WO1994029474A1 - Traitement de la lignocellulose destine a reduire au minimum la fixation de la cellulase - Google Patents

Traitement de la lignocellulose destine a reduire au minimum la fixation de la cellulase Download PDF

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Publication number
WO1994029474A1
WO1994029474A1 PCT/US1994/006592 US9406592W WO9429474A1 WO 1994029474 A1 WO1994029474 A1 WO 1994029474A1 US 9406592 W US9406592 W US 9406592W WO 9429474 A1 WO9429474 A1 WO 9429474A1
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WIPO (PCT)
Prior art keywords
lignin
substrate
fermentation
peroxidase
cellulose
Prior art date
Application number
PCT/US1994/006592
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English (en)
Inventor
Norman D. Hinman
Michael E. Himmel
Karel Grohmann
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Midwest Research Institute
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Filing date
Publication date
Application filed by Midwest Research Institute filed Critical Midwest Research Institute
Publication of WO1994029474A1 publication Critical patent/WO1994029474A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates generally to a process for treating a lignocellulosic slurry with a proteinaceous material capable of binding the lignins in the lignocellulosic material and thereby occupying sites that cellulase would normally bind upon addition of cellulase to the lignocellulosic slurry.
  • the present invention relates to a process for treatment of a lignocellulosic substrate with a proteinaceous material in order to bind the lignins in the lignocellulosic material and thereby occupy sites that cellulase would normally bind when using cellulase to convert a lignocellulosic biomass to ethanol in a simultaneous saccharification fermentation process.
  • this treatment process the amount of cellulase available to hydrolyze cellulose to glucose in a process for conversion of lignocellulosic biomass to ethanol will not be lessened.
  • Cellulose is the most widely occurring organic compound on earth, and is essentially composed of repeating subunits of the disaccharide of D-cellobiose, linked by ⁇ -(l-4)-glycosidic bonds. Total hydrolysis yields D-glucose, and partial hydrolysis gives the disaccharide cellobiose, which is ⁇ -D-glucopyranosyl- ⁇ - glucopyranosyl- ⁇ -(l-4)-D-glycopyranose. Therefore, cellulose is a ⁇ -l,4-glucan. Cellulose constitutes the major storage form of photosynthesized glucose and the major component of solar energy which has been converted to biomass. As worldwide demand for energy and food supplies increases, cellulose has become an attractive raw material. The glucose subunits of cellulose can be used for production of energy or for use in the production of protein.
  • enzyme-catalyzed saccharification of cellulose is a more promising alternative to chemical degradation which can achieve a high efficiency conversion of cellulose to glucose.
  • Enzymatic conversion of cellulose to glucose using cellulase enzymes is superior to chemical dissolution because it proceeds at moderate temperature and pressure, provides recyclable catalysts and frees the environment from undesirable side products associated with chemical hydrolysis.
  • Cellulase is a complex of enzymes which act cooperatively, or synergistically, in degrading crystalline cellulose. These enzymes are endoglucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91) or glucohydrolase, and cellobiase ( ⁇ -glucosidase EC 3.2.1.21).
  • Lignins are complex, irregular phenylpropane polymers that represent approximately 20% by weight of the available polymeric content of hardwood tree stems.
  • Wood lignin precursors are the compounds 4-hydroxy-3-methoxycinnamyl (coniferyl) alcohol, 4-hydroxy-3,5-dimethoxycinnamyl (sinapyl) alcohol, and p-hydroxycinnamyl (p-coumaryl) alcohol.
  • lignins Unlike other natural polymers, lignins cannot be degraded to yield structurally intact precursors. The presence of many reactive sites in these molecules results in condensation reactions during hydrolytic treatments used to depolymerize polysaccharide structures often associated with lignins, such as the commonly used dilute acid hydrolysis pretreatment of hardwoods and agricultural residues prior to SSF.
  • Lignin is known for its ability to interact with a wide variety of chemical compounds, including chromatography supports, materials of containment, and other biomacromolecules, primarily proteins.
  • the mechanism of adsorption is that of classical hydrophobic interaction, via van der Waals type atomic forces. Enzymes having hydrophobic surfaces are therefore especially susceptible to loss on native lignin surfaces.
  • Cellulases are enzymes that fall into this category, as cellulases have a spear-shaped terminal peptide referred to as the cellulose binding domain (CBD).
  • CBD cellulose binding domain
  • This peptide is known to have one side, or face, which is very hydrophobic, thus permitting efficient binding of the enzyme to the surface of cellulose (Kraulis et al. 1989. Biochemistry. 28:7241-7257).
  • the Klason lignin fraction in pretreated biomass increases from an approximate starting value of 20%, to a final level of about 65% (Tatsumoto et al. 1988. Appl. Biochem. Biotechnol. 18:159-174).
  • This fraction with a high native lignin content is known to adsorb enzymes with hydrophobic surfaces or domains, such as cellulases (Tatsumoto et al. 1988, suprah Therefore, it would be beneficial to discover low-cost additives, more precisely adsorbents, for addition to the post pretreatment process stream, prior to SSF, that would block the cellulase-lignin binding sites existing in pretreated biomass.
  • a process for microbial saccharification of a cellulosic substrate is disclosed in U.S. Patent 4,628,029, wherein the saccharification proceeds by inoculating an aqueous nutrient medium with a cellulase- producing microorganism culture.
  • This patent also pretreats the cellulosic substrate by strong acid delignification in order to enhance microbial digestion.
  • U.S. Patent 3,862,901 discloses purifying waste water containing high molecular weight organic compounds such as protein polypeptides by bringing a waste water solution containing said polypeptides into contact with a sulfite pulp resulting from sulfite digestion of lignocellulosic material.
  • thermophilic bacterial microorganism to digest an aqueous mixture containing lignocellulose is disclosed in U.S. Patent 4,292,328; however, this process attacks the lignin in the lignocellulosic material and frees the cellulose cells for digestion.
  • U.S. Patent 4,938,972 disclose a process for microbial bioconversion of cereal milling bi-products into proteinaceous material for human consumption by aerobically fermenting these by-products in a culture of the fungus Neurosp ra sitophila. Pretreatment with caustic solution in conjunction with high temperatures partially delignifies the lignocellulose so that the biodegradability of the lignocellulose is increased because of either partial or full removal of lignin.
  • U.S. Patent 4,957,599 disclose a process for delignifying and bleaching lignocellulosic materials into products that are digestible by ruminants and ingestible by humans.
  • a need is extant in the art of hydrolyzing lignocellulose to glucose to find a means for pretreatment of a lignocellulosic slurry with a low cost material that will bind to the lignin and occupy the sites that cellulase would ordinary bind when cellulase is added to a lignocellulosic substrate to enzymatically convert cellulose to glucose in the lignocellulosic biomass conversion of a lignocellulosic substrate to ethanol.
  • one object of the invention is provide a low-cost additive to inactivate binding sites on native lignin, a major component of biomass, that otherwise binds valuable cellulase enzymes normally used in simultaneous saccharification and fermentation.
  • a further object of the invention is to provide a low-cost proteinaceous material to inactivate binding sites on native lignin, a major component of biomass, that otherwise binds values cellulase enzymes normally used in simultaneous saccharification and fermentation.
  • a yet further object of the invention is to provide low -cost proteinaceous, non-cellulase ligninases to bind native lignins, a major component of biomass, that otherwise binds valuable cellulase enzymes normally used in simultaneous saccharification and fermentation.
  • the process of the invention is accomplished by the addition of ligninases blocking agents to the pretreatment pulp stream before delivery to the SSF tanks, or after delivery to the tanks, but before initiation of SSF; however, the characteristics of ligninases (lignin peroxidases) are such that fungal biomass preparation cannot be added to the pretreated wood pulp at temperatures above about 40 °C, nor at pH conditions outside the range of about pH 3 to about 5.
  • the time of exposure of the pretreated biomass to the ligninases must be a minimum of about 30 minutes.
  • Figure 1 depicts a flow chart of the biomass to ethanol process incorporating a lignin blocking agent of the invention.
  • the process for production of ethanol from biomass is based on the dilute acid hydrolysis of hardwoods and agricultural residues, followed by separation of the hydrolysate liquor and pretreated pulp, and finally, the use of simultaneous saccharification and fermentation technology to produce ethanol.
  • the current scheme for simple production of ethanol by fermentation from biomass has been modified by the innovation of the present invention, in that, a lignin-blocking agent has been added to provide lignin-binding sites after the acid pretreatment of the pulp stream before delivery to the simultaneous saccharification fermentation tanks, or after delivery to the tanks, but before initiation of simultaneous saccharification and fermentation.
  • the acid hydrolysis step is practiced using an acid concentration of about 2 to about 10% by volume (preferably sulfuric acid), and the hydrolysis temperature may range from about 120°C or less.
  • the residence time in the acid hydrolysis step is from about 1 to about 3 hours, and the treated biomass slurry will have a biomass solids to hquid ratio of about 20/100 to about 40/100 on a volume basis. While it is not critical, it is preferred that the particle size of the biomass range from about 1 to about 4 mm.
  • Example 1 Cellulose media is added to a 6 L vessel containing 1 L of water, and the volume is brought up to about 2,500 ml in order to leave enough room for the inocula.
  • the media is mixed in the fermenter and a lipid stock of 5 ml/L of Ergosterol and 30 ml/L of oleic acid is added, after which the mixture is autoclaved at about 120°C for about 35 to 40 minutes.
  • a mixture of antibiotics containing (500 mg of 10 mg/L) of penicillin and 500 mg (of 10 mg/L) of streptomycin is added and the pH is checked to insure that it is between about 4.5 to about 5.0.
  • a 2% by volume solution of sulfuric acid is added to effect acid hydrolysis for a period of 1 to 3 hours.
  • the acid pretreated slurry is lowered to a temperature of about 40°C and the pH is adjusted to a range between about 3 to 5, whereupon the ligninase or lignin peroxidase from Phanerochaete chryxo. ⁇ orium is added in the amount of 0.1 to 1 mg ligninase to 1 gram pretreated wood for at least 30 minutes.
  • the enzyme cellulase is added in sufficient amount to bring the volume up to the 3L mark with sterile H 2 0.
  • Fig. 1 shows the biomass to ethanol process incorporating the lignin blocking step to block cellulase binding sites on lignin.
  • ligninases proteinacous lignin peroxidases
  • This second lignin blocking agent can be any low molecular weight, aromatic compound of unidentified structure obtained from the hot water, or alcoholic extraction of biomass (i.e., normally referred to as "extractives").
  • extractives are utilized in the same way as the lignin- binding enzymes of ligninases, with the exception that, these low molecular weight aromatic compounds of unidentified structure are less temperature sensitive than the ligninase protein - as such, addition of the second class of agents may be made to the acid pretreated pulp at temperatures as high as 100 °C.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Dans un procédé simultané de saccharification et de fermentation destiné à produire de l'éthanol à partir de la biomasse, l'amélioration proposée consiste: à former un substrat à partir de matériaux tirés de la biomasse sélectionnés parmi la cellulose, l'hémicellulose et l'amidon; à ajouter audit substrat, comme agent de prétraitement, de l'acide hydrolitique; à ajouter une quantité de lignine peroxydase fixant la lignine, et destinée à bloquer les sites de fixation de la lignine dans ledit matériau; à ajouter de la cellulase à ce substrat pour obtenir une saccharification et une fermentation simultanées dans des conditions favorables à la viabilité des cellules et à la conversion des hydrolysats en éthanol; puis à prélever l'éthanol dudit substrat.
PCT/US1994/006592 1993-06-11 1994-06-10 Traitement de la lignocellulose destine a reduire au minimum la fixation de la cellulase WO1994029474A1 (fr)

Applications Claiming Priority (2)

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US7437693A 1993-06-11 1993-06-11
US074,376 1993-06-11

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0884391A1 (fr) * 1997-06-09 1998-12-16 Iogen Corporation Amélioration du procédé de prétraitement pour la conversion de cellulose en carburant d'éthanol
WO2005024037A2 (fr) * 2003-03-19 2005-03-17 The Trustees Of Dartmouth College Bloqueurs de lignine et leurs utilisations
ES2303792A1 (es) * 2007-02-15 2008-08-16 Industrias Mecanicas Alcudia S.A. Un procedimiento para la revalorizacion energetica de la fraccion organica de residuos solidos, urbanos e instalacion.
WO2011097713A1 (fr) 2010-02-11 2011-08-18 Iogen Energy Corporation Modules de liaison de glucide avec une liaison réduite à la lignine
US8337663B2 (en) 2008-12-19 2012-12-25 Novozymes, Inc. Methods for increasing hydrolysis of cellulosic material
US8580541B2 (en) 2003-03-19 2013-11-12 The Trustees Of Dartmouth College Lignin blockers and uses thereof
CN105861569A (zh) * 2016-06-01 2016-08-17 卞佳林 一种利用木质纤维植物制备燃料乙醇的方法
WO2017088892A1 (fr) 2015-11-24 2017-06-01 Inbicon A/S Compositions de bitume comprenant de la lignine

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4232845A (en) * 1978-04-20 1980-11-11 Aquila Steel Company, Ltd. On-site modular support systems for supporting cables, conduit and the like
US4292328A (en) * 1978-08-21 1981-09-29 Coulthard T Lionel Thermophilic aerobic digestion process for producing animal nutrients and other digested products
US4687741A (en) * 1985-07-15 1987-08-18 Repligen Corporation Novel enzymes which catalyze the degradation and modification of lignin
US4830708A (en) * 1987-11-30 1989-05-16 Pulp And Paper Research Institute Of Canada Direct biological bleaching of hardwood kraft pulp with the fungus Coriolus versicolor
US4889807A (en) * 1984-12-12 1989-12-26 Institut National de la Recherche Aronomique Microorganisms of the Phanerochaete chrysosporium strain and their use
US5149648A (en) * 1989-03-16 1992-09-22 Kabushiki Kaisha Kobe Seiko Sho Enzymes employed for producing pulps

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4232845A (en) * 1978-04-20 1980-11-11 Aquila Steel Company, Ltd. On-site modular support systems for supporting cables, conduit and the like
US4292328A (en) * 1978-08-21 1981-09-29 Coulthard T Lionel Thermophilic aerobic digestion process for producing animal nutrients and other digested products
US4889807A (en) * 1984-12-12 1989-12-26 Institut National de la Recherche Aronomique Microorganisms of the Phanerochaete chrysosporium strain and their use
US4687741A (en) * 1985-07-15 1987-08-18 Repligen Corporation Novel enzymes which catalyze the degradation and modification of lignin
US4830708A (en) * 1987-11-30 1989-05-16 Pulp And Paper Research Institute Of Canada Direct biological bleaching of hardwood kraft pulp with the fungus Coriolus versicolor
US5149648A (en) * 1989-03-16 1992-09-22 Kabushiki Kaisha Kobe Seiko Sho Enzymes employed for producing pulps

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0884391A1 (fr) * 1997-06-09 1998-12-16 Iogen Corporation Amélioration du procédé de prétraitement pour la conversion de cellulose en carburant d'éthanol
WO2005024037A2 (fr) * 2003-03-19 2005-03-17 The Trustees Of Dartmouth College Bloqueurs de lignine et leurs utilisations
WO2005024037A3 (fr) * 2003-03-19 2005-06-23 Dartmouth College Bloqueurs de lignine et leurs utilisations
US7604967B2 (en) 2003-03-19 2009-10-20 The Trustees Of Dartmouth College Lignin-blocking treatment of biomass and uses thereof
US7875444B2 (en) 2003-03-19 2011-01-25 The Trustees Of Dartmouth College Lignin blockers and uses thereof
US8580541B2 (en) 2003-03-19 2013-11-12 The Trustees Of Dartmouth College Lignin blockers and uses thereof
US8399228B2 (en) 2007-02-15 2013-03-19 Industrias Mecanicas Alcudia, S.A. Method for recovering energy from the organic fraction of solid urban waste and associated facility
ES2303792A1 (es) * 2007-02-15 2008-08-16 Industrias Mecanicas Alcudia S.A. Un procedimiento para la revalorizacion energetica de la fraccion organica de residuos solidos, urbanos e instalacion.
WO2008099038A3 (fr) * 2007-02-15 2008-11-20 Ind Mecanicas Alcudia S A Procédé de revalorisation énergétique de la fraction organique de résidus solides urbains et installation
US8337663B2 (en) 2008-12-19 2012-12-25 Novozymes, Inc. Methods for increasing hydrolysis of cellulosic material
WO2011097713A1 (fr) 2010-02-11 2011-08-18 Iogen Energy Corporation Modules de liaison de glucide avec une liaison réduite à la lignine
US9206406B2 (en) 2010-02-11 2015-12-08 Iogen Energy Corporation Carbohydate binding modules with reduced binding to lignin
US9885028B2 (en) 2010-02-11 2018-02-06 Iogen Energy Corporation Carbohydrate binding modules with reduced binding to lignin
WO2017088892A1 (fr) 2015-11-24 2017-06-01 Inbicon A/S Compositions de bitume comprenant de la lignine
EP3447086A1 (fr) 2015-11-24 2019-02-27 Inbicon A/S Compositions de bitume comprenant de la lignine
CN105861569A (zh) * 2016-06-01 2016-08-17 卞佳林 一种利用木质纤维植物制备燃料乙醇的方法

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