Elsevier

Algal Research

Volume 69, January 2023, 102940
Algal Research

Recovery of lipids and carotenoids from Dunaliella salina microalgae using deep eutectic solvents

https://doi.org/10.1016/j.algal.2022.102940Get rights and content

Highlights

  • DESs as pretreatment of microalgal biomass increased solvent permeability.

  • Ch-U stood out as a pretreatment for the recovery of lipids and carotenoids.

  • Some DESs act as carotenoid extraction solvents.

  • The one-pot method can reduce the extraction steps and still results in high yields.

Abstract

Microalgae are natural sources of bioactive compounds used for numerous applications such as pharmaceutical, nutraceutical, cosmetic, and recently in the production of biofuels. Achieving sustainability in some biotechnological processes still requires overcoming important challenges including the fatty acid biomass extraction processes. Methanol and chloroform are generally used in the traditional extraction processes, which are toxic and present environmental risks. Thus, studies involving environmentally sustainable solvents which are less aggressive to health are necessary. In this context, deep eutectic solvents (DESs) have shown attractive characteristics. DESs stand out as adjuvants in cell disruption and biomolecule extraction stages, working as non-toxic, non-volatile, and renewable agents. This work aimed to evaluate the effect of different DESs (pure and aqueous based on choline chloride) on extracting lipids and carotenoids from Dunaliella salina. The best total lipid recovery was 67.41 % ± 6.07 achieved using a DES synthetized with choline chloride and urea (Ch-U treatment) from dry biomass by two-step extraction. On the other hand, a significant migration to the DES phase (pre-treatment) was observed for carotenoids, and consequently lower recovery values. Thus, the one-pot method was evaluated to solve this problem and reduce the process steps, and 84.06 ± 1.22 % of carotenoids were recovered from wet biomass with Ch-U treatment and ethyl acetate and ethanol (EAE) solvent. It was also observed that the treatment performed with choline chloride and oxalic acid (Ch-Ox treatment) presented one of the highest reductions in carbohydrate content among the evaluated treatments, and the most in protein content of the microalgal biomass. Finally, the strategies tested herein were validated by comparing the results with traditional methodologies (ultrasonic and ball mill). It was generally found that DESs enhanced the solvent permeability in the microalgae cell wall and the efficiency and sustainability of the extractive process can be increased depending on the chosen strategy.

Introduction

Microalgae have attracted the interest of researchers worldwide for applications in sustainable biotechnological processes that seek to supply global demands on the use of products from natural resources, caused by the growing search for healthy habits and fundamental sustainable development [1], [2]. The potential of microalgae as a reliable and sustainable raw material for producing biofuels and valuable bioresources, such as polysaccharides, lipids, proteins, enzymes, vitamins, and carotenoids has been validated by several scientific studies, valuing the 7000 tons of dry algae biomass that is produced worldwide annually [3], [4], [5].

Dry algae biomass is a great natural supplier of neutral lipids as a potential fuel reserve and has some characteristics which contribute to their being widely exploited, such as a high growth rate, simple cultivation requirements, and the ability to adapt to adversity. Furthermore, the biochemical composition of microalgae can be modified using different cultivation conditions, and therefore the biosynthesis of lipids and carotenoids can be maximized [2], [6]. Dunaliella salina microalgae are among the main sources of natural β-carotene. This carotenoid is used in the food, cosmetic and pharmaceutical industries as a dye, antioxidant, and even as an anticancer agent [7], [8].

The challenges associated with the use of microalgae biomass compounds on a large scale are still immense. It is necessary to make improvements in the cultivation system, harvesting techniques, and mainly in the bio-compound extraction and purification stages to reduce the costs of the final product, since these steps are crucial to the quality of the final product. Traditional extraction techniques are costly processes that use conventional solvents which are volatile, flammable, poorly biodegradable, and generate toxic hazardous waste; additionally, they could compromise the application of compounds produced for food and pharmaceutical purposes [2], [9].

Extracting bioactive compounds from microalgae requires rupturing the cellular structure, which is a difficult process due to the complex composition of the cells which are formed by proteins, polysaccharides, cellulose, and lipids, offering strong resistance to mechanical and chemical treatments. Organic solvents such as chloroform/methanol, acetone, and ethanol are generally used in the lipid and carotenoid extraction processes. As a result, a wide range of pretreatments for microalgae biomass has been studied [10], [11].

The sustainability of microalgae biofuel production is directly associated with cell disruption and extraction of lipids. The polar part extraction process includes monoglycerides, diglycerides, and triglycerides that are important for biodiesel production, involving the use of solvents, chloroform, and methanol. The non-polar part, phospholipids, and glycolipids are extracted with hexane and have wide applications in the pharmaceutical and food areas [12].

Some problems associated with the use of polar solvents involve the simultaneous extraction of chlorophyll and lipids. Regarding the extraction of non-polar lipids, hexane is unable to cross the membrane which is composed of phospholipids that bind to proteins. [13]. In addition, these solvents are toxic and flammable which can be harmful to health and the environment. The development of lipo-compounds extraction processes using appropriate methods and environmentally sustainable solvents is a challenge that deserves attention.

Paliwal et al. [14] demonstrated that lutein from Chlorella saccharophila remains 3 times more stable in ionic liquid than in conventional solvent methanol at 60 and 90 °C. This is another problem faced in carotenoid extraction, which can be significantly mitigated with the use of alternative solvents.

A class of solvents has emerged as an alternative to conventional organic solvents, called deep eutectic solvents (DESs), to meet the need to prioritize product quality, extraction efficiency, and care for the environment [15], [16]. DESs have several benefits, including a simple preparation, low toxicity, and high biodegradability, and can also act as adjuvants for weakening the microalgae cell wall and enhancing a subsequent intracellular compound extraction [17], [18].

Due to the attractive characteristics of DESs, many studies in the literature have focused on the use of DESs to recover lipids and carotenoids from different natural matrices or by-products of the food and agricultural industry [10], [19], [20], [21].

Lu et al. [20] demonstrated that DESs significantly increased the extraction efficiency of lipids from Chlorella sp. biomass and also that DESs are promising ionic liquids for the pretreatment of microalgal biomass, capable of potentiating the extraction of lipids on the scale for biodiesel production. Yu et al. [22] demonstrated that green solvents, including DESs, are promising for carotenoid extraction, with their potentiality being associated with their constituents and concentration, as well as the extraction method.

In this context, this study aimed to evaluate the use of different DESs in the recovery of lipids and carotenoids from Dunaliella salina microalgae. It was also sought to improve the results through modifications in the extractive process by studying two extraction types named here the two-step method and the one-pot method. They were also compared with well-established methodologies to validate the obtained results.

Section snippets

Microalgae cultivation, harvesting, and dehydration

The Dunaliella salina strain was obtained from microalgae collection from the Laboratory of Reef Environments and Microalgae Biotechnology (LARBIM) kindly provided by Dr. Roberto Sassi from the Federal University of Paraíba (UFPB). The Dunaliella salina was cultivated in Conway medium [23] for 17 days in a photobioreactor (5 L) equipped with air compressors (Boyu S-510 4 L·min−1), LED light bulbs (750 μmol·s−1·m−2) and a 12:12 h photoperiod.

The turbidity of aliquots under different

Two-step extraction method

The recovery rate of lipids and carotenoids after DES treatment was calculated concerning the content of these constituents in the respective pre-treated biomass. In addition, the lipids and carotenoids contained in each of them were quantified according to already well-established methodologies in the literature.

Conclusion

Green extraction of lipids and carotenoids from Dunaliella salina was favored after treatment with various DESs. The eutectic urea choline chloride proved to be an effective treatment to recover lipids from the two-step method (74.99 ± 0.13 %) and to recover carotenoids from the wet biomass using the one-pot method (84.06 ± 0.13 %) (1.22 %). Comparative studies have proven the effectiveness of these strategies against the ultrasonic and ball mill treatment.

Studies related to the

Ethical statement

Ethics approval was not required for this research.

CRediT authorship contribution statement

Estéfani Asevedo: Conceptualization, Methodology, Writing - Original draft preparation, Software, Statistic. Bruna Chagas: Conceptualization, Writing - Original draft preparation, Data curation, Reviewing and editing. Sérgio Oliveira Júnior: Writing - Original draft preparation, Data curation. Everaldo Santos: Writing - Original draft preparation, Funding acquisition, Supervision Writing.

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgement

This work was supported by the Brazilian National Council for Research (CNPq) [grant number 304844/2020-9] and Coordination for the Improvement of Higher Education Personnel (CAPES) [grant number 001].

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