CA1188237A - High ethanol producing derivatives of thermoanaerobacter ethanolicus - Google Patents
High ethanol producing derivatives of thermoanaerobacter ethanolicusInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
Abstract of the Disclosure Derivatives of the newly discovered microorganism Thermoanaerobacter ethanolicus 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. The process includes the use of an aqueous fermentation medium, containing the substrate at a substrate concentration greater than 1% (w/v).
Description
HIGH ETHANOL PRODUCING DERIVATIVES OF THERMOANAEROBACTER ETHANOLICUS
Technical Field This invention relates to derivative stralns of a ne~ly discovered microorganism Thermoanaerobacter ethanolicus, a thermophilic anaerobe.
More speciflcally, this invention relates to producing ethanol using derivatives of the microorganism Thermoanaerobacter 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 bacteria, which ferment substrates such as starch, cellobiose, glucose, xylose, and other sugars to ethanol as the main product, have been reported. The microorganism Thermoanaerobacter ethanolicus is described in U.S. patent No. 492929407, September 29, 1981 of Ljungdahl et al. A related patent is U.S. patent No. 4,292,406, September 29, 1981 of LJungdahl et al.
Disclosure of Invention The present invention overcomes problems of fermentations yielding low concentrations o-F ethanol using the microorganism Thermoana robacter ethanolicus.
The present invention produces ethanol at a substrate concen-tration in a fermentation medium greater than 1~ (w/v) using bio-loyically 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 31g37, 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-yenesis 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 tATCC 31550). These biologically pure cultures of the representative derivative strains, speciEically, strain JW200L-Large of the microorganism Thermoanaerobacter _hanolicus (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 fermentation 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 JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550), tolerates ethanol concentrations of up to 10~ (v/v) ethanol in the fermentation medium only if given time to grow in low concentrations first. The above-referenced derivative strains do not show this characteristic of the parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550).
Accordingly, the present invention seeks to produce ethanol at substrate concentrations in a fermentation medium above 1% (w/v) using biologically pure cultures of derivatives of the microorganism Thermoanaerobacter ethanolicus.
Further, the invention seeks to continuously produce recoverable amounts of ethanol from substrates such as starch, cellobiose, glucose, xylose, and other sugars.
The invention in one aspect pertains to a biologically pure culture of a derivative of the microorganism Thermoanaerobacter ethanolicus having the ability to continuously produce recoverable amounts of ethanol such that a substrate can be added to a fermentation and the ethanol can be removed therefrom during a fermentation under anaerobic and thermophilic conditions in an aqueous nutrient medium containing the substrate at a 3~
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, monosaccharides, and disaccharides.
Another aspect of the invention comprehends a process for continuously producing recoverable amounts oE 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, monosaccharides, and disaccharides, under anaerobic and thermophilic conditions to the fermentation action of a derivative of the microorganism Thermoanaerobacter ethanolicus.
These and other aspects and advantages of this invention will become apparent from a consideration of the accompanying specification and claims.
Modes for Carrying out the Invention The parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus ~ATCC 31550), described in U.S. patent No. 4,292,40~ referred to above 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, lactate, acetate and hydrogen gas. When growing in an aqueous nutrient medium containing 10 g per liter (1% w/v) or less of substrate 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 ethanolicus (ATCC
31550) under optimal conditions, produces 1.8 mol of ethanol per mol of glucose. ~or example, at a concentration of 10 g per liter of glucose, one approximately obtains 0.5% (w/v) ethanol. This level of ethanol is not easily removed by known means of distillation; therefore, derivatives of the parent strain, strain JW200 of the microorganism Ther _anaerobacter 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-I.arge of thc microorganism li rmoan_erobclc~cr ethanolicus (ATCC 31936) strain JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus (ATCC 31937), and JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus (ATCC 31938), produce ethanol at levels in the fermentation medium greater than 1.5% (w/v).
The following are microorganisms, culture methods, fermentations, 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 thanolicus (ATCC 31550), the parent strain, and its high ethanol producing derivatives~
specifically, strain JW200L-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 Thermoanaerobacter ethanolicus (ATTC 31938).
(b) Culture Methods. The foregoing strains are routinely grown or cultivated under anaerobic and thermophilic conditions, specifically 68DC
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 referred to above and Arch. Microbiol. 128, 343 - 348, 1981 and which is known in the art. Extruded cracked 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 derivative 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 sucll as cellobiose, sucrose, lactose, and maltose, monosaccharides sucil as glucose, fructose, mannose, galactose, xylose, and ribose, and glycerol and pyruvate wherein the substrates are fermented to cthanol in the aqueous nutrient ~;
~L
medium.
(c) Fermentations. Fermentations of the aforementioned substrates using the derivative strains continuously produce recoverable 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. Fermentations are conducted under anaerobic and thermophilic conditions or under anaerobic and extreme thermophilic conditions. Extreme thermophilic conditions are understood to mean that fermentations are at 70C or higher. Fermentations are conducted at a pH
range of about 5.7 to 8.6 and at a temperature range between about 40C
and 78C. Thus 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 is provided. This process comprises subjecting 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 Thermoanaerobacter ethanolicus (~TTC 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 tile fermentation medium under anaerobic and thermophilic conditions, or an inert carrier gas such as nitrogen bubbled through the fermentation vessel may carry the ethanol from the fermentation medium into a condensation unit.
(d) Strain ~laintenance Procedure. Stock cultures of all of the aforementioned strains were maintained Eor 1 to 2 months at room tempcrature, about 25C, after growth at 68C in the aqueous nutriellt mediurn supplemented with 20% (w/v) cracked corn or by adding an equal amoullt of sterile anaerobic glycerol to a growing culture and storing the mixture at 20C for at least 18 months.
(e) Morphology and Taxonomy of the Derivatives. All of the derivative strains exhi~it the morphologic and taxonomic characteristics of the parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550) described in U.S. patent No. 4,292,407 referred to above.
(f) Isolation of the Parent Strain, Strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550). The parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550) can be isolated by the methods described in U.S. patent No. 4,292,407 referred to above and Arch. Microbiol. 1 , 343 - 348, 1981, 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, 12301 Parklawn Drive, Maryland, 20852, U.S.A.
Example I
Ultraviolet Light Mutagenesis Strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC
31550), the parent strain, was grown in the aqueous nutrient medium with 1% (wtv) 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 microorganisms known in the art) in 2% (w/v) agar containing 2% (w/v) soluble corn starch.
Individual colonies were analyzed randomly since most produced alcohol Strain JW200L of the microorganism Thermoanaerobacter 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 ethanolicus (ATCC 31936) was derived from strain JW200L of the microorganism Thermoanaerobacter _thanolicus and further selected for good growth on pyruvate.
~ AJ3~7 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 ethanolicus (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 60C for 30 minutes. At that time 250 ug/ml FezS34 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 ~8C and assayed for ethanol after 24 hours. Cells from the culture strain JW200Fe of the microorganism Thermoanaerobacter ethanolicus, derived as described above, from 2CG (w/v) starch produced 80 mM
ethanol and 4 mM acetate over 24 hours.
The production of ethanol by strain J~200 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.
9 1~ 3~
.
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 (~TCC 31550).
Strains of Thermoanaerobacter ethanolicus . .
(ATCC 31550) JW200 JW200L JW200Fe 10STARCH g/l ETHANOL mM ETHANOL mM ETHANOL mM
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 nicroorganism 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 transferred to the aclueous nutrient medium with increasing concentrations 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 stralns, specifically strain JW200L-Large of the microorganism Thermoanaerobacte 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), can be derived as described in Example I, II and III above or samples can be obtained from the American Type Culture Collection (ATCC) Rockville, 12301 Parklawn Drive, Maryland, 20852, U.S.A.
Table II
E~hanol production from starch for derivatives of strain JW200 of themicroorganism Thermoanaerobacter ethanolicus (ATCC 31550).
Derivative Strains of Thermoanaerobacter ethanolicus (ATCC 31937)(ATCC 31936)(ATCC 31938) JW200Fe(3) JW200L-LargeJW200L-Fe(7) STARCH g_ ETHANOL mM ETHANOL mM ETHANOL mM
31 336 l75 315 -~ L~L8 ~3~3~
Strain JW200L-Large of the microorganism Thermoanaerobacter ethanolicus tATCC 31q36), 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: KH2P04, 1.5 9/l; Na2HP04 12H20, 4.2 9/1; NH4Cl, 0.5 9/1; ~IgC12, 0.18 gil; yeast extract (Difco),
Technical Field This invention relates to derivative stralns of a ne~ly discovered microorganism Thermoanaerobacter ethanolicus, a thermophilic anaerobe.
More speciflcally, this invention relates to producing ethanol using derivatives of the microorganism Thermoanaerobacter 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 bacteria, which ferment substrates such as starch, cellobiose, glucose, xylose, and other sugars to ethanol as the main product, have been reported. The microorganism Thermoanaerobacter ethanolicus is described in U.S. patent No. 492929407, September 29, 1981 of Ljungdahl et al. A related patent is U.S. patent No. 4,292,406, September 29, 1981 of LJungdahl et al.
Disclosure of Invention The present invention overcomes problems of fermentations yielding low concentrations o-F ethanol using the microorganism Thermoana robacter ethanolicus.
The present invention produces ethanol at a substrate concen-tration in a fermentation medium greater than 1~ (w/v) using bio-loyically 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 31g37, 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-yenesis 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 tATCC 31550). These biologically pure cultures of the representative derivative strains, speciEically, strain JW200L-Large of the microorganism Thermoanaerobacter _hanolicus (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 fermentation 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 JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550), tolerates ethanol concentrations of up to 10~ (v/v) ethanol in the fermentation medium only if given time to grow in low concentrations first. The above-referenced derivative strains do not show this characteristic of the parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550).
Accordingly, the present invention seeks to produce ethanol at substrate concentrations in a fermentation medium above 1% (w/v) using biologically pure cultures of derivatives of the microorganism Thermoanaerobacter ethanolicus.
Further, the invention seeks to continuously produce recoverable amounts of ethanol from substrates such as starch, cellobiose, glucose, xylose, and other sugars.
The invention in one aspect pertains to a biologically pure culture of a derivative of the microorganism Thermoanaerobacter ethanolicus having the ability to continuously produce recoverable amounts of ethanol such that a substrate can be added to a fermentation and the ethanol can be removed therefrom during a fermentation under anaerobic and thermophilic conditions in an aqueous nutrient medium containing the substrate at a 3~
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, monosaccharides, and disaccharides.
Another aspect of the invention comprehends a process for continuously producing recoverable amounts oE 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, monosaccharides, and disaccharides, under anaerobic and thermophilic conditions to the fermentation action of a derivative of the microorganism Thermoanaerobacter ethanolicus.
These and other aspects and advantages of this invention will become apparent from a consideration of the accompanying specification and claims.
Modes for Carrying out the Invention The parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus ~ATCC 31550), described in U.S. patent No. 4,292,40~ referred to above 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, lactate, acetate and hydrogen gas. When growing in an aqueous nutrient medium containing 10 g per liter (1% w/v) or less of substrate 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 ethanolicus (ATCC
31550) under optimal conditions, produces 1.8 mol of ethanol per mol of glucose. ~or example, at a concentration of 10 g per liter of glucose, one approximately obtains 0.5% (w/v) ethanol. This level of ethanol is not easily removed by known means of distillation; therefore, derivatives of the parent strain, strain JW200 of the microorganism Ther _anaerobacter 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-I.arge of thc microorganism li rmoan_erobclc~cr ethanolicus (ATCC 31936) strain JW200Fe(3) of the microorganism Thermoanaerobacter ethanolicus (ATCC 31937), and JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus (ATCC 31938), produce ethanol at levels in the fermentation medium greater than 1.5% (w/v).
The following are microorganisms, culture methods, fermentations, 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 thanolicus (ATCC 31550), the parent strain, and its high ethanol producing derivatives~
specifically, strain JW200L-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 Thermoanaerobacter ethanolicus (ATTC 31938).
(b) Culture Methods. The foregoing strains are routinely grown or cultivated under anaerobic and thermophilic conditions, specifically 68DC
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 referred to above and Arch. Microbiol. 128, 343 - 348, 1981 and which is known in the art. Extruded cracked 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 derivative 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 sucll as cellobiose, sucrose, lactose, and maltose, monosaccharides sucil as glucose, fructose, mannose, galactose, xylose, and ribose, and glycerol and pyruvate wherein the substrates are fermented to cthanol in the aqueous nutrient ~;
~L
medium.
(c) Fermentations. Fermentations of the aforementioned substrates using the derivative strains continuously produce recoverable 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. Fermentations are conducted under anaerobic and thermophilic conditions or under anaerobic and extreme thermophilic conditions. Extreme thermophilic conditions are understood to mean that fermentations are at 70C or higher. Fermentations are conducted at a pH
range of about 5.7 to 8.6 and at a temperature range between about 40C
and 78C. Thus 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 is provided. This process comprises subjecting 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 Thermoanaerobacter ethanolicus (~TTC 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 tile fermentation medium under anaerobic and thermophilic conditions, or an inert carrier gas such as nitrogen bubbled through the fermentation vessel may carry the ethanol from the fermentation medium into a condensation unit.
(d) Strain ~laintenance Procedure. Stock cultures of all of the aforementioned strains were maintained Eor 1 to 2 months at room tempcrature, about 25C, after growth at 68C in the aqueous nutriellt mediurn supplemented with 20% (w/v) cracked corn or by adding an equal amoullt of sterile anaerobic glycerol to a growing culture and storing the mixture at 20C for at least 18 months.
(e) Morphology and Taxonomy of the Derivatives. All of the derivative strains exhi~it the morphologic and taxonomic characteristics of the parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550) described in U.S. patent No. 4,292,407 referred to above.
(f) Isolation of the Parent Strain, Strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550). The parent strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550) can be isolated by the methods described in U.S. patent No. 4,292,407 referred to above and Arch. Microbiol. 1 , 343 - 348, 1981, 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, 12301 Parklawn Drive, Maryland, 20852, U.S.A.
Example I
Ultraviolet Light Mutagenesis Strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC
31550), the parent strain, was grown in the aqueous nutrient medium with 1% (wtv) 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 microorganisms known in the art) in 2% (w/v) agar containing 2% (w/v) soluble corn starch.
Individual colonies were analyzed randomly since most produced alcohol Strain JW200L of the microorganism Thermoanaerobacter 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 ethanolicus (ATCC 31936) was derived from strain JW200L of the microorganism Thermoanaerobacter _thanolicus and further selected for good growth on pyruvate.
~ AJ3~7 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 ethanolicus (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 60C for 30 minutes. At that time 250 ug/ml FezS34 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 ~8C and assayed for ethanol after 24 hours. Cells from the culture strain JW200Fe of the microorganism Thermoanaerobacter ethanolicus, derived as described above, from 2CG (w/v) starch produced 80 mM
ethanol and 4 mM acetate over 24 hours.
The production of ethanol by strain J~200 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.
9 1~ 3~
.
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 (~TCC 31550).
Strains of Thermoanaerobacter ethanolicus . .
(ATCC 31550) JW200 JW200L JW200Fe 10STARCH g/l ETHANOL mM ETHANOL mM ETHANOL mM
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 nicroorganism 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 transferred to the aclueous nutrient medium with increasing concentrations 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 stralns, specifically strain JW200L-Large of the microorganism Thermoanaerobacte 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), can be derived as described in Example I, II and III above or samples can be obtained from the American Type Culture Collection (ATCC) Rockville, 12301 Parklawn Drive, Maryland, 20852, U.S.A.
Table II
E~hanol production from starch for derivatives of strain JW200 of themicroorganism Thermoanaerobacter ethanolicus (ATCC 31550).
Derivative Strains of Thermoanaerobacter ethanolicus (ATCC 31937)(ATCC 31936)(ATCC 31938) JW200Fe(3) JW200L-LargeJW200L-Fe(7) STARCH g_ ETHANOL mM ETHANOL mM ETHANOL mM
31 336 l75 315 -~ L~L8 ~3~3~
Strain JW200L-Large of the microorganism Thermoanaerobacter ethanolicus tATCC 31q36), 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: KH2P04, 1.5 9/l; Na2HP04 12H20, 4.2 9/1; NH4Cl, 0.5 9/1; ~IgC12, 0.18 gil; yeast extract (Difco),
2.0 9/1; glucose, 8.9 g/l; 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.~ and 7.8, preferably 7.3, and is adjusted as required with a sterile, anae-robic NaOH or HCl solution.
(h) Thermoanaerobacter ethanolicus Morphology and Toxonomy.
The following more particularly describes (e) ~lorphology and Tox-onomy, abcve:
(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 ~m long. Young cell rods during logarithmic growth often show pointed ends. These rods are 4 to 8 ~m long and 0.6 to 0.9 ~m thick. Cell rods of the late logarithmic growth phase can grow up to 200 ~m 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--12- ~L~L~ o~ ~
lized via pyruva-te-ferredoxin reductase system. Two anaerobic ferredoxins and a rubredoxin are present; and (iv) Taxonomy: The strains have some characteristics similar to Clostridiuln thermohydrosulFllricum, the only other known extreme thermophilic, 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 t~anual ofDeterminative 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 sprinss 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 hereinabovei the samples were incubated at 74 C.; after
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.~ and 7.8, preferably 7.3, and is adjusted as required with a sterile, anae-robic NaOH or HCl solution.
(h) Thermoanaerobacter ethanolicus Morphology and Toxonomy.
The following more particularly describes (e) ~lorphology and Tox-onomy, abcve:
(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 ~m long. Young cell rods during logarithmic growth often show pointed ends. These rods are 4 to 8 ~m long and 0.6 to 0.9 ~m thick. Cell rods of the late logarithmic growth phase can grow up to 200 ~m 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--12- ~L~L~ o~ ~
lized via pyruva-te-ferredoxin reductase system. Two anaerobic ferredoxins and a rubredoxin are present; and (iv) Taxonomy: The strains have some characteristics similar to Clostridiuln thermohydrosulFllricum, the only other known extreme thermophilic, 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 t~anual ofDeterminative 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 sprinss 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 hereinabovei 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.
3~
The foregoing examples illustrate specific embodiments within the scope of this invention and are not to be construed as limi-ting 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.
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.
3~
The foregoing examples illustrate specific embodiments within the scope of this invention and are not to be construed as limi-ting 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.
Claims (40)
1. A biologically pure culture of a derivative of the microorganism Thermoanaerobacter ethanolicus having the ability to continuously produce recoverable amounts of ethanol such that a substrate can be added to a fermentation and the ethanol can be removed therefrom during a fermentation under anaerobic and thermophilic conditions in 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, monosaccharides, and disaccharides.
2. The biologically pure culture of the derivative of claim 1 wherein the derivative is derived from strain JW200 of the microorganism Thermoanaerobacter ethanolicus having the characteristics of ATCC 31550.
3. The biologically pure culture of the derivative of claim 1 wherein the derivative is selected from the group consisting of strain JW200L-Large of the microorganism Thermoanaerobacter ethanolicus 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 characteristics of ATCC 31938.
4. The biologically pure culture of the derivative of claim 1 wherein the derivative is derived through mutagenesis means and selected for ethanol production in substrate concentrations of 2% (w/v).
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 ethanolicus 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 characteristics 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 ethanolicus 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 ethanolicus 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 characteristics 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, monosaccharides, and disaccharides, under anaerobic and thermophilic conditions to the fermentation action of a derivative of the microorganism 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 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 characteristics of ATCC 31938.
23. A process according to claim 20 wherein the derivative is derived through mutagenesis means and selected for ethanol production in substrate concentrations of 2% (w/v).
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 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.
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 319379 and strain JW200L-Fe(7) of the microorganism Thermoanaerobacter ethanolicus 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000415173A CA1188237A (en) | 1982-11-09 | 1982-11-09 | High ethanol producing derivatives of thermoanaerobacter ethanolicus |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000415173A CA1188237A (en) | 1982-11-09 | 1982-11-09 | High ethanol producing derivatives of thermoanaerobacter ethanolicus |
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| Publication Number | Publication Date |
|---|---|
| CA1188237A true CA1188237A (en) | 1985-06-04 |
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1982
- 1982-11-09 CA CA000415173A patent/CA1188237A/en not_active Expired
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