Atmospheric and room temperature plasma and adaptive evolution were combined to generate Escherichia coli mutants, which can simultaneously and efficiently utilize glucose and xylose to produce succinic acid in chemically defined medium under exclusively anaerobic condition. Compared to the parent strain BA305, a pflB, ldhA, ppc, and ptsG deletion strain overexpressing ATP-forming phosphoenolpyruvate (PEP) carboxykinase (PEPCK), the sugar consumption rate and succinic acid productivity of mutant BA408 were significantly improved with a marked increase in the key enzyme activities. Subsequent anaerobic fermentation of BA408 with corn stalk hydrolysate produced a final succinic acid concentration of 23.1 g L-1 with a yield of 0.85 g g(-1) sugar mixture. The observed synthesis of succinic acid from the corn stalk hydrolysate showed a great potential usage of renewable biomass as a feedstock for an economical succinic acid production using E. coli. (C) 2014 Published by Elsevier Inc.