TWI418628B - Lignocellulose Pretreatment Method for Agricultural Waste - Google Patents

Description

Lignocellulosic pretreatment method for agricultural waste

The present invention relates to a lignocellulose pretreatment method for agricultural and forestry waste. More particularly, a lignocellulosic pretreatment process using white rot fungi is used, characterized in that the white rot fungus selectively decomposes lignin but does not decompose cellulose.

Lignin is a component of plant content that is second only to cellulose and hemicellulose, generally accounting for 15-20% of the dry weight of plants, and about 30% in wood. The chemical structure of lignin is very complicated, but in nature, there are still some microorganisms that can decompose such substances. Among them, basidiomycetes have the strongest decomposition ability. However, while basidiomycetes break down lignin, they often break down substances such as cellulose and hemicellulose. White rot fungi are mostly Basidiomycota (Basidiomycota), only a small portion of the phylum Ascomycota (Ascomycota), which has a unique extracellular lignin decomposition system, capable of secreting a lignin decomposition enzymes (ligninase).

The invention utilizes white rot fungus to decompose lignin. Firstly, it is inoculated with semi-solid medium containing agricultural waste by white rot fungus Perenniporia tephropora , and it is known that the white rot fungus can selectively remove lignin. According to the activity analysis, the white rot fungus can effectively reduce the amount of lignin, thereby increasing the effect of agricultural waste being decomposed by other fiber enzymes to produce sugar, or facilitating the remaining cellulose as papermaking, producing alcohol or animal feed. raw material.

Accordingly, the present invention relates to a lignocellulose pretreatment method for selectively removing lignin by a white rot fungus, the method comprising: inoculating a white rot fungus Perenniporia tephropora TFRI 707 strain in a semi-solid medium comprising: 60 mM buffer solution (pH 4.5~6.5), 10~30 g/L glucose, 0.1~0.4 mM CuSO ₄ ‧5H ₂ O solution, 0.01~1.0% (w/v) Tween 80, 1 ml/L trace element solution And 35~70g/500mL of agricultural waste and adjust and maintain the pH at 4.0 with 1M HCl solution.

The trace element solution contains 0.1 g of Na ₂ B ₄ O ₇ ‧10 H ₂ O per liter, 0.01 g of MnSO ₄ ‧7 H ₂ O, 0.05 g of FeSO ₄ ‧7 H ₂ O, 0.07 g of ZnSO ₄ ‧7 H ₂ O, 0.01 g MnSO ₄ ‧7 H ₂ O, 0.01 g (NH ₄ ) ₆ Mo ₇ O ₂₄ ‧4H ₂ O).

The buffer solution may be one of a sodium phosphate buffer solution, a potassium phosphate buffer solution, and a sodium tartrate buffer solution; the agroforestry waste may be rice straw, wood shaving or tree wood (wood chips) Or core of tree), bark, corn cob, sugar cane bagasse, silver grass, and other wood fiber-containing waste or weeds.

In a specific embodiment of the present invention, the fresh agricultural waste is dried or dried at 60 ° C for 3 days, and the dried agricultural waste is first cut into segments, and then the high speed pulverizer (stainless steel grinder) The mixture is pulverized into a fan shape, sieved through a sieve, and then weighed and loaded into a culture vessel, and used as a solid substrate of the semi-solid medium, sterilized by high-pressure moist heat, and then dried for use.

In another embodiment of the invention, the surfactant inducer is Tween 20 or Tween 80. In still another embodiment of the present invention, the culture of the inoculated strain is placed in a thermostatic chamber and cultured for 13 to 30 days without illuminating.

In view of the various embodiments of the present invention, the various instruments, devices, methods, and related results described below are not intended to be limited to the scope of the present invention. In addition, certain theories presented and disclosed herein, but whether they are right or wrong, should be limited as long as the creation is implemented in accordance with the present invention without regard to any particular theory or action plan. It is within the scope of the invention.

The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

Example Example 1. Perenniporia tephropora TFRI 707 strain selection Sexual removal of lignin from agricultural waste Strain preservation

The obtained white rot fungus Perenniporia tephropora TFRI 707 Taiwan native strain (registration number BCRC 930104) was inoculated to potato dextrose agar (PDA) and cultured at 30 ° C for 4-5 days until the fungus hyphae grew over the whole medium. After the surface, it was sealed with a parafilm, placed in a refrigerator at 4 ° C, and re-transplanted to a new PDA medium every 30 days. Another preservation method is to take the above-mentioned mycelium-rich PDA flat medium, the hyphae at the outer edge of the medium, and take 3-4 pieces of white rot fungal hyphae into 2 mL with a hole punch with an inner diameter of 7 mm. In the storage tube, add 1.8 mL of sterilized water, seal and place at room temperature.

Preparation of semi-solid medium

In the present invention, the semi-solid medium for culturing Perenniporia tephropora TFRI 707 strain comprises: 60 mM buffer solution (pH 4.5-6.5), 0.1-0.4 mM CuSO ₄ ‧5H ₂ O solution, and a trace amount of metal ion solution (1 mL/ L), 0-0.5% (w/v) surfactant inducer, 10-30 g/L glucose, and 35-70 g/500 mL of agricultural waste and adjusted and maintained at pH 4.0 with 1 M HCl solution.

In the present example, the buffer solution may be selected from one of a sodium phosphate buffer solution, a potassium phosphate buffer solution, and a sodium tartrate buffer solution; and the surfactant inducer is Tween 20 or Tween 80.

First, it may be selected from rice straw, wood chips or core of tree, bark, corn cob sugar cane bagasse or miscanthus ( The fresh agricultural waste of silver grass is dried or dried at 60 ° C for 3 days. The dried agricultural waste is first cut into segments, and then pulverized into a fan shape by a stainless steel grinder. After being sieved through a sieve, it is weighed and loaded into a culture vessel, and then sterilized by high-pressure moist heat sterilization. Dry standby as a solid substrate for the semi-solid medium.

Method for culturing lignin in agricultural waste by using Perenniporia tephropora TFRI 707 strain

In this example, Perenniporia tephropora TFRI 707 strain was inoculated with semi-solid medium of different agricultural wastes, and the ratio of 11 7 mm hyphae was inoculated into each 35 mL medium, and placed in a thermostatic chamber, and the culture was not allowed to stand still for 13-30. Days, and the strain uses the hyphae or its secreted lignin decomposing enzyme to treat lignin in agricultural waste. The agricultural wastes used in this embodiment are rice straw, mulberry wood, mulberry bark and corn cob.

Semi- solid culture with Perenniporia tephropora TFRI 707 strain Measurement of enzyme activity in liquid Analysis method of activity of lacquer oxidase:

The semi-solid culture supernatant (or its dilution) is added to the enzyme reaction solution: 0.5 μmol of 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS) per mL (dissolved in 60 mM sodium acetate buffer, At pH 4.5), the mixture was uniformly mixed and the absorbance at 436 nm was measured at room temperature. The absorbance was converted to enzyme activity, ie, how many μmole of ABTS was oxidized in 1 minute at room temperature.

Endoglucanase activity assay method:

Add 0.20 mL of the above diluted reaction solution to 0.25 mL of DNS reagent (1.0 g of 3,5-dinitrosalicylic acid, 30.0 g of K-Na tartrate, add 20 mL of 1 N NaOH, add distilled water to 100 mL). After mixing at 100 ° C, the reaction was heated in a boiling water bath for 10 minutes, diluted with 4.5 mL of distilled water, and uniformly mixed, and the absorbance at 540 nm was measured (Miller, 1959). The measured absorbance is converted to a substantial amount of reducing sugar using a glucose standard curve. The glucose test method is to take 0.25 mL of glucose solution with concentration of 1.0, 0.8, 0.5, 0.2 and 0.1 mg/mL, and add 0.25 mL of DNS reagent to measure the absorbance under the above conditions to draw the standard amount. In addition, 0.25 mL of Day 0 enzyme solution was taken in the blank group, 0.25 mL of DNS was added, mixed, and treated under the same reaction conditions as above. The amount of reducing sugar measured by the reaction group minus the amount of blank reducing sugar, the difference obtained is the amount of reducing sugar released by the enzyme reaction. At 55 ° C, when the transforming enzyme hydrolyzes 1 μmol of sucrose per minute to produce 1 μmol of glucose and 1 μmol of fructose, the activity of the cellulase is defined as 1 U.

Method for analyzing the activity of β-glucosidase:

Take 1 ml of the semi-solid culture supernatant (or its dilution) in a sterile tube, add 100 μL of 5 mM p -nitrophenyl BD-glucose-glycoside, and place at 37 ° C for 15 minutes. . 50 μL of 1 M sodium carbonate (Na ₂ CO ₃ ) was added to stop the reaction, and its absorbance at a wavelength of 400 nm was detected by a spectrophotometer. The absorbance is converted to enzyme activity, ie how many μmole of p-nitrophenol is released in 1 minute at 37 °C.

Cellulose hydrolase activity analysis method:

The reaction solution contains 250 μL of 0.5 M 4-nitrophenyl-β-D-cellobioside ( p NPC) (Sigma) dissolved in 50 mM acetate buffer (pH 5.6) and 50 μL of extracellular fluid (or Diluent). After reacting at 45 ° C for 15 minutes, the reaction was stopped by adding 250 μL of 2 M Na ₂ CO ₃ solution, and the amount of p-nitrophenol released from pNPC was calculated by spectrophotometric measurement of its absorption at a wavelength of 405 nm. A U means that 1 μmole of p-nitrophenol is produced in 1 minute.

The results are shown in Fig. 1, which shows that the agricultural waste used in the present example was treated with Perenniporia tephropora TFRI 707 strain, and all of the lignin degrading enzyme oxidase activity was produced. That is, it indicates that Perenniporia tephropora TFRI 707 strain can decompose lignin in agricultural waste.

Figure 2 shows the lignin-degrading enzyme (laccase oxidase Laccase) and cellulolytic enzyme (ie, β-glucosidase, cellobiohydrolase, produced by white rot fungus (TFRI707) during agricultural waste cultivation. Activity of endoglucanase). In the present example, it was observed that when the mulberry bark was subjected to semi-solid culture with Perenniporia tephropora TFRI 707 strain, the activity of the cellulose enzyme in the culture supernatant was changed. As a result (see Fig. 2), only lignin-degrading enzyme (laccase oxidase Laccase) was found to exhibit enzyme activity during the culture, while the remaining cellulolytic enzymes β-glucosidase, cellobiohydrolase Only very weak or no enzyme activity is shown with endoglucanase. It indicates that the semi-solid culture of white rot fungus Perenniporia tephropora TFRI707 and agricultural waste only selectively decomposes lignin but does not decompose cellulose.

Measurement and comparison of lignin content after treatment and lignin content before treatment

The treated or untreated agricultural waste is first sterilized, dried at 60 ° C, pulverized, and then extracted with a Soxhlet apparatus using a mixture of ethanol and benzene in a ratio of 1:2 for 6 to 8 hours. After extracting with pure alcohol for 4 hours, the resin in the agricultural waste was completely extracted, and then the lignin content was detected by the Klasson method (Dence, 1992; Test Method T 222 om-06).

The Klasson method uses lignified wood without extracts as material. 1g of defatted wood powder extracted with alcohol benzene is used, and wood powder is gelatinized with 72% strong sulfuric acid at 10~15 °C for 2 hours to dissolve cellulose. With hemicellulose, add water to dilute to 3% sulfuric acid concentration, continue to boil for 4 hours or autoclave at high temperature, autoclave at high pressure for 30 minutes (121 ° C, 1.2 kg / cm ² ), after standing, to 30 Mol 1G3 (sibata) glass filter is filtered, and the residue in the filter is washed with hot water until the filtrate is not acidic. The filter cup is taken out and dried, and the acid-insoluble lignin is determined according to formula (1). The filtrate was measured for its absorbance at a wavelength of 208 nm by a spectrophotometer, and the acid soluble lignin was determined according to the formula (2). Acid soluble lignin plus insoluble lignin is the lignin content.

Total lignin content%=lignin content%÷(1-ol benzene extract %)

Formula 1):

Acid insoluble lignin% = (acid insoluble lignin weight / wood powder absolute dry weight) × 100%

Formula (2):

Acid soluble lignin%=(D×V×abs)/(a×w)×100%

Where D: dilution factor; V: filtrate volume (L); a: 113 Lg ^-1 (at 208 nm); w: absolute dry weight of wood flour

Table 1 shows the results of determination of the remaining lignin content in agricultural waste after two white rot fungi strains ( Ceriporiopsis subvermispora , CS and Perenniporia tephropora TFRI707, TFRI707) were cultured with six different agricultural wastes for 30 days. It can be seen from the table that the total lignin content of the agricultural waste (absolute dry weight 1 g) after treatment with the white rot fungus was significantly lower than that of the control group treated without the white rot fungus. Further, Perenniporia tephropora TFRI707 the present invention with another strain having selective decomposition of lignin by white rot true strain of Ceriporiopsis subvermispora comparison, used in the method of the present invention strains of benzene extraction and alcohol lignin decomposition have better effect.

Measurement of phenolic compound content

Filter the culture medium with sterile gauze, then centrifuge 1 mL of the culture solution, and add 0.1 mL of the upper layer solution to 0.2 mL of Folin-Ciocalteu reagent (refer to Singleton et al., 1999) and 2 mL. Ionized water and 1 mL of 15% Na ₂ CO ₃ were mixed and allowed to stand at room temperature for 2 hours. The absorbance was detected at a wavelength of 765 nm. The concentration of phenolic compounds was mg mL ^-1 gallic acid. Expressed as a unit. In addition, the detection of gallic acid (GAE) as a standard, according to the above steps to analyze, make a standard calibration curve. The selection of gallic acid is mainly due to the high purity and stability of the phenolic compound and is economical compared to other phenolic compounds.

It is shown in Fig. 3 that the white rot fungus Perenniporia tephropora TFRI 707 was cultured under different agricultural wastes on the 13th day, and the phenolic compound content of agricultural waste was lower than that when cultured without TFRI 707 co-culture. The content of phenolic compounds. It is indicated that the Perenniporia tephropora TFRI 707 strain is effective in removing phenolic compounds from agricultural waste, which is in line with expectations.

Example 2. Structural differences between wood fibers of microscopic examination and agricultural waste without white rot fungus

In this example, microscopic examination was carried out, and the agricultural waste at the 13th day of culture was implanted and not implanted with white rot fungus Perenniporia tephropora TFRI 707, and the change in the structure of the lignocellulosic structure was observed. In the present embodiment, the agricultural waste in the semi-solid medium is taken out, and the hyphae attached to the culture is picked out, washed with a sodium phosphate buffer solution, and observed under a dissecting microscope to magnify the agricultural waste under the field of 70 times. Structure and photographs.

The results are shown in Fig. 4, wherein (a) is rice straw, (b) is mulberry wood, (c) phylogenetic tree bark, (d) is bagasse, and (e) is corn cob. Microscopic examination revealed that the agricultural waste in the semi-solid medium without the Perenniporia tephropora TFRI 707 strain was observed, and the lignin structure with a thick structure and a dark brown color was observed. In the semi-solid medium in which Perenniporia tephropora TFRI 707 strain is implanted, the fibrous structure of the agricultural waste is exposed, and the appearance is whitened. It may be that the lignin structure in the plant has been produced by the fungus. Decomposed, so compared with the agricultural waste not implanted with the strain, it was observed that the lignin structure with thick structure and dark brown color was less obvious.

Example 3. Measuring the efficiency of decomposition of agricultural waste by cellulase

This example is to evaluate whether agricultural waste which has been partially lignin removed but retains whole cellulose is pretreated by white rot fungus, and is easily decomposed by cellulase. After the agricultural waste is not subjected to fungal treatment (control group) and solid-state culture treatment (experimental group) by white rot fungus, a part (about 0.1 g of cellulose) is taken out and transferred into a 20 ml glass column (with plastic) Cap), add 5.0 mL of 0.1 M sodium citrate buffer, pH 4.8, and 40 μL (400 μg) of tetracycline. Sterile distilled deionized water was then added to bring the final volume to 10 mL. The glass column was placed in a shaking incubator at 50 ° C. After the temperature reached 50 ° C, cellulase (about 15, 25 or 60 FPU / g cellulose, respectively) was added, and the reaction was cultured at 50 ° C. About 72~168 hours, then measure the glucose production.

The glucose test method is to take 0.25 mL of glucose solution with concentration of 1.0, 0.8, 0.5, 0.2 and 0.1 mg/mL, and add 0.25 mL of DNS reagent to measure the absorbance under the above conditions to draw the standard amount. In addition, 0.25 mL of Day 0 enzyme solution was taken in the blank group, 0.25 mL of DNS was added, mixed, and treated under the same reaction conditions as above. The amount of reducing sugar measured by the reaction group minus the amount of blank reducing sugar, the difference obtained is the amount of reducing sugar released by the enzyme reaction. At 55 ° C, when the transforming enzyme hydrolyzes 1 μmol of sucrose per minute to 1 μmol of glucose and 1 μmol of fructose, the activity of the cellulase is defined as 1 U.

The results are shown in Table 2 below. After treatment with white rot fungus, some lignin has been removed but the agricultural waste containing whole cellulose has been preserved. After decomposing with cellulase, the concentration of glucose produced is relative to the control group. Significant increase.

As a result of the above examples, the selective lignocellulose pretreatment method of the present invention can effectively decompose lignin in agricultural waste without decomposing cellulose. Therefore, the agricultural waste treated by the method of the present invention can not only effectively expose the cellulose, but also increase the effect of being decomposed by other fibrous enzymes to produce sugar, and can be applied to the remaining of the pretreatment of the present invention. Cellulose, used as a raw material for papermaking, alcohol or animal feed.

Other specific aspects

All of the features disclosed in this specification can be combined in any combination. Thus, the individual features disclosed in this specification can be replaced by alternative features that are the same, equivalent, or similar. Therefore, the various features disclosed are merely examples of a series of equivalents or similar features, unless otherwise clearly indicated.

From the foregoing description, those skilled in the art can readily determine the essential features of the invention, and various changes and modifications of the invention can be made to adapt to various different uses and conditions without departing from the scope thereof. Therefore, other specific aspects are included in the scope of patent application.

Figure 1 is a graph showing changes in oxidase activity in culture medium when white rot fungi are cultured with different agricultural wastes.

Fig. 2 is a graph showing changes in activity of cellulase and lignin degrading enzyme when white rot fungus is cultured in mulberry bark.

Figure 3 is a graph comparing the content of phenolic compounds in agricultural wastes implanted with and without white rot fungi.

Fig. 4 is a photograph showing the structural changes of fibers of different agricultural wastes before and after treatment with white rot fungi by dissecting microscope.

Claims (5)

A lignocellulose pretreatment method for selectively removing lignin by a white rot fungus, the method comprising: (1) inoculating a white rot fungus Perenniporia tephropora TFRI 707 strain in a semi-solid medium, wherein the medium contains (per liter): 35 ~70g agricultural and forestry waste, 60mL buffer solution (pH 4.5~6.5) 500mL, 7mL of 320μM CuSO ₄ ‧5H ₂ O, trace metal ion solution (0.1 g Na ₂ B ₄ O ₇ ‧10 H ₂ O per liter 0.01 g MnSO ₄ ‧7 H ₂ O, 0.05 g FeSO ₄ ‧7 H ₂ O, 0.07 g ZnSO ₄ ‧7 H ₂ O, 0.01 g MnSO ₄ ‧7 H ₂ O, 0.01 g (NH ₄ ) ₆ Mo ₇ O ₂₄ ‧4H ₂ O), 0.01~0.5% (w/v) surfactant inducer, with 10~30g/L glucose (or other sugars), adjusted and maintained at pH 4.0 with 1M HCl solution; (2) The culture is placed in a constant temperature chamber, and the culture is allowed to stand still for 13 to 30 days, and the obtained semi-solid culture selectively decomposes the lignin therein. According to the method of claim 1, wherein the agricultural waste is selected from a rice straw, a wood chip or a core of tree, a bark, and a corn cob ( At least one of corn cob), silver grass, and sugar cane bagasse. According to the method of claim 1, wherein the white rot fungus Perenniporia tephropora TFRI 707 produces an extremely high and stable lignin decomposing enzyme oxidase at 12-30 days, and has almost no cellulolytic activity. The semi-solid medium according to the first aspect of the patent application, wherein the buffer solution is one selected from the group consisting of a sodium phosphate buffer solution, a potassium phosphate buffer solution, and a sodium tartrate buffer solution. A semi-solid medium according to the first aspect of the patent application, wherein the surfactant inducer is Tween 20 or Tween 80.