This article describes a systematic study of the nutritional requirements of a patented recombinant ethanologenic Escherichia coli (11303:pLOI297) and provides cost-effective formulations that are compatible with the production of fuel ethanol in fermentations of lignocellulosic prehydrolysate characterized by-high xylose conversion efficiency. A complex and nutrient-rich laboratory medium, Luria bro th (LB), provided the benchmark with resp ect to fermentation performance standard. Xylose fermentation performance was assessed in terms of the target values for operational process parameters established by the US National Renewable Energy Laboratory (NREL)-final ethanol concentration (25 g/L), xylose-to-ethanol conversion efficiency (90%), and volumetric productivity (0.52 g/L . h). Biomass prehydrolysates that are rich in xylose also contain acetic acid, and in anticipation of a need to reduce acetic acid toxicity, the fermentors were operated with a PH control set-point of 7.0 Growth and fermentation in the minimal defined salts (DS) medium was only about 15%-compared to the reference medium. Amendment of the minimal medium containing 6 wt% xylose with both vitamins and amino acids resulted in improved growth, but the volume productivity (0.59 g/L . h) was still only about 54% of that with LB (1.1 g/L . h). Formulations directed at cost reduction through the use of less expensive commercial complex nutritional supplements were within 90% of the NREL process target with respect to yield and provided a productivity at about 80% of the LB medium, but were not economical. Corn steep liquor (CSL) at about 7-8 g/L was shown to be a complete source of nutritional requirements and supported a fermentation performance approaching that of LB. At a cost of CSL of $50/t (dry wt), the economic impact of using this amount CSL as the sole nutritional supplement in a cellulosic ethanol plant was estimated to be about 4 cent/gal of ethanol.