United States Patent (19) 11) Patent Number: 4,525,218 Chen et al. 45) Date of Patent: Jun. 25, 1985

54 SELECTIVE HYDROLYSIS OF CELLULOSE FOREIGN PATENT DOCUMENTS TO GLUCOSE WITHOUT DEGRADATION OF GLUCOSE USING ZINC CHLORIDE 44622 1/1982 European Pat. Off...... 127/37 57-22695 2/1982 Japan ...... 435/99 75 Inventors: Li F. Chen, West Lafayette, Ind.; Che-Ming Yang, Clark, N.J. OTHER PUBLICATIONS 73) Assignee: Purdue Research Foundation, West Chem. Abstracts 98: 91246X; Chen and Gong; 1982. Lafayette, Ind. Primary Examiner-Kenneth M. Schor (21) Appl. No.: 377,078 Attorney, Agent, or Firm-Cushman, Darby & Cushman 22 Filed: May 11, 1982 57 ABSTRACT (51) Int. Cl...... C13K 1/02 Cellulose is selectively hydrolyzed to glucose without (52 U.S. Cl...... 127/37; 435/99; the formation of degradation by-products by pretreat 435/105 ing the cellulose to form soluble cellodextrins through 58 Field of Search ...... 127/37; 435/99, 105 treatment with concentrated (60-80%) solutions of zinc 56 References Cited chloride. Zinc chloride is then separated from the mix ture by extraction with attendant precipitation of the U.S. PATENT DOCUMENTS cellodextrin material which is the hydrolyzed, chemi 3,479,248 11/1969 Nobile ...... 127/37 X cally or enzymatically to glucose. 4,018,620 4/1977 Pengue ...... 127/37 4,237,226 12/1980 Grethlein ...... 435/99 4,304,649 12/1981 Han et al...... 127/37 X 8 Claims, No Drawings 4,525,218 1. 2 centrated zinc chloride to liquify the cellulose, thereaf SELECTIVE HYDROLYSIS OF CELLULOSE TO ter reducing the zinc chloride concentration (e.g. by GEUCOSE WITHOUT DEGRADATION OF dilution) and completing acid hydrolysis to form glu GLUCOSE USING ZINC CHLORIDE cose. While that process provides hydrolyzed yields of over 90%, the separation of zinc chloride and glucose is BACKGROUND OF THE INVENTION costly. Much research has been conducted in the area of Accordingly, it is the primary object of the present cellulose hydrolysis to produce fermentable sugars, invention to provide a means for effectively pretreating such as glucose therefrom. Cellulose is the most abun cellulose with zinc chloride and thereafter separating dant polymer on earth, and is characterized as a straight 10 the zinc chloride from the glucose produced. chain polymer composed of glucose with beta 1,4-link This and other objects of the present invention will be ages. Cellulose may exist in crystalline or amorphous more apparent from the discussion which follows. forms. Generally speaking, one can easily hydrolyze SUMMARY OF THE INVENTION amorphous cellulose with dilute acid or enzymes. Crys talline cellulose, on the other hand, is difficult to hy 15 Cellulose is selectively hydrolyzed to glucose with drolize presumably due to a tight physical packing of out the formation of degradation by-products by pre the cellulose molecules. As a result, degradation of the treating the cellulose to form soluble cellodextrins hydrolysis products is significant as represented by the through treatment with concentrated (60-80%) solu following scheme: tions of zinc chloride. Zinc chloride is then separated 20 from the mixture by extraction with attendant precipita tion of the cellodextrin material, which is then hydro H2O -- celluloset Seglucose Hit Sedegradation lyzed, chemically or enzymatically to glucose. products The process according to the present invention gen erally comprises the steps of: Various methods have been touted for decrystallizing 25 (a) forming a mixture of cellulose together with zinc cellulose through the use of solvents to precipitate it in chloride, said zinc chloride being in the form of an an amorphous form. However, there methods all utilize aqueous solution containing from about 60 to about cellulose which is solid, albeit amorphous. 80% (preferably about 65 to 76%) by weight of zinc Penque U.S. Pat. No. 4,018,620 describes a method of hydrolyzing cellulose using calcium chloride and dilute 30 chloride; acid at a temperature of 100° C. to form a colloid sus (b) heating the mixture formed in step (a) at a temper pension of the cellulose which is the hydrolyzed at a ature of from about 70° to about 180° C. (preferably temperature of 120° C. for a period of 30 minutes. Con from about 100 to about 145° C.) for a period of time trary to Penque's findings, and due apparently to an sufficient to convert the cellulose to a liquid form; error in the unit and chemistry of Penque's analysis, we 35 (c) removing the zinc chloride from the mixture by have found that the claimed method does provide a solvent extraction thereby precipitating the partially complete conversion of cellulose to glucose. According hydrolyzed cellulose in the form of cellodextrins; and to Penque, 10% (w/v) of newsprint (which contains (d) separating the precipitated cellodextrins from the cellulose and hemicellulose) was hydrolyzed, thereby extraction media; obtaining a 10% (w/v) reducing sugar solution which is 40 (e) hydrolyzing the precipitated cellodextrin to glu equivalent to 50% of the total reducing sugar. COSes Because the hemicellulose fraction is very easy to It is important that the zinc chloride be removed as hydrolyze, and since newsprint generally contains at soon as the cellulose has been liquified-i.e. as soon as least 15% hemicellulose, one must subtract this value the cellulose has been partially hydrolyzed to form from the yield of glucose from cellulose fraction 45 soluble cellodextrins to avoid glucose degradation and thereby getting a yield of only 20%. In addition, Penque formation of such undesirable by-products as hydrox used Clinitest tablets to quantitate the sugar. These ymethylfurfural. tablets are also reactive to the degraded glucose, (Hy We have found that if cellulose is only partially hy droxymethyl furfural) and do not provide a true reading drolyzed to soluble cellodextrins, the ZnCl2 may be of reducing sugars. On the other hand, analyzing with 50 recovered by the addition of H2O, acetone, ethanol, "Tes-tape' or glucose analyzer, which is specifically ether, or other organic solvents. In the presence of such reactive to glucose, would provide a different and more solvents, the cellodextrin precipitates and ZnCl2 re accurate result. mains in the solvents. Summarizing the present process It is thus, desirable to hydrolyze cellulose in a liquid provides an improved means for producing glucose state. Unfortunately, conventional cellulose swelling 55 with removal of zinc chloride prior to glucose forma reagents and cellulose solvents are either too severe for tion by: glucose or unable to catalyze the cellulose hydrolysis. 1. Liquifying cellulose with ZnCl2/H-- or ZnCl2 and Zinc chloride is known as a cellulose swelling rea partial hydrolyzing cellulose to a water soluble cel gent, and Swells the cellulose at a concentration range lodextrin. from 60 to 80%, with maximum effect at 75% and 65%. 60 2. Recovering ZnCl2 by extraction with H2O, ace The pH of ZnCl2 at this range on concentration is 0 to tone, methanol ethanol, ether or other suitable solvents. -2, and thus is able to provide a catalytic function of 3. Hydrolyzing water soluble dextrins to glucose by cellulose hydrolysis. However, under such conditions dilute acid or enzyme action. glucose is also degraded at a faster rate. DETAILED DESCRIPTION OF THE In our concurrently filed application Ser. No. 65 377,077, titled "Quantitative Hydrolysis of Cellulose to INVENTION Glucose Using Zinc Chloride', we describe a method We have found that glucose can be dissolved in etha for hydrolyzing cellulose to pretreating same with con nol, acetone and other organic solvents in the presence 4,525,218 3 4 of a high concentration of zinc chloride, but cellodex trin or higher glucose polymers do not dissolve in ace EXAMPLE 1 tone, ethanol and other organic solvents. Thus, zinc Material and Methods chloride can readily be separated from the partially Avicel was used as noted as a source of cellulose. hydrolyzed cellulose, and the partially hydrolyzed cel Cellulase from Trichoderma verdi was used for enzy lulose (i.e. cellodextrins) can be further hydrolyzed to matic hydrolysis. This enzyme was fractionated by 50% glucose in the absence of zinc chloride. Tests indicate saturated ammonium sulfate to remove glucan. The that only water soluble cellodextrin can be readily hy protein content of the purified enzyme is 20% deter drolyzed to glucose after the separation of zinc chlo mined by Lowry's method with hovine serium albumia ride. The present process therefor provides a means for as standard. the recycling of zinc chloride. The hydrolysis of cellulose to form the cellodextrin Pretreatment of Cellulose can be carried out with and without the presence of Avicel 10gm was wetted with 12 ml of water. 50 ml acid, since the cellulose is a solution it can be hydro of 74% ZnCl2 solution containing 0.5% (w/v) HCl was lyzed randomly. The distribution of molecular weight 15 added to the wetted avicel. The cellulose solution were at certain reaction times is governed by the hydrolysis then subjected to heating with the temperature ranging rate of cellulose and degradation rate of glucose. The from 100° C. to 145° C. The heating time ranging from hydrolysis rate and degradation rate is a function of 6 minutes to 20 minutes. The heated cellulose now re temperature and the concentrations of acid and Zinc 20 duced to cellodextrin is then cooled by setting at room chloride as discussed below. temperature or cooled by plunging the reactor cell in For the convenience of recycling zinc chloride, the the ice slurry. The cellodextrin is precipitated by adding reaction may be stopped at a point where the fraction of 25 ml of acetone per gram of cellodextrin to the cellu soluble cellodextrin is at the maximum. These points lose solution. The precipitated cellodextrin was washed 25 with 25 ml of acetone per gram of avicel for 4 times. depend on the temperature and concentration of zinc The cellodextrin was then vacuum dried to remove chloride and acids, and are easily determined by the acetone. The cellodextrin thus obtained was in lumps chemist. which were then resuspended in water and freeze dried. The solution of partially hydrolyzed cellulose is then The freeze dried samples are powdery particles. 0.8 gm added to acetone or ethanol (or other organic solvents). cellodextrin was suspended in 2 ml of sodium acetate All of the cellodextrins will precipitated out with the buffer (pH 4.8, 0.05M) and 2 ml of enzyme in buffer exception of glucose. solution was then added to this suspension with the final Zinc chloride is soluble in acetone, ethanol, ether, enzyme concentration of 0.01%, 0.1%, 0.5%, 2.5% and and some other organic solvents. These organic sol 5% (w/v). The samples were incubated at 48 C. in a vents can then evaporated and recycled if desired. Zinc 35 shaker bath. 8 tiny glass beads were added to assist the chloride and glucose may be further heated. Upon heat agitation with and form glucose. ing, glucose forms active charcoal with the evolution of gas, and zinc chloride can be separated easily and then EXAMPLE 2 recycled. Alternatively chloride may be recycled in the One gram of Avicel is swollen and hydrolyzed in presence of glucose. 40 65% ZnCl2 aqueous solution. After 4 hours of heating The cellodextrin precipitate may then be subjected to to 100° C., 80% of cellulose becomes water soluable a stripping of solvent by either steam or air. Acid solu dextrin. Fifteen percent of the cellulose is hydrolyzed tion can then be added to the cellulose for further hy after acetone extraction to glucose using dilute hydro drolysis. chloric acid. In forming the initial mixture of cellulose and zinc 45 The invention having been thus described, it will be chloride solution, we have found that the maximum appreciated that various departures may be therefrom amount of cellulose which may be added to the concen within the scope of the claims which follow. trated zinc chloride solution is about 1 gram of cellulose We claim: for each 2 ml of zinc chloride solution. 1. A process for the selective hydrolysis of cellulose As noted above, we have also found that the degrada 50 solids to glucose without degradation of the glucose, tion rate of glucose is affected by temperature, the con which process comprises the steps of: centration of ZnCl2, and acid. The rate of glucose deg (a) forming a mixture of cellulose solids together with radation can be expressed as: zinc chloride, said zinc chloride being in the form of an aqueous solution containing from about 60 to Koeg -2.23 x 10'(ZnCl)4.53+4.62(H) 05')xe 55 about 80% by weight of zinc chloride; -2.185X 104/RT-20.85 (H+5 (b) heating the mixture formed in step (a) at a temper ature of from about 70° to about 180° C. for a per This means that lower acid, ZnCl2 concentration, and iod of time sufficient to convert the cellulose to a low temperature stabilizes glucose. However the con liquid form without appreciable formation of glu centration of ZnCl2 that can dissolve cellulose is detri 60 COSe; mental to the glucose. Fortunately, the data indicates (c) removing the zinc chloride from the mixture by that the dissolved and partially hydrolyzed cellulose solvent extraction thereby precipitating the par can remain in solution at a lower concentration of tially hydrolyzed cellulose in the form of cellodex ZnCl2 achieved in accordance with the present inven trins; and tion. 65 (d) separating the precipitated cellodextrins from the The following examples are offered to more fully extraction media; illustrate the invention, but are not to be construed as (e) hydrolyzing the precipitated cellodextrin to glu limiting the scope thereof. COSC. 4,525,218 5 6 5. The process of claim 1, claim 3, or claim 4 wherein 2. The process of claim 1 wherein the concentration the solvent used for extraction in step (c) is an organic of zinc chloride in step (a) ranges from about 65 to 76% solvent. by weight. 6. The process of claim 5 wherein said solvent is 3. The process of claim 1 wherein step (e) is carried acetone, ether, methanol, or ethanol. out chemically using an acid selected from the group 7. The process of claim 1 wherein the zinc chloride solution contains up to 5% acid. consisting of hydrochloric, sulfuric, nitric, phosphoric 8. The process of claim 7 wherein said acid is selected and acetic acid. from the group consisting of hydrochloric, sulfuric, 4. The process of claim 1 wherein step (e) is carried 10 nitric phosphoric and acetic acid. out enzymatically. ck :: *k -k sk

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