Detailed kinetic studies have been reported in the literature for several re­combinant strains of Z. mobilis from NREL capable of utilizing both glucose and xylose. The initial evaluation by Zhang et al. [10] involved the batch culture growth of the strain CP4 (pZB5) on medium containing 25 g L-1 glu­cose and 25 gL-1 xylose. Batch and continuous culture studies on strain 39676 (pZB4L) were reported subsequently by Lawford et al. [31,33,34]. This strain was derived from the host ATCC 39676 transformed with a plasmid derived from pZB4. Final product values for 40 gL-1 glucose/40 gL-1 xylose medium included 4.04 g L-1 xylitol as well as 36.6 g L-1 ethanol [49] although it should be noted that xylitol levels with this particular recombinant strain were unusually high. Further studies reported by Lawford and Rousseau [35] focused on kinetic and energetic evaluations of strain CP4 (pZB5) in batch and fed-batch fermentations. Kinetic characterization of the chromosoma­lly integrated xylose/arabinose strain AX101 (derived from ATCC 39676) was also reported [37,38].

To determine which of the strains was likely to be most suitable for larger scale ethanol production, a comparative evaluation in batch and continuous

culture of strains CP4(pZB5) and ZM4(pZB5) was carried out by Joachimsthal et al. [28]. From the results it was found that ZM4(pZB5) was capable of con­verting a mixture of 65 g L-1 glucose and 65 g L-1 xylose to more than 60 g L-1 ethanol in 48 h in batch culture with an ethanol yield of 0.46 gg-1, with this latter strain demonstrating superior specific sugar uptake and ethanol pro­duction rates. The results for ZM4(pZB5) are shown in Fig. 3 together with the values of comparative kinetic parameters in Table 2. Higher sugar con­centrations (75 gL-1 each sugar) resulted in incomplete xylose utilization (80 h) presumably due to increasing ethanol inhibition of xylose assimila — tion/metabolism at ethanol concentrations of 65-70 gL-1.

The results for continuous culture with ZM4 (pZB5) and medium contain­ing 40gL-1 glucose and 40 gL-1 xylose are shown in Fig. 4 [28]. While the concentration of glucose was close to zero at dilution rates up to D = 0.15 h-1, increasing residual xylose at dilution rates higher than 0.08 h-1 indicated that the maximum volumetric rate of xylose uptake for the culture had been exceeded. The maintenance energy coefficient (m) under these conditions was estimated by extrapolation as 1.6±0.2gg-1 h-1 (within 95% confidence limits) based on linear regression analysis of the data from Fig. 4a for the maximum specific sugar uptake rate (glucose and xylose) vs. dilution rate (D) (Fig. 4b). A “true biomass yield” of 0.044 gg-1 was determined from the inverse of the gradient of this linear plot. For similar experimental con­ditions, closely related values were observed by Lawford and Rousseau for strain CP4 (pZB5) [34]. However, Lawford and Rousseau noted, when ob-

Mm: maximum specific growth rate (h-1)

(qs)m: maximum specific sugar uptake rate (gg-1 h-1)

(qp)m: maximum specific ethanol production rate (gg-1 h-1) (Yx/s): overall cell yield (based on total sugar utilized) (gg-1) (Yp/s): overall ethanol yield (based on total sugar utilized) (gg-1)

served over the lower dilution rate range of D = 0.04-0.08 h-1, that both strain CP4 (pZB5) and a biomass hydrolysate adapted variant of 39676(pZB4L) exhibited values of m and “true biomass yield” that were significantly lower [35].

Results with a potentially high productivity cell recycle system using a membrane bioreactor are shown in Fig. 5 [29]. From Fig. 5(a), at sugar con­centrations of 50 gL-1 glucose and 50 gL-1 xylose and D = 0.1 h-1, an ethanol productivity of 5 g L-1 h-1 was achieved with an ethanol yield based on total sugars utilized (Yp/s) = 0.50 gg-1. No decline in specific ethanol productivity was evident up to 70 h, however as shown in Fig. 5(b), a decrease in total vi­able cells was observed after an initial steady state (40-50 h). This indicates that for effective longer term operation, high cell concentrations should be

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Fig.4 (a) Kinetics of ethanol production by Z. mobilis ZM4 (pZB5) in continuous culture on medium containing 40 gL-1 glucose and 40 gL-1 xylose (T = 30 °C, pH = 5.0). Sym­bols: biomass •; glucose ♦; xylose □; ethanol ▲ (b) Effect of dilution rate on specific rates of total sugar uptake (qs) and ethanol production (qp). Estimation of maintenance energy (m) value at D = 0 by extrapolation. Symbols: qs °; qp A achieved by less stressful methods than membrane-based cell recycling (e. g., by use of flocculent cells and cell settling).

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