US20110059497A1 - Apparatus and process for fermentation of biomass hydrolysate - Google Patents

Apparatus and process for fermentation of biomass hydrolysate Download PDF

Info

Publication number
US20110059497A1
US20110059497A1 US12/856,566 US85656610A US2011059497A1 US 20110059497 A1 US20110059497 A1 US 20110059497A1 US 85656610 A US85656610 A US 85656610A US 2011059497 A1 US2011059497 A1 US 2011059497A1
Authority
US
United States
Prior art keywords
immobilized
process according
hydrolysate
biomass
biomass hydrolysate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US12/856,566
Other languages
English (en)
Inventor
Lisa Beckler Andersen
John H. Evans, IV
Christine A. Singer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Geosynfuels LLC
Original Assignee
Geosynfuels LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Geosynfuels LLC filed Critical Geosynfuels LLC
Priority to US12/856,566 priority Critical patent/US20110059497A1/en
Assigned to GEOSYNFUELS, LLC reassignment GEOSYNFUELS, LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BECKLER ANDERSON, LISA, EVANS, JOHN H., IV, SINGER, CHRISTINE A.
Publication of US20110059497A1 publication Critical patent/US20110059497A1/en
Priority to US13/869,842 priority patent/US20140080191A1/en
Priority to US13/897,143 priority patent/US20140127775A1/en
Priority to US14/717,824 priority patent/US9523103B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • 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
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • 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
    • 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
    • C12P7/14—Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
    • 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 patent document relates to an apparatus and process for fermentation of biomass hydrolysate.
  • biomass is a renewable resource typically rich in polymers of hexoses and pentoses, it is a promising substrate for fermentation.
  • Biomass for such conversion processes may be potentially obtained from numerous different sources, including, for example: wood, paper, agricultural residues, food waste, herbaceous crops, and municipal and industrial solid wastes to name a few.
  • Biomass is made up primarily of cellulose and hemicellulose bound up with lignin.
  • the lignin inhibits the conversion of the biomass into ethanol or other biofuels, and, as a result, typically a pretreatment step is required to expose the polysaccharides, cellulose and hemicellulose.
  • saccharification either enzymatic or chemical, may be performed to break the polysaccharides into their constituent monosaccharide monomers.
  • Pretreatment and saccharification are used, therefore, to break down the long polysaccharide chains and free the sugars before they are fermented for biofuel production. Fermentation can begin once free sugars are present, either because they are naturally present or because a portion of the biomass has been reduced to its component sugars, or both.
  • inhibitory secondary products produced as a result of the degradation of hemicellulose pentoses and hexoses include furfural and 5-hydroxymethylfurfural (HMF), respectively. Furfural and HMF may further be broken down into levulinic, acetic, and formic acids.
  • Other inhibitory secondary products include phenolic compounds produced from the degradation of lignin and acetic acid produced by cleavage of acetyl groups within the hemicellulose. Concentrations of inhibitory secondary products in the hydrolysate will vary based on the source of the biomass and the hydrolysis method used.
  • Other secondary products are not formed from chemical decomposition, but may be extracted from the biomass during pretreatment and hydrolysis. These extracted secondary products include terpenes, sterols, fatty acids, and resin acids. These extracted compounds may also be inhibitory to fermentation.
  • Inhibitory secondary products may be detrimental to the fermentation process, particularly as their concentration increases.
  • a process could be developed that allows specific microbes, like yeast for example, to efficiently convert biomass hydrolysate into biofuels, such as ethanol, in the presence of inhibitory secondary products formed during pretreatment and hydrolysis.
  • inhibitory products have compound impacts when present with other inhibitory compounds; thus, a non-inhibitory amount of a certain compound may become inhibitory in the presence of a second inhibitory compound. Furthermore, even following partial recovery and/or removal of inhibitory secondary products, the remaining concentrations may be inhibitory to fermentation due to these synergies. Thus, it would be advantageous if a process could be developed that allows specific microbes, like yeast for example, to efficiently convert biomass hydrolysate into biofuels, such as ethanol, in the presence of inhibitory secondary products formed during pretreatment and hydrolysis, even when the concentrations of the individual inhibitory secondary products are below their respective inhibitory concentration level but their combined concentration is inhibitory.
  • Cellulose is a homogeneous polysaccharide composed of linearly linked glucose units.
  • Glucose is a hexose, which may be readily fermented by a number of microbes including Saccharomyces cerevisiae (traditional baker's yeast) and Kluyveromyces marxianus .
  • Yeast cells are especially attractive for cellulosic ethanol processes, as they have been used in biotechnology for hundreds of years, are tolerant to high ethanol and inhibitor concentrations, and can grow at low pH values. A low pH value helps avoid bacterial contamination and is therefore advantageous.
  • hemicellulose is a heterogeneous polymer of pentoses, hexoses, and uronic acids.
  • the saccharides principally found in hemicellulose are the pentoses xylose and arabinose and the hexoses glucose, mannose and galactose.
  • the relative amounts of different pentoses and hexoses vary with the biomass type.
  • the hemicellulose content of some cellulosic biomass may reach as high as 38% or more of the total dry biomass weight. Therefore, hemicelluloses, and the pentoses and hexoses they contain, may comprise a substantial portion of the convertible sugars available in the biomass.
  • biofuel such as ethanol.
  • biomass hydrolysate is currently subjected to a conditioning process after pretreatment and hydrolysis to reduce the concentration of inhibitory secondary products.
  • This conditioning process adds complexity and cost to the overall process and reduces the efficiency and cost-effectiveness of the conversion process.
  • the greater the required reduction in the concentration levels of the inhibitory secondary products the greater the complexity and cost.
  • a need therefore, exists for a process in which microbes, such as different yeast strains, could more effectively convert pentoses, as well as hexoses, into ethanol and other biofuels in the presence of inhibitors formed during the pretreatment and hydrolysis process.
  • an object according to one aspect of the present patent document is to provide an improved apparatus and process for converting biomass hydrolysate into ethanol or other biofuel.
  • the apparatus and process address, or at least ameliorate one or more of the problems described above.
  • a process for converting biomass hydrolysate into biofuel comprises the steps of: obtaining a biomass hydrolysate solution comprising monosaccharides; immobilizing a fermentative microbe contacting the solution with the immobilized fermentative microbe; and recovering a fermented biofuel.
  • the recovered biofuel preferably comprises alcohol, and more preferably comprises ethanol.
  • a process for converting biomass hydrolysate into biofuel comprising the steps of: contacting a biomass hydrolysate solution with immobilized fermentative microbe strain for a sufficient reaction time to convert monosaccharides in the biomass hydrolysate to biofuel; and recovering biofuel from the fermented hydrolysate.
  • the fermentative microbe is Pachysolen tannophilus and Pachysolen tannophilus is immobilized in calcium alginate.
  • the calcium alginate may be in the form of beads ranging from 0.1 mm to 5 mm in diameter, and are more preferably about 2 mm to 3 mm in diameter.
  • the calcium alginate is not required to be in bead form and may be in any other form that permits the Pachysolen tannophilus to be immobilized but still allows the sugar substrates in the biomass hydrolysate to kinetically interact with the yeast.
  • the calcium alginate may be in a sponge or mesh form.
  • the Pachysolen tannophilus /calcium alginate mixture may be applied as a coating to a natural or synthetic matrix to increase the surface area per mass of Pachysolen tannophilus /calcium alginate mixture.
  • the immobilized culture of Pachysolen tannophilus is periodically treated with a yeast growth medium to restore metabolic efficiency to the Pachysolen tannophilus .
  • the metabolic efficiency may be lost over long periods of use, especially in connection with continuous flow bioreactors.
  • the immobilized fermentative microbe strain is at least one microbe selected from a group consisting of Pichia, Candida, Klyveromyces and Zymomonas mobilis NREL strain 8b.
  • the alginate used to immobilize the culture of Pachysolen tannophilus is periodically recovered and recycled by treating the Pachysolen tannophilus /calcium alginate with a calcium chelator and monovalent counter-ion, such as sodium citrate.
  • a calcium chelator and monovalent counter-ion such as sodium citrate.
  • the resulting dialysis of the solution with an inorganic salt, such as sodium chloride regenerates sodium alginate, from which calcium alginate may be regenerated.
  • the biomass hydrolysate contains a substantial amount of secondary products that inhibit fermentation.
  • the hydrolysate solution may contain furfural levels in the range of about 0.01 to 10 g/L, 5-hydroxymethylfurfural levels in the range of about 0.01 to 10 g/L, and acetic acid levels in the range of about 0.05 to 20 g/L, or even 0.5 to 20 g/L.
  • the hydrolysate solution may contain phenolic compounds in the range of about 0.01 to 10 g/L. These levels of furfural, HMF, phenolic compounds, and acetic acid may occur in combination or in isolation. Other inhibitors may also be present.
  • more than 80% of the monosaccharides in the solution are converted to ethanol.
  • the biomass hydrolysate is obtained from the biomass by pressing.
  • the biomass and biomass hydrolysate may be subjected to a high pressure press capable of squeezing the sugar-containing liquid forming the biomass hydrolysate out of the biomass residue.
  • the biomass hydrolysate may be conditioned by passing the hydrolysate over activated carbon, a strong acid ion exchange resin and/or a weak base ion exchange resin.
  • the biomass hydrolysate solution may contains inhibitory secondary products sufficient to prevent more than 50% conversion of pentoses by the fermentative microbes in their “free” state.
  • a process for converting biomass hydrolysate into biofuel comprising the steps of: contacting the biomass hydrolysate solution with a first immobilized microbe strain; contacting the biomass hydrolysate solution with a second immobilized microbe strain; and recovering a fermented biofuel.
  • the first immobilized microbe strain is a bacterium and the second immobilized microbe strain is a yeast. Further, the first immobilized microbe strain may be contained in a first reactor and the second immobilized microbe strain may be contained in a second reactor. In an alternative embodiment, both immobilized microbe strains may be in the same reactor. If implemented so both strains are in the same reactor, the first immobilized microbe strain and the second immobilized microbe strain may also be immobilized together within the same immobilization medium.
  • the immobilization medium is a calcium alginate bead, but other immobilization mediums may also be used.
  • the first immobilized microbe strain may be immobilized in a first immobilization medium and the second immobilized microbe strain may be immobilized in a second immobilization medium.
  • the second immobilized microbe strain is capable of fermenting mannose to a biofuel.
  • a process for converting biomass hydrolysate into biofuel comprising the steps of: flowing a biomass hydrolysate solution comprising monosaccharides and one or more inhibitory secondary products through a continuous flow reactor containing an immobilized microbe strain and contacting the immobilized microbe strain with the biomass hydrolysate; and recovering a fermented biofuel.
  • the flow rate of the biomass hydrolysate is set to exceed the sedimentation rate of the immobilized microbe strain in a “free” condition.
  • the continuous flow reactor is an upflow reactor, but other continuous reactors may also be used.
  • the productivity of the biofuel conversion process is at least 0.3 g/L ⁇ h for a flow rate corresponding to a 10 hour retention time. In still another embodiment, the productivity of the biofuel conversion process is at least 0.42 g/L ⁇ h for a flow rate corresponding to a 5 hour retention time.
  • a medium for fermenting biomass hydrolysate comprises calcium alginate beads ranging from 0.1 mm to 5 mm in diameter, and a microbe strain capable of fermenting pentoses immobilized in the calcium alginate beads, wherein the immobilized microbe strain is capable of converting at least 70% of available pentoses in a biomass hydrolysate to a biofuel.
  • a medium for fermenting biomass hydrolysate comprising an immobilization substance capable of providing a micro environment for a microbe strain; and a microbe strain capable of fermenting pentoses into a biofuel immobilized in the immobilization substance, wherein the microbe strain comprises about 5% by volume of the immobilization substance.
  • FIG. 1 illustrates an overview of one embodiment of a process for the conversion of biomass into a biofuel such as ethanol.
  • FIG. 2 illustrates an overview of another embodiment of a process for the conversion of biomass into biofuels such as ethanol.
  • FIG. 3 illustrates a process for recycling a calcium alginate immobilization medium.
  • FIG. 4 illustrates a view of one embodiment of a bioreactor for performing submerged fermentation of biomass hydrolysate using immobilized microbes.
  • FIG. 5A illustrates a side view of another embodiment of a bioreactor for performing submerged fermentation of biomass hydrolysate using immobilized microbes.
  • FIG. 5B illustrates a front view of the bioreactor shown in FIG. 5A .
  • FIG. 6 illustrates an up-flow reactor for performing submerged fermentation of biomass hydrolysate using immobilized microbes.
  • FIG. 7 is a graph illustrating ethanol yield of regenerated calcium alginate beads with immobilized fermentative microbes over a series of fermentations.
  • biomass is used herein to refer to living and recently dead biological material including carbohydrates, proteins and/or lipids that may be converted to fuel for industrial production.
  • biomass refers to plant matter, including, but not limited to switchgrass, sugarcane bagasse, corn stover, corn cobs, alfalfa, Miscanthus , poplar, and aspen, biodegradable solid waste such as dead trees and branches, yard clippings, recycled paper, recycled cardboard, and wood chips, plant or animal matter, and other biodegradable wastes.
  • the present patent document teaches new and improved processes and apparatuses for fermenting biomass hydrolysate. Processes used to convert polysaccharides in biomass into hexoses and pentoses often create inhibitory secondary products that prevent or hinder fermentation. Furthermore, the combinations of inhibitory secondary products found in actual biomass hydrolysate are more toxic to ferments than any single inhibitory secondary product added to a defined, artificial medium.
  • the present patent document teaches novel processes that increase the tolerance of the fermentative microbes to inhibitory secondary products found in biomass hydrolysate by immobilizing the microbes. In certain embodiments, fermentation of hemicellulose hydrolysate containing inhibitory secondary products is carried out using immobilized Pachysolen tannophilus . In some embodiments, fermentation of hemicellulose hydrolysate is carried out using an immobilized microbe, even though the concentration of an individual secondary product or the combined concentration of secondary products in the biomass hydrolysate would be inhibitory to the microbe in its free state.
  • Immobilization confers an increased resistance on microbes to inhibitory secondary products.
  • immobilization in a calcium alginate greatly reduces the susceptibility of the yeast Pachysolen tannophilus to inhibitors contained in softwood hydrolysate.
  • the benefits of immobilization are not limited to Pachysolen tannophilus .
  • numerous different microbes may benefit from immobilization including, for example, yeasts from the genera Pichia, Candida , and Klyveromyces .
  • bacterium microbes such as Zymomonas mobilis , NREL strain 8b, also show an increased resistance to inhibitory secondary products when immobilized.
  • the calcium alginate, or other material used to immobilize the microbes is in a form with a high surface area such as in bead, sponge, or mesh form.
  • the immobilized microbe or combination of microbes should also be able to ferment monosaccharides found in hemicellulose hydrolysates—including the hexoses mannose, galactose and glucose and the pentoses xylose and arabinose—to biofuel with high efficiency.
  • FIG. 1 illustrates a general overview of one embodiment of a process for converting biomass to ethanol or other biofuels.
  • the primary steps include pretreatment 100 , hydrolysis 102 , fermentation 104 , and biofuel recovery 106 .
  • FIG. 2 illustrates another embodiment of a process for converting biomass to ethanol or other biofuels. The process in FIG. 2 differs from that in FIG. 1 in that it also includes a solid/liquid separation step 108 , an optional evaporation step 112 , an optional conditioning step 110 , and an optional secondary product recovery step 114 . If the biomass hydrolysate is provided from another source instead of generated on site, the process of the present patent document may be condensed to performing step 104 or steps 104 in combination with step 106 .
  • pretreatment is designed to reduce the recalcitrance of the biomass to enzymatic or chemical saccharification of the cellulose and hemicellulose, therein.
  • the pretreatment step 100 may occur through a number of methods, including for example, in a pressure reactor. Table 1 lists appropriate ranges for temperature, dwell time, and moisture content suitable for pretreatment in a pressure reactor. However, other operating conditions may also be suitable.
  • Effectiveness of the pretreatment step 100 may be increased by adding one or more reagents.
  • Reagents may include, but are not limited to: nitric acid, phosphoric acid, hydrochloric acid, sulphuric acid, sulphur dioxide, and sodium sulphite.
  • Other reagents that reduce the recalcitrance of the biomass to hemicellulose removal may also be added.
  • pretreatment 100 may be performed using a number of other methods, including acid prehydrolysis, steam cooking, alkaline processing, rotating augers, steam explosion, ball milling, or any other method that reduces the recalcitrance of the biomass to saccharification of the cellulose and hemicellulose contained therein.
  • the polysaccharides are broken down into their monosaccharide components so they can be fermented.
  • the Hydrolysis step 102 is used for converting the polysaccharides into fermentable sugars. In some of the harsher pretreatments 100 , hydrolysis 102 may occur simultaneously with the pretreatment step 100 and a separate hydrolysis step 102 is not required.
  • the two basic forms of hydrolysis 102 are thermo-chemical and enzymatic. Thermo-chemical hydrolysis is typically performed using a concentrated acid such as sulfuric acid or hydrochloric acid at relatively low temperatures or by using a dilute acid at relatively high temperatures.
  • a biomass hydrolysate solution comprising monosaccharides will typically be obtained by pressing and/or washing the biomass residue.
  • the obtained biomass hydrolysate is then fermented ex-situ in fermentation step 104 .
  • Solid-liquid separation may be performed using a number of methods including, but not limited to, centrifuging or pressing.
  • pressing may be accomplished with a hydraulic press.
  • numerous types of mechanical or machine presses may be used.
  • a mechanical press such as a conventional screw press, a hydro-mechanical press, a pneumatic press or any other type of press that can apply the necessary pressure to remove the hemicellulose hydrolysate from the cellulose/lignin residue may be used.
  • the press may have a range of capabilities and configurations for pressing out the hemicellulose hydrolysate.
  • the press can generate from at least about 10.5 kg/cm 2 to about 21.1 kg/cm 2 . In other embodiments, it is desirable if the press can generate at least approximately 1,410 kg/cm 2 .
  • Pressing has additional advantages because the biomass residue (which will comprise cellulose and lignin at this point) may be more valuable as a coal replacement if its density can be maximized and its moisture content minimized, thereby increasing its energy density.
  • biomass residue which will comprise cellulose and lignin at this point
  • Pulp quality is measured based on its fiber length, among other variables, but not moisture content. However, if a high energy density fuel replacement is made instead of paper pulp, reducing the moisture content is an important factor.
  • the final product that the biomass residue is to eventually be used for may determine what size and kind of press to use for solid/liquid separation.
  • a lower pressure such as in the range of 10.5 kg/cm 2 to 21.1 kg/cm 2 may be advantageous to minimize damage to the cellulose fibers.
  • higher pressures may be used to minimize the moisture content, without regard to fiber quality.
  • pressures within the range of 10.5 kg/cm 2 to 21.1 kg/cm 2 may still be used, as presses generating these types of pressures are readily available and comparatively inexpensive as compared to presses that are capable generating about 1410 kg/cm 2 of pressure.
  • presses that generate between about 10.5 kg/cm 2 and 21.1 kg/cm 2 of pressure are routinely used in the wine and olive oil industries to press grapes and olives, respectively.
  • Pressing is also advantageous because it reduces dilution from wash water.
  • Using wash water to separate the hydrolysate from the biomass will dilute the sugar stream and thus lower the resulting ethanol concentration in the fermented hydrolysate.
  • dilution of the sugar stream may be mitigated by the use of evaporators or similar machinery to reduce water content in the hydrolysate through optional evaporation step 112 , shown in FIG. 2 .
  • the recovered water from evaporation may be recycled into subsequent wash processes. Addition of an evaporation step 112 as a process step increases the sugar concentration of the hydrolysate and thus the ethanol concentration resulting from fermentation, which in turn reduces the costs of distillation.
  • the biomass hydrolysate comprises a cellulose hydrolysate, so as to include glucose (which is a hexose)
  • the glucose in the hydrolysate may be fermented by a number of yeast strains including Saccharomyces cerevisiae (traditional baker's yeast) and Kluyveromyces marxianus to name a few.
  • the biomass hydrolysate comprises a hemicellulose hydrolysate
  • the hydrolysate will include the pentoses xylose and arabinose, and a lower concentration of hexoses, except in the case of softwood hydrolysate.
  • the hexose mannose is the major saccharide and the pentose xylose is the next most abundant.
  • Microbes that can convert the combination of pentoses and hexoses found in hemicellulose hydrolysate into ethanol are not as abundant as those available for cellulose hydrolysate.
  • microbes that can convert both five-carbon and six-carbon sugars are preferably utilized so that all of the available constituent sugars of the hemicellulose hydrolysate may be converted to ethanol or other biofuels.
  • the biomass hydrolysate comprises a combination of cellulose hydrolysate and hemicellulose hydrolysate.
  • Microbes that can ferment hexoses and pentoses may be derived from the genera Pachysolen, Kluyveromyces, Pichia , and Candida.
  • Pachysolen tannophilus is preferably used in fermentation of a liquid hydrolysate comprising a hemicellulose hydrolysate. In particular, when immobilized, Pachysolen tannophilus has been found to effectively ferment hemicellulose hydrolysate produced from softwood.
  • immobilized bacterium may also be used to ferment hexose and pentose sugars in biomass hydrolysate.
  • immobilized bacterium Zymomonas mobilis NREL recombinant 8b
  • NREL recombinant 8b may be used to ferment hemicellulose hydrolysate produced from softwood, hardwood, and/or herbaceous sources.
  • Microbes with complementary metabolic properties may also be combined in the same fermentation process in step 104 to allow their complementary properties and abilities, such as complementary hexose and pentose fermentation capabilities or complimentary metabolic rates, to be used together.
  • complementary properties and abilities such as complementary hexose and pentose fermentation capabilities or complimentary metabolic rates.
  • recombinant Zymomonas is unable to ferment mannose, the most prevalent sugar contained in softwood hydrolysate
  • the recombinant Zymomonas mobilis is preferably paired with a complementary yeast or bacterium that is able to effectively ferment the hexose mannose to ethanol or another biofuel when it used to ferment softwood hydrolysate.
  • microbes are also possible including pairing different bacterium together, pairing different yeasts together, pairing various yeasts and bacterium together, or pairing or combining any number of microbes with complimentary features including using any number of microbes at the same time. As the number of combined microbes increases, however, their capabilities may begin to overlap significantly and thereby reduce the additive value of the additional microbes.
  • the pretreatment step 100 and hydrolysis step 102 may yield soluble sugars from the biomass in the form of xylose, mannose, arabinose, galactose, and glucose ready for fermentation in step 104 .
  • other secondary products which are inhibitory to the fermentation step 104 , are also produced or extracted from the biomass.
  • concentrations of fermentation inhibitors that form in converting biomass to fermentable hexoses and pentoses will vary depending on the source of the biomass and the methods used for the pretreatment step 100 and the hydrolysis step 102 .
  • acetic acid is produced by cleavage of acetyl groups from hemicellulose.
  • Phenolic and polyphenolic compounds are also formed from the degradation of lignin. While the generated Phenolic Compounds, furfural, HMF, and acetic acid are all potentially valuable compounds, they are also fermentation inhibitors, and may prevent or inhibit fermentation, particularly as their concentrations increase.
  • Phenolic and polyphenolic compounds produced from hydrolysis of wood hemicellulose and the concomitant lignin degradation include guaiacol, vanillin, phenol, vanillic acid, syringic acid, salicylic acid, gentisic acid, and others. Many of these compounds, for instance vanillin and vanillic acid, are known to inhibit the growth of and/or fermentation with microbial yeasts, such as Pachysolen and Saccharomyces.
  • other molecules may be extracted from biomass by the pretreatment and/or saccharification conditions during the pretreatment step 100 and/or hydrolysis step 102 .
  • These extracted compounds may include terpenes, sterols, fatty acids, and resin acids.
  • These extracted compounds can also be inhibitory to metabolic processes, including fermentation, in yeast and other microbes, such as bacteria.
  • metal cations including calcium, aluminum, potassium, and sodium are found in hemicellulose hydrolysate and heavy metals may be present from degradation of the metal vessels due to hydrolysis. The presence of such metal cations may also be inhibitory above certain concentrations.
  • a method commonly used to ameliorate the toxicity of hydrolysates by reducing HMF and furfural concentration is pH adjustment through “overliming” with calcium hydroxide. Overliming is the process whereby lime is added beyond that necessary for pH adjustment. Even after overliming, however, high levels of inhibitors may still exist. In addition, overliming precludes recovery of secondary products that have high value from the hydrolysate.
  • the present patent document teaches processes to protect the fermentation microbes from the degradation effects of the inhibitors by immobilizing the microbes, and more preferably immobilizing the microbes in calcium alginate. Immobilization of microbes is the attachment or inclusion in a distinct solid phase, such as calcium alginate, that permits exchange of substrates, products, inhibitors, etc. with the microbe, but at the same time separates the microbes from the bulk biomass hydrolysate environment.
  • the microenvironment surrounding the immobilized microbes is not necessarily the same as that which would be experienced by their free-cell counterparts.
  • the present patent document teaches processes for immobilizing Pachysolen tannophilus and for fermenting pentoses and hexoses in the presence of inhibitors found in hemicellulose hydrolysate, even at concentrations that would inhibit the fermentative microbe in its free state.
  • conditioning to reduce the concentration of inhibitors may be omitted in some embodiments, or, as shown in FIG. 2 , included as an optional conditioning step 110 .
  • Conditioning the biomass hydrolysate in conditioning step 110 to reduce the concentration of inhibitory secondary products may still be desirable where, for example, the concentration of the secondary products (either individually or in combination) is sufficiently high to interfere with the fermentation of sugars even by the immobilized microbe(s). In such cases, however, the concentration of the inhibitory secondary products will generally not need to be reduced to the same levels as necessary for fermentation using free microbes and thus a less severe and less costly conditioning process may be employed. To offset the costs associated with the overall fermentation process, it may also be desirable to recover secondary products having a high value through an optional high value secondary product recovery step 114 shown in FIG. 2 .
  • the concentrations of these products may remain sufficiently elevated within the hydrolysate, particularly considering the synergistic nature of the inhibitors, to interfere with fermentation of sugars to ethanol or other biofuel by the fermentative microbe(s) in their free state. Accordingly, the use of immobilized fermentative microbe(s) in fermentation step 104 is an important aspect of the processes described herein, even when the optional conditioning step 110 is employed to reduce the concentration of secondary products contained in the biomass hydrolysate.
  • conditioning step 110 it may also be desirable to perform conditioning step 110 even when the concentration of inhibitory secondary products is insufficient to inhibit fermentation by the immobilized microbe(s) where, for example, the secondary products have high value and thus it is desirable to separately recover the high value secondary products through high value secondary product recovery step 114 .
  • This may be desirable, for example, where the net value of the recovered high value secondary products may be used to offset, and hence lower, the costs associated with the overall fermentation process.
  • conditioning step 110 there are numerous methods of performing the conditioning step 110 to reduce the concentrations of inhibitory secondary products. Employing different conditioning methods for conditioning step 110 will result in different concentration levels of inhibitory secondary products remaining in the hydrolysate.
  • the method of conditioning chosen for conditioning step 110 may depend on a variety of factors, including the sensitivity of the microbe used during fermentation to inhibitory secondary products, costs, and whether there is a desire to recover high value secondary products during a recovery step 114 . The more sensitive the microbe, the more desirable it will be to reduce the concentration of the inhibitory products from the biomass hydrolysate during conditioning of the hydrolysate in step 110 .
  • conditioning step 110 may reduce the concentration of secondary products and thus may reduce the complexity and costs incurred during conditioning step 110 .
  • Some of the conditioning methods that may be employed in conditioning step 110 to reduce the concentration of secondary products include, but are not limited to: 1) overliming of hydrolysate; 2) activated carbon (AC) treatment followed by pH adjustment; 3) ion exchange followed by overliming; 4) AC treatment followed by ion exchange; and 5) AC treatment followed by nanofiltration.
  • the secondary products may be recovered in step 114 from the hydrolysate and subsequently used for other purposes.
  • Some of the high-value secondary products that may be recovered in step 114 include, but are not limited to, the mineral acid used in the pretreatment process 100 , such as sulfuric acid, acetic acid hydrolyzed from hemicellulose polymers, anti-oxidant molecules (phenolic and polyphenolic compounds) liberated from the partial hydrolysis of lignin during hydrolysis step 102 , other organic acids, nutraceutical, cosmeceutical, or pharmaceutical products, and different furans and furan derivatives, such as 5-hydroxymethylfurfural and furfural.
  • the mineral acid used in the pretreatment process 100 such as sulfuric acid, acetic acid hydrolyzed from hemicellulose polymers, anti-oxidant molecules (phenolic and polyphenolic compounds) liberated from the partial hydrolysis of lignin during hydrolysis step 102
  • other organic acids nutraceutical, cosmeceutical, or pharmaceutical products
  • High value secondary product recovery step 114 may be accomplished by adsorption of the secondary products to different matrices, including activated carbon, ion exchange resin, ion exchange membrane, organic molecule “scavenging” resins, polystyrene beads, or any other similar type medium with a high surface area. High value secondary product recovery step 114 may also be accomplished by separating the secondary product(s) from the soluble hexoses and pentoses through ion exclusion chromatography, pseudo-moving bed chromatography, high performance liquid chromatography or by filtration methods including micro-, nano-, and ultrafiltration using hollow fiber or membrane technologies. High value secondary product recovery step 114 may include several of the aforementioned processes in series to recover different molecular species.
  • the recovery process(es) employed in step 114 may be tailored to recover specific secondary products according to the nature of the starting biomass. Because many of the recovered secondary products (acetic acid, furans and their derivatives, phenolic and polyphenolic compounds, levulinic acid, formic acid, and others) are inhibitory to yeast and bacterial fermentation of sugars to ethanol, recovery of high value secondary products in step 114 may both increase the economics of the entire process and allow for more efficient fermentation in step 104 of the pentoses and hexoses.
  • microbes may be immobilized for fermentation 104 of biomass hydrolysate in step 104 using a number of different methods.
  • Microbes may be bound to a matrix material or, more preferably, immobilized by entrapment in the matrix material.
  • microbes may be immobilized by entrapment using a drop-forming procedure.
  • the resultant beads may be of different size and possess different pore sizes.
  • the beads may range in size from 0.1 mm to 5 mm in diameter, more preferably the beads may range from 2 mm to 3 mm in diameter, and more preferably the beads are about 3 mm in diameter.
  • the drop-forming procedure may be enhanced through a number of processes.
  • the beads may be hardened to different degrees and may have coatings applied to withstand shear forces in a reactor and to reduce cell loss. For example, if calcium alginate is used, the beads may be dried to increase compression stress.
  • the beads may also be hardened by glutaraldehyde treatment or coated with catalyst-free polymer to enhance their stability.
  • the beads may be recoated with plain alginate as a double layer to enhance their gel stability.
  • the beads may have a polyacrylamide coating to enhance their structural stability.
  • the beads may also be coated with a copolymer acrylic resin to increase diffusion and reduce cell leakage.
  • other additions to the drop forming procedure may be incorporated to enhance the effectiveness of the matrix.
  • a Pachysolen tannophilus /calcium alginate or other microbe/calcium alginate mixture may be applied as a coating to a natural or synthetic, high surface area, support structure.
  • the support structure only need be able to support the microbe/immobilization medium and itself.
  • the support structure may comprise a ceramic sponge, honeycomb, reactor packing material or other support structure to increase the surface area per mass of the microbe/immobilization medium when it is applied.
  • the mixture may also, or in the alternative, be applied to parts of the reactor surfaces, such as, the walls or the surface of the mixing devices.
  • the microbes may be immobilized by other methods including adsorption, cross-linking, or immobilized by any other means capable of providing a micro-environment for the microbe.
  • microbes A variety of different materials may be used to immobilize microbes. If the microbes are immobilized using entrapment calcium alginate, a natural product from brown algae (seaweed) may be preferably used. However, other materials, both natural and synthetic, may also be used to immobilize microbes using entrapment including carrageenan, xanthan gums, agarose, agar and luffa , cellulose and its derivatives, collagen, gelatin, epoxy resin, photo cross-linkable resins, polyacrylamide, polyester, polystyrene and polyurethane.
  • entrapment calcium alginate a natural product from brown algae (seaweed) may be preferably used.
  • other materials both natural and synthetic, may also be used to immobilize microbes using entrapment including carrageenan, xanthan gums, agarose, agar and luffa , cellulose and its derivatives, collagen, gelatin, epoxy resin, photo cross-linkable resins
  • Other materials that may be used to immobilize microbes using adsorption or other immobilization methods include kieselguhr, wood, glass ceramic, plastic materials, polyvinyl acetate, and glass wool.
  • the microbes When combining microbes with complimentary properties, the microbes may be combined within the same immobilization vehicle, or the microbes may be immobilized separately and the separately immobilized microbes combined in the same fermentation reactor. For example, if calcium alginate beads are used as the immobilization vehicle, different complimentary microbes may be combined within the same bead.
  • NREL strain 8b which ferments glucose and xylose to ethanol
  • Saccharomyces cerevisiae which ferments mannose and galactose
  • separate beads can be made containing each microbe and then the beads may be combined in the fermentation reactor.
  • the fermentation of the hexoses and pentoses to fuel may be performed by combining beads composed of different microbial species with complementary hexose and pentose specificities, metabolic rates, or the like.
  • different microbes are immobilized in separate reactors and the biomass hydrolysate is then run through each reactor to expose the biomass hydrolysate to each microbe.
  • different immobilization methods may be combined with different microbes.
  • immobilizing the microbes become more stable and bioreactors may be run in a continuous mode instead of batch mode. Running the bioreactor in a continuous mode is advantageous for efficiency reasons but the microbes may begin to lose metabolic efficiencies after long periods of use.
  • immobilized microbes may be periodically treated with yeast growth medium. For example, Pachysolen tannophilus and other fermentative microbes immobilized in calcium alginate may be periodically treated with a yeast growth medium to restore metabolic efficiency.
  • microbe immobilization Another advantage of microbe immobilization is that the microbe biomass may be better retained within a continuous fermentation reactor.
  • a continuous fermentation process involving a high flow rate such as that which may be experienced during the continuous running of a columnar up-flow reactor, free cells will tend to wash out. Wash out reduces the number of cells in the reactor and thus lowers the rate of the fermentation reaction. To maintain the rate of fermentation, new cells must be propagated and added to the reactor, increasing costs.
  • Table 2 below demonstrate the advantages of using immobilized microbes in a continuous fermentation process under wash out conditions (i.e., under a flow rate that would cause wash out of more than 5% of the free cells.)
  • the data in Table 2 illustrates the benefits of immobilization to prevent wash out for one particular fermentative microbe.
  • the example in Table 2 demonstrates the improvement of biofuel (e.g., ethanol) yield for immobilized Pachysolen tannophilus (NRRL Y2460) over free cells of the same microbe during continuous fermentation in a column up-flow reactor.
  • biofuel e.g., ethanol
  • immobilization can be used to prevent wash out for any type of fermentative microbe in any continuous flow bioreactor and thereby increase ethanol or other biofuel yield.
  • the data presented in Table 2 was generated by adding 8.38 ⁇ 10 11 cells of Pachysolen tannophilus to two identical up-flow reactors.
  • the cells were immobilized in 2-3 mm calcium alginate beads.
  • the cells were added free in solution.
  • Both reactors were connected to the same reservoir of artificial medium and the same peristaltic pump was used to pump the artificial medium through the reactors during the continuous fermentation process.
  • the artificial medium within the reservoir contained 10 g/L yeast extract, 20 g/L peptone, 7.2 g/L glucose, and 42.5 g/L xylose. The artificial medium was pumped into the bottom of both reactors simultaneously at the same rate and both reactors were incubated at 30° C.
  • a first test the two reactors were each run at a flow rate corresponding to a retention time of 10 hours. The reactors were each run for a total of 20 hours or for a total of 2 ⁇ the retention time.
  • a second test set up as indicated above, the two reactors were each run at a flow rate corresponding to a retention time of 5 hours. In the second test, the reactors were run for a total of 10 hours, or again for a total of 2 ⁇ the retention time.
  • the ethanol content of the first and second reactor's effluent was analyzed for ethanol content at the end of the 2 ⁇ retention time period for each test. Hence, ethanol content of effluent was determined for each reactor at two separate flow conditions. The productivity (ethanol production per hour) was also determined for each flow condition. The results are reported in Table 2.
  • Table 2 reveals that the ethanol concentration at the end of 20 hours for the 10 hour retention time flow rate was much greater for the reactors containing immobilized Pachysolen than free Pachysolen, 3.03 versus 1.84 g/L, respectively. The corresponding productivity was also greater for the immobilized Pachysolen .
  • the ethanol concentration in the effluent of the reactor containing immobilized cells was 2.08 g/L at the end of 10 hours or 2 ⁇ the retention time, but the productivity increased by 40% over that in the first test due to the faster flow rate.
  • Table 2 illustrate that immobilizing fermentative microbes decreases wash out and increases biofuel, such as ethanol, productivity in the reactor.
  • biofuel such as ethanol
  • the flow rate of the medium exceeded the sedimentation rate of the free Pachysolen tannophilus (at both flow rates tested) and the concentration of the cells in the free state reactor decreased to a low level causing the ethanol concentration and ethanol productivity to also decrease.
  • the Pachysolen tannophilus that was immobilized in the calcium alginate beads remained in the reactor and the reactor was able to increase the ethanol productivity with the increased flow rate.
  • Certain microbes that can be used in conversion of sugars to biofuels are motile; that is, they possess cilia and/or flagella and swim in fermentation medium. Another advantage of immobilization is that the motile microbe biomass may be better retained within a continuous fermentation reactor, even in fermentation process involving a low flow rate. Motile cells in the free state will tend to wash out in all flow conditions. Wash out reduces the number of cells in the reactor and thus lowers the rate of the fermentation reaction. To maintain the rate of fermentation, new cells must be propagated and added to the reactor, increasing costs.
  • Another advantage of immobilizing microbes is the ability to obtain a high biomass concentration in a continuous fermentation process.
  • a column upflow reactor as a non-limiting example, more than half, preferably about two thirds to about three quarters of the reactor volume will be composed of the bead material and the rest will be inter particle void volume when the fermentative microbes are immobilized in beads of about 2 mm to 3 mm in diameter.
  • yeast as the fermenting microbe, where 5% of the volume of the bead is yeast biomass, the reactor will effectively contains about 3.3 to 3.75% by volume yeast biomass, which is a relatively high yeast concentration for a fermentor.
  • yeast and bacteria immobilization by entrapment in calcium alginate over free cells in suspension include greater ethanol tolerance, possibly due to changes in cell membrane composition; greater specific ethanol production, increased rate of ethanol production due to increased glucose uptake and lower dissolved CO 2 in solution, and increased thermo-stability of bacteria.
  • the microbes are initially immobilized in sodium alginate which is then converted to calcium alginate.
  • Sodium alginate can have different viscosities when a given amount is dissolved in an aqueous solution. Viscosities for different sodium alginate products range from 100 or 200 mPa, to even as much as 1236 mPa.
  • alginate with medium-low viscosity of about 324 mPa is used to produce beads, although alginates with different viscosities may be used for different biomass hydrolysates or for solid-state ferments.
  • the sodium alginate is prepared by adding from 0.05 to 10%, or preferably about 3.5% (w/v) sodium alginate to deionized water.
  • the sodium alginate can be dissolved into growth medium, into a mixture of vitamins, including biotin, or into growth medium supplemented with vitamins, or into a natural solution containing biotin.
  • the initial sodium alginate concentration will depend on the final concentration desired to produce beads and on the volume added by mixing with a concentrated microbe slurry.
  • the mixture may be heated and stirred on a stir plate.
  • heating alginate polymers may cause some amount of hydrolysis of the alginate and thereby change the properties of the alginate solution, including its viscosity.
  • Cells may be cultivated in their respective media, and pelleted by centrifugation.
  • a mass of Pachysolen or other in fermentative microbe may be propagated in at least a 10 L, or more preferably at least a 200 L, or even more preferably at least a 2000 L bioreactor to a concentration of about 1 to about 20 grams wet mass per liter growth medium.
  • the resulting biomass may then be concentrated using, for example, a tangential flow filtration device to produce a 20-70% wet mass slurry of Pachysolen cells. This technique is particularly well suited for the production of large volumes of calcium alginate beads having one or fermentative microbes, such as Pachysolen , immobilized therein.
  • the concentrated cells are then recovered and thoroughly mixed with the sodium alginate medium.
  • Mixing the alginate with the microbial cells can occur in the same device as is used for the resuspension of the alginate or in a separate device. The mixing continues to homogenity of the mixture.
  • Mixing of the microbes with the highly viscous sodium alginate solution requires a mixing method that does not shear the microbes, such as a reciprocating disc mixer.
  • the cell loading into the sodium alginate medium is both organism and substrate dependent. For example, a suitable target loading for Pachysolen tannophilus in hydrolysate is at least 5 g cells/100 mL sodium alginate medium.
  • Calcium alginate beads are produced by extruding the sodium alginate medium/cells into a sterile calcium chloride solution.
  • a peristaltic pump and sterilized Master-flex Bulk-Packed Silicone Tubing that has an attached sterile 18 G needle may be used in the extruding process. The entire process is preferably done aseptically.
  • a sterile 96 hollow 19 gauge pin device may be used in place of an 18 gauge needle.
  • the beads may then be produced by extrusion and gravity dropping.
  • Other methods may include a so-called Jet Cutter to produce beads from a continuous stream of an alginate/microbe slurry.
  • Other modifications of producing beads from a continuous stream include using electrostatic attraction to produce droplets, using vibration to produce droplets, using air to produce droplets, and using a rotating disk atomizer, to name a few.
  • beads are dropped in a solution containing calcium chloride.
  • a 0.22M solution of calcium chloride dihydrate is also prepared in deionized water to receive sodium alginate/microbe mixture.
  • the sodium alginate medium and calcium chloride solution may both be autoclaved for sterilization purposes.
  • the beads may be kept at 4° C. in the calcium chloride solution for about 60 minutes to harden. Once the beads have hardened, they are preferably rinsed several times with sterile deionized water.
  • the beads are dropped into sterile growth medium containing 0.1 to 0.25 M calcium chloride.
  • the growth medium may also contain different vitamins or biotin. After about 30 minutes of hardening, the beads may be either used immediately in a fermentation or may be stored at 4° C. until use. There is no need to rinse beads prior to use or prior to storage when hardening is carried out in such a growth medium.
  • the solid calcium alginate used to immobilize microbes in beads or on a support structure may delaminate, break-up, shear, or otherwise physically degrade after prolonged use.
  • the microbe/calcium alginate mixture may also become degraded and discolored through repeated use due to the trapping of contaminants such as extractives, microbial inhibitors, and other materials. Degradation of the structure, whether due to physical and/or chemical degradation affects the performance of the fermentation process. To overcome deleterious effects of this degradation, new or fresh microbe/calcium alginate mixture may be used in the bioreactor to improve the reactors performance. However, the frequent replacement of the mixture may be uneconomical both in terms of the material costs associated with production of the calcium alginate, but also due to the cost of the lost microbes.
  • FIG. 3 illustrates a process 140 for recycling calcium alginate used in the microbe immobilization process.
  • the calcium alginate from the beads used to immobilize the microbes may be recovered and recycled using process 140 .
  • the degraded microbe/calcium alginate mixture 148 is dissociated with a calcium chelator complexed with a monovalent ion 150 , such as sodium citrate or potassium citrate.
  • Step 150 of process 140 dissociates the alginate and liberates the microbes (bacteria or yeast cells).
  • step 150 is accomplished by stirring the microbe/calcium alginate mixture in 20 g/L sodium citrate or potassium citrate with a pH 8.2. at room temperature for 15 minutes.
  • the solution is filtered to remove the large particulate and microbes (bacteria or yeast cells) in step 152 .
  • the filtered solution is then dialyzed, step 154 , against a sodium salt 156 , such as sodium chloride, to remove the calcium citrate, extractives, and soluble microbial inhibitors 158 .
  • the resulting dialysis of the filtered solution with an inorganic salt, such as sodium chloride regenerates sodium alginate.
  • the toxic materials are removed as waste stream 160 .
  • the sodium alginate is concentrated during dialysis and then used again to produce calcium alginate in steps 142 , 144 , and 146 as described above.
  • the sodium alginate is used to immobilize Pachysolen tannophilus in calcium alginate beads as taught in the above process.
  • the processes of the present patent document may be used in conjunction with other processes.
  • the paper-pulping process usually burns or discards the hemicellulose portion of the biomass.
  • the hemicellulose may be separated and removed from the biomass and processed into ethanol, or other biofuel.
  • the processes of the present patent document provide an efficient, cost-effective means for converting hemicellulose into ethanol, or other biofuels, in the paper-pulping, and other, industries.
  • the processes disclosed in the present patent document may also be used to ferment monosaccharides, both hexose and pentose, obtained from the saccharification of sugarcane bagasse.
  • Fermentation may occur using a number of methods.
  • the biomass hydrolysate is removed and fermented ex-situ.
  • a variety of bioreactor designs including a traditional non-stirred fermenter or stirred fermenter, may be used for the fermentation of the biomass hydrolysate using immobilized microbes.
  • the reactor may be a submerged reactor or other type of liquid reactor. In order to provide the highest yield, a submerged reactor is preferable to ferment five-carbon sugars.
  • a packed bed reactor could be utilized, or a tankage system similar to that employed for carbon-in-pulp processes in the gold mining industry could be used. In the latter, beads would be moved counter-current to the solution flow and could be easily recovered for regeneration. Thin film reactors may also work well with immobilized microbes.
  • solid/liquid contactors may be used with immobilized microbes.
  • reactors include ion exchange columns, packed bed reactors, trickle flow reactors, and rotating contactors.
  • Other reactors that may be used are fluidized-bed and upflow type reactors.
  • the microbes may be incorporated into a bioreactor using a number of different methods.
  • the matrix/microbe gel may be applied to a support structures to increase the effective surface area. These configurations may include coating paddle structures, used in stirred tank reactors, rotating contactors, and thin film reactors.
  • the microbes could also be incorporated in large three-dimensional open-cell supports for use in trickle flow reactors or fluidized-bed and upflow reactors.
  • FIG. 4 illustrates a view of one embodiment of a bioreactor for performing submerged fermentation of biomass hydrolysate using immobilized microbes.
  • Bioreactor 200 which may be referred to as a rotating disk contactor, comprises vessel 202 , input 204 , rotating stir stick 206 , outputs 208 and 210 , stators 212 , and rotors 214 .
  • vessel 202 is shown in a vertical configuration it may also be horizontal or in some other orientation.
  • Vessel 202 preferably includes a large opening.
  • vessel 202 may be made of two separable halves in order to facilitate maintenance access to the stators 212 or rotors 214 located within vessel 202 .
  • microbes immobilized in a matrix substance such as calcium alginate, are applied to the stators 212 and the rotors 214 .
  • biomass hydrolysate flows through the vessel 202 from input 204 and through outputs 208 and 210 .
  • the rotating stir stick 206 may be rotated to provide agitation to the biomass hydrolysate as it flows through the bioreactor 200 .
  • the bioreactor 200 is designed for continuous flow fermentation.
  • FIG. 5A and FIG. 5B illustrates a side and front view of another embodiment of a bioreactor for performing submerged fermentation of biomass hydrolysate using immobilized microbes.
  • Bioreactor 300 comprises motor 302 , rotating shaft 304 , media disk panels 306 , biomass hydrolysate 308 , vessel 310 , and optional air tube 312 .
  • Biomass hydrolysate 308 is added to the bioreactor 300 for fermentation.
  • Vessel 302 of bioreactor 300 is shown as only a bottom half, but vessel 302 may completely encapsulate the rotating media disks 306 .
  • microbes immobilized in a matrix substance may be applied to the media disk panels 306 .
  • Motor 302 rotates the media disks 306 through the biomass hydrolysate 308 .
  • bioreactor 300 includes an optional air tube 312 that may be used to further agitate the biomass hydrolysate 308 and increase fermentation by injecting air below the rotating media disk panels 306 .
  • FIG. 6 illustrates an upflow reactor.
  • Upflow reactor 400 contains sludge bed or sludge blanket 402 .
  • sludge bed 402 comprises immobilized microbes.
  • Sludge bed 402 may be comprised of one or more fermented microbes immobilized in any of the various medium described above.
  • sludge bed 402 may be comprised of Pachysolen tannophilus immobilized in calcium alginate beads.
  • Upflow reactor 402 further comprises inlet(s) 404 for influent.
  • Inlet(s) 404 may be a single inlet or more preferably a plurality of inlets across the bottom of the upflow reactor 400 to distribute the influent evenly underneath the sludge bed 402 .
  • Inlet(s) 404 allow the biomass hydrolysate to enter the upflow reactor from beneath the sludge bed 402 . As the biomass hydrolysate is fermented, biogas 406 rises to the surface of the reactor and is collected at the top 408 of the upflow reactor 400 . Effluent 410 is removed from the reactor and recycled through the inlet(s) 404 .
  • the upflow reactor 400 is a columnar upflow reactor with a low aspect ratio between the range of about 1:1 to 2:1 height to width.
  • Carbon dioxide gas produced by the fermentation process disrupts the packing of the beads loaded in the column and promotes a ‘self-fluidizing’ bed, similar to the effect achieved by a gas-lift type of reactor.
  • two or more ‘self-fluidizing’ bed columnar upflow reactors 400 can be run in series.
  • the beads in each reactors may contain the same or different microbes, so as to ferment different sugars in different reactor stages.
  • An increase in the number of reactors placed in series will reduce the sugar/ethanol variation within any given reactor, which in turn will promote better microbe performance.
  • Bioreactors based on immobilized microbes offer several advantages over ‘free cell’ systems.
  • One advantage is the increased feasibility to employ a continuous fermentation system. Immobilization ensures no loss of cell mass, such as occurs with batch fermentation and with continuous fermentation where the flow rate is such that the free cells are washed out of the reactor with the product. Continuous fermentation also decreases production down-time compared to batch fermentation. Continuous fermentation using microbes immobilized in beads increases the flow rate and the ethanol productivity possible with, for example, an upflow reactor. Immobilization also ensures no loss of cell mass of motile cells, where the flow rate is either high or low, where the inherent motility of the cell leads to loss of cell mass.
  • Pachysolen tannophilus was either immobilized in calcium alginate beads with about a 3 mm diameter (generated using the method describe above) or was in a free cell state.
  • Tables 3 and 4 below summarize the improvement of ethanol yield, and in glucose and xylose conversion resulting from the reactor design employed according to the present example.
  • the present example demonstrates the improvement of ethanol yield, and in glucose and xylose conversion, for calcium alginate-immobilized Pachysolen tannophilus in two different softwood hydrolysates (‘A’ and ‘B’) over free (i.e. unrestricted) Pachysolen tannophilus .
  • the hydrolysates were pH adjusted or overlimed and pH adjusted.
  • the Pachysolen tannophilus strain NRRL Y2460 was used in carrying out the experiment; however, other adapted or mutated strains of Pachysolen tannophilus may also be immobilized in calcium alginate and used in processes according to the present patent document.
  • the pine was transformed into a softwood hydrolysate by dilute acid hydrolysis.
  • the hydrolysate was either simply pH adjusted with sodium hydroxide or ‘overlimed’.
  • overliming with calcium hydroxide is commonly used to ameliorate the toxicity of hydrolysates.
  • the resulting solutions were fermented using Pachysolen tannophilus immobilized in 3 mm calcium alginate beads.
  • YPD Yeast Peptone Dextrose
  • the solution was adjusted to pH 6.0 with 8M potassium hydroxide, followed by filter sterilization.
  • Preparation of overlimed and pH adjusted hydrolysate required overliming to pH 10.0 with calcium oxide, followed by a 30 minute hold at 50° C. under stirring conditions.
  • the overlimed hydrolysate was then filtered to remove the solids. Following re-acidification to pH 6.0, the hydrolysate was filter sterilized.
  • Serum vials were aseptically prepared to obtain a final concentration of 95% hydrolysate with the following nutrient additions: 0.2% urea w/v, 0.2% yeast extract, and 0.05% potassium dihydrogen phosphate.
  • the inoculation rate for immobilized beads was 0.2 g beads per mL.
  • the free cells were inoculated at a rate of 0.3 OD 600 nm per mL. All experimental conditions were set up in triplicate serum vials. The vials were aseptically vented and incubated for 72 hours at 30° C. and 75 rpm prior to sampling for analysis.
  • Table 4 shows similar results to Table 3.
  • pH adjusted hydrolysate “B” as shown in Table 4, ‘free’ Pachysolen was unable to convert sugars to ethanol and no xylose was utilized.
  • Immobilized Pachysolen converted a majority of the sugars (57%) to ethanol.
  • ethanol yield (% theoretical) is based on glucose and xylose only and is calculated from total glucose and xylose concentrations before treatment. Other monosaccharides are not considered. All sugar utilization data is calculated using YSI results for glucose and xylose. Sugar utilization calculations do not differentiate between end products (i.e., includes ethanol, xylitol, biomass) and is calculated as follows (accounting for lost sugars after treatment like overliming, autoclaving, etc.):
  • Tables 5 and 6 illustrate the improvement in ethanol yield, and in glucose and xylose conversion, for calcium alginate-immobilized Zymomonas mobilis NREL strain 8b, Pachysolen tannophilus (NRRL Y2460), and Pichia stipitis (NRRL Y7124) in sugarcane hydrolysate over free cells of the same. Similar to the examples in tables 3 and 4, pH adjusted hydrolysate was compared against another conditioning method for both free and immobilized microbes.
  • hydrolysate used for the examples shown in tables 5-7 used hydrolysate derived from sugarcane bagasse instead of hydrolysate derived from softwood.
  • Tables 5 and 6 illustrate the benefit of immobilization on a variety of microbes including both yeasts and bacterium.
  • Table 5-7 were conducted by transforming sugarcane bagasse into a bagasse hydrolysate by dilute acid hydrolysis.
  • the hydrolysate was conditioned by either simply pH adjusting with sodium hydroxide or by treating the hydrolysate with activated carbon and the two ion exchange resins mentioned above. Namely, the bagasse hydrolysate was passed over a column containing activated carbon, over a column containing a strong acid cation exchange column, and a weak base anion exchange column.
  • the resulting solutions were further separated into three separate solutions each to be fermented by three different microbes, Zymomonas mobilis NREL strain 8b, Pachysolen tannophilus (NRRL Y2460), and Pichia stipitis (NRRL Y7124) respectively.
  • Zymomonas mobilis NREL strain 8b Zymomonas mobilis NREL strain 8b
  • Pachysolen tannophilus NRRL Y2460
  • Pichia stipitis NRRL Y7124
  • the two differently-conditioned bagasse hydrolysates contained different amounts of the inhibitors acetic acid, formic acid, 5-hydroxyfurfural (5-HMF), and furfural.
  • the measured values are reported in Table 7. These inhibitor levels are for the particular batch of sugarcane bagasse hydrolysate used in the experiments summarized above for which the results are reported in Tables 5 and 6.
  • the beads used for immobilizing the different microbes were incubated in a flask of Yeast Peptone Dextrose (YPD) broth for 22 hours at 30° C. and 75 rpm. Similarly, the free cells were cultured from a working slant into a flask of YPD broth and incubated for 24 hours at 30° C. and 175 rpm.
  • YPD Yeast Peptone Dextrose
  • Serum vials were aseptically prepared to obtain a final concentration of 95% hydrolysate with the following nutrient additions: 0.2% urea w/v, 0.2% yeast extract, and 0.05% potassium dihydrogen phosphate.
  • the inoculation rate for beads was 0.2 g beads per mL.
  • the free cells were inoculated at a rate of 0.01 g (wet weight) per mL for P. tannophilus and P. stipitis , and 0.006 g (wet weight) per mL for Z. mobilis 8b. All experimental conditions were set up in triplicate serum vials. The vials were aseptically vented and incubated for 6 days at 30° C. and 75 rpm prior to sampling for analysis.
  • Xylose utilization in the fermentations generally mirrored the extent of fermentation of glucose and xylose to ethanol.
  • Immobilized Zymomonas utilized 31% of xylose in pH adjusted hydrolysate and 75% in AC/IE conditioned hydrolysate, while the free cells utilized only 18% of the xylose in both conditions (Table 6).
  • Immobilized Pachysolen utilized 24% of xylose in pH adjusted hydrolysate and 96% in AC/IE conditioned hydrolysate, while the free cells utilized only 12% and 56% of the xylose, respectively (Table 6).
  • Immobilized Pachysolen utilized 25% of xylose in pH adjusted hydrolysate and 64% in AC/IE conditioned hydrolysate, while the free cells utilized only 6% and 50%, respectively.
  • Immobilized Pichia utilized 16% of xylose in pH adjusted hydrolysate and 67% in AC/IE conditioned hydrolysate, while the free cells utilized no xylose in pH adjusted hydrolysate, but 62% in AC/IE conditioned hydrolysate (Table 6).
  • microbe/calcium alginate beads were re-used in sequential fermentations and the microbes in the beads were metabolically ‘regenerated’ between fermentations to increase ethanol yield.
  • FIG. 7 illustrates the decreased ethanol yield in Fermentations 2 and 3 compared to Fermentation 1.
  • FIG. 7 illustrates that the regeneration of the Pachysolen /calcium alginate in culture medium restored the fermentative ability of the Pachysolen to produce ethanol.
  • FIG. 7 illustrates that immobilized microbes may be used in sequential fermentations and that the Pachysolen in the beads can be metabolically regenerated.
  • the present example employs a regeneration step after 3 or 4 consecutive uses of the immobilized microbes, it is possible to regenerate the microbes more or less often. It is expected that if a greater number of beads are used in sequential fermentations (i.e. fermenting under conditions of a saturating yeast concentration), the ethanol yields would remain at a higher level in successive fermentations before requiring metabolic regeneration.
  • the immobilization medium for example calcium alginate
  • the immobilization medium can degrade due to use. If the microbes are regenerated and re-used according to the present example, it may be necessary to recycle the immobilization medium as taught above.

Landscapes

  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Processing Of Solid Wastes (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
US12/856,566 2009-08-13 2010-08-13 Apparatus and process for fermentation of biomass hydrolysate Abandoned US20110059497A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US12/856,566 US20110059497A1 (en) 2009-08-13 2010-08-13 Apparatus and process for fermentation of biomass hydrolysate
US13/869,842 US20140080191A1 (en) 2009-08-13 2013-04-24 Apparatus and process for fermentation of biomass hydrolysate
US13/897,143 US20140127775A1 (en) 2009-08-13 2013-05-17 Apparatus and process for fermentation of biomass hydrolysate
US14/717,824 US9523103B2 (en) 2009-08-13 2015-05-20 Apparatus and process for fermentation of biomass hydrolysate

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US23382109P 2009-08-13 2009-08-13
US12/856,566 US20110059497A1 (en) 2009-08-13 2010-08-13 Apparatus and process for fermentation of biomass hydrolysate

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/869,842 Continuation US20140080191A1 (en) 2009-08-13 2013-04-24 Apparatus and process for fermentation of biomass hydrolysate

Publications (1)

Publication Number Publication Date
US20110059497A1 true US20110059497A1 (en) 2011-03-10

Family

ID=43586365

Family Applications (4)

Application Number Title Priority Date Filing Date
US12/856,566 Abandoned US20110059497A1 (en) 2009-08-13 2010-08-13 Apparatus and process for fermentation of biomass hydrolysate
US13/869,842 Abandoned US20140080191A1 (en) 2009-08-13 2013-04-24 Apparatus and process for fermentation of biomass hydrolysate
US13/897,143 Abandoned US20140127775A1 (en) 2009-08-13 2013-05-17 Apparatus and process for fermentation of biomass hydrolysate
US14/717,824 Expired - Fee Related US9523103B2 (en) 2009-08-13 2015-05-20 Apparatus and process for fermentation of biomass hydrolysate

Family Applications After (3)

Application Number Title Priority Date Filing Date
US13/869,842 Abandoned US20140080191A1 (en) 2009-08-13 2013-04-24 Apparatus and process for fermentation of biomass hydrolysate
US13/897,143 Abandoned US20140127775A1 (en) 2009-08-13 2013-05-17 Apparatus and process for fermentation of biomass hydrolysate
US14/717,824 Expired - Fee Related US9523103B2 (en) 2009-08-13 2015-05-20 Apparatus and process for fermentation of biomass hydrolysate

Country Status (16)

Country Link
US (4) US20110059497A1 (enrdf_load_stackoverflow)
EP (1) EP2464734A4 (enrdf_load_stackoverflow)
AP (1) AP2012006162A0 (enrdf_load_stackoverflow)
AR (1) AR077920A1 (enrdf_load_stackoverflow)
AU (2) AU2010282976A1 (enrdf_load_stackoverflow)
BR (1) BRPI1005180A2 (enrdf_load_stackoverflow)
CA (1) CA2770439A1 (enrdf_load_stackoverflow)
CL (1) CL2012000366A1 (enrdf_load_stackoverflow)
CO (1) CO6531410A2 (enrdf_load_stackoverflow)
DO (1) DOP2012000040A (enrdf_load_stackoverflow)
EC (1) ECSP12011728A (enrdf_load_stackoverflow)
IN (1) IN2012DN02172A (enrdf_load_stackoverflow)
MX (1) MX2012001743A (enrdf_load_stackoverflow)
PE (1) PE20130048A1 (enrdf_load_stackoverflow)
PH (1) PH12012500282A1 (enrdf_load_stackoverflow)
WO (1) WO2011019403A1 (enrdf_load_stackoverflow)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110056126A1 (en) * 2009-08-13 2011-03-10 Harvey J T Process for producing high value products from biomass
WO2013019822A1 (en) * 2011-08-01 2013-02-07 Cobalt Technologies, Inc. Removal of microbial fermentation inhibitors from cellulosic hydrolysates or other inhibitor-containing compositions
US20130312738A1 (en) * 2011-02-18 2013-11-28 Toray Industries, Inc. Method for producing sugar solution
US20140308712A1 (en) * 2010-12-09 2014-10-16 Toray Industries, Inc. Method for producing concentrated aqueous sugar solution
US20150191801A1 (en) * 2012-08-10 2015-07-09 Toray Industries, Inc. Method of producing sugar solution
US20150203927A1 (en) * 2012-08-10 2015-07-23 Toray Industries, Inc. Method of producing sugar solution
US9278314B2 (en) 2012-04-11 2016-03-08 ADA-ES, Inc. Method and system to reclaim functional sites on a sorbent contaminated by heat stable salts
US9352270B2 (en) 2011-04-11 2016-05-31 ADA-ES, Inc. Fluidized bed and method and system for gas component capture
US10682586B2 (en) 2017-10-03 2020-06-16 Quinton Downe Devices, systems and methods for capturing energy in distilling operations

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140316161A1 (en) 2011-11-23 2014-10-23 Segetis, Inc. Process to prepare levulinic acid
US9073841B2 (en) 2012-11-05 2015-07-07 Segetis, Inc. Process to prepare levulinic acid
CN109136216A (zh) * 2018-09-26 2019-01-04 太原理工大学 一种包埋有细菌的3d自组装泡沫的制作方法及其应用

Citations (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US70485A (en) * 1867-11-05 Benjamin C Tilghman Improved mode of treating vegetable substances for making paper-pulp
US4359534A (en) * 1981-04-28 1982-11-16 The United States Of America As Represented By The Secretary Of Agriculture Conversion of D-xylose to ethanol by the yeast Pachysolen tannophilus
JPS5813391A (ja) * 1981-07-13 1983-01-25 Res Assoc Petroleum Alternat Dev<Rapad> 微生物固定化膜
US4436586A (en) * 1982-01-22 1984-03-13 Kamyr, Inc. Method of producing kraft pulp using an acid prehydrolysis and pre-extraction
US4475984A (en) * 1981-08-17 1984-10-09 International Paper Co. Process for pretreating wood chips with monoperoxy sulfuric acid or its salts prior to alkaline pulping
US4477569A (en) * 1982-02-18 1984-10-16 Canadian Patents & Development Limited Pentose fermentation with selected yeast
US4612286A (en) * 1980-02-19 1986-09-16 Kamyr, Inc. Acid hydrolysis of biomass for alcohol production
US4996150A (en) * 1984-10-29 1991-02-26 Amoco Corporation Biocatalyst immobilization in a gel of anionic polysaccharide and cationic polymer
US5366558A (en) * 1979-03-23 1994-11-22 Brink David L Method of treating biomass material
US5395455A (en) * 1992-03-10 1995-03-07 Energy, Mines And Resources - Canada Process for the production of anhydrosugars from lignin and cellulose containing biomass by pyrolysis
US5589033A (en) * 1994-01-24 1996-12-31 Sunds Defibrator Pori Oy Production of prehydrolyzed pulp
US5700684A (en) * 1994-05-27 1997-12-23 Agrano Ag Process for preparing a biomass, use of the biomass so prepared, and panification ferment
US6139746A (en) * 1999-02-22 2000-10-31 Kopf; Henry B. Method and apparatus for purification of biological substances
US6645488B2 (en) * 1999-06-16 2003-11-11 Yilong Xue Microencapsulated pheochromocyte of ox adrenal medulla as medicine for curing pain
US6692578B2 (en) * 2001-02-23 2004-02-17 Battelle Memorial Institute Hydrolysis of biomass material
US20040203115A1 (en) * 2001-10-09 2004-10-14 Giardina Steven L Methods and compositions for production and purification of recombinant staphylococcal enterotoxin b (rseb)
US20050238746A1 (en) * 2002-12-31 2005-10-27 E.I. Dupont De Nemours & Company Apparatus, system and method for making hydrogel particles
US20060014260A1 (en) * 2004-05-07 2006-01-19 Zhiliang Fan Lower cellulase requirements for biomass cellulose hydrolysis and fermentation
US20060094033A1 (en) * 2004-05-21 2006-05-04 Carl Abulencia Screening methods and libraries of trace amounts of DNA from uncultivated microorganisms
US7101996B2 (en) * 2003-09-23 2006-09-05 Corn Products International, Inc. Process for preparing purified fractions of hemicellulose and cellulose-hemicellulose complexes from alkali treated fiber and products made by the process
US7198695B2 (en) * 2001-02-28 2007-04-03 Rhodia Acetow Gmbh Method for separating hemicelluloses from a biomass containing hemicelluloses and biomass and hemicelluloses obtained by said method
US20070086986A1 (en) * 2003-11-03 2007-04-19 Daniele Vigo Preparation of three-dimensional mammalian ovarian follicular cell and ovarin follicle culture systems in a biocompatible matrix
US20070172846A1 (en) * 2005-11-12 2007-07-26 Introgen Therapeutics, Inc. Methods for the Production and Purification of Adenoviral Vectors
US20070190629A1 (en) * 2002-03-19 2007-08-16 Forskarpatent I Syd Ab Metabolic engineering for improved xylose utilisation of saccharomyces cerevisiae
US7270472B2 (en) * 2005-02-23 2007-09-18 Bose Corporation Resonant shaking
US7344876B2 (en) * 2003-01-24 2008-03-18 Phage Biotechnology, Inc. Kluyveromyces strains metabolizing cellulosic and hemicellulosic materials
US7455997B2 (en) * 2002-08-05 2008-11-25 Ciba Specialty Chemicals Water Treatments Ltd Production of fermentation product
US7520958B2 (en) * 2005-05-24 2009-04-21 International Paper Company Modified kraft fibers
US7520988B2 (en) * 2000-01-05 2009-04-21 Sartorius Stedim Biotech Gmbh Cross-flow filter cassette
US7531344B2 (en) * 2006-01-30 2009-05-12 Georgia State University Research Foundation, Inc. Induction and stabilization of enzymatic activity in microorganisms
US20090155238A1 (en) * 2006-02-14 2009-06-18 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
US20090181433A1 (en) * 2002-02-08 2009-07-16 Genencor International, Inc. Methods for producing end-products from carbon substrates
US20090311765A1 (en) * 2005-05-10 2009-12-17 Tim Maguire Alginate poly-L-Lysine encapsulation as a technology for controlled differentiation of embryonic stem cells
US7666637B2 (en) * 2006-09-05 2010-02-23 Xuan Nghinh Nguyen Integrated process for separation of lignocellulosic components to fermentable sugars for production of ethanol and chemicals
US7785379B2 (en) * 2005-05-16 2010-08-31 Evergreen Biofuels Inc. Agricultural fibre fuel pellets
US20110056126A1 (en) * 2009-08-13 2011-03-10 Harvey J T Process for producing high value products from biomass
US20110171709A1 (en) * 2007-11-01 2011-07-14 Mascoma Corporation Product Recovery From Fermentation of Lignocellulosic Biomass
US8227219B2 (en) * 2008-07-29 2012-07-24 Tommy Mack Davis Method and apparatus for bio-fuel seeding

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2405861A (en) 1941-09-19 1946-08-13 Albright & Wilson Production of alginate solutions
AT367473B (de) * 1980-04-10 1982-07-12 Kanzler Walter Verfahren zur gewinnung von furfurol, ameisensaeure, essigsaeure aus sauren hydrolysaten von pflanzen
JP3769734B2 (ja) * 2003-04-07 2006-04-26 アサヒビール株式会社 砂糖及び有用物質を製造する方法
CN101255446B (zh) * 2007-12-18 2010-09-29 大连理工大学 一种利用固定化酵母细胞与渗透蒸发膜耦合连续发酵葡萄糖木糖的方法

Patent Citations (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US70485A (en) * 1867-11-05 Benjamin C Tilghman Improved mode of treating vegetable substances for making paper-pulp
US5366558A (en) * 1979-03-23 1994-11-22 Brink David L Method of treating biomass material
US4612286A (en) * 1980-02-19 1986-09-16 Kamyr, Inc. Acid hydrolysis of biomass for alcohol production
US4359534A (en) * 1981-04-28 1982-11-16 The United States Of America As Represented By The Secretary Of Agriculture Conversion of D-xylose to ethanol by the yeast Pachysolen tannophilus
JPS5813391A (ja) * 1981-07-13 1983-01-25 Res Assoc Petroleum Alternat Dev<Rapad> 微生物固定化膜
US4475984A (en) * 1981-08-17 1984-10-09 International Paper Co. Process for pretreating wood chips with monoperoxy sulfuric acid or its salts prior to alkaline pulping
US4436586A (en) * 1982-01-22 1984-03-13 Kamyr, Inc. Method of producing kraft pulp using an acid prehydrolysis and pre-extraction
US4477569A (en) * 1982-02-18 1984-10-16 Canadian Patents & Development Limited Pentose fermentation with selected yeast
US4996150A (en) * 1984-10-29 1991-02-26 Amoco Corporation Biocatalyst immobilization in a gel of anionic polysaccharide and cationic polymer
US5395455A (en) * 1992-03-10 1995-03-07 Energy, Mines And Resources - Canada Process for the production of anhydrosugars from lignin and cellulose containing biomass by pyrolysis
US5589033A (en) * 1994-01-24 1996-12-31 Sunds Defibrator Pori Oy Production of prehydrolyzed pulp
US5700684A (en) * 1994-05-27 1997-12-23 Agrano Ag Process for preparing a biomass, use of the biomass so prepared, and panification ferment
US6139746A (en) * 1999-02-22 2000-10-31 Kopf; Henry B. Method and apparatus for purification of biological substances
US6645488B2 (en) * 1999-06-16 2003-11-11 Yilong Xue Microencapsulated pheochromocyte of ox adrenal medulla as medicine for curing pain
US7520988B2 (en) * 2000-01-05 2009-04-21 Sartorius Stedim Biotech Gmbh Cross-flow filter cassette
US6692578B2 (en) * 2001-02-23 2004-02-17 Battelle Memorial Institute Hydrolysis of biomass material
US7198695B2 (en) * 2001-02-28 2007-04-03 Rhodia Acetow Gmbh Method for separating hemicelluloses from a biomass containing hemicelluloses and biomass and hemicelluloses obtained by said method
US20040203115A1 (en) * 2001-10-09 2004-10-14 Giardina Steven L Methods and compositions for production and purification of recombinant staphylococcal enterotoxin b (rseb)
US20090181433A1 (en) * 2002-02-08 2009-07-16 Genencor International, Inc. Methods for producing end-products from carbon substrates
US20070190629A1 (en) * 2002-03-19 2007-08-16 Forskarpatent I Syd Ab Metabolic engineering for improved xylose utilisation of saccharomyces cerevisiae
US7455997B2 (en) * 2002-08-05 2008-11-25 Ciba Specialty Chemicals Water Treatments Ltd Production of fermentation product
US20050238746A1 (en) * 2002-12-31 2005-10-27 E.I. Dupont De Nemours & Company Apparatus, system and method for making hydrogel particles
US7344876B2 (en) * 2003-01-24 2008-03-18 Phage Biotechnology, Inc. Kluyveromyces strains metabolizing cellulosic and hemicellulosic materials
US7101996B2 (en) * 2003-09-23 2006-09-05 Corn Products International, Inc. Process for preparing purified fractions of hemicellulose and cellulose-hemicellulose complexes from alkali treated fiber and products made by the process
US20070086986A1 (en) * 2003-11-03 2007-04-19 Daniele Vigo Preparation of three-dimensional mammalian ovarian follicular cell and ovarin follicle culture systems in a biocompatible matrix
US20060014260A1 (en) * 2004-05-07 2006-01-19 Zhiliang Fan Lower cellulase requirements for biomass cellulose hydrolysis and fermentation
US20060094033A1 (en) * 2004-05-21 2006-05-04 Carl Abulencia Screening methods and libraries of trace amounts of DNA from uncultivated microorganisms
US7270472B2 (en) * 2005-02-23 2007-09-18 Bose Corporation Resonant shaking
US20090311765A1 (en) * 2005-05-10 2009-12-17 Tim Maguire Alginate poly-L-Lysine encapsulation as a technology for controlled differentiation of embryonic stem cells
US7785379B2 (en) * 2005-05-16 2010-08-31 Evergreen Biofuels Inc. Agricultural fibre fuel pellets
US7520958B2 (en) * 2005-05-24 2009-04-21 International Paper Company Modified kraft fibers
US20070172846A1 (en) * 2005-11-12 2007-07-26 Introgen Therapeutics, Inc. Methods for the Production and Purification of Adenoviral Vectors
US7531344B2 (en) * 2006-01-30 2009-05-12 Georgia State University Research Foundation, Inc. Induction and stabilization of enzymatic activity in microorganisms
US20090155238A1 (en) * 2006-02-14 2009-06-18 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
US7666637B2 (en) * 2006-09-05 2010-02-23 Xuan Nghinh Nguyen Integrated process for separation of lignocellulosic components to fermentable sugars for production of ethanol and chemicals
US20110171709A1 (en) * 2007-11-01 2011-07-14 Mascoma Corporation Product Recovery From Fermentation of Lignocellulosic Biomass
US8227219B2 (en) * 2008-07-29 2012-07-24 Tommy Mack Davis Method and apparatus for bio-fuel seeding
US20110056126A1 (en) * 2009-08-13 2011-03-10 Harvey J T Process for producing high value products from biomass

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
Abbi et al "Bioconversion of Pentose Sugars to Ethanol by Free and Immobilized Cells of Candida shehatae (NCL-3501): Fermentation Behavoiur. Process Biochemistry Vol 31 No 6 1996, 555-560 *
DERWENT abstract of Noguchi, JP 58-013391 *
Gottschalk "The effect of Temperature of the Fermentation of d-Mannose by Yeast", Biochemical Journal 1947, Vol 41(2) 276-280 *
Grizzi "Calcium Alginate Dressing -- I. Physico-chemical characterization and effect of sterilization" Journal of Biomaterials Science. Polymer Edition. Vol 9 Issue 2 1998, 189-204 *
Li "Fermentation of corn stalk hydrosylate by co-immobilized multi-microorganisms" Beijing Huagong Daxue Xuebao-Journal of Beijing University of Chemical Technology, 2008 Vol 35, 74-77 *
Liu "Draft Genome Sequence of the Yeast Pachysolen Tannophilus CBS 4044/NRRL Y-2460" Eukaryotic Cell 2012 11(6) 827 *
Nowak "Comparison of Polish Industrial Distillery Yeast with Ethanol Producing Zymomonas Mobilis" Electronic Journal of Polish Agricultural Universities, 2001, Vold 4 Issue 2, online pages 1-12 *
Ravinder "Fermentation of Wheat Straw Hydolyzate to Ethanol by Pachysolen tannophilus: A comparision of Batch and Continuous Culture Systems" Biological Wastes, 30, 1989 301-308 *
Schwartz "Desalting and Buffer Exchange by Dialysis, Gel Filtration, or Diafiltration" Pall Corporation, available online at www.pall.com/main/Laboratory/Literature-Library-Details.page?id=42217, capture of google search indicating the reference has been available since 2004 included *
Slininger "Continuous Conversion of D-Xylose to Ethanol by Immobilized Pachysolen tannophilus" Biotechnology and Bioengineering, Vol 24, 1982, 2241-2251 *
Slininger "Continuous Fermentation of Feed Streams Containing D-Glucose and D-Xylose in a Two-Stage Process Utilizing Immobilized Saccharomyces cerevisiae and Pachysolen tannophilus" Biotechnology and Bioengineering Vol 32 1988 1104-1112 *
Smidsrod "Alginate as immobilization matrix for cells" Trends in Biotechnology, Vol 8, 1990, 71-78 *
Uemura "Effect of Calcium Alginate coating on the performance of Immobilized Yeast cells in calcium alginate beads" Chemical Engineering Communications, Vol 177 issue 1, 2000, 1-14 *
Wooley "A Nine-Zone Simulating Moving Bed for the Recovery of Glucose and Xylose from Bimass Hydrolyzate" Industrial and Engineering Chemistry Research, 1998, 37, 3699-3709 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110056126A1 (en) * 2009-08-13 2011-03-10 Harvey J T Process for producing high value products from biomass
US20140308712A1 (en) * 2010-12-09 2014-10-16 Toray Industries, Inc. Method for producing concentrated aqueous sugar solution
US20130312738A1 (en) * 2011-02-18 2013-11-28 Toray Industries, Inc. Method for producing sugar solution
US9598740B2 (en) * 2011-02-18 2017-03-21 Toray Industries, Inc. Method for producing sugar solution
US9352270B2 (en) 2011-04-11 2016-05-31 ADA-ES, Inc. Fluidized bed and method and system for gas component capture
WO2013019822A1 (en) * 2011-08-01 2013-02-07 Cobalt Technologies, Inc. Removal of microbial fermentation inhibitors from cellulosic hydrolysates or other inhibitor-containing compositions
US9278314B2 (en) 2012-04-11 2016-03-08 ADA-ES, Inc. Method and system to reclaim functional sites on a sorbent contaminated by heat stable salts
US20150191801A1 (en) * 2012-08-10 2015-07-09 Toray Industries, Inc. Method of producing sugar solution
US20150203927A1 (en) * 2012-08-10 2015-07-23 Toray Industries, Inc. Method of producing sugar solution
US9863011B2 (en) * 2012-08-10 2018-01-09 Toray Industries, Inc. Method of producing sugar solution
US9951393B2 (en) * 2012-08-10 2018-04-24 Toray Industries, Inc. Method of producing sugar solution
US10682586B2 (en) 2017-10-03 2020-06-16 Quinton Downe Devices, systems and methods for capturing energy in distilling operations

Also Published As

Publication number Publication date
EP2464734A4 (en) 2014-01-08
BRPI1005180A2 (pt) 2015-09-01
US20150252387A1 (en) 2015-09-10
WO2011019403A1 (en) 2011-02-17
CL2012000366A1 (es) 2012-10-19
US20140127775A1 (en) 2014-05-08
CO6531410A2 (es) 2012-09-28
PH12012500282A1 (en) 2012-10-22
EP2464734A1 (en) 2012-06-20
CA2770439A1 (en) 2011-02-17
PE20130048A1 (es) 2013-02-04
AU2016201973A1 (en) 2016-04-21
IN2012DN02172A (enrdf_load_stackoverflow) 2015-08-21
ECSP12011728A (es) 2012-04-30
AP2012006162A0 (en) 2012-04-30
AU2010282976A1 (en) 2012-04-05
MX2012001743A (es) 2012-04-11
AR077920A1 (es) 2011-10-05
US20140080191A1 (en) 2014-03-20
DOP2012000040A (es) 2012-06-30
US9523103B2 (en) 2016-12-20

Similar Documents

Publication Publication Date Title
US9523103B2 (en) Apparatus and process for fermentation of biomass hydrolysate
CN102753674B (zh) 由生物量生产高价值产品的方法
Xavier et al. Second-generation bioethanol from eucalypt sulphite spent liquor
CA2722560C (en) Processing biomass
EP2794902B1 (en) Processing biomass for use in fuel cells
US9809867B2 (en) Carbon purification of concentrated sugar streams derived from pretreated biomass
US20160348134A1 (en) Integrated Biorefinery
WO2015005589A1 (ko) 목질계 바이오매스로부터 당, 바이오에탄올 또는 미생물 대사산물을 제조하는 방법
Buyukoztekin et al. Enzymatic hydrolysis of organosolv-pretreated corncob and succinic acid production by Actinobacillus succinogenes
Chong et al. Third-generation L-lactic acid biorefinery approaches: exploring the viability of macroalgae detritus
KR101449552B1 (ko) 목질계 바이오매스로부터 발효당을 제조하는 방법
US20120301939A1 (en) Methods of treating biomass
KR101504197B1 (ko) 목질계 바이오매스로부터 바이오에탄올을 제조하는 방법
Dehkhoda Concentrating lignocellulosic hydrolysate by evaporation and its fermentation by repeated fedbatch using flocculating Saccharomyces cerevisiae
Chen et al. Enhanced repeated-batch bioethanol fermentation of red seaweeds hydrolysates using microtube array membrane-encapsulated yeast
Han et al. Ultrasound-assisted regeneration of ion exchange resins for efficient detoxification of lignocellulosic hydrolysate in biorefinery
KR101447534B1 (ko) 목질계 바이오매스로부터 초산의 독성이 경감된 발효당의 제조방법
Kumneadklang Development of bioethanol production process from oil palm trunk with ethanol membrane separation.
Chen Applications of lignocellulose biotechnology in the chemical industry
KR101536132B1 (ko) 미생물 억제물질이 제거된 목질계 바이오매스 원료 발효당의 제조 방법
Singh et al. Development of Technology for Production of Second Generation Biofuel Ethanol from Bagasse
AU2012258807A1 (en) Methods of treating biomass
BR102014023395B1 (pt) Sistema catalítico e processo de obtenção de bioetanol 2g a partir de xilana/oligômeros de xilose

Legal Events

Date Code Title Description
AS Assignment

Owner name: GEOSYNFUELS, LLC, COLORADO

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BECKLER ANDERSON, LISA;EVANS, JOHN H., IV;SINGER, CHRISTINE A.;REEL/FRAME:025400/0107

Effective date: 20101122

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION