HighlightsPigment production and accumulation is dependent of high C:N ratios in F. oxysporum and A. chevaleri.Red pigment content of F. oxysporum in terms of Absorbance units per gram of biomass increased in 191% through use of blue light. Different light wavelengths stimulate synthesis of additional pigments in A. chevalieri with highest accumulation in red and UV-light.Stimulation of pigment production in co-culture is species – specific, being only accomplished in A. chevalieri. With a rise higher that 500% of a pigment obtained in green light.

Understanding population dynamics is a key factor for optimizing co‐culture processes to produce valuable compounds. However, the measurement of independent population dynamics is difficult, especially for filamentous organisms and in presence of insoluble substrates like cellulose. We propose a workflow for fluorescence‐based online monitoring of individual population dynamics of two filamentous microorganisms. The fluorescent tagged target co‐culture is composed of the cellulolytic fungus Trichoderma reesei RUT‐C30—mCherry and the pigment‐producing bacterium Streptomyces coelicolor A3(2)—mNeonGreen (mNG) growing on insoluble cellulose as a substrate. To validate the system, the fluorescence‐to‐biomass and fluorescence‐to‐scattered‐light correlation of the two strains was characterized in depth under various conditions. Thereby, especially for complex filamentous microorganisms, microbial morphologies have to be considered. Another bias can arise from autofluorescence or pigments that can spectrally interfere with the fluorescence measurement. Green autofluorescence of both strains was uncoupled from different green fluorescent protein signals through a spectral unmixing approach, resulting in a specific signal only linked to the abundance of S. coelicolor A3(2)—mNG. As proof of principle, the population dynamics of the target co‐culture were measured at varying inoculation ratios in presence of insoluble cellulose particles. Thereby, the respective fluorescence signals reliably described the abundance of each partner, according to the variations in the inocula. With this method, conditions can be fine‐tuned for optimal growth of both partners along with natural product formation by the bacterium.

Streptomyces species are intensively studied for their ability to produce a variety of natural products. However, conditions influencing and leading to product formation are often not completely recognized. Therefore, in this study, high-throughput online monitoring is presented as a powerful tool to gain indepth understanding of the cultivation of the model organism Streptomyces coelicolor A3(2). Through online measurements of oxygen transfer rate and autofluorescence, valuable information about availability of nutrients and product formation patterns of the pigments actinorhodin and undecylprodigiosin can be obtained and explained. Therefore, it is possible to determine the onset of pigmentation and to study in detail the influencing factors thereof. One factor identified in this study is the filling volume of the cultivation vessel. Slight variations led to varying pigmentation levels. By combining optical and metabolic online monitoring techniques, the correlation of the filling volume with pigmentation could be explained as a result of different growth trajectories caused by varying specific power inputs and their influence on the pellet formation of the filamentous system. Finally, experiments with the addition of supernatant from unpigmented and pigmented cultures could highlight the applicability of the presented approach to study quorum sensing and cell-cell interaction.