阅读说明：本技术 一种低共熔溶剂协同二氧六环预处理生物质的方法 (Method for pretreating biomass by using eutectic solvent and dioxane ) 是由 王挥 张�林 徐春燕 熊凤 陈介南 詹鹏 陈德明 于 2021-10-25 设计创作，主要内容包括：本发明公开了一种低共熔溶剂协同二氧六环预处理生物质的方法。本发明以生物质为原料,首次使用低共熔溶剂与二氧六环在酸性条件下联合预处理的方式,在有效脱除木质素的同时实现了纤维素的高效转化。此预处理方法具有成本低廉、绿色无毒、生物相容性好以及易于回收利用等优点,并在能耗较低的条件下实现了较高葡萄糖收率。(The invention discloses a method for pretreating biomass by using a eutectic solvent and dioxane. According to the method, the biomass is used as a raw material, and the high-efficiency conversion of the cellulose is realized while the lignin is effectively removed by using a mode of combined pretreatment of a eutectic solvent and dioxane under an acidic condition for the first time. The pretreatment method has the advantages of low cost, environmental protection, no toxicity, good biocompatibility, easy recycling and the like, and realizes higher glucose yield under the condition of lower energy consumption.)

1. The method for pretreating biomass by the eutectic solvent and dioxane is characterized in that the biomass is pretreated by a mixed solution of the eutectic solvent and the dioxane under an acidic condition.

2. The method of claim 1, wherein the biomass comprises lignin-containing organisms, further comprising green plants or microorganisms.

3. The method according to claim 1, wherein the volume ratio of the eutectic solvent to the dioxane is 1:1-4:1, preferably 1: 1-1: 2; further preferably 1: 1.

4. The method of claim 1, 2 or 3, wherein the acidic condition is formed by adding an acidic solution, and the pH value is adjusted to 0.00-3.00.

5. The process according to claim 1, 2 or 3, characterized in that the solid-to-liquid ratio is 1:10 to 1:20(g/ml), preferably 1: 15-20, and more preferably 1: 20.

6. The process according to claim 1, characterized in that the reaction temperature is 60-140 ℃, preferably 100-140 ℃, and the reaction time is 20-100min, preferably 50-100 min.

7. The method according to claim 1, characterized in that the eutectic solvent selects a hydrogen bond acceptor as choline chloride and a hydrogen bond donor as acetic acid or glycerol, preferably: hydrogen bond acceptor and hydrogen bond donor are present in a ratio of 1: 1.8-1: 2.5, reacting at 75-85 deg.C for 1.5-3h until a transparent homogeneous solution is formed.

8. The method according to claim 1, wherein the pretreated solid residue is obtained as a raw material for the enzymatic saccharification reaction to obtain an enzymatic hydrolysate containing glucose, and the pretreated liquid is recovered and recycled.

9. The method of claim 8, wherein the mixture after the pretreatment reaction is subjected to solid-liquid separation by vacuum filtration, and the solid residue is washed with deionized water until neutral and dried for use.

10. The method as claimed in claim 8 or 9, wherein 1-5g of the solid residue is weighed, 40-60ml of sodium citrate buffer solution is added, 10-30FPU/g of cellulase of the substrate is added, and the mixture is placed in a constant temperature water bath shaker at 40-60 ℃ and 100-150rpm/min for reaction for 60-90 h.

Technical Field

The invention belongs to the field of biomass pretreatment, and particularly relates to a method for pretreating biomass under an acidic condition by using a eutectic solvent and dioxane.

Background

Under the pressure of economic, social and environmental problems caused by the over-use to the gradual exhaustion of fossil energy, environment-friendly alternative energy is actively developed and used, and is a necessary choice for realizing the sustainable development of the economy and the society. Biomass energy has attracted people's attention because of its wide source and renewable property. The biomass energy is derived from photosynthesis of green plants, and is characterized in that organic matters such as plants and microorganisms are used as energy carriers, and solar energy is stored in the form of chemical energy and can be considered as another expression form of solar energy. Biomass resources on the earth are rich, 1000-1250 million tons of biomass yield only on land can be obtained every year, and the biomass yields can be divided into five categories according to different sources: municipal solid waste, agricultural resources, domestic sewage and industrial organic wastewater, forestry resources and livestock and poultry manure. The energy contained in the annually renewable biomass is 10-20 times of the total energy consumption of the whole world.

Compared with fossil energy, biomass is an inexhaustible renewable resource on one hand, and on the other hand, the application of biomass energy can not only reduce the use cost of energy, but also effectively reduce the emission of greenhouse gases and relieve the environmental pressure. In order to use biomass resources more efficiently, it is necessary to understand the structural composition and characteristics. Taking forestry resources in common biomass as an example, lignocellulose which is a main structural component of the biomass is composed of lignin, cellulose, hemicellulose, pectin, inorganic ash and the like. Wherein, cellulose, hemicellulose and lignin are main components, and the three substances respectively account for the dry weight of the whole biomass as follows: 35-50% of cellulose, 15-20% of hemicellulose and 15-20% of lignin. The three substances are different in composition unit and molecular structure, cellulose and hemicellulose mainly comprise macromolecular polymers with hexose and pentose connected together through glycosidic bonds, and lignin is a high polymer formed by crosslinking different phenylpropane. Cellulose and hemicellulose are connected by hydrogen bonds, and internal molecules of the cellulose are connected by hydrogen bonds to form a huge hydrogen bond network; hemicellulose and lignin are connected to form a network structure mainly through chemical bonds such as ester bonds, ether bonds and the like, and cellulose is wrapped inside. The three are intertwined and complexly crosslinked to form a compact and stable three-dimensional structure, so that the lignocellulose has strong capability of resisting external biodegradation or non-biological destruction. The lignocellulose is directly subjected to enzyme hydrolysis, so that the enzyme dosage is large, the enzymolysis efficiency is very low and is usually lower than 20 percent, the sugar yield is influenced, and the economic cost is very high. This therefore poses a significant obstacle to the efficient use of biomass energy.

Research shows that the pretreatment effect of lignocellulose, namely the change of various physicochemical properties of a substrate, also determines the enzymolysis efficiency of the cellulose to a great extent. Therefore, pretreatment of lignocellulose is very important for improving the enzymolysis saccharification efficiency and reducing the use cost of enzyme. The eutectic solvent has wide application prospect in the field of biomass processing, has excellent biocompatibility and degradability, particularly has better lignin removal performance, can improve the stability and activity of a biological enzyme-catalyst, and provides conditions for effectively catalyzing biomass conversion. Meanwhile, the preparation process is relatively simple, the composite material can be prepared by mixing, stirring and heating the components, further purification is not needed during use, and a better recycling effect is achieved. But the effect of removing lignin is still not ideal. While dioxane is generally used as solvent, reaction medium and extractant. According to the method, the eutectic solvent is combined with dioxane to pretreat the biomass containing lignocellulose under an acidic condition, so that a very good effect of synergistically removing lignin and part of hemicellulose is achieved, and the change of cellulose is small.

Disclosure of Invention

The invention aims to provide a method for pretreating a biomass raw material containing lignocellulose in an environment-friendly manner, low in cost, simple in process and low in energy consumption manner, so that lignin and partial hemicellulose are well removed, the cellulose content is not greatly influenced, and the enzymatic hydrolysis yield of the biomass raw material is greatly improved.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for pretreating biomass by using a eutectic solvent and dioxane comprises the following steps: and (3) pretreating the biomass under an acidic condition by using a mixed solution of the eutectic solvent and dioxane.

Because the effect of a pure eutectic solvent for removing lignin is not ideal, a great amount of exploratory attempts are made to find a method for improving the eutectic solvent to degrade lignin, and finally, the addition of dioxane is surprisingly found out, and dioxane is generally used as a solvent, a reaction medium and an extracting agent, and no report about the simultaneous reaction of the two is available.

Therefore, the invention firstly discovers that the synergistic effect of the eutectic solvent and the dioxane can greatly improve the effect of removing the lignin, and also firstly proposes that the eutectic solvent and the dioxane are combined for treating the biomass containing the lignocellulose. In addition, acidic conditions also have a relatively important influence on the achievement of the effect.

Further, the biomass includes lignin-containing organisms, further including green plants or microorganisms.

The volume ratio of the eutectic solvent to the dioxane is 1:1-4:1, preferably 1: 1-1: 2; further preferably 1: 1.

According to the invention, through a large number of exploratory tests, the mixed solution of the eutectic solvent and the dioxane in the proportion range has more excellent lignin degradation or removal effect.

Further, the acidic condition is formed by adding an acidic solution, and the pH value is adjusted to be 0.00-3.00.

The acidic solution comprises: hydrochloric acid, preferably 12mol/L hydrochloric acid solution, and the addition amount in the reaction system is preferably 0.2-1.6 wt%; preferably 0.4 to 1.5 wt%, and more preferably 1.0 to 1.5 wt%.

The invention also finds that the effect is more outstanding under the acidic condition.

Further, the solid-to-liquid ratio is 1:10 to 1:20(g/ml), preferably 1: 15-20, and more preferably 1: 20.

The reaction temperature of the invention is 60-140 ℃, preferably 100-140 ℃, and the reaction time is 20-100min, preferably 50-100 min.

The hydrogen bond acceptor is selected from the eutectic solvent as choline chloride, and the hydrogen bond donor is acetic acid or glycerol.

Of course, hydrogen bond acceptors and hydrogen bond donors include, but are not limited to, those listed above.

The preparation process of the eutectic solvent is that a hydrogen bond acceptor and a hydrogen bond donor are adopted, and the ratio of 1: 1.8-1: 2.5, reacting at 75-85 deg.C for 1.5-3h until a transparent homogeneous solution is formed. Any other method by which a eutectic solvent can be prepared is also included.

By the above method, lignin in the raw material is mostly degraded, hemicellulose is partially degraded, and cellulose is not greatly affected. Such reaction products can be used for subsequent cellulose preparation or enzymatic hydrolysis.

Further, the pretreated solid residue is obtained and used as a raw material for enzymolysis saccharification reaction to obtain an enzymolysis liquid containing glucose, and the pretreatment liquid is recycled.

Specifically, solid-liquid separation is carried out on the mixture after the pretreatment reaction is finished by adopting vacuum filtration, the solid residue is washed to be neutral by deionized water, and the solid residue is dried for later use.

The enzymatic saccharification reaction comprises the following specific processes: weighing 1-5g of solid residue, adding 40-60ml of sodium citrate buffer solution, adding 10-30FPU/g of substrate cellulase, and placing in a constant temperature water bath shaker at 40-60 ℃ and at 100-150rpm/min for reaction for 60-90 h.

And optimizing the process conditions of the pretreatment reaction through an orthogonal experiment to obtain the optimal enzyme hydrolysis yield. Recovering the pretreatment liquid by rotary evaporation and separating out lignin.

The invention has the beneficial effects that:

the inventor of the invention firstly uses the mode of combining the eutectic solvent and dioxane to pretreat the biomass (such as poplar) containing lignocellulose under the acidic condition, effectively removes lignin and partial hemicellulose in the poplar raw material, and retains the cellulose to a greater extent, thereby greatly improving the enzymatic hydrolysis yield of the poplar.

Compared with the prior pretreatment technology, the eutectic solvent used in the invention has the advantages of simple preparation, low cost, no toxicity and good biocompatibility, can be recovered and recycled together with dioxane in modes of rotary evaporation and the like, and has unique chemical properties and green properties.

Compared with the prior art, the pretreatment reaction temperature is higher, the reaction time is longer, the reaction condition is mild, the temperature is lower, the reaction time is shorter, the reaction energy consumption is low, and the loss of sugar can be reduced.

The operation method is simple, the treatment solution can be recycled, the cost is obviously reduced, and the enzymatic hydrolysis yield of cellulose can be effectively improved.

Drawings

FIG. 1 shows the influence of different hydrochloric acid mass fractions (a), reaction temperatures (b) and reaction times (c) on lignin removal rate, hemicellulose removal rate and cellulose retention rate in acetic acid eutectic solvent + dioxane pretreatment.

FIG. 2 shows the influence of different hydrochloric acid mass fractions (a), reaction temperatures (b) and reaction times (c) on the cellulose hydrolysis yield in acetic acid eutectic solvent + dioxane pretreatment

FIG. 3 is a scanning electron microscope image of a poplar raw material (a1-a3), a pretreated (acetic acid eutectic solvent + dioxane) sample (b1-b3) and an enzymolysis sample (c1-c 3).

Fig. 4 shows the composition changes of cellulose, hemicellulose and lignin of a sample (glycerin eutectic solvent + dioxane) after pretreatment of poplar by an orthogonal experiment.

Table 1 shows orthogonal experimental conditions of pretreatment of poplar with glycerol eutectic solvent and dioxane.

Detailed Description

The following examples are intended to further illustrate the invention without limiting it.

The method for measuring hemicellulose, lignin, cellulose, glucose and xylose comprises the following steps: the contents of cellulose, hemicellulose and lignin in the poplar raw material and the pretreated poplar residue, as well as glucose and xylose after enzymatic hydrolysis, were determined by reference to the analysis method of the united states renewable energy laboratory (NREL).

In the formula: c₁、H₁And L₁The content (%) of cellulose, hemicellulose and lignin in the raw material; c₂、H₂And L₂The content (%) of cellulose, hemicellulose and lignin in the pretreated poplar residue.

In the formula: 0.9 is the conversion factor for glucose to cellulose and 0.88 is the conversion factor for xylose to hemicellulose. The saccharification rate in the invention is the enzymatic hydrolysis yield.

Comparative example 1

Accurately weighing choline chloride and acetic acid according to a molar ratio (1: 1), adding the choline chloride and the acetic acid into a reagent bottle, fully and uniformly mixing, and putting the reagent bottle into a magnetic constant-temperature oil bath kettle.

Stirring at 80 deg.C for 2 hr until colorless transparent liquid is formed in the reagent bottle, and closing the magnetic constant-temperature oil bath. The prepared eutectic solvent is preserved.

Adding 5g of dried and crushed (80 meshes) poplar into a 250mL three-neck flask, and adding 100mL of eutectic solvent; 1mL of 12mol/L HCl was added to the reaction system (reaction system pH 0.92).

The solution in the three-neck flask was subjected to oil bath to control the reaction temperature (120 ℃ C.) and magnetic stirring, and the reaction apparatus was equipped with a condensation pressure-reducing device.

After the reaction (80min) is finished, cooling the reactant to room temperature, carrying out vacuum filtration on the solid residue, washing the solid residue with a mixed solution of absolute ethyl alcohol and deionized water (v/v is 1:2) until the filtrate is clear and colorless, and sealing for later use after drying.

Weighing 1g of solid residue, adding 50ml of sodium citrate buffer solution, adding 30FPU/g of substrate cellulase, and placing in a constant temperature water bath shaker at 50 ℃ and 120rpm/min for reaction for 72 h.

After washing and drying, the removal rates of hemicellulose and lignin of the poplar are respectively 55.43 percent and 31.60 percent, and the retention rate of cellulose is 87.17 percent; the yield of enzymatic hydrolysis was 29.59%.

Comparative example 2

Accurately weighing choline chloride and glycerol according to a molar ratio (1: 1), adding the choline chloride and the glycerol into a reagent bottle, fully and uniformly mixing, and putting the reagent bottle and the glycerol into a magnetic constant-temperature oil bath pot.

Stirring at 80 deg.C for 2 hr until colorless transparent liquid is formed in the reagent bottle, and closing the magnetic constant-temperature oil bath. The prepared eutectic solvent is preserved.

5g of dried and pulverized (80 mesh) poplar was taken and put into a 250mL three-necked flask, and 100mL of the eutectic solvent was added. 1mL of 12mol/L HCl was added to the reaction system (reaction system pH 0.92).

The solution in the three-neck flask is subjected to oil bath to control the reaction temperature to 120 ℃ and is subjected to magnetic stirring, and a reaction instrument is provided with a condensation pressure reduction device.

After the reaction is finished for 80min, cooling the reactant to room temperature, carrying out vacuum filtration on the solid residue, washing the solid residue with a mixed solution of absolute ethyl alcohol and deionized water (v/v is 1:2) until the filtrate is clear and colorless, and sealing for later use after drying.

Weighing 1g of solid residue, adding 50ml of sodium citrate buffer solution, adding 30FPU/g of substrate cellulase, and placing in a constant temperature water bath shaker at 50 ℃ and 120rpm/min for reaction for 72 h.

After washing and drying, the removal rates of hemicellulose and lignin of the poplar are respectively 10.64 percent and 11.77 percent, and the retention rate of cellulose is 91.75 percent; the yield of enzymatic hydrolysis was 23.51%.

Example 1

Accurately weighing choline chloride and acetic acid according to a molar ratio (1: 1), adding the choline chloride and the acetic acid into a reagent bottle, fully and uniformly mixing, and putting the reagent bottle into a magnetic constant-temperature oil bath kettle.

Stirring at 80 deg.C for 2 hr until colorless transparent liquid is formed in the reagent bottle, and closing the magnetic constant-temperature oil bath. The prepared eutectic solvent is preserved.

5g of dried and pulverized (80 mesh) poplar was taken and put into a 250mL three-necked flask, and 50mL of a eutectic solvent and 50mL of a dioxane solution were added and mixed. 1mL of 12mol/L HCl was added to the system.

The solution in the three-neck flask is subjected to oil bath to control the reaction temperature to 120 ℃ and is subjected to magnetic stirring, and a reaction instrument is provided with a condensation pressure reduction device.

After the reaction is finished for 80min, cooling the reactant to room temperature, carrying out vacuum filtration on the solid residue, washing the solid residue with a mixed solution (1:2 ═ v/v) of absolute ethyl alcohol and deionized water until the filtrate is clear and colorless, and sealing for later use after drying.

Weighing 1g of solid residue, adding 50ml of sodium citrate buffer solution, adding 30FPU/g of substrate cellulase, and placing in a constant temperature water bath shaker at 50 ℃ and 120rpm/min for reaction for 72 h.

In this example, only a single change in conditions (12mol/L hydrochloric acid addition amount, reaction temperature, reaction time) was investigated without changing other conditions, and the results including hemicellulose removal rate, lignin removal rate, cellulose retention rate, and glucose yield were shown in fig. 1 and 2.

Example 2

Accurately weighing choline chloride and glycerol according to a molar ratio (1: 1), adding the choline chloride and the glycerol into a reagent bottle, fully and uniformly mixing, and putting the reagent bottle and the glycerol into a magnetic constant-temperature oil bath pot.

Stirring at 80 deg.C for 2 hr until colorless transparent liquid is formed in the reagent bottle, and closing the magnetic constant-temperature oil bath. The prepared eutectic solvent is preserved. 12mol/L HCl is added into the system.

5g of dried and pulverized (80 mesh) poplar was taken and put into a 250mL three-necked flask, and 50mL of a eutectic solvent and 50mL of a dioxane solution were added and mixed.

The solution in the three-neck flask is subjected to oil bath to control the reaction temperature and is magnetically stirred, and a condensation pressure-reducing device is arranged in a reaction instrument.

After the reaction is finished, cooling the reactant to room temperature, carrying out vacuum filtration on the solid residue, washing the solid residue with a mixed solution (1:2 ═ v/v) of absolute ethyl alcohol and deionized water until the filtrate is clear and colorless, drying and sealing for later use.

In this example, the other conditions were not changed, and the results of the three-factor three-level orthogonal experiment conducted with the addition amount of 12mol/LHCl, the reaction temperature, and the reaction time are shown in Table 1, and the contents of cellulose, hemicellulose, and lignin in the treated residue are shown in FIG. 4.

When the treatment temperature was 100 ℃ and the treatment time was 80min with 1mL of 12mol/L HCl (pH 0.92 in the reaction system), the enzymatic hydrolysis yield of the residue was 90.44%. When the HCl content was 1mL of 12mol/L HCl (pH 0.92 in the reaction system), the treatment temperature was 140 ℃ and the treatment time was 70min, the enzymatic hydrolysis yield of the obtained residue was 99.15%. The yield of lignin in the treated black liquor reaches 34 percent.

The recovery rate of the eutectic solvent in the first circulation is 88.20 percent; the recovery of the second cycle was 60.60%.

TABLE 1