A cold-resistant composite bacterial agent for low-temperature decomposition of rice straw and its application

Abstract

The present invention discloses a cold-resistant composite bacterial agent for decomposing rice straw at low temperature and its application, belonging to the field of microbial technology. It is composed of Flavobacterium F1, Flavobacterium F2, Flavobacterium F3, Oxalobacterium 01, Oligotrophomonas S1 and Pseudomonas P1. There is no obvious antagonistic inhibition between the strains of the cold-resistant composite bacterial agent, and each strain has the ability to produce cellulase and filter paper enzyme under low temperature environment, among which the cellulase activity reaches up to 16.48U / mL, and the filter paper enzyme activity reaches up to 21.71U / mL. When the composite bacterial agent is used to degrade rice straw at low temperature, it can significantly reduce the mechanical strength of the straw and significantly reduce the cellulose, hemicellulose and lignin content in the straw. Under low temperature conditions, the cold-resistant composite bacterial agent can stably produce lignocellulose hydrolyzing enzymes, especially cellulase, accelerate the decomposition of rice straw and the release of nutrients, and has good development and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a cold-resistant composite bacterial agent for decomposing rice straw at low temperature and application thereof. Background Art

[0002] Crop straw is the largest renewable resource on Earth, with the annual output of various types of straw steadily reaching hundreds of billions of tons. Due to the enormous amount of straw produced and its difficulty in quickly decomposing and utilizing it, most of it is burned or discarded on-site, resulting in serious resource waste and ecological problems. Rice is a major crop and a common biomass resource worldwide, rich in cellulose, hemicellulose, and lignin. my country currently leads the world in rice production, generating a large amount of rice straw annually. Returning rice straw to the fields can return nutrients to the soil, alleviating nutrient deficiencies and increasing soil fertility. Increasing the comprehensive utilization of rice straw resources is of great significance for promoting sustainable agricultural development, alleviating environmental pressures, and achieving the "dual carbon" goals of agriculture.

[0003] Returning straw to the fields after decomposition is currently the most natural, ecological, and economical way of utilization. Microorganisms play a key role in the decomposition process after straw is returned to the fields. However, in my country, it is difficult for crop straw to fully decompose during the winter after it is returned to the fields. The long-term low temperature environment in autumn and winter will produce a continuous inhibition on the activity of microorganisms, resulting in extremely slow degradation of lignocellulose and the inability to achieve an effective decomposition state, which seriously restricts the effect of returning straw to the fields to fertilize the soil and the farming of the following year. The biodegradation process of the main components of rice straw, such as cellulose and hemicellulose, is very complex and requires the synergistic action of multiple enzymes to complete. Obtaining straw-degrading strains with high enzyme activity in low-temperature environments can improve the decomposition efficiency of complex macromolecular organic matter and is the key to promoting the comprehensive utilization of rice straw.

[0004] Therefore, screening cold-loving microorganisms that can efficiently decompose straw in autumn and winter and constructing composite microbial agents that can efficiently decompose straw in low-temperature environments are key means to solve the problem of straw decomposition and return to the field in my country. It is expected to solve the ecological pain points of straw decomposition and return to the field, and has important practical significance for realizing efficient resource utilization of straw and solving problems such as food and resource shortages, environmental pollution, etc. in my country. Summary of the Invention

[0005] The purpose of the present invention is to provide a cold-resistant composite bacterial agent for decomposing rice straw at low temperature and its application, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a cold-resistant composite bacterial agent for low-temperature decomposition of rice straw, which is composed of Flavobacterium sp. F1, Flavobacterium sp. F2, Flavobacterium sp. F3, Oxalbacteraceae sp. O1, Stenotrophomonas sp. S1 and Pseudomonas sp. P1.

[0008] The second technical solution of the present invention is a method for preparing the cold-resistant composite bacterial agent, comprising the following steps: activating and culturing each strain separately and collecting the bacterial liquid; mixing the bacterial liquids of Flavobacterium F1, Flavobacterium F2, Flavobacterium F3, Oxalobacter 01, Stenotrophomonas S1 and Pseudomonas P1 in a volume ratio of 2:9:1:1:1:1 to obtain the cold-resistant composite bacterial agent.

[0009] The third technical solution of the present invention is the use of the cold-resistant composite bacterial agent in degrading rice straw.

[0010] A fourth technical solution of the present invention is a product for promoting the degradation of rice straw, which comprises the cold-resistant composite bacterial agent.

[0011] A fifth technical solution of the present invention is a method for degrading rice straw, which utilizes the cold-resistant composite bacterial agent to degrade rice straw.

[0012] Based on the above technical solution, the present invention has the following technical effects:

[0013] The present invention provides a cold-resistant composite bacterial agent for decomposing rice straw at low temperatures and its application. There is no obvious antagonistic inhibition between the strains of the cold-resistant composite bacterial agent, and each strain has the ability to produce cellulase and filter paper enzyme under low temperature conditions, wherein the cellulase activity reaches a maximum of 16.48U / mL, and the filter paper enzyme activity reaches a maximum of 21.71U / mL. When the composite bacterial agent is used to degrade rice straw at low temperatures, it can significantly reduce the mechanical strength of the straw and significantly reduce the cellulose, hemicellulose and lignin content in the straw. Under low temperature conditions, the cold-resistant composite bacterial agent can stably produce lignocellulose hydrolyzing enzymes, especially cellulase, accelerate the decomposition of rice straw and the release of nutrients, and has good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 The morphology of each strain, among which A is Flavobacterium F1, B is Flavobacterium F2, C is Flavobacterium F3, D is Oxalobacter 01, E is Stenotrophomonas S1, and F is Pseudomonas P1;

[0016] Figure 2 is the phylogenetic tree of each strain, where A is Flavobacterium F1, B is Flavobacterium F2, C is Flavobacterium F3, D is Oxalobacter 01, E is Stenotrophomonas S1, and F is Pseudomonas P1;

[0017] Figure 3 Cellulase (A) and filter paper enzyme (B) activity determination of each strain at 10°C;

[0018] Figure 4 This is a comparison of the decomposition of rice straw by the composite bacterial agent of the present invention for 60 days at low temperature, where A is a real picture and B is a grayscale processed picture;

[0019] Figure 5 Grayscale analysis of the color change of rice straw decomposed by the composite bacterial agent of the present invention, where A is 0 days, B is 28 days, and C is 60 days;

[0020] Figure 6 The mechanical strength analysis of the composite bacterial agent decomposed rice straw;

[0021] Figure 7 This is the chemical content analysis of rice straw decomposed by the composite bacterial agent of the present invention, where A is the cellulose content, B is the hemicellulose content, and C is the lignin content;

[0022] Figure 8 This is a colonization analysis of the composite bacterial agent of the present invention on the surface of rice straw. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0029] The embodiment of the present invention provides a cold-resistant composite bacterial agent for low-temperature decomposition of rice straw, which is composed of Flavobacterium sp. F1, Flavobacterium sp. F2, Flavobacterium sp. F3, Oxalbacteraceae sp. O1, Stenotrophomonas sp. S1 and Pseudomonas sp. P1.

[0030] In some specific embodiments, the volume ratio of the bacterial liquids of Flavobacterium F1, Flavobacterium F2, Flavobacterium F3, Oxalobacterium 01, Stenotrophomonas S1 and Pseudomonas P1 is 2:9:1:1:1:1.

[0031] In a low-temperature environment, the strains have strong growth and reproduction capabilities, can produce abundant cellulase, and efficiently degrade rice straw.

[0032] In some specific embodiments, the Flavobacterium F1 was deposited in the China Center for Type Culture Collection on July 1, 2024, at Wuhan University, Wuhan, China, with the deposit number CCTCC M 20241422;

[0033] The Flavobacterium F2 was deposited in the China Center for Type Culture Collection on July 1, 2024, at Wuhan University, Wuhan, China, with the deposit number CCTCC M 20241423;

[0034] The Flavobacterium F3 was deposited in the China Center for Type Culture Collection on July 1, 2024, at Wuhan University, Wuhan, China, with the deposit number CCTCC M 20241424;

[0035] The Oxalobacter 01 was deposited in the China Center for Type Culture Collection on July 1, 2024, at Wuhan University, Wuhan, China, with the deposit number CCTCC M 20241425;

[0036] The Stenotrophomonas S1 was deposited on July 1, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC M 20241426;

[0037] The Pseudomonas P1 was deposited in the China Center for Type Culture Collection on July 1, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC M 20241427.

[0038] An embodiment of the present invention also provides a method for preparing the cold-resistant composite bacterial agent, comprising the following steps: activating and culturing each strain separately and collecting the bacterial liquid; mixing the bacterial liquids of Flavobacterium F1, Flavobacterium F2, Flavobacterium F3, Oxalobacter 01, Stenotrophomonas S1, and Pseudomonas P1 in a volume ratio of 2:9:1:1:1:1 to obtain the cold-resistant composite bacterial agent.

[0039] The embodiment of the present invention also provides the use of the cold-resistant composite bacterial agent in degrading rice straw.

[0040] An embodiment of the present invention further provides a product for promoting the degradation of rice straw, wherein the product comprises the cold-resistant composite bacterial agent.

[0041] The embodiment of the present invention further provides a rice straw degradation method, which utilizes the cold-resistant composite bacterial agent or the product to degrade the rice straw.

[0042] In some specific embodiments, the conditions for the degradation treatment are: temperature of 5-15° C. and soil moisture content of 60%.

[0043] The materials and culture medium in the embodiment of the present invention are as follows:

[0044] Vancomycin was produced by Haibo Biotechnology, HB8744a;

[0045] Congo red solid medium: (NH4)2SO42.0g, MgSO4·7H2O 0.5g, K2HPO41.0g, NaC10.5g, CMC-Na 2.0g, Congo red 0.2g, agar 20.0g, distilled water 1000mL, natural pH, sterilized at 121℃ for 20min;

[0046] LB medium composition: yeast powder 0.5 g, peptone 1.0 g, NaCl 1.0 g, natural pH, distilled water 1000 mL (for solid culture, add agar 20.0 g), sterilize at 121°C for 20 min;

[0047] Enzyme production fermentation medium components: CMC-Na 2.0 g, KH2PO4 0.2 g, (NH4)2SO4 1.4 g, distilled water 100 mL;

[0048] Rice straw was purchased online from Lianyungang City, Jiangsu Province; rice soil was collected from the rice fields of Hunan Academy of Agricultural Sciences;

[0049] Urea is 10023218 Shanghai test, analytical grade AR, 500g, content ≥99.0%.

[0050] Example 1

[0051] Strain screening, isolation and identification

[0052] 1. Screening and purification of strains: Weigh 5g of fresh meadow soil from each alpine habitat (the soil comes from Haibei, Qinghai), put it into a 250mL conical flask, add 100mL of sterile water, and shake it at 10°C and 150rpm for 1h to evenly disperse the soil particles into a suspension. Let it stand for 3-4h, and then take 30mL of the supernatant and filter it to obtain the seed liquid. The seed liquid was treated in three ways, including original seed liquid, enrichment treatment, and vancomycin treatment. In order to isolate and culture bacteria using 96-well cell culture plates, the optimal dilution concentration of the treated seed liquid was determined.

[0053] To determine the optimal dilution concentration of the original seed solution, pipette different amounts of seed solution into 500 mL of 10-fold diluted LB liquid medium into an Erlenmeyer flask, shake well, transfer to a 96-well cell culture plate, and culture at 10°C for 10-14 days. Measure the absorbance and determine if microbial growth is observed in approximately 30% of the wells. The optimal dilution concentration is 1500-fold.

[0054] To determine the optimal dilution concentration for the enrichment treatment, add 50 mL of LB liquid medium diluted 10-fold to the seed solution. Incubate for 3 days. Then, determine the optimal dilution concentration using the method for determining the optimal dilution concentration of the original seed solution. The optimal dilution concentration is 3 million-fold.

[0055] The method for determining the optimal dilution concentration of the above antibiotic treatment is consistent with the optimal dilution concentration determined by the original seed solution, which is 1500 times.

[0056] Based on the determined optimal dilution concentration, the treated seed solution was added to a 96-well cell culture plate (200 μL / well) and cultured at 10°C for 2-3 weeks. The absorbance was measured to determine the culture wells where microorganisms grew, and the plate streak method was used for separation and purification.

[0057] 2. Strain re-screening: The strains obtained above were cultured on Congo red solid medium, with each strain replicated 3 times. After inverted culture at 10°C for 5-7 days, the hydrolysis zone around the strain was observed and the diameter of the transparent hydrolysis zone (D, mm) and the colony diameter (d, mm) and their ratio (D / d) were recorded. Finally, 6 cold-resistant strains with strong cellulose degradation ability were obtained ( Figure 1 ).

[0058] 3. Strain Identification

[0059] Fresh bacterial suspensions of the six strains were used as DNA templates for PCR amplification using primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'. The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for 16S rRNA gene sequencing. The sequencing results were submitted to the National Center for Biotechnology Information (NCBI) for BLAST homology sequence analysis. Sequence alignment was performed using MEGA11 software, and a phylogenetic tree was constructed using the neighbor-joining method. Figure 2 ).

[0060] The results were as follows: F1 was Flavobacterium sp., with accession number CP062159.1; F2 was Flavobacterium sp., with accession number CP130042.1; F3 was Flavobacterium sp., with accession number CP150845.1; O1 was Oxalbacteraceae sp., with accession number CP099651.1; S1 was Stenotrophomonas sp., with accession number OP986745.1; and P1 was Pseudomonas sp., with accession number MG757961.1.

[0061] Example 2

[0062] 1. Antagonism test of each strain

[0063] The cross-streak plate method was used to determine the inter-strain antagonism of the above 6 bacterial strains. The specific method is as follows: the bacteria were cross-streaked on LB solid culture medium in pairs, and inverted cultured at a constant temperature of 10°C for 7 days, and whether a sterile zone was formed at the intersection of the two bacterial streaks.

[0064] The results showed that there was no or no obvious antagonistic inhibition between the strains.

[0065] 2. Determination of Cellulase and Filter Paper Enzyme Activity of Strain

[0066] The 3,5-dinitrosalicylic acid (DNS) colorimetric method was used to determine the activity of cellulase and filter paper enzyme produced by the strain. The specific method is as follows:

[0067] 2.1 Preparation of crude enzyme solution: 6 strains were inoculated into enzyme-producing fermentation medium respectively, cultured at 10°C and 180 rpm for 3-4 days, and then centrifuged at 4°C and 8000 rpm for 10 minutes. The supernatant was used as the crude enzyme solution to be tested.

[0068] 2.2 Determination of cellulase activity: Preheat a 10mL stoppered test tube in a 50℃ water bath for 5min, add 0.5mL crude enzyme solution and 2.0mL 1% sodium carboxymethyl cellulose solution as reaction substrates, mix thoroughly and react in a 50℃ water bath for 30min. Immediately after taking out, add 2.5mL DNS reagent to terminate the enzyme reaction, place in a boiling water bath and heat for 5min, take out and cool to room temperature, and dilute to 10mL with distilled water. The blank control does not undergo a 50℃ water bath reaction, and DNS reagent is added to deactivate the enzyme activity. The rest of the treatment is the same. Determine its OD 540 Finally, the cellulase activity was calculated by comparing with the glucose standard curve and enzyme activity calculation formula.

[0069] 2.3 FPase activity assay: Preheat a 10 mL stoppered test tube in a 50°C water bath for 5 min, add 0.5 mL of crude enzyme solution and 2.0 mL of 0.1 mol / L (pH = 4.5) citric acid buffer, mix well, preheat in a 50°C water bath for 5-10 min, then add 50 mg of starch-free filter paper strips (about 1 cm x 6 cm), and keep warm in a 50°C water bath for 60 min. The remaining steps are the same as the cellulase activity assay method.

[0070] By measuring the cellulase and filter paper enzyme activities of the six strains, it was found that the strains of the composite bacterial agent of the present invention have the ability to produce cellulase and filter paper enzyme under low temperature conditions, among which the highest cellulase activity reached 16.48 U / mL and the highest filter paper enzyme activity reached 21.71 U / mL ( Figure 3 ).

[0071] Example 3

[0072] 1. Construction of composite bacterial agent

[0073] Each strain was inoculated into LB liquid medium diluted 10-fold and cultured at 10°C with shaking for 7 days before the culture suspension was collected. Six bacterial cultures of Flavobacterium sp. F1, Flavobacterium sp. F2, Flavobacterium sp. F3, Oxalobacter sp. 01, Stenotrophomonas sp. S1, and Pseudomonas sp. P1 were mixed at a volume ratio of 2:9:1:1:1:1, with a minimum volume of 0.5 mL for each culture to create a composite inoculum. Each composite inoculum was 7.5 mL.

[0074] 2. Application of the above cold-resistant composite bacterial agent

[0075] Soak the rice straw in sterile water for 24 hours, remove soluble organic matter, and then rinse it with sterile water several times. After rinsing, dry it at 85°C to constant weight, then cut the straw into 2-3 cm small segments and sterilize it at 121°C for 20 minutes before use.

[0076] Twenty round-bottom plastic boxes (15.0 cm (top diameter) × 10.5 cm (bottom diameter) × 8.5 cm (height)) were prepared and sterilized. Each box was filled with 400 g of rice soil. Sterile water was sprayed to adjust soil moisture to maintain a moisture content of approximately 60%. The soil surface was then covered with pretreated rice straw. The decomposition experiment consisted of three experimental groups: ① a blank control group uninoculated with the composite bacterial consortium, labeled CK; ② a group spray-inoculated with the composite bacterial consortium, labeled S; and ③ a group spray-inoculated with the composite bacterial consortium and urea, labeled SN. Each treatment was replicated four times. Urea was used as an exogenous nitrogen fertilizer in combination with the composite bacterial consortium to investigate and compare the decomposition effects of rice straw under low-temperature conditions. Urea was added at a rate of 2.0 g / kg of soil.

[0077] The samples were placed in a cool, well-ventilated area at a natural temperature of 5°C-15°C to simulate field temperature fluctuations. Sterile water was added every 20 days to maintain soil moisture. Color changes on the straw surface were observed and recorded. After 60 days, analysis of the straw surface color changes, mechanical strength, chemical composition, and bacterial composition was completed to comprehensively evaluate the decomposition effect of the composite bacterial consortium on rice straw under low-temperature conditions.

[0078] Result analysis:

[0079] 2.1 Color Changes of Decomposing Straw: Observe the color changes of the decomposing straw and take photos to record the results. Use ImageJ software to perform grayscale analysis of the images. Set the software parameters as follows: select 8-bit for Type; 50.0 pixels for Substrate background; and pixel of unit of length for Set scale. Keep all other settings as default. Compare the color changes of the straw during decomposition through grayscale analysis.

[0080] After 60 days of low-temperature decomposition, the color of the rice straw changed significantly, and the degree of decomposition gradually deepened. At 0d, the straw was bright in color, showing light green or light yellow. The straw surface was smooth, the structure was intact and stable, and it was straight and not deformed. At 28d, the straw color deepened, and the overall color was yellow-brown, and some of the straw turned black. Among them, the color of the S-treated rice straw deepened most significantly, followed by the CK and SN straws. Some rice straws became soft and curved. At 60d, the straw in each experimental group turned black as a whole, and only a small amount of straw surface showed yellow, among which the S and CK straws turned significantly black in color. The straw is soft and fragile, and is easy to tear when picked up with tweezers ( Figure 4 ).

[0081] Grayscale analysis results show that ( Figure 5 ), at 0d, the gray values ​​of each experimental group were similar and there was no significant difference. At 28d, the gray value of S straw decreased significantly and was lower than that of CK treated straw, with a difference of 3448841. There was no significant difference in the gray values ​​of the two straws. The gray value of SN straw was higher than that of CK, with a difference of 1754037. Similarly, there was no significant difference in the gray values ​​of the two straws. The gray value of S straw was the lowest and significantly lower than that of SN straw (p<0.05). At 60d, the gray values ​​of each treated straw decreased further, and the gray value of S straw was significantly lower than that of SN and CK straws (p<0.05). However, the gray value of SN straw was still higher than that of CK. The composite bacterial agent showed a better effect of low-temperature decomposition of rice straw, and was better than the straw treated with urea.

[0082] 2.2 Analysis of mechanical strength of decomposed straw: Rice straws of similar size and length were randomly selected from the samples to be tested. Twelve pieces were selected from each test group. The hanging load-bearing measurement was completed and the weight (g) of the straw when it was bent and fell was recorded to indicate the mechanical strength of the straw.

[0083] The results of straw mechanical strength measurement showed that the average load-bearing capacity of straw at CK, S and SN was 38.6g, 16.3g and 29.2g respectively. Figure 6After 60 days of incubation, the mechanical strength of both S and SN straws was lower than that of CK, with the S straw having a significantly lower mechanical strength than both CK and SN (p < 0.05). The composite inoculant exhibited a better low-temperature decomposition effect on rice straw and was superior to straw treated with urea.

[0084] 2.3 Analysis of chemical composition of decomposed straw: The straw after 60 days of cultivation was dried at 45 °C to constant weight, mechanically crushed and passed through a 40-mesh sieve for the determination of cellulose, hemicellulose and lignin contents (a group of fresh rice straw was added for comparison, marked as FS).

[0085] Method for determining cellulose content: weigh 0.1 g of pretreated straw into a test tube, add 5 mL of a mixture of acetic acid and nitric acid in a volume ratio of 1:1, cover and place in a boiling water bath for 25 minutes with constant stirring; remove, dilute with sterile water, centrifuge after cooling, remove the supernatant, repeat 2-3 times and then dry; take the dried sample into a test tube, add 10 mL of a mixture of 10% sulfuric acid and 0.1 mol / L potassium dichromate solution, shake well, place in a boiling water bath for 10 minutes, and transfer it to a conical flask; after cooling, add 5 mL of 20% potassium iodide solution, add 0.2 mol / L Na2S2O3 dropwise until the solution just turns blue and does not change color within half a minute, and make another set of blank controls without adding straw.

[0086] Method for determining hemicellulose content: weigh 0.1 g of pretreated straw into a beaker, add 10 mL of 70% calcium nitrate solution, boil for 5 min, filter, wash with water, and then dry; add 10 mL of 2 mol / L hydrochloric acid, place in a boiling water bath for 45 min, add 1 drop of phenolphthalein and neutralize with sodium hydroxide until it just turns rose color, then filter, wash the filter residue, and determine the total volume of the filtrate and washing liquid.

[0087] Add 1.5 mL of DNS to 2 mL of the mixed filtrate and washings, shake well, and place in a boiling water bath for 5 minutes. Cool to room temperature, then dilute to 15 mL. Measure the OD value at 540 nm. Compare the value to a glucose standard curve and multiply by 0.9 to determine the hemicellulose content.

[0088] Method for determining lignin content: weigh 0.1 g of pretreated straw into a beaker, add 10 mL of 1% acetic acid solution by volume and soak for 45 minutes, filter and wash the residue and then dry; add 4 mL of a mixture of ethanol and ether, soak for a few minutes, remove the supernatant and wash the precipitate, evaporate to dryness in a water bath, add 3 mL of 72% sulfuric acid solution by mass, shake well and let stand for 16 hours, add water and shake well, and boil in a water bath for a few minutes; add 0.5 mL of 10% barium chloride solution by mass, centrifuge to remove the supernatant and wash, and evaporate to dryness in a water bath; add 10 mL of a mixture of 10% sulfuric acid and 0.1 mol / L potassium dichromate solution to the precipitate, shake well and boil in a water bath for 15 minutes, cool, add 5 mL of 20% potassium iodide solution, add 0.2 mol / L Na2S2O3 dropwise until the solution just turns blue and does not change color within half a minute, and make another group of blank controls without adding straw.

[0089] After 60 days of low-temperature decomposition, the changes in the cellulose, hemicellulose, and lignin contents of rice straw are shown in Tables 1 and Figure 7 As shown in Figure 2 . The cellulose, hemicellulose, and lignin contents of fresh rice straw (FS) were 43.7%, 28.1%, and 21.8%, respectively. The cellulose, hemicellulose, and lignin contents of CK straw decreased by 18.6%, 3.7%, and 2.7%, respectively. The cellulose, hemicellulose, and lignin contents of S straw decreased by 30.6%, 11.4%, and 5.4%, respectively. The cellulose, hemicellulose, and lignin contents of SN straw decreased by 24.8%, 6.6%, and 4.6%, respectively.

[0090] Table 1 Changes in cellulose, hemicellulose and lignin content

[0091]

[0092] Note: The reduced content is the initial content (FS) minus the content after decomposition.

[0093] 2.4 Analysis of bacterial composition on the surface of decomposed straw (composite bacterial agent colonization analysis): Decomposed straw samples were collected on the 28th and 60th day of straw decomposition and sent to Novogene Bioinformatics Technology Co., Ltd. for 16S rRNA gene high-throughput sequencing to analyze the bacterial composition and colonization.

[0094] At 28 days, the top 10 bacterial genera in CK primarily included Paludibacter, Desulfovibrio, and Dyadobacter. After inoculation with the composite bacterial consortium, the top 10 bacterial genera in S were primarily Flavobacterium, Desulfovibrio, and Pseudomonas. Inoculation with the composite bacterial agent altered the bacterial composition, particularly the relative abundances of Flavobacterium and Pseudomonas, which increased by 4-fold and 5-fold, respectively. The top 10 bacterial genera in SN primarily included Devosia, Pedobacter, and Brevundimonas. Exogenous nitrogen application primarily increased the abundance of Brevundimonas (418-fold) and Stenotrophomonas (78-fold).

[0095] At 60 days, the top 10 bacterial species in CK and S straw differed significantly from those at 28 days of decomposition. The top 10 bacterial genera in CK primarily included Flavobacterium, Pseudomonas, and Stenotrophomonas. Without inoculation with a complex bacterial consortium, the relative abundance of these bacteria only began to increase in the later stages of natural straw decomposition. Compared with CK, S primarily increased the relative abundance of Devosia, Hyphomicrobium, and Chryseobacterium. In SN, the top 10 bacterial species primarily included Pedobacter, Flavobacterium, and Brevundimonas.

[0096] In general, the main bacterial communities in the composite inoculant, Flavobacterium and Pseudomonas, colonized well on the surface of S straw and were more active in the early stages of straw decomposition. Stenotrophomonas colonized well on SN straw.

[0097] In summary, the cold-resistant composite bacterial agent of the present invention can efficiently decompose rice straw under low-temperature conditions.

[0098] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A cold-resistant composite bacterial agent for low-temperature decomposition of rice straw, characterized in that: It is composed of Flavobacterium sp. F1, Flavobacterium sp. F2, Flavobacterium sp. F3, Oxalbacteraceae sp. O1, Stenotrophomonas sp. S1 and Pseudomonas sp. P1; The volume ratio of the bacterial liquids of Flavobacterium F1, Flavobacterium F2, Flavobacterium F3, Oxalobacter 01, Stenotrophomonas S1 and Pseudomonas P1 is 2:9:1:1:1:1; The Flavobacterium F1 was deposited in the China Center for Type Culture Collection on July 1, 2024, at Wuhan University, Wuhan, China, with the deposit number CCTCC M 20241422; The Flavobacterium F2 was deposited in the China Center for Type Culture Collection on July 1, 2024, at Wuhan University, Wuhan, China, with the deposit number CCTCC M 20241423; The Flavobacterium F3 was deposited in the China Center for Type Culture Collection on July 1, 2024, at Wuhan University, Wuhan, China, with the deposit number CCTCC M 20241424; The Oxalobacter 01 was deposited in the China Center for Type Culture Collection on July 1, 2024, at Wuhan University, Wuhan, China, with the deposit number CCTCC M 20241425; The Stenotrophomonas S1 was deposited on July 1, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC M 20241426; The Pseudomonas P1 was deposited in the China Center for Type Culture Collection on July 1, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC M 20241427.

2. The method for preparing the cold-resistant composite bacterial agent according to claim 1, wherein: The following steps are involved: Each strain was activated and cultured separately, and bacterial liquid was collected; bacterial liquids of Flavobacterium F1, Flavobacterium F2, Flavobacterium F3, Oxalobacter 01, Stenotrophomonas S1 and Pseudomonas P1 were mixed in a volume ratio of 2:9:1:1:1:1 to obtain the cold-resistant composite bacterial agent.

3. Use of the cold-resistant composite bacterial agent as claimed in claim 1 in degrading rice straw.

4. A product for promoting the degradation of rice straw, characterized in that: The product includes the cold-resistant composite bacterial agent according to claim 1.

5. A method for degrading rice straw, characterized in that: Rice straw is degraded using the cold-resistant composite bacterial agent described in claim 1 or the product described in claim 4.

6. The degradation method according to claim 5, characterized in that: The conditions for the degradation treatment are: temperature of 5-15° C. and soil moisture content of 60%.

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