A strain that degrades multiple lignin-derived aromatic compounds and its application in detoxification of hydrolysate.

Abstract

This invention discloses a strain that degrades various lignin-derived aromatic compounds and its application in detoxification of hydrolysate. The strain is classified as *Rhodococcus ethereans*. Rhodococcus ether-eating Strain N1, deposited at the China Center for Type Culture Collection (CCTCC) on August 11, 2022, with accession number CCTCC NO: M 20221270, can degrade p-hydroxybenzoic acid, p-coumaric acid, ferulic acid, vanillin, coniferyl alcohol, and syringaldehyde. After lignin depolymerization, derivative phenolic inhibitors are formed, mainly divided into three types: S-type (syringaldehyde), G-type (ferulic acid), and H-type (p-coumaric acid), which affect the fermentation of the strain. The Rhodococcus bacterium in this invention can efficiently degrade SGH-type monomers, achieving a removal efficiency of up to 68.4% when applied to the biological detoxification of hydrolysate inhibitors. Strain N1 has significant application value in improving the conversion efficiency of lignocellulose hydrolysate and reducing the detoxification cost in industrial production.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain that degrades a variety of lignin-derived aromatic compounds and its application in detoxification of hydrolysate. Background Technology

[0002] Currently, society faces the challenge of finding alternative and renewable energy sources to replace the widely used conventional energy sources (fossil fuels). The current energy crisis necessitates the development of processes based on renewable substrates. Fuels and chemicals extracted from biomass are considered environmentally friendly alternatives to petroleum products. To produce second-generation biofuels and chemicals from lignocellulosic biomass, it is necessary to isolate and utilize all plant biomass components to develop environmentally and economically viable biorefining processes.

[0003] The highly crystalline and complex structure of lignin-protected cellulose makes biomass materials extremely resilient, making depolymerization a challenging task. Therefore, various lignocellulose pretreatment methods have been developed and applied. Lignocellulose pretreatment methods can be broadly classified into four categories: chemical, physical, physicochemical, and biological pretreatment. The chemical, thermal, and mechanical processes involved in biomass conversion not only require high energy input but also generate various inhibitors, primarily phenolic compounds, acids, and furans. Among these, phenolic inhibitors mainly interfere with cell membrane synthesis and function by altering the protein ratio of the cell membrane, thereby inhibiting cell growth.

[0004] Detoxification or regulation of lignocellulose hydrolysates and slurries is one of the most effective methods to combat inhibition problems. This strategy includes physical, chemical, and biological detoxification. The aim of these detoxification strategies is to reduce inhibitory compounds to non-inhibitory levels. Many studies have reported reducing inhibitor concentrations through physicochemical methods, which typically involve high temperature and pressure and increase operating costs. Therefore, compared to chemical or physical methods, biological detoxification offers advantages such as milder reaction conditions, avoidance of further use of toxic and corrosive chemicals, fewer side effects and toxic byproducts, and lower energy requirements, making it significant for improving the conversion efficiency of lignocellulose hydrolysates. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a strain that degrades a variety of lignin-derived aromatic compounds.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned strains in the biological detoxification of lignocellulose hydrolysate.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A strain that degrades a variety of lignin-derived aromatic compounds has been classified as *Rhodococcus ethereans*.Rhodococcus aetherivorans N1 has been deposited at the China Center for Type Culture Collection (CCTCC) on August 11, 2022, with accession number CCTCC NO: M 20221270 and address: Wuhan University, Wuhan, China.

[0009] The nucleotide sequence of the 16S rDNA of strain N1 described in this invention is shown in SEQ ID NO: 1 in the sequence listing.

[0010] The present invention Rhodococcus aetherivorans The N1 screening method is as follows: soil from the leaf litter deposits in the forest park of Nanjing University of Technology is screened in a culture medium with alkali lignin as the sole carbon source.

[0011] Specifically, 5.0 g of soil sample was added to five 100 mL Erlenmeyer flasks containing inorganic salt culture medium and labeled. Glass beads were added to thoroughly break up the soil. The flasks were then placed in a constant temperature shaker at 30 ℃ and 180 rpm for approximately 10 h. 5 mL of the soil mixture was then added to each of the five 100 mL Erlenmeyer flasks and cultured at 30 ℃ and 180 rpm for 5 days to enrich the soil, serving as the stock solution for the next generation of enrichment culture. After four consecutive subcultures, the enriched solution was diluted to different concentration gradients (10T). -3 -10 -8 Take 0.1 mL of each culture and spread it onto LB solid medium. Incubate at 30 °C for several days (to ensure as many colonies as possible grow). Inoculate the enriched strains into 100 mL of alkali lignin medium, using alkali lignin as the sole carbon source, and measure their biomass. Select the strain with the highest biomass for verification.

[0012] The inorganic salt culture medium formula is as follows (g / L): ammonium sulfate 1.0, potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate trihydrate 1.5, sodium chloride 1.0, magnesium sulfate heptahydrate 0.2, trace elements 1 mL, pH 7.0.

[0013] Alkali lignin medium (g / L): ammonium sulfate 1.0, potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate trihydrate 1.5, sodium chloride 1.0, magnesium sulfate heptahydrate 0.2, alkali lignin 2.0, trace elements 1 mL, pH 7.0.

[0014] LB medium: yeast extract 5.0, peptone 10, sodium chloride 5.0, trace elements 1 mL, pH 7.0, solid medium with 2% agar powder added.

[0015] The present invention Rhodococcus aetherivoransAfter 2 days of growth on LB solid medium, N1 colonies were orange-yellow, round, with neat edges and a raised surface. Strain N1 was Gram-positive. Under a microscope, strain N1 appeared as short rods without flagella.

[0016] The aforementioned Rhodococcus aetherivorans N1 can utilize lignin as its sole carbon source for growth and exhibits highly efficient degradation capabilities for its derived aromatic compounds, which then enter the tricarboxylic acid cycle through a series of enzymatic catalysis. This strain demonstrates strong degradation capabilities for ferulic acid, p-hydroxybenzoic acid, vanillic acid, and coumaric acid, and can directly perform biological detoxification of lignocellulose hydrolysates, improving the conversion efficiency of lignocellulose hydrolysates and reducing detoxification costs in industrial production, thus possessing significant application value.

[0017] A strain containing the lignin-derived aromatic compounds described in this invention Rhodococcus aetherivorans Cloning vector for N1 16S rDNA sequence.

[0018] The recombinant cloning vector is preferably pMD19T as the starting vector.

[0019] Contains the strains described above Rhodococcus aetherivorans Genetically engineered bacteria with N1 16S rDNA sequence Escherich coli DH5α (pMD19T-16S).

[0020] The genetically engineered bacteria Escherich coli DH5α construction method: The 16S rDNA of strain N1 was amplified using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACCTTGTTACGACTT-3'), and then ligated into the cloning vector pMD19T via T / A cloning to construct the recombinant cloning vector pMD19T-16S. This vector was then transformed into the cloning host strain. Escherich coli DH5α to obtain recombinant microorganisms Escherich coli DH5α (pMD19T-16S) was used to sequence the obtained recombinant microbial exogenous fragment. The 16S rDNA sequence was compared with the NCBI database, identifying strain N1 at the molecular level. Rhodococcus aetherivorans Fungi.

[0021] Genome sequencing of strain N1 revealed similar metabolic pathways for ferulic acid and coumaric acid. Ferulic acid and coumaric acid are converted to feruloyl-CoA and coumaroyl-CoA via coenzyme A synthase (Fcs) or p-hydroxycinnamoyl-CoA synthase (CouL). Local blast analysis of key gene sequences determined the location of the Fcs sequence within the entire genome of strain N1, confirming the location of the ferulic acid-CoA synthase gene Fcs-N1 on the chromosomal genome. In the degradation pathway, ferulic acid is converted to vanillin under the catalysis of Fcs and Ech (Enoyl-CoAhydratase / aldolase). Vanillin dehydrogenase (Vdh) then catalyzes the conversion of vanillin to vanillic acid via NAD-dependent oxidation, subsequently flowing to the central metabolite protocatechuic acid under the catalysis of Vanillate o-demethylase oxygenase subunit and oxidoreductase (VanAB). Coumaric acid is converted to p-hydroxybenzoic acid under the catalysis of Fcs and Ech, and subsequently to protocatechuic acid under the catalysis of PobA (4-hydroxybenzoate-3-hydroxylase). In the degradation pathway of syringaldehyde, syringaldehyde is mainly converted to syringic acid via aldehyde dehydrogenases Yfmt (Benzaldehyde dehydrogenase) and DesV (Aldehyde dehydrogenase). All key genes in the degradation pathway can be recombinantly cloned and heterologously expressed. After genome sequencing... Rhodococcus aetherivorans The N1 strain contains all the genes for the degradation of SGH-type monomers of lignin-derived aromatic compounds, making it a wild-type strain capable of completely degrading all three types of SGH monomers.

[0022] Application of the above-mentioned strains in lignocellulose hydrolysate.

[0023] The lignocellulose hydrolysate is a corn cob dilute acid hydrolysate.

[0024] Preparation method of corn cob hydrolysate: After crushing the corn cob, it is screened through a 40-mesh sieve. The obtained corn cob is mixed with 3% H2SO4 by mass, with a solid-liquid ratio (m / V) of 1:7.5. Hydrolyze at 126 ℃ for 2.5 h. Filter the solid to obtain corn cob dilute acid hydrolysate. The phenolic inhibitor content is 3.4 g / L, which seriously inhibits the fermentation of succinic acid.

[0025] Strain N1 was inoculated into corn cob hydrolysate at an inoculation rate of 1%~30% v / v, and cultured at 25~30 ℃ with stirring or shaking for 100~144 h (preferably 120 h) to detoxify. The removal efficiency of phenols in hydrolysate by strain N1 was 68.4% within 7 days.

[0026] Verification of the effectiveness of the detoxified hydrolysate: The detoxified hydrolysate was centrifuged to remove bacterial cells and used to prepare the culture medium in place of the pure water in the Suc260 medium. The experimental method was the same for the undetoxified hydrolysate. The prepared Suc260 medium was dispensed into anaerobic bottles, 27 mL per bottle. CO2 was bubbled into each bottle for 4 min, and the culture was incubated at 121 °C for 20 min. 3 mL of Suc260 seed culture was added to each bottle using a sterile syringe, and the culture was incubated at 37 °C for 72 h.

[0027] When the detoxified hydrolysate was applied to the fermentation of succinic acid, 10.5 g / L of succinic acid was produced within 72 hours, which is 2.6 times the yield of the undetoxified hydrolysate.

[0028] The fermentation medium for Suc260 (g / L) contained: betaine 0.12, diammonium hydrogen phosphate 2.6, ammonium dihydrogen phosphate 0.87, potassium chloride 0.15, magnesium sulfate heptahydrate 0.37, trace elements 1 mL, and basic magnesium carbonate 48.0.

[0029] Beneficial effects: This invention uses soil from leaf litter deposits in forest parks as the isolation material, and through a series of screening, isolation, and purification processes, obtains a strain that can utilize lignin as the sole carbon source for growth. Rhodococcus aetherivorans Strain N1 can degrade various derived aromatic compounds. Furthermore, it can degrade phenolic inhibitors in the hydrolysate, improving the conversion efficiency and reducing detoxification costs in industrial production, offering valuable insights for industrial applications. Strain N1 achieved a 68.4% removal efficiency of phenols from the hydrolysate within 7 days. Applying the detoxified hydrolysate to succinic acid fermentation produced 10.5 g / L of succinic acid within 72 hours, 2.6 times the yield of the undetoxified hydrolysate. Strain N1 is currently the only reported strain in this genus capable of directly detoxifying lignocellulose hydrolysate, demonstrating significant application value for improving lignocellulose conversion efficiency in industrial production. Attached Figure Description

[0030] Figure 1 HPLC analysis of the degradation of various phenolic substances by Rhodococcus N1;

[0031] Figure 2 Analysis of phenolic substances in simulated degradation hydrolysate of Rhodococcus;

[0032] Figure 3 Analysis of the detoxification of lignocellulose hydrolysate by Rhodococcus N1;

[0033] Figure 4 Analysis of succinic acid production from fermentation of detoxified and non-detoxified hydrolysates. Detailed Implementation

[0034] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims. Example 1

[0035] Growth strains using lignin as the sole carbon source Rhodococcus aetherivorans N1 separation screening:

[0036] Weigh 5 g of soil sample from the leaf litter accumulation site in the Central Forest Park of Nanjing University of Technology, dilute it with inorganic salt medium, and then dilute the enriched solution into different concentration gradients (10T) using a gradient dilution method. -3 -10 -8 0.1 mL of each culture was spread onto LB medium and incubated at 30 °C for several days. The enriched strains were then inoculated into 100 mL of alkali lignin medium, using alkali lignin as the sole carbon source, and their biomass was measured. The strain with the highest biomass was selected to screen for strains that can directly utilize lignin.

[0037] Filtered Rhodococcus aetherivorans Ecological characteristics of strain N1: Colonies are orange-yellow, round, with neat edges and a raised surface; strain N1 is Gram-positive. Under a microscope, strain N1 appears as short rods without flagella.

[0038] The above inorganic salt culture medium formula (g / L) is as follows: ammonium sulfate 1.0, potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate trihydrate 1.5, sodium chloride 1.0, magnesium sulfate heptahydrate 0.2, trace elements 1 mL / L, pH 7.0.

[0039] Alkali lignin medium (g / L): ammonium sulfate 1.0, potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate trihydrate 1.5, sodium chloride 1.0, magnesium sulfate heptahydrate 0.2, alkali lignin 2.0, trace elements 1 mL / L, pH 7.0.

[0040] LB medium (g / L): yeast extract 5.0, peptone 10, sodium chloride 5.0, trace elements 1 mL / L, pH 7.0, solid medium with 2% agar powder added.

[0041] Trace element solution (g / L): Ferric chloride tetrahydrate 1.5, Cobalt chloride hexahydrate 0.19, Manganese chloride tetrahydrate 0.1, Zinc chloride 0.07, Boric acid 0.006, Sodium molybdate dihydrate 0.036, Nickel chloride hexahydrate 0.024, Copper chloride dihydrate 0.002 Example 2

[0042] A strain that degrades multiple lignin-derived aromatic compounds Rhodococcus aetherivorans N1 culture conditions and degradation characteristics.

[0043] strains Rhodococcus aetherivorans N1 can utilize glucose, xylose, fructose, lactose, and sucrose as carbon sources for growth.

[0044] LB plate medium: yeast extract 5.0, peptone 10, sodium chloride 5.0, trace elements 1 mL, pH 7.0, solid medium with 2% agar powder added.

[0045] Will Rhodococcus aetherivorans N1 was inoculated into LB agar plates and incubated at 30 °C for 48 h.

[0046] A single colony of strain N1 was picked from the plate and inoculated into 100 mL of fermentation medium. It was cultured at 30 °C and 180 rpm for 48 h. Then, it was inoculated into degradation medium at an inoculation rate of 5% v / v and cultured with shaking at 30 °C and 180 rpm for 24 h. The degradation of lignin monomers was detected by HPLC.

[0047] The above fermentation medium formula (g / L) is as follows: glucose 10, urea 2.0, potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate trihydrate 1.5, sodium chloride 1.0, magnesium sulfate heptahydrate 0.2, trace elements 1 mL, pH 7.0, sterilized at 121 ℃ for 15 min.

[0048] The degradation medium formula (g / L) is as follows: glucose 10, urea 2.0, potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate trihydrate 1.5, sodium chloride 1.0, magnesium sulfate heptahydrate 0.2, trace elements 1 mL, pH 7.0. 0.1 g / L of lignin monomers (p-hydroxybenzoic acid, p-coumaric acid, ferulic acid, vanillin, coniferyl alcohol, syringaldehyde) are added to the degradation medium respectively, and sterilized at 121 ℃ for 15 min.

[0049] Trace element solution (g / L): ferric chloride tetrahydrate 1.5, cobalt chloride hexahydrate 0.19, manganese chloride tetrahydrate 0.1, zinc chloride 0.07, boric acid 0.006, sodium molybdate dihydrate 0.036, nickel chloride hexahydrate 0.024, copper chloride dihydrate 0.002.

[0050] HPLC detection method: The bacterial reaction solution was incubated at 12,000 rpm for 1 min. 1 mL of the supernatant was then filtered through a 0.22 µM nylon membrane before HPLC detection. The HPLC system was a Dionex UltiMata 3000; the column was a C18 reversed-phase column (4.6 × 250 nm, 5 µM); the mobile phase was methanol:ultrapure water (1% acetic acid) = (70:30 v / v); the flow rate was 1 mL / min; the detection wavelength was 230 nm; the column temperature was 40 °C; and the injection volume was 10 µL.

[0051] Depend on Figure 1 As shown, strain N1 exhibited varying degradation abilities for seven monomers within 24 hours. It completely degraded p-hydroxybenzoic acid, p-coumaric acid, and ferulic acid, while vanillin, coniferyl alcohol, and syringaldehyde showed significant degradation effects. This further indicates that strain N1 possesses strong potential for lignin degradation and can be used as a potential strain for future research on phenol inhibitor degradation. Example 3

[0052] Detection and simulated degradation analysis of phenolic substances in corn cob hydrolysate

[0053] Preparation method of corn cob hydrolysate: After crushing the corn cob, it is screened through a 40-mesh sieve. The obtained corn cob is mixed with 3% H2SO4 by mass, with a solid-liquid ratio (m / V) of 1:7.5. Hydrolyze at 126 ℃ for 2.5 h. Filter the solid to obtain the corn cob dilute acid hydrolysate. The total phenolic inhibitor content is 3.4 g / L. The ratio of each component is: p-hydroxybenzoic acid: vanillin: syringaldehyde: ferulic acid: p-coumaric acid = 16:68:50:102:147.

[0054] Dilute 1 mL of corn cob hydrolysate twice, centrifuge at 12,000 rpm for 1 min, and then take 1 mL of the supernatant and filter it through a 0.22 µM nylon membrane for liquid chromatography analysis. Similarly, prepare 100 mg / L furfural, 5-hydroxymethylfurfural, p-hydroxybenzoic acid, vanillin, syringaldehyde, p-coumaric acid, and ferulic acid solutions, filter them through a 0.22 µM nylon membrane, and then analyze them in liquid chromatography. Using 2 g / L as the total phenol content, add various phenolic substances to a 100 mL simulated culture medium according to the specified proportions. The inoculum size is 2%, the fermentation temperature is 30℃, and each experiment is designed with three replicates. The culture period is 12 days, and the biomass is monitored. Samples are taken every 24 h, and the degradation efficiency of p-hydroxybenzoic acid, vanillin, ferulic acid, p-coumaric acid, and syringaldehyde is determined by HPLC. Figure 2 ).

[0055] The above-mentioned simulated culture medium formula (g / L) is as follows: urea 2.0, potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate trihydrate 1.5, sodium chloride 1.0, magnesium sulfate heptahydrate 0.2, trace elements 1 mL, pH 7.0, sterilized at 121 ℃ for 15 min. The preparation method of trace elements is the same as in Example 2.

[0056] Detection conditions for phenolic inhibitors in hydrolysate: Mobile phase A: 0.02 mol / L NaH2PO4 containing 5% acetonitrile (pH adjusted to 2.9 with H3PO4); Mobile phase B: acetonitrile / methanol = 1:1 (v:v); Flow rate 1 mL / min; Detection wavelength 270 nm; Column temperature 30 ℃; Gradient elution mode: 0-13.8 min, 100% A; 13.8-45 min, 66% A, 34% B.

[0057] like Figure 2 As shown, in the 2 g / L aromatic compound simulation experiment, within 6 days, the degradation rate of ferulic acid was 38.2%, the degradation rate of p-hydroxybenzoic acid was 74.6%, the degradation rate of vanillin was 0.5%, the degradation rate of p-coumaric acid was 59.4%, and the degradation rate of syringaldehyde was 47.0%; the degradation rate of vanillin was the slowest, and it took 10 days to completely degrade. Example 4

[0058] strains Rhodococcus aetherivorans N1 uses corn cob hydrolysate as a culture medium to detoxify phenolic inhibitors.

[0059] The method for pretreating corn cob hydrolysate with dilute acid is shown in Example 3. An appropriate amount of hydrolysate was taken and the pH adjusted to neutral. 100 mL of hydrolysate was added to each bottle and incubated at 121 °C for 20 min. Several 50 mL sterile centrifuge tubes were used, with an inoculation volume of 20%. Before inoculation, the tubes were rinsed twice with inorganic salt culture medium. Each experiment was designed with three replicates, and the culture period was 7 days. Biomass was monitored, with samples taken every 24 h to detect changes in biomass, reducing sugars, and total soluble phenols (TPC). Figure 3 ).

[0060] The above-mentioned corn cob hydrolysate pretreatment method is as follows: after crushing the corn cob, it is screened through a 40-mesh sieve. The obtained corn cob is mixed with 3% H2SO4 by mass, with a solid-liquid ratio (m / V) of 1:7.5. Hydrolyze at 126 ℃ for 2.5 h, and filter the solid to obtain the corn cob dilute acid hydrolysate.

[0061] The inorganic salt culture medium is the same as described in Example 1.

[0062] Biomass detection method: The biomass of the bacterial strain was determined using a UV spectrophotometer. The strain was diluted to a certain extent with pure water to ensure that the strain was within the OD range. 600Within a limited measurement range, record its reading.

[0063] Determination of soluble total phenols (TPC): The modified Folin-Ciocalteu method was used. Construction of the standard curve: Accurately weigh 10 mg of the standard (vanillin) and place it in a 100 mL volumetric flask. Add an appropriate amount of water and shake to dissolve; this is the standard solution. Take 1.5 mL, 1.25 mL, 1 mL, 0.75 mL, 0.5 mL, and 0.25 mL of the standard solution respectively and place them in volumetric flasks. Add 0.5 mL of Folin-Ciocalteu reagent and mix thoroughly. Then add 1 mL of 15% Na₂CO₃ solution and mix to a final volume of 25 mL. Incubate at 55 ℃ for 5 min. Cool to room temperature and measure the absorbance at 760 nm. Repeat three times. Plot the standard curve with the mass concentration of the standard in the reaction system as the x-axis and the absorbance as the y-axis. To determine the total phenol content in the sample solution, accurately measure 1 mL of the test solution into a 25 mL volumetric flask, add 9.5 mL of distilled water, shake well, then add 0.5 mL of Folin-Ciocalteu reagent, mix well, add 1 mL of 15% Na₂CO₃ solution, mix thoroughly, and bring to volume. Incubate in a 55 ℃ water bath for 5 min, cool to room temperature, and measure the absorbance at 760 nm. Calculate the concentration of phenolic compounds in the sample based on the absorbance value and the standard curve.

[0064] Depend on Figure 3 As shown, within 6 days, the removal rate of phenolic substances was 68.4%, and the consumption of reducing sugar was 11.77 g / L. In dilute acid hydrolysate, when the strain was inoculated for 4 days, the removal rate of phenolic substances reached 42.0%, indicating that strain N1 has a significant effect on the removal of phenolic substances. Example 5

[0065] strain Rhodococcus aetherivorans Application of N1 detoxified hydrolysate:

[0066] Centrifuge the detoxified hydrolysate to remove bacterial cells, and use it to prepare the culture medium in place of the pure water in the Suc260 medium. The experimental method is the same for the undetoxified hydrolysate. Dispense the prepared Suc260 medium into anaerobic flasks, 27 mL per flask. Purge each flask with CO2 for 4 min, incubate at 121 °C for 20 min. Inoculate each flask with 3 mL of Suc260 seed culture using a sterile syringe, and incubate at 37 °C for 72 h. Samples are taken every 12 h. Each experiment is designed with three replicates. Figure 4 ).

[0067] The above Suc260 fermentation medium (g / L) contains: betaine 0.12, diammonium hydrogen phosphate 2.6, ammonium dihydrogen phosphate 0.87, potassium chloride 0.15, magnesium sulfate heptahydrate 0.37, trace elements 1 mL, and basic magnesium carbonate 48.0.

[0068] The results are as follows Figure 4 As shown, the detoxified hydrolysate on the fourth day contained 30 g / L reducing sugar, and the content of phenolic inhibitors decreased from 3.6 g / L to 2.1 g / L, with a phenolic removal rate of 42%. The undetoxified hydrolysate contained 36 g / L reducing sugar. Using the hydrolysate as a carbon source for anaerobic fermentation of succinic acid, it was found that using the detoxified hydrolysate as a carbon source produced 10.5 g / L of succinic acid within 72 hours, which was 2.6 times that of the undetoxified hydrolysate.

Claims

1. Rhodococcus ethereans ( Rhodococcus aetherivorans The application of N1 in the detoxification of lignocellulose hydrolysate, wherein the *Rhodococcus ethereans* ( Rhodococcus aetherivorans N1, deposited at the China Center for Type Culture Collection on August 11, 2022, with accession number CCTCC NO: M 20221270; the lignocellulose hydrolysate is a corn cob dilute acid hydrolysate; the corn cob dilute acid hydrolysate is prepared by mixing corn cobs with 3% H2SO4 at a solid-liquid ratio of 1:7.5, hydrolyzing at 126 °C for 2.5 h, and filtering the solids, which contains phenolic inhibitors.

2. The application according to claim 1, characterized in that, Inoculate strain N1 into the lignocellulose hydrolysate at an inoculation rate of 1%–30% v / v, and culture at 25–30 °C with stirring or shaking for 100–144 h to detoxify the hydrolysate.

Images