Acinetobacter baumannii degrading lignin and application thereof

Abstract

A strain Y26 for degrading lignin, characterized in that the strain Y26 is Acinetobacter baumannii (ATCC 19606T) Acinetobacter baumannii ), preserved in the China Center for Type Culture Collection, located at No. 299, Bajiyuan Road, Wuchang District, Wuhan City, Hubei Province, China, with a preservation number of CCTCCM20242944 and a preservation date of December 30, 2024. The Acinetobacter baumannii Y26 in the present application has excellent ability to degrade lignocellulose, with a Lac enzyme activity of 60.38±2.5 U / L, a Mnp enzyme activity of 2612.14±25 U / L, a Lip enzyme activity of 1460.62±14 U / L, and a lignin degradation rate in corn stalks of 38.93%. In the present application, the corn stalks are pretreated by using the Acinetobacter baumannii Y26 in combination with maleic acid, so that the cellulose conversion rate of the corn stalks reaches 87.41%, the glucose yield reaches 0.668 g / g of stalks, and the prepared saccharification liquid has small inhibition effect on microbial fermentation, effectively improving the ethanol yield.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, and particularly relates to a strain of Acinetobacter baumannii capable of degrading lignin and application thereof. BACKGROUND

[0002] Biofuel ethanol is an ideal gasoline filler and is attracting global attention as a gasoline blending transportation fuel. Lignocellulosic biomass (LCB) is considered as an effective substitute for fossil fuels as one of the most abundant renewable resources in the world, and plays an increasingly important role in circular economy and sustainable development. Crop straw is one of the world's biomass rich in lignocellulose, mainly including wheat hull, rice straw, sugarcane residue and corn stalk, and is a potential raw material for the production of the second generation of ethanol. Among them, corn stalk is the main economic crop, with an annual output of 2884.23 million tons, and corn stalk (CS) has gradually become an unavoidable byproduct in the processing process. Using corn stalk to prepare bioenergy helps to solve the energy crisis, reduce biofuel shortage and reduce production cost, and promotes the development of circular economy.

[0003] However, the cellulose, hemicellulose and lignin in corn stalk form a dense structure that is difficult to separate, which seriously hinders its effective utilization. Therefore, in order to open the structure of lignocellulose, it is necessary to destroy the cross-linked polymer matrix structure and improve the efficiency of saccharification and fermentation through pretreatment methods, and lignin degradation is the first choice to destroy the matrix structure. At present, pretreatment mainly includes physical pretreatment (ball milling, steam explosion, etc.), chemical pretreatment (acid, alkali treatment), ionic liquid and biological pretreatment (fungi, bacteria). Biological pretreatment has gradually become the focus of attention due to its mild reaction conditions, low energy consumption, low cost and environmental friendliness. In the process of lignin degradation, bacteria have the advantages of short processing cycle and easy cultivation compared with fungi, and it can completely degrade lignin into CO2 and H2O through the secretion of a series of extracellular enzymes such as lignin peroxidase (Lip), manganese peroxidase (MnP) and laccase (Lac). Due to the influence of enzyme species and enzyme activity, the efficiency of bacterial lignin degradation is low, so it is necessary to explore more microorganisms that can efficiently degrade lignin. SUMMARY

[0004] Based on the above technical problems, the present application aims to provide a strain of Acinetobacter baumannii capable of degrading lignin.

[0005] Another object of the present application is the application of the above-mentioned Acinetobacter baumannii in the production of ethanol.

[0006] The object of the present application is achieved by the following technical solutions:

[0007] A strain Y26 for degrading lignin, characterized in that the strain Y26 is Acinetobacter baumannii Acinetobacter baumannii , preserved in the China Center for Type Culture Collection, located at No. 299, Bajiyuan Road, Wuchang District, Wuhan, Hubei, China, with a preservation number of CCTCC NO: M 20242944, and a preservation date of December 30, 2024.

[0008] The above-mentioned Acinetobacter baumannii Y26 is obtained by ARTP mutagenesis, and the laccase (Lac) enzyme activity of the Acinetobacter baumannii Y26 obtained by mutagenesis is 60.38 ± 2.5 U / L, the manganese peroxidase (Mnp) enzyme activity is 2612.14 ± 25 U / L, and the lignin peroxidase (Lip) enzyme activity is 1460.62 ± 14 U / L.

[0009] The Acinetobacter baumannii Y26 has excellent genetic stability, and the abilities of producing Lac, Mnp and LiP can be stably inherited for more than 10 generations.

[0010] The above-mentioned Acinetobacter baumannii Y26 is applied in the production of ethanol with corn stalks as raw materials.

[0011] A method for producing ethanol with corn stalks as raw materials, characterized in that the corn stalks are subjected to a first step of pretreatment by using the Acinetobacter baumannii Y26, maleic acid is added after the end of the pretreatment for a second step of pretreatment, the pretreated solid material is collected, cellulase is added for saccharification to prepare a saccharification liquid, and ethanol-producing microorganisms are added in the saccharification liquid for fermentation to produce ethanol.

[0012] The biological pretreatment process can selectively remove lignin from biomass to reduce or prevent sugar loss, but a large amount of cellulose loss and a long pretreatment culture hinder its industrial application. Therefore, in order to improve the productivity of the whole process, biological treatment is combined with chemical treatment for pretreatment. In the present application, the first step of pretreatment is performed by using the Acinetobacter baumannii Y26 to remove a large amount of lignin, and at the same time, the structure of the lignocellulose of the corn stalks and the degree of decomposition of the lignocellulose are changed. In the second step of maleic acid pretreatment, hemicellulose and residual part of lignin and its derivatives are further degraded, and at the same time, the loss of cellulose in the process is reduced, the accessibility of cellulase to cellulose is increased, and the inhibition of cellulase in the system is also reduced, thereby improving the glucose yield of cellulose. The prepared saccharification liquid has less inhibition effect on the fermentation of microorganisms in the fermentation process, and is more conducive to the fermentation synthesis of ethanol.

[0013] Further, the first step of pretreatment is inoculated with the Acinetobacter baumannii Y26 for degradation pretreatment in a culture medium with corn stalks as the only carbon source, the concentration of the stalks is 15-55 g / L, the inoculation amount of Y26 is 2-10%, and the pretreatment time is 3-11 days.

[0014] Further preferably, the first step pretreatment is a degradation pretreatment in which Acinetobacter baumannii Y26 is inoculated in a culture medium with corn straw as the sole carbon source, the straw concentration is 15-25 g / L, the inoculation amount of Y26 is 6-8%, and the pretreatment time is 7-9 days.

[0015] Furthermore, the second pretreatment step is to add maleic acid with a concentration of 5-6% to the straw after the first pretreatment, with a solid-liquid ratio of 1:15, a pretreatment temperature of 105-130°C, and a pretreatment time of 60-180 min.

[0016] Further preferably, the second pretreatment step is to add 5-6% maleic acid to the straw after the first pretreatment, with a solid-liquid ratio of 1:15, a pretreatment temperature of 120-130°C, and a pretreatment time of 100-140 min.

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

[0018] The Acinetobacter baumannii Y26 strain of the present invention has excellent ability to degrade lignocellulose, with Lac enzyme activity of 60.38±2.5 U / L, Mnp enzyme activity of 2612.14±25 U / L, and Lip enzyme activity of 1460.62±14 U / L, and the degradation rate of lignin in corn straw reaches 38.93%.

[0019] The present invention uses Acinetobacter baumannii Y26 and maleic acid to jointly pretreat corn straw, thereby further improving the degradation of hemicellulose and lignin, while reducing cellulose loss, reducing the production of harmful inhibitors, and reducing the inhibition of cellulase activity, so that the cellulose conversion rate of the corn straw reaches 87.41%, and the glucose yield reaches 0.668g / g straw. The prepared saccharification liquid has little inhibitory effect on microbial fermentation, thereby effectively increasing the ethanol yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Electrophoresis identification and phylogenetic tree of the original strain C1 (a: electrophoresis identification diagram, b: phylogenetic tree).

[0021] Figure 2 : Comparison of laccase, manganese peroxidase and lignin peroxidase activities of the strains obtained after mutation of the original strain C1.

[0022] Figure 3: Genetic stability of laccase, manganese peroxidase and lignin peroxidase of mutant Y3, Y5, Y8 and Y26 (a, b, c), and changes of 7d enzyme activity of laccase, manganese peroxidase and lignin peroxidase of mutant Y26 and original strain C1 (d, e, f).

[0023] Figure 4 : Effects of straw addition amount, inoculation amount and treatment time on changes of lignocellulose components (a, b, c), lignin degradation rate and solid recovery rate (d, e, f) in corn straw.

[0024] Figure 5 : Effects of maleic acid concentration, treatment time and treatment temperature on changes of lignocellulose components and solid recovery rate (a, b, c), effects on lignin degradation rate and hemicellulose degradation rate (d, e, f), and effects on glucose yield and cellulose conversion rate (g, h, i).

[0025] Figure 6 : Scanning electron microscope images of corn straw after different pretreatments (a, b, c, d), FT-IR (e) and XRD (f) of corn straw before and after pretreatment.

[0026] Figure 7 : Effects of different glucose concentrations, fermentation time and pH on sugar production efficiency of saccharification liquid. DETAILED DESCRIPTION

[0027] The application will be described in detail below by examples. It is necessary to point out here that the following examples are only used to further illustrate the application and cannot be understood as limiting the protection scope of the application. Those skilled in the art can make some non-essential improvements and adjustments to the application according to the above content of the application.

[0028] The corn straw used in the application is from the experimental field of Jilin Agricultural University (40 mesh), and the cellulose, lignin and hemicellulose contents are 37.94%, 22.39% and 21.39% respectively. Saccharomyces cerevisiae ) 53356, purchased from Zhiteli Zhongte Biological Technology Co., Ltd.

[0029] Lignin degradation medium: (NH4)2SO4: 2 g / L, KH2PO4: 1 g / L, K2HPO4: 1 g / L, MgSO4: 0.2 g / L, CaCl2: 0.1 g / L, FeSO4: 0.05 g / L, MnSO4: 0.02 g / L.

[0030] Ethanol fermentation medium: peptone 10 g / L, KH2PO4 2 g / L, MgSO4 2 g / L, saccharification liquid: 1 L.

[0031] Example 1

[0032] Screening, isolation and identification of original strain

[0033] (1) Screening and isolation

[0034] 10 g sample was taken from the compost in the experimental field of Jilin Agricultural University, placed in a 100 mL sterilized conical flask, 50 mL of 0.9% normal saline was added, and placed in a 37°C constant temperature shaker at 160 rpm for 3 h. The above liquid was diluted according to the dilution gradient (1x10 -1 ~1x10 -9 ) and inoculated into the primary screening alkaline lignin solid culture medium, and incubated at 37°C for 24 h. The culture medium with good growth was picked for single colony streaking and inoculation in LB plate culture medium for purification. The purified single colony was inoculated in aniline blue rescreening medium and placed in a 37°C constant temperature incubator. The size of the transparent circle appearing in the aniline blue rescreening medium was observed every 24 h, and the strain with larger transparent circle was selected for glycerol preservation.

[0035] (2) The strain was sent to Bioengineering (Shanghai) Co., Ltd. for 16s rDNA gene sequencing analysis, and a sequence length of 1473 bp was obtained. Phylogenetic analysis showed that C1 strain was closely related to Acinetobacter baumannii Acinetobacter baumannii, therefore, the strain was named Acinetobacter baumannii Acinetobacter baumannii C1, and its electrophoresis identification chart and phylogenetic tree chart are shown in Figure 1 .

[0036] Example 2

[0037] Mutagenesis of original strain

[0038] (1) ARTP mutagenesis treatment

[0039] 10 μL of the above bacterial solution was used as a sample and placed on a sterilized copper sheet, and the operation parameters were set as follows: 10 L min −1The pure helium flow rate was maintained at a distance of 2 mm from the plasma torch nozzle, the temperature was maintained at 25℃, and the optimal holding time (ranging from 10, 20, 30, 40, to 100 s) was tested to obtain a better mutagenesis duration. After treatment, the sample was eluted from the copper sheet with 1 mL of sterile water into a 1.5 mL centrifuge tube, and 100 μL of the sample was spread on LB medium and incubated at 37℃ for 24 h. The single colony strains grown on LB medium after mutagenesis were inoculated into the enzyme-producing medium of a 24-well plate and incubated at 220 rpm and 37℃ for 7 d. The enzyme activities of laccase (Lac), manganese peroxidase (MnP), and peroxidase (LiP) in the fermentation broth were determined, with the enzyme activity of the original strain without mutagenesis as a control. Mutant strains with improved activities of the three enzymes compared to the wild-type strain were screened, and the screened mutagenized bacteria were stored in glycerol.

[0040] Acinetobacter baumannii C1 was subjected to ATRP mutagenesis, and 9 intervals (10-90 s) were set. The ATRP results showed that when the mutagenesis time was 30 s, the lethality rate of C1 bacteria was 89.14%, and the lethality rate reached 100% after 40 s. Therefore, 30 s was selected for ATRP mutagenesis.

[0041] In this experiment, Acinetobacter baumannii C1 was used as the starting strain, and 29 mutant strains with significant differences in lignin-degrading enzyme activity from the original strain were obtained by ATRP mutagenesis as shown in Table 1. Figure 2 The enzyme activity of Y26 was the highest, with Lac enzyme activity of 60.38 ± 2.5 U / L, Mnp enzyme activity of 2612.14 ± 25 U / L, and Lip enzyme activity of 1460.62 ± 14 U / L. Compared with the original strain C1, the laccase (48.71 ± 2.72 U / L), manganese peroxidase (2240.85 ± 11.32 U / L), and peroxidase enzyme activity (584.59 ± 5.27 U / L) were increased by 23.96%, 16.57%, and 149.85%, respectively.

[0042] The mutant strains Y3, Y5, Y18, and Y26 with the best overall enzyme-producing performance were subjected to genetic stability detection. The activity of Lac was measured on the 4th day of each generation, the activity of Lip was measured on the 5th day, and the activity of Mnp was measured on the 7th day, to verify the genetic stability of the enzyme-producing ability of the strains. The results are shown in Table 2. Figure 3 a, b, c, the ability of Y26 to produce Lac, Mnp, and LiP can be stably inherited for more than 10 generations, and the activities of Lac, Mnp, and Lip are at a high level, while the initial Lac and Lip production ability of Y5 is significantly decreased after 10 generations of culture.

[0043] The mutant Y26 and the original strain C1 were inoculated into enzyme production medium, and the enzyme activity was determined for 7 days. In the same inoculation time, the enzyme activity of the mutant Y26 was higher than that of the original strain C1, as shown in Figure 3 d, e, and f. Therefore, the mutant Y26 was identified as a potential strain with high lignin-degrading enzyme activity.

[0044] The mutant Y26 was biologically preserved, specifically at the China Center for Type Culture Collection, located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with the preservation number CCTCC NO: M 20242944 and the preservation date of December 30, 2024.

[0045] Example 3

[0046] Effect of mutant Y26 on main components of corn stalks

[0047] The strain Y26 was inoculated into a lignin-degrading medium with corn stalks as the only carbon source. The corn stalks were pretreated by the strain Y26 at 37°C and 160 rpm. Different amounts of stalks (15 g / L, 25 g / L, 35 g / L, 45 g / L, 55 g / L), different inoculation amounts (2%, 4%, 6%, 8%, 10%), and different times (3d, 5d, 7d, 9d, 11d) were used for treatment. After the reaction, the stalks were washed and dried with water. The components of the corn stalks were analyzed using the method established by the National Renewable Energy Laboratory (NREL). The solid recovery rate and lignin degradation rate were calculated using the following formula:

[0048]

[0049] The results are shown in Figure 4 As can be seen from Figure 4 a, when the amount of stalks increased from 15 g / L to 25 g / L, there was no significant difference in lignin degradation rate. When the amount of stalks exceeded 25 g / L, the solid recovery rate gradually increased, while the lignin degradation rate decreased significantly. As shown in Figure 4 e, the strain Y26 could not further degrade more lignin in the corn stalks. As shown in Figure 4 b, the effect of different inoculation amounts on corn stalk lignin was compared. With the increase of inoculation amount, the lignin in the corn stalks was further removed. When the inoculation amount exceeded 6%, the lignin degradation rate did not change significantly, so 6% inoculation amount was selected for subsequent experiments. As shown in Figure 4 c and Figure 4fCompared with different fermentation times, when the straw addition amount was 25 g / L, the concentration was 6%, and the fermentation time was 7 days, the relative content of lignin decreased from 22.39% to 18.81%, and the lignin degradation rate reached 38.93%. The results showed that the strain Y26 could effectively degrade the lignin components in corn straw.

[0050] Removing lignin exposes more cellulose. To further explore cellulose accessibility, cellulase was used to hydrolyze the solid residue of corn straw pretreated with Y26 bacteria, resulting in a cellulose conversion rate of 54.36% and a glucose yield of 0.292 g / g. However, the steric hindrance of lignin macromolecules and lignin derivatives produced during the pretreatment process negatively impacts subsequent enzymatic hydrolysis. Lignin removal during pretreatment produces a large number of lignin degradation products and phenolic compounds, such as organic acids, phenolic compounds, furfurals, and hydroxymethylfurfural, which inhibit subsequent cellulase activity.

[0051] Example 4

[0052] Effect of combined pretreatment of Y26 and maleic acid on enzymatic hydrolysis and sugar production using corn straw as raw material:

[0053] Based on the first step pretreatment of Y26 in Example 3 (straw addition amount 25 g / L, Y26 inoculation concentration 6%, fermentation time 7 d), maleic acid (MA) was used for the second step pretreatment. In order to explore the effectiveness of MA combined with mutant bacteria Y26 in removing cellulose, hemicellulose and retaining cellulose during lignocellulose pretreatment, different concentrations of MA were used for lignocellulose pretreatment ( Figure 5 a, 5d), compared with the original corn straw directly pretreated with MA, at a concentration of 2% to 5%, with the increase of concentration, the straw solid recovery rate decreased from 55.12% to 49.44%, the hemicellulose degradation rate increased from 68.56% to 77.81%, and the lignin degradation rate increased from 43.10% to 53.54%. This is because after MA pretreatment, lignin and hemicellulose in the straw were effectively removed. The solid residue after pretreatment was enzymatically hydrolyzed ( Figure 5 g), it can be seen that the cellulose conversion rate after enzymatic hydrolysis increased from 76.86% to 86.81%, and the glucose yield increased from 0.51 g / g to 0.62 g / g. When the MA concentration exceeded 5%, the cellulose conversion rate and glucose yield decreased, probably because the excessively high concentration of acid would increase the inhibitory substances (such as furfural) during the pretreatment process. Therefore, 5% concentration of MA was selected for subsequent experiments. Subsequently, the changes in each component and the enzymatic hydrolysis effect under different pretreatment times were explored. When the pretreatment was 2.5 h ( Figure 5h), the glucose yield reached 0.629 g / g, and further prolonging the time did not significantly increase the sugar yield of corn stalks, so 2.5 h was selected as the optimal treatment time. Finally, the influence of MA at different temperatures on the chemical composition of lignocellulose and the enzymatic hydrolysis efficiency was explored Figure 5 c, f, i), under the same conditions (MA concentration of 5%, time of 2.5 h), when the temperature was 125℃, compared with Y26 alone pretreatment, the relative content of cellulose was increased from 48.41% to 69.54%, the conversion rate of cellulose was increased from 54.89% to 87.41%, the sugar yield was increased from 0.292 g / g of stalks to 0.668 g / g of stalks, which was 6.6 times higher than that of the original stalks Figure 5 i). Compared with traditional pretreatment, the two-step pretreatment used in the present application achieved a higher efficiency of hemicellulose degradation rate (80.69%) and lignin degradation rate (60.54%) Figure 5 f), which allowed more cellulose to remain (the relative content of cellulose was 69.54%), greatly improved the conversion rate of cellulose (87.41%), and induced higher glucose production, laying a foundation for efficient utilization of lignocellulosic carbohydrate polymers. In addition, the higher glucose concentration provided a better basis for subsequent bioethanol.

[0054] The structure and morphology of corn stalks after different pretreatments were compared by scanning electron microscopy, which could more directly reflect the degree of damage of corn stalks, and further prove whether the lignin and hemicellulose could be effectively degraded. As shown in Figure 6 a, the surface of untreated corn stalks was smooth and dense, as shown in Figure 6 b, after being pretreated with Y26 alone, the surface of the stalks was broken, rough and had a porous structure, as shown in Figure 6 c, after being pretreated with maleic acid alone, the stalks appeared to be broken and curled, as shown in Figure 6 d, the surface of corn stalks after two-step pretreatment with Y26+maleic acid was damaged to a greater extent, because the lignin and hemicellulose in the structure of corn stalks were effectively degraded, thereby increasing the accessibility of cellulase and enhancing the further enzymatic saccharification.

[0055] The changes in the functional groups on the surface of the samples were tested by FT-IR, as shown in Figure 6 e, two-step pretreatment had a significant effect on the structure of corn stalks, and the absorption peak at 1000~1080 cm -1 was the characteristic spectrum band of C-O-H stretching of cellulose primary and secondary alcohol. After two-step pretreatment, the absorption peak at this position was enhanced, indicating that the content of cellulose after two-step pretreatment was increased. After two-step pretreatment, the absorption peaks at 1423~1426 cm -1 and 1510~1516 cm -1The peaks at the bands near (CC in lignin) decrease and the peaks at 1600–1604 cm -1 The decrease of the characteristic peak of benzene ring indicates that lignin has been degraded to a certain extent. -1 The peak intensity at 1734-1735 cm-1 increased after the two-step pretreatment. -1 The nearby bands disappeared, which was due to the reduction of C=O conjugates in hemicellulose, indicating that hemicellulose was effectively removed.

[0056] XRD was used to observe changes in the cellulose crystal structure before and after pretreatment. After biological pretreatment, the relative crystallinity of CS increased from 42.15% to 58.51%. The relative crystallinity of CS pretreated with maleic acid alone was 52.72%, while after two-step pretreatment, the relative crystallinity of CS increased to 59.12%. This may be because the amorphous region of cellulose was destroyed, resulting in the removal of non-crystalline regions such as lignin polysaccharides. These results show that the two-step pretreatment method effectively removed lignin and hemicellulose from the straw, allowing more cellulose to be exposed and used in the subsequent production of saccharification liquid.

[0057] Example 4

[0058] The method for producing ethanol using corn stalks as raw materials comprises the following specific steps:

[0059] According to the optimal process parameters determined in Example 3, corn straw was pretreated in the first step using Acinetobacter baumannii Y26. The straw concentration in 1L of culture medium was 25g / L, the inoculum size of Y26 was 6%, and the pretreatment time was 7 days. After the treatment, maleic acid was added to the straw pretreated in the first step at a solid-liquid ratio of 1:15 for the second step pretreatment. The added concentration of maleic acid was 5%, the pretreatment temperature was 125°C, and the pretreatment time was 120min. The pretreated solid matter was collected, and 4mL0.05mol (pH=4.8) citric acid buffer solution and 73.342mL distilled water were added to 3.158g of solid matter. The mixture was sterilized at 121°C for 20min, cooled to 50°C, and 1mL of Novozymes 3rd generation cellulase with an enzyme activity of 30 FPU / mL was added. After enzymatic hydrolysis at 50°C and 160°C for 72h, the mixture was centrifuged at 12000 rpm for 10min. The supernatant was collected as the saccharification liquid.

[0060] The saccharification rate of cellulose is the primary condition for achieving high efficiency ethanol production, directly using a strain of Saccharomyces cerevisiae 53356 for fermentation, collecting the saccharified liquid after enzymatic hydrolysis, and inoculating 2% of Saccharomyces cerevisiae 53336 for ethanol fermentation. The ethanol content under different initial sugar concentrations (15 g / L, 30 g / L, 45 g / L, 60 g / L, 75 g / L, 90 g / L, 120 g / L) (the saccharified liquid with different concentrations is obtained by evaporation and concentration, and the glucose is used as an index in the saccharified liquid), different fermentation times (12 h, 24 h, 36 h, 48 h, 60 h, 72 h) and different pH (4, 4.5, 5, 5.5, 6) conditions was detected by gas chromatography.

[0061] The results are shown in Figure 7 As shown in the table, the ethanol yield increases first and then decreases with the increase of initial concentration, and when the initial sugar concentration is 75 g / L, the ethanol yield reaches the maximum, and the residual glucose is 2.21 g / L. When the initial sugar concentration exceeds 75 g / L, Saccharomyces cerevisiae 53356 cannot utilize more glucose, but inhibits the production of ethanol, and the residual glucose increases with the increase of initial sugar concentration. Figure 7 a). The ethanol yield under different pH was also observed, and the optimum pH was 5.5. The results show that when the initial sugar concentration is 75 g / L, the time is 48 h, and the pH is 5.5, the ethanol yield reaches the maximum of 49.46 g / L, and the residual glucose is 2.21 g / L. Figure 7 b). Under different time conditions, the ethanol yield increases first and then decreases, and the residual glucose decreases first and then tends to be flat. Figure 7 c).

[0062] Comparative Example 1

[0063] An equal amount of corn straw was pretreated with Y26 alone, maleic acid alone (the pretreatment conditions were the same as those of maleic acid pretreatment in the combined pretreatment of Example 4), and then subjected to cellulase hydrolysis, concentrated to the same 75 g / L concentration of saccharified liquid, and then an equal amount of Saccharomyces cerevisiae 53356 was inoculated in an equal amount of saccharified liquid, and ethanol fermentation was carried out under the same fermentation conditions. The ethanol yield and glucose residual amount after 48 h of fermentation are shown in Table 1.

[0064] Table 1:

[0065]

[0066] It can be seen that the ethanol yield of the saccharification liquid prepared by Y26 pretreatment alone and maleic acid pretreatment alone is obviously decreased, and the residual glucose amount is obviously increased, under the same conditions using Saccharomyces cerevisiae 53356 for fermentation, which indicates that more harmful inhibitors exist in the saccharification liquid to inhibit microbial fermentation, so that Saccharomyces cerevisiae fermentation is hindered, more glucose cannot be utilized to synthesize ethanol, and the ethanol yield is inhibited. In the present application, the microstructure and surface group distribution of the straw are changed by the first step of Y26 pretreatment, and in this process, macromolecules that may be converted into inhibitors are decomposed into small molecules, so that the content of inhibitors generated by the degradation of maleic acid in the second step of maleic acid pretreatment is reduced, and the inhibition effect on microbial fermentation in the obtained saccharification liquid is reduced.

[0067] Comparative Example 2

[0068] In the pretreatment process, an equal amount of corn straw is pretreated with citric acid instead of maleic acid in Comparative Example 1, and Y26+citric acid combined pretreatment (the pretreatment conditions are consistent with the maleic acid pretreatment conditions in the combined pretreatment in Example 4) is used, and then cellulase hydrolysis is carried out, and the saccharification liquid is concentrated to the same 75g / L concentration, and then an equal amount of Saccharomyces cerevisiae 53356 is inoculated in an equal amount of saccharification liquid, and ethanol fermentation is carried out under the same fermentation conditions. The ethanol yield and glucose residual amount after 48h of fermentation are shown in Table 2.

[0069] Table 2:

[0070]

[0071] It can be seen that when citric acid is used for single pretreatment, the cellulose conversion rate, sugar yield and ethanol yield of the treated straw are all improved compared with maleic acid, but when Y26 and citric acid are used for combined pretreatment, the cellulose conversion rate, sugar yield and ethanol yield of the straw are all obviously decreased compared with Y26+maleic acid combined pretreatment. It is indicated that after the straw pretreated by Y26 is further pretreated by citric acid, inhibitors inhibiting the activity of cellulase are synthesized, the sugar yield is decreased, and in addition, more harmful inhibitors exist in the saccharification liquid to inhibit microbial fermentation, so that Saccharomyces cerevisiae fermentation is hindered, more glucose cannot be utilized to synthesize ethanol, and the ethanol yield is inhibited.

[0072] It should be noted that the strain used for ethanol fermentation in the present application is Saccharomyces cerevisiae 53356, and if other microbial strains with higher efficiency of utilizing glucose for ethanol fermentation are used in the saccharification liquid prepared by the combined pretreatment process, the effect will be better.

Claims

1. A lignin-degrading strain Y26, characterized by: The strain Y26 is classified as Acinetobacter baumannii ( Acinetobacter baumannii ), deposited in the China Center for Type Culture Collection, the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC NO: M 20242944, and the deposit date is December 30, 2024.

2. Use of Acinetobacter baumannii Y26 as claimed in claim 1 in producing ethanol using corn stalks as raw material.

3. A method for producing ethanol using corn stalks as raw materials, characterized in that: Corn stalks were pretreated in the first step using Acinetobacter baumannii Y26. Maleic acid was added to the corn stalks for the second step of pretreatment after the treatment. The solid matter after the pretreatment was collected and saccharified with cellulase to prepare a saccharification liquid. An ethanol-producing microorganism was added to the saccharification liquid to ferment and produce ethanol. The classification name of the strain Y26 is Acinetobacter baumannii ( Acinetobacter baumannii ), deposited in the China Center for Type Culture Collection, the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC NO: M20242944, and the deposit date is December 30, 2024.

4. The method for producing ethanol using corn stalks as raw materials according to claim 3, wherein: The first step of pretreatment is to inoculate Acinetobacter baumannii Y26 in a culture medium with corn straw as the only carbon source for degradation pretreatment. The straw concentration is 15~55g / L, the inoculation amount of Y26 is 2~10%, and the pretreatment time is 3~11 days.

5. The method for producing ethanol using corn stalks as raw materials according to claim 3, wherein: The second pretreatment step is to add maleic acid with a concentration of 2-6% to the straw after the first pretreatment, with a solid-liquid ratio of 1:15, a pretreatment temperature of 120-130°C, and a pretreatment time of 60-180 min.

Images