OA17197A - Methods of processing lignocellulosic biomass using single-stage autohydrolysis and enzymatic hydrolysis with C5 bypass and posthydrolysis. - Google Patents

Methods of processing lignocellulosic biomass using single-stage autohydrolysis and enzymatic hydrolysis with C5 bypass and posthydrolysis. Download PDF

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OA17197A
OA17197A OA1201400577 OA17197A OA 17197 A OA17197 A OA 17197A OA 1201400577 OA1201400577 OA 1201400577 OA 17197 A OA17197 A OA 17197A
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pretreatment
biomass
activity
hydrolysis
feedstock
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OA1201400577
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Jan Larsen
Niels Nielsen Poulsen
Martin Dan JEPPESEN
Kit Kellebjerg MOGENSEN
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Inbicon A/S
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Abstract

The invention relates, in general, to methods of processing lignocellulosic biomass to fermentable sugars and to methods that rely on hydrothermal pretreatment. In particular, the invention relates to a method of processing lognocellulosic biomass comprising: providing soft lignocellulosic biomass feedstock, pretreatment of the feedstock in a single-stage pressurized hydrothermal pretreatment to very low severity, separation of the pretreated biomass into an solid fraction and a liquid fraction, hydrolyzing the solid fraction using enzymatic hydrolysis catalysed by an enzyme miture, and subsequently mixing the separated liquid fraction and the hydrolysed solid fraction, whereby xylo-oligomers in the liquid fraction are degraded to xylose monomers.

Description

Methods of processing lignocellulosic biomass using single-stage autohydrolysis and enzymatic hydrolysis with C5 bypass and post-hydrolysis.
Field.
The Invention relates, In general, to methods of processing lignocellulosic biomass to fermentable sugars and, in particular, to methods that rely on hydrothermal pretreatment.
Background.
Historical reliance on petroleum and other fossil fuels has been assodated with dramatic and alarming increases In atmospheric levels of greenhouse gases. International efforts are underway to mitigate greenhouse gas accumulation, supported by formai policy directives In many countries. One central focus of these mitigation efforts has been development of processes and technologies for utilization of renewable plant biomass to replace petroleum as a source of precursors for fuels and other chemlcal products. The annual growth of plant-derived biomass on earth Is estimated to approximate 1 x 10*11 metric tons per year dry weight. See Ueth and Whittaker (1975). Biomass utilization is, thus, an ultimate goal in development of sustainable economy.
Fuel éthanol produced from sugar and starch based plant materials, such as sugarcane, root and grain crops, Is already In wide use, with global production currently topplng 73 billion liters per year. Ethanol has always been consldered an acceptable alternative to fossil fuels, being readily usable as an additive In fuel biends or even directly as fuel for personal automobiles. However, use of éthanol produced by these first génération bloethanol technologies does not actually achieve dramatic réduction In greenhouse gas émission. The net savlngs Is only about 13% compared with petroleum, when the total fossil fuel Inputs to the final éthanol outputs are all accounted. See Farreli et al. (2006). Moreover, both économie and moral objections hâve been raised to the first génération bloethanol market. This effectively places demand for crops as human food Into direct compétition with demand for personal automobiles. And Indeed, fuel éthanol demand has been assodated with Increased grain prices that hâve proved troublesome for poor, graln-lmporting countries.
Great Interest has arisen In developing biomass conversion Systems that do not consume food crops - so-called second génération blorefining, whereby bioethano! and other products can be produced from lignocellulosic biomass such as crop wastes (stalks, cobs, pits, stems, shells, husks, etc...), grasses, straws, wood chips, waste paper and the like. In second génération technology, ferm en table 6-carbon (C6) sugars derived primarily from cellulose and fermentable 5-carbon (C5) sugars derived from hemlcellulose are liberated from biomass polysaccharide polymer chalns by enzymatic hydrolysis or, In some cases, by pure chemical hydrolysls. The fermentable sugars obtained from biomass conversion In a second génération biorefinery can be used to produce fuel éthanol or, altematively, other fuels such as butanol, or lactic acid monomers for use In synthesis of bioplastics, or many other products.
The total yield of both C5 and C6 sugars Is a central considération in commercialization of lignocellulosic biomass processtng. In the case of éthanol production, and aiso production of lactate or other chemlcals, It can be advantageous to combine both C5 and C6 sugar process streams Into one sugar solution. Modified fermentive organlsms are now avallable which can effidently consume both C5 and C6 sugars In éthanol production. See e.g. Madhavan et al. (2012); Dumon et al. (2012); Hu et al. (2011); Kuhad et al. (2011); Ghosh et al. (2011); Kurian et al. (2010); Jojlma et al. (2010); Sanchez et al. (2010); Bettlga et al. (2009); Matsushika et al. (2009).
Because of limitations of Its physlcal structure, lignocellulosic biomass cannot be effectively converted to fermentable sugars by enzymatic hydrolysls without some pretreatment process. A wide variety of different pretreatment schemes hâve been reported, each offering different advantages and disadvantages. For review see Agbor et al. (2011); Girio et al. (2010); Alvira et al. (2010); Taherzadeh and Kariml (2008). From an envlronmental and renewability perspective, hydrothermal pretreatments are especialfy attractive. These utillze pressurized steam/liquid hot water at températures on the order of 160 - 230 °C to gently meit hydrophobie lignin that Is Intricately associated with cellulose strands, to solubilize a major component of hemlcellulose, rich in C5 sugars, and to disrupt cellulose strands so as to Improve accesslbility to productive enzyme binding. Hydrothermal pretreatments can be convenlently Integrated with existing coal- and blomass-fired electrical power génération plants to effidently utilize turbine steam and excess power production capadty.
In the case of hydrothermal processes, It 1s well known In the art, and has been widely discussed, that pretreatment must be optimîzed between confllcting purposes. On the one hand, pretreatment should ideally preserve hemiceliuiose sugar content, so as to maxlmize the ultimate yield of monomeric hemicellulose-derived sugars. Yet at the same time, pretreatment should suffidently expose and pre-condition cellulose chalns to susceptibllity of enzymatic hydrolysis such that reasonable yields of monomeric ceîlulose-derived sugars can be obtalned with minimal enzyme consumptlon. Enzyme consumptlon is also a central considération in commerdalization of biomass processlng, which teeters on the verge of économie profitability* In the context of 'global market économies* as these are currently defined. Notwlthstanding d rama tic Improvements In recent years, the high cost of commerdally available enzyme préparations remalns one of the highest operatlng costs In biomass conversion.
As hydrothermal pretreatment températures and reactor résidence times are Increased, a greater proportion of C5 sugars derived from hemiceliuiose Is irretrievably lost due to chemical transformation to other substances, induding furfurai and products of condensation reactions. Yet higher températures and résidence times are required in orderto properiy condition cellulose libers for effîdent enzymatic hydrolysis to monomeric 6-carbon glucose.
In the prior art, an often used parameter of hydrothermal pretreatment severity* is *Ro,’ which Is typically referred to as a iog value. Ro reflects a composite measure of pretreatment température and reactor résidence time according to the équation: R<,= t*EXP[(T-100)/14.75] where t Is résidence time in minutes and T is reaction température In degrees centigrade. We hâve developed an alternative measure of pretreatment severity, ‘xylan number,* which provides a négative iinear corrélation with dasslcal Iog Ro, even at very low levels of *severity.’ Unlike Ro, which Is a purely emplrical description of pretreatment conditions, xylan number is a functionally slgnificant physical parameter. Xylan number provides a measure of pretreatment degree that permlts comparison of divergent biomass feedstocks, in terms of C5 recoveries, regardless of the Ro severity to which they hâve been subjected.
Whether hydrothermal pretreatment severity is expressed in terms of “xylan number* or *Ro, the optimization of pretreatment conditions for any given biomass feedstock Inherently requires some compromise between demande for high monomeric C5 sugar yields from hemiceliuiose (îow severity) and the demande for high monomeric C6 sugar yields from cellulose (high severity).
Hemlcellulose-derived C5 sugars solubilized during hydro thermal pretreatment typically Inciude a large fraction of xylo-oligomers, which strongly Inhlbit celluiase enzyme catalysis. See Shi et ai. (2011); Quing and Wyman (2011); Quing et al. (2010). Other soluble byproducts of pretreatment,
Induding acetic acid and phenollc compounds derived from solubilized lignin, are also known to
Inhlbit celluiase enzyme catalysis. See Kothari and Lee (2011); Xlmenes et al. (2010). The presence of effective levels of enzyme Inhibltors Increases enzyme consumption required to achieve a given degree of hydrolysis. Accordingly, “économie profitabiiity of commercial scale biomass conversion favors minimlzation of celluiase Inhibition by soluble compounds derived from 10 pretreatment
A variety of different hydrothermal pretreatment strategies hâve been reported for maximizing sugar yields from both hemicellulose and ceiiuiose and for minlmizing xylo-oligomer Inhibition of celluiase catalysis. In some cases, exogenous adds or bases are added In order to catalyse hemicellulose 15 dégradation (add; pH < 3.5) or irgnin solubilisation (base; pH > 9.0). In other cases, hydrothermal pretreatment Is conducted using only very mild acetic add derived from lignocellulose Itself (pH 3.59.0). Hydrothermal pretreatments under these mild pH conditions hâve been termed “autohydrdysis processes, because acetic add llberated from hemicellulose esters itself further catalyses hemicellulose hydrolysis.
Add catalysed hydrothermal pretreatments, known as “diiute add' or “add Imprégnation* treatments, typically provide high yields of C5 sugars, since comparable hemicellulose solubilisation can occur at lower températures In the presence of acid catalyst. Total C5 sugar yields after diiute add pretreatment fdlowed by enzymatic hydrolysis are typically on the order of 25 75% or more of what could theoretlcaliy be liberated from any given biomass feedstock. See e.g.
Baboukaniu et ai. (2012); Won et al. (2012); Lu et al. (2009); Jeong et al. (2010); Lee et al. (2008); Sassner et al. (2008); Thomsen et al. (2006); Chung et al. (2005).
Autohydrolysis hydrothermal pretreatments, In contrast, typically provide much lower yields of C5 sugars, since higher température pretreatment Is required In the absence of add catalyst. With the exception of autohydrolysis pretreatment conducted at commerclally unrealistic low dry matter content, autohydrolysis treatments typically provide C5 sugar yields <40% theoretical recovery. See e.g. Diaz et al. (2010); Dogaris et al. (2009). C5 yields from autohydrolysis as hlgh as 53% β
hâve been reported In cases where commerdally unrealistic reactions times and extreme high enzyme doses were used. But even these very high C5 ylelds remaln well beneath ievels routineiy obtained using dilute add pretreatment. See e.g. Lee et al. (2009); Ohgren et al. (2007).
As a conséquence of Iower C5 ylelds obtained wîth autohydrolysis, most reports conceming hydrothermal pretreatment in commerdai biomass conversion Systems hâve focused on dilute add processes. Hemicellulose-derived C5 sugar yields on the order of 85% hâve been achieved through use of so-called “two-stage* dilute acid pretreatments. In two-stage pretreatments, a Iower Initial température Is used to solubilize hemlcellulose, where a fier the C5-rich liquid fraction is separated. In the second stage, a higher température is used to condition cellulose chains. See e.g. Mesa et al. (2011 ); Kim et al. (2011 ); Chen et al. (2010); Jin et al. (2010); Monavari et al. (2009); Soderstrom et al. (2005); Soderstrom et al. (2004); Soderstrom et al. (2003); Kim et ai. (2001); Lee et ai. (1997); Paptheofanous et al. (1995). One elaborate ’two-stage* dilute add pretreatment System reported by the US National Renewable Energy Laboratory (NREL) daims to hâve achieved C5 yields on the order of 90% using corn stover as feedstock. See Humbird et al. (2011).
Xylo-oligomer inhibition of cellulase catalysis Is avoided In dilute add Systems because hydrolysls of xylo-oligomers to monomeric xylose is catalysed by the added add. The add catalysed hydrolysis of xylo-oligomers also occurs within a separate process stream from that stream in which resldual solids are subject to enzymatic hydrolysis.
Notwithstanding the Iower C5 yields which it provides, autohydrolysis continues to offer compétitive advantages over dilute add pretreatments on commerdai scale.
Most notable amongst the advantages of autohydrolysis processes is that the resldual, unhydrolysed lignin has greatly enhanced market value compared with lignin recovered from dilute add processes. First, the sulphuric add typically used in dilute add pretreatment Imparts a resldual sulphur content. This renders the resulting lignin unattractive to commerdai power plants which would otherwise be Indined to consume sulphur-free lignin fuel peilets obtained from autohydrolysis as a green* alternative to coal. Second, the sulfonation of lignin which occurs during sulphuric add-catalysed hydrothermal pretreatments renders it comparatively hydrophilic, thereby Increasing its mechanical water holding capadty. This hydrophiliaty both Increases the cost of drying the β
lignin for commercial use and also renders It poorfy sulted for outdoor storage, given Its propensity to absorb molsture. So-called ‘techno-economlc model s of NREL's process for lignocellulosic biomass conversion, with dilute add pretreatment, do not even account for lignin as a saleable commodity-onlyas an internai source offuel forprocess steam. See Humblrd étal. (2011). In 5 contrast, the économie profitability of process schemes that rely on autohydrolysis indude a slgnificant contribution from robust saie of dean, dry lignin pellets. This is espedally slgnificant In that typlcal soft lignocellulosic biomass feedstocks comprise a large proportion of lignin, between 10 and 40% of dry matter content. Thus, even where process sugar yields from autohydrolysis Systems can be diminished relative to dilute add Systems, overall profitability” can remaln équivalent or even better.
Autohydrolysis processes also avoid other well known disadvantages of dilute add. The requirement for sulphuric acid diverges from a philosophical orientation favouring green processlng, Introduces a substantiel operating cost for the add as process Input, and créâtes a 15 need for elaborate waste water treatment Systems and also for expensive anti-corrosive equlpment.
Autohydrolysis Is also advantageously scalable to modest processing scénarios. The dilute add process described by NREL Is so complex and elaborate that It cannot realistically be established on a smaller scale - only on a gigantic scale on the order of 100 tons of biomass feedstock per 20 hour. Such a scale Is only appropriate In hyper-centralized biomass processlng scénarios. See
Humblrd et al. (2011). Hyper-centralized biomass processlng of corn stover may well be appropriate In the USA, which has an abundance of genetically-engineered corn grown In chemlcally-enhanced hyper-production. But such a system is less relevant elsewhere in the worid. Such a system 1s Inappropriate for modest biomass processlng scénarios, for example, on-slte processlng at sugar cane or palm oil or sorghum fields, or régional processing of wheat straw, which typically produces much less biomass per hectare than corn, even with genetic-engineering and chemical-enhancements.
Autohydrolysis Systems, in contrast with dilute acid, are legîtimately green, readîly scalable, and 30 unencumbered by requlrements for elaborate waste water treatment Systems. It Is accordingly advantageous to provide Improved autohydrolysis Systems, even where these may not be obvlously advantageous over dilute acid Systems In terms of sugar ylelds alone.
The problem of poor C5 monomer yields with autohydrolysls has generally driven commercial providers of lignocelluloslc biomass processing technology to pursue other approaches. Some “two-stage pretreatment Systems, designed to provide improved C5 yields, hâve been reported with autohydrolysis pretreatments. See W02010/113129; US2010/0279361; WO 2009/108773; US2009/0308383; US8,057,639; US20130029406. In these two stage pretreatment schemes, some C5-rich liquid fraction ls removed by solïd/liquld séparation after a lower température pretreatment, followed by a subséquent, higher température pretreatment of the solid fraction. Most of these published patent applications did not report actual experimental results. In Its description of two-stage autohydrolytic pretreatment In WO2010/113129, Chemtex Italia reports a total of 26 experimental examples using wheat straw with an average C5 sugar recovery of 52%. These C5 recovery values do not distinguish between C5 recovery per se and monomer sugar yields, which ls the substrate actually consumed In fermentation to éthanol and other useful products.
The Introduction of a second pretreatment stage Into a scheme for processing lignocelluloslc biomass Introduces additlonai complexifies and costs. It ls accordingiy advantageous to substantlally achieve the advantages of two-stage pretreatment using a simple single-stage autohydrolysis System.
We hâve discovered that, where single-stage autohydrolysis pretreatment ls conducted to very low severity, unexpectedly hlgh final C5 monomer yields of 60% theoretical yield and higher can be achieved following enzymatic hydrolysis, while still achieving reasonabie glucose yields. Where biomass feedstocks are pretreated to xylan number 10% and higher, a large amount of the original xylan content remains within the solid fraction. Contrary to expectations, this very high residual xylan content can be enzymatically hydrolysed to monomer xylose, with high recovery, while sacrifidng only a very small percentage of cellulose conversion to glucose.
At these very low severity levels, the production of soluble by-products that affect cellulase activity or fermentiva organisais is kept so low that the pretreated material can be used directly in enzymatic hydrolysis, and subséquent fermentation, typically without requirement for any washlng or other de-toxification step.
Inhibition of cellulase catalysis by xylo-oligomers or by other soluble products In the liquid fraction can be easily avoided in the process. A solrd/liquid séparation step following pretreatment generates a liquid fraction and a solid fraction. The C5-rich liquid fraction is maintained separately in bypass from the solid fraction during enzymatic hydrolysis. Following enzymatic hydrolysis of the solid fraction, liquid fraction Is added to hydrolysate and subjectto post-hydrolysis by remainlng active xylanase enzymes. Xylo-oligomers within the liquid fraction are In this manner hydrolysed to xylose monomers only after cellulase activity is no longer necessary. The resulting combined hydrolysate and post-hydolysate comprising both C5 and C6 monomer sugars derived from both cellulose and hemicellulose can be directly fermented to éthanol by modified yeast.
Brief description of the figures.
Figure 1 shows xylan number as a fonction of pretreatment severity factor for soft lignocelluloslc biomass feedstocks subject to autohydrolysis pretreatment.
Figure 2 shows C5 recovery in soluble and insoluble form as a fonction of xylan number for soft lignocelluloslc biomass feedstocks subject to autohydrolysis pretreatment.
Figure 3 shows total C5 recovery as a fonction of xylan number for soft lignocellulosic biomass feedstocks subject to autohydrolysis pretreatment.
Figure 4 shows production of acetic acid, furfural and 5-HMF as a fonction of xylan number for soft lignocellulosic biomass feedstocks subject to autohydrolysis pretreatment.
Figure 5 shows the effect of removal of dissolved solids on cellulose conversion for soft lignocellulosic biomass feedstocks subject to very low severity autohydrolysis pretreatment.
Figure 6 shows HPLC characterization of liquid fraction from soft lignocellulosic biomass feedstocks subject to very low severity autohydrolysis pretreatment.
Figure 7 shows C5 sugar recovery as a fonction of time where solid fraction is subject to enzymatic hydroiysis foilowed by introduction of liquid fraction for post-hydroiysis.
Figure 8 shows fermentation profile of éthanol fermentation by a modified yeast strain using wheat straw that was pretreated by very low severity autohydrolysis, enzymatically hydrolysed and used as combined liquid and solid fraction without de-toxification to remove fermentation inhibitors.
Figure 9 shows a process scheme for one embodiment.
Detaîled description of embodiments.
In some embodiments the invention provides methods of processing lignocellulosic biomass comprising:
- Providing soft lignocellulosic biomass feedstock,
- Pretreating the feedstock at pH within the range 3.5 to 9.0 in a singie-stage pressurized hydrothermal pretreatment to very low severity such that the pretreated biomass Is characterized by having a xylan number of 10% or higher,
- Separating the pretreated biomass Into a solid fraction and a liquid fraction,
- Hydrolyslng the solid fraction with or without addition of supplémentai water content using enzymatic hydroiysis catalysed by an enzyme mixture comprising endoglucanase, exoglucanase, B-glucosidase, endoxylanase, xylosldase and acetyl xylan esterase activities, and
- Subsequently mixing the separated liquid fraction and the hydrolysed solid fraction, whereby xylo-oligomers In the liquid fraction are degraded to xylose monomers by the action of enzyme activities remainlng within the hydrolysed solid fraction.
As used herein, the following terms hâve the following meanlngs:
“About as used herein with référencé to a quantitative number or range refers to +/-10% in relative terms of the number or range referred to.
Autohydrolysis refers to a pretreatment process In which acetic acid liberated by hemicellulose hydrolysis during pretreatment further catalyzes hemicellulose hydrolysis, and applies to any hydrothermal pretreatment of lignocelluloslc biomass conducted at pH between 3.5 and 9.0. Commerdaily avallable cellulase préparation optlmîzed for llgnoceilulosic biomass conversion refers to a commerdaily avaiiable mixture of enzyme activities that is suffident to provide enzymatic hydrolysis of pretreated lignocelluloslc biomass and that comprises endocellulase (endoglucanase), exocellulase (exoglucanase), endoxytanase, acetyl xylan esterase, xylosldase and B-glucosidase activities. The term optimized for lignocellulosic biomass conversion refers to a product development process In which enzyme mixtures hâve been selected and/or modified for the spécifie purpose of Improvlng hydrolysis yields and/or redudng enzyme consumption In hydrolysis of pretreated lignocellulosic biomass to fermentable sugars.
Conducting pretreatment at a dry matter level refers to the dry matter content of the feedstock at the start of pressurized hydrothermal pretreatment. Pretreatment ls conducted “at a pH where the pH of the aqueous content of the biomass ls that pH at the start of pressurized hydrothermal pretreatment.
“Dry matter, also appearing as DM, refers to total solids, both soluble and Insoluble, and effectively means non-water content. Dry matter content is measured by drying at 105°C until constant welght ls achleved.
Fiber structure* ls maintalned to the extent that the average size of fiber fragments following pretreatment ls >750 um.
•Hydrothermal pretreatment refers to the use of water, either as hot liquid, vapor steam or pressurized steam comprising high température liquid or steam or both, to cook biomass, at températures of 120° C or higher, either with or without addition of acids or other chemicals.
Single-stage pressurized hydrothermal pretreatment’ refers to a pretreatment in which biomass ls subject to pressurized hydrothermal pretreatment In a single reactor configured to heat biomass In a single pass and in which no further pressurized hydrothermal pretreatment ls applied following a solid/liquld séparation step to remove liquid fraction from feedstock subject to pressurized hydrothermal pretreatment.
Solid/llquld séparation refers to an active mechanical process whereby liquid is separated from solid by application of force through pressing, centrifugal or other force.
“Soft Iignocelluloslc biomass refers to plant biomass other than wood comprising cellulose, hemiceliulose and lignin.
Solid fraction* and Liquid fraction* refer to fractionation of pretreated biomass In solid/liquld séparation. The separated liquid Is collectively referred to as liquid fraction. The resldual fraction comprising considérable Insoluble solid content Is referred to as “solid fraction.* A “solid fraction* wiil hâve a dry matter content and typlcally will also comprise a considérable residual of ‘liquid fraction.’ 'Theoretical yield* refers to the molar équivalent mass of pure monomer sugars obtained from poiymeric cellulose, or from polymeric hemiceliulose structures, In which constituent monomeric sugars may also be esterified or otherwise substituted. *C5 monomer yields* as a percentage of theoretical yield are determlned as follows: Prior to pretreatment, biomass feedstock Is analysed for carbohydrates using the strong add hydrolysis method of Sluiter et al. (2008) using an HPLC coiumn and elution System In which galactose and mannose co-elute with xylose. Examples of such Systems indude a REZEX ™ Monossacharide H+ coiumn from Phenomenex and an AMINEX HPX 87C ™ coiumn from Blorad. During strong add hydrolysis, esters and add-labile substitutions are removed. Except as otherwise spedfied, the total quantity of Xylose* + Arabinose determlned in the un-pretreated biomass Is taken as 100% theoretical C5 monomer recovery, which can be termed collectively *C5 monomer recovery.* Monomer sugar déterminations are made using HPLC characterization based on standard curves with purified extemal standards. Actual C5 monomer recovery is determlned by HPLC characterization of samples for direct measurement of C5 monomers, which are then expressed as a percent of theoretical yield.
Xylan number* refers to a characterization of pretreated biomass determlned as follows: Pretreated biomass is subject to solid/liquîd séparation to provide a solid fraction at about 30% total solids and a liquid fraction. Thls solid fraction is then partiaily washed by mixing with 70° C water In the ratio of total solids (DM) to water of 1:3 wt:wt. The solid fraction washed in this manner 1s then pressed to about 30% total solids. Xylan content of the solid fraction washed in this manner is determlned using the method of A. Sluiter, et al., Détermination of structural carbohydrates and lignln In biomass,“ US National Renewable Energy Laboratory (NREL) Laboratory Analytical Procedure (LAP) with Issue date April 25,2008, as described In Technlca! Report NREL/TP-51042618, revised April 2008. An HPLC column and elution System Is used In which galactose and mannose co-eiute with xylose. Exampies of such Systems Include a REZEX ™ Monossacharide H+ column from Phenomenex and an AMINEX HPX 87C ™ column from Blorad. This measurement of xylan content as described will include some contribution of soluble material from residua! liquid fraction that Is not washed out of solid fraction under these conditions. Accordingly, “xylan number* provides a “welghted combination measurement of residuai xylan content wlthln Insoluble solids and of soluble xylose and xylo-oligomer content within the liquid fraction.
Any suitable soft lignocellulosic biomass may be used, including biomasses such as at least wheat straw, corn stover, corn cobs, empty fruit bunches, rice straw, oat straw, barley straw, canota straw, rye straw, sorghum, sweet sorghum, soybean stover, switch grass, Bermuda grass and other grasses, bagasse, beet pulp, corn fiber, or any combinations thereof. Lignocellulosic biomass may comprise other lignocellulosic materials such as paper, newsprint, cardboard, or other municipal or office wastes. Lignocellulosic biomass may be used as a mixture of materials originatlng from different feedstocks, may be fresh, partially dried, fully dried or any combination thereof. In some embodiments, methods of the invention are practiced using at least about 10 kg biomass feedstock, or at least 100 kg, or at least 500 kg.
Lignocellulosic biomass comprises crystalline cellulose fibrils Intercalated within a loosely organized matrix of hemicellulose and sealed within an environment rich in hydrophobie lignln. While cellulose ttseff comprises long, straight chain poiymers of D-glucose, hemicellulose Is a heterogeneous mixture of short, branched-chain carbohydrates including monomers of ail the 5carbon aldopentoses (C5 sugars) as well as some 6-carbon (C6) sugars including glucose and mannose. Ugnln Is a hlghly heterogeneous polymer, lacklng any particular primary structure, and comprising hydrophobie phenylpropanoid monomers.
Suitable lignocellulosic biomass typically comprises cellulose in amounts between 20 and 50 % of dry mass prior to pretreatment, lignin In amounts between 10 and 40 % of dry mass prior to pretreatment, and hemicellulose in amounts between 15 and 40%.
In some embodiments, biomass feedstocks may be subject to partiels size réduction and/or other mechanical processlng such as grinding, milling, shredding, cutting or other processes prior to hydrothermal pretreatment. In some embodiments, biomass feedstocks may be washed and/or leached of valuable salts prior to pressurized pretreatment, as described In Knudsen et al. (1998). In some embodiments feedstocks may be soaked prior to pressurized pretreatment at températures up to 99° C.
In some embodiments the feedstock Is first soaked In an aqueous solution prior to hydrothermal pretreatment In some embodiments, It can be advantageous to soak the feedstock In an acetic add contalnlng liquid obtained from a subséquent step In the pretreatments, as described In US 8,123,864. It Is advantageous to conduct treatment at the highest possible dry matter content, as described In US 12/935,587. Conducting pretreatment at high dry matter avolds expenditure of process energy on heating of unnecessary water. However, some water content is requlred to achieve optimal eventual sugar yields from enzymatic hydrolysis. Typically It Is advantageous to pretreat biomass feedstocks at or dose to their Inhérent water holding capadty. This Is the level of water content that a given feedstock will attaln after soaklng In an excess of water followed by pressing to the mechanical limlts of an ordinary commerdal screw press - typically between 30 and 45% DM. In some embodiments, hydrothermal pretreatment Is conducted at DM content at least 35%. It will be readily understood by one skilled In the art that DM content may decrease during hydrothermal pretreatment as some water content Is added during heating. In some embodiments, feedstocks are pretreated at DM content at least 20%, or at least 25%, or at least 30%, or at least 40%, or 40% or less, or 35% or less, or 30% or less.
In some embodiments, soaking/wetting with an aqueous solution can serve to adjust pH prior to pretreatment to the range of between 3.5 and 9.0, which Is typically advantageous for autohydrolysls. It will be readily understood that pH may change during pretreatment, typically to more acidic levels as acetic add is liberated from solubilized hemicellulose.
In some embodiments, hydrothermal pretreatment Is conducted without supplémentai oxygen as required for wet oxidation pretreatments, or without addition of organic solvent as required for organosoiv pretreatment, or without use of microwave heating as required for microwave pretreatments. In some embodiments, hydrothermal pretreatment Is conducted at températures of 140° C or higher, orat 150° C or higher, orat 160° C or higher, or between 160 and 200° C, or between 170 and 190°C, or at 180°C or lower, or at 170° C or iower.
In some embodiments, some C5 content may be removed by a soaklng step prior to pressurized pretreatment In some embodiments, the single reactor may be configured to heat biomass to a single target température. Altematively, the single reactor may be configured to affect a température gradient within the reactor such that biomass is exposed, during a single passage, to more than one température région. In some embodiments, it may be advantageous to partially remove some solubîlized biomass components from within the pressurized reactor during the course of pretreatment.
Suitable hydrothermal pretreatment reactors typically Include most pulping re a et ors known from the pulp and paper Industry. In some embodiments, hydrothermal pretreatment Is administered by steam within a reactor pressurized to 10 bar or lower, or to 12 bar or lower, or to 4 bar or higher, or 8 bar or higher, or between 8 and 18 bar, or between 18 and 20 bar. In some embodiments, the pretreatment reactor Is configured for a continuous Inflow of feedstock.
In some embodiments, wetted biomass is conveyed through the reactor, under pressure, for a certain duration or “résidence time.* Résidence time is advantageously kept brief to fadlitate higher biomass throughput. However, the pretreatment severity obtained is determined both by température and also by résidence time. Température during hydrothermal pretreatment Is advantageously kept lower, not only because methods of the invention seek to obtain a very low pretreatment severity, but also because lower températures can be accomplished using lower steam pressures. To the extent that pretreatment température can be at levels of 180° C or lower, and accordingly, saturated steam pressures kept to 10 bar or lower, lower tendency for corrosion is experienced and much lower grade pressure fittings and steel compositions may be used, which reduces plant capital costs. In some embodiments, the reactor Is configured to heat biomass to a single target température between 160 and 200° C, or between 170 and 190° C. Résidence times in some embodiments are less than 60, or less than 30, or less than 20, or less than 15, or less than 14, or less than 13, or less than 12, or less than 10, or less than 8, or less than 5 minutes. Biomass feedstocks may be loaded from atmospheric pressure into a pressurized reactor by a variety of means. In some embodiments, a sluice-type “partide pump System may be used to load biomass feedstocks, such as the System described in US 13/062,522. In some embodiments, It may be advantageous to load a pretreatment reactor using a so-called “screw plug feeder. Pretreated biomass may be unioaded from a pressurized reactor by a variety of means. In some embodiments, pretreated biomass is unioaded in such manner as to preserve the fiber structure of the material. Preservingthe fiberstructureofthe pretreated biomass isadvantageous becausethis permlts the solid fraction of the pretreated material to be pressed during solid/liquid séparation to comparatîvely high dry matter levels using ordinary screw press equipment, and thereby avolding the added expense and complexity of membrane filter press Systems.
Fiber structure can be maintained by removing the feedstock from the pressurized reactor in a manner that is non-explosive. In some embodiments, non-explosive removal may be accompiished and fiber structure thereby maintained using a sluice-type system, such as that described In US 13/043,486. In some embodiments, non-explosive removal may be accompiished and fiber structure thereby maintained using a hydrocyclone removal system, such as those described In US 12/996,392.
In some embodiments, pretreated biomass can be removed from a pressurized pretreatment reactor using “steam explosion, which Involves explosive release of the pretreated material. Steam-exploded, pretreated biomass does not retain Its fiber structure and accordingly requires more eiaborate solid/liquid séparation Systems In order to achieve dry matter content comparable to that which can be achieved using ordinary screw press Systems with pretreated biomass that re tains Its fiber structure.
The biomass feedstock is pretreated to very low severity, such that the pretreated biomass Is characterized by having a xylan number of 10% or hlgher. In some embodiments, the biomass is pretreated to a xylan number of 11% or higher, or 12% or hlgher, or 13% or higher, or 14% or hlgher, or 15% or higher, or 16% or higher, or 17% or higher. The parameter xylan number refers to a composite measurement that reflects a weighted combination of both residuai xylan content remaining within insoluble solids and also the concentration of soluble xylose and xylo-oligomers within the liquid fraction. At lower Ro severity, xylan number Is higher. Thus, the highest xylan number refers to the lowest pretreatment severity. Xylan number provides a négative linear corrélation with the convention^ severity measure log Ro even to very iow severity, where residuai xylan content within Insoluble solids Is10% or higher.
Xylan number Is particulariy useful as a measure of pretreatment severity In that different pretreated biomass feedstocks having équivalent xylan number exhibit équivalent C5 monomer recovery. In contrast, conventional Ro severity is simply an empirical description of pretreatment conditions, which does not provide a rational basis for comparisons between different biomass feedstocks. For example, single-stage autohydroiysis to severity log Ro= 3.75 provides pretreated sugar cane bagasse and com stover having a xylan number of between 6-7%, while with typical wheat straw stralns, the resulting xylan number of pretreated feedstock is about 10%.
It Is advantageous that biomass feedstocks be pretreated to very low severity wherein xylan number of the pretreated feedstock Is 10% or greater. This very low severity level corresponds to a process In which the total hemicellulose content of the feedstock before pretreatment that Is either solubîlized or irretrievably lost during pretreatment 1s mlnîmlzed. At xylan number 10% and higher, with typical stralns of wheat straw, sugar cane bagasse, sweet sorghum bagasse, corn stover, and empty fruit bunches (from oll paim), at least 60% of the original C5 content of the feedstock can be recovered after single-stage autohydrolysls pretreatment, where both xylan In the solid fraction and also soluble xylose and xylo-oligomers In the liquid fraction are accounted for.
We hâve unexpectedly dlscovered that high final C5 monomer yields of at least 55% theoretlcal, or at least 60%, or at least 65%, can be obtained without appréciable loss of C6 monomer yields after enzymatic hydrolysis of feedstocks pretreated to very low severity by single-stage autohydrolysls. At very low severity levels, a large fraction of the feedstock's hemicellulose content remains within the solid fraction after pretreatment, where It can subsequently be hydrolysed to C5 monomers with high recovery using enzymatic hydrolysis.
It should be noted that reports conceming “xylose recovery” are often expressed In terms that are not comparable to the xylose recoveries reported here. For example, Ohgren et al. (2007) and Lee et al. (2009) report high xylose recoveries. But these values refer only to xylose recovery from pretreated biomass, not expressed as a percentage of the original hemicellulose content of the feedstock prior to pretreatment. Or for example WO2010/113129 refers to hemicellulose recovery as a percentage of hemicellulose content of the feedstock prior to pretreatement, but does not specify the monomer yield, which Is Invariable smaller than the total hemicellulose recovery.
Another startling feature of biomass that has been pretreated by single-stage autohydrolysls to very low severity levels is that the concentrations of pretreatment by-products that serve as Inhibitors of fermentive organisme are kept to very low levels. As a conséquence, It Is typicaliy possible to use hydrolysed biomass obtained by methods of the Invention dîrectly In fermentations, without requirement for any washing or other de-toxification step.
As Is well known In the art, autohydrolysls hydrothermal pretreatment typicaliy produces a variety of soluble by-products which ad as “fermentation Inhibitors, 1n that these Inhibît growth and/or metabolism of fermentive organlsms. Different fermentation Inhibitors are produced In different amounts, dependtng on the propertles of the lignocellulosic feedstock and on the severity of pretreatment. See Klinke et al. (2004). At least three categories of fermentation Inhibitors are typicaliy formed during autohydrolysls pretreatment: (1) furans, primarily 2-furfural and 5-HMF (5 hydroxymethylfurfural) which are dégradation produds from mono- or oligo-saccharides; (2) π
monomeric phénols, which are dégradation products of the lignin structure; and (3) small organic acids, primarily acetic acid, which origlnate from acetyl groups in hemlcelluloses, and lignin. The mixture of different Inhibitors has been shown to act synerglstically in bloethanol fermentation using yeast strains, see e.g. Palmqulst et al. (1999), and, also, using ethanolîc Escherichla coli, see e.g. Zaldivar et al. (1999). In some embodiments, It can be advantageous to subject pretreated biomass to flash évaporation, using methods well known in the art, In order to reduce levels of volatile Inhibitors, most notably furfural. Using autohydroiysis with typlcal strains of biomass feedstocks such as wheat straw, sweet sorghum bagasse, sugar cane bagasse, corn stover, and empty fruit bunches, pretreated to xylan number 10% or hlgher, In our expérience only acetic acid and furfural levels are potentîally inhibitory of fermentive organlsms. Where biomass feedstocks are pretreated at DM 35% or hlgher to xylan number 10% or hlgher, and where solid fraction is subsequently hydroiysed enzymatically at 25% or lower DM, with added water to adjust DM but without washing steps, furfural levels In the hydrolysate can typlcally be kept under 3 g/kg and acetic acid levels beneath 9 g/kg. These levels are typlcally acceptable for yeast fermentations using spedalized strains. During enzymatic hydroiysis, some additional acetic acid Is released from dégradation of hemlcellulose In the solid fraction. In some embodiments, it may be advantageous to remove some acetic acid content from liquld fraction and/or hydroiysed solid fraction using electrodialysis or other methods known in the art.
Different feedstocks can be pretreated using single-stage autohydroiysis to xylan number 10% or greater by a variety of different combinations of reactor résidence times and températures. One skliled In the art will readiiy détermine through routine expérimentation an appropriate pretreatment routine to apply with any glven feedstock, using any glven reactor, and with any given biomass reactor-loading and reactor-unloading system. Where feedstocks are pretreated using a continuous reactor, loaded by elther a slulce-system or a screw-plug feeder, and unloaded by either a partiels pump slulce System or a hydrocyclone system, very low severity of 10% or greater xylan number can be achieved using typlcal strains of wheat straw or empty fruit bunches by a température of 180° C and a reactor résidence time of 24 minutes. For typlcal strains of corn stover, sugar cane bagasse, and sweet sorghum bagasse, very low severity of 10% or greater xylan number can typlcally be achieved using a température of 180° C and a reactor résidence time of 12 minutes, or using a température of 175° C and a reactor résidence time of 17 minutes. It will be readiiy understood by one skliled In the art that résidence times and températures maybe adjusted to achleve comparable levels of Ro severity.
Following pretreatment, pretreated biomass is separated Into a solid fraction and a liquid fraction by a solid/liquid séparation step. it will be readily understood that solid fraction and liquid fraction may be further subdivided or processed. In some embodiments, biomass may be removed from a pretreatment reactor concurrentiy with solid/liquid séparation, in some embodiments, pretreated blomass Is subject to a solid/liquid séparation step after It has been unloaded from the reactor, typically using a simple and low cost screw press System, to generate an solid fraction and a liquid fraction. Cellulase enzyme activities are inhiblted by liquid fraction, most notably due to xylooligomer content but posslbly also due to phénol content and/or other compounds not yet identified. It Is accordingly advantageous to achieve the highest practicable levels of dry matter content in the solid fraction or, altematively, to remove the highest practicable amount of dissolved soiids from the solid fraction. In some embodiments, solid/liquid séparation achleves a solid fraction having a DM content of at least 40%, or at least 45%, or at least 50%, or at least 55%. Solid/liquid séparation using ordinary screw press Systems can typically achieve DM levels as high as 50% in the solid fraction, provided the biomass feedstock has been pretreated in such manner that fiber structure Is malntalned. In some embodiments, It may be advantageous to Incur hlgher plant capital expenses In order to achieve more effective solid/liquid séparation, for example, using a membrane filter press System. In some embodiments, dissolved soiids can be removed from a solid fraction by serial washlng and pressing or by displacement washlng techniques known in the pulp and paper art. In some embodiments, either by solid/liquid séparation directly, or by some combination of washlng and solid/liquid séparation, the dissolved soiids content of the solid fraction Is reduced by at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%.
Enzymatic hydrolysis of feedstocks pretreated to xylan number 10% or higher can typically be conducted with commerdaily reasonable enzyme consumption, without requirement for spécifie washlng or de-toxification steps, where the solid fraction Is pressed to at least 40% DM, or where dissolved soiids content of the solid fraction is reduced by at least 50%.
The liquid fraction obtained from solid/liquid séparation Is maintained separatelyfrom solid fraction during enzymatic hydrolysis of the solid fraction. We term this temporary séparation C5 bypass. Liquld fraction obtained from soft lignocellulosic biomass feedstocks such as typical strains of wheat straw, sugar cane bagasse, sweet sorghum bagasse, corn stover, and empty fruit bunches pretreated by singie-stage autohydrolysîs to xylan number 10% or higher typically comprise a small component of C6 monomers (1 x). primarily glucose with some other sugars; a larger component of
1» soluble C6 oligomers (about 2x - 7x); a larger comportent of C5 monomers (about 4x- 8x), primarily xylose with some arabinose and other sugars; and a much larger component of soluble xyto-oligomers (about 18x - 30x). Soluble xylo-oligomers typically include primarily xylohexose, xylopentose, xylotetraose, xylotriose and xylobiose with some higher chain oligomers.
The solid fraction Is subject to enzymatic hydrolysis using a mixture of enzyme activities. As will be readily understood by one skilled In the art, the composition of enzyme mixtures suitable for practldng methods of the invention may vary within comparatlveiy wide bounds. Suitable enzyme préparations include commerdally available cellulase préparations optimized for lignocelluloslc biomass conversion. Sélection and modification of enzyme mixtures during optimization may Include genetic engineering techniques, for example such as those described by Zhang et al. (2006) or by other methods known In the art. Commerdally available cellulase préparations optimized for lignocelluloslc biomass conversion are typically identifïed by the manufacturer and/or purveyor as such, These are typically dlstind from commerdally available cellulase préparations for general use or optimized for use In production of animal feed, food, textiles détergents or in the paper Industry. In some embodiments, a commerdally available cellulase préparation optimized for lignocellulosic biomass conversion Is used that is provided by GENENCOR ™ and that comprises exoglucanases, endoglucanases, endoxylanases, xylosidases, acetyl xylan esterases and beta glucosidases Isolated from fermentations of genetically modified Trichoderma reesei, such as, for example, the commercial cellulase préparation sold under the trademark ACCELLERASE TRIO ™. In some embodiments, a commerdally available cellulase préparation optimized for lignocellulosic biomass conversion is used that Is provided by NOVOZYMES ™ and that comprises exoglucanases, endoglucanases, endoxylanases, xylosidases, acetyl xylan esterases and beta glucosidases, such as, for example, the commercial cellulase préparations sold under elther of the trademarks CELLIC CTEC2 ™ or CELLIC CTEC3 ™.
The enzyme activities represented in three commerdally available cellulase préparation optimized for lignocellulosic biomass conversion were analysed in detail. Each of these three préparations, ACCELLERASE TRIO ™ from GENENCOR ™ and CELLIC CTEC2 ™ and CELLIC CTEC3 ™ from NOVOZYMES ™, was shown to be effective at enzyme dose levels within the manufacturera' suggested range, in providing combined C5/C6 wheat straw hydrolysate prepared according to methods of the Invention in which C5 monomer yields were at least 60% and cellulose C6 conversion yields were at least 60%. For each of these commercial cellulase préparations, levels of twelve different enzyme activities were characterized and expressed per gram protein. Experimental details are provided in Example 8. Results are shown In Table 1.
Table 1. Selected activity measurements in commercial cellulase préparations optimlzed for 5 lignocellulosic biomass conversion.
CTEC3 Activity ACTrio CTEC2 Substrate
CBHI 45412.5 U/g 17110.4 U/g 381121 U/g MeUmb-3- 1 μι
CBH II* Not measurable Not measurable Not measurable
Endo-1,4-p-glucanase 466131 U/g 149121 U/g 173115 U/g Avicel PH-101 1 pr
β-glucosidase 3350175 U/g 891160 U/g 2447170 U/g Cellobiose
Endo-1,4-p-xy1anase 278110 U/g 799155 U/g 306141 U/g WEAX (medium vise.) 1pr
β-xylosidase 27917.0 U/g 431122 U/g 8710.2 U/g WEAX (medium vlsc.1
β-L- arabînofuranosldase 2011.0 U/g 9.410.4 U/g 1210.1 U/g WEAX (medium visc.l
Laccase No activity No activity No activity Syringaldazine
Amyloglucosidase ÎAMG1 1813.6 U/g 2910.1 U/g 1811.5 U/g Com starch (soluble)
o-amylase 2.710.1 U/g 3.410.5 U/g 4.711.4 U/g Com starch (soluble) 1 pr
Acetyl xylan esterase 3.810^1910-5 U/o 3.1-10^1110^ U/o 4.2-10^14.2-10-4 U/o pNP-acetate 11
Ferulic add esterase No activity No activity No activity Methy! ferulate
In some embodiments, enzyme préparations may be used that hâve similar relative proportions as those exhibited by the commercial préparations described In Table 1 between any of the endoglucanase, exoglucanase, B-glucosidase, endoxylanase, xylosidase and/or acetyl xylan esterase activités.
Enzyme mixtures that are effective to hydrolyse lignocellulosîc biomass can alternative^ be obtained by methods well known in the art from a variety of mlcroorganisms, Induding aérobic and anaérobie bacteria, white rot fungi, soft rot fungl and anaérobie fungl. See e.g. Singhania et al. (2010). Organisms that produce cellulases typically secrete a mixture of different enzymes In appropriate proportions so as to be suitable for hydrolysls of lignocellulosîc substrates. Preferred sources of celluiase préparations useful for conversion of lignocellulosîc biomass Indude fungl such as spedes of Trichoderma, Pénicillium, Fusarium, Humicola, Aspergillus and Phanerochaate.
One fungus spedes In particular, Trichoderma reesel, has been extenslvely studied. Wild type Trichoderma reesei sécrétés a mixture of enzymes comprising two exoceliuiases (cellobiohydroiases) with respective specificities for reducing and non-reducing ends of cellulose chains, at least five different endocellulases having differing cellulose récognition sites, two Bglucosldases as well as a variety of endoxylanases and exoxylosidases. See Rouvinen, J., et al. (1990); DIvne, C., et al. (1994); Martinez, D., et al. (2008). Commercial celluiase préparations typically also Indude alpha-arablnofuranosldase and acetyt xylan esterase activltles. See e.g. Vinzant, T.( et al. (2001).
An optimized mixture of enzyme activities in relative proportions that differ from the proportions presented In mixtures naturally secreted by wild type organisms has prevlously been shown to produce higher sugar yields. See Rosgaard et al. (2007). Indeed, it Is has been suggested that optimizations of enzyme blends induding as many as 16 different enzyme proteins can be advantageously determined separateiy for any given biomass feedstock subject to any given pretreatment. See Billard, H., et al. (2012); Banerjee, G., et ai. (2010). As a commerdal practicality, however, commerdal enzyme providers typically seek to produce the smallest practicable number of different enzyme blends, In order that économies of scale can be obtained in large-scale production.
In some embodiments, it can be advantageous to supplément a commerdally avaiiable cellulase préparation optimlzed for lignocellulosic biomass conversion with one or more additional or supplémentai enzyme activities. In some embodiments, it may be advantageous simply to Increase the relative proportion of one or more component enzymes présent in the commerdai préparation. In some embodiments, lt may be advantageous to Introduce spedalized additional activities. For example, In practiclng methods of the Invention using any given biomass feedstock, particular unhydrolysed carbohydrate linkages may be identified that could be advantageously hydrolysed through use of one or more supplémentai enzyme activities. Such unhydrolysed linkages may be identified through charaderization of oligomeric carbohydrates, using methods weil known in the art, in soluble hydrolysates or in insoluble unhydrolysed residual. Unhydrolysed linkages may also be Identified through comprehensïve mlcroarray polymer profiling, using monodonal antibodîes dlrected against spécifie carbohydrate linkages, as described by Nguema-Ona et al. (2012). In some embodiments it can be advantageous to supplément a commerdally avallable celiuiase préparation optimized for lignocellulosic biomass conversion using any one or more of additional endoxylanase, B-glucosidase, mannanase, glucouronidase, xylan esterase, amylase, xylosidase, glucouranyl esterase, or arabinofuranosidase.
In some embodiments, it can altematively be advantageous to produce enzymes on-site at a lignocellulosic biomass processing fadiity, as described by Humbird et al. (2011). In some embodiments, a commerdally avallable cellulase préparation optimlzed for lignocellulosic biomass conversion may be produced on-slte, with or without customlzed supplémentation of spedfic enzyme activities appropriate to a particular biomass feedstock.
In some embodiments, whether or not a commerdally avaiiable cellulase préparations optimlzed for lignocellulosic biomass conversion is used, and whether or not enzymes are produced on-site at a biomass processing plant, advantages of the Invention can be obtained using soft lignocellulosic biomass feedstocks subject to autohydrolysis pretreatment to very low severity xylan number 10% or greater using an enzyme mixture that comprises the following: (1) Exocellulase (cellobiohydrolase) activities (EC 3.2.1.91), optionally inciuding at least two enzymes with spedfidties for redudng and non-redudng ends of cellulose chalns; (2) endoceiiulase activity (EC 3.2.1.4): (3) B-glucosldase activity (EC 3.2.1.21); (4) B-1,4 endoxylanase activity (EC 3.2.1.8); (5) acetyl xylan esterase activity (EC 3.1.1.72); and optionally (6) B-1,3 xylosidase activity (EC 3.2.1.72); and optionally (7) B-1,4 xylosidase activity (EC 3.2.1.37); and optionally (8) alpha 1,3 and/or alpha 1, 5 arablnofuranosidase activity (EC 3.2.1.23). In some embodiments, the enzyme mixture is further characterized by having relative proportions of enzyme activities as foilows: 1 FPU cellulase activity Is assodated with at least 30 CMC U endoglucanase activity and with at least at least 28 pNPG U beta glucosidase activity and with at least 50 ABX U endoxylanase activity. It will be readiiy understood by one skilled In the art that CMC U refers to carboxymethycellulose units, where one CMC U of activity libérâtes 1 umol of redudng sugars (expressed as glucose équivalents) in one minute under spedfic assay conditions of 50° C and pH 4.8; that pNPG U refers to pNPG units, where one pNPG U of activity libérâtes 1 umol of nitrophenol per minute from paranitrophenyl-B-D-glucopyranoside at 50° C and pH 4.8; and that ABX U refers to birchwood xylanase units, where one ABX U of activity liberales 1 umol of xylose redudng sugar équivalent In one minute at 50° C and pH 5.3. It will be further readiiy understood by one skilled In the art that FPU refers to filter paper units, which provides a measure of total cellulase activity Induding any mixture of different cellulase enzymes. As used herein, FPU refers to filter paper units as determlned 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).
in some embodiments the enzyme mixture may further Include any one or mor of mannosldases (EC 3.2.1.25), a-D-galactosidases (EC 3.2.1.22), a-L-arablnofuranosidases (EC 3.2.1.55), a-Dglucuronldases (EC 3.2.1.139), dnnamoyl esterases (EC 3.1.1.·), or feruloyl esterases (EC 3.1.1.73).
One skllled In the art will readiiy détermine, through routine expérimentation, an appropriate dose level of any given enzyme préparation to apply, and an appropriate duration for enzymatic hydrolysis. It Is generally advantageous to maintain lower enzyme dose levels, so as to minimize enzyme costs. In some embodiments, It can be advantageous to use a high enzyme dose. In pradidng methods of the Invention, one skilled In the art can détermine an économie optimisation of enzyme dose In considération of relevant factors Induding local biomass costs, market prices for product streams, total plant capital costs and amortization schémas, and other factors. In embodiments where a commerciaily available cellulase préparation optimlzed for lignocellulosic biomass conversion Is used, a general dose range provided by manufacturera can be used to détermine the general range within which to optimlze. Hydrolysis duration in some embodiments Is at least 48 hours, or at least 64 hours, or at least 72 hours, or at least 96 hours, or for a time between 24 and 150 hours.
As ls well known In the art, cellulase catalysls ls more efficient where hydrolysis ls conducted at low dry matter content. Higher solids concentration effectively Inhlbits cellulase catalysis, although the précisé reasons for this well known effect are not fuily understood. See e.g. Kristensen et al. (2009).
In some embodiments, it may be advantageous to conduct hydrolysis at very high DM > 20%, notwithstandlng some resulting Increase in enzyme consumption. It is generally advantageous to conduct hydrolysis at the hlghest practicabie dry matter ievel, both In order to minlmize water consumption and waste water treatment requirements. It ls additionally advantageous In fermentation Systems to use the hlghest practicabie sugar concentrations. Higher sugar concentrations are produced where hydrolysis ls conducted at higher dry matter levels. One skilled In the art will readily détermine, through routine expérimentation, a DM Ievel at which to conduct enzymatic hydrolysis that ls appropriate to achieve given process goals, for any glven biomass feedstock and enzyme préparation. In some embodiments, enzymatic hydrolysis of the solid fraction may be conducted at 15% DM or gréa ter, or at 16% DM or greater, or at 17% DM or greater, or at 18% DM or greater or at 19% DM or greater, or at 20% DM or greater, or at 21% DM or greater, or at 22% DM or greater, or at 23% DM or greater, or at 25% DM or greater, or at 30% DM or greater, or at 35% DM or greater.
In some embodiments, solid fraction ls recovered from solid/liquid séparation at 40% DM or greater, but additional water content is added so that enzymatic hydrolysis may be conducted at lower DM levels. It will be readily understood that water content may be added In the form of fresh water, condensate or other process solutions with or without additives such as polyethyiene glycol (PEG) of any molecular weight or surfactants, salts, chemlcals for pH adjustment such as ammonia, ammonium hydroxlde, calcium hydroxide, or sodium hydroxide, anti-bacterial or anti-fungal agents, or other materials.
After the solid fraction has been enzymatically hydrolysed to a desired degree of conversion, the liquid fraction, which has been maintained in C5 bypass, ls mixed with the hydrolysate mixture for post-hydrolysis. In some embodiments, ali of the recovered liquid fraction may be added at one
2β time, while in other embodiments, some component ofthe liquid fraction may be removed and/or liquid fraction may be added Incrementally. In some embodiments, prior to mlxing with liquid fraction, the solid fraction Is hydrolysed to at least 50%, or at least 55%, or at least 60% cellulose conversion, meanlng that at least the spedfied theoretical yield of glucose monomers Is obtained. A substantial portion of xylo-oligomers présent In liquid fraction can typically be hydrolysed to xylose monomers by action of xylanase and other enzymes that remain active within the hydrolysate mixture. In some embodiments post-hydrolysis is conducted for at least 6 hours, or for a time between 15 and 50 hours, or for at least 24 hours. In some embodiments, at least 60%, or atleast65%, orat least70%, or at least 75%, orat least80%, oratleast85%, orat least 90% by mass of xylo-ollgomers présent In the liquid fraction are hydrolysed to xylose monomers during post-hydrolysis by action of xylanase and other enzymes that remain active within the hydrolysate mixture. In some embodiments, the liquid fraction Is mlxed with hydrolysate directiy, without further addition of chemical additives. In some embodiments, some components of liquid fraction such as acetic add, furfural or phénols may be removed from liquid fraction prior to mlxing with hydrolysate.
In some embodiments, enzymatic hydrolysis ofthe solid fraction and/or post-hydrolysis ofthe liquid fraction may be conducted as a simultaneous saccharification and fermentation (SSF) process. As is well known In the art, when SSF can be conducted at the same température as that which Is optimal for enzymatic hydrolysis, enzyme consumption can be mlnimized because a fermentive organism introduced during the course of enzymatic hydrolysis consumes glucose and xylose monomers and thereby reduces product Inhibition of enzyme catalyzed reactions. In some embodiments, post-hydrolysis Is only conducted after the fiber fraction has been hydrolysed, without addition of fermentive organism, to at least 60% cellulose conversion.
Where biomass feedstocks such as typical strains of wheat straw, sugar cane bagasse, sweet sorghum bagasse, com stover or empty fruit bunches are pretreated at 35% or greater DM by single-stage autohydroiysis to xylan number 10% or greater, where solid fraction ofthe pretreated biomass Is obtained having at least 40% DM or having at least 50% removal of dîssolved solids, where solid fraction Is subsequently subject to enzymatic hydrolysis at DM between 15 and 27% using a commercially available celiulase préparation optimized for lignocellulosic biomass conversion, where enzymatic hydrolysis Is conducted for at least 48 hours, where liquid fraction Is added to the solid fraction hydrolysate after at least 50% glucose conversion has been obtained, and where the added liquid fraction Is subject to post-hydrolysis for a period of at least 6 hours, It Is typlcally possible to achieve C5 monomer concentrations in the combined C5/C6 hydrolysate that correspond to C5 monomer yields of 60% or greater of the theoretical maximal xylose yield.
In some embodiments, the combined C5/C6 hydrolysate can be directly fermented to éthanol using one or more modified yeast strains.
Figure 9 shows a process scheme for one embodiment. As shown, soft Iignocelluloslc biomass is soaked, washed or wetted to DM 35% or greater. The biomass is pretreated at pH within the range of 3.5 to 9.0 using pressurized steam in single-stage autohydrolysis to a severity characterized by xylan number 10% or greater. The pretreated biomass is subject to solid/liquid séparation produdng a liquid fraction and a solid fraction having DM content 40% or greater. The solid fraction is adjusted to an appropriate DM content then subject to enzymatic hydrolysis at DM content 15% or greater to a degree of cellulose conversion 60% or greater. The separated liquid fraction is subsequently mlxed with the hydrolysed solid fraction and subject to post-hydroiysls, whereby a substantiel quantity of xylo-oligomers présent In the iiquid fraction are hydrolysed to monomeric xylose. After the end of hydrolysis and post-hydroiysis as described, the C5 monomer yield Is typlcally at least 60% while the cellulose conversion Is similarly at least 60%.
Examples:
Example 1. ’Xyian number* characterization of solid fraction as a measure of pretreatment severity.
Wheat straw(WS), com stover (CS), Sweet sugarcane bagasse (SCB) and Empty Fruit Bunches (EFB) were soaked with 0-10 g acetic acid/kg dry matter biomass, pH > 4.0, prior to pretreatment at 35-50% dry matter About 60 kg DM/h biomass was pretreated at températures from 170-200°C with a résidence time of 12-18 minutes. The biomass was loaded Into the reactor using a slutce System and the pretreated material unloaded using a slulce system. The pressure within the pressurized pretreatment reactor corresponded to the pressure of saturated steam at the température used. The pretreated biomass was subject to solid/liquid séparation using a screw press, produdng a iiquid fraction and a solid fraction having about 30% dry matter. The solid fraction was washed with about 3 kg water/kg dry biomass and pressed to about 30% dry matter agaln. Details concemlng the pretreatment reactor and process are further described In Petersen et ai. (2009).
Raw feedstocks were anaiysed for carbohydrates according to the methods described in Sluiterel al. (2005) and Slulter et al. (2008) using a Dionex Ultimate 3000 HPLC system equlpped with a Rezex Monossacharide H+ coiumn from Phenomenex. Samples of liquid fraction and solid fraction were collected after three hours of continuous pretreatment and samples were collected three times over three hours to ensure that a sample was obtained from steady state pretreatment. The solid fractions were anaiysed for carbohydrates according to the methods described in Slulter et al. (2008) with an Ultimate 3000 HPLC system from Dionex equlpped with a Rezex Monossacharide H+ Monosaccharide coiumn. The liquid fractions were anaiysed for carbohydrates and dégradation products according to the methods described In Slulter et al. (2006) with an Ultimate 3000 HPLC system from Dionex equlpped with a Rezex Monossacharide H+ Monosaccharide coiumn. Dégradation products In the solid fraction were anaiysed by suspension of the solid fraction In water with 5mM sulphuric add In a ratio of 1:4 and afterward anaiysed according to the methods described In Sluiter et al. (2006) with an Ultimate 3000 HPLC system from Dionex equlpped with a Rezex Monossacharide H+ coiumn. The dry matter content and the amount of suspended solids was anaiysed according to the methods described in Weiss et al. (2009). Mass balances were set up as described In Petersen et al. (2009) and cellulose and hemicellulose recoveries were determlned. The amount of sugars which were degraded to 5-HMF orfurfural and the amount of acetate released from hemlcelleulose during pretreatment per kg of biomass dry matter was quantified as well, although loss of furfural due to flashing ls not accounted for.
The severity of a pretreatment process ls commonly described by a severity factor, first developed by Overend et al. (1987). The severity factor ls typlcally expressed as a log value such that log(Ro)=t*eksp((T-Tref)/14.75), where Ro is the severity factor, t ls the résidence time in minutes, T ls the température and T„f is the référencé température, typlcally 100°C. The severity factor ls based on klnetics of hemicellulose solubilisation as described by Belkecemi et al. (1991), Jacobsen and Wyman (2000) or Lloyd et al. (2003). The severity of a pretreatment ls thus related to resldual hemicellulose content remalnlng In the solid fraction after pretreatment.
Solid fractions prepared and washed as described were anaiysed for C5 content according to the methods described by Sluiter et al. (2008) with a Dionex Ultimate 3000 HPLC system equlpped with a Rezex Monossacharide H+ column from Phenomenex. The xylan content In the solid fraction produced and washed as described above Is linearly depended upon the severity factor for soft lignocelluloslc biomasses such as for example wheat straw, com stover of EFB when pretreating by hydrothermal autohydrolysis. The définition of severity as the xylan content in a solid fraction prepared and washed as described above is transférable between pretreatment setups. Xylan number Is the measured xylan content In the washed solid fractions, which Includes some contribution from soluble material. The dependence of xylan number on pretreatment severity iog(Ro) Is shown In Figure 1 for wheat straw, com stover, sugarcane bagasse and empty fruit bunches from palm oli processing.
As shown, there existe a ciear, négative linear corrélation between xylan number and pretreatment severity for each of the tested biomass feedstocks pretreated by single-stage autohydrolysis.
Exampie 2. C5 recovery as a fonction of pretreatment severity.
Biomass feedstocks were pretreated and samples characterlzed as described In example 1. Figure 2 shows the C5 recoveries (xylose + arablnose) as a fonction of xylan number for experiments where wheat straw was pretreated by autohydrolysis. C5 recoveries are shown as water Insoluble solids (WIS), water soluble solids (WSS) and total recovery. As shown, C5 recovery as both water Insoluble and water soluble solids Increases as xylan number increases. As xylan number increases over 10%, C5 recovery as water soluble solids diminishes while C5 recovery as water Insoluble solids continues to Increase
Typical strains of wheat straw tested contained about 27% hemlcellulose on dry matter basls prior to pretreatment Figure 3 shows total C5 recovery after pretreatment as a fonction of Xylan number for wheat straw, com stover, sugarcane bagasse and EFB pretreated by autohydrolysis. Typical strains of com stover, sweet sugarcane bagasse and EFB tested contained about 25%, 19% and 23% respectively of C5 content on dry matter basls prior to pretreatment As shown, for ali feedstocks, total C5 recovery after pretreatment is dépendent upon pretreatment severity as defïned by xylan number. As shown, where 90% of C5 content recovered after pretreatment can be fully hydroiysed to C5 monomer, a 60% final C5 monomer yield after enzymatic hydrolysis can be expected where pretreatment severity Is characterlzed by produclng a xylan number of 10% or higher.
Example 3. Production of dégradation products that Inhibit enzymes and yeast growth as a fonction of pretreatment severity.
Biomass feedstocks were pretreated and samples characterized as described In example 1. Figure 4 shows the dependence of acetic acid release and production of furfural and 5-hydroxy-methylfufural (5-HMF) as a fonction of xylan number for experiments where wheat straw was pretreated by single-stage autohydrolysis. As shown, production of these dégradation products, which are well known to Inhibit fermentive yeast and which In some cases also Inhibit cellulase enzymes, exhibits an exponential Increase at xylan numbers lower than 10%. At xylan number 10% and higher, the levels of furfural and acetic add fall within ranges that permit fermentation of pretreated biomass without requirement for de-toxification steps. In the case of acetic add, levels are further Increased during enzymatic hydrolysis of biomass pretreated to xylan number 10% and higher, although typlcaliy to levels that are well tolerated by yeast modified to consume both C5 and C6 sugars.
Example 4. Inhibition of cellulase enzymes by material remalnlng In solid fraction as a fonction of DM% of solid fraction.
Experiments were conducted In a 6-chamber free fall reactor working In princlple as the 6-chamber reactor described and used In W02006/056838. The 6-chamber hydrolysis reactor was designed In order to perform experiments with liquéfaction 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 mixlng/agîtation. A1.1 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 température up to 80’C.
The experiments used wheat straw, pretreated by single-stage autohydrolysis. The biomass was wetted to a DM of > 35% and pretreated at pH > 4.0 by steam to xylan number 10.5%.. The pretreatment was conducted In the Inblcon pilot plant In Skærbæk, Denmark. The biomass was loaded Into the pretreatment reactor using a sluice system and the pretreated biomass removed from the reactor using a sluice system. The pretreated biomass was, In some cases, subject to solid/liquid séparation using a screw press, producing a liquid fraction and a soiid fraction. The solid fraction had a DM content of about 30%, contained the majority of initial cellulose and lignin, part of the hemicellulose and a total of about 25% ofthe dissolved solids.
The chambers of the 6 chamber reactor were filled with either total pretreated biomass comprising ail dissolved and undissolved solids or pressed soiid fraction comprislng about 25% of total dissolved solids. Dry matter content was adjusted to 19 % DM. The pretreated biomass was then hydrolyzed at 50’C and pH 5.0 to 5.3 using 0.08 ml CTec2 w from Novozymes / g glucan or 0.20.3 ml Acceilerase TRIO w from Dupont, Genencor/g glucan. These dose levels of these commerdaliy available cellulase préparations optimized for lignocellulosic biomass conversion were well within the range suggested by the manufacturers. Enzymatic hydrolysis experiments were conducted for 96 hours at a mlxlng speed of 6 rpm.
Figure 5 shows cellulose conversion after enzymatic hydrolysis under these conditions as a fonction of % dissolved solids removed prior to enzymatic hydrolysis. As shown, removal of 75% dissolved solids at these enzyme dose levels Improves cellulose conversion by 10-20% in absolute terms. Thus, It Is advantageous to press solid fraction to DM content at least 40% or to otherwise reduce dissolved solids content by at least 50% prior to enzymatic hydrolysis, since this will provide Improved enzyme performance.
Example 5. Sugar content and hydrolysis of liquid fraction from biomass pretreated to xylan number > 10%.
Wheat straw, corn stover, and sugar cane bagasse were pretreated to xylan number 11.5% (WS), 12.3% (SCB) and 15.5% (CS) and subject to solid/liquid séparation to produce a iiquid fraction and a solid fraction, as described In example 5. The liquid fractions were anaiysed for carbohydrates and dégradation products according to the methods described In (Sluiter, Hames et al. 2005) using a Dionex Ultimate 3000 HPLC system equipped with a Rezex Monosaccharide column. Table 2 shows the sugar content of liquid fractions expressed as a percent of DM content broken down Into categories of oligomeric and monomeric glucose/glucan, xylose/xylan and arabinose/arabinan. As shown, whüe some glucose content Is présent In both monomeric and oligomeric form, the buik of the sugar content Is oligomeric xylan. The prédominance of xylan oligomers In liquid fraction obtained using autohydrolysis is In noted contrast with the liquid fraction obtained using dilute acid pretreatment In biomass pretreated by dilute acid hydrothermal pretreatment, the liquid fraction Is typically hydrolysed to monomerlc constituents by actions of the acid catalyst.
Table 2. Sugar content of liquid fractions In biomass pretreated to xylan number >10%.
Ollgomerlc glucan Monomerlc glucose Ollgomerlc xylan Monomerlc xylose Ollgomerlc arablnan Monomerlc arabinose Other DM
ws 5,5% 2,1% 40,4%J 8,6% 1;1% 4,8% 37%
SCB 8,2% 3,1% 39,1% 8,7% 0,7% 3,1% 37%
SC 6,2% 1,9% 37,0% 5,3% 2,8% 3,9% 43%
The liquid fraction from pretreated wheat straw was further characterized by HPLC analysis using a Thermo Sdentific Dionex CarboPacTM PA200 column using a modular Dionex ICS-5000 chromatographie System. The analytes were separated using NaOH/NaOAc-gradient conditions and measured by Integrated and pulsed amperometric détection (IPAD) using a gold electrode. Figure 6 shows an HPLC chromatogram In which the elution profile of xyloblose (X2), xylotriose (Xj), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X«) standards Is super-imposed as the upper trace over the lower trace, which depids the elution profile of liquid fraction. As shown, liquid fraction of the autohydroiysed biomass contalns a mixture comprising a small amount of xylose monomer and comparatively larger amounts of xyloblose (X2), xylotriose (X3), xylotetraose (X<), xylopentaose (Xs), and xylohexaose (Xe), along with other materials.
Example 6. Enzymatic hydrolysis of solid fradion and addition of liquid fraction after the fibre hydrolysis from biomass pretreated to xylan number > 10% and pressed to > 40% DM followed by post hydrolysis.
Expérimente were conducted in a 6-chamber free fall reactor as described In example 4.
The experiments used wheat straw, corn stover, or sugar cane bagasse pretreated by single-stage autohydrolysis to xylan numbers ranging from 11.5 to 15.6%. The biomass was eut and wetted to a DM of > 35% and pretreated by steam at 170-190‘C for 12 min. The pretreatment was conducted In the Inbicon pilot plant in Skærbæk, Denmark. The pretreated biomass was subject to solid/liquid 25 séparation using a screw press to produce a solid fraction having > 40% DM.
The chambers of the 6 chamber reactor were filled with about 10 kg pressed pretreated biomass and adjusted by water addition to 19-22 % DM. The pretreated biomass was hydrolyzed at 50*C and pH 5.0 to 5.3 using ACCELLERASE TRIO ™ from GEN ENCOR-DuPONT. The mixing speed was 6 rpm. The hydrolysis experiments were run for 96 hours and afterwards the liquid fraction pressed from the solid fraction after pretreatment was added and the post hydrolysis was run for 48 hours at 50*C and pH 5.0 to 5.3.
HPLC samples were taken daily to follow the conversion of cellulose and hemicellulose and analysed for glucose, xylose and arabinose using a Dionex Ultimate 3000 HPLC system equipped with a Rezex Monosaccharide column with quantification through use of extemal standard.
Figure 7 shows hydrolysis data for conversion of hemicellulose with addition of liquid fraction after 96 hours hydrolysis of solid fraction using sugar cane bagasse pretreated to xylan number 12.3% and hydrolysed using 0.3 ml Accellerase Trio w (Genencor) per g glucan. Shown Is a typical hydrolysis profile. C5 monomer recovery is expressed as a percent of theoretlcal yield from the material présent in the hydrolysis reaction. Most of the hemicellulose within the solid fraction has been converted to monomeric sugars within the first 24 hours In hydrolysis of the solid fraction. Addition of liquid fraction after 96 hours increases the theoretlcal potential yield, which explalns the drop In C5 conversion observed Just after liquid fraction Is added. Within the first 24 hours most of the C5 from liquid fraction Is converted to monomers. Comparing the C5 conversion just before liquid fraction Is added with the end point of the hydrolysis. it Is possible to calculate the C5 conversion In the liquid fraction as 90 % when using sugar cane bagasse under these conditions.
Table 3 shows hydrolysis data for different biomasses pretreated under different circumstances and hydrolysed using different dose levels of a commercially available cellulase préparation optlmlzed for lignocellulosic biomass conversion, Accellerase Trio ™ (Genencor). AH enzyme dose levels used were within the range suggested by the manufacturer. As shown, using single-stage autohydrolysis and enzymatic hydrolysis with C5 bypass and post-hydrolysis. C5 monomer ylelds of 60% or greater can be achieved using manufacturera’ recommended doses of commercially available cellulase préparations optimized for lignocellulosic biomass conversion while still achlevlng cellulose conversion of 60% or greater.
Table 3. Hydrolysis yields using very low severity single-stage autohydrolysis with C5 bypass and post-hydrolysis.
WS SCB SCB CS CS EFB
Dry matter after soaking [wt%] 40% 39% 39% 40% 40% 39%
Résidence time [min] 12.0 12.0 12.0 12.0 12.0 12.0
Température [’C] 183.0 182.7 182.7 174.5 174.5 185.2
Pretreatment severity [logRo] 3.52 3.51 3.51 3.27 3.27 3.58
C5 recovery from pretreatment [%] 74% 87% 87% 88% 88% 84%
Xylan number 11.5% 12.3% 12.3% 15.6% 15.6% 15.5%
Enzyme dosage [mL Ac. TRIO/g glucan] 0.2 0.3 0.3 0.3 0.2 0.4
%TS in fiber hydrolysis 22% 22% 22% 19% 22% 22%
Cellulose conversion after hydrolysis (96h) 78% 64% 66% 68% 58% 69%
Hemiceliuiose conversion (C5 recovery) after hydrolysis (96h) 80% 73% 73% 61% 61% 75%
%TS in second hydrolysis 18% 17% 17% 16% 18% 18
Cellulose conversion after post hydrolysis (144h) 78% 65% 67% 67% 61% 72%
Hemiceliuiose conversion (C5 recovery)after post hydrolysis (144h) 90% 79% 78% 71% 68% 83%
Overall cellulose conversion 78% 65% 67% 67% 61% 72%
Overall C5 monomer yield 67% 69% 68% 63% 60% 70%
Example 7. Co-fermentation to éthanol of C5 and C6 sugars in combined hydrolysate by modified yeast.
As an example on the use of a hydrolysate produced from soft lignocellulosic biomass (in this case wheat straw) prepared by single-stage autohydrolysis pretreatment to a xylan number > 10%, Figure 8 shows data for a fermentation performed without détoxification or any other process steps before fermentation with GMO yeast able to convert both C5 and C6 sugars (strain V1 from TERRANOL ™). The hydrolysate was adjusted to pH 5.5 with KOH pellets before fermentation and supplemented with 3 g/L urea. The fermentation was conducted as a batch fermentation. The initial cell concentration In the reactor was 0.75 g dw/L. The fermentations were controlled at pH 5.5 using automatic addition of 10% NH3. The température was kept at 30’C and the stirring rate was 300 rpm. As shown, glucose and xylose are readily consumed and éthanol readily produced, notwithstanding the presence of acetic acid, furfural and other compounds that would typically prove Inhibitory at hlgher levels of pretreatment severity.
Example 8. Experimental détermination of activity levels In commercial cellulase préparations.
Commercial préparations of ACCELLERASE TRIO ™ from GENENCOR ™ and CELLIC CTEC2 ™ and CELLIC CTEC3 ™ from NOVOZYMES ™ were diluted so that protein concentrations were roughly équivalent In sample préparations tested. Equivalent volumes of diluted enzyme préparations were added and assay déterminations made In duplicate or triplicate.
Assay of CBHI (exocellulase) activity was conducted In 50 mM NaOAC buffer at pH 5,25° C, for 25 minutes. Activity was determlned in triplicate by following continuous rate of 4üMethylumbelliferon release (Abs: 347nm) from the model substrate 4nmethylumbeliiferylnpncellobloside. Activity unit was 1 umole MeUmb equlvaient/mlnute. Protein concentrations were 0.16, 0.14, 0.17mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 0.5mg/ml.
Assay of Endo-1,4-P-glucanase activity was conducted In 50 mM NaOAC buffer, pH 5; 50 ’C, for 60 minutes. Activity was determined in triplicate by following absorbance change associated with génération of reducing ends from the model substrate Avicel PH-101. Activity unit was 1 pmole glucose equivalent/min. Protein concentrations were 0.80, 0.67, 0.79 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 80 mg/ml.
Assay of β-glucosldase activity was conducted In 50 mM NaOAC buffer, pH 5; 50 *C, for 20 minutes. Activity was determined in triplicate by following absorbance change assodated with release of glucose from model substrate cellobiose. Activity unit was 2 pmole glucose/min. Protein concentrations were 0.1,0.12, 0.12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 1.7 mg/ml.
Assay of Endo-1,4-p-xylanase activity was conducted in 50 mM NaOAC buffer, pH 5; 50 °C, for 60 minutes. Activity was determined In triplicate by following absorbance change assodated with génération of redudng ends from the model substrate water extractable arabinoxylan. Activity unit was 1 pmole glucose equivalent/min. Protein concentrations were 1.12,0.97,1.12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 10 mg/ml.
Assay of β-xylosidase activity was conducted In 50 mM NaOAC buffer, pH 5; 50 ’C, for 60 minutes. Activity was determined in duplicate by following release of xylose assodated with hydroiysis of the model substrate water extractable arablonxyfan. Activity unit was 1 pmole xylose/min. Protein concentrations were 1,12,0.97,1.12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 10 mg/ml.
Assay of β-L-arablnofuranosidase activity was conducted in 50 mM NaOAC buffer, pH 5; 50 ‘C, for 60 minutes. Activity was determined In triplicate by following release of arabinoase assodated with hydroiysis of the model substrate water extractable arablonxylan. Activity unit was 1 pmole arablnose/min. Protein concentrations were 1.12,0.97, 1.12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 10 mg/ml.
Assay of Amyloglucosldase (AMG) activity was conducted In 50 mM NaOAC buffer, pH 5; 50 °C, for 80 minutes. Activity was determined In triplicate by following absorbance change assodated with glucose release from the model substrate soluble com starch. Activity unit was 1 pmole glucose/mln. Protein concentrations were 1.12,0.97,1.12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 10 mg/ml.
Assay of anamylase activity was conducted In 50 mM NaOAC buffer, pH 5; 50 C, for 60 minutes. Activity was determined In triplicate by following absorbance change associated with génération of reducing ends from the model substrate soluble com starch. Activity unit was 1 pmole glucose equlvalent/min. Protein concentrations were 1.12,0.97,1.12 mg/ml respectively for CTEC3,
A CTri o, and CTEC2 assays. Substrate concentration was 10 mg/ml.
Assay of acetyl xylan esterase activity was conducted In 100 mM Sucdnate buffer, pH 5; 25 °C, for minutes. Activity was determined In triplicate by following continuous rate of 4DNitrophenyl release (Absi 410 nm) from the mode! substrate 4 40Nitrophenyl acetate. Activity unit was 1 pmole 10 pNP equivalent/mln. Protein concentrations were 0.48, 0.42, 0.51 mg/ml respectively for CTEC3,
ACTrio, and CTEC2 assays. Substrate concentration was 10 mg/mi.
Results of the activity déterminations are shown in Table 1.
The embodiments and examples are descriptive only and not Intended to ilmit the scope of the daims.
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Claims (24)

  1. Clalms:
    1. A method of processlng lignocelluloslc biomass comprising:
    - Providing soft lignocellulosic biomass feedstock,
    - Pretreating the feedstock at pH within the range 3.5 to 9.0 In a single-stage pressurized
    5 hydrothermal pretreatment at a DM content of at least 35% at températures between 160 and 200°C for résidence times of less than 60 minutes such that the pretreated biomass is characterized by having a xylan number of 10% or higher,
    - Separating the pretreated biomass Into an solid fraction and a liquid fraction,
    - Hydrolysing the solid fraction with or without addition of supplémentai water content at 15 %
    10 DM or greater for a time between 24 and 150 hours using enzymatic hydrolysis catalysed by an enzyme mixture comprising endoglucanase, exoglucanase, B-glucosidase, endoxylanase, xylosldase and acetyl xylan esterase activities, and
    - Subsequently mixing the separated liquid fraction and the hydrolysed solid fraction after at least 50% glucose conversion has been obtained and further conducting post-hydrolysis for
    15 a period of at least 6 hours, whereby xylo-oligomers In the liquid fraction are degraded to xylose monomers by the action of enzyme activities remalning within the hydrolysed solid fraction.
  2. 2. The method of claim 1, wherein the single-stage pressurized hydrothermal pretreatment is
    20 carried out as a single-stage pressurized autohydrolysis pretreatment, In which acetic add liberated by hemicellulose hydrolysis during pretreatment further catalyzes hemicellulose hydrolysis,
  3. 3. The method according to daim 1 or 2, with the proviso that sulphuric add ls not added to the 25 feedstock during the pretreatment step.
  4. 4. The method of claim 1 wherein the feedstock is wheat straw, corn stover, sugar cane bagasse, sweet sorghum bagasse, or empty fruit bunches.
    30
  5. 5. The method of claim 1 wherein the feedstock is washed and/or leached prior to pressurized pretreatment.
  6. 6. The method of daim 1 wherein the feedstock is soaked in an acetic add containîng iiquîd from a subséquent step of the pretreatment prior to pressurized pretreatment.
  7. 7. The method of daim 1 wherein the fiber structure ofthe feedstock Is maîntaîned during pretreatment.
  8. 8. The method of daim 1 wherein pressurized pretreatment is conducted at a pressure of 10 bar or iower.
  9. 9. The method of daim 1 wherein the feedstock is removed from the pressurized pretreatment reactor using a hydrocydone System.
  10. 10. The method of daim 1 wherein the feedstock is removed from the pressurized pretreatment reactor using a slulce-type System.
  11. 11. The method of daim 1 wherein the feedstock Is pretreated to a severity such that the biomass is characterized by having a xylan number 12% or hlgher.
  12. 12. The method of daim 1 wherein the solid fraction has a dry matter content of 40% or higher.
  13. 13. The method of daim 1 wherein the monomer xylose yield after post-hydrolysis Is at least 60% of the theoretical maximal yield.
  14. 14. The method of daim 1 wherein the monomer glucose yield after hydrolysls is at least 60% of the theoretical maximal yield.
  15. 15. The method of daim 1 wherein enzymatic hydrolysis Is conducted using a commerdally avaiiable cellulase préparation optimized for lignocellulosic biomass conversion used at an enzymatic dose level within the manufactura'suggested range.
  16. 16. The method of claim 1 wherein enzymatic hydrolysis Is conducted for at ieast 96 hours.
  17. 17. The method of claim 1 wherein enzymatic hydrolysis is conducted at between 15 and 23% dry matter content.
  18. 18. The method of claim 1 wherein enzymatic hydrolysis is conducted at 20% or higher dry matter content
  19. 19. The method of cialm 1 wherein enzymatic hydrolysis is conducted using an enzyme mixture comprislng exocellulase activées (EC 3.2.1.91); endocellulase activity (EC 3.2.1.4); B-glucosidase activity (EC 3.2.1.21); B-1,4 endoxylanase activity (EC 3.2.1.8); and acetyl xylan esterase activity (EC 3.1.1.72), and wherein the enzyme mixture is further characterized by having relative proportions of the enzyme activities such that for every 1 FPU cellulase activity there is at ieast 30 CMC U endoglucanase activity and with at ieast 28 pNPG U beta glucosidase activity and at least 50 ABX U endoxylanase activity.
  20. 20. The method of cialm 19 wherein the enzyme mixture further comprises B-1,3 xylosidase activity (EC 3.2.1.72); B-1,4 xylosidase activity (EC 3.2.1.37); and alpha 1,3 and/or alpha 1, 5 arabinofuranosidase activity (EC 3.2.1.23).
  21. 21. The method of cialm 1 further characterized in that a combined C5/C6 hydrolysate recovered after post-hydrolysis of the iiquid fraction is directly fermented to éthanol using one or more modified yeast strains.
  22. 22. The method of claim 1 wherein the solid fraction comprises insoluble solids with greater than 50% of associated dissolved solids removed.
  23. 23. The method of claim 1 wherein at least 85% of xylo-oligomers présent In liquid fraction are hydrolysed to xylose monomers during post-hydrolysis.
  24. 24. The method of claim 15 wherein the commercially available cellulase préparation optimized for lignocellulosic biomass conversion is supplemented with one or more additional enzyme activity.
OA1201400577 2012-08-01 2013-08-01 Methods of processing lignocellulosic biomass using single-stage autohydrolysis and enzymatic hydrolysis with C5 bypass and posthydrolysis. OA17197A (en)

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US61/678,130 2012-08-01

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