A method for microwave-assisted pretreatment and enzymatic hydrolysis fermentation of lignocellulosic biomass

Abstract

This invention discloses a microwave-assisted pretreatment and enzymatic fermentation method for lignocellulose biomass, comprising: collecting and drying biomass raw materials, pulverizing them, mixing them with an alkaline solution to form a solid-liquid mixture, and performing alkaline pretreatment under microwave conditions to obtain a pretreated solid sample. The biomass raw materials are grape pomace and corn stalks. For grape pomace, microwave pretreatment with sodium hydroxide solution is used, and the pretreated solid sample is further subjected to simultaneous enzymatic fermentation to produce ethanol. For corn stalks, microwave-assisted deacetylation treatment with sodium carbonate solution is used, and the treated sample is further subjected to dilute sulfuric acid pretreatment and enzymatic hydrolysis. This method can improve substrate accessibility and subsequent bioconversion performance for different biomass raw materials. Specifically, the grape pomace route can improve lignin removal rate and ethanol yield; the corn stalk route can improve acetyl removal rate, reduce the potential for subsequent acetic acid formation, and increase the yield of enzymatically hydrolyzed glucose.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, specifically to a microwave-assisted method for the pretreatment and enzymatic fermentation of lignocellulose biomass. Background Technology

[0002] Lignocellulosic biomass can be used to produce green and renewable energy, reducing dependence on fossil fuels and is considered one of the alternatives to petroleum-based products. Grape pomace and corn stalks are both typical lignocellulosic biomass, but their compositional characteristics and suitable conversion pathways differ, thus requiring the development of appropriate pretreatment and subsequent conversion processes. Grape pomace, as a lignocellulosic biomass, is rich in lipids, proteins, and various carbohydrates, and can replace starch as a raw material for microbial fermentation to produce high-value chemicals such as lactic acid. However, its high lignin content hinders its efficient conversion, necessitating the development of suitable pretreatment and fermentation processes. Grape pomace is mainly used as livestock feed or discarded directly, failing to achieve high-value utilization and easily causing environmental pollution. In corn stalks, the acetyl groups on the hemicellulose easily release acetic acid during subsequent dilute acid pretreatment, thus affecting subsequent enzymatic hydrolysis and bioconversion processes. Using milder sodium carbonate for microwave-assisted deacetylation can remove acetyl groups with minimal carbohydrate loss, reducing the potential for subsequent acetic acid formation and improving enzymatic hydrolysis performance after dilute acid pretreatment.

[0003] Pretreatment can degrade lignin, alter the structure of biomass, overcome the stubbornness of cellulose, and make it easier for enzymes to break it down into fermentable sugars. Commonly used pretreatment methods include alkali treatment, ultrasonic treatment, microwave treatment, hydrogen peroxide oxidation, photocatalysis, and microbial treatment. Alkali treatment can degrade lignin into smaller fragments, making lignin dissolution more significant. It has advantages such as mild preparation conditions and high production efficiency, making it one of the most popular methods in bioethanol production. Microwave pretreatment is considered one of the most promising pretreatment methods. It utilizes thermal and non-thermal effects to drive physical, chemical, or biological reactions, achieving uniform heating, high rate, high efficiency, selectivity (lignin removal), hot spot formation, high precision, and controllable heating. Microwave-assisted processes have advantages in waste treatment and food drying, such as small footprint, high throughput, fast reaction rate, high yield, and high purity, and have attracted increasing attention in recent years. Microwave heating can form high temperatures in a very short time. Microwave-assisted alkali treatment can significantly shorten reaction time, reduce energy consumption, and improve experimental efficiency.

[0004] Traditional lignocellulose pretreatment and enzymatic fermentation conversion processes involve complex enzymatic fermentation steps, product inhibition by enzymes, substrate inhibition in the early stages of fermentation, long production cycles, low yields, and high energy consumption.

[0005] Therefore, this invention proposes a microwave-assisted method for pretreatment and enzymatic fermentation of lignocellulose biomass. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a microwave-assisted method for the pretreatment and enzymatic fermentation of lignocellulose biomass. Employing a microwave-assisted alkali treatment process, it achieves the biotransformation of lignin, efficiently removes lignin, effectively deacetylates, increases carbohydrate content, and improves raw material conversion rate. This facilitates subsequent enzymatic fermentation. Furthermore, the simultaneous enzymatic fermentation process is simple, eliminating the need for separate steps. This overcomes enzyme product inhibition and avoids substrate inhibition in the early stages of fermentation. It offers advantages such as shortened production cycles, reduced equipment investment, increased yield, and reduced energy consumption. This method solves the problems of traditional lignocellulose biomass pretreatment and enzymatic fermentation methods, which involve complex simultaneous enzymatic fermentation steps requiring separate steps, enzyme product inhibition, substrate inhibition in the early stages of fermentation, long production cycles, low yields, and high energy consumption.

[0007] The specific technical solution of the present invention is as follows:

[0008] A microwave-assisted method for pretreatment and enzymatic fermentation of lignocellulose biomass includes the following steps:

[0009] Step 1: Collect dried lignocellulose biomass raw materials; wherein, the biomass raw materials are grape pomace or corn stalks;

[0010] Step 2: Crush and pass through a 40-80 mesh sieve to obtain biomass powder;

[0011] Step 3: Mix the biomass powder with alkaline solution to obtain a solid-liquid mixture, and then perform microwave pretreatment on the solid-liquid mixture;

[0012] Step 4: Obtain the pretreated solid sample and perform subsequent conversion according to the type of raw material.

[0013] in:

[0014] Route 1: When the biomass raw material is grape skins and pomace:

[0015] Grape pomace was pretreated with sodium hydroxide solution using microwaves, then washed until neutral before simultaneous enzymatic fermentation to produce ethanol. This microwave-assisted alkali treatment method for grape pomace biomass pretreatment achieves a high lignin removal rate and increases ethanol yield.

[0016] Preferably, in step two, the pulverization is carried out in a pulverizer.

[0017] Appropriate pretreatment temperature and time can effectively disrupt the structure of lignocellulose and increase the content of fermentable sugars. In step three, the pretreatment is as follows: microwave pretreatment temperature is 50-90℃, and pretreatment time is 30-60 min. Preferably, the pretreatment temperature is 90℃ and the time is 45 min.

[0018] Choosing an appropriate concentration can achieve a high lignin removal rate and a good lignin removal effect. Therefore, in step three, the weight ratio of grape pomace to sodium hydroxide alkaline solution in the solid-liquid mixture is selected as 1:10-20, preferably 1:15; the concentration of sodium hydroxide solution is selected as 1-3%, preferably 2-3%.

[0019] Because Novozymes CTEC3 cellulase has high enzymatic hydrolysis efficiency, high conversion rate, and good hydrolysis effect; and because ATCC55124 Escherichia coli fermentation yields high ethanol conversion rate and high ethanol production, resulting in good fermentation effect, Novozymes CTEC3 cellulase was selected as the cellulase in step four, and ATCC55124 Escherichia coli was selected as the fermentation strain.

[0020] Preferably, in step four, the sample pretreated in step three is washed until neutral and dried at 100-110℃ for 4-8 hours, preferably at 105℃ for 6 hours. Then, the sample is inoculated into a fermenter with a solids addition of 5-15% (w / v), an enzyme addition of 10-20 FPU / g substrate, an inoculum addition of 5-15% (v / v), and a yeast extract addition of 10-20 g / L. The fermentation temperature is controlled at 35-40℃, and the stirring speed is 150-250 rpm. Preferably, the sample is inoculated into a fermenter with a solids addition of 10% (w / v), an enzyme addition of 15 FPU / g substrate, an inoculum addition of 10% (v / v), and a yeast extract addition of 15 g / L. The fermentation temperature is controlled at 37℃, the stirring speed is 200 rpm, and the pH is adjusted to approximately 7 using CaCO3 as the pH adjuster. Adding yeast extract during fermentation can improve the microbial growth environment, increase fermentation efficiency, and thus increase ethanol production.

[0021] Route 2: When the biomass raw material is corn stalks:

[0022] Corn stalks were subjected to microwave-assisted deacetylation treatment with sodium carbonate solution. After treatment, they were washed until neutral, and then pretreated with dilute sulfuric acid and enzymatically hydrolyzed. The sodium carbonate black liquor could be further reused.

[0023] Preferably, in step two, the pulverization is carried out in a pulverizer.

[0024] Preferably, in step three, the pretreatment involves heating the solid-liquid mixture using a microwave heater; the preferred microwave pretreatment temperature is 30-70℃, and the pretreatment time is 0.5-2.5 hours. Using microwave-assisted alkali treatment for corn straw biomass pretreatment can improve the acetyl removal rate, reduce the potential for subsequent acetic acid formation, and increase the yield of enzymatically hydrolyzed glucose.

[0025] Choosing an appropriate concentration can achieve a high lignin removal rate and a good lignin removal effect. Therefore, in step three, the solid-liquid ratio of the solid to the sodium carbonate alkaline solution in the solid-liquid mixture is selected to be 1:10-20, preferably 1:15; the concentration of the sodium carbonate solution is selected to be 1-5%, preferably 3%.

[0026] Preferably, after the pretreatment in step three, the sample is washed until neutral and dried at 100-110℃ for 4-8 hours, preferably at 105℃ for 6 hours.

[0027] Preferably, in step four, the dilute acid pretreatment conditions are as follows: the deacetylated sample is pre-hydrolyzed with 0.5% by mass dilute sulfuric acid at 150°C for 0.5 h, and the solid-liquid ratio is 1:10.

[0028] Preferably, in step four, the enzymatic hydrolysis conditions are as follows: enzymatic hydrolysis with 2%-10% (w / v) solids content using deacetylated and dilute acid-pretreated corn stalks, wherein the enzyme is Novozymes CTEC3 cellulase, the enzyme addition amount is 10-20 FPU / g substrate, preferably 15 FPU / g substrate, the reaction is carried out at 50°C with constant temperature shaking for 72 hours, and the stirring speed is 150 rpm.

[0029] Compared with the prior art, the present invention provides a microwave-assisted method for pretreatment and enzymatic fermentation of lignocellulose biomass, which has the following beneficial effects:

[0030] 1. For the grape pomace route, microwave-assisted sodium hydroxide pretreatment achieved the bioconversion of lignin, improving the lignin removal rate and increasing the carbohydrate content, which is beneficial for subsequent research on simultaneous enzymatic hydrolysis and fermentation conversion processes. Simultaneous enzymatic hydrolysis and fermentation is simple, requiring no separate steps, overcoming both enzyme product inhibition and substrate inhibition in the early stages of fermentation. This approach aims to shorten the production cycle, save on equipment investment, increase yield, and reduce energy consumption.

[0031] 2. For corn stalks, microwave-assisted sodium carbonate treatment helps improve the removal rate of acetyl groups, increase carbohydrate content, reduce the potential for subsequent acetic acid formation, and improve enzymatic hydrolysis efficiency, providing a less inhibitory environment for subsequent fermentation. Attached Figure Description

[0032] Figure 1: Schematic diagram of Example 1;

[0033] Figure 2 Simultaneous enzymatic fermentation was carried out under the conditions of 1% sodium hydroxide concentration, 50°C, and 45 min in Example 1.

[0034] Figure 3 Example 1: Ethanol concentration and yield produced from grape pomace after pretreatment with 1% sodium hydroxide concentration;

[0035] Figure 4 Example 1: Ethanol concentration and yield produced from grape pomace after pretreatment with 2% sodium hydroxide;

[0036] Figure 5 Example 1: Ethanol concentration and yield produced from grape pomace after pretreatment with 3% sodium hydroxide;

[0037] Figure 6 Schematic diagram of Example 2;

[0038] Figure 7 Example 2: Enzymatic hydrolysis results of corn straw after deacetylation and dilute acid pretreatment with 2% solids addition;

[0039] Figure 8 Example 2: Enzymatic hydrolysis results of corn straw after deacetylation and dilute acid pretreatment with 10% solids addition. Detailed Implementation

[0040] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be described in detail and clearly below with reference to the accompanying drawings. Unless otherwise specified, the reagents and instruments used in the following embodiments are all commercially available products, and the experimental methods in the embodiments without specific conditions are generally performed under conventional conditions. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Example 1:

[0042] Microwave-assisted sodium hydroxide pretreatment and simultaneous enzymatic fermentation of grape pomace include the following steps (see treatment procedures). Figure 1 ):

[0043] Step 1: Collect dried grape pomace samples;

[0044] Step 2: Grind the grape skins and pomace in a grinder and pass them through a 40-80 mesh sieve to obtain grape skin and pomace powder;

[0045] Step 3: Pre-treat the solid-liquid mixture of grape pomace powder and sodium hydroxide alkaline solution (concentrations of 1%, 2%, and 3%) at a mass ratio of 1:15 using a microwave heating instrument (pre-treatment temperature (50℃, 70℃, and 90℃); pre-treatment time (30 min, 45 min, and 60 min)); wash the pre-treated sample until neutral and dry it at 105℃ for 6 hours.

[0046] Step 4: Simultaneous fermentation by enzyme-producing bacteria to produce ethanol;

[0047] Specifically, the pretreated sample was washed until neutral and then inoculated into a fermenter with 10% (w / v) solids, 10% (v / v) bacterial inoculum, and 15 g / L yeast extract. The fermentation temperature was controlled at 37℃, and the stirring speed was 200 rpm. Samples were taken periodically (0h, 6h, 9h, 12h, 24h, 36h, 48h, 96h) to determine the sugar and ethanol concentrations. The specific simultaneous enzymatic fermentation method in step four was as follows: Novozymes CTEC3 cellulase was selected, with an enzyme addition of 15 FPU / g substrate; ATCC55124 Escherichia coli was selected, with a 10% (v / v) bacterial inoculum; and CaCO3 was used as the pH adjuster. 3， Adjust the pH to approximately 7; repeat the experiment twice.

[0048] Step 5: Record the lignin removal rate of the pretreated sample, as well as the ethanol concentration and final yield produced during the synchronous enzyme fermentation process.

[0049] Simultaneous enzymatic fermentation experiments were conducted on pretreated grape pomace. The concentrations of various sugars in the grape pomace and the concentration of ethanol produced were measured at different time points during the experiment, and the ethanol yield was calculated and compared. After microwave-assisted alkaline pretreatment with sodium hydroxide, the cellulose content, hemicellulose content, lignin content, lignin removal rate, ash content, recovery rate, and moisture content of the grape pomace were determined using standard methods.

[0050] The lignin removal rate is calculated as follows:

[0051] The cellulose, hemicellulose, lignin, and ash content in grape pomace were determined according to the standard procedure of NREL [Reference: Sluiter, A. (2008). Determination of structural carbohydrates and lignin in biomass. Lab. Anal. Proced. 1617, 1–16]. The pretreated grape pomace was dried in an oven at 105℃ for 6 hours, and the sample recovery rate was determined by weighing. The moisture content was measured using a laboratory moisture meter. The lignin removal rate was calculated from the recovery rate and the mass of the grape pomace before and after pretreatment.

[0052] The methods for calculating ethanol concentration and yield are as follows:

[0053] First, standard samples of various ethanol concentrations were prepared, and their peak times and heights were determined using liquid chromatography. A standard curve of ethanol concentration versus peak height was then constructed, and the formula for calculating ethanol concentration from peak height was determined. One ml of the mixture was taken from the fermentation flask, sterilized in a metal bath, and centrifuged. One ml of the supernatant was collected and analyzed using liquid chromatography. The peak height was measured and substituted into the above formula to calculate the ethanol concentration in the sample. The theoretical ethanol yield was then calculated.

[0054] Different pretreatment processes and test results are shown in Tables 1 and 2:

[0055] Table 1. Composition of grape pomace after different pretreatment processes

[0056] ;

[0057] Table 2 Ethanol yield under different pretreatment processes

[0058] ;

[0059] The experimental results for Sample 1 showed that the lignin removal rate of the pretreated sample was 73.84%. During simultaneous enzymatic fermentation, the ethanol concentration steadily increased over time, with a larger increase in the first 24 hours, eventually stabilizing at 11.03 g / L, achieving a theoretical ethanol yield of 65.92% (e.g., ...). Figure 2 ).

[0060] like Figure 3 , 4 5. After pretreatment with 1%, 2%, and 3% alkali solutions, the cellulose content, hemicellulose content, and lignin removal rate of grape pomace showed an increasing trend with prolonged treatment time; the lignin content showed a decreasing trend. During simultaneous enzymatic fermentation, the ethanol concentration steadily increased with time, with a larger increase in the first 24 hours, eventually stabilizing within a certain range. The theoretical ethanol yield of the pretreated grape pomace was higher after 96 hours of simultaneous enzymatic fermentation; the 3% alkali solution pretreatment was more effective, with a higher lignin removal rate and higher ethanol yield.

[0061] The optimal pretreatment process is: 2% sodium hydroxide solution concentration, 70℃, 45min pretreatment. Under these conditions, the ethanol concentration and yield obtained by simultaneous enzymatic fermentation are the highest, with an average concentration of 14.04 g / L and an average yield of 92.8%.

[0062] Example 2:

[0063] Microwave-assisted sodium carbonate deacetylation treatment of corn stalks and subsequent enzymatic hydrolysis include the following steps (see treatment procedures). Figure 6 ):

[0064] Step 1: Collect corn stalks and air dry them naturally until the moisture content is below 10%;

[0065] Step 2: Crush and pass through a 20-60 mesh sieve to obtain a corn stalk sample;

[0066] Step 3: Mix the corn stalk sample with sodium carbonate solution at a solid-liquid ratio of 1:15, with a sodium carbonate concentration of 1-5 wt%.

[0067] Step 4: Process in a microwave reactor at a temperature of 30-70℃ for 0.5-2.5 hours;

[0068] Step 5: After processing, solid and liquid separation is performed. The obtained solid sample is washed with distilled water until neutral to obtain deacetylated corn stalks.

[0069] Step 6: Pre-treat the deacetylated corn stalks with dilute sulfuric acid under the following conditions: 0.5wt% sulfuric acid, 150℃, 0.5h, and solid-liquid ratio of 1:10.

[0070] Step 7: Perform enzymatic hydrolysis on the solid after dilute acid pretreatment. The enzyme addition amount is 15 FPU / g substrate, and the solid content is 2% or 10% (w / v).

[0071] The composition of corn stalk raw material was determined according to the NREL standard analytical method. Its main components were: cellulose 33.72%, hemicellulose 24.97%, lignin 20.14%, acetyl groups 2.48%, and ash 3.28%. The corn stalk sample was mixed with sodium carbonate solution for deacetylation treatment. This treatment was carried out in a microwave reactor with a sodium carbonate solution concentration of 1-5 wt%, a solid-liquid ratio of 1:15, a treatment temperature of 30-70℃, and a treatment time of 0.5-2.5 h. After treatment, solid-liquid separation was achieved by suction filtration using a Buchner funnel. The filtrate was retained for subsequent liquid chromatography analysis, and the solid residue was repeatedly washed with distilled water until neutral, then dried to obtain the deacetylated corn stalk sample. The obtained sample was used for subsequent component analysis and dilute sulfuric acid pretreatment experiments.

[0072] This study investigated the effects of temperature, time, and sodium carbonate concentration on deacetylation at a solid-liquid ratio of 1:15. Specific conditions included: 30℃, 40℃, 50℃, 60℃, and 70℃, with a fixed time of 1.5 h and a fixed sodium carbonate concentration of 3 wt%; 0.5 h, 1.0 h, 1.5 h, 2.0 h, and 2.5 h, with a fixed temperature of 50℃ and a fixed sodium carbonate concentration of 3 wt%; and 1 wt%, 2 wt%, 3 wt%, 4 wt%, and 5 wt%, with a fixed temperature of 50℃ and a fixed time of 1.5 h. All experiments were replicated, and the average values ​​were used for analysis. Component analysis was performed on corn stalk samples before and after deacetylation treatment. Cellulose, hemicellulose, lignin, and acetyl groups were measured according to the NREL standard procedure, and the acetyl removal rate was calculated. The changes in corn stalk components under different deacetylation conditions are shown in Table 3.

[0073] The deacetylated corn stalk samples were further pretreated with dilute sulfuric acid (0.5 wt% H₂SO₄, 150 °C, 0.5 h, solid-liquid ratio 1:10) to improve the accessibility of cellulose in the subsequent enzymatic hydrolysis. The pretreatment was carried out in a Parr reactor. After the reaction, solid-liquid separation was performed in the same manner as described above, and the solid residue was collected, dried, and used for subsequent enzymatic hydrolysis. To investigate the effect of sodium carbonate deacetylation pretreatment on the enzymatic hydrolysis of corn stalks, corn stalks pretreated with deacetylated dilute acid were enzymatically hydrolyzed at solid contents of 2% (w / v) and 10% (w / v). Enzymatic hydrolysis was carried out in conical flasks, with CTEC3 enzyme added at 15 FPU / g substrate. 0.05 mol / L sodium citrate buffer was added to the conical flasks to make the total volume of the enzymatic hydrolysis system 50 ml. After all sample reagents were added, the mixture was thoroughly mixed and sealed, and the reaction was carried out at 150 rpm and 50 °C for 72 h. After sampling, the enzyme was inactivated, pH adjusted, and centrifuged. The supernatant was then filtered through a 0.22 μm filter membrane into a chromatographic bottle to determine the glucose concentration in the enzyme-hydrolyzed sugar solution.

[0074] The calculation methods for glucose concentration and dextran conversion rate are as follows:

[0075] First, standard samples of various glucose concentrations are prepared, and their peak times and heights are determined using liquid chromatography. A standard curve of glucose concentration versus peak height is then constructed, and the formula for calculating glucose concentration from peak height is determined. 1 ml of the mixture is taken from an Erlenmeyer flask, centrifuged, and 1 ml of the supernatant is collected. This supernatant is then analyzed using liquid chromatography, and the peak height is measured. This value is then substituted into the above formula to calculate the glucose concentration in the sample. The enzymatic conversion rate of dextran is further calculated.

[0076] The results of compositional analysis under different deacetylation conditions are as follows:

[0077] Table 3. Composition of corn stalks after different pretreatment processes

[0078] ;

[0079] Table 3 shows that, within the tested conditions, microwave-assisted sodium carbonate treatment effectively reduced the acetyl content in corn stalks. The acetyl content in the treated solid decreased from 2.48% to 0.38%-1.48%, corresponding to an acetyl removal rate of 51.27%-88.53%. Simultaneously, the hemicellulose loss rate was controlled within the range of 3.11%-9.32%, indicating that the treatment effectively removed acetyl groups while retaining carbohydrate components.

[0080] The results on the effect of temperature showed that, with a sodium carbonate concentration of 3 wt% and a treatment time of 1.5 h, as the treatment temperature increased from 30 °C to 70 °C, the acetyl removal rate increased from 54.51% to 88.53%, and the hemicellulose loss rate increased from 3.11% to 9.32%. This indicates that increasing the temperature is beneficial for promoting acetyl removal, but it also leads to a certain degree of hemicellulose loss.

[0081] From the results of the time effect, under the conditions of 50℃ and 3wt% sodium carbonate, as the treatment time was extended from 0.5h to 2.0h, the acetyl removal rate increased from 56.56% to 85.70%, indicating that appropriately extending the treatment time is beneficial to the removal of acetyl groups; when the time was further extended to 2.5h, the acetyl removal rate did not change much, while the loss of hemicellulose continued to increase.

[0082] The results on the effect of sodium carbonate concentration showed that, under the conditions of 50℃ and 1.5h, as the sodium carbonate concentration increased from 1wt% to 5wt%, the acetyl removal rate increased from 51.27% to 86.64%, and the hemicellulose loss rate correspondingly increased from 5.42% to 8.20%. Considering both the acetyl removal efficiency and component retention, 3wt% sodium carbonate showed a better balancing effect.

[0083] Among all the conditions, the preferred deacetylation conditions are: 70℃, 1.5h, 3wt% Na2CO3, and a solid-liquid ratio of 1:15. Under these conditions, the acetyl removal rate is 88.53%, the lignin removal rate is 28.93%, and the hemicellulose loss rate is 9.32%.

[0084] Table 4. Enzymatic hydrolysis results of different pretreatment processes at 2% solids addition.

[0085] ;

[0086] Table 5. Enzymatic hydrolysis results of different pretreatment processes at 10% solids addition.

[0087] ;

[0088] Combination Figure 7 , 8 The results showed that, under the condition of 2% solid content, the sample that was microwave-assisted deacetylated with sodium carbonate and then pretreated with dilute acid had a glucose concentration of 10.55 g / L and a dextran conversion rate of 72.27% after 72 h.

[0089] Under conditions of 10% solid content, the sample that was deacetylated and then pretreated with dilute acid showed an increase in glucose concentration to 54.49 g / L and dextran conversion rate to 72.98% after 72 hours.

[0090] Among them, the sample obtained under the deacetylation conditions of 70℃, 1.5h, and 3wt% Na2CO3 showed better performance under the enzymatic hydrolysis conditions of 10% solid content, with a glucose concentration of 54.49g / L and a dextran conversion rate of 72.98% after 72h.

[0091] The results of this embodiment show that microwave-assisted sodium carbonate treatment can effectively remove acetyl groups from corn stalks under relatively mild conditions and, to some extent, remove lignin, while preserving the main structures of cellulose and hemicellulose relatively well. Since acetyl groups in corn stalks are mainly attached to hemicellulose, they are easily released into acetic acid during subsequent dilute acid pretreatment, thus affecting the subsequent enzymatic hydrolysis. Therefore, by adding a microwave-assisted sodium carbonate deacetylation step before dilute acid pretreatment, the acetyl content in the raw material can be reduced, thereby decreasing the potential for subsequent acetic acid formation.

[0092] Meanwhile, the partial removal of lignin and the loosening of the structure during deacetylation also facilitate the entry of subsequent enzyme molecules into the substrate, improving cellulose accessibility. Especially under 10% solids content conditions, the deacetylated sample still maintained a high dextran conversion rate, indicating that the pretreatment method has a positive effect on improving enzymatic hydrolysis performance under higher solids content conditions.

[0093] In summary, this embodiment demonstrates that microwave-assisted sodium carbonate pretreatment of corn stalks can effectively remove acetyl groups and improve the effects of subsequent dilute acid pretreatment and enzymatic hydrolysis, making it suitable as a pretreatment step in the bioconversion process of corn stalks.

[0094] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A microwave-assisted lignocellulosic biomass pretreatment and enzymatic hydrolysis fermentation method, characterized in that, Includes the following steps: Step 1: Collect dried lignocellulose biomass raw materials; wherein, the biomass raw materials are grape pomace or corn stalks; Step 2: Crush and pass through a 40-80 mesh sieve to obtain biomass powder; Step 3: Mix the biomass powder with alkaline solution to obtain a solid-liquid mixture, and then perform microwave pretreatment on the solid-liquid mixture; Step 4: Obtain the pretreated solid sample and carry out subsequent enzymatic hydrolysis and fermentation or simultaneous enzyme and bacteria fermentation.

2. The method of claim 1, wherein, In step two, the pulverization is carried out in a pulverizer; after the pretreatment in step three, the sample is washed until neutral and dried at 100-110℃ for 4-8 hours, preferably at 105℃ for 6 hours.

3. The method of claim 2, wherein, In step three, the pretreatment conditions are as follows: the microwave pretreatment temperature is 50-90℃, and the pretreatment time is 30-60min; in the solid-liquid mixture, the weight ratio of grape pomace to sodium hydroxide alkaline solution is selected as 1:10-20, preferably 1:15; the concentration of sodium hydroxide solution is selected as 1-3%, preferably 2-3%.

4. The method of claim 3, wherein, When the biomass raw material is grape pomace: the grape pomace is pretreated with sodium hydroxide solution using microwave, washed until neutral, and then fermented with enzymes to produce ethanol.

5. The method of claim 4, wherein, In step four, the cellulase selected is Novozymes CTEC3 cellulase, and the strain used for fermentation is ATCC55124 Escherichia coli.

6. The method according to claim 5, characterized in that, In step four, the conditions for simultaneous enzyme-microbe co-fermentation are as follows: The solids content is 5-15% (w / v), the enzyme content is 10-20 FPU / g substrate, the inoculum content is 5-15% (v / v), and the yeast extract content is 10-20 g / L. The fermentation temperature is controlled at 35-40℃, and the stirring speed is 150-250 rpm. Preferably, the solids content is 10% (w / v), the enzyme content is 15 FPU / g substrate, the inoculum content is 10% (v / v), and the yeast extract content is 15 g / L. The fermentation temperature is controlled at 37℃, the stirring speed is 200 rpm, and the pH is adjusted to 7 using CaCO3 as the pH adjuster.

7. The method of claim 1, wherein, When the biomass raw material is corn stalks: the corn stalks are subjected to microwave-assisted deacetylation treatment with sodium carbonate solution, washed until neutral, and then pretreated with dilute sulfuric acid and enzymatically hydrolyzed.

8. The method of claim 7, wherein, In step three, the pretreatment is as follows: the solid-liquid mixture is heated using a microwave heater; preferably, the microwave pretreatment temperature is 30-70℃ and the pretreatment time is 0.5-2.5h; in the solid-liquid mixture, the solid-liquid ratio of the solid to the sodium carbonate alkaline solution is selected as 1:10-20, preferably 1:15; the concentration of the sodium carbonate solution is selected as 1-5%, preferably 3%.

9. The method according to claim 7 or 8, characterized in that, In step four, the dilute acid pretreatment conditions are as follows: the deacetylated sample is pre-hydrolyzed with 0.5% by mass dilute sulfuric acid at 150°C for 0.5 hours, with a solid-liquid ratio of 1:

10.

10. The method according to any one of claims 7-9, characterized in that, In step four, the enzymatic hydrolysis conditions are as follows: the corn stalks pretreated with deacetylated and dilute acid are used for enzymatic hydrolysis with a solid content of 2%-10% (w / v), wherein the enzyme is Novozymes CTEC3 cellulase, the enzyme addition amount is 10-20 FPU / g substrate, the preferred enzyme addition amount is 15 FPU / g substrate, the reaction is carried out at a constant temperature of 50°C with shaking for 72 hours, and the stirring speed is 150 rpm.

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