Method for promoting enzymatic hydrolysis efficiency of lignocellulose

Abstract

The invention provides a method for promoting the enzymatic hydrolysis efficiency of lignocellulose. The method comprises the following steps: (1) firstly, respectively preparing biomass primary pulp of a lignocellulose raw material by using a sulfate method; (2) holocellulose is prepared from the biomass primary pulp through a bleaching method, the bleaching time is 15-20 min, and the content of combined lignin in the holocellulose is 5% or below; (3) carrying out enzyme hydrolysis on holocellulose of which the content of bound lignin is less than 5% to obtain hydrolysate; the cellulose conversion rate of the hydrolysate is 90% or more. The method is low in cost and simple in preparation, the removal time of lignin in a reaction substrate is shortened, invalid adsorption of lignin to cellulase can be effectively reduced, catalytic hydrolysis of lignocellulase is promoted, and the conversion rate of cellulose is increased to 96.95%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of lignocellulose biomass conversion, and particularly relates to a method for promoting the efficiency of lignocellulose enzymatic hydrolysis. BACKGROUND

[0002] Biomass is a potential raw material for future bioethanol and the only renewable energy that can fix carbon, and its development, efficient conversion and clean utilization are increasingly valued by researchers, which can effectively replace fossil fuels. Lignocellulose is one of the most abundant and cheapest biomass resources in nature, and up to 85 billion tons of lignocellulose raw materials are produced in the world every year, accounting for more than half of the total global biomass. The main components include various agricultural and forestry residues, pulp and paper mill waste and energy crops, etc. At present, most of the lignocellulose raw materials are not effectively utilized and discarded, causing great resource waste and environmental accumulation problems. This kind of raw material contains more than 75% of carbohydrates, which can be used as an important source for producing bioethanol, liquid fuel and chemical products, and shows great potential in cellulose ethanol.

[0003] Lignocellulose is mainly composed of cellulose, lignin and hemicellulose, so the cellulase hydrolysis process is essential for converting lignocellulose into products with added value. Because lignin will hinder the contact between cellulase and the active sites on the surface of cellulose in space. In addition, during the cellulase hydrolysis process, lignin will also non-productively bind to cellulase through hydrophobic, electrostatic and hydrogen bonding interactions, thereby inhibiting the enzymatic hydrolysis efficiency of lignocellulose and increasing the consumption and production cost of cellulase.

[0004] Although pretreatment can significantly improve the enzyme digestion rate of lignocellulose biomass and achieve the purpose of removing lignin, the production cost of completely removing lignin is high and the time is long, resulting in low production efficiency. It will also cause high loss of carbohydrates. If the lignin is not completely removed, non-productive adsorption will still occur between the remaining lignin and cellulase, resulting in weak improvement effect of enzyme hydrolysis.

[0005] During the enzyme hydrolysis process, lignocellulose will be hydrolyzed, and the lignin released in the solution in a free state is called free lignin, and the lignin remaining in lignocellulose is called bound lignin. In recent years, a large amount of research has been conducted on lignin to clarify the mechanism of lignin inhibiting enzyme hydrolysis. However, the focus of the research is mainly on the reaction mechanism of various lignin types, modified lignin, addition of non-ionic surfactants, anionic surfactants, etc. in the enzyme hydrolysis process, and there is little research on the inhibition difference of the same lignin in different states on lignocellulose enzyme hydrolysis. SUMMARY

[0006] The technical problem solved by the present application is to provide a method for promoting the hydrolysis efficiency of lignocellulose enzyme, which is low in cost, simple in preparation, reduces the removal time of lignin in the reaction substrate, effectively reduces the invalid adsorption of lignin on cellulase, promotes the catalytic hydrolysis of lignocellulose enzyme, and increases the cellulose conversion rate to 96.95%.

[0007] The present application is implemented as follows:

[0008] A method for promoting the hydrolysis efficiency of lignocellulose enzyme, the steps of which are as follows:

[0009] (1) First, a biomass raw pulp of lignocellulose raw material is prepared by a sulfite method;

[0010] (2) The biomass raw pulp is used to prepare holocellulose by bleaching, and the bleaching time is 15-20 min, and the content of bound lignin in the holocellulose is 5% or less;

[0011] (3) The holocellulose with a bound lignin content of 5% or less is subjected to enzyme hydrolysis to obtain a hydrolysate; the cellulose conversion rate of the hydrolysate is 90% or more;

[0012] The cellulase and cellobiase are included, and the dosage ratio is 5:1-1:1.

[0013] Further, the lignocellulose raw material includes coniferous wood, broad-leaved wood and herbaceous plants.

[0014] The dosage ratio of cellulase and cellobiase in the hydrolysis enzyme is 3:1.

[0015] Further, the step (1) is specifically as follows:

[0016] (1) Preparation of biomass raw pulp:

[0017] (i) The biomass raw pulp is prepared by a sulfite method on lignocellulose raw material: the dry wood chips and (NaOH+9H2O·Na2S) solution are filled in the digester, the solid-liquid ratio is set to 1:4 (w / v), the temperature is raised to the highest temperature of 100-170℃ at a rate of 2℃ / min, and the temperature is kept at the highest temperature for 30-120 min;

[0018] (ii) After the holding time at the highest temperature, the slurry and waste liquid in the digester are immediately poured into a 100-150 mesh pulp bag for separation, and the corresponding pulp substrate and waste liquid are collected;

[0019] (iii) The solid slurry is washed with water until the pH is neutral, so as to ensure that the dissolved compounds and residual chemicals in the solid slurry have been completely removed;

[0020] (iv) The washed solid slurry is treated by a mill machine, and then is dissociated by a cellulose dissociator for 1-3 min; finally, the treated solid slurry is bagged and stored in a 4℃ refrigerator for standby.

[0021] Further, the step (2) is specifically as follows:

[0022] (2) The solid slurry in step (1) is dried by sunning, and then is bleached to prepare holocellulose with a combined lignin content of 5% or less:

[0023] (i) The absolutely dry solid slurry, distilled water, sodium chlorite and glacial acetic acid are mixed and placed in a 75℃, 150rpm water bath shaker for bleaching experiment, the bleaching time is controlled for 15-20 min, and then the bleached wood pulp is washed by filtration until the filtrate is neutral, and then is air-dried and bagged for standby.

[0024] (ii) Preparation of free lignin: the wood pulp sample is washed by centrifugal washing until neutral, and then is air-dried. Finally, the free lignin sample is obtained after ball milling and sieving.

[0025] Further, the step (3) is specifically as follows:

[0026] (3) Enzymatic hydrolysis: the absolutely dry wood pulp obtained in step (2) is mixed with HAc-NaAc buffer solution (0.05M, pH=4.8), and is fully shaken, then 12%-20% cellulase, 0.1%-0.4% Tween 80 and 0.15%-0.30% tetracycline are added, and the mixture is hydrolyzed in an air shaker at 35℃-55℃ for 60-96h, to obtain a lignocellulose hydrolysate.

[0027] Further, in the sulfite pulping conditions, the dosages of NaOH+9H2O·Na2S for coniferous wood, broad-leaved wood and herbaceous plants are 17-23%, 15-21% and 9-15% of the mass of the aqueous solution respectively, the highest temperatures are 140-170℃, 120-150℃ and 100-130℃ respectively, and the holding times are 90-120min, 60-90min and 30-60min respectively.

[0028] Further, the dosage ratio of absolutely dry wood pulp to HAc-NaAc buffer solution is 8-14g: 50-200mL.

[0029] The present application has the following advantages:

[0030] The free lignin prepared by the present application is tested by heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR), and the HSQC-NMR analysis shows that the free lignin of bamboo, masson pine and poplar is composed of G-S-H, G-H and G-S lignin units respectively, and the concentration of S type unit in bamboo is the highest. The side chain region of the free lignin of the three kinds is composed of methoxyl and various linkages. Since the side chain region of masson pine is prone to form β-0-4, β-5 and β-β bonds and the like, contains more branches and has a relatively firm structure, the concentration of the linkages in the side chain region of masson pine is higher than that of the free lignin of the other two types (bamboo and poplar). The cellulose conversion rate and enzyme substrate adsorption capacity of the hydrolysate are determined by a blood glucose meter (BGM) and an ultraviolet spectrophotometer (UV). In the enzyme hydrolysis of the combined lignin, with the decrease of the lignin content, the cellulose conversion rate and the enzyme substrate adsorption capacity both show an increasing trend. In the enzyme hydrolysis of the free lignin, the lignin content has no effect on the cellulose conversion rate and the enzyme substrate adsorption capacity. The results show that the inhibition of the free lignin on the enzyme hydrolysis is more serious than that of the combined lignin, and this trend is independent of the type of biomass. And for the three different lignocelluloses (bamboo, masson pine and poplar), the order of the inhibition of the combined lignin on the enzyme hydrolysis is bamboo > poplar > masson pine. The present application aims to provide a theoretical basis and technical framework for the development of ultra-high efficient enzyme hydrolysis technology. In addition, it can also provide technical support for the removal of lignin in the reaction substrate in the pretreatment stage of lignocellulose. Therefore, the production of bioethanol by lignocellulose enzyme hydrolysis has great development potential and can alleviate the rapid growth of fossil fuel consumption in the world.

[0031] In summary, in the process of enzyme hydrolysis, compared with the lignin in the combined state, the lignin in the free state and the control of different contents of free lignin play an obvious role in improving the efficiency of enzyme hydrolysis. The method of the present application has obvious effect on the improvement of the cellulose conversion rate and enzyme adsorption capacity in the lignocellulose enzyme hydrolysis liquid. When the lignin is removed from the biomass slurry by bleaching method, the bleaching time is controlled within 15-20 min, and the content of the combined lignin prepared is 2-5%, which can ensure that the cellulose hydrolysis rate in the later stage is as high as 96.95%, greatly saving the time of the bleaching step and the hydrolysis efficiency of cellulose; and it also has the advantages of simple preparation process, easy operation and low cost. BRIEF DESCRIPTION OF DRAWINGS

[0032] The present application will be further described below with reference to the accompanying drawings and examples.

[0033] Figure 1 The schematic diagram of the side chain region in the HSQC-NMR spectrum of the three kinds of free lignin in the examples of the present application, wherein from left to right are bamboo, masson pine and poplar, and from top to bottom are S, G and H type units.

[0034] Figure 2 Schematic diagram of the aromatic region in HSQC-NMR spectra of three free lignins in the embodiments of the present application, wherein from left to right are bamboo, masson pine and poplar in turn.

[0035] Figure 3 Schematic diagram of the cellulose conversion rates of bound / free lignins in bamboo (16.93%, 11.29%, 6.26% and 2.34%), masson pine (16.15%, 9.58%, 5.60% and 2.45%) and poplar (16.39%, 11.36%, 6.65% and 2.86%) with different lignin contents during enzymatic hydrolysis in the embodiments of the present application.

[0036] Figure 4 Schematic diagram of the cellulose conversion rates of bound and free lignins (lignin content of about 2%) in three different biomasses (bamboo, masson pine and poplar) during enzymatic hydrolysis in the embodiments of the present application, wherein from left to right are bound and free lignins in turn.

[0037] Figure 5 Schematic diagram of the enzyme adsorption amounts of bound and free lignins in bamboo (16.93%, 11.29%, 6.26% and 2.34%), masson pine (16.15%, 9.58%, 5.60% and 2.45%) and poplar (16.39%, 11.36%, 6.65% and 2.86%) with different lignin contents during enzymatic hydrolysis in the embodiments of the present application.

[0038] Figure 6 Schematic diagram of the enzyme adsorption amounts of bound and free lignins (lignin content of about 2%) in three different biomasses (bamboo, masson pine and poplar) during enzymatic hydrolysis in the embodiments of the present application, wherein from left to right are bound and free lignins in turn.

DETAILED DESCRIPTION

[0039] The technical solutions of the present application will be described clearly and completely in the following combined with the drawings of the embodiments of the present application and the specific embodiments. Figures 1-6 All other embodiments obtained by those skilled in the art based on the embodiments in the present application without making any creative labor fall within the protection scope of the present application. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not mentioned by the manufacturers are all conventional products that can be purchased in the market.

[0040] Example 1

[0041] (1) Preparation of biomass slurry: (i) Preparation of biomass slurry of coniferous wood, broad-leaved wood and herbaceous plant by Kraft process in a circulating laboratory digester. A certain amount of absolutely dry wood chips and a certain amount of (NaOH + 9H2O-Na2S) solution (the amount of NaOH + 9H2O-Na2S in each raw material accounts for 17% of the mass of the aqueous solution for coniferous wood, 15% for broad-leaved wood, and 9% for herbaceous plant) were filled in each digester, and the solid-liquid ratio was set to 1:4 (w / v). The temperature was raised to the highest temperature (140°C for coniferous wood, 120°C for broad-leaved wood, and 100°C for herbaceous plant) at a rate of 2°C / min, and maintained at the highest temperature for a certain period of time (90 min for coniferous wood, 60 min for broad-leaved wood, and 30 min for herbaceous plant); (ii) After the end of the holding time at the highest temperature, the slurry and waste liquid in the digester were immediately poured into a 100-150 mesh pulp bag for separation, and the corresponding slurry substrate and waste liquid were collected;

[0042] (iii) The solid slurry was washed with tap water until the pH was neutral (measured by pH paper comparison), to ensure that the dissolved compounds and residual chemicals in the solid slurry had been completely removed; (iv) The washed solid slurry was processed by a mill machine, and then dissociated by a cellulose dissociator for 1-3 min. Finally, the processed solid slurry was bagged and stored in a 4°C refrigerator for standby use.

[0043] (2) Preparation of holocellulose, bound and free lignin: The solid slurry described in step (1) was dried and used to prepare holocellulose, bound and free lignin by bleaching and enzymatic hydrolysis, respectively; (i) Preparation of holocellulose and bound lignin: 2 g of oven-dried wood pulp, 65 mL of distilled water, 0.6 g of sodium chlorite and 0.5 mL of glacial acetic acid were placed in a conical flask, which was placed in a water bath shaker at 75 °C and 150 rpm for bleaching experiments. By controlling different bleaching times (0, 1, 10, 100 and 300 min), wood pulp samples were obtained under different bleaching intensities. After bleaching, the wood pulp was washed by filtration until the filtrate was neutral, then it was air-dried and bagged for use. A two-step acid hydrolysis method was used to determine the lignin content in the wood pulp raw material, and finally the bound lignin with different lignin content gradients was obtained. In addition, when the bleaching time is long enough, there is no lignin in the wood pulp, so the completely bleached wood pulp is called holocellulose; (ii) Preparation of free lignin: 8 g of oven-dried wood pulp and 50 mL of HAc-NaAc buffer solution (0.05 M, pH = 4.8) were mixed and placed in a conical flask, then 1.2 mL (103.3 FPU / mL) of cellulase, 0.1% Tween 80 and 0.15% tetracycline were added to the conical flask, and the hydrolysis was carried out in a horizontal constant temperature shaker at 150 rpm for 72 h. After the hydrolysis was completed, the wood pulp sample was washed by centrifugation until it was neutral, and then it was air-dried. Finally, the free lignin sample was obtained after ball milling and sieving.

[0044] (3) Lignocellulose enzymatic hydrolysis system: (i) Effect of bound lignin on enzymatic hydrolysis: The bound lignin on the enzymatic hydrolysis process was carried out according to the process described in (ii) of step (2). During the enzymatic hydrolysis process, 1 mL of hydrolysate was taken at different time nodes (1, 3, 5, 7, 12, 24, 36, 48, 60, 72, 84 and 96 h), which was inactivated at 100 °C, and then centrifuged in a 10,000 rpm centrifuge for 10 min. Finally, the supernatant of the hydrolysate was analyzed; (ii) Effect of free lignin on enzymatic hydrolysis: As mentioned before, these samples were prepared by mixing holocellulose and bound lignin samples containing equal amounts of free lignin. The ratio of free lignin to holocellulose in these samples can be seen in Table 1. After mixing, the samples were subjected to the enzymatic hydrolysis process, which was consistent with (i).

[0045] Example 2

[0046] (1) Preparation of biomass pulp: (i) The preparation of biomass pulp was carried out by Kraft pulping process in a circulating laboratory digester. A certain amount of absolutely dry wood chips and a certain amount of (NaOH + 9H20-Na2S) solution (the amount of NaOH + 9H20-Na2S in each raw material accounted for 19% of the mass of the aqueous solution for coniferous wood, 17% for broad-leaved wood, and 11% for herbaceous plants) were filled in each digester, and the solid-liquid ratio was set to 1:4 (w / v). The temperature was raised to the maximum temperature (coniferous wood: 150°C, broad-leaved wood: 130°C, herbaceous plants: 110°C) at a rate of 2°C / min, and maintained at the maximum temperature for a certain time (coniferous wood: 100 min, broad-leaved wood: 70 min, herbaceous plants: 40 min); (ii) After the end of the holding time at the maximum temperature, the pulp and waste liquid in the digester were immediately poured into a 100-150 mesh pulp bag for separation, and the corresponding pulp substrate and waste liquid were collected; (iii) The solid pulp was washed with tap water until the pH was neutral (measured by pH paper), ensuring that the dissolved compounds and residual chemicals in the solid pulp had been completely removed; (iv) The washed solid pulp was processed by a mill and then dissolved by a cellulose dissolver for 1-3 min. Finally, the treated solid pulp was bagged and stored in a 4°C refrigerator for future use.

[0047] (2) Preparation of holocellulose, bound and free lignin: The solid pulp described in step (1) was dried and then used to prepare holocellulose, bound and free lignin by bleaching and enzymatic hydrolysis, respectively; (i) Preparation of holocellulose and bound lignin: 2 g of absolutely dry wood pulp, 65 mL of distilled water, 0.6 g of sodium chlorite, and 0.5 mL of glacial acetic acid were placed in a conical flask, and the conical flask was placed in a 75°C, 150 rpm water bath shaker for bleaching experiments. By controlling different bleaching times (0, 1, 15, 120, and 300 min), wood pulp samples with different bleaching intensities were obtained. After bleaching, the wood pulp was washed by filtration until the filtrate was neutral, then it was air-dried and bagged for future use. A two-step acid hydrolysis method was used to determine the lignin content in the wood pulp raw material, and finally a gradient of bound lignin with different lignin contents was obtained. In addition, when the bleaching time is long enough, there is no lignin in the wood pulp, so the completely bleached wood pulp is called holocellulose; (ii) Preparation of free lignin: 10 g of absolutely dry wood pulp and 100 mL of HAc-NaAc buffer solution (0.05 M, pH = 4.8) were mixed and placed in a conical flask. After shaking well, 1.6 mL (103.3 FPU / mL) of cellulase, 0.2% Tween 80, and 0.20% tetracycline were added to the conical flask, and the hydrolysis was carried out in a 150 rpm horizontal constant temperature shaker for 72 h. After the hydrolysis was completed, the wood pulp sample was washed to neutral by centrifugal washing, and then it was air-dried. Finally, the free lignin sample was obtained after screening.

[0048] (3) Lignocellulolytic enzyme hydrolysis system: (i) Effect of bound lignin on enzyme hydrolysis: The enzyme hydrolysis process of the bound lignin was carried out according to the process described in (ii) of step (2). During the enzyme hydrolysis process, 1 mL of the hydrolysate (residue and supernatant) was taken at different time points (1, 3, 5, 7, 12, 24, 36, 48, 60, 72, 84 and 96 h) and inactivated at 100 °C, followed by centrifugation at 10,000 rpm for 10 min, and finally the supernatant of the hydrolysate was analyzed; (ii) Effect of free lignin on enzyme hydrolysis: As described above, these samples were prepared by mixing the holocellulose and bound lignin samples containing an equal amount of free lignin, and the ratio of free lignin to holocellulose in these samples can be seen in Table 1. After mixing, the enzyme hydrolysis process was carried out on these samples, and the steps were consistent with (i).

[0049] Example 3

[0050] (1) Preparation of biomass raw pulp: (i) The preparation of biomass raw pulp was carried out by the Kraft process in a circulating laboratory digester for coniferous wood, deciduous wood and herbaceous plants, respectively. A certain amount of absolutely dry wood chips was filled in each digester, and a certain amount of (NaOH + 9H2O·Na2S) solution (the amount of NaOH + 9H2O·Na2S in each raw material accounted for 21% of the mass of the aqueous solution for coniferous wood, 19% for deciduous wood, and 13% for herbaceous plants) was added, and the solid-liquid ratio was set to 1:4 (w / v). The temperature was raised to the maximum temperature (coniferous wood: 160 °C, deciduous wood: 140 °C, herbaceous plants: 120 °C) at a rate of 2 °C / min, and maintained at the maximum temperature for a certain time (coniferous wood: 110 min, deciduous wood: 80 min, herbaceous plants: 50 min); (ii) After the end of the holding time at the maximum temperature, the pulp and waste liquid in the digester were immediately separated in a 100-150 mesh pulp bag, and the corresponding pulp substrate and waste liquid were collected; (iii) The solid pulp was washed with tap water until the pH was neutral (measured by pH paper comparison), to ensure that the dissolved compounds and residual chemicals in the solid pulp were completely removed; (iv) The washed solid pulp was processed by a millstone machine, and then disintegrated by a cellulose disintegrator for 1-3 min. Finally, the treated solid pulp was packed and stored in a 4 °C refrigerator for standby use.

[0051] (2) Preparation of holocellulose, bound and free lignin: The solid slurry described in step (1) was dried and used to prepare holocellulose, bound and free lignin by bleaching and enzymatic hydrolysis, respectively; (i) Preparation of holocellulose and bound lignin: 2 g of oven-dried wood pulp, 65 mL of distilled water, 0.6 g of sodium chlorite and 0.5 mL of glacial acetic acid were placed in a conical flask, which was placed in a water bath shaker at 75 °C and 150 rpm for bleaching experiments. Wood pulp samples were obtained at different bleaching intensities by controlling different bleaching times (0, 5, 35, 160 and 300 min), and after bleaching, the wood pulp was washed by filtration until the filtrate was neutral, then it was air-dried and bagged for use. A two-step acid hydrolysis method was used to determine the lignin content in the wood pulp raw material, and finally the bound lignin with different lignin content gradients was obtained. In addition, when the bleaching time is long enough, there is no lignin in the wood pulp, so the completely bleached wood pulp is called holocellulose; (ii) Preparation of free lignin: 12 g of oven-dried wood pulp and 150 mL of HAc-NaAc buffer solution (0.05 M, pH = 4.8) were mixed and placed in a conical flask, then 2.0 mL (103.3 FPU / mL) of cellulase, 0.3% Tween 80 and 0.25% tetracycline were added to the conical flask, and the hydrolysis was carried out in a horizontal constant temperature shaker at 150 rpm for 72 h. After the hydrolysis was completed, the wood pulp sample was washed by centrifugation until it was neutral, and then it was air-dried. Finally, the free lignin sample was obtained after screening.

[0052] (3) Lignocellulose enzymatic hydrolysis system: (i) Effect of bound lignin on enzymatic hydrolysis: The bound lignin on the enzymatic hydrolysis process was carried out according to the process described in (ii) of step (2). During the enzymatic hydrolysis process, 1 mL of hydrolysate (residue and supernatant) was taken at different time nodes (1, 3, 5, 7, 12, 24, 36, 48, 60, 72, 84 and 96 h), which was inactivated at 100 °C, and then centrifuged in a 10,000 rpm centrifuge for 10 min. Finally, the supernatant of the hydrolysate was analyzed; (ii) Effect of free lignin on enzymatic hydrolysis: As mentioned earlier, these samples were prepared by mixing holocellulose and bound lignin samples containing equal amounts of free lignin. The ratio of free lignin to holocellulose in these samples can be seen in Table 1. After mixing, the samples were subjected to the enzymatic hydrolysis process, which was consistent with (i).

[0053] Example 4

[0054] (1) Preparation of biomass pulp: (i) The preparation of biomass pulp was carried out by Kraft pulping process in a circulating laboratory digester. A certain amount of absolutely dry wood chips and a certain amount of (NaOH + 9H20-Na2S) solution (the amount of NaOH + 9H20-Na2S in each raw material accounted for 23% of the mass of the aqueous solution for coniferous wood, 21% for broad-leaved wood, and 15% for herbaceous plants) were filled in each digester, and the solid-liquid ratio was set to 1:4 (w / v). The temperature was raised to the maximum temperature (coniferous wood: 170°C, broad-leaved wood: 150°C, herbaceous plants: 130°C) at a rate of 2°C / min, and maintained at the maximum temperature for a certain time (coniferous wood: 120 min, broad-leaved wood: 90 min, herbaceous plants: 60 min); (ii) After the end of the holding time at the maximum temperature, the pulp and waste liquid in the digester were immediately poured into a 100-150 mesh pulp bag for separation, and the corresponding pulp substrate and waste liquid were collected; (iii) The solid pulp was washed with tap water until the pH was neutral (measured by pH paper), ensuring that the dissolved compounds and residual chemicals in the solid pulp had been completely removed; (iv) The washed solid pulp was processed by a mill and then dissolved by a cellulose dissolver for 1-3 min. Finally, the treated solid pulp was bagged and stored in a 4°C refrigerator for future use;

[0055] (2) Preparation of holocellulose, bound and free lignin: The solid pulp described in step (1) was dried and then used to prepare holocellulose, bound and free lignin by bleaching and enzymatic hydrolysis, respectively; (i) Preparation of holocellulose and bound lignin: 2 g of absolutely dry wood pulp, 65 mL of distilled water, 0.6 g of sodium chlorite, and 0.5 mL of glacial acetic acid were placed in a conical flask, and the conical flask was placed in a 75°C, 150 rpm water bath shaker for bleaching experiments. By controlling different bleaching times (0, 10, 60, 200, and 300 min), wood pulp samples under different bleaching intensities were obtained, and after washing the bleached wood pulp to neutral, it was dried and bagged for future use. A two-step acid hydrolysis method was used to determine the lignin content in the wood pulp raw material, and finally a gradient of bound lignin with different lignin contents was obtained. In addition, when the bleaching time is long enough, there is no lignin in the wood pulp, so the completely bleached wood pulp is called holocellulose; (ii) Preparation of free lignin: 14 g of absolutely dry wood pulp and 200 mL of HAc-NaAc buffer solution (0.05 M, pH = 4.8) were mixed and placed in a conical flask, then 2.4 mL (103.3 FPU / mL) of cellulase, 0.4% Tween 80, and 0.30% tetracycline were added to the conical flask, and the mixture was hydrolyzed in a 150 rpm horizontal constant-temperature shaker for 72 h. After the hydrolysis was completed, the wood pulp sample was washed to neutral by centrifugal washing, and then dried. Finally, the free lignin sample was obtained after screening.

[0056] (3) Lignocellulose enzyme hydrolysis system: (i) Effect of bound lignin on enzyme hydrolysis: The enzyme hydrolysis process of the bound lignin was carried out according to the process described in (ii) of step (2). During the enzyme hydrolysis process, 1 mL of the hydrolysis liquid (residue and supernatant) was taken at different enzyme hydrolysis time nodes (1, 3, 5, 7, 12, 24, 36, 48, 60, 72, 84 and 96 h), inactivated at 100°C, and then centrifuged in a 10,000 rpm centrifuge for 10 min, and finally the supernatant of the hydrolysis liquid was analyzed; (ii) Effect of free lignin on enzyme hydrolysis: As described above, these samples were prepared by mixing the free lignin in the holocellulose and bound lignin samples containing an equal amount of free lignin, and the ratio of free lignin to holocellulose in these samples can be seen in Table 1. After mixing, the enzyme hydrolysis process was carried out on these samples, and the steps were consistent with (i).

[0057] Table 1 Ratio of free lignin and holocellulose in bamboo, pine and poplar in Examples 1-4

[0058]

[0059] Example 5

[0060] The free lignin in the three biomasses was analyzed by using the heteronuclear single quantum coherence nuclear magnetic resonance technology (HSQC-NMR) (see Figure 1 , 2 ). HSQC-NMR is a relatively mature two-dimensional nuclear magnetic characterization technology, which can provide detailed molecular structure of lignin. Figure 1 and Figure 2 are the HSQC-NMR spectra of the free lignin in bamboo, pine and poplar. From the figures, it can be seen that the HSQC-NMR spectra of the free lignin in the three biomasses can be divided into side chain region (δC / δH=50-90 / 2.5-5.0 ppm) and aromatic region (δC / δH=100-135 / 6.0-8.0 ppm). The side chain region is composed of methoxyl and various bonds, such as β-0-4, β-5, β-β. At δC / δH=55.9 / 3.7 ppm, the cross peak is more obvious due to the resonance of methoxyl. The order of the resonance of methoxyl in the three biomasses is: bamboo > poplar > pine. The aromatic region of lignin mainly has H, S and G type unit signals. As shown in Figure 1 , the free lignin in bamboo, poplar and pine is mainly composed of G-S-H type, G-S type and G-H type units, respectively. Only the free lignin in bamboo and poplar contains S type units, and the concentration of S type units in bamboo is the highest. As shown in Figure 2As shown, the side chain regions of the three types of free lignin are composed of methoxyl groups and various linkages. The concentration of the side chain region in the free lignin of Pinus massoniana is higher than that of the other two types of free lignin. Pinus massoniana is prone to form β-0-4, β-5 and β-β linkages, and contains more branches, and has a complex and firm structure. Therefore, the concentration of the linkages in the side chain of Pinus massoniana is higher than that of the other two types of free lignin (i.e. Phyllostachys pubescens and poplar).

[0061] Example 6

[0062] In the process of lignocellulose enzyme hydrolysis, the cellulose in the lignocellulose is hydrolyzed into glucose. Therefore, the glucose concentration in the hydrolysate (supernatant) is detected by BGM, and the cellulose conversion rate is calculated according to the following formula (1) (see Figure 3 ,4).

[0063]

[0064] In the formula, c is the glucose concentration of the hydrolysate (supernatant), mg / mL;

[0065] v is the volume of the HAc-NaAc (0.05M, pH = 4.8) buffer, mL;

[0066] m is the cellulose content, g;

[0067] 0.90 is the hydrolysis factor of cellulose to glucose.

[0068] Figure 3 is the cellulose conversion rate of the combined and free lignin with different lignin contents in the process of enzyme hydrolysis of Phyllostachys pubescens (16.93%, 11.29%, 6.26% and 2.34%), Pinus massoniana (16.15%, 9.58%, 5.60% and 2.45%) and poplar (16.39%, 11.36%, 6.65% and 2.86%). From Figure 3 It can be observed that, with the increase of the enzyme hydrolysis time, the cellulose conversion rate of the combined lignin is increasing, and the free lignin basically remains unchanged. Therefore, compared with the free lignin, the combined lignin has a relatively weak inhibitory effect on enzyme hydrolysis.

[0069] In the presence of lignin, the cellulose conversion rate of the raw bamboo pulp with 16.93% lignin content was 6.77% at the initial stage of enzymatic hydrolysis. With the increase of enzymatic hydrolysis time to 96 h, the cellulose conversion rate could be increased to 56.72%. When the lignin content was reduced to 6.26%, the cellulose conversion rate was increased to 89.31%. When the lignin content was reduced to 2.34%, the cellulose conversion rate was significantly increased to 96.95%. Therefore, the cellulose conversion rate of the sample with more bound lignin content was lower than that of the sample with less bound lignin content. With the increase of enzymatic hydrolysis time, some lignin would migrate from the inside of lignocellulose to the surface of the fiber, resulting in the redistribution of the whole biomass substrate. Some lignin was also separated from lignocellulose and dissolved in the solution. These dissolved lignin would re-agglomerate to form small droplets and deposit on the active sites on the surface of cellulose, hindering the binding of cellulase on cellulose and further inhibiting its hydrolysis. Therefore, after the increase of enzymatic hydrolysis to 24 h, the cellulose conversion rate curve gradually leveled off and finally remained basically unchanged.

[0070] In the presence of free lignin, the cellulose conversion rate was basically maintained at 7-10%, indicating that the change of lignin content did not affect the change of cellulose conversion rate. Under weak acidic conditions (pH = 4.8), free lignin had higher hydrophobicity than cellulose. After adding a certain amount of cellulase to the mixture of cellulose and free lignin, the free lignin would preferentially bind with the cellulase, and most of the cellulase would be rapidly inactivated, resulting in low enzymatic hydrolysis efficiency. Therefore, if there is free lignin in lignocellulose, the cellulose conversion rate is much lower than that of the sample with bound lignin. It can be seen that even if the content of free lignin changes between 2.34-16.93%, the cellulose conversion rate curve of the sample always remains in the range of 7-10%. In addition, it can be seen that the cellulose conversion rate of the sample with free lignin is much lower than that of the sample with bound lignin. Figure 3 Figure 3 It can be seen that the pine and poplar also showed the same trend as the bamboo. When the enzymatic hydrolysis time was 96 h, and the lignin content was reduced to 5.60% and 6.65% respectively, the cellulose conversion rates of the pine and poplar were 88.23% and 75.21% respectively. When the lignin content was reduced to 2.45% and 2.86% respectively, the cellulose conversion rates of the pine and poplar were 92.11% and 95.71% respectively. The results showed that the effects of bound and free lignin in the three lignocelluloses on enzymatic hydrolysis were consistent.

[0071] In addition, in order to compare the effects of different types of lignocellulose on enzymatic hydrolysis, samples with an equal amount of lignin (bound and free lignin) were selected for comparison and analysis (see Table 1).

[0072] Figure 4 ​The following are the cellulose conversion curves of bound and free lignin in three types of lignocellulose (bamboo, masson pine and poplar) during enzymatic hydrolysis. Figure 4 As shown in the figure, in the state of binding lignin, the cellulose conversion rates of these three types of lignocellulose are all improved. However, as the enzymatic hydrolysis time increases, there are certain differences in the cellulose conversion rates. The cellulose conversion rates of these three types of lignocellulose are in the order of bamboo>poplar>Pinus massoniana. Bamboo has the highest content of S-type units, poplar is in the middle, and Pinus massoniana has the lowest (Example 5). During the enzymatic hydrolysis process, S-type units are more easily detached than G-type units and H-type units. Therefore, the enzyme hydrolysis efficiency of samples containing more S-type units is higher than that of samples containing fewer S-type units. However, the cellulose conversion rates of all free lignin samples (bamboo, poplar and Pinus massoniana) remain essentially unchanged (about 10%). This is because cellulase is rapidly adsorbed by free lignin, causing its activity to eventually decrease during the hydrolysis process.

[0073] Example 7

[0074] In order to further verify the conclusions drawn in Example 6, the present invention also carried out an enzyme adsorption test on the hydrolyzate (supernatant). Enzyme adsorption refers to the adsorption of cellulase by the substrate. The adsorption amount and binding strength of cellulase can be used to evaluate the accessibility of the enzyme substrate to cellulose, that is, the stronger the adsorption of the enzyme, the higher the enzyme hydrolysis efficiency. The Coomassie Brilliant Blue method (Bradford method) was used to measure the adsorption amount of cellulase in the hydrolyzate (supernatant), and the Langmuir model was used to explain the isothermal adsorption process of cellulase on these three types of lignocellulose. The Langmuir model is a method for describing the adsorption of cellulase at a specific position on the fiber surface, which can further determine the adsorption process of cellulase on the substrate. The Langmuir adsorption equation was simulated with Origin software to determine the Langmuir constant (K L ,mL / mg), maximum adsorption capacity (q max , mg protein / g substrate) was calculated by the following formula (2), and the binding strength of the enzyme-substrate adsorption amount (T, mL / mg) was calculated by the following formula (3).

[0075]

[0076] T=q max ·K L (3);

[0077] Where: C e ——The amount of free cellulase in the solution system, mg / mL;

[0078] K LLangmuir adsorption equilibrium constant, mL / mg, indicating the strength of adsorption capacity, i.e. the strength of chemical binding between adsorbent and adsorbate, which is related to its binding sites;

[0079] q e Amount of cellulase adsorbed by substrate at equilibrium, mg / g;

[0080] q m Maximum amount of cellulase adsorbed by substrate, mg / g.

[0081] Figure 5 is the amount of cellulase adsorbed by substrate at equilibrium, mg / g; and Figure 5 As shown in FIG. 1, with the increase of enzyme hydrolysis time, the amount of cellulase adsorbed by bound lignin increased, while the amount of cellulase adsorbed by free lignin remained basically unchanged. In addition, the amount of cellulase adsorbed by bound lignin was generally higher than that adsorbed by free lignin. Therefore, compared with free lignin, the inhibition of bound lignin on enzyme hydrolysis was relatively weak.

[0082] In the state of bound lignin, the initial amount of cellulase adsorbed by raw bamboo pulp with a lignin content of 16.93% was 1.74 mg protein / g substrate. With the lignin content decreasing to 6.26%, the amount of cellulase adsorbed increased significantly to 2.81 mg protein / g substrate; with the lignin content decreasing to 2.34%, the amount of cellulase adsorbed increased significantly to 3.81 mg protein / g substrate. With the increase of enzyme hydrolysis time, the space network structure of lignocellulose was opened, and the cellulose sites were exposed. The adsorption of cellulase on the substrate (cellulose) increased, and lignin was separated from lignocellulose and deposited in the solution. The substrate (free lignin part in raw material) could adsorb part of cellulase, leading to its inactivation or inhibition of cellulase activity. At the same time, the hydrophobicity of lignin served as the main adsorption mechanism in the process of enzyme adsorption. When part of free lignin and cellulose coexist in the solution, free lignin will preferentially adsorb cellulase. Therefore, with the increase of enzyme hydrolysis time, the amount of cellulase adsorbed by substrate gradually tends to be flat, and finally reaches the adsorption equilibrium state. In the present application, the amount of enzyme adsorbed is determined by measuring the content of enzyme in the hydrolyzate (supernatant). Since free lignin is more inclined to adsorb cellulase than cellulose, the unadsorbed cellulase is dispersed in the hydrolyzate (supernatant), leading to higher adsorption of enzyme in the measurement. Therefore, the sample with less bound lignin adsorbs less cellulase than the sample with more bound lignin.

[0083] The change of lignin content in free lignin state did not affect the adsorption amount of enzyme, which was basically maintained at about 1.0 mg protein / g substrate. The free lignin in solution could adsorb more surface active sites of cellulase, thereby affecting the adsorption of enzyme in the substrate. Figure 5 It is further indicated that even if the free lignin content changes between 2.34-16.93%, the adsorption amount of cellulase on free lignin is maintained at about 1.0 mg protein / g substrate, and the free lignin content (2.34-16.93%) is still much higher than the cellulase content (1.6%) during the enzyme hydrolysis. Therefore, the cellulase is completely adsorbed by free lignin, leaving only a little hydrolysis product (supernatant).

[0084] Table 2-4 is the K L , q max and T data obtained by simulating the Langmuir model. From Table 2-4, it can be seen that the Langmuir model has a good fitting effect on the experimental data, and the R 2 under each group of conditions is above 0.95, indicating that the Langmuir model can accurately explain the isothermal adsorption process of cellulase on bound and free lignin. From Table 2-4, it can be seen that K L , q max and T increase with the decrease of lignin content, and the binding strength of free lignin is about 3 times that of bound lignin. This data better proves that free lignin has stronger adsorption strength on cellulase than bound lignin.

[0085] Table 2 Maximum adsorption amount (q max ), adsorption constant (K L ) and binding strength (T=q max ·K L ) of bound and free lignin in bamboo

[0086]

[0087]

[0088] Table 3 Maximum adsorption amount (q max ), adsorption constant (K L ) and binding strength (T=q max ·K L ) of bound and free lignin in Masson pine

[0089]

[0090] Table 4 Maximum adsorption amount (q max ), adsorption constant (K L ) and binding strength (T=q max• K L )

[0091]

[0092]

[0093] Appendix Figure 5 It is also shown that Pinus massoniana and Populus tomentosa exhibit the same trend as Phyllostachys edulis. When the enzyme hydrolysis time is increased to 96 h, the lignin content is reduced (5.60% and 6.65%), and the enzyme adsorption amounts of Pinus massoniana and Populus tomentosa reach 3.09 and 2.90 mg protein / g substrate, respectively. Appendix 3 and Appendix 4 are the K L , q max and T data of the bound and free lignin in Pinus massoniana and Populus tomentosa. From Appendix 3 and Appendix 4, it can be seen that the Langmuir adsorption isotherm model of Pinus massoniana and Populus tomentosa is consistent with that of Phyllostachys edulis. Through the analysis of the enzyme adsorption amount and adsorption kinetics, the present application further proves the influence of the types of lignocellulose, lignin content, bound and free lignin on the cellulose conversion rate, and further understands the enzyme hydrolysis efficiency.

[0094] At the same time, in order to compare the influence of the same amount of lignin (bound and free lignin) in different lignocelluloses during enzyme hydrolysis, samples with a lignin content of about 2% (Appendix 1) are selected for comparative analysis. Appendix Figure 6 shows the enzyme adsorption of the bound and free lignin in three lignocelluloses (Phyllostachys edulis, Pinus massoniana and Populus tomentosa) during enzyme hydrolysis. As shown in Appendix Figure 6 , in the state of bound lignin, the enzyme adsorption amounts of the three lignocelluloses all show an upward trend. However, with the increase of enzyme hydrolysis time, there is a certain degree of difference in the enzyme adsorption amounts of the three lignocelluloses, and the enzyme adsorption amounts of the three lignocelluloses are in the order of Phyllostachys edulis > Populus tomentosa > Pinus massoniana. The content of G-type unit in Pinus massoniana is the highest, that in Populus tomentosa is in the middle, and that in Phyllostachys edulis is the lowest. According to the literature, G-type unit is more inclined to adsorb cellulase than S-type and H-type units. Therefore, the enzyme hydrolysis efficiency of the sample containing more G-type units is lower than that of the sample containing less G-type units. However, as shown in Appendix Figure 6 , the enzyme adsorption amounts of the three free lignins (Phyllostachys edulis, Pinus massoniana and Populus tomentosa) remain unchanged (about 1.20 mg protein / g substrate). This is because the cellulase is rapidly adsorbed by the free lignin, and its activity is eventually reduced in the hydrolysis process.

[0095] In summary, the free lignin prepared by the present application is tested by heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR), and the HSQC-NMR analysis shows that the free lignin of bamboo, masson pine and poplar is composed of G-S-H, G-H and G-S lignin units respectively, and the concentration of S-type unit in bamboo is the highest. The side chain region of the free lignin of the three kinds is composed of methoxyl and various linkages. Since the side chain region of masson pine is prone to form β-0-4, β-5 and β-β bonds, etc., contains more branches, and the structure is relatively firm, the concentration of the linkages in the side chain region of masson pine is higher than that of the free lignin of the other two types (bamboo and poplar). The cellulose conversion rate and enzyme substrate adsorption capacity of the hydrolysate are determined by blood glucose meter (BGM) and ultraviolet spectrophotometer (UV). In the enzyme hydrolysis of combined lignin, with the decrease of lignin content, the cellulose conversion rate and enzyme substrate adsorption capacity increase. In the enzyme hydrolysis of free lignin, the lignin content has no effect on the cellulose conversion rate and enzyme substrate adsorption capacity. The results show that the inhibition of free lignin on enzyme hydrolysis is more serious than that of combined lignin, and this trend is independent of the type of biomass. The inhibition of combined lignin on enzyme hydrolysis of the three different lignocelluloses (bamboo, masson pine and poplar) is in the order of bamboo > poplar > masson pine. The present application aims to provide a theoretical basis and technical framework for the development of ultra-high efficiency enzyme hydrolysis technology. In addition, it can also provide technical support for the removal of lignin in the reaction substrate of the pretreatment stage of lignocellulose. Therefore, the production of bioethanol by lignocellulose enzyme hydrolysis has great development potential and can alleviate the rapid growth of fossil fuel consumption in the world.

[0096] In summary, in the process of enzyme hydrolysis, compared with lignin in the combined state, lignin in the free state and control of different contents of free lignin play an obvious role in improving the efficiency of enzyme hydrolysis. The method of the present application has obvious effect on the increase of cellulose conversion rate and enzyme adsorption capacity in the hydrolysate of lignocellulose enzyme hydrolysis. When the lignin in the biomass raw pulp is removed by bleaching method, the bleaching time is controlled in 15-20 min, and the content of combined lignin prepared is 2-5% of holocellulose, which can ensure that the cellulose hydrolysis rate in the later stage is as high as 96.95%, greatly saving the time of bleaching step and the hydrolysis efficiency of cellulose; and it also has the advantages of simple preparation process, easy operation and low cost.

[0097] Although the specific embodiments of the present application are described above, those skilled in the art should understand that the specific examples described are only illustrative, and are not intended to limit the scope of the present application, and equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present application should be covered within the scope of the claims of the present application.

Claims

1. A method for promoting the enzymatic hydrolysis efficiency of lignocellulose, characterized in that: The method steps are as follows: (1) First, a kraft method is used to prepare biomass pulp of lignocellulosic raw materials; (2) the biomass pulp is bleached to prepare holocellulose, the bleaching time is 15-20 minutes, and the bound lignin content in the holocellulose is less than 5%; (3) enzymatically hydrolyzing the holocellulose having a bound lignin content of less than 5% to obtain a hydrolyzate; wherein the cellulose conversion rate of the hydrolyzate is greater than 90%; The hydrolytic enzymes used in the enzymatic hydrolysis include cellulase and cellobiase, and the usage ratio thereof is 5:1 to 1:

1.

2. The method for promoting enzymatic hydrolysis of lignocellulose according to claim 1, wherein: The lignocellulosic raw materials include coniferous wood, broadleaf wood and herbaceous plants; The usage ratio of cellulase to cellobiose in the hydrolase is 3:

1.

3. The method for promoting enzymatic hydrolysis of lignocellulose according to claim 1, wherein: The step (1) is specifically as follows: (1) Preparation of biomass pulp: (i) Preparation of biomass pulp from lignocellulosic feedstock by the kraft process: a digester was filled with dry wood chips and a (NaOH + 9H2O·Na2S) solution at a solid-liquid ratio of 1:4 (w / v). The temperature was raised at a rate of 2°C / min to a maximum temperature of 100-170°C and maintained at the maximum temperature for 30-120 min. (ii) After the holding time at the highest temperature is completed, the slurry and waste liquid in the cooking tank are immediately poured into a 100-150 mesh slurry bag for separation, and the corresponding slurry substrate and waste liquid are collected; (iii) washing the solid slurry with water until the pH is neutral to ensure that dissolved compounds and residual chemicals in the solid slurry are completely eliminated; (iv) The cleaned solid slurry is processed by a grinding machine, and then dissociated by a cellulose dissociator for 1 to 3 minutes; finally, the processed solid slurry is bagged and stored in a refrigerator at 4°C for later use.

4. The method for promoting enzymatic hydrolysis of lignocellulose according to claim 1, wherein: The step (2) is specifically as follows: (2) The solid slurry in step (1) is dried in the sun and then bleached to prepare holocellulose with a bound lignin content of less than 5%: (i) Absolutely dry solid pulp, distilled water, sodium chlorite, and glacial acetic acid were mixed and placed in a water bath shaker at 75°C and 150 rpm for bleaching. The bleaching time was controlled to be 15-20 minutes. The bleached wood pulp was filtered and washed until the filtrate was neutral, and then air-dried and bagged for later use. (ii) Preparation of free lignin: The wood pulp sample was washed to neutrality by centrifugal washing and air-dried. Finally, the free lignin sample was obtained by ball milling and sieving.

5. The method for promoting the enzymatic hydrolysis efficiency of lignocellulose according to claim 1, characterized in that: The step (3) is specifically as follows: (3) Enzymatic hydrolysis: The absolute dry wood pulp obtained in step (2) is mixed with a HAc-NaAc buffer solution (0.05 M, pH = 4.8), shaken thoroughly, and then 12%-20% of cellulase, 0.1%-0.4% of Tween 80, and 0.15%-0.30% of tetracycline based on the weight of the absolute dry wood pulp are added. The mixture is hydrolyzed in an air shaker at 35°C-55°C for 60-96 hours to obtain a wood fiber hydrolyzate.

6. The method for promoting enzymatic hydrolysis of lignocellulose according to claim 3, wherein: In the kraft pulping conditions, the amounts of NaOH+9H2O·Na2S used for coniferous wood, broadleaved wood, and herbaceous plants account for 17-23%, 15-21%, and 9-15% of the mass of the aqueous solution, respectively; the maximum temperatures are 140-170°C, 120-150°C, and 100-130°C, respectively; and the holding times are 90-120 minutes, 60-90 minutes, and 30-60 minutes, respectively.

7. The method for promoting enzymatic hydrolysis efficiency of lignocellulose according to claim 5, characterized in that: The usage ratio of absolute dry wood pulp to HAc-NaAc buffer solution is: 8-14 g: 50-200 mL.

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