Acinetobacter baumannii for degrading lignin and application of acinetobacter baumannii

Abstract

The invention relates to a strain Y26 for degrading lignin, which is characterized in that the classification name of the strain Y26 is Acinetobacter baumannii, the strain Y26 is preserved in the China Center for Type Culture Collection, the preservation address is No. 299, eight road, Wuchang District, Wuhan City, Hubei Province, China, the preservation number is CCTCCM20242944, and the preservation date is December 30, 2024. The acinetobacter baumannii Y26 has excellent lignocellulose degradation capacity, the Lac enzyme activity is 60.38 + / -2.5 U / L, the Mnp enzyme activity is 2612.14 + / -25 U / L, the Lip enzyme activity is 1460.62 + / -14 U / L, and the degradation rate of lignin in corn straw reaches 38.93%. The acinetobacter baumannii Y26 and maleic acid are combined to pretreat the corn straw, so that the cellulose conversion rate of the corn straw reaches 87.41%, the glucose yield reaches 0.668 g / g straw, the prepared saccharification liquid has a small inhibition effect on microbial fermentation, and the yield of ethanol is effectively increased.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and particularly relates to an Acinetobacter baumannii strain capable of degrading lignin and its application. Background Art

[0002] Biofuel ethanol is an ideal gasoline extender and has received global attention as a transportation fuel for gasoline blending. Lignocellulosic biomass (LCB), as one of the most abundant renewable resources in the world, is considered an effective alternative to fossil fuels and plays an increasingly important role in the circular economy and sustainable development. Crop straw is one of the biomass with abundant lignocellulose in the world, mainly including wheat husks, rice straw, sugarcane bagasse and corn straw, and is a potential raw material for the production of second-generation ethanol. Among them, corn straw is the main cash crop with an annual output of up to 28.8423 million tons. Corn straw (CS) has gradually become an inescapable by-product in the processing process. Using corn straw to prepare bioenergy helps solve the energy crisis, reduces the shortage of biofuels and lowers the production cost, and promotes the development of the circular economy.

[0003] However, the cellulose, hemicellulose and lignin in corn straw form a dense structure that is difficult to separate, seriously hindering its effective utilization. Therefore, in order to open the lignocellulose structure, pretreatment methods are needed to destroy its cross-linked polymer matrix structure and improve the efficiency of saccharification and fermentation. Degrading lignin is the primary choice for destroying the matrix structure. Currently, pretreatment mainly includes physical pretreatment (ball milling, steam explosion, etc.), chemical pretreatment (acid, alkali treatment), ionic liquids 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, environmental friendliness and other advantages. In the process of lignin degradation, bacteria have the advantages of a short treatment cycle and easy cultivation compared with fungi. It mainly secretes a series of extracellular enzymes, such as lignin peroxidase (Lip), manganese peroxidase (MnP) and laccase (Lac), etc. to completely degrade lignin into CO2 and H2O. Due to the influence of bacteria on the types and activities of enzymes produced, the lignin degradation efficiency of bacteria is relatively low. Therefore, it is necessary to explore more microorganisms that can efficiently degrade lignin. Summary of the Invention

[0004] Based on the above technical problems, the purpose of the present invention is to provide an Acinetobacter baumannii strain capable of degrading lignin.

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

[0006] The object of the present invention is achieved by the following technical solutions: A strain Y26 capable of degrading lignin, characterized in that: the taxonomic name of the strain Y26 is Acinetobacter baumannii (Acinetobacter bauerii ), which is preserved in the China Center for Type Culture Collection, with the preservation address at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the preservation number being CCTCC M 20242944, and the preservation date being December 30, 2024.

[0007] The above-mentioned Acinetobacter baumannii Y26 was obtained by ARTP mutagenesis. The laccase (Lac) enzyme activity of the mutagenized Acinetobacter baumannii Y26 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.

[0008] Acinetobacter baumannii Y26 has excellent genetic stability, and the abilities to produce Lac, Mnp, and LiP can be stably inherited for more than 10 generations.

[0009] The application of the above-mentioned Acinetobacter baumannii Y26 in the production of ethanol using corn stover as a raw material.

[0010] A method for producing ethanol using corn stover as a raw material, which is characterized in that: Acinetobacter baumannii Y26 is used for the first-step pretreatment of corn stover. After the treatment is completed, maleic acid is added for the second-step pretreatment. The pretreated solid material is collected, cellulase is added for saccharification to prepare a saccharified solution, and ethanol-producing microorganisms are added to the saccharified solution for fermentation to produce ethanol.

[0011] The biological pretreatment process can selectively remove lignin from biomass to reduce or prevent sugar loss. However, a large amount of cellulose loss and long pretreatment culture have hindered its industrial application. Therefore, in order to improve the productivity of the whole process, a combined pretreatment of biological treatment and chemical treatment is selected. In the present invention, Acinetobacter baumannii Y26 is used for the first-step pretreatment to remove a large amount of lignin, and at the same time, it changes the structure of corn stover lignocellulose and the degree of decomposition of lignocellulose. During the second-step maleic acid pretreatment, hemicellulose and the remaining part of lignin and its derivatives are further degraded, and at the same time, the cellulose loss in this process is reduced, increasing the accessibility of cellulase to cellulose, reducing the inhibition of cellulase in the system, and increasing the glucose yield of cellulose. The finally prepared saccharified solution has less inhibition on the microbial fermentation during the fermentation process and is more conducive to the fermentation synthesis of ethanol.

[0012] Furthermore, the first-step pretreatment is to inoculate Acinetobacter baumannii Y26 in a medium with corn stover as the sole carbon source for degradation pretreatment. The straw concentration is 15 - 55 g / L, the inoculation amount of Y26 is 2 - 10%, and the pretreatment time is 3 - 11 days.

[0013] Further preferably, further, in the first-step pretreatment, Acinetobacter baumannii Y26 is inoculated in a medium with corn straw as the sole carbon source for degradation pretreatment, the straw concentration is 15 - 25 g / L, the inoculation amount of Y26 is 6 - 8%, and the pretreatment time is 7 - 9 days.

[0014] Further, in the second-step pretreatment, maleic acid with a concentration of 5 - 6% is added to the straw after the first-step pretreatment, the solid-liquid ratio is 1:15, the pretreatment temperature is 105 - 130 °C, and the pretreatment time is 60 - 180 min.

[0015] Further preferably, in the second-step pretreatment, maleic acid with a concentration of 5 - 6% is added to the straw after the first-step pretreatment, the solid-liquid ratio is 1:15, the pretreatment temperature is 120 - 130 °C, and the pretreatment time is 100 - 140 min.

[0016] The present invention has the following technical effects: In the present invention, Acinetobacter baumannii Y26 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, Lip enzyme activity of 1460.62 ± 14 U / L, and the degradation rate of lignin in corn straw reaches 38.93%.

[0017] By using Acinetobacter baumannii Y26 and maleic acid for combined pretreatment of corn straw, the present invention further improves the degradation of hemicellulose and lignin, reduces the loss of cellulose, reduces the generation of harmful inhibitors, reduces the inhibition of cellulase activity, enables the cellulose conversion rate of corn straw to reach 87.41%, the glucose yield to reach 0.668 g / g straw, and the prepared saccharified liquid has little inhibitory effect on microbial fermentation, effectively improving the ethanol yield. Description of the Drawings

[0018] Figure 1 : Electrophoretic identification and phylogenetic tree diagram of the original strain C1 (a: electrophoretic identification diagram, b: phylogenetic tree).

[0019] Figure 2 : Enzyme activity comparison diagram of laccase, manganese peroxidase and lignin peroxidase of the strain obtained after mutagenesis of the original strain C1.

[0020] Figure 3 : Genetic stability of laccase, manganese peroxidase and lignin peroxidase of mutant strains Y3, Y5, Y8 and Y26 for more than 10 generations (a, b, c), and 7-day enzyme activity changes of laccase, manganese peroxidase and lignin peroxidase of mutant strain Y26 and original strain C1 (d, e, f).

[0021] Figure 4 : Effects of straw addition amount, inoculum amount, and treatment time on the changes in lignocellulose components (a, b, c) in corn straw, lignin degradation rate, and solid recovery rate (d, e, f).

[0022] Figure 5 : Effects of maleic acid concentration, treatment time, and treatment temperature on the changes in lignocellulose components and solid recovery rate (a, b, c) in corn straw, effects on lignin degradation rate and hemicellulose degradation rate (d, e, f), and effects on glucose yield and cellulose conversion rate (g, h, i).

[0023] 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.

[0024] Figure 7 : Effects of different glucose concentrations, fermentation time, and pH on sugar production efficiency of saccharified liquid. Specific implementation manners

[0025] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the protection scope 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 content of the present invention.

[0026] The corn straw used in the present invention comes from the experimental field of Jilin Agricultural University (40 mesh), with cellulose 37.94%, lignin 22.39%, and hemicellulose 21.39%. Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) 53356 was purchased from Zhili Zhongte Biotechnology Co., Ltd.

[0027] 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.

[0028] Ethanol fermentation medium: peptone 10 g / L, KH2PO4 2 g / L, MgSO4 2 g / L, saccharified liquid: 1 L. Example 1 Screening, isolation, and identification of original strains (1) Screening and isolation Take 10 g of the compost sample from the experimental field of Jilin Agricultural University, place it in a 100 mL sterilized conical flask, add 50 mL of 0.9% normal saline, put it in a constant temperature shaker at 37 °C with a speed of 160 rpm, and culture for 3 h. Dilute the above liquid according to the dilution gradient (1×10 -1 ~1×10 -9 ), inoculate it into the primary screening alkaline lignin solid medium, and culture it upside down at 37 °C for 24 h. Take the medium with good colony growth, pick single colonies for streaking, inoculate them into the LB plate medium for purification, inoculate the purified single colonies into the aniline blue rescreening medium, place it in a constant temperature incubator at 37 °C, observe the size of the transparent circle that appears in the aniline blue rescreening medium every 24 h, select the strains with larger transparent circles, and preserve them with glycerol.

[0029] (2)Send the strain to Bioengineering (Shanghai) Co., Ltd. for 16s rDNA gene sequencing analysis, and obtain a sequence length of 1473 bp. Through phylogenetic analysis, strain C1 has a relatively close genetic relationship with Acinetobacter baumannii , so the strain is named Acinetobacter baumannii ( Acinetobacter baumannii )C1, and its electrophoresis identification diagram and phylogenetic tree diagram are as shown in Figure 1 .

[0030] Example 2 Mutation of the original strain (1)ARTP mutagenesis treatment Use 10 μL of the above bacterial liquid as a sample and place it on a sterilized copper sheet. Set the operating parameters as follows: input a pure helium flow rate of 10 L min −1 at a radio frequency power of 100 W, keep a distance of 2 mm from the plasma torch nozzle, keep the temperature at 25 °C, and test the optimal holding time (ranging from 10, 20, 30, 40 to 100 s) to obtain a better mutagenesis duration. After treatment, elute the sample from the copper sheet with 1 mL of sterile water into a 1.5 mL centrifuge tube, and spread 100 μL of the sample on the LB medium and culture it at 37 °C for 24 h. Inoculate the monoclonal strains grown on the LB medium after mutagenesis into the enzyme-producing medium in a 24-well plate, and culture them in a shaker at 220 rpm and 37 °C for 7 d. Measure the enzyme activities of laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) in the fermentation broth. Taking the enzyme activity of the original strain without mutagenesis as a control, screen the mutant strains with increased enzyme activities of the three enzymes compared to the wild-type strain, and preserve the screened mutagenized bacteria with glycerol.

[0031] Acinetobacter baumannii C1 was subjected to ARTP mutagenesis, and 9 intervals (10 - 90 s) were set. The ARTP results showed that when the mutagenesis time was 30 s, the lethality rate of C1 bacteria was 89.14%, and after 40 s, the lethality rate reached 100%. Therefore, 30 s was selected for ATRP mutagenesis.

[0032] In this experiment, Acinetobacter baumannii C1 was used as the starting strain, and 29 mutant bacteria with significantly different lignin-degrading enzyme activities from the original bacteria were obtained through the above ARTP mutagenesis. The results are as Figure 2 shown. Among them, the mutant strain Y26 had the highest enzyme activity. The Lac enzyme activity was 60.38 ± 2.5 U / L, the Mnp enzyme activity was 2612.14 ± 25 U / L, and the Lip enzyme activity was 1460.62 ± 14 U / L. Compared with the original strain C1's laccase (48.71 ± 2.72 U / L), manganese peroxidase (2240.85 ± 11.32 U / L), and peroxidase enzyme activities (584.59 ± 5.27 U / L), they were increased by 23.96%, 16.57%, and 149.85% respectively.

[0033] The mutant strains Y3, Y5, Y18, and Y26 with the best overall enzyme production performance were subjected to genetic stability detection. The Lac activity was measured on the 4th day of each generation, the Lip activity was measured on the 5th day, and the Mnp activity was measured on the 7th day to verify the genetic stability of the strain's enzyme production ability. The results are as Figure 3 shown in a, b, and c. The abilities of Y26 to produce Lac, Mnp, and LiP could be stably inherited for more than 10 generations, and the activities of Lac, Mnp, and Lip were all at relatively high levels. However, the abilities of Y5, which had the best initial Lac and Lip production abilities, to produce Lac and Lip decreased significantly after 10 generations of cultivation.

[0034] The mutant strain Y26 and the original strain C1 were respectively inoculated into the enzyme production medium, and the enzyme activity was measured for 7 d. Within the same inoculation time, the enzyme activity of the mutant strain Y26 was higher than that of the original strain C1, as specifically shown in Figure 3 d, e, and f. Therefore, the mutant strain Y26 was identified as a potential strain with high lignin-degrading enzyme activity.

[0035] The mutant strain Y26 was biopreserved, specifically preserved in the China Center for Type Culture Collection, with the preservation address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the preservation number being CCTCC M 20242944, and the preservation date being December 30, 2024.

[0036] Example 3 The effects of mutant strain Y26 on the main components of corn straw The strain Y26 was inoculated into a lignin degradation medium with corn stover as the sole carbon source. The strain Y26 pretreated the corn stover at 37 °C and 160 rpm. Through treatments with different corn stover addition amounts (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 (3 d, 5 d, 7 d, 9 d, 11 d), after the reaction was completed, the corn stover was washed with water and dried, and the composition of the corn stover was analyzed using the method established by the National Renewable Energy Laboratory (NREL). The following formulas were used to calculate the solid recovery rate and the lignin degradation rate:

[0037] The results are as Figure 4 shown. As can be seen from Figure 4 a, when the corn stover addition amount increased from 15 g / L to 25 g / L, there was no significant difference in the lignin degradation rate. When it exceeded 25 g / L, the solid recovery rate gradually increased, while the lignin degradation rate decreased significantly, indicating that the strain Y26 could not further degrade more lignin in the corn stover, as Figure 4 shown in e. Comparing the effects of different inoculation amounts on the lignin in the corn stover, as Figure 4 shown in b, as the inoculation amount increased, the lignin in the corn stover was further removed. When the inoculation amount exceeded 6%, there was no obvious change in the lignin degradation rate. Therefore, an inoculation amount of 6% was selected for subsequent experiments. As Figure 4 shown in c and Figure 4 f compared different fermentation times. When the corn stover addition amount was 25 g / L, the concentration was 6%, and the fermentation time was 7 d, the relative lignin content decreased from 22.39% to 18.81%, and the lignin degradation rate reached 38.93%. The results showed that the mutant strain Y26 could effectively degrade the lignin component in the corn stover.

[0038] The removal of lignin could expose more cellulose. To further explore the accessibility of cellulose, the solid residue of the corn stover pretreated by the strain Y26 was enzymatically hydrolyzed using cellulase, and the cellulose conversion rate was 54.36%, and the glucose sugar yield was 0.292 g / g. However, the steric hindrance of the lignin macromolecules and lignin derivatives generated during the pretreatment had a negative impact on the subsequent enzymatic hydrolysis. After the pretreatment removed lignin, a large amount of lignin degradation products and phenolic compounds such as organic acids, phenolic compounds, furfural substances, and hydroxymethylfurfural substances would inhibit the activity of the subsequent cellulase.

[0039] Example 4 Taking corn stover as the raw material, the effect of the combined pretreatment of Y26 and maleic acid on sugar production by enzymatic hydrolysis: On the basis of the first-step pretreatment with Y26 in Example 3 (straw addition amount 25 g / L, inoculation concentration of Y26 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 strain Y26 in removing lignin, hemicellulose and retaining cellulose during the pretreatment of lignocellulose, different concentrations of MA were used for the pretreatment of lignocellulose ( Figure 5 a, 5 d). Compared with the direct pretreatment of raw corn straw with MA, at concentrations of 2% - 5%, as the concentration increased, the solid recovery rate of straw 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 pretreatment with MA, lignin and hemicellulose in the straw were effectively removed. The pretreated solid residue was enzymatically hydrolyzed ( Figure 5 g), and 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 too high a concentration of acid would increase the inhibitors (such as furfural, etc.) during the pretreatment process. Therefore, a 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 also explored. When the pretreatment time was 2.5 h ( Figure 5 h), the glucose yield reached 0.629 g / g, and further extending the time did not significantly increase the sugar yield of corn straw. Therefore, 2.5 h was selected as the optimal treatment time. Finally, the effects of MA on the chemical composition and enzymatic hydrolysis efficiency of lignocellulose at different temperatures were explored ( Figure 5 c, f, i). Under the same conditions (MA concentration 5%, time 2.5 h), when the temperature was 125°C, compared with the pretreatment with Y26 alone, the relative cellulose content increased from 48.41% to 69.54%, the cellulose conversion rate increased from 54.89% to 87.41%, and the sugar yield increased from 0.292 g / g of straw to 0.668 g / g of straw, which was 6.6 times higher than the sugar yield of the original straw ( Figure 5 i). Compared with traditional pretreatment, the two-step pretreatment adopted in the present invention achieved a relatively high hemicellulose degradation rate (80.69%) and lignin degradation rate (60.54%) ( Figure 5 f), enabling more cellulose to be retained (relative cellulose content 69.54%), greatly improving the cellulose conversion rate (87.41%), inducing higher glucose production, and laying a foundation for the efficient utilization of carbohydrate polymers in lignocellulose. In addition, the relatively high glucose concentration provides a better basis for subsequent bioethanol.

[0040] Comparing the structural and morphological changes of corn stover after different pretreatments by scanning electron microscopy can more intuitively reflect the degree of damage to corn stover, and further prove whether its lignin and hemicellulose can be effectively degraded. As Figure 6 shown in a, the surface of untreated corn stover is smooth and dense. As Figure 6 shown in b, after pretreatment with Y26 bacteria alone, the surface of the stover is broken, rough and has a porous structure. As Figure 6 shown in c, after pretreatment with maleic acid alone, the stover shows fractures and curls. As Figure 6 shown in d, the surface of corn stover after two-step pretreatment with Y26 + maleic acid is damaged to a greater extent. This is because the lignin and hemicellulose in the structure of corn stover are effectively degraded, thereby increasing the accessibility of cellulase and enhancing further enzymatic saccharification.

[0041] By FT-IR testing the changes in surface functional groups of the samples, as Figure 6 shown in e, the two-step pretreatment has a significant effect on the structure of corn stover. The broad band at 1000 - 1080 cm -1 is the characteristic band of the C-O-H stretching of cellulose primary and secondary alcohols. After two-step pretreatment, the absorption peak here increases, indicating an increase in cellulose content after two-step pretreatment. After two-step pretreatment, the peaks at the bands near 1423 - 1426 cm -1 and 1510 - 1516 cm -1 (C-C in lignin) decrease and the benzene ring characteristic peak at 1600 - 1604 cm -1 decreases, indicating that lignin is degraded to a certain extent. The peak intensity increases at 1220 - 1240 cm -1 (C-O-C between lignin phenylpropane monomers), and these peaks represent substances such as ethers, alcohols and esters produced after lignin is decomposed. At the same time, after two-step pretreatment, the band near 1734 - 1735 cm -1 disappears, which is due to the reduction of C=O conjugates in hemicellulose, indicating that hemicellulose is effectively removed.

[0042] Observing the changes in the cellulose crystal structure before and after pretreatment by XRD. After biological pretreatment, the relative crystallinity of CS increased from 42.15% to 58.51%. The relative crystallinity of CS with single maleic acid pretreatment is 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 is damaged, resulting in the removal of non-crystalline regions such as lignin polysaccharides. The above results show that the two-step pretreatment method effectively removes lignin and hemicellulose in the stover, making more cellulose exposed and available for the production of subsequent saccharification liquid.

[0043] Example 4 A method for producing ethanol from corn straw, the specific steps are as follows: According to the optimal process parameters determined in Example 3, Acinetobacter baumannii Y26 was used to perform the first-step pretreatment on corn straw. The straw concentration in 1 L of the medium was 25 g / L, the inoculation amount of Y26 was 6%, the pretreatment time was 7 days. After the treatment, maleic acid was added to the straw pretreated in the first step according to 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 120 min. The pretreated solid matter was collected. In 3.158 g of the solid matter, 4 mL of 0.05 mol (pH = 4.8) citric acid buffer solution and 73.342 mL of distilled water were added, sterilized at 121 °C for 20 min, cooled to 50 °C, and 1 mL of Novozym 3-generation cellulase with an enzyme activity of 30 FPU / mL was added. After enzymatic hydrolysis at 50 °C and 160 °C for 72 h, centrifugation was carried out at 12000 rpm for 10 min. The supernatant was collected as the saccharified liquid; The saccharification rate of cellulose is the primary condition for realizing high-titer ethanol production. Directly use a strain of Saccharomyces cerevisiae 53356 that can efficiently utilize glucose for fermentation. The saccharified liquid after enzymatic hydrolysis was collected, and 2% of Saccharomyces cerevisiae 53336 was inoculated 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 above-mentioned saccharified liquids with different concentrations were obtained by evaporation and concentration, and glucose was used as the index in this saccharified liquid), different fermentation times (12 h, 24 h, 36 h, 48 h, 60 h, 72 h) and different pH values (4, 4.5, 5, 5.5, 6) was detected by gas chromatography.

[0044] The results are as Figure 7 shown. The ethanol yield first increased and then decreased with the increase of the initial concentration. When the initial sugar concentration was 75 g / L, the ethanol yield reached the maximum at this time, and the residual glucose amount was 2.21 g / L. When the initial sugar concentration exceeded 75 g / L, Saccharomyces cerevisiae 53356 could not utilize more glucose, but instead inhibited the ethanol yield, and the residual glucose amount increased with the increase of the initial sugar concentration ( Figure 7 a). The ethanol yield at different pH values was also observed, and its optimal pH was 5.5. The results showed that when the initial sugar concentration was 75 g / L, the time was 48 h, and the pH was 5.5, the ethanol yield reached the maximum of 49.46 g / L, and the residual glucose amount was 2.21 g / L ( Figure 7 b). Under different time conditions, the ethanol yield first increased and then decreased, and the residual glucose amount first decreased and then leveled off ( Figure 7 c).

[0045] Comparative Example 1 Equal amounts of corn straw were pretreated separately with Y26 alone and maleic acid alone (the pretreatment conditions were the same as those of maleic acid pretreatment in the combined pretreatment in Example 4), and then subjected to cellulase hydrolysis, concentrated into saccharified liquid with the same concentration of 75 g / L, and then equal amounts of Saccharomyces cerevisiae 53356 were inoculated into the equal amounts of saccharified liquid, and fermentation was carried out to produce ethanol under the same fermentation conditions. The results of ethanol production and glucose residual sugar content after 48 h of fermentation are shown in Table 1.

[0046] Table 1:

[0047] It can be seen that for the saccharified liquid prepared by separate pretreatment with Y26 alone and maleic acid alone, when Saccharomyces cerevisiae 53356 was used for fermentation under the same conditions, the ethanol production decreased significantly, and the glucose residual sugar content increased significantly, indicating that there were more harmful inhibitors in the saccharified liquid that inhibited microbial fermentation, which blocked the fermentation of Saccharomyces cerevisiae and prevented it from utilizing more glucose to synthesize ethanol, thus inhibiting the ethanol production. In the present invention, through the first-step pretreatment with Y26, the microstructure and surface group distribution of the straw were changed, and during this process, macromolecules that might be converted into inhibitors were decomposed into small molecules, so that during the second-step pretreatment with maleic acid, the content of inhibitors generated by the degradation of maleic acid decreased, and the inhibitory effect on microbial fermentation in the obtained saccharified liquid decreased.

[0048] Comparative Example 2 During the pretreatment process, equal amounts of corn straw were pretreated with citric acid instead of maleic acid in Comparative Example 1, and were pretreated separately with citric acid alone (the pretreatment conditions were the same as those of maleic acid pretreatment in the combined pretreatment in Example 4) and Y26 + citric acid combined pretreatment (the pretreatment conditions were the same as those of maleic acid pretreatment in the combined pretreatment in Example 4), and then subjected to cellulase hydrolysis, concentrated into saccharified liquid with the same concentration of 75 g / L, and then equal amounts of Saccharomyces cerevisiae 53356 were inoculated into the equal amounts of saccharified liquid, and fermentation was carried out to produce ethanol under the same fermentation conditions. The results of ethanol production and glucose residual sugar content after 48 h of fermentation are shown in Table 2.

[0049] Table 2:

[0050] It can be seen that when citric acid is used for single pretreatment, compared with maleic acid, the cellulose conversion rate, sugar yield, and ethanol yield of the pretreated straw are all increased. However, when Y26 and citric acid are used for combined pretreatment, the cellulose conversion rate, sugar yield, and ethanol yield of the straw are significantly decreased compared with the Y26 + maleic acid combined pretreatment group. This shows that when citric acid pretreatment is further carried out on the straw pretreated with Y26, inhibitors that inhibit the activity of cellulase are synthesized, resulting in a decrease in sugar yield. In addition, there are more harmful inhibitors in the saccharified liquid that inhibit microbial fermentation, which hinders the fermentation of Saccharomyces cerevisiae and makes it unable to utilize more glucose to synthesize ethanol, thus inhibiting the ethanol yield.

[0051] It should be noted that the strain used for ethanol fermentation in this invention is Saccharomyces cerevisiae 53356. Under the saccharified liquid prepared by this combined pretreatment process, if it is replaced with other microbial strains that have a higher efficiency of fermenting glucose to produce ethanol, the effect will be better.

Claims

1. A lignin-degrading strain Y26, characterized in that: The taxonomic name of the strain Y26 is Acinetobacter baumannii ( Acinetobacter bauerii ), which is 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 M 20242944, and the deposit date is December 30, 2024.

2. Use of Acinetobacter bauerii Y26 as claimed in claim 1 in the production of ethanol using corn stover as raw material.

3. A method for producing ethanol from corn straw, characterized in that: Perform the first-step pretreatment on corn stover using Acinetobacter bauerii Y26. After the treatment is completed, add maleic acid for the second-step pretreatment. Collect the pretreated solid material, add cellulase for saccharification to prepare a saccharified solution, and add ethanol-producing microorganisms to the saccharified solution for fermentation to produce ethanol.

4. The method for producing ethanol from corn straw as claimed in claim 3, wherein: The first-step pretreatment is to inoculate Acinetobacter bauerii Y26 in a medium with corn stover as the sole carbon source for degradation pretreatment. The concentration of the stover is 15 - 55 g / L, the inoculation amount of Y26 is 2 - 10%, and the pretreatment time is 3 - 11 days.

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

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