Method for improving yield of glucose by pretreating corn straws through two-step method

Abstract

The invention relates to a microbial strain for degrading lignin, the classification name of the strain is Cosakoniasanii P23, the strain is preserved in the China Center for Type Culture Collection (CCTCC), the preservation address is number 299 of eight road, Wuchang District, Wuhan City, Hubei Province, China, the preservation number is CCTCC M 20242945, and the preservation number is CCTCC M 20242945. According to the method disclosed by the invention, the corn straws are subjected to first-step pretreatment by adopting coxakella cocori P23, and then are subjected to second-step pretreatment by combining with glycollic acid with a specific concentration, so that lignin and hemicellulose structures in the corn straws are effectively damaged, meanwhile, the damage to cellulose in the pretreatment process is reduced, more pairs of cellulose are retained, and the yield of corn straws is improved. And the cellulose crystallinity is inhibited from being improved, so that the cellulase enzymolysis efficiency is improved, the corn straw enzymolysis sugar production efficiency is improved, and the glucose yield is improved by 19.7% compared with that of a single glycollic acid pretreatment group.

Description

Technical Field

[0001] The invention relates to the technical field of corn straw pretreatment, and in particular to a two-step method for pretreating corn straw to increase glucose yield. Background Art

[0002] Corn straw is an abundant renewable resource. After enzymatic saccharification and degradation into glucose, it can be used for fermentation to produce energy sources such as ethanol or other higher-value products. However, the structural composition of corn straw is a core factor restricting the efficient production of glucose. The mass proportion of cellulose in corn straw reaches 35-45%. The essence of enzymatic hydrolysis of corn straw to produce glucose is the enzymatic hydrolysis of cellulose. However, cellulose is wrapped by a complex of lignin and hemicellulose, forming a physical barrier that hinders the reaction. The pretreatment process changes the structure of the lignocellulosic material by destroying the cell wall and exposing the cellulose, so that the cellulase can more effectively bind to and degrade the cellulose component. Therefore, the corn straw needs to be pretreated to break through this physical barrier.

[0003] Common methods for pretreatment of corn stover include physical pretreatment (steam explosion, microwave / ultrasound, etc.), chemical pretreatment (acid / alkali treatment, oxidation pretreatment, etc.), and biological pretreatment. However, physical pretreatment requires stringent conditions, and biological pretreatment requires a long treatment time. Chemical pretreatment offers the advantages of high efficiency and low cost. Organic acid pretreatment offers a new and efficient method with advantages such as high recovery efficiency, low cost, and sustainable utilization. However, this pretreatment is prone to excessive hydrolysis, which in turn inhibits the efficiency of cellulase enzymatic hydrolysis to produce sugars. Summary of the Invention

[0004] Based on the above problems, the present invention aims to provide a microbial strain that can degrade lignin.

[0005] Another object of the present invention is to provide a two-step method for pretreating corn straw to increase glucose yield based on the above-mentioned microbial strain.

[0006] The purpose of the present invention is achieved through the following technical solutions: A microbial strain for degrading lignin, characterized in that the strain is classified as Cossackia coli ( Kosakoniacowanii ) P23, deposited in China Center for Type Culture Collection (CCTCC), the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC M 20242945, and the deposit date is December 30, 2024.

[0007] The strain P23 can synthesize enzymes related to the degradation of lignocellulose, including laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP).

[0008] Further, the enzyme activities of strain P23 were laccase activity of 40.15U / L, manganese peroxidase activity of 2707.61U / L, and lignin peroxidase activity of 900.34U / L.

[0009] Based on the above strain P23, a two-step method for pretreating corn straw to increase glucose yield was designed.

[0010] A two-step pretreatment method for corn straw to increase glucose yield is characterized in that: first, Cossackia coli is used for pretreatment in the first step, and then glycolic acid is used for pretreatment in the second step. The strain is classified as Cossackia coli ( Kosakoniacowanii ) P23, deposited in China Center for Type Culture Collection (CCTCC), the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC M 20242945, and the deposit date is December 30, 2024.

[0011] Furthermore, the first step of pretreatment is to prepare corn straw into a degradation medium, inoculate the strain P23, pretreat at 36-38° C. and 150-180 rpm, and filter to obtain straw 1 after the pretreatment.

[0012] Furthermore, the concentration of corn straw in the culture medium is 10-50 g / L, the inoculation amount of the strain P23 is 2-10%, and the pretreatment time is 3-11 days.

[0013] Further preferably, in the first step of pretreatment, the concentration of corn straw is 30 g / L, the P23 inoculation amount is 6%, and the pretreatment time is 7 days.

[0014] Furthermore, the second step of pretreatment is to add a glycolic acid solution with a mass concentration of 2-6% to the straw 1, with a solid-liquid ratio of 1:8-10, and pretreat at 105-130° C. for 30-90 minutes.

[0015] Further preferably, the second step of pretreatment is to add a 3% by mass glycolic acid solution to the straw 1 at a solid-liquid ratio of 1:10, and pretreat at 128° C. for 65 minutes.

[0016] Most specifically, a two-step method for pretreating corn straw to increase glucose yield is characterized by comprising the following steps: (1) Cut corn stalks into small pieces and crush them, then pass them through a 40-mesh sieve. Prepare a culture medium with a concentration of 10-50 g / L with the corn stalks. Inoculate the culture medium with Cossackia coli P23 at an inoculation rate of 2-10%. Pretreat the culture medium at 36-38°C and 150-180 rpm for 3-11 days, and then filter to obtain straw 1. (2) Add 2-6% ethanolic acid solution to the straw 1 with a solid-liquid ratio of 1:8-10, and pretreat at 105-130°C for 30-90 min.

[0017] The present invention has the following technical effects: The present invention uses Cossackia coli P23 to perform a first-step pretreatment on corn straw, and then combines it with a specific concentration of glycolic acid for a second-step pretreatment, thereby effectively destroying the lignin and hemicellulose structures in the corn straw, while reducing the damage to cellulose during the pretreatment process, retaining more cellulose, and inhibiting the increase in cellulose crystallinity, thereby improving the enzymatic hydrolysis efficiency of cellulase and the sugar production efficiency of corn straw enzymatic hydrolysis. The glucose yield is increased by 19.7% compared with the single glycolic acid pretreatment group. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Colony morphology and Gram staining of strain P23 of the present invention.

[0019] Figure 2 : Phylogenetic tree of 16SrDNA sequence of strain P23 in the present invention.

[0020] Figure 3 : The growth curve of strain P23 among the present invention.

[0021] Figure 4 : Determination of Lac enzyme activity of strain P23 in the present invention.

[0022] Figure 5 : Lip enzyme activity determination of strain P23 in the present invention.

[0023] Figure 6 : Determination of Mnp enzyme activity of strain P23 in the present invention.

[0024] Figure 7 : The influence of strain P23 on alkali lignin concentration and degradation rate in the present invention.

[0025] Figure 8 : The effects of different time, straw concentration and inoculum amount on lignin degradation rate during the pretreatment process of strain P23 in the present invention.

[0026] Figure 9 : The effects of different concentrations of glycolic acid on corn straw during the second step pretreatment process of the present invention, a is the recovery rate and component changes (%) of corn straw after pretreatment, b is the effect of pretreatment on glucose yield; c is the effect of pretreatment on cellulose conversion after enzymatic hydrolysis.

[0027] Figure 10: Effects of different glycolic acid pretreatment temperatures on corn straw during the second step pretreatment process of the present invention, a is the recovery rate and component changes (%) of corn straw after pretreatment, b is the effect of pretreatment on glucose yield; c is the effect of pretreatment on cellulose conversion after enzymatic hydrolysis.

[0028] Figure 11 : The effect of different glycolic acid pretreatment times on corn straw during the second step pretreatment process of the present invention, a is the recovery rate and component changes (%) of corn straw after pretreatment, b is the effect of pretreatment on glucose yield; c is the effect of pretreatment on cellulose conversion after enzymatic hydrolysis.

[0029] Figure 12 : Effects of different pretreatments on BET specific surface area, mesopore volume and area of ​​corn straw. DETAILED DESCRIPTION

[0030] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to 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-mentioned contents of the present invention.

[0031] Example 1 Isolation, screening and identification of strains The samples came from the experimental shed B01 of Jilin Agricultural University. Compost samples were collected during the high temperature period, sealed in ziplock bags, and placed at -20℃ for later use.

[0032] Culture medium formula: (1) LB enrichment medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, H2O 1 L (121°C, 20 min); (2) Alkali lignin screening medium: alkali lignin 3 g / L, (NH4)2SO4 2 g / L, MgSO4 0.5 g / L, K2HPO4 1 g / L, NaCl 0.5 g / L, H2O 1 L (121℃, 20 min); (3) Aniline blue rescreening medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, H2O 1L, aniline blue 0.4 g / L (121°C, 20 min); (4) Alkali lignin degradation medium: alkali lignin 3 g / L, (NH4)2SO4 2 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, MgSO4 0.2 g / L, CaCl2 0.1 g / L, FeSO4 0.05 g / L, MnSO4 0.02 g / L, H2O 1 L (121℃, 20 min).

[0033] 1. Isolation, purification and screening of lignin-degrading bacteria (1) Enrichment culture: Weigh 5 g of thermophilic compost sample and dissolve it in 100 mL of 0.9% saline. Incubate at 37°C and 160 rpm for 60 min. Let it rest for 10 min, take 10 mL of the supernatant and add it to 100 mL of liquid LB enrichment medium. Incubate at 37°C and 160 rpm for 24 h. (2) Initial screening of lignin-degrading bacteria: 5 mL of the enrichment solution was inoculated into the alkali lignin screening liquid medium. After two consecutive subcultures every 12 h, the dilution was performed in a gradient manner, with the dilution multiples being 10-4, 10-5, 10-6, 10-7, 10-8, 10-9, and 10-10. 100 μL of the bacterial solution from each gradient was spread onto the alkali lignin screening solid medium, with three replicates, and incubated inverted at 37°C in the dark for 24 h.

[0034] (3) Isolation and purification: Take single colonies with good growth, inoculate them into LB solid culture medium for isolation and purification, and culture them in an inverted manner at 37°C in the dark for 24 h.

[0035] (4) Rescreening of lignin-degrading bacteria: Inoculate the purified single colony into the aniline blue rescreening medium and place it in a 37°C constant temperature incubator. Observe the fading zone around the colony in the aniline blue rescreening medium every 24 hours.

[0036] (5) Preservation of bacterial strains: Screen out the strain that can produce peroxidase and manganese peroxidase, number it P23, and preserve it in glycerol.

[0037] 2. Strain Identification (1) After strain P23 was cultured overnight on the culture medium, the results were observed as follows: Figure 1 The colonies were individually arranged, yellow, round, with a smooth, moist surface, slightly raised colonies, neat edges, and easy to pick. Under an optical microscope, strain P23 was observed to be a Gram-negative bacterium with a short rod shape.

[0038] (2) The strain was subjected to physiological and biochemical identification. The measured physiological and biochemical indicators are shown in Table 1. "+" represents positive and "-" represents negative. The results are shown in Table 1.

[0039] Table 1:

[0040] (3) After PCR amplification of 16S rRNA from P23 colony, the PCR product was sent to Bio-Tech for sequencing. Blast was used to compare with all available 16S rRNA sequences in the NCBI database. The analysis and comparison showed that strain P23 and Cossackia coli strains had high consistency and homology. The genus was determined by combining colony morphology, color and Gram staining results. The results are as follows: Figure 2 , the bacteria belongs to the genus Coxsackie.

[0041] The strain P23 was deposited as a biological deposit and its classification name was Cossackia coli ( Kosakoniacowanii ) P23, deposited in China Center for Type Culture Collection (CCTCC), the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC M 20242945, and the deposit date is December 30, 2024.

[0042] Example 2 Growth Characteristics and Enzyme Activity Analysis of Cossackia coli P23 1. Growth Characteristics Analysis The growth characteristics of the isolated and purified strain P23 were evaluated. A 1% inoculum of activated logarithmic phase bacterial suspension of strain P23 was inoculated into LB medium and cultured in a shake flask at 37°C and 160 rpm in a shaking incubator. The growth characteristics of strain P23 were evaluated based on the bacterial concentration (OD600). The results are shown in Figure 2. Figure 3 The P23 strain grows faster and enters the logarithmic phase after 2 hours of culture. At this time, the bacteria can make full use of the carbon source, nitrogen source and other nutrients in the culture medium, reproduce rapidly, and accelerate the metabolic rate. The OD600 increases rapidly and reaches a peak in the logarithmic phase. After 18 hours of culture, the bacterial growth enters a relatively stable period. At this time, the bacterial metabolism enters a slow period, and enters a decline period after 24 hours.

[0043] 2. Enzyme activity determination The preserved strain P23 was activated and cultured in LB medium at 37°C for 12 hours, and then subcultured twice. 1% of the inoculum was inoculated into an alkali lignin degradation medium and cultured continuously for 7 days. 1 ml of the culture solution was taken every 24 hours and centrifuged at 12,000 rpm for 10 minutes. The supernatant was taken as the crude enzyme solution, and the changes in the activities of laccase, lignin peroxidase, and manganese peroxidase in the crude enzyme solution were measured. At the same time, the changes in the alkali lignin concentration in the supernatant were measured. The results are as follows: Figure 4 、 5 , 6: The maximum enzyme activity of peroxidase on the 6th day was 900.34U / L; the maximum enzyme activity of manganese peroxidase on the 6th day was 2707.61U / L; the maximum enzyme activity of laccase on the 7th day was 40.15U / L.

[0044] Example 3 A two-step method for pretreating corn straw to increase glucose yield comprises the following steps: (1) Cut corn straw into small pieces and crush them, then pass them through a 40-mesh sieve, and prepare a culture medium with a concentration of 10-50 g / L. The culture medium formula is as follows: 30-50 g / L corn straw, 2 g / L (NH4)2SO4, 1 g / L K2HPO4, 1 g / L KH2PO4, 0.2 g / L MgSO4, 0.1 g / L CaCl2, 0.05 g / L FeSO4, 0.02 g / L MnSO4, and 1 L H2O (sterilized at 121°C for 20 min). Inoculate the culture medium with Cossackia coli P23 at an inoculation rate of 2-10%, pretreat at 36-38°C and 150-180 rpm for 3-11 days, and then filter to obtain straw 1. (2) Add 2-6% ethanolic acid solution to the straw 1 with a solid-liquid ratio of 1:8-10, and pretreat at 105-130°C for 30-90 min.

[0045] (3) Add 4 mL of 50 mM citric acid buffer solution (pH = 5) and 73.34 mL of distilled water to 3.158 g of straw 2 to obtain an enzymatic hydrolysis system. Adjust the pH of the enzymatic hydrolysis system to 4.8. After sterilization and cooling, add 1 mL of diluted cellulase with an activity of 300 FPU / mL. Incubate the system at 50 °C and 160 rpm for 72 h, then centrifuge at 12,000 rpm for 10 min. Collect the supernatant to test the glucose content.

[0046] In order to further verify the effect of each single factor on the enzymatic sugar production efficiency after pretreatment of corn straw, the effects of P23 pretreatment time, straw concentration and inoculation amount on the lignin degradation rate in corn straw were studied during the pretreatment process. Figure 8 As shown in Figure a, the lignin degradation rate of strain P23 in liquid fermentation medium first increased and then decreased with time. On the 7th day, the degradation rate of corn straw lignin reached a peak of 28.93%. Figure 8 As shown in Figure b, the degradation rate of corn straw lignin gradually decreased with the increase of straw addition. When the straw addition amount was 10g / L-30g / L, there was no significant difference in the lignin degradation rate. When it exceeded 30g / L, strain P23 was unable to further degrade more lignin in corn straw. Therefore, when the straw addition amount was 30g / L, it was the optimal addition amount. At this time, the lignin degradation rate reached 27.38%. The effect of the inoculation amount of strain P23 on the degradation of lignin is shown in Figure 2. Figure 8As shown in Figure c, the degradation rate of corn straw lignin first increased and then decreased with the increase of the inoculation amount. When the inoculation amount reached 6%, the absolute content of lignin in corn straw decreased from 18.89% to 12.63%, and the degradation rate reached a peak of 33.16%.

[0047] After the first pretreatment, the second pretreatment was carried out to evaluate the effects of different glycolic acid concentrations, pretreatment temperature and time on the pretreatment and the final sugar yield of corn straw. Figure 9 As shown, the pretreatment effect of treatment at 115 ° C for 60 minutes. From the chemical composition point of view, the composition of corn straw has undergone tremendous changes. The relative contents of the components of corn straw after pretreatment with Cossaccharomyces cerevisiae P23 are cellulose (43.89%), hemicellulose (30.18%), and lignin (19.93%). After pretreatment with different concentrations of glycolic acid, the relative content of hemicellulose decreased by 18.99%-26.97%. It can be seen that hemicellulose is separated under the action of glycolic acid. This shows that different concentrations of glycolic acid play a role in the pretreatment process. When the glycolic acid concentration is 3%, it is sufficient to meet the enzymatic hydrolysis of cellulase to produce sugar. At this time, the cellulose conversion rate reaches 66.94%. Too high a concentration will inhibit the enzymatic hydrolysis and sugar production. The pretreatment temperature is an important influencing factor in the pretreatment process. Therefore, the optimal temperature for improving the enzymatic hydrolysis efficiency was explored in the range of 105 ° C-130 ° C. The results are shown in the figure. Figure 10 As shown in the figure, under the conditions of 3% pretreatment ethanolic acid concentration and 60 minutes, as the temperature gradually increased, the relative content of hemicellulose decreased by 10.24%-29.66% compared with the pretreatment with Cossackia coli P23, while the relative content of cellulose increased by 24.12%-49.75% compared with the original straw, which is the same as the different concentrations of ethanolic acid pretreatment. When the pretreatment temperature reached 125℃, the sugar production of the enzymatically hydrolyzed straw reached a peak of 0.40g / g corn straw. When the temperature continued to increase, the sugar production of the enzymatically hydrolyzed straw decreased. This also proves that the increase in temperature may inhibit the effect of cellulose enzymatic hydrolysis. At this time, the cellulose conversion rate was 70.73%. Under the conditions of 125℃ and 3% ethanolic acid concentration, the effect of the change in pretreatment time on the pretreatment process is shown in the figure. Figure 11As shown, the chemical composition of corn straw changes with time. The relative content of cellulose increases significantly compared to the initial straw, while the hemicellulose decreases significantly. When the pretreatment time exceeds 75 minutes, the rate of increase / decrease slows significantly. This may be because the pretreatment process has already destroyed the corn straw structure when the pretreatment time is 75 minutes. From the glucose yield, it can also be seen that the sugar yield of solid residues after cellulase hydrolysis gradually increases with the increase of pretreatment time, from 0.36g / g corn straw at 30 minutes to 0.40g / g at 75 minutes. The cellulose conversion rate increases from 16.49% without pretreatment to 74.55%, and the enzymatic hydrolysis efficiency increases by 3.5 times.

[0048] Example 4 (1) Cut corn straw into small pieces and crush them, then pass them through a 40-mesh sieve. Prepare a culture medium with a concentration of 30 g / L using the corn straw. The culture medium formula is as follows: 30 g / L corn straw, 2 g / L (NH4)2SO4, 1 g / L K2HPO4, 1 g / L KH2PO4, 0.2 g / L MgSO4, 0.1 g / L CaCl2, 0.05 g / L FeSO4, 0.02 g / L MnSO4, and 1 L H2O (sterilized at 121°C for 20 min). Inoculate the culture medium with Cossackia coli P23 at an inoculum size of 6%, pretreat at 37°C and 160 rpm for 7 days, and then filter to obtain straw 1. (2) Add 3% ethanolic acid solution to straw 1 at a solid-liquid ratio of 1:10, pretreat at 128°C for 65 minutes, and then filter to obtain straw 2.

[0049] (3) Add 4 mL of 50 mM citric acid buffer solution (pH = 5) and 73.34 mL of distilled water to 3.158 g of straw 2 to obtain an enzymatic hydrolysis system. Adjust the pH of the enzymatic hydrolysis system to 4.8. After sterilization and cooling, add 1 mL of diluted cellulase with an activity of 300 FPU / mL. Incubate the system at 50 °C and 160 rpm for 72 h, then centrifuge at 12,000 rpm for 10 min. Collect the supernatant to test the glucose content.

[0050] In Example 4, corn straw was pretreated under optimal parameters and then enzymatically hydrolyzed to produce glucose, resulting in a glucose yield of 0.42 g / g corn straw. Table 2 shows the chemical composition changes in the straw after different pretreatments. The glycolic acid and citric acid treatments represent a single acid pretreatment, the P23 pretreatment represents a single pretreatment using strain P23, the P23+glycolic acid pretreatment represents a first pretreatment using P23, followed by a second pretreatment using glycolic acid (the remaining P23+acid pretreatment groups also followed the same pretreatment sequence), and the glycolic acid+P23 pretreatment represents a first pretreatment using glycolic acid, followed by a second pretreatment using strain P23.

[0051] Table 2:

[0052] As shown in Table 2, the glucose yield from direct enzymatic hydrolysis of raw corn straw was extremely low, but glucose yield was significantly increased after single pretreatment. Furthermore, a two-step pretreatment of corn straw with strain P23 and organic acid resulted in a glucose yield of 0.42 g / g straw. The citric acid pretreatment group had a higher glucose yield than the glycolic acid pretreatment group, compared to the citric acid pretreatment group alone. However, the glucose yield in the glycolic acid pretreatment group was significantly higher than that in the citric acid pretreatment group. This suggests that P23 pretreatment, combined with glycolic acid pretreatment, inhibits lignin healing during the glycolic acid pretreatment process. This P23+glycolic acid pretreatment produces a synergistic effect on the straw pretreatment, further increasing the glucose yield from enzymatic hydrolysis. Furthermore, swapping the glycolic acid and P23 pretreatment order resulted in a decrease in cellulose conversion during enzymatic hydrolysis and a significant reduction in glucose yield.

[0053] From the effects of different pretreatments on the structure of corn straw (specific surface area, mesopore area and pore volume), Figure 12 As shown in the figure (GA is single glycolic acid pretreatment, GP is glycolic acid first and then P23 pretreatment, PG is P23 first and then glycolic acid pretreatment), the BET surface area of ​​corn straw increased significantly after glycolic acid pretreatment, and the specific surface area of ​​corn straw increased from 3.82 m 2 / g increased to 4.91 m 2 / g. This is because glycolic acid pretreatment removes part of the hemicellulose and lignin, causing the substrate structure to change and forming more cavities. Further combined with P23 pretreatment, the specific surface area of ​​corn straw is further increased to 5.18 m 2 / g, the specific surface area of ​​the enzymatic substrate increases, which helps the enzyme to better utilize the cellulose structure. The mesoporous area of ​​the raw material after glycolic acid pretreatment is 3.57 m 2 / g increased to 4.89 m 2 / g, while the mesopore area of ​​P23 further increased to 5.09 m after treatment with glycolic acid. 2 / g, and the mesopore volume is reduced from the original 0.006 cm 3 / g increases to 0.010 cm 3 / g, and the mesopore volume of the P23+glycolic acid pretreatment group increased to 0.011 cm 3 / g, the size of the mesopores is the size accessible to cellulase, and the size of the pore volume and mesopore area can further indicate that the substrate can be better utilized by cellulase.

Claims

1. A microbial strain for degrading lignin, characterized in that: The strain is classified as Cossackia coli ( Kosakoniacowanii ) P23, deposited in China Center for Type Culture Collection (CCTCC), the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC M 20242945, and the deposit date is December 30, 2024.

2. A two-step method for pretreating corn straw to increase glucose yield, characterized by: The first step of pretreatment was performed with Cossackia coli, and then the second step of pretreatment was performed with glycolic acid. The strain was classified as Cossackia coli ( Kosakoniacowanii ) P23, deposited in China Center for Type Culture Collection (CCTCC), the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC M 20242945, and the deposit date is December 30, 2024.

3. The method for increasing glucose yield by pretreating corn straw in two steps according to claim 2, wherein: The first step of pretreatment is to prepare corn straw into a degradation medium, inoculate the strain P23, pretreat at 36-38° C. and 150-180 rpm, and filter to obtain straw 1 after the pretreatment.

4. The method for increasing glucose yield by pretreating corn straw in two steps according to claim 2 or 3, wherein: The concentration of corn straw in the culture medium is 10-50 g / L, the inoculation amount of the strain P23 is 2-10%, and the pretreatment time is 3-11 days.

5. A two-step method for pretreating corn straw to increase glucose yield according to any one of claims 2 to 4, characterized in that: The second step of pretreatment is to add a glycolic acid solution with a mass concentration of 2-6% to the straw 1, with a solid-liquid ratio of 1:8-10, and pretreat at 105-130° C. for 30-90 minutes.

6. A two-step method for pretreating corn straw to increase glucose yield, characterized in that: The steps include: (1) Cut corn stalks into small pieces and crush them, then pass them through a 40-mesh sieve. Prepare a culture medium with a concentration of 10-50 g / L with the corn stalks. Inoculate the culture medium with Cossackia coli P23 at an inoculation rate of 2-10%. Pretreat the culture medium at 36-38°C and 150-180 rpm for 3-11 days, and then filter to obtain straw 1. (2) Add 2-6% ethanolic acid solution to the straw 1 with a solid-liquid ratio of 1:8-10, and pretreat at 105-130°C for 30-90 min.

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