Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1998-05-13
2002-03-19
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S161000, C435S150000, C435S252700
Reexamination Certificate
active
06358717
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a fermentation process for producing butanol using a mutant strain of
Clostridium beijerinckii.
BACKGROUND OF THE INVENTION
The acetone/butanol/ethanol (ABE) fermentation process has received considerable attention in recent years as a prospective process for the production of commodity chemicals, such as butanol and acetone, from biomass. The ABE fermentation is the most widely studied among the anaerobic fermentation processes and is a model for complex primary metabolism fermentations.
The fermentation of carbohydrates to acetone, butanol, and ethanol by solventogenic clostridia is well known. More specifically, it is known in the art that
Clostridium acetobutylicum
can be used in ABE fermentation. For example, U.S. Pat. No. 5,192,673, describes an improved fermentation process for producing high levels of butanol using a mutant strain of
Clostridium acetobutylicum
designated
Clostridium acetobutylicum
ATCC 55025.
Value-added fermentation processes are attractive for several economic and environmental reasons. Prominent among the economic factors is the current surplus of agricultural waste or by-products that can be utilized as inexpensive fermentation substrates.
Butanol is an important industrial chemical. Compared to the currently popular fuel additive, ethanol, butanol is more miscible with gasoline and diesel fuel, has a lower vapor pressure, and is less miscible with water, all of which makes butanol a superior fuel extender. Butanol is currently used as a feedstock chemical in the plastic industry and as a foodgrade extractant in the food and flavor industry. Because of the potential for carcinogen carry-over, the use of petroleum-derived butanol is not desirable for food applications.
In 1989, approximately 10 billion pounds of butanol were produced by petrochemical processes. If this amount of butanol were produced by fermentation using Clostridium and corn as the substrate, this would translate into 136 million additional bushels of corn being utilized. This additional non-food market for corn would have a price stabilizing effect on this commodity. In addition to 7.7 pounds of butanol per bushel of corn, the ABE fermentative process also produces 3.7 pounds of acetone and 1.9 pounds of ethanol. Modeling studies based on butanol at $0.30/pound, corn at $3.00 per bushel and propylene at $0.20/pound suggest that butanol production from corn starch is economically competitive with current petrochemical processes. These studies were dependent on the use of a stable, high-yielding strain of
C. acetobutylicum.
The greatest limitation to the fermentative production of butanol by clostridia is the final concentration of butanol in the fermentation broth. This has been a significant problem since ABE fermentation was first described by Chaim Weizmann in 1912 (Jones, D. T., Woods, D. T., (1986)
Microbiol. Rev.
50:484-524). The problem is caused by an additional rate limiting step in the fermentative route to butanol that is efficient in hydrolyzing starch which affects the final concentration of butanol in the fermentation broth. For example, the wildtype strain of
Clostridium beijerinckii
produces only between 10-12 g/L of butanol. Therefore, in order to make butanol production economical, a need exists for new methods of increasing the levels of butanol produced during fermentation.
SUMMARY OF THE INVENTION
The present invention relates to a method of producing solvents such as butanol, acetone and ethanol by the fermentation of a mutant strain of
Clostridium beijerinckii.
The method involves anaerobically culturing a biologically pure culture of
Clostridium beiierinckii
BA101 in a nutrient medium containing assimilable carbohydrates and optionally, one or more organic acids, such as acetate and butyrate and then recovering the butanol, acetone and ethanol. The inoculated medium is then fermented at a temperature of about 30° C. to about 40° C. for a period of about 30 hours to about 275 hours, preferably from about 45 to about 265 hours. Either batch or continuous fermentation or a combination of both methods can be employed in the methods of the present invention. The butanol, acetone and ethanol produced by the fermentation are then recovered.
The amount of butanol produced by the mutant strain according to the present invention is from about 50% to about 100%, preferably from about 60% to about 85%, greater than the amount of butanol produced by the wildtype strain
Clostridium beijerinckii
under similar fermentation conditions. Furthermore, the process of the present invention produces from about 18.0 g/L to about 21.0 g/L of butanol and the butanol yield resulting from the process of the present invention is from about 30% to about 45% and a solvent yield of from about 45% to about 60%. Preferably, the butanol yield is from about 32% to about 35% and the solvent yield is from about 48% to about 50%.
REFERENCES:
patent: 4521516 (1985-06-01), Lemme et al.
patent: 4757010 (1988-07-01), Hermann et al.
patent: 5063156 (1991-11-01), Glassner et al.
patent: 5192673 (1993-03-01), Jain
patent: 5210032 (1993-05-01), Kashket
ATCC Catalogue of Bacteria, 1996, pp. 94-95.*
Annous, B.A., (1991), “Genetic Enhancement, Regulation, Cellular Localization and Purification of Amylolytic Enzymes Produced byClostridium acetobutylicumATCC 824,” Ph.D. Thesis, 118 pp.
Blaschek, H.P. and White, B.A. (1995), “Genetic systems development in the clostridia,” FEMS Microbiol. Rev. 17:349-356.
Blaschek, H.P. et al. (1994), “Clostridium acetobutylicumMutants with Superior Saccharification and Fermentation Capability for the Conversion of Corn Starch to Butanol,” 7thInternational Symposium on Genetics of Industrial Microorganisms, Jun. 1997, Quebec, Canada.
Chen, C.-K. and Blaschek, H.P. (1999), “Acetate enhances solvent production and prevents degeneration inClostridium beijerinckiiBA!01,” Appl. Microbiol. Biotechnol. 52:170-173.
Chen, C.-K. and Blaschek, H.P. (1999), “Effect of Acetate on Molecular and Physiological Aspects ofClostridium beijerinckiiNCIMB 8052 Solvent Production and Strain Degeneration,” Appl. Environ. Microbiol. 65(2):499-505.
Chen, C.-K. and Blaschek, H.P. (1999), “Examination of Physiological and Molecular Factors Involved in Enhanced Solvent Production byClostridium beijerinckiiGA101,” Appl. Environ. Microbiol. 65(5):2269-2271.
Lin and Blascheck (1983), “Butanol Production by a Butanol-Tolerant Strain ofClostridium acetobutylicimin Extruded Corn Broth,” Appl. Environ. Microbiol. 45:966-973.
Baer, Shirley H., et al., Effect of Butanol Challenge and Temperature on Lipid Composition and Membrane Fluidity of Butanol-TolerantClostridium acetobutylicum, Applied and Environmental Microbiology, 53(12):2854-2861, (1987).
Annous, B., et al., Isolation and Characterization ofClostridium acetobutylicumMutants with Enhanced Amylolytic Activity,Applied and Environmental Microbiology, 57(9):2544-2548, (1991).
Annous, B, et al., Regulation and Localization of Amylolytic Enzymes inClostridium acetobutylicumATCC 824,Applied and Environmental Microbiology, 56(8):2559-2561, (1990).
Jones, David T., et al., Origins and relationships of industrial solvent-producing clostridial strains.FEMS Microbiology Reviews, 17:223-232, (1995).
Johnson, J.L., et al., Taxonomic relationships among strains ofClostridium acetobutylicumand other pheontypically similar organisms,FEMS Microbiology Reviews, 17:233-240, (1995).
Marlett, John A., et al., Acetone-Butanol Fermentation Process Development and Economic Evalution,Biotechnolgy Progress, 2(1):23-28, (1986).
Formanek, J. et al. (1997), “Enhanced Butanol Production byClostridium beijerinckiiBA101 Grown in Semidefined P2 Medium Containing 6 Percent Maltodextrin or Glucose,” Appl. Environ. Microbiol. 63(6):2306-2310.
George, H.A. and Chen, J.-S. (1983), “Acidic Conditions are not Obligatory for Onset of Butanol Formation byClostridium beijerinckii(Synonym, C. butylicum),” Appl. Environ. Microbiol. 46(2):321-327.
Gottschal, J.C. and Morris, J.G. (1981), “The induction of ace
Annous Bassam
Blaschek Hans
Chen Chih-Kuang
Formanek Joseph
Greenlee Winner and Sullivan P.C.
Marx Irene
The Board of Trustees of the University of Illinois
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