EP3397772A1 - Procédés de production de produits de fermentation à partir de matière première - Google Patents

Procédés de production de produits de fermentation à partir de matière première

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Publication number
EP3397772A1
EP3397772A1 EP16823468.0A EP16823468A EP3397772A1 EP 3397772 A1 EP3397772 A1 EP 3397772A1 EP 16823468 A EP16823468 A EP 16823468A EP 3397772 A1 EP3397772 A1 EP 3397772A1
Authority
EP
European Patent Office
Prior art keywords
trehalase
sugar
fermenting
molasses
amylase
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.)
Withdrawn
Application number
EP16823468.0A
Other languages
German (de)
English (en)
Inventor
Gang Duan
Vipul GOHEL
Xuan Liu
Ying Qian
Kannan Ranganathan
Xiaoping Zhang
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.)
Danisco US Inc
Original Assignee
Danisco US Inc
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 Danisco US Inc filed Critical Danisco US Inc
Publication of EP3397772A1 publication Critical patent/EP3397772A1/fr
Withdrawn legal-status Critical Current

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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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • 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
    • 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
    • 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
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01028—Alpha,alpha-trehalase (3.2.1.28)
    • 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 disclosure relates to methods and compositions for production of one or more fermentation products such as alcohol from feedstock.
  • the disclosure particularly relates to a composition comprising trehalase for production of the fermentation products.
  • Bio-alcohols are receiving increasing attention worldwide as a result of high energy prices, the growing focus on clean energy technologies and concern about global climate change. Also, bio-alcohols are important renewable fuels contributing to the reduction of negative environmental impacts generated by the worldwide utilization of the fossil fuels.
  • Bio-alcohols can be produced from renewable resources such as raw materials containing sugars such as molasses, corn stover, corncob, bagass, rice straw, sugarcane, sugar beet etc; starch such as grains, cereals, potato, corn, rice etc; and cellulose such as lignocellulosic biomass comprising cellulose,
  • hemicellulose hemicellulose, and lignin.
  • Molasses are the waste thick by-product obtained from sugar industries during preparation of sucrose by repeated evaporation, crystallization, and centrifugation of sugarcane or sugar beet juice etc. They are economical raw materials and readily available for fermentation. For instance, in the year 2005, molasses production globally was estimated at 50.7 million tons. About 48% of the total molasses was produced in Asia, and the major share of that was produced in India, China, and Thailand. The molasses produced from cane and beet each have a similar sugar composition. Both types of molasses contain both fermentable and non-fermentable sugars.
  • beet molasses contains a lower concentration of fermentable sugars and a higher concentration of non-fermentable sugars than cane molasses.
  • Sugarcane molasses is a dark viscous fluid with pH value of 5 and very rich in nutrients required by most microorganisms. It is composed of 68.36% sucrose, 18.50% glucose, and 1 3.14% maltose. Sugarcane molasses is rich in fermentable sugars approximately 40-55% (wt%) and non-fermentable sugars recorded approximately 5% (wt%).
  • sources of molasses such as sugar cane, sugar beet, starch, maize, sweet sorghum and citrus juice etc. that are rich source of carbohydrates. Such carbohydrates can be consumed by a large number of microorganisms to produce various products.
  • One aspect of the present disclosure relates to a method for enhanced production of one or more fermentation products, comprising contacting a sugar containing medium obtained from a feedstock with an effective amount of a composition comprising trehalase.
  • One aspect of the present disclosure relates to a method for enhanced production of one or more fermentation products, comprising fermenting a sugar containing medium obtained from a feedstock in the presence of a fermenting organism and a composition comprising trehalase.
  • Another aspect of the disclosure relates to a method for enhanced production of ethanol comprising contacting a sugar containing medium obtained from a feedstock with an effective amount of a composition comprising trehalase and fermenting the sugar containing medium in presence of a fermenting organism to produce ethanol.
  • Another aspect of the present disclosure relates to a method for enhanced production ethanol comprising fermenting a sugar containing medium obtained from a feedstock in the presence of a fermenting organism and a composition comprising trehalase.
  • Figure 1 shows effect of trehalase on ethanol production from molasses.
  • sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. ⁇ 1 .821 -1 .825.
  • the Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the lUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021 -3030 (1985) and in the Biochemical J. 219 (2):345-373 (1984) which are herein incorporated by reference.
  • the symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. ⁇ 1 .822.
  • SEQ ID NO: 1 corresponds to the amino acid sequence of Trehalase. DETAILED DESCRIPTION
  • the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion.
  • a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus.
  • “or” refers to an inclusive “or” and not to an exclusive “or”. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
  • compositions, a process, a method, a structure, or a portion of a composition, a process, a method, or a structure are described herein using an open- ended term such as "comprising,” unless otherwise stated the description also includes an embodiment that "consists essentially of” or “consists of” the elements of the composition, the process, the structure, or the portion of the composition, the process, or the structure.
  • contacting refers to the placing of the respective enzymes in sufficiently close proximity to the respective substrate to enable the enzymes to convert the substrate to the end product.
  • Those skilled in the art will recognize that mixing solutions of the enzyme with the respective substrates can effect contacting.
  • Saccharide encompasses fermentable and non- fermentable sugars. It includes any chemically-defined sugar, i.e., a
  • sugar containing medium refers to extract obtained from sugar-containing sources. Preparation of the sugar-containing medium used for fermentation is well known to those skilled in the art, and generally includes extraction of a juice using crushing of the sugar-containing source. The common method employed for extraction includes but is not limited to crushing, grinding, hot water extraction, diffusion, or combination thereof.
  • Various biomass feedstocks are currently being screened for fuel and chemical applications.
  • the sugar content released through hydrolysis is of particular importance and is traditionally measured with HPLC.
  • the applicants have used Ion Chromatography (IC) for determination of sugar content of molasses and surprisingly it was found that the molasses unexpectedly contained about 1 .52% w/v trehalose which was the main sugar composition except for the sucrose, glucose, and fructose. Molasses has heretofore not been known to contain trehalose.
  • Microorganisms play crucial part in fermentation process. Yeast is widely used microorganism in fermentation technology. However, other microorganism such as bacterium and fungi other than yeast are also used for fermentation.
  • the term "fermenting organism” refers to any microorganism, including bacterium, a fungus, yeast, and algae, suitable for producing desired fermentation end products like alcohol such as ethanol and butanol, amino acids, organic acids such as lactic acid, citric acid, succinic acid, monosodium glutamate, and 1 -3 propane diol.
  • the microorganism may be bacteria,
  • Suitable microorganisms capable of producing product alcohol via a biosynthetic pathway include a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus,
  • microorganisms such as dried yeast or Saccharomyces cerevisiae, S. diastaticus Kluyveromyces marxianus, Pichia kudriavzevii; Escherichia coli strain and Klebsiella oxytoca strain, and Zymomonas mobilis may be used for production of alcohols.
  • the fermenting organism includes fungal organisms such as yeast.
  • Yeast includes, but is not limited to strains of the genus
  • the yeast is
  • Saccharomyces cerevisiae Saccharomyces cerevisiae are known in the art and are available from a variety of sources including, but not limited to, American Type Culture Collection (Rockville, MD), Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, LeSaffre, Gert Strand AB, Ferm Solutions, North
  • S. cerevisiae include, but are not limited to, BY4741 , CEN.PK 1 13-7D, Ethanol Red® yeast, Ferm ProTM yeast, Bio- Ferm® XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMaxTM Green yeast, FerMaxTM Gold yeast, Thermosacc® yeast, BG-1 , PE-2, CAT-1 , CBS7959, CBS7960, and CBS7961 .
  • the fermenting organism is a bacterium selected from a group consisting of strains of Escherichia, Zymomonas, and Klebsialla, Brevibacterium, Corynebacterium, Bacillus, Clostridium and Streptomyces.
  • the fermenting organism is a fungus other than yeast selected from a group consisting of Penicillium Sp, Acremonium Sp, Trichoderma sp, and Aspergillus sp.
  • the microorganism may be immobilized or encapsulated.
  • the microorganism may be immobilized or encapsulated using alginate, calcium alginate, or polyacrylamide gels, or through the induction of biofilm formation onto a variety of high surface area support matrices such as diatomite, celite, diatomaceous earth, silica gels, plastics, or resins.
  • high surface area support matrices such as diatomite, celite, diatomaceous earth, silica gels, plastics, or resins.
  • ISPR in situ product removal
  • encapsulation may minimize the effects of the process conditions such as shearing on the microorganisms.
  • recombinant microorganism refers to a host cell transfected, transformed, or infected in vivo or in vitro with a recombinant vector, recombinant DNA construct or a polynucleotide of interest.
  • a host cell which comprises a recombinant vector of the interest is a "recombinant host cell", “recombinant cell”, or “recombinant microorganism”.
  • a “recombinant” microorganism typically comprises one or more exogenous nucleotide sequences, such as in a plasmid or vector.
  • Standard recombinant DNA and molecular cloning techniques are well known in the art and are described by Sambrook, et al. (Sambrook, J., Fritsch, E. F. and Maniatis, T. (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989, here in referred to as Maniatis) and by Ausubel, et al. (Ausubel, et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-lnterscience, 1987) and may be used for creating recombinant microorganisms.
  • the metabolic pathways of microorganisms may be genetically modified to produce one or more fermentation product such as alcohol. In some embodiments these pathways may also be modified to reduce or eliminate undesired product, and thereby improve yield of the desired product.
  • recombinant microorganisms may be selected from the group consisting of Escherichia coli, Alcaligenes eutrophus, Bacillus lichenifonnis, Paenibacillus macerans, Rhodocuccus erythropolis, Pseudomonas putida,
  • the recombinant microorganism is yeast.
  • the recombinant yeast is a crabtree-positive yeast selected from
  • Saccharomyces Zygosaccharomyces, Schizosaccharomyces, Dekkera, Torulopsis, Brettanomyces, and some species of Candida.
  • Species of crabtree-positive yeast include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Saccharomyces bayanus, Saccharomyces mikitae, Saccharomyces paradoxus, Saccharomyces uvarum, Saccharomyces castelli, Zygosaccharomyces rouxii, Zygosaccharomyces bailli, and Candida glabrata.
  • Recombinant microorganisms which produce alcohol are also known in the art (e.g., Ohta et al., Appl. Environ. Microbiol. 57:893-900 (1991 ); Underwood et al., Appl. Envrion. Microbiol. 68:1071 -81 (2002); Shen and Liao, Metab. Eng. 1 0:312-20 (2008); Hahnai et al., Appl. Environ. 73:7814-8 (2007); U.S. Patent No. 5,514,583; U.S. Patent No. 5,71 2,133; International Publication No. WO 1995/028476;
  • Biomass refers to a sugar containing source, including any cellulosic or lignocellulosic material and materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides, disaccharides, and/or monosaccharaides. Biomass can also comprise additional components, such as protein and/or lipids.
  • Some of the most common biomasses are grains and starch crops, agricultural residues, food waste, forestry materials, animal by-products, energy crops, urban and suburban wastes.
  • Biomass can be derived from a single source, or biomass can comprise a mixture derived from more than one source.
  • biomass can comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves.
  • Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, and sludge from paper manufacture, yard waste, wood, and forestry waste.
  • biomass examples include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, rye, wheat straw, barley, barley straw, hay, rice, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, cassava, milo, millet, soy, components or by products obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.
  • crop residues such as corn husks, corn stover, grasses, wheat, rye, wheat straw, barley, barley straw, hay, rice, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, cassava, milo, millet, soy, components or by products obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal
  • biomass also include aquatic or marine biomass, fruit-based biomass such as fruit waste, and vegetable-based biomass such as vegetable waste, among others.
  • aquatic or marine biomass include, but are not limited to, kelp, giant kelp, seaweed, algae, and marine microflora, microalgae, sea grass, and the like.
  • biomass does not include fossilized sources of carbon, such as hydrocarbons that are typically found within the top layer of the Earth's crust (e.g., natural gas, nonvolatile materials composed of almost pure carbon, like anthracite coal, etc).
  • feedstocks A wide variety of feedstocks are available. Most feedstocks can be made into biofuels, heat, electric power, and/or bio-based products. This makes feedstocks a flexible and widespread resource.
  • feedstock means a raw material or mixture of raw materials containing sugars and carbon source used during fermentation process.
  • a carbon source such as biomass or the carbon compounds derived from biomass are a feedstock for microorganism that produces one or more fermentation products in a fermentation process.
  • a feedstock may contain nutrients other than a carbon source.
  • Example of feedstock includes but is not limited to molasses, corn, corn mash, corn cobs, corn grain, corn husks, corn stover, sugar cane, powercane mash, sugar cane bagasse, sugar beets, date palm, sorghum, sugar maple, wheat, wheat straw, rye, barley, barley straw, hay, rice, rice straw, switchgrass, orchard prunings, waste paper, soy, sweet potatoes, mango kernel, palm kernel, palm kernel cake, cassava, components obtained from milling or grinding of grains including "enriched fiber fraction, stillage, wet cake, distillers dry grains (DDG) and distillers dry grains with solubles (DDGs), trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, cellulosic material, lignocellulosic material, waste produce, food processing waste, logging residues, forest thinnings, tallow, fish oil, manure, switch
  • Molasses is the by-product in sucrose industry. Molasses can be used as raw material for production of fermentation products such as ethanol.
  • molasses refers to a concentrate obtained from mother liquor from sugar crystallization process.
  • molasses source include but are not limited to sugar cane, sugar beet, starch containing sources or feedstocks, maize, sweet sorghum, sugar cane juice, and citrus juice.
  • composition refers a composition comprising trehalase.
  • blend refers to mixture of a composition comprising trehalase and at least one other enzyme including amylase, a-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase,
  • endoglucanase endoglucanase, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a- glucosidase, ⁇ -glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, ⁇ -amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.
  • the term encompasses mixture of trehalase enzyme and at least one other enzyme including amylase, a-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a-glucosidase, ⁇ -glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, ⁇ -amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.
  • amylase a-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase,
  • composition comprises trehalase enzyme.
  • composition comprises trehalase enzyme having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof.
  • the composition comprises blend of trehalase and at least one other enzyme including amylase, a-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a-glucosidase, ⁇ -glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, ⁇ -amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.
  • amylase a-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellula
  • composition or a blend comprising trehalase enzyme in combination with at least one other enzyme selected from a group consisting of amylase, a-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a-glucosidase, ⁇ -glucosidase,
  • transglucosidase glucose isomerase, xylose isomerase, phosphatase, ⁇ -amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.
  • compositions comprising trehalase, glucoamylase, and a-amylase.
  • composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, and at least one enzyme including glucoamylase, and a-amylase.
  • compositions comprising trehalase, glucoamylase, and ⁇ -amylase and protease.
  • composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, at least one enzyme including glucoamylase, a-amylase, and protease.
  • compositions comprising trehalase, glucoamylase, a-amylase, and ⁇ -glucosidase.
  • compositions comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, at least one other enzyme including glucoamylase, a-amylase, and a- glucosidase.
  • composition comprising trehalase, glucoamylase, a-amylase, cellulase and ⁇ -glucosidase.
  • compositions comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, at least one other enzyme including glucoamylase, a-amylase, cellulase, and ⁇ -glucosidase.
  • compositions comprising trehalase, glucoamylase, a-amylase, xylanase, cellulose, and ⁇ -glucosidase.
  • composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, at least one other enzyme including glucoamylase, a- amylase, xylanase, cellulase, and ⁇ -glucosidase.
  • composition or a blend comprising trehalase, glucoamylase, a-amylase, a-glucosidase, xylanase, cellulose, and ⁇ -glucosidase.
  • compositions comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, at least one other enzyme including amylase, a-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a-glucosidase, ⁇ -glucosidase,
  • transglucosidase glucose isomerase, xylose isomerase, phosphatase, ⁇ -amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.
  • microorganism that expresses trehalase.
  • the microorganism may be a fermenting organism or may grow in the presence of a fermenting organism.
  • the microorganism may optionally produce additional enzymes, such as glucoamylase, a-amylase, pullulanase, protease, and the like.
  • the trehalase and other enzymes are preferably expressed and secreted.
  • the microorganism may be grown under condition that increase expression and/or secretion of the enzymes, or that increase the permeability of cell membranes resulting in the release of enzymes, accumulated sugars, or other cell contents into the fermentation broth. Such conditions include the presence of detergents, solvents, or lysogenic or pore-forming proteins. Fermentation products
  • the term "fermentation product” or “fermentation yield” includes any desired product of interest, produced by a fermentation process including, but not limited to alcohol (e.g. ethanol, methanol, propanol and butanol), organic acids such as citric acid, lactic acid, succinic acid, acetic acid and gluconic acid), and amino acids such as glutamic acid, tryptophan, threonine, lysine, serine, tyrosine and methionine.
  • alcohol e.g. ethanol, methanol, propanol and butanol
  • organic acids such as citric acid, lactic acid, succinic acid, acetic acid and gluconic acid
  • amino acids such as glutamic acid, tryptophan, threonine, lysine, serine, tyrosine and methionine.
  • the fermentation product may be, but is not limited to, metabolites, such as citric acid, lactic acid, succinic acid, acetic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, itaconic acid and other carboxylic acids, glucono delta-lactone, sodium erythorbate, glutamic acid, tryptophan, threonine, methionine, lysine and other amino acids, omega-3 fatty acid, isoprene, 1 ,3-propanediol, ethanol, methanol, propanol, butanol, other alcohols, and other bio-chemicals and biomaterials.
  • metabolites such as citric acid, lactic acid, succinic acid, acetic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, itaconic acid and other carboxylic acids,
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological, microbiological and biochemical arts.
  • the disclosure provides methods for enhanced production of one or more fermentation products such as alcohol, metabolites, and other bio-chemicals and biomaterials using a composition comprising trehalase during or prior to
  • embodiments of the methods include methods of contacting sugar containing medium obtained from feedstock with an enzyme composition comprising trehalase resulting in the hydrolysis of trehalose.
  • the method can also include contacting the sugar containing medium with trehalase in combination with one or more other enzymes including amylase, ⁇ -amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a-glucosidase, ⁇ -glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, ⁇ -amylase, lipase, cutinase,
  • enzymes including amylase, ⁇ -amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellulase, xylanase, cello
  • isoamylase redox enzyme
  • esterase redox enzyme
  • transferase redox enzyme
  • pectinase pectinase
  • Some embodiments of the disclosure provides methods for enhanced production of one or more fermentation products from sugar containing medium obtained from molasses comprising contacting the medium with trehalase enzyme to obtain the fermentation product such as ethanol.
  • the method can also include contacting the sugar containing medium obtained from molasses with trehalase in combination with one or more other enzymes including amylase, a-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a- glucosidase, ⁇ -glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, ⁇ -amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transfera
  • the methods can also include fermenting the sugar containing medium obtained from feedstock to end products in the presence of fermenting
  • the contacting and fermenting can occur simultaneously or the contacting can be a pretreatment.
  • the methods comprise contacting a sugar containing medium obtained from a feedstock with a composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof.
  • the methods further comprise fermenting the sugar containing medium in presence of a fermenting organism.
  • the methods comprise contacting a sugar containing medium obtained from a feedstock with a composition comprising trehalase, glucoamylase, and a-amylase. In some embodiments the methods comprise contacting the sugar containing medium obtained from a feedstock with a composition comprising trehalase, glucoamylase, and a-amylase. In some embodiments the methods comprise contacting the sugar containing medium obtained from a feedstock with a
  • composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, and at least one other enzyme including glucoamylase, and ⁇ -amylase.
  • methods further comprise fermenting the sugar containing medium in presence of a fermenting organism.
  • the methods comprise contacting a sugar containing medium obtained from a feedstock with a composition comprising trehalase, glucoamylase, and a-amylase and protease.
  • the methods comprise contacting the sugar containing medium obtained from a feedstock with a composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, and at least one other enzyme including glucoamylase, a-amylase, and protease.
  • the methods further comprise fermenting the sugar containing medium in presence of a fermenting organism.
  • the methods comprise contacting a sugar containing medium obtained from a feedstock with a composition comprising trehalase, glucoamylase, an a-amylase, and a-glucosidase.
  • the methods comprise contacting the sugar containing medium obtained from a feedstock with a composition comprising trehalase having amino acid sequence as set forth SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, and at least one other enzyme including glucoamylase, ⁇ -amylase anda-glucosidase.
  • the method further comprises fermenting the sugar containing medium in presence of fermenting organism.
  • the methods comprise contacting a sugar containing medium obtained from a feedstock with a composition comprising trehalase, glucoamylase, a-amylase, cellulase and ⁇ -glucosidase.
  • the methods comprise contacting the sugar containing medium obtained from a feedstock with a composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, and at least one other enzyme including glucoamylase, a-amylase, cellulase, and ⁇ -glucosidase.
  • the method further comprises fermenting the sugar containing medium in presence of fermenting organism.
  • the methods comprise contacting a sugar containing medium obtained from a feedstock with a composition comprising trehalase, glucoamylase, ⁇ -amylase, xylanase, cellulose, and ⁇ -glucosidase.
  • the methods comprise contacting the sugar containing medium obtained from a feedstock with a composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, and at least one other enzyme including glucoamylase, a-amylase, xylanase, cellulase, and ⁇ -glucosidase.
  • the method further comprises fermenting the sugar containing medium in presence of fermenting organism.
  • the methods comprise contacting a sugar containing medium obtained from a feedstock with a composition comprising trehalase, and at least one other enzyme including glucoamylase, a-amylase, a-glucosidase, xylanase, cellulose, other hemicellulases, and ⁇ -glucosidase.
  • the methods comprise contacting the sugar containing medium obtained from a feedstock with a composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, and at least one other enzyme including glucoamylase, a-amylase, a-glucosidase, xylanase, cellulase, and ⁇ -glucosidase.
  • the method further comprises fermenting the sugar containing medium in presence of fermenting organism.
  • the contacting and fermenting occur simultaneously. In some embodiments the contacting occurs as a pretreatment.
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase.
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase having amino acid sequence as set forth SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof.
  • the methods further comprise fermenting the molasses in presence of a fermenting organism.
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase, and at least one other enzyme including glucoamylase, and a- amylase.
  • the methods comprise contacting the molasses with a composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, and at least one other enzyme including glucoamylase, and a-amylase.
  • the methods further comprise fermenting the molasses in presence of a fermenting organism.
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase, and at least one other enzyme including glucoamylase, and a- amylase and protease.
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, and at least one other enzyme including glucoamylase, a-amylase, and protease.
  • the methods further comprise fermenting the molasses in presence of a fermenting organism.
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses a composition
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof.
  • the methods further comprise fermenting the molasses in presence of a fermenting organism.
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase, and at least one other enzyme including glucoamylase, a- amylase, cellulase and ⁇ -glucosidase.
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, glucoamylase, a-amylase, cellulase, and ⁇ -glucosidase.
  • the methods further comprise fermenting the molasses in presence of a fermenting organism.
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase, glucoamylase, a-amylase, xylanase, cellulose, and ⁇ - glucosidase.
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, glucoamylase, a-amylase, xylanase, cellulase, and ⁇ - glucosidase.
  • the methods further comprise fermenting the molasses in presence of a fermenting organism.
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase, glucoamylase, a-amylase, a-glucosidase, xylanase, cellulose, and ⁇ -glucosidase.
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, glucoamylase, a-amylase, a-glucosidase, xylanase, cellulase, and ⁇ -glucosidase.
  • the methods further comprise fermenting the molasses in presence of a fermenting organism.
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase, and at least one other enzyme including amylase, a-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a- glucosidase, ⁇ -glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, ⁇ -amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.
  • amylase a-amylase, glucoamylase, pullulanase, and other starch degrading
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, and at least one other enzyme including amylase, a-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a-glucosidase, ⁇ -glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, ⁇ -amylase, lipase, cutinase,
  • isoamylase redox enzyme
  • esterase redox enzyme
  • transferase redox enzyme
  • pectinase pectinase
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase, and at least one other enzyme including amylase, a-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a- glucosidase, ⁇ -glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, ⁇ -amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.
  • amylase a-amylase, glucoamylase, pullulanase, and other starch degrading
  • a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase having amino acid sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof, and at least one other enzyme including amylase, a-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a-glucosidase, ⁇ -glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, ⁇ -amylase, lipase, cutinase,
  • isoamylase redox enzyme, esterase, transferase, and pectinase.
  • the methods further comprise fermenting the molasses in presence of a fermenting organism.
  • the aforementioned contacting occurs as a pre- treatment followed by fermenting step.
  • the contacting and/or pre-treatment temperature is in the range of ' ⁇ 5°C to 60 °C. In some embodiments, the contacting and/or
  • pretreatment temperature is below about 60 °C. In some embodiments, the fermentation temperature is from about 15°C to 40 °C. In some embodiments, the fermentation, contacting and pretreatment temperature is from about 15 to 40 °C. In some embodiments the contacting and/or pretreatment is conducted at a pH from about 2.0 to 7.0 or alternatively 3.5 to 7.0. In some embodiments the fermentation is conducted at a pH from about 2.0 to 7.0 or 3.5 to 7.0 or alternatively 2.0 to 9.0.
  • the fermentation products of the disclosure may be prepared by the process as described herein.
  • Fermenting organisms of the disclosure are contacted with suitable carbon source, typically in fermentation media.
  • Additional carbon source may include, but are not limited to, monosaccharaides such as fructose, oligosaccharides such as lactose, maltose, galactose, or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt.
  • Other carbon source can include ethanol, lactate, succinate, or glycerol.
  • the carbon source may also be one-carbon source such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated.
  • methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity.
  • methylotrophic yeasts are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32, Editors: Murrell, J. Collin, Kelly, Don P.; Publisher: Intercept, Andover, UK).
  • Candida will metabolize alanine or oleic acid (Suiter et al., Arch. Microbiol. 153:485-489 (1990)).
  • the source of carbon utilized in the present disclosure may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.
  • the carbon sources are glucose, fructose, and sucrose, or mixtures of these with C5 sugars such as xylose and/or arabinose for yeasts cells modified to use C5 sugars.
  • Sucrose may be derived from renewable sugar sources such as sugar cane, sugar beets, cassava, sweet sorghum, and mixtures thereof.
  • Glucose and dextrose can be derived from renewable grain sources through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, oats, rice and mixtures thereof.
  • fermentable sugars can be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Patent Application Publication No. 2007/0031918 A1 , which is herein incorporated by reference.
  • Biomass when used in reference to carbon source, refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, Iignin, starch, oligosaccharides and/or monosaccharides. Biomass can also comprise additional components, such as protein and/or lipid.
  • Biomass can be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves.
  • Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste.
  • biomass examples include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover grasses, wheat, wheat straw, barley, barley straw, hay, rice, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, cassava, milo, millet, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.
  • corn grain corn cobs
  • crop residues such as corn husks, corn stover grasses, wheat, wheat straw, barley, barley straw, hay, rice, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, cassava, milo, millet, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.
  • fermentation media may contain 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 an enzymatic pathway described herein.
  • cells are grown at a temperature in the range of about 1 5°C to about 60 °C in an appropriate medium.
  • the cells are grown at a temperature of 15 , 20 °C, 22 °C, 25 °C, 27 °C, 30 °C, 32 °C, 35 °C, 37 °C or 40 ⁇ O, 42°C, 45 ⁇ ⁇ , 47°C, 50°C, 52°C, 57°C, 60°C.
  • the cells are grown at a temperature of about 25°C to about 40 °C in an appropriate medium. In one embodiment the temperature is 30 °C to 34°C.
  • Suitable growth media for growth of the fermenting organisms are common and commercially available media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Medium (YM) broth or broth that includes yeast nitrogen base, ammonium sulfate, and dextrose (as the carbon/energy source) or YPD Medium, a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most Saccharomyces cerevisiae strains.
  • Other defined or synthetic growth media can also be used, and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science.
  • agents known to modulate catabolite repression directly or indirectly e.g., cyclic adenosine 2'
  • cAMP 3'-monophosphate
  • Suitable pH ranges for the fermentation are between pH 2.0 to pH 9.0, where pH 6.0 to pH 8.0 may be preferred for the initial condition.
  • Suitable pH ranges for the fermentation of yeast are typically between about pH 3.0 to about pH 9.0. In one embodiment, about pH 5.0 to about pH 8.0 is used for the initial condition.
  • Suitable pH ranges for the fermentation of other microorganisms are between about pH 2.0 to about pH 7.5. In one embodiment, about pH 4.5 to about pH 6.5 is used.
  • Fermentations can be performed under aerobic or anaerobic conditions. In one embodiment, anaerobic or microaerobic conditions are used for fermentation.
  • the culture conditions are such that the fermentation occurs without respiration.
  • cells can be cultured in a fermenter under micro-aerobic or anaerobic conditions.
  • Alcohol such as ethanol, or other fermentation products can be produced using a batch method of fermentation.
  • Classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation.
  • a variation on the standard batch system is the fed-batch system.
  • Fed-batch fermentation processes are also suitable in the present disclosure and comprise a typical batch system with the exception that the substrate is added in increments at the fermentation progresses.
  • Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media.
  • Batch and fed-batch fermentations are common and well known in the art and examples can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA. or Deshpande, Mukund V., Appl.
  • Alcohol such as ethanol, or other fermentation products, may also be produced using continuous fermentation methods.
  • Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing.
  • Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
  • Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.
  • Alcohol such as ethanol, or other fermentation products
  • production of Alcohol can be practiced using batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable.
  • cells can be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for alcohol production.
  • One embodiment of the present disclosure provides a method for enhanced production of one or more fermentation products, comprising contacting a sugar containing medium obtained from a feedstock with an effective amount of a composition comprising trehalase.
  • Another embodiment of the present disclosure provides a method for enhanced production of one or more fermentation products, comprising contacting a sugar containing medium obtained from a feedstock with an effective amount of a composition comprising trehalase, wherein the method further comprising fermenting the sugar containing medium in the presence of fermenting microorganisms.
  • Yet another embodiment of the present invention provides a method for enhanced production of one or more fermentation products, comprising contacting a sugar containing medium obtained from a feedstock with an effective amount of a composition comprising trehalase, wherein the contacting temperature is in the range of 15°C to 60 °C. In some embodiments the contacting temperature is in the range of 15°C to 40 °C.
  • Another embodiment of the present disclosure provides a method for enhanced production of one or more fermentation products, comprising contacting a sugar containing medium obtained from a feedstock with an effective amount of a composition comprising trehalase, wherein the method further comprising fermenting the sugar containing medium in the presence of fermenting microorganisms, wherein the fermenting temperature is in the range of 15°C to 40°C.
  • the contacting temperature is in the range of 15°C to 40°C and the fermenting temperature is in the range of 15°C to 40 °C.
  • the fermenting is conducted at a pH from about 2.0 to 9.0.
  • the trehalase is acid trehalase. In some embodiments the trehalase is from Trichoderma species.
  • amino acid sequence of the trehalase is as set forth in SEQ ID NO: 1 or an active fragment thereof, or 85% or greater identity to SEQ ID NO: 1 or an active fragment thereof.
  • One embodiment of the present disclosure provides a method for enhanced production of one or more fermentation products, comprising fermenting a sugar containing medium obtained from a feedstock in the presence of a fermenting organism and a composition comprising trehalase.
  • Another embodiment of the present disclosure provides a method for enhanced production of one or more fermentation products, comprising fermenting a sugar containing medium obtained from a feedstock in the presence of a fermenting organism and a composition comprising trehalase and at least one enzyme selected from the group consisting of amylase, a-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a-glucosidase, ⁇ -glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, ⁇ -amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.
  • amylase a-
  • feedstock selected from a group consisting of: molasses, corn, corn mash, corn cobs, corn grain, corn husks, corn stover, sugar cane, sugar cane bagasse, sugar beets, date palm, sorghum, sugar maple, wheat, wheat straw, rye, barley, barley straw, hay, rice, rice straw, switchgrass, orchard prunings, waste paper, soy, sweet potatoes, mango kernel, palm kernel, palm kernel cake, cassava, components obtained from milling or grinding of grains (including "enriched fiber fraction, stillage, distillers dry grains (DDG) and distillers dry grains with solubles (DDGs), trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, cellulosic feedstocks, waste produce, food processing waste, logging residues, forest thinnings, tallow, fish oil, manure, switchgrass, miscanthus, poplar, will
  • Another embodiment relates to sources of molasses, wherein the molasses is obtained from a source selected from a group consisting of sugar cane, sugar beet, starch containing sources or feedstocks, maize, sweet sorghum, sugar cane juice, and citrus juice.
  • Further embodiment provides fermentation products selected from the group consisting of alcohol, metabolites, and other bio-chemicals and biomaterials. Yet another embodiment provides to the alcohol selected from the group consisting of ethanol, butanol, methanol, propanol, iso-amyl alcohol, 1 ,3-propanediol, and other alcohols.
  • Further embodiment provides metabolites selected from a group consisting of citric acid, lactic acid, succinic acid, acetic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, itaconic acid and other carboxylic acids, glucono delta-lactone, sodium erythorbate, glutamic acid, tryptophan, threonine, methionine, lysine, serine, tyrosine and other amino acids, omega-3 fatty acid, isoprene, ergosterol, vitamin and its precursor, and antibiotic, enzymes, other Biochemicals or Biomaterials.
  • Another embodiment relates to fermenting organism selected from a group consisting of is yeast, a bacterium, a fungus and an algae. Another embodiment relates to the fermenting organism, wherein the fermenting organism is a
  • yeast selected from a group consisting of a Saccharomyces sp., a Candida sp., a Pichia sp., a Dekkera sp., a Hanseniaspora sp., a Pseudozyma sp., a Sacharromycodes sp., a
  • Zygosaccharomyces sp. a Torulaspora sp., a Debaryomyces sp., a Zygoascus sp., a Issatchenkia sp., a Williopsis sp., and a Brettanomyces sp.
  • the bacterium selected from a group consisting of strains of Escherichia, Zymomonas, and Klebsialla, Brevibacterium, Corynebacterium, Bacillus, Clostridium and Streptomyces.
  • Yet another embodiment relates to the fungus selected from a group consisting of Penicillium Sp, and Acremonium Sp., Trichoderma sp, and Aspergillus sp.
  • Another embodiment provides a method of producing ethanol comprising contacting a sugar containing medium obtained from a feedstock with an effective amount of a composition comprising trehalase and fermenting the sugar containing medium in the presence of a fermenting organism to produce ethanol.
  • the contacting and fermenting occur simultaneously or the contacting occurs as a pretreatment.
  • the contacting temperature is in the range of 15°C to 60 °C.
  • the contacting temperature is in the range of 15°C to 40 °C.
  • the fermenting temperature is in the range of 15°C to 40 °C.
  • the fermenting is conducted at a pH from about 2.0 to 9.0.
  • Further embodiment of the present disclosure relates to a method of producing ethanol comprising fermenting a sugar containing medium obtained from a feedstock in presence of a fermenting organism and a composition comprising trehalase.
  • Yet another embodiment of the present disclosure relates to a method of producing ethanol comprising fermenting a sugar containing medium obtained from a feedstock in the presence of a fermenting organism and a composition comprising trehalase, wherein the method yields at least 2% more ethanol as compared with feedstock fermentation carried out without using the composition comprising trehalase.
  • C is Celsius
  • [iL” is microliter
  • “ml_” is milliliter
  • “M” is molar
  • “mm” is milimeter
  • “nm” is nanometer
  • “ ⁇ ” is micron
  • “min” is minute(s)
  • "g” is gram(s)
  • "v/v” is volume by volume
  • "w/w” is weight by weight
  • wt % means weight percent
  • “Temp” is temperature
  • "OD” mean optical density
  • "MW” means average molecular weight
  • “hr” means hour.
  • Sugarcane molasses is used as a carbon source for fermentation studies. The molasses was stored at 4°C to 25 °C until use.
  • Saccharomyces cerevisiae alcohol dry yeast commercially available from Fali Yeast, AB Mauri was used.
  • Molasses Medium was prepared by diluting 333g to 400 g molasses with water to final weight of 1000 g (w/w). To this 400 mg of urea was added as a Nitrogen source.
  • Saccharomyces cerevisiae was used in the form of active dry yeast (ADY) at a concentration of 3 to 6% w/w.
  • Enzyme composition • OPTIMASHTM TREHALASE from Genencor, International Inc., US
  • Dionex ICS-5000 Column: CarboPac PA 200; Column temperature: 30C;
  • HPLC pressure/performance liquid chromatographic method
  • composition 1 comprising trehalase (OPTIMASHTM trehalase from Genencor, International Inc., US) at a concentration ranging from 0 to 60 ppm followed by 3 g of active dry yeast into 1 00 ml molasses media in 250 mL Erienmeyer flasks. Fermentation was carried out at 32°C ⁇ 2°C in incubator shaker at 100-200 rpm until glucose value observed was less than 0.1 % w/v. Thereafter, the samples were analysed for ethanol content.
  • trehalase OPTIMASHTM trehalase from Genencor, International Inc., US

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Abstract

La présente invention concerne des procédés et des compositions de production améliorée d'au moins un produit de fermentation résultant de la fermentation d'un milieu contenant du sucre obtenu à partir d'une ou de plusieurs matières premières. Les compositions de l'invention comprennent de la tréhalase.
EP16823468.0A 2015-12-28 2016-12-20 Procédés de production de produits de fermentation à partir de matière première Withdrawn EP3397772A1 (fr)

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