WO2015050809A1 - Procédés de production de produits de fermentation - Google Patents

Procédés de production de produits de fermentation Download PDF

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Publication number
WO2015050809A1
WO2015050809A1 PCT/US2014/057997 US2014057997W WO2015050809A1 WO 2015050809 A1 WO2015050809 A1 WO 2015050809A1 US 2014057997 W US2014057997 W US 2014057997W WO 2015050809 A1 WO2015050809 A1 WO 2015050809A1
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Prior art keywords
fermentation
mash
lipoamide
lipoate
starch
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PCT/US2014/057997
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English (en)
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Jason Holmes
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Novozymes A/S
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Publication of WO2015050809A1 publication Critical patent/WO2015050809A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • 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 processes of producing fermentation products from plant material.
  • the invention also relates to processes of fermenting sugars derived from plant materials using a fermenting organism.
  • the invention also relates to compositions that can be used in processes of the invention.
  • alcohols e.g. , ethanol, methanol, butanol, 1 ,3-propanediol
  • organic acids e.g. , citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid, gluconate, lactic acid, succinic acid, 2,5-diketo-D-gluconic acid
  • ketones e.g. , acetone
  • amino acids e.g. , glutamic acid
  • gases e.g. , H 2 and C0 2
  • complex compounds including, for example, antibiotics (e.g.
  • Fermentation is 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.
  • the present invention relates to processes of producing fermentation products from plant materials.
  • the invention provides processes of producing fermentation products, such as especially ethanol, in increased yield, from sugars derived from plant material by fermentation using a fermenting organism.
  • the starting material may be any plant material or a part or constituent thereof.
  • the starting material is starch-containing material.
  • the starting material is lignocellulose-containing material.
  • the sugars, to be fermented into a desired fermentation product may be derived from starch-containing material (i.e. mainly starch) or lignocellulose-containing material (i.e. mainly cellulose or hemicellulose). Fermentation process of the invention
  • the invention relates to processes of fermenting sugars derived from plant material into fermentation products using a fermenting organism, wherein lipoic acid, lipoate and/or lipoamide, or blends thereof, is(are) present in fermentation and/or added before and/or during fermentation.
  • lipoic acid or lipoate, or blends thereof is(are) present and/or dosed into fermentation in a concentration the range from 0.001-1 .8 mM/g mash, preferably 0.001 - 1 .5 mM/g mash, such as 0.01-1 .2 mM/g mash, such as 0.02-1.0 mM/g mash, preferably 0.03-0.8 mM/g mash, such as 0.04-0.6 mM/g mash, such as around 0.4 mM/g mash.
  • lipoamide is present and/or dosed into fermentation in a concentration in the range from 0.001 -10 mM/g mash, preferably 0.001 - 8 mM/g mash, such as 0.01 -6 mM/g mash, such as 0.02-4 mM/g mash, preferably 0.03-2 mM/g mash, such as 0.04-1 mM/g mash, such as around 0.4 mM/g mash.
  • lipoic acid and/or lipoate together with lipoamide are present and/or dosed into fermentation in a concentration in the range from 0.001 -0.80 mM/g mash, preferably 0.001 - 0.7 mM/g mash, such as 0.01 -0.6 mM/g mash, such as 0.02-0.5 mM/g mash, preferably 0.03-0.4 mM/g mash, such as 0.04-0.3 mM/g mash, such as around 0.2 mM/g mash.
  • the ratio between lipoic acid or lipoate and lipoamide is between 1 :9 and 9:1 , such as between 1 :5 and 5:1 , such as around 1 :1 .
  • lipoic acid, lipoate and/or lipoamide is(are) present in an increased concentration in fermentation compared to a similar fermentation wherein no lipoic acid, lipoate and/or lipoamide is(are) present and/or has been added.
  • the fermentation product yield such as especially ethanol yield
  • the fermentation product yield is boosted, i.e., increased compared to when no lipoic acid, lipoate and/or lipoamide is(are) present or added.
  • Lipoic acid, lipoate and/or lipoamide may be added before and/or during fermentation.
  • lipoic acid, lipoate and/or lipoamide is(are) added into the fermentation medium, e.g., in form of a composition of the invention further comprising one or more enzymes, preferably glucoamylase and/or one or more fermenting organisms, preferably yeast.
  • Lipoic acid, lipoate or lipoamide, or blends thereof, is(are) according to the invention used as fermentation product yield boosting compounds, which means compounds that when present in fermentation, using a fermenting organism, result in increased yield of the desired fermentation product compared to a corresponding fermentation process where no lipoic acid, lipoate and/or lipoamide is(are) present and/or has been added.
  • the invention relates to processes of producing fermentation products, preferably ethanol, from starch-containing material comprising the steps of:
  • fermentation is carried out in accordance with a fermentation process of the invention, i.e., wherein lipoic acid, lipoate and/or lipoamide, or blends thereof, is(are) present in fermentation and/or added before and/or during fermentation.
  • the invention relates to processes of producing fermentation products, preferably ethanol, from starch-containing material, preferably granular starch, comprising the steps of:
  • fermentation is carried out in accordance with a fermentation process of the invention, i.e., wherein lipoic acid, lipoate and/or lipoamide, or blends thereof, is(are) present in fermentation and/or added before and/or during fermentation.
  • saccharification and fermentation steps are carried out simultaneously.
  • the invention relates to processes of producing fermentation products, preferably ethanol, from lignocellulose-containing material, comprising the steps of:
  • step (b) hydrolyzing the material obtain in step (a);
  • fermentation is carried out in accordance with a fermentation process of the invention, i.e., wherein lipoic acid, lipoate and/or lipoamide, or blends thereof, is(are) present in fermentation and/or added before and/or during fermentation.
  • the invention also relates to compositions comprising lipoic acid, lipoate or lipoamide, or blends thereof, and further one or more enzymes and/or one or more fermenting organisms.
  • invention also relates to the use of lipoic acid, lipoate or lipoamide, or blends thereof for propagating fermenting organisms and for use in a fermentation process.
  • Fig. 1 shows the percentage (%) ethanol yield improvement over a Control after 54 hours corn mash fermentation in the presence of 2 mM and 10 mM lipoic acid or lipoamide, respectively, and 1 mM and 5 mM lipoic acid and lipoamide, and 0.6 AGU/g TS Glucoamylase E.
  • Fig. 2 shows the percentage (%) glycerol produced over a Control after 54 hours corn mash fermentation in the presence of 2 mM and 10mM lipoic acid or lipoamide, respectively, and 1 mM and 5 mM lipoic acid and lipoamide and 0.6 AGU/g TS Glucoamylase E.
  • Fig. 3 shows the percentage (%) residual glucose over a Control after 54 hours corn mash fermentation in the presence of 2 mM and 10mM lipoic acid or lipoamide, respectively, and 1 mM and 5 mM lipoic acid and lipoamide and 0.6 AGU/g TS Glucoamylase E.
  • the present invention relates to processes of producing fermentation products from plant material, including a fermentation step using a fermenting organism.
  • the present invention provides a means of increasing the fermentation product production yield, such as especially ethanol production yield, in fermenting organisms through the alteration of the metabolic pathway. This is done by adding one or more enzymatic cofactors selected from the group of lipoic acid, lipoate and/or lipoamide, or blends thereof, in fermentation.
  • Lipoic acid, lipoate and lipoamide are enzymatic cofactors involved in the metabolic pathway of fermenting organisms, such as yeast.
  • Lipoic acid (6,8-dithiooctanoic acid) is the free form of a catalytic cofactor important in the generation and transfer of acyl groups formed by the pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase complexes, which carry out two central oxidative decarboxylations in metabolism.
  • the carboxyl group of lipoic acid is joined to dihydrolipoamide transacetylase via an amide bond linking the carboxyl group of lipoic acid to a lysine s-amino group of the enzyme.
  • the resulting reactive species is an amide, called lipoamide (6,8- dithiooctanoic amide).
  • Lipoate is the conjugate base of lipoic acid, i.e., base formed by removing a proton from lipoic acid.
  • the ethanol yield is increased when 2 mM (i.e., 2 mmol) lipoic acid (LA); 2 mM or 10 mM lipoamide (LAM); or 1 mM LA and LAM is added to 5 g corn mash (35.74% TS (Total Solids)) together with 0.6 AGU/g TS glucoamylase, and fermented for 54 hours, compared to when no lipoic acid, lipoate and/or lipoamide is(are) added.
  • a dose of 10mM lipoic acid/5g mash caused a marked reduction in ethanol production.
  • Lipoamide did not show a decrease in ethanol production at 10mM, however when lipoic acid and lipoamide were combined at 5mM a strong reduction in ethanol production was noted. Fermentation Process of the Invention
  • the invention relates to processes of fermenting sugars derived from plant material into fermentation products using a fermenting organism, wherein lipoic acid, lipoate, or lipoamide, or blends thereof, is(are) present and/or added before and/or during fermentation.
  • Lipoic acid, lipoate and/or lipoamide, or blends thereof may according to the invention be added before and/or during fermentation in an effective amount/concentration.
  • the lipoic acid, lipoate and/or lipoamide concentration is according to the invention higher compared to when no lipoic acid, lipoate and/or lipoamide is present or added in fermentation.
  • lipoic acid, lipoate and/or lipoamide has(have) a yield boosting effect when producing fermentation products, such as especially ethanol, from starch-containing material in a process including a fermentation step, such as a conventional simultaneous saccharification and fermentation step (SSF step).
  • a fermentation step such as a conventional simultaneous saccharification and fermentation step (SSF step).
  • effective amounts include embodiments where lipoic acid or lipoate, or blends thereof, is(are) present and/or dosed into fermentation in a concentration in the range from 0.001-1 .8 mM/g mash, preferably 0.001 -1 .5 mM/g mash, such as 0.01 -1 .2 mM/g mash, such as 0.02-1 .0 mM/g mash, preferably 0.03-0.8 mM/g mash, such as 0.04-0.6 mM/g mash, such as around 0.4 mM/g mash.
  • effective amounts include embodiments where lipoamide is present and/or dosed into fermentation in a concentration in the range from 0.001 -10 mM/g mash, preferably 0.001 - 8 mM/g mash, such as 0.01-6 mM/g mash, such as 0.02-4 mM/g mash, preferably 0.03-2 mM/g mash, such as 0.04-1 mM/g mash, such as around 0.4 mM/g mash.
  • effective amounts include embodiments where lipoic acid and/or lipoate together with lipoamide are present and/or dosed into fermentation in a concentration in the range from 0.001-0.80 mM/g mash, preferably 0.001- 0.7 mM/g mash, such as 0.01 -0.6 mM/g mash, such as 0.02-0.5 mM/g mash, preferably 0.03-0.4 mM/g mash, such as 0.04-0.3 mM/g mash, such as around 0.2 mM/g mash.
  • the ratio between lipoic acid or lipoate and lipoamide is between 1 :9 and 9:1 , such as between 1 :5 and 5:1 , such as around 1 :1 .
  • the fermenting organism is yeast, filamentous fungus, or a bacterium.
  • the fermenting organism is derived from a strain of Saccharomyces, such as a strain of Saccharomyces cerevisiae.
  • sugars derived from plant materials are derived from starch- containing material.
  • starch-containing materials can be found in the "Starch- Containing Materials' -section below.
  • the starch-containing material is corn.
  • the starch-containing material is a small grain such as wheat.
  • the sugars derived from plant materials are derived from lignocellulose-containing material.
  • lignocellulose-containing materials can be found in the "Lignocellulose-Containing Materials (Biomass)"-section below.
  • the lignocellulose-containing material is selected from the group of corn cobs, corn stover, wheat fiber, and bagasse.
  • the fermentation product is an alcohol, preferably ethanol, especially fuel ethanol, potable ethanol and/or industrial ethanol.
  • ethanol especially fuel ethanol, potable ethanol and/or industrial ethanol. Examples of contemplated fermentation products can be found in the "Fermentation Products"-section below. Fermenting Organisms
  • fermenting organism refers to any organism, including bacterial and fungal organisms, including yeast and filamentous fungi, suitable for producing a desired fermentation product.
  • suitable fermenting organisms according to the invention are able to ferment, i.e., convert sugars, such as glucose, fructose, maltose, xylose, mannose and/or arabinose, directly or indirectly into the desired fermentation product.
  • Examples of fermenting organisms include fungal organisms, such as yeast.
  • Preferred yeast includes strains of the genus Saccharomyces, in particular a strain of Saccharomyces cerevisiae or Saccharomyces uvarum; a strain of Pichia, in particular Pichia stipitis or Pichia pastoris; a strain of the genus Candida, in particular a strain of Candida utilis, Candida arabinofermentans, Candida diddensii, Candida sonorensis, Candida shehatae, Candida tropicalis, or Candida boidinii.
  • yeast includes strains of Hansenula, in particular Hansenula polymorpha or Hansenula anomala; strains of Kluyveromyces, in particular Kluyveromyces marxianus or Kluyveromyces fagilis, and strains of Schizosaccharomyces, in particular Schizosaccharomyces pombe.
  • Preferred bacterial fermenting organisms include strains of Escherichia, in particular
  • Escherichia coli strains of Zymomonas, in particular Zymomonas mobilis, strains of Zymobacter, in particular Zymobactor palmae, strains of Klebsiella in particular Klebsiella oxytoca, strains of Leuconostoc, in particular Leuconostoc mesenteroides, strains of Clostridium, in particular Clostridium butyricum, strains of Enterobacter, in particular Enterobacter aerogenes and strains of Thermoanaerobacter, in particular Thermoanaerobacter BG1 L1 (Appl. Microbiol. Biotech.
  • Lactobacillus is also envisioned as are strains of Corynebacterium glutamicum R, Bacillus thermoglucosidaisus, and Geobacillus thermoglucosidasius.
  • the fermenting organism is a C6 sugar (hexose) fermenting organism, such as a strain of, e.g., Saccharomyces cerevisiae.
  • C5 sugar (pentose) fermenting organisms are also contemplated. Most C5 sugar fermenting organisms also ferment C6 sugars. Examples of C5 sugar fermenting organisms include strains of Pichia, such as of the species Pichia stipitis. C5 sugar fermenting bacteria are also known. Also some Saccharomyces cerevisae strains ferment C5 (and C6) sugars. Examples are genetically modified strains of Saccharomyces spp that are capable of fermenting C5 sugars include the ones concerned in, e.g., Ho et al., 1998, Applied and Environmental Microbiology, p. 1852-1859 and Karhumaa et al., 2006, Microbial Cell Factories 5:18.
  • the fermenting organism is added in fermentation so that the viable fermenting organism, such as yeast, count per mL of fermentation medium is in the range from 10 5 to 10 12 , preferably from 10 7 to 10 10 , especially about 5x10 7 .
  • yeast includes, e.g., RED STARTM and ETHANOL REDTM yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACCTM fresh yeast (available from Ethanol Technology, Wl, USA), BIOFERM AFT and XR (available from NABC - North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).
  • RED STARTM and ETHANOL REDTM yeast available from Fermentis/Lesaffre, USA
  • FALI available from Fleischmann's Yeast, USA
  • SUPERSTART and THERMOSACCTM fresh yeast available from Ethanol Technology, Wl, USA
  • BIOFERM AFT and XR available from NABC - North American Bioproducts Corporation, GA, USA
  • GERT STRAND available from Gert Strand AB, Sweden
  • FERMIOL available from DSM Specialties
  • the fermenting organism capable of producing a desired fermentation product from fermentable sugars including glucose, fructose maltose, xylose, mannose, and/or arabinose
  • the inoculated fermenting organism pass through a number of stages. Initially growth does not occur. This period is referred to as the "lag phase” and may be considered a period of adaptation.
  • the growth rate gradually increases. After a period of maximum growth the rate ceases and the fermenting organism enters "stationary phase”. After a further period of time the fermenting organism enters the "death phase" where the number of viable cells declines.
  • lipoic acid, lipoate or lipoamide, or blends thereof is(are) added to the fermentation medium when the fermenting organism is in the lag phase.
  • lipoic acid, lipoate or lipoamide, or blends thereof is(are) added to the fermentation medium when the fermenting organism is in exponential phase.
  • lipoic acid, lipoate or lipoamide, or blends thereof is(are) added to the fermentation medium when the fermenting organism is in stationary phase.
  • Fermentation product means a product produced in a process including a fermentation step using a fermenting organism.
  • Fermentation products contemplated according to the invention include 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 C0 2 ); antibiotics (e.g. , penicillin and tetracycline); enzymes; vitamins (e.g. , riboflavin, B 12 , 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., ace
  • the fermentation product is ethanol, e.g. , fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g. , beer and wine), dairy industry (e.g. , fermented dairy products), leather industry and tobacco industry.
  • Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer.
  • the sugars derived from plant material used in fermentation in a process of the invention may be derived from starch-containing material and/or lignocellulose-containing material.
  • the fermentation conditions are determined based on, e.g. , the kind of plant material, the fermentable sugars, the fermenting organism and/or the desired fermentation product.
  • One skilled in the art can easily determine suitable fermentation conditions.
  • the fermentation may according to the invention be carried out at conventionally used conditions. Preferred fermentation processes are anaerobic processes.
  • fermenting organisms may be used for fermenting sugars derived from starch-containing material. Fermentations are often carried out using yeast, such as Saccharomyces cerevisae, as the fermenting organism. However, bacteria and filamentous fungi may also be used as fermenting organisms. Some bacteria have higher fermentation temperature optimum than, e.g. , Saccharomyces cerevisae. Therefore, fermentations may in such cases be carried out at temperatures as high as 75°C, e.g. , between 40-70°C, such as between 50-60°C. However, bacteria with a significantly lower temperature optimum down to around room temperature (around 20°C) are also known. Examples of suitable fermenting organisms can be found in the "Fermenting Organisms"-section above.
  • the fermentation may in one embodiment go on for 24 to 96 hours, in particular for 36 to 72 hours.
  • the fermentation is carried out at a temperature between 20 to 40°C, preferably 28 to 36°C, in particular around 32°C.
  • the pH is from pH 3 to 6, preferably around pH 4 to 5.
  • SSF simultaneous hydrolysis/saccharification and fermentation
  • the lipoic acid, lipoate or lipoamide, or blends thereof may also be added separately.
  • the temperature is preferably between 20 to 40°C, preferably 28 to 36°C, in particular around 32°C when the fermentation organism is a strain of Saccharomyces cerevisiae and the desired fermentation product is ethanol.
  • the process of the invention may be performed as a batch or as a continuous process.
  • the fermentation process of the invention may be conducted in an ultrafiltration system where the retentate is held under recirculation in the presence of solids, water, and the fermenting organism, and where permeate is the desired fermentation product containing liquid.
  • Equally contemplated are processes conducted in a continuous membrane reactor with ultrafiltration membranes and where the retentate is held under recirculation in presence of solids, water, the fermenting organism and where the permeate is the fermentation product containing liquid.
  • the fermenting organism may be separated from the fermented slurry and recycled.
  • Fermentations are typically carried out at a pH in the range between 3 and 7, preferably from pH 3.5 to 6, such as around pH 5. Fermentations are typically ongoing for 24-96 hours.
  • fermenting organisms may be used for fermenting sugars derived from lignocellulose-containing materials. Fermentations are typically carried out by yeast, bacteria, or filamentous fungi, including the ones mentioned in the "Fermenting Organisms"-section above. If the aim is C6 fermentable sugars the conditions are usually similar to starch fermentations as described above. However, if the aim is to ferment C5 sugars (e.g., xylose) or a combination of C6 and C5 fermentable sugars the fermenting organism(s) and/or fermentation conditions may differ.
  • C5 sugars e.g., xylose
  • the fermenting organism(s) and/or fermentation conditions may differ.
  • Bacteria fermentations may be carried out at higher temperatures, such as up to 75°C, e.g., between 40-70°C, such as between 50-60°C, than conventional yeast fermentations, which are typically carried out at temperatures from 20-40°C.
  • bacteria fermentations at temperature as low as 20°C are known.
  • Fermentations are typically carried out at a pH in the range between 3 and 7, preferably from pH 3.5 to 6, such as around pH 5. Fermentations are typically ongoing for 24-96 hours.
  • the fermentation product may be separated from the fermented slurry.
  • the slurry may be distilled to extract the desired fermentation product or the desired fermentation product may be extracted from the fermented slurry by micro or membrane filtration techniques. Alternatively the fermentation product may be recovered by stripping. Methods for recovery are well known in the art.
  • Processes for producing fermentation products from gelatinized starch-containing material relates to processes of producing fermentation products, especially ethanol, from starch-containing material, which processes includes a liquefaction step and sequentially or simultaneously performed saccharification and fermentation steps.
  • the invention relates to processes for producing fermentation products from starch- containing material comprising the steps of:
  • fermentation is carried out in accordance with the fermentation process of the invention, i.e., wherein lipoic acid, lipoate or lipoamide, respectively, or a blends thereof, is(are) present in fermentation and/or added before and/or during fermentation.
  • the lipoic acid, lipoate and/or lipoamide, or a blends thereof is(are) added before and/or during the fermentation step in an effective amount/concentration.
  • lipoic acid, lipoate or lipoamide, or blends thereof may be added in a separate saccharification step, such as pre-saccharification step, before fermentation.
  • lipoic acid, lipoate and/or lipoamide, or a blends thereof is(are) added directly to the fermentation medium or to the fermenting organism propagation medium.
  • the lipoic acid, lipoate and/or lipoamide, or blends thereof, concentration is according to the invention higher compared to when no such compound(s) is/are added in fermentation.
  • lipoic acid, lipoate and/or lipoamide, or a blends thereof When added in an effective amount lipoic acid, lipoate and/or lipoamide, or a blends thereof, has(have) a yield boosting effect when producing fermentation products, such as especially ethanol, from starch-containing material in a process including a fermentation step, such as a simultaneous saccharification and fermentation step (i.e., SSF step).
  • a fermentation step such as a simultaneous saccharification and fermentation step (i.e., SSF step).
  • effective amounts include embodiments where lipoic acid or lipoate, or blends thereof, is(are) present and/or dosed into fermentation in a concentration in the range from 0.001 -1 .8 mM/g mash, preferably 0.001- 1.5 mM/g mash, such as 0.01-1.2 mM/g mash, such as 0.02-1.0 mM/g mash, preferably 0.03-0.8 mM/g mash, such as 0.04-0.6 mM/g mash, such as around 0.4 mM/g mash.
  • effective amounts include embodiments where lipoamide is present and/or dosed into fermentation in a concentration in the range from 0.001 -10 mM/g mash, preferably 0.001 - 8 mM/g mash, such as 0.01-6 mM/g mash, such as 0.02-4 mM/g mash, preferably 0.03-2 mM/g mash, such as 0.04-1 mM/g mash, such as around 0.4 mM/g mash.
  • effective amounts include embodiments where lipoic acid and/or lipoate together with lipoamide are present and/or dosed/added into fermentation in a concentration in the range from 0.001-0.80 mM/g mash, preferably 0.001- 0.7 mM/g mash, such as 0.01 -0.6 mM/g mash, such as 0.02-0.5 mM/g mash, preferably 0.03-0.4 mM/g mash, such as 0.04-0.3 mM/g mash, such as around 0.2 mM/g mash.
  • the ratio between lipoic acid and/or lipoate and lipoamide is between 1 :9 and 9:1 , such as between 1 :5 and 5:1 , such as around 1 :1 .
  • the fermentation product such as especially ethanol, may optionally be recovered after fermentation, e.g., by distillation.
  • Suitable starch-containing starting materials are listed in the section "Starch-Containing Materials"-section below.
  • Contemplated enzymes are listed in the “Enzymes' -section below.
  • the liquefaction is carried out in the presence of an alpha-amylase, preferably a bacterial alpha-amylase, especially Bacillus alpha-amylase.
  • the fermenting organism is preferably yeast, preferably a strain of Saccharomyces, especially a strain of Saccharomyces cerevisae.
  • Suitable fermenting organisms are listed in the "Fermenting Organisms"-section above.
  • steps ii) and iii) are carried out sequentially or simultaneously ⁇ i.e., as SSF process).
  • the process of the invention further comprises, prior to liquefaction step i), the steps of:
  • the aqueous slurry may contain from 10-55 wt.-% dry solids, preferably 25-45 wt.-% dry solids, more preferably 30-40 wt.-% dry solids of starch-containing material.
  • the slurry is heated to above the initial gelatinization temperature.
  • Alpha-amylase preferably bacterial alpha-amylase, may be added to the slurry.
  • the slurry is also jet-cooked to further gelatinize the slurry before being subjected to an alpha-amylase in liquefaction step i).
  • liquefaction is carried out as a three-step hot slurry process.
  • the slurry is heated to between 60-95°C, preferably between 80-90°C, and alpha-amylase is added to initiate liquefaction (thinning).
  • the slurry is jet-cooked at a temperature between 95- 140°C, preferably 105-125°C, for 1-15 minutes, preferably for 3-10 minutes, especially around 5 minutes.
  • the slurry is cooled to between 60-95°C, preferably between 80-90°C, such as around 85°C, and more alpha-amylase is added to finalize hydrolysis (secondary liquefaction).
  • the liquefaction process is usually carried out at pH 4.5-6.5, in particular at a pH between 5 and 6. Milled and liquefied starch is known as "mash".
  • the saccharification in step ii) may be carried out using conditions well known in the art. For instance, a full saccharification process may last up to from about 24 to about 72 hours.
  • a pre-saccharification step is done for 40-90 minutes at a temperature between 30-65°C, typically at about 60°C, followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation step (i.e., SSF step). Saccharification is typically carried out at temperatures from 30-70°C, such as 55-65°C, typically around 60°C, and at a pH between 4 and 5, normally at about pH 4.5.
  • the most widely used process in fermentation product production is simultaneous saccharification and fermentation (i.e., SSF process), in which there is no holding stage for the saccharification.
  • SSF process simultaneous saccharification and fermentation
  • the fermenting organism such as yeast, enzymes and lipoic acid, lipoate and/or lipoamide, or blends thereof, may be added together.
  • SSF may typically be carried out at a temperature between 25°C and 40°C, such as between 28°C and 36°C, such as between 30°C and 34°C, such as around 32°C, when the fermentation organism is yeast, such as a strain of Saccharomyces cerevisiae, and the desired fermentation product is ethanol.
  • Other fermentation products may be fermented at conditions and temperatures, well known to the skilled person in the art, suitable for the fermenting organism in question. According to the invention the temperature may be adjusted up or down during fermentation.
  • protease is added during fermentation.
  • proteases can be found in the "Proteases"-section below. Protease dosing can easily be determined by a person skilled in the art.
  • the invention relates to processes for producing a fermentation product from starch-containing material without gelatinization of the starch-containing material (i.e., uncooked starch-containing material).
  • the desired fermentation product such as ethanol, can be produced without liquefying the aqueous slurry containing the starch-containing material.
  • a process of the invention includes saccharifying (milled) starch-containing material, especially granular starch, below the initial gelatinization temperature, preferably in the presence of a carbohydrate-source generating enzyme, preferably a glucoamylase, to produce sugars that can be fermented into the desired fermentation product by a suitable fermenting organism.
  • a carbohydrate-source generating enzyme preferably a glucoamylase
  • the desired fermentation product is produced from un-gelatinized (i.e., uncooked) milled starch-containing material, especially granular starch.
  • the invention relates to processes of producing a fermentation product from starch-containing material, comprising the steps of:
  • fermentation is carried out in accordance with the fermentation process of the invention, i.e., wherein lipoic acid, lipoate and/or lipoamide, or blends thereof, is(are) present in fermentation and/or added before and/or during fermentation.
  • steps (a) and (b) are carried out simultaneously (i.e., one step fermentation) or sequentially.
  • Lipoic acid, lipoate and/or lipoamide, or blends thereof may be added before and/or during the fermentation step, especially during simultaneous saccharification and fermentation in an effective amount/concentration.
  • lipoic acid, lipoate and/or lipoamide, or blends thereof is(are) added directly to the fermentation medium or to the fermenting organism propagation medium.
  • the lipoic acid, lipoate and/or lipoamide, or blends thereof, concentration is according to the invention higher compared to when no lipoic acid, lipoate and/or lipoamide, or blends thereof, is(are) added in fermentation.
  • lipoic acid, lipoate and/or lipoamide, or blends thereof has(have) a yield boosting effect when producing fermentation products, such as especially ethanol, from starch-containing material, especially granular starch, in a process including a fermentation step, such as an one-step process of fermenting ungelatinized starch (i.e., no- cook process).
  • effective amounts include embodiments where lipoic acid or lipoate, or blends thereof, is(are) present and/or dosed into fermentation in a concentration in the range from 0.001 -1 .8 mM/g mash, preferably 0.001- 1.5 mM/g mash, such as 0.01-1.2 mM/g mash, such as 0.02-1.0 mM/g mash, preferably 0.03-0.8 mM/g mash, such as 0.04-0.6 mM/g mash, such as around 0.4 mM/g mash.
  • effective amounts include embodiments where lipoamide is present and/or dosed into fermentation in a concentration in the range from 0.001 -10 mM/g mash, preferably 0.001 - 8 mM/g mash, such as 0.01-6 mM/g mash, such as 0.02-4 mM/g mash, preferably 0.03-2 mM/g mash, such as 0.04-1 mM/g mash, such as around 0.4 mM/g mash.
  • effective amounts include embodiments where lipoic acid and/or lipoate together with lipoamide are present and/or dosed/added into fermentation in a concentration in the range from 0.001-0.80 mM/g mash, preferably 0.001- 0.7 mM/g mash, such as 0.01 -0.6 mM/g mash, such as 0.02-0.5 mM/g mash, preferably 0.03-0.4 mM/g mash, such as 0.04-0.3 mM/g mash, such as around 0.2 mM/g mash.
  • the ratio between lipoic acid or lipoate and lipoamide is between 1 :9 and 9:1 , such as between 1 :5 and 5:1 , such as around 1 :1 .
  • the fermentation product such as especially ethanol, may optionally be recovered after fermentation, e.g., by distillation.
  • Suitable starch-containing starting materials are listed in the section "Starch-Containing Materials"-section below.
  • Contemplated enzymes are listed in the “Enzymes”-section below.
  • a glucoamylase and/or an alpha-amylase may be present.
  • Alpha- amylases used are preferably acidic, preferably acid fungal alpha-amylases.
  • the fermenting organism is preferably yeast, preferably a strain of Saccharomyces. Suitable fermenting organisms are listed in the "Fermenting Organisms' -section above.
  • the term "below the initial gelatinization temperature” means below the lowest temperature where gelatinization of the starch commences.
  • Starch heated in water typically begins to gelatinize between 50°C and 75°C; the exact temperature of gelatinization depends on the specific starch, and can readily be determined by the skilled artisan.
  • the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions.
  • the initial gelatinization temperature of a given starch-containing material is the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein and Lii, 1992, Starc /Starke 44 (12): 461 -466.
  • step (a) a slurry of starch-containing material, such as granular starch, having
  • the slurry may include water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side stripper water from distillation, or other fermentation product plant process water. Because the process of the invention is carried out below the initial gelatinization temperature and thus no significant viscosity increase takes place, high levels of stillage may be used if desired.
  • the aqueous slurry contains from about 1 to about 70 vol.-% stillage, preferably 15-60% vol.-% stillage, especially from about 30 to 50 vol.-% stillage.
  • the starch-containing material may be prepared by reducing the particle size, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably 0.1-0.5 mm. After being subjected to a process of the invention at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or preferably at least 99% of the dry solids of the starch-containing material is converted into a soluble starch hydrolyzate.
  • the process of the invention is conducted at a temperature below the initial gelatinization temperature.
  • the temperature at which step (a) is carried out is between 30-75°C, preferably between 45-60°C.
  • step (a) and step (b) are carried out as a simultaneous saccharification and fermentation process.
  • the process is typically carried at a temperature between 25°C and 40°C, such as between 28°C and 36°C, such as between 30°C and 34°C, such as around 32°C.
  • the temperature may be adjusted up or down during fermentation.
  • simultaneous saccharification and fermentation is carried out so that the sugar level, such as glucose level, is kept at a low level such as below 6 wt.-%, preferably below about 3 wt.-%, preferably below about 2 wt.-%, more preferred below about 1 wt.-%., even more preferred below about 0.5%, or even more preferred 0.25% wt.-%, such as below about 0.1 wt.-%.
  • a low levels of sugar can be accomplished by simply employing adjusted quantities of enzyme and fermenting organism.
  • the employed quantities of enzyme and fermenting organism may also be selected to maintain low concentrations of maltose in the fermentation broth. For instance, the maltose level may be kept below about 0.5 wt.-% or below about 0.2 wt.-%.
  • the process of the invention may be carried out at a pH in the range between 3 and 7, preferably from pH 3.5 to 6, or more preferably from pH 4 to 5.
  • a protease is adding during fermentation.
  • proteases can be found in the "Proteases"-section below. Protease dosing can easily be determined by a person skilled in the art.
  • sugars may be derived from starch-containing materials.
  • Any suitable starch-containing starting material including granular starch, may be used according to the present invention.
  • the starting material is generally selected based on the desired fermentation product.
  • starch-containing starting materials suitable for use in a process of present invention, include whole grains, corns, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, beans, and sweet potatoes, or mixtures thereof, or cereals, or sugar-containing raw materials, such as molasses, fruit materials, sugar cane or sugar beet, potatoes. Contemplated are both waxy and non-waxy types of corn and barley.
  • granular starch means raw uncooked starch, i.e., starch in its natural form found in cereal, tubers or grains. Starch is formed within plant cells as tiny granules insoluble in water. When put in cold water, the starch granules may absorb a small amount of the liquid and swell. At temperatures up to 50°C to 75°C the swelling may be reversible. However, with higher temperatures an irreversible swelling called “gelatinization" begins.
  • Granular starch to be processed may in an embodiment be a highly refined starch, preferably at least 90%, at least 95%, at least 97% or at least 99.5% pure, or it may be a more crude starch containing material comprising milled whole grain including non-starch fractions such as germ residues and fibers.
  • the raw material such as whole grain, is milled in order to open up the structure and allowing for further processing.
  • Two milling processes are preferred according to the invention: wet and dry milling. In dry milling whole kernels are milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein) and is often applied at locations where the starch hydrolyzate is used in production of syrups. Both dry and wet milling is well known in the art of starch processing and is equally contemplated for the process of the invention.
  • the starch-containing material may be reduced in particle size, preferably by dry or wet milling, in order to expose more surface area.
  • the particle size is between 0.05 to 3.0 mm, preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fit through a sieve with a 0.05 to 3.0 mm screen, preferably 0.1-0.5 mm screen.
  • the invention relates to processes of producing desired fermentation products from lignocellulose-containing material.
  • Conversion of lignocellulose-containing material into fermentation products, such as ethanol, has the advantages of the ready availability of large amounts of feedstock, including wood, agricultural residues, herbaceous crops, municipal solid wastes etc.
  • Lignocellulose-containing materials primarily consist of cellulose, hemicellulose, and lignin and are often referred to as "biomass”.
  • lignocellulose is not directly accessible to enzymatic hydrolysis. Therefore, the lignocellulose-containing material has to be pre-treated, e.g., by acid hydrolysis under adequate conditions of pressure and temperature, in order to break the lignin seal and disrupt the crystalline structure of cellulose. This causes solubilization of the hemicellulose and cellulose fractions.
  • the cellulose and hemicelluloses can then be hydrolyzed enzymatically, e.g., by cellulolytic enzymes, to convert the carbohydrate polymers into fermentable sugars which may be fermented into a desired fermentation product, such as ethanol.
  • the fermentation product may be recovered, e.g., by distillation.
  • the invention relates to a process of producing a fermentation product from lignocellulose-containing material, comprising the steps of:
  • Lipoic acid, lipoate and/or lipoamide, or blends thereof may be added before and/or during fermentation.
  • lipoic acid, lipoate and/or lipoamide, or blends thereof is(are) added to the fermentation medium.
  • Fermentation step (c) may be carried out in accordance with the fermentation process of the invention. In preferred embodiments the steps are carried out as SSF, HHF, or SHF process steps which will be described further below.
  • the lignocellulose-containing material may according to the invention be pre-treated before being hydrolyzed and fermented.
  • the pre-treated material is hydrolyzed, preferably enzymatically, before and/or during fermentation.
  • the goal of pre- treatment is to separate and/or release cellulose, hemicellulose and/or lignin and this way improve the rate of enzymatic hydrolysis.
  • pre-treatment step (a) may be a conventional pre-treatment step known in the art. Pre-treatment may take place in aqueous slurry.
  • the lignocellulose- containing material may during pre-treatment be present in an amount between 10-80 wt. %, preferably between 20-50 wt. %.
  • the lignocellulose-containing material may according to the invention be chemically, mechanically and/or biologically pre-treated before hydrolysis and/or fermentation.
  • Mechanical treatment (often referred to as physical pre-treatment) may be used alone or in combination with subsequent or simultaneous hydrolysis, especially enzymatic hydrolysis, to promote the separation and/or release of cellulose, hemicellulose and/or lignin.
  • chemical, mechanical and/or biological pre-treatment is(are) carried out prior to the hydrolysis and/or fermentation.
  • the chemical, mechanical and/or biological pre- treatment is carried out simultaneously with hydrolysis, such as simultaneously with addition of one or more cellulolytic enzymes, or other enzyme activities mentioned below, to release fermentable sugars, such as glucose and/or maltose.
  • the pre-treated lignocellulose-containing material is washed and/or detoxified before or after hydrolysis step (b).
  • This may improve the fermentability of, e.g., dilute-acid hydrolyzed lignocellulose-containing material, such as corn stover.
  • Detoxification may be carried out in any suitable way, e.g., by steam stripping, evaporation, ion exchange, resin or charcoal treatment of the liquid fraction or by washing the pre-treated material.
  • chemical pre-treatment refers to any chemical treatment which promotes the separation and/or release of cellulose, hemicellulose and/or lignin.
  • suitable chemical pre-treatment steps include treatment with; for example, dilute acid, lime, alkaline, organic solvent, ammonia, sulphur dioxide, carbon dioxide.
  • wet oxidation and pH-controlled hydrothermolysis are also contemplated chemical pre-treatments.
  • the chemical pre-treatment is acid treatment, more preferably, a continuous dilute and/or mild acid treatment, such as, treatment with sulfuric acid, or another organic acid, such as acetic acid, citric acid, tartaric acid, succinic acid, or mixtures thereof. Other acids may also be used.
  • Mild acid treatment means in the context of the present invention that the treatment pH lies in the range from 1-5, preferably from pH 1-3. In a specific embodiment the acid concentration is in the range from 0.1 to 2.0 wt. % acid, preferably sulphuric acid.
  • the acid may be mixed or contacted with the material to be fermented according to the invention and the mixture may be held at a temperature in the range of 160-220°C, such as 165-195°C, for periods ranging from minutes to seconds, e.g., 1-60 minutes, such as 2-30 minutes or 3-12 minutes.
  • Addition of strong acids, such as sulphuric acid, may be applied to remove hemicellulose. This enhances the digestibility of cellulose.
  • Cellulose solvent treatment also contemplated according to the invention, has been shown to convert about 90% of cellulose to glucose. It has also been shown that enzymatic hydrolysis could be greatly enhanced when the lignocellulosic structure is disrupted.
  • Alkaline H 2 0 2 , ozone, organosolv (uses Lewis acids, FeCI 3 , (AI) 2 S0 4 in aqueous alcohols), glycerol, dioxane, phenol, or ethylene glycol are among solvents known to disrupt cellulose structure and promote hydrolysis (Mosier et al., 2005, Bioresource Technology 96: 673-686).
  • Alkaline chemical pre-treatment with base e.g., NaOH, Na 2 C0 3 and/or ammonia or the like
  • base e.g., NaOH, Na 2 C0 3 and/or ammonia or the like
  • Pre-treatment methods using ammonia are described in, e.g., WO 2006/1 10891 , WO 2006/1 10899, WO 2006/1 10900, WO 2006/1 10901 , which are hereby incorporated by reference.
  • oxidizing agents such as: sulphite based oxidizing agents or the like.
  • solvent pre-treatments include treatment with DMSO (Dimethyl Sulfoxide) or the like.
  • Chemical pre-treatment is generally carried out for 1 to 60 minutes, such as from 5 to 30 minutes, but may be carried out for shorter or longer periods of time dependent on the material to be pre-treated.
  • mechanical pre-treatment refers to any mechanical or physical pre-treatment which promotes the separation and/or release of cellulose, hemicellulose and/or lignin from lignocellulose-containing material.
  • mechanical pre-treatment includes various types of milling, irradiation, steaming/steam explosion, and hydrothermolysis.
  • Mechanical pre-treatment includes comminution (mechanical reduction of the particle size). Comminution includes dry milling, wet milling and vibratory ball milling. Mechanical pre- treatment may involve high pressure and/or high temperature (steam explosion).
  • high pressure means pressure in the range from 300 to 600 psi, preferably 400 to 500 psi, such as around 450 psi.
  • high temperature means temperatures in the range from about 100 to 300°C, preferably from about 140 to 235°C.
  • mechanical pre-treatment is a batch-process, steam gun hydrolyzer system which uses high pressure and high temperature as defined above. A Sunds Hydrolyzer (available from Sunds Defibrator AB (Sweden) may be used for this.
  • both chemical and mechanical pre-treatments are carried out involving, for example, both dilute or mild acid pretreatment and high temperature and pressure treatment.
  • the chemical and mechanical pretreatment may be carried out sequentially or simultaneously, as desired.
  • the lignocellulose-containing material is subjected to both chemical and mechanical pre-treatment to promote the separation and/or release of cellulose, hemicellulose and/or lignin.
  • pre-treatment is carried out as a dilute or mild acid pre- treatment step.
  • pre-treatment is carried out as an ammonia fiber explosion step (or AFEX pre-treatment step).
  • biological pre-treatment refers to any biological pre-treatment which promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from the lignocellulose-containing material.
  • Biological pre-treatment techniques can involve applying lignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC, 179-212; Ghosh, P., and Singh, A., 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of lignocellulosic biomass, Adv. Appl.
  • the pre-treated lignocellulose-containing material Before and/or during fermentation the pre-treated lignocellulose-containing material may be hydrolyzed in order to break the lignin seal and disrupt the crystalline structure of cellulose. In a preferred embodiment hydrolysis is carried out enzymatically.
  • the pre-treated lignocellulose-containing material to be fermented may be hydrolyzed by one or more hydrolases (class E.C. 3 according to Enzyme Nomenclature), preferably one or more carbohydrases including cellulolytic enzymes and hemicellulolytic enzymes, or combinations thereof.
  • protease, alpha-amylase, glucoamylase and/or the like may also be present during hydrolysis and/or fermentation as the lignocellulose-containing material may include some, e.g., starchy and/or proteinaceous material.
  • the enzyme(s) used for hydrolysis may be capable of directly or indirectly converting carbohydrate polymers into fermentable sugars, such as glucose and/or maltose, which can be fermented into a desired fermentation product, such as ethanol.
  • carbohydrase(s) has(have) cellulolytic and/or hemicellulolytic enzyme activity.
  • hydrolysis is carried out using a cellulolytic enzyme composition further comprising one or more polypeptides having cellulolytic enhancing activity.
  • polypeptide(s) having cellulolytic enhancing activity is(are) of family GH61 origin.
  • suitable and preferred cellulolytic enzyme compositions and polypeptides having cellulolytic enhancing activity are described in the "Cellulases or Cellulolytic Enzymes"-section and “Cellulolytic Enhancing Polypeptides” sections below.
  • Hemicellulose polymers can be broken down by hemicellullolytic enzymes and/or acid hydrolysis to release its five and six carbon sugar components.
  • the six carbon sugars such as glucose, galactose, arabinose, and mannose, can readily be fermented to fermentation products such as ethanol, acetone, butanol, glycerol, citric acid, fumaric acid, etc. by suitable fermenting organisms including yeast.
  • Yeast is the preferred fermenting organism for ethanol fermentation.
  • Preferred are strains of Saccharomyces, especially strains of the species Saccharomyces cerevisiae, preferably strains which are resistant towards high levels of ethanol, i.e., up to, e.g., about 10, 12, 15 or 20 vol. % or more ethanol.
  • Enzymatic hydrolysis is preferably carried out in a suitable aqueous environment under conditions which can readily be determined by one skilled in the art. In a preferred embodiment hydrolysis is carried out at suitable, preferably optimal, conditions for the enzyme(s) in question.
  • Suitable process time, temperature and pH conditions can readily be determined by one skilled in the art.
  • hydrolysis is carried out at a temperature between 25 and 70°C, preferably between 40 and 60°C, especially around 50°C.
  • the step is preferably carried out at a pH in the range from 3-8, preferably pH 4-6.
  • Hydrolysis is typically carried out for between 12 and 96 hours, preferable 16 to 72 hours, more preferably between 24 and 48 hours. Fermentation of Lignocellulose derived Material
  • Fermentation of lignocellulose derived material is carried out in accordance with a fermentation process of the invention as described above, wherein lipoic acid, lipoate and/or lipoamide, or blends thereof, is(are) present in fermentation and/or added before and/or during fermentation.
  • Lignocellulose-containing material may be any material containing lignocellulose.
  • the lignocellulose-containing material contains at least 50 wt. %, preferably at least 70 wt. %, more preferably at least 90 wt. % lignocellulose.
  • the lignocellulose-containing material may also comprise other constituents such as cellulosic material, such as cellulose, hemicellulose and may also comprise constituents such as sugars, such as fermentable sugars and/or un-fermentable sugars.
  • Lignocellulose-containing material is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees.
  • Lignocellulosic material can also be, but is not limited to, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, and pulp and paper mill residues. It is understood herein that lignocellulose-containing material may be in the form of plant cell wall material containing lignin, cellulose, and hemi-cellulose in a mixed matrix.
  • the lignocellulose-containing material is corn fiber, rice straw, pine wood, wood chips, poplar, wheat straw, switchgrass, bagasse, paper and pulp processing waste.
  • corn stover corn cobs
  • corn fiber hardwood such as poplar and birch
  • softwood softwood
  • cereal straw such as wheat straw, switch grass, Miscanthus, rice hulls
  • MSW municipal solid waste
  • industrial organic waste office paper, or mixtures thereof.
  • the lignocellulose-containing material is corn stover or corn cobs. In another preferred embodiment, the lignocellulose-containing material is corn fiber. In another preferred embodiment, the lignocellulose-containing material is switch grass. In another preferred embodiment, the lignocellulose-containing material is bagasse. SSF, HHF and SHF
  • hydrolysis and fermentation is carried out as a simultaneous hydrolysis and fermentation step (SSF).
  • SSF simultaneous hydrolysis and fermentation step
  • hydrolysis step and fermentation step are carried out as hybrid hydrolysis and fermentation (HHF).
  • HHF typically begins with a separate partial hydrolysis step and ends with a simultaneous hydrolysis and fermentation step.
  • the separate partial hydrolysis step is an enzymatic cellulose saccharification step typically carried out at conditions (e.g., at higher temperatures) suitable, preferably optimal, for the hydrolyzing enzyme(s) in question.
  • the subsequent simultaneous hydrolysis and fermentation step is typically carried out at conditions suitable for the fermenting organism(s) (often at lower temperatures than the separate hydrolysis step).
  • hydrolysis and fermentation steps may be carried out as separate hydrolysis and fermentation, where the hydrolysis is taken to completion before initiation of fermentation. This is often referred to as "SHF".
  • an alpha-amylase may be used any alpha-amylase.
  • the alpha-amylase is an acid alpha-amylase, e.g. , fungal acid alpha- amylase or bacterial 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 optimum at a pH in the range of 3 to 7, preferably from 3.5 to 6, or more preferably from 4-5.
  • bacterial alpha-amylase means any bacterial alpha-amylase classified under
  • a bacterial alpha-amylase used according to the invention may, e.g., be derived from a strain of the genus Bacillus, which is sometimes also referred to as the genus Geobacillus.
  • Bacillus alpha-amylase is derived from a strain of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus sp. TS-23, or Bacillus subtilis, but may also be derived from other Bacillus sp.
  • bacterial alpha-amylases include the Bacillus stearothermophilus alpha-amylase of SEQ ID NO: 3 in WO 99/19467, the Bacillus amyloliquefaciens alpha-amylase of SEQ ID NO: 5 in WO 99/19467, and the Bacillus licheniformis alpha-amylase of SEQ ID NO: 4 in WO 99/19467 and the Bacillus sp. TS-23 alpha-amylase disclosed as SEQ ID NO: 1 in WO 2009/061380 (all sequences are hereby incorporated by reference).
  • the bacterial alpha-amylase may be an enzyme having a degree of identity of at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to any of the sequences shown in SEQ ID NOS: 3, 4 or 5, respectively, in WO 99/19467 and SEQ ID NO: 1 in WO 2009/061380.
  • the alpha-amylase may be an enzyme having a degree of identity of at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to any of the sequences shown in SEQ I D NO: 3 in WO 99/19467.
  • the alpha-amylase is derived from Bacillus stearothermophilus.
  • the Bacillus stearothermophilus alpha-amylase may be a mature wild-type or a mature variant thereof.
  • the mature Bacillus stearothermophilus alpha-amylases may be naturally truncated during recombinant production.
  • the mature Bacillus stearothermophilus alpha-amylase may be a truncated so it has around 491 amino acids (compared to SEQ ID NO: 3 in WO 99/19467), such as from 480-495 amino acids.
  • the Bacillus alpha-amylase may also be a variant and/or hybrid.
  • BSG alpha-amylase Bacillus stearothermophilus alpha-amylase (often referred to as BSG alpha-amylase) variants having a deletion of one or two amino acids at positions R179, G180, 1181 and/or G182, preferably a double deletion disclosed in WO 96/23873 - see, e.g., page 20, lines 1 -10 (hereby incorporated by reference), preferably corresponding to deletion of positions 1181 and G182 compared to the amino acid sequence of Bacillus stearothermophilus alpha-amylase set forth in SEQ ID NO: 3 disclosed in WO 99/19467 or the deletion of amino acids R179 and G180 using SEQ ID NO: 3 in WO 99/19467 (which reference is hereby incorporated by reference).
  • BSG alpha-amylase Bacillus stearothermophilus alpha-amylase
  • Bacillus alpha- amylases especially Bacillus stearothermophilus alpha-amylases, which have a double deletion corresponding to a deletion of positions 181 and 182 and further comprise 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.
  • the bacterial alpha- amylase may also have a substitution in a position corresponding to S239 in the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4 in WO 99/19467, or a S242 variant of the Bacillus stearot ermop ilus alpha-amylase of SEQ ID NO: 3 in WO 99/19467.
  • the variant is a S242A, E or Q variant, preferably a S242Q variant, of the Bacillus stearothermophilus alpha-amylase.
  • the variant is a position E188 variant, preferably E188P variant of the Bacillus stearothermophilus alpha-amylase.
  • Bacillus sp. TS-23 variant disclosed in WO2009/061380 especially variants defined in claim 1 of WO2009/061380.
  • the bacterial alpha-amylase may also be a hybrid bacterial alpha-amylase, e.g., an alpha-amylase comprising 445 C terminal amino acid residues of the Bacillus licheniformis alpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) and the 37 N terminal amino acid residues of the alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467).
  • this hybrid has one or more, especially all, of the following substitutions:
  • variants having one or more of the following mutations (or corresponding mutations in other Bacillus alpha- amylases): H154Y, A181 T, N190F, A209V and Q264S and/or the deletion of two residues between positions 176 and 179, preferably the deletion of E178 and G179 (using SEQ ID NO: 5 of WO 99/19467 for position numbering).
  • the bacterial alpha-amylase is the mature part of the chimeric alpha- amylase disclosed in Richardson et al., 2002, The Journal of Biological Chemistry 277(29):. 267501 -26507, referred to as BD5088 or a variant thereof.
  • This alpha-amylase is the same as the one shown in SEQ ID NO: 2 in WO 2007134207.
  • the mature enzyme sequence starts after the initial "Met" amino acid in position 1 .
  • the bacterial alpha-amylase is dosed in an amount of 0.0005-5 KNU per g DS (dry solids), preferably 0.001-1 KNU per g DS, such as around 0.050 KNU per g DS.
  • Fungal Alpha-Amylase is dosed in an amount of 0.0005-5 KNU per g DS (dry solids), preferably 0.001-1 KNU per g DS, such as around 0.050 KNU per g DS.
  • Fungal alpha-amylases include alpha-amylases derived from a strain of the genus Aspergillus, such as, Aspergillus oryzae, Aspergillus niger and Aspergillis kawachii alpha- amylases.
  • a preferred acidic fungal alpha-amylase is a Fungamyl-like alpha-amylase which is derived from a strain of Aspergillus oryzae.
  • the term "Fungamyl-like alpha-amylase” indicates an alpha-amylase which exhibits a high identity, i.e., more than 70%, more than 75%, more than 80%, more than 85% more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or even 100% identity to the mature part of the amino acid sequence shown in SEQ ID NO: 10 in WO 96/23874.
  • Another preferred acidic alpha-amylase is derived from a strain 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 and described in WO 89/01969 (Example 3).
  • a commercially available acid fungal alpha- amylase derived from Aspergillus niger is SP288 (available from Novozymes A/S, Denmark).
  • wild-type alpha-amylases include those derived from a strain of the genera Rhizomucor and Meripilus, preferably a strain of Rhizomucor pusillus (WO 2004/055178 incorporated by reference) or Meripilus giganteus.
  • the alpha-amylase is derived from Aspergillus kawachii and disclosed by Kaneko et al., 1996, J. Ferment. Bioeng. 81 : 292-298, "Molecular-cloning and determination of the nucleotide-sequence of a gene encoding an acid-stable alpha-amylase from Aspergillus kawachir; and further as EMBL:#AB008370.
  • the fungal alpha-amylase may also be a wild-type enzyme comprising a starch-binding domain (SBD) and an alpha-amylase catalytic domain (i.e., non-hybrid), or a variant thereof.
  • SBD starch-binding domain
  • alpha-amylase catalytic domain i.e., non-hybrid
  • the wild-type alpha-amylase is derived from a strain of Aspergillus kawachii.
  • the fungal acid alpha-amylase is a hybrid alpha-amylase.
  • Preferred examples of fungal hybrid alpha-amylases include the ones disclosed in WO 2005/00331 1 or U.S. application publication no. 2005/0054071 (Novozymes) or U.S. application no. 60/638,614 (Novozymes) which is hereby incorporated by reference.
  • a hybrid alpha- amylase may comprise an alpha-amylase catalytic domain (CD) and a carbohydrate-binding domain/module (CBM), such as a starch binding domain, and optional a linker.
  • Specific examples of contemplated hybrid alpha-amylases include those disclosed in Table 1 to 5 of the examples in U.S. application no.
  • 60/638,614 including Fungamyl variant with catalytic domain JA1 18 and Athelia rolfsii SBD (SEQ ID NO:100 in US 60/638,614), Rhizomucor pusillus alpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ ID NO:101 in US 60/638,614), Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD (which is disclosed in Table 5 as a combination of amino acid sequences SEQ ID NO: 20, SEQ ID NO: 72 and SEQ ID NO: 96 in U.S. application no.
  • contemplated hybrid alpha-amylases include those disclosed in U.S. application publication no. 2005/0054071 , including those disclosed in Table 3 on page 15, such as Aspergillus niger alpha-amylase with Aspergillus kawachii linker and starch binding domain.
  • alpha-amylases which exhibit a high identity to any of above mention alpha-amylases, i.e., more than 70%, more than 75%, more than 80%, more than 85% more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or even 100% identity to the mature enzyme sequences.
  • An acid alpha-amylases may according to the invention be added in an amount of 0.1 to 10 AFAU/g DS, preferably 0.10 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS.
  • Preferred commercial compositions comprising alpha-amylase include MYCOLASE from DSM (Gist Brocades), BANTM, TERMAMYLTM SC, FUNGAMYLTM, LIQUOZYMETM X and SANTM SUPER, SANTM EXTRA L (Novozymes A/S) and CLARASETM L-40,000, DEX-LOTM, SPEZYMETM FRED, SPEZYMETM AA, SPEZYMETM DELTA AA, SPEZYME XTRATM (Genencor Int., USA), FUELZYMETM (from Verenium Corp, USA) and the acid fungal alpha-amylase sold under the trade name SP288 (available from Novozymes A/S, Denmark).
  • carbohydrate-source generating enzyme includes glucoamylase (being glucose generators), beta-amylase and maltogenic amylase (being maltose generators).
  • a carbohydrate-source generating enzyme is capable of producing a carbohydrate that can be used as an energy-source by the fermenting organism(s) in question, for instance, when used in a process of the invention for producing a fermentation product, such as ethanol.
  • the generated carbohydrate may be converted directly or indirectly to the desired fermentation product, preferably ethanol.
  • a mixture of carbohydrate-source generating enzymes may be used.
  • mixtures are mixtures of at least a glucoamylase and an alpha-amylase, especially an acid amylase, even more preferred an acid fungal alpha-amylase.
  • the ratio between acid fungal alpha-amylase activity (AFAU) per glucoamylase activity (AGU) (AFAU per AGU) may in an embodiment of the invention be at least 0.1 , or at least 0.16, such as in the range from 0.12 to 0.50 or more.
  • the ratio between acid fungal alpha-amylase activity (FAU-F) and glucoamylase activity (AGU) may in an embodiment of the invention be between 0.1 and 100, in particular between 2 and 50, such as in the range from 10-40.
  • 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 fungal or bacterial origin, selected from the group consisting of Aspergillus glucoamylases, in particular A. niger G1 or G2 glucoamylase (Boel et al., 1984, EMBO J. 3 (5): 1097-1 102), or variants thereof, such as those disclosed in WO 92/00381 , WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A.
  • awamori glucoamylase disclosed in WO 84/02921 , A. oryzae glucoamylase (Agric. Biol. Chem. , 1991 , 55 (4): 941-949), or variants or fragments thereof.
  • Other Aspergillus glucoamylase variants include variants with enhanced thermal stability: G137A and G139A (Chen et al., 1996, Prot. Eng. 9: 499-505); D257E and D293E/Q (Chen et al., 1995, Prot. Eng. 8: 575-582); N182 (Chen et al., 1994, Biochem. J.
  • glucoamylases include Athelia rolfsii (previously denoted Corticium rolfsii) glucoamylase (see U.S. Patent No. 4,727,026 and Nagasaka et al., 1998, "Purification and properties of the raw-starch-degrading glucoamylases from Corticium rolfsii, AppI Microbiol Biotechnol 50:323-330), Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (U.S. Patent No. Re. 32,153), Talaromyces duponti, and Talaromyces thermophilus (U.S. Patent No. 4,587,215).
  • Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 86/01831 ).
  • Contemplated fungal glucoamylases include Trametes cingulata, Pachykytospora papyracea; and Leucopaxillus giganteus all disclosed in WO 2006/069289; and Peniophora rufomarginata disclosed in WO2007/124285; or a mixture thereof.
  • hybrid glucoamylase are contemplated according to the invention. Examples include the hybrid glucoamylases disclosed in WO 2005/045018. Specific examples include the hybrid glucoamylase disclosed in Table 1 and 4 of Example 1 (which hybrids are hereby incorporated by reference).
  • the glucoamylase is derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus as described in WO 201 1/066576 (SEQ ID NOs 2, 4 or 6), or from a strain of the genus Gloephyllum, in particular a strain of Gloephyllum as described in WO 201 1/068803 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16) or a strain of the genus Nigrofomes, in particular a strain of Nigrofomes sp. disclosed in WO 2012/064351 (SEQ ID NO: 2) (all references hereby incorporated by reference).
  • glucoamylases which exhibit a high identity to any of the above-mentioned glucoamylases, i.e., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to any one of the mature parts of the enzyme sequences mentioned above.
  • compositions comprising glucoamylase include AMG 200L; AMG 300 L; SANTM SUPER, SANTM EXTRA L, SPIRIZYMETM PLUS, SPIRIZYMETM FUEL, SPIRIZYMETM B4U, SPIRIZYME ULTRATM and AMGTM E (from Novozymes A/S, Denmark); OPTIDEXTM 300, GC480TM and GC147TM (from Genencor Int., USA); 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, especially between 1-5 AGU/g DS, such as 0.1 -2 AGU/g DS, such as 0.5 AGU/g DS or in an amount of 0.0001-20 AGU/g DS, preferably 0.001-10 AGU/g DS, especially between 0.01 -5 AGU/g DS, such as 0.1-2 AGU/g DS.
  • the a beta-amylase (E.C 3.2.1.2) is the name traditionally 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. Fogarty and C.T. Kelly, 1979, Progress in Industrial Microbiology 15: 1 12-1 15). 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 NOVOZYMTM WBA from Novozymes A/S, Denmark and SPEZYMETM BBA 1500 from Genencor Int., USA. Maltogenic amylase
  • the 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 amylopectin to maltose in the alpha-configuration.
  • a maltogenic amylase from Bacillus stearothermophilus strain NCIB 1 1837 is commercially available from Novozymes A S. Maltogenic alpha-amylases are described in U.S. Patent Nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby incorporated by reference.
  • the maltogenic amylase may in a preferred embodiment be added in an amount of 0.05- 5 mg total protein/gram DS or 0.05- 5 MANU/g DS.
  • a protease used in accordance with the present invention may be any protease, such as of microbial or plant origin.
  • the protease is an acid protease of microbial origin, preferably of fungal or bacterial origin.
  • Suitable proteases include microbial proteases, such as fungal and bacterial proteases.
  • Preferred proteases are acidic proteases, i.e., proteases characterized by the ability to hydrolyze proteins under acidic conditions below pH 7.
  • Contemplated acid fungal proteases include fungal proteases derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Sclerotiumand Torulopsis.
  • proteases derived from Aspergillus niger see, e.g., Koaze et al., 1964, Agr. Biol. Chem. Japan 28: 216), Aspergillus saitoi (see, e.g., Yoshida, 1954, J. Agr. Chem. Soc. Japan 28: 66), Aspergillus awamori (Hayashida et al., 1977, Agric.
  • Contemplated are also neutral or alkaline proteases, such as a protease derived from a strain of Bacillus.
  • a particular protease contemplated for the invention is derived from Bacillus amyloliquefaciens and has the sequence obtainable at Swissprot as Accession No. P06832.
  • proteases having at least 90% identity to amino acid sequence obtainable at Swissprot as Accession No. P06832 such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% identity.
  • proteases having at least 90% identity to amino acid sequence disclosed as SEQ.ID.NO: 1 in the WO 2003/048353 such as at 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% identity.
  • papain-like proteases such as proteases within E.C. 3.4.22. *
  • cyste protease such as EC 3.4.22.2 (papain), EC 3.4.22.6 (chymopapain), EC 3.4.22.7 (asclepain), EC 3.4.22.14 (actinidain), EC 3.4.22.15 (cathepsin L), EC 3.4.22.25 (glycyl endopeptidase) and EC 3.4.22.30 (caricain).
  • the protease is a protease preparation derived from a strain of Aspergillus, such as Aspergillus oryzae.
  • the protease is derived from a strain of Rhizomucor, preferably Rhizomucor miehei.
  • the protease is a protease preparation, preferably a mixture of a proteolytic preparation derived from a strain of Aspergillus, such as Aspergillus oryzae, and a protease derived from a strain of Rhizomucor, preferably Rhizomucor mehei.
  • aspartic acid protease include, e.g., those disclosed in Berka et al., 1990, Gene 96: 313; Berka et al., 1993, Gene 125: 195-198; and Gomi et al., 1993, Biosci. Biotech. Biochem. 57: 1095-1 100, which are hereby incorporated by reference.
  • the protease may be present in an amount of 0.0001 -1 mg enzyme protein per g DS, preferably 0.001 to 0.1 mg enzyme protein per g DS.
  • the protease may be present in an amount of 0.0001 to 1 LAPU/g DS, preferably 0.001 to 0.1 LAPU/g DS and/or 0.0001 to 1 mAU-RH/g DS, preferably 0.001 to 0.1 mAU-RH/g DS or 0.1 -1000 AU/kg DM (dry matter), preferably 1-100 AU/kg DS and most preferably 5-25 AU/kg DS.
  • cellulases or “cellulolytic enzymes” as used herein are understood as comprising the cellobiohydrolases (EC 3.2.1.91 ), e.g., cellobiohydrolase I and cellobiohydrolase II, as well as the endo-glucanases (EC 3.2.1 .4) and beta-glucosidases (EC 3.2.1.21 ). See relevant sections below with further description of such enzymes.
  • cellulose In order to be efficient, the digestion of cellulose may require several types of enzymes acting cooperatively. At least three categories of enzymes are often needed to convert cellulose into glucose: endoglucanases (EC 3.2.1 .4) that cut the cellulose chains at random; cellobiohydrolases (EC 3.2.1 .91 ) which cleave cellobiosyl units from the cellulose chain ends and beta-glucosidases (EC 3.2.1.21 ) that convert cellobiose and soluble cellodextrins into glucose.
  • endoglucanases EC 3.2.1 .4
  • cellobiohydrolases EC 3.2.1 .91
  • beta-glucosidases EC 3.2.1.21
  • cellobiohydrolases are the key enzymes for the degradation of native crystalline cellulose.
  • cellobiohydrolase I is defined herein as a cellulose 1 ,4-beta-cellobiosidase (also referred to as Exo-glucanase, Exo-cellobiohydrolase or 1 ,4-beta-cellobiohydrolase) activity, as defined in the enzyme class EC 3.2.1.91 , which catalyzes the hydrolysis of 1 ,4-beta-D-glucosidic linkages in cellulose and cellotetraose, by the release of cellobiose from the non-reducing ends of the chains.
  • the definition of the term “cellobiohydrolase II activity” is identical, except that cellobiohydrolase II attacks from the reducing ends of the chains.
  • the cellulases may comprise a carbohydrate-binding module (CBM) which enhances the binding of the enzyme to a cellulose-containing fiber and increases the efficacy of the catalytic active part of the enzyme.
  • CBM is defined as contiguous amino acid sequence within a carbohydrate-active enzyme with a discreet fold having carbohydrate-binding activity.
  • the cellulases or cellulolytic enzymes may be a cellulolytic preparation or cellulolytic enzymes composition as defined in WO 2008/151079 or WO 2013/028928, which are both hereby incorporated by reference.
  • the cellulolytic preparation comprising a polypeptide having cellulolytic enhancing activity (GH61 ), preferably a GH61A polypeptide disclosed in WO 2005/074656.
  • the cellulolytic preparation may further comprise a beta-glucosidase, such as a beta-glucosidase derived from a strain of the genus Trichoderma, Aspergillus, or Penicillium, including the fusion protein having beta- glucosidase activity disclosed in WO 2008/057637 (Novozymes).
  • the cellulolytic preparation may also comprises a CBH II, preferably Thielavia terrestris cellobiohydrolase II (CEL6A).
  • the cellulolytic preparation also comprises a cellulase enzymes preparation, preferably the one derived from Trichoderma reesei.
  • the cellulolytic activity may, in a preferred embodiment, be derived from a fungal source, such as a strain of the genus Trichoderma, preferably a strain of Trichoderma reeser, or a strain of the genus Humicola, such as a strain of Humicola insolens.
  • a fungal source such as a strain of the genus Trichoderma, preferably a strain of Trichoderma reeser, or a strain of the genus Humicola, such as a strain of Humicola insolens.
  • the cellulolytic enzyme preparation comprises a polypeptide having cellulolytic enhancing activity (GH61A) disclosed in WO 2005/074656; a cellobiohydrolase, such as Thielavia terrestris cellobiohydrolase II (CEL6A), a beta-glucosidase (e.g., the fusion protein disclosed in WO 2008/057637 and cellulolytic enzymes, e.g., derived from Trichoderma reesei.
  • G61A cellulolytic enhancing activity
  • CEL6A Thielavia terrestris cellobiohydrolase II
  • beta-glucosidase e.g., the fusion protein disclosed in WO 2008/057637
  • cellulolytic enzymes e.g., derived from Trichoderma reesei.
  • the cellulolytic preparation comprises a polypeptide having cellulolytic enhancing activity, e.g., a GH61A polypeptide disclosed in WO 2005/074656; a beta- glucosidase, e.g., a fusion protein disclosed in WO 2008/057637 and cellulolytic enzymes, e.g., derived from Trichoderma reesei.
  • a polypeptide having cellulolytic enhancing activity e.g., a GH61A polypeptide disclosed in WO 2005/074656
  • beta- glucosidase e.g., a fusion protein disclosed in WO 2008/057637
  • cellulolytic enzymes e.g., derived from Trichoderma reesei.
  • the cellulolytic enzyme composition is a Trichoderma reesei cellulolytic enzyme composition, further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499).
  • the cellulolytic enzyme composition is a Trichoderma reesei cellulolytic enzyme composition further comprising Penicillium emersonii GH61A polypeptide disclosed as SEQ ID NO: 2 in WO 201 1/041397, and Aspergillus fumigatus beta-glucosidase disclosed as SEQ ID NO: 2 in WO 2005/047499, or a variant thereof, which variant has one of, preferably all of, the following substitutions: F100D, S283G, N456E, F512Y.
  • the cellulolytic enzyme composition further comprises a cellobiohydrolase I and cellobiohydrolase II derived from Aspergillus fumigatus.
  • the cellulolytic enzyme composition comprises the Aspergillus fumigatus Cel7A CBH I disclosed as SEQ ID NO: 6 in WO201 1/057140 and the Aspergillus fumigatus CBH II disclosed as SEQ ID NO: 18 in WO 201 1/057140.
  • the cellulolytic enzyme composition may also comprise some hemicellulase, such as up to 20% hemicellulase, such as up to 15%, such as up to 10%, such as xylanase and/or xylosidase, such as beta-xylosidase.
  • the cellulolytic enzyme composition comprises Aspergillus fumigatus GH10 xylanase, such as the one disclosed as SEQ ID NO: 6 (Xyl III) in WO 2006/078256, and Aspergillus fumigatus beta-xylosidase, such as the one disclosed in WO 2013/028928 (Example 16 and 17).
  • CELLUCLAST® 1.5L CELLUZYMETM, CELLIC CTECTM, CELLIC CTEC2TM, CELLIC CTEC3TM (all available from Novozymes A/S, Denmark), ACCELLERASE 1000TM, ACCELLERASE 1500TM, ACCELLERASE DUETTM, ACCELLERASE TRIOTM (all available from DuPont, USA).
  • Cellulases or cellulolytic enzymes can easily be determined by the skilled person in the art.
  • Cellulases or cellulolytic enzymes may typically be dosed in the range from 0.1-100 FPU per gram total solids (TS), preferably 0.5-50 FPU per gram TS, especially 1-20 FPU per gram TS.
  • Endoglucanases (EC No. 3.2.1 .4) catalyses endo hydrolysis of 1 ,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxy methyl cellulose and hydroxy ethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3 glucans such as cereal beta-D-glucans or xyloglucans and other plant material containing cellulosic parts.
  • the authorized name is endo- 1 ,4-beta-D-glucan 4-glucano hydrolase, but the abbreviated term endoglucanase is used in the present specification. Endoglucanase activity may be determined using carboxymethyl cellulose (CMC) hydrolysis according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268.
  • endoglucanases may be derived from a strain of the genus Trichoderma, preferably a strain of Trichoderma reesei; a strain of the genus Humicola, such as a strain of Humicola insolens; or a strain of Chrysosporium, preferably a strain of Chrysosporium lucknowense.
  • CBH Cellobiohydrolase
  • cellobiohydrolase means a 1 ,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 ), which catalyzes the hydrolysis of 1 ,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1 ,4-linked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain.
  • cellobiohydroloses examples include CBH I and CBH II from Trichoderma reseei; Humicola insolens, Aspergillus fumigatus, and CBH II from Thielavia terrestris cellobiohydrolase (CELL6A).
  • the cellulolytic enzyme composition comprises the Aspergillus fumigatus Cel7A CBH I disclosed as SEQ ID NO: 6 in WO201 1/057140 and the Aspergillus fumigatus CBH II disclosed as SEQ ID NO: 18 in WO 201 1/057140.
  • Cellobiohydrolase activity may be determined according to the procedures described by Lever et al. , 1972, Anal. Biochem. 47: 273-279 and by van Tilbeurgh et al. , 1982, FEBS Letters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288.
  • the Lever et al. method is suitable for assessing hydrolysis of cellulose in corn stover and the method of van Tilbeurgh et al. is suitable for determining the cellobiohydrolase activity on a fluorescent disaccharide derivative.
  • beta-glucosidases may be present during hydrolysis.
  • beta-glucosidase means a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21 ), which catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose.
  • beta-glucosidase activity is determined according to the basic procedure described by Venturi et al., 2002, J. Basic Microbiol. 42: 55-66, except different conditions were employed as described herein.
  • beta-glucosidase activity is defined as 1 .0 ⁇ of p-nitrophenol produced per minute at 50°C, pH 5 from 4 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodium citrate, 0.01 % TWEEN® 20.
  • beta-glucosidase is of fungal origin, such as a strain of the genus
  • beta-glucosidase is a derived from Trichoderma reesei, such as the beta-glucosidase encoded by the bgll gene (see Fig. 1 of EP 562,003).
  • the beta-glucosidase is derived from Aspergillus oryzae (recombinantly produced in Aspergillus oryzae according to WO 02/095014).
  • beta-glucosidase is derived from Aspergillus fumigatus such as the one disclosed as SEQ ID NO: 2 in WO 2005/047499, or a variant thereof, which variant has one of, preferably all of, the following substitutions: F100D, S283G, N456E, F512Y.
  • cellulolytic enhancing activity is defined herein as a biological activity that enhances the hydrolysis of a lignocellulose derived material by proteins having cellulolytic activity.
  • cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or in the increase of the total of cellobiose and glucose from the hydrolysis of a lignocellulose derived material, e.g.
  • pre-treated lignocellulose- containing material by cellulolytic protein under the following conditions: 1 -50 mg of total protein/g of cellulose in PCS (pre-treated corn stover), wherein total protein is comprised of 80- 99.5% w/w cellulolytic protein/g of cellulose in PCS and 0.5-20% w/w protein of cellulolytic enhancing activity for 1 -7 day at 50°C compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1-50 mg of cellulolytic protein/g of cellulose in PCS).
  • the polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a lignocellulose derived material catalyzed by proteins having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis preferably at least 0.1 -fold, more at least 0.2-fold, more preferably at least 0.3-fold, more preferably at least 0.4-fold, more preferably at least 0.5-fold, more preferably at least 1 -fold, more preferably at least 3-fold, more preferably at least 4-fold, more preferably at least 5-fold, more preferably at least 10-fold, more preferably at least 20-fold, even more preferably at least 30-fold, most preferably at least 50-fold, and even most preferably at least 100-fold.
  • the hydrolysis and/or fermentation is carried out in the presence of a cellulolytic enzyme in combination with a polypeptide having enhancing activity.
  • the polypeptide having enhancing activity is a family GH61A polypeptide.
  • WO 2005/074647 discloses isolated polypeptides having cellulolytic enhancing activity and polynucleotides thereof from Thielavia terrestris.
  • WO 2005/074656 discloses an isolated polypeptide having cellulolytic enhancing activity and a polynucleotide thereof from Thermoascus aurantiacus.
  • U.S. Application Publication No. 2007/0077630 discloses an isolated polypeptide having cellulolytic enhancing activity and a polynucleotide thereof from Trichoderma reesei.
  • the cellulolytic enzyme composition comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656).
  • the cellulolytic enzyme composition comprising Penicillium emersonii GH61A polypeptide disclosed as SEQ ID NO: 2 in WO 201 1/041397.
  • Hemicellulose can be broken down by hemicellulases and/or acid hydrolysis to release its five and six carbon sugar components.
  • the lignocellulose derived material may be treated with one or more hemicellulases.
  • hemicellulase suitable for use in hydrolyzing hemicellulose, preferably into xylose may be used.
  • Preferred hemicellulases include xylanases, beta-xylosidase, arabinofuranosidases, acetyl xylan esterase, feruloyl esterase, glucuronidases, galactanase, endo-galactanase, mannases, endo or exo arabinases, exo-galactanses, pectinase, xyloglucanase, or mixtures of two or more thereof.
  • the hemicellulase for use in the present invention is an exo-acting hemicellulase, and more preferably, the hemicellulase is an exo-acting hemicellulase which has the ability to hydrolyze hemicellulose under acidic conditions of below pH 7, preferably pH 3-7.
  • the hemicellulase is a xylanase.
  • the xylanase is of microbial origin, such as of fungal origin (e.g., Trichoderma, Meripilus, Humicola, Aspergillus, Fusarium) or from a bacterium (e.g., Bacillus).
  • the xylanase is derived from a filamentous fungus, preferably derived from a strain of Aspergillus, such as Aspergillus aculeatus or Aspergillus fumigatus; or a strain of Humicola, preferably Humicola lanuginosa.
  • the xylanase may preferably be an endo-1 ,4-beta-xylanase, more preferably an endo-1 ,4-beta- xylanase of GH10 or GH1 1.
  • the xylanase is an Aspergillus aculeatus GH10 xylanase, such as the one disclosed as SEQ ID NO: 2 (Xyl II) in WO W09421785.
  • the xylanase is an Aspergillus fumigatus GH10 xylanase, such as the one disclosed as SEQ ID NO: 6 (Xyl III) in WO 2006/078256.
  • the hemicellulase is a beta-xylosidase.
  • the beta- xylosidase is an Aspergillus fumigatus beta-xylosidase, such as the one disclosed in WO 2013/028928 (Example 16 and 17).
  • Examples of commercial xylanases include SHEARZYMETM and CELLIC HTECTM, CELLIC HTEC2TM CELLIC HTEC3TM from Novozymes A/S, Denmark.
  • Arabinofuranosidase (EC 3.2.1 .55) catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides.
  • Galactanase (EC 3.2.1.89), arabinogalactan endo-1 ,4-beta-galactosidase, catalyses the endohydrolysis of 1 ,4-D-galactosidic linkages in arabinogalactans.
  • Pectinase (EC 3.2.1 .15) catalyzes the hydrolysis of 1 ,4-alpha-D-galactosiduronic linkages in pectate and other galacturonans.
  • Xyloglucanase catalyzes the hydrolysis of xyloglucan.
  • the hemicellulase may be added in an amount effective to hydrolyze hemicellulose, such as, in amounts from about 0.001 to 0.5 wt.-% of total solids (TS), more preferably from about 0.05 to 0.5 wt.-% of TS.
  • TS total solids
  • Xylanases may be added in amounts of 0.001-1.0 g/kg DM (Dry Matter) substrate, preferably in the amounts of 0.005-0.5 g/kg DM substrate, and most preferably from 0.05-0.10 g/kg DM substrate.
  • the invention relates to a composition
  • a composition comprising lipoic acid, lipoate and/or lipoamide, or blends thereof, and further one or more enzymes and/or one or more fermenting organisms.
  • the ratio between lipoic acid and/or lipoate and lipoamide is between 1 :9 and 1 :9, such as between 1 :5 to 5:1 , such as around 1 :1 .
  • composition of the invention may comprise an enzyme from the class of hydrolases (class EC 3 according to Enzyme Nomenclature).
  • the enzyme is selected from the group consisting of cellulase, hemicellulase, protease, alpha-amylase, and glucoamylase, or a mixture thereof.
  • the enzyme is a glucoamylase, such as one listed in the "Glucoamylase"-section above.
  • composition may also comprise a fermenting organism, such as yeast or another fermenting organism mentioned in the "Fermenting OrganisrrT'-section above.
  • the invention relates to the use of lipoic acid, lipoate and/or lipoamide, or blends thereof, for propagating fermenting organisms, such as yeast, especially yeast derived from a strain of Saccharomyces, such as Saccharomyces cerevisiae.
  • invention also relates to the use of lipoic acid, lipoate and/or lipoamide, or blends thereof, in a fermentation process or a process of the invention.
  • lipoic acid and/or lipoate is(are) present and/or dosed into fermentation in a concentration in the range from 0.001 -1.8 mM/g mash, preferably 0.001 - 1.5 mM/g mash, such as 0.01 -1 .2 mM/g mash, such as 0.02-1 .0 mM/g mash, preferably 0.03-0.8 mM/g mash, such as 0.04-0.6 mM/g mash, such as around 0.4 mM/g mash.
  • lipoamide is present and/or dosed into fermentation in a concentration in the range from 0.001-10 mM/g mash, preferably 0.001 - 8 mM/g mash, such as 0.01-6 mM/g mash, such as 0.02-4 mM/g mash, preferably 0.03-2 mM/g mash, such as 0.04-1 mM/g mash, such as around 0.4 mM/g mash.
  • a process of producing a fermentation product from starch-containing material comprising the steps of:
  • the alpha-amylase is from the genus Bacillus, such as a strain of Bacillus stearothermophilus, in particular a variant of a Bacillus stearothermophilus alpha-amylase, such as the one shown in SEQ ID NO: 3 in WO 99/019467.
  • the Bacillus stearothermophilus alpha-amylase or variant thereof is truncated, preferably to have around 491 amino acids, such as from 480-495 amino acids.
  • glucoamylase is derived from a strain of Gloeophyllum, such as a strain of Gloeophyllum sepiarium or Gloeophyllum trabeum, such as one disclosed in WO 201 1/068803 as any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16, preferably SEQ ID NO: 2 in WO 201 1/068803 in WO 201 1/068803.
  • step (b) hydrolysis in step (b) and fermentation in step (c) is carried out as a SSF, HHF or SHF process.
  • a composition comprising lipoic acid, lipoate and/or lipoamide, or blends thereof, and one or more enzymes and/or one or more fermenting organisms.
  • composition of paragraph 50 wherein the enzyme(s) is(are) one or more hydrolases (class EC 3 according to Enzyme Nomenclature) selected from the group consisting of cellulases, hemicellulases, proteases, alpha-amylases, glucoamylases, or a mixture thereof.
  • hydrolases class EC 3 according to Enzyme Nomenclature
  • composition of paragraph 50 or 51 wherein the fermenting organism is selected from the group of yeast, filamentous fungus and/or a bacteria.
  • composition of any of paragraphs 50-52, wherein the ratio between lipoic acid and/or lipoate and lipoamide in the composition is between 1 :9 and 9:1 , such as between 1 :5 and 5:1 , such as around 1 :1.
  • lipoic acid, lipoate, and/or lipoamide, or blends thereof in a fermentation process.
  • Lipoic acid Alpha-lipoic acid sourced from Sigma Product# T5625-5G
  • Lipoamide Alpha-lipoamide sourced from Sigma Product# T5875-5G
  • Glucoamylase E Enzyme blend comprising Talaromyces emersonii glucoamylase disclosed as SEQ ID NO: 34 in W099/28448, Trametes cingulata glucoamylase disclosed as SEQ ID NO: 2 in WO 06/69289, and Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD disclosed as V039 in Table 5 in WO 2006/069290 (ratio about 75:20:5).
  • the Novo Glucoamylase Unit 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 autoanalyzer 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.
  • the 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. Initially, 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
  • AFAU Acid alpha-amylase activity
  • the activity of an acid alpha-amylase may be measured in FAU-F (Fungal Alpha-Amylase Unit) or AFAU (Acid Fungal Alpha-amylase Units).
  • FAU-F Fungal Alpha-Amylase Units (Fungamyl) is measured relative to an enzyme standard of a declared strength.
  • AFAU Acid alpha-amylase activity
  • Acid alpha-amylase activity may be measured in AFAU (Acid Fungal Alpha-amylase Units), which are determined relative to an enzyme standard. 1 AFAU is defined as the amount of enzyme which degrades 5.260 mg starch dry matter per hour under the below mentioned standard conditions.
  • Acid alpha-amylase, an endo-alpha-amylase (1 ,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1 ) hydrolyzes alpha-1 ,4-glucosidic bonds in the inner regions of the starch molecule to form dextrins and oligosaccharides with different chain lengths. The intensity of color formed with iodine is directly proportional to the concentration of starch. Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under the specified analytical conditions.
  • IODINE 40 pH 2,5 > DEXTRINS + OLIGOSACCHARIDES
  • Substrate Soluble starch, approx. 0.17 g/L Buffer: Citrate, approx. 0.03 M
  • Iodine (I2) 0.03 g/L
  • a rolled filter paper strip (#1 Whatman; 1 X 6 cm; 50 mg) is added to the bottom of a test tube (13 X 100 mm).
  • Enzyme dilutions are designed to produce values slightly above and below the target value of 2.0 mg glucose.
  • the tubes are incubated for 60 mins. at 50°C ( ⁇ 0.1 °C) in a circulating water bath.
  • the tubes are removed from the water bath, and 3.0 mL of DNS reagent is added to each tube to stop the reaction. The tubes are vortexed 3 seconds to mix.
  • a reagent blank is prepared by adding 1.5 mL of citrate buffer to a test tube.
  • a substrate control is prepared by placing a rolled filter paper strip into the bottom of a test tube, and adding 1 .5 mL of citrate buffer.
  • Enzyme controls are prepared for each enzyme dilution by mixing 1 .0 mL of citrate buffer with 0.5 mL of the appropriate enzyme dilution.
  • Glucose standard tubes are prepared by adding 0.5 mL of each dilution to 1.0 mL of citrate buffer.
  • glucose standard tubes are assayed in the same manner as the enzyme assay tubes, and done along with them.
  • each tube is diluted by adding 50 microL from the tube to 200 microL of ddH 2 0 in a 96-well plate. Each well is mixed, and the absorbance is read at 540 nm.
  • a glucose standard curve is prepared by graphing glucose concentration (mg/0.5 mL) for the four standards (G1 -G4) vs. A 540 . This is fitted using a linear regression (Prism Software), and the equation for the line is used to determine the glucose produced for each of the enzyme assay tubes.
  • the proteolytic activity may be determined with denatured hemoglobin as substrate.
  • Anson-Hemoglobin method for the determination of proteolytic activity denatured hemoglobin is digested, and the undigested hemoglobin is precipitated with trichloroacetic acid (TCA).
  • TCA trichloroacetic acid
  • the amount of TCA soluble product is determined with phenol reagent, which gives a blue color with tyrosine and tryptophan.
  • One Anson Unit is defined as the amount of enzyme which under standard conditions (i.e., 25°C, pH 5.5 and 10 min. reaction time) digests hemoglobin at an initial rate such that there is liberated per minute an amount of TCA soluble product which gives the same color with phenol reagent as one milliequivalent of tyrosine.
  • the AU(RH) method is described in EAL-SM-0350 and is available from Novozymes A S
  • the proteolytic activity may be determined with denatured hemoglobin as substrate.
  • Anson-Hemoglobin method for the determination of proteolytic activity denatured hemoglobin is digested, and the undigested hemoglobin is precipitated with trichloroacetic acid (TCA).
  • TCA trichloroacetic acid
  • the amount of TCA soluble product is determined with phenol reagent, which gives a blue color with tyrosine and tryptophan.
  • One Anson Unit is defined as the amount of enzyme which under standard conditions (i.e., 25°C, pH 7.5 and 10 min. reaction time) digests hemoglobin at an initial rate such that there is liberated per minute an amount of TCA soluble product which gives the same color with phenol reagent as one milliequivalent of tyrosine.
  • LAPU Protease assay method
  • LAPU 1 Leucine Amino Peptidase Unit
  • LAPU is described in EB-SM-0298.02/01 available from Novozymes A/S Denmark on request.
  • MANU Medianal Amylase Novo Unit
  • a target of 30 million cells/g mash of hydrated RED STARTM yeast was added to each tube before being incubated in a 32°C water bath for 54 hours. After a 54 hour fermentation time tubes were centrifuged and the liquid fraction was syringe filtered for HPLC analysis of ethanol and glucose.
  • LA lipoic acid
  • LAM lipoamide
  • Fig. 2 shows that glycerol production is lowered or unchanged despite having increased amounts of ethanol thus indicating that carbohydrates are being utilized more efficiently. Increased levels of glucose in addition to higher ethanol titer indicate a more efficient fermentation is taking place (See Fig. 3).

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Abstract

L'invention porte sur un procédé de fermentation de sucres issus de matières végétales en produits de fermentation à l'aide d'un organisme de fermentation, l'acide lipoïque, le lipoate et/ou le lipoamide, ou des mélanges de ces derniers, étant présents pendant la fermentation et/ou ajoutés avant et/ou pendant la fermentation. L'invention porte également sur des procédés de production de produits de fermentation ; sur des compositions comprenant de l'acide lipoïque, du lipoate et/ou du lipoamide, ou des mélanges de ces derniers ; et sur l'utilisation de tels composés et mélanges comprenant de tels composés.
PCT/US2014/057997 2013-10-01 2014-09-29 Procédés de production de produits de fermentation WO2015050809A1 (fr)

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WO2017087488A1 (fr) * 2015-11-17 2017-05-26 Premier Research Labs, Lp Procédé pour la production et l'extraction de l'acide dihydrolipoïque
US11680250B2 (en) 2015-07-14 2023-06-20 University Of Copenhagen Light-driven system and methods for chemical modification of an organic substrate

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11680250B2 (en) 2015-07-14 2023-06-20 University Of Copenhagen Light-driven system and methods for chemical modification of an organic substrate
WO2017087488A1 (fr) * 2015-11-17 2017-05-26 Premier Research Labs, Lp Procédé pour la production et l'extraction de l'acide dihydrolipoïque

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