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Title: DEVELOPMENT OF NEW ETHANOLOGENIC ESCHERICHIA COLI STRAINS FOR FERMENTATION OF LIGNOCELLULOSIC BIOMASS

Author
item Dien, Bruce
item Nichols, Nancy
item O Bryan, Patricia
item Bothast, Rodney

Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/20/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: In 1997, over one billion gallons of fuel alcohol were produced from corn starch. Demand for alcohol as a fuel oxygenate is expected to grow substantially. As a consequence, alcohol producers are interested in developing "lignocellulosic" biomasses as an alternate feedstock to corn starch. However, these biomasses are made up of a mixture of sugars, some of which microbes (e.g., beer yeast) currently used to ferment corn starch to alcohol do not ferment. The major technical hurdle to producing alcohol from biomass is to engineer biocatalysts that will convert all of these sugars to alcohol. We have developed a series of novel strains that do just this. In this report, we describe two new strains. Both of these strains have been specially designed to be used in continuous processes, as currently used by alcohol producers.

Technical Abstract: Two new ethanologenic strains (FBR4 and FBR5) of Escherichia coli were constructed and used to ferment corn fiber hydrolysate. The strains carry the plasmid pLOI297, which contains the genes from Zymomonas mobilis necessary for efficiently converting pyruvate into ethanol. Both strains selectively maintained the plasmid when grown anaerobically. Each culture was serially transferred 10 times in anaerobic culture with sugar limited medium containing xylose, but no selective antibiotic. An average of 93% and 95% of the FBR4 and FBR5 cells, respectively, maintained pLOI297 in anaerobic culture. The fermentation performances of the repeatedly transferred cultures were compared to cultures freshly revived from stock in pH controlled batch fermentations with 10% w/v xylose. Fermentation results were similar for all of the cultures. Fermentations were completed within 60 h and ethanol yields were 86-92% of theoretical. Maximal ethanol lconcentrations were 3.9-4.2% w/v. The strains were also tested for their ability to ferment corn fiber hydrolysate, which contained 8.5% w/v total sugars (2.0% arabinose, 2.8% glucose, and 3.7% xylose). Escherichia coli FBR5 produced more ethanol than FBR4 from the corn fiber hydrolysate. E. coli FBR5 fermented all but 0.4% (w/v) of the available sugar, while strain FBR4 left 1.6% unconsumed. The fermentation with FBR5 was completed within 55 h and yielded 0.46 g ethanol per g available sugar, 90% of the maximum obtainable.