阅读说明：本技术 一种稳定木质纤维素酶解过程的方法 (Method for stabilizing lignocellulose enzymolysis process ) 是由 李勉 胡昌辉 王静 曾徐浩 柳志强 杨世辉 于 2020-07-03 设计创作，主要内容包括：本发明涉及一种稳定木质纤维素酶解过程的方法,在向酶解罐的生物质物料中加入纤维素酶液进行酶解之前,先向生物质物料中加入有机酸盐溶液和有机酸复配液,其中,生物质物料为秸秆、玉米芯、甘蔗渣、木糖渣中任意一种,有机酸盐溶液包括甲酸钠、乙酸钠、柠檬酸钠或琥珀酸钠溶液中的一种,有机酸复配液包括柠檬酸、琥珀酸、乙酸、酒石酸、乙二酸、苹果酸、乳酸溶液中的至少一种。本发明利用多种有机酸进行复配,在木质纤维素高固酶解过程中加入复配有机酸,在木质纤维素物料高固酶解过程中能够保持纤维素酶蛋白活性,使得纤维素酶解转化率增加。(The invention relates to a method for stabilizing the enzymolysis process of lignocellulose, which comprises the steps of adding an organic acid salt solution and an organic acid complex solution into a biomass material before adding a cellulase solution into the biomass material in an enzymolysis tank for enzymolysis, wherein the biomass material is any one of straw, corncobs, bagasse and xylose residues, the organic acid salt solution comprises one of sodium formate, sodium acetate, sodium citrate or sodium succinate solution, and the organic acid complex solution comprises at least one of citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution. According to the invention, multiple organic acids are compounded, and the compounded organic acid is added in the high-solid enzymolysis process of the lignocellulose, so that the activity of the cellulase protein can be maintained in the high-solid enzymolysis process of the lignocellulose material, and the cellulose enzymolysis conversion rate is increased.)

1. A method for stabilizing the enzymolysis process of lignocellulose is characterized in that before cellulose enzyme liquid is added into biomass materials in an enzymolysis tank for enzymolysis, organic acid salt solution and organic acid compound solution are added into the biomass materials, wherein the biomass materials are any one of straws, corncobs, bagasse and xylose residues, the organic acid salt solution comprises one of sodium formate, sodium acetate, sodium citrate or sodium succinate solution, and the organic acid compound solution comprises at least one of citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution.

2. The method for stabilizing the lignocellulose enzymolysis process as claimed in claim 1, wherein after the organic acid salt solution and the organic acid complex solution are added, the pH of the enzymolysis system is maintained to be 4.6-5.6, so that the dry matter content of the biomass material in the enzymolysis system is 15% -25%, and the adding amount of the cellulase solution is 4% -6%.

3. The method of stabilizing a lignocellulosic enzymatic process of claim 2 wherein the cellulase is Novoxil Cellic CTec 2.

4. The method for stabilizing the enzymolysis process of the lignocellulose as recited in claim 1, wherein during the enzymolysis, the control temperature of the enzymolysis tank is 45-50 ℃, the stirring speed is 180-220 rpm, and the enzymolysis is carried out for 48-96 h.

5. The method for stabilizing the lignocellulose enzymolysis process as claimed in claim 1, wherein 70mL of 0.1mmol/L sodium citrate organic acid salt solution and 82 g-85 g of organic acid compound solution are respectively added into 15g of the corncob waste residue biomass material, wherein the organic acid compound solution is citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution which are mixed according to equal mass, 0.6g of Novesson Cellic CTec2 is added, the pH of the enzymolysis system is adjusted to 5.5, the temperature of the enzymolysis tank is controlled at 50 ℃, the stirring speed is 200rpm, and the enzymolysis is carried out for 96 hours.

6. The method for stabilizing the lignocellulose enzymolysis process as claimed in claim 1, wherein 65mL of 0.1mmol/L sodium citrate organic acid salt solution and 70-80 g of organic acid compound solution are respectively added into 20g of the corncob waste residue biomass material, wherein the organic acid compound solution is citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution which are mixed according to equal mass, 0.8g of Novesson Cellic CTec2 is added, the pH of the enzymolysis system is adjusted to 5.5, the temperature of the enzymolysis tank is controlled to be 50 ℃, the stirring speed is 200rpm, and the enzymolysis is carried out for 96 hours.

7. The method for stabilizing the lignocellulose enzymolysis process as claimed in claim 1, wherein 60mL of 0.1mmol/L sodium citrate organic acid salt solution and 72 g-75 g of organic acid compound solution are respectively added into 25g of the corncob waste residue biomass material, wherein the organic acid compound solution is citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution which are mixed according to equal mass, 1.0g of Novesson Cellic CTec2 is added, the pH of the enzymolysis system is adjusted to 5.5, the temperature of the enzymolysis tank is controlled at 50 ℃, the stirring speed is 200rpm, and the enzymolysis is carried out for 96 hours.

8. The method for stabilizing the lignocellulose enzymolysis process as claimed in claim 1, wherein 70mL of 0.1mmol/L sodium formate organic acid salt solution and 82 g-85 g of organic acid compound solution are respectively added into 15g of the corncob waste residue biomass material, wherein the organic acid compound solution is citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution which are mixed according to equal mass, 0.6g of Novoxil Cellic CTec2 cellulase is added, the pH of the enzymolysis system is adjusted to 4.6, the temperature of the enzymolysis tank is controlled to be 45 ℃, the stirring speed is 180rpm, and the enzymolysis is carried out for 96 hours.

9. The method for stabilizing the lignocellulose enzymolysis process as claimed in claim 1, wherein 60mL of 0.1mmol/L sodium acetate organic acid salt solution and 72 g-75 g of organic acid compound solution are respectively added into 25g of the corncob waste residue biomass material, wherein the organic acid compound solution is citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution which are mixed according to equal mass, 0.8g of Novoxil Cellic CTec2 cellulase is added, the pH of the enzymolysis system is adjusted to 5.5, the temperature of the enzymolysis tank is controlled to be 50 ℃, the stirring speed is 220rpm, and the enzymolysis is carried out for 48 h.

Technical Field

The invention belongs to the technical field of lignocellulose enzymolysis, and particularly relates to a method for stabilizing a lignocellulose enzymolysis process.

Background

China is a big country in agriculture and forestry, and endless agricultural wastes (straws, corncobs, bagasse and the like) and forestry wastes (woods, branches and leaves and the like) are generated every year. The high-value utilization of these wastes is a very promising research direction.

The lignocellulose materials produced by the nature mainly comprise three components of cellulose (32-36%), hemicellulose (35-40%) and lignin (25%). Wherein, the hemicellulose component in the corncob can be developed to produce xylose, xylo-oligosaccharide, furfural, xylitol and the like, and the cellulose component can be subjected to enzymolysis by cellulase to produce monomeric glucose. The enzymolysis liquid can be used for producing glucose syrup products through a subsequent refining process, and meanwhile, the enzymolysis liquid can be used for producing high-value products such as fuel ethanol and the like through microbial fermentation.

The key step of utilizing cellulose components in lignocellulose materials is cellulase enzymolysis, and in the process of cellulase enzymolysis, due to the change of a complex and high-solid enzymolysis system of the components of the enzymolysis materials, the activity of cellulase protein in the enzymolysis process can be reduced and even inactivated. The limiting conditions for the cellulase to exert high activity mainly comprise temperature, pH value, growth of microorganisms, adsorption of lignin in lignocellulose on enzyme protein, oxidation inactivation of enzyme protein and the like. Currently, many targeted studies are being conducted to maintain the activity of enzyme proteins.

Researches find that sodium azide and some antibiotics (tetracycline) have the bacteriostatic action, can inhibit the growth of mixed bacteria and reduce the loss of enzyme activity; sodium thiosulfate prevents the oxidative inactivation of the enzyme protein. The methods can maintain the activity of the cellulase protein to a certain degree, but in the industrial production of biomass fuel ethanol, the amount of cellulase required in a large-scale high-solid enzymatic hydrolysis system is huge, and more substances for maintaining the activity of the cellulase protein are required. And the substances such as sodium azide, antibiotics and sodium thiosulfate have low economic benefit, have single function and have weak effect of maintaining the activity of the cellulase protein.

The prior wood fiber utilization technical field has the following problems:

1. during the high-solid enzymolysis process of lignocellulose, cellulase can fully exert the effect only under the condition of proper pH value, but due to overhigh viscosity and difficult pH value control, a proper buffer system is needed for adjustment;

2. the lignocellulose high-solid enzymolysis system is complex, microbes such as bacteria and mold are easy to breed, antibacterial substances are required to be added, but the cost of sodium azide and antibiotic substances (tetracycline) is too high in large-scale industrialization, and a suitable industrialized antibacterial method needs to be found;

3. the lignocellulose enzymolysis process is 3-5 days long, the used cellulase is easy to oxidize and denature and loses enzymolysis capacity, and at present, enzyme protein denaturation can be prevented by using antioxidants such as Vc and sodium thiosulfate, but the cost is too high in large-scale industrialization, and the functions of the substances are single.

The problems of regulating the pH value, inhibiting microorganisms and resisting oxidation are the problems of inhibiting the activity of enzyme protein in the process of high-solid enzymolysis of the lignocellulose, and no method can simultaneously solve the three problems at present. The aim of the present invention is therefore to develop a process which makes use of organic acids to solve the three problems mentioned above.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for stabilizing the enzymolysis process of lignocellulose, which can keep the activity of cellulase protein in the high-solid enzymolysis process of lignocellulose materials, so that the enzymolysis conversion rate of the cellulose is increased.

The invention is realized in such a way, and provides a method for stabilizing the lignocellulose enzymolysis process, before adding cellulose enzyme solution into biomass materials in an enzymolysis tank for enzymolysis, organic acid salt solution and organic acid complex solution are firstly added into the biomass materials, wherein the biomass materials are any one of straws, corncobs, bagasse and xylose residues, the organic acid salt solution comprises one of sodium formate, sodium acetate, sodium citrate or sodium succinate solution, and the organic acid complex solution comprises at least one of citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution.

Further, after the organic acid salt solution and the organic acid compound solution are added, the pH value of the enzymolysis system is maintained to be 4.6-5.6, so that the dry matter weight of the biomass material in the enzymolysis system is 15% -25%, and the adding amount of the cellulase solution is 4% -6% of the dry matter weight of the biomass material.

Further, the cellulase was Cellic CTec2 Novov.

Further, during enzymolysis, the temperature of the enzymolysis tank is controlled to be 45-50 ℃, the stirring speed is 180-220 rpm, and the enzymolysis is carried out for 48-96 h.

Further, 70mL of 0.1mmol/L sodium citrate organic acid salt solution and 82-85 g of organic acid compound solution are respectively added into 15g of the corncob waste residue biomass material, wherein the organic acid compound solution is prepared by mixing citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution according to equal mass, 0.6g of Novoxil Cellic CTec2 is added, the pH of an enzymolysis system is adjusted to 5.5, the temperature of an enzymolysis tank is controlled to be 50 ℃, the stirring speed is 200rpm, and enzymolysis is carried out for 96 hours.

Further, 65mL of 0.1mmol/L sodium citrate organic acid salt solution and 70-80 g of organic acid compound solution are respectively added into 20g of the corncob waste residue biomass material, wherein the organic acid compound solution is prepared by mixing citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution according to equal mass, 0.8g of Novoxil Cellic CTec2 is added, the pH value of an enzymolysis system is adjusted to 5.5, the temperature of an enzymolysis tank is controlled to be 50 ℃, the stirring speed is 200rpm, and enzymolysis is carried out for 96 hours.

Further, 60mL of 0.1mmol/L sodium citrate organic acid salt solution and 72-75 g of organic acid compound solution are respectively added into 25g of the corncob waste residue biomass material, wherein the organic acid compound solution is prepared by mixing citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution according to equal mass, 1.0g of Novoxil Cellic CTec2 is added, the pH of an enzymolysis system is adjusted to 5.5, the temperature of an enzymolysis tank is controlled to be 50 ℃, the stirring speed is 200rpm, and enzymolysis is carried out for 96 hours.

Further, 70mL of 0.1mmol/L sodium formate organic acid salt solution and 82-85 g of organic acid compound solution are respectively added into 15g of the corncob waste residue biomass material, wherein the organic acid compound solution is citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution which are mixed according to equal mass, 0.6g of Novesson Cellic CTec2 cellulase is added, the pH value of an enzymolysis system is adjusted to 4.6, the temperature of an enzymolysis tank is controlled to be 45 ℃, the stirring speed is controlled to be 180rpm, and enzymolysis is carried out for 96 hours.

Further, 60mL of 0.1mmol/L sodium acetate organic acid salt solution and 72-75 g of organic acid compound solution are respectively added into 25g of the corncob waste residue biomass material, wherein the organic acid compound solution is citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution which are mixed according to equal mass, 0.8g of Novesson Cellic CTec2 cellulase is added, the pH value of an enzymolysis system is adjusted to 5.5, the temperature of an enzymolysis tank is controlled to be 50 ℃, the stirring speed is controlled to be 220rpm, and enzymolysis is carried out for 48 hours.

Compared with the prior art, the method for stabilizing the lignocellulose enzymolysis process has the main contents that various organic acids are compounded, the compounded organic acids are added in the lignocellulose high-solid enzymolysis process, the cellulase protein activity can be kept in the lignocellulose material high-solid enzymolysis process, and the cellulose enzymolysis conversion rate is increased.

The invention also has the following characteristics:

firstly, adding organic acid into an enzymolysis system, and simultaneously adding a weak acid salt, such as a citric acid-trisodium citrate system, to form a buffer system, so as to maintain the pH value of the enzymolysis system to be stable;

secondly, the combined action of a plurality of organic acids can achieve the effect of inhibiting the growth of mixed bacteria at a higher pH value (4-6);

thirdly, the organic acid has strong oxidation resistance and can prevent the enzyme protein from being oxidized and losing activity in an enzymolysis system.

Drawings

FIG. 1 is a comparison schematic diagram of the bacteriostatic action of the organic acid complex liquid in the lignocellulose enzymolysis process in example 2 of the invention;

FIG. 2 is a comparative graphical representation of the ability of the organic acid complex liquid of example 2 of the present invention to stabilize pH during the enzymatic hydrolysis of a lignocellulosic enzyme;

FIGS. 3a and 3b are schematic diagrams showing the comparison of the glucose concentration and the cellulose conversion rate of the organic acid complex liquid in example 2 of the present invention during the lignocellulose enzymolysis process, respectively;

FIGS. 4a and 4b are schematic diagrams showing the comparison of the glucose concentration and the cellulose conversion rate of the organic acid complex liquid in example 3 of the present invention during the lignocellulose enzymolysis process, respectively;

FIGS. 5a and 5b are schematic diagrams showing the comparison of the glucose concentration and the cellulose conversion rate of the organic acid complex liquid of example 4 of the present invention during the lignocellulose enzymolysis process, respectively;

FIG. 6 is a comparison of the enzymatic hydrolysis-promoting effects of different types of organic acid complex solutions according to example 5 of the present invention.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In a preferred embodiment of the method for stabilizing the lignocellulose enzymolysis process, before adding the cellulase solution into the biomass material in the enzymolysis tank for enzymolysis, an organic acid salt solution and an organic acid complex solution are added into the biomass material, wherein the biomass material is any one of straw, corncob, bagasse and xylose residue, the organic acid salt solution comprises one of sodium formate, sodium acetate, sodium citrate or sodium succinate solution, and the organic acid complex solution comprises at least one of citric acid, succinic acid, acetic acid, tartaric acid, oxalic acid, malic acid and lactic acid solution.

After the organic acid salt solution and the organic acid compound solution are added, the pH value of the enzymolysis system is maintained to be 4.6-5.6, so that the dry matter weight of the biomass material in the enzymolysis system is 15% -25%, and the adding amount of the cellulase solution is 4% -6% (w/w) of the dry matter weight of the biomass material.

The solution of organic acid salt comprises at least one of 0.1mmol/L sodium formate, 0.1mmol/L sodium acetate, 0.1mmol/L sodium citrate or 0.1mmol/L sodium succinate solution, and the cellulase adopts Novoxin Cellic CTec 2.

During enzymolysis, the temperature of the enzymolysis tank is controlled to be 45-50 ℃, the stirring speed is 180-220 rpm, and the enzymolysis is carried out for 48-96 h.

The method for stabilizing the lignocellulose enzymolysis process according to the invention is further illustrated below with reference to specific examples.