Advanced Extraction of Lipids with DHA from Isochrysis galbana with Enzymatic Pre-Treatment Combined with Pressurized Liquids and Ultrasound Assisted Extractions

Abstract

Microalgal biomass is a sustainable and valuable source of lipids with omega-3 fatty acids. The efficient extraction of lipids from microalgae requires fast and alternative extraction methods, frequently combined with biomass pre-treatment by different procedures. In this work, Pressurized liquid extraction (PLE) was optimized and compared with traditional lipid extraction methods, Folch and Bligh and Dyer, and with a new Ultrasound Assisted Extraction (UAE) method for lipids from microalgae Isochrysis galbana. To further optimize PLE and UAE, enzymatic pre-treatment of microalga Isochrysis galbana was studied with commercial enzymes Viscozyme and Celluclast. No significant differences were found for lipid yields among different extraction techniques used. However, advanced extraction techniques with or without pre-treatment are a green, fast, and toxic solvent free alternative to traditional techniques. Lipid composition of Isochrysis was determined by HPLC-ELSD and included neutral and polar lipids, showing that each fraction comprised different contents in omega-3 polyunsaturated fatty acids (PUFA). The highest polar lipids content was achieved with UAE (50 °C and 15 min) and PLE (100 °C) techniques. Moreover, the highest omega-3 PUFA (33.2%), eicosapentaenoic acid (EPA) (3.3%) and docosahexaenoic acid (DHA) (12.0%) contents were achieved with the advanced technique UAE, showing the optimized method as a practical alternative to produce valuable lipids for food and nutraceutical applications.

Biobased Solvents for Pressurized Liquid Extraction of Nannochloropsis gaditana Omega-3 Lipids

“…Since different mixtures of hexane with ethanol or with isopropanol showed good results for extraction by UAE of lipids and other microalgae compounds such as carotenoids [ 19 ], the first purpose was to substitute hexane by a biobased solvent like 2-MeTHF. An additional goal was to achieve tunable polarity using mixtures of this renewable ether with other green solvents like alcohols, beginning with ethanol.…”

“…Although it was an important component, this microalga was also the one with the major TAG proportion (26.09%) and FFA content (14.01%) in its extracts, which was in agreement with other works on I. galbana . The relatively large proportion of FFA may be due to different causes:effects of culture medium on microalgal metabolism, lipid breakdown in stressed or drying cells, and hydrolytic degradation of the biomass [ 19 , 25 ]. Lipid composition of T. chuii and N. gaditana analyzed by HPLC was quite similar, where glycolipids was the main peak in both microalgae, reaching a remarkably high percentage, as previously described for N. gaditana , with high glycolipid and omega-3 concentration [ 23 ].…”

“…Samples were weighed (0.5 g of dry biomass) and the selected solvent was added in a ratio of 1:10. Extractions for each solvent were carried out at 50 °C and 30 min, based on previous works on microalgae extraction [ 19 ]. Extraction solvents used were ethanol, 2-MeTHF and mixtures of hexane:ethanol (3:4), and 2-MeTHF:ethanol (1:1, 1:2, 1:3, and 1:4).…”

“…HPLC-ELSD analyses were performed using an Agilent 1260 Infinity HPLC equipped with an Agilent 385 (Palo Alto, CA, USA) ELSD and DAD instruments. The chromatographic separation of the different species of lipids (neutral and polar lipids) [ 19 ] was carried out with a silica normal-phase ACE (250 mm × 4.6 mm i.d., 5 μm) column maintained at 30 °C using a ternary gradient as follows: 0–2 min, 99.5% A and 0.5% B; at t = 6.5 min, 70% A and 30% B; at t = 11 min, 63% A, 27% B and 10% C; at t = 18 min, 99.5% A and 0.5% B; and at t = 20 min, 99.5% A and 0.5% B. Eluent A consisted of 2,2,4-trimethylpentane, eluent B consisted of methyl tert-butyl ether, and eluent C consisted of 2-propanol. The flow rate was 2.0 mL/min except for minutes 13 to 16, which was 1.0 mL/min.…”

“…Lipid species were identified using commercial standards for neutral lipids like TAG, DAG, MAG, and FFA. Glycolipids were identified using a standard isolated by the research group from spinach leaves, as previously described [ 4 , 6 , 19 ]. Results were expressed as the individual relative percentage of each lipid species present in the sample (normalized areas).…”