An evolved Escherichia coli strain for producing hydrogen and ethanol from glycerol

Glycerol is an attractive feedstock for biofuels since it accumulates as a byproduct during biodiesel operations; hence, here we consider converting glycerol to hydrogen using the formate hydrogen lyase system of Escherichia coli which converts pyruvate to hydrogen. Starting with E. coli BW25113 frdC that lacks fumarate reductase (this mutation reduces repression of hydrogen synthesis during glycerol fermentation) and by using both adaptive evolution and chemical mutagenesis combined with a selection method based on increased growth on glycerol, we obtained an improved strain, HW2, that produces 20-fold more hydrogen in glycerol medium (0.68 ± 0.16 mmol/L/h). HW2 also grows 5-fold faster (0.25 ± 0.01/h) than BW25113 frdC on glycerol, so it achieves a reasonable anaerobic growth rate. Corroborating the increase in hydrogen production, glycerol dehydrogenase activity in HW2 increased 4-fold compared to BW25113 frdC. In addition, a whole-transcriptome study revealed that several pathways that would decrease hydrogen yields were repressed in HW2 (fbp, focA, and gatYZ) while a beneficial pathway which encodes enolase was induced. Ethanol production was also increased 5-fold in the evolved HW2 strain.