WO2007149502A2

WO2007149502A2 - Method of producing ethanol with respiration-deficient yeast

Info

Publication number: WO2007149502A2
Application number: PCT/US2007/014407
Authority: WO
Inventors: Richard A. Hurt
Original assignee: Atom Sciences, Inc.
Priority date: 2006-06-21
Filing date: 2007-06-20
Publication date: 2007-12-27
Also published as: WO2007149502A3

Abstract

The present invention is drawn to a process for the industrial production of ethanol as a biofuel by fermenting glucose with an isolated strain of respiration-deficient yeasts from genus Kazachstania to produce ethanol; and purifying the ethanol to at least 90% purity.

Description

METHOD OF PRODUCING ETHANOL WITH RESPIRATION-DEFICIENT YEAST

Field of the Invention

The present invention pertains to the field of renewable energy production through bioprocesses. The invention utilizes a respiration-deficient yeast that has been demonstrated to efficiently produce ethanol.

Background on the Invention

Because of the depleting resources and rising costs of fossil fuels, as well as the negative environmental impact associated with obtaining fossil fuels, the development of alternative energy sources is both desirable and necessary. One alternative energy source, which is proving to be of importance as a successful replacement for oil/gasoline, is ethanol. Ethanol produced from crops has several advantages a fuel over fossil fuels, including its renewable nature and reduced air emissions upon burning. Ethanol as a biofuel can be made from a number of renewable crops including, miscanthus, switchgrass, corn, sweet sorghum, sugar cane and even algae.

The bioconversion of any natural feedstock to ethanol requires two steps: saccharification, which is the conversion of the feedstock to sugar, and fermentation, which is the conversion of sugars to ethanol.

Conversion of sugars into ethanol through fermentation by yeasts yields two molecules of adenosine triphosphate (ATP), two molecules of CO₂, and two molecules of ethanol. The first step, called glycolysis, involves breaking a 6-carbon sugar (glucose) into two molecules of glyceraldehyde-3-phosphate (G3P) which each then react with the nicotinamide adenine dinucleotide (NAD⁺) molecule and inorganic orthophosphate (Pj) to form two 3 -carbon pyruvate molecules (CHs(CO)₂OH).

C₆Hi₂O₆ + 2ATP → 2G3P (equation 1 a)

2G3P + 2NAD⁺ + 2Pi → 2CH₃(CO)₂OH + 2NADH + 2H⁺ + 4ATP (equation Ib) The net glycolysis reaction is

C₆Hi₂O₆ + 2NAD⁺ + 2Pi → 2CH₃(CO)₂OH + 2NADH + 2H⁺ + 2ATP (equation Ic)

Glycolysis occurs in the cytoplasm of cells and does not require oxygen. The pyruvate products can be further broken down, or catabolyzed, in a number of different reactions. One of these reactions is alcohol fermentation wherein, under anaerobic conditions in yeast, each pyruvate reacts with a molecule of NADH to create ethanol and carbon dioxide.

CH₃(CO)₂OH + NADH + H⁺ → C₂H₅OH + CO₂ + NAD⁺ (equation 2)

The net equation for the overall glycolysis plus alcohol fermentation process is

C₆Hi₂O₆ → 2C₂H₅OH + 2CO₂ + 2ATP (equation 3)

In this pathway, each sugar molecule is converted to two ethanol molecules and two ATP molecules. In contrast, under aerobic conditions, electron transport in the cell allows the pyruvate to be completely oxidized through a series of reactions. This oxidative process is compartmentalized in the mitochondria and yields from 36 to 38 molecules of ATP plus 6 molecules of CO₂. The energy stored in ATP is utilized by the cell to do work, but cannot be harvested as an energy source to power manmade machinery. The oxidative pathway can occur only if sufficient ADP, Pi, and O₂ are available. Thus, the alcohol fermentation pathway can be selected by maintaining an environment with low oxygen content. Similarly, the alcohol fermentation pathway is inhibited by a high oxygen environment, an effect known as the "Pasteur Effect". But even though oxygen inhibits the fermentation pathway, molecular oxygen is required by yeasts for metabolic processes other than respiration. Several studies have shown that molecular oxygen is essential for growth [Andreasen et al., 1953a, Andreasen et al., 1953b], plasma membrane integrity and ethanol tolerance [Alexandre et al., 1994, Thomas et al., 1978], and maintenance of high glycolytic and ethanol fermentation rates [Casey et al., 1984; Rosefeld et al., 2003]. Thus, one of the considerations for the industrial production of ethanol is the conflicting roles of oxygen of a) inhibiting fermentation, which decreases the ethanol yield, but b) at the same time being necessary for yeast viability. This problem of oxygen being required, but too much oxygen inhibiting the desired product, is often addressed by use of a batch process. For example, with brewery fermentation, oxygen is added during the growth phase to improve biomass accumulation [Kirsop et al., 1974], and to stimulate sluggish fermentation after the end of the cell growth phase [Sablayrolles et al., 1996].

Another solution to the problem caused by oxygen promoting a respiration pathway that does not lead to ethanol production is to use yeasts that are incapable of following the respiration pathway. In respiration-deficient yeasts, the Pasteur effect is eliminated and continuous production of ethanol is possible even in an oxygen-rich environment. Faber et al. described a continuous process for ethanol production using a respiration-deficient mutant of Saccharomyces uvarum [U.S. Patent 4,567,145]. More recently, two respiratory-deficient nuclear petites, FY23Δpetl91 and FY23Δcox5a, of Saccharomyces cerevisiae were generated using polymerase chain reaction mediated gene disruption [Hutter and Oliver, 1998]. These mutant respiratory-deficient strains produced ethanol at rates respectively 43% and 30% higher than the respiratory-sufficient parent strain but were less ethanol tolerant. A 100% respiration deficient strain of S. cerevisiae has been shown to increase ethanol productivity by 48% [Oner et al., 2005].

One of the main costs in the production of ethanol as a biofuel is the cost of the crop used for the fermentation process. In addition, one concern associated with the use of ethanol produced from crops as a biofuel is the removal of those crops as a food source for humans and/or live stock, leading to increased food costs. Thus, high efficiency methods of making ethanol as a biofuel are desired and the present invention is directed to methods of high efficiency production of ethanol as a biofuel. By "high efficiency" it is meant a high yield of ethanol from the crop source.

Detailed Description of the Invention

There is a group of yeasts that exist as naturally occurring psychrophobic, respiration- deficient yeasts. These yeasts were recently transferred from the genus Candida to the genus Kazachstania [Kurtzman, 2005]. At least some strains of two species of these yeasts, K. pintolopesii and K. slooffiae, are entirely respiration deficient. Although they grow slowly on complex agar media, as do all respiration deficient yeasts, they grow rapidly in well-aerated culture [Hurt, 1997] and rapidly ferment glucose to ethanol. K. pintolopesii ATCC 22998 is likely a naturally occurring rho⁰ (lacking a mitodhondrial genome) representative of the species [Hurt, 1997], and neither this strain nor K. slooffiae ATCC 22978 are expected to have a functional mitochondrial ATPase since both are persistently respiration deficient.

The present invention is directed to a method of using yeasts of the Kazachstania genus in the production of biofuels. In the methods of the invention a fermentation process is conducted wherein the medium is supplied with glucose and warm air is bubbled through to supply oxygen needed for growth and to remove the ethanol product. With the present invention, the ethanol yield is increased over that yield possible using respiration sufficient yeasts. Aside from the property of respiration deficiency, described above, Kazachstania yeast have additional advantageous properties, which result in improved biofuel production. One of these additional advantageous properties is that Kazachstania yeasts grow only by asexual reproduction. This is important to retaining the characteristics of the yeast without genetic contamination from respiration-competent species. Another characteristic that helps these yeasts remain isolated and makes them unique is that they have adapted to a 4O⁰C environment, which is too warm for most other yeasts to thrive. Not only does this feature help retain the genetic identity of the yeast strain but it also helps remove ethanol from the mixture by raising the vapor pressure of the ethanol produced.

It is possible to artificially induce at least two of the advantages that K. pintolopesii and K. slooffiae have acquired naturally. As previously mentioned, recently, two respiratory- deficient nuclear petites, FY23Δpetl91 and FY23Δcox5a, of S. cerevisiae were generated using polymerase chain reaction mediated gene disruption [Hutter and Oliver, 1998]. S. cerevisiae also forms mit^" respiration deficient (mitochondrial DNA segment deletion) mutants when grown in the presence 50 μg-ml^""1 ethidium bromide [Tzagoloff, 1982]. Several mit^" mutants have been characterized. S. cerevisiae is a diploid homothallic yeast that expresses either of the mating factors Mat-a and Mat-α, which are short non-branched peptide hormones expressed from the mating-type (MAT) locus on chromosome III [Butler et ah, 2004; Herskowitz et al._> 1992]. S. cerevisiae is able to switch mating type by copying genetic material from silent cassette loci HMLa and HMRa. located near the left and right telomeres of chromosome III respectively [Ravindra et αl, 1999]. Thus, rendering a S. cerevisiae strain genetically isolated by removing genes encoding mating-type pheromone production would require deletion of multiple genetic loci. Additional factors that control yeast fusion have been identified through genetic analysis of cell fusion defective mutants

(Fus^~) [Fitch, et al, 2004]. Cell fusion associated proteins FUSl [McCaffrey et al, 1987], FUS2 [Elion et al, 1995], RVS161 [Brizzio et al, 1998], FIGlznά FIG2 [Erdman et al, 1998], and PRMI [Heiman and Walter, 2004] are proteins that are induced by mating pheromone and were found to localize to the zone of cell fusion. Genetic modification or deletion of the genes encoding these functions results in cell fusion deficiency. While the use of recombinant organisms is not excluded from the invention, these yeasts can be obtained and used as naturally occurring strains if so desired. The use of an isolated naturally occurring organism has some advantages over a genetically engineered version. With a naturally occurring organism, the issue of plasmid instability, which has been recognized in artificially created respiration-deficient yeasts [Oner et al, 2005] is eliminated. Also, the prohibition on or reluctance to use genetically modified organisms that exists in many parts of the world, will not apply to naturally occurring strains of Kazachstania that can be used in the present invention, such as K. pintolopseii and K. slooffiae.

The present invention is drawn to a method of making ethanol biofuels in a cost- efficient fermentation process with a yeast strain of genus Kazachstania. One advantage of the present invention is the ability to use strains of Kazachstania that are: 1) respiration deficient, 2) isolated by lack of sexual reproductive capacity; 3) adapted to thrive at temperatures near 40⁰C; and 4) are naturally occurring. Furthermore, these naturally occurring yeasts may be genetically modified to provide additional desirable properties. Because these yeast strains are respiration deficient, there is no requirement for maintenance of anoxic conditions to select for fermentative metabolic activity. The resulting fermentative yields improve energy conversion efficiency and ethanol production as compared to respiration-competent yeasts. As the cost of feedstock is a major cost of ethanol production, the use of a respiration-deficient yeast strain is a significant cost savings per unit ethanol production. In addition, the fact that warm air or other oxygen-containing gas can be bubbled through the reaction mixture at all times serves to supply the yeast with needed oxygen continuously and helps to efficiently remove the ethanol product so that the reaction can be performed for extended periods in a continuous manner without costly batch processing.

In a preferred embodiment, K. pintolopesii is used in the method of making a biofuel of the present invention. Glucose for the fermentation process can come from saccharification of starch, cellulose, or hemicellulose from corn, corn stover, switch grass, sugar cane, sorghum, or other natural feedstock. After saccharification, the source material preferably contains mostly glucose, but also may contain other sugars. For example, hydrolysis of cellulose generates glucose but hydrolysis of hemi-cellulose generates xylose (a 5-carbon sugar). Fermentation of glucose by Kazachstania yeast requires sugar, a source of nitrogen, and appropriate vitamins and minerals. Nitrogen can be supplied by ammonium salts, urea, or peptone. Vitamins and minerals, if not already present in the sugar source, can be supplied by yeast extract.

In most cases, fermentable sugars are prepared from more complex carbohydrates found in nature in a saccharification process that is separate from the fermentation reaction. However, simultaneous saccharification and fermentation has been explored in the past [e.g. U.S. Pat. No. 3,990,944] and is actively being pursued today [e.g. U.S. Patent Application No. 20060110812]. The FY23Δpetl91 respiration-deficient strain mentioned previously has been modified to secrete a bifunctional fusion protein that contains both Bacillus subtilis α- amylase and Aspergillus awamori glucoamylase activities [Oner et al, 2005; Oner et ah, 2006]. This allowed direct single-step conversion of starch to ethanol and gave a 48% higher yield than the respiration-sufficient parent. Genetic engineering techniques known to those skilled in the art could be used to modify Kazachstania yeasts for similar purposes. These modified yeasts would still take advantage of the natural properties of Kazachstania and are encompassed in this patent.

Most industrial ethanol for biofuel is presently produced by fermentation of starch or other sugar-based feedstock in much the same way ethanol for alcoholic beverages has been made for centuries. For example, corn can be ground into flour, which is referred to in the industry as "meal", and processed without separating out the various component parts of the grain. The meal is slurried with water to form "mash." Enzymes are added to the mash to convert the starch to D-glucose (also called dextrose), a simple sugar. Ammonia is added for pH control and as a nutrient to the yeast. The mash is processed in a high-temperature cooker to reduce bacteria levels before fermentation. The mash is cooled and transferred to fermenters where yeast is added and the conversion of sugar to ethanol and carbon dioxide (CO₂) begins. With traditional yeasts (usually S. cerevisiae), the mash is kept cool to facilitate the activity of the yeast. With the Kazachstania yeasts, however, the fermentation process proceeds at a higher temperature (40⁰C). This higher fermentation temperature is advantage associated wit the present invention because the vapor pressure of pure ethanol is significant (about 120 mm Hg based on interpolation of data in the CRC Handbook of Chemistry and Physics^'). As the mole fraction of ethanol in the fermentation solution approaches 5%, the partial pressure of ethanol above the mixture should be about 6 mm Hg according to Raoult's Law.

Bubbling a gas through the fermentation mixture humidifies the gas with ethanol and other volatile materials. Lightner pointed this out in U.S. Patent Appl. 20030143704 where he used CO₂ to bubble through a fermentation mixture. While air would be much less expensive to use as the gas, it was not used in U.S. Patent Appl. 20030143704 because with traditional yeasts, such as those used in ethanol, oxygen present in air would inhibit the ethanol production. Thus, another advantage of the present invention is the fact that with Kazachstania yeast, warm air (instead of CO₂) can be used as the gas bubbled through the mixture because respiration-deficient Kazachstania produce ethanol in oxygen rich environments, thus, decreasing the costs associated with the biofuel production. The gas bubble through the fermentation mixture serves two purposes with the present invention: (1) the bubbling action keeps the yeast in suspension and (2) ethanol is removed from the liquid mixture before it reaches concentrations that are toxic to the yeast. The ethanol can be recovered from the humidified air by passing the vapor over cooling coils so that the ethanol and other volatiles are condensed. Following condensation the ethanol is further purified, e.g. with a distillation process, to reach a purity of at least 90%, preferably at least 95%, followed by a molecular sieve system to reach a purity of 100%. Following purification appropriate fuel additives are added to the ethanol using standard practices in the industry.

If a cellulosic feedstock is used instead of a sugar- or starch-based feedstock in the method of making ethanol biofuel of the present invention, an additional step is required to convert the long-chain polymers of cellulose and hemi-cellulose to their individual sugar molecules. This is done typically by either acid or enzymatic hydrolysis, the result of which is glucose in the .case of cellulose hydrolysis, and xylose (a 5-carbon sugar) in the case of hemi-cellulose hydrolysis. Since Kazachstania does not ferment 5-carbon sugars and a separate process may be included to ferment the 5-carbon sugars that build up in the fermentation mixture over time.

References cited

U.S. Patent Documents 3,990,944 Nov, 1976 Gauss et al 435/165 4,567,145 Jan, 1986 Faber et al 435/161; 435/255; 435/940 20060110812 May, 2006 O'Neal Ingram et al. 435/161 20030143704 July 31, 2003 Lightner, Gene E. 435/161; 568/913

Other References

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Claims

CLAIMS:

1. A process for the industrial production of ethanol as a biofuel, which comprises fermenting glucose with an isolated strain of respiration-deficient yeasts from genus Kazachstania to produce ethanol; andpurifying the ethanol to at least 90% purity.

2. The method of claim 1, wherein the glucose is derived from starch, cellulose, lignin or hemicellulose.

3. The process of claim 1, wherein the respiration-deficient yeast is a naturally occurring isolated strain of Kazachstania.

4. The process of claim 1, wherein the respiration-deficient yeast is a genetically- modified isolated strain of Kazachstania.

5. The method of claim 4, wherein the genetically modified isolated strain of Kazachstania has been modified to simultaneously catalyze a saccharification process and fermentation process.

6. The process of claims 1, wherein the yeast reproduces asexually.

7. The process of claim 3 wherein the yeast is K. pintolopesii.

8. The process of claim 3 wherein the yeast is K. slooffi.

9. The process of claim 3, wherein the glucose used as the starting material for the fermentation process is from saccharification of starch, cellulose, lignin or hemicellulose derived from corn, corn stover, switch grass, sorghum, sugar cane, or other natural feedstock or algae.

10. The process of claim 9 wherein glucose is separated from non-fermentable sugars.

11. The process of claim 1, wherein fermentation is conducted continuously with stoppages only for removing buildup of feedstock or products.

12. The process of claim 1 wherein ethanol is removed from the reaction in a continuous fashion by bubbling a warm gas through the reaction mixture and condensing the resulting ethanol vapor.

13. The process of claim 12 wherein the temperature of the gas is from 25 to 45 ⁰C.

14. The process of claim 13 wherein the gas comprises oxygen.

15. The process of claim 12 wherein the gas is air.

16. The method of claim 2, wherein the starch, cellulose, lignin or hemicellulose is obtained from corn, corn stover, switch grass, sorghum, sugar cane or other natural feedstock or algae.

17. The method of claim 1, wherein the ethanol is further purified with a distillation process.

18. The method of claim 17, which further comprises removing residual water from the distilled ethanol with a molecular sieve.