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WO2014075694A1 - Procédés de traitement de rafles de régimes de fruits (efb) en sucres fermentables à l'aide d'une hydrolyse enzymatique en plusieurs étapes - Google Patents

Procédés de traitement de rafles de régimes de fruits (efb) en sucres fermentables à l'aide d'une hydrolyse enzymatique en plusieurs étapes Download PDF

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WO2014075694A1
WO2014075694A1 PCT/DK2013/050388 DK2013050388W WO2014075694A1 WO 2014075694 A1 WO2014075694 A1 WO 2014075694A1 DK 2013050388 W DK2013050388 W DK 2013050388W WO 2014075694 A1 WO2014075694 A1 WO 2014075694A1
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hydrolysis
stage
efb
conversion
enzyme
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Jan Larsen
Martin Dan JEPPESEN
Kit Kellebjerg MOGENSEN
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Inbicon AS
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Inbicon AS
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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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • 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
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source

Definitions

  • the invention relates to methods of processing oil palm empty fruit bunches (EFB) to fermentable sugars using enzymatic hydrolysis.
  • EFB oil palm empty fruit bunches
  • Ethanol has always been considered an acceptable alternative to fossil fuels, being readily usable as an additive in fuel blends or even directly as fuel for personal automobiles.
  • use of ethanol produced by these "first generation" bioethanol technologies does not actually achieve dramatic reduction in greenhouse gas emission.
  • the net savings is only about 13% compared with petroleum, when the total fossil fuel inputs to the final ethanol outputs are all accounted.
  • both economic and moral objections have been raised to the "first generation" bioethanol market. This effectively places demand for crops as human food into direct competition with demand for personal automobiles. And indeed, fuel ethanol demand has been associated with increased grain prices that have proved troublesome for poor, grain-importing countries.
  • second generation bioethanol produced from lignocellulosic biomass such as crop wastes (stalks, cobs, pits, stems, shells, husks, etc . ), grasses, straws, wood chips, waste paper and the like.
  • lignocellulosic biomass such as crop wastes (stalks, cobs, pits, stems, shells, husks, etc . ), grasses, straws, wood chips, waste paper and the like.
  • fermentable 6-carbon and 5-carbon sugars are liberated from biomass polysaccharide polymer chains by enzymatic hydrolysis or, in some cases, by pure chemical hydrolysis.
  • the fermentable sugars obtained from biomass conversion in a “second generation” biorefinery can be used to produce fuel ethanol or, alternatively, other fuels such as butanol, or lactic acid monomers for use in synthesis of bioplastics, or many other products.
  • hydrothermal pretreatments are especially attractive. These utilize pressurized steam/liquid hot water at temperatures on the order of 160 - 230 o C to gently melt hydrophobic lignin that is intricately associated with cellulose strands, to solubilize a major component of hemicellulose, rich in 5 carbon sugars, and to disrupt cellulose strands so as to improve
  • Enzyme preparations that can "saccharify" pretreated lignocellulosic feedstocks have recently emerged as a new commercial market.
  • Enzymatic hydrolysis of pretreated biomass that is, enzymatic hydrolysis of cellulose to glucose and of hemicellulose to constituent oligo- and monosaccharides is typically conducted using a mixture of different hydrolytic enzyme activities.
  • many fungi and bacteria have been identified which degrade lignocellulosic biomass in nature.
  • Preparations of enzymes obtained from such organisms typically provide a variety of different enzyme activities that, collectively and synergistically, provide effective hydrolysis of lignocellulosic materials.
  • Such enzyme preparations are typically termed “cellulase” preparations, since they comprise cellulytic enzymes that catalyse hydrolysis of glucosidic linkages in cellulose, including endoglucanases, which introduce nicks in the cellulosic polymer chain thereby exposing reducing ends, and cellobiohydrolases or exoglucanases, which catalyze from reducing and non-reducing ends release of oligosaccharide products from the cellulosic polymer chain.
  • Such preparations further typically comprise B-glucosidases, which catalyse hydrolysis of oligosaccharide products to fermentable monosaccharides as well as a variety of hemicellulases, including endoxylanases, exoxylanases, exoxylosidases, mannosidases, acetyl xylan esterases, and other activities.
  • B-glucosidases catalyse hydrolysis of oligosaccharide products to fermentable monosaccharides as well as a variety of hemicellulases, including endoxylanases, exoxylanases, exoxylosidases, mannosidases, acetyl xylan esterases, and other activities.
  • Enzyme preparations comprising a multi-enzyme mixture sufficient to "saccharify" lignocellulosic biomass may be obtained by a variety of methods known in the art from a variety of microorganisms, including at least bacteria such as species of Clostridium, Cellumonas,
  • Thermonospora Bacillus, Bacteriodes, Ruminococcus, Erwinia, Acetovibrio, Mocrobispora, and Streptomyces and at least fungi such as species of Trichoderma, Penicillium, Fusarium, Humicola, Aspergillus and Phanerochaete.
  • the term "commercially available cellulase preparation” refers to a mixture of enzyme activities that is sufficient to provide enzymatic saccharification of pretreated lignocellulosic biomass and that comprises cellulase, xylanase and B-glucosidase activities.
  • the term "optimized for lignocellulosic biomass conversion” refers to a product development process in which enzyme mixtures have been selected and modified for the specific purpose of improving hydrolysis yields and/or reducing enzyme consumption in hydrolysis of pretreated lignocellulosic biomass to fermentable sugars. Selection and modification of enzyme mixtures may include genetic engineering techniques, for example such as described in (Zhang et al., 2006) or by other methods known in the art.
  • cellulase preparations optimized for lignocellulosic biomass conversion are typically identified by the manufacturer and/or purveyor as such. These are typically distinct from commercially available cellulase preparations for general use or optimized for use in production of animal feed, food, textiles detergents or in the paper industry.
  • hydrolysis yields can be substantially improved by the simple expedient of conducting enzymatic hydrolysis in at least two distinct stages, with solid/liquid separation between stages followed by re-suspension of the solid residual for further enzymatic hydrolysis.
  • Figure 1 shows the effect on hydrolysis yield of washing pretreated wheat straw and EFB to remove cellulase inhibitors prior to hydrolysis using a commercially available cellulase preparation optimized for lignocellulosic biomass conversion.
  • Figure 2 shows a comparison of hydrolysis yields obtained from pretreated washed EFB using a commercially available cellulase preparation optimized for lignocellulosic biomass conversion (A) in a two-stage hydrolysis process and in a single-stage hydrolysis process with equivalent water content, and (B) in a two-stage hydrolysis process with enzyme supplementation in the second stage and in a single-stage hydrolysis process with equivalent water content.
  • Figure 3 shows the effect on hydrolysis yields obtained from pretreated washed EFB using a commercially available cellulase preparation optimized for lignocellulosic biomass (A) in the presence of liquid hydrolysate obtained from the first stage of a two-stage hydrolysis process, and (B) in the presence of liquid hydrolysate obtained from the second stage of a two-stage hydrolysis process.
  • Figure 4 shows release of enzyme protein to solution as a function of degree of hydrolysis of pretreated wheat straw using a commercially available cellulase preparation optimized for lignocellulosic biomass conversion.
  • beta-glucosidase activity catalyses the hydrolysis of the soluble disaccharide cellobiose to glucose monomers. Because beta-glucosidase activities were always soluble, the enzyme activity would typically follow the liquid fraction during solid/liquid separation. Accordingly, some modification of beta-glucosidases so that they would bind to the lignin-rich solid residual (US 2010/0068768) or some recovery of soluble enzymes (Yang et al. 2010) was previously considered necessary for multiple-stage hydrolysis.
  • hydrolysis yields as a function of % removal of dissolved solids from the pretreated "solid" fraction in a hydrolysis reaction conducted at 20% dry matter (DM).
  • dry matter refers to total dissolved and undissolved solids.
  • Hydrolysis was conducted using a current "generation" commercially available cellulase preparation optimized for lignocellulosic biomass conversion (the preparation offered by NOVOZYMES Tm under the trade name CELLIC CTEC3 Tm). Results are shown in Figure 1.
  • the maximal improvement in hydrolysis yields which could be achieved by washing to remove soluble cellulase inhibitors was at most about 8% in absolute terms, where "% yield” is expressed as percent theoretical yield, calculated as a percentage of the amount of glucose that could theoretically be obtained based on the cellulose content of the material assuming 1.1 1 g glucose per g cellulose. If instead of washing, a two-step hydrolysis process were to be used to remove soluble cellulase inhibitors, the maximal removal of dissolved solids which could be achieved in the solid/liquid separation step following a first partial initial hydrolysis stage would be a function both of the dry matter content of the partial initial hydrolysis reaction and also of the extent to which the solid residual from the partial initial hydrolysis reaction could be pressed to a high dry matter content.
  • two-step hydrolysis as a means of overcoming negative effects of soluble cellulase inhibitors generated by pretreatment typically produces less than maximal improvements, perhaps due to loss of enzyme activity during transfer to the second stage, or for other reasons not currently understood.
  • hydrolysis yields obtained with pretreated wheat straw in a 20% DM two-step reaction with yields obtained in a 20% DM one-step reaction to which a volume of additional water was added equivalent to that used for resuspension of the residual solid in the second hydrolysis stage.
  • the residual solid remaining after partial initial hydrolysis in a multiple-stage hydrolysis scheme will typically retain beta glucosidase activities as well as all other enzyme proteins that comprise current "generations" of commercially available cellulase preparations optimized for lignocellulosic biomass conversion.
  • a previous "generation" of commercially available cellulase preparations optimized for lignocellulosic biomass conversion which include the preparation offered by NOVOZYMES Tm under the trade name CELLIC CTEC2 Tm and the preparation offered by GENNENCOR Tm under the trade name ACCELLERASE 1500 Tm, comprise modified beta glucosidase activities and other enzyme proteins configured such that effectively all enzyme proteins will adhere to the solid fraction during solid/liquid separation. (Data not shown).
  • the invention provides a method for processing empty fruit bunches (EFB) to fermentable sugars comprising
  • “Hydrothermal pretreatment” refers to the use of water, either as hot liquid, vapour steam or pressurized steam comprising high temperature liquid or steam or both, to "cook” biomass, at temperatures of 120o C or higher, either with or without addition of acids or other chemicals.
  • the pH at which biomass is hydrothermally pretreated refers to the pH of the wetted biomass/biomass slurry as it enters a pretreatment reactor.
  • hydrothermally pretreated refers to the combined w/w percentage of water insoluble and water soluble solids present in the wetted biomass/biomass slurry as it enters a pretreatment reactor.
  • Partial initial hydrolysis refers to an enzymatic hydrolysis reaction in which cellulase, B- glucosidase and hemicellulase activities are used to hydrolyse biomass to a percent conversion that is 80% or less followed by solid/liquid separation and resuspension of the solid residual in one or more subsequent hydrolysis steps.
  • the "liquid hydrolysate” obtained after partial initial hydrolysis refers to the liquid fraction remaining after solid/liquid separation of the partial initial hydrolysis mixture.
  • the "solid residual” obtained from partial initial hydrolysis refers to the solid fraction remaining after solid/liquid separation of the partial initial hydrolysis mixture. As will be readily understood by those skilled in the art, the “solid residual” will typically comprise considerable liquid content, where the total solids content of the solid residual is typically within the range of 20 to 50%.
  • the solids content of the solid residual comprises primarily lignin and unhydrolysed cellulose and
  • Subsequent hydrolysis step refers to a continued enzymatic hydrolysis of biomass that has been subject to partial initial hydrolysis.
  • the % conversion achieved in subsequent hydrolysis steps refers to the cumulative % conversion of the biomass over initial partial hydrolysis and any number of subsequent hydrolysis steps.
  • liquid fraction obtained after pretreatment refers to the liquid phase of pretreated biomass as it exists immediately after pretreatment and after equilibration to 25o C and atmospheric pressure. It will be readily understood that “liquid fraction” exists whether or not any physical solid/liquid separation step is used. Liquid fraction comprises primarily water and water soluble material.
  • % conversion refers to conversion of cellulose into glucose.
  • % conversion refers to % of the amount that could theoretically be obtained based on the cellulose content of the material. 100% theoretical recovery of glucose from cellulose is 1.1 10 g glucose per g cellulose.
  • EFB feedstock may be used in the form in which it exists after palm oil processing.
  • EFB may be subject to methods for removal of lipid-soluble substances and/or lignin including initial hot water treatment or to any of the methods described in WO2011002329, which is hereby incorporated by reference in entirety.
  • EFB or EFB previously subject to some initial thermal or chemical processing may be subject to milling, grinding, shredding or other forms of particle size reduction.
  • EFB feedstock is subject to hydrothermal pretreatment, typically within a pressurized pretreatment reactor.
  • the "severity" of hydrothermal pretreatment refers to the harshness of conditions to which biomass feedstock has been subject.
  • a variety of different composite parameters have been proposed that provide a scalar index by which different pretreatments schemes may be compared.
  • Classic "severity, " Ro is defined as (residence time in minutes) x (EXP[(pretreatment temperature - 100) / 14.75]) and is typically referred to as a logarithmic value, log Ro.
  • Many hydrothermal pretreatment schemes such as dilute acid pretreatment and acidic steam explosion require relatively strong acid conditions but can typically be practiced at lower temperatures.
  • a second severity parameter, Ro' is often used which includes a pH dimension such that, expressed as a log value, log Ro' is simply log Ro - pH.
  • pretreatment severity can be expressed in terms of residual xylan content.
  • xylan number refers to residual xylan content measured as follows: Pretreated biomass is subject to solid/liquid separation to provide a solid fraction at 30% total solids which is swelled with liquid fraction obtained after pretreatment. This solid fraction is then mixed with water in the ratio of total solids to water of 1 :3. The solid fraction washed in this manner is then pressed to 30% total solids. Xylan content of the solid fraction washed in this manner is determined using the method of A.
  • Hydrothermal pretreatment may be conducted by a variety of methods well known in the art.
  • Steam pretreatment typically may be conducted either as a "steam explosion” or using high pressure steam without explosive release of pretreated biomass. Steam pretreatment is typically conducted at high temperatures, between 170 and 220° C, and at high pressures, between 4 and 20 bar, where water exists as a mixture of liquid and vapour.
  • lignocellulosic biomass is pretreated by hydrothermal pretreatment at temperatures between 170 and 200° C.
  • biomass feedstocks may be subject to particle size reduction and/or other mechanical processing such as grinding, milling, shredding, cutting or other processes prior to hydrothermal pretreatment.
  • biomass feedstocks may be washed and/or leached of valuable salts prior to pretreatment, as described in Knudsen et al. 1998.
  • Authors are N.O. Knudsen, P.A. Jensen, B. Sander and K. Dam-Johansen.). It is advantageous to conduct pretreatment at the highest possible levels of total solids that can be used, without resulting in diminished ultimate hydrolysis efficiency.
  • hydrothermal pretreatment is conducted without supplemental oxygen as required for wet oxidation pretreatments, or without addition of organic solvent as required for organosolv pretreatment, or without use of microwave heating as required for microwave pretreatments. In some embodiments, hydrothermal pretreatment is conducted at temperatures of 140o C or higher, or at 150o C or higher, or at 160o C or higher, or 220o C, or lower.
  • Hydrothermal pretreatments may be conducted at different degrees of wetting, i.e., at different "total solids” content.
  • the raw biomass feedstock comprises some quantity of water as well as a "dry weight” that remains after all water is removed.
  • Total solids refers to both water insoluble solids and water soluble solids.
  • the total solids content of hydrothermally pretreated biomass comprises both a liquid component (hereafter "liquid fraction") and a water insoluble “fiber” component.
  • the "fiber” component comprises insoluble solids that are swelled with “liquid fraction.”
  • the "fiber” component comprises insoluble solids that do not actually retain fibrous structure perse.
  • the "liquid fraction” comprises solubilized hemicellulose, comprising predominantly C5 sugars with some C6 sugars, as well as byproducts of the pretreatment reaction that can inhibit yeast fermentation, most notably furfural, 5-hydroxy- methyl-furfural (5HMF), and acetic acid.
  • Hydrothermal pretreatment may be conducted at any pH level. In some embodiments,
  • pretreatment may be conducted at pH > 8.0. In some embodiments, pretreatment is conducted at pH lower than 8.0, in some embodiments, between 2.5 and 8.0, or in some embodiments between 0.5 and 2.5, or in some embodiments between 2.5 and 5.0. In some embodiments, hydrothermal pretreatment is conducted without addition of acids, bases or other chemicals other than those derived from biomass processing steps. Biomass feedstocks are typically pretreated to severity log Ro between about 3.3 and 4.5, or log Ro' between about -1.5 and 1.5. In some embodiments, biomass is pretreated at pH between 3.5 and 8.0 to severity Ro ⁇ 4.2, or less than 3.8, or greater than 3.4.
  • the liquid fraction and water insoluble solid component may be separated by means of solid/liquid separation methods known in the art.
  • solid/liquid separation may be achieved using a relatively inexpensive screw press, which can typically achieve total solids content of the solid residual to levels up to 45%.
  • a twin roll press or a twin wire press may be used, which can typically achieve total solids of the solid residual to levels in the range 30-45%.
  • a belt press, drum filter, centrifuge, decanter centrifuge or other device known in the art may be used.
  • pretreated biomass is removed from a hydrothermal pretreatment reactor either by sluice system, by steam explosion, or by other techniques that provide pretreated biomass as a combined mixture of both "liquid fraction" component and water insoluble "fiber” component
  • this combined mixture can be directly used in enzymatic hydrolysis, with or without flashing or further dilution.
  • unwashed water insoluble "fiber” component of pretreated biomass is used in enzymatic hydrolysis, with or without flashing or further dilution.
  • "washing" of the water isoluble "fiber” component involves not merely dilution but exposure to a volume of water or aqueous solution that is not included in enzymatic hydrolysis
  • pretreated biomass may be further subject to a de-lignification step, such as sodium hydroxide treatment or other methods known in the art.
  • Enzymatic hydrolysis of pretreated biomass that is, enzymatic hydrolysis of cellulose to glucose and of hemicellulose to constituent oligo- and monosaccharides is typically conducted using a mixture of different hydrolytic enzyme activities.
  • many fungi and bacteria have been identified which degrade lignocellulosic biomass in nature.
  • Preparations of enzymes obtained from such organisms typically provide a variety of different enzyme activities that, collectively and synergistically, provide effective hydrolysis of lignocellulosic materials.
  • Such enzyme preparations are typically termed “cellulase” preparations, since they comprise cellulytic enzymes that catalyse hydrolysis of glucosidic linkages in cellulose, including endoglucanases, which introduce nicks in the cellulosic polymer chain thereby exposing reducing ends, and cellobiohydrolases or exoglucanases, which catalyze from reducing and non-reducing ends release of oligosaccharide products from the cellulosic polymer chain.
  • Such preparations further typically comprise B-glucosidases, which catalyse hydrolysis of oligosaccharide products to fermentable monosaccharides as well as a variety of hemicellulases, including endoxylanases, exoxylanases, exoxylosidases, mannosidases, acetyl xylan esterases, and other activities.
  • B-glucosidases catalyse hydrolysis of oligosaccharide products to fermentable monosaccharides as well as a variety of hemicellulases, including endoxylanases, exoxylanases, exoxylosidases, mannosidases, acetyl xylan esterases, and other activities.
  • a multi-enzyme mixture sufficient to hyrdrolyse pretreated biomass may be obtained by a variety of methods known in the art from a variety of microorganisms, including at least bacteria such as species of Clostridium, Cellumonas, Thermonospora, Bacillus, Bacteriodes, Ruminococcus, Erwinia, Acetovibrio, Mocrobispora, and Streptomyces and at least fungi such as species of Trichoderma, Penicillium, Fusarium, Humicola, Aspergillus and Phanerochaete. It will be readily understood by one skilled in the art that individual or groups of component enzyme activities may be isolated from such preparations and used separately, either in combination or as supplements to isolated multi-enzyme mixtures.
  • an enzyme preparation is preferably used which has a substantially reduced susceptibility to glucose end-product inhibition of catalysis, meaning that the enzyme preparation exhibits less than 5% loss in absolute conversion yield due to the presence of 40 g/L glucose after 80 hours hydrolysis of wheat straw that has been hydrothermally pretreated to at least severity log Ro 3.5.
  • Pretreated EFB is first subject to partial initial hydrolysis, preferably using an enzyme preparation having substantially reduced susceptibility to glucose end-product inhibition of catalysis.
  • Suitable enzyme preparations that may be used to practice disclosed embodiments include commercially available cellulase preparations optimized for lignocellulosic biomass conversion.
  • commercially available cellulase preparation refers to a mixture of enzyme activities that is sufficient to provide enzymatic hydrolysis of pretreated lignocellulosic biomass and that comprises cellulase, xylanase and B-glucosidase activities.
  • optimal for lignocellulosic biomass conversion refers to a product development process in which enzyme mixtures have been selected and modified for the specific purpose of improving hydrolysis yields and/or reducing enzyme consumption in hydrolysis of pretreated lignocellulosic biomass to fermentable sugars. Selection and modification of enzyme mixtures may include genetic engineering techniques, for example such as described in (Zhang et al., 2006) or by other methods known in the art.
  • cellulase preparations optimized for lignocellulosic biomass conversion are typically identified by the manufacturer and/or purveyor as such. These are typically distinct from commercially available cellulase preparations for general use or optimized for use in production of animal feed, food, textiles detergents or in the paper industry.
  • any suitable commercially available cellulase preparation optimized for lignocellulosic biomass conversion may be used singly or in combination to practice the disclosed embodiments.
  • Initial enzyme activity of such preparations typically comprises at least exoglucanases, endoglucanases, hemicellulases, and beta glucosidases.
  • Such preparations typically comprise endoglucanase activity such that 1 FPU cellulase activity is associated with at least 31 CMC U endoglucanase activity and further typically comprise beta glucosidase activity such that 1 FPU cellulase activity is associated with at least at least 7 pNPG U beta glucosidase activity.
  • CMC U refers to carboxymethycellulose units.
  • One CMC U of activity liberates 1 umol of reducing sugars (expressed as glucose equivalents) in one minute under specific assay conditions of 50° C and pH 4.8.
  • pNPG U refers to pNPG units.
  • One pNPG U of activity liberates 1 umol of nitrophenol per minute from para-nitrophenyl-B-D-glucopyranoside at 50° C and pH 4.8.
  • FPU of "filter paper units" provides a measure of cellulase activity.
  • FPU refers to filter paper units as determined by the method of Adney, B. and Baker, J., Laboratory Analytical Procedure #006, "Measurement of cellulase activity", August 12, 1996, the USA National Renewable Energy Laboratory (NREL), which is expressly
  • cellulase preparations optimized for lignocellulosic biomass conversion and provided by GENENCOR Tm may be used to practice disclosed embodiments.
  • One specific example of such a cellulase preparation is sold under the tradename ACCELLERASE TRIO Tm.
  • xylanase preparations optimized for lignocellulosic biomass conversion may be used to practice disclosed embodiments.
  • xylanase preparations optimized for lignocellulosic biomass conversion and provided by NOVOZYMES may be used to practice disclosed embodiments.
  • One specific example of such a xylanase preparation is sold under the tradename CELLIC HTEC2 Tm.
  • commercially available xylanase preparations optimized for lignocellulosic biomass conversion and provided by GENENCOR Tm may be used to practice disclosed embodiments.
  • xylanase preparation Two specific examples of such a xylanase preparation are sold under the tradename ACCELLERASE XY Tm and ACCELLERASE XC Tm.
  • B-glucosidase preparations provided by NOVOZYMES may be used to practice the disclosed embodiments.
  • NOVOZYMES 188 One specific example is the B-glucosidase preparation sold under the trade name NOVOZYMES 188.
  • B-glucosidase helps reduce product inhibition of cellulose hydrolytic reactions, and, accordingly, it may be advantageous to supplement commercially available cellulase preparations optimized for lignocellulosic biomass with additional B-glucosidase activity.
  • some specific enzyme preparations, prepared by methods known in the art have been reported to offer advantages as supplements to commercially available cellulase preparations optimized for lignocellulosic biomass conversion. See e.g.
  • Partial initial hydrolysis is stopped at incomplete conversion, in some embodiments at a point between 20 and 80% conversion, or between 30 and 70% conversion, or between 40 and 50% conversion.
  • the solid residual fraction is resuspended in water or aqueous solution and the hydrolysis reaction allowed to proceed in a subsequent hydrolysis stage.
  • a commercially available cellulase preparation optimized for lignocellulosic biomass conversion is used, the bulk of enzyme activity from initial partial hydrolysis can be simply transferred to subsequent hydrolysis because it will be associated with the solid residual. In some cellulase preparations, it may be advantageous to, optionally, supplement the subsequent hydrolysis reaction with B-glucosidase activity.
  • Partial initial hydrolysis may be conducted under a variety of conditions. In some embodiments, partial initial hydrolysis may be conducted at comparatively low total solids content, for example, at 10% total solids or lower. In some embodiments, partial initial hydrolysis may be conducted at higher total solids content, for example, greater than 10%, or greater than 18%, or greater than 19%, or greater than 20%. In other embodiments, partial initial hydrolysis may be conducted at total solids content between 20% and 40%, or between 40% and 60%. All numerical values presented here should be understood to apply to measured quantities with rounding to the number of significant digits shown. In some embodiments, partial initial hydrolysis is conducted using at least 1 kg of pretreated biomass, or at least 10 kg, or at least 100 kg, or at least 1000 kg.
  • Partial initial hydrolysis can be conducted to conversion levels of between 20 and 80%. In general, it is advantageous to stop partial initial hydrolysis at conversion levels associated with minimal loss of enzyme activity to the supernatant.
  • enzyme preparations obtained from cellulytic organisms as well as commercially available cellulase preparations optimized for lignocellulosic biomass conversion will relatively quickly bind to lignocellulosic materials.
  • B-glucosidases and some specialized auxiliary enzymes most of the component enzymes catalyse reactions on an undissolved surface, releasing a soluble reaction product. Most of the component enzymes are, accordingly, found associated with the insoluble "fiber" component of pretreated biomass.
  • reaction temperatures of 40-45o C typically provide the greatest long term stability with reasonable reaction yields.
  • Partial initial hydrolysis can be stopped by a solid/liquid separation step, which produces both a liquid hydrolysate and a solid residual.
  • Solid/liquid separation after partial initial hydrolysis can be achieved using a variety of means well known in the art, including decanter centrifuges, belt presses, vacuum presses, screw presses, filter presses, drum dilters and other devices.
  • a solid/liquid separation technique is selected so as to provide optimal removal of suspended solids from the liquid hydrolysate, in order that subsequent fermentation processes may be conducted using well clarified solutions, which facilitate recycling of fermentive organisms.
  • the solid residual remaining after partial initial hydrolysis is then resuspended and subsequent hydrolysis conducted.
  • the resuspension can be conducted using water, process solutions or buffers. In some embodiments, it can be advantageous to resuspend so as to achieve the highest practicable levels of total solids in subsequent hydrolysis.
  • a supplemental enzyme dose may be applied in a subsequent hydrolysis step.
  • two or more subsequent hydrolysis steps may be conducted, with a solid/liquid separation between each step followed by resuspension and continued hydrolysis.
  • the hydrolysate obtained after solid/liquid separation of the partial initial hydrolysis mixture comprising C5 and C6 sugars and, optionally, comprising "fermentation inhibitors" and other pretreatment byproducts, can be directly fermented in a single fermentation reaction.
  • Comparisons between two-stage hydrolysis yields and one-stage hydrolysis yields are made by conducting one stage hydrolysis with equivalent pretreated EFB at lower dry matter content, by adding initially in the one-stage comparison an amount of water or aqueous solution corresponding to the amount of water added in the second hydrolysis stage.
  • cellulose conversion can always be enhanced at equivalent enzyme dose by conducting hydrolysis at lower DM.
  • a single- step hydrolysis can be conducted having the same amount of biomass, but with an amount of water corresponding to the water used in both the first and second stages of a two-step hydrolysis. Samples can be analysed according to the method described by (Kristensen et al. 2009).
  • conversion yields are improved at least 5%, or at least 6%, or at least 7%, or at least 8%, or at least 9%, or at least 10%, or at least 11 %, or at least 12%, or at least 13% in absolute terms through use of multiple-stage hydrolysis, meaning that the hydrolysis yield in equivalent time and at equivalent enzyme dose is improved at least 5% in absolute terms over the corresponding yield obtained with equivalent EFB subject to hydrolysis in a single-stage hydrolysis to which is added a quantity of water equivalent to that used for re-suspension in subsequent hydrolysis stages.
  • conversion yields are improved at least 5%, or at least 6%, or at least 7%, or at least 8%, or at least 9%, or at least 10%, or at least 11 %, or at least 12%, or at least 13% in absolute terms through use of multiple-stage hydrolysis, meaning that the hydrolysis yield in equivalent time and at equivalent enzyme dose is improved at least 5% in absolute terms over the improved conversion that is achieved through relief from glucose product inhibition of cellulase enzymes that is achieved by the two step hydrolysis, that is, improved in absolute terms by an amount, expressed as a percentage, corresponding to (two stage hydrolysis yield in absolute terms) - [(two stage hydrolysis in absolute terms obtained with equivalent EFB subject to hydrolysis in a two-stage hydrolysis to which is added in the second stage a quantity of glucose
  • Danish wheat straw was wetted to a DM of > 35% and pretreated at pH > 4.0 by steam to xylan number 10%, which corresponds to severity log Ro approximately 3.75.
  • the pretreatment was conducted in the Inbicon pilot plant in Skasrbask, Denmark.
  • the biomass was loaded into the pretreatment reactor (approx. 50 kg DM/h in continuous mode) using a sluice system and the pretreated biomass was removed from the reactor again using a similar sluice system.
  • the pretreated biomass was subject to a washing step (agitator mixer with water addition of approx. 3 kg/kg DM) followed by a solid/liquid separation using a screw press, producing a liquid fraction and a solid fraction.
  • the solid fraction had a DM content of about 30%, contained the majority of initial cellulose and lignin.
  • washing degree of the produced material was varied by pressing the material in a screw press to a dry matter of approximately 60% followed be re-addition of various amounts of filtrate, Washing degree is defined as amount of dissolved solids removed.
  • EFB from Malaysia was pretreated at DM of > 35% and at pH > 4.0 by steam to xylan number 4- 6%, which corresponds to severity log Ro approximately 4.0.
  • the pretreatment was conducted in the Inbicon pilot plant in Skasrbask, Denmark.
  • the biomass was loaded into the pretreatment reactor (approx. 50 kg DM/h in continuous mode) using a sluice system and the pretreated biomass was removed from the reactor again using a similar sluice system.
  • the pretreated biomass was subject to a washing step (agitator mixer with water addition of approx. 3 kg/kg DM) followed by a solid/liquid separation using a screw press, producing a liquid fraction and a solid fraction.
  • the solid fraction had a DM content of about 30-40%, contained the majority of initial cellulose and lignin.
  • the washing degree of the produced material was varied by pressing the material in a screw press to a dry matter of approximately 60% followed by re-addition of various amounts of filtrate.
  • Solid fractions of washed pretreated wheat straw and EFB were subject to enzymatic hydrolysis as follows: Experiments were conducted in a 6-chamber free fall reactor working in principle as the 6- chamber reactor described and used in WO2006/056838.
  • the 6-chamber hydrolysis reactor was designed in order to perform experiments with liquefaction and hydrolysis at solid concentrations above 20 % DM.
  • the reactor consists of a horizontally placed drum divided into 6 separate chambers each 24 cm wide and 50 cm in height.
  • a horizontal rotating shaft mounted with three paddles in each chamber is used for mixing/agitation.
  • a 1.1 kW motor is used as drive and the rotational speed is adjustable within the range of 2.5 and 16.5 rpm.
  • the direction of rotation is programmed to shift every second minute between clock and anti-clock wise.
  • a water-filled heating jacket on the outside enables control of the temperature up to 80°C.
  • the chambers of the 6 chamber reactor were filled with app. 10 kg of pre-treated biomass adjusted to a dry matter content of 23,5 % WIS (water insoluble solids) for wheat straw and 14 % WIS for EFB.
  • the pretreated biomass was then hydrolysed at 50°C and pH 5.0 to 5.3 using 0.033 ml Cellic CTec3TM from Novozymes / g glucan or 0.1 ml Accellerase TRIOTM from Dupont, Genecor / g glucan Enzymatic hydrolysis experiments were conducted for 24-120 hours at a mixing speed of 6 rpm. Samples were analysed according to the method described by (Kristensen et al. 2009).
  • the content of monosaccharides and disaccharides in the hydrolyzed samples was quantified on a Dionex Ultimate 3000 HPLC system equipped with a Shimadzu Rl-detector. The separation was performed in a Phenomenex Rezex RHM column at 80°C with 5 mM H 2 S0 4 as eluent at a flow rate of 0.6 mL min ⁇ 1 . Samples were diluted with eluent and filtered through a 0.22 pm filter before analysis on HPLC.
  • Figure 1 shows the effect of washing degree (removed dissolved solids - DS) on hydrothermal pretreated wheat straw and EFB.
  • the figure illustrates cellulose conversion as a function of washing efficiency measured as percentage removal of dissolved solids in the washing step between pretreatment and hydrolysis.
  • the black line refers to Wheat straw 0.033 ml CTec3 / g glucan, 24 hours hydrolysis, 23.5 % SS;
  • the gray line refers to EFB 0.1 ml Ac TRIO/ g glucan, 119 hours hydrolysis, 14 % SS.
  • EFB from Malaysia was pretreated at a DM of > 35% and pH > 4.0 by steam to xylan number 4- 6%, which corresponds to severity log Ro approximately 4.0.
  • the pretreatment was conducted in the Inbicon pilot plant in Skasrbask, Denmark.
  • the biomass was loaded into the pretreatment reactor (approx. 50 kg DM/h in continuous mode) using a sluice system and the pretreated biomass was removed from the reactor again using a similar sluice system.
  • the pretreated biomass was subject to a washing step (agitator mixer with water addition of approx. 3 kg/kg DM) followed by a solid/liquid separation using a screw press, producing a liquid fraction and a solid fraction.
  • the solid fraction had a DM content of about 30-40% and contained the majority of initial cellulose and lignin.
  • the degree of washing was found to be approximately 70%.
  • the two-step enzymatic hydrolysis of the solid fraction of washed pretreated EFB was conducted as follows: The first hydrolysis stage, or partial initial hydrolysis, was conducted in the 6 chamber reactor referred to in example 1. Chambers in the 6 chamber reactor were filled with 5.7 kg of pretreated EFB adjusted to 18% DM by addition of water. The pretreated biomass was then hydrolysed at 50°C and pH 5.0 to 5.3 using 0.1 ml Accellerase TRIOTM from Dupont, Genenncor / g glucan Enzymatic hydrolysis was conducted for 168 hours at a mixing speed of 6 rpm.
  • a sample for use in subsequent hydrolysis was taken out of the chamber and separated by centrifugation and pressing of the pellet in a laboratory press. The separation resulted in a press cake with approx. 60% DM and a liquid phase.
  • the press cake was re-suspended in 50 mM citrate buffer at pH 5.3 to a dry matter of 28% in a conical shake flask. pH was adjusted to 5.0-5.3 and the shake flask was incubated without further addition of enzyme on a shake table at 250 rpm at 50°C as the second hydrolysis stage.
  • cellulose conversion can always be enhanced at equivalent enzyme dose by conducting hydrolysis at lower DM. Accordingly, as a control to evaluate the experimental effect of two-step hydrolysis, a single-step hydrolysis was conducted having the same amount of biomass, but with an amount of water corresponding to the water used in both the first and second stages of a two-step hydrolysis. Total dry matter in a two-step hydrolysis conducted as described would correspond to 15% DM in a single-step hydrolysis control.
  • the one-step enzymatic hydrolysis of the solid fraction of washed pretreated EFB was conducted as follows: The hydrolysis was conducted in the 6 chamber reactor referred to in example 1.
  • Chambers in the 6 chamber reactor were filled with 5.7 kg of pretreated EFB adjusted to 15% DM.
  • the pretreated biomass was then hydrolysed at 50°C and pH 5.0 to 5.3 using 0.1 or 0.22 ml Accellerase TRIOTM from Dupont, Genecor / g glucan Enzymatic hydrolysis was conducted for 144 hours at a mixing speed of 6 rpm.
  • Results are shown in Figures 2a and 2b.
  • the two gray lines show cellulose conversion as a function of time for one step hydrolysis with 0.1 ml Ac TRIO / g glucan enzyme, 15 % DM (triangles) and 0.22 ml Ac TRIO / g glucan enzyme, 15 % DM (stars).
  • the black line shows cellulose conversion for a two-step hydrolysis: Black square: first hydrolysis, 0.1 ml Ac TRIO / g glucan, 18 % DM and Black circles: second hydrolysis without added enzyme, 28 % DM.
  • the two- step hydrolysis shows a large enhancement of cellulose conversion relative to the lower DM control.
  • Accellerase Trio/g glucan respectively results in a cellulose conversion of 66 and 80% respectively.
  • a cellulose conversion of 80% can be achieved using only 0.1 ml Accellerase Trio/g glucan in 168 hours of retention time.
  • enzyme consumption can be reduced by a factor of 2x using two-step rather than single-step hydrolysis of hydrothermal pretreated EFB.
  • enzyme proteins are sufficiently retained by the residual solid fraction remaining after partial initial hydrolysis to support subsequent hydrolysis.
  • the gray line in Figure 2b shows cellulose conversion as a function of time for a one step hydrolysis; 0.16 ml Ac TRIO / g glucan; 16 % DM.
  • the black line shows a two-step hydrolysis: Black square: first hydrolysis, 0.1 ml Ac TRIO / g glucan, 20 % DM and Black circles: second hydrolysis additional enzyme added 0.1 ml Ac TRIO / g remaining glucan, 27% DM.
  • the DM and enzyme dose is equivalent overall for the one- and two-step hydrolysis reactions: 16 % DM and an enzyme dose of 0.16 ml Ac TRIO / g glucan.
  • EFB from Malaysia was pretreated at a DM > 35% and a pH > 4.0 by steam to xylan number 4-6%, which corresponds to severity log Ro approximately 4.0.
  • the pretreatment was conducted in the Inbicon pilot plant in Skasrbask, Denmark.
  • the biomass was loaded into the pretreatment reactor (50 kg DM/h in continuous mode) using a sluice system and the pretreated biomass was removed from the reactor again using a similar sluice system.
  • the pretreated biomass was subject to a washing step (agitator mixer with water addition of approx. 3 kg/kg DM) followed by a solid/liquid separation using a screw press, producing a liquid fraction and a solid fraction.
  • the solid fraction had a DM content of about 30-40% and contained the majority of initial cellulose and lignin.
  • the degree of washing was found to be approximately 70%.
  • Filtrate from the first hydrolysis step was produced as follows: The first hydrolysis stage, or partial initial hydrolysis, was conducted in the 6 chamber reactor referred to in example 1. A chamber in the 6 chamber reactor was filled with 10 kg of pretreated biomass comprising about 30-40 % DM adjusted to 18% DM by addition of water. The pretreated biomass was then hydrolysed at 50°C and pH 5.0 to 5.3 using 0.1 ml Accellerase TRIOTM from Dupont, Genecor /g glucan Enzymatic hydrolysis was conducted for 96 hours at a mixing speed of 6 rpm.
  • H1 was then tested for possible inhibitory effect on the cellulase enzyme preparation as follows: Pretreated EFB was diluted to 14% WIS with 50 mM citrate buffer pH 5.3 or H1 in a conical shake flask. The pretreated biomass was then hydrolyzed at 50°C and pH 5.0 to 5.3 using 0.16 ml Accellerase TRIOTM from Dupont, Genecor / g glucan Enzymatic hydrolysis was conducted for 48 hours at a mixing speed of 250 rpm in an incubation table. Filtrate from the second hydrolysis step was produced as follows: The first hydrolysis stage, or partial initial hydrolysis, was conducted in the 6 chamber reactor referred to in example 1.
  • a chamber in the 6 chamber reactor was filled with 10 kg of pretreated biomass comprising about 30- 40% DM adjusted to 20% DM by adding water.
  • the pretreated biomass was then hydrolysed at 50°C and pH 5.0 to 5.3 using 0.1 ml Accellerase TRIOTM from Dupont, Genecor /g glucan Partial initial hydrolysis was conducted for 71 hours at a mixing speed of 6 rpm. After 71 hours of partial initial hydrolysis, a sample for use in subsequent hydrolysis was taken out of the chamber and separated by centrifugation and the solid fraction was pressed in a laboratory press into a press cake with approximately 60% DM and a filtrate.
  • the press cake was re-suspended in 50 mM citrate buffer with pH 5.3 to a dry matter of 27% in a conical shake flask, pH was adjusted to 5.0-5.3 and 0.1 ml Ac TRIO / g glucan was added.
  • the shake flask was incubated on a shake table without further addition of enzyme at 250 rpm at 50°C for the second hydrolysis stage.
  • the hydrolysate was separated by centrifugation and the pellet was pressed in a laboratory press into a press cake with approximately 60% DM and a liquid phase. This liquid phase is named H2 - filtrate after hydrolysis 2.
  • H2 was then tested for possible inhibitory effect on the cellulase enzyme preparation as follows: Pretreated EFB was diluted to 18% WIS with 50 mM citrate buffer pH 5.3 or H2 . The pretreated biomass was then hydrolysed in a conical shake flask at 50°C and pH 5.0 to 5.3 using 0.075 ml Accellerase TRIOTM from Dupont, Genecor /g glucan Enzymatic hydrolysis was conducted for 84 hours at a mixing speed of 250 rpm, 50°C in an incubation table. Results are shown in Figures 3a and 3b. As shown in Figure 3a, when hydrolysis is conducted in buffer, 50 g/kg of glucose is released.
  • Wheat straw was soaked prior to pretreatment at 20-35% dry matter with aprox. 10 g acetic acid/kg dried weight biomass. Approx. 60 kg/h wheat straw or corn stover were pretreated at temperature about 185°C with a residence time of 12 minutes. Biomass was removed from the pretreatment reactor using a sluice device or "particle pump.” The solid and liquid fraction of the pretreated biomass was afterwards separated by screw press resulting in a solid fraction having aproximately 30% total solids (including both insoluble fiber and dissolved solutes). The solid fraction was washed with 3 kg water/kg dry biomass and pressed again to 30% total solids. Washed fiber was hydrolysed using a commercially available cellulase preparation optimized for lignocellulosic biomass conversion provided by NOVOZYMES Tm and provided under the tradename CELLIC CTEC2.
  • Figure 4 shows the percentage of enzyme protein free in solution at different hydrolysis times corresponding to different degrees of conversion. As shown, using this enzyme preparation, enzyme protein loss to supernatant can be kept lower than 10% where partial initial hydrolysis is stopped at 60% conversion.

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Abstract

La présente invention concerne des procédés permettant de traiter des rafles de régimes de fruits (EFB pour Empty Fruit Bunches) à l'aide d'une hydrolyse enzymatique de la cellulase en plusieurs étapes. Les rendements de l'hydrolyse sont améliorés dans un procédé en plusieurs étapes consistant en au moins une étape de séparation solides/liquides dans une mesure étonnamment importante, bien plus que par l'atténuation de l'inhibition du produit glucose des enzymes cellulases.
PCT/DK2013/050388 2012-11-18 2013-11-18 Procédés de traitement de rafles de régimes de fruits (efb) en sucres fermentables à l'aide d'une hydrolyse enzymatique en plusieurs étapes Ceased WO2014075694A1 (fr)

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WO2015017869A1 (fr) * 2013-08-01 2015-02-05 Novozymes A/S Procédé de conversion enzymatique d'une biomasse lignocellulosique
WO2016062646A1 (fr) * 2014-10-21 2016-04-28 Dsm Ip Assets B.V. Procédé d'hydrolyse enzymatique de matériau lignocellulosique, et de fermentation de sucres
EP3098320A1 (fr) * 2015-05-29 2016-11-30 Clariant International Ltd Procede pour l'hydrolyse d'une biomasse
DK202200370A1 (en) * 2022-04-22 2023-12-14 Biovantage Dk Aps Method and apparatus for pretreatment of a biomass comprising lignocellulosic fibers

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WO2006056838A1 (fr) * 2004-11-29 2006-06-01 Elsam Engineering A/S Hydrolyse enzymatique de biomasses ayant une teneur en matieres seches elevee
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015017869A1 (fr) * 2013-08-01 2015-02-05 Novozymes A/S Procédé de conversion enzymatique d'une biomasse lignocellulosique
WO2016062646A1 (fr) * 2014-10-21 2016-04-28 Dsm Ip Assets B.V. Procédé d'hydrolyse enzymatique de matériau lignocellulosique, et de fermentation de sucres
US10435718B2 (en) 2014-10-21 2019-10-08 Dsm Ip Assets B.V. Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars
US10865427B2 (en) 2014-10-21 2020-12-15 Dsm Ip Assets B.V. Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars
US11319560B2 (en) 2014-10-21 2022-05-03 Dsm Ip Assets B.V. Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars
EP3098320A1 (fr) * 2015-05-29 2016-11-30 Clariant International Ltd Procede pour l'hydrolyse d'une biomasse
WO2016192955A1 (fr) * 2015-05-29 2016-12-08 Clariant International Ltd Procédé d'hydrolyse de biomasse
EA033749B1 (ru) * 2015-05-29 2019-11-21 Clariant Int Ltd Способ гидролиза биомассы
US10876141B2 (en) 2015-05-29 2020-12-29 Clariant International Ltd. Process for the hydrolysis of biomass
DK202200370A1 (en) * 2022-04-22 2023-12-14 Biovantage Dk Aps Method and apparatus for pretreatment of a biomass comprising lignocellulosic fibers
DK181708B1 (en) * 2022-04-22 2024-10-24 Clean Vantage Llc Method and apparatus for pretreatment of a biomass comprising lignocellulosic fibers

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