3.4. CO2 Supercritical Extraction Experiments

The SF-CO₂ extraction method was carried out by using a bench scale extraction unit, as described in our previous studies [16,18]. The extraction unit had a heating capacity up to 250 °C and CO₂ compression capacity up to 680 bar. The extraction unit can control the inlet and the outlet pressure with an accuracy of 0.6 mbar, and the CO₂ flow rate was controlled by means of an LPN/S80 ALG 2.5, Sacofgas, Italy flow meter. The inlet flow rate was adjustable until 25 mL/min and controlled using the expanded gas. The temperature was monitored using thermocouples, where micrometric valves control the inlet and outlet flow streams. The cylindrical extraction vessel had a capacity of 50 mL (D = 1.35 cm, H = 35 cm), which was filled with pretreated biomass, including diatomaceous earth, and 44 g glass beads of 3 mm to increase the contact of carbon dioxide with microalgae, and, at the same time, to avoid the biomass caking. Moreover, at the bottom of the extraction vessel, metal frit filters were used with a pore diameter of 5 μm to avoid the biomass transfer. The extraction unit was equipped with acoustic and visual high-pressure alerts and, as a primary security system, a rupture disk was installed. All the parameters were controlled through a Distributed Control System (DCS). A schematization of bench scale extraction unit are reported in Figure 4.

Bench scale SF-CO₂ extraction unit schematization. Note: HE: Heat exchanger; MFM: Mass flow meter; MV: Manual valve; DCS: Distributed control system; PC: Pressure control; TC: Temperature control.

The effect of the variation of operative conditions, such as pressure (P) in the range 100–550 bar, CO₂ flow rates 7.24 and 14.48 g/min, and temperature (T) in the range 50–75 °C on EPA and DHA extraction from Nannochloropsis sp. was investigated, while the biomass loading was kept constant (~2.0 g). The detailed experimental conditions are shown in Table 5. For each experimental test, five extraction cycles of 20 min each (extraction time = 20–100 min) were carried out. However, the results of the whole extraction process have been presented in this article.

The experimental condition during SF-CO₂ extraction for lipid recovery from Nannochloropsis sp. biomass.

Note: Biomass loading was expressed on dry basis.

The influence of the adopted operating conditions was studied on the recovery of total lipid, EPA and DHA. The amount of the extracted compounds from microalgae was expressed in terms of weight of the compound/weigh of dry biomass:

where W_C,i is the weight of the extracted EPA/DHA (mg); and, W_M is the weight of microalgae on a dry basis (g). Moreover, for each class, the recovery was compared with respect to the theoretical content.

The recovery percentage EPA/DHA was calculated from Equations (2).

where W_T is the theoretical weight of EPA/DHA (mg).

Furthermore, the purity percentage of lipid was also calculated, by using Equation (3)

where W_L is the weight of total lipid extracted (mg), and W_E is the total weight of the extract (mg).

The percentage of FAs extracted was calculated by using Equation (4):

where SFAE, MUFAE and PUFAE are the total amount of SFA extracted, the total amount of MUFA extracted, and the total amount of PUFA extracted, respectively (Table 1), and FAsT is the total theoretical content of FAs (Table 2).

Each experimental condition was investigated three times, and for each condition, the standard deviation (SD) value was calculated. After the SF-CO₂ extraction, the extracts were stored in the dark at −80 °C before analyzing the total EPA contents using GC-FID.2.5. Analytical Methods

After the mechanical pre-treatment, total lipids were analyzed by gravimetric analysis, and, more precisely, by following the Bligh and Dyer method [50]. Since a mixture of non-polar/low polarity organic solvent and polar organic solvent allows to extract a higher amount of lipids, a chloroform/methanol mixture (1:2 v/v) was used as extractant.

FAs compositions were identified by GC separation of the corresponding methyl esters (FA methyl esters C8-C24). Lipids fraction was methylated through base-catalyzed transmethylation, according to BDS EN ISO 12966-2:2017 standard method [51]. More precisely, NaOH solution in methanol (0.5 M, 6 mL) and a spatula of boiling chips were added to a known quantity of extract (about 100 mg). The sample was transferred to a 50 mL one-mark volumetric Erlenmeyer flask that was connected to a reflux condenser to boil the sample for about 10 min. At the end of boiling, the apparatus was removed from the heat source, and 6 mL of n-hexane from the top of the condenser and followed by 7 mL of the BF3 catalyst in methanol (14%) (B1252 Aldrich) were added. The sample boiled again for 30 min, and 5 mL of isooctane were added at the end of the reaction. A 20 mL sample of a saturated NaCl solution was added and swirled, and the second aliquot of saturated NaCl solution was added until it filled up to the neck of the flask. The total upper layer (2−4 mL) was taken and then transferred to a GC glass vial.

The chromatographic analysis was carried out using a 7820A GC-FID that was equipped with an HP-88 100 mt × 0.25 mm × 0.2 µm column (Agilent) for SFAs, MUFAs and PUFAs measurements.

This chromatographic is composed of a high polarity bis (Cyanopropyl) siloxane stationary phase, and it was chosen for its high resolution of positional and geometric isomers of fatty acid methyl esters. According to the chromatographic conditions reported in the standard method UNI ISO 12966-4 [52], the injector, as well as the detector temperature, was maintained at 250 °C. The column was maintained at 120 °C for 5 min, followed by temperature ramping at 4 °C/min to 240 °C and held for a further 10 min at 240 °C. Nitrogen (purity ≥ 99.9999%) was used as carrier gas with a linear velocity of 30 cm/s (flow rate approx. 1.0 mL/min) and a split ratio of 1:100. The sample injection volume was 1 µL. The FAs characterization was carried out for each extraction condition, and an internal analytical standard of the tricosanoic acid (C:23) was used for the quantification of fatty acid methyl esters. A mixture of 37 fatty acid methyl esters (C4–C24) (Supelco FAME 37, {"type":"entrez-protein","attrs":{"text":"CRM47885","term_id":"1048595075","term_text":"CRM47885"}}CRM47885) was purchased from SIGMA-Aldrich (H5149), St Louis, MO, USA and was used for the quantitative analysis.