Cascade processing of S. almeriensis

The water fraction containing the released carbohydrates was removed by centrifugation in a similar manner as described in 5.4.1. The samples were then stored in -20 °C until processing. After thawing, the carbohydrate content was determined following the same procedure with 5.2.3.

Enzymatic hydrolysis was performed on wet biomass at concentration of 100 g·L⁻¹, either directly after the PEF-treatment or, after centrifugation, removal of the supernatant and replacement by an equivalent volume of water (in case the water fraction was previously extracted, as in this case).

The protocol of enzymatic hydrolysis itself was described in full detail in [15]. In brief, enzymatic hydrolysis took place in 50 mL glass tubes with screw caps (Roth, Germany). Temperature was fixed at 50 °C in a water bath, placed atop a magnetic stirrer with heating function (neoLab, Germany) which also provided constant agitation. The pH was adjusted at 8 using sodium hydroxide (1 M). As enzymes, Alcalase (subtilisin) 2.5 L (Novozyme, Denmark) and Flavourzyme 1000 L (Novozyme, Denmark) were added at 3% (vol/w) each with regard to cell dry weight of the biomass. The hydrolysis reaction lasted for 3 h and samples were removed every 1 h with immediate deactivation of enzymes by heating at 80 °C for 10 min. Centrifugation at 10,000 × g for 10 min separated the water fraction along the free amino acids from the biomass and the amino acid content was determined spectrophotometrically using ortho-phtaldialdehyde (OPA) assay, with serine as standard. The ratio of the number of cleaved peptide bonds over the total number of peptide bonds in the sample, also defined as Degree of Hydrolysis (DH) offers an indication of the reaction rate. High pressure homogenization (HPH) was used as a benchmark. HPH took place in an EmulsiFlex-C3 homogenizer (Avestin Europe GmbH, Germany) at 2 MPa for 5 passes.

After 3 h of hydrolysis, approximately 3 mL microalgae suspension per sample for all conditions, were measured precisely into Teflon tubes. As mentioned in Sect. 5.5.2, the free amino acids were separated from the rest of the biomass through centrifugation. For lipid extraction, the following step of the cascade, the residual biomass pellet was re-suspended in 22.7 mL ethanol: hexane co-solvent 1: 0.41 vol/vol. Lipid extraction then took place as described in Sect. 5.4.1 (system A).