Combination of Synergic Enzymes and Ultrasounds as an Effective Pretreatment Process to Break Microalgal Cell Wall and Enhance Algal Oil Extraction

Abstract

Microalgal biomass is a sustainable source of bioactive lipids with omega-3 fatty acids. The efficient extraction of neutral and polar lipids from microalgae requires alternative extraction methods, frequently combined with biomass pretreatment. In this work, a combined ultrasound and enzymatic process using commercial enzymes Viscozyme, Celluclast, and Alcalase was optimized as a pretreatment method for Nannochloropsis gaditana, where the Folch method was used for lipid extraction. Significant differences were observed among the used enzymatic pretreatments, combined with ultrasound bath or probe-type sonication. To further optimize this method, ranges of temperatures (35, 45, and 55 °C) and pH (4, 5, and 8) were tested, and enzymes were combined at the best conditions. Subsequently, simultaneous use of three hydrolytic enzymes rendered oil yields of nearly 29%, showing a synergic effect. To compare enzymatic pretreatments, neutral and polar lipids distribution of Nannochloropsis was determined by HPLC–ELSD. The highest polar lipids content was achieved employing ultrasound-assisted enzymatic pretreatment (55 °C and 6 h), whereas the highest glycolipid (44.54%) and PE (2.91%) contents were achieved using Viscozyme versus other enzymes. The method was applied to other microalgae showing the potential of the optimized process as a practical alternative to produce valuable lipids for nutraceutical applications.

Cross-Linked Enzyme Aggregates and Their Application in Enzymatic Pretreatment of Microalgae: Comparison Between CLEAs and Combi-CLEAs

“…To further compare between different immobilization conditions (ethanol at 90%, acetone 90% and 70%, and PEG 50% and 60%), stability assays were performed in different temperature and pH conditions comparing free enzymes and CLEAs. The conditions were chosen in regard to the optimum conditions of previous works on N. gaditana pretreatment using the combination of the three enzymes ( Mahdy et al, 2014 ; Safi et al, 2017 ; Blanco-Llamero et al, 2021 ), using this model as a proof of concept of the derivatives activity on microalgae and taking it as the final objective of the present study.…”

“…As a proof of concept, on the basis of the previous works on US-assisted microalgae pretreatment and extraction ( Bautista et al, 2015 ; Sánchez-Bayo et al, 2019 ; Sheldon, 2019 ; Blanco-Llamero et al, 2021 ), enzymatic reactions were carried out with optimum CLEAs and combi-CLEAs (using ethanol at 90%, acetone at 90%, and PEG at 80%) combined with US and compared to soluble Alcalase®, Viscozyme®, and Celluclast® also combined with US. All preparations could be compared in terms of oil yield because they had the same amount of enzyme.…”

“…The protocol of the optimized enzymatic pretreatment of N. gaditana biomass was previously described in literature ( Blanco-Llamero et al, 2021 ). One gram of dry microalgal biomass was resuspended in 10 ml of sodium citrate buffer 0.1 M pH 5.0 containing soluble enzymes (20 mg/ml containing the three soluble commercial enzymes), single CLEAs (containing 20 mg/ml of each enzyme in the separated CLEAs), or combi-CLEAs (combined CLEAs containing a mixture of the three commercial enzymes at a final concentration of 20 mg/ml) per gram of biomass.…”

“…The physical methods such as ultrasounds (USs) accelerate the action of the enzymes, showing a synergic effect of both pretreatments, enzymatic and US, on the break of N. gaditana cell wall. When each pretreatment was applied separately on the microalgae biomass the oil yield was fewer than the one obtained by their combination, obtaining an effective combined pretreatment previously optimized in other works of the research group ( Zuorro et al, 2016 ; Chen et al, 2017 ; Zhang et al, 2017 ; Zhang et al, 2018a ; Dixon and Wilken, 2018 ; Wang et al, 2019 ; Blanco-Llamero et al, 2021 ).…”

“…N. gaditana cell wall contains mainly proteins and other biopolymers such as cellulose or pectin, among others. Thus, the cell wall must be properly disrupted to efficiently recover intracellular bioactive compounds ( Blanco-Llamero et al, 2021 ). Several methods for cell disruption could be applied.…”