A method for preparing pyruvic acid using forest wood and its application

Abstract

The present invention relates to a method for preparing pyruvic acid by using forest wood and its application. The method comprises: subjecting the forest wood to steam explosion treatment and hydrothermal treatment, then enzymolyzing the treated product by using a composite enzyme, and finally fermenting the enzymolyzed product by using pyruvic acid-producing Escherichia coli genetic engineering bacteria to obtain pyruvic acid. The present invention adopts a pretreatment method of combining steam explosion treatment with hydrothermal treatment to improve the degradation degree of hemicellulose, make the structure loose, increase more enzyme binding sites for subsequent enzymolysis, and improve the yield of fermentable sugar. Then, the treated product is enzymolyzed by using a composite enzyme to form a high-concentration fermentable sugar solution mixed with pentose and hexose. And using this as a carbon source, using a pyruvic acid-producing Escherichia coli engineered strain as a fermentation strain, fermentation obtains high-concentration pyruvic acid. The preparation method can reduce the cost of pyruvic acid and realize the high value of forest waste resources.

Description

Technical Field

[0001] The invention belongs to the technical field of pyruvic acid preparation, and relates to a method for preparing pyruvic acid by utilizing forest resources and application thereof. Background Art

[0002] Forestry waste refers to waste generated during forestry production, forest renewal and forest product processing, including logging residues, forest clearing and tending residues and wood processing residues. According to statistics, about 140 million tons of forestry waste is generated by logging and wood processing each year, and 100 million tons of forestry waste is generated by tree pruning. If such a large amount of forestry waste can be used at a high value, it will greatly improve the economic efficiency of forest resources and enhance the forest industry.

[0003] Pyruvic acid, also known as a-oxopropionic acid, is an important intermediate in the sugar metabolism of all biological cells and the mutual conversion of various substances in the body. Because its molecules contain activated ketones and carboxyl groups, it is widely used as a basic chemical raw material in the fields of chemistry, pharmaceuticals, food, agriculture and environmental protection. Its preparation methods are divided into chemical synthesis, enzyme conversion and microbial fermentation.

[0004] For example, CN106111173A discloses a catalyst for preparing pyruvate from lactate and a method for preparing pyruvate: the catalyst is selected from one or more of iron carbide, zirconium carbide, vanadium carbide, iron phosphate, copper oxyphosphate, zirconium oxyphosphate, and vanadyl phosphate, and lactate is used as a raw material and oxygen or air is used as an oxidant. Pyruvate is prepared by catalytic oxidation reaction under the action of the above catalyst; the catalytic reaction can be carried out under normal pressure conditions, and no solvent is required, the product yield is high, and the catalyst can be recovered and reused.

[0005] Compared with chemical synthesis and enzyme conversion, the synthesis of pyruvic acid by microbial fermentation is more advantageous due to its wide source of raw materials, low energy consumption, less pollution and low cost. The strategies for synthesizing pyruvic acid by microbial fermentation are still relatively limited. It would be very meaningful to develop a new method for synthesizing pyruvic acid by microbial fermentation. Summary of the invention

[0006] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for preparing pyruvic acid using forest resources and its application.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing pyruvic acid using wood, the method comprising: subjecting the wood to steam explosion and hydrothermal treatment, then enzymolyzing the treated product using a composite enzyme, and finally fermenting the enzymolysis product using pyruvic acid-producing genetically engineered Escherichia coli to obtain pyruvic acid.

[0009] Since forest biomass has a denser structure than herbaceous biomass and is difficult to degrade, the present invention adopts a pretreatment method of combining steam explosion treatment with hydrothermal treatment. First, weak steam explosion treatment is used to destroy the dense structure of the forest biomass cell wall, making the structure loose, which is conducive to strengthening the subsequent hydrothermal pretreatment; then hydrothermal treatment is used to increase the degradation degree of hemicellulose and make the structure looser, thereby increasing more enzyme binding sites for subsequent enzymatic hydrolysis and increasing the yield of fermentable sugars.

[0010] After steam explosion and hydrothermal treatment, the composition of the forest wood changes greatly, especially the hemicellulose content decreases significantly. Then, the treated product is enzymatically hydrolyzed by a composite enzyme to form a high-concentration fermentable sugar solution mixed with pentose and hexose. Using this as a carbon source, a pyruvic acid-producing Escherichia coli engineered strain is used as a fermentation strain to obtain high-concentration pyruvic acid. This preparation method can reduce the cost of pyruvic acid and realize the high value of forest waste resources.

[0011] In addition, most of the current pyruvate fermentation strains are yeasts, and since yeasts lack a pentose utilization pathway, it is difficult to efficiently utilize pentose. Therefore, the present invention uses an Escherichia coli engineered bacterium that can utilize pentose and hexose at the same time to produce pyruvate, which can achieve sugar-acid conversion to the greatest extent.

[0012] The above-mentioned steps are closely linked and promoted, and together a method for preparing pyruvic acid with low cost and high yield is obtained.

[0013] Preferably, the forest wood comprises forest waste.

[0014] Since a large amount of forestry waste is generated every year by logging, wood processing and tree pruning, the use of forestry waste as the raw material for the preparation of the present invention can realize the high value of forestry waste resources, which has great practical significance.

[0015] Preferably, the complex enzyme comprises cellulase and hemicellulase.

[0016] Preferably, the cellulase comprises any one of β-glucosidase, endoglucanase, exoglucanase or pectinase, or a combination of at least two of them; the combination of at least two of them is for example a combination of β-glucosidase, endoglucanase and exoglucanase, a combination of β-glucosidase and pectinase, and any other combination can be selected and will not be described one by one here.

[0017] Preferably, the hemicellulase comprises xylanase and / or laccase.

[0018] Further preferably, the complex enzyme comprises β-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase.

[0019] If the composite enzyme used in the method of the present invention comprises β-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase at the same time, the enzymatic hydrolysis effect is better and the concentration of the fermentable sugar solution obtained by enzymatic hydrolysis is higher.

[0020] Preferably, the ratio of filter paper enzyme activity, β-glucosidase activity, pectinase activity, xylanase activity and laccase activity in the complex enzyme is (3-5):(1-2):(0.5-1):(1-2):(0.5-1).

[0021] If the enzyme components of the complex enzyme used in the method of the present invention are mixed according to the above-mentioned specific ratio, the enzymatic hydrolysis effect is better and the concentration of the fermentable sugar solution obtained by enzymatic hydrolysis is higher.

[0022] The specific point values ​​in the above (3-5) can be 3, 3.5, 4, 4.5, 5, etc.

[0023] The specific point values ​​in the above (1-2) can be 1, 1.2, 1.5, 1.8, 2, etc.

[0024] The specific point values ​​in the above (0.5-1) can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc.

[0025] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0026] Preferably, the solid content in the enzymatic hydrolysis system is 15-30%, such as 15%, 16%, 18%, 20%, 22%, 23%, 24%, 26%, 28%, 30%, etc. Other specific values ​​in this numerical range can be selected and will not be described one by one here. Preferably, it is 20-25%.

[0027] Preferably, the enzymatic hydrolysis temperature is 40-60°C, for example, 40°C, 42°C, 45°C, 47°C, 50°C, 52°C, 55°C, 58°C, 60°C, etc., and the enzymatic hydrolysis time is 20-40h, for example, 20h, 25h, 30h, 35h, 40h, etc. Other specific point values ​​in this numerical range can be selected, and they will not be described one by one here.

[0028] Preferably, the wood is crushed before being subjected to steam explosion treatment to obtain wood particles.

[0029] Preferably, the particle size of the particles is 0.1-50 mm, such as 0.1 mm, 5 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, etc. Other specific values ​​in this numerical range can be selected and will not be described here one by one. More preferably, 5-10 mm.

[0030] Preferably, the solid content of the steam explosion treatment system is 50-80%, such as 50%, 60%, 70%, 75%, 80%, etc. Other specific values ​​in this numerical range can be selected and will not be described here one by one. More preferably, 70-75%.

[0031] Preferably, the system pressure of the steam explosion treatment is 0.8-2.0 MPa, such as 0.8 MPa, 1.0 MPa, 1.2 MPa, 1.4 MPa, 1.5 MPa, 1.6 MPa, 2.0 MPa, etc. Other specific values ​​in this numerical range can be selected and will not be described one by one here. More preferably, 1.4-1.6 MPa.

[0032] Preferably, the duration of the steam explosion treatment is 1-10 minutes, such as 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 8 minutes, 10 minutes, etc. Other specific values ​​in this numerical range can be selected, which will not be described one by one here. More preferably, 3-5 minutes.

[0033] The above steam explosion treatment parameters cooperate with each other to achieve a better steam explosion effect, loosen the cell wall structure, and are more conducive to strengthening the subsequent hydrothermal pretreatment.

[0034] Preferably, the solid content of the hydrothermal treatment system is 30-50%, such as 30%, 35%, 40%, 45%, 50%, etc. Other specific values ​​in this numerical range can be selected and will not be described here one by one. More preferably, 40-45%.

[0035] Preferably, the hydrothermal treatment system contains an acid, and the concentration of the acid is 0.1-3.0%, such as 0.1%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, etc. Other specific values ​​in this numerical range can be selected and will not be described here one by one. More preferably, 1.0-1.5%.

[0036] Preferably, the temperature of the hydrothermal treatment is 140-200°C, such as 140°C, 150°C, 160°C, 170°C, 180°C, 190°C, 200°C, etc., and the time is 20-60min, such as 20min, 30min, 40min, 50min, 60min, etc. Other specific point values ​​in this numerical range can be selected, and they will not be repeated here. It is further preferred that the temperature is 160-180°C and the time is 30-40min.

[0037] The above-mentioned hydrothermal treatment parameters cooperate with each other to achieve better hydrothermal effect, improve the degree of hemicellulose degradation, increase more enzyme binding sites for subsequent enzymatic hydrolysis, and increase the yield of fermentable sugars.

[0038] Preferably, the initial fermentable sugar concentration in the fermentation system is 10-40 g / L or more, such as 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, etc. Other specific values ​​in this numerical range can be selected and will not be described here one by one. Preferably, it is 25-35 g / L.

[0039] During the fermentation process, sugar solution is added according to the residual sugar content in the fermentation tank. When the residual sugar content is lower than 5.0g / L, sugar solution is added to maintain the sugar concentration in the fermentation tank above 10g / L. The pH value of the fermentation liquid is regulated by adding sodium hydroxide.

[0040] Preferably, the pH value of the fermentation system is 6.5-7.5, such as 6.5, 7.0, 7.5, etc. Other specific points in this numerical range can be selected and will not be described in detail here.

[0041] Preferably, the fermentation temperature is 30-40°C, for example, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, etc., and the fermentation time is 24-90h, for example, 24h, 36h, 48h, 52h, 60h, 70h, 80h, 90h, etc. Other specific point values ​​in this numerical range can be selected, and they will not be described here one by one.

[0042] The above fermentation treatment parameters cooperate with each other to achieve better fermentation effect and obtain higher concentration of pyruvic acid.

[0043] Preferably, the inoculation amount of the bacterial species in the fermentation system is 1-10%, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc. Other specific values ​​within this numerical range can be selected and will not be described one by one here. More preferably, 5-7%.

[0044] Preferably, the fermentation system also contains nitrogen sources, inorganic salts and trace elements.

[0045] Preferably, the nitrogen source comprises any one of yeast extract, peptone, corn steep liquor, molasses, ammonia water, ammonium salt or urea, or a combination of at least two of them. The combination of at least two of them is, for example, a combination of yeast extract and peptone, a combination of ammonium salt and urea, and other specific point values ​​in the numerical range can be selected, which will not be described one by one here.

[0046] The inorganic salt may be, for example, (NH4)2HPO4, NH4H2PO4, KCl, NH4Cl, MgSO4·7H2O, etc.; the trace amount of flavor enhancer may be, for example, FeCl3·6H2O, CoCl2·6H2O, CuCl2·2H2O, ZnCl2, Na2MoO4·2H2O, H3BO3, MnCl2·4H2O, etc.

[0047] In a second aspect, the present invention provides an application of the method described in the first aspect in preparing pyruvic acid.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] Since forest biomass has a denser structure than herbaceous biomass and is difficult to degrade, the present invention adopts a pretreatment method of combining steam explosion treatment with hydrothermal treatment. First, weak steam explosion treatment is used to destroy the dense structure of the forest biomass cell wall, making the structure loose, which is conducive to strengthening the subsequent hydrothermal pretreatment; then hydrothermal treatment is used to increase the degradation degree of hemicellulose and make the structure looser, thereby increasing more enzyme binding sites for subsequent enzymatic hydrolysis and increasing the yield of fermentable sugars.

[0050] After steam explosion and hydrothermal treatment, the composition of the forest wood changes greatly, especially the hemicellulose content decreases significantly. Then, the treated product is enzymatically hydrolyzed by a composite enzyme to form a high-concentration fermentable sugar solution mixed with pentose and hexose. Using this as a carbon source, a pyruvic acid-producing Escherichia coli engineered strain is used as a fermentation strain to obtain high-concentration pyruvic acid. This preparation method can reduce the cost of pyruvic acid and realize the high value of forest waste resources.

[0051] The above-mentioned steps are closely linked and promoted, and together a method for preparing pyruvic acid with low cost and high yield is obtained. DETAILED DESCRIPTION

[0052] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0053] The pyruvic acid-producing Escherichia coli genetically engineered bacteria involved in the following examples are Escherichia coli YP01 strains with a deposit number of CGMCC No. 10691, deposited at the General Microbiological Center of China National Microbiological Culture Collection Committee on April 7, 2015, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0054] The formula of the seed culture medium involved in the following examples is: carbon source: glucose 1%; ammonium chloride: 2 g / L; macroelements (mmol / L): (NH4)2HPO4 19.92, NH4H2PO4 7.56, KCl 2.00, NH4Cl 7.50, MgSO4·7H2O 1.50; trace elements (μmol / l): FeCl3·6H2O 8.88, CoCl2·6H2O 1.26, CuCl2·2H2O 0.88, ZnCl2 2.20, Na2MoO4·2H2O 1.24, H3BO3 1.21, MnCl2·4H2O 2.50.

[0055] The formula of the fermentation medium involved in the following examples is: carbon source: hydrolyzed sugar mother liquor; ammonium chloride: 5 g / L; macroelements (mmol / L): (NH4)2HPO4 19.92, NH4H2PO4 7.56, KCl 2.00, NH4Cl 7.50, MgSO4·7H2O 1.50; trace elements (μmol / L): FeCl3·6H2O 8.88, CoCl2·6H2O 1.26, CuCl2·2H2O 0.88, ZnCl2 2.20, Na2MoO4·2H2O 1.24, H3BO3 1.21, MnCl2·4H2O 2.50.

[0056] The β-glucosidase involved in the following examples was purchased from Sigma, model G0395; the endoglucanase was purchased from Sigma, model E2164; the exoglucanase was purchased from Aladdin, model D298997; the pectinase was purchased from Aladdin, model P128776; the xylanase was purchased from Sigma, model X3876; and the laccase was purchased from Aladdin, model L304691.

[0057] Example 1

[0058] This embodiment provides a method for preparing pyruvic acid using pine wood processing waste, as follows:

[0059] (1) Pine processing waste is crushed to a particle size of 10 mm; pine wood particles are watered to a moisture content of 30%, soaked for 2 hours, loaded into a steam explosion machine for steam explosion treatment, the treatment conditions are set to a pressure of 1.6 MPa, and the maintenance time is 3 minutes, and the pine wood particles after steam explosion are collected; water is added to the steam-exploded pine wood particles to a moisture content of 60%, acetic acid is added to make the acetic acid concentration reach 1% (v / v), the treatment conditions are a temperature of 180°C, and the treatment time is 30 minutes; pretreated pine wood particles and pretreatment solutions are obtained by solid-liquid separation, and the composition changes of the pine wood particles and the content of monosaccharides and oligosaccharides in the solution are determined;

[0060] The results showed that the components of pine wood changed significantly, especially the hemicellulose content, which dropped from 25% to 17%; the xylose concentration in the solution reached 5.8g / L, and the xylobiose concentration reached 3.2g / L. The data showed that after pretreatment, the structure of pine wood was destroyed and the easily degradable components had been degraded into monosaccharides or oligosaccharides.

[0061] (2) hydrolyzing the pretreated solution having a solid content of 25% by a composite enzyme, wherein the composite enzyme is a mixture of β-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase, wherein the ratio of filter paper enzyme activity, β-glucosidase activity, pectinase activity, xylanase activity and laccase activity in the composite enzyme is 4:2:1:2:1;

[0062] First, adjust the pH of the enzymatic hydrolysis system to 5.0 with sodium hydroxide; add a composite enzyme with a filter paper enzyme activity of 20 FPU / g, set the enzymatic hydrolysis temperature at 50°C, and perform enzymatic hydrolysis for 36 hours. After the end, obtain the hydrolyzed sugar mother liquor through sedimentation and filtration, and measure the concentration of monosaccharides such as glucose and xylose in the solution;

[0063] It was determined that the glucose concentration in the solution reached 95 g / L and the xylose concentration reached 35 g / L, and this sugar concentration fully met the requirements for fermenting pyruvic acid.

[0064] (3) Producing pyruvate using the genetically engineered Escherichia coli strain YP01;

[0065] (3.1) Seed culture: 100 mL of seed culture medium was sterilized at 115°C for 30 min in a 250 mL Erlenmeyer flask. After cooling, strain YP01 was inoculated into the seed culture medium at a rate of 1% (v / v), and cultured at pH = 7.0, 37°C, and 200 rpm for 12 h to obtain seed solution;

[0066] (3.2) Fermentation culture: The volume of the fermentation medium in a 5L fermenter is 3L, sterilized at 121°C for 20 min, and the hydrolyzed sugar mother solution obtained in step (2) that has been sterilized at high temperature is added to make the monosaccharide concentration in the fermenter 5%. The seed liquid is inoculated into the fermentation medium at an inoculation rate of 5% (v / v), and cultured for 24 hours at pH = 7.0, 37°C and 400rpm, and then the hydrolyzed sugar mother solution is added to make the monosaccharide concentration in the fermenter reach 5%. After 48 hours of culture, the fermentation liquid is obtained. During the culture period, the pH of the fermentation system is adjusted with 20% sodium hydroxide to maintain it at about 7.0;

[0067] High performance liquid chromatography was used to quantitatively analyze the components in the fermentation broth, and the concentrations of glucose and organic acids in the fermentation broth were determined using an Aminex HPX-87H organic acid analysis column. According to the analysis, 75 g / L of pyruvic acid can be synthesized, the sugar-acid conversion rate reaches 0.69 g / g monosaccharide, and the concentration of byproduct acids is very low, including 1.5 g / L of α-ketoglutaric acid, 0.3 g / L of fumaric acid, and 0.5 g / L of citric acid.

[0068] Example 2

[0069] This embodiment provides a method for preparing pyruvic acid using eucalyptus processing waste, as follows:

[0070] (1) The eucalyptus processing waste was crushed to a particle size of 5 mm; the eucalyptus particles were mixed with water to a water content of 25%, soaked for 2 hours, loaded into a steam explosion machine for steam explosion treatment, the treatment conditions were set to a pressure of 1.4 MPa, and the maintenance time was 5 minutes, and the eucalyptus particles after steam explosion were collected; the steam-exploded eucalyptus particles were mixed with water to a water content of 55%, acetic acid was added to make the acetic acid concentration reach 1.5% (v / v), the treatment conditions were a temperature of 160°C, and the treatment time was 40 minutes; the pretreated eucalyptus particles and the pretreated solution were obtained by solid-liquid separation, and the composition changes of the eucalyptus particles and the content of monosaccharides and oligosaccharides in the solution were determined;

[0071] The results showed that the components of eucalyptus wood changed significantly, especially the hemicellulose content, which dropped from 21% to 18%; the xylose concentration in the solution reached 4.9g / L, and the xylobiose concentration reached 1.2g / L. The data showed that after pretreatment, the structure of eucalyptus wood was destroyed, and the easily degradable components had been degraded into monosaccharides or oligosaccharides.

[0072] (2) hydrolyzing the pretreated solution having a solid content of 20% by a composite enzyme, wherein the composite enzyme is a mixture of β-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase, wherein the ratio of filter paper enzyme activity, β-glucosidase activity, pectinase activity, xylanase activity and laccase activity in the composite enzyme is 3:1:0.5:1:0.5;

[0073] First, adjust the pH of the enzymatic hydrolysis system to 5.0 with sodium hydroxide; add a composite enzyme with a filter paper enzyme activity of 20 FPU / g, set the enzymatic hydrolysis temperature at 60°C, and perform enzymatic hydrolysis for 33 hours. After the end, the mother liquor of hydrolyzed sugars is obtained through sedimentation and filtration, and the concentration of monosaccharides such as glucose and xylose in the solution is determined;

[0074] It was determined that the glucose concentration in the solution reached 81 g / L and the xylose concentration reached 33 g / L, which fully met the requirements for fermenting pyruvic acid.

[0075] (3) Producing pyruvate using the genetically engineered Escherichia coli strain YP01;

[0076] (3.1) Seed culture: 100 mL of seed culture medium was sterilized at 115°C for 30 min in a 250 mL Erlenmeyer flask. After cooling, strain YP01 was inoculated into the seed culture medium at a rate of 1% (v / v), and cultured at pH = 7.0, 37°C, and 200 rpm for 12 h to obtain seed solution;

[0077] (3.2) Fermentation culture: The volume of the fermentation medium in a 5L fermenter is 3L, sterilized at 121°C for 20 min, and the hydrolyzed sugar mother solution obtained in step (2) that has been sterilized at high temperature is added to make the monosaccharide concentration in the fermenter 5%. The seed liquid is inoculated into the fermentation medium at an inoculation rate of 5% (v / v), and cultured for 36 hours at pH = 6.5, 35°C and 400rpm, and then the hydrolyzed sugar mother solution is added to make the monosaccharide concentration in the fermenter reach 5%. After 48 hours of culture, the fermentation liquid is obtained. During the culture period, the pH of the fermentation system is adjusted with 20% sodium hydroxide to maintain it at about 7.0;

[0078] High performance liquid chromatography was used to quantitatively analyze the components in the fermentation broth, and the concentrations of glucose and organic acids in the fermentation broth were determined using an Aminex HPX-87H organic acid analysis column. According to the analysis, 74.1 g / L of pyruvic acid can be synthesized, the sugar-acid conversion rate reaches 0.65 g / g monosaccharide, and the concentration of byproduct acids is very low, including 1.3 g / L of α-ketoglutaric acid, 0.3 g / L of fumaric acid, and 0.5 g / L of citric acid.

[0079] Example 3

[0080] This embodiment provides a method for preparing pyruvic acid using poplar wood processing waste, as follows:

[0081] (1) The poplar processing waste is crushed to a particle size of 8 mm; the poplar wood particles are watered to a water content of 50%, soaked for 2 hours, loaded into a steam explosion machine for steam explosion treatment, the treatment conditions are set to a pressure of 1.2 MPa, and the maintenance time is 8 minutes, and the poplar wood particles after steam explosion are collected; the steam-exploded poplar wood particles are watered to a water content of 65%, acetic acid is added to make the acetic acid concentration reach 2.0% (v / v), the treatment conditions are a temperature of 140°C, and the treatment time is 40 minutes; the pretreated poplar wood particles and the pretreated solution are obtained by solid-liquid separation, and the composition changes of the poplar wood particles and the content of monosaccharides and oligosaccharides in the solution are determined;

[0082] The results showed that the composition of poplar wood changed significantly, especially the hemicellulose content, which dropped from 25% to 21%. The xylose concentration in the solution reached 4.6g / L, and the xylobiose concentration reached 2.9g / L. The data showed that after pretreatment, the structure of poplar wood was destroyed, and the easily degradable components had been degraded into monosaccharides or oligosaccharides.

[0083] (2) hydrolyzing the pretreated solution having a solid content of 30% by a composite enzyme, wherein the composite enzyme is a mixture of β-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase, wherein the ratio of filter paper enzyme activity, β-glucosidase activity, pectinase activity, xylanase activity and laccase activity in the composite enzyme is 5:1:1:2:0.5;

[0084] First, adjust the pH of the enzymatic hydrolysis system to 5.0 with sodium hydroxide; add a composite enzyme with a filter paper enzyme activity of 20 FPU / g, set the enzymatic hydrolysis temperature at 40°C, and perform enzymatic hydrolysis for 40 hours. After the end, obtain the hydrolyzed sugar mother liquor through sedimentation and filtration, and measure the concentration of monosaccharides such as glucose and xylose in the solution;

[0085] It was determined that the glucose concentration in the solution reached 91 g / L and the xylose concentration reached 29 g / L, which fully met the requirements for fermenting pyruvic acid.

[0086] (3) Producing pyruvate using the genetically engineered Escherichia coli strain YP01;

[0087] (3.1) Seed culture: 100 mL of seed culture medium was sterilized at 115°C for 30 min in a 250 mL Erlenmeyer flask. After cooling, strain YP01 was inoculated into the seed culture medium at a rate of 1% (v / v), and cultured at pH = 7.0, 37°C, and 200 rpm for 12 h to obtain seed solution;

[0088] (3.2) Fermentation culture: The volume of the fermentation medium in a 5L fermenter is 3L, sterilized at 121°C for 20 min, and the hydrolyzed sugar mother solution obtained in step (2) that has been sterilized at high temperature is added to make the monosaccharide concentration in the fermenter 5%. The seed liquid is inoculated into the fermentation medium at an inoculation rate of 5% (v / v), and cultured for 36 hours at pH = 6.5, 35°C and 400rpm, and then the hydrolyzed sugar mother solution is added to make the monosaccharide concentration in the fermenter reach 5%. After 48 hours of culture, the fermentation liquid is obtained. During the culture period, the pH of the fermentation system is adjusted with 20% sodium hydroxide to maintain it at about 7.0;

[0089] High performance liquid chromatography was used to quantitatively analyze the components in the fermentation broth, and the concentrations of glucose and organic acids in the fermentation broth were determined using an Aminex HPX-87H organic acid analysis column. According to the analysis, 70 g / L of pyruvic acid can be synthesized, the sugar-acid conversion rate reaches 0.65 g / g monosaccharide, and the concentration of byproduct acids is very low, including 1.6 g / L of α-ketoglutaric acid, 0.4 g / L of fumaric acid, and 0.5 g / L of citric acid.

[0090] Example 4

[0091] This example provides a method for preparing pyruvic acid using pine wood processing waste, and the operation thereof is different from that of Example 1 only in that the composition of the composite enzyme is different, and pectinase is not contained, and the reduced amount thereof is distributed to the mass of β-glucosidase, endoglucanase, and exoglucanase according to the proportion of Example 1. Other conditions remain unchanged.

[0092] In step (2), it was determined that the glucose concentration in the solution reached 81 g / L and the xylose concentration reached 25 g / L.

[0093] According to analysis, in step (3), 64.1 g / L of pyruvic acid can be synthesized, the sugar-acid conversion rate reaches 0.60 g / g monosaccharide, and the concentration of by-product acids is very low, including 1.1 g / L of α-ketoglutaric acid, 0.3 g / L of fumaric acid, and 0.5 g / L of citric acid.

[0094] Example 5

[0095] This example provides a method for preparing pyruvic acid using pine wood processing waste, and the operation thereof is different from that of Example 1 only in that the composition of the composite enzyme is different, and it does not contain β-glucosidase, endoglucanase, and exoglucanase, and the reduced amount thereof is allocated to the mass of pectinase. Other conditions remain unchanged.

[0096] In step (2), it was determined that the glucose concentration in the solution reached 30 g / L and the xylose concentration reached 15 g / L.

[0097] According to analysis, in step (3), 27 g / L of pyruvic acid can be synthesized, the sugar-acid conversion rate reaches 0.61 g / g monosaccharide, and the concentration of by-product acids is very low, including 0.3 g / L of α-ketoglutaric acid, 0.1 g / L of fumaric acid, and 0.1 g / L of citric acid.

[0098] Example 6

[0099] This example provides a method for preparing pyruvic acid using pine wood processing waste, and the operation thereof is different from that of Example 1 only in that the composition of the composite enzyme is different, and laccase is not contained, and the reduced amount thereof is allocated to the mass of xylanase. Other conditions remain unchanged.

[0100] In step (2), it was determined that the glucose concentration in the solution reached 78 g / L and the xylose concentration reached 31 g / L.

[0101] According to analysis, in step (3), 71.0 g / L of pyruvic acid can be synthesized, the sugar-acid conversion rate reaches 0.65 g / g monosaccharide, and the concentration of by-product acids is very low, including 1.3 g / L of α-ketoglutaric acid, 0.5 g / L of fumaric acid, and 0.4 g / L of citric acid.

[0102] Example 7

[0103] This example provides a method for preparing pyruvic acid using pine wood processing waste, and the operation thereof is different from that of Example 1 only in that the composition of the composite enzyme is different, and the composite enzyme does not contain xylanase, and the reduced amount thereof is allocated to the mass of laccase. Other conditions remain unchanged.

[0104] In step (2), it was determined that the glucose concentration in the solution reached 85 g / L and the xylose concentration reached 15 g / L.

[0105] According to analysis, in step (3), 62.0 g / L of pyruvic acid can be synthesized, the sugar-acid conversion rate reaches 0.62 g / g monosaccharide, and the concentration of by-product acids is very low, including 1.2 g / L of α-ketoglutaric acid, 0.3 g / L of fumaric acid, and 0.5 g / L of citric acid.

[0106] Example 8

[0107] This example provides a method for preparing pyruvic acid using pine wood processing waste. The difference between the operation and that of Example 1 is only in the fermentation technical parameters:

[0108] (3.2) Fermentation culture: The volume of the fermentation medium in a 5L fermenter is 3L, sterilized at 121°C for 20 min, and the hydrolyzed sugar mother solution obtained in step (2) that has been sterilized at high temperature is added to make the monosaccharide concentration in the fermenter 5%. The seed liquid is inoculated into the fermentation medium at an inoculation rate of 5% (v / v), and cultured for 24 hours at pH = 5.5, 20°C and 400rpm, and then the hydrolyzed sugar mother solution is added to make the monosaccharide concentration in the fermenter reach 5%. After 48 hours of culture, the fermentation liquid is obtained. During the culture period, the pH of the fermentation system is adjusted with 20% sodium hydroxide to maintain it at about 5.5;

[0109] According to analysis, in step (3), 69 g / L of pyruvic acid can be synthesized, the sugar-acid conversion rate reaches 0.63 g / g monosaccharide, and the concentration of by-product acids is very low, including 1.3 g / L of α-ketoglutaric acid, 0.5 g / L of fumaric acid, and 0.6 g / L of citric acid.

[0110] Comparative Example 1

[0111] This comparative example provides a method for preparing pyruvic acid using pine wood processing waste. The difference between the operation and Example 1 is that the pine wood processing waste is not subjected to steam explosion treatment, and other conditions remain unchanged:

[0112] (1) Pine wood processing waste is crushed to a particle size of 10 mm; water is added to the pine wood particles to a moisture content of 60%, and acetic acid is added to make the acetic acid concentration reach 1% (v / v), and the treatment conditions are a temperature of 180°C and a treatment time of 30 minutes; pretreated pine wood particles and a pretreated solution are obtained by solid-liquid separation, and the composition changes of the pine wood particles and the content of monosaccharides and oligosaccharides in the solution are determined;

[0113] It was found that the hemicellulose content in the components of pine wood dropped from 25% to 21%; the xylose concentration in the solution reached 5.1g / L, and the xylobiose concentration reached 2.0g / L.

[0114] Comparative Example 2

[0115] This comparative example provides a method for preparing pyruvic acid using pine wood processing waste. The operation thereof is different from that of Example 1 only in that the pine wood processing waste is not subjected to hydrothermal treatment, and other conditions remain unchanged:

[0116] (1) Pine wood processing waste was crushed to a particle size of 10 mm; pine wood particles were mixed with water to a moisture content of 30%, soaked for 2 hours, and loaded into a steam explosion machine for steam explosion treatment. The treatment conditions were set to a pressure of 1.6 MPa and a holding time of 3 minutes. Pretreated pine wood particles and pretreated solutions were obtained by solid-liquid separation, and the composition changes of the pine wood particles and the contents of monosaccharides and oligosaccharides in the solution were determined;

[0117] It was found that the hemicellulose content in the components of pine wood dropped from 25% to 20%; the xylose concentration in the solution reached 4.9g / L, and the xylobiose concentration reached 2.1g / L.

[0118] Comparative Example 3

[0119] This comparative example provides a method for preparing pyruvic acid using pine wood processing waste. The operation is different from that of Example 1 only in that the genetically engineered Escherichia coli strain YP01 is replaced with an equal amount of yeast (CCTCC M202019), and other conditions remain unchanged.

[0120] According to analysis, in step (3), 60 g / L of pyruvic acid can be synthesized, the sugar-acid conversion rate reaches 0.46 g / g monosaccharide, and the concentration of by-product acids is very low, including 1.9 g / L of α-ketoglutaric acid, 0.8 g / L of fumaric acid, and 0.5 g / L of citric acid.

[0121] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the method for preparing pyruvic acid using forest resources and its application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0122] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0123] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing pyruvic acid using forest wood, characterized in that: The method comprises: subjecting the wood to steam explosion and hydrothermal treatment, then enzymolyzing the treated product with a composite enzyme, and finally fermenting the enzymolyzed product with pyruvic acid-producing Escherichia coli genetically engineered bacteria to obtain pyruvic acid; the Escherichia coli genetically engineered bacteria is the Escherichia coli YP01 strain with a preservation number of CGMCC No.10691; The wood is crushed before the steam explosion treatment to obtain wood particles, the solid content of the steam explosion treatment system is 50-80%, the pressure of the steam explosion treatment system is 0.8-2.0 MPa, and the duration of the steam explosion treatment is 1-10 minutes; The solid content of the hydrothermal treatment system is 30-50%, the hydrothermal treatment system contains acid, the concentration of the acid is 0.1-3.0% v / v, the temperature of the hydrothermal treatment is 140-200° C., and the time is 20-60 min; The complex enzyme is β-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase; the ratio of filter paper enzyme activity, β-glucosidase activity, pectinase activity, xylanase activity and laccase activity in the complex enzyme is (3-5):(1-2):(0.5-1):(1-2):(0.5-1); The solid content of the enzymatic hydrolysis system is 15-30%, the enzymatic hydrolysis temperature is 40-60°C, and the enzymatic hydrolysis time is 20-40h; The pH value of the fermentation system is 6.5-7.5, the fermentation temperature is 30-40° C., the fermentation time is 24-90 h, and the inoculation amount of the bacterial strain in the fermentation system is 1-10% v / v.

2. The method according to claim 1, characterized in that The forest includes forest waste.

3. The method according to claim 1, characterized in that The solid content in the enzymatic hydrolysis system is 20-25%.

4. The method according to claim 1, characterized in that The particle size of the particles is 0.1-50 mm.

5. The method according to claim 4, characterized in that The particle size of the particles is 5-10 mm.

6. The method according to claim 1, characterized in that The solid content of the steam explosion treated system is 70-75%.

7. The method according to claim 1, characterized in that The system pressure of the steam explosion treatment is 1.4-1.6 MPa.

8. The method according to claim 1, characterized in that The duration of the steam explosion treatment is 3-5 min.

9. The method according to claim 1, characterized in that The solid content in the hydrothermal treatment system is 40-45%.

10. The method according to claim 1, characterized in that The concentration of the acid is 1.0-1.5% v / v.

11. The method according to claim 1, characterized in that The temperature of the hydrothermal treatment is 160-180° C., and the time is 30-40 min.

12. The method according to claim 1, characterized in that The initial xylose fermentable sugar concentration in the fermentation system is 10-40 g / L.

13. The method according to claim 12, characterized in that The initial xylose fermentable sugar concentration in the fermentation system is 25-35 g / L.

14. The method according to claim 1, wherein: The inoculation amount of the bacterial species in the fermentation system is 5-7% v / v.

15. The method according to claim 1, wherein: The fermentation system also contains nitrogen sources, inorganic salts and trace elements.

16. The method according to claim 15, characterized in that The nitrogen source includes any one of yeast extract, peptone, corn steep liquor, molasses, ammonia water, ammonium salt or urea, or a combination of at least two thereof.

17. Use of the method according to any one of claims 1 to 16 in the preparation of pyruvic acid.