Automated Online Flow Cytometry Advances Microalgal Ecosystem Management as in situ, High-Temporal Resolution Monitoring Tool

Abstract

Microalgae are emerging as a next-generation biotechnological production system in the pharmaceutical, biofuel, and food domain. The economization of microalgal biorefineries remains a main target, where culture contamination and prokaryotic upsurge are main bottlenecks to impair culture stability, reproducibility, and consequently productivity. Automated online flow cytometry (FCM) is gaining momentum as bioprocess optimization tool, as it allows for spatial and temporal landscaping, real-time investigations of rapid microbial processes, and the assessment of intrinsic cell features. So far, automated online FCM has not been applied to microalgal ecosystems but poses a powerful technology for improving the feasibility of microalgal feedstock production through in situ, real-time, high-temporal resolution monitoring. The study lays the foundations for an application of automated online FCM implying far-reaching applications to impel and facilitate the implementation of innovations targeting at microalgal bioprocesses optimization. It shows that emissions collected on the FL1/FL3 fluorescent channels, harnessing nucleic acid staining and chlorophyll autofluorescence, enable a simultaneous assessment (quantitative and diversity-related) of prokaryotes and industrially relevant phototrophic Chlorella vulgaris in mixed ecosystems of different complexity over a broad concentration range (2.2–1,002.4 cells ⋅μL–1). Automated online FCM combined with data analysis relying on phenotypic fingerprinting poses a powerful tool for quantitative and diversity-related population dynamics monitoring. Quantitative data assessment showed that prokaryotic growth phases in engineered and natural ecosystems were characterized by different growth speeds and distinct peaks. Diversity-related population monitoring based on phenotypic fingerprinting indicated that prokaryotic upsurge in mixed cultures was governed by the dominance of single prokaryotic species. Automated online FCM is a powerful tool for microalgal bioprocess optimization owing to its adaptability to myriad phenotypic assays and its compatibility with various cultivation systems. This allows advancing bioprocesses associated with both microalgal biomass and compound production. Hence, automated online FCM poses a viable tool with applications across multiple domains within the biobased sector relying on single cell–based value chains.

Soil bacterial biodiversity characterization by flow cytometry: The bottleneck of cell extraction from soil

“…These developments are supported by data analysis pipelines (e.g. Rubbens & Props, 2021), enabling online and also real‐time evaluations (Favere et al, 2020; Haberkorn et al, 2021). Among the reviewed articles focused on soil, the analyses of community structures based on cytometric fingerprinting, considering both gate and bin methods, have been reported for microbial photoautotroph (Menyhárt et al, 2018).…”

“…Recent advances in microbial community analysis from machine learning of FCM data have also been reported (Özel Duygan & van der Meer, 2022). These technological advancements enable automated data acquisition and analysis prompting research on microbial biodiversity (De Vrieze et al, 2021; Haberkorn et al, 2021; Özel Duygan & van der Meer, 2022; Pereira et al, 2022).…”

“…These technological advancements enable automated data acquisition and analysis prompting research on microbial biodiversity (De Vrieze et al, 2021;Haberkorn et al, 2021;Özel Duygan & van der Meer, 2022;Pereira et al, 2022).…”

Chlorella Vulgaris Photobioreactor for Oxygen and Food Production on a Moon Base—Potential and Challenges

“…A modular approach and proper analysis would allow to identify and reject any running reactor that might not fulfil the requirements for human consumption. Automated flow cytometry with advanced data analysis relying on phenotypic fingerprinting could contribute to a continuous monitoring of the microbial community (Haberkorn et al, 2021). The use of flow cytometry in a known non-axenic culture can enable the understanding of population dynamics and their response to external events.…”

Optimized Protocol for Microalgae DNA Staining with SYTO9/SYBR Green I, Based on Flow Cytometry and RSM Methodology: Experimental Design, Impacts and Validation

“…Well-known examples are SYBR Green I (Ex 497 nm/Em 520 nm), DAPI (Ex 358 nm/Em 461 nm), PicoGreen (Ex 480 nm/Em 520 nm) and SYTO 9 (Ex 483 nm/Em 503 nm), which are extensively used due to their good membrane permeability and their compatibility with almost all bench-top flow cytometers [ 4 , 12 , 15 , 16 , 17 , 36 , 37 , 38 , 39 ]. However, a review of the current literature revealed a wide range of discrepancies in staining methods, with varying results and limited comparability [ 21 , 22 , 23 , 26 , 27 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 ], highlighting the need for standardized, reproducible protocols. Moreover, only a small number of protocols give an explanation of why staining parameters such as dye concentration, staining time and temperature were chosen, or how stable the added fluorochromes were [ 16 ].…”