WO2011123055A1

WO2011123055A1 - Process for preparing ethanol from crude glycerol using novel bacteria

Info

Publication number: WO2011123055A1
Application number: PCT/SG2010/000121
Authority: WO
Inventors: Won Jae Choi
Original assignee: Agency for Science Technology and Research Singapore
Current assignee: Agency for Science Technology and Research Singapore
Priority date: 2010-03-29
Filing date: 2010-03-29
Publication date: 2011-10-06
Anticipated expiration: 2012-09-29

Abstract

The invention provides an isolated microorganism, i.e., Kluyvera cryocrescens (ATCC Deposit Designation No. PTA-10600) capable of growing on crude glycerol as a sole carbon source, which is capable of producing ethanol with high yield and high productivity from crude glycerol or a range of other carbon feedstocks. The present invention further provides for anaerobic and aerobic reaction mixtures and reaction conditions for ethanol production using the isolated Kluyvera cryocrescens bacterial strain.

Description

PROCESS FOR PREPARING ETHANOL FROM CRUDE GLYCEROL

USING NOVEL BACTERIA

FIELD OF THE INVENTION

[0001] The present invention provides a newly isolated bacteria, methods for bacterial production of ethanol and reaction mixtures for bacterial production of ethanol. The present invention further provides methods for bacterial production of ethanol from crude glycerol that is obtained as a by-product of the biodiesel industry.

BACKGROUND OF THE INVENTION

[0002] Industrial biotechnology currently accounts for about 5% of global chemical sales, primarily through ethanol, pharmaceutical intermediates, citric acid, and amino acids. This share of sales is expected to reach as high as 20% in the near future, depending on consumer acceptance and cost. This quick growth has occurred due to the development of more cost- effective and more efficient microbes. The economic advantages of developing green technologies will help keep this growth pattern active. The advantages associated with biotechnology include reducing the number of synthesis steps, lowering consumption of raw materials, increasing energy efficiency, lowering emissions and, finally, reducing production costs. There are numerous possibilities for replacing chemical techniques with

biotechno logical methods based on renewable resources. In the case of bulk chemicals, the product price is affected mainly by raw material costs. The introduction of new technologies for the efficient use of such raw materials is currently being promoted. The utilization of crude glycerol, for example, provides great potential.

[0003] Historically, ethanol has been produced mainly from sugars and carbohydrates via microbial fermentation. Considering the worldwide surplus of crude glycerol, ethanol fermentation using glycerol as a feedstock is a promising alternative process to supply bioethanol. Ethanol production from corn-derived sugars is likely more costly and less economical when compared with ethanol production from glycerol in terms of the type of manufacturing facility required, the feedstock required, and the operational costs associated with each (see, Curr. Opin. BiotechnoL, 18:213 (2007)). As such, ethanol produced from glycerol can be more economical than producing ethanol from corn, which is currently the main feedstock for ethanol production in the United States. Furthermore, ethanol produced from glycerol rather than cellulose, which is made out of wood chips and waste vegetable resources is also more economically efficient. For ethanol production from corn-derived sugars, the layout of a facility is more complex, leading to more capital intensive production methods. Additionally, the operational costs are almost 40% lower in the production of ethanol from glycerol than those for producing ethanol from corn.

[0004] Furthermore, global trends are driving the development of agricultural products into new sources of energy and materials. In Singapore for example, there is easy access to abundant palm oil feedstock from the neighboring countries, such as Malaysia and Indonesia. Singapore is also the location for major chemical logistics players that provide terminalling services and shipping to markets worldwide. With agricultural neutrality, excellent infrastructure, and the potential for downstream integration, Singapore presents an ideal location for biodiesel production and more importantly for its co-product, crude glycerol. Based on these competitive advantages, several biodiesel producers have setup plants in Singapore and as a result, Singapore is a readily accessible market for crude glycerol that can be obtained from these biodiesel plants.

[0005] The rapidly expanding market for biodiesel and bioethanol is remarkably altering the cost and availability of glycerol. In general, approximately 10 pounds of crude glycerol are formed for every 100 pounds of biodiesel produced (Applied Catalysis A: General,

306: 128 (2006), Angew. Chem. Int. Ed., 46:4434 (2007), Environmental Progress, 26:338

(2007), Biotechnology Advances, 27:30 (2009)). Bioethanol processes also generate glycerol as a by-product, including glycerol by-product formation of up to 10% (w/w) of the total sugar consumed (Biotechnol. Bioeng., 100: 1088 (2008)) and 17% (w/w) of the ethanol produced (Biotech Lett, 10:677 ( 1988), J Sci Food Agric, 82:309 (2002)). Crude glycerol has thus been widely recognized as an attractive sustainable resource for oil and chemical industries.

[0006] There have been few reports on microbial conversion of glycerol to ethanol by use of wild-type strains. Pachysolen tannophilus, Paenibacillus macerans and Enterobacter aerogenes are the only wild-type strains that have been previously known to ferment glycerol to produce ethanol as a major metabolite. Most work has been directed to ethanol production from glycerol using genetically modified Escherichia coli. In batch fermentation using minimal medium and recombinant E. coli expensive antibiotics are required and inducers to keep the reconsituted E. coli active for ethanol production (Biotechnol. Bioeng., 94:821 (2006), Appl. Environ. Microbiol., 74: 1 124, (2008), Biotechnol. Bioeng., 103 : 148 (2009), Metabolic Eng., 10:340 (2008)).

[0007] Glycerol fermentation has been primarily conducted using recombinant E. coli with pure glycerol as the carbon feedstock. Recently, there have been intensive efforts to describe methods for efficient conversion of glycerol to ethanol via metabolic pathway engineering of E. coli. These efforts have focused on minimizing the synthesis of by-products or using microaerobic conditions for ethanol production. However, ethanol productivity is not as compatible with those obtained from conventional corn-based ethanol processes.

[0008] The engineered E. coli strain produced 21 g/L of ethanol from 60g L of pure glycerol with the volumetric productivity of 0.216 g/L h under microaerobic conditions

{Biotechnol. Bioeng., 103 : 148 (2009)). In the case of crude glycerol obtained from the biodiesel industry, 10 g/L of ethanol was produced by the engineered E. coli from 22 g/L of crude glycerol with a productivity of 0.09 g/L h under anerobic conditions (Metabolic Eng. , 10:340 (2008)). Using a wild-type strain of Paenibacillus macerans, synthesis of ethanol and 1 ,2-propanediol from glycerol was described {Appl Environ Microbiol, 75: 18 (2009)). This bacterial strain was obtained from Belgian Co-ordinated Collections of Microorganisms (BCCM LMG) and was capable of producting ethanol and 1 ,2-propanediol as major metabolites at 3 g/L and 0.8 g/L, respectively during anaerobic fermentation with 10 g/L of pure glycerol. [0009] Previously, yeast Pachysolen tannophilus could convert glycerol to ethanol at 4 g/L with a yield of 0.4 mol/mol glycerol during aerobic growth (Enzyme Microb. Technoi , 4:349 ( 1982)). Enterobacter aerogenes HU- 101 , an isolate from methanogenic sludge, can also anaerobically convert crude glycerol obtained from biodiesel wastes to ethanol with a yield of 0.85 mol/mol glycerol (J. Biosci. Bioeng. , 100:260 (2005)). Dharmadi et al. have identified the metabolic processes and conditions that allow a known strain of Escherichia coli to convert glycerin into ethanol through an anaerobic fermentation process (Biotechnol. Bioeng., 94:821 (2006), Appl. Environ. Microbiol., 74: 1 124 (2008)). Gonzalez claimed anaerobic fermentation of glycerol for the production of ethanol, succinic acid, acetic acid, 1 ,2- propanediol and formic acid by use of E. coli (WO 2007/1 15228). Burd et al. have demonstrated a glycerol utilizing E. coli mutant with high increased resistance to glycerol toxicity and high uptake of glycerol (WO 2008/006037). Ethanol was also produced together with hydrogen by Enterobacter aerogenes NBRC 12010 from biodiesel waste glycerol in a bioelectrochemical reactor with thionine under anaerobic conditions (Biotechnol. Bioeng. , 98:340 (2007)). Ethanol and formate were also produced during anaerobic glycerol fermentation by mixed cultures {Biotechnol. Bioeng., 100: 1088 (2008)).

(0010] Glycerol has been utilized as carbon source for many microorganisms, particularly in anaerobes. As shown in FIG. 1 , under anaerobic conditions, glycerol can be either oxidized to dihydroxyacetone or dehydrated to 3-hydroxypropionaldehyde.

Dihydroxyacetone can be further metabolized into pyruvate, which is one of the main metabolic intermediate found in glycolysis pathway and subsequently converted to various fermentation products such as lactic acid, acetate, ethanol, butyrate and succinic acid. 1 ,3- Propanediol is a common product during anaerobic fermentation of glycerol because its production requires NADH that can be accumulated during biomass production. Glycerol can also be assimilated via glycerol 3-phosphate to pyruvate under aerobic conditions.

Dihydroxyacetone phosphate is an isomer of glyceraldehyde 3-phosphate, thus it can be linked to common glycolysis pathway. In certain cases, dihydroxyacetone phosphate can be further converted to 1 ,2-propanediol. [0011] Glycerol-based biorefinery microbial fermentation processes use inexpensive and readily available glycerol as the raw material to produce fuels and chemicals. A major challenge in fermentation of the low-grade crude glycerol is to obtain microbial strains tolerant to undesirable inhibitory components such as salts and organic solvents that are present in crude glycerol. Ethanol production from glycerol is more attractive compared to the more costly and challenging ethanol production from corn-derived sugars in terms of the type of manufacturing facility required, the feedstock required and the operational costs associated with each (Curr. Opin. Biotechnol , 18:213 (2007)). As such, development of glycerol based ethanol production methods could provide an economically efficient alternative. [0012] In view of the foregoing, there is a need for new microorganisms to be employed as biocatalysts for the production of ethanol from crude glycerol. The present invention satisfies this need by advantageously providing a novel microorganism isolated from soil. Based on its ability to utilize crude glycerol as a sole carbon source, this microorganism can be used as a biocatalyst for the production of ethanol. This isolated microorganism has been identified as non-pathogenic Kluyvera cryocrescens. BRIEF SUMMARY OF THE INVENTION

[0013) The present invention provides an isolated microorganism Kluyvera cryocrescens (ATCC Deposit Designation No. PTA- 10600), which can utilize crude glycerol as well as byproduct glycerol formed in conventional yeast fermentation and convert it to make ethanol. The invention provides methods for direct use of crude glycerol as feedstock to produce ethanol, as well as methods for improving ethanol yield in conventional bioethanol processes. Furthermore, when this new bacterial strain was cultivated together with ethanol producing yeast, it helped to dissimilate glucose so as to produce ethanol much faster. At the same time it could also convert glycerol synthesized by yeast during glucose fermentation to ethanol. As a result of this synergistic co-fermentation, there was a remarkable improvement in ethanol productivity.

[0014] Moreover, the isolated bacteria of the present invention provides a method for highly efficient production of renewable fuels and chemicals, as well as for production of chemicals from glycerol-based biorefinery methods by-products. Advantageously, the new isolate undergoes fermentation with low-grade crude glycerol and is tolerant to inhibitory components such as salts and organic solvents.

[0015] In some embodiments, the present invention provides a method for bacterially producing ethanol, wherein the method comprises incubating an isolated Kluyvera

cryocrescens (ATCC Deposit Designation No. PTA-10600) bacterial strain with a carbon feedstock to produce ethanol. In some embodiments, ethanol production occurs by incubation of the isolated Kluyvera cryocrescens in an aerobic or anaerobic reaction mixture. In further embodiments, the aerobic or anaerobic reaction mixture comprises a carbon feedstock, a nutrient composition, a buffer, and a gas.

[0016] In some embodiments, the carbon feedstock is selected from the group of a C₃-C₈ monosaccharide, a disaccharide, or a sugar alcohol. In some preferred embodiments, the carbon feedstock is glycerol. In some more preferred embodiments, the glycerol is crude glycerol obtained as a by-product from the production of biodiesel.

[0017] In some embodiments, the final concentration of crude glycerol in the reaction mixture is in the range from 25 g/L to 100 g/L. [0018] In some embodiments of the present invention, the nutrient composition is selected from the group of yeast extract, polypeptone, tryptone, corn steep liquor or mixtures thereof. [0019] In some embodiments of the present invention, the buffer comprises one or more components selected from the group of ₂HP0₄, NaH₂P0₄, EDTA, ZnS0₄*7H₂0,

CaCl₂ ^«2H₂0, FeS0₄ ^»7H₂0, Na₂Mo0₄ ^«2H₂0, CuS0₄ ^«5H₂0, CoCl₂ ^»6H₂0, MnCl₂ ^»4H₂0, MgCl₂ ^«6H₂0, or (NH₄)₂S0 . [0020] In preferred embodiments where the reaction is aerobic, the gas comprises an air and nitrogen mixture. In embodiments where the reaction is anaerobic, the gas comprises C0₂, N₂, or a mixture thereof.

[0021] In another embodiment, the present invention provides a method for production of ethanol by co-fermentation, comprising: (i) incubating a composition comprising a yeast strain capable of ethanol production and a carbon feedstock; and (ii) inoculating the yeast and feedstock composition from (i) with an isolated Kluyvera cryocrescens (ATCC Deposit Designation No. PTA- 10600) bacterial strain under fermentation conditions. In additional embodiments, co-fermentation increases ethanol production compared to ethanol production from fermentation resulting from incubating a composition comprising a yeast strain capable of ethanol production and a carbon feedstock in the absence of an isolated Kluyvera cryocrescens bacterial strain. In some embodiments, co-fermentation increases the ethanol yield by 6% or more.

[0022] In other embodiments, the present invention provides for a co-fermentation reaction mixture comprising an isolated Kluyvera cryocrescens (ATCC Deposit Designation No. PTA- 10600) and a yeast capable of producing ethanol by fermentation. In some preferred embodiments, the carbon feedstock is glucose. In additional preferred embodiments, the co- fermentation reaction mixture provides an ethanol yield that is increased by 6% or more.

[0023] In further embodiments, the present invention provides an isolated Kluyvera cryocrescens (ATCC Deposit Designation PTA-10600). In other embodiments, the invention provides for an isolated bacterial strain with sequence similarity (99.3%) in the 16S rRNA gene with the closest known strain of Kluyvera cryocrescens. In some embodiments, the isolated Kluyvera cryocrescens produces ethanol with an ethanol production rate greater than 0.216 g L/h. In still other embodiments, the isolated Kluyvera ctyocrescens bacterial strain produces ethanol from crude glycerol with an ethanol molar yield of greater than or equal to 80%. In yet another embodiment, the present invention provides a use of an isolated Kluyvera cryocrescens bacterial strain having ATCC Deposit Designation No. PTA-10600 in a fermentation reaction. [0024] These and other aspects, objects and embodiments will become more apparent when read with the detailed description and drawings that follow.

BRIEF DESCRIPTION OF THE DRAWINGS [0025] FIG. 1 illustrates the microbial metabolic pathways of glycerol processing for the production of fuels and other chemicals.

[0026] FIG. 2 illustrates the phylogenetic tree of isolated Kluyvera cryocrescens bacterial strain S26 and related organisms based on analysis of 16S rRNA sequences.

[0027] FIG. 3 illustrates a graph of the HPLC analysis of fermentation metabolites.

Metabolites examined include: CA: citric acid, Glu: glucose, PA: pyruvic acid, SA: succinic acid, LA: lactic acid, Gly: glycerol, FA: formic acid, AA: acetic acid, 12PDO: 1 ,2- propanediol, 13PDO: 1 ,3-propanediol, PRA: propionic acid, EtOH: ethanol, and BA: butyric acid.

[0028] FIG. 4 illustrates fermentative production of ethanol from crude glycerol by Kluyvera cryocrescens bacterial strain S26. Cell concentration ( A), glycerol (□), ethanol (■), and succinic acid (·).

[0029] FIG. 5 illustrates conversion of glycerol obtained from bioethanol processes into ethanol by Kluyvera cryocrescens bacterial strain S26. The dotted lines represent the control experiment reaction that does not contain the K. cryocrescens bacterial strain S26. Glycerol (□) and ethanol (■).

[0030] FIG. 6 illustrates co-fermentation of yeast Saccharomyces pastorianus ATCC 26602 and bacterial strain Kluyvera cryocrescens S26. The dotted lines represent the single fermentation profile by Saccharomyces pastorianus ATCC 26602 only in the absence of Kluyvera cryocrescens S26. The solid lines represent the co-fermentation experiments. Glycerol (o), glucose (♦ ), and ethanol (■). DETAILED DESCRIPTION OF THE INVENTION

I. DEFINITIONS

[0031) The term "bacterial production of ethanol" and variations thereof include any means for producing ethanol from a culture of isolated bacteria. In some embodiments, ethanol is produced by the isolated Kluyvera cryocrescens (ATCC Deposit Designation No. PTA- 10600) bacterial strain described herein, also designated Kluyvera cryocrescens S26. In some embodiments, ethanol is produced by a bacterial strain with 99.3% identity or greater in the 16S rRNA gene to the isolated Kluyvera cryocrescens of the present invention.

[0032] The term "capable of producing ethanol" and variants thereof include the feature that the bacterial strain produces ethanol under the following reaction conditions: the bacterial strain is incubated with a carbon feedstock, optionally a nutrient composition and optionally a buffer at a temperature allowing for bacterial growth. Exemplary temperatures include, but are not limited to, 15-55°C, such as 15°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and 55°C. The buffer can comprise one or more of the following components: ₂HP0₄, NaH₂P0₄, EDTA, ZnS0₄ ^«7H₂0, CaCl₂*2H₂0, FeS0₄ ^«7H₂0, Na₂Mo0₄ ^«2H₂0, CuS0₄ ^«5H₂0, CoCl₂-6H₂0, MnCl₂ ^»4H₂0, MgCl₂'6H₂0, or (NH₄)₂S0₄.

[0033] The term "fermentation" and variants thereof include the enzymatic, anaerobic, and aerobic breakdown of organic substances by microorganisms to produce simpler organic products. In some embodiments, fermentation refers to the utilization of carbohydrates by microorganisms {e.g. , bacteria) involving an oxidation-reduction metabolic process that takes place under anaerobic conditions and in which an organic substrate serves as the final hydrogen acceptor (i.e., rather than oxygen). Although fermentation occurs under anaerobic conditions, it is not intended that the term be solely limited to strict anaerobic conditions, as fermentation also occurs in the presence of oxygen (including aerobic and microaerobic conditions). Fermentation processes can include, but are not limited to, fermentation processes used to produce products including alcohols (e.g., arabinitol, butanol, ethanol, glycerol, methanol, 1 ,3 -propanediol, sorbitol, and xylitol); organic acids (e.g. , acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5- diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid, and xylonic acid); ketones (e.g., acetone); amino acids (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); and gases (e.g., methane, hydrogen (H₂), carbon dioxide (C0₂), and carbon monoxide (CO)).

[0034] The term "under fermentation conditions" and variants thereof include reaction conditions wherein fermentation occurs. In one embodiment, "under fermentation conditions" can refer to reaction conditions comprising utilization of carbohydrates by microorganisms (e.g. , bacteria) involving an oxidation-reduction metabolic process that takes place under anaerobic conditions and in which an organic substrate serves as the final hydrogen acceptor (i.e. , rather than oxygen). Although "under fermentation conditions" can refer to anaerobic conditions, it is not intended that the term be solely limited to strict anaerobic conditions, as fermentation also occurs in the presence of oxygen (including aerobic and microaerobic conditions). Additionally, "under fermentation conditions" can include fermentation processes used to produce fermentation products including alcohols (e.g., arabinitol, butanol, ethanol, glycerol, methanol, 1 ,3 -propanediol, sorbitol, and xylitol); organic acids (e.g. , acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5- diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid, and xylonic acid); ketones (e.g. , acetone); amino acids (e.g. , aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); and gases (e.g., methane, hydrogen (H₂), carbon dioxide (C0₂), and carbon monoxide (CO)). [0035] The term "isolated" and variants thereof with respect to bacterial strains include a substantially pure bacterial strain that is free, or "substantially free" from other contaminating bacterial strains, as well as from its natural environment such as soil. The terms "isolated" and "purified" and variants thereof include situations wherein a nucleic acid, amino acid, or bacteria is removed from at least one component with which it is naturally associated. [0036] The term "Kluyvera cryocrescen^ refers to the novel bacterial strain isolated by the methods of the present invention and described herein and assigned ATCC Deposit

Designation: PTA-10600. The isolated strain of the present invention is 99.3% identical, based on 16S rRNA gene sequencing, to the closest known strain of Kluyvera cryocrescens.

[0037] The terms "ethanol" or "EtOH" and variants thereof include the chemical compound CH3CH2OH. Ethanol also refers to ethyl alcohol, pure alcohol, grain alcohol, drinking alcohol, and fuel alcohol. [0038] The term "ethanol production rate" and variants thereof include the rate at which a microorganism produces alcohol. The ethanol production rate is expressed for example in grams of ethanol produced per liter of culture per hour.

[0039] The term "ethanol molar yield" and variants thereof include the molar amount of ethanol produced per mole of carbon feedstock initially added to the reaction mixture and is typically, but not required to be, expressed as a percentage.

[0040] The term "carbon feedstock" and variants thereof include any carbon containing substance which an isolated bacteria of the presence invention can utilize for ethanol production. Examples of carbon feedstocks include, but are not limited to, glycerol

(including crude and pure), C₃-C₈ monosaccharides, disaccharides, sugar alcohols, sorghum, pearl millet, sugar cane, and sugar beets. In some embodiments, the monosaccharides are C₆ monosaccharides. In some embodiments, the C₅ monosaccharides (pentoses) are selected from xylose, arabinose, lyxose, ribose, ribulose, and xylulose. In some embodiments, the monosaccharides are C₆ monosaccharides (hexoses) which are selected from glucose, allose, altrose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose, and tagatose. In some embodiments, the disaccharide (which includes any compound that comprises two covalently linked monosaccharide units) includes, but is not limited to, sucrose, lactose, cellobiose and maltose. In some other embodiments, the sugar alcohol is selected from sorbitol and mannitol. In still other embodiments, the carbon feedstock is pure glycerol. In some preferred embodiments, the carbon feedstock is crude glycerol. In more preferred embodiments, the carbon feedstock is crude glycerol obtained as a by-product from the production of biodiesel.

[0041] The term "glycerol," "glycerin" or "glycerine" and variants thereof include a chemical compound with the general formula C₃H₈0₃, or alternatively

HOCH₂CH(OH)CH₂OH. Glycerol is colorless, odorless, viscous liquid and is widely used in pharmaceutical formulations. Glycerol is also a 10% by-product of biodiesel products. Glycerol can also be generated from the transesterification of plant oils. Glycerol can additionally be obtained from yeast based ethanol fermentations.

[0042] The term "final concentration" and variants thereof include determining the grams of metabolite per liter of the final reaction mixture at the end of the reaction incubation period. In some embodiments, the metabolite is ethanol. [0043] The term "nutrient composition" and variants thereof include yeast extracts, polypeptone, tryptone and corn steep liquor, as well as other nutrient compositions useful for the progression of reaction mixtures.

[0044] The term "buffer" and variants thereof include any solutions included in the reaction mixtures of the present invention. Buffers can include salt solutions, water-based solutions, and ionic solutions.

[0045] The term "reaction mixture" and variants thereof include any solutions of the present invention used for the production of ethanol. In some embodiments reaction mixtures can include carbon feedstocks, nutrient compositions, and buffers, as well as other components useful in the production of ethanol. In some embodiments, the reaction mixtures are aerobic. In some other embodiments, the reaction mixtures are anaerobic.

[0046] The term "culturing" refers to fermentative bioconversion of a carbon substrate to the desired end-product within a reactor vessel. In particularly preferred embodiments, culturing involves the growth of microorganisms under suitable conditions for the production of the desired end-product(s) (e.g. , ethanol).

[0047] The term "aerobic" and variants thereof include processes for producing ethanol wherein the production occurs in presence of oxygen. The term aerobic can further encompass oxygen-limited or microaerobic conditions, wherein the amount of oxygen is in limited supply. In some embodiments, the reaction mixtures of the present invention contain oxygen. In some embodiments, the production of ethanol occurs in the presence of oxygen.

[0048] The term "anaerobic" and variants thereof include a process for producing ethanol wherein the process occurs in the absence of oxygen. To maintain anaerobic environments, enclosed reaction chambers can be used and supplemented with oxygen free gases, such as C0₂ or N₂. These methods are well-known in the art and any known method for maintaining an anaerobic environment can be employed. In some embodiments, the reaction mixtures of the present invention are prepared in the absence of oxygen. In some embodiments, production of ethanol occurs in the absence of oxygen. In some embodiments, C0₂, N₂ and mixtures thereof are utilized to maintain the anaerobic environment. In some embodiments the term "anaerobic" indicates that the level of oxygen is below the level of detection. [0049] The term "constant temperature" and variants thereof describe a process of producing ethanol wherein the process occurs at steady temperature. In some embodiments, the reaction mixtures of the present invention are incubated at a specific temperature during the ethanol production period. In some embodiments, the reaction mixtures of the present invention are incubated at about 30°C during the ethanol production period.

[0050] The term "oxygen source" and variants thereof can include air and pure oxygen gas, as well as other gas mixtures that comprise oxygen as a component. In some embodiments, the oxygen source is under pressure or in a pressurized container.

[0051] The term "air" and variants thereof can include standard breathing air, as well as other compositions that comprise oxygen. In some embodiments, the air is under pressure or in a pressurized container.

[0052] The term "co-fermentation" and variants thereof include incubation of yeast cultures with an isolated Kluyvera cryocrescens bacterial strain. Co-fermentation can also include inoculation of a yeast mixture with an isolated Kluyvera cryocrescens bacterial strain of the present invention.

[0053] The term "yeast capable of alcohol production" can include any variety of yeast capable of ethanol production. Yeast capable of ethanol production can include, for example, Saccharomyces pastorianus and Saccharomyces cerevisiae.

[0054] The term "fuel" and variants thereof include gasoline, diesel, biodiesel and other fuel sources.

[0055] The term "bio fuel" and variants thereof include gasoline admixed with ethanol, biodiesel, or bioethanol, as well as other liquid fuels derived from plant materials to which ethanol can be added.

II. Bacterial Isolation

[0056] The present invention relates in part to microbial processes for the preparation of ethanol using crude glycerol originated from, for example, the biodiesel industry. The present invention further relates to processing of crude glycerol by an isolated bacterial strain. The present invention additionally provides for isolation of a superior biocatalyst bacterial strain of Kluyvera cryocrescens. This strain was isolated from nature and examined for its ability to undergo microbial fermentation of crude glycerol in order to produce ethanol with high productivity. The bacteria is extremely useful for improving conventional bioethanol processes due to its exceptional ability to synthesize ethanol from glycerol, as well as other carbon feedstocks. In the methods of the present invention, the isolated Kluyvera cryocrescens bacterial strain is a wild-type bacterial strain capable of producing ethanol from glycerol, such as crude glycerol without the need for costly media or expensive antibiotics for inducing ethanol production.

[0057) Typically, bacteria can be readily isolated and these methods are known to those of skill in the art. Such methods for isolation have been well described (see, e.g., Huntley, Ed., Current Protocols in Microbiology (2009), incorporated herein by reference for all purposes in its entirety). Samples from which bacteria can be isolated can include, but are not limited to, soil and sludge, as well as other environmental samples.

III. Fermentation Processes

[0058] Bacterial fermentation processes have been described for a variety of

microorganisms utilizing a wide-range of substrates. Furthermore, glycerol has been utilized as carbon source for many microorganisms, particularly for anaerobes. As shown in FIG. 1 , under anaerobic conditions, glycerol can be processed by a variety of pathways and can be converted into many metabolites, including ethanol.

[0059] Any methods for fermentation and apparatuses used in fermentation that are known to one of skill in the art can be configured to operate in accordance with the present invention. Generally, fermentation conditions are selected that provide an optimal pH and temperature for promoting the best growth kinetics of the bacteria and the optimal catalytic conditions for enzymes produced by the bacteria. The fermentation reaction conditions may include parameters such as pH, temperature, buffer solutions (including for example, ion concentrations), salt concentrations, and the like, can be optimized by one skilled in the art using routine methods in order to adapt well-known fermentation conditions for use with the methods of the present invention.

[0060] For the methods of present invention, any standard fermentation reaction conditions as well as fermentors or bioreactors can be employed. Large scale fermentors, such as pilot plant fermentors, as well as bench-top models are suitable for use in the present invention. The fermentation reactions of the present invention can be anaerobic reactions or aerobic reactions. Fermentation reactions can be carried out in any suitable bioreactor, such as for example, a stirred tank reactor (continuous stirred tank reactor; CSTR) or a trickle bed reactor (TBR). Fermentation methods can also include, but are not limited to, solid state

fermentation, batch fermentation, continuous fermentation, batch-stirred reactor, continuous flow-stirred reactor, continuous flow-stirred reactor with ultrafiltration, continuous plug-flow column reactor, stirred tank reactors, single-stage air lift reactor, multistage air lift reactor, a dry tube type fermentor, a packing type fermentor, or a fluidized bed reactor.

[0061] For additional references regarding fermentation methods and apparatuses, see, e.g., Biotechnol. Bioeng. 22(9): 1907-1928 (2004); U.S. Patent No. 4,286,066; U.S. Pat. No.

6,664,095; WO 2010/0101 16; U.S. Patent No. 4,286,066; U.S. Patent No 5,925,563; WO 2009/043012; U.S. Patent No. 5,925,563; Cysewski, G. R. and Wilke, C. R., Fermentation kinetics and process economics for the production of ethanol, Lawrence Berkeley Lab Technical Report, LBL-4480, March 1976; Biotechnol. Bioeng. 20(9): 1421-1444 (1978); U.S. Publication No. 2009/0082600; WO 2008/021 141 ; Biotechnol. Progress, 9:5323-538 (1993); U.S. Pat. No. 5,487,989; U.S. Pat. No. 5,554,520; J. Bacteriol., 162(l ):344-352

(1985); U.S. Publication No. 2009/0286293; US. Pat. No. 5,424,202; U.S. Pat. No. 5,916,78; U.S. Publication No. 2006/0051848; Enz. Microb. Technoi, 7: 346-352 (1985); Biotechnol. Bioeng., 25: 53-65 (1983); Biochem. Biotechnol, 56: 141 -153 (1996); Biotechnology and Bioengineering, 16(1 1 ): 1471-1493 (1974); Biotechnology and Bioengineering,

18(9): 1315-1323 (1976); / /. Eng. Chem., 28, 430-433 (1936); Arch. Microbiol, 1 14: 1 -7 (1977); Biotechnol. Bioeng. Symp., 8: 103-1 14 (1978); Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212 (1996); Biotechnol Prog., 15:817- 827 (1996); Microbiol. Mol. Biol. Reviews, 66: 506-577 (2002); WO 2007/005646; and WO 2008/072184. All of the foregoing are incorporated herein by reference in their entirety.

[0062] In some embodiments, the present invention is practiced using batch processes, while in other embodiments, fed-batch or continuous processes, as well as any other suitable modes of fermentation are used. Additionally, in some other embodiments, cells are immobilized on a substrate as whole-cell catalysts and are subjected to fermentation conditions for the appropriate end-product production.

[0063] In addition, in some preferred embodiments of the invention, the bioreactor can comprise a first growth reactor in which the microorganisms are cultured, and a second fermentation reactor to which broth from the first growth reactor is fed and wherien most of the fermentation product (ethanol, for example) is produced. The fermentation will result in a fermentation broth comprising a desirable product (such as ethanol) and/or one or more byproducts as well as bacterial cells, in a nutrient medium.

[0064] In a preferred embodiment of the present invention, fermentation reaction conditions comprise an isolated Kluyvera cryocrescens of the present invention, a carbon feedstock, a culturing medium (nutrient composition) and optionally a buffer. Under anaerobic conditions, the reaction mixture further comprises the use of an inert gas such as C0₂, N₂, or mixtures thereof to maintain the anaerobic environment of the reaction. Those of skill in the art will know of other inert gases suitable for use in the present invention. Under aerobic conditions, the reaction mixture further comprises oxygen. (See, e.g., Appl.

Microbiol. Biotechnol. 63:258 -266 (2003); J. of General Microbiology, 139: 1033- 1040 (1993); as well as methods disclosed in U.S. Publication No. 2009/0082600 all of which are herein incorporated by reference in their entirety.)

[0065] The fermentation should desirably be carried out under appropriate conditions for the substrate-to-ethanol fermentation to occur. In certain instances, reaction conditions that may optionally be considered include temperature, media flow rate, pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum substrate concentrations and rates of introduction of the substrate to the bioreactor to ensure that substrate level does not become limiting, and maximum product concentrations to avoid product inhibition.

[0066] In some embodiments, the reaction mixture comprises a carbon feedstock, a nutrient composition, a buffer and a gas. In further embodiments the gas is oxygen and the reaction mixture is an aerobic reaction mixture. In yet other embodiments, the gas is C0₂, N₂ or a mixture thereof and the reaction mixture is an anaerobic reaction mixture. [0067] In other embodiments, the fermentation media or culturing media optionally contains suitable nitrogen source(s), minerals, salts, co factors, buffers and other components suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for the production of the desired end-product {e.g., ethanol). In some embodiments, salts and/or vitamin or precursors thereof find use in the methods of the present invention. [0068] In certain aspects, the culture medium can optionally comprise one or more of the following components: ₂HP0₄, NaH₂P0₄, EDTA, ZnSCv7H₂0, CaCl₂ ^»2H₂0,

FeSCv7H₂0, Na₂Mo0₄'2H₂0, CuS0₄ ^»5H₂0, CoCl₂-6H₂0, MnCl₂ ^«4H₂0, MgCl₂*6H₂0, and (NH₄)₂S0₄. In some embodiments, the ₂HP0₄ concentration is about 1 to 2 g/L. In some embodiments, the NaH₂P0₄ concentration is about 0.1 to 1 g/L. In other embodiments, the EDTA concentration is about 5 to 20 mg/L. In still some other embodiments, the

ZnS0₄*7H₂0 concentration is about 1 to 10 mg/L. In another embodiment, the CaCl₂ ^»2H₂0 concentration is about 1 to 10 mg/L. In other embodiments, the FeSC I- O concentration is about 1 to 10 mg/L. In some embodiments, the Na₂Mo0₄ ^»2H₂0 concentration is about 0.1 to 0.3 mg/L. In some other embodiments, the CuS0₄*5H₂0 concentration is about 0.1 to 0.3 mg/L. In still some embodiments, the CoCl₂*6H₂0 concentration is about 0.1 to 0.5 mg/L. In some embodiments, the MnCl₂ ^»4H₂0 concentration is about 1 to 10 mg/L. In yet some other embodiments, the MgCl₂ ^»6H₂0 concentration is about 0.01 to 0.2 g/L. In yet further embodiments, the (NH₄)₂S0₄ concentration is 0.5 to 4 g/L. In some preferred embodiments, the buffer comprises the following component concentrations: EDTA (10 mg/L),

ZnSC H₂0 (2 mg/L), CaCl₂ ^»2H₂0 (1 mg/L), FeS0₄ ^«7H₂0 ( 5 mg/L), Na₂Mo0₄'2H₂0 (0.2 mg/L), CuSCV5H₂0 (0.2 mg/L), CoCl₂ ^»6H₂0 (0.4 mg/L), MnCl₂ ^»4H₂0 (1 mg/L),

MgCl₂'6H₂0 (0.1 g/L), and (NH₄)₂S0₄ (2 g/L). [0069] Preferred growth media utilized in the present invention include common commercially prepared media. Other defined or synthetic growth media can also be used, as appropriate. Appropriate culture conditions are well-known to those skilled in the art.

[0070] Fermentation reactions typically occur at a constant temperature that allows for bacterial growth and nutrient processing. In some preferred embodiments, incubation of the isolated Kluyn>era cryocrescens bacterial strain reaction mixture at 30°C allows for ethanol production in excellent yield. In some alternate embodiments, the temperature is between 15°C and 55°C. In some other embodiments, the temperature is between 20°C and 40°C, such as between 25°C and 35°C. In still some other embodiments, the temperature is between 28°C and 32°C, such as about 30°C. IV. Feedstock Sources and Ethanol Production

[0071] Moreover, the isolated Kluyvera cryocrescens bacterial strain provides a wild-type strain for use in ethanol production from a wide-variety of carbon feedstocks. The isolated bacterial strain of the present invention is found to uptake and process a broad range of carbon sources in addition to glycerol, including, but not limited to, glycerol (pure or crude), a C₃-C₈ monosaccharide, a disaccharide, and a sugar alcohol. In some embodiments, the monosaccharides are C₆ monosaccharides. This broad substrate range allows the bacterial strain of the present invention to employ a variety, as well as combinations, of carbohydrates as carbon feedstocks for the production of ethanol. In a preferred aspect, the isolated Kluyvera cryocrescens bacterial strain of the present invention generates ethanol from pure and/or crude glycerol, as well as other carbon feedstocks. (See, for example, Example 5 and Table 4.) A. High level of ethanol production from pure or crude glycerol

[0072] There have been prior attempts to produce ethanol from glycerol using microbial fermentation. However, in most cases pure glycerol has been utilized as the preferred substrate due to the toxicity of crude glycerol towards microbial strains (Enzyme Microb. Technol. , 4:349 ( 1982); Biotechnol. Bioeng. , 94:821 (2006); Appl. Environ. Microbiol. ,

74: 1 124, (2008); WO 2007/1 15228; Biotechnol. Bioeng. , 103: 148 (2009); and Appl Environ Microbiol, 75: 18 (2009)). Advantageously, the isolated Kluyvera cryocrescens bacterial strain of the present invention is capable of producing ethanol from either pure glycerol or even crude glycerol as the sole carbon feedstock. [0073] Kluyvera cryocrescens is a wild-type strain, isolated from nature and is cultivated more efficiently in large volumes when compared to recombinant E. coli, which requires expensive antibiotics and inducers to keep the recombinant E. coli active for ethanol production. In one aspect of the present invention, the isolated wild-type bacteria, Kluyvera cryocrescens produces ethanol with a production rate of at least 0.61 g/L/h (designating total amount of ethanol in grams for a given solution, divided by the total volume in liters of the ethanol producing reaction, and then divided by the time in hours for which the ethanol producing reaction mixture was incubated, as further described below). Further, during ethanol production there was no detectable amount of 1 ,3-propanediol production, a main byproduct commonly found in glycerol fermentation. Ethanol production from crude glycerol, discharged from a biodiesel plant, occurs using either aerobic or anaerobic reaction conditions using the isolated bacteria of the present invention. In one aspect, using in-batch fermentation under aerobic conditions and minimal medium, glycerol, 75 g/L, was completely consumed at 44 hours, leading to 27 g/L of ethanol with a molar yield of 80%.

[0074] In a preferred aspect, the carbon feedstock is glycerol. Glycerol is colorless, odorless, viscous liquid and is widely used in pharmaceutical formulations. It can be obtained from a wide- variety of sources and in a wide-range of purities. Current biodiesel supplies are often made from triglycerides or fats, found in vegetable oils. These processes often generate crude glycerol with a glycerol content from about 60 to 80% (w/w). This low- grade glycerol may also contain water, salts, other organic materials including residual methanol and free fatty acids. The composition of non-glycerol components of crude- glycerol can vary widely depending on the feedstocks used for the biodiesel process, such as rapeseed, canola, palm and soybean. [0075] The methods of the present invention can employ a glycerol composition, wherein the glycerol composition is any solution or substrate comprising glycerol. The glycerol composition can be a by-product from the manufacture of biodiesel. In certain aspects, the isolated bacterial strain produces ethanol from crude glycerol obtained directly from a biodiesel plant with a high production rate. In other embodiments, crude glycerol can be processed and impurities can be removed by conventional separation techniques to provide a higher concentration of glycerol in the by-product, prior to employing the biodiesel byproduct as a glycerol composition for the production of ethanol from the isolated Kluyvera cryocrescens bacterial strain of the present invention. [0076] The conditions for ethanol production using the isolated bacterial strain of the present invention can be carried out using known microbial fermentation procedures.

Bacterial production of ethanol from glycerol can be performed using a variety of fermentation methods, as described above, and any method for fermentation known to one of skill in the art. In certain aspects, one of skill in the art will recognize that routine experimentation can be used to adapt prior methods for use with the methods of the present invention. (See, e.g., WO 2007/1 15228 and WO 2008/006037.)

[0077] A glycerol composition can be a solution comprising glycerol, wherein the solution is not 100% pure glycerol. A crude glycerol composition can be a composition having a glycerol purity of less than 100% relative to a contaminating component. A glycerol composition can have one or more components that are not glycerol {i.e. , one or more contaminating components). A glycerol composition having 98% - 99% glycerol can be referred to as technical grade glycerol. In certain instances, biodiesel supplies generate crude glycerol with a glycerol content from 60% to 80%o (w/w). This low-grade glycerol also contains water, salts, and other organic materials. [0078] In certain embodiments of the present invention, the glycerol-containing feedstock can contain any level of glycerol in combination with one or more additional substances or contaminants. In one preferred embodiment, the glycerol feedstock contains for example, from approximately 10% to approximately 80% glycerol (weight per volume of by-product). Suitable glycerol feedstocks include, but are not limited to, by-products of palm kernel oil production, by-products of sunflower biodiesel production and/or by-products from biodiesel production from fat tallow. In preferred embodiments, the glycerol-containing by-product contains at least approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more glycerol (weight per volume of by-product). In a preferred aspect, the glycerol feedstock is about 60% to about 80% glycerol, such as 60%, 65%, 70%, 75% or 80% glycerol.

[0079] In some further embodiments, the crude glycerol solution comprises 60-95% glycerol, 1 -10% non-glycerol organics, 1 -2% methanol, 1 -10% salts and 5-20% water. In still further embodiments, the crude glycerol solution comprises 80-85% glycerol, 2% non- glycerol organics, 0.5% methanol, 5-6% salts and 10% water. In some preferred

embodiments, the crude glycerol solution comprises about 80-85% glycerol, about 2% non- glycerol organics, about 0.5% methanol, about 5-6% salts and 10% water.

Ethanol production conditions for glycerol [0080] In the present invention, the isolated bacterial strain can produce ethanol from crude glycerol with a production rate higher than that previously reported in the literature under aerobic conditions, such as for example, the engineered E. coli strain which produced 21 g L of ethanol from 60 g L of pure glycerol with the volumetric productivity of 0.216 g/L/h under microaerobic conditions (Biotechnol. Bioeng., 103:148 (2009)). In further embodiments, the bacterial strain of the present invention exhibits high ethanol productivity and high ethanol yield during production of ethanol from crude glycerol.

[0081] In some embodiments, the reaction mixture for ethanol production from glycerol comprises glycerol, a nutrient composition, a buffer and a gas. In other embodiments, the gas is CO2, N₂, other inert gases, or gases not containing oxygen or a mixture thereof and the reaction mixture is an anaerobic reaction mixture. In further embodiments the gas is oxygen and the reaction mixture is an aerobic reaction mixture.

[0082] In some embodiments of the methods of the present invention, the reaction mixture for production of ethanol from crude glycerol comprises 50 g L crude glycerol (glycerol content, 80% (w/v)), 2 g/L yeast extract, 1.55 g/L K₂HP0₄, 0.85 g/L NaH₂P0₄, a trace element solution. In some embodiments, the stock trace element solution comprises EDTA ( 1.0 g/L), ZnSCV7H₂0 (0.2 g/L), CaCl₂ ^«2H₂0 (0.1 g/L), FeS(V7H₂0 (0.5 g/L),

Na₂MoCv2H₂0 (20 mg/L), CuSCv5H₂0 (20 mg/L), CoCl₂'6H₂0 (40 mg/L), MnCl₂*4H₂0 (0.1 g/L), MgCl₂'6H₂0 (10 g/L), and (NH₄)₂S0₄ (200 g/L). Upon mixing with the other reaction components, in one embodiment the final trace element concentrations in the reaction mixture comprise EDTA (10 mg/L), ZnS0₄ ^»7H₂0 (2 mg/L), CaCl₂ ^»2H₂0 (1 mg/L), FeSCV7H₂0 (5 mg/L), Na₂Mo0₄ ^»2H₂0 (0.2 mg/L), CuS0₄ ^»5H₂0 (0.2 mg/L), CoCl₂ ^«6H₂0 (0.4 mg/L), MnCl₂ ^«4H₂0 ( 1 mg/L), MgCl₂ ^«6H₂0 (0.1 g/L), and (NH₄)₂S0₄ (2 g/L). [0083] In the methods of the present invention, the isolated Kluyvera cryocrescens bacterial strain produces ethanol with a very high molar yield of ethanol. In some embodiments, the ethanol molar yield produced by the isolated bacterial strain is greater than or equal to 50%, such as in the range from 50% to 100%. [0084] In some embodiments, the crude glycerol solution comprises 70-95% glycerol, 1 - 10% non-glycerol organics, 1 -2% methanol, 1 -10% salts and 5-20% water. In some preferred embodiments, the crude glycerol solution comprises 80-85% glycerol, 2% non- glycerol organics, 0.5% methanol, 5-6% salts and 10% water.

[0085] In certain instances, the reaction conditions have a temperature of about 15°C to about 55°C. In some embodiments, the reaction conditions have a temperature of about 20°C to about 40°C. In some preferred embodiments, the reaction conditions have a temperature of about 25°C to about 35°C. In still some other embodiments, the temperature is between 28°C and 32°C, such as about 30°C. Preferably, the methods take place at atmospheric pressure, although higher pressures are suitable. [0086] In operation, if the pH of the broth was adjusted during recovery of ethanol and/or by-products, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor. Suitable pH ranges for fermentation are between pH 4.5 to pH 9.0. In certain instances, the pH of the reaction is between pH 6 and pH 8, more preferably between pH 6.5 and pH 7.8, and most preferably between pH 6.8 and pH 7.5 such as 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, or 7.5.

[0087] In some aspects, the method of producing ethanol from glycerol using the isolated Kluyvera cryocrescens is performed under anaerobic conditions. In some embodiments, the method of producing ethanol from glycerol using the isolated Kluyvera cryocrescens is performed under aerobic conditions. a) Anaerobic ethanol production

[0088] The isolated Kluyvera cryocrescens of the present invention is capable of metabolizing glycerol to produce ethanol under anaerobic conditions, i.e. in the absence of oxygen (see, for example, Example 7 and Table 6). In the absence of oxygen, the presence of increasing amounts of yeast extract (such as for example increasing from 2 g/L to 10 g L of yeast extract in the reaction mixture) can facilitate ethanol production. (0089] In some embodiments of the present invention, the fermentation can be anaerobic fermentation. Any method of anaerobic fermentation known to those of skill in the art can be employed in the methods of the present invention. For small scale fermentations, reactions can occur by standing still or shaking in a culture vessel that is sealed with a suitable stopper. For larger scale, a fermentor apparatus, such as one of those referenced above or any other known to one of skill in the art, can be employed where it is possible to make an anaerobic environment by replacing oxygen in the apparatus with an inert gas, such as C0₂, N₂, mixtures thereof, or any other suitable inert gas.

[0090] In the present invention, the isolated Kluyvera cryocrescens bacterial strain produced ethanol from glycerol in the absence of oxygen with an ethanol production rate greater than 0.076 g/L/h. In still other embodiments, the ethanol production rate is in the range between 0.076 g/L/h to 0.523 g/L/h. In some other embodiments, the ethanol production rate is in the range between 0.1 g/L/h to 0.5 g/L/h, such as between 0.2 g/L/h to 0.3 g/L/h. In some preferred embodiments, the ethanol production rate is 0.076 g/L/h. In yet another preferred embodiment, the ethanol production rate is 0.523 g/L/h.

[0091] In some embodiments, the isolated Kluyvera cryocrescens bacterial strain produced 23 g/L of ethanol at a rate of 0.523g/L/h under anaerobic conditions in the presence of 1 Og/L yeast extract and 50 g/L crude glycerol. In some embodiments, the isolated Kluyvera cryocrescens bacterial strain produced 14.4 g/L of ethanol at a rate of 0.076 g/L/h in the presence of 2 g/L yeast extract and 50 g/L crude glycerol.

[0092] In some embodiments, anaerobic conditions include conditions where there is significantly less oxygen than the level that is present in the aerobic environment. In some embodiments, the anaerobic environment is substantially free of oxygen (e.g., the oxygen level is below detection). In some embodiments, C0₂, N₂, inert gases, other gases not containing oxygen, or a mixtures thereof is employed to maintain the anaerobic environment. b) Aerobic ethanol production

[0093] The isolated Kluyvera cryocrescens of the present invention is capable of metabolizing glycerol to produce ethanol under aerobic conditions, i.e. in the presence of oxygen (see, for example, Example 7 and Table 6). The use of oxygen facilitates glycerol metabolism by generating more energy (ATP) via the TCA cycle, resulting in an enhanced ethanol production rate. Advantageously, reaction conditions using oxygen can also eliminate the need for the use of expensive nutritional components in culture medium or expensive apparatuses for maintaining an anaerobic environment.

[0094] In the present invention, the isolated Kluyvera cryocrescens bacterial strain produced ethanol from glycerol in the presence of oxygen with an ethanol production rate greater than 0.216 g/L/h. In some embodiments, the ethanol production rate is in the range between 0.216 g/L/h to 1.0 g/L/h. In some other embodiments, the ethanol production rate is in the range between 0.3 g/L/h to 0.9 g/L/h, such as between 0.4 g/L/h to 0.8 g/L/h, or between 0.5 g/L/h to 0.7 g/L/h. In still some other embodiments, the ethanol production rate is 0.42 g/L/h, or 0.52 g/L/h, or 0.55 g/L/h. In some other preferred embodiments, the ethanol production rate is 0.61 g/L/h. In some preferred embodiments, the ethanol production rate is 0.64 g/L/h. In yet another preferred embodiment, the ethanol production rate is 0.92 g/L/h.

[0095] In some preferred embodiments, the isolated Kluyvera cryocrescens bacterial strain produces ethanol with a very high molar yield of ethanol in the presence of oxygen. In additional preferred embodiments, the ethanol molar yield produced by the isolated bacterial strain in the presence of oxygen is greater than or equal to 60%, such as from 60% to 100%. In yet other preferred embodiments, the ethanol molar yield is in the range from 70% to 90% or more, or from 66% to 84%, or 66%, or 76%, or even 84%. In still other preferred embodiments, the ethanol molar yield is in the range from about 80% to 100% such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0096) In a preferred embodiment of the present invention, the isolated Kluyvera cryocrescens bacterial strain produced 27 g/L of ethanol from crude glycerol directly obtained from biodiesel plants with a high production rate of 0.61 g/L/h. In some other embodiments, the isolated Kluyvera cryocrescens bacterial strain produced 18.3 g/L of ethanol from crude glycerol with a production rate of 0.92 g/L/h. In still other embodiments, the isolated Kluyvera cryocrescens bacterial strain produced 9.1 g/L of ethanol from crude glycerol with a production rate of 0.89 g/L/h. In yet other embodiments, the isolated

Kluyvera cryocrescens bacterial strain produced 27 g/L of ethanol from crude glycerol with a production rate of 0.61 g/L/h. [0097] In one embodiment, the isolated Kluyvera cryocrescens bacterial strain produced at least 27 g/L of ethanol from crude glycerol directly obtained from biodiesel plants. In other embodiments, the isolated Kluyvera cryocrescens bacterial strain produced about 15.2 g/L of ethanol from a reaction mixture with an initial crude glycerol concentration of 100 g/L. In yet other embodiments, the isolated Kluyvera cryocrescens bacterial strain produced about 27 g/L of ethanol from a reaction mixture with an initial crude glycerol concentration of 75 g/L. In still other embodiments, the isolated Kluyvera cryocrescens bacterial strain produced 18.3 g/L of ethanol from a reaction mixture with an initial crude glycerol concentration of 50 g/L. In another embodiment, the isolated Kluyvera cryocrescens bacterial strain produced 9.1 g/L of ethanol from a reaction mixture with an initial crude glycerol concentration of 25 g/L.

[0098] In the present invention, the isolated Kluyvera cryocrescens bacterial strain produced 18.3 g/L of ethanol from glycerol under aerobic conditions from an initial crude glycerol concentration of 50 g/L. In some embodiments, the isolated Kluyvera cryocrescens bacterial strain produced 16.6 g/L of ethanol from a reaction mixture with an initial crude glycerol concentration of 50 g/L with 12.5% air. In still other embodiments, the isolated Kluyvera cryocrescens bacterial strain produced 14.7 g/1 of ethanol from a reaction mixture with an initial crude glycerol concentration of 50 g/L with 25% air. In still other

embodiments, the isolated Kluyvera cryocrescens bacterial strain produced 18.3 g/1 of ethanol from a reaction mixture with an initial crude glycerol concentration of 50 g/L with 50% air. In one embodiment, the isolated Kluyvera cryocrescens bacterial strain produced 10.4 g/L of ethanol from a reaction mixture with an initial crude glycerol concentration of 50 g/L with 75% air. In still other embodiments, the isolated Kluyvera cryocrescens bacterial strain produced 1 1 g/L of ethanol from a reaction mixture with an initial crude glycerol

concentration of 50 g/L with 100 % air.

[0099] In some embodiments, aerobic conditions include conditions where there is significantly more oxygen than the level that is present in the aerobic environment. In some embodiments the aerobic environment contains a detectable level of oxygen. In some embodiments, there is 40%, 50%, 60%, 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% oxygen present in the environment. In some other embodiments the aerobic conditions further comprise some level of nitrogen. In some further

embodiments, there is 40%, 50%, or 60% nitrogen present in the aerobic environment.

B. Ethanol production from other feedstocks

[0100] In addition to the processing of glycerol, the isolated Kluyvera cryocrescens bacterial strain of the present invention is further capable of processing a broad range of feedstocks (e.g., carbon feedstocks). The feedstocks of the present invention also include, but are not limited to, monosaccharides (e.g., glucose and fructose), disaccharides (e.g., lactose, sucrose and cellobiose), as well as mixtures thereof, and unpurified mixtures from renewable feedstocks such as sugar beet molasses, and barley malt. The ability of Kluyvera cryocrescens to process a broad range of substrates allows this strain to employ a variety of carbohydrates as feedstock to produce bioethanol. [0101] In some embodiments the feedstocks can include for example C₆ monosaccharides, C₅ monosaccharides, disaccharides, and sugar alcohols. In other embodiments, feedstocks include, but are not limited to, glucose, galactose, fructose, xylose, arabinose, lyxose, mannose, maltose, sucrose, cellobiose, sorbitol, mannitol and mixtures thereof (see, for instance, Example 5 and Table 4). [0102] In still other embodiments, the carbon feedstock is selected from glycerol, a C₃-C₈ monosaccharide (such as C₃, C₄, C5, C₆, C₇, Cg monosaccharides or mixtures thereof) a disaccharide, or a sugar alcohol. In some embodiments, the monosaccharides can be C₆ monosaccharides. In further embodiments, the C₆ monosaccharides are glucose, mannose, tagatose, galactose, fructose, or mannose. In some embodiments, the monosaccharides are C₅ monosaccharides. In still further embodiments, the C5 monosaccharides are xylose, arabinose, lyxose or ribose. In some further embodiments, the disaccharide is maltose. In another embodiment, the sugar alcohol is selected from the group of sorbitol and mannitol. In yet another embodiment, the feedstock is pure glycerol. In certain embodiments, a mixture of carbon feedstocks can be employed, such as a mixture of two or more carbon feedstocks. Ethanol production conditions for other carbon feedstocks

[0103] In some further embodiments, the reaction mixture used for ethanol production comprises an isolated Kluyvera cryocrescens, a carbon feedstock, optionally a nutrient composition and optionally a buffer. In some aspects, the method of producing ethanol using the isolated Kluyvera cryocrescens is performed under anaerobic conditions. In some embodiments, the method of producing ethanol using the isolated Kluyvera cryocrescens is performed under aerobic conditions. In further embodiments the gas is oxygen and the reaction mixture is an aerobic reaction mixture. In yet other embodiments the gas in C0₂, N₂ or a mixture thereof and the reaction mixture is an anaerobic reaction mixture.

[0104] In some embodiments of the methods of the present invention, the reaction mixture for production of ethanol comprises a carbon feedstock, yeast extract, 1.55 g/L K₂HP0₄, 0.85 g/L NaH₂P0₄, and a trace element solution. In some embodiments, the stock trace element solution comprises EDTA, ZnS0₄ ^«7H₂0, CaCl₂ ^»2H₂0, FeS0₄ ^»7H₂0, Na₂Mo0₄ ^«2H₂0, CuS0₄'5H₂0, CoCl₂ ^»6H₂0, MnCl₂'4H₂0, MgCl₂'6H₂0, and (NH₄)₂S0₄.

[0105] In certain instances, reaction conditions have a temperature of about 15°C to about 55°C. In some embodiments, the reaction conditions have a temperature of about 20°C to about 40°C. In some preferred embodiments, the reaction conditions have a temperature of about 25°C to about 35°C. In still some other embodiments, the temperature is between 28°C and 32°C, such as about 30°C. Preferably, the methods take place at atmospheric pressure, although higher pressures are suitable.

[0106] In operation, if the pH of the broth was adjusted during recovery of ethanol and/or by-products, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor. Suitable pH ranges for fermentation are between pH 4.5 to pH 9.0. In certain instances, the pH of the reaction is between pH 6 and pH 8 more preferably between pH 6.5 and pH 7.8, and most preferably between pH 6.8 and pH 7.5 such as 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, or 7.5. C. Measuring ethanol production rate

[0107] The calculation for measuring ethanol production rate is generally known and these methods can be employed. Ethanol production rates can be measured by any methods well- known to one of skill in the art. Generally, ethanol is measured as a metabolite as set forth in the example section. Once the total amount of ethanol is determined in grams for a given solution, this number is then divided by the total volume in liters of the ethanol producing reaction. This calculation provides the final concentration of ethanol in the reaction mixture. The concentration is then divided by the time in hours for which the ethanol producing reaction mixture has been incubated. This final number is then described as the ethanol production rate measured in grams/liter/hour (g/L/h). (See, e.g., Bioresource Technology, 98(3):677-685 (2007) and Applied Biochemistry and Bioprocesses, 78:373-388 (2007); incorporated herein by reference in their entirety).

D. Measuring ethanol molar yield

[0108] The calculation for measuring ethanol molar yield is generally well-known and any of these methods can be employed. Generally, ethanol is measured as a metabolite as described in the example section. Once the total amount of ethanol in the reaction mixture is determined in grams, grams are then converted to moles of ethanol produced by standard calculations. (See, e.g., Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., New York, 1982 and Ausubel, et al. Editor, Current Protocols in Molecular Biology, USA, 1984-2008; all of which are incorporated herein by reference in their entirety). Once the moles of ethanol produced are calculated, this number is divided by the number of moles of carbon feedstock used in the reaction. The final number is expressed as a percentage. More complex methods of calculating ethanol molar yield can also be employed, such as those described in San Martin, et al. (J. of General Microbiology,

139: 1033-1040 (1993); all of which are incorporated herein by reference in their entirety).

E. Co-fermentation of glucose and glycerol [0109] It is well-known to those of skill in the art that glycerol is synthesized during conventional yeast fermentation when the yeast are being utilized to produce ethanol. The main reason for glycerol formation is to maintain redox balance during fermentative assimilation of glucose to ethanol. The level of the glycerol by-product could be as high as 13g/L (140mM) in current bioethanol processes (J. Sci. Food Agric, 82:309 (2002)). [0110] Ethanol fermentation from a feedstock such as glucose can be facilitated when yeast cells are inoculated in combination with the isolated Kluyvera cryocrescens of the present invention as this bacteria is capable of metabolizing both glycerol and glucose to produce ethanol. As such, the methods of present invention find further use for improving ethanol yield in conventional bioethanol processes wherein the isolated Kluyvera cryocrescens of the present invention can utilize by-product glycerol formed in conventional yeast fermentation to produce ethanol. The isolated Kluyvera cryocrescens can convert the glycerol synthesized by yeast cells during glucose fermentation into ethanol. As a result of this synergistic co- fermentation, ethanol productivity can be remarkably improved.

[0111] Upon cultivation of the bacterial strain of the present invention with ethanol producing yeast, Kluyvera cryocrescens can convert the glycerol by-product, synthesized by the yeast cells during glucose fermentation, to ethanol. In some embodiments, the isolated Kluyvera cryocrescens can utilize by-product glycerol formed during conventional yeast fermentation and process the glycerol to make more ethanol, resulting in 6-7% increase in ethanol yield. [0112] Overall, this co-fermentation process additionally results in a two-fold enhancement in ethanol productivity. In some embodiments, the ethanol productivity is increased by greater than one-fold, or even increased by two-fold. [0113] In some embodiments, the methods for production of ethanol using isolated Kluyvera ctyocrescens can comprise co-fermentation with a yeast strain capable of ethanol production. In some embodiments, the co-fermentation method comprise the steps of (i) incubating a composition comprising a yeast strain capable of ethanol production with a carbon feedstock; and (ii) inoculating the yeast and feedstock composition from (i) with an isolated Kluyvera cryocrescens bacterial strain under fermentation conditions. In some embodiments, the isolated Kluyvera cryocrescens of the present invention and the yeast can be mixed with the carbon feedstock simultaneously. In other embodiments, the yeast and feedstock can be mixed prior to the addition of the isolated Kluyvera cryocrescens. In some other embodiments, the yeast and feedstock can be mixed and allowed to incubate and begin ethanol production prior to the addition of or inoculation with the isolated Kluyvera ctyocrescens bacterial strain of the present invention. In some other embodiments, the yeast and feedstock can be mixed and allowed to incubate and the yeast removed from the media prior to the addition of or inoculation with the isolated Kluyvera cryocrescens bacterial strain of the present invention. In another embodiment, the method of co-fermentation increases ethanol production when compared to fermentation resulting from incubating a composition comprising a yeast strain capable of ethanol production and a carbon feedstock in the absence of an isolated Kluyvera cryocrescens bacterial strain of the present invention. In some other embodiments, the co- ferment at ion method increases the ethanol production rate by greater than or equal to two-fold. (See Example 8 and FIG. 6.)

F. The genome of Kluyvera cryocrescens

[0114] The isolated Kluyvera cryocrescens of the present invention contains genetic material that allows for high ethanol productivity and high ethanol molar yield in both anaerobic and aerobic reaction environments. Genes involved in glycerol fermentation and ethanol production can include, for example, glycerol dehydrogenase; phosphoenylpyruvate (PEP)-dependent DHA kinases; NADH-linked 1 ,3-PDO dehydrogenase; anaerobic and aerobic glycerol-3-phosphate dehyrdogenases; and alcohol/acetaldehyde dehydrogenase, as well as others. (See, e.g., Appl. Micro. Biotech., 75(18):5871 -5883 (2009) and Virology Journal, 5: 122-127 (2008), incorporated herein by reference in their entirety.) [0115] The above genes, as well as others involved in bacterial ethanol production can be isolated from the Kluyvera cryocrescens of the present invention. Once isolated, genes can be cloned into vectors and inserted into other bacteria to allow for genetic engineering of additional strains of bacteria capable of high ethanol productivity and high ethanol molar yield in both anaerobic and aerobic reaction environments. Methods for isolation, cloning, and insertion of gene sequences into nucleic acid vectors are well-known in the art and standard methods can be employed. For exemplary techniques, see, Maniatis, et al.,

Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., New York, 1982; Innis, et al., Ed., PCR Protocols: A Guide to Methods and Applications, California, 1990; and Ausubel, et al. Editor, Current Protocols in Molecular Biology, USA, 1984-2008; all of which are incorporated herein by reference in their entirety.

[0116] A variety of bacterial vectors are commercially available and any suitable vector can be employed as a cloning vector. Further, methods for the use of vectors for bacterial cell transformation, as well as selectable markers for use in selection of transformed bacteria, are well-known in the art. For exemplary techniques, see, Mol. Gen. Genet., 216(1): 175-7 ( 1989); Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., New York, 1982; Ausubel, et al. Editor, Current Protocols in Molecular Biology, USA, 1984-2008; Sambrook, et al, Molecular Cloning, A Laboratory Manual (3rd ed. 2001 ); and Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); all of which are incorporated herein by reference in their entirety.

[0117] In some embodiments, the genes involved in glycerol fermentation and/or ethanol production from the isolated Kluyvera cryocrescens of the present invention can be isolated. In further embodiments, genes involved in glycerol fermentation and/or ethanol production can be cloned and inserted into a nucleic acid vector. In certain aspect, the present invention provides a vector comprising or consisting of a gene involved in glycerol fermentation and/or ethanol production from the isolated Kluyvera cryocrescens of the present invention or a fragment thereof. In other embodiments, one gene can be isolated from Kluyvera

cryocrescens. In still other embodiments, more than one gene can be isolated from Kluyvera cryocrescens. In another embodiment, one or more genes can be introduced into other bacterial strains. In a further embodiment, introduction of isolated genes into other bacterial strains can provide for high ethanol productivity and high ethanol molar yield in both anaerobic and aerobic reaction environments.

V. EXAMPLES Example 1 : Isolation of crude gl cerol-utilizing microorganisms

[0118] Soil samples were collected at various locations in Singapore. The surface soil was removed and the layer underneath was then collected. Water was added to the sample and the solution vortexed. The mixture was then allowed to settled for approximately 10 minutes. After settling, the liquid phase was inoculated onto glycerol-based agar plates as described in Table 1. Agar plates were incubated at room temperature for 3 days under anaerobic conditions provided by a commercially available anaerobic chamber (Anaerogen™ , Oxoid, UK).

Table 1

Medium composition for screening of glycerol-utilizing strains.

Component Cone.

Crude glycerol (glycerol content, 80% (w/v))) 25 g/L

Yeast extract 2.0 g/L

K₂HP0₄ 1.55 g/L

NaH₂P0₄ 0.85 g/L

Trace element solution¹ 10 ml/1

Agar 15 g L

'Trace element solution (EDTA 1.0 g L, ZnS0₄ ^»7H₂0 0.2 g/L, CaCl₂ ^»2H₂0 0.1 g/L, FeS0₄'7H₂0 0.5 g/L, Na₂Mo0₄ ^»2H₂0 20 mg/L, CuS0₄'5H₂0 20 mg/L, CoCl₂ ^«6H₂0 40 mg/L, MnCl₂'4H₂0 0.1 g/L, MgCl₂'6H₂0 10 g/L, (NH₄)₂S0₄ 200 g/L)

[0119] Crude glycerol was obtained from a palm oil-based biodiesel plant in Malaysia operated by Singaporean company. The composition of the crude biodiesel solution obtained is summarized in Table 2.

Table 2

The composition of crude glycerol

Component Cone % (w/v)

Glycerol 80-85

Non-glycerol organics 2

Methanol 0.5

Salts 5-6

Water 10 [0120] Strains isolated via agar plate culture were subsequently transferred to liquid culture with the same medium composition as described in Table 1 , minus the agar component, for measurement of growth and metabolite analysis. The liquid culture was incubated at 30°C for 3 days in a screw-capped bottle ( 100 ml size) with a gas inlet port and a gas outlet port, allowing for the continuous supply of either C0₂ or N₂ in order to maintain anaerobic conditions.

Example 2: Screening of ethanol producing microorganism

[0121] Ethanol can be purified using a variety of methods well-known to one of skill in the art. Ethanol or a mixed alcohol stream containing ethanol and one or more other alcohols, can be recovered from the fermentation broth by a variety of methods, such as fractional distillation or evaporation, pervaporation, extractive fermentation, in situ product removal, saline extractive distillation, separation using fatty acids and reactive distillation. For use in fuels, ethanol often undergoes dehydration processes to reduce the water content. For additional methods of ethanol purification, see the Handbook of Alternative Fuels (Lee, et al., CRC Press 2007), as well as Alcohol Production and Recovery (Maiorella, et al., Bioenergy 20:43-92 (1981); Rao et al., ("Continuous Biocatalytic Processes," Org. Process Res. Dev., 13(3):607-616 (2009)); Pinto et al. ("Saline extractive distillation process for ethanol purification," Computers & Chemical Engineering, 24(2-7): 1689- 1694 (2000); O'Brien et al. ("Ethanol production in a continuous ferment at ion/membrane pervaporation system," Appl. Micro. Biotech., 44(6):699-704 (1996); and Boudreau et al. ("Improved ethanol-water separation using fatty acids," Process Biochemistry, 41 (4): 980-983 (2006)); all of which are incorporated herein by reference in their entirety.

[0122] In the present invention, the liquid culture broth of crude glycerol-utilizing strains was prepared for HPLC analysis in order to quantify the metabolites generated by the culture during the incubation period. Metabolites analyzed include glycerol, ethanol, and other organic acids (as shown in FIG. 3). The culture was first centrifuged for 10 minutes at 10,000 x g and 4°C to remove cells and the supernatant was analyzed by HPLC. HPLC was performed using a Shimadzu Prominence system (Shimadzu Scientific Instruments,

Columbia, MD) equipped with UV and refractive index detector. Samples were injected (ly L) with 30mM aqueous sulfuric acid solution as mobile phase at the rate of 0.6 mL/min under oven temperature of 42°C. Metabolites were analyzed by using a Aminex® HPX-87H column (7.8mm x 30cm, BIO-RAD). The commercially available glycerol, ethanol, and other organic acids were employed as a standard for quantitative analysis. As shown in FIG. 3, the mixture of thirteen standards was injected under this condition and each of the metabolites was successfully analyzed.

[0123] Among about six hundred soil samples tested, 57 microbial strains were isolated based on their ability to grow on crude glycerol as the sole carbon source. Through metabolite analysis of these candidates by HPLC, 9 strains were found to produce ethanol as major metabolite under anaerobic condition (as shown in Table 3). Strain S26 was finally selected for further investigation based on its high yield of ethanol formation.

Table 3

Selected strain for ethanol production from crude glycerol.

13PDO EtOH

Strain DCW¹ Yield²

(mM) (mM)

S26 1.2 0 86.6 94

S29 1.1 0 73 85

S39 1.4 1.53 105 72

S257 1.6 0 91.6 49

S280 1.2 0.425 86.3 53

S344 1.1 0 65.4 70

S362 1.3 0 87.5 79

S233 1.1 0 90 65

S76 1.2 0 63 60

DCW : dry cell weight (mg) from 50ml liquid culture

2Yield : ethanol formed (mole)/glycerol consumed (mole)

Example 3: Metabolite Screening and Purification

[0124] Bacterial strains can be screened for end products (metabolites), such as ethanol, using a variety of methods. Ethanol production as well as screening for other metabolites can be analyzed and monitored as described below.

[0125] Methods for analysis of metabolites from bacterial strains can include for example, high-pressure liquid chromatography (HPLC), mass spectrometry (MS), liquid

chromatography-mass spectrometry (LC-MS), gas chromatography, real-time

metabolotyping, as well as others (see, e.g., "Detection of Metabolites Using High

Performance Liquid Chromatography and Mass Spectrometry", Current Protocols in Toxicology (2000); J. Microbiol. Biotechnol. \ 9{\ ) 5 \ -4 (2009); and Morgan, Ed., Current Protocols in Toxicology (2009); Spectroscopy 21 (6) (2006); Deng, et al., "GC rapid analysis of methanol and ethanol during high cell density culture of recombinant methylotrophic yeast" Industrial Microbiology (2001 ); all incorporated herein by reference in their entirety.) Additional methods of metabolite analysis using a method called real-time metabolotyping (RT-MT), which performs sequential Ή-NMR profiling and two-dimensional (2D) Ή, ^{l 3}C- HSQC (heteronuclear single quantum coherence) profiling during bacterial growth in an NMR tube (PLOS, 4(3):e4893 (2009)) are also suitable.

[0126] Computation methods can also be employed to optimize bioethanol purification processes. (See, e.g, Hoch and Espinosa, "Optimisation of a Bio-ethanol Purification Process Using Conceptual Design and Simulation Tools," 18th European Symposium on Computer Aided Process Engineering (2008); incorpo rated herein by reference in its entirety.)

[0127] Depending on the cell culture system employed, the end product can be purified from culture medium or a cell lysate by any method capable of separating the compound from one or more components of the host cell or culture medium. The compound or compounds can be separated from host cell and/or culture medium components that would interfere with the intended use of the compound. As a first step, the culture medium or cell lysate can be centrifuged or filtered to remove cellular debris. The supernatant can then be concentrated or diluted to a desired volume or diafiltered into a suitable buffer to condition the preparation for further purification. The end product can then be further purified using well-known techniques. The technique chosen will vary depending on the properties of the end product.

[0128] Additional end products that can be analyzed can include, propionic acid, ethanol, 1 ,3-propanediol (l ,3PDO), 1 ,2-propanediol, 3-hydroxypropionic acid, poly (3-hydroxy- butyrate), poly (3-mercapto-propionate), hydrogen, succinate, dihydroxyacetone, butyric acid, acetic acid, polyglutamic acid, cinnamic acid, rhamnolipids, 3-hydroxacetone, omega-3 polyunsaturated fatty acids, malate, oxaloacetate, fumarate, aconitate, citrate, citric acid (CA), glucose (Glu), pyruvic acid (PA), succinic acid (SA), lactic acid (LA), glycerol (Gly), formic acid (FA), acetic acid (AA), 1 ,2-propanediol (l ,2PDO), propionic acid (PA), ethanol (EtOH) butyric acid (BA), isocitrate, 2-ketoglutarate, glycerol-3- phosphate, pyruvate, L- lactate, D-lactate, and formate. In addition, amino acids, nucleobases, vitamins, antibiotics, and/or propylene glycol can also be produced as end products. See, e.g., U.S. Pat. No.

5,356,812; U.S. Pat. No. 5,008,473; and WO 2008/006037; all of which are incorporated herein by reference in their entirety. [0129| Ethanol can be removed by any means well-known to one of skill in the art.

Ethanol and by-products can be recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (e.g., by filtration), and recovering ethanol and optionally acid from the broth. Ethanol can be recovered for example by distillation, and acids may be recovered for example by adsorption on activated charcoal. The separated microbial cells are preferably returned to the fermentation bioreactor. The cell free permeate remaining after the alcohol(s) and acid(s) have been removed is also preferably returned to the fermentation bioreactor. Additional nutrients if any, can be added to the cell free permeate to replenish the nutrient medium before it is returned to the bioreactor. In some embodiments of the invention, ethanol is recovered from the fermentation reaction using extractive fermentation procedures in which ethanol is recovered into an oil phase in the reactor. A skilled person in the art will know of other methods to extract ethanol as well as the product. Example 4: Microbial identification

[0130] Genomic DNA extraction, PCR and sequencing of 16S rRNA gene were conducted by the general procedures described elsewhere. The 16S rRNA gene (SEQ ID NO: 1 ) was amplified from genomic DNA by PCR using the bacterial primers. The sequences of the primers used for amplification were 5 '-AGAGTTTGATCATGGCTCAG-3 ' (SEQ ID NO:2) and 5 '-AAGG AGGTGATCCAGCCGCA-3 ' (SEQ ID NO:3), corresponding to positions 8-27 and 1 ,544-1 ,525, respectively, of the Escherichia coli 16S rRNA sequence (E.

Stackebrandt and M. Goodfellow, Nucleic acid techniques in bacterial systematics, Wiley, New York, 1991 ; incorporated herein by reference in its entirety). The PCR product was purified using a Wizard PCR DNA purification system (Promega, US) and sequenced using an ABI PRISM 310 genetic analyzer (1st base Pte Ltd, Singapore). The 16S rRNA gene sequence (1 ,030 bases) was compared with the sequence data in GenBank database by using the BLAST algorithm ( arlin and Altschul, PNAS, 87:2264-2268( 1990)). The BLAST analysis for sequence similarity indicated that the closest relatives of the strain S26 were Kluyvera cryocrescens 169 (99.3%), Kluyvera ascorbata 4105 (99.2%), and Kluyvera cryocrescens WAB1904 (99.2%). The phylogenetic analysis was carried out by the neighbor-joining method using the PHYLIP program package, version 3.68 (Department of Genome Sciences and Department of Biology, University of Washington) (as shown in FIG. 2). On the basis of the sequence similarity as well as the phylogenetic analysis, it was concluded that isolated bacterial strain S26 belongs to Kluyvera cryocrescens. (See, e.g., Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., New York, 1982; and Innis, et al. Ed., PCR Protocols: A Guide to Methods and Applications, California, 1990; and Ausubel, et al. Editor, Current Protocols in Molecular Biology, USA, 1984-2008; all of which are incorporated herein by reference in their entirety.)

Example 5: Utilization of other carbon sources by isolated Kluyvera cryocrescens S26

[0131] As Kluyvera cryocrescens S26 is isolated, glycerol may not be the only carbon source the bacterial strain can utilize to produce ethanol. Other carbohydrates (carbon feestocks) may also be acceptable as substrates for ethanol fermentation. The liquid culture was incubated in a screw-capped bottle at 30°C for 3 days under anaerobic condition with 100 mM of each carbon source. As described in Table 4, Kluyvera cryocrescens S26 was capable of utilizing a number of carbohydrates as sole carbon source for its growth and producing ethanol as major metabolite. C₆ or C₅ monosaccharides, C₆-sugar alcohols, and disaccharides were tested for both microbial dissimilation and ethanol production. In addition to glycerol, Kluyvera cryocrescens S26 also exhibited ethanol production for the carbohydrates (carbon feedstocks) tested.

Table 4

Ethanol fermentation from various carbohydrates by Kluyvera cryocrescens S26

Carbon source Ethanol (mM)

Crude glycerol (C3) 38

D-Glucose (C6) 30

D-Galactose (C6) 29

Maltose (glucose+glucose) 23

D-Sorbitol (C6) 59

D-Mannitol (C6) 37

D-Fructose (C6) 37

Sucrose (glucose+ fructose) 29

Cellobiose (glucose+glucose) 25

D-Arabinose (C5) 3.5

L-Arabinose (C5) 15

D-Xylose (C5) 39

Example 6: Effect of nutrients on ethanol production

[0132] Some nutritional components present in complex nitrogen sources can be essential for cell growth and metabolite production. The effect of several complex nitrogen sources on cell growth and ethanol production was investigated (Table 5). K. cryocrescens S26 was cultivated at 30°C for 3 days in liquid medium containing crude glycerol (30 g/L) as the sole carbon source, supplemented with various nutrients under anaerobic condition (total volume of 50 mL). Yeast extract, polypeptone and tryptone resulted in improvement in cell growth and ethanol production, compared with culture without nutrient addition. When more yeast extract was added, cell growth and ethanol production were both enhanced significantly. Table 5

Effect of nutrients on the cell growth and ethanol production.

Corn steep

Yeast extract Polypeptone Tryptone

Control¹ liquor

(10 g/L) (10 g L) ( 10 g/L)

(l Og/L)

Maximum

1.2 2.1 1.4 1.7 1.4

DCW (mg)

Ethanol

4 10 7.6 8.1 3.9

(g L)

Yield (%)² 32 84 61 66 31

Culture medium without nutrient supplementation.

2Ethanol produced (mole)/glycerol initial (mole)

Example 7: Aerobic ethanol fermentation by isolated Kluyvera cryocrescens S26

Effect of dissolved oxygen

[0133] In general, glycerol fermentation has been conducted under anaerobic conditions (in the absence of electron acceptors). The strict anaerobic conditions require significant effort to avoid oxygen contamination in practical bioprocess operation. Furthermore, anaerobic fermentation needs to be supplemented with expensive nutrients such as yeast extract, polypeptone and tryptone. Glycerol fermentation in the presence of oxygen was exploited as a means to avoid the need for rich nutrients while maintaining a high level of ethanol production. Experiments were performed in an on-line controlled fermentation system.

Kluyvera cryocrescens S26 was cultivated in the liquid culture medium (total volume, 1 L; as described in Table 1 with 50 g/L of crude glycerol). Each reaction further contained either 2 g/L or 10 g/L of yeast extract. Gas was supplied at the rate of 0.25 L/min and the oxygen level was controlled by adjusting the ratio between air and nitrogen at the gas inlet.

Fermentation was carried out at 30°C until the ethanol concentration reached the maximum level. The supply of a limited amount of oxygen enhanced the ethanol production rate and yield as well as cell growth significantly. When glycerol fermentation was performed under aerobic (e.g., microaerobic) conditions, there was a remarkable enhancement in ethanol productivity even when using nutrient-free culture medium. That improvement indicated that a low level of oxygen enabled redox balance by consuming the excess reducing equivalents generated by the incorporation of glycerol into the cell mass. Table 6

Effect of oxygen on glycerol fermentation.

Air content in inlet gas (%)

O¹ 0 12.5 25 50 75 100

Maximum

3.26 1.53 3.54 4.28 4.22 4.42 4.57 DCW (mg)

Glycerol (g/L) 2.4 7.4 0 0 0 0 0

Ethanol (g/L) 23 14.4 16.6 14.7 18.3 10.4 1 1

Yield (%)² 86 58 76 66 84 46 46

Productivity

0.523 0.076 0.415 0.639 0.915 0.52 0.55

(g/L/h)

Unless otherwise noted, all reactions contain 2g/L yeast extract.

'Culture medium is supplemented with yeast extract (l Og/L).

²Ethanol produced (mole)/glycerol initial (mole)

Effect of initial crude glycerol

[0134) Glycerol fermentation was conducted to optimize initial glycerol level. Kluyvera cryocrescens S26 was cultivated in an on-line controlled fermentation system with the liquid culture medium containing varying amounts of crude glycerol (total volume 1 L for each reaction). Gas was supplied at the rate of 0.25 L/min, including air and nitrogen with 50% each. Fermentation was carried out at 30°C until ethanol concentration reached maximum level. Ethanol production was increased as the amount of glycerol increased up to 75 g/L. Synthesis of ethanol and co-products by Kluyvera cryocrescens S26 was described in FIG. 4. The isolated, Kluyvera cryocrescens produced 27 g/L of ethanol from crude glycerol directly obtained from biodiesel plant with a distinguished high production rate of 0.61 g/L/h. These results are described in Table 7 as well as FIG. 5. Table 7

Effect of initial crude glycerol concentration on ethanol production.

Initial crude glycerol concentration (g/L)

25 50 75 100

Maximum DCW

2.12 4.22 4.85 3.69

(mg)

Glycerol (g/L) 0 0 0 0

Ethanol (g/L) 9.1 18.3 27 15.2

Yield (%)* 84 84 80 33

Productivity (g/L/h) 0.891 0.915 0.607 0.27

Ethanol produced (mole)/glycerol initial (mole)

Example 8: Improvement in conventional bioethanol process

Conversion of glycerol obtained from bioethanol process

[0135] In ethanol fermentation by yeast strain, a number of by-products are produced in addition to biomass and carbon dioxide. Glycerol and organic acids are representative by- products. Approximately 5% of the carbon source was converted into glycerol in

conventional bioethanol processes. That undesirable glycerol formation led to increases in the overall operational cost of ethanol process. Glycerol synthesis in ethanol fermentation was required to maintain redox balance as well as to minimize osmotic stress. In order to increase ethanol yield in conventional ethanol fermentation, conversion of glycerol obtained from bioethanol processes into ethanol was exploited by isolated Kluyvera cryocrescens S26. Ethanol fermentation broth was prepared by Saccharomyces pastoriamis ATCC 26602 using glucose as the sole carbon source (sole carbon feedstock). The wet cell pellet of K.

cryocrescens S26 was added to fermentation broth without yeast cells and incubated at 30°C without supplementation with nutrients. Glycerol formed in ethanol fermentation could be utilized by K. cryocrescens S26 and converted into ethanol, resulting in increased ethanol level. These results are described in FIG. 6.

Co-fermentation of glucose and glycerol

[0136] Ethanol fermentation from glucose could be facilitated when yeast cells were inoculated in combination with Kluyvera cryocrescens S26 because this bacterial strain was capable of metabolizing both glycerol and glucose to produce ethanol. Co-fermentation of both strains was carried out under microaerobic condition in liquid medium containing 70 g/L glucose as the sole carbon source (sole carbon feedstock). As shown in FIG. 6, glucose was processed by both yeast and bacteria to synthesize ethanol. Glycerol formed by yeast was also converted into ethanol by K, cryocrescens S26, resulting in a higher ethanol

concentration than that obtained in single fermentation by yeast only. The ethanol production rate was increased two-fold by co-fermentation and residual glycerol was significantly reduced, leading to a higher ethanol yield from a glucose carbon feedstock.

[0137] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS: 1. A method for bacterially producing ethanol, said method comprising incubating an isolated Kluyvera cryocrescens (ATCC Deposit Designation No. PTA-10600) bacterial strain with a carbon feedstock under fermentation conditions to produce ethanol.

2. The method for bacterially producing ethanol of claim 1, wherein said ethanol production occurs by incubation of the isolated Kluyvera cryocrescens in an aerobic or anaerobic reaction mixture.

3. The method for bacterially producing ethanol of claim 2, wherein said aerobic or anaerobic reaction mixture comprises a carbon feedstock, a nutrient composition, a buffer, and a gas.

4. The method for bacterially producing ethanol of claim 3, wherein said carbon feedstock is selected from the group consisting of a C₃-C₈ monosaccharide, a disaccharide, and a sugar alcohol.

5. The method for bacterially producing ethanol of claim 3, wherein said carbon feedstock is selected from the group consisting of glycerol.

6. The method for bacterially producing ethanol of claim 5, wherein said glycerol is selected form the group consisting of crude glycerol and pure glycerol.

7. The method for bacterially producing ethanol of claim 6, wherein said crude glycerol is obtained as a by-product from the production of biodiesel.

8. The method for bacterially producing ethanol of claim 4, wherein said C monosaccharide is selected from the group consisting of D-glucose, D-galactose, D- fructose, and D-mannose.

9. The method for bacterially producing ethanol of claim 4, wherein said disaccharide comprises C₆ monosaccharides.

10. The method for bacterially producing ethanol of claim 4, wherein said disaccharide is selected from the group consisting of maltose, sucrose, and cellobiose.

11. The method for bacterially producing ethanol of claim 4, wherein said disaccharide comprises C₅ monosaccharides.

12. The method for bacterially producing ethanol of claim 4, wherein said monosaccharide is selected from the group consisting of D-xylose and L-arabinose.

13. The method for bacterially producing ethanol of claim 4, wherein said sugar alcohol is selected from the group consisting of D-sorbitol, and D-mannitol.

14. The method for bacterially producing ethanol of claim 5, wherein the final concentration of glycerol in the reaction mixture is in the range from 25 g/L to 100 g/L.

15. The method for bacterially producing ethanol of claim 3, wherein said nutrient composition is selected from the group consisting of a yeast extract, polypeptone, tryptone, corn steep liquor and mixtures thereof.

16. The method for bacterially producing ethanol of claim 3, wherein said buffer comprises one or more components selected from the group consisting of ₂HP0₄, NaH₂P0₄, EDTA, ZnS0₄'7H₂0, CaCl₂'2H₂0, FeSCV7H₂0, Na₂Mo0₄ ^«2H₂0, CuS0₄ ^»5H₂0, CoCl₂*6H₂0, MnCl₂'4H₂0, MgCl₂'6H₂0, and ( H₄)₂S0₄.

17. The method for bacterially producing ethanol of claim 3, wherein said gas for an aerobic reaction mixture comprises a mixture of air and nitrogen.

18. The method for bacterially producing ethanol of claim 17, wherein said gas for an aerobic reaction mixture comprises 50% nitrogen and 50% air.

19. The method for bacterially producing ethanol of claim 2, wherein said gas for an anaerobic reaction mixture is selected from the group consisting of C0₂, N₂, or a mixture thereof.

20. The method for bacterially producing ethanol of claim 3, wherein said reaction mixture is incubated at a temperature in the range of 15°C to 55°C.

21. The method for bacterially producing ethanol of claim 1, wherein said bacterial strain produces ethanol at a rate greater than 0.216 g/L/h.

22. The method for bacterially producing ethanol of claim 1, wherein said bacterial strain produces ethanol at a rate in the range between 0.216 g/L/h to 1.0 g/L/h.

23. The method for bacterially producing ethanol of claim 1, wherein said bacterial strain provides for an ethanol molar yield of greater than or equal to 80%.

24. A reaction mixture comprising an isolated Kluyvera cryocrescens (ATCC Deposit Designation No. PTA-10600) and a carbon feedstock.

25. The reaction mixture of claim 24, wherein the reaction mixture is an anaerobic or an aerobic reaction mixture.

26. The reaction mixture of claim 24, wherein the reaction mixture comprises: a carbon feedstock, a nutrient composition, a buffer and a gas.

27. The reaction mixture of claim 26, wherein said carbon feedstock is selected from the group consisting of a C₃-C₈ monosaccharide, a disaccharide, a

polysaccharide, and a sugar alcohol.

28. The method for bacterially producing ethanol of claim 26, wherein said carbon feedstock is glycerol.

29. The method for bacterially producing ethanol of claim 28, wherein said glycerol is selected form the group consisting of crude glycerol and pure glycerol.

30. The method for bacterially producing ethanol of claim 29, wherein said crude glycerol is obtained as a by-product from the production of biodiesel.

31. A method for production of ethanol by co-fermentation, said method comprising: (i) incubating a composition comprising a yeast strain capable of ethanol production and a carbon feedstock; and (ii) inoculating the yeast and feedstock composition from (i) with an isolated Kluyvera cryocrescens (ATCC Deposit Designation No. PTA-10600) under fermentation conditions.

32. The method of claim 31, wherein co-fermentation increases ethanol production compared to ethanol production from fermentation resulting from incubating a composition comprising a yeast strain in the absence of an isolated Kluyvera cryocrescens bacterial strain (ATCC Deposit Designation No. PTA-10600).

33. The method of claim 32, wherein co-fermentation increases the ethanol yield by greater than or equal to 6%.

34. A reaction mixture comprising an isolated Kluyvera cryocrescens and a yeast capable of producing ethanol by fermentation.

35. The reaction mixture of claim 34, wherein said reaction mixture further comprises a carbon feedstock.

36. The reaction mixture of claim 35, wherein the carbon feedstock is glucose.

37. The reaction mixture of claim 34, wherein said reaction mixture produces ethanol with an increased ethanol yield compared to a composition comprising yeast and a carbon feedstock in the absence of an isolated Kluyvera cryocrescens bacterial strain.

38. The reaction mixture of claim 37, wherein the ethanol yield is increased by greater than or equal to 6%.

39. An isolated Kluyvera cryocrescens bacterial strain having ATCC Deposit Designation No. PTA-10600.

40. Use of an isolated Kluyvera cryocrescens bacterial strain having ATCC Deposit Designation No. PTA-10600 in a fermentation reaction.