A method for improving the degradation efficiency of corn stalk at low temperature

Abstract

A method for improving the low-temperature decomposition efficiency of corn stalks includes the use of *Pseudomonas fruticosa* (… Pseudomonas fragi Cell-free culture medium PfCFs prepared by strain D12 participated in the fermentation and degradation of corn straw. The taxonomic name of strain D12 is *Pseudomonas fruticosa*. Pseudomonas fragi Cell-free culture medium of *Pseudomonas frutescens* D12, deposited at the China Center for Type Culture Collection (CCTCC), No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M2025126 and deposit date of January 14, 2025, can promote the growth of various room-temperature bacteria and fungi at low temperatures, improving the cold resistance of functional bacteria. The cell-free culture medium (CFS) of *Pseudomonas frutescens* D12 also contains enzymes that can degrade lignin. At low temperatures, the CFS simultaneously promotes the co-growth of *Bacillus subtilis* and *Alternaria alternata*, thereby improving the degradation efficiency of lignocellulose in corn stalks by functional bacteria.

Description

[0001] This invention is a divisional application of patent 202510712812.7 entitled "A type of Pseudomonas berries producing microbial low-temperature growth-promoting protein and its application". Technical Field

[0002] This invention relates to the field of microbial technology, and more specifically to a method for improving the low-temperature decomposition efficiency of corn stalks. Background Technology

[0003] Microorganisms play a vital role in modern agriculture and biotechnology applications, serving as biofertilizers, plant growth promoters, and food fermentation agents. However, low temperatures often limit the activity and growth rate of beneficial microorganisms, thus affecting their practical application. For example, in cold climates, the activity of plant rhizosphere microbial communities decreases, leading to a decline in plant stress resistance.

[0004] To address the aforementioned issues, researchers have explored various methods to enhance the growth capacity of beneficial bacterial strains under low-temperature conditions. One effective strategy is to utilize the metabolites or signaling molecules secreted by the strains to promote their growth through cellless fermentation (CFS). Cellless fermentation involves fermenting the strains and then removing cellular components using methods such as centrifugation and filtration, retaining only the liquid portion secreted by the strains. This liquid contains various bioactive substances, such as enzymes, metabolites, antibiotics, and signaling molecules. Previous studies have shown that certain... Pseudomonas CFS of certain strains has the ability to promote the growth of other microorganisms. For example, existing technologies have reported... Pseudomonas fluorescens The secreted metabolites have been shown to enhance the stress resistance and growth activity of plant rhizosphere microorganisms. Furthermore, there are also literature reports... Pseudomonas aeruginosa CFS (Continuous Fermentation Promoter) can promote the growth of certain lactic acid bacteria and improve fermentation efficiency under certain conditions. However, there is currently limited research on highly efficient CFS strains that can broadly promote the growth of various microorganisms under low-temperature conditions. For example, CN117736931A discloses a low-temperature resistant version of *Pseudomonas berries*, but does not disclose its ability to promote the growth of other microorganisms at low temperatures. Patent CN113832067A discloses the preparation of a low-temperature growth promoter for room-temperature bacteria using low-temperature *Pseudomonas berries*, but its target is only bacteria, and there is no record of its effect on fungi. Existing CFS applications are mostly focused on room temperature or specific temperatures, lacking research results on their stable function in low-temperature environments.

[0005] Furthermore, existing methods for promoting bacterial growth using CFS typically rely on the selection of specific strains and the optimization of culture conditions. However, these methods have limited applicability across different low-temperature conditions and target strains, and often require complex preparation processes, increasing the difficulty and cost of application. Therefore, there is an urgent need to develop a CFS-producing strain that can efficiently and broadly promote the growth of multiple bacteria under low-temperature conditions to meet the needs of agriculture and biopharmaceutical production. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a strain of *Pseudomonas berries*. The cell-free culture medium (CFS) produced by this strain during cultivation can significantly enhance the growth ability of various microorganisms whose growth is inhibited at low temperatures (below 15°C) under low-temperature conditions. This solves the problem in the prior art that it is impossible to promote the co-growth of various bacteria, fungi, and other microorganisms under low-temperature conditions, and has significant innovation and broad application prospects.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A strain that produces microbial low-temperature growth-promoting proteins, characterized in that: the strain is classified as *Pseudomonas fruticosa* (…). Pseudomonas fragi D12, deposited at the China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, accession number CCTCC NO: M2025126, deposited on January 14, 2025.

[0009] The *Pseudomonas berries* D12 is a Gram-negative, short rod-shaped bacterium with a length of 1.8-2.2 μm. The colony morphology is irregularly round with a concave center and serrated edges. The color changes to milky yellow and translucent. *Pseudomonas berries* D12 can grow efficiently at temperatures below 15°C, and its cell-free culture medium has growth-promoting activity against a variety of bacteria, fungi, and other microorganisms at low temperatures.

[0010] Experiments have shown that the CFS of *Pseudomonas berries* D12 at 10℃ inhibits the growth of various target strains that are inhibited at low temperatures, such as... Bacillus subtilis (Bacillus subtilis) Bacillus cereus (Bacillus cereus) Bacillus megaterium (Bacillus megaterium) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), fungi such as Aspergillus niger (Aspergillus niger) Trichoderma reesei (Trichoderma reesei) Trametes versicolor (White rot fungus) and Irpex lacteus The growth and metabolism of functional strains such as (Bacillus albus) are significantly promoted, showing broad application potential and good stability.

[0011] In addition, during the cultivation process, *Pseudomonas berries* D12 can use lignin as the sole carbon source for growth and metabolism, thereby achieving the decomposition of lignin.

[0012] Furthermore, the growth temperature of the aforementioned *Pseudomonas berryensis* D12 is 4~30℃.

[0013] Furthermore, the working temperature of the cell-free culture medium produced by the above-mentioned *Pseudomonas berries* D12 is 0~15℃, preferably 10℃.

[0014] Pseudomonas berries D12 and its prepared cell-free culture medium can be applied to low-temperature agricultural production to improve the growth efficiency and activity of the target strain under low-temperature conditions.

[0015] Specifically, the application of *Pseudomonas berries* D12 and its cell-free culture medium in promoting the use of low-temperature-intolerant microorganisms in low-temperature fermentation, specifically in low-temperature composting fermentation processing, wherein the low-temperature fermentation temperature is 0~15℃.

[0016] More preferably, the low-temperature fermentation temperature is 10°C.

[0017] The present invention has the following technical effects:

[0018] The CFS produced by *Pseudomonas berries* D12 of this invention can promote the growth of various cold-sensitive bacteria and fungi at temperatures below 15°C, improving their cold resistance. Secondly, it can enhance the metabolic capacity of these functional bacteria and increase the low-temperature enzyme activity of enzymes that degrade lignocellulose. Furthermore, *Pseudomonas berries* D12 can use lignin as its sole carbon source for growth and metabolism, and the CFS of *Pseudomonas berries* D12 also contains enzymes that can degrade lignin, further improving the degradation efficiency of lignocellulose at low temperatures. Attached Figure Description

[0019] Figure 1 This invention provides a pulsed-field gel electrophoresis pattern of Pseudomonas berryis D12.

[0020] Figure 2 Growth curves of Pseudomonas berryis D12 at different temperatures.

[0021] Figure 3 OD values ​​of PfCFs for different bacteria after culturing at 10℃ for 52 h.

[0022] Figure 4 Effects of PfCFs on the dry weight (DW) of different fungi after culturing at 10℃ for 96 h.

[0023] Figure 5 :PfCFs Bacillus subtilisEffects of cellulase and xylanase activities after culturing at 10℃ for 52 h.

[0024] Figure 6 Growth status of *Pseudomonas berryii* D12 in a lignin-based carbon source medium.

[0025] Figure 7 Growth curves and lignin concentration changes of Pseudomonas berries D12 in lignin-based carbon source media.

[0026] Figure 8 Different molecular weights of PfCFs have the effect on Bacillus subtilis OD value after culturing at 10℃ for 52 h.

[0027] Figure 9 Different substances in PfCFs Bacillus subtilis Effect of OD value after culturing at 10℃ for 52 h.

[0028] In the attached figure, “***” and “****” indicate the range of significant differences: ***: 0.0001 < p < 0.001; ****: p < 0.0001. Detailed Implementation

[0029] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0030] The low-temperature intolerant microorganisms mentioned in this invention refer to microorganisms whose growth and metabolism are inhibited, whose activity is poor, or even whose growth and metabolism cease at temperatures below 15°C, especially functional bacteria such as fungi and bacteria that degrade lignocellulose.

[0031] Example 1

[0032] Isolation, screening and identification of the original strain

[0033] (a) Isolation of strains

[0034] (1) Collect 5g of soil from the forest area of ​​Changbai Mountain Nature Reserve, dissolve it in 100mL of prepared salt solution, and culture it at 4℃ and 180 rpm for 4 h with shaking. The salt solution is composed of 0.006 mM FeSO4·7H2O; 0.01 mM CaCO3·7H2O; 0.08 mM MgSO4·7H2O; 0.07 mM MnSO4·7H2O and 0.006 mM ZnSO4·7H2O.

[0035] (2) 1 mL of the well-mixed soil sample salt solution was subjected to different gradients (10) -2 10 -3 10 -4 Dilute the solution and spread 200 μL of the diluted solution onto R2a solid plates. Incubate the plates at 4°C for 10 days. Each plate contains 0.5 g yeast extract, 0.5 g peptone, 0.5 g casein hydrolysate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g potassium dihydrogen phosphate, 0.024 g anhydrous magnesium sulfate, 0.3 g sodium pyruvate, 15.0 g agar, and 1000 mL of distilled water. The final pH is 7.2 ± 0.2.

[0036] (3) Then, single colonies are selected based on their morphology, color, size and gloss.

[0037] (II) Screening of the original strain D12

[0038] (1) Dissolve the selected single colonies in physiological saline (0.8% sodium chloride solution), use an inoculation loop to take an appropriate amount of bacterial solution and streak it on LB solid medium. After multiple subcultures, combined with microscopic observation, select single colonies with uniform morphology and state and place them in LB liquid medium. Shake flask culture at 160 rpm and 4℃ for 3 days to obtain fermentation broth.

[0039] (2) The fermentation broth obtained above was centrifuged (6000 rpm, 15 min, 4 ℃) and filtered through a 0.22 μm filter membrane to obtain cell-free culture medium.

[0040] (3) Add the above cell-free culture medium to a liquid culture medium containing the indicator strain Bacillus subtilis, culture at 0~15℃ and observe the growth. At the same time, set up a control group without cell-free culture medium.

[0041] (4) By measuring the growth curve of the indicator strain, the strain that can produce a cell-free culture medium that promotes bacterial growth was named D12. The selected strain was inoculated into LB liquid medium and cultured with shaking at 160 rpm and 4°C. The logarithmic phase bacterial culture was preserved with glycerol (400 uL of 50% glycerol was added to 600 uL of bacterial culture).

[0042] The growth curve of the indicator strain shows that the screened original strain D12 has the ability to produce bacterial growth factors under low temperature conditions.

[0043] Example 2

[0044] New strains obtained by mutagenesis of the original strain and their identification

[0045] (I) ARTP mutagenesis treatment (1) Take the logarithmic phase bacterial culture of the original strain D12, wash it with sterile physiological saline, and prepare a solution with a concentration of 1×10 8 (2) The bacterial suspension was treated with an ARTP mutagenesis system (power 120 W, helium flow rate 10 SLM). The lethality curve was plotted through a preliminary experiment, and the optimal treatment time was determined to be 90 s (lethality rate 85±3%). (3) The mutagenized bacterial suspension was spread on LB selective plates containing 0.1% sodium dodecyl sulfate (SDS). After culturing at 4℃ for 5 days, morphologically modified single colonies (diameter increased by 30%-40% compared with the original strain, and radial wrinkles appeared at the edge) were picked. (4) Genetic stability was verified by three generations of subculture, and the phenotypic stable mutant strain D12 (ARTP mutant strain) was selected.

[0046] (II) Analysis of physiological and biochemical characteristics of the strains before and after mutagenesis (1) Morphological observation: Gram staining showed that the original strain D12 was a Gram-negative short rod-shaped bacterium with a length of about 2 μm. The colony shape was round, slightly raised in the middle, with smooth edges, and the colony color was opaque white. The mutant strain D12 maintained the Gram-negative short rod shape (1.8-2.2 μm), but the colony shape changed to an irregular round shape with a concave center and serrated edges. The color changed to milky yellow and translucent.

[0047] (2) Changes in key metabolic indicators:

[0048] Physiological identification was performed on the strains before and after mutagenesis. The measured physiological indicators are shown in Table 1. "+" represents positive and "-" represents negative.

[0049] Table 1:

[0050]

[0051] (3) Functional verification: The cell-free culture medium of mutant strain D12 was added to the culture medium containing Bacillus subtilis under the same conditions as described above. After culturing at 10℃ for 48 h, the biomass increased by 42% compared with the original D12 treatment group (OD600 0.86 vs 0.61), and the indicator bacteria lag period was shortened by 3.5 h.

[0052] (III) Molecular identification

[0053] (1) By comparing the mutant strain D12 with all available 16S rDNA sequences in the NCBI database using blastn, the mutant strain D12 showed 99.8% similarity to the original strain D12, and its sequence listing is shown in SEQ ID NO.1. Finally, based on morphological observation and physiological and biochemical tests, it was speculated that the mutant strain D12 after mutagenesis was *Pseudomonas berryae*, and the mutant strain D12 was finally named *Pseudomonas berryae* D12 (… Pseudomonas.fragiD12);

[0054] (2) Whole-genome resequencing revealed an A214G point mutation in the gyrB gene, which upregulated the expression of the sigma factor rpoS gene, which is associated with phenotypic changes, by 3.7-fold.

[0055] (3) Pulsed-field gel electrophoresis, such as Figure 1 As shown, combined with whole-genome sequencing comparison, the chromosome size increased from 5.2 Mb to 5.4 Mb before and after the mutation, suggesting the presence of genomic island insertion.

[0056] The mutant strain *Pseudomonas berries* D12 obtained by mutagenesis in this invention is deposited at the China Center for Type Culture Collection and classified as *Pseudomonas berries*. Pseudomonas fragi D12, with the address at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, accession number CCTCC NO: M2025126, and accession date January 14, 2025.

[0057] Example 3: Growth of *Pseudomonas berries* D12 and preparation of cell-free culture medium

[0058] (a) Growth characteristics of Pseudomonas berries D12

[0059] (1) The mutagenized *Pseudomonas berries* D12 was inoculated into LB liquid medium and cultured with shaking at 180 rpm and different temperatures (4, 10, 15, 25, 30℃). Samples were taken at fixed points to measure the OD of the bacterial culture. 600 The values ​​were used to plot growth curves at different temperatures, and the results are as follows: Figure 2 As shown.

[0060] (II) Preparation of cell-free culture medium for *Pseudomonas berries* D12

[0061] After removing *Pseudomonas berryae* D12 from -80℃ storage, single colonies were first isolated by streaking on LB agar plates containing 10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone, and 2% agar powder. Then, single colonies were picked and inoculated into LB liquid medium for rejuvenation culture. Next, 1 mL of the rejuvenation culture was transferred to 100 mL of liquid LB medium and cultured at 30℃ for 5 days at 160 rpm. After the incubation period, the cells were separated from the supernatant by centrifugation at 12000 rpm for 10 minutes at 4℃. The supernatant was then filtered through a 0.22 μm sterile filter to obtain cell-free culture medium (PfCFs) of *Pseudomonas berryae* D12.

[0062] Example 4

[0063] The promoting effect of cell-free culture medium (PfCFs) on the low-temperature growth of functional strains

[0064] Select functional strains (including bacteria and fungi) that have cellulose degradation capabilities but whose growth is limited at temperatures below 15°C. Bacteria, such as… Bacillus subtilis (Bacillus subtilis) Bacillus cereus (Bacillus cereus) Bacillus megaterium (Bacillus megaterium) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), fungi such as Aspergillus niger (Aspergillus niger) Trichoderma reesei (Trichoderma reesei) Trametes versicolor (White rot fungus) and Irpex lacteus (Bacterium tumefaciens), by adding 25% PfCFs and culturing at 10℃, with the control group being cultured at 10℃ without PfCFs, was used to evaluate the promoting effect of PfCFs on the low-temperature growth of the above functional bacteria (the optimal growth temperature for the above functional bacteria is 25~35℃, and growth is significantly inhibited below 15℃). The experimental results are as follows: Figure 3 and Figure 4 As shown, under low temperature conditions, the growth rate of functional bacterial strains and the number of functional fungal cells in the experimental group with added PfCFs were significantly higher than those in the control group.

[0065] Example 5

[0066] Changes in the activity of key enzymes in functional strains and their functional characteristics at low temperatures

[0067] Under low-temperature conditions, the activities of key enzymes in functional strains are often inhibited, thus limiting their biodegradation capacity. Therefore, the effects of PfCFs on the enzyme activities of functional bacteria were tested at temperatures as low as 10°C.

[0068] (1) This invention systematically evaluated the changes in the activity of key enzymes—cellulase and xylanase—of functional strains at low temperatures by adding cell-free culture medium of Pseudomonas berries D12 (PfCFs) to a culture medium containing Bacillus subtilis.

[0069] Experimental results are as follows Figure 5 As shown, the addition of 25% PfCFs significantly enhanced the activities of cellulase and xylanase at a low temperature of 10℃. At 10℃, cellulase activity increased by 283.61% compared to the control group, and xylanase activity increased by 313.79%. This enhanced enzyme activity not only improved the degradation efficiency of cellulose and xylan at low temperatures but also significantly improved the overall metabolic activity and growth rate of the strain.

[0070] (2) Degradation of lignin by Pseudomonas berries D12

[0071] The culture medium using lignin as the sole carbon source is prepared as follows:

[0072] Lignin degradation medium (g / L): 1.0 g lignin, 1.0 g KH₂PO₄, 1.0 g K₂HPO₄, 1 g NaNO₃, 1 g (NH₄)₂SO₄, 1 g NaCl, 0.5 g MgSO₄·7H₂O, 0.05 g CaCl₂, diluted to 1 L with distilled water, and the pH adjusted to 6.8. In the above medium with lignin as the sole carbon source, the inoculum size was 1% (6.1 × 10⁻⁶ g / L). 8 CFU / mL) of *Pseudomonas berries* D12 was inoculated and cultured with shaking at 10°C for 48 h. The growth of *Pseudomonas berries* D12 in the lignin-based carbon source medium is as follows: Figure 6 As shown.

[0073] *Pseudomonas fruticosa* D12 was inoculated into the culture medium with lignin as the sole carbon source and cultured with shaking at 10°C for 48 hours. Samples were taken every 12 hours, and the supernatant was diluted 10-fold with distilled water. Using distilled water as a control, the absorbance was measured at 280 nm using a UV spectrophotometer. The absorbance values ​​were then substituted into the regression equation to calculate the lignin degradation rate. The formula is as follows:

[0074] Lignin degradation rate (%) = A1 - A2 / A1 × 100%

[0075] Where A1 represents the concentration of lignin in the non-inoculated lignin degradation medium (control).

[0076] A2 represents the concentration of lignin in the sample to be tested, and the concentration of lignin remaining in the culture medium at a certain moment.

[0077] The results are as follows Figure 7 As shown, the OD value trend is a rapid increase followed by a slow increase to reach its peak. *Pseudomonas berries* D12 grows rapidly in the first 24 hours of culture, with the fastest OD value increase, reaching 1.886 at 36 hours. Growth slows down between 24 and 36 hours, reaching its peak at 36 hours with an OD value of 2.117, at which point the viable cell count increases by 7.0 × 10⁻⁶. 8 CFU / mL.

[0078] The trend of lignin degradation rate was observed by examining changes in lignin concentration. The initial lignin content was approximately 0.51 g. With increasing culture time, the lignin content gradually decreased, while the degradation rate gradually increased. At the beginning of culture (12 hours), the lignin degradation rate was 15%, reaching its maximum at 24 hours (22.6%), with a lignin content of 0.397 g in the solution. The degradation rate then stabilized and remained relatively constant over the next 24-48 hours. This indicates that *Pseudomonas berries* D12 can grow using lignin as its sole carbon source under low-temperature conditions, and it can synthesize enzymes related to lignin degradation under these conditions, thus achieving its lignin-degrading ability.

[0079] Low-temperature enzyme activity is closely related to the functional characteristics of strains at low temperatures. The results above show that the addition of CFS to Pseudomonas berries D12 improves the growth and metabolism of functional bacteria at temperatures below 15℃, increases the enzyme production efficiency of the strain, significantly improves the low-temperature enzyme activity of the corresponding enzymes, and improves the growth and metabolism of functional strains at low temperatures, thus verifying its potential value in practical applications.

[0080] Example 6

[0081] Preliminary exploration of growth-promoting factors in cell-free culture medium of *Pseudomonas berries* D12

[0082] To preliminarily identify the key factors promoting the growth of functional strains of *Pseudomonas fructus* D12 in cell-free culture medium, *PfCFs* were separated into components of different molecular weights and subjected to *Bacillus subtilis* at 10°C. The experimental results are as follows: Figure 8 As shown, under the conditions of PfCFs components with molecular weights of 5-30 kDa, the OD value of Bacillus subtilis increased significantly, indicating that the components with molecular weights of 5-30 kDa have extremely significant growth-promoting activity for bacteria at room temperature. However, the OD values ​​of other molecular weight components remained basically unchanged, indicating that the growth-promoting activity of PfCFs components with molecular weights less than 5 kDa and greater than 30 kDa is poor, and they have basically no growth-promoting effect on functional bacteria at low temperatures.

[0083] To investigate the growth-promoting factors of the 5-30 kDa molecular weight fraction, the growth-promoting activities of biomolecules such as polysaccharides, nucleic acids, and proteins in this fraction of PfCFs were further validated:

[0084] Nucleic acid extraction involves using a specialized kit to isolate genomic DNA and total RNA from PfCFs. The polysaccharide extraction process includes steps such as strain activation, expansion culture, cell removal, concentration, protein removal, alcohol precipitation, and dialysis, ultimately yielding the crude polysaccharide product.

[0085] The results were verified through experiments, as follows: Figure 9The results showed that the crude polysaccharide and nucleic acid extracts from cell-free culture medium of *Pseudomonas fruticosa* D12 did not promote the growth of the functional strain. Following the testing sequence, after confirming that PfCF nucleic acid and extracellular polysaccharides had no significant promoting effect on the low-temperature growth of the functional strain, proteinase K was directly used to enzymatically digest some proteins in PfCFs to observe the effect of PfCF proteins on the functional bacteria. This eliminated the complex protein extraction steps of precipitation, centrifugation, and dialysis desalting. The figures show that the growth-promoting activity of the cell-free culture medium treated with proteinase K, which disrupted some protein structures, was lower than that of the untreated cell-free culture medium, indicating that proteins play a key role in the growth-promoting effect.

[0086] In summary, the proteins in the cell-free culture medium of *Pseudomonas berries* D12 effectively promoted the growth and metabolism of the functional strain at low temperatures and enhanced the activity of key enzymes. Furthermore, the CFS of *Pseudomonas berries* D12 also contains enzymes that can degrade lignin, further improving the degradation efficiency of lignocellulose at low temperatures. These functions provide an effective solution for biomass degradation under low-temperature environments and have broad application prospects.

[0087] The verification of the above-mentioned functions of Pseudomonas berries D12 and its CFS further clarifies that D12 and its CFS can be applied to agricultural production, such as microbial fermentation under low-temperature conditions (e.g., compost fermentation, feed fermentation, biomass fermentation for sugar production, ethanol production and other biotransformations).

[0088] Example 7

[0089] CFS prepared by *Pseudomonas fruticosa* D12 was applied to straw degradation:

[0090] A bacterial culture of Bacillus subtilis that produces cellulase and xylanase at room temperature but is inhibited at low temperatures (optimal growth temperature 30℃, viable count 2.0 × 10⁻⁶). 8 Add 25% PfCFs (cfu / mL, which basically stops growth and metabolism below 6℃) to form a compound microbial agent. The volume ratio of PfCFs to the microbial liquid is 1:100. Sprinkle the compound microbial agent into the pretreated corn stalks and ferment in a natural environment of 0~6℃.

[0091] Meanwhile, corn stalks pretreated with Bacillus subtilis culture without the addition of PfCFs were fermented under the same conditions as a control group without PfCFs.

[0092] Comparative Example 1

[0093] A strain of *Pseudomonas spp.* was purchased, and CFS was prepared. A compound microbial agent was then prepared using the same method as in Example 7, and the same corn stalks were fermented under the same conditions. The *Pseudomonas spp.* strain was purchased from Beijing BioBio Biotechnology Co., Ltd., with platform number bio-02547 and original number L3-1.

[0094] Example 8

[0095] CFS prepared by *Pseudomonas fruticosa* D12 was applied to straw degradation:

[0096] The bacterial culture of Bacillus subtilis, which produces cellulase and xylanase at room temperature but is inhibited at low temperatures (optimal growth temperature 30℃, viable count 2.0 × 10⁻⁶), was used to extract the bacteria. 8 The bacterial culture of *Alternaria alternata* (cfu / mL, essentially ceasing growth and metabolism below 6℃) and *Alternaria alternata*, which produces lignin-degrading enzymes that degrade lignin at room temperature but are inhibited at low temperatures (optimal growth temperature 28℃, viable count 1.0 × 10⁻⁶) was obtained. 8 (cfu / mL, basically ceasing growth and metabolism below 6℃) are mixed in a 1:1 volume ratio to form a compound bacterial agent. Then, 25% PfCFs are added to form a compound bacterial agent. The volume ratio of PfCFs to the mixed bacterial agent is 1:100. The compound bacterial agent is sprinkled into the treated corn stalks and fermented in a natural environment of 0~6℃.

[0097] Meanwhile, corn stalks pretreated with a mixed bacterial solution (composed of Bacillus subtilis and Bacillus albus in a 1:1 volume ratio) without the addition of PfCFs were fermented in the same environment as control group 2 without PfCFs. The corn stalks were fermented at room temperature of 30℃ and at temperatures of 0~6℃, respectively.

[0098] After 30 days of fermentation, the degradation efficiency of cellulose, hemicellulose, and lignin in the straw of each group was statistically analyzed to reflect the degree of straw fermentation. The degradation rates of lignin, cellulose, and hemicellulose in the fermented corn straw were determined using chemical analysis methods (such as the Van der Waals detergent method). The specific steps are as follows:

[0099] (a) Determination of neutral detergent fiber (DNF)

[0100] 1. Weigh 1g of the air-dried sample, place it in a beaker, add 100mL of neutral detergent and a few drops of n-octanol.

[0101] 2. Boil and reflux for 1 hour, then filter while hot using a glass frit crucible of known weight.

[0102] 3. Rinse the residue with boiling water until the filtrate is neutral, and dry it together with the crucible residue at 105°C to constant weight. Record the weight m1 (DNF + crucible weight m0).

[0103] (II) Determination of Acid Detergent Fiber (ADF)

[0104] 1. Take the DNF from (I), add 100mL of acidic detergent and a few drops of n-octanol, and boil under reflux for 1 hour.

[0105] 2. While still hot, filter the mixture using a glass frit crucible of known weight. Rinse the residue with boiling water until the filtrate is neutral. Dry the mixture, along with the crucible, at 105°C until constant weight. Record the weight m2 (ADF + crucible weight m0).

[0106] (III) Determination of Acid-Washed Lignin (ADL)

[0107] 1. Take the dry ADF from (II), add 5 mL of 72% sulfuric acid, and stir at room temperature for 2 hours to digest and dissolve the cellulose.

[0108] 2. Dilute with water to a sulfuric acid concentration of approximately 3%, filter, rinse with hot water until neutral, and dry the crucible and residue at 105°C to constant weight. The weight is recorded as m3 (ADL + ash content + crucible weight m0).

[0109] 3. Ash in a muffle furnace at 550℃ for 2 hours, cool and weigh, and record as m4 (acid-insoluble ash + crucible weight m0).

[0110] NDF = (m1 - m0) / sample dry weight × 100%.

[0111] ADF = (m2 - m0) / sample dry weight × 100%.

[0112] Hemicellulose (%) = D = NDF - ADF

[0113] Lignin content = (m3 – m4) / sample dry weight × 100%.

[0114] Cellulose content (%) = ADF - DNF - Acid-insoluble ash

[0115] Formulas for calculating the degradation rate of each component:

[0116]

[0117] The *Bacillus subtilis* and *Bacillus albus* strains mentioned above were all self-selected. The final results are shown in Table 2.

[0118] Table 2: Effect of PfCFs on the fermentation efficiency of ambient-temperature bacteria at low temperatures

[0119]

[0120] The degradation results of lignocellulose in corn stalks show that in the control group 1 without PfCFs, Bacillus subtilis completely ceased growth and metabolism at temperatures below 6°C, resulting in extremely low degradation efficiency of lignocellulose in the stalks, all below 10%. However, in Example 7, the addition of the PfCFs of this invention significantly improved the degradation rates of lignin, hemicellulose, and cellulose. This indicates that the addition of PfCFs enhanced the growth and metabolism of Bacillus subtilis at low temperatures, thereby achieving the degradation of cellulose and hemicellulose. In Comparative Example 1, CFS also improved the degradation efficiency of cellulose and hemicellulose in the stalks, but the overall efficiency was lower than in Example 7, and it did not promote the degradation efficiency of lignin. In Example 7, in addition to higher degradation efficiency of cellulose and hemicellulose, the PfCFs also achieved a lignin degradation rate of 16.16%, indicating that the PfCFs of this invention contain enzymes that can degrade lignin at low temperatures, thus further improving the degradation efficiency of lignin in the stalks.

[0121] In control group 2, without the addition of PfCFs, the degradation efficiency of lignin, hemicellulose, and cellulose bacteria in corn stalks decreased to below 10% at temperatures below 6°C. This indicates that at this temperature, both *Bacillus subtilis* and *Bacillus thuringiensis* ceased growth and metabolism, and could not effectively participate in the degradation of lignin and cellulose. Example 8 further added PfCFs to control group 2. It can be seen that at low temperatures, PfCFs simultaneously promoted the co-growth of the bacteria *Bacillus subtilis* and the fungus *Bacillus thuringiensis*, thereby improving the degradation efficiency of lignin and cellulose in corn stalks by the functional bacteria. Since PfCFs contain enzymes that can degrade lignin at low temperatures, in Example 8, they worked together with *Bacillus thuringiensis*, which produces lignin-degrading enzymes, increasing the concentration and activity of lignin-degrading enzymes in the degradation system, further improving the degradation efficiency of lignin. The lignin degradation rate reached 27.37%, thus altering the overall degradation effect of the compound microbial agent on lignin and cellulose in corn stalks.

Claims

1. A method for preparing a cell-free culture medium that promotes the low-temperature growth of microorganisms, characterized in that: The cell-free culture medium is a cell-free culture medium PfCFs of *Pseudomonas berrieseri* D12, and the *Pseudomonas berrieseri* is *Pseudomonas berrieseri* (…). Pseudomonas fragilis D12, deposited at the China Center for Type Culture Collection (CCTCC), No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M2025126 and deposit date of January 14, 2025, is a single colony of *Pseudomonas berrieseri* D12 isolated by streaking on an LB agar plate containing 10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone, and 2% agar. The colony was then picked and inoculated into LB liquid medium for rejuvenation culture. 1 mL of the rejuvenation culture was then transferred to 100 mL of liquid LB medium and incubated at 30°C for 5 days at 160 rpm. After incubation, the culture was... Centrifuge at rpm for 10 minutes, separate the bacterial cells and supernatant at 4°C, and then filter the supernatant through a 0.22 μm sterile filter membrane to obtain cell-free culture medium PfCFs of Pseudomonas berries D12.

2. A method for improving the low-temperature degradation efficiency of corn straw based on the cell-free culture medium prepared according to claim 1, characterized in that: Bacillus subtilis, which produces cellulase and xylanase at room temperature but whose production is inhibited at low temperatures (… Bacillus subtilis Add 25% PfCFs by mass to the bacterial solution to form a compound bacterial agent. Sprinkle the compound bacterial agent into the pretreated corn stalks and ferment them in a natural environment at 0~6℃.

3. The method for improving the low-temperature decomposition efficiency of corn stalks as described in claim 2, characterized in that: The volume ratio of PfCFs to bacterial culture was 1:100, the optimal growth temperature of Bacillus subtilis was 30℃, and the viable cell count was 2.0 × 10⁻⁶. 8 cfu / mL.

4. A method for improving the low-temperature degradation efficiency of corn stalks based on the cell-free culture medium prepared according to claim 1, characterized in that: Bacillus subtilis, which produces cellulase and xylanase at room temperature but is inhibited at low temperatures, Bacillus subtilis The bacterial culture of *Aureobasidium leucopsis* and the lignin-degrading enzyme that degrades lignin at room temperature but is inhibited at low temperatures. Milkweed The bacterial solution is composed of a mixed bacterial solution, and then 25% PfCFs is added to form a compound bacterial agent. The compound bacterial agent is sprinkled into the treated corn stalks and fermented in a natural environment of 0~6℃.

5. The method for improving the low-temperature decomposition efficiency of corn stalks as described in claim 4, characterized in that: The mixed bacterial solution contained Bacillus subtilis and Alternaria alternata in a 1:1 volume ratio, PfCFs in a 1:100 volume ratio, and Bacillus subtilis had an optimal growth temperature of 30°C and a viable count of 2.0 × 10⁻⁶ cells / day. 8 The optimal growth temperature for *Bacillus thuringiensis* was 28℃, with a cfu / mL concentration and a viable count of 1.0 × 10⁻⁶. 8 cfu / mL.

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