## 7 New Approaches to Monitoring and Optimisation of Microalgae Cultivation Systems
### Two-Dimensional and Infrared Spectroscopy  
Two-dimensional spectroscopy and infrared (IR) spectroscopy are advanced analytical tools that enable precise monitoring of biotechnological processes, including the cultivation of microalgae. These technologies enable non-invasive analysis of the biochemical composition of biomass and the tracking of metabolic changes in real time. IR spectroscopy is an optical technique that can be used to detect a wide range of organic compounds and thus offers significant potential for online monitoring of bioprocesses [20]. Organic molecules have characteristic signatures in the IR region of the electromagnetic spectrum. The most useful parts of this region for monitoring purposes are the mid-infrared region (MIR; 200-4000 cm\(-\)1) and the near-infrared region (NIR; 4000-13,000 cm\(-\)1). In addition, NIR and MIR spectroscopic sensors can detect multiple compounds simultaneously [226]. Due to the complex nature of the spectral signals obtained with these techniques, the application of multivariate analysis methods such as principal component analysis (PCA) and partial least squares (PLS) regression is essential to obtain relevant information. NIR spectra or absorbance at specific wavelengths were used to analyse processed and dried microalgae samples. Two-dimensional spectroscopy, including techniques such as two-dimensional correlation infrared spectroscopy (2D-IR) and two-dimensional optical spectroscopy, enables the analysis of protein, lipid, and carbohydrate structures in microalgae. The use of two-dimensional methods enables the unfolding of overlapping absorption bands and thus improves the differentiation of individual cellular components. Studies suggest that 2D-IR is particularly useful to assess the dynamics of structural changes of proteins and lipids under environmental stress conditions such as nutrient deprivation or elevated CO\({}_{2}\) concentrations [227]. Infrared spectroscopy, in particular Fourier transform infrared spectroscopy (FTIR), is frequently used to analyse the biochemical composition of microalgae and facilitates the identification and quantitative assessment of key biomass components such as proteins, lipids, and polysaccharides. FTIR in ATR (attenuated total reflectance) mode enables \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Monitoring Method** & \begin{tabular}{c} **Monitored Variable** \\ **(Concentration)** \\ \end{tabular} & **Sensor Type** & **Off-Line/On-Line** & **Refs.** \\ \hline IR radiation (MIR, & Lipid \\ NIR, FTIR) & Protein & ATR flow system \\ Carbohydrate content & Fibre-optic probe & Off-line and on-line & [216; 217] \\ \hline FC & Lipid content & Flow cytometer & Off-line and on-line & [218; 219] \\ \hline ISM & Cell number concentration & & & \\ Cell mass concentration & & & & \\ Cell morphology & Microscope + CCD & On-line & [220] \\ Population composition & (contamination) & & & \\ \hline Absorbance spectrum & \begin{tabular}{c} Pigments \\ Fatty acids \\ \end{tabular} & Spectrophotometer & Off-line & [221; 222] \\ \hline OD, turbidity & Cell mass concentration & \begin{tabular}{c} OD sensor \\ Turbidity sensor \\ \end{tabular} & Off-line and on-line & [19; 222] \\ \hline Colour analysis & Cell mass concentration & CCD camera & Off-line & [223; 224] \\ \hline Fluorometry & \
\begin{tabular}{c} Photosynthetic efficiency \\ Quantum yield \\ Lipids \\ \end{tabular} &
\begin{tabular}{c} Pulse amplitude \\ modulated fluorometer \\ \end{tabular} & Off-line and on-line & [225] \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Methods for monitoring microalgae cultivation variables. rapid and non-invasive monitoring of changes in chemical composition during cultivation. Analysing FTIR spectra enables the determination of lipid accumulation in microalgae cells, which is crucial for biofuel production research [228].