WO2008072184A1 - Cultures de fermentation et procédés les utilisant - Google Patents

Cultures de fermentation et procédés les utilisant Download PDF

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Publication number
WO2008072184A1
WO2008072184A1 PCT/IB2007/055025 IB2007055025W WO2008072184A1 WO 2008072184 A1 WO2008072184 A1 WO 2008072184A1 IB 2007055025 W IB2007055025 W IB 2007055025W WO 2008072184 A1 WO2008072184 A1 WO 2008072184A1
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WO
WIPO (PCT)
Prior art keywords
fermentation
yeast
ethanol
culture
lactic acid
Prior art date
Application number
PCT/IB2007/055025
Other languages
English (en)
Inventor
Volkmar Passoth
Johanna Blomqvist
Johan SCHNÜRER
Original Assignee
Volkmar Passoth
Johanna Blomqvist
Schnuerer Johan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Volkmar Passoth, Johanna Blomqvist, Schnuerer Johan filed Critical Volkmar Passoth
Priority to EP07849425A priority Critical patent/EP2099922A1/fr
Publication of WO2008072184A1 publication Critical patent/WO2008072184A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C12P7/14—Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
    • 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

Definitions

  • the present invention is directed to fermentation cultures and methods employing such fermentation cultures, for example, for ethanol production, for improving the stability of a running fermentation, and/or for producing distiller's grain.
  • Industrial ethanol which has a broad range of uses, e.g. for drinking and as a car fuel, is basically produced according to well known fermentation techniques which have been modified over time to provide higher yields, improved purity, and the like.
  • ethanol can also be produced by a fed batch process, where the substrate is continuously added to a fermentor, but the product is only removed after the fermentation is finished. After removal of the ethanol product, yeast can be recycled to the fermentor, keeping the biomass concentration high in the fermentor and saving energy for building up the biomass.
  • One example of such processes is the Biostil® process of Chematur Engineering AB of Karlskoga, Sweden.
  • the fermentation is usually run by the fermentation yeast Sac char omyces cerevisiae, the classical baking and brewing yeast.
  • the continuous or fed batch processes enable an efficient conversion of the substrate to ethanol, because the processes are run with a sugar limitation, i.e., sugars are added in amounts that are below the uptake capacity of the yeasts. This leads to a sugar concentration of close to zero in the fermentor. [0003]
  • Such a process can encounter difficulties in that it often results in starvation stress for the yeasts. Additionally, the yeasts are challenged by low oxygen concentrations, ethanol concentrations above 6% (VfV), and, in some cases, pH values lower than 4. Another problem is the frequent occurrence of infections by wild yeasts and/ or lactic acid bacteria.
  • Wild yeasts may produce killer toxins, which can kill the production strain, see, for example, Marquina et al, "Biology of killer yeasts," Int. Microbiol., 5:65-71 (2002). Lactic acid bacteria has been found to decrease the ethanol yield and the yeast viability in the fermentation, as described, for example, by Schell et al, "Contaminant occurrence, identification and control in a pilot-scale corn fiber to ethanol conversion process," Bioresour. Technol, 98:2942-2948 (2007), and Thomas et al, "Effect of lactobacilli on yeast growth, viability and batch and semi-continuous alcoholic fermentation of corn mash," /. Appl. Microbiol., 90:819-828 (2001). In certain processes, e.g. wine production, by-products from the fermentation process, may, even at very low concentrations, have a negative impact on the quality of the final product.
  • by-products from the fermentation process may, even at very low
  • the present invention provides improved fermentation cultures, which may be used in a variety of methods.
  • the invention is directed to a fermentation culture for ethanol production, comprising a yeast of the genus Dekkera and a lactic acid bacteria.
  • the invention is directed to methods employing the fermentation cultures.
  • the invention is directed to a method for producing ethanol, comprising fermenting a substrate using a culture comprising a yeast of the genus Dekkera and a lactic acid bacteria.
  • the invention is directed to a method for improving the stability of a running fermentation for producing ethanol wherein a substrate is fermented using a yeast culture, the method comprising adding to the running fermentation a yeast of the genus Dekkera and a lactic acid bacteria.
  • the invention is directed to a method for producing distiller's grain, comprising fermenting a substrate using a culture comprising a yeast of the genus Dekkera and a lactic acid bacteria.
  • the present invention is directed to fermentation cultures and methods employing such fermentation cultures, for example, for ethanol production, for improving the stability of a running fermentation, and/or for producing distiller's grain.
  • the fermentation culture comprises a fermentation culture for ethanol production comprising a yeast of the genus Dekkera and a lactic acid bacteria.
  • yeasts of the genus Dekkera and lactic acid bacteria are known in the art and strains thereof are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
  • ATCC American Type Culture Collection
  • DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • the yeast comprises Dekkera bruxellensis, also known as Brettanomyces bruxellensis.
  • This yeast is frequently occurring in various fermented foods and in contaminated red wine where it can produce 4-ethylphenol, which gives the wine an off-flavor.
  • the yeast also belongs to the natural microflora of several fermented foods like Iambic beer or sourdough. It has been shown in a model fermentation that the yeast can produce ethanol and by-products (aldehydes and others) in accordance with the rules of the Brazilian legislation for cachaca, a national distilled beverage. Strains of this species are readily accessible to the public, for example, from CBS, 74, and NRRL, Y-12961, although other isolates of D. bruxellensis may be employed as well.
  • a strain CBS-2499 is disclosed in detail by Woolfit et al, Eukaryotic Cell, 6(4):721-733 (2007).
  • the lactic acid bacteria comprises Lactobacillus vini, Lactobacillus mobilis, or a mixture thereof.
  • the biomass of the lactic acid bacteria in the combination of yeast and bacteria in the culture is about 1-15%, generally correlating to a cell number ratio of the yeast to the bacteria of from about 1:3 to about 15:1. In a more specific embodiment, the cell number ratio of the yeast to the bacteria is from about 1:3 to about 5:1.
  • the fermentation cultures as described are suitable for use in ethanol production. Various methods for ethanol production are known in the art and may be employed herein. Typically, ethanol is produced by fermentation of a substrate in a fermentor using a fermentation culture. Examples include those methods described in U.S. Patent No. 4,886,751, incorporated herein by reference, and the known Biostil® process of Chematur Engineering AB.
  • the fermentation cultures of the invention may be used in any such methods known in the art.
  • the present methods provide a stable fermentation start up process, particularly in the first weeks of the process, and therefore overcome disadvantages of prior art processes which are often subject to instability in the first several weeks, resulting in wasted materials, time and cost.
  • the present methods also provide a stable fermentation reaction over extended periods of time, for example, months and/or years.
  • the substrate comprises molasses, wheat starch, or combinations thereof.
  • Other substrates suitable for releasing sugar for fermentation are also known in the art and may be employed in such methods as well.
  • the ethanol production method may be initiated by mixing the yeast and bacteria with the selected substrate in a fermentor.
  • a typical fermentor has a volume of about 100 m 3 and for such a volume about 1 metric ton of fermentation culture comprising a yeast of the genus Dekkera and a lactic acid bacteria as described may be employed.
  • the fermentation reaction is conducted with sugar limitation, oxygen limitation, or both sugar limitation and oxygen limitation.
  • sugar limitation defines the fermentation medium as having little or no excess, measurable sugar. The amount of sugar which is added is below the uptake capacity of the yeast.
  • the fermentation medium contains less than about 1 g/1 of glucose, more specifically, less than about 0.1 g/1 of glucose.
  • oxygen limitation defines the fermentation medium as having little or no excess, measurable oxygen. This does not require that the fermentation is anaerobic, only that the measurable oxygen concentration is low or zero as the amount of oxygen added is below the uptake capacity of the process.
  • the fermentation is conducted at a temperature above room temperature, i.e., above about 25 0 C, more specifically above about 3O 0 C, and in one embodiment, at about 35 0 C.
  • the pH of the fermentation medium may be selected based on other process conditions. In one embodiment, the pH is less than about 5, and more specifically, less than about 4.
  • the fermentation may be conducted as either a batch process or a continuous process, or using a combination to obtain a semi-continuous process.
  • the fermentation is a continuous process wherein substrate is continuously added to the fermentor and the ethanol product is continuously removed.
  • the fermentation can be conducted as a fed batch process, where the substrate is continuously added to a fermentor, but the product is only removed after the fermentation is finished.
  • yeast can be recycled to the fermentor.
  • a continuous process an ethanol and yeast-containing stream is continuously removed from a fermentor in which the fermentation is conducted, ethanol is separated from the removed stream, and the remaining yeast is recycled to the fermentor.
  • the fermentation culture may be employed to stabilize a running fermentation.
  • a yeast of the genus Dekkera and a lactic acid bacteria as described in detail above are added to the running fermentation.
  • the running fermentation may employ any suitable yeast, including the conventional S. cerevisiae or a yeast of the genus Dekkera. The addition may stabilize the running fermentation against infection and/or against process condition stress.
  • a mixture of Dekkera yeast and lactic acid bacteria as described herein is added to the running fermentation.
  • This addition inhibits the infecting organisms and restabilizes the fermentation.
  • the amount of the inventive fermentation culture to be added depends on the size of the fermentor and the total load and is easily determined by one of ordinary skill in the art, without undue experimentation. In one embodiment, the amount of fermentation culture of yeast and bacteria added is about 1 metric ton for a fully utilized 100 m 3 fermentor. Where a running fermentation is exhibiting process stress, addition of the fermentation culture can provide a more robust process.
  • a method for producing distiller's grain comprises fermenting a substrate using a culture comprising a yeast of the genus Dekkera and a lactic acid bacteria, as described in detail above.
  • Distiller's grain is currently used as animal feed.
  • the fermentation process may be similar to that described above for the production of ethanol, regulated to obtain more biomass at the cost of ethanol.
  • the aeration may be enhanced, and/or a higher input of nitrogen may be employed, for instance by using raw material with a higher protein content and/or by adding additional N- sources like urea, although this alternative may be undesirable depending on the required purity of the product.
  • the fermentation should be run under carbon limitation to avoid ethanol formation.
  • Cultivation conditions may also be modified depending on the end use of the distillers' grain. For example, when using the distiller's grain for biogas production, a high protein content is not desired, so a decrease in nitrogen content to a certain extent may be suitable.
  • An improved sugar utilization has three major impacts: (i) a better ethanol yield in the production and thus an improved process economy, (ii) a better utilization of the chemical energy that is stored in the substrate, thus leading to a more beneficial environmental impact of the entire process, and (iii) less substrate leftovers that might be used by potentially infecting organisms. Additional advantages will be apparent to those skilled in the art.

Abstract

La présente invention concerne une culture de fermentation pour la production d'éthanol comprenant une levure du genre Dekkera et une bactérie d'acide lactique. La culture de fermentation peut être utilisée dans des procédés de production d'éthanol, pour améliorer la stabilité d'une production d'éthanol par fermentation continue et/ou pour produire des céréales à distillerie.
PCT/IB2007/055025 2006-12-11 2007-12-11 Cultures de fermentation et procédés les utilisant WO2008072184A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP07849425A EP2099922A1 (fr) 2006-12-11 2007-12-11 Cultures de fermentation et procédés les utilisant

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US87409306P 2006-12-11 2006-12-11
US60/874,093 2006-12-11

Publications (1)

Publication Number Publication Date
WO2008072184A1 true WO2008072184A1 (fr) 2008-06-19

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EP (1) EP2099922A1 (fr)
WO (1) WO2008072184A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5100791A (en) * 1991-01-16 1992-03-31 The United States Of America As Represented By The United States Department Of Energy Simultaneous saccharification and fermentation (SSF) using cellobiose fermenting yeast Brettanomyces custersii
WO1994029475A1 (fr) * 1993-06-11 1994-12-22 Midwest Research Institute Masse cellulaire provenant de cuves de fermentation et utilisee comme source de nutrition dans la conversion de biomasse en ethanol
DE19921561A1 (de) * 1999-05-11 2000-11-16 Nicolas Ratzmann Verfahren zur Gewinnung von in Symbiose lebenden, aus Hefestämmen und Bakterien bestehenden Mikroorganismen

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5100791A (en) * 1991-01-16 1992-03-31 The United States Of America As Represented By The United States Department Of Energy Simultaneous saccharification and fermentation (SSF) using cellobiose fermenting yeast Brettanomyces custersii
WO1994029475A1 (fr) * 1993-06-11 1994-12-22 Midwest Research Institute Masse cellulaire provenant de cuves de fermentation et utilisee comme source de nutrition dans la conversion de biomasse en ethanol
DE19921561A1 (de) * 1999-05-11 2000-11-16 Nicolas Ratzmann Verfahren zur Gewinnung von in Symbiose lebenden, aus Hefestämmen und Bakterien bestehenden Mikroorganismen

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GADAGA T H ET AL: "The growth and interaction of yeast and lactic acid bacteria isolated from Zimbabwean naturally fermented milk in UHT milk", INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 68, 1 January 2001 (2001-01-01), pages 21 - 32, XP003008974, ISSN: 0168-1605 *
MARTENS H ET AL: "Microbiological aspects of a mixed yeast-bacterial fermentation in the production of a special Belgian acidic ale", JOURNAL OF THE INSTITUTE OF BREWING, vol. 103, no. 2, 1997, pages 85 - 91, XP001536932, ISSN: 0046-9750 *
PASSOTH VOLKMAR ET AL: "Dekkera bruxellensis and Lactobacillus vini form a stable ethanol-producing consortium in a commercial alcohol production process", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 73, no. 13, July 2007 (2007-07-01), pages 4354 - 4356, XP002479448, ISSN: 0099-2240 *
RENOUF V ET AL: "Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking", JOURNAL OF APPLIED MICROBIOLOGY, vol. 100, no. 6, June 2006 (2006-06-01), pages 1208 - 1219, XP002479450, ISSN: 1364-5072 *

Also Published As

Publication number Publication date
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