CN111676257A - A method for improving the efficiency of hardwood cellulose high-concentration enzymatic hydrolysis to produce fermentable sugar - Google Patents

Abstract

The invention relates to a method for improving the efficiency of cellulose high-concentration enzymatic hydrolysis of fermentable sugars in broad-leaved fast-growing wood. High-concentration hydrolysis of the solid substrate after washing: a certain concentration is dispersed in the acetic acid-sodium acetate buffer solution, and then the composite surfactant and cellulase composed of sodium lignosulfonate or sodium lignosulfonate and other surfactants are added to carry out hydrolysis. Reducing sugars and lignocellulose residues are obtained. The invention combines metal chloride pretreatment with high-concentration milling to treat lignocellulosic raw materials, uses pulp and papermaking waste-sodium lignosulfonate as surfactant and is compounded with other types of surfactants, and introduces it into cellulase high-concentration In the hydrolysis system, the yield of fermentable sugars by enzymatic hydrolysis is improved. The high-efficiency and high-concentration enzymatic hydrolysis of lignocellulose is realized, and the production cost of bioethanol can be greatly saved.

Description

A method for improving the efficiency of hardwood cellulose high-concentration enzymatic hydrolysis to produce fermentable sugar

technical field

The invention relates to the technical field of hardwood resource conversion and utilization, in particular to a comprehensive treatment method that can effectively improve the efficiency of hydrolysis of cellulose high-concentration enzymes in hardwood fast-growing wood to produce fermentable sugars.

Background technique

Lack of resources, energy shortage and environmental degradation have become the three major bottlenecks restricting the sustainable development of human society, which will seriously affect the rapid and sustained growth of the national economy. Using resource-rich lignocellulose as raw material to realize the full-component utilization of biomass resources, especially the technology of producing fuel ethanol by microbial fermentation, is increasingly attracting attention. Lignocellulose is one of the main components of plant cell walls. The total amount of lignocellulose produced by photosynthesis on the earth can reach 100 billion tons every year. For example, the total amount of lignocellulose that can be collected every year in the United States can reach 1 billion tons. Up to 800 million tons, but only a small part is effectively used by humans. During the long-term evolution, lignocellulose has evolved a more complex biological structure to resist the threat of microorganisms and animals, which is called the "resistance" of lignocellulose. This "resistance" of lignocellulose will affect the penetration and mass transfer of enzyme reagents or chemical reagents into lignocellulosic raw materials, as well as the accessibility and activity of cellulase. Therefore, pretreatment is a key technology for the production of fuel ethanol from lignocellulosic feedstocks.

The pretreatment of lignocellulosic raw materials refers to breaking the encapsulation of cellulose by hemicellulose and lignin through physical, mechanical, chemical and microbial means, improving the dense structure of cellulose, releasing more free hydroxyl groups, making it easier to interact with microorganisms and fibers. A direct contact reaction occurs with the enzyme, thereby overcoming the natural "resistance" of lignocellulosic raw materials and improving the conversion efficiency of biomass enzymatic hydrolysis. Metal chlorides can selectively remove hemicellulose from lignocellulose cell walls, and at the same time, the lignin linked to hemicellulose will be partially dissolved, thereby exposing cellulose, which is conducive to the direct contact between cellulase and cellulose . At the same time, studies have shown that some metal salts can increase the activity of cellulase, thereby improving the hydrolysis efficiency of cellulase.

In addition to the price of cellulase and the natural recalcitrance of lignocellulose, the high cost of ethanol distillation (about 30% of the entire ethanol production investment) is also one of the key factors restricting the commercialization of bioethanol. The main reason for the high cost of ethanol distillation is that the concentration of ethanol in the fermentation broth is too low. Therefore, if the enzyme hydrolysis system with high substrate concentration can be effectively used, the problem of excessive energy consumption in subsequent ethanol distillation can be fundamentally solved. Studies have shown that when the substrate concentration is higher than 10%, the cellulosic raw materials cannot be effectively converted, and the upper limit of one feeding is 12%-15%. The possible reasons are: as the substrate content increases, the final product and inhibitor increase accordingly, the viscosity of the slurry increases, and the substrate cannot be effectively stirred, resulting in the inability of the enzyme to fully contact it, the inhibitory effect of high concentration is gradually enhanced, and the cellulose is converted. rate decreased accordingly.

SUMMARY OF THE INVENTION

In order to relieve the natural recalcitrance of lignocellulose to cellulase, the present invention utilizes metal chlorides to pretreat lignocellulosic raw materials to selectively degrade hemicellulose and part of lignin therein, thereby exposing cellulose from the dense structure come out. By adding surfactant to the enzymatic hydrolysis system, the rheological property of the hydrolyzed substrate is improved, so as to achieve the purpose of improving the enzymatic hydrolysis efficiency.

The method for improving lignocellulose high-solid enzymatic hydrolysis of the present invention is characterized in that, comprising the following steps:

(1) choose 2-3 year old eucalyptus wood after peeling and slicing, water immersion, single screw extrusion dehydration and air drying, as raw material to carry out subsequent pretreatment and enzymatic hydrolysis;

(2) metal chloride pretreatment, its technical conditions are: put the eucalyptus wood raw material obtained in step (1) into a closed small steel tank with high temperature and high pressure resistance, then add metal chloride pretreatment solution to the tank, cover tightly cover and place the jar in the air bath cooker. The pretreatment temperature is 170-190°C, the temperature control error is ±2°C, and the pretreatment time is 20min. The solution required for the above pretreatment is the currently prepared solution, and the concentration is 0.1-0.3mol/L. After the pretreatment is completed, stop heating immediately, remove the small tank, cool it to room temperature with running water, then place the solid and liquid in the small tank in a nylon mesh bag, and use a spin dryer to separate the solid and liquid to obtain the pretreatment liquid and solid substrate;

(3) Fine fibrillation, the technical conditions are: the solid matrix obtained in step (2) is washed with deionized water to neutrality, and the concentration is adjusted to about 20% with deionized water, and then the solid matrix is treated with a high-consistency refiner. The matrix is further fibrillated to obtain powder with a mesh number of about 60-80 mesh, which is air-dried and used for later use;

(4) high-concentration enzymatic hydrolysis of solid substrate, its technical condition is: the solid substrate obtained in step (3) is mixed with a certain amount of pH value of 4.8 (±0.1) acetic acid/sodium acetate buffer solution, and the concentration of the substrate is 10 % (% is the ratio of mass to volume, g:ml), the dosages of cellulase and cellobiase are 15-20FPU/g and 22.5-30CBU/g absolute dry biomass respectively. And add a drop of ethyl acetate to prevent the generation of microorganisms and miscellaneous bacteria during the hydrolysis process. Then an appropriate amount of suitable surface aptamer is added to the enzymatic hydrolysis system to promote the enzymatic hydrolysis. The above samples were placed in a constant temperature incubation shaker and reacted at 50±2°C for 48-72 hours, during which the rotational speed of the shaker was maintained at 150-200 r/min. After the reaction, it was heated in a water bath at 90° C. for 10 min to inactivate the cellulase, and the enzymatic hydrolyzed solution was separated by a vacuum filter pump, and finally the enzymatic hydrolyzed solution and solid residue were obtained. The glucose content in the pretreatment solution was then determined by HPLC.

Preferably, the metal chloride salt in step (2) is one of MgCl ₂ , FeCl ₂ , FeCl ₃ , KCl and NaCl.

Preferably, the surfactant added in step (4) is sodium lignosulfonate, and its combination with Tween 40, Tween 80, cetyltrimethylammonium bromide, polyethylene glycol (2000, 4000 , 6000, 8000) in a compound.

Beneficial effects:

1. The present invention applies the residue-sodium lignosulfonate in the pulp and paper enterprise as an auxiliary agent in the cellulase hydrolysis system, which plays a role in improving the efficiency of the pulp and paper mill, and simultaneously plays a role in improving the output of fuel ethanol. Purpose.

2. The present invention adds a small amount of sodium lignosulfonate and its compounding auxiliaries to the cellulase high-concentration hydrolysis system, which can significantly improve the enzymatic hydrolysis efficiency of cellulose, especially when the substrate is not added. The higher the concentration, the more obvious the efficiency improvement.

Description of drawings

Fig. 1 is a process flow diagram of the present invention.

Detailed ways:

The following examples are to further illustrate the present invention, but not to limit the present invention.

Example 1

(1) choose 2-3 year old eucalyptus wood after peeling and slicing, water immersion, single screw extrusion dehydration and air drying, as raw material to carry out subsequent pretreatment and enzymatic hydrolysis;

(2) metal chloride pretreatment, its specific technical conditions are: the raw material obtained in step (1) is put into a closed high temperature and high pressure small steel tank, then MgCl is added to the tank _The pretreatment solution is closed tightly, and the small steel tank is closed. Tanks are placed in an air bath cooker. The pretreatment temperature was 170°C, the temperature control error was ±2°C, the pretreatment time was 20min, the pretreatment solution concentration was 0.2mol/L, and the solid-liquid ratio was 1:6. After the pretreatment is completed, stop heating immediately, remove the small tank, cool it to room temperature with running water, then place the solid and liquid in the small tank in a nylon mesh bag, and use a spin dryer to separate the solid and liquid to obtain the pretreatment liquid and solid substrate;

(3) Fine fibrillation, the specific technology is as follows: the solid substrate obtained in step (2) is washed to neutrality, and the concentration is adjusted to about 20% with deionized water, and then the solid substrate is treated with a KRK high-consistency disc refiner. Further fibrillation was carried out to obtain powder with a mesh number of about 80, which was air-dried for later use.

(4) Enzymatic hydrolysis of solid substrate, its specific technology is: mixing the solid substrate obtained in step (3) with a certain amount of 0.1mol/L pH value of 4.8 (±0.1) acetic acid/sodium acetate buffer solution, to the concentration of the substrate is 10% (% is the ratio of mass to volume, g:ml), and the dosages of cellulase and cellobiase are 15FPU/g and 22.5CBU/g absolute dry biomass respectively. And add a drop of ethyl acetate to prevent the generation of microorganisms and miscellaneous bacteria during the hydrolysis process. The above samples were placed in a constant temperature incubation shaker and reacted at 50° C. for 48 hours, during which the rotational speed of the shaker was maintained at 150 r/min. After the reaction, it was heated in a water bath at 90° C. for 10 min to inactivate the cellulase, and the enzymatic hydrolyzed solution was separated by a vacuum filtration pump, and finally the enzymatic hydrolyzed solution and solid residue were obtained. The glucose content in the pretreatment solution was then determined by HPLC. The final yield of enzymatic hydrolysis of glucose was 81.22%.

Example 2

(1) choose 2-3 year old eucalyptus wood after peeling and slicing, water immersion, single screw extrusion dehydration and air drying, as raw material to carry out subsequent pretreatment and enzymatic hydrolysis;

(2) metal chloride pretreatment, its specific technical conditions are: the raw material obtained in step (1) is put into a closed high temperature and high pressure small steel tank, then MgCl is added to the tank _The pretreatment solution is closed tightly, and the small steel tank is closed. Tanks are placed in an air bath cooker. The pretreatment temperature was 170°C, the temperature control error was ±2°C, the pretreatment time was 20min, the pretreatment solution concentration was 0.2mol/L, and the solid-liquid ratio was 1:6. After the pretreatment is completed, stop heating immediately, remove the small tank, cool it to room temperature with running water, then place the solid and liquid in the small tank in a nylon mesh bag, and use a spin dryer to separate the solid and liquid to obtain the pretreatment liquid and solid substrate;

(3) Fine fiberization, the specific technology is: washing the solid matrix obtained in step (2) to neutrality, and adjusting the concentration with deionized water to be about 20%, and then using a KRK high-consistency disc refiner to carry out the solid matrix. Further fibrillation is carried out to obtain a powder with a mesh number of about 80, which is air-dried and used for later use.

(4) Enzymatic hydrolysis of solid substrate, its specific technology is: mixing the solid substrate obtained in step (3) with a certain amount of 0.1mol/L pH value of 4.8 (±0.1) acetic acid/sodium acetate buffer solution, to the concentration of the substrate is 10% (% is the ratio of mass to volume, g:ml), the dosages of cellulase and cellobiase are 15FPU/g and 22.5CBU/g absolute dry biomass respectively. And add a drop of ethyl acetate to prevent the generation of microorganisms and miscellaneous bacteria during the hydrolysis process. Then, 0.1% -0.5% (relative to the substrate mass) of sodium lignosulfonate is added to the enzymatic hydrolysis system to promote the enzymatic hydrolysis. The above samples were placed in a constant temperature incubation shaker and reacted at 50°C for 48h, during which the rotational speed of the shaker was maintained at 150r/min. After the reaction, it was heated in a 90°C water bath for 10 min to inactivate the cellulase. The enzymatic hydrolysis solution is separated by a vacuum filtration pump, and finally the enzymatic hydrolysis solution and solid residue are obtained. The glucose content in the pretreatment solution was then determined by HPLC. The final enzymatic hydrolysis of glucose yield is shown in Table 1. It shows that the addition of sodium lignosulfonate can promote the enzymatic hydrolysis of cellulose.

Table 1 Effects of different dosages of sodium lignosulfonate on cellulase hydrolysis

Example 3

(1) choose 2-3 year old eucalyptus wood after peeling and slicing, water immersion, single screw extrusion dehydration and air drying, as raw material to carry out subsequent pretreatment and enzymatic hydrolysis;

(2) metal chloride pretreatment, its specific technical conditions are: the raw material obtained in step (1) is put into a closed high temperature and high pressure small steel tank, then MgCl is added to the tank _The pretreatment solution is closed tightly, and the small steel tank is closed. Tanks are placed in an air bath cooker. The pretreatment temperature was 170°C, the temperature control error was ±2°C, the pretreatment time was 20min, the pretreatment solution concentration was 0.2mol/L, and the solid-liquid ratio was 1:6. After the pretreatment is completed, stop heating immediately, remove the small tank, cool it to room temperature with running water, then place the solid and liquid in the small tank in a nylon mesh bag, and use a spin dryer to separate the solid and liquid to obtain the pretreatment liquid and solid substrate;

(3) Fine fibrillation, the specific technology is: washing the solid substrate obtained in step (2) with deionized water to neutrality, and adjusting the concentration to about 20% with deionized water, and then using a KRK high-consistency disc refiner. The solid matrix is further fibrillated to obtain a powder with a mesh number of about 80, which is air-dried for later use.

(4) Enzymatic hydrolysis of solid substrate, its specific technology is: mixing the solid substrate obtained in step (3) with a certain amount of 0.1mol/L pH value of 4.8 (±0.1) acetic acid/sodium acetate buffer solution, to the concentration of the substrate is 5-20% (% is the ratio of mass to volume, g:ml), the dosage of cellulase and cellobiase is 15FPU/g and 22.5CBU/g absolute dry biomass respectively. And add a drop of ethyl acetate to prevent the generation of microorganisms and miscellaneous bacteria during the hydrolysis process. In the enzymatic hydrolysis system, 0.4% (relative to the mass of the substrate) sodium lignosulfonate was added. The above samples were placed in a constant temperature incubation shaker and reacted at 50°C for 48h, during which the rotational speed of the shaker was maintained at 150r/min. After the reaction, it was heated in a water bath at 90° C. for 10 min to inactivate the cellulase, and the enzymatic hydrolyzed solution was separated by a vacuum filter pump, and finally the enzymatic hydrolyzed solution and solid residue were obtained. The glucose content in the pretreatment solution was then determined by HPLC. Table 2 lists glucose yields at substrate concentrations of 5-20%. The higher the substrate concentration, the more obvious the promoting effect of sodium lignosulfonate on cellulase hydrolysis.

Table 2 Influence of the addition of sodium lignosulfonate on substrate concentration cellulase hydrolysis

Example 4

(1) choose 2-3 year old eucalyptus wood after peeling and slicing, water immersion, single screw extrusion dehydration and air drying, as raw material to carry out subsequent pretreatment and enzymatic hydrolysis;

(2) metal chloride pretreatment, its specific technical conditions are: the raw material obtained in step (1) is put into a closed high temperature and high pressure small steel tank, then MgCl is added to the tank _The pretreatment solution is closed tightly, and the small steel tank is closed. Tanks are placed in an air bath cooker. The pretreatment temperature was 170°C, the temperature control error was ±2°C, the pretreatment time was 20min, the pretreatment solution concentration was 0.2mol/L, and the solid-liquid ratio was 1:6. After the pretreatment is completed, stop heating immediately, remove the small tank, cool it to room temperature with running water, then place the solid and liquid in the small tank in a nylon mesh bag, and use a spin dryer to separate the solid and liquid to obtain the pretreatment liquid and solid substrate;

(3) Fine fiberization, the specific technology is: washing the solid matrix obtained in step (2) to neutrality, and adjusting the concentration with deionized water to be about 20%, and then using a KRK high-consistency disc refiner to carry out the solid matrix. Further fibrillation is carried out to obtain a powder with a mesh number of about 80, which is air-dried and used for later use.

(4) Enzymatic hydrolysis of solid substrate, its specific technology is: mixing the solid substrate obtained in step (3) with a certain amount of 0.1mol/L pH value of 4.8 (±0.1) acetic acid/sodium acetate buffer solution, to the concentration of the substrate is 10% (% is the ratio of mass to volume, g:ml), and the dosages of cellulase and cellobiase are 15FPU/g and 22.5CBU/g absolute dry biomass respectively. And add a drop of ethyl acetate to prevent the generation of microorganisms and miscellaneous bacteria during the hydrolysis process. Then add 0.4% (relative to the mass of the substrate) sodium lignosulfonate to the enzymatic hydrolysis system, and add cationic surfactant-hexadecyl in a certain mass ratio (relative to the mass of the sodium lignosulfonate) Trimethylammonium bromide to facilitate enzymatic hydrolysis. The above samples were placed in a constant temperature incubation shaker and reacted at 50°C for 48h, during which the rotational speed of the shaker was maintained at 150r/min. After the reaction, it was heated in a water bath at 90° C. for 10 min to inactivate the cellulase, and the enzymatic hydrolyzed solution was separated by a vacuum filter pump, and finally the enzymatic hydrolyzed solution and solid residue were obtained. The glucose content in the pretreatment solution was then determined by HPLC. The final enzymatic hydrolysis of glucose yield is shown in Table 3. Compared with the simple use of sodium lignosulfonate, the addition of the cationic surfactant cetyltrimethylammonium bromide can significantly improve the conversion efficiency of the enzymatic hydrolysis reaction system.

Table 3 Effect of sodium lignosulfonate and cationic surfactant on cellulase hydrolysis

Example 5

(1) choose 2-3 year old eucalyptus wood after peeling and slicing, water immersion, single screw extrusion dehydration and air drying, as raw material to carry out subsequent pretreatment and enzymatic hydrolysis;

(2) metal chloride pretreatment, its specific technical conditions are: the raw material obtained in step (1) is put into a closed high temperature and high pressure small steel tank, then MgCl is added to the tank _The pretreatment solution is closed tightly, and the small steel tank is closed. Tanks are placed in an air bath cooker. The pretreatment temperature was 170°C, the temperature control error was ±2°C, the pretreatment time was 20min, the pretreatment solution concentration was 0.2mol/L, and the solid-liquid ratio was 1:6. After the pretreatment is completed, stop heating immediately, remove the small tank, cool it to room temperature with running water, then place the solid and liquid in the small tank in a nylon mesh bag, and use a spin dryer to separate the solid and liquid to obtain the pretreatment liquid and solid substrate;

(3) Fine fibrillation, the specific technology is as follows: the solid substrate obtained in step (2) is washed to neutrality, and the concentration is adjusted to about 20% with deionized water, and then the solid substrate is subjected to a KRK high-consistency disc refiner. Further fibrillation is carried out to obtain powder with a mesh number of about 80, which is air-dried and used for later use.

(4) Enzymatic hydrolysis of solid substrate, the specific technique is: mixing the solid substrate obtained in step (3) with a certain amount of 0.1mol/L pH value of 4.8 (±0.1) acetic acid/sodium acetate buffer solution in step (3), When the concentration of the substrate is 10% (% is the ratio of mass to volume, g:ml), the dosage of cellulase and cellobiase is 15-20FPU/g and 22.5-30CBU/g absolute dry biomass, respectively. And add a drop of ethyl acetate to prevent the generation of microorganisms and miscellaneous bacteria during the hydrolysis process. Then, 0.4% (relative to the mass of the substrate) sodium lignosulfonate was added to the enzymatic hydrolysis system , and a nonionic surfactant-Tween 80 was added in a certain mass ratio (relative to the mass of the sodium lignosulfonate) Compounding is carried out to facilitate enzymatic hydrolysis. The above samples were placed in a constant temperature incubation shaker and reacted at 50°C for 48h, during which the rotational speed of the shaker was maintained at 150r/min. After the reaction, it was heated in a water bath at 90° C. for 10 min to inactivate the cellulase, and the enzymatic hydrolyzed solution was separated by a vacuum filter pump, and finally the enzymatic hydrolyzed solution and solid residue were obtained. The glucose content in the pretreatment solution was then determined by HPLC. The final enzymatic hydrolysis of glucose yield is shown in Table 4. The compounding of Tween and sodium lignosulfonate in a mass ratio of 1:1 can convert almost all the cellulose in the solid matrix into glucose, thereby improving the utilization rate of cellulose.

Table 4 Sodium lignosulfonate and Tween 80 compound improve enzymatic hydrolysis efficiency

Claims (4)

1. A method for improving the efficiency of producing fermentable sugar by high-concentration enzymatic hydrolysis of broad-leaved wood cellulose comprises the following specific steps:

(1) peeling and slicing 2-3 years old eucalyptus, soaking in water, performing single-screw extrusion dehydration, and then air-drying to obtain a raw material for subsequent pretreatment and enzymatic hydrolysis;

(2) the metal chloride salt is used for assisting the pretreatment of high-temperature water, and the technical conditions are as follows: and (2) putting the eucalyptus raw material prepared in the step (1) into a small closed high-temperature-resistant high-pressure-resistant steel tank, adding the pretreatment solution into the tank, tightly covering the tank, and putting the small tank into an air bath cooking pot. The pretreatment temperature is 170-190 ℃, the temperature control error is +/-2 ℃, the pretreatment time is 10-30min, the solution required by the pretreatment is the prepared metal chloride solution, and the concentration is 0.1-0.3 mol/L. After the pretreatment is finished, immediately stopping heating, removing the small tank, cooling to room temperature by using flowing water, then placing solid and liquid in the small tank into a nylon mesh bag, and separating the solid and the liquid by using a spin dryer to obtain a pretreatment liquid and a solid matrix;

(3) the dispersion and the fibrillation treatment of the solid matrix have the technical conditions that: washing the solid matrix obtained in the step (2) with deionized water to be neutral, adjusting the concentration to be about 15-20% by using deionized water, further performing fibrillation on the solid matrix to obtain powder with the mesh number of about 60-80, and air-drying the powder for later use.

(4) The high-solid enzyme hydrolysis of the solid matrix has the technical conditions that: adding a certain amount of acetic acid-sodium acetate buffer solution with the pH of 4.8 (+ -0.1) into the solid matrix obtained in the step (3) serving as a substrate, and then addingA certain amount of mixed liquid of cellulase and cellobiase is added with a drop of ethyl acetate to prevent the generation of microorganisms and mixed bacteria in the hydrolysis process. And finally, diluting the enzymolysis substrate to a certain concentration by using an acetic acid-sodium acetate buffer solution. Adding appropriate additives to the enzymatic hydrolysis systemSurface active agentTo facilitate the enzymatic hydrolysis. The sample is placed in a constant temperature culture oscillator and reacts for 48-72h under the condition of 50 (+ -2) DEG C, and the rotating speed of the oscillator is kept at 150-. After the reaction is finished, heating the mixture in a water bath at 90-95 ℃ for 5-10min, inactivating the cellulase, and separating the enzymolysis liquid by using a vacuum filter pump to obtain the enzymolysis liquid and solid residues. Glass beads are added into the reaction system during high-concentration enzyme hydrolysis to promote the reaction to be fully carried out.

2. The method for improving the efficiency of high-concentration enzymatic hydrolysis of hardwood cellulose for production of fermentable sugars as claimed in claim 1 wherein: the metal chloride salt for assisting the pretreatment of the high-temperature water in the step (2) comprises: ferric chloride, ferrous chloride, magnesium chloride, sodium chloride, potassium chloride.

3. The method for improving the efficiency of high-concentration enzymatic hydrolysis of hardwood cellulose for production of fermentable sugars as claimed in claim 1 wherein: and (3) refining cellulose by adopting one or combination of a KRK high-concentration disc refiner and a double-helix press.

4. The method for improving the efficiency of high-concentration enzymatic hydrolysis of hardwood cellulose for production of fermentable sugars as claimed in claim 1 wherein: the surfactant in the step (4) is one or more of Tween 40, Tween 80, sodium lignosulphonate, cetyl trimethyl ammonium bromide and polyethylene glycol (2000, 4000, 6000 and 8000).

Images