# NaDES-based biorefinery of Spirulina (Arthrospira platensis): A new path for sustainable high value-added metabolites

Soukaina Hilali, Louise van Gheluwe, Mervé Yagmur, Laura Wils, Myriam Phelippe, Barbara Clément-Larosière, Bénédicte Montigny, Johan Jacquemin, Emilie Thiery, Leslie Boudesocque-Delaye

► To cite this version:

Soukaina Hilali, Louise van Gheluwe, Mervé Yagmur, Laura Wils, Myriam Phelippe, et al. NaDES-based biorefinery of Spirulina (Arthrospira platensis): A new path for sustainable high value-added metabolites. Separation and Purification Technology, 2024, 329, pp.125123. 10.1016/j.seppur.2023.125123. hal-04213157

HAL Id: hal-04213157

https://hal.science/hal-04213157v1

Submitted on 21 Sep 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
NaDES-based biorefinery of Spirulina (Arthrospira platensis): a new path for sustainable high value-added metabolites

3

4 Soukaina Hilali,ª Louise Van Gheluwe,ª Mervé Yagmur,ª,ᶜ Laura Wils,ª Myriam Phelippe,ᵇ Barbara Clément-

Larosière,ᵇ Bénédicte Montigny,ᶜ Johan Jacquemin,ᵈ Emilie Thiery,ª and Leslie Boudesocque-Delayeª,\*

a. EA 7502 SIMBA, Université de Tours, Faculté de Pharmacie, 31 avenue Monge 37200 Tours, France

b. Aqua Eco Culture, 7 rue d'Armor Maroué, 22400 Lamballe, France

c. EA 6299 PCM2E, Université de Tours, Faculté des Sciences et Techniques, Bât J, Avenue Monge, 37200 Tours, France

d. MSN Department, Mohammed VI Polytechnic University, Lot 660, Hay Moulay Rachid, Ben Guerir 43150, Morocco

\*Correspondence: leslie.boudesocque@univ-tours.fr; +33(0)2.47.36.71.75.

11

12

Abstract

To meet the criteria of modern extraction, it is essential to use solvents that simultaneously reach the standards of green chemistry and allow the extraction of a wide spectrum of bio-compounds.

Natural Deep Eutectic Solvents (NaDES), as a green solution, meet this difficult task and represent a satisfactory alternative to organic solvents. Microalgae are recognized as a sustainable resource to produce polar and non-polar metabolites, such as carotenoids, free fatty acids (FFA) and phycobiliproteins. Among them, Spirulina (Arthrospira platensis) is one of the most studied, due to its

strong potential for food, health and cosmetic. In this work, two biorefinery scenarios were investigated using polar and non-polar NaDES. The first one is a combination of a polar/non-polar sequential solid/liquid extraction together with a liquid-liquid extraction of the polar extract. The second scenario implements an innovative triphasic solid/liquid/liquid approach, based on the simultaneous use of polar and non-polar NaDES in the solid/liquid extraction step. Several NaDES

were screened in this study to identify the best polar/non-polar NaDES pair. According to the results, the use of the triphasic approach allowed an increase of the productivity by a factor 4 for chlorophylls and FFA, and by a factor 2 for carotenoids. It was also interesting to note that the phycocyanin-enriched polar fraction achieves much higher levels of purity than the corresponding
single-phase extraction for both applied biorefinery scenarios. These results pave the way for the first time for a microalgae biorefinery entirely based on the use of NaDES and industrially credible.

31

### Keywords

Natural deep eutectic solvents; microalgae; phycobiliprotein; free fatty acids; biorefinery; Spirulina

34

## 1. Introduction

Since the last decade, many studies have been conducted on deep eutectic solvents (DES) for organic synthesis, biomass dissolution, formulation of pharmaceuticals or extraction of biocompounds among others [1]. Based on their unique physico-chemical properties, DES have been considered as a new class of alternative solvents suitable to substitute petrochemical solvents, which are very well-known for their toxicity and negative impacts on the environment [2]. However, the formulation of DES was not always fully satisfying these ecological and toxicological criteria, and in fact, more natural alternatives were then sought.

Inspiring by extremophiles plants, the concept of Natural Deep Eutectic Solvents (NaDES) was first described in 2013 by Dai et al. [3] in which NaDES were considered as the third biological liquid in plants beside water and lipids. NaDES were formed using abundant natural molecules, such as sugars, amino-acids, organic acids, polyols, etc.[3,4] Briefly, DES and NaDES exhibit a lower melting point than their pure components taken separately, due to hydrogen bond, van der Waals and/or electrostatic interactions.[3,5] NaDES, because of their great potential, have focused the attention of a large number of scientists working in very different fields and disciplines, ranging from thermodynamics, pharmacology to extractive sciences. The definition of the term NaDES is still intensely debated, especially within the thermodynamics community.[6] Indeed, if the abbreviation NaDES is commonly used in the field of extractive sciences to designate mixtures based on natural compounds, which in a well-defined ratio are liquid at room temperature. Of course, such a description does not really correspond, in fact, to a proper definition of a eutectic point.[7] Nevertheless, to
facilitate understanding between disciplines, we have chosen to use the term NaDES to designate the selected formulations described therein, even if it is not certain that each formulation corresponds to a eutectic (invariant) point in the strict sense. However, from an environmental point of view, such alternative solvents represent therefore an interesting and attractive alternative to conventional organic solvents, especially by considering their natural origin, low toxicity and high biodegradability.[1-3] In the literature, the most common used compounds for the formulation of NaDES are choline chloride, glycerol, ethylene glycol and sugar but organic acids, fatty acids, and amino-acids were also reported.[8]

Most of the NaDES described in the literature are hydrophilic, even if hydrophobic NaDES have also recently emerged.[5] Among all hydrophobic NaDES, those based on menthol appear as the most popular. This abundant and cheap molecule is generally extracted from *Mentha* species (~€60/kg),[5] and was used for the formulation of NaDES in numerous studies for the extraction of different bio-compounds from different biomasses, including microalgae.[9-11]

Microalgae, as renewable resources of molecules with high added value, have focused the interest of both academic and industrial researchers. These photosynthetic organisms have the ability to adapt to various environmental conditions, being able to be oriented to facilitate the synthesis of high value-added compounds in substantial quantities.[12,13]

Microalgae are also recognized as a rich source of polyunsaturated fatty acids (PUFA) of pharmaceutical or nutraceutical interest, such as eicosapentaenoic acid, docosahexaenoic acid, linoleic acid and γ-linolenic acid.[12,13] One of these microalgae with high potential is the cyanobacterium *Spirulina* (*Arthrospira platensis*). It was an excellent source of nutrients, which ranks first in the microalgae market and it is also considered as "super food".[14]

Therefore, this microalgae is of major commercial interest in the cosmetic, pharmaceutical, food and nutraceutical fields. This popularity is essentially due to its richness in proteins (60-
81 70% dry matter) and in pigments, mainly in carotenoids, chlorophylls and phycocyanin. [15-18]

82 Phycocyanin is a hydrophilic pigment characterized by its intense blue color and has been broadly referred to as a valuable dye due to its protein-based structure and uncommon natural blue color. The biological activities of this pigment include antioxidant, antiviral, anticancer and antitumor properties. [13] Carotenoids, another abundant pigments found in spirulina, are lipid-soluble pigments that are accountable for the yellow, orange, and red coloration in numerous vegetables, plants and micro-organisms. Both Humans and animals are unable to synthesize the carotenoids and get them through alimentation. They are used for coloring and flavouring in food and feed, and for nutraceutical application as a provitamin.

91 A source. [15-17] Many researches were carried out in order to isolate and purify the phycocyanin; however, in most cases the lipophilic bio-compounds, such as carotenoids, chlorophylls, and fatty acids were generally wasted with the residual biomass. [19]

94 Chlorophylls are also known for their masking and pigmenting properties. Free fatty acids (FFA) are of great interest in the cosmetics field, acting as regulators of the cutaneous microbiome (reactive or acne-prone skin) or as anti-aging agents. [11]

97 The extraction of various bioactive compounds has been commonly carried out using conventional extraction methods, such as maceration and Soxhlet extraction techniques.

99 However, such methods come with numerous drawbacks including high time and solvent consumption with low extraction yield. Consequently, researchers have shifted their attention toward novel extraction techniques, like microwave assisted extraction, supercritical fluid extraction, ultrasound assisted extraction (UAE). Among those techniques, UAE stand out as an inexpensive and quite straight forward substitute to conventional extraction processes. [20-22] This process uses acoustic cavitation to disrupt the cell wall allowing the penetration of the solvent into the plant material, increasing thus both the contact surface area and the mass transfer. [17-22] Consequently, UAE increases the
extraction yield, rate of extraction and reduces the extraction time without the need of applying high temperatures.[21] Numerous applications of UAE for the extraction of lipids, proteins, carotenoids, and phycocyanin are already reported in the literature.[11,15,21,23] Although UAE gives many advantages, there is no report available on biphasic extraction from Spirulina using this technique, to date. In fact, almost all the methods reported in the literature for the purification of phycocyanin require numerous of expensive and time-consuming steps, such as precipitation, centrifugation, dialysis, ion-exchange chromatography, gel filtration chromatography.[24-26] Moreover, their purification cost ranges between 50-90% of the entire production.[27] Hence, the development of more effective and cost-efficient techniques that allow both high purity and recovery is considered of high priority.

To echo this need, herein, a novel extraction approach is proposed. This approach is based on a biphasic extraction allowing, for the first time, both the extraction and purification of phycocyanin, as well as, the extraction of lipophilic compounds in a single extraction step. Furthermore, recently our group highlighted the high potential of glycerol based-NaDES for phycocyanin recovery and stabilization using UAE.[15] Especially glycerol/glucose (2:1, mol/mol) with 20% of water was highlighted as the most performant medium considering the extraction and stability enhancement of phycocyanin.[15] Additionally, menthol-based NaDES have been described as an interesting alternative option for FFA and pigments extraction from Spirulina,[11] even if its total recovery was lower than those of fatty acids based NaDES, as the selectivity towards polyunsaturated FFA (PUFA) could be dramatically improved using them. During this study, these particular NaDES, i.e. those based on polar glycerol/glucose and non-polar menthol-based systems, were selected to build up sustainable biorefinery scheme, in order to propose a new sustainable path for Spirulina pigments and FFA valorisation.
Therefore, the main objective of this work is to investigate and compare two biorefinery scenarios (sequential coupled with liquid-liquid extraction and triphasic solid/liquid/liquid (SLL) extraction) with different NaDES formulations to reach high phycocyanin extraction and purification. More importantly, this pioneer work was performed to pave the way toward a time and energy saving green process for the valorisation of numerous bio-compounds from Spirulina.

## 2. Material and methods

### 2.1 Raw Material.

Spirulina (Arthrospira platensis) biomass was kindly provided by Aqua Eco Culture (Lamballe, France). The fresh biomass (20.16% dry matter) was frozen just after harvesting at -20 °C and was stored at the same temperature prior to be used. All extractions were performed between January and May 2022, in SIMBA Laboratory (Tours, France). Octanoic acid 98%, α-D-(+)-glucose 99%, lauric acid 98%, 1,3-propanediol 98%, 1,2-octanediol 98% were purchased from Acros Organics (Geel, Belgium). Glycerol 99% was purchased from Fisher Scientific SAS (Illkirch, France). Ascorbic acid and Menthol 99% were purchased from COOPER (Melun, France). Water was purified using a Milli-Q system (Millipore Corporation, Bedford, MA, USA). Ethanol 96% analytical grade and methanol HPLC grade were purchased from Carlo Erba (Val de Reuil, France). Fatty acids standard kits (saturated and unsaturated) was purchased from Sigma Aldrich (St Louis, MO, USA).

### 2.2. NaDES preparation and analysis.

#### 2.2.1 NaDES preparation

NaDES were prepared by mixing a fixed ratio of hydrogen bound donor (HBD) and hydrogen bound acceptor (HBA) as shown in Table 1. The mixture was stirred and heated at 80°C for hydrophilic NaDES and at 50°C for hydrophobic NaDES until a colorless solvent was obtained.

Insert here Table 1

#### 2.2.2.¹H and ¹³C NMR
¹H NMR spectra were recorded on a Bruker® Avance 300 (300 MHz) NMR spectrometer, using DMSO-d₆ as internal solvent and reference (δ<sub>H</sub> = 2.50 ppm). ¹³C NMR was recorded at 75 MHz on the same instrument, using the DMSO-d₆ solvent, with the reference peak at δ<sub>C</sub> = 39.52 ppm.

The NMR tubes were prepared by diluting 20 or 50 µL of NaDES in 480 or 450 µL of DMSO-d₆, respectively.

### 2.2.3. COSMO-RS component calculations

During this work, the sigma profile and the sigma surface of each species were directly visualized by using the COSMOthermX software (version BIOVA 2021) along with their BP-TZVP COSMO files when available into the BIOVA COSMObase 2021 database. However, if such files are not already available into this database, like in the case of the α-D-glucose and L-menthol, the following calculations were realized to obtain suitable 3D descriptors for a wide range of conformers for each investigated molecule.

In such a case, the structure of the species was firstly optimized, with a convergence criterion of 10⁻⁸ Hartree in the gas phase, by using DFT calculations within the Turbomole 7.5.1 program package[28] utilizing the BP68 functional with the def-TZVP basis set,[29]and combining the Resolution of Identity (RI) approximation.[30] Each resultant optimized 3D structure was then used as an input for the generation of the COSMO file within the sigma profile and the sigma profile of each conformer of each species using the COSMOconfX program (version BIOVA 2021). [31]

The sigma profile and the sigma surface of the most stable conformer (i.e. presenting the lowest energy conformer) was used to represent the 3D charge distribution onto the surface of each molecule (i.e. reflecting their sigma profile and sigma profile) to localize HBA and HBD sites, as the sigma surface represents the charge distribution onto the surface of each molecule.
183 In fact, within the COSMO-RS method, HBA areas are represented as negative surfaces
184 (shown in red color in Fig. S1 of the ESI), while HBD are expressed as positive surfaces, shown
185 in blue color (Fig. S1 of ESI). The green area represents the non-polar part of the molecule,
186 mainly corresponding to Van der Waals-type interactions.

187 Similarly, on each graph presenting a sigma profile, a peak localized with a positive surface
188 charge density (σ, e/Å²) corresponds to HBA groups, while a peak observed in negative values
189 corresponds to HBD groups. A peak observed at around 0 e/Å² mainly corresponds to
190 nonpolar moieties causing hydrophobic van der Waals-type interactions.

191 During this work, both sigma profile and sigma surface were used as descriptors to provide
192 information on molecule's association driven by their apolar (hydrophoci) and polar HBAs and
193 HBDs moieties.

2.3. *Extraction process*.

195 The ultrasound-assisted extraction (UAE) was carried using an ultrasonic bath RK-100H
196 (Bandelin electronic, Berlin, Germany).

197 Each extraction was performed in triplicate. Two strategies were investigated herein, as displayed in
198 Figure 1.

199 *Insert here figure 1*

200 The first one (Figure 1A) is based on a sequential extraction (SE) of Spirulina. First, biomass
201 was extracted with hydrophilic NaDES using UAE at room temperature for 90 min at a given
202 biomass/solvent ratio (1:5, w/w). The resulting polar extract was then centrifuged during 20
203 min at 16 200 g (Rotanta 460R, Hettich, Kirchlengern, Germany). After its centrifugation, the
204 supernatant was submitted to a liquid-liquid extraction (LLE) using a non-polar NaDES (1:1,
205 vol. ratio) and the pellet (algae) was reused for a second 90 min UAE using the same non-
206 polar NaDES.
The second scenario, specially developed for this work, is based on a biphasic extraction (Figure 1B).
Herein, 3 g of hydrophilic and 3 g of hydrophobic NaDES were simultaneously used along with 0.6 g
of microalgae (1:5, w/w ratio per NaDES) for a 90 min UAE at ambient temperature. After each 30
min, samples were vortex for 2 min. The resulting extract was then centrifuged during 20 min at 16
200 g, resulting, thus, in two separated phases, which were then further analyzed. To highlight the
potential interest to mix these two NaDES into this single extraction step, reference monophasic
extraction processes were carried under the same condition, i.e. for 90 min UAE, by mixing 3 g of a
particular NaDES with 0.6 g of biomass (1:5, w/w ratio).

2.4. *Extract analysis.*

216 2.4.1. *Free Fatty acid (FFA) analysis*

217 FFA qualitative and quantitative were determined by liquid chromatography-mass
218 spectroscopy (LC-ESI-MS), according to Wils et al.[25] These analyses were performed using
219 an Acquity H-Class coupled with an SQD detector (Waters, Saint Quentin in Yvelines, France).
220 The system was fitted with a BEH C18 (50 mm × 2.1 mm; 1.7 µm). The column oven was set at
221 60°C. The mobile phases were (A) 0.01% formic acid (aq.) containing 0.2 mM ammonium
222 formate and (B) 50% isopropanol in acetonitrile containing 0.01% formic acid (aq) and 0.2
223 mM ammonium formate. The flow rate was 0.24 mL/min and gradient was set as follows:
224 initial solvent B content was 50%, raised to 98% in 16 min and maintained for 4 min. The
225 column was then re-equilibrated to initial conditions. ESI was in negative mode and
226 performed with cone voltage at 50 V with capillary voltage of 2.8 kV. The NaDES extracts
227 required a prior pre-treatment, which was carried by solid-phase extraction (SPE) using C18-
228 silica gel (Ait, 40–60 µm Fischer Scientific, Waltham, MA, USA). The elution gradient ranged
229 from 50 to 100% water:methanol. Herein, only the 100% methanolic fractions were
230 analyzed.[25] Samples were all prepared in methanol. Nervonic acid (C 20:4) at 10 µg/mL was
231 added as internal standard. All FFAs were identified by comparison of retention time and
232 mass spectra using commercial standards. FFA titration was performed using a calibration
curve of each standard ranging from 2.5 µg/mL to 12.5 µg/mL (1 µL injection) and using
nervonic acid as internal standard. Linear regression and calculation were performed of SIR
chromatogram using Targetlynx (Waters).

2.4.2. Pigment titration of non-polar extract

The pigment titration was performed according to Wils et al.[25] Briefly, 100 mg of the
extract were diluted by 0.8 mL of methanol; 290 µL of the resulting solute were used for both
the carotenoid and chlorophyll titration. The absorption spectra were measured using UV-vis
microplates spectrophotometer reader (Multiskan GO, Thermo Fisher Scientific, SAS) and the
carotenoids (C<sub>Ca</sub>) and chlorophyll (C<sub>Ch</sub>) contents were then calculated using the absorbances
at 450, 645 and 663 nm by applying the following equations 1 and 2:

$$
C_{ch} = [(20.2 \times OD_{645}) + (8.2 \times OD_{663})] \times FD \quad \text{Eq. 1}
$$

$$
C_{ca} = (4 \times OD_{450}) \times FD \qquad \text{Eq. 2}
$$

where C<sub>Ch</sub> (µg/ml) is the chlorophyll concentration, C<sub>Ca</sub> (µg/ml) the carotenoids concentration,
FD the dilution factor, and OD<sub>645</sub>, OD<sub>663</sub>, and OD<sub>450</sub> the extract optical density at 645, 663 and
640 nm, respectively.

2.4.3. Pigment titration of polar extract

The pigment titration was performed according to Wils et al.[25] Briefly, 100 mg extract was
diluted by 0.8 mL of distilled water; 250 µL of the resulting solute was used for the
phycocyanin titration. The absorption spectra were measured using UV-vis microplates
spectrophotometer reader (Multiskan GO, Thermo Fisher Scientific, SAS). The phycocyanin
(C<sub>PC</sub>) contents was obtained using the absorbances at 620 and 652 nm by applying the
equation 3 as proposed by Bennett et al.[27]

$$
C_{PC} = \frac{OD_{620} - (0.474 \times OD_{652})}{5.34} \quad \text{Eq. 3}
$$

where, C<sub>PC</sub> (mg/mL) is the phycocyanin concentration, and OD<sub>620</sub> and OD<sub>652</sub> is the extract
optical density at 620 and 652 nm, respectively.
The phycocyanin yield (YPC) was then calculated using equation 4, as follows:

$$Y_{PC} = \frac{C_{PC} \times 10 \times V_{NADES}}{M_{DM}} \qquad \text{Eq. 4}$$

where VNADES is the NaDES volume, MDM the mass of dried matter and YPC the extraction yield expressed in milligram of phycocyanin per gram of dried matter (mg/g DM).

### 2.4.4. Colorimetry in CIELAB color space.

Colorimetric measurements were performed on the monophasic extract of GG and compared to the biphasic extract of the same NaDES coupled with MC8. The equipment used is a handheld chroma meter C400 (Konika Minolta, Tokyo, Japan) able to read translucid samples in a cuvette. All the measurements were performed at ambient temperature and performed in triplicates.

CIELAB coordinates (L*a*b) of all samples were measured and significant differences were highlighted using equation 5.

$$\Delta E = (\Delta L)^2 + (\Delta a)^2 + (\Delta b)^2 \qquad \text{Eq. 5}$$

### 2.4.5. Statistical analyses

The statistical significance was evaluated by the one-way ANOVA. Differences were considered to be significant at p < 0.05.

## 3. Results and discussion

### 3.1. Smart NaDES selection strategy

The selection of the raw materials used during the preparation of the so-called NaDES is a crucial step in order to truly design a formulation which respects Green Chemistry principles and could be then used as green solvents. Indeed, like all non-volatile solvents, the solvent/metabolite separation requires purification or de-extraction steps using conventional organic solvents, biobased or not, which drastically reduces the environmental interest of using these matrices. One of the solutions frequently mentioned in the literature, and use in industry, is the recycling and re-equipping of NaDES after the de-extraction of the metabolites
282 of interest.[32,33] However, this approach further complicates the extraction scheme by
283 multiplying the number of unit operations and in fact increases the overall cost and footprint
284 of the whole process.

285 Another approach, which we have applied herein, consists in selecting raw materials
286 biocompatible with the targeted application of the extract produced. In fact, by applying such
287 an approach, no further extraction scheme is requested as the NaDES will be further used as a
288 passive ingredient presents in final formulation containing the targeted and extracted
289 product. As in the present case, the targeted application being associated with the cosmetic
290 market, the raw materials were preselected according to the following criteria: i)
291 compatibility with the European and Chinese cosmetic regulations; and ii) availability of
292 biosourced resources. The main constraint of the cosmetic market concerning NaDES is the
293 prohibition of choline chloride, i.e. the most used ingredient for the preparation of this class
294 of solvents.[1]

295 Thus, all raw materials used during this work were firstly selected as they are compatible with
296 cosmetics, which allows their integration into the finished product. This singularity further
297 allows to deeply simplify the overall extraction process as separation units and steps including
298 metabolite recovery and solvent removal/recycling could be vanished. More importantly, this
299 possibility provides a real sense of the utilisation of NaDES, respecting thus Green Chemistry
300 principles by proposing, also, a sustainable solution with lower footprint than current
301 benchmarks.[34]

302 The ingredients of these NaDES are functional ingredients used in current cosmetic
303 formulation as emollient, solvent or texture agent. In fact, by keeping the NaDES in the
304 finished product further allows to decrease the use of additional formulation ingredients,
305 reinforcing the overall hypothesis that the proposed approach is greener and more
306 sustainable than conventional ones.
The biocompatible character of NaDES was also tested previously on keratinocyte cell culture and all formulations do not show significant toxicity at tested concentrations.[11]

Among all NaDES described in the literature, GG and combination of Menthol and fatty acids or thymol were in fact selected.[35-37] The choice of menthol partner in hydrophobic NaDES was mainly guided thanks to the sigma profile of preselected biocompatible compounds (see Figure S1 of the Electronic Supporting information). In fact, each decision was based on either the formulation or on a molecule selection, was also driven thanks to simulation calculations.

Herein, the COSMOthermX software was used to visualize the sigma profile and the 3D mapping of the charges' distribution onto each preselected molecule. Both of them were used therein as descriptors to further validate decisions made. In fact, these descriptors could be used to depict the compatibility of the menthol with selected partners. Selected partners are defined as: i) hydrophobic species mainly dominated by van der Waals-type interactions, with a HBD group as observed with C8 and C12 fatty acids; ii) hydrotropes with variable chain length to provide a modulated hydrophobic character along with both HBA and HBD moieties, as observed with 1,3-propanediol and 1,2-octanediol; and, iii) a molecule mimicking the 3D mapping and the sigma profile of the menthol like the thymol (see Figure S1 of the Electronic Supporting information).

As reported in Table 1, another advantage for formulating hydrophobic NaDES based on Menthol is that their relatively low density data (on average around 0.9 g/mL) and their low viscosity (on average between 20 and 70 mPa.s),[38] making these NaDES easy to handle.

In contrary, the tested glucose-based NaDES has a density and viscosity close to that of glycerol, making its handling more tedious but still experimentally and industrially feasible.[34]
Reference extractions were performed with different NaDES; polar (GG) and non-polar (MC8, MC12, MO, MP, and MT). Figure 2 displays the pigments (Fig. 2A) and FFA (Fig. 2B) content in the extracts after each monophasic extraction.

Insert here figure 2

Regarding GG extract, the main pigment extracted was the water-soluble protein phycocyanin with 90.85 ± 2.50 mg/g DM. Although GG was considered as polar NaDES, a minority of chlorophylls and carotenoids were extracted (respectively, 3.72 ± 0.09, and 1.71 ± 0.04 mg/g DM). Regarding the non-polar NaDES, the NaDES MO exhibited the highest selectivity toward non-polar pigments with 8.9 ± 0.1 mg/g DM of chlorophylls and 3.8 ± 0.1 mg/g DM of carotenoids.

Concerning the FFA content of reference extracts, the profile was substantially the same for each; with: myristic acid (C14), palmitic acid (C16), palmitoleic acid (C16:1), stearic acid (C18), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3). FFA profile was mostly dominated by PUFA. The polar GG extract was surprisingly enriched in FFA with a total amount of 26.55 mg/g DM, compared to 36.06 mg/g DM in MC12 extract. In fact, sugar-based NaDES are less polar than water with polarity close to methanol.[37] Thus, belonging to the category of NaDES that exhibit solvent duality (lipophilicity and hydrophilicity), explaining why GG can extract also a high quantity of FFA.

### 3.3. Sequential extraction.

Two strategies were initially considered: 1) an extraction with a polar NaDES followed by a non-polar NaDES or 2) an extraction with a non-polar NaDES followed by a polar NaDES. Regarding this last strategy, it was abandoned since no metabolite was extracted with polar NaDES after the non-polar extraction (data not shown as its concentration was always lower than the detection limit of the equipment used). Based on our observation, it appears that the non-polar NaDES clogged the hydrophilic NaDES access points, limiting thus the extraction of the targeted compounds. Subsequently, solely the strategy 1) was then used. First, polar NaDES (GG) was used to extract metabolites, mostly polar, from Spirulina. After UAE extraction and
centrifugation, the polar extract exhibited a green-blue color due to the extracted pigment phycocyanin (Fig. 2A). For sequential extraction, this green-blue extract has been set aside and the pellet biomass (called GG pellet) was reused for the extraction of non-polar metabolites with non-polar NaDES (MC8, MC12, MO, MP, and MT). Figure 3 displays the pigments (Fig. 3A) and FFA (Fig. 3B) content of non-polar extracts after the sequential extraction (abbreviated as Seq). These results were compared with reference monophasic extractions (abbreviated as MonoP).

Insert here Figure 3

After the sequential extraction of pellet biomass with non-polar NaDES, the results showed a decrease of non-polar pigments (chlorophylls and carotenoids) and FFA (especially linoleic acid) compared to the references monophasic extraction. For example, regarding the sequential extraction with the NaDES MO, the content of chlorophylls and carotenoids decreased, respectively, by 71% and 78%, compared to the reference extract. Regarding FFA, linoleic acid content was 17.74 ± 1.23 for the MO reference extract and was zero for sequential extract. In view of the FFA content in GG extracts (Fig2.B), one could point-out that a significant percentage of these non-polar metabolites was already extracted via the polar NaDES GG.

Therefore, the polar extract GG was submitted to a liquid-liquid extraction (LLE) with non-polar NaDES intended for both non-polar compound extraction and phycocyanin purification. The non-polar phase obtained was pooled with the non-polar extract from the sequential extraction to form the so-called: pooled extract. Figure 4 displays the phycocyanin content (mg/g DM) in the polar extracts after liquid-liquid extraction with non-polar NaDES.

Insert here Figure 4

Regardless of the non-polar NaDES used to perform liquid-liquid extraction on the polar extract, the amount of phycocyanin in this phase remains approximately the same: e.g. 82 mg/g DM; i.e. a result close to the reference GG extract close to 90.8 ± 2.5 mg/g DM.
Therefore, it could be speculated that the liquid-liquid extraction using a menthol based NaDES did not affect the polar composition of the polar fraction. Figure 5 displays the non-polar pigment (Fig. 5A) and FFA (Fig. 5B) content in the extracts after a sequential extraction (abbreviated as Seq), liquid-liquid extraction (abbreviated as LL), and in the non-polar extract called "pooled". Regarding the pigment profiles of non-polar extracts, as shown in Fig. 5A, one can see that using a liquid-liquid extraction it is possible to recover a large majority of the chlorophylls and carotenoids pigments. For example, the recovery rate of chlorophylls varied between 61% and 80% with MC12 and MO, respectively. However, in the case of carotenoids, its recovery rate is between 26% and 35% using MT and MO, respectively.

### Insert here figure 5

The recovery of the green chlorophyll and orange carotenoid pigments allowed the purification of GG phase restoring thus the bright blue color related to the high phycocyanin content as shown in Figure 6.

### Insert here figure 6

In the case of FFA (Fig. 5B), the collected results indicate that only 12%, 13%, 11%, 16%, and 17% of FFA were left in the GG hydrophilic extract after MC8, MC12, MO, MP, and MT liquid-liquid extraction, respectively. NaDES composition in each liquid phase was determined after the LLE extraction using ¹H and ¹³C NMR. No significant variation of the NaDES concentration in its rich phase was notified. More importantly, no trace of non-polar NaDES component transfer to GG was observed (Figure S2 to S5, Electronic Supporting Information). It was important to assess this point, as GG can solubilize non-polar metabolites, and could then disturb the non-polar NaDES equilibrium during the LLE process. By using the sequential extraction combined with liquid-liquid extraction, it was then possible to obtain selectively two distinct extracts: i) the purified polar with mainly water-soluble phycocyanin, and ii) the pooled non-polar extract composed of non-polar metabolites: chlorophylls, carotenoids and
407 FFA. Collected data could be used as a first proof of concept that LLE using solely NaDES
408 matrixes is in fact possible. This further open the way to multistep biorefinery or purification
409 procedure, using such promising alternative solvents. However, reference extractions with
410 hydrophobic NaDES remain better and less time-consuming than the sequential extraction
411 followed by liquid-liquid extraction.

412 Nevertheless, in the literature most of LLE processes including NaDES described a
413 NaDES/organic solvent or water systems, either for metabolites recovery from NaDES, [38-42]
414 or liquid-liquid chromatography. [43] Herein and for the first time, we described an extraction
415 process using solely NaDES-based solvents, generating two different enriched NaDES-based
416 extracts. However, in order to improve the productivity of the process, we then decided to
417 investigate a triphasic S/L/L approach.

### 3.3. Solid/Liquid/Liquid strategy.

419 An innovative solid/liquid/liquid (SLL) process was considered as an alternative biorefinery
420 scenario allowing the extraction of polar and non-polar compounds into two easily separated
421 fractions. Herein, the polar fraction (GG) was characterized by a bright blue color (rich in
422 phycocyanin) while the non-polar fraction with a greenish yellow coloration (chlorophylls,
423 carotenoids, and FFA). Figure 7 displays the phycocyanin content (mg/g DM) in each polar
424 extract (abbreviated as BiP GG) after a biphasic extraction using non-polar NaDES.

425

Insert here figure 7

426 Whatever the NaDES used, similar phycocyanin content was collected back, i.e. about 75
427 mg/g DM. Contrary to monophasic extract (Figure 6), after the biphasic extraction, the polar
428 phase exhibits a bright blue color. This might imply that it is possible to both extract and
429 purify the phycocyanin using this extraction technique.
In order to confirm these hypotheses, the content of non-polar pigments (Fig.8B) and FFA (Fig.8B) was investigated in the polar fraction (abbreviated GG SLL) and in the non-polar fraction (abbreviated as "non-polar NaDES" SLL).

Insert here figure 8

It was found that chlorophylls, carotenoids, and FFA concentrations were more than 80% lower in the GG polar fraction of the biphasic extract than values collected using the monophasic GG for all used couplings. After the biphasic extraction the contents of pigment and FFA in the non-polar extract were either higher or comparable with data found using the monophasic extraction, used herein as a reference. Based on data collected, MO was found to be the best lipophilic NaDES for the extraction of non-polar metabolites.

Extraction performances of FFA were slightly improved for all NaDES compared to the reference extraction and GG resulting extracts exhibited all a bright blue color. Thus, this biphasic strategy appears to be a relevant scenario to produce in one step both polar and non-polar enriched extracts.

The non-polar fraction from the LLE and SLL processes could well be incorporated into anti-aging cosmetic preparations, thanks to the compatibility of NaDES with cosmetic regulations and its richness in carotenoids and FFA of interest. The presence of menthol will not be a limitation, as the active ingredient content of finished products is generally around 1%. Cytotoxicity results against keratinocytes and cutaneous microbiota carried out on a panel of NaDES, including Menthol-based NaDES, showed good safety at cosmetic doses.[11]

### 3.4. Colorimetric analysis

Here, it was assumed that the biphasic extraction allowed both the extraction and purification of phycocyanin. This hypothesis was argued by both the blue color of the extracts and the important decrease in the chlorophylls, carotenoids, and FFA contents in the polar fraction. To further investigate this hypothesis, a colorimetry analysis using CIELAB color
system was performed using GG extracts as reference, GG coupled with MC8 as the biphasic
extracts, and GG after liquid-liquid extraction with MC8. The extracts were all diluted with
water to 1:20 ratio for a better reading of colorimetry parameters. The system is composed of
one channel for light (L*) and two color channels (a* and b*). The L*axis goes from 0 (black
to 100 (white), the a* from green (-a*) to red (+a*), and b from blue (-b*) to yellow (+b*). In
the case of the GG extract, i.e. the reference, collected values were: a*= 3.66 ± 0.11, b*= 1.12
± 0.01, and L*= 23.43 ± 1.34, representing a greener blue. In the case of the biphasic extract,
the following values were collected: a*= -0.2 ± 0.01, b*= -7.06 ± 0.01, and L*= 27.29 ± 0.02
representing a pure light blue similar to the one found after the liquid-liquid extract (e.g. a*=
0.38 ± 0.01, b*= -6.78 ± 0.01, and L*= 27.06 ± 0.02). This was further confirmed thanks to the
calculation of ΔE, which was significant, i.e. >2, in comparison with the selected references:
e.g. the sequential GG (ΔE = 96.7) or biphasic GG (ΔE = 86.3), but not significant when
comparing with the value collected for the Sequential and Biphasic extract (ΔE = 0.5).

It was noted here that both biphasic and liquid-liquid extractions allow the purification of
phycocyanin as confirmed by the blue-sky color of the extract. Even if the biphasic extraction
has a lower yield that the monophasic extraction, it allows, for all the tested coupling, both
the extraction and purification of phycocyanin. This could potentially eliminate a further
purifications step prior to the incorporation of the blue extracts in cosmetic, nutraceutical or
food products.

Moreover, as GG was highlighted as powerful stabilization media for phycocyanin, using this
biphasic approach extraction, purification and stabilization of the high value pigment could be
performed in one step.

### 3.5. Sequential/LLE vs SLL approach

Considering the polar extracts and therefore the phycocyanin content, similar performances
were obtained with Seq/LLE and triphasic SLL extraction. Nonetheless, the SLL process slightly
improved the extraction of non-polar compounds in comparison with the sequential
extraction followed by a liquid-liquid extraction. Moreover, this biphasic process has an extraction time three times shorter than the other. Figure 9 summarizes the results obtained for the NaDES MC12 and MO, which allows an easy comparison of the two tested approaches in terms of their productivity.

Insert here Figure 9

As shown in Figure 9, the use of the biphasic process increases the productivity for all tested metabolites. Indeed, with the sequential approach, solely 100 mg of chlorophyll could be extracted per hour, while 250 and 500 mg/h could be extracted using the biphasic approach with GG/MC12 and GG/MO, respectively. For the FFAs, ~0.5 g would be extracted per hour for the sequential approach compared to more than 2 g/h for the biphasic, corresponding to a four-fold increased productivity. Considering carotenoids, major high value-added pigments, productivity was increased only of a factor 2, which is still a very good result.

Data collected therein using the NaDES extracts with ultrasonic extraction processes were then compared with those reported in the literature, to further highlight their competitive levels and thus the validity of the proposed strategy. In fact, the levels observed using laboratory scale processes (0.26 mg/mL solvent) [44] or industrial ones (0.17 mg/ml solvent) [45], are about ten times less concentrated than those achieved therein by following the NaDES protocols (reaching 3.0 mg/mL NaDES on average). However, the non-volatile nature of NaDES makes subsequent concentration steps very difficult, unlike aqueous extracts.

However, it is important to note that biological activities related to cosmetic applications (tyrosinase, antioxidant, anti-inflammatory) have shown active phycocyanin concentrations oscillating between 20 and 100 µg/mL [46] These concentrations would correspond to a use of our extracts between 0.5 and 3% in a cosmetic product, which is in line with industrial practices in this field.[47]

### 3.6. Biorefinery approaches sustainability assessment.
The SLL extraction process, appearing then as a major improvement in microalgae biorefinery processes panel, was then analyzed according to the principles of green extraction as established by Chemat et al. [48] This original and green charter in the form of the “six principles of green extraction” allows an innovation that is not only limited to the process but to all the solid-liquid extraction aspects. More recently, new indicators derived from analytical chemistry have appeared, such as the ComplexGAPI method. [49] This approach makes it possible to assess the greenness of an analytical process by considering the entire workflow, including the steps preliminary to the analysis itself. This approach has been specially developed on the basis of the GAPI and CHEM 21 approaches, to take into account the specific features of emerging solvents such as ionic liquids or deep eutectic solvents. [49] The ComplexGAPI approach simplifies access to green performance indicators through the use of freely available online software. Rente et al. recently used this tool to assess the comparative impact of extraction processes involving NaDES. [47] Minimal adaptation of some parameters makes the tool very easy to use. We therefore applied the ComplexGAPI strategy to both our sequential/LLE and SLL approaches. The parameters used to generate the pictograms are summarized in Table 2, and the pictograms generated are shown in Figure 10.

Insert here Table 2 & Figure 10

Indicators of good environmental performance appear in green, those of average impact in yellow and the most negative impacts in red.

The parameters grouped together under the term "pre-extraction procedure" represent the steps involved in preparing NaDES prior to be used as an extraction solvent. In the pictogram, these indicators are located in the center of the graph. Logically, the pictograms are identical for both processes, as the NaDES involved are the same. Next come parameters relating to sample preparation (left), solvents and reagents (center), then instrumentation (right). The major difference between the two processes lies in energy consumption, which is drastically reduced in the SLL
approach, enabling the indicator to change from red to yellow. Interestingly enough, the
consumption indicators are suitable for sample analysis and can be quite low when considering an
extractive process. Nevertheless, the ComplexGAPI approach allows us to visually highlight the
environmental impact of our processes. Selecting NaDES using our Smart NaDES approach eliminates
the need for numerous extract processing steps saving, thus, energy and limiting waste generation.
Overall, both approaches have good environmental impacts, with the SLL process having a more
favorable impact. This greenness indicator, combined with increased productivity, makes the SLL
process a prime choice for the development of economically and environmentally sustainable
biorefinery schemes.

## 4. Conclusions

Whether sequential/LLE or SLL strategies, polar and non-polar metabolites could be extracted
from the microalgae Spirulina using exclusively NaDES. The SLL process fully fulfilled the eco-
extraction guidelines allowing a dramatic reduction of unitary operation combined with an
improved mass transfer and pre-purification of phycocyanin. When compared to the
sequential extraction, the productivity of this process increases four-fold and doubled the
content of FFA and carotenoids, respectively. In the light of collected results, the biphasic
approach assisted by ultrasound extraction could be seen as a promising approach for the
extraction, purification, and valorisation of Spirulina high value-added compounds, covering a
wide range of polarity.

These results describe for the first time a full NaDES-based biorefinery scheme of Spirulina,
opening the way to alternative extractions solvent for developing promising biomasses
valorisation. Extension to other microalgae species and to plant biomasses is currently under
investigation, as well as, innovative alternative extraction processes.

555
556 **Author Contributions**

557 Conceptualization, L.B.D.; Methodology, L.B.D., E.T., J.J. and B.C.L.; investigation, S.H., L.V.G., E.T., L.W., and M.Y.;
558 resources, M.P. and B.C.L.; writing—original draft preparation, S.H., and L.V.G.; writing—review and editing, E.T.,
559 L.B.D., J.J. and B.M.; supervision, L.B.D., J.J. and B.M.; project administration, L.B.D.; funding acquisition, L.B.D. All
560 authors have read and agreed to the published version of the manuscript.

561 **Conflicts of interest**

562 There are no conflicts to declare

563 **Acknowledgements**

564 This work was supported by the French National Research Agency (ANR-19-CE43-0001-01), the Région Centre Val
de Loire (L. Wils, PhD Grant) and Université de Tours (M. Yagmur, PhD grant, les Rabelasiennes).

566

567 **Supplementary material**

568 Supplementary material is available online at

569 **References**

570 [1] K. M. Jeong, J. Ko, J. Zhao, Y. Jin, D. E. Yoo, S. Y. Han, J. Lee, Multi-functioning deep eutectic
571 solvents as extraction and storage media for bioactive natural products that are readily applicable to
572 cosmetic products, J. Clean. Prod. 151 (2017) 87–95. https://doi.org/10.1016/j.jclepro.2017.03.038.

573 [2] B. Socas-Rodríguez, Á. Santana-Mayor, A. V. Herrera-Herrera, M. Á. Rodríguez-Delgado, Deep
574 eutectic solvents, in A. M. Inamuddin, S. K. Asiri (Eds.) Green Sustainable Process for Chemical and
575 Environmental Engineering and Science, Elsevier, 2020, pp. 123-177, https://doi.org/10.1016/B978-0-12-817386-2.00005-6

577 [3] Y. Dai, G.-J. Witkamp, R. Verpoorte, Y. H. Choi, Natural Deep Eutectic Solvents as a New Extraction
578 Media for Phenolic Metabolites in Carthamus tinctorius L., Anal. Chem. 85 (2013) 6272–6278
579 https://doi.org/10.1021/ac400432p

580 [4] Y. H. Choi, J. van Spronsen, Y. Dai, M. Verberne, F. Hollmann, I. W.C.E. Arends, G.-J. Witkamp, R.
581 Verpoorte, Are Natural Deep Eutectic Solvents the Missing Link in Understanding Cellular Metabolism
582 and Physiology?, Plant Physiol., 156 (2011) 1701–1705, https://doi.org/10.1104/pp.111.178426
583 [5] B. D. Ribeiro, C. Florindo, L. C. Iff, M. A. Z. Coelho, I. M. Marrucho, Menthol-based Eutectic Mixtures: Hydrophobic Low Viscosity Solvents. ACS Sustain. Chem. Eng. 3 (2015) 2469–2477.
584
585 https://doi.org/10.1021/acssuschemeng.5b00532

586 [6] B. B. Hansen, S. Spittle, B. Chen, D. Poe, Y. Zhang, J. M. Klein, A. Horton, L. Adhikari, T. Zelovich, B. W. Doherty, B. Gurkan, E. J. Maginn, A. Ragauskas, M. Dadmun, T. A. Zawodzinski, G. A. Baker, M. E. Tuckerman, R. F. Savinell, J. R. Sangoro Deep Eutectic Solvents: A Review of Fundamentals and Applications, Chem. Rev. 121 (3) (2021) 1232-1285 https://doi.org/10.1021/acs.chemrev.0c00385

590 [7] G. Ah-Iung, B. Flamme, F. Ghamouss, M. Maréchal, J. Jacquemin, Guidelines for designing highly concentrated electrolytes for low temperature applications. Chem. Commun., 56 (2020) 9830-9833
591
592 https://doi.org/10.1039/D0CC03963B

593

594 [8] M. Benlebna, M. Ruesgas-Ramón, B. Bonafos, G. Fouret, F. Casas, C. Coudray, E. Durand, M. Cruz Figueroa-Espinoza, C. Feillet-Coudray Toxicity of Natural Deep Eutectic Solvent Betaine:Glycerol in Rats. J Agric Food Chem. 66(24) (2018) 6205-6212. https://doi.org/10.1021/acs.jafc.8b01746

597 [9] W. Pitacco, C. Samorì, L. Pezzolesi, V. Gori, A. Grillo, M. Tiecco, M. Vagnoni, P. Galletti. Extraction of astaxanthin from Haematococcus pluvialis with hydrophobic deep eutectic solvents based on oleic acid. Food Chem. 379 (2022) 132156. https://doi.org/10.1016/j.foodchem.

600 [10] C. Crampon, C. Nikitine, M. Zaier, O. Lépine, C. Dejoye Tanzi, M. Abert Vian, F. Chemat, E. Badens, Oil extraction from enriched Spirulina platensis microalgae using supercritical carbon dioxide. J. Supercrit. Fluids 119 (2017) 289–296 https://doi.org/10.1016/j.supflu.2016.10.006.

603 [11] L. Wils, C. Leman-Loubière, N. Bellin, B. Clément-Larosière, M. Pinault, S. Chevalier, C. Enguehard-Gueiffier, C. Bodet, L. Boudesocque-Delaye Natural deep eutectic solvent formulations for spirulina: Preparation, intensification, and skin impact. Algal Res. 56 (2021) 102317
604
605
606 https://doi.org/10.1016/j.algal.2021.102317
607 [12] X. Yang, Y. Li, Y. Li, D. Ye, L. Yuan, Y. Sun, D. Han, Q. Hu Solid Matrix-Supported Supercritical CO2 Enhances Extraction of γ-Linolenic Acid from the Cyanobacterium Arthrospira (Spirulina) platensis and Bioactivity Evaluation of the Molecule in Zebrafish. Mar. Drugs 17 (2019) 203 https://doi.org/10.3390/md17040203

611 [13] N. Seyidoglu, S. Inan, C. Aydin, A Prominent Superfood: Spirulina platensis. in N. Shiomi, V. Waisundara (Eds.) Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine, InTech, 2017. https://doi.org/10.5772/66118.

614 [14] M. Raczyk, K. Polanowska, B. Kruszewski, A. Grygier, D. Michałowska, Effect of Spirulina (Arthrospira platensis) Supplementation on Physical and Chemical Properties of Semolina (Triticum durum) Based Fresh Pasta. Molecules 27 (2022) 355 https://doi.org/10.3390/molecules27020355

617 [15] S. Hilali, L. Wils, A. Chevalley, B. Clément-Larosière, L. Boudesocque-Delaye, Glycerol-based NaDES as green solvents for ultrasound-assisted extraction of phycocyanin from Arthrospira platensis—RSM optimization and ANN modelling. Biomass Convers. Biorefinery 12 (2022) 157–170 https://doi.org/10.1007/s13399-021-02263-6

621 [16] W.S. Park, H.-J. Kim, M. Li, D.H. Lim, J. Kim, S.-S. Kwak, C.-M. Kang, M.G. Ferruzzi, M.-J. Ahn Two Classes of Pigments, Carotenoids and C-Phycocyanin, in Spirulina Powder and Their Antioxidant Activities. Molecules 23 (2018) 2065 https://doi.org/10.3390/molecules23082065

624 [17] R. Sharma, B. Bhunia, A. Mondal, T. K. Bandyopadhyay, I. Devi, G. Oinam, R. Prasanna, G. Abraham, O. N. Tiwari, Statistical optimization of process parameters for improvement of phycobiliproteins (PBPs) yield using ultrasound-assisted extraction and its kinetic study. Ultrason. Sonochem. 60 (2020) 104762 https://doi.org/10.1016/j.ultsonch.2019.104762

628 [18] L. Wils, S. Hilali, L. Boudesocque-Delaye, Biomass Valorization Using Natural Deep Eutectic Solvents: What's New in France? Molecules 26 (2021) 6556 https://doi.org/10.3390/molecules26216556
631 [19] S. Marzorati, A. Schievano, A. Idà, L. Verotta, Carotenoids, chlorophylls and phycocyanin from
632 Spirulina: supercritical CO2 and water extraction methods for added value products cascade. Green
633 Chem. 22 (2020) 187–196 https://doi.org/10.1039/C9GC03292D

634 [20] F. Chemat, N. Rombaut, A.G. Sicaire, A. Meullemiestre, A.S. Fabiano-Tixier, M. Abert-Vian
635 Ultrasound assisted extraction of food and natural products. Mechanisms, techniques, combinations,
636 protocols and applications. A review. Ultrason. Sonochem. 34, 540–560 (2017)
637 https://doi.org/10.1016/j.ultsonch.2016.06.035

638 [21] S. Dey, V. K. Rathod, Ultrasound assisted extraction of β-carotene from Spirulina platensis.
639 Ultrason. Sonochem. 20 (2013) 271–276 https://doi.org/10.1016/j.ultsonch.2012.05.010

640 [22] M. Jacotet-Navarro, N. Rombaut, A.S. Fabiano-Tixier, M. Danguien, A. Bily, F. Chemat.
641 Ultrasound versus microwave as green processes for extraction of rosmarinic, carnosic and ursolic
642 acids from rosemary. Ultrason. Sonochem. 27 (2015) 102–109
643 https://doi.org/10.1016/j.ultsonch.2016.06.035

644 [23] L. Vernès, M. Abert-Vian, M. El Maâtaoui, Y. Tao, I. Bornard, F. Chemat Application of ultrasound
645 for green extraction of proteins from spirulina. Mechanism, optimization, modeling, and industrial
646 prospects. Ultrason. Sonochem. 54 (2019) 48–60 https://doi.org/10.1016/j.ultsonch.2019.02.016

647 [24] F.S. Figueira, C. C. Moraes, S. J. Kalil, C-phycocyanin purification: multiple for different
648 applications. Braz. J. Chem. Eng. 35 (2018) 1117–1128 https://doi.org/10.1590/0104-6632.20180353s20170160

650 [25] L. Wils, M. Yagmur, M. Phelippe, B. Montigny, B. Clément-Larosière, J. Jacquemin, L.
651 Boudesocque-Delaye, Alternative Solvents for the Biorefinery of Spirulina: Impact of Pretreatment on
652 Free Fatty Acids with High Added Value. Mar. Drugs 20 (2022) 600
653 https://doi.org/10.3390/md20100600
654 [26] G. Patil, S. Chethana, A. S. Sridevi, K. S. M. S. Raghavarao, Method to obtain C-phycocyanin of
655 high purity. J. Chromatogr. A 1127 (2006) 76–81 https://doi.org/10.1016/j.chroma.2006.05.073

656 [27] A. Bennett, L. Bogorad, Complementary chromatic adaptation in a filamentous blue-green alga.
657 J. Cell Biol. 58 (1973) 419–435 https://doi.org/10.1083/jcb.58.2.419

658 [28] TURBOMOLE User's Manual, Version 7.5.1; COSMOlogic GmbH & Co. KG: Leverkusen, Germany,
659 **2021**.

660 [29] A. Schäfer, C. Huber, R. Ahlrichs, Fully Optimized Contracted Gaussian Basis Sets of Triple Zeta
661 Valence Quality for Atoms Li to Kr. J. Chem. Phys.,100 (1994) 5829-5835
662 https://doi.org/10.1063/1.467146

663 [30] F. Weigend, M. Häser, RI-MP2: First Derivatives and Global Consistency. Theor. Chem. Acc., 97
664 (1997) 331-340 https://doi.org/10.1007/s002140050269

665 [31] BIOVIA COSMOconf 2021 User Guide, Version 2021; Dassault Systèmes Customer Support :
666 Vélizy-Villacoublay, France, 2021

667 [32] E. Rodríguez-Juan, C. Rodríguez-Romero, J. Fernández-Bolaños, M.C. Florido, A. Garcia. Phenolic
668 compounds from virgin olive oil obtained by natural deep eutectic solvent (NADES): effect of the
669 extraction and recovery conditions. J Food Sci Technol 58 (2021) 552–561
670 https://doi.org/10.1007/s13197-020-04567-3

671 [33] K.M. Jeong, M.S. Lee, M.W. Nam, J. Zhao, Y. Jin, D.K. Lee, S.W. Kwon, J.H. Jeong, J. Lee Tailoring
672 and recycling of deep eutectic solvents as sustainable and efficient extraction media, J. Chromatogr.
673 A, 1424, 10-17 (2015) https://doi.org/10.1016/j.chroma.2015.10.083

674 [34] E. Hendriks, G. M. Kontogeorgis, R. Dohrn, J.-C. de Hemptinne, I. G. Economou, L. F. Žilnik, V.
675 Vesovic Industrial Requirements for Thermodynamics and Transport Properties Ind. Engin. Chem.
676 Res. 49 (22) (2010) 11131-11141 https://doi.org/10.1021/acs.iecr.0c05356
677 [35] N. Schaeffer, D. O. Abranches, L.P. Silva, M. A.R. Martins, P. J. Carvalho, O. Russina, A. Triolo, L. Paccou, Y. Guinet, A. Hedoux, J. A.P. Coutinho, Non-Ideality in Thymol + Menthol Type V Deep Eutectic Solvents, ACS Sust. Chem. Engin. 9 (5) (2021) 2203-2211
680 https://doi.org/10.1021/acssuschemeng.0c07874

681 [36] T. Hložek, T. Bosáková, Z. Bosáková, P. Tůma, Hydrophobic eutectic solvents for endocrine disruptors purification from water: Natural and synthetic estrogens study, Sep. Purif. Technol., 303, 122310 (2022) https://doi.org/10.1016/j.seppur.2022.122310

684 [37] D. van Osch, C. Dietz, S. Warrag, M. Kroon, The Curious Case of Hydrophobic Deep Eutectic Solvents: A Story on the Discovery, Design, and Applications, ACS Sust. Chem. Engin. 8 (29) (2020) 10591-10612 https://doi.org/10.1021/acssuschemeng.0c00559

687 [38] Y. Dai, J. van Spronsen, G.-J. Witkamp, Verpoorte, Y.H. Choi, Natural deep eutectic solvents as new potential media for greentechnology, Analytica Chimica Acta, 766, 61–68 (2013)
688 https://doi.org/10.1016/j.aca.2012.12.019

690 [39] Y. L. Kua, S. Gan, Natural Deep Eutectic Solvent (NADES) as a Greener Alternative for the Extraction of Hydrophilic (Polar) and Lipophilic (Non-Polar) Phytonutrients. Key Eng. Mater. 797 (2019) 20–28 https://doi.org/10.4028/www.scientific.net/KEM.797.20

693 [40] D. Smink, S. R. A. Kersten, B. Schuur, Recovery of lignin from deep eutectic solvents by liquid-liquid extraction. Sep. Purif. Technol. 235 (2020) 116127
694 https://doi.org/10.1016/j.seppur.2019.116127

696 [41] R. Verma, T. Banerjee, Liquid-Liquid Extraction of Lower Alcohols Using Menthol-Based Hydrophobic Deep Eutectic Solvent: Experiments and COSMO-SAC Predictions. Ind. Eng. Chem. Res. 57 (2018) 3371–3381 https://doi.org/10.1021/acs.iecr.7b05270

699 [42] H. Ebadnezhad, M.R. Afshar Mogaddam, A. Mohebbi, M.A. Farajzadeh, M. Nemati, M. Torbati
700 Combination of temperature-assisted ternary phase homogenous liquid-liquid extraction with deep
701 eutectic solvent-based dispersive liquid-liquid microextraction for the extraction of phytosterols
702 from cow milk and cream samples. J. Sep. Sci. 44 (2021) 1482-1489
703 https://doi.org/10.1002/jssc.202001012

704 [43] J.-B. Chagnoleau, N. Papaiconomou, M. Jamali, D. O. Abranches, J. A. P. Coutinho, X. Fernandez, T. Michel Toward a Critical Evaluation of DES-Based Organic Biphasic Systems: Are Deep Eutectic Solvents so Critical? ACS Sustain. Chem. Eng. 9 (2021) 9707-9716
705
706
707 https://doi.org/10.1021/acssuschemeng.1c01628

708 [44] S. P. Kamble, R. B. Gaikar, R. B. Padalia, K. D. Shinde, Extraction and purification of C-phycocyanin from dry Spirulina powder and evaluating its antioxidant, anticoagulation and prevention of DNA damage activity. Journal of Applied Pharmaceutical Science, 3(08) (2013) 149-153. https://doi.org/10.7324/JAPS.2013.3826
709
710
711

712 [45] Wachda, H. Hadiyanto, G. D. Harjanto, M. L. Huzain, R. W. Ajiet Production of antioxidant C-phycocyanin using extraction process of Spirulina platensis in large scale industry IOP Conf. Ser.: Mater. Sci. Eng. 633 (2019) 012025 https://doi.org/10.1088/1757-899X/633/1/012025
713
714

715 [46] R. Fernandes, J. Campos, M. Serra, J. Fidalgo, H. Almeida, A. Casas, D. Toubarro, A. Barros. Exploring the Benefits of Phycocyanin: From Spirulina Cultivation to Its Widespread Applications.
716
717 Pharmaceuticals 16(4) (2023) 2023592. https://doi.org/10.3390/ph16040592

718 [47] D. Rente, M. Cvjetko Bubalo, M. Panić, A. Paiva, B. Caprin, I. Radojčić Redovniković, A. R. C. Duarte, Review of deep eutectic systems from laboratory to industry, taking the application in the cosmetics industry as an example, J.Clean. Prod. 380 (2022) 135147
719
720
721 https://doi.org/10.1016/j.jclepro.2022.135147

722 [48] F. Chemat, M. A. Vian, G. Cravotto, Green Extraction of Natural Products: Concept and Principles.
723 Int. J. Mol. Sci. 13 (2012) 8615-8627 https://doi.org/10.3390/ijms13078615
724 [49] J. Płotka-Wasylka, W. Wojnowski, Complementary green analytical procedure index

725 (ComplexGAPI) and software. Green Chem. 23 (2021) 8657–8665

726 https://doi.org/10.1039/D1GC02318G

727
<table><caption>729 Table 1: Studied hydrophilic and hydrophobic NaDES compositions</caption><thead><tr><th colspan="2">Eutectic mixtures</th><th>Component 1</th><th>Component 2</th><th>Molar Ratio</th><th>Water content (wt%)</th><th>Density (g/mL)</th></tr></thead><tbody><tr><td rowspan="6">Non-polar</td><td>GG</td><td>Glycerol</td><td>Glucose</td><td>2:1</td><td>20%</td><td>1.303 ± 0.002</td></tr><tr><td>MC8</td><td rowspan="5">Menthol</td><td>Octanoic acid</td><td>5:1</td><td>-</td><td>0.885 ± 0.003</td></tr><tr><td>MC12</td><td>Lauric acid</td><td>4:3</td><td>-</td><td>0.881 ± 0.003</td></tr><tr><td>MO</td><td>1,2-Octanediol</td><td>1:1</td><td>-</td><td>0.907 ± 0.001</td></tr><tr><td>MP</td><td>1,3-Propanediol</td><td>1:2</td><td>-</td><td>0.963 ± 0.005</td></tr><tr><td>MT</td><td>Thymol</td><td>1:1</td><td>-</td><td>0.931 ± 0.002</td></tr></tbody></table>

730

<caption>731 Table 2: Complex GAPI indicator for sequential/LLE and SLL processes considering 1g of biomass</caption>

732 engaged

<table><thead><tr><th>GAPI Parameter</th><th>Sequential /LLE</th><th>SLL</th></tr></thead><tbody><tr><td><strong>Pre-extraction processes</strong></td><td></td><td></td></tr><tr><td>Yield and conditions</td><td></td><td></td></tr><tr><td>Yield</td><td>100 %</td><td>100 %</td></tr><tr><td>Temperature/time</td><td>max 1h 80°C</td><td>max 1h 80°C</td></tr><tr><td>Relation to green economy</td><td></td><td></td></tr><tr><td>Number of rules met</td><td>5-6</td><td>5-6</td></tr><tr><td>Reagents and solvents</td><td></td><td></td></tr><tr><td>Health hazard</td><td>No special hazards</td><td>No special hazards</td></tr><tr><td>Safety hazard</td><td>No hazard</td><td>No hazard</td></tr><tr><td>Instrumentation</td><td></td><td></td></tr><tr><td>Technical set-up</td><td>Common set up</td><td>Common set up</td></tr><tr><td>Energy</td><td>0.5 kWh/sample</td><td>0.5 kWh/sample</td></tr><tr><td>Occupational hazard</td><td>Hermetization</td><td>Hermetization</td></tr><tr><td>Work up and purification</td><td></td><td></td></tr><tr><td>End product work up purification</td><td>No work up</td><td>No work up</td></tr><tr><td>Purity</td><td>&gt; 98%</td><td>&gt; 98%</td></tr><tr><td>E-factor</td><td></td><td></td></tr><tr><td>E-factor input</td><td>0</td><td>0</td></tr><tr><td><strong>Sample preparation and extraction</strong></td><td></td><td></td></tr><tr><td>Sample preparation</td><td></td><td></td></tr><tr><td>Collection</td><td>n.a.</td><td>n.a.</td></tr><tr><td>Preservation</td><td>None</td><td>None</td></tr><tr><td>Transport</td><td>None</td><td>None</td></tr><tr><td>Storage</td><td>Under normal conditions</td><td>Under normal conditions</td></tr><tr><td>Type of method</td><td>No sample preparation</td><td>No sample preparation</td></tr><tr><td>Scale of extraction</td><td>n.a.</td><td>n.a.</td></tr><tr><td>Solvent/reagent used</td><td>Green solvents</td><td>Green solvents</td></tr><tr><td>Additional treatment</td><td>none</td><td>none</td></tr><tr><td>Reagents and solvents</td><td></td><td></td></tr><tr><td>Amount</td><td>&lt; 10 g</td><td>&lt; 10 g</td></tr><tr><td>Health hazard</td><td>No special hazards</td><td>No special hazards</td></tr><tr><td>Safety hazard</td><td>No hazards</td><td>No hazards</td></tr><tr><td>Instrumentation</td><td></td><td></td></tr><tr><td>Energy</td><td>2.36 kWh per sample</td><td>0.79 kWh per sample</td></tr><tr><td>Occupational hazard</td><td>Hermetic</td><td>Hermetic</td></tr><tr><td>Waste</td><td>No waste</td><td>No waste</td></tr><tr><td>Waste treatment</td><td>n.a.</td><td>n.a.</td></tr><tr><td>Method type</td><td></td><td></td></tr><tr><td>Type of analysis</td><td>n.a.</td><td>n.a.</td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>Figure 1: Extraction protocol for the sequential (A) and triphasic solid/liquid/liquid (B) extraction strategy</figcaption></figure>

734

736

<figure><img src="image_3.png" /></figure>
737 Figure 2: Pigment profile (A) and FFA profile (B) for reference extractions

738
<figure><img src="image_4.png" /><figcaption>Figure 3: Pigment profile (A) and FFA profile (B) for reference solid/liquid extraction vs sequential</figcaption></figure>

739

<figure><img src="image_5.png" /><figcaption>solid/liquid extraction of GG pellet</figcaption></figure>

740

741

<caption>Figure 4: Phycocyanin content after liquid-liquid extraction of the polar phase GG</caption>
A

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>

742

743 Figure 5: Pigment profile (A) and FFA profile (B) for sequential extraction compared to reference

<figure><img src="image_8.png" /></figure>

744 extraction

745 Figure 6: Typical color difference between monophasic, LLE and biphasic extracts.

746
747

748
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>Figure 7: Phycocyanin content of the polar phase GG after SLL extraction</figcaption></figure>

749

<caption>Figure 8: Pigment profile (A) and FFA profile (B) for biphasic extraction</caption>

750
A Non Polar pigments and productivity

<figure><img src="image_11.png" /></figure>

B FFA and productivity

<figure><img src="image_12.png" /></figure>

751

<figure><img src="image_13.png" /><figcaption>752 Figure 9: Comparison of the sequential/LLE and SLL approach in terms of productivity</figcaption></figure>

753
Figure 10: Sustainability analyses of sequential LLE strategy vs SLL approach using using ComplexGAPI
tool ⁴¹ Pictograms were drawn by a freeware software available at:

https://mostwiedzy.pl/en/justyna-plotka-wasylka,647762-1/complexgapi (accessed on 06/30/2023)

(red fields indicate steps that may be hazardous to the environment, yellow fields indicate steps with
medium environmental impact, and green fields reflect eco-friendly steps).