Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

Microbial Cell Factories

Research

Robert Hänsch¹  Manfred Rohde²  Florian David³  Antje Berger³  Ezequiel Franco-Lara³ 

[1] Botanical Institute, Technische Universität Braunschweig, Humboldtstraße 1, 38106, Braunschweig, Germany;Helmholtz Centre for Infection Research Inhoffenstraße 7, 38124, Braunschweig, Germany;Institute of Biochemical Engineering, Technische Universität Braunschweig, Gausstrasse 17, 38106, Braunschweig, Germany;

关键词: Propidium Iodide;    Production Intensity;    Antibody Fragment;    Bioreactor Cultivation;    Single Cell Analysis;   

DOI  :  10.1186/1475-2859-10-23

 received in 2011-02-02, accepted in 2011-04-15,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundSingle cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively.ResultsHere different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied.ConclusionsIt was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development.

【 授权许可】

Unknown   
© David et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.