Note: Descriptions are shown in the official language in which they were submitted.
3~ -- 1 -- PROCESS FOR TREATING CELLULOSIC MATERIALSAND OBTAINING GLU('OSE THEREFROM A process is disclosed fo]A treating cellulosic materials and obtaining glucose therefrom, which process can include recovering of cellulose from cellulosic materials. Cellulose is recovered by treating the cellulosic materials to remove hemicellulose, then dissolving the cellulosic materials in a solvent, after which lignin is removed and the cellulose is then precipitated from the remaining solution. The cellulose may be recovered in sheets or in long threads, fibres or as a flocculent 10 material, and may then be hydrolyzed to provide glucose by subjecting the cellulose to a cellulase enzyme and may also include subjection to an acid. A yield of glucose may also be obtained by hydrolyzing the cellulose materials that have been dissolved in a solvent and then 15 separating lignin from the glucose. This invention relates to a process for treating cellul- osic materials, and, more particularly, relates to a process for yielding glucose from cellulosic materials. The utilization of cellulosic waste materials, such as 20 cornstalks, sawdusts, straws, bagasse, and the like, has been the subject of strong interest recently, particul. arly with respect to utilization of such waste materials for developing alternate sources of fuels, chemicals and other useful products. .. ~ 3130 cellulosic materials include three principal components - cellulose, hemicellulose, and lignin. Methods for extraction of hemicellulo~e have heretofore been sugges- ted and/or utilized and such extracted hemicellulose can be utilized for many ~xisting methods involving hydroly- sis, fermentation, pyrolysis and the like. Lignin has likewise hereto~ore been isolated from cellul- osic materials. Lignin has been found to be higher in hydrogen and carbon and lower in oxygen content than lO cellulose and hemicellulose and has the highest heating value of the three. Isolated lignin can be burnt directly to generate steam and electricity and can also be used to produce a number of useful products including vanillin, dimethylsulfoxide, dimethyl sulfide, methyl mercaptan, 15 and catechol. Recovery of cellulose and/or utilization of the same as by hydrolyzing to provlde glucose has presented a problem heretofore, however, primarily due to the cryst- alline structure of the cellulose molecules and the 20 presence therein of a lignin seal. . In addition, attempts have heretofore been made to hydrolyze cellulose, and have included the use of acids or enzymes in so doing. But such attempts have not been ; completely successful, at least in providing an econom- , . ' 31~3~ --3-- ically attractive method that is useful in providing a satisfactorily high yield of glucose from the cellulose in such cellulosic materials. This invention provides a process for providing a yield of glucose from cellulosic materials, whic~ contain hemicellulose. lignin and cellulose, said process comprising the steps of: removing hemicellulose from cellulosic mat- erials; dissolving the cellulose contained in the cellulosic materials having said hemicellulose removed therefrom in a solvent at room conditions, said solvent comprising Cadoxen; reprecipitating the dissolved cellulose; and hydrolyzing the reprecipitated cellulose to yield glucose at a pH of ~bout 5. As suggested hereinabove, cellulose molecules form highly ordered crystalline structures. In addition, in cellulosic materials, lignin in middle lamella poses as a physical seal surrounding cellulose fibers in such materials. In one embodiment of the process of this invention, selective solvent extraction is utilized to fractionate hemicellulose, cellulose and lignin (the three major components of all cellulosic materials). This fraction- r~ '' ' ' ~;'~ : ; ~ . 111~33~ ation is necessary because it has been found that aftercellulose is dissolved in solution, it is no longer protected by its crystalline structure or by the lignin seal and cellulose can thereafter be easily precipitated. The recovered cellulose can then be utilized in a number of ways, including, for example, use as a "synthetic cotton", as "new paper", and has been found to be partic- ularly useful when the cellulose i9 hydrolyzed to provide a high yield of glucose. 10 An example of the embodiment of our process for recover-` ing cellulose from cellulosic materials, and utilizing the same, including utilization by hydrolyzing to provide glucose from cellulose follows. In this embodiment of our process, the cellulosic mater- 15 ials, after being physically reduced to a suitable size if necessary or desirable, are first extracted with a ' dilute acid or alkali to remove hemicellulose. The resi- due after hemicellulose removal contains cellulose and lignin. ; 20 The residue is then dissolved in a suitable solvent. ; There are a number of solvents reported in literature capable of dissolving cellulose. After examination of several, a solvent known as Cadoxen was found to give ' the' best results. Cadoxen was first reported by Jayme 3~ and ~euschaffer (see Jayme~ G. and Neuschaffer, K., 1957~ Dle ~aturwissenschaf-ter, 44 (3): 62-63, and Jayme, G. and Neuschaffer, K., 1'357, Dle Makromolekulare Chemie, 28:71-83). Cadoxen is made of 25-3~/c ethylenedi- amine and 70-75% water, and also about 4.5 to P/0 cadmium (added as oxide or hydroxide). cadoxen has good solvat- ing properties, is a clear, colorless, nearly odorless liquid, is stable for an almost unlimited time, causes little degradation of cellulose and is relatively easy 10 to prepare. , The fact that water is a part of cadoxeh formulation is of great importance. Most cellulosic wastes contain moisture. Cadoxen requires no pre-drying of the wastes in dissoIving cellulose, which avoids an otherwise costly 15 operation~ ` About 10 grams of cellulose was dissolved in 100 grams of Cadoxen as an example to show the effect thereof. By filtration or centrifugation, lignin which does not dissolve can be separated. 20 The remaining cellulose-Cadoxen solution is then further processed to recover cellulose, as for example by one of ; the following: a. The cellulose-Cadoxen solution can be spun through a nozzle into a suitable liquid to regenerate ~il8380 cellulose in the form o~ long threads. This could be a source of "synthetic cotton". b. The cellulose-Cacloxen solution can be spread into a suitable liquid to regenerate cellulose in the form of a sheet. SUch sheets should have many applica- tions. Regenerated cellulose will recrystallize, which ~ill reduce the transparency. After adding fillers and coating materials, a new type of paper can therefore be made. c. The cellulose-Cadoxen solution can be spun through a nozzle into a suitable liguid to regenerate cellulose in the form of long fibres. This could be the basis of a "pulping" process which is essentially for removal of lignin and hemicellulose from woody materials. 15 The regenerated cellulose fibre might be made into paper like the ordinary pulp. The cellulose-cadoxen solution is physically stable but this stability apparently requires;a close balance among cellulose, water, ethylenediamine and cadmium. The exact 20 nature of the balance is not precisely known, but if, for example, excess water is added, cellulose will re-precip- itate from the solution. cadoxen i~ of high pH, and upon addition of an acid, cellulose also re-precipitates from the solution. The re-precipitated cellulose~ like 25 other linear polymers, may spontaneously re-crystallize. The re-precipitated cellulose can then be-readily hydrol- 380 -- 7 -- yzed to give a high yield of ~lucose by either an acidor cellulose enzymes. For example, by adding an acid to a cellulose-Cadoxen solution to bring its pH down to about 5 (whereupon cellulose re-precipitates), followed by adding a Tricoderma viride cellulase solution, the re-precipitated cellulose is hydrolyzed to glucose. The ethylenediamine and cadmium salt in the solution apparently do not inactivate the enzyme. In order to improve the economics of the process~ Cadoxen 10 needs to be recovered and reused. By different recycle techniquesj the process can be further divided into several alternatives which are described as follows: (a) Adding water to precipitate cellulo~e from a cellulose-Cadoxen solution. Separate cellulose by filtr- 15 ation or centrifugation. The cellulose is then hydrol- yzed to glucose by acids or enzymes or both. The liquid can be added with C0 to precipitate cadmium carbonate which in turn~can be converted into cadmium oxide for reuse. The amine left in the aqueous solution can be 20 recovered by standard methods such as di~tillation or extraction. (b); Adding C0 directly into a cellulose-cadoxen solution wheraupon both cellulose and cadmium carbonate will precipitate. After separation from the liquid 25 which c'ontains ethylenediamine that is to be recovered as in ~a), the mixture of the two solid precipitates can - 8 - be treated with cellulases to hydrolyze and dissolve away cellulose to produce glucose. Cadmium carbonate precipitate left can be converted into cadmium oxide for reuse. (c) Adding a water immicible solvent to extract ethylenediamine for recycle, whereupon cellulose will precipitate. ThUs, we can in one step separate the mixture into 3 fractions: amine in an organic layer~ cadmium in an aqueous layer and cellulose as a precipi- 10 tate. The 3 fractions are to be processed separately. (d) The re-precipitated cellulose can be hydrolyzed to glucose by either an acid-enzyme two-step process or an all-enzyme process. An acid or a cellulase enzyme ~most likely an endo-l, 4-beta-glucanase but the cellul- ` 15 ase mechanisms are still a subject of considerable cont- roversy) can "solubilize" cellulose (by forming cellod- extrins). To complete the hydrolysis to glucose, either free or immobilized cellulases are then utilized. (e) cellulose re-precipitated from cadoxen has been 20 observed to spontaneously form beads if left to stand in solution for several days. Such beads may have potential as a sùpport for enzyme immobLlization. In a further embodiment of the process of our invention, cellulose is dissolved and re-precipitated "in situ" an~ 25 therefore never physically removed from the lignin. The process is now thought to be preferred where separation 11iL838~ of cellulose is not desired. In this process, the cellulose is hydrolyzed to glucose and the lignin in solid form which remains is filtered from the resulting glucose solution. This has been found to be easier than is separating the lignin from the cellulose and then hydrolyzing the cellulose. Enzyme can be used to hydrolyze the cellulose, and it is believed that acid hydrolysis can also be utilized. For this process, the following steps are utilized: lQ 1. Treat cellulosic materials to remove hemicellu- lose (this treatment can be the same as described herein- above with respect to the first discussed embodiment); 2. Dissolve the cellulose materials in solvent (hereagain, this step can be identical to that described 15 hereinabove with respect to the first discussed embodi- ment); 3. Add water, acid, or another liquid such as methanol to re-precipitate cellulosc; 4. Hydrolysis with enzyme or acid to yield glucoseg 20 leaving behind only water insoluble lignin; and 5. Separation of lignin from glucose syrup by filtration. . Examplès of our process are as ~ollows: 33~ -- 10 -- EX~P~E 1 Pretreatment and HydrolysLs of - Cellulose (a) Preparation of 1:he Solvent (Cadoxen): Ethylene diamine, 25 to 30~/c in water, was saturated by adding an excess of cadmium oxide which was previously oven dried. Cadmium oxide which did not dissolve formed a hydroxide which appeared as a white precipitate. The precipitate was filtered or centri- fuged off. The procedure of adding cadmium oxide and separating out the hydroxide was performed twice to lO ensure that a solution saturated with cadmium oxide resulted. In this way an aqueous solution of 25 to 30~/O ethylenediamine and 4.5 to P/~ cadmium resulted. Due to its cadmium and ethylene diamine content, this solvent is commonly known as Cadoxen. (b) Dissolution of ~-Cellulose ~ B (1) Crystalline ~-cellulose (Avicell~ O l g~ was added to lO ml of the solvent, prepared as in (a), and mixed. Dissolution of the cellulose was complete within lO minute~s at room conditions. 20~ ~t2) Dissolution tests were repeated for O.l g~ ~0.2~g, 0.3 g, 0.4 g, and 0.5 g portions of crystalline cellulose (Avicell), each in lO ml solvent~ by a procedure where the cellulose~was mixed with solvent at room conditions and then cooled by placing on ice. 25 Times required for complete dissolution to occur are summarized in Table l. ~, _ ` ~ ' 38~ 11 -- TAsLE 1 % Avicell Dissolution in Solvent Time 1 2 minutes 2 2 minutes 3 7 minutes 4 9 minutes 15 minutes (3) Dissolution tests, performed as described in (2) above, were repeated. In this case, however~ amorphous cellulose,~instead of crystalline cellulose, was dissolved. Using the same quantities of cellulose, 10 and the same procedures as in (3j, the dissolution times summarized in Table 2 were observed. ~ TABLE 2 % cellulose Dissolution in Solvent Time 1 2 minutes 2 ~ 2 minutes 3 7 minutes 4 9 minutes 15 minutes (4) Solutions containing greater amounts of cellulose were made by modifying the compo~ition of the solvent and by altering the dissolution procedure. 20 Sodium hydroxide was added to the solvent in (a) to give a solution which is 0.5 M in sodium hydroxide. To 1 3~ - 12 - milliliter of the modifiecl solvent, 300 milligrams ~vicell were added, followed by an excess of CdO. Mixing of this solution was carried out resulting in an almost clear, very viscous solution having a cellulose concentration of approximately 30D/ (c) Preparation of Enzyme. (1) Cellulase enzyme from the Enzyme Development Corporation, New York, was processed as follows to give enzyme preparation "CS". Enzyme~ 10 g, was added to 10 25 ml water. Using an ultrafiltration membrane the enzyme solution was concentrated and diluted with excess water and then concentrated to a volume of 25 milliliters again. This was repeated several times until the salt and carbohydrate level o the enzyme preparation was 15 negligibleO (2) Enzyme preparation "CW" was made as follows. Enzyme, 10 gram, was dissolved in 100 milliliters water. Mext 57 gram ammonium sulfate was added. Upon mixing, the ammonium sulfate dissolved and a white precipitate 20 formed. This precipitate was separated by centrifugation and re-dissolved in 30 milliliters of w~ er. The solution B was then desalted using Sephadex G-25 ~Pharmacia Corpor- ation) and made up to a final volume of about 100 ml. (d) Hydrolysis of Crystalline ~-Cellulose. ~5 (1) To 1 volume of ~/c crystalline ~-cellulose dissolved in the solvent described in (a~, 2 volumes of 3~/0 HCl was added causing the dissolved cell~lose to 38~:) - 13 - re-precipitate. This was followed by addition of 5 volumes of sodium acetate buffer and 1 volume ~nzyme preparation "CS'. Upon mixing and incubation at 50C~ up to 500/O conversion of cellulose to glucose was obtained in 30 minutes. Apparently, the presence of cadmium and ethylene diamine did not inactivate the enzyme. (2) One volume of 3% Avicell in solvent (aj was mixed with 2.5 volumes water causing the cellulose to 10 re~precipitate. The re-precipitated cellulose was washed with water after which the cellulose concentrat- ion was 0.5%. After adjusting the solution to pH 5 wit~ buffer, sufficient en~yme preparation "CW" was added td give an enzyme volume- cellulose weight ratio of 18 : 1. 15 Incubation of this solution at 50C gave 80/c conversion to glucose in 5 hours and 90k in 50 hours. In comparison, a control experiment using cellulose which had not been dlssolved in solvent and re-precipitated, i.e., untreated cellulose, gave conversions of only 15% after 5 hours 20 and 47% after 50 hours. EX~MPLE 2 Pretreatment~and Hydrolysis of Agricultural Residues Preparation of the solvent and enzyme solutions was done exactly as described previously in Example 1. (a) Dissolution of cellulose in corn cob. Corn 25 cobs~ ground to 40 mesh size particles, were combined with solvent in a weight ration of 1:55 corncob: cadoxen. o - 14 - After stlrring for 2.5 hours, the solid and liquid phases were separated~ The solid phase was washed with water, dried and weighed. Weight loss, based on initial and final weights of dry solids, was as high as 77%. This indicated all cellulosic material had been dissolved~ (b) Dissolution of cellulose in corn residue. Corn residue, ground to 40 mesh, was combined with solvent in a weight ratio of 1:10 corn residue: Cadoxen. 10 The stirring, washing and recovering of solids was performed as described in Example 2(a). weight loss was estimated to be between 44% and 94% (dry basis). (c) In Situ dissolution and hydrolysis of corn residue. Corn residue containing 3~/0~ -cellulose was 15 mixed with Cadoxen in a 1:4.2 weight ratio - corn residue: Cadoxen. After sitting 12 hours, buffer, water~ and enzyme preparation "CW" wexe added to give a 2.5% solution of residue at pH 5. Hydrolysis of the mixture at 45C gave 72% conversion of the ~-cellulose to glucose 20 in 19 hours. Since the solvent pretreatment and subse- guent cellulose re-precipitation was done without first separating the solvent and dis601ved cellulose from the solid residue, this technique was referred to as "in situ" dissolution ~and re-precipitation). ~d) In Situ dissolution and hydrolysis of bagasse (sugar cane residue). Bagasse residue containing 33% ~-ce}lulose was mixed with cadoxen in a 1:4.2 weight ' .~' ~ ' - 15 - ratio - bagasse: Cadoxen. using the same conditions as in Example 2(c), 7~/0 conversion was obtained in 19 hours. EYAMPLE 3 Hydrolysis with Immobilized Enzyme (a) Preparation of immobilized enzyme. A filter disk of chemically activated porous PVC membrane material (supplied by Amerace Corporation~ Butter ~ew Jersey) was submersed in a solution of l~/o gluteralde- hyde buffered with phosphate to pH 7 for 2 hours at room conditions. After washing with water and sodium acetate 10 buffer (pH 5), the disk was submersed in enzyme prepara- tion "CW" for 12 hours at 4C. The disk was then washed with buf~er, placed in a column apparatus and used as described below. (b) Hydrolysis of cellodextrins using immobilized 15 enzyme was completely solubilized with 7~/c being converted to glucose and the balance to cellodextrins. This clear solution was passed through the immobilized enzyme disk 5 times resulting in an increase in glucose conversion from 7~/0 to 81%. Glucose was formed due to hydrolysis ~0 of solu~le cellooligosaccharides to glucose~ A control study using only soluble enzyme was conducted, consecu- tively. In this run~ glucose conversion stayed constant at 7~/0 during the same time period. EXAMPLE 4 Formation of Cellulose Beads 25 A solution of 2% Avicell in Cadoxen was combined with 3~ - 16 - 50O/c Hcl in a 1:1.5 volume ratio of solution: acid. This caused the cellulose to precipitate. The mixture was heated to 50C for 30 minutes and then cooled to room temperature. Upon standing for twelve hours the flocculent cellulose re-precipitate had spontaneously formed small cellulose beads. As can be appreciated from the foregoing, this invention provides a process for treating cellulosic materials and yielding glucose therefrom, with one disclosed process 10 dissolving and re-precipitating cellulose "in situ" while another disclosed proces~ recovers cellulose from cellulosic materials which can then be utilized to provide a high yield of glucose. ~ ' : ~ .
