Rieckenberg Fabian, Götz Katharina, Hilterhaus Lutz, Liese Andreas, Zeng An-Ping
Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, TUHH, V-1, Denickestrasse 15, 21073, Hamburg, Germany,
Bioprocess Biosyst Eng. 2014 Dec;37(12):2475-82. doi: 10.1007/s00449-014-1225-2. Epub 2014 Jun 4.
The hyperthermophilic archaeon Pyrococcus furiosus is an interesting organism for research and application, especially owing to its unique NADPH-dependent hydrogenase I. However, mass production of P. furiosus through fermentation is susceptible to fault because of its sensitivity to oxygen, a short exponential and stationary phase and a rapid cell lysis in typical cultivation process. In this study, significant improvement for pilot plant scale production processes for P. furiosus biomass was made by investigations of the fermentation process with subsequent hydrogenase I enzyme purification. Scale-up in a 300-L stirred tank bioreactor was successfully achieved. A repeated-batch cultivation process with high reproducibility and productivity was realized. Furthermore, the enzyme hydrogenase I was purified, and its activity tested and verified. The improvements in this production process for the production of large amount of P. furiosus biomass and hydrogenase I have been achieved, especially by successfully implementing the following key measures and steps: unsterile cultivation setup, skipping typical intermediate preculture and inoculation steps, accelerating the cultivation process by defining an optimal state of the inoculation, optimal time point of biomass harvesting and finally by choosing a one-step purification procedure for enzyme recovery.
嗜热古菌激烈火球菌是一种在研究和应用方面都很有趣的生物，特别是因其独特的依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的氢化酶I。然而，通过发酵大规模生产激烈火球菌容易出现故障，因为它对氧气敏感，在典型培养过程中指数生长期和稳定期较短，且细胞快速裂解。在本研究中，通过对发酵过程进行研究并随后纯化氢化酶I，对激烈火球菌生物量的中试规模生产工艺有了显著改进。成功实现了在300升搅拌罐生物反应器中的放大培养。实现了具有高重现性和生产力的重复批次培养过程。此外，对氢化酶I进行了纯化，并对其活性进行了测试和验证。通过成功实施以下关键措施和步骤，尤其实现了大量生产激烈火球菌生物量和氢化酶I的生产工艺改进：非无菌培养设置、跳过典型的中间预培养和接种步骤、通过确定接种的最佳状态和生物量收获的最佳时间点来加速培养过程，最后通过选择一步纯化程序来回收酶。
