Classical and Modern Genetic Manipulations for Improving Butanol Production by Clostridium Acetobutylicum in Extruded Corn Broth (Tolerance, Transformation, Dnase)
Lin, Yun-Long
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https://hdl.handle.net/2142/70075
Description
Title
Classical and Modern Genetic Manipulations for Improving Butanol Production by Clostridium Acetobutylicum in Extruded Corn Broth (Tolerance, Transformation, Dnase)
Author(s)
Lin, Yun-Long
Issue Date
1984
Department of Study
Food Science
Discipline
Food Science
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Abstract
By employing serial enrichment, a derivative of Clostridium acetobutylicum ATCC 824 was obtained which grew at concentrations of butanol that prevented growth of the wild-type strain. The SA-1 mutant produced consistently higher concentrations of butanol (from 5 to 14%) and lower concentrations of acetone (12.5 to 40%) than the wild-type strain in 4-20% extruded corn broth (ECB). SA-1 demonstrated higher conversion efficiency of extruded corn to butanol than the wild type strain at every concentration of ECB tested. Characterization of the wild-type and SA-1 strain in 6% ECB demonstrated the superiority of the latter in terms of growth rate, time of onset of butanol production, carbohydrate utilization, pH resistance, and final butanol concentration in the fermentation broth.
Development of a plasmid gene transfer system for Clostridium acetobutylicum SA-1 is a prerequisite for plasmid-genetic manipulation of this microorganism. Heat treatment of C. acetobutylicum SA-1 protoplasts at 55(DEGREES)C for 15 min prior to transformation, resulted in expression in this microorganism of the kanamycin (km) resistance determinant associated with pUB110. Agar and agarose gel enzymic assay indicated that treatment at 55(DEGREES)C for 15 min completely or significantly inactivated the deoxyribonuclease (DNase) activity associated with SA-1 protoplasts. Plasmid pUB110 DN was isolated from km('r) SA-1 transformant cultures using a modification of the procedure employed for Clostridium perfringens. For inactivation DNase activity, diethyl pyrocarbonate was incorporated into protocols. Restriction studies further verified the presence of pUB110 DNA in km('r) transformants of SA-1.
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