WO2005118827A2 - Fermentation process - Google Patents
Fermentation process Download PDFInfo
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- WO2005118827A2 WO2005118827A2 PCT/US2005/018421 US2005018421W WO2005118827A2 WO 2005118827 A2 WO2005118827 A2 WO 2005118827A2 US 2005018421 W US2005018421 W US 2005018421W WO 2005118827 A2 WO2005118827 A2 WO 2005118827A2
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- Prior art keywords
- starch
- fermentation
- amylase
- alpha
- containing material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- the present invention relates to fermentation processes for producing fermentation products, such as ethanol, from starch-containing material.
- Fermentation processes are used for making a vast number of commercial products, including alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glu- tamic acid); gases (e.g., H 2 and CO 2 ), and more complex compounds, including, for example, antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B 12 , beta- carotene); hormones, and other compounds which are difficult to produce synthetically.
- alcohols e.g., ethanol, methanol, butanol
- organic acids e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid
- ketones e.g., acetone
- amino acids
- Fermentation processes are also commonly used in the consumable alcohol (e.g., beer and wine), dairy (e.g., in the production of yogurt and cheese), leather, and tobacco industries. There is a need for further improvement of fermentation processes and for improved processes which include a fermentation step.
- the present invention provides improved fermentation processes for producing a fermentation product, such as ethanol, from a starch-containing starting material.
- a fermentation process of the invention utilizes the starch-containing material more efficiently than a traditional fermentation process and/or boosts the fermentation rate.
- the invention relates to a process of fermenting starch-containing material, which process comprises: i) fermenting said starch-containing material by subjecting said material to a fermenting microorganism, ii) reducing the particle size of the starch-containing material obtained from step i) and/or liquefying the starch-containing material obtained from step i), and iii) fermenting.
- the starch-containing material has been subjected to a carbohydrate-source generating enzyme prior to and/or simultaneously with fermentation step i)
- the starch-containing material may preferably be mash, especially corn mash or un-gelatinized starch.
- the invention relates to a process for producing a fermentation product from starch-containing material, comprising (a) reducing the particle size of starch-containing material; (b) liquefying the material obtained in step (a); (c) subjecting the liquefied material obtained in step (b) to a fermentation process of the invention.
- the invention relates to a process for producing a fermentation product from starch-containing material, comprising (a) reducing the particle size of starch-containing material; (b) subjecting the material in step (a) to a fermentation process of the invention in the presence of an acid alpha-amylase.
- the fermentation product may optionally be recovered, preferably by distillation.
- the fermentation product is ethanol.
- Fig. 1 shows an integral fermentation-liquefaction system suitable for carrying out the invention.
- Fig. 2 shows the conversion percentage of residual starch containing material collected in a 2800 micro meter mesh sieve after initial SSF subjected to additional milling and/or liquefac- tion followed by additional SSF.
- the present invention provides improved fermentation processes which are suitable for producing fermentation products, especially ethanol, from a starch-containing starting ma- terial.
- fermentable sugar(s) i.e., DP 1-3 sugar(s)
- the fermentable sugar uptake rate of the fermenting microorganism is lower than the enzymatic sugar release rate.
- concentration of the fermenting microorganism increases and the sugar uptake rate becomes greater than the enzymatic sugar release rate.
- the release of fermentable sugar(s) decreases. This results in a decreased fermentation rate.
- the overall fermentation performance decrease is prevented or at least reduced. This is accomplished by milling and/or liquefying the fermentation medium after a period of time or after fermentation as an integral part of the fermentation process.
- the inventors have found that improved conversion rates of starch present in corn mash may be attained by securing that starch found in corn mash is made accessible to enzymatic degradation. Residual starch measured at the end of the fermentation seems to be inaccessible to enzymatic attack.
- the invention concerns a fermentation process comprising: i) fermenting starch-containing material by subjecting said material to a fermenting microorganism, ii) reducing the particle size of the starch-containing material obtained from step i) and/or liquefying the starch-containing material obtained from step i), and iii) fermenting.
- the starch-containing material has been subjected to a carbohydrate-source generating enzyme prior to fermentation step i), simulta- neously with fermentation step i), or prior to and simultaneously with fermentation step i).
- the fermentation process of the invention may be a batch fermentation process carried out in a fermentation tank or the like. According to preferred embodiments of the invention a partial or especially the entire fermentation medium volume is liquefied over a period of time after initial fermentation has taken place.
- the starch- containing particles in the fermentation medium volume to is to be reduced in size and/or liquefied in step ii) may be the entire or a fractionated part of the initially fermented starch- containing material from step i) , e.g., a liquid fraction containing enzymes and/or a fraction containing the remaining starch-containing material.
- the additional step(s) is(are) preferably initiated at the time when the fermenting microorganism has consumed significantly all read- ily available fermentable sugars.
- the additional liquefaction step is initiated when the concentration of DP 1-3 sugars, preferably glucose and/or maltose, in the fermentation medium reaches below 15 g/L fermentation medium, preferably below 10 g/L, even more preferred below 5 g/L.
- the additional step(s) is(are) initiated when the fermentation product production rate has decreased to below 85%, preferably below 75%, even more preferred 65% of the maximal rate.
- the additional step(s) i.e. step ii) may be carried out by subjecting a side-stream of the initially fermented material to particle size reduction and/or liquefaction at elevated temperatures for a period of time and thereafter saccharifying and/or fermenting said material further, preferably as a SSF step.
- additional liquefaction is carried out by (slowly) transferring the fermentation medium to and/or through a liquefaction holding tank, where the additional particle size reduction is carried out and/or liquefaction is carried out at typically between 40 and 80°C, preferably between 50-70°C for 1 to 60 minutes, preferably for between 20 to 40 minutes, such as around 30 minutes.
- an effective amount of alpha-amylase is added.
- the alpha-amylase may be any of the alpha-amylases mentioned below in the section "Alpha-Amylases".
- the alpha- amylase is of bacterial origin, preferably a Bacillus alpha-amylase, or a fungal alpha- amylase, preferably an acid Aspergillus alpha-amylase.
- the material is recycled or introduced into the same or an additional fermentation tank.
- the fermenting microorganism might be inactivated (killed) during the additional step(s) (e.g. due to elevated temperatures) the period of time for treating the entire fermentation volume should be at least as long as the doubling time of the fermenting microorganism.
- the fermenting microorganism is yeast this typically means that the entire fermentation volume can be additionally liquefied in 12 hours or more.
- FIG. 1 illustrates an example of an integral fermentation-liquefaction system. It should be understood that Fig. 1 only illustrates one example of a system suitable for carry- ing out a process of the present invention. It is to be understood that a remaining starch- containing material after initial fermentation may also be separated, the particles reduced in size and/or liquefied using other means well know to the skilled person in the art.
- step i) the fermenting microorganism consumes and converts easily accessible fermentable sugars into desired fermentation product. This en- sures that essentially no fermentable sugars are lost or wasted due to the heating/elevated temperatures during the additional step ii).
- the additional step ii) may lead to increased fermentation rates, shorter fermentation times, higher starch/ethanol yields, i.e., higher conversion percentage, and/or less residual starch.
- Fermentation refers to any fermentation process comprising a fermentation step.
- a fermentation process of the invention includes, without limitation, fermentation methods or processes used to produce alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H 2 and CO 2 ); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B ⁇ 2 , beta-carotene); and hormones.
- alcohols e.g., ethanol, methanol, butanol
- organic acids e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid
- ketones e.g., acetone
- amino acids e.g
- Fermentation processes also include fermentation processes used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry.
- the process of the present invention may be used in combination with a saccharifi- cation process, e.g. SSF, in which additional enzymatic activities, such as esterase, such as lipase and/or cutinase, phytase, laccase, cellulase, xylanase, alpha-amylase, glucoamylase, glucosidase, protease, cellobiase, or mixtures thereof, may be used in processing the substrate, e.g., a starch substrate.
- the fermentation process of the invention is used for the production of ethanol.
- Fermentation medium refers to the environment in which the fermentation is carried out and which includes the fermentation substrate, that is, the carbohydrate source that is metabolized by the fermenting microorganism(s).
- the fermentation medium including fermentation substrate and other raw materials used in the fermentation process of the invention may be processed, e.g., by milling and/or liquefaction or other desired processes prior to the fermentation.
- the fermentation medium can refer to the medium before or after the fermenting microorganism(s) is(are) added, such as, the medium in or resulting from a liquefaction step, as well as the medium which comprises the fermenting microorganisms, such as, the medium used in a simultaneous saccharification and fermentation process (SSF) .
- SSF simultaneous saccharification and fermentation process
- Fermenting microorganism refers to any organism, including bacterial and fungal organisms, suitable for use in a desired fermentation process. Especially suitable fermenting microorganisms are according to the invention able to ferment, i.e., to convert, sugars, such as DP L3 sugars, especially glucose or maltose, directly or indirectly into the desired fermen- tation product. Examples of fermenting organisms include fungal organisms, such as yeast. Preferred yeast includes strains of Saccharomyces spp., and in particular Saccharomyces cerevisiae.
- yeast Commercially available yeast include, e.g., RED STAR®/Lesaffre Ethanol Red (available from Red Star/Lesaffre, USA), SUPERSTART (available from Alltech), GERT STRAND (available from Gert Strand AB, Sweden), FALI yeast (available from Fleisch- mann's Yeast, USA), and FERMIOL (available from DSM Specialties).
- the fermenting organism is preferably yeast, which is applied to the mash.
- Preferred yeast is derived from Saccharomyces spp., more preferably, from Saccharomyces cerevisiae. The yeast is applied to the starting material and the fermentation is ongoing for 24-96 hours, such as typically 35-60 hours.
- the temperature is generally be- tween 26-34°C, in particular about 32°C, and the pH is generally from pH 3-6, preferably around pH 4-5.
- Yeast cells are preferably applied in amounts of 10 5 to 10 12 , preferably from 10 7 to 10 10 , especially 5x10 7 viable yeast count per ml of fermentation medium. During the ethanol producing phase the yeast cell count should preferably be in the range from 10 7 to 10 10 , especially around 2 x 10 8 . Further guidance in respect of using yeast for fermentation can be found in, e.g., "The alcohol Textbook" (Editors K. Jacques, T.P. Lyons and
- any suitable substrate or raw material may be used according to the present invention.
- the substrate is generally selected based on the desired fermentation product and the process employed, as is well known in the art.
- substrates suitable for use in the processes of present invention include starch-containing plant materials, such as tubers, roots, whole grains, corns, cobs, wheat, barley, rye, milo or cereals, sugar-containing raw materials, such as molasses, fruit materials, sugar, cane or sugar beet, potatoes, and cellulose-containing materials, such as wood or plant residues.
- liquefied mash includes any of the above raw materials, which have been subjected to liquefaction using any method known in the art. Preferred is enzymatically mash, especially liquefied corn mash, and un-gelatinized starch (i.e., uncooked starch).
- Carbohydrate-Source Generating Enzyme includes glucoamylase (being a glucose generator), and beta-amylase and maltogenic amylase (being maltose generators). Other enzymes producing other carbohydrates suitable for the fermenting microorganism in question are also contemplated according to the invention.
- a carbohydrate-source generating enzyme is capable of providing energy to the fermenting microorganism(s) used in the process of the invention and/or may be converting directly or indirectly to the desired fermentation product, such as ethanol.
- the carbohydrate-source generating enzyme may be a mix- ture of enzymes falling within the definition.
- glucoamylase and alpha-amylase especially acid amylase, even more preferred fungal acid alpha-amylase.
- the ratio between acid fungal alpha-amylase activity (AFAU) per glucoamylase activity (AGU) (AFAU per AGU) may in one embodiment of the invention be at least 0.1 , in particular at least 0.16, such as in the range from 0.12 to 0.50 or even higher such as between 0.5 and 1.0.
- contemplated glucoamylases, alpha-amylases and beta-amylases are set forth in the sections below. It is to be understood that the enzymes used according to the invention should be added in effective amounts. Effective amount can easily be determined by a person skilled in the art.
- Glucoamylase A glucoamylase used according to the invention may be derived from any suitable source, e.g., derived from a microorganism or a plant.
- Preferred glucoamylases are of fun- gal or bacterial origin, selected from the group consisting of Aspergillus glucoamylases, in particular A. nigerG or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102), or variants thereof, such as disclosed in WO 92/00381 , WO 00/04136 add WO 01/04273 (from Novozymes, Denmark); the A.
- awamori glucoamylase (WO 84/02921), A. oryzae (Ag- ric. Biol. Chem. (1991), 55 (4), p. 941-949), or variants or fragments thereof.
- Other Aspergillus glucoamylase variants include variants to enhance the thermal stability: G137A and G139A (Chen et al. (1996), Prot. Eng. 9, 499-505); D257E and D293E/Q (Chen et al. (1995), Prot. Engng. 8, 575-582); N182 (Chen et al. (1994), Biochem. J. 301 , 275-281); disulphide bonds, A246C (Fierobe et al.
- glucoamylases include Corticium rolfsii glucoamylase (US patent no. 4,727,046) also referred to as Althelia rolfsii glucoamylase, Talaromyces glucoamylases, in particular, derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (US patent no. Re. 32,153), Talaromyces duponti, Talaromyces thermophilus (US patent no.
- Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfu- ricum (WO 86/01831).
- compositions comprising glucoamylase include AMG 200L; AMG 300 L; SANTM SUPER, SANTM EXTRA L, SPIRIZYMETM PLUS, SPIRIZYMETM FUEL, SPIRIZYMETM B4U and AMGTM E (from Novozymes A/S); OPTIDEXTM 300 (from Genencor Int.); AMIGASETM and AMIGASETM PLUS (from DSM); G-ZYMETM G900, G-ZYMETM and G990 ZR (from Genencor Int.).
- Glucoamylases may in an embodiment be added in an amount of 0.02-20 AGU/g DS, preferably 0.1-10 AGU/g DS, such as 0.4-4 AGU/g DS, such as around 2 AGU/g DS.
- Alpha-Amylases are of fungal or bacterial origin. More preferably, the alpha-amylase is a Bacillus alpha-amylase, such as, derived from a strain of B. licheniformis, B. amyloliquefaciens, B. sultilis and ⁇ . stearothermophilus. Other alpha-amylases include alpha-amylase derived from a strain of the Bacillus sp.
- the Bacillus alpha-amylase may also be a variant and/or hybrid, especially one de- scribed in any of WO 96/23873, WO 96/23874, WO 97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documents hereby incorporated by reference). Specifically contemplated alpha-amylase variants are disclosed in US patent nos.
- Bacillus alpha-amylases especially Bacillus stearothermophilus alpha-amylase, which have a double deletion corresponding to delta(181-182) and further comprises a N193F substitution (also denoted 1181 * + G182* + N193F) compared to the wild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467.
- a hybrid alpha-amylase specifically contemplated comprises 445 C-terminal amino acid residues of Bacillus licheniformis alpha-amylase (shown as SEQ ID NO: 4 in WO 99/19467) and the 37 N-terminal amino acid residues of the alpha-amylase derived from Ba- cillus amyloliquefaciens (shown as SEQ ID NO: 3 in WO 99/194676), with one or more, especially all, of the following substitution: G48A+T49I+G107A+H156Y+A181T+N190F+I201 F+A209V+Q264S (using the Bacillus licheniformis numbering).
- variants having one or more of the following mutations (or corresponding mutations in other Bacillus alpha-amylase backbones): H154Y, A181T, N190F, A209V and Q264S and/or deletion of two residues between positions 176 and 179, preferably deletion of E178 and G179 (using the SEQ ID NO: 5 numbering of WO 99/19467).
- Other alpha-amylase includes alpha-amylases derived from a strain of Aspergillus, such as, Aspergillus oryzae and Aspergillus niger alpha-amylases. In a preferred embodiment, the alpha-amylase is an acid alpha-amylase.
- the acid alpha-amylase is an acid fungal alpha-amylase or an acid bacterial alpha-amylase. More preferably, the acid alpha-amylase is an acid fungal alpha-amylase derived from the genus Aspergillus. A commercially available acid fungal amylase is SP288 (available from No- vozymes A/S, Denmark) or alternatively SP288 comprising a starch-binding domain. In an embodiment, the alpha-amylase is an acid alpha-amylase.
- the term "acid alpha- amylase" means an alpha-amylase (E.C.
- 3.2.1.1 which added in an effective amount has activity at a pH in the range of 3.0 to 7.0, preferably from 3.5 to 6.0, or more preferably from 4.0-5.0.
- One preferred acid fungal alpha-amylase is a Fungamyl-like alpha-amylase.
- the term "Fungamyl-like alpha-amylase” indicates an alpha-amylase which exhibits a high homology, i.e. more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% or even more than 90% homology (identity) to the amino acid sequence shown in SEQ ID No. 10 in WO96/23874.
- the alpha-amylase is an acid alpha-amylase, preferably from the genus Aspergillus, preferably of the species Aspergillus niger.
- the acid fungal alpha-amylase is the one from A. niger disclosed as "AMYA_ASPNG" in the Swiss- prot/TeEMBL database under the primary accession no. P56271.
- a preferred acid alpha-amylase for use in the present invention may be derived from a strain of B. licheniformis, B.
- amyloliquefaciens and B. stearothermophilus.
- Preferred commercial compositions comprising alpha-amylase include MYCOLASE from DSM (Gist Brochades), BANTM, TERMAMYLTM SC, FUNGAMYLTM, LIQUOZYMETM X and SANTM SUPER, SANTM EXTRA L (Novozymes A S) and CLARASETM L-40,000, DEX-LOTM, SPEYME FRED, SPEZYMETM AA, and SPEZYMETM DELTA AA (Genencor Int.), and the acid fungal alpha-amylase sold under the trade name SP 288 (available from Novozymes A/S, Denmark).
- the alpha-amylase may be added in amounts as are well-known in the art.
- the bacterial alpha-amylase may be added in amounts as are well-known in the art.
- the alpha-amylase activity is preferably present in an amount of 0.5-5,000 NU/g of DS, in an amount of 1-500 KNU/kg of DS, or more preferably in an amount of 5-1 ,000 KNU/kg of DS, such as 10-100 KNU/kg DS.
- the acid alpha-amylase activity is preferably present in an amount of 5-50,0000 AAU/kg of DS, in an amount of 500-50,000 AAU/kg of DS, or more preferably in an amount of 100-10,000 AAU/kg of DS, such as 500-1 ,000 AAU/kg DS.
- Fungal acid alpha-amylases are preferably added in an amount of 10-10,000 AFAU/kg of DS, in an amount of 500-2,500 AFAU/kg of DS, or more preferably in an amount of 100-1 ,000 AFAU/kg of DS, such as approximately 500 AFAU/kg DS.
- Maltogenic amylase may also be a maltogenic alpha-amylase.
- a "maltogenic alpha-amylase” (glucan 1 ,4-alpha-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyze amylose and amy- lopectin to maltose in the alpha-configuration.
- a maltogenic alpha-amylase from Bacillus stearothermophilus strain NCIB 11837 is commercially available from Novozymes A/S under the tradename NOVAMYLTM. Maltogenic alpha-amylases are described in EP patent no. 120,693, US Patent nos.
- the maltogenic alpha-amylase is used in a raw starch hydrolysis process, as described, e.g., in WO 95/10627, which is hereby incorporated by reference.
- fungal alpha-amylases may be added in an amount of 0.001-1.0 AFAU/g DS, preferably from 0.002-0.5 AFAU/g DS, preferably 0.02- 0.1 AFAU/g DS.
- Beta-amylase At least according to the invention the a beta-amylase (E.C 3.2.1.2) is the name tradi- tionally given to exo-acting maltogenic amylases, which catalyze the hydrolysis of 1 ,4-alpha- glucosidic linkages in amylose, amylopectin and related glucose polymers. Maltose units are successively removed from the non-reducing chain ends in a step-wise manner until the molecule is degraded or, in the case of amylopectin, until a branch point is reached. The maltose released has the beta anomeric configuration, hence the name beta-amylase.
- Beta-amylases have been isolated from various plants and microorganisms (W.M. Fo- garty and C.T. Kelly, Progress in Industrial Microbiology, vol. 15, pp. 112-115, 1979). These beta-amylases are characterized by having optimum temperatures in the range from 40°C to 65°C and optimum pH in the range from 4.5 to 7.
- a commercially available beta-amylase from barley is SPEZYMETM BBA 1500 from Genencor Int., USA.
- growth Stimulators In an embodiment of the process of the invention one or more growth stimulators are added to further improve the fermentation, and in particular, the performance of the fermenting organism, such as, rate enhancement and product yield.
- Preferred stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D, and E.
- minerals include minerals and mineral salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.
- the enzymes may be derived or obtained from any origin, including, bacterial, fungal, yeast or mammalian origin.
- the term "derived” means in this context that the enzyme may have been isolated from an organism where it is present natively, i.e. the identity of the ami ⁇ o acid sequence of the enzyme are identical to a native enzyme.
- derived also means that the enzymes may have been produced recombinantly in a host organism, the recombinant produced enzyme having either an identity identical to a native enzyme or having a modified amino acid sequence, e.g., having one or more amino ac- ids which are deleted, inserted and/or substituted, i.e., a recombinantly produced enzyme which is a mutant and/or a fragment of a native amino acid sequence or an enzyme produced by nucleic acid shuffling processes known in the art.
- a native enzyme are included natural variants.
- the term "derived” includes enzymes produced synthetically by, e.g., peptide synthesis.
- the term “derived” also encompasses en- zymes which have been modified e.g. by glycosylation, phosphorylation, or by other chemical modification, whether in vivo or in vitro.
- the term “obtained” in this context means that the enzyme has an amino acid sequence identical to a native enzyme.
- the term encompasses an enzyme that has been isolated from an organism where it is present natively, or one in which it has been expressed recombinantly in the same type of organism or another, or enzymes produced synthetically by, e.g., peptide synthesis. With respect to recombinantly produced enzymes the terms “obtained” and “derived” refers to the identity of the enzyme and not the identity of the host organism in which it is produced recombinantly.
- the enzymes may also be purified.
- the term “purified” as used herein covers enzymes free from other components from the organism from which it is derived.
- the term “pu- rified” also covers enzymes free from components from the native organism from which it is obtained.
- the enzymes may be purified, with only minor amounts of other proteins being present.
- the expression “other proteins” relate in particular to other enzymes.
- the term “purified” as used herein also refers to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the enzyme of the inven- tion.
- the enzyme may be "substantially pure,” that is, free from other components from the organism in which it is produced, that is, for example, a host organism for recombinantly produced enzymes.
- the enzymes are at least 75% (w/w) pure, more preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure. In another preferred embodiment, the enzyme is 100% pure.
- the enzymes used in the present invention may be in any form suitable for use in the processes described herein, such as e.g. in the form of a dry powder or granulate, a non- dusting granulate, a liquid, a stabilized liquid, or a protected enzyme. Granulates may be produced, e.g. as disclosed in US Patent Nos.
- Liquid enzyme preparations may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, lactic acid or another organic acid according to established methods.
- stabilizers such as a sugar, a sugar alcohol or another polyol, lactic acid or another organic acid according to established methods.
- Protected enzymes may be prepared according to the method disclosed in EP 238,216.
- the fermentation process of the invention can be used in any process of producing a fermentation product.
- a preferred application of the fermentation process of the invention described herein is in an ethanol production process (e.g., for use as a fuel or fuel additive).
- the invention relates to a process for producing a fermen- tation product from starch-containing material, comprising (a) reducing the particle size of starch-containing material; (b) liquefying the product of step (a); (c) subjecting the liquefied material obtained in step (b) to a fermentation process of the invention.
- the raw material such as whole grain, preferably corn
- particle size e.g., by milling in order to open up the structure and allow for further processing.
- Two processes are preferred according to the invention: wet milling and dry milling. Most used for, e.g., ethanol production is dry milling where the whole kernel is milled and used in the remaining part of the process. Wet milling may also be used and gives a good separation of germ and meal (starch granules and protein) and may advantageously, with a few exceptions, be applied at locations where there is a parallel production of, e.g., syrups. Both wet and dry milling processes are well known in the art. Other particle size reducing technologies such as emulsifying technology, rotary pulsation may also be used.
- ethanol production processes generally involves the steps of liquefac- tion, saccharification, fermentation, and optionally recovery, e.g., by distillation.
- liquefaction step (b) e.g., milled (whole) grain raw material is broken down (hydrolyzed) into malto- dextrins (dextrins).
- Hydrolysis may be carried out by acid treatment or enzymatically by alpha-amylase treatment, in particular a Bacillus alpha-amylase described above in the "alpha- amylase"-section.
- the raw material is in one preferred embodiment of the process of the in- vention milled whole grain. However, a side stream from starch processing may also be used.
- the starting material may be a slurry of from about 25 to about 45 wt-% milled whole grain, and water.
- enzymatic liquefaction is carried out as a three- step hot slurry process.
- the slurry is heated to between 60-95°C, preferably 80-85°C, and the enzyme(s) is(are) added to initiate liquefaction (thinning).
- the enzyme(s) is(are) added to initiate liquefaction (thinning).
- at least an alpha- amylase is added.
- the slurry may in one embodiment be jet-cooked at a temperature between 95-140°C, preferably 105-125°C to complete gelatinization of the slurry.
- the liquefaction process is carried out at around pH 4.5-6.5, in particular at a pH between 5 and 6. Milled and liquefied whole grains are known as mash.
- the liquefaction step (b) may be performed in the presence of any alpha-amylase preferably one mentioned above in the section "Alpha-Amylase".
- Preferred alpha-amylases are of fungal or bacterial origin. Bacillus alpha-amylases, variant and hybrids thereof, are specifically contemplated according to the invention.
- the alpha-amylase may be added in effective amounts well-known in the art.
- the bacterial alpha-amylase may be added in amounts as are well-known in the art.
- the alpha-amylase activity is preferably present in an amount of 0.5-5,000 NU/g of DS, in an amount of 1-500 KNU/kg of DS, or more preferably in an amount of 5-1 ,000 KNU/kg of DS, such as 10-100 KNU/kg DS.
- the acid alpha-amylase activity is preferably present in an amount of 0.005-500 AAU/g DS, in an amount of 0.500-50 AAU/g DS, or more preferably in an amount of 0.1-10 AAU/g of DS, such as 0.5-1 AAU/g DS.
- Fungal alpha-amylases may be added in an amount of 0.001-1.0 AFAU/g DS, preferably from 0.002-0.5 AFAU/g DS, preferably 0.02-0.1 AFAU/g DS.
- Bacillus alpha-amylases may be added in effective amounts well known to the person skilled in the art.
- Saccharification and Fermentation To produce low molecular fermentable sugars, i.e., carbohydrate source that can be metabolized by a fermenting microorganism, such as yeast, the maltodextrin from the lique- faction step must be further hydrolyzed. Hydrolysis is performed enzymatically using a carbohydrate-source generating enzyme, such as preferably glucoamylase. Alternatively, e.g., alpha-glucosidases, beta-amylase, Maltogenic alpha-amylase or acid alpha-amylases may be used. According to the invention saccharification and fermentation are carried out simultaneously, i.e., as a SSF process. According to this aspect of the invention step (c) of the process for producing a fermentation product is carried out using the fermentation process of the invention as described above.
- a carbohydrate-source generating enzyme such as preferably glucoamylase.
- the fermentation product is recovered, e.g. by distillation using any method know in the art.
- the fermented material may be distilled to extract the fermentation product, in particular ethanol.
- the end product, obtained according to an ethanol production process of the invention may be used as, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol.
- the invention relates to a process for producing a fermentation product from starch-containing material, comprising (a) reducing the particle size of starch-containing material; (b) carrying out the fermentation process of the invention
- the starch-containing material may according to this aspect of the invention be un- gelatinized starch (i.e. uncooked starch).
- the starting material comprises about 25 to about 45 wt-% starch-containing material, e.g., milled whole grain and water.
- starch-containing material e.g., milled whole grain and water.
- Bacterial Alpha-amylase A Bacillus stearothermophilus alpha-amylase variant with the mutations: I181*+G182*+N193F disclosed in US patent no. 6,187,576 and available on request from Novozymes A/S, Denmark.
- Glucoamylase TN Glucoamylase derived from Talaromyces emersonii and disclosed as SEQ ID NO: 7 in WO 99/28448 with side activity of Aspergillus niger glucoamylase and As- pergillus niger acid alpha-amylase and available on request from Novozymes A/S, Denmark.
- Alpha-amylase activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Ini- tially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which is compared to a colored glass standard.
- KNU Kilo Novo alpha amylase Unit
- FAU Fungal Alpha-Amylase Unit
- Acid alpha-amylase activity is measured in AFAU (Acid Fungal Alpha-amylase Units), which are determined relative to an enzyme standard.
- the standard used is AMG 300 L (from Novozymes A/S, Denmark, glucoamylase wild-type Aspergillus niger G1 , also disclosed in Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102) and WO 92/00381).
- the neutral alpha-amylase in this AMG falls after storage at room temperature for 3 weeks from approx. 1 FAU/mL to below 0.05 FAU/mL.
- the acid alpha-amylase activity in this AMG standard is determined in accordance with the following description.
- 1 AFAU is defined as the amount of enzyme, which degrades 5.260 mg starch dry matter per hour under standard conditions. Iodine forms a blue complex with starch but not with its degradation products. The intensity of colour is therefore directly proportional to the concentration of starch.
- Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under specified analytic conditions.
- Alpha-amylase Starch + Iodine ⁇ Dextrins + Oligosaccharides 40°C, pH 2.5 Blue/violet t 23 sec.
- Substrate Starch, approx.
- Acid Alpha-amylase Units The acid alpha-amylase activity can be measured in AAU (Acid Alpha-amylase Units), which is an absolute method.
- AAU Acid Amylase Unit
- One Acid Amylase Unit (AAU) is the quantity of enzyme converting 1 g of starch (100% of dry matter) per hour under standardized conditions into a product having a transmission at 620 nm after reaction with an iodine solution of known strength equal to the one of a color reference. Standard conditions/reaction conditions: Substrate: Soluble starch. Concentration approx. 20 g DS/L. Buffer: Citrate, approx.
- the starch should be Lintner starch, which is a thin-boiling starch used in the laboratory as colorimetric indicator. Lintner starch is obtained by dilute hydrochloric acid treatment of native starch so that it retains the ability to color blue with iodine.
- Glucoamylase activity (AGh Glucoamylase (equivalent to amyloglucosidase) converts starch into glucose.
- the amount of glucose is determined here by the glucose oxidase method for the activity determination.
- the method described in the section 76-11 Starch Glucoamylase Method with Subsequent Measurement of Glucose with Glucose Oxidase in "Approved methods of the American Association of Cereal Chemists". Vol.1-2 AACC, from American Association of Cereal Chemists, (2000); ISBN: 1-891127-12-8.
- AGI glucoamylase unit
- the starch should be Lintner starch, which is a thin-boiling starch used in the laboratory as colorimetric indicator. Lintner starch is obtained by dilute hydrochloric acid treatment of native starch so that it retains the ability to color blue with iodine.
- Glucoamylase activity The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute under the standard conditions 37°C, pH 4.3, substrate: maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes.
- An auto-analyzer system may be used. Mutarotase is added to the glucose dehydrogenase reagent so that any alpha-D-glucose present is turned into beta-D-glucose.
- Glucose dehydrogenase reacts specifically with beta-D-glucose in the reaction mentioned above, forming NADH which is determined using a photometer at 340 nm as a measure of the original glucose concentration.
- polypeptide "homology” is understood as the degree of "identity" between two sequences indicating a derivation of the first sequence from the second.
- the homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453. The following settings for amino acid sequence comparison are used: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.
- Examples 1 The purpose of the experiment was to investigate the accessibility of residual starch contained in corn mash particles after initial SSF. Corn mash was sieved through 2800 micrometer sieve by washing with Dl-water. The obtained starch containing particles were treated as follows: 1) One part was milled (wet) to smaller size particles. 2) Second part was milled and re-liquefied using Bacterial Alpha-Amylase A (BAAA) at a dose of 100 NU/g DS for 90 minutes at 85°C.
- BAAA Bacterial Alpha-Amylase A
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US11/597,941 US20070202583A1 (en) | 2004-05-27 | 2005-05-24 | Fermentation Process |
EP05754253A EP1753867A4 (en) | 2004-05-27 | 2005-05-24 | Fermentation process |
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US (1) | US20070202583A1 (en) |
EP (1) | EP1753867A4 (en) |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008026932A1 (en) | 2006-08-30 | 2008-03-06 | Cambi As | Method and device for thermal enzymatic hydrolysis of ligno cellulose |
US8728773B2 (en) | 2005-11-28 | 2014-05-20 | Matthias Boy | Fermentative production of organic compounds using substances containing dextrin |
CN104328143A (en) * | 2014-10-24 | 2015-02-04 | 哈尔滨艾博雅食品科技开发有限公司 | Method for producing alcohol from fresh tobacco leaves |
Families Citing this family (6)
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US7727726B2 (en) * | 2007-06-01 | 2010-06-01 | Syngenta Participations Ag | Process for starch liquefaction and fermentation |
US7915020B2 (en) * | 2007-06-01 | 2011-03-29 | Syngenta Participations Ag | Process for starch liquefaction and fermentation |
US7914993B2 (en) * | 2007-06-01 | 2011-03-29 | Syngenta Participations Ag. | Process for starch liquefaction and fermentation |
US7662617B2 (en) * | 2007-11-03 | 2010-02-16 | Rush Stephen L | Systems and processes for cellulosic ethanol production |
US7514247B2 (en) * | 2007-11-03 | 2009-04-07 | Wise Landfill Recycling Mining, Inc. | Systems and processes for cellulosic ethanol production |
WO2014159929A1 (en) | 2013-03-14 | 2014-10-02 | Abengoa Bioenergy New Technologies, Llc | Method for adding enzymes to obtain high ethanol yield from cereal mash |
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US4243750A (en) * | 1979-05-29 | 1981-01-06 | National Distillers And Chemical Corp. | Process for the hydrolysis of starch and the continuous fermentation of the sugars obtained therefrom to provide ethanol |
US4286058A (en) * | 1979-11-06 | 1981-08-25 | Wenger Manufacturing | Enzymatic conversion of high moisture shear extruded and gelatinized grain material |
US4316956A (en) * | 1980-02-06 | 1982-02-23 | Novo Industri A/S | Fermentation process |
US5231017A (en) * | 1991-05-17 | 1993-07-27 | Solvay Enzymes, Inc. | Process for producing ethanol |
KR0152482B1 (en) * | 1995-09-29 | 1998-10-01 | 최차용 | Process for continuous preparation of strain metabolic product by fermentation |
WO2002038787A2 (en) * | 2000-11-10 | 2002-05-16 | Novozymes A/S | Secondary liquefaction of starch in ethanol production |
US20040063184A1 (en) * | 2002-09-26 | 2004-04-01 | Novozymes North America, Inc. | Fermentation processes and compositions |
-
2005
- 2005-05-24 CN CNA2005800255419A patent/CN1993471A/en active Pending
- 2005-05-24 WO PCT/US2005/018421 patent/WO2005118827A2/en active Application Filing
- 2005-05-24 US US11/597,941 patent/US20070202583A1/en not_active Abandoned
- 2005-05-24 EP EP05754253A patent/EP1753867A4/en not_active Withdrawn
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8728773B2 (en) | 2005-11-28 | 2014-05-20 | Matthias Boy | Fermentative production of organic compounds using substances containing dextrin |
WO2008026932A1 (en) | 2006-08-30 | 2008-03-06 | Cambi As | Method and device for thermal enzymatic hydrolysis of ligno cellulose |
CN104328143A (en) * | 2014-10-24 | 2015-02-04 | 哈尔滨艾博雅食品科技开发有限公司 | Method for producing alcohol from fresh tobacco leaves |
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EP1753867A2 (en) | 2007-02-21 |
EP1753867A4 (en) | 2011-09-21 |
CN1993471A (en) | 2007-07-04 |
WO2005118827A3 (en) | 2006-09-14 |
US20070202583A1 (en) | 2007-08-30 |
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