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Wilke et al.
 CONVERSION OF CELLULOSIC
Aug. 3, 1976
MATERIALS TO SUGAR Inventors: Charles R. Wilke, El Cerrito; Gautam Mitra, Berkeley, both of Calif.
Assignee: The United States of America as
lases and Their Applications, Gould Ed. A.C.S. Publi cations Washington, D.C. (1969) pp. 391-396.
Primary Examiner-A. Louis Monacell
Assistant Examiner—'l‘homas G. Wiseman Attorney, Agent, or Firm~Dean E. Carlson; F. A. Robertson; Irene S. Croft
represented by the United States Energy Research and Development Administration, Washington, DC.  Filed: June 28, 1974  Appl. No.: 484,010
 US. Cl ................................. .. 195/33; 195/100;
 ABSTRACT A process for the production of sugar, mainly glucose,
by the enzymatic degradation of cellulosic materials, particularly cellulosic wastes, which comprises hydro
lyzing the cellulosic material in the presence of cellu
lase enzyme to produce a sugar solution and recover
 Int. Cl.2 .................... .. C12D 13/02; C12B 3/08
ing from the hydrolysis products a major proportion of
the cellulase enzyme used in the hydrolysis reaction
Field of Search ............... .. 195/33, 115, 99, ll,
195/7, 104, 4, 8, 9, 10, 66 R, 63, 68, 100
for re-use. At least a portion of the required makeup cellulase enzyme is produced in a two-stage operation wherein, in the ?rst stage, a portion of the output
sugar solution is utilized to grow a cellulase-secreting
UNITED STATES PATENTS
microorganism, and, in the second stage, cellulase en zyme formation is induced in the microorganism
. . . ..
. . . ..
. . . . . . . ..
Bellamy ............................ .. 195/104
Dasinger . . . . .
Ghose . . . . . . . . .
Metzger . . . . . . . . .
containing culture medium by the addition of an ap propriate inducer, such as a cellulosic material. Cellu
lase enzyme is precipitated from the culture liquid by
the addition of an organic solvent material, such as a
Hulme . . . . . . . . . . . . . . .
. . . ..
Mandels et a1.
Bellamy et al. ..................... .. 195/33
low molecular weight alkyl ketone or alcohol, and the cellulase precipitate is then fed to the hydrolysis reaction.
OTHER PUBLICATIONS Mandels et al., “The Production of Cellulose,” Cellu
CELLULOSlC 12 WASTES " 511115110511 w 14
9 Claims, 1 Drawing Figure
15 ABSORPTION STAGE
PRODUCT SUGAR SOLUTION
FURNACE -44 ASH
sr5111uz511 11511 EXCHANGER
In accordance with the present invention. particularly cellulosic wastes.4 glucosyl bonds are split to produce a mixture of simple 55 gation by various workers. gasoline consumption. Eng. The liquid sheer magnitude of this potential source dictates the sugar-containing stream from the adsorption step is the product stream of the process. 247. or they can be converted to methane which can be a useful source of gaseous fuel. a major industrial step. Chem. F.68. and recovering from the hydrolysis products cellulase enzyme for recycling to the hydroly sis reaction and a solution containing product sugar.1 3. The solid phase is washed with water for recovery of enzyme adsorbed thereon. acid hydrolysis requires costly materi als of construction because of the inherent problems of corrosion.000) showing various provide a process for the production of sugar by the types of activities with respect to different kinds of enzymatic hydrolysis of cellulosic materials. ' hydrolysis vessel. expressed either as the heat of combustion of cellulose or of the glucose or alcohol theoretically 0b 20 tainable therefrom. 29. it is an object of this invention to 60 herein to designate a complex system of enzymes (mo lecular weight range 12. according to the fol lowing equation: SUMMARY OF THE INVENTION It is an object of this invention to provide an ef?cient and economical method for the utilization of cellulosic The terms “cellulase” and “cellulase enzyme” are used materials. Or the hydrolysis products can be used to manufacture various organic chemicals pres ently produced from petroleum. J. having a very high cellulose content. Hydrolysis of cellulose yields a mixture of simple comprises hydrolyzing the cellulosic material in the presence of cellulase enzyme.000 . whereas in wood cellulose it is about 8000 — 10.4-linked D-glucose residues. particularly cellulosic wastes. the hydrolysis reaction to provide makeup enzyme. a portion of the product sugar stream is utilized to pro duce at least a portion of the required makeup enzyme and commercial solid wastes containing approximately ?fty percent paper and other cellulosic materials are by the steps comprising growing a cellulase-synthesiz ing microorganism thereon and inducing cellulase en 35 produced in the United States. Contract No. there is provided a process for the conversion of cellulosic CONVERSION OF CELLULOSIC MATERIALS TO SUGAR The invention disclosed herein was made in the course of. It is still a further object of the invention to produce the necessary makeup enzyme for the hydrolysis of the intense study for the last 25 years.35 lb of gasoline (7200 BTU). During enzymatic hydrolysis by cellulase enzyme. mainly glucose. In terms of available energy. reducing sugars. BacterioL. 59. W-7405-ENG-48 materials. it is preferable to feed makeup en. More speci?cally. However. to sugar which with the United States Atomic Energy Commission. B. In the North Amer carbon are ?xed every year by photosynthesis.more. Further wash water used.. out of enzyme used in the hydrolysis reaction can thus be which about half appears in the form of cellulose. particu larly cellulosic wastes. 485 . Most early efforts in this ?eld were geared to the acid The cellulose molecule is a high molecular weight the 1930’s (Bergius.. The liquid sugar-contain ing phase is contacted with incoming cellulosic solids necessity of improving the scienti?c and technological capabilities for cellulose utilization. cellulose degradation through enzymatic means has been a subject of investi polymer of B~1. thereby converting the cellulose to sugar. BACKGROUND OF THE INVENTION This invention relates to the enzymatic hydrolysis of cellulosic materials. A major proportion of the cellulase ican continent certain cellulosic materials are available for economic processing at this time. The 25 recovered from the hydrolysis products. For chemical pulp and ?lter paper the degree of polymerization generally varies between 500 and 1000. institutional In a particular mode of operation of the invention. Ind. In spite of these vari ous sources of available cellulose. These altogether contain about one hundred and twenty million tons of zyme formation in the microorganism-containing cul ture medium by the addition of an appropriate inducer such as a cellulosic material. mainly glucose. Precipitation of the makeup cellulase enzyme from the culture medium after the removal of solids hydrolysis of wood products to sugars. The most nota ble process in this context was the Bergius process in alcohol fermentation process can only be successful if therefrom is accomplished by organic solvent extrac the reducing sugars are available at a suf?ciently low 45 tion. the degree of polymerization varying widely depending upon its 50 origin. a pound of cellulose is equivalent whereby the enzyme contained therein is adsorbed on to 0. maximize the amount of water utilized in the washing lase enzyme recovered by the above-described washing increase the supply signi?cantly. In addition. Both of these phases contain cellulase enzyme from the hy drolysis stage. - substrates.972. the exact mechanism thereof is not yet entirely clear. in the present invention.C.000. and the result reducing sugars. the energy equivalence of which is about a of unhydrolyzed solids is a function of the amount of ?fth of the current US. 1937). 2. These hydrolysis products can be converted to ethyl alcohol which can be used as a liquid fuel to replace petroleum.l . More speci?cally. the solid material which is then introduced into the On the land area of the earth about 1.6 X 1010 tons of ing wash water containing recovered enzyme is then fed to the hydrolysis reaction. about sixty million tons of bagasse are available.7r theory proposed by Reese et al.3 billion tons per year of agricultural wastes in zyme in solid form to the hydrolysis reaction in order to 40 the United States. or under. the prod ucts of the enzymatic hydrolysis of the cellulosic mate rial are separated into a liquid sugar-containing phase and a solid phase containing unhydrolyzed solids. In recent years. Two hundred ninety million tons per year of residential. Although the degradation of crystalline cellulose by enzymatic means has been the object of 65 It is a further object of the invention to recover for recycling a major proportion of the enzyme used in the hydrolysis of cellulosic materials. The most accepted postulate in this area has been the two step C1 . Since the amount of cellu cellulose.775 cellulosic materials by utilizing a portion of the output sugar solution. The precipitated enzyme is then introduced into price.
for the obtainable from US.. viride QM6a with a linear accelerator and which is ably a plural stage countercurrent mixer-?lter. which is prefer which is a mutant strain obtained by irradiating conidia of T.1 ml of 0. J. the fungus The ?ltered unhydrolyzed solids leave ?lter 21 by Trichoderma viride. particularly T. In hydrolysis section 19. and the like. is introduced by means of line 11 into shredder l2 and then discharged via line 13 into grinder 14 where it is ground to a suit~ able particle size if necessary. 83. of the order of from sub both C. The cellulase enzyme used in the hydrolysis step of the present invention is synthesized by certain cellulo 25 above. 30 stantially 6% to substantially 10%. Strains.972. 269 (1957). is introduced via line 32 and mixed with incoming sugar solution from erally used in solid waste treatment in sanitary engi line 23. which are discharged via version process of the present invention. Cellulase activities are measured by interacting a given substrate with the enzyme and measuring the amount of reducing sugar produced in a given time. The upper limit of cellulase concentration is determined by the solubility of the enzyme protein in solution while the lower limit is dictated by economic considerations. Bacteriol. and C.8. The cellulosic solids sugar concentration determined by dinitrosalicylic acid with the recovered enzyme protein adsorbed on them test. According to this theory the C1 component of the enzyme ?rst disintegrates the cellulose chain prior to its solubilization. The subsequent hydrolytic action at the B-l. and J. 1 is added to sugar-containing stream from ?lter 21 enters adsorp a mixture of 1 ml of appropriately diluted enzyme and . the concentration should be such as to provide an enzyme activity of from substantially 0. The water necessary for dilution. Adsorption stage 15 is preferably a train of countercurrent mixer-?lters.. 40 heated to process temperature in heat exchanger 26 by BRIEF DESCRIPTION OF THE DRAWING the makeup enzyme stream. Ninth Edition. 1970. This group of microorgan isms includes Trichoa'erma viride. to Reaction is carried out at'50°C for l hour and reducing recover cellulase enzyme in the liquid phase by adsorp tion on incoming cellulosic solids. the ground waste is culture growth of a cellulase-producing fungal microor contacted with cellulase enzyme protein at a tempera ganism such as Trichoderma viride are reported by ture from substantially 30°C to substantially 60°C. Bacteriol. One such test is the Filter Paper activity test which measures combined Cl — CI activity. is introduced into wash train 25 by way of line 27. which can be tive in the synthesis of cellulase. mainly lytic microorganisms which are capable of producing glucose. therefore. 50 mg Following hydrolysis. Trichoderma koningii. centration of sugar in the product solution is generally above about 5% by weight which has been found to be too high a concentration to effectively support growth of the cellulolytic microorganism. A portion of the output stream.4 linked glucosyl bonds of the solubilized 4 zyme.775 (1950). The ground waste is then fed to adsorption stage 15 by way of line 16 where it is contacted with ?ltrate solution from the hydrolyzer reaction. the effluents are discharged by way of line 20 to ?lter 21 where a liquid sugar-contain ing phase is separated from a solids phase.2 to substan tially 5 mg/ml of enzyme protein. less than about 15% by weight. The ferred to dilute the solution to approximately 0. of the total supplied to the hydrolysis section. 79. The solution in the hydrolysis section is maintained preferably on the acid side. The wash water. The results of various other workers in the ?eld com bine to postulate an alternative hypothesis according to which it is the C1 component which delivers the initial attack on the cellulose chain by random action. can be used as a source of fuel for the reboiler Referring now to the drawing in detail. The con tion stage by way of line 17. (exo) mechanism producing cellobiose residues. 72. Temperatures much above sub (1962).) Of these. together agitated cylindrical concrete digesters of the type gen 60 with a suitable nutrient composition.05 M sodium citrate buffer at pH of 4. and the spent solids. as noted above. It has been found that an enzyme activity of from substantially 2 to substantially 3 Filter Paper units is most ef?cacious. Army Natick Laboratories in recovery of adsorbed enzyme by washing with process water at a temperature and pH corresponding to that in Massachusetts. 816 (1960). Overall cellulase enzyme recovery in adsorption stage 15 and wash train 25 amounts to at least 75%. The liquid stream containing The invention is illustrated in the accompanying drawing wherein the single FIGURE is a flow diagram desorbed enzyme is then fed to hydrolyzer 19 by way of of a particular mode of operation of the cellulose con line 28. Catalogue of described. Filter Paper activity is expressed as milligrams of are then fed to hydrolyzer 19 via line 18 as noted reducing sugar produced by the undiluted enzyme sam ple. such as waste paper. for the growth of fungal microorganism as hereinafter (See American Type Culture Collection. is removed by means of line 23 and utilized in the enzyme makeup section Fusarium solani. The amount of time required for hydrolysis de pends upon the concentration of the cellulase enzyme present. F iltrate solution is introduced into the adsorp 55 furnace in the enzyme makeup section and for steam/ power generation. Penicillium funiculosum. The liquid (l X 6 cm) of Whatman Filter Paper No. viria'e QM9414 way of line 24 and enter wash train 25. has been found to be especially produc the hydrolysis section. for a period of time suf?cient to 65 Mandels and Reese. waste cellu losic material. at a pH of from about 4 to about 6.. ' DETAILED DESCRIPTION OF THE INVENTION 45 line 29. and as much as about 85%.5 chain is attributed to the CI component of the enzyme. The liquid stream leaving adsorption section 15 via line 22 is the product stream of the process and consists of a mixture of simple reducing sugars.r components. that is. for example. Nutrient compositions suitable for submerged neering. effect conversion of a substantial proportion of the Bacteriol. In this test. 400 cellulose into sugar. Generally. J.3. pre . 20 tion stage 15 by means of line 17.5 ~ 3% ground waste is then conveyed by way of line 18 to hydrolyzer 19 which consists preferably of a plurality of by weight. pro— ducing fragments of lower molecular weight which can then be acted upon by the C1 component by an endwise to substantially 6 Filter Paper units which corresponds to a concentration of from substantially 0. Another suitable nutrient material comprising stantially 60°C will tend to denature the cellulase ‘en a mixture of the inorganic chemicals (NH4)2SO4. as noted above. Makeup enzyme is produced in fermentors 30 and 31 utilizing that portion of the output sugar solution re moved by means of line 23. It is. pref erably around 50°C.
3 — 1.. 95. Column bottoms are discharged via the following claims. The distillation feed can be preheated by heat exchange with column bottoms in heat exchanger 43 and can be further heated in reboiler-furnace 44 before its entry to the distillation column. to terial being substantially 10 mg/ml. an amount suf?cient to provide a cellulose concentration the fungus. of the output sugar solution is diluted to an approximately 1% solu tion and mixed with a nutrient composition comprising cottonseed oil and a commercial fertilizer comprising superphosphate. The spent ularly methyl or ethyl alcohol. is added to the cooled. To recover the adsorbed enzyme the filtered solids are washed in a series of countercurrent mixer-settlers and Cooling is preferably effected'by heat exchange with wash water entering wash train 25.. corresponding to substantially 82% conversion. for 40 hours is maintained. The plant feed is sterilized prior to introduction an amount of sterilized plant feed sufficient to provide to the fermentor. Following growth of the fungus the culture of about 0. The sterile medium is then fed to heat exchanger 26 via line 34 where it is cooled to a temperature suitable for fermentation. hydrolyzing the cellulosic material by the degrad liquid stream from the distillation column top before ing action of cellulase enzyme under conditions being reused for solvent precipitation. the concentration of the nutrient ma priate inducer which is introduced by way of line 35. (NH2)2CO2. partic is recovered from the ?ltrate by distillation.. waste paper containing substantially 6% moisture and having a cel lulose content of substantially 61% is shredded and ground to. the enzyme protein is precipi discharged from fermentor 31 by means of line 37.58% solution.775 KH2PO4.5% by weight in fermentor 31 is pre is transferred to an enzyme growth fermentor to which ‘ ferred. The presence of a cellulosic material is required.3. by heat exchange with condensing steam. can also be used. The growth feed is sterilized ‘at'a temperature of substantially 144°C and cooled to a'ternperature of substantially 30°C. is fed into mycelium growth fermentor 30 for a period of time suf?cient to exhaust essentially all of the sugars for growth of the 25 enzyme activity in the liquid phase is recovered by countercurrentadsorption on the incoming solid feed to the hydrolysis stage. Heating to a temperature of about 145°C will effec tively sterilize the growth medium. The precipitated enzyme is then fed is the preferred organic solvent. The 50 solids from the hydrolyzer following washing are uti precipitated enzyme protein is separated in ?lter 39 lized as fuel for the reboiler-furnace for heating the and fed into hydrolyzer 19 by means of line 40.200. described above. the volume ratio of solvent to liquid being of line 38 for enzyme precipitation. is utilized for solvent recovery in distillation column 42. A continuous process for enzymatically converting a cellulosic material to soluble sugar which comprises: Makeup solvent is added by means of line 46 to the a. Ser. prefer ably around 30°C. a portion of the feed from grinder 14 is 35 most feasibly utilized as the inducer. but for the pre sent process. Although acetone substantially 3:1. The hydrolysis temperature is maintained at substantially 50°C and pH at substantially 4. Enzyme is produced at substan tially 1. sterile medium. The cooled. 1 ' cellulase-secreting microorganism. The reboiler generation. Adv. The nutrient ma EXAMPLE . For an enzyme activity of 2. mesh size before being utilized for hydrolysis. line 45 and can be cooled by-preheating distillation What we claim is: l feed in heat exchanger 43 and then recycled to the wash water stream for introduction to wash train 25. myce lium solids are separated from the enzyme-containing liquid phase in ?lter 36. and thus it is not the organic solvent is recovered from the top of the intended to limit the invention except by the terms of distillation column. I a cellulose concentration of substantially 1% is added as an enzyme inducer. The growth medium is introduced into sterilizer 33 where it is heated to a suitably high temperature.The cellu losic material can be from any source. Following enzyme production.Solid concentration in the hydrolysis vessel is 10%. substantially 7%. Effluents from the hydrolyzer are passed through a ?lter to remove the unreacted solids from the enzyme 5 and glucose solution. sterile feed just prior to intro duction to a mycelium growth fermentor for growth of induce formation of the cellulase enzyme. Substantially all of the acetone than about 6 carbon atoms) ketones or alcohols. 65 1. The from substantially 20°C to substantially509C. Generally. An 45 tated from the culture broth by the addition thereto of organic solvent is added to the liquid stream by means acetone. temperatureof the-wash water fed back to the hydrolysis vessel. 391 (1969‘). > example. An inoc ulum of Trichoderma viride QM9414.972. the rest being adsorbed on spent solids.3 Filter Paper activity. a residence time of terial is added preferably in a concentration of substan tially 5 — 12 mg/ml of appropriately diluted sugar solu tion. . discharged from ?lter 39 by means of line 41. —. The feed to the distillation column and for steam/power liquid stream. The fermentation reaction is carried out at a. Various modi?cations and changes in the process of the present invention will fumace can be fueled by combustion of spent solids from wash train 25 as noted above. 55 Although the invention has been described speci? cally with respect to the enzymatic degradation of waste paper.Considering now a speci?c example. The liquid stream from the adsorption step is the product output of reducing sug ars as 5. After ?ltration to re The liquid stream containing the makeup enzyme is move mycelium solids. CaClz and PIO teose peptone is described by Mandels and Weber. it has b?emfound that a mixture of cottonseed oil as a protein “source and 6 a superphosphate-containing fertilizer of the type readily available on the commercial market is equally suitable and much more economical. Approximately 51% of the hydro lyzer enzyme activity remains in the liquid phase after ?ltration.‘ to which an inoculum of the desired microorganism is added in order to initiate growth. Substantially all of 60 be obvious to those skilled in the art without departing from the true spirit and scope thereof.8. in Chem. it will be appreciated that the present invention is applicable to the treatment of cellulosic materials from other sources. other lower alkyl (less to the hydrolysis vessel. thereby heating the wash water to hydrolysis -reaction temperature. . ‘Mycelium from fermentor 30 is fed into enzyme growth fermentor 31 ‘for production of enzyme in the presence of an appro ' A minor portion.7 Filter Paper.
5 to substantially 6 Filter hydrolysis stage to replenish the cellulosic material converted to hydrolysis products. and h. thereby recovering cellulase adsorbed on said sol 20 growing the cellulase-synthesizing microorganism in ids by solution in water. recovering a stream of product sugar solution from 6. adding the water containing recovered cellulase enzyme from step (f) to the hydrolysis stage. A process according to claim 7 further de?ned by sugar solution. d. contacting the liquid sugar-containing phase from step (b) with the added fresh cellulosic material Paper activity. f. g. c. in a second zone. A process according to claim 1 further de?ned by utilizing in the hydrolysis ‘reaction cellulase enzyme in an amount suf?cient to provide an enzyme activity in the range from substantially 0. phase containing unhydrolyzed spent solids. A process according to claim 7 wherein the or ganic solvent is acetone.7 3. A process according to claim 1 wherein the cellu hydrolysis stage. ing a liquid sugar-containing phase and a solid 5 3. thereby recovering the cellulase enzyme 15 maintaining the temperature during microorganism contained in the liquid sugar-containing phase by growth and during cellulase enzyme formation in the adsorption on fresh cellulosic material. and 2. prior to the introduction thereof to the hydrolysis 5. * * * * * 35 45 50 55 60 65 . ' b. range from substantially 20°C to substantially 50°C. e. said hydrolysis products compris losic material is waste cellulosic material.972. A process according to claim 1 further de?ned by stage.775 supportive of growth of the cellulase-secreting which promote conversion of cellulose to soluble sugar. 9. 8. growing a cellulase-synthesizing microorganism on a minor portion of the product the presence of a nutrient composition comprising superphosphate and cottonseed oil. washing the solid phase containing unhydrolyzed spent solids from step (b) with a stream of water. A process according to claim 1 further de?ned by recovering the makeup cellulase enzyme from the cul ture medium by adding to the liquid phase thereof an organic solvent to precipitate the cellulose enzyme therefrom. adding a stream of fresh cellulosic material to the 2. A process according to claim 1 further de?ned by step (d). providing a stream of makeup cellulase enzyme for 25 the hydrolysis stage by the steps including: 1. removing a stream of hydrolysis products from the microorganism. in a ?rst zone. 4. 7. contacting the microorganism containing medium with an amount of cellulosic material sufficient to induce formation of cellu lase enzyme under conditions substantially non 30 recovering organic solvent from the liquid phase by distillation and utilizing spent unhydrolyzed solids as fuel to supply process heat for the distillation step. A process according to claim 1 further de?ned by maintaining the temperature of the hydrolysis reaction in the range from substantially 30°C to substantially 60°C. both of said phases containing cellulase enzyme from the hydrolysis stage.
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