*2.5. Microalga Cultures*

The heterotrophic marine microalga *C. cohnii* ATCC® 30772™ (American Type Culture Collection) was used to study the production of omega-3 fatty acids. Stock cultures and growth procedure were performed according to ATCC guidelines and protocols, as previously described [16]. Initially, the microalgal cells were incubated in standing precultures at 27 ◦C in the dark, on a medium containing 9 g/L glucose, 25 g/L sea salts and 2 g/L yeast extract. The pH of the medium was set at 6.5. After 4 days of growth, the standing cultures were used as inoculum for shaken cultures.

The medium for the shaken cultures contained 25 g/L sea salts and 2 g/L yeast extract, while hydrolysates after enzymatic treatment of the solid pulps (6 samples) or the liquid fractions (3 samples) were employed as a carbon source. In order to increase the total sugar content of the hydrolysates from solid pulps, enzymatic reactions took place at 12% (*w*/*v*) initial solids, while all other parameters (temperature, enzyme loading, pH, agitation) were the same as those mentioned in Section 2.3. The total culture volume was 30 mL in 100 mL shake flasks, 27 mL of either 2-times diluted enzymatic hydrolysate from solid fraction or undiluted enzymatically treated liquid fraction and 3 mL inoculum, as it had to consist of 10% (*v*/*v*) of the final culture volume. The pH of the culture was set at 6.5 with NaOH solution. The temperature of the culture was set at 27 ◦C and agitation was essential for homogeneity, namely 160 rpm [27]. Each experiment was performed in duplicate. The growth of cells was supervised on a daily basis by measuring the optical density (OD) at 685 nm and the TRS concentration with the DNS method. Moreover, to track carbon consumption more accurately, an HPLC analysis was performed with an Aminex HPX-87H column, as mentioned above. At the end of cultivation, cells were harvested by centrifugation, washed thoroughly with dH2O contacting 25 g/L sea salts, lyophilized and weighed, in order to determine the biomass concentration.
