TO ETHANOL

RELATED APPLICATION

[001] This application claims the benefit of U.S. Provisional Application No.

61/458,545, filed on November 26, 2010. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[002] The present disclosure generally relates to fermentation media and fermentation conditions that result in the efficient conversion of glycerol to ethanol by microorganisms.

[003] The efficient conversion of a carbon source, such as glycerol or glucose, into ethanol relies on many conditions, including the composition of the nutrients supplied to the fermentor and the reaction conditions maintained in the fermentor. Yeast fermentations, which have been practiced for decades at industrial scale, have been highly optimized to allow the fermentation to reach ethanol titers in excess of 150 g/L within 60 to 80 hours. Likewise, ethanologenic strains of Escherichia coli, such as KOI 1, have been optimized for the conversion of sugars, including sugars found in hemicelluloses and sweet whey (York, S.W. and Ingram, L.O., "Soy-based medium for ethanol production by Escherichia coli KOI 1," J. Indust. Microbiol. (1996) v. 16: 374 - 376; Asghari, A., Bothast, R. J., Doran, J. B., and Ingram, L.O., "Ethanol production from hemicellulose hydrolysates of agricultural residues using genetically engineered Escherichia coli strain KOI 1," J. Ind. Microbiol.

(1996) v. 16: 42 - 47).

[004] Recent advances in the production of biodiesel, alcohol esters of fatty acids derived from renewable resources, have led to a surplus of glycerol. Traditionally, glycerol has commanded a very high value, making it prohibitively expensive for use as a carbon source in industrial-scale fermentations. Furthermore, very few organisms can ferment glycerol under anaerobic conditions in the absence of an external electron acceptor. With the rapid expansion of biodiesel production, glycerol is now a low-cost, abundant alternative to sugars or amino acids as carbon sources for industrial-scale fermentations. Recent work by Gonzalez and colleagues has demonstrated the use of glycerol as a carbon source to produce many chemical intermediates, including ethanol, and lactate (Yazdani, S.S., and R. Gonzalez, "Engineering Escherichia coli for the efficient conversion of glycerol to ethanol and co- products," Metab. Eng. (2008) v. 10: 340-351; Mazumdar, S., Clomburg, J.M., and R.

Gonzalez, "Escherichia coli Strains Engineered for Homofermentative Production of D- Lactic Acid from Glycerol," Appl. Env. Microbiol. (2010) v. 76: 4327-4336). Furthermore, Northrop and colleagues, referring to Paenibacillus macerans as Bacillus acetoethylicum, determined that P. macerans produces a mixture of acetone and ethanol from a variety of sugars, but from glycerol it produces only ethanol (Northrop, J.H., Ashe, L.H., and J.K. Senior, "Biochemistry of Bacillus acetoethylicum with Reference to the Formation of Acetone," J. Biol. Chem. (1919) v. 39: 1-21) and Gupta and colleagues confirmed these results (Gupta, A., Murarka, A., Campbell, P., and R. Gonzalez, "Anaerobic Fermentation of Glycerol in Paenibacillus macerans: Metabolic Pathways and Environmental Determinants," Appl. Env. Microbiol. (2009) v. 75: 5871-5883).

[005] Although fermentation media based on soy hydro lysates have been proposed for the production of ethanol by bacterial strains (e.g., U.S. Patent 6,130,076 and York, S.W. and Ingram, L.O., "Soy-based medium for ethanol production by Escherichia coli KOI 1," J. Indust. Microbiol. (1996) v. 16: 374 - 376), the production of the hydrolysate required the processing of soy meal by the addition of expensive enzyme preparations prior to the start of fermentation. This step adds additional time, equipment, and expense to the fermentation process and the cost of the ethanol produced by this process. Furthermore, these references teach to the use of sugar-based carbon sources (glucose, hemicellulose hydrolysates, and sweet whey) without mention of the use of glycerol as a carbon source for the production of ethanol.

[006] One alternative strategy is to perform a first fermentation to convert glycerol to ethanol, and a second fermentation to convert glucose or another appropriate sugar source to ethanol, followed by mixing the two separate fermentations to achieve a final ethanol titer that is high enough to distill economically. However, this approach requires running separate fermentation lines. A more practical approach would be to combine the benefits of these two separate fermentations into a single process using the same equipment.

[007] There remains a need for a low-cost fermentation medium capable of supporting the growth of microorganisms on glycerol as a carbon source for the conversion of glycerol into ethanol wherein the microorganism can consume non-hydrolyzed nitrogen sources. SUMMARY OF THE INVENTION

[008] The present invention provides methods of converting a carbon source into ethanol efficiently and at reduced costs compared to prior art processes. In one embodiment, the method comprises culturing an ethanologenic microorganism that produces an

extracellular enzyme such as an exogenous (i.e. extracellular) protease capable of

hydrolyzing unhydrolyzed nitrogen sources, in a suitable culture medium, wherein the culture medium comprises a mixture of a carbon source such as glycerol, sugars or complex carbohydrates and an unhydrolyzed nitrogen nutrient source such as soybean meal, soybean flour, cottonseed flour or other nitrogen-rich, materials, wherein the microorganism converts the carbon source into ethanol. In one embodiment, the method comprises converting a carbon source into ethanol using ethanologenic microorganisms wherein the method does not require the addition of enzymes to the cell culture or cell culture medium that are produced outside the cell culture or cell culture medium such as the addition of semi-purified or purified enzyme preparations. Thus in one preferred embodiment, all of the enzymes needed for the fermentation process of converting the carbon source to ethanol are inherent to the ethanologenic microorganism.

[009] In one embodiment, the invention provides a method for the production of ethanol from glycerol comprising culturing an ethanologenic microorganism that produces at least one extracellular enzyme capable of converting an unhydrolyzed nitrogen source into a hydrolyzed nitrogen source suitable for use by the microorganism as a nutrient for growth, in a suitable culture medium comprising a mixture of glycerol and an unhydrolyzed nitrogen source, thereby converting the ethanol to glycerol, preferably in the absence of adding additional enzyme to the cell culture medium.

[010] In one embodiment the invention provides a method for the production of ethanol from sugars and complex carbohydrates comprising culturing an ethanologenic

microorganism that produces at least one extracellular enzyme capable of converting an unhydrolyzed nitrogen source into a hydrolyzed nitrogen source suitable for use by the microorganism as a nutrient for cell growth, in a suitable culture medium comprising a mixture of an unhydrolyzed nitrogen source, and one or more of sugars and complex carbohydrates, preferably in the absence of the addition of enzymes produced outside the cell culture medium including purified or semi-purified hydrolases for the conversion of complex carbohydrates to mono-, di-, tri- and/or tetrasaccharides. [Oi l] In certain embodiments, the unhydrolyzed nitrogen source is a plant-based unhydrolyzed nitrogen source including but not limited to, soybean meal, soybean flour, cottonseed meal, cotton seed flour, or a combination thereof.

[012] In one embodiment, the unhydrolyzed nitrogen source includes sources other than plants such as fish meal and certain meat extracts or a combination thereof.

[013] In one embodiment, the ethanologenic microorganism is a member of the genus Bacillus, Clostridium, Lactobacillus, Lactococcus, or Paenibacillus.

[014] In one embodiment, the amount of unhydrolyzed nitrogen source in the culture medium is about 1 to about 50 grams per liter and preferably about 5 to about 40 grams per liter.

[015] In one embodiment, glycerol is the carbon source and the glycerol in the culture medium is about 1 to about 200 grams per liter, preferably about 5 to about 100 grams per liter, preferably about 10 to about 100 grams per liter and more preferably about 20 to about 50 grams per liter.

[016] In one embodiment, the present invention provides a method of producing ethanol by culturing an ethanologenic microorganism that produces an extracellular protease that is capable of converting an unhydrolyzed nitrogen source into a hydrolyzed nitrogen source suitable for use by the microorganism as a nutrient for cell growth, in a suitable culture medium comprising a mixture of glycerol, yeast extract, and one or more of soybean meal, soybean flour, cottonseed meal, and cottonseed flour.

[017] In one embodiment, the present invention provides a culture medium for culturing an ethanologenic microorganism to convert glycerol into ethanol, the culture medium comprising an ethanologenic microorganism that produces at least one extracellular enzyme capable of converting an unhydrolyzed nitrogen source into a hydrolyzed nitrogen source suitable for use by the microorganism as a nutrient for growth, yeast extract and one or more of soybean meal, soybean flour, cottonseed meal and cottonseed flour, preferably with the proviso that the cell culture medium does not comprise additional enzymes produced outside the cell culture. BRIEF DESCRIPTION OF THE DRAWINGS

[018] So that the manner in which the above recited features of the present disclosure can be understood in detail, a more particular description of the disclosure, briefly

summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

[019] Figure 1 shows ethanol production from glycerol in various fermentation media at 24 and 48 hour (h) time points during the fermentation. The various media include MM1 , MM14, MM15, simplified MM15 ("Simpl. MM 15") and MM17, as described below.

[020] Figure 2 shows ethanol production from glycerol in MM 17 fermentation medium with alterations as follows: MM 17 - SBM refers to MM 17 with the soybean meal omitted; MM 17 - YE refers to MM 17 with the yeast extract omitted; MM 17 - NG refers to MM 17 with the NutriGo 1500 omitted; MM 17 + CM refers to MM 17 in which the soybean meal has been replaced with an equal weight of cottonseed flour.

[021] Figure 3 shows the ethanol production from glycerol of a non-sporulating mutated isolate of Paenibacillus macerans Strain LMG 13285 on MM23 fermentation medium which uses soybean flour as the unhydrolyzed nitrogen source.

DETAILED DESCRIPTION

[022] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention, as claimed. In this application, the use of the singular includes the plural, the word "a" or "an" means "at least one", and the use of "or" means "and/or", unless specifically stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements or components comprising one unit and elements or components that comprise more than one unit unless specifically stated otherwise.

[023] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[024] As used herein "ethanologenic microorganism" refers to a microorganism with the ability to convert at least a portion of a carbon source including, but not limited to, glycerol, to ethanol. The ethanologenic microorganisms are ethanologenic by virtue of their ability to express one or more enzymes that individually or together convert at least a portion of a carbon source such as glycerol to ethanol. Examples of ethanologenic organisms include yeast, bacteria or fungi. Examples of suitable microorganisms include, but are not limited to, a member of the genus Bacillus, Clostridium, Lactobacillus, Lactococcus, or Paenibacillus. In one preferred embodiment, the ethanologenic microorganism is Paenibacillus macerans. One preferred strain of Paenibacillus macerans is Strain LMG13285 of the Belgian Coordinated Collections of Micro-Organisms. By way of example, enzymes suitable produced by ethanologenic microorganisms include but are not limited to: glycerol dehydrogenase and dihydroxyacetone kinase (conversion of glycerol to dihydroxyacetone phosphate, a common intermediate with the glucose pathway); a CoA-linked aldehyde dehydrogenase and an alcohol dehydrogenase (Gupta, et al. (2009) Appl. Env. Microbiol. 75: 5871-5883).

[025] In a preferred embodiment, ethanologenic microorganisms in accordance with the invention also express extracellular enzymes that are particularly suitable for utilizing unhydrolyzed nitrogen sources. Examples of such extracellular enzymes include proteases capable of converting unhydrolyzed proteins to hydrolyzed nitrogen sources such as amino acids suitable for use as a nitrogen source by the microorganisms. Suitable proteases expressed by preferred microorganisms of the invention include, but are not limited to serine proteases which are typically present in the Bacillus/Paenibacillus species and glutamic acid proteases also known to be present in certain Bacillus species In a preferred embodiment, the microorganism used in the culture system express sufficient enzyme to convert adequate amounts of unhydrolyzed nitrogen sources to a nitrogen source to support growth of the organisms in the absence of the addition of proteases and other enzymes, purified enzymes or purified enzyme components from sources outside the cell culture system.

[026] Ethanologenic microorganisms may also be genetically engineered to express the desired enzymes suitable for use in the present invention. Examples of such genetically engineered organisms include those expressing and secreting one or more enzymes particularly suitable for converting a carbon source such as glycerol to ethanol and/or organisms expressing enzymes particularly suitable for utilizing unhydrolyzed, nitrogen sources. The microorganism may be genetically engineered to overexpress a suitable enzyme native to the organism, or to express/overexpress an enzyme that is heterologous.

[027] As used herein an "exogenous protease" is a protease produced or expressed by an ethanologenic microorganism that is secreted by the microorganism into the extracellular space. The term "exogenous protease" is used interchangeably herein with the terms "extracellular protease" and "secreted protease".

[028] As used herein the phrase "culturing an ethanologenic microorganism that produces an exogenous protease in a suitable culture medium" refers to growing a population of microorganisms under suitable conditions in a liquid or solid medium in a fermentation process that involves the enzymatic and anaerobic breakdown of carbon based substances by microorganisms to produce simpler end products such as ethanol. While fermentation occurs under anaerobic conditions, it is not intended that the term be solely limited to strict anaerobic conditions, as fermentation may also occur in the presence of oxygen. One skilled in the art is aware of the of the various "suitable conditions" for culturing and fermentation such as suitable temperatures, and components of the nutrient medium best suited for the microorganisms which may be used in the fermentation process to produce the desired end products such as ethanol.

[029] The methods and techniques utilized for culturing or generating the

microorganisms disclosed herein are known to the skilled worker trained in microbiological and recombinant DNA techniques. Methods and techniques for growing microorganisms (e.g., bacterial cells), transporting isolated DNA molecules into the host cell and isolating, cloning and sequencing isolated nucleic acid molecules, knocking out expression of specific genes, etc., are examples of such techniques and methods. These methods are described in many items of the standard literature, which are incorporated herein in their entirety: "Basic Methods In Molecular Biology" (Davis, et al, eds. McGraw-Hill Professional, Columbus, OH, 1986); Miller, "Experiments in Molecular Genetics" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1972); Miller, "A Short Course in Bacterial Genetics" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1992); Singer and Berg, "Genes and Genomes" (University Science Books, Mill Valley, CA, 1991); "Molecular Cloning: A Laboratory Manual," 2 ^nd Ed. (Sambrook, et al, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989); "Handbook of Molecular and Cellular Methods in Biology and Medicine" (Kaufman, et al, eds., CRC Press, Boca Raton, FL, 1995); "Methods in Plant Molecular Biology and Biotechnology" (Glick and Thompson, eds., CRC Press, Boca Raton, FL, 1993); and Smith-Keary, "Molecular Genetics of Escherichia coli" (The Guilford Press, New York, NY, 1989).

[030] As defined herein, a knocked-out gene is a gene whose encoded product, e.g., a protein, does not or substantially does not perform its usual function or any function. A knocked-out gene can be created through deletion, disruption, insertion, or mutation. As defined herein, microorganisms that lack one or more indicated knocked-out genes are also considered to have knock outs of the indicated gene(s). The microorganisms themselves may also be referred to as knock outs of the indicated gene(s). Such knock outs can also be conditional or inducible, using techniques that are well-known to those of skill in the art. Also contemplated are "knock ins", in which a gene, or one or more segments of a gene, are introduced into the microorganism in place of, or in addition to, the endogenous copy of the gene. Once again, many techniques for creating knock in microorganisms are known to those of ordinary skill in the art.

[031] The present invention offers several advantages over currently employed methods of producing ethanol in a fermentation process. One advantage is the economy of scale offered by the use of less expensive nitrogen sources employed in the fermentation medium. The preferred methods according to the invention do not require the addition of any commercially generated enzymes such as those enzymes necessary for converting

unhydrolyzed nitrogen sources to nitrogen sources suitable for cell growth. In accordance with the invention, the enzymes employed are preferably produced in sufficient amounts by the ethanologenic microorganisms used in the cell culture system. As commercially generated enzymes are expensive, the invention is more economical than those methods which do require additional commercial enzymes.

[032] Yet another advantage of the methods of the invention is that the entire process can be conducted in a single fermentation process which yields almost entirely ethanol.

[033] The methods of the invention also advantageously employ by-products of other processes (such as those generating glycerol) and avoid the need for hydrolysis of nitrogen sources which have their own waste disposal issues. [034] Examples

[035] Example 1 -Materials

[036] A description of media tested according to embodiments of the invention is provided below. Although certain chemical supply companies and their products are mentioned above and below, it is noted that embodiments of the invention are not limited to these companies and products. Other products, having similar compositions, from the same or different companies may be used.

[037] Cottonseed Flour-based Minimal Medium (MM1 + Proflo)

[038] Proflo is a cottonseed protein product obtained from Archer Daniels Midland (Decatur, IL). All other chemicals were obtained from Sigma Chemical Company (St. Louis, MO).

[039] MM1 was made by the following procedure. An MM1 concentrate was prepared by mixing 80 ml of 1.0 M Tricine (adjusted to pH 7.4 by NaOH), 20 ml of 10 mM FeS0 ₄ , 100 ml of 1.9 M NH ₄ C1, 20 ml of 276 mM K ₂ S0 ₄ , 20 ml of 0.5 mM CaCl ₂ , 20 ml of 528 mM MgCl ₂ , 200 ml of 5 M NaCl, 2 ml of a micronutrient solution, and purified water up to 2 L. The micronutrient solution contained 30 μΜ (ΝΗ ₄ )Μθ7θ ₂ 4, 4 mM H ₃ B0 ₃ , 300 μΜ CoCl ₂ , 100 μΜ CuS0 ₄ , 800 μΜ MnCl ₂ , 100 μΜ ZnS0 ₄ .

[040] Additionally, the following solutions were made: 3 M NH ₄ C1, 400 mM Na ₂ HP0 ₄ , 500 mM (NH ₄ ) ₂ S0 ₄ , and 1 mM sodium selenite. Per liter of medium, we added 100 ml of the MM1 concentrate, 10 ml of 3 M NH ₄ C1, 10 ml of 400 mM Na ₂ HP0 ₄ , 10 ml of 500 mM (NH ₄ ) ₂ S0 ₄ , 1 ml of ImM sodium selenite, 10 g Proflo, and 869 ml of purified water. All parts of the medium were filter sterilized.

[041] Soy Peptone-based Medium (MM14)

[042] All chemicals were obtained from Sigma Chemical Company (St. Louis, MO). Per liter, MM14 contained 3 g Na ₂ HP0 ₄ , 12 g NaH ₂ P0 ₄ , 0.5 g (NH ₄ ) ₂ S0 ₄ , 0.5 g K ₂ S0 ₄ , 30 g tryptone, 25 g soy peptone enzymatic digest, and 3 g yeast extract. The medium was sterilized by autoclave.

[043] Soybean Meal-based Medium (MM15)

[044] Soybean meal was obtained from Hieden Feed and Supply (Houston, TX), yeast extract was obtained from Ohly Americas (Hutchison, MN), and all other chemicals were obtained from Sigma Chemical Company (St. Louis, MO). Per liter, MM 15 contained 0.25 g Na ₂ HP0 ₄ , 1 g NaH ₂ P0 ₄ , 1 g K ₂ S0 ₄ , 6 g Ohly KAT, and 36.8 g soybean meal. The medium was sterilized by autoclave, and 20 ml of a Minerals /Vitamins mix was added.

[045] The Minerals /Vitamins mix was a 50/50 v/v mixture of a Minerals solution and a Vitamins solution. The Minerals solution was 1.22 g MgS0 ₄ and 108 mg FeCl ₃ 6H ₂ 0 in 25 ml water. The Vitamins solution was 100 mg thiamine HCl, 2 mg biotin, and 1 ml Trace mix in 1 L of water. The Trace mix was 40 mg calcium pantothenate, 20 mg pyridoxine HCl, and 8 mg cyanocobalamin in 1 L of water. The Minerals /Vitamins mix was filter sterilized.

[046] Simplified MM15

[047] Soybean meal was obtained from Hieden Feed and Supply (Houston, TX), yeast extract was obtained from Ohly Americas (Hutchison, MN), and NutriGo 1500 was obtained from Nutrients Incorporated (Manitowoc, WI). Per liter, Simplified MM 15 contained 36.8 g soybean meal, 6 g yeast extract, and 1.8 g NutriGo 1500. The medium was sterilized by autoclave.

[048] Soybean Meal-based Medium (MM17)

[049] Soybean meal was obtained from Hieden Feed and Supply (Houston, TX), yeast extract was obtained from Ohly Americas (Hutchison, MN), and NutriGo 1500 was obtained from Nutrients Incorporated (Manitowoc, WI). Per liter, MM 17 contained 36.8 g soybean meal, 6 g yeast extract, and 0.3 g NutriGo 1500. Soybean meal and yeast were sterilized by autoclave. NutriGo 1500 was pasteurized for 1 min at 105°C.

[050] Example 2-Experimental Methods

[051] A description of fermentation conditions tested according to embodiments of the invention is provided below. According to one embodiment of the invention, P. macerans, strain LMG 13285 was sourced from the Belgian Coordinated Collections of Microorganisms and is capable of converting glycerol to ethanol in high yields. This strain was maintained on plates containing Luria-Bertani (LB) medium (10 g tryptone, 5 g yeast extract, 10 g NaCl, and 15 g agar per liter). Liquid Nutrient Broth Yeast Extract (NBYE, 8 g/1 Difco nutrient broth, 5 g/1 Difco yeast extract and 5 g/1 NaCl) supplemented with 40 g/1 glycerol, was used for overnight culture and fermentation. CaC0 ₃ (10 g/1) was added to all shake-flask cultures for pH control. Chemicals were obtained from Fisher Scientific (Pittsburgh, PA) and Sigma- Aldrich Co. (St Louis, MO). [052] Cell culture optical density was measured at 600 nm (OD ₆ oo) using a Biochrom Libra S22 spectrophotometer (Biochrom Ltd.). 10 μΐ of 10 M HC1 was used to remove CaCC"3 residuals when measuring OD. Ethanol, glycerol, and organic acid concentrations were determined using a Shimadzu LC-20AD HPLC equipped with a UV-monitor (210 nm) and refractive index detector (RID). Products were separated using an Aminex HPX-87H column (Bio-Rad Laboratories) with 2.5 mM H ₂ SO ₄ as the mobile phase (0.6 ml min ^"1 , 55° C).

[053] The fermentation seed culture was prepared as follows: the cultures (stored as glycerol stocks at -80°C) were streaked onto LB plates and incubated overnight at 42°C. Single colonies growing on these plates were used to inoculate 40 ml (NBYE medium as described above) seed cultures in 250 ml baffled flasks grown aerobically at 42°C and 175 rpm overnight. These cultures were used to provide inocula in the 0.5-L fermentor.

[054] Anaerobic fermentations using various fermentation media as described above were conducted in 0.5-L fermentation systems under the following conditions: temperature control, maintained at 42°C; pH control, maintained at pH (6.0) using 5 M sodium hydroxide; and agitation (200 rpm) provided by a magnetic stir bar and magnetic stir plate.

Fermentations were carried out for 48 hours. Samples were collected at 24 and 48 hours and analyzed for nutrients and metabolites, as described above.

[055] P. macerans, strain LMG13285, was tested for baseline performance in the MM1 medium, which resulted in 7.7 g/L of ethanol in 48 h (Figure 1). Although the MM1 medium is low-cost, the productivity of the culture in this medium was low. Plackett-Burman experiments (Strobel, R.J. and G.R. Sullivan, "Experimental Design for Improvement of Fermentations," in Demain, A.L. and J.E. Davies (ed.), Manual of Industrial Microbiology and Biotechnology, 2 ^nd Ed., ASM Press, Washington, D.C.) were designed to test various factors contributing to the performance of the fermentations: temperature, pH, aeration, carbon source, nitrogen supply, phosphorus supply, mineral supply, and vitamin supply. The data from this series of experiments identified the optimum temperature (42°C), the optimum pH (6.0), and the optimum carbon source (glycerol at a starting concentration of 60 g/L). The optima for the other variables were undetermined; response surface methodology (Strobel, R.J. and G.R. Sullivan, "Experimental Design for Improvement of Fermentations," in Demain, A.L. and J.E. Davies (ed.), Manual of Industrial Microbiology and Biotechnology, 2 ^nd Ed., ASM Press, Washington, D.C.) was employed to further test the remaining variables. [056] Example 3 -Preparation of optimized fermentation medium using hydro lyzed nitrogen sources

[057] Several nitrogen sources were tested (enzymatically hydrolyzed soy peptone, acid- hydrolyzed soy peptone, Trypticase Soy Broth (TSB) powder, tryptone, ammonium chloride, and urea) as were various concentrations of sodium phosphate, ammonium sulfate, potassium sulfate, magnesium chloride, calcium chloride, and yeast extract. The result of these experiments was formulation of medium MM 14. Although the medium was not very cost- effective, the productivity of the culture increased dramatically to 34.1 g/L ethanol production in 48 h.

[058] Soy peptone present in MM 14 is an enzymatically hydrolyzed soy protein product. The hydrolysis is necessary for many organisms that do not produce sufficient levels of protease to degrade the soy proteins and liberate free amino acids and short oligopeptides to use as nutrients. The hydrolysis step, whether by acid hydrolysis or protease hydrolysis, and subsequent purification steps add additional cost to the soy protein.

Therefore, various other nitrogen components, including unhydrolyzed protein sources, were tested for their effects on ethanol production and cell growth.

[059] Example 4-Preparation of optimized fermentation medium using unhydrolyzed nitrogen sources

[060] To lower the cost of the MM 14 medium, the expensive medium components were replaced with less expensive, less refined alternatives. Using a combination of Plackett- Burman and response surface experiments (as described in Strobel, R.J. and Sullivan, G.R., "Experimental Design for Improvement of Fermentations " In: Demain, A.L. and Davies, J.E., editors in chief. " Manual of Industrial Microbiology and Biotechnology (Second

Edition)" (1999) Washington DC: ASM Press) to make decisions regarding inclusion of and amount of each medium component, various alternate nitrogen sources were tested: casein hydrolysate, whey protein hydrolysate, corn steep powder, soy peptone, several commercial yeast extracts, rice protein isolate, corn steep liquor, urea, soy powder, soy protein

concentrate, and soybean meal. Soybean meal and a low-cost yeast extract (Ohly KAT, obtained from Ohly Americas (Hutchison, MN)) were the most effective combination of nutrients, resulting in medium MM 15, a combination of soybean meal, yeast extract and trace minerals and vitamins. This medium cost $0.09/L; the productivity of the LMG13285 culture achieved 27.6 g/L ethanol produced from glycerol in 48 h. Although the media cost was reduced by two orders of magnitude, the amount of ethanol produced was less than desired.

[061] To simplify the preparation of MM 15 when used in large-scale fermentations, attempts were made to reduce the number of components in the medium. The complex trace minerals and vitamins were replaced with 1.8 g/L NutriGo 1500. Pasteurization of the NutriGo, instead of autoclaving, reduced the amount used to 0.3 g/L. In this medium formulation, MM 17, the productivity of the LMG 13285 culture reached 35.3 g/L ethanol produced in 48 h for a price of $0.09/L; thus, a simple medium consisting of an unhydrolysed nitrogen source (soybean meal), yeast extract, and NutriGo 1500 achieved good performance with a low cost.

[062] Examination of the MM 17 cultures under the microscope revealed that the cells began sporulating approximately 24 hours into the fermentation. The addition of hydrolyzed casein to the medium prevented sporulation. The substitution of soy flour for soybean meal reduced the size of the solids in the medium. This resulted in the MM 19 formulation. The price is higher ($0.34/L), but the cells stay productive for a longer period of time and reach a titer of 43 g/L ethanol.

[063] As demonstrated in Figure 2, each of the components affects the efficient conversion of glycerol to ethanol by LMG 13285. The omission of soybean meal (MM 17 - SBM, Figure 2) reduced productivity by over 50%. The omission of yeast extract (MM17 - YE, Figure 2) reduced productivity even more; performance fell from 35.3 g/L in 48 h to 6.9 g/L of ethanol; the absence of NutriGo 1500 had only a marginal impact on performance, reducing the ethanol titer to 29 g/L at 48 h.

[064] Soybean meal may be replaced by other unhydrolyzed nitrogen sources, such as cotton flour (Proflo). At an equal weight (36.8 g/L), cotton flour can substitute soybean meal with comparable productivity: 31 g/L ethanol at 48 h.

Example 5 -Lower cost medium/culture combinations

[065] In response to the sporulating cultures on MM 17 as discussed above, MM 19 was prepared with hydrolyzed casein to prevent sporulation in MM 19. However, the use of hydrolyzed casein results in a price increase. Therefore, MM23 was prepared without the use of casein or NutriGo 1500 supplement to be more economical. For MM23, the soy flour was obtained from Honeyville Food Products (Rancho Cucamonga, CA) and the Ohly KAT was obtained from Ohly Americas (Hutchison, MN). Per liter, MM23 contained 36.8 g soy flour and 6 g Ohly KAT. The soy flour and Ohly KAT were sterilized by autoclave.

[066] In order to more effectively exploit the most economical medium, it was determined that a culture of ethanologenic bacteria that did not sporulate would eliminate the need for additional components to prevent sporulation.

[067] To create a non-sporulating mutant of P. macerans, strain LMG 13285, a culture of the cells was exposed to nitrosoguanidine using standard microbiological techniques. These cells were plated and non-sporulating colonies were selected by visual inspection. Approximately 50 colonies were tested for performance, and Isolate 170 was selected as the best performer.

[068] The lack of sporulating phenotype allowed us to eliminate casein hydrolysate from the medium formulation and further review of the medium eliminated the NutriGo 1500 based on price for performance resulting in the MM23 medium. The performance of LMG13285-Spo- Isolate 170 on MM23 medium is shown in Figure 3. This combination of culture and medium achieves a titer of 38 g/L in 72 h on a medium that costs $0.09/L.

[069] Ohly KAT is an expensive component of MM23, accounting for $0.07/L out of the $0.09/L total cost. The soy flour component of the medium accounts for the other $0.02/L. We tested many yeast products in an effort to replace Ohly KAT and cut medium costs. They included many samples of yeast extract or yeast autolysate (the extract is refined autolysate) and one sample of dried whole yeast. The table below lists all of our costs for media made with each yeast product. The ethanol production is listed, as well as the cost per ton ethanol for the medium. The Hy-Yest 413 provides yeast extract provides the best compromise between cost and ethanol titer. Yeast Product Cost of $/L for $/L for Total EtOH 72 h $/te

Prod/kg Product Medium (g/L) EtOH

Hebei YE $0.91 $0,005 $0,025 15.5 g/L $1,612

ZhengZheng $3.30 $0.02 $0.04 23.8 g/L $1,681

Hy-Yest 413 $7.50 $0,045 $0,065 29 - 33.5 g/L $2,080

Ohly Provesta 027 $5.81 $0,035 $0,055 25.4 g/L $2,165

Lieber E $7.54 $0,045 $0,065 28.0 g/L $2,321

Lieber S $7.15 $0.04 $0.06 25.7 - 28 g/L $2,335

Hy-Yest 412 $9.00 $0.05 $0.07 28.2 g/L $2,482

Angel YE FM802 $7.83 $0.05 $0.07 27.7 g/L $2,527

Ohly KAT $11.55 $0.07 $0.09 28 - 38 g/L $2,686

Dried Yeast $1.10 $0.01 $0.03 10.4 g/L $2,885

CNSane $4.34 $0.03 $0.05 9 - 17.7 g/L $3,846

Hy-Yest 101 $6.50 $0.04 $0.06 8.8 g/L $6,818

[070] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[071] While the foregoing is directed to embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.