WO2015175308A1 - Hydrolyse enzymatique améliorée de biomasse - Google Patents

Hydrolyse enzymatique améliorée de biomasse Download PDF

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Publication number
WO2015175308A1
WO2015175308A1 PCT/US2015/029648 US2015029648W WO2015175308A1 WO 2015175308 A1 WO2015175308 A1 WO 2015175308A1 US 2015029648 W US2015029648 W US 2015029648W WO 2015175308 A1 WO2015175308 A1 WO 2015175308A1
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WIPO (PCT)
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saccharification
dissolved oxygen
biomass
reactor
mixture
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PCT/US2015/029648
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English (en)
Inventor
Michael Bodo
Jeffrey David Cohen
Chuanbin Liu
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Danisco Us Inc.
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Publication of WO2015175308A1 publication Critical patent/WO2015175308A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

Definitions

  • an improved method or process of enzymatic hydrolysis of lignocellulosic biomass materials provides for improved hydrolysis efficacy and/or efficiency, and improved yields of fermentable sugars.
  • an apparatus or reactor that can be used to practice such a method or process.
  • Cellululosic and lignocellulosic plant biomass provide an abundant and renewable feedstock for the production of valuable products such as fuels and other chemicals, replacing petroleum feedstock, which is non-renewable and increasingly costly and scarce.
  • Ethanol and other fuel alcohols have many desirable features that made them ideal petroleum substitutes.
  • most of the ethanol presently in the market place is produced from food-related resources such as corn grain and sugar cane juice, which is not seen as economically feasible and sustainably practical in the long run given the rapid rise in fuel and energy demands around the globe.
  • lignocellulosic sources of biomass are seen as a potentially inexpensive and feasible substitute feedstock, which at the same time would help avoid feedstock conflict with the prevalent food industry.
  • Cellulose and hemicellulose are the most abundant plant materials produced by photosynthesis. Lignocellulosic biomass materials are made up of three major organic fractions, cellulose, hemicellulose and lignin.
  • cellulose and hemicelluloses together can constitute as much as three quarters of overall biomass composition, and both of these can be degraded or converted to sugars, which can in turn be used as an energy source by numerous microorganisms ⁇ e.g., bacteria, yeast and fungi) that produce extracellular enzymes capable of hydrolysis of the polymeric substrates to monomeric sugars (Aro et al., (2001 ) J. Biol. Chem., 276: 24309-24314). Monomeric sugars can then be metabolized or fermented by ethanologen microorganisms into ethanol, by other microorganisms into chemicals, or simply used as building blocks to be converted into useful materials with chemical processes.
  • microorganisms e.g., bacteria, yeast and fungi
  • Monomeric sugars can then be metabolized or fermented by ethanologen microorganisms into ethanol, by other microorganisms into chemicals, or simply used as building blocks to be converted into useful materials with chemical processes.
  • the lignin Before they can be effectively degraded, however, the lignin will typically first need to be permeabilized, for example, by various pretreatment methods, and the hemicelluloses disrupted such that the complex carbohydrate cellulose polymers become more readily accessible to celluloytic hydrolysis enzymes. Afterwards, the pretreated mixture is subject to a hydrolysis/saccharification step whereby the enzymes solubilize the pretreated biomass, breaking it down into oligomeric saccharides and further into monosaccharides that are fermentable. In order for the entire biomass to fermentation product(s) process to be economically conducted, it is desirable that the saccharification or enzymatic hydrolysis product contains a high concentration of fermentable sugars. For most types of lignocellulosic biomass, economic viability of the industrial process also would require a high biomass dry matter level prior to the saccharification step. At such high dry matter levels, efficient enzymatic hydrolysis can be a challenge.
  • Enzymatic hydrolysis of lignocellulosic biomass materials is thus comparatively more attractive in that there tends to be a higher potential of generating greater yields of fermentable sugars, and less inhibitors.
  • enzymatic hydrolysis has its limitations as well. Due to the complexity of the plant biomass materials, pretreatment is typically necessary to render or disrupt the cellulosic structure of the biomass and make it more accessible to the enzymes. Many enzymatic activities may need to be present in a consortium or at least applied together to the lignocellulosic biomass material, delicately balanced in order to achieve effective synergism and more complete breakdown of the materials. Moreover typically a large amount of enzymes is required in order to achieve reasonable and commercially viable rate and yields.
  • cellulases which are enzymes that hydrolyze cellulose (comprising beta-1 ,4-glucan or beta D-glucosidic linkages) resulting in the formation of glucose, cellobiose,
  • EG endoglucanases
  • exoglucanases exoglucanases
  • CBH cellobiohydrolases
  • BG beta-glucosidases
  • Endoglucanases act mainly on the amorphous parts of the cellulose fiber, whereas cellobiohydrolases are also able to degrade crystalline cellulose (Nevalainen and Penttila, (1995) Mycota, 303-319).
  • Beta-glucosidase acts to liberate D-glucose units from cellobiose, cello- oligosaccharides, and other glucosides (Freer, (1993) J. Biol. Chem., 268: 9337-9342).
  • Enzymatic hydrolysis of the complex lignocellulosic structure and rather recalcitrant plant cell walls involves the concerted and/or tandem actions of a number of different endo-acting and exo-acting enzymes (e.g., cellulases and hemicellulases).
  • endo-acting and exo-acting enzymes e.g., cellulases and hemicellulases.
  • Beta-xylanases and beta-mannanases are endo-acting enzymes
  • beta-mannosidase beta-glucosidase
  • alpha-galactosidases are exo-acting enzymes.
  • xylanases together with other accessory proteins (non-limiting examples of which include L-a-arabinofuranosidases, feruloyl and acetylxylan esterases, glucuronidases, and ⁇ -xylosidases) can be applied.
  • accessory proteins non-limiting examples of which include L-a-arabinofuranosidases, feruloyl and acetylxylan esterases, glucuronidases, and ⁇ -xylosidases
  • mannanases galactanases ⁇ e.g., endo- and exo-galactanases
  • arabinases ⁇ e.g., endo-arabinases and exo-arabinases
  • ligninases amylases
  • glucuronidases proteases
  • esterases ⁇ e.g., ferulic acid esterases, acetyl xylan esterases, coumaric acid esterases or pectin methyl esterases
  • lipases other glycoside hydrolases
  • xyloglucanases CIP1 , CIP2, swollenins, expansins, and cellulose disrupting proteins.
  • the cellulose disrupting proteins are cellulose binding modules.
  • the saccharification reactor typically would comprise a mixing means such as an agitator.
  • the slurry or saccharification mixture is brought to the desirable temperature by either heating or cooling, whereas the temperature desired is set based on the temperature optima for the saccahrification enzymes to be used to hydrolyze the biomass being processed, in order to achieve the best possible saccharification raction rate.
  • the slurry or saccahrification mixture is brought to the desired pH through the addition of acid or base as required, depending on the initial pH of the pretreated biomass material, which can vary depending on the pretreatment used.
  • the specific pH that is desired is based on the pH optima of the saccharification enzymes to be used with the particular type of biomass being processed.
  • the mixing allows a substantially uniform pH to be achieved throughout the biomass and enzyme mixture, which in turn allows optimal functioning of the enzymes.
  • the concentration of dissolved oxygen in the saccharification mixture comprising a lignocellulosic biomass material and an enzyme product, can become substantially depleted during the enzymatic saccharification reaction. As a result, the saccharification performance is reduced and so are the yields of the fermentable sugars.
  • the present description provides an improved method or process for saccharifying a pretreated biomass at a high dry weight of biomass to produce fermentable sugars.
  • the method or process of the invention uses a fed batch reactor system whereby the enzymatic hydrolysis step is carried out, at least partially, in a saccharification reactor that is sufficiently aerated and/or mixed such that the dissolved oxygen concentration of the saccharification mixture is maintained at above a certain threshold level.
  • the method or process comprises:
  • a loading step comprising introducing the lignocellulosic biomass material and an enzyme composition into a reactor;
  • the lignocellulosic biomass material has been subject to one or more pretreatment or size reduction steps.
  • pretreatment or size reduction steps are selected from one or a combination of one or more of (1 ) a mechanical pretreatment, (2) an acidic pretreatment, (3) a steam and/or heating and/or pressure- based pretreatment, (4) a cryopretreatment, (5) an alkaline pretreatment, and/or (6) an enzymatic pretreatment.
  • the lignocellulosic biomass material is suitably one that comprises at least about 3 wt.% lignin, for example, at least about 5 wt.%, at least about 7 wt.%, at least about 9 wt.%, at least about 10 wt.%, at least about 15 wt.% or even at least about 20 wt.% lignin, referencing the total weight of carbohydrate polymers present in the biomass material.
  • the enzyme composition comprises at least one cellulase.
  • the enzyme mixture comprises two or more cellulases.
  • the enzyme mixture further comprises one or more hemicellulases.
  • the enzyme mixture further comprises one or more accessory enzymes.
  • the enzyme composition comprises a number of enzymes in amounts sufficient to cause hydrolysis of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or even at least 60% or more of the cellulose (glucan) in the biomass substrate.
  • the enzyme composition comprises a number of enzymes in amounts sufficient to cause hydrolysis of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or even at least 60% or more of the
  • hemicelluloses (xylan) in the biomass substrate hemicelluloses (xylan) in the biomass substrate.
  • the enzymes of the enzyme composition are introduced into the reactor during the loading step in two or more separate mixtures.
  • the enzymes in these separate mixtures can be the same or different enzymes.
  • the separate mixtures may comprise a different amount of a same enzyme.
  • the enzymes of the enzyme are introduced into the reactor during the loading step in two or more separate mixtures.
  • the enzymes of the enzyme composition are present in a single enzyme mixture.
  • the enzymes of the enzyme composition are produced by a single microorganism.
  • the microorganism can be one that natively produce such an enzyme mixture.
  • the microorganism can be genetically engineered to produce such an enzyme mixture, wherein certain of the enzymes of the enzyme mixture are heterologous to the microorganism, or are expressed at different than native levels as compared to the levels of these enzymes that would be produced by the native microorganism.
  • the mixing step comprises stirring or agitating the content of the reactor at a rate sufficient to cause effective mixing of the biomass and the enzyme composition.
  • the mixing or agitation can be continuous or intermittent, and whether or not it is continuous may depend on the dry solids weight of the biomass material and the relative ease or difficulty in keeping the solids in suspension, the size of the reactor, and/or the available means for temperature control.
  • the amount of lignocellulosic biomass material present in the reactor during the mixing step is at a level of at least 10%, at least 15%, at least 17%, at least 19%, or even at least 21 % dry solids weight.
  • the dry solids weight of the lignocellulosic biomass material present in the reactor during the mixing step is at about 10% to about 40% dry solids weight, or at about 15% to about 35% dry solids weight, or at about 17% to about 30% dry solids weight, or about 19% to about 25% dry solids weight.
  • the saccharification step takes place at a pH of about 3 to about 9, or at a pH of about 3.5 to about 7.5, or at a pH of about 4 to about 7, or at a pH of about 4.5 to about 6.5, or even at a pH of about 5 to about 6.
  • the saccharifcation step takes place for a period of at least 1 hour, or at least 5 hours, or at least 10 hours, or at least 15 hours, or at least 24 hours, or at least 24 hours, or even at least 72 hours.
  • the saccharification step takes place for a period of 1 to 120 hours, or 5 to 96 hours, or 10 to 85 hours, or 15 to 80 hours, or 24 hours to 72 hours, or 48 to 65 hours.
  • the saccharification step takes place at a temperature of at least about 20°C, or at least about 25°C, or at least about 30°C, or at least about 35°C, or at least about 40°C, or at least about 45°C, or at least about 50°C, or at least about 55°C, or at least about 60°C.
  • the saccharification step takes place at a temperature within the range of 20°C to 65°C, or the range of 25°C to 60°C, or the range of 30°C to 58°C, or the range of 35°C to 55°C.
  • the concentration of dissolved oxygen in the saccharification mixture is maintained at a level of above 1 .5%, preferably at a level of above 2%, more preferably at a level of above 2.5%, or above 3.0%, or above 3.5%, or above 4.0%, or above 4.5%, or above 5.0%, or above 5.5%, or above 6.0%, or above 6.5%, or above 7.0%, or above 7.5%, or even above 8.0% of the saturating dissolved oxygen concentration of such a mixture at the saccharification temperature immediately prior to the saccharification step.
  • the level of dissolved oxygen in the saccharification mixture is maintained at a level within the range of about 1 .5% to about 35%, of about 3% to about 30%, or about 5% to about 25%, or about 7.5% to about 20% of the saturating dissolved oxygen concentration of such a mixture at the saccharification temperature immediately prior to the saccharification step.
  • the dissolved oxygen level in the saccharification mixture is measured using a dissolved oxygen probe. In certain embodiments, the dissolved oxygen probe is pre-calibrated.
  • the disclosure provides an apparatus or reactor suitable for carrying out the enzymatic hydrolysis of a lignocellulosic biomass substrate, wherein the reactor comprises an off-gas condenser, an agitator, a pH probe, a temperature sensor and a dissolved oxygen probe.
  • the reactor further comprises means of adjusting such operational conditions as pH, temperature, dissolved oxygen concentrations, etc., such that the saccharification conditions can be maintained at certain preferred levels to insure a successful outcome.
  • the reactor is loaded with a lignocellulosic biomass material and an enzyme composition, which constitutes a sacharification mixture.
  • the saccharification mixture has a total volume of at least 5%, at least 10%, at least 15%, or even at least 20% less than the volume of the reactor.
  • the reactor after the saccharification mixture is loaded and sufficiently mixed to become a substantially homogenous slurry, has a headspace that is at least about 5%, or at least about 10%, or at least about 15%, or at least about 20% of the total volume of the reactor.
  • the reactor further comprises a gas inlet, which can be placed at or near the headspace of the reactor or at a position that would be
  • a sterile airflow is introduced into the reactor through the gas inlet.
  • the sterile airflow is introduced into the reactor at a rate of about 50 to about 400 M 3 per hour, for example, at a rate of about 50 M 3 per hour to about 350 M 3 per hour, or about 100 M 3 per hour to about 300 M 3 per hour, or about 150 M 3 per hour to about 280 M 3 per hour.
  • the reactor has a vertical, cylindrical geometry.
  • the agitator enters the reactor from the top of the reactor, optionally but preferably in a centered location.
  • the agitation or mixing effectuated by the agitator creases a downward-flow direction in the center of the biomass-enzyme slurry or suspension, and an upward flow near the wall of the reactor.
  • the invention pertains to the improved and higher levels of fermentable sugars in the product resulting from practicing the method of the first aspect, in a reactor of the second aspect. The thus-produced fermentable sugars can then be used for the production of high value chemicals, fuels and/or other useful products
  • FIGURE 1 Time courses of glucose, xylose and arabinose products at varying levels of dissolved oxygen concentrations in the saccharification mixture in accordance with the small, laboratory scale experiment of Example 1 .
  • FIGURE 2 Dissolved oxygen concentration throughout a large, industrial scale saccharification run in accordance with Example 2. This dissolved oxygen concentration profile was generated during and throughout the saccharification run.
  • FIGURE 3 Time courses of glucose, and xylose/arabinose products during saccharification runs. This figure depicts the comparison of the yields of such sugars in a large, industrial scale but poorly aerated reactor, with a small, laboratory scale and fully aerated reactor, as detailed in Example 2.
  • FIGURE 4 Time course of glucose, and xylose/arabinose products during saccharification runs. This figure depicts the comparison of the yields of such sugars in a large, industrial scale, and well aerated reactor, with a small laboratory scale, fully aerated reactor, and detailed in Example 3.
  • FIGURE 5 Dissolved oxygen levels in the saccharification mixtures of
  • FIGURE 6 Time course of glucose, and xylose/arabinose products during the saccharification runs of Reactors 1 -4 of Example 4.
  • FIGURE 7 Dissolved oxygen levels in the saccharification mixtures of Reactors 5-8 of Example 5.
  • FIGURE 8 Dissolved oxygen levels in a mixture of size-reduced dilute ammonia pretreated corn stover and water over time, in accordance with Example 5.
  • FIGURE 9 Dissolved oxygen levels over time in a saccharification mixture prepared with Accellerase® TRIOTM and a whPCS from NREL, in accordance with Example 5.
  • FIGURE 10 Dissolved oxygen levels over time in a saccharification mixture prepared with Accellerase ® TRIOTM and an Avicel, in accordance with Example 5.
  • Described is a method of improving the enzymatic saccharification of a lignocellulosic biomass material and increasing the yield of fermentable sugars from such lignocellulosic biomass materials using the same enzyme composition and dose. More specifically the improved method is one that involves the maintenance of dissolved oxygen level in the saccharification mixture and throughout the
  • a saccharification reactor comprising a dissolved oxygen probe installed in such a way as to enable the measurement and monitoring of dissolved oxygen levels in the saccharification mixture during the saccharification step of the biomass to fermentable sugar process.
  • the reactor is optionally loaded with a volume of the saccharification mixture that is at most about 95% of its total volume so as to leave a sufficiently large head space.
  • the reactor may further comprises a means for introducing an oxygen flow into the reactor, such that, when the dissolved oxygen level in the saccharification mixture falls below a certain threshold level, new oxygen can be added to the slurry to insure optimal saccharification efficiency and efficacy.
  • the improved method or process of the invention comprises:
  • a loading step comprising introducing a lignocellulosic biomass material and an enzyme composition into a vessel;
  • a mixing step comprising stirring or agitating the content of the reactor such that the enzyme composition and the lignocellulosic biomass material are sufficiently mixed into a substantially homogenous slurry that is a saccharification mixture;
  • a saccharification step comprising incubating the saccharification mixture under conditions that allow the hydrolysis of the biomass material into soluble
  • the concentration of dissolved oxygen in the saccharification mixture during the saccharification step of (c) is maintained at a level above about 1 .5%, for example, above about 1 .5%, above about 2.5%, above about 5%, above about 10%, above about 20%, or even above about 30%, of the saturating oxygen level of such a saccharification mixture at the saccharification temperature immediately prior to the saccharification step.
  • the lignocellulosic biomass material saccharified using such an improved method or process of the invention is suitably one that comprises at least 3 wt.% lignin, for example, at least about 3 wt.%, at least about 5 wt.%, at least about 10 wt.%, at least about 15 wt.%, or even at least about 20 wt.%, referencing the total weight of polymeric carbohydrates in the biomass material.
  • substantially purified or "clean" cellulose model biomass materials such as Avicel would be unsuitable for the purpose of the invention herein.
  • the lignocellulosic biomass material is also preferably pretreated before they are introduced into the reactor via the loading step.
  • the amount of lignocellulosic biomass material loaded to the reactor is such that the resulting saccharification mixture immediately before the saccharification step has a dry solids weight level of at least about 10%, for example, at least about 10%, at least about 15%, at least about 20%, or even at least about 25%.
  • the enzyme composition comprises at least one cellulase. In some embodiments,
  • the enzyme composition comprises two or more cellulases. In certain embodiments, the enzyme composition further comprises one or more hemicellulases. In further embodiments, the enzyme composition comprises one or more accessory enzymes. In certain embodiments, the enzyme composition is provided to the reactor in an amount that is sufficient to convert at least 40% of the cellulose (glucan) in the lignocellulosic biomass substrate in the saccharification mixture to glucose. In some embodiments, the enzyme composition is provided to the reactor in an amount that is sufficient to convert at least 25% of the hemicelluloses (xylan) in the lignocellulosic biomass substrate in the saccharification mixture to xylose.
  • the enzyme composition comprises at least one cellulase and at least one hemicellulase.
  • the enzyme composition may be produced by a single microorganism. Or the enzyme composition may be one that is an admixture of various enzymes produced by different microorganism.
  • the mixing step comprises stirring or agitating the reactor in such a way that the biomass material is sufficiently mixed and evenly exposed to the enzyme composition in the saccharification mixture, which is typically a substantially homogenous slurry.
  • the saccharification step is carried out at a pH of about 3 to about 9, for example, about 3.5 to about 8.5, about 4 to about 8, about 4.5 to about 7.5 or even about 5 to about 6.5.
  • the pH of the saccharification mixture in the reactor is adjusted by the addition or acid or base as needed, and the pH level of the saccharification mixture is measured and continuously monitored such that, whenever the pH of the saccharification mixture deviates from the preferred range, which is pre-determined based on the pH optima of the enzymes in the enzyme composition, an acid or a base is added to bring the pH back within the desired range.
  • the sacharification step is carried at a temperature of at least about 25 °C, for example, at least about 25 °C, about 30 °C, about 35 °C, at least about 40 °C, at least about 45 °C, or at least about 50 °C.
  • the saccharification step is charried out at a temperature within the range of about 25 °C to about 65 °C, for example, about 25 °C to about 62 °C, about 30 °C to about 60 °C, about 35 °C to about 58 °C, or about 40 °C to about 55 °C.
  • the temperature of the saccharification mixture is measured and continuously monitored such that whenever the temperature of the saccharification mixture deviates from the preferred range, which is predetermined based on the temperature optima of the enzymes in the enzyme composition, the cooling or heating means attached to the reactor is engaged to adjust the temperature back to within the desired range.
  • the saccharification step is conducted for a period of at least 1 hour, for example, about 2 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 30 hours, about 36 hours or longer. In certain embodiments, the saccharification step is conducted for a period within the range of 1 hour and 120 hours, for example, about 2 hours to about 1 10 hours, about 5 hours to about 100 hours, about 10 hours to about 96 hours, about 12 hours to about 90 hours, about 12 hours to about 84 hours, about 24 hours to about 80 hours, about 30 hours to about 72 hours, or even about 36 hours to about 68 hours.
  • the saccharification step is conducted for a period sufficient to convert or enzymatically hydrolyze at least 30% of the glucan in the lignocellulosic biomass material into glucose. In certain embodiments, the saccharification is conducted for a period sufficient to convert or enzymatically hydrolyze at least 20% of the xylan in the lignocellulosic biomass material to xylose. In certain further embodiments, the saccharification step is conducted for a period sufficient to convert at least 30% of the glucan and at least 20% of the xylan in the lignocellulosic biomass material into fermentable monomeric sugars.
  • the dissolved oxygen level in the saccharification mixture is at least about 1 .5%, preferably at least about 2.5%, or about 5%, or about 7.5%, or at least about 10% of the of the saturation dissolved oxygen level in a given saccharification mixture before the saccharification.
  • the saturation dissolved oxygen can be readily measured using a dissolved oxygen probe, immersed in a
  • the level of dissolved oxygen in the saccharification mixture can be monitored continuously throughout the saccharification step, or alternatively, the level of dissolved oxygen in the saccharification mixture can be monitored at pre-determined intervals.
  • the desired dissolved oxygen level can be maintained by continuously directing a certain flow of oxygen or air into either the headspace of the saccharification reactor or through an aeration inlet that is submerged when the saccharification mixture is placed in the reactor.
  • the headspace is suitably at least 5% of the volume of the reactor, to which preferably is affixed a gas inlet.
  • the influx of air or oxygen can be intermittent or as needed, triggered by the detection of a dissolved oxygen level below a certain threshold.
  • the influx of air or oxygen is suitably pre-sterilized to prevent the introduction of contaminating materials such as
  • Another aspect of the invention is an apparatus or reactor comprising at least a gas inlet affixed to the wall of either the headspace or at a position that is submerged when the saccharification mixture is placed in the reactor, a dissolved oxygen probe that is inserted in such a way to allow immersion of the probe in the saccharification mixture/slurry during saccharification, and monitoring and reporting out of dissolved oxygen level in the saccharification mixture continuously or intermittently.
  • an aeration means is suitably attached to the gas inlet of the reactor, which allows for continuous air or oxygen flow into the reactor/slurry, or for intermittent air or oxygen flow into the reactor/slurry when the dissolved oxygen level in the saccharification mixture drops below a certain threshold level, as reported by the read out of the dissolved oxygen probe.
  • the reactor further comprises a mixing means which can be suitably an agitator with an impeller or a stirrer or a mixer that can otherwise cause effective and even mixing of the saccharificaction mixture/slurry, maintain solids suspension throughout the saccharification step.
  • the mixing means can be kept at a constant mixing speed throughout the saccharification step or it can have varying mixing speeds.
  • the reactor further comprises a temperature sensor together with a cooling and a heating means. The temperature sensor is suitably affixed to the reactor in such a way to allow measurement, monitoring and reporting out of the temperature of the saccharification mixture/slurry during the saccharification step.
  • the cooling and/or heating means may be any suitable methodology used in industrial practices involving temperature-controlled fermentation or incubation tanks, such as, for example, running water or other fluids at certain elevated or reduced temperatures to achieve heating or cooling, respectively.
  • the cooling or heating means of the invention is preferably triggered to turn on when the temperature readout from the temperature probe is above or below a predetermined preferred temperature range for the
  • the preferred temperature range is determined based on the temperature optima of the various enzymes used in the saccharification step, but from time to time, a lower or reduced temperature may be applied to the
  • the reactor further comprises a pH sensor together with certain fluid inlet(s) or sampling outlet(s).
  • the pH sensor can be suitably affixed to the reactor in such a way to allow measurement, monitoring and reporting out of the pH of the saccharification mixture/slurry during the saccharification step.
  • the pH of the saccharification can be measured by sampling the saccharification slurry through the sampling outlets. If and when the pH of the saccharification slurry deviates beyond a predetermined preferred range, an amount of acid or base can be introduced to the saccharification slurry through the fluid inlet to bring the pH of the saccharification mixture back into the preferred range.
  • the preferred pH range is determined based on the pH optima of the various enzymes used in the saccharification step.
  • the pH of the slurry Prior to the start of the saccharification step, the pH of the slurry is adjusted to be within the preferred pH range.
  • the amount of acid or base required to adjust pH may vary, much dependent on the particular lignocellulosic biomass material and the particular pretreatment method applied to the material.
  • compositions or inventive concept wherein the component(s) or element(s) after the term is in the presence of other known component(s) or element(s) in a total amount that is less than 30% by weight or by significance of the total composition or inventive concept and do not contribute to or interferes with the actions or activities of the component(s) or element(s).
  • composition or inventive concept comprising the component(s) or element(s) may further include other non-mandatory or optional component(s) or element(s).
  • component(s) are present in the composition.
  • biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising
  • hemicelluloses hemicelluloses, lignin, starch, polysaccharides, oligosaccharides, and/or
  • Biomass may also comprise additional components, such as proteins and/or lipids.
  • biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves.
  • Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solids wastes.
  • biomass examples include, without limitation, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetable, fruits, flowers and animal manure.
  • biomass that is useful for the invention include biomass that has a relatively high carbohydrate value, is relatively dense, and/or is relatively easy to collect, transport, store, and/or handle.
  • the biomass that is useful includes corn cobs, corn stover, sugarcane bagasse, wheat straws, and other abundantly and readily available plant-based materials.
  • the biomass suitably applicable to the invention includes those that comprises at least about 2.5 wt.%, for example, at least about 2.5 wt.%, at least about 5 wt.%, at least about 7.5 wt.%, or even at least about 10 wt.% of lignin among the total weight of polymeric carbohydrates.
  • pretreated biomass refers to biomass materials that have been subject to a treatment or pretreatment prior to saccharification.
  • biomass materials may be pretreated by any method known to one skilled in the art, such as with acid, base, organosolvent, oxidizing agent, or other chemicals.
  • biomass materials may be pretreated with one or more chemicals in combination with steam, or heat, or with steam or heat alone. Suitable pretreatment may also include mechanical disruption such as by crushing, grinding or chopping, as well as application of other disrupting physical energies such as ultrasound, microwave or pressure.
  • biomass materials may be used with the method or process herein or in the reactor as described, but more suitable is the use of biomass materials that has been pretreated to enhance subsequent enzymatic hydrolysis or saccharification.
  • the biomass material may initially, prior to the saccharification reaction/step, be in a form of high dry solids weight level (or have a dry appearance), or alternatively the biomass material may initially be in a more dilute form such as in the case of stillage.
  • the term “Ngnocellulosic” refers to a composition comprising both lignin and cellulose. Lignocellulosic material may further comprise hemicelluloses.
  • the biomass materials suitably applicable to the invention herein are accordingly lignocellulosic materials.
  • cellulosic refers to a composition comprising cellulose.
  • Cellulosic material may further comprise hemicelluloses.
  • Cellulosic material may further comprise lignin and other polymeric carbohydrates.
  • cellulose or “cellulosic materials” refers to materials containing cellulose.
  • lignocellulose or “lignocellulosic materials” refers such cellulosic materials that also contain lignin. It is known that the largest component polysaccharides constituting the cell walls of plant biomass include cellulose, hemicelluloses and pectin.
  • Cellulose is an organic compound with the formula (C 6 H 0 O 5 )n, representing a polysaccharide consisting of a linear chain of ⁇ (1 ->4) linked D-glucose units.
  • Hemicellulose refers to any of several heteropolymers (matrix
  • Hemicellulases may be xylan, glucuronoxylan,xyloglucans, arabinoxylans,
  • glucomannan and mannans.
  • the monomers may include xylose, mannose, galactose, rhamnose, and arabinose, which are mostly D-pentose (C-5 sugars), and occasionally small amounts of L-sugars as well.
  • Xylose is in most cases the most abundant sugar monomer, although in softwoods mannose can be the most abundant sugar. Not only regular sugars can be found in hemicellulose, but also their acidified form, for instance glucuronic acid and galacturonic acid can be present.
  • Cellulose and lignocellulose are found in various plants and plant-derived materials, including stems, leaves and cobs, various parts of grains, including, for example, corn fiber, wheat hull, etc.
  • Cellulosic materials or lignocellulosic materials can also be materials produced from plants and plant parts, such as paper and pulp.
  • sacharification refers to the production of fermentable sugars from polysaccharides or polymeric carbohydrates, such as those contained by certain cellulosic materials or lignocellulosic materials.
  • hydrolysate refers to the product of saccharification, which contains the sugars produced in the saccharification process or step, the remaining unhydrolyzed biomass, and the enzymes and breakdown products of such enzymes used for saccharification.
  • slurry refers to a mixture of insoluble material and a liquid.
  • substantially homogenous slurry refers to a slurry that is sufficiently mixed so that substantially the same composition exists throughout the slurry composition under the action of the agitation means to which it is subjected. This term is used interchangeably herein with “thoroughly mixed slurry.”
  • dry weight or “dry solids weight” of biomass refers to the weight of the biomass having all or essentially all water removed. Dry weight is typically measured according to the American Society for Testing and Materials (ASTM) standard E1756-01 (Standard Test Method for Determination of Total Solids in
  • dry weight of biomass concentration refers to the total amount of biomass dry weight added into a fed batch system reactor, calculated at the time of addition, as a percent of the total weight of the reacting composition in the reactor at the end of the run.
  • reaction conditions refers to the time, temperature, pH and reactant concentrations which are described in details herein.
  • Reaction conditions can further include parameters such as mixing or stirring by the action of an agitator system in the reactor, including without limitation to impellers.
  • the mixing or stirring may be continuous or intermittent, with, for example, interruptions resulting from adding additional components or for temperature and pH assessment.
  • Enzymes have traditionally been classified by substrate specificity and reaction products. In the pre-genomic era, function was regarded as the most amenable (and perhaps most useful) basis for comparing enzymes and assays for various enzymatic activities have been well-developed for many years, resulting in the familiar EC classification scheme.
  • Cellulases and other glycosyl hydrolases which act upon glycosidic bonds between two carbohydrate moieties (or a carbohydrate and non- carbohydrate moiety--as occurs in nitrophenol-glycoside derivatives) are, under this classification scheme, designated as EC 3.2.1 .-, with the final number indicating the exact type of bond cleaved. For example, according to this scheme an endo-acting cellulase (1 ,4- -endoglucanase) is designated EC 3.2.1 .4.
  • carbohydrase modules which is available in the form of an internet database, the Carbohydrate-Active enZYme server (CAZy), available at http://afmb.cnrs- mrs.fr/CAZY/index.html (Carbohydrate-active enzymes: an integrated database approach. See Cantarel et ai, (2009) Nucleic Acids Res. 37 (Database issue):D233- 38).
  • CAZy defines four major classes of carbohydrases distinguishable by the type of reaction catalyzed: Glycosyl Hydrolases (GH's), Glycosyltransferases (GT's), Polysaccharide Lyases (PL's), and Carbohydrate Esterases (CE's).
  • the enzymes of the disclosure are glycosyl hydrolases.
  • GH's are a group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety.
  • a classification system for glycosyl hydrolases, grouped by sequence similarity, has led to the definition of over 85 different families. This classification is available on the CAZy web site.
  • protein includes proteins, polypeptides, and peptides.
  • cellulases refer to all enzymes that hydrolyzes cellulose, i.e., any of its components, e.g., 1 ,4-beta-D-glycosidic linkages in cellulosic materials such as those found in various plants and plant-related or -derived materials, such as grains, seeds, cereals, etc., or plant cell walls.
  • cellulase comprises at least the enzymes classified in E.C. 3.2.1 .4 (cellulase/endocellulases or endoglucanases), E.C. 3.2.1 .91
  • endocellulases examples include endo-1 ,4-beta-glucanase, carboxymethyl cellulase (CMCase), endo-1 ,4-beta-D-glucanase, beta-1 ,4-glucanase, beta-1 ,4-endoglucan hydrolase, celludextrinase, and various endoglucanases such as those produced by naturally- occurring wood-rotting fungi.
  • exocellulases include cellobiohydrolases, which in turn includes those that cleave the 1 ,4-beta-D-glycosidic linkages from the reducing ends of the cellulose chain and those that cleaves the same linkages from the non-reducing ends.
  • Cellulases may also refer to complete enzyme systems that are useful for efficiently converting crystalline cellulose to glucose. Such complete cellulase system typically would comprise components from each of the cellobiohydrolase,
  • Endo-1 ,4-beta-glucanases (EG) and exo- cellobiohydrolases (CBH) catalyze the hydrolysis of cellulose to cellooligosaccharides (cellobiose as a main product), while beta-glucosidases (BGL) convert the
  • Cellulases may further refer to complete enzyme systems that comprises not only cellulases but also certain hemicellulases, or any combination thereof.
  • a number of commercial cellulase compositions are available and suitable for use in the methods/processes and/or with the reactors described herein, including, for example, products of Genencor, Danisco US Inc., such as ACCELLERASE® 1000 and ACCELLERASE® 1500, ACCELLERASE® BG, ACCELLERASE® DUET, and
  • ACCELLERASE® TRIOTM products of Novozymes, such as its Celluclast, Novozyme 188, Cellic CTec2, Cellic CTec3; products of AB Enzymes, such as its Flashzyme; products of Codexis, such as its CodeXyme® cellulase products; products of Dyadic, such as its CMax® products.
  • Certain of the commercial compositions as listed above also contains hemicellulases. For example, about 1 /5 to 1 /4 of the total proteins of ACCELLERASE® DUET are hemicellulases, and about 1 /3 of the proteins in
  • ACCELLERASE® TRIOTM are hemicellulases.
  • the term "endoglucanase” as used herein refers to an enzyme of classification E.C. 3.2.1 .4, which catalyzes the hydrolysis of 1 ,4-beta-D-glycosidic linkages that are found in cellulosic materials. Methods of measuring endoglucanase activities are known, including, for example, the one measuring the hydrolysis of carboxymethyl cellulose (CMC) as described by Ghose, Pure & App. Chem, (1987) 59:257-268.
  • CMC carboxymethyl cellulose
  • cellobiohydrolase refers to an enzyme with cellobiohydrolase activity or capable of catalyzing the hydrolysis of a particular glocosidic linkage in cellulose.
  • the cellobiohydrolase (CBH) activity may be CBH class I (CBH I) or CBH class II (CBH II) activity or a combination of both CBH I and CBH II.
  • the cellobiohydrolase may hydrolyse (1 ⁇ 4) ⁇ -D-glucosidic linkages in cellulose and cellotetraose, releasing cellobiose from the non-reducing ends of the chains.
  • cellobiohydrolase activity may be exo-cellobiohydrolase activity or cellulose 1 ,4 ⁇ -cellobiosidase activity.
  • the cellobiohydrolase II activity can be classified under E.C. classification EC. 3.2.1 .91 .
  • the cellobiohydrolase I activity can be classified under E.C. classification EC. 3.2.1 .176.
  • beta-glucosidase refers to an enzyme having beta- glucosidase activity or one that is capable of catalyzing the hydrolysis of terminal non- reducing ⁇ -D-glucosyl residues and release of monomer ⁇ -D-glucose from cellobiose.
  • ⁇ -glucosidase activity can be classified under E.C. classification E.C. 3.2.1 .21 .
  • hemicellulase refers to a group of enzymes capable of catalyzing the hydrolysis of a hemicellulosic materials.
  • hemicellulases refer to three major types of enzymes: beta-xylosidases, L-cc- arabinofuranosidases, and xylanases. Those enzymes include, for example,
  • arabinases arabinofuranosidases, certain acetylmannan esterases, acetylxylan esterases, ferulyoyl esterases, mannanases, mannosidases, xylanases, and
  • Hemicellulases can be from many different glycosyl hydrolase families, including, without limitation, beta-xylosidases of GH3; beta-xylosidases of GH39; L-a-arabinofuranosidase (EC 3.2.1 .55), ⁇ -xylosidase (EC 3.2.1 .37), endo- arabinanase (EC 3.2.1 .99), and/or galactan 1 ,3- -galactosidase (EC 3.2.1 .145) of beta-xylosidases of GH3; beta-xylosidases of GH39; L-a-arabinofuranosidase (EC 3.2.1 .55), ⁇ -xylosidase (EC 3.2.1 .37), endo- arabinanase (EC 3.2.1 .99), and/or galactan 1 ,3- -galactosidase (EC 3.2.1 .145) of
  • GH43 L-a-arabinofuranosidase (EC 3.2.1 .55) of GH51 , as well as the xylanases of GH10 and GH1 1 , and the beta-xylosidases of GH30, for example.
  • xylanase refers to a 1 ,4-beta-D-xylan xylohydrolase of E.C. 3.2.1 .8, which catalyzes the hydrolysis of 1 ,4-beta-D-xylosidic linkages in xylan.
  • Xylanase activities can be measured, for example, by the PHBAH assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem. 47:273-279.
  • ⁇ -xylosidase activity may hydrolyse successive xylose residues from the non- reducing termini of (1 ⁇ 3) ⁇ -D-xylans, e.g. the ⁇ -xylosidase may be a 1 ,3 ⁇ -D- xylosidase. 1 ,3 ⁇ -D-xylosidases may be classified under E.C. classification E.C.
  • the ⁇ -xylosidase may be a 1 ,4 ⁇ - xylosidase.
  • 1 ,4 ⁇ -xylosidases may be classified under E.C. classification E.C. 3.2.1 .37.
  • L-alpha arabinofuranosidases may hydrolyse (1 ⁇ 6) ⁇ -D-galactosidic linkages in arabinogalactan proteins and (1 ⁇ 3):(1 ⁇ 6) ⁇ -galactans to yield galactose and (1 ⁇ 6) ⁇ -galactobiose.
  • L-alpha-arabinofuranosidases may be classified under E.C. classification E.C. 3.2.1 .164.
  • sacharification enzyme refers to an enzyme that can catalyze conversion of a component of biomass to fermentable sugars.
  • microorganism refers to any bacterium, yeast, or fungal species.
  • ethanologen and “ethanologenic microorganism” are used interchangeably to refer to a microorganism with the ability to convert a sugar or oligosaccharide to ethanol.
  • the ethanologenic microorganism are ethanologenic by virtue of their ability to express one or more enzymes that individually or collectively convert soluble sugars to ethanol.
  • Such an ethanolgen can also be referred as an "ethanol producing
  • microorganism which is an organism or cell that is capable of producing ethanol from a hexose or a pentose.
  • ethanol producing cells would contain at least one alcohol dehydrognase and a pyruvate decarboxylase.
  • examples of ethanol producing microorganisms include fungal microorganisms such as yeast, such as, for example, the species and strains of Saccharomyces, e.g., S. cerevisiae.
  • heterologous with reference to a polynucleotide or
  • polypepide/protein refers to a polynucleotide or polypeptide/protein, or an enzyme that does not naturally occur in a host cell.
  • the protein is a commercially important industrial protein. It is intended that the term encompass proteins that are encoded by naturally occurring genes, mutated genes, and/or synthetic genes.
  • endogenous refers to a polynucleotide or polypeptide/protein that occurs naturally in the host cell.
  • fermentation refers to the enzymatic and/or anaerobic breakdown of organic substances by microorganisms to produce simpler organic compounds. Although fermentation occurs under anaerobic conditions, the term “fermentation” as used herein is not intended to be limited to strict anaerobic conditions, because fermentation also can occur in the presence of oxygen at various levels. Accordingly, in the context of the present invention, fermentation encompasses at least some fermentative conversion of a soluble cellulosic fermentable sugar into an end product.
  • contacting refers to placing of the enzyme(s) in a reactor, vessel or the like, such that the enzymes can come into sufficiently close proximity to the substrate so as to enable the enzymes to convert the substrate to the end product.
  • an enzyme e.g., in a solution form
  • one or more substrates whether in a relatively pure or crude form, constitutes contacting.
  • yield with reference to the ethanol production refers to the production of a compound, e.g., ethanol, from a certain amount of a starting material, e.g., a lignocellulosic based biomass feedstock.
  • a starting material e.g., a lignocellulosic based biomass feedstock.
  • yield is also suitably used herein with reference to the production of fermentable sugars, and in that context, it refers to the amount of fermentable sugars produced from a given lignocellulosic biomass materials.
  • Yield may be expressed as the product formed over a particular amount of time from the starting material.
  • the invention provides an improved method or process for enzymatic hydrolysis or saccharfication of a lignocellulosic biomass material.
  • the improved method or process when applied can lead to an increased yield of fermentable sugars from a given biomass material.
  • the invention further provides an improved apparatus or reactor wherein the improved method or process as above can be carried out.
  • corn stover is the most abundant agricultural residue produced in the United States each year, making it a highly suitable feedstock for fermentable sugars, cellulosic fuels, and chemicals production.
  • corn stover composition can vary with climate conditions, harvest seasons, location, or the plant variety, which all affects the content of cellulose, hemicellulose, lignin and other components. Many of these components can confound the efforts to convert these materials, some by being recalcitrant, while other negatively affect conversion by being inhibitory to certain biological processes.
  • the first approach is acid hydrolysis. It is a relatively inexpensive and simple process, but the involvement of acids, typically also accompanied by heating, makes the process and the equipment used to carry out the process challenging. For example, the equipment and connectors must be made of materials that are corrosion resistant in an acidic, humid and heated environment for sustained periods of time. The used acids and other process wastes are hazardous and must also be handled with substantial care.
  • the conversion can be unsatisfactory in that the resulting sugars can be further degraded under high temperature.
  • High concentration of inhibitors can also form, including, for example, furfural, which are inhibitors to fermenting organisms or ethanologens involved in downstream processing of the sugars produced by the saccharification step. Removal of such inhibitors can be costly and cumbersome.
  • the second known approach is enzymatic hydrolysis. Such processes are typically carried out in mild, physiological conditions, having the potential of achieving high yields of fermentable sugars that are not subsequently degraded. Handling of the materials used in the saccharification step as well as the waste, unrelated residual biomass, is also much less cumbersome. On the other hand, the costs of producing enzymes, which are required in high quantities in order to sustain cellulosic
  • One way of making enzymatic hydrolysis of lignocellulosic biomass more effective and efficient is to pretreat the biomass feedstock, in order to render or disrupt the lignin tightly wound around the lignocellulosic structure and make the cellulose and hemicellulose part of the biomass more readily accessible to the enzymes.
  • a biomass material Prior to saccharification, a biomass material is preferably subject to one or more pretreatment step(s) in order to render xylan, hemicellulose, cellulose and/or lignin material more accessible or susceptable to enzymes and thus more amenable to hydrolysis by the enzyme(s) and/or enzyme blends/compositions of the disclosure.
  • Pretreatment may include chemical, physical, and biological pretreatment.
  • physical pretreatment techniques can include without limitation various types of milling, crushing, steaming/steam explosion, irradiation and hydrothermolysis.
  • Chemical pretreatment techniques can include without limitation dilute acid, alkaline, organic solvent, ammonia, sulfur dioxide, carbon dioxide, and pH-controlled
  • Bio pretreatment techniques can include without limitation applying lignin-solubilizing microorganisms.
  • the pretreatment can occur from several minutes to several hours, such as from about 1 hour to about 120.
  • any of the methods or processes provided herein may further comprise pretreating the biomass material, such as pretreating the biomass with acid or base.
  • the acid or base may be ammonia, sodium hydroxide, or phosphoric acid.
  • the method may further comprise pretreating the biomass material with ammonia.
  • the pretreatment may be steam explosion, pulping, grinding, acid hydrolysis, or
  • the pretreatment may be by elevated temperature and the addition of either of dilute acid, concentrated acid or dilute alkali solution.
  • pretreatment solution can added for a time sufficient to at least partially hydrolyze the hemicellulose components and then neutralized
  • the pretreatment entails subjecting biomass material to a catalyst comprising a dilute solution of a strong acid and a metal salt in a reactor.
  • the biomass material can, e.g., be a raw material or a dried material.
  • pretreatment can lower the activation energy, or the temperature, of cellulose
  • Another example of a pretreatment method entails hydrolyzing biomass by subjecting the biomass material to a first hydrolysis step in an aqueous medium at a temperature and a pressure chosen to effectuate primarily depolymerization of hemicellulose without achieving significant depolymerization of cellulose into glucose. This step yields a slurry in which the liquid aqueous phase contains dissolved monosaccharides resulting from depolymerization of hemicellulose, and a solid phase containing cellulose and lignin.
  • the slurry is then subject to a second hydrolysis step under conditions that allow a major portion of the cellulose to be depolymerized, yielding a liquid aqueous phase containing dissolved/soluble depolymerization products of cellulose. See, e.g., U.S. Patent No. 5,536,325.
  • a further example of method involves processing a biomass material by one or more stages of dilute acid hydrolysis using about 0.4% to about 2% of a strong acid; followed by treating the unreacted solid lignocellulosic component of the acid
  • Another example of pretreatment method comprises prehydrolyzing biomass ⁇ e.g., lignocellulosic materials) in a prehydrolysis reactor; adding an acidic liquid to the solid lignocellulosic material to make a mixture; heating the mixture to reaction temperature; maintaining reaction temperature for a period of time sufficient to fractionate the lingo-cellulosic material into a solubilized portion containing at least about 20% of the lignin from the lignocellulosic material, and a solid fraction containing cellulose; separating the solubilized portion from the solid fraction, and removing the solubilized portion while at or near reaction temperature; and recovering the solubilized portion.
  • the cellulose in the solid fraction is rendered more amenable to enzymatic digestion. See, e.g., U.S. Patent 5,705,369.
  • Pretreatment can also comprise contacting a biomass material with
  • Pretreatment can also comprise contacting a lignocellulose with a chemical ⁇ e.g., a base, such as sodium carbonate or potassium hydroxide) at a pH of about 9 to about 14 at moderate temperature, pressure, and pH. See PCT Publication WO2004/081 185.
  • a chemical ⁇ e.g., a base, such as sodium carbonate or potassium hydroxide
  • Ammonia may be used in a pretreatment method.
  • Such a pretreatment method comprises subjecting a biomass material to low ammonia concentration under conditions of high solids. See, e.g., U.S. Patent Publication 20070031918, PCT publication WO 061 10901 .
  • Saccharification enzymes which also may be referred to as a saccharification enzyme consortium, are used to hydrolyze the biomass releasing oligosaccharides and/or monosaccharides in a hydrolysate. Saccharification enzymes are reviewed in Lynd, L. R., et al. (Microbiol. Mol. Biol. Rev. (2002) 66:506-577).
  • a saccharification enzyme consortium comprises one or more enzymes selected primarily, but not exclusively, from the group “glycosidases” which hydrolyze the ether linkages of di-, oligo-, and polysaccharides and are found in the enzyme classification EC 3.2.1 .x (Enzyme Nomenclature 1992, Academic Press, San Diego, Calif, with Supplement 1 (1993), Supplement 2 (1994), Supplement s (1995,
  • Glycosidases useful in the present method can be categorized by the biomass component that they hydrolyze. Glycosidases useful for the present method include cellulose-hydrolyzing glycosidases (for example, cellulases,
  • hemicellulases endoglucanases, exoglucanases, cellobiohydrolases, ⁇ -glucosidases), hemicellulose- hydrolyzing glycosidases, called hemicellulases, (for example, xylanases,
  • glycosidases for example, amylases, a-amylases, ⁇ -amylases, glucoamylases, a-glucosidases, isoamylases.
  • peptidases EC 3.4.x.y
  • lipases EC 3.1 .1 .x and 3.1 .4.x
  • ligninases EC 1 .1 1 .1 .x
  • feruloyl esterases EC 3.1 .1 .73
  • microorganism may comprise a group of enzymes, all of which may contribute to the cellulose-degrading activity.
  • Commercial or non-commercial enzyme preparations such as cellulase, may comprise numerous enzymes depending on the purification scheme utilized to obtain the enzyme.
  • the saccharification enzymes used in the present method comprise at least one "cellulase", and this activity may be catalyzed by more than one enzyme.
  • the saccharification enzymes used in the present method may comprise at least one hemicellulase, generally depending on the type of pretreated biomass used in the present process. For example, hemicellulase is typically not needed when saccharifying biomass pretreated with acid and is typically included when saccharifying biomass pretreated under neutral or basic conditions.
  • Saccharification enzymes may be obtained commercially, such as Spezyme® CP cellulase (Genencor International, Rochester, N.Y.) and Multifect® xylanase (Genencor).
  • Other commercial cellulase compositions are available and suitable for use in the methods/processes and/or with the reactors described herein, including, for example, products of Genencor, Danisco US Inc., such as ACCELLERASE® 1000 and ACCELLERASE® 1500, ACCELLERASE® BG, ACCELLERASE® DUET, and
  • ACCELLERASE® TRIOTM products of Novozymes, such as its Celluclast, Novozyme 188, Cellic CTec2, Cellic CTec3; products of AB Enzymes, such as its Flashzyme; products of Codexis, such as its CodeXyme® cellulase products; products of Dyadic, such as its CMax® products.
  • Certain of the commercial compositions as listed above also contains hemicellulases. For example, about 1 /5 to 1 /4 of the total proteins of ACCELLERASE® DUET are hemicellulases, and about 1 /3 of the proteins in
  • ACCELLERASE® TRIOTM are hemicellulases.
  • saccharification enzymes may be produced biologically, including using recombinant microorganisms. New saccharification enzymes may be developed, which may be used in the present process.
  • saccharification is performed at or near the pH and temperature optima for the saccharification enzymes being used.
  • the pH optimum can range from about 3 to about 9, but is more typically between about 4.5 and about 7.
  • the temperature optimum can range between about 20 °C to about 80 °C, and is more typically between about 25 °C and about 60 °C.
  • biomass in the slurry becomes partially hydrolyzed.
  • the slurry becomes less viscous, allowing additional biomass to be added to the slurry while maintaining the mixability of the slurry with the agitator in the reactor, even with a fairly rudimentary and inexpensive agitator system.
  • the additional portion of biomass adds more solids and thus increases the percent of total solids loaded in the saccharifying slurry.
  • the pH and temperature are controlled within the preferred ranges while mixing and the saccharification reaction continues.
  • the thorough mixing of the slurry allows control of pH in a narrow range as more biomass is added and acid or base is added to make pH adjustments.
  • the tight pH control may help to improve saccharification enzyme function.
  • the thorough mixing of the slurry allows better control of the temperature of the reactor contents in a narrow range as more biomass is added, which also improves saccharification enzyme function.
  • Sources or means of heating or cooling that may be used are well known to one skilled in the art, and may include a jacket on the reactor, internal coils in the reactor, or a heat exchanger through which the reactor contents is pumped.
  • the tight temperature control enhances saccharification by allowing the saccharification to run at the highest temperature possible without overshooting the reactor temperature and thermally inactivating the enzymes.
  • Additional portions of a saccharification enzyme consortium may optionally be added following one or more new biomass loadings. Each added portion of a
  • saccharification enzyme consortium may include the same enzymes as in the initially added saccharification enzyme consortium, or it may include a different enzyme mixture.
  • the first added saccharification enzyme consortium may include only or primarily cellulases, while a later added saccharification enzyme consortium may include only or primarily hemicellulases.
  • Any saccharification enzyme consortium loading regime may be used, as determined to be best at saccharifying the specific biomass in the reactor.
  • One skilled in the art can readily determine a useful
  • Liquefaction of biomass results from further saccharification, thereby again reducing biomass slurry viscosity, allowing addition of more biomass while retaining mixability.
  • additional biomass may be added following a fed batch system, while maintaining stirring by the agitator.
  • the additional biomass feedings may be semi- continuous, allowing periods of liquefaction between additions.
  • the biomass feeding may be continuous, at a rate that is slow enough to balance the continuous liquefaction occurring during saccharification. In either case, mixability of the slurry is monitored and biomass addition is controlled to maintain thorough mixing as determined by the agitator system overcoming the yield stress of the slurry.
  • the particle size of the non-soluble biomass can be repeatedly further reduced during the saccharification step.
  • particle size reduction can be achieved by multiple applications of mechanical force for this purpose.
  • a mechanical particle size reduction mechanism may be, for example, a blender, grinder, shearer, chopper, sheer disperser, disperser, or roto-stat.
  • Particle size reduction may also be imposed by other non-mechanical methods, such as ultrasonic methods.
  • the particle size may be reduced prior to initial production of a slurry for saccharification, prior to addition of pretreated biomass to an existing saccharifying slurry, and/or during saccharification of a slurry.
  • the saccharification may be run until the final percent solids target is met and then the saccharifying biomass may be transferred to a fermentation process, where
  • SSF simultaneous saccharification and fermentation
  • Fermentable sugars produced in the present process may be fermented by suitable microorganisms that either naturally or through genetic manipulation are able to produce substantial quantities of desired target chemicals.
  • Target chemicals that may be produced by fermentation include, for example, acids, alcohols, alkanes, alkenes, aromatics, aldehydes, ketones, biopolymers, proteins, peptides, amino acids, vitamins, antibiotics, and pharmaceuticals.
  • Alcohols include, but are not limited to methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, propanediol, butanediol, glycerol, erythritol, xylitol, and sorbitol.
  • Acids may include acetic acid, lactic acid, propionic acid, 3-hydroxypropionic acid, butyric acid, gluconic acid, itaconic acid, citric acid, succinic acid and levulinic acid.
  • Amino acids may include glutamic acid, aspartic acid, methionine, lysine, glycine, arginine, threonine, phenylalanine and tyrosine.
  • Additional target chemicals include methane, ethylene, acetone and industrial enzymes.
  • Biocatalysts may be microorganisms selected from bacteria, filamentous fungi and yeast. Biocatalysts may be wild type microorganisms or recombinant microorganisms, and may include
  • biocatalysts may be
  • biocatalysts used in fermentation to produce target chemicals have been described and others may be discovered, produced through mutation, or engineered through recombinant means. Any biocatalyst that uses fermentable sugars produced in the present method may be used to make the target chemical(s) that it is known to produce by fermentation.
  • biocatalysts that produce biofuels including ethanol and butanol.
  • Isobutanol, 1 -butanol or 2-butanol may be produced from fermentation of hydrolysate produced using the present process by a microbial host following the disclosed methods.
  • E. coli Genetically modified strains of E. coli have also been used as biocatalysts for ethanol production (Underwood et al., Appl. Environ. Microbiol. (2002) 68:6263-6272).
  • a genetically modified strain of Zymomonas mobilis that has improved production of ethanol is described in US 2003/0162271 A1 .
  • a further engineered ethanol-producing strain of Zymomonas mobilis and its use for ethanol production are described in co- owned US Patent Nos. 7,741 ,1 19 and 7,741 ,084, respectively, which are herein incorporated by reference.
  • Ethanol may be produced from fermentation of hydrolysate produced using the present process by Zymomonas mobilis following the disclosed methods.
  • the present process may also be used in the production of 1 ,3-propanediol from biomass.
  • Recombinant strains of E. coli have been used as biocatalysts in fermentation to produce 1 ,3 propanediol (U.S. Pat. No. 6,013,494, U.S. Pat. No.
  • Hydrolysate produced by saccharification using the present process may be fermented by E. Coli to produce 1 ,3-propanediol as described in Example 10 of co- owned US Patent No. 7,781 ,191 , which is herein incorporated by reference.
  • Lactic acid has been produced in fermentations by recombinant strains of E. Coli (Zhou et al., Appl. Environ. Microbiol. (2003) 69:399-407), natural strains of Bacillus (US20050250192), and Rhizopus oryzae (Tay and Yang, Biotechnol.
  • a mutant of Propionibacterium acidipropionici has been used in fermentation to produce propionic acid (Suwannakham and Yang, Biotechnol. Bioeng. (2005) 91 :325- 337), and butyric acid has been made by Clostridium tyrobutyricum (Wu and Yang, Biotechnol. Bioeng. (2003) 82:93-102).
  • Propionate and propanol have been made by fermentation from threonine by Clostridium sp. strain 17cr1 (Janssen, Arch. Microbiol. (2004) 182:482-486).
  • a yeast-like Aureobasidium pullulans has been used to make gluconic acid (Anantassiadis et al., Biotechnol. Bioeng. (2005) 91 :494-501 ), by a mutant of Aspergillis niger (Singh et al., Indian J. Exp. Biol. (2001 ) 39:1 136-43).
  • 5-keto- D-gluconic acid was made by a mutant of Gluconobacter oxydans (Elfari et al., Appl Microbiol. Biotech. (2005) 66:668-674), itaconic acid was produced by mutants of Aspergillus terreus (Reddy and Singh, Bioresour. Technol.
  • citric acid was produced by a mutant Aspergillus niger strain (Ikram-UI-Haq et al., Bioresour. Technol. (2005) 96:645-648), and xylitol was produced by Candida guilliermondii FTI 20037 (Mussatto and Roberto, J. Appl. Microbiol. (2003) 95:331 -337).
  • 4- hydroxyvalerate-containing biopolyesters also containing significant amounts of 3- hydroxybutyric acid 3-hydroxyvaleric acid, were produced by
  • Phenylalanine was also produced by fermentation in Eschericia coli strains ATCC 31882, 31883, and 31884. Production of glutamic acid in a recombinant coryneform bacterium is described in U.S. Pat. No. 6,962,805. Production of threonine by a mutant strain of E.
  • Methionine was produced by a mutant strain of Corynebacterium lilium (Kumar et al, Bioresour. Technol. (2005) 96: 287-294).
  • Biocatalysts Useful peptides, enzymes, and other proteins have also been made by biocatalysts (for example, in U.S. Pat. No. 6,861 ,237, U.S. Pat. No. 6,777,207, U.S. Pat. No. 6,228,630).
  • Target chemicals produced in fermentation by biocatalysts may be recovered using various methods known in the art. Products may be separated from other fermentation components by centrifugation, filtration, microfiltration, and nanofiltration. Products may be extracted by ion exchange, solvent extraction, or electrodialysis. Flocculating agents may be used to aid in product separation. As a specific example, bioproduced 1 -butanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see for example, Durre, Appl. Microbiol.
  • Amino acids may be collected from fermentation medium by methods such as ion-exchange resin adsorption and/or crystallization.
  • Example 1 Dissolved Oxygen (DO) Levels in Saccharification Mixtures at
  • the gas proportions and gas flow rates were adjusted to achieve a set of time- weighted average dissolved oxygen concentrations in the saccharification mixtures of 0.3%, 13%, 49%, 78% and 92% of the saturation dissolved oxygen level, respectively.
  • the dissolved oxygen concentrations were maintained throughout the remainder of the saccharification step until it is terminated at 70.1 hours.
  • saccharification mixture had a low 0.3% dissolved oxygen concentration. At DO levels above 13%, xylose release was faster but the levels of xylose release were
  • Millimeter-sized particles of corn stover were thermo-chemically pretreated using water and ammonia in a steam and air environment.
  • the pretreated corn stover was enzymatically saccharified in a reactor equipped with process controls including controls and monitoring of temperature, pH, agitation intensity, head pressure and sterile air-flow rate through the vessel's head space.
  • the rector was equipped also with a dissolved oxygen probe.
  • the reactor used had a vertical, cylindrical geometry with the shaft of the agitation means entering the vessel from the top of the vessel at a centered location on the head plate of the reactor.
  • the agitation produced a downward flow direction in the center of the biomass solids suspension formed with the enzyme solution, and an upward flow near the walls of the vessel.
  • the reactor was further equipped with an external, pumped, re-circulation loop containing a heat exchanger for temperature control.
  • the reactor had a total volume of about 90 M 3 .
  • the batch volume used for this example was about 65 M 3 after the addition of all components, including water, pretreated corn stover, acid and enzymes.
  • the saccharification temperature was maintained at 47 °C in both the large, industrial scale saccharification reaction, as well as the small, laboratory scale saccharification reaction.
  • the sterile air flow was maintained in a range of 50 to 100 M 3 /hour at standard pressure and temperature (STP) 0 °C and 1 atm pressure
  • Example 3 The same experimental conditions of Example 2 (above) were used for this experiment under Example 3, with the exception that the sterile air flow rates into the large, industrial scale reactor and the small, laboratory scale reactor were maintained at 250 M 3 /hour at STP. The dissolved oxygen concentrations were measured throughout the saccharification step, and it was determined that at this sterile air flow, the dissolved oxygen concentration in the saccharification mixture never dropped below about 4,800 ppb.
  • Reactor 5 was dosed with Enzyme 1 , and was sealed.
  • Reactor 6 was dosed with Enzyme 2, and was also sealed.
  • Reactors 7 was dosed with Enzyme 2, but with addition of 1 mg of T. reesei GH61 enzyme; the reactor was also sealed.
  • Reactor 8 was dosed with Enzyme 2, which had been previously heat inactivated; and the reactor was also sealed. [00189] The levels of dissolved oxygen concentrations in those reactors were monitored and shown in FIGURE 7. It was apparent that the T. reesei GH61 enzyme contributes only to a limited extent to the depletion of dissolved oxygen in the
  • Accellerase® TRIOTM was used to saccharify a dilute acid pretreated corn stover biomass obtained from NREL (Schell DJ et al., DILUTE-

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Abstract

L'invention concerne une méthode ou un procédé amélioré(e) pour la saccharification d'une charge de biomasse lignocellulosique en sucres fermentables, comprenant le maintien de taux d'oxygène dissous dans le mélange de saccharification. L'invention concerne également un appareil ou un réacteur amélioré utile pour mettre en oeuvre la méthode ou le procédé amélioré(e).
PCT/US2015/029648 2014-05-13 2015-05-07 Hydrolyse enzymatique améliorée de biomasse WO2015175308A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NO20160022A1 (en) * 2016-01-06 2017-07-07 Hofseth Biocare Asa A new method to improve enzyme hydrolysis and resultant protein flavor and bio-activity of fish offcuts
WO2018085370A1 (fr) * 2016-11-02 2018-05-11 Novozymes A/S Procédés de réduction de la production de primevérose pendant la saccharification enzymatique de matière lignocellulosique
US10081802B2 (en) 2013-07-29 2018-09-25 Danisco Us Inc. Variant Enzymes
CN111218491A (zh) * 2018-11-27 2020-06-02 南京理工大学 提高木质纤维素转化效率的蒸汽-氨联合预处理工艺

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WO2014072393A1 (fr) * 2012-11-09 2014-05-15 Dsm Ip Assets B.V. Procédé d'hydrolyse enzymatique de matière lignocellulosique et de fermentation de sucres
WO2014130812A1 (fr) * 2013-02-21 2014-08-28 Novozymes A/S Procédés de saccharification et de fermentation d'un matériau cellulosique

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WO2014072393A1 (fr) * 2012-11-09 2014-05-15 Dsm Ip Assets B.V. Procédé d'hydrolyse enzymatique de matière lignocellulosique et de fermentation de sucres
WO2014130812A1 (fr) * 2013-02-21 2014-08-28 Novozymes A/S Procédés de saccharification et de fermentation d'un matériau cellulosique

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10081802B2 (en) 2013-07-29 2018-09-25 Danisco Us Inc. Variant Enzymes
US10167460B2 (en) 2013-07-29 2019-01-01 Danisco Us Inc Variant enzymes
US10479983B2 (en) 2013-07-29 2019-11-19 Danisco Us Inc Variant enzymes
NO20160022A1 (en) * 2016-01-06 2017-07-07 Hofseth Biocare Asa A new method to improve enzyme hydrolysis and resultant protein flavor and bio-activity of fish offcuts
NO342626B1 (en) * 2016-01-06 2018-06-25 Hofseth Biocare Asa A new method to improve enzyme hydrolysis and resultant protein flavor and bio-activity of fish offcuts
US10827767B2 (en) 2016-01-06 2020-11-10 Hofseth Biocare Asa Process to improve enzyme hydrolysis and resultant protein flavor and bio-activity of fish offcuts
WO2018085370A1 (fr) * 2016-11-02 2018-05-11 Novozymes A/S Procédés de réduction de la production de primevérose pendant la saccharification enzymatique de matière lignocellulosique
CN111218491A (zh) * 2018-11-27 2020-06-02 南京理工大学 提高木质纤维素转化效率的蒸汽-氨联合预处理工艺

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