*4.5. Fourier Transform Infrared Spectroscopy*

Fourier transform infrared (FTIR) spectra of biomass were recorded using Vertex 70 coupled with the HTS XT microplate reader (Bruker Optik GmbH, Ettlingen, Germany) Sample aliquots were pipetted on a 384 well microplate dried and recorded in the frequency range of 4000–600 cm<sup>−</sup>1. Omega fatty acids were identified by absorption bands at ~1743 cm−<sup>1</sup> and ~3012 cm−1. Due to the experimental setup and amount of accumulated biomass during the cultivation processes, samples for FTIR were collected only on day 14.

For data analyses only spectra with absorption limits between 0.25 and 0.80 were used, and therefore, in accordance with the Lambert-Bouger-Beer law, the concentration of a component is proportional to the intensity of the absorption band. Spectra were vector normalized, and therefore the intensity of the vibration band was proportional to the amount of band vibrations (i.e., the intensity is proportional to concentration).

#### **5. Conclusions**

FTIR spectra of *C. cohnii* cells clearly showed the medium-induced metabolic responses, including variations of the produced total lipids/FA, PUFAs, and DHA. Further studies of the concentrations and composition of PUFAs produced under various cultivation conditions together with the cell growth data would allow us to identify the most efficient cultivation medium composition. Nevertheless, current observations point out to the positive effect (both in terms of process economy and process efficiency) of supplementing the standard *C. cohnii* cultivation medium with recycled components (e.g., dinoflagellate extracts and extraction ethanol).

**Author Contributions:** Conceptualization, K.D. and E.D.; methodology, E.D., M.G. and A.S. (Anastasija Suleiko); formal analysis, K.D.; investigation, E.D., M.G., K.S. and A.S. (Anastasija Suleiko); resources, J.V.; writing—original draft preparation, E.D.; writing—review and editing, K.D., A.S. (Arturs Suleiko), A.S. (Anastasija Suleiko), M.G., J.V. and E.D.; visualization, A.S. (Arturs Suleiko), K.D. and K.S.; supervision, K.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by European Regional Development Fund, grant number 1.1.1.1/18/A/022.

**Institutional Review Board Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

