Brauneck Gesa, Heykena Mareke Sophie, Schütterle Dorothea M, Mann Marcel, Schlembach Ivan, Rosenbaum Miriam A, Magnus Jorgen Barsett
AVT - Biochemical Engineering, RWTH Aachen University, Forckenbeckstraße 51, Aachen, 52074, Germany.
Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Jena, Germany.
Appl Biochem Biotechnol. 2025 Sep 9. doi: 10.1007/s12010-025-05378-y.
Microbial co-cultures provide significant advantages over commonly used axenic cultures in biotechnological processes, including increased productivity and access to novel natural products. However, differentiated quantification of the microorganisms in co-cultures remains challenging using conventional measurement techniques. To address this, a fluorescence-based approach was developed to enable the differentiated online monitoring of microbial growth in co-cultures. Co-cultures of Streptomyces species (Streptomyces sp./spp.) with Trichoderma reesei (T. reesei) were investigated. T. reesei was tagged with the fluorescent protein mCherry, while Streptomyces spp. were used as untagged wild-type strains. First, two-dimensional (2D) fluorescence spectra of the individual microorganisms were recorded using an in-house-built device to identify characteristic fluorescence areas for online monitoring of Streptomyces spp. These spectra revealed areas of strong autofluorescence for Streptomyces spp., while T. reesei exhibited little to no autofluorescence, making these areas promising for differentiating the microorganisms in co-cultures. Time-resolved measurements further refined these results, identifying the wavelength combination 405/580 nm as particularly useful for online monitoring the growth of Streptomyces spp. To online monitor the co-culture, autofluorescence for Streptomyces spp. and mCherry fluorescence for T. reesei were used. This approach allowed for differentiated online monitoring of the co-cultures, confirmed by microscopic images. Additionally, the developed method was applied to screen varying inoculation ratios of the microorganisms in co-culture, revealing dynamic changes in the co-culture composition. This highly promising method enables reliable, differentiated online monitoring of co-cultures and has the capacity for expansion to other species, making further validation essential to confirm its potential.
在生物技术过程中，微生物共培养相对于常用的无菌培养具有显著优势，包括提高生产力和获得新型天然产物。然而，使用传统测量技术对共培养物中的微生物进行差异定量仍然具有挑战性。为了解决这个问题，开发了一种基于荧光的方法，以实现对共培养物中微生物生长的差异在线监测监测。研究了链霉菌属（链霉菌种/菌株）与里氏木霉（T. reesei）的共培养。里氏木霉用荧光蛋白mCherry标记，而链霉菌属用作未标记的野生型菌株。首先，使用自制设备记录单个微生物的二维（2D）荧光光谱，以确定用于在线监测链霉菌属的特征荧光区域。这些光谱显示链霉菌属有很强的自发荧光区域，而里氏木霉几乎没有或没有自发荧光，这使得这些区域有望用于区分共培养物中的微生物。时间分辨测量进一步完善了这些结果，确定波长组合405/580 nm对在线监测链霉菌属的生长特别有用。为了在线监测共培养物，使用了链霉菌属的自发荧光和里氏木霉的mCherry荧光。这种方法允许对共培养物进行差异在线监测，显微镜图像证实了这一点。此外，所开发的方法被应用于筛选共培养物中微生物的不同接种比例，揭示了共培养物组成的动态变化。这种非常有前景的方法能够对共培养物进行可靠的、差异的在线监测，并且有扩展到其他物种的能力，因此进一步验证以确认其潜力至关重要。
