EP2099922A1

EP2099922A1 - Fermentation cultures and methods employing the same

Info

Publication number: EP2099922A1
Application number: EP07849425A
Authority: EP
Inventors: Volkmar Passoth; Johanna Blomqvist; Johan SCHNÜRER
Original assignee: Individual
Current assignee: Individual
Priority date: 2006-12-11
Filing date: 2007-12-11
Publication date: 2009-09-16
Also published as: WO2008072184A1

Abstract

A fermentation culture for ethanol production comprises a yeast of the genus Dekkera and a lactic acid bacteria. The fermentation culture is suitable for use in methods for ethanol production, for improving the stability of a running fermentation production of ethanol, and/or for distiller's grain production.

Description

FERMENTATION CULTURES AND METHODS EMPLOYING THE SAME

FIELD OF THE INVENTION

[0001] 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.

BACKGROUND OF THE INVENTION

[0002] 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. Today, ethanol is mainly produced in continuous processes, wherein substrate is continuously added to the fermentor and the ethanol product is continuously removed. Alternatively, 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, however, 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.

[0004] In commercial practices, it has also been observed in continuous processes such as the Biostil® process that after inoculation with bakers' yeast (S. cerevisiae), it normally takes up to three weeks until the fermentation runs stable. This is a serious drawback for a process intended for efficient commercial use.

[0005] Further, in some cases, stress factors and/or infections can lead to incomplete fermentation, so called stuck and sluggish fermentation, see, for example, Bisson et al, "Stuck and sluggish fermentations," Am. J. Enol. Viticult., 50:107-119. (1999). This can lead to substantial economic losses; commonly, when this occurs, the whole fermentor content (typically up to 100 m³ or even more) will have to be discarded and the fermentation is reinitiated. [0006] With an increasing focus on ethanol as a renewable energy source for various applications, it would be desirable to employ a fermentation culture in ethanol production that is robust against both stress factors in the fermentation process and potential infecting microorganisms, and that allows a stable fermentation from the time of inoculation. SUMMARY OF THE INVENTION

[0007] The present invention provides improved fermentation cultures, which may be used in a variety of methods.

[0008] More particularly, in one embodiment, the invention is directed to a fermentation culture for ethanol production, comprising a yeast of the genus Dekkera and a lactic acid bacteria.

[0009] In other embodiments, the invention is directed to methods employing the fermentation cultures. In one embodiment, 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. In another embodiment, 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. In yet another embodiment, 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.

[0010] The fermentation cultures and methods of the invention are advantageous in providing stable and robust production processes. Further, the fermentation cultures and methods my exhibit stable fermentation from the beginning of the fermentation process. These and additional advantages provided by various embodiments of the invention will be more apparent in view of the following detailed description. DETAILED DESCRIPTION

[0011] 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.

[0012] In a specific embodiment, the fermentation culture comprises a fermentation culture for ethanol production comprising a yeast of the genus Dekkera and a lactic acid bacteria. Both 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). [0013] In a further embodiment, 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).

[0014] In a further embodiment, the lactic acid bacteria comprises Lactobacillus vini, Lactobacillus mobilis, or a mixture thereof. L. vini strains have been isolated from wine fermentation, and at least one strain of L. mobilis has been isloated from lactic acid- containing beverages. Strains of these species are also readily accessible to the public, for example, L. mobilis as EMBL accession number AB242320 and L. vini as DSM20605. See also www.ncbi.nim.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=357762. [0015] Various combinations of the yeast of the genus Dekkera and the lactic acid bacteria may be employed. In a specific embodiment, 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. [0016] 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. [0017] In one embodiment of the method for producing ethanol according to the present invention, 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.

[0018] 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 m³ 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. Although any desired fermentation conditions may be employed, as desired, in one embodiment, the fermentation reaction is conducted with sugar limitation, oxygen limitation, or both sugar limitation and oxygen limitation. As will be recognized by one of ordinary skill in the art, 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. In one embodiment, the fermentation medium contains less than about 1 g/1 of glucose, more specifically, less than about 0.1 g/1 of glucose. As will also be recognized by one of ordinary skill in the art, 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. In further embodiments, the fermentation is conducted at a temperature above room temperature, i.e., above about 25⁰C, more specifically above about 3O⁰C, and in one embodiment, at about 35⁰C. 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.

[0019] The fermentation may be conducted as either a batch process or a continuous process, or using a combination to obtain a semi-continuous process. In one embodiment, the fermentation is a continuous process wherein substrate is continuously added to the fermentor and the ethanol product is continuously removed. Alternatively, 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. After removal of the ethanol product, yeast can be recycled to the fermentor. For example, in one embodiment of 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.

[0020] In another embodiment of the invention, the fermentation culture may be employed to stabilize a running fermentation. For example, in a method for improving the stability of a running fermentation for producing ethanol, wherein a substrate is fermented using a yeast culture, 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. For example, in a case wherein an infection occurs, which may become obvious by a decreasing ethanol productivity and/or the production of non-desired side products like organic acids, 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 m³ fermentor. Where a running fermentation is exhibiting process stress, addition of the fermentation culture can provide a more robust process.

[0021] In yet another embodiment of the invention, 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. Thus, 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. Additionally, 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.

[0022] There are a number of advantages with the use of a fermentation culture of Dekkera yeast and Lactobacillus (lactic acid bacteria) for ethanol production and in the other methods described herein. Both organisms may occupy different ecological niches, which are thus closed for eventual infections. Additionally, D. bruxellensis has been described as having a higher affinity for glucose than the conventional ethanol production yeast, S. cerevisiae, see, Renouf et al, "Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of wine making," /. Appl. Microbiol., 100:1208-1219 (2006). Thus, an improved sugar utilization, especially in continuous cultures where the sugar concentrations are usually low, may be obtained. 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.

[0023] The specific illustrations and embodiments described herein are exemplary only in nature and are not intended to be limiting of the invention defined by the claims. Further embodiments and examples will be apparent to one of ordinary skill in the art in view of this specification and are within the scope of the claimed invention.

Claims

WHAT IS CLAIMED IS:

1. A fermentation culture for ethanol production, comprising a yeast of the genus Dekkera and a lactic acid bacteria.

2. The fermentation culture of claim 1, wherein the yeast comprises Dekkera bruxellensis .

3. The fermentation culture of claim 1, wherein the lactic acid bacteria comprises Lactobacillus vini, Lactobacillus mobilis, or a mixture thereof.

4. The fermentation culture of claim 1, wherein a cell number ratio of the yeast to the bacteria is from about 1:3 to about 15:1.

5. The fermentation culture of claim 4, wherein a cell number ratio of the yeast to the bacteria is from about 1:3 to about 5:1.

6. A method for producing ethanol, comprising fermenting a substrate using a culture comprising a yeast of the genus Dekkera and a lactic acid bacteria.

7. The method of claim 6, wherein the yeast comprises Dekkera bruxellensis.

8. The method of claim 6, wherein the lactic acid bacteria comprises Lactobacillus vini, Lactobacillus mobilis, or a mixture thereof.

9. The method of claim 6, wherein a cell number ratio of the yeast to the bacteria is from about 1:3 to about 15:1.

10. The method of claim 9, wherein a cell number ratio of the yeast to the bacteria is from about 1:3 to about 5:1.

11. The method of claim 6, wherein the substrate comprises molasses, wheat starch, or a mixture thereof.

12. The method of claim 6, wherein the fermentation is conducted in a medium comprising less than about 1 g/1 of glucose.

13. The method of claim 12, wherein the fermentation is conducted in a medium comprising less than about 0.1 g/1 of glucose.

14. The method of claim 6, wherein the fermenting step is conducted at a temperature of about 35⁰C and a pH of less than about 4 and with sugar and oxygen limitation.

15. The method of claim 6, wherein the fermentation is a continuous process.

16. The method of claim 15, wherein 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.

17. 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.

18. The method of claim 17, wherein the addition stabilizes the running fermentation against infection.

19. The method of claim 17, wherein the addition stabilizes the running fermentation against process condition stress.

20. 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.