阅读说明：本技术 一种提高木质纤维素糖化效率的预处理方法 (Pretreatment method for improving saccharification efficiency of lignocellulose ) 是由 陈跃辉 田文静 贺倩妮 陈玥如 于 2019-11-27 设计创作，主要内容包括：本发明公开了一种提高木质纤维素糖化效率的预处理方法。该方法是将乙二酸、氯化胆碱、丙三醇加入水中反应,得到改性低共熔溶剂,采用改性低共熔溶剂对木质纤维素原料进行预处理,该方法能够在更低的温度条件下实现对木质纤维素的预处理,更有利于提高木质纤维素的糖化效率,能大幅提升木质纤维素水解过程中释放碳水化合物总量。(The invention discloses a pretreatment method for improving saccharification efficiency of lignocellulose. According to the method, oxalic acid, choline chloride and glycerol are added into water to react to obtain a modified eutectic solvent, and the modified eutectic solvent is adopted to pretreat the lignocellulose raw material.)

1. A pretreatment method for improving saccharification efficiency of lignocellulose is characterized by comprising the following steps: adding oxalic acid, choline chloride and glycerol into water for reaction to obtain a modified eutectic solvent, and pretreating the lignocellulose raw material by using the modified eutectic solvent.

2. The pretreatment method for improving saccharification efficiency of lignocellulose as recited in claim 1, wherein the pretreatment method comprises the following steps: the molar ratio of the oxalic acid to the choline chloride to the glycerol is 0.25-0.5: 1-1.5: 1.

3. The pretreatment method for improving saccharification efficiency of lignocellulose as recited in claim 2, wherein the pretreatment method comprises the following steps: the molar ratio of the oxalic acid to the choline chloride to the glycerol is 0.25-0.5: 1:1.

4. The pretreatment method for improving saccharification efficiency of lignocellulose as recited in claim 1, wherein the pretreatment method comprises the following steps: the water accounts for 4-16% of the mass of the modified eutectic solvent.

5. The pretreatment method for improving saccharification efficiency of lignocellulose as recited in any one of claims 1 to 4, wherein the pretreatment method comprises the following steps: adding oxalic acid, choline chloride and glycerol into water, and reacting at 75-105 ℃ until the reaction system is transparent.

6. The pretreatment method for improving saccharification efficiency of lignocellulose as recited in claim 1, wherein the pretreatment method comprises the following steps: the mass of the lignocellulose raw material accounts for 8-16% of the total mass of the modified eutectic solvent and the lignocellulose raw material.

7. The pretreatment method for improving saccharification efficiency of lignocellulose as recited in claim 1 or 6, wherein: the pretreatment process of the lignocellulose raw material by adopting the modified eutectic solvent comprises the following steps: adding lignocellulose into the modified eutectic solvent, and pretreating for 1.5-10 hours at the temperature of 80-120 ℃.

8. The pretreatment method for improving saccharification efficiency of lignocellulose as recited in claim 7, wherein: the pretreatment is carried out for 6-8 h at the temperature of 110-120 ℃.

Technical Field

The invention relates to a lignocellulose pretreatment method, in particular to a method for improving enzymolysis saccharification efficiency by utilizing a novel ternary modified eutectic solvent of oxalic acid/glycerol/choline chloride to pretreat lignocellulose, and belongs to the technical field of biofuel preparation.

Background

Lignocellulosic biomass such as agricultural and forestry crops and energy crops has the characteristics of environmental protection, renewability, large production capacity, rich carbohydrate and the like, and the use of fossil fuel can be reduced to a great extent by fully utilizing the new energy to produce the biofuel. The lignocellulose mainly comprises cellulose, hemicellulose and lignin, and the three are tightly interwoven together through hydrogen bonds, covalent bonds and non-covalent bonds to form a firm protective umbrella, so that the resistance of the lignocellulose in the enzymolysis saccharification process is enhanced, and the conversion of the lignocellulose into fermentable sugar is the bottleneck stage for producing the biofuel. The pretreatment technology can greatly change the physical and chemical structural characteristics of the lignocellulose biomass, improve the enzymolysis saccharification efficiency and further improve the total output of the biomass for producing the biofuel.

Lignocellulosic pretreatment methods are numerous. The physical method is to reduce the crystallinity and polymerization degree of cellulose by adjusting the thickness and size of biomass through mechanical means such as crushing, radiation, ball milling and the like, but lignin attached to the surface of the structure still hinders the accessibility of cellulase, so that the sugar yield is not high. The physical and chemical methods include wet oxidation, CO₂Blasting, steam blasting and the like, so that the area contact of the enzyme and the cellulose surface is wider, the cost is high, the requirement on equipment is strict, and the defects that inhibitors can be generated in subsequent fermentation are overcome. The pretreatment by the biological method utilizes actinomycetes, white rot fungi and brown rot fungi to decompose lignin in biomass, and the method has the advantages of mild conditions and environmental friendliness, but has no controllable treatment system with short time period. The chemical method utilizes chemical reagents such as ionic liquid, acid, alkali, organic solvent and the like to dissolve one of cellulose, hemicellulose and lignin, and separates out a cellulose part which can be basically saccharified completely. The ionic liquid has high thermal stability and chemical stability, but is expensive, high in toxicity and difficult to apply industrially. The acid and alkali methods have ideal pretreatment effects, and although the acid and alkali methods are corrosive, byproducts inhibiting the enzyme activity cannot be generated in the pretreatment process. Although the traditional organic solvent method can greatly improve the lignin removal rate and reduce the cellulose loss rate, the cost is high and the difficulty is high. In recent years, an organic solvent method represented by a eutectic solvent (DES) has become one of the important methods for pretreating lignocellulose, wherein the solvent is synthesized from two or more components, the freezing point of the solvent is significantly lower than the melting point of each component, and if the components are all naturalThe organic matter is called as a natural eutectic solvent (NADES), has physicochemical properties similar to those of ionic liquid, can greatly improve the enzymolysis and saccharification efficiency of the lignocellulose, is nontoxic and environment-friendly, is easy to biodegrade, and has simple and convenient operation method.

Although the pretreatment method is not exhaustive, the cost reduction of the treatment reagent, the low energy consumption of the operation process, the simplification of the operation method and the non-toxic and harmless release of pollutants are still the most urgent requirements for improving the enzymolysis and saccharification efficiency of the lignocellulose and further producing the biofuel in a large scale. Ten years ago, Lee J W et al showed (Lee J W, Rodrigues R C L B, Jeffries T W. Simultaneous analysis and ethanol transfer of oxidative acidic pretreated contacted with reactive surface method [ J]BioresourcTechnol, 2009,100(24):6307-6311.) oxalic acid can directly catalyze the hydrolysis of cellulose and hemicellulose, is one of the strongest known organic acids, and organic acids such as maleic acid and oxalic acid can overcome the defects of generation inhibitors and corrosion of equipment, and can be used as a substitute for traditional acids such as sulfuric acid; lee J W et al (Lee J W, J effries TW. efficients of acids in the hydrolysics of lignocellulosic biobased devices a range of combined sensitivity factors [ J W]Bioresource Technology,2011,102(10):5884-Of microorganismsHas lower toxicity than acetic acid or sulfurous acid, and can not inhibitGlycolysisHarmful odor is not generated, hemicellulose can be effectively degraded, and a large residual quantity of cellulose is left; therefore, the oxalic acid which is cheap, non-toxic and environment-friendly is one of the potential pretreatment organic solvents.

Therefore, in recent years, many researchers have tried to develop their own green advantages by using oxalic acid or oxalic acid as one of the components of the natural eutectic solvent, and hope that more energy sources can be obtained from biomass. In 2011, Li X et al (Li X, Cai Z, Horn E, et al. Oxalic acid precursor of edge strand particulate and blocking pin chips: release of heterocyclic hydrocarbons [ J]Tappijournal,2011 (5):41-45.) the results of the study showed that pretreatment of straw particles with oxalic acid resulted in carbohydrate extractionThe temperature is obviously increased by 2.3 times, but the temperature needs to reach 160 ℃; in 2016, Zhang C W et al (Zhang C W, Xia S Q, Ma P S. simple prediction of lignocelluosic biological using regenerative solvents [ J]Bioresource Technology,2016: S0960852416309907) pretreatment of corn cobs with three deep eutectic solvents, monocarboxylic acid/choline chloride, dicarboxylic acid/choline chloride and polyol/choline chloride (DESS), characterization showed that DESS was removed by removal of DESSHemicelluloseAnd lignin to decompose the structure of the corncob so as to obtain higher glucose yield, the removal rate of the oxalic acid/choline chloride to the lignin can reach 98.5 percent, and the glucose yield obtained after enzyme hydrolysis is only 45.2 percent; 2017, Hou X D et al (Hou X D, Feng G J, Ye M, et al, Significantlyen modified enzymic hydrolysics of edge line via a high-performance two-step stationary solutions synthetic prediction [ J]Bioresource Technology,2017,238:139-146.) straw was treated first with oxalic acid/choline chloride and then with choline chloride/urea, the enzymatic hydrolysis of straw was significantly enhanced by the synergistic effect of the two-stage deep eutectic solvent, although the glucose yield could reach 90.2%, which is undoubtedly a cumbersome experimental procedure; therefore, at the present stage, oxalic acid is used as one of organic reagents for pretreating lignocellulose, and the defects of harsh treatment conditions, low saccharification efficiency, complicated operation process and the like exist.

While DES systems that do not contain oxalic acid are described, for example, in Kumar AK et al (Kumar A K, Parikh B S, prazakram. Natural deep electronic solution mediated prediction of edge strand w: biological catalysis of protein extract and enzymatic hydrolysis of predicted biological stress [ J]In the NADES reagents tested by Environmental Science and Pollution Research,2016,23(10):9265-^-1The conversion efficiency of reducing sugar is not high; such as Liu D et al (Liu D, Yan X, Zhuo S, et al, Pandoraea, sp.B-6 assays the deep electronic solution prediction of edge strand via promoting lignin polymerization [ J]Bioresource Technology,2018,257:62-68) continuing the experiments to optimize Kumar A K et al, biomass strawPretreating with lactic acid/choline chloride at a molar ratio of 5:1 at 140 deg.C for 8h, named as DES-RS, activating PandoraeaSP.B-6 seeds, inoculating, culturing, and collecting in DES-RS sterile mineral salt culture medium to obtain final sugar yield of 73.1%. .

In summary, the existing NADES system at the present stage has the disadvantages of harsh treatment conditions or incapability of achieving ideal saccharification efficiency, etc. The prior latest patent technology (CN 107904266 a) discloses a pretreatment method for improving the saccharification effect of lignocellulose, specifically discloses a pretreatment method for improving the saccharification effect of lignocellulose by using choline chloride, citric acid and glycerol according to the ratio of 1: 0.5: 1.5 mixing to obtain NADES, pretreating the rice straw at 130 deg.C for 4 hr to reach peak reducing sugar yield of 840mg/g, and conversion efficiency of 91.3%. However, the method still has the technical problems of high treatment temperature and low reducing sugar yield, and needs to be further improved.

Disclosure of Invention

In order to solve the problems of the existing lignocellulose pretreatment technology, the invention aims to provide a novel DES system with a ternary combination of oxalic acid, glycerol and choline chloride, and compared with the existing choline chloride, citric acid and glycerol, the DES system can realize pretreatment of lignocellulose at a lower temperature, is more favorable for improving the saccharification efficiency of lignocellulose and can greatly improve the total amount of released carbohydrates in the hydrolysis process of lignocellulose.

In order to achieve the technical purpose, the invention provides a pretreatment method for improving the saccharification efficiency of lignocellulose.

In a preferable scheme, the molar ratio of the oxalic acid, the choline chloride and the glycerol is 0.25-0.5: 1-1.5: 1. The most preferred molar ratio is 0.25 to 0.5:1:1. The molar ratio of the oxalic acid, the choline chloride and the glycerol has obvious influence on the pretreatment effect of the lignocellulose raw material, for example, the introduction of the oxalic acid can obviously improve the pretreatment effect of the lignocellulose raw material compared with the combined use of the choline chloride and the glycerol, the preferable molar ratio of the oxalic acid, the choline chloride and the glycerol is 0.25-0.5: 1-1.5: 1, and the best pretreatment effect can be achieved when the ratio of the choline chloride and the glycerol is 1:1.

In the preferable scheme, the water accounts for 4-16% of the mass of the modified eutectic solvent.

According to the preferable scheme, oxalic acid, choline chloride and glycerol are added into water to react at 75-105 ℃ until the reaction system is transparent.

In the preferable scheme, the lignocellulose raw material accounts for 8-16% of the total mass of the modified eutectic solvent and the lignocellulose raw material.

In the preferred scheme, the pretreatment process of the lignocellulose raw material by using the modified eutectic solvent comprises the following steps: adding lignocellulose into the modified eutectic solvent, and pretreating for 1.5-8.5 h at the temperature of 80-120 ℃. The most preferred pretreatment conditions are: pretreating for 6-8 h at 110-120 ℃. The pretreatment temperature and the pretreatment time have influence on the pretreatment effect of the lignocellulose raw material, the influence of the temperature is most obvious, the pretreatment effect is obviously improved along with the increase of the temperature, but the temperature is close to an ideal value to 120 ℃, the effect is unexpected, compared with other eutectic solvents in the prior art, the pretreatment temperature is obviously reduced, the preferred pretreatment temperature is 110-120 ℃, and the best pretreatment temperature is 120 ℃. The pretreatment time is not as obvious as the temperature on the pretreatment effect, but reaches the peak value within 6-8 h.

Preferably, the lignocellulosic material is rice straw.

The invention discloses a pretreatment method for improving saccharification efficiency of lignocellulose, which comprises the following steps:

1) cleaning the lignocellulose raw material with the particle size of 180-400 mu m by using clear water, and drying the lignocellulose raw material at 55 ℃ to constant weight.

2) Mixing 4-16% (W/W) of water content and oxalic acid/glycerol/choline chloride prepared in a certain proportion to form a novel organic solvent, sealing, putting into a constant-temperature drying oven, and completely forming liquid for later use.

3) Mixing the rice straw content of 8-16% (W/W) with the liquid in the step 2), oscillating for 1.5-10 h at 80-120 ℃ in a shaking table, cleaning filter residues until the washing liquid is neutral, and drying the filter residues to obtain a pretreated lignocellulose sample.

4) Adding 8-38 FPU/g substrate cellulase and 3) in (pH4.8, 0.1mol/L) citrate buffer solution, carrying out enzymolysis for 22-72 h at 45-55 ℃ at 100-180 rpm, and sampling to calculate the saccharification efficiency.

Compared with the prior art, the technical scheme of the invention has the advantages that:

1) compared with the prior art, the lignocellulose pretreated by the oxalic acid/glycerol/choline chloride modified eutectic solvent has higher enzymolysis saccharification efficiency at lower pretreatment temperature, for example, the content of reducing sugar released by rice straws pretreated at 120 ℃ after enzymolysis is increased from 350.661mg/g to 961.524mg/g, and the enzymolysis saccharification efficiency is increased from 42.58% to 98.26% which is 2.308 times that of the straws which are not pretreated, so that the lignocellulose pretreated by the oxalic acid/glycerol/choline chloride modified eutectic solvent has obvious technical advantages compared with the prior art.

2) The treatment process for pretreating lignocellulose by using the oxalic acid/glycerol/choline chloride modified eutectic solvent is simple and convenient, the equipment requirement is low, and the raw materials are cheap, easy to obtain and green.

Drawings

FIG. 1 shows the reducing sugar yield before and after pretreatment of lignocellulose at different temperatures;

FIG. 2 shows the variation of reducing sugar yields before and after pretreatment of lignocellulose at different molar ratios;

FIG. 3 shows the variation of reducing sugar yield before and after lignocellulose pretreatment at different times;

FIG. 4 is a graph of optimal choline chloride/oxalic acid (CC/OA) pretreated lignocellulosic reducing sugar yield versus optimal present invention and non-pretreated reducing sugar yield;

FIG. 5 shows the surface structure change before and after lignocellulose pretreatment; a. b, c are not pretreated, d, e, f are example 2 pretreated.

Detailed Description

The invention is further illustrated by the following examples of rice straw research with reference to the accompanying drawings, but the invention is not limited thereto.