We developed a novel xylose-utilizing ethanologenic Escherichia coli strain FBR3 with the potential for fermentation of mixed sugars from lignocellulosic hydrolysates into ethanol in an antibiotic-free media, This stain carries the plasmid pLOI297 which contains the genes from Zymomonas mobilis necessary for efficiently converting pyruvate into ethanol. Strain FBR3 selectively maintains pLOI297 when grown anaerobically. Cultures of strain FBR3 were serially transferred ten times in aerobic and anaerobic cultures containing either glucose or xylose with no selective antibiotic. An average of 97.4 +/-3.5% of the cells maintained pLOI297 in anaerobic cultures. In contrast, the plasmid quickly disappeared from aerobic cultures. Plasmid maintenance depends upon deletion of two enzyme activities : pyruvate formate lyase (pfl) and lactate dehydrogenase (Idh). The stability of the pfl mutation was confirmed by the absence of hydrogen gas production, an indirect assay for pfl activity, in each of the cultures. The FBR3 strain was transferred on xylose-containing medium and tested in pH-controlled batch fermentations for efficient conversion of pentoses and hexoses into ethanol. The batch medium contained either 10% (w/v) arabinose, glucose, xylose, or a mixture of these sugars. Fermentations were completed in 70-80 h and ethanol yields were 90-91% of theoretical; maximum ethanol concentrations were 4.38-4.66% (w/v). The fermentation performance of the new FBR3 strain compared favorably to the previously reported performance of strain K011. (C) 1998 Elsevier Science Inc.