CN116855401A - Bacterial strain for degrading various lignin-derived aromatic compounds and application of bacterial strain in detoxification of hydrolysate - Google Patents

Bacterial strain for degrading various lignin-derived aromatic compounds and application of bacterial strain in detoxification of hydrolysate Download PDF

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CN116855401A
CN116855401A CN202310427912.6A CN202310427912A CN116855401A CN 116855401 A CN116855401 A CN 116855401A CN 202310427912 A CN202310427912 A CN 202310427912A CN 116855401 A CN116855401 A CN 116855401A
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信丰学
姜万奎
高海燕
孙敬翔
蒋羽佳
章文明
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Nanjing Tech University
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Abstract

The invention discloses a strain for degrading various lignin-derived aromatic compounds and application thereof in detoxification of hydrolysate, wherein the strain is classified as rhodococcus aetheriae @, and the strain is prepared from the strainRhodococcus aetherivorans) N1, which is preserved in China center for type culture Collection, with a preservation date of 2022, 8 months and 11 days, and a preservation number of: cctccc NO: m20221270. The strain N1 can degrade p-hydroxybenzoic acid, p-coumaric acid, ferulic acid and vanillaAldehyde, coniferyl alcohol and syringaldehyde. After lignin depolymerization, derived phenolic inhibitors are formed and fall into three main categories: s-type (syringaldehyde), G-type (ferulic acid) and H-type (p-coumaric acid) influence the fermentation of the strain. The rhodococcus in the invention can degrade SGH type monomer with high efficiency, and the removal efficiency is up to 68.4% when the rhodococcus is applied to biological detoxification of hydrolysate inhibitor. The strain N1 has important application value in improving the conversion efficiency of lignocellulose hydrolysate and reducing the detoxification cost of industrial production.

Description

Bacterial strain for degrading various lignin-derived aromatic compounds and application of bacterial strain in detoxification of hydrolysate
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a strain for degrading various lignin-derived aromatic compounds and application of the strain in detoxification of hydrolysate.
Background
Currently, society is faced with the difficulty of finding alternative and renewable energy sources to replace the widely used conventional energy sources (fossil fuels), and current energy crisis requires development of renewable substrate-based processes. Fuels and chemicals extracted from biomass are considered as environmentally friendly alternatives to petroleum products. In order to produce second generation biofuels and chemicals from lignocellulosic biomass, it is necessary to separate and use all plant biomass components to develop an environmentally and economically viable biorefinery process.
The highly crystalline, structurally complex lignin-protected cellulose renders biomass materials highly recalcitrant, making their depolymerization a difficult task. Therefore, various methods of lignocellulose pretreatment have been developed and applied. Pretreatment methods of lignocellulose can be largely classified into four types of chemical, physical, physicochemical, and biological pretreatment. The chemical, thermal and mechanical processes involved in biomass conversion not only require high energy input but also produce a variety of inhibitors, mainly phenolic, acid and furanic compounds. Wherein, the phenolic inhibitor mainly interferes with the synthesis and the function of the cell membrane by changing the protein ratio of the cell membrane, and inhibits the cell growth.
Detoxification or modulation of lignocellulosic hydrolysates and slurries is one of the most effective methods for combating inhibition problems. The strategy includes physical, chemical and biological detoxification. The goal of these detoxification strategies is to reduce the level of inhibitory compounds to non-inhibitory levels. Many studies report that the inhibitor concentration is reduced by physicochemical means, which generally involves high temperatures and pressures and increases the operating costs. Thus, advantages of biological detoxification over chemical or physical methods include mild reaction conditions, avoidance of further use of toxic and corrosive chemicals, fewer side effects of toxic products, and less energy requirements, which are significant for increasing the conversion efficiency of lignocellulosic hydrolysates.
Disclosure of Invention
The invention aims to provide a strain for degrading various lignin-derived aromatic compounds.
The invention also solves the technical problem of providing the application of the strain in the biological detoxification of lignocellulose hydrolysate.
In order to solve the technical problems, the invention adopts the following technical scheme:
a strain for degrading various lignin-derived aromatic compounds is classified and named as rhodococcus aetheriae @Rhodococcus aetherivorans) N1, which is preserved in China center for type culture Collection, with a preservation date of 2022, 8 months and 11 days, and a preservation number of: cctccc NO: m20221270, the preservation address is: chinese university of Wuhan and Wuhan.
The nucleotide sequence of the 16S rDNA of the strain N1 is shown as SEQ ID NO in a sequence table: 1.
According to the inventionRhodococcus aetherivorans The N1 screening method comprises the following steps: the soil from the forest park leaf pile-up of the university of Nanjing industry was screened in a medium with alkali lignin as the sole carbon source.
Specifically, 5.0. 5.0 g soil samples were taken and added to 5 triangular flasks of 100 mL inorganic salt medium and numbered, glass beads were added to thoroughly break the soil, and the flasks were placed in a constant temperature shaker at 30℃and 180 rpmAnd is sufficiently oscillated about 10 a h a. Each of the 5 mL soil mixtures was added to 5 inorganic salt media of 100 mL, and enriched 5 d was cultured in a shaker at 30℃and 180 rpm as mother liquor for the next-generation enrichment culture. After four successive passages, the enrichment was diluted by gradient dilution to different concentration gradients (10 -3 -10 -8 ) Respectively, 0.1. 0.1 mL of the strain is coated on LB solid medium, and the strain is placed in a constant temperature incubator at 30 ℃ for culturing for a plurality of days (ensuring that as many colonies as possible grow out). Inoculating the strain obtained by enrichment culture into an alkali lignin culture medium containing 100 mL, taking alkali lignin as a unique carbon source, measuring the biomass of the alkali lignin, and selecting the highest biomass for verification.
The formula of the inorganic salt culture medium is (g/L): ammonium sulfate 1.0, monopotassium phosphate 0.5, dipotassium phosphate trihydrate 1.5, sodium chloride 1.0, magnesium sulfate heptahydrate 0.2, trace elements 1mL, pH 7.0.
Alkali lignin medium (g/L): ammonium sulfate 1.0, monopotassium phosphate 0.5, dipotassium phosphate trihydrate 1.5, sodium chloride 1.0, magnesium sulfate heptahydrate 0.2, alkali lignin 2.0, trace elements 1mL and pH 7.0.
LB medium: yeast powder 5.0, peptone 10, sodium chloride 5.0, trace elements 1mL, pH 7.0, and agar powder 2% added to the solid medium.
According to the inventionRhodococcus aetherivorans After growing 2 d on LB solid medium, the colony is orange, round, neat in edge and convex on the surface, and the strain N1 is positive in gram staining. Under the microscope, the strain N1 is in a short rod shape and has no flagella.
The said processRhodococcus aetherivorans N1 can grow by using lignin as the sole carbon source and has high-efficiency degradation capability on the derived aromatic compounds, and enters tricarboxylic acid cycle through catalysis of a series of enzymes. The strain has strong degradation capability on ferulic acid, parahydroxybenzoic acid, vanillic acid and coumaric acid, can directly carry out biological detoxification on lignocellulose hydrolysate, improves the conversion efficiency of the lignocellulose hydrolysate, reduces the detoxification cost of industrial production, and has important application value.
A Chinese medicinal composition comprising the sameThe strain capable of deriving aromatic compounds by utilizing various ligninRhodococcus aetherivorans Cloning vector for N1 16S rDNA sequence.
The recombinant cloning vector is preferably pMD19T as a starting vector.
Comprising said strainRhodococcus aetherivorans Genetically engineered bacterium of N1 16S rDNA sequenceEscherich coliDH5α ( pMD19T-16S )。
The genetically engineered bacteriumEscherich coliDH5 alpha construction method: using primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R:5'-TACCTTGTTACGACTT-3' amplifying 16S rDNA of strain N1, connecting to cloning vector pMD19T by means of T/A cloning, constructing recombinant cloning vector pMD19T-16S, transforming it into cloning host bacteriumEscherich coliDH5 alpha obtaining recombinant microorganismEscherich coli DH5 alpha (pMD 19T-16S), sequencing the obtained exogenous fragment of recombinant microorganism, and comparing the 16S rDNA sequence with NCBI database to identify the strain N1 at molecular levelRhodococcus aetherivoransThe genus bacteria.
The strain N1 provided by the invention has similarity to the metabolic pathways of ferulic acid and coumaric acid through genome sequencing, and the ferulic acid and the coumaric acid are converted into Feruloyl-CoA and coumaric acyl-CoA through coenzyme a synthase (Fcs Feruloyl-CoA synthase) or p-hydroxycinnamoyl-CoA synthase (CouL). The position of the Fcs sequence in the pathway in the whole genome of strain N1 was determined by performing Local blast on the key gene sequence, and the ferulic acid coa synthetase gene Fcs-N1 was determined in the genome chromosome. In the degradation pathway, ferulic acid is converted to vanillin under Fcs and Ech (Enoyl-CoA hydroatase/aldolase) catalysis, vdh (Vanillin dehydrogenase) catalyzes the conversion of vanillin to vanillic acid by NAD-dependent oxidation, followed by VanAB (vanilla o-demethylase oxygenase subunit and oxidoreductase) catalysis towards the central metabolite protocatechuic acid. Coumaric acid is converted to p-hydroxybenzoic acid under the catalysis of Fcs and Ech, followed by conversion to protocatechuic acid under the catalysis of PobA (4-hydroxybenzoate-3-hydroxyase). In the degradation path of syringaldehyde, the syringaldehyde is mainly prepared by aldehyde dehydrogenase Yfmt (Benzaldehyde dehydrogenase) and DesV (Aldehyde dehydrogenase) convert syringaldehyde to syringic acid. All key genes in the degradation path can be recombined and cloned for heterologous expression. Through the sequence-based on the genome,Rhodococcus aetherivorans the N1 strain contains all genes for degrading SGH type monomers of lignin derived aromatic compounds, and can completely degrade wild strains of SGH three types of monomers.
The application of the strain in lignocellulose hydrolysate.
The lignocellulose hydrolysate is corn cob dilute acid hydrolysate.
The preparation method of the corncob hydrolysate comprises the following steps: pulverizing corncob, sieving with 40 mesh sieve, mixing the obtained corncob with 3% H by mass 2 SO 4 Mixing, hydrolyzing at 126 deg.C and solid-liquid ratio (m/V) of 1:7.5 and 2.5. 2.5 h, and vacuum filtering to obtain diluted acid hydrolysis solution of cob with phenolic inhibitor content of 3.4 g/L, which can severely inhibit succinic acid fermentation.
The strain N1 is inoculated into the corncob hydrolysate with the inoculum size of 1% -30% v/v, the temperature of 25-30 ℃ is 25-30 ℃, the mixture is stirred or shake-cultured, the fermentation is 100-144 h (preferably 120-h), the detoxification is carried out, and the removal efficiency of the strain N1 on phenols in the hydrolysate within 7 days is 68.4%.
And verifying the effect of the hydrolysate after detoxification: and (3) centrifuging the detoxified hydrolysate to remove thalli, replacing pure water in a Suc260 culture medium to prepare the culture medium, wherein the experimental method of the non-detoxified hydrolysate is consistent. Subpackaging the prepared Suc260 culture medium into anaerobic bottles, each bottle being 27 and mL, and introducing CO into each bottle 2 4 min,121 ℃ and 20 min. Each flask was inoculated with 3 mL of Suc260 seed solution by a sterile syringe and incubated at 37℃for 72 h.
The detoxified hydrolysate is applied to the fermentation of succinic acid, and 10.5 g/L succinic acid is produced within 72 hours, which is 2.6 times of the yield of the non-detoxified hydrolysate.
Wherein, suc260 fermentation medium (g/L): betaine 0.12, diammonium phosphate 2.6, monoammonium phosphate 0.87, potassium chloride 0.15, magnesium sulfate heptahydrate 0.37, trace element 1mL and basic magnesium carbonate 48.0.
The beneficial effects are that: the invention relates to a forest park rotThe soil at the leaf accumulation position is used as a separating material, and a series of screening, separating and purifying are carried out to obtain a strain capable of growing by using lignin as a unique carbon sourceRhodococcus aetherivorans N1, and is capable of degrading a wide variety of derived aromatic compounds. In addition, the strain N1 can degrade phenolic inhibitors in the hydrolysate, improve the conversion efficiency of the hydrolysate, reduce the detoxification cost of industrial production and has certain reference value for industrial production. The removal efficiency of the strain N1 on the phenols of the hydrolysate within 7 days was 68.4%. The detoxified hydrolysate is applied to the fermentation of succinic acid, and 10.5 g/L succinic acid is produced within 72 hours, which is 2.6 times of the yield of the non-detoxified hydrolysate. The strain N1 is the only strain reported so far for the strain to directly utilize lignocellulose hydrolysate for detoxification, improves the lignocellulose conversion efficiency for industrial production, and has important application value.
Drawings
FIG. 1 is an HPLC analysis of Rhodococcus N1 to degrade various phenolic substances;
FIG. 2 is a simulated degradation hydrolysate phenolic analysis of rhodococcus;
FIG. 3 is a detoxification analysis of lignocellulosic hydrolysate by rhodococcus N1;
FIG. 4 is an analysis of succinic acid production by fermentation of detoxified and non-detoxified hydrolysis solutions.
Detailed Description
The invention will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the invention as described in detail in the claims.
Example 1
Growth strain using lignin as unique carbon sourceRhodococcus aetherivorans Isolation and screening of N1:
weighing 5 g soil sample at the accumulation place of the humic acid in central forest park of Nanjing university of Industrial university, diluting with inorganic salt culture medium, and diluting the concentrated solution into different concentration gradients (10) -3 -10 -8 ) Respectively spreading 0.1. 0.1 mL on LB medium, and culturing in a constant temperature incubator at 30deg.C for several days. Inoculating the strain obtained by enrichment cultureThe biomass was measured in an alkali lignin medium containing 100 mL with alkali lignin as the sole carbon source. Selecting the strain with the highest biomass, thereby screening out the strain capable of directly utilizing lignin.
Screening to obtainRhodococcus aetherivorans N1 strain ecology characteristics: the bacterial colony is orange, round, neat in edge and convex in surface, and the bacterial strain N1 is gram-positive. Under the microscope, the strain N1 is in a short rod shape and has no flagella.
The formula (g/L) of the inorganic salt culture medium comprises the following components: ammonium sulfate 1.0, monopotassium phosphate 0.5, dipotassium phosphate trihydrate 1.5, sodium chloride 1.0, magnesium sulfate heptahydrate 0.2, trace elements 1mL/L, and pH 7.0.
Alkali lignin medium (g/L): ammonium sulfate 1.0, monopotassium phosphate 0.5, dipotassium phosphate trihydrate 1.5, sodium chloride 1.0, magnesium sulfate heptahydrate 0.2, alkali lignin 2.0, trace elements 1mL/L and pH 7.0.
LB medium (g/L): yeast powder 5.0, peptone 10, sodium chloride 5.0, trace elements 1mL/L, pH 7.0, and agar powder 2% added to the solid medium.
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
Bacterial strain for degrading various lignin-derived aromatic compoundsRhodococcus aetherivorans N1 culture conditions and degradation characteristics.
StrainRhodococcus aetherivorans N1 can be grown by using glucose, xylose, fructose, lactose and sucrose as carbon sources.
LB plate medium: yeast powder 5.0, peptone 10, sodium chloride 5.0, trace elements 1mL, pH 7.0, and agar powder 2% added to the solid medium.
Will beRhodococcus aetherivorans N1 is inoculated in LB plate medium, cultured at 30 ℃ for 48 h,
single colonies of the strain N1 were picked from the plates and inoculated into 100 mL fermentation medium, incubated at 30℃at 180 rpm for 48 h, then inoculated into degradation medium at an inoculum size of 5% v/v, incubated at 30℃with shaking at 180 rpm for 24 h, and then tested for monolignol degradation by HPLC.
The formula (g/L) of the fermentation medium 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 1mL, pH 7.0, and sterilizing at 121deg.C for 15min.
The formula (g/L) of the degradation culture medium 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 1mL, pH 7.0, 0.1 g/L lignin monomers (p-hydroxybenzoic acid, p-coumaric acid, ferulic acid, vanillin, coniferyl alcohol, syringaldehyde) were added to the degradation medium, respectively, and sterilized at 121℃for 15min.
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.
HPLC detection method: the thallus reaction solution is filtered by a nylon filter membrane of 0.22 mu M for liquid phase detection after 12,000 rpm for 1 min, and then 1mL supernatant is taken. The model of the liquid chromatograph is Dionex UltiMata 3000; the chromatographic column is a C18 reversed phase chromatographic column (4.6X1250 nm,5 mu M); the mobile phase is methanol: ultrapure water (1% acetic acid) = (70:30 v/v); the flow rate is 1 mL/min, the detection wavelength is 230 nm, the column temperature is 40 ℃, and the sample injection amount is 10 mu L.
As shown in FIG. 1, the strain N1 has different degradation capacities for seven monomers in 24 h, wherein three monomers of parahydroxybenzoic acid, paracoumaric acid and ferulic acid can be completely degraded, and four monomers of vanillin, coniferyl alcohol and syringaldehyde have obvious degradation effects. Further shows that the strain N1 has stronger lignin degradation potential and can be used for researching subsequent phenolic inhibitor degradation strains.
Example 3
Detection and simulated degradation analysis of phenolic substances in corncob hydrolysate
CornThe preparation method of the core hydrolysis liquid comprises the following steps: pulverizing corncob, sieving with 40 mesh sieve, mixing the obtained corncob with 3% H by mass 2 SO 4 Mixing, hydrolyzing at 126 ℃ for 2.5 h with a solid-liquid ratio (m/V) of 1:7.5, and carrying out suction filtration on the solid to obtain the corn cob dilute acid hydrolysate, wherein the content of total phenolic inhibitor is 3.4 g/L, and the proportion of each component is as follows: p-hydroxybenzoic acid: vanillin: syringaldehyde: ferulic acid: p-coumaric acid=16: 68:50:102:147.
taking 1mL corn cob hydrolysate, diluting for 2 times, centrifuging at 12,000 rpm for 1 min, and then taking 1mL supernatant, filtering with a nylon filter membrane of 0.22 mu M, and detecting liquid phase. 100 mg/L of furfural, 5-hydroxymethylfurfural, p-hydroxybenzoic acid, vanillin, syringaldehyde, p-coumaric acid and ferulic acid are also prepared, and are filtered through a nylon filter membrane of 0.22 mu M and then subjected to liquid phase detection. With 2 g/L as total phenol content, various phenol substances are proportionally added into a simulated liquid culture medium with the total volume of 100 mL, the inoculation amount is 2%, the fermentation temperature is 30 ℃, each experiment design is repeated for 12 days, 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 measured by HPLC (figure 2).
The formula (g/L) of the simulated liquid culture medium is as follows: urea 2.0, monopotassium phosphate 0.5, dipotassium phosphate trihydrate 1.5, sodium chloride 1.0, magnesium sulfate heptahydrate 0.2, trace elements 1ml, pH 7.0, sterilizing at 121 ℃ for 15min. The trace elements were prepared in the same manner as in example 2.
Detection conditions of phenolic inhibitor components in the hydrolysate: mobile phase a: 0.02 mol/L NaH with 5% acetonitrile 2 PO 4 (with H 3 PO 4 pH adjusted to 2.9), mobile phase B: acetonitrile/methanol=1:1 (v: v); the flow rate is 1 mL/min, the detection wavelength is 270 nm, the column temperature is 30 ℃, and a gradient elution mode is adopted, wherein the flow rate is 0-13.8 min, and the detection wavelength is 100% A;13.8-45 min,66% A,34% B.
As shown in fig. 2, in the 2 g/L aromatic compound simulation experiment, the degradation rate of ferulic acid was 38.2%, the degradation rate of parahydroxybenzoic acid was 74.6%, the degradation rate of vanillin was 0.5%, the degradation rate of paracoumaric acid was 59.4%, and the degradation rate of coumaric aldehyde was 47.0% within 6 days; vanillin degradation rate was the slowest and only completely degraded on day 10.
Example 4
StrainRhodococcus aetherivorans N1 uses corncob hydrolysate as a culture medium to detoxify the phenolic inhibitor.
The method for treating the corn cob hydrolysate pretreated by dilute acid is shown in example 3, a proper amount of hydrolysate is taken and the pH value is regulated to be neutral, and each bottle of hydrolysate is 100 mL at 121 ℃ for 20 min.50 The mL sterilized centrifuge tubes were several, with 20% inoculum size, rinsed twice with mineral salt medium before inoculation, three replicates per experimental design, and the incubation period was 7 days, the biomass was monitored, samples were taken every 24 th h, and the change in data for biomass, reducing sugars, and soluble Total Phenols (TPC) was detected (fig. 3).
The pretreatment method of the corncob hydrolysate comprises the following steps: pulverizing corncob, sieving with 40 mesh sieve, mixing the obtained corncob with 3% H by mass 2 SO 4 Mixing, hydrolyzing at 126 deg.C and solid-liquid ratio (m/V) of 1:7.5 and 2.5. 2.5 h, and vacuum filtering to obtain diluted acid hydrolysis solution of cob.
The inorganic salt medium was as described in example 1.
The biomass detection method comprises the following steps: the determination of the biomass of the strain utilizes an ultraviolet spectrophotometer to dilute the strain to a certain extent by pure water, thereby ensuring that the strain is at OD 600 The readings are recorded within a limited measurement range.
Determination of soluble Total Phenols (TPC): drawing of standard curves, measured by the modified Folin-Ciocalteu method: taking a standard substance (vanillin) 10 mg, precisely weighing, placing in a 100 mL volumetric flask, adding a proper amount of water, and oscillating to dissolve to obtain a standard solution. Placing standard solution 1.5 mL,1.25 mL,1 mL,0.75 mL,0.5 mL,0.25 mL into volumetric flask, adding 0.5 mL Fu Lin Fen reagent, mixing, and adding 1mL 15% Na 2 CO 3 Mixing the solutions to 25 mL, and placing in a 55 ℃ constant-temperature water bath kettle for 5min. Cooling to room temperature, measuring absorbance at 760 and nm wavelength, repeating for 3 times, and drawing a standard curve by taking the mass concentration of the standard substance in the reaction system as an abscissa and the absorbance as an ordinate. Sample ofMeasuring total phenol content in the solution, accurately measuring the solution 1mL to be measured in a 25 mL volumetric flask, adding 9.5 mL distilled water, shaking, adding 0.5 mL Fulin reagent, mixing, adding 1mL 15% Na 2 CO 3 The solution is fully and evenly mixed, fixed in volume, placed in a constant temperature water bath kettle at 55 ℃ for heat preservation for 5min, cooled to room temperature, and then measured for light absorption value under 760 and nm. And calculating the concentration of the phenolic compound in the sample to be detected according to the absorbance value and the standard curve.
As shown in FIG. 3, the removal rate of the phenolic substance was 68.4% and the consumption of reducing sugar was 11.77 g/L within 6 days. In the dilute acid hydrolysate, when the strain is inoculated on the 4 th day, the removal rate of the phenolic substances can reach 42.0%, and the removal effect of the strain N1 on the phenolic substances can be found to be remarkable.
Example 5
StrainRhodococcus aetherivorans Application of hydrolysate after N1 detoxification:
and centrifuging the detoxified hydrolysate to remove thalli, and replacing pure water in a Suc260 culture medium to prepare the culture medium, wherein the experimental method of the non-detoxified hydrolysate is consistent. Subpackaging the prepared Suc260 culture medium into anaerobic bottles, each bottle being 27 and mL, and introducing CO into each bottle 2 4 min,121 ℃ and 20 min. Each vial was inoculated with 3 mL of Suc260 seed solution using a sterile syringe, incubated at 37℃for 72 h, sampled every 12 h, and repeated for each experimental design (FIG. 4).
Suc260 fermentation medium (g/L) above: betaine 0.12, diammonium phosphate 2.6, monoammonium phosphate 0.87, potassium chloride 0.15, magnesium sulfate heptahydrate 0.37, trace element 1mL and basic magnesium carbonate 48.0.
As shown in FIG. 4, the detoxified hydrolysate on the fourth day contains 30. 30 g/L of reducing sugar, the content of phenolic inhibitor is reduced from 3.6 g/L to 2.1 g/L, and the phenolic substance removal rate is 42%; the non-detoxified hydrolysate contains 36 g/L reducing sugar, the hydrolysate is used as a carbon source for anaerobic fermentation of succinic acid, and the fact that the non-detoxified hydrolysate is used as the carbon source to generate 10.5 g/L succinic acid in 72 hours is 2.6 times that of the non-detoxified hydrolysate.

Claims (8)

1. Strains for degrading various lignin-derived aromatic compounds, which are classifiedNamed as rhodococcus ether feeding%Rhodococcus aetherivorans) N1, which is preserved in China center for type culture Collection, with a preservation date of 2022, 8 months and 11 days, and a preservation number of: cctccc NO: m20221270.
2. The strain utilizing a plurality of lignin-derived aromatic compounds according to claim 1 wherein: the lignocellulose derived aromatic compound is at least one of ferulic acid, coumaric acid, syringaldehyde, vanillin, p-hydroxybenzoic acid and coniferyl alcohol.
3. Use of the strain of claim 1 for detoxification of lignocellulosic hydrolysate.
4. The use according to claim 3, wherein the lignocellulosic hydrolysate is a dilute acid hydrolysate of corncob.
5. The method according to claim 3, wherein the strain N1 is inoculated into the lignocellulose hydrolysate at an inoculum size of 1% -30% v/v, the temperature is 25-30 ℃, the mixture is stirred or shake-cultured, and the hydrolysate is subjected to 100-144 h fermentation for detoxification.
6. The use according to claim 4, wherein the diluted acid hydrolysis solution of corncob is: corncob and 3% by mass of H 2 SO 4 Mixing, hydrolyzing at 126 deg.C at solid-liquid ratio (m/V) of 1:7.5 and 2.5. 2.5 h, and vacuum filtering to obtain the final product containing phenolic inhibitor.
7. A strain comprising the lignin-derived aromatic compound according to claim 1Rhodococcus aetherivoransCloning vector for N1 16S rDNA sequence.
8. Comprising the strain of claim 1Rhodococcus aetherivorans Genetically engineered bacterium of N1 gene sequenceEscherich coli DH5α。
CN202310427912.6A 2023-04-20 2023-04-20 Bacterial strain for degrading various lignin-derived aromatic compounds and application of bacterial strain in detoxification of hydrolysate Pending CN116855401A (en)

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