Continuous fermentation studies with xylose-utilizing recombinant Zymomonas mobilis

This study examined the continuous cofermentation performance characteristi cs of a dilute-acid "prehydrolysate-adapted" recombinant Zymomonas 39676:pZ B4L and builds on the pH-stat batch fermentations with this recombinant tha t we reported on last year. Substitution of yeast extract by 1% (w/v) corn steep liquor (CSL) (50% solids) and Mg (2 mM) did not alter the cofermentat ion performance. Using declared assumptions, the cost of using CSL and Mg w as estimated to be 12.5 cent/gal of ethanol with a possibility of 50% cost reduction using fourfold less CSL with 0.1% diammonium phosphate. Because o f competition for a common sugar transporter that exhibits a higher affinit y fur glucose, utilization of glucose was complete whereas xylose was alway s present in the chemostat effluent. The ethanol yield, based on sugar used , was 94% of theoretical maximum. Altering the sugar ratio of the synthetic dilute acid hardwood prehydrolysate did not appear to significantly change the pattern of xylose utilization. Using a criterion of 80% sugar utilizat ion for determining the maximum dilution rate (D-max), changing the composi tion of the feed from 4% xylose to 3%, and simultaneously increasing the gl ucose from 0.8 to 1.8% shifted D-max from 0.07 to 0.08/h. With equal amount s of both sugars (2.5%), D-max was 0.07/h. By comparison to a similar inves tigation with rec Zm CP4:pZB5 with a 4% equal mixture of xylose and glucose , we observed that at pH 5.0, the D-max was 0.064/h and shifted to 0.084/h at pH 5.75. At a level of 0.4% (w/v) acetic acid in the CSL-based medium wi th 3% xylose and 1.8% glucose at pH 5.75, the D-max for the adapted recombi nant shifted from 0.08 to 0.048/h, and the corresponding maximum volumetric ethanol productivity decreased 45%, from 1.52 to 0.84 g/(L.h). Under these conditions of continuous culture, linear regression of a Pirt plot of the specific rate of sugar utilization vs D showed that 4 g/L of acetic acid di d not affect the maximum growth yield (0.030 g dry cell mass/g sugar), but did increase the maintenance coefficient twofold, from 0.46 to 1.0 g of sug ar/(g of cell.h).