Experimental Design and Optimization of Recovering Bioactive Compounds from Chlorella vulgaris through Conventional Extraction

Abstract

Microalgae contain an abundance of valuable bioactive compounds such as chlorophylls, carotenoids, and phenolics and, consequently, present great commercial interest. The aim of this work is the study and optimization of recovering the aforementioned components from the microalgae species Chlorella vulgaris through conventional extraction in a laboratory-scale apparatus using a “green” mixture of ethanol/water 90/10 v/v. The effect of three operational conditions—namely, temperature (30–60 °C), duration (6–24 h) and solvent-to-biomass ratio (20–90 mLsolv/gbiom), was examined regarding the extracts’ yield (gravimetrically), antioxidant activity, phenolic, chlorophyll, and carotenoid contents (spectrophotometric assays), as well as concentration in key carotenoids, i.e., astaxanthin, lutein, and β-carotene (reversed-phase–high-performance liquid chromatography (RP–HPLC)). For this purpose, a face-centered central composite design (FC-CCD) was employed. Data analysis resulted in the optimal extraction conditions of 30 °C, for 24 h with 37 mLsolv/gbiom and validation of the predicted models led to 15.39% w/w yield, 52.58 mgextr/mgDPPH (IC50) antioxidant activity, total phenolic, chlorophyll, and carotenoid content of 18.23, 53.47 and 9.92 mg/gextr, respectively, and the total sum of key carotenoids equal to 4.12 mg/gextr. The experimental data and predicted results were considered comparable, and consequently, the corresponding regression models were sufficiently reliable for prediction.

Extraction of Bioactive Compounds from C. vulgaris Biomass Using Deep Eutectic Solvents

“…The control solvent is the best among the four tested for carotenoid extraction. This finding is in accordance with the literature since ethanol/water mixtures are known to be particularly efficient solvents in extracting carotenoids from micro and macro algae [37,49,50].…”

“…These findings imply that the extracted carotenoids and phenolics sustained degradation under prolonged exposure to oxygen and light [49,55]. An initially increasing trend of the carotenoids content, followed by a decreasing one versus extraction time, was also reported for the C. vulgaris extractions using a conventional solvent [37]. A maximum extraction time above which the TPC of the C. vulgaris extracts decreases due to oxidation of the phenolics compounds was found by Mahmood et al, 2019 [40]; and Zakaria et al, 2017 [55], as well.…”

“…According to the analysis that preceded, at low extraction temperatures, extracts with a relatively lower content of carotenoids are obtained. Moreover, the application of a high solvent-to-biomass ratio, i.e., the use of a larger amount of solvent at a low extraction temperature, might have led to the extraction of other molecules present in the C. vulgaris cells that did not contribute to the antioxidant activity of the extracts [ 37 ]. Due to the presence of these compounds, along with the lower carotenoid concentrations, the extracts’ antioxidant potency was reduced, and higher IC50 values were measured.…”

“…The ethanol/water 70/30 w / w mixture (EtOH/w), a non-toxic, efficient, conventional solvent, was used for comparison [ 49 ]. The extraction conditions for the solvent screening were selected based on the relevant published work of our scientific group [ 37 , 49 ]. Specifically, the extractions were carried out at a 20:1 solvent-to-biomass ratio (r, [g SW g –1 DW ]) for a time period of three hours (t, [h]).…”

“…Chlorella species are verified to have one of the highest percentages of phenolic compounds and carotenoids compared to other microalgae strains [ 35 ]. The recovery of compounds with antioxidant activity, such as polyphenols and carotenoids, from C. vulgaris using conventional solvents is well documented [ 36 , 37 , 38 , 39 ]. On the contrary, the use of DESs for the same purpose has hardly been addressed.…”