CN112553261A

CN112553261A - Method for high-solid enzymolysis of lignocellulose

Info

Publication number: CN112553261A
Application number: CN202011230691.6A
Authority: CN
Inventors: 龚晓贝; 刘浩; 付时雨; 孙成武
Original assignee: Anhui Chengsheng Biotechnology Co ltd; South China University of Technology SCUT
Current assignee: Anhui Chengsheng Biotechnology Co ltd; South China University of Technology SCUT
Priority date: 2020-11-06
Filing date: 2020-11-06
Publication date: 2021-03-26

Abstract

The invention relates to a method for high-solid enzymatic hydrolysis of lignocellulose, comprising the steps of (1) material selection: selecting the raw material of the king palm leaf sheath, washing it first, and cutting it into slices with a length of no more than 20 mm and a thickness of no more than 5 mm, Step (2) NSSC cooking; Step (3) dissolving; Step (4) grading; Step (5) drying; Step (6) Enzymolysis: the thin-walled component particles obtained in Step (5) , cellulase, 0.04% tetracycline, pH 4.8 citric acid buffer in a 12ml conical flask, sealed and placed in a constant temperature shaking box for enzymatic hydrolysis reaction; reaction conditions: temperature 50°C, speed 150rpm, reaction time 72h . The present invention can effectively solve the problem that the high-solid enzymatic hydrolysis of lignocellulose needs to be dried, but the drying causes keratinization and reduces the efficiency of cellulase.

Description

Method for high-solid enzymolysis of lignocellulose

Technical Field

The invention relates to the field of lignocellulose high-solid enzymolysis, in particular to a method for lignocellulose high-solid enzymolysis.

Background

Cellulosic ethanol is a new generation of renewable clean energy, and is always expected to achieve the goal of reducing carbon emission, replace first generation sugar and starch ethanol, maintain the safety of grain energy and the like [1 ]. The raw material of the cellulosic ethanol mainly comprises agricultural and forestry wood waste, and is generally subjected to three steps of pretreatment, enzymolysis and fermentation to produce fermentation liquor with the ethanol concentration of about 5-6% (v/v, about 40-48 g/L), and then the fermentation liquor is subjected to reduced pressure distillation and molecular sieve treatment to obtain absolute ethanol with the volume concentration of more than 99.5%, so as to further produce fuel ethanol for mixed gasoline. The industrialization of cellulosic ethanol still faces a plurality of technical bottlenecks so far, and the most prominent is the enzymolysis difficulty of high solid content.

At present, pilot plant devices and techniques are used at home and abroad, the mature production facilities and techniques of ethanol of the first generation are generally adopted, wherein the distillation operation requires that the ethanol concentration of the fermentation liquor at least reaches 40g/L < 3 >, and if the energy consumption of reduced pressure distillation is reduced as much as possible, the ethanol concentration of the fermentation liquor needs to be increased to 8-10% (v/v, which is equivalent to 64-80 g/L). Therefore, the concentration of the accumulated glucose in the enzymolysis solution is not lower than 125-157g/L, and correspondingly, the initial solid substrate of single batch of enzymolysis needs to reach the solid content of 20-40% (w/w, solid material/water). The high-solid enzymolysis is carried out according to the conditions, and the rheological problem, namely the enzymolysis bottom material loses fluidity due to overhigh viscosity, mass transfer is difficult, and the efficiency of the cellulase is reduced, is firstly overcome. Zhang et al started from two aspects of increasing the cellulose content of raw materials and improving a reactor, and carried out enzymolysis with high solid content of 20 wt% in a self-made reactor with a stirrer by using poplar (cellulose accounts for 80%) treated by a solvent method as a bottom material, wherein the glucose concentration of an enzymolysis solution reaches 158g/L after 48 hours. From the pretreatment stage, the Boehringer team of the university of eastern science develops a dry dilute acid pretreatment technology under the condition of high solid content, and fermentation liquor with the concentration of 101.1 g/L (or 12.8% v/v) of ultrahigh ethanol is prepared from straw raw materials. More researchers have made improvements in enzyme digestion control by keeping the viscosity of the enzyme digestion reaction mixture low through a fed-batch manner. Ellison et al studied waste copy paper fed-batch semi-synchronous saccharification (SSSF) using a specially made high shear mixing bioreactor, resulting in a batch feed to total substrate solids equivalent to 65% (w/w), requiring only 3.7 FPU/g substrate of total enzyme dosage to produce high concentration ethanol (11.6%, v/v). In addition, there have been research groups that have conducted studies on enzyme preparations, such as the use of thermostable cellulases without a cellulose binding domain (CBM) as a catalyst. Therefore, the improvement of the characteristics of the raw materials (the content of cellulose), the pretreatment method, the enzymolysis equipment, the enzymolysis program and the enzyme preparation are feasible ways for overcoming the rheological obstacle under the high-solid condition, realizing the high-solid conversion of the cellulose and finally improving the distillation efficiency of the product.

However, it is not sufficient to solve the "rheology" problem, and the "keratinization" problem is also an important cause of difficulty in mass production of high-solids enzymatic hydrolysis technology. Both in single batch charging and in batch feeding, a bed charge of relatively high dry matter concentration has to be prepared, which requires pressure drying, air drying or stoving of the pretreated wet material. The drying process may lead to collapse of the internal pores of the woody substrate particles, known as "hornification", which is often irreversible and can significantly reduce the accessibility of the substrate to the enzyme, ultimately affecting the rate and conversion of the cellulase hydrolysis reaction.

Disclosure of Invention

The invention aims to provide a method for high-solid enzymolysis of lignocellulose, which solves the problems that the existing high-solid enzymolysis of lignocellulose needs dried raw materials, but the drying causes keratinization and reduces the efficiency of cellulase.

The technical scheme adopted by the invention for solving the technical problems is as follows: a method for high-solid enzymolysis of lignocellulose comprises the following steps:

selecting materials in step (1): selecting a royal palm leaf sheath raw material, cleaning the royal palm leaf sheath raw material, and cutting the royal palm leaf sheath raw material into slices with the length not more than 20mm and the thickness not more than 5mm to obtain royal palm leaf sheath slices;

step (2) NSSC cooking: putting the palm leaf sheath slices into a boiler, and cooking by using neutral sulfite with the liquid-solid ratio of 4: 1; when in cooking, the temperature is firstly raised for 60min, and then the temperature is kept for 30 min; the heat preservation temperature is 150-170 ℃;

step (3) fluffing: putting the cooked royal palm leaf sheath slices into a fluffer, and fluffing for 5000 turns;

step (4) grading: putting the defibered royal palm leaf sheath thin slices into a standard Baore sieving instrument for fiber grading, and separating parenchyma cell components from fiber bundles;

and (5) drying: drying the separated parenchymal cell components until the water content is reduced to be below 10 percent, and obtaining irregular granular parenchymal tissue particles with the length of less than 2 mm;

step (6) enzymolysis: placing the parenchyma granules obtained in the step (5), cellulase, 0.04% tetracycline and a pH4.8 citric acid buffer solution into a 12ml hammer-shaped bottle, sealing, and placing into a constant-temperature shaking box for enzymolysis reaction; reaction conditions are as follows: the temperature is 50 ℃, the rotating speed is 150rpm, and the reaction time is 72 h;

wherein, the feeding mode: feeding materials in a mode of feeding materials for multiple times, in equal proportion and at equal time intervals, wherein the total reaction time is 72 hours, and the total feeding amount of the dried parenchyma particles is 36-45%; simultaneously, cellulase was supplemented at 10FPU/g solids cellulase per feed.

Further, a three-time feeding mode is adopted in the enzymolysis in the step (6), initial one-time feeding is carried out, 24-hour secondary feeding is carried out, 48-hour three-time feeding is carried out, and the reaction is completed within 72 hours; the content of parenchyma granules after each drying is 12-15%, and enzyme liquid is supplemented by solid cellulase according to 10FPU/g in each feeding.

Further, in the enzymolysis in the step (6), 0.7 to 0.9 percent of Tween 80 is also added into the hammer-shaped bottle.

Further, the fluffer is a PTI95568 fluffer of Austria PTI company, and the boiler is a Japanese bamboo straw principle horizontal rotary boiler.

And (3) further, when drying in the step (5), naturally drying in air or drying in an oven at 105 ℃ until the water content is reduced to below 10%.

Further, the temperature of the NSSC cooking in the step (2) is 170 ℃.

A preparation method of a raw material for high-solid cellulose enzymolysis comprises the following steps:

and (5) drying: drying the separated parenchymal cell components until the water content is reduced to be below 10 percent, and obtaining irregular granular parenchymal tissue particles with the length of less than 2 mm.

The invention has the beneficial effects that: according to the method, parenchyma granules prepared by drying parenchyma components extracted from the royal palm leaf sheath raw material are subjected to enzymolysis in a multi-feeding mode, the sugar content of an enzymolysis liquid can reach 150 g/L-170 g/L, and the problems that the existing lignocellulose high-solid enzymolysis needs dried raw materials, but the drying causes keratinization and the efficiency of cellulase is reduced are effectively solved.

Drawings

FIG. 1 shows the ratio of the sheath fiber and the thin-walled component of the royal palm leaves obtained under different cooking conditions.

FIG. 2 shows the cellulose hydrolysis rate of the thin-walled fraction of the sheaths of the royal palm leaves obtained under different cooking conditions.

FIG. 3 is a graph of the effect of dry keratosis on parenchymal and fibrous components.

FIG. 4 is a graph showing the enzymolysis process of the thin-walled fraction.

FIG. 5 is a plot of regression line analysis of wetmass enzymatic hydrolysis rates.

FIG. 6 is a graph showing the progress of enzymatic digestion of a fiber fraction ground and sieved through a 200 mesh sieve.

FIG. 7 is a graph showing single and multiple feed enzymatic hydrolysis of the wang palm leaf sheath thin-walled component oven dried material at 36% total solids for different feed modes.

FIG. 8 is a Duobian plot of single and multiple feed enzymatic hydrolysis curves for 45% total solids versus 36% Elaeis guineensis leaf sheath thin wall fraction oven-dried material.

Detailed Description

The embodiment provides a method for high-solid enzymolysis of lignocellulose, which comprises the following steps:

the thin-walled component dried material is more suitable for high-solid enzymolysis than the wetting material. The wetting material has strong water absorption capacity, the mixed materials of the enzymolysis system have high viscosity, the liquefaction in the oscillation incubator is slow, the reaction is not uniform, and cohesive lumps exist at the bottom. The dry material was not, and the initially dry granules did not absorb water efficiently, delaminated from the water, and liquefied quickly after addition of the enzyme solution (8 h). Moreover, the wet material contains a large amount of water, so that the wet material is difficult to be used for high-solid enzymolysis in practical operation.

The fluffer is a PTI95568 fluffer of Austria PTI company, and the boiler is a Japanese bamboo hat principle horizontal rotary boiler.

And (5) during drying, naturally drying the mixture or drying the mixture in a 105 ℃ oven until the water content is reduced to below 10 percent.

The temperature for NSSC cooking in the step (2) is 170 ℃.

Example 2, in the step (6), a three-time feeding mode is adopted in the enzymolysis, wherein the initial one-time feeding is performed, the reaction is performed for 24 hours for two times, the reaction is performed for 48 hours for three times, and the reaction is completed for 72 hours; the content of parenchyma granules after each drying is 12-15%, and enzyme liquid is supplemented by solid cellulase according to 10FPU/g in each feeding. The rest is the same as example 1.

In the step (6) of enzymolysis, 0.7 to 0.9 percent of Tween 80 is also added into the hammer-shaped bottle in the embodiment 3. The rest is the same as example 2.

The higher the cooking temperature is, the lower the total solid yield is, the cooking heat preservation temperature is improved from 150 ℃ to 180 ℃, and the total solid yield is reduced from about 70% to about 50%. According to the grading measurement, the yield of the thin-wall component (P200) is generally maintained between 20% and 23%, when no chemical agent is used, the yield rises and then falls along with the increase of the temperature, and the yield is higher (23.4%) at 170 ℃; the yield is higher (25.9%) at 160 ℃ by adding 6% of sodium carbonate for cooking; with NSSC cooking, the thin-walled fraction yield decreased slightly with increasing temperature, from 23.4% at 150 ℃ to 20.6% at 180 ℃. Compared with the thin-wall component, the fiber component (R14) is influenced by the cooking condition more obviously, the temperature is increased from 150 ℃ to 180 ℃, the yield of the fiber component is reduced by more than 30 percent, from the perspective of comprehensive biorefinery, the thin-wall component is suitable for biotransformation, the fiber component is suitable for pulping and papermaking, and the degradation loss of the fiber component is avoided as much as possible. Furthermore, the fraction of the intermediate fraction (P14R200), that is, that which passes through a 14 mesh screen but is retained by a 200 mesh screen, is contaminated with fibers and undispersed parenchyma, the lower the fraction yield, indicating that cooking separates the different cell types of the plant more thoroughly, and from FIG. 2, NSSC cooking at 170 ℃ gives the least amount of intermediate fraction (yield 2%), while at 180 ℃ the more intermediate fraction reflects partial fiber bundle degradation and size reduction, consistent with the reduction in the R14 fraction.

NSSC cooking is more beneficial to the biotransformation of cellulose than water or sodium carbonate cooking, after NSSC cooking is carried out at 170 ℃, the CED value of thin-wall component enzymolysis reaches 94.6 percent, the higher cooking temperature is 180 ℃, the CED is equal to the CED value, and the CED obtained by 160 ℃ cooking is 90.3 percent.

The royal palm leaf sheath fiber and thin wall components obtained after pretreatment and classification are still in a wet state, drying is needed for high solid enzymolysis, the solid material is dried at 105 ℃, then conventional enzymolysis with 4% solid content is carried out, the concentration of the reaction product glucose is measured after 72 hours, and the CED is calculated. FIG. 3 shows the keratinization effect caused by drying, and it is evident that the dried material is more resistant to enzymatic hydrolysis than the moistened material at the same enzyme dosage. In a lower cellulase dosage range (5-10 FPU/g solid material), the cornification effect caused by drying reduces the CED value of thin walls and fiber components. However, the thin-walled component was significantly more resistant to keratinization than the fibrous component, and by regression line analysis 3, the 72h CED of the thin-walled and fibrous components remained 97.2% and 71.0%, respectively, after drying. Sufficient enzyme and time to reduce drying-induced CED reduction of the thin-walled fraction, hydrolysis with 15FPU/g solids cellulase for 72h, the dried thin-walled fraction had a CED (91.9%) close to that of the wet control sample (93.3%). Referring to FIG. 3/4/5, unlike the thin-walled fraction, the oven-dried induced keratosis of the royal palm fibers is increasingly pronounced as the amount of enzyme is increased. The enzyme progress curve obtained by grinding the fiber fraction to a size similar to P200 is shown in fig. 6, where decreasing the size improves the accessibility of the enzyme to the substrate, and the CED is about doubled, both wet and dry. But abrasive fibers do not ultimately overcome the hornification effect as do thin walled components.

The inhibition phenomenon of the cellulase caused by dry keratinization can be attributed to irreversible change of water absorption performance of solid materials, firstly, the microporous structure of wood fiber cells collapses, a pore passage is closed, dry materials cannot absorb water effectively after entering an enzymolysis reaction system, enzyme cannot enter the interior of solid material particles along with diffusion of water molecules, the accessibility of the cellulase is reduced, and the enzymolysis rate is reduced. Water Retention Value (WRV) is commonly used to quantify the irreversible loss of water absorption in wood fibers. As shown, the WRV of the thin wall and fiber components decreased after oven drying, with a trend that closely matched the hydrolysis results. Wherein the WRV of the thin-walled fraction is reduced by about 50%, and correspondingly, the CED after 2h is reduced by 44.9% when 5FPU/g solids cellulase is used (FIG. 4). even if a sufficient amount of cellulase (e.g., 15FPU/g solids) is used, the reduction in CED is still significant at the initial stage of the enzymatic hydrolysis (FIG. 4), but finally, as the particle structure of the thin-walled fraction is destroyed, the keratinization inhibition is gradually overcome as the enzymatic hydrolysis time is prolonged. WRV corresponds to the initial CED of dry solids enzymatic hydrolysis, the higher the WRV, the higher the rate of enzymatic hydrolysis in the initial stage, but at the later stage of enzymatic hydrolysis, with sufficient enzyme, the keratinization can be overcome, as with dry and wet thin-walled components, the WRV does not correspond exactly to the final CED value.

Table 1 water retention values of the royal palm leaf sheath solids samples.

Keratinization caused by oven drying reduces the rate of cellulase hydrolysis (CEDs) of the fiber and thin-walled components of the sheaths of the royal brown leaves, but under sufficient enzymatic and time conditions, the thin-walled components can overcome this keratinization effect. Although the dried thin-walled component has difficulty in recovering the water retention property (WRV value) in an enzymolysis system, the enzymolysis rate of the dried thin-walled component can still be close to that of the wetting material. Unlike the fiber component, the fiber component is still inhibited by dry keratinization-induced enzymatic degradation even when ground to a size similar to that of the thin-walled component.

Taking the royal palm leaf sheath thin-wall component drying material as a raw material, adopting a single feeding mode, a feeding mode with two batches of feeding (18 percent of solid content each time) and a feeding mode with three batches of feeding (12 percent of solid content each time), supplementing enzyme liquid by solid material cellulase according to 10FPU/g each time, and finally realizing enzymolysis with the total solid content of 36 percent. The results are shown in figure 1, and the reaction is carried out for a long time (120h) by feeding in three different ways, and finally, the enzymolysis liquid with the glucose concentration of more than 150g/L can be produced. However, from a performance point of view, batch feeding is advantageous to overcome the "rheology" problem, allowing the enzymatic process to progress earlier to high sugar levels. As can be seen from the 24h sampling point in FIG. 1, the difference between the glucose output by single feeding with 36% solid content and the 24h output by double enzymolysis with 18% solid content is not great, and the high viscosity of the bottom material hinders the diffusion of the product and the movement of the enzyme. By adopting a strategy of feeding twice, the concentration of glucose liquid accumulated in 72h of enzymolysis reaches 150.3g/L, and the time can be saved by 2 days after the enzymolysis is finished. The high sugar level (144.5g/L) was also entered at 72h with three feeds.

Further improves the total solid content to 45 percent, and can obtain the enzymolysis liquid with higher sugar concentration. Two feeding strategies of feeding three times according to the solid content of 12% and feeding three times according to the solid content of 15% are compared with the attached figure 2, and as can be seen from the enzymolysis result, the material consumption of the latter is increased to 45% of the total solid content, which is equivalent to that 45g of dried material is mixed with 100g of liquid containing enzyme, and the solid material can absorb 95.4g of water according to the water retention value of 2.12g/g of the thin-wall component dry material in the table 1. If a single feed is used, effective enzymolysis cannot be realized. The problem is solved by feeding the thin-walled component drying material in batches, and the enzymolysis liquid containing 171.4g/L of glucose is finally obtained, and the result also shows that the problem of high-solid enzymolysis rheology can be solved by fully utilizing the dry cutinization effect.

The pretreated non-wood thin-wall component drying material is an advantageous raw material for finally realizing high-solid cellulose enzymolysis and downstream ethanol fermentation industrialization.

The above embodiments are not to be considered from a limiting point of view, but rather from an illustrative point of view. The scope of the invention is indicated by the appended claims rather than by the foregoing description, and all differences within the scope and range of equivalents thereof will be construed as being included in the present invention. Various insubstantial improvements are made by adopting the method conception and the technical scheme of the invention; the present invention is not limited to the above embodiments, and can be modified in various ways.

Claims

1. A method for high-solid enzymolysis of lignocellulose is characterized by comprising the following steps: the method comprises the following steps:

and (5) drying: drying the separated parenchymal cell components until the water content is reduced to be below 10 percent, and obtaining irregular granular parenchymal component granules with the length of less than 2 mm;

step (6) enzymolysis: placing the thin-walled component particles obtained in the step (5), cellulase, 0.04% tetracycline and a citric acid buffer solution with the pH value of 4.8 into a 12ml conical flask, sealing, and placing into a constant-temperature oscillation box for enzymolysis reaction; reaction conditions are as follows: the temperature is 50 ℃, the rotating speed is 150rpm, and the reaction time is 72 h;

wherein, the feeding mode: feeding materials in a mode of feeding materials for multiple times, in equal proportion and at equal time intervals, wherein the total reaction time is 72 hours, and the total feeding amount of the dried thin-wall component particles is 36-45%; at the same time, 10FPU/g is fed in each time_{Solid material}The cellulase supplements the cellulase.

2. The method of ligno-cellulosic high-solids enzymatic hydrolysis as claimed in claim 1, wherein: in the step (6), a three-time feeding mode is adopted in enzymolysis, primary feeding is carried out initially, secondary feeding is carried out after 24 hours of reaction, tertiary feeding is carried out after 48 hours of reaction, and the reaction is finished for 72 hours; the content of the thin-wall component particles after each drying is 12-15 percent, and the feeding amount is 10FPU/g_{Solid material}The cellulase supplements the enzyme solution.

3. The method of high-solids enzymatic hydrolysis of lignocellulose according to claim 1 or 2, characterized in that: in the enzymolysis in the step (6), 0.7 to 0.9 percent of Tween 80 is also added into the conical flask.

4. The method of ligno-cellulosic high-solids enzymatic hydrolysis as claimed in claim 3, wherein: the fluffer is a PTI95568 fluffer of Austria PTI company, and the boiler is a Japanese bamboo hat principle horizontal rotary boiler.

5. The method of ligno-cellulosic high-solids enzymatic hydrolysis as claimed in claim 4, wherein: and (5) during drying, naturally drying the mixture or drying the mixture in a 105 ℃ oven until the water content is reduced to below 10 percent.

6. The method of ligno-cellulosic high-solids enzymatic hydrolysis as claimed in claim 5, wherein: the temperature for NSSC cooking in the step (2) is 170 ℃.

7. A preparation method of a raw material for high-solid cellulose enzymolysis is characterized by comprising the following steps: the method comprises the following steps:

and (5) drying: drying the separated parenchymal cell component until the water content is reduced to below 10 percent to obtain irregular granular parenchymal component granules with the length less than 2 mm.