Source: UNIVERSITY OF ILLINOIS submitted to
IMPROVEMENT AND CHARACTERIZATION OF CLOSTRIDIUM BEIJERINCKII BA101
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0185853
Grant No.
(N/A)
Project No.
ILLU-698-316
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2000
Project End Date
Sep 30, 2006
Grant Year
(N/A)
Project Director
Blaschek, H. P.
Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
FOOD SCIENCE & HUMAN NUTRITION
Non Technical Summary
Currently 2.58 billion pounds of butanol is produced in the United States. If this is produced from corn, this would create a market for 509 million bushel of corn annually. Development of such a large corn market would improve economic conditions for farmers. Butanol can be produced from corn by fermentation using Clostridium beijerinckii BA101. We intend to develop superior strains for efficient production of butanol from corn. This project would improve economic conditions of farmers. It is anticipated that newly developed strains would ferment corn efficiently.
Animal Health Component
(N/A)
Research Effort Categories
Basic
30%
Applied
30%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
51115102020100%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
1510 - Corn;

Field Of Science
2020 - Engineering;
Goals / Objectives
1. Clostridium beijerinckii BA101 strain development for solvent production. 2. Use of AFLP to identify genetic markers associated with various derivatives of C. beijerinckii 8052. 3. Examination of Non-PTS based sugar transport in C. beijerinckii BA101 and 8052. 4. Physiological characterization of the newly constructed C. beijerinckii mutant and recombinant strains. 5. Downstream processing of butanol from fermentation broth.
Project Methods
We will use modern molecular techniques to identify those genes which are responsible for increased butanol production by Clostridium beijerinckii BA101. Through metabolic engineering, critical biochemical pathways involved in butanol production will be manipulated, creating strains with increased butanol production. In addition to metabolic engineering, we will use novel butanol recovery processes to recover butanol from the fermentation broth economically. Overall this will develop a culture, fermentation process, and a butanol recovery technique that will increase the economic vaibility of fermentative butanol production.

Progress 10/01/00 to 09/30/06

Outputs
The availability of the genomic sequence for the butanol hyper producing Clostridium beijerinckii BA101 strain has allowed for genomic characterization of important genes in the butanol bio-synthetic pathway. Specifically, transcriptional microarray technology analysis of this microorganism has allowed for identification and characterization of unique industrial strain attributes. These results have resulted in a 'roadmap' for construction of future second-generation butanol-producing clostridia.

Impacts
The availability of genomic sequence information for Clostridium beijerinckii will allow for sequence wide comparison with the only other sequenced solventogenic clostridium, namely C. acetobutylicum. Genomic differences can be related to performance attributes and will allow, for the first time, the applications of systems biology to the re-construction of second generation strains for the production of butanol and higher alcohols from biomass.

Publications

  • Lee, J., Mitchell, W.J., Tangney, M. and Blaschek, H.P. 2005. Evidence for the presence of an alternative glucose transport system in Clostridium beijerinckii NCIMB 8052 and the solvent hyper-producing mutant BA101. Appl. Environ. Microbiol. 71:3384-3387.
  • Ezeji, T., Karcher, P.M., Qureshi, N. and Blaschek, H.P. 2005. Improving performance of a gas stripping-based recovery system to remove butanol from Clostridium beijerinckii fermentation. Bioprocess and Biosystems Engineering. 27:207-214.
  • Ezeji, T., Qureshi, N. and Blaschek, H.P. 2005. Industrially Relevant Fermentations. Chapter In: Handbook on Clostridia (Ed: P. Durre) CRC Press p.799-814.
  • Karcher, P., Ezeji, T.C., Qureshi, N. and Blaschek, H.P. 2005. Microbial production of butanol: Product recovery by extraction. In: Microbial Diversity: Current Perspectives and Potential Applications (Ed. Satyanarayana, T.). Kluyver Academic Publishers. p. 865-880.


Progress 01/01/05 to 12/31/05

Outputs
A draft sequence of the Clostridium beijerinckii 8052 genome was completed and annotated. This work is significant because it allows for an examination of global gene regulation of solvent production by this anaerobic fermentative bacterium and the development of superior strains which are tailored for production of second generation biofuels such as butanol from the corn biorefinery.

Impacts
The expected impact of this work relates to the development of second generation strains of Clostridium beijerinckii that have potential for co-product utilization in a dry milling based biorefinery environment thereby improving the overall economics.

Publications

  • Qureshi, N. and Blaschek, H.P. 2005. Butanol production from agricultural biomass. In: Food Biotechnology, Shetty, K., Pomett, A. and Levin, R.E., eds., p.525-549.
  • Karcher, P.M., Ezeji, T.C., Qureshi, N. and Blaschek, H.P. 2005. Microbial production of butanol: Product recovery by extraction. In: Microbial Diversity: Current Perspectives and Potential Applications, Satyanarayana, T. and Johri, B.N., eds., p. 865-880.
  • Ezeji, T.C., Karcher, P.M., Quresh, N. and Blaschek, H.P. 2005. Improving performance of a gas-stripping based recovery system to remove butanol from Clostridium beijerinckii fermentation. Bioprocess Biosys. Eng. 27:207-214.
  • Qureshi, N., Karcher, P., Cotta, M.A. and Blaschek, H.P. 2005. High productivity continuous biofilm reactor for butanol production: Effect of acetic and butyric acids and CSL on bioreactor performance. Appl. Biochemistry and Biotechnology Vol. 113-116, p. 713-721.


Progress 01/01/04 to 12/31/04

Outputs
We continue to examine the application of improved downstream technologies for improved recovery of butanol from the fermentation broth and integrated gas stripping appears to show promise. Use of low value agricultural substrates or co-products also appears to hold promise for imacting the economics of this biobased process.

Impacts
The impact of this work relates to the commercialization of the ABE process which is currently underway with a large midwestern corn wet miller. If butanol was produced from corn, this would create a large market demand. Development of such a large corn market would improve economic conditions for farmers and rural communities.

Publications

  • Ezeji, T.C., Qureshi, N. and Blaschek, H.P. 2003. Production of butanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping. World J. Microbiol. and Biotechnol. 19:595-603.
  • Ezeji, T.C., Qureshi, N. and Blaschek, H.P. 2003. Continuous production of butanol from starch based packing peanuts. Appl. Biochem. Biotechnol. 105-108:375-382.
  • Qureshi, N. and Blaschek, H.P. 2004. Butanol production from agricultural biomass. In: Food Biotechnology (Eds. Shetty, K., Pometto, A. and Paliyath, G.). Marcel Dekker, New York (In Press).


Progress 01/01/03 to 12/31/03

Outputs
Systems for genetic manipulation of C. beijerinckii were developed which are based on a filamentous virus-like particle. These systems offer the opportunity for further improvement of the acetone, butanol, ethanol (ABE) fermentation microroganism for commercial exploitation and conversion of agricultural by-products to value added products. Furthermore, use of by-products such as degermed corn from the corn wet and dry milling industries offer additional opportunities to improve the economic competitiveness of biological production of solvents.

Impacts
The impact of this work relates to the commercialization of the ABE process which is currently underway with a large midwestern corn wet miller.

Publications

  • Li, Y. and Blaschek, H.P. 2002. Molecular characterization and utilization of the CAK1 filamentous viruslike particle derived from Clostridium beijerinckii. J. Industrial Microbiol. and Biotechnol. 28:118-126.
  • Campos, E.J., Qureshi, N. and Blaschek, H.P. 2002. Production of acetone butanol ethanol (ABE) from degermed corn using Clostridium beijerinckii BA101. Appl. Biochem. and Biotechnol. 98:553-561.
  • Blaschek, H.P., Annous, B.A., Formanek, J. and Chen, C.K. 2002. A method of producing butanol using a Mutant Strain of Clostridium beijerinckii. University of Illinois at Urbana-Champaign. Patent #6,358, 717, March 19, 2002.


Progress 01/01/02 to 12/31/02

Outputs
An economic evaluation of the ABE fermentation using Clostridium beijerjinckii BA101 was carried out. In addition, various substrates with respect to their ability to be employed in the production of butanol. Gas stripping was evaluated as an appproach for the continuous removal of toxic products from the fermentation medium with the goal of producing butanol by continuous fermentation.

Impacts
Systems have been developed which improve the potential for commercialization of the ABE fermentation using Clostridium beijerinckii BA101.

Publications

  • Qureshi, N., Lolas, A. and Blaschek, H.P. 2001. Soy molasses as fermentation substrate for production of butanol using Clostridium beijerinckii BA101. J. Industrial Microbiol. and Biotechnol. 26:290-295.
  • Qureshi, N. and Blaschek, H.P. 2001. ABE production from corn: A recent economic evaluation. J. Industrial Microbiol. and Biotechnol. 27:292-297.
  • Qureshi, N. and Blaschek, H.P. 2001. Recent advances in ABE fermentation: Hyper-butanol producing Clostridium beijerinckii BA101. J. Industrial Microbiol. and Biotechnol. 27:287-291.
  • Qureshi, N. and Blaschek, H.P. 2001. Evaluation of recent advances in butanol fermentation, upstream, and downstream processing. Bioprocess Biosystems Engineering. 24:219-226.
  • Qureshi, N. and Blaschek, H.P. 2001. Recovery of butanol from fermentation broth by gas stripping. Renewable Energy. 22:557-564.


Progress 01/01/01 to 12/31/01

Outputs
The solvent-forming genes, constituting the sol operon, have been cloned from both the Clostridium beijerinckii NCIMB 8052 and C. beijerinckii BA101 strains. Restriction enzyme digests indicate that the sequence of the C. beijerinckii BA101 operon is different than the wild-type. Glucose uptake in C. beijerinckii BA101 has also been characterized. It was found that C. beijerinckii BA101 is defective in PTS activity and that the strain compensates for this defect with enhanced glucokinase activity.

Impacts
The sequence differences found in the C. beijerinckii BA101 sol operon may provide an explanation for the enhanced solvent production by this strain. These genes are now being used to construct new strains, with the goal of increasing butanol production.

Publications

  • No publications reported this period


Progress 01/01/00 to 12/31/00

Outputs
The AFLP fingerprints of C. beijerinckii NCIMB 8052, BA101, A10, SA1 and SA2 are >99% identical. This confirms that these strains are derived from a common parent strain, C. beijerinckii 8052. Very few polymorphic fragments were observed, but those that were observed are unique to strains C. beijerinckii BA101, A10 and SA1. It is hoped that these markers for these strains may lead to a genetic explanation for the differing morphologies of these strains.

Impacts
The AFLP results confirm that C. beijerinckii BA101 is derived from NCIMB 8052. The unique DNA polymorphisms for the mutant strain may lead to the genetic alteration that is responsible for the over-production of butanol by this strain.

Publications

  • No publications reported this period