FI66429B - FOERFARANDE FOER PRODUKTION AV GLUKOS FRAON CELLULOSA-MATERIALSOM INNEHAOLLER HEMICELLULOSA OCH LIGNIN - Google Patents

Description

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M MuTLAOON.NGSSItlllFT 6 6429 ^ (51) K * Jlu / l * .a.3 C 13 K 1/02, C 12 P 19/02 FINLAND — FINLAND 790790 (niHdMkHM-./MMv*! 07.03.79 (23 ) AHcafiM — GHtlglMadag 07-03-79 (41) TafctfidwlMl - MMt offwdlt 09.09.79 ytM-μ (44) MW ...... ,, 1 ,, 29.06.84 htwt- och mhnwtywlm 'AmMm «l * d odk MUkrMMi p »Mearad 7 (32) (33) (31) * rr *« * r «**» * B «t * <prior ** 08.03.78 USA (US) 884 ^ 79 Proven-Styrkt (71) Purdue Research Foundation, Graduate House East, West Lafayette,

Indiana, USA (72) George T. Tsao, West Lafayette, Indiana,

Michael R. Ladisch, West Lafayette, Indiana,

Christine M. Ladisch, West Lafayette, Indiana,

Teh-an Hsu, West Lafayette, Indiana, USA (US) (7 ^) Oy Kolster Ab (5 ^) Method for the production of glucose from cellulosic materials containing hemicellulose and lignin - For the production of glucose from cellulosic material, in which case hemlcel-lulosa och 1ign in

The invention relates to a process for the production of glucose from cellulosic materials containing hemicellulose and lignin, wherein the hemicellulose is removed from the cellulosic materials; the cellulose contained in the cellulosic materials is dissolved at room temperature in a solvent which is a mixture of ethylenediamine, cadmium oxide and water.

The utilization of cellulosic waste materials such as corn stalks, sawdust, straw, bagasse, etc. has recently received a lot of interest, especially with regard to the utilization of such waste materials for the development of backup sources of fuels, food, chemicals and other useful products.

The cellulosic ingredients contain three main components, which are cellulose, hemicellulose and lignin. Methods for extracting hemicellulose have been proposed and / or used in the past, and such extracted hemicellulose can be utilized in many known methods, such as hydrolysis, fermentation, pyrolysis, etc.

Lignin has also been isolated from cellulosic materials in the past. Lignin has been found to have a higher hydrogen and carbon content and a lower oxygen content than cellulose and hemicellulose, and has the highest calorific value of the three. The isolated lignin can be burned directly to generate steam and electricity and can also be used in the preparation of a number of useful substances such as vanillin, dimethyl sulfoxide, dimethyl sulfide, methyl mercaptan and catechol.

However, the recovery and / or utilization of cellulose, for example by hydrolysis to produce glucose, has hitherto been a problem, mainly due to the crystalline structure of the cellulose molecules and the lignin barrier therein.

In addition, attempts have been made in the past to hydrolyze cellulose using acids or enzymes. However, such attempts have not been entirely successful; at the very least, no economically attractive method has been obtained which would be useful for providing a satisfactorily high yield of glucose from the cellulose in such cellulosic materials.

The method of this invention for recovering glucose from cellulosic materials is characterized in that the dissolved cellulose is reprecipitated and the reprecipitated cellulose is hydrolyzed with a cellulase enzyme to obtain glucose at a pH of about 5.

As discussed above, cellulose molecules form highly organized crystal structures. In addition, in cellulosic materials, lignin in the middle lamella settles as a physical barrier surrounding the cellulosic fibers in such materials.

The process of this invention uses selective solvent extraction to fractionate hemicellulose, cellulose and lignin (the three main components of cellulosic materials). This fractionation is necessary because it has been found that after being dissolved in solution in the cellulose, it is no longer protected by the crystal structure or lignin barrier and the cellulose can then be easily precipitated. The recovered cellulose is hydrolyzed to give a high yield of Glu.

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After the cellulosic materials have been physically reduced to a size of up to 40 mesh, if necessary or desired, they are first extracted with dilute acid or alkali to remove hemicellulose. After removal of the hemicellulose, the residue contains cellulose and lignin.

The residue obtained is dissolved in a suitable solvent. Numerous solvents capable of dissolving cellulose have been reported in the literature. After several such studies, the solvent known as cadoxene was found to give the best results. The cadoxene solvent was first proposed by Jayme and Neuschaffer (see Jayme, G. and Neuschaffer, K., 1957, Die Naturwissenschafter, 44 (3): 62-63, and Jayme, G. and Neuschaffer, K., 1957, The Macromolecular Chemistry, 28: 71-83). Cadoxene is made from 25-30% ethylenediamine and 70-75% water as well as about 4.5-7% cadmium (as added oxide or hydroxide). Cadoxin has good dissolution properties, is a clear, colorless, almost odorless liquid, is stable for almost indefinite time, causes only slight shortening of cellulosic fibers, and is relatively easy to prepare.

The fact that water is one part of the cadoxene combination is very important. Most cellulosic waste contains moisture. Cadoxene does not require pre-drying of the waste when dissolving the cellulose, thus avoiding an otherwise expensive work step.

About 10 g of cellulose was dissolved in 100 g of cadoxene as an example to demonstrate its effect. Insoluble lignin can be separated by filtration or centrifugation.

The cellulose-cadoxene solution is physically stable, but this stability apparently requires a closed balance between cellulose, water, ethylenediamine, and cadmium. The exact nature of this equilibrium is not known, but if, for example, excess water is added, the cellulose precipitates back out of solution. Cadoxin has a high pH, and after the addition of acid, the cellulose also precipitates back from solution. Recrystallized cellulose, as well as other linear polymers, can self-recrystallize. The reprecipitated cellulose can then be easily hydrolyzed to give a high glucose yield by the cellulase enzyme. For example, by adding an acid to a cellulose-cadoxene solution to lower its pH to about five (after which the cellulose precipitates back) 4 66429 and then adding the Tricoderma viride cellulase solution, the precipitated cellulose is hydrolyzed to glucose. The ethylenediamine and cadmium salt in solution do not appear to inactivate the enzyme.

To improve the economics of the method, it is necessary to recover and reuse cadoxene.

By various recovery methods, the process can be further divided into several alternatives, which are described as follows: (a) Water is added to precipitate cellulose from the cellulose-cadoxene solution. The cellulose is separated by filtration or centrifugation. The cellulose is then hydrolyzed to glucose by enzymes. The liquid can be added with CCl 4 to precipitate cadmium carbonate, which in turn can be converted to cadmium oxide for reuse. The amine remaining in the aqueous solution can be recovered by standard methods such as distillation or extraction.

(b) CC> 2 is added directly to the cellulose-cadoxene solution, after which both cellulose and cadmium carbonate precipitate. After separation from the liquid containing ethylenediamine, which is recovered as in (a), a mixture of the two solid precipitates can be treated with cellulases to hydrolyze and dissolve the cellulose to give glucose. The remaining cadmium carbonate precipitate can be converted to cadmium oxide for reuse.

(c) A water-immiscible solvent is added to extract the ethylenediarine for recovery, after which the cellulose precipitates. Thus, the mixture can be divided in one step into three fractions: an amine in the organic layer, cadmium in the aqueous layer and cellulose as a precipitate. These three verses are dealt with separately.

(d) The reprecipitated cellulose is hydrolyzed to glucose by an enzymatic process. The cellulase enzyme (most likely en-do-1,4-beta-glucanase, but the mechanisms of cellulase are still highly controversial) can "dissolve" cellulose (by forming cellodextrins). To complete hydrolysis to glucose, either free or immobilized cellulases can be used.

(e) Cellulose reprecipitated from cadoxine has been found to spontaneously form small spheres if allowed to stand in solution for 5 days. Such beads can serve as a support when the enzyme is immobilized.

After the cellulose has been hydrolyzed to glucose, the remaining lignin in solid form can be separated from the resulting glucose solution by filtration or centrifugation.

The following steps are used for this method: 1. The cellulosic materials are treated to remove hemicellulose; 2. The cellulosic ingredients are dissolved in a solvent; 3. Add water, acid, or other liquid, such as methanol, to reprecipitate the cellulose: 4. Hydrolyze with an enzyme to give glucose, leaving only water-insoluble lignin; and 5. Separate the lignin from the glucose syrup by filtration.

The following examples of the method are given:

Example 1 Pretreatment and hydrolysis of oi-cellulose (a) Preparation of solvent (cadoxene) 25-30% ethylenediamine in water was saturated by adding an excess of cadmium oxide previously dried in an oven. Cadmium oxide, which did not dissolve, formed a hydroxide which appeared as a white precipitate. The precipitate was filtered or centrifuged off. The addition of cadmium oxide and the separation of hydroxide were performed twice to ensure that a solution saturated with cadmium oxide was obtained. In this way, an aqueous solution of 25-30% ethylenediamine and 4.5-7% cadmium was obtained. Based on its cadmium and ethylenediamine content, this solvent is generally known as cadoxene.

(b) Dissolution of ol-cellulose (1) Crystalline α-cellulose (Avicell), 0.1 g, was added to 10 ml of the solvent prepared according to (a) and mixed. The cellulose was completely dissolved in 10 minutes under room conditions.

(2) Dissolution tests were repeated with 0.1 g, 0.2 g, 0.3 g, 0.4 g and 0.5 g portions of crystalline α-cellulose (Avicell), each in 10 ml of solvent, by a method in which the cellulose was mixed with the solvent at room temperature and then cooled by placing 6 66429 on ice. The times required for complete dissolution are summarized in Table 1.

Table 1% in Avicell solvent Dissolution time 1 2 minutes 2 2 minutes 3 7 minutes 4 9 minutes 5 15 minutes (3) The dissolution test performed as described in (2) above was repeated. In this case, however, amorphous cellulose was dissolved instead of crystalline cellulose. Using the same amounts of cellulose and the same methods as in (3), the dissolution times summarized in Table 2 were observed.

Table 2% cellulose in solvent Dissolution time 1 2 minutes 2 2 minutes 3 7 minutes 4 9 minutes 5 15 minutes (4) Solutions containing larger amounts of cellulose were made by varying the composition of the solvent and changing the dissolution procedure. Sodium hydroxide was added to the solvent prepared in (a) to give a solution of 0.5 M relative to sodium hydroxide. To 1 ml of the modified solvent was added 300 mg of Avicell, followed by excess CdO. After mixing the solution, an almost clear, very viscous solution with a cellulose content of approx.

30%.

(c) Enzyme production (1) Cellulase enzyme (Enzyme Development Corporation,

New York) was treated as follows to give the enzyme preparation "CS". The enzyme, 10 g, was added to 25 ml of water. Using an ultrafiltration membrane, the enzyme solution was concentrated and diluted with excess water and then concentrated again to a volume of 25 ml. This was repeated several times until the salt and carbohydrate content of the enzyme preparation was negligible.

(2) The enzyme preparation "CW" was prepared as follows. The enzyme, 10 g, was dissolved in 100 ml of water. Next, 57 g of ammonium sulfate was added. After stirring, the ammonium sulfate dissolved and a white precipitate formed. This precipitate was separated by centrifugation and redissolved in 30 ml of water. The solution was then desalted using Sephadex®b-25 (Pharmacia Corporation) and made up to a final volume of about 100 ml.

(d) Hydrolysis of crystalline (β-cellulose) (1) To 1 volume of 2% crystalline β-cellulose dissolved in the solvent described in (a) was added 2 volumes of 30% HCl, which caused reprecipitation of dissolved cellulose. 5 volumes of sodium acetate buffer and 1 volume of the enzyme preparation "CS" were added, and after mixing and incubation at 50 ° C, the cellulose was converted to glucose up to 50% in 30 minutes, apparently in the absence of cadmium and ethylenediamine. -activated the enzyme.

(2) One volume of 3% Avicell in solvent (a) was mixed with 2.5 volumes of water, which caused cellulose to reprecipitate. The reprecipitated cellulose was washed with water, after which the consistency of the cellulose was 0.5%. After the pH of the solution was adjusted to 5 with buffer, sufficient enzyme preparation "CW" was added to give a volume ratio of enzyme to cellulose weight of 18: 1. Incubation of this solution at 50 ° C gave 80% conversion to glucose in 5 hours and 90% As a comparison, a control experiment using cellulose that had not been dissolved in a solvent and reprecipitated, i.e., untreated cellulose, gave only a 15% conversion after 5 hours and a 47% conversion after 50 hours.

Example 2

Pre - treatment and hydrolysis of agricultural residues

The solvent and enzyme solutions were prepared exactly as described in Example 1 above.

8 66429 (a) Dissolving cellulose in corn cobs

Corn cones ground into 40 mesh particles were contacted with dilute acid to remove hemicellulose. The residue and solvent were combined in a weight ratio of 1:55, corn cob / cadoxene. After stirring for 2.5 hours, the solid and liquid phases were separated. The solid phase was washed with water, dried and weighed. The weight loss, calculated from the initial and final weights of the dry solid, was as high as 77%. This indicated that all of the cellulosic material had dissolved.

(b) Dissolution of cellulose in maize waste

Corn waste that had been ground to a mesh size of 40 mesh. was contacted with dilute acid to remove hemicellulose. The residue and solvent were combined in a weight ratio of 1:10, corn waste: cadoxene. Mixing, washing and solid recovery were performed as described in Example 2 (a). Weight loss was estimated to be between 44-94% (calculated on a dry basis).

(c) In situ dissolution and hydrolysis of corn residue

A corn residue containing 38% Λ-cellulose was contacted with dilute acid to remove hemicellulose. The residue and cadoxene were combined in a corn residue weight ratio of cadoxene of 1: 4.2. After standing for 12 hours, buffer, water and the enzyme preparation "CW" were added to give a 2.5% solution of the residue at pH 5. Hydrolysis of the mixture at 45 ° C gave 72% conversion of β-cellulose to glucose. per hour. Since the pretreatment as a solvent and the subsequent reprecipitation of cellulose were first performed without separating the solvent and dissolved cellulose from the solid residue, this method was termed "in situ" dissolution (and reprecipitation).

(d) In situ dissolution and hydrolysis of bagasse (sugar cane residue)

The Bagassi residue containing 33% Λ-cellulose was contacted with dilute acid to remove hemicellulose. The residue was mixed with cadoxin in a bagasse: cadoxene weight ratio of 1: 4.2. Using the same conditions as in Example 2 (c), a 70% conversion was obtained in 19 hours.

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Example 3

Hydrolysis by immobilized enzyme (a) Preparation of immobilized enzyme

A filter plate made of chemically activated porous PVC membrane material (supplied by Amerace Corporation, Butter New Jersey) was immersed for two hours at room temperature in a solution of 10% glutaraldehyde buffered with phosphate to pH 7. After washing with water and sodium acetate buffer (pH 5) the plate was immersed in the enzyme preparation "CW" for 12 hours at 4 ° C. The plate was then washed with buffer, placed in a column apparatus and used as described below.

(b) Hydrolysis of cellodextrins

Hydrolysis of cellodextrins with immobilized ethnzyme resulted in complete dissolution, with 79% being converted to glucose and the remainder to cellodextrins. This clear solution was passed five times through an immobilized enzyme plate, resulting in an increase in glucose conversion from 79% to 81%. Glucose was formed from the hydrolysis of soluble cello-oligosaccharides to glucose. A comparative study was performed using only soluble enzyme, sequentially.

In this experiment, glucose conversion remained unchanged at 79% for the same length of time.

Claims (10)

A process for producing glucose from cellulosic material containing hemicellulose and lignin, wherein the hemicellulose is removed from the cellulosic materials; the cellulose contained in the cellulose materials is dissolved at room temperature in a solvent which is a mixture of ethylenediamine, cadmium oxide and the water, characterized in that the dissolved cellulose is precipitated again and the precipitated cellulose is hydrolyzed to a cellulase enzyme. glucose while the pH is about 5. A method according to claim 1, characterized in that the cellulose materials are physically reduced to a size not exceeding 40 mesh prior to the removal of the hemicellulose. 3. A method according to claim 1, characterized in that the hemicellulose is removed by extraction with a dilute acid or alkali. Process according to claim 1, characterized in that the lignin is separated from the cellulose materials by filtration or centrifugation after the cellulose has been hydrolyzed to obtain glucose. Process according to any of claims 1-4, characterized in that the cellulase enzyme added is endo-1,4-beta-glucanase. Method according to claim 5, characterized in that the cellulase enzyme added is a Tricoderma viride cellulase solution. Process according to any of claims 1-6, characterized in that the solvent is recovered for use again. Process according to Claim 7, characterized in that the solvent is recovered by the addition of CO2 to precipitate cadmium carbonate which can be converted to cadmium oxide for ether use. 9. A process according to claim 7, characterized in that the solvent is recovered by adding a water-immiscible solvent to extract ethylenediamine for ether circulation to separate the obtained mixture.