HIGH ETHANOL PRODUCING DERIVATIVES OF THERMOANAEROBACTER ETHANOLICUS

Abstract: Derivatives of the newly discovered microorganism Thermoanaerobacter ehtanolicus which under anaerobic and thermophilic conditions continuously ferment substrates such as starch, cellobiose, glucose, xylose and other sugars to produce recoverable amounts of ethanol solving the problem of fermentations yielding low concentrations of ethanol using the parent strain of the microorganism Thermoanaerobacter ethanolicus are disclosed. These new derivatives are ethanol tolerant up to 10 % (v/v) ethanol during fermentation.

Description: HIGH ETHANOL PRODUCING DERIVATIVES OF THERMOANAEROBACTER ETHANOLICUS Description This application claims a priority filing date based on an application entitled "High Ethanol Producing Derivatives of Ther¬ moanaerobacter ethanolicus," Serial No. 317,702, filed November 2, 1981 (02.11.81), in the United States Patent and Trademark Office as a national application. The United States Government has rights in this invention pursuant to Contract No. DE-AS09-79-ER10499-A001 awarded by the U. S. Department of Energy. Technical Field This invention relates to derivative strains of a newly dis¬ covered microorganism Thermoanaerobacter ethanolicus, a thermo- phiTic anaerobe. More specifically, this invention relates to producing ethanol using derivatives of the microorganism Thermo¬ anaerobacter ethanolicus. Background Art The microorganism Thermoanaerobacter ethanolicus has been described as an extreme thermophilic, non-spore-forming anaerobic bacterium which ferments a variety of carbohydrates to ethanol as the main product (Wiegel, J. and Ljungdahl, L. G. , Arch. Microbiol. 128, 343-348, 1981). Relatively few thermophilic anaerobic bac¬ teria, which ferment substrates such as starch, cellobiose, glucose, xylose, and other sugars to ethanol as the main product, have been reported. The microorganism Thermoanaerobacter ethan¬ olicus is described in U. S. Patent No. 4,292,407 which is incor¬ porated herein by reference. A related patent is U. S. Patent No. 4,292,406 which is incorporated herein by reference. Disclosure of Invention The present invention overcomes problems of fermentations yielding low concentrations of ethanol using the microorganism Thermoanaerobacter ethanolicus. The present invention produces ethanol at a substrate concen¬ tration in a fermentation medium greater than 1% (w/v) using bio¬ logically pure cultures of derivatives of the microorganism Ther¬ moanaerobacter ethanolicus. The present invention provides deriv- atives having the ability to ferment substrates such as starch, cellobiose, glucose, xylose and other sugars to ethanol under anaerobic and thermophilic conditions in an aqueous, nutrient medium obtaining ethanol concentrations high enough to make contin¬ uous fermentations possible. Specifically, substrate can be added to a fermentation medium and ethanol can be removed therefrom during the same continuous fermentation. Microorganism strains of Thermoanaerobacter ethanolicus desig¬ nated by the following accession numbers: ATCC 31550, ATCC 31936, ATCC 31937, and ATCC 31938, respectively, are on deposit with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. The strain designated ATCC 31550 was deposited with the American Type Culture Collection on August 9, 1979. Strains designated ATCC 31936, ATCC 31937, and ATCC 31938, respectively, were all deposited with the American Type Culture Collection on August 11, 1981. All of the above-referenced deposits were made pursuant to the Budapest Treaty. The derivative strains, produced both through induced muta- genesis and multiple selections for spontaneous mutations (de¬ scribed in Examples I, II, and III below) differ from the parent strain, strain JW200 of the microorganism Thermoanaerobacter etha¬ nolicus (ATCC 31550), in that the production of ethanol continues at a high rate although the ethanol concentration in the fermenta¬ tion medium is greater than 1% (w/v) and substrate concentrations in the fermentation medium are greater than 1% (w/v). The follow- ing derivative strains are representatives of a large number of derivative strains obtained from " the parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550). These biologically pure cultures of the representative derivative strains, specifically, strain JW200l-Large of the microorganism Thermoanaerobacter ethanolicus (ATCC 31936) representative of ultraviolet light mutagenesis and pyruvate selection; strain JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus (ATCC 31937) representative of iron deprivation selection and low growth on pyruvate; and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus (ATCC 31938) representative of ultraviolet light mutagenesis, iron deprivation and low growth on pyruvate, are ethanol tolerant up to 10% (v/v) ethanol in the fer¬ mentation medium. The foregoing representative derivative strains produce ethanol at ethanol concentrations in a fermentation medium greater than 1% (w/v) whereas the parent strain does not produce ethanol at ethanol concentrations in a fermentation medium greater than 1% (w/v). The parent strain, strain J 200 of the microor¬ ganism Thermoanaerobacter ethanolicus (ATCC 31550), tolerates ethanol concentrations of up to 10% (v/v) ethanol in the fermenta¬ tion medium only if given time to grow in low concentrations first. The above-referenced derivative strains do not show this charac¬ teristic of the parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550). It is an object of the present invention to produce ethanol at substrate concentrations in a fermentation medium above 1% (w/v) using biologically pure cultures of derivatives of the micro¬ organism Thermoanaerobacter ethanolicus. It is a further object to continuously produce recoverable amounts of ethanol from substrates such as starch, cellobiose, glucose, xylose, and other sugars. These and other objects, aspects and advantages of this invention will become apparent from a consideration of the accom¬ panying specification and claims. Modes for Carrying Out the Invention The parent strain, strain JW200 of the microorganism Thermo¬ anaerobacter ethanolicus (ATCC 31550), described in U. S. Patent 5 No. 4,292,407 which is incorporated herein by reference, is an extreme thermophilic, non-spore-forming anaerobic bacterium which ferments a variety of substrates such as starch, cellobiose, glucose, xylose, and other sugars to ethanol, carbon dioxide, lac- tate, acetate and hydrogen gas. When growing in an aqueous 10 nutrient medium containing 10 g per liter (1% w/v) or less of sub¬ strate the major products are ethanol and carbon dioxide. If higher substrate concentrations are used, there is a shift away from ethanol production to production of the other products. Strain JW200 of the microorganism Thermoanaerobacter ethanol- 15 icus (ATCC 31550) under optimal conditions, produces 1.8 mol of ethanol per mol of glucose. For example, at a concentration of 10 g per liter of glucose, one approximately obtains 0.5% (w/v) etha¬ nol. This level of ethanol is not easily removed by known means of distillation; therefore, derivatives of the parent strain, 20 strain JW20Q of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550), altered to grow at high substrate concentrations to yield ethanol above 1% (w/v) are useful. The representative derivatives mentioned above, strain JW200L-Large of the micro¬ organism Thermoanaerobacter ethanolicus (ATCC 31936), strain -25 JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus .• (ATCC 31937), and JW200L-Fe(7) of the microorganism Thermoanae¬ robacter ethanolicus (ATCC 31938), produce ethanol at levels in the fermentation medium greater than 1.5% (w/v). The following are microorganisms, culture methods, fer enta- 30 tions, strain maintenance procedures, morphology and taxonomy of the derivatives, isolation of the parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus. (a) Organisms. The organisms are cultures of the parent strain, strain JW200 of the microorganism Thermoanaerobacter etha- 35 no!icus (ATCC 31550), the parent strain, and its high-ethanol pro¬ ducing derivatives, specifically, strain uW200L-Large of the microorganism Thermoanaerobacter ethanolicus (ATCC 31936), strain of JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus (ATCC 31937), and strain JW200L-Fe(7) of the microorganism Thermo- anaerobacter ethanolicus (ATTC 31938). (b) Culture Methods. The foregoing strains are routinely grown or cultivated under anaerobic and thermophilic conditions, specifically 68°C and pH between 5.7 to 8.6 in anaerobic tubes (Bellco Glass Co., Vineland, N. J. no. 2046-18142) in the aqueous nutrient medium described in U. S. Patent No. 4,292,407 which is incorporated herein by reference (Arch. Microbiol. 128, 343-348, 1981 which is incorporated herein by reference), and which is known in the art. Extruded crack.ed corn was used at concentrations of 20% (w/v) as a rapid indicator of hydrolysis of starch by the derivative strains. When concentrations of substrates exceeded 5% (w/v) yeast extract in the aqueous nutrient medium was increased to .6% (w/v). All of the aforementioned derivative strains produce mainly ethanol and only small amounts of lactate and acetate as fermentation products. Additionally, all the aforementioned deriv- ative strains produce ethanol at a substrate concentration in the fermentation medium greater than 1% (w/v). Routine growth includes but is not limited to the following substrates: soluble and insoluble starch, pectin, disaccharides such as cellobiose, sucrose, lactose, and maltose, monosaccharides such as glucose, fructose, annose, galactose, xylose, and ribose, and glycerol ■ and pyruvate wherein the substrates are fermented to ethanol in the aqueous nutrient medium. (c) Fermentations. Fermentations of the aforementioned sub¬ strates using the derivative strains continuously produce recover- able amounts of ethanol. More specifically, substrate can be added to a fermentation using the derivative strains, and ethanol can be removed therefrom during the same continuous fermentation. Fer¬ mentation. Fermentations are conducted under anaerobic and thermo¬ philic conditions or under anaerobic and extreme thermophilic con- ditions. Extreme thermophilic conditions are understood to mean that fermentations are at 70°C or higher. Fermentations are con- OM ducted at a pH range of about 5.7 to 8.6 and at a temperature range between about 40°C and 78°C. Thus a process for continuously pro¬ ducing recoverable amounts of ethanol such that a substrate can be added to a fermentation and the ethanol can be removed therefrom during a fermentation is provided. This process comprises sub¬ jecting the aqueous nutrient medium containing the substrate at a substrate concentration in the fermentation medium, greater than 1% (w/v), wherein the substrate includes but is not limited to the following substrates: soluble and insoluble starch, pectin, disaccharides such as cellobiose, sucrose, lactose, and maltose, monosaccharides such as glucose, fructose, mannose, galactose, xylose, and ribose, and glycerol and pyruvate, to the fermentation action of a derivative of strain JW200 of the microorganism Ther- oanaerobacter ethanolicus (ATTC 31550) wherein the derivative is derived through both induced and spontaneous mutagenesis. The process includes recovering ethanol from the fermentation medium. Distillation means may be used to recover ethanol from the fermen¬ tation medium under anaerobic and thermophilic conditions, or an inert carrier gas such as nitrogen bubbled through the fermenta¬ tion vessel may carry the ethanol from the fermentation medium into a condensation unit. (d) Strain Maintenance Procedure. Stock cultures of all of the aforementioned strains were maintained for 1 to 2 months at room temperature, about 25°C, after growth at 68°C in the aqueous nutrient medium supplemented with 20% (w/v) cracked corn or by adding an equal amount of sterile anaerobic glycerol to a growing culture and storing the mixture at -20°C for at least 18 months. (e) Morphology and Taxonomy of the Derivatives. All of the derivative strains exhibit the morphologic and taxono ic charac¬ teristics of the parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550) described in U. S. Patent No. 4,292,407 which is incorporated herein by reference. (f) Isolation of the Parent Strain, Strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550). The parent strain, strain JW200 of the microorganism Thermoanaero- bacter ethanolicus (ATCC 31550) can be isolated by the methods described in a U. S. Patent No. 4,292,407 which is incorporated herein by reference (Arch. Microbiol. Y__, 343-34E-, 1981 which is incorporated herein by reference) and which is known in the art. Samples of the parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550) can also be obtained from the American Type Culture Collection (ATCC) Rockville, MD. Example I Ultraviolet Light Mutagenesis Strain JW200 of the microorganism Thermoanaerobacter ethanol- icus (ATCC 31550), the parent strain, was grown in the aqueous nutrient medium with 1% (w/v) soluble corn starch to early log phase, then transferred to a 3 ml quartz cuvette pregassed with argon and rinsed with reducing solution. The cells were held under a bacteriostatic UV light source at 10 cm for times up to 3 minutes. Samples were withdrawn every 30 seconds and diluted and. rolled out (a type of plating technique for anaerobic microorgan¬ isms known in the art) in 2% (w/v) agar containing 2% (w/v) solu- ble corn starch. Individual colonies were analyzed randomly since most produced alcohol. Strain JW200L of the microorganism Thermo¬ anaerobacter ethanolicus was derived from a UV treatment of 60 seconds. It produced in excess of 80 mM ethanol at 2% (w/v) starch. This strain was further selected for spontaneous mutations by iron deprivation and low growth on pyruvate to give strain JW200L-Fe(7) of the microorganism Thermoanaerbacter ethanolicus (ATCC 31938). Strain JW200L-Large of the microorganism Thermoanaerobacter etha¬ nolicus (ATCC 31936) was derived from strain JW200L of the micro¬ organism Thermoanaerobacter ethanolicus and further selected for good growth on pyruvate. OMP Example II Iron Deprivation Ferredoxins, which are iron containing proteins, have been reported to be involved in ethanol production in some thermophilic anaerobic bacteria; therefore, 0.1 ml of a culture of strain JW200 of the microorganism Thermoanaerobacter ethanol cus (ATCC 31550) was transferred to 1 ml of the aqueous nutrient medium supplemented with 1% (w/v) starch and containing no iron. The culture was incubated at 60°C for 30 minutes. At that time 250 ug/ml was added and incubation was continued for three days. Growth after 3 days was low. These cells were diluted 1/100 into the aqueous nutrient containing 2% . (.w/v) starch grown at 68°C and assayed for ethanol after 24 hours. Cells from the culture strain JW200Fe of the microorganism Thermoanaerobacter ethanolicus, derived as described above, from 2% (w/v) starch produced 80 mM ethanol and 4 mM acetate over 24 hours. The production of ethanol by strain JW200 of the microorgan¬ ism Thermoanaerobacter ethanolicus (ATCC 31550), by the strain JW200L (described in Example I) of the microorganism Thermoanae¬ robacter ethanolicus and by strain JW200Fe of the microorganism Thermoanaerobacter ethanolicus with increasing starch concentra¬ tions is given in Table I below. OM ~ TABLE 1 Ethanol production from starch at increasing concentrations by two derivatives of strain JW200 of the microorganism Thermoanaerobac¬ ter ethanolicus (ATCC 31550) and the parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550). Strains of Thermoanaerobacter ethanolicus (ATCC 31550) JW200 JW200L JW200Fe STARCH aZl ETHANOL mM ETHANOL M ETHANOL M 5 58 59 59 10 82 98 " 82 20 25 210 90 50 8 320 110 100 5 242 245 140 11 142 360 Example III Pyruvate Selection Strain JW200L (derived as described in Example I) of the microorganism Thermoanaerobacter ethanolicus, strain JW200Fe (derived as described in Example II) of the microorganism Thermo¬ anaerobacter ethanolicus, and strain JW200L-Fe (derived by treat¬ ment described in Example I followed by treatment described in Example II) of the microorganism Thermoanaerobacter ethanolicus, were grown in the aqueous nutrient medium with 2% (w/v) solid agar supplemented with 1% (w/v) pyruvate and both 0.05% (w/v) glucose and xylose. Large and small colonies of strain JW200L of the microorganism Thermoanaerobacter ethanolicus, of strain JW200Fe of the microorganism Thermoanaerobacter ethanolicus, and of strain JW200L-Fe of the microorganism Thermoanaerobacter ethanolicus were selected. Each colony was grown in the aqueous nutrient medium supplemented with both 1% (w/v) ' cellobiose and 1% (w/v) xylose. The best strains were then transferred to the aqueous nutrient medium with 20% (w/v) cracked corn to test for the ability to grow at high substrate concentrations. The best cultures were trans¬ ferred to the aqueous nutrient medium with increasing concentra¬ tions of soluble corn starch. The ethanol production was measured after four days and is shown in Table II below for representative strains (derivative strains). Samples of these derivative strains, specifically strain JW200L-Large of the microorganism Thermoanae¬ robacter ethanolicus (ATCC 31936), strain JW200Fe(3) of the micro¬ organism Thermoanaerobacter ethanolicus (ATCC 31937) and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus (ATCC 31938), can be derived as described in Example I, II and III above or samples can be obtained-from the American Type Culture Collection (ATCC) Rockv lle, MD. Table II Ethanol production from starch for derivatives of strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550). Derivative 'strains of Thermoanaerobacter ethanolicus (ATCC 31937) (ATCC 31936) (ATCC 31938) JW200Fe(3) JW200L-Large JW200L-Fe(7) STARCH g/1 ETHANOL M ETHANOL mM ETHANOL mM 31 366 175 315 62 470 246 195 200 416 210 150 242 272 205 180 Strain JW200L-Large of the microorganism Thermoanaerobacter ethanolicus (ATCC 31936), strain JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus (ATCC 31937), and strain JW200L-Fe- (7) of the microorganism Thermoanaerobacter ethanolicus (ATCC- 31938) have been maintained without reversion for a period of over six months. (g) Aqueous Nutrient Medium. The aqueous nutrient medium referred to in (b) Culture Methods, above, used for isolation, growth or cultivation, and to maintain the isolated parent and derivative strains of Thermoanaerobacter ethanolicus has the following preferred composition: KH^PO * , 1.5 g/1 ; Na 2 HP0^ * 12H 2 0, 4.2 g/1; NH 4 C1 , 0.5 g/1; MgClg, .0.18 g/1; yeast extract (Difco), 2.0 g/1; glucose, 8.9 g/1; and Wolfe's mineral solution, 5 ml. The medium is prepared under anaerobic conditions and must be stored under an atmosphere of an inert gas, such as nitrogen or argon. The pH of the medium is in the range of about 6.8 and 7.8, preferably 7.3, and is adjusted as required with a sterile, anae¬ robic NaOH or HC1 solution. (h) Thermoanaerobacter ethanolicus Morphology and Toxonomy. The following more particularly describes (e) Morphology and Tox¬ onomy, above: (i) Morphology: Cells grown at 60° C. show tumbling motility. Older cells, or grown in liquid or at higher tempera- tures, such as 75° C, show less motility although they are flag¬ ellated. The flagellation is of the retarded peritrichous type, with between 1 and 10 flagella that are up to 80 ym long. Young cell rods during logarithmic growth often show pointed ends. These rods are 4 to 8 ym long and 0.6 to 0.9 ym thick. Cell rods of the late logarithmic growth phase can grow up to 200 ym long, which then may divide into chains of bacteria during the beginning of the stationary growth phase; (ii) Spores: Spores are not formed; (iii) Other Characteristics: The strains are strictly anaerobic, gram-variable, catalase-negative, pyruvate is metabo- lized via pyruvate-ferredoxin reductase system. Two anaerobic ferredoxins and a rubredoxin are present; and (iv) Taxonomy: The strains have some characteristics similar to Clostridium thermohydrosulfuricum, the only other known extreme thermophil c, anaerobic, glycolytic bacteria. However, the new strains do not form spores, thus excluding them from the genus Clostridium. Other properties described in this application appear to exclude the new strains from previously identified gen- era that are described in the 8th edition of Bergey's Manual of Determinative Bacteriology (Williams and Wilkins Comp. Baltimore) 1974. Therefore, the new strains , represent a new genus and new species that has been named Thermoanaerobacter ethanolicus, wherein ATCC 31550 is representative of these strains. (i) Isolation of Thermoanaerobacter ethanolicus. The spe¬ cific method of isolation, (f) Isolation of the Parent Strain, Strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550), referred to above, is more particularly described as follows: samples from the hot springs of Yellowstone National Park were collected under sterile anaerobic conditions; one g of samples were used to incubate 100 ml of the nutrient medium described hereinabove; the samples were incubated at 74° C; after 3 days dilution rolls were made using anaerobic tubes in accord- ance with the Hungate technique as modified by Bryant and Robinson; after 2 days incubation at 74° C, agar shakes were made from the tubes of the highest dilution which exhibited growth; the nutrient medium, supplemented with 2% agar was used for the agar cultures; the agar shakes were rolled out and incubated at 60° C. in a 30° slanted position; and final strain cultures were obtained by selecting single colonies and repeating the agar shake procedure several times. OMP The foregoing examples illustrate specific embodiments within the scope of this invention and are not to be construed as limiting said scope. While the invention has been described herein with regard to certain specific embodiments, it is not so limited. It is to be understood that variations and modifications thereof may be made by those skilled in the art without departing from the scope of the invention. Industrial Applicability This invention is useful in fermenting starch, cellobiose, glucose,, xylose, and other sugars to ethanol. OMP

Claims: Clai s What is claimed is: 1. A biologically pure culture of a derivative of the microor- ganism Thermoanaerobacter ethanol cus having the ability to con¬ tinuously produce recoverable amounts of ethanol such that a sub¬ strate can be added to a fermentation and the ethanol can be removed therefrom during a fermentation under anaerobic and ther¬ mophilic conditions in an aqueous nutrient medium containing the substrate at a substrate concentration in the fermenat on medium greater than 1% (w/v) wherein the substrate is selected from the group consisting of starch, pectin, glycerol, pyruvate, monosac- charides, and disaccharides. 2. The biologically pure culture of the derivative of claim 1 wherein the derivative is derived from strain JW200 of the micro¬ organism __]_____________ _____l__ι_ι having the characteristics of ATCC 31550. 3. The biologically pure culture of the derivative of claim 1 wherein the derivative is selected from the groups consisting of strain 0W200L-Large of the microorganism Thermoanaerobacter etha¬ nolicus having the characteristics of ATCC 31936, strain JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the character- isties of ATCC 31938. 4. The biologically pure culture of the derivative of claim 1 wherein the derivative is derived through mutagenesis means. 5. The biologically pure culture of the derivative of claim 4 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter etha¬ nolicus having the characteristics of ATCC 31936, strain JW200Fe(3) of the microorganism Thermoanaerobacter ethanol cus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the character- isties of ATCC 31938. 6. The biologically pure culture of the derivative of claim 4 having the ability to produce ethanol at an ethanol concentration in the fermentation medium greater than 1.5% (w/v). 7. The biologically pure culture of the derivative of claim 6 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter etha¬ nolicus having the characteristics of ATCC 31936, strain JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanol icus having the character¬ istics of ATCC 31938. 8. The biologically pure culture of the derivative of claim 6 wherein the derivative is ethanol tolerant up to 10% (v/v) ethanol in the fermentation medium. 9. The biologically pure culture of the derivative of claim 8 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter etha¬ nolicus having the characteristics of ATCC 31936, strain JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the character¬ istics of ATCC 31938. 10. The biologically pure culture of the derivative of claim 8 ■ wherein the fermentation is conducted under extreme thermophilic conditions. 11. The biologically pure culture of the derivative of claim 10 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter etha- nolicus having the characteristics of ATCC 31936, strain JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the character¬ istics of ATCC 31938. 12. The biologically pure culture of the derivative of claim 8 wherein the fermentation is conducted at a pH range of about 5.7 to 8.6. 13. The biologically pure culture of the derivative of claim 12 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter etha¬ nolicus having the characteristics of ATCC 31936, strain JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the character¬ istics of ATCC 31938. 14. The biologically pure culture of the derivative of claim 12 wherein the fermentation is conducted at a temperature range between about 40°C and 78°C. 15. The biologically pure culture of the derivative of claim 14 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter etha¬ nolicus having the characteristics of ATCC 31936, strain JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the character¬ istics of ATCC 31938. 16. The biologically pure culture of the derivative of claim 14 ' wherein the monosaccharides are selected from the group consisting of glucose, fructose, mannose, galactose, xylose, and ribose. 17. The biologically pure culture of the derivative of claim 16 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter etha- nolicus having the characteristics of ATCC 31936, strain JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the character¬ istics of ATCC 31938. 18. The biologically pure culture of the derivative of claim 14 wherein the disaccharides are selected from the group consisting of cellobiose, sucrose, lactose and maltose. 19. The biologically pure culture of the derivative of claim 18 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter etha¬ nolicus having the characteristics of ATCC 31936, strain JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the character¬ istics of ATCC 31938. 20. A process for continuously producing recoverable amounts of ethanol such that a substrate can be added to a fermentation and the ethanol can be removed therefrom during a fermentation which comprises subjecting an aqueous nutrient medium containing the substrate at a substrate concentration in the fermentation medium greater than 1% (w/v) wherein the substrate is selected from the group consisting of starch, pectin, glycerol, pyruvate, onosac- charides, and disaccharides, under anaerobic and thermophilic con¬ ditions to the fermentation action of a derivative of the micro¬ organism Thermoanaerobacter ethanolicus. 21. A process according to claim 20 wherein the derivative is derived from strain JW200 of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31550. 22. A process according to claim 20 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter ethanolicus having the character¬ istics of ATCC 31936, strain JW200Fe(3) of the microorganism Therm- oanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31938. 23. A process according to claim 20 wherein the ' derivative is derived through mutagenesis means. . _ O PI 24. A process according to claim 23 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter ethanolicus having the character- istics of ATCC 31936, strain JW200Fe(3) of the microorganism Therm- oanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31938. 25. A process according to claim 23 wherein ethanol is produced at an ethanol concentration in the fermentation medium greater than 1.5% (w/v). 26. A process according to claim 25 wherein the derivative is selected from the group consisting o strain JW200L-Large of the microorganism Thermo'anaerobacter ethanolicus having the character- istics of ATCC 31936, strain JW200Fe(3) of the microorganism Therm- oanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanol cus having the characteristics of ATCC 31938. 27. A process according to claim 25 wherein the derivative is eth- anol tolerant up to 10% (v/v) ethanol in the fermentation medium. 28. A process according to claim 27 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter ethanolicus having the character¬ istics of ATCC 31936, strain JW200Fe(3) of the microorganism Therm- • oanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanol cus having the characteristics of ATCC 31938. 29. A process according to claim 27 conducted under extreme ther¬ mophilic conditions. 30. A process according to claim 29 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter ethanolicus having the character¬ istics of ATCC 31936, strain JW200Fe(3) of the microorganism Therm¬ oanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31938. • 31. A process according to claim 27 conducted at a pH range of about 5.7 to 8.6. 32. A process according to claim 31 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter ethanolicus having the character¬ istics of ATCC 31936, strain JW200Fe(3) of the microorganism Therm- oanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31938. 33. A process according to claim 31 conducted at a temperature range between about 40°C and 78°C. 34. A process according to claim 33 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter ethanolicus having the character¬ istics of ATCC 31936, strain JW200Fe(3) of the microorganism Therm- oanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31938. 35. A process according to claim 33 wherein ethanol is recovered. 36. A process according to claim 35 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter ethanolicus having the character¬ istics of ATCC 31936, strain JW200Fe(3) of the microorganism Therm- oanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31938. 37. A process according to claim 35 wherein the monosaccharides are selected from the group consisting of glucose, fructose, man- nose, galactose, xylose, and ribose. 38. A process according to claim 35 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter ethanolicus having the character- istics of ATCC 31936, strain JW200Fe(3) of the microorganism Therm- oanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31938. 39. A process according to claim 35 wherein the disaccharides are selected from the group consisting of cellobiose, sucrose, lactose and maltose. 40. A process according to claim 39 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter ethanolicus having the character- istics of ATCC 31936, strain JW200Fe(3) of the microorganism Therm- oanaerobacter ethanolicus having the characteristics of ATCC 31937, and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31938.

Inventors: CARREIRA LAURA H, LJUNGDAHL LARS G, CARREIRA, LAURA, H., LJUNGDAHL, LARS, G.
Applicants: UNIV GEORGIA, UNIVERSITY OF GEORGIA RESEARCH FOUNDATION, INC.
Application year: 1982
Main classification: C12P7
IPC codes: C12N1, C12N15, C12P7