**2. Results**

To access the possibility of replacing YE with cheaper alternative sources of nitrogen and nutrients for the cultivation of *C. cohnii* and DHA production, we used extracts obtained from de-oiled dinoflagellate biomass, as well as ethanol, which was used as part of one of the oil extraction methods. Two main methods were used to extract oil from the dinoflagellate biomass (see Scheme 2). In the first case, the oil was extracted from lyophilized biomass using hexane. Thus, obtaining a SCO, which after esterification can be separated into FAs. A waste product of this process is de-oiled microalgae biomass, which is hydrolysed to obtain dinoflagellate extract (DE) (see Scheme 2A). In the second case, the oil extraction is carried out through saponification of fats in wet biomass with KOH in the presence of ethanol. Thus, obtaining the hydroalcoholic phase, containing soaps, and de-oiled microalgae biomass. Ethanol, which is used in this process, extracts multiple components from the biomass and can serve as a source of carbon and organic nitrogen, vitamins, nutrients, and salts for subsequent cultivations (see Scheme 2B).

The first method requires additional biomass processing before the oil extraction (e.g., freezing and drying (lyophilization). On the contrary, the second method requires an additional extraction step. The obtained DEs by the first and second method were called DEA and DEB, respectively. Experiments on the effect of the DE on the growth of *C. cohnii* were carried out in mediums with glucose as the main carbon source. EE was used as an alternative source of carbon.

**Scheme 2.** Dinoflagellate extract, extraction ethanol, and free fatty acid (FFA) methyl ester acquisition from (**A**) lyophilized biomass and (**B**) wet biomass.

## *2.1. Experiments with Glucose as the Carbon Source*

Multiple cultivation experiments were carried out using a complex medium containing glucose, sea salt, and YE and/or DE to study the effect of DE on the growth rate, biomass, and lipids including DHA by *C. cohnii*. The media composition used in the experiments with glucose as the main carbon source are summarized in Table 2. The mediums under study contained either only YE, only DE, or YE and DE (25/75 *<sup>w</sup>*/*w*). Selection of the initial glucose concentration (10 g·L−1) is justified by the fact that the mentioned amount of substrate is enough for the biomass to fully consume 1 g·L−<sup>1</sup> of YE. Furthermore, de Swaaf et al. [17], Jiang et al. [26], and Diao et al. [27], as part of previously reported studies, have shown that the maximum biomass growth rate is achieved if the glucose concentration is maintained in the range of 5–25 g·L−1. Additionally, de Swaaf et al. has demonstrated, that biomass growth inhibition begins at glucose concentrations of 20–25 g·L−1. The specific biomass growth rates and yields in different mediums are shown in Table 2. The maximum specific biomass growth rate and yield from glucose were observed in the medium containing exclusively DEA, and were equal to 1.012 and 0.601 g·g<sup>−</sup>1, respectively. The lowest specific growth rate (0.655 h−1) was observed with the medium, which contained only DEB. A similar growth rate (0.615 h−1) was observed with the reference medium with no added extracts.

The addition of 25% YE to 75% DEB into the cultivation medium (DEB75) increased the specific biomass growth rate by 37% (up to 0.901 h<sup>−</sup>1), while the yield of biomass remained mostly unchanged (0.398 g·g−1). However, the addition of 25% of YE to DEA (medium DEA75) lowered the specific biomass growth rate to 0.9 h<sup>−</sup>1, in comparison to the mediums containing only DE extracts.


**Table 2.** The medium compositions and the growth parameters of *C. cohnii* with glucose as carbon source.

Where *μ*max is the specific biomass growth rate and Yx/s is the biomass yield from a substrate.

Figure 1 shows the biomass growth (A) and glucose consumption (B) curves in mediums given in Table 2. It can be seen that all mediums containing YE ensured complete assimilation of glucose in 7–14 days, while in mediums containing DE, only half of the initially supplemented substrate was assimilated until the 14th experiment day.

**Figure 1.** Cultivation of *C. cohnii* with glucose as a carbon source—(**A**) optical density (OD) change over time and (**B**) glucose concentration change over time.

Almost all growth curves, except the DEA medium, reached the lag phase during the first day of cultivation. The medium containing only YE showed the highest average specific growth rate for the first three days until all glucose was consumed. After that, the growth passed into the stationary phase and cyst formation began. In other mediums, the maximum specific growth rate was observed only on the first cultivation day, after which the biomass growth remained constant.
