## 7 New Approaches to Monitoring and Optimisation of Microalgae Cultivation Systems
### Multiparameter Flow Cytometry  
Multiparameter flow cytometry (MFC) is an advanced analytical tool that enables rapid and precise monitoring of microalgae cultivation processes by simultaneously analysing multiple physiological and biochemical parameters of individual cells. The MFC provides information on cell size and cellular components by measuring the light scatter and, if desired, the fluorescence of individual cells stained with fluorescent dyes. Flow cytometry can be used as a rapid and reliable off-line method for monitoring lipid content [233]. This technique allows the assessment of population heterogeneity, the identification of metabolic changes, and the determination of microalgae responses to variable environmental conditions, making it extremely useful for microalgae biotechnology. A major application of MFC in the cultivation of microalgae is the analysis of cell viability and metabolic activity. The use of fluorophores, which selectively colour cellular structures, enables the simultaneous assessment of membrane integrity, mitochondrial potential, and enzymatic activity. Dyes such as SYTOX Green or propidium iodide allow differentiation between dead and living cells, while the JC-1 probe facilitates the assessment of mitochondrial activity, which is valuable in studies investigating the effects of environmental stress on microalgae [234]. Another important application of MFC in the cultivation of microalgae is the assessment of cell cycle dynamics. The analysis of DNA content with intercalating dyes such as Hoechst 33,342 or DAPI allows the determination of the proportion of cells in different cell cycle phases (G0/G1, S, G2/M). This method can be used to evaluate the proliferation rates of microalgae depending on the cultivation conditions, e.g., changes in nutrient availability or light intensity [235]. MFC is also used to analyse the accumulation of industrially relevant compounds such as lipids, pigments and polysaccharides. Fluorescent dyes such as Nile red and BODIPY 505/515 allow a quantitative assessment of lipid content in individual cells, which is critical when researching microalgae as a raw material source for biofuel production. In addition, the measurement of chlorophyll autofluorescence enables the assessment of photosynthetic efficiency and the overall health of the microalgae [236]. A notable advantage of MFC is the ability to analyse oxidative stress in microalgae cultures, which is important for both basic research and applied studies. Fluorogenic probes such as H2 DCF-DA allow the detection of reactive oxygen species (ROS) and facilitate the assessment of microalgae responses to stress factors such as high light intensity, salinity, or the presence of environmental toxins [237]. Flow cytometry methods for monitoring lipid content, enzymatic activity, cell viability, and cell number in microbial biofuel production have been reviewed [238]. Total cellular lipid content was measured after staining with Nile red and calibration of fluorescence signals using standard methods. The cell size and shape distribution of the microalgae were determined by forward scatter measurements in flow cytometry [239]. Due to the staining required, the implementation of at-line and on-line monitoring has been challenging, and, until recently, off-line applications in microalgae cultures have been reported. The first commercial system capable of working on-line in microalgae bioprocessing, including automated sampling and dilution, was announced by FlowCam in January 2013 [231]. Other online flow cytometry monitoring systems, such as the Cytosense flow cytometer [240], are designed for monitoring low to very low cell densities in marine or freshwater ecosystems. In yeast fermentation monitoring, flow cytometry has been used in combination with an autosampler and a flow injection system for sample processing in at-line mode [241].