Reliable online measurement of population dynamics for filamentous co‐cultures

Abstract

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.

Tunable population dynamics in a synthetic filamentous coculture

“…To validate the conclusions from this experiment, similar cocultures were conducted with an S. coelicolor strain tagged with an mNG fluorescence protein as reported by Palacio‐Barrera et al ( 2022 ). This work demonstrates a good correlation of the mNG fluorescence intensity to the dry biomass of S. coelicolor and, hence, enables more direct monitoring of individual population dynamics of both organisms.…”

“…Although the OTR only delivers a sum signal of the coculture respiration activity, the metabolic contribution of each partner can be estimated by comparison to axenic cultures. Further, we have developed a fluorescence‐based procedure for online monitoring of population dynamics in filamentous cocultures, which is applied here (Palacio‐Barrera et al, 2022 ). Additionally, we previously could show that fluorescence monitoring can help estimate the onset of pigment biosynthesis for S. coelicolor (Finger et al, 2022 ).…”

“…T. reesei RUT‐C30‐mCherry as well as S. coelicolor A3(2) DSMZ 40783 and Streptomyces coelicolor A3(2)–mNG. Construction of tagged strains was previously reported (Palacio‐Barrera et al, 2022 ). Unless stated otherwise, cultivations were inoculated with 10 6 spores/ml.…”

“…Unless stated otherwise, cultivations were inoculated with 10 6 spores/ml. S. coelicolor spores were prepared similarly to Hobbs et al ( 1989 ) and as mentioned in previous works (Finger et al, 2022 ; Hobbs et al, 1989 ; Palacio‐Barrera et al, 2022 ). Briefly, a spore suspension was spread on soy‐flour mannitol agar plates, which consisted of 20 g/L mannitol, 20 g/L soy flour, and 20 g/L agar.…”

“…The medium for all cultivations was an LNP minimal medium, which is described also in previous works (Finger et al, 2022 ; Palacio‐Barrera et al, 2022 ). For the carbon source, 5 g/L glucose and 30 g/L α‐cellulose were added.…”