymes secreted by microorganisms to degrade lignocellulose has attracted much attention due to its environmental friendliness and high added value of products. However, the degradation ability of a single microorganism is limited and it is easily affected by environmental factors. Therefore, constructing a composite bacterial agent that can act synergistically and efficiently degrade lignocellulose has become the focus of research.

[0006] Although there are currently some research reports on composite bacterial agents for lignocellulose degradation, there are still some problems to be solved. For example, further research and exploration are still needed in aspects such as the selection of the types and proportions of microorganisms in the composite bacterial agent, the optimization of culture conditions, and the actual application effect.

[0007] Therefore, developing a composite bacterial agent for promoting lignocellulose degradation has important practical application value and theoretical significance. Summary of the Invention

[0008] In view of this, the main object of the present invention is to provide a composite microbial agent for promoting the degradation of lignocellulose. The various strains of the composite microbial agent provided by the present invention act synergistically with each other and can efficiently and stably degrade lignocellulose.

[0009] To achieve the above object, the technical solution of the present invention is as follows:

[0010] A composite microbial agent for promoting the degradation of lignocellulose, comprising Brevundimonas sp. AFBⅠ, Alcaligenes faecalis AFBⅡ, Achromobacter sp. AFBⅢ, Staphylococcus sp. GBSⅠ, Glutamicibacter sp. GBSⅡ and Bacillus sp. GBSⅢ. Among them, the genome of Brevundimonas sp. AFBⅠ includes the nucleotide sequence shown in SEQ ID NO.1, the genome of Alcaligenes faecalis AFBⅡ includes the nucleotide sequence shown in SEQ ID NO.2, the genome of Achromobacter sp. AFBⅢ includes the nucleotide sequence shown in SEQ ID NO.3, the genome of Staphylococcus sp. GBSⅠ includes the nucleotide sequence shown in SEQ ID NO.4, the genome of Glutamicibacter sp. GBSⅡ includes the nucleotide sequence shown in SEQ ID NO.5, and the genome of Bacillus sp. GBSⅢ includes the nucleotide sequence shown in SEQ ID NO.6.

[0011] Among them, the Brevundimonas sp. AFBⅠ is deposited in the China General Microbiological Culture Collection Center, with the deposit number of CGMCC No. 33411 and the deposit date of January 14, 2025; the Alcaligenes faecalis AFBⅡ is deposited in the China General Microbiological Culture Collection Center, with the deposit number of CGMCC No. 33412 and the deposit date of January 14, 2025; the Achromobacter sp. AFBⅢ is deposited in the China General Microbiological Culture Collection Center, with the deposit number of CGMCC No. 33413 and the deposit date of January 14, 2025; the Staphylococcus sp. GBSⅠ is deposited in the China General Microbiological Culture Collection Center, with the deposit number of CGMCC No. 33414 and the deposit date of January 14, 2025; the Glutamicibacter sp. GBSⅡ is deposited in the China General Microbiological Culture Collection Center, with the deposit number of CGMCC No. 33415 and the deposit date of January 14, 2025; the Bacillus sp. GBSⅢ is deposited in the China General Microbiological Culture Collection Center, with the deposit number of CGMCC No. 33416 and the deposit date of January 14, 2025.

[0012] Further, the mass ratio of the Brevundimonas sp. AFBⅠ, Alcaligenes faecalis AFBⅡ, Achromobacter sp. AFBⅢ, Staphylococcus sp. GBSⅠ, Glutamicibacter sp. GBSⅡ and Bacillus sp. GBSⅢ is (0.9 - 1.1):(0.9 - 1.5):(0.9 - 1.5):(0.9 - 1.1):(0.9 - 1.1):(0.9 - 1.5).

[0013] Further, the mass ratio of the Brevundimonas sp. AFBⅠ, Alcaligenes faecalis AFBⅡ, Achromobacter sp. AFBⅢ, Staphylococcus sp. GBSⅠ, Glutamicibacter sp. GBSⅡ and Bacillus sp. GBSⅢ is 1:1:1:1:1:1.

[0014] The second object of the present invention is to provide a preparation method of the above - mentioned composite microbial agent for promoting lignocellulose degradation, including the following steps:

[0015] 1) Sampling and processing of samples: Select farmland humus soil, remove the surface soil, use the five - point sampling method to collect soil with a depth of 5 - 10 cm, mix well, put it into a sterilized self - sealing bag, and store it refrigerated.

[0016] 2) Enrichment of degrading bacteria: After sterilizing the prepared enrichment medium, inoculate it, inoculate the collected soil sample into the enrichment medium, and conduct enrichment culture in a constant - temperature shaker.

[0017] 3) Gradient dilution and spread - plate culture: Use sterile water to gradient - dilute the enriched bacterial liquid to 10-6 dilution, and then take 10 -4 , 10 -5 , 10 -6 gradient dilutions were spread on CMC-Na medium plates and incubated in an inverted position in a constant temperature incubator for 7 - 10 days, and the colony growth was observed and recorded;

[0018] 4) Purification of the degrading bacteria: Select strains with good growth and a colony diameter of more than 5 mm for three-zone streak isolation and purification. Stenotrophomonas AFBⅠ, Alcaligenes faecalis AFBⅡ, Achromobacter AFBⅢ, Staphylococcus GBSⅠ, Glutamicibacter GBSⅡ, and Bacillus GBSⅢ were obtained. The Stenotrophomonas AFBⅠ, Alcaligenes faecalis AFBⅡ, Achromobacter AFBⅢ, Staphylococcus GBSⅠ, Glutamicibacter GBSⅡ, and Bacillus GBSⅢ obtained by cultivation were mixed in proportion to obtain a compound bacterium agent.

[0019] Furthermore, in step 2), the components of the enrichment medium include: peptone 8 - 12 g / L, K2HPO4 0.8 - 1.2 g / L, Na2CO3 3 - 6 g / L, MgSO4·7H2O 0.08 - 0.12 g / L, FeSO4·7H2O 0.01 - 0.02 g / L, MnSO4 0.03 - 0.06 g / L, yeast extract 8 - 12 g / L, and the solvent is distilled water.

[0020] Furthermore, in step 3), the components of the CMC-Na medium include: CMC-Na 8 - 12 g / L, KNO3 0.8 - 1.2 g / L, K2HPO4 0.3 - 0.8 g / L, NaCl 1 - 2 g / L, MgSO4·7H2O 0.3 - 0.8 g / L, agar 10 - 16 g / L, and the solvent is distilled water.

[0021] The present invention also aims to provide the application of the above compound bacterium agent for promoting the degradation of lignocellulose in the degradation of lignocellulose and / or cellulose.

[0022] The beneficial effects of the present invention at least include:

[0023] The compound bacterium agent provided by the present invention has no antagonistic effect among various strains and synergizes with each other, and can efficiently and stably degrade lignocellulose. When decomposed for 90 days, the compound bacteria can increase the decomposition rate of Chinese fir by 27.53% and that of Moso bamboo by 29.45%, and accelerate the release of nitrogen and cellulose. Description of the Drawings

[0024] Figure 1 They are the growth state diagrams of each colony.

[0025] Figure 2 They are the experimental results diagrams of Congo red staining of GBSⅢ strain.

[0026] Figure 3 It is the result diagram of the filter paper strip disintegration test for GBSⅠ strain.

[0027] Figure 4 It is the result diagram of the antagonistic test for the degrading bacteria.

[0028] Figure 5 It is the phylogenetic tree of AFBⅠ constructed based on the 16S rDNA gene sequence.

[0029] Figure 6 It is the phylogenetic tree of AFBⅡ constructed based on the 16S rDNA gene sequence.

[0030] Figure 7 It is the phylogenetic tree of AFBⅢ constructed based on the 16S rDNA gene sequence.

[0031] Figure 8 It is the phylogenetic tree of GBSⅠ constructed based on the 16S rDNA gene sequence.

[0032] Figure 9 It is the phylogenetic tree of GBSⅡ constructed based on the 16S rDNA gene sequence.

[0033] Figure 10 It is the phylogenetic tree of GBSⅢ constructed based on the 16S rDNA gene sequence.

[0034] Figure 11 It is the column chart of the change in the mass loss rate of Phyllostachys edulis leaves.

[0035] Figure 12 It is the column chart of the change in the mass loss rate of Cunninghamia lanceolata leaves.

[0036] Figure 13 It is the diagram of the change in the total nitrogen content of Phyllostachys edulis leaves.

[0037] Figure 14 It is the diagram of the change in the total nitrogen content of Cunninghamia lanceolata leaves.

[0038] Figure 15 It is the diagram of the change in the carbon-nitrogen ratio of Cunninghamia lanceolata leaves.

[0039] Figure 16 It is the trend diagram of the change in the lignin concentration of Phyllostachys edulis leaves.

[0040] Figure 17 It is the trend diagram of the change in the lignin concentration of Cunninghamia lanceolata leaves.

[0041] Figure 18 It is the trend diagram of the change in the cellulose concentration of Phyllostachys edulis leaves.

[0042] Figure 19 It is the trend diagram of the change in the cellulose concentration of Cunninghamia lanceolata leaves.

[0043] Figure 20 It is a trend chart of the change in the ratio of lignin to nitrogen in Chinese fir leaves.

[0044] Figure 21 It is a trend chart of the change in the lignin degradation rate of bamboo leaves.

[0045] Figure 22 It is a trend chart of the change in the lignin degradation rate of Chinese fir leaves.

[0046] Figure 23 It is a trend chart of the change in the cellulose degradation rate of bamboo leaves.

[0047] Figure 24 It is a trend chart of the change in the cellulose degradation rate of Chinese fir leaves.

[0048] Figure 25 It is an effect diagram of the degradation of Chinese fir leaves. Among them, (a) is before degradation, and (b) is after degradation. Detailed implementation mode

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0050] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement it. When the combination of technical solutions conflicts with each other or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the protection scope required by the present invention.

[0051] The following specifically illustrates the solutions proposed by the present invention through specific embodiments:

[0052] Embodiment 1

[0053] Prepare the culture medium:

[0054] Enrichment medium: peptone 10.0 g, K2HPO4 1.0 g, Na2CO3 5.0 g, MgSO4·7H2O 0.1 g, FeSO4·7H2O 0.015 g, MnSO4 0.05 g, yeast extract 10 g, made up to 1000 mL with distilled water;

[0055] LB liquid medium: peptone 10 g, yeast extract 5 g, NaCl 10 g, made up to 1000 mL with distilled water;

[0056] Hutchinson's inorganic salt medium: KH2PO4 1.0 g, NaCl 0.1 g, MgSO4·7H2O 0.3 g, NaNO3 2.5 g, FeCl3 0.01 g, CaCl2 0.1 g, made up to 1000 mL with distilled water;

[0057] Filter paper disintegration medium: 40 mL of Hutchinson's inorganic salt medium, 2 filter papers of 1 cm × 6 cm;

[0058] Carboxymethyl cellulose sodium (CMC) medium: CMC-Na 10.0 g, KNO3 1.0 g, K2HPO4 0.5 g, NaCl 1.5 g, MgSO4·7H2O 0.5 g, agar 13 g, made up to 1000 mL with distilled water;

[0059] Enzyme-producing medium: (NH4)SO4 2.0 g, KH2PO4 3.0 g, CaCl2 0.5 g, MgSO4 0.5 g, CoCl2 3.0 mg, FeSO4·7H2O 7.5 mg, ZnSO4·7H2O 2.0 mg, MnSO4·H2O 2.5 mg, made up to 1000 mL with distilled water.

[0060] Collection and treatment of samples:

[0061] Soil samples were collected from the farmland humus soil of the Huanjiang Karst Ecosystem Observation and Research Station. When collecting samples, the surface soil was removed, and the samples at a depth of 5 - 10 cm were collected using the five-point sampling method, mixed evenly, and put into a sterilized self-sealing bag. Each sample had 4 replicates and was stored in a 4°C refrigerator for later use.

[0062] Enrichment of degrading bacteria:

[0063] The prepared enrichment medium was divided into bottles, 90 mL per bottle, and sterilized in an autoclave. After cooling to room temperature, 10 g of soil samples were added to each bottle on a clean bench. The conical flask mouths were sealed with sealing film and enriched in a constant temperature shaker at 180 r·min -1 and cultured at 28°C for 7 days.

[0064] Gradient dilution and spread plate culture:

[0065] The enriched bacterial solution was diluted to 10 -6 dilution with sterile water. 100 μL of the diluted solutions at 10 -4 , 10 -5 , 10 -6 gradients were pipetted and spread on the CMC-Na medium plates. Each dilution had 3 replicates and was incubated in an inverted position in a constant temperature incubator at 28°C for 7 - 10 days. The colony growth was observed and recorded.

[0066] Purification of Degrading Bacteria:

[0067] According to the diameter of the colonies in the medium with CMC-Na as the sole carbon source, the degradation and utilization effect of the strains on cellulose was preliminarily evaluated. Strains with better growth and larger diameters were selected for three-zone streak isolation and purification to obtain 6 colonies, which were respectively recorded as AFBⅠ, AFBⅡ, AFBⅢ, GBSⅠ, GBSⅡ, and GBSⅢ. Among them, the growth of each colony was as Figure 1 described, and the diameters of each colony are shown in Table 1 below.

[0068] Screening of Degrading Bacteria (Congo Red Staining Experiment):

[0069] The purified strains (AFBⅠ, AFBⅡ, AFBⅢ, GBSⅠ, GBSⅡ, and GBSⅢ) were spot-inoculated on the CMC medium, with three replicates for each strain. They were incubated in an inverted position in an incubator at 30 °C for 2 - 3 d. After staining with 1 g / L Congo red solution for 15 min, they were rinsed and decolorized with 1 mol / L NaCl solution. The cellulose degradation ability of the strains was judged by the size of the transparent circle Dc value (Dc = diameter of the hydrolysis circle (D, mm) / diameter of the colony (d, mm)), and they were marked and preserved. The Dc values of each transparent circle are shown in Table 1 below. Figure 2 The result experiment for strain GBSⅢ.

[0070] Filter Paper Disintegration Test:

[0071] Add 2 sterile filter papers of 1×6 cm to 40 mL of Hutchinson's inorganic salt medium, and inoculate 1 mL of bacterial solution for constant-temperature shaking respectively. The control group was added with an equal amount of sterilized LB culture solution. The above operations were all carried out on a clean bench. To avoid the filter paper breaking due to shaking, a constant-temperature shaker at 130 r·min -1 and 28 °C was used for incubation. The degradation effect was judged according to the degree of breakage of the filter paper: (+) for the edge of the filter paper swelling; (++) for the filter paper swelling neatly and bending; (+++) for the filter paper being amorphous; (++++) for being in a mass-like paste; (+++++) for being semi-clear. Among them, the degradation effect of strain GBSⅠ was as Figure 3 described, and the disintegration effects of each strain on the filter paper are shown in Table 1 below.

[0072] Table 1: Data Sheet of the Performance Results of Different Strains

[0073]

[0074]

[0075] Example 2 Antagonism Test of Degrading Bacteria

[0076] The superior degrading single strains obtained after purification in Example 1 were streaked intersecting on an LB medium, and cultured in an inverted position at 28 °C. Wait until the strains grow plump and check whether there is an antibacterial zone at the intersection of the strains. The absence of an antibacterial zone indicates that the strains have no antagonistic effect, and the preparation of the compound bacterium agent can be carried out. The results show that as Figure 4 shown, it can be seen from the experimental results that there is no antagonistic effect among the strains AFBⅠ, AFBⅡ, AFBⅢ, GBSⅠ, GBSⅡ, and GBSⅢ, and they can be jointly used to prepare the compound bacterium agent.

[0077] Identification of the strains in Example 3

[0078] The above-mentioned strains (AFBⅠ, AFBⅡ, AFBⅢ, GBSⅠ, GBSⅡ, and GBSⅢ) that obtained obvious degradation effect and can be used for the preparation of the compound bacterium agent in Example 1 were inoculated into an LB medium, and cultured under constant temperature oscillation at 120 r / min -1 and 28 °C for 18 h, and then molecular biology identification was carried out.

[0079] Extraction of strain DNA: The genomic DNA of the strains was extracted according to the operating steps of the genomic extraction kit. The extracted DNA was detected for concentration (μg / ml) and purity (A260 / A280) using NanoDrop2000, and the integrity of the DNA was detected by 1.0% agarose gel electrophoresis.

[0080] PCR amplification and sequencing: Using the extracted genomic DNA as a template, universal bacterial primers were selected for PCR amplification. Among them, the upstream primer 27F (SEQ ID NO.7): 5'-AGTTTGATCMTGGCTCAG-3', and the downstream primer 1492R (SEQ ID NO.8): 5'-GGTTACCTTGTTACGACTT-3'). Amplification system (25 μl): 0.5 μl of DNA template, 0.5 μl of each upstream and downstream primer, 12.5 μl of PCR MasterMix, and dd H2O was added to make up 25 μl. Reaction procedure: Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 90 s, for a total of 35 cycles; final extension at 72 °C for 7 min.

[0081] After amplification, sequencing was carried out, and the nucleotide sequence of the genome of AFBⅠ was as shown in SEQ ID NO.1, the nucleotide sequence of the genome of AFBⅡ was as shown in SEQ ID NO.2, the nucleotide sequence of the genome of AFBⅢ was as shown in SEQ ID NO.3, the nucleotide sequence of the genome of GBSⅠ was as shown in SEQ ID NO.4, the nucleotide sequence of the genome of GBSⅡ was as shown in SEQ ID NO.5, and the nucleotide sequence of the genome of GBSⅢ was as shown in SEQ ID NO.6.

[0082] The sequencing results were submitted to the GenBank database of the National Center for Biotechnology Information (NCBI). The basic local alignment search tool (BLAST) was used for homology alignment search. The 16S rDNA gene sequences of the model strains with higher homology and a typical species of a neighboring family and genus were selected as exogenous bacteria. The neighbor-joining (NJ) method in MEGA-X software was used for 1000 times of Bootstraps tests to construct a phylogenetic tree. The results are as Figures 5 - 10 shown.

[0083] Among them:

[0084] AFBⅠ: Brevundimonas sp.

[0085] AFBⅡ: Alcaligenes faecalis

[0086] AFBⅢ: Achromobacter sp.

[0087] GBSⅠ: Staphylococcus sp.

[0088] GBSⅡ: Glutamicibacter sp.

[0089] GBSⅢ: Bacillus sp.

[0090] Example 4

[0091] Based on the indoor antagonism test, it was found that there was no antagonism among the 6 selected highly efficient lignocellulose-degrading bacteria, and the preparation of compound microbial agents could be carried out. This example provided the determination of the laccase activity of the fermentation broth of single bacteria and mixed bacteria. The bacterial suspensions of A1 (AFBⅠ), A2 (AFBⅡ), A3 (AFBⅢ), A4 (GBSⅠ), A5 (GBSⅡ), A6 (GBSⅢ), A7 (AFBⅠ + GBSⅢ), A8 (AFBⅢ + GBSⅡ), A9 (AFBⅡ + GBSⅠ), A10 (AFBⅠ + AFBⅡ + AFBⅢ), A11 (GBSⅠ + GBSⅡ + GBSⅢ), A12 (AFBⅠ + GBSⅠ + AFBⅢ), A13 (GBSⅠ + GBSⅢ + AFBⅢ), A14 (GBSⅡ + AFBⅡ + GBSⅢ), A15 (equal amount mixture of 6 strains) were inoculated into the enzyme-producing medium at an inoculation amount of 3%, and the control group (CK) was added with an equal amount of sterile water. Homologous strains were purchased from Huizao Biotechnology Co., Ltd.; Brevundimonas sp. with the number HZB119338, Alcaligenes faecalis with the number HZB223880, Achromobacter sp. with the number HZB224282, Staphylococcus sp. with the number HZB282363, Glutamicibacter sp. with the number HZB218287, Bacillus sp. with the number HZB112061, and they were mixed equally for treatment B16. Each treatment had three replicates. After culturing at 28°C and 130 r / min for 5 d, centrifugation was carried out at 4000 r / min for 10 min, and the supernatant was used as the crude enzyme solution. The laccase (Lac) activity was determined by the guaiacol method. One enzyme activity unit (U) was defined as the amount of enzyme required to catalytically oxidize 1 nmol of guaiacol within 1 min under specific conditions.

[0092] The determination results of the laccase activity of the fermentation broth of single bacteria and mixed bacteria are shown in Table 2. It can be seen from Table 2 that the laccase activity of the compound bacteria was significantly higher than that of the single bacteria, among which the mixture of 6 strains was the most significant, and it was significantly higher than the strains sold on the market, indicating that there may be a synergistic effect among different strains, and the combination screened under natural conditions was significantly stronger than the artificial combination purchased on the market, and the internal regulatory mechanisms of microorganisms coordinated with each other to promote the degradation of lignocellulose.

[0093] Table 2: Data table of laccase activity results for different treatments

[0094]

[0095] Example 5

[0096] Indoor Degradation Effect of Composite Degrading Bacteria

[0097] Based on indoor antagonism tests and enzyme activity experiments, this example carried out an indoor decomposition experiment of Cunninghamia lanceolata using a mixed bacterial solution (H6) of 6 cellulose-degrading bacteria, namely Alcaligenes faecalis, Achromobacter, Brevundimonas, Staphylococcus, Bacillus, and Glutamicibacter.

[0098] The collected moso bamboo and Cunninghamia lanceolata litter were cut into 1 - 2 cm segments and dried to a constant weight in an oven at 60 °C. 100 mL of sterilized Hutchinson's inorganic salt medium was added to a conical flask, and an equal amount of the 6 bacterial solutions was inoculated at an inoculation amount of 3%. The control group was added with an equal amount of sterilized LB culture solution. It was cultured at 37 °C and 180 r / min, with 4 replicates for each treatment. Fermentation broth was obtained at 20 d, 60 d, and 90 d of culture respectively, filtered through filter paper, and then the residue was dried at 65 °C and weighed. The weight loss rate of Cunninghamia lanceolata leaves was calculated by the weight loss method ((initial dry weight - dry weight at measurement time) / initial dry weight), and the lignin and cellulose contents were determined by the acid detergent fiber method.

[0099] Lignin and cellulose degradation rate:

[0100] E(%) = (M0 × C0 - M t × C t ) / (M0 × C0) × 100%

[0101] M0 is the initial dry weight of the sample (g), M t is the dry weight of the sample at the t sampling time (g), C0 is the initial lignin or cellulose concentration of the sample (mg·g -1 ), C t is the lignin or cellulose concentration of the sample at the t sampling time (mg·g -1 ).

[0102] The experimental results are as Figures 11 to 25 shown;

[0103] The research results show that at 90 days of decomposition, the H6 mixed bacterial solution can increase the decomposition rate of Cunninghamia lanceolata by 27.53% and that of moso bamboo by 29.45%, and accelerate the release of nitrogen and cellulose.

[0104] At 90 days of decomposition, the lignin degradation rate of Cunninghamia lanceolata under the treatment of the H6 mixed bacterial solution was 29.87%; the cellulose degradation rate was 71.95%; the lignin degradation rate of moso bamboo was 65.87%; all were significantly higher than those of the CK group.

[0105] It should be noted that in this document, the terms "include", "comprise" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising one..." does not exclude the presence of additional identical elements in the process, method, article or device comprising such element.

[0106] The serial numbers of the embodiments of the present invention above are only for description and do not represent the superiority or inferiority of the embodiments.

[0107] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A composite bacterial agent for promoting the degradation of lignocellulose, characterized in that: Including Brevundimonas sp., Alcaligenes faecalis, Achromobacter sp., Staphylococcus GBS I, Glutamicibacter sp. and Bacillus sp., wherein the Brevundimonas AFB I is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 33411 and a deposit date of January 14, 2025; the Alcaligenes faecalis AFB II is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 33412 and a deposit date of January 14, 2025; the Achromobacter AFB III is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with a deposit number of CGMCC No.33413, the preservation date is January 14, 2025. The Staphylococcus GBSⅠ is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with the preservation number CGMCC No.33414, and the preservation date is January 14, 2025. The glutamicum Bacillus GBSⅡ is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with the preservation number CGMCC No.33415, and the preservation date is January 14, 2025. The Bacillus GBSⅢ is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with the preservation number CGMCC No.33416, and the preservation date is January 14, 2025.

2. The composite bacterial agent for promoting lignocellulose degradation according to claim 1, characterized in that: The mass ratio of Brevundimonas AFBⅠ, Alcaligenes faecalis AFBⅡ, Achromobacter AFBⅢ, Staphylococcus GBSⅠ, Bacillus glutamicum GBSⅡ and Bacillus GBSⅢ is (0.9-1.1):(0.9-1.5):(0.9-1.5):(0.9-1.1):(0.9-1.1):(0.9-1.5).

3. The composite bacterial agent for promoting lignocellulose degradation according to claim 1, characterized in that: The mass ratio of Brevundimonas AFBⅠ, Alcaligenes faecalis AFBⅡ, Achromobacterium AFBⅢ, Staphylococcus GBSⅠ, Bacillus glutamicum GBSⅡ and Bacillus GBSⅢ is 1:1:1:1:1:1:

1.

4. Use of any composite bacterial agent for promoting lignocellulose degradation according to claim 1 to 3 in degrading lignocellulose.

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