碳中和背景下全生物基乙醇酸得到了广泛的关注,但其高昂发酵成本、匮乏的下游分离纯化工艺研究,限制了后续的工业生产应用。该研究以重组大肠杆菌为对象,采用统计学方法在摇瓶水平上对发酵培养基成分进行优化,并基于5 L发酵罐进行初步放大验证和下游分离纯化研究。最终利用低廉的玉米芯水解液等成分,摇瓶水平乙醇酸产量达到4.77 g/L,较初始培养基提高2.58倍;5 L罐乙醇酸产量达42 g/L,提高至优化前的1.4倍;通过初步的生物分离纯化,成功获得生物基乙醇酸,晶体纯度达到99.3%,符合市售标准要求。该研究通过发酵培养基优化、5 L发酵罐放大验证以及下游生物分离纯化,初步构建了生物基乙醇酸的低成本生产体系,为生物基乙醇酸的工业化生产应用奠定基础。
The whole biological synthesis of glycolic acid has the characteristics of green and high efficiency. In the context of carbon neutralization, fully bio-based glycolate has received extensive attention, but its high fermentation cost and lack of downstream separation and purification technology have limited subsequent industrial applications. In this study, recombinant Escherichia coli was used as the object, the suitable glycolate production strains were screened by the high-throughput screening platform which constructed using the glycolate biosensor pGBS-PffS-sfgfp. The fermentation medium components were optimized by statistical method at the shaking flask level using cheap corncobs hydrolysate and other substrates, and preliminary amplification verification and downstream separation and purification were conducted based on 5 L fermenter. Finally, the optimal fermentation medium component was given as 6.66 mg/L CaCl2, 12 mg/L MgSO4·7H2O, 0.13 mg/L ZnCl2, 0.04 mg/L MnCl2·4H2O, 4.5 g/L KH2PO4, 13 g/L corncobs hydrolysate, 0.78 g/L NH3·H2O, 1.5 g/L yeast, 8.5 g/L tryptone, 0.5 g/L NaCl, 25.45 g/L Na2HPO4, the yield of glycolate reached 4.77 g/L, which was 2.58 times higher than the initial medium; the yield of 5 L tank was 42 g/L to 1.4 times before optimization. Preliminary biological separation and purification of glycolate fermentation solution was conducted by means of activated carbon decolorization, calcium hydroxide precipitation and cooling crystallization, and bio-based glycolate was successfully obtained, and the purity of glycolate was detected by HPLC. The results showed that the final glycolate crystal purity reached 99.3%, which met the commercial standard. In this study, through optimization of fermentation medium, amplification verification in 5 L fermentation tank, and downstream separation and purification, a low-cost production system of bio-based glycolate was initially constructed, which laid the foundation for industrial production of bio-based glycolate.
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