CN101875953B - Method for performing steam explosion of wood fiber raw material, directional enzymatic dissociation and alcoholic fermentation - Google Patents

Abstract

A method for steam explosion, directional enzymolysis separation and ethanol fermentation of lignocellulosic raw materials, characterized in that steam explosion, directional enzymolysis of xylan, solid-liquid separation, deep enzymolysis of cellulose, ethanol fermentation of xylose and gas and glucose Anaerobic ethanol fermentation method. Due to the adoption of a new process of secondary enzymatic hydrolysis, and the introduction of β-glucosidase and a small amount of cellulase into xylanase, the effect of directional enzymolysis and separation of xylose in raw materials has been significantly improved, and it is a lignocellulosic raw material. The effective separation of xylose and glucose and high-efficiency ethanol fermentation provide a new method that significantly reduces the production cost of ethanol fermentation of lignocellulosic raw materials.

Description

Methods of steam explosion, directional enzymatic separation and ethanol fermentation of lignocellulosic raw materials

1. Technical field

The invention belongs to the technical field of enzymatic hydrolysis and biotransformation of biomass raw materials in biochemistry, and in particular relates to a method for preparing fuel ethanol through steam explosion pretreatment of lignocellulosic raw materials, directional enzymolysis of xylan, xylose fermentation and glucose fermentation.

2. Background technology

Cellulose and xylan in lignocellulosic raw materials can be hydrolyzed to glucose and xylose, and then fermented by microorganisms to produce many bio-based platform compounds, such as fuel ethanol, organic acids and polyols. Among many lignocellulosic raw materials, agricultural and forestry waste straws and grasses are rich in glucosyl and xylose, with huge yield and low price, which is the focus of the current development of biomass raw materials.

In straw and grass natural raw materials, cellulose, xylan and lignin form a tight complex through complex chemical and physical bonding, which forms an obstacle to enzymatic hydrolysis and utilization, and certain pretreatment processes must be taken to destroy them. The natural structure fully exposes the macromolecules of cellulose and xylan, reduces their degree of polymerization, and then improves the efficiency of enzymatic hydrolysis. At present, steam explosion is one of the mainstream pretreatment technologies for lignocellulosic raw materials, especially suitable for straw and grass raw materials.

Glucose and xylose account for more than 60% and 35% of the total sugar substances in the sugar solution obtained from lignocellulosic raw materials through steam explosion pretreatment and direct enzymatic hydrolysis. The strains of glucose ethanol fermentation - Saccharomyces cerevisiae and its production process are very mature. Compared with them, the strains of xylose fermentation are rare. At present, there are only a few strains such as Candida shohata and Pichia dry yeast, and their Industrial production performance is far less than Saccharomyces cerevisiae. These strains used for xylose fermentation have low tolerance to sugar and fermentation products such as ethanol during fermentation, and the utilization of xylose is inhibited by glucose, and precise restrictions need to be controlled during xylose fermentation Sexual oxygen supply measures. Due to the different fermentation strains and fermentation process conditions of these two types of sugars, the industrial scale-up of the synchronous fermentation technology of hexose and pentose sugars in the enzymatic hydrolysis liquid of lignocellulosic raw materials is seriously restricted, and there are problems such as high manufacturing and operating costs of fermentation equipment, wind loss Large size, complex process control, low yield and many other shortcomings have formed the bottleneck of industrial production.

3. Contents of the invention

The purpose of the present invention is to provide a low chemical consumption and environment-friendly separation of lignocellulosic raw materials by steam explosion and directional enzymatic hydrolysis according to the technical principles of steam explosion and xylan enzymatic hydrolysis according to the composition and structural characteristics of lignocellulosic raw materials and methods for fermenting xylose, especially for ethanol fermentation of xylose and glucose.

In lignocellulosic raw materials, glucose as the main sugar component accounts for more than 60% of the total sugar, while xylose generally accounts for about 35%. If the glucose base and xylose base can be separated and then fermented separately, the gas consumption can be greatly reduced, the load of xylose fermentation can be significantly reduced, the concentration of glucose alcohol fermentation can be doubled, and the overall utilization efficiency of the equipment can be improved. Reducing the production cost is an effective measure to alleviate the production bottleneck of the synchronous fermentation technology of hexose and pentose in the enzymatic hydrolysis solution of lignocellulosic raw materials.

The separation of xylose and glucose in lignocellulosic raw materials can be carried out by alkaline extraction or dilute acid hydrolysis of xylan, and the method of steam explosion combined with xylanase directional enzymatic hydrolysis is a modern method based on enzyme engineering. Bioprocessing technology can greatly reduce the consumption of acid and alkali chemicals, and can significantly improve the economic benefits of the fermentation process, and has the advantages of simple process and environmental affinity.

Concrete technical solution of the present invention is as follows:

A method for steam explosion, directional enzymatic separation and ethanol fermentation of lignocellulosic raw materials, characterized in that it adopts raw material preparation, steam explosion, directional enzymolysis of xylan, solid-liquid separation, deep enzymolysis of cellulose, xylose and gas ethanol fermentation And the method for glucose anaerobic ethanol fermentation, its concrete steps are as follows:

a. Cut the straw or grass raw materials into 3-5cm segments for steam explosion, the charging coefficient is 65-75%, the temperature is 195-205°C, and the holding time is 60-180 seconds;

b. The first round of directional enzymolysis of xylan: the pH value of the neutralized slurry is 4.5-5.2, the total solids concentration is adjusted to 3.0-5.0% (w/v), and xylanase 10-20U/g is added Slurry, cellulase 1.0-2.0FPU/g slurry and β-glucosidase 5-10U/g slurry, reaction temperature 48-50°C, stirring speed 120-150rpm, reaction 48-50h, solid-liquid separation to obtain Enzymolysis solution I and enzymolysis residue I;

c. The second round of cellulose deep enzymolysis: adjust the total solids concentration of enzymolysis slag I to 10-15% (w/v), pH4.5-5.2, add cellulase 15-20FPU/g cellulose and β-glucosidase 30～40IU/g cellulose, control reaction temperature 48～50℃, stirring speed 100～150rpm, react for 48～50h, separate solid-liquid to obtain enzymolysis liquid II and enzymolysis residue II;

d. Concentrate the enzymatic solution I to a xylose concentration ≥ 30.0g/L, add Candida shohata to ferment for 12-14 hours under aerobic conditions, and reuse the yeast;

e. Concentrate the enzymatic solution II to a glucose concentration ≥ 150.0 g/L, add Saccharomyces cerevisiae to ferment for 16-18 hours under anaerobic conditions, and reuse the yeast.

4. Description of drawings

Fig. 1 is a schematic flow chart of steam explosion of lignocellulosic raw materials, directional enzymatic separation and ethanol fermentation;

Figure 2 is a comparison of the yields of total sugars by enzymatic hydrolysis in different ways before and after straw raw materials are pretreated by steam explosion. The enzymolysis conditions are: the total solid concentration of the raw material is 3.0-5.0% (w/v), and the pH is 4.8-5.2. Total enzyme dosage: xylanase 40-80U/g xylosyl, cellulase 15-20FPU/g glucosyl and β-glucosidase 30-40U/g glucosyl. The reaction temperature is 48-52° C., the stirring rate is 100-150 rpm, and the reaction is 48-50 hours. in:

Bar graph 1 shows the result of one-step enzymatic hydrolysis of straw powder (0.2mm ~ 0.5mm) without steam explosion directly adding xylanase, glucose and cellulase;

Bar graph 2 shows the result of one-step enzymatic hydrolysis of steam-exploded straw by adding xylanase, cellulase and β-glucosidase;

Bar graph 3 shows the results of the first round of directional enzymolysis in which steam-exploded straw was added with xylanase, β-glucosidase and a small amount of cellulase for two-step enzymolysis;

Bar graph 4 shows the results of the second round of deep enzymolysis in which cellulase and β-glucosidase are added to the enzymolysis residue of 3 for two-step enzymolysis;

The histogram (3+4) represents the cumulative results of 3 and 4 two-step enzymatic hydrolysis.

Fig. 3 is the effect of the combination of enzymes on the directional enzymatic hydrolysis of steam-exploded straw. The enzymolysis conditions are as follows: the total solid concentration of raw materials is 3.0-5.0% (w/v), pH 4.8-5.2, reaction temperature 48-50° C., stirring speed 100-150 rpm, and reaction for 48-50 hours. The total enzyme dosage is: xylanase 10-20U/g slurry, cellulase 5-20FPU/g slurry and beta-glucosidase 10-40U/g slurry. Wherein, the abscissa indicates the combination of enzymes, X is xylanase; BG is β-glucosidase; C is cellulase.

Figure 4 is the effect of the amount of cellulase added on the directional enzymolysis effect of xylanase. The enzymolysis conditions are as follows: the total solid concentration of raw materials is 3.0-5.0% (w/v), pH 4.8-5.2, reaction temperature 48-50° C., stirring speed 100-150 rpm, and reaction for 48-50 hours. Xylanase 10-20U/g slurry and β-glucosidase 5-10U/g slurry. Wherein, the abscissa represents the added amount of cellulase (FPU/g slurry).

Figure 5 shows the effect of the solid concentration of steam-exploded straw on the directional enzymatic hydrolysis of xylan. The enzymolysis conditions are: pH 4.8-5.2, reaction temperature 48-50°C, stirring speed 100-150rpm, reaction 48-50h. Add 10-20 U/g slurry of xylanase, 1.0-2.0 FPU/g slurry of cellulase and 5-10 U/g slurry of β-β-glucosidase. The abscissa represents the total solids concentration (%, w/v) of the steam-exploded straw.

5. Specific implementation

After the straw and grass raw materials are air-dried, they are cut into 3-5cm lengths by a rotary cutting device and stored for later use.

The cut straw or grass raw materials are added to the intermittent automatic steam explosion device, and the natural accumulation method is used for filling to ensure the uniformity of steam heating, and the volume filling coefficient is controlled to be 65-75%. After sealing, steam is introduced to directly heat the material to a temperature of 195-205°C. After using steam to keep warm for 60-180 seconds, the automatic discharge valve is quickly opened by the automatic control system to blast and discharge to obtain slurry. The dry weight yield of the slurry reaches more than 80% of the raw material, among which the yield of glucose group is higher than 85%, accounting for 43.0-45.0% of the dry weight of the slurry, and the yield of xylose group reaches more than 60%, accounting for more than 85% of the dry weight of the slurry. 12.0-15.0% of the weight.

Use Ca(OH) ₂ or ammonia water to neutralize the slurry to pH 4.5-5.2, adjust the total solids concentration to 3.0-5.0% (w/v), add xylanase 10-20U/g slurry, cellulase 1.0-2.0 FPU/g slurry and β-glucosidase 5-10U/g slurry, the reaction temperature is controlled at 48-50°C, and the stirring speed is 100-150rpm for the first round of directional enzymolysis of xylan. After reacting for 48-50 hours, use centrifugation (5000 rpm for 15 minutes) or bag filter to separate the enzymatic hydrolysis system to obtain enzymatic hydrolysis solution I and enzymatic hydrolysis residue I. In the enzymatic solution I, the yield of xylose accounts for more than 65.0% of the xylosyl in the slurry, and the yield of glucose accounts for less than 25.0% of the glucosyl in the slurry.

Add an appropriate amount of water to the enzymatic residue I, adjust the total solids concentration to 10-15 (w/v) and pH 4.5-5.2, then add cellulase 15-20 FPIU/g cellulose and β-glucosidase 30-40IU/g cellulose, the reaction temperature is controlled at 48-50°C, and the stirring rate is 100-150rpm for the second round of deep enzymatic hydrolysis. After reacting for 48-60 hours, use centrifugation (5000 rpm for 15 minutes) or bag filter to separate the enzymatic hydrolysis system to obtain enzymatic hydrolysis liquid II and enzymatic hydrolysis residue II. In the enzymatic solution II, the yield of glucose accounts for about 50.0% of the glucose group in the slurry.

Concentrate the enzymolysis solution I by vacuum decompression method to xylose concentration ≥ 30.0g/L, insert activated Candida shohata to cell concentration OD600nm ≥ 15.0, ferment for 12-14 hours under concurrent gas conditions, and use centrifugation Centrifuge at 3000rpm for 5-10min to recycle the yeast for re-fermentation. The sugar utilization rate is more than or equal to 90.0%, and the ethanol yield reaches more than 85.0% of the theoretical yield.

Concentrate the enzymolysis solution II by vacuum decompression method to glucose concentration ≥ 150.0g/L, insert activated Saccharomyces cerevisiae to cell concentration OD600nm ≥ 15.0, ferment under anaerobic conditions for 16-18 hours, and use centrifugation at 3000rpm Centrifuge for 5-10 minutes to reuse the yeast for further fermentation. The sugar utilization rate is more than or equal to 95.0%, and the ethanol yield reaches more than 92.0% of the theoretical yield.

It can be seen from Figure 2 that the enzymatic hydrolysis yield of xylan and cellulose in straw can be significantly improved by adopting the steam explosion pretreatment method. Under the same dosage of xylanase, cellulase and β-glucosidase, the yields of xylan and cellulose of untreated straw were only 14.2% and 24.1% by one-step enzymatic hydrolysis, while After steam explosion, their enzymatic hydrolysis yields can reach 80.6% and 67.8%, respectively, so steam explosion pretreatment can significantly promote the enzymatic hydrolysis of xylan and cellulose in lignocellulosic raw materials. Adding a small amount of cellulase on the basis of xylanase and β-glucosidase can improve the yield of directional enzymolysis of xylan. In the first round of directional enzymolysis process, the enzymatic hydrolysis yield of xylose group can reach 66.5.%, while the enzymatic hydrolysis yield of glucosyl group is only 24.8%; The enzymatic hydrolysis yield of the method reaches 50.5%, and the total yield of the two rounds of enzymatic hydrolysis of the glucose base reaches 75.3%, which is nearly 11.1% higher than that of the one-step method of 67.8%. This shows that, first, cellulase and xylanase can promote each other in enzymatic hydrolysis of xylan and cellulose; Most of the xylose groups are also conducive to the subsequent deep enzymatic hydrolysis of cellulose, which improves the yield of glucose enzymatic hydrolysis.

It can be seen from Figure 3 that xylanase and β-glucosidase alone cannot effectively enzymatically hydrolyze xylan, but the addition of cellulase can significantly increase the enzymatic hydrolysis yield of xylosyl.

It can be seen from Figure 4 that adding a small amount of cellulase on the basis of xylanase and β-glucosidase can significantly increase the enzymatic hydrolysis yield of xylosyl in steam-exploded straw. When the added amount of cellulase is at 2.0U/g slurry, the enzymatic hydrolysis yield of xylosyl can be increased from 49.5% to 66.5%, and the rate of improvement reaches 34.%, while the enzymatic hydrolysis yield of glucosyl can be increased Control within 24.5%. Exceeding the added amount has little effect on promoting the xylosyl enzymatic hydrolysis yield, but it will lead to a rapid increase in the glucosyl enzymatic hydrolysis yield, affecting the effect of directional enzymatic hydrolysis. Therefore, the appropriate amount of cellulose added is 1.0-2.0 U/g pulp.

It can be seen from accompanying drawing 5 that when the total solids concentration is higher than 5.0-6.0% (w/v), the enzymatic hydrolysis yield of xylose in the steam-exploded stalk shows a trend of obvious decline. Considering the steam-exploded - It is advisable to take 3.0-5.0% (w/v) as the total solid content of enzymatic hydrolysis for directional enzymatic hydrolysis to separate xylose.

The analysis and determination of glucose base (cellulose) and xylose base (xylan) in the described lignocellulosic raw material and the calculating method of enzymatic hydrolysis yield all adopt the standard method (A .SLUITER, B.HAMES, R.RUIZ, C.SCARLATA, J.SLUITER, D.TEMPLETON, and D.CROCKER. Laboratory Technical Report NREL/TP-510-42618, 2008).

The enzyme activity assay method of described xylanase, cellulase and β-glucosidase adopts the assay method (GHOSE, T.K.and BISARIA V.S. of International Union of Pure Chemistry and Applied Chemistry, (1987) Pure & Appl.Chem., 59(12), 1739-1752; GHOSE, T.K. and BISARIA V.S., (1987) Pure & Appl. Chem., 59(2), 257-268);

The mensuration of glucose, xylose and ethanol in the described enzymolysis solution all adopts high-performance liquid chromatography (HPLC) to analyze, and the system of chromatograph and operating condition are: Agilent 1200 chromatograph and workstation, sugar column Bio-RadAminex HPX-87H , the column temperature was 55° C., the mobile phase was 0.5 mmol/L H ₂ SO ₄ , the flow rate was 0.6 mL/min, and the detector was a refractive index detector (RID).

Beneficial effects: Due to the adoption of steam explosion and secondary enzymatic hydrolysis technology, especially the introduction of β-glucosidase and a small amount of cellulase into xylanase, the orientation of xylose in lignocellulosic raw materials is significantly improved. The effect of enzymatic hydrolysis and separation makes the ratio of enzymatic hydrolysis yield of xylose group and glucose group in raw materials exceed 65% and 25%, and at the same time promotes the enzymatic hydrolysis of cellulose, increasing the total yield of enzymatic hydrolysis of glucose group by 10% It provides a low chemical consumption and environmentally friendly technical method for the effective separation of xylose and glucose in lignocellulosic raw materials and high-efficiency ethanol fermentation, which can significantly save the production cost of cellulose ethanol fermentation.

Claims (1)

1. a steam explosion of wood fiber raw material, directional enzymatic separate and the method for ethanol fermentation, it is characterized in that adopting get the raw materials ready, steam explosion, xylan directional enzymatic, solid-liquid separation, Mierocrystalline cellulose degree of depth enzymolysis, wood sugar are held concurrently the method for gas ethanol fermentation and glucose anaerobism ethanol fermentation, its concrete steps are as follows:

A. stalk or grass as raw material are cut into 3～5cm fragment and carry out steam explosion, coefficient 65～75%, 195～205 ℃ of temperature, soaking time 60～180 seconds;

B. first round xylan directional enzymatic: in and the pH value of slurry be 4.5～5.2, regulating total solid concentration is 3.0～5.0% (w/v), add zytase 10～20U/g slurry, cellulase 1.0～2.0FPU/g slurry and beta-glucosidase 5～10U/g slurry, 48～50 ℃ of temperature of reaction, stir speed (S.S.) 120～150rpm, reaction 48～50h, solid-liquid separation obtains enzymolysis solution I and Production With Residue of Enzymatic Hydrolysis I;

C. second take turns Mierocrystalline cellulose degree of depth enzymolysis: the total solid concentration to 10 of adjusting Production With Residue of Enzymatic Hydrolysis I～15% (w/v), pH4.5～5.2, add cellulase 15～20FPU/g Mierocrystalline cellulose and beta-glucosidase 30～40IU/g Mierocrystalline cellulose, 48～50 ℃ of control temperature of reaction, stir speed (S.S.) 100～150rpm, reaction 48～50h, solid-liquid separation obtains enzymolysis solution II and Production With Residue of Enzymatic Hydrolysis II;

D. concentrated enzymolysis solution I is to xylose concentration 〉=30.0g/L, and the shehatae candida after the access activation is to cell concn OD600nm 〉=15.0, at double gas condition bottom fermentation 12～14h, backstocking;

E. concentrated enzymolysis solution II is to glucose concn 〉=150.0g/L, and the yeast saccharomyces cerevisiae after the access activation is to cell concn OD600nm 〉=15.0, the 16～18h that under anaerobic ferments, backstocking.

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