**3. Discussion**

The main components of the culture medium for *C. cohnii* are the substrate (glucose, glycerol, ethanol, acetic acid), yeast extract, and sea salt (artificial or natural). The biomass yield from each component of the cultivation medium was determined during bioreactor cultivations and reached 0.7 g·g−<sup>1</sup> for glucose, 5.3 g·g−<sup>1</sup> for yeast extract, and 0.87 g·g−<sup>1</sup> for sea salt (results not shown). Despite the fact that, compared to other components, a small amount (in terms of mass) of yeast extract is used, its price is the highest (8–10 USD/kg) and makes up more than half of the cultivation medium cost. Considering that the costs of raw materials for the production of SCO is about half of all production expenses, the reduction of the necessary amounts of cultivation medium components poses a significant effect on the overall process economy.

The obtained results of this study show that DEA stimulated a similar biomass growth rate when compared to YE, while the biomass yield was significantly higher if glucose was used as a carbon source. The absence of a lag phase during cultivation in mediums containing only DEA, both with glucose or ethanol, unlike mediums with YE, indicates that the mineral and vitamin composition of DEA is more preferable for proliferation of *C. cohnii* cells. However, a decrease of the biomass growth rate in mediums with DEA should be noted. The increase in biomass titres after cultivation for four days (media with DEA) and seven days (media with DEA75) becomes constant and equal to that obtained in the control medium (with glucose). Therefore, despite the high initial biomass growth rates, the efficiency of DEA is approximately two times lower when compared to that of YE. The latter can be compensated by increasing the initial concentrations of DEA or the addition of small amounts of YE to the cultivation medium, which in itself depends on the economic feasibility of the process.

The use of DEB as a nitrogen source, in the case of both glucose and ethanol, had a minimal effect on the growth rate and biomass yield. Obviously, many thermal and chemical biomass processing steps, which are necessary to obtain DEB, completely or to a larger extent degrade the initial vitamins present in *C. cohnii* biomass.

EE, although it did not significantly affect the biomass growth rate, showed the same biomass yield when compared to the control sample (pure ethanol), which indicates the feasibility of using it as a carbon source for *C. cohnii* cultivation.

Cross comparison of the FTIR spectral data showed that the growth medium components clearly affect the biochemical composition of *C. cohnii* cells. When grown in mediums with YE, EE-YE, EE-DEA75, or EE-DEB75, *C. cohnii* cells contained more proteins (compared to the cells grown in any other studied mediums) but the absorption band at 1745 cm−<sup>1</sup> (ester C=O bonds of lipids/FA) was not detected, thus indicating that the cells did not overproduce lipids/FA. In the context of searching for the growth conditions promoting the accumulation of PUFAs/DHA the most promising results were acquired when *C. cohnii* was cultivated in the mediums with DEA75 and glucose (Figure 4), EE-DEA, EE-DEB, EE, or ethanol (Figure 5). However, further quantitative analyses of the main cell macromolecular components (carbohydrates, proteins, and lipids) are needed to identify the most efficient growth media that promotes the overproduction of PUFAs/DHA by *C. cohnii*.

To summarize the above mentioned, DEA and EE can both be successfully used as alternative sources of nitrogen, nutrients, and carbon to reduce the costs of conventional SCO and DHA production processes.

Moreover, the production of DEA can be optimized through the use of hydrochloric acid and sodium hydroxide. The hydrolysate obtained in this way, after neutralization with acid, can be directly used to create a suitable dinoflagellate cultivation medium. Furthermore, the use of the above-mentioned hydrolysate will make it possible to exclude such energy-demanding steps of the production process as evaporation and simultaneously significantly reduce the amount of sea salt, which otherwise should be added in the cultivation medium in large quantities.
