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WO2009108748A2 - Procédés pour la production de 1,3-propanediol - Google Patents

Procédés pour la production de 1,3-propanediol Download PDF

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Publication number
WO2009108748A2
WO2009108748A2 PCT/US2009/035229 US2009035229W WO2009108748A2 WO 2009108748 A2 WO2009108748 A2 WO 2009108748A2 US 2009035229 W US2009035229 W US 2009035229W WO 2009108748 A2 WO2009108748 A2 WO 2009108748A2
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Prior art keywords
glycerol
fermentation
acid
producing
fermentation product
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WO2009108748A3 (fr
Inventor
Jiyin Liu
Chee Leong Soong
Yongming Zhu
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Novozymes AS
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Novozymes AS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric

Definitions

  • This invention comprises a process for producing 1 ,3- ⁇ ropanedio!
  • 1 3-Propanedio! is a substance of great industrial importance, used mainly in the industrial production of detergents and polymers
  • 1 ,3-prapaned ⁇ l is used in liquid detergents as a stabilizing agent for amylases, lipases, and proteases as well as "protective softener” in the hquid detergents for dishwashing by hand
  • 1 ,3-propaned ⁇ ol is used in the industrial production of polymers, more particularly as a monomer used to synthesize polyesters, polyethers or poiyurethanes
  • l ,3-propaned ⁇ ol is mainly produced by chemical synthesis involving hydration in an acid medium of acrolein to 3-hydroxyprop ⁇ onaldehyde which is then reduced to 1 ,3-prapaned ⁇ l by catalytic hydrogenation
  • Another synthetic route consists of hydrocarbonylation of ethylene oxide by carbon monoxide and hydrogen under high pressure in the presence of catalysts and solvents 1 ,3-Propaned ⁇ o! also can be produced from the fermentation of glycerol
  • the present invention relates to a process of producing 1 3-propaned ⁇ ol, comprising fermenting glycerol in a fermentation medium with one or more microorganisms which convert glycerol to 1 ,3-propaned ⁇ l. wherein the glycerol is from whole stillage thin stillage and/or syrup or from a glycerol stream produced by a process for producing a fermentation product from lignocelluiosic material
  • the present invention also relates to a process of producing a fermentation product and 1 ,3-propaned ⁇ ol, comprising the steps of (a) converting a starch material to dext ⁇ ns,
  • the present invention also relates to a process of producing a fermentation product and 1 ,3-pro ⁇ aned ⁇ oi, comprising the steps of (a) converting a lignocelluiosic materia! to a saccharified materia!,
  • the present invention also relates to a process of producing biodiese! and 1 ,3-pro ⁇ aned ⁇ ol, comprising the steps of
  • glycosyrene kinase means a polypeptide responsible for an enzyme that catalyzes the conversion of glycerol and ATP to giyceroi-3-phosphate and ADP ATP may be replaced by physiological substitutes (e g , phosphoenolpyruvate)
  • Glycerol kinase is encoded for example, by GUT1 (GenBank U11583x19) and glpK (GenBank L 19201 ) (see WO
  • glycerol dehydrogenase means a polypeptide responsible for an enzyme that catalyzes the conversion of glycerol to dihydroxyacetone (E C 1 1 1 6) or glycerol to glyceraldehyde (E C 1 1 1 72) Glycerol dehydrogenase may be dependent upon NADH (E C 1 1 1 6), NADPH (E C 1 1 1 72), or other cofactors (e g , E C 1 1 99 22)
  • An NADH- dependent glycerol dehydrogenase is encoded, for example, by gidA (GenBank U00006) (see
  • dehydratase enzyme or “dehydratase” means any enzyme that catalyzes the conversion of glycerol to 3-hydroxy ⁇ rop ⁇ onaldehyde
  • Dehydratase enzymes include glycerol dehydratases (E C 4 2 1 30) and dio! dehydratases (E C 4 2 1 28) having preferred substrates of glycerol and 1 ,2-propaned ⁇ oi, respectively
  • Genes encoding dehydratase enzymes can be obtained from Qtrohacier freundu, Clostridium pasteu ⁇ anum, Klebsiella oxytoca, Klebsiella pneumoniae, and Salmonella typhimunum
  • dehydratase reactivation factor refers to those proteins responsible for reactivating the dehydratase activity
  • dehydratase reactivating activity refers to the phenomenon of converting a dehydratase not capable of catalysis of a substrate to one capable of catalysis of a substrate or to the phenomenon of inhibiting the inactivation of a dehydratase or the phenomenon of extending the useful half-life of the dehydratase enzyme in vivo
  • Two proteins have been identified as being involved as the dehydratase reactivation factor (see WO 98/21341 (U S Patent No 6,013 494 ⁇ and references therein, which are
  • the terms "1 ,3-propaned ⁇ oi oxidoreductase”, " I 3-propanedioi dehydrogenase” or “DhaT” mean the poly ⁇ e ⁇ t ⁇ de(s) responsible for an enzyme that is capable of catalyzing the interconversion of 3-hydroxyprop ⁇ onaldehyde and 1 ,3-propaned ⁇ ol
  • Genes encoding a 1 ,3-propaned ⁇ ol oxidoreductase include dhaT from Ciirobacter freundu, Clostridium pasteu ⁇ anum and Klebsiella pneumoniae Each of these genes encodes a polypeptide belonging to the family of type IN alcohol dehydrogenases, exhibits a conserved iron-binding motif and has a preference for the NAD + ZNADH linked intorconvorsion of 3-hydroxy ⁇ rop ⁇ onaldehyde and 1 3-propaned ⁇ o!
  • dha reguion means a set of associated genes or open reading frames encoding various biological activities, including but not limited to a dehydratase a reactivation activity and a 1 ,3-pro ⁇ anedsol oxidoreductase Typically a dha reguion comprises the open reading frames dhaR, orfY, dhaT, orfX, orfW dhaB1 , dhaB2, dhaB3, and orfZ as described herein
  • host cell or "host microorgan
  • foreign gene means genetic materia! native to one organism that has been placed within a host microorganism by various means
  • the gene of interest may be a naturally occurring gene, a mutated gene, or a synthetic gene
  • transformation and “transfection” mean the introduction of new genes in a cell after the incorporation of nucleic acid
  • the genes may be integrated into chromosomal DNA or introduced as extrachromosoma! replicating sequences
  • transformation refers to the product of a transformation
  • isolated refers to a protein or DNA sequence that ⁇ s removed from at least one component with which it is naturally associated
  • isolated nucleic acid molecule means a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases
  • isolated nucleic acsd molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA
  • the term "whole stallage” means the side-product coming from the distillation of fermented mash (the main product is the fermentation product, e g , ethanol)
  • thin stiliage means the supernatant of the centrifugation of the whole stillage Typically, the thin stiliage contains 4-6% DS (mainly starch and proteins) and has a temperature of about 60-90 13 C Thin stiliage is viscous and difficult to handle
  • syrup means the syrup of suspended solids which are concentrated after the removal of water in an evaporation process of the thin stiliage stream, e g after the backset is sent to the head of the plant Typically, the syrup contains a solids content of 20 to 50 percent solids by weight
  • the present invention relates to a process of producing 1 3-propaned ⁇ ol, comprising fermenting glycerol in a fermentation medium with one or more microorganisms which convert glycerol to 1 3-propaned ⁇ ol, wherein the glycerol is from whole stiliage, thin stallage and/or syrup or from a glycerol stream produced by a process for producing a fermentation product from lignocellul ⁇ sic materia!.
  • microorganisms for use in the process can be any microorganism which converts glycerol to 1 ,3-propanedioi. Glycerol is converted to 1 ,3-propanediol via the intermediate 3-hydroxypropionaidehyde.
  • the conversion of glycerol to 3-hydroxy ⁇ ropionaldehyde is catalyzed by a dehydratase, which can be a glycerol dehydratase (E.G. 4.2.1.30), dioi dehydratase (E. C. 4.2.1.28) or any other enzyme able to catalyze this transformation.
  • a dehydratase which can be a glycerol dehydratase (E.G. 4.2.1.30), dioi dehydratase (E. C. 4.2.1.28) or any other enzyme able to catalyze this transformation.
  • Glycerol dehydratase is encoded by the dha regulon.
  • 1 ,3-Propanediol is produced from 3-hydroxypropionaidehyde by a NAD T - (or NADP " ) linked host enzyme.
  • This reaction can be catalyzed by a 1 ,3-propanediol dehydrogenase (E.G. 1.1.1.202), another alcohol dehydrogenase, 1 ,3-propanedioi oxidoreduciase, or 3-hydroxy ⁇ ropionaldehyde reductase.
  • Suitable microorganisms may be either prokaryotic or eukaryotic. Furthermore, the microorganism may be a wild-type strain or a recombinant strain.
  • Microorganisms which convert glycerol to 1 ,3-propanedioi include strains of Aerohacter, Aspergillus, Bacillus, Candida, Gtrobacter, Clostridium, Debaryomyces, Enterobacter, Escherichia, Hansenula, llyobacter, Klebsiella, Kluyveromyces, Lactobacillus, Methylobacter, Mucor, Peiobacter, Pichia, Pseudomonas, Saccharomyces, Salmonella, Schizosacchammyces, Streptomyces, Torulopsis, and Zygosaccharomyces.
  • the microorganism is a strain of Aerobacter, Escherichia biattae. Escherichia coii, Citrobacter, or Klebsiella.
  • the microorganism may further comprise one or more mutations which block alternate pathways for intermediates of the 1 ,3-propanediol production pathway.
  • mutations which block alternate pathways for intermediates of the 1 ,3-propanediol production pathway.
  • the elimination of glycerol kinase prevents glycerol from being converted to glycerol 3-phos ⁇ hate at the expense of ATP.
  • the elimination of glycerol dehydrogenase prevents glycerol from being converted to dihydroxyacetone.
  • Mutations can be directed toward a structural gene so as to impair or improve the activity of an enzyme or can be directed toward a regulatory gene, including promoter regions and ribosome binding sites, so as to modulate the expression level of an enzyme.
  • the microorganism can be a wild-type strain or a mutant strain.
  • the microorganism may a recombinant host cell which is transformed with a nucleic acid construct comprising one or more polynucleotides encoding one or more enzymes involved in the 1 ,3-propaned ⁇ ol pathway (e g , a dehydratase and/or a 1 ,3-propaned ⁇ ol dehydrogenase) operabiy linked to one or more (several) control sequences that direct the expression of the coding sequence(s) in the host cell under conditions compatible with the control sequences
  • a nucleic acid construct comprising one or more polynucleotides encoding one or more enzymes involved in the 1 ,3-propaned ⁇ ol pathway (e g , a dehydratase and/or a 1 ,3-propaned ⁇ ol dehydrogenase) operabiy linked to one or more (s
  • the polynucleotide ⁇ may be manipulated in a variety of ways to provide for expression of the p ⁇ iypeptsdefs) Manipulation of the polynucleotides' sequencefs) prior to its insertion into a vector may be desirable or necessary depending on the expression vector
  • the techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art
  • the control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide
  • the promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide
  • the promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hyb ⁇ d promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either native or foreign to the host cell
  • suitable promoters for directing the transcription of the nucleic acid constructs are the promoters obtained from the E coli lac operon, Streptomyces coelicolor agarase gene ⁇ dagA), Bacillus subtihs ievansucrase gene (sacB) Bacillus hcheniformis aipha-amyiase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM) Bacillus amyloiiquefaciens aipha-amylase gene ⁇ anv/Q), Bacillus hcheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et a/ 1978 Proceedings of the National Academy of Sciences USA 75 3727-3731 ), as well as the tec promoter
  • useful promoters are obtained from the genes for Saccharomyces cerevisiae enoiase (ENO-1 ) Saccharomyces cerevisiae galactokinase (GAL1 ), Sacchaiomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phos ⁇ hate dehydrogenase (ADH 1 , ADH2/GAP), Saccharomyces vievsssae t ⁇ ose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1 ), and Saccharomyces cerevisiae 3- ⁇ hosphogiycerate kinase
  • ENO-1 Saccharomyces cerevisiae enoiase
  • GAL1 Saccharomyces cerevisiae galactokinase
  • ADH 1 , ADH2/GAP
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription
  • the terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide Any terminator that is functional in the host cell of choice may be used
  • Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger giucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger aipha-giucosidase, and Fusanum oxysporum trypsin-like protease
  • Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enoiase, Saccharomyces cerevissae cytochrome C (CYC1 ) and Saccharomyces cerevsssae glyceraldehyde-3-phos ⁇ hate dehydrogenase
  • Other useful terminators for yeast host cells are described by Romanos et al supra
  • the control sequence may also be a suitable leader sequence a nontranslaied region of an mRNA that is important for translation by the host cell
  • the leader sequence is operabiy linked to the 5' terminus of the nucleotide sequence encoding the polypeptide Any leader sequence that is functional in the host ceil of choice may be used
  • Preferred leaders for filamentous fungal host ceils are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase Suitable leaders for yeast host ceils are obtained from the genes for Saccharomyces cerevisiae enoiase (ENO- 1 ), Saccharomyces cerevisiae 3-phos ⁇ hogiycerate kinase, Sacchatomyces cerevisiae alpha-factor, and Saccharomyces cetevisiae alcohol dehydrogena8e/giycera!debyde-3-phospbate dehydrogenase (ADH2/GAP)
  • the control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3 ' terminus of the nucleotide sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenossne residues to transcribed mRNA Any
  • Preferred polyadenylation sequences for filamentous fungal host ceils are obtained from the genes for Aspergillus oryzae TAKA amylase Aspergillus niger giucoamyiase. Aspergillus nidulans anthraniiate synthase Fusanum oxysporum trypsin-iike protease, and Aspergillus mger aipha-giueossdase
  • control sequence may also be a signal peptide coding sequence that codes for an amino acid sequence linked to the ammo terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway
  • the 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the secreted polypeptide Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence
  • the foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence Alternatively the foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide However, any signal peptide coding sequence that directs the expressed polypeptide
  • Effective signal peptide coding sequences Tor bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 1 1837 maltogeme amylase, Bacillus stearothetmophilus alpha-amylase, Bacillus bcheniformis subtilism, Bacillus licheniformis beta-lactamase Bacillus stearothermophilus neutral proteases (nptT nprS, nprM), and Bacillus subtnis prsA Further signal peptides are described by Simonen and Paiva, 1993, Microbiological Reviews 57 109- 137
  • Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger giucoamyiase, Rhizomucot miehei aspartic proteinase, Humicola insolens celiulase, Humicoia insolens endogiucanase V, and Humicola lanuginosa lipase
  • Usefui signal peptides for yeast host cells are obtained from the genes for Saccharornyces cerevtstae alpha-factor and Saccharoniyces cerevisiae snvertase Other usefui signal peptide coding sequences are described by Romanos et a/ supra
  • the control sequence may also be a propeptide coding sequence that codes for an amsno acid sequence positioned at the amino terminus of a polypeptide
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases)
  • a propeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide
  • the propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (ap?E), Bacillus subtilis neutral protease (nprT), Saccharornyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836)
  • regulatory- systems are those that cause the expression of the gene to be turned on or off in response to a chemscal or physical stimulus, including the presence of a regulatory compound Regulatory systems in prokaryotsc systems include the lac tac, and trp operator systems In yeast, the ADH2 system or GAL1 system may be used In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niget giucoamyiase promoter, and Aspergillus oryzae giucoamyiase promoter may be used as regulatory sequences Other examples of regulatory sequences are those
  • the host ceil also may be transformed with a recombinant expression vector comprising a polynucleotide encoding one or more enzymes involved in the 1 ,3-propaned ⁇ ol pathway, a promoter and transcriptional and transiationa!
  • stop signals The various nucleic acids and control sequences described herein may be joined together to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites Alternatively, the polynucleotide sequence may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression
  • the recombinant expression vector may be any vector (e g a plasmid or vsrus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleotide sequence
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced
  • the vectors may be linear or closed circular plasmids
  • the vector may be an autonomously replicating vector ; e , a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e g , a plasrnid, an extrachromosomal element, a mmichromosome, or an artificial chromosome
  • the vector may contain any means for assuring self-replication
  • the vector may be one that, when introduced into the host cell is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated
  • a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used
  • the vectors preferably contain one or more (several) selectable markers that permit easy selection of transformed, transacted, transduced, or the like cells
  • a selectable marker is a gene the product of which provides for biocide or viral resistance resistance to heavy metals, prototrophy to auxotrophs, and the like
  • bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis or markers that confer antibiotic resistance such as ampicsiliii, kanamycm, chloramphenicol or tetracycline resistance
  • Suitable markers for yeast host cells are ADE2, HIS3, LEU2 LYS2, MET3, TRP1 , and URA3
  • Selectable markers for use in a filamentous fungal host cell include, but are not limited to, arndS (acetamidase), argB ⁇ ornithine carbamoyitransferase), bar (phosphinoihncin aceiyitransferase), hph (hygromycin phosphotransferase), maD (nitrate reductase), pyrG (orotidine-S'-pnosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthramlate syntha
  • the vectors preferably contain an eiement(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome
  • the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or nonhomologous recombination
  • the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise iocat ⁇ on(s) in the chromosome(s)
  • the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10 000 base pairs, preferably 400 to 10 000 base pairs and most preferably 800 to 10,000 base pairs, which have a high degree of identity to the corresponding target sequence to enhance the probability of homologous recombination
  • the integrationai elements may be any sequence that is homologous with the target sequence in the genome of the host cell
  • the integrational elements may be non-encoding or encoding nucleotide sequences
  • the vector may be non-encoding or encoding nucleotide sequences
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question
  • the origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell
  • the term "origin of replication' or "'piasmid replicator' is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo
  • bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E coll, and pUBHO, pE194, ⁇ TA1060, and pAM ⁇ i permitting replication in Bacillus
  • origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1 ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6
  • AMA 1 and ANSI examples of origins of replication useful in a filamentous fungal cell are AMA 1 and ANSI (Gems et a/ 1991 , Gene 98 61-67, Cullen et a/ , 1987, Nucleic Acids Research 15 9163-9175, WO 00/24883 ⁇ isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883
  • More than one copy of a polynucleotide may be inserted into a host cell to increase production of the gene product
  • An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide can be selected for by cultivating the cells in the presence of the appropriate selectable agent
  • cassettes e g containing the genes encoding dehydratase, and dehydratase reactivation factor into the host cell may be accomplished by known procedures such as by transformation (e g using calcium- permeabilized cells, electroporatson), or by transfection using a recombinant phage virus
  • the fermentation medium for fermenting glycerol preferably contains suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for 1 ,3-propaned ⁇ ol production
  • the media comprises CoHI) salts and/or vitamin B 1? or precursors thereof
  • Adenosyi-cobaiamin (coenzyme B 1? ) is an essential cofactor for dehydratase activity Synthesis of coenzyme B- is found in prokaryotes, some of which are able to synthesize the compound de novo, for example Qirohacter species Clostridium species, Escherichia hlattae, and Klebsiella species, while others can perform partial reactions E cols, for example, cannot fabricate the corrin ring structure, but is able to catalyze the conversion of cobinamide to cor ⁇ noid and can introduce the 5'-deoxyadenosy! group
  • a coenzyme B 1? precursor such as vitamin B 12 , needs to be provided in E co// fermentations
  • Vitamin B 12 additions to E coli fermentations may be added continuously, at a constant rate or staged as to coincide with the generation of cell mass or may be added in single or multiple bolus additions
  • Preferred ratios of vitamin B 1 ⁇ (mg) fed to cell mass (OD550) are from Q 08 io 0 80
  • Most preferred ratios of vitamin B 12 (mg) fed to cell mass (OD550) are from 0 12 to 0 48
  • microorganisms capable of de novo B 12 biosynthesis may be suitable production cells and the addition of B 12 to these microorganisms will be unnecessary
  • Preferred media are common commercially prepared media such as Luna Bertani (LB) broth, Sabo ⁇ raud Dextrose (SD) broth or Yeast medium (YM) broth
  • LB Large Bertani
  • SD Sabo ⁇ raud Dextrose
  • YM Yeast medium
  • agents known to modulate catabolite repression directly or indirectly e g , cyclic adenosine 2' 3'- monophosphate
  • agents known to modulate enzymatic activities e g , methyl viologen
  • agents known to modulate enzymatic activities e g , methyl viologen
  • Fermentation is earned out at a pH of 5 to 9, e g , 6 to 8 Reactions may be performed under aerobic or anaerobic conditions where aerobic, anoxic, or anaerobic conditions are preferred based on the requirements of the microorganism
  • the process of the present invention may be practiced using batch fed-batch or continuous processes and any known mode of fermentation is suitable
  • 1 ,3-propaned ⁇ ol can be obtained by subjecting the reaction mixture to extraction with an organic solvent distillation, and column chromatography (U S Patent No 5,356 812)
  • a particularly good organic solvent for this process is cydohexane (U S Patent No 5,008,473)
  • the present invention also relates to a process of producing a fermentation product and 1 ,3-propaned ⁇ ol, comprising the steps of (a) converting a starch material to dextrins;
  • 1 ,3-propanediol is produced from glycerol contained in whole stiiiage.
  • 1 ,3-propanedioi is produced from glycerol contained in thin stiiiage in another embodiment
  • 1 ,3-propanedio! is produced from glycerol contained in syrup in another embodiment, the whole stiiiage and thin stiiiage are blended, and
  • 1 ,3-pro ⁇ anedioi is produced therefrom.
  • the whole stiiiage and syrup are blended, and 1 ,3-pro ⁇ anedioi is produced therefrom.
  • the thin stiiiage and syrup are blended, and 1 ,3-propaned ⁇ ol is produced therefrom in another embodiment, the whole stiiiage, thin stiiiage and syrup are blended and 1 ,3-pro ⁇ anedioi is produced therefrom.
  • the glycerol produced in a biodsesei production process described below is added to the whole stiiiage, thin stiiiage, syrup, and/or any combination thereof.
  • the "fermentation product” may be any product produced by a process that includes a fermentation step using a fermenting organism in an embodiment, the fermentation product is a biofuei such as butanoi or ethanoi
  • Other fermentation products include acetic acid ascorbic acid, citric acid, 2.5-diketo-D-glucon ⁇ c acid, fumaric acid, gluconic acid, gluconate, glycerol, and lactic acid, and succinic acid.
  • starch-containing starting mate ⁇ al including granular starch
  • the starting material is selected based on the dessred fermentation product
  • starch-containing starting materials suitable for use in the process include tubers, roots, stems whole grains, corns, cobs, wheat, barley, rye. m ⁇ !o, sago, cassava, tapioca sorghum, rice peas, beans, or sweet potatoes, or mixtures thereof or cereals Waxy and non-waxy types of corn and barley may aiso be used
  • Granular starch means uncooked starch, / e , starch in its natural form found in cereal tubers or grams Starch is formed within plant cells as tiny granules insoluble in water When put in cold water, the starch granules may absorb a small amount of the liquid and swell At temperatures up to 50 0 C to 75°C the swelling may be reversible However at higher temperatures an irreversible swelling called ' gelatinization" begins Granular starch to be processed may be a highly refined starch quality, preferably at least 90%, at least 95%, at least 97% or at least 99 5% pure or it may be a more crude starch- containing materials comprising milled whole gram including non-starch fractions such as germ residues and fibers
  • the starch-containing material is converted to dext ⁇ ns by liquefaction, e g , by an alpha-amylase, and the dext ⁇ ns are converted to a saccharified material by saccha ⁇ fication, e g , by a glucoamylase, and the saccharified material is then converted to the fermentation product du ⁇ ng fermentation Saccha ⁇ fication and fermentation may be earned out sequentially or simultaneously
  • liquefaction may be earned out as a three-step hot slurry process
  • the slurry ss heated to between 80-95 C, preferably 80-85 0 C, and an alpha-amylase is added to initiate liquefaction (thinning)
  • the slurry may be jet-cooked at a temperature between 95-140 0 C, preferably 105-125 11 C 1 for 1-15 minutes preferably for 3-10 minutes especially around 5 minutes
  • the slurry is cooled to 60-95 0 C and more alpha-amylase is added to finalize hydrolysis (secondary liquefaction)
  • the liquefaction process is usually carried out at a pH of 4 5-6 5, in particular at a pH between 5 and 6 Milled and liquefied whole grams are known as mash
  • saccha ⁇ fication may be earned out using conditions well known in the art
  • a full saccharsfication process may last up to from about 24 to about 72 hours, however, it is common only to do a pre-saccharification of typically 40-90 minutes at a temperature between 30-65 0 C, typically about 60 0 C, followed by complete saccharification during fermentation in a simultaneous saccha ⁇ fication and fermentation process (SSF process) Saccharification is typically carried out at a temperature of 20-75°C, preferably of 40-70 0 C, typically around 60 0 C, and at a pH between 4 and 5, normally at about 4 5
  • SSF simultaneous saccha ⁇ fication and fermentation
  • the process for producing a fermentation product from starch-containing materia! occurs without gelatimzation (often referred to as 'cooking") of the starch-contammg material
  • the desired fermentation product is produced without liquefying the aqueous slurry containing the starch-containing material
  • the starch-coniaining materia! e g granular starch, is treated below the initial gelatimzation temperature preferably in the presence of an a!pha-arny!ase and/or a carbohydrate-source generating enzyme to produce sugars that can be fermented into the desired fermentation product by a suitable fermenting organism
  • the conversion of the starch-containing material to dext ⁇ ns, saccha ⁇ fication and fermentation are earned out simultaneously (/ e , one step fermentation)
  • the term "initial gelatimzation temperature” means the lowest temperature at which gelatimzation of the starch commences in genera! starch heated in water begins to gelatinize between 50°C and 75 O C, the exact temperature of gelatsnszation depends on the specific starch and can readily be determined by the skilled artisan Thus the initial gelatinszation temperature may vary according to the plant specses, to the particular variety of the plant species as well as with the growth conditions
  • the initial gelatimzation temperature of a given starch-contasnsng materia! may be determined as the temperature at which birefringence is lost in 5% of the starch granules using the method described by Go ⁇ nstein and Ln, 1992, Starch/Starke 44 (12) 461-466
  • saccha ⁇ fication and fermentation are earned out as a simultaneous saccha ⁇ fication and fermentation process
  • the process is typically earned ai a temperature between 25X and 40 0 C, such as between 28X and 35°C, such as between 30"C and 34"C preferably around 32°C
  • simultaneous saccharification and fermentation is earned out so that the sugar level such as glucose ievel, is kept at a low level such as below 6 wt °/, preferably below about 3 wt %. preferably below about 2 wt %, more preferred below about 1 wt % , even more preferred below about 0 5%, or even more preferred 0 25% wt %, such as below about 0 1 wt %
  • a low levels of sugar can be accomplished by simply employing adjusted quantities of enzyme and fermenting organism
  • the employed quantities of enzyme and fermenting organism may also be selected to maintain low concentrations of maltose in the fermentation broth For instance the maltose level may be kept beiow about 0 5 wt % or below about 0 2 wt %
  • the process may be carried out at a pH of 3 to 7, preferably 3 5 to 6, or more preferably 4 to 5
  • the starch containing material is milled, e g , dry milled or wet milled, prior to the conversion of the starch material to dextnns
  • Dry milling comprises the steps of x) reducing the particle size of the starch-containing material, and y) forming a slurry comprising the starch-containing material and water
  • the aqueous slurry may contain from 10-55 wt % dry solids preferably 25-45 wt % dry solids (DS), more preferably 30-40% dry solids of starch-containing material
  • the raw materia! such as whole gram is reduced in particle see in order to open up the structure and allowing for further processing
  • whole kernels are milled and used
  • the particle size is reduced to between 0 05 to 3 0 mm preferably 0 1-0 5 mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the siarch-containing material fit through a sieve with a 0 05 to 3 0 mm screen, preferably 0 1-0 5 mm screen C Process for Producing a Fermentation Product and 1 ,3-Pr ⁇ panediol from a
  • the present invention also relates to a process of producing a fermentation product and 1 ,3-pro ⁇ aned ⁇ oi, comprising the steps of (a) converting a lignocellulosic materia! to a saccharified materia!,
  • the glycerol produced in a biodiese! production process described below is added to the glycerol containing stream
  • the hgnocellulose-containing materia! may be any material containing lignocellulose
  • the lignoce ⁇ ulosG-eontaimng materia! contains at least 50 wt %, preferably at least 70 wt %, more preferably at least 90 wt % lignocellulose
  • the hgnocellulose-containing materia! may a!so comprise other constituents such as ce ⁇ ulosic material such as cellulose, hemiceilulose and may aiso comprise constituents such as sugars, such as fermentable sugars and/or un-fermentable sugars
  • Lignoceiluloso-containing materia! is generally found for example, in the stems leaves, hulls husks, and cobs of plants or leaves, branches, and wood of trees
  • Lignoceilulosic material can also be but is not limited to, herbaceous material, agricultural residues forestry residues, municipal solid wastes, waste paper, and pulp and paper mil! residues
  • Lignocellulose-containing materia! may be in the form of plant cell wall materia! containing iignin, cellulose, and hemi-celluloso in a mixed matrix
  • the hgnocellulose-containing materia! is corn fiber, rice straw, pine wood, wood chips poplar, wheat straw, switchgrass, bagasse, paper and pulp processing waste
  • corn stover corn fiber hardwood, such as poplar and birch, softwood, cereal straw, such as, wheat straw, switchgrass, municipal solid waste (MSW), industrial organic waste, office paper or mixtures thereof
  • the materia! is corn stover
  • the materia! is corn fiber Production of Fermentation Products from Lignoceliuiose-Containing Material
  • Lignoceliulose- containing materials primarily consist of cellulose, hemicellulose, and lignin and are often referred to as "biomass”.
  • the structure of iignoceliuiose is not directly accessible to enzymatic hydrolysis. Therefore, the ⁇ gnoce ⁇ uiose-containing materia! has to be pre-treated, e.g., by acid hydrolysis under adequate conditions of pressure and temperature, in order to break the iignin sea! and disrupt the crystalline structure of cellulose. This causes solubilization of the hemicelluiose and cellulose fractions.
  • the cellulose and hemicelluiose can then be hydroiyzed enzymaticaily, e.g., by celiuioiytic enzymes, to convert the carbohydrate polymers into fermentable sugars which may be fermented into desired fermentation products, such as ethanol.
  • the steps are carried out as a simultaneous hydrolysis and fermentation step (SHF) or a hybrid hydrolysis and fermentation (HHF) which are described further below.
  • SHF simultaneous hydrolysis and fermentation step
  • HHF hybrid hydrolysis and fermentation
  • SHF means that combined/simuitaneous hydrolysis and fermentation are carried out at conditions (e.g., temperature and/or pH) suitable, preferably optimal, for the fermenting organism in question.
  • HHF typically begins with a separate hydrolysis step and ends with a simultaneous hydrolysis and fermentation step.
  • the separate hydrolysis step is an enzymatic cellulose hydrolysis step typically carried out at conditions (e.g., at higher temperatures) suitable. preferably optimal, for the hydroiyzing enzyme(s) in question.
  • the following simultaneous hydrolysis and fermentation step is typically carried out at conditions suitable for the fermenting organism (often at lower temperatures than the separate hydrolysis step).
  • the ⁇ gnocelluiose-containing material may be pre-treated before being hydroiyzed and/or fermented, in a preferred embodiment, the pre-treated material is hydroiyzed, preferably enzymaticaily, before and/or during fermentation.
  • the goal of pre-treatment is to separate and/or release cellulose, hemiceilulose and/or iignin and thus improve the rate of enzymatic hydrolysis.
  • Pre-irealme ⁇ l may be a conventional pre-treatment step known in the art Pre- treatment may take place m an aqueous slurry
  • the lignoceilulose-contasning material may during pre-treatment be present in an amount between 10-80 wt %, preferably between 20-50
  • the hgnoceliulose-contaimng material may be chemically, mechanically, biologically and/or thermally pre-treated before hydrolysis and/or fermentation Mechanical treatment
  • physical pre-treatment may be used alone or in combination with subsequent or simultaneous hydrolysis, especially enzymatic hydrolysis, to promote the separation and/or release of cellulose, hemicellulose and/or lignin
  • the chemical, mechanical biological and/or thermal pre-treatment is carried out prior to hydrolysis and/or fermentation
  • the chemical, mechanical, biological and/or thermal pre-treatment is carried out simultaneously with hydrolysis, such as simultaneously with addition of one or more cellulolytic enzymes, or other enzyme activities mentioned below, to release fermentable sugars, such as glucose and/or maltose
  • the pre-treated hgnoceilulose-containing material is washed and/or detoxified before hydrolysis
  • This may improve the fermentability of, e g dilute-acid hydrolyzed hgnocellulose-containing material, such as corn stover Detoxification may be carried out in any suitable way, e g , by steam stripping, evaporation, son exchange, ressn or charcoal treatment of the liquid fraction or by washing the pre-treated material
  • Chemical pre-treatment means any chemical treatment which promotes the separation and/or release of cellulose, hemicellulose and/or lignin
  • suitable chemical pre- treatment steps include treatment with, for example, dilute acid, lime alkaline organic solvent, ammonia sulfur dioxide carbon dioxide
  • wet oxidation and pH-controlied hydroihermolysis also are chemical pre-treatmenis
  • the chemical pre-treatment is acid treatment, more preferably a continuous dilute and/or mild acid treatment, such as, treatment with sulfuric acid, or another organic acid, such as acetic acid, citric acid, tartaric acid, succinic acid or mixtures thereof Other acids may also be used
  • Mild acid treatment means that the treatment is at a pH of 1-5 preferably 1-3
  • the acsd concentration is from Q 1 to 2 0 wt % acid, preferably sulphuric acid
  • the acsd may be mixed or contacted with the material to be fermented and the mixture may be held at a temperature of 160-220 0 C such as 165-195°C, for periods ranging from minutes to seconds e g , 1-60 minutes, such as 2-30 minutes or 3- 12 minutes
  • Addition of strong acids, such as sulphu ⁇ c acid may be applied to remove hemicellulose This enhances the digestibility of cellulose
  • Cellulose solvent treatment may be used to convert cellulose, e g , about 90% of cellulose, to glucose It has also been shown that enzymatic hydrolysis could be greatly enhanced when the lignoceliuiosic structure is disrupted Alkaline H 2 O 2 , ozone, organosolv (uses Lewis acids, FeCI-,, (AI)-SO 4 in aqueous alcohols) glycerol dioxane, phenol, or ethylene glycol are among solvents known to disrupt cellulose structure and promote hydrolysis (Mosier er a/ 2005, Bioresource Technology 96 673-686)
  • the chemical pre-treatment may be an alkaline chemical pre-treatment with base, e g ,
  • oxidizing agents such as sulphite based oxidizing agents or the like
  • solvent pre-treatments include treatment with DMSO (dimethyl sulfoxide) or the like
  • Chemical pre-treatment is generally carried out for 1 to 60 msnutes, such as from 5 to 30 minutes but may be earned out for shorter or longer periods of time dependent on the material to be pre-treated
  • mechanical pre-treatment means any mechanical or physical pretreatment which promotes the separation and/or release of cellulose, hemicellulose and/or lignin from iignoceilulose-containing material
  • mechanical pre-treatment includes various types of milling, irradiation, steamsng/steam explosion and hydrothermolysis
  • Mechanical pre-treatment includes comminution (mechanical reduction of the particle size) Comminution includes dry milling, wet milling and vibratory ball milling
  • Mechanical pre- treatment may involve high pressure and/or high temperature (steam explosion)
  • high pressure means pressure of 300 to 600 psi, preferably 400 to 500 psi, such as around 450 psi
  • high temperature means a temperature of about 100 to 300 0 C, preferably from about 140 to 235°C
  • mechanical pre- treatment is a batch-process, steam gun hydrolyzer system which uses high pressure and high temperature as defined above A Sunds Hydrolyzer (available from Sunds Defibrator AB (Sweden)) may be used for this
  • both chemical and mechanical pre-treatments are earned out involving, for example, both dilute or mild acid pretreatment and high temperature and pressure treatment
  • the chemical and mechanical pretreatment may be carried out sequentially or simultaneously, as desired
  • the lignoceilulose-containing material is subjected to both chemical and mechanical pretreatment to promote the separation and/or release of cellulose, hemiceliuiose and/or lignm in a preferred embodiment the pre-treatment is earned out as a dilute and/or mild acid steam explosion step In another preferred embodiment pre-treatment is earned out as an ammonia fiber explosion step (or AFEX pretreatment step)
  • biological pretreatment means any biological pretreatment which promotes the separation and/or release of cellulose hemiceilulose, and/or iignin from the lignoceliuiose- contasnsng material
  • Biological pre-treatment techniques can involve applying hgmn- solubihzing microorganisms (see, for example, Hsu, 1996. Pretreatment of Biomass, in Handbook on Bioethano! Production and Utilization, Wyman, ed Taylor & Francis Washington, DC, 179-212, Ghosh and Singh, 1993 "Physicochemica!
  • the pre-treated hgnoceilulose-contaimng materia! may be hydroiyzed in order to break the lignin sea! and disrupt the crystalline structure of cellulose in a preferred embodiment, hydrolysis is earned out enzymatically
  • the pre- treated lignocellulose-containing material may be hydroiyzed by one or more hydrolases (class EC 3 according to the Enzyme Nomenclature), preferably one or more carbohydrases including cellulolytic enzymes and hemice ⁇ uiolytic enzymes, or combinations thereof
  • alpha-amylase, glucoamylase, protease, and/or the like may also be present during hydrolysis and/or fermentation as the hgnoceliuiose-containmg material may include some, e g , starchy and/or proteinaceous material
  • the enzyme(s) used for hydrolysis ⁇ s(are) capable of directly or indirectly converting carbohydrate polymers into fermentable sugars, such as glucose and/or maltose which can be fermented into a desired fermentation product, such as ethanoi in a preferred embodiment, the carbohydrase(s) has(have) ce ⁇ ulolytic and/or hemiceliulolytic enzyme activity
  • hydrolysis is earned out using a ceiluloiytic enzyme preparation further comprising one or more polypeptides having celluiolytic enhancing activity in a preferred embodiment, the polypept ⁇ de(s) having celiuiolytic enhancing activity ⁇ s(are) of family GH61A origin
  • suitable and preferred ce ⁇ ulolytic enzyme preparations and polypeptides having ceiluloiytic enhancing activity are described below
  • Hemicellulose polymers can be broken down by hemiceliuiioiytic enzymes and/or acid hydrolysis to release its five and six carbon sugar components The six carbon sugars
  • hexoses such as glucose, galactose, arabmose, and mannose
  • suitable fermenting organisms including yeast
  • Yeast is the preferred fermenting organism for ethanoi fermentation
  • Enzymatic hydrolysis is preferably earned out in a suitable aqueous environment under conditions which can readily be determined by one skilled in the art in a preferred embodiment, hydrolysis is earned out at suitable, preferably optimal, conditions for the enzyme(s) in question
  • Suitable process time temperature and pH conditions can readily be determined by one skilled in the art
  • hydrolysis is earned out at a temperature between 25 and 70' C, preferably between 40 and 6Q°C, especially around 50 0 C.
  • the step is preferably earned out at a pH of 3-8, preferably 4-6.
  • Hydrolysis is typically earned out for between 12 and 96 hours, preferable 16 to 72 hours, more preferably between 24 and 48 hours
  • the enzyme(s) used in the processes of the present invention for producing fermentation products are used in an effective amount
  • Alpha-Amylases Any alpha-amylase may be used for converting the starch material to dextrins
  • the alpha-amylase is an acid alpha-amylase, e g., acid fungal alpha- amylase or acid bacterial alpha-amylase.
  • the term "acid alpha-amylase' ' means an aipha- amylase (E C 3.2 1.1 ) which added in an effective amount has activity optimum at a pH of 3 to 7, preferably 3 5 to 6, or more preferably 4-5.
  • an alpha-amylase is present before and/or during fermentation
  • the aipha-amylase is an alpha-amylase that has less than 70%, preferably less than 60%. more preferably less than 50%, even more preferably less than 40%, more preferably less than 30% residual activity left at a pH below 6, preferably pH 5, especially below pH 4 5 compared to its maximum activity (/ e.. 100%)
  • the bacterial alpha-amylase is preferably derived from the Bacillus in a preferred embodiment, the Bacillus aipha-amylase is derived from a strain of Bacillus licheniformis. Bacillus amyloliquefaciens, Bacillus subtilis or Bacillus stearothermophilus, but may also be derived from other Bacillus sp Specific examples of alpha-amyiases include the Bacillus licheniformis alpha-amylase shown in SEQ ID NO 4 in WO 99/19467, the Bacillus amyloliquefaciens alpha-amylase SEQ ID NO 5 in WO 99/19467 and the Bacillus stearothermophilus aipha-amylase shown in SEQ ID NO 3 in WO 99/1946/' (all sequences hereby incorporated by reference).
  • the aipha-amylase may be an enzyme having a degree of identity of at least 60%, preferably at least 70%, more preferred at least 80%, even more preferred at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to any of the sequences of SEQ ID NOs. 1 , 2 and 3 in WO 99/19467
  • the Bacillus alpha-amylase may also be a variant and/or hybrid, especially one described in any of WO 96/23873, WO 96/23874, WO 97/41213, WO 99/19467.
  • WO 00/60059, and WO 02/10355 all documents hereby incorporated by reference
  • aipba-arnyiase variants are disclosed sn U S Patent No 6,093 562, 6 297,038 or 6,187,576 (hereby incorporated by reference) and include Bacillus stearoihermophilus alpba-amyiase (BSG alpba-amyiase) variants having a deletion of one or two ammo acid in positions R179 to G182, preferably a double deletion disclosed in WO 1996/023873 - see e g , page 20, lines 1-10 (hereby incorporated by reference), preferably corresponding to delta(181-182) compared to the wild-type BSG alpha-
  • a specific hybrid alpha-amylase comprises 445 C-termma! amino acid residues of the Bacillus licheniformis alpha-amylase (shown in SEQ ID NO 4 of WO 99/ 19467) and the 37 N-termina! amino acsd residues of the alpha-amylase derived from Bacillus amylohquefaciens (shown in SEQ ID NO 5 of WO 99/19467), with one or more, especially all, of the following substitution
  • variants having one or more of the following mutations for corresponding mutations in other Bacillus alpha-amylase backbones) H154Y, A181T, N190F, A209V and Q264S and/or a deletion of two residues between positions 176 and 179 preferably a deletion of E 178 and G179 fusing the SEQ ID NO 5 numbering of WO 99/19467)
  • the bacterial alpha-amylase is dosed in an amount of 0 0005-5 KNU per g DS preferably 0 001-1 KNU per g DS, such as around 0 050 KNU per g DS
  • Fungal alpha-amylases include alpha-amylases derived from a strain of Aspergillus, such as, Aspergillus oryzae, Aspergillus niger and Aspergillis kawachu alpha-amylases
  • a preferred acidsc fungal alpha-amylase is an alpha-amylase which is derived from a strain of Aspergillus oryzae, preferably, an alpha-amylase which exhibits a high identity. » e , at least rQ%, at least /5%, at least 80% at least 85% at least 90%, at least 95%, at least 96% at least 97%, at least 98% at ieast 99% or even 100% identity to the mature part of the amino acid sequence shown in SEQ ID NO 10 in WO 96/238/4
  • Another preferred acid aipha-amylaso is derived from an Aspetgillus nigot strain in a preferred embodiment the acid fungal alpha-amylase is the one from Aspergillus niger disclosed as "AMYA ASPNG" in the Swiss-prot/TeEMBL database under the primary accession no P56271 and described in WQ 89O1989 (Example 3 - incorporated by reference)
  • a commercially available acid fungal alpha-amylase derived from Aspergillus nigot is SP288 (available from Novozymes A/S, Denmark)
  • Other wild-type alpha-amyiases include those derived from a strain of the genera
  • Rhizomucor and Me ⁇ pilus preferably a strain of Rhizomucor pusillus (WO 2004/0551 /8 which is incorporated herein by reference) or Me ⁇ pilus giganteus in a preferred embodiment
  • the alpha-amyiase is derived from Aspergillus kawachu and disclosed by Kanoko Qt al , 1998, J Ferment Bioeng 81 292-298, "Molecular-cloning and determination of the nucleotide-sequence of a gene encoding an acid-stabie alpha- amyiase from Aspetgillus kawachii", and further as EMBL #AB008370
  • the fungal alpha-amylase may also be a wild-type enzyme comprising a starch- binding domain (SBD) and an alpha-amylase catalytic domain 0 e , non-hybrid), or a variant thereof
  • the wild-type alpha-amyiase is derived from a strain of Aspergillus ⁇ av,>achi ⁇ in a preferred embodiment
  • the fungal acid alpha-amyiase is a hybrid alpha-amylase
  • Preferred examples of fungal hybrid alpha-amyiases include the ones disclosed in WO 2005/00331 1 or U S Application Publication no 2005/0054071 (Novozymes) or U S Application No 60/638,614 (Novozymes) which is hereby incorporated by reference
  • a hybnd alpha-amyiase may comprise an alpha-amylase catalytic domain (CD) and a carbohydrate-bindsng domain/module (CBM), such as
  • hybnd aipha-amylases include those disclosed in U S Application Publication no 2005/00540/1 including those disclosed in Table 3 on page 15 such as Aspetgillus niget alpha-amylase with Aspergillus kawachn iinker and starch binding domain
  • the alpha-amylase also may be an alpha-amyiase which exhibits a high identity to any of above mention alpha-amylases, / e , at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 96%, at least 9/%, at least 98%, at least 99% or oven 100% identity to the mature enzyme sequences
  • An acid alpha-amylase may be added in an amount of 0 001 to 10 AFAU/g DS, preferably from 0 01 to 5 AFAU/g DS, especially 0 3 to 2 AFAU/g DS or 0 001 to 1 FAU-F/g DS, preferably 0 01 to 1 FAU-F/g DS
  • compositions comprising alpha-amylase include MYCOLASE from DSM (Gist Brocades;, BANTM TERMAMYL ⁇ SC, FUNGAMYLTM , LIQUOZYME TM X LiQUOZYMETM SC and SANTM SUPER, SANTM EXTRA L (Novozymes AJS) and CLARASETM L-40,000, BEX-LOTM SPEZYMETM FRED, SPEZYMETM AA and SPEZYME TM DELTA AA (Genencor hit ), FUELZYME (Verenium), and the acid fungal alpha-amylase sold under the trade name SP288 (available from Novozymes AiS, Denmark)
  • carbohydrate-source generating enzyme includes beta-amyiase (a maltose generator) glucoamyiase (a glucose generator), and maltogemc amylase (another maltose generator), as well as potiulanase and alpha-glucosidase
  • a carbohydrate-source generating enzyme is capable of producing a carbohydrate that can be used as an energy- source by the fermenting organ ⁇ sm(s) when used in a process for producing a fermentation product, such as ethanoi
  • the generated carbohydrate may be converted dsrectiy or indirectly to the desired fermentation product preferably ethanoi
  • a mixture of carbohydrate- source generating enzymes may be used Examples are mixtures of at least a glucoamyiab ⁇ and an alpha-amyjase especialiy an acid amylase even more preferred an acid fungal alpha-amylase
  • a glucoamyiase may be denved from any suitable source, e g derived from a microorganism or a plant Preferred glucoamyiases are of funga! or bacteria! origin, selected fiom the group consisting of Aspergillus giucoamyiases in particular Aspergillus niger G1 or G2 glucoamylase (Boel ef a/ 1984, FMBO J 3(5) 1097-1 102) or variants thereof such as those disclosed in WO 92/00381 WO 00/04 136 and WO 01/042 ⁇ 3 (from Novozymes Denmark) the A awamo ⁇ glucoamylase disclosed in WO 84/02921 Aspergillus oryzae glucoamvlase (Agn ⁇ Biol Chem 55 ( 4) 941-949 ( 1991 )) or variants or fragments thereof
  • Other Aspergillus glucoamylaso variants include variants with enhanced
  • glicoamyiases include giucoamyiases from Clostridium in particular C thermoamyolyticum [EP 135 138) and C thermohydrosuifuncum (WO 86/01831 ) and Trametes ⁇ ngulata Pdcnykytospora papyraced, and Leucopaxillus giganteus all disclosed in WO 2006/069289 oi Pen ⁇ hora rufomarginata disclosed in international application no PCT/US200 /7066618 or a mixture thereof
  • the giucoamyiase also may be a hybrid glucoamylase Examples of hybrid glucoamylasos arc disclosed in WO 2005/045018, in particular the hybud giucoamyiases disclosed in Tables 1 and 4 of Example 1 (which hybrids are hereby incorporated by reference)
  • the giucoamyiases may exhibit a high identity to any of above mention giucoamyiases i e at least 70% at least 75% at least 80%, at least 85% at least 90% at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzymes sequences mentioned above
  • compositions comprising glucoamyiase include AMG 200L, AMG 300 L, SANTM SUPER, SAN Tf/ EXTRA L SPIRIZYMETM PLUS 1 SPIRIZYME Tf/ FUEL SPiRIZYME 111 -' B4U and AMG 1 '" 1 E (from Novozymes A/S), OPTIDEX 1 '" 1 300 (from Genencor lnt ⁇ , AM IGASE TM and AMiGASETM PLUS (from DSM), G-ZYME TM G900, G-ZYMETM and G990 ZR (from Genencor lnt )
  • Giucoamylases may be added in an amount of 0 0001-20 AGU/g DS preferably 0 001-10 AGU/g DS, especially between 0 01-5 AGU/g DS, such as 0 1-2 AGU/g DS
  • a beta-amylase (E C 3 2 1 2) is traditionally an exo-actsng maltogenic amylase, which catalyzes the hydrolysis of aipha-1 ,4-glucosidic linkages in amyiose, amylopectin and related glucose polymers Maltose units are successively removed from the non-reducing chain ends in a step-wise manner until the molecule is degraded or. in the case of amylopectin, until a branch point is reached The maltose released has the beta anome ⁇ c configuration, hence the name beta-amyiase
  • Beta-amylases have been isolated from various plants and microorganisms ⁇ Fogarty and Kelly 1979, Progress in industrial Microbiology 15 1 12-1 15) These beta-amylases are characterized by having optimum temperatures of 40 1 C to 65' C and optimum pH of 4 5 to 7
  • a commercially available beta-amylase from barley is NOVOZYM TM WBA from Novozymes A'S, Denmark and SPEZYMETM BBA 1500 from Genencor lnt , USA
  • amylase may also be a maltogenic alpha-amyiase
  • a 'maltogenic alpha- amylase" (glucan 1 ,4-al ⁇ ha-maltohydrolase, E C 3 2 1 133) is able to hydrolyze amyiose and amylopectin to maltose in the alpha-configuration
  • a maitogenic amylase from Bacillus stearothetmophilus strain NCiB 1 1837 is commercially available from Novozymes
  • A/S Maitogenic alpha-amyiases are described in U S Patent Nos 4,598,048, 4,604 355 and 6,162,628, which are hereby incorporated by reference
  • the maltogenic amylase may be added in an amount of 0 05-5 mg total protein/gram DS or 0 05- 5 MANU/g DS Ceiiuiolytic Activity
  • the term ' viiuiolytic activity as used herein are understood as comprising enzymes having cellobiohydroiase activity (EC 3 2 1 91 ), e g , cellobiohydroiase i and ceilobiohydrolase Ii, as well as endoglucanase activity (EC 3 2 1 4) and beta-glueosidase activity (EC 3 2 1 21 )
  • At least three categories of enzymes are important for converting cellulose into fermentable sugars endoglucanases (EC 3 2 1 4) that cut the cellulose chains at random, ceilobiohydrolases (EC 3 2 1 91 ) which cleave ceilobiosyi units from the cellulose chain ends and beta-glucosidases (EC 3 2 1 21 ) that convert ceilobiose and soluble cellodextrins into glucose
  • ceilobiohydroiases seem to be the
  • the DCiuiolytic activity may, in a preferred embodiment, be in the form of a preparation of enzymes of fungai origin, such as from a strain of Tnchoderma, preferably a strain of Tnchoderma reeses, a strain of Humicola, such as a strain of Humicola tnsolens, or a strain of Chrysosponum, preferably a strain of Chi ⁇ sosponum lucknowense in a preferred embodiment the DCiuiolytic enzyme preparation contains one or more of the following activities DCiulase, hemicellulase ce ⁇ ulolytic enzyme enhancing activity, beta- glucosidase activity, endogiucanase, cellubsohydrolase or xylose-isomerase in a preferred embodiment, the celluloiytic enzyme preparation is a composition disclosed in U S Application No 60/941 ,251 , which ss hereby incorporated by reference in a preferred embodiment, the celluloiytic enzyme preparation
  • E ⁇ doglucanasos (EG)
  • endoglucanase ' means an endo-1 4-(1 3,1 4)-beta ⁇ D-g!ucan
  • E C 3 2 1 4 4-glucanohydroiase (E C 3 2 1 4), which catalyzes endo-hydroiysis of 1 ,4-beta-D-glycos ⁇ dic linkages in cellulose, cellulose derivatives (such as carboxymethy! cellulose and hydroxyethy! cellulose) lichensn beta-1 4 bonds in mixed beta-1 ,3 gl ⁇ cans such as cereal beta- D-g I u can s or xyloglucans and other plant material containing cellulosic components
  • Endoglucanase activity may be determined using carboxymethyl cellulose (CMC) hydrolysis according to the procedure of Ghose, 198/, Pure and Appl Chem b9 2b /-268
  • endoglucanases may be derived from a strain of Trichodetma preferably a strain of Tnchoderma reesei, a strain of Humicola, such as a strain of Humicola msolens, or a strain of Chrysospo ⁇ um preferably a strain of Chrysospo ⁇ uni lucknowense
  • cellobiohydroiase means a 1 ,4-beta-B-glucan cellobiohydroiase (E C 3 2 1 91 ), which catalyzes the hydrolysis of 1 4-beta-D-giucossd ⁇ c linkages in cellulose, celloohg ⁇ saccha ⁇ des, or any beta-1 ,4-l ⁇ nked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain
  • CBH I and CBH Il from Tnchoderma seseei Humicola msolens and CBH Il from Thielavsa terrestnts (CELL6A)
  • Cellobiohydrolase activity may be determined according to the procedures described by Lever et a/ , 1972, Anal Biochem 47 273-279, van Tilbeurgh et a/ , 1982, FFBS L etters 149 152-156 and van Tilbeurgh and Claeyssens, 1985 FEBS Letters 187 283-288
  • the Lever et a/ method is suitable for assessing hydrolysis of cellulose in corn stover and the method of van Tilbeurgh et a/ is suitable for determining the cellobiohydrolase activity on a fluorescent disaccharide derivative
  • beta-glucosidase means a beta-D-glucoside glucohydrolase (E C 3 2 1 21 ) which catalyzes the hydrolysis of terminal non-reducing beta-D-gluc ⁇ se residues with the release of beta-D-giucose Beta-giucosidase activity is determined according to the procedure described by Ventu ⁇ et a!
  • beta- giucosidase activity ss defined as 1 0 ⁇ mole of p-nitrophenol produced per minute at 50°C, pH 5 from 4 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodium Citrate 0 01 % TWE EN® 20 in an embodiment, the beta-giucosidase is of fungal origin, such as a strain of
  • the beta-giucosidase is a derived from Trichoderma reesei such as the beta-giucosidase encoded by the bgi1 gene ⁇ see Fig 1 of EP 582003) in another embodiment, the beta-giucosidase is derived from Aspergillus oiyzae (recombmantiy produced in Aspergillus oryzae according to WO 02/095014), Aspergillus funiigatus (recombmantiy produced in Aspergillus oryzae according to Example 22 of WO 02/095014) or Aspergillus niger (J Appl 3 157-163 (1981 ))
  • Hemicelluloiytic enzymes The pre-treated lignocellulose-containing materia! may further be subjected to one or more hemiceliuioiytic enzymes, e g , one or more hemice ⁇ ulases
  • Hemiceliuiose can be broken down by hemiceilulases and/or acsd hydrolysis to release its hve and six carbon sugar components in an embodiment the iignoceiiuiose derived material may be treated with one or more hemicellulases
  • hemiceliulase suitable for use in hydrolyzing hemiceliulose, preferably into xylose may be used
  • Preferred hemiceiiulases include xylanases arabinofuranosidases, acetyl xyian esterases, feruioyl esterases, glucuronidases, endo-galactanase mannases endo or exo arabsnases exo-gaiactanses, and mixtures of two or more thereof
  • the hemiceliulase is an exo-acting hemicellulase.
  • the hemiceliulase is an exo- actsng hemiceliulase which has the ability to hydrolyze hemiceliuiose under acidic conditions of beiow pH 7, preferably pH 3-7
  • An example of a suitable hemicellulase is VISCGZYMETM ⁇ available from Novozymes A/S, Denmark) in an embodiment, the hemicellulase is a xyianase
  • the xylanase may be of microbial origin, such as of fungal origin (e g , Aspergillus, Fusanum, Humicola, Menptlus, Tnchodetma,) or from a bacterium (e g , Bacillus)
  • the xylanase is derived from a filamentous fungus, preferably derived from a strain of Aspergillus, such as Aspergillus aculeatus, or a strain of Humicola,
  • the hemiceliulase may be added in an amount effective to hydrolyze hemicelluiose, such as. in amounts from about 0.001 to 0 5 wt % of total solids (TS), more preferably from about 0.05 to 0 5 wt % of TS.
  • TS total solids
  • Xyianases may be added in amounts of 0 001-1 0 g/kg DM (dry matter) substrate, preferably in the amounts of 0.005-0.5 g/kg DM substrate, and most preferably from 0.05-0.10 g/kg DM substrate
  • ceiiuioiytic enhancing activity is defined herein as a biological activity that enhances the hydrolysis of a lignoceliuiose derived material by proteins having DCiuioiytic activity
  • Ceiiuioiytic enhancing activity is determined by measuring the increase in reducing sugars or in the increase of the total of celiobiose and glucose from the hydrolysis of a hgnocellulose derived material, e.g , pre-treated hgnoce ⁇ ulose-containing material by DCiuioiytic protein under the following conditions: 1-50 mg of total protein/g of cellulose in PCS (pre-treated corn stover), wherein total protein is comprised of 80-99.5% w/w DCiuioiytic protein/g of cellulose in PCS and 0 5-20% w/w protein of DCiuioiytic enhancing activity for 1-7 day at 50 0 C compared to a control hydrolysis with equal total protein loading without DCiuioi
  • the polypeptides having DCiuioiytic enhancing activity enhance the hydrolysis of a iignoceiiuiose derived material catalyzed by proteins having DCiuioiytic activity by reducing the amount of DCiuioiytic enzyme required to reach the same degree of hydrolysis preferably at least 0.1 -fold, more at least 0.2-foid, more preferably at least 0 3-fold, more preferably at ieast 0 4-fold, more preferably at least 0 5-fold, more preferably at least 1-fold, more preferably at ieast 3-fold, more preferably at ieast 4-fold, more preferably at ieast 5-fold, more preferably at ieast 10-fold, more preferably at ieast 20-fold, even more preferably at ieast SO-ToId, most preferably at ieast 50-fold, and even most preferably at ieast 100-fold in an embodiment, the hydrolysis and/or fermentation is earned out in the presence of a
  • WO 2005/0 /'4656 discloses an isolated polypeptide having DCiuioiytic enhancing activity and a polynucleotide thereof from Thermoascus aurantiacus.
  • U.S. Application Publication No 2007/0077630 discloses an isolated polypeptide having celiuioiytsc enhancing activity and a polynucleotide thereof from T ⁇ choderma reeses
  • protease may be added during sacchanficatioiVhydraiysis fermentation or simultaneous saccharification/hydrolysis and fermentation
  • the protease may be any protease in an embodiment the protease is an acid protease of microbial origin, preferably of fungal or bacterial origin An acid fungal protease is preferred, but also other proteases can be used
  • proteasos include microbial proteases, such as fungal and bacto ⁇ a! proteases
  • Preferred proteases are acidic proteases, i e , proteases characterized by the ability to hydrolyze proteins under acidic conditions below pM ⁇
  • Acsd fungal proteases include fungal proteases derived from AspetgiHus Candida Conolus Endothia, Enthomophtta Irpex Mucot Pen/album, Rhizopus, Scletotium and Torulopsis Especially preferred arc proteasos derived from Aspergillus niger (see e g , Koazo et al , 1984 Agr Biol Chem Japan 28 216) Aspergillus saitoi (see, e g Yoshida, 1954, J Agr Chem Soc Japan 28 66), Aspergillus av/amon (Hayashida et al 1977 Agric Bio!
  • the protease may be a neutral or alkaline protease, such as a protease derived from a strain of Bacillus A particular protease is derived from Bacillus amyionquetaciens and has the sequence obtainable at Swissprot as Accession No P06832
  • the proteases may have at least 90% identity to amino acsd sequence obtainable at Swissprot as Accession No P06832 such as at least 92%, at least 95%, at least 96%, at least 9f%, at least 98%, or particularly at least 99% identity
  • Other proteases include proteases having at least 90% identity to amino acid sequence disclosed as SEQ ID NO 1 in the WO 2003/048353 such as at 92%, at least 95% at ieast 96% at least 97%, at least 98%, or particularly at least 99% identity
  • Papain-iike proteases such as proteases within E C 3 4 22 * ⁇ cysteine protease), such as EC 3 4 22 2 (papain), EC 3 4 22 6 (chymopapain), EC 3 4 22 7 (asclepain), EC 3 4 22 14 (actinidain), EC 3 4 22 1b (cathepsin L), EC 3 4 22 25 (giycyi endopeptidase) and EC 3 4 22 30 (ca ⁇ cain) aiso may bo used
  • the protease is a protease preparation derived from a strain of Aspergillus, such as Aspergillus oryzae in another embodiment the protease is derived from a strain of Rhizomucor, preferably Rhizomucor rnehei in another embodiment, the protease is a protease preparation, preferably a mixture of a proteolytic preparation derived from a strain of Aspergillus, such as Aspergillus oryzae, and a protease derived from a strain of Rhizomucor, preferably Rhizomucor miehei
  • Aspartic acid proteases are described in, for example, Handbook of Proteolytic Enzymes, Edited by Barrett Rawhngs and Woessner, Aeadermc Press, San Diego, 1998 Chapter 270) Suitable examples of aspartic acid protease include, e g , those disclosed m Berka et a/ , 1990, Gene 96 313, Berka et a/ , 1993, Gene 125 195-198, and Gomi et a/ , 1993, Biosc!
  • Biotech Biochem 57 1095-1 100 which are hereby incorporated by reference
  • Commercially available products include ALCALASE®, ESPERASETM , FLAVOURZYMETM , PROMIXTM , NEUTRASE®, RENNILASE®, NOVOZYMTM FM 2 OL, and NOVOZYM 7 " 1 50008 (available from Novozymes A/S, Denmark) and GC106 111 -' and SPEZYMETM FAN from Genencor int , lnc , USA
  • the protease may be present in an amount of 0 0001-1 mg enzyme protein per g DS, preferably 0 001 to Q 1 mg enzyme protein per g DS Alternatively, the protease may be present in an amount of 0 0001 to 1 LAPU/g DS preferably 0 001 to 0 1 LAPU/g DS and/or 0 0001 to 1 mAU-RH/g DS, preferably 0 001 to 0 1 mAU-RH/g DS
  • fermenting organism refers to any organism, including bacteria! and fungal organisms including yeast and filamentous fungi, suitable for producing a desired fermentation product Especially suitable fermenting organisms are able to ferment, / e convert, sugars, glucose, xylose, fructose and/or maltose, directly or indirectly into the desired fermentation product
  • yeast Preferred yeast includes strains of Saccharomyces, in particular a strain of Saccharomyces cerevisiae or Saccharomyces uvarum a strain of Pichia, in particular Pichia stipitis such as Pichia stipitis CBS 5773, or Pichia pastons, a strain of Candida, in particular a strain of Candida boidinii Candida diddensn, Candida utilis
  • yeast includes strains of Zymomonas and Hansenula, in particular Hansenula anomala, strains of Klyveromyces, in particular Klyveromyces fragi
  • fermenting organisms include strains of Escherichia in particular Escherichia coli strains of Leuconostoc, in particular Leuconostoc mesenteroides, strains of Clostridium, in particular Clostridium butyncum, strains of Enterobacter, in particular Enterobacter aerogenes and strains of Thermoanaerobacter, in particular Thermoanaerobacter BG1 L1 ⁇ Appl Microbiol Biotech 77 61-86) and Thermoanarobacte?
  • the fermenting organism is added io the fermentation medium so that the viable fermenting organism, such as yeast, count per mL of fermentation medium is from 10* to 10 12 , preferably from 10 :/ to 1Q 1Q , especially about 5x10 7 .
  • yeast includes, e.g , ETHANOL RED I Iv! yeast (available from Fermentis/Lesaffre, USA), FAL! (available from Fleischmann's Yeast, USA), SUPERSTAR! and THERMOSACCTM fresh yeast (available from Ethanol Technology, Wl, USA), BIOFERM AFT and XR (available from NABC - North American Bioproducts Corporation, GA. USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).
  • the fermenting organism capable of producing a desired fermentation product from fermentable sugars, such as, e.g., glucose, fructose and/or maltose is preferably grown under precise conditions at a particular growth rate.
  • the inoculated fermenting organism pass through a number of stages, initially growth does not occur. This period is referred to as the "lag phase” and may be considered a period of adaptation During the next phase referred to as the “exponential phase” the growth rate gradually increases. After a period of maximum growth the rate ceases and the fermenting organism enters "stationary phase”. After a further period of time the fermenting organism enters the "death phase" where the number of viable cells declines.
  • Fermentation media must contain suitable carbon and nitrogen substrates which are well known in the art.
  • Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose and oligosaccharides such as lactose, mannose or sucrose.
  • the fermentation may be carried out at conventionally used conditions Preferred fermentation processes are anaerobic processes.
  • the fermentation may in one embodiment go on for 8 to 120 hours, in particular 24 to 96 hours.
  • the fermentation is earned out at a temperature between 25 to 4OX, preferably 28 to 35°C, such as 30 0 C to 34°C, and in particular around 32°C.
  • the pH when initiating fermentation is from 3 to 6. preferably around 4 to 5
  • the process comprises a simultaneous hydrolysis/saccharification and fermentation (SHF/SSF) where there is no separate holding stage for the
  • hydroiysis/saccharification meaning that the hydroiyzing/saccharifying enzyme(s) and the fermenting organism are added together
  • the temperature is preferably between 25 to 40°C, preferably 28 to 35°C such as 30 0 C to 34°C in particular around 32°C when the fermentation organism is a strain of Saccharomyces cerevisiae and the desired fermentation product is ethanes!
  • the process of the invention may be performed as a batch or as a continuous process
  • the fermentation process of the invention may be conducted in an ultrafiltration system where the retentate ss held under recirculation in the presence of solids, water, and the fermenting organism, and wherein the permeate is the desired fermentation product containing liquid
  • the process is conducted in a continuous membrane reactor with ultrafiltration membranes and wherein the retentate is held under recirculation in presence of solids, water, the fermenting organism and wherein the permeate ss a liquid containing the fermentation product
  • the fermenting organism may be separated from the fermented slurry and recycled to the fermentation medium
  • the fermentation product may be separated from the fermentation medium e g , by distillation and/or cent ⁇ fugation
  • the slurry may be distilled to extract the desired fermentation product or the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques Alternatively the fermentation product may be recovered by stripping Methods for recovery are well known in the art
  • Liquid fermentation products are recovered from the fermented mash (often referred to as "beer mash") e g , by distillation, which separates the desired fermentation product from other liquids and/or solids
  • the remaining fraction referred to as whole stillage which is typically in the range of 1 1% to 15% solids, contains all of the other non-starch components of the corn kernel that pass through the process (germ, protein, gluten, hull & fiber etc )
  • the whole stillage is dewatereci and separated into a solid and a liquid phase e g by cent ⁇ fugation
  • the solid phase is referred to as 'wet cake" (or 'wet grains”) and the liquid phase (supernatant) is referred to as thin stillage
  • Dewatered wet cake is dried to provide "Distillers Dried Grains ' (DDG) used as nutrient in animal feed
  • Thin sti ⁇ age is typically evaporated to provide condensate of 4-6% DS (mainly of star
  • the present invention also relates to a process of producing biodiesel and 1 ,3-propanediol, comprising the steps of
  • esters e g., methyl esters
  • the reaction usually requires a catalyst, e g., a strong base such as sodium or potassium hydroxide, or an acid such as sulfuric acid.
  • the glycerol from a biodiesei production process is added to the whole stiliage. thin stillage, syrup, and/or any combination thereof or to a glycerol containing stream described above.
  • Thin sliilage obtained from a dry milling process was condensed by 4-5 folds in laboratory using a rotoevaporator operated at 80 0 C
  • the condensed stillage was used to simulate the thick stillage from the industry
  • the condensed stillage was neutralized with 50% (w/v) sodium hydroxide to pH 7 before inoculation
  • Corn steep liquor purchased from Sigma-Aldrich was cent ⁇ fuged at 200 rpm for 10 mm to separate the solids, and the supernatant was decanted and neutralized with 50% (w/v) sodium hydroxide to pH 7
  • the neutralized solids-free corn steep hquor was added as the nitrogen source in certain experimental runs
  • the 10X mineral solution was prepared according to Huang et a!
  • Seed culture was grown aerobicaiiy in a 250-mL Pyrex flask at 32°C for 16 h before inoculation
  • the seed medium contains 0 125% yeast extract, 0 25% casein hydr ⁇ lyzate, 0 175% K 2 HPQi 0 075% KH 2 PO 4 0 0625% MgSO 4 , 0 125% K 2 SO ⁇ , and 2 5% (v/v) glycerol (all units are w/v except glycerol)
  • Table 1 shows the bottle preparations and Table 2 summarizes the yield of 1 ,3-pro ⁇ aned ⁇ ol from the preparations
  • the product yield was only 0 27 mol 1 3-pro ⁇ aned ⁇ ol /mol initial glycerol at 48 h, and increased to 0 32 after 72 h
  • the addition of nitrogen source did not bring positive impact to the yield, and surprisingly, it markedly inhibited the 1 ⁇ -propanediol production before 24 h in contrast, the addition of minerals (Bottle 5, 6) significantly gave rise to an increase in the 1 ,3-propaned ⁇ ol yield, which was 0 38 at 24 h and 0 42 at 72 h
  • the fermentation media supplemented with both corn steep liquor and minerals Bottle 7, 8) didn't show significant difference in 1 ,3-propaned ⁇ o
  • a process of producing 1 ,3-propaned ⁇ ol comprising fermenting glycerol in a fermentation medium with one or more microorganisms which convert glycerol to 1 ,3-propaned ⁇ ol, wherein the glycerol is from whole siiilage, thin stiliage and/or syrup or from a glycerol stream produced by a process for producing a fermentation product from hgnoceilulosic material
  • the recombinant hosts comprise a nucleic acid encoding a glycerol dehydratase and a nucleic acid encoding a 1 3-propaned ⁇ ol dehydrogenase which are native to the microorganism
  • microorganisms are independently a strain of Aerobacter Aspergillus, Bacillus Candida, Citrobacter, Clostridium, Debaryomyces, Enterobacter, Escherichia Hansenula, llyobacter, Klebsiella, Kluyveromyces, Lactobacillus, Meihylobacter, Mucor Pelobacter, Pichia, Pseudomonas, Saccharomyces Salmonella, Schizosaccharomyces, Streptomyces, Torulopsis, and Zygosaccharornyces
  • a process of producing a fermentation product and 1 ,3-propaned ⁇ ol comprising the steps of
  • the fermentation product is an alcohol (butanol, ethanol (fuel ethanol, potable ethano! and/or industrial ethano!) ⁇ acetic acid ascorbic acid, citric acid, 2,5-d ⁇ keto-D-glucon ⁇ c acid fumaric acid, gluconic acid, gluconate, glycerol, and lactic acid or succinic acid
  • alpha-amyiase is a fungal alpha-amylase, preferably derived from Aspergillus, preferably a strain of Aspergillus awamo ⁇ , Aspergillus kawachn Aspergillus niger, or Aspergillus oiyzae, or a strain of Rhizomucor, preferably a strain of Rhizomucor pusillus, or a strain of Menpilus, preferably a strain of Menpilus giganteus
  • the fungai alpha-amylase is a hybrid enzyme comprising an aipha-amylase catalytic domain (CD) and a carbohydrate-binding module/domain (CBM) and optionally linker or a wild-type fungal acsd alpha-amyiase catalytic domain (CD) and a carbohydrate-binding module (CBM) and optionally a linker
  • the hybrid alpha-amylase is selected from the group of a Rhizomucor pusiilus alpha-amylase wsth an Atheha roifsn glucoamyiase linker and starch binding domain (SBD), a Rhizomucor pus'llus alpha-amyiase with an Aspergillus niger glucoamyiase linker and SBD, a Menpilus giganteus alpha-amyiase with an Atheha roifsn glucoamyiase linker and SBD an Aspergillus niger alpha-amylase with an Aspergillus kawachi! linker and SBD, and an Aspergillus niger alpha-amyiase with an Aspergillus kawachn linker and starch binding domain (SBD)
  • SBD Rhizomucor pusiilus alpha-amylase wsth an Atheha
  • hybrid aipha-amyiase is an Aspergillus niger alpha-amyiase with an Aspergillus kawachn linker and starch binding domain (SBD)
  • carbohydrate-source generating enzyme is selected from the group consisting of beta-amyiaso, glucoamylaso, alpha-glucosidase maltogenic amylase, pu ⁇ ulanase, or a mixture of two or more thereof
  • the glucoamylase is derived from a strain of Aspergillus, preferably Aspergillus awamo ⁇ or Aspergillus n/ger, a strain of Atheha preferably Athelia rolfsu, a strain of Leucopaxillus preferably Leucopaxillus gsganteus, a strain of Pachykytospora, preferably a strain of Pachykytospo ⁇ a papyracea, a strain of Peniophota preferably a strain of Peniophota tufomarginata, a strain of Talaromycos preferably Talaiomyces emersonii, a strain of Trametes, preferably Trameies csngulaia, or a mixture thereof
  • the one or more fermenting organssms are a fungal strasn, such as a filamentous fungus strain, a yeast strain, or a bacterial strain
  • yeast is derived from a strain of Pichia, preferably a strain of Pichia st ⁇ itis or Pschia pastons. or a strain of Saceharomyces, preferably a strain of Saceharomyces cerevisiae.
  • the fermentation product is an alcohol fbutanol, ethanol (fuel ethanol, potabie ethanoi and/or industrial ethane! ⁇ ), acetic acid, ascorbic acid, citric acid, 2,5-d ⁇ keto-D-gluconic acid fumaric acid, gluconic acid, gluconate, glycerol, and lactic a ⁇ d, or succinic acid

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Abstract

La présente invention concerne des procédés destinés à la production de 1,3-propanediol consistant à faire fermenter des résidus solubles de distillation, des jus de distillation bruts, ou du sirop dans un milieu de fermentation en présence d'un ou de plusieurs microorganismes qui convertissent en 1,3-propanediol le glycérol.
PCT/US2009/035229 2008-02-27 2009-02-26 Procédés pour la production de 1,3-propanediol Ceased WO2009108748A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010128070A3 (fr) * 2009-05-05 2011-02-17 Metabolic Explorer Culture continue pour la production de 1,3-propanediol à l'aide d'une concentration en glycérine élevée
US20110130586A1 (en) * 2008-06-02 2011-06-02 University Of Saskatchewan Recovery of multiple compounds and recyclable water from thin stillage
WO2011137147A1 (fr) * 2010-04-28 2011-11-03 E. I. Du Pont De Nemours And Company Fabrication de sirop à teneur élevée en matières solides à partir de bouillon de fermentation d'hydrolysat de biomasse lignocellulosique
WO2012045179A1 (fr) * 2010-10-08 2012-04-12 University Of Saskatchewan Procédé pour la conversion de glycérol en 1,3-propanediol
US8906235B2 (en) 2010-04-28 2014-12-09 E I Du Pont De Nemours And Company Process for liquid/solid separation of lignocellulosic biomass hydrolysate fermentation broth

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2692281B1 (fr) * 1992-06-15 1995-07-21 Agronomique Inst Nat Rech Procede pour l'obtention de produits a activite bacterienne, capables de transformer le glycerol en 1,3-propanediol, souches correspondantes et application a la production industrielle de 1,3-propanediol.
CN100427605C (zh) * 2005-06-17 2008-10-22 清华大学 一种由粗淀粉原料生产1,3-丙二醇和2,3-丁二醇的方法

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110130586A1 (en) * 2008-06-02 2011-06-02 University Of Saskatchewan Recovery of multiple compounds and recyclable water from thin stillage
US8835665B2 (en) * 2008-06-02 2014-09-16 University Of Saskatchewan Recovery of multiple compounds and recyclable water from thin stillage
WO2010128070A3 (fr) * 2009-05-05 2011-02-17 Metabolic Explorer Culture continue pour la production de 1,3-propanediol à l'aide d'une concentration en glycérine élevée
WO2011137147A1 (fr) * 2010-04-28 2011-11-03 E. I. Du Pont De Nemours And Company Fabrication de sirop à teneur élevée en matières solides à partir de bouillon de fermentation d'hydrolysat de biomasse lignocellulosique
CN102858989A (zh) * 2010-04-28 2013-01-02 纳幕尔杜邦公司 从木质纤维素生物质水解产物发酵液中生产高固体糖浆
US8721794B2 (en) 2010-04-28 2014-05-13 E I Du Pont De Nemours And Company Production of high solids syrup from lignocellulosic biomass hydrolysate fermentation broth
US8906235B2 (en) 2010-04-28 2014-12-09 E I Du Pont De Nemours And Company Process for liquid/solid separation of lignocellulosic biomass hydrolysate fermentation broth
CN102858989B (zh) * 2010-04-28 2016-08-03 纳幕尔杜邦公司 从木质纤维素生物质水解产物发酵液中生产高固体糖浆
WO2012045179A1 (fr) * 2010-10-08 2012-04-12 University Of Saskatchewan Procédé pour la conversion de glycérol en 1,3-propanediol

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