EP3397772A1

EP3397772A1 - Methods for the production of fermentation products from feedstock

Info

Publication number: EP3397772A1
Application number: EP16823468.0A
Authority: EP
Inventors: Gang Duan; Vipul GOHEL; Xuan Liu; Ying Qian; Kannan Ranganathan; Xiaoping Zhang
Original assignee: Danisco US Inc
Current assignee: Danisco US Inc
Priority date: 2015-12-28
Filing date: 2016-12-20
Publication date: 2018-11-07
Also published as: KR102784155B1; KR20180114006A; WO2017116840A1; BR112018013214A2

Abstract

Methods and compositions for enhanced production of at least one fermentation product resulting from the fermentation of sugar containing medium obtained from one or more feedstocks are disclosed herein. The compositions disclosed herein comprise trehalase.

Description

METHODS FOR THE PRODUCTION OF FERMENTATION PRODUCTS

FROM FEEDSTOCK

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] The present application claims the benefit of International Application

No. PCT/CN2015/099147, filed December 28, 2015, which is hereby incorporated by reference.

TECHNICAL FIELD

[002] 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.

BACKGROUND

[003] 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.

[004] 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, and lignin.

[005] 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. However, 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%). There are various 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.

[006] Production of alcohol and other fermentation products from molasses by industrial microorganisms such as yeast (Saccharomyces cerevisiae) is of great commercial importance. Most of the readily available sugars such as sucrose, glucose and fructose in molasses can be consumed by the yeast during

fermentation. The fermentation efficiency or the carbon conversion into end products during yeast fermentation depends on several factors, i.e. pH, and temperature, dry solids content, organic acid and most importantly glycerol. The production of unproductive yeast, often referred to as wild yeast is undesirable because it produces only biomass at the cost of end product. State of the art describes various processes for production of fermentation products including bio-alcohol. However, efforts to enhance production of fermentation products such as alcohol production have fallen short of expectation.

[007] Thus, there is a need in the industry to develop methods and compositions for enhanced production of fermentation products including bio-alcohol such as ethanol.

SUMMARY

[008] 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. [009] 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.

[0010] 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.

[0011] 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings, which are incorporated herein and form part of the specification, illustrate the present disclosure and, together with the description, further serve to explain the principles of the disclosure and to enable a person skilled in the relevant art(s) to make and use the disclosure . In the drawings, like reference numbers, letters, or renderings indicate identical or functionally similar elements.

[0013] Figure 1 shows effect of trehalase on ethanol production from molasses.

SEQUENCE LISTING

[0014] The 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.

[0015] SEQ ID NO: 1 corresponds to the amino acid sequence of Trehalase. DETAILED DESCRIPTION

[0016] Unless otherwise defined, all technical and scientific terms used in the present specification and the appended claims have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0017] The following definitions are provided as an aid to understand the present disclosure.

[0018] It is to be understood that the disclosure is not limited to particular embodiments, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, terms in the singular and the singular forms "a", "an" and "the", for example, include plural referents unless the content clearly dictates otherwise. As used herein, where the indefinite article "a" or "an" is used with respect to a statement or description of the presence of a step in a process disclosed herein, unless the statement or description explicitly provides to the contrary, the use of such indefinite article is not intended to limit the presence of the step in the process to one in number.

[0019] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used for testing of the subject matter recited in the current disclosure, the preferred materials and methods are described herein. In describing and claiming the subject matter of the current disclosure, the following terminology will be used in accordance with the definitions set out below.

[0020] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains" or "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, 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. Further, unless expressly stated to the contrary, "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).

[0021] As used herein, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed.

Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the disclosure be limited to the specific values recited when defining a range.

[0022] When materials, methods, or machinery are described herein with the term "known to those of skill in the art", "conventional" or a synonymous word or phrase, the term signifies that materials, methods, and machinery that are conventional at the time of filing the present application are encompassed by this description. Also encompassed are materials, methods, and machinery that are not presently conventional, but that will have become recognized in the art as suitable for a similar purpose.

[0023] When a composition, a process, a method, a structure, or a portion of a composition, a process, a method, or a structure, is 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.

[0024] Further in this connection, certain features of the disclosure which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination. [0025] The term "optionally" is used herein to mean "is provided in some

embodiments and not provided in other embodiments". Any particular embodiment of the disclosure may include a plurality of "optional" features unless such features conflict.

[0026] Throughout this application, various embodiments of the disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and should not be construed as a limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0027] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "in the range/ranging/ranges between" a first indicated number and a second indicated number and "ranging/ranges from" a first indicated number "to" a second indicated number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[0028] The term "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.

[0029] The term "Sugar," as used herein, encompasses fermentable and non- fermentable sugars. It includes any chemically-defined sugar, i.e., a

monosaccharide, disaccharide, tri-saccharide, or oligosaccharide that is suitable to be fermented to produce a fermentation product, in particular, ethanol.

[0030] The term "sugar containing medium" as used herein 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.

[0031] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the compositions, films, and methods described herein. In this application, the use of the singular includes the plural unless specifically state otherwise. Also, the use of "or" means "and/or" unless state otherwise.

[0032] The following discussion is directed to the embodiments of the present disclosure only, and nothing within the following disclosure is intended to limit the overall scope of this disclosure. The scope of the present disclosure is to be defined solely by the claims, as presented at the end of this specification.

[0033] Determination of the sugar content of biomass feedstock

[0034] 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. However, in the present disclosure 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

[0035] 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.

[0036] 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. In some embodiments the microorganism may be bacteria,

cyanobacteria, filamentous fungi, or yeasts. 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,

Enterococcus, Alcaligenes, Paenibacillus, Arthrobacter, Corynebacterium,

Brevibacterium, Schizosaccharomyces, Kluveromyces, Yarrowia, Pichia,

Zygosaccharomyces, Debaryomyces, Candida, Brettanomyces, Pachysolen, Hansenula, Issatchenkia, Trichosporon, Yamadazyma, or Saccharomyces. In some embodiments, 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.

[0037] In some embodiments, the fermenting organism includes fungal organisms such as yeast. Yeast includes, but is not limited to strains of the genus

Saccharomyces, Pichia, Candida. Dekkera, Hanseniaspora, Pseudozyma,

Sacharromycodes, Zygosaccharomyces, Zygosaccharomyces, Zygoascus spp, Issatchenkia, Williopsis, Torulaspora, Debaryomyces, Zygosaccharomyces,

Brettanomyces and Brettanomyces. In some embodiments, 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

American Bioproducts, Martrex, and Lallemand. S. cerevisiae include, but are not limited to, BY4741 , CEN.PK 1 13-7D, Ethanol Red® yeast, Ferm Pro™ yeast, Bio- Ferm® XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMax™ Green yeast, FerMax™ Gold yeast, Thermosacc® yeast, BG-1 , PE-2, CAT-1 , CBS7959, CBS7960, and CBS7961 .

[0038] In some embodiments, the fermenting organism is a bacterium selected from a group consisting of strains of Escherichia, Zymomonas, and Klebsialla, Brevibacterium, Corynebacterium, Bacillus, Clostridium and Streptomyces.

[0039] In some embodiments, the fermenting organism is a fungus other than yeast selected from a group consisting of Penicillium Sp, Acremonium Sp, Trichoderma sp, and Aspergillus sp.

[0040] In some embodiments, the microorganism may be immobilized or encapsulated. For example, 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. In some embodiments, "in situ product removal" (ISPR) may be used in combination with immobilized or encapsulated microorganisms. This combination may improve productivity such as specific volumetric productivity, metabolic rate, product alcohol yields, and tolerance to product alcohol. In addition, immobilization and

encapsulation may minimize the effects of the process conditions such as shearing on the microorganisms.

Recombinant microorganisms

[0041] The term "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.

[0042] 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. For example, 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.

[0043] In one embodiment, recombinant microorganisms may be selected from the group consisting of Escherichia coli, Alcaligenes eutrophus, Bacillus lichenifonnis, Paenibacillus macerans, Rhodocuccus erythropolis, Pseudomonas putida,

Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium,

Enterococcus faecalis, Bacillus subtilis, Candida sonorensis, Candida methanosorbosa, Kluyveromyces lactis, Kluyveromyces marxianus, Kluveromyces thermotolerans, Issatchenkia orientalis, Debaryomyces hansenii, and

Saccharomyces cerevisiae.

[0044] In one embodiment, the recombinant microorganism is yeast. In one embodiment, 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.

[0045] 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;

Feldmann et al., Appl. Microbiol. Biotechnol. 38:354-61 (1992); Zhang et al., Science 267:240-3 (1995); U.S. Patent Publication No. 2007/0031918A1 ; U.S. Patent No. 7,223,575; U.S. Patent No. 7,741 ,1 19; U.S. Patent Publication No. 2009/0203099A1 ; U.S. Patent Publication No. 2009/0246846A1 ; and International Publication No. WO 2010/075241 , which are herein incorporated by reference).

Biomass

[0046] The term "Biomass" as used herein 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.

[0047] 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.

[0048] Biomass can be derived from a single source, or biomass can comprise a mixture derived from more than one source. For example, 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. Examples of biomass 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.

[0049] Examples of "biomass" also include aquatic or marine biomass, fruit-based biomass such as fruit waste, and vegetable-based biomass such as vegetable waste, among others. Examples of aquatic or marine biomass include, but are not limited to, kelp, giant kelp, seaweed, algae, and marine microflora, microalgae, sea grass, and the like. In certain aspects, 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).

Feedstock

[0050] 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.

[0051] The term "feedstock" used herein means a raw material or mixture of raw materials containing sugars and carbon source used during fermentation process. For example, 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.

[0052] Example of feedstock includes but is not limited to molasses, corn, corn mash, corn cobs, corn grain, corn husks, corn stover, sugar cane, sugercane 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, switchgrass, miscanthus, poplar, willow, algae, municipal solid wastes (MSW), lawn wastes, wastewater treatment sludge, urban wood wastes, disaster debris, trap grease, yellow grease, and waste cooking oil and combinations thereof.

[0053] Molasses is the by-product in sucrose industry. Molasses can be used as raw material for production of fermentation products such as ethanol.

[0054] The term "molasses" refers to a concentrate obtained from mother liquor from sugar crystallization process. Examples of 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.

Compositions and Blends

[0055] In this specification, the "composition" refers a composition comprising trehalase.

[0056] The terms "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, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a- glucosidase, β-glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, β-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, β-glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, β-amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase. [0057] In one aspect a composition comprises trehalase enzyme. In another aspect the 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.

[0058] In some aspects 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, β-glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, β-amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.

[0059] Another aspect provides a 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, β-glucosidase,

transglucosidase, glucose isomerase, xylose isomerase, phosphatase, β-amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.

[0060] Another aspect provides a composition comprising trehalase, glucoamylase, and a-amylase. Another aspect provides 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 enzyme including glucoamylase, and a-amylase.

[0061] Another aspect provides a composition comprising trehalase, glucoamylase, and α-amylase and protease. Another aspect provides 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, at least one enzyme including glucoamylase, a-amylase, and protease..

[0062] Another aspect provides a composition comprising trehalase, glucoamylase, a-amylase, and α-glucosidase. Another aspect provides 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, at least one other enzyme including glucoamylase, a-amylase, and a- glucosidase. [0063] Another aspect provides a composition comprising trehalase, glucoamylase, a-amylase, cellulase and β-glucosidase. Another aspect provides 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, at least one other enzyme including glucoamylase, a-amylase, cellulase, and β-glucosidase.

[0064] Another aspect provides a composition comprising trehalase, glucoamylase, a-amylase, xylanase, cellulose, and β-glucosidase. Another aspect provides 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, at least one other enzyme including glucoamylase, a- amylase, xylanase, cellulase, and β-glucosidase.

[0065] Another aspect provides a composition or a blend comprising trehalase, glucoamylase, a-amylase, a-glucosidase, xylanase, cellulose, and β-glucosidase. Another aspect provides 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, 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, β-glucosidase,

[0066] Another aspect provides a 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

[0067] 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.

[0068] 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.

Methods

[0069] As used herein the term "method" or "process" 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.

[0070] The different aspects and embodiments of the present compositions and methods can be combined to further increase the fermentation product.

[0071] 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

fermentation. The methods are based on the surprising finding of presence of trehalose in molasses as described above. Presence of trehalose in the molasses was not expected. Further experiments showed that addition of trehalase during or prior to fermentation increases fermentation yield. Thus, 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, α-amylase, glucoamylase, pullulanase, and other starch degrading enzymes, protease, phytase, endoglucanase, cellulase, xylanase, cellobiohydrolase, other hemicellulase, a-glucosidase, β-glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, β-amylase, lipase, cutinase,

isoamylase, redox enzyme, esterase, transferase, and pectinase.

[0072] 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, β-glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, β-amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.

[0073] The methods can also include fermenting the sugar containing medium obtained from feedstock to end products in the presence of fermenting

microorganisms. The contacting and fermenting can occur simultaneously or the contacting can be a pretreatment.

[0074] In some embodiments, 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. In some embodiments the methods further comprise fermenting the sugar containing medium in presence of a fermenting organism.

[0075] In some embodiments, 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 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 α-amylase. In some embodiments the methods further comprise fermenting the sugar containing medium in presence of a fermenting organism.

[0076] In some embodiments, the methods comprise contacting a sugar containing medium obtained from a feedstock with a composition comprising trehalase, glucoamylase, and a-amylase and protease. 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, a-amylase, and protease. In some embodiments the methods further comprise fermenting the sugar containing medium in presence of a fermenting organism.

[0077] In some embodiments, the methods comprise contacting a sugar containing medium obtained from a feedstock with a composition comprising trehalase, glucoamylase, an a-amylase, and a-glucosidase. 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 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, α-amylase anda-glucosidase. In some embodiment the method further comprises fermenting the sugar containing medium in presence of fermenting organism.

[0078] In some embodiments, the methods comprise contacting a sugar containing medium obtained from a feedstock with a composition comprising trehalase, glucoamylase, a-amylase, cellulase and β-glucosidase. 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, a-amylase, cellulase, and β-glucosidase. In some embodiment the method further comprises fermenting the sugar containing medium in presence of fermenting organism.

[0079] In some embodiments, the methods comprise contacting a sugar containing medium obtained from a feedstock with a composition comprising trehalase, glucoamylase, α-amylase, xylanase, cellulose, and β-glucosidase. 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, a-amylase, xylanase, cellulase, and β-glucosidase. In some embodiment the method further comprises fermenting the sugar containing medium in presence of fermenting organism.

[0080] In some embodiments, 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 β-glucosidase. 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, a-amylase, a-glucosidase, xylanase, cellulase, and β-glucosidase. In some embodiment the method further comprises fermenting the sugar containing medium in presence of fermenting organism.

[0081] In some embodiments, the contacting and fermenting occur simultaneously. In some embodiments the contacting occurs as a pretreatment.

[0082] In one embodiment, there is provided a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase. In one embodiment, there is provided 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. In some embodiments the methods further comprise fermenting the molasses in presence of a fermenting organism.

[0083] In one embodiment, there is provided 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. In some embodiment 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. In some embodiments the methods further comprise fermenting the molasses in presence of a fermenting organism.

[0084] In one embodiment, there is provided 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. In one embodiment, there is provided 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. In some embodiments the methods further comprise fermenting the molasses in presence of a fermenting organism.

[0085] In one embodiment, there is provided a method for enhanced production of ethanol from molasses comprising contacting the molasses a composition

comprising trehalase, and at least one other enzyme including glucoamylase, a- amylase, and a-glucosidase. In one embodiment, there is provided 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. In some embodiments the methods further comprise fermenting the molasses in presence of a fermenting organism.

[0086] In one embodiment, there is provided 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 β-glucosidase. In one embodiment, there is provided 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 β-glucosidase. In some embodiments the methods further comprise fermenting the molasses in presence of a fermenting organism.

[0087] In one embodiment, there is provided a method for enhanced production of ethanol from molasses comprising contacting the molasses with a composition comprising trehalase, glucoamylase, a-amylase, xylanase, cellulose, and β- glucosidase. In one embodiment, there is provided 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 β- glucosidase. In some embodiments the methods further comprise fermenting the molasses in presence of a fermenting organism.

[0088] In one embodiment, there is provided 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 β-glucosidase. In one embodiment, there is provided 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 β-glucosidase. In some embodiments the methods further comprise fermenting the molasses in presence of a fermenting organism.

[0089] In one embodiment, there is provided 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, β-glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, β-amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase. In one embodiment, there is provided 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, β-glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, β-amylase, lipase, cutinase,

isoamylase, redox enzyme, esterase, transferase, and pectinase.

[0090] In one embodiment, there is provided 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, β-glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, β-amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase. In one embodiment, there is provided 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, β-glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, β-amylase, lipase, cutinase,

isoamylase, redox enzyme, esterase, transferase, and pectinase. In some

embodiments the methods further comprise fermenting the molasses in presence of a fermenting organism.

[0091] In some embodiments the aforementioned contacting and fermenting occurs simultaneously.

[0092] In some embodiments the aforementioned contacting occurs as a pre- treatment followed by fermenting step.

[0093] In some embodiments, 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.

Fermentation Process

[0094] The fermentation products of the disclosure may be prepared by the process as described herein.

[0095] 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.

[0096] Additionally 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. In addition to one and two carbon sources, 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. For example, 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). Similarly, various species of Candida will metabolize alanine or oleic acid (Suiter et al., Arch. Microbiol. 153:485-489 (1990)). Hence, it is contemplated that 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.

[0097] Although it is contemplated that all of the above mentioned carbon source and mixtures thereof are suitable in the present disclosure, in some embodiments, 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. In addition, 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. Examples of biomass 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.

[0098] In addition to an appropriate carbon source, 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.

Culture conditions

[0099] Typically cells are grown at a temperature in the range of about 1 5°C to about 60 °C in an appropriate medium. In some embodiments, 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 ^<Ό, 47°C, 50°C, 52°C, 57°C, 60°C. In some embodiments, 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. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2',

3'-monophosphate (cAMP), can also be incorporated into the fermentation medium.

[00100] 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.

[00101] Fermentations can be performed under aerobic or anaerobic conditions. In one embodiment, anaerobic or microaerobic conditions are used for fermentation.

[00102] In some embodiments, the culture conditions are such that the fermentation occurs without respiration. For example, cells can be cultured in a fermenter under micro-aerobic or anaerobic conditions.

Industrial batch and continuous fermentations

[00103] 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.

Biochem. Biotechnol., 36:227, (1992), herein incorporated by reference.

[00104] 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.

[00105] It is contemplated that the production of Alcohol such as ethanol, or other fermentation products, can be practiced using batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells can be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for alcohol production.

[00106] 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.

[00107] 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.

[00108] 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.

[00109] 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. In some embodiments, 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. In some embodiments, the fermenting is conducted at a pH from about 2.0 to 9.0.

[00110] In one embodiment the trehalase is acid trehalase. In some embodiments the trehalase is from Trichoderma species.

[00111] In some embodiments the 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.

[00112] 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.

[00113] 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, β-glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, β-amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.

[00114] Another embodiment relates to 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, willow, algae, municipal solid wastes (MSW), lawn wastes, wastewater treatment sludge, urban wood wastes, disaster debris, trap grease, yellow grease, and waste cooking oil and combinations thereof.

[00115] 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.

[00116] 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.

[00117] 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

recombinant organism. Further embodiment relates to the 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. Yet another embodiment relates to 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.

[00118] 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. In some embodiments, the contacting and fermenting occur simultaneously or the contacting occurs as a pretreatment. In some embodiments 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. In some embodiments the fermenting temperature is in the range of 15°C to 40 °C. In some embodiments the fermenting is conducted at a pH from about 2.0 to 9.0.

[00119] 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.

[00120] 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.

[00121] The following Examples are offered by way of illustration, not limitation.

EXAMPLES

[00122] The methods disclosed herein are illustrated in the following examples. From the above discussion and these examples, one skilled in the art can ascertain the various embodiments of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the methods and compositions disclosed herein to adapt it to various uses and conditions. [00123] The following additional abbreviations are used: "C" is Celsius, " [iL" is microliter, "ml_" is milliliter, "M" is molar, "mM" id milimolar, "mm" is milimeter, "nm" is nanometer, "μ" is micron, "min" is minute(s), "g" is gram(s), "v/v" is volume by volume, "w/w" is weight by weight, "wt" is weight, "wt %" means weight percent, "Temp" is temperature, "OD" mean optical density, "MW" means average molecular weight and "hr" means hour.

Materials

[00124] The following materials were used in the experiments. All commercial reagents were used as received unless otherwise noted.

Feedstock

[00125] Sugarcane molasses is used as a carbon source for fermentation studies. The molasses was stored at 4°C to 25 °C until use.

Source:

• Jiangnan University, No.1 800, Lihu Avenue, Binhu District, Wuxi city, Jiangsu, China.

• COFCO Bio-chemical Energy (Longjiang) Co., Ltd, Baishan Industrial Park, Longjiang, Qiqihaer, Heilongjiang, China.

Yeast

[00126] Saccharomyces cerevisiae, alcohol dry yeast commercially available from Fali Yeast, AB Mauri was used.

Media

[00127] 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.

Microorganism and Inoculum Preparation

[00128] Saccharomyces cerevisiae was used in the form of active dry yeast (ADY) at a concentration of 3 to 6% w/w.

Enzyme composition: • OPTIMASH™ TREHALASE from Genencor, International Inc., US

Analytical methods

A. Sugar content of molasses we determined by using Ion chromatography - Pulsed amperometric Detector (IC-PAD) method.

Dionex ICS-5000; Column: CarboPac PA 200; Column temperature: 30C;

Flow rate: 0.5 ml/min; Injection volume: 25ul; Detection: PAD.

Gradient:

• 0-10min, 50mM NaOH

• 10-1 5min, 50-1 OOmM NaOH, curve 6

· 15-30min, 0-90mM NaAc, 100mM NaOH, curve 7

• 30-35min, 90-250mN NaAc, 100mM NaOH

• 35.1 -39min, 250mMNaAc, 100mM NaOH

• 39.1 -48min, 50mM NaOH

B. Ethanol content determination was done by using the HPLC method.

[00129] The composition of the reaction products was measured by high

pressure/performance liquid chromatographic method (HPLC) (Agilent Isocratic system 1 200, USA with Rl detector) by injecting 20 μ\- of appropriately diluted samples onto a Aminex Column HPX-87H (catalogue number 1250140, Bio-Rad) maintained at 60°C using 0.01 N sulphuric acid as a mobile phase at a flow rate of 0.7 mL/min. The detection was carried out using Refractive index detector (RID).

Example 1

Determination of the sugar content of molasses

[00130] The molasses was analyzed by ion chromatography (IC). The results are shown in Table 1 , below:

[00131] As shown in Table 1 , the molasses unexpectedly contained about 1 .52% w/v trehalose. Molasses has heretofore not been known to contain trehalose.

Presence of trehalose in molasses was further confirmed by treating the spent wash obtained from the molasses fermentation (without any enzymatic treatment) with trehalase that results in reduction content of trehalose. The results are summarised in Table 2. Table 1 : Results of IC analysis of molasses

Table 2: Confirmation of presence of Trehalose in molasses

Example 2

Fermentation of molasses in the presence of Composition 1

[00132] Batch fermentation was initiated by addition of the composition 1 comprising trehalase (OPTIMASH™ 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.

[00133] It was observed that increasing the concentration of composition 1 in molasses fermentation results in increased ethanol production (Figure 1 and Table 3) as compared to the molasses fermentation carried out without the composition 1 .

Table 3: Effect of Trehalase on Ethanol production

[00134] While the disclosure has been described with reference to several exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the disclosure without departing from the essential scope thereof. Therefore, it is intended that the disclosure not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out this disclosure, but that the disclosure will include all embodiments falling within the scope of the appended claims.

Claims

CLAIMS What is claimed is

1 . 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.

2. The method of claim 1 , wherein the method further comprising fermenting the sugar containing medium in the presence of fermenting microorganisms.

3. The method of claim 1 , wherein the contacting temperature is in the range of 15°C to 60 °C.

4. The method of claim 3, wherein the contacting temperature is in the range of 15°C to 40 °C.

5. The method of claim 1 , wherein the fermenting temperature is in the range of 15°C to 40 °C.

6. The method of claim 2, wherein the fermenting is conducted at a pH from about 2.0 to 9.0.

7. The method of claim 1 , wherein the method optionally comprises contacting the sugar containing medium with 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, β-glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, β- amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.

8. The method of claim 1 , wherein the trehalase is acid trehalase.

9. The method of claim 1 , wherein the trehalase is from Trichoderma species.

10. The method of claim 1 , wherein the fermentation product is selected from the group consisting of alcohol, metabolites, and other bio-chemicals and

biomaterials.

1 1 . The method of claim 8, wherein the alcohol is selected from the group consisting of ethanol, butanol, methanol, propanol, iso-amyl alcohol, 1 ,3-propanediol, and other alcohols.

12. The method of claim 10, wherein the metabolites are 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.

13. The method of claim 1 , wherein the feedstock is 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, willow, algae, municipal solid wastes (MSW), lawn wastes, wastewater treatment sludge, urban wood wastes, disaster debris, trap grease, yellow grease, and waste cooking oil and combinations thereof.

14. The method of claim 13, 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.

15. The method of claim 2, wherein the fermenting organism is yeast, a bacterium, a fungus or algae.

16. The method of claim 2, wherein the fermenting organism is a recombinant microorganism.

17. The method of claim 15, wherein the yeast is 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.

18. The method of claim 15, wherein the bacterium is selected from a group

consisting of strains of Escherichia, Zymomonas, and Klebsiella, Brevibacterium, Corynebacterium, Bacillus, Clostridium and Streptomyces.

19. The method of claim 15, wherein the fungus is selected from a group consisting of Penicillium Sp, Acremonium Sp, Trichoderma sp, and Aspergillus sp.

20. 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 presence of a fermenting organism to produce ethanol.

21 . The method of claim 20, wherein the contacting and fermenting occur

simultaneously or the contacting occurs as a pretreatment.

22. The method of claim 20, wherein contacting temperature is in the range of 15°C to 60°C.

23. The method of claim 20, wherein the contacting temperature is in the range of 15°C to 40 °C.

24. The method of claim 20, wherein the fermenting temperature is in the range of 15°C to 40 °C.

25. The method of claim 20, wherein the fermenting is conducted at a pH from about 2.0 to 9.0.

26. The method of claim 20, wherein the method optionally comprises contacting the sugar containing medium with 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, β-glucosidase, transglucosidase, glucose isomerase, xylose isomerase, phosphatase, β- amylase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, and pectinase.

27. The method of claim 20, wherein the trehalase is acid trehalase.

28. The method of claim 20, wherein the trehalase is from Trichoderma species.

29. The method of claim 20, wherein the fermenting organism is yeast, a bacterium or a fungus.

30. The method of claim 24, wherein the fermenting organism is a recombinant

microorganism.

31 . The method of claim 24, wherein the yeast is 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

32. The method of claim 24, wherein the bacterium is selected from a group

33. The method of claim 24, wherein the fungus is selected from a group consisting of Penicillium Sp, Acremonium Sp, Trichoderma sp, and Aspergillus sp.

34. The method of claim 20, wherein the feedstock is selected from a group

35. The method of claim 34, wherein the molasses is obtained from a source