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    «2 United States Patent ‘Yang et al. 'US007604967B2 (10) Patent No.: US 7,604,967 B2 (35) Date of Patent: ‘Oct. 20, 2009 6 IGNIN-BLOCKING TREATMENT OF BIOMASS AND USES THEREOF (75) Inventors: Bin Yang, Hanover, NH (US); Charles E. Wyman, Norwich, VT (US) (73) Assignce: The Trustees of Dartmouth College, Hanover, NH (US) (4) Notice: Suibjoct to any dislaimer the tem of this patent is extended or adjusted under 35 US. 154(b) by 422 days. (21) Appl. Nos 10391,740 (22) Filed: Mar. 19, 2003 ws) Prior Publication Data US 200801855421 Sep. 23, 2004 (1) Ince CHP 706 (2006.01) (2) US.CL ABS/161: 530378; 435/68.1 435/99 (58) Field of Classification Search 435/99, 45/165 ‘Se application ile for complete search history. 66) References Cited USS. PATENT DOCUMENTS 4000075 4° 21977 Hoge ansiee 4257225 A 121980. Gretein 4586430 A 121985 Comverse tal ‘oo8-40 —“S1987 Shennan 408745 4111987 Hinge AR46A01 A S195. Roberts a SSOLT2L A KISSD. Waterbury ea Su2s977 A 61902 Grohmann el 5258203 A 11/1903. Lyad eal 528417 A 6.1995 Torget et 5503996 A 411996 Torgt $529,663 A 6.1996 Springer 5536328 A 71906 Bink Sloso74 A * 11/1997 Choietal ase 5705369 A V1998 Torgt 837.506 A LL/1908. Lym etal GrA19 A 22000 Towgte {606204 A $2000, Hester et a GAS9S10 122000 Lieak 718218 B2* 32006 Marcus etal 44322 FOREIGN PATENT DOCUMENTS @ 0.268 575 2 * S988 GB DTS A IWS Wo wossdsaT4 121994 OTHER PUBLICATIONS Minami etal Characterization of eared mediam fom subrerse and semisolid culivation of OF Aspergillus awamori NRRISLI2 By sizexchsion chromatography. Braz. J. Chem. Eng. vl. 16 No.2 un. 1999. pp. 17 ‘Wyman, hares E, “Biomass Ethanol: Techical Progress, Ope ‘unie, and Comercial Challenges". Anau, Rev. Eaetgy Eaton 1999, V 24 pp. 189-26, Wyman, Charles E. Spindles, Diane D., and Grohans, Kae. “Simulaneus Sacchaifcstion and Fermentaion of Sever Lignocellulosc Feedstocks 0 Fuel Ethanol", Bomass and Benen 1992, sol 3. No. Ss pp. 31-307, Pergamon Press Li Great Batin ‘Wyman, Chases F “Ethanol fom Lignocsluose Biomass Teck solos. Eeonomicssani Opportunities Bioresource Techaology 0, {99a pp. So, Flevier Seenee Limited, Gre Bran Wyman. CE. Spindle, DID, Grohmann, K. and Lasik SM *Stnulanous Sacharfation ant Fermentation of Celie with the Yast Brntanomsces lewseni™ Bitochnolog an Bioenineet Ing Symp. No. 17.1986, pp. 221-238 Wyman, Charles E, “Twenty Yeas of Tis, Tibulations, and Research Progress in Bioethanol Technology, Appi Biochom try and Botethnology, 001, 91-93; pp. 2k, The Humana Press Ine ‘righ John D., Woman, Chases End Grohmann, Karel, “Simul tandous Sacehaifcaion sod Permeation of gnocelllone, 18, Pp. 75.90, The Human Press Ine Spindler, Diane D, Wyman, Charles E.. Mohagheshi, CGroluna, Katel, “Thermotolerat Yeas’ fe Sinllzncous ‘harication and Fermentation of Celalose to Ethan” 1088, 279.295, The Humana Press Ine Spindler DianeD, Wyma, Cares F.Grohsnn, Kael nd Philip: pls, Goome P, "Ealution of the Ceobiose-Fenmenting Yeast Fistononscer easier inthe Simntanens Stechanision ant ant Feamonttion of Clllose", Brtechnolopy Tetes, May 1992,V. 14 No.5. pp. 03-407 Spindle, Diane D, Wyman, Charles End Grohmann, Karel, “The Slnuitancous ‘Scchaifation. and. Fermentation of Prsicted ‘Woo Copsto Fihana”, Applied Bichemisty and Biotechnology. fof Pereted Wheat Sra Hino th Selected Yeas! Stesns ad BGhucosidase "Supplementation". Appliod Biochemistry and Biotechnology 199, vol 2021, p, $2954, Spindler Diane, Wyman, Charles and Grohmann Karel “Eaaion ‘of Pretrete Herbceous Crops forthe Simultancus Scacchacic- tion and Fementtion Process, Apglied Blochemisty” and Bietchnoloy 199, vl. 2428, pp. 278-285. ‘Yang Bin, Boussnd, Abdel, Mafeld, Shawn D Grows, Davi J ant Sad, John N “Fast and Eficint Allain Pero Test ‘ment to Enhance the Enzymati Digest of Steam Exploded wood Subsuates, Biotechnology and biocapinerng, Mat 20, 2002, sa. 77,No. 6, pp. 678-684 (Coutinved) Primary Exaniner—Anish Gupta (74) Antorney, Agent, or Fiem—Lathrop & Gage LLP 6 ABSTRACT Disclosed isa method for converting celulose ina Tignocel- Indosie biomass. The method provides fora lignin-blocking polypeptide andlor protein treatment of high lignin solids ‘The treatment enbunces cellulase availability in cellulose conversion. Collulase eflciencies areimprovedytheprotein ‘orpolypeptde treatment, The treatment may be used in com- bination with steam explosion and acid pretydrotysis tech- niques. Hydrolysis yields from ign containing biomass are enhanced 5-20%%, and enzyme utilization is increased from 10% to 50%, Thus, a more ellciet and economical method of processing lignin containing biomsss materials wiles & polypeptcprotein treatment step that efletively blocks lig- nin binding of cellulase 20.Claims, 3 Drawing Sheets US 7,604,967 B2 Page 2 OTHER PUBLICATIONS Sevalt VIM. Glaser, W.G.and Beauchemin KA, Lignin Impact ‘on Fiber Degradation, 3. Reversal of Inhibition of Enzymatic iutoysis by Chemical Modification of Ligminand by Addives” ‘erie, Food Chem, 1997, vo. 4S, No.5, pp. 1823-1828, American ‘Chemieal Society Mohagheohi. 4. Tucker, M. Grohmann, K.and Wyman, C.. “High SoldeSimulancourStchaificationand Feeattion of rotated Wheat Saw to haga”, Applied Biochemistry and Bioechaolo, 1092, 01 33,99, 6781, The Homann Pres Ine Lu, Yanpin, Vang, Bin, Greps. David. Seder Joba N. and Mansfeld, Shawn D, “Cellule Adsorption and an Evaluation of Enayme Resyele Dring Hyolysis of Steam-Pxploded Softwood Residues”, Applied Bicchemstry and Biotechnology, 2002, vo. 98100, pp 641-664, Humana Pres Ine nksson Tony. Borjsin, Johan ant Temel, Foe, “Mechanism of surfactant effect in enzymatic hydrolysis of lignoelulose” Enzyme and Microbial Feshnology 2002,» 31, pp 353-364, Flsevict Science [ne mel, Mand Grohmann, Ks “ile Acid Prtreat Rotation Herbaceous Crops”, Applied Biochem= ‘ery and Bitechalogy 192. vol 435, pp. 115-133, Tho uaa Pr Ie Sutil, Roger and Sadi, John N, “The role of Lignin inthe ‘Ausorption of Celllases during Enzymatic Teeatmeat of Lignocel- lulosie Materia”, Biotachnology and Bioengineeing Symp. No.7 6p. 789-702 ‘Wo, Michael M, Chang, Kevin, Gregg: David Boussid, Abe easton, Rodge Pan Salt, Ja N. "Opiimizaton of Steam Explosion to. Enbance Hemicellslore Recovery and. Enzymatic inoys of Cellulose in Sofood” Applied Biochemistry and Biotechnology 1999, vl. 7-79, p. 47-4, The Humana Press Ic. ‘Grohmann, K Tong, Rand Himmel, M, “Dilute Acid Pretest ‘meat of Biomass at High Solids Concentrations, Biotechnology and Bioengincering Symp. No. 17,1986, pp. 138-51 ‘Chernoghzoy, Vladimir Ermolora,OkfV.and Klyossy, Anatole ‘A, "Adsorption of highpurify endo-L4-Prgcanases rm Tichoderma reeset on component of igncellosc materials cel- Tulse ann. and xan", Enzyme MGctob Technol, 1988, vol. 10 ‘August pp. 503507 ‘Boussaid, At, Robinson, Jamie, Cal, Visn, Greg, Davi J and SaddJohn N, "Fonneatbity ofthe Homiclllose Derived Sus aus Gm Steun-Explodod Softwood (Douglas Fu), Tat Cont ‘Biotechnol Pulp Pap Ind, 7 198, pp. €239.282 (Goold, Michael "Alkaline Peroxide elignfaton of rill [Residues 10 Eabance Enzymatic Sacchsfestion” Biotschaslogy ain Bioenginoering. 1984, vl. XXVI, pp. 046-083 lon Wiley 8 Sons, ne (Ghose, TK. Roychoodhuy, PK. and Ghosh, P, “Simultaneous Sas haifcation and Fermentation (SSF) of Lignocellulsis to Ehnol Under Vacuum Cyeling and Step Feoliag™, Biotechnology and Bioengineering, 1984 vol. XXVInpp. 577-181 John Wiley Sons Ine (Overeml, Pan Chomet, , “Factionaton of Ligocelalosics by ‘Steam-Aqucous Prctrstinents, Pileophial Transactions of the Royal Sosy of London, Series A, Mathematical ant Physical Se sence Apr 30 987, vl. 321, se 6. Technoony inthe 190 Unlzation of Eianocellulosic Wastes, p. 523-536, The Royal Soc 1 Sehwvald, W, Smid, Chan, M. Brevi, C. and Sadler, N. The influence of SO, impregnation an factionton om product recovery and enzymic hols of steamteated sprucenood", 1980p. 231-282 Goughian, MP, Elsivie NY ‘Kala Join Chang How Min, and Joo, Hasan, “The Reactions of Lignins “with High Temperature Hytiogen | Peroxide" oeforschung. 1999, vo. 53.No, 3p. 277-284, Waller de Graver, BeslinNew York, ‘Brooks, Ronald F and Belly, W. Dexter “Bioconversion of lat ‘Biomass to Ethanol Pye. Ann Fuels Biomass Symp. 2, 1978, pp PS1sP-S30 Boussaid, A Jarvis, 3, Greg DJ. and Saddles, 1N., Optimization oftlemicllulose Sugar Recovery fom Stem xplode Softwood (Douglas Fin, roe. Biomass Con ofthe Amerias, 2% Monte, ‘ug 2429, 1997, pp 87381 Oce Action dated Sep. 11, 2007 issue in related US. Appl. No, 1129817 International Search Report and Wrien Opinion dated Ju. 27, 2008 Issued in related PCT Patent Applicaton Seal No. PCT S208 costs. Tena. Engl Translation of EPO2SSS7SA2, May 25, 1988 * cited by examiner US 7,604,967 B2 Sheet 1 of 3 Oct. 20, 2009 US. Patent 1old | Suiss2001g " aekoay, / a ue L spios i sousptey mie cen on J | a U.S. Patent Oct. 20, 2009 Sheet 2 of 3 US 7,604,967 B2 100) 40 alpha-cellulose | ‘¢—alpha-cellulose plus BSA conversion,.% Time, hours FIG.2 Me Relative FPA acti wp BO ceo ro seg Time, hours FIG.3 U.S. Patent Oct. 20, 2009 Sheet 3 of 3 US 7,604,967 B2 _2diding cellulase alpha-cellulose plus BSA —e- alpha-celluiose plus 8SA and cellulase 10 20 20 a so Time, hours HIG 14 1240 hh E10 \\ ED een e 08 5 3 ‘ —=— C81 plus BSA and cellulase | 08 \ @— CS1 plus cellulase j Boa 2 Bd Time, hours FIG.5 US 7,604,967 B2 1 LIGNIN-BLOCKING TREATMENT OF BIOMASS AND USES THEREOF GOVERNMENTAL INTEREST The United States Government may have certain tights i the present invention as research relevant to its development was funded by United States Department of Energy (DOF) ‘contract umber DE FC36-O0GO010889 and DE. FC36- 1GOL1075, BACKGROUND OF THE INVENTION |. Field ofthe Invention ‘The present invention pertains tothe field of biomass pro- «essing to produce fuels, chemicals and other useful products ‘and, mote specifically 1o saccharifying lignocellulosic bio- ‘mass materials to produce sgars for conversion to ethanol and other produetsby enhancing the effectiveness ofeelulase through selective binding or blocking ofthe lignin compo- nent. Use of a protein wash enhances bioconversion effi ciency by increasing the availability of cellulase and other ‘enzymes to cellulose 2, Description ofthe Related Art Cellulosic biomas is useful for generating ethanol, Such materials specially known as lignocellulosic materials, of biomass, (eg. wood and solid wastes), have been used as source material to generate carbohydrates, which in ten may be used to produce ethanol, 2s well as other products. Lignocellulosic biomass isa complex structureof cellulose fibers wrapped ina lignin and hemicelhulose sheath. Te ratio ‘of the three components varies depending on the ‘ype of biomass. Typical ratios ae a fllows TABLE 1 cone ‘CORN SOFTWOOD COR ROE _ STOVER ‘Table | is only an approximation, For example, wood dif {ers in composition, depending on the particular type of ‘wood, where softwoods, (gymnosperms) generally have ‘more ghicommanans and less ghucuronoxylans than do hard- woods. Cellose isa polymer of D-ghicose monomer with f-1-4- Finkages benweed each monomer Forming.eins of abou 500 ‘10,000 D-glucose units. Hemicellulose is a polymer of sugars, primarily D-xylose with other pentoses and some hexoses, also with i-1-4-inkages. Lignin is & complex ran- ‘dom polyphenolic polymer. Lignocellulose biomass repre sents an inexpensive and readily available substrate for the preparation of sugars. These sugars may be use alone, fer- mented to produce sleohols and industrial chemicals, oF ‘chemically converted to other compounds Ethanol is one of the alcohols that may be produced using ‘carbohydrate derived froma lignocellulosic biomass, and has ‘numberof industrial and fuel uses. OF particular interest is the use of edhanol as a gasoline additive that boosts octane, redees pollution, and partially replaces gasoline in fel mix- tures. Fthanol-blended gasoline formulations are well-known ‘commercial preduets commonly called “gasohol” has heen, 0 o 2 proposed to eliminate gasoline almost completely from the fuel and to bur ethanol in high concentrations ‘Conversion of cellulose biomass ino renewable els and chemicals often involves chemical andor enzymatic re ‘ment of the biomass with cellulase or other enzymes. In particular, cellulase enzymes hydrolyze cellulose to D-ghi- ‘cose, whieh sa simple sugar. In high lignin content ignocel- Tnlosic biomass, high doses of cellulase are nceded to degrade the cellulose with high yields because the lignin binds pref= creataly withthe cellulase, thereby reducing access of cel Tulase to cellulose. Consequently, when processing high lig- in content biomass materials, less cellulase is available to degrade cellulose because the lignin coating ofthe cellulose Iihersseavenges cellulase Ths, the effectiveness ofthe pro- ‘cess for digesting cellulose is reduced. TBioconversion of cellulose biomass to ethanol has been studied since the 1940's. However, the cellulose-to-ethanol process is not yet economical compared to prodcing petro- eum products by existing technology. Enzymatic hydrolysis isa firly slow process. The costs of cellulases are high, and the required amount of cellulases is also high, which increases processing eosts. Reduction in the amount of cel Inlase nected to obtain a satisfactory sagar yield can have a significant impact on process economies. Therefore improv- ing the efficiency of enzyme use is major need in the bio- conversion process ‘The mechanism ofhydrolysisand the relationship between te stricture and function of various cellulases have been extensively studied, Several factors are thought to influence enzymatic hydrolysis of cellulose, These feetors inlude lige tin conteat, hemicellulose content, acetyl content, surface area of cellulose and cellulose erystllinity. Its generally ‘understood that the lignin present in complex substrates, sch as steam-exploied wood, especially softwood, has a nega tive effect on cellulase activity. The exact reasons are poorly ‘understood because the complexity of biomass is suet hat ‘ecg one harrier to digestion ean enhance or disguise the ‘importance of others. For example, cellulose hydrolysis has been showin to improve with increasing lignin removal, although differences are reported in the degree of lignin ‘removal needed. ‘A variety of factors may be associated withthe deleterious effets of lignin upon saccharification. The ratio of syringyl moiety to guaiaeyl moiety inthe lignin may alfet soectar- ication, Although the exact role of iin ia Fimiting bydeoly- sis has beon difficult to define its probable that one of the ‘ost significant limitations isthe effect of Tignin on her swelling nd its resulting influence on cellulose accessibility. ‘The removal of lignin inewases accessibility of cellulose and allows more cellulase activity: Lignin complexes may be physically and chemically resistant to enzyme attack. While Some lignin components are water soluble, others are insoluble und may preipitate from solution, Condensed lig- nin has the ability to adsorb protein from aqueous solitons. Lignin removal may open more surface area for enzymat attack and reduce the amount of eellase that is non-3 cally adsorbed on the lignocellulosic substrate. Studies ‘volving aid pretreated softwood report a positive correla- ‘ion between digestibility and the extent of deisnifcation, ‘but the results are eompicate by the presence of hemicel Jose. Some substrates require higher temperatures for hemi cellulose removal to be effective, suggesting that hemicel- Jose isnot the only additional factor impacting digestibility, And other evidence does not suppor a ole for hemicellulose in changing cellulose digestibility. ‘Although celllose crystallinity is generally reasoned to impede enzymes, rates slow with increasing crystallinity US 7,604,967 B2 3 some studies but increase in other studies, The degree of ‘rptallinty may not significantly change ove an extended hydrolysis time Crstalinity seems les important than Fig- nin rmovel and to impact rates more than yield, Several states have focused on explaining celulose digestibility by the accesibiity of cellulose to enzymes. Corsations have heen developed o relate rates to poe volume and aeessible surface area However the complex shape of celles may ‘test dificult in penetrating sc pores, nd concems have boon mised sbout substrate changes daring these measte= ments Cellulases are composed of a mixture of enzymes having stiffen activites, and the enzyme strstre differs boseeen ‘nieroorganisss, While the mectsnisms of hydrolysis and the relationship between the strvture and funeton of various ceaulses have been extensively stdied, many details of ‘enzymic activity ar stl poorly understood. The enzymatic hydrolysis of cellulose substrates is tong allected by end- product inhibition and enzyme festres. Low specifi el Ins activity on ellulese isan important factor ta iit the effectiveness of hydrops One way to citumvent this low Speci activity so recycle and reuse the enzyme. However on-peodctve cellulase adsorption plays an important role in the development of ways to reuse enzymes and allets recycle efficiency Besides the complexity ofthe diferent types of cellulase, activity on the substrate is also complicated by substrate ‘characterises, Deo esistance fom te complex strut and composition oF nat ellloic biomass the ignocel- Tulse substrate shouldbe preteatedto make ita susceptible as possible tothe action othe enzymes. Many prtreatmcat methods have been developed. Forexample, incensed aces "bility of Ranceelislone substrate canbe achived hy so Bilizng hemicellulose in has ie conditions Callas adsorption om igcelllosic subsites conte ‘ing high content of natural materials has nt been extensively stati. Typically igocelilsie substatos contain a mich higher content of ignin compared to "me ceuose sub- stats. Ligni may inhibit enzymatic hydrolysis of gnoce- Jufosc material Celfulases are nt only adsorbed 10 the cel Jolosie prt of the substrate, but als adsorbed othe igi Lignin ho only shel the clilose but alo acts s a come polite adsorbeot. However lignin docs aot pear to restit the extent of hydrolysis of the carbohydrate moiety if suf ‘ial cllulase is prsent. Cellllytic enzymes bind tral tolignin, Whenadsortion piles are compared, much more ceazyme protein is associated with hydrolyzed resides of Fanocooosie materials than that of mod elulose Bal ‘etsidase has ahighafnty fb various nin faeGons, wile it doesnot bind to polysaccharides. The imeversible asorp~ tion of specific cellule components isnot observed in the prong hydrolysis of stand shirakamba wood eontain- dng abundant ignin, I unclear whether the adsorption of ‘llulses on lignin results om pectic ornon-spocifibind- ing. When the Hain is extracted from a seam-exploded aspen wood with water and alkali prio o hydrolysis elle Jase is sil found tobe adsorbed 0 the Hin ‘ignin plays an important ole in enzymatic hydrolysis of Fgnoceolsie material, as reported in Suelife & Saddle, Biotechnol. Bioeng. Symp. 8%, 17:749-62 (1986). Compars- tive sorption profiles demonsete that much more enzyme ‘vas retained with hyd esidass, compared to that of model pure cellulose, a reported in Abdel & Sadler, Int Conf. Biotechnol. Pulp Pap. Ind, 7, C239-C242 (1998) In ‘a study hy Chemoglaaow et. a Enzyme Mier, Teco 10803-507 (1988) endoglucanases that sorbed on Hani Jost activity. The inaetivating effect of Tian was observed 0 o 4 also with steam-exploded substrate, but not ifthe latter was acid-treated, nor with the lignocarbohydrate complex. Su life et al, Biotechnol, Bioeng., 17: 749-762 (1986) report that adsorption of cellulases on different lignin preparations from steam-treated hardwood is influenced by the nature of the lignin and Peglicosidase was most affected by lignin Tus, different types of Tgnin an forms of Hignin may inf cence cellulase adsorption. Also the form afte liga, which ‘contains distinct lignin and ignocarbohydrate complexes, socms to influence celislases differently. It is generlly ‘agreed that dhe form and positioning of mos ignin changes falter steam-explosion, such that de lignin separates from cellutoge to form agglomerates ‘Several proposals have heen made foe saving the problem of ineffective andor inefficient enzyme degradation of high Tignin containing biomass materials. One of these isa pre- treatment step that degrades or removes atleast portion of the hemicellulose andior lignin from the biomass. For example, a combination of heat and acid pre-treatment ofthe lignocellulosic mass for a period of time has been used to hydrolyze hemicellulose. However, this process provides for only very Timited removal of lignin, as reported in Grohmann ft al. Biotechnol. Bioeng. Symp. 17. Symp. Biotechnol Puels Chem, 8", 135-151 (1986) and Torget eta, Applied Biochemistry and Iiotechnology, 34-35:115-123 (1992). Lignin removal from cellulosic ibers has also beea peo posed though using # caustic alkali, such as in Krat pulping ‘and paper making, However, this process does not produce simple sugars and does not separate the hemicelose from the calles, ‘USS. Pat, No. 4,668,340 issued to Sherman relates to bio mass hydrolysis processing that produces almost exclusively ‘hemicellulose sugars. Acid is intreduced to the biomass, and js removed from each stage to be fed to the next in its soquence. The hydrolysis of cellulose is minimized in the process, and results in a cellulosic pulp containing over 90% ofthe feed a-cellose USS, Pat. No, 4,708,746 issued to Hinger relates to the specific hydrolysis of cellulase followed by treatment with high-pressure steam. However, the use of high steam alone doesnot provide forthe complete hydrolysis af the cellslose substrate US. Pat. No. $125,977 issued to Grohmann eta. and US. at. No, 5,424,417 issved to Torget et a, relate tothe prehy- rolysis ofa lignocellulosie biomass to solubilize the hem cellulosic sugars with concomitant release of some soluble lignin, Prebydrolysis renders the remaining cellulose more readily digestible with enzymes or other chemical means US. Pat. No. $424,417 describes a process wherein ligno- cellulose is subjectecl to prehydrolysis step by passing an acidic or alkaline solution tough solid of lignocelilosic particles, with the continuous removal of soluble reaction products The technique permits less severe combination of DH, temperature, and time than conventional prehydrolyss Extraction of hemicellulose and lignin occurs simultaneously ‘nthe same resctor and under the same eokitons. US. Pat, No. 6,022,419 issned 10 Torget etal. relates to a process in which a lignocellesie biomass is fractionated by using a dilute aid, e dilue sulfur acd at 007 wt %,t0 convert cellulose into monomeric sugars in relatively high yields. However, cellulose hydrolysis sing an acid catalysts ‘costly and requires special equipment. Inedliton the desired sugars are labile to the harsh conditions, and significant mounts of unwanted and toxi by product typically form. IF exposed 100 long, the glucose derived from the cellulose ‘depradesinto hydroxymethy frfarl, which further degrades into unwanted degradation products ineluding levulinic acid US 7,604,967 B2 5 ‘and formic acid, The acidic conditions similaely degrade xylose, which i formed from hemicellulose ‘WO 9429474 issued to Hinman relates to a process in ‘which a treatment of lignocellulose minimizes binding of cellulase. A subsirate is Formed of cellulose, hemicellulose, and starch. A hydrolytic acid pretrestment agent is added 10 the substrate, aia lignin peroxidase to block lignin binding sites in the biomass. Cellulase is added to the substrate using Simultaneous Sacclurifcaton and Fermentation (SSP) pro- ‘cess conditions favorable for ell vibilty and conversion of ‘ethanol ‘Kacal eta, $3: 277-284 (1999) relates to the we of per- ‘oxide treatments to remove lignin under alkaline conditions ‘during pulp bleaching. Under alkaline conditions, hydrogen peroxide reacts with both aliphatic and aromatic strictures of Jignin, leading to depolymerization and subsequent removal with water washing. Gould, Biotechnol. Bioeng., 26:46-52 (1984), reports the use of alkaline peroxide to remove lignin ‘and improve enzymatic hydrolyzability of herbaceous res ‘dues. Ramos eta, Holzfoeschung 46:149-154 (1992), report theuse of alkaline peroxide o steam explode hardwood. Yang ‘et al, Biotechnology and Bincngincering 7716): 678-684 (2002), report the use of alkaline peroxide treatment 10 ‘enhance the enzymatic digestibility of steam-exploded soft wood substrates ‘Generally softwoods have been considered the worst-case scenarios as @ feedstock for the biocoaversion processes bocanse ther highly calitrant lignin reduces the efiiency ‘ofenzymatic hydmolyss. Schwald etal, Enzyme Systems for Lignocelluosic Degradation, Googhlan, M. P, Elsiviee,N-Y. pp. 231-242 (1089), and Wu etal. App. Biochem. Biotel~ nol, 77-79, 47-54 (1998), report that compromise in the pre-ircalment conditions wil likely be required, i softwood residues are 19 be considered as a potential feedstock for biomass processing. ie, amedium severity processisneeded between those optimized for high hemivelislose recovery and elfcient cellulose hydrolysis, According to the aforementioned pretreatment processes, cellulose substrates produced by pretreatment at medium severity (about log R,-3.76) containahigh lignin conten that Timits cellulase accessibility to celulose. The term “R,” is used in the industry as an indicator of the relative severity of ‘treatment method forthe processing ofa biomass. Specifi- aly, nthe field of ignocelinlosics and fractionation of wood ‘componeats,"R,."has been used to define a “severity param- ‘eter This equation is desribed in Overend, R.P.&Chomet, E, (1987 Fractionation of lignocelluosies by steam-agueous protreatments. Phil. Trans R. Soc. Lond, 523-36. oreo P10)1478} o where isthe severity factor and is optimized a 3.8 forthe prehyrolysis of hemicelulose ts time of exposure in min- Utes, and Tis temperature in degrees Centigrade, SUMMARY OF THE INVENTION ‘The present invention advances the at and overcomes the problems outlined above by providing an improved and more ‘ffcient method for enzymatically hydrolyzing high lignin= ‘content biomass. These advantages ar obtained without nec ‘essarly subjecting the biomass to harsh of osher reaction ‘conditions and, further by a process that avoids significant production of toxie and unwanted degradation by-products In one embodiment, the method utilizes a protein andor polypeptide that preferentially binds with ignin more readily than cellulose. A high lignin-content biomass is treated with s 0 6 the lginin blocking protein andor polypeptide, for example by wathing the biomass with a composition that comprises the lgnin-blocking protein and/or polypeptide or by adding such materials toa saccharfication broth. The lignin-block- ‘ng polypeptide and/or protein preferentially. bind and thereby impede the lignin from further binding. Celhlose- hydrolyzing enzymes, such as celobiohydrolase and gh casidase, may then hydrolyze cellulose more efficiently and rapidly. Without teatment of the lignin-contsining biomass ‘with a lignin-blocking polypeptide and/or protein, lignin in the biomass imeversibly binds portion of the cellulose hydrolyzing enzymes, rendering them unable wo hydrolyze cellulose, Protein andior polypeptide treatment effectiveness ‘through lignin binding, thus reducing and/or eliminating ron. roxuetive adsorption ofthe enzymes, The treatment of bio nass with a lignin-blocking protein and/or polypeptide thus ‘improves processing of relatively high lignin substrates by circumventing alliity of lignin for the enzymes. The polypeptide wash reduces enzyme use andor improves per- formance because the enzymes do not become bound to the lignin, and remain available to hydrolyze the biomass. Tn one aspect the present method reduces enzyme loading in hydrolysis of high lignin content hiomass. The amonnt of enzyme, sch as celine, that s needed to provide bydeoly- sisi significantly reduced though tating the biomass with a Tignin-blocking protein and/or polypeptide. These avan- tages reduce the overall costs of biomass conversion pro- “According to one embodiment, the method enhanees the ‘enzymatic digestibility ofellolose. This method includes the stops of treating a high lignin biomass witha lignin-blocking polypeptide andr protein to provide treated biomass hav- ng # blocked lignin component, and exposing the treated biomass to an effective amount ofa hydrolyzing enzyme, By ‘way of example, the hydrolyzing enzyme comprises Brghi- cosidase, cellobiohydrolase, endoglucanase, or a combina tion thereof ignin-blocking polypeptides and/or proteins that are use ful oe these poepases inclade any polypeptide andlor protein, or lignin-blocking fragment thereof, having an affinity for lignin, and especially, for example, bovine serum albumin (BSA), soybean protein, amylase, chicken egg albumin, and combinations thereof, Lignin-blocking polypeptides andlor proteins may be any polypeptide or protein that doesnot hve “appreciable binding lliniyforcelluose. By way of example, Tignin-blocking polypeptides andor proteins may have 3 ‘molecular weight ranging from 2,000 Daltons t© 300,000 Daltons. In some embodiments the range may be that of a relatively high molecular weight, ranging from $5,000 Dal- fons to 80,000 Daltons, eg, that of an albumin, However, lignin-blocking polypeptides andor proteins having alower molecular Weight are als envisioned as useful in the practice ff the present methods, These smaller lignin-blocking polypeptides, for example, may comprise a peptide fragment ‘comprised of amino acids thats capable of effectively Block: ‘ng or otherwise interfering with binding sites onthe li "The lignin-blocking materials, suchas polypeptides, ro teins, and fragments thereof, are not molecules tht are oth- envise intrinsically available toa igain-contaning biomass. ‘he lignin-blocking materials are usually provided ina rel tively purified and isolated preparation of such materials and jn concentrations that are not present in nature. Ths, an incidental presence of protein andor peptide, eg, ina sae~ charification or fermentation media, wotld not provide the lignin-blocking action ofthe herein defined preparations. The lignin-blocking polypeptides, proteins and/or lignin-block- ing fragments thereof are provided tothe Biomass asan exter US 7,604,967 B2 7 nally supplied source of material not inherent to the native milieu of « biomass under onfinary circumstances, absent Jnervention by he hand of man ‘The lignin-blocking polypeptides and proteins may be pre pared in a composition with water, for example, The ighin- blocking polypeptide or protein that is used inthe testing sep may include a relatively low concentration of lignin- blocking polypeptide and/or protein, far example, 1% ofthe Jignin-blocking polypeptide andr protein by weight of the ‘composition, or from 1% to 5% by weight ofthe composition, “The methodology employs compositions of ignin-block- ing polypeptide andior protein, as well as composition of a cellulose hydrolyzing enzyme, such as cellulase. As used here, 2 composition is defined as including a colloidal sus- pension, lgtid phaseof a mist, liqudlsolid mist suspensions, Vapor mixtures, and/or a solution that includes the lignin blocking protein andor polypeptide or @ lignin-blocking fragment theroof, ign is @ phenolic polymer that can be derived by the dehydrogenative polymerization of eonifery|aleohol andi singpyl alcohol. Lignin has water-soluble and non-water foluble forms, Both water-insoluble and water-soluble Fignins absorb polypeptide and protein, Lignin presents no0- specitie adsorption sites for polypeptide and protein binding ‘with, for example, lignin-teating polypeptides and proteins Tike bovine serum albumin and chicken exg alfumin. Con ‘dense lignin has the ability to absorb polypeptide and pro~ ‘ein from aqueous solutions, Dikydroxyphenyl groups and Phenolic hydroxy! groups ofthe lignin molecule form bind- {ng sites that may e sed to bind with andlor precipitate protein, Many different proteins can, therefore, be wsed to bind lignin and enhance enzyme access to cellulose in a biomass ‘By way of example, lignocellulosic biomass having high lignin contents defined a a biomass that comprises at last '5%% by weipht lignin, atleast 10% by weight lignin, atleast 20% by weight lignin, atleast 40% by weight lignin, irom 5% ‘to 50% lignin, or from 10% to 50% by weight lignin, Process ‘conditions for hydrolyzing cellulose ar, generally, a tem= perature ranging from about 120° C. to 240° C.,a pressure Fanging from about 12 psig to about 470 psig, and acid con- ‘centration ranging from 0 105% by weight TInvarious embodiments the lignocellulosic biomass com- prises wood, agricultural and Toresey residues, grasses, ‘municipal wastes (paper mill eMlvent, newspaper cardboard ‘etc ), oF combinations thereof, For example, the lignocell Josit’ material may comprise birch, Dougls fir, corn stove, siraw or combination thereof. These materials may be sub- Jected to other preprocessing that decreases or increases the Fignin content, for example, eflvent fom a paper mill. Thus, the method is upplicable to environmental remediation pro- ‘esses, a well as those intended to produce ethanol from fel ‘The ignin-blocking polypeptide and/or proven treatment of biomass may occur simltaneousy with the addition of @ cellulose-hydrolyzing enzyme to the biomass. A lesser ‘advantage in conversion efiiency may be provided. Tis ‘eavisioned however, that fist teatinga biomass witha lignin- blocking polypeptide and/or protein, o lignin-blocking. a= ment thereof, ad then adding the cellulose hydrolyzing ‘enzyme provides the most eflicieney in eolulose conversion. ‘Treating a biomass witha lignin-blocking polypeptide and! ‘or protcin, eg. by washing with a protein solution, may be followed by aiding cellulase, or an enzyme of similar cello- Jose hydrolyzing activity. This treating step produces hydrolysis yield from the cellulose that may be measured as percentage improvement in cellulase eonversion efficiency. By way of example, a 20% improvement in pereentage con 0 o 8 version ofthe total cellulose wo carbohydrate may be obtained in comparison to the hydrolysis yild from cellulose of biomass thats ot treated witha lignin-blocking polypeptide ndior protein. As used herein, the term “a lignin-blocking polypeptide andior protein” means any protein capable of providing a comparative improvement in cellulase conver sion efficiency by binding with lignin to ineresse the svi ability of hydrolyzing enzyme. Saccharifeation of high lige ‘in content substeaes often benefits by at least a 5% ‘improvement in conversion eficiency. Sill other embodiments pertain 1 improved processes for producing an organie compouad rom a high lignin-contain- {ng lignocelllosie biomass, The washing or lignin-blocking polypeptide andor protein treating step may be precesd, for ‘example, by a hydrolyzing step of contacting the lignocelh- Tose biomass with acid and steam To provide a treated said biomass with a grester lignin component. The hydrolyzed biomans is then washed and trated with a lignin-blocking polypeptide andor protein. This lignin-blockng treatment is followed by adding an effective amount of « hydrolyzing tenzyme unter conditions that are suitable for hydrolysis of the cellulose to produce carbohydrate at an efficient high ate ‘The effective amount of hydrolyring enzyme for a lignin- blocking polypeptide andlor protein-teated biomass, for example, is at least 25% less than the effetive amount of hydrolyzing enzyme require for a similar conversion yield roma lignocellulosic biomass that is not weated with Hignin- blocking polypeptide andor protein. ‘Process steps in adliton to the hydrolyzing step or steps ‘may include extacting the carbohydrate, fermenting the cat boiydrate in the presence ofan ethanol-converting microor- gzanism fora period of ime and under suitable conditions ina “ng ethanol andl extracting the ixture, Extraction may ooct, for ‘example, by ultrafiltration andr frctional distillation. Cel Iufase-performance measured as a minimum cellulase eon- centration required o achieve atime-to-targetealulose com version are improved rom 5% to 73% oF fom 20% 10 75%, ensured asa percentage difference compared to other pro ‘cesses that donot provide fora lignin-blocking proteinandor polypeptide treatment of the biomass, ‘Additional embodiments ofthe method comprise mixing particulate biomass having a hgh lignin content with a sul- ficient amouat of am aqueous acid to produce a wet meal of Tignocellulosi biomass, heating the biomass to remove hen celfose, cooling and washing the sof, introducing a sufi cient amount ofa lignin-blocking polypeptide and/or protein {0 the residual solids to produce a treated biomass with a blocked lignin component, and adding an effective amount oF a hydrolyzing enzyme to the teated biomass to provide car boiydrte Substrates protested under higher severity are more aoees- sible to cellulase enzyme, but have lower recovery of the hemicllulose-derived sugars. By contrast, pretreatment st lower severity conditions generally liberates hemicellulose- erived sugars, but generate solid reside that i pt readily amenable to the hydrolysis of eellulose. Primarily, adding protein in the cellulase solution can increase stability and prevent denaturation of eelulase. This effect in Hignocellulose hydrolysis is explained by the pro- ‘ein's ability to block the non-specific adsorption sites of ron-cellulose fraction of the substrate and enhance the Amount a cellulase availabe to absorb onthe celllose fine- ‘ion. Lignin afinityforeeulase may be blockes by protein in the three ways (1) lose physical association with Hignin; 2) hydrophobie groups adsorption to lignin; and US 7,604,967 B2 9 @) precipitation involving dihydroxyphenyl groups and phenolic hydroxy! groups of lignin As co tbe latter mechanism, lignin isa complex phenolic polymer that may result Irom ibe dehydrogenative polymer ‘ration of conifer alcohol andor sinapyl alcohol. Both ‘water-insoluble and water-soluble lignin adsorb protein. Tae ‘sorption capacities vary depending on the different pre- treatment methods and feedstocks, Furthermore ests show that added protein at low concentrations does not effect the rateothydrolysis, which suggests that protein has no effecton the catalytic mechanism of the celluolyti enzymes, There- or itis key tat protein blocks the non-specific adsorption sites on lignin to prevent tnprodctive binding of celases ‘on lignin. The resulting improvement in bydrolysis may ‘occur by introducing negative charges onto the Hignin surface ‘due to adsorption of protein. In tur, the negative charzes prevent binding of negatively charged hydrolyzing enzymes. ‘Without being bound by theory, ts believed that nonspecific binding of protein to ignin decreases unproductive binding oF cellulases to lignin surfaces, Use of protein treatment in 3 process for lignocellslose conversion advantageously fac tates a lowering ofthe cellulase loading level to acbieve the same tanget conversion percentage. For example, in the stud- Jes reported below, it was possible to lower the enzyme load- ing by 50% to achive the same level of hydrolytic cellulose ‘conversion with addition of proein at 2 pL to pretreated Tignocelfulose substrates. [BRIEF DESCRIPTION OF THE FIGURES FIG.1 isa schematic diagram showing process equipaient that may be used according to one embodiment that uses BSA protein washing for Iignocelfulose conversion FIG, 2 shows solution concentration changes that result ‘rom hydrolysis of @-cellulose, in comparison with and with- ‘out protein (BSA) adalition. IG. 3 shows filter paper activity (FPA) comprising ‘changes during hydrolysis of corn sfover with and without protein addition, FIG. 4 shows total protein in supematant during hydrolysis, ‘of excelislose with and without protein addition FIG, 8 shows protein in supematant during hydrolysis of ‘corn stovee with and without protein adiion DPTAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS, ‘There will now be shown and described a process for increasing process efficiency in making useful products out of high lignin content lignocellulosic biomass. Eificieney is improved by treating the biomass with a lignin-binding pro- ‘einand orpolypeptide.Insome embodiments, this saccom- plished with a protein wash ofthe biomass, Protein binding to Jignin renders the lignin less available to bind cellulase or ‘ther cellulose-hydrolyzing enzyme. Thus, more cellulase is available te hydrolyze cellulose ina protetetreted biomass, fn less celle is ultimately needed to provide a higher ‘yield of component sigas from the biomass. The process is ius much more efficient than those inthe prior ut In adie tion, hydrolytic aetvity occurs with greater speed “The following discussion provides specific instances of this process demonstating the instrumentalities according t0 the various embodiments by vay of example, and not by nmitation, TIG. 1 shows one embodiment of reactor system 100 that ‘may be used for biomass conversion. pile of lignoceilosic ‘material 102i conveyer to chopper mill 104 by the action af ‘levator 196 The chopper mill 104 chops andor grinds mite rialof the lignocellulosic biomass pile 1020 predetermined size that is suitable for downstream processing, A serew 0 o 10 extruder 108 transfers the chopped lignocellulosic material trom chopper mill 104. Steam 110 may be added to serew exinider 108, which may be configured to proloce a steam ‘explosion in the lignacelhulosie material 102 for example, by pacessing the lignocelhulosie material at high pressure sul- Ticket to prevent boiling and temperature of 120°C. 0 240° C. fora time ranging from one minute to sixty minutes oF more. The sew exter 108 optionally slurries the chopped lignocellulosic material with an acidification solution 112 that contains, for example, from 1% t0 5% by weight of sulfuric wed mixed to homogeneity in water eg. to produce fa pllof 1.210 14, The dischange fro serew extruder 108 is ‘ished info residence tank 114, which is maintained at a temperature below 100" C, to cool the material and slop Trther reaeto Residence tank 114 discharges into serew conveyor 116, ata first three Way mixing station 118 mixes the slurry witha lime solution 120, eg, one with sulicient Time to impart a predetermined pli of 10 to 11. The slurry is di charged into a solids holding tank 122 where it resides for an fppropriate ime permiting the lime to remove deleterious byproducts of acid hydrolysis. Additional seid 124, such as sulfuric acid, may beadded into the solids balding tank 12210 Aust pH intoa ringe ftom 50 7. The solids holding tank 122 discharges into a second three way mixing station 126 for jurther mixing witha prewash solution 128 that contains @ Jignin-blocking protein and/or polypeptide, eg, one impan- ing a 1% to 3% lignin blocking protein andor polypeptide content by weight of the slany. Turlher mixing occurs {hough turbufator 130, which discharges into a thin! three way mixing station 140. Tn tum, the third three way mixing station 140 introduces ‘an enzymatic solution 142 that contains & prehydrolyzing fenzyie, for example, cellulase or a mixture of cellulase and ‘ther enzymes including glucosidase. Alternatively, the enzy~ ‘atic soliton 142 contsins a inoewlim and growth meses ‘including 2 microorganism capable of saccharifying the sluny for hydrolysis of eelluloseby the in vivo prodiction of such enzymes, The slurry travels toa heated hydrolysis rene- {or vessel 144, which may be one ofa series of such reactor vessels, foran appropriate residence time permitting hydeoly~ sisof the slurry. Forexample, this residence ime may be rom ‘one fo three days. A series (not shown) of hydrolysis reactor vessels 144 may permit continuous batch processing. ‘Slury discharge from the hydrolysis reactor 144 may be subjected to additional mixing ea fourth mixing station 184, ‘whieh adds second enzymatic solution 186, such asamicro- ‘onganism-containing en¢ymatie solution or an aqueous solt- ‘don with addtional enzymes useful for conversion processes, ice the eonversion of ars int aleohols. The second enzy- ‘matie solution 156 reacts in @ converter reactor 158, lor ‘example, to convert sugars into alcohol or other organic com pouinds, Discharge from converter reactor 158 may be sh- ited fo a vortex separator 160, which discharges solids to "Waste disposal where the solids may, for example, be used as ‘boiler fe, Liquids from vortex separstor ae submitted to cally, protein pretreatment followed by hydrolysis using a lower cellulase concentration (mg/ml) was able wo achieve the same conversion eliciency as did a higher cellulase concen- tration in eases where there Was no protein pretreatment. Relatively greater amounts of enzyme were saved. With increasing amounts of Iignin content of the substrate, The resull fom this study demonsirate that the proein treatment improved the level of cellulase enzyme hydrolysis of Jose even inthe most recalitrant of lignocellulosic biomass materials. It is shown here that protein treatment saves 10-25% FPU activity. Forexample, inthe lowihrough reactor ith 0.1% sulfuric acid, a 20 FPUig cellulose of calulase ‘concentration produced a conversion efficiency of 81% with- ‘out BSA prewash, This is compared to essentially the same ‘conversion elficieney of 804% being provided by acelulase ‘concentration of 15 FPUig cellulose as enabled by 1% BSA, prewash, Similarly, the cellulase hydrolysis yield per unit of ‘ellulase is enhanced rom $% t0 20%. In relation to the ‘conversion of om stover, about 30% conversion was achieved using 20 FPU'g eslalose of cellulase when the biomass was not teated with 19% protein wash, while only 12 FPUlg eellose of cellulase prexiced essentially the same ‘amount of conversion when the biomass was trated with 3 1% protein (BSA) wash, This is about a S0% reduction in the ‘amount of required enzyme. Thus, using the herein disclosed process of protein treatment, cellulase is decreased 5% to 50%, of 20° to 30%, of 20% to 40%, 10 provide essentially the same yield measured as percentage conversion of celli- Jose to carbohydrate EXAMPLE2 Protein Treatment of High Lignin-containing Biomass ‘The present example demonstrates the uty ofthe inven- tive process for enhancing cellose degradation and the ef- ciency of cellulase, or other cellulose-

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