Elsevier

Process Biochemistry

Volume 121, October 2022, Pages 240-247
Process Biochemistry

Microalgal fractionation for lipids, pigments and protein recovery

https://doi.org/10.1016/j.procbio.2022.07.002Get rights and content

Highlights

  • he presence of neutral lipids favors the extraction of pigments by supercritical CO2.

  • The addition of ethanol allows the extraction of all lipids and pigments.

  • The extraction of proteins is improved by delipidation.

  • Lipid extraction allows extraction of unextracted proteins without delipidation.

Abstract

Microalgae fractionation was investigated combining supercritical carbon dioxide extraction of lipids and pigments and protein extraction from the residues, on Chlorella vulgaris and Nannochloropsis oculata. The results indicated that for N. oculata, chlorophyll (a) is extracted rapidly and selectively apart from Chlorophyll (b), representing 34% and 0.41% in 30 min at 450 bar and 50 °C and up to 43% and 0.48% at 750 bar at the same time and temperature, respectively. The extraction profile of chlorophyll (a) is similar to of neutral lipids. For C. vulgaris, the results emphasized the importance of adding a co-solvent during the supercritical extraction to achieve 30% of chlorophyll (a), while the extraction of other pigments was lower especially for chlorophyll (b) extraction yield reaching 0.1% of its total content. Then for the second time, the extraction of proteins was studied on N. oculata delipidated cake. The results showed that the higher the level of delipidation, the higher the extraction yield, with an increase from 54% for crude cake to 73% for delipidated cake.

Introduction

Microalgae provide a pool of possibilities for the isolation of natural substances of significant commercial interest in industries such as pharmaceutical drugs, dietary or beauty products [1], [2]. These include lipids, proteins, polyphenols, carbohydrates, and pigments like chlorophylls and carotenoids. Typically, the extraction of lipids from microalgal biomass can be achieved after complete cell lysis or as a result of liquid extraction using chemical solvents [1]. The previous methods are nonselective and generally accompanied by cell-wall destruction, leading to complex extracts of hydrophilic and hydrophobic components, including pigments that limit the purity of the extract [3]. Such treatment requires additional expensive phases in downstream processing for phase separation and compound purification. An attractive solution could be to develop a selective procedure to extract separately lipids and pigments. Liquid extraction can be more selective, but it requires the use of organic solvents which can have a significant environmental impact. Alternative procedures have been applied to achieve high extraction of the pigments, among which water in subcritical conditions [3] or supercritical carbon dioxide (ScCO2) extraction. Furthermore, solvent-free products, compatible with the use of the “natural” label, can be easily obtained using CO2, the most widely used supercritical solvent [4].

Countless studies have tested ScCO2 to extract pigments from inside the microalgal cell, applied for instance to carotenoids recovery from Nannochloropsis and Chlorella. Pressure and temperature have a large influence on the selectivity of the process [4], [5]. But given that ScCO2 is a non-polar solvent, a polar co-solvent is used frequently to increase its extraction capacity when the targeted molecules have a higher polarity. The most widely applied as a viable green solvent is ethanol [6]. The pressure, temperature and the solvent flowrate are important parameters that will define the selectivity of the process [4]. Notwithstanding the numerous studies, the influence of the structure and composition of microalgae on the extraction yield of intracellular pigments and on the selectivity of extraction is not well described. Moreover, scarce information on the mechanisms of pigments extraction using ScCO2 is available [7], [8].

Valorization of the residue is usually neglected since the lipid phase, which is the most important, is removed after ScCO2 extraction. Nevertheless, proteins remained in the press residue can be also valorized, with the advantage that the pigments have been removed as well. Among numerous microalgae species, Nannochloropsis oculata is one of few species likely to be used as a source of proteins for food or feed formulations [9]. To improve the nutritional value of proteins fraction, it should be isolated from the remaining biomass components. However, proteins extraction from N. oculata is often prevented by the intrinsic rigidity of its cell wall mainly composed of cellulose and hemicellulose [10]. To overcome this barrier, cell disruption should be used to facilitate proteins extraction [11]. Numerous disruption methods involving ultra-sonication, high pressure homogenization or bead-beating have been investigated [11], [12], [13], [14]. The main drawbacks in all the aforementioned works could be summed-up by: (i) the high cost of cell disintegration process, (ii) the non-selectivity of the extraction and the co-release of undesirable components, (iii) the difficulty in the downstream separation processes due to the micronization of cell debris [15], [16].

This study aims at understanding how lipids and pigments could be selectively extracted during ScCO2 extraction of microalgae. To achieve this goal, the co-extraction of lipids and four pigments (chlorophyll (a), chlorophyll (b), lutein and β-carotene) was carried out in different conditions, with or without co-solvent. These extractions were conducted on two microalgal species, one at high concentration of neutral lipids (Nanochloropsis oculata) and one at low concentration of neutral lipids (Chlorella vulgaris) without any prior disintegration of the cells. Influence of defatting pretreatment on protein extraction was studied afterwards on Nanochloropsis residues using a different solvent for lipid extraction to achieve a different level of delipidation.

Section snippets

Microalgae

Chlorella vulgaris and Nannochloropsis oculata were supplied as a frozen paste from Alpha Biotech (Asserac, France). Both species were cultivated in 10-L tubular air-lift photobioreactors. Sueoka medium, with a high N-nutrient concentration, was used for the cultivation of C. vulgaris. Conway medium, deficient in N nutrients to bring about the accumulation of lipids, was used for the growth of N. oculata. The two cultures were grown at a constant temperature of 25 ± 1 °C and constant pH of 7.5.

Lipid content

The total lipid content for N. oculata was 399 mg/g, representing 39.9 ± 1.6% DW, which falls within the range of total lipid content in N. oculata, composing 31–68% of total biomass as reported by Spolaore et al. [29]. The corresponding value for C. vulgaris was 24.4 ± 1.1% DW, in accordance with the results reported by Liu et al. [30]. Fractionation by SPE proved that the lipids of N. oculata are mainly composed of neutral lipids, with a concentration of 175 mg/g DW (representing 44.1 ± 1.6%

Conclusions

The aim of this study was to ascertain the influence of the microalga structure and composition on the extraction yield of intracellular pigments and their selective extraction. These extractions were managed on two kinds of microalga, one at high concentration in neutral lipid (Nanochloropsis occulata) and one at low concentration in neutral lipid (Chlorella vulgaris) without any prior disintegration of the cells. Total chlorophyll a was selectively extracted at 43% of under 750 bar from

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors’ contribution

All authors have contributed equally and substantially to the conception of the final manuscript and to its drafting and approval. Sara Obeid, PhD student, has made the experiment, Nicolas Beaufils supervised the experimental work, Jerome Peydecastaing supervised the analytical experiments, Severine Camy supervised supercritical CO2 experiments, Pierre-Yves Pontalier: supervised extraction experiments, Hosni Takache supervised experimental work and Ali Ismail: supervised extraction experiments.

Declaration of Interest

None.

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