*2.1. Comparison of Growth, Substrate Consumption, and Accumulation of PUFAs with Glucose, Ethanol and Glycerol*

Batch cultivation results with a single carbon substrate, or their combinations with glycerol, are shown in Figures 1 and 2, respectively. The growth on glycerol was compared to the growth on glucose and ethanol within a range of substrate concentrations. As seen in Figure 1, at all concentrations, the tested growth and substrate consumption with glycerol were roughly comparable to those with ethanol but proceeded significantly slower than with glucose. In contrast, Taborda et al. [12] and Safdar et al. [10] reported the growth and uptake rates for glycerol as comparable or even surpassing those with glucose. Apparently, growth parameters might vary depending on the strain, inoculum size and other cultivation parameters. Glycerol can be applied in a wide range of concentrations without

any significant variations in its uptake kinetics or growth inhibition. Ethanol, in contrast, is demonstrated to inhibit growth at concentrations above 5 g/L [20]. Clearly, our data confirm that glycerol could serve as the sole carbon substrate for *C. cohnii* cultivation. At the same time, it could potentially be used as a co-substrate for mixotrophic cultivations. Under mixotrophic growth conditions (Figure 2), the uptake of glycerol and glucose occurred simultaneously, although at the initial stage of cultivation, glycerol slightly slowed down glucose consumption (compared with the growth on glucose as the sole carbon source, shown in Figure 1). Additionally, ethanol could be taken up simultaneously with glycerol (Figure 2).

**Figure 1.** Growth and substrate consumption of *C. cohnii* on media with ethanol (**a**), glycerol (**b**), or glucose (**c**).

The early-stage accumulation of PUFAs in the *C. cohnii* biomass, cultivated on each of the three carbon sources, was monitored by FTIR spectroscopy, following the approach used in Didrihsone et al. [21]. FTIR was chosen as a rapid analytical method, requiring a small sample size and no complex pretreatment steps. The validity of infrared spectroscopy to estimate the content of saturated, monounsaturated and polyunsaturated fatty acids has

been reported previously [22–24]. Yoshida and Yoshida [25] evaluated the FTIR spectra of synthetic and dietary triglyceride oils with various PUFAs, including DHA. The secondderivative spectra for the alkene (-HC=CH-) C-H stretching vibrational mode of several synthetic triglycerides and dietary PUFA oils showed that the peak position corresponded to the peak position in raw spectra, and the position was changed from 3005 to 3013 cm−<sup>1</sup> when the extent of unsaturation was increased from mono-ene to hexa-ene. Particularly in spectra of DHA oils, the alkene peak position was at 3013.4 cm−1. Here, the secondderivative spectra revealed a small peak at 3014 cm−<sup>1</sup> as a simple, separate spectral feature, and accordingly, could be ascribed to the =CH- stretching of cis-alkene in PUFAs of the *C. cohnii* cells (Figure 3). The vast evidence accumulated so far on the fatty acid composition of *C. cohnii* cells indicates that DHA is the dominant PUFA in this species [26–30]. Apart from DHA (C22:6), there is a small amount of C22:5, while the rest of its fatty acid fraction is composed of C18:1, and of C12-C18 saturated fatty acids. Notably, DHA is the only representative of hexa-enes at measurable quantities; therefore, the spectral feature at 3014 cm−<sup>1</sup> can be specifically related to *C. cohnii* DHA.

**Figure 2.** Mixotrophic growth of *C. cohnii* on glycerol with ethanol (**a**) or with glucose (**b**).

The strongest absorbance at 3014 cm−<sup>1</sup> was found in the glycerol-grown cells. Notably, the accumulation of PUFAs with glycerol was already well-pronounced after 28 h of cultivation. At this early time point, hardly any absorbance was seen in the glucose-grown cells, despite the fact that glucose enabled faster growth. The absorbance of the ethanolgrown cells was more similar to that of the glycerol culture; nevertheless, after 70 h of cultivation, the glycerol-grown cells had accumulated significantly more PUFAs (Figure 3). Previously, we performed a chromatographic analysis of the fatty acid composition of *C. cohnii* CCMP 316 biomass grown in fed-batch mode with ethanol [30]. Following the same methodology, we also analyzed the DHA content of the same strain, grown in batch mode on 40 g L−<sup>1</sup> glucose under conditions similar to those of the present study (unpublished data). The DHA content in these cultivations was in the range of 3.0–3.5% of

the biomass dry weight. Here, this value would correspond to the black lines at the top panel of Figure 3, providing a rough absolute scale for the change of DHA content, seen in the spectra.

**Figure 3.** Vector-normalized, second-derivative FTIR spectra of *C. cohnii* biomass, showing relative amounts of accumulated PUFAs when grown with glycerol vs. glucose (**a**) or with glycerol vs. ethanol (**b**). Spectra obtained from cultivations with three concentrations of each carbon source are presented: with 5 g/L, 10 g/L and 40 g/L of glucose; 8 g/L, 14 g/L and 27 g/L of glycerol; and 0.7 g/L, 1.5 g/L and 3 g/L of ethanol.
