Grünberger Alexander, Schöler Katja, Probst Christopher, Kornfeld Georg, Hardiman Timo, Wiechert Wolfgang, Kohlheyer Dietrich, Noack Stephan
Institute of Bio- and Geosciences IBG-1: Biotechnology Forschungszentrum Jülich Jülich Germany.
SU Development Anti-Infectives Sandoz GmbH Biochemiestrasse 10 Kundl Tyrol Austria.
Eng Life Sci. 2016 Jul 27;17(1):86-92. doi: 10.1002/elsc.201600083. eCollection 2017 Jan.
Development times for efficient large-scale production, utilizing fungal species, are still very long. This is mainly due to the poor knowledge of many important variables related to fungal growth and morphogenesis. We specifically addressed this knowledge gap by combining a microfluidic cultivation device with time-lapse live cell imaging. This combination facilitates (i) studying population heterogeneity at single-cell resolution, (ii) monitoring of fungal morphogenesis in a high spatiotemporal manner under defined environmental conditions, and (iii) parallelization of experiments for statistical data analysis. Our analysis of , the workhorse for antibiotic production worldwide, revealed significant heterogeneity in size, vitality and differentiation times between spore, mycelium and pellets when cultivated under industrially relevant conditions. For example, the swelling rate of single spores in complex medium ( ) and the formation rate of higher branched mycelia in defined glucose medium ( ) were estimated from broad time-dependent cell size distributions, which in turn were derived from computational image analysis of 257 and 49 time-lapse series, respectively. In order to speed up the development of new fungal production processes, a deeper understanding of these heterogeneities is required and the presented microfluidic single-cell approach provides a solid technical foundation for such quantitative studies.
利用真菌物种进行高效大规模生产的开发时间仍然很长。这主要是由于对许多与真菌生长和形态发生相关的重要变量了解不足。我们通过将微流控培养装置与延时活细胞成像相结合，专门解决了这一知识空白。这种结合有助于：（i）在单细胞分辨率下研究群体异质性；（ii）在确定的环境条件下以高时空方式监测真菌形态发生；（iii）使实验平行化以进行统计数据分析。我们对全球抗生素生产的主力军[具体菌种未提及]进行分析后发现，在工业相关条件下培养时，孢子、菌丝体和菌球在大小、活力和分化时间上存在显著的异质性。例如，从广泛的随时间变化的细胞大小分布中估计了复杂培养基中单个孢子的肿胀率（[具体数值未提及]）和限定葡萄糖培养基中高度分支菌丝体的形成率（[具体数值未提及]），这些分布分别来自对257个和49个延时序列的计算图像分析。为了加快新型真菌生产工艺的开发，需要更深入地了解这些异质性，而本文提出的微流控单细胞方法为这种定量研究提供了坚实的技术基础。
