Sáez Juan Carlos, Schell Daniel J, Tholudur Arun, Farmer Jody, Hamilton Jenny, Colucci José A, McMillan James D
Department of Chemical Engineering, University of Puerto Rico, Mayagüez Campus, PO Box 9046 Mayagüez, Puerto Rico
Biotechnol Prog. 2002 Nov-Dec;18(6):1400-7. doi: 10.1021/bp0200292.
A methodology is described and applied for performing carbon mass balances across cellulase enzyme production processes using both soluble sugar and insoluble cellulose substrates. The fungus Trichoderma reesei was grown on either glucose, lactose, or cellulose in aerobic batch mode, and the evolution of the main carbonaceous components (cell mass, cellulose, soluble protein, adsorbed protein, sugars, and carbon dioxide) was followed. A variety of analytical techniques were utilized to measure these components, including (i) gravimetric analysis, (ii) near-infrared spectroscopy, (iii) bicinchoninic acid based soluble protein measurement, (iv) gas mass spectrometry and flow rate, (v) CHNS/O elemental analyses, and (vi) high-performance liquid chromatography. The combined set of measurements allowed carbon mass balances across the cellulase production process to be assessed to determine the consistency of the underlying kinetic data. Results demonstrate the capability to determine the levels and distribution of all major carbonaceous components during the cellulase production process on both soluble and insoluble substrates. Average carbon mass balance closures were near 100% during early stages (<72 h) of the cultivations using glucose, lactose, or cellulose as the substrates, but carbon mass closures trended high later in the cultivation. Analysis of carbon allocation results suggests that an error in the gas mass flow rate measurement was the primary cause for carbon mass balance closures to exceed 110% late in the process.
本文描述并应用了一种方法，用于在使用可溶性糖和不溶性纤维素底物的纤维素酶生产过程中进行碳质量平衡分析。里氏木霉在有氧分批模式下，分别以葡萄糖、乳糖或纤维素为底物进行培养，并跟踪主要含碳成分（细胞质量、纤维素、可溶性蛋白质、吸附蛋白质、糖类和二氧化碳）的变化情况。利用了多种分析技术来测量这些成分，包括：（i）重量分析；（ii）近红外光谱法；（iii）基于二喹啉甲酸的可溶性蛋白质测量；（iv）气质联用仪和流速测量；（v）CHNS/O元素分析；以及（vi）高效液相色谱法。这一系列测量结果能够评估纤维素酶生产过程中的碳质量平衡，以确定基础动力学数据的一致性。结果表明，该方法能够确定在以可溶性和不溶性底物进行纤维素酶生产过程中所有主要含碳成分的水平和分布情况。在以葡萄糖、乳糖或纤维素为底物的培养早期阶段（<72小时），碳质量平衡平均闭合率接近100%，但在培养后期碳质量闭合率呈上升趋势。对碳分配结果的分析表明，气质流速测量误差是导致后期碳质量平衡闭合率超过110%的主要原因。
