Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
  • C12N1/18—Baker's yeast; Brewer's yeast
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
  • C12M21/12—Bioreactors or fermenters specially adapted for specific uses for producing fuels or solvents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M43/00—Combinations of bioreactors or fermenters with other apparatus
  • C12M43/02—Bioreactors or fermenters combined with devices for liquid fuel extraction; Biorefineries
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
  • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present invention relates to a system for the production of cellulosic ethanol and recovery of other bioproducts .
• the present invention also relates to a system for management of yeast to facilitate the production of ethanol.
• the present invention further relates to a method of propagating ethanologen for use in the production of a fermentation product from biomaes.
• Ethanol can be produced from grain-based feedstocks (e.g. corn, sorghum/milo, barley, wheat, soybeans, etc.), from sugar (e.g. from sugar cane, sugar beets, etc.), and from biomass (e.g. from lignocellulosic feedstocks such as switchgrass, corn cobs and stover, wood or other plant material) .
• grain-based feedstocks e.g. corn, sorghum/milo, barley, wheat, soybeans, etc.
• sugar e.g. from sugar cane, sugar beets, etc.
• biomass e.g. from lignocellulosic feedstocks such as switchgrass, corn cobs and stover, wood or other plant material
• Biomass comprises plant matter that can be suitable for direct use as a fuel/energy source or as a feedstock for processing into another bioproduct (e.g., a biofuel such as cellulosic ethanol) produced at a bioreflnery (such as an ethanol plant) .
• Biomass may comprise, for example, corn cobs and stover (e.g., stalks and leaves) made available during or after harvesting of the com kernels, fiber from the corn kernel, switchgrass, farm or agricultural residue, wood chips or other wood waste, and other plant matter (grown for processing into bioproducts or for other purposes) .
• biomass will be harvested and collected from the field and transported to the location where it is to be used or processed.
• ethanol is produced from starch.
• Corn kernels are cleaned and milled to prepare starch-containing material for processing, (corn kernels can also be fractionated to separate the starch-containing material (e.g. endosperm) from other matter (such as fiber and germ).)
• the starch-containing material is slurried with water and liquefied to facilitate saccharification where the starch is converted into sugar (e.g. glucose) and fermentation where the sugar is converted by an ethanologen (e.g. yeast) into ethanol.
• sugar e.g. glucose
• an ethanologen e.g. yeast
• the fermentation product is beer, which comprises a liquid component containing ethanol and water and soluble components, and a solids component containing unfermented particulate matter (among other things) .
• the fermentation product is sent to a distillation system.
• the fermentation product is distilled and dehydrated into ethanol.
• the residual matter e.g. whole stillage
• the residual matter comprises water, soluble components, oil and unfermented solids (i.e. the solids component of the beer with substantially all ethanol removed that can be dried into dried distillers grains (DDQ) and sold as an animal feed product) .
• Other co-products for example Byrup (and oil contained in the syrup) , can alBo be recovered from the stillage. Water removed from the fermentation product in distillation can be treated for re-use at the plant ,
• Lignocellulosic biomass typically comprises cellulose, heraicellulose and lignin.
• Cellulose a type of glucan
• Hemicellulose is a branched chain polysaccharide that may comprise several different pentose (C5) sugar monomers (such as xylose and arabinose) and small amounts of hexose (C6) sugar monomers in branched chains .
• the biomass is prepared so that sugars in the lignocellulosic material (such as glucose from the cellulose and xylose from the hemicellulose) can be made accessible and fermented into a fermentation product from which ethanol can be recovered. After fermentation the fermentation product is sent to the distillation system, where the ethanol is recovered by distillation and dehydration. other bioproducts such as lignin and organic acids may also be recovered as by-products or co-products during the processing of biomass into ethanol. Determination of how to more efficiently prepare and treat the biomass for production into ethanol will depend upon the source and type or composition of the biomass. Biomass of different types or from different sources is likely to vary in properties and composition (e.g. relative amounts of cellulose, hemicellulose, lignin and other components) . For example the composition of wood chips will differ from the composition of corn cobs or switchgrass.
• the present invention relates to a method of propagating ethanologen for use in the production of a fermentation product from biomass.
• the method comprises the steps of providing a medium for propagation of ethanologen; supplying a first cell mass of ethanologen to the medium; supplying xylose to the medium as a carbon source for the ethanologen; and maintaining the medium comprising the first cell mass of ethanologen at a pH of between about 5.0 and 6.0 and at a temperature of between about 26 and about 37 degrees Celsius so that the first cell mass of ethanologen is propagated into a second cell mass of ethanologen.
• the second cell mass of ethanologen is larger than the first cell mass of ethanologen.
• the present invention also relates to a method of propagating ethanologen for use in the production of a fermentation product from biomass.
• the method comprises the steps of providing a medium for propagation of ethanologen; supplying a first cell mass of ethanologen to the medium; providing an agent to the medium; providing a component obtained from the biomass to the medium as a carbon source for the ethanologen; and maintaining the medium comprising the first cell mass of ethanologen at a pH of between about 5.0 and 6.0 and at a temperature of between about 26 and about 37 degrees Celsius so that the first cell mass of ethanologen ie propagated into a second cell mass of ethanologen.
• the second cell mass of ethanologen is larger than the cell mass of the first amount of ethanologen.
• the biomass comprises lignocellulosic material; the lignocellulosic material comprises at least one of corn cobs, com plant husks, corn plant leaves and com plant stalks .
• the component is obtained from the 13 042927
• the component comprises pentose.
• Pentose comprises the carbon source for the ethanologen.
• the ethanologen comprises yeast cells capable of fermenting pentose into ethanol.
• the present invention further relates to a system for propagating ethanologen for use in the production of a fermentation product from biomass in a fermentation system.
• the system comprises a first stage comprising a first vessel configured to maintain a medium comprising ethanologen; a second stage comprising a second vessel configured to maintain a medium supplied from the first stage; a source of xylose to be provided to the medium as a carbon source for ethanologen in the first stage; and a source of xylose to be provided to the medium as a carbon source for the ethanologen in the second stage.
• the ethanologen has a first cell mass when supplied to the first stage and the ethanologen has a second cell mass when supplied from the first stage to the second stage and the ethanologen has a third cell mass when supplied from the second stage.
• the medium in the first vessel is maintained at a pH of between about 5.0 and 6.0 and at a temperature of between about 26 and about 37 degrees Celsius so that ethanologen can be propagated into the second cell mass; the medium in the second vessel is maintained at a pH of between about 5.0 and 6.0 and at a temperature of between about 26 and about 37 degrees Celsius so that ethanologen can be propagated into the third cell mass .
• the second cell mass is at least 200 times larger than the first cell mass .
• the third cell mass is at least 20 times larger than the second cell mass.
• FIGURE 1A is a perspective view of a biorefinery comprising a cellulosic ethanol production facility.
• FIGURE IB is a perspective view of a biorefinery comprising a cellulosic ethanol production facility and a corn-based ethanol production facility.
• FIGURE 2 is a schematic diagram of a system for receipt and preparation of biomass for a cellulosic ethanol production facility.
• FIGURE 3 is a schematic block diagram of a system for the production of ethanol from biomass.
• FIGURES 4A, 4B and 4C are schematic block diagrams of systems for treatment and processing of components from the production of ethanol from biomass .
• FIGURES 5 ⁇ and 5B are schematic diagrams of the process flow for systems for the production of ethanol from biomass.
• FIGURE 6A is a schematic block diagram of apparatus used for preparation, pre-treatment and separation of biomass.
• FIGURE 6B is a perspective view of apparatus used to pre-treat and separate the biomass.
• FIGURE 7A is a schematic block diagram of the process flow for a system for the production of a fermentation product from a liquid component (C5) .
• FIGURE 7B is a schematic block diagram of the process flow of a system according to an exemplary embodiment .
• FIGURE 7C is a schematic block diagram of the process flow of a system according to an exemplary embodiment.
• FIGURE 8A is a schematic block diagram of the process flow of a system according to an exemplary embodiment.
• FIGURE 8B is a schematic block diagram of the process flow of a system according to an exemplary embodiment .
• FIGURE 9 is schematic diagram of a system according to an exemplary embodiment.
• FIGURE 10A is a line graph Bhowing the growth of ethanologen using xylose.
• FIGURE 10B is a line graph showing the growth of ethanologen using xylose.
• FIGURE 11A is a line graph showing the growth of ethanologen.
• FIGURE 11B is a line graph showing the growth of ethanologen.
• FIGURE 12A is a line graph showing the growth of ethanologen using xylose.
• FIGURE 12B is a line graph showing the growth of ethanologen using xylose and a biomass liquid component.
• FIGURE 12C is a line graph showing the growth of ethanologen using xylose and a treated biomass liquid component.
• FIGURE 13A is a line graph showing the growth of ethanologen using xylose during the second stage of a two-stage propagation.
• FIGURE 13B is a line graph showing the growth of ethanologen using xylose during the second stage of a two-stage propagation.
• FIGURE 14A is a line graph showing the growth of ethanologen using an initial loading of xylose during the second stage of a two- stage propagation.
• FIGURE 14B is a line graph showing the growth of ethanologen using a continuous xylose feed during the second stage of a two-stage propaga ion.
• FIGURE 15A is a line graph showing xylose consumption and ethanol production by ethanologen both with and without aeration.
• FIGURE 15B is a line graph showing the growth of ethanologen both with and without aeration.
• FIGURE 16A is a graph showing the growth of ethanologen using xylose .
• FIGURE 16B is a graph showing growth of ethanologen using xylose.
• FIGURE 16C is a graph showing growth of ethanologen using glucose .
• TABLES 1A and IB list the composition of biomass comprising lignocellulosic plant material from the corn plant according to exemplary and representative embodiments.
• TABLES 2A and 2B list the composition of the liquid component of pre-treated biomass according to exemplary and representative embodimen ⁇ .
• TABLES 3A and 3B list the composition of the solids component of pre-treated biomass according to exemplary and representative embodiments .
• TABLE 4 lists the growth medium composition according to Examplee 1 through Example 4.
• TABLES 5A and 5B list the results of use of a system according to Example 1A.
• TABLES 6A and 6B list the results of use of a system according to
• ABLES 7A, 7B and 7C list the results of use of a system according to Example 2.
• TABLES 8A and 8B list the results of use of a system according to Example 3.
• TABLES 9A and 9B list the results of use of a system according to Example .
• TABLES 10A and 10B list the results of use of a system according to Example 5.
• FIGURE 1A a biorefinery configured to produce ethanol from biomass is shown.
• the biorefinery is configured to produce ethanol from biomass in the form of a lignocellulosic feedstock such as plant material from the com plant (e.g. com cobs and co stover) .
• a lignocellulosic feedstock such as plant material from the com plant (e.g. com cobs and co stover) .
• Lignocellulosic feedstock such as lignocellulosic material from the com plant comprises cellulose (from which C6 sugars such as glucose can be made available) and/or hemicellulose (from which C5 sugars such as xylose and arabinose can be made available) .
• the biorefinery comprises an area where biomass is delivered and prepared to be supplied to the cellulosic ethanol production facility.
• the cellulosic ethanol production facility comprises apparatus for preparation, pre-treatment and treatment of the biomass into treated biomass suitable for fermentation into fermentation product in a fermentation system.
• the facility comprises a distillation system in which the fermentation product is distilled and dehydrated into ethanol.
• the biorefinery may also comprise a waste treatment system (shown as comprising an anaerobic digester and a generator) .
• the waste treatment system may comprise other equipment configured to treat, process and recover components from the cellulosic ethanol production process, such as a solid/waste fuel boiler, anaerobic digester, aerobic digester or other biochemical or chemical reactors.
• a biorefinery may comprise a cellulosic ethanol production facility (which produces ethanol from lignocellulosic material and components of the corn plant) co-located with a corn-based ethanol production facility (which produces ethanol from starch contained in the endosperm component of the corn kernel) .
• a cellulosic ethanol production facility which produces ethanol from lignocellulosic material and components of the corn plant
• a corn-based ethanol production facility which produces ethanol from starch contained in the endosperm component of the corn kernel
• Com fiber (a component of the co kernel) , which can be made available when the com kernel is prepared for milling (e.g.
• a biorefinery e.g. a cellulosic ethanol production facility
• a biorefinery may be co-located with other types of plants and facilities, for example an electric power plant, a waste treatment facility, a lumber mill, a paper plant or a facility that processes agricultural products .
• the biomass preparation system may comprise apparatus for receipt/unloading of the biomass, cleaning (i.e. removal of foreign matter), grinding (i.e. milling, reduction or densification) , and transport and conveyance for processing at the plant.
• biomass in the form of com cobs and stover may be delivered to the biorefinery and stored (e.g. in bales, piles or bins, etc.) and managed for use at the facility.
• the biomaes may comprise at least 20 to 30 percent corn cobs (by weight) with corn stover and other matter.
• the preparation system of the biorefinery may be configured to prepare any of a wide variety of types of biomass (i.e. plant material) for treatment and processing into ethanol and other bioproducts at the plant .
• biomass comprising plant material from the corn plant is prepared and cleaned at a preparation system. After preparation, the biomass is mixed with water into a slurry and is pre-treated at a pre- treatment system. In the pre-treatment system, the biomass is broken down (e.g. by hydrolysis) to facilitate separation into a liquid component (e.g. a stream comprising the C5 sugars) and a solids component (e.g. a stream comprising cellulose from which the C6 sugars can be made available) .
• a liquid component e.g. a stream comprising the C5 sugars
• a solids component e.g. a stream comprising cellulose from which the C6 sugars can be made available
• the C5-sugar-containing liquid component (C5 stream) and C6-sugar-containing solids component (C6 stream) can be treated (as may be suitable) and fermented in a fermentation system. Fermentation product from the fermentation system is supplied to a distillation system where the ethanol is recovered.
• removed components from treatment of the C5 stream can be treated or processed to recover by-products, Vietnamese as organic acids and furfural.
• removed components from treatment of the C6 stream such as lignin or other components, can be treated or processed into bioproducts or into fuel (such as lignin for a solid fuel boiler or methane produced by treatment of residual/removed matter such as acids and lignin in an anaerobic digester) .
• fuel such as lignin for a solid fuel boiler or methane produced by treatment of residual/removed matter such as acids and lignin in an anaerobic digester
• components removed during treatment and production of ethanol from the biomasB from either or both the C5 stream and the C6 stream (or at distillation) may be processed into bioproducts (e.g.
• removed components from the distillation system such as stillage or removed solids
• from the treatment of the fermentation product before distillation e.g. removed solids and particulate matter, which may comprise residual lignin, etc.
• bioproducts or fuel e.g. methane produced in an anerobic digester
• the biomass comprises plant material from the corn plant, such as com cobs, husks and leaves and stalks; the composition of the plant material (i.e. cellulose, hemicellulose and lignin) will be approximately as indicated in TABLES 1A and IB.
• the plant material comprises corn cobs, husks/leaves and stalks (i.e.
• the plant material may comprise (by weight) up to 100 percent cobs, up to 100 percent husks/leaves, approximately 50 percent cobs and approximately 50 percent husks/leaves, approximately 30 percent cobs and approximately 50 percent husks/leaves and approximately 20 percent stalks, or any other combinations of cobs, husks/leaves and stalks from the corn plant. See TABLE 1A.
• corn stalks comprise the upper half or three-quarters portion of the stalk.
• the lignocellulosic plant material may comprise fiber from the corn kernel (e.g. in some combination with other plant material) .
• the lignocellulosic plant material of the biomass (from the corn plant) will comprise (by weight) cellulose at about 30 to 55 percent, hemicellulose at about 20 to 50 percent, and lignin at about 10 to 25 percent; according to a particularly preferred embodiment, the lignocellulosic plant material of the biomass (cobs, husks/leaves and stalk portione from the corn plant) will comprise (by weight) cellulose at about 35 to 45 percent, hemicellulose at about 24 to 42 percent, and lignin at about 12 to 20 percent.
• pre- treatment of the biomass will yield a liquid component that , comprises (by weight) xylose at no less than 1.0 percent and a solids component that comprises (by weight) cellulose (from which glucose can be made available) at no less than 45 percent.
• biomass is pre-treated in a pre-treatment system and then separated into a liquid component and a solids component.
• an acid will be applied to the prepared biomass to facilitate the break down of the biomass for separation into the liquid component (C5 stream from which fermentable C5 sugars can be recovered) and the solids component (C6 stream from which fermentable C6 sugars can be accessed) .
• the acid can be applied to the biomass in a reaction vessel under determined operating conditions (i.e. acid concentration, pH, temperature, time, pressure, solids loading, flow rate, supply of process water or steam, etc.) and the biomass can be agitated/mixed in the reaction vessel to facilitate the break down of the biomass.
• an acid such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, etc. (or a formulation/mixture of acids) can be applied to the biomass.
• sulfuric acid will be applied to the biomaBS in pre- treatment .
• the liquid component (C5 stream) comprises water, dissolved sugars (such as xylose, arabinose and glucose) to be made available for fermentation into ethanol, acids and other soluble components recovered from the hemicellulose.
• dissolved sugars such as xylose, arabinose and glucose
• the liquid component may comprise approximately 5 to 7 percent solids (i.e. suspended/residual solids such as partially-hydrolyzed hemicellulose, cellulose and lignin) .
• the liquid component will comprise at least 2 to 4 percent xylose (by weight) ,- according to other exemplary embodiments, the liquid component will comprise no less than 1 to 2 percent xylose (by weight) .
• TABLES 2A and 2B list the composition of the liquid component of pre-treated biomass (from prepared biomass as indicated in TABLES 1A and IB) according to exemplary and representative embodiments.
• the solids component (C6 stream) comprises water, acids and solids such as cellulose from which sugar, such as glucose, can be made available for fermentation into ethanol, and lignin.
• the solids component may comprise approximately 10 to 40 percent solids (by weight) (after separation) ; according to a particularly preferred embodiment, the solids component will comprise approximately 20 to 30 percent solids (by weight) .
• the solids in the solids component comprise no less than 30 percent cellulose and the solids component may also comprise other dissolved sugarB (e.g. glucoee and xylose) .
• TABLES 3A and 3B list the composition of the solids component of pre-treated biomass (from prepared biomass as indicated in TABLES 1A and IB) according to exemplary and representative embodiments .
• the severity of operating conditions may cause formation of components that are inhibitory to fermentation.
• C5 sugars such as xylose or arabinose
• HMF hydroxymethylfurfural
• Acetic acid may also be formed, for example when acetate is released during the break down of celluloee in pre-treatment .
• Sulfuric acid which may be added to prepared biomass to facilitate pre-treatment, if not removed or neutralized, may also be inhibitory to fermentation.
• the formation of inhibitors can be reduced or managed; according to other exemplary embodiments, components of the pre-treated biomass may be given further treatment to remove or reduce the level of .inhibitors (or other undesirable matter) .
• the C5 stream and the C6 stream are processed separately; as shown, the C5 stream and the C6 stream may be processed separately prior to co-fermentation (C5/C6 fermentation as shown in FIGURE 5A) or processed separately including separate fermentation (separate C5 fermentation and C6 fermentation as shown in FIGURE 5B) .
• Treatment of the C5 stream (liquid component) of the biomass may be performed in an effort to remove components that are inhibitory to efficient fermentation (e.g. furfural, HMF, sulfuric acid and acetic acid) and residual lignin (or other matter) that may not be fermentable from the C5 sugar component so that the sugars (e.g. xylose, arabinose, as well as other sugars such as glucose) are available for fermentation.
• the C5 sugars in the C5 stream may also be concentrated to improve the efficiency of fermentation (e.g. to improve the titer of ethanol for distillation) .
• Treatment of the C6 stream (solids component) of the biomaes may be performed to make the C6 sugars available for fermentation.
• hydrolysis such as enzyme hydrolysis
• treatment may also be performed in an effort to remove lignin and other non-fermentable components in the C6 stream (or to remove 3 042927
• the C5 stream and the C6 stream can be treated separately and subsequently combined after treatment (e.g. as a slurry) for co-fermentation in the fermentation system to produce a C5/C6 fermentation product from the available sugars (e.g. xylose and glucose) ; the C5/C6 fermentation product can (after treatment, if any) be supplied to the distillation system for recovery of the ethanol (e.g. through distillation and dehydration).
• the C5 stream and the CS stream can each be separately processed through fermentation and distillation (after treatment, if any) to produce ethanol.
• a suitable fermenting organism will be used in the fermentation system; the selection of an ethanologen may be based on various considerations, such as the predominant types of sugars present in the slurry. Dehydration and/or denaturing of the ethanol produced from the C5 stream and the C6 stream may be performed either separately or in combination.
• FIGURE 6A and 6B show the apparatus used for preparation, pre- treatment and separation of lignocellulosic bioraass according to an exemplary embodiment.
• biomass is prepared in a grinder (e.g. grinder or other suitable apparatus or mill) .
• Pre-treatment of the prepared biomass is performed in a reaction vessel (or set of reaction vessels) supplied with prepared biomass and acid/water in a predetermined concentration (or pH) and other operating conditions.
• the pre-treated biomass can be separated in a centrifuge into a liquid component (C5 stream comprising primarily liquids with some solids) and a solids component (CS stream comprising liquids and solids such as lignin and cellulose from which glucose can be made available by further treatment) .
• C5 stream comprising primarily liquids with some solids
• CS stream solids component
• a liquid component (C5 stream) is introduced to a treatment system.
• the C5 stream may be treated by filtration and/or by concentration into a treated liquid component that comprises sugars available for fermentation.
• the treated liquid component is supplied to a fermentation system to produce by fermentation of the sugars with an ethanologen (i.e. an organism such as yeast) a fermentation product (that comprises ethanol) .
• an ethanologen i.e. an organism such as yeast
• a fermentation product that comprises ethanol
• the ethanologen comprises a yeast derived from genetically modified recombinant Saccharomyces cerevisiae.
• the ethanologen is a strain of Saccharomyces cerevisiae yeast altered to convert xylose and glucose to ethanol (a genetically modified yeast derived from an organism as described in U.S. Patent No. 7,622,284, assigned to Royal Nedalco B.V. ) .
• yeast is supplied with a growth medium (e.g. water and a carbon source, such as sugar) and a agent (e.g. nutrients, etc.) to facilitate the growth of a sufficient amount of yeast (i.e. yeast cell mass) for inoculation (i.e. yeaet inoculum to be supplied) to the fermentation system.
• a growth medium e.g. water and a carbon source, such as sugar
• a agent e.g. nutrients, etc.
• the growth medium for the propagation system will comprise, for example, a sterile yeast extract-peptone medium, xylose as the carbon source, and other agents (e.g. nutrients).
• Agents supplied with the ethanologen may include antibiotics, supplemental or accessory enzymes, nutrients or other components providing nutritional or other benefits to the organism.
• Nutrients may comprise yeast extract, urea, diammonium phosphate, magnesium sulfate, zinc sulfate or other salts, etc.
• the yeast inoculum is incubated under conditions comprising a temperature of about 30 degrees Celsius and a pH of about 2013/042927
• the temperature may be maintained in a range of about 28 to 32 degrees Celsius and the pH in a range of about 5.2 to 5.8 or a time of at least 12 hours .
• FIGURES 7C and 9 an exemplary embodiment of the propagation system for the ethanologen (shown as yeast) employing two stages is shown.
• a yeast culture can be grown into an initial yeast inoculum that can be provided to the first stage of the propagation syetem.
• the initial yeast inoculum is transferred to a larger vessel and diluted (e.g. by 250x) .
• the initial yeast inoculum and a portion of the C5 stream i.e. liquid component comprising C5/other sugars
• water may be supplied along with agents (such as nutrients) and fresh yeast.
• yeast is grown in the first propagation stage under conditions comprising a temperature of about 30 degrees Celeius and a pH of about 5.5 for about 24 hours.
• the temperature may be maintained in a range of about 26 to 37 degrees and the pH in a range of about 3.5 to 6.5 for a time of at least 24 hours.
• the yeast inoculum from the first propagation stage is transferred to a larger vessel and diluted (e.g. by lOx) .
• the yeast inoculum from the first propagation stage and a portion of the C5 stream (i.e. liquid component comprising C5/other eugars) and water may be supplied along with agents (such as nutrients) and fresh yeast.
• yeast is grown in the second propagation stage under conditions comprising a temperature of about 30 degrees Celsius and a pH of about 5.5 for about 24 hours.
• the temperature may be maintained in a range of about 26 to 37 degrees and the pH in a range of about 3.5 to 6.5 for a time of at least 24 hours.
• the fermentation, product (which may also be referred to as beer or fermentation broth or as comprising beer or fermentation broth) will comprise ethanol and water as well as unfermented matter (e.g. any unfermented sugars) and non-fermentable matter (e.g. residual lignin and other solids) .
• the fermentation product will also comprise in the form of particulate matter the ethanologen (i.e. yeast cells) that was used to produce ethanol as well as other components produced by the fermentation system, for example, such as glycerol (a product of fermentation) and acetic acid.
• a treatment system for the fermentation product may also be provided.
• the treatment system can comprise separation of the fermentation product into a liquid component (i.e. a treated fermentation product, which will comprise substantially ethanol and water) and a solids component (which will comprise substantially solids matter such as the ethanologen/yeast cells) .
• the separation of the fermentation product into the liquid component and solids component can be performed on a centrifuge.
• the solids component from treatment comprising the yeast cells can be supplied to and re-used in the fermentation system (i.e. recycled for use in a fermentation tank) along with additional or fresh yeast cells (if necessary) .
• the yeast cells may be treated in a yeast cell treatment system.
• the yeast cell treatment system may comprise washing the yeast cells and separating the yeast cells prior to recirculation to a fermentation tank or the fermentation product stream.
• the yeast propagation system will provide for the growth of yeast into a suitable yeast cell 'mass at a suitable rate to be supplied to the fermentation system.
• the system will allow for the growth of yeast using xylose as a carbon source for growth.
• the system will allow for the selective growth of yeast that can use xylose as a carbon source (i.e. yeast that will propagate in a medium comprising xylose) even if other yeast is present (i.e. as a contaminant); in a medium that provides xylose as a sole carbon source (i.e.
• yeast that are capable of propagating using xylose as a carbon source will propagate and other/contaminant yeast that may not be as capable of propagating using xylose as a carbon source (such as more common forms of yeast that typically propagate in a medium containing glucose) will not propagate at the same rate (or at all) .
• the yeast will be capable of fermenting both xylose and glucose into ethanol.
• the yeast propagation system will provide a growth medium and environment in which the yeast will convert sugar (e.g.
• the system will facilitate the efficient growth of yeast cell mass into an inoculum that can be provided to the fermentation system in a biorefinery.
• the yeast cell mass will grow by about 200 to 500 fold in the first stage and about 20 to 40 fold in the second stage.
• FIGURES 7B, 7C and 9 A series of Examples were conducted according to exemplary embodiments of the yeast propagation system (as shown, for example, in FIGURES 7B, 7C and 9) to evaluate ethanologen growth under various conditions.
• the ethanologen used in the Examples was a strain of Saccharomyces cerevisiae yeast able to convert xylose and glucose to ethanol (a genetically modified yeast derived from an organism as described in U.S. Patent No. 7,622,284, by Royal Nedalco B.V., for example, strain No. RWB218; Strain No. RN1001 and Strain No. RN1014) .
• Data from the Examples is shown in FIGURES 10A through 16C and TABLES 5A through 11C.
• TABLE 4 lists the composition of the growth medium (including added agents such as nutrients) according to Examples 1A through .
• yeast inoculum An ethanologen culture was grown into an initial inoculum (yeast inoculum) using a sterile yeast extract-peptone (YP) medium (with 12.5 grams yeast extract per liter of medium and 10 grams peptone per liter) .
• the inoculum was incubated at 30 degrees Celsius for approximately 17 to 18 hours.
• An inoculum to media ratio of 1:250 was used.
• a pump was used to control the xylose feed rate.
• the pH of the medium was able to be maintained at 5.5 (by adding a 45 percent by weight solution of potassium hydroxide) .
• the samples were periodically analyzed for yeast growth (cell mass) , sugars, organic acids and ethanol.
• the optical density was measured (at 600 nanometers using a spectrophotometer) as an indication of the amount (i.e. cell mass) of yeast in the sample. (HPLC was used to analyze other components.)
• the propagation system was used in Example 1A to evaluate the effect of pH regulation on the growth of the ethanologen.
• the ethanologen was yeast (Strain No. RN1001) .
• Samples were prepared in two separate reaction vessels. The samples comprised a medium (e.g. water and agents, such as nutrients, as indicated in TABLE 4) and the yeast inoculum. The samples also comprised xylose at a concentration of 30 grams per liter (of medium) .
• the pH of the samples in each reaction vessel was adjusted to 5.5.
• the temperature of each reaction vessel was held at 30 degrees Celsius.
• the yeast was propagated in each reaction vessel for 54 hours. In one of the reaction vessels, the H was maintained (regulated) at 5.5; in the other reaction vessel the pH was unregulated.
• Example IB The propagation system was used in Example IB to evaluate the effect of feeding xylose at different rates on the growth of the ethanologen.
• the ethanologen was yeast (Strain No. RN1001) .
• Samples were prepared in two separate reaction vessels. The samples comprised a medium (e.g. water and agents, such as nutrients, as indicated in TABLE 4) and the yeast inoculum. The samples also comprised xylose at a concentration of 20 grams per liter (of medium) .
• the temperature of each reaction vessel was held at 30 degrees Celsius.
• the yeast was propagated in each reaction vessel for 24 hours.
• a first biomass liquid component was prepared by dilute acid pre-treatment of com cobs (see FIGURE 6A and TABLES 2A and 2B) .
• a treated biomass liquid component was prepared by treating the first liquid component by ion exchange chromatography.
• the ethanologen was yeast (Strain No. RN1001) .
• samples were prepared in three separate reaction vessels. The samples comprised a medium (e.g. water and agents, such as nutrients, as indicated in TABLE 4) and the yeast inoculum. The samples also comprised xylose at a concentration of 20 grams per liter (of medium) .
• each sample was adjusted to and maintained (regulated) at 5.5.
• the temperature of each reaction vessel was held at 30 degrees Celsius.
• the yeast was propagated in each reaction vessel for 24 hours .
• the sample in the first reaction vessel was supplied with xylose solution (50 percent by weight, along with additional nutrients) at 0.19 milliliters per minute for 30 hours resulting in a total amount of xylose of about 106 grams per liter.
• the propagation of the yeast continued for another 66 hours.
• the sample in the second reaction vessel was supplied with xylose solution (50 percent by weight, along with additional nutrients) at 0.26 milliliters per minute for about 22 hours and after that with the biomass liquid component at 2.8 milliliters per minute for about 2.6 hours resulting in a total amount of xylose of about 95 grams per liter.
• the propagation of the yeast continued for another 71 hours.
• the sample in the third reaction vessel was supplied with xylose solution (50 percent by weight, along with additional nutrients) at 0.19 milliliters per minute for about 22 hours and after that with the treated biomass liquid component at 3.0 milliliters per minute for about 2 hours resulting in a total amount of xylose of about 92 grams per liter.
• the propagation of the yeast continued for another 72 hours.
• the samples were tested and analyzed for yeast growth (by dry weight) , ethanol concentration, xylose concentration and optical density (at 600 nanometers [OD 600 ] ) to evaluate xylose conversion. It was observed that xylose obtained from biomass (lignocellulosic plant material) could be used to propagate yeast (within the indicated operating conditions) .
• the results are shown in FIGURES 12A through 12C and TABLES 7A through 7C.
• the propagation system as indicated in FIGURES 7C and 9 was used in Example 3 to evaluate the growth of the ethanologen in a two-stage propagation system with differing xylose levels.
• the ethanologen was yeast (Strain No. RNlOOl) .
• the sample comprised medium (e.g. water and agents, such as nutrients, as indicated in TABLE 4) and the yeast inoculum.
• the sample also comprised xylose at a concentration of 20 grams per liter (of medium) .
• the yeast was propagated for 24 hours . Af er 24 hours 300 mL of the Bample from the first stage was used to inoculate each of two samples in two separate reaction vessels (stage two) .
• the samples comprised medium (e.g. water and agents, such as nutrients, as indicated in TABLE 4) so that the total sample volume was 3 liters in each reaction vessel of the second stage.
• medium e.g. water and agents, such as nutrients, as indicated in TABLE 4
• the sample in the first vessel of the second stage comprised xylose at a concentration of 40 grams per liter (of medium)
• the sample in the second vessel of the second stage comprised xylose at a concentration of 30 grams per liter (of medium) .
• the temperature of each reaction vessel was held at 30 degrees Celsius.
• the yeast was propagated in each reaction vessel for 12 hours.
• the pH of each sample was adjusted to and maintained (regulated) at 5.5.
• the sample in the first reaction vessel of the second stage was supplied with xylose solution (50 percent by weight, along with additional nutrients) at 0.26 milliliters per minute for 12 hours resulting in a total amount of xylose of about 64 grams per liter.
• the propagation of the yeast continued for another 30 hours.
• the sample in the second reaction vessel of the second stage was supplied with xylose solution (50 percent by weight, along with additional nutrients) at 0.30 milliliters per minute for about 12 hours resulting in a total amount of xylose of about 64 grams per liter.
• the propagation of the yeast continued for another 30 hours. It was observed that the total time to propagate yeast (e.g.
• yeast cell mass grown from approximately 0.04 grams to 20 grams in the first stage and from approximately 2 grams to 75 grams in the second stage ⁇ could be reduced in a two-stage propagation system (i.e. using approximately 78 hours) as compared to a one-stage propagation system (i.e. using approximately 100 to 120 hours). See also Examples 1A, IB and 2. The results are shown in FIGURES 13A and 13B and TABLES 8A and 8B .
• the propagation system as indicated in FIGURES 7C and 9 was used in Example 4 to evaluate the effect of xylose feeding on the growth of the ethanologen.
• the ethanologen was yeast (Strain No. RN1014) .
• the sample comprised medium (e.g. water and agents, such as nutrients, as indicated in TABLE 4) and the yeast inoculum.
• the sample also comprised xylose at a concentration of 20 grams per liter (of medium) .
• the yeast was propagated for 24 hours. After 24 hours 300 mL of the sample from the first stage was used to inoculate each of two samples in two separate reaction vessels (stage two) .
• the samples comprised medium (e.g.
• the propagation system was used in Example 5 to evaluate the effect of aeration on the growth of the ethanologen.
• the ethanologen was yeast (Strain No. RWB218) .
• Samples were prepared in two separate reaction vessels. The samples comprised medium (e.g. water and agents, such as nutrients, ae indicated in TABLE 4) and the yeast inoculum. The samples also comprised xylose at a concentration of 30 grams per liter (of medium) .
• medium e.g. water and agents, such as nutrients, ae indicated in TABLE 4
• the samples also comprised xylose at a concentration of 30 grams per liter (of medium) .
• One reaction vessel was aerated at 5 liters per minute; the other reaction vessel was not aerated.
• the yeast was propagated in each reaction vessel at 32 degrees Celsius for 53 hours.
• the samples were tested and analyzed for yeast growth (by dry weight) , ethanol concentration, xylose concentration and optical density (OD SO o) to evaluate xylose conversion. It was observed that the growth of yeast could be improved when the Baraple was aerated (within the indicated operating conditions) insofar as the yeast used the supplied xylose for cell raass growth rather than for the production of ethanol. The results are shown in FIGURES 15A and 15B and TABLES 10A and 10B.
• the propagation system was used in Example 6 to evaluate the effect of xylose and glucose concentrations on the growth of the ethanologen.
• the ethanologen was yeast (Strain No. RWB218) .
• Samples were prepared in three separate reaction vessels .
• the samples comprised medium (e.g. water and agents, such as nutrients, as indicated in TABLE 11A) and the yeast inoculum.
• the temperature of each reaction vessel was held at 32 degrees Celsius.
• Each reaction vessel was aerated at 4.5 liters per minute .
• the first and second reaction vessels were supplied with xylose at a concentration of 60 grams per liter; the third reaction vessel was supplied with glucose at a concentration of 60 grams per liter.
• the pH of the samples in the second and third reaction vessels was adjusted to and maintained (regulated) at 5.5.
• the yeast was propagated in the first and second reaction vessel at 32 degrees Celsius for 120 hours.
• the yeast was propagated in the third reaction vessel at 32 degrees Celsius for 52 hours. It was observed that the growth of yeast could be improved when the sample was aerated insofar as the yeast used the supplied xylose for cell mass growth rather than the production of ethanol, notwithstanding the concentration of xylose (within the indicated operating conditions) .
• the results are shown in FIGURES 16A through 16C and TABLES 11A through 11C. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

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