Development and Initial Characterization of Ethanol-Furfural Dual Resistant Derivatives of Escherichia Coli Fbr5

The future of bioethanol as a viable  alternative fuel
source will be dependent in part on developing a process that utilizes inexpensive
feedstocks for fermentation.  Corn stover, when compared to other feedstocks,
is a relatively economical domestic raw material. To use corn stover, a complex
pretreatment is required to release usable sugars from the hemicellulose. 
During this pretreatment process toxic byproducts are produced. One major
inhibitory toxin produced during this process is the aldehyde furfural. 
Furfural is detrimental to the growth of cells and therefore negatively impacts
the production of ethanol.  Ethanol, the desired product, is also harmful to
the cell and inhibits fermentation. These toxins are one of many reasons the development
of the hemicellulose bioethanol industry has been hindered.

In an attempt to produce a biocatalyst capable of overcoming
the inhibition of these toxins, dually resistant derivatives of FBR5 have been
isolated. These derivatives were isolated by a sequential selection process. 
First, furfural resistant mutants of FBR5 were isolated from media supplemented
with toxic concentrations of furfural.  Two individual furfural resistant derivatives
were then exposed to several rounds of liquid enrichment in ethanol containing
media.  To initially characterize these dual resistance mutants, an experiment
comparing growth after 24 hours at varying ethanol concentrations was conducted.
The preliminary results suggest that dual resistant mutants exhibit improved
growth when compared to FBR5.  Additionally, a membrane permeability assay is
being developed to determine what affects ethanol and furfural have on membrane
permeability.  Qualitative plate assays also suggest that the furfural
resistance phenotype has not been compromised by the selection of the ethanol
resistance phenotype.  Pilot scale fermentations will also be conducted to
compare the dual resistant strain performance as a biocatalyst to the furfural
resistant strains, the ethanol resistant strains, and FBR5.