Roca E, Meinander N, Hahn-Hägerdal B
Chemical Engineering Department, University of Santiago de Compostela, Santiago de Compostela, Spain.
Biotechnol Bioeng. 1996 Aug 5;51(3):317-26. doi: 10.1002/(SICI)1097-0290(19960805)51:3<317::AID-BIT7>3.0.CO;2-G.
Continuous xylitol production with two different immobilized recombinant Saccharomyces cerevisiae strains (H475 and S641), expressing low and high xylose reductase (XR) activities, was investigated in a lab-scale packed-bed bioreactor. The effect of hydraulic residence time (HRT; 1.3-11.3 h), substrate/cosubstrate ratio (0.5 and 1), recycling ratio (0, 5, and 10), and aeration (anaerobic and oxygen limited conditions) were studied. The cells were immobilized by gel entrapment using Ca-alginate as support and the beads were treated with Al(3+) to improve their mechanical strength. Xylose was converted to xylitol using glucose as cosubstrate for regeneration of NAD(P)H required in xylitol formation and for generation of maintenance energy. The stability of the recombinant strains after 15 days of continuous operation was evaluated by XR activity and plasmid retention analyses. Under anaerobic conditions the volumetric xylitol productivity increased with decreasing HRT with both strains. With a recycling ratio of 10, volumetric productivities as high as 3.44 and 5.80 g/L . h were obtained with the low XR strain at HRT 1.3 h and with the high XR strain at HRT 2.6 h, respectively. However, the highest overall xylitol yields on xylose and on cosubstrate were reached at higher HRTs. Lowering the xylose/cosubstrate ratio from 1 to 0.5 increased the overall yield of xylitol on xylose, but the productivity and the xylitol yield on cosubstrate decreased. Under oxygen limited conditions the effect of the recycling ratio on production parameters was masked by other factors, such as an accumulation of free cells in the bioreactor and severe genetic instability of the high XR strain. Under anaerobic conditions the instability was less severe, causing a decrease in XR activity from 0.15 to 0.10 and from 3.18 to 1.49 U/mg with the low and high XR strains, respectively. At the end of the fermentation, the fraction of plasmid bearing cells in the beads was close to 100% for the low XR strain; however, it was significantly lower for the high XR strain, particularly for cells from the interior of the beads.
在实验室规模的填充床生物反应器中，研究了使用两种表达低和高木糖还原酶（XR）活性的不同固定化重组酿酒酵母菌株（H475和S641）连续生产木糖醇的情况。研究了水力停留时间（HRT；1.3 - 11.3小时）、底物/共底物比例（0.5和1）、循环比（0、5和10）以及通气（厌氧和氧限制条件）的影响。使用海藻酸钙作为载体通过凝胶包埋法固定细胞，并使用Al(3+)处理珠子以提高其机械强度。以葡萄糖作为共底物将木糖转化为木糖醇，用于木糖醇形成过程中所需的NAD(P)H再生以及维持能量的产生。通过XR活性和质粒保留分析评估连续运行15天后重组菌株的稳定性。在厌氧条件下，两种菌株的木糖醇体积生产率均随HRT的降低而增加。循环比为10时，在HRT为1.3小时的低XR菌株和HRT为2.6小时的高XR菌株中，木糖醇体积生产率分别高达3.44和5.80 g/L·h。然而，在较高的HRT下，木糖醇在木糖和共底物上的总产率最高。将木糖/共底物比例从1降至0.5会增加木糖醇在木糖上的总产率，但生产率和木糖醇在共底物上的产率会降低。在氧限制条件下，循环比对生产参数的影响被其他因素掩盖，例如生物反应器中游离细胞的积累以及高XR菌株的严重遗传不稳定性。在厌氧条件下，不稳定性不太严重，低XR菌株和高XR菌株的XR活性分别从0.15降至0.10和从3.18降至1.49 U/mg。发酵结束时，低XR菌株珠子中携带质粒的细胞比例接近100%；然而，高XR菌株的这一比例明显较低，特别是珠子内部的细胞。
