*2.2. Experiments with Ethanol as the Carbon Source*

The EE, collected after the lipid extraction from *C. cohnii* biomass, was used as the main carbon source in complex mediums containing YE and DE as sources of nitrogen and nutrients. The media compositions, the specific biomass growth rates, and biomass yields are summarized in Table 3.

The highest specific growth rate and biomass yield were obtained in mediums containing YE and reached 0.757 h−<sup>1</sup> and 0.282 g·g−1, respectively. In mediums with DE, the growth rates were slightly lower than using YE and reached 0.701 h−<sup>1</sup> for DEA and 0.651 h−<sup>1</sup> for DEB. The biomass yield on the 14th day of cultivation in mediums containing DEA and DEB, reached 0.234 and 0.221 g·g<sup>−</sup>1, respectively.


**Table 3.** The medium compositions and the growth parameters of *C. cohnii* with extraction ethanol (EE) as carbon source.

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

From Figure 2 it can be observed that in the case of YE and DEA, the specific biomass growth rate reached the maximum and remained constant until the 4th cultivation day until the substrate was not entirely consumed. In turn, the biomass growth rate in mediums containing only DEB was relatively high only on the first day of cultivation, after which it gradually decreased. It should be noted that during cultivation in mediums containing YE, similarly as in the glucose experiment, the lag phase was observed during the first day of cultivation.

**Figure 2.** The optical density (OD) change over time of *C. cohnii* in mediums with extraction ethanol (EE) as a carbon source.

Experiments on pure and extraction ethanol (EE) were conducted to evaluate their effect on the biomass growth rate and yield. The specific growth rate in the case of EE was 0.470 h−1, which is for 20% more than in pure ethanol (0.383 h−1) and 50% more than in mediums containing YE or DE. The maximum biomass yield in both cases was very similar (0.124 g·g−<sup>1</sup> for EE and 0.122 g·g−<sup>1</sup> for pure ethanol), but with EE it was reached on the seventh day, and with pure ethanol on the 10th cultivation day.

#### *2.3. Evaluation of Lipid/FA and PUFA Accumulation in C. cohnii Biomass by FTIR*

FTIR is a rapid method, particularly used for monitoring the relative content of each macromolecular component under varying growth conditions [28–31]. FTIR spectroscopy of *C. cohnii* biomass was used to evaluate the growth medium-induced production of lipids/FA and PUFAs. Fish oil supplements naturally contain about 30% of EPA and DHA in the form of triacylglycerols (TAGs), a tri-ester [32,33]. The FTIR spectrum of fish oil (Figure 3) reveals three high-intensity absorption bands at 2925, 2854 cm−<sup>1</sup> (CH3 and CH2 vibrations, respectively), and 1745 cm−<sup>1</sup> (C=O vibrations of lipid esters) that are indicative of lipids, FAs or triglycerides and therefore are indicative of total lipids. The spectrum also revealed a smaller peak at 3011 cm−<sup>1</sup> (olefinic group = CH), which is typical for unsaturated fatty acids (PUFAs/DHA) [34–39].

**Figure 3.** FTIR spectrum of fish oil food supplement (LYSI HF, Iceland). 10 mL contains: FA (2155 mg) incl. EPA (690 mg) and DHA (920 mg), and vitamins: E (9,2 mg), A (460 μg), and D (20 μg).

The characteristic absorption bands of the major cell components in the FTIR spectra are at 1080 cm−<sup>1</sup> of carbohydrates; 1250 cm−<sup>1</sup> of nucleic acids; 1650 and 1545 cm−<sup>1</sup> of proteins (Amide I and Amide II, stretching vibrations of C=O bond of amide and bending vibrations of the N-H bond, respectively); triplet bands in 2800–3000 cm−<sup>1</sup> and 1744 cm−<sup>1</sup> of lipids/FA (C-H stretching in CH3 and CH2 and C=O of esters/ester carbonyl, respectively) and ~3014 cm−<sup>1</sup> of PUFAs/DHA (olefinic HC=CH stretching mode) [28,40]. The position and intensities of particular absorption bands allow to monitor or evaluate the macromolecular composition of cells as well as the accumulation of lipids/FAs and PU-FAs [40–43]. PUFAs in the FTIR spectrum show a peak in the range of 3005–3013 cm−1, particularly the specific peak of DHA oils is at ~3013,4 cm−<sup>1</sup> [44].

Samples for FTIR were collected only on day 14, due to the amount of accumulated biomass in the experimental setup. For data analysis of *C. cohnii* cells, 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. The 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. Therefore, the latter allows to crosscompare the cell biomass composition, accumulation, and number of macromolecular components (e.g., proteins, carbohydrates, FAs, PUFAs, DHA, etc). This is an especially

valuable FTIR spectroscopy approach for quick and informative evaluation of large sample sets to select the best growth conditions for the production/accumulation of the targeted metabolites. Further quantitative and qualitative analyses of the most relevant samples can be carried out more precisely by FTIR spectroscopy, chromatography, mass spectroscopy, etc. Therefore, even though FTIR is a semi-quantitative method and does not provide precise values, it remarkably saves resources and time for evaluation of different biotechnological processes.

FTIR spectra of *C. cohnii* grown in mediums with YE, DEA, DEA75, DEB75, or glucose (Figure 4) showed that the macromolecular composition of cells is different depending on the growth medium composition. The spectrum profile of the *C. cohnii* cells grown in medium containing YE was noticeably different from others of this experimental set. Spectra of the cells grown with YE showed similar amounts of total carbohydrates but higher content of proteins and lower content of total lipids than in cells grown in mediums with DEA, DEA75 DEB75, or glucose. The vector normalized spectra of cells grown without YE showed similar content of the total carbohydrates and proteins, but the content of lipids/FA and PUFAs/DHA varied. The highest number of total lipids/FAs (2925, 2854, and 1745 cm−1) was detected in cells grown in mediums with glucose but lower with DEA75 and DEB75. However, a higher amount of PUFAs/DHA (3014 cm<sup>−</sup>1) was detected in cells grown in medium with DEA75.

**Figure 4.** Vector normalized FTIR spectra of *C. cohnii* biomass after 14 days of growth in mediums, with YE, DEA, DEA75, DEB75, or glucose.

FTIR spectra of *C. cohnii* cultivated in mediums with EE-YE, EE-DEA, EE-DEB, EE-DEA75, EE-DEB75, EE, or pure ethanol are shown in Figure 5. FTIR spectra showed different cell macromolecular compositions, which can be grouped into two clusters. The first group EE-YE, EE-DEA75, and EE-DEB75 produce relatively high amounts of proteins, low amounts of total carbohydrates, and lesser amounts of total lipids than cells grown with EE-DEA, EE-DEB, EE, or pure ethanol. The FTIR spectra of the second group (i.e., cells grown in EE-DEA, EE-DEB, EE, or pure ethanol showed more total lipids/FA and PUFAs/DHA compared to those of the first group). The highest content of FAs and PUFAs/DHA was detected in *C. cohnii* grown in mediums with EE/EE-DEA and EE-DEB.

**Figure 5.** Vector normalized FTIR spectra of *C. cohnii* biomass after 14 days of growth in seven different mediums with ethanol.
